Three 2-oxoglutarate-dependent dioxygenases required for flavonoid biosynthesis were characterized in Arabidopsis thaliana. Genes encoding flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and leucoanthocyanidin dioxygenase (LDOX) were cloned and sequenced. The predicted proteins encoded by each of these Arabidopsis genes shared high homology with all F3H, FLS, or LDOX sequences available in Genbank. Low-stringency DNA blot analysis indicated that F3H and LDOX are encoded by a single gene in Arabidopsis, while FLS may be encoded by two or three genes. RNA blot analysis was performed to determine the expression patterns of these three genes relative to previously-cloned genes encoding flavonoid biosynthetic enzymes. Light-induction experiments and analysis of regulatory mutants showed that the CHS, CHI, F3H, and FLS1 are coordinately regulated in Arabidopsis seedlings, encode enzymes acting near the beginning of the pathway, and are therefore referred to as "early" genes. The same experiments showed that DFR and LDOX are regulated distinctly from "early" genes, share similar expression patterns in response to light, and are not expressed in the ttg mutant. DFR and LDOX are therefore referred to as "late" genes due to the timing of expression in response to light and the fact that they encode enzymes acting late in flavonoid biosynthesis. To determine whether any of the previously-identified transparent testa mutants were defective in F3H, FLS, or LDOX, the chromosomal locations of these genes in the Arabidopsis genome were determined. The positions of these genes suggested that no previously-identified tt mutant was defective in the cloned FLS or LDOX structural genes, while tt6 was potentially the F3H locus. The coding region of F3H was amplified by PCR from tt6 genomic DNA and sequenced, and several point mutations were found in the coding region of this allele, three of which are predicted to result in amino acid substitutions. Polyclonal antibodies were also developed using four different purified, recombinant flavonoid enzymes as antigens. These antibodies were used to determine the pattern of accumulation of flavonoid enzymes in developing seedlings. Immunoblot analysis was also performed to determine whether mutations in genes encoding specific flavonoid enzymes or an enzyme in pathways that compete for or provide substrate for flavonoid biosynthesis (mutants defective in tryptophan or ferulic acid biosynthesis) affect the levels of flavonoid enzymes. These analyses showed that mutant seedlings which lacked specific flavonoid or tryptophan biosynthetic enzymes accumulated higher steady-state levels of other enzymes in the pathway. These results suggest that the accumulation of specific flavonoid intermediates or indole can lead directly or indirectly to higher levels of flavonoid enzymes. / Ph. D.

The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. 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. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. 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 a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis. / Ph. D.