Intracellular compartmentation and plant cell signalling

reviews 35 Le Guen, L. et al. (1992) Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase, Gene 120, 249-254 36 Muranaka, T., Banno, H. and Machida, Y. (1994) Characterization of tobacco protein kinase NPK5, a homolog ofSaccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase ofS. cerevisiae, Mol. Cell. Biol. 14, 2958-2965 37 Wilson, W.A., Hawley, S.A. and Hardie, G. (1996) Glucose repression/depression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP : ATP ratio, Curr. Biol. 6, 1426-1434 38 Treitel, M.A. and Carlson, M. (1995) Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein, Proc. Natl. Acad. Sci. U. S. A. 92, 3132-3136 39 Jacobsen, S.E., Binkowski, K.A. and Olszewski, N.E. (1996) SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis, Prec. Natl. Acad. Sci. U. S. A. 93, 9292-9296 40 Deng, X-W. et aL (1992) COP1, anArabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a G~ homologous domain, Cell 71, 791-801 41 Knight, J.S. and Gray, J.C. (1994) Expression of genes encoding the tobacco chloroplast phosphate translocator is not light-regulated and is repressed by sucrose, Mol. Gen. Genet. 242, 586-594 42 Roitsch, T., Bittner, M. and Godt, D.E. (1995) Induction of apoplastic invertase of Chenopodium rubrum by D-giucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation, Plant Physiol. 108, 285-294 43 Martin, T. et al. (1997) Identification of mutants in metabolically regulated gene expression, Plant J. 11, 53-62 44 German, M.S. (1993) Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and the giycolytic intermediates, Proc. Natl. Acad. Sci. U. S. A. 90, 1781-1785

45 0zcan, S. et al. (1996) Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression, Proe. Natl. Acad. Sci. U. S. A. 93, 12428-12432 46 Zenke, F T. et al. (1996) Activation of Gal4p by gatactose-dependent interaction of galactokinase and Gal80p, Science 272, 1662-1665 47 Toyoda, Y. et al. (1994) Evidence for glucokinase translocation by glucose in rat hepatocytes, Biochem. Biophys. Res. Commun. 204, 252-256 48 Lam, H-M. et al. (1996) The molecular-genetics of nitrogen assimilation into amino acids in higher plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 569-593 49 Huppe, H.C. and Turpin, D.H. (1994) Integration of carbon and nitrogen metabolism in plant and algal cells, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 577-607 50 Vincentz, M. et al. (1993) Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites, Plant J. 3, 315-324 51 Sonnewald, U., Wilke, I. and Herbers, K. (1995) Plant responses to sugar accumulation in transgenic tobacco plants, in Carbon Partitioning and Source-Sink Interactions in Plants (Madore, M.A. and Luoas, W.J., eds), pp. 246-257, American Society of Plant Physiologists 52 Szekeres, M. et al. (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis, Cell 85, 171-182 53 Somerville, S. and Somerville, C. (1996)Arabidopsis at 7: still growing like a weed, Plant Cell 8, 1917-1933

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Intracellular compartmentation and plant cell signalling

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Jacques Joyard, Alair Pugin and Raou Ranjeva

Compartmentation is an essential feature of eukaryotic cells, and is crucial for the regulation of cell metabolism. Recent progress has significantly improved the understanding of signal transduction pathways in plants, including the activation of lightsignalling networks and the tightly controlled generation of the calcium message. Cell compartmentation is important for the regulation and proliferation of these signalling processes. C

ompartmentation is necessary for the functional organization of eukaryotic cells, because it allows metabolic pathways to be sequestrated within membrane-bound organelles, and allows the independent regulation and control of metabolic fluxes in response to cellular metabolism 1. The interaction between metabolic pathways is achieved through membranes whose highly selective permeability limits the number of intermediates transported from one compartment to another. Of all the eukaryotic organisms, metabolic compartmentation is probably at its most complex in plant cells: in addition to the organeltes found in other 21 4

