Flavonol Synthase Research Articles

Analysis of the transcriptome and metabolome reveals phenylpropanoid mechanism in common bean (Phaseolus vulgaris) responding to salt stress at sprout stage

AbstractCommon bean (Phaseolus vulgaris) seeds are important legume crops and an important source of dietary proteins and carbohydrates. Therefore, it is important to develop strategies to improve salt tolerance in common beans. In this study, transcriptome and metabolome analyses were conducted on local common bean variety under salt stress at the sprout stage for a period of 0, 12, and 24 h. Results showed that phenylpropanoid pathways (including phenylpropanoid biosynthesis and phenylalanine metabolism) and flavonoid pathways (including flavonoid biosynthesis and flavone and flavonol biosynthesis) played an important role in controlling responses to salt stress as evidenced by analysis of differentially expression genes, common expression patterns, WCGNA, and differentially altered metabolites (DAMs) analyses. In addition, exploration of the activities of 4‐coumarate‐CoA ligase (4CL), caffeoyl‐CoA O‐methyltransferase (CCoAOMT), peroxidase (POD), chalcone isomerase (CHI), dihydroflavonol‐4‐reductase (DFR), and flavonol synthase (FLS) further showed that phenylpropanoid and flavonoid pathways participate in plant responses to salt stress. Moreover, the phenylpropanoid pathways and flavonoid pathways were found to be potential pathways regulating plant response to salt stress based on transcriptome and metabolome analysis. The activities of 4CL, CCoAOMT, POD, CHI, DFR, and FLS revealed that these pathways are crucial to the regulation of plant responses to salt stress. These findings provided theoretical basis for further improvement of salt tolerance in common bean.

Physiological response and molecular regulatory mechanism reveal a positive role of nitric oxide and hydrogen sulfide applications in salt tolerance of Cyclocarya paliurus.

As a multifunctional tree species, Cyclocarya paliurus leaves are rich in bioactive substances with precious healthy values. To meet the huge requirement of C. paliurus leaf production, sites with some environmental stresses would be potential land for developing its plantations due to the limitation of land resources in China. Nitric oxide (NO) and hydrogen sulfide (H2S) are common gas messengers used to alleviate abiotic stress damage, whereas the mechanism of these messengers in regulating salt resistance of C. paliurus still remains unclear. We performed a comprehensive study to reveal the physiological response and molecular regulatory mechanism of C. paliurus seedlings to the application of exogenous NO and H2S under salt stress. The results showed that the application of sodium hydrosulfide (NaHS) and sodium nitroprusside (SNP) not only maintained the photosynthetic capacity and reduced the loss of leaf biomass, but also promoted endogenous NO synthesis and reduced oxidative damage by activating antioxidant enzyme activity and increasing the content of soluble protein and flavonoids. Moreover, transcriptome and metabolome analysis indicated the expression of genes encoding phenylalanine ammonia lyase (PAL), cytochromeP450 (CYP), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) in flavonoid biosynthesis pathway was all up-regulated by the application of NO and H2S. Meanwhile, 15 transcriptional factors (TFs) such as WRKY, ERF, bHLH and HY5 induced by NO were found to regulated the activities of several key enzymes in flavonoid biosynthesis pathway under salt stress, via the constructed co-expression network. Our findings revealed the underlying mechanism of NO and H2S to alleviate salt stress and regulate flavonoid biosynthesis, which provides a theoretical basis for establishing C. paliurus plantations in the salt stress areas.

