UFGT Research Articles

매향 딸기로부터 anthocyanin 합성 유전자의 분리 및 과실발달 과정에서의 발현 분석

매향' 딸기의 안토시아닌 생합성은 개화 후 26일째 시작되어 과실의 성숙기 동안 계속된다. 딸기로부터 안토시아닌의 생합성에 관여하는 주요 유전자를 분리하였다. 각각의 유전자에 대해, 다양한 식물체의 유사 유전자의 염기서열을 비교하여 PCR (polymerase chain reaciton) primer를 제작하였다. 숙기의 딸기에서 분리된 total RNA로부터 합성된 CDNA와 각 primer를 이용하여 RT (reverse transcriptase)-PCR을 수행하였다. 각 CDNA clone의 염기서열을 작성하여 분석한 결과, 이들은 안토시아닌 생합성에 관여하는 phenylalanine ammonia lyase (PAL), 4-cummarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidine synthase (ANS) 그리고 UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT) 효소에 해당되었다. Northern blot 분석 결과, 이들 유전자는 과실 발달과정에서 시기적으로 조절되었다. 특히 PAL을 제외한 모든 유전자는 과실에서만 주로 발현되었다. PAL, DFR 그리고 ANS유전자는 과실 초기 발달 단계인 개화 후 10일에 검출된 후 감소하다가, 22일에 다시 증가하기 시작하여 34일에 최대가 되었다. 한편, 다른 유전자들은 초기에는 발현되지 않다가, 안토시아닌이 축적되기 시작하는 개화 후 <TEX>$22{\sim}30$</TEX>일에 처음으로 검출되었다. 본 연구를 통해, 딸기 과실 발달과정에서 안토시아닌 생합성 과정에 관여하는 여러 유전자가 과실 숙기에 함께 조절되는 현상을 알 수 있다. 이러한 연구 결과는 안토시아닌 합성과정을 제어하는 조절 유전자가 존재한다는 것을 시사한다. 그리고 딸기의 안토시아닌 생합성 유전자의 발현패턴을 크게 두 가지로 나눌 수 있는 것으로 보아, 딸기의 안토시아닌 생합성에는 적어도 두 가지 서로 다른 조절 기작이 관여하여 색소 발달 과정을 제어할 것으로 보인다. Anthocyanin synthesis in strawberry (Fragaria x ananassa cv Maehyang) begins approximately 26 days postflowering and continued throughout fruit ripening. A set of cDNA clones encoding the anthocyanin biosynthetic enzymes were isolated from strawberry. A pair of primers were designed for polymerase chain reaction (PCR) through the comparison of the nucleotide sequences of homologous genes from diverse plants. Reverse transcriptase-PCRs were performed using cDNA synthesized from ripe fruit total RNA and the primers corresponding to each gene. Eight genes of the anthocyanin pathway were cloned and confirmed by sequencing to code for phenylalanine ammonia lyase (PAL), 4-cummarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidine synthase (ANS), UDP-glucose:flavonoid-3-O-glucosyl-transferase (UFGT). Northern analyses showed that the corresponding genes were differentially expressed during the fruit development process. All genes except PAL were predominantly expressed in fruit. Expression of PAL, DFR and ANS was detected 10 days postflowering at the early stage of fruit development, declined for a while and sharply increased 22 days postflowering then showed a peak 34 days postflowering. The other genes, however, were not expressed up to 22 or 30 days postflowering when the initial fruit ripening events occur at the time of initiation of anthocyanin accumulation. The onset of anthocyanin synthesis in ripening strawberry coincides with a coordinated induction of the anthocyanin pathway genes, suggesting the involvement of regulatory genes. We propose that at least two different regulatory mechanisms playa role in the biosynthesis of anthocyanin during color development of strawberry.

Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit

Anthocyanin biosynthesis is strongly up-regulated in ripening fruit of grapevines (Vitis vinifera L.) grown under drought conditions. We investigated the effects of long-term water deficit on the expression of genes coding for flavonoid and anthocyanin biosynthetic enzymes and related transcription factors, genes sensitive to endogenous [sugars, abscisic acid (ABA)] and environmental (light) stimuli connected to drought stress, and genes developmentally regulated in ripening berries. Total anthocyanin content has increased at harvest in water-stressed (WS) fruits by 37-57% in two consecutive years. At least 84% of the total variation in anthocyanin content was explained by the linear relationship between the integral of mRNA accumulation of the specific anthocyanin biosynthetic gene UDP-glucose : flavonoid 3-O-glucosyltransferase (UFGT) and metabolite content during time series from véraison through ripening. Chalcone synthase (CHS2, CHS3) and flavanone 3-hydroxylase (F3H) genes of the flavonoid pathway showed high correlation as well. Genes coding for flavonoid 3',5'-hydroxylase (F3'5'H) and O-methyltransferase (OMT) were also up-regulated in berries from dehydrated plants in which anthocyanin composition enriched in more hydroxylated and more methoxylated derivatives such as malvidin and peonidin, the grape anthocyanins to which human gastric bilitranslocase displays the highest affinity. The induction in WS plants of structural and regulatory genes of the flavonoid pathway and of genes that trigger brassinosteroid hormonal onset of maturation suggested that the interrelationships between developmental and environmental signalling pathways were magnified by water deficit which actively promoted fruit maturation and, in this context, anthocyanin biosynthesis.

Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines

BackgroundFruit coloration of red-skinned grapevines is mainly due to anthocyanin pigments. We analysed a panel of nine cultivars that included extreme phenotypes for berry colour, ranging from green (absence of anthocyanins) to red, purple, violet and blue. Expression of six genes of the anthocyanin pathway coding for flavanone-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT), glutathione-S-transferase (GST), O-methyltransferase (OMT) and four transcription factors (MybA, MybB, MybC, MybD) was analysed by quantitative RT-PCR at four developmental stages from before the onset of ripening until full maturity and compared to anthocyanin metabolites.ResultsTotal anthocyanin content at full maturity correlated well with the cumulative expression of F3H, UFGT and GST throughout ripening. Transcripts of the last two genes were absent in the green-skinned cultivar 'Sauvignonasse', also known as 'Tocai friulano', and were at least 10-fold less abundant in pale red cultivars, such as 'Pinot gris' and 'Gewürztraminer', compared to fully coloured cultivars. Predominance of tri-hydroxylated anthocyanins (delphinidin, petunidin and malvidin) in cultivars bearing dark berries with violet and blue hue was associated with higher ratios of F3'5'H/F3'H transcription, compared to red-skinned cultivars. Higher levels of OMT transcripts were observed in berries of cultivars that accumulated methoxylated forms of anthocyanins more abundantly than non-methoxylated forms.ConclusionColour variation of the grape berry conforms to a peculiar pattern of genotype-specific expression of the whole set of anthocyanin genes in a direct transcript-metabolite-phenotype relationship. Cumulative mRNA levels of the structural genes and their relative abundance throughout ripening explained per se the final phenotype for anthocyanin content, anthocyanin composition, colour intensity and colour hue of grapes at berry maturity.

Functional characterization of Nicotiana benthamiana homologs of peanut water deficit-induced genes by virus-induced gene silencing

Determining the functional role of genes that are differentially regulated during a stress response is challenging. In this study, few water deficit-induced genes from peanut were characterized in Nicotiana benthamiana using virus-induced gene silencing (VIGS) and their relevance for stress adaptation was validated. Twenty-five cDNA clones from peanut water deficit stress-induced cDNA library that had more than 50% nucleotide similarity with N. benthamiana or tomato homologs were selected. VIGS in peanut is not yet feasible and therefore we characterized these 25 genes in N. benthamiana. Increased membrane damage was seen under water deficit stress in most of the silenced plants signifying that many of these stress-induced genes are important to confer drought tolerance. Among the genes tested, silencing by homolog of flavonol 3-O-glucosyltransferase (F3OGT), homolog of alcohol dehydrogenase, homologous to salt inducible protein, and homolog of heat shock protein 70 showed more visible wilting symptoms compared with the control plants during water deficit stress. Interestingly, down-regulation of two genes, homologous to aspartic proteinase 2, and homolog of Jumonji class of transcription factor showed relative drought tolerant phenotypes. F3OGT silenced plants showed more wilting symptoms, membrane damage and chlorophyll degradation than any other silenced plants during water deficit. Our results demonstrate that VIGS approach can be used to characterize and assess the functional relevance of water deficit stress-induced cDNAs in a heterologous species.

