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.

Discovery of a novel flavonol O-methyltransferase possessing sequential 4'- and 7-O-methyltransferase activity from Camptotheca acuminata Decne.

The biosynthetic route for flavonol in Camptotheca acuminata has been recently elucidated from a chemical point of view. However, the genes involved in flavonol methylation remain unclear. It is a critical step for fully uncovering the flavonol metabolism in this ancient plant. In this study, the multi-omics resource of this plant was utilized to perform flavonol O-methyltransferase-oriented mining and screening. Two genes, CaFOMT1 and CaFOMT2 are identified, and their recombinant CaFOMT proteins are purified to homogeneity. CaFOMT1 exhibits strict substrate and catalytic position specificity for quercetin, and selectively methylates only the 4'-OH group. CaFOMT2 possesses sequential O-methyltransferase activity for the 4'-OH and 7-OH of quercetin. These CaFOMT genes are enriched in the leaf and root tissues. The catalytic dyad and critical substrate-binding sites of the CaFOMTs are determined by molecular docking and further verified through site-mutation experiments. PHE181 and MET185 are designated as the critical sites for flavonol substrate selectivity. Genomic environment analysis indicates that CaFOMTs evolved independently and that their ancestral genes are different from that of the known Ca10OMT. This study provides molecular insights into the substrate-binding pockets of two new CaFOMTs responsible for flavonol metabolism in C. acuminata.

In Vitro Comparative Analysis of the Effect and Structure-Based Influencing Factors of Flavonols on Lipid Accumulation.

Flavonols represented by quercetin have been widely reported to have biological activities of regulating lipid metabolism. However, the differences in flavonols with different structures in lipid-lowering activity and the influencing factors remain unclear. In this study, the stability, transmembrane uptake ratio, and lipid metabolism regulation activities of 12 flavonol compounds in the 3T3-L1 cell model were systematically compared. The results showed that kaempferide had the highest cellular uptake ratio and the most potent inhibitory effect on adipogenesis at a dosing concentration of 20 µM, followed by isorhamnetin and kaempferol. They inhibited TG accumulation by more than 65% and downregulated the expression of PPARγ and SREBP1c by more than 60%. The other four aglycones, including quercetin, did not exhibit significant activity due to the structural instability in the cell culture medium. Meanwhile, five quercetin glucosides were quite stable but showed a low uptake ratio that no obvious activity was observed. Correlation analysis also showed that for 11 compounds except galangin, the activity was positively correlated with the cellular uptake ratio (p < 0.05, r = 0.6349). These findings may provide a valuable idea and insight for exploring the structure-based activity of flavonoids at the cellular level.

Integrated metabolomics and transcriptomics reveal molecular mechanisms of corolla coloration in Rhododendron dauricum L.

Rhododendron dauricum L. is a semi-evergreen shrub of high ornamental and medicinal values in Northeast China. To study the molecular mechanisms of corolla coloration in R. dauricum, integrated metabolomics and transcriptomics were performed in R. dauricum featuring purple flowers and R. dauricum var. album featuring white flowers. Comparative metabolomics revealed 25 differential metabolites in the corolla of the two distinct colors, enriched in flavonoids that are closely related to pigmentation in the flower. Differential analysis of the transcriptomics data revealed enrichment of structural genes for flavonoid biosynthesis (99 up- and 58 down-regulated, respectively, in purple corollas compared to white ones). Significantly, CHS and CHI, key genes in the early stage of anthocyanin synthesis, as well as F3H, F3'H, F3'5'H, DFR, ANS, and UFGT that promote the accumulation of pigments in the late stage of anthocyanin synthesis, were up-regulated in R. dauricum (purple color). In R. dauricum var. album, FLS were key genes determining the accumulation of flavonols. In addition, transcriptome-metabolome correlation analysis identified 16 R2R3 MYB transcription factors (out of 83 MYBs) that are important for corolla coloration. Five negative and four positive MYBs were further identified by integrated transcriptional and metabolic network analysis, revealing a key role of MYBA and MYB12 in regulating anthocyanins and flavonols, respectively. Moreover, we validated the function of RdMYBA by creating stable transgenic plants and found that RdMYBA promotes anthocyanin biosynthesis. In summary, we systematically characterized the transcriptome and metabolome of two R. dauricum cultivars with different flower colors and identified MYBs as key factors in modulating corolla coloration.

