Our long term goal is to elucidate the mechanisms that control gene expression during plant development. As a first step toward understanding the basis for this regulation, we propose to study isoprenoid biosynthesis by taking advantage of our ability to isolate mutants with biochemical defects in the pathway and to acquire cloned genes for these activities. In plants, a wide range of isoprenoid compounds, including hormones, sterols and photosynthetic pigments, play vital roles in metabolism and development. All isoprenoids are derived from a common precursor, mevalonic acid (MVA), which is synthesized by HMG CoA reductase. This enzyme plays a key role in regulating and coordinating isoprenoid biosynthesis, but the basis of this control remains obscure. Our immediate objective is to elucidate the mechanisms of HMG CoA reductase regulation by physiological, molecular and genetic analyses. To this end, we will study the organization and regulation of HMG CoA reductase using molecular probes. Two structural genes for HMG CoA reductase have been identified in Arabidopsis and isozyme-specific antibodies will be prepared against HmG CoA reductase fusion proteins expressed in E. coli. Regulation of reductase will be examined by Northern and Western blot analysis and in situ hybridization in plants responding to environmental changes, metabolic feedback and developmental cues. In addition, anti-reductase antibodies will be used to determine the localization of the two isozymes within the cell by fractionation and immunolocalization. As a complementary approach, we have devised a number of schemes to isolate isoprenoid mutants in Arabidopsis using both conventional and molecular genetics: (1) conditional and non-conditional MVA auxotrophs will be identified in mutant plant populations on the basis of a nutritional requirement for MVA, (2) anti-sense RNA will be used to inhibit HMG CoA reductase expression, (3) mutants in the plant reductase gene will be identified initially in yeast, and subsequently transformed into Arabidopsis for testing, (4) attempts will be made to disrupt the chromosomal copies of HMG CoA reductase using emerging gene targeting technologies. The characterization of these mutants will help us elucidate the steps in the isoprenoid pathway in plants and provide a genetic basis for studying isoprenoid functions during plant development.
