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Progress in Botany 62 Springer-Verlag Berlin Heidelberg GmbH 62 PROGRESS IN BOTANY Genetics Physiology Systematics Ecology Edited by K. Esser, Bochum U. Llittge, Darmstadt J. W. Kadereit, Mainz W. Beyschlag, Bielefeld , Springer With 53 Figures ISSN 0340-4773 ISBN 978-3-642-52378-6 DOI 10.1007/978-3-642-56849-7 ISBN 978-3-642-56849-7 (eBook) The Library of Congress Card Number 33-15850 This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 2001 Originally published by Springer-Verlag Berlin Heidelberg New York in 2001 Softcover reprint of the hardcover 1st edition 2001 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: Design & Production, Heidelberg Typesetting: M. Masson-Scheurer, Heidelberg SPIN 10723375 3113130 - 5432 1 0 - Printed on acid-free paper Contents Contents Review 50 Years of Translocation in the Phloem of Plants, With Reference to Trees ........................................................................ Walter Eschrich (With 5 Figures) 3 Genetics Introns, Splicing and Mobility ............................................................. Ralf Sagebarth and Ulf Stahl (With 4 Figures) 15 1 2 3 4 5 15 15 18 Introduction ................................................................................... Intron Structure and Splicing Pathway....................................... Intron Distribution ........................................................................ Protein-Assisted Splicing .............................................................. Intron Mobility .............................................................................. a) Protein-Assisted Homing ..................................................... b) Transposition to Ectopic Sites ............................................. 6 Future Prospects ............................................................................ References ............................................................................................ 20 22 22 25 28 29 Barley Mutagenesis ................................................................................ Anders Falk, Alan H. Schulman, S0ren K. Rasmussen, and Christer Jansson 34 1 2 34 34 34 Introduction ................................................................................... Construction and Utilization of Barley Mutant Libraries a) Barley Mutants Induced by Radiation or Chemicals ........ b) Molecular Analysis of Barley Mutants Induced by Radiation or Chemicals ................................................... c) Fast Forward Genetics; Chromosome Landing Refined ... d) Barley Reverse Genetics ....................................................... e) Extending the Mutant Banks of Barley: Contribution from Arabidopsis ........................................... 35 36 38 39 VI Contents 3 Transposable Elements As Major Contributors and Tools in Genomic Mutagenesis .............................................................. a) The Mutagenic Impact and Application of DNA Transposons ............................................................ b) The Nature of Retro-Transposons ...................................... c) The Mutagenic Impact and Application of Retro-Transposons ........................................................... 4 Mutational Breeding in Barley: an Example. Improving Nutritional Qualities .................................................. References ............................................................................................ Extranuclear Inheritance: Cytoplasmic Linear Double-Stranded DNA Killer Elements of the Dairy Yeast Kluyveromyces lactis .............................................. FriedheIm Meinhardt and Raffael Schaffrath (With 4 Figures) 1 2 3 Introduction ................................................................................... Genetic Organization of the Killer Plasmids .............................. Zymocin Toxin .............................................................................. a) Structure ................................................................................ b) Biogenesis, Assembly and Secretion ................................... c) Immunity ............................................................................... d) Mode of Action ..................................................................... e) Resistance .............................................................................. 4 Replication ..................................................................................... 5 Gene Expression ............................................................................ a) Transcription ......................................................................... b) Translation ............................................................................ 6 Phylogeny ....................................................................................... 7 Conclusions and Outlook ............................................................. References ............................................................................................ Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria ..................................................................................... Karlheinz Esser, Thomas Lisowsky, Georg Michaelis, and EIke Pratje (With 4 Figures) 1 2 3 Introduction ................................................................................... Mitochondrial Genomes ............................................................... a) Saccharomyces cerevisiae ..................................................... b) Petite-Positive and Petite-Negative Yeasts ......................... c) Linear Mitochondrial Genomes .......................................... Evolution of the Mitochondrial Genomes .................................. a) Origin of Mitochondria ........................................................ b) Transfer of Mitochondrial Genes to the Nucleus .............. c) Gene Transfer to Mitochondria ........................................... 39 39 41 42 43 46 51 51 52 53 53 54 55 56 57 59 61 61 63 63 65 65 71 71 71 71 72 72 72 72 73 74 Contents VII 4 74 76 Cross-Talk Between Mitochondria, Cytosol and the Nucleus .. a) Retrograde Regulation ........................................................ . b) Proteins with Dual Function and/or Dual Localization ... . c) New Aspects ......................................................................... . 5 Mitochondrial Protein Transport in Higher Plants .................. . a) Pre-Sequences ...................................................................... . b) The Translocases of the Mitochondrial Outer and Inner Membranes ......................................................... . c) Processing Peptidases .......................................................... . d) Chaperones ........................................................................... . e) Developmentally Regulated Protein Import ..................... . f) Differences Between Plants and Yeast ............................... . g) Protein Export ...................................................................... . References ........................................................................................... . Genetics of Phytopathogenic Bacteria ............................................... . Jutta Ahlemeyer and Rudolf Eichenlaub 1 2 3 4 5 6 Introduction .................................................................................. . The hrp Genes of Phytopathogenic Bacteria ............................ .. The Type III Secretion System .................................................... . Regulation of hrp Genes .............................................................. . Harpins and Avr Proteins ............................................................ . Plant Defense ................................................................................ . a) Recognition of the Pathogen .............................................. . b) Signal Transduction ............................................................ . c) Hypersensitive Cell Death and Other Locally Induced Defense Reactions ................. . 7 Outlook .......................................................................................... . References 78 79 80 81 82 84 85 85 86 87 89 98 98 99 99 102 103 105 105 105 106 108 108 Plant Biotechnology: Transgenic Crops for the Third Millennium ..................................... . 114 Frank Kempken (With 1 Figure) 1 2 Introduction .................................................................................. . Methods for Gene Transfer and Expression of Foreign DNA in Higher Plants ............................................................................ . a) Agrobacterium-Mediated Transformation ....................... . b) Transformation of Protoplasts ........................................... . c) Biolistic Transformation ..................................................... . d) Regeneration, Selection and Verification of Transformed Plants ......................................................... . e) Targeted Gene Expression .................................................. . f) Silencing of Transformed Genes ........................................ . g) Removal of Marker Genes ................................................... . 114 116 117 117 117 118 119 120 120 VIII Contents 3 Improvements in Agriculture ...................................................... a) Enhancing Plant Resistance ................................................. ex) Herbicide Resistance ..................................................... 13) Insect Resistance ........................................................... y) Resistance to Viral Pathogens ...................................... 8) Resistance Against Bacterial and Fungal Pathogens ................................................... b) Artificially Introduced Male Sterility to Produce Hybrid Seeds ...................................................... c) Improving Plant Micronutrients ......................................... d) Modified Carbohydrates in Transgenic Crops .................. e) Biodegradable Plastics from Transgenic Plants ................ f) Production of Vaccines ........................................................ 4 Current Use of Genetically Engineered Plants and Future Prospects .................................................................... 5 Recent Controversies Regarding the Safety of GM Plants ........ References ............................................................................................ 121 121 122 122 123 Modification of Oilseed Quality by Genetic Transformation Steffen Weber, Karim Zarhloul, and Wolfgang Friedt (With 3 Figures) 140 1 2 3 140 141 144 144 147 149 150 151 151 154 156 157 161 164 166 Introduction ................................................................................... Biosynthesis and Composition of Vegetable Oil ........................ Regeneration Capacity of Oilseed Plants .................................... a) Oilseed Rape (B. napus L.) .................................................. b) Sunflower (H. annuus) ......................................................... 4 Tools for Genetic Transformation of Oil Plants ......................... a) Biolistic Approach ................................................................ b) Agrobacterium-Mediated Transformation ........................ ex) Oilseed Rape .................................................................. 13) Sunflower ....................................................................... 5 State of the Art ............................................................................... a) Transgenic Oilseed Plants with Novel Traits ..................... b) Release of Transgenic Oilseeds into the Field .................... 6 Future Trends and Perspectives ................................................... References ............................................................................................ 123 124 125 125 126 126 127 129 131 Physiology Significance of Phloem-Translocated Organic Sulfur Compounds for the Regulation of Sulfur Nutrition ................................................ 177 Cornelia Herschbach and Heinz Rennenberg (With 1 Figure) 1 2 Introduction ................................................................................... 177 Phloem-Translocated Organic Sulfur Compounds ................... 179 Contents 3 Consequences of Phloem Transport of Sulfur for the Organic Sulfur Content ofthe Roots ............................... 4 Regulation of Sulfate Uptake ....................................................... 5 Consequences of Phloem Translocation for the Regulation of Sulfate Uptake ........................................................................... 6 Conclusions .................................................................................... References ............................................................................................ IX 181 183 186 188 189 Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) ............................................................................... 194 Hartmut Gimmler (With 7 Figures) 1 2 3 Introduction ................................................................................... General Aspects ............................................................................. Mutualistic Relationships Between Algae and Fungi in Acidic Habitats .......................................................................... a) Stimulation of Growth .......................................................... b) Benefit of Algae Association with Fungi ............................ a.) Vitamins ......................................................................... 13) Dissolved Inorganic Carbon ........................................ y) Abscisic Acid .................................................................. &) Amino Acids .................................................................. c) Benefit of Fungi or Yeasts in Associations with Algae ...... a.) Carbon Source ............................................................... 13) Oxygen ............................................................................ 4 Mutualistic Relationships Between Algae and Bacteria ............ 5 The Geosiphon Association .......................................................... References ............................................................................................ 194 195 196 196 199 199 203 204 204 204 204 206 207 210 212 The Extracellular Matrix of the Plant Cell: Location of Signal Perception, Transduction and Response ............ 215 Karl-Josef Dietz (With 2 Figures) 1 2 The Extracellular Matrix is a Dynamic Component of the Plant Cell .............................................................................. a) The Cell-Wall Structure and the Proteins of the ECM ...... b) The Chemical Composition ofthe Apoplast ...................... c) Remodeling ofthe ECM During Development .................. d) Responses to Environmental Stimuli ................................. e) Pathogenesis-Related Deconstruction or Adaptation of the Cell Wall ...................................................................... Signal Perception in the Extracellular Space .............................. a) Receptor Kinases ................................................................... b) Seven-Trans membrane-Domain Receptors ....................... c) Evidence for the Existence and Involvement of Integrin-Like Proteins ...................................................... 215 216 218 219 220 221 222 222 225 225 x Contents d) Arabinogalactan Proteins .................................................... e) Ion Channels ......................................................................... 3 Signal Transduction ...................................................................... 4 ECM Formation and Remodeling ................................................ a) Enzymatic Activities in the Apoplast .................................. b) Proteases ................................................................................ c) Expansins ............................................................................... 5 Perspectives .................................................................................... References ............................................................................................ 227 227 228 228 228 229 231 232 232 Photosynthesis: Carbon Metabolism from DNA to Deoxyribose Grahame J. Kelly (With 3 Figures) 238 1 2 Introduction ................................................................................... The Chloroplast ............................................................. ................ a) Ribulose-Bisphosphate Carboxylase/Oxygenase ............... b) Other Calvin-Cycle Enzymes ............................................... c) Transitory Starch Metabolism ............................................. 3 The Photosynthetic Cell ................................................................ a) Uptake ofInorganic Carbon ................................................ b) Sucrose Biosynthesis ............................................................ c) The Enigma of Glucose and DNA ....................................... d) Mitochondrial Respiration and the Oxidative Pentose-P Pathway ................................ e) Photorespiration ................................................................... f) C4 Photosynthesis ................................................................. g) Crassulacean Acid Metabolism ........................................... 4 The Whole Plant ............................................................................ a) Translocation ........................................................................ b) CO 2 Fixation by Stressed Plants .......................................... c) CO 2 Fixation when CO 2 Supply is Abundant ..................... References ............................................................................................ 238 239 239 241 243 244 244 246 246 The Metabolic Diversity of Plant Cell and Tissue Cultures Otto Grather and Bernd Schneider 266 1 2 266 Introduction and Objectives ........................................................ Metabolic Diversity of Higher Plants and Their In Vitro Cultures .......................................................... 3 Novel Natural Products from Cell and Tissue Cultures of Higher Plants ............................................................................. 4 Strategies to Induce the Formation of Natural Products in Plant-Cell and Tissue Cultures ................................................ 5 Conclusions .................................................................................... References ............................................................................................ 248 249 250 251 253 253 254 255 256 267 269 293 295 296 Contents XI Systematics Molecular Systematics: 1997-1999 ....................................................... 307 Kenneth J. Sytsma and William J. Hahn Introduction ................................................................................... Progress from 1997 to 1999 .......................................................... 3 Advances in Methodology ............................................................ a) DNA Extraction ..................................................................... b) New Genes for Phylogenetics .............................................. c) DNA Fingerprinting ............................................................. d) Data Analysis ......................................................................... 4 Systematic Progress in Non-Angiosperms ................................. 5 Systematic Progress in Angiosperms .......................................... a) Basal Angiosperms (Excluding Monocots) ........................ b) Basal Angiosperms: Monocots ............................................ c) Basal Eudicots ....................................................................... d) CaryophylIids ........................................................................ e) Rosids ..................................................................................... f) Asterids...... .......................... ................ .................................. 6 Hybridization and Introgression ................................................. 7 Polyploid Origins ........................................................................... 8 Biogeography and Phylogeography ............................................. 9 Interfacing Ecology and Systematics ........................................... 10 Interfacing Development and Systematics ................................. 11 Future Prospects and Problems ................................................... References ............................................................................................ 1 2 307 308 310 310 310 311 311 313 314 315 315 316 317 318 319 321 322 322 324 325 326 327 Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny............................................................... 340 Michael Melkonian Introduction ................................................................................... Genomics Meets Phylogeny ......................................................... Bonsai Genomics: the Phylogeny of Mitochondria, Plastids and Nucleomorphs ........................................................................ a) Mitochondria/Hydrogenosomes ......................................... b) Plastids and Nucleomorphs ................................................. References ............................................................................................ 340 341 Systematics of Bryophytes Patricia Geissler 383 1 2 3 1 2 3 4 General Aspects ............................................................................. Jubilee (Festschrift) and Special Volumes .................................. Phylogeny ....................................................................................... Speciation, Population Biology .................................................... 347 347 354 370 383 383 384 387 XII Contents 5 6 Taxonomy, Revisions .................................................................... Flora Checklists ............................................................................. 7 Conservation .................................................................................. References ............................................................................................ 388 390 391 392 Ecology The Search for Generality in Studies of Disturbance and Ecosystem Dynamics ..................................................................... 399 Peter S. White and Anke Jentsch (With 11 Figures) 1 2 3 4 Introduction ................................................................................... Why Study Disturbances? Why Seek Generality? ..................... a) Patchiness and Heterogeneity ............................................. b) Biodiversity, Adaptation and Ecosystem Response .......... c) Human Effects on Natural Disturbance Regimes .............. d) Novel Human Disturbances ................................................. e) Habitat Fragmentation ......................................................... f) Traditional Land Uses as Disturbance Regimes ................ g) Climate Change ..................................................................... h) Exotic-Species Invasions ...................................................... i) Why Seek Generality? ........................................................... Disturbances and Disturbance Regimes ..................................... a) Problems with the Relative Definition ................................ b) The Absolute Definition ....................................................... c) Diffuse and Discrete Disturbances ...................................... d) Site Potential and Class-I and Class-II Disturbances ........ e) Other Definition Issues ........................................................ f) Heterogeneity, Homogeneity and Scale ............................. g) From Disturbance Event to Disturbance Regime with Spatio-Temporal Dimensions ..................................... What Hinders the DeVelopment of Generality in Disturbance Ecology? ............................................................... a) Spatial and Temporal Variation in Disturbance Events ... a) Disturbances Interact with a Unique Topography Template ......................... 13) Disturbances Vary with Climate .................................. y) Disturbances Vary with Soil Development ................. 8) Disturbances Vary with Feedback and Interactions .. b) Spatial and Temporal Variation in the Effects of Disturbance and Ecosystem Responses to Disturbance ....................................................................... a) Disturbances Vary in the Heterogeneity They Create .................................................................... 13) Disturbances Vary in Patch Size .................................. 399 399 400 400 401 401 402 402 403 404 404 404 406 407 409 409 411 412 412 414 414 415 416 416 416 418 418 419 Contents XIII Disturbances Differ in Intensity and Severity and, Hence, in Ecosystem Legacy ............................... . c) Rates of Response and Species Adaptations Vary Among Ecosystems .............................................................. . d) Methods of Sampling and Analysis ................................... . a) The Scale of Observation Affects Conclusions Because Disturbances Are Episodic and Patchy ....... . 13) Surrogate Variables Are Often the Only Ones Measured ............................................. . y) Disturbances Vary Both Stochastically and Deterministically ................................................... . Approaches to Generality in Disturbance Ecology ................... . 5 a) Approaches to Generality at the Patch Scale .................... . a) Absolute Ecosystem Characteristics and Disturbance Effects ............................................... . 13) Legacies Produced Disturbance .................................. . y) Relativizing Patch Conditions to Ecosystem Charateristics ........................................ . 8) Comparing Disturbances with Historic Precedents .............................................. . E) Disturbance Effects on Site Quality and Ecosystem Trajectory ........................................... . b) Approaches to Generality at the Multiple-Patch Scale .... . a) Patch Dynamics and Dynamic Equilibrium .............. . 13) Disturbance Architecture ............................................ . y) Classifying Disturbance Regimes ............................... . c) Approaches to Generality Through the Classification of Species Roles .................................................................... . a) Successional Roles and the Intermediate-Disturbance Hypothesis ......... . 13) Response of Functional Groups to Disturbance ....... . y) Resilience to Disturbance ............................................ . 8) Dominant Growth Forms ............................................ . 6 Conclusions ................................................................................... . a) Choosing a Focus of Interest, Level of Resolution and Degree of Abstraction .................................................. . b) Establishing the Spatial and Temporal Frame of Reference .......................................................................... . c) Describing Disturbance ....................................................... . d) Determining Patterns in Disturbance RegimesCorrelation of Spatial and Temporal Parameters ............ . References y) 420 421 422 423 424 424 425 426 426 428 428 429 429 430 430 433 434 434 434 438 439 439 439 440 440 441 442 443 XIV Contents Heterogeneous Soil-Resource Distribution and Plant Responsesfrom Individual-Plant Growth to Ecosystem Functioning ............... 451 Elisabeth Huber-Sannwald and Robert B. Jackson 1 2 3 4 5 6 Introduction ................................................................................... Background .................................................................................... Abiotic Causes of Soil-Resource Heterogeneity ......................... Biotic Causes of Soil-Resource Heterogeneity ........................... Patterns of Heterogeneity ............................................................. Response Variables at Different Levels of Organization ........... a) Plant Responses .................................................................... b) Plant-Fungus Interaction .................................................... c) Plant-Plant Interactions Within and Between Populations of Different Species .......................... d) Plant Community/Ecosystem Responses ........................... 7 Conclusions and Future Directions ............................................. References ............................................................................................ 451 452 453 454 456 457 458 461 462 464 466 467 The Existence of Bark and Stem Photosynthesis in Woody Plants and Its Significance for the Overall Carbon Gain. An Eco-Physiological and Ecological Approach ................................ 477 Hardy Pfanz and Guido Aschan (With 8 Figures) 1 2 3 4 5 Introduction ................................................................................... Anatomy and Morphology ofthe Bark and Rhytidome ............ a) The Hidden Chlorenchyma: Nomenclature Problems ..... b) Location and Anatomy......................................................... a) The Sub-Corticular Chlorenchyma ............................. (3) The Lenticels .................................................................. The Chlorophyll Content of Stems: as Green as Leaves? .......... a) How Much Chlorophyll Is It? ............................................... Microclimatic Considerations ...................................................... a) The Micro- and Nano-Climates ofRhytidome and Bark ................................................................................. b) Bark Temperatures ............................................................... c) How Much Light Reaches the Chlorenchyme? ................. a) Peridermal and Rhytidomal Light Transmission ...... (3) Light Penetration Through Lenticels .......................... y) Light Penetration: Spectral Discrimination ................ The Source of CO 2: Stem-Internal or -External CO 2? ................ a) How Much CO 2 Is Inside the Stem? .................................... b) CO 2 Flux in Stems: from Inside to Outside or Vice Versa? ........................................................................ a) Diffusion of CO 2 Out ofthe Stem ................................ (3) Aqueous Transport of CO 2 ........................................... y) CO 2 Consumption Via Bark Photosynthesis .............. 477 481 481 483 483 483 484 485 486 486 487 487 488 488 488 490 490 492 492 493 494 Contents 6 XV Measurements of Bark Photosynthesis ....................................... a) Corticular Photosynthesis and Diffusion Problems .......... b) Light Response of Bark Photosynthesis ............................. c) Can Extremely High CO2 Partial Pressure Negatively Affect Corticular Photosynthesis? .................... d) Wood Photosynthesis: Evidence of a Fairy Tale ................ 7 Ecological Considerations ............................................................ a) Corticular Photosynthesis and Stress ................................. a) Interactions with Epiphytes ......................................... (3) Air Pollution .................................................................. b) Corticular Photosynthesis at the Whole-Plant Level ........ c) Contributions of Corticular Photosynthesis to the Carbon Balance .......................................................... 8 Open Questions and Aims for Further Studies .......................... References ............................................................................................ 494 494 496 Ecological Aspects of Clonal Growth in Plants Hansjorg Dietz and Thomas Steinlein 511 1 2 511 512 512 513 515 516 517 517 519 520 521 521 Introduction ................................................................................... Sexual Versus Clonal Propagation .............................................. a) Allocation Plasticity and Costs ............................................ b) Dispersal and Population Development ............................. c) Genetic Diversity ................................................................... 3 Implications of Herbivory and Disease for Clonal Growth ...... 4 Processes Within Clones (Clonal Fragments) ............................ a) Physiological Integration ..................................................... b) Division of Labor .................................................................. c) Foraging ................................................................................. 5 Processes Between Clones (Clonal Fragments) .......................... a) Competition .......................................................................... b) Patterns in the Development of Clonal Plant Populations at the Community Level ....................................................... 6 Conclusions .................................................................................... References ............................................................................................ 496 497 498 498 498 499 501 501 503 504 523 524 525 Subject Index .......................................................................................... 531 List of Editors Professor Dr. Dr. h. c. mult. K. Esser Lehrstuhl fur Allgemeine Botanik, Ruhr Universitat Postfach 10 21 48 44780 Bochum, Germany Phone: +49-234-32-22211; Fax: +49-234-32-14211 e-mail: [email protected] Professor Dr. U. Wttge TU Darmstadt, Institut fur Botanik, FB Biologie (10) SchnittspahnstraBe 3-5 64287 Darmstadt, Germany Phone: +49-6151-163200; Fax: +49-6151-164808 e-mail: [email protected] Professor Dr. J. W. Kadereit Institut fUr Spezielle Botanik und Botanischer Garten Universitat Mainz, SaarstraBe 21 55099 Mainz, Germany Phone: +49-6131-392533; Fax: +49-6131-393524 e-mail: [email protected] Professor Dr. W. Beyschlag Fakultat fUr Biologie, Lehrstuhl fUr Experimentelle Okologie und bkosystembiologie Universitat Bielefeld, UniversitatsstraBe 25 33615 Bielefeld, Germany Phone: +49-521-106-5573; Fax: +49-521-106-6038 e-mail: [email protected] Walter Eschrich was born on October 4,1924, in Breslau Silesia. He went to school in Silesia. His father, Dr. Friedrich Eschrich, was head of a high school (Oberstudiendirektor), and his mother, Elisabeth Eschrich (nee Florian), was from Breslau. There was an interruption of his school time in 1942 due to the draft for war-time army service. He had his preliminary graduation exams in Breslau. He was wounded in Russia (amputation of the lower leg) in May 1944. Thereafter, he repeated his graduation exam (Abitur) in Rheinhausen, Lower Rhine. Beginning in 1945, he studied biology at the University of Bonn. He received his doctorates in botany, chemistry and zoology. He has been married to Dr. Berthilde Eschrich (nee Zimmermann) of St. Wendel, Saar since 1955; they have a son, Ivo, born in 1969. Dr. Eschrich began teaching pharmacognosy at the University of Bonn after obtaining his doctorate in botany in 1953. Since 1968, he has been teaching botany at the University of G6ttingen. He has been a Professor Emeritus since 1993. Review 50 Years of Translocation in the Phloem of Plants, With Reference to Trees Walter Eschrich Fifty years ago botanists were convinced that the photosynthetically produced sucrose in the green leaves of autotrophic plants is transported into the sieve elements and translocated via the symplast to the sinks, where it is used to make cytoplasm and carbohydrates, especially those of the cell walls. Anatomic investigations appeared to be necessary. When I asked Walter Schumacher in Bonn for a thesis topic, he introduced me to the phloem as the tissue which takes care of sucrose transport in plants. Sucrose gives rise to all polymer products of plant life: cell walls, wood, cytoplasm" protein, enzymes, starch, slimes, dyes. Sucrose and its galactosides, raffinose, stachyose and verbascose, were found in phloem exudates. Also, a few other oligosaccharides, such as ketose and fructans, were found in plant juices, but they constitute vacuolar contents and not phloem exudates. The origin of any phloem exudate is the sieve tube or the sieve cell. Sucrose is transported to these locations. It is the sap that moves inside the phloem elements and supplies the living cells with water, salts, and nutrients. However, energy for life processes also moves through the phloem, similar to the electric current in a wire; phloem exudate can be regarded as the blood of the higher plant. As in animals, this "blood" can be shed by wounding; however, wounds can also be healed by forming wound sieve tubes (Eschrich 1953). When searching for other characteristics of the sieve elements, it was found that the nucleus is missing in sieve elements and that callose occurs in sieve elements. Callose is a B-1,3-gluco-polysaccharide. It appears regularly in sieve tubes, but its function is still unknown (Eschrich 1954, 1956). Callose is well defined by its staining behavior: it produces a bright yellow fluorescence with dilute aniline blue; with resorcinol blue the callose plugs stain blue (Eschrich 1953). In fact, staining reactions help to recognize, but not to clarify, the function of a polysaccharide, probably with the notable exception of the well-known iodine-starch reaction. Callose is a plant product, but it is not restricted to the phloem. Deposits of callose are the plugs in pollen tubes, and in hyphae of some Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 4 Review fungi. The wall of the pollen mother cells is known to consist of callose; other sources of callose are the walls of laticifers. Callose appears in anthers, pollen tubes, and pollen grains. In root hairs, trichomes, cystoliths, pit channels, lenticels, and even green algae, for example Caulerpa prolifera, callose is present (Eschrich 1956), but a unifying function is not known, except that callose is frequently deposited in channels, where the protoplasmic connection from cell to cell is narrowing as in pits. Later, I was mainly concerned with the question regarding the function of the sieve tube, where callose seemed to be important. It was accepted that the answer could be found by observations on the sieve tube itself. Some dyes were known to move in the sieve tubes. One of them is potassium-fluorescein when applied as a watery solution to phloem bundles. This was the beginning of fluorescence microscopy, a technique that has been extended to innumerable organic dyes in recent years. Additional results were obtained with radioactive tracers. Since 14C02 is assimilated by the green leaves, all compounds deriving from the labeled sugars, 14C0 2 must have been available as a gas under photosynthetic light conditions and, since photosynthesis primarily delivers sucrose in green leaves, it was clear that all labeled compounds synthesized in this way must have been produced by photosynthesis. Biochemical techniques easily separate the labeled compounds (Eschrich and Kating 1964; Kating and Eschrich 1964). Eventually, the labeling technique was extended to the localization of labeled tissue and cells (Fritz and Eschrich 1970; Fig. 1). Transposing this technique to microscopic dimensions and covering the tissue section with a photographic emulsion lead to microautoradiographs, in which the dissolution can be so low that single starch grains or mitochondria can be discriminated under the light microscope by their label (Fig. 1). These first steps into experimental plant physiology opened a field to me, which made me curious, and it is still an exciting field of research for me. Fig.!. Schematic representation of MUnch's (1930) original experiment, in which sacs 1 and 2 were semipermeable membranes containing different concentrations of impermeant solutes (C 1<C2 ) Arrows indicate direction of water flow 50 Years of Translocation in the Phloem of Plants, With Reference to Trees 5 How to Determine What Is Translocated in the Phloem There is no doubt that sieve elements translocate sugar and several other products. Which sugar? Probably the same sugar that is produced by photosynthesis in the leaves. It is sucrose, and the honeydew of aphids, which feed on sieve tubes, also consists of sucrose. Aphids have very fine tools for piercing a single sieve tube, and the sieve-tube exudate emanating from the opened sieve tube penetrates the plasmalemma for a long period before the wound is plugged. This sweet honeydew is collected and sold as a delicious syrup in wood-rich areas, e.g. in the north German Harz Mountains. Naturally, wood honey is pure sieve-tube or sieve-cell sap. If labeled with 14C, its radioactivity indicates from where it comes. Certainly, leaves fumigated with 14C0 2 will produce labeled sucrose, which can be extracted and chromatographed. Another question pertains to how the sieve-tube sap moves into and inside the sieve tube. Does this translocation always go in the same direction, up or down, and what happens on a ramification of the path? One of the most interesting problems in translocation physiology is: How will the sucrose be moved in sieve tubes: by suction? by pressure? Ernst Munch (1930) developed the concept of pressure flow, which he called "Druckstrom" (Fig. 2). However, experimental support was missing. Therefore, in our lectures, we use a model which looks quite simple (Young et a1.1973; Figs. 3-5). A twig of spruce, infected with spruce aphids, is exposed to 14C0 2 • Using illumination, the twig will photosynthesize, and the green cells will produce 14C-Iabeled sucrose. The latter can be obtained as wood honey and, after freeze-drying the twig, microtome sections of the needles can be coated with photographic emulsion, which - after exposure - will produce micro auto radiographs, which show where 14C0 2 has been assimilated. Structure - Function Relations A new technique, called "patch clamp" has been recently developed by Neher and Sackman in the Max-Planck Institute in Gottingen. This technique allows physical analyses across the tonoplast. A description understandable for botanists was published by Hedrich and Schroeder in 1989. Diversity and persistence of the higher plants rely on seed dispersal. The vegetative propagation by buds, roots, tubers, and rhizomes would cause less diversity of plants; it would be invariable cloning. In contrast to herbaceous plants, trees, in general, start to flower late in their onto- 6 Fig. 2 Review 50 Years of Translocation in the Phloem of Plants, With Reference to Trees 7 rs 91 sl rs Fig. 3. Device used to observe movement of sugar-dye solutions (stippled) inside semipermeable tubes. b Length of semipermeable membrane; ds disposable syringe; gt glass tube; rs rubber stopper; sc stopcock; srt silicone rubber tube; st semipermeable tube; x position of front above outlet of stopcock genetic development, in extreme cases not before 30 years of growth. In the meantime, survival of a tree species depends on vegetative growth. This includes food acquisition for growth in height and circumference, and it requires water, light, and acclimatization to environmental conditions such as drought or cold. Despite these abilities, structures have been developed by plants that are based mainly on cambial growth. For the evaluation of cambial growth, tree-like organisms, such as palms, which have no secondary growth, can be observed. Palms do not grow in thickness. Their stem is ... Fig. 2. Microautoradiographs of a series of 1-flm-thick cross sections of a phloem bundle of the stem of Vicia faba. The blade of the superimposed leaf was exposed for 35 min to 130 flCi 14C02. Sections were stained with gentian violet. Exposure time 131 days; brightfield illumination: 190x. a-e Subsequent sections; developed for 20 min at 20 DC. f Developed for 6 min at 20 DC. g Part of a cross section of a vascular bundle from a Vida faba stem, which was labeled as in a. Freezing in dry ice caused rupture of cell walls in the cambial region (ca) by ice crystals. Exposure time was 216 days; stained with gentian violet; developed for 20 min at 20 DC; 220x. ca Cambial region; cc companion cell; st sieve tube; x metaxylem Review 8 Fig.4. Setup used for demonstration of bidirectional front movement in a single semipermeable tube (st). 1.5 M Sucrose-amidoschwarz solution introduced at A; 1.5 M sucrose-tartrazine solution introduced at B gtp st Fig. 5. Setup for simulation of narrow tube. A glass tube (gt) with 6 mm outer diameter was inserted into semipermeable tube (st) leaving two narrow channels, as seen in sectional view. The glass tube was open at both ends 50 Years of Translocation in the Phloem of Plants, With Reference to Trees 9 composed entirely of leaf traces that have no stabilizing elements, thus differing from those occurring in leaf veins. Since secondary growth does not occur in palms, only primary phloem elements are functional, obviously for many years. Seasonal changes of structures do not occur in palms; the palms are designed to continue with primary growth. The origin of the cambial meristem can be seen in "primary" meristems with longevity, which are restricted to tree-like perennials. Some mono cots like Dracaena, Cordyline, Yucca, and Aloe produce new stem bundles by monopleuric cambia, but they do not increase in thickness. The commonly applied explanation for this growth is the basipetal extension of the apical meristem in the form of a mantel with a lateral meristem. This would fit the definition of the concentric stem bundles being the lower ends of leaf traces. However, since the stem bundles are embedded in "secondary" parenchyma, deriving from the monopleuric cambium, this parenchymatous tissue forms no medullary rays, since a medulla is lacking. Therefore, the term conjunctive parenchyma seems to be appropriate (Eschrich 1995). A similar situation is given in the stems of Bougainvillea spectabilis and other Nyctaginaceae. There, the stem bundles are also produced by a monopleuric cambium, but they develop centripetally into the open collateral bundles, which means they develop longitudinal stripes of bipleuric cambium. Pteridophytes, on the other hand, produce hadrocentric bundles, which occur in the stems of Cyathea, Sphaeropteris, and other tree ferns, or in extended branched rhizomes (Pteridium aquilinum). These bundles develop no stripes of cambial tissue; pteridophytes do not seem to constitute a step in the phylogenetic development of the cambium. Trees with cambial growth are designed for life in climates with winter and summer, standing 100 or more years in the spot where they germinated. They adapt to environmental circumstances. When they grow in the open, trees keep their lower branches, which may reach the ground, while trees growing in dense forest often shed the lower branches with increasing height of the forest canopy, where subcoronary light intensity becomes insufficient for net photosynthesis. Since the upper side of the branch insertion remains wet from rain, wooddestroying fungi develop there and cause the dead branches to break off (Butin and Kowalski, pers. commun.). Photosensitivity The orthotropic growth of tree seedlings indicates that light is perceived in the same way as in herbaceous plants. The photoreceptors in trees act similarly to the phytochrome in herbaceous plants. 10 Review When the daily radiation in early spring has increased, starting in January, maple trees (Acer pseudoplatanus) begin to produce cambial sap when 13,000 J cm-2 have reached the crown region. In birch (Betula pendula), 22,000 J cm- 2 is necessary for cambial sap production (Essiamah 1982; Essiamah and Eschrich 1995). Many tree buds have brown scales. The outer bud scales of the beech (Fagus sylvatica) transmit light only from the red (700 nm) region of the spectrum and increasingly toward the infrared (>800 nm) (Eschrich 1995). Tree buds are initiated in the previous summer. At this early stage it is decided whether a bud will develop into a flower or whether a vegetative bud will be initiated. In some trees, which flower prior to leaf emergence (cherry, apple, maple, red bud), the flower buds have already formed during the previous year. Even here, determination of a flower bud does not seem to be correlated with short or long days. Assimilate Transport In contrast to annual herbaceous plants, a tree provides assimilates not only for growth and seed dispersal, but to a great extent also for the deposition of reserves. The latter are used in periods of frost and drought. However, the dimension of a tree and its longevity require stability by incorporation of cellulose, hemicellulose, and lignin in quantities that are unthinkable in herbaceous plants. The yearly reactivation requires a dynamic complementation of assimilates. Naturally, the sucrose needs water for its distribution in the plant, independently of its movement in the symplast, in the apoplast, or in both transport compartments. The water intake starts in the primary root, because other parts of the plant are unable to absorb water. The motor for the water uptake into root hairs is the osmotic potential made effective by sucrose. It is counteracted by the water potential of the soil. In trees, much more than in herbs, osmotic potential and water potential tend to equilibrate by many different water sinks and water sources, and over long distances. Leaf Initiation in Trees With the expanding leaf, the axillary bud of next year's shoot is initiated. In temperate zones, the leaf primordia of a bud are designed to adapt to external light conditions, developing into sun or shade leaves. In a forest, the external light conditions are transmitted differently through the bud scales. In Fagus sylvatica, the mode of construction in both sun and shade leaves is completed by the end of July. Removing a shelter tree will 50 Years of Translocation in the Phloem of Plants, With Reference to Trees 11 expose the shaded buds to full light. Up to the end of June, the shaded buds will adapt to the new conditions and turn into sun-lit buds. Later, at the end of July, all leaf primordia are completed. Adaptation to new, more intensive light is no longer possible, and in May of the following year, the emerging leaves appear red, reduced in size, and are unable to carry out net photosynthesis. When complete shelter is removed, no reserves will be available for the formation of new buds and leaves. Removal of shelterwood in late summer will sooner or later cause dieback of the light-exposed younger trees. Characteristically, the density of veins in the adult sun leaf is high, up to 31.4% of the leaf blade, and the chlorophyll content amounts to 19-411lg cm-2, while the density of veins in a shaded leaf is as low as 18.5% of the leaf blade, and the chlorophyll content is reduced to 15-32 Ilg cm-2• Consequently, a sucrose molecule being transported to the phloem for loading has to move 155 Ilm or less in the sun leaf, but in the shade leaf, it may move up to 240 Ilm for phloem loading (Eschrich et al. 1988). Periodicity of Leaf Formation Deciduous trees are common in climates with alternating summer and winter. In contrast to the great and heavy leaves of most palms and similar perennials, deciduous trees develop leaves that are functional only for the season, and constructed with the least material. The periodicity of leaf formation in most broad-leaved trees and some gymnosperms (Metasequoia, Larix) follows an annual cycle, while some gymnosperms (Picea) replace the leaves in longer intervals. New leaves are generally initiated in axillary buds. The leaf primordia are completed in early autumn, but the leaves themselves do not grow before bud burst in spring. Up to this time of the season, cambium and secondary phloem of Acer pseudoplatanus, Fraxinus excelsior, and Salix caprea show increased activity of endo-l,3-B-D-glucartase (callase), which may be related to reactivation of the phloem (Krabel et al. 1993). For leaf renewal, it is necessary for old leaves to be shed. This is usually carried out by an abscission layer between petiole and branch or between leaflet and rhachilla (Eschrich 1972). In beech, it was found that heavy metals (Fe, Mn, Zn, Pb) are transported to the browning autumn leaves (Fromm et al. 1987). Other elements, essential for growth, like Mg, K, and P, were found to be retrieved via the phloem in the leaf-supporting branches, prior to the shedding of the leaves (Eschrich et al. 1988). However, beeches and some oaks are known to keep their old and dry leaves for a long time throughout the winter and up to the emergence of new leaves. During a 5-year observation period, it was found that autumn-leaf retention occurred regularly every year on the same beech 12 Review trees, while other beech-trees of the same stand and age regularly shed their leaves in October. By measuring the water content, it was found that those beeches shedding their leaves in October had a somewhat higher water content in the twigs than that of beeches which kept their withered leaves over winter. It is possible that the reduced water content prohibited the completion of the abscission layer on schedule (Krabel et al. 1995). Characteristic for many broad-leaved trees is the autumn coloration (Eschrich 1972), which in fact is an elimination of phenolic substances that may interfere with enzyme activities by protein denaturation. Most perennial trees growing in summer/winter climates are acclimatized to frost periods. References Eschrich W (1953) Beitrage zur Kenntnis der Wundsiebrohrenentwicklung bei Impatiens holsti. Planta 43:37-74 Eschrich W (1954) Ein Beitrag zur Kenntnis der Kallose. Planta 44:532-542 Eschrich W (1956) Kallose (ein kritischer Sammelbericht). Protoplasma 47:487-530 Eschrich W (1995) Funktionelle Pflanzenanatomie. Springer, Berlin Heidelberg New York Eschrich W, Fritz E (1972) Microautoradiography of water-soluble organic compounds. In: Luttge U (ed) Microautoradiography and electron probe analysis, chapter 4. Springer, Berlin Heidelberg New York Eschrich W, Fromm J. Evert RF (1988) Transmission of electric signals in sieve tubes of zucchini plants. Bot Acta 101:327-331 Essiamah SK, Eschrich W (1982) Die Dynamik der Fruhjahrssaftbildung. Forstarchiv 53:133-135 Fromm J, Essiamah S. Eschrich W (1987) Displacement of frequently occurring heavy metals in autumn leaves of beech. Trees 1:164-171 Hedrich R, Schroeder JI (1989) The physiology of ion channels and electrogenic pumps in higher plants. Annu Rev Plant Physiol40:539-569 Munch E (1930) Die Stoffbewegungen in der Pflanze. Gustav Fischer, Jena Young H, Evert RF, Eschrich W (1973) On the volume-flow mechanism of phloem transport. Planta 113: 355-366 Communicated by U. Luttge Prof. Dr. Walter Eschrich Ludwig-Beck-StraBe 7 37075 Gottingen, Germany Genetics Genetics Introns, Splicing and Mobility Ralf Sagebarth and Ulf Stahl 1 Introduction Introns are found in the nucleus, in bacteria, in phages and in organelles. All known organellar introns can be classified into two distinct classes according to their structures and splicing pathways: group-I and group-II introns. However, it has to be noted that the distributions of both classes are not limited to organellar genomes. Both classes contain self-splicing introns. Furthermore, some of the introns are mobile. Group-II introns are remarkable because their splicing mechanism resembles that of nuclear pre-messenger RNA (mRNA) introns and because of their much more complex mobility pathway than group-I introns. Also, in contrast to group-I introns, the mobility of group-II introns is strongly linked to their biological role as introns (i.e., splicing). Here, we review recent findings regarding the splicing, distribution and mobility of group-II introns. 2 Intron Structure and Splicing Pathway Group-II introns can be classified on the basis of their structural properties and splicing mechanisms. Because their primary sequences are not well conserved, these introns are folded into a characteristic secondary structure (Fig. lA) first formulated by Michel and co-workers (Michel et al. 1982). This model is characterized by a central core of approximately 30-40 nucleotides from which six major substructures (domains I-VI) radiate (Fig. lA). Subsequently, a further subdivision into IIA and lIB was necessary (Michel et al. 1989), because some structural differences located in domains I and VI were obvious. Some group-II introns encode for a protein whose main protein-coding sequence is always inserted in domain IV (Fig. lA). The open reading frames (ORFs) are either freestanding within the intron (Fig. lB) or are in frame with the upstream exon. Several tertiary interactions involving only a single domain or different domains have been identified and are known to be essential for Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Genetics 16 5'exon B ~~--~~--~~~~~I~ J{T t '( Zil q. I YADD Fig.!. A Model of the secondary structure of group-II introns. As an example, the structure of the Podospora intron coxl-Il is shown. The long, continuous lines represent intron sequences, and the open boxes indicate exon sequences. The six domains radiating from the central core are numbered 1-VI. The open reading frame (ORP) is mostly outlooped in domain IV. Intron and exon binding sites (IBSI, IBS2, EBSI and EBS2) are marked by thicker curved lines. The single A within domain VI represents the branch-point adenosine (Schmidt et al. 1993). B Schematic representation of a group-II intron with a freestanding ORF. Cross-hatched boxes represent the upstream (5'E) and downstream exons (3'E). The thin line represents intron sequences, and the intronic ORF is indicated by an open box. Protein domains are indicated by marked areas (RT reverse-transcriptase domain, X maturase domain, Z Z domain, Zn Zn 2+-finger-like domain). The essential YADD motif is marked by an arrow function (Michel et al. 1989; Michel and Ferat 1995; Costa et al. 1997a). One of the most important interactions for the determination of the 5' splice site (SS) is the pairing of two intron sequences (each usually six nucleotides long) located in domain I (exon binding sites 1 and 2, EBS1 and EBS2), with the first 12 nucleotides preceding the 5' SS (intron binding sites 1 and 2, IBS1 and IBS2; Fig. 1A). A good candidate that may reveal unknown tertiary interactions is the self-splicing intron Pl.LSU-I2 identified in the LSUrRNA gene of the brown alga Pylaiella littoralis (Fontaine et al. 1995). This intron readily forms homogenous populations of molecules with a native conformation (Costa et al. 1997b), making it suitable for physical-chemical studies (eg. Introns, Splicing and Mobility 17 footprinting analysis) of higher-order structure at low salt concentrations resembling those found in vivo. The splicing reaction of group-II introns consists of two consecutive trans esterification reactions (Fig. 2A). After complete folding of the intron active site (Costa and Michel 1999), the 5' exon is bound by the intron via EBSII2-IBS1I2 interactions. In the first step, the phosphodiester bond at the 5' SS is cleaved by the attack of the 2' hydroxide (OH) of an intron internal bulged adenosine located seven (group IIA) or eight nucleotides (group lIB) upstream of the 3' SS within domain VI. The free 5' exon remains attached to the intron/3'-exon intermediate via the IBSEBS interaction. In the second step, the 3' OH of the free 5' exon attacks the 3' SS, resulting in exon ligation and release of the intron in a lariat form. This in vitro splicing reaction was demonstrated to be fully reversible in vitro (Augustin et al. 1990; Morl and Schmelzer 1990). Furthermore, the intron lariat can also integrate into any RNA enabling an appropriate IBS/EBS interaction (Morl and Schmelzer 1990). Only a few A branchpoint splicing IBS 111 5 'SS 5'E .. B 3'SS EBS A A 3'E I . - - - - - - - - -- tl,,~, 3'SS A 3'E I OH +Step I 3'SS 3'SS C1 q~"E +" u+ ... RBS H2O orOH- I . 5'E IBS "'SS I OH lariat hydrolytic splicing 5'E III 3'E tl'"" 3'E ligated exons (/+ linear intron 5'E III 3'£ ligated exons Fig. 2 A,B. Splicing pathways of group-II introns. Intronic sequences are drawn as thin lines, while exon sequences (3'E, S'E) are drawn as thicker lines. The intron binding site/exon binding site tertiary interactions are indicated. The single A represents the branch-point adenosine. A Branch-point splicing. The nucleophile attack of the 2' OH group contributed by the bulged adenosine at the 5' splice site (5'55) is indicated by a broken line. The nucleophile attack of the 5' exon at the 3' splice site (3'55) is also indicated by a broken line. B Hydrolytic splicing. The nucleophilic attack of a free water molecule or OH- ion at 5'SS is indicated by a broken line. See text for further details. (Daniels et al. 1996) 18 Genetics group-II introns are able to self-splice in vitro, predominantly under essentially non-physiological conditions; this suggests a protein dependence of splicing reactions in vivo (Perlman and Podar 1996). Alternatively, the first step of the splicing reaction can also be initiated by the nucleophilic attack of a free water molecule or OH- ion (Fig. 2B; Peebles et al. 1987; Jarrell et al. 1988a,b). Nevertheless, the intron can perform the second step of splicing. However, the intron is then released in a linear form instead of a lariat form. It was demonstrated in 1996 (Daniels et al. 1996) that this hydrolytic pathway occurs parallel to and competes with the branching pathway in vitro. Interestingly, under physiological ionic conditions, the hydrolytic pathway prevails, indicating a possible in vivo relevance. It was proven by Perlman and coworkers (Podar et al. 1998) that group-II introns are capable of in vivo 5' hydrolysis, depending on whether they have an appropriate genetic background. The reason for 5' hydrolysis might be that the activation or inhibition of the hydrolytic pathway serves a regulatory purpose. For example, accelerating the hydrolysis cells might reduce the mobility of introns (for details, see below) by producing higher levels of linear introns that are unable to perform reverse splicing. Furthermore, the hydrolytic pathway might be the major splicing pathway for several groupII introns lacking a branched adenosine (Michel et al. 1989). As already mentioned, several group-II introns encode proteins that can consist of four or fewer conserved domains of different functions (Fig. IB), including a reversetranscriptase domain (RT domain) with an essential motif composed of a tyrosine, an alanine and two aspartic acids (YADD motif), followed by a maturase (X domain) and a Zn 2+-finger-like domain (Zn domain). Phylogenetic comparisons indicate that the RT domain is most closely related to the non-long terminal repeat (LTR) class of retroelements (Doolittle et al. 1989; Xiong and Eickbush 1990; McClure 1991). In addition to the RT domain, group-II introns also contain a conserved domain (domain Z) upstream of the RT domain; this domain is also a characteristic ofnon-LTR elements of unknown function (McClure 1991). While the maturase domain promotes splicing, the RT and Zn domain contribute to intron mobility (Kennell et al. 1993; Zimmerly et al. 1995; Matsuura et al. 1997). With the discovery of two ORF-containing group-lIB introns in the brown alga P. littoralis (Fontaine et al. 1995), it was possible to establish two different lineages of ORFs ("lineage a" and "lineage b"; Fontaine et al. 1997). While "lineage a" contains subgroupIIA introns, "lineage b" is comprised of subgroup-lIB introns, thus indicating the coevolution of proteins and introns. 3 Intron Distribution Compared with the widespread distribution of group-I introns, the distribution of group-II introns was first thought to be limited to the organelles of plants and fungi. With the detection of group-II introns in cyanobacteria and proteobacteria, the putative ancestors of chloroplasts and mitochondria (Ferat and Michel 1993; Ferat et al. 1994), it was obvi- Introns, Splicing and Mobility 19 ous that their distribution might be as wide as the distribution of group-I introns. After this, group-II introns were detected in Pseudomonas (Yeo et al. 1997), agrobacteria and rhizobia (Knoop et al. 1994; Martinez-Abarca et al. 1998), Lactococcus (Mills et al. 1996; Shearman et al. 1996), Clostridium (Mullany et al. 1996) and Bacillus (Huang et al. 1999), indicating a wide distribution of group-II introns throughout bacteria. Strikingly, all group-II introns detected in bacteria are associated with elements with putative or proven mobility. At least for the Lactococcus intron LUtrB, mobility independent of its surrounding element (i.e., the conjugative plasmid) could be demonstrated (Matsuura et al. 1997; see below), thus indicating that bacterial group-II introns possess their own mobility. It is possible that group-II introns use, or even require, the mobility of their host elements as a vector in order to spread within the bacteria. It remains to be elucidated whether the association of group-II introns with mobile elements is simply a coincidence or is the result of restrictions limiting the horizontal transfer between bacteria. An unusual association of a group-II intron-like ORF consisting only of a RT domain was detected in Serratia (Kulaeva et al. 1998). Instead of being associated with a group-II intron, this ORF obviously resides in a bacterial ret ron element (Inouye and Inouye 1995). Because protozoa encompass most of the phylogenetic breadth of the eukaryotic lineage, it is very interesting to reveal their intron content. Sequencing projects examining protozoan mitochondrial DNA (mtDNA) revealed not only remarkably few group-I introns but also very few group-II introns (Gray et al. 1998). Only a total of seven group-II introns in five of 23 completely sequenced chondriom sequences could be detected. This sporadic distribution and the evidence of horizontal transfer of some of the introns make the wholesale acquisition of group-II introns by the eukaryotic cell via a-proteobacteria endosymbionts unlikely (Gray et al. 1998). However, the jakobid flagellate Reclinomonas americana, a very ancestral mitochondriacontaining protozoan representing a very early off-shoot of the main eUkaryotic line, contains a single ORF-less group-II intron (Lang et al. 1997). This suggests a long presence of this now immobile intron in this protozoan, and a possible vertical inheritance from a a-proteobacteria. The only ecological niche in which group-II introns have not been found is the genomes of phages, archaebacteria, metazoa and the nuclei of eukaryotes. Beagley et al. (1996) reported that a group-I intron is present in the mitochondria of the sea anemone Metridium senile. This provides evidence that introns are not generally absent from multi-cellular animals, thus indicating that group-II introns might also be present in some primitive metazoa. 20 Genetics 4 Protein-Assisted Splicing As already noted, group-II introns require one or more proteins for efficient in vivo splicing. Aside from nuclear encoded proteins, the main protein needed for splicing is the intron-encoded protein itself (at least for the splicing of ORF-containing introns). Genetic experiments with yeast (Carignani et al. 1983; Moran et al. 1994) and two bacterial introns (Matsuura et al. 1997; Martinez-Abarca et al. 1998) demonstrated that the intron-encoded protein functions as a maturase and demonstrated an association of this activity with domain X (Fig. 1B). The first biochemical evidence of maturase activity could be established for the Lactococcus intron ltrB (Matsuura et al. 1997). While the intron does not self-splice under physiological conditions, the addition of highly purified ribonucleoprotein (RNP) particles consisting of mRNA and intron-encoded protein (Lambowitz and Perlman 1999) isolated from Escherichia coli established an easily detectable splicing reaction with the accumulation of free intron lariats and ligated exons. The protein-assisted in vitro splicing reaction does not require adenosine triphosphate. Further analysis revealed that no additional proteins from the host are required (Zimmerly et al. 1999) for an efficient splicing reaction and that the protein is highly sufficient for its own intron (Saldanha et al. 1999). Most recent studies reveal that the primary binding site of the Il1aturase encoded by Ll.ltrB is a region comprising the ribosome-binding site and the start codon located in domain IV of the intron RNA (Wank et al. 1999). This binding is only enhanced by other structural elements, such as domain I and the IBS-EBS interaction. These results are surprising, because these elements are known to be very important for the selfsplicing reaction (Michel and Ferat 1995; Perlman and Podar 1996); in contrast, domain IV is dispensable (Koch et al. 1992). The primary binding site of the intron-encoded protein within domain IV was further confined (Wank et al. 1999). The resulting minimal binding site can be folded into a short bifurcating stem-loop structure and is present not only in Ll.ltrB and the Saccharomyces introns Sc.coxl-Il (coxl-Il for cytochrome c oxidase subunit 1 intron 1) and Sc.coxl-I2 (Wank et al. 1999), but also in introns Pa.coxl-Il from Podospora and Ec.intB from E. coli (Sagebarth, unpublished). Therefore, this structure might be a conserved motif in ORF-containing group-II introns. It still has to be elucidated whether this structural element is also required for in vivo splicing. It is unknown whether host proteins further enhance in vivo splicing. Possible candidates for such proteins, at least in E. coli, might be the ribosomal protein S12 (rps12; Coetzee et al. 1994) and the protein StpA (Zhang et al. 1995). These proteins are able to bind RNA with a broad specificity and may act as general RNA chaperones. For instance, they are known to enhance the splicing of a group-I intron by resolving mis- Introns, Splicing and Mobility 21 folded RNAs and promoting the assembly of the precursors into an active conformation; however, they are not required for the catalytic step itself (Coetzee et al. 1994; Zhang et al. 1995). It could be that these proteins also initiate the folding of group-II introns before the intronencoded proteins proceed. Interestingly, although a rps12 gene is absent from animal and fungal mtDNAs, it is mostly present in protozoan mtDNA and in the organelles of plants. Furthermore, it is supposed that nuclear encoded mitochondrial proteases (m-AAA protease and PIMI protease) are essential for splicing in yeast mitochondria (Arlt et al. 1998; Van Dyck et al. 1998). Although intron-encoded proteins present in yeast must be translated as fusion proteins composed of the upstream exon and the intron ORF, the expected full-length proteins could not be detected. Instead, only shorter proteins could be observed, and the putative processing site seems to be located within or in front of domain Z (Bergantino and Carignani 1990; Moran et al. 1994; Zimmerly et al. 1999). Furthermore, activities essential for splicing and mobility could only be assigned to a processed protein (Zimmerly et al. 1999). The biological scope of these findings is manifold. First, because the putative processing site is located within the intron, the target sites are always available for the proteases. Therefore, processing of the intron-encoded protein is independent of the integration site of the intron. Second, the distribution of protein-encoding group-II introns might be limited to species that can contribute a suitable protease; otherwise, the splicing of the intron is abolished. Depending on the integration site of the intron, this can lead to a lethal phenotype. This apparent strong dependence of splicing on genuine proteases might limit their distribution. However, introns possessing a freestanding ORF might be predestined to invade new taxa. This might reflect the distribution of both kinds of introns. While introns with ORFs linked to their upstream exons are limited to fungi, introns possessing a free-standing ORF can be found in such divergent taxa such as plants, algae and bacteria. With regard to nuclear encoded splicing factors, approximately 20 genes were found to be essential for splicing mitochondrial introns in yeast. However, a direct effect on splicing has not been established for any of them (Atkin et al. 1995; Gottschalk et al. 1998; Koehler et al. 1998; Bui et al. 1999). Most (if not all) of them, in addition to their effect on splicing, have a second cellular function (eg. mitochondrial biogenesis, mitochondrial import, assembly). It has even been suggested that their participation in splicing was acquired secondarily (Lambowitz and Perlman 1990, 1999). In yeast and bacteria, the intron-encoded maturase activity appears to be highly specific for its own intron (Moran et al. 1994; Saldanha et al. 1999); however, the situation seems to be different in the plastids of higher plants. Chloroplasts of higher plants contain up to 20 group-II introns. However, only the intron within the tnrK gene coding for 22 Genetics transfer RNA LysuUU contains the only chloroplast reading frame with homology to maturase-like proteins of group-II introns (Neuhaus and Link 1987). This intron-encoded protein, designated matK, is expressed in chloroplasts (du Jardin et al. 1994) and has RNA-binding activity (Liere and Link 1995). A detailed analysis of splicing in barley chloroplasts revealed that splicing of all the group-IIA introns and one lIB intron depends on protein synthesis in the chloroplast (Hess et al. 1994; Htibschmann et al. 1996; Jenkins et al. 1997; Vogel et al. 1999). This indicates that matK might have evolved to function in the splicing of multiple group-II introns. The evolution of intronencoded maturases from an intron-specific to a general splicing factor seems to reach an end point when the separation of the maturase from the intron sequence has been accomplished (Ems et al. 1995). 5 Intron Mobility Some group-II introns are mobile genetic elements. One example is the group-II intron coxl-Il in the mitochondria of the ascomycete P. anserina; this intron can exist in two different molecular phases, indicating a mobility of introns. In juvenile mycelia, it is an integrated part of the mtDNA and, during aging, it is amplified as a covalently closed circular molecule (plDNA; Stahl et al. 1978; Klick et al. 1989). Due to this unusual feature of coxl-Il, Esser and co-workers introduced the term "mobile intron" (Osiewacz and Esser 1984). The first genetic evidence of intron mobility came from crosses between yeast strains with intronless and intron-containing alleles. During these crossings, group-II introns are capable of invading the intronless alleles (Meunier et al. 1990; Skelly et al. 1991). This homing of group-II introns is very efficient and occurs with frequencies up to almost lOO%. In addition to homing, it was presumed that group-II introns might be transposed to other genomic locations. The first indication of transposition came from the discovery of twintrons in the plastids of Euglena (Copertino and Hallick 1991) and recently, in the cryptomonad alga Pyrenomonas salina (Maier et al. 1995). Twintrons are introns within introns that are sequentially spliced (Copertino et al. 1991) and are thought to be formed via the transposition of a mobile intron into a pre-existing intron (Copertino and Hallick 1991). a) Protein-Assisted Homing The catalytic activities of both the intron RNA and the intron-encoded protein are needed for an efficient homing process (Zimmerly et al. 1995a,b; Yang et al. 1996; Matsuura et al. 1997). The latter consists of three activities (i.e., maturase, RT and endonuclease) and is provided by the intron-encoded protein. The major homing pathway occurs via a so- Introns, Splicing and Mobility A 23 Bacteria (i.e. Lactococcus) B rzzz:1zZZ/1 Yeast (i.e. Saccharomyces) IZZZNzZZI fZZhi rZZhi "'r" rZZJ7i l ~ IZZ/fii )'1 '1' l',"11 ;\\I.k'hll1l'llt P;/?h "I PZZl )'] PZZl fJWH fZZJ 'I Fig. 3 A,B. Homing mechanisms in bacteria and yeast. DNA exon sequences of the recipient are represented as striped open boxes, while DNA exon sequences of the donor are shown as cross-hatched open boxes. Intron RNA sequences are shown as thin lines with intron binding site/exon binding site interactions as indicated. Wavy lines represent complementary DNA. A Homing mechanism in bacteria. B Homing mechanism in yeast. The co-converted tract within the 5'exon is indicated with an open arrow. DSBGR doublestrand break gap repair. See text for further details (Eickbush 1999) called target DNA-primed reverse-transcription mechanism. The intron lariat, excised by a splicing reaction, remains associated with the intronencoded protein, forming a very stable RNP particle (Zimmerly et al. 1999). The homing process is initiated by a complete reverse splicing reaction of the excised intron lariat, thus integrating the intron RNA into the DNA target site (Fig.3A; Yang et al. 1996; Matsuura et al. 1997; Zimmerly et al. 1999). In the next step, the endonuclease activity provided by the Zn domain (Fig. IB) of the intron-encoded protein cleaves the anti-sense strand at position + 10 or +9 (in yeast introns or in bacterial intron Ll.ltrB, respectively) of the 3' exon (Zimmerly et al. 1995b; Yang et al. 1996; Matsuura et al. 1997). The complete recognition sequence encompasses approximately 30 bp surrounding the DNA target site (Guo et al. 1997; Cousineau et al. 1998). 24 Genetics The anti-sense cleavage generates a free 3' OH that is used as a primer for complementary DNA (cDNA) synthesis by the RT domain (Fig. lB) of the intron-encoded protein. The integrated intron RNA serves as a template for cDNA synthesis. When the cDNA synthesis extends into the 5'exon, the anti-sense strand is probably displaced by the action of a helicase. After degradation of the intron RNA and synthesis of the sense strand, the homing event is completed. These last two steps are accomplished by DNA replication and repair enzymes provided by the host. This straightforward pathway can be found in the Lactococcus intron ItrB (Fig. 3A; Mills et al. 1997; Cousineau et al. 1998). In contrast, homing in yeast is accompanied by a co-conversion that is very efficient for upstream exon sequences, while downstream sequences are poorly co-converted (Lazowska et al. 1994; Moran et al. 1995; Eskes et al. 1997). Because this asymmetric co-conversion cannot be explained by the bacterial mechanism, the homing of group-II introns in yeast seems to be more complicated and requires additional features, such as double-strand break gap repair (DSBGR) mechanisms (Fig. 3B; Eskes et al. 1997; Zimmerly et al. 1999). After complete reverse splicing, anti-sense cleavage and initiation of eDNA synthesis DSBGR may be initiated by strand invasion of single-stranded 3' eDNA tails into sequences of the donor DNA (Eskes et al. 1997). After sufficient extension of the eDNA, integration into the recipient DNA is completed by DSBGR, resulting in the coconversion of upstream exon sequences. Alternatively, after initial cDNA synthesis using the integrated intron as a template, a template switch to donor pre-mRNA might occur. After extension into donor exons, the cDNA could replace the recipient DNA by DSBGR (Eskes et al. 1997). Although the initial steps of the homing mechanism in yeast and bacteria are identical, the final steps in yeast convert (Eskes et al. 1997), leading to the efficient co-conversion of upstream exon sequences via a DSBGR mechanism. In addition to this strict RT -dependent pathway, group-II introns in yeast possess a RT-independent mechanism (Eskes et al. 1997). RTindependent homing is not a simple crossover at the DNA level, because homing is completely blocked when the mutual recognition of IBS and EBS is disrupted. Therefore, an attractive hypothesis might be that the process is initiated at the RNA level by reverse splicing of the intron lariat and is continued at the DNA level. It was estimated that, in a yeast system, up to 38% of recombinant progenies in crosses appear to result from RT-independent events, but only when the target site differs from the original sequence, leaving only the EBSlIIBSl interaction intact (Eskes et al. 1997). According to available data, homing in yeast is a mixture of RTdependent and RT -independent events and is more complex than homing in bacteria. Homing at the DNA level seems to be favored when cDNA synthesis is blocked or the sequence of the cleavage site is altered Introns, Splicing and Mobility 25 (Eskes et al. 1997), thus providing a mechanism for transposition to ectopic sites (see below). The demonstration of RT activity could indicate a mobile group-II intron if the activity can be assigned to a suitable ORF. The first RT activity in plant mitochondria was detected in potatoes (Moenne et al. 1996) and was assigned to the mat-r ORF encoded by a group-II intron in the nadl gene [for reduced nicotinamide adenine dinucleotide (NADH)-dehydrogenase subunit I; Begu et al. 1998]. This ORF lacks some essential features usually exhibited by functional RTs. First, the conserved YADD motif is changed to a YADN motif (Tyr-Ala-Asp-Asn). It is known that substitutions of the two asparagines within this motif abolish RT activity completely. Second, the expression of the mat-r ORF is uncertain, because the ORF lacks a proper translation-initiation signal. A missing start codon seems to be an attribute of all known mat-r ORFs in plant mitochondria, but the translation of none of them has been proven. Although the mat-r transcripts are extensively edited, improving the homology to fungal RT sequences, the expression of mat-r is still in question, because editing is known from putative pseudogenes (Aubert et aI. 1992; Brandt et aI. 1993; Giege et aI. 1998). b) Transposition to Ectopic Sites In addition to homing (the invasion of intronless alleles by introns into their homing sites), group-II introns are able to invade new genomic locations. Transposition to ectopic sites in mitochondria could be demonstrated for yeast intron Sc.coxl-11 (Muller et al. 1993), intron Sp.cob11 in S. pombe (cob for cytochrome b; Sagebarth et al. 1994) and intron coxl-11 in P. anserina (Sellem et al. 1993; Schmidt et al., unpublished). In contrast to homing, no co-conversion of flanking exon sequences could be found (Schmidt et al. 1994). All new integration sites are preceded by an IBS-like motif, thus indicating an RNA-mediated transposition pathway. This data shows that transposition follows a pathway at least slightly different from homing, leading to a duplication of the intron separated by genomic sequences of different lengths. Homologous recombination between the two intron copies finally results in the formation of sub-genomic circles. Therefore, mobile group-II introns increase the instability of mtDNA. It was initially presumed that the intron lariat is able to reverse splice into transcripts with a sufficient IBS-like motif (Fig. 4A). After cDNA synthesis, RNA degradation and second-strand synthesis, the introncontaining sequence integrates into its cognate genomic location via homologous recombination, thus displacing the intronless copy. Instead of integration into transcripts, the intron lariat may be able to reverse splice directly into the ectopic DNA sites, followed by reverse transcription of the inserted intron RNA. Genetics 26 A transposition u B 1'"' ' ' ' ' ' ' homing-like transposition IllS " u 1,,,,,,,,,,,,,,, 1 1 rnl'r\l'fr;lll\l'IIPII\lll 'iCl'\)lld \lr:ll1d \\ Illlw"I" iJ1tl'~Llli('n ill ~l'Il\\ml' V771 V771 ~ h<>Il1<>I"~"l" Ic",,,"h;IlClll<m "\lh~l'11l)1llil' circk ... r77/1 t77/1 ~ IH'Il1"I<>~<>l" 1,',<>1111';""11<'" frl'l' l'irclilar ill11\ III Fig. 4 A,B. Transposition pathway of group-II introns. As an example, the proposed mechanism for the transposition (via reverse splicing into transcripts) is illustrated. Exon RNA sequences are shown as thicker lines, while intronic RNA is shown as thin lines. Intronic DNA sequences are drawn as opened boxes, while exon DNA sequences are drawn as hatched open boxes. A Transposition pathway to ectopic sites leading to subgenomic circles. B Homing-like transposition pathway leading to tandem duplicated introns and the release of circular intron molecules. See text for further details This assumption was recently confirmed. At least in vitro, the yeast intron Sc.coxl-Il is able to reverse splice into ectopic DNA transposition sites (Yang et al. 1998). Efficiency ranges up to 3%, compared with those at the normal homing site. However, no anti-sense cleavage (and, therefore, no reverse transcription) could be detected using these ectopicDNA target sites (Yang et al. 1998). This is probably due to the lack of adequate sequences necessary to generate the primer for reverse transcription via anti-sense cleavage, thus indicating that a RT -independent pathway might be used for transposition. Consequently, the cognate IBS motif at the original 5' exon-intron junction should be a perfect target site for transposition. This homing-like transposition would lead to the formation of tandem duplicated introns. Subsequent homologous recombination between the two intron copies should liberate one entire intron sequence with a precise ligation of the intron boundaries. Both tandem introns and free intron DNA circles could be detected in the mitochondria of P. anserina not only for coxl- II but also for coxl-I4 (Sainsard-Chanet et al. 1994; Sagebarth et al., unpublished). Free intron circles could also be detected for intron cob-Il in s. pombe and intron coxl-Il in S. cerevisiae (Schmidt et al. 1994). However, no tandems or free circles could be detected for the mitochondrial Podospora intron nad5-14 (nad5 for NADH dehydrogenase subunit 5; Sainsard-Chanet et al. 1994; Sagebarth et aI., unpublished). The protein encoded by this intron is probably the cause of the immobility, because the YADD motif is changed to YANV and the Zn2+ -finger-like motif is missing (Mohr et al. 1993). Introns, Splicing and Mobility 27 As for transposition, two mechanisms for the homing-like transposition explaining the release of intron circles (Fig. 4B) are possible: 1. The intron lariat integrates into unspliced transcripts. 2. The lariat integrates directly into the genomic copy of the intron. The free intron lariat reverse splices into a DNA target site comprising a 5' exon-intron junction with efficiency up to 34% compared with the original homing site, i.e., the 5' exon-3' exon junction site (Yang et al. 1998). Again, no anti-sense strand cleavage and no reverse transcription could be detected, raising the question of how the eDNA synthesis is initiated. It is obvious that the formation of tandem duplicated introns itself does not account for the origin of free intron circles by reverse splicing. Tandem introns can also be formed by re-integrating free intron circles into their genomic copies, thus providing a causality problem. This is further complicated by the observation that free intron DNA circles are already present in ascospores of Podospora, thus indicating a vertical transmission of intron DNA circles (Sagebarth et aI., unpublished). Therefore, it is possible that the de novo formation of tandem introns exclusively originate via the integration of inherited intron DNA circles. In this case, the first intron DNA circles could be derived via the reverse transcription offree intron RNA (Kennell et al. 1993). These results emphasize that the formation of free intron circles is a general feature of ORF-containing group-II introns and that the term "mobile intron" (Osiewacz and Esser 1984) was correct. For all of the analyzed introns, these DNA circles can only be detected by peR; however, intron coxl-Il from P. anserina is a special case, because it can be found in much higher amounts (Stahl et al. 1978). The reason for the underlying amplification is still unclear. Some experimental data indicates that replication processes take place at free intron circles of coxl-Il (Wedde and Stahl, personal communication) and might be responsible for the observed amplification. Another reason for amplification might be a more efficient transposition into the 5' exon-intron junction compared with other introns. Free circular introns are excluded from the transcription machinery of the cell and are probably not able to continue with mobility. Therefore, in terms of mobility resulting from an irregular reverse-splicing step, free circular introns are most likely "dead-end" molecules. 28 Genetics 6 Future Prospects Much work has been done to reveal the homing process of group-II introns during the last 5 years. In contrast to homing, the transposition pathway of group-II introns is not well understood. Transposition is of outstanding biological interest, because it influences the actual distribution of group-II introns. Another neglected field of research is the effect of group-II introns on their hosts. Transposition to ectopic sites within a host increases the instability of the genomes by increasing the probability of homologous recombination between the duplicated introns. Therefore, a negative effect of group-II introns on their hosts can be expected. Surprisingly, the absence of group-II introns, e.g., in S. cerevisiae, had no growth effect (Seraphin et al. 1987) while, in S. pombe, a prolonged generation time was observed (Schafer et al. 1991). The only negative effect of a group-II intron on the viability of its host was recently detected in P. anserina. The precise deletion of the intron coxl-Il resulted in life span prolonged twofold (Begel et al. 1999). It can be speculated that this contrary effect in yeast and Podospora might be due to the different life cycles of the host cells. Whereas S. cerevisiae is a unicellular organism with a limited number of generations, Podospora is a multi-cellular organism with a much higher number of propagation steps. Therefore, increased accumulation of deleterious effects caused by group-II introns is possible in Podospora and might influence the viability of the organism. These negative effects are suspected to be much more pronounced in multi-cellular organisms with well-defined cell differentiation accompanied by a loss of omnipotence. Therefore, in higher organisms, deleterious effects caused by mobile group-II introns cannot easily be genetically compensated (as in yeast or fIlamentous fungi). If further analysis of transposition in multi-cellular organisms confirms a negative effect of group-II introns on their hosts, it might be a selective advantage for the host to avoid invasion of group-II introns. This selective advantage might be an explanation of the complete absence of introns in higher animals. One strategy the organism could adopt to prevent invasion of group-II introns is the deletion of nuclear encoded splicing factors. Acknowledgements. 