RUI: Collaborative Research: SAC9, A Novel, Plant-Specific Phosphoinositide Essential for Membrane/Cytoskeleton Dynamics in Arabidopsis

了解植物细胞脂质信号对于了解植物生理的许多方面的调节至关重要。磷酸肌苷（phosphoinosidtides, pi）在植物对逆境的响应、维管发育、保护细胞动力学以及细胞的生长和形态中起着至关重要的作用。植物已经进化出不同于动物和酵母的新的PI信号传导机制和新的PI修饰途径。本项目的研究将对PI在植物细胞中的功能和新的工具和方法产生新的见解，这将大大加强植物PI信号的研究，并最终导致开发具有更强的非生物和生物胁迫耐受性的工程植物的新方法。主要通过与蛋白质的相互作用，pi调节包括囊泡运输和膜/细胞骨架相互作用在内的功能。PI的积累部分受PI磷酸酶（包括sac结构域蛋白）的作用调节。SAC9结构域蛋白在真核生物中是保守的，但一个新的独特家族成员SAC9仅在植物和绿藻中发现。植物和藻类SAC9蛋白与其他SAC9结构域蛋白（包括植物中发现的其他SAC9结构域蛋白）有许多保守的差异。Williams博士的研究小组的初步研究表明，SAC9对植物中PtdIns(4,5)P2水平和肌动蛋白细胞骨架分布的调节都很重要。进一步表征新的SAC9蛋白和SAC9突变植物的表型，将为真核生物中SAC9结构域蛋白的研究提供信息，并有助于理解pi、肌动蛋白细胞骨架和植物细胞功能之间的相互作用。本项目将(1)通过体外和体内表达表征AtSAC9的活性和细胞定位；(2)研究了植物特异性c端结构域和sac9特异性WW结构域在AtSAC9功能和细胞分布中的作用；(3)通过瞬时和诱导表达研究来检测SAC9在体内的功能；(4)通过荧光蛋白和免疫定位检测SAC9、PtdIns(4,5)P2和肌动蛋白丝的共定位，验证我们的SAC9功能模型。更广泛的影响两位主要研究人员都有丰富的经验，并成功地与本科生进行研究，他们相信，在课堂上和研究实验室为本科生提供令人兴奋的研究机会，有助于将他们带入科学管道。Drs。威廉姆斯和德瓦尔德已经开发出了一些方法，以确保他们实验室的本科生将接受复杂方法的技术培训，并参与研究设计和执行。这项研究将主要由哈维马德学院（HMC）和犹他州立大学（USU）的本科生进行。南大的一名研究生将与本科生合作并参与本科教学。该项目建立在HMC和USU的本科生正在进行的成功合作的基础上。

Understanding plant cell lipid signaling is critical for understanding the regulation of many aspects of plant physiology. Phosphoinositides (PIs) play critical roles in plant responses to stress, vascular development, guard cell dynamics and cell growth and morphology. Plants have evolved novel mechanisms of PI signaling and novel PI modifying pathways that are distinct from those in animals and yeast. Studies undertaken in this project will lead to new insights into the function of PIs in plants cells and new tools and methods that will greatly enhance study of plant PI signaling, and ultimately lead to the development of novel approaches for engineering plants with greater tolerances to abiotic and biotic stresses. Primarily through their interactions with proteins, PIs regulate functions including vesicle trafficking and membrane/cytoskeleton interactions. PI accumulation is regulated in part by the action of PI phosphatases, including the Sac-domain proteins. Sac-domain proteins are conserved across eukaryotes, but a novel distinct family member, SAC9, is found only in plants and green algae. Plant and algal SAC9 proteins have many conserved differences from the other Sac-domain proteins, including the other Sac-domain proteins found in plants. Dr. Williams's group's initial studies have demonstrated that SAC9 is important for regulation of both PtdIns(4,5)P2 levels and actin cytoskeleton distribution in plants. Further characterization of the novel SAC9 protein, and phenotypes of sac9 mutant plants, will both inform studies on Sac-domain proteins across eukaryotes, and contribute to an understanding of the interactions between PIs, the actin cytoskeleton and plant cell functions. This project will (1) characterize the activity and cellular localization of AtSAC9 through in vitro and in vivo expression; (2) characterize the roles of the plant-specific C-terminal domain and SAC9-specific WW domain in AtSAC9 function and cellular distribution; (3) examine SAC9 function in vivo through transient and inducible expression studies; and (4) test our model of SAC9 function by examining co-localization of SAC9, PtdIns(4,5)P2 and actin filaments using fluorescent proteins and immunolocalization.Broader ImpactBoth principal investigators have extensive experience and demonstrated success conducting research with undergraduates, and believe that providing exciting research opportunities to undergraduates in the classroom and the research lab is instrumental for bringing them into the science pipeline. Drs. Williams and Dewald have developed ways that ensure the undergraduates in their laboratories will be trained technically in sophisticated approaches and will participate in research design and execution. The research will be carried out principally by undergraduate students at both Harvey Mudd College (HMC) and Utah State University (USU). One graduate student at USU will collaborate with the undergraduates and participate in undergraduate teaching. This project builds on an ongoing and successful collaboration that has involved undergraduates at both HMC and USU.