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.

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.

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.

FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

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.

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.

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.

Weaker photosynthetic acclimation to fluctuating than to corresponding steady UVB radiation treatments in grapevines.

The effects of transient increases in UVB radiation on plants are not well known; whether cumulative damage dominates or, alternately, an increase in photoprotection and recovery periods ameliorates any negative effects. We investigated photosynthetic capacity and metabolite accumulation of grapevines (Vitis vinifera Cabernet Sauvignon) in response to UVB fluctuations under four treatments: fluctuating UVB (FUV) and steady UVB radiation (SUV) at similar total biologically effective UVB dose (2.12 and 2.23 kJ m-2 day-1), and their two respective no UVB controls. We found a greater decrease in stomatal conductance under SUV than FUV. There was no decrease in maximum yield of photosystem II (Fv/Fm) or its operational efficiency (ɸPSII) under the two UVB treatments, and Fv/Fm was higher under SUV than FUV. Photosynthetic capacity was enhanced under FUV in the light-limited region of rapid light-response curves but enhanced by SUV in the light-saturated region. Flavonol content was similarly increased by both UVB treatments. We conclude that, while both FUV and SUV effectively stimulate acclimation to UVB radiation at realistic doses, FUV confers weaker acclimation than SUV. This implies that recovery periods between transient increases in UVB radiation reduce UVB acclimation, compared to an equivalent dose of UVB provided continuously. Thus, caution is needed in interpreting the findings of experiments using steady UVB radiation treatments to infer effects in natural environments, as the stimulatory effect of steady UVB is greater than that of the equivalent fluctuating UVB.

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.

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.

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.

Kaempferol-3-rhamnoside overaccumulation in flavonoid 3'-hydroxylase tt7 mutants compromises seed coat outer integument differentiation and seed longevity.

Seeds slowly accumulate damage during storage, which ultimately results in germination failure. The seed coat protects the embryo from the external environment, and its composition is critical for seed longevity. Flavonols accumulate in the outer integument. The link between flavonol composition and outer integument development has not been explored. Genetic, molecular and ultrastructural assays on loss-of-function mutants of the flavonoid biosynthesis pathway were used to study the effect of altered flavonoid composition on seed coat development and seed longevity. Controlled deterioration assays indicate that loss of function of the flavonoid 3' hydroxylase gene TT7 dramatically affects seed longevity and seed coat development. Outer integument differentiation is compromised from 9 d after pollination in tt7 developing seeds, resulting in a defective suberin layer and incomplete degradation of seed coat starch. These distinctive phenotypes are not shared by other mutants showing abnormal flavonoid composition. Genetic analysis indicates that overaccumulation of kaempferol-3-rhamnoside is mainly responsible for the observed phenotypes. Expression profiling suggests that multiple cellular processes are altered in the tt7 mutant. Overaccumulation of kaempferol-3-rhamnoside in the seed coat compromises normal seed coat development. This observation positions TRANSPARENT TESTA 7 and the UGT78D1 glycosyltransferase, catalysing flavonol 3-O-rhamnosylation, as essential players in the modulation of seed longevity.

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.

The Tapetal Major Facilitator NPF2.8 Is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana.

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Although the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that a member of the nitrate/peptide transporter family is required for the accumulation and transport of pollen-specific flavonol 3-o-sophorosides, characterized by a glycosidic ß-1,2-linkage, to the pollen surface of Arabidopsis (Arabidopsis thaliana). Ectopic, transient expression in Nicotiana benthamiana epidermal leaf cells demonstrated localization of this flavonol sophoroside transporter (FST1) at the plasmalemma when fused to green fluorescent protein (GFP). We also confirmed the tapetum-specific expression of FST1 by GFP reporter lines driven by the FST1 promoter. In vitro characterization of FST1 activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1 insertion mutant by complementation with an FST1 genomic fragment restored the accumulation of flavonol glycosides in pollen grains to wild-type levels, corroborating the requirement of FST1 for transport of flavonol-3-o-sophorosides from the tapetum to the pollen surface.

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

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

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.

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.

Comprehensive Analysis of Metabolome and Transcriptome in Fruits and Roots of Kiwifruit.

Kiwifruit (Actinidia chinensis) roots instead of fruits are widely used as Chinese medicine, but the functional metabolites remain unclear. In this study, we conducted comparative metabolome analysis between root and fruit in kiwifruit. A total of 410 metabolites were identified in the fruit and root tissues, and of them, 135 metabolites were annotated according to the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway. Moreover, 54 differentially expressed metabolites (DEMs) were shared in root and fruit, with 17 DEMs involved in the flavonoid pathway. Of the 17 DEMs, three flavonols (kaempferol-3-rhamnoside, L-Epicatechin and trifolin) and one dihydrochalcone (phloretin) showed the highest differences in the content level, suggesting that flavonols and dihydrochalcones may act as functional components in kiwifruit root. Transcriptome analysis revealed that genes related to flavonols and dihydrochalcones were highly expressed in root. Moreover, two AP2 transcription factors (TFs), AcRAP2-4 and AcAP2-4, were highly expressed in root, while one bHLH TF AcbHLH62 showed extremely low expression in root. The expression profiles of these TFs were similar to those of the genes related to flavonols and dihydrochalcones, suggesting they are key candidate genes controlling the flavonoid accumulation in kiwifruit. Our results provided an insight into the functional metabolites and their regulatory mechanism in kiwifruit root.

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.