拟南芥CML24参与花粉萌发及花粉管极性生长的分子机理

The successful germination and rapid tip elongation of pollens are essential for sexual reproduction of flowering plants, and it is well known that both external and cytosolic calcium (Ca2+) are important for the both processes. Consequently, the investigation of Ca2+-mediated signaling pathways in pollens and pollen tubes is indispensable for better understanding of male gametophyte signal transduction during fertilization. Recently, Calmodulin-Like-proteins (CMLs) are found with EF-hand Ca2+-binding domain and predicted as putative Ca2+ sensors, but their function in plant fertilization is completely unknown. By using in-vitro and in-vivo pollen germination assays, we found that the germination rate and pollen tube length are dramatically reduced in cml24-1, a functional null mutant of CML24. CML24 is highly expressed in mature pollens and pollen tubes, so the functional investigation of molecular mechanisms of CMLs will be helpful for better understanding of plant cell Ca2+ signaling during polarized cell growth and plant fertilization. Previous studies suggested that CML24 is involved in plant Nitric Oxide (NO) signaling, and external K+ is required for both pollen germination and elongation, so we hypothesize that CML24 may utilize NO and cytosolic Ca2+ signaling to modulate transmembrane ion transportation, and thus regulate pollen germination and pollen tube elongation. To certify our hypothesis, the dynamics of cytosolic NO and Ca2+ will be inspected by using specific fluorescent dyes and examined with confocal microscopes. In addition, in order to uncover the involvement of CML24 in Ca2+ regulation of ion channels, patch clamp technique will also be employed to study K+, Cl- and Ca2+ channels of pollens and pollen tubes. Furthermore, the interacting proteins functioning with CML24 in pollen germination and pollen tube elongation will also be screened for dissection of CML24 mediated-signaling-pathways and putative molecular mechanisms.

花粉是植物雄配子体的载体，其正常萌发和快速极性生长，对于开花植物成功受精并繁殖后代至关重要。钙调素类似蛋白（Calmodulin Like Protein，CML）家族是近年新发现的植物细胞Ca2+响应元件，在拟南芥中有50个基因。利用CML24的功能缺失突变体，我们发现CML24参与调控拟南芥的花粉萌发及花粉管生长，但其具体分子机理及相关信号转导途径还不清楚。鉴于已有结果显示该基因与NO及 Ca2+信号转导途径相关，而K+为花粉萌发及花粉管快速生长所必需。我们推测，CML24调控花粉萌发及花粉管生长是通过改变NO及Ca2+胞内信号，影响花粉及花粉管的跨膜离子转运（如K+等）实现的。因此，利用NO及Ca2+荧光标记、电生理膜片钳技术及蛋白互作等研究手段，验证我们的假说，明确CML24调控花粉萌发及花粉管生长的作用机制，对于了解植物生殖调控机理、完善植物细胞信号转导网络具有重要的意义。

花粉是植物雄配子体的载体，其正常萌发和快速极性生长，对于开花植物成功受精并繁殖后代至关重要，该过程中的Ca2+信号转导途径起到非常重要的作用。钙调素类似蛋白（Calmodulin Like Protein，CML）家族是近年新发现的植物细胞Ca2+响应元件，在拟南芥中有50 个基因。利用CML24 的功能缺失突变体，我们发现CML24 是有活性的Ca2+结合蛋白，参与调控拟南芥的花粉萌发及花粉管生长，且与NO 及 Ca2+信号转导途径相关，是胞质Ca2+调控K+跨膜转运进而影响花粉萌发及花粉管快速生长所必需的。相关的研究成果发表于Plant Molecular Biology(2014. 86(3). 225-236.)及Plant Cell & Environment(2015. 38(11), 2372–2386.)等杂志，对于了解植物生殖调控机理、完善植物细胞信号转导网络具有重要的意义。