CuZn-superoxide dismutase is differentially modified in localization and expression by three abiotic stresses in miniature rose bushes.

Three abiotic stresses, copper application (CS), mechanical rubbing (MS) and water deprivation (WS) applied on miniature rose bushes specifically activate the expression of the CuZn-Superoxide dismutase (SOD). The Cu/Zn-SOD protein immunodetected in the 4th internode was shown engaged in lignification in phloem, cambium and xylem cells. The SOD occurrence was detailed in the vessel associated cells (VACs), using immunogold labeling observed in transmission electron microscopy. The enzyme was detected in mitochondria, plastids, Golgi vesicles, endoplasmic reticulum and plasma membrane. In addition, in pit-fields without plasmodesmata linking vessel associated cells to vessels, the abiotic stresses increased the transfer apparatus volume. The content in unmethylatedpectins increased in wall ingrowths after CS and MS, but not in WS. In addition to the different localization, the SOD was differentially overexpressed according to the applied stress: an isoform detected at 17 kDa under CuSO4 application, two isoforms respectively detected at 20 and 17 kDa under MS and detected at 17 and 15 kDa under WS. Notably, the only 17 kDa isoform was detected in plasma membrane vesicles from plants submitted to the three stresses. Thus, by increasing the transfer apparatus development, the key role of VACs was emphasized in establishing an adaptative response to abiotic stresses, in miniature rose bushes. Additionally, it has been observed that the differential SOD localization under such stresses sustained the regulatory function of VACs in the transitory sink function of xylem.

Fungal wood-degrading enzymes in esca-diseased grapevine and effects of carbohydrate environment on fungal development.

In esca disease affecting grapevines, Phaeomoniella chlamydospora and Phaeoacremonium minimum colonize the woody parts of the trunks and arms, where they obtain nutrition from xylem sap and, potentially, from residues resulting from the enzymatic breakdown of lignified cell walls, particularly osidic residues. We quantified the secretion of lignin peroxidase, manganese peroxidase and laccase by these fungi in woody tissues of selectively infected cuttings using immunolabeling and transmission electron microscopy. Our results indicated that the detection of these enzymes was generally higher in tissues infected with Phaeoacremonium minimum. These data were confirmed through immunodetection of enzymes secreted by hyphae of fungi grown in vitro. Additionally, we observed that the supply of various carbohydrates (mono, di, tri and tetrasaccharides and polymers) differentially influenced fungal growth and polypeptide secretion. Since some secreted polypeptides display detrimental effects on grapevine cells, these results raise the question of whether the carbohydrate environment could be a factor affecting the aggressiveness of these pathogens.

Implication of actin in the uptake of sucrose and valine in the tap root and leaf of sugar beet.

Actin microfilaments (F-actin) are major components of the cytoskeleton essential for many cellular dynamic processes (vesicle trafficking, cytoplasmic streaming, organelle movements). The aim of this study was to examine whether cortical actin microfilaments might be implicated in the regulation of nutrient uptake in root and leaf cells of Beta vulgaris. Using antibodies raised against actin and the AtSUC1 sucrose transporter, immunochemical assays demonstrated that the expression of actin and a sucrose transporter showed different characteristics, when detected on plasma membrane vesicles (PMVs) purified from roots and from leaves. The in situ immunolabeling of actin and AtSUC1 sites in PMVs and tissues showed their close proximity to the plasma membrane. Using co-labeling in protoplasts, actin and sucrose transporters were localized along the internal border and in the outermost part of the plasma membrane, respectively. This respective membrane co-localization was confirmed on PMVs and in tissues using transmission electronic microscopy. The possible functional role of actin in sucrose uptake (and valine uptake, comparatively) by PMVs and tissues from roots and leaves was examined using the pharmacological inhibitors, cytochalasin B (CB), cytochalasin D (CD), and phalloidin (PH). CB and CD inhibited the sucrose and valine uptake by root tissues in a concentration-dependent manner above 1 µM, whereas PH had no such effect. Comparatively, the toxins inhibited the sucrose and valine uptake in leaf discs to a lesser extent. The inhibition was not due to a hindering of the proton pumping and H+ -ATPase catalytic activity determined in PMVs incubated in presence of these toxins.

