JXB Advance Access originally published online on May 23, 2006
Journal of Experimental Botany 2006 57(9):2025-2035; doi:10.1093/jxb/erj153
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RESEARCH PAPER |
The contribution of extensin network formation to rapid, hydrogen peroxide-mediated increases in grapevine callus wall resistance to fungal lytic enzymes
1Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal
2Instituto Gulbenkian de Ciências, Apartado 14 2781-901 Oeiras, Portugal
*To whom correspondence should be addressed. E-mail: Phil{at}itqb.unl.pt
Received 17 August 2005; Accepted 31 January 2006
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Abstract |
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Grapevine (Vitis vinifera cv. Touriga) callus cell walls contain a high level of the monomeric extensin, GvP1. Hydrogen peroxide stimulus of these cultures causes the rapid loss of monomeric GvP1, concomitant with marked increases in insoluble GvP1 amino acids and wall resistance to digestion by fungal lytic enzymes. JIM11 immunolocalization studies indicated that monomeric and network GvP1 were evenly distributed in the callus cell wall. These primary cell walls were used to investigate the specific contribution of extensin and other ionically bound cell-wall proteins to hydrogen peroxide-mediated increases in resistance to fungal lytic enzymes. This was performed by removing ionically-bound proteins and assaying for hydrogen peroxide-enhanced resistance after the addition of selected protein fractions. The results indicate that hydrogen peroxide-induced increases in resistance to digestion by fungal lytic enzymes require a co-operative action between network extensin formation and the electrostatic interaction of additional wall proteins with the extracellular matrix.
Key words: Disease resistance, extensin network, fungal lytic enzymes, primary cell wall, protoplasts
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Introduction |
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The rapid production of reactive oxygen species in plant cells, including hydrogen peroxide (H2O2), is an essential component of the plant defensive reaction to microbial attack (Lamb and Dixon, 1997) and also to some forms of abiotic stress (Cazalé et al., 1998). The oxidative burst in many plant species originates from NAD(P)H oxidases (Auh and Murphy, 1995; Papadakis and Roubelakis-Angelakis, 1999) or apoplastic peroxidases (Bolwell et al., 1998) and is important as a signalling agent (Levine et al., 1994) for the mobilization of later gene expression associated with antioxidant defences, phytoalexin biosynthesis, and the development of systemic acquired resistance (Tenhaken et al., 1995; Mittler, 2002). However, stress-related production of reactive oxygen species is also required for the rapid, transcription-independent cross-linking of plant cell wall components, including hydroxyproline-rich glycoproteins (HRGPs), which form part of the plant's primary defence (Bradley et al., 1992; Brownleader et al., 1997; Wojtaszec et al., 1997; Jackson et al., 2001).
The group of hydroxyproline-rich glycoproteins (HRGPs) contains extensins, arabinogalactan proteins (AGPs), proline-rich proteins (PRPs), and solanaceous lectins (Sommer-Knudsen et al., 1998). Extensins are abundant in some dicot cell walls and are the most studied family of HRGPs (Cassab, 1998). Extensins have a poly(II) Pro like configuration giving them a rod-like shape (Cooper et al., 1987), which can reach 50–80 nm in length (Cooper et al., 1987; Brownleader et al., 1996). The polypeptide backbone of these 60–90 kDa proteins contains many repeats of structural, Ser(Hyp)4–6 motifs which are heavily glycosylated with 1–4 arabinose residues O-linked to contiguous stretches of Hyp residues (Shpak et al., 1999), and most of the serine residues are O-galactosylated (Cooper et al., 1987). These structural motifs are often flanked by short (4–5 aa) sequences rich in Tyr, Lys, Val, and His, which are thought to contain the sites of extensin cross-linking (Kieliszewski and Lamport, 1994).
