β-Glucosidases and hydroxamic acid glucosides

The thesis is an investigation of a proposed defense system in the Poaceae, consisting of glycosylated substrates being activated by β-glucosidase enzymes upon tissue rupture. The cyclic hydroxamic acids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) are secondary metabolites that occur in high abundance as glucosides in the Poaceae, among them the cereals maize (Zea mays), wheat (Triticum aestivum) and rye (Secale cereale). Upon tissue disruption the hydroxamic acid glucoside is cleaved by β-glucosidase to release a toxic aglucone that is unstable and decomposes to the reactive benzoxazolinones. To protect the intact plant itself from the toxic products released by hydrolysis of the glucosides, the glucoside substrates and the enzyme need to be compartmentalized. In this study, the β-glucosidases in rye, wheat and maize seedlings are shown to be located in the epidermis and in cells associated with the vascular tissues. At the cellular level, β-glucosidase enzyme was located to plastids, cell walls and cytoplasm in all three species. To locate the hydroxamic acid glucoside substrates in rye, wheat and maize seedlings, tissue sections were stained with ferric chloride. In all three species staining was observed in the vascular bundles of shoots and coleoptiles, in the vascular cylinder in roots and in the outer cell layers of shoots, coleoptiles and roots. In order to characterize the enzymatic part of the defense system a rye β-glucosidase cDNA was isolated. It was identified as a gene for a plastid located β-glucosidase (EC3.2.1.21) of glycoside hydrolase family 1. The sequence corresponding to the mature protein was expressed in Escherichia coli, purified and compared to purified intracellular β-glucosidase of rye. The recombinant protein was found to be homologous to the intracellular rye enzyme. In addition a cell wall enzyme from rye has been purified and identified as a glucan 1,3-β-glucosidase (EC3.2.1.58) of glycoside hydrolase family 3. Both the plastidic and the cell wall enzyme exhibited activity with DIBOA-glucoside and DIMBOA-glucoside as substrates and may therefore both be involved in the activation of the defense response.