Molecular Dissection of Metabolic Channels for Sterol and Sesquiterpene Metabolism in Tobacco

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Isoprenoids are ubiquitous in nature, essential for life, and mediate a number of interactions between organisms and their environment. In addition, plants are a rich source for pharmaceutically important isoprenoids. Yet, our understanding of the isoprenoid biosynthetic pathway in plants is limited. Recent studies indicate that tobacco cell suspension cultures are uniquely suited for detailed physiological and biochemical studies of one important but putative branch point in isoprenoid metabolism. When fungal elicitors are added to the cell cultures, the cultures cease sterol production and instead synthesize and secrete anti-microbial sesquiterpenoids. The decline in sterol biosynthesis has been correlated with suppression of squalene synthetase enzyme activity, and the induction of sesquiterpenoid biosynthesis wit the induction of a sesquiterpene cyclase. Because these two enzymes are positioned at a putative branch point in the pathway, the induction of one enzyme and the suppression of the other were interpreted as an important regulatory mechanism controlling carbon flow and hence, end product formation. This interpretation suggested that isoprenoid metabolism was occurring in a homogeneous environment with intermediates mixing freely and accessible to other to successive or competing enzymes. Recent results suggest otherwise and indicate that independently regulated arrays of isozymes dedicated to the production of specific end products might be aligned along a surface or membrane.. This hypothesis of discrete metabolic channels predicts selective transcription of genes coding for enzymes of sesquiterpene biosynthesis in elicitor-treated cells versus genes for sterol metabolism in control cells, and that the enzyme proteins must have targeting information for insertion into the correct metabolic channel or unit. The goals of this work are to test these predictions using a molecular approach including cloning of the sesquiterpene cyclase and squalene synthetase genes, analysis of the cis-elements directing their expression, and fusion of their putative targeting signal onto a reporter gene which will either alter the kind or amount of isoprenoid formed. Results from these studies will help elucidate the mechanisms regulating isoprenoid metabolism in plants, and will have important impact on for future manipulations of this pathway using genetic engineering technology.//