Journal of Experimental Botany, Vol. 51, No. 352, pp. 1799-1811,
November 1, 2000
© 2000 Oxford University Press
Original Papers |
The fungal elicitor cryptogein induces cell wall modifications on tobacco cell suspension
1 UMR 692, INRA-Université de Bourgogne, Laboratoire de Phytopharmacie et Biochimie des Interactions Cellulaires, BV 1540, 21034 Dijon cedex, France
2 INRA, Service Commun de Microscopie Electronique, BV 1540, 21034 Dijon cedex, France
3 IRD, GeneTrop Unité de Phytopathologie, BP 5045, 34032 Montpellier, France
4 Laboratoire de Phytoparasitologie, INRA/CNRS, Centre de Microbiologie du Sol et de l'Environnement, BV 1540, 21034 Dijon cedex, France
Received 3 February 2000; Accepted 27 June 2000
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Abstract |
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Upon addition of the fungal elicitor cryptogein, suspension cells of tobacco (Nicotiana tabacum cv. Xanthi) aggregated in clusters. Cytochemical experiments indicated that elicited cells displayed fibrillar expansions of pectin along the primary cell wall. Immunocytochemical detection of pectin epitopes indicated that the fibrillar material surrounding the treated cells was mostly composed of low methylated galacturonan sequences, but the use of the cationic probe did not reveal the presence of negatively charged carboxyl groups: the presence of important amounts of calcium ions in these pectic fibrillar expansions accounts for these observations. These data indicate that tobacco cells treated with cryptogein show a cell wall altered by the presence of a calcium pectate gel, resulting from the reorganization of pectin in the middle lamellae. These results are consistent with a drastic reduction in wall digestibility, partially reversed by increasing the pectolyase concentration in the hydrolytic solution. Diphenylene iodonium, an inhibitor of the oxidative burst triggered by cryptogein on tobacco cells, partially prevents elicited cell walls from this loss of digestibility, suggesting a possible role of active oxygen species in the cell wall strengthening. This work represents a new element of the signal transduction cascade triggered on tobacco cells by cryptogein.
Key words: Cell walls, cryptogein, pectic fibrillar expansions, Nicotiana tabacum, tobacco.
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Introduction |
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Plants are exposed to a great number of pathogenic microorganisms, but a relatively small proportion of them are able to invade plants and cause diseases. They defend themselves against pathogens by triggering a wide range of mechanisms, including the hypersensitive response (HR), which leads to lesions associated with cell death at infection sites, thus limiting pathogen growth to restricted areas (Keen, 1986
; Morel and Dangl, 1997). Mechanisms underlying the HR include (i) generation and recognition of extracellular signals, (ii) intracellular signalling, and (iii) activation of specific defence responses of targeted cells.
Elicitins, a family of low molecular weight proteins secreted by many species of the Oomycete Phytophthora (Ricci et al., 1989), induce an hypersensitive-like response in tobacco (Ricci et al., 1993), Raphanus (Keizer et al., 1998), and several Brassica species (Bonnet et al., 1996; Roussel et al., 1999). In order to understand the biochemical processes triggered by elicitins, the effects of cryptogein on tobacco cell suspensions have been studied for several years. The early events detected in cells following elicitin treatment include binding of the elicitor to a high affinity site on the plasma membrane, alkalinization of the extracellular medium (Blein et al., 1991), potassium and chloride efflux (Blein et al., 1991; Pugin et al., 1997), rapid and important calcium influx (Tavernier et al., 1995), and transient production of active oxygen species (AOS) (Rustérucci et al., 1996; Simon-Plas et al., 1997). Early changes in gene expression have also been reported a few minutes after addition of cryptogein to tobacco cells (Petitot et al., 1997; Suty et al., 1995, 1996). These responses are blocked by the protein kinase inhibitor staurosporine (Viard et al., 1994), indicating that phosphorylation is involved in the initial signal transduction; in parallel, activation of MAPK homologues was also demonstrated (Lebrun-Garcia et al., 1998; Zhang et al., 1998). Furthermore, it has been recently demonstrated that elicitins constitute a new class of extracellular sterol carrier proteins (Mikes et al., 1997, 1998) able to pick up sterols from purified plasma membranes or from tobacco cells (Vauthrin et al., 1999).
The data available in the literature concerning wall modifications of suspension cells following treatments with elicitors of defence reaction pointed out the oxidative cross-linking of proteins in walls of soybean suspension cells (Bradley et al., 1992; Brisson et al., 1994). Although accumulation of pectin material in vessels was recently evidenced by microscopy, following infiltration of cryptogein in cotyledons of B. napus (Roussel et al., 1999), little information is available on interaction between cell wall molecules and elicitins. It seemed thus interesting to examine whether changes might occur during the elicitation of tobacco cells by cryptogein.
