Protein Glycosylation in Pollen Tube Growth and Fertilization

大多数粮食作物是开花植物，需要成功受精才能产生谷物和果实。受精需要花粉落在雌花上并形成花粉管，花粉管是一种快速拉长的单细胞突起，在雌性组织中生长，在某些情况下长达几厘米。这种花粉管能够根据化学引导信号重新调整生长方向，将雄性生殖细胞传递给雌性。与其他植物细胞一样，花粉管被细胞壁包围，细胞扩张需要适当分泌新的细胞壁物质，这种物质专门针对生长顶端的特殊、灵活的细胞壁。内部分泌机制和细胞壁必须相互适当协调，并与伸长方向相协调，才能成功地持续生长。尽管这一过程对农业生产很重要，但在我们对外部细胞壁和细胞内分泌装置如何协调以保持稳定但反应灵敏的尖端区域的理解中存在着重大差距。这个项目将使用遗传学、生物化学和细胞成像方法来阐明这两个隔室之间的联系。这项研究还将支持研究生和本科生的培养。研究活动将被整合到植物生物学实验室入门课程中，为学生提供真实的研究体验。此外，该项目将与密歇根大学自然历史博物馆合作，向公众提供互动的动手活动。该项目由综合组织系统分部的植物、真菌和微生物发育机制计划以及分子和细胞生物科学部的细胞动力学和功能计划共同资助。开花植物的育性取决于花粉管的持续快速伸长和它们重新定向生长以有效地瞄准接受胚珠的能力。适当的花粉管伸长需要羟脯氨酸(Hyp)O-阿拉伯糖基化，即翻译后将阿拉伯糖链加到羟脯氨酸上。阿拉伯糖基转移酶突变体表现出更短、更宽的花粉管，有时还会有分支，这表明顶端区域的稳定性受损。花粉管伸长所需的分泌物取决于花粉管顶端高度动态的F-肌动蛋白结构。F-肌动蛋白的组织部分受控于1类福尔马林同源蛋白的成核活性。这些蛋白质的胞外区含有预测的阿拉伯糖基化基序，这些基序与细胞壁的相互作用有关。这个项目将检验这样的假设，即花粉管细胞壁和肌动蛋白细胞骨架是通过1类福尔曼同源蛋白的Hyp O-阿拉伯糖基化来协调的，通过与覆盖的细胞壁的相互作用，将它们的肌动蛋白组织活性限制在适当的皮质区域。在突变体和野生型中，将检测细胞壁极性和肌动蛋白组织的改变。对于这一蛋白质家族的三个重要成员，包括两个已知的花粉管肌动蛋白调节器，将确定1类福尔马林的体内糖基化状态。此外，还将确定胞外结构域对花粉管中蛋白质迁移率和定位的影响。这项研究将在花粉管极化和生长所需的两个主要细胞成分之间建立一个重要的连接点。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The majority of food crops are flowering plants that require successful fertilization to produce grains and fruits. Fertilization requires pollen to land on the female flower and form a pollen tube, a rapidly elongating single-celled projection which grows through the female tissue, in some cases for several centimeters. This pollen tube is able to reorient its growth in response to chemical guidance cues to deliver the male reproductive cells to the female. The pollen tube, like other plant cells is surrounded by a cell wall and cell expansion requires the proper secretion of new cell wall material that is specifically targeted to the specialized, flexible cell wall at the growing tip. The internal secretory machinery and the cell wall must be appropriately coordinated with each other and with the direction of elongation for successful sustained growth. Despite the importance of this process for agricultural production, a major gap exists in our understanding of how the external cell wall and the intracellular secretory apparatus coordinate to maintain a stable but responsive tip region over time. This project will elucidate the linkage between these two compartments using genetics, biochemistry and cell imaging approaches. The research will also support the training of graduate and undergraduate students. Research activities will be integrated into an introductory plant biology lab course giving students an authentic research experience. In addition, the project will provide interactive, hands-on activities to the general public in collaboration with the University of Michigan Museum of Natural History. This project was co-funded by the Plant, Fungal and Microbial Developmental Mechanisms Program in the Division of Integrative Organismal Systems and the Cellular Dynamics and Function Program in the Division of Molecular and Cellular Biosciences. Flowering plant fertility depends on the sustained rapid elongation of pollen tubes and their ability to reorient growth to effectively target receptive ovules. Proper pollen tube elongation requires hydroxyproline (hyp) O-arabinosylation, the posttranslational addition of arabinose sugar chains to hydroxyprolines. Arabinosyltransferase mutants exhibit shorter, wider and sometimes branched pollen tubes, suggesting impaired tip-region stability. The secretion required for pollen tube elongation depends on the highly dynamic F-actin structures at the pollen tube tip. F-actin organization is controlled, in part, by the nucleation activity of class 1 formin homology proteins. The extracellular domains of these proteins contain predicted arabinosylation motifs which have been implicated in interaction with the cell wall. This project will test the hypothesis that the pollen tube cell wall and actin cytoskeleton are coordinated via hyp O-arabinosylation of the class 1 formin homology proteins, limiting their actin organizing activity to appropriate cortical domains though interaction with the overlying cell wall. Altered cell wall polarity and actin organization will be assayed in the mutants and wild type. The in vivo glycosylation status of the class 1 formins will be determined for three important members of this protein family, including two known regulators of pollen tube actin. Furthermore, the effect of the extracellular domains on protein mobility and localization in the pollen tube will be determined. This research will establish an essential point of connection between two major cell components required for pollen tube polarization and growth.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.