JXB Advance Access originally published online on May 31, 2008
Journal of Experimental Botany 2008 59(9):2317-2324; doi:10.1093/jxb/ern096
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RESEARCH PAPER |
Early events induced by chitosan on plant cells
University of Poitiers, Laboratoire de Biochimie, Physiologie et Biologie Moléculaires Végétales, FRE CNRS 3091, 40, Avenue du Recteur Pineau, F-86022 Poitiers cedex, France
* To whom correspondence should be addressed. E-mail: gabriel.roblin{at}univ-poitiers.fr
Received 16 February 2008; Revised 7 March 2008 Accepted 10 March 2008
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Abstract |
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Chitosan (a polymer of β-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 µg 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 µg 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.
Key words: Chitosan, elicitor, H+-ATPase, membrane potential
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chitosan (β-1,4-linked glucosamine) is a deacetylated derivative of chitin found in the composition of cell walls of many fungi (Bartnicki-Garcia, 1968). From data in previous reports, two biological roles can be ascribed to this compound. First, at defined concentrations, it presents antifungal properties as shown by its inhibitory action on the mycelial growth of a number of pathogenic fungi, including root pathogens, such as Fusarium oxysporum and Pithium phanidermatum (Leuba and Stossel, 1986; Benhamou, 1992; El Ghaouth et al., 1994; El Hassni et al., 2004), and also by its inhibitory effect on spore germination (Hadwiger and Beckman, 1980). Secondly, it acts as a potent elicitor, therefore enhancing plant resistance against pathogens (Benhamou and Thériault, 1992; El Ghaouth et al., 1994). It is thus an effective inducer of phytoalexin synthesis in various plant cells (Hadwiger and Beckman, 1980; Köhle et al., 1984), and triggers callose formation (Köhle et al., 1985; Conrath et al., 1989), lignification responses in wounded wheat leaves (Pearce and Ride, 1982), and the production of proteinase inhibitors (Walker-Simmons et al., 1984; Pena-Cortes et al., 1988). To date, only the long-term induced events have been investigated. Therefore, the aim of this work was to focus attention on the early events possibly triggered by chitosan at the cell plasma membrane level. To this end, the modification of the bioelectrical transmembrane potential on the motor cell membrane of Mimosa pudica was monitored. In most cases, application of an elicitor triggers a rapid depolarization of the membrane (Mathieu et al., 1966; Katou et al., 1982; Pelissier et al., 1986; Mayer and Ziegler, 1988; Kuchitsu et al., 1993; Thain et al. 1995). This event is the macroscopic visualization of ion migrations, namely the influx of Ca2+ and H+ and the efflux of Cl– and K+ (Dixon et al., 1994; Cervone et al., 1997), resulting from activation of ion channels. In this regard, by measurement of pH variations in the bathing medium of pulvinar tissues and by observations of effects on plasma membrane vesicles (PMVs), it has been shown that a primary site of action of chitosan is the plasma membrane H+-ATPase. In addition, it was observed that at relatively high concentrations, chitosan inhibited H+-sustained nutrient uptake and K+-driven motor cell reactions. These results are discussed in the light of the cytotoxicity of the compound.
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Materials and methods |
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Plant materials
The sensitive plants (M. pudica L.) and Cassia fasciculata Michx. were grown in an organic compost watered daily. They were kept in climate-controlled chambers at 27.5±0.5 °C and 65±5% relative humidity. Illumination was regulated so as to give 16 h of light (photophase 06.00–22.00 h) provided by fluorescent tubes (mixing Osram fluora and Osram day-light types) with a photon flux density (400–700 nm) of 36 µmol m–2 s–1 at the plant apex. Plants of M. pudica and C. fasciculata were used when they bore 10 developed leaves.
Electrophysiological measurements
The transmembrane electrical potential was measured by the classical electrophysiological method using microelectrodes (tip diameter <1 µm, tip resistance from 5 to 30 M
). For details, see Moyen et al. (2007). Briefly, leaf was excised from the stem, and the pulvinus was fixed to the bottom of a 4 ml plexiglas chamber filled with a medium containing 1 mM NaCl, 0.1 mM KCl, and 0.1 mM CaCl2, buffered at pH 5.5 with 10 mM MES/NaOH (Abe, 1981). The glass microelectrode was impaled into a motor cell of the abaxial (‘extensor half’) of the pulvini. Under these conditions, the resting transmembrane potential averaged at –124±19 mV from 32 assays.
