Four glycosyltransferase genes are responsible for synthesis and accumulation of different flavonol glycosides in apple tissues.

Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.

Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

Integrated metabolome, full-length sequencing, and transcriptome analyses unveil the molecular mechanisms of color formation of the canary yellow and red bracts of Bougainvillea × buttiana 'Chitra'.

Bougainvillea is a typical tropical flower of great ornamental value due to its colorful bracts. The molecular mechanism behind color formation is not well-understood. Therefore, this research conducted metabolome analysis, transcriptome analysis, and multi-flux full-length sequencing in two color bracts of Bougainvillea × buttiana 'Chitra' to investigate the significantly different metabolites (SDMs) and differentially expressed genes (DEGs). Overall, 261 SDMs, including 62 flavonoids and 26 alkaloids, were detected, and flavonols and betalains were significantly differentially accumulated among the two bracts. Furthermore, the complete-length transcriptome of Bougainvillea × buttiana was also developed, which contained 512 493 non-redundant isoforms. Among them, 341 210 (66.58%) displayed multiple annotations in the KOG, GO, NR, KEGG, Pfam, Swissprot, and NT databases. RNA-seq findings revealed that 3610 DEGs were identified between two bracts. Co-expression analysis demonstrated that the DEGs and SDMs involved in flavonol metabolism (such as CHS, CHI, F3H, FLS, CYP75B1, kaempferol, and quercetin) and betacyanin metabolism (DODA, betanidin, and betacyanins) were the main contributors for the canary yellow and red bract formation, respectively. Further investigation revealed that several putative transcription factors (TFs) might interact with the promoters of the genes mentioned above. The expression profiles of the putative TFs displayed that they may positively and negatively regulate the structural genes' expression profiles. The data revealed a potential regulatory network between important genes, putative TFs, and metabolites in the flavonol and betacyanin biosynthesis of Bougainvillea × buttiana 'Chitra' bracts. These findings will serve as a rich genetic resource for future studies that could create new color bracts.

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 and targeted metabolomic analyses provide insights into the flavonoids biosynthesis in the flowers of Lonicera macranthoides.

BACKGROUND: Flavonoids are one of the bioactive ingredients of Lonicera macranthoides (L. macranthoides), however, their biosynthesis in the flower is still unclear. In this study, combined transcriptomic and targeted metabolomic analyses were performed to clarify the flavonoids biosynthesis during flowering of L. macranthoides. RESULTS: In the three sample groups, GB_vs_WB, GB_vs_WF and GB_vs_GF, there were 25, 22 and 18 differentially expressed genes (DEGs) in flavonoids biosynthetic pathway respectively. A total of 339 flavonoids were detected and quantified at four developmental stages of flower in L. macranthoides. In the three sample groups, 113, 155 and 163 differentially accumulated flavonoids (DAFs) were detected respectively. Among the DAFs, most apigenin derivatives in flavones and most kaempferol derivatives in flavonols were up-regulated. Correlation analysis between DEGs and DAFs showed that the down-regulated expressions of the CHS, DFR, C4H, F3'H, CCoAOMT_32 and the up-regulated expressions of the two HCTs resulted in down-regulated levels of dihydroquercetin, epigallocatechin and up-regulated level of kaempferol-3-O-(6''-O-acetyl)-glucoside, cosmosiin and apigenin-4'-O-glucoside. The down-regulated expressions of F3H and FLS decreased the contents of 7 metabolites, including naringenin chalcone, proanthocyanidin B2, B3, B4, C1, limocitrin-3,7-di-O-glucoside and limocitrin-3-O-sophoroside. CONCLUSION: The findings are helpful for genetic improvement of varieties in L.macranthoides.

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.

The IAA17.1/HSFA5a module enhances salt tolerance in Populus tomentosa by regulating flavonol biosynthesis and ROS levels in lateral roots.

