【作者】 李莉；

【导师】 章文华；

【作者基本信息】 南京农业大学 ， 细胞生物学， 2015， 博士

【摘要】 土壤盐渍化是环境中主要的非生物胁迫,威胁全球农业种植,严重影响农作物的生长发育和产量。高盐环境下导致植物体内产生渗透胁迫和离子毒害作用,植物为了抵御盐胁迫带来的伤害,在这种高盐环境下生存,逐步形成了一系列能够应对盐胁迫的机制。磷脂分子不仅是细胞膜的主要组成部分,还是细胞中重要的第二信使,调节植物生长发育,响应外界环境胁迫。磷脂酶包括磷脂酶A (PLA)、磷脂酶C (PLC)和磷脂酶D (PLD),能够水解细胞膜上的磷脂组分,产生一系列的产物信号分子,在细胞响应外界信号过程中起着重要的作用。根据作用底物的不同,植物PLC可以分为磷脂酰肌醇特异性PLC( PI-PLC)和底物非特异性的PLC( NPC )。传统的研究认为植物PI-PLC水解磷脂酰肌醇4,5-二磷酸(PtIns(4,5)P2)同时产生两个重要的第二信使分子1,2-二酰甘油(DAG)和1,4,5-肌醇三磷酸(InsP3)。PI-PLC信号途径在植物适应各种胁迫中有着重要的作用。水稻是典型的单子叶植物,含有四个PI-PLC基因,分别命名为OsPLC1-4。本研究利用OsPLC1敲除突变体和回补、过表达等转基因株系结合生理生化实验阐述了水稻OsPLC1在盐胁迫下的作用和相关的调控机制。主要的研究方法和结论如下:1 .鉴定了水稻中一个Tos1 7插入的OsPLC1突变体osplc1,发现缺失OsPLC 1的突变体的耐盐性显著降低。盐处理后osplcI比WT积累更多的Na+,呈现较高的Na+/K+比,死亡率增加,PLC总酶活降低。向突变体中回补OsPLC1可以将其耐盐性恢复至野生型水平,而过表达OsPLC1后植株的耐盐性得到了提高。说明OsPLC1在植物响应盐胁迫中有着不可忽视的作用。2.利用OsPLC1自身启动子融合GUS报告基因在水稻植株中进行组织表达模式分析,GUS染色显示OsPLC1在水稻的根、茎、叶、种胚中均有表达,并特异性表达在叶片表皮的保卫细胞中。3.在水稻原生质体和烟草表皮细胞中表达OsPLC1-GFP融合蛋白,荧光主要集中在胞质中,并且在盐诱导下荧光会有上膜的趋势。结合OsPLC1的抗体和免疫分析法检测植株胞质和膜上OsPLC1蛋白含量,证实在正常生长情况下,OsPLC1主要在细胞质中,当植株受到盐胁迫时,OsPLC1会迅速被募集到质膜上,水解质膜上的底物行使功能,产生下游信号分子,参与盐胁迫。4. OsPLC1具有典型PI-PLC的结构和水解功能。OsPLC1体外水解作用需要微摩尔级的Ca2+参与,并在100 μM时达到最高水解活性;将OsPLC1催化区保守部位重要的氨基酸突变掉能够大大降低OsPLC1活性。5. OsPLC1在体外同时具有水解PtdIns4P和PtdIns(4,5)P2的作用,且对PtdIns4P的水解活性高于PtdIns(4,5)P2;同时应用PtdIns4P生物探针观察OsPLC1在活体细胞内对PtdIns4P的水解情况,结果显示OsPLC1可以在植物体内水解质膜上的PtdIns4P。6.提取osplcl和WT植株总磷脂,通过薄层层析分离显示osplc1总PIP含量明显高于野生型,而在盐处理下只有WT出现明显的磷脂酰肌醇单磷酸(PIPs)含量下调,再次说明了 OsPLC1在植物体内对PtdIns4P的水解作用。在WT和osplc1的原生质体中添加PtdIns4P、PtdIns(4,5)P2、InsP2, InsP3, DAG能在一定程度上的增加盐处理后细胞的存活率,降低盐对它们的伤害,而添加磷脂酰丝氨酸(PS)时却没有任何效果。7.为了进一步了解水稻OsPLC1参与盐胁迫的调控机制,进行了 WT和osplc1中下游DAG和InsP3的含量的测定。盐胁迫下,OsPLC1的缺失直接影响了 DAG和InsP3的积累。通过在WT和osplcl中表达Ca2+探针NES-YC3.6来检测胞质内Ca2+的变化情况,发现在osplc1中胞内Ca2+响应灵敏度和波动幅度明显低于WT,OsPLC1的缺失会间接影响Ca2+调控的下游信号通路。利用钙离子染料Fluo-3AM检测不同磷脂孵育后的WT和osplc1的原生质体经盐处理后的Ca2+波动差异,发现经过PtdIns4P、PtdIns(4,5)P2孵育后,Ca2+波动幅度比对照都有了明显提高。综上,本研究表明了 OsPLC1可水解植株体内PtdIns4P和PtdIns(4,5)P2而更倾向水解PtdIns4P,直接调控下游InsP2、InsP3、DAG这些第二信使的含量变化,间接影响Ca2+响应外界压力的灵敏度和波动幅度,进而参与盐胁迫过程,帮助植株适应盐害,提高耐盐能力。更多还原