Analysis of flavonoid metabolism of compounds in succulent fruits and leaves of three different colors of Rosaceae.

Red flesh apple (Malus pumila var. medzwetzkyana Dieck), purple leaf plum (Prunus cerasifera Ehrhar f), and purple leaf peach (Prunus persica 'Atropurpurea') are significant ornamental plants within the Rosaceae family. The coloration of their fruits and leaves is crucial in their appearance and nutritional quality. However, qualitative and quantitative studies on flavonoids in the succulent fruits and leaves of multicolored Rosaceae plants are lacking. To unveil the diversity and variety-specificity of flavonoids in these three varieties, we conducted a comparative analysis of flavonoid metabolic components using ultra-high-performance liquid phase mass spectrometry (UPLC-MS/MS). The results revealed the detection of 311 metabolites, including 47 flavonoids, 105 flavonols, 16 chalcones, 37 dihydroflavonoids, 8 dihydroflavonols, 30 anthocyanins, 14 flavonoid carbon glycosides, 23 flavanols, 8 isoflavones, 11 tannins, and 12 proanthocyanidins. Notably, although the purple plum and peach leaves exhibited distinct anthocyanin compounds, paeoniflorin and corythrin glycosides were common but displayed varying glycosylation levels. While the green purple leaf peach fruit (PEF) and red flesh apple leaf (AL) possessed the lowest anthocyanin content, they exhibited the highest total flavonoid content. Conversely, the red flesh apple fruit (AF) displayed the highest anthocyanin content and a diverse range of anthocyanin glycosylation modifications, indicating that anthocyanins predominantly influenced the fruit's color. Purple PLF, PLL, and PEL showcased varying concentrations of anthocyanins, suggesting that their colors result from the co-color interaction between specific types of anthocyanins and secondary metabolites, such as flavonols, flavonoids, and dihydroflavonoids. This study provides novel insights into the variations in tissue metabolites among Rosaceae plants with distinct fruit and leaf colors.

Fisetin suppresses chondrocyte senescence and attenuates osteoarthritis progression by targeting sirtuin 6.

Osteoarthritis (OA) is the most common type of arthritis and is an age-related joint disease that is particularly prevalent in subjects over 65 years old. The chronic rise of senescent cells has a close correlation with age-related diseases such as OA, and the senescence-associated secretory phenotype (SASP) is implicated in OA cartilage degeneration pathogenesis. Sirtuin 6 (SIRT6) is likely to be a key senescence-related regulator. Fisetin (FST) is a natural flavonol of the flavonoid family that is recommended as a senolytic drug to extend health and lifespan. However, the potential chondroprotective effects of FST on OA rats are largely unclarified. The aim of this study is to investigate the ameliorative effects of FST on OA joint cartilage and the relationship with SIRT6 and the detailed mechanisms from anti-inflammatory and anti-senescent perspectives. Rats were subjected to destabilization of the medial meniscus (DMM) surgery as a means of inducing the experimental OA model in vivo. Chondrocytes treated with IL-1ß were utilized for mimicking the OA cell model in vitro. Intra-articular injection of FST, OSS_128,167 (OSS, SIRT6 inhibitor), and MDL800 (MDL, SIRT6 agonist) in vivo or administering them in IL-1ß-induced rat chondrocytes in vitro were performed in order to determine the effects FST has on OA and the link with SIRT6. This study found SIRT6 level to be negatively correlated with OA severity. SIRT6 downregulation was validated in the joint cartilages of DMM rats and IL-1ß-treated chondrocytes. It was also notably demonstrated that FST can activate SIRT6. Both the administration of FST and activation of SIRT6 using MDL were found to rescue cartilage erosion, decrease extracellular matrix (ECM) degradation, prevent cartilage from apoptosis, and improve detrimental senescence-related phenotype. The alleviative effects of FST against inflammation, ECM degradation, apoptosis, and senescence in IL-1ß-stimulated chondrocytes were also confirmed. SIRT6 loss occurs in articular cartilage in OA pathogenesis, which is linked to aging. FST attenuates injury-induced aging-related phenotype changes in chondrocytes through the targeting of SIRT6.

