Flavonol Synthase Research Articles

Exogenous Melatonin Application Delays Senescence of Kiwifruit Leaves by Regulating the Antioxidant Capacity and Biosynthesis of Flavonoids.

Melatonin, a multiple signal molecule, plays important roles in delaying senescence during the development of plants. Because few species have been studied for the effect of exogenous melatonin on anti-aging, the plausible mechanism of melatonin of anti-aging effects on other plant species has remained largely unknown. In the present study, the effects of exogenous melatonin on leaf senescence in kiwifruit were examined during natural aging after melatonin (200 μM) or water (Control) pretreatment. The decreased membrane damage and lower hydrogen peroxide (H2O2) content due to the enhanced scavenging activity of antioxidant enzymes peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) demonstrated that melatonin effectively delayed the aging of kiwifruit leaves. Likewise, owing to up-regulated expression of chlorophyll a/b-binding protein (CAB) gene in the sampled leaves pretreated with melatonin, chlorophyll degradation decreased. Therefore, osmoregulatory substances in sampled leaves accumulated (e.g., soluble sugar and soluble protein) and seedling cell environment stability was maintained. Simultaneously, melatonin decreased H2O2 concentration owing to increased glutathione (GSH) and ascorbate (AsA) content, and the expression levels of glutathione reductase (GR), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) were up-regulated by melatonin application, indicating that the increase of GSH and AsA was attributed to the expression of these genes. In addition, a large amount of flavonoids accumulated in seedlings pretreated with melatonin, and transcript levels of eight genes involved in flavonoid synthesis, including phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxymate (C4H), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonol synthase (FNS), leucoanthocyanin reductase (LAR), anthocyanin reductase (ANR), flavonoid 3-O-glucosyltransferase (UFGT) were enhanced in response to melatonin application. These results indicated that melatonin delayed aging of kiwifruit leaves by activating the antioxidant capacity and enhancing flavonoid biosynthesis. All of these results can provide clear proof that melatonin plays a key roles in delaying leaf senescence.

Physiological characterization of leaf and internode after bud break in Japanese indigenous Koshu grape by comparative RNA sequencing analysis.

Koshu is indigenous to Japan and considered the most important wine grape in Japan. Koshu grape berry possesses characteristics that make it unique from European V. vinifera as wine grape. However, the physiological characteristics of Koshu leaf and internode remain unknown. An understanding of those characteristics would contribute to improvements in Koshu cultivation, thereby enhancing grape berry and wine quality. To identify the genes responsible for the physiological characteristics of Koshu, we comprehensively analyzed leaf and internode differences at the transcriptome level between Koshu and Pinot Noir by RNA sequencing. A total of 248 and 131 differentially expressed genes (DEGs) were detected in leaves and internodes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of these DEGs revealed that “flavonoid biosynthesis” and “glutathione metabolism” pathways were significantly enriched in Koshu leaves. On the other hand, when internodes were compared, “flavonoid”-related GO terms were specifically detected in Koshu. KEGG pathway enrichment analysis suggested that the expression of such genes as leucoanthocyanidin reductase and flavonol synthase in the flavonoid biosynthesis pathway was higher in Koshu than Pinot Noir. Measurement of the relative expression levels of these genes by RT-qPCR validated the results obtained by RNA sequencing. The characteristics of Koshu leaf and internode, which are expected to produce flavonoids with antibacterial activity and UV protection function, would suit Japanese climate as a survival strategy.

