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Biol Res 32: 3 5 - 6 0 , 1999 Signal transduction networks and the biology of plant cells* 1 2 3 MAARTEN J C H R I S P E E L S L O R E T O HOLUIGUE , RAMON LATORRE , SHENG LUAN , ARIEL O R E L L A N A H U G O PENA-CORTES , NATASHA V RAIKHEL , PAME LA C RONALD & ANTHONY TREWAVAS 4 3 7 5 6 8 'Department of Biology, University of California San Diego, La Jolla, CA 92093-0116, USA department of Molecular Genetics and Microbiology, Catholic University of Chile, Santiago, Chile 'Department of Biology, Faculty of Science, University of Chile, Santiago, Chile department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA department of Biology, Faculty of Chemistry and Biology, University of Santiago de Chile, Santiago, Chile d O E Plant Research Lab, Michigan State University, East Lansing, MI 48824-1312 Plant Pathology Department, University of California Davis, Davis, CA 95616-8680 "Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, United Kingdom 7 ABSTRACT The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories. * This paper contains a summary of research results and relevant background information that was presented at a US-Chile workshop on Plant Cell Biology and Signal Transduction, held in Santiago de Chile November 14-16, 1998. The workshop was organized by Maarten Chrispeels and Ariel Orellana. Support for the workshop was provided by the National Science Foundation (USA), the Fundación Andes, Conicyt. Fundación para Biología Celular, and the University of Chile The authors are listed alphabetically. ** Corresponding author: Dr. Maarten J. Chrispeels, Department of Biology, University of California San Diego, La Jolla, CA 92093-0116, USA, e-mail: [email protected], or Dr. Ariel Orellana, Departamento de Biología, Facultad de Ciencias, Las Palmeras 3425 - Casilla 653, Universidad de Chile, Santiago, Chile, e-mail: [email protected] Received 26.3.99. Accepted 14.4.99 36 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 I. THE EXTRACELLULAR MATRIX: STRUCTURE, BIOGENESIS AND ROLE IN SIGNALING /. The extracellular matrix or cell wall of the plant cell is a multicomponent struc ture. We currently view the cell wall as a threed i m e n s i o n a l n e t w o r k of c e l l u l o s e mi crofibrils and cross-linking polysaccharides (hemicellulose) embedded in a gel matrix of acidic polysaccharides rich in galacturonic acid (pectins) and held together by calcium bridges. The two major groups of flowering plants, the monocots and the dicots, differ in their hemicellulosic poly mers; monocots have arabinoxylans, and dicots have xyloglucans (see Carpita and Gibeaut, 1993). This network is enmeshed with a variety of structural proteins and glycoproteins and partially cross-linked by aromatic substances (monolignols). En zymes needed for the biosynthesis or modi fication of the network {e.g., peroxidase, xyloglucan endotransglycosylase, afucosidase, glucanase) or for the modifi cation of metabolites (e.g., invertase, phos phatase, ascorbic acid oxidase) may be ionically or covalently bound to the hemi cellulose network or the pectin matrix. Assembly of the cell wall requires the coordination of the activities of large cel lulose synthase complexes on the outer face of the plasma membrane with those of many different glycosyltransferases in the Golgi that assemble the pectin and hemicellu losic polymers. These polymers are trans ported to the plasma membrane in secre tory vesicles, as are the cell wall proteins and glycoproteins, and their cross-linking is catalyzed by cell wall enzymes. In spite of this cross-linking, the walls of young cells remain flexible and can extend through breaking and reforming of cross-links to a c c o m m o d a t e the e n o r m o u s volume changes that characterize the growth of plant cells. In highly differentiated cells such as xylem vessels or fibers, the walls become thick, stiff and incompressible af ter impregnation with lignin, a polymer that is formed extracellularly by polymer ization of secreted monolignol subunits (see Boudet, 1998). Cell differentiation is accompanied by the synthesis and secretion of different com ponents of the ECM of plant cells, as visu alized by the interaction of monoclonal antibodies against cell wall epitopes. This process has recently been studied in Zinnia cells. When exposed to the hormones auxin and c y t o k i n i n , Z i n n i a m e s o p h y l l cells transdifferentiate into tracheary elements. This transdifferentiation is accompanied by a new pattern of gene expression and changes in the repertoire of proteins and polysaccharides at the cell surface (Stacey etal, 1995). 2. The extracellular matrix is a source signals for development and defense. of Two types of cell wall macromolecules appear to generate signals for plant cells: the arabinogalactan proteins and the hemicelluloses. Arabinogalactan proteins (AGPs) are extracellular or plasma mem brane-anchored proteins that are typically more than 90% carbohydrate. The polypep tide backbone has a domain that is rich in Ala, Ser, Thr, and Hyp, and the carbohy drate moiety consists of short arabinosides attached to Hyp and arabinogalactans Olinked to Hyp or Ser. The arabinogalactan sidechains have 30 to 150 sugar residues, and most have a pure B(l->3)-D galactan backbone with terminal L-arabinofuranosyl residues on short sidechains (Du et al, 1996). AGPs have been shown to promote the differentiation of single, densely cyto plasmic carrot cells into somatic embryos. The AGPs are not produced by the cells that differentiate into embryos, but need to be present in the culture medium or pro duced by neighboring cells (McCabe et al, 1997). Completely different signals, small oli gosaccharides of 5-15 sugar residues, are involved in defense responses and in the loosening of the hemicellulosic network that permits plant cells to grow in size as a result of their internal osmotic pressure. Oligosaccharides involved in defense re a c t i o n s r e s u l t from the b r e a k d o w n of polysaccharides by plant or fungal enzymes. These oligosaccharides bind to receptors C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 Xyloglucan PGA junction zone RGI with arabinogalactan side-chains 37 Extensin Figure I. Model of a type 1 cell wall in the process of expanding. A model of possible alterations in structure that first permit microfibril separation and then lock them into place. Microfibrils of a single layer align parallel in a helical arrangement around elongating cells. Xyloglucans can separate from the microfibrils under the action of expansins and be cleaved by glycosidases or endo-transglycosylases. When displacement caused by turgor pressure within the cell is complete, extensin molecules, inserted radially, interlock the separated microfibrils to arrest further stretching. Additional proteins may also be inserted to cross-link extensin, forming a heteropeptide network. Formation of intramolecular covalent bonds among the indivi dual wall proteins signals the end of elongation. From Carpita and Gibeaut, 1993, reprinted by permission of the publisher, Blackwell Science. 38 C H R I S P E E L et al. Biol Res 3 2 , 1 9 9 9 , 3 5 - 6 0 on the plasma membrane and this signal is transduced to the nucleus, where a battery of plant defense genes are induced (see below) (Hahn, 1996). Oligosaccharides in volved in growth are the result of the action of e n d o g l u c a n a s e s o n t h e c e l l w a l l xyloglucans. They are broken down by exoglucanases and may provide substrates for xyloglucan endo-transglycosidases that are thought to modify the xyloglucan net work. 3. A gene that encodes a subunit of cellu lose synthase has at last been identified. Cellulose is a linear polymer made of 6-1,4 linked glucose units. In the primary cell wall the degree of polymerization reaches over 2,000. Fifty to 80 of these linear poly mers lie in parallel and bind tightly to each other through hydrogen bonds, creating a microfibril. Electron microscope studies of freeze-fractured membranes provided the first suggestion that a rosette-shaped pro tein complex located in the plasma mem brane may be involved in cellulose biosyn thesis. Purification of the protein(s) in volved in the synthesis of cellulose proved to be difficult and was unsuccessful at least for plants. However cellulose synthetase was purified successfully from extracts of Acetobacter xylinum, a bacterium that syn thesizes cellulose. This allowed the cloning of the gene that encodes the catalytic subunit of cellulose synthase. Use of this gene as a probe to search for homologous genes led to the identification of putative cellu lose synthases in plants. Analyzing the Acetobacter sequence and the structure of the gene and hydrophobic cluster analysis allowed the identification of a signature s e q u e n c e ( D . . . D . . . D . . . Q x x R W ) that is p r e s e n t in a g r o u p of p r o c e s s i v e 8glycosyltransferases, which could be present in cellulose synthases from other organisms as well (Saxena et al, 1995). This information helped Delmer's group to identify two ESTs from cotton, obtained by random sequencing of a cottonhair cDNA library, as the first two potential genes encoding cellulose synthase from higher plants (Pear et al, 1996). Although direct evidence that these two genes indeed en code cellulose synthase was not provided, all the circumstantial evidence strongly supported this conclusion. The identification of a gene that encodes a cellulose synthase of higher plants came from studies with Arabidopsis thaliana. Arioli et al (1998) identified after exten sive screening an Arabidopsis mutant that has impaired cellulose synthesis. The phenotype of this mutant, radial swelling (rsw) of the roots mimics the response of wild type roots to cellulose synthesis inhibitors such as dichlorobenzonitrile. Genetic map ping allowed the identification of the locus RSW1 and the further cloning of the gene. Analysis of the RSW1 amino acid sequence showed that it contains the D...D...D...QxxRW motif and is homologous to the cel-A genes from cotton. The deficiency of cellulose in the R S W mutant could be complemented by transformation with the RSW1 cDNA. The sequences encoded by these cellu lose synthase genes have been used to search for homologous genes through out plant databases, and a number of different genes potentially involved in cellulose biosyn thesis have been identified. Thus, a vast field of research to understand the molecu- UDP-Glc UDP-Glc transporter Glucosyl transferase Figure 2. Proteins needed for the biosynthesis of a glucan in the Golgi. The cycle begins with the uptake of UDP-Glc by a transporter that also permits uridine monophosphate to leave. UDP-Glc is the substrate of a membrane-anchored glucosyltransferase that adds a glucose (G) to a growing chain, and produces UDP, which is cleaved by UDPase to produce P. and UMP. Whether the glucan is anchored to a protein (as shown) is not known. C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 lar basis of cellulose biosynthesis has just emerged. 4. The biosynthesis of hemicellulosic polysaccharides and the carbohydrate moieties of proteoglycans and glycoproteins occurs in the Golgi apparatus. In contrast to cellulose biosynthesis, which takes place in the plasma membrane, hemicelluloses, pectin, and the glycosidic chains of proteoglycans are synthesized intracell u l a r ^ in the Golgi apparatus (Driouch et al, 1993). The formation of polysaccharides in the lumen of the Golgi requires the following enzymes and transporters: (1) glycosyltransferases that use sugar nucleotide diphosphate as substrate and cause the p o l y s a c c h a r i d e chain to g r o w ; (2) UDPases that degrade nucleotide diphosphate produced by the glycosyltransferases; (3) a transporter that removes inorganic phosphate; (4) a transporter that exchanges sugar nucleotide diphosphate for nucleotide monophosphate (see Fig 2). Little is known about the glycosyltransferases involved in this process, and so far the only enzyme that has been purified and cloned is the xyloglucan oc-l,2-fucosyltransferase, an enzyme involved in the fucosylation of xyloglucan, one of the most abundant hemicelluloses in dicots (Perrin et al, 1999). The sequence of this enzyme predicts a single transmembrane domain close to the amino terminus, resembling the structure of mammalian glycosyltransferases that participate in protein glycosylation in the Golgi. T o p o l o g i c a l analysis of glucan synthase I, an e n z y m e involved in x y l o g l u c a n bios y n t h e s i s , indicates that its active site is located in the lumen of the Golgi cisternae ( M u ñ o z et al, 1996). U n p u b l i s h e d r e s u l t s from O r e l l a n a ' s and M o h n e n ' s g r o u p s i n d i c a t e that the active site of x y l o g l u c a n a - 1 , 2 - f u c o s y l t r a n s f e r a s e and polygalacturonate a-4-galacturonosyltransferase, e n z y m e s i n v o l v e d in xyloglucan ( h e m i c e l l u l o s e ) and h o m o g a l a c turonan (pectin) biosynthesis respectively, would also be p r e s e n t in the lumen of Golgi c i s t e r n a e . 