Poster: Late and Moved Abstracts

Abs # 1030: Examination of the Flavonol Synthase Isoforms in Arabidopsis thaliana

Presenter:	Owens, Daniel K., dowens@vt.edu
Authors	Owens, Daniel K. (A)   Walton, Anne B. (A)   Winkel, Brenda S.J. (A)
Affiliations:	(A): Virginia Tech

Flavonols are a ubiquitous class of plant secondary metabolites that serve essential physiological functions in plants and have potent activities when ingested by animals. Six genes were identified in Arabidopsis that appear to encode flavonol synthase (FLS), a 2-oxoglutarate-dependant dioxygenase required for the synthesis of these compounds. We tested the hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation. Analysis of promoter-GUS fusion constructs identified distinct expression patterns for AtFLS1, 2, 3, and 5. AtFLS4 and 6 do not appear to be expressed based on EST and MPSS data. These two genes, as well as AtFLS2, also contain premature stop codons that eliminate essential residues. An HPLC-based activity assay was utilized to examine the substrate specificities of the three other FLS isoforms. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. Homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanidin synthase as a template to identify structural elements that may contribute to biochemical activity. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest that Arabidopsis contains a single catalytically-active form of FLS. The possibility that the expressed pseudogenes, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production is being explored using RNAi and yeast two-hybrid analyses.