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eukaryotes, plant cells contain structures such as the vacuole, a variety of plastids and the cell wall. This dictates the need for carefully integrated intracellular signalling pathways to coordinate the sequential events involved in signal transduction between different compartments. As plant cell compartmentation is unique, comparative analyses of signal transduction processes in animals or fungi cannot be relied on for wholly understanding the mechanisms in plants. This review considers the consequences of compartmentation for signalling in plants, and focuses on two examples: the signalling networks by which light induces pleiotropic effects

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Fig. 1. Compartmentation of PhyA ('phytochrome') signal transduction pathways as determined by microinjection experiments. Single-cell assays in a PhyA-deficient mutant [tomato aur ('aurea')] were used to demonstrate that phytochrome signalling pathways involve the ~ subunit (G~) of heterotrimeric G proteins, cyclic guanosine 3'-5'-cyclic monophosphate (cGMP), Ca2+ and calmodulin5'6. (a) In wild-type cells, light triggers full chloroplast development and anthocyanin accumulation within the vacuole. Components of the signalling pathways shown include: cab ('chlorophyll a/b-binding protein'); fnr ('ferredoxin:NADP-oxidoreductase'); and chs ('chalcone synthase'). (b) Microinjection of PHYA protein, or of both Ca2÷or calmodulin and cGMP, in a mutant hypocotyl cell, mimics the effect of light in the wild type. The Ca2÷/calmodulinand the cGMP pathways can be inhibited by trifluoperazine and genistein, respectively. (c) Microinjection of Ca2÷or calmodulin induces the expression of some photosynthesis genes, such as cab, from the light-harvesting complex, but only partial chloroplast development. In addition, no anthocyanin accumulation is observed (broken lines show inactive pathways). (d) Microinjection of cGMP induces the expression of chs and therefore anthocyanin synthesis and accumulation within the vacuole. In this case, no chloroplast development is observed. The expression of some photosynthesis genes, such as fnr, requires the addition of both Ca2+or calmodulin and cGMP. An uncharacterized 'plastidic factor' produced by the developing plastids is necessary for complete chloroplast biosynthesis 3'14.Distinct arrows represent different signalling pathways; headless arrows show inhibition.

on cell compartments; and the compartmentation and regulation of concentration of Ca 2÷involved as a second messenger in multiple response pathways.

Light signal transduction, compartmentation and plastid biogenesis Light is not only necessary as the energy source for photosynthesis, but is also important for all stages of plant growth and development, from seed germination to flowering 2. At the cellular level, light has pleiotropic effects on various compartments, from transcriptional activation of photosynthesis-related genes to anthocyanin accumulation within the vacuole. In addition, chloroplast biogenesis is regulated by an intricate series of interactions between the nuclear and chloroplast genomes, which respond to extrinsic signals, such as light, and also to intrinsic signals as a part of normal plant development3. Therefore, the light regulatory network and other signalling pathways are probably interlinked. In this context, a family of light-sensing proteins that mediate plant responses to red and far-red light the phytochromes - has been extensively studied 4, with a view to establishing their role in the transduction pathway.

Transduction of the light signal The possible involvement of heterotrimeric G proteins in the light-regulated expression of several nuclear and chloroplast genes has been suggested by experiments involving the microinjection of GTP~S and cholera toxin A-chain into hypocotyl cells of the aur ('aurea') mutant of tomato ~. This mutant lacks phytochrome A and is devoid of functional chloroplasts. Indeed, microinjection of various putative signalling molecules, or antagonists/agonists of these molecules, was used to establish three distinct phytochrome signal transduction pathways (Fig. 1) (Refs 5 and 6): • A Ca2+-dependent pathway that regulates the transcription of photosystem II genes, ATP synthase structural genes, Cab ('chlorophyll a/b-binding protein'), Rbcs and partial chloroplast development. • A cyclic GMP (cGMP)-dependent pathway that regulates the expression of the gene encoding chalcone synthase and the production of anthocyanin. • A pathway dependent on both Ca 2+ and cGMP that regulates the expression of genes encoding components of photosystem I and cytochrome bJf complexes, and is required for complete chloroplast development. June 1997,Vol.2, No. 6

