Title page for ETD etd-546192639761151

Type of Document Dissertation
Author Pelletier, Matthew K.
Author's Email Address mpelleti@vt.edu
URN etd-546192639761151
Title Molecular and Biochemical Genetics of 2-Oxoglutarate-Dependent Dioxygenases Required for Flavonoid Biosynthesis in Arabidopsis thaliana
Degree PhD
Department Biology
Advisory Committee
Advisor Name Title
Cramer, Carole L.
Esen, Asim
Falkinham, Joseph O. III
Rutherford, Charles L.
Winkel, Brenda S. J. Committee Chair
  • flavanone 3-hydroxylase
  • flavonol synthase
  • cross-pathway regulation
  • metabolic regulation
  • leucoanthocyanidin dioxygenase
Date of Defense 1997-04-24
Availability unrestricted
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.

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