Cysteine_ A multifaceted amino acid involved in

Plant Physiology and Biochemistry 129 (2018) 77–89

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

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

T

Gabriel Roblina, Stéphane Octavea,b, Mireille Fauchera, Pierrette Fleurat-Lessarda, Jean-Marc Berjeauda,∗ a

Université de Poitiers, Ecologie & Biologie des Interactions, UMR CNRS 7267, 1 rue Georges Bonnet, TSA51106, 86073 Poitiers cedex 9, France Current address: Sorbonne Universités, Université de Technologie de Compiègne, UMR CNRS 7025, Génie Enzymatique et Cellulaire, Rue du Docteur Schweitzer CS 60319, 60203 Compiègne Cedex, France b

A B S T R A C T

Early effects induced by cysteine were monitored using the model of Mimosa pudica pulvinar cells. Rapid dosedependent 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.

1. Introduction Sulfur is an essential macronutrient for plant growth and development. S is predominantly available to higher plants in the form of SO42− taken up from the soil by the roots. A series of reactions in which sulfate is reduced to sulfide led to cysteine (Cys), a key product in the sulfur assimilation pathway (Rausch and Wachter, 2005). Cys is not only a protein component, but also a primary substrate in the synthesis of methionine and many other sulfur-containing compounds such as thionins, defensins, glucosinolates and S-containing phytoalexins (Rausch and Wachter, 2005). In addition, among these compounds, glutathione is of particular importance regarding its roles in cellular redox homeostasis, in the detoxification of xenobiotics (Xiang et al., 2001; Maughan and Foyer, 2006) and in the protection against heavy metal toxicity (Mendoza-Cózalt et al., 2008; Cobbett and Goldsbrough, 2002; Sharma and Dietz, 2006 and references therein). Most of the Cys is formed and accumulated in the cytosol (Krueger

∗

Corresponding author. E-mail address: [email protected] (J.-M. Berjeaud).

https://doi.org/10.1016/j.plaphy.2018.05.024 Received 20 March 2018; Received in revised form 4 May 2018; Accepted 22 May 2018 Available online 23 May 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.

et al., 2009) but it is also found in plastids and mitochondria where it has different functions (Romero et al., 2014). Thus, in mitochondria, the molecule plays a central role in the detoxification of cyanide, and in the cytosol, Cys is considered a determinant of the oxidative capacity (López-Martín et al., 2008a; b) and has specific functions in plant-pathogen interactions (Alvarez et al., 2012). In fact, the Cys content is correlated with plant resistance and is required for the hypersensitive response (Alvarez et al., 2012). The implication of Cys has been considered in these relatively long lasting events, but the short-term physiological effects of the compound have not yet been examined. Therefore, the first aim of the present study was to examine whether Cys may be involved in an early signaling function for plant cells. Ionic perturbations at the plasma membrane level have been described as pivotal and primary processes linked to the recognition of stress application by plant cells (Dixon et al., 1994; Cervone et al., 1997). In this line, we monitored modifications of the membrane potential and induction of proton fluxes triggered after a short time on pulvinar motor


G. Roblin et al.

chambers at 27.5 ± 0.5 °C and 65% relative humidity. Illumination was regulated to give 16 h of light (photophase from 06 a.m. till 22 p.m.) provided by fluorescent tubes (a mix of Osram fluora and Osram day-light types) with a photon fluence rate of 80 μmol m−2 s−1 at the plant apex. Cuttings of Vitis vinifera L. cv. Cabernet Sauvignon were grown in a greenhouse under natural day-light conditions and fed daily with Snyder solution. In the experiments concerning the translocation of Cys, the 35–45 cm high vine cuttings bearing 8 well-developed leaves were transferred a week before the assays from greenhouse to climate-controlled chambers in conditions described above.

cells of Mimosa pudica leaf by exogenous application of Cys. Additionally, we showed its implication on other more long lasting modifications of ionic equilibrium as indicated by the changes induced on the osmocontractile reaction of pulvinar motor cells caused by a shock on M. pudica leaves or a period of darkness on Cassia fasciculata leaves. We also examined the development of the characteristic necrotic effect observed on blades when Cys was applied at concentrations higher than 1 mM and observed the corresponding structural changes in pulvinar cells. Elemental sulfur (S0) has been used for a long time in plant disease control and by the early 20th century, it was the most widely used fungicide until the synthesis of systemic S-compounds (Cooper and Williams, 2004). S0 might act at multiple sites in cells since fungal resistance has not yet been reported following its application (Beffa et al., 1987). Furthermore, the observation that sulfur metabolites are involved in plant disease resistance has led to the concept of sulfur-induced resistance (Bloem et al., 2007; and references therein). Thus, S0 is accumulated in the xylem of resistant genotypes of Theobroma cacao infected by Verticillium dahliae (Williams et al., 2002) and this accumulation is followed by an increase in sulfate, glutathione and Cys (Cooper et al., 1996; Williams et al., 2002). Furthermore, Bloem et al. (2004) reported that the Cys content increased in the leaf disc samples of Brassica napus 2.3 fold in the course of infection with Pyrenopeziza brassicae. Considering the above-mentioned observations, we address the question of whether Cys may be used in the control of particular plant diseases concerning the fungal-induced degradation of their wood tissues. We focused particularly on Eutypa dieback and esca disease which affect grapevines worldwide, resulting in serious economic losses (Dubos et al., 1983; Munkvold et al., 1994). These diseases share similar general features. In both cases, the fungal pathogens invade the vascular system of the trunks and shoots through pruning wounds leading to characteristic necrosis of woody tissues. The colonization phase in wood tissues induces particular symptoms appearing on the leaves after an incubation period of three or more years and, finally, leads to the grapevine death. Attempts to control the disease are based on treatments using chemical compounds (e.g. mancozeb, cymoxanil, fosetyl-Al …) or natural molecules (e.g. chitosan, salicylic acid) (Bertsch et al., 2013). However, these treatments are not yet completely effective. In the course of a previous work (Octave et al., 2005), we stressed that among S-containing compounds, Cys caused a marked inhibitory effect on the mycelial growth of E. lata (El) the agent responsible for Eutypa dieback. In this model, we determined the optimal conditions to inhibit the in vitro development of the fungus. In the present work, we test and confirm the antifungal effect of Cys on Phaeomoniella chlamydospora P.W. Crous & W. Gams (Pch) and Phaeoacremonium minimum W. Gams, P.W. Crous, M.J. Wingfield and L. Mugnai (Pmi), pathogens involved in the development of esca disease. A major problem in the wood-degrading diseases is linked to the internal localization of the pathogens which constitute an important impediment that should be carefully taken into account in treatments with exogenously applied compounds that should reach the fungal target. In this scope, we monitored the translocation of [35S] cysteine in grapevine cuttings following its application by leaf spraying to analyze whether Cys may be a convenient agent to control the development of the disease. Finally, we discuss the data of the events described here in the scheme built from previous results obtained on the effects of Cys on general metabolic fates and its possible antifungal utilization.

2.2. Electrophysiological measurements and determination of proton fluxes The transmembrane potential of pulvinar motor cells of M. pudica leaves of 2-month-old plants was measured by the classical electrophysiological method using microelectrodes with tip diameter < 1 μm and tip resistance in the range 5–30 MΩ, drawn from capillaries provided with an internal microfibre (GC 150F15; Clark Electromedical Instruments, Pangbourne, UK). For details, see Amborabé et al. (2001) and Saeedi et al. (2013). Leaf was excised and pulvinus was fixed to the bottom of a 4-ml Plexiglas chamber filled with a buffered medium (10 mM MES/NaOH, pH 5.2, containing 1 mM NaCl, 0.1 mM KCl, and 0.1 mm CaCl2 (Abe, 1981). The glass microelectrode was inserted into a motor cell of the abaxial (“extensor”) half of the organ. Under these conditions, the resting transmembrane potential (Ψ0) was in the range −110 to −130 mV. The calculated value from 24 assays gave −117 ± 3 mV (SEM). The pH variations in an incubation medium composed of 0.50 mM CaCl2 and 0.25 mM MgCl2 were read on a pH meter provided with combined electrodes (Futura micro-combination, Beckman Coulter) and linked to a potentiometric recorder. Transverse sections of primary pulvini of M. pudica (400 mg), treated as previously described (Amborabé et al., 2008), excreted protons in their bathing medium. In order to quantify the amount of mobilized protons, titration was made 90 min after the application of cysteine in 2 ml incubation medium with 5 × 10−3 M HCl. The experiments were repeated 3 times.