Extensins are secreted into the apoplast as soluble monomers where the positively charged lysine and protonated histidine residues are thought to interact ionically with the negatively charged uronic acids of pectins (Cooper et al., 1987; Showalter, 1993). Their solubility to saline extraction rapidly decreases after external stimuli such as wounding, infection or the presence of elicitors (Brisson et al., 1994; Brownleader et al., 1997; Wojtaszec et al., 1997; Otte and Barz, 2000; Jackson et al., 2001) as a result of oxidative extensin cross-linking. The resultant formation of an extensin network is a peroxidase-mediated and peroxide-dependent process (Cooper and Varner, 1984) which has been proposed to involve the peroxidase-mediated coupling of extensin tyrosine residues, to form isodityrosine linkages (Fry, 1982). The network may be further cross-linked by the formation of larger tyrosine oligomers such as di-isodityrosine (Brady et al., 1996) or pulcherosine (Brady et al., 1998) at pre-formed intra- or inter-extensin isodityrosine linkages in the network.
Models of the dicot primary cell wall describe a complex nanostructure composed of relatively rigid cellulose microfibrils coated and cross-linked with xyloglucans and embedded in a mobile pectin network (Carpita and Gibeaut, 1993; Jarvis and McCann, 2000). Electron microscopy studies of the primary cell wall in onion have indicated thin walls, c. 100 nm thick, composed of 3–4 laminate layers of 8–15 nm thick microfibrils coated with xyloglucan, and spaced 20–40 nm apart (McCann et al., 1990). This suggests that the length of monomeric extensin (50–80 nm; Cooper et al., 1987; Brownleader et al., 1996) is theoretically sufficient to span inter-microfibrillar distances in primary cell walls. It is therefore conceivable that the formation of a dense extensin network (10–20% of the wall) with multiple cross-links can help immobilize wall polymers to increase cell-wall rigidity and resistance to pathogen ingress.
Some studies have, in fact, demonstrated a relationship between HRGP accumulation in cell walls and increased resistance to plant disease (Esqerre-Tugaye et al., 1979; Raggi, 2000). Bradley et al. (1992) have reported that elicitation of soybean results in the rapid, transcriptional independent, oxidative cross-linking of cell-wall structural proteins, including an extensin-like HRGP. This deposition was concomitant with an increase in cell-wall resistance to enzymatic digestion (Brisson et al., 1994). The possible contributions of additional, peroxide-driven reactions to the observed increase in wall resistance were not excluded, however. The peroxidase catalysed cross-linking of, for example, feruloylated pectins (Zimmerlin et al., 1994; Sanchez et al., 1996) could contribute to increased resistance. Hydrogen peroxide is also capable of driving the non-enzymatic, oxidative scission of polysaccharides (Fry, 1998; Chen and Schopfer, 1999), which could alter cell-wall properties. It therefore remains unclear whether extensin network formation during rapid, defensive responses is a direct, causal event in changes to primary cell-wall resistance, or whether it merely contributes to a more complex alteration in wall properties, involving multiple, alternative reactions.
The work presented in this paper explores the contribution of extensin network formation to peroxide-mediated increases in cell-wall resistance to fungal lytic enzymes. By extracting and selectively restoring fractions of extracellular proteins to the cell wall, it was possible to isolate the contribution of extensin network formation to changes in wall resistance to digestion. It is demonstrated that extensin network formation does enhance wall resistance to fungal lytic enzymes, but requires the interaction of additional wall proteins with the extracellular matrix to be effective.
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Materials and methods |
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Growth conditions of grapevine callus
Grapevine callus was induced from leaf explants (0.5 cm2) of grapevine cv. Touriga, placed on Murashige and Skoog (1962) based tissue culture medium, containing 4.4 g l–1 Murashige and Skoog basal salt mixture (Sigma, Madrid, Spain) 5 g l–1 of sucrose (Duchefa), 100 mg l–1 of casein hydrolysate, 20 g l–1 of polyvinylpyrrolidone 40T, 0.5 mg l–1 of 2,4-dichlorophenoxyacetic acid, 0.22 mg l–1 of kinetin, and 2 g l–1 of Gelrite (Sigma, Madrid, Spain). The cultures were kept at 24 °C in the dark and were subcultured every 2 weeks.