MicroRNAs constitute an additional layer in plant response to simultaneous bio- and abiotic stresses as exemplified by UV-B radiation and flg22-treatment on Arabidopsis thaliana.

Plants are confronted with various environmental stresses and develop sophisticated adaptive mechanisms. Our previous work demonstrated that the crosstalk of flg22 and ultraviolet (UV)-B-induced signalling cascades reprograms the expression of flavonol pathway genes (FPGs), benefiting plant defence responses. Although several transcription factors have been identified to be involved in this crosstalk, the underlying mechanism is largely unclear. Here, we analyzed microRNAs (miRNAs) and identified 126, 129 and 113 miRNAs with altered abundances compared to untreated control in flg22-, UV-B- and flg22/UV-B-treated seedlings, respectively. Two distinct modules were identified: The first consists of 10 miRNAs repressed by UV-B but up-regulated by flg22, and the second with five miRNAs repressed by flg22 but up-regulated by UV-B. In Arabidopsis, the knockdown of miR858a, a representative of module I, increased the abundance of CHS (a marker gene for FPGs), whereas its overexpression reduced CHS. Conversely, knockout of miR164b from module II decreased CHS and its overexpression increased CHS transcript levels. These data suggest a decisive role of miRNAs in the crosstalk. In the next, we described the interaction between miR858a and its target MYB111 (a positive regulator of FPGs) from module I in detail. We showed that MYB111 was profoundly post-transcriptionally regulated by miR858a during the crosstalk, whose expression was specifically but antagonistically controlled by UVR8- and FLS2-mediated signallings. Moreover, transcriptional monitoring using the GUS reporter gene demonstrates that miRNA-mediated posttranscriptional regulation is the main driving force in reprogramming the expression of FPGs and regulates plant adaptation to multiple concurrent environmental stresses.

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.

An LcMYB111-LcHY5 Module Differentially Activates an LcFLS Promoter in Different Litchi Cultivars.

Flavonol synthase (FLS) is the crucial enzyme of the flavonol biosynthetic pathways, and its expression is tightly regulated in plants. In our previous study, two alleles of LcFLS,LcFLS-A and LcFLS-B, have been identified in litchi, with extremely early-maturing (EEM) cultivars only harboring LcFLS-A, while middle-to-late-maturing (MLM) cultivars only harbor LcFLS-B. Here, we overexpressed both LcFLS alleles in tobacco, and transgenic tobacco produced lighter-pink flowers and showed increased flavonol levels while it decreased anthocyanin levels compared to WT. Two allelic promoters of LcFLS were identified, with EEM cultivars only harboring proLcFLS-A, while MLM cultivars only harbor proLcFLS-B. One positive and three negative R2R3-MYB transcription regulators of LcFLS expression were identified, among which only positive regulator LcMYB111 showed a consistent expression pattern with LcFLS, which both have higher expression in EEM than that of MLM cultivars. LcMYB111 were further confirmed to specifically activate proLcFLS-A with MYB-binding element (MBE) while being unable to activate proLcFLS-B with mutated MBE (MBEm). LcHY5 were also identified and can interact with LcMYB111 to promote LcFLS expression. Our study elucidates the function of LcFLS and its differential regulation in different litchi cultivars for the first time.

Effects of Zinc Oxide Nanoparticles on Growth, Development, and Flavonoid Synthesis in Ginkgo biloba.

