The Impact of Light Wavelength and Darkness on Metabolite Profiling of Korean Ginseng: Evaluating Its Anti-Cancer Potential against MCF-7 and BV-2 Cell Lines.

Korean ginseng is a source of functional foods and medicines; however, its productivity is hindered by abiotic stress factors, such as light. This study investigated the impacts of darkness and different light wavelengths on the metabolomics and anti-cancer activity of ginseng extracts. Hydroponically-grown Korean ginseng was shifted to a light-emitting diodes (LEDs) chamber for blue-LED and darkness treatments, while white fluorescent (FL) light treatment was the control. MCF-7 breast cancer and lipopolysaccharide (LPS)-induced BV-2 microglial cells were used to determine chemo-preventive and neuroprotective potential. Overall, 53 significant primary metabolites were detected in the treated samples. The levels of ginsenosides Rb1, Rb2, Rc, Rd, and Re, as well as organic and amino acids, were significantly higher in the dark treatment, followed by blue-LED treatment and the FL control. The dark-treated ginseng extract significantly induced apoptotic signaling in MCF-7 cells and dose-dependently inhibited the NF-κB and MAP kinase pathways in LPS-induced BV-2 cells. Short-term dark treatment increased the content of Rd, Rc, Rb1, Rb2, and Re ginsenosides in ginseng extracts, which promoted apoptosis of MCF-7 cells and inhibition of the MAP kinase pathway in BV-2 microglial cells. These results indicate that the dark treatment might be effective in improving the pharmacological potential of ginseng.

Enhancement of the flavone contents of Scutellaria baicalensis hairy roots via metabolic engineering using maize Lc and Arabidopsis PAP1 transcription factors.

Baicalin, baicalein, and wogonin are valuable natural flavonoid compounds produced by Scutellaria baicalensis. In this study, we showed that the maize transcription factor Lc can enhance the production of these three flavonoids in hairy root cultures of S. baicalensis by comprehensively upregulating flavonoid biosynthesis pathway genes (SbPAL1, SbC4H, and Sb4CL) and baicalein 7-O-glucuronosyltransferase (UBGAT), ultimately yielding total flavonoid contents of up to 80.5 ± 6.15 mg g-1 dry weight, which was 322% greater than the average value of total flavonoid contents produced by three GUS-overexpressing lines. Similarly, the Arabidopsis transcription factor PAP1 was found to enhance flavonoid accumulation by upregulating SbPAL1, SbPAL2, SbPAL3, SbC4H, Sb4CL, SbCHI, and UBGAT, ultimately yielding total flavonoid contents of up to 133 ± 7.66 mg g-1 dry weight, which was 532% greater than the average value of total flavonoid contents produced by three GUS-overexpressing lines. These findings indicate that metabolic engineering in S. baicalensis can be achieved using Agrobacterium rhizogenes-mediated transformation and that the production of baicalin, baicalein, and wogonin can be enhanced via the overexpression of ZmLc and AtPAP1 in hairy root cultures. These results also indicate that ZmLc and AtPAP1 can be used as positive regulators of the flavonoid biosynthetic pathway of S. baicalensis hairy root cultures.

Effect of Light and Dark on the Phenolic Compound Accumulation in Tartary Buckwheat Hairy Roots Overexpressing ZmLC.

Fagopyrum tataricum 'Hokkai T10' is a buckwheat cultivar capable of producing large amounts of phenolic compounds, including flavonoids (anthocyanins), phenolic acids, and catechin, which have antioxidant, anticancer, and anti-inflammatory properties. In the present study, we revealed that the maize transcription factor Lc increased the accumulation of phenolic compounds, including sinapic acid, 4-hydroxybenzonate, t-cinnamic acid, and rutin, in Hokkai T10 hairy roots cultured under long-photoperiod (16 h light and 8 h dark) conditions. The transcription factor upregulated phenylpropanoid and flavonoid biosynthesis pathway genes, yielding total phenolic contents reaching 27.0 ± 3.30 mg g-1 dry weight, 163% greater than the total flavonoid content produced by a GUS-overexpressing line (control). In contrast, when cultured under continuous darkness, the phenolic accumulation was not significantly different between the ZmLC-overexpressing hairy roots and the control. These findings suggest that the transcription factor (ZmLC) activity may be light-responsive in the ZmLC-overexpressing hairy roots of F. tataricum, triggering activation of the phenylpropanoid and flavonoid biosynthesis pathways. Further studies are required on the optimization of light intensity in ZmLC-overexpressing hairy roots of F. tataricum to enhance the production of phenolic compounds.