2.3. Observation of the pulvinar movements on Mimosa pudica and Cassia fasciculata Seedlings of M. pudica bearing the first fully developed leaf constituted the experimental model for the observation of the seismonastic motor reaction, characterized by a leaf drooping in about 2 s and a recovery lasting about 20 min. The experimental conditions have been extensively described previously (Saeedi et al., 2013). As a reminder, seedlings were excised and dipped in distilled water for 4 h. After this time lapse, distilled water was replaced by the compounds to be assayed dissolved in a medium buffered with 2.5 mM MES at pH 5.0. Leaf position was determined by measuring the angle formed by the vertical and the petiole with a transparent protractor. The angle was determined before and after stimulation by a touch applied on the abaxial half of the organ at 1-h intervals for 4 h. Each experiment was carried out 3 times on 10 leaves in each experimental set (n = 30). Leaves of C. fasciculata were excised from three months old plants bearing 15 well-developed leaves. The leaves were transferred to test solutions for 3 h and then were put into darkness in the middle of the photophase (at 2 p.m.). Leaflet movements were measured under a dimgreen safelight by measuring the distance between the leaflet tips with a caliper square. This linear measurement was then converted into angular values as previously described (Bonnemain et al., 1978). Each experiment was carried out 3 times on 8 leaves in each experimental set (n = 24).

2. Materials and methods 2.1. Plant growth conditions Seedlings and older plants of Mimosa pudica and Cassia fasciculata were grown in a compost watered daily and kept in climate-controlled 78


G. Roblin et al.

Purified plasma membrane vesicles (PMVs) were prepared by phase partitioning of microsomal fractions from the primary pulvini of M. pudica according to Lemoine et al. (1991) with some minor modifications. Vesicles were frozen in liquid nitrogen and stored at −80 °C. They were put in the inside-out position by adding 0.05% Brij in the assayed medium. Vanadate-sensitive ATPase activity of the vesicles was measured in a medium buffered with 50 mM MES/TRIS at pH 6.5. Proton movement was determined by the decrease of 9-aminoacridine absorbance at 495 nm in a medium buffered with 10 mM MES/TRIS at pH 6.5. For details, see Amborabé et al. (2008). Treatments of PMVs with Cys, methionine and ethionine at the given concentration were made 3 min before the beginning of the assay. In the 6 batches of vesicles used in this work, about 68% of the enzyme activity can be attributed to plasma membrane functioning as indicated by the effect of the plasma membrane H+-ATPase inhibitor sodium orthovanadate used at 0.25 mM (Table 3).

Mycelial growth was measured in the dark at 20 °C on cultures grown in Petri dishes containing the solid medium brought to pH 4.8 with 10 mM N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES)/KOH buffer. Cys, other amino acids and different sulfate salts were added as a powder thoroughly mixed during cooling of the culture medium (at 50 °C) to obtain the desired final concentration. The inoculum consisted of a 3-mm diameter mycelial disk taken from a solid culture by means of a cork-borer. The diameter of the colony was measured daily in six identical wells for a period corresponding to complete mycelial invasion of the dishes in the controls. Experiments were carried out at least three times. The assays of spore germination were conducted on 96 well microplates. The liquid culture medium was the same used in mycelial growth. A total of 200 μl of medium in each well was sown with 50000 spores. The microplates were shaken at 100 rpm at 22 °C in the dark. The absorbance of the cultures was measured 3 d after sowing at 595 nm using a multiplate reader (Sunrise reader, Tecan). Experiments were carried out at least three times.

2.5. Necrosis experiments

2.8. Translocation of cysteine in vine cuttings

The possible toxic effect of Cys on the C. fasciculata model was quantified by a “necrosis index” defined in a five stage gradation and represented by the extent of necrosis expressed as a percentage of the total leaf area. Unaltered leaves (first stage) correspond to 0% necrosis and the fifth to leaflet abscission stages correspond to 100% necrosis. For details, see Roblin and Fleurat-Lessard (1983). Additional experiments have been conducted on vine cuttings in greenhouses to verify whether additive treatments with Cys may induce some necrotic phenomena on blades. In this aim, leaves of cuttings were sprayed in the morning for 4 consecutive days at the end of June with a solution buffered with MES 2.5 mM at pH 5.0 and composed of Cys at various concentrations (from 1 to 50 mM) and a wetting agent (Etaldyne 95, Fertiligène, Rhône-Poulenc) at 0.5 ml l−1. Observations were made a week after the treatment.

A week before the assays, the 35–45 cm high vine cuttings, bearing 8 well-developed leaves, were transferred from greenhouse to climatecontrolled chambers at 25 ± 0.5 °C and 65 ± 5% relative humidity. To monitor the uptake and translocation of Cys, leaves were sprayed with [35S] Cys (460 kBq ml−1) at a final concentration of 5 mM (buffered in MES 2.5 mM at pH 5) and supplemented with the wetting agent Etaldyne 95 at 0.5 ml l−1. Two types of experiments were carried out. First, a leaf in the middle part of the cutting was sprayed to monitor the translocation in the upward and downward directions and second, all the leaves above the two more basal leaves were sprayed to mimic a treatment applicable in the field. The labelling was determined in different parts of the cuttings as follows. Twenty foliar disks (6 mm diameter) were harvested at random in the blade, taking care however to avoid the areas at the tips of the leaf because of a possible accumulation of the labeled compound due to the dripping evidenced by autoradiographic determinations (see Fig. 5). Fragments (1 cm) of internodes, arms and stem were harvested in the middle part of the organs. Fragments (1 cm) (grouped by 5) of roots were harvested at random in 4 areas of the large root system developed in the cuttings at this stage of development. The samples were weighed and then put in a solution containing perchloric acid (56%), H2O2 (27%) and 0.1% Triton X100 (17%). The experimental tubes were placed in an oven at 60 °C for 12 h. Then, 4 ml of scintillation liquid (Ecolyte TM, ICW) was added and stirred. Radioactivity was measured on a scintillation reader (TRI-CARB, 1900 TR, Packard). In the case of autoradiographic assays, the treated leaves were placed in perforated aluminum foil and put into lead plates in a freezer at −20 °C to avoid redistribution of the Cys. After freezing, the leaves were lyophilized, placed on a Mylar sheet (7 μm thick) and placed between glass plates in contact with an autoradiographic film (Kodak biomax MR film) for 2 months in darkness at room temperature. Then, the films were developed (Kodak LX 24) and fixed (Kodak AL 4). In each protocol, the experiments were carried out on 5 cuttings for each experimental sampling.

2.4. Preparation and use of plasma membrane vesicles

2.6. Microscopy Controls and cysteine-treated M. pudica primary pulvini were treated for transmission electron microscopy as follows. Chemical fixation occurred at 25 °C for 13 min in a mixture of 1.5% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M Sörensen phosphate buffer at pH 6.8. After washing in six baths, each of 10 min, in 0.1 M Sörensen phosphate buffer with 7.5% sucrose, postfixation occurred for 3 min in 1% (V/V) OsO4 in the washing buffer. Dehydration was carried out in increasing ethanol series (10%, 50% and 100%), each applied for 5 min. Next, samples were impregnated in LR White (LRW) resin/ethanol mixtures (1/3 resin plus 2/3 alcohol for 20 min and 2/3 resin plus 1/3 alcohol twice for 30 min) and finally in pure LRW for 1 h and placed overnight at 4 °C. Embedding in LRW was followed by polymerization at 56 °C in the oven. Thin sections (600 nm) of the blocks, stained with uranyl acetate and lead citrate were observed using a Jeol 1010 microscope equipped with a camera that allowed digitalized pictures to be obtained. 2.7. Fungal growth conditions

2.9. Recapitulative view of biological models and analyzed traits The fungi were grown on a growth medium composed with yeast nitrogen base minimal (YNBm, Difco™ 233520, USA) at 1.7 g l−1 supplemented with glucose 50 mM and glutamine 40 mM. The medium was solidified with agar at 20 g l−1 and sterilized under 0.5 bars at 110 °C for 15 min (Fleurat-Lessard et al., 2014). The strains of Phaeomoniella chlamydospora P.W. Crous & W. Gams (PC-PC 37), of Phaeoacremonium minimum W. Gams, P.W. Crous, M.J. Wingfield and L. Mugnai (PA-PC 24) were isolated in the Cognac area. These strains were kindly provided by Dr. P. Larignon (IFVV Rodihlan, France).