The present paper focuses on tobacco cell wall modifications using immunocytochemical probes and shows that cryptogein treatment results in the reorganization of the middle lamellae as evidenced by the presence of pectic expansions rich in calcium.
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Materials and methods |
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Plant material
Tobacco cell suspension cultures (N. tabacum cv. Xanthi) were grown in Chandler's medium (Chandler et al., 1972
), at a constant temperature of 24 °C and a continuous illumination, on a rotary shaker (125 rpm). They were sampled for experiment in the exponential phase of growth (every 4 d, 25 ml of cells are subcultured in 100 ml of fresh medium).
Cells were elicited 4 d after subculture by the addition of cryptogein in aqueous solution (20 nM). Cryptogein, the fungal elicitor from Phytophthora cryptogea was purified (according to Ricci et al., 1989) and kindly provided by Dr M Ponchet (INRA, Antibes, France).
Determination of AOS production
H2O2 production was monitored in the culture medium of tobacco cells elicited or not with cryptogein, by chemiluminescence of luminol using a luminometer (B CL book). Every 10 min, a 250 µl aliquot of the medium was added to 50 µl of 0.3 mM luminol and 300 µl of assay buffer (175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, 150 mM MES, pH 6.5). Diphenylene iodonium (DPI) was purchased from Sigma, and added to the cell suspensions 5 min before cryptogein treatment.
Wall digestibility assay
Untreated and elicited cells at their maximal rate of AOS production were collected on a sintered glass, washed twice for 5 min in the culture medium pH 5.6, containing 0.4 M mannitol. One gram of filtered cells was suspended for incubation in 10 ml of protoplasting enzyme mixture containing 1.5% (w/v) of cellulase Y-C, 1% (w/v) of macerozyme and 0.1% (w/v) of pectolyase Y-23 (Onozuka, Seishin Pharmaceutical Co., Tokyo, Japan), in the culture medium supplemented with 0.4 M mannitol. This protoplast isolation procedure was adapted from basic conditions previously described for suspension-cultured cells (Brisson et al., 1994; Satiat-Jeunemaître et al., 1996). The cells were stirred (125 rpm) at 24 °C to prevent their sedimentation. At various times (5, 10, 30, 45, 60, 90 min), aliquots of cells (300 µl) were collected in microtubes, stored at 4 °C for sedimentation of the cells and washed three times with the culture medium to stop the enzymatic digestion. Moreover, different concentrations (0.2, 0.5 and 1% (w/v)) of pectolyase Y-23 were tested in the enzymatic mixture. A 0.1% aqueous solution of Calcofluor White (Fluorescent Brightener 28, Sigma) was used to label the cell walls (Hugues and McCully, 1975). Protoplasts were distinguished from intact cells by their spherical shape and lack of Calcofluor White staining. The protoplast/cell ratio was evaluated under microscope (DMRB, Leica) using bright field or epifluorescence microscopy with a Leica A filter set: excitation 340–380 nm, barrier filter 425 nm. Four experiments were achieved for each enzymatic treatment. For each sampling, about 1000 cells were analysed.
Histochemistry
Untreated or elicited suspension cells at their maximal rate of AOS production were collected by filtration. Packed cells were deposited in polypropylene molds (Histomold, Leica), previously filled with Tissue-Tek OCT embedding medium (Miles, Bayer Diagnostics, France). Cells were frozen by diving the molds in isopentane cooled by liquid nitrogen, and 20 µm thick sections were cut at -18 °C on a Reichert 1800 Cryostat.
Sectioned cell wall fragments were stained using different reagents, and were examined under a light microscope (DMRB, Leica).
Pectic compounds were visualized after incubation in aqueous solution of ruthenium red (1/5000) (Sigma) (Jensen, 1962). Phenolic compounds were detected by their autofluorescence under UV illumination using the Leica A filter. Lignin and suberin stainings were performed with the phloroglucinol–HCl reagent (Prolabo) (lignin red) and the Black Sudan (Sigma) (suberin black) in alcoholic solution, respectively (Jensen, 1962). Detection of callose in cell walls was performed under UV light after staining with 0.05% (w/v) aniline blue (Merck) in 50% (v/v) ethanol (bright fluorescence) (O'Brien and McCully, 1981).
