A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

UGT73C6 and UGT78D1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana

Flavonol glycosides constitute one of the most prominent plant natural product classes that accumulate in the model plant Arabidopsis thaliana. To date there are no reports of functionally characterized flavonoid glycosyltransferases in Arabidopsis, despite intensive research efforts aimed at both flavonoids and Arabidopsis. In this study, flavonol glycosyltransferases were considered in a functional genomics approach aimed at revealing genes involved in determining the flavonol-glycoside profile. Candidate glycosyltransferase-encoding genes were selected based on homology to other known flavonoid glycosyltransferases and two T-DNA knockout lines lacking flavonol-3-O-rhamnoside-7-O-rhamnosides (ugt78D1) and quercetin-3-O-rhamnoside-7-O-glucoside (ugt73C6 and ugt78D1) were identified. To confirm the in planta results, cDNAs encoding both UGT78D1 and UGT73C6 were expressed in vitro and analyzed for their qualitative substrate specificity. UGT78D1 catalyzed the transfer of rhamnose from UDP-rhamnose to the 3-OH position of quercetin and kaempferol, whereas UGT73C6 catalyzed the transfer of glucose from UDP-glucose to the 7-OH position of kaempferol-3-O-rhamnoside and quercetin-3-O-rhamnoside, respectively. The present results suggest that UGT78D1 and UGT73C6 should be classified as UDP-rhamnose:flavonol-3-Orhamnosyltransferase and UDP-glucose:flavonol-3-O-glycoside-7-O-glucosyltransferase, respectively.     

苦荞转录因子FtMYBF的克隆、亚细胞定位及表达分析

R2R3-MYB转录因子SG7亚家族成员在植物黄酮醇生物合成中具有极其重要的调控作用.探究苦荞中SG7 R2R3-MYB转录因子在黄酮醇生物合成中的功能,为苦荞黄酮醇生物合成的分子调控机制研究奠定基础.采用RT-PCR技术从苦荞中克隆到一个前期经转录组与代谢组联合分析鉴定到的SG7 R2R3-MYB转录因子,命名为Ft...     

Genomic organization of a UDP-glucosyltransferase gene determines differential accumulation of specific flavonoid glucosides in tepals

Glycosylation plays a major role in the chemical diversity of flavonoids. The wide diversity of the family-1 glycosyltransferase (UGT) impairs the determination of the biochemical function solely from its primary sequence. Here we combined differential expression and target metabolomic analysis in various Crocus species to identify a gene that is key in determining the flavonoid composition of Crocus species that belong to the Crocus series. UGT703B1 recognizes isorhamnetin and kaempferol as substrates in vitro. In addition, UGT703B1 expression was found to be highly correlated with the presence of kaempferol 7-O-biglucoside-3-O-β-glucoside and isorhamnetin-3,7-O-diglucoside. These flavonols were present in C. sativus and C. cartwrightianus albus, both from series Crocus but absent in Crocus species from the other series analyzed. Further, the presence of both flavonols was associated with the expression of UGT703B1, and this expression was correlated with the presence of the UGT703B1 coding gene, with the exception of C. cancellatus, whose genomic sequence was present but contained a shorter intronic sequence and promoter alterations, suggesting the presence of regulatory sequences in the deleted part of that intron and promoter important for UGT703B1 expression. Overall, the data obtained supports the involvement of UGT703B1 in the formation of specific kaempferol and isorhamnetin glucosides, while demonstrating that the integration of metabolomic and differential expression analysis is a versatile tool for understanding a multigene family of UGTs in Crocus.     

Four glucosyltransferases from rice: cDNA cloning, expression, and characterization

Four UDP-dependent glucosyltransferase (UGT) genes, UGT706C1, UGT706D1, UGT707A3, and UGT709A4 were cloned from rice, expressed in Escherichia coli, and purified to homogeneity. In order to find out whether these enzymes could use flavonoids as glucose acceptors, apigenin, daidzein, genistein, kaempferol, luteolin, naringenin, and quercetin were used as potential glucose acceptors. UGT706C1 and UGT707A3 could use kaempferol and quercetin as glucose acceptors and the major glycosylation position was the hydroxyl group of carbon 3 based on the comparison of HPLC retention times, UV spectra, and NMR spectra with those of corresponding authentic flavonoid 3-O-glucosides. On the other hand, UGT709A4 only used the isoflavonoids genistein and daidzein and transferred glucose onto 7-hydroxyl group. In addition, UGT706D1 used a broad range of flavonoids including flavone, flavanone, flavonol, and isoflavone, and produced at least two products with glycosylation at different hydroxyl groups. Based on their substrate preferences and the flavonoids present in rice, the in vivo function of UGT706C1, UGT706D1, and UGT707A3 is most likely the biosynthesis of kaempferol and quercetin glucosides.     

Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida

Petunia hybrida mutants, homozygous recessive for one of the genes An1, An2, An6, or An9 do not show anthocyanin synthesis in in vitro complementation experiments per se (see also Kho et al. 1977). Extracts of flowers of these mutants all provoke anthocyanin synthesis in isolated petals of an an3an3 mutant. Mutants homozygous recessive for one of the genes An1, An2, An6, or An9 and homozygous recessive for F1 accumulate dihydroflavonols in comparable amounts. The synthesis of dihydromyricetin is blocked in an1an1 mutants, which indicates a regulating effect of the gene An1 on the gene Hfl. Similar mutants, but dominant for F1, accumulate flavonols (kaempferol and quercetin) instead of dihydroflavonols. Myricetin is accumulated in minor amounts and not at all in an1an1 mutant. Furthermore, the synthesis of this flavonol is not controlled by the gene F1. The synthesis of cyanidin (derivatives) is greatly reduced when flavonols are synthesized (F1 dominant). In mutants dominant for Ht1 and Hf1 and thus able to synthesize cyanidin (derivatives) and delphinidin (derivatives), predominantly delphinidin (derivatives) are synthesized. The results indicate that kaempferol (derivatives), quercetin (derivatives), and delphinidin (derivatives) are the main endproducts of flavonoid biosynthesis in Petunia hybrida.     

7-Rhamnosylated Flavonols Modulate Homeostasis of the Plant Hormone Auxin and Affect Plant Development

Flavonols are a group of secondary metabolites that affect diverse cellular processes. They are considered putative negative regulators of the transport of the phytohormone auxin, by which they influence auxin distribution and concomitantly take part in the control of plant organ development. Flavonols are accumulating in a large number of glycosidic forms. Whether these have distinct functions and diverse cellular targets is not well understood. The rol1-2 mutant of Arabidopsis thaliana is characterized by a modified flavonol glycosylation profile that is inducing changes in auxin transport and growth defects in shoot tissues. To determine whether specific flavonol glycosides are responsible for these phenotypes, a suppressor screen was performed on the rol1-2 mutant, resulting in the identification of an allelic series of UGT89C1, a gene encoding a flavonol 7-O-rhamnosyltransferase. A detailed analysis revealed that interfering with flavonol rhamnosylation increases the concentration of auxin precursors and auxin metabolites, whereas auxin transport is not affected. This finding provides an additional level of complexity to the possible ways by which flavonols influence auxin distribution and suggests that flavonol glycosides play an important role in regulating plant development.     

Functional analysis of a predicted flavonol synthase gene family in Arabidopsis

The genome of Arabidopsis (Arabidopsis thaliana) contains five sequences with high similarity to FLAVONOL SYNTHASE1 (AtFLS1), a previously characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under nonstressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell type-specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus, it is possible that these "pseudogenes" have alternative, noncatalytic functions that have not yet been uncovered.     

Structural basis for acceptor‐substrate recognition of UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.     

Flavonols accumulate asymmetrically and affect auxin transport in Arabidopsis

Flavonoids represent a class of secondary metabolites with diverse functions in plants including ultraviolet protection, pathogen defense, and interspecies communication. They are also known as modulators of signaling processes in plant and animal systems and therefore are considered to have beneficial effects as nutraceuticals. The rol1-2 (for repressor of lrx1) mutation of Arabidopsis (Arabidopsis thaliana) induces aberrant accumulation of flavonols and a cell-growth phenotype in the shoot. The hyponastic cotyledons, aberrant shape of pavement cells, and deformed trichomes in rol1-2 mutants are suppressed by blocking flavonoid biosynthesis, suggesting that the altered flavonol accumulation in these plants induces the shoot phenotype. Indeed, the identification of several transparent testa, myb, and fls1 (for flavonol synthase1) alleles in a rol1-2 suppressor screen provides genetic evidence that flavonols interfere with shoot development in rol1-2 seedlings. The increased accumulation of auxin in rol1-2 seedlings appears to be caused by a flavonol-induced modification of auxin transport. Quantification of auxin export from mesophyll protoplasts revealed that naphthalene-1-acetic acid but not indole-3-acetic acid transport is affected by the rol1-2 mutation. Inhibition of flavonol biosynthesis in rol1-2 fls1-3 restores naphthalene-1-acetic acid transport to wild-type levels, indicating a very specific mode of action of flavonols on the auxin transport machinery.     

Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.