Dissertation
BIOCHEMICAL CHARACTERIZATION OF HYDROXYCINNAMOYL COA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) AND HYDROXYCINNAMOYL COA:QUINATE HYDROXYCINNAMOYL TRANSFERASE (HQT): EFFECTS OF GENE MODULATION IN PHENYLPROPANOID PATHWAY ON LIGNIN AND CHLOROGENIC ACID BIOSYNTHESIS
Doctor of Philosophy (PhD), Washington State University
01/2010
Handle:
https://hdl.handle.net/2376/2814
Abstract
To comprehensively investigate: effects of phenylpropanoid pathway modulation to anti-oxidant
chlorogenic acid and lignins in tobacco (<italic>Nicotiana tabacum<italic>), and whether metabolic cross-talk occurred;
substrate versatilities of hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferases
(HCT/HQT); and effects of manipulating monolignol pathway steps on lignin primary structures and
vascular plant cell wall properties in <italic>Arabidopsis thaliana<italic>.
<italic>HQT RNAi<italic> transgenic tobacco plants were generated with similar growth/development and lignin
deposition patterns to WT; however, CGA levels were drastically reduced by ~99%, ~92% and ~70% in
stem tissue, apical and basal leaves. For <italic>HCT RNAi<italic> lines, phenotypes were severely stunted, with
partially reduced lignin levels slightly H-enriched. Although no HCT transcripts were detected, lines still
produced G- and S- lignins. No significant effect on chlorogenic acid levels occurred. Data suggest HCT
and HQT branches of metabolism are independent, with additional factors involved in G/S monolignol
deposition.
Kinetic studies of HCT and HQT were carried out. HCT readily converted <italic>p<italic>-coumaryl CoA and
caffeoyl CoA in presence of shikimic acid into corresponding shikimate esters, but was also able to
slowly catalyze reverse reaction. HQT uses <italic>p<italic>-coumaryl CoA and caffeoyl CoA as preferred substrates
with quinic acid, but reverse reaction was not readily catalyzed. Following HCT crystal structure
determinations, site-directed mutagenesis established that His153 functioned as general base, whereas
alanine substitution of two other residues resulted in reduced catalytic activity.
Second area of emphasis was to investigate lignin macromolecular configuration and assembly.
Gene families included <italic>p<italic>C3H, 4CL (4-coumarate CoA ligase), F5H (ferulate 5-hydroxylase), "caffeic
acid <italic>O<italic>-methyl transferase" (COMT), CCR (cinnamoyl CoA reductase) and CAD (cinnamyl alcohol
dehydrogenase), respectively. While such modulations resulted in H-,G- and S- enriched lignins, albeit
with lignin amounts varying substantially, we established a linear relationship between estimated lignin
levels versus cleavable monomer release independent of monomeric composition. This relationship was
established for <italic>p<italic>C3H, 4CL, F5H, CCR and CAD lines. One exception was COMT knockout, which had
a significant difference in cleavable monomer release relative to estimated lignin contents. However,
when modified 8-<italic>O<italic>-4´ (benzodioxane) inter-unit linkage frequencies were taken into account, the same
relationships held indicative of control over lignin primary structures
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Details
- Title
- BIOCHEMICAL CHARACTERIZATION OF HYDROXYCINNAMOYL COA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) AND HYDROXYCINNAMOYL COA:QUINATE HYDROXYCINNAMOYL TRANSFERASE (HQT): EFFECTS OF GENE MODULATION IN PHENYLPROPANOID PATHWAY ON LIGNIN AND CHLOROGENIC ACID BIOSYNTHESIS
- Creators
- Claudia L. Cardenas-Ardila
- Contributors
- Norman G Lewis (Advisor)John A Browse (Committee Member)Amit Dhingra (Committee Member)Sonja Roje (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 366
- Identifiers
- 99900581549501842
- Language
- English
- Resource Type
- Dissertation