The general description of the experiments carried out in the different biological models is given in Table 1. 2.10. Chemicals Buffers and cysteine were purchased from Sigma-Aldrich Chimie, radiolabelled L-[35S]-cysteine (NEG-022T) from NEN Radiochemicals and Etaldyne 95 from Rhone-Poulenc. 79


G. Roblin et al.

Table 1 Overview of aims and analyses developed to study the effects of cysteine on different biological models. Biological materials

Aims/Analyses Early membrane effects Deleterious effects

Leaf models

Fungal pathogens of Vitis

Cuttings

Mimosa pudica L. Cassia fasciculata Michx

Phaeomoniella chlamydospora Phaeoacremonium minimum

Vitis vinifera

Cell membrane potential (motor cell of MpL) Proton fluxes (motor cell of MpL) H + -ATPase activity (PMVs From motor cells of MpL) osmocontractile reaction of motor cells i Mpl and CfM Ultrastructural damage (motor cell of MpL) Necrosis development on blades (CfM)

Translocation

3. Results

3.1.1. Cysteine effect on the bioelectrical membrane potential The addition of Cys to the bathing medium of isolated M. pudica primary pulvini triggered a long lasting depolarization of the pulvinar cell membrane in a concentration-dependent manner from 0.1 to 10 mM, which was triggered after a short lag time lasting some minutes (Table 2). Typical recordings of the time course of the bioelectrical events show that the concentration-dependent decrease of the membrane potential occurred until a plateau was reached in approximately 20 min. Fusicoccin, a fungal toxin known to trigger membrane hyperpolarization, was added at the end of each experiment to ensure that the observed bioelectrical variation resulted truly from a biological effect rather than an artifactual one (Fig. 1A). By comparison, the addition of 10 mM glycine (Gly) triggered a transient depolarization of similar amplitude but after a shorter latency. In this case, repolarization was achieved in approximately 30 min (Fig. 1A).

Table 2 Effects of cysteine (Cys) applied at various concentrations and of glycine (Gly) applied at 10 mM on the characteristics of the transmembrane potential variation of the pulvinar motor cell and on the characteristics of the proton fluxes measured in the bathing medium of excised primary pulvini of Mimosa pudica, Calculations on ΔΨ and on the amount of mobilized protons were respectively made 20 min and 90 min after addition of the product. Mean ± SD; Nb: number of assays.

Control Cys 0.1 Cys 1 Cys 10 Gly 10

ΔΨ (mV)

Nb

Latency (min)

H+ (nmole)

Nb

– 186 ± 36 150 ± 30 96 ± 24 19 ± 8

−1 ± 1 −3 ± 3 −22 ± 3 −32 ± 6 −39 ± 4

5 4 4 5 6

– 13 ± 1 7±1 3±1 2±1

– 17 ± 6 80 ± 10 145 ± 35 210 ± 25

3 3 3 3 3

35S cys redistribution in grapevine organs

3.2. Deleterious effects induced by cysteine on plant cells 3.2.1. Development of necrosis on C. fasciculata leaflets A more or less pronounced effect, first appearing at the leaflet tips, was observed following a treatment of the isolated leaves by Cys (Fig. 3A). The necrosis developed after a latency and was a function of the dose applied. Thus, at 1 mM, a slight necrosis was clearly seen only after 7 d and only increased the ageing effect observed on the controls. Characteristic larger necrosis was induced by Cys 5 mM and Cys 10 mM after 3 and 2 days, respectively. The leaflets were extensively damaged the next day following the application at very high dose (50 mM) (Fig. 3A). The data in Fig. 3B show that a treatment as short as 30 min at 50 mM produced significant necrosis detected 2 d after the application and that a treatment lasting 3 h led to a complete necrosis of the leaflets.

protons absorbed

Latency (s)

Absence of leaf necrosis

3.1.3. Cysteine effect on ion-driven turgor-mediated pulvinar reactions Due to its disturbing action on ionic distribution, as indicated by the bioelectrical events and the H+ mobilization, Cys is expected to affect biological models implicating ions in their functioning. In this line, the effect of Cys transported from the cut section of the hypocotyls to the pulvinar tissues was analyzed on the rapid seismonastic and slower dark-induced ion-driven osmocontractile reactions of pulvinar cells. As seen in Fig. 2A, the leaf drooping of M. pudica was inhibited in a dose-dependent concentration and observed as early as 1 h after the addition of the compound in the test tubes. After 2 h of Cys action, the inhibition was 11% at 0.5 mM, 21% at 1 mM and 74% at 10 mM. The data in Fig. 2B indicate that Cys acted differentially by considering other sulfur-containing compounds applied at 10 mM since inhibition by methionine was only by 13% and ethionine by 21%. Comparatively, glycine did not show any significant effect on the amplitude of the movement (+5%). Lastly, compared to the L-form, the D-form was also very inhibitory although its action was slightly slower (50% inhibition). The application of Cys on leaves of C. fasciculata induced a low amplitude spontaneous closure of the leaflets maintained in the light visible 1 h after application and an inhibition of the large closure movement induced by darkness application. These effects were concentration dependent. Thus, the dark-induced closure was inhibited after 1 h darkness by 17% at 1 mM, 45% at 5 mM and 59% at 10 mM (Fig. 2C).

3.1.2. Cysteine effect on H+ fluxes and proton pump activity It has been previously reported that sections of M. pudica primary pulvini induced a spontaneous acidification of the incubation medium as a result of the proton pump activity (Amborabé et al., 2008) as argued by the activating effect of the fusicoccin (Otsiogo-Oyabi and Roblin, 1984). Fig. 1B shows the time course of the pH variation recorded in the bathing medium of pulvinar sections after the addition of Cys at various concentrations. A concentration-dependent pH increase was observed after the addition of Cys at concentrations higher than 0.1 mM after a concentration-dependent latency. Gly application triggered a similar time course but induced a larger proton influx (Table 2). The major characteristic is that these pH increases were long lasting. For example, when Cys and Gly at 10 mM were applied, the pH

bioelectrical potential

Inhibition of mycelial growth (compared to other amino acids and sulfate salts) Inhibition of spore germination

increased respectively by 0.5 and 0.7 pH unit in comparison to the control and returned to the level of the control in approximately 24 h even in the presence of the compound (not shown, see Otsiogo-Oyabi and Roblin, 1984). Note that the latency is longer than that observed in the depolarization (Table 2). These data raise the question of whether Cys may act directly on the plasma membrane H+-ATPase activity. As deduced from Table 3, the effect of Cys at the assayed concentrations on PMVs cannot be ascribed to a direct inhibitory effect on the activity of the plasma membrane H+ATPase or to a protonophoric effect.

3.1. Early biological effects induced by cysteine on plant cells

Treatment (mM)

Sprays of leaves

80


G. Roblin et al.

Table 3 Determinations of the net proton movement (measured by absorbance variation from the control absorbance at 495 nm) and of the vanadate-sensitive ATPase activity in plasma membrane vesicles purified from Mimosa pudica pulvini treated with cysteine, other sulfur compounds and vanadate. Values are mean ± SD; number of assays in parentheses. Product

Concentration (mM)

Absorbance variation −1

(unit mg prot Control Cysteine

Methionine Ethionine Vanadate

– 1 5 10 5 5 0.25

0.42 0.47 0.40 0.43 0.47 0.46 0.14

± ± ± ± ± ± ±

0.07 0.05 0.07 0.08 0.07 0.05 0.06

min

Vanadate-sensitive ATPase

−1

)

(19) (8) (6) (8) (4) (6) (9) (- 67%)

activity (nmol Pi mg prot−1 min−1) 331 320 328 333 331 306 105

± ± ± ± ± ± ±

16 (11) 24 (4) 40 (4) 40 (6) 7 (4) 3 (4) 25 (11) (- 68%)

pulvini compared to controls (b). Detailed pictures in the cytoplasm and organelles indicated that in the control the dense nucleus matrix contained numerous and large chromatin clusters (c), whereas it was particularly damaged in treated pulvini, being mostly transparent to electrons, with only small clusters of chromatin occurring along the nucleus membrane (d). In the control, Golgi stacks were surrounded by many vesicles (e), whereas, in treated cells, the cytoplasmic matrix was of lower density and contained stretched Golgi stacks with few secreted vesicles (f). The cytoplasm in the control had long endoplasmic reticulum profiles lining the plasma membrane (g), whereas highly dilated profiles of rough endoplasmic reticulum and less abundant polysomes occurred in treated plants (h). Mitochondria in the controls presented large cristae in their dense matrix (i) whereas they were irregularly shaped and deeply modified in treated cells showing large distorted cristae in a clear matrix (j). 3.3. Antifungal effect of cysteine on Pch and Pmi The results of Fig. 4A show that, among amino acids, Cys added to the growth medium drastically inhibited the colony development of Pch and Pmi. Beside Cys, Arg, Asp and Leu inhibited both fungi in a lower extent. Differential effects were also observed: growth of Pch was also inhibited in media containing Glu, Gly, His, and Trp, whereas growth of Pmi was inhibited by Lys, Met and Tyr. The induced inhibition by Cys was concentration-dependent (Fig. 4B). Thus, a low inhibitory effect on the mean growth rate was observed at 0.5 mM (5% for Pch, 11% for Pmi) so that it can be considered near the threshold concentration. At 5 mM, a nearly similar inhibition was observed for both fungi (33% and 37%, respectively), and a complete inhibition was obtained at 15 mM on Pch and 20 mM on Pmi, indicating that Pch was more sensitive to the treatment than Pmi. The results of Fig. 4C show that the effect of Cys is not due to sulfur in the form of sulfate. Indeed, sulfate salts of K and Mg present no significant action on fungal development, whereas NH4 sulfate only slightly inhibited it (5% for Pch, 17% for Pmi). In contrast, mycelial growth was completely halted following treatments with Cu and Fe salts. The spore germination of both fungi was inhibited by Cys in the same range of concentrations as the mycelia. Pch was inhibited by 31% and Pmi by 25% at 5 mM. However, Pch appeared more sensitive than Pmi to the treatment since complete inhibition was obtained at 15 mM on Pch and 20 mM on Pmi (Fig. 4D).

