Accumulation of sterols and sesquiterpenoids have been correlated with changes in the activity level of particular branch point enzymes of the isopernoid biosynthetic pathway, squalene synthetase and sesquiterpene cyclase. The next obvious question is whether these enzyme activities are regulated by the absolute amounts of the enzyme proteins. Both enzymes are to be purified, antibody probes for each generated, and the absolute amount of each protein determined. The subcellular distribution of an enzyme activity common to both pathways, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and those activities unique to each, squalene synthetase and sesquiterpene cyclase, will be determined in tobacco cells fractionated by conventional biochemical methods. Experiments measuring the in vivo incorporation rate of radioactive mevalonate, isopentenyl pyrophosphate, and farnesyl pyrophosphate into sterols and sesquiterpenoids, and subsequent in vitro enzyme assays will identify other regulated, and thus potentially limiting, enzyme activities.%%% Plant isoprenoids comprise a wide range of compounds of physiological importance (ie. plant growth hormones), as well as of agricultural (ie. flavor and aroma constituents) and pharmaceutical (ie. oral contraceptives) interest. Our understanding of how plants regulate the production of these compounds is still incomplete. The proposed work is to examine 3 putative mechanisms regulating the biosynthesis of two classes of isoprenoids, sterols and sesquiterpenoids. Information of this type should provide additional strategies for the future genetic and biotechnological improvement of plants for agriculture and industry.***//
