Arabidopsis CYP98A3 Mediating Aromatic 3-Hydroxylation. Developmental Regulation of the Gene, and Expression in Yeast

Abstract

The general phenylpropanoid pathways generate a wide array of aromatic secondary metabolites that range from monolignols, which are ubiquitous in all plants, to sinapine, which is confined to crucifer seeds. The biosynthesis of these compounds involves hydroxylated and methoxylated cinnamyl acid, aldehyde, or alcohol intermediates. Of the three enzymes originally proposed to hydroxylate the 4-, 3-, and 5-positions of the aromatic ring, cinnamate 4-hydroxylase (C4H), which converts trans-cinnamic acid to p-coumaric acid, is the best characterized and is also the archetypal plant P450 monooxygenase. Ferulic acid 5-hydroxylase (F5H), a P450 that catalyzes 5-hydroxylation, has also been studied, but the presumptive 3-hydroxylase converting p-coumarate to caffeate has been elusive. We have found that Arabidopsis CYP98A3, also a P450, could hydroxylate p-coumaric acid to caffeic acid in vivo when expressed in yeast (Saccharomyces cerevisiae) cells, albeit very slowly. CYP98A3 transcript was found in Arabidopsis stem and silique, resembling both C4H and F5H in this respect. CYP98A3 showed further resemblance to C4H in being highly active in root, but differed from F5H in this regard. In transgenic Arabidopsis, the promoters of CYP98A3 and C4H showed wound inducibility and a comparable developmental regulation throughout the life cycle, except in seeds, where the CYP98A3 promoter construct was inactive while remaining active in silique walls. Within stem and root tissue, the gene product and the promoter activity of CYP98A3 were most abundant in lignifying cells. Collectively, these studies show involvement of CYP98A3 in the general phenylpropanoid metabolism, and suggest a downstream function for CYP98A3 relative to the broader and upstream role of C4H.Plants synthesize thousands of secondary metabolites from offshoots of primary metabolism (Croteau et al., 2000). In most cases the biosynthetic routes are unknown and even in some of the well-studied pathways many aspects remain uncertain. Phenylpropanoid metabolism generates phenolic intermediates and end products that include lignin monomers, flavonoids, isoflavonoids, lignans, tannins, quinones, and sinapate esters (Strack, 1997; Dixon and Steele, 1999;Nair et al., 2000). Lignin constitutes approximately 15% to 30% of the dry weight in woody plants, and contributes about 30% of the organic carbon in plant biomass in general (Lewis and Yamamoto, 1990; Douglas, 1996; Boudet, 2000). Thus, lignin assembly places a huge demand on phenylpropanoid supply. Lignification is considered a biochemical adaptation to provide mechanical strength and "non-seeping" water transport channels as plants adopted terrestrial habitats. The biosynthetic pathways appear to have been further diversified and recruited to supply metabolites for a variety of other end uses such as attraction of pollinators for promoting sexual propagation, pest deterrence, pathogen resistance, UV radiation protection, and allelopathic exclusion of potentially competing plants (Dixon et al., 1996). The inherent ...

A coumaroyl-ester-3-hydroxylase Insertion Mutant Reveals the Existence of Nonredundant meta-Hydroxylation Pathways and Essential Roles for Phenolic Precursors in Cell Expansion and Plant Growth

“…It is accompanied by a strong increase in p-coumaroyl esters, H lignin units, and flavonoids. This confirms the central role of CYP98A3 as the C3#H in the general phenylpropanoid pathway (Schoch et al, 2001;Franke et al, 2002aFranke et al, , 2002bNair et al, 2002). However, the persistence of detectable amounts of SM in cyp98A3 null plants and the presence of G and S lignin units mainly in their roots support the hypothesis of an alternative meta-hydroxylase pathway independent of CYP98A3.…”

“…Three hydroxylations are necessary to form these three units, which are catalyzed by three different cytochrome P450 monooxygenases: cinnamate 4-hydroxylase (C4H or CYP73), p-coumaroyl ester 3#-hydroxylase (C3#H or CYP98), and ferulate 5-hydroxylase (F5H or CYP84; Humphreys and Chapple, 2002). The 3-hydroxylation step was only recently shown to involve the cytochrome P450 CYP98A3 in Arabidopsis (Schoch et al, 2001;Franke et al, 2002aFranke et al, , 2002bNair et al, 2002). The meta-hydroxylation (or 3-hydroxylation) is not catalyzed on the free p-coumaric acid as anticipated, but on its conjugates with shikimic or quinic acids (Schoch et al, 2001).…”

Genes encoding enzymes of the lignin biosynthesis pathway in Eucalyptus

“…EGSBRT3314B10.g (30 reads, 17 in WD2) is a full-length gene and EGEQCL2001E08.g (11 reads, 8 in WD2) encodes 253 N-terminal amino acids of the protein. The protein encoded by cluster EGSBRT3314B10.g shows 77.2% sequence identity to the functionally characterized A. thaliana C3H (Schoch et al, 2001;Franke et al, 2002;Nair et al, 2002), while the peptide encoded by EGEQCL2001E08.g shows an identity of 70.7%. PSORT indicates ER localization for both proteins.…”

Genetically Engineered Crops for Biofuel Production: Regulatory Perspectives

“…CYP98A3, a candidate gene for C3H activity, converted p-coumaroyl shikimate and p-coumaroyl quinate into their corresponding caffeic acid conjugates and did not prefer p-coumaric as a substrate (Schoch et al, 2001). Further, characterization of Arabidopsis, alfalfa and poplar CYP98A3 by mutant or transgenic approaches showed that CYP98A3 is involved in lignification (Franke et al, 2002;Nair et al, 2002;Reddy et al, 2005, Coleman et al, 2008. A gene that encodes for hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) activity, which converts p-coumaryl CoA to p-coumaroyl shikimate or p-coumaroyl quinate was also identified and its role in lignin biosynthesis was unequivocally established (Hoffmann et al, 2003;.…”