Hot Air Treatment Induces Disease Resistance through Activating the Phenylpropanoid Metabolism in Cherry Tomato Fruit.

To explore the effects of hot air (HA, 38 °C for 12 h) treatment on the phenylpropanoid metabolism in cherry tomatoes, phenylpropanoid metabolite levels and the activities and expression of key enzymes were analyzed in HA-treated fruit. HA treatment enhanced phenylpropanoid metabolism, as evidenced by elevated levels of phenolics and flavonoids, higher activities of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase, and upregulated expression of LeCHS, LeCHI, LeF3H, and LeFLS. Levels of several phenylpropanoid metabolites were higher after HA treatment, including p-coumaric acid, caffeic acid, chlorogenic acid, isoquercitrin, quercetin, and rutin. These metabolic changes may be related to the reduced disease incidence and smaller lesion diameters observed in HA-treated fruit inoculated with Alternaria alternata (black mold) or Botrytis cinerea (gray mold). The results suggest that HA treatment induces disease resistance by activating the phenylpropanoid pathway in cherry tomato fruit.

Regulation of the Phenylpropanoid Pathway: A Mechanism of Selenium Tolerance in Peanut (Arachis hypogaea L.) Seedlings.

To clarify the mechanisms of selenium (Se) tolerance in peanut seedlings, we grew peanut seedlings with sodium selenite (0, 3, and 6 mg/L), and investigated the phenylpropanoids metabolism in seedling roots. The results showed that selenite up-regulated the expression of genes and related enzyme activities involving in the phenylpropanoids biosynthesis cascade, such as phenylalanine ammonia-lyase, trans-cinnamate-4-hydroxylase, chalcone synthase, chalcone isomerase, and cinnamyl-alcohol dehydrogenase. Selenite significantly increased phenolic acids and flavonoids, which contributed to the alleviation of selenite-induced stress. Moreover, selenite enhanced the formation of endodermis in roots, which may be attributed to the up-regulation of lignin biosynthesis mediated by the selenite-induced changes of H2O2 and NO, which probably regulated the selenite uptake from an external medium. Accumulation of polyphenolic compounds via the phenylpropanoid pathway may be one of the mechanisms of the increasing selenite tolerance in plants, by which peanut seedlings survived in seleniferous soil, accompanied by accumulation of Se.

Chlorogenic Acid Biosynthesis Appears Linked with Suberin Production in Potato Tuber (Solanum tuberosum).

Potato (Solanum tuberosum L.) is a good source of dietary antioxidants. Chlorogenic acid (CGA) and caffeic acid (CA) are the most abundant phenolic acid antioxidants in potato and are formed by the phenylpropanoid pathway. A number of CGA biosynthetic routes that involve hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and/or hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) have been proposed, but little is known about their path in potato. CA production requires a caffeoyl shikimate esterase (CSE), and CA serves as a substrate of lignin precursor ferulic acid via the action of caffeic/5-hydroxyferulic acid O-methyltransferase (COMT I). CGA is precursor of caffeoyl-CoA and, via caffeoyl-CoA O-methyltransferase (CCoAOMT), of feruloyl-CoA. Feruloyl-CoA is required for lignin and suberin biosynthesis, crucial for tuber development. Here, metabolite and transcript levels of the mentioned and related enzymes, such as cinnamate 4-hydroxylase (C4H), were determined in the flesh and skin of fresh and stored tubers. Metabolite and transcript levels were higher in skin than in flesh, irrespective of storage. CGA and CA production appear to occur via p-coumaroyl-CoA, using HQT and CSE, respectively. HCT is likely involved in CGA remobilization toward suberin. The strong correlation between CGA and CA, the correspondence with C4H, HQT, CCoAOMT2, and CSE, and the negative correlation of HCT and COMT I in potato tubers suggest a major flux toward suberin.

[Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis].

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.

Overexpression of cinnamate 4-hydroxylase and 4-coumaroyl CoA ligase prompted flavone accumulation in Scutellaria baicalensis hairy roots.

Scutellaria baicalensis Georgi, a species of the Lamiaceae family, is considered as one of the 50 fundamental herbs used in traditional Chinese medicine. In order to enhance flavone (baicalein, baicalin, and wogonin) content in S. baicalensis roots, we overexpressed a single gene of cinnamate 4-hydroxylase (C4H) and 4-coumaroyl coenzyme A ligase (4CL) using an Agrobacterium rhizogenes-mediated system. SbC4H- and Sb4CL-overexpressed hairy root lines enhanced the transcript levels of SbC4H and Sb4CL compared with those in the control and also increased flavones contents by approximately 3- and 2.5-fold, respectively. We successfully engineered the flavone biosynthesis pathway for the production of beneficial flavones in S baicalensis hairy roots. The importance of upstream gene C4H and 4CL in flavone biosynthesis and the efficiency of metabolic engineering in promoting flavone biosynthesis in S. baicalensis hairy roots have been indicated in this study.

Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

[Effect of different developmental stage on plant growth and active compounds in Scutellaria baicalensis].

OBJECTIVE: To study the developmental phase on the growth and active compounds in Scutellaria baicalensis. METHOD: 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. RESULT: 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. CONCLUSION: Seedlings and withered periods may be the key phase affecting the growth and active compounds in S. baicalensis.

Differential expression of anthocyanin biosynthetic genes and anthocyanin accumulation in tartary buckwheat cultivars 'Hokkai t8' and 'Hokkai t10'.

Six genes involved in anthocyanin biosynthesis in tartary buckwheat have been cloned, namely, FtC4H, Ft4CL, FtCHI, FtF3H, FtF3'H, and FtANS, which encode cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone isomerase (CHI), flavones 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), and anthocyanidin synthase (ANS), respectively. Then, these cDNAs were used, along with previously isolated clones for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), to compare gene expression in different organs, flowering stages, and maturing seeds of tartary buckwheat cultivars 'Hokkai T8' and 'Hokkai T10'. Quantitative real-time polymerase chain reaction analysis showed that these anthocyanin biosynthetic genes were most highly expressed in the stems and roots of Hokkai T10. The FtANS gene was more highly expressed than other genes during flowering and maturing seeds. In addition, the anthocyanin concentration was higher in 'Hokkai T10' than in 'Hokkai T8'; however, naringenin chalcone, a flavonoid, was absent from 'Hokkai T10' seedlings based on fluorescence microscopy.

Differential expression of flavonoid biosynthesis genes and accumulation of phenolic compounds in common buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum) is a short-season grain crop that is a source of rutin and other phenolic compounds. In this study, we isolated the cDNAs of 11 F. esculentum enzymes in the flavonoid biosynthesis pathway, namely, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) 1 and 2, chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonol synthase (FLS) 1 and 2, and anthocyanidin synthase (ANS). Quantitative real-time polymerase chain reaction analysis showed that these genes were most highly expressed in the stems and roots. However, high performance liquid chromatography analysis indicated that their flavonoid products, such as rutin and catechin, accumulated in the flowers and leaves. These results suggested that flavonoids may be transported within F. esculentum. In addition, light and dark growth conditions affected the expression levels of the biosynthesis genes and accumulation of phenolic compounds in F. esculentum sprouts.

Molecular cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in the phenylpropanoid biosynthesis pathway in garlic (Allium sativum).

The cDNAs encoding phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were cloned from garlic (Allium sativum) using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE) PCR. Amino acid sequence alignments showed that AsPAL and AsC4H have more than 70% amino acid identity with their homologues in other plants. The expression of AsPAL and AsC4H transcripts was highest in the roots but surprisingly low in the bulbils, where phenylpropanoid compounds are most concentrated. These results suggest that some phenylpropanoids are synthesized in the roots and subsequently transported to the bulbils of A. sativum .

Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis.

The initial reactions of the phenylpropanoid pathway convert phenylalanine to p-coumaroyl CoA, a branch point metabolite from which many phenylpropanoids are made. Although the second enzyme of this pathway, cinnamic acid 4-hydroxylase (C4H), is well characterized, a mutant for the gene encoding this enzyme has not yet, to our knowledge, been identified, presumably because knock-out mutations in this gene would have severe phenotypes. This work describes the characterization of an allelic series of Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, each of which harbor mis-sense mutations in C4H (At2g30490). Heterologous expression of the mutant proteins in Escherichia coli yields enzymes that exhibit P420 spectra, indicative of mis-folded proteins, or have limited ability to bind substrate, indicating that the mutations we have identified affect protein stability and/or enzyme function. In agreement with the early position of C4H in phenylpropanoid metabolism, ref3 mutant plants accumulate decreased levels of several different classes of phenylpropanoid end-products, and exhibit reduced lignin deposition and altered lignin monomer content. Furthermore, these plants accumulate a novel hydroxycinnamic ester, cinnamoylmalate, which is not found in the wild type. The decreased C4H activity in ref3 also causes pleiotropic phenotypes, including dwarfism, male sterility and the development of swellings at branch junctions. Together, these observations indicate that C4H function is critical to the normal biochemistry and development of Arabidopsis.

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea.

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA(3)) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.

Phenylalanine ammonia lyase functions as a switch directly controlling the accumulation of calycosin and calycosin-7-O-beta-D-glucoside in Astragalus membranaceus var. mongholicus plants.

Previously it had been shown that calycosin and calycosin-7-O-beta-D-glucoside (CGs) accumulate in whole plants, mainly in leaves, of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus) plants in response to low temperature. In this work, it was demonstrated that the influences of different conditions on CGs biosynthesis, by examining the changes in CGs content, as well as the expression of related genes, including phenylalanine ammonia lyase (PAL1), cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), chalcone reductase (CHR), chalcone isomerase (CHI), isoflavone synthase (IFS), and isoflavone 3'-hydroxylase (I3'H). The seven gene mRNAs accumulated in leaves of A. mongholicus upon exposure to low temperature in a light-dependent manner, though they exhibited different expression patterns. Transcriptions of CHS, CHR, CHI, IFS, and I3'H of the calycosin-7-O-beta-D-glucoside pathway were all up-regulated when plants were transferred from 16 degrees C to 2 degrees C or 25 degrees C or from 2 degrees C (kept for 24 h) to 25 degrees C. However, fluctuations in temperature influenced differently the transcriptions of PAL1 and C4H of the general phenylpropanoid pathway in leaves. Moreover, the amount of PAL1 expression changed sharply up and down, consistent with the variation of the content of CGs. PAL enzyme activity appears to be the limiting factor in determining the CGs levels. The inhibitor of PAL enzyme, L-alpha-aminooxy-beta-phenylpropionic acid, almost entirely shut down CGs accumulation at low temperature. All these results confirmed that PAL1, as a smart gene switch, directly controls the accumulation of CGs in A. mongholicus plants, in a light-dependent manner, during low temperature treatment.