Measurement of pH variations
Primary pulvini [
400 mg fresh weight (FW)] were excised from leaves, divided in transverse sections, and treated as previously described (Moyen et al., 2007): unbuffered incubation medium composed of 0.50 mM CaCl2, 0.25 MgCl2, variations of pH in the incubation medium read on a pH-meter provided with a micro-combination electrode (Futura, Beckman Coulter) and linked to a potentiometric recorder. The experiments were repeated at least three times.
Preparation and use of PMVs
Purified PMVs were prepared by phase partitioning of microsomal fractions from the primary pulvini of M. pudica leaves according to Lemoine et al. (1991). Proton pumping and ATPase activity were measured as described in Noubahni et al. (1996) with some minor modifications. PMVs were put in the inside-out configuration by adding 0.05% brij in the assay medium (Vianello et al., 1982). In treated sets, chitosan was added at the given concentration 10 min before the beginning of the assay. Proton pumping activity was monitored as the decrease in the absorbance of acridine orange at 495 nm. The assay medium consisted of 50 mM KCl, 10 mM MES/TRIS (pH 6.5), 3 mM ATP, 1 mM dithiothreitol, 1 mg ml–1 bovine serum albumin, 20 µM acridine orange, 5 mM valinomycin, 300 mM sorbitol, and 75 µg of protein in a final volume of 1 ml. The reaction was started by addition of MgSO4 to a final concentration of 3 mM. ATPase hydrolysis activity of the PMVs was measured by the phosphate released from ATP by the method of Ames (1966) in a medium containing 3 mM MgSO4, 100 mM KCl, 50 mM MES/TRIS (pH 6.5), 3 mM ATP, 250 mM sucrose, and 40 µg of protein. After 5, 10, 15, and 20 min, 100 µl aliquots were sampled and mixed with the molybdate reagent which stops the reaction. In the six batches of vesicles used in this study, treatment with 0.25 mM sodium orthovanadate showed that 50–70% of the enzyme activity can be attributed to plasma membrane functioning.
PMVs either treated with chitosan as described above or non-treated (control) were fixed at room temperature (25 °C) in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 45 min. The next steps performed at 4 °C consisted of extensive washing in the buffer followed by post-fixation in 1% (v/v) osmium tetroxide for 5 min and dehydration in an increasing ethanol series. Embedding occurred in London Resin White (LRW). Polymerization was conducted at 60 °C for 24 h. Thin sections (80 µm thick) of these chemically fixed PMVs were contrasted in uranyl acetate and lead citrate, and were observed by transmission electron microscopy using a JEM 1010 Jeol microscope operating at 80 kV.
Uptake assay of sucrose and valine
Excised primary pulvini were divided into two parts by longitudinal sections and distributed so that 10 sections constitute an experimental set, four such sets being considered for each experimental point. The tissues were pre-incubated and incubated in a medium containing 0.5 mM CaCl2 and 0.25 mM MgCl2 buffered with 10 mM MES brought to pH 5.5 with 1 M NaOH. The pulvinar sections were rinsed in 20 ml of fresh medium for 1 h after excision and then transferred to a medium containing chitosan at the assayed concentration. After a defined incubation time, 10 sections were placed in the presence of 1 mM [14C]sucrose solution (final specific activity of the medium 11 kBq ml–1) and 1 mM [3H]valine solution (final specific activity of the medium 9.25 kBq ml–1). After incubation with the labelled substrate for 30 min, the apoplastic label was removed by three washes of 3 min each in the incubation medium. The sections were then dried, weighed, and digested in a mixture of H2O2, perchloric acid, Triton X-100 (2/1/1 by vol.) overnight at 55 °C. The radioactivity was then counted after addition of a scintillation medium (Ecolite, ICN) by liquid scintillation spectrometry (Packard Instr.).
Procedure for observation of dark- and light-induced turgor movements
The dark- and light- induced movements were observed on excised leaves of C. fasciculata. After excision, the cut petioles were dipped in distilled water for 1 h to recover. The leaves were then transferred to the assayed solution buffered at pH 5.5 with 2.5 mM MES/NaOH. The leaflet closure movements were induced by darkening the plants in the middle of the photophase and the opening movements were induced by lighting the plants in the middle of the night phase. The movements were followed by measuring the distance between the leaflets tips with a calliper square allowing the determination of angular values (Bonmort and Roblin, 1996). The dark- and light-induced movements observed on the C. fasciculata leaflets are similar to those triggered on M. pudica leaflets, but handling is easier since C. fasciculata is much less sensitive to mechanical shock.