Auxinic herbicide conjugates with an α-amino acid function: Structural requirements for biological activity on motor cells.

Two Fabaceae exhibiting rapid osmocontractile pulvinar movements were used in this study because this activity is modified by natural auxin and dramatically by 2,4D. A short chain with a carboxylic group being required for auxinic properties, a critical point to analyze is whether the recently synthesized proherbicide ε-(2,4-dichlorophenoxyacetyl)-L-Lys (2-4D-L-Lys) maintains some biological activity despite the increase in length of the chain and the substitution of the carboxyl group by an α-amino acid function. No trace of 2,4D could be detected in the pulvinar tissues treated for 1 h with 2,4D-L-Lys. Complementary approaches (electrophysiology, pH measurements, use of plasma membrane vesicles) suggest that it was less efficient than 2,4D to activate the plasma membrane H+-ATPase (PM-H+-ATPase). However, it modified the various ion-driven reactions of Mimosa pudica and Cassia fasciculata pulvini in a similar way as 2,4D. Additionally, it was much more effective than fusicoccin to inhibit seismonastic movements of M. pudica leaves and, at low concentrations, to promote leaflet opening in dark, indicating that its mode of action is more complex than the only activation of the PM-H+-ATPase. Various substitutions on 2,4D-L-Lys affected its activity in correlation with the molecular descriptor "halogen ratio" of these derivatives. Conjugation with D-Lys also led to a decrease of pulvinar reaction, suggesting that 2,4D-Lys maintains the main signaling properties of 2,4D involved in pulvinar movements providing that the terminal zwitterion is in a suitable orientation. Our data guide future investigations on the effect of 2,4D and 2,4D-L-Lys on the vacuolar pump activity of motor cells.

Characteristic early membrane effects induced by tryptophan in pulvinar motor cells.

Tryptophan at concentrations higher than 0.1 mM, triggered characteristic early physiological effects such as rapid (within 5 min) dose-dependent membrane hyperpolarization in Mimosa pudica motor cells and modification of the time course of the spontaneous proton efflux monitored in the incubation medium of pulvinar tissues. The rapid modifications of the leaf turgor-mediated movements seen on the primary pulvini of M. pudica following a shock and on Cassia fasciculata leaflets during a transition from light to darkness indicate that tryptophan disturbed the ionic migrations involved in the electrophysiological events and in the osmocontractile reaction of the motor cells. These reactions were specific to tryptophan compared to those induced by serine and 5-hydroxytryptophan. The tryptophan mode of action cannot be linked to a direct modification of the plasma membrane H+-ATPase activity as monitored on purified pulvinar plasma membrane vesicles. The tryptophan metabolism-linked products tryptamine and indole also inhibited the motile reactions, activated in a continuous manner the H+ secretion of pulvinar tissues and showed properties of a protonophore and an ATPase activity inhibitor on plasma membrane vesicles, respectively. The specific behavior of tryptophan in the reaction studies here is discussed in light of the previously reported action of phytohormones.

Cysteine: A multifaceted amino acid involved in signaling, plant resistance and antifungal development.