Fig. 1. (A) Typical recordings of transmembrane potential variations in parenchyma cells of Mimosa pudica primary pulvinus induced by application of cysteine (C) at various concentrations (expressed in mM) and glycine (G) at 10 mM; Co indicates control. Compounds were added at time 0 (black arrow) and 10 μM fusicoccin was added at black arrowheads. The number of assays is indicated in Table 1. (B) Representative time courses of pH variations monitored in the bathing medium of pulvinar tissues of Mimosa pudica induced following application of cysteine (C) at various concentrations as indicated in mM and of 10 mM glycine (G), compared to control (Co). Compounds were added as indicated by the black arrow.

3.2.2. Damage in motor cells of primary pulvini of Mimosa pudica The previous data indicated that some preponderant cell processes were strongly affected. Observations on the effects of Cys were carried out to determine the cell compartment(s) that are attacked. The images in Fig. 3C were obtained in motor cells of the primary pulvinus of the first leaf, in control (a, c, e, g, i) and Cys-treated (b, d, f, h, j) M. pudica plants. The general images from electron microscopy showed spherically shaped motor cells, surrounded by a thin wall and containing a large central vacuole surrounded by a narrow layer of cytoplasm in which the organelles are spread (a). At this scale, the nucleus and cytoplasm appear more transparent to electrons in the section of treated

3.4. Translocation of cysteine in cuttings of grapevine Fig. 5A shows the translocation of the labeling in the cuttings upwards and downwards from a leaf located in the intermediary part of the cuttings sprayed with 5 mM [35S] Cys. The data measured in the different parts of the cuttings after 24 and 48 h is discussed here. First, the amount of radioactivity noted in the sprayed leaf was the same, indicating that there is no increase in the uptake capacity of the leaves in this time lapse. Second, although a similar amount of radioactivity 81


G. Roblin et al.

Fig. 2. Effects of sulfur compounds on the osmocontractile reactions of motor organs. (A) Modification of the amplitude of the seismonastic reaction of primary pulvini of Mimosa pudica as a function of the cysteine concentration. (B) Modification of the amplitude of the seismonastic reaction of primary pulvini of Mimosa pudica by methionine, ethionine and glycine, and comparative effects of L- and D-cysteine, applied at 10 mM concentration. Mean ± SE; n = 30. (C) Modification of the time course of the closing movements of Cassia fasciculata pinnules induced by darkness as a function of the cysteine concentration. Mean ± SE; n = 24.

proportion of labeling was found in the lower parts (arm, stock, roots), and after 72 h, the relative proportions of labeling were more uniform in the cuttings. This means that a redistribution occurred during this time lapse. A similar problem as for blade is linked to the random harvesting of the root fragments in a very developed root apparatus and was also tentatively reduced by increasing the numbers of samples. An additional experiment has been carried out to check whether application on blades of Cys at relatively high doses may induce damaging effects on the grapevines. As observed in Fig. 7, the leaves of cuttings sprayed at different developmental stages did not show any external symptoms of toxicity at concentrations ranging from 1 to 50 mM, when observed 1 week after 4 applications. Note that a white deposit was observed at the higher concentrations (50 mM in particular), probably resulting from the formation of cystine (Leustek and Sato, 1999). To conclude, the important observation is that Cys was transported downwards from leaves to the roots and accumulated in the trunk which is the area where the fungal pathogens (Pch and Pmi) are localized in the course of the development of esca disease.

was measured in the entire cutting, a modification in the repartition was noted. In particular, the level of radioactivity increased in internodes (+30%) but decreased in the blade disks (−79%) in the samples harvested at 48 h compared to those harvested at 24 h (Fig. 5B). Third, the labelling was different according to the position on the stem. In fact, the side of stems bearing leaves on the orthostichy opposite to the donor leaf presented only 30% radioactivity of the parts bearing leaves in the same orthostichy at 24 h. This dissymmetry was reduced in the samples taken at 48 h (60% on the opposite line). A similar result was observed on the samples harvested on blades, and the dissymmetry noted at 24 h was abolished at 48 h, indicating that a transversal translocation occurred during this time lapse. Fourth, the labeling measured in trunk after 24 h increased more than two fold after 48 h, whereas the accumulation noted in the arms after 24 h was considerably reduced after 48 h. Caution should be taken when considering the data obtained on the blade based on the random harvesting of the disks. In fact, as seen on the autoradiographs in Fig. 5A, labeling may not be uniformly distributed showing some areas with high labeling, particularly at the edges as a consequence of dripping after sprays. Nevertheless, the high number of disks harvested on each blade (20) might reduce the risk of erroneous data. Under the condition of sprays on many leaves of the cutting (8 in our assays), the amount of radioactivity increases with time in the different parts of the cuttings (Fig. 6A). In particular, the amount found in internodes increased considerably at 72 h after the sprays. However, it should also be stressed that the labeling increased significantly in stocks and roots (approximately 10 times higher than at 24 h). Fig. 6B shows the relative proportions of the labeling monitored in the various parts of cuttings (apart blades) measured 24, 48 and 72 h after sprayings. After 24 h, the internodes in the middle part presented a high labeling, and interestingly, stocks are also labeled. After 48 h, the major

4. Discussion 4.1. Cysteine as a multifunctional compound in the life of the cell Sulfur is available to plants in the form of sulfate present in the soil. Transported from the roots and distributed throughout the plant, sulfate undergoes a multiple step reductive pathway leading to the end product Cys (Leustek and Sato, 1999; Takahashi et al., 2011) concentration of which was estimated to be greater than 300 μM in the cytosol (Krueger et al., 2009). Cys occupies a pivotal role in plant cell life by its involvement in many important processes. First, Cys intervenes in the 82


G. Roblin et al.

Fig. 3. Representative time courses of the development of necrosis observed on pinnules of Cassia fasciculata (A) treated for 6 h by increasing concentrations of cysteine (in mM) and (B) treated by cysteine at 50 mM for various durations (in h). (C) Modifications of the structural arrangement in motor cells of primary pulvini of Mimosa pudica leaf induced by 10 mM cysteine applied for 3 h. General views: transmission electron microscopy of motor cells showing a large central vacuole (V) surrounded by a narrow layer of cytoplasm, in which the nucleus (N), tannin vacuoles (arrow), plastids (P) and mitochondria (m) are spread in (a) control and (b) cysteine-treated motor cells. Detailed views: (c), in control, nucleus (N) with a dense matrix, large nucleolus (nu) and chromatin clusters (chr) near the nuclear membrane, rough profiles of endoplasmic reticulum (er) in the dense cytoplasm. (d), in treated motor cells, clear cytoplasmic matrix, large nucleolus with an irregular border and small chromatin clusters; in adjacent cytoplasm, dilated profiles of er. (e) in control, Golgi stacks surrounded by emitted large vesicles, whereas in treated plants (f) Golgi with stretched stacks with few vesicles around. (g) in control, long profiles of rough er along plasma membrane, whereas dilated er profiles in treated plants (h). (i) in control, mitochondria with cristae in a dense matrix, whereas in treated plants, damage in the irregularly-shaped mitochondria containing many distorted cristae in a clear matrix (j). Scale bars: a, b = 10 μm; c-j = 1 μm.