215

reviews As differential regulation of the pathways probably regulates the relative levels of photosynthetic complexes and anthocyaninss, the importance of compartmentation of all the regulatory factors, enzymes, ions or metabolites involved in these pathways should also be considered. For example, are the G proteins involved in the different pathways soluble in the cytosol, can they partition between the nucleus and the cytosol, or are they membrane-bound, like the blue-light-activated G protein present in the plasma membrane of etiolated pea seedlingsT? The implication that Ca 2÷ is required for the phytochrome signal transduction pathway is further illustrated by the operation of Ca 2÷sensitive protein phosphorylation in response to red light s, but its involvement in the control of potassium channels by phytochrome has yet to be demonstrated9.

Analysis of photomorphogenesismutants One of the most effective methods of dissecting signal transduction pathways for light responses is the analysis of mutants exhibiting anomalous photoresponses 2. In Arabidopsis, mutations in positive regulators of photomorphogenesis result in the hy ('hypocotyl elongated') phenotype in plants grown in the light, whereas mutations in negative regulators result in the cop ('constitutive photomorphogenic') or det ('de-etiolated') phenotypes in plants grown in the dark. In other mutants, such as fus ('fusca'), an additional phenotype is the accumulation of anthocyanin, providing further evidence for a link between the lightregulated synthesis of vacuolar compounds and chloroplast development. Using mutants for positive regulatory elements of the phytochrome signal transduction pathway, it has been possible to characterize a unique far-red-light response that results in the inability of cotyledons to green upon illumination; this response is suppressed by sucrose~°. This block in greening is the result of severe repression of the nuclearencoded genes encoding the plastid enzyme protochlorophyltide reductase, coupled with irreversible plastid damage. In addition to compartmentation of the response, these observations highlight the possibility of cross-talk occurring between light signalling, sugar sensing, and other signalling pathways. When grown in darkness, mutants for negative regulators of photomorphogenesis (cop, det and fus mutants) exhibit almost all the phenotypic characteristics normally observed in light-grown, wild-type seedlings. However, such mutants show various signs of incomplete chloroplast development. For example, the plastids in the detl, copI and cop9 mutants develop an extended thylakoid membrane network, but lack chlorophyll11. Tissue specificity for plastid differentiation is also affected - ectopic expression of photosynthesis-related genes has been observed in roots ~2. The observation that both nuclear-encoded (e.g. Rbcs, Cab and PetF) and chloroplast-encoded (e.g. rbcL, psbA and psaA) photosynthesis-related genes are constitutively expressed in the dark and in dark-adapted plants suggests that COP and DET gene products are components of a signal transduction pathway responsible for coupling light perception to chloroplast and cell developmentH. These gene products act downstream of photoreceptors, as shown by analyses of double mutants between the pleiotropic cop, det and fus mutations and the photoreceptor mutations. The cloning of copl, fus6/copll, detl and cop9 (Ref. 11)has generated the necessary molecular tools with which to analyze the signalling pathways. It is known that the corresponding gene products 21 6

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are present in all tissues and that their accumulation is independent of light activation. They are therefore proposed to act together, within the nucleus, as negative regulators of photomorphogenic development. Light signals may disrupt these nuclear interactions, and thereby permit the expression of photomorphogenesis. Thus, it is likely that light modulates their activities post-translationally, through protein-protein interactions, protein modification, subcellular localization or a combination of these processesit. Whereas the nuclear localization of the COP9 complex and DET1 seems to be independent of the light conditions, the nucleocytoplasmic partitioning of COP1 is regulated by light in a cell type-dependent manner ~3.Interestingly, COP1 contains some homology to a ~ subunit of heterotrimeric G proteins, which further illustrates the flexible localization of such transduction proteins. For example, when dark-grown Arabidopsis seedlings are exposed to light, a ghicuronidase (GUS)-COP1 fusion protein slowly partitions from a nuclear to a cytoplasmic location in hypocotyl cells, and the opposite happens for light-grown seedlings transferred into darkness. In root cells, however, GUS-COP1 fusion protein is constitutively nuclear, which is consistent with the established role of COP1 in suppressing chloroplast development in roots in both light and dark 11.