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781 bp per gene in 82.473 Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

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.

Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

Enhancement of chlorogenic acid production in hairy roots of Platycodon grandiflorum by over-expression of an Arabidopsis thaliana transcription factor AtPAP1.

To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA.

O- and N-Methyltransferases in benzylisoquinoline alkaloid producing plants.

BACKGROUND: Secondary metabolites such as benzylisoquinoline alkaloids (BIA) have attracted considerable attention because of their pharmacological properties and potential therapeutic applications. Methyltransferases (MTs) can add methyl groups to alkaloid molecules, altering their physicochemical properties and bioactivity, stability, solubility, and recognition by other cellular components. Five types of O-methyltransferases and two types of N-methyltransferases are involved in BIA biosynthesis. OBJECTIVE: Since MTs may be the source for the discovery and development of novel biomedical, agricultural, and industrial compounds, we performed extensive molecular and phylogenetic analyses of O- and N-methyltransferases in BIA-producing plants. METHODS: MTs involved in BIA biosynthesis were isolated from transcriptomes of Berberis koreana and Caulophyllum robustum. We also mined the methyltransferases of Coptis japonica, Papaver somniferum, and Nelumbo nucifera from the National Center for Biotechnology Information protein database. Then, we analyzed the functional motifs and phylogenetic analysis. RESULT: We mined 42 O-methyltransferases and 8 N-methyltransferases from the five BIA-producing plants. Functional motifs for S-adenosyl-L-methionine-dependent methyltransferases were retained in most methyltransferases, except for the three O-methyltransferases from N. nucifera. Phylogenetic analysis revealed that the methyltransferases were grouped into four clades, I, II, III and IV. The clustering patterns in the phylogenetic analysis suggested a monophyletic origin of methyltransferases and gene duplication within species. The coexistence of different O-methyltransferases in the deep branch subclade might support some cases of substrate promiscuity. CONCLUSIONS: Methyltransferases may be a source for the discovery and development of novel biomedical, agricultural, and industrial compounds. Our results contribute to further understanding of their structure and reaction mechanisms, which will require future functional studies.

Impact of Sodium Silicate Supplemented, IR-Treated Panax Ginseng on Extraction Optimization for Enhanced Anti-Tyrosinase and Antioxidant Activity: A Response Surface Methodology (RSM) Approach.

Ginseng has long been widely used for its therapeutic potential. In our current study, we investigated the impact of abiotic stress induced by infrared (IR) radiations and sodium silicate on the upregulation of antioxidant and anti-tyrosinase levels, as well as the total phenolic and total flavonoid contents of the Korean ginseng (Panax ginseng C.A. Meyer) variety Yeonpoong. The RSM-based design was used to optimize ultrasonic-assisted extraction time (1-3 h) and temperature (40-60 °C) for better anti-tyrosinase activity and improved antioxidant potential. The optimal extraction results were obtained with a one-hour extraction time, at a temperature of 40 °C, and with a 1.0 mM sodium silicate treatment. We recorded maximum anti-tyrosinase (53.69%) and antioxidant (40.39%) activities when RSM conditions were kept at 875.2 mg GAE/100 g TPC, and 3219.58 mg catechin/100 g. When 1.0 mM sodium silicate was added to the media and extracted at 40 °C for 1 h, the highest total ginsenoside content (368.09 mg/g) was recorded, with variations in individual ginsenosides. Ginsenosides Rb1, Rd, and F2 were significantly affected by extraction temperature, while Rb2 and Rc were influenced by the sodium silicate concentration. Moreover, ginsenoside F2 increased with the sodium silicate treatment, while the Rg3-S content decreased. Interestingly, higher temperatures favored greater ginsenoside diversity while sodium silicate impacted PPD-type ginsenosides. It was observed that the actual experimental values closely matched the predicted values, and this agreement was statistically significant at a 95% confidence level. Our findings suggest that the application of IR irradiation in hydroponic systems can help to improve the quality of ginseng sprouts when supplemented with sodium silicate in hydroponic media. Optimized extraction conditions using ultrasonication can be helpful in improving antioxidant and anti-tyrosinase activity.