Auxin signaling provides a promising approach to controlling root system architecture and improving stress tolerance in plants. However, how the auxin signaling is transducted in this process remains unclear. The Aux indole-3-acetic acid (IAA) repressor IAA17.1 is stabilized by salinity, and primarily expressed in the lateral root (LR) primordia and tips in poplar. Overexpression of the auxin-resistant form of IAA17.1 (IAA17.1m) led to growth inhibition of LRs, markedly reduced salt tolerance, increased reactive oxygen species (ROS) levels, and decreased flavonol content. We further identified that IAA17.1 can interact with the heat shock protein HSFA5a, which was highly expressed in roots and induced by salt stress. Overexpression of HSFA5a significantly increased flavonol content, reduced ROS accumulation, enhanced LR growth and salt tolerance in transgenic poplar. Moreover, HSFA5a could rescue the defective phenotypes caused by IAA17.1m. Expression analysis showed that genes associated with flavonol biosynthesis were altered in IAA17.1m- and HAFA5a-overexpressing plants. Furthermore, we identified that HSFA5a directly activated the expression of key enzyme genes in the flavonol biosynthesis pathway, while IAA17.1 suppressed HSFA5a-mediated activation of these genes. Collectively, the IAA17.1/HSFA5a module regulates flavonol biosynthesis, controls ROS accumulation, thereby modulating the root system of poplar to adapt to salt stress.

Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana.

Flavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. However, their effects on global gene expression and signaling pathways are unclear. To explore the roles of flavonol metabolites in signaling, we performed comparative transcriptome and targeted metabolite profiling of seedlings from the flavonol-deficient Arabidopsis loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). RNA-seq results indicated that flavonols modulate various biological and metabolic pathways, with significant alterations in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor-mediated up-regulation of biosynthesis genes. Interestingly, upstream amino acid biosynthesis genes involved in methionine and tryptophan synthesis were altered under flavonol deficiency and exogenous supplementation. Quercetin treatment significantly up-regulated aliphatic glucosinolate biosynthesis genes compared with kaempferol and rutin. In addition, expression and metabolite analysis of the transparent testa7 mutant, which lacks hydroxylated flavonol derivatives, clarified the role of quercetin in the glucosinolate biosynthesis pathway. This study elucidates the molecular mechanisms by which flavonols interfere with signaling pathways, their molecular targets, and the multiple biological activities of flavonols in plants.

Quercetin for the treatment of COVID-19 patients: A systematic review and meta-analysis.

Currently approved therapies for COVID-19 are mostly limited by their low availability, high costs or the requirement of parenteral administration by trained medical personnel in an in-hospital setting. Quercetin is a cheap and easily accessible therapeutic option for COVID-19 patients. However, it has not been evaluated in a systematic review until now. We aimed to conduct a meta-analysis to assess the effect of quercetin on clinical outcomes in COVID-19 patients. Various databases including PubMed, the Cochrane Library and Embase were searched from inception until 5 October 2022 and results from six randomized controlled trials (RCTs) were pooled using a random-effects model. All analyses were conducted using RevMan 5.4 with odds ratio (OR) as the effect measure. Quercetin decreased the risk of intensive care unit admission (OR = 0.31; 95% confidence interval (CI) 0.10-0.99) and the incidence of hospitalisation (OR = 0.25; 95% CI 0.10-0.62) but did not decrease the risk of all-cause mortality and the rate of no recovery. Quercetin may be of benefit in COVID-19 patients, especially if administered in its phytosome formulation which greatly enhances its bioavailability but large-scale RCTs are needed to confirm these findings.

Novel flavonol derivatives containing benzoxazole as potential antiviral agents: design, synthesis, and biological evaluation.

A series of flavonol derivatives containing benzoxazole were designed and synthesized, and the structures of all the target compounds were determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The structure of X2 was further confirmed by single crystal X-ray diffraction analysis. The results of the bioactivity tests showed that some of the target compounds possessed excellent antiviral activity against tobacco mosaic virus (TMV) in vivo. In particular, the median effective concentration (EC50) values for the curative and protective activities of X17 against TMV were 127.6 and 101.2 µg/mL, respectively, which were superior to those of ningnanmycin (320.0 and 234.6 µg/mL). The results of preliminary mechanism study indicated that X17 had a strong binding affinity for TMV coat protein (TMV-CP), which might hinder the self-assembly and replication of TMV particles. In addition, X17 was able to effectively inhibit tobacco leaf membrane lipid peroxidation and facilitate the removal of O2- from the body, thereby improving the disease resistance of tobacco plants. Therefore, the design and synthesis of flavonol derivatives containing benzoxazole provides value for the development of new antiviral drugs.

Flavonol Derivatives Containing a Quinazolinone Moiety: Design, Synthesis, and Antiviral Activity.