39 The question is then, how do the nucleotide sugars get into the lumen of the Golgi cisternae? Studies using pea Golgi vesicles show that UDP-Glc can be transported into the lumen of Golgi vesicles by a protein mediated process (Munoz et al, 1996). This membrane transporter seems to be specific for UDP-Glc; therefore it seems possible that different transporters would be responsible for the incorporation of the substrates used by the different Golgi glycosyltransferases involved in both polysaccharides and proteoglycans biosynthesis, as well as protein glycosylation. After the transfer of the sugar into the oligosaccharide, the nucleoside diphosphate (UDP) is quickly transformed into UMP and i n o r g a n i c p h o s p h a t e by a N u c l e o s i d e Diphosphatase present in the lumen of the Golgi apparatus (Neckelmann and Orellana, 1998). The hydrolysis of UDP eliminates a potential inhibitor for the transferases and also drives the transfer reaction toward the polymerization of sugars. U M P leaves the Golgi cisternae by a process stimulated by UDP-Glc, suggesting that the UDP-Glct r a n s p o r t e r would be a U D P - G l c / U M P antiporter. On the other hand, inorganic phosphate is not accumulated in the cisternae and is transported out of the cisternae. 5. The ECM-plasma membrane-cytoskeleton continuum plays a role in signal transduction. Recent evidence indicates that receptor kinases are important players in signal transduction pathways. Receptor kinases are proteins that have an extracellular domain that receives the signal and a cytoplasmic domain that transmits the signal. Some other plasma membrane anchored proteins also have large extracellular domains. For example, some AGPs are anchored by a GPI anchor. Recently a m e m b r a n e - a n c h o r e d glucanase was discovered that plays a major role in regulating cell elongation (Nicol et al, 1998). When plant cells are plasmolyzed, the plasma membrane does not pull evenly away from the cell wall, but the two remain in close contact at very specific adhesion points called Hechtian strands. These 40 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 Hechtian strands are not found when cells are incubated with short peptides that con tain an Arg-Gly-Asp (RGD) motif. Isolated protoplasts of plant cells readily agglutinate when they are incubated with a genetically engineered protein that contains numerous RGD motifs, suggesting that plasma mem branes contain RGD receptors. In animal cells, adhesion is mediated by plasma mem brane receptors called integrins that interact with extracellular adhesion proteins (e.g., fibronectin and vitronectin) via the tripeptide Arg-Gly-Asp (RGD), a motif that is conserved in adhesive proteins. On the cy toplasmic side of the membrane integrins interact with proteins of the cytoskeleton, thereby establishing the ECM-plasma membrane-cytoskeleton continuum. There is as yet very little evidence that in plants transplasma membrane proteins interact with the cytoskeleton, but we know that a large num ber of signals induce rapid cytoskeleton re organization, leading to the suggestion that a similar continuum exists in plant cells. II. THE SECRETORY SYSTEM PLAYS AN IMPORTANT ROLE IN SORTING PROTEINS FOR DIFFERENT CEL LULAR DESTINATIONS /. Vacuolar targeting requires vacuolar sorting signal. a specific The secretory system is a series of or ganelles that includes the e n d o p l a s m i c reticulum (ER), Golgi apparatus, plasma membrane and vacuole. Soluble proteins containing an N-terminal signal sequence are co-translationally inserted into the ER lumen, and after their synthesis is complete they are transported through the secretory system in transport vesicles that bud from one compartment and fuse with the next. F r o m the ER, proteins are transported through the Golgi cisternae to the transGolgi network, where a major sorting event occurs. Proteins are either secreted to the outside of the cell (the default pathway) or they are targeted to the vacuole via a prevacuolar compartment, a route that is be lieved to be mediated by membrane-bound receptors to which the cargo proteins bind so that they can be properly sorted. For a soluble protein to reach the vacu ole, it must contain a specific vacuolar sorting signal. Three types of vacuolar sorting signals have been identified (Fig 3). Some proteins contain a propeptide, which comprises the sorting signal and which is removed from the protein upon deposition in the vacuole. Several pro teins are known to contain a propeptide at their N - t e r m i n u s , including sweet potato sporamin and barley aleurain (reviewed in Chrispeels and Raikhel, 1992). Deletion and mutagenesis studies have shown that the N - t e r m i n a l propeptide is required for transport to the vacuole and have defined a conserved motif within the propeptides, which is essential for proper sorting and Signal Peptide Sporamin, Aleurain NTPP CTPP Tobacco Chitinase, Barley Lectin Phytohemaglutinin Part of mature protein Figure 3. Location of vacuolar targeting signals in plant proteins. Vacuolar targeting signals can be propeptides at the C-terminus (CTPP) or the N-terminus (NTPP) of a protein, or be part of the mature protein. 41 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 thus is thought to be recognized by the sorting machinery. The second type of propeptide signal is found at the C-terminus of several vacuolar proteins, including barley lectin and to bacco chitinase (reviewed in Chrispeels and Raikhel, 1992). No common motif can be identified between these sorting signals, and while the propeptides have been shown to be both necessary and sufficient for vacu olar transport, extensive mutagenesis studies have determined that there is no sequence motif that is required for their function (reviewed in Bar-Peled et al, 1996). It is still not known whether a receptor protein is present at the trans-Golgi network that recognizes this type of signal. Finally, a region of the mature protein can also serve as a sorting signal. Bean phytohemagglutinin contains this type of signal (reviewed in Chrispeels and Raikhel, 1992). although the nature of the signal means that it is very difficult to precisely identify the residues responsible for tar geting information. Legumin also appears to contain vacuolar sorting information within the mature protein, and the entire legumin a chain is required for efficient sorting (BarPeled et al, 1996). The mechanism by which these signals work is not known. 2. Cells have more than one type of vacuole and probably have multiple paths for pro teins to reach the vacuole. Studies using the fungal m e t a b o l i t e wortmannin, an inhibitor of phospholipid metabolism in tobacco cells, have shown that proteins containing an N-terminal sig nal are transported to the vacuole by a different mechanism than proteins contain ing a C - t e r m i n a l signal ( r e v i e w e d in Bassham and Raikhel, 1997). In the pres ence of wortmannin, proteins containing the barley lectin C-terminal propeptide were secreted, whereas those containing the sporamin N-terminal signal were correctly sorted to the vacuole. In addition, mem brane proteins are transported by a diffe rent pathway compared to soluble proteins (reviewed in Bassham and Raikhel, 1997). Therefore, it appears that multiple path- ways to the vacuole exist within a single plant cell. In fact, some specialized cell types have been shown to contain two dis tinct types of vacuoles, which have their own d i s c r e t e c o m p l e m e n t of p r o t e i n s (Neuhaus and Rogers, 1998). In mature vegetative cells, these vacuoles are thought to fuse to form a single large central vacu ole upon which all the vacuolar targeting pathways converge (reviewed in Bassham and Raikhel, 1997). 3. Vacuolar targeting requires transmem brane cargo receptors that function in traf fic between the trans-Golgi network and the prevacuolar compartment. Using an affinity column with immobilized peptide corresponding to the aleurain Nterminal propeptide, a protein was isolated from pea clathrin-coated vesicles that is thought to represent a sorting receptor for vacuolar proteins that have this signal (re viewed in Beevers and Raikhel, 1998). This protein (BP-80) is an integral membrane protein that can also bind to other N-terminal signals, but not to the barley lectin Cterminal propeptide. An Arabidopsis homologue of BP-80 (named AtELP) was identified by searching the DNA sequence AtVTIla (v-SNARF.) AtELP (Vacuolar Cargo Receptor) AtPKPUp (t-SNARK) Prevacuolar Compartment Figure 4. Schematic diagram of targeting and fusion of Golgi-derived clathrin-coated vesicles with the prevacuolar compartment. Only three components of the machinery are depicted in this figure: a putative vacuolar sorting receptor, AtELP, a vSNARE, AtVTI 1 A, and a tSNARE, AtPEP 12p. Other members of the machinery that are important for vesicle targeting and target membrane fusion, including soluble and membrane receptors, and proteins that constitute the clathrin coat, are not shown on this diagram. 42 C H R I S P E E L etal. Biol Res 32, 1999, 3 5 - 6 0 database using motifs common to sorting receptors from various eukaryotic species (reviewed in Beevers and Raikhel, 1998). Both the pea and Arabidopsis proteins have been localized by electron microscopy to the Golgi apparatus, and in addition AtELP was found in the pre-vacuolar compart ment (Sanderfoot et al, 1998) (Fig 4). These receptor-like proteins are proposed to bind to their cargos (proteins containing an Nterminal vacuolar sorting signal) at the trans-Golgi network and package them into clathrin-coated vesicles for transport to the prevacuolar compartment. After the formation of vesicles contain ing vacuolar cargo proteins from the transGolgi network, the vesicles have to be di rected to the prevacuolar compartment and fuse with that organelle. A number of pro teins have been identified from various organisms that are required for the correct targeting, docking and fusion of a transport vesicle with its target membrane. As some components for vesicle targeting and fu sion appear to be similar between many different organisms and different stages of the secretory pathway, the basic mecha nisms for these processes are probably con served. Information from the better studied yeast and mammalian systems can there fore be very useful in the study of vesicle trafficking in plants. 