Overexpression of Ginkgo biloba Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase 2 gene (GbHDR2) in Nicotiana tabacum cv. Xanthi.

2-C-Methyl-d-erythrol-4-phosphate (MEP) pathway in plant supplies isoprene building blocks for carotenoids and chlorophylls essential in photosynthesis as well as plant hormones such as gibberellin and abscisic acid. To assess the effect of overexpression of the terminal enzyme of the MEP pathway, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR), transgenic Nicotiana tabacum overexpressing class 2 HDR from Ginkgo biloba (GbHDR2) under the control of 35S promoter was constructed. Contents of chlorophylls a and b in transgenic tobacco were enhanced by 19 and 7%, respectively, compared to those of the wild type. The carotenoid level was also 18% higher than that in the control plant. As a result, photosynthetic rate of the transgenic tobacco was increased by up to 51%. Diterepenoid duvatrienediol content of transgenic tobacco was also elevated by at least sixfold. To explore the molecular basis of the enhanced isoprenoid accumulation, transcript levels of the key genes involved in the isoprenoid biosynthesis were measured. Transcript levels of geranylgeranyl diphosphate synthase (GGPP), kaurene synthase (KS), gibberellic acid 20 oxidase (GA20ox), and phytoene desaturase (PD) genes in the transgenic tobacco leaves were about twofold higher compared to the wild type. Therefore, upregulation of down-stream genes involved in biosynthesis of di- and tetraterpenoids due to GbHDR2 overexpression was responsible for elevated production of isoprenoids and enhanced photosynthetic rate. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02887-5.

Natural Korean Medicine Dang-Gui: Biosynthesis, Effective Extraction and Formulations of Major Active Pyranocoumarins, Their Molecular Action Mechanism in Cancer, and Other Biological Activities.

Angelica gigas Nakai (AGN) is a crucial oriental medicinal herb that grows especially in Korea and the Far-East countries. It contains chemically active compounds like pyranocoumarins, polyacetylenes and essential oils, which might be useful for treatment of several chronic diseases. It has been used for centuries as a traditional medicine in Southeast Asia, but in Western countries is used as a functional food and a major ingredient of several herbal products. The genus Angelica is also known as 'female ginseng' due to its critical therapeutic role in female afflictions, such as gynecological problems. However, it is well-documented that the AGN pyranocoumarins may play vital beneficial roles against cancer, neurodisorders, inflammation, osteoporosis, amnesia, allergies, depression, fungi, diabetes, ischemia, dermatitis, reactive oxygen species (ROS) and androgen. Though numerous studies revealed the role of AGN pyranocoumarins as therapeutic agents, none of the reviews have published their molecular mechanism of action. To the best of our knowledge, this would be the first review that aims to appraise the biosynthesis of AGN's major active pyranocoumarins, discuss effective extraction and formulation methods, and detail the molecular action mechanism of decursin (D), decursinol angelate (DA) and decursinol (DOH) in chronic diseases, which would further help extension of research in this area.

Molecular cloning of an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase involved in saponin biosynthesis from the medicinal plant Centella asiatica.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

Expression of Terpenoid Biosynthetic Genes and Accumulation of Chemical Constituents in Valeriana fauriei.

Valeriana fauriei (V. fauriei), which emits a characteristic and unpleasant odor, is important in traditional medicine. In this study, the expression of terpenoid biosynthetic genes was investigated in different organs that were also screened for volatile compounds including valerenic acid and its derivatives. Specific expression patterns from different parts of V. fauriei were observed using quantitative real-time PCR (qRT-PCR). The highest transcript levels of biosynthetic genes involved in mevalonic acid (MVA) and methylerythritol phosphate (MEP) production were found in the stem. Although the amounts of volatile compounds were varied by organ, most of the volatile terpenoids were accumulated in the root. Gas chromatography mass spectrometry (GC-MS) analysis identified 128 volatile compounds, which represented 65.33% to 95.66% of total volatiles. Certain compounds were only found in specific organs. For example, isovalerenic acid and valerenic acid and its derivatives were restricted to the root. Organs with high transcript levels did not necessarily have high levels of the corresponding chemical constituents. According to these results, we hypothesize that translocation may occur between different organs in V. fauriei.

