Control of polar plant cell growth: functional characterization of regulators and effectors of the tobacco pollen tube Rac/Rop GTPase Nit-Rac5

本研究的目的是通过控制细胞尖端的Rac/Rop激活或该事件下游的细胞过程来阐明调节极性花粉管生长的分子机制。这一研究结果将有助于进一步了解Rac/Rop信号，以及极性植物细胞生长、植物发育和受精过程中花粉管生长方向的分子调控。花粉管是一种大细胞，它以严格的极性方式通过雌花组织快速生长，并在受精过程中将封闭在细胞质中的精子细胞运输到卵细胞。烟草花粉管是研究极地植物细胞生长的一个极好的模型系统，因为它们在培养中正常伸长，易于实验操作，可以在体外大量生长，并且遗传上易于处理（瞬时和稳定转化，正在进行的基因组测序项目）。我们发表的研究表明，Rac/Rop gtpase是Rho家族gtpase的植物近亲，具有明显的多样性和很大程度上未被表征的功能，它们与花粉管顶端的质膜有关，是花粉管细胞极性生长的关键调节因子。在此，我们建议使用多种方法对酵母与烟草花粉管Rac/Rop GTPase Nt-Rac5双杂交相互作用的蛋白质进行功能表征。基于酵母双杂交实验、瞬时过表达和GFP标记的大量初步数据表明，这些蛋白可以作为Nt-Rac5的调节剂或效应器。

The goal of the proposed study is to elucidate molecular mechanisms regulating polar pollen tube growth by controlling localized Rac/Rop activation at the tip of these cells, or cellular processes downstream of this event. Results obtained will enhance current knowledge concerning Rac/Rop signaling, and the molecular control of polar plant cell growth, of plant development and of the direction of pollen tube growth during fertilization. Pollen tubes are large cells that grow rapidly in a strictly polar manner through female flower tissue and transport sperm cells enclosed in their cytoplasm to egg cells during fertilization. Tobacco pollen tubes are an excellent model system to investigate polar plant cell growth, because they elongate normally in culture, are amenable to experimental manipulation, can be grown in vitro in large quantities and are genetically tractable (transient and stable transformation, ongoing genome sequencing project). Our published work has shown that Rac/Rop GTPases, plant relatives of Rho family GTPases with apparently diverse and largely uncharacterized functions, are associated with the plasma membrane specifically at the tip of pollen tubes and are key regulators of the polar growth of these cells. Here, we propose to functionally characterize using a variety of approaches proteins that show yeast two-hybrid interaction with the tobacco pollen tube Rac/Rop GTPase Nt-Rac5. Substantial preliminary data based on yeast two-hybrid assays, transient over-expression and GFP tagging indicate that these proteins act either as regulators or effectors of Nt-Rac5.