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Cell 82:545-554 Zimmerly S, Moran IV, Perlman PS, Lambowitz AM (1999) Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase activity by the intron RNA. I Mol Bioi 289:473-490 Communicated by K. Esser Dipl. BioI Ralf Sagebarth, Prof. Dipl.-Ing. Dr. UlfStahl Technische Universitat Berlin (Berlin University of Technology) Dept. of Microbiology and Genetics, TIB 4/4-1 Gustav-Meyer-Allee 25 13355 Berlin, Germany e-mail: [email protected] Genetics Barley Mutagenesis Anders Falk, Alan H. Schulman, S0ren K. Rasmussen, and Christer Jansson 1 Introduction The construction and utilization of mutants have a long history in plant breeding. In this review, we discuss two novel approaches to barley mutagenesis. First, we describe the powerful approach of neutron radiation for the production of deletion libraries in barley. With this method, deletions in the range of 100-10,000 bp can be generated. Based on the known number of neutron-induced mutations in barley, one can expect that between 10,000 and 20,000 mutagenized plants will be required in order to achieve a reasonable (>90%) probability of identifying at least one deletion mutant per gene. The second approach deals with transposon mutagenesis. Since the rediscovery of Mendel's laws, genetic linkage, the genetic code and the molecular nature of the gene, three fundamental findings have changed our views of genetics: genome imprinting and other epigenetic phenomena, the existence of transposable elements, and the presence of repetitive DNA as the major component of the genome. During the past 20 years, it has become apparent that the last two discoveries are inextricably linked; much of the repetitive DNA in eukaryotes is composed of transposable elements and their transpositionally inert relics. In the final section of the review, we give an example of ongoing mutational breeding in barley. 2 Construction and Utilization of Barley Mutant Libraries a) Barley Mutants Induced by Radiation or Chemicals When plant breeding was in its infancy, barley (Hordeum vulgare 1.) and other crop plants were mutagenized with the intention of generating plants with more agronomically favorable traits. The mutagenic treatments involved radiation such as X-rays, y rays, neutrons and chemicals, such as ethyl methane sulfonate and sodium azide. The mutagenized seed was grown for one generation, called the M1 generation. The screen Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Barley Mutagenesis 35 for mutants was performed during the next generation, called the M2 generation, in which mutations occurred in the homozygous condition and mutated plants could be identified by their phenotype. While relatively few mutants of importance for plant breeding were isolated, it was realized that the mutant approach could reveal the functions of individual genes of the plant. Since then, the analysis of mutants has been a popular method in plant biology. A large collection of mutants, especially in barley, has therefore been collected during recent decades. The total number of barley mutants in these banks is estimated to be approximately 10,000 (Lundqvist 1992), although not all are at different loci. Several different classes of mutants have been isolated. Examples of viable mutations are the eceriferum (waxiess), erectoides and praematurum (early-heading) mutants. The eceriferum mutants were shown to be localized to 79 different loci (Lundqvist and Lundqvist 1988). Mutants whose biosynthesis of various biomolecules (such as the anthocyanin-deficient mutants; Kristiansen and Rohde 1991; Olsen et al. 1993) or level of resistance against pathogens (Freialdenhoven et al. 1994, 1996; Jorgensen 1996) are affected have also been identified. Of the lethal mutants, many have affected pigment synthesis, as in the albina, xantha, viridis and tigrina mutants (Henningsen et al. 1993; Hansson et al. 1997), which define more than 100 different loci. Although they are powerful techniques for the induction of mutations, chemicals and radiation have the drawback that they do not easily lend themselves to the cloning of the genes that have been mutated. Taking into account the availability of large mutant banks, the development of techniques that facilitate the cloning of the mutated genes is highly desirable and would make the existing mutant banks a valuable source for basic plant-biology research. This review will concentrate on methods that can be used to clone the genes mutated in these mutant banks and on the progress achieved to date. Basically, the techniques are of two different kinds: forward and reverse genetics. b) Molecular Analysis of Barley Mutants Induced by Radiation or Chemicals One option available for the cloning of a mutated gene is the method of map-based cloning (or chromosome landing as it is now popularly called; Tanksley et al. 1995). The technique has been most successfully applied to Arabidopsis, mainly due to the favorable relationship between genetic and physical distance in this species. However, two barley genes identified from mutant screens were recently cloned via the chromosome-landing approach (Buschges et al. 1997; Lahaye et al. 1998a,b). When mutated, the Mlo gene confers a durable, non-race-specific resistance to the powdery mildew fungus (Erysiphe graminis f. sp. hordei) in barley (Jorgensen 1992). Amplified fragment-length polymorphism (AFLP) analysis of bulked segregants identified a set 36 Genetics of AFLP markers that were polymorphic between the resistant and susceptible pools. Using a mapping population of 2000 resistant F2 plants, one AFLP marker was found to co-segregate with the Mlo gene (Simons et aI. 1997). Eventually, a 30,000-bp region was sequenced, and a likely candidate gene was identified. The final proof of cloning was found by analyzing rare intragenic recombinants in Mlo (Biischges et aI. 1997). Similar procedures were applied to the cloning of the RARI gene although, in this case, fewer AFLP primer combinations were screened due to the identification of a co-segregating marker at an early stage during the mapping process (Lahaye et al. 1998a). These clonings represent milestones in the analysis of barley mutants. Obviously, chromosome landing is still a tedious and labor-intensive process that needs to be much refined to make it applicable to largescale cloning of the genes mutated in the existing mutant banks. One advantage of chromosome landing is that the method is applicable to all identified mutants. Other cloning possibilities include extensive analysis of the phenotype of the mutant, with the hope of thereby finding clues to the identity of the mutated gene. In such cases, similarities to mutants isolated from other organisms can prove useful. In this way, the xantha-f, -g and -h genes were shown to encode Mg-chelatase (Jensen et al. 1996), and the ant-I8 gene was shown to encode dihydroflavonol-4-reductase (Kristiansen and Rohde 1991; Olsen et aI. 1993). Extensive phenotypic analysis can be successful for the cloning of some structural genes but is less efficient for the cloning of regulatory genes, such as transcription factors. For instance, extensive analysis of the tigrina mutants was not successful for the cloning of these genes (Hansson et al. 1998). Unfortunately, it is usually not possible to tell from the phenotype of a mutant whether the mutated gene is a structural gene or a regulatory gene. c) Fast Forward Genetics; Chromosome Landing Refined Large-scale AFLP analysis of mutants can be expected to improve the process of chromosome landing. It is now possible to analyze AFLPs on automated sequencers with fluorescently labeled primers, thereby significantly speeding the screening of AFLP markers. A new method for the rapid AFLP-based mapping of mutants was recently described (Castiglione et al. 1998). A number of AFLP markers were placed on the barley linkage map using di-haploid F2 lines from the Proctor x Nudinka cross. Linkage to these markers can be tested by means of AFLP analysis of F2 segregants from crosses of the type "mutant x Proctor" and "mutant x Nudinka". Barley Mutagenesis 37 When linkage to a mapped AFLP marker has been established, further mapping can be done using known cleaved, amplified polymorphic sequence (CAPS) or restriction fragment-length polymorphism markers. AFLP bands identified from bulked segregant analysis can also be quickly located on the right barley chromosome arm using a set of ditelosomic wheat-barley addition lines (Cannell et al. 1992). Again, further mapping should be done using known markers from this chromosome arm. AFLP markers could alternatively be converted into more convenient markers, such as CAPS markers. The polymorphic AFLP band is then sequenced from both parent cultivars used in the mapping. However, because AFLPs are not more than a few hundred base pairs long, the probability of finding polymorphic sites that can be used for CAPS construction is quite low. A more attractive solution would be to detect single-nucleotide polymorphisms using denaturing high-performance liquid chromatography (DHPLC). DHPLC is a recently developed method of detecting small deletions or single-nucleotide polymorphisms (Liu et al. 1998; Giordano et al. 1999). In this way, a single-nucleotide polymorphism detected within a sequenced AFLP band could be used as a co-dominant marker. DHPLC can be fully automated and is therefore suitable for screening large mapping populations. A further attractive approach is the utilization of deletion mutants for chromosome landing. For instance, one can land directly on deletions closely linked to a mutated gene using the AFLP analysis of bulked segregants. If the bulked segregants are mutant and wild-type F2 plants from a back-cross of the mutant to the wild type, all polymorphisms between the bulks are expected to be consequences of the mutation process. If methylation-sensitive enzymes (such as PstI or TaqI) are used, expressed regions of the genome would be preferentially screened in the AFLP analysis. In this way, one might even land on the very deletion that caused the mutant phenotype. Obviously, many AFLP markers would have to be screened for this approach, but the utilization of automatic sequencers would probably make this possible. Obtained messenger RNA or complementary DNA (cDNA) from a deletion mutant could also be hybridized to a cDNA micro-array to identify the transcript lacking in the mutant. Obviously, the cloning of regulatory genes may be difficult with this approach, because several genes can be expected to be down-regulated as a consequence of a mutation in a regulatory gene. The preferred method of inducing deletion mutants in plants is neutron radiation. The sizes of neutron-induced deletions have been extensively investigated only in Arabidopsis, where the size distribution indicated that most deletions are more than 8 kb in size (Bruggeman et al. 1996). However, a fraction of neutron mutants do not seem to be large deletions; thus, it is safest to have a set of independent mutants for each 38 Genetics investigated locus. For many loci, such series of independent mutants do exist. d) Barley Reverse Genetics The advent of large-scale genomic sequencing and expressed sequence tags (ESTs) provides the basis of reverse genetics. The use of radiationinduced deletion mutants to obtain a reverse-genetics system in barley is intriguing. To detect deletion mutants, DNA fragments would be amplified from DNA pools of M2 plants, and pools with amplified fragments smaller than the expected wild-type fragment would be further analyzed to identify the M2 plant that contains the deletion. Similar techniques were used to detect deletions in the nematode Caenorhabditis elegans, although the deletions were induced by transposons (Zwaal et al. 1993). Hybridization-based methods may also be used to detect deletions in reverse genetics. Individual preparations of M2-plant DNA could be immobilized on a (genomic) DNA micro-array, and probes for genes of interest would then be hybridized to the array. The generation of large numbers of plant DNA preparations can be done using various automated techniques. Obviously, it will be important to optimize the signalto-noise ratio in hybridizations to a genomic DNA micro-array. The DHPLC (explained above) method may be used to detect small deletions or point mutations in polymerase chain reaction (PCR) fragments in M2-plant DNA pools. This method may be especially applicable in the detection of point mutations in essential genes, because deletions may not yield viable mutants in these cases. Also, point mutations can be more informative (for instance, about the contributions of individual amino acids to protein function). Therefore, a reverse-genetics system based on point mutations is desirable. In barley, point mutations are most effectively induced by sodium azide (Olsen et al. 1993). For all PCR-based methods, the maximum pool size that can be used for the efficient detection of mutants is an important factor to consider. An attractive pool size would be 96 plants per pool, because this is the maximum number of samples accepted by most PCR machines. Then 96x96=9216 M2 plants can be grown in a square, and DNA preparations from all rows and columns in the square can be made, yielding 2x96 DNA preparations. Mutations would be identified by a row coordinate and a column coordinate. Before embarking on these adventures, it is necessary to consider the expected mutation frequency per gene, which varies among different genes and different mutagens. The mutation frequency in barley is calculated according to the spike-progeny method, which is the fraction of spike progenies that segregate for a certain phenotype or a certain gene. Generally the mutation frequency is less than one mutant per gene per 15,000 spike progenies. Barley Mutagenesis 39 e) Extending the Mutant Banks of Barley: Contribution from Arabidopsis Since the days of mutation breeding, the mutant approach has also been applied to the model plant Arabidopsis. The contribution of Arabidopsis research is clearly seen in the invention of a number of new screening techniques used to identify mutants, especially in the field of plantpathogen interactions. Using very fine-tuned screens, many Arabidopsis mutants that are either compromised in their resistance to pathogens (Glazebrook et al. 1996) or exhibit an enhanced level of resistance to pathogens (Frye and Innes 1998) have now been identified. These new screening techniques are also readily applicable to barley and other crop plants. Using the new screening methods and the emerging methods for reverse genetics, the mutant banks of barley can be significantly increased. Eventually, when the barley genome has been completely sequenced, all genes will be identified and their function analyzed by a combination of forward- and reverse genetics techniques. Many technical problems remain to be solved in the outlined methods. Barley is still a difficult plant to transform, and many of the described methods need an efficient system for the complementation of mutants. It is not likely that the method of intragenic recombination, as seen in the case of Mlo, could be generally used to establish the identity of a gene. In some cases, such as the resistance genes against powdery mildew (Shirasu et al. 1999), a transient expression system may prove sufficient for complementation tests. 3 Transposable Elements As Major Contributors and Tools in Genomic Mutagenesis a) The Mutagenic Impact and Application of DNA Transposons Transposable elements are, in contrast to the genes recognized by classical methods, self-mobilizing, independent genetic units that comprise a dynamic, fluid, rapidly evolving fraction of the genome. Transposable elements comprise two classes: the class-I elements or retrotransposons, which replicate via an RNA intermediate, and the class-II or DNA transposons, which move as DNA via a cut-and-paste mechanism (Finnegan 1990). The class-II elements or transposons were the first to be actively studied in plants. Work on chromosome breakage in maize by McClintock during the 1930s (McClintock 1939) led to her pioneering proposal of the existence of "controlling elements" (McClintock 1956). The genetics of controlling elements has perhaps been best characterized in maize (Fedoroff 1983). Characteristically, they are found in two states: 40 Genetics autonomous and non-autonomous. Autonomous elements excise, fully or partially restoring gene function and giving rise to sectors if the excision is somatic, then re-integrate at another locus. Non-autonomous elements are stably integrated unless mobilized by the presence of an autonomous element (Dellaporta and Chomet 1985). In addition, the autonomous elements may undergo a phase change in which they become "cryptic" or inactive. During the 1980s, the molecular nature of controlling elements was determined to be that of mobile DNA transposons (Shure et al. 1983; Doring and Starlinger 1984), a conceptual revolution recognized when McClintock received the Nobel Prize (McClintock 1984). All DNA transposons share a similar organization: short, terminal, inverted repeats of approximately 10 bp and a central region encoding a transposase required for the cutting and pasting of the transposon at its termini during mobilization. Autonomous versions are generally 4-10 kb, whereas nonautonomous forms are smaller deletion derivatives of the autonomous elements, ranging to less than 400 bp. The best-characterized DNA transposons have been the AciDs (paired as autonomous/non-autonomous), En or Spm/I or dSpm, Mutator of maize, and the Tam elements of snapdragon (Antirrhinum majus; Doring and Starlinger 1986; Gierl and Saedler 1986). Analysis of these systems in both species has been aided by the insertion of the elements into genes involved in the expression of easily scored phenotypic traits, such as anthocyanin and amylose biosynthesis. This has enabled simultaneous exploration of the effects of transposon insertions on gene expression and regulation, and examination of the mechanisms affecting transposon activity (Martin and Lister 1989; Weil and Wessler 1990; Fedoroff et al. 1995; Fedoroff 1999; Girard and Freeling 1999) Given the spectacular phenotypes generated by transposon insertion and excision, perhaps it is not surprising that molecular characterization of the elements led to their development as tools for gene tagging and mutagenesis. The first successful transposon tagging was that of the bronze gene in maize, on the anthocyanin-biosynthesis pathway (Fedoroff et al. 1984). Combining the recombinational-genetic analysis of insertionally induced mutants and their excision-generated revertants, transposons were applied in their native hosts in many similar efforts thereafter. The real beginning of the widespread application of transposons for gene tagging and mutagenesis came with the transfer of well-characterized maize elements to other species (beginning with the easily transformed tobacco) and the demonstration of the elements' mobilities in those species (Baker et al. 1986). As in maize, the successful tagging of genes in heterologous species has relied on easily scored and cell-autonomous phenotypes, such as a yellow-leaf phenotype in tomato (Peterson and Yoder 1993) or corolla color in petunia (Chuck et al. 1993). Due to the power of the genetics of Barley Mutagenesis 41 Arabidopsis thaliana, it was natural to move maize transposons to this plant (Van Sluys et al. 1987). Successful gene tagging strategies for A. thaliana based both on Spm (Aarts et al. 1993) and on modified Ac vectors with increased transposase expression in trans with a Ds element (Bancroft et al. 1993) were developed. Two-element tagging strategies, particularly employing Ac transposase under a strong promoter and a Ds element modified to be selectable or screenable, have become increasingly popular (Osborne and Baker 1995; Fitzmaurice et al. 1999). Such systems have proven effective in the monocot rice (Izawa et al. 1997; Chin et al. 1999) and are being used in other monocots previously recalcitrant to transformation, such as barley (McElroy et al. 1997) and wheat (Takumi et al. 1999). b) The Nature of Retro-Transposons Unlike the type-II DNA transposable elements, such as Ac and En, integrated copies of retro-transposons are not excised as a part of transposition. Instead, transposition is a replicative process and would be better described as propagation. The retro-transposons may be divided into two main classes: long terminal repeat (LTR)-bearing retro-transposons (Grandbastien 1992; Bennetzen 1996; Kumar and Bennetzen 1999), and the long- and short-interspersed elements (LINEs and SINEs, respectively), which do not bear LTRs (Schmidt 1999). The LTR retrotransposons resemble retroviruses in their organization, encoded gene products and life cycle. The life cycle for both retro-transposons and the retroviruses involves successive transcription, reverse transcription and integration back into the genome (Boeke and Chapman 1991). These two groups are highly likely to have been in existence in the last common ancestor of the fungi, plants and animals, or were laterally transferred into each group shortly thereafter. Retro-transposons are functionally distinguished from retroviruses by their lack of infectivity in mammals, depending on the env or envelope gene. Both major groups of retrotransposons, the copia-like (Flavell et al. 1992; Voytas et al. 1992) and the gypsy-like (Suoniemi et al. 1998a), are ubiquitous in plants. Each transcript of a retro-transposon has the potential (as eDNA) to be re-integrated into the genome, thereby giving rise to additional transcripts following integration. These new copies are inherited if the integrations occur in cells ultimately giving rise to gametes. Therefore, perhaps it is not surprising that retro-transposons are highly prevalent in many plant genomes, where the germ line is formed only following many somatic divisions. Retro-transposons may even contribute half of the total DNA content in some plants (Pearce et al. 1996a; San Miguel et al. 1996) and comprise a major part of the repetitive DNA component of the genome. Their replicative dynamics appear, at least in some cases (Rai 42 Genetics and Black 1999; Vicient et al. 1999), to be a major factor contributing to genome size variations in plants. c) The Mutagenic Impact and Application of Retro-Transposons Due to their invasiveness, promoter activity and sheer copy numbers, retro-transposons can have many effects on the genome and organism. Insertional gene inactivation or mutagenesis by SINEs, LINEs and retrotransposons has been documented (Varagona et al. 1992; Britten 1997; Hirochika 1997). Changes in promoter specificity or activation pattern by LTR retro-transposons have also been demonstrated in plants (Marillonnet and Wessler 1997). The sheer quantity of DNA comprising particularly prevalent retro-transposon families can affect the genome size (Vicient et al. 1999). The genome size, in turn, is thought have many physiological, ecological and developmental consequences through effects on the size of the cell nucleus, cell-cycle time and the time to maturity. For several reasons, genotypic change due to retro-transposon activity can be much more rapid than change due to mutations of single-copy genes or small gene families. Retro-transposon insertions have great mutagenic potential, because they are kilobase-scale alterations in the surrounding DNA. Each element contains transcriptional control elements that can cause major perturbations in the activity of adjacent genes. Furthermore, retro-transposons are known to be activated by stress in plants (Wessler 1996; Grandbastien 1998; Takeda et al. 1998). Retro-transposon insertions create joints between genomic DNA and their own conserved termini; therefore, they can also serve as convenient tools for tracking the changes they induce. Several techniques that produce marker bands from retro-transposon insertion loci have been developed (Waugh et al. 1997; Ellis et al. 1998; Flavell et al. 1998; Gribbon et al. 1999; Kalendar et al. 1999). Retro-transposon insertions are unidirectional, leading to progressive genome diversification, which can be subjected to pedigree analysis (Shimamura et al. 1997; Ellis et al. 1998; Flavell et al. 1998). Mutagenesis by retro-transposons, due to the linkage of retrotransposon activity to experimentally induced stress, may also be connected to environmental stimuli. When such environmental stress factors display eco-geographical variation, the genomic effects in natural populations may be detectable in the retro-transposon prevalence and insertion patterns. Moreover, retro-transposon replication is error prone, because it relies on reverse transcriptase; therefore, the retrotransposon fraction of the genome evolves comparatively rapidly. This may result in even greater variation in transposition rates between or- Barley Mutagenesis 43 ganisms and may cause genome diversification both within populations and between them. Thus, plant genomes may be viewed as being in dynamic flux, with retro-transposons playing a considerably greater role than DNA transposons in genomic modification. The development of these retrotransposons as gene-tagging tools, however, has not been widespread. Several reasons may be cited: The high copy number of most retro-transposon families makes genetic analysis difficult. This problem is increased by the lack of reversion due to the absence of retro-transposon excision. Furthermore, many retro-transposon families, as demonstrated for maize and barley, exhibit a nested insertion pattern; they are found preferentially inserted into other retro-transposon families (San Miguel et al. 1996; Suoniemi et al. 1997). However, particularly low-copy-number families of retro-transposons appear to be far more mutagenic than those present in greater numbers; perhaps for this reason, they are found in fewer copies. Nevertheless, gene tagging and mutagenesis are possible with retrotransposons. The first demonstrably active plant retro-transposon, Tnt1, was isolated by selecting for insertion into a nitrate-reductase gene (Grandbastien et al. 1989), arguably a tagging procedure. The Tos17 retro-transposon of rice, highly active in tissue cultures, is used in a large-scale program that screens for mutants among regenerated plants (Hirochika 1997). The replicative nature of retro-transposition has selected for highly effective integration mechanisms catalyzed by the enzyme integrase. Integrase is highly conserved, and plant integrase appears to closely match retroviral integrases in structure (Suoniemi et al. 1998b). Hence, the applicability of retroviruses and retro-transposons as vectors for gene therapy and transformation has been well recognized (Kingsman et al. 1991; Ivics et al. 1993; Bushman 1994; Katz et al. 1996). Due to the increasing understanding of the life cycle of plant retrotransposons (JaaskeHiinen et al. 1999), there is no reason these cannot be developed for gene ablation or specific tagging using the same principles. 4 Mutational Breeding in Barley: an Example. Improving Nutritional Qualities Mutational breeding in barley was initiated to reduce the anti-nutritional effect of phytic acid [myoinositol 1,2,3,4,5,6-hexakisphosphate (IP6)] and to provide plant material for biochemical studies of the biosynthesis of IP6 via the sequential addition of phosphate to myoinositol. Barley, like other cereals, stores up to 80% of the total grain phosphate as phytin (Raboy and Gerbasi 1996). The remaining phosphate is free phosphate, phospholipid nucleic acids, etc. Most of the phytin is stored as electron- 44 Genetics dense particles in protein bodies in the aleuron cell layer of barley, and the remaining (-10%) is stored in the embryo. This is in contrast to maize, which stores most of its phytin in the embryo (O'Dell et al. 1972). The anti-nutritional effect of phytic acid is due to the lack of phytase activity in monogastic animals, such as pigs, chicken, fish and humans; hence, phytin passes undegraded through the intestines and stomach and is released with the feces. In the soil, phytin is degraded and thus contributes to the pollution of freshwater streams, because phosphate leads to the eutrophic growth of algae. Phytate also forms salts with cations, such as zinc, calcium and iron, making these unavailable for uptake. Limited bio-availability of particulate iron is of great concern in human nutrition, as detailed in the World Food Summit held in Rome in 1996. Modern agriculture compensates for phytate effects by adding phosphate to fodder and supplementing it with minerals, or by adding microbial phytase as a feed enzyme. The initial screening for low phytate grains was an indirect measurement based on the assumption that reduced phytate results in increased free phosphate. This allowed the use of a simple molybdate staining procedure for free phosphate; grains staining blue were taken as indicative of reduced phytate content. Initially, 2000 M2 half grains from sodium-azide-treated Pallas POI grains were screened; for those that scored positive, the embryo-containing part was germinated, and M3 spikes were harvested. To confirm that these actually were low in phytate, a thin-layer chromatography (TLC) system was used to detect and separate phytic acid from free phosphate and intermediates of phytate, and 18 mutants were confirmed to be lowphytate varieties (Rasmussen and Hatzack 1998). There was an unexpectedly high number of mutants, which could indicate that phytate biosynthesis has a hot spot for mutations. The selected mutants were tentatively diveded into two classes: the A-type (with less than 10% phytate) and the B-type (with 50-60% phytate, compared with 100% for the non-mutated strain). Tests for allelism showed that the mutations are located in unlinked loci. DNA gel blot analyses using inositol 1,3,4-trisphosphate 5/6-kinase and myoinositol phosphate synthase as probes did not indicate a mutation at these loci (Rasmussen, unpublished). The TLC system was further refined to detect myoinositol with less than six phosphate groups. The selected lines have been field grown in inspection plots at several locations in Denmark for 2 years, and many exhibited normal vigor and normal or almost normal seed set. As expected, in some cases, pleiotropic effects could be noted (such as shriveled kernels, particularly with the A type). These and other effects might be due to additional mutations in the raw mutants, which could be eliminated by cross-breeding. Several lines were also propagated in New Zealand during 1998-1999 and in Denmark during 1999 to increase the amount available for animal feed trails. Barley Mutagenesis 45 The mutant lines have been investigated with respect to phosphorus nutrition in 201 pots, with rock wool as the growth medium. The plants were grown to maturity outdoors with an automated siphon airlift watering system. The grain yield of the B-type low-phytate mutants was the same as for the parent varieties. The grain yield for the A-type was severely reduced at all phosphate levels. To test whether nutritional improvements had been achieved, tests were initiated with rats, because these were previously successfully used in tests for high lysin and nitrogen nutrition by Eggum (1973). The tests showed that the apparent digestibility of phosphate was improved in mutant lines and that more zinc was taken up from these lines by the rats (Poulsen et al. 2000). This indicated that mutational breeding is feasible and suggested that feeding trials should be repeated with piglets and broilers. Several high-lysin barley mutants have been generated and analyzed genetically, biochemically, nutritionally and in breeding programs. Although much effort has been spent on attempts to breed barley for high lysin content, the yield has always been 5-10% lower than those used in national tests. This could be because of lower starch content and relatively higher amounts of non-starch carbohydrates in the mutants. It was generally accepted that it might not be possible to breed highyielding barley with a high lysin content. Genetically, there is no simple relationship between improved lysin content and lower grain yield (Doll 1975). From the genetic analyses, it was evident that many loci on different chromosomes could be mutated to yield high-lysin barley. It was also known that high lysin content was obtained due to an increase in albumin at the expense of the concentration of the storage protein hordein. Therefore, a new strategy was used by Jensen (1991): screening for lowhordein mutants with a minimal yield reduction as an indirect way of finding mutants with a high lysin content. The so-called turbidity test, which gives a simple reflection of the content of alcohol-soluble storage proteins, was used. Twenty low-hordein barley mutants were scored from 49,000 M2 grains. Several of these had improved lysin content, minimal yield depression and a kernel weight similar to that of the mother variety Sultan (Eggum et al. 1995). In balanced feeding trials with rats, the mutants resulted in an increase in biological value of up to 20%. Thus, it seems to be possible to develop high-lysin barley cultivars. 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Acad Sci USA 90:7431-7435 Dr. Anders Falk Dr. Christer Jansson Department of Plant Biology The Swedish University of Agricultural Sciences P.O. Box 7080 75007 Uppsala, Sweden Dr. Alan H. Schulman Institute of Biotechnology University of Helsinki Plant Genomics Laboratory P.O. Box 56 (Viikinkaari 6) 00014 Helsinki, Finland Communicated by K. Esser Dr. Seren K. Rasmussen Rise National Laboratory Roskilde, Denmark Genetics Extranuclear Inheritance: Cytoplasmic Linear Double-Stranded DNA Killer Elements of the Dairy Yeast Kluyveromyces lactis FriedheIm Meinhardt and Raffael Schaffrath 1 Introduction Plasmids were originally considered to be extra-chromosomal, autonomously replicating, covalently closed, circular, double-stranded DNA (dsDNA) elements restricted to prokaryotes (Lederberg 1952). However, the detection of the 2-Jlm circular plasmid in the yeast Saccharomyces cerevisiae (Sinclair et al. 1967) required broadening of the definition, because the occurrence of extra-chromosomal DNA was no longer limited to bacteria. Subsequently, similar circular dsDNA elements were also found in fIlamentous fungi (Esser et al. 1986) and other yeast genera, such as Zygosaccharomyces (Tohe and Utatsu 1985) and Kluyveromyces (Falcone et al. 1986). When linear dsDNA elements were detected a dozen years later in the mitochondria of Zea mays (Pring et al. 1977), the term plasmid was again used. Linear plasmids have been found in a great number of micro-organisms since then; we refer to previous contributions in this series (Meinhardt and Rohe 1993; Schriinder and Meinhardt 1995b) and a mini-review (Meinhardt et al. 1997) for compilations of such elements. This chapter focuses on the cytoplasmically localized linear yeast elements that have been isolated from numerous genera as diverse as Botyroascus, Kluyveromyces, Pichia, Saccharomyces, Trichosporon and Winge a (Cong et al. 1994; Fukuhara 1995). The genetic organization of yeast linear elements appears to be quite uniform (Kitada and Hishinuma 1984; Meinhardt et al. 1998), with the killer plasmid pair pGKLl and pGKL2 (also termed kl and k2) characterized the most thoroughly (Gunge 1995; Meinhardt et al. 1997; Fig. 1). Reviews describing yeast linear plasmids in general and the K. lactis killer system in particular have been written during the past 10 years (Gunge 1986, 1995; Meinhardt et al. 1990, 1997; Stark et al. 1990; Schriinder and Meinhardt 1995b; Fukuhara 1995). However, since the last review in this series (Schriinder and Meinhardt 1995b) and, more importantly, the recently established advances towards pGKLl- and pGKL2-specific manipulation techniques (including allelic replacement, gene shuffling and site-specific muta- Progress in Botany, Vo!' 62 © Springer-Verlag Berlin Heidelberg 2001 52 pGKL1 I PGKL2 ~ 1 2 terminal protein I DNA-polymerase toxin a+l) 1 dispensable ~ , Genetics terminal protein I DNA-polymerase cappingenzyme 2 3 ~ K f- helicase SSB 4 ill ~ immunity RNApolymerase 6 toxin y RNA-pol.subunit TRFl ~9~ Fig. 1. Genetic organization of pGKLl (8.9 kb) and pGKL2 (l3.4 kb). Open reading frames are numbered according to Hishinuma et al. (1984) and Tommasino et al. (1988). Directions of the genes correspond to the transcriptional orientation. When known, gene functions are also indicated. For details, see text genesis in vivo; Schaffrath et al. 1999), our knowledge of this peculiar system has greatly increased at the molecular-genetic level. 2 Genetic Organization of the Killer Plasm ids Kluyveromyces lactis cells containing the linear elements pGKLl and pGKL2 differ from plasmidless cells in that they are able to kill other sensitive yeasts belonging to either the same or a different species (Gunge et al. 1982). Because curing resulted in the concomitant loss of the killer ability, it soon became evident that these cytoplasmically localized linear elements constitute the physical basis of the killer phenotype (Gunge et al. 1982; Starn et al. 1986). pGKLl carries four open reading frames (ORFs), which code for a pGKLl-specific DNA-polymerase, a, J3 and y subunits of the toxin, and immunity against it (Sect. 3.a). pGKL2 has 11 ORFs. No function could be assigned to ORF 1; ORF 2 encodes the plasmid-specific DNA polymerase including the terminal protein. ORF 3 exhibits similarities to capping enzymes (Larsen et al. 1998); ORF 4 presumably encodes a helicase. ORF 5 is likely to encode a single-stranded binding protein. ORF 6 codes for the RNA polymerase that transcribes plasmid-based genes; ORF 7 may be a subunit of the latter (Schaffrath et al. 1996). No function is known for ORFs 8 and 9; ORF 10 encodes a terminal recognition factor that binds to the terminal inverted repeats (TIRs) of both plasmids. Regarding ORFs 1-10, we refer to reviews of the killer system and linear plasm ids for detailed information and citations of original articles (Stark et al. 1990; Gunge 1995; Meinhardt et al. 1997; Schaffrath et al. 1999). We recently detected another ORF of unknown function Extranuclear Inheritance 53 (ORF 11) on pGKL2 (Meinhardt et al. 1998; Larsen and Meinhardt, unpublished); thus, the genetic organization of the K. lactis killer plasmids is as outlined in Fig. 1. 3 Zymocin Toxin The killer phenomenon of K. lactis differs, both genetically and biochemically, from those of other well-known yeast systems, particularly those specified by dsRNA viruses from Saccharomyces cerevisiae (Wickner 1996). Unlike the ionophore toxins of the latter, the pGKLl and pGKL2 dsDNA plasmid-encoded zymocin system of K. lactis inhibits the growth of a variety of sensitive yeast genera via GI-specific cellcycle arrest (Stark et al. 1990; Butler et al. 1991a; Schaffrath et al. 1997c). As a consequence, for purposes of molecular mycology and vector construction in yeast-cell factory research, the plasmid-encoded zymocin system of K. lactis is of considerable interest in the growing nonconventional yeast community (Wesolowski-Louvel et al. 1996). a) Structure The zymocin secreted by killer strains of K. lactis is a heterotrimeric protein complex with polypeptide subunits of 99, 30 and 28 kDa, respectively (Stark et al. 1990). Using sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) analysis of culture fIltrates from nonkiller and zymocin-producing strains, it became evident that all the subunits (termed a, ~ and y, in decreasing order of their Mr) are encoded by pGKLl; ORF 2 encodes the a and ~ subunits, whereas ORF 4 specifies the y polypeptide (Stark and Boyd 1986; Schriinder and Meinhardt 1995a). Zymocin biosynthesis can be prevented in the presence of tunicamycin, suggesting that the complex is glycosylated (Sugisaki et al. 1985). As judged from endo-~-N-acetylglucosaminidase H (endo-H), the digestion of purified zymocin preparations and subsequent SDS-PAGE analysis, the a subunit exhibits an Mr decrease of approximately 3.4 kDa (Stark and Boyd 1986). Together with the observation that mannose-rich moieties can only be detected for the a subunit using a concanavalin-Aconjugated, horseradish-peroxidase-based chromogenic assay (Sugisaki et al. 1984), this body of evidence indicates the presence of a single, Nlinked oligosaccharide chain associated with the a polypeptide. In contrast, none of the other subunits appears to be N- nor O-glycosylated, and both are endo-H resistant (Stark and Boyd 1986). The structural integrity of the a~ glycoprotein complex is maintained by internal cysteine bonds within the a subunit and an intermolecular disulfide bridge between the ~ and y polypeptides. Hence, treatment of purified zymocin 54 Genetics with -SH reagents, such as l3-mercaptoethanol, causes subunit disintegration and completely abolishes biological activity (Stark et al. 1990). b) Biogenesis, Assembly and Secretion As shown by amino-acid-sequence analysis of the individual zymocin subunits, the N-terminal residues of the mature a, 13 and y polypeptides are A30 (ORF 2), GS95 (ORF 2) and A19 (ORF 4), respectively. Hence, the two larger subunits are the processed products of a single gene, ORF 2 of pGKLl, and result from the proteolysis of an al3 precursor approximately two-thirds of the way along the primary al3 translation product. The y subunit, however, must be the single processed product encoded by ORF 4 on pGKLl (Stark and Boyd 1986). As for the latter, its mature N-terminus (A 19 ) co-localizes with the first of three potential predicted signal-peptidase-recognition sites (C 16AA -1-A-1-A-1-). In contrast, despite the existence of a potential recognition site within the ORF 2 leader region (V 19 QG-1-), the N-terminus ofthe a subunit (A30 of ORF 2) in the al3 precursor does not appear to result from signal-peptidase cleavage. Instead, it results from the proteolytic procession of another endopeptidase (Stark et al. 1990). The mature a and 13 subunits both commence at positions that are immediately preceded by the paired basic residues Lys-Arg (a=KR29, I3=KRs94 )' a motif that is known to be utilized for the Kex2p-specific procession of secreted proteins from S. cerevisiae (Julius et al. 1984). Interestingly, the K. lactis kexl mutation, which results in a deficiency of zymocin secretion, can be rescued by single-copy complementation with the KEX2 gene from S. cerevisiae and vice versa. The K. lactis KEXI gene is able to functionally complement a kex2.1. gene knockout in baker's yeast (Tanguy-Rougeau et al. 1988; WesolowskiLouvel et al. 1988). Kex2p localizes to the Golgi and is essentially involved in the biogenesis of secreted proteins, such as pre-pro-a-pheromone and the dsRNA-encoded Kl killer-toxin precursor. Not only do pGKLl- and pGKL2-carrying S. cerevisiae strains process and secrete the zymocin as authentically as the original K. lactis host strain, but the kex2.1. mutant of baker's yeast is also unable to produce secretable zymocin (Tokunaga et al. 1990; Stark et al. 1990). Thus, zymocin production appears to be very similar in both yeast species, and processing is obligatorily coupled with secretion during its biogenesis andlor assembly. Consistently, in the absence of al3 secretion, the y subunit fails to be secreted but accumulates intracellularly (Tokunaga et al. 1989, 1990). Similarly, a zymocin-resistant S. cerevisiae strain harboring a conditional galactose-regulatable ORF 4 secretion vector does not allow y subunit export under inducing conditions. Instead, despite its functional secretion signal, which has been shown to efficiently direct the secretion of heterologous proteins, the y subunit also accumu- Extranuclear Inheritance 55 lates intracellularly (Baldari et al. 1987; Tokunaga et al. 1987a, 1988, 1993, 1997). However, its secretion can be effectively restored by genetically engineering a heterologous pre-pro sequence from S. cerevisiae in-frame between the y leader peptide and its mature coding region (Tokunaga et al. 1989, 1990, 1992). If this foreign pro sequence can circumvent the requirement that the a/3 precursor promote y-subunit export, why might a/3 be needed for during biogenesis? Explanations may include a role in assisting the correct folding of the y polypeptide or a requirement that the a/3 precursor assemble the y subunit into a secretable holo-zymocin by facilitating the formation of the intermolecular /3-y disulfide bridge (see above) and/or by providing the unglycosylated y subunit with a glycosylated entity typical of yeast exo-proteins. c) Immunity Not surprisingly, a K. lactis killer strain also expresses immunity towards its own secreted exo-zymocin. Immunity is strictly dependent on the maintenance of pGKLl: as shown by curing experiments, pGKLl-free isolates of K. lactis that still contain pGKL2 are phenotypically sensitive towards zymocin and are indistinguishable from plasmid-free non-killer strains (Niwa et al. 1981). In contrast, non-killer strains carrying the pGKLl-derived orf21:!. knock-out plasmids kl-NK2, pGKLlS or pGKLlD retain immunity but fail to produce zymocin (because they are deleted in the a/3-subunit structural gene, ORF 2; Gunge 1995). Because strains that carry the pGKLl-based orf2-41:!. deletion plasmids Fl and F2 fail to confer both auto-immunity and zymocin expression (Kikuchi et al. 1984), all this evidence indicates that pGKLl ORF 3 codes for immunity. In fact, on cloning ORF 3 into a K. lactis ARS vector and retransformation into a pGKLl-free host strain, immunity towards zymocin was partially restorable as long as the cell co-maintained pGKL2 (Tokunaga et al. 1987b). Therefore, Stark et al. (1990) postulated that pGKL2 is essential for ORF-3 gene expression on the ARS vector. Alternatively, pGKL2 might encode another immunity factor (in addition to pGKLl ORF 3). Although there is sequence homology between pGKLl ORF 3 and pGKL2 ORF 1 (Stark et al. 1990), a role for the latter in immunity has been nearly disproved by targeted gene disruption; strains carrying a pGKL2based orfll:!.::LEU2 null mutation on hybrid plasmid pRKL2 are indistinguishable from wild-type killer strains in the expression of both killer and immunity phenotypes (Schaffrath et al. 1992). Immunity is also seen in S. cerevisiae strains towards expression of intracellular y-toxin subunits (see above; Tokunaga et al. 1989), evidence that may indicate that immunity does not simply work by excluding zymocin from the cell. Instead, it suggests that immunity functions via the interaction of the 56 Genetics Orf3p determinant with the y subunit itself or its putative intracellular target(s} (Stark et al. 1990). d} Mode of Action The K. lactis zymocin is active against the growth of a variety of sensitive yeast genera, including Saccharomyces, Kluyveromyces and Candida, but not Schizosaccharomyces (Gunge and Sakaguchi 1981; Gunge et al. 1981); moreover, it differs biochemically from the S. cerevisiae ionophore killer toxins (Wickner 1996), i.e., it is not able to elicit rapid efflux of K+ ions from treated cells, nor does it cause sensitive cells to shrink (Butler et al. 1991a). Instead, treated cells increase in volume, suggesting that zymocin inhibits cell division rather than cellular growth (Butler et al. 1991a). Indeed, sensitive S. cerevisiae cells arrest at the unbudded G1 stage of the cell-division cycle, and this block typically requires approximately 10,000 zymocin molecules (Butler et al. 1991a). In addition, fluorescence-activated cell-sorter analysis demonstrates a pre-replicative DNA content (1n) indicative of a G1 cell-cycle stage prior to START (Butler et al. 1991a). Further evidence that the zymocin acts specifically in G1 is provided by the observation that cells that have been chemically arrested by hydroxyurea during S-phase prior to zymocin treatment are able to complete one round of cell division. The cells are arrested in the new, unbudded G1 cell-cycle stage once they have been released from the chemical S-block in the presence of zymocin (Butler et al. 1991a). Consistently, as shown by the incorporation of radiolabelled precursors into RNA and protein, treated cells are still metabolically active, allowing transient macromolecular biosynthesis. Also, they increase in volume by approximately 30-50%, similar to the increase caused by bona fide preSTART Gl arrests induced by the mating-pheromone cascade (Leberer et al. 1997) or the growth of cdc28 ts strains at the non-permissive temperature (Nasmyth 1996). Earlier reports that zymocin functions by inhibiting the CDC35 gene product, adenylate cyclase, thus abolishing the roles of cyclic adenosine monophosphate (cAMP) essential for mitotic growth and cell division (Sugisaki et al. 1983), have been disproved by knocking out BCYl, which encodes the regulatory subunit of cAMP-dependent protein kinases. The bcyl!l null mutant, which no longer requires cAMP to undergo cellular division, grows in the absence of a functional CDC35 gene product but is sensitive to (and is Gl-arrestable by) the exo-zymocin as its wild-type parent strain (White et al. 1989). Thus, it is highly unlikely that zymocin exerts its toxicity by inhibiting Cdc35p; however, studying its mode of action may be a useful tool for dissecting stage-specific events in G1. To execute START, a sufficient level of Cdc28p-kinase activity is required, and its amount is mainly determined by the levels of Gl cyclins (Cross Extranuclear Inheritance 57 and Tinkelenberg 1991). Although it is attractive to speculate that zymocin might act by antagonizing this essential G1 cyc1in function, it is noteworthy that neither a hyperactive dominant CLN3-1 allele nor the over-expression of another G1 cyc1in, CLN2, can significantly reduce zymocin sensitivity (Butler et al. 1994; Schaffrath et al. 1997a). Therefore, the mode of zymocin action still remains unclear, and we anticipate that molecular-genetic analyses of zymocin-resistant mutants (see below) rather than a biochemical approach will provide a more direct route to identify and characterize its cellular target(s). Interestingly, the toxic activity of the zymocin complex appears to reside solely within the y subunit; conditional expression of its structural gene (ORF 4 from pGKLl) from tightly regulated yeast promoters (UAS GAL or UAS MET) mimics exogenous treatment of the holo-zymocin and is fully lethal to sensitive strains of S. cerevisiae under inducing conditions (Tokunaga et al. 1989; Butler et al. 1991b; Schaffrath et al. 1997a). Endogenous expression of the mature y-subunit structural gene (Le., without its native secretion leader) results in biologically active y toxin, whereas exogenously applied y polypeptide is not able to inhibit cell growth (Tokunaga et al. 1989). As for the roles played by the a and 13 subunits within the secreted exo-zymocin, this strongly implies that the holo form must assist the y-toxin subunit to be taken up by the cell. Therefore, as a precondition for y-subunit entry and action, the holozymocin is expected to first bind to the cell surface in order to signal its toxicity (Stark et al. 1990). Because the a subunit has been shown to be similar to carbohydrate-binding domains of wheat-germ agglutinin (Stark et al. 1990) and has been shown to exhibit an essential exochitinase activity in vitro that can be specifically inhibited by 13allosamidin (Butler et al. 1991c), it is attractive to speculate that its role is to promote zymocin binding to cell-wall-associated chitin. Together with the extreme hydrophobicity predicted for the 13 subunit, it suggests that interaction with the cell membrane assists y-subunit uptake and entry into the sensitive cell (Stark et al. 1990). e) Resistance Based on their ability to grow in the presence of the holo form, zymocinresistant mutants [termed skt (sensitivity to K. lactis toxin), iki (insensitive to killer) and kti (K. lactis toxin insensitive)] have been isolated independently by three groups (Kawamoto et al. 1990; Butler et al. 1994; Kishida et al. 1996; Schaffrath et al. 1997b). The sensitivity of these mutants toward the intracellular, conditional expression ofthe y subunit from inducible promoters (see above) can distinguish zymocin binding/uptake (class I) from y-toxin target site mutants (class II). Studies on chs (chitin synthesis deficient) S. cerevisiae mutants (Takita and Cas- 58 Genetics tilho-Valavicius 1993) are consistent with the role the a. subunit may play in assisting the docking of zymocin to the cell surface due to its exochitinase activity (see above). The majority of these behave as class-I mutants; i.e., they are resistant to exo-zymocin but are sensitive to endogenous expression of the toxic y subunit. Moreover, KTI2 is allelic with CHS3, and KTIlO may correspond to CHS6 (Stark, personal communication). CHS3 codes for the catalytic subunit of chitin synthase III (CSIII), the in vivo activity of which is lacking in chs6 mutants and usually amounts to 90% of a wild-type cell's chitin synthesis (Cos et al. 1998; Ziman et al. 1998). In addition, knocking out CHS4 (isoallelic to SKT5), which encodes a post-translational activator of CSIII, renders cells resistant to exo-zymocin (Kawamoto et al. 1992, 1993; Trilla et al. 1997). Although the class-I gene KTI6 is non-allelic with CHS3, CHS4 and CHS6, its mutation obviously affects zymocin binding or y-toxin uptake (Stark, personal communication). Thus, it is possible that it corresponds to either CHS5 or CHS7, two genes that were recently shown to function in the targeting and trafficking of Chs3p from the endoplasmic reticulum to chitosomes (Ziman et al. 1996; Santos and Snyder 1997; Trilla et al. 1999). In summary, we propose that the primary interaction of holo-zymocin with its sensitive cell is facilitated by docking to cellwall chitin. Thus, chitin may serve as a receptor molecule and all scenarios that lead to a reduction to chitin synthesis due to the abolishing, reducing and/or mis-trafficking of CSIII activity may render cells resistant to the toxic effects of zymocin. The presence of as many as ten distinct target-site class-II mutants (see above) suggests that a complex pathway trans duces zymocin's inhibitory effect (Butler et al. 1994). Although some of them may be involved in the expression of targets inhibited by the y toxin, a number of proteins could also participate in the process that is blocked by it. These might act as a biochemical pathway or, alternatively, may form a complex containing several components. To identify y-toxin target(s), one exploits genetic screens involving two-hybrid interaction trapping and gene-knockout transposon tagging (Schaffrath et al. 1997a). As for the latter, a pool of yeast transformants that have a Tn3::1acz::LEU2 minitrans po son randomly inserted into the genome are screened for viability and y-toxin resistance by conditionally switching on y-subunit expression under UAS GALl control. Candidate clones are then subjected to plasmid rescue in Escherichia coli and are characterized via DNA sequencing. In this way, we and others have identified several resistant yeast disruptants. To date, five genes (IKIl, IKI3, KTIl2, SIT4 and SAP155) have been implicated to affect intracellular y-toxin action. Ikilp is part of an insoluble fraction in cell extracts, and Iki3p is predicted to possess a membrane-spanning region, raising the possibility that an insoluble Ikil p/Iki3p-containing compartment is involved in toxin action (Yajima Extranuclear Inheritance 59 et al. 1997). Depletion or over-production of Kti12p confers resistance towards the toxic y subunit; thus, Kti12p may be a potential y-toxin target (Butler et al. 1994). If absent (kti12D) or mutated (as in the kti12 background), the y toxin cannot bind to Kti12p; high KTI12 gene dosage might lead to excess unbound Kti12p, which competes for a downstream effector molecule, thereby diluting the toxic signal of the y subunit (Schaffrath et al. 1997a). Class-II mutations (ktill and kti13) can be partially suppressed by high-copy KTI12, suggesting that both genes may act upstream of KTI12 and may limit the available Kti12p pool when mutated (Butler et al. 1994; Schaffrath et al. 1997a). Sap155p associates in a cell-cycle-dependent manner with the Sit4p phosphatase, which functions late in G1 for progression into S phase (Luke et al. 1996). Sit4p is required for the execution of START, and sit4ts strains arrest late in Gl prior to START, in part due to the role of St4p in expressing the G1 cyclin genes CLNl and CLN2. Interestingly, sit4i1 null mutants, which are viable only in certain SSD-v backgrounds, are fully resistant to partially purified zymocin (Stark 1996). Thus, the Sit4p phosphatase is required for cells to respond to the K. lactis zymocin. 4 Replication The presence of (1) TIRs that differ in both pGKLl and pGKL2, (2) the covalently linked proteins at the 5' ends and (3) genes on pGKLl and pGKL2 encoding a DNA-polymerase of the viral B type is reminiscent of some DNA viruses, such as adenoviruses and phages with linear genomes (Tommasino et al. 1988; Salas et al. 1995). Because mutations in one of the polymerase loci cannot be complemented by the respective homologous gene of the other plasmid (Schaffrath et al. 1995a), each DNA polymerase is evidently plasmid specific. Data obtained from the sequence analysis of purified terminal proteins of pGKL2 (Takeda et al. 1996) show that it is descended from the N-terminus of the pGKL2encoded DNA polymerase. Thus, plasmid-specific replication is presumably caused by the initial interaction of the TIR and the respective terminal protein, which is (as outlined for pGKL2) part of the DNA polymerase. In the linear bacteriophages, such as <D29 of Bacillus subtilis and in adenoviruses, the terminal protein and the DNA polymerase are encoded by separate, successively arranged genes. Thus, it is tempting to speculate that an in-frame fusion of the gene encoding a terminal protein with the respective polymerase-encoding trait may be the reason for the genetically proven plasmid specificity of the DNA polymerases of linear plasmids. This is merely a speculation; it is not known whether processing of the terminal protein happens via autocatalytic cleavage of the precursor protein or via the action of a separate (plasmid-encoded?) protease. Irrespective of the processing mode, the fate of the terminal 60 Genetics protein and its corresponding polymerase is quite obvious: they are typical single-use polypeptides and, as such, may play a key role in plasmid copy-number control. In addition to the terminal proteins and DNA polymerases, other plasmid-encoded proteins were identified as involved in autonomous replication. These include the gene product of ORF 10, a terminal-recognition factor (TRF1) binding specifically to the TIRs of both plasmids (McNeel and Tamanoi 1991; Tommasino 1991) and the polypeptide predicted for ORF 5, which is presumably a singlestranded binding protein, SSB for short (Schaffrath and Meacock 1995, 1996). The presumed replication mechanism is outlined in Fig. 2. Initiation 5' - 3' 3 ~~~~ l J Tennination p~ Fig. 2. Hypothetical mechanism for plasmid replication via protein priming (Schaffrath and Meinhardt 1998). ori Origin of replication. The rounded triangle represents the terminal recognition factor (TRF) encoded by open reading frame 10. Solid lines correspond to the terminal inverted repeat (TIR). White circles represent single-stranded binding proteins (SSBs). 1 Replication is initiated by the binding of the TRF to the origin, located within the TIR; the DNA polymerase (DNP) initiates replication using the terminal protein (TP) as the primer. 2 During elongation, the displaced single strand is covered with SSBs.3 During the termination of replication, SSBs and DNP dissociate from the DNA Extranuclear Inheritance 61 5 Gene Expression Cytoplasmic compartmentalization of the yeast linear killer plasmids indicates the existence of an independent expression machinery that is not under the control of the nucleus or other compartments, such as the mitochondria. Genes governed by nuclear promoters cannot be expressed when integrated into linear plasmids (Kamper et al. 1989a; Gunge et al. 1995) and vice versa; pGKL-based genes are not expressed in the nucleus (Kamper et al. 1989b, 1991; Romanos and Boyd 1988). a) Transcription The pGKL2 ORF-6-encoded RNA polymerase plays a key role in the expression of the cytoplasmic genetic information. The predicted enzyme resembles the 13 and 13' subunits of prokaryotic RNA polymerases, but there are also striking similarities to the largest subunits of the eukaryotic RNA polymerases II and III (Wilson and Meacock 1988; Schaffrath et al. 1995b). Essentially, all conserved domains of the multi-subunit RNA polymerases are present in pORF6, which thus appears to be related to the complex eukaryotic enzymes (however, with a much simpler Table 1. Comparison of upstream conserved sequence (UCS) elements from different yeast cytoplasmic linear elements. (Schaffrath et al. 1999) Yeast Element ORFs UCS References Kluyveromyces lactis pGKLl 4 NATNTGA pGKL2 11 NATNTGA Romanos and Boyd (1988); Wilson and Meacock (1988); Schickel et al. (1996); Larsen and Meinhardt (unpublished) Saccharomyces kluyveri pSKL 10 NATNTGA Hishinuma and Hirai (1991) Pichia acaciae pPacl-2 TATTTGA Bolen et al. (1994) Pichia etchellsij pPEI-B pPEI-A 10 2 NATNTGA NATNTGA Meinhardt (unpublished) Meinhardt (unpublished) Debaryomyces hansenij pDHLl 2 {A/dAT{A/G)T GA Fukada et al. (1997) Consensus ORF, open reading frame. NATNTGA 62 Genetics architecture). Homologous genes are also present on other yeast cytoplasmic elements, i.e., pSKL of Saccharomyces kluyveri (Hishinuma and Hirai 1991) and pPEIB of Pichia etchellsii (Meinhardt, unpublished). All of the genes present on pGKLl and pGKL2 are likely to be expressed independently (Meinhardt et al. 1994); each is preceded by a seven-nucleotide-spanning upstream conserved sequence (UeS), which apparently constitutes the essential part of cytoplasmic promoters (Schickel et al. 1996; Table 1). Transcription generally appears to be .... -.................................... ,''''" 1 pGJU.2 pSJU. HCV SN VAC -'SF 2 3 olU"3 OlU"3 -19-m-19 - -25- -i 21 ' -21- ' 1-20- ' LT-20- V v VL ~-2 8 - ! I{il-22- 5 VI III IlIa IV V I 4 C.E - 137 CE - 34 CE -23CE .......... ,,' guany IyItransferase triphosphatase pGJU.2 OlU"3 --16-v(DJi< J6 - 1i\DJK - 5252-1 - ll -i RLP-llO- FT- 8- ' N E - 14 - ' -13- ' -109P-109L- Ill-I pSK.L 0 L I ORr) con:!St!'n!Su5sequence ........................•. , methy Itrans ferase SAM-BS g l :ijAKccDili ...,. LKFEKI~PN I!!l e xccDjJilKOIYHLKFKKiMil'!'lDPI g ,e .G, ,LS, .L ...... 1.SLE . D lIE T A !Ii 1 11 TI W )1 A P V M F V F Y M ~ m .~GM Y VF V H C III IIIa IV V VI pG"-1.'ORn pSJU. ORF3 CKV Col CE CE CE CE CE CE Sp CE SC CE HCV SFV VAC ASF Fig. 3. Structure of the viral-like capping enzyme encoded by pGKL2 open reading frame 3. Conserved motifs of messenger RNA triphosphatases correspond to Arabic numerals. Roman numerals indicate characteristic motifs of guanylyl transferases. A putative Sadenosylmethionine binding site (SAM-BS; PROSITE reference no. PDOC00871) of the methyltransferase corresponds to the roller jewel. ASP African swine-fever virus, CE capping enzyme, Cel Caenorhabditis elegans, CHV Paramecium bursaria chlorella virus, MCV Molluscum contagiosum virus, PGKL2 putative capping enzyme of linear plasmids from Kluyveromyces lactis, pSKL putative capping enzyme of linear plasmids from Saccharomyces kluyveri, Sc S. cerevisiae, SPV Shope fibroma virus, Sp Schizosaccharomyces pombe, VAC Vaccinia virus. Conserved amino acids are highlighted Extranuclear Inheritance 63 weak, as estimated from assays based on a reporter gene (Schriinder and Meinhardt 1995; Schriinder et al. 1996). Expression of the reporter gene can vary up to 12-fold, depending on the UCS used. Deletion of the UCS leads to complete lack of expression (Schaffrath et al. 1996; Schickel et al. 1996). This balanced level of expression might also be indicative of a postulated viral ancestor (see below), because it ensures stable propagation and the viability of the host cell. b) Translation Eukaryotic messenger RNA (mRNA) can only be translated efficiently when the transcripts carry the typical 5' guanyl cap. Capping requires three enzymatically catalyzed reactions (Bisaillon and Lemay 1997): 1. Removal of Pi from the 5'end by mRNA triphosphatase, resulting in di-phosphorylated RNA, pp(N)n 2. Transfer of guanosine monophosphate from guanosine triphosphate to the 5'-diphosphate end by RNA guanylyl transferase, leading to G(5')ppp(5')N(N)n 3. The transfer of a methyl group from S-adenosylmethionine to N-7 of the guanine by RNA (guanine-7-)methyltransferase, eventually giving rise to the mature cap, (m7G-(5')ppp(5')N(N)n) As for replication and transcription, cytoplasmic linear plasmids do not have access to the nuclear capping system either. Indeed, conserved motifs of capping enzymes reside in the predicted polypeptide e:t:J.coded by pGKL2 ORF 3, a gene essential for the maintenance of the system (Larsen et al. 1998; Fig. 3). Data obtained with a heterologously expressed ORF 3 gene product, which was found to have guanylyltransferase and triphosphatase activities, are consistent with these observations (Meinhardt and Tiggemann, unpublished). 6 Phylogeny It has repeatedly been stated throughout this chapter that linear plas- mids generally share similarities with genomes of protein-primed replicating viruses (adenoviruses and linear phages) that, like the plasmids, have TIRs with covalently attached proteins and viral B-type DNA polymerases. Comparative alignments of DNA and RNA polymerases and concomitant phylogenetic distance estimations made it obvious that such present-day manifestations as vertebrate adenoviruses, bacterial phages, fungal and plant mitochondrial elements and cytoplasmic yeast linear plasmids probably derive from a common ancestor (Meinhardt and Rohe 1993). However, the cytoplasmic linear yeast elements, includ- Genetics 64 Ii L r-I l L- shope fibroma vIrus V3c(;inia virus mollu scum contaglOsum virus pGKL2 pSKL pPEIB african swine fever virus l r Cuenurhubdl/is eiegul1S Homo sapiens ~ Candida albicans ~ I Saccharomyces cerev ISlQe S(.'hi~osacch(lrom}'ce spOil/be Chlorella vIrus I I Cl'llllIdra ja;nculala Trypanosoma bruce I Fig. 4. Phylogenetic tree of guanylyl transferases. Source of sequences: pPEIB (Meinhardt, unpublished); other sequences were from the Swiss Prot and European Molecular-Biology Laboratory databases. Calculation was done by the TREE program provided by the Husar Genius Net Service of DKFZ (Heidelberg, Germany) ing pGKLl and pGKL2, represent a relatively isolated group much more closely related to the fungal and plant mitochondrial linear plasmids than to adenoviruses (Kempken et al. 1992; Rohe et al. 1992). The recent identification of a viral-like capping enzyme (Larsen et al. 1998; Fig. 3) not only denotes the plasmid-specific cytoplasmic capping of transcripts but adds further strong evidence for viral ancestry. Both the capping enzyme encoded by ORF 3 and the putative helicase predicted for ORF 4 of pGKL2 are homologous to corresponding polypeptides encoded by cytoplasmic viruses, such as the African Swine Fever virus (ASFV). From phylogenetic distance estimations based on guanylyltransferases, ASFV appears to be the nearest relative of yeast killer plasmids known (Fig. 4). Irrespective of the taxon in which cytoplasmic DNA elements occur, their compartmentalization necessitates nucleus-independent replication, transcription and, as a prerequisite for translation, the capping of mRNAs. All these rather complex processes are encoded by both cytoplasmic viruses (Poxviridae, lridoviridae) and yeast cytoplasmic plasmids. It is hardly likely that these pathways have evolved in parallel, but it strongly suggests a common ancestor. Extranuclear Inheritance 65 7 Conclusions and Outlook During the past decade, analysis of this novel class of genetic elements has yielded considerable insight into the biology of these so-called linear plasmids. The development of gene shuffles and knockout strategies allows one to ask questions regarding the functions of various plasmid genes. The questions to be addressed in the future include the target site of the y-toxin subunit, which eventually results in the Gl arrest. Another important mechanism to be elucidated is the uptake of the y toxin by the sensitive cell, a process evidently mediated by a and ~ subunits of the heterotrimeric toxin. Genes encoding DNA and RNA polymerases, helicases and a capping enzyme suggest that the killer plasmids are of viral ancestry. The nearest known relatives are the cytoplasmically localized, harmful pox viruses, such ASFV, Vaccinia virus and Molluscum contagiosum virus. Thus, it is quite evident that so-called cytoplasmic yeast linear plasmids are virallike elements rather than plasmids. Accordingly, in addition to the elucidation of the killer system, Le., its molecular mechanism and cellular target(s), these genetic traits offer fascinating approaches for studying viral functions in a virus/host system that is safe and easy to handle. Acknowledgements. We thank M. Larsen and S. Jeske for their help with the artwork. This work has been supported by an Alexander von Humboldt "Feodor Lynen Fellowship" (FLF-DEU/I037031) to Raffael Schaffrath and Deutsche Forschungsgemeinschaft grants to Raffael Schaffrath (Scha 750/2-1) and Friedheim Meinhardt (Me 1142/3-1). 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Esser Dr. Raffael Schaffrath Martin-Luther-Universitat Institut fur Genetik Domplatz 1 06108 Halle/SaaIe, Germany Genetics Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria Karlheinz Esser, Thomas Lisowsky, Georg Michaelis, and Elke Pratje 1 Introduction This review continues our previous article in this series (Lisowsky et al. 1999) and focuses on the evolution of mitochondrial genomes, nuclearmitochondrial interactions and protein transport in mitochondria. Additional aspects of mitochondria are covered by recent reviews of mitochondrial DNA (mtDNA) repair (Croteau et al. 1999; Sawyer and Van Houten 1999), the machinery of mitochondrial inheritance (Yaffe 1999), mitochondria and life span control in fungi (Osiewacz and Kimpel 1999), mitochondrial assembly in yeast (Grivell et al. 1999), organellar RNA polymerases (Hess and Borner 1999), mitochondrial transcription signals (Brennicke et al. 1999b), RNA editing (Brennicke et al. 1999a; Mulligan et al. 1999), mitochondrial ribosomal proteins (Graack and Wittmann-Liebold 1998) and the alternative functions of mitochondria (Zorov et al. 1997). 2 Mitochondrial Genomes a) Saccharomyces cerevisiae The yeast Saccharomyces cerevisiae was the first eukaryotic organism to have its complete nuclear genome sequence determined. However, the available mtDNA sequence was incomplete, contained many errors and was derived from different polymorphic strains. Recently, Foury et al. (1998) reported the complete mtDNA sequence of the S. cerevisiae strain used for nuclear genome sequencing. The data assemble into a circular map of 85,779 bp and include 10 kb of new sequence. Seven small, hypothetical open reading frames are listed. Hot spots for point mutations are found in exons near the insertion of optional mobile group-I intronrelated sequences. These data suggest that the shuffling of mobile elements plays an important role in remodeling yeast mitochondrial genomes. Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 72 Genetics b) Petite-Positive and Petite-Negative Yeasts S. cerevisiae can form "petite colonie" mutants with deletions (p-) or complete loss (pO) of mtDNA, and early studies on these mutants contributed to the field of cytoplasmic inheritance (Chen and Clark-Walker 2000). Clark-Walker and co-workers addressed the question of how the petite-positive S. cerevisiae tolerates the elimination of mtDNA and how the loss of mtDNA leads to lethality in petite-negative yeasts, such as Kluyveromyces lactis and Schizosaccharomyces pombe. Recent investigations have revealed that pO lethality can be suppressed by specific mutations in the a, f3 and y subunits of the mitochondrial Fl adenosine triphosphatase (ATPase) in K. lactis. In contrast, the inactivation of genes coding for Fl ATPase a and f3 subunits and the disruption of several other genes in S. cerevisiae convert this petite-positive yeast into a petite-negative form. Thus, the authors showed that a function associated with the Fl ATPase and distinct from its role in energy transduction is required in the petite-positive phenotype of S. cerevisiae (Chen and Clark-Walker 1999,2000). c) Linear Mitochondrial Genomes At variance with the common belief that mitochondrial genomes are represented by circular DNA molecules, a large number of organisms have been found to carry linear mtDNA. They belong to a variety of taxonomic groups: ciliates, protozoa, algae, slime molds, oomycetous fungi and yeasts (Nosek et al. 1998). These linear genomes are characterized by linear restriction maps and telomere-like terminal structures. Their study might contribute to the maintenance and functioning of mitochondrial telomeres and to the evolutionary history of organelle genomes. 3 Evolution of the Mitochondrial Genomes a) Origin of Mitochondria Several articles and reviews deal with the origin of mitochondria and the eukaryotic cell. The endosymbiosis theory, which assumes that mitochondria originate from a bacterial endosymbiont, is generally accepted. Data from sequence analyses strongly indicate a single mitochondrial origin from an eubacterial ancestor related to a-proteobacteria (Gray et al. 1999; Lang et al. 1999a). Phylogenetic analysis suggests that the three a-proteobacteria Bradyrhizobium, Rhodospirillum and Rickettsia are most closely related to mitochondria (Lang et al. 1999b). The hypothesis Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 73 that a nucleus-containing eukaryotic host has taken up an eubacterial endosymbiont was first proposed by Margulis (1970). Recently, two new symbiosis hypotheses have been discussed: the hydrogen hypothesis and the syntrophy hypothesis. Both hypotheses associate the acquisition of mitochondria with the origin of the eukaryotic cell. The hydrogen hypothesis (Martin and Muller 1998; Muller and Martin 1999) proposes a symbiosis between a methanogenic archaeon (as the host) and a hydrogen-producing a-proteobacterium (as the symbiont). Due to this symbiosis, the strictly anaerobic host became independent of exogenous hydrogen and carbon dioxide sources but became dependent on the symbiont. The migration of genes for carbohydrate metabolism from the symbiont to the host made the endosymbiosis irreversible. This hypothesis is in agreement with the fact that eukaryotes contain eubacterial-like metabolic enzymes, even in amitochondrial eukaryotic organisms; hydrogenosomal proteins are similar to the respective mitochondrial proteins. The syntrophy hypothesis speculates that 8-proteobacteria (rather than a-proteobacteria) were taken up by the methanogenic archaea (Moreira and L6pes-Garcia 1998; L6pes-Garcia and Moreira 1999). The 8-proteobacteria are ancestral sulfate-reducing myxobacteria producing hydrogen and carbon dioxide. The syntrophy hypothesis is based on the finding that many myxobacterial genes have specific homologs in eukaryotic signaling pathways. As a second endosymbiont, a-proteobacteria took part in the symbiosis, increasing the rate of methanogenesis. Mitochondria are derived from this second endosymbiont. However, Andersson and Kurland (1999) argued that the available genome sequences do not support the hydrogen and syntrophy hypotheses for the origin of mitochondria. Finally, Karlin et al. (1999) suggested a fusion of a Clostridium-like eubacterium and a Sulfolobulus-like archaebacterium as the ancestor of animal mitochondria and primitive amitochondrial eukaryotes. This analysis is based on the dinucleotide abundance, which is similar in both prokaryotes. b} Transfer of Mitochondrial Genes to the Nucleus The majority of genes from the original endosymbiont have been lost or were transferred to the nucleus. The mitochondrial genomes sequenced to date vary in size and contain between five genes (Plasmodium falciparum; Feagin et al. 1992) and 94 genes (Reclinomonas americana; Lang et al. 1997; Lisowsky et al. 1999). The gene for eubacterial-like RNA polymerase (RPO) is one example of the loss of a mitochondrial gene in most eukaryotes. Several mitochondrial RPO genes have only been identified in jakobid protists, such as R. americana (Lang et al. 1999a,b). 74 Genetics Many examples demonstrate the migration of mitochondrial genes to the nucleus. This includes genes encoded by mtDNA in some species and by nuclear DNA in others. Most intracellular gene migration took place early during eukaryotic evolution. Recent gene transfers were reported for the COX2 gene in Fabaceae. Subunit 2 of cytochrome oxidase is mitochondrially encoded in all taxa analyzed to date, with the exception of legumes (Nugent and Palmer 1991; Covello and Gray 1992; Adams et al. 1999). Adams et al. (1999) examined the presence and expression of mitochondrial and nuclear COX2 genes in 392 legume genera. Nine legumes contained both a nuclear and a mitochondrial copy, and seven of them transcribed both genes. The inactivation of COX2 occurred several times due to loss of transcription, RNA editing, or partial or complete gene loss. The mitochondrial and nuclear copies are inactivated with similar frequencies. c) Gene Transfer to Mitochondria The discussion of the earliest land plants has focused on liverworts, hornworts and bryophytes. Recently, liverworts have been identified as the earliest land plants by the analysis of group-II introns in mitochondrial genes (Qui et al. 1998). Palmer and co-workers examined the gain of three introns in the genes for COX2 and NADl of 352 land plants. These introns are present in most mosses, hornworts and vascular plants but are absent from liverworts, green algae and red algae, indicating that liverworts are the earliest land plants. The integration of a group-I intron in the mitochondrial COXl gene is an example for a recent horizontal transfer of genetic material in plant species. This mobile self-splicing sequence is widespread among angiosperm COXl genes (Cho et al. 1998; Cho and Palmer 1999). Southernblot analysis of 335 genera of land plants and extrapolation to all angiosperm genera indicate that this intron has invaded the COXl gene over 1000 times during angiosperm evolution. A more detailed analysis has been performed within the Araceae family. Six of 14 genera tested contain the COXl intron, and the authors argued that the invasion occurred via at least three (or even five) separate horizontal transfer events. 4 Cross-Talk between Mitochondria, Cytosol and the Nucleus One of the most important regulatory aspects connected with mitochondria is the integration of the functions of these organelles into the complex cellular network (Butow et al. 1988; Grivell et al. 1999). Thus, the elucidation of the mechanisms used for communication of mitochondria Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 75 with cytosolic and/or nuclear functions is of interest (Poyton and McEwen 1996). This especially concerns the export of regulatory signals from mitochondria to the cytosol or nucleus (Chelstowska et al. 1999). Today, it is obvious that this task is facilitated by small molecules generated or processed by different mitochondrial pathways (Jia et al. 1997; Lill et al. 1999). With the rapid accumulation of detailed data about the size, structure and gene content of complete mitochondrial genomes from different species (Unseld et al. 1997) new insights into this complex regulatory network between mitochondria and other cellular functions are possible (Grivell et al. 1999). One important general conclusion from the mitochondrial genome projects, which use cells from species ranging from plants to humans, is that the mitochondrial genomes only contribute to the process of oxidative phosphorylation. The relatively small number of mitochondrially encoded RNAs or proteins are all directly or indirectly involved in the assembly and function of the oxidative chain ,, I Nucleus signals for regulation of nuclear genes and cytosolic functions I 11 I AlP ROS I Ca 2+ Fel S ...... _---- - oxidative phosphorylation - TCA cycle - steps of amino acid biosynthesis - steps of nulceotide biosynthesis - biogenesis of iron sulfur proteins Fig.!. Cross-talk between mitochondrion, cytosol and nucleus. Important biosynthetic pathways inside mitochondria deliver small molecules that mediate signals for the cytosol or the nucleus to regulate specific cellular functions. In addition to respiration that is linked to the function of the mitochondrial DNA, there are other essential pathways, such as the tri-carboxylic acid (rCA) cycle, some steps of the biosynthesis of amino acids and nucleotides, and the assembly of iron-sulfur proteins, which also take place in the mitochondrial matrix. All these pathways are directly or indirectly involved in the generation of small molecules that are exported from mitochondria and serve as signals for receptor molecules in the ~osol or nucleus. ATP generation of adenosine triphosphate by the oxidative chain; Ca + regulated export of calcium from mitochondria; PelS biogenesis and assembly of iron-sulfur proteins for mitochondrial and cytosolic function; ROS generation of reactive oxygen species 76 Genetics (Boumans et al. 1998). Nevertheless, mitochondria contain a number of other biosynthetic pathways that are vital for all aspects of cellular functions. The most important pathways are the TCA cycle, different steps in the biosynthesis of amino acids or nucleotides (Zelenaya-Troitskaya et al. 1995), and the biosynthesis and assembly of iron-sulfur proteins (Li et al. 1999; Lill et al. 1999). The pathways that recently got the most attention (and some of the small molecules that are generated or processed and used to communicate with other cellular compartments) are summarized in Fig. 1. The latest research data for these pathways, the small molecules generated by them, and their impact on the regulation of other cellular functions are addressed in the following sections. a) Retrograde Regulation The oxidative chain generates a number of small molecules that are used as sensors to determine the functional state of mitochondria. Most obvious are ATP, protons and the production of reactive oxygen species (ROS; Nakamura et al. 1997; Charizanis et al. 1999; Kowaltowski and Veresci 1999). A number of cellular receptors and nuclear promoter elements that interact with these molecules have already been identified. Therefore, the feedback control between the respiratory status of mitochondria and other cellular functions was named retrograde regulation (Butow et al. 1988; Chelstowska et al. 1999). Meanwhile, different nuclear genes that are involved in this specific regulatory pathway have been identified, and they have consequently been named RTG (retrograde regulation) genes (Butow et al. 1988; Jia et al. 1997; Rothermel et al. 1997; Chelstowska et al. 1999). Most of them encode proteins that are nuclear transcription factors (Table 1). For example the RTGI and RTG3 genes encode DNA-binding proteins that interact with binding sites in upstream-activating promoter elements of enzymes essential for mitochondrial and peroxisomal function (Liao and Butow 1993; Chelstowska and Butow 1995). Changes in the respiratory state of mitochondria result in changes of the expression level of those genes, and this response is called the retrograde response (Margossian and Butow 1996). Another gene of this class, RTG2, encodes a protein containing an hsp70-like ATP-binding domain and is necessary for the expression of genes regulated by RTGI and RTG3 (Velot et al. 1996). Thus, this regulatory mechanism is employed by the cell in sensing the status of oxidative phosphorylation and the intactness of mitochondrial genomes. Therefore, many of these genes also affect mtDNA stability and other nuclear genes that are essential for the maintenance and intactness of the organellar genomes. As mitochondrial genomes become dysfunctional, an increase especially in ROS transduces signals to the Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 77 Table 1. Important nuclear genes for retrograde regulation between the mitochondria, cytosol and nucleus or with dual functions for mitochondria and the cell Gene symbol Function of the protein RTGI Nuclear transcription factor, regulates GIT2 Nucleus (citrate synthase) RTG2 Nuclear transcription factor, regulates GIT2 Nucleus (citrate synthase) RTG3 Nuclear transcription factor, regulates GIT2 Nucleus (citrate synthase) ILV5 Branched-chain amino acid biosynthesis, maintenance of intact mitochondrial genomes Mitochondrial matrix ERVI Morphology of mitochondria, intact mt genomes, vegetative growth, survival of the cell Mitochondria, cytosol NPSI Generation of elementary sulfur for the biogenesis of PelS proteins for mitochondria and the cytosol Mitochondrial matrix ATMI Export of PelS proteins from mitochondria to the cytosol or nucleus Outer mitochondrial membrane TRX Thioredoxin proteins, diverse redox functions Cytosol, nucleus, mitochondria Localization nucleus and induces the expression of genes involved in retrograde regulation (Charizanis et al. 1999; Kowaltowski and Vercesi 1999). The most recent data demonstrate that this process is essential for the control of life span and longevity (Kirchmann et al. 1999; Osiewacz and Kimpel 1999); therefore, it is also responsible for the replicative capacity, which is the number of times an individual cell divides. Therefore, the deletion of a gene required for the retrograde response (RTG2) also eliminates the increased replicative capacity of the cell (Velot 1996; Kirchmann et al. 1999). This indicates that the molecular mechanisms of aging are linked to the retrograde response and emphasizes their general applicability. Another essential factor for retrograde signaling was identified as calcium ions, which can be released from mitochondria through regulated channels, such as the porin complex of the mitochondrial membrane (Hansford 1994; Biswas et al. 1999). This pathway is activated in response to mitochondrial genetic and metabolic stresses; thus, it defines a novel mode of inter-organelle cross-talk (Babcock et al. 1997) that integrates mitochondria into the intracellular calcium-signaling network (Babcock et al. 1997; Ichas et al. 1997). Genetics 78 b) Proteins with Dual Function and/or Dual Localization In addition to retrograde regulation, there are other mechanisms that are not directly linked to genetic feedback regulation but that connect nuclear, cytosolic and mitochondrial functions because the same proteins are shared by different cellular compartments. The most important class of genes involved in such mechanisms encodes mitochondrial proteins that have dual functions and/or dual localizations and that are not related to the protein-import machinery of mitochondria (Baker and Schatz 1991; Lisowsky 1992; Lill et al. 1999). Selected representatives of these genes are included in Table 1. Important examples, functions of the proteins and their modes of action will be discussed in the following section. The most recent example represents genes essential for the assembly of iron-sulfur proteins in mitochondria (Fig. 2). Iron-sulfur proteins are essential for nuclear, cytosolic and mitochondrial functions of the cell. In yeast, all iron-sulfur proteins are exclusively assembled inside mitochondria and are distributed from there to specific compartments of the cell (Kispal et al. 1999; Li et al. 1999). Two key factors for this process were recently identified: Nfs1p (a mitochondrial-matrix protein that catalyzes the release of sulfur from cysteine molecules) and cytosolic or nuclear function of Fe/S proteins I export of Fe/S proteins iron export (?) - - - - - - - - \ ' - iron import Ala + 5 Nfs1p Mitochondrion Fig. 2. Iron-sulfur proteins for mitochondrial, cytosolic and nuclear functions are all assembled inside mitochondria. The most recent research (Kispal et al. 1999) identified the Nfslp protein as the catalyst for the generation of elementary sulfur from cysteine (Cys). In that reaction, alanine (Ala) and sulfur (S) are produced. In a number of additional synthesis steps, the sulfur is used together with imported iron molecules for the biogenesis of iron-sulfur centers of proteins. The assembled proteins with a function in the cytosol or the nucleus are then exported from mitochondria. The export of iron from mitochondria has been discussed but not yet proven (Kispal et al. 1999). The essential functions of iron-sulfur proteins in different compartments of the cell, and the functions of different cellular receptors and sensors that measure the intracellular amounts of iron, offer many new possibilities for the feedback regulation of mitochondrial, cytosolic and nuclear functions. (Beinert et al. 1997; Lange et al. 1999; Li et al. 1999) Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 79 Atmlp (a protein of the outer membrane that facilitates the export of the assembled iron-sulfur proteins; Kispal et al. 1999). The biogenesis of iron-sulfur proteins offers many physiological feedback control mechanisms, because the cell contains a number of sensors and receptors for the measurement of iron content, iron import and the distribution of iron molecules to different compartments (Lange et al. 1999). The functions of iron-sulfur proteins in the oxidative chain, in the regulation of translation in the cytosol, and in the nucleus demonstrate the regulatory interdependence of the different cellular compartments (Beinert et al. 1997). Many of the proteins with dual localization and/or function are involved in the genome stability of mtDNA. For example, the nucleusencoded IL vs protein is localized in the mitochondrial matrix, has a specific enzymatic function in the synthesis of branched-chain amino acids and is essential for the stability of mtDNA (Zelenaya-Troitskaya et al. 1995). In contrast, the yeast ERVI protein, which also has a dual function in mitochondrial genome stability, mitochondrial morphology and vegetative growth (Lisowsky 1992; Hofhaus et al. 1999), is found in mitochondria and the cytosol (Becher et al. 1999). The facilitation of different functions by different localizations of proteins also occurs for the diverse group of thioredoxin proteins. These small redox proteins are found in the cell at different locations, from the nucleus to mitochondria. They act by modifying special sulfur groups in different target proteins, thus regulating the activity of enzymes and nuclear transcription factors in response to the redox status of the cell (Nakamura et al. 1997). c) New Aspects One question for discussion is whether large molecules, special proteins or protein fragments that are exported from mitochondria act as specific signals. No evidence in favor of this hypothesis has been found, with the exception of the special case of cytochrome-c release from mitochondria for the initiation of apoptosis in animal cells (Goel and Khanduja 1998; Bossy-Wetzel and Green 1999). Because this mechanism does not exist in plants and fungi, and because apoptosis is a very complex phenomenon that has been addressed in many other reviews, we will not concentrate on this subject. As a consequence of the detailed data regarding the regulatory network between mitochondria, the cytosol and the nucleus, there is no need to postulate a secondary export of other mitochondrial proteins or protein fragments to explain the observed regulatory effects. The identified small molecules are sufficient to explain all the effects observed in vivo. The nuclear genes and their promoters, which are directly or indirectly regulated by the small molecules, allow (1) efficient 80 Genetics evaluation of the functional status of mitochondria and (2) optimized feedback control of the different cellular functions and growth conditions. Most of the genes and mechanisms essential for nucleusmitochondria cross-talk were first identified in yeast or other lower fungi. However, the rapid identification of homologous genes from plants and other higher organisms indicates their general importance for all cells and organisms. There is even evidence that cross-talk between plastids and mitochondria also occurs in plants (Hedtke et al. 1999). Another important topic is the rapidly evolving field of iron-sulfur protein biogenesis in mitochondria. Regarding that topic, it will be especially interesting to find out how plant cells generate iron-sulfur proteins for plastids and whether there is exchange between mitochondria and chloroplasts for that purpose. Thus, the major tasks for the future are: - The investigation of iron-sulfur protein biogenesis in plastids - Cross-talk between mitochondria and plastids in plants - The identification of additional small molecules essential for crosstalk - The isolation of new nuclear genes for retrograde regulation - The determination of mitochondrial proteins with dual functions and dual localizations - The identification of homologous genes from other species 5 Mitochondrial Protein Transport in Higher Plants Mitochondrial protein import has been intensively characterized in the model organism yeast during the last 10 years (Voos et al. 1999). Less is known about the components and pathways of plant mitochondrial protein import. Recent studies have revealed similarities and certain differences between mitochondrial protein import in yeast and in plants (Braun and Schmitz 1999; Whelan 1999); these studies allow one to make a provisional but rather comprehensive description of plant mitochondrial protein import. That description is summarized in this chapter. The differences between mitochondrial protein import in plants and in yeast are probably caused by the more complex situation in plant cells, which contain plastids as additional organelles and exhibit more sophisticated development. In addition, aspects of mitochondrial protein export (which is being increasingly studied) are summarized in this section. Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 81 a) Pre-Sequences Import studies indicate that, in plants, at least five different mitochondrial protein-import pathways exist (Whelan 1999), but some components of the distinct pathways are used in common by certain proteins. Mitochondrially imported proteins following the general import pathway are usually synthesized as precursors with N-terminal presequences that are recognized by certain receptors during import and are finally removed by a signal peptidase. Detailed analyses of the known plant signal sequences (in comparison with those of yeast mitochondrial proteins) revealed that plant mitochondrial targeting sequences are slightly extended and have an increased serine content (Glaser et al. 1998; Sjoling and Glaser 1998; Tanudji et al. 1999). The presequences of the known mitochondrial proteins in plants have an average length of 40 amino acid residues (varying from 13 to 85 residues) and are rich in basic, hydroxylated residues. A typical plant mitochondrial pre-sequence is characterized by a domain structure with an Nterminal amphiphilic a-helix and a C-terminal processing domain comprised of a helix flanked by basic residues. Arginine residues near the processing sites are important for pre-sequence recognition. Similar characteristics are found in most mitochondrial and plastid targeting sequences of different species; therefore, certain mechanisms must exist to avoid mis-targeting mitochondrial proteins into plastids. Localizations in different cell compartments were reported for several proteins (Small et al. 1998); this may reflect a function of these proteins in both compartments. Nevertheless, the targeting of proteins imported into organelles in vivo is highly specific (Glaser et al. 1998) despite the similarity of mitochondrial and plastid targeting sequences, and cases of mis-targeting seem to be exclusively reported for heterologous systems, in vitro import assays or chimeric constructs used in import studies. It seems probable that cytosolic chaperones (such as Hsp70) and import receptors at the outer face of the mitochondria prevent mis-targeting by recognizing and binding precursors destined for mitochondrial import. In addition, pre-sequences of several proteins destined for import into chloroplasts have been reported to undergo specific phosphorylation prior to targeting, followed by de-phosphorylation prior to proteolytic processing (Waegemann and Soll 1996; Soll and Tien 1998). This phosphorylation/de-phosphorylation cycle may represent a regulatory mechanism and may enhance the specificity of targeting. In the case of the pre-sequence of the mitochondrially imported Fl ~ ATPase subunit of Nicotiana, an unspecified covalent modification has been reported (Von Stendingk et al. 1999), indicating that post-translational modifications of pre-sequences for organellar import may be more frequent in plants. 82 Genetics Table 2. Components of the plant mitochondrial protein import Location Class Component Molecular weight a (kDa) Outer membrane Import receptors Tom70 b Tom20 70 23 c Components of translocation channel Tom40 Tom9b Tom8 b Tom7 Tom6 b 36c 9 8 7c 6 Components of translocation channel Tim23 20 C Tim17 2S C uMpp I3Mp~ Impl Imp2 b SId S3-SSd Chaperones mtHsp70 mtHsp60 mtHsplO Mge Mdj 70 d 60 d 10 26 d 40 Peptidases Mppb Inner membrane Peptidases Matrix -e aMolecular mass determined from the sequence or from the apparent molecular weight. bPlant components that cannot be definitely assigned to certain yeast proteins. cComponents of the translocase complex that are reported to be homologous to fungal counterparts. dTwo isoforms are reported. ePlant expressed-sequence tags possibly encoding homologous proteins of the fungal counterparts. b) The Translocases of the Mitochondrial Outer and Inner Membranes Mitochondrial protein import occurs at translocase complexes that form import channels in the outer and inner mitochondrial membranes (TOM, TIM; Table 2; Fig. 3) and cooperate with: 1. Cytosolic factors and receptors at the surface of mitochondria that recognize proteins destined for import 2. Chaperones that support import by influencing pre-protein folding 3. Signal peptidases removing the pre-sequences of imported proteins As in yeast mitochondria, import across the inner membrane is dependent on matrix ATP and membrane potential (.'l'l'). Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria precursor proteins 83 import cytosol TOM complex OM IMS TIM complex 1M processing peptidase matrix Fig. 3. Protein-import apparatus of plant mitochondria. 1M Inner membrane; IMS intermembrane space; OM outer membrane; for abbreviations, see text and Table 2 Recently, the translocase of the outer mitochondrial membrane (TOM) complex of potato was purified and analyzed (Jansch et al. 1998). The total molecular weight of the complex was estimated to be only 230 kDa, while the fungal TOM complex is described to have a molecular weight of approximately 500 kDa. Seven subunits weighing 70, 36, 23, 9, 8, 7 and 6 kDa were identified, three of which (Tom36, Tom23 and Tom7) were found to be homologous to the yeast proteins Tom40, Tom20 and Tom7, respectively, as determined using partial amino acid sequences. The plant TOM complex was shown to contain an additional low-molecular-weight subunit. Counterparts of a yeast second-receptor sub-complex (which is composed of the Tom22 and Tom37 proteins and is proposed to function in response to a different subset of proteins) do not seem to exist in plants (Perryman et al. 1995; Heins and Schmitz 1996; Jansch et al. 1998; Braun and Schmitz 1999). The lack of the Tom22/Tom37 sub-complex is supported by the experimental data. Antibodies against Tom20 inhibit only the import of proteins using this pathway in yeast; however, in plants, mitochondrial import is completely inhibited (Heins and Schmitz 1996). 84 Genetics Only two subunits of the plant translocase of the inner mitochondrial membrane (TIM) complex have been identified; they are homologs of the yeast Tim23 and Tim17 proteins, but with molecular masses of 20 kDa and 25 kDa, respectively (Bomer et al. 1996). Other TIM components identified in fungi are Tim44, the additional small proteins Tim8, 9, 10, 12 and 13, and a second TIM sub-complex for the import of hydrophobic carrier proteins; this complex consists of Tim22 and Tim54. Plant counterparts have not yet been identified. c) Processing Peptidases The matrix processing peptidase (MPP) consists of two subunits, u- and j3-MPP, of which j3-MPP represents the proteolytically active subunit (Yang et al. 1988; Kitada et al. 1995). In plants, the peptidase was reported to consist of membrane-bound multi-functional proteins that are core subunits of the cytochrome b/c l complex and are involved in electron transport and the proteolytic processing of precursor proteins. Recent studies showed that, in plants, two pairs of isoforms of the two MPP subunits are present (Braun et al. 1995); these may be encoded by four separate genes. Furthermore, the composition of the cytochrome b/c l complex may be different for different tissues, developmental stages or growth conditions (Hinsch et al. 1995; Glaser and Dessi 1999). Recently, in soybean and spinach, a matrix-localized MPP that may represent an unbound form or an additional soluble enzyme was also identified (Szigyarto et al. 1998). In Neurospora, the j3-MPP, encoded by a single gene, was shown to occur in different locations: 70% is membrane bound in the cytochrome blcl complex, and 30% is soluble in the matrix (Schulte et al. 1989; Glaser and Dessi 1999). The co-evolution of cytochrome c reductase and the MPP has been investigated in different organisms, including the lower plants Platycerium, Equisetum and Polytomella (Brumme et al. 1998). It is assumed that the membrane-bound, bi-functional MPP of plants represents the ancient evolutionary situation, and the soluble MPP of yeast is the more developed derivative (Braun and Schmitz 1997). MPP is an important signal peptidase in plants, because it is responsible for the proteolytic cleavage of several hundred imported proteins (Glaser et al. 1996; Glaser and Dessi 1999). Recently, the cleavage specificity of MPP has been analyzed in different organisms (Schneider et al. 1998), revealing several types of cleavage sites; however, the sites are only loosely defined and exhibit no detectable species specificity. Homologs of the mitochondrial intermediate peptidase, a matrixlocalized signal peptidase (Kalousek et al. 1992) that has been identified in yeast, are not found in plants, and there is no indication that this type of peptidase is present in plant mitochondria. The occurrence of plant Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 85 homologs of the inner-membrane peptidase (Imp), another yeast signal peptidase (Pratje et al. 1994), has not yet been experimentally identified but seems probable for several reasons (see below). d) Chaperones Although the cytosolic chaperones Hsp70 and pCsDnaJ have been identified in plants (Rochester et al. 1986; Preisig-Miiller and Kind11993; Mooney and Harmey 1996; Guy and Li 1998), it is not yet clear whether they are involved in mitochondrial protein targeting, as described for their yeast homologs, Hsp70 and Ydj I1Mas5. From yeast mitochondria, it is known that the mitochondrial chaperones Hsp60 and Hspl0 are involved in the folding of mitochondrial proteins and that the chaperones mtHsp70, Mdj and Mge cooperate in folding and importing proteins. The mitochondrial chaperone mtHsp70 has been identified in several plant species (Watts et al. 1992; Neumann et al. 1993; Vidal et al. 1993; Guy and Li 1998), and it participates in mitochondrial protein import in a manner similar to that of its fungal counterpart (by binding mitochondrial pre-sequences and pulling them into the matrix space; Zhang et al. 1999). There is evidence that two highly homologous isoforms of mtHsp70 exist in plants, as seen in spinach and Nicotiana. Similarly, the plant mitochondrial Hsp60, which has been identified in different species, was shown to be present in pumpkin as two isoforms with 95% identity (Prasad and Steward 1992; Tsugeki et al. 1992). Plant homologs of other mitochondrial chaperones were also identified recently: the proteins Mdj, two isoforms of Mge (Kroczynska et al. 1996; Padidam et al. 1999) and Hspl0 of barley, potato and Arabidopsis (Hartman et al. 1992; Burt and Leaver 1994; Koumoto et al. 1996). The genes MDJ and HSPlO of Arabidopsis were shown to complement Escherichia coli deletion mutants of DnaJ and GroES, respectively. e) Developmentally Regulated Protein Import There is evidence from different studies that plant mitochondrial protein import is regulated during development and is dependent on growth conditions (Dessi et al. 1996, 1998; Dessi and Whelan 1997; Leon et al. 1998; Whelan 1999). The generation of specific targeting signals by alternate transcription start sites, differential splicing or cyclic adenosine monophosphate-dependent phosphorylation are reported in a number of cases (Jaussi 1995; Neupert 1997; Isenmann et al. 1998; Anandatheerthavarada et al. 1999). It is known that import efficiency (but not matrix-localized processing activity) can decrease, depending on the age of the plants (Dudley et al. 1997; Huang et al. 1998; Wood et al. 1998). 86 Genetics McCabe et al. (1998) and Murcha et al. (1999) reported that the importing of alternative oxidase (AOX) and FAd precursor proteins is regulated age dependently in different tissues and that the amount of AOX is always closely matched with the amount of Tom20 protein in soybeans. Thompson et al. (1998) describe differences in the expression of a large number of mitochondrial proteins, comparing the basal meristem with green leaves of barley. Pavlov and Glaser (1998) demonstrated the inhibition of protein import by amphiphilic cations, possibly affecting connections between proteins of the inner-mitochondrial-membrane channel. From the study of von Stedingk et al. (1997), it was suggested that the reducing status of the cell influences the activities of import complexes, probably due to the redox and conformational statuses of the SH groups ofTim17 or other proteins at the outer face of the inner mitochondrial membrane. In addition, the development of plant cells has been shown to be affected by the expression of mitochondrial chaperones in several cases: mtHsp70 overexpression is reported to enhance the growth of transgenic Nicotiana plants (Wood et al. 1998) and, in the seed germination of Arabidopsis and maize, a developmentally regulated expression ofHsp60 was reported (Prasad and Steward 1992). In plants, many components, especially chaperones and MPP proteins, exist as different isoforms that seem to be expressed tissue specifically or depend on the developmental stage. Mitochondrial protein import probably plays an important regulatory role in plants and may consist of different mechanisms, such as the modification of pre-sequences, differential expression of the isoforms of proteins, changing of the compositions of protein complexes, and other mechanisms. f) Differences Between Plants and Yeast The plant mitochondrial protein import, described in the preceding sections, can be briefly characterized as follows: - In comparison with fungi and mammals, plants possess slightly extended mitochondrial targeting sequences with a significantly higher serine content. The TOM complex is smaller than the yeast Tom22/Tom37 receptor sub-complex, may be formed of fewer subunits, and lacks homologs. However, one additional small subunit was identified. The second TIM sub-complex of yeast, specific for mitochondrial carrier proteins, does not seem to exist in plants. The import receptors of plant mitochondria may have broader substrate specificity. Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 87 - The two MPP subunits of plants are membrane-bound core subunits of the cytochrome b/,! complex. The presence of an additional soluble-processing-peptidase activity has been described. - Isoforms of several components (chaperones, MPP) have been found, and they seem to be differentially expressed, depending on the developmental stage or tissue. g) Protein Export Mitochondrial protein export includes: 1. The possible delivery of proteins and peptides from mitochondria into other compartments ofthe cell. 2. The release of cytochrome c from mitochondria during apoptosis in mammalian cells, a mechanism that does not seem to exist in plants and yeasts and is therefore not discussed in this chapter. 3. The export of mitochondrial proteins into the inner membrane or the inter-membrane space, a process that has been described for several mitochondrially encoded and imported proteins. The first case documented by some recent studies refers to proteins that were thought to be located exclusively in mitochondria and can now also be found in other compartments (Soltys and Gupta 1999a,b). The cytosolic forms of the enzyme fumarase in yeast, human aspartate aminotransferase and the chaperones mtHsp60 and mtHsp70 were shown to be processed by the mitochondrial MPP. Therefore, at least their Nterminal pre-sequences must have entered the mitochondrion prior to their re-export into the cytosol (Stein et al. 1994; Singh et al. 1997; Khan et al. 1998; Zhou et al. 1998). Most of the fumarase protein is thought to reverse its import direction after cleavage of the targeting sequence in the mitochondrial matrix (Ungermann et al. 1994). An export of proteins and peptides derived from mitochondrially encoded proteins has been reported in plant and animal cells (Abad et al. 1995; Dabhi and Fischer Linda11995; Bhuyan et al. 1997). The delivery of mitochondrial proteins to other cellular compartments often implies an additional function (Poyton et al. 1992), as described for the mitochondrially transmitted factors that act as histocompatibility antigens (Fischer-Lindahl et al. 1991; Bhuyan et al. 1997). It is assumed that specific mitochondrial protein export mechanisms exist in plants and yeasts, but the components and functions are poorly understood. Gram-negative bacteria possess several distinct mechanisms for protein export. In mitochondria, only a small number of proteins that are suggested to be involved in export processes have been identified. No counterparts of the sec-dependent major bacterial export 88 Genetics cytosol a OM export of gene product N IMS 1M matrix ----1 COX 2 ARG 8 '" f-- chimeric mitochondrial gene cytosol b OM no export of gene product IMS 1M N ----1 matrix COX 2 ARG 8 '" f-- chimeric mitochondrial gene Fig. 4 a,b. Selection system for mutants defective in mitochondrial protein export (He and Fox 1999). In wild-type strains of yeast, the gene product of the nuclear ARGS gene is imported into the mitochondrial matrix, where it functions in the arginine biosynthesis of the cell. a Mitochondria of a yeast strain with a deletion of the nuclear ARGS gene were transformed with a chimeric gene comprised of the mitochondrial COX2 gene fused to ARGS m , which was changed to the mitochondrial code at several codons. For the transformation of the organelle, a particle gun was used. The resulting yeast strain was respiratory competent (PEn but could not grow on media lacking arginine (Arg-), because the gene product was removed from the matrix by export across the inner membrane. b Mitochondria of the same yeast strain after the occurrence of a secondary mutation affecting mitochondrial protein export. This strain was respiratory defective (pet) due to its impaired export across the inner membrane, but it could grow on media lacking arginine (Arg+), because the ARG8 ffi gene product is localized in the matrix Extranuclear Inheritance: Genetics and Biogenesis of Mitochondria 89 system were found in mitochondria, but the yeast mitochondrial Imp, which consists of the two subunits Impl and Imp2, is homologous to the E. coli signal peptidase (Lep), an essential component of the bacterial protein-export pathway (Behrens et al. 1991; Nunnari et al. 1993; Pratje et al. 1994). Plant Imp homologs have not been reported but are suggested by the sequence similarity of expressed-sequence tag (EST) sequences from Arabidopsis, tomato, Chlamydomonas and Zea mays and by the occurrence of mitochondrial imported proteins with bipartite pre-sequences (such as cytochrome 'I, which may be cleaved by an Imphomologous signal peptidase; Braun et al. 1992). Recently, Bogsch et al. (1998) described the novel bacterial TAT export system (twin arginine translocation pathway), which is capable of the pH-dependent exporting of proteins in folded conformations. Possible homologs of the components TatC and TatD have been found to be encoded in the mitochondrial and plastid DNAs of plant cells and in the genomes of Reclinomonas, yeast, humans, nematodes and plants (Bogsch et al. 1998; Marzo et al. 1998; Weiner et al. 1998). However, to date, there are no experimental data demonstrating their function in mitochondrial protein export. The yeast Oxal protein, which is required for export of the N- and Ctermini of the mitochondrially encoded cytochrome oxidase subunit 2 (Cox2; He and Fox 1997; Hell et al. 1997), represents another characterized mitochondrial protein-export component. Oxal homologs have been identified in different plant species, both experimentally (Hamel et al. 1997; Sundberg et al. 1997; Moore et al. 2000) and as EST sequences. 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Esser Elke Pratje Institut fur Allgemeine Botanik Universitat Hamburg OhnhorststraBe 18 22609 Hamburg, Germany Genetics Genetics of Phytopathogenic Bacteria Jutta Ahlemeyer and Rudolf Eichenlaub 1 Introduction Only a small proportion of bacteria are plant pathogenic and have developed mechanisms to invade and colonize their host plants and cause disease. However, resistant host-plant cultivars and certain non-host plants are able to recognize and combat phytopathogenic bacteria. These resistant plants react with a localized induced cell death at the site of infection; this is termed hypersensitive response (HR) and is induced by so-called elicitors, such as avirulence proteins (Avr proteins). These are recognized by corresponding receptor proteins in the plant. It has been shown that the ability to cause disease in compatible interactions with host plants and the induction of HR in incompatible interactions both depend on the ability of the bacteria to express a cluster of genes termed hrp (hypersensitive reaction and pathogenicity; Lindgren et al. 1986). Thus, hrp mutants of plant pathogenic bacteria cause no detectable reactions in either host or non-host plants. Rrp genes seem to be a common feature of all Gram-negative plant pathogenic bacteria. Some of the hrp genes encode a protein-secretion mechanism known from animal pathogenic bacteria (the type III secretion system) which apparently enables them to direct proteins into plant cells. The type III secretion system differs markedly from the earlier discovered type I protein secretion [which involves adenosine triphosphate (ATP)-binding cassette transporters] and the signal-peptide/sec-dependent type II secretion system (Salmond and Reeves 1993; Lee 1997). In this review, we will report current knowledge of the different classes of hrp genes and their function and regulation. Because we have already dealt with this subject in a previous review (Bahro et al. 1997), we will confine ourselves here to completing the picture as far as possible. For those readers who become more deeply interested in the subject, a number of excellent reviews on hrp genes, the type III secretion system and other aspects of plant-microbe interactions, such as pathogeninduced resistance reactions, will be cited in the respective sections of this review. Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Genetics of Phytopathogenic Bacteria 99 2 The hrp Genes of Phytopathogenic Bacteria Hrp genes have been described for phytopathogenic, Gram-negative bacteria of the genera Erwinia, Pseudomonas, Ralstonia and Xanthomonas. They are organized in 25- to 40-kb clusters consisting of 20-27 genes located either on the chromosome or on a megaplasmid (Alfano and Collmer 1997; He 1998). When expressed in Escherichia coli or P. jluorescens, the complete hrp clusters of Erwinia amylovora resp. P. syringae pv. syringae allow these non-pathogenic bacteria to induce a HR in tobacco, indicating that not only are hrp clusters necessary for the induction of HR but, at least in some cases, they are sufficient (Huang et al. 1988; Beer et al. 1991). In all compatible interactions investigated to date, however, hrp genes were required for the induction of disease but did not suffice on their own. Depending on gene homologies and the order of gene loci, hrp clusters can be divided into two groups (Alfano and Collmer 1997). Within the genera Erwinia and Pseudomonas (group I) and the genera Ralstonia and Xanthomonas (group II), almost all hrp genes are homologous. Furthermore, operon structures and regulation systems share many common features within these groups. Among both groups of hrp clusters, at least nine genes are homologous. Because these genes are not only found in phytopathogenic bacteria but are also highly conserved in Gram-negative animal pathogenic bacteria, they were termed hrc (hrp and conserved; Bogdanove et al. 1996). The hrc genes encode components of the so-called type III secretion system (Salmond and Reeves 1993), which enables pathogenic bacteria to apparently transfer proteins directly into eukaryotic cells (Lee 1997). In addition to these type III secretion system genes, the hrp clusters also encode HR-eliciting proteins termed harpins and components of a pilus structure termed hrp pilus, along with regulatory elements involved in the regulation of hrp cluster genes and certain avr genes. 3 The Type III Secretion System The type III secretion system was first described for Gram-negative animal-pathogenic bacteria, such as Yersinia pestis, Salmonella typhimurium, Shigella jlexneri and enteropathogenic E. coli (Bonas and Ackerveken 1999). The best-investigated and -understood of these is the type III secretion system of Yersinia, which mediates the translocation of certain Yops (Yersinia outer proteins). These have various functions in pathogenicity, e.g. the cytotoxin YopE (Rosquist et al. 1991), the protein tyrosine phosphatase YopH (Guan and Dixon 1990) and the serine/threonine kinase YopO (Galyov et al. 1993). These proteins, which function in pathogenicity, are secreted into the host cell by the action of Genetics 100 approximately 22 proteins encoded by the genes yscA-U (Bergman et al. 1994) and lcrD (Plano et al. 1991). Counterparts of some ysc genes are found in the hrp genes of plant pathogenic bacteria, e.g. in P. syringae, where at least 16 genes are involved in a type III secretion mechanism and 12 exhibit extensive sequence homology to correspondingysc genes. As mentioned previously, the nine so-called hrc genes are conserved among all type III secretion systems of Gram-negative animal and plant pathogenic bacteria. Surprisingly, eight of these nine genes share striking homologies with genes of the flagellar assembly apparatus of Grampositive and Gram-negative bacteria. By comparison with the flagellar apparatus, the location of these components of the type III secretion apparatus can be supposed (He 1998; Bonas and Van den Ackerveken 1999). Supposed location of components of the type III secretion system Flagellar proteins FlhA Flil FliN/FliY FliP FliQ FliR FliB FliF Type III secretion system Yersinia sp. Phytopathogens Location HrcV HrcN HrcQ HrcR HrcS HrcT HrcU HrcJ HrcC Inner membrane ATPase Inner membrane Inner membrane Inner membrane Inner membrane Inner membrane Periplasm spanning Outer membrane LcrD YscN YscQ YscR YscS YscT YscU YscJ YscC Gene products of hrcR-V are probably located in the inner membrane, and HrcJ is possibly a lipoprotein that spans the periplasm and is linked to the inner and outer membranes. The cytoplasmic component HrcN has homologies to ATPases, suggesting that this protein delivers energy for the type III secretion system. HrcC is one of the outer-membrane channel-forming proteins involved in type II secretion of proteins and fIlamentous phages (Kumar et al. 1980). This supposed configuration was confirmed by experiments indicating that an hrcC mutant of P. syringae pv. syringae accumulates the HrpZ harpin in the periplasm while, in an hrpU mutant, the harpin remains in the cytoplasm (Charkowski et al. 1997). Thus, the type III secretion system consists of components mediating transport through both membrane systems of Gram-negative bacteria. In addition to the harpin HrpZ, four of the seven to eight extracellular proteins secreted by P. syringae pv. tomato are translocated by the type III secretion system (Yuan and He 1996). One of these proteins, with a molecular weight of 10 kDa, is HrpA, which forms an Hrp pilus of ap- Genetics of Phytopathogenic Bacteria 101 proximately 2-llm length and 6- to 8-nm diameter (Roine et al. 1997). Purified HrpA protein can self-assemble into a pilus-like structure, and a non-polar mutation in hrpA leads to the absence of a pilus and deficiency in disease induction or HR triggering. Although the exact function of the pilus in the transfer process is poorly understood, HrpA is required for the secretion of putative virulence proteins and is also thought to be involved in the regulation of the type III secretion system of P. syringae pv. tomato (Wei et al. 2000). Recently, an in vitro model system was developed for the secretion of Avr proteins by X. campestris pv. vesicatoria (Rossier et al. 1999). It could be demonstrated that two Avr proteins were secreted by the type III system without leakage of cytoplasmic proteins into the medium. It was also shown that the type III secretion system of X. campestris pv. vesicatoria was able to transport the harpins PopA from R. solanacearum, AvrB from P. syringae pv. glycinae and, most strikingly, the cytotoxin YopE of Y. pseudotuberculosis. Thus, functional conservation of the type III secretion systems of plant and animal pathogens appears to require common secretion signals. Although no sequence homologies in the N-terminal region of the secreted proteins could be found in this study, it has been shown that a 15-amino-acid fragment from the Nterminus of YopE, joined to a reporter gene, allowed ysc-dependent secretion (Sory et al. 1995). Therefore, further work is required to fully understand the selectivity of the secretion mechanism. Although all Gram-negative phytopathogenic bacteria investigated carry hrp genes (with the exception of Agrobacterium tumefaciens), there is no evidence to date that these genes are also present in Grampositive phytopathogenic bacteria. Hybridization of the total DNA of the Gram-positive, tomato pathogen Clavibacter michiganensis ssp. michiganensis against a DNA probe of hrcV of X. campestris pv. vesicatoria, and of a probe carrying selected parts of the hrp cluster of E. herbicola pv. gypsophila, gave no indications for the presence of hrp-like genes. Moreover, polymerase chain reaction with degenerate primers designed from highly conserved regions of the genes hrcN, hrcR, hrcU, and hrcV was unsuccessful in producing the expected amplification products (Ahlemeyer and Eichenlaub, unpublished). Additionally, genes analogous to hrp genes have not yet been located in Gram-positive animal pathogenic bacteria. In Listeria, with the exception of one protein (internalin A), all virulence proteins have a signal-peptide sequence on the N-terminus, which is typical for proteins excreted by the type II secretion system (Engelbrecht et al. 1998). Furthermore, the complete chromosomal DNA sequence of Mycobacterium tuberculosis provides no information regarding the presence of a type III-like secretion system in this Gram-positive animal pathogen. 102 Genetics 4 Regulation of hrp Genes Generally, the expression of hrp genes is suppressed when the pathogenic bacteria are grown in complex media rich in various nutrients, while it is induced in plants and in minimal media mimicking the apoplast (Wengelnik et al. 1996a). The regulatory system for controlled expression of hrp genes in phytopathogenic Pseudomonads, consisting of the genes hrpR, hrpS and hrpL, is best understood. These genes are part of a multi-component regulation system governing the expression of other hrp and the avr genes. HrpR and HrpS are related to members of the NtrC family of two-component signal-transduction systems (Xiao et al. 1994), which consist of a sensor protein and a response regulator mediating activation of gene expression. The requirement for an alternative sigma factor, which is provided by hrpL, is common to the NtrC family. HrpL belongs to the ECF (extra cytoplasmic function) family of 0" factors. The regulatory cascade for the transcription of P. syringae hrp genes first requires the activation of transcription at the hrpL promoter by HrpS and HrpR, together with the alternative 0" factor 0"54 RpoN (Xiao et al. 1994). The HrpL 0" factor then allows the transcription of the hrp genes hrpK, hrpJ, hrpU, hrpC and hrpZ, and various avr genes. Common to all these genes is the presence of a consensus sequence in the promoter region, termed the hrp or avr box (Innes et al. 1993; Xiao et al. 1994). However, slight differences in the regulatory system may occur within the same species. In P. syringae pv. phaseolicola, HrpR seems to function as a transcriptional activator ofHrpS (Grimm et al. 1995). E. amylovora possesses genes homologous to hrpS and hrpL (Wei and Beer 1995). The HrpL 0" factor of Erwinia controls the transcription of five hrp genes that also share a common motif in their promoter region similar to the P. syringae hrp box. Despite this similarity, attempts to complement a hrpL mutant of E. amylovora with the hrpL gene from P. syringae failed (Wei and Beer 1995). Although the regulatory systems of these two groups of bacteria have some components in common, they are still too different to be exchanged. A different regulatory system has been described for X. campestris pv. vesicatoria; this system may also be common to other phytopathogenic Xanthomonas strains. It consists of two genes, hrpG and hrpX, which are linked to each other but do not map within the chromosomal hrp cluster (Wengelnik and Bonas 1996; Wengelnik et al. 1999). These genes control the expression of the five transcription units hrpB-hrpF (Bonas et al. 1991), but there is no requirement for RpoN (Horns and Bonas 1996). Because expression of all hrp genes is suppressed in a hrpG mutant, it appears that this is the first gene in the regulatory system of X. campestris pv. vesicatoria (Wengelik et al. 1996b). HrpG is homologous to response regulators of two-component regulatory systems and activates Genetics of Phytopathogenic Bacteria 103 the transcription of hrpX (and hrpA; Wengelnik et al. 1996b). In turn, HrpX, an AraC-type transcriptional activator, stimulates transcription of the hrpB-hrpF operons (Wengelik and Bonas 1996). In accordance with two-component regulatory systems, another component must be postulated, i.e. a sensor protein monitoring the bacterial environment, the medium or the plant apoplast. This protein triggers the regulatory cascade leading to hrp-gene expression. The question regarding the kinds of effectors crucial for the induction of hrp-gene expression remains unanswered. Initial reports that plant components are required (Schulte and Bonas 1992) may be misleading, because the same group has meanwhile developed synthetic media in which hrp genes are expressed (Wengelnik et al. 1996a). 5 Harpins and Avr Proteins In addition to the secretion of hrp pilus components, the translocation of proteins directly affecting the plant cell also seems to be dependent on a functional type III secretion system. These proteins include the harpins encoded by genes of the hrp clusters in several Erwinia species (hrpN), P. syringae pathovars (hrpZ) and R. solanacearum (PopA), along with several Avr proteins. Harpins comprise relatively small (28-44 kDa), glycine-rich, cysteinefree, hydrophilic proteins inducing an HR in an incompatible interaction. Purified harpins retain their elicitor activity even after heat treatment. Several harpins have been shown to induce the expression of plant defense genes. Recently, a salicylic acid dependent induction of systemic acquired resistance (SAR) by harpin HrpN of E. amylovora was also shown using transgenic Arabidopsis plants (Dong et al. 1999). Nevertheless, the role and possible function of harpins in bacteriaplant interactions is not yet fully clear. Whereas harpins are homologous within a genus, they are not conserved among different genera (Lindgren 1997). Furthermore, although an hrpNEa mutant of E. amylovora was unable to induce an HR in tobacco and was inefficient in disease induction on host plants (Wei et al. 1992), an hrpZpss deletion mutant of P. syringae pv. syringae was still able to induce an HR (Alfano et al. 1996). Additionally, popA mutants of R. solanacearum are not affected in their ability to cause an HR (ArIat et al. 1994). There is strong evidence that the translocation of Avr proteins is also dependent on the type III secretion system (Alfano and Collmer 1997; Lee 1997; Vivian and Gibbon 1997). Avr proteins are elicitors of defense reactions in incompatible interactions. According to the gene-for-gene concept (Flor 1971; Keen 1992), it is supposed that these proteins are recognized in the plant by receptors encoded by R genes. Thus, incompatibility in the bacteria-plant interaction is mediated by a bacterial avr 104 Genetics gene together with a corresponding R gene in the plant. If the host plant and pathogen do not carry corresponding Rand avr genes, there is no recognition, the reaction is compatible, and disease symptoms develop. Some Avr proteins have been shown to act as virulence factors in the absence of corresponding host-plant R genes (Lorang et al. 1994; Ritter and DangI1995). Although no Avr protein has yet been detected in a plant cell, some experiments strongly support the idea that Avr proteins are directly translocated into the plant cell by means of the type III secretion system. Some Avr proteins are only active when located within the plant cell (Van den Ackerveken at al. 1996). For AvrB of P. syringae pv. syringae, it has been shown that the purified Avr protein does not induce an HR in plants carrying the corresponding R gene (rpml), whereas transient expression of avr B in Arabidopsis thaliana possessing rpml leads to cell death (Gopalan 1996). In support of this, E. coli expressing avrB constitutively only induce an HR in A. thaliana when they carry the intact hrp gene cluster of P. syringae pv. syringae, which allows the functional expression of the type III secretion system (Pirhonen et al. 1996). The function of harpins in triggering an HR in a non-host plant and the function of Avr protein in inducing a resistance reaction in plants carrying a corresponding R gene raise the question of why such genes, which clearly reduce the chance of the pathogen to successfully parasitize the plant, are found in phytopathogenic bacteria. An answer to this question may be that these genes initially played a role in virulence or in fitness when infecting susceptible plants (He 1998). Thus, proteins secreted by the type III secretion system, whether they are found in the medium or are transported into the plant cell, may all have initially been virulence factors. However, during the co-evolution of plants and bacterial pathogens, plants have learned to recognize some of these gene products as elicitors for their defense in order to establish an equilibrium between host and pathogen. Investigations of the P. syringae pv. syringaelsnap bean pathosystem indicate that the role of the type III secretion system in the compatible interaction might be completely mediated by its requirement for the growth of the bacteria in the phyllosphere (Hirano et al. 1999). The population size of hrcC and hrc] mutants on the leaf surface and in the intercellular space was clearly diminished in comparison to that of a wild type strain. The mutants in the type III secretion system were nevertheless able to cause disease symptoms under certain conditions. Genetics of Phytopathogenic Bacteria 105 6 Plant Defense In the plant, pathogenic bacteria are recognized directly or indirectly, and various local and systemic defense responses are activated through the action of several components mediating the recognized signal. a) Recognition of the Pathogen Several elicitors of plant defense responses are known. Two kinds of elicitors are generally distinguished. The first are pathogen-derived substances, such as Avr proteins and harpins, along with chitin and lipopolysaccharides. The second type are fragments of plant material that, e.g. originate from the hydrolysis of plant cell-wall polymers by enzymes of the pathogen; these are termed endogenic elicitors (Ebel and Casio 1994). It is suggested that elicitors are recognized by specific receptors of the plant. Although highly specific binding of elicitors to certain membrane fractions has been demonstrated (Shibuya et al. 1993; Nurnberger et al. 1994), and a f3-glucan binding protein has been purified from soybean (Umemoto et al. 1997), a plant receptor that binds the effector (elicitor) and mediates signal transmission to the cytosol has not been found. The current explanation of the recognition of Avr proteins delivered into the plant cell by the type III secretion system is that Avr proteins interact with corresponding R-gene products of the plant. Some R proteins are characterized by domains with 12-21 leucine-rich repeats (LRRs), nucleotide-binding domains and serine-threonine domains (HammondKosak and Jones 1997). LRRs have also been described in the animal system, where they mediate protein-protein interactions and ligand binding in proteins involved in signal transduction (Kobe and Deisenhofer 1994; Jones and Jones 1997). Thus, it is currently thought that R proteins with LRRs act as receptors for Avr proteins (Baker et al. 1997; Parker and Coleman 1997; Hutcheson 1998). b) Signal Transduction To date, only fragmentary knowledge of the signal-transduction chain between pathogen recognition and the expression of genes involved in plant defense is available. As a result of elicitor recognition, an influx of H+ is observed in plant-cell suspension cultures; this results in an alkalization of the medium (Atkinson et al. 1985) and lowers the pH in the cytoplasm. Lowering the cytoplasm pH by adding propionic acid in rice cell cultures was shown to induce the expression of phenylalanine ammonium lyase (PAL), the key enzyme for phytoalexin biosynthesis (He 106 Genetics et al. 1998). The same effect was obtained with a phosphatase inhibitor, indicating that a cascade of protein phosphorylations is part of the signal-transduction pathway. Treating tomato cells with the phosphatase inhibitor calyculin A led to a phosphorylation of various proteins that were also phosphorylated after elicitation (Felix et al. 1994). The influx of Ca2+ is also observed directly after the elicitation of plant cells (Knight et al. 1991). The essential role of Ca2+ accumulation in the cell is demonstrated by treating cells with Ca2+ ionophores and Ca 2+-channel blockers, which either activate phytoalexin synthesis or abolish the induction of an HR (Stab and Ebe11987; He et al. 1994). The Ca2+- and NO-mediated production of superoxide radicals 2-) by reduced nicotinamide dinucleotide phosphate oxidase during the oxidative burst is another link in the signal-transduction chain (Chandra and Low 1997; Jabs et al. 1997; Wojtaszek 1997; Delledonne et al. 1998). The superoxide radical may be a second messenger for the activation of defense genes and the expression of pathogenesis-related (PR) proteins in the SAR (Chen et al. 1993; Tenhaken et al. 1995). (.° c) Hypersensitive Cell Death and Other Locally Induced Defense Reactions It has been repeatedly mentioned in this review that plants can recognize attack by a bacterial pathogen and react in various ways. The most spectacular defense mechanism is the HR, which can be induced by the pathogen in host or non-host plants. The HR is a fast, active cell death in the plant tissue at the site of pathogen invasion; it prevents the spread of the pathogen. The HR has been described for various incompatible reactions between different plant species and phytopathogenic viruses, bacteria and fungi. In a bacterial infection, as occurs in nature, a HR can hardly be detected, but the infIltration of leaves with an inoculum of more than 106 bacteria per milliliter usually leads to a clearly visible HR within 24 h. For some time, it was not clear whether the HR resulted from the killing of plant cells by the pathogen or was a reaction of the plant cells in the form of localized cell death. Although neither the mechanism nor the genes involved are known, it is currently believed that the HR is a form of programmed cell death (PCD; Dangl et al. 1996; Greenberg 1996; Mittler and Lam 1996; Heath 1998). That the HR is genetically controlled is suggested by mutants of Arabidopsis, maize and barley in which an HR can be triggered without interaction with a pathogen (Dietrich et al. 1994; Greenberg et al. 1994; Mittler and Lam 1996). In plant-cell suspension cultures, a number of morphological and ultrastructural alterations resembling those observed during the programmed cell death of animal cells occur. In plant cells treated with an HR-inducing elicitor, plasmolysis and a condensation of the nucleus is Genetics of Phytopathogenic Bacteria 107 observed (Yano et al. 1998). Endonucleases are activated, and the typical cleavage of chromosomal DNA into discrete DNA fragments (DNA laddering), as observed during the apoptosis of animal cells, is also seen during the HR of plant cells (Ryerson and Heath 1996). In the interaction with an obligate biotrophic pathogen, it is feasible that localized cell death during the HR is a sufficient defense against the pathogen. However, in addition to cell death, the cells surrounding the site of pathogen invasion also activate a number of defense reactions that allow the plant to control saprophytic or necrotrophic pathogens (Hammond-Kosack and Jones 1996). Due to callose apposition (Parker et al. 1993), the cross-linking of hydroxyproline-rich glycoproteins (HPRGs; Bradley et al. 1992), the production of papillae (Heath 1980) and lignification (Whetten and Sederoff 1995), cell walls can be reinforced to render the spreading and attack of the pathogen more difficult and to reduce the flow of nutrients to the pathogen. Phytoalexins, a group of chemically heterogeneous, low-molecularweight, lipophilic, anti-fungal compounds with a broad spectrum of anti-microbial effects (Morrissey and Osbourn 1999), are also produced. However, the specific role of phytoalexins, which can be considered plant antibiotics, is not clear for most host-pathogen interactions. On contact with a pathogen, or as a reaction on elicitor application, plants accumulate PR proteins in the vacuole, cell wall or intercellular space (Stintzi et al. 1993). Most of these proteins resist acid pH and proteolytic cleavage. Their action seems to be mostly directed against fungi, because some exhibit chitinase and 1,3-13-glucanase activities (Colinge et al. 1993; Melchers et al. 1994), while the function of others is unknown. One of the earliest responses of the plant against a pathogen is the production of reactive oxygen species (ROSs) in a reaction termed oxidative burst (Baker and Orlandi 1995; Tenhaken et al. 1995; Wojtaszek 1997). In addition to functioning as a second messenger in the pathogeninduced cellular signal transduction (Desikan et al. 1998), ROSs probably also directly kills pathogens. During the oxidative burst in plants, H20 2 concentrations toxic to microorganisms have been detected (Peng and Kuc 1992). Furthermore, ROSs are essential for the formation of lignin polymers and the cross-linking ofHPRGs (Showalter 1993). As described above, in addition to these local defense reactions, a defense mechanism termed SAR is often induced. In this reaction, mediated by an accumulation of salicylic acid, the non-infected parts of the plant also exhibit an elevated disease resistance against various pathogens (Sticher et al1997; Pieterse et al. 1998). All or some of these defense mechanisms are triggered simultaneously in various plants on contact with a microbial pathogen. Some of the defense reactions are also activated in a compatible interaction; however, because this usually occurs too late to prevent the progress of infection, disease symptoms still develop (Alfano and Collmer 1996). 108 Genetics 7 Outlook Future work on pathogenic bacteria will focus on understanding the mechanism by which bacterial proteins are transferred into the plant cell and elucidating the plant proteins with which they interact. Additionally, the signal-transduction pathway that triggers plant defense needs to be investigated in detail. 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Mol Plant-Microbe Interact 9:704-712 Wengelnik K, Rossier 0, Bonas U (1999) Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes. J Bacteriol 181 :6828-6831 Whetten R, SedroffR (1995) Lignin biosynthesis. Plant Cell 7:1001-1013 Wojtaszek P (1997) Oxidative burst: an early plant response to pathogen infection. Biochern J 322:681-692 Xiao Y, Hue J, Yi J, Lu Y, Hutcheson SW (1994) Identification of a putative alternative (J factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes. J BacterioI176:1025-1036 Genetics of Phytopathogenic Bacteria 113 Yano A, Suzuki K, Uchimiya H, Shinshi H (1998) Introduction of hypersensitive cell death by a fungal protein in cultures of tobacco cells. Mol Plant Microbe Interact 11:115-123 Yuan J, He SY (1996) The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. J Bacteriol 178:6399-6402 Dipl. BioI. Jutta Ahlemeyer Institut fiir Pflanzenbau und Pflanzenziichtung II Justus-Liebig -Universitat Giessen Ludwigstrasse 27 35390 Giessen, Germany Communicated by K. Esser Prof. Dr. Rudolf Eichenlaub Lehrstuhl Gentechnologie/Mikrobiologie Universitat Bielefeld Universitatsstrasse 25 33501 Bielefeld, Germany e-mail: [email protected] Genetics Plant Biotechnology: Transgenic Crops for the Third Millennium Frank Kempken 1 Introduction During recent years, the use of plant biotechnology and genetically modified (GM) crops has increased considerably. In 1999, transgenic soybean with engineered herbicide resistance accounted for more than 50% of the soybean grown in the us. Maize and cotton carrying an endotoxin gene from Bacillus thuringensis (Bt) exhibiting insect resistance are now widely used. A rice plant that produces enough p-carotene to supplement the daily requirements of a human and serves to enhance iron uptake was recently presented. Some of the most important milestones in the development of transgenic crops are summarized in Table 1. The data given there highlight the enormous developments that have occurred during the last two decades of this last millennium. Consequently, one may conclude that the future of GM crops during the first decade of the third millennium should be bright. However, recent years have also witnessed increasing anti-GM-crop activity. This is not only true in Europe; resistance is also growing notably overseas (Lehrman 1999), making the fate of transgenic crops somewhat uncertain. In most European countries, consumer acceptance of GM-based food products is low. Certainly, the controversies surrounding Pusztai's work on transgenic potatoes (Enserink 1999; Ewen and Pusztai 1999; Loder 1999a) and the proposed toxic effect of pollen from Bt-maize on the monarch butterfly (Losey et al. 1999; Shelton and Roush 1999) have also contributed to the negative image of GM crops. Consequently, this review will not only highlight some of the advances in the making and use of GM plants, it will also focus on the heated debate concerning the safety of GM crops. Finally, for those readers not familiar with the basic techniques utilized in the generation of GM plants, some technical remarks have been included. Due to the size limitations of this text, only a limited selection of applications is covered. Progress in Botany, Vo!' 62 © Springer-Verlag Berlin Heidelberg 2001 Plant Biotechnology: Transgenic Crops for the Third Millennium 115 Table 1. Achievements in plant biotechnology (Kempken 1997; Kempken and Kempken 2000). References are given for original scientific papers only Year Milestones References 1980 First transfer of bacterial DNA from Agrobacterium tumefaciens to a plant Hernalsteens et al. (1980) 1983 Selective markers available and Ti plasmid disarmed Bevan et al. (1983); Fraley et al. (1983); Herrera-Estrella et al. (1983) 1984 First plant protoplast transformation Hain et al. (1985) 1985 Herbicide resistance available Comai et al. (1985) 1986 Viral resistance available First field tests with GM plants Powell-Abel et al. (1986) 1987 Engineered insect resistance Fischhoff et al. (1987); Vaeck et al. (1987) Sanford et al. (1987) Biolistic transformation possible 1988 Control of tomato fruit ripening Sheehyet al. (1988); Smith et al. (1988) 1989 Production of antibodies in plants Hiatt et al. (1989) 1990 Artificial male sterility Mariani et al. (1990) 1991 Modified carbohydrate content Oakes et al. (1991) 1992 Modified fatty-acid composition Knutzon et al. (1992); Voelker et al. (1992) Vasil et al. (1992) Poirier et al. (1992) Biolistic wheat transformation Biodegradable plastic from plants 1994 "FlavrSavr" tomato commercially available (first GM-plant-based food) 1998 More than ten genes simultaneously cloned into a plant. Worldwide, more than 48 GM plants commercially approved Chen et al. (1998) British researcher claims GM potatoes unsafe GM rice with better nutritional value Worldwide area of cultivation of GM crops larger than 40 million ha Ewen and Pusztai (1999)8 1999 To date, more than 9000 field trials in OECD countries "Monarch butterfly" controversy Losey et al. (1999); Shelton and Roush (1999) GM, genetically modified; OEeD, Organization for Economic Cooperation and Development. 8This paper was published more than 1 year after a public television statement; for comments regarding the paper, see Enserink (1999) and Loder (1999a). Genetics 116 2 Methods for Gene Transfer and Expression of Foreign DNA in Higher Plants The first successful transformation of higher plants was reported for tobacco in 1980; only a few years later, dominant selectable markers were in use (Hernalsteens et al. 1980; Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983). Since then, numerous higher plants have been transformed (Table 2), and a number of reliable techniques and protocols for transformation have been developed (Potrykus 1991; Birch 1997; Hansen and Wright 1999). Three methods are most commonly used; these include Agrobacterium-mediated gene transfer, direct transfer of genes into protoplasts and the introduction of foreign DNA by the biolistic or "particle gun" approach. Table 2. Examples of transformed plants: (Gasser and Fraley 1989; Krautwig and Lorz 1995; Kempken and Kempken 2000) Fruits Vegetables Cereals Other crops Woody plants Ornamental Other plants Apple Asparagus Barley Alfalfa Eucalyptus Carnation Banana Beans Maize Atropa Pine Chrysanthemum Cherry Broccoli Rice Cotton Poplar Geranium Spruce Gerbera Cranberry Cabbage Rye Flax Grape Carrot Sorghum Liquorice Marigold Kiwi Cauliflower Wheat Mangold Morning glory Melon Chicory Oilseed rape Petunia Orange Cucumber Pepper Rose Papaya Eggplant Soybean Turf grass Pear Horseradish Sugar beet Plum Lettuce Sugar cane Raspberry Pea Strawberry Potato Sweet potato Tomato Sunflower Tobacco Arabidopsis Plant Biotechnology: Transgenic Crops for the Third Millennium 117 a) Agrobacterium-Mediated Transformation Agrobacterium tumefaciens is the etiological agent of crown gall disease and produces tumorous crown galls on infected species. Virulent strains of A. tumefaciens contain large Ti (tumor-inducing) plasmids, which are responsible for gene transfer and tumor induction. The Ti plasmids contain a region of DNA (the T-DNA), which is actually transferred into the plant. The usefulness of this system for plant genetic engineering is based on the fact that heterologous DNA can be introduced into the TDNA region and, thus, can be transferred into a plant (Potrykus 1991; Birch 1997; Hansen and Wright 1999). This technique is particularly useful with dicot species; it is less useful with monocots (Hooykaas-Van Slogteren et al. 1984; Bytebier et al. 1987; Schafer et al. 1987) and is extremely difficult with cereals, although some reports of successful transformation do exist (Raineri et al. 1990; Chan et al. 1993). Apparently, this technique works only in plants, which exhibit a proper wound response (Protrykus 1991). b) Transformation of Pro top lasts Protoplasts of plant cells are ideally suited for DNA uptake in the presence of polyethylene glycol or mediated by electroporation (Shillito et al. 1985; Fromm et al. 1986). However, the regeneration of plants from protoplasts is often very difficult, and no general protocol applies. Therefore, regeneration has to be determined empirically for each case. Nevertheless, the regeneration of transformed protoplasts was successfully achieved for a number of plants (Hansen and Wright 1999), including maize (Rhodes et al. 1988; Sukhapinda et al. 1993) and rice (Gupta and Pattanayak 1993). c) Biolistic Transformation The introduction of "particle-gun" or high-velocity microprojectile technology was a very significant breakthrough in plant transformation. DNA is targeted through the cell wall into intact cells or tissues using small metal particles (0.5-5 ~m) as carriers, which are accelerated to very high speed. This technique was developed by Sanford and coworkers (Sanford et al. 1987); a detailed description is provided by Klein et al. (1992). The procedure is particularly useful for obtaining the transformation of a particular tissue or for cereals, which are difficult to transform with other techniques. Because the regeneration of plants from immature embryos is possible (Vasil 1988), immature embryos from cereals are usually chosen as the target. 118 Genetics Transgenic barley (Wan and Lemaux 1994), corn (Fromm et al. 1986; Gordon-Kamm et al. 1990; Koziel et al. 1993; Murry et al. 1993), sorghum (Cassas et al. 1993), rice (Christou et al. 1991; Li et al. 1993) and wheat (Vasil et al. 1992, 1993; Weeks et al. 1993; Becker et al. 1994; Nehra et al. 1994) were realized with this method. Biolistic transformation also enables the transformation of plastids of higher plants. This approach has a number of biotechnological applications (Bock and Hagemann 2000) and may be particularly useful in avoiding pollen-based horizontal gene transfer from transgenic plants to weedy relatives. Horizontal transfer of plastid-based genes is limited because, in most plants, plastids are only inherited maternally (Hagemann 1992). The technique of plastid transformation is based on inserting genes into the plastid DNA. This has been achieved for a number of genes, including neomycin phosphotransferase (conferring kanamycin resistance; Carrer et al. 1993), Bacillus thuringensis endotoxin (conferring insect resistance; McBride et al. 1995) and 5-enol-pyruvyl shikimate-3-phosphate synthase (conferring herbicide resistance; Daniell et al. 1998). d) Regeneration, Selection and Verification of Transformed Plants Most of the above-mentioned gene-transfer techniques are performed on isolated cells, embryonic tissue or leaf disks. Tissue-culture techniques are required to culture these plant materials and to achieve the regeneration of intact plants. Detailed protocols have been developed for many plant species (Vasil and Thorpe 1994; Gamborg and Phillips 1995). The selection of transformed plants can be achieved with a number of marker and reporter genes (Bowen 1993). In particular, kanamycin resistance has frequently been used to generate GM plants. However, the use of antibiotic-resistance genes has raised critical questions regarding the safety of transgenic plants. Recently, a new method that does not require antibiotic-resistance genes was published. Instead, it is based on the overexpression of the isopentenyl transferase gene from the Ti plasmid of A. tumefaciens (Kunkel et al. 1999). The increase in cytokinin levels leads to shoot generation from transformed plant cells. In combination with an inducible promoter, this system can be useful for the selection of transgenic regenerants (Kunkel et al. 1999). To prove expression of the desired phenotypes, it is very important to verify the transformation of plants by molecular techniques (such as Southern hybridization) and by phenotypic assays (Birch 1997). Analysis of the offspring can also be a helpful tool (Potrykus 1991) but may not always be feasible (for example, in transformed trees, where sexual reproduction is very slow). Plant Biotechnology: Transgenic Crops for the Third Millennium 119 e) Targeted Gene Expression Cell- or tissue-specific gene expression is easily achieved due to the availability of a number of cell- or tissue-specific promoters (Table 3). New promoters can be isolated by using reporter genes or from ongoing expressed-sequence-tag sequence projects. This is a far-reaching achievement with respect to plant biotechnology, because complex changes in plant metabolism cannot be performed with ectopic promoters; they require targeting to the correct tissue sites. Intracellular targeting of direct-translation products into particular organelles (chloroplasts; Zoubenko et al. 1994) or into the secretory pathway (Chrispeels 1991) is achieved by using the correct leader sequences. A recent application of this mechanism is the secretion of proteins by plant roots (Borisjuk et al. 1999). This technique may have a wide range of applications, because it allows easy collection of novel plant-produced proteins. Table 3. Cell- and tissue-specific promoters Cell or tissue type Name of promoter or gene Source Reference Epidermis Itpl Tobacco Canevascini et al. (1996) Flower and root tip CHS15 Bean Faktor et al. (1996) Guard cells 0.3-kb fragment of Potato AGPase Muller-Rober et al. (1994) Meristem PCNA Kosugi et al. (1995) Phloem Sh Maize Fragment ofRTBV Rice Phloem, vascular parenchyma and bundle-sheath cells RolC Agrobacterium Graham et al. (1997) Pollen LAT52 Tomato Twell et al. (1990) Seed Puroindolin-b Wheat Digeon et al. (1999) Tapetum TA29 Tobacco Mariani et al. (1990) Rice Graham et al. (1997) Yin et al. (1997) PCNA, proliferating-cell nuclear antigen; RTBV, rice tungro bacilliform virus. 120 Genetics f) Silencing of Transformed Genes Although the production of transgenic plants is routinely performed for at least some plant species, a number of problems remain to be solved. The level of expression of transformed genes is often unpredictable and may vary among individual transformants. An ever-larger problem arises from the loss of transgene expression caused by transgene inactivation (Finnegan and McElroy 1994; Stam et al. 1997). Transgene silencing or inactivation was reported for single trans genes (Linn et al. 1990) and for independent transgenes in the same plant. This can be caused by differences in either the copy number (Hobbs et al. 1990) or the integration site (Prals and Meyer 1992). In addition, crossbreeding transgenic plants may result in the inactivation of one or more of the transformed genes (Matzke et al. 1989). Two kinds of gene silencing are known: first, transcriptional gene silencing (TGS), which is caused by promoter inactivation, and second, post-transcriptional gene silencing (PTGS). The latter occurs when the promoter is active, but the messenger RNA does not accumulate (Stam et al. 1997). Many cases of TGS, which is often caused by methylation, have been reported (Linn et al. 1990; Meyer et al. 1994; Vaucheret 1994). In plant DNA, 5-methylcytidine is found in both CpG and CpNpG symmetrical and asymmetrical sites (Ingelbrecht et al. 1994; Meyer et al. 1994; Park et al. 1996). Methylation of promoter regions seems to cause TGS, whereas methylation in coding regions appears to contribute to PTGS (Stam et al. 1997). PTGS is often more complicated than TGS. For example, co-suppression describes the inactivation of transformed additional copies of an endogenous gene. Ultimately, not only the introduced genes but also the endogenous genes are silenced (Napoli et al. 1990). The PTGS mechanism appears to act on any RNA that is homologous to the activating transgene (English et. al. 1996) and has an interesting application in the protection of transgenic crops from viral attack (Sect. 3.a.y). The hallmark of PTGS is a strongly reduced accumulation of specific transcripts; this may be due to a sequence-specific RNA degradation process (Stam et al. 1997). The mechanisms by which transgenes are recognized and subsequently inactivated by the cell are unknown; however, some strategies to circumvent transgene silencing have been proposed. These include selection for single-copy transgene insertion or the development of site-specific recombination systems (Finnegan and McElroy 1994). g) Removal of Marker Genes Finally, to increase consumer acceptance, the removal of marker genes from commercially used crop lines is recommended by some governmental agencies (for example, the German Robert Koch Institute). The Plant Biotechnology: Transgenic Crops for the Third Millennium 121 potential risks posed by selectable marker genes to the consumer of transgenic plants have been intensively studied and were found to be extremely low (Fuchs et al. 1993). These studies mainly were focused on markers such as neomycin phosphotransferase. This marker is commonly used and is present in Calgen's FlavrSavr tomatoes (Redenbaugh et al. 1992), for example. Although there is no scientific reason to restrict the use of this or other selectable markers (Yoder and Goldsbrough 1994), it may be desirable to remove selectable markers from transgenic plants. A number of techniques that may help remove marker sequences from transgenic plants are available (Yoder and Goldsbrough 1994). These include co-transformation, site-specific recombination, targeted gene replacement or the use of transposable elements that could reposition transgenes for subsequent elimination (Goldsbrough et al. 1993). Unfortunately, targeted gene replacement is very inefficient in higher plants (Halfter et al. 1992; Kempin et al. 1997), with which its use is hampered. A new and very promising method of marker-gene removal is based on the use of the ere recombinase (Gleave et al. 1999). The marker genes are flanked by the ere binding sites loxP. Once transgenic plants are generated, transient expression of ere initiates the removal of sequences flanked by loxP. 3 Improvements in Agriculture During recent years, many new crops with GM characteristics have been generated and field tested, and some have even been commercially released. A selection of the most important achievements is discussed in detail below. a) Enhancing Plant Resistance Losses caused by weeds or pathogens can be very severe. According to SchlOsser (1997), between 1988 and 1990, the pathogen-related loss of yield was to 15% in rice, 16.3% in potato and 12.4% in wheat. Similarly, weeds can cause declining yields; this problem particularly affects crops such as maize and soybean. Genetic engineering has led to promising crop improvements with regard to the control of pathogens and weeds. In this review, some of the most significant achievements will be discussed. Genetics 122 a) Herbicide Resistance Today, the use of herbicides is a common practice, and several herbicides are often used simultaneously to reduce the loss of yield incurred by weeds. Biodegradable herbicides, such as phosphothricine (Basta) and glyphosate (Round-Up) have been developed (Duke 1996). However, the use of these herbicides was strongly limited because of their toxicity to crops and weeds alike. With the advent of genetic engineering, however, plants exhibiting resistance to phosphothricine and glyphosate could be produced. Resistance to phosphothricine, which inhibits glutamine synthetase, could be achieved with the use of the bacterial bar gene encoding phosphinothricin acetyltransferase (De Block et al. 1987). Glyphosate (N-phosphonomethylglycine) prevents biosynthesis of aromatic amino acids by inhibiting 5-enol pyruvyl shikimate-3-phosphosynthase (EPSPS) in the plant (Comai et al. 1995). Resistance in plants can be achieved by: 1. Overexpression of plant EPSPS (Shah et al. 1986) together with a bacterial oxidoreductase (Barry et al. 1994) 2. The use of a modified EPSPS from Salmonella typhimurium (Comai et al. 1995) or the A. tumefaciens strain CP4 (Barry et al. 1994) 3. The use of a mutagenic plant EPSPS (Monsanto 1997) The gene derived from S. typhimurium was used for experimentation only, whereas the other genes have been introduced in a number of plant species for commercial purposes (Shah et al. 1995). According to a recent study conducted by the United States Department of Agriculture (1999), the use of herbicide-resistant crops has significantly contributed to a reduction of the total amount of herbicides used. In addition, yield increased in some of the regions where the tests were performed. The ecological advantages of transgenic herbicide-resistant crops are obvious: phosphothricine and glyphosate, which are biodegradable, can be safely applied, and the total amount of herbicides used is lower. Furthermore, soil erosion, an additional environmental factor, is usually caused by frequent and deep plowing of agriculturally used areas. However, using the GM plants, soil erosion is reduced by 90%, because the need for frequent plowing is eliminated. 13) Insect Resistance Gene transfer into crop plants to impart insect resistance is another approach and has already found commercial application. Bacillus thuringensis genes, which encode insect-toxic proteins, have been introduced into many plants (tomatoes, cotton and maize) in a modified form with adapted codon usage (Delannay et al. 1989; Wilson et al. 1992; Shah et al. Plant Biotechnology: Transgenic Crops for the Third Millennium 123 1995). Different B. thuringiensis strains are known to produce more than 100 different insect toxins. These toxins are made during sporulation and are called o-endotoxins. Four different classes are known; they exhibit toxicity to lepidoptera (cry!), lepidoptera and diptera (cryII), coleoptera (cry/II) and diptera (cry/V; Fischhoff et al. 1987; Vaeck et al. 1987). As with herbicides, the use of Bt plants has led to a reduction of pesticide applications in agriculture (United States Department of Agriculture 1999). While the advantages gained with these plants have been clearly demonstrated, the safety of Bt plants to benign insects was recently challenged (Losey et al. 1999; Chap. 5). In addition to the use of the B. thuringiensis toxin, a number of different strategies have been used. For example, the expression of an uamylase inhibitor in a common bean led to its resistance to burchid beetles (Shade et al. 1994). y) Resistance to Viral Pathogens The prospect of intervening in viral disease development dates back to 1929, when it was shown for the first time that prior inoculation of tobacco with a mild infectious tobacco mosaic virus (TMV) could prevent infection with more severe strains (Scholthof et al. 1993). Sanford and Johnson (1985) proposed that the expression of viral proteins in a host would disrupt the normal balance of viral propagation and, consequently, may lead to protection of the crop against the virus. This event was termed cross protection. Experimental proof was delivered in 1986, when Beachy and co-workers demonstrated protection against viral infection in transgenic tobacco that expressed a TMV coat protein (Beachy et al. 1990). This approach was originally called coat-protein-mediated protection and has been shown to be effective against several viruses in many other plant species (Dempsey et al. 1998). However, this type of protection can also be achieved with other viral genes and, in fact, resembles a special case of co-suppression (Sect. 2.f). Other strategies against viruses are based on the use of anti-sense RNA (Pang et al. 1993), the use of antibodies (Tavladoraki et al. 1993; Schouten 1998) or even the expression of mammalian interferoninduced anti-viral gene products (Truve et al. 1993). Recently, the use of naturally occurring plant-resistance genes has also become feasible (Dempsey et al. 1998; Salmeron and Vernooij 1998). 8) Resistance Against Bacterial and Fungal Pathogens Resistance against fungal pathogens is another target of genetic crop improvement. Transgenic tobacco co-expressing chitinase and gluca- 124 Genetics nase genes exhibit enhanced protection against fungal attack (Zhu et al. 1994). Similarly, Jach et al. (1995) demonstrated the expression of antifungal genes from barley in transgenic tobacco. Disease resistance from the expression of foreign phytoalexin (from grapevine) was also shown in transgenic tobacco (Hain et al. 1993). The expression of a bar gene in transgenic rice not only led to herbicide resistance but was also shown to prevent infection by the fungus Rhizoctonia solani, the etiological agent of sheath blight (Uchimiya 1993). Specific chitinases and glucanases have been used to generate anti-fungal activity (Sela-Buurlage et al. 1993). In addition, numerous plant proteins exhibiting anti-fungal activity and anti-fungal toxins are known, thus providing a source for future disease-prevention strategies (Dempsey et al. 1998; Salmeron and Vernooij 1998). Finally, the use of single dominant resistance genes (R) of the plant disease-resistance signaling pathway provides promising strategies for the future generation of transgenic plants with microbial-disease resistance (Salmeron and Vernooij 1998). b) Artificially Introduced Male Sterility to Produce Hybrid Seeds The sale of hybrid corn (Crow 1998) is a multi-million-dollar business, because farmers prefer to plant seed derived from the crossing of two parent lines that will give high-yielding FI plants. Such hybrid vigor (or heterosis) is also important for many other crops (e.g. tomato or sunflower). It is important to avoid self-fertilization of the parental lines. In some cases, a genetically determined system of pollen sterility makes it possible to produce hybrid seed but, for some crops, hand emasculation of the female line is necessary, making seed production rather expensive. Therefore, artificial systems that introduce male sterility in higher plants are desired. In recent years, many strategies to genetically engineer male sterility have been published. These approaches are particularly useful where cytoplasmic male sterility systems are not available. Pollen ablation may be caused by anther-specific expression of ribonucleases. Mariani et al. (1990, 1992) made use of Barnase, a gene for an extracellular RNase from Bacillus amyloliquefaciens. This bacterium uses Barnase as a defense system against competing bacteria. B. amyloliquefaciens also expresses Barstar, a specific inhibitor of Barnase; it can be used to obtain fertile FI plants. In a similar strategy, the Osg6B promoter from rice fused to an endo-p-1,3-glucanase was employed. The construct was transformed into tobacco and, during the formation of tetrads, was expressed in the tapetum cells, leading to a significant reduction of the amount of fertile pollen (Tsuchiya et al. 1995). Yet another strategy is based on the induction of male sterility by tapetum-specific de-acetylation of externally applied nontoxic N-acetyl-L-phosphinothricin (Kriete et al. 1996). Plant Biotechnology: Transgenic Crops for the Third Millennium 125 c) Improving Plant Micronutrients The nutritional health and well being of humans and animals depends on plant food, which provides the majority of all essential vitamins, minerals and health-promoting phytochemicals. However, these micronutrients are generally contained at low concentration in staple crops. Therefore, research to understand and manipulate the synthesis and content of plant micro nutrients is underway (DellaPenna 1999). The current status of vitamin-E enrichment in crops was recently reviewed (Grusak 1999). A major breakthrough was the generation of a rice plant by Potrykus and collaborators; it contains high amounts of f3-carotene and is iron-rich (Gura 1999; Ye et al. 2000). This new GM rice can be used to prevent vitamin A and iron deficiency, both common among humans depending on rice as a major food source. To achieve this goal, seven genes were transferred into ordinary rice. Four genes were needed to produce f3-carotene from geranylgeranyl pyrophosphate, and three genes contributed to iron supplementation. In the future, as a result of the ongoing genomic-sequence projects, all genes responsible for pathways leading to important micro nutrients are expected to be known. This should provide the tools for better and healthier nutrition, particularly for people who depend mostly on staple foods for their daily diet (DellaPenna 1999). d) Modified Carbohydrates in Transgenic Crops Starch is used not only for human and animal foods and feed but is also an important resource for industrial applications. Because starch can be isolated easily from a number of crop plants, including maize and potato, starch production can be modified in transgenic plants. With respect to different applications, the amount of starch can be increased and, more importantly, the composition of starch - i.e., its amylose (unbranched) and amylopectin (branched) contents - is of interest. Overexpression of adenosine diphosphate (ADP) glucose pyrophosphorylase, a key enzyme of starch biosynthesis, led to a 20% increase in starch production (Stark et al. 1992). In contrast, on introduction of a chimeric ADP glucose pyrophosphorylase gene into potato, sucrose was produced at the expense of starch production, which was greatly reduced (MullerRober et al. 1992). These early examples demonstrate that plant carbohydrates can be manipulated. However, carbohydrates are produced in the photosynthetically active plant leaves (i.e., source), mainly in form of hexamers, and are mostly consumed or stored in flowers, seeds and roots (i.e., sinks). Consequently, the transport of carbohydrates needs to be considered, too. Therefore, efforts to efficiently manipulate carbohy- 126 Genetics drate content should be directed at specific tissues (Stitt and Sonnewald 1995). Other applications include potatoes that are better suited to storage under cold conditions. This was achieved by ectopic expression of a tobacco invertase-inhibitor-like protein, which prevented the cold-induced sweetening of potato tubers (Greiner et al. 1999). In addition to starch, cellulose synthesis has become a target of genetic modification (Chapple and Carp ita 1998). e) Biodegradable Plastics from Transgenic Plants Biodegradable plastics have a number of industrial applications. Poly-(3hydroxybutyric acid; PHB) and other, related aliphatic polyesters from bacteria (polyhydroxalkanoic acids; PHA) are produced by microbial fermentation (Steinbiichel and Fiichtenbusch 1998). However, PHA production in transgenic plants poses an attractive alternative for future production. The production of biodegradable plastics in plants on an agricultural scale could reduce costs and, in the long term, could even rival the production of petroleum-derived plastics (Poirier et al. 1995; Steinbiichel and Fiichtenbusch 1998). In a first attempt, biosynthetic genes for PHB from the bacterium Ralstonia eutrophus were transformed and expressed in A. thaliana; however, the levels of PHB were low, and the plants exhibited retarded growth (Poirier et al. 1992). Redirection of the PHB biosynthetic pathway from the cytoplasm to the plastids by using appropriate targeting signals led to a 100-fold increase in PHB production (up to 10 mg/g fresh weight) in pre-senescing leaves (Nawrath et al. 1994). The homopolymer PHB is somewhat brittle, whereas the co-polymers poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) are more flexible and, consequently, are more suitable for industrial applications. The current problem is that, with the exception of acetyl-coenzyme A (acetyl-CoA), metabolites are not available. However, by expressing four trans genes and diverting metabolic pools of acetyl-CoA and threonine, the co-polymer was produced in A. thaliana and in seeds of oilseed rape (Slater et al. 1999). Together with improved extraction protocols (Steinbiichel and Fiichtenbusch 1998), these transgenic plants may become commercially attractive in the future. t) Production of Vaccines The idea of producing immunogenic proteins and antibodies in higher plants is still rather new (Conrad and Fiedler 1994; Ma and Vine 1999), but its future prospects are tremendous. The guaranteed absence of Plant Biotechnology: Transgenic Crops for the Third Millennium 127 contaminating pathogenic viruses is one of the most important advantages afforded by plant-produced vaccines. The first vaccine candidate to be expressed in a plant was the streptococcal antigen IIII from Streptococcus mutans, which accumulated to levels of 0.02% of the total leaf protein in tobacco (Curtiss and Cardineau 1990). S. mutans is the main bacterial culprit of tooth decay, and immunization may ultimately prevent tooth decay. Other examples include the B subunit of the heat-labile enterotoxin of E. coli, which was expressed in transgenic potato (Haq et al. 1995). Mice that were fed with these potatoes developed the corresponding antibodies. Many more examples were reviewed by Ma and Vine (1999). However, plants are preferred for the treatment of humans, because they can be consumed in the form of raw fruits and vegetables, such as tomatoes or banana. The rabies-virus glycoprotein was successfully expressed in tomato, albeit at a low level of only 0.001% of the level of soluble protein (McGarvey et al. 1995). In addition, it is possible to express and assemble intact functional antibodies in transgenic plants; these antibodies could be used for passive immunotherapy. This was first demonstrated more than 10 years ago (Hiatt et al. 1989). Furthermore, a wide range of antibody fragments have been expressed in plants, including single-chain Fv (Owen et al. 1992) and Fab (De Neve et al. 1993) production in Arabidopsis and tobacco. Originally, only low levels of expression were observed but, by using an endoplasmic-reticulum retention signal (Schouten et al. 1996) or by expression in seeds (Fiedler et al. 1997), much higher expression was observed. An interesting example of a plant-expressed antibody is "Guy's 13", a murine immunoglobulin GI that binds to the previously mentioned adhesion protein of S. mutans. The antibody is expressed at high levels in tobacco and can easily be purified in large quantities (Ma et al. 1994). In a human trial, the "Guy's 13" antibody from plants prevented oral colonization by S. mutans (Ma et al. 1998). This was the first demonstration of a therapeutic application of plant-derived antibodies. 4 Current Use of Genetically Engineered Plants and Future Prospects An increasing number of field trials with transgenic plants have been performed. In 1986, only five such experiments were performed. In 1990, 120 were performed, and 332 were performed in 1993 (Ahl-Goy and Duesing 1995). By 1999, the number exceeded 9000. Field trials were performed not only with plants but also with animals and microorganisms (Fig. 1). As shown in Fig. lA, most experiments were conducted with GM maize (37.1%). Other species were potato (11.8%), oil- 128 Genetics A maize 37.4% potato 11 .9% other 4.4% alfalfa 1.0% melon 1.2% wheat 1.2% rice 1.3% sugar beet 3.0% oilseed rape 11 .0% soybean 8.7% tomato 8.2% cOlton 6.5% B herbicide & insect resistance 8.6% her bicide resistance 32 .5% herbicide & virus resistance 1.2% herbicide resistance & other tra its 3.5% herbicide resistance & male sterility 10.3% other 14.0% insect res istance 5.5% virus resistance 6.6% bacterial & fungal resistance 3.6% modified fatty acids 14.0% modified carbon hydrates 6.9% c other 4.7% Italy 1.2% Belgium 1.2% UK 1.3% Netherlands 1.5% Canada 8.5% France 3.4% Plant Biotechnology: Transgenic Crops for the Third Millennium 129 seed rape (10.9%), soybean (8.6%), tomato (8.1%) and cotton (6.4%). In addition, only a few trials were performed employing other species. Until 1999, the most frequently tested genetic trait was herbicide resistance (Fig. IB), often combined with other features, such as microbial resistance or male sterility. However, GM crops with introduced changes other than resistance genes will become more important in the near future. Most trials were performed in the USA (78.17%; Fig. lC). European countries have contributed only a few experiments. For example, 2.39% of all experiments were carried out in France, and less than 1% in Germany. Today, approximately 48 transgenic crops are commercially available worldwide. However, not all are approved by every country. Examples of GM crops approved by the European Union are shown in Table 4. Crops approved in the USA were listed in a review by Birch (1997). 5 Recent Controversies Regarding the Safety of GM Plants The safety of GM plants was analyzed extensively prior to commercialization by the respective companies, and approval was given by the appropriate governmental institutions; however, since 1998, the public has witnessed an ever-growing resistance to foods containing GM-plant products. In addition, the environmental safety of transgenic crops has been questioned. In this review, only two of these controversies can be addressed. On public television in 1998, the researcher Pusztai claimed that he had found evidence that transgenic potatoes expressing lectin would stunt the growth of rats. Ever since then, this program has been a source of continuing furor in Great Britain and everywhere else in Europe. The British Royal Society reviewed Pusztai's data and concluded that the study was based on poor experimental design and analysis (Loder 1999b; Masood 1999). In the meantime, Pusztai published his work (Ewen and Pusztai 1999). Notably, he no longer claimed that the growth of rats was impaired, but he still notes a side effect of the GM potato which may not be linked to the introduced lectin gene itself. Nevertheless, many Fig. 1 A-C. Field trials with genetically engineered plants. A Most frequently used plant species. B Most frequently used genetic traits. C Countries were the trials have been performed. The data shown in A and C are based on numbers for 1998 given by the Organization for Economic Cooperation and Development and the United Nations Industrial Development Organization. Numbers in B are from the European Union only Genetics 130 Table 4. European Union (EU) approved commercial transgenic crops. Data from June 1999; in addition, a number of proposals have been submitted but have not yet been approved Company EU country were the proposal was submitted Plant Genetic trait introduced Year of approval Seita France, 1993 Tobacco Herbicide resistance (bromoxynil) 1994 Plant Genetic Systems UK, 1994 Oilseed rape Male sterility and herbicide resistance (phosphinothricine) 1996 a Ciba Geigy France, 1994 Maize Insect resistance and 1997 herbicide resistance (phosphinothricine) Bejo Zaden B.V. Netherlands, 1994 Radicchio Insect resistance and 1996a herbicide resistance (phosphinothricine) Monsanto UK, 1994 Soybean Herbicide resistance (glyphosate) 1996 a Plant Genetic Systems France, 1995 Oilseed rape Male sterility and herbicide resistance (phosphinothricine) 1997 AgrEvo UK 1995 Oilseed rape 1998a France 1995 Maize Herbicide resistance (phosphinothricine) Herbicide resistance (phosphinothricine) Monsanto France, 1995 Maize Insect resistance 1998 Northrup UK, 1996 Maize Insect resistance 1998 a Florigene Europe B.V. Netherlands, 1996 Carnation Netherlands, 1997 Carnation Netherlands, 1997 Carnation Changed color of 1998 flower Prolonged flower life 1998 time Changed color of 1998 flower 1998 aRestrictions apply; for details, see http://www.rki.de/GENTEC/GENTEC.HTM. researchers criticized the paper for its faulty experimental approach (Enserink 1999). The second example to be discussed here is the "Monarch butterfly" controversy. In 1999, Losey et al. published a paper entitled "Transgenic Pollen Harms Monarch Larvae". To demonstrate the toxic effect of the Bt pollen, the authors applied pollen from Bt maize to milkweed (Asclepias curassavica), which is consumed by the Monarch butterfly (Danaus Plant Biotechnology: Transgenic Crops for the Third Millennium 131 plexippus). In this laboratory study, approximately 50% of the monarch butterflies died within 4 days. This study had a profound impact when reported by the press; it led to significant protests by environmentalists. Shelton and Roush (1999) replied to this paper with their own entitled "False Reports and the Ears of Man", stressing that the impact of Btmaize pollen on non-target insects is very low in the field. In fact, Btmaize fields contain even more insect species than their conventional counterparts (Shelton and Roush 1999). Both of the cases discussed above share scientific controversies, which in itself is not unusual. However, in a multimedia world, reports are often used by the press and published without proper background information. As is the nature of bad news, it is spread much more easily and, arguably (for obvious reasons), with a greater will. Consequently, at the end of the second millennium, a few, possibly poorly designed studies appear to have put the future of GM crops at stake. However, considering the tremendous advantages of GM crops, it is likely that the public will eventually realize the benefits that they provide. To achieve this goal, as was recently noted by Beachy (1999), the active participation of scientists is required. Thus, in addition to performing research, producing grant proposals and publications, and teaching students, the education of the public may necessarily become a higher priority for scientists during the third millennium. This will be necessary in order to underwrite an appropriately informed level of biological knowledge among the general population. 