Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis / 中国中药杂志

The study aimed to clone the open reading frame of cinnamate 4-hydroxylase (C4H) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. One unique sequence containing C4H domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of C4H was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. C4H expression profiles in responds to MeJA (methyl jasmonate) application were analyzed by real-time PCR. The length of C4H open reading frame (ORF) was 1 515 bp, encoding 514 amino acids. The GenBank accession number is KF134783. Inducible-experiments showed that the genes were induced by mechanical wound as well as MeJA induction, and reached the highest expression level at 8 h and 20 h, respectively. The full-length cDNA of C4H and its expression patterns will provide a foundation for further research on its function in the molecular mechanisms of aromatic compounds and flavonoids biosynthesis.

Effect of different developmental stage on plant growth and active compounds in Scutellaria baicalensis / 中国中药杂志

<p><b>OBJECTIVE</b>To study the developmental phase on the growth and active compounds in Scutellaria baicalensis.</p><p><b>METHOD</b>Seeds of wild plants were collected from Laiwu and sowed in Fangshan (Beijing) and Laiwu (Shandong). Samples of aerial and underground parts were collected in five growth periods of sprouts, seedlings, flowering, seed drop and withered periods respectively. The length of taproot, fresh weight of root, diameter of taproot and the length of stem were determined. The content of active compounds and total flavonoids were determined by HPLC and ultraviolet spectrophotometry respectively. The transcripted level of PAL1, PAL2, PAL3, C4H, 4CL, CHS, GUS and UBGAT were analyzed with RT-PCR.</p><p><b>RESULT</b>The results showed that the aerial part of S. baicalensis grew quickly before flowering stage, and the underground part grew mostly between the periods of flowering and withered. In the whole growing developmental periods, the content of total flavonoids was not changed significantly, the content of baicalin was increased gradually and the content of baicalein was decreased gradually. Expression level of PAL and 4CL was the highest in withered period, CHS was increased between flowering and seed drop and decreased in withered period.</p><p><b>CONCLUSION</b>Seedlings and withered periods may be the key phase affecting the growth and active compounds in S. baicalensis.</p>

Studies on cinnamic acid-4-hydroxylase from wounded potato tissue: isolation and characterization of cinnamic acid-4-hydroxylase activation factor.

Cinnamic acid-4-hydroxylase activation factor has been found to be located in the supernatant fraction of wounded potato tissue homogenate in phosphate buffer. The factor has been purified to homogeneity as judged by SDS polyacrylamide gel electrophoresis, by heat treatment on boiling water-bath for 7.5 min followed by dialysis with cut off limit of 8 kDa and final separation by gel filtration on Sephadex G-50 column. Gel filtration resolved this into three active fractions of molecular mass 12500, 10000 and 8500 Da conjugated to a fluorescent compound and subsequently identified as a folate derivative. The amino acid analysis of polypeptide chains of these fractions revealed that the polypeptides were rich in glutamic and aspartic acids. The fluorescent moiety of the complex released from polypeptide chain of molecular weight 10000 by mild acid hydrolysis was able to support the growth of Lactobacillus casei ATCC 7469 which requires folic acid for its growth. On storage, this compound degraded into a number of fluorescent products identified as p-amino benzoic acid, p-amino benzoyl glutamic acid, pteroic acid and 6-methyl pterin indicating that the activation factor is a folic acid derivative conjugated with the polypeptide chain.

The phenyl propanoid pathway enzymes in Solanum tuberosum exist as a multienzyme complex.

The elution profile of the core sequence enzymes of the phenyl propanoid pathway, namely phenyl alanine ammonia lyase, t-cinnamic acid 4-hydroxylase and p-coumaryl CoA ligase, on AcA 34 column suggested the existence of a high molecular form (P1) and a low molecular form (P2) for all the three enzymes. All the P1 forms eluted together in same fractions, while the P2 forms eluted out according to their respective molecular mass. Rechromatography of P1 form under identical conditions showed a similar elution profile (Q1 and Q2 forms). Further, the Q1 form did not show any significant increase in specific activity when compared to the P1 form. These results suggested the possibility of these enzymes existing as a protein cluster. Further confirmation was obtained on repeated column chromatography of the Q1 form in presence of 0.1 M KCl which did not result in complete dissociation of the complex to its individual enzyme components. The identification of the subunit polypeptide of the individual enzyme components in the multi enzyme complex and the in vitro demonstration of the phenyl propanoid core pathway reaction sequence using phenylalanine alone as a substrate supplementing the required cofactors for appropriate reactions substantiated that at least the core enzymes of the phenyl propanoid sequence existed as a multi enzyme complex.

Identification of a non-microsomal cinnamic acid 4-hydroxylase from potato tuber (S. tuberosum) and its partial purification.

The cytoplasmic localisation of cinnamic acid 4-hydroxylase (CA4H) has been shown by isolation and subcellular fractionation of the enzyme in Hepes buffer. The enzyme was purified by ammonium sulphate fractionation followed by AcA-34 molecular sieve chromatography. The enzyme existed as a high molecular mass which dissociated to a lower form on dilution on the column. The pH optimum, sulphydryl requirement, molecular and preliminary kinetic characteristics were investigated.

Studies on the induction of cinnamic acid 4-hydroxylase in potato tuber.

The change in activity of cinnamic acid 4-hydroxylase (CA4H) in potato parenchyma tissue exposed to various conditions has been examined. Maximum induction of CA4H activity was obtained at 18 hr of incubation. Though CA4H induction can occur in dark, over 100% increase in enzyme activity was obtained on exposure of the tissue to light. Actinomycin D and cycloheximide inhibited the induction process. Mn2+, though known to cause an induction of CA4H in Jerusalem Artichoke, strongly inhibited potato CA4H induction. Dithiothreitol enhanced the CA4H activity due to either activation or protection of the enzyme. CA4H induction was significantly regulated at very low concentrations of trans-cinnamate and paracoumarate.