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Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki 28 / 2007 Lignin biosynthesis in Norway spruce: from a model system to the tree Sanna Koutaniemi Department of Applied Biology Faculty of Agriculture and Forestry Department of Biological and Environmental Sciences Division of Plant Biology Faculty of Biosciences Finnish Graduate School in Plant Biology University of Helsinki Finland Academic dissertation To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public criticism, in Viikki, Auditorium B2 (Forest Sciences building, Latokartanonkaari 9), on December 14th 2007, at 12 o'clock noon. Supervised by Professor Teemu Teeri Department of Applied Biology University of Helsinki Reviewed by Professor Hely Häggman Department of Biology University of Oulu Docent Markku Keinänen Faculty of Biosciences University of Joensuu Opponent Doctor Deborah Goffner Centre National de la Recherche Scientifique Université Paul Sabatier Tolouse, France Custos Professor Jaakko Kangasjärvi Department of Biological and Environmental Sciences University of Helsinki Cover: Norway spruces in Viikki in November 2004, after the first snowfall. ISSN 1795-7079 ISBN 978-952-10-4291-1 (paperback) ISBN 978-952-10-4292-8 (pdf) http://ethesis.helsinki.fi Helsinki University Printing House Helsinki 2007 This page intentionally left blank CONTENTS ABBREVIATIONS ORIGINAL PUBLICATIONS ABSTRACT 1 INTRODUCTION .........................................................................................................................1 2 REVIEW OF THE LITERATURE..................................................................................................3 2.1 Timing of lignification .............................................................................................................3 2.2 Composition and structure of lignin ......................................................................................3 2.2.1 Lignin in reaction wood.......................................................................................................4 2.3 Monolignol biosynthesis ........................................................................................................6 2.3.1 Monolignol biosynthetic enzymes .......................................................................................6 2.3.2 Biosynthesis of ferulic and sinapic acids...........................................................................10 2.3.3 Arabidopsis and Populus ‘ lignification toolboxes’..............................................................10 2.3.4 Functional divergence of monolignol biosynthetic genes...................................................12 2.3.5 Control of lignin biosynthesis ............................................................................................13 2.3.6 Effects of the genetic down-regulation of monolignol biosynthetic genes...........................17 2.4 Storage and transport of the monolignols into the apoplast ..............................................21 2.5 Polymerisation of monolignols ............................................................................................22 2.5.1 Dirigent-like proteins and template polymerisation ............................................................24 2.5.2 Combinatorial chemical coupling model............................................................................24 2.5.3 Initiation sites for lignin polymerisation..............................................................................25 2.6 Enzymes for the formation of monolignol radicals .............................................................26 2.6.1 Peroxidases .....................................................................................................................26 2.6.2 H2O2 producing enzymes .................................................................................................29 2.6.3 Laccases .........................................................................................................................30 2.6.4 Other phenoloxidases ......................................................................................................31 2.6.5 Redox shuttles .................................................................................................................31 2.7 Tissue cultures as models for lignin biosynthesis..............................................................32 2.7.1 Xylogenic Zinnia elegans tissue culture ............................................................................32 3 AIMS OF THE STUDY ...............................................................................................................34 4 MATERIALS AND METHODS ...................................................................................................35 5 RESULTS AND DISCUSSION...................................................................................................36 5.1 The localisation and role of dibenzodioxocin structures in lignin (II).................................36 5.2 Lignin polymerisation in the Norway spruce tissue culture (I, III) ......................................37 5.2.1 Peroxidase activity is needed for lignin formation (I) .........................................................37 5.2.2 Coniferin -glucosidase activity in the culture medium (I)..................................................38 5.2.3 NADH oxidase activity in the culture medium (I) ...............................................................38 5.2.4 Functional characterisation of the secreted peroxidases (III) and laccase .........................39 5.2.5 The cell wall matrix and other regulatory factors contribute to the structural outcome of lignin polymerisation (III) ...........................................................................................................43 5.2.6 Lignin-bound peroxidases and laccases ...........................................................................44 5.3 Norway spruce EST collection (IV).......................................................................................45 5.3.1 Monolignol biosynthetic genes and peroxidase/laccase genes..........................................46 5.3.2 Genes of potential H2O2 producing enzymes ....................................................................47 5.4 Expression profiling of lignin biosynthetic genes using real-time RT-PCR (IV) ................47 5.4.1 Coordinated, high expression in developing xylem samples as a marker of lignin biosynthetic genes ....................................................................................................................48 5.4.2 Up-regulation of lignin biosynthetic gene expression under stress.....................................50 5.4.3 Lignin biosynthetic gene expression in roots, phloem and needles....................................51 5.4.4 Lignin biosynthesis in the A3/85 tissue culture line - true native spruce lignin?..................51 6 CONCLUDING REMARKS ........................................................................................................53 ACKNOWLEDGEMENTS.............................................................................................................55 REFERENCES .............................................................................................................................57 ABBREVIATIONS 4CL ABC ADHG AEOMT ATP ATPA2 C3H C4H CA CAD CCOMT CCR CHS CoA COMT DHP ELISA EST F5H G GA H HCT HRP IEF MW MWL NAD(P)H NMR 4-NPG OMT PAL pCA pI RACE RSCL RT-PCR S SA SAD SAM SDS-PAGE SOD TAL UDP 4-coumarate:CoA ligase ATP-binding cassette aldehyde dehydrogenase acid/ester O-methyltransferase adenosine triphosphate Arabidopsis thaliana peroxidase A2 p-coumarate 3-hydroxylase cinnamate 4-hydroxylase coniferyl alcohol cinnamyl alcohol dehydrogenase caffeoyl-CoA O-methyltransferase cinnamoyl-CoA reductase chalcone synthase coenzyme A caffeate/5-hydroxyconiferaldehyde O-methyltransferase dehydrogenation polymer enzyme-linked immunosorbent assay expressed sequence tag ferulate/coniferaldehyde 5-hydroxylase guaiacyl gibberellin p-hydroxyphenyl hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase horseradish peroxidase isoelectric focusing molecular weight milled wood lignin nicotinamide adenine dinucleotide (phosphate) nuclear magnetic resonance 4-nitrophenyl glucoside O-methyltransferase phenylalanine ammonia-lyase p-coumaryl alcohol isoelectric point rapid amplification of cDNA ends released suspension culture lignin reverse transcriptase - polymerase chain reaction syringyl sinapyl alcohol sinapaldehyde dehydrogenase S-adenosyl methionine sodium dodecyl sulphate - polyacrylamide gel electrophoresis superoxide dismutase tyrosine ammonia-lyase uridine 5’-diphosphate ORIGINAL PUBLICATIONS This thesis is based on the following four articles. They are referred to in the text by their Roman numerals. In addition, some unpublished data is included. I Kärkönen A, Koutaniemi S, Mustonen M, Syrjänen K, Brunow G, Kilpeläinen I, Teeri TH, Simola LK (2002) Lignification related enzymes in Picea abies suspension cultures. Physiologia Plantarum 114: 343-353 II Kukkola EM, Koutaniemi S, Gustafsson M, Karhunen P, Ruel K, Lundell TK, Saranpää P, Brunow G, Teeri TH, Fagerstedt KV (2003) Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopyimmunogold labeling. Planta 217: 229-237 III Koutaniemi S, Toikka MM, Kärkönen A, Mustonen M, Lundell T, Simola LK, Kilpeläinen IA, Teeri TH (2005) Characterization of basic p-coumaryl and coniferyl alcohol oxidising peroxidases from a lignin-forming Picea abies suspension culture. Plant Molecular Biology 58: 141-157 IV Koutaniemi S, Warinowski T, Kärkönen A, Alatalo E, Fossdal CG, Saranpää P, Laakso T, Fagerstedt KV, Simola LK, Paulin L, Rudd S, Teeri TH (2007) Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RTPCR. Plant Molecular Biology 65: 311-328 The articles are reprinted with a kind permission from the copyright holders, Blackwell Publishing (I) and Springer Science and Business Media (II to IV). ABSTRACT Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the ligninforming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the upregulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses. 1 INTRODUCTION The evolution of land plants and their ability to colonise the earth required a mechanism that allowed the transport of water and nutrients from the roots into the aerial parts. This was achieved by the evolution of water-conducting elements, which are hollow, interconnected cells that form a continuum from the roots to the leaves and to the shoot apex. These water-conducting cells contain a thick secondary cell wall that mainly consists of parallel chains of cellulose, which are organised through hydrogen bonding to microfibrils and further lamellar structures. In addition to cellulose, hemicelluloses are also incorporated. To allow water conduction through the cells, the hydrophilic carbohydrates are incrusted with lignin, which makes the cell walls hydrophobic and impermeable to water. It also gives compressive strength to the vascular elements to withstand the negative pressure that is thought to be generated by transpiration and the cohesive movement of water along the vascular elements (Steudle, 2001). Compressive strength is also needed to support the weight of the stem and the crown, especially in trees. Therefore, lignin is indispensable to land plants. In addition to the vascular elements, a few other cell types, including the supportgiving fibres and sclereids, are also lignified in plants (Esau, 1960). Lignified fibres are long cells, in which the lumen is nearly filled with secondary cell wall. They are found, for example, between the vascular elements in angiosperm xylem, where they support the upright growth habit. Sclereids also contain a secondary cell wall, but they are smaller in size than fibres, and they are found in, for example, the protective layers of the seeds and stem in some species. Lignin also functions as a defence barrier against invading pathogens, and can be formed in response to wounding. On the other hand, lignin hinders the utilisation of the cellulosic cell walls of plants as forage or in pulp and paper industry, and thus has a tremendous economical impact. Occurrence of lignin in the plant kingdom is restricted to vascular plants. It is found from pteridophytes and higher plants but not from bryophytes, although some phenylpropanoid metabolites have been detected in mosses, too (Lewis and Yamamoto, 1990). Majority of lignin is synthesised in trees, in which most of the secondary xylem is lignified. In gymnosperms, the xylem consists of lignifying tracheids, which act as both water-conducting and supporting structures, and ray parenchyma that is responsible for horizontal transport of nutrients (Figure 1A; Esau, 1960). Ray tracheids are associated with ray parenchyma, which can also have a lignified secondary cell wall. In angiosperm xylem, the water-conducting cells are the large vessel elements that contain secondary thickenings, as well as the less abundant tracheids, whereas fibres act as supporting structures (Figure 1B). Both axial and ray parenchyma exist in angiosperms (Esau, 1960). Lignin biosynthesis is a part of the phenylpropanoid metabolism, which includes also the diverse flavonoids, coumarins, suberin and lignans. The biosynthetic pathways to these compounds are partly shared, which has complicated our view on lignin biosynthesis. The research on lignin biosynthesis has focused on a few model species, which include both trees and herbaceous plants, traditionally, for example, tobacco (Nicotiana tabacum L.). Of the tree species, poplars are commonly used due to the available genomic sequence from black cottonwood (Populus trichocarpa Torr. & Gray) (Tuskan et al., 2006) and to the relative ease of the generation of transgenic plants and 1 clonal material. Species of Eucalyptus and Pinus have also been utilised. The herbaceous model plant Arabidopsis thaliana (L.) Heynh. is widely used for studies on both secondary cell wall development and lignin biosynthesis (Nieminen et al., 2004; Groover, 2005). In addition to the lignified vascular elements, the Arabidopsis inflorescence stem contains interfascicular fibres that have thickened secondary cell walls with both G and S lignin (Figure 1C; Chapple et al., 1992; Ehlting et al., 2005). Moreover, under certain conditions, the hypocotyl, inflorescence stem and roots can undergo secondary growth (reviewed in Nieminen et al., 2004). The next chapters will describe the existing knowledge on the lignin polymer and its biosynthesis, including the synthesis of the precursors, their transport into the apoplast and polymerisation into lignin. The current views on the regulation of the lignification process will also be discussed. Figure 1 Cross-sections of (A) Norway spruce (Picea abies; gymnosperm) xylem, (B) silver birch (Betula pendula; angiosperm) xylem and (C) Arabidopsis thaliana inflorescence stem. (D) Ultrastructure of a gymnosperm tracheid, adapted from Cote (1967). C, cambium; T, tracheid; R, ray parenchyma; P, phloem; V, vessel element; F, fibre; IF; interfascicular fibre; SE, phloem sieve element; X, xylem element. The pictures are reprinted with a kind permission from Kurt Fagerstedt (A,B), Martin Bonke (C) and Pekka Saranpää (D). 2 2 REVIEW OF THE LITERATURE 2.1 Timing of lignification Lignification takes place during the growing season. In tree species, the extensive secondary xylem is formed by the vascular cambium, which is an undifferentiated layer of cells, equivalent to the shoot apical meristem (Groover, 2005). It surrounds the stem and produces xylem inwards and phloem outwards as the cambial cells divide. This division is followed by the differentiation process, including cell expansion and elongation through the turgor-driven expansion of the wall and the deposition of new primary cell wall material, mainly pectin, hemicelluloses and cellulose. After the final dimensions of the cell have been reached, the deposition of the secondary cell wall begins, followed closely by lignification. In the final phase, the cells undergo programmed cell death (Plomion et al., 2001). In the Finnish gymnosperm species, Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.), secondary growth and lignification take place between late May and August. In deciduous trees, such as silver birch (Betula pendula Roth.), this occurs in a shorter timeframe, in June and July (Marjamaa et al., 2003), allowing time for the formation of photosynthesising new leaves in the spring, and for the recycling of the leaf nutrients towards the autumn before the dormant season. The formation of a thick secondary cell wall is typical for all lignifying cell types. The wall usually contains three layers, S1 to S3, in which the cellulose microfibril angle changes relative to the longitudinal cell axis (Figure 1D), resulting in increased tensile strength in multiple dimensions (Donaldson, 2001; Plomion et al., 2001). Lignification starts already during the cellulose synthesis, beginning from the cell corners and middle lamella, and proceeding towards the lumen (Terashima et al., 1988). This first stage begins after the start of S1 formation. During the second, slow stage, lignin deposition into the S2 layer is started while polysaccharide deposition still continues. The main lignification takes place after the formation of the S3 layer (Terashima et al., 1988). During lignification, the water-filled pores between the cellulose microfibrils are filled with lignin, which replaces water and is thought to form covalent linkages with the carbohydrates. In the mature wall, the secondary cell wall contains up to 80% of the total lignin because of its greater volume. However, the lignin concentration is highest in the middle lamella, up to 85% of weight (Donaldson, 2001). 2.2 Composition and structure of lignin Lignin is a hydrophobic polymer that consists of three hydroxycinnamyl alcohols, pcoumaryl, coniferyl and sinapyl alcohol (pCA, CA and SA, respectively), which differ in their methoxylation degree on the aromatic ring (Figure 2A). They give rise to phydroxyphenyl (H), guaiacyl (G) and syringyl (S) units in lignin. Gymnosperm lignin is composed mainly of G units, whereas in angiosperm lignin both G and S units are incorporated. H units are a minor component in both gymnosperm and angiosperm 3 lignin, while lignin in monocotyledons has relatively more H units. These hydroxycinnamyl alcohols are also found in pteridophyte lignin, but the composition varies between species (Lewis and Yamamoto, 1990). Other phenolic subunits can also be incorporated to lignin. For example, p-coumaric and ferulic acids that are incorporated in grass cell walls through ester and ether linkages (Carpita, 1996), can also be esterified to lignin, and could thus function as bridges between carbohydrates and lignin (Iiyama et al., 1990; Ralph et al., 1995). In poplar lignin, esters of p-hydroxybenzoate have been observed (Meyermans et al., 2000), and dihydroconiferyl alcohol is incorporated in softwood lignins (Ralph et al., 2004) (Figure 2A). The monomer composition of lignin varies also at cellular level. For example, in Betula papyrifera (Marsh.) and Arabidopsis, lignin in the vessel walls is mainly G lignin, while in the supporting fibres and the ray parenchyma, both G and S units are incorporated (Fergus and Goring, 1970; Chapple et al., 1992). However, variation has been observed among angiosperms (Donaldson, 2001). Within the xylem cells, H units are found mainly in the compound middle lamella and cell corners in both softwoods and hardwoods (Whiting and Goring, 1982; Terashima and Fukushima, 1988). Within the angiosperm secondary cell wall, G units are incorporated throughout the lignification process, whereas S units are incorporated only in the later phases (Terashima et al., 1986). When the precursors are polymerised to form lignin, covalent C-C (condensed) and C-O (non-condensed) linkages are formed between the monomers. The main substructures in lignin are -O-4 (aryl ether), -5 (phenyl coumaran), 5-5-O-4 (dibenzodioxocin) and (pinoresinol) (Figure 2B; Lewis et al., 1999). -O-4 is the most abundant structure in wood lignin, representing over 50% of the linkages. The chemical properties of lignin are in part defined by the nature of these linkages: the condensed C-C structures, such as 5-5 and -5, are more difficult to degrade, for example during pulping, than the non-condensed C-O structures like -O-4. Linkage pattern also affects the three-dimensional structure of the lignin polymer, as -O-4 structures tend to form elongated chains, in which the aromatic rings lie in the same plane with each other and the lamellar microfibrils (Donaldson, 2001). The monomer composition also affects the linkage pattern. For example, the 5-position in SA is methoxylated and cannot take part in linkage formation. Therefore, the proportion of non-condensed bonds increases with increasing lignin methoxylation. The proportion of non-condensed substructures also increased towards the later stages of lignification and cell wall maturity, e.g., in pine and in growing maize (Zea mays L.) internodes (Terashima and Fukushima, 1988; Joseleau and Ruel, 1997). It therefore appears that increased methoxylation is typical for later developmental stages. 