Antifungal Hydrolases in Pea Tissue: I. Purification and Characterization of Two Chitinases and Two β-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Abstract

Chitinase and beta-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 beta-1,3-glucanases. The different molecular forms of chitinase and beta-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and beta-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 beta-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both beta-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 [(3)H]chitin, as well as in their relative lysozyme activity. Similarly, the two beta-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.