Nam Il Park

Molecular cloning and characterization of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis

The involvement of genes in flavones biosynthesis was investigated in different organs and suspension cells obtained from Scutellaria baicalensis. Three full-length cDNAs encoding phenylalanine ammonia-lyase isoforms (SbPAL1, SbPAL2, and SbAPL3) and one gene encoding cinnamate 4-hydroxylase (SbC4H) from S. baicalensis were isolated using rapid amplification of cDNA ends (RACE)-PCR. These cDNAs were used together with previously-isolated clones for 4-coumaroyl CoA ligase (4CL) and chalcone synthase (CHS) to show the expression level in different organs of S. baicalensis. These genes were upregulated in suspension cells of S. baicalensis with biotic/abiotic stress factors. The baicalin and baicalein contents in roots were 22 and 107 times higher than those in flowers, respectively. The treatment of suspension cells with methyl jasmonate (MeJa) enhanced the major flavones in S. baicalensis. Cumulatively, the results of this study should advance ability to biosynthesize important and useful medicinal compounds from a variety of plant species.

Anthocyanin accumulation and expression of anthocyanin biosynthetic genes in radish (Raphanus sativus).

Radish [Raphanus sativus (Rs)] is an important dietary vegetable in Asian countries, especially China, Japan, and Korea. To elucidate the molecular mechanisms of anthocyanin accumulation in radish, the gene expression of enzymes directly involved in anthocyanin biosynthesis was analyzed. These genes include phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS). RsDFR and RsANS were found to accumulate in the flesh or skin of two radish cultivars (Man Tang Hong and Hong Feng No.1). Radish skin contained higher CHS, CHI, and F3H transcript levels than radish flesh in all three cultivars. In the red radish, 16 anthocyanins were separated and identified by high-performance liquid chromatography (HPLC) and elctrospray ionization-tandem mass spectrometry (ESI-MS/MS). Some of them were acylated with coumaroyl, malonoyl, feruoyl, and caffeoyl moieties. Furthermore (-)-epicatechin and ferulic acid were also identified in the three cultivars.

Metabolic Profiling of Glucosinolates, Anthocyanins, Carotenoids, and Other Secondary Metabolites in Kohlrabi (Brassica oleracea var. gongylodes)

We profiled and quantified glucosinolates, anthocyanins, carotenoids, and other secondary metabolites in the skin and flesh of pale green and purple kohlrabis. Analysis of these distinct kohlrabis revealed the presence of 8 glucosinolates, 12 anthocyanins, 2 carotenoids, and 7 phenylpropanoids. Glucosinolate contents varied among the different parts and types of kohlrabi. Glucoerucin contents were 4-fold higher in the flesh of purple kohlrabi than those in the skin. Among the 12 anthocyanins, cyanidin 3-(feruloyl)(sinapoyl) diglucoside-5-glucoside levels were the highest. Carotenoid levels were much higher in the skins than the flesh of both types of kohlrabi. The levels of most phenylpropanoids were higher in purple kohlrabi than in pale green ones. trans-Cinnamic acid content was 12.7-fold higher in the flesh of purple kohlrabi than that in the pale green ones. Thus, the amounts of glucosinolates, anthocyanins, carotenoids, and phenylpropanoids varied widely, and the variations in these compounds between the two types of kohlrabi were significant.

Carotenoid content and expression of phytoene synthase and phytoene desaturase genes in bitter melon (Momordica charantia)

Momordica charantia, a tropical plant, produces a fruit that has a β-carotene concentration five times higher than that of carrot. To elucidate the molecular basis of β-carotene accumulation in M. charantia, the gene expression levels of phytoene synthase (McPSY) and phytoene desaturase (McPDS) were determined. These levels were particularly high in the flowers of M. charantia. During fruit maturation, the expression levels of McPSY and McPDS decreased during the mid-stages but increased in the fully mature fruit. In addition, carotenoids accumulated as the peel changed from green to orange. Thus, McPSY and McPDS expression correlated with carotenoid accumulation during fruit maturation. Principal component analysis (PCA) also was used to evaluate the differences among the profiles of seven carotenoids identified in the fruit at several maturation stages. Riper fruits had higher carotenoid concentrations than less ripe fruits.

Enhancement of flavone levels through overexpression of chalcone isomerase in hairy root cultures of Scutellaria baicalensis.

A complementary DNA (cDNA) encoding Scutellaria baicalensis chalcone isomerase (SbCHI) was isolated using rapid amplification of cDNA ends polymerase chain reaction. After the treatment of wounding or methyl jasmonate, SbCHI transcripts were increased in S. baicalensis cell suspensions. SbCHI-overexpressed and SbCH-silenced transgenic hairy root lines were established by using an Agrobacterium rhizogenes-mediated system. SbCHI-overexpressed hairy root lines not only enhanced SbCHI gene expression but also produced more flavones (i.e., baicalin, baicalein, and wogonin) than the control hairy root line. In contrast, SbCHI-silenced hairy root lines reduced SbCHI transcripts and flavone production compared to those of the control hairy roots. In addition, the amount of wogonin in all hairy root cultures was increased compared to that of wild-type roots of S. baicalensis. Finally, this study showed the importance of CHI in flavone biosynthesis and the efficiency of metabolic engineering in S. baicalensis hairy roots.