Quantitative Real-time Reverse Transcriptase-PCR Profiling of Anthocyanin Biosynthetic Genes during Orange Fruit Ripening

Genes encoding chalcone synthase (CHS), anthocyanidin synthase (ANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT), some of the enzymes of anthocyanin biosynthetic pathway, were assayed in two different experiments using quantitative real-time reverse transcriptase (RT)-PCR, in order to test their transcription levels in the flesh of blood and common orange [Citrus sinensis (L.) Osbeck] fruit, and to investigate their role in anthocyanin accumulation in the same tissue. The first experiment compared a blood orange and a common orange cultivar during seven different fruit maturation stages. This was followed by the test of 11 different genotypes at the end of the winter season. Data collected from the first experiment, over the blood orange cultivar, were statistically analyzed using the Pearson correlation coefficient. Results show that CHS, ANS, and UFGT mRNA transcripts are up- and co-regulated in the blood orange cultivar, whereas they are down-regulated in the common orange cultivar. There is evidence of correspondence between the target genes expression level and the content of the pigment assessed. The second test confirms this correlation and shows that enzyme synthesis levels and pigment accumulation, in plants grown under the same environmental conditions, are dependent on the differences occurring among the genotypes tested. These results suggest that the absence of pigment in the common orange cultivars may be caused by the lack of induction on the structural genes expression. This is the first report on the characterization of the relationships between biosynthetic genes expression and fruit flesh anthocyanin content in blood oranges.

Reciprocal regulation ofArabidopsis UGT78D2 and BANYULS is critical for regulation of the metabolic flux of anthocyanidins to condensed tannins in developing seed coats

Anthocyanins are major color pigments in plants. Their biosynthetic pathways are well established, and the majority of these biosynthetic enzymes have been identified in model plants such asArabidopsis, maize, and petunia. One exception inArabidopsis is UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT). This enzyme is known as Bronze-1 (Bz1 ) in maize, where it converts anthocyanidins to anthocyanins. Phylogenetic sequence analysis of theArabidopsis thaliana UDP-glycosyltransferase (UGT) family previously indicated that UGT78D1, UGT78D2, and UGT78D3 cluster together with UF3GTs from other species. Here, we report thatUGT78D2 encodes a cytosolic UGT that is functionally consistent with maize Bz-1. Biochemically, UGT78D2 catalyzes the glucosylation of both flavonols and anthocyanidins at the 3-OH position. A T-DNA-insertedugt78d2 mutant accumulates very little anthocyanin and lacks 3-O-glucosylated quercetin. Expression analysis indicated thatUGT78D2, in opposite toBANYULS, is highly expressed in anthocyanin-accumulating seedlings but repressed in condensed tannin-accumulating seed coats. This suggests that the reciprocal regulation of these two genes is important in directing the metabolic flux to either anthocyanins or condensed tannins. Consistent with this, the ectopic expression of UGT78D2 produces purple-colored seed coats due to the accumulation of anthocyanins. Taken together, our data indicate thatUGT78D2 encodes an enzyme equivalent to maize Bz1, and that the reciprocal regulation of UGT78D2 and BANYULS is critical for the regulation of metabolic flux of anthocyanidins inArabidopsis.

Expression analysis of anthocyanin biosynthetic genes in apple skin: Effect of UV-B and temperature