4. The SNARE hypothesis for vesicle fusion and the discovery of plant homologues that function in this process. A c c o r d i n g to the S N A R E h y p o t h e s i s , vesicle docking requires the interaction of a vesicle SNARE (vSNARE) with a SNARE on the target membrane (tSNARE) (Beevers and Raikhel, 1998). The first v S N A R E (syntaxin 1) was isolated from the synaptic plasma membrane of neurons, where it is involved in neurotransmitter release. It has been shown to form a complex with several soluble and integral membrane proteins that arc all believed to function in vesicle dock ing and fusion by mechanism as yet un known. A number of syntaxin homologues have now been identified in non-nerve cells and in various organisms. They have simi lar sequences and predicted secondary structures to syntaxin 1, and therefore may function in a similar way in a variety of vesicle fusion events. It has been hypoth esized that a different syntaxin isoform may be required for each transport step of the s e c r e t o r y p a t h w a y ( R o t h m a n and Sollner, 1997). Typical syntaxin homologues are type II transmembrane proteins. Most of the pro tein is hydrophilic and faces the cytosol, except for a hydrophobic domain about 2030 amino acids long at the C-terminus of the protein. The hydrophilic part is thought to interact with other factors involved in vesicle trafficking, whereas the hydropho bic domain is responsible for anchoring the protein in the membrane. Several syntaxin homologues have now been identified in plants that are also pre sumably involved in transport through the secretory pathway. By functional comple mentation of the yeast (Saccharomyces cerevisiae) secretory pathway mutant pep 12. researchers have isolated a cDNA clone (AtPEPl 2) from Arabidopsis thaliana ecotype Columbia that encodes a protein homologous to yeast P e p l 2 p and other members of the syntaxin family (reviewed in Bassham and Raikhel, 1997). The yeast pep 12 mutant is defective in transport of some soluble proteins to the vacuole and is thought to function in transport between the trans-Golgi network and the prevacuolar compartment (PVC). AtPEPl 2p may there fore play a role in transport to the vacuole in plants. A t P E P l 2 p has been localized by electron microscopy to a PVC in Ara bidopsis roots and may act as a receptor for trans-Golgi network-derived transport vesicles en route to the vacuole (Fig 4). By genetic analysis, another Arabidopsis syntaxin homologue, KNOLLE (reviewed in Beevers and Raikhel, 1998), was identi fied. It is presumably involved in cytokin esis in the embryo, and is located at the forming cell plate during cell division (re viewed in Beevers and Raikhel, 1998). Sev eral syntaxin homologues therefore exist in Arabidopsis, and each of them may be involved in separate steps of the secretory system or function at different develop mental stages, or in different tissues. Re- 43 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 cently, a v S N A R E protein, A t V T I l a , that most likely interacts with AtPEP12 has been identified (H. Zheng and N. Raikhel, unpublished data) (Fig 4). III. THE PLASMA MEMBRANE AND THE TONOPLAST CONTAIN CHANNELS AND TRANSPORTERS WITH IMPORTANT SIGNALING FUNCTIONS 1. Calcium is an important second mes senger in many signal transduction networks. 2+ The concentration of free C a in the cyto plasm is very low (10 to 200 nM) and most of the cellular calcium is sequestered in calcium stores in cytoplasmic organelles or in the cell wall (Fig 5). The movement of C a from the cytoplasm into the stores requires energy in the form of ATP and is carried out by calcium dependent C a 2+ 2+ ATPases. Movement in the other direction (release into the cytosol) occurs via cal cium channels down the electrochemical calcium gradient. Both types of proteins, pumps and channels, are particularly abun dant in the plasma membrane and the tonoplast, but also occur in other membranes such as the ER and the mitochondria. In 1957, Hodgkin and Keynes performed a simple but seminal experiment. They in jected radioactive calcium and potassium into the cytoplasm of an axon. After sev eral hours they measured the distribution of the two radioactive ions and found that potassium had diffused throughout the cy toplasm, but the radioactive calcium had barely moved from the site of injection. The inescapable conclusion was that cal cium is immobile in the cytoplasm. When calcium is artificially elevated in the cyto plasm, the extra calcium will bind to pro- extracellular Ca 2+ fi wall binding sites. lmM (pectins, proteins) membrane phospholipid binding sites ATPase and Ca /H antiport 2+ + Figure 5. Summary of the major calcium relationships in a plant cell as presently understood. The figure indicates the presence of channels and calcium-ATPases and estimates of concentration in the main organelles. The presence of cytoplasmic binding sites inhibits direct calcium diffusion in the cytoplasm and necessitates the production of calcium waves using IP -calcium sensitive channels in the ER and vacuole. 3 44 C H R I S P E E L et al. Biol Res 3 2 , 1 9 9 9 , 3 5 - 6 0 2+ teins and is sequestered into C a stores. These stores are in the cell wall, the vacu ole, the ER and the mitochondria. Calcium channel proteins permit the flow of cal cium from the stores back into the cyto plasm. These channels open when cells are signaled in some way, and calcium then enters the cytoplasm down in its electro chemical gradient. Families of calcium channels are known to exist: some are activ ated by changes in membrane potential, some by membrane stretch and yet others by second messengers. A single channel can transmit 10 atoms of C a per second. Elevation of calcium at the channel mouth can be rapid, and concentration here can reach 100 m M . The channels rapidly close when cytosolic calcium is elevated and calcium is then pumped back into the intra cellular stores or into the wall. Transient elevations of calcium, sometimes called spikes, may last anywhere from a few sec onds to many minutes, depending on the characteristics of the stimulating signal. 6 2+ 2. How does calcium make waves, and how do the waves move? Calcium itself does not move through the cytoplasm; however, this calcium spike can propagate itself as a wave (Malho et al, 1998). This propagation depends on the second messenger, inositol 1,4,5 triphos phate (IP ). This molecule is generated through the action of the plasma membrane bound enzyme, phospholipase C. When phospholipase C is activated it hydrolyzes phosphatidyl inositol diphosphate (PIP ) to yield diacylglycerol and I P . Unlike cal cium, I P is freely mobile in the cytoplasm, and I P - s e n s i t i v e calcium channels are found in organelles throughout the cyto plasm. When I P is present, the calcium channels open and calcium enters the cyto plasm. The role of I P in mobilizing cal cium was demonstrated in plant cells a decade ago (Alexandre et al, 1990; Gilroy et al, 1990), and I P binding to the channels was discovered more recently (Allen et al, 1995). These observations help us under stand how calcium makes a wave. The I P sensitive calcium channels need both I P 3 2 3 3 3 3 3 3 3 3 and calcium to open (Marchant and Taylor, 1997). When I P binds to a channel, a cal cium binding site is briefly exposed. If calcium is absent, the channel rapidly inac tivates. If calcium is present, the calcium binding site is occupied and the channel opens. However, the binding of calcium only serves to delay the eventual IP3-induced channel inactivation. Mobilization of calcium through a single IP3-dependent calcium channel is therefore brief and selflimiting. So how does the wave move? When an IP3-dependent calcium channel opens, the calcium concentration near ad jacent channels will increase. The calciumbinding sites of these channels will be oc cupied and they will briefly open, in turn enabling the opening of others. Calcium is therefore responsible for inducing further calcium release and this release underpins wave movement. The wave is not a forward movement of calcium, but a forward move ment of calcium release. IP3-induced inac tivation of channels generates direction in calcium wave movement away from the point of origin. Because these channels are located in membranes, the wave moves across the face of the membrane. 3 3. Plasma membrane potassium play fundamental roles in signal tion processes in plants. channels transduc Potassium transport processes play a fun damental role in plant cell physiology. Po tassium ion m o v e m e n t s across the cell membrane are involved in leaf movement, cell elongation, plant growth and develop ment and in the regulation of stomatal ap erture (for reviews see Schroeder et al, 1994; Cherel et al, 1996; Fox and Guerinot, 1998). Potassium is the dominant cation within the vacuole, and accumulation of K inside this organelle is essential for gene rating the turgor pressures that drive cell expansion. Potassium transport in plants is mediated by carriers and channels and is customarily divided into high- and lowaffinity K uptake (Schroeder et al, 1994). High-affinity K transport is defined as the K influx that occurs when the external K concentration is in the range of 1 to 200 + + + + + 45 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 lnM (e.g., Fox and Guerinot, 1998) and was thought to be mediated exclusively by car riers. Low-affinity K uptake is mediated by voltage-dependent inward rectifier K channels when the external concentration of K is > 0.3 mM. These channels belong to a superfamily of ion channels designated the S4 superfamily, which includes Na , C a , and K voltage-dependent channels (Jan and Jan, 1992). In all of these chan nels, the fourth transmembrane domain (S4) contains several positively charged amino acids in every third position (Fig 6B). KAT1 and AKT1 (Fig 6A) were the first inward rectifier K channels cloned from the plant Arabidopsis thaliana (Schroeder et al, 1994). The KAT1 channel is ex pressed in guard cells and the vascular system of the stem and plays a crucial role in stomatal opening (Fox and Guerinot, 1998). AKT1 is present in roots and plays an important role in K uptake from soil. Mutants from Arabidopsis thaliana in which + + + + 2+ + + + the AKT1 channel gene was disrupted show reduced K uptake and grow poorly on media containing < 100 mM K (Hirsch et al, 1998). This result suggests that inward rectifier channel activity might also medi ate high-affinity uptake (Schroeder et al, 1994). K S T 1 , a gene-encoding a guard cell K channel, was cloned from a Solanum tuberosum cDNA library based on its simi larity to K A T 1 . KST1 is also present in flowers, albeit at a low level. Four more inward rectifier K channels genes have been identified in Arabidopsis thaliana and Solanum tuberosum: AKT2 and AKT3 and SKT2 and S K T 3 . Two outward rectifier K channel genes have also been cloned from Arabidopsis thaliana: SKOR (Gaymard et al, 1998) and K C O l . SKOR is present in root stelar tissues and its role is to release K into the xylem sap. K C O l is expressed in seedlings and at low levels in leaves and flowers. For original references see Fox and Guerinot (1998). + + + + + + B Pore Plant K channels subunits Gene KAT1 KST1 Spedes A. Thelma S. tuberosum K Transport Subunit structure Inward SKT2/3 S. tuberosum Inward W WA V I S MBWS s B *w V 1?M T Pr V WR№'5!' • T13 ¥ S,t0 1 T | [:LT| A LJJWS 1' T i • L a vv I Shaker l_ V I L V RATI LSM L L» AKT1 F N M l LU SKOR L L L I LT Inward A. thaliana D A F • TI 1 '•v, ft ? " 1 |à y VF L L VH I F L S HS G LO I . ¥ S SLFA L E DI F V G A L F Ä LE D N F V I L F F H IKE D I I ' SKOR A. thaliana Outward KCOl A. thaliana Outward 90 S4 Inward AKT1/2/3 Shaker KÄT1 AKT1 SKOR Bum a Nucleotide binding Ankyrin lepejt domain Figure 6 A. Cloned plant potassium channels and their subunit structure. B. Primary sequence alignment of the pore region (top) and S4 segment (bottom) for three plant channels in comparison to Shaker. Potassium channel fingerprint GYGD is highlighted. Conserved positively-charged residues in the S4 segment are indicated in white by the amino acid number. C. Proposed transmembrane structure of one potassium channel subunit indicating membrane spanning segments SI through S6 and the pore region (top), and tetrameric subunit assembly to form one channel (bottom). 