Impact of Light and Dark Treatment on Phenylpropanoid Pathway Genes, Primary and Secondary Metabolites in Agastache rugosa Transgenic Hairy Root Cultures by Overexpressing Arabidopsis Transcription Factor AtMYB12.

Agastache rugosa, otherwise called Korean mint, has a wide range of medicinal benefits. In addition, it is a rich source of several medicinally valuable compounds such as acacetin, tilianin, and some phenolic compounds. The present study aimed to investigate how the Tartary buckwheat transcription factor AtMYB12 increased the primary and secondary metabolites in Korean mint hairy roots cultured under light and dark conditions. A total of 50 metabolites were detected by using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The result showed that the AtMYB12 transcription factor upregulated the phenylpropanoid biosynthesis pathway genes, which leads to the highest accumulation of primary and secondary metabolites in the AtMYB12-overexpressing hairy root lines (transgenic) than that of the GUS-overexpressing hairy root line (control) when grown under the light and dark conditions. However, when the transgenic hairy root lines were grown under dark conditions, the phenolic and flavone content was not significantly different from that of the control hairy root lines. Similarly, the heat map and hierarchical clustering analysis (HCA) result showed that most of the metabolites were significantly abundant in the transgenic hairy root cultures grown under light conditions. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed that the identified metabolites were separated far based on the primary and secondary metabolite contents present in the control and transgenic hairy root lines grown under light and dark conditions. Metabolic pathway analysis of the detected metabolites showed 54 pathways were identified, among these 30 were found to be affected. From these results, the AtMYB12 transcription factor activity might be light-responsive in the transgenic hairy root cultures, triggering the activation of the primary and secondary metabolic pathways in Korean mint.

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.

Enhancement of rutin in Fagopyrum esculentum hairy root cultures by the Arabidopsis transcription factor AtMYB12.

Common buckwheat (Fagopyrum esculentum Moench) is rich in phenolic compounds and may be useful for the treatment of metabolic syndrome in humans. To improve the production of rutin in buckwheat, we overexpressed the flavonol-specific transcription factor, AtMYB12 using Agrobacterium rhizogenes into hairy root culture systems. This induced the expression of flavonoid biosynthetic genes encoding phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone isomerase, flavone 3-hydroxylase, flavonoid 3'-hydroxylase, and flavonol synthase. This led to the accumulation of rutin in buckwheat hairy roots up to 0.9 mg/g dry wt. PAP1 expression, however, did not correlate with the production of rutin.

Comparison of yield and metabolites according to the types of hilling materials utilized during Glehnia littoralis sprout vegetable cultivation.

Various hilling materials (rice hulls, pine sawdust, and perlite) were compared to produce sprout vegetables using beach silvertop (Glehnia littoralis Fr. Schm. ex Miq.). We have investigated the yield, total phenolic content, total flavonoid content, antioxidant capacities (DPPH, ABTS), phenolic compounds, and volatile compounds of G. littoralis sprout vegetables. Comparing the yield and phenolic compounds according to the hilling materials, the rice hulls treatment was the most and followed by sawdust and perlite. The leaves and stems of G. littoralis sprout vegetable contain approximately 27 volatile compounds. The sawdust treatment had a pine scent even during the hilling process, and these scent components were entirely absorbed by the stem. The result suggested that sawdust treatment, like rice hulls, had a high yield and high content of beneficial compounds, but the stem of G. littoralis had a pine tree scent, reducing the inherent scent of G. littoralis.