We investigated the expression of the five anthocyanin biosynthetic genes, chalcone synthase ( CHS), flavanone 3-hydroxylase ( F3H), dihydroflavonol 4-reductase ( DFR), anthocyanidin synthase ( ANS), and UDP-glucose: flavonoid 3-O-glucosyltransferase ( UFGluT) during ripening in five early ripening apple cultivars (‘Iwai’, ‘Sansa’, ‘Tsugaru’, ‘Homei-Tsugaru’, and ‘Akane’), as well as under UV-B and temperature treatments. Anthocyanin biosynthesis increased progressively during ripening, with the onset of rapid accumulation occurring at 107 days after full bloom (DAFB) in the four apple cultivars except ‘Iwai’. Generally, increase in the expression levels of all five biosynthetic genes in fruit skin coincided with increase of anthocyanin concentration. When the fruit was bagged about 1 month prior to commercial harvest to prevent anthocyanin biosynthesis, the expression of CHS, ANS and UFGluT was substantially depressed in fruit skin, whereas that of all five genes including CHS, ANS and UFGluT was enhanced by UV-B and low temperature treatments with the accumulation of anthocyanins. The accumulation level of the main anthocyanin pigment, cyanidin 3-galactoside, in the fruits under UV-B and low temperature treatment, was less than that in the field-ripened fruits, while cyanidin 3-arabinoside was relatively the same level. Our results showed that UV-B and low temperature were important factors for anthocyanin accumulation in apple fruit skin by inducing the expression of the anthocyanin biosynthetic genes, especially CHS, ANS and UFGluT genes.

Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition

In grape cultivation, high night temperature generally reduces anthocyanin accumulation in berry skin. To clarify the regulatory mechanism of anthocyanin biosynthesis under the conditions of high night temperatures, we examined flavonoid accumulation, the activities of related enzymes and gene expression in the anthocyanin biosynthetic pathway in grape berries grown under cool and warm night conditions. Anthocyanin accumulation in the skin of berries grown at high night temperatures (30 °C continuous) was reduced as compared to that of berries grown at low night temperatures (30 °C in daytime/15 °C in night time). On the other hand, flavonol levels in the skin of berries were not significantly different between low and high night temperature conditions. High night temperatures also inhibited the gene expression of chalcone synthase ( CHS), flavanone 3-hydroxylase ( F3H), dihydroflavonol 4-reductase ( DFR), leucoanthocyanidin dioxygenase ( LDOX) and UDP-glucose: flavonoid 3-O-glucosyltransferase ( UFGT) at veraison and an increase in UFGT activity after veraison. These results suggest that the inhibition of anthocyanin biosynthesis in grape berries grown under high night temperature condition could be caused by lower expression levels of anthocyanin biosynthetic genes at an early stage of ripening and lower activities of anthocyanin biosynthetic enzymes, particularly UFGT.

Regulatory Mechanism of Anthocyanin Biosynthesis in 'Kyoho' Grape Berries Grown under Different Temperature Conditions

To clarify the mechanism regulating anthocyanin biosynthesis in ‘Kyoho’ grape berries grown under high temperature, we examined flavonoids accumulation and the activities of related enzymes as well as photoassimilates translocation on the berries. Anthocyanin content in the skin of berries of grape was lowered at 30 days after veraison when grown under high temperature condition (at consistently 30°C) as compared with berries at consistently 25°C or 30°C in the day and 15°C in the night concomitantly. Phenylalanine concentration in the skin and the photoassimilates partitioning to berry were also lower in those grown at 30°C than vines at 25°C or 30/15°C. While, phenylalanine ammonia lyase (PAL) activity in berries grown at 25°C was higher than that in berries grown at 30°C or 30/15°C. Berries grown at 30/15°C had the highest UDP-glucose : flavonoid 3-O-glucosyltransferase (UFGT) activity among three temperature conditions 30 days after veraison. These results suggest that grape berries grown at successive high temperature decrease anthocyanin accumulation in the skin by virtue of the decrease of the UFGT activities and the concentration of phenylalanine and photoassimilates partitioning to berry. On one hand, the grape berry grown under cool night temperature condition would result in high level of anthocyanin accumulation due possibly to great induction of UFGT activity and high phenylalanine accumulation.

Exogenous ethylene stimulates the long‐term expression of genes related to anthocyanin biosynthesis in grape berries

