Molecular cloning and yeast expression of cinnamate 4-hydroxylase from Ornithogalum saundersiae baker.

OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5' non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-MS and nuclear magnetic resonance (NMR) analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.

Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

Characterization of cytochrome P450 monooxygenases isolated from trichome enriched fraction of Artemisia annua L. leaf.

CYPs have major role in the biosynthesis and modification of secondary metabolites. Predicting the possible involvement of CYPs in secondary metabolism, 20 partial sequences were amplified from the cDNA of trichome enriched tissue of Artemisia annua. Seven CYPs were converted to full length and assigned to different families based on sequence homology. These were co-expressed with CPR in Saccharomyces cerevisiae and microsome fractions were assayed for conversion of sesquiterpenes, phenols and fatty acid substrates. CIM_CYP02(c73) and CIM_CYP05(c81) converted trans-cinnamic acid to p-coumaric acid; and capric acid, lauric acid to their hydroxylated products, respectively. Higher expression of CIM_CYP71AV1, CIM_CYP03(c72a), CIM_CYP06(c72b), CIM_CYP02(c73) and CIM_CYP04(c83) was observed in the mature leaf, whereas expression of CIM_CYP05(c81) was more in the seedling. CIM_CYP71AV1, CIM_CYP02(c73) and CIM_CYP04(c83) expressed more in the flower bud compared to the leaf, with minor expression in stem. All CYPs' expression increased progressively with time after wounding except for CIM_CYP07(c92). These results relate involvement of CIM_CYP02(c73) to phenyl-propanoid metabolism in the leaf and CIM_CYP05(c81) to fatty acid metabolism in the seedling. Expression of CIM_CYP71AV1 and CIM_CYP02(c73) significantly increased when sprayed with trans-cinnamic acid indicating a relationship between phenylpropanoid and artemisinic acid pathways.

cDNA cloning and expression pattern of cinnamate-4-hydroxylase in the Korean black raspberry.

Cinnamate-4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, which is responsible for synthesizing a variety of secondary metabolites that participate in development and adaptation. In this study, we isolated a full-length cDNA of the C4H gene from the Korean black raspberry (Rubus sp.) and found that this gene existed as a single gene. By comparing the deduced amino acid sequence of Rubus sp. C4H with other sequences reported previously we determined that this sequence was highly conserved among widely divergent plant species. In addition, quantitative real time PCR studies indicated that the C4H gene had a differential expression pattern during fruit development, where gene expression was first detected in green fruit and was then remarkably reduced in yellow fruit, followed by an increase in red and black fruit. To investigate the two peaks in expression observed during fruit development and ripening, we measured the flavonoid content. The content of the major flavanol of Korean black raspberry fruits was determined to be highest at the beginning of fruit development, followed by a gradually decrease according to the developmental stages. In contrast, the content of anthocyanins during the progress of ripening was dramatically increased. Our results suggest that the C4H gene in Korean black raspberry plays a role during color development at the late stages of fruit ripening, whereas the expression of C4H gene during the early stages may be related to the accumulation of flavanols.