Pollen-pistil communication in flowering plants: a role for endocytosis?

开花植物的繁殖成功是由花粉和雌蕊之间的交流事件决定的，这些交流事件导致受精，并最终发育出有活力的种子。授粉后，只有自己的花粉会被接受，并继续通过雌蕊，而所有其他“外来”花粉将被拒绝。授粉后，花粉粒产生一根管子，用于将精细胞准确地输送到雌蕊子房深处的雌性胚囊中，胚囊中存放着等待受精的卵细胞。这些所谓的花粉管显示极化生长，这是一种细胞生长的类型，其特征是仅在生长的顶端区域延伸。不同雌蕊组织提供的信号在相对较短的时间内引导这些花粉管在较远的距离内朝着正确的方向前进。生长中的花粉管如何感知这些信号并将其转化为（物种特异性的）生长反应目前尚不清楚。花粉管中两极分化的生长有两个过程：胞吐作用和胞吞作用。在包括植物在内的其他真核细胞类型中，内吞过程已被证明在细胞和组织信号传导中发挥重要作用。然而，内吞作用在种内和种间花粉管与周围雌蕊组织的交流中所起的作用尚未确定。这个博士项目包括广泛的最先进的研究技术：从蛋白质生物化学，分子生物学，遗传学，显微镜和成像分析，到数学建模。与Emyr Lloyd-Evans博士合作，成功的候选人将识别和功能表征花粉管内吞噬所吸收的花粉和雌蕊蛋白，并建立在极化尖端生长过程中发现的内吞噬活性与体内花粉管生长效率之间的联系。在轮转项目1期间，该学生将在我的实验室工作，他/她将在分子生物学、蛋白质分析（分离、重组表达、PAGE和Western Blotting）、植物和共聚焦显微镜（与Walter Dewitte博士合作）中使用更通用和基本的工具进行强化培训。我们将生成T-DNA构建体，用于开发含有新型花粉特异性细胞器GFP标记蛋白的转基因植物品系，包括拟南芥。对于轮换项目2，该学生将与卡迪夫大学的E lloyd - evans博士合作，将他们的哺乳动物内溶酶体纯化方法应用于植物细胞，即花粉管。对于这个轮换的第一部分，学生将确定并完善感兴趣的蛋白质组分的最佳分离策略，例如（蔗糖）梯度的超离心，分析将包括荧光显微镜，电子显微镜和western blotting。其次，学生将确定内体Ca2+稳态对花粉管生长的影响。EL-E实验室已经开发了许多技术来确定Ca2+的局部变化对多种哺乳动物溶酶体蛋白的影响，并最终确定它们的细胞功能。这些技术将适用于植物细胞，其中Ca2+尖端梯度已知是花粉管生长所必需的。

Reproduction success in flowering plants is unequivocally determined by the species-specific communication events between pollen and pistil resulting in fertilisation, and the development of viable seeds ultimately. Upon pollination, only own pollen will be accepted and continue their journey through the pistil whereas all other 'foreign' pollen will be rejected. After pollination, pollen grains produce a tube needed for the accurate delivery of sperm cells to the female embryo sacs deep down in the pistil ovary, harbouring the egg cells that are waiting to be fertilised. These so-called pollen tubes show polarized growth, a type of cell growth which is characterised by extension at the growing apical domain only. Signals provided by the different female pistil tissues guide these pollen tubes in the right direction over long distances in a relatively short time. How growing pollen tubes perceive these signals & translate them into a (species-specific) growth response is currently not known. Two processes of membrane trafficking at the apex characterize this polarized growth in pollen tubes: exocytosis and endocytosis. In other eukaryote cell types, including in plants, the process of endocytosis has been demonstrated to play an important role in cell and tissue signalling. However, a role for endocytosis in intra and inter-specific pollen tube communication with the surrounding pistil tissues has not been established yet.This PhD-project includes a wide range of state-of-the-art research techniques: from protein biochemistry, molecular biology, genetics, microscopy and imaging analysis, to mathematical modelling. In collaboration with Dr Emyr Lloyd-Evans, the successful candidate will identify and functionally characterise pollen and pistil-proteins that are taken up by pollen tube endocytosis, and establish a link between the endocytic activity found during the polarised tip growth with pollen tube growth efficiency in vivo. During Rotation project 1, the student will work in my lab and s/he will obtain intensive training in the more general & basic tools used in molecular biology, protein analysis (isolation, recombinant expression, PAGE and Western Blotting), working with plants and confocal microscopy (in collaboration with Dr Walter Dewitte). We will generate T-DNA constructs used for the development of transgenic plant lines, including Arabidopsis thaliana, containing novel pollen-specific organelle GFP marker proteins. For Rotation Project 2, the student will work with the Dr E Loyd-Evans at Cardiff University to adapt their mammalian endo-lysosomal purification methodologies for use in plant cells, i.e., pollen tubes. For the first part of this rotation the student will determine and then refine the best separation strategy of protein fractions of interest, e.g. ultracentrifugation of (sucrose) gradients, analysis will include fluorescence microscopy, electron microscopy and western blotting. Secondly, the student will determine what influence endosomal Ca2+ homeostasis has on pollen tube growth. The EL-E lab has developed a number of techniques to determine the impact of localised changes in Ca2+ on a multitude of mammalian lysosome proteins and ultimately their cellular function. These techniques will be adapted for use in plant cells where Ca2+ tip gradients are known to be essential for pollen tube growth.