Gabriel Roblin

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.

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.

Structural modifications induced by Eutypa lata in the xylem of trunk and canes of Vitis vinifera

Eutypa dieback, a devastating disease in grapevines, is caused by the fungal pathogen Eutypa lata, a wood-inhabiting fungus. E. lata acts by degrading wood tissues in the colonisation areas, and produces foliar symptoms. These striking symptoms have been attributed to the production of toxic metabolites by the pathogen, the most widely studied being eutypine. The aims of the study were to compare the effects of E. lata on xylem structure at the site of infection and in remote tissues. In healthy Vitis vinifera, the vessel-associated cells (VACs) in the trunk have a protective layer that covers the entire lignified wall and forms a transfer apparatus in pits located at the VAC / vessel interface. This apparatus occurs similarly in VACs in the basal part of canes but is less developed in the apical part. In the presence of E. lata, which is found only in the trunk and the cordons, the VACs initiated a program of secretory activity that led to the enlargement of the transfer apparatus, which is formed by tightly associated fibrils. This secretory activity was followed by VAC death. Furthermore, the hypertrophy of the transfer apparatus spread according to an acropetal gradient in the canes. Treatment with eutypine also induced the development of the transfer apparatus in VACs of basal and apical parts of canes excised from healthy vines. However, this apparatus was formed by loosely packed fibrils in VACs that were not completely damaged. Therefore, metabolites other than eutypine are expected to be involved in the VAC degeneration observed in infected vines.

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.

Effects of glycine on dark-and ligh-induced pulvinar movements and modifications of proton fluxes in the pulvinus of Mimosa pudica during glycine uptake

Glycine (1–50 mM) increases the rate of the dark-induced (scotonastic) movements and decreases the amplitude and the rate of the light-induced (photonastic) movements of the secondary pulvini of Mimosa pudica leaves. The uptake of glycine is accompanied by a long-lasting dose-dependent increase in the alkalinity of the bathing medium of the excised pulvini. The data are in agreement with a H+-glycine co-transport mechanism within the pulvinar cells. Fusicoccin (50 μM), known to promote H+−K+ exchange, antagonizes the effects of glycine on the movements and the alkalization of the bathing medium of the excised pulvini. The present results argue for the hypothesis that proton fluxes mediate the scotonastic and photonastic pulvinar movements.

Redistribution of potassium, chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K+ and Cl- shift towards the adaxial half of the motor organ whereas Ca2+ shifts towards the abaxial half. Compounds known to affect K+ transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.

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 [Ca²⁺](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 [Ca²⁺](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 [Ca²⁺](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.

PHYTOCHROME‐ and BLUE PIGMENT‐MEDIATED LEAF MOVEMENTS: EXPERIMENTAL APPROACH IN THE SIGNAL TRANSDUCTION

Abstract— Leaflet movements of Cassia fasciculata are induced by transferring leaves from light to darkness or from darkness to light. Phytochrome mediates the dark‐induced closure whereas a blue and far red light absorbing pigment (cryptochrome?) is the photoreceptor triggering the light‐induced opening. These movements are the result of reversible turgor variation driven by ionic migrations (H+, K+, Cl−) in cortical parenchyma cells of motor organs (“pulvini”) localized at the leaflet base. Calcium plays a predominant role in the regulation of the movements as shown by the inhibitory effects of chelators (EDTA, EGTA), intracellular antagonist TMB‐8 and by the promoting effect of ionophore A 23187. Compounds known as calcium channel blockers (LaCl3, verapamil and nifedipine) inhibited whereas Bay K 8644, a calcium channel activator, promoted the phytochrome‐mediated movement. In contrast, all these calcium channel modulators had no effect on the blue pigment‐mediated movement. From these results, it is suggested that calcium is not mobilized in the same manner in the two types of movements: possibly from external stores in the phytochrome‐mediated response and from internal stores in the blue pigment‐mediated response. Calcium acts possibly through calmodulin as suggested by a modification in the kinetics of the movements induced by inhibitors of calmodulin action (trifluoperazine, R 24571, W‐7). The unexpected promotion of the movements by these inhibitors shows that calmodulin action on the ion migrations is not simple and direct. Experimental observations suggested that regulation might be done through cAMP metabolism. db‐cAMP promoted the movements. Compounds known either to activate adenylate cyclase (prostaglandins, forskolin) or to inhibit phosphodiesterase (imidazolidinones, ICI 58301) induced the same modifications as db‐cAMP. By contrast, a phosphodiesterase activator (imidazole) inhibited the movements.

Changes in membrane potential related to glycine uptake in the motor cell of the pulvinus of Mimosa pudica

Summary Earlier work suggested that glycine uptake by the pulvinus motor cell of Mimosa pudica is a H + -cotransport mechanism. This hypothesis is sustained by the present investigations showing that glycine is transported electrogenically across the motor cell membrane as evidenced by concentration — dependent depolarizations observed upon addition of glycine in the range 1–10 mM. On the contrary, fusicoccin (0.1–10μM)elicited fast and great hyperpolarization. This antagonistic action of glycine and fusicoccin on membrane potential is in agreement with the antagonistic effects of these substances on the pulvinus movements previously observed. The data corroborate the assumption that glycine is taken up by pulvinus tissue by a H + -cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma and provide support for the model proposed previously on pulvinus functioning involving proton fluxes.

Effects of prostaglandins E, precursors and some inhibitors of prostaglandin biosynthesis on dark- and light-induced leaflet movements in Cassia fasciculata Michx.

Prostaglandin E1 and prostaglandin E2 speed up the dark-induced (scotonastic) and light-induced (photonastic) leaflet movements of Cassia fasciculata. The precursors of prostaglandin biosynthesis, homo γ-linolenic and arachidonic acids, and an intermediary product, prostaglandin-interm-5, act in the same manner on these movements. Inhibitors of prostaglandin biosynthesis, indomethacin and phenylbutazone, inhibited the scotonastic but promoted the photonastic movements in an unexpected way. Since the pulvinar movements are mediated by water and ion migrations, the observed modifications of these movements indicate that prostaglandins and their precursors may affect, as in animal cells, processes linked to a variation of membrane permeability.