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Esser Assistant Professor Frank Kempken Lehrstuhl ffir Allgemeine und Molekulare Botanik Ruhr-Universitat Bochum 44780 Bochum, Germany e-mail: [email protected] Tel.: +49-234-3222465 Fax: +49-234-3214184 Genetics Modification of Oilseed Quality by Genetic Transformation Steffen Weber, Karim Zarhloul, and Wolfgang Friedt 1 Introduction Oil crops, like oilseed rape and sunflower, are important sources of energy, both for human consumption and for feeding livestock. They are also raw materials for a wide range of industrial products for many nonedible purposes. Modification of the fatty-acid composition to make oil crops more competitive in various segments of the food and industrial oil markets has recently been an important objective of molecular genetics and plant breeding. Consequently, one of the most important objectives of oilseed breeding is the genetic modification of seed storage oil by maximizing the proportion of specific or functional fatty acids in order to obtain tailor-made raw materials suitable for various industrial purposes. However, the quality of vegetable food products has acquired an increased relevance for human nutrition with the advent of so-called functional food. Regarding the specific properties of such nutritives, genetic engineering has the ability to adapt plant storage lipids to meet specific nutritional and even therapeutic requirements. Oilseed rape (Brassica napus 1.) has recently become the world's third most important annual crop for vegetable oil production due to substantial progress in breeding and cultivation practices. Rapeseed oil is unique in having a large spectrum of uses and good properties for food and non-food applications. Regarding genetic engineering, oilseed rape is easy to handle in tissue-culture systems; this has allowed the establishment of a number of well-documented gene-transfer protocols. Thus, oilseed rape has been the subject of numerous attempts to alter the biochemical pathways of storage-lipid biosynthesis. Genetic engineering of plant lipid biosynthesis in rapeseed has already led to commercialization; since 1995, transgenic varieties with genetically modified fatty-acid patterns have been released. In sunflower (Helianthus annuus 1.), hybrid varieties are almost exclusively used for commercial oilseed production. Native sunflower oil is mainly used for human consumption, because it contains a large amount of essential linoleic acid (006 CI8:2), which gives sunflower-seed oil a high nutritional value. In addition, "high-oleic" varieties have been creProgress in Botany, Vo!' 62 © Springer-Verlag Berlin Heidelberg 2001 Modification of Oilseed Quality by Genetic Transformation 141 ated via mutagenesis; oleic acid «(09 CI8:!) accumulates to 85% in the seed oil. This oil can be used for oleochemistry, because it offers a broad range of applications. However, the natural variation of important agronomic traits, such as resistance to pathogens and stresses, is rather limited (although some progress has been achieved in broadening the genetic variation and improving the currently available germ plasm via interspecific hybridization). Clearly, the application of genetic engineering would increase the number of possible ways to improve the potential of the crop. In contrast to rapeseed and soybean, an efficient transformation system is lacking for sunflower due to the recalcitrance of the plant to tissue-culture techniques suitable for gene transfer. Therefore, to a large extent, efforts to transform sunflower still address the improvement and optimization of the methods of regeneration and gene transfer. Nevertheless, transgenic sunflowers have already been released in the field. Genetic engineering is considered a powerful tool for practical plant breeding, because the transfer of specific traits to a target genotype is possible without changing the phenotype and agronomic performance of the recipient. The isolation of a majority of genes encoding enzymes of storage-oil synthesis made this basic metabolism one of the first targets of gene transfer to plants. This review highlights both the achievements made and the impediments observed with regard to the modification of oilseed quality via genetic transformation. In order to give an overview of the technical aspects and efforts (ranging from the regeneration of fertile plants from tissue cultures to the final selection and field testing of the transform ant with the desired phenotype), they are discussed for the two oilseed crops oilseed rape and sunflower. These differ with regard to their amenability to gene transfer. 2 Biosynthesis and Composition of Vegetable Oil Vegetable oils consist of triacylglycerols (TAGs), which are, at the cellular level, deposited and stored in oil bodies until germination, at which time they are catabolized as an energy source. TAGs chemically represent esters of glycerol and fatty acids, where the latter determine the physical and chemical properties of the lipid molecule via their chain lengths and the presence or absence of double bonds and/or functional groups. Distinct oils and fats are characterized by their specific fattyacid composition, which determines their usefulness for both food and non-food applications. The biochemical pathways of plant lipid synthesis are well understood and have been comprehensively reviewed by a number of authors (Ohlrogge et al. 1991; Topfer et al. 1995; Murphy 1999). ~I Capric IC Myristic IC 14 ,o Lauric ICIa 10 :0 Caprylic [ C~:O +4U·g- C 1S;O C1a ;o Cwo Caprylic Capric Lauric Myristic Palmitic Stearic C2. :1 Nervonlc IC1e:o Palmitic - J m /}) Acyl-CoA Pool Membrane synthesis If. """ / / / C1a: 1-OH Ricinoleic (TAGs) ~ Fig. 1. Plant storage-lipid biosynthesis. Substrates and metabolites are shown in white rectangles, enzymes are black ellipses, coenzyme A-activated fatty acid residues are gray rectangles Cytosol :rl' Ca:o C 10;O C I2 ;O Triacylglycerols '" ~. ::l ~ Cl tv ~ - Modification of Oilseed Quality by Genetic Transformation 143 We will now give a short overview of the most important steps of plant-lipid biosynthesis and the major enzymes involved in this basic metabolic pathway. In plants, consecutive steps of de novo fatty-acid biosynthesis take place in different cell compartments (Fig. 1). Fatty-acid synthesis starts in the stroma of the plastids, where malonyl-coenzyme A (CoA) is synthesized from acetyl-CoA and carbonate by an acetylCoA carboxylase. Condensing enzymes [ketoacyl acetyl carrier protein (ACP) synthases; KASs], as part of the fatty-acid synthase multi-enzyme complex, elongate the initial malonyl-CoA consecutively by adding C2 units derived from malonyl-CoA with the release of CO 2, During this process, the growing fatty-acid chain is bound to an ACP. After seven condensation cycles, a C16 acyl thioester (palmitoyl-ACP) is elongated (by another specific KAS) to stearoyl-ACP, which then is desaturated to oleoyl-ACP by the action of a ~9-stearoyl-ACP desaturase. The three latter acyl residues are released from ACP via hydrolization by acyl-ACP thioesterases (TEs), resulting in palmitic (CI6:0), stearic (CI8:0) and oleic (CI8:1) acids as the primary fatty acids. Acyl-ACP TEs are grouped by their evolutionary origin into FatA and FatB TEs. The FatA types have unsaturated acyl groups as substrates, while FatB types prefer saturated acyl-ACPs (Jones et al. 1995). In certain plant species (Cuphea sp.), specific FatB TEs terminate the chain length of storage-lipid fatty acids by hydrolyzing acyl-ACPs before they reach a length of 18 carbons, resulting in short- to medium-chain fatty acids: capric (C8:0), caprylic (10:0), lauric (CI2:0) and myristic (C14:0) acids. However, fatty acids hydrolyzed from ACP are exported into the cytoplasm, where they are activated with CoA by a membrane-bound acyl-CoA synthase In some plant species, such as Brassica sp., oleoyl-CoA is subject to further elongation, which results in long-chain fatty-acid residues [eicosenoyl (C20:1), erucoyl (C22:1) and nervonoyl (C24:1)-CoA] that complete the acyl-CoA pools of the respective plant species (Fig. 1). At the endoplasmic reticulum (ER), the assembly of TAGs is catalyzed by acyltransferases, which convert CoA-activated acyls and glycerol-3-phosphate into TAGs with the release of inorganic phosphate. In the first step, lysophosphatidic acid (LPA) is formed by glycerol-3-phosphate acyltransferase, which is then converted into diacylglycerol (DAG)-phosphate by the action of a substrate-specific LPA acyltransferase (LPA-AT). The substrate specificity of LPA-AT results in the occupation of the central sn2 position of the resulting TAG by a specific fatty acid, which has an impact on the fatty-acid pattern of the seed oil. For the complementation of TAG synthesis, the phosphate group bound to the DAG is released by phosphatidic acid phosphatase prior to the conversion of the DAG into the final TAG by DAG acyltransferase (DAG-AT), the only enzyme unique to storage-lipid synthesis (Murphy 1999). Alternatively, DAG can be reversibly converted to phosphatidyl choline (PC; Fig. 1), which may be used in membrane lipid synthesis or as a substrate of membrane-bound desaturases, which then de-saturate the acyl residues at positions ~9, M2 and M5, or ~6. Additionally, in some plant species, functional groups, e.g., hydroxyl residues in castor seeds (Ricinus communis 1.), are added to the PC-bound fatty-acid residue prior to de-saturation. Due to acyl exchange, PCs with such altered fatty-acid residues can then be re-converted into various DAGs that complement the substrate pool for DAG-AT used in the TAG synthesis of storage lipids (Fig. I). At present, a wide range of various genes encoding enzymes involved in plant storage-lipid synthesis have been isolated and cloned (Martinez de Ilarduya et al. 1999; Mekhedov et al. 1999) and thus are available to genetically engineer the fatty-acid composition of seed oils. In the following discussion, we will focus on the progress already achieved and the state of the art concerning genetic engineering of storage lipid biosynthesis of two major oil crops, rape seed (B. napus L.) and sunflower 144 Genetics (H. annuus 1.), by specifically highlighting recent results obtained in our working group. 3 Regeneration Capacity of Oilseed Plants The regeneration of fertile plants from explants in vitro is the most important prerequisite for stable genetic transformation. In any genetransfer system, single cells are transformed; they subsequently have to be selected and regenerated into fertile plants to allow transmission of the transgene to progenies. Regeneration systems that allow selection at a very early stage of tissue culture are most favorable. This is particularly true for indirect regeneration pathways that include a callus phase allowing selection at the level of cell proliferation from explants rather than fast, direct regeneration pathways where shoots regenerate quickly and, thus, often escape the selection process. A number of selectable marker genes are available for selection purposes (Wilmink and Dons 1993; Metz and Nap 1997). In contrast, efficient regeneration systems meeting the requirements for the successful establishment of transgenic plants are restricted to a limited number of species and, therefore, represent the dominant difficulty for most crop species. Therefore, in the following discussion, we will mainly devote our attention to regeneration from explants due to gene transfer. We will compare oilseed rape, which is comparatively easy to regenerate via a broad range of tissue-culture techniques, and sunflower, a species known to be recalcitrant to the application of in vitro cultures. a) Oilseed Rape (B. napus 1.) Because rapeseed is easy to handle in various tissue-culture systems it is highly suitable for recombinant DNA technology. Nevertheless, factors controlling regeneration in vitro have to be optimized when establishing tissue-culture protocols. The impact of the most important factors on shoot -regeneration efficiency in B. napus and related species will be discussed with regard to genetic transformation. The genotype dependence of tissue-culture systems is a well-documented phenomenon in all plant species that have been subjected to in vitro cultures. For Brassica species, this has been observed, for example, in B. carinata (Barro and Martin 1999), B. oleracea (Henzi et al. 1999) and B. campestris (Lim et al. 1998). In oilseed rape (B. napus), genotypic effects have been reported for almost all tissue-culture systems, e.g., hypocotyl (Damgaard et al. 1997), cotyledon (Ono et al. 1994), microspore (Weber et al. 1995) and protoplast cultures (Parihar et al. 1995). In our own investigations with hypocotyl explants as the target tissue for Agrobacterium-mediated transformation, we also observed a strong influence of the genotype. For genetic transformation and the subsequent selection steps, the method of De Block et al. (1989) was applied with minor modifications 145 Modification of Oilseed Quality by Genetic Transformation Table 1. Regeneration of kanamycin-resistant shoots from B. nap us genotypes "RS306" and "Drakkar" after co-cultivation with Agrobacterium tumefaciens strain GV3101, which bears the binary vector pASBnDES1 affected by antibiotics to eliminate Agrobacterium tumefaciens. Data are from two independent experiments. Experiment I consisted of six replications comprising 200 hypocotyl explants (eight dishesx25 explants); experiment II consisted of five replications of 225 co-cultivated explants Regeneration Factor Genotype Antibiotic RS306 Betabactyl Drakkar Betabactyl Betabactyl Carbenicillin Experiment I II II Shoots/replication Frequency (%) 13.6 6.88 1.5 1.4 2.6 0.8" 0.6 1.2 8Significant difference between the two genotypes (P=0.05). (Zarhloul et al. 1999a). We co-cultured etiolated hypocotyl segments of the B. napus genotypes "Drakkar" (spring type) and the re-synthesized rapeseed line "RS306" (winter type) using the Agrobacterium strain GV3101IpMP90RK. The results of the experiment revealed a significant difference between the two genotypes regarding their regeneration response after co-cultivation with A. tumefaciens (Table O. However, a broad variation in regeneration efficiency was found in "RS306", ranging from 1% to 14% in six replications. The frequency of shoot regeneration from "Drakkar" was significantly lower and did not exceed 3%, although this cultivar is known for its superior transformation and regeneration responses (Schaffert et al. 1996). Consequently, different genotypes are variably suitable under different culture conditions. The supplementation of the culture medium with plant hormones is indispensable to obtain plant regeneration; the concentration and type of phytohormone have been shown to be important factors influencing the frequency of regeneration from various explants. Takasaki et al. (1996) stated that 6-benzylaminopurine (BAP) and unaphthaleneacetic acid (NAA) were indispensable for shoot regeneration from B. campestris cotyledons. In hypocotyl explants, different cauliflower (B. oleracea var. botrytis 1.) genotypes had varying reactions to BAP concentrations ranging from 1 mg/I to 5 mg/I (Ding et al. 1998). In our investigations, we had similar results. With an optimized transformation protocol of De Block et al. (1989), we investigated the regeneration of potentially transformed plants from hypocotyl segments co-cultivated with agrobacteria using "Drakkar" and the line "RS306" on AS media differing in their concentrations of the supplemented plant hormones BAP and NAA. The genotypic response was modified by the level of the respective phytohormone and by the BAP:NAA ratio (Zarhloul et al. 1999b). For "Drakkar", the frequency of regeneration was increased by augmentation of the NAA concentration from 0.1 mg/I to 0.5 mg/I, irrespective of the BAP concentration (Fig. 2). In contrast, "RS306" shows a very variable response to different combinations of BAP and NAA. The highest regeneration frequency, with up to 70% regenerating explants, was obtained when 3-5 mg/I BAP were supplied together with 0.3 mgll NAA (Fig. 2). The use of silver nitrate (AgN0 3 ) as an ethylene inhibitor is reported to be a prerequisite for plant regeneration in tissue cultures (Burnett et al. 1994; Pua et al. 1996; Zhang et al. 1998; Kuvshinov et al. 1999), because ethylene is considered to be involved in plant morphogenesis in vitro. Sealing the culture containers with porous paper tape instead of parafilm decreases the ethylene concentration and humidity during culture, which leads to better shoot regeneration. Usage of porous paper tape in combination Genetics 146 Regenerated plantlets per explant plated (%) -------------------A----------~__- - - _ _ _ l D 80 • 70 60 Escape Positive < 50 30 20 10 o RS 306 1 : 0.1 3 : 0.3 5 : 0.1 3 : 0.1 1 : 0.3 1 : 0.5 5 : 0.3 5 : 0.5 3 : 0.5 BAP NAA (mgll) Fig. 2. Influence of the hormone composition on the regeneration and transformation of the spring B. napus cv. "Drakkar" and re-synthesized rapeseed line RS306 after cocultivation with Agrobacterium tumefaciens with AgN03 increased the regeneration from 0-5% to 80% in B. rapa, which is known to be rather recalcitrant to tissue-culture techniques. As a consequence, the transformation efficiency also was increased (Kuvshinov et al. 1999). The optimum concentration for shoot regeneration is 5-10 mg AgNOil. It is interesting that supplementation of the media with AgN0 3 first leads to an increase of ethylene production (Zhang et al. 1998); this is thought to be due to stress induced by silver (Pua and Chi 1993). However, silver ions inhibit the ethylene action by competitively binding to ethylene receptors (Beyer 1976); thus, they interrupt the ethylene signal-transduction pathway (Zhang et al. 1998). In transgenic Brassica plants, evidence of the ethylene regulation of in vitro shoot regeneration has been described by introducing an anti-sense oxidase gene (Pua and Lee 1995). High agar concentrations prevent the formation of ethylene, leading to a better regeneration of shoots (Zhang et al. 1998). In contrast, a very high concentration of gelling agent in the media decreases the water potential, which results in poor delivery of water, nutrients and growth regulators to the explant and, therefore, causes a very low regeneration efficiency (Zarhloul, unpublished data). Antibiotics used to eliminate agrobacteria after the co-culture step can further influence the regeneration response of the co-cultivated explants, because they decrease the frequency of shoot differentiation, especially if kanamycin is used as a selective agent (Ling et al. 1998). It is reported that Betabactyl (ticarcillin potassium clavunate), unlike the widely used carbenicillin and cefotaxime, is light stable and is resistant to inactivation by J3-lactamase (Ling et al. 1998). However, it is recommended that one use carbenicillin, which prevents the medium from turning brown and eliminates the toxic effects of the prolonged use of AgN0 3 on plant tissue (De Block et al. 1989). Therefore, we have conducted an experiment to investigate the influence of carbenicillin and Betabactyl on shoot regeneration. The results show a clear difference (based on the number of regener- Modification of Oilseed Quality by Genetic Transformation 147 ants); however, the difference is not significant, due to the small number of regenerants obtained (Table 1). In conclusion, pronounced interactions of the genotype with culture conditions have to be taken into consideration when establishing a transformation system depending on tissue cultures, even in B. nap us, because any given genotype requires specific optimal culture conditions. b) Sunflower (H. annuus) During recent decades, many attempts to establish efficient tissueculture techniques for various tissues of sunflower suitable for rapid propagation, screening in vitro or gene transfer have been made (Friedt 1992; Alibert et al. 1994; Hahne 1994; Hahne 2000). However, of all tissues tested, only three explant sources meet requirements regarding efficiency and reproducibility: immature zygotic embryos (IZEs), cotyledons of young seedlings and apical meristems of young seedlings (shoot tip explants). These explant sources share direct and fast regeneration pathways without an intermediate callus phase; thus, they are problematic regarding the selection of transformed cells (Hahne 2000). While these explants regenerate plants via direct morphogenesis, regeneration from apical meristems also takes place via multiple shoot formation from the meristematic tissue. Regarding mature cotyledons, regeneration from this type of explant was obtained via somatic embryogenesis (Fiore et al. 1997; Gurel and Kazan 1999) and direct organogenesis (Sarrafi et al. 1996; Nestares et al. 1998). However, the choice of genotype plays an important role, because the organogenic potential of cotyledon explants has been shown to be under additive genetic control in sunflowers (Deglene et al. 1997; Berrios et al. 1999) and can also be influenced by nucleus-cytoplasm interactions (Nestares et al. 1998). Regarding its usefulness for Agrobacterium-mediated transformation, this explant suffers from the fact that most of the regenerated shoots originate from cells located in the palisade parenchyma of the cotyledon, which is not accessible to agrobacteria (Burrus et al. 1993; Laparra et al. 1995). Therefore, this explant is more suitable for the use of the biolistic approach, discussed below. Sunflower IZEs have been the subject of various studies where the influences of different factors on plant regeneration were examined. This explant type regenerates plants via both somatic embryogenesis (Jeannin et al. 1993) and direct organogenesis (Jeannin et al. 1995, Ivanov et al. 1998), depending on the osmotic pressure. The osmotic pressure is provided by the sucrose content of the induction medium, which affects endogenous levels of phytohormones (Charriere and Hahne 1998). Although genotypic effects are reported using IZEs, fertile plants have been obtained from a broad range of genotypes. However, this explant is laborious to obtain, and the regenerable area shared by both morphogenic pathways is restricted to a small number of cells at the junction of the hypocotyl and radicula. As a consequence, Laparra et al. (1995) did not derive transformed plantlets by subjecting IZEs to both Agrobacterium-mediated and biolistic transformation. However, a conference abstract indicates progress in this area (Milller et al. 1998). 148 Genetics The use of shoot apical meristems or embryonic axes for Agrobacterium-mediated transformation is a method proposed earlier for plant species recalcitrant to tissueculture techniques, such as soybean (Christou and McCabe 1992), lupins (Molvig et al. 1997), chickpea (Kar et al. 1996) and peanut (Heatley and Smith 1996). The shoot apical meristem has been widely used for sunflower transformation and represents the only type of explant that has allowed the recovery of stable transformants (MaloneSchoeneberg et al. 1991; Knittel et al. 1994; Burrus et al. 1996). However, Knittel et al. (1994) concluded that the improvement of the transformation efficiency requires higher percentages of adventitious shoot formation de novo. In addition, Burrus et al. (1996) showed that shoots do not originate from a single transformed cell by analyzing transformation patterns of transgenic shoots emerging from apical meristems. They showed that such shoots originate from transformation events that occur within the meristem. Therefore, the transformation efficiency of shoot apices might be enhanced using genotypes that regenerate both types (meristem-derived and adventitious shoots) at a reasonably high frequency. It has been reported repeatedly that wild Helianthus species seem to have a higher regeneration potential in vitro than the cultivated sunflower (Friedt et al. 1997; Nurhidayah et al. 1997). Therefore, it is possible that the use of interspecific hybrids helps to elevate the transformation efficiency in sunflowers. Regarding Agrobacterium-mediated transformation, we screened interspecific hybrid progenies previously derived by embryo rescue (Krauter et al. 1991) for their ability to regenerate shoots from apical meristems (Weber et al. 2000a). Because of the high proportion of alien genome in young generations of interspecific hybrids (Rieseberg et al. 1996) and the hypothesis that selection during the domestication process may have led to a loss of genes necessary for the in vitro regeneration of cultivated sunflowers (Alibert et al. 1994), we determined that progenies of interspecific crosses in early generations are the most promising material with which to examine the regeneration potential in vitro. Agronomic characterization 4.0 Sh 0015 IE xplanl I Origin of Shoots 3.5 D 3.0 merislematic de novo 2.5 2.0 1.5 mean 1.0 05 0 - I - I -- - -- - I - - - I I. - Fig. 3. Regeneration from shoot tip explants of interspecific hybrid sunflowers. Progenies of interspecific hybrids are indicated by the wild parent of each cross combination (Weber et al. 2000) Modification of Oilseed Quality by Genetic Transformation 149 and evaluation of such progenies should provide seed stocks that combine sufficient agronomic performance and a high regeneration potential in vitro for use in breedingoriented research (Weber et al. 2000a). The interspecific hybrids used in our study revealed a fairly large variation of regeneration potential among (and, to some extent, within) single cross combinations. Nine lines responded better than the commercial hybrid cultivar "Albena", and 50% of the interspecific progenies were superior to the parental inbred lines "HA89" and "Baso" regarding this trait. Progenies of crosses of the commercial line "HA89" with the wild species H. decapetalus, H. giganteus, H. strumosus and H. mollis had the best results. Regarding the origin of the shoots, interspecific hybrids exhibiting a superior ability to regenerate shoots de novo were identified (Fig. 3). We assume that the superior potential to regenerate shoots is due to the introgression of "alien" alleles into the cultivated sunflower. Obviously, high potential for regeneration in vitro can be found in various interspecific hybrid combinations, but not in each progeny (Weber et al. 2000a). This is in accordance with results obtained by other groups; organogenesis in mature sunflower cotyledons is under additive genetic control (Deglene et al. 1997; Berrios et al. 1999). In addition, the regeneration potential of progenies derived from crosses of high-oleic inbred lines with interspecific hybrids was investigated. Genotypes originating from crosses to the wild parents H. giganteus and H. tuberosus exhibited a superior ability to regenerate shoots in vitro in relation to adapted, inbred lines and commercial hybrids, which served as controls (Weber, unpublished data). Interspecific hybrid progenies selected for their superior regeneration potential represent a promising starting material for transformation experiments employing shoot apical meristems, because they deliver meristem-derived and adventitious shoots at a high frequency. 4 Tools for Genetic Transformation of Oil Plants For plant transformation, two approaches are the most promising: Agrobacterium-mediated transformation (Horsch et al. 1985) and direct gene transfer using the bombardment of intact plant tissues by DNA-coated microprojectiles (biolistic approach) (Klein et al. 1992). For a more detailed discussion of various transformation techniques and specific aspects of the two dominant methods, see the reviews by De Block (1993), Klein and Zhang (1994), Walden and Wingender (1995), Christou (1995, 1997), Luthra et al. (1997), Furth (1997) and Birch (1997). Prior to the regeneration of fertile plants from transformed cells, which has been shown to be a critical step, the transfer of DNA into the host cell and the subsequent proper integration of the transferred sequences in the genome have to be performed by choosing the gene-delivery method. Therefore, in the following discussion, we will discuss the development of transformation strategies regarding oilseed rape, which is now transformed routinely, and sunflower, a species that still faces a number of impediments to transformation. 150 Genetics a) Biolistic Approach The biolistic approach was developed for plant species that defied efforts to genetically transform them, Le., legumes, or that are not susceptible to infection with A. tumefaciens, i.e., monocots (including cereals; Christou 1995; Jaehne et al. 1995). In fact, a wide range of target explants were used, including intact leaves (Schaeffer et al. 1995), immature zygotic embryos (Fitch et al. 1990; Hunold et al. 1995; Lappara et al. 1995), somatic embryos (Fitch et al. 1990; Scorza et al. 1995), hypocotyl sections (Fitch et al. 1990, 1992) and microspores (Jaehne et al. 1994; Harwood et al. 1995). Although very high transformation frequencies (up to 26%) can be achieved after bombarding highquality tissues, e.g., in oats (Avena sativa L.; Cho et al. 1999), as a means of direct DNA delivery, the biolistic approach often results in multiple and linked integration events (Pawlowski and Somers 1996). These require the selection of transform ants containing a limited number of trans gene copies (De Block 1993). However, in rice (Oryza sativa 1.), it was recently shown that such multiple-copy events are made up of single transgenic loci formed due to a "two-step integration" of the foreign DNA, with the establishment of "integration hotspots" (especially due to particle bombardment; Kohli et al. 1998). Other features often considered disadvantages of direct gene-transfer methods in general are arbitrary or imprecise integration and the integration of non-functional or "junk" DNA, i.e., vector sequences (De Block 1993). The inactivation of gene expression due to nucleicacid interactions (Matzke and Matzke 1995) is often discussed in context with multiple gene copies. However, none of these shortcomings leads to decreased transgene expression following particle bombardment (compared with Agrobacterium-mediated transformation), because biolistics is a powerful tool for many plant species that cannot be transformed by A. tumefaciens (Christou 1995). In oilseed rape and other Brassica species, the biolistic approach has not played an important role, due to the feasibility of the highly efficient Agrobacterium-mediated transformation (Poulsen 1996). Efforts to establish or improve biolistic transformation in rapeseed are scarce, because the overall transformation efficiency does not equal that of the Agrobacterium approach. Therefore, particle bombardment in Brassica was mainly used to study and characterize promoters and genes via transient-expression assays (White et al. 1994; Kost et al. 1996). Chen and Beversdorf (1994) reported an improved transformation efficiency of 2% by combining microprojectile bombardment and desiccation/DNA imbibition in a transformation that utilized microspore-derived embryo hypocotyls. More recently, using biolistics, Fukuoka et al. (1998) developed a transformation procedure for microspores. This approach was remarkable, because all the offspring of primary transformants expressed the transgene, indicating complete homozygosity for the transgene. In sunflowers, Bidney (1990) first used particle bombardment on shoot apices. Although transient expression was obtained, no fertile plants could be regenerated. The same was the case for the investigations of Hunold et al. (1995), who used immature zygotic embryos and mature cotyledons as target explants for biolistic gene transfer. In a histological study, Hunold et al. (1993) showed that transient expression is often due to dying cells. Laparra et al. (1995) conducted a comparative study with different approaches to sunflower transformation. Regarding the highly regenerative cotyledon explants, it has been mentioned already that regeneration occurs directly from a small number of cells located in sub-epidermal layers in the palisade parenchyma (Burrus et al. 1993); thus, they are hardly accessible to agrobacteria (Laparra et al. 1995). In order to circumvent this barrier, Vischi et al. (1996) bombarded cotyledon explants with DNAcoated gold particles. Regenerants that exhibited amplification products specific for the transferred DNA were obtained after polymerase chain reaction (PCR) analysis. How- Modification of Oilseed Quality by Genetic Transformation 151 ever, this approach did not result in a reviewed publication. More recently, the authors reported the utilization of a focussing device for the particle gun to direct and concentrate the particles to the regenerable area, which is located close to the embryonic axis (Vischi et al. 1999). We established the biolistic transformation of sunflower cotyledons as a putative alternative to Agrobacterium-mediated shoot-tip transformation. Preliminary results employing the transient expression of 13glucuronidase (GUS) and the green fluorescent protein (GFP) indicate that, in addition to the appropriate preparation of the particle delivery device (PDS-I000/He Particle Delivery System, Bio-Rad Laboratories), the physical state of the target explant is a crucial factor. This is because transient GUS expression strongly depends on the duration of the preculture period. However, using a conventional set up, we observed shoots that expressed GUS in a chimeric fashion 4 weeks after bombardment (Weber et al. 2000b). Very recent results obtained by transiently expressing GFP indicate that the focussing device described by Vischi et al. (1999) represents a powerful accessory that directs particles to the regenerable area of the sunflower cotyledon explant. Although the utilization of biolistics have yielded no published results regarding stable sunflower transformation, this approach merits further research because of the difficulties of Agrobacterium-mediated transformation of sunflowers, discussed below. b) Agrobacterium-Mediated Transformation Agrobacterium-mediated transformation is the most widespread method of plant transformation, especially for dicot species, due to the benefits of this transformation strategy. It is simple, efficient, inexpensive and, in contrast to the direct DNA-delivery systems, allows precise integration of the DNA of interest, which is clearly defined by the T-DNA border sequences. This is achieved by the complex "natural" mechanism of gene transfer to plant cells, which is mediated by agrobacteria (Zupan and Zambryski 1995; Sheng and Citovsky 1996; Christie 1997; De la Riva et al. 1998). Another advantage of the Agrobacterium-mediated transformation (as compared with direct gene transfer) is the limited number of integrated transgenes that can be linked or unlinked, allowing subsequent segregation via sexual recombination (De Block 1993). a) Oilseed Rape There is a large number of factors that influence transformation efficiency using A. tumefaciens. The factors can be divided into those that influence the gene transfer and those that influence the regeneration of 152 Genetics transgenic plants from the transformed cell. The first problem mostly arises if one of the following prerequisites are not fulfilled: successful bacterial colonization, induction of bacterial virulence, generation of the T-DNA transfer complex, T-DNA transfer and the integration ofT-DNA into the plant genome (De la Riva et al. 1998). The latter represents a tissue-culture aspect and depends on a range of factors: selectable markers, antibiotics for agrobacteria elimination, culture conditions (light intensity, sub-culture interval, concentration and combination of the applied growth phytohormone, etc.). The culture conditions have to be arranged and optimized with regard to the genotype and plant species used in order to recover transgenic plants. A large number of studies describing various transformation protocols for oilseed rape have been published. In these studies, all possible factors eventually influencing the transformation and regeneration efficiency were investigated. The first transformation in the genus Brassica was conducted with the aim of investigating the virulence of different Agrobacterium strains (Holbrook and Miki 1985; Doms et al. 1985). The most virulent agrobacteria for B. napus were nopaline strains, followed by succinamopine strains; the efficiency of octopine strains was very weak (Charest et al. 1989). A recent study in which different A. tumefaciens strains were compared in terms of their transformation efficiency in B. rapa showed that the best results were achieved with the nopaline strain LBA4404 (pAL4404), which harbors a Ti plasmid derived from the native octopine strain pTiAch5 (Kuvshinov et al. 1999). This result is in accordance with findings published by Charest et al. (1989) and indicates an interaction between the chromosomal background of the Agrobacterium strain and the helper plasmid. The Agrobacterium-mediated transformation and regeneration abilities of a large number of tissue-explant sources from B. napus and related species (B. campestris, B oleracea) were investigated. Using A. tumefaciens-mediated gene transfer, it was possible to obtain plants from various explant types from the genus Brassica: hypocotyls (Radke et al. 1988; De Block et al. 1989; Damgaard et al. 1997; Babic et al. 1998; Bhalla and Smith 1998; Ding et al. 1998; Cao et al. 1999; Paul and Sikdar 1999), cotyledonary petioles (Moloney et al. 1989; Jun et al. 1995; Damgaard et al. 1997; Bhalla and Smith 1998; Babic et al. 1998), thin cell layers (Charest et al. 1988), stem segments (Fry et al. 1987; Kuvshinov et al. 1999), pro-embryos (Pechan 1989) and petioles (Cao et al. 1999). However, hypocotyles and cotyledons are still the most widely used explants. Although stable transformation was achieved for both explant types, the efficiencies differ among the species. Cotyledon petioles responded better than hypocotyl explants in six B. napus winter cultivars, because the use of cotyledon petioles resulted in successful transformation with all cultivars tested, whereas hypocotyl segments were successfully employed only for one cultivar (Damgaard et al. 1997). Similar results have been obtained for B. carinata (Babic et al. 1998). The suitability of the target explant for transformation also depends on the cultivar. Bhalla and Smith (1998) have demonstrated that regeneration from hypocotyls is rapid, with a higher frequency than with cotyledons. An evaluation of explant sources showed that cotyledons were the most promising targets for the transformation of cauliflower (B. oleracea var. botrytis). However, interactions between the genotype and the explant source were observed (Ding et al. 1998). Selection for transformed cells requires a selectable marker gene, which is introduced together with the gene of interest, enabling only transformed cells to grow and to form plants. The use of the selective agent has to be optimized to inhibit regeneration from non-transformed tissue, which results in the recovery of non-transgenic plants (so-called escapes). The most popular selectable marker in plant transformation is the neomycin Modification of Oilseed Quality by Genetic Transformation 153 phosphotransferase gene (nptII), which confers resistance to various aminoglycosides, such as kanamycin, gentamycin and paromomycin. However, the efficiency of selection using the nptII gene as a selectable marker is very variable in Brassica. Depending on the aminoglycoside used for selection, Kuvshinov et al. (1999) observed a rate of 90% escapes under kanamycin selection in B. rapa, compared with 10% under hygromycin. However, transgenic shoots were regenerated from 4-9% of the explants plated, irrespective of the selective agent applied. Comparable results have been obtained in B. juncea (Paul and Sikdar 1999). Another highly efficient type of selectable marker is that conferring resistance to herbicides. The most popular marker in this group is the bar gene, which confers resistance to phosphinotricin (De Block et al. 1989). Regarding the selection efficiency, Babic et al. (1998) have observed that, in a medium with phosphinotricin, selection was more stringent than selection with kanamycin; however, shoots regenerated at a lower frequency. The authors concluded that phosphinotricin probably kills un-transformed tissue, and the subsequent release of toxic substances may inhibit the regeneration of transformed tissue. In B. oleracea, a transformation efficiency of up to 35% was obtained by employing A. rhizogenes as a vector (Henzi et al. 1999). In B. oleracea and B. campestris, transformation rates exceeding 15% were reported using rhizogenes-mediated transformation (Christey et al. 1997). These results demonstrate that, with A. rhizogenes, high transformation efficiencies can be achieved. However, such transgenic plants may exhibit an altered phenotype (reduced fertility, wrinkled leaves, reduced apical dominance, shortened internode, late flowering and plagiotropic roots) due to the expression of rolloci (Christey et al. 1997). However, segregation of the rol gene from the gene of interest can be achieved in subsequent generations by recombination to obtain phenotypically normal transgenic plants. In Section 3.a, it was demonstrated that the genotype has a strong influence on the regeneration frequencies of various tissue-culture techniques. This is also true for transformation. We reported above that the rape cultivars "Drakkar" and "RS306" differ in their regeneration response after co-cultivation with A. tumefaciens (Table 1). We have also observed that an augmentation of phytohormone concentration allowed sufficient regeneration in "Drakkar" and a very high regeneration frequency in "RS306" (Fig. 3). In addition, an increase of phytohormone concentration had a strong influence on the transformation frequency. For "Drakkar", the highest transformation frequency (9911 of explants regenerating transformed shoots) was achieved by applying 5 mg BAP/I together with 0.5 mg NAAn, which is the hormone combination at which the best regeneration rate was achieved. In "RS306", a transformation rate of 34% was obtained by the phytohormone combination 3 mg BAP/I plus 0.5 mg NAAlI, which indicates a very high susceptibility of this genotype to A. tumefaciens infection and an outstanding ability to regenerate transformed plants from cells to which the gene was transferred. Furthermore, the highest frequencies of transformation were achieved when high concentrations ofNAA were present (Fig. 3). "RS306" has also shown an excellent regeneration response using Agrobacterium-mediated transformation in combination with other gene constructs (Weier et al. 1997; Zarhloul et al. 1999a). However, these results clearly demonstrate that the genotype strongly influences the response in terms of obtaining transformed plants in rapeseed. Furthermore, the genotype interacts with conditions present during the subsequent subculturing step, i.e., the supplementation ofphytohormones. 154 Genetics The results of Table 1 and Fig. 2 indicate the importance ofthe genotype, the culture conditions after transformation and the media supplements; this is in accordance with other publications (Ono et al. 1994; Poulsen 1996; Takasaki et al. 1996; Zhang et al. 1998). Consequently, any given transformation protocol has to be adapted to the genotype employed to allow the regeneration of a sufficient number of transgenic plants. 13) Sunflower As early as 1946, sunflowers were shown to be susceptible to A. tumefaciens infection (De Ropp 1946). During the 1980s, Agrobacteriummediated transformation was first used in sunflower for the expression of foreign genes (Murai et al. 1983; Matzke et al. 1984; Helmer et al. 1984; Goldsborough et al. 1986). Later, the first report of fertile, transgenic progeny was published by Everett et al. (1987). Hypocotyl segments were used as explants to transfer the nptII gene conferring kanamycin resistance, and the authors reported the regeneration of transgenic plants from callus selected for resistance to kanamycin. Unfortunately, this approach could not be reproduced, because it suffered from poor reliability due to the fact that regeneration from callus in sunflowers is difficult and is restricted to very few genotypes (Hahne 1994; Hahne 2000). These obstacles led to the use of half-shoot apices as target explants for sunflower transformation (Schrammeijer et al. 1990). Although plants expressing the trans gene (uidA, GUS) in a chimeric fashion have been recovered, this approach suffered from a very low efficiency, because only 0.1 % of the explants co-cultured yielded plants transmitting the transgene to their progeny. However, this system was optimized by Malone-Schoeneberg et al. (1991) and Bidney et al. (1992), who subjected the explants to bombardment with uncoated microprojectiles prior to Agrobacterium inoculation and obtained a transformation efficiency of 2%. The latter authors related these results to the creation of microlesions by the bombardment, which allowed improved access of the agrobacteria to the meristematic tissue. Knittel et al. (1994) further improved this approach and found that - depending on the genotype - up to 7% of the explants gave rise to plants exhibiting transgene expression via an improved Agrobacterium infection system and stringent selection. However, this approach did not lead to reports of the successful transformation of sunflower with agriculturally relevant genes, at least in the public domain. Nevertheless, it is very notable that researchers from Pioneer Hi-bred succeeded in transforming sunflowers using the system described (Anonymous 1991). It appears that the shoot-apex approach encounters problems originating from the na- ture of the explant, because mainly chimeric shoots regenerate and, as a consequence, the transgene is often not transmitted to the progeny. This was validated by Burrus et al. Modification of Oilseed Quality by Genetic Transformation ISS (1996) by analyzing expression patterns using uidA (GUS) as a reporter gene. However, using shoot apices, tissue disruption seems to be a necessity, because Grayburn and Vick (1995) succeeded in causing a A. tumefaciens-mediated gene transfer to sunflower by subjecting this explant to shaking with glass beads prior to Agrobacterium inoculation. In addition, the authors reduced the in vitro culture efforts to a minimum. This approach was used recently by Sankara-Rao and Rohini (1999), who excised and selected transgenic shoots from germinated seedlings wounded by removing a cotyledon prior to inoculation with agrobacteria. Conference abstracts indicate that efficient tissue disruption (as a prerequisite for successful Agrobacterium infection) can be achieved biochemically using macerating enzymes in addition to mechanical or physical methods. Our own investigations were focussed on this topic and led to an improvement of the frequency of explants producing shoots with transgene expression (Weber et al. 1998a,b). We expected the macerating enzymes to have two effects: 1. An increase of the area where agrobacteria can attach to the plant cells. 2. The digestion of the cell wall should result in a release of compounds capable of inducing bacterial virulence. We studied the effects of cellulase, pectinase and macerozyme on the GUS expression of shoot-apical meristems using the sunflower line HA300B and the A. tumefaciens strain GV2260 harboring the binary vector p35S-GUS-INT (Vancanneyt et al. 1989). The shoot-apex explants were treated with enzymes dissolved in the Agrobacterium suspension during the co-culture period (3 days). The first experiments demonstrated that the concentration of the enzyme plays an important role. Pectinase and cellulase had beneficial effects on GUS expression at concentrations of 0.05% and 0.1 %, respectively, while macerozyme proved to be unsuitable at all concentrations tested. We obtained enhancement of the frequency at which explants produced shoots with GUS expression by applying cellulases of different origins, although this enhancement occurred with specific efficiencies. Substantial progress was achieved by applying a combination of cellulase and pectinase (ES2) at optimum levels; using this protocol, 5.7% of explants had shoots expressing GUS Table 2. Influence of treatment with ES2 (0.1% cellulase and 0.05% pectinase) on the stable p-glucuronidase (GUS) expression of sunflower shoot apices. Mean values of four independent experiments. (Weber et al. 1998b) GUS expression Explants (%) with GUS-positive Explants Morphogenic Shoots Treatment n (%) structures Uniform Chimeric Control ES2 248 244 50.4 51.7 0.4 2.0 0.0 5.7 3.6 9.1 156 Genetics uniformly, while no such shoots were observed in the non-treated control. In addition, the percentage of explants regenerating shoots chimeric for GUS expression was elevated from 3.6% in the control to 9.1% in the ES2-treated explants (Table 2). In accordance with our results. Alibert et al. (1998) increased the efficiency of the protocol developed by Grayburn and Vick (1995) by including treatment of apical meristems with pectolytic enzyme prior to Agrobacterium inoculation. The authors concluded from competition experiments using 3H-labeled bacteria that the beneficial effect of enzyme treatment does not arise from an increased adhesion of bacteria to the plant cells (Alibert et al. 1998). However. our experiments indicate that enzyme treatment is inferior to vir induction on the level of transient expression but is more effective than the latter regarding stable expression. This led us to the assumption that the positive effect of enzyme treatment is due to an increase of the overall area where agrobacteria can attach (Weber et al. 1998b). In addition. the beneficial effect of enzyme treatment appeared to be restricted to shoot-apex explants (particularly the meristematic tissue). because it was not successful when applied to Agrobacterium-mediated transformation employing cotyledons from mature seeds as explants. Because the selection of transform ants using kanamycin appears to be insufficient. selection using the GFP derived from the jellyfish Aequorea victoria as a vital marker (Elliot et al. 1999) is currently being evaluated. As a non-antibiotic marker. GFP offers a number of advantages. one of which is especially important for sunflower. i.e .• the detection of plants chimeric for the transgene. Transient and stable expression of this reporter gene has been demonstrated in sunflowers (Weber 1998). Finally. after successful transformation. selection and regeneration of transgenic sunflower shoots. the recovery of plantlets is a major problem. Shoots regenerated from cocultured explants under the selective pressure of kanamycin are small. and the rooting of such shoots has rarely been reported. Therefore. the shoots have to be grafted onto rootstocks (Fischer et al. 1992). which requires considerable fine-tuning (Hahne 2000). However. according to our observations. this represents another critical step in the sunflower transformation procedure. because the success of grafting is variable. According to the method of Krasnyanski and Menczel (1993). an improvement can be achieved by employing young hypocotyls as the rootstock (Weber. unpublished). Little success has been achieved regarding the use of alternative sunflower explants for Agrobacterium-mediated gene transfer. Laparra et al. (1995) conducted a comparative study and showed that neither IZEs nor cotyledonary explants allow the recovery of stable transform ants, although considerable transient expression is detectable. This result is primarily due to the specific features of regeneration in these explants (Sect.2.b). 5 State of the Art Major goals of the genetic modification of crop plants are the internal, end-product-oriented alteration of quality for industrial or nutritional purposes, and changes that may alter the plants' reaction to external environmental factors, i.e., novel agronomic traits. Plants engineered to have an altered food quality (fatty acid, storage-protein or carbohydrate Modification of Oilseed Quality by Genetic Transformation 157 composition) have to be distinguished from transgenic plants with altered agronomic traits, where the major aim is to improve yield per se or the stability of the yield (for example, through insect and herbicide resistance; Dunwell 1998). Currently, various plant species with novel or improved traits are grown in field trials or as commercial crop varieties worldwide. Most of the genetically modified plants have transgenes coding for herbicide resistance or insect tolerance. In the following discussion, we will focus on the two oil crops rapeseed and sunflower, which were furnished with novel traits regarding the modification of oil quality and their release into the field. a) Transgenic Oilseed Plants with Novel Traits The quality of oils and fats is determined by their composition of fatty acids, their chain length, degree of de-saturation, functional groups, etc. For industrial purposes, oils or fats with high amounts of a single or unique fatty acid, or vegetable oils containing unusual fatty acids or novel compositions are needed (Friedt and Luhs 1998). Due to corresponding limitations of genetic variation in the available germ plasm, these aims can hardly be achieved by conventional breeding. Unusual fatty acids are common to many plant species that do not have agronomic characteristics (such as high yield, shatter resistance, etc.) and that cannot be sexually crossed to crop plants in order to transfer the desired trait sexually (Friedt and Luhs 1998). Therefore, modifications of the fatty-acid composition (i.e., the introduction of novel or unusual fatty acids or an increase of the content of an existing fatty acid in order to facilitate industrial processing of the raw material) are the most important objectives of genetic engineering in oil crops. Aiming at a defined breeding objective, the fatty-acid composition of an oil can be engineered by modifying the content of one or a few fatty acid(s). The modification wanted may be obtained by either increasing or reducing the expression of enzyme activities (Knutzon et al. 1992; Cartea et al. 1998). As shown above, rapeseed can be considered one of the more easily transformable plants with a high susceptibility to A. tumefaciens. This has resulted in a large number of studies with this species. In sunflower, the commercially oriented genetic engineering of specific traits has not been possible, due to the lack of an efficient transformation system. However, recent conference abstracts indicate success in this area. The first report of the development of a stable transformation system in sunflowers was published during the early 1990s (Anonymous 1991), when researchers from Pioneer Hi-bred (Des Moines, Iowa, USA) succeeded in using the combined partide-bombardmentlAgrobacterium approach developed by Malone-Schoeneberg et al. (1991). Researchers intended to 158 Genetics use this system to engineer traits like seed-oil quality, protein quality and disease resistance (Anonymous 1991). In 1995, researchers from VanderHave (Rilland, The Netherlands) claimed to have accomplished the transition from procedure establishment to product development regarding sunflower transformation (Peerbolte et al. 1995). The first successful alteration of fatty-acid composition in oilseed rape concerned de-saturation and was accomplished by Knutzon et al. (1992). Through the anti-sense expression of ~9-stearoyl-ACP-desa turase (~9-DES; Fig. 1), the amount of stearic acid was increased to approximately 40% of the total fatty-acid level. This effect was due to the fact that the anti-sense ~9-DES transcript hybridized with the RNA of the native ~9-desaturase and therefore led to a suppression of the endogenous ~9-DES expression. This resulted in an accumulation of stearoyl-ACP, from which stearic acid can be released by the activity of stearoyl-ACP-TE (Topfer et al. 1995). A conference abstract provided by researchers from Pioneer Hi-bred (Coughlan et al. 1999) gives phenotypic information regarding the seed-specific inactivation of ~9-DES, probably by an anti-sense construct in sunflowers. The authors report a transgenic event with a 35% stearate fatty-acid content that exhibits regular germination at low temperatures, in contrast to high-stearate canola and soybean. It concludes with an analysis of phospholipids purified from the endomembranes (predominately ER) of developing seeds. They conclude that the ability of sunflower to tolerate high amounts of saturated fatty acids is not due to an exclusion of stearate from the membrane lipids (i.e., phospholipids). This is reflected by a 25% stearate content of PC (Fig. 1) in transgenic lines, compared with 3% in the wild type. The total unsaturated fatty-acid content (i.e., palmitic and stearic) was elevated to 40% of the total fatty-acid content by crossing transgenic high-stearate lines with high-oleic (009 CI8:1) sunflowers. Furthermore, the authors expected that the high level of saturated fatty acids may be raised by crossing such lines with transgenic progeny over-expressing a soybean FatA ACP TE (Coughlan et al. 1999). Another objective has been the alteration of polyunsaturated fatty acids, such as linoleic (006 CI8:2) and a-linoleic acids (003 CI8:3), in order to increase the oxidative stability and hydrogenation costs (Hitz et al. 1995) or to maximize the a-linoleic acid content for use in the nonfood sector, e.g., as a drying agent for paints and varnishes (Ohlrogge 1994). Using the anti-sense technique, Hitz et al. (1995) produced rapeseed lines that accumulated 88% oleic at the expense of linoleic acid by crossing transgenic rapeseed lines engineered with an anti-sense complementary DNA (cDNA) for the oleoyl-ACP-desaturase gene and a mutant line possessing 77.8% oleic acid in its seed oil. In a similar approach, researchers at DuPont (Wilmington, Del., USA) produced transgenic soybean lines with an a-linoleic acid content of only 2% (Kinney 1998). In the food sector, dietary supplementation of y-linoleic acid Modification of Oilseed Quality by Genetic Transformation 159 (006 18:3) has been shown to be beneficial under many physiological and pathological conditions. Oil from borage (Borago officinalis 1.) contains 25-40% y-linoleic acid, whereas it is absent from rapeseed. Transgenic rapeseed lines containing 20.8% oleic, 23.4% linoleic and 43% y-linoleic acid were produced by researchers from Calgene (Davis, Calif., USA; Huang et al. 1999), probably by introducing a .M-desaturase isolated from borage. Another interesting approach has been contributed by Cahoon et al. (1999), who expressed a divergent form of ~12-0Ieic acid desaturase in somatic soybean embryos. The sequences were derived from expressed sequence tags generated from cDNA libraries of Momordica charantia and Impatiens balsamica, which accumulate high amounts of a-eleostearic and a-parinaric acid in their seed oils. These fatty acids contain conjugated double bonds and, therefore, are valuable drying agents, because they require less oxygen than, e.g., linoleic acid. The transformed soybean somatic embryos accumulated up to 17% (weight/weight) of these fatty acids. The change of fatty-acid chain lengths has been another aim of lipidcomposition modification. A major success was achieved when seeds of transgenic Arabidopsis thaliana plants engineered with a TE (Fig. 1) gene from California bay (Umbellularia californica), a plant containing 70% lauric acid in its oil (Pollard et al. 1991), exhibited an accumulation of up to 25% of this fatty acid (Voelker et al. 1992). The expression of the same gene in rapeseed resulted in a similar accumulation of lauric acid (Voelker et al. 1996). Another achievement regarding the production of medium-chain fatty acids in rapeseed was reported by Voelker et al. (1997). The expression of FatB TEs from nutmeg (Myristica fragrans) and elm (Ulmus americana) in rapeseed leads to an enrichment of C1418 and CI0-18 saturated fatty acids, respectively. The expression of aTE from the tropical tree mangosteen (Garcinia manostana) in transgenic rapeseed leads to an accumulation of approximately 20% stearic acid (Hawkins and Kridl 1998). Another example of altering the fatty-acid chain length was achieved by introducing TEs from Cuphea lanceolata into rapeseed. In the transgenic progeny, a distinct, altered fatty-acid composition of the T2 seeds was obtained depending on what TE gene was expressed; ClFatB3 caused 1% and 3% caprylic and capric acids, respectively, whereas ClFatB4 led to the formation of 7% myristic and 15% palmitic acids in the storage oil (Tapfer et al. 1995). Regarding sunflower, the first report of transformation with genes involved in the fatty-acid metabolism concerned the modification of the seed-oil composition for industrial applications (Molinier and Hahne 1998). The latter authors report the transformation of sunflower with coding sequences of an acyl-ACP TE from Cuphea spp., under the control of its own promoter or a seed-specific promoter from sunflower. This produced an oil with elevated levels of medium-chain (myristic and palmitic) fatty acids. Recently, we achieved the Agrobacterium-mediated 160 Genetics transformation of sunflowers with the FatB3 gene from C. lanceolata (Ait.), which encodes the acyl-ACP TE C1FatB3 (Topfer et al. 1995) under the control of a sunflower-seed-specific promoter (Prieto-Dapena et al. 1999). The presence of the T-DNA was demonstrated by PCR, and integration was confirmed by Southern analysis of T2 seeds obtained from the T 1 primary transformants. Data on the fatty-acid profIles will be available soon. Seed oil of the Mexican shrub C. hookeriana is composed of up to 75 mol% caprylic acid (C8:0) and capric acid (ClO:0). Expression of a cDNA cloned from C. hookeriana encoding a medium-chain-specific TE (ChFatB2) in B. napus resulted in the production of C8:0 and ClO:0 fatty acids in seeds. In selected transgenic rapeseed progeny lines, the levels of caprylic and capric acids reached up to 50% of the amount measured in C. hookeriana seeds (Froman et al. 1999). Another objective represents the introduction of fatty acids with functionalities, such as unique double-bond positions or functional groups, Le., hydroxyl groups (Friedt and Luhs 1998). Such fatty acids were produced by Arabidopsis plants engineered with the oleate 12hydroxylase gene from castor beans (Ricinus comunis L.). The transgenic seeds accumulated up to 17% hydroxyl fatty acids, Le., ricinoleic (C18:1OH), densipolic (C18:2-0H) and lesquerolic acids (C20:1-0H; Broun and Somerville 1997). The understanding of the mechanism by which novel chain fatty acids are directed into seed storage oil is a challenge because, in total, the levels of newly introduced fatty acids never exceed 40% (Friedt and Luhs 1998; Murphy 1999). In rapeseed, it was observed that lauric acid is not incorporated in the second position of the TAG, because the native LPAAT (Fig. 1) is not specific for laurate. This led to the engineering of downstream metabolic pathways, Le., the introduction of specific enzymes involved in the TAG synthesis. A major increase (up to 60%) of lauric acid in the rapeseed oil could be achieved by expressing a gene from coconut that encodes an LPA-AT specific for lauric acid (Murphy 1996). However, super-transformation of laurate oilseed rape with this LPA-AT resulted in a lauric acid accumulation of up to only 50% (Knutzon 1999). Another aspect of research in rapeseed has been an increase in the amounts of very long chain fatty acids. In the production of high levels of erucic acid (HEAR) via classical breeding methods, an erucic acid content of 66% represents the theoretical upper limit, because erucic acid is not normally incorporated in the second position of the TAG (Friedt and Luhs 1998). Oil with an erucic acid content greater than 90% could be attractive as a substitute for petrochemicals (Topfer et al. 1995). Analogous to the novel formation of lauric acid in laurate rapeseed, the integration of a LPA-AT specific for erucic acid and other very long mono-unsaturated fatty acids may eventually lead to an increase of eru- Modification of Oilseed Quality by Genetic Transformation 161 cic acid from its current level (-45-50%) to approximately 70% (Friedt and Luhs 1999). For this purpose, several LPA-ATs from different organisms, such as the LPA-ATs from meadowfoam (Limnanthes douglasii; Weier et aI. 1997; Lassner et al. 1995), yeast (Zou et al. 1997) and Escherichia coli (Weier et al. 1998), were transferred and expressed in HEAR rapeseed genotypes. The "tri-erucin" content was increased to 13% of the total TAG content in some re-synthesized HEAR lines. However, the total content of erucic acid in the oil was not increased simultaneously (Weier et al. 1997). Therefore, further genes encoding enzymes that further limit the biosynthesis of erucic acid must be identified in order to achieve the aim of maximum C22:1 content in oilseed rape. Regarding the engineering of TAG synthesis, an unexpected result was obtained when a yeast sn2-acyltransferase was expressed in transgenic Arabidopsis and rapeseed. The resulting transgenic lines exhibited an increase of their total oil content by 8-48% (Zou et al. 1997). Although this result is assumed to be primarily due to the use ofthe constitutive CaMV35S promoter (Murphy 1999), it indicates the importance of downstream metabolic pathways for the channeling of fatty acids to storage lipids and for the overall fatty-acid yield. In summary, specific pathways of plant lipid biosynthesis have been altered in various plant species by engineering separate steps. However, most transgenic lines have been reported to accumulate relatively low levels of the novel fatty acid(s) introduced (Murphy 1999). In addition, the introduction of a yeast sn2-acyltransferase in Arabidopsis had quite unexpected quantitative effects. These results demonstrate the importance of two aspects that have to be considered regarding the modification of seed oil by genetic engineering: (1) interactions between apparently distinct metabolic steps and pathways, and (2) specific responses of different species to the introduction of foreign enzymes. Finally, conventional amelioration following gene transfer is required in order to stabilize the desired phenotype and develop commercial varieties. b) Release of Transgenic Oilseeds into the Field The first transgenic plants released into the field with the aim of commercialization were a virus-resistant tobacco in 1990 and a virus resistant tomato in China (James and Krattiger 1996). Since then, the worldwide list of field trials has grown tremendously (Table 3). The USA was the first Western country to grant permission for the sale of the genetically modified Flavr-Savr delayed-ripening tomato (in May 1994; James and Krattiger 1996). In 1998, the dominant transgenic crops released into the field were soybean (52% of the global area cultivated with transgenic crops), corn (30%), cotton and canola (9% each; James 1998). It is 162 Genetics Table 3. Global area of transgenic crops in 1997 and 1998 by trait groups, in millions of hectares. (James 1998) 1998>1- Percent 63 19.8 36 7.7 <0.1 <1 0.3 Quality traits <0.1 <1 <0.1 <1 <0.1 Global totals 11 100 27.8 100 16.8 Trait 1997" Percent Herbicide tolerance 6.9 Insect resistance 4.0 Insect resistance and herbicide tolerance Increase Ratio 71 12.9 2.9 28 3.7 1.9 0.2 2.5 "Excluding China. obvious that oil seeds cover the largest area, where herbicide resistance accounts for almost 75% ofthe area (Table 3). Regarding genetically modified oil crops, the number of field trials and field releases reflects the technical advantage of oilseed rape compared with other species. The use of genetic engineering has focussed on the improvement of agronomic traits, Le., the introduction of resistance to herbicides, fungal pathogens and insects. The cultivation area of herbicide-resistant canola in Canada is an excellent example: it is expected to reach 4 million hectares and to occupy 80% of the total rapeseed area Table 4. Field trials with transgenic Canola worldwide until 1999, according to the Organization for European Cooperative Development's Database of Field Trials http://www.olis.oecd.orglbiotrack.nsf) Characteristic Trait Number of field trials Marker GUS/nptII 102 Seed quality Protein Oil Other 25 145 46 Disease resistance/tolerance Fungal Insect Viral 26 18 4 Herbicide resistance Glufosinate Glyphosate Bromoxynil 2,4-D Sulfonylurea 153 125 21 24 2 Other Abiotic stress tolerance Male sterility 11 96 Total GUS, l3-glucuronidase. 798 Modification of Oilseed Quality by Genetic Transformation 163 forecasted (Anonymous 1999). The market leader is AgrEvo (now Aventis, Frankfurt/Main, Germany) with its LibertyLink rapeseed (75% of the market), followed by Monsanto (St. Louis, Mo., USA) who compete with their Round-Up-ready Canola (25%). Nevertheless, the transgenic rapeseed plants tested in field trials differ in their modified traits, including marker genes (such as GUS), genes for antibiotic resistance (kanamycin), herbicide tolerance and disease resistance, and genes that contribute to product quality. The distribution of transgenic canola according to traits is shown in Table 4. A database search for the traits displayed resulted in a list of nearly 800 field trials performed with transgenic canola. Although 40% of these trials were performed with plants engineered for tolerance to various herbicides, the modification of seed-oil quality accounts for nearly 20% of the plants tested. The next most frequent field trials were those with plants having marker genes and those with plants engineered for male sterility, while 10% of the field trials represented rapeseed plants engineered for biotic stress tolerance. Although sunflower transformation is still far from routine, field trials with transgenic lines started as early as 1991 in the USA and were conducted by Pioneer Hi-bred with plants engineered for seed storage proteins (Table 5). In Europe, the first field trials with transgenic sunflowers were performed in 1994 by Limagrain Genetics SA (Mas Grenier, France) and VanderHave with plants carrying uidA (GUS) as a marker gene. Together with two field trials performed by Rustica Prograin Genetique (Mondonville, France), the activities of these three companies account Table 5. Field trials with transgenic sunflower worldwide until 1999 (Hahne 2000), according to the Robert Koch Institut (http://www.rki.de/GENTEC/GENTEC.HTM). the Joint Research Centre of the European Commission (http://biotech.jrc.itlgmo.htm) and the Organization for European Cooperative Development's Database of Field Trials http://www.olis.oecd.orglbiotrack.nsf) Character Trait Number of field trials Marker GUS 7 Seed quality Seed protein Seed oil 4 Disease resistance/tolerance Fungal Insect Viral 8 5 Herbicide resistance Glufosinate Chlorsulphuron 2 Other Total Drought tolerance Male sterility 2 1 1 2 33 164 Genetics for all the field releases of transgenic sunflowers worldwide. According to the sources screened, little more than 30 field trials have been conducted with sunflower to date, with emphasis on the engineering of resistance to fungal pathogens and insects (Table 5). It is interesting to note that only two field trials were conducted with plant progenies whose the seed-oil quality was engineered. 6 Future Trends and Perspectives Man has been modifying plants for thousands of years simply by adapting to the requirements of a sustained agriculture. Conventional methods of plant breeding and - in recent years - modern biotechnology have evolved into powerful tools for developing crop species and novel varieties. The new techniques of genetic modification that have been developed during the last 20 years do not fundamentally differ from conventional breeding in their objectives. Moreover, gene transfer to plants should allow the introduction of novel traits from foreign species or taxa (or even different kingdoms) without changing the agricultural performance of a given genotype. From a methodological point of view, the transformation of oilseed rape can be considered a routine practice. Nevertheless, there are still factors to be evaluated and optimized when establishing a transformation protocol, i.e., the regeneration capacities of genotypes and their interaction(s} with culture conditions. In contrast, sunflower still faces a number of major impediments regarding transformation because of its recalcitrance to in vitro cultures. Nevertheless, innovations that resulted in improvements in the genetic engineering of the cultivated sunflower, such as pre-bombardment or enzyme treatment for improved access of A. tumefaciens, can also be employed for other recalcitrant species, thus facilitating the progress of genetic engineering in crop plants. In addition, sunflower transformation merits further research, especially for the modification of lipid synthesis, because initial results indicate that common approaches that alter specific steps of the biochemical pathway(s} yield variable results in different species. Regarding the modification of seed-oil quality by genetically engineering metabolic pathways, important prerequisites have been elaborated: genes encoding key enzymes of plant lipid synthesis have been isolated and can be used in plant transformation, and corresponding systems for the modification of various oil crops have been established (albeit with different efficiencies). Oilseed rape has evolved into one of the model crop-plant species in terms of the modification of plant lipid synthesis. It has yielded useful information concerning the basic understanding of biochemical mechanisms and has already led to commercialized varieties with novel fatty-acid patterns. However, transformation Modification of Oilseed Quality by Genetic Transformation 165 experiments in rapeseed have also provided results that indicate the limits of seed-oil modification via the engineering of separate steps because, in a number of cases, the expected or desired phenotype was not obtained, although the genes were properly integrated and expressed. It appears that such obstacles may occur because the enzymes engineered may not be limiting in the target metabolic pathway. In some cases, the alteration of single steps of plant lipid biosynthesis affected other (agronomic) traits, such as seed germination or total oil content. In addition, recent results in sunflowers indicate that different species respond to the engineering of specific metabolic pathways in different ways. As a consequence, further and better knowledge of plant lipid biosynthesis and the traits affected by the alteration of a specific metabolic step is required. This should result in novel and more effective strategies for the modification of seed-oil quality via genetic engineering. At present, most field releases of genetically modified plants concern crops engineered for herbicide resistance. The modification of oilseed quality is of minor importance compared with the total number of field releases of genetically modified organisms and the acreage of varieties with novel traits. This reflects the obstacles mentioned above; the strategies employed to alter specific pathways of plant lipid synthesis are not always suited to the objective. It is clear that genetic engineering cannot replace conventional breeding methods (especially with regard to the modification of oilseed quality) because, in many cases, the desired or improved phenotype can only be obtained via the subsequent selection or crossing of plant prototypes with other genotypes. Field trials with plants having genetically engineered oil quality provide further knowledge of the physiology and environmental control of plant lipid metabolism and, therefore, probably reflect scientific interests rather than commercial interests. The use of transgenic crop plants for both food and non-food uses will depend on broad acceptance by the public. In addition to the scientific and commercial interests, this should be taken into serious consideration. The increased knowledge provided by genetic engineering and field trials with transgenic crops should be used to decrease reluctance. 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Plant Cell 9:909-923 Zupan JR, Zambryski P (1995) Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physioll07:1041-1047 Dr. Steffen Weber Dipl.-Ing. agr. Karim Zarhloul Professor Wolfgang Friedt Institut flir Pflanzenbau und Pflanzenziichtung, IFZ Heinrich-Buff-Ring 26-32 35392 Giessen, Germany e-mail: [email protected] Communicated by Tel.: +49-(0)641-9937420 K. Esser Fax: +49-(0)641-9937429 Physiology Physiology Significance of Phloem-Translocated Organic Sulfur Compounds for the Regulation of Sulfur Nutrition Cornelia Herschbach and Heinz Rennenberg 1 Introduction Sulfur is an essential nutrient of all living organisms. In plants, it is fifth or sixth in order of elemental abundance, after hydrogen, oxygen, carbon, nitrogen and phosphorus (Pitman and Cram 1977; Raven 1980; Cram 1990). Reduced-sulfur, i.e. sulfur in the oxidation state -2, is the most important form of sulfur in living cells. It supports the specific conformations and functions of enzymes and structural proteins via reactive sulfide moieties and disulfide bonds. Sulfur is available for plants mainly as sulfate at the roots (Rennenberg 1984). Therefore, sulfate has to be activated, reduced to sulfide and incorporated into carbohydrate skeletons by assimilatory sulfate reduction before it can be used in protein synthesis (Brunold 1990, 1993). The final product of assimilatory sulfate reduction in plants is cysteine. From this amino acid, all other reduced-sulfur compounds, including methionine (Giovanelli 1990), glutathione (Bergmann and Rennenberg 1993), the S-alkykysteine sulfoxides of Liliaceae, the isothiocyanates of Brassicaceae (Schnug 1990) and phytochelatins (Rauser 1995), are synthesized in a whole set of metabolic pathways. In most plant species, a specific sulfur/nitrogen (S/N) ratio of approximately 1120 reflects the relationship of these macro-nutrients in protein (Dijkshoorn and Van Wijk 1967). The SIN ratio is significantly higher only in taxa (Brassicaceae, Liliaceae) that accumulate secondary sulfur compounds (Evans 1975). SIN ratios higher than 1120 are also observed in plant tissues that accumulate sulfur-rich storage proteins (Bohlmann 1993), whereas significantly lower values are found in tissues of plants, such as grain legumes and cereals, that accumulate sulfur-poor storage proteins (Muntz et al. 1997; Tabe et al. 1997). The published literature clearly indicates that sulfate uptake is adapted to nitrogen nutrition (Clarkson et al. 1989, 1993, 1999; Karmoker et al. 1991; Kreuzwieser et al. 1996; Kreuzwieser and Rennenberg 1998) and to the sulfur status of plants (Smith 1980; Lee 1982; Clarkson et al. 1983; Datko and Mudd 1984a,b; Deane-Drummond 1987; Herschbach and Rennenberg 1991; Hawkesford et al. 1993; Yildiz et al. 1994; Herschbach et al. 1995a,b; Matsuda and Colman 1995; Kreuzwieser Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 178 Physiology et al. 1996; Lappartient and Touraine 1996; Takahashi et al. 1997; Hatzfeld et al. 1998; Kreuzwieser and Rennenberg 1998; Lappartient et al. 1999). This adaptation may be interpreted as means of maintaining the species-specific SIN ratio and, simultaneously, avoiding accumulation of excess nitrogen and sulfur. In herbaceous plants, assimilatory sulfate reduction is thought to be localized mainly in photosynthetically active tissues (Brunold 1990). However, this pathway also seems to be active in heterotrophic cells, because isolated root tissues were found to reduce-sulfate (Brunold and Suter 1989) and grow without the addition of reduced-sulfur compounds in culture solutions (Hawkesford and Belcher 1991). The messenger RNA (mRNA) levels of adenosine triphosphate (ATP) sulfurylase and adenosine phosphosulfate reductase in roots were found to be induced by sulfur deprivation (Yildiz et al. 1996; Takahashi et al. 1997; Bolchi et al. 1999; Lappartient et al. 1999) and conditions of enhanced demand for reduced-sulfur as a consequence of cadmium exposure (Heiss et al. 1999; Lee and Leustek 1999). Assimilatory sulfate reduction in mycorrhizal and non-mycorrhizal roots has been suggested to contribute to the overall need for reduced-sulfur in deciduous trees (Herschbach and Rennenberg 1997; Hartmann et al. 2000a). Still, it is not clear which portion of the plant's total demand for reduced-sulfur is contributed by assimilatory sulfate reduction in the roots. Irrespective of the distribution of assimilatory sulfate reduction between leaves and roots, part of the sulfate taken up by the roots has to be allocated to the leaves for reduction, and reduced-sulfur compounds produced in the leaves and the roots have to be allocated from these sites of sulfate reduction to tissues that require reduced-sulfur for the synthesis of proteins and other reduced-sulfur compounds. In particular, the meristematic tissues of the shoot and roots can be considered major sinks for reduced-sulfur. Thus, uptake of sulfate, sulfate reduction and the allocation of sulfate and reduced-sulfur compounds by long-distance transport processes have to be coordinated and regulated in order to meet the reduced-sulfur demands of plants undergoing growth and development. The initial step of the sulfur nutrition of plants is the membrane transport of sulfate at the soil-root interface. Rennenberg (1995) and Lappartient and Touraine (1996) suggested that demand-driven control adapts sulfate uptake by the roots to the sulfur demand of the whole plant. They proposed that glutathione transported in the phloem acts as a signal that regulates sulfate uptake by the roots. However, recent studies demonstrated that demand-driven control of sulfate uptake by glutathione does not operate in beech (Kreuzwieser et al. 1996; Kreuzwieser and Rennenberg 1998) and is more complex in transgenic poplars that over-express y-glutamylcysteine synthetase (Herschbach et al. 2000). Therefore, the present review is focused on the phloem allocation of Significance of Phloem-Translocated Organic Sulfur 179 sulfur compounds and its consequences for (1) the roots' sulfur budget and (2) sulfate uptake by the roots. This approach is adopted in order to elucidate the extent to which the model of demand-driven control of sulfur nutrition must be improved on the basis of current results. 2 Phloem-Translocated Organic Sulfur Compounds Almost half a century ago, investigations with radio-labeled sulfur demonstrated that sulfur is cycled within plants, but the sulfur compounds involved were not identified (Biddulph et al. 1956, 1958). Ziegler (1975) suggested that reduced-sulfur transport in the phloem may be more important than sulfate translocation. The first clear evidence of phloem allocation of organic sulfur was published 1979. After feeding 35S sulfate to a mature tobacco leaf, reduced 35S in the form of cysteine, methionine and glutathione was identified along the stem basipetal and apical to the leaf (Rennenberg et al. 1979). These sulfur compounds were also labeled in phloem exudates of Ricinus plants subjected to similar feeding experiments (Bonas et al. 1982). Hocking (1980) detected cystine in phloem exudates of the tobacco tree Nicotiana glauca. Magnicol and Bergmann (1984) showed that homo-glutathione plays an important role in reduced-sulfur transport to developing bean seeds, and glutathione was detected in phloem exudates from Curcubita (Alosi et al. 1988). Cysteine, methionine and glutathione were also identified in phloem exudates from different deciduous tree species (beech: Kreuzwieser 1997; poplar: Fig. 1; Herschbach et al. 1998; 2000; oak: Rennenberg 1999). In both deciduous trees and herbaceous plants, glutathione was the main reduced-sulfur compound transported that was identified, but considerable amounts of unidentified sulfur compounds were found to be allocated in numerous experiments. Recently, Bourgis et al. (1999) has identified S-methylmethionine in phloem exudates from several herbaceous plant species and found that S-methylmethionine and glutathione were present in similar amounts in phloem exudates of wheat. Because S-methylmethionine was not analyzed in previous studies of phloem transport of reduced-sulfur, the role of glutathione as the main reduced-sulfur compound allocated in the phloem will have to be reevaluated in future studies. If reduced-sulfur compounds transported in phloem signal the sulfur demand of the shoot to the root, reduced-sulfur concentrations of phloem exudates should vary at different levels of sulfur and nitrogen nutrition. Lappartient and Touraine (1996) showed that glutathione (but not cysteine or sulfate) levels in the phloem decreased during sulfur starvation. In young, non-mycorrhizal beech trees, glutathione concentrations of the phloem increased with increasing sulfate supply during Physiology 180 sulfate shoot leaves total reduced sulfur shoot phloem root phloem roots root xylem shoot xylem - cys met y-glu-cys GSH shoot leaves shoot phloem root phloem roots root xylem shoot xylem shoot leaves shoot phloem nd. roots phloem n,d. ~ roots root xylem ~ . shoot xylem o 50 100 150 200 250 percentage of control 0 50 100 150 200 250 300 percentage of control Fig. 1. Sulfur concentrations in poplar leaves, roots, xylem and phloem after additional sulfate supply during growth in commercial soil. Wild-type poplars were micropropagated and transferred in soil, as described by Strohm et al. (1995). During growth on soil, poplar trees were fertilized every 2 weeks with a commercial fertilizer, as described by Herschbach et al. (2000), with 0 (control), 1.6 (dark gray), 8.0 (light gray) and 16 mM (cross-hatched) sulfate added as MgS0 4 • After 7-8 weeks on soil, phloem exudates, leaf and root samples, xylem sap and phloem exudates were sampled and subjected to sulfate, thiol and methionine analyses, as described by Herschbach et al. (2000). Data given represent the percentage of the control without additional sulfate. Asterisks indicate significant differences (P<0.5) from the control. cys Cysteine, y-glu-cys y-glutamylcysteine, GSH glutathione, met methionine, n.d. not detected Significance of Phloem -Translocated Organic Sulfur 181 short-term exposure (3 days) in hydroponic culture solutions (Kreuzwieser 1997). Changes in sulfate, methionine or S-methylmethionine concentrations were not investigated in that study. In poplar, sulfate, cysteine, y-glutamylcysteine, glutathione and methionine were analyzed in phloem exudates (in combination with tissue and xylem sap concentrations) after long-term supply with different levels of sulfur nutrition (Fig. 1). In contrast to the results of short-term experiments with beech, the content of reduced-sulfur did not increase in phloem exudates of poplar as a consequence of additional sulfate supply. The slightly increased glutathione concentrations in phloem exudates of the shoot were not present in roots. Similar results were obtained with beech grown in commercial soil and subjected to additional sulfate supply for a period of 2 months (Herschbach, unpublished). Therefore, it can be assumed that the level of reduced-sulfur compounds (such as glutathione) initially responds to changes in sulfate supply to the roots but is backregulated to its original level once a new steady state is achieved in the roots. This view is supported by the results of short-term sulfur-fumigation experiments with wild-type and transgenic poplar plants. After 48 h of exposure to atmospheric H2S, cysteine, y-glutamylcysteine and glutathione levels were increased in wild-type poplar leaves. Correspondingly, cysteine, y-glutamylcysteine and glutathione increased in phloem exudates, whereas methionine and sulfate remained unaffected (Herschbach et al. 2000). Apparently, a surplus of reduced-sulfur in the shoot results in enhanced allocation of reduced-sulfur from the shoot to the roots in the phloem. Similar results were obtained in transgenic poplars that over-expressed bacterial y-glutamylcysteine synthetase. The transgenic poplar plants had higher y-glutamylcysteine and glutathione but similar methionine and sulfate concentrations in the leaves compared with the concentrations in wild-type plants. As a consequence, elevated y-glutamylcysteine and glutathione concentrations were observed in phloem exudates of the transgenic plants (Herschbach et al. 1998, 2000). H2S fumigation further enhanced leaf and phloem-exudate y-glutamylcysteine and glutathione concentrations. Correlation analysis revealed that the glutathione concentration in the leaves largely determined the glutathione concentration of the phloem exudates (Herschbach et al. 2000). 