Transcriptome and metabolome analysis in shoot and root of Valeriana fauriei.

BACKGROUND: Valeriana fauriei is commonly used in the treatment of cardiovascular diseases in many countries. Several constituents with various pharmacological properties are present in the roots of Valeriana species. Although many researches on V. fauriei have been done since a long time, further studies in the discipline make a limit due to inadequate genomic information. Hence, Illumina HiSeq 2500 system was conducted to obtain the transcriptome data from shoot and root of V. fauriei. RESULTS: A total of 97,595 unigenes were noticed from 346,771,454 raw reads after preprocessing and assembly. Of these, 47,760 unigens were annotated with Uniprot BLAST hits and mapped to COG, GO and KEGG pathway. Also, 70,013 and 88,827 transcripts were expressed in root and shoot of V. fauriei, respectively. Among the secondary metabolite biosynthesis, terpenoid backbone and phenylpropanoid biosynthesis were large groups, where transcripts was involved. To characterize the molecular basis of terpenoid, carotenoid, and phenylpropanoid biosynthesis, the levels of transcription were determined by qRT-PCR. Also, secondary metabolites content were measured using GC/MS and HPLC analysis for that gene expression correlated with its accumulation respectively between shoot and root of V. fauriei. CONCLUSIONS: We have identified the transcriptome using Illumina HiSeq system in shoot and root of V. fauriei. Also, we have demonstrated gene expressions associated with secondary metabolism such as terpenoid, carotenoid, and phenylpropanoid.

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.

The Mechanism Underlying the Antibacterial Activity of Shikonin against Methicillin-Resistant Staphylococcus aureus.

Shikonin (SKN), a highly liposoluble naphthoquinone pigment isolated from the roots of Lithospermum erythrorhizon, is known to exert antibacterial, wound-healing, anti-inflammatory, antithrombotic, and antitumor effects. The aim of this study was to examine SKN antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The SKN was analyzed in combination with membrane-permeabilizing agents Tris and Triton X-100, ATPase inhibitors sodium azide and N,N'-dicyclohexylcarbodiimide, and S. aureus-derived peptidoglycan; the effects on MRSA viability were evaluated by the broth microdilution method, time-kill test, and transmission electron microscopy. Addition of membrane-permeabilizing agents or ATPase inhibitors together with a low dose of SKN potentiated SKN anti-MRSA activity, as evidenced by the reduction of MRSA cell density by 75% compared to that observed when SKN was used alone; in contrast, addition of peptidoglycan blocked the antibacterial activity of SKN. The results indicate that the anti-MRSA effect of SKN is associated with its affinity to peptidoglycan, the permeability of the cytoplasmic membrane, and the activity of ATP-binding cassette (ABC) transporters. This study revealed the potential of SKN as an effective natural antibiotic and of its possible use to substantially reduce the use of existing antibiotic may also be important for understanding the mechanism underlying the antibacterial activity of natural compounds.

Phenylalanine and LED lights enhance phenolic compound production in Tartary buckwheat sprouts.

The present study aimed to investigate the effects of different l-phenylalanine (l-Phe) concentrations and various light-emitting diodes (LEDs) on the accumulation of phenolic compounds (chlorogenic acid, vitexin, rutin, quercetin, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside) in Tartary buckwheat sprouts. We found that 5mM was the optimum l-Phe concentration for the synthesis of total and individual phenolic compounds. The highest rutin (53.09 mg/g DW) and chlorogenic acid (5.62 mg/g DW) content was observed with Red+Blue and white lights. Comprehensive differences in total and individual anthocyanin content were observed between different lights; however, the total anthocyanin content (9.12 mg/g DW) was 1.5-fold higher in blue light. The expression levels of regulatory genes, such as FtDFR and FtANS, were 7.1-fold higher with l-Phe treatment. Gene expression results showed that the phenolic compounds in Tartary buckwheat sprouts increased with the use of l-Phe and LED lights.

Production of Triterpenoid Sapogenins in Hairy Root Cultures of Silene vulgaris.

