UDP-glucose:flavonoid 3-O-glucosyltransferase Gene Research Articles

MYBA2 gene involved in anthocyanin and flavonol biosynthesis pathways in grapevine.

MYBA2 transcription factor (Myb-related gene) affects the coloring in grapevine berry and plays an important role in the biosynthesis of anthocyanin. The MYBA2 gene was cloned from Vitis vinifera L. cv. Cabernet Sauvignon and polyclonal antibodies for VvmybA2 were prepared. The VvmybA2 gene expression patterns were observed in seven tissues (the leaf, stem, flower, bud, root, berry, and tendril) and during the berry development stage at transcriptional and translational levels, respectively. The results indicated that the expression of VvmybA2 was approximately 11-fold higher in the berry than that in the other six tissues, and increased rapidly from 60 days after full bloom reaching a maximum on day 80. Furthermore, both the anthocyanin content and UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT) gene expression levels increased rapidly 60 days after full bloom. Moreover, correlation analysis indicated that the transcriptional and translational expression levels of the VvmybA2 gene were significantly positively correlated with not only UFGT and DFR genes but also with the anthocyanin content during berry development. These results suggested that VvmybA2 could not only regulate the transcription of both UFGT and DFR but also is involved in the expression of the UFGT gene associated with color determination in grape berries.

English

The expression of the UDP glucose: flavonoid-3-Oglucosyltransferase (UFGT), which encoded the last enzyme of the anthocyanin pathway, was under developmental control as well as affected by external stimuli such as ABA. Three fragments of the 1.38 kb upstream region of the UFGT gene from Litchi (Litchi chinesis Sonn.) were fused to the GUS-coding region, and the expression of these constructs was analyzed in onions. To characterize the cis-regulatory functions of the promoters for enzymes in anthocyanin biosynthesis, we examined onions carrying a series of nested UFGT promoter-β-glucuronidase (GUS) fusion for GUS activity by histochemical staining. The AE-box and ELI-box in anthocyanin biosynthesis of angiosperm were found in UFGT promoter. MRE which was MYB binding site involved in light responsiveness, control the transcription of genes in anthocyanin biosynthesis. The region of -910 to -344 in UFGT promoter showed high activities in the three parts. Although the expression characteristics were indistinguishable from those of the full-length promoter, we observed differences in UFGT promoter regulation for the different construct. The results suggested that region of 910 to 344 of UFGT promoter had multiple functions in the expression under the various developmental stages and stress conditions in litchi.   Key words: Flavonoid-3-Oglucosyltransferase (UFGT), promoter, litchi, transient expression.

‘Zhengmei’: A new early-ripening table grape

Introduction: As a result of natural hybridization and human selection over centuries, the grape berry skin colour has become greatly diversified. The colour of grape skins is determined by the accumulation of anthocyanins (HARBONE et al. 2000). Anthocyanin content is a trait of major interest in Vitis vinifera L. They have very important role in wine and grape industry, confer colour and contribute to other sensory characteristics (BOSS et al. 1996). Molecular analyses of structural genes involved in the anthocyanin biosynthetic pathway showed that expression of the UDP glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene is a critical point in the control of berry colour (KOBAYASHI et al. 2001). It was shown that Myb-related gene, VvMybA1, regulates anthocyanin biosynthesis in grapes via UFGT gene expression (KOBAYASHI et al. 2004, 2005) and the colouring of grape skin depends on the genotype of VvMybA1 (LIJAVETZKY et al. 2006, AZUMA et al. 2007). Recently, it has been shown that the presence of Gret1, a Ty3–gypsy-type retrotransposon, in the promoter region of VvMybA1 gene, is associated with white-berry cultivars when present in a homozygous state (KOBAYASHI et al. 2004). Coloured cultivars hold at least one allele at the VvMybA1 locus not containing this large insertion: VvMybA1b or VvMybA1c (KOBAYASHI et al. 2004, AZUMA et al. 2007, YAKUSHIJI et al. 2006). It was revealed that the VvMybA1b and VvMybA1c alleles are functional. The VvMybA1b allele has a single long terminal repeat (LTR) of Gret1 in the 5 flanking region near the coding region of VvMybA1 gene (AZUMA et al. 2007). VvMybA1c, the wild type allele, is the original sequence of VvMybA1 gene before the insertion of Gret1 (YAKUSHIJI et al. 2006). In most examined white-skinned grapes, the colour locus is homozygous for VvMybA1a, whereas the genotype of colour skinned berries are heterozygous (VvMybA1a/VvMybA1b or VvMybA1a/ VvMybA1c) or are homozygous (VvMybA1c) (AZUMA et al. 2008). Due to the lack of data for Armenian grapevines, the present study has the objective to analyze the role of VvmybA1 gene in berry color variation. In order to ascertain the existent allelic composition for this gene and its association with berry color variation, thirty autochthonous Armenian grapevine cultivars were analyzed.