Antioxidant Activities of Transgenic Flower Over-Expressing FLS and TT8 Involving in Flavonoid Biosynthesis

Transgenic differently over-expressed cDNA involving in flavonoid biosynthesis (FLAVONOL SYNTHASE: FLS and TRANSPARENT TESTA 8: TT8) and wild type plants were grown in the culturing room radiated with fluorescent light. Flowers of each transgenic and WT were collected and extracted with acidic methanol. The flower extracts were analyzed for the relative content of each flavonoid subgroup by spectrophotometry. The result showed that the both transgenic extracts contained flavanone and flavone higher than WT extract did. The FLS extract contained the highest amount of flavonol content (1.4 times) following with the TT8 extracts (1.25 times) comparing to WT extract. The TT8 extract has anthocyanin content approximately 3 times, while the FLS extract has only 0.5 times of WT. The extract was used to evaluate the antioxidant capacity by ABTS and DPPH methods. The result showed that, both transgenic extracts have a better antioxidant capacity than WT extract. Both transgenic extracts have similar results of EC50 performed by DPPH method, however the TT8 extracts showed the highest antioxidant capacity under ABTS experimental condition. Therefore, types and concentration of flavonoids in the extract could directly affect the antioxidant capacity.

Genome-Wide Association and Metabolic Pathway Analysis of Corn Earworm Resistance in Maize.

Maize ( L.) is a staple crop of economic, industrial, and food security importance. Damage to the growing ears by corn earworm [ (Boddie)] is a major economic burden and increases secondary fungal infections and mycotoxin levels. To identify biochemical pathways associated with native resistance mechanisms, a genome-wide association analysis was performed, followed by pathway analysis using a gene-set enrichment-based approach. The gene-set enrichment exposed the cumulative effects of genes in pathways to identify those that contributed the most to resistance. Single nucleotide polymorphism-trait associations were linked to genes including transcription factors, protein kinases, hormone-responsive proteins, hydrolases, pectinases, xylogluconases, and the flavonol synthase gene (in the maysin biosynthesis pathway). The most significantly associated metabolic pathways identified included those that modified cell wall components, especially homogalacturonan, wax esters, and fatty acids; those involved in antibiosis, especially 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), flavonoids, and phenolics; and those involved in plant growth, including N uptake and energy production. The pathways identified in this study, and especially the cell wall-associated pathways, identified here for the first time, provide clues to resistance mechanisms that could guide the identification of new resistant ideotypes and candidate genes for creation of resistant maize germplasm via selection of natural variants or gene editing.

Great Cause-Small Effect: Undeclared Genetically Engineered Orange Petunias Harbor an Inefficient Dihydroflavonol 4-Reductase.

A recall campaign for commercial, orange flowering petunia varieties in spring 2017 caused economic losses worldwide. The orange varieties were identified as undeclared genetically engineered (GE)-plants, harboring a maize dihydroflavonol 4-reductase (DFR, A1), which was used in former scientific transgenic breeding attempts to enable formation of orange pelargonidin derivatives from the precursor dihydrokaempferol (DHK) in petunia. How and when the A1 cDNA entered the commercial breeding process is unclear. We provide an in-depth analysis of three orange petunia varieties, released by breeders from three countries, with respect to their transgenic construct, transcriptomes, anthocyanin composition, and flavonoid metabolism at the level of selected enzymes and genes. The two possible sources of the A1 cDNA in the undeclared GE-petunia can be discriminated by PCR. A special version of the A1 gene, the A1 type 2 allele, is present, which includes, at the 3′-end, an additional 144 bp segment from the non-viral transposable Cin4-1 sequence, which does not add any functional advantage with respect to DFR activity. This unequivocally points at the first scientific GE-petunia from the 1980s as the A1 source, which is further underpinned e.g., by the presence of specific restriction sites, parts of the untranslated sequences, and the same arrangement of the building blocks of the transformation plasmid used. Surprisingly, however, the GE-petunia cannot be distinguished from native red and blue varieties by their ability to convert DHK in common in vitro enzyme assays, as DHK is an inadequate substrate for both the petunia and maize DFR. Recombinant maize DFR underpins the low DHK acceptance, and, thus, the strikingly limited suitability of the A1 protein for a transgenic approach for breeding pelargonidin-based flower color. The effect of single amino acid mutations on the substrate specificity of DFRs is demonstrated. Expression of the A1 gene is generally lower than the petunia DFR expression despite being under the control of the strong, constitutive p35S promoter. We show that a rare constellation in flavonoid metabolism—absence or strongly reduced activity of both flavonol synthase and B-ring hydroxylating enzymes—allows pelargonidin formation in the presence of DFRs with poor DHK acceptance.