Preparation of specimens for transmission electron microscopy
Suspension cells elicited with cryptogein, or untreated cells, were harvested by filtration as described above, washed with 0.1 M sodium phosphate buffer pH 7.2, and suspended in a fixative mixture. Throughout the two protocols described below, cells were allowed to sediment between each step.
Aliquots of cells were fixed in 0.1 M buffered sodium phosphate–3% glutaraldehyde–2% paraformaldehyde (pH 7.2) for 16 h at 4 °C, post-fixed with 1% osmium tetroxide for 1 h at 4 °C and embedded in Epon (TAAB, England) according to the usual procedure (Luft, 1961).
Alternatively, cells were fixed in 0.1 M phosphate buffer (pH 7.2) containing 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde for 2 h at 4 °C. After fixation, cells were washed several times in the same buffer over a period of 1 h. To block the non-specific attachment of antibodies to free aldehyde groups for indirect immunocytochemical labelling, cells were then treated with 50 mM ammonium chloride in 10 mM phosphate buffer for 30 min. After dehydration in an ethanol series, cells were progressively infiltrated in LR White Medium Grade (Oxford Instruments, Orsay, France) in a Reichert AFS (Automatic Freeze substitution System) according to a Progressive Low Temperature protocol at -19 °C (Vanden Bosch, 1991). Resin, to which 0.5% benzoin methyl ether (Sigma) was added, was polymerized for 48 h at -19 °C, then 3 h at 10 °C with UV light.
Ultra-thin sections (90 nm) were cut on an ultramicrotome (Reichert, Ultracut E) and observations were made with a Hitachi H600 transmission electron microscope operating at 75 kV.
Immunocytochemistry
Ultra-thin sections from cells embedded in LR White were collected on formvar-coated nickel grids and then successively incubated as follows for pectin localization: 30 min with 50 mM Tris-HCl buffer pH 7.4 with 0.9% (w/v) NaCl (TBS), containing 1% (w/v) dried milk and 10% (v/v) normal goat serum, then 1 h at 37 °C, with the monoclonal antibody (JIM 5 diluted 1/5 or JIM 7 diluted 1/10, in TBS-1% (w/v) dried milk) (Knox et al., 1990). After washes in the same buffer, the grids were incubated 1 h at room temperature with a goat secondary immunoglobulin G conjugate (EM GAT-15 BioCell Research Laboratory, Cardiff, UK), labelled to 15 nm colloidal particles and diluted in TBS (pH 8.2)-1% (w/v) dried milk (dilution 1/25). The grids were then rinsed 5x10 min in the same buffer pH 8.2 and 3x10 min in distilled water and counterstained with 1% (w/v) aqueous uranyl acetate for 10 min.
Specificity of labelling was assessed through the following control experiments: (i) omission of the primary antibody incubation step, (ii) incubation with the antiserum previously adsorbed with the corresponding antigen for 3 h at room temperature: JIM 5 diluted 1/5 in a solution of 1 mg of polygalacturonic acid per ml, JIM 7 diluted 1/10 in a solution of 1 mg of pectin per ml.
Cytochemistry
PATAg-based polysaccharide visualization:
Detection of polysaccharides with the PATAg method (periodic acid, thiocarbohydrazide, silver proteinate) was performed on ultra-thin sections from untreated or elicited cells embedded in Epon mounted on gold grids as previously described (Roland and Vian, 1991).
Enzyme-gold labelling of ß-1,4-glucans:
An exoglucanase-gold complex (15 nm in diameter) with affinity for ß-(1,4)-D-glucans was prepared (as described by Benhamou et al., 1987). The enzyme (provided by C Breuil, University of British Columbia, Canada) was used to detect cellulosic material. Thin sections of Epon-embedded cells, collected on formvar-coated nickel grids, were floated on a drop of 10 mM phosphate-buffered saline (PBS), pH 6.5, containing 0.02% (w/v) polyethylene glycol (PEG 20 000, Sigma) for 10 min and then incubated with the enzyme-gold complex diluted 10-fold in PBS-PEG for 20 min (Nicole and Benhamou, 1991). After several washes in PBS and distilled water, the sections were stained with uranyl acetate (Valentines, 1961). Control was carried out incubating the sections with the exoglucanase-gold complex previously adsorbed with 5 mg ml-1 of commercial substrate, ß-(1,4)-D-glucans from barley.
