Vacuolar Sequestration of Anthocyanin in Maize

开花植物合成多种代谢物，其中许多储存在液泡中。 许多这样的次级代谢产物是有毒的或高度反应性的，必须从细胞中隔离以防止损伤;有趣的是，许多这些储存的化合物是有用的药物，因为它们与酶和其他细胞成分的特定相互作用。 为了了解植物如何管理有毒代谢物，花色素苷色素和前体类黄酮化合物的液泡隔离机制将使用遗传学和生物化学相结合的方法进行分析。 花色素苷是理想的模型化合物，因为已经知道很多关于它们的生物化学和它们的合成的遗传控制。 这些色素赋予许多花红色/蓝色/紫色;色素是广泛分布在绿色植物中的一个大的化学类别黄酮类化合物的成员。 黄酮类化合物是抵御紫外线伤害的天然防晒剂，传粉昆虫的引诱剂，植物-微生物相互作用的信号，以及成功的花粉-柱头相互作用所需的分子。 调味品也是健康人类饮食的重要组成部分。 尽管它们具有良好的特性，但类黄酮通过氧化蛋白质和插入DNA增加突变频率而引起细胞损伤;如果类黄酮被有效地隔离到液泡区室中，则可以防止这种损伤。 目前，已知玉米的BZ 2蛋白和矮牵牛的AN 9蛋白在花青素从细胞质转移到液泡的早期步骤中是必需的;缺乏这些蛋白的突变体植物在细胞质中积累花青素。 在本项目中，将测试MRP 29在移动花色素苷穿过液泡膜中的作用。 目前的模型是，BZ 2是花青素的载体蛋白;它将这种货物运送到MRP 29转运蛋白，MRP 29转运蛋白又将货物转移到液泡中。 MRP 29是一种ATP盒结合蛋白转运蛋白，由激活花青素途径生物合成基因所需的相同转录因子调节。 研究将侧重于确定MRP 29是否定位于液泡的液泡膜，MRP 29突变对花青素螯合的影响，MRP 29与BZ 2和相关蛋白在体内和体外的相互作用，以及在研究项目期间建立的体外测定中花青素螯合的要求。 将转化缺乏所有ABC转运蛋白的突变酵母细胞以表达玉米MRP 29蛋白;使用从这样的酵母菌株制备的液泡，可以纯化仅含有一种植物ABC转运蛋白的液泡。 这种材料将用于在更精细的生化水平上测试模型。

Flowering plants synthesize diverse metabolites, many of which are stored in the vacuoles. Many such secondary metabolites are toxic or highly reactive and must be sequestered from the cell to prevent damage; interestingly, many of these stored compounds are useful drugs, because of their specific interactions with enzymes and other cellular constituents. To understand how plants manage toxic metabolites the mechanism of vacuolar sequestration for anthocyanin pigments and the precursor flavonoid compounds will be analyzed using a combination of genetics and biochemistry. Anthocyanins are ideal model compounds because so much is already known about their biochemistry and the genetic control of their synthesis. These pigments confer the red/blue/purple colors in many flowers; the pigments are members of a large chemical class, the flavonoids, widely distributed in green plants. Flavonoids are natural sunscreens against UV damage, attractants for pollinators, signals in plant-microbe interaction, and molecules required for successful pollen-stigma interaction. Flavonoids are also an important component of a healthy human diet. Despite their good features, flavonoids cause cellular damage by oxidizing proteins and intercalating into DNA increasing mutation frequency; such damage is prevented if the flavonoids are sequestered efficiently into the vacuolar compartment. Presently, it is known that the BZ2 protein of maize and the AN9 protein of Petunia are required at an early step in the transfer of anthocyanin from the cytoplasm to the vacuole; mutant plants lacking these proteins accumulate anthocyanin in the cytoplasm. In this project the role of MRP29 in moving anthocyanin across the tonoplast membrane will be tested. The current model is that BZ2 is a carrier protein for anthocyanin; it delivers this cargo to the MRP29 transporter, which in turn transfers the cargo into the vacuole. MRP29, an ATP Cassette Binding Protein transporter, is regulated by the same transcription factors that are required to activate the biosynthetic genes of the anthocyanin pathway. Research will focus on establishing whether MRP29 is localized to the tonoplast membrane of the vacuole, on the impact of mutation at MRP29 on anthocyanin sequestration, on the interaction of MRP29 with BZ2 and related proteins in vivo and in vitro, and on the requirements for anthocyanin sequestration in an in vitro assay that will be established during the research project. Mutant yeast cells, lacking all ABC transporters, will be transformed to express the maize MRP29 protein; using vacuoles prepared from such yeast strains, tonoplasts containing just one plant ABC transporter can be purified. This material will be used to test the model at a more refined biochemical level.