Solar UV light regulates flavonoid metabolism in apple (Malus x domestica).

Ultraviolet-B light (UV-B) is one environmental signal perceived by plants that affects the flavonoid pathway and influences the levels of anthocyanins, flavonols, and proanthocyanidins. To understand the mechanisms underlying UV exposure, apple trees were grown under spectral filters that altered transmission of solar UV light. Fruit analysis showed that UV induced changes in physiology, metabolism, and gene expression levels during development over a season. These changes were sustained after storage. Under low UV, ripening was delayed, fruit size decreased, and anthocyanin and flavonols were reduced. Expression analysis showed changes in response to UV light levels for genes in the regulation and biosynthesis of anthocyanin and flavonols. Transcription of flavonol synthase (FLS), ELONGATED HYPOCOTYL 5 (HY5), MYB10, and MYB22 were down-regulated throughout fruit development under reduced UV. Functional testing showed that the FLS promoter was activated by HY5, and this response was enhanced by the presence of MYB22. The MYB22 promoter can also be activated by the anthocyanin regulator, MYB10. As ambient levels of UV light vary around the globe, this study has implications for future crop production, the quality of which can be determined by the response to UV.

Transcriptome analysis of filling stage seeds among three buckwheat species with emphasis on rutin accumulation.

Buckwheat is an important minor crop with pharmaceutical functions due to rutin enrichment in the seed. Seeds of common buckwheat cultivars (Fagopyrum esculentum, Fes) usually have much lower rutin content than tartary buckwheat (F. tartaricum, Ft). We previously found a wild species of common buckwheat (F. esculentum ssp. ancestrale, Fea), with seeds that are high in rutin, similar to Ft. In the present study, we investigated the mechanism by which rutin production varies among different buckwheat cultivars, Fea, a Ft variety (Xide) and a Fes variety (No.2 Pingqiao) using RNA sequencing of filling stage seeds. Sequencing data generated approximately 43.78-Gb of clean bases, all these data were pooled together and assembled 180,568 transcripts, and 109,952 unigenes. We established seed gene expression profiles of each buckwheat sample and assessed genes involved in flavonoid biosynthesis, storage proteins production, CYP450 family, starch and sucrose metabolism, and transcription factors. Differentially expressed genes between Fea and Fes were further analyzed due to their close relationship than with Ft. Expression levels of flavonoid biosynthesis gene FLS1 (Flavonol synthase 1) were similar in Fea and Ft, and much higher than in Fes, which was validated by qRT-PCR. This suggests that FLS1 transcript levels may be associated with rutin accumulation in filling stage seeds of buckwheat species. Further, we explored transcription factors by iTAK, and multiple gene families were identified as being involved in the coordinate regulation of metabolism and development. Our extensive transcriptomic data sets provide a complete description of metabolically related genes that are differentially expressed in filling stage buckwheat seeds and suggests that FLS1 is a key controller of rutin synthesis in buckwheat species. FLS1 can effectively convert dihydroflavonoids into flavonol products. These findings provide a basis for further studies of flavonoid biosynthesis in buckwheat breeding to help accelerate flavonoid metabolic engineering that would increase rutin content in cultivars of common buckwheat.

Cloning and Sequence Analysis of Gene Encoding Flavonol Synthase from Trung Du Teas Growing in Thai Nguyen

In this study, we conducted the cloning and sequencing gene encoding FLS from the two green and purple Trung Du tea cultivars. The length of FLS gene is 996 bp, encodes 331 amino acid. Results of FLS gene analysis showed that green and purple Trung Du cultivars has 13 nucleotide variants total as compare with FLS sequence published on Genbank. Nucleotide sequence differences lead to change amino acid sequence of key functional motives in FLS like motif characterizes the 2OG-Fe (II) oxygenase superfamily, the PxxxIRxxx-EQP motif at the N-terminal (18→29) determines the FLS activity, the CPQ/RPxLAL motif) is the binding site of 2-oxoglutarate. How amino acid position changes affect FLS activity need further research. FLS gene isolates are sources for further research to aim elucidating the function of this enzyme.

