Two glycosyltransferases involved in anthocyanin modification delineated by transcriptome independent component analysis in Arabidopsis thaliana.
ABSTRACT: To identify candidate genes involved in Arabidopsis flavonoid biosynthesis, we applied transcriptome coexpression analysis and independent component analyses with 1388 microarray data from publicly available databases. Two glycosyltransferases, UGT79B1 and UGT84A2 were found to cluster with anthocyanin biosynthetic genes. Anthocyanin was drastically reduced in ugt79b1 knockout mutants. Recombinant UGT79B1 protein converted cyanidin 3-O-glucoside to cyanidin 3-O-xylosyl(1?2)glucoside. UGT79B1 recognized 3-O-glucosylated anthocyanidins/flavonols and uridine diphosphate (UDP)-xylose, but not 3,5-O-diglucosylated anthocyanidins, indicating that UGT79B1 encodes anthocyanin 3-O-glucoside: 2''-O-xylosyltransferase. UGT84A2 is known to encode sinapic acid: UDP-glucosyltransferase. In ugt84a2 knockout mutants, a major sinapoylated anthocyanin was drastically reduced. A comparison of anthocyanin profiles in ugt84a knockout mutants indicated that UGT84A2 plays a major role in sinapoylation of anthocyanin, and that other UGT84As contribute the production of 1-O-sinapoylglucose to a lesser extent. These data suggest major routes from cyanidin 3-O-glucoside to the most highly modified cyanidin in the potential intricate anthocyanin modification pathways in Arabidopsis.
Project description:Purple carrots accumulate abundant cyanidin-based anthocyanins in taproots. UDP-glucose: sinapic acid glucosyltransferase (USAGT) can transfer the glucose moiety to the carboxyl group of sinapic acid thereby forming the ester bond between the carboxyl-C and the C1 of glucose (1-O-sinapoylglucose). 1-O-sinapoylglucose can serve as an acyl donor in acylation of anthocyanins and generate cyanidin 3-xylosyl (sinapoylglucosyl) galactoside in purple carrots. This final product helps stabilize the accumulation of anthocyanins. In this study, a gene named DcUSAGT1 encoding USAGT was cloned from 'Deep purple' carrot taproots. Enzymatic activity was determined using high performance liquid chromatography (HPLC). The optimal temperature and pH value were 30°C and 7.0, respectively. Kinetic analysis suggested a Km (sinapic acid) of 0.59 mM. Expression profiles of DcUSAGT1 showed high expression levels in the taproots of all the three purple carrot cultivars but low expression levels in those of non-purple carrot cultivars. The USAGT activity of different carrots in vitro indicated that crude enzyme extracted from the purple carrot taproots rather than non-purple carrot taproots exhibited USAGT activity. These results indicated that DcUSAGT1 may influence anthocyanin biosynthesis of purple carrot taproots.
Project description:Sweet potato anthocyanins are water-soluble pigments with many physiological functions. Previous research on anthocyanin accumulation in sweet potato has focused on the roots, but the accumulation progress in the leaves is still unclear. Two purple sweet potato cultivars (Fushu No. 23 and Fushu No. 317) with large quantities of anthocyanin in the leaves were investigated. Anthocyanin composition and content were assessed with ultra-performance liquid chromatography diode-array detection (UPLC-DAD) and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and the expressions of genes were detected by qRT-PCR. The two cultivars contained nine cyanidin anthocyanins and nine peonidin anthocyanins with an acylation modification. The acylation modification of anthocyanins in sweet potato leaves primarily included caffeoyl, p-coumaryl, feruloyl, and p-hydroxy benzoyl. We identified three anthocyanin compounds in sweet potato leaves for the first time: cyanidin 3-p-coumarylsophoroside-5-glucoside, peonidin 3-p-coumarylsophoroside-5-glucoside, and cyanidin 3-caffeoyl-p-coumarylsophoroside-5-glucoside. The anthocyanidin biosynthesis downstream structural genes DFR4, F3H1, anthocyanin synthase (ANS), and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT3), as well as the transcription factor MYB1, were found to be vital regulatory genes during the accumulation of anthocyanins in sweet potato leaves. The composition of anthocyanins (nine cyanidin-based anthocyanins and nine peonidin-based anthocyanins) in all sweet potato leaves were the same, but the quantity of anthocyanins in leaves of sweet potato varied by cultivar and differed from anthocyanin levels in the roots of sweet potatoes. The anthocyanidin biosynthesis structural genes and transcription factor together regulated and controlled the anthocyandin biosynthesis in sweet potato leaves.
Project description:Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.
