MOLECULAR MECHANISM OF POLLEN TUBE ELONGATION

本提案的目标是了解分子机制 花粉管伸长的基础。花粉管伸长是一个过程 对植物有性繁殖至关重要当花粉沉积在 雌性生殖器官雌蕊的可接受柱头表面， 雄配子通过花粉管伸长运输到子房， 使卵细胞受精。花粉管伸长是一种定向生长 从柱头表面的花粉粒到子房， 位于雌蕊的基部末端，是雌蕊的数百到数千倍。 花粉粒的直径。花粉管伸长常被用来比较 因为它是一种定向的细胞运动， 基于雌蕊细胞外基质中的尖端生长过程， 组织中与理解轴突生长的机制类似， 对花粉管伸长的研究面临着同样的挑战， 很难破译的机制，是基于特定的 趋化性引诱剂，或表面结构粘附，或缺乏 有意的吸引和花粉管遵循阻力最小的路径。 我们已经分离并鉴定了第一个蛋白组分（TTS 蛋白质）和其cDNA， 促进花粉管伸长，并显示出可能 有助于雌蕊引导花粉管向 卵巢TTS蛋白是一种阿拉伯半乳聚糖蛋白， 在花柱传导组织的细胞间基质中， 特殊的媒介，通过它花粉管从柱头延伸到 卵巢我们的结果表明TTS蛋白到达其花粉管 通过粘附于表面和生长促进活性 花粉管顶端吸收和水解糖残基， 这些蛋白质。TTS蛋白也显示出增加的梯度， 从柱头端到卵巢端的糖基化水平 传递组织，与花粉管生长方向相同。的 拟议的研究重点特别是阐明分子 TTS蛋白与花粉相互作用的机制 管，确定其花粉管表面粘附的贡献 特性和其糖成分的移动由花粉管， 其促进花粉管伸长的活性，并在检测 相关糖基化水平梯度的功能意义 这种蛋白质。分子、细胞、生物化学和 将使用转基因方法。涉及的生化参数 TTS蛋白与花粉管之间的相互作用将被确定。 TTS蛋白的结构-功能关系将通过以下方法进行检查： 产生修饰的TTS蛋白，并确定这些变化如何影响 它们的花粉管生长促进活性及其与 花粉管TTS蛋白在花粉定向中的作用 还将在体内和体外检查管伸长。拟议 实验应该能清楚地了解一个主要的 传递组织蛋白介导其功能，并证明是否 与该蛋白质相关的糖梯度是化学活性的， 花粉管定向伸长显著。 了解花粉 管伸长应该扩展我们对细胞迁移的一般知识。

The goal for the present proposal is to understand the molecular mechanism underlying pollen tube elongation. Pollen tube elongation is a process central to plant sexual reproduction. After pollen is deposited on the receptive stigmatic surface of the female reproductive organ pistil, the male gametes are transported by pollen tube elongation to the ovary to fertilize the egg cell. Pollen tube elongation is a directed outgrowth from the pollen grain on the stigmatic surface toward the ovary which is located at the basal end of the pistil, hundreds to thousand times the diameter of pollen grains away. Pollen tube elongation is often compared to axon outgrowth in animal systems since it is a directed cell movement based on a tip growth process in the extracellular matrix of pistillate tissues. Similar to understanding the mechanism underlying axon outgrowth, studies on pollen tube elongation face the same challenges of the difficulty to decipher among mechanisms that are based on specific chemotactic attractants, or to surface structure adhesion, or to a lack of deliberate attraction and pollen tubes follow a path of least resistance. We have isolated and characterized the first protein component (TTS protein) and its cDNAs from pistillate tissues of tobacco which can promote pollen tube elongation and display properties suggesting it may contribute to the overall pistil mechanism of guiding pollen tubes towards the ovary. TTS protein is an arabinogalactan protein specifically located in the intercellular matrix of the stylar transmitting tissue, a specialized medium through which pollen tubes elongate from the stigma to the ovary. Our results show that TTS protein attains its pollen tube growth promotion activity by adhering to the surface and to the growth tips of pollen tubes which uptake and hydrolyze the sugar residues on these proteins. TTS proteins also display a gradient of increasing glycosylation levels from the stigmatic to the ovarian end of the transmitting tissue, the same direction as pollen tube growth. The proposed research focuses specifically on elucidating the molecular mechanism underlying the interactions between TTS protein and pollen tubes, on determining the contribution of its pollen tube surface adhesion properties and the mobilization of its sugar components by pollen tubes to its activity in promoting pollen tube elongation, and on examining the functional significance of the gradient of glycosylation levels associated with this protein. A combination of molecular, cellular, biochemical and transgenic approaches will be used. The biochemical parameters involved in the interaction between TTS protein and pollen tubes will be determined. The structure-functional relationship of TTS protein will be examined by generating modified TTS proteins and determining how these changes affect their pollen tube growth promoting activity and their interactions with pollen tubes. The participation of TTS proteins in the direction of pollen tube elongation will also be examined in vivo and in vitro. The proposed experiments together should yield a clear understanding of how a major transmitting tissue protein mediates its functions and demonstrate whether a sugar_gradient associated with this protein is chemotactically significant in directional pollen tube elongation. Understanding pollen tube elongation should extend our knowledge on cell migration in general.