3 Consequences of Phloem Transport of Sulfur for the Organic Sulfur Content of the Roots In herbaceous plants, phloem-translocated sulfur is mainly transported to the roots (Rennenberg et al. 1979; Bonas et al. 1982). In perennial plants like deciduous trees, sulfur transported in the phloem is also un- 182 Physiology loaded in considerable amounts along the transport path and may be incorporated into the storage tissue of the trunk (Herschbach and Rennenberg 1995, 1996; Schulte et al. 1998; Hartmann et al. 2000a). In splitroot experiments with Brassica napus, feeding glutathione to a part of the root revealed that glutathione is cycled within the plant and that glutathione transport in the phloem can enhance the glutathione pool of the roots (Lappartient and Touraine 1996). In spinach, atmospheric sulfur, supplied to the shoot in the forms of H2S and S02' increased the levels of glutathione and other thiols in the shoot and the roots at low and sufficient sulfate supply (Herschbach et al. 1995a). Apparently, H2S exposure resulted in a surplus of reduced-sulfur in the shoot; this was partially transported (in form of glutathione in the phloem) to the roots and determined the roots' glutathione content. Similar results were observed with poplar (Herschbach et al. 2000). The elevated glutathione concentrations in phloem exudates of poplar plants subjected to H 2S fumigation resulted in higher glutathione concentrations of the roots. A direct correlation between phloem and root glutathione concentrations was observed for both wild-type poplar plants and those that overexpressed y-glutamylcysteine synthetase (Herschbach et al. 2000). Transgenic poplar that over-expressed y-glutamylcysteine synthetase in the cytosol showed enhanced glutathione levels in phloem exudates and, correspondingly, elevated glutathione concentrations in the roots. Apparently, glutathione transport in the phloem determined the glutathione levels of the root (Herschbach et al. 2000). However, this correlation was circumstantial, and it is possible that enhanced sulfate reduction and glutathione synthesis in the roots (as found in the shoot; Hartmann et al. 2000b) contributed significantly to the pool of reducedsulfur compounds in the roots of transgenic poplar. When the roots of poplar trees were grown with an additional sulfate supply, the pool of soluble sulfur compounds of the shoot was not increased. The glutathione concentration of the phloem was slightly enhanced in the shoot but not in the root (Fig. 1). Apparently, glutathione was removed from the phloem during long-distance transport. However, sulfate and reduced-sulfur concentrations were considerably increased in the root tissues. Sulfate concentrations were possibly increased due to enhanced vacuolar storage (Bell et al. 1994, 1995); reduced-sulfur concentrations were possibly increased because of enhanced assimilatory sulfate reduction in the roots. The enhanced cysteine, methionine, yglutamylcysteine and glutathione concentrations observed in the root and shoot xylem sap of poplars subjected to sulfate fertilization may also originate from enhanced assimilatory sulfate reduction in the roots. Because the additional reduced-sulfur in the xylem did not result in elevated thiol concentrations of the leaves, it may either be stored during transport along the trunk or may be used in the leaves for additional protein synthesis (Fig. 1). Significance of Phloem-Translocated Organic Sulfur 183 In summary, there is substantial evidence that phloem allocation of glutathione and other reduced-sulfur compounds can increase the reduced-sulfur concentration of the roots. However, published investigations lack analysis of S-methylmethionine. Because S-methylmethionine is allocated in amounts similar to that of glutathione in the phloem of wheat (Bourgis et al. 1999), it is possible that the transport of this sulfur compound from the shoot to the roots is also important in determining the reduced-sulfur pool ofthe roots. 4 Regulation of Sulfate Uptake It is well established that sulfate uptake is an energy-dependent process driven by the proton motive force of the plasma membrane (Lass and Ullrich-Eberius 1984; Cram 1990; Clarkson et al. 1993; Hawkesford et al. 1993). In a number of studies, changes in sulfate uptake in response to sulfur starvation were investigated (Jeanjean and Brodo 1977; Smith 1980; Lee 1982; Clarkson et al. 1983, 1999; Datko and Mudd 1984a,b; Deane-Drummond 1987; Herschbach and Rennenberg 1991; Hawkesford et al. 1993; Yildiz et al. 1994; Herschbach et al. 1995a,b; Matsuda and Colman 1995; Kreuzwieser et al. 1996; Lappartient and Touraine 1996; Takahashi et al. 1997; Hatzfeld et al. 1998; Kreuzwieser and Rennenberg 1998; Lappartient et al. 1999). Independent of the duration and the species analyzed, sulfur deprivation increased sulfate uptake by two- to tenfold, and re-supply of sulfate repressed sulfate uptake. Therefore, it has been concluded that sulfate uptake, as the first step in sulfur nutrition, must be strictly related to the sulfur demand of the plant. In earlier investigations, regulation of sulfate uptake was analyzed in physiological experiments involving the use of metabolic inhibitors. A large body of evidence from these studies supports the regulation of sulfate uptake by negative feedback control mediated either by sulfate itself (Smith 1980; Jensen and Konig 1982; Cram 1983a,b; Bell et al. 1995) or by a product of the assimilatory sulfate reduction, such as cysteine, methionine or glutathione (Hart and Filner 1969; Ferrari and Renosto 1972; Maggioni and Renosto 1977; Clarkson et al. 1983; Datko and Mudd 1984a,b; Rennenberg et al. 1988, 1989; Herschbach and Rennenberg 1991, 1994; Kreuzwieser et al. 1996; Lappartient and Touraine 1996; Kreuzwieser and Rennenberg 1998). Cacco et al. (1980) proposed that regulation of sulfate uptake is achieved by changes in the availability of sulfate porters. Investigations with the translation inhibitors cycloheximide and puromycine indicated that glutathione reduced and sulfur deficiency increased the de novo synthesis of sulfate porters in tobacco cell cultures (Rennenberg et al. 1989). Similar results were obtained with excised tobacco roots (Gunz, Herschbach and Rennenberg, unpublished). From the turnover of sulfate porters, half-lives of 1.5-2.5 h were 184 Physiology calculated (Rennenberg et al. 1989). In addition, Clarkson et al. {1992, 1993} observed that sulfate uptake depended on continuous protein synthesis. In their study, the presence of cycloheximide during sulfur deprivation prevented the de-repression of sulfate uptake in barley roots. Nevertheless, the controlling factor and its mechanism of action remains unclear. From experiments with the pre-translational inhibitors a-amanitin and cordycepin, it was concluded that the turnover rate of sulfate transporter mRNA was much slower than the turnover of the sulfate transporter itself (Rennenberg et al. 1989). Apparently, the inhibition of sulfate uptake by glutathione is achieved by changes in transporter-protein synthesis rather than changes in mRNA synthesis. Since the first plant sulfate transporter was identified (Smith et al. 1995), studies on the regulation of sulfate uptake have been extended to the molecular level. The first two high-affinity sulfate transporters (shstl and shst2) isolated from plants (Stylosanthes hamata) are expressed exclusively in roots, and mRNA levels increase during sulfur starvation (Smith et al. 1995; Hawkesford and Smith 1997). Similar results were observed in barley for the high-affinity sulfate transporter hvstl (Smith et al 1997; Hawkesford and Smith 1997). In maize, the transcript of a high-affinity sulfate permease (ZmSTl) increased during sulfur deprivation in roots and in the shoot (Bolchi et al. 1999). From Arabidopsis thaliana, only low-affinity sulfate transporters have been sequenced (Takahashi et al 1996, 1997). In contrast to the high-affinity sulfate transporters from tropical legumes and barley (shstl, shst2 and hvstl), the low-affinity sulfate transporters from A. thaliana (AST68 and AST56) and Brassica juncea (LAST) were expressed in roots and shoots (Takahashi et al 1996, 1997; Heiss et al. 1999). The authors suggested that low-affinity sulfate transporters (shst3 from S. hamata, AST68 and AST56 from A. thaliana and LAST from B. juncea) are involved in internal and/or sub-cellular compartmental transport rather than sulfate uptake (Smith et al. 1995; Takahashi et al. 1997; Heiss et al. 1999). However, the mRNA level of the low-affinity sulfate transporter AST68 from A. thaliana increased in sulfur-starved roots from Arabidopsis and B. napus (Takahashi et al. 1997; Lappartient et al. 1999). Apparently, mRNA levels of this sulfate-transporter gene are related to the sulfate demand of the plant. Although sulfate uptake increased simultaneously, the investigations lack western analysis to provide information about the sulfate-transporter protein level. Taken together, the results of physiological and molecular studies indicate that the regulation of sulfate uptake can take place at both the translational and the pre-translational levels. Recently, experiments were carried out to investigate the effect of reduced-sulfur compounds on the transcription level of sulfate transporters. In Arabidopsis and Brassica, the transcript of the low-affinity sulfate transporter AST68 appears to be repressed specifically by glutathione. Significance of Phloem-Translocated Organic Sulfur 185 Repression was achieved by glutathione and cysteine exposure, but repression by cysteine treatment was counteracted by buthionine sulfoximine (BSO), a specific inhibitor of the initial step of glutathione synthesis catalyzed by y-glutamyleysteine synthetase (Lappartient et al. 1999). From these results, the authors concluded that glutathione, rather than cysteine or another reduced-sulfur compound, regulates sulfate uptake. Previously, a similar conclusion was drawn from physiological experiments. When cysteine was applied in the presence of BSO, the reduction of sulfate uptake by cysteine was counteracted in excised tobacco roots and whole tobacco plants (Herschbach and Rennenberg 1991, 1994). However, in situ hybridization experiments demonstrated that the AST68 sulfate transporter is expressed in the central cylinder but not in the xylem, endodermis, cortex or epidermis of leaves and roots (Takahashi et al. 1996). Therefore, it seems more likely that this transporter is not directly involved in sulfate uptake by the roots and, thus, in the regulation of sulfur nutrition. Also Heiss et al. (1999) concluded that the low-affinity sulfate transporter is not involved in the regulation of sulfate uptake. The authors showed that the expression of the lowaffinity sulfate transporter decreased during cadmium exposure of B. juncea roots, i.e., under conditions of increased sulfate requirement for glutathione synthesis. They concluded that low-affinity sulfate transporters are involved in the regulation of sulfate transport to the shoot and that this transport was diminished under conditions of enhanced sulfate demand by the roots. However, sulfate uptake was not analyzed by the authors. Thus, these results suggest that low- and high-affinity sulfate transporters are regulated differently. From experiments with maize, Bolehi et al. (1999) proposed that the high-affinity sulfate-uptake system is regulated by L-cyst(e)ine, whereas the low-affinity system may be regulated by glutathione. In sulfur-deficient maize seedlings, the transcript of a high-affinity sulfate permease (ZmSTl) was downregulated by L-cyst(e)ine, both in the presence and the absence of BSO, a specific inhibitor of glutathione synthesis. In earlier studies with barley roots, glutathione and cysteine decreased during sulfate starvation, when sulfate uptake rates increased (Smith et al. 1997). Simultaneously, the mRNA level of the high-affinity sulfate transporter (hvstl) increased, probably due to decreasing repression by glutathione, cysteine or sulfate. Sulfate uptake is not only dependent on sulfur demand but is also adapted to the nitrogen nutrition of plants. Brunold (1993) proposed that O-acetylserine is the best candidate for coupling nitrogen and sulfur metabolism. O-Acetylserine enhanced the transcript of the high-affinity sulfate transporter hvstl in barley roots (Smith et al. 1997); as a consequence, glutathione and cysteine levels increased. Therefore, it was concluded that cysteine and glutathione act as negative regulators, whereas 186 Physiology O-acetylserine mediates positive control, counteracting glutathione and cysteine repression of the high-affinity sulfate transporter. Accordingly, sulfate uptake increased during O-acetylserine supply to maize cell cultures (Clarkson et al. 1999) and in excised beech roots (Kreuzwieser and Rennenberg 1998). These investigations demonstrate that sulfate uptake is related to the sulfur demand of the plants and that regulation at the pre-translational level is involved. However, the protein level of the sulfate transporter was not determined in addition to the mRNA levels in these studies. Therefore, it can not determined whether increasing sulfate uptake rates were the result of enhanced mRNA levels of the sulfate transporter or were caused by a combined increase of mRNA synthesis and translation. 5 Consequences of Phloem Translocation for the Regulation of Sulfate Uptake To maintain appropriate sulfur nutrition at the whole-plant level, sulfate uptake and sulfate reduction have to be adapted to the sulfur demand of the shoot and the roots. Therefore, allocation of a signal (from the shoot to the roots) that regulates sulfur nutrition has been suggested (Rennenberg 1984, 1995; Rennenberg and Lamoureux 1990). From the observations that glutathione (1) reduced sulfate uptake in heterotrophic but not in photo-heterotrophic tobacco cell cultures (Rennenberg et al. 1988) and (2) is the main reduced-sulfur compound allocated from mature tobacco leaves to the roots (Rennenberg et al. 1979), it was suggested that glutathione acts as a regulatory signal. Physiological experiments on glutathione transport from the shoot to the roots and its influence on sulfate uptake and sulfate transport to the shoot support this hypothesis. Feeding glutathione to a mature tobacco leaf diminished sulfate uptake by the roots up to 46% (Herschbach and Rennenberg 1994). H2S and S02 exposure oftobacco and spinach provided additional (but indirect) evidence of an interaction between the phloem allocation of glutathione and sulfate uptake. These gaseous sulfur compounds are taken up by leaves and partially metabolized into glutathione, which may be transported in the phloem to the roots (De Kok 1990; Rennenberg and Lamoureux 1990). In the roots, glutathione may be responsible for the downregulation of sulfate uptake (Rennenberg and Herschbach 1996). In spinach, reduced-sulfur levels of the roots increased as a consequence of H2S and S02 exposure of the leaves (Herschbach et al. 1995a) and, in both spinach and tobacco, sulfate uptake andlor sulfate transport to the shoot were reduced, depending on the level of sulfur nutrition (Herschbach et al. 1995a,b). A similar regulation of nitrate uptake by phloem-allocated glutamine was observed as a consequence of ammonia fumigation of beech trees (Gessler et al. 1998). Significance of Phloem-Translocated Organic Sulfur 187 In a split-root experiment, Lappartient and Touraine (1996) directly demonstrated that glutathione can act as a phloem-allocated signal that regulates sulfate uptake in B. napus. In this experiment, one part of the root system was subjected to sulfur starvation, whereas the other part was exposed under sulfur-sufficient conditions. Similar to the case in sulfur-starved roots, sulfate uptake increased (and glutathione levels decreased) in sulfur-sufficient roots of the split-root system. When glutathione was fed to one part of the roots in the split-root system of sulfur-sufficient B. napus seedlings, sulfate uptake of the other part was inhibited (Lappartient and Touraine 1996). Because glutathione levels decreased in phloem exudates after sulfur starvation and increased after feeding glutathione to sulfur-sufficient roots, the authors concluded that glutathione is the inter-organ signal regulating sulfate uptake. With the same split-root system, Lappartient et al. (1999) showed that the transcript level of the low-affinity sulfate transporter AST68 increased in sulfur-sufficient roots of B. napus and A. thaliana when the other parts of the roots were starved of sulfate. De-repression of the sulfate transporter was accompanied by low glutathione concentrations but not by low cysteine or sulfate concentrations in phloem exudates (Lappartient and Touraine 1996). Therefore, de-repression may be specifically attributed to the additional glutathione transported in the phloem (Lappartient et al. 1999). Investigations with poplar lead to the conclusion that the relationship of sulfur nutrition to the sulfur demand is more complex. Transgenic poplars that over-expressed y-glutamylcysteine synthetase in the cytosol exhibited higher cysteine, y-glutamylcysteine, glutathione and sulfate concentrations in phloem exudates than wild-type plants (Herschbach et al. 1998,2000). Taking into account the fact that glutathione is thought to repress sulfate uptake in many plants (Herschbach and Rennenberg 1991, 1994; Herschbach et al. 1995a,b; Lappartient and Touraine 1996; Lappartient et al. 1999), downregulation of sulfate uptake must be expected under these conditions. However, enhanced glutathione synthesis by the transgenic plants will require elevated sulfate uptake. Experiments with detached roots showed that the increased sulfate demand of the shoot was met by enhanced sulfate uptake by the roots in transgenic (compared with wild-type) poplars, irrespective of the enhanced glutathione level (Herschbach et al. 2000). These results clearly demonstrated that a high glutathione transport level in the phloem does not necessarily reduce sulfate uptake by the roots. Increased sulfate uptake rates of the roots of transgenic poplar plants were accompanied by enhanced sulfate concentrations in the phloem, demonstrating that sulfate could not be the phloem-mobile signal that reduces sulfate uptake by the roots (Herschbach et al. 2000). Thus, in poplar, neither phloem-allocated glutathione nor phloem-allocated sulfate seems to regulate sulfate uptake by the roots under conditions of enhanced demand. 188 Physiology However, enhanced glutathione transport in the phloem reduced sulfate transport to the shoot under conditions of decreased demand mediated by H 2S fumigation of the leaves in poplar plants (Herschbach et al. 2000). H2S exposure of poplar shoots increased glutathione concentrations in phloem exudates and, as a consequence, reduced sulfate transport to the shoot (Herschbach et al. 2000). Correlation analysis revealed that the sulfate-to-glutathione ratio in the phloem may be the inter-organ signal regulating sulfate uptake and the xylem loading of sulfate (Herschbach et al. 2000). High sulfate and low glutathione concentrations in the phloem seem to signal a surplus of oxidized sulfur, and sulfate uptake and sulfate transport to the shoot are diminished. Low sulfate-to-glutathione ratios in the phloem mediate increased sulfate uptake by the roots and increased sulfate transport to the shoot. Therefore, the hypothesis that the sulfate-to-glutathione ratio in the phloem can regulate sulfur nutrition may be a new starting point for future experiments on the regulation of sulfur nutrition at the wholeplant level. 6 Conclusions Published investigations indicate that sulfate uptake is adapted to the sulfur demand of the plant and that this demand is signaled to the roots by sulfur transported from the shoot. The sulfur compound(s) involved in signaling the sulfur demand of the shoot to the root are still a matter of debate, irrespective of whether physiological experiments or molecular analyses of sulfate transporters are considered. Physiological studies revealed that glutathione is involved in signaling the sulfur demand of the shoot to the root under conditions of reduced demand, but it may not operate as a signal when increased sulfate uptake by the root is required. Recent studies led to the conclusion that the sulfate-toglutathione ratio in the phloem may determine sulfur nutrition under conditions of either enhanced or reduced demand. Molecular approaches indicated that the transcription of high- and low-affinity sulfate transporters is regulated in different ways. Apparently, only high-affinity sulfate transporters are responsible for sulfate uptake and are adapted to the sulfate demand of the whole plant. The low-affinity sulfate transporter may be involved in the regulation of internal and sub-cellular transport processes. 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FEBS Lett 392:95-99 Takahashi H, Yamazaki M, Sasakura N, Watanabe A, Leustek T, De Almeida-Engler J, Engler G, Van Montagu M, Saito K (1997) Regulation of cysteine biosynthesis in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana. Proc Nat! Acad Sci USA 94:11102-11107 Yildiz FH, Davies JP, Grossman AR (1994) Characterization of sulfate transport in Chlamydomonas reinhardtii during sulfur-limited and sulfur-sufficient growth. Plant Physiol 104:981-987 Yildiz FH, Davies JP, Grossman A (1996) Sulfur availability and the SACI gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii. Plant PhysioII12:669-675 Ziegler H (1975) Nature of transported substances. In: Zimmermann MH, Milburn JA (eds) Encyclopedia of plant physiology, vol I. Springer, Berlin Heidelberg New York, pp 59-100 Communicated by U. LUttge Dr. Cornelia Herschbach Prof. Dr. Heinz Rennenberg Albert -Ludwigs- U niversitat Freiburg Institut fUr Forstbotanik und Baumphysiologie Professur flir Baumphysiologie Am Flughafen 17 79085 Freiburg, Germany e-mail: [email protected] Tel.: +49-761-2038303 Fax: +49-761-2038302 Physiology Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) Hartmut Gimmler "Where are all the undescribed fungi?" Hawksworth and Rossman (1997) 1 Introduction Mutualistic associations of microbes are widespread in nature, particularly in aquatic habitats. In such associations, two (or more) systematically distinct organisms mutually benefit from exchanges of food, protection, habitat or transport (Duchateau-Nguyen et al. 1995). The evolution of phototrophs featured the repeated emergence of mutualistic associations with fungi (Selosse and Le Tacon 1995). For example, Cyanophyta are involved in lichens, Rhodophyta and Chromophyta form some mycophycobioses, and Chlorophyta interacted during evolution repeatedly with fungi in the form of lichens or mycophycobioses. Interactions between fungi and green algae have already been described for the Devonian period (Taylor et al. 1992). Associations reach their highest level in the form of lichens or endosymbioses (Reisser 1992a; Hawksworth 1994; Kappen 1994). In lichens, unicellular algae (or cyanobacteria) are in direct contact with fungi. Both symbiotic partners form well-defined structural and functional units. The lichen lifestyle is found in various representatives of Dicaryomycotina. Analysis of small-subunit ribosomal DNA suggests at least five independent origins of lichens in distinct groups of Ascomycetes and Basidiomycetes (Gargas et al. 1995). Because parasitic, mycorrhizal and free-living saprobic fungi are involved, authors construe mutualism and parasitism as endpoints in the evolution of the lichen lifestyle. Endosymbiosis is characterized by the localization of the phycobiont in the cytoplasm of the host cell (which is usually a plant or animal cell, but in rare cases can also be a fungal cell; Mollenhauer 1992). Information regarding both types of symbiosis is legion (Reisser 1992a), and the topic is beyond the scope of this chapter. Instead, this review focuses on mutualistic relationships between freeliving algae and fungi coexisting in the same habitat without direct structural contact with each other. Such interactions have seldom been discussed in relevant reviews (compare, for example, Goff 1983; Reisser 1992a). However, the ecology and physiology offungalJalgal associations found in fresh and marine waters in which the fungi live as epiphytes on algae (preferentially seaweed; Van Donk and Bruning 1992) are beyond Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) 195 the scope of this review. In general, such associations have a saprobic or parasitic character and lack mutualistic relationships. Finally, because of obvious similarities, some mutualistic relations between free-living algae and bacteria will also be discussed. At the end of this chapter, we will discuss Geosiphon pyriforme, an endosymbiotic association between a Glomus-like fungus and strains of the cyanobacteria genus Nostoc. In this case, the phycobiont Nostoc is certainly not free-living. However, from the endosymbiotic association, an endosymbiotic algal partner can be isolated and cultivated, and its properties can be investigated separate from the fungal partner. 2 General Aspects Associations between free-living algae and fungi/yeasts, as reviewed in this article, take place exclusively in aquatic habitats. According to Jorgensen (1993), they reflect phase 1 of the evolution of terrestrial associations. This phase is characterized by facultative, commensalistic relationships. Flavonoids that protect against ultraviolet light were not yet required (Jorgensen 1993). The mutualistic relationships between the partners of such associations began with solute exchange in the water phase. Chemotactic attraction may be involved only occasionally, but it eventually leads to physical contact. A selective recognition allows a host to discriminate true symbionts from commensalists (Duchateau-Nguyen et al. 1995); recognition is a prerequisite for the evolution of real symbiotic or endosymbiotic relationships. Basic mechanisms of signal exchange, recognition, specificity and regulation of associations with direct physical contact have been discussed by Reisser (1992b) and Ahmadjian (1992). The associations discussed in this review very likely lack recognition systems, except for the Geosiphon system (see below). The best experimental proof of mutualistic interactions without physical contact and recognition systems comes from experiments in which one of the partners is separated from its associate by enclosure in a dialysis bag (Nakatsu and Hutchinson 1988). Mutualistic relationships between free-living microbes reflect an early evolutionary step during the development of real symbiotic or endosymbiotic relationships. Nevertheless, such relationships have been studied much less than the latter, probably for one of the following reasons: 1. It seems to be self-evident that mutualistic relationships between free-living algae and fungi are intermediate evolutionary steps on the way to the development of lichen symbiosis, so they have not been subjects of intense study. 2. The experimental analysis of mutualistic relationships of two distinct microbes coexisting in the same habitat is difficult, especially in the field. 196 Physiology 3. During recent decades, physiological processes in algae and fungi have usually been studied in axenic cultures. This tendency was necessarily strengthened by the expansion of molecular biology. However, this leads to the elimination of microbial associations in the laboratory. Consequently, there was apparently no need to investigate the biological significance of mutualistic relationships between free-living, unicellular algae and fungi. This review summarizes information scattered in the literature about this topic. It emphasizes the view that mutualistic relationships between free-living algae and fungi may improve the chances of both partners to conquer extreme habitats, a property that is often attributed to association between algae and fungi at the level of lichens only. Instead of giving a complete review of the recent literature, we focus on the description of mutualistic relationships under extreme environmental conditions based mainly on two case studies. 3 Mutualistic Relationships between Algae and Fungi in Acidic Habitats The survival of the fittest can be studied best under extreme conditions. However, as shown by a mathematical model (Weisbuch and Duchateau 1993), biological selection occurs not only at the level of individual organisms, but also at the level of their mutualistic associations. Therefore, in extreme environments, such as habitats with a highly acidic pH, biochemical and biophysical adaptations of individual species to high proton concentrations are not the only beneficial adaptations. Mutualistic relationships between distinct species may also improve competition during the evolutionary conquest of a habitat with harsh conditions, such as a pH of 1.0. In the following case study, two examples of the latter are described, one discovered in the laboratory and one during field studies. a) Stimulation of Growth The acid-resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) grows better in non-sterile than in axenic cultures (Fig. lA). This effect is thought to be due to chemical interactions between the alga and microbial contaminants. In fact, using classical microbiological techniques, it was possible to isolate an acid-resistant yeast and an acidtolerant, fIlamentous fungus from non-sterile cultures of D. acidophila. The latter is a new fungus imperfectus belonging to the family of Desmatiaceae (Moniliales). It is registered at the Centraalbureau voor Schim- Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) 10' 197 , A Dunaliella acidophila B Yeast present 10' , ~ I ., ~ ~ ~ OJ .a .a E ::s E ::J t: OJ U Euglena mutabilis (Red pond) non sterile culture t: OJ 10' U axenic culture Yeast absent 10' 100 Time [hI 200 • 10 15 20 Time [dl Fig. 1 A-B. Growth of the acid-tolerant alga Dunaliella acidophila (A) and Euglena mutabilis (B) in the presence or absence of fungal contaminants. D. acidophila was grown at pH 1.0 in an inorganic medium (Albertano et al. 1981) under non-sterile or axenic conditions (data of the author). E. mutabilis was grown at pH 2.0 in a culture medium complemented with 0.1 % proteose peptone. (Fig. 2c of Nakatsu and Hutchinson 1988) melcultures (Netherlands) as Bispora sp. (collection number 335.97). It is remarkable that this microbial contaminant is also present in D. acidophila cultures of various algal culture collections, although samples are sold as "axenic". This may indicate that some fungal material (spores?) is tightly attached to the surface of the alga and is difficult to separate from it by conventional microbiological techniques. In fact, at the surface of D. acidophila (but not of other Dunaliella species), reflection electron microscopy and transmission electron microscopy pictures show little "knobs", which have not yet been identified. The fungal contaminants of D. acidophila were detected in our laboratory cultures and in cultures of the Weizmann Institute of Sciences (Israel; Pick, pers. commun.). Euglena mutabilis and an associated yeast isolated from strongly acidic tundra ponds at the Smoking Hills (Northwest Territories, Canada) tolerate pH values between 1.8 and 2.0 (Nakatsu and Hutchinson 1988). The tolerance to acid pH is remarkably enhanced when E. mutabilis and the yeast, presumably a Cryptococcus species, were co-cultivated (Fig.lB). This mutualism occurred even between E. mutabilis from one field location and a yeast from another one. In every field collection made of Euglena, the Cryptococcus species was also present (Nakatsu and Hutchinson 1988). It is relatively easy to show that growth of D. acidophila or E. mutabilis is improved in the presence of the fungus relative to the yeast (Fig. 1). The experimental proof that the growth of the latter is also stimulated by the presence of the algae is more difficult because, for 198 I-:;';;;~;"P ~. . 1 u ~I~ L I [ I I I I [ I I I I [ I I I I I I 0 Alga present Alga absent E 0,5 .... c: Gl .0 0 0 E ::J c: u '0 '" :t'" Q; i ~ I 8,0 x 10' t NE >. ·1 Physiology ~ j I Cryptococcus sp. 1 1 --1 4,0 x 10' () j 0 0,1 0 5 Time 10 [d 1 15 0 5 10 Time [d 15 20 1 Fig. 2. A Growth of the filamentous fungus Bispora on agar with 2% glycerol as the carbon source (pH 1.0, 3% agar; composition of the medium as in Fig. 1A). B Growth of the yeast Cryptococcus in liquid culture in the presence or absence of Euglena mutabilis (pH 2.0, medium as in Fig. 1B; Fig. 1d of Nakatsu and Hutchinson 1988) obvious reasons, the heterotrophic organisms Bispora and Cryptococcus do not grow at all in the absence of organic carbon sources. The fllamentous Bispora is able to utilize glycerol as a carbon source (pH 1.0; Fig.2A). Glycerol is the main osmotic solute of D. acidophila (which contains between 30 and 3000 mM glycerol, depending on the salinity of the medium; Fuggi et al. 1988; Gimmler and Weis 1992). The permeability coefficient of the plasma membrane of Dunaliella cells varies between 3X10- 11 mls and 5XlO-13 mls (Gimmler and Hartung 1988). This value, together with the high internal concentration, is sufficient to permit the leaching of significant amounts of glycerol into the medium. Concentrations of up to 3 mM have been measured in the culture medium. Therefore, it is possible to assume that glycerol leakage from D. acidophila cells supports the growth of Bispora if co-cultured with the alga. Attempts to measure the growth ofthe fllamentous fungus in liquid culture in the presence of D. acidophila failed for technical reasons. For example, the growth of the fungus cannot be quantified by microscopy techniques. With respect to the EuglenalCryptococcus association, the methodological situation is better, because microscopy can be used to count cells. Figure 2B demonstrates that the unicellular Cryptococcus grows slightly better in the presence than in the absence of E. mutabilis at pH 2 (Nakatsu and Hutchinson 1988). Because, in this particular experiment, the medium was complemented with proteose peptone as the nitrogen and carbon source, the growth of Cryptococcus is already reasonably Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) 199 high in the absence of E. mutabilis. The stimulation of growth by the presence of Euglena must be due to some specific compounds that leach from the alga into the medium. b) Benefit of Algae by Association with Fungi The stimulation of algal growth by the presence offungi or yeasts (Fig. 1) is explained best by the assumption that fungi produce compounds of potential benefit to algae and excrete them into the medium. Then these solutes are taken up by the algae, followed by growth stimulation. In order to verify this chain of events, it must be demonstrated that the compound X: 1. 2. 3. 4. Is synthesized by the fungus or the yeast Is excreted into the medium Is taken up by D. acidophila or E. mutabilis Stimulates algal growth It is also feasible that a compound X is synthesized and excreted by the alga and causes inhibition of algal growth if it accumulates in the medium. Consumption of such a compound by the fungus would improve algal growth. In the following, two cases (vitamins and CO2 ) of the former assumption and one case (02) of the latter assumption are discussed. a) Vitamins Some fungi are well known for their ability to excrete vitamins into their medium, whereas others require the addition of certain growth factors to the medium. The composition of water-soluble vitamins of Bispora is shown in Table 1 (Gimmler and Carandang 1998). Concentrations are mostly in the nanomolar range. The main vitamin is B2, followed by B1• The contents of folic acid and vitamin B12 are very low; vitamin B6 was not detected. This does not mean that vitamin B6 is totally absent, but it may be present only in minor concentrations. All these vitamins could also be detected in the medium, with concentrations ranging from 40 pM to 0.2 11M. The relative concentrations in the medium correspond to the relative internal concentrations. Concentration gradients across the plasma membrane permit a passive efflux of vitamins into the medium, according to their chemical gradient. Much less is known about vitamins of the Cryptococcus species investigated by Nakatsu and Hutchinson (1988). Growth of most (but not all) investigated species of Cryptococcus is entirely absent in the absence of vitamins. Thiamine or biotin have to be added to the culture medium of 200 Physiology Table 1. Internal vitamin concentrations and concentrations in the medium of the filamentous fungus Bispora grown at pH 1.0 (Dunaliella acidophila culture medium; Gimmler and Carandang 1988) Vitamins Vitamin in Bispora sp. Concentration (nM) in the culture medium Gradient across the plasma membrane (VitamincytIVitaminned) Vitamin B2 990±610 211±10 4.7 Vitamin Bl 400±100 82±27 4.9 Folic acid 60±S6 0.42±0.042 143 Vitamin B12 0.039±0.0062 0.OS3±0.016 0.7 Vitamin B6 Not measured <6 many Cryptococcus species to obtain optimal growth rates (Lodder 1970; Barnett and Pankhurst 1974). However, this is not valid for all species. Because we are dealing with a taxonomically new Cryptococcus species, it is possible that (1) the strain isolated by Nakatsu and Hutchinson is one that does not require vitamins for growth and (2) certain vitamins leak from the yeast into the medium. If Cryptococcus is physically separated from E. mutabilis cells by growth in a dialysis bag, growth of the alga is still stimulated (Nakatsu and Hutchinson 1988). This demonstrates the involvement of a water-soluble, low-molecular-weight compound released by the yeast during the stimulation process. If vitamins playa role in the mutualistic relationships between acidresistant algae and fungi, then growth of axenic cultures of D. acidophila and E. mutabilis should be stimulated by the addition of vitamins. Indeed, many unicellular algae are auxotrophic and require vitamins, such as cyanocobalamin, thiamine and biotin, for optimal growth. Euglena gracilis, for example, is so sensitive to vitamin B12 that it is often used in bioassays for the quantitative determination of this vitamin in natural water (Kim et al. 1983). In addition, the growth of many Dunaliella species and of E. mutabilis is stimulated by vitamins under standard conditions, which include a neutral pH (Provasoli and Carlucci 1974). However, neither organism responded to the addition of selected vitamins in the expected way when cultured at an acidic pH. This seems to be a contradiction to the working hypothesis of mutualistic relationships. However, considering the conditions vitamins of the medium are exposed to during a cultivation experiment, it is clear that the danger of decomposition of vitamins is quite large. The high proton concentration, for example, may decompose vitamin B12, vitamin B2 and folic acid. These vitamins are also photo-labile and will be subject to photo-decomposi- 0.47 0.26 0.012 2x10-7 0.44 0.55 0.29 0.01 2.4xlO-7 0.10 Vitamin Bl Folic acid Vitamin B12 Vitamin B6 Culture medium Non-sterile Axenic 6.l±s.63 0.001±0.0001 s.4s±3.87 ±1.39 4.43±2.48 545 11.4 8.1 Non-sterile 0.s4±0.Os 61 1.2 3000 54 5.2 7.2 Axenic 0.2 1.2 0.8 1.1 1.2 11.3 1.6 8.4 2.4 1.3 Vitamionon.steriliVitaminaxenic Medium D. acidophila Gradient across the Vitamin accumulation plasma membrane (Vitamincy/Vitamin ned ) 0.0006±0.0004 4170 0.6s±0.34 1.36±0.02 3.4±1.8 In D. acidophila Non-sterile Axenic Vitamin concentration (flM) Vitamin B2 Vitamins oms 10.8 3.4 3.4 Alga/fungus Axenic Table 2. Internal vitamin concentrations and concentrations in the medium of the acid-resistant algae Dunaliella acidophila grown at pH 1.0 under non-sterile and axenic conditions (Gimmler and Carandang 1988). The vitamin gradients and accumulations were calculated from data of Table 1, except for the last column, which reflects a comparison of the vitamin concentrations of D. acidophila and those of Bispora sp. (Table 1), both grown separately under axenic conditions (pH 1.0) o .... IV ~ ~ 9- ~ oq &. ::s [a:: Cfl. a:: ::s 'TI p.. §"' ~ ~ I t::I:l >6' en f3- o·~ ::s ~ n 0. en ~ 2 s:: 202 Physiology tion during the 14-h light period of the light-dark regime applied during the cultivation of algae. In fact, it could be demonstrated experimentally that some water-soluble vitamins decompose in illuminated D. acidophila medium (pH 1.0; not shown). It is supposed that one clue during the support of algal growth by fungal vitamins in acid-tolerant associations may be the depot function of fungal cells; inside the fungal cells, vitamins are stabilized because of the neutral cytosolic pH. After excretion into the acid medium, a small part is immediately taken up by the algae, whereas the rest decomposes with time. However, the continuous excretion from the fungal depot also assures a continuous supply for the algae. Obviously, one critical parameter in this respect is the distance between the excreting and adsorbing organisms. The smaller the distance, the better are the chances to catch some of the excreted material before decomposition. Un stirred water bodies occurring in nature are expected to allow better transfer of solutes than strongly mixed cultures typically found in the laboratory. In Table 2, vitamin concentrations of D. acidophila are summarized. The order of the sequence corresponds approximately to that of Bispora (Table 1). The considerable vitamin gradient across the plasma membrane indicates that any uptake of vitamins from the medium into the algal cells occurs against the chemical gradient and requires special uptake mechanisms. Table 2 also shows that, except for vitamin B6, there is hardly any difference between the concentrations of non-sterile and o .£:: >. .c o c. e0 :E u ~bI E "0 E -0- Folie acid 0,5 ___ Biotin c: Q) -'" CIS • C. :l c: '...e CIS :> 0 0 50 100 Vitamin concentration [!-1 M 1 Fig. 3. Uptake of 14C-Iabeled biotin and folic acid into Dunaliella acidophila as a function of vitamin concentration (pH 1.0,20 °C, 30 min; Carandang and Gimmler 1998) Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) 203 axenic media. However, internal vitamin concentrations are higher inside cells cultured non-sterile than in cells cultured axenically. The concentrations of vitamin B2, vitamin BI and folic acid are higher in D. acidophila than in Bispora, whereas the opposite is the case for vitamin B12 . If the supposed uptake of water-soluble vitamins from the culture medium is facilitated, the vitamin incorporation should exhibit substrate saturation. This is indeed the case; the uptake of 14C-labeled folic acid and biotin into D. acidophila cells was saturated at vitamin concentrations close to 50 jlM (Fig. 3; Carandang and Gimmler 1998). This is in agreement with the working hypothesis of a transfer of vitamins from the fungus to the alga. Moreover, the experimentally determined vitamin concentrations in the media (Tables 1, 2) and the ranges of Km values for vitamin uptake that can be extracted from Fig. 3 indicate that the uptake systems are operating far below their Km values. The Euglena uptake mechanism for vitamin B12 has been particularly studied (Watanabe et al. 1988). In this system, a cobalamin-binding protein at the outer surface of Euglena plays a role in the uptake of vitamin B12. It has a molecular mass of 56,000 kDa and a high binding affinity for cobalamin (Ks=1.1 nM). (3) Dissolved Inorganic Carbon Respiratory rates of Bispora (20°C, pH 1.0) are in the range of 25 jlmol CO2 produced g-I fresh weight h-I. All CO 2 produced during fungal respiration will first equilibrate according to the intracellular cytosolic pH of 7.3 and will eventually occur in the culture medium (pH 1.0) exclusively as CO 2, Maximal rates of D. acidophila are in the range of 4000 jlmol CO 2 fixed g-I fresh weight h-I. In other words, in terms of rates, fungal CO 2 supply from respiration is 160-fold lower than the algal CO 2 demand for photosynthesis. The actual CO 2 balance in the culture vessel will depend on the rate of CO2 influx from the air into the culture medium and the individual contributions of biomass from both partners in the association. Under realistic culture conditions, the biomass contribution from the fungus may be greater than that of the alga. Because the rate of photosynthesis of air-adapted D. acidophila cells is high but the Km value of photosynthesis for the CO 2 species is unusual [approximately tenfold lower (21jlM) than that of other Dunaliella species (0.53 jlM); Gimmler and Slovik 1995], it is realistic to expect a stimulation of photosynthesis in the alga due to the respiratory CO2 of the fungus. In addition, for the acidophilic E. mutabilis, a limitation of photosynthesis by inorganic carbon was found in un stirred batch cultures, which may mimic field conditions better than cultures bubbled with air (or air enriched with 5% CO 2; Olaveson and Stokes 1989). 204 Physiology y) Abscisic Acid The phyto-hormone abscisic acid (ABA) is known to take part in signal transduction during stress. The fungus releases significant amounts of the phyto-hormone into the culture medium (not shown). This also applies to D. acidophila (Hirsch et al. 1989). Because no convincing physiological role of ABA in unicellular algae has yet been detected (Hartung and Gimmler 1994), whether or not ABA takes part in mutualistic relationships between Bispora and D. acidophila was not investigated. 0) Amino Acids Bispora does not require an organic nitrogen source; it grows well with either ammonium (standard medium) or nitrate as the only nitrogen source. However, inorganic nitrogen can be replaced by organic nitrogen, e.g., by the non-protein amino acid y-aminobutyric acid (Gimmler 2000). Analysis of the standard culture medium for amino acids did not present evidence of any significant efflux of amino acids from the fungus. This means that Bispora does not support the growth of D. acidophila by excreting amino acids. With respect to the E. mutabilislCryptococcus association, this point has not been clarified, because the media applied by Nakatsu and Hutchinson (1988) always contained proteose peptone. c) Benefit of Fungi or Yeasts in Associations with Algae a) Carbon Source The benefits fungi get by coexisting with algae are much more obvious that those the algae get by living together with a fungus. Fungi and yeasts are heterotrophic and require carbon sources for growth. The occupation of an acidic habitat by a yeast or a fungus is limited by the presence of a carbon source in this ecological niche. There are two possible options for such carbon sources: 1. Contamination ofthe habitat with organic debris. 2. Previous occupation of the site by an autotrophic organism, which releases organic matter into its environment. The term "release" must be used in a rather general way. It does not necessarily have to reflect a special efflux mechanism. 205 Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) The dying of cells in the acid medium and the subsequent extraction would also supply the required organic material. With respect to the association between D. acidophila and Bispora, it is clear that: 1. The major osmotic solute ofthe algae is glycerol. 2. The permeability coefficient of the plasma membrane is 3xlO- ll mls (Gimmler and Weis 1992). 3. The glycerol concentrations in the culture medium reach levels of up to3mM. 4. Bispora utilizes glycerol as carbon source (Fig. 2). For the uptake, it is assumed to express a glycerol uptake mechanism in the plasma membrane. For E. mutabilis, organic osmotica and the excretion of organic solutes into the medium have been less studied. Trehalose, maltose and sucrose are the major low-molecular-weight carbohydrates of Euglena, but nothing is known regarding the excretion of these solutes (Hellebust 1974). Under certain conditions, Euglena excretes significant amounts of glycolate into the medium. Yeasts usually do not grow with this substrate as the only carbon source (Lodder 1970). However, Euglena excretes amino acids (Hellebust 1974), which can support the growth of Cryptococcus . .... ;,!! 0 0 0 ....... 150 C '; o<J ._I:: 100 I:: II CI> ... «-e I:: 50 ~ 0 0..- ~ c A 1 h N, ..... 4 h N, ...... (5 -+- 24 h N, .... 48 h N, I:: .... 0 150 0 0 -, 0 '0 40 20 60 ~ 0 ...c: 100 - Time of nitrogen bubbling [ h) (\) o=' 100 c: B 0 0 :;:'0 "''-c a.. 0 a.. '- o <J «0 ~ 50 ~ 50 0 I=~ « 0 0 0 20 40 Time of nitrogen bubbling (hI 60 ---- 1 20 40 60 Time [h] Fig. 4 A-C. Effect of anerobiosis on the adenosine triphosphate (ATP) pool (A) and the ATP/adenosine diphosphate ratio (B) in the acid-resistant filamentous fungus Bispora (pH 1.0). C Recovery of the system after various times of anaerobiosis by oxygenation. (Carandang. unpublished) Physiology 206 f3) Oxygen Bispora is an obligate aerobic organism and requires 02 for growth. Under anaerobic conditions, the adenosine triphosphate (ATP) pool and the ATP/adenosine diphosphate ratio drop drastically (Fig.4A,B) and recover rapidly on re-aeration (Fig. 4C). However, prolonged times in the absence of 02 strongly decrease the extent of recovery of the fungus (Fig.4C). In addition, all Cryptococcus species investigated so far are unable to perform fermentative reactions (Barnett and Pankhurst 1974). Therefore, both organisms are very likely to be stimulated by the photosynthetic 02 evolution of coexisting algae if fungal respiration is not saturated by 02. A decrease in the 02 concentration in the environment of the algae by fungal respiration could exert another beneficial effect on the algae: it could increase carbon efficiency by lowering photorespiration. In Fig. 5, all available information about mutualistic relationships between D. acidophila and Bispora coexisting at pH 1.0 is summarized. Using all indicated rates and rate constants, future computer models will Ambient air Culture medium (pH 1.0) :--...- + ... Glycerol",..., c~ \1 :' Vito",., _->:I,_ _ _......L.I_l_-_;um____,_, - .. Glycerol.",.,,' RlplratlOn I G~"",,"_ pH 7.3 "'~-Vilm.dlum --~- Vl~ - -~it~~~- DIC""",", DJIHvoI __ • ': JFUNGUS Glycerolmodlum - - - . - :- Glycerol",_ : Dead cell$ __ _ _ I Fig. 5. Compilation of putative mutualistic interactions between Dunaliella acidophila and the filamentous fungus Bispora coexisting in an inorganic medium of pH 1.0. Growth is symbolized by the formation of daughter cells; microbial death is symbolized by compartments surrounded by dotted lines. Black dots at membranes are supposed sites of catalyzed translocation. D Decomposed (physiologically inactive) vitamins, DIG dissolved inorganic carbon. In principle (but not in all details), the model is also valid for the Euglena mutabilislGryptococcus association Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) 207 allow us to prove or disprove predictions made by our working hypotheses. Of special importance will be extrapolations to field conditions. In principle, the model can also be applied to the E. mutabilislCryptococcus association. However, much required information is missing. This particularly applies to the carbon source excreted by Euglena and utilized by the yeast. 4 Mutualistic Relationships Between Algae and Bacteria Reports of mutualistic relationships between algae and bacteria are rare in comparison with those described between different bacterial species. In lake waters, bacteria are frequently associated with Anabaena heterocysts (Pearl and Kellar 1978). The association is initiated by chemotactic responses of the bacteria. Experimental evidence suggests that the bacteria benefit by utilizing algal excretion products. In return, the bacteria stimulate nitrogen fixation of the heterocysts by removing 02 through respiration in micro-zones around the heterocysts during periods of high ambient 02 concentration in the lake water (Fig. 6). It could be shown that, in the presence of bacteria, Anaebaena rapidly overcomes nitrogenase inhibition by elevated 02 concentrations, whereas axenic Anabaena oscil/arioides + bacteria ;!. 150 o o ~ c:o ~ 100 ~---- --- - 50 Anabaena oscil/arioides, axeni ?;- :~ u "' CI> ::: c: CI> Cl .~ Z o o 2 4 Time (h] Fig. 6. Nitrogenase activity in axenic and non-sterile cultures of Anaebaena oscillarioides, as affected by the transfer from ambient 02 concentration (control) to elevated 02 levels (p02=0.40 atm; Pearl and Kellar 1978). On the x-axis, the time after replacement of the gas phase is displayed. Nitrogenase was measured in vivo by the reduction of acetylene 208 Physiology Anabaena cultures exhibit a stronger initial inhibition of nitrogenase by 02 and a longer time for recovery from this inhibition. In another study, Steinberg and Bach (1996) reported that the growth of Scenedesmus subspicatus was only stimulated by groundwater fulvic acid from lignite leachate if bacteria were present. This effect occurred even though fulvic acids clearly quenched the light available for photosynthesis of Scenedesmus. It was concluded that fulvic acids were utilized by the bacteria as a carbon source that produced and released organic substances (vitamins?) that stimulated the growth ofthe algae. Interactions between algae and bacteria are also observed in terrestrial coenoses (for example, on limestone, where they exhibit a trophic structure; Zenova et al. 1990). Green algae (Chlorella, Hormidium, Pleurococcus, Chlorococcus) and cyanobacteria (Synechococcus, Phormidium, Oscillatoria) function as autotrophic producers; gram-positive and gram-negative bacteria (i.e., Streptomyces) function as reducers of minerals and consumers. In their metabolic reactions, algae/bacteria associations differ from those of the same micro-organisms cultivated separately in axenic cultures. Some interactions between acidophilic bacteria will be briefly discussed, because some of these interactions involve specific aspects of acidophile metabolism in acidic environments (Johnson 1998; Norris and Johnson 1998). For example, the chemolithotrophic extreme acidophiles Thiobacillus thiooxidans and T. ferrooxidans are phylogenetically closely related (Lane et al. 1992) and coexist in the same habitat. Thiobacillus oxidizes elemental sulfur or H2S to sulfuric acid. Strongly acidic environments, however, are not only ecological niches for acidophilic algae such as D. acidophila; they are also required for iron-oxidizing bacteria, such as T. ferrooxidans. The substrate for the energy metabolism of the latter is ferrous iron, which is stable in oxygenated solutions only at high acidity (Norris and Johnson 1998). Mutualistic interactions have also been observed between the chemolithotrophic, acidophilic iron oxidizers, such as Leptospirillum ferrooxidans or T. ferrooxidans, and heterotrophic acidophiles, such as Acidiphilum or Ferromicrobium acidophilus (Johnson 1998; Fig. 7). The iron oxidizers tend to release organic acids into the medium; these are entirely protonated at acidic pHs. Whenever they accumulate to higher levels, a feedback inhibitory mechanism occurs; plasma membranes are highly permeable to protonated organic acids, but not to the anions. Therefore, the acids (after re-entry in the protonated form) accumulate inside the cells, causing a collapse of the intracellular pH via indirect proton transfer. This inhibits cellular metabolism and, therefore, growth. However, in the presence of acidophilic heterotrophs, organic acids are utilized as a carbon source (Fig. 7) and, therefore, are kept at a low level. This mechanism is also thought to be the reason L. ferrooxidans and T. ferrooxidans remain viable for much longer periods in resting cultures 209 Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) Ambient air Thiobacillus ferrooxidans Leptospirillum ferrooxidans Acid water phase (Chemolithotrophic iron oxidizers) O2 CO2 ~------ C02 ~--I'----- C02 1 calvin~ Cycle Respiration Glycolysis TCA -{YCle pH 1.0 (55 0 C) 02 ~ ~esPiration Glycolysis TCA -{YCle pH 7.0 / H+ + ROO' ~ ROOH ~'--------.----'---___ ~ ROOH ~ ROO' + H+ H+ + ROO' ~ ROOH --,---+ ROOH Acidophilium sp. Ferromicrobium acidophilus (Heterotrophic acidophiles) Fig.7. Compilation of putative mutualistic interactions between chemotrophic acidophilic iron oxidizers and heterotrophic acidophiles coexisting in an acid habitat (pH 1.0, SSOC). The dotted box symbolizes the pool of organic acids in the environment of the bacteria. The pool is filled by organic acids from autotrophic, chemolithotrophic acidophiles. Heterotrophs use these organic acids as a carbon source. The absence of heterotrophic acidophiles causes a considerable increase in the external organic acid concentration, followed by re-entry of the organic acids into the autotrophic acidophiles, subsequent interference with the internal pH (indirect proton transfer) and inhibition of growth. Black dots at membranes are supposed sites of catalyzed translocations. The scheme is drawn according to the information presented by Johnson (1998) and the references therein when the culture contains contaminants, such as Acidiphilum or F. acidophilus (Johnson and Roberto 1997). Natural acidic habitats are usually low in dissolved organic compounds (DOCs) and are considered to be oligotrophic. In contrast, in acidic mine drainage water, a large number of various organic inputs can be identified. Microbial settlement in the natural acid habitats depends on the presence or absence of light. In the absence of light (i.e., in the absence of photosynthetic acidophiles), primary production is restricted to sulfur- and iron-oxidizing bacteria in the manner described above. Because these bacteria may excrete low-molecular-weight compounds, and because dead bacterial cells may increase the level of DOCs, heterotrophic, acidophilic bacteria, such as Acidiphilum or F. acidophi- 210 Physiology lus, may benefit from the bacterial production of DOCs (Johnson 1998). In the presence of light, acidophilic algae are likely to contribute to the DOC levels of acid habitats. This enables not only the growth of fungi (as described above) but also that of heterotrophic bacteria. For acidophilic heterotrophic bacteria, the advantage of coexisting with phototrophic algae rather than chemolithotrophic bacteria is the photosynthetic production of 02 by the algae. Most acidophilic bacteria are described as obligate aerobes (Johnson 1998). In summary, the benefit acidophilic heterotrophs receive by coexisting with chemolithotrophic bacteria and/or phototrophic algae is quite obvious. However, many more studies will be required to elaborate the supposed mutualistic relationships between acidophilic algae and bacteria. At present, it is reasonable to assume that relationships similar to those described between acidophilic algae and fungi may exist. 5 The Geosiphon Association Contrary to the associations of free-living microbes described so far, G. pyriforme is an endosymbiotic association that includes direct physical contact (Schnepf 1964). It consists of a terrestrial Glomus-like fungus as the macrobiont and the cyanobacterium Nostoc as the microbiont (Mollenhauer 1992). It is considered in this review because the Nostoc strain involved differs from many other endosymbiotic cyanobacteria in that it can be propagated after isolation from the host. This indicates a rather primitive stage of endocyanosis. The photosynthetic carbon assimilation of G. pyriforme (Kluge et al. 1991) and its photosynthetic capacity (Bilger et al. 1994) have been studied. Results indicate that the photosynthetic capacity of the endosymbiotic Geosiphon system is higher than that of the isolated cyanobacterium, which demonstrates that the latter benefits from coexistence with the fungus. The mechanistic reason for this observation is not known. The benefit of the fungus from endocyanosis is much more obvious. If infected with Nostoc, it can grow in the light on a totally inorganic medium (Kluge et al. 1991). When the fungus lacks its endosymbionts, it does not grow at all under the same conditions. G. pyriforme expresses a nitrogenase in the heterocysts of Nostoc; therefore, it is able to fix N2 (Kluge et al. 1992). Heterocysts of endosymbiotic Nostoc are significantly larger than those of freeliving Nostoc. A special transport system for glutamate and aspartate is localized in the plasma membrane of this Nostoc strain (Strasser and Falkner 1986). It operates as counter-exchange mechanism in light. However, in the dark, unidirectional release of glutamate causing a net release of this amino acid is observed. Data imply that the N2 fixation of the cyanobacterium provides a considerable ecological advantage to Geosiphon. The fungal respiratory activity may increase the efficiency of Mutualistic Relationships Between Algae and Fungi (Excluding Lichens) 211 nitrogenase by lowering the partial 02 pressure in the close environment of the endosymbiotic Nostoc and may thereby offer a benefit to the phycobiont. Further studies demonstrated that the fungus of the Geosiphon association has some properties resembling those of fungi forming arbuscular mycorrhiza (Schussler et al. 1994, 1996; Gehrig et al. 1996). Recently, specific recognition of the establishment of the Geasiphon endosymbiosis by incorporation of Nostoc into the fungus was also studied (Schussler et al. 1997). The observation that the cell wall of the fungus is an effective diffusion barrier against release or uptake of carbohydrates such as glucose or sucrose is of great interest for forthcoming studies of solute exchange in Geosiphon (Schussler et al. 1995). This implies that the fungus has some difficulty during the uptake of external carbon sources and depends heavily on carbohydrates produced internally by the endosymbiotic Nastoc. However, it must be remembered that the mycobionts of lichens also synthesize a proteinaceous and highly hydrophobic cell-wall surface layer (Honegger 1997). This material spreads over the wall surface of the phycobiont of the lichen during the first contact of fungal hyphae with juvenile algal cells. This hydrophobic coat in the thalline interior of a lichen can be considered an equivalent of a cuticle. Therefore, solute exchange and metabolic interaction at the mycobiontphycobiont interface of lichens must be much more complex than assumed earlier. Perhaps this phenomenon represents a link between the terrestrial Geosiphon association and lichens adapted to terrestrial habitats. However, it also has an important difference from mutualistic associations between free-living microbes in aquatic habitats, particularly acidic habitats. The latter requires massive solute exchange across the surface of the involved organisms. Any hydrophobic surface layer would reduce the efficiency of exchange (Burgstaller 1997). It has to be remembered that the efficiency of solute exchange between partners of an association is much lower in associations of free swimming microbes, because the volume separating the microbes is larger than in the Geosiphon system or in lichens, where no (or only relatively small) volumes separate mycobionts and phycobionts. Acknowledgements. I am grateful to Prof. W. Hartung for critical reading of the manuscript. Part of the work summarized in this review was supported by the Deutsche Forschungsgemeinschaft (SFB 251, TP A2). 212 Physiology References Ahmadjian V (1992) Basic mechanisms of signal exchange, recognition and regulation in lichens. In: Reisser W (ed) Algae and symbiosis. Biopress Limited, Bristol, pp 675-698 Albertano P, Pinto G, Santisi S, Taddei R (1981) Spermatozopssis acidophila Kalina (Chlorophyta, Volvocales), a little known alga from highly acidic environments. G Bot ItalI15:65-76 Barnett JA, Pankhurst (1974) A new key to the yeasts. 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Luttge Prof. Dr. Hartmut Gimmler Julius-von-Sachs-Institut fur Biowissenschaften Lehrstuhl Botanik I der Universitat Wurzburg Julius-von-Sachs-Platz 2 97082 Wurzburg, Germany e-mail: [email protected] Physiology The Extracellular Matrix of the Plant Cell: Location of Signal Perception, Transduction and Response Karl-Josef Dietz 1 The Extracellular Matrix is a Dynamic Component of the Plant Cell The plant protoplast is enclosed by a rigid corset mainly built from carbohydrates that, once formed during cell development, apparently remain invariable. This misleading perception is supported by the static appearance of plant cells in microscopic studies. For instance, a leaf typically will not change its habitus following full expansion. It is a module built for specific physiological functions and will be shed under sub-optimal conditions. However, during recent years, a large body of knowledge proving that the extracellular matrix (ECM) is responsive to a large array of internal and external stimuli (some of which will be discussed below) has accumulated. In the following discussion, "internal signals" are considered to be produced endogenously in the plant, whereas environmental parameters may act as external stimuli. The ECM's potential to remodel is illustrated by some examples: 1. Almost any plant tissue can be used for plant regeneration in situ; 2. 3. 4. 5. 6. this involves a major reconstruction of the ECM and adaptation of apoplastic biochemistry. The man-made grafting of organs. Similar inter-plant connections occasionally form in nature following tight contact of two "compatible" plants. The shedding ofleaves or fruits. The formation oflateral roots. Wound healing. The development of functional junctions between parasite plants and host plants. Thus, the microscopic invariability of typical plant cells reflects the lack of developmental requirements, the de-activation of the corresponding programs and the absence of appropriate stimuli. However, even apparently inert plant cells rapidly and specifically respond to growth conditions by changing the apoplastic milieu and the structure of the ECM. Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Physiology 216 Apoplasl A) chemical stimulus Cytosol @ B) chemical stimulus @ downstream events <® C) physicaJ stimulus Fig. 1 A-C. Simplified scheme of signal transfer from the extracellular space to the symplast. A A stimulus activates a receptor which transduces the signal to the cytoplasm. B The stimulus is transported to the cytoplasm. C A signal is transmitted to the symplast via the cell-wall cytoskeleton continuum. In each of the three cases, downstream events inside the symplast trigger the response Furthermore, the ECM and the plasma membrane facing the apoplast are sites of signal perception. The perceived signals are transformed into biochemical or genetic responses that usually help the plant to adapt to the prevailing growth conditions. These events depend on specific signal-transduction pathways downstream of the stimulus perception. Figure 1 summarizes three pathways involved in ECM-cytoplasm signaling; these will be discussed below. The field of signal transduction has developed with great speed due to the employment of genomic approaches. Therefore, in this review, the dynamics and multiplicity of ECM function can only be addressed with selected examples and cannot be covered exhaustively. a) The Cell-Wall Structure and the Proteins of the ECM The plant cell wall forms a rigid network of polysaccharides, proteins, lignins and other minor compounds. The cellulose micro-fibrils are coated with xyloglucan. The micro-fibrils are inter-connected with arabinoglucan and xyloglucan molecules. The network is embedded in a matrix of polygalacturonic acid derivatives that are salt-bridged (in part) by Ca2+. Other polymers, such as rhamnogalacturonan and various structural proteins [for example, hydroxyproline rich proteins and arabinogalactan proteins (AGPs)], are attached to the basic framework. These basic constituents of the cell wall have been investigated at the chemical and molecular levels (Carpita and Gibeaut 1993; Showalter The Extracellular Matrix of the Plant Cell: Location of Signal Perception 217 1993). The structural significance of the diverse components of the plant cell wall is being analyzed using selected mutant phenotypes and genetically engineered plants (Reiter 1998). Additionally, there are two new fields of cell-wall structure and function. 1. It has been recognized that the cell wall is attached to the plasma membrane and the cytoskeleton, thereby creating an ECM-cytoplasm continuum in plants similar to that in animal cells. Macromolecules involved in the formation of attachment sites are AGPs with glycosylphosphatidyl inositol (GPI) anchors (Youl et al. 1998), cell-wallassociated kinases (Braun and Walker 1996) and integrin-like proteins with arginine-glycine-aspartic acid (RGD)-binding sites (Reuzeau and Pont-Lezica 1995). The cell-wall!cytoplasm connection has strong implications for signaling during development and adaptation and will be discussed below. 2. New biochemical mechanisms that allow remodeling of the cell-wall structure in response to developmental or environmental stimuli have been elucidated. Three examples are given here: 1. Xyloglucan endotransglycosylases, which cleave xyloglycan backbones, are suggested to form new linkages, thereby remodeling the cell-wall structure (Thompson et al. 1997). 2. Expansins, which show no detectable glycanase activity, facilitate cell-wall extension in vitro and in vivo (Cosgrove 1999). 3. An increasing number of extracellular proteases that exhibit celland development-specific expression are being identified (Jorda et al. 1999). They are likely to function in specific developmental contexts and are not (or at least not exclusively) associated with pathogen defense, as previously hypothesized (Vera and Conejera 1988). The protein content of the cell-wall fraction is usually approximately 10%. In addition to the cell-wall-bound proteins, a fraction of soluble proteins with an apparently low complexity of fewer than 100 polypeptides can be extracted from the apoplast. However, it should be noted that a thorough analysis of minor apoplastic proteins has not been performed and is critical due to the contamination of intercellular washing fluid with cytosolic material. The genomic approaches will catalyze important progress by allowing the identification of genes with putative signals for secretion. In addition to proteins with structural functions or lytic or synthetic activity, the apoplast is the site of accumulation of inhibitors in the context of either metabolic regulation or defense. Inhibitors of invertase (Weil et al. 1994), polygalacturonidase (Leckie et al. 1999) and proteases (Ojima et al. 1997) illustrate the diversity of inhibitor proteins secreted into the extracellular space. 218 Physiology b) The Chemical Composition of the Apoplast The chemistry of the apoplast was reviewed previously (Dietz 1997). Various techniques and methods have been employed to study the pH of the bulk water phase of the apoplast and have shown that the apoplastic pH is usually slightly acidic in differentiated tissues, ranging from pH 5 to pH 6.5 (Dietz 1997). Successful methods were pH-sensitive electrodes (Felle 1998) and various pH-sensitive non-permeating fluorophores, which offer time and spatial resolution (Hoffmann and Kosegarten 1995; Miihling et al. 1995; Taylor et al. 1996). The apoplastic pH changes with the developmental state of the tissue (Taylor et al. 1996) and with environmental factors (Miihling et al. 1995) and reveals a micro-heterogeneity (for example, in the vicinity of the stomata and in the xylem vessels of leaves; Canny 1995; Hoffmann and Kosegarten 1995). As discussed below, the apoplastic pH is an important factor triggering signal transduction from the ECM. Its influence on channel-gating properties is well established and may be postulated for enzyme activity and receptor binding. The apoplastic ion concentrations are low under non-saline growth conditions (total cation and anion concentration in the below 100-mM range) but increase with salinity stress (Speer and Kaiser 1994; Dietz 1997). Accumulation of inorganic ions in the apoplast are assumed to playa significant role in the progress of damage in glycophytic species (Speer et al. 1994). It has been shown in vitro that cell-wall enzymes from glycophytes and halophytes do not differ in salt tolerance (Thiyagarajah et al. 1996). In addition to proteins and inorganic ions, the apoplastic fluid contains sugars, amino acids, ascorbic acid, hormones and hormone conjugates at low but physiologically relevant concentrations (Dietz 1997). Important compounds defining the redox chemistry of the apoplast are the reactive oxygen species (ROSs) superoxide and hydrogen peroxide and the ascorbic-acid pool. ROSs are involved in the cross-linking of cell-wall components and in defense reactions. Interestingly, in vitro cross-linking of proteins to the cell-wall fabric can also be replicated in a test system containing cell-wall peroxidase, cysteine (as the reductant) and alkaline buffer (Wojtaszek et al. 1997). It should be noted that the dynamics of the chemical composition of the apoplast have not been studied in detail during plant development, nor has the dependence of the dynamics on environmental parameters. Due to the relatively small volume of the apoplast (usually between 40 and 100 ~1!g fresh weight), homeostatic mechanisms are likely to control the ionic and metabolic compositions of the ECM, as has been shown for the extracellular phosphate concentration (Mimura et al. 1992). In addition, it is not known whether the apoplast is an "oxidizing" milieu throughout tissue development or whether more reducing conditions occur and allow specific biochemical steps (such as expansin-dependent The Extracellular Matrix of the Plant Cell: Location of Signal Perception 219 cell expansion) to proceed at some developmental stages. This will have to be investigated. c) Remodeling of the ECM during Development The development of a plant cell includes the synthesis of the primary wall as the first step. In a second step, the meristemic cells expand and develop their final shape, a process that might be defined as remodeling of the ECM. However, even fully differentiated cells undergo remodeling, as is evident during fruit ripening or the formation of graft junctions or lateral roots. Such processes depend on positional information and the induction of appropriate developmental programs. Two examples may illustrate this relationship. Attachment of expansin-coated beads to the apical meristems of tomato plants induced bulging and the formation of leaf-like structures (Fleming et al. 1997). In agreement with these results, LeExp18, an expansin gene, was shown to be expressed specifically in cells at the site of primordium formation and to function as a molecular marker for leaf initiation (Didier et al. 1998). The authors suggested a model postulating a crucial role for expansins in leaf-primordium initiation. This example shows that the differentiation involves cross-talk between the ECM and the symplast. Another example for such an interaction is the formation of secondary plasmodesmata at graft junctions (Fig. 2). During this process, two separate internodes of compatible species establish a functional anatomical connection. On the microscopic level, walls of neighboring cells are lytically degraded at sites of close attachment between the endoplasmic reticulum (ER) and the plasma membrane. The formation of functional plasmodesmata rather than C) D) Fig. 2 A-D. Schematic representation of the formation of secondary plasmodesmata at graft junctions (Kollmann and Glockmann 1991). The cell walls of (A) two adjacent cells are degraded at (B) endoplasmic reticulum (ER)-plasma membrane attachment sites, and median cavities are formed. C The ER strands of both cells come into contact and fuse. D By reconstructing the cell wall, fully functional secondary plasmodesmata are formed 220 Physiology dead ends depends on the mutual exchange of information regarding the site of cell-wall lysis and the coordinated development of fusion sites (Kollmann and Glockmann 1991). Freely diffusible factors are not suitable candidates for signal molecules transmitting information with sufficient spatial precision. Conversely, physical stimuli derived from the cell wall can provide the required spatial resolution. Similar developmental coordination is required for vascular differentiation in the graft union (Wang and Kollmann 1996) and for many other developmental processes (for example, for the development of functional connections between cormophytic parasites and their host plants). d) Responses to Environmental Stimuli Adaptation to adverse growth conditions, such as high salinity, atmospheric pollutants, low or high temperatures, heavy-metal contamination or pathogen attack, frequently involves the induction of defense reactions and adaptive responses on the gene level. A subset of pathogenesis related proteins is secreted to the ECM and serves various functions there. Reinforcement of the cell wall, deposition of wax material at the cuticular surface, increase in lytic capacity, anti-microbial and antifungal activities, degradation of foreign proteins, and detoxification of 0 3 and S02 exemplify the mUltiple apoplastic functions of plant tissues in response to environmental parameters (Dietz 1997; Sakurai 1998). Heavy-metal and salt stress induce changes in the apoplastic-protein composition (Brune et al. 1994; Ramanjulu et al. 1999). In both cases, it was shown that de novo synthesis is responsible for the stress-dependent accumulation of apoplastic proteins (Blinda et al. 1997; Ramanjulu et al. 1999). These studies also demonstrated that the apoplast is rapidly supplied with newly synthesized proteins, which is important for rapid responses to external growth parameters. In the presence of toxic cadmium concentrations, the increased expression of lipid-transfer protein correlates with the increased deposition of waxes on the cuticular surfaces of barley leaves (Hollenbach et al. 1997). Abscisic acid was recognized as the putative signal coupling the heavy-metal stress to the increased expression of lipid-trans fer-protein genes. Heavy metals interfere with root development and the water status of the plant. Therefore, increased synthesis of cuticular material may be involved in decreasing cuticular transpiration. Salt-sensitive plants reveal inhibited rates of root elongation. This is accompanied by changes in cell-wall properties. Increased yield thresholds of the cell walls of salt-treated maize roots in psychrometric analyses (Neumann et al. 1994) and the decreased extensibility (but lower mechanical strength) of cell walls of NaCI-adapted tobacco cells indicate altered cell-wall composition (Bressan et al. 1990). The contents of both insoluble and soluble proteins increased threefold The Extracellular Matrix of the Plant Cell: Location of Signal Perception 221 and sevenfold, respectively, in walls of tobacco cells during salt adaptation (Bressan et al. 1990). In addition to the proteins of control cells, new apoptastic proteins were synthesized under salt conditions. These observations demonstrate the flexible response of the ECM to environmental conditions. A recent observation concerns the structural rearrangement of the ECM/plasma-membrane interface during adaptation to cold stress. In Jerusalem artichokes, remodeling of the ECM/plasma-membrane attachment sites takes place during long-term acclimation to sub-freezing temperatures (Murai and Yoshida 1998). This suggests that the development of frost hardiness is related to a reduction in cell-wall/plasmamembrane attachment sites and may be related to the prevention of mechanical damage to the plasma membrane during freezing. e) Pathogenesis-Related Deconstruction or Adaptation of the Cell Wall Plant pathogens and saprophytes have to relate to the plant cell wall as site of first contact and colonization. Therefore, they secrete a battery of cell-wall-degrading enzymes, such as pectinases, cutinases, cellulases, xylanases and proteases (Walton 1994). However, products of this degradation process can be exploited as signals by the plant cell and can be transduced into proper defense responses. Receptors facing the apoplast perceive these signals, and complementary DNA (eDNA) sequence information regarding pathogenesis-related receptors are available (Scheel 1998). An important defense reaction of plant cells involves the production of toxic ROSs to induce local cell death (Baker et al. 1997). Plant plasma membranes contain reduced nicotinamide adenine dinucleotide oxidases, which reduce 02 to the superoxide anion radical (°2--) at the extracellular site. Either spontaneously or catalyzed enzymatically by superoxide dis mutase, 2-- is disproportionated to H20 2 and 02. ROSs are substrates for cross-linking reactions in the cell wall, defense-gene activation, or the triggering of programmed cell death. The latter process is the hypersensitive response. Frequently, the hypersensitive response is followed by the development of systemic acquired resistance (SAR) in plant parts not previously exposed to the pathogen. During a screening for mutants that exhibited the hypersensitive response but are unable to develop SAR, DIR1, a lipid-trans fer-protein homolog, was identified (Cameron et al., pers. commun.). In another approach using activationtagged lines, two related genes were identified that, when overexpressed, cause either constitutive disease resistance (CDR1) or constitutive disease susceptibility (CDS1; Xia et al., personal communication). Both cDNAs are 40% homologous and encode putative aspartate proteases. The biochemical functions of the gene products, particularly given ° 222 Physiology the opposite effects on disease development, are not yet understood. Pathogenesis-related defense reactions, signal transduction events and remodeling of the cell wall represent the best-analyzed examples demonstrating the dynamics of the ECM, but they cannot be covered in any detail in this review. 2 Signal Perception in the Extracellular Space As described above, the apoplast is a cellular space with a distinct chemical composition and physical context. Accordingly, a number of extracellular parameters may serve as signals to induce developmental or adaptive responses of adjacent cells. Table 1 summarizes some extracellular stimuli that are perceived in the apoplast by different mechanisms (which will be discussed in the following paragraphs); 1. The role of (some) receptor kinases in signal transduction is well established. 2. The first plant cDNA sequences that code for G-protein-coupled receptors have been reported. 3. Biochemical evidence of the existence of integrin-like proteins is available, although homologous genes have not yet been isolated from plants. 4. An important regulatory role in development is becoming evident for AGPs. 5. Regulation of ion channels dependent on apoplastic signals represents the last mechanism, which will be discussed in the context of apoplast-symplast signaling. a} Receptor Kinases Cell-to-cell interaction during development and adaptive responses to prevailing environmental conditions depend on signal perception and transduction from the ECM to the intracellular space. This eminent role is fulfilled by receptor-like kinases (RLKs) and other membraneassociated protein kinases (Lease et al. 1998; Satterlee and Sussman 1998). RLKs are comprised of an extracellular sensing domain and an intracellular transducing element. Both elements are connected by a single transmembrane domain. The intracellular activity of plant RLKs is constituted by a serine/threonine-kinase domain. The total number of RLK genes in the Arabidopsis genome is estimated to be on the order of 400. However, few extracellular signals that bind to specific RLKs and trigger downstream events have been identified. Examples are brassinosteroids, a new class of plant hormone (Li and Chory 1997), and the self- The Extracellular Matrix of the Plant Cell: Location of Signal Perception 223 Table 1. Examples of extracellular stimuli that provoke cellular responses Interacting or perceiving structure8 Response or suggested function Reference Chemical stimulus pH K+ channel Development and stress response, stomatal opening, phloem transport Ca2+ GCAC1-anion channel Guard-cell volume regulation K+ -channel inhibition Growth regulation Malic acid Anion channel Stomatal closure Abscisic acid Anion channel Guard-cell closure Anion channel Solubilization of xyloglucans Guard-cell opening Amtmann et al. (1999); Hoth and Hedrich (1999); Marten et al. (1999) Schulz-Lessdorf et al. (1996) Thiel et al. (1996) Hedrich and Marten (1993) Schwarz and Schoeder (1998) Tominaga et al. (1999) Marten et al. (1991) Receptor kinase BRIl Growth promotion Li andChory (1997) Cytokinin response Expression of glutathione S-transferase Cell coordination in meristems PlakidouDymock et al. (1998) Gamble et al. (1998) Clayton et al. (1999) Fletcher et al. (1999) Pathogen response Programmed cell death Somatic embryo genesis He et al. (1999) Gao and Showalter (1999) McCabe et al. (1997) Extracellular signal Auxin Brassinosteroids (?) Cytokinin Ethylene GCR (seventransmembranedomain receptor) ETR1 histidine kinase Ozone Ca2 + CLAVAT A receptor kinase Physical stimulus Cell-wall strain (?) WAK Arabinogalactan proteins Cell division 8BRI, brassinosteroid-insensitive mutant; CLAY ATA, organ-number mutant; ETR, ethylene receptor; GCAC, guard-cell anion channel; GCR, guanosine triphosphate-bin dingprotein-coupled receptor; WAK, wall-associated kinase. 224 Physiology incompatibility (SI) glycoprotein Ste20-like kinase (Walker and Zhang 1990; Table 1). For some RLKs, an important role in developmental regulation has been established without knowledge of the ligand. The leucine-rich receptor kinase HAESA controls the abscission of floral organs. Reporter-gene activity under control of the HAESA promoter shows expression in the abscission zone in cells that remain attached to the plant shoot (Walker et al. 1999). Mutations in another RLK result in the crinkly phenotype in maize. The epidermal cells have altered shapes and sizes, and the leaves develop graft-like fusions (Becraft et al. 1996), indicating that RLKs are important for the control of meristem activity and leaf development (Fletcher et al. 1999). Another group of serine/threonine kinases is represented by wallassociated kinases (W AKs), which are suggested to connect the cell-wall networks to the intracellular space. The five genes of the WAK family in Arabidopsis differ in their extracellular N-terminal domains, but they all have cysteine-rich stretches homologous to epidermal growth factor repeats from animals (He et al. 1999). WAKI is extracted from plant tissue in the crude cell-wall fraction and dissociates from the glycans only under harsh conditions, such as boiling in buffers containing 4% sodium dodecyl sulfate and dithiothreitol. The WAK proteins so extracted contain pectin residues, as indicated by the binding of antipectin antibodies to the WAK1-protein in Western blots. This suggests that WAKs are attached to the pectins of the cell wall. The members of the WAK-protein family exhibit tissue-specific expression (He et al. 1999). Reporter-gene expression under the control of WAK promoters revealed that they are expressed in cells arranged in a ring-like fashion at organ junctions, such as the root-hypocotyl transition, at the bases of trichomes, hydathodes and root tips, and along veins. WAKI and WAK2 are induced by wounding. Thus, they apparently have overlapping and distinct functions in plant development and defense. Plants with modified levels of WAK expression exhibit disturbance of cell expansion (i.e., the dwarf phenotype) or higher susceptibility to pathogens. The mutant plants are disturbed during the development of SAR (He et al. 1998). WAKs are likely to function in cell-wall attachment and in the transduction of extracellular signals to the cytoplasm. Another group of RLKs, namely leucine-rich repeat transmembrane protein kinases 1 and 2, was identified in developing maize endosperm and contained a distinct extracellular domain, including leucine-rich repeats, a proline-rich region, a putative protein-degradation sequence (PEST) and a serine-rich stretch (Li and Wurtzel 1998). Their temporal and spatial pattern of expression suggests a role in mediating signals associated with seed development. A well-established system of ECM signaling and function during development is the SI reaction in Brassica. Successful pollination involves five steps: The Extracellular Matrix of the Plant Cell: Location of Signal Perception 1. 2. 3. 4. 5. 