Ginkgo biloba is a highly valuable medicinal plant known for its rich secondary metabolites, including flavonoids. Zinc oxide nanoparticles (ZnO-NPs) can be used as nanofertilizers and nano-growth regulators to promote plant growth and development. However, little is known about the effects of ZnO-NPs on flavonoids in G. biloba. In this study, G. biloba was treated with different concentrations of ZnO-NPs (25, 50, 100 mg/L), and it was found that 25 mg/L of ZnO-NPs enhanced G. biloba fresh weight, dry weight, zinc content, and flavonoids, while 50 and 100 mg/L had an inhibitory effect on plant growth. Furthermore, quantitative reverse transcription (qRT)-PCR revealed that the increased total flavonoids and flavonols were mainly due to the promotion of the expression of flavonol structural genes such as GbF3H, GbF3'H, and GbFLS. Additionally, when the GbF3H gene was overexpressed in tobacco and G. biloba calli, an increase in total flavonoid content was observed. These findings indicate that 25 mg/L of ZnO-NPs play a crucial role in G. biloba growth and the accumulation of flavonoids, which can potentially promote the yield and quality of G. biloba in production.

A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.

To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase CtUGT4, with flavonoid-O-glycosyltransferase activity, was identified in safflower. The fusion protein of CtUGT4 was heterologously expressed in Escherichia coli, and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7-O-glucoside, and kaempferol to form kaempferol-3-O in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. CtUGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3-O-glucoside, and quercetin to quercetin-3-O-glucoside. After overexpressing CtUGT4 in safflower, the content of quercetin-3-O-rutinoside in the safflower florets increased significantly, and the content of quercetin-3-O-glucoside also tended to increase, which preliminarily confirmed the function of CtUGT4 flavonoid-O-glycosyltransferase. This work demonstrated the flavonoid-O-glycosyltransferase function of safflower CtUGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of UGT genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.

Overexpression of JsFLS5 in calli improves salinity tolerance by maintaining active oxygen balance and reducing Na+ toxicity in Juglans sigillata.

The escalating global climate change significantly threatens plant growth, development, and production through salinity stress. Flavonoids, a crucial category of secondary metabolites, have been extensively studied for their role in modulating plant growth and development mechanisms in the face of biological and abiotic stress. The flavonol synthetase (FLS) gene plays a key role in the biosynthesis and accumulation of flavonoids. To investigate the correlation between salt tolerance and flavonol synthesis, JsFLS5 was overexpressed in the callus of Juglans sigillata cv. "Qianhe-7." This study shows that the upregulation of JsFLS5 significantly elevates the overall flavonoid content by modulating the expression of genes associated with flavonoid synthesis under salinity stress conditions. Additionally, the overexpressing callus exhibited enhanced resistance to salt stress compared to the wild-type callus, as evidenced by reduced levels of reactive oxygen species accumulation, electrolyte leakage, and malondialdehyde content in the overexpressing callus relative to the wild type (WT). Moreover, the overexpressing callus showed higher antioxidant enzyme activity and a more efficient ascorbic acid-glutathione cycle. Furthermore, the concentration of Na+ in the overexpressing callus was lower than WT, resulting in a decreased Na+ /K+ ratio. These findings suggest that JsFLS5 overexpression in calli effectively mitigates the oxidative damage induced by osmotic stress and reduces Na+ toxicity by enhancing flavonoid synthesis under salt stress conditions. Consequently, this study offers a novel perspective for comprehending the role of JsFLS5 in the response to abiotic stress in J. sigillata.

Metabolomic analysis and bioactivities of Arbutus unedo leaves harvested across the seasons in different natural habitats of Sardinia (Italy).