Determining cell wall digestibility
One hundred mg of cells was teased apart into small clumps and incubated in a solution composed of 2% (w/v) cellulase, 1% (w/v) macerozyme, 4.4 g l–1 Murashige and Skoog (1962) basal salt mixture, 0.8 M sorbitol (Duchefa) in an orbital shaker (Abalab, Agitorb 160 E) at 25 °C, 80 rpm. Unless otherwise stated in the text, the time of incubation was 75 min. The digest was then centrifuged at 10 000 g for 5 min and the supernatant discarded. The pellet was resuspended in 1 ml of 0.4 M sorbitol containing 0.05% (w/v) calcofluor white staining and counted in a haemocytometer, under UV and visible light of a fluorescence microscope. Protoplasts were distinguished by their spherical shape and lack of fluorescence from calcofluor white staining of cell wall material. The ratio of protoplasts:intact cells was determined from three replicates of at least three independent digests. Comparative data were analysed by Student's t test.
Stimulation and inhibition of extensin cross-linking
Grapevine callus (1.5 g) was vacuum infiltrated with sodium acetate buffer (15 mM, pH 4.5) containing 100 µM H2O2 for 15 min at 24 °C to cause GvP1 insolubilization. Inhibition of extensin insolubilization was obtained by adding 5 mM ascorbate to the infiltration buffer. As a control, callus tissues were infiltrated with sodium acetate buffer alone. The level of soluble and insoluble GvP1 in these cells was then assayed by Superose-12 chromatography as described below.
Preparation of ionically bound, extracellular matrix proteins from cell walls
The removal of ionically bound proteins from cell walls was accomplished by gently separating callus pieces into small clumps and washing them extensively in 15 mM sodium acetate buffer (pH 4.5), followed by vacuum-assisted infiltration with 15 mM sodium acetate buffer (pH 4.5) containing 1 M KCl for 15 min at 24 °C. The saline eluate was collected by vacuum-assisted filtration through No. 1 filter paper (Whatman, Clifton, NJ), clarified by centrifugation at 10 000 g for 5 min, equilibrated in 15 mM sodium acetate (pH 4.5), and concentrated by pressure-assisted filtration through a 10 kDa cutoff membrane (Diaflow, Amicon, Beverly, MA).
To prepare GvP1-deficient saline eluates, concentrated saline eluate was loaded onto Sepharose SP in a XK 16 column (Amersham-Pharmacia Biotech, Uppsala) equilibrated with degassed 15 mM sodium acetate buffer (pH 4.5), and washed with the same buffer at a flow rate of 2 ml min–1 until all non-binding material that absorbed at 280 nm had been removed and stored. Bound proteins were then eluted within a 0–0.5 M NaCl gradient over 45 min. GvP1-enriched fractions (eluting between 62–74 ml) were identified by Superose-12 chromatography (described below) and discarded. All remaining fractions were pooled and equilibrated in sodium acetate buffer (15 mM, pH 4.5). Pure GvP1 was obtained as described previously (Jackson et al., 2001)
Binding of ECM proteins to saline extracted cell walls
Native cells were processed in order to remove ionically bound components as described above. Cells were then collected and washed with 15 mM sodium acetate buffer (pH 4.5). Whole eluates or selected fractions of cell wall proteins were then added to desired levels and incubated for 5 min before washing twice in 15 mM sodium acetate (pH 4.5) to remove unbound material. The efficiency of incorporation and hydrogen peroxide-induced insolubilization of these materials was monitored by Superose-12 chromatography as described below.
Quantification of GvP1 in saline extracts of cell walls
The content of GvP1 in saline eluates was determined on a Superose-12 gel-filtration column equilibrated with degassed 0.1 M sodium acetate buffer (pH 5.0) at a flow rate of 0.5 ml min–1. The eluate was monitored at 280 nm using a 2100 UV monitor linked to a PC via a data acquisition card with analogue to digital conversion (PC-512, National Instruments, Madrid). The relation of GvP1 peak height to µg GvP1 injected onto a Superose-12 column was determined from known quantities (5–60 µg) of pure GvP1. To determine the quantity of GvP1 in cell wall eluates, their Superose-12 chromatograms were subject to Gaussian analysis. The contribution of proximal components to the GvP1 peak was subtracted, and the adjusted GvP1 peak height was used to quantify GvP1. The results were expressed as µg GvP1 mg–1 DW cell wall.