Chemicals
All chemicals were purchased from Sigma-Aldrich Chimie, Saint-Quentin-fallavier, France. Chitosan from crab shells was prepared according to a method previously described by Young et al. (1982). A stock solution of fusicoccin (FC) at 1 mM final concentration was prepared in methanol (7% final concentration).
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Results |
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Electrophysiological modifications induced by chitosan
The addition of chitosan to the bathing medium of M. pudica pulvini caused a transient depolarization of the motor cell membrane in a concentration-dependent manner at concentrations >10 µg ml–1 and up to 100 µg ml–1. Figure 1 shows representative time courses of the bioelectrical events recorded in this range of concentrations: the depolarization occurred after a latency period (in the order of some minutes), decreasing as the applied concentration was increased, peaked at
10–15 min, and returned to its initial resting potential after 30–40 min even in the presence of chitosan. FC (10 µM) added at the end of each experiment triggered membrane hyperpolarization which ascertained the bioelectrical viability of the impaled cell. Quantitative results of this experiment are given in Table 1. Experiments conducted at 250 µg ml–1 did not give conclusive results since the motor cell reaction was so intense that the microelectrodes were expelled from the cells.
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Effect of chitosan on the H+-ATPase activity
Particular attention was paid to the proton-translocating ATPase on account of previous data showing that this enzyme is a characteristic target in the elicitation chain. Chitosan effects were therefore observed, first on the induced modification of proton fluxes in pulvinar tissues and, secondly, on the activity of the enzyme measured on PMVs.
As the result of proton pump activity, pulvinar tissues spontaneously acidified their incubation medium until an equilibrium near pH 5.0 was attained (Otsiogo-Oyabi and Roblin, 1984). Figure 2 shows representative time courses of the pH variations recorded in the bathing medium of pulvinar tissues after addition of chitosan at various concentrations (from 25 µg ml–1 to 250 µg ml–1). After application of chitosan, a transient concentration-dependent pH rise was clearly seen at concentrations >25 µg ml–1. The pH variation started after a latency period ranging from 30 s (250 µg ml–1) to 3 min (25 µg ml–1), peaked in the range 10 min (25 µg ml–1) to 15 min (250 µg ml–1), and returned to its initial value in 2 h even in the presence of chitosan. It has been verified that this effect was not due to a modification in the buffering capacity of the compound. These data suggest that the plasma membrane H+-ATPase may be inhibited by a chitosan treatment. In order to verify the hypothesis, proton pumping assays and determination of ATPase catalytic activity were performed following chitosan treatment of PMVs purified from primary pulvini tissues. As previously reported (Moyen et al., 2007), 10 µM FC, known to activate the vanadate-sensitive ATPase, activated both processes. In contrast, data in Table 2 show that the vanadate-sensitive ATPase activity of PMVs was inhibited by chitosan in a dose-dependent manner at concentrations >10 µg ml–1, a maximal inhibition of 50% being reached at concentrations >100 µg ml–1. It should be stressed that the proton pumping was more sensitive to chitosan application than the catalytic activity since it was inhibited at concentrations as low as 10 µg ml–1 and up to 50 µg ml–1. At higher concentrations, measurement were no longer possible, indicating that PMVs were damaged. This assumption was verified by electron microscope observations. Indeed, control PMVs appeared in cross-sections as circle-shaped elements (Fig. 3A, D) whereas PMVs treated with chitosan at 50 µg ml–1 and 250 µg ml–1 were deflated, but not disrupted (Fig. 3B, C, E). This modification therefore explained the absence of proton pumping activity since the stain was no longer able to be absorbed by the vesicles in this induced configuration.
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Subsequent effects on physiological membrane-related phenomena
Due to this inhibitory action on the plasma membrane H+-ATPase, different processes regulated by this enzyme are expected to be disturbed by treatment with chitosan. In order to verify this expectation, two models were chosen.
First, the effect of chitosan was observed on the membrane transport of assimilates. Indeed, as a consequence of the effect observed above on the H+-ATPase, the uptake of assimilates co-transported with H+ is expected to be hindered. From the data in Table 3, it appears that the dose-dependent inhibitory effect of chitosan was higher and more rapid on valine uptake than on sucrose uptake.