detoxification (Mendoza-Cozalt et al., 2008; Dixon et al., 2002). Cys is essential to the initiation of the hypersensitive response during effector-triggered immunity characterized by an induction of the oxidative burst (Alvarez et al., 2012) showing a maximal effect at 0.2 mM on suspensionn-cultured cells (Bolwell et al., 2002). If the implication of Cys is now well documented concerning its regulatory role during plant-pathogen interactions by the observation on processes triggered many hours after the application of the stress, then the early signals induced by Cys at the plasma membrane level

structure of proteins through the formation of disulfide bridges essential for the stability and function of the molecules (Haag et al., 2012). Second, Cys is the precursor molecule used in the synthesis of S-containing compounds such as methionine, vitamins and cofactors (Droux, 2004) and many plant defense compounds such as glucosinolates, thionins defensins and S-containing phytoalexins (De Lucca et al., 2005; Rausch and Wachter, 2005). Of particular importance is the synthesis of glutathione, which is determinant in cellular redox homeostasis (Noctor et al., 2012) and in processes of heavy metal and xenobiotic

83


G. Roblin et al.

Fig. 4. Developmental effects observed on Phaeomoniella chlamydospora (Pch) (black columns) and Phaeoacremonium minimum (Pmi) (white columns). (A) Specific inhibitory effect of cysteine compared to other amino acids applied at 40 mM, on the mean rate of colony growth of Pch and Pmi. (B) Concentration-dependent effects of cysteine and (C) comparative effects of sulfate salts applied at 40 mM on both fungi. Data represent mean ± SE, n = 18. Calculations were made over an 18-d period for Pch and a 12-d period for Pmi. (D) Effects of cysteine at various concentrations on the spore germination of the fungi. Data represent mean ± SE, n = 30. Calculations were made 5 days after sowing. 1, Ala; 2, Arg; 3, Asn; 4, Asp; 5, Cys; 6, Gln; 7, Glu; 8, Gly; 9, His; 10, Ileu; 11, Leu; 12, Lys; 13, Met; 14, Phe; 15, Pro; 16, Ser; 17, Thr; 18, Trp; 19, Tyr; 20, Val.

amino acids. In particular, glycine triggered a depolarization spike after a latency reduced to seconds followed by the return to the basal potential in approximately 30 min, as observed on the M. pudica motor cells (Otsiogo-Oyabi and Roblin, 1985, Fig. 1A), on Lemna gibba cells (Fisher and Lüttge, 1980) and on mesophyll cells of barley leaf (Felle and Zimmermann, 2007). Second, as shown in Fig. 1B, Cys also modified the spontaneous proton efflux resulting from the H+ ATPase activity monitored in the bathing medium of pulvinar tissues (Amborabé et al., 2008). Thus, application of Cys induced a net proton influx occurring subsequent to the depolarization as deduced from the observed longer lag time. The time course of the pH variations also indicated that proton influx lasted many hours. It is noteworthy that similar long-lasting proton influxes were monitored following the application of glycine (Otsiogo-Oyabi and Roblin, 1984) and isoleucine (Roblin et al., 2016) on pulvinar tissues of M. pudica and the application of several amino acids on A. sativa coleoptiles (Kinraide et al., 1984). This can be related to the continuous uptake of the amino acids that are transported through specific transporters (Fischer et al., 1998) mediated by a H+-substrate cotransport mechanism (Kinraide and Etherton, 1980; Delrot et al., 2001). Plants possess a large number of amino acid transporters, many of them being able to transport Cys, some even with a high specificity (Harrington and Smith, 1977; Tegeder, 2012). The long-lasting proton redistribution suggests a possible role in long-term cellular processes, in particular in contributing to the pH homeostasis inside or outside of the cell. The data obtained on the osmocontractile reaction of pulvinar cells

have been investigated poorly so far. 4.2. Cysteine induced early modifications on ionic equilibrium The early perception of Cys was shown in pulvinar cells by the monitoring of membrane depolarization occurring after a short lag period (within min) after application of the compound. This observation indicates that ionic fluxes at the plasma membrane level were disturbed early. The depolarization could result either from the exit of a negative charge or the entry of a positive charge. Consequently, this suggests that specific ionic channels were activated by Cys. Considering the ionic species mobilized in the processes leading to the induction of action potentials evoked in particular in the pulvinar motor cells (Samejima and Sibaoka, 1980; Abe, 1981; Moran, 2007; Volkov et al., 2010)) and in the wound-induced variation potential in wheat leaves (Katicheva et al., 2014), Cl− may be implied in the depolarization phase, and the repolarizing current may be carried by K+ moving passively in response to the reduced membrane polarity. Such an interpretation has also been given for the electrical events triggered in Avena sativa coleoptiles treated with various amino acids (Kinraide et al., 1984). Felle and Zimmermann (2007) also showed the time courses of the potential variation in cells of barley leaves treated with various amino acids and monitored the temporal sequence of Cl− and K+ fluxes triggered by the amino acid application. Importantly, they showed that Ca2+ was the first ion to move and was obligatory to induce the Cl− and K+ fluxes. It should be emphasized that the time course of the depolarization triggered by Cys differs from that of other 84


G. Roblin et al.

Fig. 5. Characteristic example of repartition of 35S labelling in grapevine cuttings following spraying with 5 mM of radioactive cysteine on a leaf (marked by a red star) situated in an intermediary position on the shoot. Data obtained from 5 cuttings are expressed in pmoles.g−1 FW ± SD. Data indicated on the leaf blade were obtained from 20 disks harvested at random (see autoradiographies in insert) and data on woody parts were obtained from 0.5 mm slices. Measurements made 24 h (A) and 48 h (B) after spraying. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2007 and references therein). The inhibition of the leaf drooping of M. pudica was observed 1 h after the addition of the compound in the test tubes (Fig. 2A). A part of this latency can be related to the transport from the cut end of the hypocotyl and the accumulation of Cys in the motor cells until the concentration reached an active level. By considering the long lasting proton entry indicated by the pH increases in the bathing medium of pulvinar tissues, it should be concluded that the K+ exit has to be inhibited, in the context of the exchange H+/K+. The mechanism of this inhibition must be examined further since it did not

also indicated that Cys modified bulk ionic migration and associated water movement by considering the well-documented mechanism driving the seismonastic reaction and the dark-induced movements. It has been established that the coordinated contraction of the pulvinar motor cells resulted from a rapid turgor loss driven by a large efflux of K+ (acting as osmoticum) and Cl− (Samejima and Sibaoka, 1980; Satter and Galston, 1981; Moran, 2007). This suggests that these movements are regulated by the activity of ionic channels and the activity of aquaporins (Fleurat-Lessard et al., 1997; Temmei et al., 2005; Moran,

Fig. 6. 35S labelling of internodes, arms, trunks and roots of grapevine cuttings monitored 24 h, 48 h and 72 h after sprays with 5 mM of radioactive cysteine on leaves (marked by a red star) situated in an upper position on the shoot. (A) Total labelling and (B) Repartition of the labelling in the various parts. Data were obtained from 5 cuttings. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

85


G. Roblin et al.

Fig. 7. Absence of necrosis on the leaves of grapevines sprayed at different developmental stages with cysteine at various concentrations as indicated. Note at 50 mM cysteine a white deposit observed some days after sprayings.

circulated at a velocity of 1 cm min−1 which fit well with the velocity observed for leucine, GABA and glutamate in Soybean (Servaites et al., 1979). A second type of electrical signal depicted as a variation potential spreading in the vascular apparatus may result from cell necrosis (see Fig. 4) resulting from Cys accumulation in the plant tissues. Long-distance intercellular electrical signals linked to damaging factors have been considered as a potential mechanism of coordinate functional responses in different plant cells (Vodeneev et al., 2015). Furthermore, it has been shown that electrical signals induce the expression of pathogen response genes (Fisahn et al., 2004). Cys also acts as an indirect signal following its perception by fungi. Indeed, Cys induced an excretion of ergosterol when applied to different fungi (Kahlos and Tikka, 1994; Octave et al., 2005) and this sterol presents properties of an elicitor (Granado et al., 1995; Amborabé et al., 2003; Kasparovski et al., 2003; Vatsa et al., 2011).

result from a modification of the H+ ATPase activity nor from a protonophore effect as shown by the data on PMVs. It should be stressed that the effect of Cys is higher than that induced by other S-containing compounds such as methionine and ethionine and is specific compared to other amino acids. In fact, in the same experimental model, 2.5 mM isoleucine did not significantly modify the amplitude of the seismonastic reaction (Roblin et al., 2016) whereas glycine at 10 mM (Fig. 2A) and more conspicuously at 50 mM (Otsiogo-Oyabi and Roblin, 1984) increased the amplitude of osmoregulated movements. Note that methionine and ethionine acted similarly to Cys: inhibition of the seismonastic reaction and absence of an effect on the H+ ATPase activity of PMVs. We also emphasized that the smaller effect observed on the seismonastic reaction after the application of D-Cys (- 15%) compared to L-Cys suggests that the process is not strongly stereospecific. 4.3. Cysteine as a signal molecule in the plant-pathogen relationship