Silene vulgaris (Moench) Garcke (Caryophyllaceae) is widely distributed in North America and contains bioactive oleanane-type saponins. In order to investigate in vitro production of triterpenoid saponins, hairy root cultures of S. vulgaris were established by infecting leaf explants with five strains of Agrobacterium rhizogenes (LBA9402, R1000, A4, 13333, and 15834). The A. rhizogenes strain LBA9402 had an infection of 100% frequency and induced the most hairy roots per plant. Methyl jasmonate (MeJA)-induced changes in triterpenoid saponins in S. vulgaris hairy roots were analyzed. Accumulation of segetalic acid and gypsogenic acid after MeJA treatment was 5-and 2-fold higher, respectively, than that of control root. We suggest that hairy root cultures of S. vulgaris could be an important alternative approach to the production of saponins.

Riboflavin accumulation and molecular characterization of cDNAs encoding bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase, lumazine synthase, and riboflavin synthase in different organs of Lycium chinense plant.

Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide-essential cofactors for a wide variety of enzymes involving in numerous metabolic processes. In this study, a partial-length cDNA encoding bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase (LcRIBA), 2 full-length cDNAs encoding lumazine synthase (LcLS1 and LcLS2), and a full-length cDNA encoding riboflavin synthase (LcRS) were isolated from Lycium chinense, an important traditional medicinal plant. Sequence analyses showed that these genes exhibited high identities with their orthologous genes as well as having the same common features related to plant riboflavin biosynthetic genes. LcRIBA, like other plant RIBAs, contained a DHBPS region in its N terminus and a GCHII region in its C-terminal part. LcLSs and LcRS carried an N-terminal extension found in plant riboflavin biosynthetic genes unlike the orthologous microbial genes. Quantitative real-time polymerase chain reaction analysis showed that 4 riboflavin biosynthetic genes were constitutively expressed in all organs examined of L. chinense plants with the highest expression levels found in the leaves or red fruits. LcRIBA, which catalyzes 2 initial reactions in riboflavin biosynthetic pathway, was the highest transcript in the leaves, and hence, the richest content of riboflavin was detected in this organ. Our study might provide the basis for investigating the contribution of riboflavin in diverse biological activities of L. chinense and may facilitate the metabolic engineering of vitamin B2 in crop plants.

Accumulation of phenylpropanoids and correlated gene expression in hairy roots of tartary buckwheat under light and dark conditions.

Differential expression patterns of flavonoid biosynthetic pathway genes in the hairy roots of tartary buckwheat cultivars "Hokkai T8" and "Hokkai T10" were studied over a time course of the light-dark cycle. The Agrobacterium rhizogenes-mediated transformation system was applied for inducing hairy roots. Further, a total of six phenolic compounds and two anthocyanins were analyzed in the hairy roots which were exposed to both light and dark conditions, and their amounts were estimated by HPLC. The gene expression levels peaked on day 5 of culture during the time course of both dark and light conditions. Notably, FtPAL, Ft4CL, FtC4H, FtCHI, FtF3H, FtF3'H-1, and FtFLS-1 were more highly expressed in Hokkai T10 than in Hokkai T8 under dark conditions, among which FtPAL and FtCHI were found to be significantly upregulated, except on day 20 of culture. Significantly higher levels of phenolic compound, rutin, along with two anthocyanins were detected in the hairy roots of Hokkai T10 under both conditions. Furthermore, among all the phenolic compounds detected, the amount of rutin in Hokkai T10 hairy roots was found to be ∼5-fold (59,01 mg/g dry weight) higher than that in the control (12.45 mg/g dry weight) at the respective time periods under light and dark conditions.

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.

Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus (Bge.).