225 Adhesion Recognition of the S-locus Hydration of the pollen Germination Pollen-tube growth Three proteins are involved in triggering the SI response and are all encoded on the S-locus of the Brassica genome. A recently identified Slocus cysteine-rich (SCR) protein exhibits little sequence conservation between genotypes, contains eight cysteine residues and appears to be a promising candidate for the pollen-grain-associated factor (Stanchev et al. 1996; Stephenson et al. 1997). This factor may bind to the S-locus glycoprotein (SLG), which is present in the ECM ofthe epidermal cells of the recipient stigma. Following its formation, the SLG-SCR complex binds to the S-locus receptor kinase (SRK), and the downstream events are activated. The pollen is rejected. In another case, the SCR is not able to bind to SLG, and the SRK-mediated SI response is not triggered (McCubbin and Kao 1999). Although only a few RLKs have been investigated, the available studies illustrate the important roles and the "potential" of RLKs in the regulation of plant development and adaptation and promise exciting new knowledge from the investigation of other receptor kinases. b) Seven-Transmembrane-Domain Receptors Members of the large super-family of guanosine triphosphate (GTP)binding-protein-coupled receptors are well characterized in animal and fungal systems and have been cloned from plants using sequence information available from the expressed-sequence-tag database (Josefsson and Rask 1997; Plakidou-Dymock et al. 1998). The family of receptors with seven transmembrane domains trans duces extracellular signals to downstream targets via trim eric G proteins. Anti-sense suppression of one of them (glucocorticoid receptor I; GCRI) reduced sensitivity to the plant growth substances cytokinins in shoots and roots of Arabidopsis thaliana (Plakidou-Dymock et al. 1998), suggesting that GCRI may be involved in hormone signaling from the ECM to the cytoplasm. c) Evidence for the Existence and Involvement ofIntegrin-Like Proteins The interaction of the plasma membrane and cytoskeleton with the ECM as a mechano-sensitive transmitter is well established in animal cells. Integrins are proteins that mediate the ECM-cytoskeleton continuum (Longhurst and Jennings 1998) and are involved in numerous cellular Physiology 226 Table 2. Immunological evidence of the existence of integrin-like proteins in various species. Plant fractions were separated by sodium dodecyl sulfate polyacrylamide-gel electrophoresis and analyzed by Western blotting Plant species/genus Molecular mass of the cross-reactive polypeptides (kDa) Reference Chara contra ria 58,80 Katembe et al. (1997) Fucus 92 Quatrano et al. (1991) Arabidopsis thaliana 58,100 Swatzell et al. (1999) 58,84-116 Katembe et al. (1997) Zea mays 55,76 Quatrano et al. (1991) Nicotiana 110 Lynch et al. (1998) Allium cepa 105-l25 Gens et al. (1996) processes, such as tissue organization, cell growth and inflammation. Integrins are heterodimers with extra- and intracellular domains connected via single transmembrane helices. The receptor domain in the extracellular structure recognizes and binds to proteins with the exposed tri-peptide motif arginine-glycine-aspartic acid (RGD). The interaction with RGD domains is essential for integrin function. In animal cells, integrin-dependent processes are blocked after addition of RGD peptide. Similar experiments in plants and algae have provided evidence that RGD-recognition sites are also conserved in plants (Canut et al. 1998; Wayne et al. 1992). Addition of RGD peptides inhibits gravisensing in Chara (Wayne et al. 1992). High-affinity binding of RGD peptides to A. thaliana plasma membranes was demonstrated using an iodinated RGD-containing hepta-peptide (Canut et al. 1998). A number of papers have demonstrated the presence of integrin-like proteins in plantmembrane preparations. These papers mainly used two antibodies: a commercially available monoclonal antibody and a polyclonal antibody raised against the C-terminal domain of the 131-integrin from chicken (Marcantonio and Hynes 1988; Table 2). The detected molecular masses of cross-reactive polypeptides ranged between 55 and 125 kDa and were identified as integrin-like proteins and their degradation products, respectively. Swatzell et al. (1999) showed the cross-reactivity of a lOO-kDa polypeptide from A. thaliana plasma membranes with an avian 13integrin antibody directed against the cytoplasmic domain of the integrin. Thus, an increasing body of evidence that plant cells also rely on RGD-binding sites and integrin-like proteins is accumulating, although the integrin function still has to be determined in plants. The Extracellular Matrix of the Plant Cell: Location of Signal Perception 227 d) Arabinogalactan Proteins AGPs constitute a family of glycosylated and structurally complex extracellular proteins of rod-like or globular shape. According to the domain structure of the protein backbone, AGPs are grouped into three subfamilies: two classes of "classical" AGPs and one class of "non-classical" AGPs (Schultz et al. 1998). The classical AGPs with the simplest domain structure are comprised of an N-terminal signal peptide for secretion, an amino-acid stretch rich in hydroxyproline, alanine, serine and threonine, and a C-terminal signal sequence for the attachment of GPI. GPI constitutes a membrane anchor (Nothnagel 1997; Schultz et al. 1998). There is no indication of the presence of GPI anchors in "nonclassical" AGPs. More than 17 genes encoding AGPs are present in the A. thaliana genome, approximately ten of which have been sequenced. AGPs are implicated in cell-fate determination during embryogenesis (Samaj et al. 1999), in the induction of somatic embryogenesis (Kreuger and Van Holst 1993; McCabe et al. 1997), in cell expansion and proliferation (Serpe and Nothnagel 1994), and in the induction of programmed cell death (Gao and Showalter 1999). Evidence of a role for AGPs in cell-to-cell signaling and ECM functions comes from experiments with antibodies or Yariv reagent (which is a dye specifically interacting with AGPs) and from an Arabidopsis AGP mutant that is disturbed during its interaction with Agrobacterium (Schultz et al. 1998). Further approaches have to be developed to elucidate AGP functions in plants. e) Ion Channels Ligand- and effector-dependent gating of ion channels or other transporters provides an efficient mechanism to transmit information from the apoplast to the symplast. Candidate transporters are K+ channels, anion channels, Ca2 + channels and H+-pumping adenosine triphosphatases. Anion channels were recently discussed with respect to their role in signal transduction in guard cells and root-soil interaction (Schroeder 1995). pH, Ca2+ concentration, malic acid, abscisic acid and auxins are apoplastic parameters that have been shown to affect ionchannel activity and have been implicated in ion-channel-mediated physiological responses of plant cells (Table 1). Experiments on K+ channels (Lemtiri-Chlieh and MacRobbie 1994; Amtmann et al. 1999; Hoth et al. 1999) and anion channels in guard cells (Schulz-Lessdorf et al. 1996; Schwarz and Schroeder 1998; Marten et al. 1999) and K+ channels in coleoptiles (Thiel et al. 1996) revealed the broad importance of pH -sensitive gating of ion channels, and the molecular basis is beginning to be understood (Hoth and Hedrich 1999). It is likely that other, uni- 228 Physiology dentified extracellular factors also affect transporter activities and are involved in ECM -cytoplasm signaling. 3 Signal Transduction Most of the addressed primary sensing events in the apoplast and initial sensor responses are coupled to downstream signaling cascades. Most of the basic components known to be involved in signal transduction in animal and fungal cells also have been identified in plants. Trimeric G proteins (Hooley 1998; Lee and Assmann 1999), small GTP-binding proteins (Palme et al. 1999), protein kinases and phosphatases, microtubule-associated protein kinase pathways (Jonak et al. 1999), 14-3-3 proteins (Moorhead et al. 1999) and other signaling mechanisms have been shown to be involved in transducing apoplastic signals to response events, such as the induction of gene expression or the activation of metabolic pathways. 4 ECM Formation and Remodeling ECM formation and remodeling depend on the presence of enzyme activity, substrates and a proper chemical milieu in the apoplast. In this section, apoplastic enzyme activities will be discussed briefly, with emphasis on two groups of enzymes: extracellular proteases and expansins. a) Enzymatic Activities in the Apoplast The apoplast is an extra-plasmatic compartment of the plant cell, and its composition is governed by two pathways: plasma membrane transport and vesicular processes, i.e., exo- and endocytosis. As discussed above, delivery of newly synthesized proteins to the ECM is fast. The basic enzyme activities of the apoplast have been discussed in a recent review of the function of the apoplast under stress (Dietz 1997). Typical apoplastic enzymes are hydrolases and peroxidases. Their activity changes depending on the growth conditions (Brune et al. 1994; Blinda et al. 1997; Ramanjulu et al. 1999). At this point, it should be stressed that probably not all apoplastic proteins and enzymes have been identified and that it is difficult to prove the extracellular association of a specific enzyme activity, particularly if the same activity is also present inside the cell. In the ideal case, three independent indications should demonstrate the apoplastic location: The Extracellular Matrix of the Plant Cell: Location of Signal Perception 229 1. Identification of the enzyme activity in a highly purified cell-wall fraction or in intercellular washing fluid, and demonstration of enrichment of specific activity in the ECM fractions (as compared with crude cell extracts) 2. cDNA cloning and DNA-sequence determination showing the presence of a suitable sequence address for secretion 3. Heterologous expression of the cloned gene for antibody generation and immunological detection of the antigen in the ECM or ECM fractions In the following sections, the review will only focus on two groups of enzyme activities whose extracellular occurrence has been proven, although their precise mode of action and physiological function is only beginning to be understood. b) Proteases The number of characterized proteins associated with the extracellular space has increased continuously during recent years; the identification of these proteins has been based either on purification from cell walls or apoplast fractions or on the cloning of genes with sequence information for secretion (Table 3). These proteins are subjected to processing and turnover by extracellular proteases. In fact, three major types of proteinases have been described to occur in the ECM: serine proteases, aspartic acid proteases and metalloproteases. Evidence of the existence of cysteine proteases in the extracellular space is not unequivocal. A cysteine protease appears to be synthesized at the ER, particularly under conditions of stress. However, this particular cysteine protease appears to be directed to the vacuole and not to the ECM (Forsthoefer et al. 1998). Another gene, named CyplSa, was isolated from stressed pea plants; it has sequence similarity to cysteine proteases. Immunocytochemical detection showed that the CyplSa gene product is located in cell walls of stem cortical cells. Its protease activity has not yet been determined (Jones and Mullet 1995). Extracellular proteases are suggested to function in establishing, maintaining and remodeling the cell-matrix structure, in apoplastic metabolism, in signal perception and transduction, in the processing of precursor proteins and in defense reactions. Table 3 summarizes some results on extracellular proteases; these results suggest the role of the proteases in diverse physiological processes, such as pathogenesis, nodule formation and organ differentiation. Apparently, various proteases are associated with the cell wall and the apoplast of plant cells, but their functions in development and adaptation still need to be established. It Organ expansion Unknown function in development Lateral root formation Degradation of PRP Actinorrhizal nodule development Fruit development Gamete fusion Rodrigo et aL (1989) Xia et al., personal communication Jones and Mullet (1995) McGeehan et al. (1992) Kinoshita et al. (1992) Ribeiro et aL (1995) Lycopersicon esculentum Arabidopsis thaliana Pisum sativum Yamagata et aL (1994) Vera and Conejero (1988); Jorda et al. (1999) Jorda et al. (1999) Neuteboom et aL (1999) Messdaghi and Dietz (2000) Cucumis melD Lycopersicon esculentum Lycopersicon esculentum A. thaliana A. thaliana, Hordeum vulgare, Nicotiana tabacum and other species PMSF- and chymostatin-sensitive; maximum activity in young leaves and fruits; pI=7.3 Chlamydomonas rheinhardtii Alnus glutinosa Glycine max Reference Plant species/genus eDNA and protein of "cucumisin"; thermostable; 79-kDa preproenzyme; mature size of 67 kDa; optimum at pH 10 eDNA and protein; induction by viroid infection; 69-kDa mature protein (P69-B, -C); pathogenesis induced; DTT activated; pCMB sensitive; slight inhibition by PMSF; pI=10 eDNA; P69-A; P69-D; tissue- and cell-specific constitutive expression eDNA; auxin induced, Sequence similarity to cysteine proteases eDNA, sensitive to inhibition by TIMP and phenanthroline eDNA; 70-kDa pre-proenzyme; 50kDa mature protease Subtilisin-like; eDNA Optimum at pH 3; PMSFinsensitive; pepstatin-sensitive 40% homologous cDNAs Characteristics· .cDNA, complementary DNA; DTT, dithiothreitol; pCMB, p-chloromercuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride; PRP, pathogenesis-related protein; TIMP, tissue inhibitor of metalloproteases of mammalian cells. Serine protease Metalloprotease Cysteine protease Degradation of PRP Aspartic-acid protease Induction of constitutive disease resistance (CDR1) or constitutive disease susceptibility (CDSl) Unknown; stress adaptation under drought and salt Unknown Suggested physiological role Protease type Table 3. Selected protease activities associated with the extracellular matrix ~ 0 '" §: "0 ::r" '< tv '"0 The Extracellular Matrix of the Plant Cell: Location of Signal Perception 231 is tempting to speculate that they are as important as their animal counterparts in adjusting the ECM to the prevailing developmental and environmental conditions. However, the biochemical characterization of extracellular proteases is still beginning, because no specific substrate has been identified for any cloned protease from the plant ECM. c) Expansins Expansins constitute a family of related proteins weighing approximately 25 kDa during their maturity. Presently, 24 expansin genes are known from A. thaliana, but it is estimated that approximately 30 genes of expansins and expansin-like proteins are present in the A. thaliana genome (Cosgrove 1999). The AtExpB gene consists of three exons that encode for distinct protein domains. Exon 1 codes for the apoplastic targeting address, exon 2 codes for an endoglucanase-like core region, and exon 3 codes for a domain with structural similarity to bacterial cellulose-binding domains (Cosgrove 1999). A function of expansins in cell-wall extension has been shown in vitro and in vivo, although the biochemical mechanism of expansin action is not yet understood. The initial identification of expansins was achieved by screening cell-wall protein isolates for cell-wall extension activity in vitro using so-called extensometers (McQueen-Mason and Cosgrove 1994). In addition to the in vitro activity, a role of expansins in cell enlargement has been shown for root hairs, hypocotyls, shoot apical meristems and cell cultures. The developmental control of gene expression is consistent with the role of expansins in growth, although the correlation is not always perfect (for instance, during tomato fruit ripening; Brummell et al. 1999). This discrepancy may be the consequence of the complexity of the expansin gene family. Reporter-gene expression has revealed a highly tissue- and cell-specific pattern of expression; for instance, AtExp7 is expressed in root hairs and trichoblasts, AtExp4 is expressed in vascular tissue, and AtExp1 is expressed in guard cells. An intriguing hypothesis is that AtExp 1 may be involved in keeping the thick cell walls of the guard cells flexible for continuous opening and closing of the stomatal complex (Cosgrove 1999). Apparently, expansins have evolved for distinct functions, with concomitant specificity in spatial and temporal expression. This short description of expansins and extracellular proteases may suffice to demonstrate the fascinating complexity of ECM activities. However, it should be stressed that other extracellular proteins also offer exciting clues to the dynamics of the ECM of plant cells (for instance lipid-transfer proteins, glycosyl transferases, peroxidases and thionins) but cannot be addressed here. 232 Physiology 5 Perspectives A complex network of transport-dependent, secretory, enzymatic and non-enzymatic parameters determines the capacity for construction and deconstruction of the ECM of plant cells. Furthermore, the extracellular space is the site of initial perception of the endogenous and exogenous stimuli that are transmitted to the cell symplast and evoke specific or general metabolic and genetic responses. This review has summarized some of the emerging topics and has indicated the challenges presently faced by plant physiologists. Various extracellular proteins, such as WAKs, expansins and proteases, have been shown to constitute multigene families of closely related gene products with tightly regulated spatial and temporal patterns of expression. However, for these examples (and for many other factors that were only partly addressed in this review), substrates, ligands, downstream signaling events and responses are not yet understood. In the near future, from a combination of methods, including molecular genetics, protein chemistry, biochemistry and physiology, we can expect an exciting new understanding of: 1. The interaction between the ECM and the symplasts of plant cells 2. The important ability of the plant cell to adapt the ECM to growth conditions as a fast, active and flexible response Acknowledgements. 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Kelly "One of the first eukaryotic organisms that will be completely sequenced is the small mustard species Arabidopsis thaliana .... about 59% of the genome sequence is currently available in public databases and .... it is currently anticipated that the complete genome sequence .... will be available by the end of the year 2000." (Somerville and Somerville 1999). 1 Introduction Life, like the Calvin cycle, is a revolution that returns to its beginning. Even photosynthetic carbon metabolism is a part of this revolving life. Traditionally, photosynthesis is seen as life's energy transducer, whereby solar light energy is collected and changed into the potential energy in the covalent bonds of sugar molecules. Indeed, the fate of a large proportion of these sugar molecules is respiration so that this energy can become available to non-photosynthetic cells or to photosynthetic cells in darkness. However, a small fraction of these sugars are metabolized to form the monomers (including deoxyribose; Debnam and Emes 1999) that are linked to make the new DNA of newly emerging photosynthetic cells. This DNA will be transcribed to initiate the synthesis of the Calvincycle enzymes in these cells so that they too can contribute to the conversion of CO 2 to more sugars and, ultimately, more DNA. We should reflect on the photosynthesis-DNA link because, as the above quote indicates, we can expect to be able to view the entire nucleotide sequences of the five chromosomes of the small, photosynthetic, flowering plant Arabidopsis thaliana within the next year or two. We will also be able to determine the position (or positions) on these chromosomes where each of the 11 enzymes of the Calvin cycle are encoded. Due to the imminence of these events, this review of photosynthetic carbon metabolism concentrates on the properties of all of the proteins that make up the machinery of CO 2 fixation. However, it must be remembered that these proteins are synthesized from instructions on DNA via a process that may be influenced by the status of CO 2 fixation and that may be manipulated using the tools of modern molecular biology. The literature covered in this review has predominantly been published since the last review in volume 60 of Progress in Botany. Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 239 2 The Chloroplast One impressive advance and one surprise concerning chloroplasts have recently occurred. The advance was the successful isolation of functional chloroplasts from the diatoms Odontella sinensis and Coscinodiscus granii. These chloroplasts are unusual in that they have four envelope membranes; at least one of these must have remained intact, because the chloroplasts photosynthesized and appeared to possess a phosphate (P) translocator (Wittpoth et al. 1998). The surprise is the report that chloroplasts, and not phytochrome, mediate the photo-induction of spore germination in the liverwort Marchantia polymorpha (Nakazato et al. 1999). Such observations are reminders that there is substantial diversity in the cellular and molecular characteristics of photosynthetic organisms and that the genome of shade-loving A. thaliana will only be a guide to the genomes of other photosynthetic organisms. a) Ribulose-Bisphosphate Carboxylase/Oxygenase The focus of research on ribulose-bisphosphate (P 2 ) carboxylase/oxygenase (Rubisco), the most popular of the Calvin-cycle enzymes, has shifted toward the two DNA sequences that encode its two subunits. The higher-plant enzyme consists of eight chloroplast-encoded large subunits that contain catalytic sites, and eight nuclear-encoded small subunits whose function is poorly defined (Spreitzer 1999). Molecular approaches to the study of the enzyme have been hampered by this complex arrangement, but recent successes in producing a directed mutation in the large subunit of the tobacco enzyme (Whitney et al. 1999) suggest that there will be more of this type of research in the future. The creation of hybrid enzymes has been reported; both Arabidopsis large subunit! pea small subunit and sunflower large subunit/tobacco small subunit enzymes have been constructed (Getzoff et al. 1998; Kanevski et al. 1999). Both hybrid Rubiscos were active and, in fact, were hardly different from their normal counterparts. Investigations into the movement of entire Rubiscos from one species to another for practical purposes have also been performed. In particular, the observation (Uemura et al. 1997) that certain red algae contain a form of Rubisco that is more than twice as efficient as the higher-plant Rubisco at distinguishing between CO 2 and 02 (thereby favoring photosynthesis over photorespiration) led to a suggestion that agricultural productivity might be increased if the Rubisco in crop plants is replaced by this red-algal enzyme (Mann 1999). However, the overall lower activity of the red-algal enzyme is a drawback to this proposal (Portis and Zhang 1999). In addition, while the activity of Rubisco may be the principal determinant of the photosynthetic rate, the photosynthetic rate is 240 Physiology certainly not the principal determinant of the growth rate of crops in the field (Hansen et al. 1999). In related research, Ramage et al. (1998) attempted to improve the ability of a cyanobacterial Rubisco to distinguish between CO 2 and 02 using site-directed mutagenesis to alter its amino acid sequence so that it mimics that of the efficient red-algal enzyme. The attempt was not successful. Nevertheless, study of the relatively unique algal Rubiscos is enriching our understanding of the enzyme. In the chloroplasts of many green micro-algae, most of the enzyme is in the proteinaceous structure called the pyrenoid and performs CO 2 fixation there (Borkhsenious et al. 1998; Moroney and Chen 1998). In dinoflagellates, it is a strange enzyme; it is composed only of large subunits and has not displayed its catalytic ability in vitro (Whitney and Andrews 1998). Considerable attention has been given to the relatively complex regulation of Rubisco activity. Initially, the inactive protein is activated when CO 2 and Mg2+ bind to an activation site, an event that seems to be partially reversed near midday, when the well-known midday depression of photosynthesis is evident (Sinha et al. 1997). Secondly, the enzyme fully activated by CO 2 and Mg2+ can be inactivated again by the binding of various sugarPs, such as xylulose-P 2 and, as recently reported by Zhu et al. (1998), 3-ketoarabinitol-P2; both of these are formed when the chemical reaction follows a false route. Another recently discovered inactivator is glycero-2,3-pentodiulose-P 2, which can occur as a contaminant in stored preparations of the enzyme's substrate, ribulose-P2 (RuBP; Kane et al. 1998). However, the most physiologically relevant inactivator is 2-carboxy-arabinitol-1-P (CA1P), which appears in the leaves of some (but not all) plant species at night, binding to the enzyme and keeping it inactive. A previous report that this CAlP might not be in the same part of the leaf as Rubisco has been enthusiastically challenged by Parry et al. (1999). Of course, removal of CA1P must occur if photosynthesis is to be possible during the next day. Dissociation of CAIP from the enzyme (and dissociation of all other inhibitory sugar-Ps) is deemed to be the task of a second protein, called Rubisco activase (Hammond et al. 1998a). In a new twist to the topic of light-mediated regulation of Calvincycle enzymes (our earlier reviews), Zhang and Portis (1999) have discovered that one form of this protein undergoes a thioredoxin-f-mediated reduction of disulfide bonds. This reduction turns on its ability to interact with the Rubisco and rid the latter of the inhibitory CAlP. Subsequent dissolution of the CA1P also seems to be enhanced by illumination, because the activity of the participating phosphatase has been found (Heo and Holbrook 1999) to be markedly stimulated when the chloroplast pool of glutathione becomes totally reduced, as expected in light. The notable temperature sensitivity of the Rubisco activase, and experiments with plants engineered to contain reduced levels of the activase, have been used to provide further evidence that the activase is intimately involved in the overall light-mediated activation of Rubisco (Feller at al. 1998; Hammond et al. 1998b). However, there are tantalizing indications that the influence of the activase extends beyond this role (He et al. 1997; Li et al. 1999). This suspicion and the data of Morales et al. (1999), which imply that the productivity of maize is correlated with the content of Rubisco activase in its leaves (an observation that conflicts somewhat with the above-mentioned dogma that the photosynthetic rate is not a primary determinant of growth rate), will ensure that this mysterious protein will receive more attention from researchers during the coming years. Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 241 b) Other Calvin-Cycle Enzymes A relative resurgence of interest in the remaining ten enzymes of the Calvin cycle has occurred during the past 2 years. This should continue, because the Arabidopsis genome sequence will soon be available (Sect. 1), and a complete collection of clones of complimentary DNAs for all of the Calvin-cycle enzymes is now available; the last to be added to the list was transketolase (Flechner et al. 1996). Another stimulus is the increasingly sophisticated view of the in vivo status of the enzymes. The association of glyceraldehyde-3-P (GAP) dehydrogenase and ribulose-SP (RuSP) kinase with a small linking peptide, reported for higher plants in our previous review, has now been shown to be widespread. It occurs in mosses, green algae and even cyanobacteria (Wedel and Soll 1998). Several of the Calvin-cycle enzymes, including RuSP kinase, transketolase and RuSP 3-epimerase, seem to be arranged in a thin layer on thylakoid membrane surfaces. They are surrounded by more soluble stromal proteins, including Rubisco and the enigmatic carbonic anhydrase (Jebanathirajah and Coleman 1998; Teige et al. 1998). The 3-epimerase was also studied by Chen et al. (1998b), who found that, in spinach leaves, it is an eight-subunit protein that is extremely active but very labile. The well-known light-mediated reductive activation of several of the Calvin-cycle enzymes, including fructose-l,6-bisphosphatase (FBPase), sedoheptulose-l,7-bisphosphatase (SBPase; Dunford et al. 1998) and RuSP kinase, have been reviewed by Ruelland and MiginiacMaslow (1999), who suggested that a central feature of these enzymes is a loosening of the structure of the enzymes on reduction of the disulfide bonds, thereby increasing the flexibility of the active site and, consequently, the likelihood of catalysis. Nicotinamide adenine dinucleotide phosphate (NADP)-linked malate dehydrogenase from sorghum provides an excellent example of this phenomenon (Goyer et al. 1999). Most of the Calvin cycle's GAP dehydrogenase exists in chloroplasts as a tetramer of two "A" subunits and two slightly different "B" subunits, although a minor portion (-20%) occurs as a homotetramer of four "A" subunits (Scagliarini et al. 1998). Surprisingly, some of the GAP dehydrogenase is located in the nucleus, where it apparently has a totally different catalytic function, namely the excision of the base uracil from DNA (Wang et al. 1999b). Another surprising enzyme is aldolase (a protein responsible for two of the 13 reactions in the Calvin cycle; Flechner et al. 1999). When Haake et al. (1998) used anti-sense DNA technology to lower the aldolase activity in potato leaves, the inhibition of photosynthesis and growth was unexpectedly dramatic, given that the enzyme catalyzes a reversible reaction and possesses "no known regulatory properties" (authors' quote). It should be recalled, however, that this enzyme is susceptible to inhibition by RuBP when operating photosynthetically (i.e., catalyzing fructose-l,6- P2 formation; our 1992 review). Physiology 242 Unlike the two aldolase reactions, the Calvin cycle's two phosphatase reactions in higher-plant chloroplasts require two enzyme proteins: FBPase and SBPase. Only SBPase is specific for its reaction; the FBPase can act as an SBPase (Ashton 1998). However, the specific SBPase appears to be vital for the plant because, when its level in tobacco leaves was reduced to less than 20% of the normal level, the plants exhibited chlorosis and reduced growth (Harrison et al. 1998b). These conclusions may not pertain to cyanobacteria, however, because Tamoi et al. (1999) found that the most important FBPase in cyanobacteria is a strange, bifunctional (FBPase/SBPase) protein not found in land plants; it is quite resistant to inactivation by H 20 2 • Finally, marine micro-algae have again provided an example of a Calvin cycle enzyme with relatively unique properties: an RuSP kinase that is a tetramer of especially large subunits, from the chromophyte Heterosigma carterae (Hariharan et al. 1998). Continuous operation of the Calvin cycle requires that five-sixths of the GAP pool be used to regenerate RuBP and that no more than onesixth be diverted to sucrose or starch synthesis. It is well recognized that the chloroplast envelope's P translocator ensures that the quota for sucrose is not exceeded (Fig. 1), but the mechanism restricting starch synthesis within the chloroplast has been poorly defined. Isotope studies by Schleucher et al. (1999) have revealed that the reaction representing the i amylopectin amy+ose pentose-Ps ~ A DP-tlucose glucose-1-P ---CO erythrose-4-P t gl",0"-6-P - RUBy ~ - ~.... .... fructose-6-P 3,GA ~~ I _____ DHAP- GAP 2 S"'r' Pi1 ~ )0 GAP CHLOROPLA ST Fig. 1. Maintaining the Calvin cycle's pool of ribulose-P r The phosphate translocator (a) prevents excessive export of glyceraldehyde-3-P. The pool of the Calvin cycle's erythrose4-P may be sufficient to appreciably inhibit hexose-P isomerase (b) so that the synthesis of transitory starch (amylose and amylopectin) is not excessive. 3PGA 3-P-glycerate, DHAP dihydroxyacetone-P, S7P sedoheptulose-7-P Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 243 first step out of the Calvin cycle and toward starch (the conversion of fructose-6-P to glucose-6-P) is not at equilibrium in the chloroplast. This indicates a restriction of the activity of the enzyme hexose-P isomerase, presumably to ensure that the quota of photosynthate for starch is not exceeded. The restriction may be effected through the strong inhibition of this enzyme by erythrose-4-P (our 1982 review; Backhausen et al. 1997; Fig. 1). One last point of interest is the fascinating disappearance of enzymes specific to the Calvin cycles of tomato and sweet-pepper fruits as these fruits ripen and the population of plastids changes from predominantly chloroplasts to predominantly chromoplasts. The NADP-linked GAP dehydrogenase disappears from sweet-pepper fruits (Backhausen et al. 1998), and the plastidic FBPase disappears from tomato fruits (Buker et al. 1998). Unexpectedly, the latter enzyme remains in ripe sweet-pepper fruits (Thorn et al. 1998). c) Transitory Starch Metabolism There is currently a burgeoning interest in the enzymology of the biosynthesis of starch in plant plastids, partly due to the commercial value of starch products and the perceived possibility that molecular-biology techniques may be used to increase this value by altering the proportions and/or characteristics of the two principal components, amylose (a linear glucose polymer) and amylopectin (a branched glucose polymer). In chloroplasts, starch is synthesized and decomposed diurnally. Synthesis by day represents an important transient sink of photosynthate; without it, the rate of photosynthesis may be reduced (Sun et al. 1999a). As noted above, synthesis begins when some of the Calvin cycle's fructose-6-P is diverted and isomerized to glucose-6-P; this glucose-6-P is then converted, in two steps, to adenosine diphosphate (ADP)-glucose, the glucose donor for polymer manufacture (Fig. 1). The second of these two steps is catalyzed by ADP-glucose pyrophosphorylase, which is restricted to chloroplasts in leaves (Chen et al. 1998a). This pyrophosphorylase is a principal regulatory enzyme of starch synthesis (Greene and Hannah 1998; Kleczkowski 1999), but the importance of the preceding enzyme, P-glucomutase, should not be overlooked. Its indispensability has been demonstrated in experiments with pea leaves (Harrison et al. 1998a), and the possibility that it may contribute to regulation has been raised by Hattenbach and Heineke (1999), who found that both RuBP and FBP inhibit its activity. A collection of starch synthases catalyzes the glucosyl transfer from ADP-glucose to the growing starch molecules that constitute the insoluble starch granule in the chloroplast. Some of these are totally or predominantly soluble enzymes, and these forms add glucosyl units to 244 Physiology amylopectin (Tomlinson et al. 1998; Kossmann et al. 1999). Branching of the amylopectin molecule is achieved by specialized branching enzymes (Jobling et al. 1999). There are indications that the degree of branching is initially excessive, resulting in a highly branched, soluble, glycogen-like molecule, but this is apparently rectified by the indirect intercession of a de-branching enzyme (Greene and Hannah 1998; Zeeman et al. 1998a). The other component of starch, amylose, is synthesized within the amylopectin matrix by a special form of starch synthase that is bound to the starch granule and is therefore referred to as the granule-bound starch synthase (Tomlinson et al. 1998; Tatge et al. 1999; Wang et al. 1999a). A marine alga has provided another surprise. Nyvall et al. (1999), having noted that red algae synthesize starch in the cytosol rather than in the chloroplast, investigated Gracilaria tenuistipitata and found that its starch synthase prefers uridine diphosphate-glucose rather than ADP-glucose. The dissimulation of chloroplast starch proceeds at night. Experiments with Arabidopsis indicate that the intact starch granule is initially attacked by a novel endoamylase, whose activity is induced by the onset of darkness and then mysteriously disappears after 2 h (Kakefuda and Preiss 1997). By then, however, the granule is sufficiently eroded, so other endoamylases, including one specifically directed at starch (Zeeman et al. 1998b), can continue the degradation process, releasing small, soluble sugars. New evidence has increased the likelihood that glucose is the sugar exported to the cytosol, where it is phosphorylated by a hexokinase as a first step toward sucrose manufacture (Schleucher et al. 1998; Veramendi et al. 1999). It seems unlikely that either glucose1-P or glucose-6-P could be exported, because chloroplasts lack envelope transporter proteins for these glucose-Ps, despite the fact that nonphotosynthetic plastids (amyloplasts, chromoplasts, embryo plastids) have them (Eastmond and Rawsthorne 1998; Harrison et al. 1998a; Kammerer et al. 1998; Thorn et al. 1998). Amyloplasts even possess an ADP-glucose transporter for the import of this substrate of starch synthesis, coincident with the previously reported unusual cytosolic location of ADP-glucose pyrophosphorylase in cereal grain endosperm (Shannon et al. 1998). 3 The Photosynthetic Cell a) Uptake ofInorganic Carbon The collection of CO 2 by photosynthetic cells and the delivery of this CO 2 to Rubisco is intriguing. Cyanobacteria and algae seem to engage in this process, but land plants usually do not. Much recent research has been devoted to the elucidation of the molecular mechanisms used by algae to collect and concentrate CO 2 within themselves. Because this Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 245 research has been recently reviewed (Kaplan and Reinhold 1999; Moroney and Somanchi 1999), no more than a succinct summary is presented here. The central feature of the inorganic carbon (Cj)-uptake mechanism of these algae is the possession of a CO 2 transporter protein and/or a bicarbonate transporter protein on the plasma membrane. Recent evidence from experiments with marine red, green and brown macro-algae, a marine diatom, and a marine dinoflagellate indicate that CO 2 transport is the preferred or only option. In that case, the transporter is assisted by a carbonic anhydrase delivered to the extracellular, periplasmic space, where it catalyzes the generation of CO 2 from bicarbonate, the most abundant Cj species in seawater (Flores-Moya and Fernandez 1998; Nimer et al. 1998, 1999; Andria et al. 1999; Axelsson et al. 1999). A similar situation exists with freshwater green micro-algae, such as Chlorella and Chlamydomonas (see above reviews). Experiments with another member of this group, Eremosphaera viridis, indicate that the CO 2 transporter also performs the pumping task; Le., it hydrolyzes cytosolic adenosine triphosphate (ATP) and uses the liberated energy to pump CO 2 into the cell against the concentration gradient (Deveau et al. 1998). However, the Crconcentration process does not stop there. Additional proteins involved include CO 2 and bicarbonate transporters on the chloroplast envelopes of the green micro algae Chlamydomonas reinhardtii and Dunaliella tertiolecta (Amoroso et al. 1998), and several intracellular carbonic anhydrases, (including forms associated with pyrenoids, thylakoids and even mitochondria; Eriksson et al. 1998; Villarejo et al. 1998; Park et al. 1999). The large proteinaceous pyrenoids common in microalgae are generally considered to be a part of (and perhaps essential for) the Crconcentrating mechanism, but Morita et al. (1998) have questioned this by reporting that two of four examined species of the green-algae genus Chloromonas lack pyrenoids but have the ability to concentrate Cj. However, the observation of pyrenoids has led to the first report of an algae-like, Cr concentrating mechanism in a land plant. Primitive bryophytes in the class Anthocerotae (hornworts) possess pyrenoids and a Crconcentrating mechanism (Smith and Griffiths 1996). Exactly why this pyrenoid-based Crconcentrating mechanism was lost as land plants evolved is a mystery (Smith and Griffiths 1996). However, its loss may explain another mystery: the presence of carbonic anhydrase in land plants. Perhaps the loss was not complete, and this enzyme is simply a relic of the ancestral mechanism. It may even exist in a loose association with Rubisco in the chloroplast stroma (a relic pyrenoid?; Jebanathirajah and Coleman 1998), and plasmalemma and thylakoid-bound forms are also reported to exist (Stemler 1997; Ignatova et al. 1998). Some researchers have been tempted to conclude that it has no role in the leaves of land plants (our 1996 review). However, this might not apply to rice plants; Sasaki et al. (1998) recently proposed that rice plants suffer from Zn deficiency because of the consequent drop in the activity of carbonic anhydrase, which is a Zn metalloprotein. Incidentally, a very recent report that marine micro-algae can use Cd instead of Zn to assemble this enzyme (Cullen et al. 1999) may be relevant to the observation (Siedlecka et al. 1999) that Cd benefits the activation of Rubisco in bean leaves through a process involving carbonic anhydrase. Carbonic anhydrase may gain notoriety if the conclusions of Tchernov et al. (1998) become widely known. These authors found that the enzyme in marine phytoplankton is so effective that a large excess of CO 2 is produced; because it is not assimilated, it is simply evolved, making phytoplankton a source (rather than a sink, as popularly believed) of this greenhouse gas. 246 Physiology b) Sucrose Biosynthesis Rather surprisingly, research focussed directly on sucrose biosynthesis and its regulation in photosynthetic cells has been almost non-existent during the past 2 years. The only point of interest has been the entry of a new regulator of the penultimate enzyme, sucrose-P synthase. It seems that the regulatory proteins named "14-3-3"s, well known in mammalian cell biology, also exist in photosynthetic cells and bind to phosphorylated proteins, including sucrose-P synthase (and nitrate reductase; Toroser et al. 1998; Moorhead et al. 1999). It seems likely that the inactivation of these two enzymes by phosphorylation is not complete until a 143-3 protein has attached to the phosphorylated site. c) The Enigma of Glucose and DNA The downregulation of photosynthesis that occurs if soluble sugars accumulate in photosynthetic cells and tissues is a relatively new topic that has become both controversial and complicated. Hexokinase is thought to be the enzyme that detects the level of sugars (as hexoses) and then initiates a signal-transduction pathway that leads to a reduction in the transcription of DNA and a consequent reduction in the biosynthesis of enzymes required for CO 2 ftxation. Alteration of the cell's content of hexokinase and experiments with alternative substrates of the enzyme that are phosphorylated but not further metabolized support this role. However, it is unknown whether hexokinase performs the role directly or indirectly, through perturbations that its altered activity causes in the metabolism (Dai et al. 1999; Halford et al. 1999; Moore et al. 1999). Added to this uncertainty is the complication that more than one sugarsensing/signaling system might exist; there is evidence that hexokinaseindependent systems influence the expression of other genes not directly related to CO 2 ftxation (Chiou and Bush 1998; Cheng et al. 1999; Lalonde et al. 1999). There are also indications that environmental parameters, such as low temperature or an abundant supply of nitrogen, can reduce or cancel the system that links sugar levels to the production of photosynthetic enzymes (Huner et al. 1998; Nielsen et al. 1998). Another query concerning the hexokinase system is how the overaccumulation of sucrose (a disaccharide of glucose and fructose) could result in an increased involvement of this enzyme, for which the substrates are hexoses (glucose or fructose, but not sucrose). A solution has been supplied by Moore et al. (1999), who propose that the sucrose is hydrolyzed to the hexoses and then re-synthesized from them in a futile cycle that involves hexokinase but achieves nothing metabolically, other than ATP hydrolysis (Fig. 2). In this scheme, sucrose is hydrolyzed by the enzyme invertase, an event that might explain why photosynthetic 247 Photosynthesis: Carbon Metabolism from DNA to Deoxyribose _ _- L l - hexoses ~ .... sucrose \ export to sinks CYTOSOL CELL WALL Fig. 2. Sucrose cycling and the sugar-mediated regulation of genes encoding enzymes of photosynthesis. The thick arrows represent the standard overview of photosynthesis, with CO 2 entering chloroplasts and sucrose being exported to sink tissues. No free hexoses are involved in this pathway. However, if sucrose accumulates in the vacuoles or cell wall, it may be cycled (thin arrows). Initial hydrolysis by invertase (a) releases free hexoses, which are transported to the cytosol, where hexokinase (b) converts them into hexose-Ps, which can be used to remanufacture the sucrose via the standard pathway. The hexokinase, on becoming more active, somehow initiates a signal pathway involving other proteins ([Cln) and culminating in the repression of the DNA transcription that encodes enzymes of the photosynthetic carbon metabolism. Open circles, squares and triangles represent membrane transporter proteins (Moore et al. 1999) cells possess ample amounts of this enzyme (Kingston-Smith et al. 1999). The scheme might even be elaborated to include the enigmatic cytosolic invertase which, unlike those in the vacuole and cell wall, operates at neutral pH and is more specific for sucrose (Sturm 1999). Lastly, one wonders about the unknown molecules in the signal-transduction pathway subsequent to the enlivened hexokinase. An intriguing possibility is that trehalose (a disaccharide of two glucose units linked C1-to-C1), common in fungi and invertebrates but seldom detected in plants, is involved. By revealing the presence in plants of enzymes of trehalose metabolism, plant-cell DNA and protein analysis have indicated that this might be so (Goddijn and Smeekens 1998; Moorhead et al. 1999; Muller et al. 1999). Indeed, a link to hexokinase might be involved if trehalose6-P strongly inhibits this enzyme in plants, as it does in the fungus Aspergillus niger (Panneman et al. 1998). Physiology 248 d) Mitochondrial Respiration and the Oxidative Pentose-P Pathway There is current consensus, recently reviewed by Hoefnagel et al. (1998), that an intricate metabolic interaction occurs between mitochondria and chloroplasts in illuminated photosynthetic cells. This includes continued respiration by mitochondria in light, such that 10-15% of newly produced photosynthate is re-oxidized to CO 2 (Lawlor 1995). This respiration is considered essential for the generation of carbon skeletons (via the Krebs cycle) destined for use in the biosynthesis of amino acids. The respiration may also be essential for the oxidation of excess reducing equivalents (via the respiratory electron-transport chain; Hoefnagel et al. 1998). The latter role may be especially pertinent at the onset of illumination when, during the so-called induction phase, the Calvin cycle lacks the ability to use the available reducing power supplied by the photochemistry (Igamberdiev et al. 1998; Padmasree and Raghavendra 1999). A second route by which photosynthate may be re-oxidized to CO2 is the oxidative pentose-P pathway, in which glucose-6-P is oxidatively decarboxylated to Ru5P by two enzymes. Six molecules of this Ru5P may then be metabolized back to five molecules of glucose-6-P by eight enzymes. A report that this pathway is restricted to plastids (our 1996 review) has been supported in general; nevertheless, it has been shown ~ CO 2 ( •• Calvin cycle enzymes ~.: J,;:...... .' •.••••• t GAP glucose-6-P t ~ mRNAs + ON A t deoxyri bonucleosi de-P3s t "bOld eos; de- P3S Ru5P ~ ribose-5-P ~ribose-5-P Fig. 3. From DNA to deoxyribose: a conceptual overview. The DNA-directed synthesis of Calvin-cycle enzymes leads to the photosynthetic generation of sugar-Ps, including ribose-SoP, which is the immediate precursor of the deoxyribose in the sugar-phosphate backbone of DNA. A plastid-envelope pentose-P translocator (Debnam and Emes 1999) may be a component of this system. In reality, the events shown would predominantly occur in different cells, with photosynthesis in mature cells and DNA synthesis in meristematic cells Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 249 that it is not universal. It applies to spinach, maize and pea plastids, but not to tobacco and carrot plastids (Krook et al. 1998; Debnam and Emes 1999). A common feature, highlighted by Debnam and Emes (1999), is that the initial two enzymes that catalyze the oxidative de-carboxylation of the glucose-6-P are always found in both the plastid and the cytosol, but the subsequent two enzymes, which begin the metabolism of the RuSP, are often restricted to the plastid. This implies that a plastidenvelope pentose-P translocator must exist to permit the metabolism of any RuSP generated in the cytosol. It is not inconceivable that such a translocator may be critical for the synthesis of the deoxyribose component ofthe DNA offuture photosynthetic cells (Fig. 3). e} Photorespiration A third process by which photosynthate may be re-oxidized to CO 2 is photorespiration. It is initiated when Rubisco (Sect. 2.a) accepts 02 rather than CO 2 as its second substrate; these two small, gaseous molecules act in a straightforward competitive manner for access to the enzyme's catalytic site. When 02 is successful, the products are one molecule of glycerate-3-P (a Calvin-cycle intermediate) and one molecule of glycolate-P (not a Calvin-cycle intermediate). Subsequently, two molecules of glycolate-P are metabolized (via seven reactions distributed in four cellular compartments) to one molecule of glycerate-P and one molecule of CO 2; the latter represents a loss to plant growth. Part of this pathway was recently reconstituted in vitro from mitochondria and peroxisomes isolated from spinach leaves (Raghavendra et al. 1998). There may be some flexibility in the pathway; where the glycine-to-serine conversion was blocked in mutant plants, an alternative route involving formate was observed (Wingler et al. 1999a). Researchers have long been attracted by the concept that photorespiratory CO 2 loss could be avoided (and plant growth consequently increased) if CO 2 's competitive advantage at the active site of Rubisco could be improved. Recent attention has focussed on C4 plants, which achieve this naturally by using the C4 pathway as a CO 2 pump. Based on this phenomenon, Ishimaru et al. (1998) genetically engineered potato to express the C4 enzyme pyruvate orthophosphate (Pi) dikinase from maize, Ku et al. (1999) engineered the addition of the maize gene for Penol-pyruvate (PEP) carboxylase into rice to achieve an increased activity of that enzyme in the leaves, and Lipka et al. (1999) genetically engineered potato to simultaneously achieve expression of a bacterial PEP carboxylase and expression of a Flaveria pringlei NADP-linked malic enzyme. In the latter two cases, there was a limited indication that some reduction in photorespiration occurred, but a notable increase in plant growth rate has not been seen. With rice, increased growth may not oc- 250 Physiology cur, because this plant already seems to be at its capacity for utilizing the photosynthate that its leaves generate. Any extra production resulting from a reduction in photo respiration may only result in feedback inhibition of photosynthesis (Winder et al. 1998; Sun et al. 1999b), perhaps via the hexokinase system reviewed above (Sect. 3.c). f) C4 Photosynthesis The most impressive aspect of C4 photosynthesis is the near-total absence of concomitant photorespiration. The well-known, PEP-carboxylase-based CO 2 pump in the mesophyll cells of C4 plants raises the CO 2 concentration around the Calvin cycle's Rubisco in bundle-sheath cells 10- to 20-fold (Jenkins 1997), so the competition between CO 2 and 02 in bundle-sheath cells heavily favors CO 2 , Nevertheless, recent intensive investigations (Maroco et al. 1997, 1998; Laisk and Edwards 1998) have revealed that 02 still influences C4 photosynthesis. It initiates a minor amount of photorespiration in C4 -plant leaves in normal air but, if these leaves are exposed to air with progressively reduced levels of 02 (so that photorespiration becomes non-existent), another role emerges. This role is that of electron acceptor in the so-called pseudo-cyclic electron transport system, whereby the electron flow from water splitting to 02 boosts the supply of ATP. This event is of special benefit to C4 photosynthesis, which has a higher requirement for ATP than C3 photosynthesis. When these two effects of 02 (one positive, the other negative) are balanced, an optimum 02 concentration of approximately 7% is revealed (Maroco et al. 1997). Modern DNA technology has been applied to sorghum leaves to confirm the differential gene expression expected to produce the typical compartmentation of enzymes between mesophyll and bundle-sheath cells of C4 -plant leaves (Wyrich et al. 1998). Two surprises have arisen. The first is from Ueno (I998a), who found that pyruvate Pi dikinase, which is classically a mesophyll-cell enzyme, is also present in the bundle-sheath cells of some C4 species. The second surprise is from Wingler et al. (I999b), who have destroyed the long-standing status of maize as the traditional NADP-malic enzyme-type C4 plant by demonstrating that the release of CO 2 in the bundle-sheath cells of maize is not catalyzed exclusively by NADP-malic enzyme. Instead, it is catalyzed by a partnership of this enzyme and PEP carboxykinase. Another study with NADP-malic enzyme has shown that it is a marker for the evolution ofC4 photosynthesis. Forms of the enzyme in C3 , C3 -C4 intermediate, C4 -like and true C4 species of Flaveria were examined, and it was observed that slightly smaller forms of the enzyme with distinctive kinetic properties became more significant as C4 photosynthesis became more prominent (Drincovich et al. 1998; Casati et al. 1999). The other C4 enzyme that has received considerable attention has been PEP carboxylase. Light-mediated acti- Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 251 vation by phosphorylation of this enzyme is apparently independent of any Calvin-cycle activity (Smith et al. 1998). The activation is theoretically vital, because the enzyme is judged to be well below saturation with respect to its substrate (Mg-PEP) in cells, thereby making it more responsive to effector molecules (Tovar-Mendez et al. 1998). The phosphorylated enzyme is, therefore, more influenced by these effectors (Bakrim et al. 1998). Interestingly, it has been reported that the enzyme's other substrate, bicarbonate, also influences these regulatory phenomena when its concentration fluctuates around physiological levels (Parvathi et al. 1998). The emergence of C4 plants some seven million years ago (see our preceding review) has been blamed on increased aridity at that time and on lowered atmospheric CO 2 levels (Pagani et al. 1999). The present-day interplay between the CO 2 level, water supply and temperature has been analyzed by Collatz et al. (1998), who conclude that, with the current CO 2 level of 0.035%, a warmest-month temperature greater than 22°C favors C4 plants, whereas C3 plants are favored below this temperature. An intriguing link with the physiology of water stress has also emerged from studies of the strange amphibious sedge Eleocharis vivipara, which possesses C4 -like traits (including Kranz anatomy) when growing out of water, but not when submerged. Ueno (1998b) has shown that the C4 like traits develop in submerged plants treated with abscisic acid. The author consequently proposes that this plant hormone might be central in the differentiation of C4 photosynthetic tissue. Another link involves theC 3 plant rice; pyruvate Pi dikinase, traditionally a C4 chloroplast enzyme, appears in the cytosol of root cells of this plant when they are water stressed or exposed to abscisic acid (Moons et al. 1998). g) Crassulacean Acid Metabolism Unlike C4 plants, crassulacean acid metabolism (CAM) plants do everything in one cell. At night, with stomates open, two enzymes (PEP carboxylase and malate dehydrogenase) collect CO 2 and reductively combine it with PEP to generate malate. The malate is delivered to (and stored in) the large central vacuole that gives CAM plant tissues their succulent, fleshy character. During the day, with stomates shut, the reserve of malate is used to release the CO 2 needed to operate the Calvin cycle and permit plant growth. The remaining pyruvate is metabolized to a carbohydrate reserve, from which the next night's supply of PEP is obtained. Clearly, CAM is revolutionary in two senses: it entails the Calvin cycle supported by the diurnal/nocturnal PEP-malate-pyruvate-PEP cycle, and it enables these plants to survive in hot, dry habitats by being unconventional with respect to stomatal operation. The majority of recent research regarding CAM has highlighted the flexibility of this type of photosynthetic carbon metabolism and the diversity of hot dry habitats in which CAM plants can be found. In a recent 252 Physiology Table 1. A comparison of C3 photosynthesis, classic crassulacean-acid metabolism (CAM), and CAM variants (Cushman and Bohnert 1999). Plants engaged in CAM cycling or CAM idling accumulate smaller pools of organic acids nocturnally compared with plants performing classic CAM; such plants synthesize these organic acids from internally respired C02 Night Stomates open Net CO 2 uptake Day CAM (classic) CAM idling Night Day Night Night + + + + + + + Pool of organic acid Net reduction of CO 2 to sugars CAM cycling + Day + + Day + + + review, Cushman and Bohnert (1999) clarified the possible permutations of CAM (Table O. In addition, Cockburn (1998) has predicted that a more subtle, high-frequency and low-amplitude organic acid fluctuation termed rapid-cycling CAM may be a Cj-concentrating mechanism in some plants. However, Cockburn has also predicted that it will be difficult to detect if it indeed exists. Extensive attention has been given to the neo-tropical genus Clusia [which includes species that are obligatory CAM, C3-CAM intermediate, weak-CAM inducible and (perhaps) nonCAM (Le., C3 )], because comparisons between morphologically similar species with different potentials for CAM permit better analyses of the true value of CAM. Notably, species with CiCAM plasticity seemed to have the advantage of a greater ability to adapt their photosynthetic carbon metabolism to changes in environmental parameters, such as light availability and water supply (Borland et al. 1998; Grams et al. 1998; Herzog et al. 1999a,b; Liittge 1999). A similar conclusion has been used to explain the rather unexpected occurrence of the CAM plant Aechmea magdalenae in the shaded understory of rainforests. It appears to use brief inputs of high light more efficiently compared with similar C3 species and (as expected) grew better during the dry season (Skillman et al. 1999). There is another indication (Baattrup-Pedersen and Madsen 1999) that flexibility in CAM serves its purpose. Its intuitively unexpected occurrence in some fresh-water plants is related to the acquisition of the scarce supply of CO 2; Le., night-time collection and storage (as malate) of CO 2 becomes possible. Incidentally, it appears that CO 2 supply can also be a problem inside the leaves of classic CAM plants; their succulence raises mesophyll resistance to CO 2 diffusion (Maxwell et al. 1998). Comparison of two species of Peperomia, one CAM and the other C3, led Woerner and Martin (1999) to propose that CAM plants use water Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 253 more efficiently not only because they open their stomates when the atmospheric evaporative demand is lower (i.e., at night), but also because they have fewer stomates. Some plants only display CAM when they are water-stressed, e.g., Mesembryanthemum crystallinum. An ingenious experiment by Eastmond and Ross (1997) has indicated that the water stress is primarily detected by the roots, which presumably send a signal to the leaves to switch from C3 to CAM. However, the nature of the signal is not yet known, and Taybi and Cushman (1999) have shown that the imposition of water stress on detached leaves can initiate a Ca2+-dependent signaling pathway that promotes transcription of the gene for PEP carboxylase. A fundamental feature of CAM is the diurnal rhythm of its components (Blasius et al. 1998). Rhythmic oscillations can be detected at all levels, from whole-plant features (such as CO 2 exchange and leaf malate levels) to molecules (such as enzymes). A fine recent example is the daynight oscillation of one of the subunits of the vacuolar ATPase in a photoautotrophic cell-suspension culture from M. crystallinum (Rockel et al. 1998). 4 The Whole Plant An impressive description of whole-plant photosynthesis has recently been provided by Shishido et al. (1999). These authors prepared an account of the entire carbon balance of a young tomato plant and recorded that 42% of fixed carbon was subsequently respired, 28% remained in the source leaves and 29% went into sinks (8% to the shoot apex, 12% to the stem and 9% to the roots). a) Translocation Fascinating details concerning the movement of photosynthetically generated sucrose from leaves to sink tissues via the phloem continue to emerge. There is some contradiction concerning the capacity of this process. It appears to be saturated during the day in castor beans, as evidenced by its unchanged rate when leaf photosynthesis is stimulated 40% on doubling the CO 2 supply (Grimmer and Komor 1999). However, in an Arabidopsis mutant deficient in the ability to synthesize transitory starch, the rate of sucrose translocation to roots was doubled, apparently to compensate for the loss ofthe starch sink (Zeeman and ap Rees 1999). Excellent research during recent years has established that, in plants such as carrot and potato (Shakya and Sturm 1998; Kiihn et al. 1999), sucrose is actively loaded into the phloem by a distinctive sucrose-H+ 254 Physiology symporter of the phloem cell's plasma membrane. Subsequently, the sucrose is moved to sinks; otherwise, it is capable of initiating a signaltransduction pathway that culminates in a reduced activity of this protein (Chiou and Bush 1998), possibly caused by its phosphorylation (Roblin et al. 1998). Clearly, this method of translocation is sophisticated, but it is not universal. It has long been known that cucurbits (such as pumpkin) translocate much of their photosynthetic product as the trisaccharide raffinose or the tetra-saccharide stachyose (sucrose extended by the addition of one or two galactose units). These two sugars have been shown to constitute 85% of the translocated sugars in the culinary herb basil (Biichi et al. 1998). More startling is the discovery (Wang and Nobel 1998) that fructans (sucrose extended by the addition of one to three fructose units) can be the translocated sugars. Wang and Nobel reported the first example of this in the CAM plant Agave deserti. In both these cases, extension of the sucrose molecule was determined to occur in the vicinity of the phloem rather than in the photosynthetic cells. There is a report that the translocation performed by willow (Salix babylonica) and possibly most other temperate forest trees is not sophisticated. Apparently, sucrose simply diffuses through the abundant plasmodesmatal connections that exist between the parenchyma and phloem cells of the leaf (Turgeon and Medville 1998). b) CO 2 Fixation by Stressed Plants Chilling-resistant plants, such as wheat, rye and Arabidopsis, acclimate to cold temperatures (5°C) by synthesizing extra Calvin-cycle and sucrose-biosynthesis enzymes so that their ability to utilize the harvested light energy becomes similar to that of plants grown at moderate temperatures (23°C; Huner et al. 1998; Strand et al. 1999). Studies with rice indicate that this response may occur to some extent when a relatively minor drop in temperature takes place (Ohashi et al. 1998). Chillingsensitive plants, such as tomato and grapevine, have photosynthesis curtailed by low temperatures in the light. Even low temperatures in the dark (Le., at night) decrease daytime photosynthesis, due (at least in part) to interference in the activation of sucrose-P synthase and a proportional increase in photorespiration (Jones et al. 1998b; Flexas et al. 1999). Water stress reduces photosynthesis both directly (by inducing stomatal closure) and indirectly (by promoting reductions in the activities of certain photosynthetic enzymes; Saccardy et al. 1996; SanchezRodriguez et al. 1999; Wingler et al. 1999c). Stomatal closure has been favored as the more significant factor, but Tezara et al. (1999) have recently added the chloroplast thylakoid's ATP synthase (coupling factor) to the list of enzymes whose activities are lowered during water stress. Photosynthesis: Carbon Metabolism from DNA to Deoxyribose 255 They argue that this loss of activity is of prime importance. An odd pair of results relates to the C4 enzyme pyruvate Pi dikinase. Water stress causes its activity to plummet in leaves of the C4 plant sugarcane but causes it to unexpectedly appear in the roots of the C3 plant rice (Du et al. 1998; Moons et al. 1998). Two reports relate to current environmental issues. In the first, Jones et al. (1998a) consider that high-temperature damage to the Rubisco system of dinoflagellate micro-algae is the primary reason for the bleaching of coral reefs. In the second, Allen et al. (1998) review the likelihood that ozone depletion and the concurrent rise in ultraviolet-B irradiance might reduce the photosynthesis of crops and natural vegetation; they conclude that this effect is minimal. c) CO 2 Fixation when CO 2 is Supply Abundant The consistent increase (0.015% per year) in the concentration of CO 2 in the atmosphere due to human activities has led to voluminous research on the photosynthetic carbon metabolism of plants growing in the presence of CO 2-enriched air. Several reviews of this research have appeared recently (our 1996 review; Makino and Mae 1999; Norby et al. 1999); thus, a detailed coverage of the literature will not be made here. In the most recent work, large-scale (and technologically impressive) field experiments indicate that most agricultural species (including wheat, rice and radish) and most forest trees in their usual environments display a consistent and substantial (-40%) increase in their rates of photosynthesis when the concentration of CO 2 in the surrounding air is approximately doubled (Garcia et al. 1998; Monje and Bugbee 1998; Usuda and Shimogawara 1998; Vu et al. 1998; Norby et al. 1999). Acclimation (i.e., downregulation) of photosynthesis involves the sugar-sensing transduction pathway that culminates in the reduction of gene transcription for photosynthetic enzymes (most notably Rubisco) and the consequent reduction in photosynthetic capacity (Moore et al. 1999; Sims et al. 1999; Fig. 2). 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Photosynth Res 55:67-74 Communicated by U. Liittge Dr. Grahame J. Kelly Centre for Molecular Biotechnology School of Life Sciences Queensland University of Technology Brisbane, Queensland 4000, Australia e-mail: [email protected] Physiology The Metabolic Diversity of Plant Cell and Tissue Cultures Otto Gdither and Bernd Schneider 1 Introduction and Objectives Natural products are in high demand as lead structures in the development of new pharmaceuticals and agricultural chemicals. Establishing a pool of natural products of highest attainable chemical diversity is a major strategic approach for screening programs. As sources of natural compounds, living organisms are not able to compete with chemical synthesis (particularly combinatorial chemistry) in the number of compounds provided. However, nature is unequalled by synthetic chemists in its ability to form a tremendous diversity of complex structures. The complexity of natural products often becomes obvious during attempts to produce these compounds using synthetic chemistry. Frequently, considerable effort by numerous research groups is required to synthesize a particular natural product in the laboratory, while the same task is a simple matter for living systems. Consequently, natural products and compounds derived from them are indispensable agents in medicine and agriculture, providing a significantly greater portion of products for pharmacological applications than in the pest-control sector. On close evaluation of the potential of living systems to generate natural products, distinctive features of each group of organisms become obvious. Polyketides, though not exclusively occurring in microorganisms, can be considered as a characteristic class of compounds of microbial origin. The portion of organic halogen compounds is significantly greater in marine organisms than in other living systems. Plants are characterized by the occurrence of many different classes of compounds. Traditionally, they represent a rich source of alkaloids, various types of terpenoids, phenylpropanoids and quinones. Plant-derived cell and tissue cultures are suitable sources of novel natural products, as discussed by several authors (Zenk 1982; Constabel and Vasil 1988; Ellis 1988; Phillipson 1990; Fowler 1992). This overview intends to estimate the diversity of chemical structures accessible from plant-derived cell and tissue cultures. To some extent, approaches that can induce the formation of modified chemical structures are included. This review does not involve aspects of the biotechnological production Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 The Metabolic Diversity of Plant Cell and Tissue Cultures 267 of natural products by plant cell and tissue cultures. Strategies aimed at the development of biotechnological production processes for compounds of commercial interest are a separate research field and have been reviewed from various points of view (Barz et al. 1977; Heinstein 1985; Staba 1985; Hamill and Rhodes 1993; Banthorpe 1994; Yeoman and Yeoman 1996; Berlin 1997). The starting point of the present compilation was an earlier review in this field (Ruyter and Stockigt 1989). The literature on natural products from cell and tissue cultures of higher plants has been reviewed (to the middle of 1999) to list compounds that are not yet known from intact plants or that were first found in cell cultures and later in plants. Compounds already known from plants, even if they later were found in cell cultures, are not considered here. Natural products from lower plants, even if they are claimed to be new, are also not subject of this review. Although the individual steps involved in biotransformation reactions could provide important information regarding the assessment of the chemical potential of cell cultures, new metabolites obtained after biotransformation are also not included. An exception has been made for novel compounds isolated after precursor feeding, because these metabolites are also assumed to be present (probably) in non-treated cell cultures at trace concentrations inaccessible to the analytical techniques used. Based on the list of novel compounds, the chemodiversity of natural products from cell cultures of higher plants has been assessed. 2 Metabolic Diversity of Higher Plants and Their In Vitro Cultures The diversity of natural products found in plant cell and tissue cultures exhibits subtly differentiated features and has to be considered along with the underlying genetic diversity of the parent plant species. The origin of the metabolic diversity of cell and tissue cultures is the same as in intact plants. Basic pathways of secondary metabolism are derived from primary metabolism; these pathways are divided into subpathways. Templates originating from different pathways are combined. In this way, various metabolic processes are responsible for the formation of diverse skeletons of natural products. Depending on the plant families and genera, only a limited number of structural types or templates are found. The skeletons are often specific for these taxa and, therefore, are used as chemotaxonomic markers. Further modification of natural products proceeds via the functionalization of the skeletons. The most common types of modification reactions in secondary-metabolite biosynthesis are oxidation, hydroxylation, reduction, dimerization and rearrangement. They are comparable to the reactions found in biotransformation processes (Suga and Hirata 268 Physiology 1990). Functionalization is frequently complemented by conjugation with various carbohydrates, amino acids, acyl groups and aromatic substituents. Preferentially, functionalization occurs in peripheral regions of the molecules. Whereas the skeleton determines the classification of natural products, the type and position of function ali ties essentially contribute to biological activities. The pattern of functional groups frequently is characteristic for a distinct species, i.e., many plants are able to form unique compounds that do not occur in other species even if they are closely related. This observation is. valid for cell and tissue cultures as well. With regard to the aim of this overview, it can be expected that plant cell cultures in general are able to produce the same types of carbon skeletons the parent plants do. They are not able to perform biosynthetic reactions, which are not known in the plant kingdom. However, due to the enormous number of potential modifications, the occurrence of special compounds with particular substitution patterns is unpredictable. Moreover, because compartmentation and degradation (in addition to biosynthesis) significantly influence the accumulation of natural products, the discovery of novel natural products in plant cell or tissue cultures seems likely but unforeseeable with respect to the formation of a defined compound in a particular culture. Many natural products first isolated from cell cultures were later also identified in the parent plant. Thus, it is likely that compounds that have not been found in plants do occur in concentrations not accessible to modern analytical methodologies. Another reason for the apparent absence of such compounds in intact plants might be compartmentation to certain plant organs and absence in other tissues. Moreover, the accumulation of natural products depends on vegetation cycles, nutritional and environmental conditions, the extent and type of pathogen or herbivore attack, and other exogenous and endogenous factors. However, the expression of genes of secondary metabolism in a de-differentiated cell culture may be altered as a consequence of the selection of certain clones during long-term cultivation under artificial conditions. Reduced or lost activity of a single key enzyme involved in a biosynthetic pathway may cause the accumulation of precursors or create alternative biosynthetic routes not yet known in the intact plant. The development of more sensitive analytical methods and instrumentation, especially modern coupled techniques [gas chromatography-mass spectrometry (MS), liquid chromatography (LC)-MS and Le-nuclear magnetic-resonance spectroscopy], has contributed enormously to the detection of novel compounds occurring only in trace amounts in both plants and cell cultures. The Metabolic Diversity of Plant Cell and Tissue Cultures 269 3 Novel Natural Products from Cell and Tissue Cultures of Higher Plants In total, 322 natural products from cell and tissue cultures of higher plants, described in the literature up to the middle of 1999, have been claimed to be novel compounds. The structures of these compounds are shown in Table 1. Natural products that were not explicitly mentioned as new or that were occasionally identified during rather biochemically and biologically oriented studies may have been overlooked in some rare cases. Classification of the natural products in terms of the taxonomies of their sources has been undertaken throughout the collection of compounds shown in Table 1. This approach reveals that cell cultures of only a moderate number of plant families (48 in total) have been successfully explored for new compounds. Indeed, there has been no systematic screening of cell cultures for natural products using taxonomy as a primary selection criterion. Often, only plant species known to contain high amounts of natural products of particular commercial or scientific interest were used to establish cell cultures. The suitability of some of these cultures for the biotechnological production of secondary compounds was evaluated, and others were chosen to investigate physiological, biochemical and genetic aspects of plant metabolism in general. Thus, the selection of species for cultivation in sterile cell and tissue cultures is not representative of the plant kingdom as a whole. The vast majority of secondary products from plants is represented by terpenoids (>20,000), alkaloids (-10,000) and phenolics (-8,000) (Harborne and Baxter 1993). Brief inspection of Table 1 reveals that most novel natural products from plant cell cultures are also attributable to these chemical classes. One hundred terpenoids, 72 alkaloids (including other nitrogen-containing compounds) and 144 phenolics are listed in Table 1, whereas the remaining six compounds are classified as "miscellaneous". On closer consideration, clustering of frequently occurring structural types in a few species can be observed. In other words, the majority of novel compounds from plant cell and tissue cultures were found in a relatively small number of species. For example, more than 50% (37 compounds) of the 72 alkaloids listed in Table 1 were isolated from the Apocynaceae and are indole alkaloids (comprising several subgroups). Because plants of Catharanthus rose us are the source of the established anti-cancer drugs vincristine and vinblastine, cell cultures of the periwinkle plant and of related Apocynaceae species became popular subjects in the search for further anti-tumor agents. Twelve indole alkaloids (16-27) originated from cell cultures of two Catharanthus species, C. rose us (Kutney et al. 1980; Kohl et al. 1981, 1984; Gueritte et al. 1983) 270 Physiology Table 1. Structures of novel compounds isolated from plant cell and tissue cultures d Acanthaceae ~:~:~~::~: ~ ~!: : : ~~) OH :::;;0.-" o ~IIII' 0 0 d o Andrographis paniculata Nees (ce) (Allison et al. 1968) Panicu!ide C (3) Andrographis paniculata Nees (ee) (Allison et al. 1968) ::;;- o H°""1I"'" 0 0 Actinidiaceae R Hd'" 3P-((E)-p-Coumaroyloxy)-2a,24-dihydroxyurs-12-en-28-oic acid {4, R = (EHrCQumaroyl) Actinidia polygama (Ge) (Sashida et al. 1994) OH OH 2a,3a,24- Trihydroxyurs-11-en-13p,28-olide (S) Actinidia polygama (CC) (Sash ida et a!. 1994) Apiaceae SD C0 H UmbeUiterone-(( E)-3-hydroxymethyl-l-butenyl)-ether (6) Ammi majus L. (SC) (Hamerski et al. 1990) H H 0 OH 0 C02H Ammi majus L. (SC) (Hamerski et a!. 1990) ~ y-\b ~ HPC/\_ Umbelliferone-(( E)-3-melhyl- bula-l ,3-dienyl)-ether (7) 7-Hydroxybulylidenephtatide 7-o-glucoside (9, R =H) 7-Hydroxybutylidenephtalide 7-0-{6'-0malonylglucoside) (10, R malonyl) Petroselinum crispum L. (SC) (Hagemeier et al. 1999) = 2 J:::N ~)-j Cucumopine (8) Daucus carota (HR) (Davioud et al. 1988) ~ib 6-Benzylaminopurineriboside (11) Pimpinella anisum L, (SC) (Ernst et al. 1983) Apocynaceae Aspidochibine (12) Aspidosperma quebracho blanco Schlecht (SC) (Aimi et at. 1991b) 3-0xo-14, 15-dehydrorhazinilam (13) Aspidosperma quebracho blanco Schlecht (SC) (Aimi et ai, 1991 b) 1,6-Propano-3-ethylideno-1 ,4piperazine-2,5-dione (14) Aspidosperma quebracho blanco Schlecht (SC) (Aimi et al. 1994) The Metabolic Diversity of Plant Cell and Tissue Cultures 271 Table 1 (continued) Apocynaceae l1-Hydroxytubotaiwine (15) Aspidosperma quebracho blanco Schlecht (SC) (Aimi et al. 1994) 1O-Hydroxy-desacetyl-akuammiline (18) Catharanthus roseus G. Don (Se) (Petiard et al. 1982) (16R)-19,20-Z-lsositsirikine (21) Catharanthus roseus G. Don (Se) (Kohl 8t aL 1984) Akuammicine-,B-D-xylopyranoside (16) Catharanthus reseus G. Don (SC) (Kutney et al. 1980) 20-Hydroxytabersonine (17) Catharanthus roseus G. Don (SCl (Kohl et al. 1981) 7-Hydroxy-indolenine-ajmalicine (19) Catharanthus roseus G. Don (SC) (Petiard et al. 1982) Pseudoindoxyl-ajmalicine (20) Catharanthus roseus G. Don (SC) (Petiard et al. 1982)] 21-Hydroxy-cycfo-lochnerine (22) Catharanthus roseus G. Don (SC) (Kohl et al. 1984) = ~ I <7 ~ = = H I ' N Epchrosine (28) Ochrosia efliptica Labil!. (Se) (Pawelka et al. 1986) = = = = = Anthraserpine (23, R, OCH 3; R2 CH 3; R3 H) Dimethoxyanthraserpine (24, A"R 3 OCH 3; R2 CH 3) Pseudoanthraserpine (25, R, OCH 3; R2,R 3 H) Desanthraserpine (26, A, ,R3 H; R2 = CH 3) Dimethoxydesanthraserpine (27, R, H; R2 CH 3; R3 = OCH3) Catharanthus trichophyllus (HR) (Oavioud et al. 1989) = = Pericine (29) Picralima nitida Stapf. (SC) (Arens et aL 1982) OR2 ~ <7 I Acetylpolyneuridine (30) Rauwolfia mann;; Stapf. (Se) (Betzler et aI., see: Ruyter and Stockigt 1989) I H Rt . "'"OGle = Rauglucine (31, R, = CH3; R2 H) AcetylraugJucine (32, R, CH 3; R2 Ac) Acetylnorrauglucine (33, R, H; R2 Ac) Rauwolfia serpentina Benth. (SC) (Ruyter et al. 1988) = = = = 21-Hydroxysarpagan-p-D-glucoside (34) Rauwolfia serpentina Benth. (Se) (Ruyter et al. t 988) Physiology 272 Table 1 (continued) Apocynaceae OH Np-Melhylajmaline (35) Rauwolfia serpentina Benth. (SC) (Polz et aI., see: Ruyter and Stockigt 1989) (19S).Hydroxy-Np-melhylraumacline (39) Rauwolfia serpentina Benth. (SC) (Aimi et at. 1991 a, Takayama et al. 1992) Raumacline (36, 208) lsoraumacline (37, 20,R) Rauwolfia serpentina Benth. (SC) (Polz et al. 1990, EndreS al al. 1993) = Alkaloid G (42) Rauwolfia serpentina Benth. (SC) (EndreB et al. 1993) 6a-Hydroxy-raumacline (40, A H) 6a-Melhoxy-raumacline (41, R = CH 3) Rauwolfia serpentina Benth. (SC) (EndreS et al. 1993) ~ I N/l"Methylraumacline (38) Rauwolfia serpentina Benth. (SC) (Polz et al. 1990) OH . ~ I HO "IIIOH H 12-Hydroxyajmaline (43) Rauwolfia serpentina Benth. (HRl (Falkenhagen et al. 1993) OH 3-Qxo-isovoacangine (45) Tabemaemontana e/egans Siapf. (CC) (van der Heijden et al. 1986) Compound D (44) Rauwolfia serpentina Benth. x Rhazya stricta Decaisne (SC) (Aimi et al. 1996) Voafrine A (46, R = a-H) Voalrine B (47. R = fJ-H) Voacanga africana Stapf. (SC) (St6ckigt et al. 1983) Canganol (48) Voacanga africana Stapf. (SC) (Pawelka 1985) Araliaceae OCH3 CI OH OH H HO I H?(oH~L Chloropanaxydiol (49) Panax ginseng (CC) (Fujimoto and Satoh 1988) ,/0 H~ ~.eonjdjn H~O H OH 3-a-p..O-xylopyranosyl(1-+2)-p..o-galactopyranoside (50) Aralia cordata Thunb. (SC) (Asada et al. 1994) 273 The Metabolic Diversity of Plant Cell and Tissue Cultures Table 1 (continued) Asphodeliaceae OH OH 0 ~OR ~ OGle 3.4-Dihydro-2.4.8,9-tetrahydroxy-6-methyt-1 (2 HJanthracenone-4- o-P.D-glucopyranoside (51, R = H) 3,4-Dihydro-4,8,9-trihydroxy-2-methoxy-6-methyl-1 (2 H)anthracenone-4- Q...8-D-glucopyranoside (52, R = CH 3 ) Aloe baroadensis (eC) (Vagi at al. 1998) Balsaminaceae OCH3 OH OCH3 Methylene-3,3'-bilawsone (53) Impatiens bafsamina L. (RC) (Panichayupakaranant at at. 1995) 4,4' -8iisofraxidin (54) Impatiens balsamina L. (RC) (Panichayupakaranant et al. 1998) HO OCH3 Berberidaceae :g?= C0 H3 HO NH I~ HO~:I Gluco-Jatrorrhizine (56) "' ~ H I :::,.. Berberis stolonifera (SC) (ROffer et aI., see: Ruyter Dehydrodiconiferyl·alcohol· r ~D·g[ucoside (57) Plagiorhegma dubium Maxim. (SC) (Arens et al. 1985) and Stockigl 1989) OH OCH3 2-Norberbamunine (55) Berberis stolonifera V29 (eC) (Cassels et al. 1987) OH OH H Podoverine A (58) Podophyllum versipelle Hance (Sel Podoverine B (59, A Podoverine C (60, A (Arens et al. 1986) =OH) =H) Podophyllum versipelle Hance (SC) (Arens el al. 1986) Bignoniaceae o ~ IOIR ::... ~ yy-O~ 0 H 0 8·Hydroxydehydro·iso·a--lapachone (61, A = H) 3,8·0ihydroxydehydro·iso·a-lapachone (62, A ,8-0H) Catalpa ovata G. Don (CC) (Ueda et al. 1980) = OH 0 a·Hydroxy·2·isopropenyl· furanonaphthoquinone (63) Catalpa ovata G. Don (CC) (Ueda et al. 1980) 274 Physiology Table 1 (continued) Boraginaceae Deoxyshikonofuran (68) Ltthospermum erythrorhizon Sieb. et Zucco (SC) (Yazaki et al. 1986) Echinofuran (65, R, = OAe; R2 = H) Echium Iycopsis L. (CC) (InouyeetaL 1981) Echinone (64) Echium Iycopsis L (CC) (Inouye at al. 1981) Echinofuran B (66, R1 ,R 2 = H) Lithospermum erythrorhizon Sieb. et Zuce, (SC) (Fukui et al. 1984) r Hydroxyechinofuran B (67, R, = H; R2 = OH) Lithospermum erythrorhizon Siab. et Zucc, (RC) (Fukui et al. 1998) ~ I r -.;:: H 0 ~ o H Rhizinone (71) Lithospermum erythrorhizon Siab. et Zucco (HA) (Fukui et al. 1999) Dlhydroechinofuran (70) Lithospermum erythrorhizon Slab. et Zucco (SC) (Fukui et al. 1992) Dihydroshikonofuran (69) Lithospermum erythrorhizon Sieb. et Zucco (SC) (Yazaki et al. 1987) Brassicaceae H02C~ HO,c yvV H ~ I 0H N-(E)-4-Coumaroylaspartic acid (72) Arabidopsis thaliana L. (eC) (Mock el al. 1993) ~ 0 Cactaceae 0"" RPm"'" ~ H ~I R20 Cephalocerone (73) Cepha/ocereus senilis (SC) (Pare el al. 1991) 6,7-Dihydroxy-5-methoxyflavone 7-0-j3-0glucopyranoside {74, Rl = Glc; R2 = CH 3} 8aicalein 7- 0-(6" -Q-malonyl- ~o-glucopyranoside) (75, R, = 6-O-malonyl-j3-o-glucose; R2 = H) Cephalocereus senilis (Se) (Liu et al. 1993b, Uu et al. 1993a) 0 (25)-6,7 -Dihydroxy-5-methoxyflavanone 7- 0-P- Dglucopyranoside (76, R, = Glc; R2 = CH 3) (25')-5,6,7-Trihydroxy-f!avanone 7-0-f3-0glucopyranoside (77 ~ R, = Glc; R2 = H) Cephafocereus senifis (SC) (Liu et al. 1993b. Uu et al. 1993a) Campanulaceae OH ~OH_~ ~ RO Lobetyol (78, R = HI Lobelyoline (79, R ::: Glc) Lobetyolinine (80, R ::: Gent) Lobelia inflala (HR) (Ishimaru et al. 1991, Ishimaru et al. 1992) 275 The Metabolic Diversity of Plant Cell and Tissue Cultures Table 1 (continued) Cannabaceae ~ HoN Celastraceae # """ R, \. 1 R 2 14-Methoxyabteta-8,11 ,13-trien-3-one (82, R, :::; 0; R2 = OCH 3) Abieta-8,11,13-trien-3a-ol (83, R, = a-OH,P.H; R2 = H) 14-Methoxyabieta-8,11,13-trien-3fJ-ol (84, R, = a-H,P.OH; R2 OCH 3 ) Tripterygium wilfordii Hook F. (SC) (Kutney and Han 1996) = 7 P. 19-Dihydroxy-14-melhoxy-18( 4-t3}abeo-abieta3,8,11, 13-tetraen-18-oic acid lactone (86) Tripterygium wilfordii' Hook F. (SC) (Kutney and Han 1996) 14,B-Acetoxy-{7,8){J,(9, 11 )/3,(12,13) a-lns(epoxy)-2j3, 19-dihydroxy18(473)abeo-abieta-3-en-18-oic acid lactone (88, R = a-H,jJ-OAc) (7,8)13,(9,11 )13,(12,13) a-Tris(epoxy)-2j3, 19-dihydroxy-14-oxo18(473)abeo-abieta-3-en-18-oic acid lactone (89, R = 0) Tripterygium wilfordii Hook F. (SC) (Kulney and Han 1996) TriptocalJic acid A (91, Al = CH 3 ; R2 = H) TriplocalJic acid D (92, A, = H: A2 = CH 3) Tripterygium wilfordii var. regelii Makino (CC) (Nakano el al. 1997a, Nakano el al. 1997b) 1-(4-Hydroxyphenyl)-nonan-l-one (81) Humulus lupufus L. cv Wye Northdown (SC) (Chandra et al. 1991) 7-0xo-18(4~3)abeo-abieta-3,8, 11, 13-telraen-18-01C acid (85) Tripterygium wilfordii Hook F. (SC) (Kutney and Han 1996) (7,8)13,(9, 11)jJ-Bis(epoxy)-2P, 19-dihydroxy-14-oxo18(473)abeo-ableta-3, 12-dien-18-oic acid lactone (87) Tnpterygium wilfordil Hook F. (SC) (Kutney and Han 1996) TriptocaUol (90) Tripterygium wilfordl! var. regelii Makino (CC) (Nakano et al. 1997a) Triplocallic aCid B (93) Tripterygium wilfordii var. rege/ii Makino (CC) (Nakano et a!. 1997a) Triptocallic acid C (94) Tripterygium wilfordii var. regelii Makino(CC) (Nakano el al. 1997b) 276 Physiology Table 1 (continued) Celastraceae (continued) Triptocalline A (95) Tripterygium wilfordi; var. regelii Makino(CC) (Nakano et al. 1997b) Triptohairic acid (96) Tripterygium wilfordii var. regeli! Makino (RG) (Nakano et aL 1998) 1fJ-Acetoxy-9 a-benzoyloxy-15-cinnamoyloxy-4a,6a-dihydroxy-dihydroagalofuran (97, A::: cinnamoyl) Tripterygium wilford!! var. regeli; Makino (RC) (Nakano et al. 1998) Chenopodiaceae H~H02C 0 H HO OH ~OH I ~ Q o I ~ ~ OH H H02C - Y OCH3 H02C Q OCH 3 0 N-(E)-Feruloylaspartic acid (99) Beta vulgaris l. subsp. vulgaris var. conditiva (eC) (Bokern et al. 1991) 1- 0-( E)-Feruloyl-3- a-D-glucuronosylglycerol (98) Beta vulgaris l. subsp. vulgaris var. conditiva (Ge) (Bokern e1 al 1991) OH ~OH ~ I ~ H 0 H H02C H~ Q 0 OH 0 R 1-0-(E)-p-Coumaroyl-2-,B-O-glucuronosyl-{J-O-glucose (100, R:: H) 1-0(E)-Feruloyl-2-,8-D-glucuronosyl-,8-D-glucose (101, A = OCH 3) Chenopodium rubrum l. (SC) (Bokern e1 al. 1987) Compo sitae (2-Glyceryl)-o.coniferaldehyde (102, R 1,3-dihydroxy-2-propyl) (2-Propenal)-o.coniferaldehyde (103, R = 1-oxo-2-propenyl) Artemisia annua l. (CC) Tanacetum parthenium (l.) Schultz Bip. (CC) (Sy and Brown 1999) = ~ ~ Dihydroparthenine (106) Parthenium hysterophorus (CC) (Talwar and Kalsi 1989) 6-Acetyl-3-angeloyloxy-5-hydroxy2-isopropenyl-2,3-dihydrobenzofurane (105) Eupatorium cannabinum (HA) (Siebertz et al. 1989) Kinobeon A (104) Carthamus tinctoris l. (SC) (Kobayashi et at 1992) ~ 0 o ~/I Ho~~°g-( 14-lsobutyryloxyeuparin (107) Tagetes patula (HA) (Menelaou et al. 1991) The Metabolic Diversity of Plant Cell and Tissue Cultures 277 Table 1 (continued) Droseraceae o 3,3'-Oi-o-methylellagic acid 4,4·-di-0-P.glucoside (108) Dionaea museipula Ellis (SC) (Pakulski and Budzianowski 1996) Ebenaceae 7-Methyl-1 ,4,5-trihydroxynaphtalene 4- 0(6"-D-{3-xylopyranosyl)-P.glucopyranoside (109) Diospyros kaki Thunb. (SC) (Gonda et al. 1999) Ephedraceae 4-(6-Methyl-1-heptene-5-yl)benzoic acid (110) Ephedra distachya (SC) (Song et al. 1994) Euphorbiaceae Helioscopinolide 0 (111, A, :::: a-H,,lJ-H; R2 :::: 0; R3 :::: OH) Helioscopinolide E (112, R, :::: a-H ..8-H: R2 :::: 0; R3:::: H) Helioscopinolide F (113, R,:::: 0; R2 :::: a-H,P.H; R3 = H) Helioscopinolide I (116) Euphorbia calyptrata var. involnerata (SC) (Minghelli el al. 1996) Helioscopinolide G (114, A,.R 2 :::: a-H,,8-H; R3:::: H) a-H,P.H; R2 = a-OH,P.H; R3 = OH) Helioscopinolide H (115, R, Euphorbia calyptrata var. involnerata (SC) (Borghi et al. 1991, Crespi-Perellino el al. 1996, Minghelli el al. 1996) = o Di-(2-hydroxyelhyl)-disulfide (118) Ricinus communis (SC) (Gafni and Shechler 1981) Helioscopinolide L (117) Euphorbia calyptrata var. involnerata (SC) (Mingheni el al. 1996) Physiology 278 Table 1 (continued) Fumariaceae OCH3 OCH 3 (·)·trans-N-Methylisocorypalmine (119) Dicentra peregrina (Sel (Konishi et al. 1998) N-Methyldicentrine (120) Dicentra peregrina (SC) (Konishi et al. 1998) 7-Hydroxynordicentrine (121) Dicentra peregrina (SC) (Konishi et al. 1998) Gentianaceae GIC~ O'J=L ~C02H OCH3 OH 4-Hydroxy-2,6-dimethoxyphenol I-O-jJ-O-glucopyranoside (122) Swertia japonica Makino (RC) (Ishimaru el al. 1990b) 5-(3 '. 