The effect of Gvp1 deposition on the amino acid composition of covalently-bound cell wall protein
Grapevine callus (1.5 g) was vacuum-infiltrated with 3 ml 100 µM H2O2 for 15 min at 24 °C to cause GvP1 deposition. Callus infiltrated with 3 ml water under the same conditions was used as the untreated control. The callus was then washed in 20 ml of 1 M KCl to remove ionically-bound cell wall protein and then homogenized in liquid nitrogen. The homogenate was washed by centrifugation at 4500 g for 5 min in 1% Triton X-100, three times with 1 M KCl, and three times with distilled water. The washed cell-wall pellet was freeze-dried and acid hydrolysed in 6 M HCl containing 1 mg ml–1 phenol at 110 °C for 16 h under nitrogen. The hydrolysate was clarified by ultrafiltration through a 0.2 µM filter (Millipore, Bedford, USA). Norleucine was added as an internal calibrant. The acid hydrolysates were derivatized with phenylisothiocyanate using a Waters pico-tag system and four samples corresponding to different quantities of hydrolysate were analysed on a Waters Pico-Tag amino acid analysis system (Waters, Milford, USA) as per the manufacturer's instructions.
Immunolocalization of GvP1 in callus cell walls
Cells were washed in PBS and then blocked in 2% BSA in PBS for 30 min. They were then washed in PBS (3 times, 5 min) and incubated overnight at 4 °C with primary antibody (1:10) before a further wash in PBS. Cells were incubated with secondary antibody (1:600) (CyTM5-conjugated Affinipure goat anti-rat IgG (H+L); Jackson ImmunoResearch, USA) for 90 min in the dark and subsequently washed in PBS. Confocal images were captured from a Leica confocal spectral microscope (Leica SP2 AOBS) and constructed using ImageJ software (NIH).
Two-dimensional electrophoresis
Isoelectric focusing (IEF) was performed using an IPGphor IEF System (Amersham-Pharmacia Biotech, Uppsala). 25 µg of protein was added to 250 µl rehydration buffer and loaded onto a 13 cm Immobiline DryStrip (pH 3–10). Strip rehydration occurred over 12 h at 20 °C and 30 V. Focusing employed voltage steps of 250 V for 1 h, 500 V for 1.5 h, 1000 V for 1.5 h, 2500 V for 1 h, followed by a gradient to 8000 V over 1.5 h. IEF strips were equilibrated for 15 min in equilibration buffer [50 mM TRIS-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue] containing 10 mg ml–1 DTT, followed by 15 min in equilibration buffer containing 25 mg ml–1 iodoacetamide. After equilibration, strips were applied to 12.5% (w/v) SDS-PAGE gels and sealed with 0.5% (w/v) agarose in SDS buffer containing a few mg of bromophenol blue. Electrophoresis was carried out at 30 mA per gel with a maximum of 250 V for approximately 4 h. Gels were silver stained (Blum et al., 1987) and the image obtained from a Molecular Dynamics densitometer using ImageQuant software (Sunnyvale, Ca. USA).
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Results |
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H2O2 induces extensin deposition and increased cell wall resistance to digestibility
Grapevine callus is composed of cells measuring c. 25 nmx65 nm arranged in filaments (Fig. 1A). The incubation of these cells with commercial mixtures of fungal lytic enzymes leads to the formation of protoplasts (Fig. 1B) with complete digestion of the cell wall, as also indicated by loss of calcoflour white staining (cf. Fig. 1C, D). A time-course study of protoplast release is presented in Fig. 2. The time corresponding to c. 50% release of protoplasts from native cells (75 min) was subsequently used in all the assays.
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The incubation of grapevine cells with hydrogen peroxide caused a marked decrease in protoplast release (Table 1), clearly indicating that peroxide caused c. 30% increase in wall resistance to digestion by fungal lytic enzymes.