Regulation of chloroplast gene expression Regulation of the phytochrome signal transduction pathway may involve the release of molecules from various cell compartments into the cytosol. During chloroplast development, the expression of nuclear genes encoding chloroplast components involves a feedback mechanism from the chloroplast to the nucleus3'14. In many mutants having defective chloroplasts, nuclear genes encoding photosynthesis-related proteins are not expressed14. This has been extensively analyzed in carotenoid-deficient mutants and in plants treated with norflurazon (a carotenoid biosynthesis inhibitor), which show similar phenotypes. The most probable mechanism for explaining such observations is the production, by chloroplasts, of a positive signal required for the expression of photosynthesis-related nuclear genes, the production of this 'plastidic factor' being prevented by photooxidation3,14. To date, nothing is known about the chemical nature of the plastidic factor, despite the extensive use of inhibitors of chloroplast transcription and translation, chlorophyll synthesis and photosynthesis. The possible involvement of molecules deriving, directly or indirectly, from chloroplast metabolism cannot be entirely ruled out, because there is increasing evidence that metabolites play a role in the regulation of gene expression15. For example, hexose sugars can induce repression of light-induced, photosynthesisrelated genes 16, and also repress genes for chloroplast proteins that are not light regulated, such as the gene encoding the phosphate/triose-phosphate translocator of the chloroplast envelope3. Hexokinases may act as the sensor and signal transmitter for sugar repression in higher plants is. Interestingly, a hexokinase activity has been identified and characterized in the outer envelope membrane of chloroplasts tT. By virtue of their localization at the interface between the chloroplast and the cytos01, envelope membranes may play a pivotal role in signal transduction from the chloroplast to other cell compartments. In addition, signals produced either within or outside plastids can be perceived on either the outer or the inner envelope membrane.

reviews Once again, genetic approaches may provide clues for the identification of elements of the signal transduction pathway from chloroplasts to the nucleus, and especially the plastidic factor. For example, Arabidopsis mutants with recessive gun ('genomes uncoupled') mutations have been identified that point to the importance of coupling the expression of nuclear genes encoding photosynthesis-related proteins to the functional state of chloroplast development18. The recessive nature of the gun mutation suggests that the signal transduction pathway normally functions to repress nuclear gene expression in the absence of the plastidic factor.

Calcium compartmentation and signalling Calcium ions are now recognized as major second messengers in higher plants, where they have been shown to regulate many processes 19. Originally, research efforts concentrated on understanding the control of cytosolic Ca~*concentration ([Ca2+]~t), which is known to act as a Ca2÷signal that responds to both biotic and abiotic effectors (Fig. 2). As a consequence, the other organelles have been considered as simple Ca2÷ stores from which Ca 2÷may be mobilized to elicit Ca 2÷bursts and oscillations in the cytosol. In fact, it is becoming increasingly clear that compartments such as chloroplasts have their own free Ca2÷ concentrations that are subject to oscillations that may be crucial to the regulation of chloroplast functions2°. In essence, the Ca2÷ concentration of a given compartment depends on the balance of uptake over release. This is achieved via channels and pumps, the activities of which are controlled and finely tuned by external and/or intracellular signals. The plasma membrane, the tonoplast (delimiting the large central vacuole) and the endoplasmic reticulum contain systems working either against or down the electrochemical gradient. At the plasma membrane, for example, different types of Ca2÷ channels, such as nonselective Ca 2+ channels activated by abscisic acid21, stretch22or voltage23'24, have been characterized along with exchangers and Ca2+ATPases 19. Such a complex set of membrane-bound proteins also exists at the tonoplast, where channels operated by either inositol 1,4,5-triphosphate (IP~) (Ref. 25)or cyclic ADP-ribose (cADPR) (Ref. 26)(Fig. 2a) have been characterized, in conjunction with voltage-activated channels that are purported to contribute to Ca~÷-dependent Ca2+ release from the vacuole27. Very recently, voltage-gated channels presumably involved in mechanosensitive response have been identified at the endoplasmic reticulum - where Ca2÷ ATPases also occur2s. As regards other compartments, it is unclear how Ca 2+ permeates the membrane to flow into or out of the organetles, even if Ca2+channels are likely to exist at the nuclear envelope and Ca2÷ ATPase at the mitochondria. By and large, the number of identifiable membranebound proteins involved in Ca2+ movement is increasing with time, and it is anticipated that their functioning will be found to be tightly coordinated.

