Characterization of CsUGT73AC15 as a Multifunctional Glycosyltransferase Impacting Flavonol Triglycoside Biosynthesis in Tea Plants.

Flavonol glycosides, contributing to the health benefits and distinctive flavors of tea (Camellia sinensis), accumulate predominantly as diglycosides and triglycosides in tea leaves. However, the UDP-glycosyltransferases (UGTs) mediating flavonol multiglycosylation remain largely uncharacterized. In this study, we employed an integrated proteomic and metabolomic strategy to identify and characterize key UGTs involved in flavonol triglycoside biosynthesis. The recombinant rCsUGT75AJ1 exhibited flavonoid 4'-O-glucosyltransferase activity, while rCsUGT75L72 preferentially catalyzed 3-OH glucosylation. Notably, rCsUGT73AC15 displayed substrate promiscuity and regioselectivity, enabling glucosylation of rutin at multiple sites and kaempferol 3-O-rutinoside (K3R) at the 7-OH position. Kinetic analysis revealed rCsUGT73AC15's high affinity for rutin (Km = 9.64 µM). Across cultivars, CsUGT73AC15 expression inversely correlated with rutin levels. Moreover, transient CsUGT73AC15 silencing increased rutin and K3R accumulation while decreasing their respective triglycosides in tea plants. This study offers new mechanistic insights into the key roles of UGTs in regulating flavonol triglycosylation in tea plants.

A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

MdSCL8 as a Negative Regulator Participates in ALA-Induced FLS1 to Promote Flavonol Accumulation in Apples.

Apples (Malus domestica) are rich in flavonols, and 5-aminolevulinic acid (ALA) plays an important role in the regulation of plant flavonoid metabolism. To date, the underlying mechanism of ALA promoting flavonol accumulation is unclear. Flavonol synthase (FLS) is a key enzyme in flavonol biosynthesis. In this study, we found that ALA could enhance the promoter activity of MdFLS1 in the 'Fuji' apple and improve its expression. With MdFLS1 as bait, we screened a novel transcription factor MdSCL8 by the Yeast One-Hybrid (Y1H) system from the apple cDNA library which we previously constructed. Using luciferase reporter assay and transient GUS activity assay, we verified that MdSCL8 inhibits the activity of MdFLS1 promoter and hinders MdFLS1 expression, thus reducing flavonol accumulation in apple. ALA significantly inhibited MdSCL8 expression. Therefore, ALA promoted the expression of MdFLS1 and the consequent flavonol accumulation probably by down-regulating MdSCL8. We also found that ALA significantly enhanced the gene expression of MdMYB22 and MdHY5, two positive regulators of MdFLS. We further demonstrated that MdMYB22 interacts with MdHY5, but neither of them interacts with MdSCL8. Taken together, our data suggest MdSCL8 as a novel regulator of MdFLS1 and provide important insights into mechanisms of ALA-induced flavonol accumulation in apples.

Exploring the genes involved in biosynthesis of dihydroquercetin and dihydromyricetin in Ampelopsis grossedentata.

Dihydroquercetin (DHQ), an extremely low content compound (less than 3%) in plants, is an important component of dietary supplements and used as functional food for its antioxidant activity. Moreover, as downstream metabolites of DHQ, an extremely high content of dihydromyricetin (DHM) is up to 38.5% in Ampelopsis grossedentata. However, the mechanisms involved in the biosynthesis and regulation from DHQ to DHM in A. grossedentata remain unclear. In this study, a comparative transcriptome analysis of A. grossedentata containing extreme amounts of DHM was performed on the Illumina HiSeq 2000 sequencing platform. A total of 167,415,597 high-quality clean reads were obtained and assembled into 100,584 unigenes having an N50 value of 1489. Among these contigs, 57,016 (56.68%) were successfully annotated in seven public protein databases. From the differentially expressed gene (DEG) analysis, 926 DEGs were identified between the B group (low DHM: 210.31 mg/g) and D group (high DHM: 359.12 mg/g) libraries, including 446 up-regulated genes and 480 down-regulated genes (B vs. D). Flavonoids (DHQ, DHM)-related DEGs of ten structural enzyme genes, three myeloblastosis transcription factors (MYB TFs), one basic helix-loop-helix (bHLH) TF, and one WD40 domain-containing protein were obtained. The enzyme genes comprised three PALs, two CLs, two CHSs, one F3'H, one F3'5'H (directly converts DHQ to DHM), and one ANS. The expression profiles of randomly selected genes were consistent with the RNA-seq results. Our findings thus provide comprehensive gene expression resources for revealing the molecular mechanism from DHQ to DHM in A. grossedentata. Importantly, this work will spur further genetic studies about A. grossedentata and may eventually lead to genetic improvements of the DHQ content in this plant.

Combined transcriptomic and metabolic analyses reveal potential mechanism for fruit development and quality control of Chinese raspberry (Rubus chingii Hu).

KEY MESSAGE: Combined transcriptomic and metabolic analyses reveal that fruit of Rubus chingii Hu launches biosynthesis of phenolic acids and flavonols at beginning of fruit set and then coordinately accumulated or converted to their derivatives. Rubus chingii Hu (Chinese raspberry) is an important dual functional food with nutraceutical and pharmaceutical values. Comprehensively understanding the mechanisms of fruit development and bioactive components synthesis and regulation could accelerate genetic analysis and molecular breeding for the unique species. Combined transcriptomic and metabolic analyses of R. chingii fruits from different developmental stages, including big green, green-to-yellow, yellow-to-orange, and red stages, were conducted. A total of 89,188 unigenes were generated and 57,545 unigenes (64.52%) were annotated. Differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly involved in the biosynthesis of secondary metabolites. The fruit launched the biosynthesis of phenolic acids and flavonols at the very beginning of fruit set and then coordinately accumulated or converted to their derivatives. This was tightly regulated by expressions of the related genes and MYB and bHLH transcription factors. The core genes products participated in the biosynthesis of ellagic acid (EA) and kaempferol-3-O-rutinoside (K-3-R), such as DAHPS, DQD/SDH, PAL, 4CL, CHS, CHI, F3H, F3'H, FLS, and UGT78D2, and their corresponding metabolites were elaborately characterized. Our research reveals the molecular and chemical mechanisms of the fruit development of R. chingii. The results provide a solid foundation for the genetic analysis, functional genes isolation, fruit quality improvement and modifiable breeding of R. chingii.

The R2R3-MYB transcription factor MtMYB134 orchestrates flavonol biosynthesis in Medicago truncatula.

KEY MESSAGE: Our results provide insights into the flavonol biosynthesis regulation of M. truncatula. The R2R3-MYB transcription factor MtMYB134 emerged as tool to improve the flavonol biosynthesis. Flavonols are plant specialized metabolites with vital roles in plant development and defense and are known as diet compound beneficial to human health. In leguminous plants, the regulatory proteins involved in flavonol biosynthesis are not well characterized. Using a homology-based approach, three R2R3-MYB transcription factor encoding genes have been identified in the Medicago truncatula reference genome sequence. The gene encoding a protein with highest similarity to known flavonol regulators, MtMYB134, was chosen for further experiments and was characterized as a functional flavonol regulator from M. truncatula. MtMYB134 expression levels are correlated with the expression of MtFLS2, encoding a key enzyme of flavonol biosynthesis, and with flavonol metabolite content. MtMYB134 was shown to activate the promoters of the A. thaliana flavonol biosynthesis genes AtCHS and AtFLS1 in Arabidopsis protoplasts in a transactivation assay and to interact with the Medicago promoters of MtCHS2 and MtFLS2 in yeast 1-hybrid assays. To ascertain the functional aspect of the identified transcription factor, we developed a sextuple mutant, which is defective in anthocyanin and flavonol biosynthesis. Ectopic expression of MtMYB134 in a multiple myb A. thaliana mutant restored flavonol biosynthesis. Furthermore, overexpression of MtMYB134 in hairy roots of M. truncatula enhanced the biosynthesis of various flavonol derivatives. Taken together, our results provide insight into the understanding of flavonol biosynthesis regulation in M. truncatula and provides MtMYB134 as tool for genetic manipulation to improve flavonol synthesis.

Molecular cloning and functional characterization of NtWRKY11b in promoting the biosynthesis of flavonols in Nicotiana tabacum.

The biosynthesis of flavonols and anthocyanins is precisely regulated by different transcription factors in plants. WRKY11 promotes the biosynthesis of flavonoids in apple, but the molecular mechanism of WRKY11 regulating flavonols biosynthesis, and whether WRKY11 plays the same roles in other plants species remains to be further studied. Here, we cloned four NtWRKY11 genes from tobacco, which all contained the conserved WRKYGQK heptapeptide and a zinc-finger motif. The NtWRKY11b showed higher expression levels than the other NtWRKY11 genes in all the tobacco tissues examined, especially in tobacco leaves. Silencing of NtWRKY11b in tobacco leaves reduced the content of flavonols to 45.2 %-69.8 % of that in the WT plants, but overexpression of NtWRKY11b increased the flavonols content by 37.8 %-80.7 %. Transcriptome analysis revealed 8 flavonoids related differentially expressed genes (DEGs) between NtWRKY11b-OE and WT plants, among which the transcription of NtMYB12, NtFLS, NtGT5, and NtUFGT was significantly induced by posttranslational activation of NtWRKY11b with the presence of protein synthesis inhibitor, indicating a putative direct promotion of NtWRKY11b on the transcription of these flavonoids related genes. Chromatin immunoprecipitation assays further demonstrated that NtWRKY11b could bind to the promoter regions of NtMYB12, NtFLS, NtGT5, and NtUFGT to activate the transcription of these genes. Moreover, ectopic expression of NtWRKY11b also promoted the expression levels of NtCML38, NtCTL1, NtWRKY44, and NtCML37 genes, which have been shown to enhance plant resistance to various stresses. Our findings revealed the molecular mechanism of NtWRKY11b regulating flavonols biosynthesis, and provided a promising target for increasing flavonols content in tobacco.

COP1 mediates light-dependent regulation of flavonol biosynthesis through HY5 in Arabidopsis.

Flavonols, a class of flavonoids, accumulate as protective agents in response to various stresses. Among various environmental stimuli, light is one of the factors regulating flavonol production. MYB12/11/111, members of the R2R3 MYBs family, regulates spatio-temporal flavonol accumulation in Arabidopsis. Although various studies indicate at the involvement of an E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and ELONGATED HYPOCOTYL 5 (HY5) in flavonoid biosynthesis in response to UV-B, the regulatory roles of these components under visible light are yet to be investigated. Here, we demonstrate that flavonol accumulation in Arabidopsis is light-regulated. Furthermore, our analysis suggests that MYB12 is a HY5-dependent light-inducible gene and plays a key role in the activation of the flavonol biosynthesis in response to light. Our results indicate the involvement of COP1 in the dark-dependent repression of MYB12 expression and flavonol accumulation. In addition, results also suggest that the effect of COP1 on MYB12 is indirect and is mediated through HY5, a direct transcriptional activator of the MYB12. Together these findings indicate that COP1 acts as a master negative regulator of flavonol biosynthesis in the dark.

Nbnrp1 mediates Verticillium dahliae effector PevD1-triggered defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway in Nicotiana benthamiana.

PevD1, a fungal effector secreted by Verticillium dahliae, could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco plants. PevD1 could drastically induce the expression of Nbnrp1, which is an asparagine-rich protein (NRP) of Nicotiana benthamiana. Our previous research indicated that Nbnrp1 positively regulated PevD1-induced cell necrosis and disease resistance. In this study, we further investigated PevD1-induced immune responses in both wild-type (WT) and Nbnrp1-RNAi lines through RNA-seq, in order to reveal the underlying mechanism of Nbnrp1-modulated PevD1-induced disease resistance in N. benthamiana. Results showed that Nbnrp1-RNAi lines exhibited reduced PevD1-induced immune responses, like inhibiting H2O2 accumulation and MAPK phosphorylation. To silence Nbnrp1 inhibited the expression of PevD1-induced differential expression genes (DEGs) involved in pathways associated with sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction and phenylpropanoid biosynthesis, etc. It is worth noting that sesquiterpene phytoalexin capsidiol accumulation were obviously decreased in Nbnrp1-RNAi plants after PevD1 treatment, accompanied with the down-expression of EAS and EAH, which were two key genes related to capsidiol biosynthesis. These results suggested that Nbnrp1 mediates PevD1-induced defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway.

Functional characterization of three flavonol synthase genes from Camellia sinensis: Roles in flavonol accumulation.

Flavonol derivatives are a group of flavonoids benefiting human health. Their abundant presence in tea is associated with astringent taste. To date, mechanism pertaining to the biosynthesis of flavonols in tea plants remains unknown. In this study, we used bioinformatic analysis mining the tea genome and obtained three cDNAs that were annotated to encode flavonol synthases (FLS). Three cDNAs, namely CsFLSa, b, and c, were heterogenously expressed in E. coli to induce recombinant proteins, which were further used to incubate with three substrates, dihydrokampferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM). The resulting data showed that three rCsFLSs preferred to catalyze (DHK). Overexpression of each cDNA in tobacco led to the increase of kampferol and the reduction of anthocyanins in flowers. Further metabolic profiling of flavan-3-ols in young tea shoots characterized that kaempferol derivatives were the most abundant, followed by quercetin and then myricetin derivatives. Taken together, these data characterized the key step committed to the biosynthesis of flavonols in tea leaves. Moreover, these data enhance understanding the metabolic accumulation relevance between flavonols and other main flavonoids such as flavan-3-ols in tea leaves.