The treatment of grape berries (Vitis vinifera L. cv. Cabernet Sauvignon) with the ethylene‐releasing compound, 2‐chloroethylphosphonic acid (2‐CEPA), at veraison is a method known to enhance grape skin colour. We observed that it produced a 6‐fold increase, up to 30 pmol g−1 FW, of the cluster internal ethylene compared to untreated controls within the 24 h following treatment. This ethylene upsurge was associated with increased levels of chalcone synthase (CHS) and flavanone 3‐hydroxylase (F3H) transcripts, which persisted over the following 20 days. Transcript levels of leucoanthocyanidin dioxygenase (LDOX) and UDP glucose‐flavonoid 3‐O‐glucosyl transferase (UFGT) were similarly enhanced by 2‐CEPA, although to a lesser extent. The effect on UFGT was confirmed at the protein level by an immunoblot analysis. The transcript accumulation of dihydroflavonol 4‐reductase (DFR) was unaffected by 2‐CEPA treatment. Examination of the levels of CHS, F3H and UFGT mRNAs in berries during bunch exposure to ethylene, revealed elevated levels of each transcript within the first 6 h of treatment when compared to nonethylene‐treated controls. HPLC analyses of berry skin extracts showed that levels of each of the anthocyanins analysed (delphinidin, cyanidin, petunidin, peonidin and malvidin) increased over the 10 days following the ethylene burst, and decreased thereafter. However, anthocyanin levels at harvest were still higher in ethylene treated grapes than in controls. This data is the first evidence that ethylene triggers gene expression related to anthocyanin synthesis in grapes, and in addition, our results also confirm the existence of other regulatory modes in the anthocyanin biosynthetic pathway.

Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skin

Anthocyanin is the major color pigment in plants. In order to understand anthocyanin biosynthesis mechanism in apple, the cDNAs encoding flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3- O-glucosyltransferase (UFGT) were isolated from cDNA libraries prepared from the skin tissues of apple ( Malus domestica Borkh. cv. Fuji). Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Each gene is a member of a multigene family. The mRNAs of anthocyanin biosynthetic genes were detected preferentially in the skin tissue and expression of the genes was coordinately induced by light. The transcripts were detected abundantly in the skins of cultivars with red skin, but rarely in that of a cultivar carrying non-red fruit, suggesting that these genes have major roles in determination of apple skin color.

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.

Abscisic acid and 2,4-dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries

SummaryThe effects of abscisic acid (ABA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the expression of seven anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries were investigated. In untreated berries, the expression of the UDP-glucose-flavonoid: 3-O-glucosyltransferase (UFGT) gene was detected only at 42 d after full bloom (DAB), whereas the phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) genes were expressed throughout the growing period. ABA increased anthocyanin content in the skin and the expression of PAL, CHS, CHI, DFR and UFGT genes at 7 d after treatment. In contrast, 2,4-D inhibited the accumulation of anthocyanin and the expression of all the genes examined. The results clearly show that the anthocyanin levels resulting from the application of ABA and 2,4-D were correlated with the expression of anthocyanin biosynthetic pathway genes.

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.

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis.

A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53,094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4'-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments.

Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings.

Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.

Expression of an Antirrhinum majus UDP-glucose:flavonoid-3- O-glucosyltransferase transgene alters flavonoid glycosylation and acylation in lisianthus ( Eustoma grandiflorum Grise.)

A binary vector containing an Antirrhinum majus UDP-glucose:flavonoid-3- O-glucosyltransferase (UFGT) cDNA under the control of the cauliflower mosaic virus 35S promoter was used to transform lisianthus ( Eustoma grandiflorum Grise.). Of four independent transgenic lines recovered, one produced high levels of the UFGT transcript and synthesized 3- O-glucosylated anthocyanins novel to lisianthus, as well as enhanced levels of 3- O-glucosylated flavonols. The novel 3- O-glucosylated anthocyanins accounted for approximately 30% of the total anthocyanins in the petals. The level of 3- O-glucosylated flavonols increased by approximately 4.8 x, representing a change from the production of 3- O-galactosylated flavonols to 3- O-glucosylated flavonols of approximately 32%. In addition, the increase in glucosylation of the flavonols resulted in a corresponding decrease in acylation. Modifications at other positions of the flavonoid nuclei were unaffected. The results indicate that in lisianthus there is a high specificity shown by some flavonoid modifying enzymes, such as that for flavonol acylation, while other enzymes, such as those involved in modifications at the C-5 of the anthocyanin, are more flexible in their choice of substrate. The results also provide information on the role of flavonoid glycosylation and acylation in copigmentation in lisianthus.

Expression of anthocyanin biosynthesis pathway genes in red and white grapes.

The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.