Astragalus membranaceus is one of the most important traditional Korean and Chinese medicinal herbs because it contains triterpenoid saponins (astragaloside I, II, III, and IV), which have beneficial and pharmacological effects on health. In this study, we analyzed 10 mevalonate pathway genes that are involved in astragaloside biosynthesis using the Illumina/Solexa HiSeq2000 platform. We determined the expression levels of the 10 genes using quantitative real-time PCR, and analyzed the accumulation of astragalosides in different organs using high-performance liquid chromatography. Genes related to the mevalonate pathway were expressed in different levels in different organs. Almost all genes showed high transcript levels in the stem and leaf, with the lowest transcript levels being recorded in the root. In contrast, most astragalosides accumulated in the root. In particular, the astragaloside IV content was distributed in the following order: root (0.58 mg/g DW) > flower (0.27 mg/g DW) > stem (0.23 mg/g DW) > leaf (0.04 mg/g DW). In the root, astragaloside II exhibited the highest content (2.09 mg/g DW) compared to astragaloside I, III, and IV. Notably, gene expression did not follow the same pattern as astragaloside accumulation. We suggest carefully that astragalosides are synthesized in the leaves and stem and then translocated to the root. This study contributes towards improving our understanding of astragaloside biosynthesis in A. membranaceus.

Accumulation of flavonoids and related gene expressions in different organs of Astragalus membranaceus Bge.

Astragalus membranaceus is one of the important medicinal plant in China and Korea. It is used to increase metabolism and digestion, enhance the immune system, and promote the healing of wounds and injuries. In the present study, we used quantitative real-time PCR to investigate the expression of genes related to the biosynthesis of flavonoids, in addition to high-performance liquid chromatography to assess calycosin and calycosin-7-O-ß-D-glucoside accumulation, in the different plant organs of A. membranaceus. The transcript levels of all genes (AmPAL, AmC4H, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS, AmI3'H, and AmUCGT) involved in calycosin and calycosin-7-O-ß-D-glucoside biosynthesis were the highest in the flower. Calycosin content was ordered as follows: leaf (145.56 µg/g dry weight [DW]) > stem (18.3 µg/g DW) > root (1.64 µg/g DW) > flower (0.09 µg/g DW), whereas calycosin-7-O-ß-D-glucoside content was ordered as follows: root (4.88 µg/g DW) > stem (3.86 µg/g DW) > leaf (2.0 µg/g DW) > flower (not detected). All genes exhibited the highest transcription levels in the flower, whereas calycosin and its glycoside content were the highest in the leaf and root, respectively. Our results indicate that the enhancement of calycosin-7-O-ß-D-glucoside in the roots may originate from high calycosin accumulation in the stem and leaf. Thus, the mechanisms regulating calycosin and calycosin-7-O-ß-D-glucoside content differ in the different organs of A. membranaceus. The results are expected to provide baseline information from which the mechanism of flavonoid biosynthesis in the different organs of A. membranaceus may be elucidated.

Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 µg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

Effects of light-emitting diodes on expression of phenylpropanoid biosynthetic genes and accumulation of phenylpropanoids in Fagopyrum tataricum sprouts.

Buckwheat sprouts are a popular food item in many countries. The effects of light-emitting diodes (LEDs) on sprout growth and development, changes in mRNA transcription, and accumulation of phenylpropanoid compounds were studied in tartary buckwheat 'Hokkai T8' sprouts. The highest transcript levels were observed after 2 days of LED exposure for all genes, especially FtPAL and FtF3'H, which showed higher expression in sprouts grown under blue and white light than in those grown under red light. Catechin content in sprouts grown under red light increased dramatically throughout the 10 day time course. Maximum rutin content (43.37 mg/g dry weight (DW)) was observed in sprouts at 4 days after exposure (DAE) to blue light. Similarly, the highest cyanidin 3-O-rutinoside content (0.85 mg/g DW) was detected at 10 DAE to blue light. On the basis of these results, blue LED light is recommended as a light source for enhancing the content of phenolic compounds in tartary buckwheat sprouts.

Variation of glucosinolate accumulation and gene expression of transcription factors at different stages of Chinese cabbage seedlings under light and dark conditions.