Molecular Basis for Chemical Evolution of Flavones to Flavonols and Anthocyanins in Land Plants.

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3ß-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of (2S)-flavanones to dihydroflavonols, but its origin and evolution remain elusive. Here, we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium (Physcomitrella) patens and Selaginella moellendorffii, suggesting that they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem.

Role of Flavonol Synthesized by Nucleus FLS1 in Arabidopsis Resistance to Pb Stress.

Lead (Pb) is an important pollutant of worldwide concern with respect to extensive pollution sources and highly toxic effect. Flavonol can improve plant resistance to abiotic stress and is also responsible for the alleviating effect under Pb stress. The relationship between Pb stress and flavonol and the knowledge about the mechanisms of flavonol function are very limited. Pb affected the energy metabolism process and, thus, inhibited plant growth and development. Flavonol accumulation controlled by FLS1 (flavonol synthase) could alleviate the toxic effect. Importantly, nes (mutant of NES that allows FLS1 to enter the nucleus expression) showed better growth status and lighter oxidative damage than NES (N-terminal nucleus exclusion signal peptide prevents FLS1 from entering the nucleus expression), which indicated that nucleus flavonol synthesized by nucleus FLS1 plays a key role in plant resistance to Pb stress. Although FLS1 signals were detected in the cell membrane, cytoplasm, and nucleus, membrane flavonol, cytoplasm flavonol, and nucleus flavonol were not exercising their function in the corresponding position. The expression of nucleus FLS1 intervened in the total content and composition of flavonol. The results also revealed that nucleus flavonol could regulate the ascorbate metabolism for alleviating the damage on the chloroplast, thus maintaining the photophosphorylation pathway. Our findings provided new insights for the molecular basis of Pb tolerance and response mechanism of the plant.

Annotation of genes involved in high level of dihydromyricetin production in vine tea (Ampelopsis grossedentata) by transcriptome analysis.

BACKGROUND: Leaves of the medicinal plant Ampelopsis grossedentata, which is commonly known as vine tea, are used widely in the traditional Chinese beverage in southwest China. The leaves contain a large amount of dihydromyricetin, a compound with various biological activities. However, the transcript profiles involved in its biosynthetic pathway in this plant are unknown. RESULTS: We conducted a transcriptome analysis of both young and old leaves of the vine tea plant using Illumina sequencing. Of the transcriptome datasets, a total of 52.47 million and 47.25 million clean reads were obtained from young and old leaves, respectively. Among 471,658 transcripts and 177,422 genes generated, 7768 differentially expressed genes were identified in leaves at these two stages of development. The phenylpropanoid biosynthetic pathway of vine tea was investigated according to the transcriptome profiling analysis. Most of the genes encoding phenylpropanoid biosynthesis enzymes were identified and found to be differentially expressed in different tissues and leaf stages of vine tea and also greatly contributed to the biosynthesis of dihydromyricetin in vine tea. CONCLUSIONS: To the best of our knowledge, this is the first formal study to explore the transcriptome of A. grossedentata. The study provides an insight into the expression patterns and differential distribution of genes related to dihydromyricetin biosynthesis in vine tea. The information may pave the way to metabolically engineering plants with higher flavonoid content.

Selective Hydrogen Atom Abstraction from Dihydroflavonol by a Nonheme Iron Center Is the Key Step in the Enzymatic Flavonol Synthesis and Avoids Byproducts.

The plant non-heme iron dioxygenase flavonol synthase performs a regioselective desaturation reaction as part of the biosynthesis of the signaling molecule flavonol that triggers the growing of leaves and flowers. These compounds also have health benefits for humans. Desaturation of aliphatic compounds generally proceeds through two consecutive hydrogen atom abstraction steps from two adjacent carbon atoms and in nature often is performed by a high-valent iron(IV)-oxo species. We show that the order of the hydrogen atom abstraction steps, however, is opposite of those expected from the C-H bond strengths in the substrate and determines the product distributions. As such, flavonol synthase follows a negative catalysis mechanism. Using density functional theory methods on large active-site model complexes, we investigated pathways for desaturation and hydroxylation by an iron(IV)-oxo active-site model. Contrary to thermochemical predictions, we find that the oxidant abstracts the hydrogen atom from the strong C2-H bond rather than the weaker C3-H bond of the substrate first. We analyze the origin of this unexpected selective hydrogen atom abstraction pathway and find that the alternative C3-H hydrogen atom abstraction would be followed by a low-energy and competitive substrate hydroxylation mechanism hence, should give considerable amount of byproducts. Our computational modeling studies show that substrate positioning in flavonol synthase is essential, as it guides the reactivity to a chemo- and regioselective substrate desaturation from the C2-H group, leading to desaturation products efficiently.