Identification of differentially expressed genes in flower, leaf and bulb scale of Lilium oriental hybrid \u2018Sorbonne\u2019 and putative control network for scent genes

BackgroundLily is an economically important plant, with leaves and bulbs consisting of overlapping scales, large ornamental flowers and a very large genome. Although it is recognized that flowers and bulb scales are modified leaves, very little is known about the genetic control and biochemical differentiation underlying lily organogenesis and development. Here we examined the differentially expressed genes in flower, leaf and scale of lily, using RNA-sequencing, and identified organ-specific genes, including transcription factors, genes involved in photosynthesis in leaves, carbohydrate metabolism in bulb scales and scent and color production in flowers.ResultsOver 11Gb data were obtained and 2685, 2296, and 1709 differentially expressed genes were identified in the three organs, with 581, 662 and 977 unique DEGs in F-vs-S, L-vs-S and L-vs-F comparisons. By functional enrichment analysis, genes likely to be involved in biosynthetic pathways leading to floral scent production, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 3-ketoacyl-CoA thiolase (KAT), hydroperoxide lyase (HPL), geranylgeranyl pyrophosphate (GGPP) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HDS) and terpene synthase (TPS), and floral color genes, such as dihydroflavonol 4-reductase (DFR), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) were identified. Distinct groups of genes that participate in starch and sucrose metabolism, such as sucrose synthase (SS), invertase (INV), sucrose phosphate synthase (SPS), starch synthase (SSS), starch branching enzyme (SBE), ADP-glucose pyrophosphorylase (AGP) andβ-amylase (BAM) and photosynthesis genes (Psa, Psb, Pet and ATP) were also identified. The expression of six floral fragrance-related DGEs showed agreement between qRT-PCR results and RPKM values, confirming the value of the data obtained by RNA-seq. We obtained the open reading frame of the terpene synthase gene from Lilium ‘Sorbonne’, designated LsTPS, which had 99.55% homology to transcript CL4520.Contig5_All. In addition, 54, 48 and 50 differently expressed transcription factor were identified by pairwise comparisons between the three organs and a regulatory network for monoterpene biosynthesis was constructed.ConclusionsAnalysis of differentially expressed genes in flower, leaf and bulb scale of lily, using second generation sequencing technology, yielded detailed information on lily metabolic differentiation in three organs. Analysis of the expression of flower scent biosynthesis genes has provided a model for the regulation of the pathway and identified a candidate gene encoding an enzyme catalyzing the final step in scent production. These digital gene expression profiles provide a valuable and informative database for the further identification and analysis of structural genes and transcription factors in different lily organs and elucidation of their function.

A comparison of the molecular mechanisms underpinning high-intensity, pulsed polychromatic light and low-intensity UV-C hormesis in tomato fruit

Postharvest treatment of tomato fruit with high-intensity, pulsed polychromatic light (HIPPL) has previously been shown to induce delayed ripening and disease resistance comparable to that of low-intensity UV-C (LIUV). Little, however, is known of the mechanisms underpinning postharvest HIPPL hormesis in tomato fruit. Expression of genes involved in plant hormone biosynthesis, defence, secondary metabolism and ripening were monitored 24h post treatment (24 HPT), 10 d post treatment (10 DPT) and 12h post inoculation with Botrytis cinerea (12 HPI). All genes monitored were constitutively expressed and changes in expression profiles following treatment were highly similar for both HIPPL and LIUV treatments. Expression of pathogenesis-related proteins P4, β-1,3,-Glucanase and Chitinase 9 and a jasmonate biosynthesis enzyme (OPR3), were significantly upregulated at 10 DPT and 12 HPI. Both treatments significantly downregulated the expression of polygalacturonase and flavonol synthase at 10 DPT and 12 HPI. Ethylene biosynthesis enzyme ACO1 and β-carotene hydroxylase were significantly upregulated at 24 HPT, and phenylalanine ammonia-lyase (PAL) was significantly upregulated at 12 HPI. Both HIPPL and LIUV treatments stimulate defence responses that are mediated by salicylic acid, jasmonic acid and ethylene. This may lead to broad range resistance against both necrotrophic and biotrophic pathogens as well as abiotic stresses and herbivorous pests. Following inoculation with B. cinerea only PAL showed indication of a gene priming response for HIPPL- and LIUV-treated fruit.