2.2.1 Lignin in reaction wood Reaction wood is formed in response to gravitational stimuli in both gymnosperms and woody angiosperms. It is formed on a large scale when the shoot is inclined, and functions to regain the vertical growth position (Timell, 1986; Mellerowicz et al., 2001). However, even vertically grown wood contains some reaction wood, as it is also formed 4 Figure 2 (A) The structures of the three traditional lignin precursors p-coumaryl, coniferyl and sinapyl alcohol, and dihydroconiferyl alcohol and p-hydroxybenzoic acid that are incorporated to a lesser extent into lignin in gymnosperm and Populus species, respectively. (B) The main lignin substructures. to support branches and, e.g., in response to wind (Mellerowicz et al., 2001). The mechanism of reaction wood formation differs between softwoods and hardwoods, although in both it is characterised by an eccentric growth of the stem, which is induced by ethylene (Nelson and Hillis, 1978; Little and Eklund, 1999). Reaction wood in gymnosperms is called compression wood. It is formed on the underside of the inclined stem and is darker-coloured. It is anatomically characterised by rounder cells and extensive intercellular spaces. In chemical terms, the total lignin content and the proportion of H units and condensed linkages are higher in compression wood than in vertically grown wood (Timell, 1986). Accordingly, expression or protein levels of lignin biosynthetic genes/enzymes and the ethylenesynthesising ACC oxidase were increased during the first days of compression wood formation (McDougall, 2000; Plomion et al., 2000; Bedon et al., 2007). On the contrary, reaction wood in angiosperms is formed on the upper side of the inclined stem and branches, and it is called tension wood. It is thought to contract longitudinally to pull the stem back to the vertical position (Mellerowicz et al., 2001; Pilate et al., 2004). In some species, the fibre S3 and sometimes also the S2 layers are missing; instead they are replaced by a gelatinous G-layer that consists of highly crystalline cellulose (Timell, 1969; Pilate et al., 2004). The G-layer in tension wood is 5 mainly devoid of lignin, and less hemicelluloses have also been found compared with normal wood (Timell, 1969; Pilate et al., 2004). In concordance with the low lignin levels, transcription of lignin biosynthetic genes was down-regulated during tension wood formation in hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) (Andersson-Gunnerås et al., 2006). 2.3 Monolignol biosynthesis The hydroxycinnamyl alcohol precursors of lignin are collectively known as monolignols. They are synthesised through the phenylpropanoid pathway (Figure 3). It is connected to the primary metabolism through the shikimate/chorismate pathway that produces phenylalanine, which is the entry compound of the carbon skeletons into the phenylpropanoid metabolism (Lewis et al., 1999). As the lignin precursors are extensively methylated through S-adenosyl methionine (SAM)-dependent reactions, the biosynthesis must be coordinated with the methionine cycle. Also ATP and reducing power in the form of NADPH are consumed during biosynthesis (Lewis et al., 1999). The pathway involves a series of hydroxylations, methylations and reductions. Originally, the hydroxylations and methylations were thought to take place at the level of hydroxycinnamic acids, which would then be reduced to the corresponding alcohols through coenzyme A (CoA) intermediates. The ability of many of the enzymes to oxidise various substrates along the pathway has blurred the picture; however, according to the current view, the main monolignol biosynthetic pathway is diverted from p-coumarate to p-coumaroyl-CoA, from which the CA and SA biosynthesis branches off (Figure 3; Schoch et al., 2001; Hoffmann et al., 2004). However, experimental evidence for this exists only for a few species and exceptions have been found (see below). As many enzymes of the pathway are encoded for by small gene families, spatial and temporal association with lignification have been used as markers for the involvement of a specific gene/enzyme in lignin biosynthesis. Most enzymes utilise nearly the whole range of, e.g., acid or CoA ester intermediates in the pathway, but typically, Km values in the low µM range (1-10 µM) have been observed for the main substrates (Lewis et al., 1999). 2.3.1 Monolignol biosynthetic enzymes PAL, C4H and 4CL The first step of monolignol biosynthesis is the non-oxidative deamination of Lphenylalanine to yield trans-cinnamic acid and an ammonium ion, which is recycled for phenylalanine biosynthesis. The reaction is catalysed by phenylalanine ammonialyase (PAL; Koukol and Conn, 1961). In addition to PAL, some monocotyledons have 6 7 another entry point into the pathway. Tyrosine can be converted into p-coumaric acid through tyrosine ammonia-lyase (TAL) in a reaction analogous to PAL, bypassing the cinnamic acid hydroxylation (Neish, 1961). Cinnamic acid is hydroxylated to p-coumaric acid by cinnamic acid 4-hydroxylase (C4H), which is a heme-containing cytochrome P450 enzyme (Russell and Conn, 1967; Potts et al., 1974). The enzyme is a monooxygenase that reduces O2 into water while transferring the other oxygen to the hydroxylate-receiving cinnamic acid. The electrons needed for the reduction are supplied by another enzyme, NADPH:cytochrome P450 reductase (Benveniste et al., 1986; Urban et al., 1997). Activation of p-coumaric acid to the corresponding CoA ester is catalysed by 4coumaric acid:CoA ligase (4CL) using the energy provided by ATP hydrolysis (Walton and Butt, 1970). In kinetic studies with purified or recombinant 4CL proteins from, e.g., spruce, pine and Arabidopsis, most enzymes were able to use p-coumaric and ferulic acids, and even caffeic and 5-hydroxyferulic acids (Lüdertiz et al., 1982; Voo et al., 1995; Hamberger and Hahlbrock, 2004). Sinapic acid was utilised by only a few enzymes from, e.g., soybean (Glycine max (L.) Merr.) and Arabidopsis (Lindermayr et al., 2002; Hamberger and Hahlbrock, 2004). However, no change in lignin was observed in the corresponding Arabidopsis knockout mutant (Costa et al., 2005), casting doubt on the participation of sinapic acid in lignin biosynthesis. Mixed-substrate assays in aspen (Populus tremuloides Michx.) have shown that the utilisation of certain substrates by 4CL isoforms can be inhibited by other substrates (Harding et al., 2002). C3H, HCT, CCOMT and CCR The enzyme catalysing the 3-hydroxylation was identified as a cytochrome P450 in Arabidopsis (Schoch et al., 2001; Franke et al., 2002b; Nair et al., 2002). The key finding was, however, that the reaction took place at the ester level, after the CoA moiety of pcoumaroyl CoA was exchanged to shikimic or quinic acid (Heller and Kühnl, 1985; Schoch et al., 2001), or to a methyl group (Franke et al., 2002b). The Arabidopsis C3H preferred p-coumaroyl shikimate over the corresponding quinate as a substrate (Schoch et al., 2001). p-Coumaric acid was also hydroxylated, albeit at a slow rate (Franke et al., 2002b; Nair et al., 2002). However, differences and alternative routes for 3hydroxylation apparently exist, for example in alfalfa (Medicago sativa L.) and Arabidopsis (Guo et al., 2001; Abdulrazzak et al., 2006; Chen et al., 2006) The enzyme catalysing the formation of the shikimate/quinate ester is hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT; Rhodes et al., 1979; Ulbrich and Zenk, 1980; Hoffmann et al., 2003). The same enzyme catalyses also the reverse reaction after 3-hydroxylation has taken place, releasing caffeoyl-CoA. Either shikimic or quinic acids, or both, can be utilised in the reaction, depending on species. Accumulation of shikimate esters is rare, and it was considered a transient intermediate (Ulbrich and Zenk, 1980; Schoch et al., 2001). However, caffeoyl quinate (chlorogenic acid) is widespread in the plant kingdom (Hoffmann et al., 2003). The 3-methylation of caffeoyl-CoA to feruloyl-CoA is catalysed by caffeoyl-CoA Omethyltransferase (CCOMT). It uses SAM as the methyl donor, and acts solely on CoA esters. The activity was first found in elicited parsley suspension cultures (Pakusch et 8 al., 1989), and it was linked to lignin biosynthesis in differentiating tracheary elements of Zinnia elegans (Jacq.) (Ye et al., 1994) and in transgenic tobacco (Zhong et al., 1998). The four-electron reduction of p-coumaroyl-CoA and feruloyl-CoA to the corresponding aldehyde is catalysed by an NADHP-dependent cinnamoyl-CoA reductase (CCR; Gross et al., 1973). CCR enzymes from various species have shown high affinity to feruloyl-CoA but often lower or negligible affinity to p-coumaroyl-CoA (Lüderitz and Grisebach, 1981; Goffner et al., 1994; Li et al., 2005; Patten et al., 2005). Sinapoyl-CoA was also reduced in vitro, even with gymnosperm enzymes (Lüderitz and Grisebach, 1981). F5H and COMT Biosynthesis of SA is diverted from coniferaldehyde by a 5-hydroxylation reaction. It is catalysed by ferulate-5-hydroxylase (F5H), another cytochrome P450 enzyme (Grand, 1984; Meyer et al., 1996). The preferred substrate was later shown to be coniferaldehyde or CA instead of ferulic acid or its CoA ester (Chen et al., 1999; Humphreys et al., 1999; Osakabe et al., 1999); however, Km values for ferulic acid in the µM range have also been reported for F5H in poplar (Grand, 1984). Coniferaldehyde was also shown to inhibit the hydroxylation of ferulic acid in sweetgum (Liquidambar styraciflua L.), eliminating the route to sinapic acid in vivo (Osakabe et al., 1999). Methylation of the 5-position in 5-hydroxyconiferaldehyde is catalysed by caffeic acid/5-hydroxyconiferaldehyde O-methyltransferase (COMT) that uses SAM as the methyl group donor like CCOMT. Although COMT was initially shown to catalyse the methylation of both caffeic acid and 5-hydroxyferulic acid, it was later shown to be specifically required for S unit biosynthesis (Atanassova et al., 1995; Doorsselaere et al., 1995). Studies in sweetgum and aspen proved that the preferred substrate is in fact 5hydroxyconiferaldehyde, with a 5-fold difference to 5-hydroxyferulic acid (Osakabe et al., 1999; Li et al., 2000). For COMT also, 5-hydroxyconiferaldehyde competitively inhibited the methylation of 5-hydroxyferulic acid and caffeic acid in mixed-substrate assays (Li et al., 2000). Interestingly, a multifunctional OMT that is capable of methylating both caffeic and 5-hydroxyferulic acids and the corresponding CoA esters (acid/ester OMT; AEOMT) was cloned from loblolly pine (Pinus taeda L.) and tobacco (Li et al., 1997; Maury et al., 1999). The pine sequence was ca. 60% similar to angiosperm COMTs but had much lower sequence similarity to CCOMT genes. Based on this, the existence of dual methylation pathways, operating both at the acid and CoA ester level was proposed. The AEOMT promoter from radiata pine (Pinus radiata D. Don) directed the expression of a reporter gene into various lignifying cell types both in pine and tobacco (Moyle et al., 2002). CAD The final reduction of the monolignol aldehydes to alcohols is catalysed by an NADPHdependent cinnamyl alcohol dehydrogenase (CAD; Gross et al., 1973; Mansell et al., 9 1974). Most CAD enzymes are capable of reducing all three hydroxycinnamaldehydes, and even caffeoyl aldehyde and 5-hydroxyconiferaldehyde (Lüderitz and Grisebach, 1981; Kim et al., 2004). Also a sinapaldehyde-specific dehydrogenase, SAD, was found in aspen (Li et al., 2001). However, this has been questioned later (Anterola and Lewis, 2002), and no specific requirement for SAD has been found (Kim et al., 2004). Moreover, the aspen SAD only partly complemented the amount of S and G units in CAD-deficient Arabidopsis (Sibout et al., 2005). 2.3.2 Biosynthesis of ferulic and sinapic acids Ferulic and p-coumaric acids are esterified into the primary walls of grasses and of dicotyledonous Chenopodiaceae, and they are potentially cross-linked to lignin during lignification (Ralph et al., 1995; Carpita, 1996; Boudet, 2003). Esters of sinapic acid accumulate in Brassicaceae family, where they act as UV-absorbing components (Chapple et al., 1992). p-Coumaric acid is a component of the core monolignol biosynthetic pathway (Figure 3), but ferulic and sinapic acids fall outside of the current model. There is evidence that at least sinapic acid is synthesised through the monolignol pathway. For example, the levels of both sinapoylmalate and S units in lignin were reduced in an Arabidopsis knock-out mutant of the COMT1 gene (Goujon et al., 2003b). Instead, 5-hydroxyferuloyl malate and 5-hydroxyferuloyl glucose accumulated. Nair et al (2004) demonstrated, using an Arabidopsis reduced epidermal fluorescence 1 (ref1) mutant, that ferulic and sinapic acids are synthesised from coniferaldehyde and sinapaldehyde through direct oxidation by an aldehyde dehydrogenase (ADHG; Figure 3). However, low levels of wall-bound ferulic acid and sinapate esters remained in the ref1 mutant, indicating that an alternative route to hydroxycinnamic acids also exists. Amino acid sequences similar to REF1 gene product were also found from monocotyledons, suggesting that ferulate in grass cell walls is synthesised through a similar pathway (Nair et al., 2004). Alternative routes to ferulate in monocotyledons, and possibly sinapate in Brassicaceae, could involve a direct acylation from feruloylCoA (Yoshida-Shimokawa et al., 2001) or acylation from feruloyl-CoA via feruloylglucose (Obel et al., 2003). It is also possible that the route takes place at the level of yet unidentified esters or even free acids, especially as COMT-like enzymes with high affinity to caffeic acid and/or 5-hydroxyferulic acid have been identified from pine and alfalfa (Li et al., 1997; Inoue et al., 2000). 2.3.3 Arabidopsis and Populus ‘lignification toolboxes’ Raes et al. (2003) and Costa et al. (2003) published a genome-wide bioinformatic approach to characterise the monolignol biosynthetic gene families in Arabidopsis. Based on homology searches, 34 genes were identified as candidate monolignol biosynthetic genes, and most gene families contained multiple members. In addition, several 4CL-, CCR- and COMT-like genes were found. Expression, phylogenetic and in silico promoter analyses pinpointed 12 genes as candidates for a role in vascular lignification (Table I; Raes et al., 2003). One important criterion was the presence of a 10 conserved AC-rich promoter element that has been shown to enhance the xylem expression of several monolignol biosynthetic genes (e.g., Ohl et al., 1990; Hauffe et al., 1991; Lacombe et al., 2000), while repressing the expression in phloem (Hauffe et al., 1993; Hatton et al., 1995). Among the 12 candidate genes, this element was detected specifically in the G branch genes, except in C4H that was encoded by a single gene. The S unit specific F5H and COMT genes lacked this AC element, suggesting that it is one of the causes behind the spatial and temporal differences of G and S lignin deposition in Arabidopsis (Chapple et al., 1992). The lack of AC elements in C4H probably reflected a more relaxed regulation of the gene to allow the expression in several different tissue types, and the wider role that the single C4H gene must have in phenylpropanoid metabolism (Figure 3, Raes et al., 2003). Also for CCOMT, CCR, F5H and CAD families, a single gene was predicted responsible for developmental lignification (Table I; Raes et al., 2003). The CCR and F5H genes have been functionally characterised in the mutants irregular xylem 4 (irx4) and ferulic acid hydroxylase 1 (fah1), respectively (Chapple et al., 1992; Jones et al., 2001; Patten et al., 2005). Redundancy exists in the CAD family, as major changes in lignin content were only observed in a cad-c cad-d double mutant (Sibout et al., 2005). Additionally, AtCAD1 had a minor role in lignification in young developing stems, possibly being responsible for the remaining G and S units in the double mutant (Eudes et al., 2006). Two PAL genes were predicted responsible for developmental lignification in the inflorescence stem (Raes et al., 2003), and accordingly, changes in lignin were only observed in a pal1 pal2 double mutant (Rohde et al., 2004). PAL1 appeared more important for general phenylpropanoid metabolism, whereas PAL2 was more related to responses to light, UV light and stress (Rohde et al., 2004). Two 4CL genes were pinpointed as candidates for vascular lignification (Raes et al., 2003), but only the role of 4CL1 has been verified in transgenic plants (Lee et al., 1997). Only G units were affected, suggesting that G and S units are synthesised through different 4CL isoforms. Hierarchical clustering of the microarray data from different stages of the developing Arabidopsis inflorescence stem identified a set of coregulated genes that was more highly expressed during the later stages of stem development, when lignification is pronounced (Ehlting et al., 2005). This set included all the previously identified 'lignification toolbox' genes (Raes et al., 2003). In addition, one NADPH:cytochrome P450 reductase, ATR2 (Mizutani et al., 1997), was among the coregulated genes, suggesting that the product of this gene supports the hydroxylase activity of C4H, C3H and F5H. No genes related to SAM metabolism were among the coregulated genes. However, the SAM SYNTHETASE 3 gene is apparently needed for the methyl transfer reactions during lignin biosynthesis in Arabidopsis, based on the decreased lignin content in the methionine overaccumulation 3 (mto3) mutant (Shen et al., 2002). Comparison of the lignin biosynthetic gene families between Arabidopsis and Populus (Tsai et al., 2006; Tuskan et al., 2006) showed a reasonable conservation of the gene family size, except for HCT and CAD genes (Table I). Particularly the HCT family has expanded in Populus, and probably reflects the accumulation of hydroxycinnamoyl derivatives, e.g., p-coumaroyl, caffeoyl and feruolyl quinates, in Populus leaves (Tsai et al., 2006). Only a few monolignol biosynthetic genes of Populus trichocarpa have been functionally characterised in transgenic or mutant plants (Table I), but other examples 11 from related Populus species exist (Doorsselaere et al., 1995; Tsai et al., 1998; Zhong et al., 2000). Also the extensive collections of expressed sequence tags (ESTs) from Populus species (Sterky et al., 1998 and 2004) and the associated expression profiling studies have suggested a role for certain gene family members in lignification (e.g., Hertzberg et al., 2001; Andersson-Gunnerås et al., 2006). Table I The number of monolignol biosynthetic genes found in Arabidopsis thaliana and Populus trichocarpa. Numbers in brackets denote 'like' genes. The numbers are based on articles by Raes et al. (2003), Tsai et al. (2006) and Tuskan et al. (2006). Genes that have been functionally associated with monolignol biosynthesis are also indicated. *, indirect evidence for involvement in lignification based on promoter sequence and expression analysis (Raes et al., 2003) Gene Arabidopsis Populus Functional association to lignification PAL 4 5 AtPAL1, AtPAL2 C4H 4CL HCT C3H CCOMT 1 4 (9) 1 3 7 2 (1) 5 7 3 6 AtC4H* At4CL1, At4CL2* AtHCT* AtC3H1* AtCCOMT1*, PtCCOMT1, PtCCOMT2 AtCCR1 AtF5H1 AtCOMT, AtCAD-C, AtCAD-D, AtCAD1 CCR F5H COMT CAD 2 (5) 2 1 (13) 3 (6) 7 2 (1) 2 (6) 1 (14) Rohde et al., 2004 Lee et al., 1997 Franke et al., 2002 Meyermans et al., 2000 Jones et al., 2001 Chapple et al., 1992 Goujon et al., 2003 Sibout et al., 2005, Eudes et al., 2006 2.3.4 Functional divergence of monolignol biosynthetic genes Lignin is only one of the end-products of the phenylpropanoid metabolism. Therefore, especially the first genes of the pathway from PAL to 4CL contribute also to the biosynthesis of flavonoids, coumarins, suberin and stilbenes (Figure 3). On the other hand, the later genes are common to lignin and lignan biosynthesis. Competition between the different branches for the intermediate metabolites likely exists; for example, p-coumaroyl-CoA is a common substrate for HCT and the flavonoid-related chalcone synthese (CHS; Figure 3). For many monolignol biosynthetic gene families, two or more classes have been found (Fahrendorf and Dixon, 1993; Ehlting et al., 1999; Nedelkina et al., 1999; Raes et al., 2003) and they are thought to be functionally divergent. In support of this, different PAL and 4CL genes, for example, were expressed in lignifying and flavonoid/condensed tannin-accumulating cells in aspen (Hu et al., 1998; Kao et al., 2002). Complex formation between subsequent enzymes, i.e., metabolic channelling, has been suggested as a means to separate these competing pathways and to increase the flux through the pathway by channelling the products of one enzyme directly to the next (Winkel-Shirley, 1999). The complexes are putatively formed around the 12 membrane-attached enzymes, such as the cytochrome P450 hydroxylases C4H, C3H and F5H. In support of this, tobacco PAL1 was partially localised in the microsomes, while PAL2 was completely cytosolic (Rasmussen and Dixon, 1999; Achnine et al., 2004). Immunolocalisation of PAL1 and C4H coincided in the membranes, suggesting complex formation (Achnine et al., 2004). In fact, the channelling of cinnamic acid between PAL and C4H has been demonstrated in tobacco microsomes. This was also supported in vivo in tobacco cell suspension cultures, in which exogenously supplied cinnamic acid could not equilibrate with endogenously formed cinnamic acid (Rasmussen and Dixon, 1999). Dixon et al. (2001) have proposed that the pathways to G and S lignin could also be channelled through different enzyme complexes, possibly already early in the pathway. Biotic and abiotic stresses, including pathogen infections, can also induce the biosynthesis of various defensive phenolics (Vance et al., 1980; Dixon and Paiva, 1995). It is possible that genes involved in stress-induced and developmental lignification are only partly shared. For example, two CCR genes of Arabidopsis were differentially expressed in lignifying tissues and after pathogen infection (Lauvergeat et al., 2001). Also the PAL-C4H channelling in tobacco suspension cultures was reduced after elicitation (Rasmussen and Dixon, 1999). 