Accumulation of Tilianin and Rosmarinic Acid and Expression of Phenylpropanoid Biosynthetic Genes in Agastache rugosa

Korean mint (Agastache rugosa), a perennial, medicinal plant of the Labiatae family, has many useful constituents, including monoterpenes and phenylpropanoids. Among these, tilianin and rosmarinic acid, 2 well-known natural products, have many pharmacologically useful properties. Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first and second committed steps in the phenylpropanoid pathway of plants, leading to the production of tilianin. In this study, cDNAs encoding CHS (ArCHS) and CHI (ArCHI) were isolated from A. rugosa using rapid amplification of cDNA ends (RACE)-PCR. Amino acid sequence alignments showed that ArCHS and ArCHI shared high sequence identity and active sites with their respective orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of genes involved in tilianin and rosmarinic acid biosyntheses in the flowers, leaves, stems, and roots of A. rugosa. High-performance liquid chromatography (HPLC) revealed that the accumulation pattern of tilianin matched the expression patterns of ArCHS and ArCHI in different organs of A. rugosa. Moreover, acacetin, the precursor of tilianin, also demonstrated an accumulation pattern congruent with the expression of these 2 genes. The transcription levels of ArPAL, ArC4H, and Ar4CL were the highest in the leaves or flowers of the plant, which also contained a relatively high amount of rosmarinic acid. However, the roots showed a significant content of rosmarinic acid, although the transcription of ArPAL, ArC4H, and Ar4CL were low. The findings of our study support the medicinal usefulness of A. rugosa and indicate targets for increasing tilianin and rosmarinic acid production in this plant.

Production of phenolic compounds in hairy root culture of tartary buckwheat (Fagopyrum tataricum Gaertn).

Fagopyrum tataricum Gaertn (tartary buckwheat) is an excellent medicinal and nutrient-rich crop. It has a high content of rutin and other phenolic compounds. An experiment was conducted to investigate in vitro production of phenolic compounds from hairy root culture of tartary buckwheat. Hairy root growth was promoted by increasing culture time in MS medium. The highest hairy root growth reached up to 11.2 g/l dry weight at 18 d after placement. Transformation was confirmed by PCR using rol genes, rol A (304 bp), B (797 bp), C (550 bp), and D (1035 bp) genes which is transferred into hairy roots from the Ri-plasmid in Agrobacterium rhizogenes and is responsible for the induction of hairy root from plant species. Rutin, quercetin, (−) epicatechin, (−) catechin hydrate, gallic acid, ferulic acid, chlorogenic acid, and caffeic acid were identified both in hairy and wild type roots of tartary buckwheat. The main compound found in the both types of root was epicatechin followed by rutin. The concentration of phenolic compounds in the hairy roots of tartary buckwheat was several-fold higher compared with wild type roots of same species. Our results indicate that hairy root culture of F. tataricum is a valuable alternative approach for the production of phenolic compounds.

Cloning and Characterization of Phenylalanine Ammonia-Lyase and Cinnamate 4-Hydroxylase and Pyranocoumarin Biosynthesis in Angelica gigas

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) are important enzymes in the phenylpropanoid pathway and also in the accumulation of decursin (1) and decursinol angelate (2), which are major secondary metabolites in Angelica gigas. Using PCR with degenerate primers targeted to conserved regions of available orthologous PAL and C4H sequences, cDNAs encoding PAL and C4H from A. gigas were isolated. Both genes were used to show the comparative developmental and inducible accumulation of mRNAs in different organs and in suspension cells of A. gigas. PAL and C4H were induced most strongly in response to 300 microM methyl jasmonate treatment at 6 and 12 h, respectively, and were highly expressed in the fine roots of A. gigas. Similarly, the production of 1 and 2 was most prolific in the fine roots of the plant.

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.)

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Carotenoid accumulation and characterization of cDNAs encoding phytoene synthase and phytoene desaturase in garlic (Allium sativum).

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of AsPSY1, AsPSY2, and AsPDS in the bulbils, scapes, leaves, stems, bulbs, and roots of garlic. High-performance liquid chromatography demonstrated that carotenoids were not biosynthesized in the underground organs (roots and bulbs), but were very abundant in the photosynthetic organs (leaves) of A. sativum. A significantly higher amount of β-carotene (73.44 μg·g(-1)) was detected in the leaves of A. sativum than in the other organs.