Nitrogen form and mycorrhizal inoculation amount and timing affect flavonol biosynthesis in onion (Allium cepa L.)

Mycorrhizal symbiosis is known to be the most prevalent form of fungal symbiosis with plants. Although some studies focus on the importance of mycorrhizal symbiosis for enhanced flavonoids in the host plants, a comprehensive understanding of the relationship still is lacking. Therefore, we studied the effects of mycorrhizal inoculation of onions (Allium cepa L.) regarding flavonol concentration and the genes involved in flavonol biosynthesis when different forms of nitrogen were supplied. We hypothesized that mycorrhizal inoculation can act as a biotic stress and might lead to an increase in flavonols and expression of related genes. The three main quercetin compounds [quercetin-3,4′-di-O-β-d-glucoside (QDG), quercetin-4′-O-β-d-glucoside (QMG), and isorhamnetin-4′-O-β-d-glucoside (IMG)] of onion bulbs were identified and analyzed after inoculating with increasing amounts of mycorrhizal inocula at two time points and supplying either predominantly NO3− or NH4+ nitrogen. We also quantified plant dry mass, nutrient element uptake, chalcone synthase (CHS), flavonol synthase (FLS), and phenyl alanine lyase (PAL) gene expression as key enzymes for flavonol biosynthesis. Inoculation with arbuscular mycorrhizal fungi (highest amount) and colonization at late development stages (bulb growth) increased QDG and QMG concentrations if plants were additionally supplied with predominantly NH4+. No differences were observed in the IMG content. RNA accumulation of CHS, FLS, and PAL was affected by the stage of the mycorrhizal symbiosis and the nitrogen form. Accumulation of flavonols was not correlated, however, with either the percentage of myorrhization or the abundance of transcripts of flavonoid biosynthesis genes. We found that in plants at late developmental stages, RNA accumulation as a reflection of a current physiological situation does not necessarily correspond with the content of metabolites that accumulate over a long period. Our findings suggest that nitrogen form can be an important factor determining mycorrhizal development and that both nitrogen form and mycorrhizas interact to influence flavonol biosynthesis.

Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae

BackgroundKaempferol is a flavonol with broad bioactivity of anti-oxidant, anti-cancer, anti-diabetic, anti-microbial, cardio-protective and anti-asthma. Microbial synthesis of kaempferol is a promising strategy because of the low content in primary plant source.MethodsIn this study, the biosynthesis pathway of kaempferol was constructed in the budding yeast Saccharomyces cerevisiae to produce kaempferol de novo, and several biological measures were taken for high production.ResultsFirstly, a high efficient flavonol synthases (FLS) from Populus deltoides was introduced into the biosynthetic pathway of kaempferol. Secondly, a S. cerevisiae recombinant was constructed for de novo synthesis of kaempferol, which generated about 6.97 mg/L kaempferol from glucose. To further promote kaempferol production, the acetyl-CoA biosynthetic pathway was overexpressed and p-coumarate was supplied as substrate, which improved kaempferol titer by about 23 and 120%, respectively. Finally, a fed-batch process was developed for better kaempferol fermentation performance, and the production reached 66.29 mg/L in 40 h.ConclusionsThe titer of kaempferol in our engineered yeast is 2.5 times of the highest reported titer. Our study provides a possible strategy to produce kaempferol using microbial cell factory.