Functional characterization of 2-oxoglutarate-dependent dioxygenase gene family in chickpea

Chickpea is an important leguminous crop plant with two cultivated types, desi and kabuli. It is nutritionally enriched in flavonoid content in addition to minerals and vitamins imparting huge health benefits to human beings. Our study elucidates the functionality of 2-oxoglutarate dependent dioxygenase (2-ODD) gene family members i.e., flavanone-3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) in chickpea using heterologous bacterial system and in-planta studies in Arabidopsis. This provides information about the biosynthesis of two very significant sub-classes of flavonoids- flavonols and anthocyanins. Here, we show that all the three homologs of F3H in chickpea can utilize not just naringenin but also eriodictyol as their substrate. Moreover, we show that FLS in chickpea exhibits bifunctionality having both FLS and F3H activity. Also, our study indicates the richness of desi chickpea over kabuli type through gene expression and metabolite content analyses. Overall, our study establishes the functionality of 2-ODD gene family involved in the early and late steps of flavonoid biosynthesis pathway in chickpea. It paves way for better genetic manipulation of the pathway for direct or indirect synthesis of three major subclasses of flavonoids (flavonol, anthocyanin and proanthocyanin) to develop nutritious, environmentally stable and healthy chickpea (Cicer arietinum) crop.

The parallel biosynthesis routes of hyperoside from naringenin in Hypericum monogynum.

Hyperoside is a bioactive flavonoid galactoside in both medicinal and edible plants. It plays an important physiological role in the growth of flower buds. However, the hyperoside biosynthesis pathway has not been systematically elucidated in plants, including its original source, Hypericaceae. Our group found abundant hyperoside in the flower buds of Hypericum monogynum, and we sequenced its transcriptome to study the biosynthetic mechanism of hyperoside. After gene screening and functional verification, four kinds of key enzymes were identified. Specifically, HmF3Hs (flavanone 3-hydroxylases) and HmFLSs (flavonol synthases) could catalyze flavanones into dihydroflavonols, as well as catalyzing dihydroflavonols into flavonols. HmFLSs could also convert flavanones into flavonols and flavones with varying efficiencies. HmF3'H (flavonoid 3'-hydroxylase) was found to act broadly on 4'-hydroxyl flavonoids to produce 3',4'-diydroxylated flavanones, dihydroflavonols, flavonols, and flavones. HmGAT (flavonoid 3-O-galactosyltransferase) would transform flavonols into the corresponding 3-O-galactosides, including hyperoside. The parallel hyperoside biosynthesis routes were thus depicted, one of which was successfully reconstructed in Escherichia coli BL21(DE3) by feeding naringenin, resulting in a hyperoside yield of 25mg/l. Overall, this research not only helped us understand the interior catalytic mechanism of hyperoside in H. monogynum concerning flower development and bioactivity, but also provided valuable insights into these enzyme families.

Mutational analysis of flavonol synthase of M. pinnata towards enhancement of binding affinity: a computational approach

Millettia pinnata is an important medicinal plant that has been used as a treatment of various diseases due to presence of wide range of pharmacological properties. The plant contains quercetin, kaempferol, karanjin, pongaglabrone, kanjone, kanugin, gammatin, pongaglabol, and other bioflavonoids. Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, and antidiabetic activities etc. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols, i.e. biosynthesis of kaempferol from dihydrokaempferol. The current work examined the binding affinity-based approach to improve the enzyme catalytic activity using computational methods. Sequential site-directed mutagenesis was used to create four mutants with the goal to increase hydrogen bonds and further improving the ligand (dihydrokaempferol) binding efficiency. Simulations were done to monitor the stability of the mutants followed by molecular docking to confirm interactions with ligand. For structure validation, various dynamic analysis like RMSD, RMSF, ROG, SASA, H-bond, PCA, DCCM, and FEL were performed, which predicts the stability of wild-type (WT) proteins and mutants. The Mutant_2 and Mutant_3 showed maximum H-bonding and better stability than other mutants and WT that proved higher affinity suggesting improved catalysis. Mutant_2 and Mutant_3 exhibited binding affinities of −7.6 and −8.2 kcal/mol, respectively for the ligand. The outcome of present study will provide significant improvement in synthesis of kaempferol and other plant-based flavonoids. Communicated by Ramaswamy H. Sarma

Genomic-wide identification and expression analysis of R2R3-MYB transcription factors related to flavonol biosynthesis in Morinda officinalis

