植物光合细胞耐干燥机制中4种蛋白质在膜保护系统中的作用

Dehydration tolerance is an adaptive trait necessary for the colonization of land by plants, and remains widespread among bryophytes. We have recently shown that moss Physcomitrella patens, a model for functional genomic studies at the cellular level, is highly dehydration tolerant. Its vegetative organs (e.g leafy-shoots) survive approximately 80% water loss, which is uncommon in higher vascular plants, except among resurrection plants. Further, we verify that vegetative desiccation tolerance can be induced by the phytohormone abscisic acid (ABA) in Physcomitrella patens. Using a comprehensive proeomic analysis via DIGE, we identified 594 and 352 differentially-expressed protein spots induced by drought and ABA treatment, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defense, protein synthesis and quality control, and energy production. Of these, arabinogalactan protein, phospholipase D, phosphatidylethanolamine binding protein and pore-forming toxin are the likely dominant players in the defense system. Following the analysis of sequence and conserve domain, and current data, the four proteins are known to be associated with membrane components and membrane stabilization. However, the functional details of these proteins in membrane protection are largely limited. Our long-term goal is to elucidate the biological functions of proteins in response to drought tolerance in plant vegetative tissues. The current proposal is to clarify functional roles of the four membrane-associated proteins in membrane protection mechanism, a triggering response by ABA. The specific objectives of the proposal are to: (1) confirm subcellular localization of the selected proteins; (2) characterize functional details of these proteins in membrane protection systems upon drought and ABA treatment; (3) analyze protein-protein relationships; (4) determine the fluid-gel phase transition temperature of Physcomitrella membranes. We will generate mutants using a targeted gene disruption strategy, and then characterize the phenotype of corresponding Physcomitrella mutants; identify differentially-expressed proteins via comparative proteomics method; analyze the time course of expression of the four membrane-associated proteins in both RNA and protein levels; and probe plasma membrane phase behavior by differential scanning calorimetry or fourier transform infrared spectroscopy, in the presence of these membrane-associated proteins. Accomplishment of the proposed objectives will contribute to our knowledge of these protein functions in membrane mechanism that prevent dehydration damage. Importantly, the membrane-associated proteins, the protective expressive feature induced by exogenous ABA can be visualized and highlighted in the overall mechanism of dehydration tolerance in plant vegetative tissues. Therefore, this project will provide insights into the role of membrane-associated proteins in plant cells.

帮助绿色植物细胞耐受干旱的基因/蛋白具较高同源性，维管植物表达"抗旱基因"能力弱（除少数复苏植物），而登陆较早的苔藓植物表达"抗旱基因"能力强。我们前期研究表明，模式植物小立碗藓光合细胞组成性表达抗旱基因；感受脱水后，剧烈增加抗旱基因表达量，以此获得巨大抗旱能力（耐受80%水分丧失）；而植物激素ABA可将其抗旱能力增到"抗干燥"水平。荧光差异蛋白组学发现一条重要线索指向4种膜相关蛋白- - 阿拉伯半乳聚糖蛋白、磷酯酶D、磷脂酰乙醇胺结合蛋白和成孔蛋白可能涉及细胞脱水时的膜系统保护。推测：4种蛋白相互或共同作用增加细胞膜"塑性"，弱化细胞膜脱水时的变形或解构，使细胞在干旱、甚至"干燥"时，最大程度保全膜完整性。膜完整性是细胞耐受脱水的关键机制。本项目拟用荧光定位、靶向敲除、比较蛋白组学、膜结构和相变分析阐明这4种蛋白功能及相互关系。揭示植物光合细胞强大膜保护系统中蛋白质分子的贡献和作用机制。

模式植物小立碗藓绿色光合细胞在登陆演化过程中获得巨大的抗脱水力（耐受80%水分丧失）。本项目系统研究了4种膜相关蛋白，即阿拉伯半乳聚糖蛋白（AGP）、磷酯酶D（PLD）、磷脂酰乙醇胺结合蛋白（PEBP）和成孔蛋白（PFT）在小立碗藓光合细胞耐脱水保护机制中的作用。主要研究内容有：1）4种蛋白质与细胞膜的关系；2）4种蛋白质在干旱和ABA处理后的时间表达谱；3）制备基因敲除突变体或过表达突变体，进行差异蛋白组分析；4）显微观察细胞壁和膜结构变化；研究细胞膜相变行为和检测膜渗透性。已获得的重要结果有：1）在小立碗藓基因组中进行基因家族分析，并基于植物分子演化关系，重新命名这4种蛋白为FLA_D2，PLDα1，PEBP-like2和BP2；2）将蛋白编码序列与GFP融合，构建瞬时表达载体。在烟草表皮细胞、水稻和小立碗藓的原生质体中观察蛋白质分布定位。已确定PLDα1和BP2主要定位于细胞膜；PEBP1亦有膜定位迹象；但FLA可能是外泌蛋白; 3）在脱水胁迫或ABA诱导下，PLDα1和BP2可在5分钟内强烈上调，表明这2个基因功能可能定位于细胞脱水耐受分子网络的信息上游；4）通过同源重组策略，获得全部4个蛋白质的缺失突变体。在正常培养条件下，这4个蛋白缺失突变体的生长速率有不同程度降低。在快速脱水条件下，突变体死亡率上升，表明突变体细胞的脱水耐受力下降；5）差异蛋白组分析发现LEA蛋白是小立碗藓的绿色光合细胞耐脱水性的重要指示蛋白；但是在缺失PLDα1蛋白的突变体中，非LEA蛋白的分子保护机制已初露端倪。6）与其它突变体相比，缺失FLA_D2蛋白的植物在脱水胁迫时最早死亡，表明该基因的关键功能在于细胞膜保护；7）超微结构研究发现，小立碗藓光合细胞在ABA处理前后的超微结构已改变，细胞壁显现扰动效应，且与质膜高度缠绕。当前研究表明，小立碗藓绿色光合细胞存在一个应答水分快速变化的分子系统。除PEBP-like2外，另3个蛋白质应处于细胞脱水耐受网络的信息上游。这个苔藓脱水快速响应系统的揭示有望成为研究细胞脱水耐受机制演化的又一个基石。