46 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 4. Inward and outward rectifying potas sium channels of plants and animals have the same basic structure. As can be appreciated in Figure 6, inward rectifiers and SKOR channels share the same subunit structure consisting of six transmembrane domains (S1-S6) and a pore domain (P) linking segments S5 and S6. The S4 domain contains conserved, regu larly-spaced, positively-charged amino acid residues (Fig 6B). Many different K c h a n nel genes in metazoans have this same ba sic structure, and Shaker, the first K chan nel gene cloned from the fly Drosophila melanogaster (Tempel et al, 1987), is a classic example. In Shaker K channels, the S4 domain forms part of the voltage sensor and the P region is part of the ion conductive pore (Papazian and Bezanilla, 1997; Doyle et al, 1998). The pore region of K channels is extremely well conserved (Fig 6B). The Shaker K channel is a tetrameric structure formed by four identical subunits (Fig 6C), and KAT1 and AKT1 are also likely to form tetramers (Cherel et al, 1996). Additionally, a putative cyclic nucleotide-binding domain is present in the carboxyl terminus in both KAT1 and A K T 1 . In AKT1 an ankyrin domain is present downstream of the cyclic nucle otide-binding domain (Fig 6C). It is worth noting here that KAT1 has a great simi larity to the ether a-go-go (eag) Droso phila channel. The eag channel forms part of a large class of K channels containing a cyclical nucleotide binding domain and is activated by hyperpolarizing voltages (e.g., Trudeau et al, 1995). + + + + + + 5. Aquaporins, or water channel occur in the plasma membrane vacuolar membrane. proteins, and the How does water flow through a biological membrane? Does it simply diffuse through the lipid bilayer, or does it pass more or less unimpeded through specialized pores? Recent discoveries show that water-flow t h r o u g h m e m b r a n e s is f a c i l i t a t e d by aquaporins (AQPs), integral membrane pro teins that can increase the water permea bility of a membrane 10-20 fold (Preston et al, 1992; Maurel et al, 1993). Aquaporins are not pumps, but simply facilitate the passage of water through the membrane when the osmotic or hydrostatic pressure is higher on one side than on the other. Aquaporins belong to the MIP family of membrane proteins, which also includes glycerol channels and possibly some ion channels. These 26-28 kDa proteins have six membrane-spanning domains and the signature sequence N P A V T . The activity of water channel proteins is most conveniently assayed by expressing them in Xenopus oocytes and then mea suring the swelling rate when the oocytes are shifted to a hypotonic condition. AQPs form tetramers in the membrane, but the monomer is probably the unit that trans ports water. A molecular model suggests that two cytoplasmic loops are half-buried in the lipid membrane to form the aqueous pore. Both the abundance and the activity of aquaporins can be regulated. Some aqua porins are expressed in a tissue-specific or organ-specific pattern. This is true both in mammals and in plants, where aquaporins have been most studied. In Arabidopsis there are at least 23 different cDNAs that fall into two large groups on a cladogram, TIPs (tonoplast integral proteins) and PIPs (plasma membrane integral proteins). In addition, there is a small group of more distantly related aquaporins (Weig et al, 1997). It is important to note that all of these genes have not yet been shown to encode active aquaporins. Indeed, some of the proteins appear not to be active as aquaporins when their mRNAs are intro duced in Xenopus oocytes. This could mean that they do not encode aquaporins or that the polypeptides made in the oocytes do not behave as active aquaporins. Perhaps they do not arrive at the plasma membrane, or they need to be post-translationally mod ified or they need to interact with other proteins not present in oocytes. In any case, we cannot rule out the idea that the these m e m b e r s of the M I P family have completely different functions and may be involved in the transport of other sub stances. 47 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 Aquaporin expression can be altered by hormones or environmental signals, such as light or drought. Phosphorylation may regulate the activity of AQP as has been shown for Arabidopsis a - T I P and spinach PM28A. When spinach plants experience water deficit, the plasma m e m b r a n e aquaporin PM28A is less phosphorylated than when the leaves are turgid and this inhibition of phosphorylation may decrease the activity of the water channel. When PM28A is expressed in Xenopus oocytes, its water channel activity is inhibited by agents that prevent phosphorylation (Johansson et al, 1998). 6. Aquaporins have multiple roles in cel lular and whole plant physiology. What is the role of aquaporins in cellular physiology or in the whole plant? Deter mining the role of aquaporins is likely to take many years of research, but some hy potheses are beginning to emerge as a re sult of studying the expression patterns of aquaporins. Expression has so far been studied mostly at the mRNA level and not at the protein level, simply because it is much easier to make gene-specific probes than to make protein-specific a n t i b o d i e s when dealing with a family of homologous pro teins. Analysis of the expression pattern of the active aquaporin y-TIP from Arabi dopsis and Z m T I P l from Zea mays shows that expression is highest in meristematic and expanding cells, young root epidermis, the endodermis, the parenchyma cells that surround functional xylem vessels, and in the pedicel (Barrieu et al, 1998; Chaumont et al, 1998). What does this tell us about the possible functions of aquaporins? The high expression in the meristem and expanding cells may be related to the biogenesis of new vacuoles and the rapid expansion of vacuolar volume that accompanies cell en largement. TIPs may be needed to sustain the rapid water influx into the vacuole that sustains cell growth in size. The high ex pression in the epidermis, endodermis and pedicel suggests that TIPs may be present at high levels in cells that can undergo sudden fluxes in ions or metabolites. If ions and metabolites suddenly enter the cytoplasm, then the cell has to adjust its osmotic pressure, and the most readily available source of water is in the vacuole (Fig 7). However, it is only readily avail able if the tonoplast is truly permeable to water. High expression in the parenchyma cells that surround the active xylem vessels c o u l d i n d i c a t e an i m p o r t a n t r o l e in transcellular water flow. Water flows radi ally in and out of vessels, and this flow will be less impeded if the tonoplast is perme able to water. In this case the water perme ability of the tissue would reside in and be r e g u l a t e d by t h e p l a s m a m e m b r a n e . Whereas these are all interesting hypoth eses, they need to be supported by evidence and such evidence will probably have to c o m e from m u t a n t s that lack specific aquaporins. A recent report indicates that a A • Metabolites Channel/Transporter © T o n o p l a s t Aquaporin B • Tonoplast Aquaporin Figure 7. Schematic representation of two roles for tonoplast aquaporins. A. Tonoplast aquaporins are needed for cytoplasmic osmotic equilibration in cells that can experience rapid fluxes of metabolites or mineral nutrients. B. Tonoplast aquaporins permit rapid transcellular flow and increase the effective cross-section of the cytoplasm for symplastic flow. From Barrieu etal, 1998. Reprinted with permission of the publisher. 48 C H R I S P E E L et al. Biol Res 32, 1999, 35-60 PIP antisense mutant of Arabidopsis has an unusually large root system as if the plant were trying to compensate for slower water loss from the shoot. IV. SIGNAL TRANSDUCTION NETWORKS IN ABIOTIC STRESSES 7. Plants have both rapid sponses to water deficit. and slow re- An important difference between plants and animals is that plants cannot move around at will. This leads to a big difference when it comes to the way they interact with their environment. During evolution, animals developed mechanisms to respond to the environment by their behavior. Plants, on the other hand, developed mechanisms to respond to their e n v i r o n m e n t d e v e l o p menially. For plant biologists, it has always been important to understand how plants respond to their environment, especially the stressful environments, because this is critical for agriculture. Perhaps the most common problem for farmers worldwide is water stress, which can result from drought, salinity, or cold temperature. Plants respond to water stress by both rapid and slow pathways. The rapid pathway involves the regulation of ion channels that close the stomatal pores in the leaves by reducing guard cell turgor. Potassium fluxes through potassium channels (see below) play an important role in this process. The slow response involves cascades of gene expression that shape the developmental program of the plant. Ion channel regulation in guard cells provides a nice model for studying the early steps in the water stress pathway. Studies have shown that water stress elicits at least two early p a t h w a y s ; one involves production of a plant hormone abscisic acid (ABA), and the other is ABA-independent (Fig 8). A number of reports suggest that the A B A - d e p e n d e n t pathway further branches out to contain calcium-dependent and calcium-independent pathways, both of which regulate the activity of several ion channels in the plasma membrane and tonoplast (Assmann, 1993; Ward et al, 1995). Calcium-dependent pathways inactivate the inward K channel, and calcium-independent pathways activate the outward K channel. Both pathways may be involved in the activation of outward anion channels. The combination of these effects causes a net efflux of ions and decline in guard cell turgor, a prelude to stomatal closure. An ABA-independent pathway directly utilizes the osmotic stress as a signal that regulates ion channels through a volume or osmo-sensing mechanism (Liu and Luan, 1998). If a guard cell is in a swelling state during stomatal opening, the inward K channel is activated, while the outward K channel is inactivated. This leads to increase in the turgor pressure and accelerates the opening process. If a guard cell is in a "shrinking state" during hyperosmotic shock caused by drought or darkness, the inward K channel will be inactivated, and the outward K channel will be activated. This leads to further efflux of K and speeds up the closure process. The volume, or osmo-sensing mechanism, provides a way for both positive feedback of stomatal movements and for response to water stress. In the slow response, a water stress signal is perceived at the plasma membrane and transduced all the way to the nucleus where + + + + + + + Osmotic Sensor two component system shol-like protein oxidative burst mechanical force DgpaD fe8l[¡Í)©d][LD©ÍÍB®DT) second messengers CDPK/MAPK cascade ABA transcriptional factors Gene Expression Biochemical Processes Figure 8. Regulation of stomatal closure resulting from water deficit or osmotic stress. The three or perhaps four pathways all lead to the opening of K channels. The outflux of K causes a drop in guard cell turgor and subsequent stomate closure. + + 49 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 gene expression is initiated. It is not hard to see the rationale because most develop mental and physiological changes in a plant start with gene expression. Examples of how scientists approach this problem are given below. Hyperosmotic Stress (drought, low humidity etc.) ABA dependent pathway / 2+ Ca -dependent A B A - i n d e p e n d e n t pathway \ / 2 Ca +-independent Cytoskeleton? \ *" 2. Molecular, genetic and cellular ap proaches have elucidated the signal trans duction pathways involved in stress re sponses. When plants are exposed to water deficit or osmotic stress, they respond physiologi cally with adaptive processes. These pro cesses have a biochemical basis that in turn depends on the expression of new genes. How the water deficit signal is perceived by the hypothesized osmotic sensor and transduced to the nucleus (Fig 9) is pres ently being investigated with molecular, genetic, and cellular techniques. The first approach uses molecular biology. A gen eral strategy here is to first look for genes that are induced by water stress conditions, which can be done by a number of methods such as differential screening of cDNA li braries. The next step is to define the cis elements that are responsible for the stress induction. These cis-acting elements are usually located in the promoter region of the gene. Using this information, one can find the transcriptional factors that interact with the cis-acting elements and go up stream of the signaling pathway by finding components that regulate the transcriptional factors. One of the leaders who use this approach is Kazuo Shinozaki's group in Japan. Here are two reports that precisely d e s c r i b e the p o w e r of this a p p r o a c h . Y a m a g u c h i - S h i n o z a k i and S h i n o z a k i (1994) describe how the initial cis-acting elements were identified. Liu et al (1998) continued to identify the transcriptional factors that act on the cis-acting elements. More recently, a genetic approach was applied to the dissection of the water stresspathway. This approach starts by fusing a water stress-responsive promoter to the luciferase reporter and then asking: Can we select for mutants that have abnormal ex pression pattern of this reporter gene in ??? | / Ion channels \ "* G u a r d cell t u r g o r Stomatal aperture Figure 9. Components in water stress signaling that lead to the expression of new genes and the physiological response of the plants. ransgenic Arabidopsisl A chemical mu tagenesis procedure was used to mutagenize the seeds, and seedlings were monitored for luciferase expression under a fluores cent camera. Plants with aberrant expres sion patterns of reporter genes under var ious conditions (drought, cold, salinity) were isolated and tested further to identify those with true defects in their response to water stress. A number of mutants have been identified