2.3.5 Control of lignin biosynthesis Several reports suggest that the timing of lignin biosynthesis is controlled by carbon supply, which is directly linked to light and photosynthetic activity. For example, in Arabidopsis, the expression of the monolignol biosynthetic genes followed a diurnal cycle, during which the expression peaked twice a day (Rogers et al., 2005a). While some genes needed the dark period for light-stimulated induction, others were insensitive to changes in light, being presumably under circadian control (Harmer et al., 2000; Rogers et al., 2005a). Sucrose availability was identified as an important factor controlling the magnitude of the oscillations, and the signalling was proposed to take place through hexose derivatives of glucose and fructose, the sucrose break-down products (Rogers et al., 2005a). Light was also shown to activate phenylpropanoid metabolism in Arabidopsis roots, resulting in an ectopic accumulation of monolignol glucosides and flavonoids (Hemm et al., 2004). The photoreceptors phytochrome B (PHYB) and cryptochrome 2 (CRY2) were needed for the activation, along with the transcription factor hypocotyl elongated 5 (HY5). The carbon-nutrient balace hypothesis proposes that secondary metabolism is directed towards carbon-rich compounds in nitrogen-limited plants and to nitrogenrich compounds in carbon-limited plants (Coley et al., 1985). In support of the hypothesis, increased levels of phenylpropanoids, including lignin, have been observed under limited nitrogen supply (e.g., Gebauer et al., 1997; Fritz et al., 2006). Studies with a nitrate reductase-deficient mutant of tobacco showed that this shift resulted from the decreased nitrate concentration, which induced the expression of the early pathway genes PAL, 4CL and HCT (Fritz et al., 2006). The pal1 pal2 double mutant of Arabidopsis provided an insight into the link between primary and secondary metabolism when the flux of carbon into the 13 phenylpropanoid metabolism was reduced. In the mutant, lignin content was reduced to 30% and phenylalanine accumulated to high levels (Rohde et al., 2004). The phenylalanine pool was suggested to act as a link between the primary and secondary metabolism, resulting in the down-regulation of nearly all phenylpropanoid metabolites, up-regulated photosynthesis and sucrose degradation and imbalanced amino acid composition in the mutant (Rohde et al., 2004) Metabolic control Many of the monolignol biosynthetic genes are under metabolic regulation, particularly in the early pathway. For example, feeding of phenylalanine into a lignin-forming pine suspension culture increased the transcription of all monolignol biosynthetic genes (Anterola et al., 2002). Similarly, p-coumaric acid increased HCT activity (Lamb, 1977), probably through transcriptional activation as was shown for CHS (Loake et al., 1992). Apparently, a coupling between PAL and C4H activities exists and it is mediated by sensing the cinnamic acid pool. Increases in cinnamic acid concentration in tobacco were reflected in a decreased PAL activity by feedback regulation, which probably acts to decrease the flux into the phenylpropanoid pathway when its capacity is reduced (Bolwell et al., 1986; Blount et al., 2000). This inhibition was shown to take place both at transcriptional and post-translational level, the latter through PAL inactivation (Bolwell et al., 1986; Mavandad et al., 1990). It is possible that a low concentration of free cinnamic acid is maintained through a tight channelling between PAL and C4H; however, concentrations exceeding 100 µM would be needed for inhibition based on studies in tobacco cell cultures (Rasmussen and Dixon, 1999). Other examples of posttranslational modifications of the pathway enzymes are scarce, but for example PAL can be subjected to phosphorylation by a Ca2+ dependent protein kinase, possibly regulating its activity or stability (Cheng et al., 2001). Transcriptional control Coordinated expression of monolignol biosynthetic genes, also with SAM metabolism and shikimate pathway genes, has been observed in, e.g., Arabidopsis, loblolly pine and eucalyptus (Harmer et al., 2000; Anterola et al., 2002; Kirst et al., 2004; Ehlting et al., 2005; Gachon et al., 2005). This coordinated expression suggests regulation by transcription factors through conserved promoter elements. In Arabidopsis, for example, many genes that are under circadian control contain a sequence element that guides a rhythmic 'evening' expression pattern (Harmer et al., 2000). The xylem expression of bean PAL genes was shown to depend on AC-rich elements in their promoters (Leyva et al., 1992). These elements have been observed in the promoters of many monolignol biosynthetic genes in several species (e.g., Hauffe et al., 1991; Ye et al., 1994; Feuillet et al., 1995; Lacombe et al., 2000; Raes et al., 2003). AC elements are recognized by MYB type transcription factors, which are defined by an Nterminal DNA binding domain that contains a combination of characteristic sequence repeats called R1, R2 and R3 (Stracke et al., 2001). In plants, the R2R3-type MYBs bind 14 to AC elements (Sablowski et al., 1994; Patzlaff et al., 2003). Enhancement of lignin biosynthetic gene expression by R2R3-type MYBs has been shown, for example, in Arabidopsis by activation tagging. The identified PAP1 MYB was a master regulator of phenylpropanoid biosynthesis, activating the production of flavonoids, lignin and hydroxycinnamic acid esters. Expression of PAL, CHS and dihydroflavonol reductase (DFR) genes was up-regulated (Borevitz et al., 2000). A specific activation of lignin biosynthesis was demonstrated for pine PtMYB4. Overexpression in tobacco led to increased lignin content due to enhanced expression of C3H, CCOMT, COMT, CCR and CAD genes (Patzlaff et al., 2003). Lignin quality can also be controlled by MYB transcription factors, as an increased S/G ratio without any change in lignin content was found in transgenic tobacco overexpressing eucalyptus EgMYB2. The expression of the late pathway genes was highly induced in these plants (Goicoechea et al., 2005). MYB transcription factors also act as repressors of lignin biosynthesis. For example, overexpression of AmMYB308 or AmMYB330 from Antirrhinum majus (L.) in tobacco resulted in reduced lignin content in the stem, and decreased expression of C4H, 4CL and CAD genes (Tamagnone et al., 1998). Antisense expression of hybrid aspen PttMYB21a resulted in increased lignin content in bark and increased CCOMT expression in phloem, suggesting that it inhibits the pathway in tissues that normally do not lignify (Karpinska et al., 2004). Transcription of PttMYB21a was also up-regulated in tension wood in hybrid aspen, in which lignin biosynthesis was repressed (Andersson-Gunnerås et al., 2006). Apparently, the phenylpropanoid metabolism is controlled by several interacting MYB proteins, both repressors and activators that compete for the target sequences (Tamagnone et al., 1998; Hemm et al., 2001). Dual repressor-activator roles for single MYB proteins have also been suggested (Patzlaff et al., 2003). AC elements are also recognised by zinc finger transcription factors that contain a LIM domain. NtLIM1 from tobacco was shown to be a positive regulator of lignin biosynthesis, as antisense expression in tobacco decreased lignin content and repressed the expression of PAL, 4CL and CAD genes (Kawaoka et al., 2000). DOF-type transcription factors have also been identified as putative regulators of lignin biosynthetic genes, based on the microarray analyses of Arabidopsis ectopic lignification mutants ectopic lignification 1 (eli1), de-etiolated 3 (det3) and ectopic lignification in pith 1 (elp1; Rogers et al., 2005b). In addition, KNOX type homeodomain transcription factors BREVIPEDICELLUS (BP) from Arabidopsis (Mele et al., 2003) or ARBORKNOX1 (ARK1) from Populus (Groover et al., 2006) have been shown to affect lignin biosynthesis. Microarray analysis of mutant plants showed that BP acts as a repressor of lignin biosynthesis in Arabidopsis, and conserved binding sites for KNOX transcription factors were observed in several monolignol biosynthetic genes (Mele et al., 2003). KNOX type transcription factors are needed to maintain the meristematic identity of the cells in the shoot apical meristem and in the vascular cambium (Scofield and Murray, 2006). They were also suggested to have a dual role in cambial development, maintaining the undifferentiated nature of cambial initials while functioning in the cambial daughter cells to promote the differentiation process in combination with other proteins (Groover et al., 2006). A role in the regulation of lignin biosynthesis has also been proposed for Arabidopsis 15 MADS-box transcription factors SHATTERPROOF (SHP) 1 and 2, specifically in the dehiscence zone of seed pods (Liljegren et al., 2000), and NAC-domain transcription factors SECONDARY-WALL ASSOCIATED NAC-DOMAIN PROTEIN 1 (SND1) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) 1 and 2 in the interfascicular fibres (Zhong et al., 2006; Mitsuda et al., 2007). The defects must be partly due to the uninitiated or aberrant cell differentiation, but a role as a master regulator of the whole process of differentiation and formation of lignified secondary cell walls cannot be excluded. Decreased expression of monolignol biosynthetic genes has also been observed in mutants and transgenic plants where metabolic bottlenecks have been introduced (e.g., Rohde et al., 2004; Abdulrazzak et al., 2006). Interestingly, different results have been observed in insertion and cosuppression mutants for C3H gene in Arabidopsis, suggesting that gene silencing through cosuppression can affect the whole pathway, whereas this regulatory mechanism was not activated in the insertion mutant (Abdulrazzak et al., 2006). However, the interactions within monolignol biosynthesis appear complex, since, e.g., the expression of lignin biosynthetic genes was both upand down-regulated in the Arabidopsis pal1 pal2 mutant (Rohde et al., 2004). Hormonal control Reports on the hormonal control of lignification are scarce. Auxin and cytokinin have been shown to control the growth and differentiation of cambial cells (Uggla et al., 1998; Mähönen et al., 2006) and the transdifferentiation of Z. elegans mesophyll cells into tracheary elements (Fukuda and Komamine, 1980; Fukuda and Komamine, 1982), but how and if the secondary cell wall synthesis and lignification are coregulated at the hormonal level is not known. It has been shown, however, that auxin and gibberellin (GA) have opposite effects on the quantity of G and S units in lignin in the phloem fibres of Coleus blumei (Benth.) (Aloni et al., 1990). A role for GA in lignin biosynthesis is supported in other studies as well. For example, transgenic hybrid aspen with upregulated GA20-oxidase, a GA biosynthetic enzyme, contained more S-lignin and the expression of F5H, COMT and SAD was up-regulated. Increased growth and fibre length were also observed (Eriksson et al., 2000; Israelsson et al., 2003). Similarly, in Arabidopsis that over-expressed the GA20-oxidase gene, biomass production was upregulated and the stems contained more lignin due to the induction of monolignol biosynthetic genes (Biemelt et al., 2004). Addition of active GA3 to Arabidopsis petioles or to xylogenic Zinnia cell culture increased the lignin content, but apparently through increased polymerisation of existing phenols (Biemelt et al., 2004; Tokunaga et al., 2006). Ectopic lignification has been observed in Arabidopsis mutants eli1/cev1, elp1/pom1 and det3, in which jasmonic acid and ethylene-related pathways are up-regulated (Schumacher et al., 1999; Caño-Delgado et al., 2000; Zhong et al., 2000; Ellis et al., 2002; Caño-Delgado et al., 2003). All the mutants are defective in cell wall synthesis-related phenomena (in cellulose synthase subunit, chitinase-like and vacuolar ATPase genes, respectively), which cause aberrant lignification without the concomitant secondary cell wall synthesis. It was suggested that the impaired cell wall structure triggers 16 defence responses via jasmonate and ethylene-dependent pathways, leading to ectopic lignification through stress-induced gene family members (Ellis et al., 2002; CañoDelgado et al., 2003). However, Rogers et al. (2005b) proposed that the impaired sugar signalling related to the dark-photomorphogenic phenotype of these mutants is responsible for the constitutive activation of lignin biosynthesis. 2.3.6 Effects of the genetic down-regulation of monolignol biosynthetic genes Repression of the expression of the monolignol biosynthetic genes, leading to reduced enzyme activity, usually results in a decreased lignin content and/or altered monomer composition. Changes in monomer composition and other phenolic metabolites reflect the plasticity of the pathway to redirect the flux if one step becomes rate-limiting. They also reflect the plasticity of the lignin polymer to accommodate for the available building blocks to meet the requirements for function. In some cases, growth defects have also been observed. The degree of down-regulation usually correlates with the degree of observed changes (e.g., Bate et al., 1994; Piquemal et al., 1998). For most genes, reduction to 20 to 50% residual activity is needed to obtain a visible phenotype or changes in lignin, indicating that very few steps in monolignol biosynthesis can be considered rate-limiting during normal developmental lignification (Anterola and Lewis, 2002). The analysis of lignification in transgenic or mutant plants has proven to be challenging. The phenotypic and biochemical effects of a genetic modification can become more pronounced during later developmental stages, or when the plants are moved from the greenhouse to field conditions (Pinçon et al., 2001). Low stability of the transgene has also been observed (Bate et al., 1994). Therefore, analysis of the transgenic plants throughout the development is needed, preferably at the same developmental stage with the control plants. This is necessarily not equal to the chronological age because the genetic changes can also slow down the development (Patten et al., 2005). In addition, the quantitative and qualitative methods for the analysis of lignin have limitations, since all the used methods describe only some and possibly different fraction of lignin. Due to this, changes in the condensation degree of lignin inevitably affect the monomer fraction that can be studied with a particular method (Anterola and Lewis, 2002). Lignin quantity and structure Decreased lignin content in xylem has been observed for all monolignol biosynthetic genes when the enzyme activity is reduced below a certain threshold (e.g., Atanassova et al., 1995; Kajita et al., 1997; Sewalt et al., 1997; Piquemal et al., 1998; Marita et al., 1999; Zhong et al., 2000; Franke et al., 2002a; Hoffmann et al., 2004). CAD represents an extreme, since lignin quantity was slightly changed only in a cad null mutant of loblolly pine, which had <1% residual CAD activity (MacKay et al., 1997; Ralph et al., 1997). In general, it appears that changes in the early pathway genes lead to stronger 17 reductions in lignin quantity, whereas changes in the expression of the later genes cause qualitative differences. The impacts of genetic down-regulation on lignin quality include changes in monomer composition, reflecting the redirection of the pathway. For example, repression of C3H activity in the Arabidopsis ref8 mutant resulted in a block of G and S synthesis, and the remaining stem lignin consisted mainly of H units (Franke et al., 2002a). Similarly, S units were nearly abolished and G units prevailed in the fah1 mutant of Arabidopsis, deficient in F5H (Chapple et al., 1992; Meyer et al., 1998), and in tobacco and poplar having repressed COMT activity (Atanassova et al., 1995; Doorsselaere et al., 1995). In the case of COMT, a new lignin monomer, 5-hydroxy-CA, was incorporated (Atanassova et al., 1995; Doorsselaere et al., 1995). When the last step of monolignol biosynthesis, CAD, was down-regulated in, e.g., tobacco and pine, alcohol monomers were replaced with aldehydes (Halpin et al., 1994; MacKay et al., 1997; Ralph et al., 1997). Results of genetic down-regulation can also be additive. When aspen was simultaneously down-regulated for 4CL and up-regulated for F5H expression, total lignin content was reduced by 50% and S/G ratio increased in the transgenic plants, reflecting the properties of both single transformants (Li et al., 2003a). The changes in G and S units are not always easy to interpret, which reflects the fact that our understanding of lignin biosynthesis is far from complete. For example, opposite effects on S/G ratio were observed in transgenic tobacco and alfalfa with repressed PAL and C4H activites (Sewalt et al., 1997; Chen et al., 2006). For 4CL, either increased S/G ratio with a decrease in both G and S units, no change in S/G or a decrease in only G units have been observed in different species (Kajita et al., 1996; Kajita et al., 1997; Lee et al., 1997; Hu et al., 1999). For HCT, only S units decreased in RNAi-silenced tobacco (Hoffmann et al., 2004). These results have been interpreted as evidence for the differential regulation of the biosynthesis of G and S units already during the early steps of the pathway, possibly through different enzyme complexes (Sewalt et al., 1997; Dixon et al., 2001). This is logical, taken into account that both temporal and spatial differences in G and S lignin deposition exist (Fergus and Goring, 1970; Terashima et al., 1986; Chapple et al., 1992). It is also likely that differences in the pathway organisation exist between species. For example in alfalfa, the methylation of the 3-position appears different from other model species. Down-regulation of CCOMT activity in alfalfa decreased only G units in lignin while S units remained at the wild type level, whereas COMT downregulation mainly affected the amount of S units, as could be expected (Guo et al., 2001; Chen et al., 2006). The authors proposed that an SA-specific 3-methylation could take place at the aldehyde or alcohol level. It was supported by kinetic studies, showing that 5hydroxyconiferaldehyde and caffeoyl aldehydes were the preferred substrates for COMT (Parvathi et al., 2001). Alternatively, COMT or another OMT could introduce the 3-methoxyl group to SA at the level of caffeic acid or yet unidentified ester, as HCTactivity was needed for S unit biosynthesis (Chen et al., 2006). Existence of alternative 3-hydroxylation pathways in the roots of Arabidopsis has also been suggested (Abdulrazzak et al., 2006). 