Vacuolar Sequestration of Anthocyanin in Maize

开花植物合成不同的代谢物，其中许多储存在液泡中。许多这样的次生代谢物是有毒的或高度活性的，必须从细胞中隔离出来以防止损害；有趣的是，这些储存的化合物中有许多是有用的药物，因为它们与酶和其他细胞成分有特殊的相互作用。为了了解植物如何管理有毒的代谢物，我们将结合遗传学和生物化学来分析花青素和类黄酮前体化合物的液泡隔离机制。花青素是理想的模型化合物，因为人们对花青素的生物化学和合成的遗传控制已经有了很多了解。这些色素赋予许多花红色/蓝色/紫色；这些色素是一大类化学物质-类黄酮类的成员，广泛分布于绿色植物中。黄酮类化合物是抵御紫外线伤害的天然防晒剂，是传粉者的引诱剂，是植物-微生物相互作用的信号，也是成功的花粉-柱头相互作用所必需的分子。类黄酮也是人类健康饮食的重要组成部分。尽管黄酮类化合物具有良好的特性，但它通过氧化蛋白质和插入DNA来增加突变频率，从而导致细胞损伤；如果黄酮类化合物有效地隔离在液泡室中，这种损害是可以防止的。目前已知，玉米的BZ2蛋白和矮牵牛的AN9蛋白在花青素从细胞质向液泡运输的早期阶段是必需的，缺乏这些蛋白的突变植株在细胞质中积累花青素。在这个项目中，将测试MRP29在液泡膜上移动花青素的作用。目前的模型是BZ2是花青素的载体蛋白；它将这些货物运送到MRP29转运体，MRP29转运体又将货物转移到液泡中。MRP29是一种ATP盒结合蛋白转运体，它受相同的转录因子调控，这些转录因子激活花青素途径的生物合成基因。研究重点将集中在确定MRP29是否定位于液泡的液泡膜上，MRP29突变对花青素固定的影响，MRP29与BZ2和相关蛋白质在体内和体外的相互作用，以及在研究项目期间将建立的体外实验中对花青素固定的要求。缺乏所有ABC转运蛋白的突变酵母细胞将被转化为表达玉米MRP29蛋白；使用从这种酵母菌株制备的空泡，只包含一个植物ABC转运蛋白的液泡膜可以被纯化。这种材料将被用来在更精细的生物化学水平上测试模型。

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.