Different cell compartments and the regulation of cytosolic calcium Recent results have shown that different cell compartments may contribute to the regulation of [Ca2+]~yt through the selective activation of channels and/or transporters in response to external or internal signals. For example, microinjection of caged IP3 into guard cells results in the increase of [Ca2+]cyt(Ref. 29). Such an increase, which promotes stomatal closure, is caused by the release of vacuolar

Ca2+via IPgperated channels29. Interestingly, cytosolic Ca2+ oscillates in response to different stimuli (Fig. 2) (Ref. 30). Although no biological role has yet been attributed to such Ca2+oscillations, they have the potential to encode stimulusspecific information. An approach using genetically transformed plants expressing apoaequorin31 has allowed better insight into the importance of Ca2+ compartmentation in signal transduction. Aequorin, the luminescence of which is specific for Ca2+, shows changes in intracellular Ca2+. A recent refinement came from the development of techniques to direct aequorin expression to specific subcellular locations and thus to follow Ca2+ concentration in various cell compartments 2°'32. Using this approach, it has been shown that cold shock induces a large increase in [Ca2+]cyt in Arabidopsis seedlings 32(Fig. 2b). Such an increase is mainly caused by an influx of extracellular Ca~+. However, the use of apoaequorin targeted to the cytoplasmic face of the tonoplast, able to report on localized 'microdomain' increases in Ca2+released from the vacuole, has provided evidence that the vacuolar compartment contributes to the Ca2÷ signal generated in response to cold shock32.

Differential regulation of cytosol and chloroplast calcium It has been established that [Ca2+]cyt oscillates following a circadian pattern under continuous illumination of tobacco and Arabidopsis seedlings2°. In constant darkness, the rhythms damp rapidly and usually disappear. As light is known to control chloroplast activity and especially Ca2+ influx, it was interesting to assess whether chloroplasts are involved in these circadian changes in [Ca2+]cyt. Apoaequorin has been targeted to the chloroplasts by creating a fusion construct with the coding sequence of the transit peptide of the small subunit of Rubisco. In contrast to [Ca2+]cyt, no significant changes in chloroplast Ca 2+concentration ([Ca2+]~hz) could be detected under continuous illumination (Fig. 2c). More surprisingly, a significant increase in [Ca2*]chloccurred on switching the light off, and this was followed by occasional damped oscillations. These noninvasive and direct measurements of Ca2÷ concentrations thus revealed that [Ca2+]cyt and [Ca2+]~hI are differently regulated. The generation of apoaequorin constructs specifically targeted to organelles will allow us to decipher, in detail, the contribution that each compartment makes to the transduction process.

