Tonoplast Protein Trafficking in Arabidopsis

本研究的长期目标是确定和表征植物液泡膜蛋白运输的机制。液泡膜蛋白运输的化学抑制剂和液泡膜蛋白异常定位的突变体先前被鉴定。生物活性探针表明，多个途径，包括一个新的高尔基体独立的途径，目标液泡膜蛋白。 这个项目将确定膜蛋白靶向液泡的机制，以及这些蛋白如何有助于液泡的生物发生。这项工作计划将检验高尔基体独立途径参与液泡膜蛋白运输的假设，并将研究磷酸肌醇脂质在植物液泡生物发生中的新作用。本研究的具体目的是：1）通过对两个液泡膜蛋白定位缺陷突变体的鉴定，确定ITT4和ITT6基因在液泡膜蛋白靶向中的作用。2)确定磷脂酰肌醇脂类在液泡生物发生中的作用。3)使用活细胞的实时显微镜来表征高尔基体非依赖性途径用于液泡膜蛋白靶向的动力学。鉴于液泡膜蛋白在调节细胞膨压和液泡的储存潜力中的重要作用，这些结果可能对植物环境胁迫反应的生物学及其在农业中的应用产生未来的影响。更广泛的影响在这个项目中使用的化学和遗传筛选将有利于产生新的化学物质和突变体，将提供给科学界的领域。该项目中的一些实验适合高中和本科生的研究活动，并将促进对学生进行分子生物学和荧光高分辨率活细胞成像方面最先进技术的培训。PI将利用这些活动继续吸引北卡罗来纳州科学与数学学院的少数族裔高中生，目标是提高少数族裔学生在大学内攻读科学学位的兴趣。PI的实验室还将接待River Oaks中学学生的访问，以激发这些学生对科学相关领域的兴趣，并向公众传达科学研究的影响。

INTELLECTUAL MERITThe long-term goal of this research is to identify and characterize the mechanisms for trafficking of plant vacuole-tonoplast membrane proteins. Chemical inhibitors of tonoplast protein trafficking and mutants with abnormal localization of a tonoplast protein were previously identified. The bioactive probes indicated that multiple pathways, including a novel Golgi-independent pathway, target vacuole-membrane proteins. This project will identify the mechanisms by which membrane proteins are targeted to the vacuole, and how these contribute to its biogenesis. The work plan will test the hypothesis that a Golgi-independent pathway engages in tonoplast protein trafficking, and will investigate a novel role for phosphoinositide lipids in plant vacuole biogenesis. The specific aims for this research are: 1) To identify the roles of IMPAIRED TONOPLAST TRAFFICKING4 (ITT4) and ITT6 genes in tonoplast-protein targeting by characterization of two mutants with defects in tonoplast protein localization. 2) To determine the role of phosphoinositide lipids in vacuole biogenesis. 3) To characterize the dynamics of the Golgi-independent pathway for tonoplast-protein targeting using real-time microscopy of live cells. Given the essential roles of vacuole membrane proteins in regulating cell turgor, and the storage potential of the vacuole, these results may have future impacts on the biology of environmental stress responses in plants and its applications to agriculture. BROADER IMPACTSChemical and genetic screens employed in this project will benefit the field by generating novel chemicals and mutants that will be available to the scientific community. Some of the experiments in this project are amenable for high school and undergraduate research activities, and will promote the training of students in state-of-the-art techniques in molecular biology and fluorescent high resolution live-cell imaging. The PI will use these activities to continue to engage minority high school students from the NC School of Science and Mathematics with the goal of increasing the interest of minority students in pursuing science degrees within the University. The PI's laboratory will also host visits from River Oaks Middle School students to spark the interest of these students towards science-related fields, and to convey the impacts of scientific research to the public.