Accumulation of Tilianin and Rosmarinic Acid and Expression of Phenylpropanoid Biosynthetic Genes in <i>Agastache rugosa</i>

Korean mint (<i>Agastache rugosa</i>), 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 <i>A. rugosa</i> 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 <i>A. rugosa</i>. High-performance liquid chromatography (HPLC) revealed that the accumulation pattern of tilianin matched the expression patterns of <i>ArCHS</i> and <i>ArCHI</i> in different organs of <i>A. rugosa</i>. Moreover, acacetin, the precursor of tilianin, also demonstrated an accumulation pattern congruent with the expression of these 2 genes. The transcription levels of <i>ArPAL</i>, <i>ArC4H</i>, and <i>Ar4CL</i> 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 <i>ArPAL</i>, <i>ArC4H</i>, and <i>Ar4CL</i> were low. The findings of our study support the medicinal usefulness of <i>A. rugosa</i> and indicate targets for increasing tilianin and rosmarinic acid production in this plant

Enhanced Accumulation of Phytosterol and Triterpene in Hairy Root Cultures of Platycodon grandiflorum by Overexpression of Panax ginseng 3‑Hydroxy-3-methylglutaryl-coenzyme A Reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the rate-limiting step in the mevalonate pathway. To elucidate the functions of HMGR in triterpene biosynthesis, Platycodon grandiflorum was transformed with a construct expressing Panax ginseng HMGR (PgHMGR). We used PCR analysis to select transformed hairy root lines and selected six lines for further investigation. Quantitative real-time PCR showed higher expression levels of HMGR and total platycoside levels (1.5–2.5-fold increase) in transgenic lines than in controls. Phytosterols levels were also 1.1–1.6-fold higher in transgenic lines than in controls. Among these lines, line T7 produced the highest level of total platycosides (1.60 ± 0.2 mg g^–1 dry weight) and α-spinasterol (1.78 ± 0.16 mg g^–1 dry weight). These results suggest that metabolic engineering of P. grandiflorum by Agrobacterium-mediated genetic transformation may enhance production of phytosterols and triterpenoids

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 <i>PgMVD</i> and <i>PgFPS</i> than that by the wild-type control. Among the hairy root lines transformed with <i>PgMVD</i>, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with <i>PgFPS</i> showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of <i>PgMVD</i> produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of <i>PgFPS</i> 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 <i>PgMVD</i> and <i>PgFPS</i> genes into the hairy roots of the plant. Our results suggest that <i>PgMVD</i> and <i>PgFPS</i> play an important role in the triterpene biosynthesis of P. ginseng

Riboflavin Accumulation and Characterization of cDNAs Encoding Lumazine Synthase and Riboflavin Synthase in Bitter Melon (Momordica charantia)

Riboflavin (vitamin B₂) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotidecofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that <i>McLS</i> and <i>McRS</i> share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that <i>McLS</i> and <i>McRS</i> were constitutively expressed in <i>M. charantia</i>, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. <i>McLS</i> and <i>McRS</i> were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from <i>M. charantia</i> may aid the metabolic engineering of vitamin B₂ in crops

Accumulation of Phenylpropanoids and Correlated Gene Expression during the Development of Tartary Buckwheat Sprouts

Buckwheat sprouts are considered an excellent dietary source of phenolic compounds. The time duration and amount of light for sprouting strongly affect the nutritional quality of sprouts. In this study, these two factors were investigated in two cultivars of tartary buckwheat sprouts: Hokkai T8 and T10. The transcriptional levels of flavonoid biosynthetic genes were investigated in light/dark- and dark-treated sprouts. Among the main flavonoid biosynthesis structural genes, <i>FtPAL</i>, <i>Ft4CL</i>, <i>FtF3H</i>, <i>FtDFR</i>, and <i>FtANS</i> exhibited higher transcriptional levels than others as compared to that of a housekeeping gene (histone H3) during sprouting; <i>FtF3′H1</i>, <i>FtF3′H2</i>, <i>FtFLS2</i>, and <i>FtANS</i> were substantially upregulated at 2, 4, and 6 days in light/dark-treated T10 sprouts than in dark-treated ones. However, <i>FtDFR</i> was downregulated in 8 and 10 day old light/dark-treated sprouts of both cultivars. High-performance liquid chromatography (HPLC) analysis revealed that increasing the culture time did not affect the accumulation of flavonoids or anthocyanins. However, light contributed the production of anthocyanins in Hokkai T10 sprouts. The anthocyanins included cyanidin 3-<i>O</i>-glucoside, cyanidin 3-<i>O</i>-rutinoside, and delphinidin-3-<i>O</i>-coumarylglucoside, which were identified by HPLC and electrospray ionization–tandem mass spectrometry. Instead of anthocyanins, Hokkai T8 sprouts produced large amounts of 4 flavonoid <i>C</i>-glycosylflavone compounds in both light/dark and dark conditions: orientin, isoorientin, vitexin, and isovitexin. These results indicate that these two types of tartary buckwheat sprouts have different mechanisms for flavonoid and anthocyanin biosynthesis that also vary in light/dark and dark conditions