4.4. Cysteine as a toxic molecule

The capacity of Cys to act in different ways as a signal molecule may be related to the biological processes described here. In particular, ion fluxes at the plasma membrane level are considered as early events in the signaling cascade mediating elicitor-induced defense responses and are prerequisite for subsequent cell reactions (Colcombet et al., 2009). Notably, ion leakage elicited in the hypersensitive response of A. thaliana is dependent on the cytosolic cysteine and glutathione was not effective in increasing the intensity of the total ion leakage (Alvarez et al., 2012). Cys may also be a signal through the induced proton redistribution. It has been suggested that H+ fluxes intervene in the elicitation processes (Hagendoorn et al., 1991; Schaller and Oecking, 1999) and that cytosolic acidification consecutive to H+ entry into the cell may function as a second messenger mediating MAP kinase activation in the response of cells to stresses (Tena and Renaudin, 1998). Considering its electrogenic properties, Cys may intervene in long distance signaling by creating cell-to-cell electrical conduction in grapevines. This is possible since Cys is transported over long distances in both the stem apex and the roots (Figs. 5 and 6). The pathway for apical transport could be the xylem or the phloem, whereas transport toward root system implicated only phloem transport as shown in Nicotiana tabacum (Rennenberg et al., 1979) and Ricinus communis (Bonas et al., 1982). In these plants, cysteine, methionine and glutathione

Cysteine can present toxic properties in the case of accumulation above a threshold (Hildebrandt et al., 2015), as a result of its high chemical reactivity, leading to form species with sulfur in high oxidation state. Pivato et al. (2014) mentioned that a cytosolic concentration of Cys above 50 μM is toxic to Arabidopsis cells by irreversible thiol oxidation, a loss of sulfur and the formation of hydroxyl radicals. Cys also has the capacity to promote oxidation damage through the formation of complexes with metal ions triggering Fenton reactions (Park and Imlay, 2003). The third source of toxicity comes from the formation of hydrogen sulfide (plus ammonia and pyruvate) in the cytosol resulting from the action of L-cysteine desulfhydrase (Alvarez et al., 2010; Gotor et al., 2015). Sekiya et al. (1982) observed that L-Cys applied in the range 1–15 mM induced increasing H2S emission in the leaves of several plants. The data on the model of C. fasciculata leaves have allowed us to specify the conditions leading to necrosis of the plant tissues. It is noteworthy that the long-term development of external symptoms requires a concentration-dependent time lapse to become visible. However, ultrastructural observations show that major changes noted in many compartments of motor cells were largely expressed after 3 h of treatment. Comparatively, the treatment of the grapevine leaves 86


G. Roblin et al.

induces early cell events implicated in plant signaling, namely, modification of ionic balance at the plasma membrane level. Early ionic disturbances are considered to participate in the reactions triggering physiological steps leading to plant resistance against pathogen-induced stresses. We generalize a previous observation demonstrating its antifungal action, justifying the proposal that among the S-containing compounds, Cys appears to be a promising potential compound able to control the development of fungal pathogens. In a focusing concern and considering its redistribution from leaves throughout the grapevine, particularly in the vine stock, this compound may constitute a tool to fight microbial agents implied in wood diseases by inhibiting mycelial development and sporulation.

appears less detrimental to the plant. In this latter case, Cys is absorbed through the blade epidermis and redistributed in the various tissues which may result in a lower active concentration than in the case of C. fasciculata leaves in which Cys is absorbed directly in the transpiration stream through hypocotyl cuts. Among amino acids, the necrotic effect induced by Cys is specific since other amino acids did not induce any necrotic effect when applied in the same range of concentrations on the Cassia leaflet model. Note that Cys induces general effects regardless of the organism since the same compartments are disturbed in plant cells (Fig. 3C) and in fungal cells (Octave et al., 2005). 4.5. Fungicidal effect of cysteine and possible use to control wood degrading diseases

Contributions

Elemental sulfur (S0) has been shown to have a role in plant defense (Cooper and Williams, 2004; Resende et al., 1996). In addition, it has been reported that sulfur applied in the form of sulfate has a significant effect on the health status of plants (Klikocka et al., 2005) and that sulfur metabolites are involved in plant disease resistance, supporting the concept of sulfur-induced resistance (Bloem et al., 2005; and references therein). In addition to its effects on plant reactions, Cys has been shown to have a direct impact on the development of fungi of different genera. Cys inhibited mycelial growth in Botrytis cinerea (ascomycete) (Leroux, 1994), in Inonotus obliquus (basidiomycete) (Kahlos and Tikka, 1994) and in Scedosporium species (Galgóczy et al., 2016). In addition to the data on the ascomycete Eutypa lata (Octave et al., 2005), we added a similar impact on two other wood-degrading fungi, Pch and Pmi. In the particular concern of the wood degrading diseases, it appears that Cys may be a convenient agent to control the spread of the disease. As a positive point, we showed here that Cys applied on leaves was redistributed upwards and downwards in the entire cutting, and, in particular, accumulated in the vinestock, which is the main location of the pathogens implied in this type of disease. However, two questions must be answered. First, it should be verified whether Cys is the sole sulfur compound implied since Cys may be metabolized into other sulfur compounds (methionine and glutathione in particular) that are somewhat less efficient on fungal inhibition (Octave et al., 2005). However, these compounds can be interesting due to their own mode of action. Thus, methionine has been shown to induce H2O2 generation, a key element in plant defense signaling and to upregulate the expression of many defense-related genes (Boubakri et al., 2013). Reduced glutathione serves as the first line of defense against the production of reactive oxygen species and reacts directly with toxins and heavy metals in a reaction mediated by glutathione-Stransferases (Edwards et al., 2000). Therefore, methionine and glutathione may have a complementary role to Cys since they are also transported towards the root system (Rennenberg et al., 1979; Bonas et al., 1982). Second, in the scope of a treatment in grapevines, the conditions of use have to be specified, in particular, the number of applications, the more convenient period in the year and the doses to be sprayed on the leaves. We expected that the amounts of compounds reaching the fungal target in grapevines may be somewhat lower than the amounts sprayed on the leaves, as a function of the length of the canes. The verification that high doses (50 mM) appear to not be detrimental to the grapevine leaves is of practical importance (Fig. 7). Also interesting is the observation that spore germination is inhibited as seen in Alternaria species (ascomycete) (Daigle and Cotty, 1991) and in dermatophytes such as Aspergillus spp. and Fusarium spp. (Nguyen et al., 1981; Pandey et al., 1984) and here in Pch and Pmi (Fig. 1C). This property may be useful for hindering the dissemination of the pathogen.