Cloning and expression of UDP-glucose: flavonoid 3-O-glucosyltransferase gene in peach flowers.

To elucidate the connection between flower coloration and the expression of genes associated with anthocyanin biosynthesis, a gene encoding UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) was isolated, and the expression of the last four genes in the anthocyanin biosynthetic pathway during peach flower development was determined. The nucleotide sequence of the peach UFGT (GenBank accession No. JX149550) is highly similar to its homologs in other plants. Total anthocyanin content initially increased during peach flower development, and then decreased over time. Expression of the four anthocyanin biosynthesis genes increased until the full-bloom stage, and then decreased during late florescence. Expression of F3H, DFR, and UFGT increased dramatically at the full-bloom stage, coinciding with an increase in anthocyanin concentration. The UFGT gene may not be the only gene of the anthocyanin pathway to be differentially controlled in red peach flower tissues. Further studies are needed to genetically and physiologically characterize these genes and enzymes in peach flowers and to gain a better understanding of their functions and relationships with flower coloration.

Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors.

BackgroundFlavonoids such as anthocyanins, flavonols and proanthocyanidins, play a central role in fruit colour, flavour and health attributes. In peach and nectarine (Prunus persica) these compounds vary during fruit growth and ripening. Flavonoids are produced by a well studied pathway which is transcriptionally regulated by members of the MYB and bHLH transcription factor families. We have isolated nectarine flavonoid regulating genes and examined their expression patterns, which suggests a critical role in the regulation of flavonoid biosynthesis.ResultsIn nectarine, expression of the genes encoding enzymes of the flavonoid pathway correlated with the concentration of proanthocyanidins, which strongly increases at mid-development. In contrast, the only gene which showed a similar pattern to anthocyanin concentration was UDP-glucose-flavonoid-3-O-glucosyltransferase (UFGT), which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. Expression of flavonol synthase (FLS1) correlated with flavonol levels, both temporally and in a tissue specific manner. The pattern of UFGT gene expression may be explained by the involvement of different transcription factors, which up-regulate flavonoid biosynthesis (MYB10, MYB123, and bHLH3), or repress (MYB111 and MYB16) the transcription of the biosynthetic genes. The expression of a potential proanthocyanidin-regulating transcription factor, MYBPA1, corresponded with proanthocyanidin levels. Functional assays of these transcription factors were used to test the specificity for flavonoid regulation.ConclusionsMYB10 positively regulates the promoters of UFGT and dihydroflavonol 4-reductase (DFR) but not leucoanthocyanidin reductase (LAR). In contrast, MYBPA1 trans-activates the promoters of DFR and LAR, but not UFGT. This suggests exclusive roles of anthocyanin regulation by MYB10 and proanthocyanidin regulation by MYBPA1. Further, these transcription factors appeared to be responsive to both developmental and environmental stimuli.

Expression analysis of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene in an interspecific hybrid grape between Vitis ficifolia var. ganebu and Vitis vinifera cv. Muscat of Alexandria

Kadainou R-1, an interspecific hybrid grape derived from red (Vitis ficifolia var. ganebu) and white (V. vinifera cv. Muscat of Alexandria) grapes, accumulates high concentrations of anthocyanin in the berry skin. Hence, the expression of uridine 5′-diphosphate (UDP)-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key enzyme of the anthocyanin pathway, was examined in the berry skin of Kadainou R-1. As information on gene sequences of V. ficifolia var. ganebu and other wild grape species was unavailable, we performed GeneChip hybridization using biotin-labeled genomic deoxyribonucleic acid (DNA) to investigate how the genomic sequences of V. vinifera varieties and that of V. ficifolia var. ganebu differ. The study showed a lower correlation coefficient between V. vinifera cultivars and V. ficifolia var. ganebu than that among V. vinifera cultivars. The sequences of the UFGT gene derived from both parents of the red and white cultivars were sequenced in Kadainou R1 and revealed that both were expressed irrespective of the fact that it was not expressed in the white grape (male parent).