that are categorized into many types. Some are constitutive response mutants that express the reporter gene even under normal conditions, suggesting a mu tation in negative regulators of the path way. Some are less responsive to the stress condition, suggesting a mutation in posi tive regulators. Some are hypersensitive to the stress condition, also suggesting mu tation in negative regulators (Ishitani et al, 1997). A good example for a cellular approach that utilized a transient assay system was given by Sheen (1996). An ABA-responsive promoter was fused to the gene-encoding green fluorescent protein (GFP) and trans formed into maize mesophyll protoplast by electroporation. The expression of G F P was monitored under fluorescence micro scope after the cells were treated by ABA, cold, and high salt. All these treatments caused expression of GFP in the trans formed cells. With the idea that calcium may serve as the second messenger for A B A , c a l c i u m i o n o p h o r e was used to mimic the treatments. Indeed calcium in creases in the cells led to expression of GFP. Then the author went on to show that 50 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 a calcium-dependent protein kinase (CDPK) may mediate calcium messenger and a protein phosphatase may antagonize the CDPK function. Biochemical approaches were also used to tackle the early steps in the water stress pathway. Most noteworthy might be the finding of mitogen-activated protein kinase (MAPK) cascade in the early stage of the water stress signaling (Jonak et al, 1996). This approach involves a technique called in-gel kinase assay that can detect the activity of a protein kinase in the protein extract isolated from plant cells treated by various stress conditions. Using this technique, M A P K is found to activate rapidly and transiently following water stress. Activation of MAPK involves phosphorylation at both threonine and tyrosine residues and inactivation requires these residues to be dephosphorylated. Protein kinases and phosphatases involved in these processes have recently been identified and have opened up a new window to understanding stress-signaling pathways (Meskiene et al, 1998; Xu et al, 1998; Gupta et al, 1998; Luan, 1998). In s u m m a r y , studies using these approaches suggest that water stress signaling may have ABA-dependent and independent pathways. ABA-dependent pathways may have calcium-dependent and calciumindependent branches. ABA-independent pathways may involve osmosensing pathways that activate M A P K cascades. All pathways may play a role in rapid response, such as stomatal regulation and slow response involving gene expression. A combination of these approaches will be fruitful in dissecting the details of these p a t h w a y s ( S h i n o z a k i and Y a m a g u c h i Shinozaki, 1997). 3. Abscisic acid and jasmonic acid ate wound signal transduction. medi- Wounding of plants, as often occurs when they are attacked by insects, causes the plants to mount a defense response. New genes are expressed both at the site of wounding and after the transmission of a systemic signal throughout the plant. When this phenomenon first began to be investigated, C. A. Ryan and his group established that wounding results in the induction of proteinase inhibitors (PINs), and the induction of the genes that encode these inhibitors (pin genes) has been used as a diagnostic to investigate the signal transduction pathway that leads from wounding to gene expression. The plant hormones abscisic acid (ABA) and jasmonic acid (JA) play an important role in the conversion of many environmental signals into changes in plant gene expression. The in vivo involvement of ABA and JA in the gene activation processes that follow mechanical damage of the plant tissue is supported by the finding that endogenous ABA and JA levels rise three- to six-fold upon wounding. This increase is, moreover, not restricted to the tissue damaged directly but can also be detected in non-wounded, systemically induced tissues (Peña-Cortés et al, 1995). This phenomenon is common to several plant species including potato, tomato and tobacco (Sánchez-Serrano et al, 1991). Furthermore, in all three plant species, a correlation has been established between the ABA and JA increase and either the expression of the pin2 gene family (in potato and tomato) or the activity of an introduced reporter gene driven by the pin2 promoter in transgenic potato or tobacco plants (Peña-Cortés and Willmitzer, 1995). Further evidence for the involvement of ABA and JA in wound-induced pin2 gene expression was provided by a series of experiments in which potato plants were sprayed with ABA or JA and pin2 mRNA accumulated in the absence of any wounding (Peña-Cortés et al, 1991; Hildmann et al, 1992). Both n o n - s p r a y e d and directlysprayed l e a v e s s h o w e d i n c r e a s e d pin2 mRNA levels with a pattern identical to the one described for wounded plants. Conclusive evidence for the involvement of ABA in wound-induced activation was obtained from mutants impaired in ABA biosynthesis. In those mutants, wound induction of pin2 was not observed (Peña-Cortés et al, 1989). However, in these mutants treatment with ABA caused a return of the accumulation of pin2 mRNA to levels normally found in wild-type plants upon wounding C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 (Peña-Cortés et al, 1995). Unexpectedly, exogenous application of JA to these mutants also lead to the accumulation of pin2 mRNA, suggesting that the JA site of action is located downstream from the site affected by ABA in the signal transduction pathway mediating wound response (PeñaCortés et al, 1996). 4. An electrical current may mediate the systemic activation of wound-induced genes. The local and systemic activation of pin2 genes following wounding suggest the existence of a signal that moves from the injured tissue to the upper or lower leaves and leads to the systemic induction of pin2 gene expression. Several mediators, both chemical and physical, have been suggested as the putative systemic signal. Phytohormones such as ABA (Peña-Cortés et al 1989; Hildmann et al, 1992), JA (Farmer and Ryan, 1990, 1992; Peña-Cortés et al, 1993), ethylene (O'Donnell et al, 1996), the peptide systemin (Pearce et al, 1991), and oligosaccharides (Ryan, 1987) all have been demonstrated to be chemical signals. Moreover, hydraulic and electrical signals, classified as physical signals, have been implicated in wound-induced gene expression. The a p p e a r a n c e of variation potentials throughout most of the shoot has been reported following localized wounding by heating or burning in several plants (Wildon et al, 1989; Malone and Stankovic, 1991). Furthermore, Wildon et al (1992) reported that mechanical wounding and localized burning generate electrical signals that are propagated through the plant, thereby inducing pin2 expression systemically. Like wounding, the application of electrical current was able to initiate ABA and JA accumulation in wild-type plants but not in ABA-deficient plants (Herde et al, 1996). These results suggested that, like wounding, electrical current requires the presence of ABA for the induction of pin2 gene expression. In contrast to wounding and electrical current, the burning of leaves activated pin2 gene expression in ABAdeficient plants by directly triggering the 51 biosynthesis of JA via an alternative pathway that is independent of endogenous ABA levels (Herde et al, 1996). Analyzing tomato ABA mutants that had progressively lower ABA levels it was found that there is a t h r e s h o l d l e v e l of A B A for s t r e s s induced gene expression (Herde et al, 1999). Since pin2 mRNA was not detected upon wounding or current application in mutants with progressive attenuation in ABA content, it was suggested that a threshold value of ABA exists and functions as an on/off switch for wound-induced pin2 gene expression. Therefore, the ABA level functioning as the threshold value for the ABAdependent pathway that leads to pin2 gene expression upon wounding and current application was between 21 % and 4 7 % of that found in wild-type tomato plants. The A B A level that a c c u m u l a t e d in wounded and current-treated plants was roughly reflected by the respective JA content of wild-type and mutant plants after these treatments (Peña-Cortés et al, 1995). Unwounded wild-type and ABA-deficient plants had almost identical levels of JA, suggesting that JA biosynthesis is not influenced by endogenous levels of ABA. Harms et al (1995) reported that high endogenous level of JA in transgenic plants overexpressing a flax aliene oxide synthase (AOS) cDNA did not induce constitutive pin2 gene expression. One possible explanation for these phenomena is that endogenous ABA and JA are stored in cellular compartments different from those w h e r e A B A and JA a c c u m u l a t e upon wounding. JA biosynthesis or at least part of it, is suggested to occur in the chloroplast (Herde et al, 1995), which implies that accumulation of JA in this organelle is not sufficient to lead to changes in the expression of JA-responsive genes such as pin2. 5. The biosynthesis of J A during stress may involve two different cellular compartments, the chloroplast and the peroxisomes. The biosynthetic pathway for JA, called the octadecanoic pathway, was first elucidated by Vick and Zimmerman (1987). The 52 C H R I S P E E L et al. Biol Res 3 2 , 1999, 3 5 - 6 0 synthesis of JA starts with linoleic acid and i n v o l v e s t h e s e q u e n t i a l a c t i v i t i e s of lipoxygenase to make fatty acid hydroper oxide, hydroperoxide dehydratase or allene oxide synthase (AOS) to produce allene oxide, and allene oxidase to produce 12oxo-phytodienoic acid (PDA). 12-oxo-PDA t h e n u n d e r g o e s c h a i n s h o r t e n i n g via 6oxidation. It is assumed that these final steps of the pathway take place in the peroxi somes, because that is where the 8-oxidation e n z y m e s a r e l o c a t e d in p l a n t c e l l s . Kleiter and G e r h a r d t (1998) r e c e n t l y p r o v i d e d evidence that long-chain fatty acids can be degraded completely into their con stituent acetyl units by higher-plant per oxisomes. But where are the first steps in the pathway carried out? When Herde et al (1995) examined the location of the flax AOS in the transformed tobacco plants (see above) they found it to be located in the chloroplasts. Subsequently, Blee and Joyard (1996) showed that the first three enzymes in the pathway, up to the production of DPA, are located in the chloroplast enve lope (Fig 10). The failure of the increased levels of JA in the AOS transgenic plants to activate the Local and systemic signals Pathogen or Insect Attack or Wounding \1 PLASMA MEMBRANE -Lipase I 1 1 LOX AOS AOC PEROXISOME Reductase ß-Oxidase -12-oxo-PDA X Reductase ^ ß-Oxidase I Jasmonic Acid Gene activation T Phosphatase Figure 10. Alternative pathway for jasmonic acid biosynthesis in plants. Over-expression of the flax allene oxide synthase (AOS) cDNA leads to an increase of endogenous jasmonic acid (JA) levels in transgenic potato plants, the transgenic protein being accumulated in the chloroplast. Blee and Joyard (1996) showed the presence of this protein in the plastidial envelope allowing the formation of 12-oxo-PDA in this organelle. The last steps of JA biosynthesis involve three B-oxidation reactions that may take place in the peroxisomes. Acyl-CoA oxidase activity involved in the decarboxylation of fatty acids generates H O Plastid and cytosolic phosphatases are involved in the signaling pathway mediating JA biosynthesis and subsequently gene activation. Kinase activity seems also to be required for the wound-induced accumulation of J A but not for gene activation. Salicylic acid inhibits JA biosynthesis by preventing the wound-induced AOS gene expression (Harms et al, 1998). LOX, lipoxygenase; AOC, allene oxide cyclase; 12-HPLA, hydroperoxylinolenic acid. 2 r 53 C H R I S P E E L et at. Biol Res 32, 1999, 3 5 - 6 0 pin2 genes could be explained by an accu mulation of JA in a compartment (possibly the p e r o x i s o m e s ) that avoids the inter action of JA with its putative receptor and subsequent gene activation. Although sixweek old AOS plants show JA levels 6-10 times higher than those found in untransformed plants, they do not show phenotypic changes. However, in older plants, the older leaves of the transgenic lines show symptoms that resemble the leaf le sion found in pathogen infected leaves. The lesions become