18 Accumulation of metabolites If the flux within the phenylpropanoid pathway is restricted at some point, the carbon flow is directed to other products of the pathway. In the early part of the pathway, the increasing metabolites are often conjugates of the substrates of the down-regulated gene product. For example, wall-esterified hydroxycinnamic acids accumulated in 4CLdeficient aspen and tobacco (Kajita et al., 1997; Hu et al., 1999), and increased levels of p-coumaryl esters were observed in Arabidopsis down-regulated for C3H (Franke et al., 2002a). p-Hydroxybenzoate and phenolic glucosides accumulated in CCOMT downregulated poplar xylem (Meyermans et al., 2000; Zhong et al., 2000), whereas the levels of free and tyramin-conjugated ferulic acid increased in tobacco with repressed CCR activity (Ralph et al., 1998). The accumulated metabolites can be partly incorporated to lignin, possibly to increase the cell wall strength (Hu et al., 1999). Repression of HCT increased the levels of caffeoyl quinate in tobacco stems (Hoffmann et al., 2004). This was surprising, because HCT catalyses both the quinate/shikimate ester formation before and hydrolysis after the 3-hydroxylation, and caffeoyl quinate can be utilised in the latter reaction. However, the levels of caffeoyl quinate also reflected the expression levels of PAL, C4H and CCR genes in tobacco (Howles et al., 1996; Blount et al., 2000; Chabannes et al., 2001b). It might act as an alternative sink when flux through the pathway is blocked, while the shikimate ester could be the main C3H substrate in tobacco (Schoch et al., 2001; Hoffmann et al., 2004). The flow can also be directed towards other branches of the phenylpropanoid metabolism. For example, accumulation of flavonol glycosides and anthocyanin derivatives were observed in C3H or HCTdeficient Arabidopsis (Abdulrazzak et al., 2006; Besseau et al., 2007). Growth defects Growth defects have often, but not always, been observed when the expression of the monolignol biosynthetic genes has been down-regulated. The defects have included stunted growth and deformations or altered colour in the leaves (e.g., Elkind et al., 1990; Piquemal et al., 1998; Hoffmann et al., 2004; Abdulrazzak et al., 2006). Milder phenotypes have showed changes in xylem colour (orange-brown in tobacco after CCR, pink in poplar after COMT and red-brown in tobacco and pine after CAD downregulation; Halpin et al., 1994; Doorsselaere et al., 1995; MacKay et al., 1997; Piquemal et al., 1998), as well as changes in epidermal fluorescence when sinapoyl esters were decreased, e.g., in a C3H mutant of Arabidopsis (Franke et al., 2002a). The colour changes are likely due to incorporation of aldehydes and other phenolics in lignin. In addition, collapsed xylem elements and deformations in the xylem cell wall have been observed (Piquemal et al., 1998; Meyermans et al., 2000; Zhong et al., 2000; Jones et al., 2001; Franke et al., 2002a). Deformations are likely a result of decreased lignin deposition in the cell wall, affecting its structural integrity and mechanical strength. Interestingly, down-regulation of CCR activity in tobacco resulted in a disorganised fibre and vessel walls, specifically in the S2 sublayer (Chabannes et al., 2001a). A decrease in non-condensed subunits was observed in the same area, linking the -O-4 bonds to the cohesion of the microfibril lamella and ultrastructural organisation of the 19 cell wall. The results also supported the theory that lignification in different cell types and parts of the cell wall is tightly and independently regulated. Existence of collapsed vessel elements suggests that other observed growth defects are a result of malfunctioning xylem transport. However, there are cases where no growth abnormalities have been observed in spite of deformed vessel elements, e.g., in CCOMT-deficient tobacco and poplar (Meyermans et al., 2000; Zhong et al., 2000), suggesting that other factors may also be involved. In Arabidopsis with down-regulated CCR activity, accumulation of soluble phenolics was suggested to have toxic effects that retard growth (Goujon et al., 2003a). Recently, it was shown in HCT-silenced Arabidopsis that the growth defects associated with lignin deficiency were a consequence of flavonoid accumulation, which affected polar auxin transport and thus plant development (Besseau et al., 2007). Inhibition of the flavonoid accumulation through simultaneous silencing of the CHS gene restored the auxin transport and the wild type growth habit while the lignin-deficient phenotype persisted. Surprisingly, increased growth and/or cellulose accumulation were observed in transgenic aspen with decreased lignin content (Hu et al., 1999; Li et al., 2003a). In loblolly pine, the cad-n1 null allele that controls lignin quality coincided with a quantitative trait locus (QTL) for increased diameter growth (Wu et al., 1999). These results suggest that a decreased flow of carbon into lignin could be balanced by a greater input into carbohydrates (Hu et al., 1999; Wu et al., 1999). Association of QTL analysis with microarray data in eucalyptus showed that the expression levels of lignin biosynthetic genes, and of related shikimate and SAM metabolism genes, correlated negatively with diameter growth (Kirst et al., 2004). Moreover, two QTLs that regulated the transcription of lignin biosynthetic genes colocalised with known QTLs for growth, providing further evidence for the coregulation of growth and lignin biosynthesis. However, similar increases in growth have not been observed in, e.g., tobacco or Arabidopsis, despite numerous transgenic plants. Hence this phenomenon could be specific to trees and secondary growth, for which the investments in cellulose and lignin biosyntheses far exceed those in herbaceous plants. Effects on the pulping efficiency of wood or digestability of forage The introduced quantitative and qualitative changes in lignin have potential to increase the digestibility of forage or the pulping efficiency of wood in the pulp and paper industry (Jung and Vogel, 1986; Baucher et al., 2003). In general, lignin with increased non-condensed linkages, i.e., S units, is more easily dissolved during chemical pulping, which suggests that over-expression of S pathway genes could be beneficial for pulping (Baucher et al., 2003). This has been demonstrated, for example, with poplars that overexpressed the F5H gene (Huntley et al., 2003). CAD downregulation in either pine or poplar reduced sulfide and alkali consumption but had conflicting effects on pulp yield (Baucher et al., 1996; Lapierre et al., 1999; Pilate et al., 2002). On the other hand, COMT downregulation in poplar resulted in more condensed lignin and a corresponding increase in alkali consumption (Lapierre et al., 1999; Jouanin et al., 2000; Pilate et al., 2002). 20 Similarly, the digestibility of forage can be affected by lignin quantity (Barnes et al., 1971; Shadle et al., 2007). This has been especially evident in maize brown midrib (bm) mutants, which are characterised by a reddish-brown colour of the leaf midrib and stalk pith, and decreased levels and/or altered composition of lignin (reviewed in Barrière et al., 2004). Several bm mutants have been found in natural breeding populations. The bm3 mutant contains a defective COMT gene (Vignols et al., 1995), while the others are apparently related to mutations in regulatory sequences or genes, for example transcription factors (Barrière et al., 2004; Guillaumie et al., 2007a) 2.4 Storage and transport of the monolignols into the apoplast Once formed, the monolignols are either stored, putatively in the vacuole, as more hydrophilic and less toxic conjugates, or transported into the apoplast (Boerjan et al., 2003). Accumulation of monolignol 4-O-glucosides (p-coumaryl alcohol glucoside, coniferin and syringin) has been observed in the cambial sap of conifers and some woody angiosperms during cambial activation in the spring (Freudenberg and Harkin, 1963; Terazawa et al., 1984). They can build up to 10 mM concentrations in the developing xylem and disappear at the onset of dormancy (Savidge and Förster, 1998). Radiotracer experiments have shown that glucoside-derived monolignols can be incorporated into lignin, though unexpectedly partly through conversion into aldehyde first (Terashima et al., 1988; Tsuji and Fukushima, 2004). Savidge et al, (1998) speculated that glucoside conjugation could prevent a premature lignification during early secondary cell wall development. A role for coniferin as a quickly metabolisable precursor for defence-related phenolics was also suggested (Bednarek et al., 2005). A UDP-glucose:coniferyl alcohol glucosyltransferase activity has been identified from the cambial sap of Norway spruce and pine (Schmid and Grisebach, 1982; Savidge and Förster, 1998). The spruce enzyme preferred CA, while the pine enzyme catalysed the conjugation of both CA and SA equally well. Also the corresponding aldehydes were conjugated. The accumulation of monolignol glucosides could be a tree-specific phenomenon, as reports from herbaceous species are rare. However, three homologous glucosyltransferases that were able to glucosylate both CA and SA, and the corresponding aldehydes and acids in vitro, have been identified in Arabidopsis (Lim et al., 2001 and 2005). Simultaneous down-regulation of the three UGT72E genes reduced the light-induced monolignol glucoside accumulation in roots by 90%, while overexpression caused accumulation of monolignol glucosides both in roots and leaves. A concomitant decrease in sinapoyl malate in leaves was also found, suggesting that the total size of the phenylpropanoid pools in leaves is balanced (Lanot et al., 2006). The fact that ectopic accumulation of monolignol glucosides and flavonoids in roots was only observed after light induction suggests that the phenomenon was a photoprotective stress response, and that the accumulation of monolignol glucosides was not destined for lignin biosynthesis. For polymerisation into lignin, transport of the monolignols into the apoplast is needed. The transport form, whether glucoside or aglucone, is unknown. However, the identification and cell wall localisation of a coniferin-hydrolysing -glucosidase in, e.g., 21 spruce and pine argues for the glucoside transport (Marcinowski and Grisebach, 1978; Dharmawardhana et al., 1995 and 1999; Samuels et al., 2002). Two -glucosidases that hydrolyse monolignol glucosides have also been identified from Arabidopsis (EscamillaTrevino et al., 2006). Of these, BGLU45 was specific for coniferin and syringin, while BGLU46 had higher activity towards p-coumaryl alcohol glucoside, and hydrolysed other phenolic glucosides as well (Escamilla-Trevino et al., 2006). Their expression pattern coincided with lignifying tissues, supporting a role in lignification. The reported Km values of -glucosidases for monolignol glucosides vary from 0.18 to 7 mM (Marcinowski and Grisebach, 1978; Dharmawardhana et al., 1995; Escamilla-Trevino et al., 2006). The high values suggest that the biological relevance of -glucosidase activity might be low; however, if the 10 mM concentrations reported for coniferin in pine cambial sap are generally found, the high Km values would not be a problem. At least three transport mechanisms have been suggested. Firstly, monolignol aglucones could diffuse freely through the membranes (Boija and Johansson, 2006), provided that free monolignols do not reach toxic concentrations. Another possibility is the ATP-binding cassette (ABC) transporters that carry small-molecular-weight compounds across membranes (Yazaki, 2006). Several ABC transporters were coregulated with lignin biosynthetic genes during the development of Arabidopsis inflorescence stem (Ehlting et al., 2005). Also vesicular secretion has been suggested as a means for transport. Supporting this, radioactive label derived from phenylalanine and cinnamate was localised to Golgi-associated vesicles in wheat coleoptiles and Cryptomeria japonica (L.f.) D. Don (Pickett-Heaps, 1968; Takabe et al., 1985). In developing contorta pine (Pinus contorta Dougl.) tracheids, dark-stained, putatively phenolic vesicles appeared on the trans-Golgi network during secondary cell wall synthesis and early lignification (Samuels et al., 2002). As no hemicellulose was localised to the vesicles, they were proposed to be involved in monolignol (glucoside) secretion. According to this model, a controlled vesicular secretion of glucosides and the consecutive deglucosylation in the cell wall would provide precursors for lignification during cell wall synthesis, whereas the vacuolar rupture during the programmed cell death would release the stored monolignol glucosides from the vacuole for the final stages of lignification (Samuels et al., 2002). 2.5 Polymerisation of monolignols Lignin is a hydrophobic and optically inactive polymer, which is highly complex and heterogeneous in nature (Figure 4A). Lignin polymerisation is a radical coupling reaction, where the monolignols are first activated into phenoxy radicals in an enzymecatalysed dehydrogenation reaction. The radicals couple to form dimers, oligomers and eventually the lignin polymer (Freudenberg, 1968). The radicals are stabilised through resonance structures, which also allow the radical to form a covalent bond with another radical on several locations on the monolignol (Figure 4B), resulting in the different lignin substructures (Figure 2B). 22 Figure 4 (A) A simplified structural model of a gymnosperm lignin, adapted from Brunow et al. (1998b). (B) The resonance structures of a coniferyl alcohol radical. For the polymer to grow, radicals must also be formed on larger molecules, such as monolignol oligomers or polymeric lignin. The reaction is a stepwise addition of monolignols onto the growing polymer, so-called 'end-wise polymerisation' (Sarkanen, 1971). Several possibilities for radical generation on polymeric lignin have been suggested. First, a monolignol radical could withdraw an electron from the polymer, returning to the ground state for re-oxidation. This was shown to take place between monolignols (Takahama, 1995), and it requires that the monolignol radical has a higher redox potential than the electron-donating lignin substructure has. However, radical transfer from CA to S type polymers was shown to be inefficient (Sasaki et al., 2004). Secondly, a shuttle mechanism involving, e.g., Mn2+/Mn3+ ions could be involved in radical transfer (Önnerud et al., 2002). It is also possible that an oxidative enzyme could directly oxidise the polymer. A fungal lignin peroxidase (from Phanerochaete chrysosporium Burds.) that is involved in lignin degradation, was shown to bind to lignin-like polymer and to oxidise it directly (Johjima et al., 1999). The identification of a poplar peroxidase that oxidises polymeric lignin (Sasaki et al., 2004) suggests that a 23 direct enzymatic oxidation may be the predominant mechanism for the generation of radicals on the lignin polymer. How the polymerisation is controlled at the molecular level is a matter of controversy. The simplest model states that the polymerisation is a chemical coupling reaction which is guided by the redox potentials of the radicals and by the chemical environment, for example pH and monolignol concentration (Ralph et al., 2004). The opposite view emphasises a strict biochemical/enzymatic control through dirigent-like proteins and a template-guided polymerisation (Guan et al., 1997; Gang et al., 1999). 2.5.1 Dirigent-like proteins and template polymerisation Dirigent protein guides the stereoselective coupling of CA radicals to the (+)-isomer of pinoresinol, a β-β lignan, in Forsythia suspensa (Thunb.) Vahl (Davin et al., 1997). Lignans are often defence-related phenolic di- and oligomers that are derived from the monolignols and are usually optically active (Lewis and Davin, 1999). Dirigent proteins are thought to function by binding and orientating the radicals for coupling to obtain the needed stereochemistry (Davin et al., 1997). The formation of the (+)-pinoresinol is the only proven function of a dirigent protein, but it is possible that other lignans are also synthesised with the help of dirigent proteins. Dirigent genes have been found by homology in both gymnosperms and angiosperms, and they were expressed in the cambial region and the ray parenchyma cells in Forsythia (Burlat et al., 2001). In mature xylem, dirigent-like proteins were immunolocalised to the middle lamella and the S1 layer of the secondary cell wall (Burlat et al., 2001). They were suggested to be different from lignan forming proteins and to act as monolignol (radical) binding sites in the initial phase of lignification to guide the formation of the primary structures of lignin (Gang et al., 1999). These primary structures would then be replicated through template polymerisation, in which the preformed chain is used as a template (Guan et al., 1997; Chen and Sarkanen, 2003). However, no repeating structure has been found in lignin, casting doubt on the template polymerisation theory (Ralph et al., 2004). Dirigent family genes were expressed at a low level in the xylem of western red cedar (Thuja plicata D. Don) and Sitka spruce (Picea sitchensis (Bong.) Carr.) (Kim et al., 2002; Ralph et al., 2006a). In Sitka spruce shoot, higher expression was found in the shoot tip than in the woody shoot base, and only two dirigent genes had a secondary xylem-preferred expression pattern (Friedmann et al., 2007). The expression of most dirigent-like genes was induced after insect feeding in Sitka spruce bark and xylem, suggesting a preferential role in defence (Ralph et al., 2006a). 2.5.2 Combinatorial chemical coupling model Lignin polymerisation can also be considered in terms of simple radical coupling reactions that are under chemical control (Ralph et al., 2004). Studies on dehydrogenation polymers (DHPs), which are synthetic, in vitro made lignins, have shown the importance of the monolignol radical concentration in determining the structure of the polymer. Under high monolignol concentration, so-called bulk 24 polymerisation takes place. Monolignols dimerise and highly condensed lignin is formed, containing a large proportion of C-C bonds, phenolic end groups and cinnamyl alcohol side chains. -O-4, -5 and linkages are present in nearly equal amounts (Sarkanen, 1971; Ämmälahti and Brunow, 2000). If monolignols are added gradually to the reaction, endwise polymerisation predominates. This is thought to be similar to the process in xylem. It is characterised by a step-wise addition of monolignols into the growing polymer, resulting in a large proportion of -O-4 and less linkages (Sarkanen, 1971). The monolignol radical concentration is controlled by both the rate of monolignol secretion into the cell wall, and the amount of oxidative activity available (Ralph et al., 2004). Linkage proportions are also affected by steric factors of the transition state during radical coupling, as some combinations are more stable than others (Brunow et al., 1998b). Another factor is the pH, since the proportion of -O-4 linkages increases in lower pH (Ämmälahti and Brunow, 2000), presumably because the last step of the reaction requires an acid catalyst (Brunow et al., 1998b). Two phenoxy radicals of the same monolignol species dimerise easily, but the crosscoupling between radicals of different monolignol species is controlled by the redox potentials of the radicals (Brunow et al., 1998b). This is especially relevant during the cross-coupling of monolignols into the phenolic end groups on the polymer. In general, cross-coupling occurs if the monoligol has fewer methoxyls than the phenolic end group on the polymer. If this is not the case, the concentration of the monolignol radical must be in large excess for the cross-coupling to take place (Brunow et al., 1998b). Because DHPs are synthesised in suspension, they lack the cell wall matrix that is present during xylem lignification. The addition of pectin to DHP synthesis resulted in more soluble and higher molecular weight (MW) polymer (Cathala and Monties, 2001). Addition of lignin fractions isolated from wood also increased the size of the polymer (Guan et al., 1997). At the microscopic level, lignin in the middle lamella and cell corners of pine xylem appeared as spherical particles, whereas in the secondary cell wall, more elongated structures were observed between the cellulose microfibrils (Donaldson, 1994). These structures correspond to the empty spaces between the cell wall components in the irregular pectin network in the middle lamella and to the lamellae formed by the microfibrils in the secondary cell wall, emphasising the structural constraints caused by the other cell wall components on lignin polymerisation. 