In muro detection and quantitative analysis of anionic site:
Ultra-thin sections from LR White embedded untreated and elicited cells were collected on gold grids and labelled with a cationic gold probe to detect anionic sites mainly borne by unesterified pectins not bound to calcium ions (Skutelsky and Roth, 1986). Sections were treated with 3% (v/v) acetic acid at pH 2.6 for 15 min at room temperature then incubated with a 10 nm cationic poly-L-lysine colloidal gold complex (BioCell) diluted 300-fold in 3% (v/v) acetic acid for 1 h at room temperature (Roy et al., 1994). After rinsing in 3% (v/v) acetic acid and distilled water, sections were stained with 1% (w/v) aqueous uranyl acetate.
Control experiments were performed by preincubating the sections, prior to labelling, in (i) a 1 mg ml-1 solution of poly-L-Lysine for 1 h at room temperature, (ii) a proteinase K solution at 1 mg ml-1 in 50 mM TBS, pH 8.0 for 3 h at room temperature. Labelling was also carried out with the cationic gold probe on (i) NaOH-demethylated samples: demethylation of pectic polysaccharides was performed in a blockwise manner, during dehydratation steps, by bathing the fixed cells in a solution of NaOH 0.1 M in 60% ethanol (v/v) at 4 °C overnight and (ii) samples subjected to a chelation of calcium cations bound to uronic acids without solubilization of pectic polymers: cells were bathed during dehydration series in a 60% (v/v) alcoholic solution of EGTA-Na2 (0.5%) at 4 °C (His et al., 1997).
Density of labelling (D) over sections of both untreated or elicited cells was compared by determining the number of gold particles per µm2±standard error. The density of labelling was calculated as D=number of gold particles/primary cell wall area surface. For untreated and elicited cells, 15 microphotographs, taken from three randomly sections made on each of five blocks, were recorded and analysed with an image processing system (Samba, TITN-Alcatel, Grenoble, France). Images were digitized with a JVC video camera coupled to a Matrox MVP/AT grabber card, performing 512x512 pixel images.
Calcium cytolocalization:
Untreated or elicited cells were subjected to a potassium-pyroantimonate precipitation method, commonly used for calcium localization (Jauneau et al., 1997; Mentré and Halpern, 1988). Briefly, cells were incubated in a solution of 4 ml 5% (w/v) potassium pyroantimonate (Sigma), 1 ml 10% (w/v) paraformaldehyde and 1% (w/v) phenol, pH 7.5 (adjusted with acetic acid) for 3 h at room temperature. After rinsing, cells were treated according to the protocole described above for the Epon embedding and polymerization steps.
Cell wall preparation
The method was adapted from that described by Fry (Fry, 1988). Cell walls were prepared from untreated or elicited tobacco cells. Cells were filtered, and approximately 23 g of cells were frozen and ground in liquid N2. The cell powder was homogenized in 100 ml of buffer A (20 mM HEPES pH 7.5 with 1% (w/v) sodium deoxycholate, 5 mM sodium metabisulphite and some drops of n-octanol) for 3 min with a Polytron homogenizer. After filtration of this homogenate on muslin in a sintered glass, the residue was washed with 50 ml of the buffer B (20 mM HEPES pH 7.5 additionned with 0.5% (w/v) sodium deoxycholate and 3 mM sodium metabisulphite) and incubated in 100 ml of the buffer B, overnight at 4 °C. After filtration, the residue, corresponding to the crude cell walls, was washed twice with 100 ml of distilled water and stirred for 2 h at room temperature in 50 ml of phenol : acetic acid : water (2 : 1 : 0.5, w/v/v). To eliminate phenol, cell walls were washed with ethanol 70% (v/v). Cell wall material was then lyophilized and weighed.
Pectin extractions
The method was adapted from that described by Gross (Gross, 1984). Anhydrous cell wall material was resuspended in 100 ml of buffer C (50 mM sodium acetate and 50 mM CDTA: cyclohexanediaminetetraacetic acid pH 6.5) and incubated for 6 h at room temperature. This solution was filtered and washed with 25 ml buffer C. The filtrate was dialysed (Spectra/Por1; 6000 to 8000 MWCO) for 72 h at 4 °C against distilled water (three changes), then lyophilized for quantification of ionically-associated pectin. The residue remaining after filtration was used to extract the covalently-bound pectins. It was incubated with stirring in 40 ml of buffer D (50 mM sodium carbonate and 2 mM CDTA), overnight at 4 °C and 1 h at 25 °C. This suspension was filtered and washed with 25 ml of buffer D. The filtrate, containing covalently-bound pectins, was neutralized to pH 6.5 with glacial acetic acid (temperature kept below 35 °C), before being dialysed as described above and lyophilized for quantification of covalently-bound pectins.
