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

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9318134 Esen A cDNA library was constructed in a bacteriophage lambda vector, positive clones selected, and the entire sequence of a Beta- glucosidase allele determined. To identify the conserved and variable regions of the maize Beta-glucosidase gene and its protein product, sequences of the sorghum and teosinte genes will be determined by polymerase chain reaction based methods and compared to that of maize. The hydroxamic acid glucoside DIMBOA-glucoside was purified and shown to be the major natural substrate of maize Beta-glucosidase. Other hydroxamic acid glucosides and natural substrates will be identified by extracting Beta-glycosides from maize tissues. They will be fractionated and tested for substances that competitively inhibit the hydrolysis of artificial substrates (e.g., p-nitrophenol-Beta-glucopyranoside) by maize Beta- glucosidase and yield glucose and an aglycone when incubated with the enzyme. The inhibitory glucosides and glycosides (i.e., putative substrates) will be purified by chromatography and identified by NMR. The hydrolysis of the identified natural substrates will be studied by appropriate spectrophotometric and chromatographic assays, and their kinetic constants (e.g., Km, Vmax, Vmax/Km) will be determined. Temporal and spatial occurrence of DIMBOA-glucoside and other hydroxamic acid glucosides will be studied. The glucosides will be extracted from different organs and plant parts sampled during ontogeny. The extracts will be analyzed for quantitative and qualitative changes in the distribution of hydroxamic acid glucosides. These changes will be related to those of the enzyme in the same organs and parts. In addition, plant organs and parts will be sectioned and reacted with the Ferric Chloride Reagent, which specifically yields a purplish blue complex with hydroxamic acids, to determine histochemically the organ and tissue distribution of hydroxamic acid glucosides. Subcellular location of hydroxamic acid glucosides will be studied by franctionating cell components by differential contrifugation procedures. Determining in which tissues the enzyme occurs will be done by infusing tissue sections with chromogenic Beta-D-glucosides (e.g., 5-bromo-4-chloro-3-indolyl Beta-D-glucoside or 6-bromo-2- naphthyl Beta-D-glucoside), which produce colored precipitates when cleaved by Beta-glucosidase. Color location can then be observed under the light microscope. Temporal and spatial expression of the Glu1 gene will be studied by activity assays, immunoblotting, in situ hybridizaiton, and Northern blotting. ***