Isolation and Analysis of Cinnamic Acid 4-Hydroxylase Homologous Genes from a Hybrid Aspen,<i>Populus kitakamiensis</i>

Kawai, Shinya; Mori, Ariko; Shiokawa, Takahiro; Kajita, Shinya; Katayama, Yoshihiro; Morohoshi, Noriyuki

doi:10.1271/bbb.60.1586

Cited by 35 publications

(23 citation statements)

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“…Additional sequence comparisons indicated that this longer CYP73A9v1 clone contained two introns, the first 162-bp intron interrupting amino acid 262 and the second 1,726-bp intron interrupting amino acid 307. Both of these introns were located at positions conserved in other isolated t-CAH genes (Kawai et al, 1996;Bell-Lelong et al, 1997;Mizutani et al, 1997). Genomic DNA PCR using a 5Ј promoter region primer identical to Ϫ121 to Ϫ101 (relative to the ATG) in the 5Ј phage fragment and a 3Ј gene-specific primer complementary to ϩ1119 to ϩ1135 (relative to the ATG) in the coding sequence downstream of both introns generated a single PCR product corresponding to the size we would expect for the 2.9-kb CYP73A9v1 3Ј fragment containing two introns.…”

Section: Cyp73a9 Genomic Clonementioning

confidence: 99%

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Molecular Characterization of CYP73A9 andCYP82A1 P450 Genes Involved in Plant Defense in Pea

Whitbred

Schuler

2000

Plant Physiology

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Cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of phenylpropanoids, terpenes, and alkaloids. Two pea (Pisum sativum) P450 cDNAs (CYP73A9v1, encoding trans-cinnamic acid hydroxylase [t-CAH] in the core phenylpropanoid pathway, and CYP82A1v1, possibly encoding an activity in a late branch of the phenylpropanoid pathway) have previously been described. Of three CYP73A9 genes now isolated, the CYP73A9v1 gene is full-length with two introns at positions conserved in other t-CAH genes, and the CYP73A9v2 and CYP73A9v3 gene fragments are 5Ј-truncated and lack introns. The full-length CYP82A1v2 gene contains a single intron at an alternate position. Nucleotide searches of the CYP73A9v1 and CYP82A1v2 promoters have indicated that the regulatory sequences for these early and late phenylpropanoid transcripts are substantially different. The P-, L-, and H-boxes identified in white light-, ultraviolet light-, and elicitor-induced footprints in early phenylpropanoid promoters (phenylalanine ammonia lyase [PAL], 4-coumarate coenzyme A:ligase [4-CL], and chalcone synthase [CHS]) are conserved in the t-CAH promoter and are absent from the CYP82A1v2 promoter. Both promoters contain TCA motifs identified in stress-responsive promoters, box IV elements identified in elicitor-responsive PAL and CHS promoters, and spatially conserved woundresponse elements potentially coordinating regulation of these wound-responsive promoters.

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Section: Cyp73a9 Genomic Clonementioning

confidence: 99%

“…1). Although t-CAH proteins and cDNAs have been among the best characterized plant P450 sequences, very few genomic DNA clones encoding t-CAH (Kawai et al, 1996;Bell-Lelong et al, 1997;Mizutani et al, 1997) have been isolated and analyzed at the nucleotide level.…”

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confidence: 99%

Molecular Characterization of CYP73A9 andCYP82A1 P450 Genes Involved in Plant Defense in Pea

Whitbred

Schuler

2000

Plant Physiology

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“…is a useful model for the study of phenylpropanoid metabolism and lignin biosynthesis in trees (Douglas, 1996). Several genes encoding enzymes of phenylpropanoid metabolism and lignin biosynthesis have been cloned from poplar species, including PAL (Subramaniam et al, 1993;Osakabe et al, 1995), C4H (Ge and Chiang, 1996;Kawai et al, 1996), bispecific caffeic acid-Omethyltransferase (COMT; Bugos et al, 1991;Dumas et al, 1992), and CAD (van Doorsselaere et al, 1995b). The monomer composition of angiosperm lignin, including that of poplar, varies according to cell type and stage of tissue development (Campbell and Sederoff, 1996).…”

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confidence: 99%

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1

et al. 1998

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The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa ؋ Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.The enzyme 4CL (EC 6.2.1.12) catalyzes the formation of CoA thioesters of hydroxycinnamic acids by a two-step reaction mechanism that involves the hydrolysis of ATP (Gross and Zenk, 1974). These thioesters serve as substrates for a number of important reactions within plant phenylpropanoid metabolism. Depending on the species and tissue, formation of hydroxycinnamate esters and amides often involves CoA derivatives of 4-coumaric, caffeic, and/or ferulic acid, whereas flavonoid and stilbene biosynthesis requires cinnamoyl and/or 4-coumaroyl CoA esters as the substrates (Hahlbrock and Scheel, 1989;Dixon and Paiva, 1995;Holton and Cornish, 1995). The biosynthesis of salicylic acid from cinnamic acid may occur via oxidation of a cinnamoyl-CoA intermediate (Ryals et al., 1996), and within virtually all higher plants, generation of lignin monomers is presumed to require the conversion of 4-coumaric acid, ferulic acid, and sinapic acid to the corresponding CoA esters in preparation for side-chain reduction .In keeping with this metabolic demand for different hydroxycinnamoyl CoA esters, 4CL preparations from plants are normally found to be able to use a number of hydroxycinnamic acid derivatives as substrates (Gross and Zenk, 1974; Hahlbrock, 1975, 1977;Grand et al., 1983;Lozoya et al., 1988;Voo et al., 1995;Lee and Douglas, 1996). It has also been proposed, however, that different isoforms of 4CL might possess different patterns of substrate preference, and characterization of 4CL isoforms from some plants supports this (Knobloch and Hahlbrock, 1975;Ranjeva et al., 1976;Wallis and Rhodes, 1977;Grand et al., 1983). Such diversity could conceivably enable particular 4CL isoforms to efficiently supply an appro...