Control of anthocyanin biosynthesis pathway gene expression by eutypine, a toxin from Eutypa lata, in grape cell tissue cultures

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 micromol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOXwas not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed.

Myb -related genes of the Kyoho grape ( Vitis labruscana ) regulate anthocyanin biosynthesis

Partial cDNAs of myb-related regulatory genes were isolated from the tetraploid Kyoho grape ( Vitis labruscana: V. labrusca x V. vinifera) and the expression patterns of the corresponding genes were studied. Since MybA gene expression is closely related to coloring and/or ripening of the berry (expression increases strongly with the commencement of coloring and berry softening, and is detected only in berry skin and flesh), full-length cDNAs for the gene were isolated from a mature-berry cDNA library. Three different species of MybA were identified from the cDNA sequences. Delivery of these cDNAs to somatic embryos of grape led to the induction of reddish-purple spots and UDP-glucose:flavonoid 3- O-glucosyltransferase (UFGT) gene expression in non-colored embryos. The UFGT transcript was not detected in control embryos, while other structural genes for anthocyanin biosynthesis were expressed in both control and pigmented embryos. In addition, introduction of the UFGT gene induced the same reddish-purple spots in embryos. In contrast, treatment with the leucoanthocyanidin dioxygenase (LDOX) gene failed to induce these spots. Our results strongly suggest that MybA genes are involved in the regulation of anthocyanin biosynthesis in the grape via expression of the UFGT gene.

Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening

Recent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase, dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol treatment, whereas for UDP glucose-flavonoid 3- O-glucosyltransferase (UFGT) there was a marked increase in expression from 1 to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin accumulation following ethanol treatment.

Comparison of UDP-glucose:flavonoid 3- O-glucosyltransferase (UFGT) gene sequences between white grapes ( Vitis vinifera) and their sports with red skin

The expression of the UDP-glucose:flavonoid 3- O-glucosyltransferase (UFGT) gene has been shown to be critical for anthocyanin biosynthesis in the grape berry. Using white cultivars and bud sports with red skin, we examined the expression of seven anthocyanin biosynthetic genes including the UFGT gene and compared the coding/promoter sequences of the UFGT gene. Northern blot analysis showed that the seven anthocyanin biosynthetic genes were expressed coordinately at higher levels in the red-skin sports than in the white-skin progenitors of the sports. It was especially notable that UFGT gene expression was detected only in the red-skin sports and Kyoho. However, there were no differences in either coding or promoter sequences between Italia ( Vitis vinifera) and its red-skin sport Ruby Okuyama, or between Muscat of Alexandria ( V. vinifera) and the red-skin sport Flame Muscat. From these findings, the phenotypic change from white to red in the sports is thought to be the result of a mutation in a regulatory gene controlling the expression of UFGT.

Anthocyanin composition and anthocyanin pathway gene expression in grapevine sports differing in berry skin colour

The synthesis of anthocyanins was investigated in six grapevine sports with altered skin colour. Cabernet Sauvignon and Shiraz grapes contained anthocyanin monoglucosides as well as acetylated and coumarylated derivatives, predominantly of malvidin, whereas Pinot Noir grapes contained only anthocyanin monoglucosides. Fruit of some sports arising from varieties with black berries either lacked anthocyanins or had much lower levels than their progenitors. A bud sport of Cabernet Sauvignon (Bronze Cabernet) exhibited a 90% reduction in anthocyanin levels, but an anthocyanin composition similar to that of the normal Cabernet Sauvignon fruit. The fruit of a white-coloured bud sport of Bronze Cabernet did not contain anthocyanins. Coloured berries of sports originating on varieties with white-skinned berries also had lower levels of anthocyanins than black grapes, but like Pinot Noir contained very low levels of acylated anthocyanins. The berries of these sports also varied in the proportions of the different types of 3-monoglucosides they contained. The results suggest that control of 3-monoglucoside synthesis and modification vary in the sports. The expression of six genes from the flavonoid biosynthetic pathway was determined. Some genes were expressed in all grapes, even where little or no anthocyanins accumulated, but expression of the gene encoding a UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) was only detected in coloured grapes that synthesised anthocyanins. Southern and northern analysis of the white grapes indicated that the UFGT gene was present but was not expressed. Thus the lack of anthocyanins in white-skinned varieties or sports correlates with a lack of expression of the UFGT gene, although the expression of some other flavonoid pathway genes was also decreased.