Early effects induced by cysteine were monitored using the model of Mimosa pudica pulvinar cells. Rapid dose-dependent membrane depolarization (within seconds) and modification of proton secretion (within minutes) were triggered at cysteine concentrations higher than 0.1 mM. These effects did not result from a modification of the plasma membrane H+-ATPase activity nor from a protonophore effect as shown by assays on plasma membrane vesicles isolated from pulvinar tissues. In a 0.5-10 mM range, cysteine inhibited the ion-driven turgor-mediated seismonastic reaction of Mimosa pudica primary pulvini and the dark-induced movement of Cassia fasciculata leaflets. At concentrations higher than 1 mM, it induced a long-lasting leaflet necrosis dependent on the concentration and treatment duration. Electron microscopy showed that cysteine induced important damage in the nucleus, mitochondria, endoplasmic reticulum and Golgi of the M. pudica motor cell. Cysteine inhibited in a concentration-dependent manner, from 0.5 to 20 mM, both the mycelial growth and the spore germination of the fungal pathogens Phaeomoniella chlamydospora and Phaeoacremonium minimum implicated in esca disease of grapevines. Using [35S] cysteine, we showed that the amino acid was absorbed following leaf spraying, translocated from leaves to other parts of grapevine cuttings and accumulated within trunks and roots. Therefore, cysteine showed relevant properties to be a candidate able to control fungal diseases either by acting as an early signal directing plant host reaction or/and by acting directly on fungal development.

Modifications of the chemical structure of phenolics differentially affect physiological activities in pulvinar cells of Mimosa pudica L. II. Influence of various molecular properties in relation to membrane transport.

Early prediction of compound absorption by cells is of considerable importance in the building of an integrated scheme describing the impact of a compound on intracellular biological processes. In this scope, we study the structure-activity relationships of several benzoic acid-related phenolics which are involved in many plant biological phenomena (growth, flowering, allelopathy, defense processes). Using the partial least squares (PLS) regression method, the impact of molecular descriptors that have been shown to play an important role concerning the uptake of pharmacologically active compounds by animal cells was analyzed in terms of the modification of membrane potential, variations in proton flux, and inhibition of the osmocontractile reaction of pulvinar cells of Mimosa pudica leaves. The hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), polar surface area (PSA), halogen ratio (Hal ratio), number of rotatable bonds (FRB), molar volume (MV), molecular weight (MW), and molar refractivity (MR) were considered in addition to two physicochemical properties (logD and the amount of non-dissociated form in relation to pKa). HBD + HBA and PSA predominantly impacted the three biological processes compared to the other descriptors. The coefficient of determination in the quantitative structure-activity relationship (QSAR) models indicated that a major part of the observed seismonasty inhibition and proton flux modification can be explained by the impact of these descriptors, whereas this was not the case for membrane potential variations. These results indicate that the transmembrane transport of the compounds is a predominant component. An increasing number of implicated descriptors as the biological processes become more complex may reflect their impacts on an increasing number of sites in the cell. The determination of the most efficient effectors may lead to a practical use to improve drugs in the control of microbial attacks on plants.

Unsaturated amino acids derived from isoleucine trigger early membrane effects on plant cells.

Unsaturated amino acids (UnsAA) have been shown to affect the activity of various biological processes. However, their mode of action has been investigated poorly thus far. We show in this work that 2-amino-3-methyl-4-pentenoic acid (C2) and 2-amino-3-methyl-4-pentynoic acid (C3) structurally derived from isoleucine (Ile) exhibited a multisite action on plant cells. For one, C2 and C3 induced early modifications at the plasma membrane level, as shown by the hyperpolarization monitored by microelectrode implantation in the pulvinar cells of Mimosa pudica, indicating that these compounds are able to modify ionic fluxes. In particular, proton (H(+)) fluxes were modified, as shown by the pH rise monitored in the bathing medium of pulvinar tissues. A component of this effect may be linked to the inhibitory effect observed on the proton pumping and the vanadate-sensitive activity of the plasma membrane H(+)-ATPase monitored in plasma membrane vesicles (PMVs) purified from pulvinar tissues of M. pudica and leaf tissues of Beta vulgaris. This effect may explain, in part, the inhibitory effect of the compounds on the uptake capacity of sucrose and valine by B. vulgaris leaf tissues. In contrast, an unexpected action was observed in cell reactions, implicating ion fluxes and water movement. Indeed, the osmocontractile reactions of pulvini induced either by a mechanical shock in M. pudica or by dark and light signals in Cassia fasciculata were increased, indicating that, compared to Ile, these compounds may modify in a specific way the plasma membrane permeability to water and ions.

