Flavonoids are present in fruits, vegetables and beverages derived from plants (tea, red wine), and in many dietary supplements or herbal remedies including Ginkgo Biloba, Soy Isoflavones, and Milk Thistle. Flavonoids have been described as health-promoting, disease-preventing dietary supplements, and have activity as cancer preventive agents. Additionally, they are extremely safe and associated with low toxicity, making them excellent candidates for chemopreventive agents. The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xenobiotics. This review focuses on the flavonoid effects on cytochrome P450 (CYP) enzymes involved in the activation of procarcinogens and phase II enzymes, largely responsible for the detoxification of carcinogens. A number of naturally occurring flavonoids have been shown to modulate the CYP450 system, including the induction of specific CYP isozymes, and the activation or inhibition of these enzymes. Some flavonoids alter CYPs through binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, acting as either AhR agonists or antagonists. Inhibition of CYP enzymes, including CYP 1A1, 1A2, 2E1 and 3A4 by competitive or mechanism-based mechanisms also occurs. Flavones (chrysin, baicalein, and galangin), flavanones (naringenin) and isoflavones (genistein, biochanin A) inhibit the activity of aromatase (CYP19), thus decreasing estrogen biosynthesis and producing antiestrogenic effects, important in breast and prostate cancers. Activation of phase II detoxifying enzymes, such as UDP-glucuronyl transferase, glutathione S-transferase, and quinone reductase by flavonoids results in the detoxification of carcinogens and represents one mechanism of their anticarcinogenic effects. A number of flavonoids including fisetin, galangin, quercetin, kaempferol, and genistein represent potent non-competitive inhibitors of sulfotransferase 1A1 (or P-PST); this may represent an important mechanism for the chemoprevention of sulfation-induced carcinogenesis. Importantly, the effects of flavonoids on enzymes are generally dependent on the concentrations of flavonoids present, and the different flavonoids ingested. Due to the low oral bioavailability of many flavonoids, the concentrations achieved in vivo following dietary administration tend to be low, and may not reflect the concentrations tested under in vitro conditions; however, this may not be true following the ingestion of herbal preparations when much higher plasma concentrations may be obtained. Effects will also vary with the tissue distribution of enzymes, and with the species used in testing since differences between species in enzyme activities also can be substantial. Additionally, in humans, marked interindividual variability in drug-metabolizing enzymes occurs as a result of genetic and environmental factors. This variability in xenobiotic metabolizing enzymes and the effect of flavonoid ingestion on enzyme expression and activity can contribute to the varying susceptibility different individuals have to diseases such as cancer. As well, flavonoids may also interact with chemotherapeutic drugs used in cancer treatment through the induction or inhibition of their metabolism.

Flavonoids are ubiquitous in photosynthesising cells and are commonly found in fruit, vegetables, nuts, seeds, stems, flowers, tea, wine, propolis and honey. For centuries, preparations containing these compounds as the principal physiologically active constituents have been used to treat human diseases. Increasingly, this class of natural products is becoming the subject of anti-infective research, and many groups have isolated and identified the structures of flavonoids possessing antifungal, antiviral and antibacterial activity. Moreover, several groups have demonstrated synergy between active flavonoids as well as between flavonoids and existing chemotherapeutics. Reports of activity in the field of antibacterial flavonoid research are widely conflicting, probably owing to inter- and intra-assay variation in susceptibility testing. However, several high-quality investigations have examined the relationship between flavonoid structure and antibacterial activity and these are in close agreement. In addition, numerous research groups have sought to elucidate the antibacterial mechanisms of action of selected flavonoids. The activity of quercetin, for example, has been at least partially attributed to inhibition of DNA gyrase. It has also been proposed that sophoraflavone G and (-)-epigallocatechin gallate inhibit cytoplasmic membrane function, and that licochalcones A and C inhibit energy metabolism. Other flavonoids whose mechanisms of action have been investigated include robinetin, myricetin, apigenin, rutin, galangin, 2,4,2'-trihydroxy-5'-methylchalcone and lonchocarpol A. These compounds represent novel leads, and future studies may allow the development of a pharmacologically acceptable antimicrobial agent or class of agents.