【Abstract】 Salinity is a major abiotic stress that severely threatens plant growth and crop production. High concentration of salts in the soil influences plant growth and development through osmotic stress and ion toxic. In order to survive under high salinity,plants display complex physiology and molecular biology mechanisms. Phospholipid molecules are not just main structural components of membranes but dynamic informational elements that they have emerged as important second messengers to regulate plant growth and development, cellular responses to environmental change and stress. Phospholipase including phospholipase A (PLA), phospholipase C (PLC) and phospholipase D (PLD) are key enzymes capable of generating lipids that act as second messengers in response to external environmental stress. Two classes of PLCs have been identified in plants and categorized by the substrates which they act on.Phosphoinositide-specific phospholipase C (PI-PLC) uses primarily phosphoinositides(PIs), whereas nonspecific PLC (NPC) uses common lipid such as PC and PE. Plant PI-PLC is traditionally considered to cleave the substrate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and generates inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (DAG), both of which are second messengers in the phosphoinositide signal transduction pathways. The phosphoinositide/phospholipase pathway is supposed to an important metabolic pathway to mediate some responses.Rice,a monocot, contains four PI-PLC genes,were named OsPLC1-4 according to previous reports. In this report, we characterized the salt-responsive phosphoinositide-specific gene OsPLC1 and demonstrated that it had a critical role for the salt stress response of rice by establishing mutant, complementation and overexpression lines. The main methods and results were presented as follows:1. A homozygous Tos17 insertional mutant of OsPLC1, osplc1 (Nipponbare) was isolated, and osplc1 exhibited decreased plant tolerance to salinity. The osplc1 mutant accumulated more Na+ and exhibited an apparently increased Na+/K+ after salt stress.Meanwhile, osplcl demonstrated reduced PLC total activity and increased mortality after salinity. Complementation and overexpression lines were established by transgenic technology. When treated with salt, the complemented lines rescued the sensitive phenotype from salt damage compared to osplcl and nearly as the same as wild-type plants and the OsPLC1 overexpressed lines were observed increased salt tolerance.2. To confirm the expression pattern of OsPLC1, a β-glucuronidase (GUS) construct driven by the OsPLC1 native promoter was used to get transgenic plants. GUS signals were detected in root, stem, and embryo, specially detected in the stomata apparatus.3. Transient expression of OsPLCl and green fluorescent protein (GFP) fusion protein in rice protoplasts and Nicotiana benthamiana epidermal cells shown that fluorescence were mainly localized to the cytoplast and were found concentrating on membrane after salt treatment. For further confirmation, the display of OsPLC1 was confirmed by western boltting with OsPLC1 antibody. The results showed that OsPLC1 protein was restricted in cytoplast under normal condition; however, it was found fast recruiting on membrane with NaCl stimuli and involved in salt stress signal transduction.4. OsPLC1 contains typical structure and hydrolysis function of plant PI-PLC.OsPLC1 hydrolyzing activity in vitro was depended on the micromolar Ca2+. 100 μM Ca2+ was found to stimulate the maximum activity. Amino acid exchanges in conserved domains greatly decreased the activity of hydrolyzing.5. Based on the our data, OsPLC1 displayed a high hydrolyzing activity toward PtdIns4P and followed by PtdIns(4,5)P2 in vitro. To confirm that if it equally occurred in vivo, we used a lipid biosensor FAPP1-GFP fusion protein that monitors PtdIns4P dynamics in living plant cells. The results suggested that OsPLCl could hydrolyze PtdIns4P equally well in vivo.6. The total lipid were extracted and subjected to TLC to separate the different species.The result shown that osplcl phosphatidylinositol monophosphate (PIPs) content were extremely higher than wild type, and the PIP content was decreased in the wild type after salt treatment. Again, the results proved that OsPLC1 hydrolyze PtdIns4P in vivo. We also assayed the survival rate of protoplasts that supplement to wild type and osplc1 with InsP2, InsP3, PtdIns4P, PtdIns(4,5)P2 , DAG and phosphatidylserine (PS) before salt treatment. Except for PS, supplementation with InsP2, PtdIns4P, InsP3, PtdIns(4,5)P2 and DAG were effective in increasing salt tolerance of osplcl protoplasts.7. To further understand the mechanism that OsPLC1 regulates salt tolerance, we detected the DAG and InSP3 contents in the wild type and osplc1. Under salt stress condition, knockout of OsPLC1 directly influenced the DAG and InsP3 accumulation.We generate transgenic lines that expressing NES-YC3.6 (Ca2+ sensor) in wild type and osplcl to indicate [Ca2+]cyt. These results indicated that the response of salt induced[Ca2+]cyt increase in stomata in osplcl was insensitive and significantly lower than wild type. Using Ca2+ dye (Fluo-3AM) to detect [Ca2+]cyt changes in wild-type and osplc1 protoplasts shown that supplementation with PtdIns4P or PtdIns(4,5)P2 increased Ca2+oscillation after salt treatment.In conclusion, this study indicates that OsPLCl is involved in salt stress signal transduction pathway by hydrolyzing PtdIns4P and PtdIns(4,5)P2, regulating downstream messengers such as InsP2、InsP3、DAG, and indirectly affecting Ca2+ oscillation response to salt stress. OsPLC1 displays a positive role in rice tolerance to salt stress.更多还原