BACKGROUND: Arbutus unedo L. is a wild tree of Mediterranean regions used as food and in traditional medicine and important for afforestation programs. There is no detailed information available on the variation of A. unedo leaves metabolome across the seasons. The leaves were analyzed by Proton nuclear magnetic resonance (1 H NMR)-based metabolomics, comparing samples harvested across the seasons and in ten different natural habitats of Sardinia (Italy). RESULTS: Multivariate analysis showed the impact of seasonal variation on the metabolome: glucose and quinic acid increased in summer, while in spring sucrose was accumulated. ß-Arbutin, the main known active principle of A. unedo, generally reached the highest concentration in autumn. In winter, O-ß-methylglucose, γ-aminobutyric acid (GABA), flavonols (quercetin-3-O-α-rhamnoside, myricetin-3-O-α-rhamnoside, kaempferol-3-O-α-rhamnoside), catechin, and gallocatechin increased. Characteristic metabolomic features were found also for samples collected in different locations. For instance, trees growing at the highest altitude and exposed to lower temperatures produced less flavonols and catechins. The only sample collected on trees growing on limestones, dolomites, and dolomitic limestones type of soil showed generally the highest content of arbutin. The highest phenolics content was found during spring, while samples collected on flowering branches in winter were the ones with the highest flavonoid content. The antioxidant activity was also variated, ranging from 1.3 to 10.1 mg of Trolox equivalents (TE)/mL of extract, and it was positively correlated to both total phenolics and flavonoid content. Winter samples showed the lowest antibacterial activity, while summer and autumn ones exhibited the highest activity (IC50 values ranging from 17.3 to 42.3 µg/mL against Staphylococcal species). CONCLUSION: This work provides 1 H-NMR fingerprinting of A. unedo leaves, elucidating the main metabolites and their variations during seasons. On the basis of arbutin content, autumn could be considered the balsamic period of this taxon. Samples collected in this season were also the most active ones as antibacterial. Moreover, an interesting metabolomic profile enriched in catechins and flavonols was observed in leaves collected in winter on flowering branches which were endowed with high antioxidant potential.

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.

Ethylene inhibits ABA-induced stomatal closure via regulating NtMYB184-mediated flavonol biosynthesis in tobacco.

Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184, however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells.

MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Transcription factor VvWRKY70 inhibits both norisoprenoid and flavonol biosynthesis in grape.

Norisoprenoids and flavonols are important secondary metabolites in grape berries (Vitis vinifera L.). The former is a class of ubiquitous flavor and fragrance compounds produced by the cleavage of carotenoids, and the latter, which is derived from the flavonoid metabolic pathway, has been proposed as a general quality marker for red grapes. However, the transcriptional regulatory mechanisms underlying norisoprenoid and flavonol production are still not fully understood. In this study, we characterized a transcription factor, VvWRKY70, as a repressor of both norisoprenoid and flavonol biosynthesis in grape berries, and its expression was downregulated by light and high-temperature treatment. Overexpressing VvWRKY70 in grape calli reduced norisoprenoid and flavonol production, particularly under light exposure or at high temperature, by repressing the expression of several related genes in the isoprenoid and flavonoid metabolic pathways. VvWRKY70 downregulated ß-CAROTENE HYDROXYLASE 2 (VvBCH2) and CHALCONE SYNTHASE 3 (VvCHS3) expression based on yeast 1-hybrid analysis combined with electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR. We discuss the role of VvWRKY70 in the coordinated regulatory network of isoprenoid and flavonoid metabolism. These findings provide a theoretical basis to improve flavor, color, and other comprehensive qualities of fruit crops and their processing products.

Synthesis of Flavonol-Bearing Probes for Chemoproteomic and Bioinformatic Analyses of Asteraceae Petals in Search of Novel Flavonoid Enzymes.

This study aimed at searching for the enzymes that are responsible for the higher hydroxylation of flavonols serving as UV-honey guides for pollinating insects on the petals of Asteraceae flowers. To achieve this aim, an affinity-based chemical proteomic approach was developed by relying on the use of quercetin-bearing biotinylated probes, which were thus designed and synthesized to selectively and covalently capture relevant flavonoid enzymes. Proteomic and bioinformatic analyses of proteins captured from petal microsomes of two Asteraceae species (Rudbeckia hirta and Tagetes erecta) revealed the presence of two flavonol 6-hydroxylases and several additional not fully characterized proteins as candidates for the identification of novel flavonol 8-hydroxylases, as well as relevant flavonol methyl- and glycosyltransferases. Generally speaking, this substrate-based proteome profiling methodology constitutes a powerful tool for the search for unknown (flavonoid) enzymes in plant protein extracts.

