Molecular and genetic studies related to zeta-carotene desaturation and carotenoid biosynthesis in maize and rice.

摘要

Carotenoids are present in all photosynthetic organisms and serve several major functions: as accessory pigments for light harvesting, as photoprotectors against photooxidative damage and as precursors of the plant hormone-abscisic acid (ABA). In this work, I have isolated and sequenced a cDNA encoding a ZDS (zeta-carotene desaturase) from maize and sequenced the corresponding gene from rice, in the carotenoid biosynthetic pathway. In both plants, the gene is likely to be single copy. The maize cDNA was functionally tested in E. coli and demonstrated to encode a two-step desaturase. With the ZDS encoding cDNA, combined with RFLP analysis, transcript and HPLC (High-pressure liquid chromatography) analysis of maize carotenoid mutants, the potential function of several of the maize genetic loci that are linked to the carotenoid desaturation steps were further characterized. It was found that maize mutants vp9 and y9 accumulate zeta-carotene, while y8 mutants are completely blocked in the pathway and do not accumulate any intermediates; it appears that the vp9 locus is the likely candidate for the zds structural gene, while y9 affects zeta-carotene, desaturation in some unknown manner. Of the other desaturation mutants tested, vp2 was found to be blocked in the biosynthesis of homogentisic acid (HGA), an intermediate of plastoquinone biosynthesis, while w3 was not, despite its conditioning of phytoene accumulation.; Preliminary studies were also carried out to characterize a rice Zds cDNA and the corresponding rice genomic DNA. To raise a maize ZDS antibody, the expression of a fusion protein-ZDS construct, pET23a-Mzds348, in the expression vector pET23a, was also achieved.; A rice mutant 84NMEMdr2 was characterized by HPLC analysis. Pigments were extracted from endosperm, embryo, and leaves of the rice mutant. In embryo and albino seedlings, a carotenoid intermediate, phytoene, was found to accumulate. This suggests that this rice mutation is due to a block in the carotenoid biosynthetic pathway at the step mediated by the enzyme phytoene desaturase (PDS). This is the first rice mutant found to be blocked in the carotenoid biosynthetic pathway.