necrotic and expand on the surface leading to leaf-death and ab scission. This phenotype expands to the upper part of the plant, and after 13-weeks the transgenic lines have only a few healthy leaves at the top of the plant (the youngest leaves). At this time untransformed plants show the first senescence symptoms in the older leaves. The phenotype observed in the transgenic plants correlates with an even higher JA content, reaching levels 1220 higher than those observed in normal plants. In these older leaves pin2 gene ex pression has been turned on and other JAresponsive genes such as A O S , lipoxy genase have been activated as well. In ad dition, the expression of pathogen-related genes (PRs) is also upregulated in the transgenic plants (Pena-Cortes unpublished results). More interestingly, the transgenic plants with increased levels of JA also show elevated levels of H , O This increase cor relates with the appearance of the symp toms and with the activation of the JAresponsive genes. Treatment of control plants with H O also results in the accu mulation of pin2-, AOS-, LOX- and PRmRNA. Furthermore, mechanical wounding or JA application leads to an increase of endogenous levels of H 0 in potato leaves. There are at least two p o s s i b l e expla nations for this observed increase in H 0 . The rise in H O could simply be a byproduct of the increased JA biosynthesis because the reaction in the pathway catalyzed by acyl-CoA oxidase generates H 0 . It is also possible the JA activates other reactions that lead to the production of H 0 or in hibits reactions that scavenge H 0 Further work is clearly needed to determine how the wound signal is transmitted in plants. r 2 z 2 2 2 2 z 2 2 2 2 2 2 2 V. PLANTS RESPOND TO BIOTIC STRESSES FROM PATHOGENS AND PESTS WITH A MULTITUDE OF DEFENSE RESPONSES THAT INVOLVE COMPLEX SIGNALING NETWORKS 1. Plant pathogen interactions are gov erned by single genes in a gene for the gene system. Genetic analysis of many plant pathogen interactions has demonstrated that plants contain single resistance genes (or R genes) that confer resistance against a specific race of the pathogens that expresses a spe cific and complementary avirulence (avr) gene. If either the plant R gene mutates or the pathogen avr gene mutates, then resist ance of the plant to the pathogen is lost. This is generally referred to as the gene for gene system of plant pathogen resistance. Resistance of a plant to a pathogen mani fests itself in a variety of ways, but is often accompanied by a hypersensitive response (HR). HR is the localized induction of cell death at the site of infection, often re sulting in numerous visible necrotic spots on the infected plant organ. Pathogenicity oc curs when the plant does not detect the presence of the pathogen soon enough to mount an effective defense response. Re sistance of a plant to a pathogen sets off a series of reactions that include a rapid oxi dative burst at the plasma membrane, ion fluxes characterized by H - K exchange, crosslinking of cell wall proteins and syn thesis of phenolics, production of antimi crobial compounds (phytoalexins), and in duction of the pathogen related (PR) genes that encode proteins such as chitinases, glucanases, osmotin, etc. These responses of the plant occur in many different plant pathogen interactions (bacteria, viruses, fungi) suggesting that there is a common signal transduction network for the re sponse. In addition to this local necrotic response, the pathogen also induces a sys temic response that results in a reduction of the severity of disease symptoms after in fection by any pathogen, including a nor mally virulent one. This phenomenon is referred to as systemic acquired resistance (SAR). + + 54 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 2. Molecular identification sistance (R) genetic approaches led to the and cloning of pathogen re genes. tion of the proteins. Song et al (1995) cloned the rice Xa21 gene which confers r e s i s t a n c e to Xanthomonas campestris oryzae pv. oryzae, the organism that causes rice bacterial blight. Xa21 e n c o d e s a re c e p t o r - l i k e kinase with an LRR in its e x t r a c e l l u l a r d o m a i n . The d o m a i n struc tures of different R genes are shown in Figure 11. This domain is thought to trans duce an e x t r a c e l l u l a r signal through acti vation of the intracellular kinase domain (Ronald, 1997). Yet another class is rep resented by the rice gene Xa21D, encod ing a p r e s u m e d secreted LRR ( W a n g et al, 1998). A l t h o u g h these genes have highly c o n s e r v e d structural motifs, vir tually nothing is known about the subcel lular location of the p r o t e i n s , the identity of the ligands or the d o m a i n s essential for p a t h o g e n r e c o g n i t i o n . Once the mo lecular d e t e r m i n a n t s g o v e r n i n g these in t e r a c t i o n s are known we can begin to d e v e l o p novel strategies for disease con trol. In the past several years, disease resist ance (R) genes have been isolated from diverse species such as tomato, Arabidopsis, t o b a c c o , flax, b a r l e y and r i c e . Inter estingly, the proteins they encode share cer tain motifs that all suggest a role in signal transduction. For example, the tomato Pto gene that confers resistance to a bacterial disease encodes a serine/threonine kinase, suggesting a role in a phosphorylation cas cade. The largest group of R proteins car ries leucine-rich repeats (LLR), putative cytoplasmic signaling domains and nucle otide binding sites. These proteins are thought to reside entirely within the cell. The tomato disease R gene Cf9, on the other hand, is an extracytoplasmic glyco protein with an LRR motif in the extracel lular domain. Such LRR domains may be instrumental in bringing about dimeriza Cf : ii 3 J Xa21D Xa21 1 Pto NBS-LRR Figure II. Domain structure of different resistance (R) gene products involved in plant disease resistance. 55 C H R I S P E E L et al. Biol Res 32, 1999. 3 5 - 6 0 diffusion to surrounding cells Figure 12. Signals in plant defense response against pathogens. The defense response is triggered by specific recognition between the product of the avirulence gene from the pathogen (Avr), and the product of the resistance gene from the plant (R). After this recognition, several plant metabolites like H,0,. ethylene, salicylic acid (SA), jasmonates (JA) and nitric oxide (NO) are accumulated. These metabolites act as secondary signals for the transcriptional activation of defense genes. 3. Signaling for transcriptional activation of genes in the defense response against pathogens. A number of genes that encode for defense proteins are activated at different times after infection ( H a m m o n d - K o s a c k and Jones, 1996). These defense proteins have different functions in the defense reaction, such as antimicrobial activity, cell wall reinforcement, synthesis of phenolic com pounds, and protection against oxidative stress. Temporal coordination in the tran scription of these defense genes is con trolled by a complex network of signaling pathways (Yang et al, 1997; Blumwald et al, 1998). In recent years, significant ad vances have been obtained in the identifi cation of components involved in this net work. Thus, several plant metabolites have been identified as secondary signals, which are transiently accumulated in the plant cell at different times after the pathogen signal is perceived. These metabolites are reactive oxygen species (ROS), such as H , 0 „ salicylic acid (SA), jasmonates (JA), ethylene, and nitric oxide (NO) (Fig 12) (Durner et al, 1997; Van Camp and Van Montagu, 1998). Furthermore, classical components for intracellular signal trans duction, such as ion fluxes, G-proteins, protein phosphorylation cascades, cyclic AMP and cyclic ADP-ribose, have been also identified (Zhu et al, 1996; Blumwald et al, 1998; Durner et al, 1998). One of the signaling pathways that has been most studied in recent years is the SAmediated pathway (Durner et al, 1997). This pathway leads to the transcriptional activation of genes encoding for acidic pathogenesisrelated proteins (PR proteins) and for oxidative stress-detoxifying enzymes (like glutathione s-transferase, GST). The biosynthesis of SA occurs via the phenylpropanoid pathway, through benzoic acid (BA). Other signals such as ethylene, jasmonates, NO and H 0 activate this biosynthetic pathway, through the transcrip tional activation or the enzymatic activation of two key enzymes (phenylalanine ammonia-ly ase, PAL, and benzoic acid 2-hydroxylase, BA2H). SA is accumulated in the vacuole as a glucoside (SAG), which is inactive (Fig 13). 2 2 56 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 oxidative burst metabolism 2 2 I ->- detoxification catalases AsPx SA- A lipid peroxides? (+) (-) SAI, PA -*-BA PAL NPR SA BH2H A / (+) SAG tx defense genes (GST, GPx, acidic PR) \ ^ ^ A B P ) S A I ethylene jasmonates NO Figure 13. Perception and transmission of'SA signal. Salicylic acid (SA) is synthesized from phenylalanine (PA) and benzoic acid (BA). This biosynthetic pathway is activated by ethylene, jasmonates and NO (which induce the expression of the gene for the enzyme phenylalanine ammonia-lyase, PAL), and by H , 0 , (which activates the enzyme benzoic acid 2-hydroxylase, BA2H). SA can be accumulated as the inactive glucoside form (SAG). SA activates transcription of genes encoding for acidic pathogenesis-related proteins (acidic PR) and for the oxidative stress-detoxifying enzymes glutathione-S-transferase (GST), and glutathione peroxidase (GPx). This effect of SA can be mediated by free radicals (S A)produced by inhibition of catalases and ascorbate peroxidase (AsPx)by the product of genes nprl/sail/niml, or by a SA-binding protein (SABP2). Different approaches have been used to elucidate the mechanism by which SA is able to transduce the signal through the nucleus and activate transcription of genes. Although some of the components involved have been identified, the mechanism is still unclear. Genetic approaches have led to the isolation of the n p r l / s a i l / n i m l class of Arabidopsis mutants (npr, nonexpresser of PR gene; sai, salicylic acid insensitive; nim, noninducible immunity). Recently, the NPR1 protein was identified as a 60kDasoluble protein containing ankyrin repeats, that are thought to function in proteinprotein interactions (Cao et al, 1997). Overexpression of NPR1 leads to enhanced re sistance to diverse pathogens (Cao et al, 1998). Biochemical and molecular approaches have lead to the characterization of SAbinding proteins in tobacco (Durner et al, 1 997). One group of these SA-binding pro teins are h e m e - c o n t a i n i n g e n z y m e s in volved in detoxification of H 0 , such as catalases and ascorbate peroxidase (AsPx). Binding of SA to these proteins inhibits 2 2 their enzymatic activity, leading to an in crease in the endogenous concentration of H O The hypothesis that H 0 and other ROS could act as second messengers down stream of SA in the transcriptional acti vation of genes is now being debated. It has been also suggested that the radical SA, produced by the interaction of SA with catalases and peroxidases, could partici pate as a secondary signal through lipid peroxidation. Recently, a new soluble SAbinding protein was identified in tobacco, although its nature and function remain to be elucidated (Du and Klessig, 1997). In the nucleus, the SA signal is able to acti vate transcription of immediate early and late genes. Several immediate early genes, which transcription does not require de novo protein synthesis, have been isolated. A h o m o l o g u e to myb gene (Yang and Klessig, 1996), a gene with homology to glucosyltransferase genes (Horvath and Chua, 1996), and GST genes ( U l m a s o v et al, 1994) are a m o n g t h i s c l a s s . C i s elements that respond to SA with an imme diate early kinetics have been identified in 2 r 2 2 57 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 the promoter of some of these and other viral and bacterial genes (Qin et al, 1994). In vitro DNA-protein binding assays and in vivo transcriptional assays using transgenic plants have given some information about the mechanism by which these elements are activated by SA. Results obtained from these studies indicate that SA increases the binding of transcription factors (from the TGA family of bzip factors) to these cis elements, mediated by protein phosphory lation (Jupin and Chua, 1996; Stange et al, 1997). Interestingly, other signals like