Temporal lag between gene expression and metabolite accumulation in flavonol biosynthesis of Arabidopsis roots

The temporal lag between gene expression and metabolite accumulation has been estimated in flavonol biosynthesis, but the time difference between these events is unclear. In the present study, we investigated the expression of flavonol biosynthetic genes ELONGATED HYPOCOTYL5, MYELOBLASTOSIS PROYEIN12/PRODUCTION OF FLAVONOL GLYCOSYDES1, CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE, and FLAVONOL SYNTHASE1, and the accumulation of flavonol glycosides (kaempferol and quercetin glycosides) in time-series samples of Arabidopsis thaliana roots. All genes started to be expressed within 3h after sequential light irradiation (HAS) and reached their maximum expression levels at 12 HAS, and the accumulation of the flavonol glycosides started at 6 HAS. Metabolome analysis using liquid chromatography-mass spectrometry showed that the accumulation of kaempferol 3-O-glucoside-7-O-rhamnoside and kaempferol 3-O-rhamnosyl (1→2) glucoside-7-O-rhamnoside reached their maximum levels at 48 HAS, whereas other flavonol glycosides, such as kaempferol/quercetin 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3-O-rhamnosyl (1→2) glucoside-7-O-rhamnoside, increased gradually until 96 HAS. These results show that the expression of the flavonol genes is an early response against light exposure, and that the accumulation of the flavonol glycosides is a late response.

De novo Assembly of the Camellia nitidissima Transcriptome Reveals Key Genes of Flower Pigment Biosynthesis.

The golden camellia, Camellia nitidissima Chi., is a well-known ornamental plant that is known as “the queen of camellias” because of its golden yellow flowers. The principal pigments in the flowers are carotenoids and flavonol glycosides. Understanding the biosynthesis of the golden color and its regulation is important in camellia breeding. To obtain a comprehensive understanding of flower development in C. nitidissima, a number of cDNA libraries were independently constructed during flower development. Using the Illumina Hiseq2500 platform, approximately 71.8 million raw reads (about 10.8 gigabase pairs) were obtained and assembled into 583,194 transcripts and 466, 594 unigenes. A differentially expressed genes (DEGs) and co-expression network was constructed to identify unigenes correlated with flower color. The analysis of DEGs and co-expressed network involved in the carotenoid pathway indicated that the biosynthesis of carotenoids is regulated mainly at the transcript level and that phytoene synthase (PSY), β -carotene 3-hydroxylase (CrtZ), and capsanthin synthase (CCS1) exert synergistic effects in carotenoid biosynthesis. The analysis of DEGs and co-expressed network involved in the flavonoid pathway indicated that chalcone synthase (CHS), naringenin 3-dioxygenase (F3H), leucoanthocyanidin dioxygenase(ANS), and flavonol synthase (FLS) play critical roles in regulating the formation of flavonols and anthocyanidin. Based on the gene expression analysis of the carotenoid and flavonoid pathways, and determinations of the pigments, we speculate that the high expression of PSY and CrtZ ensures the production of adequate levels of carotenoids, while the expression of CHS, FLS ensures the production of flavonols. The golden yellow color is then the result of the accumulation of carotenoids and flavonol glucosides in the petals. This study of the mechanism of color formation in golden camellia points the way to breeding strategies that exploit gene technology approaches to increase the content of carotenoids and flavonol glucosides and to decrease anthocyanidin synthesis.

Identification of functional flavonol synthase genes from fragrant wild cyclamen (Cyclamen purpurascens)

Cyclamen purpurascens is considered suitable for horticultural breeding of cyclamens because it has an attractive fragrance that is not found in other wild species. To improve the commercial value of cyclamen flowers, this fragrance has been introduced into ornamental cultivars. However, variation in flower color is somewhat limited in these cultivars, and therefore understanding the genetic networks of flower coloration in C. purpurascens is required. We previously isolated DNA fragments of anthocyanin biosynthetic genes from C. purpurascens, broadening our understanding of the biosynthetic pathway of flavonols, which are co-pigments in flower coloration. In this study, we isolated complete open reading frames of flavonol synthase genes from C. purpurascens (CpurFLS1 and CpurFLS2) and analyzed the in planta functions of the genes by molecular complementation assay using the fls mutant of Arabidopsis thaliana. Expression patterns in several organs of C. purpurascens were also determined. The results strongly suggest that the CpurFLS genes participate in flavonol synthesis. We discuss the involvement of these two FLSs in flower coloration in C. purpurascens.