2.5.3 Initiation sites for lignin polymerisation Lignification in the cell wall begins from the cell corners and middle lamella, far away from the plasma membrane (Terashima and Fukushima, 1988; Fagerstedt et al., 1998). Evidently, some factor controls the site of the initial lignin polymerisation and prevents the monolignols from being polymerised next to the plasma membrane directly after the transport into the cell wall. Discrete sites of initiation were observed within each cell wall layer in lignifying xylem of pine (Donaldson, 1994), suggesting that the controlling factor is embedded in the wall layers during the synthesis. Several 25 possibilities for the identity of these nucleation sites have been suggested, such as cell wall structural proteins with tyrosine residues (Keller et al., 1989; Boerjan et al., 2003), or ferulates and diferulates that are cross-linked to polysaccharides, at least in grasses (Ralph et al., 1995). Lewis and coworkers suggested that the initiation sites could be composed of arrays of dirigent-like proteins guiding the initial assembly of monolignols (Gang et al., 1999). In addition, the localisation of the enzymes that catalyse the radical formation could be the controlling factor. For example, certain peroxidase isoenzymes from, e.g., zucchini (Cucurbita pepo L.) have affinity to the pectin-Ca2+ complex (Carpin et al., 1999), which is mainly located in the middle lamella and cell corners where lignification begins (Carpita and Gibeaut, 1993). The negatively charged pectins could also bind positively charged polyamines, which are substrates for di- and polyamine oxidases that are possibly involved in H2O2 production (Boerjan et al., 2003). The correct placement of the initiation sites in the cell wall was suggested to be regulated through the vesicular secretion of pectin and other hemicelluloses, and potentially associated initiation factors (Samuels et al., 2002). 2.6 Enzymes for the formation of monolignol radicals Oxidative enzymes catalyse the activation of the monolignols into radicals. Several suggestions for the identity of the enzymes exist, including peroxidases (Harkin and Obst, 1973), laccases (Sterjiades et al., 1992), other phenoloxidases (Savidge and Udagama-Randeniya, 1992) and even cytochrome c oxidase (Koblitz and Koblitz, 1964). Most of these enzymes exist as numerous isoenzymes and generally have broad substrate specificity. Because of this, high affinity to monolignols or mere temporal and spatial correlation with lignification can only be considered as suggestive for a role in lignin polymerisation. Conclusive evidence is only obtained from transgenic plants in which the enzymatic activity has been down-regulated and which show changes in lignin quantity or quality. Preferably, a promoter that restricts the expression into the vascular tissue should be used. 2.6.1 Peroxidases The plant peroxidase superfamily contains three classes of peroxidases from plants, fungi and bacteria (Welinder, 1991). Class I of the superfamily contains bacterial peroxidases, the yeast cytochrome c peroxidases and intracellular plant ascorbate peroxidases. Class II consists of fungal peroxidases, and class III of secretory plant peroxidases. The last group contains all plant peroxidases that are targeted to the endoplasmic reticulum, either for secretion to the cell wall or transport into the vacuole (Welinder, 1991). Peroxidases are heme-containing oxidoreductases that use H2O2 as the ultimate electron acceptor. The natural electron donor molecules in a peroxidase-catalysed reaction vary, and include, for example, monolignols, hydroxycinnamic acids (Zimmerlin et al., 1994), tyrosine residues in, e.g., extensins (Brownleader et al., 1995) and auxin (Hinman and Lang, 1965). In addition to this (often polymerisation26 promoting) peroxidative cycle, peroxidases are also able to produce O2•- and H2O2 through an oxidative cycle (Yokota and Yamazaki, 1965), and even produce hydroxyl radicals through a hydroxylic cycle if a suitable reductant is present (Chen and Schopfer, 1999). Therefore, in a plant, peroxidases have a plethora of functions, including the regulation of the balance between cell wall growth and cross-linking, lignification, suberisation, accumulation of heavy metals, degradation of toxic molecules and production of reactive oxygen species during wounding or pathogen attack. They are also involved in nodulation, mycorrhization and senescence (reviewed in Passardi et al., 2005). The genomic sequences of Arabidopsis (Arabidopsis Genome Initiative, 2000) and rice (Oryza sativa L.) (International Rice Genome Sequencing Project, 2005) contained 73 and 138 peroxidase genes, respectively (Tognolli et al., 2002; Welinder et al., 2002; Passardi et al., 2004). In Arabidopsis, most of the genes were expressed at least at a low level throughout the plant (Valèrio et al., 2004). However, some genes were actively transcribed, and 0.85% of all Arabidopsis ESTs encoded for class III peroxidases (Welinder et al., 2002). A majority of peroxidases were expressed in roots, and several responded to various biotic and abiotic stimuli (Hiraga et al., 2000; Welinder et al., 2002). Even highly similar genes (>70%) were differentially expressed, indicating subfunctionalisation after gene duplication (Welinder et al., 2002). The amino acid sequences of class III peroxidases vary, but the size and the general structure are conserved. The classic fold contains several -helices with two Ca2+ ions and a non-covalently bound heme that is mostly buried inside the protein (Schuller et al., 1996; Gajhede et al., 1997). Based on their isoelectric points (pIs), peroxidases can be divided into acidic, neutral and basic isoforms, but no correlation of the function with the pI of peroxidases has been observed. Isoenzymes are glycosylated to varying degree through asparagine residues, which increases the heterogeneity of peroxidases at the protein level (Veitch, 2004). Some peroxidases contain a C-terminal extension that is thought to direct the protein into the vacuole (Welinder et al., 2002). The catalytic cycle involves the reduction of H2O2 to water by the electrons from reducing substrates, like monolignols, which are oxidised to radicals (R•) (1). The active site of peroxidases contains three amino acid residues important for the catalytic mechanism. The distal His42 (according to horseradish peroxidase C1 (HRP C1) numbering) and Arg38, as well as Pro139, are involved in hydrogen bonding and proton transfers that are prerequisites for the electron transfers and O-O cleavage (Henriksen et al., 1999). In addition, several hydrophobic residues are important in creating an aromatic binding pocket for the reducing substrate (Henriksen et al., 1998; Østergaard et al., 2000). The binding pocket in most peroxidases is, however, large enough to accommodate a variety of reducing substrates, resulting in generally low substrate specificity. This, together with the putative redundancy caused by the large number of peroxidase isoenzymes, has made it difficult to relate given isoenzymes with specific physiological processes. (1) H2O2 + 2 RH 2 R•+ 2 H2O 27 Peroxidases in lignification Numerous reports on peroxidase activity or gene expression in lignin-forming tissues have appeared, but only a few isoenzymes or genes have been specifically associated with lignification (e.g., Sato et al., 1993; Quiroga et al., 2000; Christensen et al., 2001; Marjamaa et al., 2006). Although several reports on the transcriptional regulation of monolignol biosynthesis exist, not much is known for peroxidases. The promoter of the HRP C2 gene, for example, was shown to contain an AC element that are found in many monolignol biosynthetic genes (e.g., Raes et al., 2003). The promoter was activated by NtLIM1, an AC element binding transcription factor from tobacco, which was required for both basal and wound-induced expression the C2 peroxidase (Kaothien et al., 2002). It is likely that the control of the whole lignification process requires a mechanism for the coordinated expression and/or activation of the monolignol biosynthetic genes/enzymes and the radical-forming peroxidases. This was supported by the apparent coregulation of five peroxidase genes with the 'lignification toolbox' genes during Arabidopsis inflorescence stem development (Ehlting et al., 2005). Data from transgenic plants down-regulated for peroxidase activity has confirmed the role of some peroxidase isoforms in lignin polymerisation in, e.g, tobacco and Populus sieboldii (Miq.) x Populus grandidentata (Michx.) (Talas-Ogras et al., 2001; Blee et al., 2003; Li et al., 2003b). Both quantitative (up to 50% reduction) and qualitative changes were reported, but no obvious growth phenotypes, other than larger xylem elements, were found. Antisense expression of the TP60 peroxidase gene in tobacco resulted in an equal reduction of both G and S units, suggesting the existence of a feedback regulation to decrease the monolignol synthesis and transportation under reduced oxidative capacity in the apoplast (Blee et al., 2003). However, no metabolite analysis other than phloroglucinol staining was performed to confirm that the monolignols or their derivatives did not accumulate in xylem. In aspen, down-regulation of the PRXA3a gene reduced the lignin content by 20%. Incorporation of G units into lignin decreased while S units remained at the wild type level (Li et al., 2003b). This is not surprising, as most peroxidases are likely to be inefficient in SA oxidation due to structural constraints (Østergaard et al., 2000; Nielsen et al., 2001). However, peroxidases from, e.g., Populus alba (L.) and Z. elegans were shown to oxidise SA efficiently (Sasaki et al., 2004; Gabaldón et al., 2005). The Populus peroxidase, CWPO-C, was immunolocalised into the middle lamella and cell corners of poplar xylem fibre walls, coinciding partly with the S type lignin (Sasaki et al., 2006). Interestingly, CWPO-C was also found in the cytosol of ray parenchyma cells, suggesting that ray parenchyma could provide the fibre middle lamella with CWPO-C (Sasaki et al., 2006). Polymeric lignin rarely fits into the active site of a peroxidase; however, CWPO-C was also able to oxidise polymeric lignin (Sasaki et al., 2004). In a fungal lignin peroxidase from Phanerochaete chrysosporium, oxidation of polymeric lignin was shown to take place on the protein surface via long-range electron transfer (Johjima et al., 1999). It will be interesting to see if a similar mechanism is functional in secretory plant peroxidases as well. The fact that only 50% reductions at best in lignin amount have been accomplished, argues for redundancy in peroxidase activities. It is likely that in vivo several 28 isoenzymes participate in lignin polymerisation. Division of labour between the isoenzymes could also exist, both spatially between different cell wall layers and functionally, for example in the radical generation on the free monolignols versus the polymer. 2.6.2 H2O2 producing enzymes Peroxidases require H2O2 as the ultimate electron acceptor, and H2O2 synthesis may be the rate-limiting factor for lignin polymerisation (Nose et al., 1995; Gabaldón et al., 2006). It has been suggested that, in addition to the lignifying cells themselves, the nonlignifying xylem parenchyma cells could also generate H2O2 for their lignifying neighbours (Ros Barceló, 2005). This could be especially relevant during the later stages of xylem lignification. Several mechanisms and enzymes for H2O2 production have been suggested, such as diamine and polyamine oxidases, plasma membrane NADPH oxidase, germin-like oxalate oxidase and even peroxidases themselves. However, only indirect evidence for the role for any of the enzymes in lignification exists, based mainly on colocalisation. Copper-containing diamine oxidases and flavin-containing polyamine oxidases are mainly extracellular enzymes that catalyse the catabolism of putrescine, spermidine and spermine, producing H2O2 in the process (reviewed in Cona et al., 2006). Expression of diamine oxidase genes correlated with lignification and peroxidase expression in Arabidopsis seedlings (Møller and McPherson, 1998), and polyamine oxidase activity coincided with peroxidase activity in lignifying tissues in chickpea (Cicer arietinum L.) epicotyls and maize mesocotyls (Angelini and Federico, 1989). In soybean, inhibitors of diamine oxidase activity decreased both H2O2 and lignin content in the hypocotyls (Su et al., 2005). Germin-like oxalate oxidases can also generate H2O2 within the cell wall using oxalate and O2 as substrates (Lane et al., 1993). Accumulation of the oxalate oxidase transcript and protein was detected, e.g, in the vascular bundles of germinating wheat embryos (Caliskan and Cuming, 1998). Deposition of lignin-like material also correlated with increased oxalate oxidase and peroxidase activities in aluminium-stressed wheat seedlings (Hossain et al., 2005), and three germin-like genes had similar expression patterns with the monolignol biosynthetic genes during Arabidopsis inflorescence stem development (Ehlting et al., 2005). H2O2 can also be produced through plasma membrane NADPH oxidase (Lamb and Dixon, 1997). The enzyme uses cytoplasmic NADPH and apoplastic O2 as substrates to produce apoplastic superoxide O2•-, which is mutated to H2O2 either spontaneously (slow reaction) or by a CuZn-superoxide dismutase (SOD). Production of O2•- and H2O2 were colocalised in lignifying vascular tissue in spinach (Spinacia oleracea L.) and Zinnia, and inhibitor treatments suggested that O2•- was generated through an NADPH oxidase (Ogawa et al., 1997; Ros Barcelo, 1998). SOD has also been localised to the cell walls of vascular tissue, for example in spinach (Ogawa et al., 1997). Several NADPH oxidase-like genes appeared coregulated with the monolignol biosynthetic genes in Arabidopsis, further supporting their role in lignification (Ehlting et al., 2005). 29 As mentioned above, apoplastic peroxidases can also generate O2•- and subsequently H2O2 in a reaction that uses, at least in vitro, NADH as a reductant instead of the monolignols (Halliwell, 1978; Mäder and Amberg-Fisher, 1982). For this reaction to occur in vivo, NADH and a system for its regeneration should be present in the apoplast, but Otter and Polle (1997), for example, found an insufficient concentration of NADH in the apoplast of spruce needles. Other possible reductants for the peroxidasemediated O2•- production include cysteine and indole-3-acetic acid (Ferrer et al., 1990; Bolwell, 1996), but no further proof for these has been presented. Apparently, regulation of the H2O2 concentration in the cell wall is complex. Several enzyme systems for the generation exist, and it is utilised, in addition to peroxidases, also in non-enzymatic processes. One example is the H2O2 scavenging effect of ascorbate by returning the peroxidase-generated phenolic radicals into the ground state (Takahama and Oniki, 1997). H2O2 can also be used to generate hydroxyl radicals nonenzymatically via Fenton reaction for cell wall loosening (Fry, 1998). Tight regulation is needed to allow for the controlled expansion of the cells and to restrict lignification only to the cells destined to lignify, while maintaining the ability for massive production of reactive oxygen species during stress or pathogen attack. 2.6.3 Laccases Laccases belong to a family of multi-copper oxidases that includes also ascorbate oxidase, ceruloplasmin and bilirubin oxidase (Hoegger et al., 2006). They are found from plants, fungi, insects and bacteria. Plant laccases form a monophyletic group within the family (Hoegger et al., 2006). Laccases are characterised by a blue colour that results from a specific copper coordination. They contain two catalytic centres where altogether four copper ions are found. Coordination of the copper ions is based on conserved histidine motifs. Laccases oxidise most o- and p-phenols, and even monophenols, into radicals (R•), reducing molecular oxygen into water in the process (2) (Ducros et al., 1998). (2) 4 RH + O2 • 4 R + 2 H2O Plant laccases are thought to have a multiplicity of functions, such as lignin polymerisation (Sterjiades et al., 1992), wound healing (McCaig et al., 2005) and even iron oxidation (Hoopes and Dean, 2004; McCaig et al., 2005). In plants, laccases exist as multigene families (Ranocha et al., 1999; Sato et al., 2001). High expression of laccases in developing xylem has been observed in many tree species (Sterky et al., 1998; Sato et al., 2001). However, various expression patterns were observed for Arabidopsis laccases, suggesting that functions other than lignification also exist (McCaig et al., 2005). As phenoloxidases, laccases can polymerise monolignols into a lignin-like precipitate in vitro, and qualitatively the polymers are indistinguishable from peroxidase DHPs (Bao et al., 1993; Sterjiades et al., 1993). Many laccases also have high affinity to monolignols (Ranocha et al., 1999). However, transgenic poplars expressing laccase genes in antisense orientation showed no changes in lignin content or quality (Ranocha et al., 2002). Instead, soluble phenolics accumulated in the ray parenchyma cells and 30 detachments in the fibre cell wall layers were observed at the microscopic level. The authors suggested that laccase activity was needed for the polymerisation of some phenolic component(s) that was important for the cohesion of the cell wall layers (Ranocha et al., 2002). Interestingly, expression of the same laccase decreased in tension wood (Andersson-Gunnerås et al., 2006), which contains less lignin and decreased cohesion of the G-layer of the fibres (Pilate et al., 2004). Arabidopsis contains 17 laccase genes that are distributed into five phylogenetic families (McCaig et al., 2005; Cai et al., 2006). Knock-out mutant of the LAC15 gene, which is allelic to the transparent testa 10 (tt10) mutant (Pourcel et al., 2005), contained pale seeds as a result of incomplete proanthocyanidin polymerisation (Pourcel et al., 2005; Liang et al., 2006). The seed coat also contained 30% less lignin, which is the first genetic evidence for laccase involvement in lignin polymerisation (Liang et al., 2006). In addition, ferroxidase activity (Fe3+ Fe2+) has been identified for a laccase-like enzyme (Hoopes and Dean, 2004). The authors proposed that a laccase-assisted Fe2+ uptake from the apoplast would function to minimise the levels of free iron (Hoopes and Dean, 2004), which can react with H2O2 to produce reactive oxygen species (Fry, 1998). This was suggested as an alternative reason for the high expression levels of laccases that have been observed in xylem. 2.6.4 Other phenoloxidases Also other, non-blue copper phenoloxidases have been implicated in lignin polymerisation. CA oxidase activity was identified from the xylem of several conifers (Savidge and Udagama-Randeniya, 1992; Richardson et al., 1997) and later characterised as a cathecol (o-diphenol) oxidase (Udagama-Randeniya and Savidge, 1995). It is not known whether CA oxidases exist in woody angiosperms. The current view emphasises the role of peroxidases and possibly laccases in lignin polymerisation. 2.6.5 Redox shuttles The lignin-degrading fungi secrete peroxidases that oxidise either the lignin polymer directly (Johjima et al., 1999), or oxidise small MW compounds or ions that act as redox shuttles and withdraw electrons from polymeric lignin. An example of the latter is manganese peroxidase (Glenn and Gold, 1985; Jensen et al., 1996). Similarly, the use of redox shuttles in lignin polymerisation has been put forward (Westermark, 1982; Önnerud et al., 2002). According to this theory, an enzyme in the cell wall or on the plasma membrane generates an oxidising agent that is small enough to diffuse within the cell wall. It reacts with the free monolignols or the polymer, generating the respective radicals and returning to the ground state for re-oxidation. Westermark suggested this redox shuttle to be O2•- in complex with Ca2+, and demonstrated the formation of dimers from CA, although high Ca2+ concentration (50 mM) was needed (Westermark, 1982). Önnerud et al. (2002) used Mn3+, which was produced either nonenzymatically or by a fungal manganese peroxidase, in complex with oxalate for DHP synthesis from CA. The identity of the putative Mn-oxidising enzyme in the plant cell 31 wall is unclear. However, the redox shuttle-mediated oxidation appears to be chemically feasible and could be efficient in polymer oxidation, provided that a suitable enzyme–mediator pair exists in the cell wall. 2.7 Tissue cultures as models for lignin biosynthesis Some tissue culture lines of both angiosperms and gymnosperms can synthesise ligninlike polymers. In some systems, a fraction of the cells differentiate into tracheids or vessel elements, in which case lignin is partly or exclusively deposited into the secondary cell wall (Fukuda and Komamine, 1982; Eberhardt et al., 1993; Nose et al., 1995). Alternatively, extracellular lignin is formed into the culture medium (Simola et al., 1992; Messner and Boll, 1993; Lange et al., 1995; Nose et al., 1995). In most tissue culture systems, lignin formation is induced, either by fungal elicitors or water stress (Messner and Boll, 1993; Tsutsumi and Sakai, 1993; Lange et al., 1995) or a change in the growth hormones (Simola et al., 1992; Eberhardt et al., 1993). Sucrose has also been used as an inducer of lignin formation, for example in sycamore (Acer pseudoplatanus L.) and loblolly pine cell suspension cultures (Carceller et al., 1971; Nose et al., 1995), supporting the regulatory role of sucrose and the need for carbon during lignification (Rogers et al., 2005a). Tissue culture lignins appear to represent early developmental lignins, as they contain higher proportions of H units and condensed linkages than lignin isolated from wood (Brunow et al., 1990; Brunow et al., 1993; Lange et al., 1995). In this respect, they also resemble the in vitro synthetic lignins, DHPs. Still, the abundance of -O-4 and dibenzodioxocin structures in, e.g., spruce released suspension culture lignin (RSCL) argues for extensive end-wise polymerisation in the cell suspension (Brunow et al., 1998a). In this respect, tissue cultures provide an interesting model for lignin biosynthetic studies, allowing the characterisation of the polymer without harsh chemical extractions that potentially alter its structure. It can be argued that the free polymer without the cell wall context does not represent the native lignin; however, the native state in xylem is difficult to reach with the current methods. Transformation of a lignin-forming Pinus radiata tissue culture has also been reported, after which the cells retained the ability to differentiate into tracheids (Möller et al., 2003). This creates an ideal model system to study the function of the genes related to the biosynthesis of both cell wall and lignin. 