Re-interpretation of prokaryote and eukaryote signalling repertoires Unravelling the signalling pathways that plants use to respond to endogenous regulators controlling their development, or to adapt to changes in their environment, represents a longstanding goal of plant biologists 33. Although substantial progress has been made in recent years, a clear picture of plant signalling networks is still far from complete. Research has been partly based on parallels with known signalling pathways in animal systems. This has proved to be fruitful, as many of the plant genes already identified share striking sequence and functional homologies with their animal counterparts. Cell signalling paradigms thus emerge, involving conserved elements such as G proteins, protein kinases and phosphatases, and second messengers such as Ca2* or phospholipid-derived molecules 34. However, genetic studies have revealed that plants have evolved their own specific pathways, involving multifunctional proteins, based on prokaryote and eukaryote June 1997, VoL 2, No. 6

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repertoires. The best example to date is provided by the ethylene transduction cascade in Arabidopsis, which involves a membrane receptor resembling the 'two-component' regulators from bacteria, and a protein kinase closely related to the animal MAPKKKs (mitogen-activated protein kinase kinase kinases) 35. It is also clear from the examples described that the unique complexity of compartmentation in plant cells contributes to an elaboration of these pathways.

The formation of signalling molecules and the complexity of compartmentation Compartmentation of the biosynthesis of signalling molecules is one of the first regulatory events in intracellular signalling. For example, although a wide variety of plant 21 8

June1997,Vol,2, No. 6

growth regulators derived from amino acids (e.g. auxin and ethylene) or adenine (cytokinins) appear to be made in the cytosol, the cell wall is a source of oligosaccharides, and plastids produce various terpenoid- and lipid-derived signalling compounds. Also, compartmentation is required for the biosynthesis of terpenoid-derived growth regulators: brassinosteroids and phytoalexins are synthesized from the cytosolic farnesyl pyrophosphate, whereas geranylgeranyl pyrophosphate, synthesized in chloroplasts, is the precursor for abscisic acid and gibberellins 3~-~8(Fig. 3). Little is known about how these pathways are regulated by environmental and developmental signals. By analogy with animal cells, signalling cascades involving lipid-derived compounds may also operate in plants 39'~°.

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June1997,Vol.2,No.6

21 9

reviews responses; when cell compartmentation is destroyed, it allows the formation of volatile lipid-derived compounds such as jasmonate.