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Secondly, another type of observation was carried out on the effect of chitosan on the ion-driven osmocontractile reactions of pulvinar cells induced by variations in light regimes (dark- and light-induced movements). Following a period of darkness during the light phase of the photoperiodic cycle, the leaflets rapidly close as a consequence of a rapid turgor loss in the pulvinar motor cells. This turgor variation is driven by an efflux of K+ and Cl– (Satter and Galston, 1981) and an influx of protons (Otsiogo-Oyabi and Roblin, 1984). The light-induced movements are the result of inverse ion fluxes. Applied 3 h before subjecting the plants to darkness, chitosan at high concentration (250 µg ml–1) induced a partial closure of the leaflets observed after 1 h treatment. Angle variation calculated after 30 min of darkness showed that closure was unaffected at 50 µg ml–1 and enhanced by 26% at 100 µg ml–1 and 250 µg ml–1 (Fig. 4A). This result suggests that the underlying K+ efflux was activated. In contrast, the light-induced movements were strongly inhibited by the compound in a dose-dependent manner (20% at 100 µg ml–1, 50% at 250 µg ml–1) (Fig. 4B). These results suggest that the underlying, inwardly directed K+ influx was strongly inhibited.
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Discussion |
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The results of the present study show that a primary site of chitosan action is located at the plasma membrane and give additional data on the mode of action of the compound at this cellular site.
Chitosan affects physiological processes linked to the plasma membrane H+-ATPase activity
The early perception of chitosan by plant cells was shown by the transient dose-dependent membrane depolarization (Fig. 1) and the modification of the proton fluxes displayed by the observed transient pH rise in the bathing medium of pulvinar tissues (Fig. 2). The induced transmembrane potential variation and the H+ flux modification showed a close relationship in the latency period as well as in the peaking of the time courses in both processes, so that a causal link of H+ entry in the cells might correspond to the depolarization of the membrane. However, the electrical potential rapidly returned to its basal level whereas the H+ flux was more sustained, such that other compensatory ionic fluxes have to be considered to explain the discrepancy, either an efflux of cations or an influx of anions. The time course of pH variation also leads to the conclusion that H+ fluxes were disturbed in a prolonged way and therefore may intervene in the regulation of long-term processes. Experiments on PMVs demonstrated the direct action of the chitosan on the H+-ATPase plasma membrane, inducing the collapse of the proton motive force, subsequently impairing the energetics of the cells. As a consequence, it might be expected that the plasma membrane systems linked to the proton motive force would be impaired. Indeed, it was shown that application of chitosan led to the modification of the membrane transport of nutrients. However, note that valine uptake was more inhibited than sucrose uptake. This observation is in accordance with previous data showing that amino acid uptake was more sensitive to membrane depolarization and proton mobilization than sucrose uptake (Michonneau et al., 2004). The second illustrative model concerns the inhibitory effect observed on ion-sustained processes as illustrated by the modification of the H+/K+-regulated turgor motor cell reaction. It is noteworthy that the motor reactions driven by K+ efflux were poorly modified, whereas reactions driven by K+ influx were dramatically inhibited.
Chitosan effect on the plasma membrane H+-ATPase is a primary event in the elicitation chain
The above data on H+-ATPase activity have to be considered within the scope of the involvement of chitosan in plant-induced resistance. The defence processes which are induced by specific components released from the pathogen or host cell walls during the infection are characterized by several reactions including formation of mechanical barriers, activation of extracellular proteins, and production of phytoalexins with antibiotic properties (Bell, 1981, and references therein).
Chitosan has been shown to trigger several of these reactions. Thus, it elicited callose synthesis in soybean leaves (Köhle et al., 1985) and induced the lignification response in wheat leaves (Pearce and Rice, 1982). It is a powerful activator of characteristic enzyme activities, i.e. chitinase and β-1,3-glucanases (Romanazzi et al., 2002; Amborabé et al., 2004), and proteinase inhibitor in tomato (Walker-Simmons et al., 1984). It also activates the phenylpropanoid pathway by increasing the activity of phenylalanine ammonia lyase in potato suspension culture (Dornenburg and Kow, 1997) and in cotyledons of M. pudica (Rossard et al., 2006), leading to an accumulation of many phenolic compounds (Hadwiger and Beckman, 1980; Köhle et al., 1984; Reddy et al., 1999; Rossard et al., 2006). It has also been shown to intervene in upstream events by triggering the oxidative burst in relation to NADPH oxidase activation (Rossard et al., 2006), leading to the production of H2O2 (Kauss and Jeblick, 1996). In addition, chitosan also activates the octadecanoid pathway as shown by an increased level of jasmonic acid in rice (Rakwal et al., 2002) and the increased lipoxygenase activity in wheat (Rohland et al. 1997).