G. Roblin performed the experiments on pulvinar motor cells. S. Octave performed the experiments on plant necrosis and in part on the assays of the antifungal effect of cysteine. M. Faucher participated on the assays related to the transport of cysteine in grapevines. P. FleuratLessard realized the microscopic observations. J-M Berjeaud supervised the study. All the authors participated to the writing of the paper. Acknowledgments: We thank Janine Bonmort and Florence Thibault for technical help with this research. We would like to acknowledge Emile Béré for his kind assistance with microscopic observations and Dr. Estelle Luini for help with setting up assays on cysteine transport. The funding is partly granted by the following 2015–2020 programs: the State-Region Planning Contracts (CPER) and the European Regional Development Fund (FEDER). References Abe, T., 1981. Chloride ion efflux during an action potential in the main pulvinus of Mimosa pudica. Bot. Mag. Tokyo 94, 379–383. Álvarez, C., Calo, L., Romero, L.C., García, I., Gotor, C., 2010. An O-acetylserine(thiol) lyase homolog with L-cysteine desulfhydrase activity regulates cysteine homeostasis in Arabidopsis. Plant Physiol. 152, 656–669. Álvarez, C., Bermúdez, M.A., Romero, L.C., Gotor, C., García, I., 2012. Cysteine homeostasis plays an essential role in plant immunity. New Phytol. 193, 165–177. Amborabé, B.-E., Fleurat-Lessard, P., Bonmort, J., Roustan, J.-P., Roblin, G., 2001. Effects of eutypine, a toxin from Eutypa lata, on plant cell plasma membrane: possible subsequent implication in disease development. Plant Physiol. Biochem 39, 51–58. Amborabé, B.-E., Rossard, S., Pérault, J.-M., Roblin, G., 2003. Specific perception of ergosterol by plant cells. C. R. Biol. 326, 363–370. Amborabé, B.-E., Bonmort, J., Fleurat-Lessard, P., Roblin, G., 2008. Early events induced by chitosan on plant cells. J. Exp. Bot. 59, 2317–2324. Beffa, T., Pezet, R., Turian, G., 1987. Multiple-site inhibition by colloidal elemental sulfur (S0) of respiration by mitochondria from young dormant α spores of Phomopsis viticola. Physiol. Plantarum 69, 443–450. Bertsch, C., Ramírez-Suero, Magnin-Robert, M., Larignon, P., Chong, J., Abou-Mansour, E., Spagnolo, A., Clément, C., Fontaine, F., 2013. Grapevine trunk diseases: complex and still poorly understood. Plant Pathol. 62, 243–265. Bloem, E., Riemenschneider, A., Volker, J., Papenbrock, J., Schmidt, A., Salac, I., Haneklaus, S., Schnug, E., 2004. Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine desulphydrase activity in Brassica napus L. J. Exp. Bot. 55, 2305–2312. Bloem, E., Haneklaus, S., Schnug, E., 2005. Significance of sulfur compounds in the protection of plants against pests and diseases. J. Plant Nutr. 28, 763–784. Bloem, E., Haneklaus, S., Salac, I., Wickenhäuser, P., Schnug, E., 2007. Facts and fiction about sulfur metabolism in relation to plant-pathogen interactions. Plant Biol. 9, 596–607. Bolwell, G.P., Bindschedler, L.V., Blee, K.A., Butt, V.S., Davies, D.R., Gardner, S.L., Gerrish, C., Minibayeya, F., 2002. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J. Exp. Bot. 53, 1367–1376. Bonas, U., Schmitz, K., Rennenberg, H., Bergmann, L., 1982. Phloem transport of sulfur in Ricinus. Planta 155, 82–88. Bonnemain, J.-L., Roblin, G., Gaillochet, J., Fleurat-Lessard, P., 1978. Effets de l’acide abscissique et de la fusicoccine sur les réactions motrices des pulvinus du Cassia fasciculata Michx. et du Mimosa pudica L. C. R. Acad. Sci. Paris. Sér. D 286, 1681–1686. Boubakri, H., Wahab, M.A., Chong, J., Gertz, C., Gandoura, S., Mliki, A., Bertsch, C., Soustre-Gacougnolle, I., 2013. Methionine elicits H2O2 generation and defense gene expression in grapevine and reduces Plasmopara viticola infection. J. Plant Physiol.

5. Conclusion Taken together, the data presented here allow us to argue that Cys 87


G. Roblin et al.

Romero, L.C., Hoefgen, R., Gotor, C., Hesse, H., 2009. Analysis of cytosolic and plastidic serine acetyl-transferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis. Plant Cell Environ. 32, 349–367. Lemoine, R., Bourquin, S., Delrot, S., 1991. Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters. Physiol. Plantarum 82, 377–384. Leroux, P., 1994. Influence du pH, d’acides aminés et de diverses substances organiques sur la fongitoxicité du pyriméthanil, du glufosinate, du captafol, du cymoxanil et du fenpiclonil vis-à-vis de certaines souches de Botrytis cinerea. Agronomie 14, 541–554. Leustek, T., Sato, K., 1999. Sulfate transport and assimilation in plants. Plant Physiol. 1320, 637–643. López-Martín, C., Becana, M., Romero, L.C., Gotor, C., 2008a. Knocking out cytosolic cysteine synthesis compromises the antioxydant capacity of the cytosol to maintain discrete concentrations of hydrogen peroxide in Arabidopsis. Plant Physiol. 147, 562–572. López-Martín, C., Romero, L.C., Gotor, C., 2008b. Cytosolic cysteine in redox signaling. Plant Signal. Behav. 3, 880–881. Maughan, S., Foyer, C.H., 2006. Engineering and genetic approaches to modulating the glutathione network in plants. Physiol. Plantarum 126, 382–397. Mendoza-Cózalt, D.G., Butko, E., Springer, F., Torpey, J.W., Komives, E.A., Kehr, J., Schroeder, J.I., 2008. Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the longdistance transport of cadmium and the effect of cadmium on iron translocation. Plant J. 54, 249–259. Moran, N., 2007. Osmoregulation of leaf motor cells. FEBS Lett. 581, 2337–2347. Munkvold, G.P., Duthie, J.A., Marois, J.J., 1994. Reduction in yield and vegetative growth of grapevines due to Eutypa dieback. Phytopathology 84, 186–192. Nguyen, N.T., Galgóczy, J., Novák, E.K., 1981. Morphogenetic effect of L-cysteine on dermatophytes. Acta Microbiol. Acad. Sci. Hungar. 28, 347–357. Noctor, G., Mhamdi, A., Chaouch, S., Han, Y., Neukermans, J., Marquez-Garcia, B., Queval, G., Foyer, C.H., 2012. Glutathione in plants: an integrated overview. Plant Cell Environ. 35, 454–484. Octave, S., Amborabé, B.-E., Luini, E., Ferreira, T., Fleurat-Lessard, P., Roblin, G., 2005. Antifungal effects of cysteine towards Eutypa lata, a pathogen of vineyards. Plant Physiol. Biochem. 43, 1006–1013. Otsiogo-Oyabi, H., Roblin, G., 1984. Effects of glycine on dark- and light-induced pulvinar movements and modifications of proton fluxes in the pulvinus of Mimosa pudica during glycine uptake. Planta 161, 404–408. Otsiogo-Oyabi, H., Roblin, G., 1985. Changes in membrane potential related to glycine uptake in the motor cell of the pulvinus of Mimosa pudica. J. Plant Physiol. 119, 19–24. Pandey, D.K., Chandra, H., Tripathi, N.N., Dixit, S.N., 1984. Antimycotic activity of some amino acids against dermatophytes. Arzneimittelforschung 34, 554–556. Park, S., Imlay, J.A., 2003. High levels of intracellular cysteine promote oxidative DNA damage by driving the fentom reaction. J. Bacteriol. 185, 1942–1950. Pivato, M., Fabrega-Prats, M., Masi, A., 2014. Low-molecular-weight thiols in plants: functional and analytical implications. Arch. Biochem. Biophys. 560, 83–99. Rausch, T., Wachter, A., 2005. Sulfur metabolism: a versatile platform for launching defence operations. Trends Plant Sci. 10, 503–509. Rennenberg, H., Schmitz, K., Bergmann, L., 1979. Long-distance transport of sulfur in Nicotiana tabacum. Planta 147, 57–62. Resende, M.L.V., Flood, J., Ramsden, J.D., Rowan, M.G., Beale, M.H., Cooper, R.M., 1996. Novel phytoalexins including elemental sulphur in the resistance of cocoa (Theobroma cacao L.) to verticilium wilt (Verticilium dahliae Kleb.). Physiol. Mol. Plant Pathol. 48, 347–359. Roblin, G., Fleurat-Lessard, P., 1983. Dimethylsulfoxide action on dark- and light-induced leaflet movements and its necrotic effects on excised leaves of Cassia fasciculata. Physiol. Plantarum 58, 493–496. Roblin, G., Laduranty, J., Bonmort, J., Aidene, M., Chollet, J.-F., 2016. Unsaturated amino acids derived from isoleucine trigger early membrane effects on plant cells. Plant Physiol. Biochem. 107, 67–74. Romero, L.C., Aroca, M.A., Laureano-Marín, A.M., Moreno, I., García, I., Gotor, C., 2014. Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana. Mol. Plant 7, 264. Saeedi, S., Rocher, F., Bonmort, J., Fleurat-Lessard, P., Roblin, G., 2013. Early membrane events induced by salicylic acid in motor cells of the Mimosa pudica pulvinus. J. Exp. Bot. 64, 1829–1836. Samejima, M., Sibaoka, T., 1980. Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery. Plant Cell Physiol. 21, 467–479. Satter, R.L., Galston, A.W., 1981. Mechanisms of control of leaf movements. Annu. Rev. Plant Physiol. 32, 83–110. Schaller, A., Oecking, C., 1999. Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11, 263–272. Sekiya, J., Schmidt, A., Wilson, L.G., Filner, P., 1982. Emission of hydrogen sulfide by leaf tissue in response to L-cysteine. Plant Physiol. 70, 430–436. Sharma, S.S., Dietz, K.-J., 2006. The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J. Exp. Bot. 57, 711–726. Servaites, J.C., Schrader, L.E., Jung, D.M., 1979. Energy-dependent loading of amino acids and sucrose into the phloem of soybean. Plant Physiol. 64, 546–550. Takahashi, H., Kopriva, S., Giordano, M., Saito, K., Hell, R., 2011. Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes. Annu. Rev. Plant Biol. 62, 157–184.

