Functional characterization of a putative Arabidopsis thaliana phosphatidylinositol transfer protein which increases Al tolerance in yeast

铝毒是世界范围内农作物生产的主要限制因素。这项拟议的研究建立在拟南芥cDNA AtAlr54的发现基础上，AtAlr54编码一种与酵母Sec14p磷脂酰肌醇转移蛋白(PITP)同源的蛋白质，其表达导致酵母耐铝能力增强。因为AtAlr54与酵母Sec14p有同源性，并补充了酵母sec14-1TS突变体的生长缺陷，所以AtAlr54本身很可能是一个PITP。随后对肌醇磷脂代谢缺陷的酵母突变株进行的遗传实验表明，AtAlr54介导的耐铝性增强可能是通过调节肌醇磷脂的数量或分布而起作用的。这项拟议的研究致力于AtAlr54的生化特征，作为研究PITPs在铝耐受性中作用的第一步。这一特征将包括对AtAlr54脂质结合和转运特性的全面生化分析，旨在测量AtAlr54如何影响体内磷脂酰肌醇和可溶性肌醇磷酸代谢的代谢实验，确定哪些脂质信号通路参与AtAlr54介导的铝耐受性的遗传和突变实验，以及监测磷脂酰肌醇在金属胁迫下的分布的成像实验。这项研究将使用酵母作为一个系统，以了解AtAlr54是如何发挥作用的，并将指导针对AtAlr54表达改变而设计的植物的功能分析。

Aluminum (Al) toxicity is a major limiting factor for crop production worldwide. The proposed research builds on the discovery of an Arabidopsis cDNA, AtAlr54, which encodes a protein homologous to the yeast Sec14p phosphatidyl inositol transfer protein (PITP) and whose expression causes increased Al tolerance in yeast. Because AtAlr54 shares homology with yeast Sec14p and complements the growth defect of a yeast sec14-1ts mutant, AtAlr54 is itself likely a PITP. Subsequent genetic experiments with yeast mutants defective in the metabolism of phosphoinositides suggested that AtAlr54-mediated increased Al tolerance might act by regulating amount or distribution of phosphoinositides. The proposed research addresses the biochemical characterization of AtAlr54 as a first step towards investigating the function of PITPs in Al tolerance. This characterization will include comprehensive biochemical analyses of AtAlr54 lipid binding and transfer properties, metabolic experiments designed to measure how AtAlr54 affects phosphoinositide and soluble inositol phosphate metabolism in vivo, genetic and mutagenesis experiments to determine what lipid signaling pathways are involved in AtAlr54-mediated Al tolerance, and imaging experiments to monitor phosphoinositide distribution in response to metal stress. This research will employ yeast as a system to get a picture of how AtAlr54 functions, and will guide functional analyses in plants engineered for altered AtAlr54 expression.