Molecular Mechanism of Polarized Growth in Pollen Tubes

9724047阳尖生长的花粉管向胚珠定向生长，将精子输送到胚囊，因此对高等植物的有性繁殖至关重要。花粉管还为研究极化生长提供了一个有吸引力的模型系统，部分原因是它们是单倍体，易于培养。该项目的长期目标是阐明控制花粉管中极化细胞生长的分子途径。我们已经鉴定出一种Rho类型的小GTP结合蛋白Rop1，它优先在花粉和花粉管中表达，并定位于豌豆和拟南芥花粉管的顶端。抗体显微注射实验表明，Rop1可能通过调节依赖于钙离子的胞吐作用而在花粉管生长中发挥关键作用。这项拟议工作的目的是用反向遗传学方法证实Rop1在极化管生长中的作用，并研究Rop1在花粉管中功能的潜在分子和细胞机制。Rop1的作用将在转基因拟南芥植株上进行遗传测试，这些植株在花粉管特异启动子的控制下表达显性正突变和负突变的Rop1等位基因。转基因花粉管将被分析顶端生长和体内指导的变化。表达Rop1显性突变等位基因的转基因花粉管还将使用透射电子显微镜和快速冷冻和冷冻替代技术分析分泌小泡行为的变化，并使用钙离子选择性振动探针和比率离子成像分析细胞外钙离子内流和尖端聚焦的细胞内钙离子梯度的变化。这些仔细的分析将检验Rop1通过调节依赖于钙信号的极化胞吐来控制极化细胞生长的观点。为了进一步了解Rop1介导的极化生长的潜在机制，将通过使用交互式克隆技术(如酵母双杂交方法)克隆编码Rop1伙伴的基因来鉴定Rop1效应器。Rop1效应器有望与GTP结合的Rop1(显性正突变体)特异性地相互作用，但不与GDP结合的Rop1(显性负突变体)相互作用。这些研究将为我们阐明导致花粉管定向生长的分子途径的长期目标奠定坚实的基础。这项工作还可能对理解高等生物极化生长的分子基础产生广泛的影响，并为操纵高等植物的有性繁殖提供有用的信息，例如为杂交生产设计雄性不育。花粉管的极地生长是一个有趣而重要的过程，在这个过程中，萌发的花粉管向单一方向延伸，以调节植物的受精和繁殖。PI已经确定了一种与信号转导系统有关的蛋白质(ROP1，GTP结合蛋白)，该信号转导系统似乎通过钙通量控制花粉管的极地生长。以这一初步数据为基础，PI将使用ROP1的突变体来检测蛋白质的功能。利用光学显微镜分析，ROP1在诱导钙离子通量中的作用将被可视化，并与电子显微镜下延伸花粉管所需的细胞内事件相关联。ROP1必须以级联方式与其他蛋白质相互作用，通过细胞传递信号。与ROP1相互作用的蛋白质将被识别和表征。这项研究对于描述植物繁殖中的一个关键过程具有重要意义。这项研究的实际应用包括改变植物的生殖生长以创造更好的植物。***

9724047 Yang Tip-growing pollen tubes grow directionally toward the ovules to delivery sperms to the embryo sac and thus are critical for sexual reproduction in higher plants. Pollen tubes also present an attractive model system for the study of polarized growth, partly because they are haploid and can be easily cultured. The long-term goal of this project is to elucidate molecular pathways that govern polarized cell growth in pollen tubes. We have identified a rho-type small GTP-binding protein, Rop1, that preferentially expressed in pollen and pollen tubes and is localized to the apex of pea and Arabidopsis pollen tubes. Antibody microinjection experiments indicate that Rop1 plays a pivotal role in pollen tube growth likely by modulating Ca2+-dependent exocytosis. The goal of this proposed work is to confirm the role of Rop1 in polarized tube growth using a reverse genetics approach and to investigate underlying molecular and cellular mechanisms for the function of Rop1 in pollen tubes. The role of Rop1 will be genetically tested using transgenic Arabidopsis plants that express dominant positive and negative mutant Rop1 alleles under the control of a pollen tube-specific promoter. The transgenic pollen tubes will be analyzed for changes in tip growth and in vivo guidance. The transgenic pollen tubes expressing Rop1 dominant mutant alleles will also be analyzed for changes in the behavior of secretory vesicles using TEM and the rapid freeze and freeze substitution technique and for changes in the influx of extracellular Ca2+ and the tip-focused intracellular Ca2+-gradients using a Ca2+-selective vibrating probe and ratiometric ion imaging. These careful analyses will test the notion that Rop1 controls polarized cell growth by modulating Ca2+ signaling-dependent polarized exocytosis. To further understand the underlying mechanism for Rop1-mediated polarized growth, Rop1 effectors will be identified by cloning genes encoding Rop1 partners using interactive cloning techniques such as the yeast two-hybrid method. Rop1 effectors are expected to interact specifically with GTP-bound Rop1 (dominant positive mutant) but not with GDP-bound Rop1 (dominant negative mutant). These studies will establish a solid foundation for our long-term goal of elucidating molecular pathways that lead to directional pollen tube growth. This work may also generate a broad impact on understanding the molecular basis of polarized growth in higher organisms as well as produce useful information for manipulating sexual reproduction in higher plants, such as engineering male sterility for hybrid production. The polar growth of pollen tubes is an interesting and significant process in which a germinating pollen tube extends in a single direction to mediate fertilization and reproduction in plants. The PI has identified a protein (ROP1, GTP-binding protein) involved in the signal transduction system that appears to control the polar growth of pollen tubes through calcium fluxes. With this preliminary data as a base the PI will use mutants of ROP1 to examine the function of the protein. Using light microscopy assays the role of ROP1 in inducing a calcium fluxes will be visualized and correlated with electron microscopy of the intracellular events that are required to extend the pollen tube. ROP1 must interact with other proteins in cascade that transmits signals through the cell. Proteins that interact with ROP1 will be identified and characterized. This research is of fundamental importance in describing a critical process in the reproduction of plants. Practical applications of the research include modifying plant reproductive growth to create superior plants. ***