Antibiotic resistance is a major global problem and there is a pressing need to develop new therapeutic agents. Flavonoids are a family of plant-derived compounds with potentially exploitable activities, including direct antibacterial activity, synergism with antibiotics, and suppression of bacterial virulence. In this review, recent advances towards understanding these properties are described. Information is presented on the ten most potently antibacterial flavonoids as well as the five most synergistic flavonoid-antibiotic combinations tested in the last 6 years (identified from PubMed and Science-Direct). Top of these respective lists are panduratin A, with minimum inhibitory concentrations (MICs) of 0.06-2.0 mu g/mL against Staphylococcus aureus, and epicatechin gallate, which reduces oxacillin MICs as much as 512-fold. Research seeking to improve such activity and understand structure-activity relationships is discussed. Proposed mechanisms of action are also discussed. In addition to direct and synergistic activities, flavonoids inhibit a number of bacterial virulence factors, including quorum-sensing signal receptors, enzymes and toxins. Evidence of these molecular effects at the cellular level include in vitro inhibition of biofilm formation, inhibition of bacterial attachment to host ligands, and neutralisation of toxicity towards cultured human cells. In vivo evidence of disruption of bacterial pathogenesis includes demonstrated efficacy against Helicobacter pylori infection and S. aureus alpha-toxin intoxication. (c) 2011 Elsevier B. V. and the International Society of Chemotherapy.

Arabidopsis thaliana and Medicago truncatula are enabling the intricacies of the physiology of these compounds to be deduced. In the present review, we cover recent advances in flavonoid research, discuss deficiencies in our understanding of the physiological processes, and suggest approaches to identify the cellular targets of flavonoids.Buer CS, Imin N, Djordjevic MA (2010) Flavonoids: new roles for old molecules. J. Integr. Plant Biol. 52(1), 98–111.]]>

In this study, five common buckwheats and nine tartary buckwheats grown at different locations were analyzed for the contents of rutin, quercetin, and amino acids by high-performance liquid chromatography and spectrophotometry. The rutin content was higher than quercetin in buckwheat seeds. Rutin content was in the range from 0.05 (0.05 g per 100 g dry seeds) to 1.35% of buckwheat seeds. Quercetin content varied from 0.01 to 0.17% and in some common buckwheats it was even difficult to detect. Comparatively, tartary buckwheat seeds contained more rutin and quercetin than common buckwheat seeds. Meanwhile, the bran has higher rutin content than the farina in tartary buckwheat seeds, with a respective content of 0.45 to 1.19% and 0.14 to 0.67%. It was found that amino acid contents were around 1.79 to 12.65% (farina) and 5.74 to 7.89% (bran) in common buckwheats, and 1.73 to 5.63% (farina) and 2.64 to 16.78% (bran) in tartary buckwheat seeds. The highest total rutin content was found to be 1.35% in tartary buckwheat seeds from Sichuan, China. The highest total amounts of amino acid were detected to be 20.13% in tartary buckwheat seeds from Changzhi, Shanxi Province (China). Our results suggested that food products made of whole-buckwheat flour are healthier than those made of fine white flour.

Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein 'Sensitive to ABA and Drought 2' (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11(D126N) in F. tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11(D122N) in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.

Plants produce a large variety of secondary metabolites including alkaloids, glucosinolates, terpenoids and phenylpropanoids. These compounds play key roles in plant-environment interactions and many of them have pharmacological activity in humans. Jasmonates (JAs) are plant hormones which induce biosynthesis of many secondary metabolites. JAs-responsive transcription factors (TFs) that regulate the JAs-induced accumulation of secondary metabolites belong to different families including AP2/ERF, bHLH, MYB and WRKY. Here, we give an overview of the types and functions of TFs that have been identified in JAs-induced secondary metabolite biosynthesis, and highlight their similarities and differences in regulating various biosynthetic pathways. We review major recent developments regarding JAs-responsive TFs mediating secondary metabolite biosynthesis, and provide suggestions for further studies.