2.7.1 Xylogenic Zinnia elegans tissue culture Fukuda and Komamine (1980, 1982) established a Z. elegans cell culture, in which mesophyll cells were induced by a low auxin/cytokinin ratio to transdifferentiate into tracheary elements, and to eventually go through programmed cell death (Groover et al., 1997). The process was synchronous and up to 50% of the cells differentiated. Secondary thickenings became visible after 48 h of the initiation of the culture and lignification started 12 h later, correlating with increases in the transcription or activities of monolignol biosynthetic genes/enzymes (e.g., Fukuda and Komamine, 1982; 32 Church and Galston, 1988; Demura et al., 2002; Pesquet at al., 2005). Inhibitors of phenylalanine synthesis demonstrated that lignification was regulated separately from differentiation, as no effect on the formation of secondary thickenings was observed while lignification was prevented (Sato et al., 1993). Lignification of the tracheary elements was shown to continue even after they had undergone programmed cell death. The monolignols and H2O2, needed for polymerisation, were likely supplied by the non-lignifying parenchyma-like cells in the culture (Hosokawa et al., 2001; Pesquet at al., 2005; Gómez Ros et al., 2006). Similar process was suggested to take place in xylem, as expression of monolignol biosynthetic genes and production of H2O2 have also been observed in the xylem parencyma cells in, e.g., tobacco, French bean (Phaseolus vulgaris L.) and Z. elegans (Hauffe et al., 1991; Smith et al., 1994; Ros Barceló, 2005). It was also demonstrated that extracellular dilignols can be incorporated into lignin and thus function as precursors for polymerisation (Tokunaga et al., 2005). Appearance of several peroxidase isoforms has been correlated with tracheary element differentiation and lignification (Fukuda and Komamine, 1982; Church and Galston, 1988; Sato et al., 1993; Lopez-Serrano et al., 2004; Pesquet at al., 2005; Sato et al., 2006). Highly similar peroxidases from Arabidopsis were also temporally and spatially linked with lignification (Sato et al., 2006). However, a role for laccases in lignin polymerisation could not be excluded, either, since high expression of laccase genes was found during the late stages of differentiation (Demura et al., 2002; Pesquet et al., 2005). 33 3 AIMS OF THE STUDY Given their dominance in northern boreal ecosystems, gymnosperms have been underrepresented in studies on lignin biosynthesis. Norway spruce (Picea abies (L.) Karst.) is the one of the main tree species in boreal forests and it is, after Scots pine, the second most common tree species in Finland. In the Finnish forest industry, spruce wood comprises nearly half of the utilised raw material and it is used in both the pulp and paper and the wood product industries (Peltola, 2006). The knowledge of lignin biosynthesis and of the polymer properties could be utilised, for example, in the design of pulping processes, making understanding of lignin biosynthesis also of economic importance. Therefore, the aim of this study was the characterisation of the lignin biosynthetic pathway in Norway spruce. We especially focused on the final polymerisation stage, for which the enzymes and regulatory mechanisms have been poorly defined. To identify the genes that are specifically involved in lignin biosynthesis and that could, e.g., function as the targets in gymnosperm breeding programmes, the roles of individual gene family members during developmental and stress-induced lignification were studied using EST sequencing and real-time RT-PCR. The following themes were adressed in the publications: Identification of the monolignol-oxidising and H2O2-producing enzyme systems that are responsible for lignin polymerisation, using as a model a Norway spruce tissue culture line that forms extracellular lignin. Characterisation of the secreted culture medium peroxidase/laccase enzymes based on their affinities to monolignols and on possible product specificities using the dibenzodioxocin substructure as a marker. Localisation of the dibenzodioxocin substructure in Norway spruce xylem. Identification of monolignol biosynthetic genes and peroxidase/laccase genes that are responsible for developmental lignification in Norway spruce, using EST sequencing and profiling of gene expression in different lignin-forming tissues. Comparison of developmental and stress-induced lignification at the gene expression level. 34 4 MATERIALS AND METHODS The materials and methods used in this study are described in detail in the publications I to IV. A brief summary is provided in Table II. Table II The methods used in this study. The publications in brackets denote that the methods were conducted only by the co-authors in the respective publications. Method Analysis of phenolics by HPLC cDNA cloning cDNA library construction DHP production ELISA Enzyme activity assays Hapten conjugation for antibody production Isoelectric focusing NMR spectroscopy PCR Protein purification RACE Real-time RT-PCR RNA isolation SDS-PAGE Sequence analysis and bioinformatics Transmission electron microscopy Publication (I) III IV I, (III) II I, III II I, III (I), (III) III III III IV III, IV III III, IV (II) 35 5 RESULTS AND DISCUSSION 5.1 The localisation and role of dibenzodioxocin structures in lignin (II) The degradability of lignin during pulping is largely determined by its composition and structure (Baucher et al., 2003). Therefore, the quantity and localisation of dibenzodioxocin (5-5-O-4; Figure 2B) in the cell wall in part affects the pulping efficiency of wood. For the localisation of the structure, an antiserum was raised in rabbits against the dibenzodioxocin structure that was coupled to keyhole limpet hemocyanin. Specificity of the antiserum was tested using indirect competitive ELISA. Microtiter plates were coated with dibenzodioxocin, and different structural model compounds were used to compete with the immobilised antigen for binding to the antidibenzodioxocin serum. The specificity of the antiserum was at least 1000-fold, as none of the tested compounds, except dibenzodioxocin itself, could inhibit the binding (II, Fig. 1b). The antiserum was used for the immunolocalisation of the dibenzodioxocin structure in the xylem cell walls of spruce by transmission electron microscopy. The antigen was detected in the S2+S3 layer of young tracheids that contained developing cell walls. In mature tracheids, the dibenzodioxocin structures were found in more abundance from all layers of the cell wall, with the highest densities again in the S2+S3 layer (II, Fig. 3 and Table 1). Although lignification begins from the cell corners and middle lamella already during the synthesis of the S1 layer (Terashima et al., 1988), the dibenzodioxocin structures appeared in the middle lamella only in the mature tracheids. This indicates that lignification in the middle lamella and S1 layer is completed only during the final lignification phase, for example, after the vacuolar rupture during the first stages of the programmed cell death (Groover et al., 1997). Late appearance of the structure is partly explained by the coupling mechanism. The formation of dibenzodioxocin is preceded by the formation of the 5-5 linkage on the polymer, which requires the formation of two radicals on the polymer. This polymer cross-coupling takes place when the concentration of CA approaches zero in the reaction zone (Brunow et al., 1998b). Once formed, the 5-5 linkage has a low redox potential and it cross-couples easily with CA, forming dibenzodioxocin (Figure 5). Lignification of the cell wall was shown to proceed in distinct phases (Fukushima and Terashima, 1991), which may be a result of fluctuations in CA concentration in the cell wall, which could thus regulate dibenzodioxocin formation. The dibenzodioxocin structure was thought to be a branching point in lignin, a start of a new lignin chain (Brunow et al., 1998b). The idea is contradictory to the late appearance of dibenzodioxocin structures observed here. Using confocal laser-scanning immunofluorescence microscopy for the detection of the dibenzodioxocin antigens in Norway spruce xylem sections, the structure was localised more precisely to the S3 layer lining the tracheid lumen (Kukkola et al., 2004). Taking into account the difficulty of oxidising the dibenzodioxocin structure either enzymatically or non-enzymatically (Sipilä et al., 2004), the results suggest that dibenzodioxocin could, in fact, be a terminal structure in lignin. It is possible that it creates a protective layer in the tracheid lumen against, e.g., pathogens, or seals the cell wall effectively from water and solutes. 36 Figure 5 The biosynthesis of the dibenzodioxocin structure. Under low concentrations of CA, cross-coupling of polymeric end-groups is possible and 5-5 linkages are formed. These structures are easily oxidised and cross-couple with a CA radical, resulting in dibenzodioxocin formation. Adapted from Brunow et al. (1998b) 5.2 Lignin polymerisation in the Norway spruce tissue culture (I, III) The Norway spruce tissue culture line A3/85 that was initiated from an immature zygotic embryo, produces extracellular lignin-like material (Brunow et al., 1990; Simola et al., 1992). For functional characterisation of the secreted proteins that are potentially involved in lignin polymerisation, apoplastic proteins were fractionated and their catalytic properties were studied using enzyme activity assays. The proteins were also used for the synthesis of DHPs to study the possible differences in polymer structure. 5.2.1 Peroxidase activity is needed for lignin formation (I) Both peroxidase and laccase activities were found in the spent culture medium. Isoelectric focusing (IEF) gels that were stained for peroxidase activity revealed 10 to 12 peroxidase isoenzymes in the culture medium (I, Fig. 1). The isoenzymes were divided into acidic, slightly basic and highly basic peroxidases based on their isoelectric points (pI). Peroxidases that were ionically or covalently bound to the cell wall were also found. The covalently bound fraction contained only acidic isoenzymes, whereas the more numerous ionically bound peroxidases represented the whole pH range. Roughly, 15% of apoplastic peroxidase activity was either ionically or covalently bound to the cell wall, the rest remaining soluble in the medium (I, Table 1). Laccase activity in the culture medium was minimal compared with the secreted peroxidase activity (I, Table 1), and only a single laccase isoform was detected on an IEF gel (I, Fig. 2). This laccase, however, was also able to oxidise CA and form a polymer, which contained most of the typical lignin structures and could not be distinguished from the DHP produced with peroxidases (I, Fig. 3). Thus the initial DHP experiment could not distinguish whether the peroxidase or laccase activity was more important for lignin polymerisation, although the low laccase activity in the culture medium suggested a major role for peroxidases. The necessity of peroxidase activity for the production of RSCL was demonstrated in peroxidase inhibition studies, where the addition of KI, a H2O2 scavenger, into the culture medium resulted in a drastic 37 reduction in the amount of produced RSCL (Figure 6). Similarly, in a Pinus taeda suspension culture that produces extracellular lignin after induction with 8% sucrose, lignin formation was inhibited after KI addition (Nose et al., 1995). mg RSCL / 25 ml culture 8 7 6 5 4 3 2 1 0 0 2.5 5 10 KI (mM) Figure 6 The effect of potassium iodide on the formation of the released suspension culture lignin in the Norway spruce tissue culture. 5.2.2 Coniferin -glucosidase activity in the culture medium (I) -glucosidase activities in the tissue culture were studied by comparing the utilisation of a synthetic substrate, 4-nitrophenol glucoside (4-NPG), and the natural substrate, coniferin. Coniferin- -glucosidase activity was enriched in the culture medium (18.2% of the total coniferin- -glucosidase activity) compared with the 4-NPG activity (3.2% of the total 4-NPG glucosidase activity) (I, Table 1), suggesting that a -glucosidase specialised in coniferin hydrolysis exists in the culture medium. The coniferin- -glucosidase activity was three orders of magnitude lower than the total peroxidase activity in the culture medium, casting doubt on the role of coniferin-glucosidase in lignin biosynthesis. However, it is possible that only a part of the total peroxidase activity participates in lignin polymerisation and the total activities could no be compared as such. It could not be ruled out, either, that CA would be transported as an aglucone, since no coniferin was found in the culture medium 24 h after initiation (I). 5.2.3 NADH oxidase activity in the culture medium (I) Peroxidase activity in the apoplast is driven by the availability of H2O2, which is used as the ultimate electron acceptor in CA oxidation. However, peroxidases can also produce H2O2 by oxidising NADH in a reaction involving a superoxide radical (Halliwell, 1978; Mäder and Amberg-Fisher, 1982; Liszkay et al., 2003). A significant NADH oxidase activity, which could be responsible for H2O2 production, was found in the culture medium (I, Table 1). However, for this reaction to continue, NADH and a system for its regeneration should exist in the apoplast. NADH content in the culture medium could not be measured because some components in the medium interfered with the assay. One possibility for regeneration of NADH is a wall-bound malate dehydrogenase 38 utilising malate supplied by the protoplast (Gross et al., 1977). Low malate dehydrogenase activity was found from the cell wall fraction in the spruce tissue culture (I, Table 1). However, no conclusive evidence for peroxidase involvement in H2O2 production was obtained. Other possible enzymes for H2O2 generation were not tested here. 5.2.4 Functional characterisation of the secreted peroxidases (III) and laccase For the determination of catalytic properties, purification of the individual enzymes was needed. This proved to be difficult, as (putatively) phenolic compounds and carbohydrates secreted by the cells interfered with the proteins during chromatography. The use of ammonium sulphate precipitation and hydrophobic interaction chromatography decreased the unspecific binding of these compounds to the proteins. The high salt concentrations that are used in these methods may especially influence the hemicelluloses and pectin that contain charged uronate residues. Together with lectin affinity chromatography, peroxidase activity was separated from most contaminating proteins, including the laccase activity. Gel filtration separated the acidic and basic peroxidase isoenzymes based on their MW (50-60 kDa and 35-40 kDa, respectively). The basic peroxidases were the target in the further purification process for two reasons. Firstly, highly basic isoenzymes were common to both the developing xylem of Norway spruce and the lignin-forming tissue culture, whereas the other xylem peroxidases differed from the ones secreted into the culture medium (data not shown). Secondly, we had identified from the tissue culture basic peroxidase isoenzymes that were bound to RSCL (see below). PaPX4 and PaPX5 are specialised in monolignol oxidation Two highly basic peroxidases, PaPX4 and PaPX5, were purified to homogeneity using cation exchange chromatography (III, Fig. 1). Their catalytic properties were studied with enzyme activity assays using the three monolignols as substrates. pCA and CA were readily oxidised by both PaPX4 and PaPX5. The enzymes had high affinity towards both monolignols, with apparent Km values between 11.2 and 32.0 µM (III, Table 3). Based on Km and catalytic efficiency values (Vmax/Km), PaPX4 preferred CA over pCA as a substrate, with a 6-fold difference in Vmax/Km. PaPX5 oxidised both pCA and CA equally well, but the 2-fold difference in Vmax/Km suggested preferential pCA oxidation. The difference is, however, small enough to allow both monolignols to be oxidised, and other regulatory factors, such as pH in the cell wall could significantly affect catalytic preferences. In the tissue culture, the pH of the culture medium changed from ca. 5.2 to 4 during the first 24 h of culture, and was stable for the next four days (I). SA was a poor substrate for both isoforms. This could be expected, as gymnosperm lignin does not contain syringyl units. The catalytic properties of HRP were also determined to compare this model peroxidase to the spruce tissue culture peroxidases. The apparent Km values of HRP 39 (type II) for monolignols were 10-fold compared with the tissue culture peroxidases (III, Table 3), indicating a specialisation of PaPX4 and PaPX5 for monolignol oxidation. Despite the high Km values, the catalytic efficiency of HRP in monolignol oxidation was in the same range as for PaPX4 and PaPX5. This discrepancy could be explained by the open and flexible aromatic substrate binding site and the exposed heme edge of HRP C (Henriksen et al., 1998 and 1999). For small substrates, only weak interactions with the enzyme are possible, leading to promiscuity in substrate specificity. The high affinity of PaPX4 and PaPX5 for monolignols might lead to a slower exit rate of the monolignol radical from the substrate access channel, thus slowing down the reaction. The distance between the substrate binding site and the heme is also critical, as shorter distances facilitate electron transfer between the molecules. The substrate access channel in monolignol-oxidising peroxidases contains similar amino acid residues For the cDNA cloning of the PaPX4 and PaPX5 genes, the purified proteins were digested with trypsin and the resulting peptides were sequenced. After RT-PCR with degenerate primers and RACE, full-length cDNAs were cloned. The cloned sequences were 82% identical at the nucleotide level, and 80% identical and 88% similar at the amino acid level. Both sequences coded for proteins with a putative signal peptide for secretion out of the cytosol. The calculated pIs of PaPX4 and PaPX5 were 8.2 and 8.8, respectively, whereas when estimated from a SDS-PAGE gel, both had a pI of 9.5 to 10. The difference is due to the effect of the bound Ca2+ ions and heme which are known to increase the positive charge up to two units (Welinder et al., 2002). Based on sequence alignments, both PaPX4 and PaPX5 contained all the structurally conserved residues typical for class III peroxidases. The substrate specificity of enzymes is determined by the structure of the active site, i.e., amino acids. In the Arabidopsis peroxidase ATPA2, several hydrophobic contacts were observed between the enzyme and the monolignols (Østergaard et al., 2000). The amino acids shown to be in contact with pCA or CA in ATPA2 were identical to those in PaPX4 and PaPX5 (Figure 7). As all three peroxidases were efficient in CA and pCA oxidation, this suggests that a preferential structural organisation for monolignol oxidation might exist. Many of the residues were also conserved in other lignin-forming peroxidases, but not in all (Figure 7). This could reflect the evolutionary development of several structural solutions for monolignol oxidation, or suggest that some peroxidases are more likely to be involved in SA oxidation. In ATPA2, the binding of SA to the active site was sterically prevented by Ile138 and Pro139 (Østergaard et al., 2000; Nielsen et al., 2001). Based on the high conservation of Pro139 in plant peroxidases, the authors proposed that inability to oxidise SA is a general property of plant secretory peroxidases. However, peroxidases that contain Pro139 but still oxidise SA efficiently have been found, at least in poplar and Zinnia (Sasaki et al., 2004 and 2006; Sato et al., 2006). Probably these peroxidases contain a different structural organisation in their active site that accomodates SA. Alternatively, a long-range electron transfer chain from the surface of the protein to the active site may exist, similarly to fungal lignin peroxidase from Phanerochaete chrysosporium (Johjima et al., 1999). 