Metabolic Profiling in Chinese Cabbage (Brassica rapa L. subsp. pekinensis) Cultivars Reveals that Glucosinolate Content Is Correlated with Carotenoid Content

A total of 38 bioactive compounds, including glucosinolates, carotenoids, tocopherols, sterols, and policosanols, were characterized from nine varieties of Chinese cabbage (Brassica rapa L. subsp. pekinensis) to determine their phytochemical diversity and analyze their abundance relationships. The metabolite profiles were evaluated with principal component analysis (PCA), Pearson correlation analysis, and hierarchical clustering analysis (HCA). PCA and HCA identified two distinct varieties of Chinese cabbage (Cheonsangcheonha and Waldongcheonha) with higher levels of glucosinolates and carotenoids. Pairwise comparisons of the 38 metabolites were calculated using Pearson correlation coefficients. The HCA, which used the correlation coefficients, clustered metabolites that are derived from closely related biochemical pathways. Significant correlations were discovered between chlorophyll and carotenoids. Additionally, aliphatic glucosinolate and carotenoid levels were positively correlated. The Cheonsangcheonha and Waldongcheonha varieties appear to be good candidates for breeding because they have high glucosinolate and carotenoid levels

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, <i>Fagopyrum esculentum</i> 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 <i>FeFLS1</i>, were detected in stems of the high-rutin line than in stems of the original line. <i>FeCHI</i> and <i>FeFLS2</i> genes also showed higher expression levels in seeds of the high-rutin cultivar. In contrast, <i>FePAL</i>, <i>FeC4H</i>, <i>Fe4CL1</i>, <i>FeCHS</i>, <i>FeF3H</i>, <i>FeF3</i>′<i>H</i>, <i>FeFLS2</i>, and <i>FeDFR</i> 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

Metabolic Profiling in Plasma and Brain Induced by 17β-Estradiol Supplementation in Ovariectomized Mice

17β-Estradiol is an ovarian hormone that regulates energy circulation and storage by acting on the central nervous system. However, the metabolic differences between the blood and brain when stimulated by 17β-estradiol are poorly understood. Moreover, research using menopause-induced models to investigate primary metabolites in the blood and brain is limited. Thus, this study aimed to identify metabolic changes in the plasma and brain resulting from 17β-estradiol supplementation in an estrogen-deficient mouse model. Three groups of mice were utilized: sham-operated mice (Sham), ovariectomized mice (OVX), and ovariectomized mice that received a weekly supplementation of 17β-estradiol (E2). Plasma and brain samples from these mice were subjected to metabolic analysis using gas chromatography–time-of-flight–mass spectrometry. Compared with the plasma samples from the Sham and OVX groups, the plasma samples from the E2 group contained higher contents of branched-chain amino acids (BCAAs), such as valine, isoleucine, and leucine. Meanwhile, the brain samples from the E2 group contained higher contents of most metabolites, including BCAAs, neurotransmitters, tricarboxylic acid cycle intermediates, and fatty acids, than those from the two other groups. This study is the first to reveal differences in energy metabolism induced by 17β-estradiol supplementation through brain metabolic profiling of ovariectomized mice, emphasizing the importance of brain metabolic profiling in menopausal hormone research

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 <i>MaUGT78D1</i> and <i>MaLUS</i> play important regulatory roles in the rutin and betulin biosynthetic pathways

Accumulation of Charantin and Expression of Triterpenoid Biosynthesis Genes in Bitter Melon (<i>Momordica charantia</i>)

Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of <i>McSE</i> and <i>McCAS1</i>, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength