一氧化氮（NO）参与低温抑制茶树花粉管生长的信号传导机制的研究

Low temperature is one of the major factors affecting tea production and quality, so that understanding the mechanism of tea plant (Camellia sinensis L.) resisting to low temperature warrants serious attention. The mechanism of low temperature tolerance is significantly different in reproductive and vegetative tissue in plant. More interestingly, Camellia pollen germinated at low temperature (even at 3 °C), while other angiosperm pollen did not germinate at low temperature. Based on the results of my Youth Foundation Project, the resources, localization and content of NO in cold-inhibited C. sinensis pollen tubes are investigated using confocal laser scanning microscope (CLSM), electron paramagnetic resonance (EPR) spectroscopy combining with pathological methods in the present project. Furthermore, extracellular Ca2+ and H+ fluxes, cytoplasmic Ca2+ gradient and pH, plasma membrane H+-ATPase activity in cold stress- and/or other related drugs-treated pollen tubes are detected by Non-invasive Micro-test Technique (NMT), Atomic Force Microscope and phosphate determination method so as to explore the crosstalk of NO with Ca2+ and H+ in cold stress-treated pollen tubes. In order to demonstrate the effects of NO signaling pathway on biosynthesis and degradation of proline (Pro) in cold-inhibited pollen tubes, P5CS1 and ProDH genes are homologous cloned and their expression is analyzed, and then activities of P5CS and ProDH are detected. Moreover, regulating effects of NO on key factors of pollen tubes such as actin filaments, vesicles, cell wall and ultra-structure during cold stress are evaluated in the present project. The results of this project will reveal the signaling pathway of NO in cold-stressed pollen tubes, which will highlight the mechanism of cold tolerance in plant reproductive tissue.

低温是影响茶叶产量和品质的重要因素之一，茶树抗寒机制的研究意义重大。植物生殖器官抗寒机理与营养器官存在巨大差异。茶树花粉管在低温下能萌发生长，而一般被子植物花粉管在低温下不能萌发。本项目拟以茶树花粉管为材料，在青年基金研究的基础上，用激光共聚焦、电子顺磁共振（EPR）等技术，研究低温胁迫下NO的来源、定位和定量。用非损伤微测、原子力显微镜和定磷法，分析低温胁迫下， NO与胞外Ca2+流、胞内Ca2+浓度梯度、质膜H+-ATPase活性、胞外H+流和胞内pH之间的信号交叉。同源克隆P5CS1和ProDH基因并分析其在低温胁迫下的表达，测定P5CS和ProDH活性，研究低温胁迫下NO信号途径对Pro合成与降解的调控。用细胞生物学手段研究低温胁迫下NO对花粉管关键要素微丝、囊泡、细胞壁和超微结构的调控。项目旨在探讨NO参与低温抑制花粉管生长的信号转导机制，为植物生殖器官抗寒机理提供科学依据。

NO作为重要的信号分子参与茶树花粉管响应低温胁迫的过程，但其作用机理不清楚。本项目在前期研究的基础上，利用细胞生物学、转录组学和分子生物学技术研究NO参与低温抑制茶树花粉管生长的信号途径。主要结果如下：. 花粉管在常温CK、低温LT及NO处理培养后，收集并提取RNA，之后用Illumina HiSeqTM2000 System进行转录组测序。按照绝对值log2Ratio>1和可能性>0.7的标准筛选出了三个处理转录组数据之间的两两比较的差异基因。筛选出NO来源、植物激素、转录因子、泛素系统、次生代谢物质、囊泡转运、细胞壁建成等相关基因并进行分析和验证。结果表明，在低温胁迫下，细胞色素P450和精氨酸代谢相关基因等众多基因参与NO的产生；之后在NO的影响下形成了庞大复杂的信号转导途径，首先植物激素信号途径（ABA、BRs、ET和Auxin）受到响应并通过各自信号途径中的相关基因调节激素的水平，接着相关转录因子（MYB、ZFP、CAMTA和MADS-box）进行转录修饰，MADS-box还可能直接作用于NO合成代谢途径，进而调节茶树花粉管体内NO的含量，进一步就是UPS系统（E3s、26S proteasome、U-box和BTB/POZ）可能在NO的参与调节下，间接调节花粉管内翻译后的蛋白水平。另外，在NO信号途径的调控下，胞内的Ca2+浓度、囊泡的极性运输及细胞的合成代谢中的CAMTA、PI4K和COBRA-like基因也都参与了低温胁迫下的花粉管伸长。. 利用特异探针标记，在激光共聚焦显微镜下观察，结果显示，与正常生长的花粉管相比，经低温或NO处理后，花粉管顶端Ca2+浓度梯度被破坏、花粉管中ROS的含量增加、胞质内的pH酸化、细胞壁组分的分布也发生变化，而NO清除剂cPTIO一定程度上缓解低温对花粉管的影响。通过筛选转录组差异基因发现，cGMP信号、Ca2+信号、ROS信号、pH、Actin组装、细胞壁建成及MAPK级联等相关基因参与低温胁迫下NO信号转导。. 对茶树中的P5CS、ProDH及鸟氨酸δ转氨酶（δ-OAT）基因进行荧光定量发现冷胁迫诱导的NO增加了δ-OAT基因的表达并减少了ProDH基因的表达，但是却不影响P5CS基因的表达，表明在低温胁迫下，NO通过促进δ-OAT基因的表达及抑制ProDH基因的表达来调节茶树花粉中的脯氨酸的累积。