It should be stressed that all these reported events occurred after a time lapse of at least 30 min in the case of H2O2 production (Kauss and Jeblick, 1996). The present data give additional insight into the earlier events triggered by chitosan and complete the frame on the involvement of chitosan in elicitation processes. By inducing membrane depolarization, it acts in the same way as many other fungal elicitors (Mathieu et al., 1966; Katou et al., 1982; Pelissier et al., 1986; Mayer and Ziegler, 1988; Kuchitsu et al., 1993; Thain et al., 1995). Similarly, H+ influx was a characteristic reaction observed following application of various elicitors on different plant models (Dixon et al., 1994; Mathieu et al., 1996; Pugin et al., 1997). Such modification resulted from inhibition of H+-ATPase (Hagendoorn et al., 1991; Schaller and Oecking, 1999) playing the role of a switch between the elicitor recognition and the pathogen defence signalling pathways. The direct inhibitory effect reported here on H+-ATPase argues for this hypothesis.
Another step that should be addressed in the future concerns the characterization of a putative receptor. In a previous work, the possibility of such an interaction of chitosan with a receptor was discarded and its action was explained by an interaction of the compound with regularly spaced negative charges on the plasma membrane (Kauss et al., 1989). However, the desensitization process which occurred as well in H+ flux variation and electrophysiological events (Amborabé et al., 2003) constitutes a pertinent argument for the presence of a specific receptor. This hypothesis would also help towards an understanding of the fact that the membrane potential and pH value were restored to their initial state even in the presence of the compound. In this case, receptor sites would be saturated upon arrival of the chitosan molecules, leading to disruption of the signalling reaction, only restored by a new application of the compound after a refractory period as reported previously (Amborabé et al., 2003).
Chitosan induces cytotoxic effects
In some conditions, chitosan treatment resulted in the reduction and eventual loss of viability in both plant and fungal cells (Kendra and Hadwiger, 1987). This observation has prompted the evaluation of the cytotoxic effect of the compound that appeared related to the applied concentration.
In this line, two explanations can be envisaged. On the one hand, as an elicitor, chitosan may act through the hypersensitive response as shown by H2O2 synthesis observed in treated plant tissues (Kauss and Jeblick, 1996). However, this effect may be limited by considering the results of Lafontaine and Benhamou (1996) who noted no symptoms related to toxicity nor cytological alterations in their experimental conditions (the highest concentration used was 37.5 µg ml–1). In contrast, as reported in previous studies on plant material treated with higher concentrations (200 µg ml–1 and up to 1000 µg ml–1), chitosan destroyed cell organization (Köhle et al., 1984) and induced leakage of electrolytes, UV-absorbing material, and proteins (Young et al., 1982). It has been demonstrated that the leakage of this material was clearly due to the polycationic nature of chitosan which acts in a similar way to Triton X-100 (Young et al., 1982), resulting in the formation of large ‘pores’ in the membrane, explaining, therefore, the dose-dependent deflating effect seen on treated PMVs (Fig. 3).
Antifungal effects noted on in vitro assays on Botrytis cinerea (Amborabé et al., 2004) and on Fusarium oxysporum f. sp albedinis (El Hassni et al., 2004) may, therefore, be the result of a general cellular cytotoxic effect not restricted to fungal cells. As a consequence, in a practical approach, the evidence is that chitosan has to be used at concentrations that do not alter plant physiology and, according to the present results on PMV ATP-ase activity, it did not reach the plant cell membrane at concentrations higher than 100 µg ml–1. However, the actual concentration at this site is difficult to appreciate in planta from external treatment, since it depends on the absorption capacity, the transport into the treated leaves, and the binding capacity of the cell wall.
In conclusion, curative treatments using chitosan must be conducted with care since the increased permeability of host cells induced by the compound could facilitate infection by enabling fungal products to enter host cells. In contrast, the utilization of chitosan at concentrations that trigger elicitor action should be envisaged without restriction.
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