BackgroundThe R2R3-MYB transcription factors are a crucial and extensive gene family in plants, which participate in diverse processes, including development, metabolism, defense, differentiation, and stress response. In the Lingnan region of China, Morinda officinalis is extensively grown and is renowned for its use as both a medicinal herb and food source. However, there are relatively few reports on the R2R3-MYB transcription factor family in M.officinalis.ResultsIn this study, we identified 97 R2R3-MYB genes in the genome of Morinda officinalis and classified them into 32 subgroups based on phylogenetic comparison with Arabidopsis thaliana. The lack of recent whole-genome duplication events in M.officinalis may be the reason for the relatively few members of the R2R3-MYB family. We also further analyzed the physical and chemical characteristics, conserved motifs, gene structure, and chromosomal location. Gene duplication events found 21 fragment duplication pairs and five tandem duplication event R2R3-MYB genes in M.officinalis may also affect gene family expansion. Based on phylogenetic analysis, cis-element analysis, co-expression analysis and RT-qPCR, we concluded that MoMYB33 might modulate flavonol levels by regulating the expression of 4-coumarate-CoA ligase Mo4CL2, chalcone isomerase MoCHI3, and flavonol synthase MoFLS4/11/12. MoMYB33 and AtMYB111 showed the highest similarity of 79% and may be involved in flavonol synthase networks by the STRING database. Moreover, we also identified MoMYB genes that respond to methyl Jasmonate (MeJA) and abscisic acid (ABA) stress by RT-qPCR.ConclusionsThis study offers a thorough comprehension of R2R3-MYB in M.officinalis, which lays the foundation for the regulation of flavonol synthesis and the response of MoMYB genes to phytohormones in M.officinalis.

Transcriptomic Analysis Reveals the Regulatory Mechanism of Color Diversity in Rhododendron pulchrum Sweet (Ericaceae).

Rhododendron pulchrum Sweet is a traditional ornamental plant cultivated in China and presents a great variation in petal coloration. However, few studies have been performed to reveal the genes involved and the regulatory mechanism of flower color formation in this plant. In this study, to explore the underlying genetic basis of flower color formation, transcriptome analysis was performed by high-throughput sequencing techniques on four petal samples of different colors: purple, pink, light pink, and white. Results show that a total of 35.55 to 40.56 million high-quality clean reads were obtained, of which 28.56 to 32.65 million reads were mapped to the reference genome. For their annotation, 28,273, 18,054, 24,301, 19,099, and 11,507 genes were allocated to Nr, Swiss-Prot, Pfam, GO, and KEGG databases, correspondingly. There were differentially expressed genes among the four different petal samples, including signal-transduction-related genes, anthocyanin biosynthesis genes, and transcription factors. We found that the higher expressed levels of genes associated with flavonol synthase (FLS) might be the key to white formation, and the formation of red color may be related to the higher expression of flavanone 4-reductase (DFR) families. Overall, our study provides some valuable information for exploring and understanding the flower color intensity variation in R. pulchrum.

Full-length transcriptome analysis provides insights into flavonoid biosynthesis in Ranunculus japonicus.

Ranunculus japonicus Thunb. is a traditional Chinese herb. Plants in the genus Ranunculus are generally rich in flavonoids, which have antibacterial, anti-infective, and other pharmacological effects. However, owing to the lack of reference genomes, little is known about the flavonoid biosynthetic pathway in R. japonicus. In this study, PacBio isoform sequencing (PacBio iso-seq) and DNA nanoball sequencing (DNB-seq) were combined to build a full-length transcriptome database for three different tissues of R. japonicus. A total of 395,402 full-length transcripts were obtained, of which 308,474 were successfully annotated. A Kyoto Encyclopedia of Genes and Genomes analysis identified 29 differentially expressed genes encoding nine key enzymes for flavonoid biosynthesis. Correlation analysis indicated that flavanone 3-hydroxylase and flavonol synthase genes might have key roles in the accumulation of flavonoid substances in the different tissues of R. japonicus. The structures of chalcone synthase and chalcone isomerase enzymes were spatially modeled. Reverse-transcription quantitative PCR was used to verify gene expression levels of key enzymes associated with flavonoid biosynthesis. In addition, 22 MYB transcription factors involved in flavonoid biosynthesis and phenylpropanoid biosynthesis were discovered. The reliable transcriptomic data from this study provide genetic information about R. japonicus as well as insights into the molecular mechanism of flavonoid biosynthesis. The results also provide a basis for developing the medicinal value R. japonicus. This article is protected by copyright. All rights reserved.