Co-occurrence of tannin and tannin-less vacuoles in sensitive plants.

Vacuoles of different types frequently coexist in the same plant cell, but the duality of the tannin/tannin-less vacuoles observed in Mimosa pudica L. is rare. In this plant, which is characterized by highly motile leaves, the development and original features of the double vacuolar compartment were detailed in primary pulvini from the young to the mature leaf stage. In young pulvini, the differentiation of tannin vacuoles first occurred in the epidermis and progressively spread toward the inner cortex. In motor cells of nonmotile pulvini, tannin deposits first lined the membranes of small vacuole profiles and then formed opaque clusters that joined together to form a large tannin vacuole (TV), the proportion of which in the cell was approximately 45%. At this stage, transparent vacuole profiles were rare and small, but as the parenchyma cells enlarged, these profiles coalesced to form a transparent vacuole with a convexity toward the larger-sized tannin vacuole. When leaf motility began to occur, the two vacuole types reached the same relative proportion (approximately 30%). Finally, in mature cells displaying maximum motility, the large transparent colloidal vacuole (CV) showed a relative proportion increasing to approximately 50%. At this stage, the proportion of the tannin vacuole, occurring in the vicinity of the nucleus, decreased to approximately 10%. The presence of the condensed type of tannins (proanthocyanidins) was proven by detecting their fluorescence under UV light and by specific chemical staining. This dual vacuolar profile was also observed in nonmotile parts of M. pudica (e.g., the petiole and the stem). Additional observations of leaflet pulvini showing more or less rapid movements showed that this double vacuolar structure was present in certain plants (Mimosa spegazzinii and Desmodium gyrans), but absent in others (Albizzia julibrissin, Biophytum sensitivum, and Cassia fasciculata). Taken together, these observations strongly suggest that a direct correlation cannot be found between the presence of a tannin vacuole and the osmoregulated motility of pulvini.

Modifications of the chemical structure of phenolics differentially affect physiological activities in pulvinar cells of Mimosa pudica L. I. Multimode effect on early membrane events.

A study of the structure-activity relationship carried out on several benzoic acid-related phenolics indicates that this type of compounds hinders the osmocontractile reaction of pulvinar cells in the range of 0-100%. Tentatively, we tried to find a way that could explain this differential action. With this aim, the relationship between the inhibitory effect and important molecular physico-chemical parameters (namely lipophilicity and degree of dissociation) was drawn. In addition, the effect of a variety of these compounds was investigated on their capacity to modify the electrical transmembrane potential and induce modifications in proton fluxes. Finally, using plasma membrane vesicles purified from pulvinar tissues, we examined the effects of some selected compounds on the proton pump activity and catalytic activity of the plasma membrane H(+)-ATPase. Taken together, the results indicate that a modification of the molecular structure of phenolics may induce important variation in the activity of the compound on these early membrane events. Among the tested phenolics, salicylic acid (SA) and acetylsalicylic acid (ASA, aspirin) are of particuler note, as they showed atypical effects on the physiological processes studied.

Uptake and metabolic effects of salicylic acid on the pulvinar motor cells of Mimosa pudica L.

In this paper, the salicylic acid (o-hydroxy benzoic acid) (SA) uptake by the pulvinar tissues of Mimosa pudica L. pulvini was shown to be strongly pH-dependent, increasing with acidity of the assay medium. This uptake was performed according to a unique affinity system (K(m) = 5.9 mM, V(m) = 526 pmol mgDW(-1)) in the concentration range of 0.1-5 mM. The uptake rate increased with increasing temperature (5-35 °C) and was inhibited following treatment with sodium azide (NaN3) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting the involvement of an active component. Treatment with p-chloromercuribenzenesulfonic acid (PCMBS) did not modify the uptake, indicating that external thiol groups were not necessary. KCl, which induced membrane depolarization had no significant effect, and fusicoccin (FC), which hyperpolarized cell membrane, stimulated the uptake, suggesting that the pH component of the proton motive force was likely a driving force. These data suggest that the SA uptake by the pulvinar tissues may be driven by two components: an ion-trap mechanism playing a pivotal role and a putative carrier-mediated mechanism. Unlike other benzoic acid derivatives acting as classical respiration inhibitors (NaN3 and KCN), SA modified the pulvinar cell metabolism by increasing the respiration rate similar to CCCP and 2,4-dinitrophenol (DNP). Furthermore, SA inhibited the osmoregulated seismonastic reaction in a pH dependent manner and induced characteristic damage to the ultrastructural features of the pulvinar motor cells, particularly at the mitochondrial level.