Chinese cabbage is one of the most important leafy vegetables widely used in East Asian cuisines. The glucosinolate (GSL) accumulation and transcript levels of 7 transcription factors (Dof1.1, IQD1-1, MYB28, MYB29, MYB34, MYB51, and MYB122, and their isoforms) involved in the biosynthesis of aliphatic and indolic glucosinolates (GSLs) were analyzed at different stages of Chinese cabbage (Brassica rapa ssp. pekinensis) seedlings under light and dark conditions using high performance liquid chromatography and quantitative real time PCR. During seedling development, transcription of almost all transcription factors under light conditions was higher expressed than under dark conditions. Five aliphatic GSLs (progoitrin, sinigrin, glucoalyssin, gluconapin, and glucobrassicanapin) and four indolic GSLs (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrasscin) were detected. Total GSL contents under light conditions 6, 8, and 10 days after sowing (DAS) were 3.2-, 3.9-, and 6.9-fold higher, respectively than those of dark conditions. Interestingly, total GSL contents 2 {85.4 micromol/g dry weight (DW)} to 10 (7.74 micromol/g DW) DAS under dark conditions were gradually decreased. In this study, our results suggest that light affects the levels of GSL in Chinese cabbage seedlings. These results could be useful for obtaining cabbage varieties rich in GSLs.

Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

Comparative analysis of flavonoids and polar metabolite profiling of Tanno-original and Tanno-high rutin buckwheat.

Rutin is an important indicator for evaluating the quality of buckwheat. In this study, flavonoid biosynthesis was compared between two common cultivars (an original and a high-rutin line) of buckwheat, Fagopyrum esculentum Moench. Transcriptional levels of the main flavonoid biosynthetic genes were analyzed by real-time PCR, and main flavonoid metabolites were detected by high-performance liquid chromatography (HPLC); levels of gene expression varied among organs of the two cultivars. Significantly higher transcription levels of most flavonoid biosynthetic genes, except FeFLS1, were detected in stems of the high-rutin line than in stems of the original line. FeCHI and FeFLS2 genes also showed higher expression levels in seeds of the high-rutin cultivar. In contrast, FePAL, FeC4H, Fe4CL1, FeCHS, FeF3H, FeF3'H, FeFLS2, and FeDFR were highly detected in the roots of the original line. The HPLC results indicated 1.73-, 1.62-, and 1.77-fold higher accumulation of rutin (the primary flavonoid compound) in leaves, stems, and mature seeds of the high-rutin cultivar (24.86, 1.46, and 1.36 µg/mg, respectively) compared with the original cultivar (14.40, 0.90, and 0.77 µg/mg, respectively). A total of 46 metabolites were identified from seeds by gas chromatography-time-of-flight mass spectrometry. The metabolite profiles were subjected to principal component analysis (PCA). PCA could clearly differentiate the original and high-rutin cultivars. Our results indicate that the high-rutin cultivar could be an excellent alternative for buckwheat culture, and we provide useful information for obtaining this cultivar.

Molecular cloning and characterization of tyrosine aminotransferase and hydroxyphenylpyruvate reductase, and rosmarinic acid accumulation in Scutellaria baicalensis.

Rosmarinic acid (a-O-caffeoyl-3,4-dihydroxyphenylacetic acid, RA) is a caffeoyl ester widely distributed in plants. cDNA clones encoding tyrosine aminotransferase (TAT1 and 2) and hydroxyphenylpyruvate reductase (HPPR) have been isolated from Scutellaria baicalensis. The open reading frames (ORFs) of SbTAT1 and 2 were 1230 and 1272 bp long and encoded 409 and 423 amino acid residues, respectively. HPPR corresponded to a 942-bp ORF and 313 amino acid residues of translated protein. To study the molecular mechanisms of TAT and HPPR and investigate RA accumulation in S. baicalensis, we examined the transcript levels of TAT isoforms and HPPR with quantitative real-time PCR and analyzed the RA content in different organs by using high-performance liquid chromatography. The transcript levels of SbTATI SbTAT2, and SbHPPR in the flowers were higher than those in other organs. RA was also highly accumulated in the flowers and with a trace amount in the roots. No RA was detected in the leaves and stems of S. baicalensis. The amount of accumulated RA in the flowers was 28.7 times higher than that in the roots. Our results will be helpful in elucidating the mechanisms of RA biosynthesis in S. baicalensis.