Quantitation of pectins
This method was adapted from that described by Taylor (Taylor, 1993). The lyophilized samples (ionically-associated pectin and covalently-bound pectins) were resuspended in distilled water. Aliquots of successive dilutions of each sample (100 µl) were placed in borosilicated tubes. Concentrated sulphuric acid (1.5 ml) and 50 µl of carbazole reageant (0.1% (w/v) in ethanol 100%) were then added. The tubes were mixed and incubated for 60 min at 60 °C. After cooling, the absorbance of pink-red samples were read at 530 nm. Standards of 0–50 µg of polygalacturonic and galacturonic acids were treated in parallel.
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Results |
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The addition of the fungal elicitor, cryptogein (20 nM), to the tobacco cell-suspension medium induces the transient production of hydrogen peroxide (Fig. 1
). The inhibitor of mammalian phagocyte oxidase, diphenylene iodonium (10 µM), added 5 min before the elicitor, completely abolished this AOS production (Fig. 1). For all experiments described in this paper, cells were sampled at the maximal rate of AOS production, i.e. 90 min after cryptogein treatment.
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) or DPI+cryptogein (
) were added or not (
) to the cell suspension. Every 10 min, AOS production was determined by chemiluminescence as described in Materials and methods. The arrow indicates at what time of the elicitation process the samples were harvested and subsequently analysed with experiments described in Figs 2.Histochemical and cytochemical characterization of cell wall polysaccharides
Tobacco suspension cells observed by light microscopy appeared single-layered on microscope slides or organized in small clusters (Fig. 2A
). After cryptogein treatment, tobacco cells aggregated in clumps of cells joined together by non-translucent material (Fig. 2B). Histochemical, cytochemical and immunocytochemical characterization of control and elicited cell wall polysaccharides was performed in order to understand the nature of this alteration.
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Histochemical characterization of cell walls:
Histochemical investigations were performed on cryosections of untreated or elicited tobacco cells in culture medium. Following ruthenium red staining for pectin detection by bright field microscopy, walls of control cells were surrounded by a regular red layer. Most of the elicited cells displayed a red-stained fibrillar material, heterogeneous in thickness, visualized along the primary cell walls (data not shown), indicating the presence of pectin-like polymer. Attempts to identify lignin, suberin, phenolic compounds, and callose in primary cell walls histochemically provided identical results both with control and elicited cells (data not shown).
Cytochemical detection of polysaccharides by the PATAg procedure:
A positive reaction was observed on middle lamellae and walls of both untreated (Fig. 2C) and elicited cells (Fig. 2D) after staining of thin sections according to the PATAg procedure. However, the fibrillar material, 1–3 µm in thickness, that bordered the elicited cells and was closely associated with the primary cell wall (Fig. 2D), also showed a positive reaction. This indicates the polysaccharide nature of this material.
Cytochemical localization of ß-(1,4)-D-glucans:
After labelling of sections with the exoglucanase-gold probe, gold particles were found to be evenly distributed over the primary cell wall of untreated and cryptogein-elicited cells. In contrast, weak labelling was observed over the middle lamellae and the fibrillar material of elicited cells (data not shown). In both control and elicited cells, when the gold complex was incubated previously with ß-(1,4)-D-glucans from barley, only scattered gold particles occurred on the primary cell wall (data not shown).
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) or 1% (
) were used. (C) Effect of diphenylene iodonium (DPI, 10 µM) on protoplast release from control and elicited cells: (•) control cells, (
Anionic sites and Ca2+ distribution in cell wall
In muro detection of anionic sites: The cationic gold probe was used to detect negatively charged groups, e.g. COO- of pectic polymers. In both untreated or elicited cells, gold particles were uniformly distributed over the primary cell wall (Fig. 3A, B). Quantification of labelling did not reveal any significant differences in the averages of gold particles per µm2 (245±50 for control cells and 263±55 for elicited cells). On the contrary, middle lamellae of control cells and fibrillar pectic expansion of cryptogein-treated cells were almost free of labelling (Fig. 3A, B). To assess labelling specificity, preincubation of sections with unlabelled poly-L-lysine prior to treatment with the cationic gold probe resulted in a significant loss of labelling (Fig. 3C).
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Treatment with proteinase K had no significant effect on the distribution of gold particles (278±16 µm2), indicating that labelling did not result from gold-complex aspecific binding with cell wall proteins. When a chemical demethylation of embedded cells was performed, a slight increase in labelling density was noticed (341±20 µm2), confirming that methyl esterification blocked anionic binding sites in the primary cell walls. Conversely, in these experimental conditions, no labelling was observed on the fibrillar expansion of elicited cells. When sections were incubated with EGTA, a calcium chelator, density of labelling slightly increased over the primary cell walls (348±22 µm2). In these conditions, gold particles were visualized on the pectic expansion (Fig. 3D
), suggesting the presence of calcium.