40 41 PaPX4 and PaPX5 produce typical synthetic in vitro lignins different from RSCL The purified peroxidases were used to catalyse the oxidative coupling of CA to produce DHP. The conditions simulated end-wise lignin biosynthesis in xylem in the sense that both CA and H2O2 were slowly fed into the reaction. The resulting polymer was quantified using 2D HSQC 13C-NMR (Heikkinen et al., 2003). The analysis focused on the quantity of -O-4 and dibenzodioxocin structures because their formation is typical for end-wise polymerisation occurring in wood (Sarkanen, 1971; Brunow et al., 1998b). Both PaPX4 and PaPX5 formed DHP from CA, although the yield with PaPX4 was 6-fold higher than with PaPX5. This probably reflected the difference in catalytic efficiencies. Structurally the DHPs were similar, with approximately 40% -O-4, 35% -5 and 20% . The amount of dibenzodioxocin was <2% with both enzymes (III, Table 4). A reference DHP using HRP type II was also synthesised. It was structurally similar to the DHPs produced by the spruce peroxidases and no significant differences were found (III, Table 4), despite the fact that the spruce peroxidases were specialised in monolignol oxidation. The total concentrated culture medium proteins were also used for DHP synthesis, and again the structure was similar to that of other DHPs (data not shown). All DHPs differed structurally from RSCL, which contained more -O-4 and dibenzodioxocin structures, and less -5 (III, Table 4). Therefore, the structure of RSCL could not be replicated by DHP synthesis, not even using the full complement of soluble culture medium proteins. This indicates that some regulatory factors operate during the culture. One factor contributing to the structural differences between the DHPs and RSCL is the rate of monolignol feeding, which despite the slow rate during DHP production is far less controlled than monolignol secretion into the apoplast by cultured cells. Concentration of CA was shown to be a major determinant of polymer structure in cross-coupling studies (Brunow et al., 1998b). Also pH was shown to affect the structural properties of DHP by increasing the proportion of -O-4 at lower pH (Ämmälahti and Brunow. 2000). In addition to coupling chemistry, changes in pH can influence the enzymatic activity of, for example, peroxidases, and thereby regulate the polymerisation process. However, the differences in the pH between DHP synthesis and suspension culture were small, as the DHPs were synthesised at pH 4.5 whereas the pH in the culture was ca. 4 during the polymerisation phase (I). The concentrated culture medium proteins could also lack some components that contribute to the synthesis of RSCL. The MW cut-off limit during the concentration was 10 kDa. Therefore, it is likely that small MW proteins were lost during the concentration, but, e.g., dirigent-like proteins, shown to be involved in lignan biosynthesis and also suggested to guide the initial phases of lignin biosynthesis (Davin et al., 1997; Gang et al., 1999), were mostly retained (monomer MW ca. 26 kDa). Dirigent proteins are expressed in the tissue culture based on the Norway spruce EST collection, but their role is unknown. Di- and oligomers of CA were found in the culture medium (I); however, it is not known if they are true lignans that could be synthesised through dirigent proteins. Alternatively, the dirigent-like proteins could be related to a stress/defence response, which was the prevalent role of dirigent-like proteins in spruce shoot apex (Ralph et al., 2006a). However, two dirigent-like genes of 42 Sitka spruce were more strongly expressed in the woody shoot base than in the tip, and could possibly have a role in lignin biosynthesis (Friedmann et al., 2007). Thus, the DHP experiment could not conclusively determine the role of PaPX4 and PaPX5 in the polymerisation of RSCL. It is likely that several apoplastic peroxidases are involved in the polymerisation. It can also be speculated that if different isoforms are responsible for the production of monolignol radicals and radicals on the polymer, a single isoform might not be sufficient to produce native-like lignin. PaPX4 and PaPX5 could also be involved in lignan biosynthesis in the tissue culture. They were not expressed in developing spruce xylem (IV). Catalytic properties of the culture medium laccase The laccase activity in the culture medium was also purified to homogeneity using cation exchange chromatography. This oxidase activity was designated as laccase because of the blue colour of the enzyme, and due to the high homology of the Nterminal sequence to pine laccases (data not shown). The Km value of the laccase for CA was very low, 3.5 µM, and it had a pH optimum of 4 (data not shown), which is typical for the apoplast and for the culture medium here. However, the specific activity was very low compared to the purified peroxidases. Therefore, although the low Km value indicates that the laccase is specialised in monolignol oxidation, the low specific activity makes it unlikely that it could have a substantial role in CA oxidation in the tissue culture. 5.2.5 The cell wall matrix and other regulatory factors contribute to the structural outcome of lignin polymerisation (III) To compare RSCL and milled wood lignin (MWL) in more detail at the structural level, the quantitative 13C-NMR analysis was applied. The previously identified characters of RSCL (Brunow et al., 1990 and 1998a) were confirmed in this study. The linkage distribution in RSCL had features of both MWL and DHPs (III, Table 4). The high levels of -O-4, and the dibenzodioxocin levels equal to MWL showed that the synthesis of RSCL contained characters of end-wise polymerisation (Sarkanen, 1971). On the other hand, the high amount of linkages was reminiscent of DHP synthesis, which is characterised by a high degree of CA dimerisaton. Coupling of the dimers into a higher MW polymer results in a high proportion of free side chains and phenolic hydroxyl groups (Sarkanen, 1971), also found in RSCL (Brunow et al., 1993). The differences between MWL and RSCL emphasised the importance of the cell wall matrix and of the microenvironment in the reaction zone during lignin polymerisation (Brunow et al., 1993). The three-dimensional structure of the cell wall matrix has been shown to affect the three-dimensional structure of the lignin polymer (Donaldson, 1994). The potential effects of carbohydrates were also demonstrated by the addition of pectin to DHP synthesis. Compared with the control, a higher MW polymer in complex with pectin was formed (Cathala et al., 2001). 43 5.2.6 Lignin-bound peroxidases and laccases When the lignin precipitate was extracted with 1 M NaCl, several proteins were released according to SDS-PAGE (data not shown). When the extracted proteins were separated on an IEF gel and stained for peroxidase or laccase activity, basic peroxidase isoenzymes and two oxidases were found (Figure 8). Due to the high number of peroxidase isoenzymes at the alkaline, low-resolution end of the IEF gel, it could not be resolved if the lignin-bound peroxidases were the same as those in the culture medium. However, the laccases were better resolved on the gel, suggesting that one of the laccases was the same as the one in the culture medium, while the other was specifically bound to lignin. We were not able to obtain enough of the bound laccase protein for Nterminal sequencing, so no conclusive evidence for this was obtained. The peroxidase isoforms were purified and internal peptides were sequenced. According to the sequences, they were novel peroxidase isoforms (Warinowski, Koutaniemi and Teeri, unpublished). Studies on their role in DHP synthesis are currently under way. We also tested the ability of the culture medium proteins to bind to different lignin preparations. The culture medium proteins were incubated with technical lignin, which is isolated from the waste liquor of a pulping process. The lignin was washed with distilled water, and the bound proteins were released from lignin with 1 M NaCl. Based on an IEF gel stained for peroxidase activity, the basic peroxidases were enriched in the bound fraction, and none of the acidic proteins were able to bind (Figure 8). Similar results were obtained with MWL, with additional binding of the pI 8.4 peroxidases. Extraction of untreated technical lignin was not performed as a control; however, it is unlikely that any active protein could remain after the pulping process. The ability to bind to lignin is intriguing. Lignin is a hydrophobic polymer with no net charge. Although the phenolic hydroxyl group can act as an acid, its pKa is 9.9 and it is protonated and thus uncharged in the acidic pH of the cell wall. How can the proteins then be released from RSCL with a high salt concentration? Possibly, the proteins have affinity to some other component in RSCL, for example, hemicelluloses or pectin could be present. Anionic pectin is one of the few charged plant cell wall polymers, to which cationic peroxidases could bind through ionic interactions. Specific pectin-binding motifs have also been found from zucchini peroxidases (Carpin et al., 2001). Of the spruce peroxidases, at least PaPX4 contained the pectin binding motif. Affinity to pectin could direct peroxidases to certain areas in the cell wall, e.g., to middle lamella that is mainly composed of pectin. A specific affinity of the peroxidases for lignin itself is also possible. In this case, the interaction would probably be hydrophobic. These interactions could be disrupted in high salt concentration because of conformational changes in the proteins. The pectin binding site of the zucchini peroxidase was located on the surface of the enzyme, on the opposite side of the substrate access channel where it did not interfere with the activity of the enzyme (Carpin et al., 2001). Similarly, a hydrophobic patch on the peroxidase could result in an affinity for lignin, even for specific structures in lignin, and thereby create another level of control in lignin biosynthesis. 44 Figure 8 Isoelectric focusing gels of lignin-bound proteins stained for peroxidase (A,C) or laccase (B) activity. (A,B) Culture medium proteins and the proteins extracted from the released suspension culture lignin (RSCL) with 1 M NaCl. (C) Culture medium proteins were tested for their ability to bind reversibly to technical lignin in vitro. Bound proteins were extracted with 1 M NaCl. 5.3 Norway spruce EST collection (IV) For the identification of more peroxidase and other lignin biosynthetic genes, and to gain a wider perspective on lignin biosynthesis in Norway spruce, three cDNA libraries were generated for EST sequencing. Two of the libraries represented the tissue culture at 24 and 72 h after initiation of the suspension. The third library represented the developing xylem of spruce. The xylem was collected at the beginning of August, which in southern Finland is the time of active lignification and of the shift between earlywood and latewood formation (Marjamaa et al., 2003). Altogether 7444 EST sequences were obtained, the majority of them from the 5' end. The ESTs were clustered into 3831 clusters (unigenes), of which 70% contained only one sequence (IV, Table 1). For 27% of the unigenes, no match was found in the EMBL Uniprot database when the confidence limit was set to expect value 1e-5 and sequences containing <20% probable protein coding region were removed. These 1036 sequences represent potential novel unknown proteins. Among the 20 most abundant unigenes in the EST database were ubiquitin, many cytoskeleton and defence-related genes, two laccases and several genes involved in methyl transfer reactions (Table III). 45 Table III Annotations of the 20 most abundant unigenes among the 7444 individual sequences in the spruce EST database. Unigene Accession Annotation Cluster size C_K0000200010G06F1 C_K0000200026A07F1 C_K0000200017C12F1 C_K0000400028F01F1 C_K0000400034G01F1 C_K0000400069H11F1 C_K0000100015E10F1 C_K0000400050C03F1 C_K0000400053H11F1 C_K0000400053B01F1 C_K0000400003F02F1 C_K0000400063E11F1 C_K0000400057B08F1 C_K0000400058E01F1 Ubiquitin Antimicrobial peptide 65 62 Endochitinase A Caffeoyl-CoA O-methyltransferase 56 43 Tubulin alpha-3 chain Caffeic acid 3-O-methyltransferase 42 41 Methionine synthase Laccase 40 38 S-adenosylmethionine synthetase Translationally controlled tumour protein homolog 35 30 S-adenosylmethionine synthetase 29 27 Cytochrome P450 98A2 Sucrose synthase Elongation factor 1-alpha Pathogenesis-related protein Pathogenesis-related protein Caffeoyl-CoA O-methyltransferase Blue copper protein (laccase) Serine hydroxymethyltransferase 26 26 25 24 22 22 20 20 C_K0000400056E06F1 C_K0000200021F01F1 C_K0000200011D03F1 C_K0000400044D12F2 C_K0000400056G05F1 C_K0000400015E12F1 5.3.1 Monolignol biosynthetic genes and peroxidase/laccase genes The EST database contained all the genes needed for monolignol biosynthesis (IV, Table 2). For most catalytic steps, several unigenes were found. However, in each gene family, only one of the unigenes contained ESTs from both the tissue culture and the developing xylem. Moreover, the unigene common to both libraries contained 1.8 to 10 times more ESTs than all the other unigenes together, suggesting that this set of unigenes represents the main monolignol biosynthetic pathway in Norway spruce. cDNAs for nearly all monolignol biosynthetic enzymes have been cloned previously from pine (MacKay et al., 1995; Li et al., 1997; Zhang and Chiang, 1997; Anterola et al., 2002). Putative spruce orthologs with >96% similarities at the amino acid level were identified in the spruce EST database, indicating a high conservation of monolignol biosynthetic gene sequences within the gymnosperms. Amino acid sequences of the full-length or nearly full-length unigenes from 4CL, C3H, CCOMT and CCR families were used for a phylogenetic analysis, together with the pine and Arabidopsis monolignol biosynthetic genes. The spruce unigene having highest similarity to the Arabidopsis 'lignification toolbox' genes (Raes et al., 2003) was, for each family, the one that was highlighted for a role in lignin biosynthesis by the EST counts (data not shown). 46 Peroxidase and laccase ESTs were numerous and they were clustered into 13 and 6 unigenes, respectively. Here, too, only one unigene contained ESTs from both library types (IV, Table 3). However, the distribution of ESTs into different unigenes was more even and association of these isoenzymes with lignin polymerisation was difficult. Based on EST counts, lignin biosynthetic genes were strongly expressed in both tissue types, as 2.7% of the developing xylem and 1.9% of the tissue culture ESTs were annotated as monolignol biosynthetic genes. If peroxidase and laccase unigenes were included, the figures were 4.4% and 2.4%, respectively. The expression of lignin biosynthetic genes in the two tissue culture libraries was similar, suggesting that lignin biosynthesis is constitutive in the tissue culture, at least between 24 and 72 h. More differences were observed when the tissue culture was compared with the developing xylem. Based on EST counts, the early monolignol biosynthetic genes from PAL to 4CL were more strongly expressed in the tissue culture than in developing xylem (IV, Table 2). This could be related to a more pronounced role for the early genes in the biosynthesis of other phenylpropanoids (Hu et al., 1998; Kao et al., 2002). Especially the methyltransferase genes, CCOMT and COMT were more abundantly expressed in the developing xylem (IV, Table 2). A high expression of these genes appears typical for xylem, and it was thought to reflect the importance of methyl groups in directing the carbon flux into lignin (Sterky et al., 1998; Whetten et al., 2001). Laccases that are potentially involved in lignin polymerisation were almost exclusively expressed in xylem based on the ESTs (IV, Table 3). 5.3.2 Genes of potential H2O2 producing enzymes The EST database was also searched for unigenes that could be related to H2O2 generation. In general, the expression levels were lower than those of monolignol biosynthetic genes. Eight germin-like ESTs were found and they clustered into four unigenes (data not shown). In addition, two ESTs that belonged to the same cluster, annotated as NADPH oxidase, and one amine oxidase singleton were also found. Based on the slightly higher representation of the germin-like oxalate oxidases, they could be primarily responsible for H2O2 synthesis in spruce. Both germin-like genes and NADPH oxidase genes were co-regulated with the monolignol biosynthetic genes during the development of the Arabidopsis inflorescence stem (Ehlting et al., 2005). 5.4 Expression profiling of lignin biosynthetic genes using real-time RT-PCR (IV) The expression of the identified lignin biosynthetic genes was studied in more detail to verify the expression patterns suggested by the EST counts and to explore their roles under specific developmental conditions. Similar approach has been used to identify the monolignol biosynthesic genes in lignifying Sitka spruce shoots (Friedmann et al., 2007) and in different tissues of maize (Guillaumie et al., 2007b) using micro/macroarray hybridisations. We used real-time reverse transcription followed by polymerase chain reaction (real-time RT-PCR), since the method is very sensitive and specific when 47 differentiation between highly similar transcripts is needed (Huggett et al., 2005). Several tissues were analysed, including developing xylem from 1- and 40-year-old spruces (representing the development of juvenile and mature xylem types, respectively), the lignin-forming tissue culture at 72 h after initiation of the suspension, phloem and needles from the 40-year-old tree, and roots from the 1-year-old seedlings. The absolute transcript levels were quantified using standard curves for each amplified sequence. In addition to the unigenes, a few previously cloned lignin biosynthetic genes were included in the analysis. Each gene was analysed from two biological replicate samples. 5.4.1 Coordinated, high expression in developing xylem samples as a marker of lignin biosynthetic genes The EST counts suggested that certain gene family members had a primary role in lignification in Norway spruce. This was verified in the real-time RT-PCR experient. In each gene family, except PAL and CCOMT, the putative lignification-related unigene was significantly more highly expressed than the other gene family members in the developing xylem of both 1- and 40-year-old spruces (IV, Fig. 3). For PAL and CCOMT, the differences were significant only in the 1-year-old seedlings. This was also evident in a hierarchical clustering of the expression levels (Figure 9). A xylem-specific cluster of highly expressed genes included a single unigene for each catalytic step. Only for CCOMT, two unigenes were included in this xylem-expressed set. These unigenes were highly similar in sequence, and could represent a recent gene duplication. Interestingly, PaC4H3/5 and PaCCOMT1 appeared in a different cluster than the rest of the genes. This could suggest a transcriptional regulation different from the other genes. Such a mechanism could be, e.g., the AC elements in the promoter regions of the genes, which were suggested to control the expression of all genes specific to G lignin in Arabidopsis (Raes et al., 2003). Peroxidase expression levels were, in general, lower than those of monolignol biosynthetic or laccase genes. Developing xylem in both 1 and 40-year-old trees expressed a single predominant peroxidase, PaPX13 (IV, Fig. 4). In addition, PaPX8, PaPX9, PAPX14 and PaPX15 had considerable expression, the latter two only in the 40year-old spruce. These genes can be considered the strongest candidates for lignin polymerisation, although none of the peroxidases clustered together with the xylem specific monolignol biosynthetic genes in the cluster analysis (Figure 9). This kind of clustering has been observed in microarray experiments in Arabidopsis (Ehlting et al., 2005). The lack of correlation in spruce data could be due to the fact that clustering was based on absolute expression levels (which for peroxidases were lower than for monolignol biosynthetic genes), not on fold-change as in microarray experiments. Also, as peroxidases are particularly stable enzymes (Fagerstedt et al., 1998), the transcript levels may poorly reflect the amount of active protein in the cell wall. Given the high number of peroxidase genes in Arabidopsis and rice (Tognolli et al., 2002; Passardi et al., 2004), only a subset of spruce peroxidases was analysed here and other xylem-expressed isoforms likely exist. 