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“…9) For heterogeneous gene expression in yeast, the forward primer was synthesized to include a start codon (in bold) with addition of a BamHI site (italic) to aid in cloning in the yeast expression vector described below, (C4H-sense: 5Ј-CCG-GATCCAAGATAATGGATCTCCTCCTC-3Ј) and the reverse primer was also designed with addition of a BamHI site, (C4H-antisense: 5Ј-GGGATCCGCATCCAAAACCATCCCG-3Ј). All Escherichia coli manipulations were carried out in strain MV1190.…”

Section: Construction Of C4h Expression Vector In Yeastmentioning

confidence: 99%

5-Aryl-1,3,4-oxadiazole-2-thiols as a New Series of trans-Cinnamate 4-Hydroxylase Inhibitors

et al. 2004

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A series of 5-aryl-1,3,4-oxadiazole-2-thiols was found to inhibit trans-cinnamate 4-hydroxylase (C4H) from Populus kitakamiensis, which was expressed in yeast. 5-Phenyl-1,3,4-oxadiazole-2-thiol showed inhibitory activity comparable to 2-hydroxy-1-naphthoic acid, a known C4H inhibitor. Studies on the structure-activity relationship indicated that the presence of a thiol group was significant for stronger activity. Of the compounds tested, 5-(3-fluorophenyl)-1,3,4-oxadiazole-2-thiol was the most active. AE Pesticide Science Society of Japan Keywords: trans-cinnamate 4-hydroxylase, 5-aryl-1,3,4-oxadiazole-2-thiols, cytochrome P450, Populus kitakamiensis. INTRODUCTIONThe trans-cinnamate 4-hydroxylase (C4H) is a cytochrome P450 which catalyzes the hydroxylation of trans-cinnamic acid into pcoumaric acid, the first oxygenation step in the metabolism of phenylpropanoids in plants.1) Phenylpropanoid metabolism is a plant-specific pathway leading to a variety of compounds such as lignins, lignans, flavonoids, isoflavonoids and coumarins, which are essential for plant development and defense against ultraviolet light, predators and pathogens. C4H, which is highly inducible by wounding and pathogen infection, is indicated to be the major enzyme in the regulation of lignification and defenserelated reactions.2) Therefore, specific C4H inhibitors would offer an attractive means of studying the important phenylpropanoid pathway of plants. Since C4H has not been found in any invertebrate or vertebrate animals, it might be an unexploited target for development of a new herbicide. Schalk et al. described 2-hydroxy-1-naphthoic acid (1) 3) and piperonylic acid (2) 4) as a competitive inhibitor and a mechanism-based inactivator of C4H, respectively. There is no report in the literature of C4H inhibitors other than these structural analogs of cinnamic acid. By random screening, we have recently found that 5-phenyl-1,3,4-oxadiazole-2-thiol strongly inhibits C4H, CYP73a from Populus kitakamiensis, which is expressed in yeast. In the present paper we report the structure-activity relationships of a series of 5-aryl-1,3,4-oxadiazole-2-thiols. MATERIALS AND METHODS Chemicals2-Hydroxy-1-naphthoic acid and piperonylic acid were purchased from Tokyo Kasei Co. Compound 3 was purchased from Aldrich Co. 2-Methylthio-5-phenyl-1,3,4-oxadiazole (4) was prepared by methylation of 3 with methyl iodide.5) 2-Phenyl-1,3,4-oxadiazole (5) was prepared according to the reported method. 6) 5-Substituted-1,3,4-oxadiazole-2-thiols (7-30) were synthesized from the reaction of the corresponding hydrazides and carbon disulfide in the presence of potassium hydroxide according to procedures reported previously. [6][7][8] The structures of the compounds were confirmed using 1 H-NMR spectra which were recorded on a JEOL EX-400 (400 MHz) spectrometer. The following procedure for the preparation of 5-(3-fluorophenyl)-1,3,4-oxadiazole-2-thiol (23) is typical. To a solution of 2.6 g of 3-fluorobenzohydrazide and 1.2 g of potassium hydroxide in 20 ml of ethanol was add...

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Isolation and Analysis of Cinnamic Acid 4-Hydroxylase Homologous Genes from a Hybrid Aspen,Populus kitakamiensis

Cited by 35 publications

References 20 publications

Molecular Characterization of CYP73A9 andCYP82A1 P450 Genes Involved in Plant Defense in Pea

Molecular Characterization of CYP73A9 andCYP82A1 P450 Genes Involved in Plant Defense in Pea

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1

5-Aryl-1,3,4-oxadiazole-2-thiols as a New Series of trans-Cinnamate 4-Hydroxylase Inhibitors

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