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
Cardenas-Ardila, Claudia L.
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To comprehensively investigate: effects of phenylpropanoid pathway modulation to anti-oxidant chlorogenic acid and lignins in tobacco (Nicotiana tabacum), 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 Arabidopsis thaliana. HQT RNAi 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 HCT RNAi 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 p-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 p-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 pC3H, 4CL (4-coumarate CoA ligase), F5H (ferulate 5-hydroxylase), "caffeic acid O-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 pC3H, 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-O-4´ (benzodioxane) inter-unit linkage frequencies were taken into account, the same relationships held indicative of control over lignin primary structures