Understanding the molecular mechanisms underlying the effects of light intensity on flavonoid production by RNA-seq analysis in Epimedium pseudowushanense B.L.Guo.

Epimedium pseudowushanense B.L.Guo, a light-demanding shade herb, is used in traditional medicine to increase libido and strengthen muscles and bones. The recognition of the health benefits of Epimedium has increased its market demand. However, its resource recycling rate is low and environmentally dependent. Furthermore, its natural sources are endangered, further increasing prices. Commercial culture can address resource constraints of it.Understanding the effects of environmental factors on the production of its active components would improve the technology for cultivation and germplasm conservation. Here, we studied the effects of light intensities on the flavonoid production and revealed the molecular mechanism using RNA-seq analysis. Plants were exposed to five levels of light intensity through the periods of germination to flowering, the flavonoid contents were measured using HPLC. Quantification of epimedin A, epimedin B, epimedin C, and icariin showed that the flavonoid contents varied with different light intensity levels. And the largest amount of epimedin C was produced at light intensity level 4 (I4). Next, the leaves under the treatment of three light intensity levels (“L”, “M” and “H”) with the largest differences in the flavonoid content, were subjected to RNA-seq analysis. Transcriptome reconstruction identified 43,657 unigenes. All unigene sequences were annotated by searching against the Nr, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In total, 4008, 5260, and 3591 significant differentially expressed genes (DEGs) were identified between the groups L vs. M, M vs. H and L vs. H. Particularly, twenty-one full-length genes involved in flavonoid biosynthesis were identified. The expression levels of the flavonol synthase, chalcone synthase genes were strongly associated with light-induced flavonoid abundance with the highest expression levels found in the H group. Furthermore, 65 transcription factors, including 31 FAR1, 17 MYB-related, 12 bHLH, and 5 WRKY, were differentially expressed after light induction. Finally, a model was proposed to explain the light-induced flavonoid production. This study provided valuable information to improve cultivation practices and produced the first comprehensive resource for E. pseudowushanense transcriptomes.

Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum.

Epimedium species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs) and anthocyanins, predominantly contributing to the color diversity of Epimedium flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF) as well as bHLH TF and WD40 protein at the transcriptional level. MYB TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from Epimedium sagittatum, but a R2R3-MYB TF involved in regulating both these two pathways has not been functionally characterized to date in Epimedium plants. In this study, we report the functional characterization of EsMYB9, a R2R3-MYB TF previously isolated from E. sagittatum. The previous study indicated that EsMYB9 belongs to a small subfamily of R2R3-MYB TFs containing grape VvMYB5a and VvMYB5b TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of EsMYB9 in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of EsMYB9 contributes to the autoactivation activity, and EsMYB9 interacts with EsTT8 or AtTT8 bHLH regulator. Transient reporter assay shows that EsMYB9 slightly activates the expression of EsCHS (chalcone synthase) promoter in transiently transformed leaves of Nicotiana benthamiana, but the addition of AtTT8 or EsTT8 bHLH regulator strongly enhances the transcriptional activation of EsMYB9 against five promoters of the flavonoid pathway genes except EsFLS (flavonol synthase). In addition, co-transformation of EsMYB9 and EsTT8 in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The potential role of EsMYB9 in modulating the biosynthesis and accumulation of sucrose-induced anthocyanin and flavonol-derived BCs is also discussed. These findings suggest that EsMYB9 is a novel R2R3-MYB TF, which regulates the flavonoid biosynthetic pathway in Epimedium, but distinctly different with the anthocyanin or flavonol-specific MYB regulators identified previously in Epimedium plants.