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Grapevine callus cells contain high levels of a 90 kDa, monomeric extensin, denoted GvP1 (Jackson et al., 2001). Superose-12 gel filtration chromatograms of salt eluates (Fig. 3A) allowed the quantification of monomeric (salt-soluble) GvP1 in callus cell walls, which elutes as a major peak at 9.5 ml (trace 1). Prior to incubation with H2O2, these cell walls contained c. 70 µg of monomeric GvP1 mg–1 DW cell wall. The incubation of these cells in the presence of 100 µM H2O2 resulted in the substantial insolubilization of GvP1, c. 30–50 µg extensin mg–1 DW cell wall (see trace 2).
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Amino acid analyses of these cell walls also demonstrate a concomitant increase in the major amino acids of GvP1 after incubation with hydrogen peroxide and the removal of residual, ionically bound protein from walls (Fig. 4). This confirms that the hydrogen peroxide-mediated increase in wall resistance to digestion is accompanied by the formation of an insoluble extensin-like network. Ascorbate incubation of native cells prior to peroxide stimulus resulted in the loss of this resistance (Table 1) and inhibited extensin insolubilization (Fig. 3A, trace 3), which supports a relationship between extensin-network formation and increased resistance to digestion.
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In order to determine if GvP1 was uniformly distributed in these walls or localized to specific sites (e.g. the middle lamella), these cells have been probed with the anti-extensin monoclonal antibodies, JIM11 and JIM12. These antibodies showed different immunoreactivities against native and deglycosylated GvP1 (Fig. 5). JIM11 produced the strongest signal, which was markedly decreased against deglycosylated GvP1, suggesting its reactivity against extensin glycans (Fig. 5A). JIM12 produced a relatively weaker signal, but presented similar reactivities against native and deglycosylated forms, probably due to its recognition of the GvP1 polypeptidic core (Fig. 5B). Similar results with these antibodies have been obtained with carrot extensin (Smallwood et al., 1994).
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The stronger immunoreactivity of JIM11 was also evident when GvP1 was probed in situ (Fig. 6, cf A [JIM11] and B [JIM12]). The immunoreactivity with JIM11 demonstrated that GvP1 is evenly distributed in the primary cell wall. Saline extraction of these cells markedly reduced the level of GvP1 (Fig. 6C), indicating that prior to treatment with peroxide, these cell walls contain minor levels of extensin network. Hydrogen peroxide treatment resulted in the formation of substantially higher levels of insoluble network extensin, which was also uniformly deposited in the cell wall (Fig. 6D). These results confirm that the formation of the extensin network is a wall-wide phenomenon, which would be essential for its effective contribution to increased resistance in these cells to protoplasting enzymes.
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The removal of ionically-bound ECM proteins from native cells by saline elution did not significantly alter the wall digestibility (Table 1). However, the c. 30% increase in resistance to digestion observed after incubation with H2O2 was reduced to 15% after the removal of ionically-bound ECM proteins, although these walls remained 22% more resistant to digestion when compared with salt-washed, control cell walls. Moreover, the partial loss of resistance could be largely recovered by the addition of whole saline eluates to these cells. Interestingly, this regaining of resistance could also be achieved with the addition of GvP1-depleted saline eluates (see also Fig. 3, trace 4), but not after the addition of pure GvP1. This indicates that hydrogen peroxide-induced increases in resistance to protoplasting enzymes involve at least two components. About 50% of the overall increase in resistance remains salt-insoluble, and therefore could be related to peroxide/peroxidase-mediated formation of the insoluble extensin network. The remaining 50%, however, appears to be mediated by the electrostatic interaction of non-extensin ECM proteins with the peroxide-modified cell-wall structure.
