REGULATION OF MAIZE PROVITAMIN A CAROTENOID BIOSYNTHESIS

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Carotenoids, derived from plant food sources, are converted in humans to vitamin A. Vitamin A can not be synthesized de novo and it is essential for growth and development. Deficiencies manifest as xerophthalmia, blindness, increased mortality due to increased severity of childhood diseases, and increased maternal transmission of viruses such as HIV. Endosperms of food crops, such as maize and wheat, are low in pro- vitamin A as compared with non-provitamin A carotenoids; rice endosperms are completely deficient in carotenoids of any sort. About 250 million children worldwide suffer from Vitamin A deficiency that is associated with consumption of foods low in provitamin A carotenoids; it has been estimated that improved Vitamin A nutrition would eliminate approximately 1.3-2.5 million annual deaths. Vitamin A intervention programs have proven only moderately effective; an alternative approach to alleviating Vitamin A deficiency worldwide is to improve levels of provitamin A carotenoids in food stables through metabolic engineering of the carotenoid biosynthetic pathway. To metabolically engineer enhanced levels of provitamin A carotenoids, we must understand how the carotenoid biosynthetic pathway is regulated, particularly in the nutritionally important endosperm of the seed. We propose that the relative accumulation of provitamin A carotenoids is mediated by control of transcript levels for the biosynthetic enzymes; and that appropriate modification (enhancement of repression) of transcript levels in maize endosperm can lead to increased levels of provitamin A carotenoids relative to other non-provitamin A carotenoids. We propose to isolate and study of additional genes needed to test our hypothesis; we will isolate cDNA(s) encoding GGPPS (geranylgeranyl pyrophosphate synthase), a pivotal enzyme "upstream" of the pathway, and genes encoding enzymes that specifically affect the relative accumulation of the provitamin A betacarotene (LCYB, LCYE and HYDB) (lycopene betacyclase, lycopene epsilon cyclase, betacarotene hydroxylase). We will focus on cDNA isolation, transcript, protein and carotenoid analysis, followed by experiments leading to modification of transcript levels in plant tissue culture and in transgenic plants. Novel approaches for gene isolation will involve the use of "color complementation," a screening approach based on function of the gene product rather than nucleic acid homology.