48 Contrary to peroxidases, laccase genes were highly and specifically expressed in all developing xylem samples (IV, Fig. 5). Two laccases, PaLAC2 and PaLAC4, also clustered together with the monolignol biosynthetic genes (Figure 9). This could equal a role in lignin biosynthesis; alternatively, the correlation could be related to other processes in wood formation, e.g., secondary cell wall synthesis. These processes were shown to take place simultaneously both at gene expression level (Friedmann et al., 2007) and at enzymatic level (Terashima et al., 1988). However, the phenoloxidase activity of laccases is well established (Sterjiades et al., 1992; Liu et al., 1994; Ranocha et al., 2002; Pourcel et al., 2005). Therefore, if laccases are involved in the biosynthetic processes other than lignin polymerisation, it is likely related to the oxidation and cross-linking of some phenolic component in the cell wall, as suggested also by the transgenic poplars expressing a laccase antisense construct (Ranocha et al., 2002). At the gene expression level, developing xylem from the juvenile and mature trees was very similar in respect of both monolignol biosynthesis and polymerisation (Figure 9). This indicates that no major changes in lignin biosynthesis take place during the maturation of a tree. Rather, structural differences in lignin between the cell wall layers have been observed (Pinçon et al., 2001; Kukkola et al., 2003). Figure 9 Hierarchical clustering of the genes and samples based on the expression levels determined by real-time RT-PCR. The colour scheme maximum was set to 4000 for better resolution of the colours; LAC1 expression in young compression wood was ca. 8000. YCW, young compression wood from 1-year-old spruce; X, xylem from 40-year-old spruce; YVX, young vertical xylem from 1-year-old spruce; N, needle; R, root; P, phloem; Hacontrol, non-infected phloem; TC, tissue culture; Ha-infected, Heterobasidion annosum infected phloem. 49 5.4.2 Up-regulation of lignin biosynthetic gene expression under stress To compare the developmental lignification to stress-induced lignin formation, compression wood from bent 1-year-old seedlings and phloem from 32-year-old trees that were infected with Heterobasidion annosum (Fr.) Bref. (S-type) were also studied using real-time RT-PCR. Both types of stresses induce lignin formation (Vance et al., 1980; Timell, 1986). According to the real-time RT-PCR analysis, expression of the monolignol biosynthetic genes in compression wood was similar to that in vertical xylem, and only a few genes were significantly induced (IV, Fig 3). However, expression of peroxidase and laccase genes differed between the two samples. Induction of PaPAL2 and PaC4H3/5 could be responsible for an increased monolignol production leading to higher lignin content, while lack of any C3H induction could explain the more abundant H units in compression wood lignin (Figure 3; Timell, 1986). Expression of PaPX2, PaLAC1 and PaLAC2 was strongly up-regulated in compression wood (IV, Fig. 4 and 5). Although the variation in the expression levels was high for PaLAC1 and PaLAC2, the induction was significant and reproducible in both biological replicate samples. Whether the induction is needed for lignin polymerisation or for further crosslinking of other cell wall constituents is uncertain. H. annosum infection resulted in an increased lignin content in the infected bark samples compared with the non-infected controls (IV, Table V). However, similar increase in lignin was also seen in a wounded control, and therefore the lignin formation could be a response to wounding. Increased lignin levels were a result of a general up-regulation of the whole phenylpropanoid pathway, as many of the gene family members were significantly induced after H. annosum infection (IV, Fig. 6). The unigene predominant in developing xylem was in most cases also predominant in infected phloem. Apparently, the developmental and infection/wounding-induced monolignol biosynthetic pathways are mostly shared in spruce, with PaPAL2 and PaC3H2, however, having a defence-related role, since they were specifically induced in the infected phloem. Interestingly, PaC3H2 was exclusively expressed in the infected phloem sample, suggesting a primary function in defence. The total peroxidase transcript levels increased after H. annosum infection. The induced peroxidases were different from those predominant in xylem (IV, Fig. 6), indicating a specific defence response. PaPX3 was mainly responsible for the increase in peroxidase transcript levels. No induction was observed for laccase transcripts. However, laccase transcripts were induced in shoot tips of Sitka spruce after insect feeding (Ralph et al., 2006b), suggesting that some laccases have defence-related functions. Interestingly, PaPAL2 was induced both after pathogen infection and in compression wood. It appears to be responsible for the increase in the flux of phenylalanine into the phenylpropanoid pathway in different stress situations. PaPX2 and PaPX3 were also induced during both stresses, although to different extent. According to in situ hybridisation, PaPX2 was specifically expressed in the developing tracheids (Marjamaa et al., 2006), and accordingly, expression was detected in developing xylem samples also in this analysis. Thus, it could have a dual role in developmental and stress-induced lignification. PaPX3 transcripts were not detected by 50 in situ hybridisation; however, low expression in developing xylem samples was detected here, in addition to the strong induction in infected phloem. Its expression could depend more strongly on environmental cues. 5.4.3 Lignin biosynthetic gene expression in roots, phloem and needles Needles and developing phloem were also included in the real-time RT-PCR analysis, although lignin formation in these tissues is less pronounced. It is mostly confined to strands of vascular tissues, and fibres and sclerenchyma acting as supportive tissue (Esau, 1960). Roots were also analysed. Roots in, e.g., gymnosperm and angiosperm tree species undergo extensive secondary growth and lignification, but the roots analysed here were from 1-year-old seedlings, in which the lignifying tissue is less abundant. Accordingly, the expression of monolignol biosynthetic genes was mostly low in these tissues and it was difficult to distinguish the lignin-forming genes. It is also likely that in non-xylem tissues, products of the phenylpropanoid pathway other than lignin have more important roles, involving especially the PAL, C4H and 4CL genes. Within these families, the detected expression levels were more constant or even higher for the genes that were not associated with developmental lignification (IV, Fig. 4). Peroxidase expression patterns varied between the three tissues, and differed especially from xylem (IV, Fig. 5). This is in accordance with the diverse expression profiles and induction patterns observed for peroxidases in rice (Hiraga et al., 2000). Total peroxidase expression was highest in roots. A preferential expression in roots was also observed in Arabidopsis for nearly all peroxidases, suggesting that a variety of peroxidase activities are needed in the development and maintenance of root tissue (Welinder et al., 2002). Expression levels in newly formed needles were very low. Low expression levels were also found in Arabidopsis rosette (Welinder et al., 2002), suggesting a minor role for peroxidases in leaves/needles. Laccase expression in Norway spruce was confined to xylem, as only a single laccase, PaLAC3, was expressed in nonxylem tissues (IV, Fig. 5). Given the potential role of laccases in cell wall formation, a role for PaLAC3 in the formation or cross-linking of the primary cell wall could be speculated. In Arabidopsis, most laccases were expressed in a variety of tissues (McCaig et al., 2005). However, in tree species a xylem-preferred expression for laccases has been observed (Ranocha et al., 1999; Sato et al., 2001) (IV), and it could be specifically related to the formation of secondary xylem. 5.4.4 Lignin biosynthesis in the A3/85 tissue culture line - true native spruce lignin? One of the interests throughout this work was to define the validity of the tissue culture as a model for lignin biosynthesis. The real-time RT-PCR experiment established that the monolignol biosynthetic pathway in the tissue culture is similar to that in wood (IV, Fig. 4). The expression of the genes was not as high as in xylem, possibly reflecting the fact that only 10 to 15% of the cultured cells differentiate into tracheids. However, at least in Zinnia cell cultures, also mesophyll cells synthesised monolignols and could supply the tracheary elements with monolignols through the culture medium 51 (Hosokawa et al., 2001). Expression levels within gene families were also more even in the tissue culture than in the xylem, possibly indicating that the products of the other branches of the phenylpropanoid pathway, for example lignans, were actively formed. At least dimers and higher oligomers of CA were found in the culture medium (I). Whether the dimers are true lignans or soluble intermediates of lignin biosynthesis is not known. Contrary to the monolignol biosynthetic genes, a different but overlapping set of peroxidases was expressed in the tissue culture compared with the developing xylem (IV, Fig. 4). Differences in laccase expression were even more pronounced, as only a single laccase, PaLAC3, was expressed at a very low level in the tissue culture, whereas most of the other laccases were highly expressed in developing xylem (IV, Fig. 5). The differences in oxidative enzymes suggest that the polymerising enzymes differ between the tissue culture and wood. Hierarchical clustering of the samples, based on the gene expression profiles, showed that in respect of lignin biosynthetic gene expression in general, the tissue culture was most similar to H. annosum infected phloem (Figure 9). This probably reflects the stress caused by the suspension culture, i.e., possible mechanical stress due to rotation. Based on the ESTs, many stress and defence-related proteins were highly expressed in the tissue culture. Also, several defence-related proteins, such as thaumatin, osmotin and chitinase, were identified in the spent culture medium, based on N-terminal sequences of the proteins (data not shown). Thus, one definition of RSCL could be stress lignin, as suggested by the increased H units in lignin as well. Nevertheless, the structure of the lignin-like precipitate is the closest to MWL among all in vitro synthetic lignins (III). It allows the studies of a lignin-like polymer without the complexity of the cell wall. This is a major advantage compared to studies on wood lignin, the structure of which is inevitably changed during the harsh chemical isolation procedures. Based on the similarity in monolignol biosynthetic gene expression and in the structural composition of lignin, the tissue culture is a good functional model for lignin biosynthesis. It provides a versatile tool for, e.g., precursor feeding studies and monolignol glucosylation and transportation studies, and is particularly suitable for the studies on polymerisation mechanisms. 52 6 CONCLUDING REMARKS The view on lignin biosynthesis has been transformed during the last decade. Lignin is now often seen as a more plastic polymer that can compensate for a lack of certain subunits by incorporating other, also untraditional subunits. Debate on whether the resulting polymer is lignin or something unnatural still continues. Thinking from the plants' point of view, if the polymer fulfils the function of lignin by waterproofing the water-conducting elements, providing support and protecting against pathogens, it is lignin. The quantity and quality of lignin are both also of economical importance in timber and in pulp and paper industry, as well as in plant-feeding animal husbandry. This study began with the search of a lignin polymerising enzyme. It was established that peroxidases are needed for lignin polymerisation in the Norway spruce tissue culture. However, not even the full complement of culture medium proteins could reproduce the linkage pattern of RSCL in vitro. This emphasised the importance of the rate of monolignol feeding, i.e., CA concentration, in the reaction zone. The quantitative structural comparison of RSCL and MWL illustrated the significance of the cell wall matrix in lignin polymerisation. Despite the differences, RSCL resembles MWL more than any in vitro synthesised lignin-like polymers, validating the use of the tissue culture as a model for lignin biosynthesis. The most important similarities between RSCL and MWL were the amounts of -O-4 and dibenzodioxocin structures. Both are formed through end-wise polymerisation, for which oxidation of the polymer is needed. This was shown to be a direct peroxidasecatalysed reaction (Sasaki et al., 2004), and it is possibly a key factor in lignin polymerisation. The reversibly lignin-bound peroxidases identified in this study have potential to execute this, or another yet unidentified role in lignin polymerisation, and deserve further interest in research. Two of the basic culture medium peroxidases were functionally characterised at the enzymatic level. Both PaPX4 and PaPX5 had high affinity to monolignols. By comparison of the amino acid sequences of PaPX4 and PaPX5 to other peroxidase sequences, conservation of the monolignol-binding residues that were identified in Arabidopsis (Østergaard et al., 2000) was observed in several lignification-related peroxidases. The dibenzodioxocin structure was shown to be formed mostly during the final phase of cell wall lignification, and it was most abundant in the S2+S3 layers of the secondary cell walls in spruce xylem. Later, it was more specifically localised into the S3 layer lining the tracheid lumen (Kukkola et al., 2004). The late appearance of dibenzodioxocin structures and the difficulty of oxidising it (Sipilä et al., 2004) suggest that dibenzodioxocin is a terminal structure in the lignin polymer. It was less abundant in angiosperm than in gymnosperm lignin (Kukkola et al., 2004), and could, in part, be related to the higher pulping efficiency of angiosperm wood. For monolignol biosynthetic genes, expression profiling revealed the most likely gene family members responsible for developmental lignification. The same genes had also highest expression in the tissue culture, demonstrating that a similar monolignol biosynthetic pathway operates in both systems. Only a few of the studied peroxidase genes had high expression in developing xylem samples, making these candidates for 53 lignin polymerisation in wood. However, transgenic plants should be generated to obtain conclusive evidence for the participation of these genes in lignin biosynthesis. Developmental and stress-induced lignification were mostly similar at the gene expression level. In compression wood, the expression of a few monolignol biosynthetic genes and two peroxidases was up-regulated compared with vertically grown wood. The up-regulated genes, PaPAL2 and PaC4H3/5, were probably responsible for the increased flux of phenylalanine into the lignin biosynthetic pathway. H. annosum infection induced the biosynthesis of lignin in phloem, which was a result of a general upregulation of the whole phenylpropanoid pathway. Possibly, the biosynthesis of defence compounds other than lignin was also induced during the infection. Our knowledge of lignin biosynthesis has increased during the last decade; however, unclarities still exist. There appears to be differences between species in the organisation of the monolignol biosynthetic pathway, especially concerning the hydroxylation and methylation of the 3-position, and only indirect evidence of the mechanisms of monolignol transport and H2O2 generation exist. Most importantly, the factors that regulate lignification during development and their interplay with environmental signals need to be resolved for a true understanding of lignin biosynthesis in plants. 54 ACKNOWLEDGEMENTS This work was carried out during the years 1999 to 2007 at the Department of Applied Biology, the Institute of Biotechnology and the Division of Plant Biology at the Department of Biological and Environmental Sciences of the University of Helsinki. I wish to thank the heads of the departments for providing excellent research facilities. In addition, the education and connections provided by the Finnish Graduate School in Plant Biology are greatly appreciated. This study was made possible by the financial support of the Finnish Funding Agency for Technology and Innovation (Tekes), the Centre of Excellence Program of the Academy of Finland and the Finnish Society of Forest Science. I want to express my deepest gratitude to my supervisor, Prof. Teemu Teeri, for his interest and guidance during these years. He has taught me the ways of scientific thinking and stimulated discussions by his vast knowledge of molecular biology, plant secondary metabolism, and science in general. In addition, his enthusiasm for the art of crayfish parties will be remembered for years to come! I am also grateful for having worked with Prof. Liisa Simola, whose long experience in plant physiology and tissue cultures made this study possible. Her encouragement during the last years is greatly appreciated. The expertise of Prof. Ilkka Kilpeläinen in NMR spectroscopy and lignin chemistry has also been essential for this work, and is greatly acknowledged. I also wish to thank my other coauthors, Edward Alatalo, Prof. Gösta Brunow, Prof. Kurt Fagerstedt, Dr. Carl G. Fossdal, Mikaela Gustafsson, Dr. Pirkko Karhunen, Dr. Anna Kärkönen, Dr. Eija Kukkola, Tapio Laakso, Dr. Taina Lundell, Maaret Mustonen, Lars Paulin, Dr. Stephen Rudd, Dr. Katia Ruel, Dr. Pekka Saranpää, Dr. Kaisa Syrjänen, Merja Toikka and Tino Warinowski, for their invaluable expertise and contribution to the papers. Anna, especially, is also thanked for her friendship and for always having time for discussions, Tino for his organisational skills, and Maaret for her excellent technical assistance and patience with the tissue culture. I also wish to thank the pre-reviewers, Prof. Hely Häggman and Dr. Markku Keinänen, for their valuable suggestions to improve this thesis. In addition, I want to acknowledge the two consortiums that have been involved in this work: the original Lignin Biosynthesis consortium, and the later Wood Formation consortium, for creating forums for fruitful discussions and collaboration. Especially the multidiciplinary meetings during the first years opened new ways of thinking for us all. The life of a lab rat can only be truly understood by another. Therefore, my past and present colleagues in the Lignin project and in the Gerbera Laboratory deserve heartfelt thanks for creating the most enjoyable atmosphere both in and out of the lab. I am also indebted to you for the exponential increase in my knowledge of flower development soon I could master a B.Sc. in gerberology! Special thanks go to the excellent technicians, Marja Huovila, Anu Rokkanen and Eija Takala, for their help in lab practical matters, to Dr. Satu Koskela for sharing the first years as novices in the plant kingdom, and to my officemate Miia Ainasoja for sharing the ups and downs of science on the spot. I also wish to thank Prof. Paula Elomaa and Dr. Mika Kotilainen for their encouragement and interest in my work, and especially, Dr. Roosa Laitinen for the helpful and valuable discussions, and for being a wonderful friend through the toughest times. 55 The plant and forest pathology groups, the plant vaccine and strawberry groups and other staff at the Department of Applied Biology are warmly thanked for the pleasant collaboration. During the first years, I regularly visited the labs at the Division of Plant Biology, and therefore, I wish to thank the staff, especially the groups of Prof. Kurt Fagerstedt and Prof. Marjatta Raudaskoski for the welcoming attitude and collaboration. Special thanks to Kaisa Marjamaa for sharing the moments of mutual frustration during the writing process! The days at the Institute of Biotechnology, sharing the lab with the groups of Prof. Ykä Helariutta, Prof. Alan Schulman and Prof. Kristiina Mäkinen, are also warmly remembered. Dr. Nisse Kalkkinen is acknowledged for the help in proteomics, and for the possibility to work with the HPLC equipment in his laboratory. I am also grateful to all my friends and relatives, especially Tiina, Klaus & Samuli, Mikko & Nina, Hanna & Juha, Ursula, Johanna, Sanna, Outi, Anneli & Jami, Minna & Tommi, and my tai chi friends, for sharing the life outside of science, and for not asking too often when this thesis be will finished. Your friendship has been invaluable. BK-91 is thanked for the fabulous times during the years when all this began, and for keeping up the spirit thereafter. 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