Extra- or intracellular receptors? Signalling pathways are frequently initiated at the cell surface. In the case of plant-microbe interactions, the host plasma membrane is the site of recognition between cells and molecular signals (e.g. Nod factors responsible for triggering the development of symbiotic nodules 43, or elicitors of defence reactions against pathogens44). In the latter case, binding of elicitor molecules to specific receptors at the plasma membrane activates a signal transduction pathway that generates a complex network of second messengers and triggers the expression of appropriate genes involved in the Fig. 4. A model for the cascade induced in tobacco cells by cryptogein, an elicitor defence response. High-affinity binding protein from Phytophthora cryptogea. The interaction of the elicitor with highsites for various elicitors have been affinity binding sites (1) first triggers protein phosphorylation (2) and an influx of reported to occur on the plasma memCa2+ (3). Staurosporine can inhibit the protein phosphorylation (2), and La3. can brane. The putative cascade triggering inhibit Ca~÷influx. Protein phosphorylation should involve the receptor and/or a the hypersensitive response of tobacco Ca2÷ channel. The Ca2÷ influx is responsible for the activation of several plasma cell suspensions to cryptogein, an elicitor membrane proteins and for the biosynthesis of phytoalexins (4). The cytosolic Ca~÷ protein from Phytophthora cryptogea 4~, increase triggers chloride and potassium efflux (5) and the activation of a plasma is shown in Fig. 4. The elicitor-receptor membrane NADPH oxidase (6), which is responsible for a transient production of active oxygen species (7), the alkalization of the extracellular medium and the acidinteraction activates a cascade of events ification of the cytosol (8). The cytosolic pentose phosphate pathway regenerates related to the activation of plasma memthe NADPH used as a source of electrons by the oxidase. In elicitor-treated cells, brane proteins: early events involve proCa2+ and chloride channel activation is responsible for a large plasma membrane tein phosphorylation, Ca 2+ influx, chlodepolarization (9). This depolarization may trigger signal amplification as addiride and potassium efflux, plasma tional ion channels respond to depolarization. Among these channels, plasma meremembrane depolarization, transient probrane voltage-dependent Ca2+-channels may be activated. Both plasma membrane duction of activated oxygen species by a depolarization and cytosol acidification should lead to the activation of the plasma plasma membrane NADPH oxidase, membrane H+-ATPase, but this proton pump can be transiently inhibited (5') in cytosol acidification and extracellular order to prevent an excess of ATP consumption. Cytosol acidification probably alkalinization. Cytosolic Ca 2+ and pH, induces changes in the activity of many unidentified enzymes. Active oxygen species, including superoxide, hydrogen peroxide and the hydroxyl membrane potential and activated oxyradical, seem to play an important role in the elicitor-induced hypersensitive reacgen species may thus build a complex tion. Hydrogen peroxide from the transient oxidative burst triggers many defence network of second messengers involved responses, including cross-linking of proline-rich cell wall proteins and peroxidain signal amplification towards a spetive polymerization of cinnamoyl alcohols, leading to the lignification of cell walls. cific defence response. Such complexMoreover, it has been reported that H202 triggers the hypersensitive death of chality has recently been outlined by lenged cells and functions in surrounding cells as a diffusible signal for induction Hammond-Kosack and Jones 4~, who sugof defence genes encoding enzymes involved in cellular protection, such as glutagest that the hierarchy of signalling thione S-transferase, ghitathione peroxidase and polyubiquitin. Abbreviations: events contributes to the amplification DPI, diphenylene iodonium; Rp, putative receptor; Pi, inorganic phosphate; La, lanand coordination of the diverse array of thanum; SOD, superoxide dismutase. defence responses in the various cellular compartments. However, it is still unclear as to which membrane is the tarThe picture is less clear fbr receptors that mediate get for the different enzymes involved (these include phos- responses to endogenous growth regulators (with the exceppholipases A, C and D, and lipoxygenases). It is often tion of the ethylene receptor, which is known to reside on the assumed that the plasma membrane generates phospho- plasma membrane3~). For example, apparently contradictory lipid-derived second messengers, but this can also be the results have been presented on the location of abscisic acid case for other plant cell membranes. For example, phospho- receptors. This suggests that surface receptors and intracellipase D and A1 activities were detected in the tonoplast of lular receptors may well co-exist and mediate different sets Acer pseudoplatanus cells 41. Plastid envelope membranes of responses 47. In the case of auxin, the best-characterized contain a whole set of enzymes that can produce diacylglyc- auxin-binding protein (ABP1) exhibits curious features, erol, phosphatidic acid 17 and various fatty acid-derived mol- as the protein mainly resides within the endoplasmic retiecules, such as 12-0xo-phytodienoic acid 42 (the precursor of culum (where its function is unknown), but also acts as an jasmonate). T h e importance of compartmentation in the auxin receptor at the plasma membrane 4s'49. It is not regulation of signal production is highlighted during wound known how ABP1 reaches the plasma membrane or how 220

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reviews this relocalization is controlled. Recent findings that demonstrate a role for brassinosteroids in plant growth and development 3s have renewed interest in the possible existence of steroid receptors in plant cells. Further analyses of brassinosteroid-insensitive mutants will be of particular interest, as they may reveal whether the mechanism of action of brassinosteroids in plants is similar to that operating in animals. In animals, this process proceeds via diffusion of the ligand within the cell, followed by an association with cytosolic or nuclear receptors, which in turn may directly interact with promoter sequences and regulate gene expression. In addition to the few putative receptors for plant hormones identified to date, the recent literature reveals an increasing number of transmembrane protein kinases that resemble the receptor protein kinases of animals s°. These proteins, known as receptor-like protein kinases, exhibit a single transmembrane domain, an intracellular kinase domain able to catalyse autophosphorylation on serine and threonine residues, and can be classified according to the structure of their extracellular domain. Although the function of most of the proteins is still unknown, their expression in a wide variety of tissues, together with the observed variations in their structure, suggest that they may act as cell surface receptors for diverse extracellular ligands. The identity of such ligands has still to be clarified. The recent discovery that small peptides play a role in the response of cells to phytohormones 51'52 suggests that there may be a variety of interesting putative ligands for these transmembrane receptor kinases.