0- {3-D-Glucopyranosyl) benzoyloxygentisic aCId (123) Swertia japonica Makino (AC) (Ishimaru el al. 1990b) 8-O-Primverosylbellidifolin (124) Swertia japonica Makino (RC) (!shimaru et al. 1990a) Guttiferae OR Pax ant hone (125, R1 = CH 3 : R 2 ::: H) Hypericum patulum Thunb. (eG) (Ishiguro et al. 1993) Paxanthonin (127, R ::: CH 3) Hypericum patulum Thunb. (CC) (Ishiguro et al. 1995b) Paxanthone B (126. R, ::: H; R2 ::: 3-methyl-2-butenyl) Hypericum patulum Thunb. (CC) (Ishiguro et al. 1995a) Demethylpaxanthonin (128, R::: H) Hypericum patulum Thunb. (CC) (Ishiguro et al. 1996) o OH ~ H~~OR OH PagJucinol (131) Hypericum patu{um Thunb. (SC) (Ishiguro et al. 1998) Padiaxanthone (129) Hypericum patulum Thunb (eC) (Ishiguro et al. 1996) Patulone (130) Hypericum patu{um Thunb. (CC) (Ishiguro et al. 1997) OH Patuloside A (132, R::: ,6-D-Glc) Patuloside B (133, R::: jJ-O-Glc(2-)1 )-a-L-Rha) Hypericum patulum Thunb. (SC) (Ishiguro et al. 1999) 6-C-Prenylluteolin (134) Hypericum penoratum var. angustifofium (SC) (Dlas et al. 1998) 279 The Metabolic Diversity of Plant Cell and Tissue Cultures Table 1 (continued) Haemodoraceae OH HO OH JR :R2 ,p Ry I .." ~ ~ 5 R3 R4 Anigopreissin A (135) Anigozanthos preiss;; (RC) (Holscher and Schneider 1996) 3,3' -bls-Hydroxyanigorufone (140) Anigozanthos preissi; (RC) {Holscher and Schneider 1997} 2,3-Dimelhoxy-4-phenylphenalen-l-one (136, R 1 ,R2 == OCH 3 : R3 == phenyl; R4 ,Rs,Rs,R 7 H) 7.8-Dimethoxy-6-phenylphenalen-l-one (137, Rj,R 2 ,R 3 ,R 4 == H; Rs phenyl; R6 ,R 7 == OCH 3 ) 5-Hydroxy-6-methoxy-7 -phenylphenalen-l-one (138, R"R 2 ,R 3 ,R 7 = H; R4 OH; Rs = OCH 3 : As phenyl) 5-Hydroxy-2,6-dimethoxy-7 -phenylphenalen-l-one (139, R,.Rs = OCH 3 ; R2 ,R 3 .R 7 = H; R4 = OH; Rs == phenyl) Anigozanthos preissii (RC) (Holscher and Schneider 1997) = = = = Labiatae OH OH (E,E)-2-(3,4-Dihydroxyphenyl)ethenyI3(3,4-dihydroxyphenyl)-2-propenoate (141) LavanduJa angustifolia Mill. Subsp. augustifolia (AC) (Banthorpe et at 1985) l-Methylethyl 2,3-dihydro-7,8-dihydroxy-5methoxy-3,10-dimethyl-4-oxo-4f-1-naphto [2,3-bJpyran-3-butanoate (142) Lavandula vera (SC) (Nakajima el al. 1990) 5.7,2',6'-T etrahydroxyflavone 2'-0-j3-D-glucopyranoside (143) Scutellaria baiea/ensls (HA) (Zhou el al. 1997) Lamiaceae OH Cyanidin 3-0-(2-0-(6-0(E}-p-coumaroyl-j3-D-glucopyranosyl)(6-0-{E)-p-coumaroyl-j3-D-glucopyranosyl)-S-O(6-O-malonylf3-D-glucopyranoside) (144, R = (E)-p-coumaroyloxy) Ajuga reptans (SC) (Terahara et al. 1996) 280 Physiology Table 1 (continued) Lardizabalaceae R 3,6-Hydroxy-30-norolean-12,20(29) -d\en-2B-oic acid (145. R H) 3a-Hydroxy-30-norolean-12,20(29) -dien-28-oic acid (146, R = H) 3a,24-Dihydroxy-30-norolean-12.20(29) -dien-28-oic acid (147, R = OH) Akebia quinata Deena (eC) (Ikuta and Itokawa 19S6, Ikuta and Itokawa 1988b) = 3,B-Hydroxy-29(or 30)al-olean-12en-28-oic acid (148) Akebia quinata Deene (Ce) (Ikuta and Itokawa 1986) Trifoside A (153. Rl ::: P-D-Glc«2-tl)-,8-D-Xyl)(3-t1)-a-L-Ara; R2 == H; RJ = a-C02 H,,e.CHJ ) Trifoside B (154, R, = a-L-Ara((2-t1)-j3-D-XyIH3-t1)~,e.D-Glc; R2 H; R3 CH 2) Trifoside C (155, R, = Q"L·Ara«2~1)·jJ-D·Xyl)(3~1)·jJ-D·Glc; R, = OH; R3 = CH,) Akebia trifoliata Koidz. (CC) (Ikuta 1995) = = Quinatoside A (149, Rl ::: a-L-Ara; R2 ::: OH) Quinatoside 8 (150, Rl = a-L-Ara(3-'Jol)-P.O-Glc; R 2 ::: OH) Quinatoside C (151, Rl = a-L-Ara(2-tl)-,6-D-Xyl; R2 ::: OH) Quinatoside 0 (152, Rl ::: a-L-Ara(2-tl)-P-0-Xyl; R2 ::: H) Akebia quinata (eC) (Ikuta and ltokawa 1989b) 3-epi-Mesembryanthemoidigenic acid (156, R1 = a-OH; R2 == CH 20H) 3-D-Acetyl-3-epi-mesembryanthemQidigenic acid (157, R,::: a-OAc; R2= CH 20H) 3-O-Aeetylmesembryanthemoidigenic acid (158, R1 = j3-0Ac; A2 == CH 20H) 3-O-Aeetylserragenic acid (159, A, == a-OAe; R2::: C02H) 3·O-Aeetyl-3-epi-serragenie acid (160, R, = j3-0Ae; R2 == C0.2H) Stauntonia hexaphylla Deene (CC) (Ikuta and Itokawa 1989a) = Mubenoside A (161, R, jJ-D·Glc«2~1)·jJ-D·Xyl)(3~1)·a-L·Ara; R, = a-OH,jJ-CH3) Stauntonia hexaphyl/a Deene (CC) (Ikula el al. 1991) 3a-Hydroxy-11 a,12a-epoxyoleanan-28, 13fJ--olide (162) Stauntonia hexaphy/fa Deene (CC) (lkuta and Morikawa 1992) Lauraceae OCH3 Neocaryachine N-metho salt (163) Cryptocarya chinensis Hems!. (CC) (Chang al al. 1998) The Metabolic Diversity of Plant Cell and Tissue Cultures 281 Table 1 (continued) Leguminosae = = Agroastragaloside J (164, R 2,3-di-O-acetyl-,B-D-xylose) Agroastragaloside II (165. R 2-o-acelyl-,B-D-xylose) Astragalus membranaceus (HR) (Hirotani et al. 1994a, Hirotani et al. 1994b) 7-Acetylchrysophanol (16B) Cassia didymobotrya Fres (SC) (Delle Monache et af. 1991) Agroastragaloside III (166, R = 2,3-di-D-acetyl-j3-D-xylose) Agroastragaloside IV (167, R 2-Q-acelyl-j3-D-xylose) Astragalus membranaceus (HR) (Zhou el at 1995) Chrysophanol-physcion-l0, 10bianthrone (169) Cassia didymobotrya Fres (SC) (Delle Monache et al. 1991) = (E)-3'-Hydroxy-3,4,5'-lrimelhoxyslllbene (170, R = CH 3 ) (Z)-3'-Hydroxy-3,4,S'-lrimelhoxyslilbene (171, R = CH 3 ) {E)-4,3'-Dihydroxy-3,S '-dimelhoxystilbene (172, R = H) Cassia didymobotrya Fres (SG) (Delle Monache et al. 1991) OH ~ OCH3 HCO~ OH 3 7,4 '-Dihydroxy-3,S.3 '-trimethoxyflavone (173) Cassia didymobotrya Fres (SC) (Delle Monache at al. 1991) H~OCH3 7-Methylphyscione (174) Cassia occidentalis L. (Ce) (Kitanaka el al. 1985) 7-Melhyltorosachrysone (175) Cassia occidentalis L. (CC) (Kitanaka et al. 1985) ~OH OH ~ o Echinatin (176) Glycyrrhiza 8chinata L. (GC) (Furuya et al. 1971) Licodione (177) G/ycyrrhiza echinata (SC) (Furuya et at 1976) S'-Prenyl-licodione (178) Glycyrrhiza echinata (SC) (Ayabe el al. 1986) H OCH3 Ucoagrochalcone A (179) GlycYffhiza g/abra (RG) (Asada el al. 1998) Licoagrocarpin (180) Glycyrrhiza glabra (RG) (Asada el al. 1998) OCH3 Ucoagrodione (181) Glycyrrhiza glabra (RC) (U el aJ. 1998) Physiology 282 Table 1 (continued) Leguminosae Medicarpin-3- 0-fJ-o-gtucoside-6 -- O-malonate (183) (R = 6' -O-malonyl-jJ-D-glucopyranosyl) Med/caga sativa L. (SC) RXX?u1 I (Kessmann el al 1990) H3CO ~ ;7 o licoagrone (182) Gfycyrrhiza gfabra (RG) (Asada et al. 1999) ~ I HO OCH3 Afrormosin-l - 0-p..D-glucoside-6· - O-malonale (184) (R = 6 '-O-malonyl-fJ-D-glucopyranosyl) Medicaga sativa L. (SC) (Kessmann 81 al. 1990) OH HO OH (+)-(1 R,2S,SA,6S)-2,6-0i(4-hydroxyphenyl)3,7-dioxabicyclo[3.3.0Joctane (187) Vigna angu/aris (SC) (Kobayashi and Ohla 1983) , 7, GICO,%O ~, HO Kudzuisoflavone A (185) Pueran'a lobata (SC) (Hakamatsuka el at. 1992) Kudzuisoflavone B (186) Pueraria lobata (SC) (Hakamatsuka et aL 1992) ~ 2'-Hydroxydaidzein7,4'·di-0-jJ-D-glucoside (188) Vigna anguJaris (SC) (Kobayashi and Ohta 1983) Linaceae 5-MethoxypodophyJlotoxine4-jJ-O-gJucoside (189) Unum flavum L. (RC) (GBF 1985, Berlin et al. 1988) MaJpighiaceae 6-Acetoxygalphimine B (190) Galphimia glauca (CC) (Osuna et al. 1999) OGlc The Metabolic Diversity of Plant Cell and Tissue Cultures 283 Table 1 (continued) Menispermaceae ~ OCH30H .IIII~ l Dechloroacutumine (191) Menispermum dauricum (AC) (Sugimoto et al. 1998) •"'NCH3 o OCH3 Q CH 3 Moraceae Kuwanone J (192, R"R 2.R 3 ::: OH) (193, R, ::: H; A2.A 3 ::: OH) Kuwanone Kuwanone R (194, A"R J ::: OH; R2 = H) Kuwanone V (195, A"R 2 '" H; R3::: OH) Marus alba L. (Ge) (Ueda at al. 1982, Ueda et al. 1984, Ikula at at 1986) Mutberrofurane T (197) Marus alba L. (Ge) (Hana et al. 1989) Mulberrofurane E (196) Morus alba L. (Ge) (Ueda et a!. 1984) a OH = ..•' 0 H Kuwanol E (198) Motusalba L. (CC) (Hana et at 1989) ~ HO ~ ~ I H OH "" , I Glc Mulberroside D (199, R OH; (Z)-isomer) Mutberroside E (200. R::: H; (E)-isomer) Morus alba (Sel (Hana et al. 1997) I Q 0: I Glc OH Mulberroside F (201) Marus alba (Sel (Hana et al. 1997) Paeoniaceae H 3/3,23-Dihydroxy-lup-20(29)en-28-oic acid (202) Paeonia japonica (ee) (lkuta and Itokawa 1988a) H 3p,23-Dihydroxy-30-norolean12,20(29)-dien-28-oic acid (203) Paeonia japonica (ee) (Ikuta and Itokawa 1988a) 284 Physiology Table 1 (continued) Paeoniaceae 3,B-Hydroxy-oleana-l1 ,13(18)dien-28-oic acid (206, R = H) Paeonia japanica (CC) Paeonia suffruticosa (eC) 3,fl,23-Dihydroxy-oleana-l1 ,13(18)dien-28-oic acid (207, R = OH) Paeonia suffruticosa (eC) (Ikuta et af. 1995) 3,8-Hydroxy-l1-oxo-olean12-en-28-oic acid (205) Paeonia japonica (eC) Paeonia laerif/ora (Ge) (lkuta et aL 1995) 11 a,12a-Epoxy-3.B,23-dihydroxyolean·28,13,B-olide (204) Paeonia japanica (eC) Paeonia lacrmara (eC) (Ikuta et al. 1995) Papaveraceae H3CO OCH3 10-Hydroxysanguinarine (208, R 1 ,R 2 = H) 12-Hydroxychelirubine (209, Rl = CH 3 ; R2 Eschschoftzia ca/itarnica (SC) (Tanahashi and Zenk 1990) 10-Hydroxdihydrosanguinarine (210, R1 ,R 2 = H) OH) 12-Hydroxydihydrochelirubine (211, R1 CH 3 ; R2 Eschscholtzia califomica (SC) (Tanahashi and Zenk 1990) = = =OH) 10-Hydroxychelerythrine (212) Eschscholtzia califomica (SC) (Tanahashi and Zenk 1990) Pedaliaceae o ~ ~I o 2-(4-Methyr-1,3-pentadienyl)anthraquinone (213) Sesamum indicum (RC) (Ogasawara et al. 1993) 2-(4-Methyl-3-pentenyt)anthraquinone (214) Sesamum indicum (RC) (Ogasawara et at. 1993) Phytolaccaceae Esculentoside A (215, R /i-O-Xyl(2->1)-/i-O-G1c(2->1)-a-L-Rha) Esculentoside L, (216, R /i-O-Xyl(2->1)-/i-o-Glc) Phytolacca acinosa (RC) (Strauss et al. 1995) = = (2S, 12 E.1S S)Betanidin 5-0-[(5" -o-E-feruloyl)·2· - o-P.O·apiofuranosylj-,B-O-glucopyranoside (217, A a-C02H) (2 S, 12E, 15R)Betanidin 5- 0-[(5"- o-E-feruloyl)-2'-o-{J-D-apiofuranosylj-,B-D-glucopyranoside (218. A = ,B-C02H) Phytolacca americana (SC/CC) (Schliemann et al. 1996) = The Metabolic Diversity of Plant Cell and Tissue Cultures 285 Table 1 (continued) Polygalaceae (219. A = ,lJ-O-Glu((2 .. 1)-a-L-Aha(4->1),lJ-O-Xyl(3-> 1)-,lJ-O-GaI)(3 ..1)-,lJ-O-Fuc) Po/yga/a amare/fa Crantz CCG) (De.bEme et al. 1999) Rosaceae q-Q-oH OCH3 OGle OCH3 2'·Glucopyranosyl-aucuparin (220) Malus domestica cv, Liberty eSC) (Borejsza-Wysocki at al. 1999) Rubiaceae 1(or.4)-Hydroxy-2(or-3)-hydroxymethyl-5,6-dimethoxyanthraquinane (221, R1 = OH; R2 = CH20H; R3.A4 = H; As, As = OCH3 or 222. A,.A, = H; A, = CH,OH; A, = OH; As.A, = DCH,) 1,4,6-Trihydroxy-5-methoxy-2(or-3)-methylanthraquinone (223, R"R4.Rs =OH; R2 = CH 3; R3 = H; Rs = OCH3 or 1 ,5~Dimethoxy~2.3~methylendioxyanthraquinone (232. A,.As = OCH,; A,+A, = OCH,O; A,.A, = H) 1 ,2.5.6~ Tetramethoxyanthraquinone (233. A,.A,.As.A. = OC;:H,; A,.A, = H) 5-Hydroxy~2~methylanthraquinone 224. A,.A,.A, = OH; A, = H; A, = CH,; As = OCH,) 2-Hydroxy-1,3,4-trimethoxyanthraquinone (225. A,.A,.A, = OCH,; A, = OH; As.A, = H) (234. A, .A,.A,.A. = H; A, = CH,; As = OH) 2,4,6-Trihydroxy-l ,3-dimethoxyanthraquinone (235. A, .A, = OCH,; A,.A,.R. = OH; As = H) Cinchona /edgeriana (SC) (Robins at at 1986) 1,3,S-Trihydroxy-4-methoxyanthraquinone (226. A, .A,.As = OH; A, = OCH,; A,.A, = H) 1,4-Dimethoxy-2,3-methylendioxyanthraquinone (227. A,.A, =OCH,; A,+A, =OCH,O; As.A. = H) 1,3-0ihydroxy-4-methoxyanthraquinone (228. A,.A, = OH; A, = OCH,; A,.As.A. = H) 1,3-Dihydroxy-2,S-dimethoxyanthraquinone (229. A,.A, = OH; A,. As = OCH,; A,.A. = H) 2,5( or~3,5)~Dihydroxy~ 1.3,4(or~ 1.2,4)~trimethoxyanthraquin~ one (230, A"A 3,A4 OCH 3; R2.Rs OH; Ae H or 231. A,.A,.A, = OCH,; A,.As =OH; A. = H) Cinchona Jedgsriana (CC) (Wijnsma et at 1984) = = = 2~Hydroxy-l.3,4,6(or-l ,3,4, 7)~tetramethoxyanthraquinone (236, A,.R3.R 4,Re OCH3; A2 = OH; Rs.A7 = H or 237. A,.A,.A,.A, = OCH,; A, = OH; As.A, = H) = J;Ie 0 OH 1.6(or~ 1.7)~Dihydroxy~2-methyJanthraquinone R5~~R1 ~ I I A' R,O 3 R2 Robustaquinone A (244. R,.R 4 = CH3; R2 =OH; R3.RS = H; Rs = OCH3) Robustaquinone B (245. R, = CH3; R2.R3.Rs = H; R4.RS = CH 3) Robustaquinone C (246. R,.R 2.R3,Rs = H; R4 = CH3; As OCH3) Robustaquinone 0 (247. A, = CH 3 ; R2 ,R 3,R s.As H; R4 = CH3) Robustaquinone E (248, R"R 3.R s = H; R2 = OH; R4 = CH3; Rs = OCH3) Aobustaquinone F (249. R,.R 3.R4 = H; R2.Re = OH; Rs = CH3) Robustaquinone G (250. A, = CH 3; R2 = OH; R3+~ = OCH2; As = CH 3; A.=OCH,) Aobustaquinone H (251. R,.R 2 = H; Al.As = OCHl ; A4,As CH3) Cinchona robusta How. (SC) (Schripsema et al. 1999) = = = (238, A"R e = OH; R2 = CH3: A3,R4.RS.R7 = H or 239, A"A7 = OH; R2 = CH 3; R3,R4,As.Ae = H) 2,4,5~ Trihydroxy-l-methoxyanthraquinona (240. A, = OCH,; A,.A,.A s = OH; A,.A•. A, = H) 1.6(or·l.7)-Dihydroxy-3.7(or-3,6)-dimethoxyanthraquinone (241. A,.Ae = OH; A3.R7= OCH3; R2.A4.Rs = H or 242. R,.R 7 = OH; R3.Ae = OCH 3; R2,R4,R s = H) 6.7·Dihydroxy-l-mathoxy~2-methylanthraquinone (243. A, = OCH,; A, = CH,; A•• A, = OH; A,.A,.As = H) Cinchona pubescens (CC) (Wijnsma et al. 1986) 286 Physiology Table 1 (continued) Rubiaceae o OH ~OR1 ~OR2 o 2-Methyl-3,5.6-trihydroxyanthraquinone (252, R"R 2 .R 3,R 4 :::: 1-Hydroxy-2-hydroxymethyl-3-melhoxyanthraquinone (258, R, = H; R, = GH3) 2-n-ButyJoxymethyl-l,3-dihydroxyanthraquinone (259, R, = n-butyl; R, = H) Ophiorrhiza pumila Champ. (eC) (Kitajima el al. 1998) H) 2·Methyl-3,5,6-trihydroxyanthraquinone-6-~primveroside (253, R"R 2 ,R J :::: H; R4 :::: fJ-Prim) 3-Hydroxymorindone (2S4, R, ::: OH; R2,R 3 ,R4 == H) 3-Hydroxymorindone-6-,B-primveroside (255, R, :::: OH; R2,R3:::: H; R 4 :::: p-Prim) 5,6-Dihydroxy-lucidine (256, A"R 2 :::: OH; R3.R4:::: H) 5,6-Dihydroxy-lucidine-3-fJ-primveroside (257, R"R, OH; R3 ji-Prim; R, H) Morinda citrifolia L. {SC) (Inoue et al. 1981) = = = Rutaceae ~ R ~ 1 OH 1,& 1 \PH~~~H H02C~ 0 1 H3CO H H ~ rY'ay 0 OH Ptelecultinium-salt (262) Ptelsa trifoliata L. (CC) (Petit-Paly et aJ. 1987) KaempferoI3-0-jj.o-glucopyranoside6"-(3-hydroxy-3-methylglutarate) (260, R = H) Kaempferol 3- O-,B-O-glucopyranoside6 .,-(3-hydroxy-3-methylglutarate)-7 - 0P-D-glucopyranoside (261, R = Glc) Citrus aurantifofia (CC) (Berhow et al. 1994) ~ _ \pH H02~ ~ Alkaloid A6 (264, R = GI) Ruta bracteosa, R. chafepensis, R. macrophylla (CC) (Baumert et aJ. 1992) Gravacridonol monoglucoside (265, R = OGlc) Thamnosma montana (SC) (Baumert et aJ. 1994) H3Co:xxx0~o~o H~ ~ I 00 OH Rutarensin (263) Ruta chafepensis L. (SC) (Fischer et at. 1988) 8-Methoxydictamnine (266) Ruta graveofens L. (SC) (Sieck et aL 1973) Rutagravine (268) Ruta graveo/ens L. (CC) (Nahrstedt et al. 1985) 1-Hydroxyrutacridone-epoxide (261) Ruta graveo/ens L. (CC) (Nahrstedt et al. 1985) 287 The Metabolic Diversity of Plant Cell and Tissue Cultures Table 1 (continued) Scrophulariaceae c5 OH H3CO,,&OH ~H {"'" ~. ~ I OH OH ~ O~~OH ~ H~ !(I HO OH 7-Desoxy-8-epi-valerosidate (269) Penstemon serrulatus strain AC (CC,SC) (WysokinsKa and Skrzypek 1992) ~OR OH cis-Martynoside (270, R = CH J ) cis-Leucosceptoside (271, R ;::: H) Penstemon serrulatu$ Menz, (SC) (Skrzypek et aL 1999) Simaroubaceae 2-Hydroxycanthin-6-one (272, R,.R3.R,j = H; R2 = OH) 4-Hydroxycanthin-6-one (273, R T,R2 .R 4 H; R3 OH) Ailanthus a/tissima Swingle (SC) (Crespi-PereHino at al. 1986) = = 4-Hydroxy-5-methoxycanthin---6-one (274, R 1 ,R2 H; R3 = OH; R4 "" OCH 3 ) Brucea javanica (SC) (Uu et al. 1990) = 5.11-Dimethoxycanlhin-6-one (27S, R"A 4 = OCH3; R 2 ,R 3 H) Brueea javanica (SC) (Chen et a!. 1993) = 4.5-Dihydrocanthin-6-one (276) Ailanthus altissima Swingle (SC) (Crespi-Perellino et aL 1986) Solanaceae RON"" '!r~OH Tigloylpseudolropin (277) Atropa baetica Wi!lk. (HR) (Zarate 1999) )):.(R ~I IX) Hyalbidone (281) Hyoscyamus a/bus (HR) (Sauerwein et al. 1991) = 3-Hydroxy-6-propionyloxy-tropane (279, R C 2H 5) 3-Hydroxy-6-buty'YI-(or 6-isobutyryl)-oxy-tropane (280, R = C 3 H 7) Dautura candida hybrid. (HR) (Christen et al. 1990) cis-9,lO-Dihydrocapsenone (278) Capsicum annuum L. var. New Ace (SC) (Whitehead et aL 1987) H § >-- (3R,4S.5R.7S.9R)-3-Hydroxy-9·tigloyloxy· solavetivone (282. A 2-methY!'2-bulenoyJ) (3R.4S.5R. 7 S. 9R).J.Hydroxy-9-(3-methylbutenoyloxy)solavetlvone (283, R 3-methyl -2·butenoyJ) (3R.4S.SR. 7 S. 9R)-3-Hydroxy-9·isobulyryloxy· soJavetivone (284, R isobutyryl) (3R,4S,5A,7 S, 9R)-3,9-Dihydroxysolavetivone (285. R = H) Hyoscyamus a/bus l. (AG) (Kuroyanagi et a1. 1998) = = = ~OGent Isopenlylgenliobioside (286) Lycopersicon esculentum (SC) (De Rosa et al. 1996) 288 Physiology Table 1 (continued) Solanaceae HO'CI~'C(t? HO,C l-Hydroxydebneyol (288, R, = OH; A2 = H; C-7Ha) 8-Hydroxydebneyol (289, R, H; R2 = OH; C-7Ha) 7-Epi-debneyol {290, R"R2 = H; C-7HPJ Nicotiana tabacum (SC) (Whitehead et al. 1988) = j3-D-Glucosyl-isofucosterol (287) Lycopersicon esculentum (SC) (De Rosa et al. 1997) f\O~H Mikimopine (291) Nicotiana tabacum (HR) (Isogai et al. 1990) ~ H°VcO ~o~OH UOH 3a-«E)-3-Methoxymethacryloyloxy)-tropane (294) Physochlaina orientalis (SC) (Gorinova et al. 1994) Demelhylphyllalbine (293) Physochlam8 orientalis (SC) (Gorinova et al. 1994) 15-Hydroxytrichodielle (292) Nicotiana tabacum (SC) (Zook et al. 1996) H~~~ OH~O ~ o oH H OH OH sc-, (295) Solanum chrysotrichum Schldl. (SC) (Villarreal et al. 1998) Taxaceae Taxal C (296, R, = Ac: R2 = NHCOn-CsH,,) 10-Deacetyltaxol C (297, R, H: R2 NHCOn-CsH,,) N-Methyltaxol C (298, R, = Ac; R2 = N{CH 3)COn-CsH,,) Taxicultine (299, R, = Ac; R2 = NHCOn-C 3H 7) Taxus baccata (CC) (Ma et al. 1994a) = = 20,50,1 Op, 14,8-Tetraacetoxy-4(20), ll-laxadiene (300, R"R3,R4,R s 20,50,10,8- Triaceloxy-14,8-propionyloxy-4(20), ll-taxadiene (301, R"R 3,R4 = Ac: R2 = H; Rs = propionyl) 20,50,10,8-Triacetoxy-14,8-isobutyryloxy-4(20),11-taxadiene (302, R"R3,R4 Ac; R2 = H; As isobulyryl) 2a,50,10,8-Triaceloxy-14,8-(2-melhyl-bulyryloxy)-4(20),11-laxadiene (303, R"R 3,R4 = Ac; R2 H; Rs 2-melhyl-bulyryl) Taxus chinensis var. marei (SC) (Ma et al. 1994b) = =Ac: R2 =H) = = = Taxuyunnanin-7,8-01 (304, R,.R 3,R 4,R s Taxus cuspidata Sieb el Zucc, (CC) (Fedoreyev et al. 1998) =Ac; R2 =OH) 10,B-Hydroxy-2a,5 a-diacetoxy-14,8-(3-hydroxy-2-melhyl-bulyryloxy)-4(20), 11taxadiene (305, A"A 2 = H; R 3 ,A 4 = Ac; As = 3-hydroxy·2-methyl-bulyryl) 2a, lOp, 14{J-Trihydroxy-Sa-aceloxy-4{20), l1-laxadiene (306, A, ,A 2 ,A 4 ,A s =H; A3 =Ac) 2a;Hydroxy-5a, lOp, 14{J-triaceloxy-4(20), l1-taxadiene (307, R"R 3,A s Taxus yunnanensis (SC) (Cheng et al. 1996) =Ac; R2,R 4 =H) The Metabolic Diversity of Plan t Cell and Tissue Cultures 289 Table 1 (continued) Taxaceae 2a-Benzoxy-4a-acetoxy-l P.7 p, 1OfJ-trihydroxy-9-dehydro- tax-ll-ena (308, A,.R3 =H; A2 = 0; R4 = Bz; As =OH) 2a-Benzoxy-4u.l O,8-diacetoxy-l /3.7 fJ,9a-trihydroxytax-l1-ene =Ac; Rz =a-OH,P.H; R, =H; R, =Bz; Rs =OH) (309, A, 2a,4a,7 /3,9a. 10,8-Pentaacetoxytax-l1-ene (310. R"R 3 ,R4 = Ac; R2 a-OAc,p..H; Rs H) 2a-Benzoxy-4a, 7 p,9a, 1O,8-tetraacetoxy-l ,8-hydroxytax-l1-ene (311. R, ,R3 = Ac; R2 ll'-OAc,,8-H; R4 Bz; Rs OH) Taxus chinensis (Pilger) Rehd. (SC) (Men hard et al. 1 998) = 13-Deacetoxybaccatin t (312) Taxus chinensis (Pilger) Rehd. (SC) (Menhard et al. 1998) = = = = Umbelliferae OH OH H Cyanidin 3-0-(6- 0-(6- 0-( E)-feruloyl-{J-D-glucopyranosyl)-2- Q-{3-0xylopyranosyl-f3-D-g1ucopyranoside) (313, A = (E)-feruloyloxy) Glehnia fittoralis (CC) (Miura et al. 1998) st5Hoyy Valerianaceae OAc ...::::: Valdlate (314) Valenana offlcma/ls L var sambucifolia Mikan (HA) (Granicher el al. 1995) = OR3 ~"'" RP OH o i H = = = = = = = = OR, Compound M (322. R,.A3 isovaleroyJ; R2 Va/en·ana wallichii D.C. (SC) (Becker and Chavadej 1985) 7 -Desisovaleroyl-7-acetyl-homovaltrata (315. A, 3-methyl-valeroyf; R2.R3 Ac) l-Homoisovaltrate (316. R, 3-methyl-valaroyl; R2 Ac; R3 isovaleroyl) Dihomovaltrate (317. R,.R 2 = 3-methyl-valeroyl; R3 = Ac) 11/J-Hydroxy-isovallrate (318. R, = isovaleroyl; R2 Ac; R3 3-hydroxy-isovaleroyl) l-a-Ace-homovaltrate (319. A, 2-acetoxy-isovaleroyl; R2 3-methyl-valeroyl; R3 Ac) Compound E (320. R, 2-acetoxy-isovaleroyl; R2 3-acetoxy-isovaleroyl; R3 = Ac) Compound 0 (321. R, 2-acetoxy-isovaleroyl; R2 3-hydroxy-jsovaleroyl; R3 Ac) Va/eriana waffichii D.C. (SC) (Becker at at 1984. Becker and Chavadej 1985) =Ac) = = = = = = = Ac, acetyl; Ara, arabinose; Bn, benzyl; Bz, benzoyl; CC, callus culture; Gent, gentiobiose; Glc, glucose; HR, genetically transformed root culture ("hairy roots"); Prim, primverose; RC, non-transformed root culture; Rha, rhamnose; Rib, ribose; SC, suspension culture; Xyl, xylose 290 Physiology and C. trichophyllus (Davioud et al. 1989). A number of novel compounds of this type (31-42, in addition to a variety of known structures) were isolated from highly productive Rauwolfia serpentina cellsuspension cultures (Ruyter and Stackigt 1989), and another new indole alkaloid (43) was found in hairy roots of the same species (Falkenhagen et al. 1993). The alkaloids of the Apocynaceae, although they have the indole ring system in common, exhibit highly diverse structural features based on variations in the skeleton. From Rauwolfia cell or root cultures, for example, a variety of new members of indole alkaloid sub-groups, namely ajmalanes (31-33, 35, 43; Ruyter et al. 1988; Ruyter and St6ckigt 1989; Falkenhagen et al. 1993), sarpaganes (30,34; Ruyter et al. 1988; Ruyter and Stockigt 1989) and raumaclines (36-41; Polz et al. 1990; Aimi et al. 1991a; Takayama et al. 1992; EndreB et al. 1993) were identified. Known compounds of the latter subgroups and those of indolenines, heteroyohimbines, tetraphyllicines and yohimbanes were also found. Unusual structural types [such as voafrines (46, 47) from Voacanga africana (St6ckigt et al. 1983), which represent dimeric indole alkaloids] and skeletons containing medium-sized rings, e.g., aspidochibine (12) from Aspidosperma quebracho blanco (Aimi et al. 1991b), have been found in cell cultures of this family (St6ckigt et al. 1995). Five new indole alkaloids (the canthin-6-ones; 272-276) were found in the species Ailanthus altissima (Crespi-Perellino et al. 1986) and Brucea javanica (Liu et al. 1990; Chen et al. 1993) of the family Simaroubaceae. To the best of our knowledge, and in contrast to alkaloids from cell cultures of the Apocynaceae and Simaroubaceae, novel alkaloidal structures from cell cultures of other indole alkaloid-producing plant families, such as Loganiaceae and Rubiaceae, have not been reported in the literature. This clustering of novel compounds is also observed for the terpenoids. Sesqui-, di- and tri terpenoids (82-97) from the Celastraceae (Kutney and Han 1996; Nakano et al. 1997a,b, 1998), helioscopinolides (111-117) from Euphorbia calyptrata (Euphorbiaceae; Borghi et al. 1991; Crespi-Perellino et al. 1996; Minghetti et al. 1996), tri-terpenoid saponins and sapogenins (145-162) from the Lardizabalaceae (Ikuta and Itokawa 1986, 1988b, 1989a,b; Ikuta et al. 1991; Ikuta and Morikawa 1992; Ikuta 1995) and taxane diterpenoids (296-312) from Taxus species (Taxaceae; Ma et al. 1994a,b; Cheng et al. 1996; Fedoreyev et al. 1998; Menhard et al. 1998) together account for 58 of the 100 terpenoids. The remaining 42 compounds are scattered more broadly into 11 families. After the discovery ofpaclitaxel (Taxol) as an anti-tumor agent (Wall and Wani 1998), Taxus and related species have been widely examined in the search for natural products. Plant cell cultures are promising systems for the production of paclitaxel and its biogenetic precursors and, therefore, they were intensely investigated. Considering the large number of research groups involved in phytochemical studies of Taxus The Metabolic Diversity of Plant Cell and Tissue Cultures 291 plants (Parmar et al. 1999), the 17 new taxanes (296-312), which were isolated by only four groups (Ma et al. 1994a,b; Cheng et al. 1996; Fedoreyev et al. 1998; Menhard et al. 1998), represent a reasonable yield and indicate that plant cell cultures are suitable systems in which to find novel compounds. Although Taxus plants are a rich source of diverse secondary products, there are no reports of novel compounds other than taxanes from corresponding cell cultures. Obviously, the search for taxanes was highly preferred to the isolation of other new natural products that probably occur in Taxus cell cultures. Only compounds already known from other species were occasionally isolated from Tax us (Salciccioli et al. 1998). Thus, the novel structures from Taxus cell cultures are restricted to taxanes and exhibit only moderate structural diversity. The diversity of natural products hitherto known from plant cell and tissue cultures seems to reflect not only the objective potential of these sources but, at least in part, the rather subjective scientific approaches employed. In Table 1, the family Lardizabalaceae is represented by 18 novel compounds (145-162), all identified by the group of Ikuta (Ikuta and Itokawa 1986, 1988b, 1989a,b; Ikuta et al. 1991; Ikuta and Morikawa 1992; Ikuta 1995). This is another excellent example illustrating that cell cultures from suitable species are worthy of intense study. However, this example also demonstrates that the specific interests, experiences and approaches of the researchers involved may essentially determine the selection of cell cultures for phytochemical studies and, consequently, the types of novel compounds discovered. Several species of the Rubiaceae were among the first plants studied in callus and suspension cultures. Thirty-nine novel anthraquinones and anthraquinone glycosides (221-259) were found in cultures of the genera Cinchona (Wijnsma et al. 1984, 1986; Robins et al. 1986; Schripsema et al. 1999), Morinda (Inoue et al. 1981) and Ophiorrhiza (Kitajima et al. 1998). These compounds exhibited only limited diversity; this diversity was almost exclusively due to the oxygenation and glycosidation patterns. Two anthraquinones (213, 214) were found in the family Pedaliaceae (Ogasawara et al. 1993). The Boraginaceae, known for the occurrence of shikonin, which was the first natural compound produced biotechnologically via cell cultures (Tabata and Fujita 1985), are the source of further new naphtho-, benzo- and benzohydroquinones (64-70; Inouye et al. 1981; Fukui et al. 1984, 1992, 1998; Yazaki et al. 1986, 1987). In addition, a unique quinone structure (71) was isolated from Lithospermum erythrorhizon (Fukui et al. 1999). Further quinones were found in the Bignoniaceae (61-63) and Leguminosae (genus Cassia; 168, 174). In contrast, other quinone-producing families, such as the Ancistrocladaceae, Droseraceae, Iridaceae, Juglandaceae, Polygonaceae and Rhamnaceae, did not contribute novel quinones from their respective 292 Physiology cell cultures, presumably in part due to the lack of appropriate in vitro systems. Members of speciaI phenylpropanoids were found in cell cultures of both mono- and dicotyledonous plants. The kuwanones and related structures (192-198) from Morus alba (Moraceae; Ueda et aI. 1982, 1984; Ikuta et aI. 1986; Hano et aI. 1989) and the phenylphenalenones (135139) from Anigozanthos preissii (Haemodoraceae; Holscher and Schneider 1997) presumably are formed by biologicaI Diels-Alder reactions of prenylated chaIcones and diarylheptanoid intermediates, respectively (Ichihara and Oikawa 1998). Cell cultures of only a few plant families produce diverse novel compounds. These families, mainly the Leguminosae and the Solanaceae, comprise a relatively large number of genera and species, many cell and tissue cultures of which have been studied phytochemically. From cell cultures of Leguminosae species, triterpene saponins (164-167; Hirotani et al. 1994a,b; Zhou et al. 1995), a chaIcone (179; Asada et aI. 1998), chaIcone metabolites (176-178, 181; Furuya et al. 1971, 1976; Ayabe et aI. 1986; Li et aI. 1998), flavonoids (180, 183-186, 188; Kobayashi and Ohta 1983; Kessmann et aI. 1990; Hakamatsuka et aI. 1992; Asada et aI. 1998), a tetrahydroanthracene (175; Kitanaka et al. 1985), a biaurone (182; Asada et aI. 1999) and a lignan (187; Kobayashi and Ohta 1983) have been isolated. Remarkably, a suspension culture of Cassia didymobotrya was the source of a bianthrone (169), stilbenes (170-172) and a flavone (173; Delle Monache et al. 1991). Moreover, the occurrence of anthraquinones in the genus Cassia (C. didymobotrya and C. occidentalis) has been mentioned above. The natural products isolated from Solanaceae cell and tissue cultures exhibit a similarly broad diversity. Sesquiterpenes (278, 282-285, 288290,292; Whitehead et aI. 1987, 1988; Zook et aI. 1996; Kuroyanagi et al. 1998), tropane alkaIoids (277, 279, 280, 293, 294; Christen et aI. 1990; Gorinova et aI. 1994; Zarate 1999), a piperidone alkaloid (281; Sauerwein et al. 1991), a sterol glucoside (287; De Rosa et al. 1997), a steroid sapogenin (295; Villarreal et aI. 1998) and an aIiphatic glycoside (286; De Rosa et aI. 1996) have to be mentioned. A new opine (291) was also found in hairy roots of Nicotiana tabacum transformed by Agrobacterium rhizogenes (Isogai et al. 1990). Another compound (8) of that type was found in Daucus carota (Apiaceae) hairy roots (Davioud et al. 1988). Because opines are produced by infected plant tissue only, they have to be considered a consequence of integration of T-DNA from the Ti plasmid of A. rhizogenes into the plant genome rather than true plant natural products. The Metabolic Diversity of Plant Cell and Tissue Cultures 293 4 Strategies to Induce the Formation of Natural Products in Plant Cell and Tissue Cultures The majority of novel compounds from the whole spectrum of plant cell and tissue cultures have been isolated from cell suspensions (approximately 160 novel compounds). A considerable number of compounds have also been found in callus tissues (-100). Most of these compounds were found without special treatment of the cultures except the usual optimization of the culture conditions. Cell suspension cultures consist of completely de-differentiated cells. In theory, genetic totipotency enables each individual cell to synthesize the natural products of the whole plant. In practice, however, this is not generally true. The genes responsible for the formation of natural products are not expressed in each cell line. Thus, the formation of natural products in nontreated cell cultures sometimes is disappointing, mostly because conditions enabling enhanced cell growth and cell division are used. Moreover, during long-term cultivation, fast-growing cell clones with a low ability to biosynthesize secondary products can be asserted against slower-growing clones. Nevertheless, the opportunity to manipulate the medium and optimize the hormone balance for a desired purpose is one of the most important advantages of cell cultures. A typical example is the variation of the auxin:cytokinin ratio to promote the formation of non-differentiated callus tissue or to stimulate differentiation to allow root growth or shoot formation, respectively (Banthorpe 1994). Hormone composition was used, for example, to trigger cell-culture lines of Euphorbia calyptrata to produce a number of helioscopinolide diterpenoids (113, 114, 116, 117; Minghetti et al' 1996). In a number of species, some degree of morphological differentiation seems to encourage the stimulation of natural-product formation. Both non-transformed and genetically transformed (by Agrobacterium rhizogenes) root cultures ("hairy roots") possess a higher degree of differentiation than cells growing as callus or in suspension. Thus, these systems are increasingly employed to search for novel compounds. During recent years, more than 20 novel compounds were found in transformed root cultures, whereas nearly 30 further novel compounds were isolated from nontransformed root cultures (Table 1). A small number of compounds, shown in Table 1, were isolated from cells derived from particular plant tissue [from flower tissue (144; Terahara et al' 1996)] or from cultures maintained under special conditions, such as an air-lift reactor (263; Fischer et al. 1988) or immobilized cells (142; Nakajima et al. 1990). Other compounds were isolated by employing peculiar techniques, such as radioactivity-guided fractionation (118; Gafni and Shechter 1981) or the absorption of metabolites on activated carbon (66; Fukui et al' 1984). Plant in vitro cultures are maintained under sterile conditions and, therefore, do not compete with herbivores and pathogens, which fre- 294 Physiology quently trigger the production of natural products in plants as a part of their defense mechanism. This is regarded as another reason for the frequently low productivity of cultures, especially suspension cultures. Several methods, e.g., elicitor treatment and precursor feeding, have been employed to induce or stimulate the formation of secondary products in these systems. The effect of exogenous stress factors can be simulated by biotic elicitors, such as cellular fractions of pathogens, lytic enzymes and substances that either are involved by themselves in or have an influence on signal-transduction pathways. Treatment with yeast extracts, for example, produced compounds 1l0(Song et al. 1994), 185 and 186 (Hakamatsuka et al. 1992), and 208-212 (Tanahashi and Zenk 1990). Elicitation by means of cellulase or chitin led to the new compounds 288-290 (Whitehead et al. 1988) and 73 Pare et al. 1991), respectively. Moreover, chemical elicitors also stimulate the formation of novel secondary compounds in plant cell and tissue cultures. Actinomycine and colchicine were successfully employed in finding compounds 187, 188 (Kobayashi and Ohta 1983) and 315-321 (Becker et al. 1984; Becker and Chavadej 1985), respectively. Jasmonates and other intermediates of the lipoxygenase cascade were proven to be effective signaling compounds in plants and elicit secondary-product formation in cell cultures (Gundlach et al. 1992). Therefore, they may be considered as tools to simulate pathogen or herbivore attack. For example, the sesquiterpenes 282-285 (Kuroyanagi et al. 1998) and the taxane derivatives 308-312 (Menhard et al. 1998) were found after jasmonate treatment. Precursor feeding enhances the spectrum of natural products. This approach led to the isolation of 8-methoxydictamnine (266) from cell cultures of Ruta graveolens (Steck et al. 1973) and was later extensively used to induce the formation of a variety of indole alkaloids (36-42) in Apocynaceae cell cultures (Polz et al. 1990; Aimi et al. 1991a; Takayama et al. 1992; EndreB et al. 1993). It is also used to overcome rate-limiting steps and blocked biosynthetic pathways; in this way, it increases the concentration of natural products above the detectable level. A straightforward approach, the combination of biosynthetic pathways from different species, has been applied to induce the production of novel compounds. Cultured hybrid cells established from cells of two Apocynaceae species, Rauwolfia serpentina and Rhazya stricta, were found to produce a broad spectrum of indole alkaloids (Sheludko et al. 1999), including a new one (44) that was not found in either the parent plants or cell cultures (Aimi et al. 1996). Hybrid cell cultures and (presumably more efficiently) the transfer ofbiosynthetic genes between plant cells and micro-organisms and between different plant cell lines are expected to become broadly used techniques in the near future. For example, transfer of a gene encoding trichodiene synthase from a Fusarium species to tobacco cell cultures led to the in vivo formation of The Metabolic Diversity of Plant Cell and Tissue Cultures 295 a novel sesquiterpenoid, 15-hydroxytrichodiene (292; Zook et al. 1996). Molecular genetic techniques will provide the opportunity to combine biosynthetic pathways with the prospect of creating novel types of structures via combinatorial biosynthesis. 5 Conclusions In this review, it has been shown that at least 322 novel natural products from cell and tissue cultures of higher plants were hitherto described in the literature. There was a significant increase of approximately 230 structures since a comprehensive overview (Ruyter and Stockigt 1989) was published more than 10 years ago. The continuous demand for novel pharmaceuticals will enhance the role of plant in vitro systems in the search for bioactive compounds. In recent years, natural products from plant cell cultures, particularly alkaloids, were described to be of substantial pharmacological interest (Fowler 1992). It is expected that an increasing number of novel natural products will be isolated from different types of cell cultures, and the portion of natural products from hairy roots and non-transformed root cultures will probably increase above those from callus and suspension cultures. The future selection of species surely will consist of a broader spectrum of taxa than previously used. By inspecting the list in Table 1 and our own work in this field, we conclude that special approaches adapted to carefully selected individual cell lines (rather than random screening) are of considerable benefit in producing novel natural compounds from cell cultures. Cell cultures can perform modification reactions, rather than the formation of completely new skeletons; this capacity could be used to create variations of known structures, especially if pharmacological or pest-control properties of related compounds have already been realized. The more general problem of using cell cultures to form novel compounds is the translating of large genetic diversity into metabolic and, finally, structural diversity; our ability to do this is limited to specific cases. This could be overcome systematically using traditional methods of improving culture conditions and sophisticated methods stimulating the production of secondary compounds. The development of new genetic approaches in natural-products chemistry and the application of these approaches to cell and tissue cultures are other promising aspects to consider. The transfer and expression of biosynthetic genes between plant cells and micro-organisms could bring new vitality into this field. Combinatorial biosynthesis should no longer be restricted to micro-organisms; it should be extended to plant systems, where it will consequently enhance the metabolic diversity of plant cell and tissue cultures. 296 Physiology Acknowledgements. We thank Dr. H.-F. Moeschler and Dr. M. 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Luttge Present address: Bernd Schneider Max-Planck-Institute for Chemical Ecology Carl-Zeiss-Promenade 10 07745 Jena, Germany e-mail: [email protected] Otto Grather Firmenich SA Route des Jeunes 1 1211 Geneva 8, Switzerland Systematics Systematics Molecular Systematics: 1997-1999 Kenneth J. Sytsma and William J. Hahn 1 Introduction During the period of our last review of plant molecular systematics (in Progress in Botany, Volume 58; Sytsma and Hahn 1996), which covered 1994-1995, we indicated a number of areas that should emerge as important issues in the field of molecular systematics: 1. The analysis of DNA data 2. The search for alternative genes or DNA regions 3. Co-evolutionary studies using molecules to track the evolution of the different organisms involved 4. The interfacing of developmental genetics and phylogenetic systematics to understand morphological evolution 5. Adaptive radiations Since then, important progress has been made in all these areas - sometimes in spectacular fashion, as will be discussed later. As reviewed in that paper, the time period of the last review (the first half ofthe decade) also witnessed the increasing prevalence of rapid DNA sequencing and large-scale attempts to uncover relationships at all taxonomic levels in green plants. DNA-sequence data had especially provided insights into plant relationships and evolution not afforded by other means; these sequence data were increasingly used in parallel with traditional morphological, anatomical and cytological information. During the latter half of the decade, enough was known regarding flowering-plant radiation to enable the first explicitly molecular-phylogenetic classification of angiosperms to be published (Angiosperm Phylogeny Group 1998). In addition, the role of traditional morphological, anatomical and cytological information has been enhanced, as reviewed below, due to (1) their increased use in combined molecular and traditional character analyses, and (2) the use of independent molecular data to assess issues of homology in previously considered (and sometimes not previously considered) problematic morphological and anatomical characteristics. Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 308 Systematics 2 Progress from 1997 to 1999 How have we progressed since 1996? This review encompasses nearly 1000 articles and book chapters on the topic of plant molecular systematics during the time period 1997-1999; this is compared with approximately 350 articles and chapters during the last 2-year period. These citations are available as an Endnote 3.1 file on e-mail request ([email protected]). The top ten list (alphabetically arranged) during this period should again highlight the importance and farreaching impact of plant molecular systematics. They have been chosen for their extensive surveys (taxa and sequences), the resolution of longstanding systematic or evolutionary problems, and because they opened new areas of research. More details on many of these papers are included elsewhere in this review. 1. The Angiosperm Phylogeny Group (1998) presented a classification of flowering plants based extensively on recent molecular and morphological systematic data. This first-ever, molecule-based classification of a major group of organisms recognizes 462 flowering-plant families in 40 putatively monophyletic orders and a small number of monophyletic, informal higher groups: the monocots, commelinoids, eudicots, core eudicots, rosids (including eurosids I and II) and asterids (including euasterids I and II). 2. Using floral homeotic genes, Barrier et al. (1999) resolved the longstanding issue of the origin of the polyploid condition in the Hawaiian silversword alliance. These genes are found in duplicate copies in members of the Hawaiian silversword alliance and appear to have arisen as a result of interspecific hybridization between two North American tarweed species. 3. Bruneau (1997) presented chloroplast DNA (cpDNA) and morphological evidence that shifts from passerine to hummingbird pollination have occurred a minimum of four times in Erythrina L. (Leguminosae). Homology assessment reveals that petal morphology and size, and calyx and pollen morphologies differ with hummingbird pollination in these multiple lineages. Assessment also indicates that morphological characteristics, even those comprised of modifications associated with adaptive pollination systems, can provide useful phylogenetic information if carefully analyzed. 4. Cameron et al. (1999) outlined the first comprehensive analysis of the Orchidaceae using rbeL sequence data from 171 taxa representing nearly all tribes and subtribes of the family. The proposed classification based on these data divides the family into five primary monophyletic clades: apostasioid, cypripedioid, vanilloid, orchidoid and epidendroid orchids, in that order. The results should serve as a standard to which future morphological and molecular studies can be compared. Molecular Systematics: 1997-1999 309 5. Duff and Nickrent (1999) examined phylogenetic relationships among embryophytes (tracheophytes, mosses, liverworts and hornworts) with mitochondrial small-subunit (19S) ribosomal DNA (rDNA) sequences and identified a basal hornwort lineage, the placement of Equisetum and the monophyly of gymnosperms, thus demonstrating the affmities of Gnetales and conifers. 6. Hapeman and Inoue (1997) conclusively demonstrated the convergent nature oflabellum and pollinia features as a response to pollinator interactions during the radiation of Platanthera (Orchidaceae). 7. With rbcL evidence, Les et al. (1997) demonstrated that hydrophily, unisexuality and marine habit in angiosperms have evolved repeatedly; the multiple origins of hydrophilous, marine plants offer an extraordinary example of convergent evolution in angiosperms. 8. Mathews and Donoghue (1999), using a novel analysis of duplicate phytochrome genes to root the angiosperms, unambiguously placed the root near Amborella from New Caledonia and identified water lilies (Nymphaeales) and, subsequently, Austrobaileya (from Australia) as early branches. 9. With evidence from mitochondrial, plastid and nuclear genome sequences, Qiu et al. (1999) similarly resolved the basal relationships of the angiosperms. 10. Young et al. (1999) documented the monophyly of the parasitic Scrophulariaceae and Orobanchaceae, which represent a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to non-photosynthetic holoparasites; they also demonstrate that holoparasitism evolved independently at least five times. Have there been books, texts and other notable reviews since 1996? A number of books pertinent to specific areas or issues in molecular systematics are essential reading or additions to one's library: - Molecular Evolution and Adaptive Radiation (Givnish and Sytsma 1997c) examines plant and animal examples of adaptive radiation and related topics from both molecular and morphological perspectives. - Molecular Systematics of Plants II: DNA Sequencing (Soltis et al. 1998a) is the costly but more thorough update of the first edition. - Molecular Systematics and Plant Evolution (Hollingsworth et al. 1999) is the largely European competitor of the previous book. All three are wide-ranging in scope and discuss areas once considered outside the mainstream of systematics; all the books indicate the evolving, multi-disciplinary nature of molecular systematics as practiced today. Importantly, the first systematic textbook utilizing recent molecular results, Plant Systematics: a Phylogenetic Approach (Judd et al. 1999), is now available. Significant reviews include the contribution of plastid 310 Systematics rbcL DNA sequences to angiosperm phylogenetics (Chase and Albert 1998), the comparative utility of cpDNA restriction sites and DNA sequence data for phylogenetic studies in plants (Jansen et al. 1998), the phylogenetic utility of rDNA sequences (Kuzoff et al. 1998; Hershkovitz et al. 1999; Soltis et al. 1999c), the origin and evolution of plastids and their genomes (Palmer and Delwiche 1998), molecular evidence for Eastern Asian and Eastern North American disjunct distributions (Wen 1999), hybrid origins of plant species (Rieseberg 1997), gene flow from domesticated plants into their wild relatives (Ellstrand et al. 1999), assessment of congruence (Johnson and Soltis 1998), phylogenetic incongruence due to genome evolution (Wendel and Doyle 1998), polyploids (Soltis and Soltis 1999), the organization of angiosperm genomes (Bennetzen 1998), genome size and C-values in angiosperms (Leitch et al. 1998), ancient DNA (Wayne et al. 1999), the use of phylogenetic approaches for the analysis of breeding-system evolution (Weller and Sakai 1999), and adaptive radiations and molecular systematics (Givnish 1997). 3 Advances in Methodology a) DNA Extraction A survey of the preservation of DNA in plant specimens and the inactivation and re-activation of DNases in field specimens was presented (Adams et al. 1999). Examples of endophytic fungal DNA contamination of leaf tissues are increasing (Camacho et al. 1997; Zhang et al. 1997), and a s.8S nuclear ribosomal gene-sequence database-search strategy that can determine whether the target organism (rather than a contaminant) has been sequenced has been provided (Cullings and Vogler 1998). b) New Genes for Phylogenetics Continued reliance on nuclear internal transcribed spacers (ITSs) and cpDNA genes (rbcL, ndhF, matK) is the norm in most phylogenetic studies. Specific comparisons, however, demonstrate that cpDNA restriction-site analysis often provides significantly more variable characters than sequence analysis (Jansen et al. 1998; Plunkett and Downie 1999). Additional cpDNA spacers and introns are now used successfully, including rps16 introns in Caryophyllaceae (Oxelman et al. 1997), psbAtrnH intergenic spacers in sub-tribe Sonchinae (Kim et al. 1999b) and rp116 introns in bamboo (Kelchner and Clark 1997). Additional nuclear rDNA (nrDNA) regions have also been introduced, including external transcribed spacers (ETSs) in Asteraceae (Baldwin and Markos 1998) Molecular Systematics: 1997-1999 311 and SS in Leguminosae (Crisp et al. 1999). Putative coding and intron regions of single- or low-copy nuclear genes or gene families are increasingly used, including pgi in Clarkia (Gottlieb and Ford 1997), granulebound starch synthase (waxy) in Poaceae (Mason-Gamer et al. 1998), arginine decarboxylase (adc) in Brassicaceae (Galloway et al. 1998), alcohol dehydrogenase (adh) in Brassicaceae (Charlesworth et al. 1998), plant terpenoid synthases (Bohlmann et al. 1998), vicilin in Sterculiaceae (Whitlock and Baum 1999), glutamine synthetase (ncpGS) in Oxalidaceae (Emshwiller and Doyle 1999), putrescine N-methyltransferase (pmt) in Nicotiana (Riechers and Timko 1999), introns of the MCM1, Agamous, Deficiens and serum-response factor (MADS)-box gene pistillata (Bailey and Doyle 1999), knot-like (knox) genes in seed plants (Bharathan et al. 1999) and 2S albumin seed-storage protein genes from Brassicaceae (Boutilier et al. 1999). c) DNA Fingerprinting Contributions of polymerase-chain-reaction-based fingerprinting methods to plant systematics and evolutionary biology have been summarized (Wolfe and Liston 1998). Random, amplified polymorphic DNA analyses (RAPDs) continue to be used in both distance and cladistic analyses, although there are still questions regarding their validity in systematics studies (Harris 1999). Phylogenetic and phenetic analyses of RAPD polymorphisms in Senecio nebrodensis and S. viscosus (and other species) revealed a sister-group relationship between the species rather than a previously supported progenitor-derivative relationship (Purps and Kadereit 1998). A population-level-pooling strategy to discount lowfrequency allelic variations within taxa and to obtain a "cumulative genotype" was used in a RAPD phylogenetic analysis of Central American species of Pinus (Furman et al. 1997). Hyper-variable intersimple sequence repeats (ISSRs) were used to assess hybridization and diploid speciation in Penstemon (Scrophulariaceae; Wolfe et al. 1998). Polymorphic chloroplast simple-sequence repeats have been used for both population-level and systematics questions (Provan et al. 1999a,b). Amplified fragment-length polymorphism (AFLP) has been used in distance and cladistic analyses of Solanum sect. Petota (potatoes) and sect. Lycopersicum (tomatoes; Kardolus et al. 1998) and to assess putative hybridization in Salix (Beismann et al. 1997). d) Data Analysis Most papers continue to rely on Fitch parsimony, but other methods and assumptions are increasingly being examined. The long-awaited Paup* 312 Systematics computer program (Swofford 2000) makes these various options available. A summary of the ways maximum-likelihood analyses of DNA sequence data have been made practical by recent advances in models of DNA substitution, computer programs and computational speed, a description of the maximum-likelihood method and recent improvements in models of substitution, and the formulation and testing of biological hypotheses using the likelihood-ratio test statistic have been presented (Huelsenbeck and Crandall 1997). Spectral analysis has been introduced (Charleston and Page 1999), and its use and systematic bias in the evolution of chloroplasts have been examined (Lockhart et al. 1999). A number of important advances in the understanding of the amount of homoplasy, its role in phylogenetic analyses and (especially) the problem of completing parsimony searches with large data sets have occurred. Homoplasy has been shown to be greater in morphological data sets than in molecular data sets (Givnish and Sytsma 1997b). Simulation studies indicate that the probability of correct phylogenetic inference increases with the number of variable (or informative) characters and their consistency index. The probability decreases with the number of taxa when the consistency index has been standardized to eliminate its dependence on the number of taxa. Given that actual studies based on DNA data generate more characters with a higher level of consistency than comparable studies based on morphology, molecular studies may often provide a more precise guide to phylogenetic relationships (Givnish and Sytsma 1997a). Likewise, in a review of sequence data for six plant families, the percentage of supported nodes within a tree was positively correlated with the number of characters and was negatively correlated with the number of taxa (Bremer et al. 1999). However, in another study, accuracy improved dramatically with the addition of taxa and improved much more slowly with the addition of characters. Thus, if taxa can be added to break up long branches, it is much more preferable to add taxa than to add characters (Graybeal 1998). Angiospermwide analyses of multiple data sets indicate that Paup swaps to completion with the combined data set but not necessarily with the individual data sets. In addition, when there is increased signal (as in many combined analyses), the starting trees are generally much closer to the ultimate shortest trees than any of the individual analyses (Chase and Cox 1998; Soltis et al. 1998b). Functional constraints on amino acids in rubisco, the codon bias and the adaptive nature of some rubisco variations, and their implications for rbeL analyses, were summarized (Kellogg and Juliano 1997). Importantly, an analysis of 2538 rbeL sequences covering all major lineages of green plants indicates that, although they are rapidly evolving and highly homoplastic, third positions contain most of the phylogenetic structure in rbeL data (Kallersjo et al. 1999). Examples where ITSs of nrDNA have resisted homogenization by concerted evolution (and Molecular Systematics: 1997-1999 313 which thus present potential problems during phylogenetic analysis) are becoming more common (Campbell et al. 1997; Aguilar et al. 1999). 4 Systematic Progress in Non-Angiosperms The molecular systematics of green algae have been reviewed elsewhere (Chapman et al. 1998). Three mitochondrial group-II introns are present (with occasional losses) in mosses, hornworts and all major lineages of vascular plants but are entirely absent from liverworts, green algae and all other eukaryotes. These results indicate that liverworts are the earliest land plants, with the three introns having been acquired in a common ancestor of all other land plants (Qiu et al. 1998b). The basal position of liverworts is also suggested by rbeL sequences alone (Lewis et al. 1997). However, a combined analysis of plastid-coded genes and the 18S rRNA gene places hornworts basally among land plants, while mosses and liverworts each form a clade and are sister to each other (Nishiyama and Kato 1999). Likewise, a group-I intron sequence conserved in the mitochondrial DNA (mtDNA) nadS gene of all investigated mosses and liverworts (but none of the hornworts) supports a sister-group relationship between mosses and liverworts (Beckert et al. 1999). Mitochondrial small-subunit (19S) rONA sequences parsimoniously identify either liverworts or hornworts as the basal land-plant clade, although hornworts are basal with maximum likelihood (Duff and Nickrent 1999). This molecular analysis supports a sister relationship between Equisetum and leptosporangiate ferns and suggests a monophyletic lineage for gymnosperms, similar to the lineage of angiosperms. Analysis of rbeL sequences strongly supports monophyly of the Selaginellaceae and suggests that the morphologically distinctive subgenus Selaginella is a sister group to all other species in the family; leaf isophyly and reduction represent independent reversals in response to seasonal drought (Korall et al. 1999). 19S mtDNA and rONA sequences place Equisetum and leptosporangiate ferns as sister clades (Duff and Nickrent 1999). Phylogenetic studies of extant pteridophytes indicate that leptosporangiate ferns form a monophyletic group, as do Psilotaceae (Psilotum and Tmesipteris) and Ophioglossaceae (Ophioglossum and Botryehium). Osmundaceae are basal, and the water ferns are monophyletic (Wolf et al. 1998). Surprisingly, the phylogenetic positions of the two problematic and monotypic fern families Hymenophyllopsidaceae and Lophosoriaceae have been solved. RbcL supports a sister relationship between Hymenophyllopsis and Cyathea and between Lophosoria and Dieksonia; thus, both families are part of a well-supported clade of tree ferns (Wolf et al. 1999). Nuclear 18S rRNA sequences support a monophyletic clade of gymnosperms with three sub-clades (Cycadales/Ginkgoales, Gnetales and 314 Systematics Coniferales). The sequences also place the cycad/ginkgo clade as the earliest gymnosperm lineage and provide strong support for the sistergroup relationship between Gnetales and Coniferales (Chaw et al. 1997). Further molecular evidence indicates that the shared morphological characteristics of Gnetales and angiosperms are convergent rather than homologous (Duff and Nickrent 1999; Hansen et al. 1999). Analysis of rbeL sequences in Southern-Hemisphere Araucariaceae revealed that Wollemia was derived prior to Agathis and Arauearia. Morphological characteristics, such as the number of cotyledons, the position of the male cone and cuticular micro-morphologies were phylogenetically informative (Setoguchi et al. 1998). An ITS study of Pinus, representing all recognized subsections of the genus, provided weak support for the monophyly of subgenus Pinus and of subgenus Strobus, moderate support for the monophyly of a narrowly circumscribed subsection of Pinus (subsection Sylvestres) and strong support for a clade of North and Central American hard pines (Liston et al. 1999). 5 Systematic Progress in Angiosperms The most dramatic findings during the past 3 years have been the resolution of basal angiosperms and relationships of the major lineages based on rbeL, atpB and 18S rDNA sequences (Soltis et al. 1997, 1999b; Qiu et al. 1999). All studies are consistent in stating that Amborella, Nymphaeales, Austrobaileya and Illiciales are basal in angiosperms. Nandi et al. (1998) presented a combined analysis of 162 extant angiosperm taxa for which rbeL sequence data and/or non-molecular information are available. Capparales s.l. and the nitrogen-fixing clade, two novel molecular clades, are only found in the rbeL and the combined trees, not in the morphological trees. A phylogenetic analysis (primarily of eudicots) based on sequences of three genes (atpB and rbeL, and nuclear ribosomal18S DNA) provided high bootstrap values (>90%) for a number of higher taxonomic groups (Hoot et al. 1999), including eudicots, ranunculids, "core" eudicots (including caryophyllids, asterids and rosids), caryophyllids and asterids. During the past decade, it has become increasingly clear that the angiosperm subclass Hamamelidae is polyphyletic. A broad rbeL survey of dicots, including representatives of 25 families that have traditionally been placed in the Hamamelidae, formally places these disparate families and at last eliminates the subclass (Qiu et al. 1998a). Molecular Systematics: 1997-1999 315 a) Basal Angiosperms (Excluding Monocots) A phylogeny (using non-molecular, rbeL, matK and 18S rDNA data), the resulting classification and an analysis of the floral evolution of water lilies (Nymphaeaceae; Nymphaeales) were presented (Les et al. 1999). When floral features were evaluated using this well-corroborated phylogeny, the pleiomerous condition of water-lily flowers exhibited several instances of secondary derivations. An ITS, matK and a morphological study of Nuphar consistently resolved two lineages: one comprised of New W orId taxa and the other forming primarily an Old WorId lineage (Padgett et al. 1999). Most notable are the strong support for a monophyletic lineage of dwarf taxa and the lack of support for the common taxonomic practice of uniting all North American and Eurasian taxa under one species. Sequencing of various cpDNA spacers and introns indicates that the genus Magnolia is polyphyletic and contains species of Miehelia (Azuma et al. 1999). The core Laurales plus Calycanthaceae and Idiospermaceae are strongly supported (Renner 1999). In Laurales, the deepest split is between Calycanthaceae (including Idiospermaceae) and the remaining six families. Additionally, Siparuna and its sister taxon, the monotypic West African Glossoealyx, are not closely related to the remaining Monimiaceae, supporting the view that the Monimiaceae are polyphyletic (Renner et al. 1997). b) Basal Angiosperms: Monocots Despite what might appear to be difficult evolutionary transitions, hydrophiles and unisexuality have evolved repeatedly in Alismatidae, based on rbeL; marine angiosperms (known only from Alismatidae) evolved in three separate lineages (Les et al. 1997). ITS data for Cypripedioideae (Orchidaceae) demonstrate that each genus is monophyletic with the plicate-leafed genera, Cypripedium and Sebenipedium, as successive sister groups to the rest of the subfamily (Cox et al. 1997). An rbeL and trnL- F assessment of Amaryllidaceae and related asparagalean families supports the monophyly of Amaryllidaceae. Agapanthaceae is its sister family, and Alliaceae is sister to the Amaryllidaceael Agapanthaceae clade; the origin of the family appears to be western Gondwana (Africa; Meerow et al. 1999). Resolving the relationships within the Palmae continues to be made difficult by a lack of variation, this time in the trnL-trnF region (Baker et al. 1999). The traditional Commelinales has been shown to be highly polyphyletic and exhibits morphological convergence (based on rbeL), with Eriocaulaceae and Xyridaceae sisters to Poaceae and its relatives, Rapateaceae a sister to Bromeliaceae and Mayacaceae, and Commelinaceae a sister to Philydrales and its allies. In addition, Thurnia is a sister 316 Systematics to Prionium at the base of the Cyperaceae-Juncaceae clade (Givnish et al. 1999). Analysis of trnL-F corroborates these findings in placing Haemodoraceae with Philydraceae, Pontederiaceae and Commelinaceae (Hopper et al. 1999). A reconstruction of the phylogenetic history of the Pontederiaceae has been used to examine breeding-system evolution (Graham et al. 1998). The relationships of subfamilies within Bromeliaceae have been clarified with ndhF analysis (Terry et al. 1997); Broeehinia is the sister group of the remainder of the Bromeliaceae, and Puya (Pitcairnioideae) is the sister group of the Bromelioideae. An extensive survey of Cyperaceae using rbeL supports the idea that the family is monophyletic and is derived from a grade of Juncaceae. It also suggests that Oxyehloe (Juncaceae) is a sister taxon to Cyperaceae and that a broader circumscription of Cyperus is necessary. Finally, it reveals uncertainty in the generic and tribal statuses of the Scirpeae (Muasya et al. 1998). The Poaceae is fast becoming one of the best phylogenetically known large families. ITS data support the six commonly recognized subfamilies (Bambusoideae, Pooideae, Arundinoideae, Centothecoideae, Chloridoideae and Panicoideae), place the herbaceous bamboo allies Streptoehaeta and Pharus as basal taxa and, thus, indicate an origin outside South America (Hsiao et al. 1999). cpDNA restrictionsite mapping is consistent with an origin and early diversification of grasses as forest understory herbs, followed by one or more radiations into open habitats. This was followed by multiple origins of C-4 photosynthesis and specialization for wind pollination (Soreng and Davis 1998). c) Basal Eudicots Strap-shaped petals, apetaly and wind pollination have evolved independently three times in the Hamamelidaceae, based on ITS data (Li et al. 1999). Comparative cpDNA restriction-site mapping gives poor resolution and/or support for the relationships among the four chromosomal lineages of the Berberidaceae. This indicates that they may have radiated from an ancestral stock in a relatively short evolutionary time (Kim and Jansen 1998). Considerable progress has been made in understanding relationships within Papaverales. Hoot et al. (1997) provided an overview of the order based on molecular and morphological data, discussed the evolution of floral morphology and geographical distribution, and indicated that Pteridophyllum (Pteridophyllaceae) is a sister group to Fumariaceae and Papaveraceae. Importantly, whereas morphological characteristics provide some support for the molecular phylogeny found for the prickly poppies, the alkaloid characteristics appear to be quite homoplasious (Schwarzbach and Kadereit 1999). A restriction-fragmentlength polymorphism analysis of trnK of Papaveraceae suggests that Molecular Systematics: 1997-1999 317 Papaver arose polyphyletically from within a paraphyletic Meconopsis in response to Tertiary climatic aridification (Kadereit et al. 1997). An ITS phylogeny of the subfamily Chelidonioideae (Papaveraceae) shows that morphological change is concentrated in the BoeeonialMacleaya clade and is probably related to the evolution of wind pollination from insect pollination in the two genera after a habitat shift (Blattner and Kadereit 1999). Most subfamilies in the Proteaceae are essentially monophyletic, based on atpB and the non-coding spacer region between atpB and rbeL, but most tribes and subtribes are not (Hoot and Douglas 1998). Other important results include the fact that Bellendena is weakly supported as the sister group to the rest of the Proteaceae. In addition, there is limited congruence with morphological characters, and the African and South American genera are dispersed among various clades with taxa from Australia and Asia, suggesting a former Gondwanan distribution for Proteaceae. A trnL/trnF and ITS study strongly supports the origin of Dryandra within a paraphyletic Banksia; the distribution of eastern Australian taxa at derived positions on the molecular cladograms suggests considerable cladogenesis in Banksia prior to the formation of the Nullarbor Plain during the Tertiary (Mast 1998). cpDNA of the Crassulaceae strongly supports a basal division of the family, separating subfamily Crassuloideae from all other taxa, but four of the six traditionally recognized subfamilies are polyphyletic (Brochmann et al. 1998). Sedum is markedly polyphyletic and comprises taxa of most of the other genera of the family. A matK analysis within Saxifraga indicates that the major trend in gynoecial evolution has been from a superior ovary toward greater inferiority, but with several apparent reversals toward greater superiority (Mort and Soltis 1999). ITS and trnL-F analysis in Korthalsella, a genus of reduced, monoecious, Old WorId mistletoes, confirms the hypothesis that branch shape and cladotaxy are unreliable indicators of relationships (Molvray et al. 1999). d) Caryophyllids Basal relationships within the Caryophyllales remain obscure, based on cpDNA ORF2280 homolog sequences (Downie et al. 1997). However, a polyphyletic Amaranthaceae is nested within a paraphyletic Chenopodiaceae, Nyctaginaceae is allied with Phytolaccaceae, and Caryophyllaceae is sister to Chenopodiaceae and Amaranthaceae. Cactaceae is nested among other aridity-adapted lineages of the Portulacaceae (based on ITS), with an origin dated in the mid-Tertiary, approximately 30 million years ago (Hershkovitz and Zimmer 1997). The Plumbaginaceae is a strongly supported monophyletic group sister to Polygonaceae (based 318 Systematics on rbcL) and is in the same clade as Simmondsiaceae, Nepenthaceae, Droseraceae and Caryophyllales (Lledo et al. 1998). e) Rosids Phylogenetic analysis of rbcL in Leguminosae is consistent with previous hypotheses in suggesting that the family as a whole is monophyletic but that only two of its three subfamilies are natural (Doyle et al. 1997). The basal split in the family appears to involve the tribes Cercideae and/or some members ofCassieae (both in Caesalpinioideae). The remainder of the family is comprised of two clades: (1) Mimosoideae and the caesalpinioid tribe Caeasalpinieae and other Cassieae, and (2) Papilionoideae. A phylogeny of the Betulaceae using rbcL, ITS and morphological data indicates that the Betulaceae is monophyletic and Casuarinaceae is its sister group and confirms the status of the two subfamilies Betuloideae and Coryloideae (Chen et al. 1999). cpDNA significantly demonstrates that Ulmaceae s.l. is not monophyletic. It also demonstrates that distinct families (Ulmaceae and Celtidaceae) are warranted, that the Ulmaceae is the sister group to Celtidaceae and all other families in the order, and that Cannabaceae is nested within Celtidaceae (Wiegrefe et al. 1998). The enigmatic aquatic family Podostemaceae is clearly placed within the order Malpighiales, based on 18S rDNA and rbcL data (Soltis et al. 1999a). An ITS analysis of Viola supports an Andean origin for the genus, intermingles mostly stemmed, yellow-flowered, six-chromosome species with stemless, white/blue-flowered, 12-chromosome species and, surprisingly, places the largely woody Hawaiian sect. Nosphinium within the amphi-Beringian V. langsdorffii complex (Ballard et al. 1998). Two major clades in the Myrtaceae have been defined, based on rbcL: (1) the Myrtaceae lineage sister to a Melastomataceae lineage and (2) Onagraceae, Lythraceae s.l. and Combretaceae. Phenotypic characteristics suggest that the ancestor of the first clade was characterized by the acquisition of fibrous seed exotegmen, while the ancestor of the second clade had flowers with stamens inserted directly on the rim ofthe hypanthium (Conti et al. 1997). Parallel evolution of glucosinolate biosynthesis in Capparales and Drypetes (Euphorbiaceae) is inferred from congruent nrDNA and rbcL phylogenies (Rodman et al. 1998). Analysis of nuclear arginine decarboxylase (adc) in Brassicaceae strongly supports Aethionema as the basal genus, because it is the only genus without two adc genes (Galloway et al. 1998). The resulting phylogeny provides robust phylogenetic data regarding relationships within the complex mustard family and provides independent support for proposed tribal realignments based on cpDNA data. Phylogenetic analyses of rbcL robustly group Setchellanthaceae with other mustard-oil-producing plants (Karol et al. 1999). Sequence data for rbcL and atpB in Sapindales show that Molecular Systematics: 1997-1999 319 Rutaceae is paraphyletic, with Simaroubaceae and Meliaceae closest to Rutaceae (Chase et al. 1999). Extensive studies of the Malvales have now clarified the circumscription of the Malvales and its relationship to other rosids. Sequence analyses of atpB and rbeL support an order Malvales expanded beyond Sterculiaceae, Tiliaceae, Bombacaceae and Malvaceae to include the bixalean clade (Bixaceae, Diegodendraceae and Cochlospermaceae), the cis tale an clade (Cistaceae, Dipterocarpaceae and Sarcolaenaceae) and Thymelaeaceae (including Gonystyloideae and Aquilarioideae; Alverson et al. 1998). The monophyly of only one traditional family, the Malvaceae, is supported by ndhF. The other three families are not monophyletic; thus, "monothecate" anthers may have been derived at least twice, independently: in the core Bombacaceae and in the traditional Malvaceae (Alverson et al. 1999). The Dipterocarpaceae are comprised of two major clades that correspond (with one exception) to the occurrence of the base chromosome numbers 7 and 11 (Kajita et al. 1998; Dayanandan et al. 1999). f) Asterids 18S rDNA and rbeL place Polemoniaceae near sympetalous families with two staminal whorls, including Fouquieriaceae and Diapensiaceae and related "ericalean" families, rather than near sympetalous families with a single staminal whorl, such as Hydrophyllaceae and Convolvulaceae (Johnson et al. 1999). Based on matK, the Epacridaceae is sister to a clade within Ericaceae; Arbutus and pyrola branch early in Ericaceae, before the rhododendroid group (Kron 1997). The relationships indicated by matK suggest that sympetalous flowers are likely plesiomorphic within rhododendroids (Kron et al. 1999). According to matK-sequence data, Asclepiadaceae forms a monophyletic group derived from Apocynaceae, and each of the subfamilies of Asclepiadaceae is monophyletic and is based on reliable palynological characters (Civeyrel et al. 1998). Sequence variation in the rps16 intron (cpDNA) of the subfamily Rubioideae (Rubiaceae) supports monophyly of three tribes (Rubioideae, Cinchonoideae and Ixoroideae), while there is no support for the inclusion of Antirheoideae (Andersson and Rova 1999). A proposed reorganization of the genus Psyehotria and the tribe Psychotrieae (Rubiaceae) based on ITS and rbeL sequence data demonstrates that Psyehotria is broadly paraphyletic. Two groups formerly assigned to Psyehotria (sect. Notopleura and subgenus Heteropsyehotria plus Palieourea) are more closely related to other genera in the Psychotrieae than they are to other species of Psyehotria, and the Malesian epiphytic myrmecophytes of the sub-tribe Hydnophytinae are imbedded in Psyehotria (Nepokroeff et al. 1999). Hydrophyllaceae, excluding Hy- 320 Systematics drolea and Codon, is nested within a paraphyletic Boraginaceae s.l. using ndhF sequences (Ferguson 1998). Fortunately, the assessment of species boundaries with molecular data and the resulting evidence of rampant synonymy in Solanum sect. Petota (Castillo and Spooner 1997; Miller and Spooner 1999). The disintegration of Lamiales, Scrophulariales and some constituent families is finally beginning to occur. Sequences of rbcL and ndhF have resolved phylogenetic relationships in Labiatae s.l., with four clades supported: subfamilies Nepetoideae, Lamioideae, Pogostemonoideae and Scutellarioideae (Wagstaff et al. 1998). Scrophulariaceae is comprised of three major lineages, two of which are now called Scrophulariaceae and Plataginaceae; the hemisparasitic third lineage has been combined with other holoparasitic Orobanchaceae (Depamphilis et al. 1997; Reeves and Olmstead 1998; Young et al. 1999). An analysis based on rbcL and ndhF indicates that Bignoniaceae is more derived within the order Lamiales s.l. than previously believed; Paulownia and Schlegelia, previously placed in either Bignoniaceae or Scrophulariaceae, do not belong in Bignoniaceae (Spangler and Olmstead 1999). Paulownia is the sister to Gesneriaceae, with the Klugieae identified as the sister to the remainder of the family (Smith et al. 1997). Remarkably, Saintpaulia has been shown to have evolved from the Streptocarpus subgenus Streptocarpella; the striking differences in flower and vegetative characteristics is probably due to ecological adaptation, leading to the relatively rapid radiation of Saintpaulina (Moller and Cronk 1997). Acanthanceae is in need of taxonomic alignment; trnL-trnF introns and spacers do not support its monophyly, although there is strong morphological evidence for this relationship (McDade and Moody 1999). The results of rbcL analysis provide a basis for the exclusion of Adoxaceae s.l. (including Sambucus and Viburnum) from the Dipsacales (Backlund and Bremer 1997). ITS does not support the placement of Adoxa (Adoxaceae) within Sambucus, as indicated by a morphological analysis; this is a good example of morphological convergence (Eriksson and Donoghue 1997). Molecular data indicate that the evolutionary history of the Araliaceae and Apiaceae is more complex than the simple derivation of Apiaceae from within Araliaceae (Plunkett et al. 1997). Classification systems of Apioideae based on phytochemical and most morphological features, particularly at the tribal and sub-tribal levels, are unsatisfactory, considering the emerging DNA data (Downie et al. 1998; Katz-Downie et al. 1999). Based on cpDNA sequences, the three Australasian families Alseuosmiaceae, Argophyllaceae and Phellinaceae are each monophyletic, form a monophyletic group and belong within Asterales (Karehed et al. 1999). The saga of resolving tribal relationships within Asteraceae continues with trnL/trnF data, information congruent with prior molecular work (Bayer and Starr 1998). ITS data indicate that the Bidens-Coreopsis complex originated in Mexico and that Bidens has Molecular Systematics: 1997-1999 321 been derived twice within Coreopsis (Kim et al. 1999c). An ITS study of the Astereae, the largest tribe of Asteraceae in North America, surprisingly (and in conflict with morphology) indicates that all North American Astereae are members of a strongly supported clade and that a diverse group of predominately woody taxa from Africa, Australia and South America are basal Astereae (Noyes and Rieseberg 1999). 6 Hybridization and Introgression The study of hybrids, hybrid speciation and subsequent introgression is increasingly using molecular/genomic information, especially for model systems. Rieseberg (1998) summarizes the use of genetic mapping as a tool for studying hybrid speciation. The use of AFLP markers to study introgression between the cultivated sunflower and the largely sympatric Helianthus petiolaris indicates that the H. petiolaris genome may be differentially permeable to introgression. Different markers display significantly variable rates of introgression (Rieseberg et al. 1999). Analysis of cytoplasmic and nuclear markers in the Louisiana Iris hybrids indicates that the traditional view that interactions between divergent genomes are always deleterious is an oversimplification. Crosses between divergent lineages can lead to the formation of both fit and unfit hybrid genotypes (Burke et al. 1998). ISSR fingerprinting in Penstemon does not support the hybrid origin of P. spectabilis but does support the diploid-hybrid speciation origin of P. clevelandii (Wolfe et al. 1998). Variation at the single-copy nuclear-locus histone H3-D (versus cpDNA) in Glycine suggests that cpDNA haplotype polymorphisms transgress species boundaries; the pattern suggests hybridization rather than lineage sorting (Doyle et al. 1999). Not unexpectedly, cpDNA results provide strong evidence that hybridization and reticulate evolution are rampant in Eucalyptus (Jackson et al. 1999). Furthermore, cpDNA transfer across species boundaries in Eucalyptus may provide an important source of information regarding past plant distributions in Australia (McKinnon et al. 1999). Also unsurprising is the fact that the nature of the extensive introgression of cytoplasmic genomes across oak species is related to their ecology, the compatibility of interspecific crosses and the presence of related species in a population (Dumolin-Lapegue et al. 1999). Introgression in Adansonia is postulated, based on both nuclear and cpDNA analyses (Baum et al. 1998). 322 Systematics 7 Polyploid Origins Phylogenetic analysis indicates that autopolyploidy has arisen at least twice in Heuchera grossulariifolia and possibly up to as many as seven times (Segraves et al. 1999). Remarkably, two tetraploid species of Tragopogon arose during the past 50 years in eastern Washington and northern Idaho, and each is spreading, not from a population of single origin, but through repeated, independent polyploidization events that recreate the polyploid taxa (Cook et al. 1998). In contrast, the B genome of bread wheat (Triticum aestivum) is monophyletic in origin, not polyphyletic (resulting from hybridization and introgression among different polyploid species sharing a single genome; Blake et al. 1999). The nature of genome evolution following polyploidization has received increased attention. Polyploidization in Gossypium is associated with enhanced recombination, as genetic lengths of allotetraploid genomes are more than 50% greater than those of their diploid counterparts (Brubaker et al. 1999). The formation of allopolyploid wheat was accompanied by rapid, non-random changes in coding (and low-copy, non-coding) DNA sequences (Liu et al. 1998). In contrast, there was no evidence of interaction among duplicated genes in allopolyploid cotton. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation, and most duplicated genes evolved independently of each other and at the same rate as those of their diploid progenitors (Cronn et al. 1999). The first known example of an active locus (cytosolic isozyme of phosphoglucose isomerase) was reported in a tetraploid plant species (tetraploid Clarkia gracilis) that is no longer expressed in its diploid relatives (Ford and Gottlieb 1999). A method to separate homologs from each other and from more divergent crosshybridizing sequences (paralogs) in tetraploid Gossypium was reported (Cronn and Wendel 1998). 8 Biogeography and Phylogeography Molecular evidence for and against vicariance explanations for certain eastern Asian/eastern North American disjunctions has accumulated (Wen 1999). Sequence divergence of the matK gene among Liquidambar species places the divergence time of the disjunct species in the genus at 45-90 million years ago, in agreement with the fossil record (Li et al. 1997). A molecular phylogenetic perspective of Aesculus supports an Arcto-Tertiary distribution and subsequent vicariance (Xiang et al. 1998a). Relationships among slipper orchids support the previous biogeographic hypothesis of a widespread Northern-Hemisphere distribution, followed by range fragmentation due to Miocene cooling (Cox et al. 1997). Phytogeographic patterns of Actaea suggest a Tertiary origin and Molecular Systematics: 1997-1999 323 species surviving in refugia during the glacial periods of the Pleistocene (Compton et al. 1998). Gleditsia appears to have only one Asian/North American disjunction, no intercontinental species pairs and low sequence divergence between G. amorphoides and its closest Asian relatives, thus implicating long-distance dispersal, not vicariance (Schnabel and Wendel 1998). Likewise, a phylogeographical analysis of ITS indicates that Fraxinus likely originated in North America, with two subsequent events of intercontinental migration from North America to Asia (Jeandroz et al. 1997). The disjunction of Pogonia is best explained by speciation following a northward long-distance dispersal and subsequent northwestward migration via Bering land bridges during the Tertiary (Cameron and Chase 1999). A more common explanation is probably that multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distributions of Acer (Hasebe et al. 1998). A common pattern seen is that the eastern Asian species are sister to all North American species (Aralia sect. Aralia, Calycanthus and Adiantum pedatum; Xiang et al. 1998b). Apparently, all major oak lineages evolved locally at middle latitudes within the general distribution of their fossil ancestors, and the widespread white oaks of the Northern Hemisphere have a New World origin (Manos et al. 1999). Elsewhere in the Southern Hemisphere, a consensus of atpB-rbcL intergenic-spacer (Setoguchi et al. 1997) and ITS/rbcL (Manos 1997) data provide a complicated biogeographical story for the diversification of Nothofagus. Vicariance adequately explains two of the three main clade disjunctions, whereas dispersal best explains the third. Biogeographic patterns among the three principal clades of the largely Asian Caryota (Palmae) are congruent to Wallace's 1910 line or Huxley's line; species that do not honor the line are always derived elements within sub-clades (Hahn and Sytsma 1999). A maximum-likelihood analysis of branching times in Adansonia shows that the dispersal between Africa and Australia occurred well after the fragmentation of Gondwana and, therefore, involved over-water dispersal (Baum et al. 1998). Similarly, the close relationship between the highly disjunct South African and Australian species of Pelargonium is interpreted as having been caused by longrange dispersal to Australia, probably as recently as the late Pliocene (Bakker et al. 1998). cpDNA evidence indicates that the evolution of Microseris in Australia and New Zealand occurred after long-distance dispersal from western North America (Vijverberg et al. 1999). The biogeographical origins of oceanic island species have been actively examined, and surprises have been revealed. Hesperomannia, endemic to Hawaii, is related not to the tribe Mutisieae (Asteraceae), as previously thought, but to Vernonieae; it is a sister to African species of Vernonia. An estimated 17- to 26-million-year divergence time suggests that the progenitor of Hesperomannia arrived at one of the low islands of 324 Systematics the Hawaiian-Emperor chain between the late Oligocene and middle Miocene (Kim et al. 1998). Likewise, based on ndhF data, the two Hawaiian native species of Rubus are not closely related, as previously presumed, and two separate colonizations (one from western North American and the other from Pacific Rim islands) are now required (Howarth et al. 1997). ITS sequences indicate that Hawaiian Sanicula constitute a monophyletic group that is descended from a paraphyletic assemblage of mostly Californian species (Vargas et al. 1998). In another wellstudied island system, ITS sequences suggest recent colonization of the Macaronesian Islands from northern Africa for the Asteriscus alliance (Asteraceae, Inuleae; Francisco-Ortega et al. 1999) and a Mediterranean origin for the Macaronesian endemic genus Argyranthemum (Asteraceae; Francisco-Ortega et al. 1997a,b). In a montane "island" system, Knox and Palmer (1998) indicated that the giant lobelias arrived in ancient upland Tanzania (possibly the Uluguru Mountains) as colonists from the Asia/Pacific region with branched inflorescences. Phylogeographical studies at the species level also abound and are yielding fascinating results (Schaal et al. 1998). Molecular data support the hypothesis that the weedy and cosmopolitan Senecio vulgaris var. vulgaris is an evolutionary derivative of S. vulgaris ssp. denticulatus from the coasts of western Europe and montane altitudes of southern Spain and Sicily (Comes et al. 1997). Based on a phylogeographic study using the single-copy nuclear gene glyceraldehyde 3-phosphate dehydrogenase (G3pdh), Olsen and Schaal (1999) demonstrated that cassava (Manihot esculenta ssp. esculenta) is derived from wild populations along the southern border of the Amazon basin and is not derived from several progenitor species, as previously proposed. Of some importance, the post-glacial history of plants in the Northern Hemisphere is being revealed with molecular tools (Taberlet et al. 1998; Ferris et al. 1999). For example, Scots pine in western Europe is being studied via mtDNA variation (Sinclair et al. 1999), and Dryas is being studied via cpDNA (Tremblay and Schoen 1999). 9 Interfacing Ecology and Systematics The argument concerning the use of morphological characteristics in combined analyses where the character evolution of these very characters will be examined continues (Givnish 1997; Givnish et al. 1997; Luckow and Bruneau 1997). Adaptive radiations (Givnish 1997) have been extensively examined in Hawaii and Macaronesia; silverswords (Baldwin 1997; Baldwin et al. 1998), Sonchus (Kim et al. 1999a) and Argyranthemum (Francisco-Ortega et al. 1997a,b) have been studied. At least in island systems, it appears that extensive radiation and convergence characterizes island lineages, notably with respect to woodiness Molecular Systematics: 1997-1999 325 (Kim et al. 1999a; Panero et al. 1999). Extensive radiation with respect to carnivory (Givnish et al. 1997), breeding systems (Barrett and Graham 1997), parasitic plants (Nickrent et al. 1998) and xerophytes (Bakker et al. 1999) has been documented. The interface of ecology and molecular systematics is also dramatically seen in studies of floral evolution and pollination. Molecular phylogeny suggests the plausible hypothesis that hawk-moth pollination was ancestral in Adansonia and that there were two parallel switches to pollination by mammals (Baum et al. 1998). cpDNA analysis in Lopezia (Onagraceae) indicates that "snapping" stamens, a means of mechanically depositing pollen on visiting pollinators, evolved only once in the genus and that hummingbird pollination, which is plesiomorphic in the genus, may have been secondarily regained following its loss to fly pollination (Okane and Schaal 1998). Evolutionary trends of floral-scent chemistries in Magnolia were examined using molecular-phylogenetic trees (Azuma et al. 1999). Three alternative scenarios explaining how yucca moths and yuccas co-evolved were discussed, based on molecularphylogenetic histories of the two groups and a molecular-clock assumption (Pellmyr and Leebens-Mack 1999). Various traits pertaining to floral morphology in Orchis and related genera are interpreted as a result of ecological convergence related to pollinator-mediated selection (Aceto et al. 1999). A molecular analysis of Stylidiaceae indicates that the simple flowers of Oreostylidium may have evolved by reduction and paedomorphosis of the zygomorphic and sensitive flowers of a Stylidium-like ancestor, a change caused by adaptation to a new environment lacking a suitable pollinator (Laurent et al. 1998). A growing number of molecular-phylogenetic studies report that fruit characteristics are often the most homoplastic of the morphological features examined and are under strong ecological selection to change. Fruit morphology evolved in parallel, from simple to complex structures, in several lineages of Brachycome (Asteraceae; Denda et al. 1999). Reliance on fruit form or embryo characteristics has resulted in contradictory taxonomic concepts in the Brassicaceae (Mummenhoff et al. 1997; Koch et al. 1999). Correlation of molecular phylogenies with biochemical data indicates that chemotaxonomic information is more reliable than fruit type in Rutaceae (Chase et al. 1999). The morphological and anatomical characters of the fruit are highly homoplastic in Apioideae, thus explaining why the previous tribal and sub-tribal designations and relationships were unsatisfactory (Katz-Downie et al. 1999). 10 Interfacing Development and Systematics An exciting new area of molecular systematics that combines fields as diverse as comparative embryology, molecular phylogenetics and 326 Systematics genome analysis has emerged. The new discipline of evolutionary developmental biology attempts to explain how developmental processes and mechanisms become modified during evolution and how evolution has produced the vast diversity of living organisms, past and present (Holland 1999). There has been significant progress made in understanding the evolution of floral organization; these advances are reinforcing the ideas that phenotypic evolution can proceed via changes of large effect at a few loci and that promoter evolution can be an important and frequent mechanism (Baum 1998). Genes of specific interest that have been examined include Leafy (Frohlich and Meyerowitz 1997) and MADS-box floral-meristem genes (Frohlich 1999; Lawton-Rauh et al. 1999). The molecular developmental genetics and ontogenetic systematics of the angiosperm petal (Albert et al. 1998) and inferior ovary (Gustafsson and Albert 1999) have been reviewed. Developmental information, in combination with phylogenetic information, indicates a paedomorphic origin for the floral morphology of Pyrola minor (Ericaceae; Freudenstein 1999). 11 Future Prospects and Problems In our last review of molecular systematics (Progress in Botany, Volume 58; Sytsma and Hahn 1996), which covered the period 1994-1995, we outlined three major areas where the prospects for molecular-systematic involvement would be high: (1) floral development and evolution, (2) adaptive radiation and (3) species definitions, the use of paraphyletic taxa, phylogenetic history within species and coalescence. As reviewed above, all have emerged as viable areas in molecular systematics, some in more detail than others. A number of new areas (and associated problems) will be important in the future. Analysis of the geographic distribution of cpDNA and newer fingerprint markers has enabled current patterns of population differentiation to be related to post-glacial migration routes from different forest refugia (Newton et al. 1999). Such results highlight the importance of refugial areas for the conservation of intraspecific variation in species. The explosive growth of phylogeography stresses the importance of this area in evolutionary biology; predictions regarding future challenges for the field center on several facets of genealogical concordance (Avise 1998). Genome-based in situ hybridization methods are being used on cultivated plants or model organisms and will soon have an impact on systematic and evolutionary studies (Stace and Bailey 1999). Genomic affinities of species in Zea were examined with genomic in situ hybridization (GISH) and provide a useful addition to the taxonomic classification of the genus (Poggio et al. 1999). GISH in Arachis (Fabaceae) identifies the diploid wild progenitors of cultivated and related wild peanut Molecular Systematics: 1997-1999 327 species (Raina and Mukai 1999). The utility of GISH in identifying genome contributions of polyploids should be a much-anticipated prospect. Fluorescent in situ hybridization (FISH) permits long stretches of DNA (rONA, centromeric or telomeric regions) to be visualized on individual chromosomes (Gill and Friebe 1998). Using a refined fiber-FISH technique, DNA clusters as long as 1.71 Mb - more than 1% of the Arabidopsis genome - can be visualized (Jackson et al. 1998). FISH has the great ability to track specific chromosome parts among species and, in concert with molecular phylogenetic trees, it should be a powerful tool to examine chromosomal evolution and speciation in a wide group of plants. 