170, 1561–1568. Cervone, F., Castoria, R., Leckie, F., De Lorenzo, G., 1997. Perception of fungal elicitors and signal transduction. In: Aducci, P. (Ed.), Signal Transduction in Plants. Birkhaüser Verlag, Basel, Switzerland, pp. 153–177. Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothionins : roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159–182. Colcombet, J., Mathieu, Y., Peyronnet, R., Agier, N., Lelièvre, F., Barbier-Brygoo, H., Frachisse, J.-M., 2009. R-type anion channel activation is an essential step for ROSdependent innate immune response in Arabidopsis suspension cell. Funct. Plant Biol. 36, 832–843. Cooper, R.M., Williams, J.S., 2004. Elemental sulfur as an induced antifungal substance in plant defence. J. Exp. Bot. 55, 1947–1953. Cooper, R.M., Resende, M.L.V., Flood, J., Rowan, M.G., Beale, M.H., Potter, U., 1996. Detection and cellular localization of elemental sulphur in disease-resistant genotypes of Theobroma cacao. Nature 379, 159–162. Daigle, D.J., Cotty, O.J., 1991. The influence of cysteine, cysteine analogs and other amino acids on spore germination of Alternaria species. J. Can. Bot. 69, 2353–2356. Delrot, S., Rochat, C., Tegeder, M., Frommer, W., 2001. Amino acid transport. In: Lea, P.J., Morot-Gaudry, J.-F. (Eds.), Plant Nitrogen. Springer and INRA Editions, pp. 213–235. De Lucca, A.J., Cleveland, T.E., Wedge, D.E., 2005. Plant-derived antifungal proteins and peptides. Can. J. Microbiol. 51, 1001–1014. Dixon, R.A., Harrison, M.J., Lamb, C.J., 1994. Early events in the activation of plant defense response. Annu. Rev. Phytopathol. 32, 479–501. Dixon, D.P., Lapthorn, A., Edwards, R., 2002. Plant glutathione transferases. Genome Biol. 3 Reviews3004.1. Droux, M., 2004. Sulfur assimilation and the role of sulfur in plant metabolism: a survey. Photosynth. Res. 79, 331–348. Dubos, B., Bugaret, Y., Bulit, J., Roudet, J., 1983. Maladies du bois: symptômes et méthodes de lutte. Phytoma 344, 16–19. Edwards, R., Dixon, D.P., Walbot, V., 2000. Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health. Trends Plant Sci. 5, 193–198. Felle, H.H., Zimmermann, M.R., 2007. Systemic signalling in barley through action potentials. Planta 226, 203–214. Fisahn, J., Herde, O., Willmitzer, L., Pena-Cortés, H., 2004. Analysis of the transient increase of the cytosolic Ca2+ during the action potential of higher plants with high temporal resolution: requirement of Ca2+ transients for induction of jasmonic acid biosynthesis and PINII gene expression. Plant Cell Physiol. 45, 456–459. Fischer, W.-N., André, B., Rentsch, D., Krolkiewicz, S., Tegeder, M., Breitkreuz, K., Frommer, W.B., 1998. Amino acid transport in plants. Trends Plant Sci. 3, 188–195. Fisher, E., Lüttge, U., 1980. Membrane potential changes related to active transport of glycine in Lemna gibba G1. Plant Physiol. 65, 1004–1008. Fleurat-Lessard, P., Bouché-Pillon, S., Leloup, C., Bonnemain, J.-L., 1997. Distribution and activity of the plasma membrane H+-ATPase in Mimosa pudica L. in relation to ionic fluxes and leaf movements. Plant Physiol. 113, 747–754. Fleurat-Lessard, P., Luini, E., Berjeaud, J.-M., Roblin, G., 2014. Immunological detection of Phaeoacremonium aleophilum, a fungal pathogen found in esca disease. Eur. J. Plant Pathol. 139, 137–150. Galgóczy, L., Homa, M., Papp, T., Manikandan, P., Vágvölgyi, C., 2016. In vitro antifungal activity of cysteine derivatives and their combinations with antifungal agents against clinically relevant Scedosporium species. Int. J. Clin. Med. Microbiol. 1, 111. http:// dx.doi.org/10.15344/ijcmm/2016/111. Gotor, C., Laureano-Marín, A.M., Moreno, I., Aroca, Á., García, I., Romero, L.C., 2015. Signaling in the plant cytosol: cysteine or sulfide? Amino Acids 47, 2155–2164. Granado, J., Felix, G., Boller, T., 1995. Perception of fungal sterols in plants. Subnanomolar concentrations of ergosterol elicit extracellular alkalinization in tomato cells. Plant Physiol. 107, 485–490. Haag, A.F., Kerscher, B., Dall’angelo, S., Sani, M., Longhi, R., Baloban, M., Wilson, H.M., Mergaert, P., Zanda, M., Ferguson, G.P., 2012. Role of cysteine residues and disulfide bonds in the activity of a legume root-nodule-specific, cysteine-rich peptide. J. Biol. Chem. 287, 10791–10798. Hagendoorn, M.J.M., Poortinga, A.M., Wong Fong Song, H.W., Van der Plas, L.H.W., Van Walraven, H.E., 1991. Effects of elicitors on the plasma membrane of Petunia hybrida cell suspensions: role of pH in signal transduction. Plant Physiol. 96, 1261–1267. Harrington, H.M., Smith, I.K., 1977. Cysteine transport into cultured tobacco cells. Plant Physiol. 60, 807–811. Hildebrandt, T.M., Nunes Nesi, A., Araújo, W.L., Braun, H.-P., 2015. Amino acid catabolism in plants. Mol. Plant 8, 1563–1579. Kahlos, K., Tikka, V.H., 1994. Antifungal activity of cysteine, its effect on C-21 oxygenated lanosterol derivatives and other lipids in Inonotus obliquus, in vitro. Appl. Microbiol. Biotechnol. 42, 385–390. Kasparovski, T., Milat, M.L., Humbert, C., Blein, J.-P., Havel, L., Mikes, V., 2003. Elicitation of tobacco cells with ergosterol activates a signal pathway including mobilization of internal calcium. Plant Physiol. Biochem. 41, 495–501. Katicheva, L., Sukhov, V., Akinchits, E., Vodeneev, V., 2014. Ionic nature of burn-induced variation potential in wheat leaves. Plant Cell Physiol. 55, 1511–1519. Kinraide, T.B., Etherton, B., 1980. Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles. Plant Physiol. 65, 1085–1089. Kinraide, T.B., Newman, I.A., Etherton, B., 1984. A quantitative simulation model for H+amino acid cotransport to interpret the effects of amino acids on membrane potential and extracellular pH. Plant Physiol. 76, 806–813. Klikocka, H., Haneklaus, S., Bloem, E., Schnug, E., 2005. Influence of sulfur fertilization on infection of potato tubers with Rhizoctonia solani and Streptomyces scabies. J. Plant Nutr. 28, 819–833. Krueger, S., Niehl, A., López-Martin, M.C., Steinhauser, D., Donath, A., Hildebrandt, T.,

88


G. Roblin et al.

mechanisms of generation and propagation. Plant Signal. Behav. 10 e1057365–3. Volkov, A.G., Foster, J.C., Ashby, T.A., Walker, R.K., Johnson, J.A., Markin, V.S., 2010. Mimosa pudica: electrical and mechanical stimulation of plant movements. Plant Cell Environ. 33, 163–173. Williams, J.S., Hall, S.A., Hawkesford, M.J., Beale, M.H., Cooper, R.M., 2002. Elemental sulphur and thiol accumulation in tomato and defense against a fungal vascular pathogen. Plant Physiol. 128, 150–159. Xiang, C., Werner, B.L., Christensen, E.M., Oliver, D.J., 2001. The biological functions of glutathione revisited in Arabidopsis transgenic plants with altered glutathione levels. Plant Physiol. 126, 564–574.

Tegeder, M., 2012. Transporters for amino acids in plant cells: some functionings and many unknowns. Curr. Opin. Plant Biol. 15, 315–321. Temmei, Y., Uchida, S., Hoshino, D., Kanzawa, N., Kuwahara, M., Sasaki, S., Tsuchiya, T., 2005. Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation. FEBS Lett. 579, 4417–4422. Tena, G., Renaudin, J., 1998. Cytosolic acidification but not auxin at physiological concentration is an activator of MAP kinases in tobacco cells. Plant J. 16, 173–182. Vatsa, P., Chiltz, A., Luini, E., Vandelle, E., Pugin, A., Roblin, G., 2011. Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells. Plant Physiol. Biochem. 49, 764–773. Vodeneev, V., Akinchits, E., Sukhov, V., 2015. Variation potential in higher plants:

89