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Like their animal counterparts, plant glutamate receptor-like (GLR) homologs are intimately associated with Ca(2+) influx through plasma membrane and participate in various physiological processes. In pathogen-associated molecular patterns (PAMP)-/elicitor-mediated resistance, Ca(2+) fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca(2+)] ([Ca(2+)]cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense-related genes by OGs. In addition, wild-type Col-0 plants treated with the glutamate-receptor antagonist 6,7-dinitriquinoxaline-2,3-dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs-triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen-mediated plant defense signaling pathways in Arabidopsis thaliana.

Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca2+ signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca2+ signatures: both elicitors induced a biphasic cytosolic [Ca2+] elevation together with a single mitochondrial [Ca2+] elevation concomitant with the first cytosolic [Ca2+] peak. In addition, both elicitors induced a chloroplastic [Ca2+] elevation peaking later in comparison to cytosolic [Ca2+] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP3 should play an important role in Ca2+ signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca2+] variations. Our data also showed that, depending on [Ca2+] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles. (C) 2012 Elsevier Ltd. All rights reserved.

International audience; The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapid and transient increase in free cytosolic Ca2þ in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca2þ-channel blocker (La3þ) showed that in these cells, MAPK activation was dependent on the cytosolic Ca2þ increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca2þ transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity.

Plant cells use calcium-based signalling pathways to transduce biotic and/or abiotic stimuli into adaptive responses. However, little is known about the coupling between calcium signalling, transcriptional regulation and the downstream biochemical processes. To understand these relationships better, we challenged tobacco BY-2 cells with cryptogein and evaluated how calcium transients (monitored through the calcium sensor aequorin) impact (1) transcript levels of phenylpropanoid genes (assessed by RT-qPCR); and (2) derived-phenolic compounds (analysed by mass spectrometry). Most genes of the phenylpropanoid pathway were up-regulated by cryptogein and cell wall-bound phenolic compounds accumulated (mainly 5-hydroxyferulic acid). The accumulation of both transcripts and phenolics was calcium-dependent. The transcriptional regulation of phenylpropanoid genes was correlated in a non-linear manner with stimulus intensity and with components of the cryptogein-induced calcium signature. In addition, calmodulin inhibitors increased the sensitivity of cells to low concentrations of cryptogein. These results led us to propose a model of coupling between the cryptogein signal, calcium signalling and the transcriptional response, exerting control of transcription through the coordinated action of two decoding modules exerting opposite effects.

International audience; The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by β-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase–dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem.

A decade-long investigation of nitric oxide (NO) functions in plants has led to its characterization as a biological mediator involved in key physiological processes. Despite the wealth of information gathered from the analysis of its functions, until recently little was known about the mechanisms by which NO exerts its effects. In the past few years, part of the gap has been bridged. NO modulates the activity of proteins through nitrosylation and probably tyrosine nitration. Furthermore, NO can act as a Ca2+-mobilizing messenger, and researchers are beginning to unravel the mechanisms underlying the cross talk between NO and Ca2+. Nonetheless, progress in this area of research is hindered by our ignorance of the pathways for NO production in plants. This review summarizes the basic concepts of NO signaling in animals and discusses new insights into NO enzymatic sources and molecular signaling in plants.

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.

Anion effluxes are amongst the earliest reactions of plant cells to elicitors of defence responses. However, their properties and their role in disease resistance remain almost unknown. We previously demonstrated that cryptogein, an elicitor of tobacco defence responses, induces a nitrate (NO(3) (-)) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO(3) (-) efflux and clarified the mechanisms through which it contributes to cell death. Application of the discontinuous single electrode voltage-clamp technique in tobacco cells elicited with cryptogein enabled us to record the activation of slow-type deactivating anion channel currents. Cryptogein-induced plasma membrane depolarization and Ca(2+) influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO(3) (-) efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO(3) (-) efflux in mediating proteases activation was further assessed. It is shown that cryptogein induced the activation of three proteases with apparent molecular masses of 95, 190 and 240 kDa. Their activation occurred independently on the anion efflux and, together with cell death, was strongly reduced by cycloheximide and the protease inhibitor PMSF. In contrast, the NO(3) (-) efflux was shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during the HR. Collectively, our data indicate that anion efflux is an early prerequisite to morphological and biochemical events participating to cell death.