Flavonol synthase gene MsFLS13 regulates saline-alkali stress tolerance in alfalfa

Alfalfa (Medicago sativa L.) is one of the most extensively grown leguminous forage worldwide. Environmental saline-alkali stress significantly influences the growth, development, and yield of alfalfa, posing a threat to its agricultural production. However, little is known about the potential mechanisms by which alfalfa responds to saline-alkali stress. Here, we investigated these mechanisms by cloning a saline-alkali-induced flavonol synthase gene (MsFLS13) from alfalfa, which was previously reported to be significantly upregulated under saline-alkali stress, and examining its function in the saline-alkali response. Overexpression of MsFLS13 in alfalfa promoted plant tolerance to saline-alkali stress by enhancing flavonol accumulation, antioxidant capacity, osmotic balance, and photosynthetic efficiency. Conversely, MsFLS13 inhibition using RNA interference reduced flavonol synthase activity and inhibited hairy root growth under saline-alkali stress. Yeast one-hybrid and dual-luciferase reporter assays indicated that the R2R3-MYB MsMYB12 transcription factor activates MsFLS13 expression by binding to the MBS motif in the MsFLS13 promoter. Further analysis revealed that abscisic acid mediates the saline-alkali stress response partially by inducing MsMYB12 and MsFLS13 expression, which consequently increases flavonol levels and maintains antioxidant homeostasis in alfalfa. Collectively, our findings highlight the crucial role of MsFLS13 in alfalfa in response to saline-alkali stress and provide a novel genetic resource for creating saline-alkali-resistant alfalfa through genetic engineering.

Transcription factor NnMYB5 controls petal color by regulating GLUTATHIONE S-TRANSFERASE2 in Nelumbo nucifera.

Lotus (Nelumbo spp.) is an important aquatic ornamental genus in the family Nelumbonaceae comprising only two species: N. lutea with yellow flowers and N. nucifera with red or white flowers. The petal color variations between these two species have previously been associated with the potential activities of FLAVONOL SYNTHASE (FLS) and MYB5. However, the underlying genetic mechanisms of flower color divergence within the N. nucifera species remains unclear. Here, quantitative trait locus mapping led to the identification of MYB5, a candidate gene controlling petal color in N. nucifera. Genotyping of 213 natural lotus accessions revealed an 80-kb presence/absence variant (PAV) of the NnMYB5 gene that is associated with petal color variation. Transcriptome analysis, dual-luciferase and yeast one-hybrid assays showed that NnMYB5 could directly activate the anthocyanin transporter gene GLUTATHIONE S-TRANSFERASE2 (NnGST2). Heterologous expression of NnGST2 in Arabidopsis (Arabidopsis thaliana) and its overexpression in lotus petals induced anthocyanin accumulation. Deletion of the 80-kb PAV within NnMYB5 inactivated NnGST2 expression and blocked anthocyanin accumulation in white N. nucifera petals. In contrast, the anthocyanin deficiency of N. lutea occurred due to pseudogenized NlMYB5 alleles. Our results establish a regulatory link between NnMYB5 and NnGST2 in petal anthocyanin accumulation and demonstrate the independent mechanisms controlling flower coloration in Nelumbo.