Cytolocalization of calcium:
When cell suspensions were submitted to the pyroantimonate precipitation method, calcium pyroantimonate appeared as electron-dense precipitates when observed with the electron microscope. Opaque deposits with variable size were seen in the middle lamellae of both types of cells (Fig. 4A, B), and abundantly observed in the fibrillar pectic material of cryptogein-treated cells (Fig. 4B).
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Modifications of pectic polysaccharides
Immunocytochemical detection of pectin epitopes:
When indirect immunolabelling was performed on sections of untreated cells with the JIM 5 anti-pectin monoclonal antibody, which recognizes epitopes of low methylated galacturonan sequences (0–50% esterification), gold decoration was irregular over the primary cell walls. Gold particles were evenly distributed over the middle lamellae (Fig. 5A). The pattern of labelling on primary cell walls was similar on sections of control and cryptogein-elicited cells (Fig. 5B). However, the fibrillar material surrounding the treated cells exhibited an intense labelling mostly localized at the external side of the middle lamellae (Fig. 5B).
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After the use of JIM 7 monoclonal antibody which targets highly methylesterified pectins (35–90% esterification), gold particles were irregularly distributed over the primary cell walls of both untreated or elicited cells (Fig. 5C
, D). The fibrillar expansion of elicited cells (Fig. 5D) and middle lamellae of both types of cells exhibited a weak labelling. When sections were incubated with JIM 5 or JIM 7 antibodies, preadsorbed with a solution of specific substrate, an important reduction of labelling was observed.
Extraction and quantification of pectic polysaccharides:
Quantitative and qualitative changes in cell wall polysaccharides have been investigated. Dry matters of entire cell wall preparations which were measured after freeze-drying, were not significantly different as indicated in Table 1. Extractions of pectic polysaccharides of untreated or elicited suspension cells were performed on cell wall preparations; two successive soluble fractions were obtained (i) ionically-associated pectic polysaccharides and (ii) covalently-bound pectic polysaccharides. The yield of pectins extracted from control or elicited cell walls was evaluated by determining the quantity of galacturonic acid contained in each fraction (Table 1). The total amount of pectic polysaccharides obtained from 1 g of cell wall (about 100 mg) is comparable with results previously reported for several species (Jarvis, 1982). No significant differences could be observed in total amount of pectic compounds in preparations from untreated (13.4±1.6 mg) or cryptogein-treated cells (12.8±1.6 mg). However, the comparative yields of each kind of pectic material showed a modification of their proportion. In one hand, changes were noted for ionically-associated pectins which increased from 54.5±3.1% for untreated cells to 70.2±2.1% for elicited cells. On the other hand, the quantity of covalently-bound pectins decreased from 45.5±3.1% for untreated cells to 29.8±2.1% for elicited cells.
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Changes in the digestibility of elicited tobacco cells
To determine whether the pectic fibrillar proliferations associated with the elicited primary cell walls may modify cell resistance to enzymatic digestion, protoplast release from control or elicited cell suspensions was monitored. Kinetics of digestion of control tobacco cell walls with a microbial hydrolytic enzymes mixture, containing 1.5% (w/v) cellulase, 1% (w/v) macerozyme, 0.1% (w/v) pectolyase are illustrated in Fig. 6A. The protoplast/cell ratio was evaluated at 16±5% and 35±2% after 5 min and 30 min digestion, respectively, then regularly increased and reached a maximum of 77±6% after 90 min digestion.
On cryptogein-treated cells, a drastic reduction in wall digestibility was recorded. From 5 to 30 min of enzyme digestion of elicited cell suspensions, the number of spherical protoplasts increased from 3.6±1.5% to 12.8±2.5%. Nevertheless, from 30 to 90 min of enzyme treatment, aggregated elicited cells, exhibiting retention of Calcofluor White staining, appeared refractory to digestion. Thus, the protoplast release was inhibited and a maximum protoplast/cell ratio of 13% was recorded all along the experiment. In order to investigate the relationship between pectic expansion of the elicited cells and reduction of their cell wall digestibility, further enzymatic digestions were performed using combinations of enzyme mixtures containing 0.1, 0.2, 0.5 or 1% (w/v) of pectolyase. Results showed that increasing the pectolyase concentration in the hydrolytic solutions led to an increase of the digestibility of elicited cell walls (Fig. 6B). The maximum protoplast release, obtained with 0.5% or 1% of pectolyase, was estimated at 56±4% and 85±5%, respectively, after 30 min of treatment. However, estimations of the protoplast/cell ratios were limited to 30 min of digestion, since an important lysis of protoplasts was observed beyond this laps of time.