A series of flavonol derivatives containing quinazolinone were designed and synthesized, and their antiviral activities against tobacco mosaic virus (TMV) were evaluated. The results of the half maximal effective concentration (EC50 ) test against TMV showed that the EC50 value of curative activity of K5 was 139.6 µg/mL, which was better than that of the commercial drug ningnanmycin (NNM) 296.0 µg/mL, and the EC50 value of protective activity of K5 was 120.6 µg/mL, which was superior to that of NNM 207.0 µg/mL. The interaction of K5 with TMV coat protein (TMV-CP) was investigated using microscale thermophoresis (MST) and molecular docking and the results showed that K5 can combine with TMV-CP more strongly to TMV-CP than that NNM can. Furthermore, the assay measuring malondialdehyde (MDA) content indicated that K5 had the ability to improve the disease resistance of tobacco. Hence, this study offers strong evidence that flavonol derivatives have potential as novel antiviral agents.

Portable paper-based probe for on-site ratiometric fluorescence determination of total flavonol glycosides in plant extract using smartphone imaging.

A smartphone-assisted, paper-based ratio fluorescence probe is presented for the rapid, low-cost and on-site quantification of total flavonol glycosides in Ginkgo biloba extracts (GBE). The Al3+/Eu-MOF/paper-based probe utilizes lanthanide metal-organic framework (Ln-MOF) nanoparticles immobilized on Whatman filter paper along with Al3+ for detecting flavonols, which are the hydrolyzed products of flavonol glycosides. The color change of the paper-based fluorescence image from red to orange depends on the concentration of the target analyte in the sample solution. The smartphone equipped with a red, green, blue (RGB) color detector measured the fluorescence signal intensity on the paper substrate after adding flavonol. The analytical variables affecting the performance of the probe, including the addition sequence of the aluminum nitrate solution, its concentration, that of the Ln-MOF solution, the drying time of the paper probe, the reaction time and the sensitivity parameters of the mobile phone camera (ISO), were optimized. Under optimal conditions, the Al3+/Eu-MOF/paper-based probe has good linear response in the concentration range 7 ~ 80 µg mL- 1 and a lower detection limit of 2.07 µg mL- 1. The results obtained with the paper-based ratio fluorescence probe and smartphone combination were validated by comparing them with high-performance liquid chromatography (HPLC) measurements. This study provides a potential strategy for fabricating Al3+/Eu-MOF/paper-based probe used for total flavonol glycosides determination.

Health-Promoting Properties of Natural Flavonol Glycosides Isolated from Staphylea pinnata L.

Staphylea, also called bladdernuts, is a genus of plants belonging to the family Staphyleaceae, widespread in tropical or temperate climates of America, Europe, and the Far East. Staphylea spp. produce bioactive metabolites with antioxidant properties, including polyphenols which have not been completely investigated for their phytotherapeutic potential, even though they have a long history of use for food. Here, we report the isolation of six flavonol glycosides from the hydroalcoholic extract of aerial parts of Staphylea pinnata L., collected in Italy, using a solid-phase extraction technique. They were identified using spectroscopic, spectrometric, and optical methods as three quercetin and three isorhamnetin glycosides. Among the flavonol glycosides isolated, isoquercetin and quercetin malonyl glucoside showed powerful antioxidant, antimicrobial, and wound healing promoting activity and thus are valuable as antiaging ingredients for cosmeceutical applications and for therapeutic applications in skin wound repair.

Safflower CtFLS1-Induced Drought Tolerance by Stimulating the Accumulation of Flavonols and Anthocyanins in Arabidopsis thaliana.

Flavonol synthase gene (FLS) is a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily and plays an important role in plant flavonoids biosynthetic pathways. Safflower (Carthamus tinctorius L.), a key source of traditional Chinese medicine, is widely cultivated in China. Although the flavonoid biosynthetic pathway has been studied in several model species, it still remains to be explored in safflower. In this study, we aimed to elucidate the role of CtFLS1 gene in flavonoid biosynthesis and drought stress responses. The bioinformatics analysis on the CtFLS1 gene showed that it contains two FLS-specific motifs (PxxxIRxxxEQP and SxxTxLVP), suggesting its independent evolution. Further, the expression level of CtFLS1 in safflower showed a positive correlation with the accumulation level of total flavonoid content in four different flowering stages. In addition, CtFLS1-overexpression (OE) Arabidopsis plants significantly induced the expression levels of key genes involved in flavonol pathway. On the contrary, the expression of anthocyanin pathway-related genes and MYB transcription factors showed down-regulation. Furthermore, CtFLS1-OE plants promoted seed germination, as well as resistance to osmotic pressure and drought, and reduced sensitivity to ABA compared to mutant and wild-type plants. Moreover, CtFLS1 and CtANS1 were both subcellularly located at the cell membrane and nucleus; the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assay showed that they interacted with each other at the cell membrane. Altogether, these findings suggest the positive role of CtFLS1 in alleviating drought stress by stimulating flavonols and anthocyanin accumulation in safflower.