Transcriptomic and metabolomic perspectives for the growth of alfalfa (Medicago sativa L.) seedlings with the effect of vanadium exposure

Hitherto, the effect of vanadium on higher plant growth remains an open topic. Therefore, nontargeted metabolomic and RNA-Seq profiling were implemented to unravel the possible alteration in alfalfa seedlings subjected to 0.1 mg L−1 (B group) and 0.5 mg L−1 (C group) pentavalent vanadium [(V(V)] versus control (A group) in this study. Results revealed that vanadium exposure significantly altered some pivotal transcripts and metabolites. The number of differentially expressed genes (DEGs) markedly up- and down-regulated was 21 and 23 in B_vs_A, 27 and 33 in C_vs_A, and 24 and 43 in C_vs_B, respectively. The number for significantly up- and down-regulated differential metabolites was 17 and 15 in B_vs_A, 43 and 20 in C_vs_A, and 24 and 16 in C_vs_B, respectively. Metabolomics and transcriptomics co-analysis characterized three significantly enriched metabolic pathways in C_vs_A comparing group, viz., α-linolenic acid metabolism, flavonoid biosynthesis, and phenylpropanoid biosynthesis, from which some differentially expressed genes and differential metabolites participated. The metabolite of traumatic acid in α-linolenic acid metabolism and apigenin in flavonoid biosynthesis were markedly upregulated, while phenylalanine in phenylpropanoid biosynthesis was remarkably downregulated. The genes of allene oxide cyclase (AOC) and acetyl-CoA acyltransferase (fadA) in α-linolenic acid metabolism, and chalcone synthase (CHS), flavonoid 3′-monooxygenase (CYP75B1), and flavonol synthase (FLS) in flavonoid biosynthesis, and caffeoyl-CoA O-methyltransferase (CCoAOMT) in phenylpropanoid biosynthesis were significantly downregulated. While shikimate O-hydroxycinnamoyltransferase (HCT) in flavanoid and phenylpropanoid biosynthesis were conspicuously upregulated. Briefly, vanadium exposure induces a readjustment yielding in metabolite and the correlative synthetic precursors (transcripts/unigenes) in some branched metabolic pathways. This study provides a practical and in-depth perspective from transcriptomics and metabolomics in investigating the effects conferred by vanadium on plant growth and development.

Late Elongated Hypocotyl Positively Regulates Salt Stress Tolerance in Medicago truncatula.

Abiotic stress, such as drought, osmotic, and salinity stresses, seriously affects plant growth and crop production. Studying stress-resistant genes that enhance plant stress tolerance is an efficient way to facilitate the breeding of crop species with high stress tolerance. In this study, we reported that the core circadian clock component, the LATE ELONGATED HYPOCOTYL (LHY) orthologue MtLHY, plays a positive role in salt stress response in Medicago truncatula. The expression of MtLHY was induced by salt stress, and loss-of-function mutants of MtLHY were shown to be hypersensitive to salt treatment. However, overexpression of MtLHY improved salt stress tolerance through a higher accumulation of flavonoids. Consistently, exogenous flavonol application improved the salt stress tolerance in M. truncatula. Additionally, MtLHY was identified as a transcriptional activator of the flavonol synthase gene, MtFLS. Our findings revealed that MtLHY confers plant salt stress tolerance, at least by modulating the flavonoid biosynthesis pathway, which provides insight into salt stress tolerance that links the circadian clock with flavonoid biosynthesis.

A flavonol synthase (FLS) gene, GhFLS1, was screened out increasing salt resistance in cotton

BackgroundFlavonols play important roles in antioxidation and anticancer activities, longevity, and cardiovascular protection. Flavonol synthase (FLS) is a key enzyme for flavonol synthesis.ResultPhenotypic, transcriptional and metabolic data were analyzed, which showed that there was a close relationship between salt stress and flavonoids, and flavonols were significantly upregulated under salt stress. Nine, seven, four, and four FLS genes were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium raimondii, respectively. The results of subcellular localization showed that FLS existed in the nucleus and cytoplasmic. Through phylogenetic analysis, 24 FLS genes were divided into three subfamilies. The results of the RNA sequencing showed that the expression of GhFLS genes was mainly induced by salt, drought, low temperature, and heat stress. GhFLS promoter mainly comprised plant hormone response elements and abiotic stress elements, indicating that the GhFLS gene may play a key role in abiotic stress response. The proline contents of pYL156:GhFLS1 was reduced significantly compared to pYL156 under salt stress, thereby reducing the resistance of cotton to salt stress.ConclusionThis study will lay a foundation for further study on the antioxidant regulation mechanism of the FLS gene under abiotic stress.