Transcriptomes of Podophyllum hexandrum unravel candidate miRNAs and their association with the biosynthesis of secondary metabolites

Podophyllotoxin is a high value metabolite having anticancerous activity and procured from a medicinal plant Podophyllum hexandrum. The escalating demand for podophyllotoxin necessitates development of alternate production platforms. Cell cultures have been tried in the past, however, podophyllotoxin yields were very low (0.3%). Multiple genes of biosynthetic pathway have been correlated with podophyllotoxin content. Understanding regulation of podophyllotoxin is, therefore, essential to design a suitable genetic intervention strategy. Six potential miRNAs were identified in NGS transcriptomes of P. hexandrum for which eight potential mRNA targets were discerned. The identified miRNAs and their mRNA targets (UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors) were further tested for any correlation through qRT-PCR. miR396b, miR2673a, miR828b and miR2910 established negative correlation with their mRNA targets through downregulation via complementary miRNA:mRNA base pairing. This is the first report on identification, characterisation, validation and expression analysis of miRNAs and their targets in P. hexandrum.

Transcriptome-Wide Identification of Novel UV-B- and Light Modulated Flavonol Pathway Genes Controlled by VviMYBF1.

Flavonols constitute a group of flavonoids with important photoprotective roles in plants. In addition, flavonol content and composition greatly influences fruit quality. We previously demonstrated that the grapevine R2R3-MYB transcription factor (TF) VviMYBF1 promotes flavonol accumulation by inducing the expression of flavonol synthase (VviFLS1/VviFLS4), a key step of the initial flavonol pathway. Despite this, gene networks underlying flavonol modification in grapevine including both structural and regulatory genes remain poorly understood. In order to identify flavonol modifying genes and TFs acting downstream of VviMYBF1 a microarray-based transcriptome analysis was performed on grapevine hairy roots ectopically expressing VviMYBF1 or a Green Fluorescent Protein as control. VviFLS1 was induced in VviMYBF1 transgenic roots and glycosylated flavonols accumulated significantly compared with control lines. Among the differentially expressed genes, potential flavonol-modifying enzymes with predicted rhamnosyltransferase (e.g., RhaT1) or glycosyltransferase (e.g., GT3) activities were identified. In addition, important TFs of the MYB and bZIP families such as the proanthocyanidin regulator VviMYBPA1 and the UV-B light responsive HY5 homolog VviHYH were significantly altered in their expression pattern by overexpression of VviMYBF1. Co-temporal expression analysis demonstrated positive correlation of VviMYBF1 with VviFLS1, VviGT3, and VviRhaT1 during berry development and in fruits ripened with different light and UV-B radiation conditions at field. These results show that VviMYBF1 overexpression led to the identification of novel genes of the flavonol pathway and that the flavonol modifying machinery can be influenced by agricultural practices to optimize flavonol composition in grapes.

Enhancing Biosynthesis of Flavonol Protective Biomaterials Using FLS Transgenic Plants

Flavonol synthase (FLS) gene encodes an enzyme that is involved in conversion substrates into flavonols, quercetin and kaempferol. These substances are a subgroup of flavonoids which have an important role in both plant and human health. Many environmental factors such as temperature, pH and UV-A radiation have been studied and presented relationship with flavonoid synthesis. In this experiment, the combination of visible and UV-A lights was used as factors for elevating flavonoid biosynthesis of wild type (WT) plant and two lines of FLS transgenic plant under tissue culture condition. Both transgenic lines significantly enhanced the accumulation of quercetin and kaempferol substances nearly one fold higher than WT plant did. The photosynthetic pigment levels of chlorophyll A, chlorophyll B and carotenoid in transgenic lines are in the range 45.20-46.88, 16.34-17.04 and 13.63-13.46, while those of WT plants are 35.93, 13.18 and 10.55 (µg/g FW), respectively. Therefore, FLS transgenic plants containing high flavonol content showed a better in the protection photosynthetic pigments by less reductions of chlorophyll and carotenoid pigments.