RhoGTPase signalling in double fertilisation in Arabidopsis and maize (A15)

双受精是所有开花植物种子生产的关键过程，因此对作物生产至关重要。在双受精过程中，两个雄性精子细胞与两个雌性配子融合，两个雌性配子分别发育成胚胎和营养胚乳。这一过程的一个基本特征是，花粉管的顶端在花组织中定向生长，将雄配子传递给雌配子体(Bleckmann等人，2014；Dresselhaus等人，2016)。这种极尖的生长需要多个反馈环来平衡细胞扩张和稳定性。植物的RhoGTP酶(Rho)是整合这些反馈环的中心因素。它们的活性通过激活ROP鸟嘌呤核苷酸交换因子(ROPGEF)和失活GTP酶激活蛋白(GAP)来调节(Qin和Yang，2011)。在以前的研究中，只对单个蛋白质或机制进行了研究。然而，我们和其他人表明，这些因素中的多个虽然在同一个细胞中表达，但它们的作用是一致的或顺序的。此外，我们发现不同的ROPGEF调节相同ROP在根毛中的定位或活性，这是植物中的第二个极尖生长过程(Denninger等人，2019年)。基于对根毛中不同ROPGEF功能的观察，我推测ROP信号的不同方面受到花粉管中不同ROPGEF的调控。对花粉管生长过程中ROPGEF的更好的理解将有助于我们对种子生产的理解。在本项目中，我们将研究三个在拟南芥花粉管中优先表达的ROPGEF在花粉管顶端生长和配子相互作用过程中的特定功能。我们的目标是通过检测钙信号和肌动蛋白组织作为读数来确定它们在ROP定位和激活上的特定功能。为了研究植物之间的这些保守机制，我们将利用敲除突变体和敲除突变体来研究玉米花粉管中的ROPgef功能，并将其与拟南芥中的ROPgef功能进行比较。此外，我们将研究雌拟南芥配子体中的ROP信号，在那里这一途径仍然完全未知。初步结果支持在该组织中表达至少一种ROP，我们将进一步研究是否存在其他ROP信号因子，以及它们在双受精过程中在雌配子体细胞中的功能。

Double fertilisation is a key process for seed production in all flowering plants and therefore crucial for crop production. During double fertilisation, two male sperm cells fuse with two female gametes, which develop into the embryo and the nurturing endosperm, respectively. An essential feature of this process is that the tip of the pollen tube grows directionally through the floral tissue and delivers male gametes to the female gametophyte (Bleckmann et al., 2014; Dresselhaus et al., 2016). This polar tip growth requires multiple feedback loops for balanced cellular expansion and stability. The RhoGTPases RHO OF PLANTS (ROPs) are central factors to integrate these feedback loops. Their activity is regulated by activating ROP guanine nucleotide exchange factors (ROPGEFs) and inactivating GTPase-Activating Proteins (GAPs) (Qin and Yang, 2011). In previous studies, only individual proteins or mechanisms were investigated. We and others, however, showed that multiple of these factors, although expressed in the same cell, act in concert or in sequence. Additionally, we found that distinct ROPGEFs regulate either localisation or activity of the same ROP in root hairs, a second polar tip growth process in plants (Denninger et al., 2019). Based on these observations of distinct ROPGEF functions in root hairs, I hypothesise that different aspects of ROP signalling are regulated by different ROPGEFs in pollen tubes. A better understanding of ROPGEFs during pollen tube growth would enhance our understanding of seed production.In this project, we will investigate the specific functions of three, preferentially pollen-expressed ROPGEFs in pollen tubes during tip growth and gamete interaction in Arabidopsis thaliana. We aim to identify their specific functions on ROP localisation and activation by examining Ca2+ signalling and actin organisation as readouts. To study the conservation of these mechanisms between plants, we will investigate ROPGEF function in pollen tubes of Zea mays using knock-out and knock-down mutants and compare this to ROPGEF function in Arabidopsis. Additionally, we will investigate ROP signalling in the female Arabidopsis gametophyte, where this pathway remains completely unexplored. Preliminary results support expression of at least one ROPGEF in this tissue, and we will further investigate the presence of other ROP signalling factors, as well as their function in cells of the female gametophyte during double fertilisation.