The above data suggest that increases in resistance could be related to a peroxide/peroxidase-mediated event, and is in agreement with other reports describing enhanced wall resistance after hydrogen peroxide treatment of native, primary walls (Bradley et al., 1992; Brisson et al., 1994; Tire et al., 1994). The data also suggest that the formation of the extensin network may contribute to the increased resistance to fungal lytic enzymes. However, the activation of wall peroxidase could result in the formation of alternative cross-links, such as between feruloylated pectins (Fry, 2004), and peroxide or related reactive oxygen species can directly modify the pectin network via non-enzymatic reactions (Fry, 1998; Chen and Schopfer, 1999). In addition, peroxide can function as a signalling molecule for the rapid mobilization of gene expression related to plant defensive responses, including wall resistance (Lamb and Dixon, 1997). It therefore remains unclear whether extensin network formation is a major causal event in hydrogen peroxide-enhanced wall resistance to lytic enzymes, or whether other wall-modifying processes make more important contributions.
H2O2-induced increase in wall resistance is a rapid, transcription-independent process involving extensin and other ECM proteins
In order to determine if hydrogen peroxide incubation alters the levels of cell wall proteins other than GvP1, the proteins of saline eluates of control and H2O2-incubated cells have been compared by 2D electrophoresis (Fig. 7A). Extensins cannot be visualized in these gels, since its migration is limited in SDS-PAGE systems (Wojtaszec et al., 1997; Jackson et al., 2001). Thirty minutes of incubation with H2O2 was sufficient to alter the abundance of other major, wall proteins, leading to the intensification of some, and a marked reduction in others (Fig. 7A).
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In order to avoid H2O2-mediated alterations in the abundance of non-extensin cell wall proteins, which could contribute to changes in wall resistance, the walls of living callus cells were exhaustively extracted with 1 M KCl. This treatment effectively removes ionically bound proteins, and renders the cells incapable of increasing wall resistance in response to H2O2, even when peroxidase is selectively bound to the cell wall in endogenous levels (Table 2). Such cells are effectively killed by the saline extraction. However, the incubation of salt-washed cells with saline extracts leads to the successful re-attachment of the majority of ionically bound ECM proteins, although a few acidic proteins are lost (Fig. 7B). The hydrogen peroxide-induced changes in the levels of other cell-wall proteins that were observed in native cells did not occur in these cells (Fig. 7A, B). Hydrogen peroxide-treatment of these cells did cause insolubilization of extensin in a quantitatively similar way to that observed after peroxide-incubation of living cells (Fig. 3B, trace 1). Probing these walls with JIM11 (Fig. 6) demonstrated that, as in native cells, the distribution of re-attached extensin (Fig. 6E) and the extensin network formed after peroxide incubation (Fig. 6F) was homogeneous. Peroxide also caused a c. 30% increase in resistance, quantitatively comparable to that observed in H2O2-treated native cells (Table 2). Furthermore, the extraction of these cells with saline solutions after peroxide treatment resulted in a c. 10% decrease in resistance. This indicates that, as in native cells, the hydrogen peroxide-induced increase in resistance involves at least two components. In this reconstituted system, 70% of the overall increase in resistance remained salt-insoluble, whereas the remaining 30% appears to be mediated by the electrostatic interaction of non-extensin ECM proteins with the peroxide-modified cell wall structure.
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Together, these results demonstrate that the hydrogen peroxide-induced increase in resistance to fungal lytic enzymes is a transcription-independent event, which apparently does not involve the oxidative cross-linking of cell-wall proteins other than extensin. It also clearly demonstrates that one can extract and re-bind the extracted ECM proteins without irreversibly altering the walls capacity to increase resistance in response to H2O2. This therefore provided the opportunity to assay selected fractions of the ionically bound ECM proteins for their contribution to peroxide induced increases in resistance to fungal lytic enzymes.
The specific contribution of extensin network formation to cell-wall resistance
To quantify the specific impact of extensin insolubilization on cell-wall resistance to digestion, saline-extracted cells were reconstituted with endogenous levels of pure GvP1 (Fig. 6G). The extensin peroxidase, GvEP (Jackson et al., 2001) was also added to allow extensin network formation. Hydrogen peroxide treatment of these cells resulted in the formation of the extensin network at levels similar to those observed in H2O2-incubated native cells (Fig. 3B, trace 2; Fig. 6H). However, resistance of these cell walls was not enhanced, but remained unchanged from that observed in saline-extracted cells (Table 3). This strongly indicates that the formation of the extensin network in response to H2O2 is insufficient, by itself, to enhance wall resistance to lytic enzymes.
