The structural and functional continuum of the plant apoplast is the first site of contact with a pathogen and plays a crucial role in initiation and coordination of many defence responses. In this paper, we present an overview of the involvement of the plant apoplast in plant-pathogen interactions. The process of infection of French bean (Phaseolus vulgaris L.) plants by Colletotrichum lindemuthianum is analysed. The ultrastructural features of plant defence responses to fungal infection are then compared with those observed in plants or cell suspensions treated with various elicitors. Changes in cell walls and in whole plant cells responding to infection seem to be highly similar in all systems used. Model systems of French bean and white lupin (Lupinus albus L.) are then utilised to provide some biochemical characteristics of oxidative reactions in the apoplast evoked by elicitor treatment. The species specificity of various mechanisms generating reactive oxygen species is discussed, and some details of pH-dependent H2O2-generating activity of peroxidases are demonstrated. As its exocellular nature is an important feature of the oxidative burst, the major consequence of this event, i.e., the oxidative cross-linking of wall components during the papilla formation and strengthening of the walls, is analysed. Finally, the possible involvement of other wall-associated and developmentally regulated H2O2-generating mechanisms, like amine and oxalate oxidases, in plant defence is demonstrated. It is concluded that under stress conditions, such apoplastic mechanisms might be employed to increase plants' chances of survival.

Plants respond to various biotic or abiotic stimuli with different mechanisms and the mobilization of phenolic compounds is one of them. In white lupin ( Lupinus albus L.) plants, isoflavones and their glucosides were localized in cell walls where the high constitutive activity of β β β β -glucosidase (EC 3·2·1·21) was also identified. The enzyme was partially purified from root cell walls. Its polymeric active form has 180 or 200 kDa as determined by non-denaturing electrophoresis and gel filtration, respectively, and the isoelectric point is at pH 6·9. The enzyme is an exoglucosidase, preferentially hydrolysing conjugates of phenolic compounds with β β β β anomers of glucose. It is not active against purified lupin cell walls. The specific β β β β -glucosidase activity varies in different tissues with the highest one in roots, and always higher in cell walls than in protoplast. The cell wall location of the enzyme was confirmed biochemically by its activity in intercellular washing fluids. Both aglycones and glycosides were also present in these fluids. The specific β β β β -glucosidase activity correlated well with the isoflavonoid aglycone/glycoside ratios in various tissues: the higher β β β β -glucosidase activity, the higher relative aglycone content. β β β β -glucosidase activity was found to be inducible under conditions of yeast elicitor treatment. Induction of the enzyme was accompanied by changes in the isoflavone secretion and accumulation, suggesting the regulatory role of β β β β -glucosidase activity in root exudation of isoflavonoids. No correlation however, was found between changes in β β β β -glucosidase activity and the presence of isoflavonoids in root exudates of plants grown under various nitrogen nutrition regimes.

Acidic exocellular class II[ chitinase (EC 3.2.1.14) was previously identified in healthy white lupin (Lupim~s albus L.) plants and suspension-cultured cells by N-terminal microsequencing. In this study, the detection of chitinase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin derivatives is described. Chitinase activity was observed in protein fractions of cytoplasmic or exocellular origin from roots, hypoco@s, cotyledons, and leaves of healthy white hipin plants. Using isoelectrofocusing tollowed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activity was distributed fairly evenly within a plant with acidic isoforms predominating in cell walls and basic (or neutral) ones found intracellularly. Exocellular location of some chitinase isoforms were also confirmed by detection of their activities in intercellular washing fluids from white lnpin tissues. Chitinase activity was demonstrated in culture filtrates and cell walls of suspension-cultured white lupin cells.

The development of an in vitro model system to study the process of dimerization of hydroxycinnamic acids is reported. Model compounds ferulic acid (FA) or methyl ferulate were reacted with H2O2 in the presence of horseradish peroxidase (HRP) or peroxidases from Lupinus albus L. apoplastic fluids. Following solid-phase extraction, the reaction products were analysed using gas chromatography-mass spectrometry. Major analytical and biochemical parameters of model reactions were determined and optimized. Six ferulic dimers were identified and quantified. With the use of this model system we found methyl ferulate to be a better substrate for lupin and horseradish peroxidases than FA. Use of various peroxidases did not influence the qualitative composition of reaction products although it affected the rate of substrate utilization and the quantitative output of reactions. Various isoforms of lupin apoplastic peroxidases revealed differentiated specificity towards hydroxycinnamic acids or their methyl esters. The potential of isoflavonoids (major phenolic components in white lupin cell walls) to influence peroxidase-catalysed formation of ferulic dehydrodimers was also tested. Genistein (5,7,4'-trihydroxyisoflavone) and genistin (genistein-7-O-β-glucoside) did not affect the qualitative composition of the reaction products. However, genistein inhibited the rate of ferulic substrate oxidation, while genistin showed the opposite effect, stimulating both utilization of ferulic substrate by HRP and subsequent polymerization and / or degradation of dehydrodimers formed. To our knowledge, this is the first indirect evidence that isoflavonoids might play a regulatory role in the oxidative formation of intermolecular cross-links in cell walls.

A suspension culture of white lupin cells has been established, and proteins of the exocellular matrix analysed. Based on homologies of N-terminal amino-acid sequences, three stress- or defence-related proteins: acidic class III chitinase, polygalacturonase-inhibiting protein, and germin/oxalate oxidase, secreted by lupin cell culture, were identified.