Synthesis of Flavonols and Assessment of Their Biological Activity as Anticancer Agents.

A series of flavanols were synthesized to assess their biological activity against human non-small cell lung cancer cells (A549). Among the sixteen synthesized compounds, it was observed that compounds 6k (3.14 ± 0.29 µM) and 6l (0.46 ± 0.02 µM) exhibited higher potency compared to 5-fluorouracil (5-Fu, 4.98 ± 0.41 µM), a clinical anticancer drug which was used as a positive control. Moreover, compound 6l (4'-bromoflavonol) markedly induced apoptosis of A549 cells through the mitochondrial- and caspase-3-dependent pathways. Consequently, compound 6l might be developed as a candidate for treating or preventing lung cancer.

Manganese sulfate application promotes berry flavonoid accumulation in Vitis vinifera cv. 'Cabernet Sauvignon' by regulating flavonoid metabolome and transcriptome profiles.

BACKGROUND: Flavonoids are vital for the development of high-quality grapes and wine, and manganese deficiency decreases grape berry coloration. However, the effects and underlying mechanisms of action of manganese sulfate on grape metabolic profiles have not been adequately researched. In this study, three concentrations of manganese sulfate solutions, 0.5 µmol·L-1 (low, L), 5 µmol·L-1 (middle, M - the standard manganese concentration of Hoagland nutrient solution, control), and 1000 µmol·L-1 (high, H), were applied to the 'Cabernet Sauvignon' grapevine (Vitis vinifera L.) to explore the effect on berry composition. RESULTS: Manganese application improved manganese concentration effectively in grape organs. Furthermore, the concentrations of malvidin 3-O-(6-O-acetyl)-glucoside, malvidin 3-O-glucoside, malvidin-trans-3-O-(6-O-p-coumaryl)-glucoside, and peonidin 3-O-(6-O-acetyl)-glucoside increased significantly under H treatment. Weighted gene co-expression network analysis (WGCNA) revealed that the structural genes (VvDFR, VvUFGT, and VvOMT) of flavonoid biosynthesis were upregulated under H treatment, and their transcription levels correlated positively with malvidin- and peonidin-derived anthocyanin concentrations. CONCLUSIONS: This study suggested that manganese application regulates berry transcriptional and flavonoid metabolic profiles, providing a theoretical basis for improving the color of red grapes and wines. © 2023 Society of Chemical Industry.

Puncturing lipid membranes: onset of pore formation and the role of hydrogen bonding in the presence of flavonoids.

Products of lipid peroxidation induce detrimental structural changes in cell membranes, such as the formation of water pores, which occur in the presence of lipids with partially oxidized chains. However, the influence of another class of products, dicarboxylic acids, is still unclear. These products have greater mobility in the lipid bilayer, which enables their aggregation and the formation of favorable sites for the appearance of pores. Therefore, dodecanedioic acid (DDA) was selected as a model product. Additionally, the influence of several structurally different flavonoids on DDA aggregation via formation of hydrogen bonds with carboxyl groups was investigated. The molecular dynamics of DDA in DOPC lipid bilayer revealed the formation of aggregates extending over the hydrophobic region of the bilayer and increasing its polarity. Consequently, water penetration and the appearance of water wires was observed, representing a new step in the mechanism of pore formation. Furthermore, DDA molecules were found to interact with lipid polar groups, causing them to be buried in the bilayer. The addition of flavonoids to the system disrupted aggregate formation, resulting in the displacement of DDA molecules from the center of the bilayer. The placement of DDA and flavonoids in the lipid bilayer was confirmed by small-angle X-ray scattering. Atomic force microscopy and electron paramagnetic resonance were used to characterize the structural properties. The presence of DDA increased bilayer roughness and decreased the ordering of lipid chains, confirming its detrimental effects on the membrane surface, while flavonoids were found to reduce or reverse these changes.

Metabolic profile and transcriptome reveal the mystery of petal blotch formation in rose.