Project description:Glycosylation contributes to the diversity and stability of anthocyanins in plants. The process is catalysed by various glucosyltransferases using different anthocyanidin aglycones and glycosyl donors. In this study, we found that an anthocyanidin 3-O-glucoside-2″-O-glucosyltransferase (3GGT) from purple sweet potato (Ipomoea batatas) catalyses the conversion of anthocyanidin 3-O-glucoside into anthocyanidin 3-O-sophoroside, which is functionally different from the 3GGT ortholog of Arabidopsis. Phylogenetic analysis indicated regioselectivity of 3GGT using uridine-5'-diphosphate (UDP)-xylose or UDP-glucose as the glycosyl is divergent between Convolvulaceae and Arabidopsis. Homology-based protein modeling and site-directed mutagenesis of Ib3GGT and At3GGT suggested that the Thr-138 of Ib3GGT is a key amino acid residue for UDP-glucose recognition and that it plays a major role in sugar-donor selectivity. Wild-type and ugt79b1 mutants (defective in UDP carbohydrate-dependent glycosyltransferases, UGTs) of Arabidopsis plants overexpressing Ib3GGT produced the new component cyanidin 3-O-sophoroside. Moreover, Ib3GGT expression was associated with anthocyanin accumulation in different tissues during I. batatas plant development and was regulated by the transcription factor IbMYB1. Localization assays for Ib3GGT showed that glycosyl extension occurs in the cytosol and not in the endoplasmic reticulum. This study therefore reveals the function of Ib3GGT in glycosyl extension of anthocyanins and demonstrates that Thr-138 is the key amino acid residue for UDP-glucose recognition.
Project description:BACKGROUND:Anthocyanins determinate the flower color of many plants. Tobacco is a model plant for studying the molecular regulation of flower coloration. We investigated the mechanism underlying flower coloration in tobacco by profiling flavonoid metabolites,expression of anthocyanin biosynthetic structural genes and their regulator genes in the pink-flowered tobacco cultivar Yunyan 87 and white-flowered Yunyan 87 mutant. RESULT:Significant down-accumulation of anthocyanins, including cyanidin 3-O-glucoside, cyanin, cyanidin 3-O-rutinoside, pelargonidin 3-O-beta-D-glucoside, cyanidin O-syringic acid, pelargonin, and pelargonidin 3-O-malonylhexoside (log2 fold change < -?10), endowed the flower color mutation in Yunyan 87 mutant. Transcriptome analysis showed that the coordinately down-regulated anthocyanin biosynthetic genes including chalcone isomerase, naringenin 3-dioxygenase, dihydroflavonol 4-reductase and UDP-glucose:flavonoid 3-O-glucosyltransferase played critical roles in suppressing the formation of the aforesaid anthocyanins. Several genes encoding MYB and bHLH transcription factors were also found down-regulated, and probably the reason for the suppression of structural genes. CONCLUSION:This is the first study of tobacco flower coloration combining metabolome and transcriptome analyses, and the results shed a light on the systematic regulation mechanisms of flower coloration in tobacco. The obtained information will aid in developing strategies to modify flower color through genetic transformation.
Project description:Mulberry fruits are known as rich sources of anthocyanins and are consumed in syrup form after the addition of sugar and acid; however, there is little information on the anthocyanin composition and antioxidant activity of mulberries of different cultivars and their changes during processing. To address this, the antioxidant activity and anthocyanin composition of 12 cultivar mulberry fruit cultivars were investigated by high-performance liquid chromatography and ultra-high-performance liquid chromatography coupled with electrospray ionization/quadrupole time-of-flight. Additionally, different quantities of citric acid were used to evaluate antioxidant activities and anthocyanin composition of mulberry syrup. Sixteen anthocyanins were identified in mulberry fruits using accurate mass spectrometry. Several anthocyanins were tentatively identified for the first time in mulberry fruits and include: malvidin hexoside, cyanidin malonyl hexose hexoside, cyanidin pentoside, cyanidin malonyl hexoside, petunidin deoxyhexose hexoside, and cyanidin deoxyhexoside. The major anthocyanin in mulberries was cyanidin-3-O-glucoside, followed by cyanidin-3-O-rutinoside. Morus Alba L. Iksu showed the highest cyanidin-3-O-glucoside content (8.65 mg/g dry weight) among 12 mulberry fruit cultivars. As citric acid levels increased, mulberry syrup showed significantly higher antioxidant activity (p < 0.05).