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

Genome-Wide Analysis of Artificial Mutations Induced by Ethyl Methanesulfonate in the Eggplant (Solanum melongena L.).

Whole-genome sequences of four EMS (ethyl methanesulfonate)-induced eggplant mutants were analyzed to identify genome-wide mutations. In total, 173.01 GB of paired-end reads were obtained for four EMS-induced mutants and (WT) wild type and 1,076,010 SNPs (single nucleotide polymorphisms) and 183,421 indels were identified. The most common mutation type was C/G to T/A transitions followed by A/T to G/C transitions. The mean densities were one SNP per 1.3 to 2.6 Mb. The effect of mutations on gene function was annotated and only 7.2% were determined to be deleterious. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed 10 and 11 genes, which were nonsynonymous mutation or frameshift deletion in 48-5 and L6-5 involved in the anthocyanin biosynthesis or flavone and flavonol biosynthesis. QRT-PCR results showed that only the Sme2.5_06210.1_g00004.1, which was annotated as UFGT (Flavonoid galactosidase transferase), expression significantly decreased in the L6-5 mutant compared with the WT. Also, the Sme2.5_06210.1_g00004.1 expression was lower in the colorless eggplant compared with colorful eggplant in the natural eggplant cultivar. These results suggest that Sme2.5_06210.1_g00004.1 may play a key role in eggplant anthocyanin synthesis.

Molecular and Functional Characterization of Oryza sativa Flavonol Synthase (OsFLS), a Bifunctional Dioxygenase.

Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. We isolated OsFLS from the rice ( Oryza sativa) cultivar "Ilmi" OsFLS includes highly conserved 2-ODD-specific motifs and FLS-specific regions. Recombinant OsFLS exhibited both FLS and flavanone 3ß-hydroxylase (F3H) activities, converting dihydroflavonols into flavonols and flavanones into dihydroflavonols, respectively, and more efficiently used dihydrokaempferol than dihydroquercetin as a substrate. OsFLS was expressed in both nonpigmented and pigmented rice seeds and was developmentally regulated during seed maturation. Transgenic tobacco ( Nicotiana tabacum) plants expressing OsFLS produced pale pink or white flowers with significantly increased levels of kaempferol-3- O-rutinoside and dramatically reduced levels of anthocyanin in their petals. Additionally, pod size and weight were reduced compared to the wild type. Several early and late biosynthetic genes of flavonoid were downregulated in the transgenic flowers. We demonstrated that OsFLS is a bifunctional 2-ODD enzyme and functions in flavonol production in planta.

Functional characterization of flavonoid 3'-hydroxylase, CsF3'H, from Crocus sativus L: Insights into substrate specificity and role in abiotic stress.

Stress-responsive dihydroxy flavonoids exhibit capability to inhibit the accretion of reactive oxygen species (ROS). The formation of these dihydroxy flavonols is catalyzed by flavonoid hydroxylases which are among the rate limiting enzymes of flavonoid biosynthesis pathway. Although flavonoid hydroxylases have been identified in several plant species but their role in abiotic stress is not explicitly documented. In the present study we report identification of all the flavonoid biosynthesis pathway genes of Crocus sativus and their expression profiling. We also report functional characterization of flavonoid 3' hydroxylase (CsF3'H) and attempt to explore its physiological role in vitro and in planta. The results indicated that CsF3'H is 1608 bp long encoding 535 amino acids. Docking and enzyme kinetic studies revealed that CsF3'H catalyzes hydroxylation of naringenin and dihydrokaempferol to eriodictoyl and dihydroquercetin respectively, but exhibits higher affinity for naringenin. Further, CsF3'H showed comparatively higher expression in floral tissues particularly stigma and its expression was significantly enhanced in response to UV-B, dehydration and salinity stress indicative of its role in stress. The expression of CsF3'H was associated with concomitant accumulation of eriodictoyl and dihydroquercetin. Transient overexpression of CsF3'H in Nicotiana benthamiana leads to the accumulation of substantial amounts of eriodictoyl and dihydroquercetin. Further, it was observed that transient expression of CsF3'H conferred tolerance to UV-B and dehydration stress as was evident from higher chlorophyll and soluble sugar and lower MDA contents. Taken together, these results suggest that CsF3'H confers tolerance to UV-B and dehydration in planta through synthesis of dihydroflavonols.

Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth.

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A.

The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia × crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3'-hydroxylase (F3'H), and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence relatedness with F3'Hs in the CYP75B subfamily of cytochromes P450 than with those with known F3'5'H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg g-1 fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.