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To assay the contribution of other ionically bound ECM proteins to this resistance, an extensin-free fraction was prepared by selectively removing extensin from saline eluates by cation exchange chromatography (see Materials and methods). The resulting fraction of ECM proteins (Fig. 3A, trace 4), contained <2.5% (w/w protein) of the level of GvP1 in whole saline eluates. The addition of this fraction to saline-extracted cells did not restore their ability to show increased resistance in response to H2O2 (Table 3). However, when the GvP1-free fraction (contains GvEP) was re-attached to walls together with GvP1, a gain in resistance of 20% was observed after incubation with H2O2. This enhanced resistance to lytic enzymes was not observed in cells where the extensin-free fraction had been subject to denaturation by boiling in the presence of DTT, although native GvEP was present and extensin network formation occurred. Similarly, negative results were also obtained in cells where ionically bound ECM proteins were restored only after the GvP1 network had been formed in their absence.
These results demonstrate that the formation of the extensin network is an essential part of the H2O2-mediated increase in the resistance of grapevine callus primary walls to digestion by fungal lytic enzymes. However, it also demonstrates that the H2O2-enhanced resistance in these cells requires the interaction of additional ECM proteins with the wall during extensin network formation to be effective.
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Discussion |
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The role of extensin cross-linking in rapid, H2O2-mediated increases in primary wall resistance to fungal lytic enzymes has been investigated.
The primary cell wall of suspension-cultured or callus cells of many dicot species has been shown to be capable of rapidly insolubilizing extensin in a transcriptionally independent, peroxide-mediated reaction (Everdeen et al., 1988; Bradley et al., 1992; Brownleader et al., 1997). Recently, it has been shown that grapevine callus primary walls contained high levels of monomeric extensin (GvP1) which can be rapidly insolubilized in response to elicitors or exogenous peroxide in a reaction apparently mediated by the extensin peroxidase, GvEP (Jackson et al., 2001). This earlier observation has been confirmed here and the anti-extensin antibody, JIM11, has been used to show that monomeric GvP1 is evenly distributed in these walls, as is the GvP1 network formed after stimulation with H2O2. The hydrogen peroxide-induced formation of the extensin network occurred with the production of walls c. 30% more resistant to digestion with protoplasting enzymes, in agreement with other reports that the deposition of HRGPs can be associated with increased wall resistance (Esqerre-Tugaye et al., 1979; Raggi, 2000).
Extensin consists of extended, rod-like proteins measuring 50–80 nm in length (Cooper et al., 1987; Brownleader et al., 1996), and is therefore capable of spanning inter-microfibrillar distances (20–40 nm; McCann et al., 1990). As a consequence, the extensin network is potentially capable of penetrating both the pectin and the cellulose–xyloglucan networks, and could contribute to the immobilization of wall polymers resulting in a more rigid wall.
The contribution of extensin network formation to wall rigidity has never been tested experimentally. Extensin deposition has been closely associated with essential events in primary wall biosynthesis (Cooper et al., 1994), growth cessation (Cassab and Varner, 1987), and defensive wall modifications (Brisson et al., 1994). Interesting data have also been obtained which describes an important role for extensin in correct, primary wall biosynthesis (Cooper et al., 1994) and for the definition of cell shape and intercellular adhesion (De Tullio et al., 1999; Hall and Cannon, 2002). However, these studies fail to provide information of how extensin insolubilization might affect wall properties during these processes.
