9603927 Walbot Flowering plants synthesize an incredible diversity of "toxic" natural products. The PIs use many of these compounds in medicine, for example, the anti-cancer drugs taxol and colchicine, the heart medicine digitalis, and even the widely used pain relievers of aspirin and quinine are based on plant natural products. Within the plant, theses compounds and many additional pigments and secondary metabolites deter herbivores and pathogens, and are thus part of the plant's ability to survive in the natural world. Surprisingly little is known, however, about how plants can synthesize "toxic" molecules and yet avoid damaging their own cells. Biosynthesis occurs in the plant cell cytoplasm, but the toxic metabolites are stored in the plant vacuole. The biochemistry and molecular biology of anthocyanin pigment biosynthesis is the best understood secondary metabolite pathway in higher plants. Anthocyanins are the red-blue-purple pigments found in most flowers and many fruits, and their color makes them a useful "marker" for elucidating the mechanisms of vacuolar sequestration. In maize, the Bronze2 gene encodes that last genetically defined step in anthocyanin biosynthesis. This enzyme is a glutathione S-transferase (GST) that adds a chemical "tag" to cytoplasmic anthocyanin that facilitates entry into the plant vacuole. This is the first established mechanism by which plants can transport large secondary metabolites into the vacuole. In the proposed work, the investigators will define the distribution of the Bronze2 enzyme, determine if it complexes with other GSTs, and determine if this enzyme has additional in vivo substrates besides anthocyanin. Mutants in Bz2 and other maize GSTs will be used to define which enzymes perform unique roles and which have overlapping roles. Structural studies of the transiently glutathionationated anthocyanin pigment will define the site(s) of glutathione tagging and determine if subsequent anthocyanin modification in the vacuole occurs at th e same sites tagged with glutathione in the cytoplasm. Understanding the key steps of anthocyanin transfer from cytoplasm to vacuole will provide new insights into the capacity of plants to synthesize and store a wide range of toxic but very useful metabolites.