BcMYB111 Responds to BcCBF2 and Induces Flavonol Biosynthesis to Enhance Tolerance under Cold Stress in Non-Heading Chinese Cabbage.

Flavonols have been shown to respond to a variety of abiotic stresses in plants, including cold stress. Higher total flavonoid content was found in non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. chinensis) after cold stress. A non-targeted metabolome analysis showed a significant increase in flavonol content, including that of quercetin and kaempferol. Here, we found that an R2R3-MYB transcription factor, BcMYB111, may play a role in this process. BcMYB111 was up-regulated in response to cold treatment, with an accompanying accumulation of flavonols. Then, it was found that BcMYB111 could regulate the synthesis of flavonols by directly binding to the promoters of BcF3H and BcFLS1. In the transgenic hairy roots of NHCC or stable transgenic Arabidopsis, overexpression of BcMYB111 increased flavonol synthesis and accumulation, while these were reduced in virus-induced gene silencing lines in NHCC. After cold stress, the higher proline content and lower malondialdehyde (MDA) content showed that there was less damage in transgenic Arabidopsis than in the wild-type (WT). The BcMYB111 transgenic lines performed better in terms of antioxidant capacity because of their lower H2O2 content and higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities. In addition, a key cold signaling gene, BcCBF2, could specifically bind to the DRE element and activate the expression of BcMYB111 in vitro and in vivo. The results suggested that BcMYB111 played a positive role in enhancing the flavonol synthesis and cold tolerance of NHCC. Taken together, these findings reveal that cold stress induces the accumulation of flavonols to increase tolerance via the pathway of BcCBF2-BcMYB111-BcF3H/BcFLS1 in NHCC.

Impact of cooking methods of red-skinned onion on metabolic transformation of phenolic compounds and gut microbiota changes.

Herein, we investigated the stability and bioaccessibility of phenolics in differently cooked red-skinned onion (RSO) and consequently their impact on the gut microbiota and metabolism of phenolics. In fact, the different processes used to cook vegetables can modify and re-arrange the molecular profiles of bioactive compounds, such as phenolics in phenolic-rich vegetables, such as RSO. Fried and grilled RSO were compared to raw RSO and a blank control and subjected to oro-gastro-intestinal digestion and subsequent colonic fermentation. For upper gut digestion, the INFOGEST protocol was used, and for lower gut fermentation, a short-term batch model, namely, MICODE (multi-unit in vitro colon gut model), was employed. During the process, phenolic compound profile (through high-resolution mass spectrometry) and colon microbiomics (qPCR of 14 core taxa) analyses were performed. According to the results, the degradation driven by the colon microbiota of RSO flavonols resulted in the accumulation of three main metabolites, i.e., 3-(3'-hydroxyphenyl)propanoic acid, 3-(3'-hydroxyphenyl)acetic acid and 3-(3',4'-dihydroxyphenyl)acetic acid. Also, colonic fermentation of raw onions resulted in a substantial increase in beneficial taxa, which was larger compared to the heat-treated onions, particularly Lactobacillales and beneficial clostridia. Also, a higher level of inhibition of opportunistic bacteria was seen for the raw onion samples, namely, Clostridium perfringens group and Escherichia coli. Thus, our results showed that RSO, and especially the raw one, is an excellent dietary source of flavonols that are strongly metabolized by gut bacteria and can positively modulate the gut microbiota. Although additional in vivo studies are necessary, this work is one of the first to explore how RSO processed with different cooking methods can differently impact the phenolic metabolism and microbiota composition in the large intestine of humans, fine-tuning the antioxidant nature of foods.