The primary walls of saline-extracted callus cells were used as a matrix to bind selected fractions of ECM proteins. The saline extraction of intact cells rendered walls incapable of becoming more resistant to fungal lytic enzymes in response to H2O2, even when the peroxidase GvEP was present. However, the re-attachment of endogenous ECM proteins of the cell wall restored the ability of these cells to demonstrate H2O2-enhanced resistance. This indicated that the removal of ionically bound ECM proteins from the wall matrix did not irreversibly alter the walls ability to show H2O2-mediated increases in resistance, and that ionically-bound ECM proteins play an essential role in this response. This experimental system therefore provided the means to investigate how the extensin network formation can contribute to increased wall resistance to fungal lytic enzymes.
In the absence of other endogenous and ionically-bound ECM proteins, extensin network formation did not increase wall resistance to digestion. That a substantial network formation occurred in these cell walls was confirmed both by the significant loss of monomeric extensin, and an increased immuno-reactivity of JIM11 with insolubilized extensin. In cells whose ECM protein contents had been modified to lack GvP1, a lack of increase in resistance in response to peroxide was also seen. However, where non-extensin ECM proteins were present during extensin deposition, an increase in resistance to digestion, similar to that observed after peroxide incubation of native cells was obtained. This strongly indicates that formation of the extensin network is essential for rapid, H2O2-enhanced resistance to lytic enzymes in these cells, but that the presence of non-extensin ECM proteins in the extracellular matrix is required.
One explanation for this observation is that the binding of ECM proteins to the negatively charged wall (Shomer et al., 2003) results in the relaxation of the electrostatically charged pectin network, which can show marked changes in hydration in response to variations in apoplastic pH, ion content (MacDougall et al., 2001b), the level of pectin methylation (Zsivanovits et al., 2004), as well as the presence of basic peptides (MacDougall et al., 2001a). The presence of ECM proteins in the wall could, therefore, help relax the pectin network, resulting in decreased wall hydration and pectin mobility (Ha et al., 1997). Under these conditions, the formation of the extensin network could help lock the wall polymers in a more tightly packed configuration with smaller wall pore sizes, which can restrict the mobility of lytic enzymes into the wall matrix (Grignon and Sentenac, 1991), thereby enhancing resistance to digestion. This might explain why the formation of the extensin network in the absence of non-extensin ECM proteins does not appreciably increase wall resistance, since in such walls, extensin deposition could help fix the wall in a relatively more hydrated and mobile structure, which is, as a consequence, more porous.
Although H2O2-mediated increases in resistance to lytic enzymes appears to become largely locked into the wall structure after extensin network formation, 30% of the increased resistance after peroxide is apparently contributed by the electrostatic interaction of non-extensin ECM proteins with the wall matrix. This was clearly shown by the partial reduction in resistance of peroxide-treated cells after removal of these proteins and the restoration of full resistance after their replacement. These observations provide additional support for the existence of a co-operative action between these proteins and the extensin network in peroxide-mediated modification of wall resistance to lytic enzymes.
At present, the non-extensin ECM proteins which contribute to resistance to fungal lytic enzymes have yet to be characterized. It would be of interest to determine whether the proteins involved are functionally specific to this process, or consist of secreted proteins of diverse functions, yet which share the capacity to interact electrostatically with the wall matrix. However, as far as we are aware, the data that have already been collated constitute the first direct proof that extensin network formation can be a causal event in peroxide-mediated increases in primary wall resistance to digestion by lytic enzymes. Nevertheless, it is clear that the formation of the extensin network contributes to this resistance in a more complex fashion than has previously been described. How the interaction of other ECM proteins with the primary wall can alter the extensin network's functional contribution to enhanced resistance to fungal lytic enzymes can now also be considered.
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Acknowledgements |
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José Mário Leitão Ribeiro and Cristina Silva Pereira acknowledge personal grants from the PRAXIS XXI programme of the Fundação de Tecnologia e Ciências (Refs: PRAXIS XXI SFRH/BD/6486/2001 and PRAXISXXI SFRH/BD/19872/99, respectively). This work was also partially financed by FCT within the projects BME/33201/2000 and BCI/33116/1999.
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Abbreviations |
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ECM, extracellular matrix; GvP1, grapevine extensin peak 1; GvEP, grapevine extensin peroxidase; HRGP, hydroxyproline-rich glycoprotein.
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