Structure, properties, and tissue localization of apoplastic alpha-glucosidase in crucifers

Apoplastic alpha-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identi fied. Arabidopsis contains at least three alpha-glucosidase genes; Aglu-1 a nd Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tag s. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli r ecognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napu s L.). The acidic alpha-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active f ractions were resolved and both contained a 96-kD immunoreactive polypeptid e. The N-terminal sequence from the 96-kD broccoli alpha-glucosidase indica ted that it corresponds to the Arabidopsis Aglu-2 gene and that approximate ly 15 kD of the predicted N terminus was cleaved. The 81-kD protein was mor e abundant than the 96-kD protein, but it was not active with 4-methylumbel liferyl-alpha-D-glucopyranoside as the substrate and it did not bind to con canavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-alp ha-D-glucopyranoside revealed that the acidic alpha-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascula r tissue, especially in leaf traces.