Comparative Transcriptomics for Genes Related to Berberine and Berbamine Biosynthesis in Berberidaceae.

Berberine and berbamine are bioactive compounds of benzylisoquinoline alkaloids (BIAs) present in Berberis species. The contents of berbamine are 20 times higher than berberine in leaf tissues in three closely related species: Berberis koreana, B. thunbergii and B. amurensis. This is the first report on the quantification of berberine compared to the berbamine in the Berberis species. Comparative transcriptome analyses were carried out with mRNAs from the leaf tissues of the three-species. The comparison of the transcriptomes of B. thunbergii and B. amurensis to those of B. koreana, B. thunbergii showed a consistently higher number of differentially expressed genes than B. amurensis in KEGG and DEG analyses. All genes encoding enzymes involved in berberine synthesis were identified and their expressions were variable among the three species. There was a single copy of CYP80A/berbamunine synthase in B. koreana. Methyltransferases and cytochrome P450 mono-oxidases (CYPs) are key enzymes for BIA biosynthesis. The current report contains the copy numbers and other genomic characteristics of the methyltransferases and CYPs in Berberis species. Thus, the contents of the current research are valuable for molecular characterization for the medicinal utilization of the Berberis species.

APUM23, a nucleolar Puf domain protein, is involved in pre-ribosomal RNA processing and normal growth patterning in Arabidopsis.

Pumilio, an RNA-binding protein that contains tandemly repeated Puf domains, is known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. APUM23 is constitutively expressed, with higher levels in metabolically active tissues, and its expression is up-regulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth, with serrated and scrunched leaves, an abnormal venation pattern, and distorted organization of the palisade parenchyma cells - a phenotype that is reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on this localization, rRNA processing was examined. In apum23, 35S pre-rRNA, and unprocessed 18S and 5.8S poly(A) rRNAs, accumulated without affecting the steady-state levels of mature rRNAs, indicating that APUM23 is involved in the processing and/or degradation of 35S pre-rRNA and rRNA maturation by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 small nucleolar RNAs (snoRNAs) and accumulated RNAs within the nucleolus. Our data suggest that APUM23 plays an important role in plant development via rRNA processing.

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.

An efficient protocol for genetic transformation of watercress (Nasturtium officinale) using Agrobacterium rhizogenes.

Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01-0.02 µmol/g of DW) and 4-methoxyglucobrassicin (0.06-0.18 µmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06-0.21 µmol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol).

An efficient protocol for genetic transformation of Platycodon grandiflorum with Agrobacterium rhizogenes.

The balloon flower (Platycodon grandiflorum) is a popular traditional medicinal plant used in Korea to treat conditions such as bronchitis, asthma, tuberculosis, diabetes, and inflammatory diseases. Recently, immunopharmacological research identified triterpenoid and saponin as important active compounds in P. grandiflorum. To study and extract these compounds and other metabolites from P. grandiflorum, a technique was developed for producing hairy root cultures, which are a reliable source of plant compounds. To achieve this, the activity of Agrobacterium rhizogenes was exploited, which can transfer DNA segments into plant genomes after infecting them. In this study, the A. rhizogenes strain R1000 was determined that had the highest infection frequency (87.5%) and induced the most hairy roots per plant, and the concentration of antibiotics (75 mg/l kanamycin) was elucidated for selection after transformation. Wild-type and transgenic hairy roots contained various phenolic compounds, although both of them had similar concentrations of phenolic compounds. In the future, the protocols described here should be useful for studying and extracting valuable metabolites such as phenolic compounds from P. grandiflorum hairy root cultures.

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 ß-carotene concentration five times higher than that of carrot. To elucidate the molecular basis of ß-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.