Occurrence of interactions between individual Sr²âº- and Ca²âº-effects on maize root and shoot growth and Sr²âº, Ca²âº and Mg²âº contents, and membrane potential: consequences on predicting Sr²âº-impact.

Occurrence of functional interactions between Sr(2+) and Ca(2+) were investigated on maize plants grown under hydroponic conditions in presence of various mixtures of SrCl2 [0-0.01-1-10 mM] and CaCl2 [0-0.2-2-20 mM]. External [Ca(2+)] modulated the effect of Sr(2+) on the plant dry weight, and on the Sr(2+), Ca(2+) and Mg(2+) contents of roots and shoots. An intermediary functional step between external [Sr(2+)] and [Ca(2+)], and organ ion content, occurred at the plasma membrane of cortical root cells where Sr(2+) and Ca(2+) could influence ion uptake by acting on membrane potential. The decrease of the Sr(2+)-evoked membrane depolarization induced by Ca(2+) could not solely be attributed to the Ca(2+)-effect on the resting membrane potential. Most of the time the individual effects of Sr(2+) and Ca(2+) were not additive, as these two ions clearly interacted with each other to jointly affect the plant physiology. In spite of these interactions, both [Sr(2+)](ext) or [Sr(2+)](ext)/[Ca(2+)](ext) ratio values seemed to enable a correct prediction of the Sr(2+)-effects on the plant. However using the [Sr(2+)](ext)/[Ca(2+)](ext) ratio improved significantly the adequacy of prediction compared to the use of [Sr(2+)](ext) alone, as it increased up to 25% the proportion of variability accounted for by the model.

Early membrane events induced by salicylic acid in motor cells of the Mimosa pudica pulvinus.

Salicylic acid (o-hydroxy benzoic acid) (SA) induced a rapid dose-dependent membrane hyperpolarization (within seconds) and a modification of the proton secretion (within minutes) of Mimosa pudica pulvinar cells at concentrations higher than 0.1mM. Observations on plasma membrane vesicles isolated from pulvinar tissues showed that SA acted directly at the membrane level through a protonophore action as suggested by the inhibition of the proton gradient and the lack of effect on H(+)-ATPase catalytic activity. Comparative data obtained with protonophores (carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol) and inhibitors of ATPases (vanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol) corroborated this conclusion. Consequently, the collapse of the proton motive force led to an impairment in membrane functioning. This impairment is illustrated by the inhibition of the ion-driven turgor-mediated seismonastic reaction of the pulvinus following SA treatment. SA acted in a specific manner as its biosynthetic precursor benzoic acid induced much milder effects and the m- and p-OH benzoic acid derivatives did not trigger similar characteristic effects. Therefore, SA may be considered both a membrane signal molecule and a metabolic effector following its uptake in the cells.

Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²âº](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 µM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²âº](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl3, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²âº](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus.

Ergosterol triggers characteristic elicitation steps in Beta vulgaris leaf tissues.