Identification and functional characterization of a flavonol synthase gene from sweet potato [Ipomoea batatas (L.) Lam.

Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, which catalyzes the conversion of dihydroflavonols into flavonols. In this study, the FLS gene IbFLS1 was cloned and characterized from sweet potato. The resulting IbFLS1 protein showed a high similarity with other plant FLSs. The conserved amino acids (HxDxnH motifs) binding ferrous iron and residues (RxS motifs) binding 2-oxoglutarate were found in IbFLS1 at conserved positions, as in other FLSs, suggesting that IbFLS1 belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. qRT-PCR analysis showed an organ-specific pattern of expression of the IbFLS1 gene, which was predominantly expressed in young leaves. The recombinant IbFLS1 protein could catalyze the conversion of dihydrokaempferol and dihydroquercetin to kaempferol and quercetin, respectively. The results of subcellular localization studies indicated that IbFLS1 was found mainly in the nucleus and cytomembrane. Furthermore, silencing the IbFLS gene in sweet potato changed the color of the leaves to purple, substantially inhibiting the expression of IbFLS1 and upregulating the expression of genes involved in the downstream pathway of anthocyanin biosynthesis (i.e., DFR, ANS, and UFGT). The total anthocyanin content in the leaves of the transgenic plants was dramatically increased, whereas the total flavonol content was significantly reduced. Thus, we conclude that IbFLS1 is involved in the flavonol biosynthetic pathway and is a potential candidate gene of color modification in sweet potato.

Synergistic actions of three MYB transcription factors underpins the high accumulation of myricetin in Morella rubra.

Flavonols are health-promoting bioactive compounds important for human nutrition, health, and plant defense. The transcriptional regulation of kaempferol and quercetin biosynthesis has been studied extensively, while little is known about the regulatory mechanisms underlying myricetin biosynthesis, which has strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. In this study, the flavonol-specific MrMYB12 in Morella rubra preferred activating the promoter of flavonol synthase 2 (MrFLS2) (6.4-fold) rather than MrFLS1 (1.4-fold) and upregulated quercetin biosynthesis. Furthermore, two SG44 R2R3-MYB members, MrMYB5 and MrMYB5L, were identified by yeast one-hybrid library screening using the promoter of flavonoid 3',5'-hydroxylase (MrF3'5'H), and transcript levels of these R2R3-MYBs were correlated with accumulation of myricetin derivatives during leaf development. Dual-luciferase and electrophoretic mobility shift assays demonstrated that both MrMYB5 and MrMYB5L could bind directly to MYB recognition sequence elements in promoters of MrF3'5'H or MrFLS1 and activate their expression. Protein-protein interactions of MrMYB5 or MrMYB5L with MrbHLH2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. MrMYB5L-MrbHLH2 showed much higher synergistic activation of MrF3'5'H or MrFLS1 promoters than MrMYB5-MrbHLH2. Studies with Arabidopsis thaliana homologs AtMYB5 and AtTT8 indicated that similar synergistic regulatory effects occur with promoters of MrF3'5'H or MrFLS1. Transient overexpression of MrMYB5L-MrbHLH2 in Nicotiana benthamiana induced a higher accumulation of myricetin derivatives (57.70 μg g-1 FW) than MrMYB5-MrbHLH2 (7.43 μg g-1 FW) when MrMYB12 was coexpressed with them. This study reveals a novel transcriptional mechanism regulating myricetin biosynthesis with the potential use for future metabolic engineering of health-promoting flavonols.

Transcriptomic Analysis on the Peel of UV-B-Exposed Peach Fruit Reveals an Upregulation of Phenolic- and UVR8-Related Pathways.