In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.

In animals, nitric oxide (NO) is produced by a family of enzymes named nitric oxide synthases (NOSs). Although no NOS-like gene has been found in the Arabidopsis thaliana genome, biochemical studies have suggested that a NOS-like protein is likely to be activated in plants resisting pathogens. This protein has been recently identified as a variant P protein of glycine decarboxylase. This discovery means that studies of nitric oxide signalling functions in plants are now entering a new phase.

The function of nitric oxide (NO), a gaseous free radical emitted by many plants, is incompletely understood. In the present study the hypothesis that NO generation, like that of the reactive oxygen species, occurs as a general response to different environmental cues was tested. Leaf peels and mesophyll cell suspensions of Nicotiana tabacum cv. Xanthi were loaded with the NO-specific fluorophore, diaminofluorescein, and subjected to an abiotic stressor. Light stress and mechanical injury had no apparent effect on NO production. In contrast, high temperatures, hyperosmotic stress, salinity and epi-illumination in a microscope all led to rapid surges in NO-induced fluorescence. The fluorescence originated from cells of the palisade mesophyll and across all epidermal cell types, including guard cells, subsidiary cells, and long and short trichomes. Fluorescence was evident first in the plastids, then in the nucleus and finally throughout the cytosol. Nicotiana plumbaginifolia cell suspensions expressing the calcium reporter aequorin provided evidence that, under hyperosmotic stress, NO participates in the elevation of free Ca 2+ + + in the cytoplasm. The physiological significance of NO production in response to abiotic stressors is discussed.

Cryptogein is a 10-kD protein secreted by the oomycetePhytophthora cryptogea that induces a hypersensitive response on tobacco (Nicotiana tabacum var. Xanthi) plants and a systemic acquired resistance against various pathogens. The mode of action of this elicitor has been studied using tobacco cell suspensions. Our previous data indicated that within minutes, cryptogein signaling involves various events including changes in ion fluxes, protein phosphorylation, sugar metabolism, and, eventually, cell death. These results suggested that transport of sugars could be affected and, thus, involved in the complex relationships between plant and microorganisms via elicitors. This led us to investigate the effects of cryptogein on glucose (Glc) uptake and mitochondrial activity in tobacco cells. Cryptogein induces an immediate inhibition of Glc uptake, which is not attributable to plasma membrane (PM) depolarization. Conversely, cryptogein-induced valine uptake is because of PM depolarization. Inhibition of the PM Glc transporter(s) was shown to be mediated by a calcium-dependent phosphorylation process, and is independent of active oxygen species production. This inhibition was associated with a strong decrease in O2 uptake rate by cells and a large mitochondrial membrane depolarization. Thus, inhibition of Glc uptake accompanied by inhibition of phosphorylative oxidation may participate in hypersensitive cell death. These results are discussed in the context of competition between plants and microorganisms for apoplastic sugars.

Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca(2+)](cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration. The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration. Cryptogein signature is characterized by a long-sustained [Ca(2+)](cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death. The [Ca(2+)](cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx. H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in [Ca(2+)](cyt) increase and may activate calcium channels from the plasma membrane. Competition assays with different elicitins demonstrate that [Ca(2+)](cyt) increase is mediated by cryptogein-receptor interaction.

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inhibition. Secondly, since previous data suggested that beticolins could also be active against other Mg2(+)-dependent enzymes, we tested beticolin-1 on the vacuolar H(+)-PPase, which requires Mg2+ as co-substrate, and on the alkaline and acid phosphatases, which do not use Mg2+ as co-substrate. Only vacuolar H(+)-PPase is sensitive to beticolin-1, which suggests that beticolins are specific to enzymes that use a complex of Mg2+ as the substrate. The same Mg2H2B2 complex which is responsible of the plasma membrane H(+)-ATPase inhibition appears to be also involved in the inhibition of the vacuolar H(+)-PPase.

Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively. Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment. N. tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin. Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis. Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N. rustica. Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein. Phytoalexin production was lower in response to both elicitins in N. tabacum var Xanthi cells than in N. rustica cells, and capsicein was the most efficient elicitor of this response. In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium. These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses.

Treatment of suspension-cultured tobacco (Nicotiana tabacum var Xanthi) cells with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, induced a great stimulation of Ca2+ influx within the first minutes. Ca2+ influx is essential for the initiation of cryptogein-induced responses, since ethyleneglycol-bis([beta]-amino-ethyl ether)-N,N[prime]-tetraacetic acid or La3+, which block Ca2+ entrance, suppress cryptogein-induced responses such as extracellular alkalinization, active oxygen species, and phytoalexin production. Moreover, once initiated, these responses require sustained Ca2+ influx within the 1st h. A Ca2+ ionophore (A23187) was able to trigger an extracellular alkalinization but not the formation of active oxygen species and phytoalexins, even in the presence of cryptogein. Staurosporine, a protein kinase inhibitor that was recently reported to suppress cryptogein-induced responses (M.-P. Viard, F. Martin, A. Pugin, P. Ricci, J.-P. Blein [1994] Plant Physiol 104: 1245-1249), inhibited Ca2+ influx induced by cryptogein in a dose-dependent manner. These results suggest that protein phosphorylation followed by Ca2+ influx might be involved in the initial steps of cryptogein signal transduction.

In higher plants, the lipolytic enzymes and their physiological functions are not well characterized [1]. Most reports demonstrated that phospholipid catabolism in plants is achieved by the concerted actions of membrane-bound enzymes including phospholipase D, phosphatidate phosphatase, lipolytic acyl hydrolases and lipoxygenases [1,2]. With the exception of the phospholipase D, the literature on plant phospholipases is still very limited. We previously reported that tonoplast from Acer pseudoplatanus cells contains small amounts of phosphatidc acid and lysophospholipids, which were produced together with free fatty acids, particularly after addition of Ca2+[3]. These data suggested the possible involvement of phospholipase D and phospholipase A in the metabolism of vacuolar membrane lipids. The phospholipase activities were studied by following the hydrolysis of added sn-2-[14C]linoleyl-PC to tonoplast vesicles. Tonoplast was obtained by osmotic lysis of pure preparations of vacuoles isolated from protoplasts derived from Acer pseudoplatanus cells [4], This present work demonstrated clearly the presence of phospholipase D and phospholipase A1 activities associated with the tonoplast of Acer. The phospholipase A1 showed an optimal activity at pH about 6–6.5, did not necessarily require divalent cations, but was stimulated by Mg2+ and particularly by Ca2+. This work presents the first evidence for the presence of phospholipases A1 in plant cells.

The distribution of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) among the outer and inner monolayers of the vacuolar membrane of Acer pseudoplatanus was investigated using isolated vacuoles, chemical labelling agents (trinitrobenzene-sulfonate and fluorescamine), phospholipase A2 from bee venom, phospholipase C and phospholipase D. Treatments were performed with intact or sonicated vacuoles. Analysis of the transbilayer distribution of PC and PE in the vacuolar membrane of Acer was limited by phospholipid fractions which were inaccessible to the probes. Lipid-protein interactions and modification of the surface charge and surface pressure in the membrane layers during treatments may obviously exert a strong influence on labelling or hydrolysis of membrane phospholipids. However, simultaneous treatments carried out with phospholipase A2 and phospholipase C show that PE is approximately 20% more abundant in the outer monolayer than in the inner monolayer and PC is equally distributed between both leaflets of tonoplast. Compared to the phospholipids asymmetrical distribution observed in plasma membrane of erythrocyte, the vacuolar membrane of Acer is not characterized by a marked asymmetrical distribution of its major phospholipids.

We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), the production of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca(2+)](cyt) rise promoted by Ca(2+) influx through Ca(2+)-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca(2+)](cyt) similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H(2)O(2) production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H(2)O(2) production is dependent on Ca(2+) influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca(2+) influx but independent of both H(2)O(2) production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H(2)O(2) and NO levels.