The effect of diphenylene iodonium (DPI), an inhibitor of AOS production, on cell wall digestibility was also investigated. Whereas DPI alone did not significantly affect the digestibility of control cell walls, this compound prevented the strengthening of elicited cell walls (Fig. 6C): up to 30 min of enzyme treatment, the protoplast release was similar for control and elicited cells in the presence of DPI (35±2% and 30±2%, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Aggregation of tobacco cells in clusters, 90 min after addition of the fungal elicitor, cryptogein, in the culture medium, was associated with a drastic reduction in wall digestibility by a standard cocktail of hydrolytic enzymes. This phenomenon was partially reversed by increasing the pectolyase concentration in the hydrolytic solution. Cytochemical experiments also indicated that elicited cells showed fibrillar expansion of pectin attached to the primary cell wall, whereas untreated cells did not. Dry matter content of entire cell wall preparations of elicited cells were not significantly different from those of control cells, and the yield of total pectins extracted from control or elicited cell walls was similar. However, in elicited cells, the proportion of ionically-associated pectins increased whereas the quantity of covalently-bound pectin decreased. Immunocytochemical detection of pectin epitopes also indicated that the fibrillar material surrounding the treated cells was mostly composed of low methylated galacturonan sequences, although the further use of a cationic probe did not reveal the presence of negatively charged groups COO-. The presence of important amounts of calcium ions in these pectic fibrillar expansions accounted for these observations. In contrast, no apparent changes in callose or cellulose content was detected. Taken as a whole, these results indicate that tobacco cells treated with cryptogein show a cell wall modified by the presence of a calcium pectate gel, resulting from the reorganization of pectin in the middle lamellae. This represents a new phenomenon described among the early events previously reported on tobacco cells after cryptogein addition, which concerns the interface between cells and the extracellular medium.
Since the total amount of pectins is similar in both control and elicited cell walls, it can be assumed that the ionically associated pectins, very abundant in the fibrillar expansions of elicited cells, do not result from a de novo synthesis. These pectins could arise from demethylation of highly methylated polygalacturonans present in the cell wall by cell-wall-bound pectin methylesterases (PME; EC 3.1.1.11). These enzymes, widely distributed in plants, specifically hydrolyse methyl ester groups in the C6 position of galacturonic acids (Christensen et al., 1998), and can be involved in various physiological processes such as pathogenesis, fruit maturation and growth (Bordenave et al., 1996). Further, it is well known that pectin methyl esterases are highly pH regulated and that alkaline pH favours their activity, maximal at a pH close to 8 (Moustacas et al., 1986). As an alkalinization of the extracellular medium is classically observed following addition of cryptogein to a tobacco cell suspension (Blein et al., 1991), the elicitation process might create local conditions favourable to a modification of the ratio between methylated and acidic pectins in favour of the latters.
The fact that important amounts of calcium are found in the fibrillar pectic material of elicited cells is also an interesting feature. First, the association of low methylated pectins and calcium ions has already been reported by several authors (Guglielmino et al., 1997; Jarvis, 1982; Jauneau et al., 1997) and an increase in calcium-pectin binding has been shown to be correlated with an increase of unesterified pectins amount (Goldberg et al., 1996; Jarvis, 1984). Calcium ions are involved in the formation of intermolecular associations between different pectin chains containing blocks of at least nine successive carboxyl groups (Liners and Van Cutsem, 1992) leading to the formation of a gel-like matrix. Among defence responses that plants produce to react to pathogenic infection, reinforcement of cell walls close to the pathogen is likely one of the most efficient reaction to restrict host tissue colonization (Hammond-Kosack and Jones, 1996). A wide range of molecules have been shown to be involved in wall cross-linking, including lignin, suberin, callose or HRGP (Matern et al., 1995). But the role that pectic gels have in the wall consolidation remains unclear, although they have been reported to plug vessels in order to slow down or stop the progression of vascular microbes (Rioux et al., 1998). The so-called gels produced in response to pathological or physiological stimulation originated from vessel-associated parenchyma cells. Interestingly, a recent study paralleled responses of rapeseed cotyledons in fungal-induced HR, versus cryptogein-elicited necrotic lesions (Roussel et al., 1999), showing that both biotic and abiotic stresses elicit vessel obstruction by pectic gels. Cross-linking by calcium was suggested to reinforce pectins in the HR areas, increasing gel resistance to polygalacturonases secreted by many bacteria and fungi during the first phase of host–pathogen interaction (Messiaen and Van Cutsem, 1994). So, pectin rearrangement in walls of tobacco suspension cells, triggered by cryptogein, revealed close similarity with physiological processes induced in planta by this elicitor. Nevertheless, another role of these calcium-bound pectins should be examined. Indeed, pectin is considered not only as a structural component but also as the main active plant cell wall polymer in terms of signalling. In particular, pectin fragments of a determined length and a precise conformation induced by calcium ions, bind to high affinity sites on the plasmalemma and trigger the activation of plant defence responses (Messiaen and Van Cutsem, 1999). So, it could be suggested that these fibrillar expansions observed in tobacco cells elicited with cryptogein could act as ‘secondary elicitors’ for neighbouring cells, all the more that they appeared to come away from the cell wall and diffuse in the extracellular medium. The plausibility of this hypothesis remains to be tested.
