Extensin network formation in Vitis vinifera callus cells is an essential and causal event in rapid and H(2)O(2)-induced reduction in primary cell wall hydration.

BACKGROUND: Extensin deposition is considered important for the correct assembly and biophysical properties of primary cell walls, with consequences to plant resistance to pathogens, tissue morphology, cell adhesion and extension growth. However, evidence for a direct and causal role for the extensin network formation in changes to cell wall properties has been lacking. RESULTS: Hydrogen peroxide treatment of grapevine (Vitis vinifera cv. Touriga) callus cell walls was seen to induce a marked reduction in their hydration and thickness. An analysis of matrix proteins demonstrated this occurs with the insolubilisation of an abundant protein, GvP1, which displays a primary structure and post-translational modifications typical of dicotyledon extensins. The hydration of callus cell walls free from saline-soluble proteins did not change in response to H(2)O(2), but fully regained this capacity after addition of extensin-rich saline extracts. To assay the specific contribution of GvP1 cross-linking and other wall matrix proteins to the reduction in hydration, GvP1 levels in cell walls were manipulated in vitro by binding selected fractions of extracellular proteins and their effect on wall hydration during H(2)O(2) incubation assayed. CONCLUSIONS: This approach allowed us to conclude that a peroxidase-mediated formation of a covalently linked network of GvP1 is essential and causal in the reduction of grapevine callus wall hydration in response to H(2)O(2). Importantly, this approach also indicated that extensin network effects on hydration was only partially irreversible and remained sensitive to changes in matrix charge. We discuss this mechanism and the importance of these changes to primary wall properties in the light of extensin distribution in dicotyledons.

Association of hemicellulose- and pectin-modifying gene expression with Eucalyptus globulus secondary growth.

Wood properties are ultimately related to the morphology and biophysical properties of the xylem cell wall. Although the cellulose and lignin biosynthetic pathways have been extensively studied, modifications of other wall matrix components during secondary growth have attracted relatively less attention. In this work, thirty-eight new Eucalyptus cDNAs encoding cell wall-modifying proteins from nine candidate families that act on the cellulose-hemicellulose and pectin networks were cloned and their gene expression was investigated throughout the developing stem. Semi-quantitative RT-PCR revealed distinct, gene-specific transcription patterns for each clone, allowing the identification of genes up-regulated in xylem or phloem of stem regions undergoing secondary growth. Some genes, namely an endo-1,4-beta-glucanase, one mannan-hydrolase and three pectin methylesterases showed transcription in juvenile and also in mature stages of wood development. The patterns of gene expression using samples from tension and opposite wood disclosed a general trend for up-regulation in tension wood and/or down-regulation in opposite wood. Localised gene expression of two selected representative clones, EGl-XTH1 and EGl-XTH4, obtained through in situ hybridization confirms the RT-PCR results and association with secondary xylem formation. Likewise, immunolocalisation studies with the anti-pectin antibody (JIM5) also supported the idea that the development of tissue-specific pectin characteristics is important during secondary growth. These results emphasize an involvement of hemicellulose and pectin biochemistry in wood formation, suggesting that the controlled and localised modification of these polysaccharides may define cell properties and architecture and thus, contribute to determining different biophysical characteristics of Eucalyptus wood.

A proteomic analysis of the wound response in Medicago leaves reveals the early activation of a ROS-sensitive signal pathway.

Wounded Medicago truncatula leaves produce a burst of O(2)(-) (phase I) between 1 and 15 min, then of O(2)(-) and H(2)O(2) (phase II) between 1 and 3 h. Our previous results suggest reactive oxygen species (ROS) may provide signals to mobilise early (6 h), apoplastic, wound-responsive proteins (WRPs). 2DE and MALDI-TOF/TOF were used to analyse how the suppression of ROS production at different time points by diphenyleneiodonium (DPI), affects the expression of WRPs. Rapid (≤3 min) DPI inhibition of phase I O(2)(-) production suppressed the differential regulation of 7 out of 19 WRPs, which were consequently classified as ROS-dependent WRPs. DPI inhibition of only phase II ROS production failed to suppress the wound regulation of 18 out of 19 WRPs, but led to the altered expression of 1 ROS-dependent WRP and 2 non-WRPs (Group B). The data indicates Group B proteins are alternatively targeted via the modulation of phase II ROS production. This reinforces an important role for phase I O(2)(-) signalling in the early wound response, but indicates that this response is partly regulated by phase II of the oxidative burst. This data provides an informed basis for further proteomic studies aimed at identifying early activated O(2)(-) signalling components in wounded Medicago.

Associating wound-related changes in the apoplast proteome of Medicago with early steps in the ROS signal-transduction pathway.

Early wound-related changes in the leaf apoplast proteome of Medicago truncatula have been characterized by 2-DE and MALDI-TOF/TOF and the differential expression of 28/110 extracellular proteins could be reproducibly observed 6 h after wounding. Wounding induced an initial (0-30 min) burst of O2-, followed by a later (3-6 h) production of O2- and H2O2. The infiltration of 5 microM DPI

Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves.

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non-canonical natures. The presence of non-canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.

A biochemical and molecular characterization of LEP1, an extensin peroxidase from lupin.

An analysis of apoplastic extensin cross-linking activity in vegetative organs of Lupinus albus indicated that leaves contained the highest specific activity. Assays of peroxidases fractionated from this material demonstrated that this activity could be largely attributed to a soluble and apoplastic 51-kDa peroxidase, denoted LEP1. Relative to other purified peroxidases, LEP1 demonstrates high extensin cross-linking activity and can be classified as an extensin peroxidase (EP). Optimal conditions for the in vitro oxidation of other phenolic substrates included 1.5-3.0 mm peroxide at pH 5.0. EP activity of LEP1 was low under these conditions but optimal and substantially higher with 100 microm peroxide and neutral pH, suggesting that physiological changes in pH and peroxide in muro could heavily influence the extensin cross-linking activity of LEP1 in vivo. Analysis of LEP1 glycans indicated 11-12 N-linked glycans, predominantly the heptasaccharide Man3XylFucGlcNAc2, but also larger structures showing substantial heterogeneity. Comparative assays with horseradish peroxidase isoform C and peanut peroxidases suggested the high level of glycosylation in LEP1 may be responsible for the high solubility of this EP in the apoplastic space. A full-length cDNA corresponding to LEP1 was cloned. Quantitative reverse transcriptase-PCR demonstrated LEP1 induction in apical portions of etiolated hypocotyls 30-60 min after exposure to white light, prior to the onset of growth inhibition. Comparative modeling of the translated sequence indicated an unusually unobstructed equatorial cleft across the substrate access channel, which might facilitate interaction with extensin and confer higher EP activity.