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

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. Overall design: 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 – 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.

花青素（Anthocyanins）是一类类黄酮化合物，可赋予多种植物的叶片、果实与花朵红/紫色色泽。其生物合成经由多步通路完成，该通路受MYB转录因子（MYB transcription factors）调控。VvMYBA1与VvMYBA2可激活葡萄（Vitis vinifera）体内的花青素生物合成，而在白葡萄品种中这两个基因丧失功能。

本研究构建了VvMYBA基因表达水平改变的转基因葡萄，并采用微阵列（microarray）技术对浆果组织进行转录组分析。结果显示，在西拉（Shiraz）葡萄中，VvMYBA是花青素合成、修饰与运输后期阶段的正向调控因子。

上调表达基因ANTHOCYANIN 3-O-葡萄糖苷-6''-O-酰基转移酶（Vv3AT）编码一种BAHD酰基转移酶蛋白（BAHD acyltransferase protein），其所属进化枝与多数花青素酰基转移酶不同。功能研究（植株体内与体外实验）表明，Vv3AT具有宽泛的花青素底物特异性，同时可利用脂肪族与芳香族酰基供体——这是该酶家族在自然界中被发现的全新活性。

在缺乏酰基化花青素的红浆果葡萄品种黑比诺（Pinot Noir）突变体中，Vv3AT携带无义突变，编码的截短蛋白缺失了BAHD蛋白活性所需的两个结构基序。启动子激活实验证实，VvMYBA1可激活Vv3AT的转录，这一发现加深了学界对BAHD基因家族调控机制的认知。Vv3AT可利用两类酰基供体的特性，在植株体内或体外的花青素工程改造中具有应用价值。

总体实验设计：转基因霞多丽（Chardonnay）、西拉葡萄与未转化的野生型对照均种植于同一温室，在自然光条件下辅以夜间中断处理。昼夜温度分别约为27℃与22℃。为开展微阵列实验，从独立转基因株系中采集整果样本：转基因霞多丽设3个生物学重复，转基因西拉设4个生物学重复。根据平均可溶性固形物总量（TSS，以白利度°Brix为单位）判断，在接近成熟时采收果串。目标TSS范围为20~24°Brix，该数值通过每串果的子样本TSS测定确定（详见补充表S53）。单个样本由单串果的全部剩余浆果组成，若单串浆果数量少于100粒，则采用多串果合并为一个重复样本。整果样本立即置于液氮（liquid nitrogen）中速冻。对于果皮样本，先从新鲜浆果上剥离果皮，再置于液氮中速冻。所有样本均保存于-80℃环境中。