Anthocyanin and proanthocyanidin (PA) are important secondary metabolites and beneficial to human health. Their biosynthesis is induced by jasmonate (JA) treatment and regulated by MYB transcription factors (TFs). However, which and how MYB TFs regulate this process is largely unknown in apple. In this study, MdMYB9 and MdMYB11 which were induced by methyl jasmonate (MeJA) were functionally characterized. Overexpression of MdMYB9 or MdMYB11 promoted not only anthocyanin but also PA accumulation in apple calluses, and the accumulation was further enhanced by MeJA. Subsequently, yeast two-hybrid, pull-down and bimolecular fluorescence complementation assays showed that both MYB proteins interact with MdbHLH3. Moreover, Jasmonate ZIM-domain (MdJAZ) proteins interact with MdbHLH3. Furthermore, chromatin immunoprecipitation-quantitative PCR and yeast one-hybrid assays demonstrated that both MdMYB9 and MdMYB11 bind to the promoters of ANS, ANR and LAR, whereas MdbHLH3 is recruited to the promoters of MdMYB9 and MdMYB11 and regulates their transcription. In addition, transient expression assays indicated that overexpression of MdJAZ2 inhibits the recruitment of MdbHLH3 to the promoters of MdMYB9 and MdMYB11. Our findings provide new insight into the mechanism of how MeJA regulates anthocyanin and PA accumulation in apple.

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

The plant hormone jasmonate (JA) plays an important role in regulating growth, development, and immunity. Activation of the JA-signaling pathway is based on the hormone-triggered ubiquitination and removal of transcriptional repressors (JASMONATE-ZIM DOMAIN [JAZ] proteins) by an SCF receptor complex (SCF(COI1)/JAZ). This removal allows the rapid activation of transcription factors (TFs) triggering a multitude of downstream responses. Identification of TFs bound by the JAZ proteins is essential to better understand how the JA-signaling pathway modulates and integrates different responses. In this study, we found that the JAZ3 repressor physically interacts with the YABBY (YAB) family transcription factor FILAMENTOUS FLOWER (FIL)/YAB1. In Arabidopsis thaliana, FIL regulates developmental processes such as axial patterning and growth of lateral organs, shoot apical meristem activity, and inflorescence phyllotaxy. Phenotypic analysis of JA-regulated responses in loss- and gain-of-function FIL lines suggested that YABs function as transcriptional activators of JA-triggered responses. Moreover, we show that MYB75, a component of the WD-repeat/bHLH/MYB complex regulating anthocyanin production, is a direct transcriptional target of FIL. We propose that JAZ3 interacts with YABs to attenuate their transcriptional function. Upon perception of JA signal, degradation of JAZ3 by the SCF(COI1) complex releases YABs to activate a subset of JA-regulated genes in leaves leading to anthocyanin accumulation, chlorophyll loss, and reduced bacterial defense.

The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish (Raphanus sativus L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene RsTT8. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., RsCHS, RsCHI, RsF3H, RsDFR, and RsANS). Heterologous co-expression of both RsTT8 and RsMYB1 in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis.

Phenolic secondary metabolites are only produced by plants wherein they play important roles in both biotic and abiotic defense in seed plants as well as being potentially important bioactive compounds with both nutritional and medicinal benefits reported for animals and humans as a consequence of their potent antioxidant activity. During the long evolutionary period in which plants have adapted to the environmental niches in which they exist (and especially during the evolution of land plants from their aquatic algal ancestors), several strategies such as gene duplication and convergent evolution have contributed to the evolution of this pathway. In this respect, diversity and redundancy of several key genes of phenolic secondary metabolism such as polyketide synthases, cytochrome P450s, Fe2+/2-oxoglutarate-dependent dioxygenases and UDP-glycosyltransferases have played an essential role. Recent technical developments allowing affordable whole genome sequencing as well as a better inventory of species-by-species chemical diversity have resulted in a dramatic increase in the number of tools we have to assess how these pathways evolved. In parallel, reverse genetics combined with detailed molecular phenotyping is allowing us to elucidate the functional importance of individual genes and metabolites and by this means to provide further mechanistic insight into their biological roles. In this review, phenolic metabolite-related gene sequences (for a total of 65 gene families including shikimate biosynthetic genes) are compared across 23 independent species, and the phenolic metabolic complement of various plant species are compared with one another, in attempt to better understand the evolution of diversity in this crucial pathway.

Anthocyanins, synthesized via the flavonoid pathway, are a class of crucial phenolic compounds which are fundamentally responsible for the red color of grapes and wines. As the most important natural colorants in grapes and their products, anthocyanins are also widely studied for their numerous beneficial effects on human health. In recent years, the biosynthetic pathway of anthocyanins in grapes has been thoroughly investigated. Their intracellular transportation and accumulation have also been further clarified. Additionally, the genetic mechanism regulating their biosynthesis and the phytohormone influences on them are better understood. Furthermore, due to their importance in the quality of wine grapes, the effects of the environmental factors and viticulture practices on anthocyanin accumulation are being investigated increasingly. The present paper summarizes both the basic information and the most recent advances in the study of the anthocyanin biosynthesis in red grapes, emphasizing their gene structure, the transcriptional factors and the diverse exterior regulation factors.

CrORCA3 (octadecanoid-responsive Catharanthus AP2/ERF domain), methyl jasmonate (MeJA), and sodium nitroprusside (SNP) on the differentiated tissue of Catharanthus roseus hairy roots. The changes in catharanthine concentration and in the levels of mRNA transcripts of pathway genes and regulators were tracked for 192 h. ORCA3 overexpression led to a slight decrease of the accumulation of catharanthine, while MeJA treatment caused a large increase in the levels of transcripts of pathway genes and the catharanthine concentration. SNP treatment alone or SNP in combination with MeJA treatment caused a dramatic decrease of the cathanranthine concentration, while at the same time the levels of transcripts of zinc finger-binding proteins genes (ZCTs) increased. The latter treatment also caused a decrease of the levels of transcripts of type-I protein prenyltransferase gene (PGGT-I). This response of transcriptional repressors and pathway genes may explain the antagonistic effects of NO and MeJA on catharanthine biosynthesis in C. roseus hairy roots.]]>

Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0-41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.

Because structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside-5-O-malonylglucoside-3'5'-O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Anthocyanins generate the red color in the pericarp of Litchi chinensis. UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT, EC. 2.4.1.91) stabilizes anthocyanidin by attaching sugar moieties to the anthocyanin aglycone. In this study, the function of an UFGT gene involved in the biosynthesis of anthocyanin was verified through heterologous expression and virus-induced gene silencing assays. A strong positive correlation between UFGT activity and anthocyanin accumulation capacity was observed in the pericarp of 15 cultivars. Four putative flavonoid 3-O-glycosyltransferase-like genes, designated as LcUFGT1 to LcUFGT4, were identified in the pericarp of litchi. Among the four UFGT gene members, only LcUFGT1 can use cyanidin as its substrate. The expression of LcUFGT1 was parallel with developmental anthocyanin accumulation, and the heterologously expressed protein of LcUFGT1 displayed catalytic activities in the formation of anthocyanin. The LcUFGT1 over-expression tobacco had darker petals and pigmented filaments and calyxes resulting from higher anthocyanin accumulations compared with non-transformed tobacco. In the pericarp with LcUFGT1 suppressed by virus-induced gene silencing, pigmentation was retarded, which was well correlated with the reduced-LcUFGT1 transcriptional activity. These results suggested that the glycosylation-related gene LcUFGT1 plays a critical role in red color formation in the pericarp of litchi.

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6''-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6''RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6''RhaT enzyme expressed in Escherichia coli exhibited 6''-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6''RhaT converted the administered quercetin into rutin, suggesting that FeF3G6''RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6''RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6''RhaT is involved in rutin biosynthesis in common buckwheat.

Plant hormones regulate almost all aspects of plant growth and development. The past decade has provided breakthrough discoveries in phytohormone sensing and signal transduction, and highlighted the striking mechanistic similarities between the auxin and jasmonate (JA) signalling pathways. Perception of auxin and JA involves the formation of co-receptor complexes in which hormone-specific E3-ubiquitin ligases of the SKP1-Cullin-F-box protein (SCF) type interact with specific repressor proteins. Across the plant kingdom, the Aux/IAA and the JASMONATE-ZIM DOMAIN (JAZ) proteins correspond to the auxin- and JA-specific repressors, respectively. In the absence of the hormones, these repressors form a complex with transcription factors (TFs) specific for both pathways. They also recruit several proteins, among which the general co-repressor TOPLESS, and thereby prevent the TFs from activating gene expression. The hormone-mediated interaction between the SCF and the repressors targets the latter for 26S proteasome-mediated degradation, which, in turn, releases the TFs to allow modulating hormone-dependent gene expression. In this review, we describe the similarities and differences in the auxin and JA signalling cascades with respect to the protein families and the protein domains involved in the formation of the pathway-specific complexes.

