Stress responses in alfalfa (Medicago sativa L) XVII. Identification of multiple hydrolases and molecular characterization of an acidic glucanase

Abstract

Multiple chitinases and glucanases were identified in extracts from fungal infected alfalfa tissues by chromatofocusing, activity gel assays and Western blotting. At least six glucanase isoforms were detected by activity staining of isoelectric focusing gels, varying in apparent pI from 4·7 to approximately 10·0. Chitinases were purified from alfalfa extracts by affinity chromatography; several chitinases in alfalfa extracts cross-reacted on Western blots with anti-chitinase antibody prepared against a basic bean chitinase. Thus, both chitinase and glucanase families appear to be complex in alfalfa. A cDNA for an acidic glucanase Aglu1, with a deduced pI of around 6·0, was isolated from a library prepared from RNA from elicited alfalfa suspension cells. This glucanase, which possesses a putative glycosylated C-terminal extension, appears to belong to a small gene family in alfalfa. It is not strongly expressed in alfalfa plants infected with Colletotrichum trifolii (Race 1). Glucanase transcripts detected by the Aglu1 probe were expressed at low levels in control, uninfected alfalfa plants, and were strongly induced in Phoma medicaginis infected leaf tissue and elicited cell suspension cultures.