UV-B treatment deeply influences plant physiology and biochemistry, especially by activating the expression of responsive genes involved in UV-B acclimation through a UV-B-specific perception mechanism. Although the UV-B-related molecular responses have been widely studied in Arabidopsis, relatively few research reports deepen the knowledge on the influence of post-harvest UV-B treatment on fruit. In this work, a transcriptomic approach is adopted to investigate the transcriptional modifications occurring in the peel of UV-B-treated peach (Prunus persica L., cv Fairtime) fruit after harvest. Our analysis reveals a higher gene regulation after 1 h from the irradiation (88% of the differentially expressed genes-DEGs), compared to 3 h recovery. The overexpression of genes encoding phenylalanine ammonia-lyase (PAL), chalcone syntase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) revealed a strong activation of the phenylpropanoid pathway, resulting in the later increase in the concentration of specific flavonoid classes, e.g., anthocyanins, flavones, dihydroflavonols, and flavanones, 36 h after the treatment. Upregulation of UVR8-related genes (HY5, COP1, and RUP) suggests that UV-B-triggered activation of the UVR8 pathway occurs also in post-harvest peach fruit. In addition, a regulation of genes involved in the cell-wall dismantling process (PME) is observed. In conclusion, post-harvest UV-B exposure deeply affects the transcriptome of the peach peel, promoting the activation of genes implicated in the biosynthesis of phenolics, likely via UVR8. Thus, our results might pave the way to a possible use of post-harvest UV-B treatments to enhance the content of health-promoting compounds in peach fruits and extending the knowledge of the UVR8 gene network.

Integrated transcriptome and metabolome provide insight into flavonoid variation in goji berries (Lycium barbarum L.) from different areas in China

Goji berries (Lycium barbarum L.) were rich in flavonoids, showing high nutritional and medicinal value. However, a thorough evaluation and comparison of the flavonoids in goji berries from various regions and the possible biological regulation pathways with differences are scanty. Here, we investigated the flavonoid metabolites and gene expression levels of goji berries from three major production areas in China using transcriptomics sequencing and metabolomics. The total flavonoid content and total polyphenol content of goji berry in Ningxia (57.87 μg/g and 183.41 μg/g, respectively) were higher than in Qinghai (50.77 μg/g and 156.81 μg/g) and Gansu (47.86 μg/g and 111.17 μg/g). We identified the 105 differentially accumulated flavonoids (DAFs) and 1858 differentially expressed genes (DEGs) from the goji berries in three habitats. Interestingly, gossypetin-3-O-rutinoside and isorhamnetin were significantly expressed between Ningxia and Qinghai berries. The chalcone isomerase (CHI), chalcone synthase (CHS), and flavonol synthase (FLS) genes also played key roles in the regulation of flavonoid synthesis. In addition, MYB1 positively regulated the expression of quercetin-3-O-glucoside, quercetin-7-O-glucoside and isohyperoside. As a result, we speculated that CHI, CHS, FLS genes, and related transcription factors jointly controlled the variation of flavone accumulation in goji berries. These findings may provide a new perspective for understanding the accumulation and molecular mechanisms of goji flavonoids.

Yellow Petal locus GaYP promotes flavonol biosynthesis and yellow coloration in petals of Asiatic cotton (Gossypium arboreum)

Yellow Petal locus GaYP is located on chromosome 11 and encodes a Sg6 R2R3-MYB transcription factor, which promotes flavonol biosynthesis and yellow coloration in Asiatic cotton petals. Petal color is pivotal to ornamental value and reproduction of plants. Yellow coloration in plant petals is mainly attributed to colorants including carotenoids, aurones and some flavonols. To date, the genetic regulatory mechanism of flavonol biosynthesis in petals is still to be elucidated. Here, we employed Asiatic cottons with or without deep yellow coloration in petals to address this question. Multi-omic and biochemical analysis revealed significantly up-regulated transcription of flavonol structural genes and increased levels of flavonols, especially gossypetin and 6-hydroxykaempferol, in yellow petals of Asiatic cotton. Furthermore, the Yellow Petal gene (GaYP) was mapped on chromosome 11 by using a recombinant inbred line population. It was found that GaYP encoded a transcriptional factor belonging to Sg6 R2R3-MYB proteins. GaYP could bind to the promoter of flavonol synthase gene (GaFLS) and activate the transcription of downstream genes. Knocking out of GaYP or GaFLS homologs in upland cotton largely eliminated flavonol accumulation and pale yellow coloration in petals. Our results indicated that flavonol synthesis, up-regulated by the R2R3-MYB transcription activator GaYP, was the causative factor for yellow coloration of Asiatic cotton petals. In addition, knocking out of GaYP homologs also led to decrease in anthocyanin accumulation and petal size in upland cotton, suggesting that GaYP and its homologs might modulate developmental or physiological processes beyond flavonol biosynthesis.