Sealed tonoplast vesicles were prepared from vacuoles of Acer pseudoplatanus cells. The rate of contamination by plasmalemma never exceeded 3%. The vesicles of tonoplast exhibited ATP- and PPi-dependent proton translocation activity. The properties of ATP-dependent H+-transport were studied and compared to those of ATP hydrolysis previously described (A. Pugin et al., in: B. Marin (Ed.), Plant Vacuoles, NATO ASI Series, Serie A, Vol. 134, Plenum Press, 1987, pp. 135–141). H+-transport and enzymatic properties of the tonoplast ATPase were well correlated. The ATP-dependent H+-translocation into the vesicles had an optimum around pH 7.3; MgATP2− was preferentially used as a substrate with an apparent Km of 153 μM. ATP-dependent H+ transport was stimulated by anions. This effect was shown to be partly due to the ability of permeant anions to dissipate the electrical potential Δψ. When Δψ was collapsed, H+-ATPase activity was stimulated by Cl− and inhibited by NO3−. The use of three pH probes: acridine orange (AO), 9-amino-6-chloro-2-methoxyacridine (ACMA) and quinacrine confirmed the limits of acridine orange for the measurement of pH gradients in the presence of NO3−. Various chemicals were potent inhibitors of the tonoplast H+-ATPase, the sulfhydryl reagents N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), mersalyl, the lipid soluble carbodiimide N,N′-dicyclohexylcarbodiimide (DCCD), the synthetic estrogen diethylstilbestrol (DES) and vanadate which is often reported as a specific inhibitor of the plasmalemma ATPase. The inefficiency of vanadate on the tonoplastic PPi-dependent H+ transport indicated that the inhibition of the ATP-dependent H+-pump resulted from an effect on the enzyme. The sensitivity of the tonoplastic H+-ATPase to vanadate is discussed. We suggest that there are V-type ATPases which could operate via a phosphoenzyme intermediate.

When a plant cell is challenged by a well-defined stimulus, complex signal transduction pathways are activated to promote the modulation of specific sets of genes and eventually to develop adaptive responses. In this context, protein phosphorylation plays a fundamental role through the activation of multiple protein kinase families. Although the involvement of protein kinases at the plasma membrane and cytosolic levels are now well-documented, their nuclear counterparts are still poorly investigated. In the field of plant defence reactions, no known study has yet reported the activation of a nuclear protein kinase and/or its nuclear activity in plant cells, although some protein kinases, e.g. MAPK (mitogen-activated protein kinase), are known to be translocated into the nucleus. In the present study, we investigated the ability of cryptogein, a proteinaceous elicitor of tobacco defence reactions, to induce different nuclear protein kinase activities. We found that at least four nuclear protein kinases are activated in response to cryptogein treatment in a time-dependent manner, some of them exhibiting Ca2+-dependent activity. The present study focused on one 47 kDa protein kinase with a Ca2+-independent activity, closely related to the MAPK family. After purification and microsequencing, this protein kinase was formally identified as SIPK (salicyclic acid-induced protein kinase), a biotic and abiotic stress-activated MAPK of tobacco. We also showed that cytosolic activation of SIPK is not sufficient to promote a nuclear SIPK activity, the latter being correlated with cell death. In that way, the present study provides evidence of a functional nuclear MAPK activity involved in response to an elicitor treatment.

Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response.

International audience; In grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. • Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. • In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA)treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. • These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation.

Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.

International audience; Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species.Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized.Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine ( Vitis vinifera ) suspension cells, and the mutant–plant interaction resulted in the formation of hypersensitive response-like necrosis. Genetic segregation analyses revealed that the pathogenicity phenotype was linked to a single locus, but showed that the mutated gene was not tagged by the plasmid pAN7-1.Mutant A336 is the first oxalate-deficient mutant to be described in B. cinerea and it differs from all the nonpathogenic B. cinerea mutants described to date.

In animals, nitric oxide (NO) is an endogenously produced radical involved in cell communication and signal transduction. Its functions in plants are currently being discovered at an unprecedented pace, and insight into NO-derived mechanisms has mainly been gained from research on signal transduction. Numerous studies have firmly placed NO as one component of the signal perception–transduction network that connects plant responses to primary signals, including hormones, elicitors of defence responses or abiotic stresses. Protein kinases and the second messengers Ca 2+, cGMP, and cADPR convey part of the NO signal within cells. Furthermore, NO-based protein modifications are emerging as broad-based mechanisms for posttranslational regulation of protein function and might be implied in the regulation of numerous signaling pathways.