Concluding remarks A combination of experimental approaches has brought considerable progress in defining major elements of the signal transduction pathways in plant cells. At present, our understanding of the spatio-temporal aspects of signalling is still in its infancy, but an emerging picture is that the various pathways involve specific receptors, a network of signalling components with remarkably conserved functions and interaction with other pathways. Tight compartmentation is often required to ensure a specific response. Such organization allows signalling intermediates to work in parallel pathways. Understanding how the various signals perceived by the numerous and specific receptors are integrated to control cellular development and differentiation is a major goal, and the importance of compartmentation to these processes needs to be addressed. An appreciation of the long-distance regulation of metabolism may be helpful in understanding such processes, as already demonstrated in sugar sensing and signallinglh'ls: sugars are long-distance messengers of whole-organism carbohydrate status, as well as substrates for both cellular metabolism and local carbohydrate-sensing systems. The sites, timing and extent of signal-induced gene expression widely contribute to the integration of the whole plant within its environment, and are therefore involved in the regulation of physiological and developmental processes. Dedication Dedicated to Prof. Jean Guern on the occasion of his retirement. References

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Articles of interest

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42 BlUe,E. and Joyard, J. (1996) Envelope membranes from spinach chloroplasts are a site of metabolism of fatty acid hydroperoxides, Plant Physiol. 110, 445~54 43 Geurts, R. and Franssen, H. (1996) Signal transduction inRhizobiuminduced nodule formation, Plant Physiol. 112, 447~53 44 Jackson, A.O. and Taylor, C.B. (1996) Plant-microbe interactions: life and death at the interface, Plant Cell 8, 1651-1668 45 Pugin, A. and Guern, J. (1996) Mode of action of elicitors: involvement of plasma membrane fimctions, C. R. Acad. Sci. Paris 319, 1055-1061 46 Hammond-Kosack, K.E. and Jones, J.D.G. (1996) Resistance genedependent plant defense responses, Plant Cell 8, 1773-1791 47 Giraudat, J. (1995) Abscisic acid signaling, Curr. Opin. Cell Biol. 7, 232-238 48 Barbier-Brygoo, H. (1995) Tracking auxin receptors using functional approaches, Crit. Rev. Plant Sci. 14, 1-25 49 Venis, M.A. and Napier, R.M. (1995) Auxin receptors and auxin-binding proteins, Crit. Rev. Plant Sci. 14, 27-47 50 Braun D.M. and Walker, J.C. (1996) Plant transmembrane receptors: new pieces in the signaling puzzle, Trends Biochem. Sci. 21, 70-73 51 van de Sande, K. et at. (1996) Modification ofphytohormone responses by a peptide encoded by ENOD40 of legumes and a nonlegnme, Science 273, 370-373 52 Miklashevichs, E. et al. (1996) Do peptides control plant growth and development? Trends Plant Sci. 1, 411

H ~ n e Barbier-Brygoo* is at thelnstitut des Sciences Centre National dela Recherche Scientifique (CNRS)I U Propre de Recherche 40, Avenue de la Terrasse, F~93.198Gif Su~"

is at the Unit6.Associee I de Bourg~gn Phyto Biochimie de.Interactions Cellula[res:, INRA .. BV1540. F-21034 Dijon Cedex, Franoe;and Raoul Ranjeva is at,,~ theLaboratoire Signaux e,t Messages Cellutaires chez les..

*Auth~rfof c0rrespbridence(tei:-+33169 82 ~8:68i i~k:~:+33~:i/~9 823768

e-mailhelene.br/[email protected],cnrs-gif.fr~21

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