This study investigates the role of the fungal sterol ergosterol as a general elicitor in the triggering of plant innate immunity in sugar beet. Evidence for this specific function of ergosterol is provided by careful comparison with cholesterol and three plant sterols (stigmasterol, campesterol, sitosterol), which do not enable the integrity of responses leading to elicitation. Our results demonstrate the modification of H(+) flux by ergosterol, due to the direct inhibition of the H(+)-ATPase activity on plasma membrane vesicles purified from leaves. The ergosterol-induced oxidative burst is related to enhanced NADPH-oxidase and superoxide dismutase activities. The similar effects obtained with the fungal elicitor chitosan further reinforce the particular role of ergosterol in the induced defences. The involvement of salicylic acid and/or jasmonic acid signalling in the ergosterol-enhanced plant non-host resistance is also studied. The possible link between ergosterol-triggered plant innate immunity and its putative impact on the structural organization of plant plasma membrane are discussed in terms of the ability of this fungal sterol to promote the formation of lipid rafts.

Early events induced by chitosan on plant cells.

Chitosan (a polymer of beta-1,4-glucosamine residues) is a deacetylated derivative of chitin which presents antifungal properties and acts as a potent elicitor of plant resistance against fungal pathogens. Attention was focused in this study on the chitosan-induced early events in the elicitation chain. Thus, it was shown that chitosan triggered in a dose-dependent manner rapid membrane transient depolarization of Mimosa pudica motor cells and, correlatively, a transient rise of pH in the incubation medium of pulvinar tissues. By using plasma membrane vesicles (PMVs), it was specified that a primary site of action of the compound is the plasma membrane H(+)-ATPase as shown by its inhibitory effect on the proton pumping and the catalytic activity of the enzyme up to 250 microg ml(-1). As a consequence, chitosan treatment modified H(+)-mediated processes, in particular it inhibited the uptake of the H(+)-substrate co-transported sucrose and valine, and inhibited the light-induced H(+)/K(+)-mediated turgor reaction of motor cells. The present data also allowed the limit of the cytotoxicity of the compound to be established close to a concentration of 100 microg ml(-1) at the plasma membrane level. As a consequence, chitosan could be preferably used in plant disease control as a powerful elicitor rather than a direct antifungal agent.

Membrane effects of 2,4-dichlorophenoxyacetic acid in motor cells of Mimosa pudica L.

2,4-dichlorophenoxyacetic acid applied to excised leaves of Mimosa pudica L. inhibited in a dose-dependent manner the shock-induced pulvinar movement. This inhibition was negatively correlated with the amount of [(14)C] 2,4-dichlorophenoxyacetic acid present in the vicinity of the motor cells. Although 2,4-dichlorophenoxyacetic acid is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. This observation opens the question of its mode of action which may be through external signaling or following internal transport by a specific anionic form transporter. The effect was related to molecular structure since 2,4-dichlorophenoxyacetic acid>3,4-dichlorophenoxyacetic acid>2,3-dichlorophenoxyacetic acid. An essential target of 2,4-dichlorophenoxyacetic acid action lies at the plasmalemma as indicated by the induced hyperpolarization of the cell membrane. Compared to indole-3-acetic acid and fusicoccin, it induced a complex effect on H(+) fluxes. Applied to plasma membrane vesicles purified from motor organs, 2,4-dichlorophenoxyacetic acid enhanced proton pumping, but, unlike fusicoccin, it did not increase the H(+)-ATPase catalytic activity in our experimental conditions. Taken together, the data suggest that 2,4-dichlorophenoxyacetic acid acts on cell turgor variation and the concomittant ion migration, in particular K(+), by a mechanism involving specific steps compared to indole-3-acetic acid and fusicoccin.

Early changes in membrane permeability, production of oxidative burst and modification of PAL activity induced by ergosterol in cotyledons of Mimosa pudica.