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca2+](nuc)) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the beta-1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca2+](nuc) elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca2+](nuc) elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca2+](nuc) rise depends on free cytosolic calcium, IP3, and active oxygen species (AOS) but is independent of nitric oxide.

Nitric oxide (NO) is both a gaseous free radical and a versatile cell-signalling effector that plays important roles in diverse (patho)physiological processes. In ani mals, NO production is catalyzed predominantly by nitric oxide synthases (NOS), which are heme-contain ing proteins related to the cytochrome P450 family. These enzymes catalyze the conversion of L-arginine to L-citrulline and NO using NADPH and molecular oxy gen as cosubstrates, and employ FAD, FMN, tetrahy drobiopterin (BH4), and calmodulin (CaM) as cofactors (Bogdan 2001). The biological effects of NO are medi ated by posttranslational modification of cysteine resi dues and transition metal centers, a key process referred as nitrosylation (Stamler et al. 2001). Because of its high biological reactivity, NO production by NOS is tightly regulated to control the specificity of its signalling as well as to limit its toxicity (Kone et al. 2003). In recent years, NO has also become an increasingly popular target of investigation in plants. NO has been implicated in disease resistance, stomatal closure, re sponses to abiotic stress, iron homeostasis, and in vari ous developmental processes (Neill et al. 2002a, b; Wendehenne et al. 2004). A major advance in our understanding of NO functions in plants has been the identification of enzymes that catalyze NO synthesis. Nitrate reductase (NR) was the first enzymatic source of NO to be identified (Yamasaki and Sakihama 2000). In addition to its role in nitrate reduction, NR catalyzes the reduction of nitrite to NO using NAD(P)H as co-factor. As recently discussed by Meyer et al. (2004), it remains

Despite cell wall reinforcement being a well-known defence mechanism of plants, it remains poorly characterized from a physical point of view. The objective of this work was to further describe this mechanism. Vitis vinifera cv Gamay cells were treated with UV-light (254 nm), a well-known elicitor of defence mechanisms in grapevines, and physical cell wall modifications were observed using the atomic force microscopy (AFM) under native conditions. The grapevine cell suspensions were continuously observed in their culture medium from 30 min to 24h after elicitation. In the beginning, cellulose fibrils covered by a matrix surrounded the control and treated cells. After 3 h, the elicited cells displayed sprouted expansions around the cell wall that correspond to pectin chains. These expansions were not observed on untreated grapevine cells. The AFM tip was used to determine the average surface elastic modulus of cell wall that account for cell wall mechanical properties. The elasticity is diminished in UV-treated cells. In a comparative study, grapevine cells showed the same decrease in cell wall elasticity when treated with a fungal biotic elicitor of defence response. These results demonstrate cell wall strengthening by UV stress.

In mammals, nitric oxide (NO) is a reactive free radical involved in diverse physiological functions. NO and its redox-related forms NO + and NO − react with di(oxygen) and its derivatives, with metalloproteins and thiol-containing proteins. NO-mediated nitrosation of proteins represents an important cellular regulatory mechanism. Biosynthesis of NOis catalysed by nitric oxide synthase (NOS). Three isoenzymes representing distinct gene products have been identified: the inducible NOS isoform, the constitutive neuronal and endothelial isoforms. Inducible and constitutive NOSs have the same structural features, but their activities differ in their dependence to calcium and the rate of NO produced. The principal NO-mediated functions in mammals are endothelium-dependent relaxation, neurotransmission and immune response. The role of NO in the antitumor immune response comprises both regulatory and effector functions at the intra- or inter-cellular level. The first function includes inhibition of lymphocyte proliferation or participation in different transduction pathways. The second function includes pro- or anti-tumoral effects and NO-mediated cell toxicity or cell resistance to apoptosis. In plants, NO is produced via non-enzymatic reactions, by the activity of a constitutive nitrate reductase, and by a mammalian-type NOS which corresponding gene has not yet been cloned. NO was found to regulate plantgrowth, to act as antioxidant, and to function as a signaling molecule in plants challenged by pathogens. Interestingly, key proteins involved in animal NO signaling appear to be modulated by NO in plants. This finding complements the list of evidence suggesting that portions of transduction mechanisms are shared between plants and animals.

Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.

The tonoplast H(+)-PPase was previously characterized in Acer pseudoplatanus cells (Pugin et al (1991) Plant Sci 73, 23-34; Fraichard et al (1993) Plant Physiol Biochem 31, 349-359). Tonoplast vesicles were obtained from vacuoles isolated from protoplasts of A pseudoplatanus suspension cultures and used to study kinetic effects of Mg2+ and Ca2+ on PPi hydrolysis. The concentrations of ionic species (free Mg2+, free PPi, and MgPPi complexes) were calculated with apparent dissociation constants of 55.3 microM for MgPPi and 59.6 microM for CaPPi. Our results indicated that the substrate of the tonoplast PPase was a MgPPi complex and that free Mg2+ was essential for PPi hydrolysis. With fixed free Mg2+ concentrations, PPase activity showed Michaelis-Menten kinetics with respect to MgPPi. Moreover, free Mg2+ acted as an allosteric activator with a Hill coefficient of 2.4, indicating at least two Mg2+ binding sites on the enzyme. The Mg-imidodiphosphate complex was a competitive inhibitor of the substrate MgPPi but did not change significantly the allosteric activation by free Mg2+. This result confirmed the presence of Mg2+ regulatory sites. Ca2+ acted as an uncompetitive inhibitor of MgPPi hydrolysis. Furthermore, the sensitivity of the H(+)-PPase to Ca2+ increased with decrease in free Mg2+ concentration. Therefore, Ca2+ and Mg2+ may compete for a common binding site. Taken together, our results confirm that activation by free Mg2+ and inhibition by Ca2+ could be involved in the regulation of the PPase activity in vivo.

The interactions of two beticolins, Cercospora beticola toxins, and of their magnesium complexes with liposomes or plasma membrane were studied. The fluorometric pH titration curves of beticolins in liposomes and in plasma membranes reveal the presence of the dissociated form of beticolins. The concentration of the magnesium complex in these membranes increases at high pH. The partition coefficient of beticolin-1 on liposomes is 3-fold higher than that of beticolin-2 and the fluorescence of both compounds on liposomes is similar. The addition of magnesium to liposomes causes a 40-fold and 20-fold increase in the partition coefficient of beticolin-1 and -2, respectively, as a result of the interactions between membrane, magnesium and beticolins. Beticolins react to a ΔpH across the liposome membrane but the formation of the magnesium complex completely abolishes this effect.

When tobacco cells were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, changes in plasmalemma fluxes were noticed. A strong alkalinization of the culture medium, accompagnied with a leakage of potassium, were induced few minutes after treatment, and reached a plateau within 30 min. Then, they remained at the same level for several hours. This treatment also induced a rapid, but transient production of activated oxygen species (20–80 min). All these physiological responses were fully sensitive to staurosporine, a known protein kinase inhibitor. A simultaneous treatment with cryptogein and staurosporin stimulated the accumulation of capsidiol, a sesquiterpenoid phytoalexin. These different results are in agreement with those reported by Felix et al and Conrath et al and led us to study the time course of protein phosphorylation during the first min of the elicitation process. It appeared that cryptogein induced a staurosporine-sensitive phosphorylation of microsomal proteins. Therefore, these data suggest that phosphorylated proteins were essential for transduction of elicitor signals.

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.

Unlike most tonoplast ATPases, the vacuolar ATPase of Acer pseudoplatanus cells ( K m = 0.4 mM) was strongly inhibited by vanadate ( I 50 = 10 μ M). The inhibition was non-competitive. Chemicals usually added in the reaction mixture either increase (NH 4 + , K + ) or decrease (Na + , EDTA) the ATPase inhibition. However, these results do not explain the insensitivity ot vanadate of most tonoplast ATPases. We suggest that the tonoplast contains 2 classes of ATPase, one sensitive to vanadate, the other insensitive; each class should be more or less abundant (or active) according to the plant species studied or its physiological state of growth. It appears from this study that sensitivity or insensitivity of an ATPase to vanadate is not really a good criterion to distinguish between plasmalemma and tonoplast.