MeJA and auxins are also able to activate transcription of GST genes, using the same cis-elements and the same m e c h a n i s m . Therefore, it seems that these nuclear events of transcriptional activation of oxidative stress-protective genes are the convergence poi nt of different signal transduction path ways. REFERENCES A L E X A N D R E J, L A S S A L E S J P , K A D O R T ( 1 9 9 0 ) O p e n ing of c a l c i u m c h a n n e l s in isolated red beet root v a c u o l e m e m b r a n e by inositol 1,4,5 t r i p h o s p h a t e . Nature 343: 567-570 A L L E N GJ, M U I R SR, S A N D E R S D ( 1 9 9 5 ) R e l e a s e of c a l c i u m from individual plant v a c u o l e s by both InsP^ and cyclic A D P - r i b o s e . S c i e n c e 2 6 8 : 7 3 5 - 7 3 7 A R I O L I T, P E N G L, B E T Z N E R A, B U R N J, W I T T K E W, H E R T H W, C A M I L L E R I C, H O F T E H, P L A Z I N S K I J. B I R C H R, C O R K A, G L O V E R J, R E D M O N D J, W I L L I A M S O N R ( 1 9 9 8 ) M o l e c u l a r a n a l y s i s of c e l l u lose b i o s y n t h e s i s in A r a b i d o p s i s . S c i e n c e 2 7 9 : 7 1 7 720 A S S M A N N ( 1 9 9 3 ) Signal t r a n s d u c t i o n in guard c e l l s . Annu Rev Cell Biol 9: 3 4 5 - 3 7 5 B A R - P E L E D M, B A S S H A M D C , R A I K H E L NV ( 1 9 9 6 ) . T r a n s p o r t of p r o t e i n s in e u k a r y o t i c cells: m o r e q u e s tions a h e a d . Plant Mol Biol 32: 2 2 3 - 2 4 9 B A R R I E U F, C H A U M O N T F, C H R I S P E E L S MJ ( 1 9 9 8 ) . High e x p r e s s i o n of the t o n o p l a s t a q u a p o r i n Z m T I P l in c o n d u c t i n g tissues of maize. Plant Physiol 17:1 1531 163 B A S S H A M D C , R A I K H E L NV (1997) M o l e c u l a r a s p e c t s of v a c u o l a r b i o g e n e s i s . Adv Bot Res 2 5 : 4 3 - 5 8 B E E V E R S L, R A I K H E L NV ( 1 9 9 8 ) T r a n s p o r t to the vacu ole: r e c e p t o r s and trans e l e m e n t s . J Exp Bot 4 9 : 1271 1279 BLF.E E. J O Y A R D J ( 1 9 9 6 ) E n v e l o p e m e m b r a n e s from s p i n a c h c h l o r o p l a s t are a site of m e t a b o l i s m of fatty acid h y d r o p e r o x i d e s . Plant Physiol 1 1 0 : 4 4 5 - 4 5 4 B L U M W A L D E, A H A R O N G S , L A M B C - H (1998) Early signal t r a n s d u c t i o n p a t h w a y s in p l a n t - p a t h o g e n inter a c t i o n s . T r e n d s Plant Sci 3: 3 4 2 - 3 4 6 B O U D E T AM ( 1 9 9 8 ) A new view of l i g n i f i c a t i o n . T r e n d Plant Sci 3: 67-71 C A R P I T A N. G I B E A U T DM ( 1 9 9 3 ) Structural m o d e l s of p r i m a r y cell walls in f l o w e r i n g p l a n t s : c o n s i s t e n c y of m o l e c u l a r s t r u c t u r e with the physical p r o p e r t i e s of the walls d u r i n g g r o w t h . Plant J 3 : 1-30 C A O H, G L A Z E B R O O K J, C L A R K E J D , V O L K O S. D O N G XN (1997) T h e A r a b i d o p s i s N P R 1 gene that c o n t r o l s s y s t e m i c acquired r e s i s t a n c e e n c o d e s a novel protein c o n t a i n i n g a n k y r i n r e p e a t s . Cell 88: 5 7 - 6 3 C A O H, LI X, D O N G XN (1998) G e n e r a t i o n of broads p e c t r u m d i s e a s e r e s i s t a n c e by o v e r e x p r e s s i o n of an essential regulatory gene in s y s t e m i c a c q u i r e d resis tance. Proc Natl A c a d Sci USA 9 5 : 6 5 3 1 - 6 5 3 6 CHAUMONT F, B A R R I E U F. H E R M A N EM. C H R I S P E E L S MJ ( 1 9 9 8 ) C h a r a c t e r i z a t i o n of a maize t o n o p l a s t a q u a p o r i n e x p r e s s e d in z o n e s of cell divi sion and e l o n g a t i o n . Plant Physiol 117: 1 143-1 I 52 C H E R E L I, D A R A M P, G A Y M A R D F. H O R E A U C. T H 1 B A U D J - B , S E N T E N A C H ( 1 9 9 6 ) Plant K+ c h a n nels: s t r u c t u r e , activity and function. B i o c h e m Soc T r a n s 24: 9 6 4 - 9 7 0 C H R I S P E E L S MJ, R A I K H E L NV (1992) Short peptide d o m a i n s target p r o t e i n s to plant v a c u o l e s . Cell 68: 613-616 D O Y L E DA, C A B R A L J.M, P F U E T Z N E R RA, K U O A, GULBIS JM, COHEN SL, CHAIT BT, MACKINNON R ( 1 9 9 8 ) T h e s t r u c t u r e of the p o t a s s i u m c h a n n e l : m o l e c u l a r basis of K c o n d u c t i o n and selectivity. S c i e n c e 2 8 0 : 69-81 D R I O U I C H A, FA YE L, S T A E H E L I N A (1993) The plant Golgi a p p a r a t u s : a factory for c o m p l e x p o l y s a c c h a rides and g l y c o p r o t e i n s . T r e n d s B i o c h e m Sci 1 8 : 2 1 0 214 DU H, C L A R K E A E , B A C I C A (1996) A r a b i n o g a l a c t a n p r o t e i n s : a c l a s s of e x t r a c e l l u l a r m a t r i x p r o t e o g l y c a n s i n v o l v e d in plant growth and d e v e l o p m e n t . T r e n d s Cell Biol 6: 4 1 1 - 4 1 4 DU H, K L E S S I G D F (1997) Role for salicylic acid in the a c t i v a t i o n of defense r e s p o n s e s in c a t a l a s e - d e f i c i e n t t r a n s g e n i c t o b a c c o . Mol Plant M i c r o b e - I n t 7: 9 2 2 925 D U R N E R J, S H A H J, K L E S S I G D F ( 1 9 9 7 ) Salicylic acid and d i s e a s e r e s i s t a n c e in p l a n t s . T r e n d s Plant Sci 2: 266-274 D U R N E R J. W E N D E H E N N E D, K L E S S I G D F (1998) Defense gene i n d u c t i o n in t o b a c c o by nitric o x i d e , cyclic G M P , and cyclic A D P - r i b o s e . Proc Natl Acad Sci U S A 9 5 : 1 0 3 2 8 - 1 0 3 3 3 F A R M E R EE, R Y A N CA (1990) Interplant c o m m u n i c a tion: A i r b o r n e methyl j a s m o n a t e i n d u c e s s y n t h e s i s of p r o t e i n a s e i n h i b i t o r s in plant l e a v e s . Proc Natl Acad Sci USA 87: 7 7 1 3 - 7 7 1 6 F A R M E R EE, R Y A N CA ( 1 9 9 2 ) R e g u l a t i o n of e x p r e s s i o n of p r o t e i n a s e i n h i b i t o r g e n e s by methyl j a s m o n a t e and j a s m o n i c acid. Plant Physiol 9 8 : 9 9 5 - 1 0 0 2 FOX T C , G U E R I N O T ML ( 1 9 9 8 ) M o l e c u l a r b i o l o g y of c a t i o n t r a n s p o r t in p l a n t s . Annu Rev Plant Mol Biol 49: 669-696 G A Y M A R D F, P I L O T G, L A C O M B E B , B O U C H E Z D, B R U N E A U D , B O U C H E R E Z J, M 1 C H A U X F E R R I E R E H (1998) Identification and d i s r u p t i o n of a Shaker-\ike o u t w a r d c h a n n e l i n v o l v e d in K release into the xylem sap. Cell 94: 6 4 7 - 6 5 5 G I L R O Y S, R E A D N D , T R E W A V A S AJ ( 1 9 9 0 ) E l e v a t i o n of s t o m a t a l c y t o s o l c a l c i u m by p h o t o l y s i s of loaded c a g e d probes initiates stomatal c l o s u r e . N a t u r e 346: 769-771 + + 58 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 G U P T A R, H U A N G Y, K I E B E R JJ, L U A N S (1998) Identification of a dual-specificity protein p h o s p h a t a s e that i n a c t i v a t e s M A P k i n a s e from A r a h i d o p s i s . Plant J 16: 5 8 1 - 5 9 0 H A H N MG (1996) M i c r o b i a l e l i c i t o r s and their r e c e p t o r s in p l a n t s . Annu Rev P h y t o p a t h o l 34: 3 8 7 - 4 1 2 H A M M O N D - K O S A C K K E , J O N E S JD ( 1 9 9 6 ) R e s i s t a n c e g e n e - d e p e n d e n t plant defense r e s p o n s e s . Plant Cell 8: 1773-1791 H A R M S K, A T Z O R N R, B R A S H A, K U H N H , W A S T E R N A C K C, W I L L M I T Z E R L . P E N A C O R T E S H ( 1 9 9 5 ) E x p r e s s i o n of a flax a l l e n e o x i d e s y n t h a s e c D N A l e a d s to i n c r e a s e d e n d o g e n o u s j a s m o n i c acid (JA) levels in t r a n s g e n i c p o t a t o plants but not to a c o r r e s p o n d i n g a c t i v a t i o n of J A - r e s p o n d ing g e n e s . Plant Cell 7: 1 6 4 5 - 1 6 5 4 H E R D E O. F U S S H, P E N A - C O R T E S H, F I S A H N J (1995) P r o t e i n a s e inhibitor II gene e x p r e s s i o n induced by electrical s t i m u l a t i o n and control of p h o t o s y n t h e t i c activity in t o m a t o p l a n t s . Plant Cell Physiol 36: 7 3 7 742 H E R D E O, A T Z O R N S R, F I S A N H J, W A S T E R N A C K C. W I L L M I T Z E R L, P E N A - C O R T E S , H (1996) L o c a l ized w o u n d i n g by heat initiates the a c c u m u l a t i o n of p r o t e i n a s e inhibitor II in A B A - d e f i c i e n t plants by t r i g g e r i n g j a s m o n i c acid b i o s y n t h e s i s . Plant Physiol 112: 8 5 3 - 8 6 0 H E R D E O, P E N A - C O R T E S H, W A S T E R N A C K C, W I L L M I T Z E R L, F I S A N H J (1999) Electric signaling and pin2 gene e x p r e s s i o n on different stimuli d e p e n d on a distinct t h r e s h o l d level of e n d o g e n o u s abscisic acid in several abscisic a c i d - d e f i c i e n t tomato m u t a n t s . Plant Physiol 1 19: 2 1 3 - 2 1 8 H I L D M A N N T , E B N E H T M , P E N A - C O R T E S H, S A N C H E Z - S E R R A N O J, W I L L M I T Z E R L, P R A T S. (1992) G e n e r a l roles of abscisic acid and j a s m o n i c acids in gene activation as result of mechanical w o u n d ing. Plant Cell 4: 1 157-1 170 H1RSCH RE, L E W I S B D , S P A L D I N G EP, S U S S M A N MR ( 1 9 9 8 ) A role for the A K T 1 p o t a s s i u m c h a n n e l in plant n u t r i t i o n . S c i e n c e 2 8 0 : 918-921 H O D G K I N A L , K E Y N E S RD (1957) M o v e m e n t s of labelled c a l c i u m in giant squid a x o n s . J P h y s i o l 138: 253-281 H O R V A T H D, C H U A N-H (1996) Identification of an i m m e d i a t e - e a r l y salicylic a c i d - i n d u c i b l e t o b a c c o gene and characterization of induction by other c o m p o u n d s . Plant Mol Biol 3 1 : 1061-1072 ISHITAN1 M, X I O N G L. S T E V E N S O N B, Z H U J-K (1997) G e n e t i c a n a l y s i s of o s m o t i c and cold stress signal t r a n s d u c t i o n in A r a b i d o p s i s : i n t e r a c t i o n s and c o n v e r g e n c e of abscisic a c i d - d e p e n d e n t and abscisic acidi n d e p e n d e n t p a t h w a y s . Plant Cell 9: 1935-1949 J A N LY, J A N YN ( 1 9 9 2 ) T r a c i n g the roots of ion c h a n nels. Cell 6 9 : 7 1 5 - 7 1 8 J O H A N S S O N I, K A R L S S O N M , S H U K L A V K , C H R I S P E E L S M J , L A R S S O N C, K J E L L B O M P (I 998) W a t e r t r a n s p o r t activity of the p l a s m a m e m brane a q u a p o r i n P M 2 8 A is r e g u l a t e d by p h o s p h o r y l a tion at t w o different sites. Plant Cell 10: 4 5 1 - 4 6 0 J O N A K C, K I E G E R L S, L I G T E R I N K W, B A R K E R PJ, H U S K I S S O N N S , H I R T H (1996) Stress s i g n a l i n g in p l a n t s : a m i t o g e n - a c t i v a t e d protein k i n a s e p a t h w a y is a c t i v a t e d by cold and d r o u g h t . Proc Natl Acad Sci USA 9 3 : 1 1 2 7 4 - 1 1 2 7 9 J U P I N I, C H U A N - H ( 1 9 9 6 ) A c t i v a t i o n of the C a M V as1 c i s - e l e m e n t by s a l i c y l i c acid: differential D N A b i n d i n g of a factor related to T G A l a . E M B O J 15: 5679-5689 K L E I T E R A, G E R H A R D T B (1998) G l y o x y s o m a l and b e t a - o x i d a t i o n of l o n g - c h a i n fatty a c i d s : c o m p l e t e ness of d e g r a d a t i o n . Planta 2 0 6 : 125-130 LIU Q, K A S U G A M, S A K U M A Y, A B E H, M I U R A S, Y A M A G U C H I - S H I N O Z A K I K, S H I N O Z A K 1 K (1998) T w o transcription factors, D R E B 1 and D R E B 2 , with an E R E B P - A P 2 D N A b i n d i n g d o m a i n s e p a r a t e two cellular signal t r a n s d u c t i o n p a t h w a y s in d r o u g h t and l o w - t e m p e r a t u r e - r e s p o n s i v e gene e x p r e s s i o n , res p e c t i v e l y , in A r a b i d o p s i s . Plant Cell 10: 1 3 9 1 - 1 4 0 6 LIU K, LU AN S ( 1 9 9 8 ) V o l t a g e - d e p e n d e n t K+ c h a n n e l s as t a r g e t s of o s m o s e n s i n g in guard c e l l s . Plant Cell 10: 1957-1970 L U A N S (1998) P r o t e i n p h o s p h a t a s e s and s i g n a l i n g c a s c a d e s in h i g h e r p l a n t s . T r e n d s Plant Sci 3: 2 7 1 - 2 7 5 M A L H O R, M O U T I N H O A, V A N D E R L U I T A. T R E W A V A S AJ (1998) Spatial c h a r a c t e r i s t i c s of c a l c i u m s i g n a l l i n g : the c a l c i u m w a v e as a basic unit in plant cell c a l c i u m s i g n a l l i n g . Phil T r a n s Roy Soc B 3: 1 4 6 3 - 1 4 7 3 M A L O N E M, S T A N K O V I C B , ( 1 9 9 1 ) Surface p o t e n t i a l s and h y d r a u l i c s i g n a l s in wheat leaves following localized w o u n d i n g by heat. Plant Cell E n v i r 14: 4 3 1 436 M A R C H A N T J S , T A Y L O R C W (1997) C o o p e r a t i v e activation of IP^ r e c e p t o r s by sequential b i n d i n g of IP^ and C a safeguards a g a i n s t s p o n t a n e o u s activity. Curr Biol 7: 5 1 0 - 5 1 8 M A U R E L C, R E I Z E R J. S C H R O E D E