Phytomelatonin interferes with flavonols biosynthesis to regulate ROS production and stomatal closure in tobacco

Flavonols are well-known antioxidants that prevent stomatal closure via interfering with ROS signaling. Phytomelatonin regulates stomatal closure, but the signaling pathways are still largely unknown. Here, we investigated the role of flavonols in phytomelatonin-mediated stomatal closure in tobacco plants. The application of melatonin induced stomatal closure through NADPH oxidase-mediated ROS production. Transgenic tobacco plants overexpressing soybean GmSNAT1 (coding for serotonin N-acetyltransferase that catalyzes the penultimate step in phytomelatonin biosynthesis) had higher phytomelatonin concentration, accumulated more ROS in guard cells and were more sensitive to melatonin-induced stomatal closure than the wild-type plants, which was associated with the higher expression of PMTR1-homologous genes. Exogenous melatonin decreased flavonol concentrations in guard cells and the expression of flavonoid-related genes in wild-type and transgenic tobacco plants, and these inhibitory effects were more obvious in GmSNAT1-overexpressing plants than the wild type. However, the melatonin-mediated stomatal closure and ROS production were diminished by the application of kaempferol (a type of flavonol). Additionally, transgenic tobacco plants with increased expression of NtFLS (encoding flavonol synthase) were less sensitive to melatonin-induced stomatal closure. In conclusion, phytomelatonin hampers the biosynthesis of flavonols in guard cells, which results in high concentration of ROS and induces stomatal closure in tobacco plants.

Investigating the induction of polyphenol biosynthesis in the cultured Cycolocarya paliurus cells and the stimulatory mechanism of co-induction with 5-aminolevulinic acid and salicylic acid.

Background: Plant cell culture technology is a potential way to produce polyphenols, however, this way is still trapped in the dilemma of low content and yield. Elicitation is regarded as one of the most effective ways to improve the output of the secondary metabolites, and therefore has attracted extensive attention. Methods: Five elicitors including 5-aminolevulinic acid (5-ALA), salicylic acid (SA), methyl jasmonate (MeJA), sodium nitroprusside (SNP) and Rhizopus Oryzae Elicitor (ROE) were used to improve the content and yield of polyphenols in the cultured Cyclocarya paliurus (C. paliurus) cells, and a co-induction technology of 5-ALA and SA was developed as a result. Meanwhile, the integrated analysis of transcriptome and metabolome was adopted to interpret the stimulation mechanism of co-induction with 5-ALA and SA. Results: Under the co-induction of 50 μM 5-ALA and SA, the content and yield of total polyphenols of the cultured cells reached 8.0 mg/g and 147.12 mg/L, respectively. The yields of cyanidin-3-O-galactoside, procyanidin B1 and catechin reached 28.83, 4.33 and 2.88 times that of the control group, respectively. It was found that expressions of TFs such as CpERF105, CpMYB10 and CpWRKY28 increased significantly, while CpMYB44 and CpTGA2 decreased. These great changes might further make the expression of CpF3'H (flavonoid 3'-monooxygenase), CpFLS (flavonol synthase), CpLAR (leucoanthocyanidin reductase), CpANS (anthocyanidin synthase) and Cp4CL (4-coumarate coenzyme A ligase) increase while CpANR (anthocyanidin reductase) and CpF3'5'H (flavonoid 3', 5'-hydroxylase) reduce, ultimately enhancing the polyphenols accumulation Conclusion: The co-induction of 5-ALA and SA can significantly promote polyphenol biosynthesis in the cultured C. paliurus cells by regulating the expression of key transcription factors and structural genes associated with polyphenol synthesis, and thus has a promising application.