Two different populations of vacuoles were released from protoplasts of isolated Acer pseudoplatanus cells by ultracentrifugation on density gradient. It was shown by fluorescence microscopy and assays with marker enzymes that there were only traces of contamination by other organelles and by cytoplasmic components in the vacuole preparations. The vacuoles contained an ATPase with an optimum activity at pH 7.5. This vacuolar ATPase activity was much higher than the one of vacuoles isolated by the lysis of protoplasts with polybases. In fact, the ATPase activity of the vacuoles isolated by ultracentrifugation was inhibited by diethylaminoethyl dextran (DEAE-dextran) and dextran sulfate.

Le Phialophora cinerescens alcalinise nettement le milieu liquide sur lequel il est cultive durant 7 semaines; Le saccharose fourni est rapidement hydrolyse en glucose et fructose. La production des deux glycopeptides neutre et anodique — qui affectent la croissance des jeunes plantes d'Oeillet — a ete appreciee. Le glycopeptite neutre est synthetise des le debut de la culture, le glycopeptide anodique plus tardivement; les variations de la croissance du P. cinerescens cultive in vitro suggerent que les deux glycopeptides ne sont pas des produits de l'autolyse du mycelium.

Summary A new and efficient method for plant vacuole isolation using digitonin for the lysis of protoplasts is described. The plasma membrane of Acer pseudoplatanus L. is a cholesterol-rich membrane which is highly susceptible to digitonin. An effective separation of vacuoles from other cell constituents was obtained by flotation through a discontinuous Ficoll gradient, provided that the disruption of plasmalemma was performed in a dense medium (0.7 M mannitol supplemented with 10.8% (w/v) Ficoll 400). The final yield of vacuoles was about 20%. Contamination of the fraction by cytoplasm, mitochondria, and peroxisomes did not exceed 2%. The isolation procedure is rapid and would allow the preparation of vacuolar material on a large scale.

Several reports indicate that an anion-sensitive H+-ATPase insensitive to vanadate occurs in the tonoplast of higher plant cells (Lin et al., 1977; Leigh and Walker, 1980; Admon et al., 1981; Marin et al., 1981; Walker and Leigh, 1981; Aoki and Nishida, 1984; Smith et al., 1984; Mandala and Taiz, 1985). Our unability to measure an ATPase activity in vacuoles of Acer pseudoplatanus cells, prepared with DEAE-dextran and dextran sulfate as described by Alibert et al. (1982) lead us to incriminate the method used for their isolation. This hypothesis was supported by the unstability of the vacuoles out of the Ficoll used to form the density gradient. Therefore a new method for the isolation of vacuoles from Acer pseudoplatanus cells has been established. These vacuoles showed an ATPase activity, the properties of which have been studied and compared to those of other well known tonoplastic ATPases.

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Beticolin-1 is a toxin produced by the fungus Cercospora beticola. The chemical structure of this toxin was previously elucidated. The effects of beticolin-1 on purified corn root plasma membrane H+-ATPase were studied in a solubilized form or were reconstituted into liposome membranes. The ATP hydrolysis activity of the purified solubilized enzyme was inhibited by micromolar concentrations of beticolin-1, and this inhibition was noncompetitive with respect to ATP. When this purified enzyme was inserted into liposome membranes, a competitive inhibition of the H+-ATPase hydrolysis activity by beticolin-1 was observed. The effect of beticolin-1 on the formation of H+-ATPase-phosphorylated intermediate was also studied. With the purified enzyme in its solubilized form, the level of phosphorylated intermediate was not affected by the presence of beticolin-1, whereas micromolar concentrations of the toxin led to a marked inhibition of its formation when the enzyme was reconstituted into liposomes. These data suggest that (a) the plasma membrane H+-ATPase is a direct target for beticolin-1, and (b) the kinetics of inhibition and the effect on the phosphorylated intermediate are linked and both depend on the lipid environment of the enzyme.