The formation of this pectate–calcium matrix in elicited cells is associated with a drastic reduction of cell wall digestibility by a cocktail of hydrolytic enzymes (Fig. 6A). Furthermore, the use of DPI, an inhibitor of AOS production (Simon-Plas et al., 1997), prevented this strengthening of elicited cell walls. Indeed, during the first 30 min of enzymatic treatment, the quantity of protoplasts released from elicited cells in the presence of DPI, and from control cells, is similar (about 30%), whereas only 14% of protoplasts are obtained from elicited cells, in the absence of DPI. This suggests that AOS could play a role in the strengthening of tobacco cell walls elicited by cryptogein. Following elicitation of soybean cells, the oxidative cross-linking of two cell wall structural proteins leading to a reduced digestibility by hydrolytic enzymes was described (Brisson et al., 1994). However, they indicated that they could not rule out contributions from the oxidative cross-linking of other pre-existing cell wall polymers such as pectins. Last, an important accumulation of hydrogen peroxide has been microscopically evidenced within the pectic fibrillar expansions of elicited cells (data not shown). This is in agreement with the hypothesis of a direct intervention of AOS on cell wall reinforcement, possibly via cross-linking of phenolic residues present on pectins.
In conclusion, the work, reported in this paper, provides an additional contribution to the understanding of cryptogein effects on tobacco cells. The convergency of the results obtained here with some previously demonstrated in planta confirms the use of suspension cells as a functional tool to elucidate signal transduction steps responsible for physiological processes occurring on the whole plant. These elements of signal transduction cascade triggered on tobacco cells by cryptogein, are tentatively summarized in Fig. 7. Indeed the important amount of calcium ions localized within the pectic fibrillar expansion of elicited cells opens new fields of investigation concerning the analysis of the signal transduction cascade triggered by cryptogein. It has already been demonstrated, using 45Ca, that cryptogein triggered a drastic augmentation of the radioactive calcium associated with tobacco suspension cells (Tavernier et al., 1995). Although the amounts of calcium detected that way were very important (about 100 nmol g-1 of cells), they were interpreted as an influx of calcium inside the cells, necessary to initiate the signal transduction pathway leading to tobacco cell responses. The data presented here suggest that part of the calcium associated with elicited tobacco cells may be present in the cell wall. Further experiments are necessary to elicit the precise origin of this calcium in order to assess its role in the signal transduction pathway involved in elicitation processes. Moreover, preliminary experiments suggest that AOS production could be, either an intermediate in this signal transduction pathway leading to cell wall modifications, or directly involved in cell wall strengthening via an oxidative cross-linking which may be an integral component of the hypersensitive response.
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Acknowledgments |
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We thank JP Knox (John Innes Centre, Norwich, UK) for the gift of JIM 5 and JIM 7 antibodies, M Ponchet (INRA, Antibes, France) for the gift of cryptogein, A Jauneau (IFR 40-CNRS, Castanet-Tolosan, France) for his help in calcium localization, D Dubois for excellent technical assistance, and C Schneider for the quantitative analysis of labelling with the cationic gold probe. This work was supported by INRA, the Caisse Régionale du Crédit Agricole Mutuel de Côte d’Or and the Conseil Régional de Bourgogne.
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5 These authors contributed equally to this work.
6 To whom correspondence should be addressed. Fax: +33 80 69 32 65. simon{at}epoisses.inra.fr
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