Jasmonic acid is an important plant stress signalling molecule. It induces the biosynthesis of defence proteins and protective secondary metabolites. In alkaloid metabolism, jasmonate acts by coordinate activation of the expression of multiple biosynthesis genes. In terpenoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERF-domain family of plant transcription factors. Other jasmonate-regulated (secondary) metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain transcription factors. If so, such regulators could be used to improve plant fitness or metabolite productivity of plants or cell cultures.

Flavonoids synthesized by the phenylpropanoid pathway participate in myriad physiological and biochemical processes in plants. Due to the diversity of secondary transformations and the complexity of the regulation of branched pathways, single gene strategies have not been very successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis (Arabidopsis thaliana) transcription factor, AtMYB12, in tobacco (Nicotiana tabacum), which resulted in enhanced expression of genes involved in the phenylpropanoid pathway, leading to severalfold higher accumulation of flavonols. Global gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMYB12 regulated a number of pathways, leading to flux availability for the phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial microRNA reversed insect resistance of the AtMYB12-expressing tobacco plants. Our study suggests that AtMYB12 can be strategically used for developing safer insect pest-resistant transgenic plants.

Jasmonates are plant hormones that induce the accumulation of many secondary metabolites, such as rutin in buckwheat, via regulation of jasmonate-responsive transcription factors. Here, we report on the identification of a clade of jasmonate-responsive subgroup 4 MYB transcription factors, FtMYB13, FtMYB14, FtMYB15, and FtMYB16, which directly repress rutin biosynthesis in Fagopyrum tataricum. Immunoblot analysis showed that FtMYB13, FtMYB14, and FtMYB15 could be degraded via the 26S proteasome in the COI1-dependent jasmonate signaling pathway, and that this degradation is due to the SID motif in their C-terminus. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that FtMYB13, FtMYB14, and FtMYB15 interact with the importin protein Sensitive to ABA and Drought 2 (FtSAD2) in stem and inflorescence. Furthermore, the key repressor of jasmonate signaling FtJAZ1 specifically interacts with FtMYB13. Point mutation analysis showed that the conserved Asp residue of the SID domain contributes to mediating protein-protein interaction. Protoplast transient activation assays demonstrated that FtMYB13, FtMYB14, and FtMYB15 directly repress phenylalanine ammonia lyase (FtPAL) gene expression, and FtSAD2 and FtJAZ1 significantly promote the repressing activity of FtMYBs. These findings may ultimately be promising for further engineering of plant secondary metabolism.

Tartary buckwheat (Fagopyrum tataricum) not only provides a supplement to primary grain crops in China but also has high medicinal value, by virtue of its rich content of flavonoids possessing antioxidant, anti-inflammatory, and anticancer properties. Light is an important environmental factor that can regulate the synthesis of plant secondary metabolites. In this study, we treated tartary buckwheat seedlings with different wavelengths of light and found that red and blue light could increase the content of flavonoids and the expression of genes involved in flavonoid synthesis pathways. Through coexpression analysis, we identified a new MYB transcription factor (FtMYB116) that can be induced by red and blue light. Yeast one-hybrid assays and an electrophoretic mobility shift assay showed that FtMYB116 binds directly to the promoter region of flavonoid-3'-hydroxylase (F3'H), and a transient luciferase activity assay indicated that FtMYB116 can induce F3'H expression. After transforming FtMYB116 into the hairy roots of tartary buckwheat, we observed significant increases in the content of rutin and quercetin. Collectively, our results indicate that red and blue light promote an increase in flavonoid content in tartary buckwheat seedlings; we also identified a new MYB transcription factor, FtMYB116, that promotes the accumulation of rutin via direct activation of F3'H expression.