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A grapevine anthocyanin acyltransferase, transcriptionally regulated by VvMYBA, can produce most acylated anthocyanins present in grape skins

ABSTRACT: Anthocyanins are flavonoid compounds responsible for red/purple colours in the leaves, fruit and flowers of many plant species. They are produced through a multistep pathway which is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are non-functional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification and transport in Shiraz. One up-regulated gene ANTHOCYANIN 3- O-GLUCOSIDE-6”-O-ACYLTRANSFERASE (Vv3AT) encodes a BAHD acyltransferase protein, belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilise both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In V. vinifera cv. Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro.

ORGANISM(S): Vitis vinifera

PROVIDER: GSE56915 | GEO | 2015/10/16

SECONDARY ACCESSION(S): PRJNA244929

REPOSITORIES: GEO

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Project description:Anthocyanins are flavonoid compounds responsible for red/purple colours in the leaves, fruit and flowers of many plant species. They are produced through a multistep pathway which is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are non-functional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification and transport in Shiraz. One up-regulated gene ANTHOCYANIN 3- O-GLUCOSIDE-6â-O-ACYLTRANSFERASE (Vv3AT) encodes a BAHD acyltransferase protein, belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilise both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In V. vinifera cv. Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. Transgenic Chardonnay/Shiraz and non-transformed WT controls were all grown in the same glasshouse in ambient light with a night break. Day and night temperatures were approximately 27oC and 22oC respectively. For microarray experiments, whole berries were sampled from independent transgenic lines: three from transgenic Chardonnay and four from transgenic Shiraz, resulting in three and four biological replicates respectively. Bunches were harvested close to ripeness based on average total soluble solids (TSS, measured as oBrix). This was aimed to be between 20 â 24 oBrix and was determined from TTS of a subsamples from each bunch (Table S53). A sample consisted of all remaining berries from a single bunch except when there were <100 berries in which case more than one bunch was used in the one replicate. Whole berries were immediately frozen in liquid nitrogen. For skin samples, the skins were first removed from fresh berries then frozen in liquid nitrogen. All samples were stored at -80oC.

Project description:Pink-flowered strawberry is a new promising ornamental flower derived from intergeneric hybridization (Fragaria × Potentilla) with bright color, prolonged flowering period and edible fruits. However, the transcriptional events underlying anthocyanins biosynthesis pathway have not been fully characterized in its petal coloration. The pigment compounds accumulated in its fruits were the same as cultivated strawberry, but different from in its flowers. To gain insights into the regulatory networks related to anthocyanin biosynthesis and identify key genes, we performed an integrated analyses of the transcriptome and metabolomes involved in red petals at three development stages (Bud stage (L), Coloration beginning stage (Z) and Big bud stage (D)) of pink-flowered strawberry. Transcript and metabolite profiles were generated through high-throughput RNA-sequencing and high-performance liquid chromatography coupled with mass spectrometry, respectively. The results showed that the main pigments of red and dark pink petals were anthocyanins, among which cyanidins were the main compounds. There were no anthocyanins detected in white-flowered hybrids. A total of 50 285 non-redundant unigenes were obtained from the transcriptome databases, among which 59 differentially expressed genes could be identified as putative homologues of flower coloration related genes. Based on a comprehensive analysis relating pigmentation compounds to gene expression profiles, the mechanism of flower color formation was examined in pink-flowered strawberry. Furthermore, a new hypothesis explaining the lack of color phenotype of the white-flowered strawberry hybrids from the level of the transcriptome. The expression patterns of FpDFR gene and FpANS gene corresponded to the accumulation patterns of cyanidin contents in pink-flowered strawberry hybrids with different shades of pink; Whereas other anthocyanin biosynthesis genes were weakly related flower color deepened. Moreover, FpANS, FpBZ1 and FpUGT75C1 genes were the key factors that lead to the inability to accumulate anthocyanins in the white petals of PFS hybrids. Meanwhile, the competitive effect of FpFLS gene and FpDFR gene may further inhibit anthocyanin synthesis. The data presented herein are important for understanding of the molecular mechanisms underlying the petal pigmentation and will be powerful for integrating into novel genes that are potential targets for breeding new valuable pink-flowered strawberry cultivars.

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A grapevine anthocyanin acyltransferase, transcriptionally regulated by VvMYBA, can produce most acylated anthocyanins present in grape skins

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