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Mol Phylogenet EvollO:178-190 Young NO, Steiner KE, De Pamphilis CW (1999) The evolution of parasitism in ScrophulariaceaelOrobanchaceae: plastid gene sequences refute an evolutionary transition series. Ann Mo Bot Garden 86:876-893 Zhang WP, Wendel JF, Clark LG (1997) Bamboozled again! Inadvertent isolation of fungal rONA sequences from bamboos (Poaceae: Bambusoideae). Mol Phylogenet Evol 8:205-217 Kenneth J. Sytsma Botany Department University of Wisconsin Madison, WI 53706, USA e-mail: [email protected] Communicated by J.W. Kadereit William J. Hahn Center for Environmental Research and Conservation Columbia University New York, NY 10027, USA Systematics Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny Michael Melkonian 1 Introduction The enormous amount of data generated by the various genomesequencing projects has led to a plethora of studies addressing the evolutionary history oflife on Earth. Genomics meets phylogeny! But where are the algae in this scenario? With the exception of organelle and other "bonsai" genomes that sequencing centers sometimes like to sequence "in between", enthusiasm for sequencing whole algal genomes is waning. University meets industry! Algae are not causes of human disease, nor are they thought of as "models" for the study of human disease or its prevention. And who wants to eat transgenic algae? The "green yeast" turned pale. This is why sophisticated molecular-phylogenetic analyses oflarge data sets from five to eight taxa addressing "evolution from yeast to man" abound, but phylogenetic relationships between the major algal lineages, which span a greater evolutionary distance than that between fungi and animals, remain poorly understood. Molecular phylogeneticist meets taxonomist!? When it comes to phylogenetic analyses adequate taxon sampling is of the essence. This requires detailed knowledge regarding the morphology, biodiversity, systematics, occurrence and culturing of algae. However, molecular phylogeneticists and taxonomists rarely meet and, when they do, they don't speak the same language. Is the "Renaissance in algal phylogenetics" only wishful thinking? With so many meetings that fail to take place in algal research is there progress that needs to be reported regarding the phylogeny of the major algal lineages? Fortunately, yes. Some of it has been well documented in the recent reviews of H.R. Preisig in this Series (Prog Bot 60:369-412; Prog Bot 61:285-299). The present review focuses on the impact genome analyses have on eukaryote and algal phylogeny. Methods of molecularphylogenetic inference and progress regarding the phylogeny of the major algal lineages will be reviewed in a forthcoming report. For the present review, approximately 400 publications concerning the period Progress in Botany, Vol. 62 © Springer-Verlag Berlin Heidelberg 2001 Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 341 1996-2000 have been scanned. This is a personal, selective, biased account but is hopefully still useful. Almost all contemporary textbooks of biology, genetics, evolution and systematics contain useful chapters that introduce the burgeoning field of molecular evolution and phylogenetics. Several books devoted entirely to this subject have also been published recently, including Graur and Li (1999), Hillis et al. (1996), Li (1997) and Page and Holmes (1998). The reader is also referred to the brief account of molecular phylogenetics in a previous contribution to this series (Prog Bot 57:284288). In addition, some useful books dealing specifically with the systematics and phylogeny of plants and algae, and incorporating molecular studies, are now available (Bhattacharya 1997; Kenrick and Crane 1997; Soltis et al. 1998; Judd et al. 1999). 2 Genomics Meets Phylogeny The most influential single event that has shaped the field of molecular phylogenetics was the July 1995 report of all 1,830,137 DNA base pairs of the gram-negative bacterium Haemophilus influenzae, the first completely sequenced genome of a free-living organism (Fleischmann et al. 1995). Since then, in quick succession, the complete genome sequences of an archae on (Methanococcus jannaschii; -1.66 Mb), a eukaryote (Saccharomyces cerevisiae; -12 Mb) and many other taxa have been reported, including the nematode Caenorhabditis elegans (-97 Mb) in 1998 and the fruit fly Drosophila melanogaster (-180 Mb) in early 2000. Later in the year 2000, the first higher-plant nuclear genome (Arabidopsis thaliana) will be completely sequenced, with the completed human genome presumably following in 2001. Although eukaryotic algae do not appear on any list of ongoing genome projects, genome projects in several protists, including Dictyostelium, Leishmania, Plasmodium and Trypanosoma, are underway. Updated compilations of completed and ongoing "microbial" genome projects can be found on the websites of the major genome-sequencing centers (http://www.tigr.org/ tdb/mdb/mdb.html). Once the first genomes of representatives of the three domains of life (Bacteria, Archaea, Eucarya) were sequenced, phylogeneticists immediately addressed questions regarding the root of the tree of life and the nature of the last universal common (or cellular) ancestor (LUCA) of the three domains. Since the seminal discovery (Woese and Fox 1977) that the microorganisms now known as the Archaea constitute a fundamental "third domain oflife" distinct from Bacteria and Eucarya, much effort has been directed toward understanding the molecular biology and biochemistry of the Archaea and determining the phylogenetic relationships between the three domains oflife. The most widely held view of the 342 Systematics universal tree of life is based on ribosomal RNA (rRNA)-sequence comparisons, in which the root is located using formerly duplicated proteincoding genes, such as adenosine triphosphatases (ATPases), translationelongation factors and transfer RNA (tRNA) synthases. This view states that Archaea and Eucarya are "sister groups", sharing a more recent common ancestor with each other than either does with the Bacteria (Gogarten et al. 1989; Iwabe et al. 1989; Woese et al. 1990; Brown and Doolittle 1995; Baldauf et al. 1996; Lawson et al. 1996; Gribaldo and Cammarano 1998). This view was challenged even before the advent of "microbial genomics", because protein-coding genes not involved in transcription and translation often failed to reveal the alleged sistergroup relationship between the Archaea and the Eucarya (Gupta and Golding 1993; Golding and Gupta 1995; Brown and Doolittle 1997; Gupta 1998). Depending on which protein data set was used, molecular phylogenies showed various relationships between Bacteria, Archaea and Eucarya. In some cases even the topology within the three domains was no longer recovered, with archaeal and bacterial lineages intermixed (Brown and Doolittle 1997; Doolittle 1998a). Comparative genome analyses of the three domains has led to the conclusion that each domain was a mosaic of the two others, in terms of gene content (Koonin and Galperin 1997; Bell and Jackson 1998). Quite surprisingly, the Eucarya contain more bacterial genes than archaeal genes, and the Archaea contain more bacterial genes than eukaryotic genes. Finally, in Thermotoga maritima, previously thought to be one of the most slowly evolving lineages of Bacteria, 24% of the predicted 1877 genes have archaeal affinities (Nelson et al. 1999). Eighty-one of those genes are clustered in 15 regions of the genome, ranging in size from 4 to 20 kb. Because gene order is conserved in many of the clustered regions, Nelson et al. suggested that rampant lateral gene transfer presumably occurred between the thermophilic T. maritima and Archaea. Similar conclusions had been reached before with respect to the hyper-thermophilic bacterium Aquifex aeolicus (Aravind et al. 1998). Given the mosaic genome nature of the three domains of life, several molecular evolutionists have abandoned the three-domain concept; they consider only two primary domains and derive the third domain from the symbiotic merging of ancestral members of the two primary domains. In most of these scenarios, the derived domain corresponds to the Eucarya, which is thought to have originated from the merging of ancient Bacteria and Archaea (Gupta 1998; Katz 1998; Martin and Muller 1998; Moreira and L6pez-Garcia 1998). Originally proposed by Zillig (1987) as the "fusion model" (Gupta and Golding 1996), its most recent versions come under the names "hydrogen hypothesis" (Martin and Muller 1998) and "syntrophy hypothesis" (Moreira and L6pez-Garcia 1998; for a comparison of both hypotheses, see L6pez-Garcia and Moreira 1999). In the "hydrogen hypothesis", an ancient symbiosis occurred Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 343 involving a H 2 - and COr excreting a-proteobacterium (the symbiont) as one partner and a member of the Archaea (nominally the host), using H2 and CO 2 as its sole sources of energy and carbon, as the other partner. Martin and Milller (1998) suggest that, through the symbiosis, the host converted from autotrophy to heterotrophy by using genes from the symbiont to import substrates and to glycolytic ally decompose them to maintain an anaerobic energy metabolism. The symbiont was then either lost or converted to a hydrogenosome or a mitochondrion (see below). Other researchers maintain that the rRNA tree rooted in the bacterial branch is correct and that apparent contradictions observed in some phylogenies based on protein-coding genes should be explained by massive lateral gene transfers (Brown and Doolittle 1997; Woese 1998). The assumption is that proteins (and rRNA) involved in replication, transcription and translation (so-called informational proteins; Rivera et al. 1998) are generally not involved in lateral gene transfers and have evolved at a similar rate in the three domains, as indicated by application of the relative-rate test (Brown and Doolittle 1997). Doolittle (1998b) has argued that lateral gene transfers are inherent in the lifestyles of eukaryotes, because eukaryotes endocytose food involving a "gene-transfer ratchet" through which an "endocytobiont" transfers part of its DNA into the host nucleus (the "you are what you eat" hypothesis). In a new twist to the question of the root of the universal tree of life, Forterre and Philippe (1999) radically depart from the commonly held notion regarding the ancestry of prokaryotes and propose that a eukaryote-like cell (rather than a prokaryote) was the last universal common ancestor of all life forms. These authors argue that contradictions observed among universal phylogenies obtained with rRNA and various proteins do not require specific ad hoc hypotheses. They simply reflect the weakness of the tree-reconstruction methods that have been used to infer the phylogenies (Forterre 1997; Philippe and Laurent 1998). A major point emerging from different analyses of the most conserved phylogenetic markers (i.e., rRNA, actin, tubulin, elongation factors, ATPases, tRNA synthases) is that all these markers are highly mutationally saturated (Philippe and Adoutte 1998; Philippe and Forterre 1999; Roger et al. 1999). A high level of saturation (i.e., noise) means that numerous substitutions have occurred at the same position, diminishing or completely abolishing an ancient phylogenetic signal. Philippe and Forterre (1999) showed that, in the six data sets previously used to root the universal tree, most positions are saturated with respect to amino acid substitutions. This makes the relative-rate test used by Brown and Doolittle (1997) useless, because distances estimated from highly saturated sequences tend to be similar even if the substitution rates are quite different (Philippe and Laurent 1998). Given the high level of saturation, how could it be possible that presumptive ancient relationships between 344 Systematics the three domains of life were recovered in previous analyses? Philippe and Forterre (1999) argue that the inferred topology is the result of a tree-reconstruction artifact known as the long-branch attraction (LBA) phenomenon (Philippe and Laurent 1998). Species that evolve faster than others display sequences very divergent from those of their close relatives; thus, fast-evolving sequences appear to be more distantly related to their relatives than they really are. Because phylogenies inferred from saturated data sets are very sensitive to differences in evolutionary rates, fast-evolving sequences are attracted by the long branch of the outgroup that roots the tree (Philippe and Adoutte 1998). According to Philippe and Forterre (1999), the bacterial rooting of the universal tree of life could be explained by LBA, because the two longest branches are the Bacteria and that of the outgroup. Using elongation factors, Lopez et al. (1999) demonstrated that the evolutionary rate at a given position was generally not the same in different taxonomic groups, in agreement with the covarion model of evolution (Fitch and Markowitz 1970). They developed a simple method (the "H-P method"), which considers a given amino acid only when it undergoes very few changes within predefined taxonomic groups, thus limiting the analysis to slowly evolving sites. Using this method, Lopez et al. (1999) showed that the bacterial rooting of the universal tree (based on elongation factors) is clearly due to quickly evolving positions, suggesting an LBA artifact, whereas a eukaryotic rooting was supported (albeit weakly) by the slowly evolving positions. Similar results were obtained using the signal-recognitionparticle protein, the quickly evolving positions recovered the Archaea/Eucarya clade, whereas the slowly evolving positions led to a eukaryotic rooting, recovering monophyly of the two prokaryotic domains (Brinkmann and Philippe 1999). Although a eukaryotic rooting of the universal tree of life may presently seem to be too radical to be readily accepted, the idea is not without merit. In many ways, the Archaea seem to occupy an intermediate position between Eucarya and Bacteria (Edgell and Doolittle 1997; Forterre 1997; Bell and Jackson 1998). Most importantly, there is no a priori reason to suspect that the "simplicity" of the Bacteria is ancestral and not the result of reductive evolution via the streamlining of life processes toward more efficiency. In fact, in a stimulating article, Poole et al. (1999) provide compelling evidence that eukaryotes have retained more vestiges of an ancestral RNA world (and, therefore, are more likely to resemble the LUeA) than prokaryotes. Many eukaryotic features of the information systems in eukaryotes involve RNA components (such as telomerase guide RNA) and rRNA processing by small nucleolar RNAs. Poole et al. (1998, 1999) suggest that there are two selective advantages for the "simplification" of RNA metabolism and genome structure in Bacteria: r-selection and thermo reduction, the latter eliminates many thermolabile, RNA-catalyzed processing steps (Forterre Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 345 1995). Extensive streamlining of the information-processing system in Bacteria (and, to a lesser extent, in Archaea) was probably accompanied by extensive "non-orthologous gene displacements" (i.e., functional replacement of a given protein by a paralogous or unrelated protein with the same activity; Koonin and Mushegian 1996). Forterre (1999) has suggested that the source of the displacing genes was often a virus or plasmid. No matter where the root of the universal tree of life is located, the uniqueness of the three domains of life remains firmly established, despite currently fashionable hypotheses that derive domains simply by fusion of cells from other domains. This is demonstrated by recent analyses of several completely sequenced genomes from both Archaea and Bacteria, and their comparison with the yeast genome (Forterre 1997; Olsen and Woese 1997; Doolittle and Logsdon 1998; Clayton et al. 1998; Gaasterland 1999). Although unique, each domain exhibits a mosaic of genes/proteins present in the two other domains. These findings have inspired the various "fusion hypotheses" (see above) or have suggested that rampant lateral gene transfer played an essential role in cell evolution. It is undisputed that lateral gene transfer plays an important role among prokaryotes and presumably explains the presence of many a-proteobacterial genes in eukaryotes (from an a-proteobacterial endocytobiont that was the precursor of mitochondria; Martin and Herrmann 1998). However, it should also be recalled that mosaicism, such as that occurring among the three domains of life, is inherent in the evolutionary process and can originate from a variety of mechanisms. These mechanisms include different rates of character evolution, nonorthologous replacement, gene duplication and gene loss leading to unrecognized paralogy (Forterre and Philippe 1999). Despite the conspicuous mosaicism of genes/proteins in the three domains of life, the uniqueness of the three domains and their phylogenetic relationships are not obscured. This was demonstrated in a recent phylogenetic analysis based on the presence or absence of families of protein-coding genes in 11 complete genomes; the resulting tree was largely consistent with the rRNA tree (Fitz-Gibbon and House 1999). Even more importantly, analyses using informational (involved in replication, transcription and translation) or operational subsets of the genes yielded similar results. The only significant difference in the analyses of the two subsets of genes was in the relative lengths of the branches separating the Bacteria from the Eucarya (yeast) and Archaea, which was longer in the informational-gene subset (Rivera et al. 1998). Similar to the situation for rRNA trees, the deepest branches (here in the Bacteria) were difficult to resolve (Fitz-Gibbon and House 1999). Another recently hotly debated issue in genome phylogenies relates to the possible age ofthe last common ancestor of the three domains oflife. This discussion was initiated by R.F. Doolittle et al. (1996) in an analysis 346 Systematics of sequence data from 57 different proteins that were used to determine the divergence times of the major biological groups. This study suggested that prokaryotes and eukaryotes may have shared a common ancestor as recently as approximately 2 billion years ago. This conclusion is in contrast to evidence from the geological record, which indicates that oxygen-evolving cells (perhaps similar to present-day cyanobacteria) existed approximately 3.5 billion years ago (Schopf 1993). Doolittle et al. calculated evolutionary distances for pairwise comparisons of homologous sequences within and between groups; to calibrate the "protein clock", they used the distances between chordate species for which a fossil record is known. This work has been criticized for several reasons; Hasegawa and Fitch (1996) argued that a "covarion model of evolution" (where different residues are functionally constrained at different periods of time) had not been taken into consideration. In addition, even if a "covarion model" is not valid, equal mutational rates at all sites should not be implied; i.e., a y distribution would match the distribution of mutations among sites better than the Poisson distribution. Using a parametric y distribution, Miyamoto and Fitch (1996) and Gogarten et al. (1996) were able to determine that the prokaryotic/eukaryotic divergence was at least 3.5 billion years ago. Martin (1996) suggested another possible source of error; many eukaryotic genes are presumably derived from more recently acquired bacterial genes via lateral gene transfer. As a consequence, W.F. Doolittle (1997) has argued that the 2-billion-year result of Doolittle et al. (1996) does not measure the full length of the tree but just the part of the tree extending from the Eucarya down to the origin of mitochondria and back up to contemporary Bacteria bypassing the extra billion years down to the root. Feng et al. (1997) have expanded on the data of Doolittle et al (1996) to analyze 64 proteins, taking advantage of the newly sequenced archaeal genomes. Evolutionary distances were calculated according to the method of Grishin (1995), which corrects for site-to-site variations in mutation rates. For 25 proteins with both archaeal and bacterial sequences and with no evidence oflateral transfer between them, Feng et al. (1997) calculate divergence times between 3.1 and 3.8 billion years. Nevertheless, there are still serious difficulties with the fossil record, because divergences within the Bacteria extend only 2.1-2.5 billion years into the past. Brocks et al. (1999) recently showed that molecular fossils of biological lipids are well preserved in 2.7-billion-year-old shales from Australia. They conclude that both cyanobacteria and eukaryotes were presumably extant 2.7 billion years ago. What can be learned from "genome phylogenetics" for algal phylogeny? First, the only "algal" genome that has been completely sequenced is that of the cyanobacterium Synechocystis sp. PCC6803 (Kaneko et al. 1996b). It has had an enormous influence on studies of chloroplast functions and plastid phylogeny in general. Plastid genomics (see below) Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 347 is starting to address questions of algal phylogeny, but the number of completely sequenced plastid genomes is much lower than that of the larger bacterial genomes, reflecting a general lack of commercial interest in the former. "Genome phylogenetics" of prokaryotes has revealed the mosaic nature of these genomes, a characteristic which has plagued phylogenetic analyses ever since. Almost every week, a new microbial genome sequence is completed. The data are apparently generated much faster than one can make biological (phylogenetic) sense of them. Computational problems abound; some are simply related to the loss of phylogenetic signal due to mutational saturation in very ancient lineages. However, unequal rates of evolution (and, as a consequence, LBA), gene duplications accompanied by differential loss of the duplicated genes, lateral gene transfers or poor taxon sampling are common problems in any phylogenetic analysis and are likely to adversely affect the analysis of algal phylogeny in the future as they already did in the past. Another lesson to be learned is that genes coding for the replication, transcription and translation machinery ("informational genes") still form the "blueprint of the enduring cellular hardware" (Doolittle and Logsdon 1998). They presumably reflect the evolutionary history of organismic lineages more faithfully than other molecular markers. 3 Bonsai Genomics: the Phylogeny of Mitochondria, Plastids and Nucleomorphs Mitochondria, plastids and nucleomorphs contain genomes with a much reduced gene content compared to their free-living ancestors. The mechanisms of gene transfer from symbiont to host, symbiont genome reduction, and cross-talk between symbiont(s} and host are central research topics in contemporary cell and evolutionary biology with wideranging implications for eukaryote phylogeny. Since plastids and nucleomorphs are confined to the algae and their descendants (Le., embryophyte plants), the phylogeny of these bonsai genomes impinges on algal phylogeny in general. Because these genomes are at most a few hundred kilobases in size, complete genome sequences are increasingly used for phylogenetic analysis, although taxon sampling still remains a major problem for bonsai phylogenetics. a} Mitochondria/Hydrogenosomes Mitochondrial evolution and phylogeny based on comparative genomics have recently been adequately reviewed (Lang et al. 1998a, 1999a,b; Gray et al. 1999). The two most important single studies that relate to the phylogeny of mitochondria during the past 4 years were the description 348 Systematics of the complete genome sequence (1,111,523 bp) of the obligate intracellular parasite Rickettsia prowazekii by Kurland and colleagues (Andersson et al. 1998; for a discussion of these results in relation to mitochondrial evolution, Muller 1997; Muller and Martin 1999) and the publication of the complete mitochondrial sequence of the protist Reclinomonas americana, the most eubacteria-like mitochondrial genome discovered to date (Lang et al. 1997). R. prowazekii is the causative agent of typhus and is a member of the a-proteobacteriallineage of gram-negative bacteria. Mitochondrial gene sequences had previously firmly established that mitochondria had a monophyletic origin within the a-proteobacteria (Yang et al. 1985). Indeed, the a-proteobacterial ancestor of the mitochondria may well have been an ancestral member of the Rickettsiales (Gray and Spencer 1996; Gray 1998; Sicheritz-Ponten et al. 1998). As expected for an obligate intracellular parasite the genome of R. prowazekii is small compared with that of free-living bacteria containing 834 protein-coding genes only. Although small for a bacterial genome, the R. prowazekii genome still contains an order of magnitude more protein-coding genes than the largest mitochondrial genome characterized to date. The genomic parallels between mitochondria and intracellular bacteria, such as R. prowazekii, are remarkable and include massive losses of genes, extensive gene rearrangements and accelerated rates of nucleotide substitution (Andersson and Kurland 1998). The functional profile of the genes of R. powazekii is also similar to those of mitochondria: no genes for anaerobic glycolysis are found in R. prowazekii, but a complete set of genes encoding components of the tricarboxylic acid cycle, the respiratory chain and ATP production are found, as in mitochondria. Unique among bacterial genomes, a large fraction (-24%) of the genome of R. prowazekii contains non-coding DNA; these may be degraded remnants "of neutralized genes that await elimination from the genome" (Andersson et al. 1998). It is likely that the activities of functionally similar nuclear genes renders genes of the intracellular symbiont/parasite expendable and, as a consequence, they become vulnerable to elimination by mutation. The mutations are mostly deleterious, because selection cannot operate on such clonal populations (Andersson and Kurland 1998). The accumulation of deleterious but non-lethal mutations is referred to as "Muller's ratchet" (Felsenstein 1977). Although striking similarities occur in the functional profiles of genes in R. prowazekii and mitochondria, phylogenetic analyses provide no evidence that the mitochondrial genome evolved directly from an already reduced, Rickettsia-like genome (Lang et al. 1998a; Gray et al. 1999). Instead, mitochondria and the Rickettsiales are almost certainly descendants of separate processes of reductive genome evolution. Although most such genes were completely lost in R. prowazekii, during the course Systematics and Evolution of the Algae. 1. Genomics Meets Phylogeny 349 of mitochondrial evolution, most of the ancestral genes were either lost or transferred to the nuclear genome. In fact, more than 300 (lproteobacterial genes are encoded by the nuclear genome of yeast (Hodges et al. 1999; Gaasterland 1999). More than half of these genes encode proteins that are involved in bio-energetic processes and in the biosynthesis of macromolecules (Andersson and Kurland 1999). Lang et al. (1997) reported the complete mitochondrial sequence (69,034 bp) of the heterotrophic jakobid (retortamonad) flagellate R. americana (Palmer 1997a). The genome was shown to contain the largest collection of genes (97) identified in any mitochondrial DNA (mtDNA), including genes for 5S rRNA and at least 18 protein-encoding genes not previously known from mitochondrial genomes. Almost half of the 62 protein-encoding genes code for translation components, while the other half encode enzymes involved in bio-energetic processes. Gene transcription in R. americana is Bacteria-like, because the genome encodes all four components of a eubacteria-type RNA polymerase (rpo AD). In all other mitochondria known, the transcription of mtDNA is performed by a nuclear-encoded, single-subunit bacteriophage T3/T7like RNA polymerase (Gray and Lang 1998), a striking example of gene replacement during evolution. Other ancestral features of the R. americana mtDNA include the presence of a secY gene for protein sorting (as in Bacteria and plastids), Shine-Dalgarno base pairing between the 3' end of the small subunit (SSU) rRNA and the 5' end of its messenger RNAs, and a eubacterial RNaseP RNA (Lang et al. 1997, 1998a). Several other "ancestral traits" are also preserved in the mtDNA of R. americana: a universal genetic code, an almost complete set of structurally normal tRNAs, an apparent lack of RNA editing, and a eubacterial operon gene structure. During the past 7 years, many complete mtDNA sequences have been determined, mostly through the two Canadian mitochondrial sequencing programs FMGP (http://megasun.bch.umontreal.ca/people/langl FMGP) and OGMP (http://megasun.bch.umontreal.ca/ogmpl). A recent survey of the gene structure and gene content of mtDNA in 23 taxa of protists was published by Gray et al. (1998). From this, it is apparent that mtDNAs can be classified into two categories: "ancestral" and "derived" (Gray et al. 1999). The R. americana mtDNA is an ancestral pattern; animal and most fungal mtDNAs belong in the derived category, as do some highly atypical mtDNAs, such as those of Chlamydomonas spp. (Denovan-Wright et al. 1998) and Plasmodium (Feagin 1994). Derived mtDNAs are characterized by extensive gene loss, marked divergence in rDNA and rRNA structure, an accelerated rate of sequence divergence, biased codon usage and non-standard codon assignments (Gray et al. 1999). The distinction between ancestral and derived mitochondrial genomes is not clear-cut; several mtDNAs (such as that of Acanthamoeba castellanii) have both ancestral and derived traits (Burger et al. 350 Systematics 1995). Land-plant (embryophyte) mtDNAs represent a special case, because they are relatively large (>200 kb), due to a high proportion of non-coding sequences but encode only 2.5 times as many proteins as human mtDNA (32 proteins versus 13 proteins; Unseld et al. 1997). In addition, angiosperm mtDNAs have become recombinationally active, RNA editing has evolved, and approximately 1% of the mtDNA has been recruited from the chloroplast genome. As a consequence, in angiosperm mtDNA, eubacterial-like gene clusters have broken up leading to fragmentation and dispersal of protein-coding genes, the emergence of trans-splicing, gene transfer to the nucleus, and the incorporation of chloroplast DNA (cpDNA) and nuclear DNA (Gray et al. 1999). Phylogenetic reconstructions using either single-gene phylogenies (mostly SSU rRNA) or concatenated sets of genes support the notion that all mitochondrial genomes have a monophyletic origin and descended from an a-proteobacterial ancestor (Gray and Spencer 1996; Andersson et al. 1998; Lang et al. 1998a, 1999b; Gray et al. 1999). The relatively low gene content of mtDNA compared with the smallest known eubacterial genomes implies a rapid and extensive loss or transfer of genes during the early stage of mitochondrial evolution from a protomitochondrial genome. Differences in gene content among extant mtDNAs are best explained by assuming differential gene losses after the initial massive gene transfers from the protomitochondrial genome. All phylogenetic analyses of mitochondria published to date indicate a close affiliation between mitochondria and the rickettsial group of aproteobacteria. The latter are comprised of not only animal pathogens, such as Rickettsia and Ehrlichia, but also bacterial endosymbionts of protists, such as Holospora and Caedibacter (Springer et al. 1993). Some free-living a-proteobacteria (such as Paracoccus and Rhodobacter) and symbiotic/parasitic soil bacteria (such as Agrobacterium and Bradyrhizobium) are only slightly less closely related to mitochondria than are the Rickettsiaceae (Lang et al. 1998a; Gray et al. 1999). These studies suggest that the bacterial ancestor of mitochondria was a facultative aerobe and that the presence of nearly identical systems for aerobic energy production in a-proteobacteria (such as Rickettsia) and mitochondria is indicative of this common ancestry. However, ATP/adenosine diphosphate (ADP) translocases in the intracellular, parasitic Rickettsiaceae and in mitochondria have independent origins (Andersson 1998). This suggests that, although ATP production in mitochondria presumably originated from an a-proteobacterial ancestor, ATP/ADP translocation originated in the eukaryotic genome subsequent to the divergence of mitochondria and a-proteobacteria (Andersson and Kurland 1999). The "hydrogen hypothesis" (Martin and Muller 1998) focuses on hydrogen rather than ATP as the metabolic link that supported the endosymbiotic association between a facultative anaerobic a-proteobacterium and a methanogenic archaeon (see above). It draws heavily on the pres- Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 351 ence of specialized anaerobic mitochondria (hydrogenosomes; see below) in certain protists which produce hydrogen and proposes that the common ancestor of mitochondria and hydrogenosomes carried all the enzymes currently found in both these organelles. Differential gene losses then would account for the different sets of enzymes present in contemporary mitochondria and hydrogenosomes. This scenario, however, is weakened by the fact that hydrogenosomes not only occur in putatively early-branching eukaryotes, such as the trichomonads, but also in more advanced eukaryotes in which the vast majority of taxa contain normal respiratory mitochondria, such as ciliates and fungi (Finlay and Fenchel1989; Embley et al. 1995, 1997). Therefore, it is more parsimonious to assume that hydrogenosomes evolved several times, independently from mitochondria, by successive loss of coding sequences in anaerobic environments (Embley et al. 1997) than to postulate that aerobic mitochondria either independently lost unique hydrogenosomal genes several hundred times or that "silent" hydrogenosomal genes exported to the nuclear genome early during mitochondrial evolution were "re-activated" and independently targeted to the mitochondrion several times in different taxa upon the taxon's adaptation to an anaerobic environment (Embley and Martin 1998). Phylogenetic analyses of unique hydrogenosomal genes should help to distinguish between these alternatives: a single common origin of mitochondria and hydrogenosomes, with differential gene losses in the two organelles (as proposed by the hydrogen hypothesis) should lead to the clustering of ideally all unique hydrogenosomal genes from different taxa within the lineage of a-proteobacteria that during evolution, presumably gave rise to the mitochondria. A polyphyletic origin of hydrogenosomes from aerobic mitochondria explains the presence of hydrogenosomal genes by lateral gene transfer from taxa that are different from the mitochondrial ancestor. The idea that hydrogenosomes are derived from mitochondria is now generally accepted because, in phylogenetic analyses, a number of hydrogenosomal proteins, such as adenylate kinase and the heat-shock proteins Hsp 10, Hsp60 and Hsp70, group within the mitochondrial clade (Bui et al. 1996; Muller 1997; Palmer 1997b). What about the origin of unique hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO) and hydrogenase? PFO apparently has a complex phylogenetic history and belongs to a larger gene family with several paralogous genes (Rosenthal et al. 1997; Horner et al. 1999). Horner et al. (1999) concluded that PFO is likely to have a monophyletic origin in eukaryotes related to eubacterial homologs. However, no sister group to the eukaryote lineage could be identified, and PFOs of a-proteobacteria do not appear to be closely related to the eukaryote PFOs. Therefore, Andersson and Kurland (1999) concluded that PFO may have been present in the common ancestor of eukaryotes and that 352 Systematics eukaryotic lineages with mitochondria could have retained a PFO homolog with functions slightly different from those observed in extant hydrogenosomes. More insight into the origin of unique hydrogenosomal genes has been obtained from studies of the anaerobic heterotrichous ciliate Nyctotherus ovalis (Akhmanova et al. 1998). This study contains the first description of a genome in a hydrogenosome. Phylogenetic analyses using primers directed against conserved regions of the mitochondrial SSU rRNA genes from ciliates place the N. ovalis sequence among the mitochondrial SSU rRNA genes from aerobic ciliates. This supports the notion that this hydrogenosome is derived from an aerobic mitochondrion. A nuclear-encoded gene for a putative hydrogenase was also isolated and sequenced from N. ovalis. Surprisingly, the N-terminal half of the predicted polypeptide shares 35-41 % sequence identity with [Fe J-hydrogenases from Clostridium and Desulfovibrio. The middle and C-terminal parts resemble the HoxE/HoxF proteins of Synechocystis and the nuclear genes nuoE and nuoDF, which code for components of the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenases in the mitochondrial respiratory chain. The mosaic character of the N. ovalis hydrogenase suggests that it may have been assembled from genes coding for NADH dehydrogenases fused with hydrogenase-like genes from other bacteria. Since hydrogenosomes most likely evolved several times from aerobic mitochondria, it is conceivable that their hydrogenases may also have independently evolved from various gene fusions (Akhmanova et al. 1998). Such an origin of hydrogenosomal hydrogenases, if widespread, would be difficult to explain by either the hydrogen or the syntrophy hypotheses. There is essentially no need to assume that a hydrogen-metabolism link was the driving force for the origin of mitochondria; other scenarios forcing an endosymbiosis between an a.-proteobacterium and an anaerobic eukaryote can be envisaged. Andersson and Kurland (1999) recently proposed a model for the origin of mitochondria (the "ox-tox hypothesis") that is in accordance with many recent observations and is more compatible with the suggested link between eukaryotes and the RNA world (see above), and the serial-endosymbiosis theory. These authors suggest that the origin of mitochondria was based on a twophase selection process for aerobic respiration by an a.-proteobacterial symbiont. The initial function of the symbiont is postulated to have been detoxification, and the later function was the provision of ATP to the host cell. In this scenario, the sudden rise in atmospheric oxygen levels approximately 2 billion years ago is recognized as an environmental crisis for anaerobic organisms. Anaerobes could adapt to the presence of oxygen by exploiting the ability of facultative aerobic bacteria to locally detoxify the environment by consuming oxygen (Bernard and Fenchel 1994; Vellai et al. 1998). Initially, such an association between an 0.proteobacterium and an anaerobic eukaryote was extracellular and Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 353 could become more intimate if the aerobic cell moved into the host as an endocytobiont. In the second phase, massive gene transfer from the endocytobiont to the host nucleus (with gene replacement) occurred, making metabolic pathways of the cytosol more compatible with an oxygen-enriched environment, and enhancing transport functions at the host's cell boundary (Keeling and Doolittle 1997). The transport of ATP from the mitochondrion to the host cell was implemented by the acquisition of host transport (ATP/ADP transporter) and control functions. While the issue of how and when mitochondria originated is far from settled (Sogin 1997), a few conclusions may be safely made; all available evidence indicates that mitochondria are monophyletic and originated from an a-proteobacterium, probably with rickettsial affinities. The host could have been a prokaryote (as in the different fusion hypotheses) or, more likely, a eukaryote (as in classical serial-endosymbiotic theory). Presently, no extant eukaryotes that never contained mitochondria during their evolutionary history are known. The lack of mitochondria in some presumptive "ancient" eukaryotic lineages is most likely secondarily derived due to the loss of mitochondria, as in the Microsporidia (Germot et al. 1997; Keeling and McFadden 1998; Hirt et al. 1999; Fast et al. 1999), Entamoeba (Roger et al. 1996), Parabasalia (Bui et al. 1996; Germot et al. 1996; Horner et al. 1996) and diplomonads (Roger et al. 1998). Thus, the "Archezoa hypothesis" (Cavalier-Smith 1989) is presumably invalid. The way the evolution of hydrogenosomes is related to that of mitochondria is unknown, but multiple independent origins of hydrogenosomes from aerobic mitochondria is the currently preferred model. To what extent has mitochondrial genomics contributed to our current view of algal phylogeny? A concatenated, aligned data set of amino acid sequences from cob and coxl-3, representing a total of 14 (Lang et al. 1998a) or 18 (Gray et al. 1999) taxa, has been used to infer the phylogeny of mitochondria using a combination of the PROTDIST and FITCH programs and bootstrapping. Because mitochondria are thought to be monophyletic, the resulting phylogenetic tree should reflect the host phylogeny. The most significant result concerning algal phylogeny is the fact that the three red-algal taxa (Chondrus, Porphyra, Cyanidium) form a sister group to the three green-plant (Viridiplantae) taxa (Prototheca, Marchantia, Triticum). This clade is reasonably well supported by bootstraps: 84% in Lang et al. (1998a) and 95% in Gray et al. (1999). Although this would be expected if plastids evolved via a single primary endocytobiotic event, it has not been possible to demonstrate this relationship using nuclear-encoded rRNA sequence comparisons (Melkonian 1996). For other clades, supportive evidence from nuclearencoded rRNA-sequence comparisons exist, e.g., the heterokonts (stramenopiles; 83% and 96% bootstraps). It is clear, however, that 354 Systematics taxon sampling remains a serious problem in mitochondrial phylogenetics. b) Plastids and Nucleomorphs The analysis of the origin and phylogeny of plastids remains one of the most intense research areas in contemporary evolutionary biology; progress in this field has been summarized in several recent review articles (Melkonian 1996; Delwiche and Palmer 1997; Kowallik 1997; Palmer and Delwiche 1998; Douglas 1998; Delwiche 1999; Prog Bot 57:288-289). There is now almost general consensus among researchers that all plastids had a single phylogenetic origin among the cyanobacteria. This idea has been supported by phylogenetic analyses of single plastid-encoded genes, of concatenated genes from whole-plastid genomes and by genecluster analyses. While the single cyanobacterial origin of plastids is well established, fundamental questions of plastid evolution and phylogeny remain unresolved. What was the nature of the cyanobacterial ancestor of plastids? What is the relationship between the different plastid lineages, and how many times did plastids enter phagotrophic hosts during secondary endocytobioses? What were the mechanisms by which plastids were permanently incorporated into the host cell? What are the mechanisms of protein targeting in the different types of plastids, and how did they evolve? The most dramatic feature distinguishing plastids from cyanobacteria is the extreme reduction in the size and gene content of the plastid genomes. The genome of Synechocystis PCC 6803 is 3573 kb and contains a total of 3168 protein-coding genes (Kaneko et al. 1996b; Kotani and Tabata 1998), whereas that of the least reduced plastid (from the red alga Porphyra purpurea) is only 191 kb and contains approximately 250 genes. The plastid genomes of the Viridiplantae (green plants) are even more reduced. This reduction of the coding capacity of the plastid genome is the hallmark of an integrated organelle (compared with an endocytobiont). However, plastids contain many more proteins than are encoded by their genome - estimates range from 500 to 5000 (Martin and Herrmann 1998). The plastid proteins not encoded by the plastid genome are encoded in the nuclear genome and are targeted to the plastid. Three mechanisms underlie the reduction of the plastid genome: gene loss, substitution and transfer (Martin et al. 1998; Race et al. 1999). As in mitochondria (see above), Muller's ratchet presumably strongly favored gene transfer from the plastid to the nucleus. However, this has not led to a complete disappearance of the plastid genome, even in plastids that lost photosynthetic functions during evolution. The reasons genes were maintained in the plastid are controversial. Nucleotide substitution rates Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 355 in plastid genes are lower than expected, suggesting that compensatory mechanisms (such as efficient DNA repair, high ploidy levels and genetic recombination) slow down the effects of Muller's ratchet. Race et al. (1999) discuss possible reasons for maintaining plastid genomes and suggest, based on an original proposal by Allen (1993), that the expression of structural proteins needed to maintain a redox balance in bioenergetic membranes is mediated by redox signaling requiring location of the corresponding genes in the same organelle that generates the redox signal. This hypothesis fares well with a functional analysis of the genes encoded in plastids. Among the 205 different protein-coding genes contained in nine completely sequenced plastid genomes, 46 proteins have been shown to be common to each plastid genome (and the genome of Synechocystis). Twenty-four of these proteins are constituents of the photosynthetic membrane, 16 are ribosomal proteins, three are subunits of RNA polymerase, one is Rubisco and two are unknown proteins (Martin et al. 1998). When did gene loss occur during plastid evolution, and to what extent were such losses unique processes rather than occurring as parallel losses in independent plastid lineages? This question was addressed by mapping all 205 genes known to exist in nine completely sequenced chloroplast genomes onto the topology of plastid phylogeny obtained from a concatenated data set of 45 plastid-encoded proteins (Martin et al. 1998). This analysis showed that more than 90% of the genes originally present in the genome of a cyanobacterium (such as Synechocystis) were lost from the endocytobiont/plastid genome(s) either before the three lineages of primary plastids (cyanelles, rhodoplasts and chloroplasts; Prog Bot 57:293) split or, less likely, as two or more parallel losses in the different plastid lineages. If the tree topology presented by Martin et al. (1998) is regarded as true (despite the limited taxon sampling!), then mapping of the 159 genes present in only a subset of the plastid genomes analyzed indicates that unique gene-loss events account for the fate of only 58 genes, whereas the majority of genes (101) have undergone parallel losses in independent lineages (44 genes lost twice, 43 genes lost three times and 14 genes lost four times, independently). If one counts each loss of an individual gene as one event (which may be an overestimate), the ratio of parallel losses to unique losses becomes 4.7 (58 unique losses versus 273 parallel losses). In any case, the number of parallel losses is high compared with the number of unique losses, indicating that a significant selection pressure toward functional homogenization existed during the evolution of different types of plastids. Some of these genes have been lost completely, but others have been transferred to the nucleus. Martin et al. (1998) documented 44 cases of functional green-plant nuclear genes among the 210 genes examined. All of these genes acquired transit pep tides for re-import into the chloroplast, where they are still functional (Martin and Schnarrenberger 1997). The major- 356 Systematics ity (27) of these proteins are derived from genes that were lost in parallel in the different plastid lineages, suggesting that, perhaps in cyanelles/rhodoplasts, these proteins may also be re-targeted to the organelles, although specific information is lacking. There are, however, many more proteins present in plastids that are encoded in the nuclear genome and which are targeted to the plastid. Except for the large subunit (LSU) of Rubisco (rbeL) all Calvin-cycle enzymes belong to this category. The rbeL phylogeny itself has been controversial for several years (Delwiche and Palmer 1996; Delwiche 1999; Prog Bot 57:295-296). Two structurally and evolutionary distinct types of Rubisco are known: formI Rubisco consists of eight SSUs (rbeS) and eight LSUs (rbeL), forming the L8S8 holoenzyme, and form-II Rubisco consists only oflarge subunits (L 2_6 holoenzyme). In plastids, until recently, only form-I Rubisco was known. It comes in two types "R type" and "G type". The first is found in the cpDNA of rhodoplasts (and two groups of proteobacteria), and both subunits (rbeL and rbeS) are encoded by the plastid. The G type is found in chloroplasts (only rbeL is encoded by the plastid genome), cyanelles (both rbeL and rbeS are encoded by the cyanelle genome), cyanobacteria and three groups of proteobacteria. Delwiche and Palmer (1996) conducted phylogenetic analyses of all available bacterial rbeL sequences and representative plastid rbeL sequences and concluded that both (1) mUltiple events of horizontal (lateral) gene transfer and (2) an ancient-gene duplication followed by multiple differential gene losses could account for the complex topology observed. A more refined analysis of the form-I rbeL phylogeny (Watson and Tabita 1997) has shown that four groups of rbeL occur: a cyanobacteria/proteobacteria G type, a cyanobacteria/cyanelle/chloroplast G type, a proteobacteria R type and a rhodoplast R type. The cyanobacterial proteobacteria G-type clade contains a-, [3-, and y-proteobacteria, whereas the proteobacteria R-type clade contains only a- and [3-proteobacteria. The authors suggest that the two cyanobacteria (Syneehoeoccus WH7803 and Proehlorococcus marin us), which cluster with the yproteobacteria, obtained their rbeL gene from the latter via lateral gene transfer. The R-type rbeL was perhaps transferred from an a-proteobacterium to the common ancestor of the rhodoplast-containing algae (an ancestral red alga), where it presumably replaced the original G-type rbeL. While it remains possible that an ancient Rubisco-gene duplication prior to the divergence of the cyanobacteria gave rise to the R- and Gtype rbeLs (with subsequent differential gene loss accounting for the observed distribution of the two rbeL types among extant plastid types) this scenario is weakened by the fact that there is no evidence for the coexistence of form-I Rubiscos of both Rand G types in any single organism. Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 357 The form-II Rubisco is generally regarded as the more ancient Rubisco form and is presumably the progenitor of form I. The biochemical properties of form-II enzymes suggest that they are unable to sustain growth in an aerobic environment (Lorimer et al. 1993). Therefore, it came as a great surprise when it was discovered that several photosynthetic, peridinin-containing dinoflagellates contain only form-II Rubisco (Morse et al. 1995; Whitney et al. 1995; Rowan et al. 1996). Uniquely, the form-II Rubisco in dinoflagellates is nuclear-encoded by a multi-gene family (Rowan et al. 1996). The predominantly expressed Rubisco is encoded as a polyprotein consisting of three concatenated subunits. The presence of a type-II Rubisco in dinoflagellates raises many questions regarding the function of this enzyme. These include the question of how the dinoflagellates master the unfavorable oxygen/carbon-dioxide balance of the enzyme (so far investigators have been unable to measure significant enzyme activity). But in the present context, the phylogenetic origin of the dinoflagellate form-II Rubisco matters. The Symbiodinium Rubisco is 65% identical to the form-II Rubisco of Rhodospirillum rubrum, an a-proteobacterium (Rowan et al. 1996). Phylogenetic analysis indicated that the Gonyaulax form-II Rubisco groups within the aproteobacteria (Delwiche and Palmer 1996), while analysis of both dinoflagellate taxa showed a sister-group relationship between the dinoflagellates and Rhodospirillum spp. (Watson and Tabita 1997). The different scenarios that could explain the origin of the unusual form-II Rubisco in dinoflagellates have been addressed by Morse et al. (1995), Rowan et al. (1996), Melkonian (1996), Delwiche and Palmer (1996), Palmer (1996), Martin and Schnarrenberger (1997), and Watson and Tabita (1997). Basically, the form-II Rubisco of dinoflagellates could be of cyanobacterial, mitochondrial or other proteobacterial origin. Acquisition of form-II Rubisco by lateral gene transfer (from an a-proteobacterial symbiont?) is favored by several authors (Palmer 1996; Watson and Tabita 1997). The phylogenetic origin of other Calvin-cycle enzymes was reviewed and discussed by Martin and Schnarrenberger (1997). The host cell of plastid symbiosis and the cyanobacterial symbiont that preceded the evolution of plastids presumably possessed redundant sets of genes for the enzymes of core carbohydrate metabolism. Analysis of Calvin-cycle enzymes is complicated for this reason and because similar functional redundancy in such enzymes probably also existed earlier, during the origination of mitochondria from an a-proteobacterium (Nowitzki et al. 1998). In both cases, endosymbiotic gene transfer resulted in functionally redundant copies in the host's nuclear genome. The simplest interpretation that explains the preferentially eubacterial carbohydrate metabolism in eukaryotes is that, during the endosymbiotic origins of organelles, the elimination of functional redundancy in carbohydrate metabolism in the nuclear genome resulted in the preferential loss of the "eukaryotic" isoforms. Why this should be so remains unclear, but the 358 Systematics eubacterial enzymes may have been functionally more efficient in the new "oxygen-rich" cytosol generated by the metabolism of the bacterial symbionts/organelles. In summary, phosphoglycerate kinase (PGK) appears to be of cyanobacterial origin (this also holds true for the cytosolic isoenzyme that presumably replaced a more ancient PGK of putative mitochondrial origin; Brinkmann and Martin 1996). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also of cyanobacterial origin (the Calvin-cycle enzymes GapA and GapB). The cytosolic/glycolytic form of GAPDH has been suggested to be of mitochondrial origin (Henze et al. 1995), although the situation is complicated by considerable GAPDH-gene diversity among Bacteria, duplication of the nuclear gene and novel rerouting to the chloroplast (Meyer-Gauen et al. 1994), and possible lateral transfers between eukaryotes (Fagan et al. 1998). Triosephosphate isomerase (TPI) appears to be of a-proteobacterial (i.e., mitochondrial) origin (Schmidt et al. 1995; Keeling and Doolittle 1997). Fructose-l,6bisphosphate aldolase (FBA) consists of two classes (class I and class II). Class I occurs in the chloroplasts and cytosol of higher plants (Chlamydomonas reinhardtii only contains a single class-I FBA localized in the chloroplast; Schnarrenberger et al. 1994), and its origin is still unclear. The class-II FBA occurs in most eubacteria, including cyanobacteria, but only in four eukaryotes (Cyanophora paradoxa, Euglena gracilis, S. cerevisiae, and Schizosaccharomyces pombe). Phylogenetic analyses reveal that these nuclear genes have proteobacterial affinity (Plaumann et al. 1997). Fructose-l,6-bisphosphatase (FBP) appears to be of mitochondrial origin (both the Clavin cycle and the cytosolic forms). Transketolase (TKL) appears to have a cyanobacterial origin (the chloroplast and cytosol isoenzymes apparently originated from a recent gene duplication; Flechner et al. 1996). For phosphoribulokinase (PRK), classI and class-II enzymes are known; the higher-plant and Chlamydomonas nuclear-encoded PRK genes are descendants of the class-II cyanobacterial enzymes. Glucose-6-phosphate isomerase (GPI) in higher plants occurs as a chloroplast enzyme and as a cytosolic enzyme; both isoforms are encoded in the nuclear genome (Nowitzki et al. 1998). The chloroplast GPI is of cyanobacterial origin (it clusters with the GPI from Synechocystis PCC6803), whereas the cytosolic form appears to be of eubacterial (perhaps mitochondrial) origin. Taken together, the phylogenetic history of Calvin-cycle enzymes demonstrates a significant contribution of endosymbiotic gene transfer to the nuclear genome; some chloroplast isoenzymes are of cyanobacterial origin, some are of mitochondrial or unknown eubacterial origin. Cytosolic isoenzymes, when present, are also of cyanobacterial, mitochondrial or other eubacterial origin, suggesting that ancient eukaryotic (host) enzymes became redundant on endocytobioses and were replaced by endosymbiont enzymes. Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 359 What happened to endosymbiont genes that could not replace redundant host genes after transfer to the host nucleus because functionally equivalent genes were not present in the nuclear genome? Were such genes lost completely, or were they added to the gene pool of the host, enhancing the host's functional abilities? It is likely that such questions can only be addressed specifically when a plant or algal genome has been completely sequenced (Blanchard and Schmidt 1995). The question of the nature of the cyanobacterial ancestor of plastids has puzzled researchers for many years. Studies of the molecular phylogeny and systematics of cyanobacteria (and oxychlorobacteria) have been summarized during recent years (Wilmotte 1994; Turner 1997; Honda et al. 1999a; Turner et al. 1999; Prog Bot 61:285-299). Because only one cyanobacterial genome has been completely sequenced (several new cyanobacterial genome sequencing projects, including P. marin us, are in progress), phylogenetic analyses in this group of Bacteria rely on single genes (mainly SSU rRNA), using extensive taxon sampling and refined tree-reconstruction methods. The latest analysis of the phylogeny of cyanobacteria, based on 1377 aligned positions in the SSU rRNA gene and involving 53 taxa of cyanobacteria and ten plastids, was presented by Turner et al. (1999). The conclusions generally corroborate previous analyses but reveal several relationships not previously noted, whereas other relationships previously supported are contradicted. In general, plastids consistently form a monophyletic group with strong support and bear no relationship to any of the three chlorophyll bcontaining oxychlorobacteria, which are themselves not closely related to each other (Prog Bot 57:292; Bhattacharya and Medlin 1995). Gloeobacter violaceus is the deepest-branching lineage among the cyanobacteria [this is supported by studies of the phylogeny of other genes, such as DNA-dependent RNA polymerase (rpoe; Palenik and Swift 1996), tufA (Kohler et al. 1997) and group-I introns in tRNAfMet (Paquin et al. 1997)]. This placement of G. violaceus is consistent with other, nonnucleotide characteristics. Tree interference was performed using the maximum-likelihood method with correction for site-to-site variation in the evolutionary rate, and confidence was inferred using relativelikelihood support (RLS; Jermiin et al. 1997) scores. Ten clades ("sequence groups") of cyanobacteria/plastids were identified: the Oscillatoria (OSe), the SynechocystislPleurocapsalMicrocystis (SIP/M) group, which together form a sister group, the SynechococcuslPhormidium (SO/PHOR) groups, which form a sister group to the "unicellular thermophilic" (UNIT) group, and five other groups that, under a 90% RLS rule, form a radiation from a common ancestral node. Among the latter are the plastids that form a monophyletic lineage whose sister group could not be identified. Because of the observed polytomy, plastids, which in other analyses often group near the base of the cyanobacterial radiation (Nelissen et al. 1995; Urbach et al. 1998), cannot be assigned 360 Systematics such a position. In a similar study (but using a smaller taxon sampling and lacking plastids), Honda et al. (1999a) found seven lineages of cyanobacteria that basically conform to the lineages observed by Turner et al. (1999). That the three known genera of oxychlorobacteria ("prochlorophytes") are polyphyletic within the cyanobacterial radiation is supported by the finding that the chlorophyll alb-binding proteins from all three known prochlorophyte genera are closely related to cyanobacterial chlorophyll a-binding proteins and do not belong to the extended gene family encoding chlorophyll alb and chlorophyll alc light-harvesting proteins and are therefore the result of convergent evolution (La Roche et al. 1996). This does not rule out the possibility that chlorophyll band phycobilins may have been present together in the cyanobacterial ancestor of the plastids (Tomitani et al. 1999). With regard to pigments, two exciting new observations were recently made in cyanobacteria: the first refers to the oxychlorobacterium P. marinus, the smallest (0.5-0.7 ~m) and presumably most abundant photosynthetic organism on Earth (Partensky et al. 1999). Prochlorococcus uniquely contains di-vinyl chlorophylls a and b and, in some strains (e.g. CCMP1375), contains small amounts of a novel type (PEIII) of phycoerythrin that functions as a light-harvesting antenna (Hess et al. 1996; Lokstein et al. 1999). The genomic region containing the genes for PEIII was characterized and shown to encode three structural phycobiliproteins and at least three different polypeptides similar to cyanobacterial proteins involved in the linkage of the subunits or the synthesis and attachment of chromophore groups (Hess et al. 1999). The genome size of P. marin us CCMP1375 is significantly smaller (1.81 Mb) than those of all other cyanobacteria investigated so far, suggesting that P. marin us evolved from an ancestral cyanobacterium of the picoplanktonic Synechococcus group (Urbach et al. 1998) by reducing its cell and genome sizes and by recruiting a protein originally synthesized under conditions of iron depletion to construct a reduced antenna system to replace the large phycobilisomes (Partensky et al. 1999). In another twist to the discovery of novel cyanobacterial pigments, chlorophyll d, long regarded as an artifact produced by extraction of pigments from red algae, was recently identified as the major chlorophyll in a photosynthetic prokaryote squeezed out of Lissoclinum patella, a colonial ascidian from Palau (Miyashita et al. 1996). This is a curious analogy to the discovery of Prochloron 20 years earlier on the same Pacific island (Prog Bot 44:318). The organism, Acaryochloris marina, can be grown photoautotrophically with high oxygen-evolving activity when grown in far-red (712 nm) light (Miyachi et al. 1997; Miyashita et al. 1997). Trace amounts of the phycobilins phycocyanin and allophycocyanin were also found in this organism and were shown to be physically attached to photo system-II complexes (Schiller et al. 1997; Hu et al. 1999). Hu et al. (1998) demonstrated that chlorophyll d was present Systematics and Evolution of the Algae. 1. Genomics Meets Phylogeny 361 in the reaction centers of purified photosystem-I complexes and that it changed absorption after laser excitation at 740 nm (and hence was named P740). In photosystem II, chlorophyll d is a major antenna pigment (Mimuro et al. 1999,2000). It appears that the evolution of chlorophyll d as a reaction-center pigment in A. marina is another adaptation of cyanobacteria to unusual light conditions (far red). It remains to be established where A. marina belongs on the phylogenetic tree of the cyanobacteria. From studies of the molecular phylogeny of cyanobacteria, it may be concluded that, although plastids had a monophyletic origin within the cyanobacteria, their cyanobacterial sister group remains unknown. Only one recent study questioned the monophyletic origin of plastids (Valentin 1997), based on a phylogenetic analysis of SecA (a key polypeptide of the thylakoid translocation machinery). However, SecA is encoded in the nucleus in land plants and in the plastid genome in brown and red algae, and it was shown that the high AT content of the plastid sequences significantly affected the amino acid composition and the "polyphyly" between the "green" and "brown and red" protein is due to differences in nucleotide composition and the existence of covarion substitution (Barbrook et al. 1998). Three lineages of simple (two-envelope-membrane) plastids have been recognized: cyanelles (sometimes termed cyanoplasts), rhodoplasts and chloroplasts (Prog Bot 57:293). Previous phylogenetic analyses have supported the conclusion that the three lineages diverged early, with cyanelles (containing a peptidoglucan layer between the two envelope membranes) presumably the earliest divergence (Helmchen et al. 1995). These conclusions have been corroborated by additional single-gene phylogenetic analyses (Bhattacharya and Medlin 1995; Van de Peer et al. 1996; Bhattacharya and Schmidt 1997) and, more significantly, by plastid-genome phylogenies using concatenated protein sequences (11,039 amino-acid positions). Although the latter approach obviously suffers from insufficient taxon sampling, it has the advantage of using a much larger data set than single-gene phylogenies. It is concluded that both approaches will eventually converge on the same topologies, once the limitations imposed by poor taxon sampling or limited data sets are overcome and realistic models of sequence evolution are used in phylogenetic analyses (Lockhart et al. 1998). The analysis of gene clusters, particularly large ones, such as the ribosomal protein operons, have also been used to tracing phylogenetic relationships among plastids (Kowallik 1997; Ohta et al. 1997; Sugita et al. 1997; Wang et al. 1997; Leitsch et al. 1999; Stoebe and Kowallik 1999). However, a problem with using gene clusters (or indels, the number and insert positions of introns, or other sequence signatures) in phylogenetic analyses is that there is often insufficient taxon sampling. In addition, the data set is limited (each cluster essentially represents a single charac- 362 Systematics ter). This is exemplified by the SI2/SlO ribosomal gene cluster; a consensus of 12 eubacterial operons shows the SI2/SlO cluster to be linked to the 5' end ofthe L2 cluster, whereas a consensus of seven archaebacterial operons shows the two clusters to be split. In the single cyanobacterium Synechocystis PCC6803, the clusters are again split. In plastids, two arrangements can be observed; clusters are split in cyanelles and chloroplasts but, in rhodoplasts, the S12/S10 cluster is attached to the 3' end of the ribosomal gene cluster (Stoebe and Kowallik 1999). Nothing can be said regarding the ancestral state, because only one cyanobacterium was analyzed, and all other (eu)bacteria differ from the cyanobacterial arrangement. If we assume that the cyanobacterial arrangement is ancestral, the cyanelles and chloroplasts have retained this plesiomorphic character and, again, nothing can be said regarding their relationship. The rhodoplasts then have a derived (apomorphic) arrangement of the S12/S10 cluster, but whether the arrangement of the cluster in the plastid (apicoplast) of Plasmodium (which is identical to that of rhodoplasts) is indicative of a monophyletic origin of the two groups of plastids (rhodoplasts and apicoplasts) or represents a homoplasy can only be decided after a formal phylogenetic analysis involving many more characters. Simple plastids are thought to have evolved from the endocytobiosis of a cyanobacterium into a unicellular, colorless eukaryotic host equipped with a mitochondrion of the flat cristae-type. Initially, the endocytobiont must have been surrounded by four layers (from inside to outside): symbiont cytoplasmic membrane, peptidoglucan, lipopolysaccharide (outer) membrane and phagosomal (host) membrane. Except for cyanelles (see above), extant simple plastids are surrounded only by two envelope membranes. The origin of the inner membrane (which contains most of the translocator functions of the organelle) has never been in doubt; it derives from the cyanobacterial cytoplasmic (plasma) membrane. However, the origin of the outer envelope membrane has always been controversial; most biology textbooks refer to it as the former phagosomal membrane of the host. However, evidence of its lipid composition and the presence of pore-forming proteins has raised the possibility that it may have originated from the lipopolysaccharide membrane. Exciting new data obtained from studies of protein targeting to the plastid shed new light on this old question. Protein targeting to plastids has not only been studied in great detail in chloroplasts of higher plants (Heins et al. 1998; Soli and Tien 1998; Chen and Schnell 1999; Keegstra and Cline 1999; Keegstra and Froehlich 1999; McFadden 1999a), it is also beginning to be unraveled in complex plastids (Schwartzbach et al. 1998; McFadden 1999b). Nuclear-encoded proteins destined for the chloroplast are synthesized on cytosolic ribosomes with transit peptides that, together with cytosolic factors, target the pre-protein to the organellar surface. The protein-import machinery Systematics and Evolution of the Algae. 1. Genomics Meets Phylogeny 363 of chloroplasts differs in important aspects from that of other organelles. Both the outer and inner envelopes have their own translocation complexes, termed Toe (translocon at the outer membrane of chloroplasts) and Tic (translocon at the inner membrane of chloroplasts). The proteins that form these two complexes show no apparent sequence homologies to the components of protein translocases of other organelles. The Toc complex fulfills three essential functions during protein import. 1. It specifically recognizes the transit peptide. 2. After binding ofthe pre-protein, it initiates membrane translocation. 3. It participates in the formation of contact sites between the outer and inner membranes. The Toc complex contains three membrane proteins: Toc159, Toc34 and Toc75 (Keegstra and Froehlich 1999; May and SollI999). A homolog of Toc75 (SynToc75; 22% sequence identity) was identified in the Synechocystis PCC6803 genome (Bolter et al. 1998). Experimental evidence demonstrated that the cyanobacterial protein was present in the lipopolysaccharide membrane, and reconstitution of this protein in liposomes identified it as a voltage-gated, high-conductance channel with high affinity for polyamines and pep tides (Bolter et al. 1998). Reconstituted Toc75 also has channel activity (Hinnah et al. 1997). SynToc75 is an essential protein, and a homolog is also present in all gram-negative bacteria studied to date (Reumann et al. 1999). These findings suggest that Toc75 was recruited from a pre-existing channel-forming protein present in the lipopolysaccharide membrane of the cyanobacterial ancestor of plastids, indicating that the outer envelope membrane of chloroplasts (and perhaps other simple plastids) derived from the lipopolysaccharide membrane of the cyanobacterial symbiont (Bolter et al. 1998). Reumann et al. (1999) suggest that the relocation of the Toc75 gene resulted in a different (inside-out) insertion of Toc75 into the outer envelope membrane, causing it to function in reverse, i.e., it began importing instead of exporting proteins. These authors also propose that transit pep tides may derive from the original substrate of the cyanobacterial homolog of Toc75, an idea that can be tested via sequence analysis of the secretion substrate of SynToc75. More recently, Reumann and Keegstra (1999) presented evidence that Tic20 and Tic22 also have cyanobacterial homologs and concluded that the protein-import machinery of chloroplasts is mainly derived from the endocytobiotic cyanobacterium. Some components (Toe 159, Toc 34, TicllO), however, are not related to any Synechocystis proteins, suggesting that the import apparatus had at least two origins. Interestingly, none of the import proteins seems to be related to any of the four main protein secretion systems in Bacteria (Settles and Martienssen 1998). Plastid genome phylogenetics is still in its infancy. Too few complete sequences have been determined to date to address anything other than 364 Systematics basic phylogenetic questions. However, as automatic sequencing becomes ever more powerful, the number of completely sequenced plastid genomes is likely to increase significantly during the coming years, enhancing our understanding of the phylogeny and evolution of plastids and their hosts in a profound way. Some recently determined plastidgenome sequences include those of the green algae Chlorella vulgaris C27 (Wakasugi et al. 1997), Nephroselmis olivacea (Turmel et al. 1999) and Mesostigma viride (Lemieux et al. 2000), that of the glaucocystophyte C. paradoxa (UTEX555; L6ffelhardt et al. 1997) and that of the cryptophyte Guillardia theta (Douglas and Penny 1999). One of the hallmarks of plastid phylogeny has been the recognition that, although plastids presumably had a monophyletic origin, they entered eukaryotic hosts several times (the "second paradox of plastid evolution"; Prog Bot 57:288-289). One primary endocytobiosis was followed by several secondary endocytobioses in different protist lineages (Delwiche and Palmer 1997; McFadden and Gilson 1997; Douglas 1998; Palmer and Delwiche 1998; Delwiche 1999). A secondary endocytobiosis is defined as one in which both the symbiont and the host were eukaryotes; the plastid was acquired "second hand" from the symbiont (a eukaryotic alga), which obtained its plastid via the primary endocytobiotic event. The result of a secondary endocytobiotic event is a photosynthetic organelle surrounded by more than two envelope membranes, Le., a complex plastid. While this general scenario is widely accepted [for an alternative view, see Stiller and Hall (1997, 1998) and the discussion of their findings in Delwiche and Palmer (1997)], the question of how many secondary endocytobioses led to the evolution of complex plastids is still hotly debated. There is strong evidence that two different types of eukaryotic algae acted as symbionts in secondary endocytobioses, namely green and red algae. This evidence comes from phylogenetic analyses of single genes of plastids and, in two instances, of remnant symbiont nuclei (i.e., nucleomorphs, see below), and from complete plastid genomes and plastid gene clusters. Thus, the minimum number of secondary endocytobioses is two (Bhattacharya and Medlin 1995; Melkonian 1996; Palmer and Delwiche 1998). This has been most clearly shown in the cryptophytes and chlorarachniophytes, in which evidence in favor of an independent origin of their plastids was obtained by analyses of two different genomes (that of the plastid and that of the nucleomorph). Both data sets demonstrate a red-algal ancestry of the cryptophyte symbiont and a green-algal ancestry of the chlorarachniophyte symbiont (Van de Peer et al. 1996; Van de Peer and De Wachter 1997). The two host genomes have no specific relationship to each other or to the genomes of their respective symbionts. Whether there have been more than two separate endocytobioses is debatable, and the conclusions largely depend on how one interprets the significance of rRNA phylogenies and the likelihood of plastid gains or Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 365 losses. Most researchers interpret the incongruence in rRNA tree topologies between the plastid and nuclear phylogenies (Bhattacharya and Medlin 1995; Melkonian et al. 1995) as evidence for additional endocytobioses (Delwiche and Palmer 1997; Palmer and Delwiche 1998). The plastid phylogeny suggests that the plastids ofheterokonts, haptophytes, cryptophytes and perhaps the peridinin-containing dinoflagellates evolved from a red algal-like ancestor (Medlin et al. 1997; Durnford et al. 1999). The plastids of chlorarachniophytes and Euglenozoa derived from a green-algae-like ancestor (McFadden et al. 1995; Van de Peer et al. 1996; Ishida et al. 1997). In plastid phylogenies using different molecular markers (SSU rRNA, rbeL, tufA), the plastids of diverse heterokonts, haptophytes and cryptophytes, though clustering with or within the rhodoplast clade, did not form a monophyletic lineage to the exclusion of the rhodoplasts of red algae (Daugbjerg and Andersen 1997; Medlin et al. 1997). This argues that a single secondary endocytobiotic event did not lead to the complex plastids of these three lineages; however, the robustness of the observed topologies has not been adequately tested, e.g., by user-defined trees. Host gene phylogenies (mostly SSU rRNA, but also actin) have also not revealed a monophyletic lineage comprised ofheterokonts, haptophytes and cryptophytes. To the contrary, heterokonts group weakly with alveolates, to the exclusion of haptophytes and cryptophytes (the alveolate/heterokont relationship is better supported in LSU rRNA analyses; Van der Auwera and De Wachter 1998) and cryptophytes group weakly with glaucocystophytes (Bhattacharya et al. 1995; Medlin et al. 1997), although conflicting data have been presented using tubulin genes (Keeling et al. 1999); however, a sister group to haptophytes has not yet been identified. Similarly, chlorarachniophytes and euglenoids do not form a monophyletic lineage in plastid or nuclear phylogenies. Taken together no current phylogenies support a "two secondary endocytobioses only" scenario (as advocated by Cavalier-Smith 1999), although it is impossible to reject this hypothesis as long as the sequence of evolutionary divergence of the various "crown group" eukaryotes cannot be unequivocally established (which, according to the "big bang" hypothesis for eukaryote radiation, may never be possible; Philippe and Adoutte 1998). It can always be argued that some early-diverging common ancestor of a lineage that includes, e.g., the heterokonts, haptophytes, cryptophytes, alveolates and possibly other taxa, such as fungi and animals, obtained a plastid via secondary endocytobiosis of a red alga. However, note that this scenario requires that the red algae (and the Viridiplantae and glaucocystophytes, if plastids are of monophyletic origin) cannot belong to this assemblage; they must diverge earlier, because a secondary endocytobiosis requires a preceding primary endocytobiosis! Similarly, it may be argued that euglenoids and chlorarachniophytes (plus some other lineages) have a common ancestor that ob- 366 Systematics tained a plastid via secondary endocytobiosis of a green alga. Note that, according to the above scenario, such a lineage must exclude lineages with rhodoplast-type complex plastids and the Viridiplantae; the latter must be an earlier-diverging lineage (see above). What one sees in rRNA trees is, however, the opposite; the euglenoids diverged earlier than the Viridiplantae. While it may be argued that the basal position of the euglenoids in rRNA trees is artificial (i.e., caused by LBA, Stiller and Hall 1999), it is in accordance with the fact that all early-branching taxa within the kinetoplastid/Euglenozoa lineage are (primarily?) without plastids. In other words, the acquisition of a chloroplast was apparently a late event in the evolution of the kinetoplastid/Euglenozoa clade (Linton et al. 1999); hence, the Viridiplantae can diverge much later on the rRNA tree than the Euglenozoa can. Similar observations have been made in all other investigated lineages harboring complex plastids: in the heterokonts (in which several lineages of aplastidal protists diverged earlier than the photosynthetic heterokonts; Leipe et al. 1994; CavalierSmith and Chao 1996; Van de Peer and De Wachter 1997; Karpov et al. 1998; Guillou et al. 1999; Honda et al. 1999b), the cryptophytes [McFadden et al. 1994; Marin et al. 1998; Clay and Kugrens 1999; the apparently contradictory data of Cavalier-Smith et al. 1996 were shown to be due to insufficient taxon sampling (Marin et al. 1998)] and the dinoflagellates (Saunders et al. 1997; Gunderson et al. 1999). An early divergence of aplastidal taxa in lineages that also contain later-diverging taxa with complex plastids is usually explained by assuming a later acquisition of plastids through independent secondary endocytobioses in these lineages (Melkonian 1996; Delwiche and Palmer 1997). It has, however, been argued that it is "less difficult" to lose complex plastids several times than to gain them several times through secondary endocytobioses (Cavalier-Smith 1999). Is this true? To approach the question of the likelihood of multiple secondary endocytobioses, it might be useful to review the sequence of events that probably led to the establishment of complex plastids and the protein-import mechanisms that are used to target proteins into extant complex plastids (Schwartzbach et al. 1998; McFadden 1999b; Roos et al. 1999). Before dealing with complex plastids, it should be noted that no taxon in the glaucocystophytes, the red algae or the Viridiplantae (i.e., in the lineages of algae containing simple plastids) is known to lack plastids. Hosts have become so dependent on the many biosynthetic functions performed by proteins targeted to simple plastids that the plastid is not dispensable, even when photosynthesis is (as in many leucoplast-containing taxa). In a secondary endocytobiosis, the symbiont (a red or a green alga; see above) is as metabolically dependent on its plastid as the red and green algae are today (plastid phylogenies show that complex plastids originate within the rhodoplast and chloroplast lineages, respectively; see above). Thus, at the time of engulfment, the endocytobiont nucleus harbored numer- Systematics and Evolution of the Algae. 1. Genomics Meets Phylogeny 367 ous genes for plastid proteins, so the retention of the endocytobiont nucleus was presumably essential for the initial maintenance of the symbiotic association (Gilson et al. 1997). An analogous situation exists in extant symbiotic associations between dinoflagellates and cryptophytes, in which the "kleptochloroplasts" are only stable if the symbiont nucleus is not degraded (Schnepf 1993). Because the endocytobiont nucleus was presumably unable to reproduce sexually, Muller's ratchet was in operation (see above), leading to a second round of intracellular gene transfer, this time from the endocytobiont nucleus to the host nucleus (McFadden 1999b). Most researchers now favor this scenario over the "alternative scenario" of Hauber et al. (1994; Bodyl 1997), which states that the host of the secondary endocytobiotic event already contained a plastid (for a discussion of the problems with this hypothesis, see Melkonian 1996). The nucleomorph-genome projects (McFadden et al. 1997a) have failed to find a large number of genes coding for plastid proteins (Gilson and McFadden 1996; Zauner et al. 2000). Evidence for the nucleus-to-nucleus transfer of genes coding for plastid proteins (in this case, light-harvesting-complex proteins; LHCPs) in both cryptophytes and chlorarachniophytes is forthcoming (Deane et al. 2000). What happened to the genes that were transferred to the host nucleus? Initially, four membranes separated the plastid stroma from the host nucleus, the outermost representing the phagosomal membrane. The simplest solution for dispensing of such foreign proteins would be to target them to either the secretory pathway or the lysosomal pathway (Melkonian 1996). In fact, almost all genes for host nuclear-encoded plastid proteins cloned and sequenced to date (from all groups of algae with complex plastids) have a bipartite targeting-signal sequence that encodes an N-terminal endoplasmic reticulum (ER) signal peptide and a downstream plastid transit peptide (Schwartzbach et al. 1998; McFadden 1999b). As a consequence, in vitro studies have shown that such proteins are co-translationally processed in both heterologous and homologous systems (Bhaya and Grossman 1991; Kishore et al. 1993; Lang et al. 1998b; Waller et al. 1998). In Euglena, pulse-chase cellular fractionation studies (Sulli and Schwartzbach 1995) and immunogold localization (Osafune et al. 1991) have demonstrated that the (polyprotein) precursors of the chloroplast proteins LHCP-II and SSU Rubisco are transported as membrane bound intermediates from the ER to the Golgi apparatus prior to chloroplast localization. Why was the lysosomal pathway apparently favored over the secretory pathway for the "export" of plastid proteins whose genes were acquired by the host? Clues come from exciting recent work with Euglena (Sulli et al. 1999). Asparaginelinked glycosylation reporters and pre-sequence deletion constructs of the precursor to LHCP-II were used to identify pre-sequence regions translocated into the ER lumen and stop-transfer anchor domains. The authors demonstrated that the hydrophobic domain of the stromal- 368 Systematics targeting transit peptide functions as a stop-transfer domain during cotranslational insertion of the LHCP-II precursor into the ER, with most of the protein mass located in the cytosol. If all transit peptides of euglenoid plastid proteins could function as stop-transfer domains during cotranslational insertion into the ER, this would "save" soluble chloroplast proteins from being lost as secretory proteins to the extracellular environment. At the same time, it would spare the protein from proteolytic digestion in the lysosomal compartment (in dinoflagellates, however, precursors of nuclear-encoded plastid proteins may lack a membrane anchor; Sulli et al. 1999). In heterokonts, haptophytes and cryptophytes, the outer membrane of the four-membrane envelope surrounding the complex plastids bears ribosomes, and plastid proteins are co-translationally inserted into this chloroplast ER (CER; Gibbs 1979) en route to the plastid stroma (Lang et al. 1998b). Here, it is likely that plastid proteins are secreted into the ER lumen (they avoid the secretory pathway, because the CER completely engulfs the plastid). How proteins are transported through the third membrane in four-membrane plastids is unknown, as is the nature of that membrane. Usually, it is assumed to represent the former plasma membrane of the endocytobiont (Whatley et al. 1979). McFadden (1999b) noted that such a designation requires either a transport step "from outside to inside", which would be quite unusual, or an equally unlikely "porous" membrane (which is incompatible with the localization of proteins between the two inner and outer envelope membranes in cryptophytes and chlorarachniophytes). An alternative designation of the third membrane was proposed by Melkonian (1996), who suggested that both outer membranes may represent ER (a kind of autophagosomal engulfment by the ER, similar to what one can observe in extant bacterial endocytobionts of protists; Guillou et al. 1999). The exit of plastid proteins from the ER could be envisaged as reverse transport through the translocon, perhaps facilitated by improper folding of the protein. It is likely that the evolution of an ER-dependent targeting system for plastid proteins during secondary endocytobioses is the reason the additional membranes (of which there are one or two) cannot be lost without mis-targeting the plastid proteins. Is the acquisition of signal pep tides of many plastid proteins during nucleus-to-nucleus gene transfer an extremely rare event that could have occurred only twice? Signal peptides are relatively variable, in terms of consensus and size (Nielsen et al. 1997). Furthermore, several signal peptides of plastid proteins have boundaries located near introns, suggesting the possible acquisition of these targeting peptides via exon shuffling (Caron et al. 1996, Waller et al. 1998; McFadden 1999b). No new import machinery was needed to re-target plastid proteins to the organelle during secondary endocytobiosis (in contrast to the evolution of simple plastids during the primary endocytobiotic event), because the Systematics and Evolution of the Algae. I. Genomics Meets Phylogeny 369 pre-existing ER/phagosome targeting system of the host was available. Thus, there is no reason to suspect that secondary endocytobioses must have been extremely rare, as postulated by Cavalier-Smith (1999). In fact, the variability in the number of envelope membranes surrounding complex plastids (three or four), the extent to which remnants of the endocytobiont nucleus were retained (or not) and the different mechanisms by which proteins are targeted to the complex plastid (direct ER versus phagosomaI targeting; see above) argue that there were more than two independent secondary endocytobioses, each involving different endocytobionts and different hosts. Why have cryptophytes and chlorarachniophytes retained nucleomorphs, whereas no trace of the endocytobiont nucleus is left in the euglenoids, dinoflagellates, heterokonts and haptophytes? It is now clear that nucleomorphs encode at least some plastid proteins (Zauner et aI. 2000), which explains why they cannot be lost without impairing plastid function and stability. Why can't the remaining genes encoding plastid proteins be transferred to the host nucleus? Schwartzbach et al. (1998) have suggested that the genes are locked in the nucleomorph because, during the miniaturization of this genome (leading to the "bonsai" condition; McFadden et al. 1997a), the nucleomorph developed introns of such a small size (Gilson and McFadden 1996) that the host nuclear splicing apparatus could no longer cope with them (a specialized splicing apparatus is apparently present in the nucleomorphs; Gilson and McFadden 1996). It is here suggested that, in the euglenoids, heterokonts, haptophytes and peridinin-containing dinoflagellates, the endocytobiont nucleus became dispensable once all the genes encoding plastid proteins necessary for the function and stability of the plastid had been transferred from the endocytobiont nucleus to the host nucleus and had acquired signal peptides. The reason(s) the nucleomorphs apparently escaped Muller's ratchet subsequent to their genetic reduction are obscure (do they engage in recombination?). How likely is it that complex plastids have been lost many times during evolution? Let us assume that all or almost all (in the case of nucleomorphs) genes encoding plastid proteins in an endocytobiont nucleus were transferred to a host nucleus following a secondary endocytobiotic event. Furthermore, suppose the nucleomorphs were retained only because, for unknown reasons, a few plastid proteins could not be retargeted from the host nucleus to the plastid. In that case, we may conclude that plastid losses are almost as unlikely in organisms containing complex plastids as they are in organisms containing simple plastids, because the host became as dependent on plastid functions as the endocytobiont was. This is exemplified in the apicomplexan plastid (the apicoplast) which, despite its miniature size (35 kb in Plasmodium falciparum), is present in many (perhaps all) apicomplexans (McFadden et al. 1997b; Denny et aI. 1998). Interestingly, it has not been found in the 370 Systematics Perkinsozoa, the putative sister group of the Apicomplexa (Noren et al. 1999). This suggests that the apicoplast is the result of yet another secondary endocytobiosis independent of that which gave rise to the peridinin-containing complex plastid of the related dinoflagellates. The apicoplast has recently attracted much attention, because it is the likely site of several "prokaryotic" biosynthetic pathways (the genes of which are encoded in the apicomplexan nucleus). These pathways are amenable to drugs and, thus, could be useful in malaria prophylaxis and therapy (Fichera and Roos 1997; McFadden and Waller 1997; Waller et al. 1998; McFadden and Roos 1999; Roos et al. 1999). A similar dependence on the presence of complex plastids can be seen in algae that have dispensed with phagotrophy, e.g., the photosynthetic euglenoids (the colorless taxa with photosynthetic sister groups that have been studied in any detail all have leucoplasts) or the cryptophytes (the colorless Chilomonas, which is sister to a Cryptomonas species has retained leucoplast, nucleomorph and starch accumulation). It may be assumed that, in algae that have retained phagotrophy, the metabolic dependence on complex plastids may not have been as strict. 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