R JI, C H R I S P E E L S MJ ( 1 9 9 3 ) The v a c u o l a r m e m b r a n e protein y-TIP c r e a t e s w a t e r specific c h a n n e l s in Xenopus oocytes. E M B O J 12: 2 2 4 1 - 2 2 4 7 M c C A B E PF, V A L E N T I N E TA, FORSBERG LS. P E N N E L L Rl ( 1 9 9 7 ) S o l u b l e signals from cells identified at the cell wall e s t a b l i s h a d e v e l o p m e n t a l pathway in carrot. Plant Cell 9: 2 2 2 5 - 2 2 4 1 M E S K I E N E I, B O G R E L, G L A S E R W , B A L O G .1. B R A N D S T O T T E R M, Z W E R G E R K, A M M E R E R G, H I R T H ( 1 9 9 8 ) M P 2 C , a plant protein p h o s p h a t a s e 2C, functions as a n e g a t i v e r e g u l a t o r of m i t o g e n a c t i v a t e d protein k i n a s e p a t h w a y s in yeast and p l a n t s . Proc Natl A c a d Sci U S A 9 5 : 1 9 3 8 - 1 9 4 3 M U Ñ O Z P, N O R A M B U E N A L, O R E L L A N A A (1996) E v i d e n c e for a U D P - G l u c o s e t r a n s p o r t e r in Golgi a p p a r a t u s - d e r i v e d v e s i c l e s from pea and its possible role in p o l y s a c c h a r i d e b i o s y n t h e s i s . Plant Physiol 112: 1 5 8 5 - 1 5 9 4 N E C K E L M A N N G, O R E L L A N A A ( 1 9 9 8 ) M e t a b o l i s m of uridine 5 ' - d i p h o s p h a t e - g l u c o s e in Golgi vesicles from pea s t e m s . Plant Physiol 1 17: 1 0 0 7 - 1 0 1 4 N E U H A U S J M , R O G E R S JC ( 1 9 9 8 ) S o r t i n g of p r o t e i n s to v a c u o l e s in plant c e l l s . Plant Mol Biol 38: 127-144 N1COL F, HIS I, J A U N E A U A, V E R N H E T T E S S, C A N U T H, H Ó F T E H ( 1 9 9 8 ) A p l a s m a m e m b r a n e - b o u n d putative e n d o - 1 , 4 - B - D - g l u c a n a s e is r e q u i r e d for normal wall a s s e m b l y and cell e l o n g a t i o n in A r a b i d o p s i s , E M B O J 17: 5 5 6 3 - 5 5 7 6 O'DONNELL OJ, C A L V E R T C, A T Z O R N R, W A S T E R N A C K C, L E Y S E R H M O , B O W L E S DJ ( 1 9 9 6 ) E t h y l e n e as a signal m e d i a t i n g the w o u n d r e s p o n s e of t o m a t o p l a n t s . S c i e n c e 2 7 4 : 1 9 1 4 - 1 9 1 7 2 + 59 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 P A P A Z I A N D M , B E Z A N I L L A F ( 1 9 9 7 ) H o w does an ion c h a n n e l sense v o l t a g e ? N e w s Physiol Sci 12: 2 0 3 - 2 1 0 PEAR J, K A W A G O E Y, S C H R E C K E N G O S T W, D E L M E R D, S T A L K E R D ( 1 9 9 6 ) H i g h e r plants c o n t a i n h o m o logues of the b a c t e r i a l c e l A g e n e s e n c o d i n g the cata lytic s u b u n i t of c e l l u l o s e s y n t h a s e . Proc Natl Acad Sci U S A 9 3 : 1 2 6 3 7 - 1 2 6 4 2 P E A R C E G, S T R Y D O M D , J O H N S O N S, R Y A N CA ( 1 9 9 1 ) A p o l y p e p t i d e from t o m a t o l e a v e s i n d u c e s the s y n t h e s i s of w o u n d - i n d u c i b l e p r o t e i n a s e i n h i b i t o r proteins. Science 253: 895-898 P E N A - C O R T E S H, S A N C H E Z - S E R R A N O JJ, M E R T E N S R, W I L L M I T Z E R L, P R A T S ( 1 9 8 9 ) A b s c i s i c acid is i n v o l v e d in the w o u n d - i n d u c e d e x p r e s s i o n of the p r o t e i n a s e i n h i b i t o r II gene in p o t a t o and t o m a t o . Proc Natl A c a d Sci U S A 86: 9 8 5 1 - 9 8 5 5 P E N A - C O R T E S H , W I L L M I T Z E R L, S A N C H E Z S E R R A N O JJ ( 1 9 9 1 ) A b s c i c i c acid m e d i a t e s w o u n d i n d u c t i o n but not d e v e l o p m e n t a l - s p e c i f i c e x p r e s s i o n of the p r o t e i n a s e inhibitor II gene f a m i l y . P l a n t Cell 3: 9 6 3 - 9 7 2 P E N A - C O R T E S H, A L B R E C H T T, P R A T S, W E I L E R E W . W I L L M I T Z E R L, ( 1 9 9 3 ) A s p i r i n p r e v e n t s w o u n d - i n d u c e d gene e x p r e s s i o n in t o m a t o p l a n t s by b l o c k i n g j a s m o n i c acid s y n t h e s i s . P l a n t a 1 9 1 : 123128 P E N A - C O R T E S H, F I S A H N J, W I L L M I T Z E R L ( 1 9 9 5 ) S i g n a l s i n v o l v e d in w o u n d - i n d u c e d p r o t e i n a s e in hibitor II gene e x p r e s s i o n in t o m a t o and p o t a t o p l a n t s . Proc Natl A c a d Sci U S A 9 2 : 4 1 0 6 - 4 1 1 3 P E N A - C O R T E S H, W I L L M I T Z E R L ( 1 9 9 5 ) R o l e of hor m o n e s in gene a c t i v a t i o n in r e s p o n s e to w o u n d i n g . In: D A V I E S PJ (ed) P l a n t H o r m o n e s : P h y s i o l o g y , B i o c h e m i s t r y and M o l e c u l a r Biology. D o r d r e c h t , The N e t h e r l a n d s : K l u w e r A c a d e m i c Publisher, pp 4 9 5 - 5 1 4 P E N A - C O R T E S H, P R A T S, A T Z O R N R, W A S T E R N A C K C, W I L L M I T Z E R L ( 1 9 9 6 ) A b s c i s i c a c i d - d e f i c i e n t plants do not a c c u m u l a t e p r o t e i n a s e inhibitor II m R N A following s y s t e m in t r e a t m e n t . P l a n t a 198: 4 4 7 - 4 5 1 P E R R I N R, D e R O C H E R A, B A R - P E L E D M , X E N G W, N O R A M B U E N A L, O R E L L A N A A, R A I K H E L N , K E E G S T R A K (1999) Xyloglucan fucosyltransferase an e n z y m e i n v o l v e d in plant cell wall b i o s y n t h e s i s . Science 284: 1976-1979 PRESTON GM, CARROLL TP, GUGGINO WB, AGRE P ( 1 9 9 2 ) A p p e a r a n c e of w a t e r c h a n n e l s in Xenopus o o c y t e s e x p r e s s i n g red cell C H I P 2 8 p r o t e i n . S c i e n c e 256: 385-387 QIN X - F , H O L U I G U E L, H O R V A T H D M , C H U A N - H (1994) I m m e d i a t e early t r a n s c r i p t i o n a c t i v a t i o n by salicylic acid via the C a u l i f l o w e r M o s a i c V i r u s as-1 e l e m e n t . Plant Cell 6: 8 6 3 - 8 7 4 R O N A L D PC ( 1 9 9 7 ) The m o l e c u l a r basis of d i s e a s e r e s i s tance in rice. Plant M o l Biol 3 5 : 179-186 R O T H M A N J E , S O L L N E R TH ( 1 9 9 7 ) T h r o t t l e and d a m p ers: c o n t r o l l i n g the e n g i n e of m e m b r a n e fusion. Sci ence 2 7 6 : 1 2 1 2 - 1 2 1 3 R Y A N CA ( 1 9 8 7 ) O l i g o s a c c h a r i d e s i g n a l i n g in p l a n t s . Ann Rev Cell Biol 3 : 2 9 5 - 3 1 7 S A N C H E Z - S E R R A N O J J , A M A T I S, E B N E T H M , H I L D M A N N T, M E R T E N S R, P E N A - C O R T E S H, P R A T S, W I L L M I T Z E R L ( 1 9 9 1 ) T h e i n v o l v e m e n t of abscisic acid in w o u n d r e s p o n s e s of p l a n t s . In: D A V I E S W J , J O N E S H G (eds) T h e P h y s i o l o g y and B i o c h e m i s t r y of A B A . L o n d o n , E n g l a n d : Bios S c i e n tific P u b l i s h e r s L i m i t e d ( " B i o s " ) S A N D E R F O O T A A , A H M E D SU, M A R T Y - M A Z A R S D, R A P O P O R T I, K I R C H H A U S E N T, M A R T Y F, R A I K H E L N V ( 1 9 9 8 ) A p u t a t i v e v a c u o l a r c a r g o re c e p t o r p a r t i a l l y c o l o c a l i z e s with A t P E P 1 2 p on a p r e v a c u o l a r c o m p a r t m e n t in A r a b i d o p s i s r o o t s . Proc Natl A c a d Sci U S A 9 5 : 9 9 2 0 - 9 9 2 5 S A X E N A I, B R O W N M, F E V R E M , G E R E M 1 A R, H E N R I S S A T B (1995) M u l t i d o m a i n a r c h i t e c t u r e of 6 - g l y c o s y l t r a n s f e r a s e s : i m p l i c a t i o n s for m e c h a n i s m of a c t i o n . J B a c t e r i o l 177: 1 4 1 9 - 1 4 2 4 S C H R O E D E R JI, W A R D J M , G A S S M A N W ( 1 9 9 4 ) Per s p e c t i v e s on the p h y s i o l o g y and s t r u c t u r e of i n w a r d rectifying K+ c h a n n e l s in h i g h e r p l a n t s : b i o p h y s i c a l i m p l i c a t i o n s for K u p t a k e . A n n Rev B i o m o l Struct 23: 441-471 S H E E N J (1996) C a - d e p e n d e n t protein kinases and stress signal t r a n s d u c t i o n in p l a n t s . S c i e n c e 2 7 4 : 19001902 S H I N O Z A K I K, Y A M A G U C H I - S H I N O Z A K I K (1997) G e n e e x p r e s s i o n and signal t r a n s d u c t i o n in waterstress r e s p o n s e . Plant Physiol 1 15: 3 2 7 - 3 3 4 S O N G W - Y , W A N G G-L, C H E N L, K I M H - S , PI L I - Y A . G A R D N E R J, W A N G B, H O L S T E N T, ZHA1 W - X , Z H U L - H , F A U Q U E T C, R O N A L D PC ( 1 9 9 5 ) A r e c e p t o r k i n a s e - l i k e protein e n c o d e d by the rice d i s ease r e s i s t a n c e g e n e Xa21. S c i e n c e 2 7 0 : 1 8 0 4 - 1 8 0 6 S T A C E Y NJ, R O B E R T S J C , C A R P I T A N C , W E L L S B, M c C A N N M C ( 1 9 9 5 ) D y n a m i c c h a n g e s in cell sur face m o l e c u l e s are very early e v e n t s in the differen tiation of m e s o p h y l l cells from Zinnia elegans into t r a c h e a r y e l e m e n t s . Plant J 8: 8 9 1 - 9 0 6 S T A N G E C, R A M I R E Z I, G O M E Z I, J O R D A N A X, H O L U I G U E L ( 1 9 9 7 ) P h o s p h o r y l a t i o n of n u c l e a r p r o t e i n s d i r e c t s b i n d i n g to salicylic a c i d - r e s p o n s i v e e l e m e n t s . Plant J 1 1 : 1 3 1 5 - 1 3 2 4 T E M P E L B L , P A P A Z I A N D M , S C H W A R Z T L , J AN Y-N, J A N L Y ( 1 9 8 7 ) S e q u e n c e of a p r o b a b l e p o t a s s i u m channel c o m p o n e n t e n c o d e d at Shaker locus oiDrosophila. S c i e n c e 2 3 7 : 7 7 0 - 7 7 5 T R U D E A U M C , W A R M K E J W , G A N E T Z K Y B, R O B E R T S O N G ( 1 9 9 5 ) H E R G , a h u m a n inward rec tifier in the v o l t a g e - g a t e d p o t a s s i u m c h a n n e l family. Science 269: 92-95 U L M A S O V Y, H A G E N G, G U I L F O Y L E T (1994) The ocs e l e m e n t in the s o y b e a n G H 2 / 4 p r o m o t e r is activated by both active and inactive auxin and s a l i c y l i c acid a n a l o g u e s . Plant M o l Biol 2 6 : 1 0 5 5 - 1 0 6 4 V A N C A M P W , V A N M O N T A G U M ( 1 9 9 8 ) H 0 and N O : r e d o x signals in d i s e a s e r e s i s t a n c e . T r e n d s Plant Sci 3: 3 3 0 - 3 3 4 VICK BA, Z I M M E R M A N N DC (1987) Oxidative systems for the m o d i f i c a t i o n of fatty a c i d s . In: S T U M P F PK, C O N N E E (eds) T h e B i o c h e m i s t r y of P l a n t s , Vol 9. N e w York: A c a d e m i c P r e s s , pp 5 3 - 9 0 W A N G G L , R U A N D L , S O N G W Y , S I D E R I S S, C H E N LL, PI L Y , Z H A N G S P , Z H A N G Z , F A U Q U E T C, G A U T B S , W H A L E N M C , R O N A L D PC ( 1 9 9 8 ) X a 2 1 D e n c o d e s a r e c e p t o r - l i k e m o l e c u l e with a leuc i n e - r i c h r e p e a t d o m a i n that d e t e r m i n e s r a c e - s p e c i f i c r e c o g n i t i o n and is subject to a d a p t i v e e v o l u t i o n . Plant C e l l 10: 7 6 5 - 7 7 9 W A R D J M , PEI Z - M , S C H R O E D E R JI ( 1 9 9 5 ) R o l e s of ion c h a n n e l s in initiation of signal t r a n s d u c t i o n in h i g h e r p l a n t s . P l a n t Cell 7: 8 3 3 - 8 4 4 W E I G A, D E S W A R T E C, C H R I S P E E L S , M J ( 1 9 9 7 ) M o l e c u l a r a n a l y s i s of the a q u a p o r i n g e n e family in Arabidopsis thaliana. Plant P h y s i o l 114: 1347-1357 + 2 + 2 2 60 C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0 W I L D O N D C , D O H E R T Y H M , E A G L E S G, B O W L E S DJ. THA1N JF (1989) S y s t e m i c responses arising from l o c a l i z e d heat stimuli in t o m a t o plants. Ann Bot 64: 6 9 1 - 6 9 5 W I L D O N D C , T H A I N J F , M I N C H I N P E H , G U B B IR, REILLY AJ, SKIPPER YD, D O H E R T Y HM, O D O N N E L L PJ, B O W L E S DJ ( 1 9 9 2 ) Electrical sig naling and s y s t e m i c p r o t e i n a s e i n h i b i t o r i n d u c t i o n in the w o u n d e d plant. Nature 3 6 0 : 6 2 - 6 5 XU Q. FU HH, G U P T A R, L U A N S ( 1 9 9 8 ) M o l e c u l a r c h a r a c t e r i z a t i o n of a t y r o s i n e - s p e c i f i c protein p h o s p h a t a s e e n c o d e d by a s t r e s s - r e s p o n s i v e g e n e in A r a b i d o p s i s . Plant Cell 10: 8 4 9 - 8 5 7 Y A M A G U C H 1 - S H I N O Z A K I K, S H 1 N O Z A K I K ( 1 9 9 4 ) A novel c i s - a c t i n g e l e m e n t in an A r a b i d o p s i s gene is i n v o l v e d in r e s p o n s i v e n e s s to d r o u g h t , l o w - t e m p e r a ture, or h i g h - s a l t stress. Plant Cell 6: 2 5 1 - 2 6 4 Y A N G Y, K L E S S I G D F ( 1 9 9 6 ) Isolation and c h a r a c t e r i z a t i o n of a t o b a c c o m o s a i c v i r u s - i n d u c i b l e myb o n c o g e n e h o m o l o g u e from t o b a c c o . Proc Natl Acad Sei U S A 9 3 : 1 4 9 7 2 - 1 4 9 7 7 Y A N G Y, S H A H J, K L E S S I G D F ( 1 9 9 7 ) Signal p e r c e p tion and t r a n s d u c t i o n in plant d e f e n s e r e s p o n s e s . G e n e s D e v e l o p 1 1: 1 6 2 1 - 1 6 3 9 Z H U Q. D R Ö G E - L A S E R W, D I X O N R A , L A M B C ( 1996) T r a n s c r i p t i o n a l activation of plant defense g e n e s . C u r r O p i n G e n e t D e v e l o p 6: 6 2 4 - 6 3 0