Tissue-Specific Expression and Subcellular Localization of Maize Beta-Glucosidase

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Beta-glucosidase (EC 3.2.1.21) catalyses the hydrolysis of aryl- and alkaly-beta-D-glucosides, as well as cellobiose. In maize, the enzyme is encoded by a highly polymorphic locus, Glu1. The main objectives of the proposed research are to clone and characterize the gene coding for the maize beta-glucosidase enzyme; to deduce the amino acid sequence of the enzyme from the sequence of its gene; to determine in what organs and tissues this enzyme is found; to determine where in the cell this enzyme is found, and to determine the natural substrates of the enzyme. The gene will be cloned into a multipurpose expression vector; and the desired recombinant clones will be indentified by immunoscreening. The natural substrates will be determined by extracting beta-glucosides with alcohol from maize tissues and testing whether the extracts contain a substance which will competitively inhibit beta- glucosidase hydrolysis of commonly used artificial substrates (e.g., p-nitrophenol-beta-D-glucopyranoside) which are readily hydrolyzed by beta-glucosidase to produce glucose. Determining in which tissues the enzyme is found will be done by infusing tissue sections with chromogenic beta-glucosides (e.g., X-glu and 6-bromo- 2-naphthyl beta-glucoside), which produce colored precipitates when cleaved by beta-glucosidase. The location of the color can then be observed under the light microscope. Subcellular localization will be done using immunocytochemical techniques. The results of the proposed research will further knowledge of the structure, location, function, and regulation of plant beta- glucosidases; they will contribute to studies directed at finding the molecular basis for subcellular targeting and will aid in the development of markers which would be useful in maize breeding.