BACKGROUND: Petal blotch is a unique ornamental trait in angiosperm families, and blotch in rose petal is rare and has great esthetic value. However, the cause of the formation of petal blotch in rose is still unclear. The influence of key enzyme genes and regulatory genes in the pigment synthesis pathways needs to be explored and clarified. RESULTS: In this study, the rose cultivar 'Sunset Babylon Eyes' with rose-red to dark red blotch at the base of petal was selected as the experimental material. The HPLC-DAD and UPLC-TQ-MS analyses indicated that only cyanidin 3,5-O-diglucoside (Cy3G5G) contributed to the blotch pigmentation of 'Sunset Babylon Eyes', and the amounts of Cy3G5G varied at different developmental stages. Only flavonols but no flavone were found in blotch and non-blotch parts. As a consequence, kaempferol and its derivatives as well as quercetin and its derivatives may act as background colors during flower developmental stages. Despite of the differences in composition, the total content of carotenoids in blotch and non-blotch parts were similar, and carotenoids may just make the petals show a brighter color. Transcriptomic data, quantitative real-time PCR and promoter sequence analyses indicated that RC7G0058400 (F3'H), RC6G0470600 (DFR) and RC7G0212200 (ANS) may be the key enzyme genes for the early formation and color deepening of blotch at later stages. As for two transcription factor, RC7G0019000 (MYB) and RC1G0363600 (WRKY) may bind to the promoters of critical enzyme genes, or RC1G0363600 (WRKY) may bind to the promoter of RC7G0019000 (MYB) to activate the anthocyanin accumulation in blotch parts of 'Sunset Babylon Eyes'. CONCLUSIONS: Our findings provide a theoretical basis for the understanding of the chemical and molecular mechanism for the formation of petal blotch in rose.

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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: This 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.

Genome-wide analysis of UDP-glycosyltransferases family and identification of UGT genes involved in abiotic stress and flavonol biosynthesis in Nicotiana tabacum.

BACKGROUND: Uridine disphosphate (UDP) glycosyltransferases (UGTs) act upon a huge variety of highly diverse and complex substrates, such as phytohormones and specialized metabolites, to regulate plant growth, development, disease resistance, and environmental interactions. However, a comprehensive investigation of UGT genes in tobacco has not been conducted. RESULTS: In this study, we carried out a genome-wide analysis of family-1 UDP glycosyltransferases in Nicotiana tabacum. We predicted 276 NtUGT genes, which were classified into 18 major phylogenetic subgroups. The NtUGT genes were invariably distributed among all the 24 chromosomes with structural diversity in exon/intron structure, conserved motifs, and cis-acting elements of promoters. Three groups of proteins which involved in flavonoid biosynthesis, plant growth and development, transportation and modification were identified that interact with NtUGT proteins using the PPI analysis. Expression analysis of NtUGT genes in cold stress, drought stress and different flower color using both online RNA-Seq data and the realtime PCR analysis, suggested the distinct role of NtUGT genes in resistance of cold, drought and in flavonoid biosynthesis. The enzymatic activities of seven NtUGT proteins that potentially involved in flavonoid glycosylation were analyzed, and found that all seven exhibited activity on myricetin; six (NtUGT108, NtUGT123, NtUGT141, NtUGT155, NtUGT179, and NtUGT195) showed activity on cyanidin; and three (NtUGT108, NtUGT195, and NtUGT217) were active on the flavonol aglycones kaempferol and quercetin, which catalyzing the substrates (myricetin, cyanidin or flavonol) to form new products. We further investigated the enzymatic products and enzymatic properties of NtUGT108, NtUGT195, and NtUGT217, suggested their diverse enzymatic activity toward flavonol, and NtUGT217 showed the highest catalyzed efficient toward quercetin. Overexpression of NtUGT217 significantly increase the content levels of the quercetin-3-O-glucoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside in transgenic tobacco leaves. CONCLUSION: We identified 276 UGT genes in Nicotiana tabacum. Our study uncovered valuable information about the phylogenetic structure, distribution, genomic characters, expression patterns and enzymatic activity of NtUGT genes in tobacco. We further identified three NtUGT genes involved in flavonoid biosynthesis, and overexpressed NtUGT217 to validate its function in catalyze quercetin. The results provide key candidate NtUGT genes for future breeding of cold and drought resistance and for potential metabolic engineering of flavonoid compounds.