Antifungal Hydrolases in Pea Tissue

Abstract

Chitinase and 13-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv "Dot") pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were /3-1,3-glucanases. The different molecular forms of chitinase and 13-1,3-glucanase were differentially regulated. Chitinase Chl (mol wt 33,100) and .8-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and .l-1,3-glucanase Gl (mol wt 33,500) accumulated during the course of maturation.With a simple, three-step procedure, both chitinases and both j8-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [3H]chitin, as well as in their relative lysozyme activity. Similarly, the two f8-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.An attack by potential pathogens elicits profound changes in the metabolism of a plant. In recent years, particular attention has been paid to the strongly altered pattern of protein synthesis in plant cells treated with pathogens or pathogen-derived elicitors. Analysis of proteins (9), of translation products of mRNA (7,9,21), and of translation products of newly formed mRNA (7) indicate that 20 or more proteins are newly formed or strongly induced upon infection or elicitor treatment. What is the significance of these proteins for the defense of the plant against pathogens? The function of most of them is still entirely unknown. Some of them, the pathogenesis-related (PR-) proteins (21, 23) and the resistance-related proteins (9), have obtained suggestive names. However, their relation to pathogenesis or to induced resistance remains a challenging, unsolved puzzle.The function of a small number of the newly synthesized proteins has been identified. Certain Many pathogenic microorganisms attack their host plants by secreting hydrolases, such as pectinase and cellulase, which are able to degrade components of the plant cell wall. Correspondingly, plants possess hydrolases capable of attacking and degrading the cell walls of potential pathogens. The widespread occurrence of lysozyme and chitinase is particularly intriguing, since these enzymes appear to lack endogenous substrates in higher plants (2). Abeles et al. (1) have observed that ethylene induces chitinase and /3-1,3-glucanase in bean leaves. Since many fungi have cell walls rich in chitin and ,3-1,3-glucan, they hypothesized that these two hydrola...

Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

“…Differential activation of members ofsmall multigene families encoding defence related products under different stress conditions have been described for HRGP and chalcone synthase in Phaseolus vulgaris (7,21). In addition, two chitinases and two j3-glucanases showing differential regulation during development and in response to fungal infection have been described in pea tissue (16). Transcripts of some defence genes (HRGP, PR 1) have been shown to accumulate in areas distant to the site of infection whereas others may be more localized (23,24).…”

“…In plants inoculated in a patchwise fashion with X. c. vitians, increases in CHL activity were seen in both inoculated, collapsed areas and in the surrounding, apparently healthy, tissue. In contrast, changes in CHL activity in plants inoculated with E. coli were only seen in the inoculated areas; the specific activity of CHL in the surrounding tissue was the same as that ofuninoculated Multiple isoforms of chitinase and l-1,3-glucanase have been described in a number of plants and in some cases these isoforms show differential regulation in response to pathogens or with aging (16). Accordingly, we wished to determine the number ofisoforms offl-glucanase and CHL in healthy plants and whether different isoforms were specifically induced by the different treatments employed.…”

“…CHLs were separated from the dialysed extract by affinity chromatography on a column of regenerated chitin (19) at pH 8, as described by Mauch et al (16). After washing with 10 mM Tris buffer (pH 8), the enzymes were eluted from the column with 20 mm acetic acid (pH 3), and the eluate was adjusted to pH 4.3 by addition of 1 M Tris (pH 7.5).…”

“…This antifungal action is probably due to the hydrolysis of the noncrystalline chitin present at the apex of the growing fungal hypha (26). Bean and pea chitinases also possess lysozyme activity that is capable of hydrolyzing the peptidoglycan in bacterial cell walls (4,16). Thus, these hydrolases are potentially effective in defence reactions against bacterial patho-'The work described in this paper was supported by the Gatsby Foundation.…”

Plant defence genes are induced in the pathogenic interaction between bean roots and Fusarium solani, but not in the symbiotic interaction with the arbuscular mycorrhizal fungus Glomus mosseae

“…Chitinase activity was determined with [$H] chitin as a substrate as described by Boller et al (1983), and β-1,3-glucanase activity was measured according to Mauch, Hadwiger & Boller (1988 a). Protein was measured by the method of Bradford (1976) using BSA as a standard.…”