高等植物生殖过程中，精细胞依赖花粉管到达雌性生殖细胞实现受精。花粉管的生长需要顶端ROP活性在激活和失活状态间周期性的振荡，而ROP活性的振荡依赖于彼此拮抗的正反馈和负反馈信号调节。研究表明，钙离子是一个关键的ROP负反馈调节信号，但其对ROP负反馈调节的分子机理尚且未知。我们的前期结果表明，ROP负调节因子REN1的活性受钙调节；我们还发现钙依赖蛋白激酶CDPK16的突变体中钙信号介导的ROP负反馈调节被削弱，此外钙离子还能激活CDPK16在体外磷酸化 REN1蛋白。因此我们推测钙离子介导的ROP负反馈调节是通过钙依赖蛋白激酶CDPK16感受钙信号后，通过磷酸化激活ROP负调节因子REN1蛋白，来降低花粉管顶端的ROP活性实现负反馈。我们计划开展一系列实验，探索REN1蛋白是否是CDPK16蛋白的底物，以及REN1磷酸化状态对其活性的影响，进而阐明钙信号介导的ROP负反馈调节的分子机理。

In higher plants, pollen tubes penetrate through multiple female sporophytic tissues for delivering sperm cells to female reproductive cells, thus robust growth of pollen tube is essential for plant life cycle. Pollen tube growth is controlled by a signaling network that is dependent on the tip plasma-membrane localized ROP GTPase. The apical ROP activity oscillates through the antagonistic negative and positive feedback signaling pathways. Previous studies indicate that the negative feedback is controlled by both cytosolic calcium and the REN1 RopGAP, which promotes the conversion of active ROP to inactive ROP. However, the mechanism by which calcium negatively regulates ROP signaling remains unknown. Our preliminary results suggest that calcium may positively regulate REN1 through calcium-dependent protein kinases (CDPKs). CDPK16 phosphorylated REN1 in vitro in a calcium-dependent manner. Loss-of-function cdpk16 mutants displayed enhanced pollen tube growth in high calcium media, indicating attenuated negative feedback regulation of ROP signaling by calcium. We hypothesize that REN1 is a substrate for the calcium sensor CDPK16 and that CDPK16 mediated phosphorylation of REN1 activates its GAP activity for deactivating ROP GTPase. To test this hypothesis, we will study whether REN1 is the substrate of CDPK16 in response to calcium by protein-protein interaction and in vitro/in vivo phosphorylation detection, also we will investigate the role of phosphorylation on the REN1 protein plays in the regulation of ROP activity. Our research will elucidate the molecular mechanism for calcium-mediated negative feedback regulation of ROP activity in pollen tubes.