Project description:The anthocyanin extract from a domestic Perilla cultivar (Perilla frutescens var. acuta) were isolated and characterized with high mass accuracy and multi-dimensional fragmentation by means of ultra-performance liquid chromatography (UPLC) and electrospray ionization-ion trap-time of flight mass spectrometry analysis (ESI-IT-TOF-MSn). The new developed and applied LC-MS method focused on in-depth screening of anthocyanin compounds with similar structures which also provided a new approach of anthocyanin characterization without the use of external standards. Selective detection of interested anthocyanins was achieved utilizing extracted ion chromatogram (EIC) analysis, while MSn spectra were recorded to allow identification of the anthocyanin based on characteristic fragmentation patterns. Seven anthocyanins including one feruloyl (Cyanidin 3-O-feruloylglucoside-5-O-glucoside), two caffeoyl (Cyanidin 3-O-caffeoylglucoside-5-O-glucoside, Cyanidin 3-O-caffeoylglucoside-5-O-malonylglucoside) and four coumaroyl substituted anthocyanins (Cis-shisonin, Malonyl-cis-shisonin, Shisonin, and Malonyl-shisonin) were identified. Annexin-V FITC/PI flow cytometric assay was performed to analyze the influence of anthocyanin extract of P. frutescens var. acuta on cell apoptosis. The results suggested that Perilla anthocyanins can induce Hela cell apoptosis by a dose dependent manner.
Project description:The goal of the present study was to investigate the bioactive molecules (anthocyanins and fatty acids) present in the aril of pomegranate. Major anthocyanins present in the aril of pomegranate were identified by HRMS as delphinidin 3,5-diglucoside, cyanidin 3,5-diglucoside, pelargonidin 3,5-diglucoside, cyanidin 3-glucoside and delphinidin 3-glucoside. In-vitro study revealed that bioaccessibility of anthocyanin in duodenal condition was varied between 7.3 and 9.7%. Encapsulation enhances the bioaccessibility of both the phenolics to some extent in gastric as well as duodenal condition. Seed oil contains significant amount of unsaturated fatty acids especially ω-5 fatty acids. Geometrical isomers of ω-5 fatty acids were also identified by GC-MS. The spray dried anthocyanin formulation has potential for food application.
Project description:Almost all flowers of the tea plant (Camellia sinensis) are white, which has caused few researchers to pay attention to anthocyanin accumulation and color changing in tea flowers. A new purple-leaf cultivar, Baitang purple tea (BTP) was discovered in the Baitang Mountains of Guangdong, whose flowers are naturally pink, and can provide an opportunity to understand anthocyanin metabolic networks and flower color development in tea flowers. In the present study, twelve anthocyanin components were identified in the pink tea flowers, namely cyanidin O-syringic acid, petunidin 3-O-glucoside, pelargonidin 3-O-beta-d-glucoside, which marks the first time these compounds have been found in the tea flowers. The presence of these anthocyanins seem most likely to be the reason for the pink coloration of the flowers. Twenty-one differentially expressed genes (DEGs) involved in anthocyanin pathway were identified using KEGG pathway functional enrichment, and ten of these DEG's screened using venn and KEGG functional enrichment analysis during five subsequent stages of flower development. By comparing DEGs and their expression levels across multiple flower development stages, we found that anthocyanin biosynthesis and accumulation in BTP flowers mainly occurred between the third and fourth stages (BTP3 to BTP4). Particularly, during the period of peak anthocyanin synthesis 17 structural genes were upregulated, and four structural genes were downregulated only. Ultimately, eight critical genes were identified using weighted gene co-expression network analysis (WGCNA), which were found to have direct impact on biosynthesis and accumulation of three flavonoid compounds, namely cyanidin 3-O-glucoside, petunidin 3-O-glucoside and epicatechin gallate. These results provide useful information about the molecular mechanisms of coloration in rare pink tea flower of anthocyanin-rich tea, enriching the gene resource and guiding further research on anthocyanin accumulation in purple tea.
Project description:Purple carrots (Daucus carota ssp. sativus var. atrorubens Alef.) accumulate large amounts of cyanidin-based anthocyanins in their taproots. Cyanidin can be glycosylated with galactose, xylose, and glucose in sequence by glycosyltransferases resulting in cyanidin 3-xylosyl (glucosyl) galactosides in purple carrots. The first step in the glycosylation of cyanidin is catalysis by UDP-galactose: cyanidin galactosyltransferase (UCGalT) transferring the galactosyl moiety from UDP-galactose to cyanidin. In the present study, a gene from 'Deep purple' carrot, DcUCGalT1, was cloned and heterologously expressed in E. coli BL21 (DE3). The recombinant DcUCGalT1 galactosylated cyanidin to produce cyanidin-3-O-galactoside and showed optimal activity for cyanidin at 30?°C and pH 8.6. It showed lower galactosylation activity for peonidin, pelargonidin, kaempferol and quercetin. It accepted only UDP-galactose as a glycosyl donor when cyanidin was used as an aglycone. The expression level of DcUCGalT1 was positively correlated with anthocyanin biosynthesis in carrots. The enzyme extractions from 'Deep purple' exhibited galactosylation activity for cyanidin, peonidin and pelargonidin, while those from 'Kuroda' (a non-purple cultivar) did not.