Ergosterol (a fungal membrane component) was shown to induce transient influx of protons and membrane hyperpolarization in cotyledonary cells of Mimosa pudica L. By contrast, chitosan (a fungal wall component with known elicitor properties) triggered membrane depolarization. In the processes induced by ergosterol, a specific desensitization was observed, since cells did not react to a second ergosterol application but did respond to a chitosan treatment. This comparative study correspondingly shows that ergosterol and chitosan were perceived in a distinct manner by plant cells. Generation of O2*-, visualized by infiltration with nitroblue tetrazolium, was displayed in organs treated with ergosterol and chitosan. This AOS production was preceded by an increase in activity of NADPH oxidase measured in protein extracts of treated cotyledons. In all the previously described processes, cholesterol had no effect, thereby indicating that ergosterol specifically induced these physiological changes known to participate in the reaction chain activated by characteristic elicitors. Contrary to chitosan, ergosterol did not greatly activate secondary metabolism as shown by the small change in content of free phenolics and by the low modification in activity of PAL, the key enzyme of this metabolic pathway. Therefore, future studies have to clarify the signalling cascade triggered by ergosterol recognition.

Polypeptide metabolites secreted by the fungal pathogen Eutypa lata participate in Vitis vinifera cell structure damage observed in Eutypa dieback.

Eutypa dieback is a devastating disease of Vitis vinifera L. caused by the fungal pathogen Eutypa lata. This wood-inhabiting fungus degrades tissues in the trunk and cordons of infected vines and induces symptoms in the foliage. These symptoms have been attributed to the production of toxic metabolites by the pathogen, in particular eutypine. Recently, we have isolated polypeptide compounds secreted by the fungus in artificial culture. The aims of this study were to examine the effects induced in leaves by applying polypeptides and eutypine to detached canes and to compare this to the changes in leaf structure induced by E. lata in the vineyard. In leaves taken from vines infected with E. lata, the changes in mesophyll cells indicate that the fungus has an effect on tissue remote from the infected area. The size of mesophyll cells decreased by more than half, starch content was reduced and tannins were abundant. Plastids, mitochondria and cell walls were highly modified. In leaves taken from healthy canes treated with polypeptides of E. lata, the structure of mesophyll cells was also modified. The cell size did not change, but the tannin content increased and modifications in plastids and mitochondria were similar to those observed in leaves taken from infected vines. The major effect was the complete disorganisation of cell walls. Eutypine had less effect on organelle structure and did not modify the cell wall. In canes treated with polypeptides, vessel-associated cells (VACs) were also damaged. Abundant tannins occurred in the vacuoles of VACs and marked changes were noted in mitochondria, plastids and the protective layer, in particular in the pit at the vessel interface. In these pits, the protective layer, the primary wall and the middle lamella were all highly modified. In contrast, treatment with eutypine induced the development of a large transfer apparatus bordering the unmodified pectocellulose wall. These results illustrate that treatment with polypeptides produced by E. lata may cause changes in mesophyll cells in leaves and VACs in canes, that resemble changes observed in naturally infected vines. Comparatively, the differences with eutypine action were stressed. Both types of toxins may co-operate in vivo to produce the degeneration observed during the disease.

Antifungal effects of cysteine towards Eutypa lata, a pathogen of vineyards.

Cysteine inhibited mycelial growth of the pathogenic fungus affecting grapevines Eutypa lata Pers. Fr. Tul. and C. Tul. in a concentration-dependent manner. The threshold value (defined by the concentration inducing a growth inhibition higher than 5%) was 0.5 mM. A 10 mM concentration induced a complete inhibition of growth and triggered necrotic processes as evidenced by an increasing number of nuclei stained by propidium iodide. In conditions mimicking the plant environment (in particular, a pH near the apoplastic value, i.e. 5.5), 6 mM cysteine induced dramatic modifications in the structural organization of the mycelium (wall, mitochondria, vacuoles and nucleus) leading to death of the hyphae. The antifungal effect of the molecule increased at the acidic experimental pH (pH 4.1). The effect was highly specific to cysteine since modifying the molecular arrangement or masking the SH-function hindered the antifungal efficiency. Cysteine spectrum of action was broad among the various strains of E. lata tested. However, a lower efficiency was observed against fungal species intervening in other grapevine diseases (esca, black dead arm). Besides its direct antifungal effect, the role of cysteine presents particular interest in the fight against fungal pathogens since it triggered an excretion of ergosterol, a compound with elicitor properties. Therefore, cysteine may indirectly increase plant defense reactions.