Genetic dissection of phenylpropanoid metabolism in Arabidopsis

Phenylpropanoid biosynthesis has been studied for decades in many different plant species. In the last twenty years, Arabidopsis thaliana has risen to prominence as a useful model plant for studying this secondary metabolic pathway as well as myriad other biochemical and developmental processes. A large number of Arabidopsis mutants have been identified which exhibit altered flavonoid, sinapate ester and lignin phenotypes. The characterization of these plants and the cloning of the genes defective in these mutants has increased our understanding of the enzymes involved in phenylpropanoid metabolism as well as the proteins required for its regulation. A screen for Arabidopsis plants with decreased fluorescence in leaves identified several mutants that are blocked in phenylpropanoid biosynthesis. One of these reduced epidermal fluorescence mutants, ref2, contains reduced levels of a number of phenylpropanoid pathway-derived products, including sinapoylmalate in leaves, sinapoylcholine in seeds, and syringyl lignin in stems. The REF2 gene was cloned and found to encode the cytochrome P450 monooxygenase CYP83A1, a protein with homology to an enzyme involved in glucosinolate biosynthesis. Reinspection of the biochemical phenotypes of ref2 revealed that it accumulates reduced levels of all methionine-derived glucosinolates in seeds and leaves, suggesting that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates. The phenylpropanoid phenotypes of the ref2 mutant, therefore, suggest a novel metabolic link between glucosinolate biosynthesis, a secondary biosynthetic pathway found only in plants in the order Caparalles, and phenylpropanoid metabolism, a pathway found in all plants and considered essential to the survival of terrestrial plant species. During the characterization of the ref2 mutant, it was discovered that Arabidopsis roots exposed to light contain high levels of many soluble phenylpropanoids, including coniferin and syringin (coniferyl and sinapyl-4-O-glycosides) as well as a number of flavonoids. In contrast, roots of etiolated and soil-grown plants contain very low levels of soluble phenylpropanoids. To elucidate the apparent light-dependent regulation of root secondary metabolism, extracts of mutants defective in light perception, including phyA, phyB and hy4, as well as light response, such as hy5, cop9, cop1 and the det mutants, were analysed for phenylpropanoid content. The results of these assays showed that PHYA and PHYB are the primary photoreceptors involved in light-dependent soluble phenylpropanoid accumulation, although CRY1 is also involved in regulating primarily flavonoid biosynthesis. The absence of coniferin, syringin and flavonoids in roots of the hy5 mutant indicates that the HY5 transcription factor is required for phenylpropanoid accumulation. Further, the presence of phenylpropanoids in etiolated roots of det1 and cop9 mutants indicate that these proteins repress root phenylpropanoid biosynthesis in the absence of light. In contrast, the COP1 protein, which is thought to bind HY5 and mediates COPS degradation of HY5 in aerial tissues, does not play a role in regulating phenylpropanoid accumulation in roots. Characterization of this metabolic light response suggests that this is a new high irradiance response in Arabidopsis, and indicates that Arabidopsis roots may offer a novel system for investigating the regulation of phenylpropanoid biosynthesis