The general objective of this research is to develop understanding of the mechanisms operating in higher plants to regulate the biosynthesis of diterpenoid metabolites that contribute to a plant's ability to survive environmental and biological stresses. The focus is on the regulation of cyclization enzymes that catalyze the first committed step of the pathway leading to a physiologically active diterpenoid metabolite(s), and thus is a logical site for regulation. One target enzyme is casbene synthetase, which catalyzes the synthesis of the putative phytoalexin, casbene, in castor bean seedlings. The casbene synthetase gene is transcriptionally activated in response to treatment of the seedlings with pectic fragment elicitors. Genomic clones containing casbene synthetase coding sequences have been isolated. A major goal of the research is to characterize the casbene synthetase gene and the cis- and trans-acting factors that participate in the transcriptional activation of the gene in response to pectic fragment elicitors. These investigations are of importance in connection with plant disease resistance since phytoalexins are among the defensive components that appear in a resistance response. Another experimental target is kaurene synthetase, a key regulatory enzyme in the pathway leading to the gibberellin family of regulators that help mediate environmental influences on plant growth and development. A major objective is to purify kaurene synthetase to homogeneity, and to use the purified protein to prepare specific antibodies and kaurene synthetase cDNA probes. These probes will be used to measure kaurene synthetase mRNA levels in plant tissues, and changes in their levels under different environmental conditions, as an indicator of their relative potentials for gibberellin biosynthesis.
