干旱胁迫和盐胁迫是影响植物生长发育最重要的非生物胁迫因素。研究者对植物感受 及响应干旱胁迫和盐胁迫的级联途径、基因表达及生理应答做了大量研究。然而，对植物 如何感受干旱胁迫和盐胁迫的机制仍然不清楚。前期研究表明，干旱胁迫和盐胁迫对植物 造成的第一相反应是高渗胁迫，并快速引起细胞内钙离子的增加。根据此特征，裴真明实 验 室 发现了拟南芥介导 渗透胁迫 引起胞内钙离子增加 的 渗 透 胁 迫 感 受 受 体 （Hyperosmolarity-induced [Ca2+]i increase 1, OSCA1）。本论文在前期对水稻 OsOSCA 家族 进行进化和表达分析的基础上，研究该受体家族成员 OsOSCA1.4 感知高渗和高盐引起的 渗透胁迫的作用机制。主要研究结果如下：

首先，利用动物细胞 HEK293 表达水稻 OsOSCA 家族成员并结合 Fura-2 染料测定胞 内钙离子变化的方法，筛选感受高渗胁迫引起胞内钙离子增加的 OsOSCA 成员。结果表明， OsOSCA1.4 可以介导细胞感知高渗和高盐引起的渗透胁迫信号，引起胞内钙离子浓度增 加。利用 EGTA 和 LaCl3 处理转化 OsOSCA1.4 的动物细胞并测定高渗引起的胞内钙离子增 加的结果表明，OsOSCA1.4 介导胞外钙离子内流入胞质，引起胞内钙离子增加。利用膜片 钳技术测定转化 OsOSCA1.4 动物细胞的离子通透特性。结果表明，OsOSCA1.4 可能是一 种钙透过的非选择性阳离子通道，并且介导感知高渗和高盐造成的渗透胁迫信号，引起胞 外钙离子内流进入胞内，产生一个内向的全细胞电流。对 OsOSCA1.4 进行动物细胞中亚 细胞定位实验。结果表明，OsOSCA1.4 定位于 HEK293 细胞的质膜上。以上结果证明， OsOSCA1.4 编码一种钙透过非选择性阳离子通道，动物体系中该离子通道感知高渗和高盐 引起的渗透胁迫，导致胞外钙离子内流及胞内钙离子快速增加。

其次，利用转 Cameleon YC3.6 的转基因水稻检测幼苗根部位响应高渗和高盐引起胞 内钙离子变化的结果表明，野生型水稻中高渗和高盐都能够引起根细胞内钙离子的增加。 然而，水稻 T-DNA 插入突变体 ososca1.4 中，高渗胁迫引起的胞内钙离子增加受到部分抑 制，而高盐胁迫引起的胞内钙离子增加反而会稍微增加。另外，应用水稻原生质体细胞瞬 转体系对 OsOSCA1.4 的亚细胞定位分析表明，OsOSCA1.4 定位于质膜上。这些结果证明， 定位于质膜的水稻 OsOSCA1.4 介导细胞感知高渗胁迫信号而引起胞内钙离子增加；而高 盐胁迫引起的胞内钙离子增加，除 OsOSCA1.4 外可能还有其它蛋白发挥作用，存在功能 冗余的现象。

再次，利用 RNA-Seq 高通量测序技术对 OsOSCA1.4 介导的高渗和高盐诱导基因进行 了分析。本研究中，利用 125 mM sorbitol 和 75 mM NaCl 分别处理五叶期野生型水稻 ZH11和突变体 ososca1.4。在处理前（0 h）、处理后 1 h 和 36 h 分别取样，然后提取 RNA 进行 表达谱测序分析。结果表明，OsOSCA1.4 介导且共同响应高渗胁迫和高盐胁迫的胁迫响应 基因数目在处理 1 h 时是 65 个，在处理 36 h 时是 206 个。OsOSCA1.4 介导且特异响应高 渗胁迫的基因数目在处理 1 h 时是 28 个，在处理 36 h 时是 253 个。OsOSCA1.4 介导且特 异响应高盐胁迫的基因数目在处理 1 h 时是 294 个，在处理 36 h 时是 241 个。

最后，鉴于 OsOSCA1.4 蛋白介导的高渗胁迫和高盐胁迫的响应过程存在差异。因此 进一步研究了胁迫处理 36 h 时 OsOSCA1.4 介导的胁迫响应基因在“分子功能”通路上 GO 富集的差异。结果表明，OsOSCA1.4 介导的响应高渗胁迫的基因显著地富集在了 8 条重要 的“分子功能”通路中，包括“kinase activity”（激酶活性）、 “DNA binding transcription factor activity”（DNA 结合转录因子活性）以及“nucleoside phosphate binding”（核苷酸磷 酸基团结合）等等；而 OsOSCA1.4 介导的响应高盐胁迫的基因只显著地富集在了“hydrolase activity”（水解酶活性）和“catalytic activity”（催化活性）通路上。基因的启动子元件分 析结果表明，富集在相同通路上的基因具有相似的启动子元件，例如 OsOSCA1.4 介导响 应高渗胁迫的“DNA binding transcription factor activity”（DNA 结合转录因子活性）通路 上 13 表达上调基因中有 11 个基因的启动子都含有序列为“G/CGG/CCCCCACCA/C”的 元件，有 10 个基因的启动子都含有序列为“GCTTCCTCCCCC”的元件。根据以上结果 可以推测，OsOSCA1.4 功能缺失影响了其介导的重要“分子功能”通路上基因的表达，最 后阻碍了水稻植株对高渗胁迫产生及时有效的响应，例如气孔的关闭过程等。而在整个植 株产生的高盐胁迫响应过程中 OsOSCA1.4 介导的胁迫响应基因可能并没有充当重要的角 色，因此 OsOSCA1.4 的功能缺失并不会影响水稻植株对高盐胁迫信号的感知。

综上所述，OsOSCA1.4 编码一种定位于质膜的水稻钙透过非选择性阳离子通道，该离 子通道感知高渗和高盐引起的渗透胁迫，导致胞外的钙离子内流及胞内钙离子快速增加。 水稻根细胞对高渗和高盐胁迫响应机制的研究结果表明，水稻 OsOSCA1.4 介导高渗胁迫 引起的胞内钙离子增加；而除了 OsOSCA1.4 介导细胞感知高盐胁迫引起胞内钙离子增加 外，可能还有其它蛋白发挥作用，存在功能冗余的现象。根据“分子功能”通路的 GO 富 集结果也可以推测 OsOSCA1.4 介导的胁迫响应基因参与了更重要的植物高渗胁迫响应过 程，而 OsOSCA1.4 介导的胁迫响应基因在高盐胁迫响应过程中起到的作用并不是最关键 的。除了 OsOSCA1.4 基因外，OsOSCAs 基因家族的另外 10 个成员在胁迫信号感知过程中 也可能起着重要作用。OsOSCAs 基因家族的功能研究给植物细胞中高渗胁迫和高盐胁迫信 号感知通路网络的解析奠定了坚实的基础。

Drought stress and salt stress are the most important abiotic stress factors that affect plant growth and development. Perception of drought stress and salt stress triggered the different signaling cascade pathway, gene expression and physiological responses in plant. However, the mechanisms on plant perceiving drought stress and salt stress remain unclear. Previous studies showed that the first phase of drought stress and salt stress is the hyperosmolality stress, which induced the rapid elevation of cytoplasmic Ca2+ ([Ca2+]i). According to this, Yuan et al had found an osmosensing receptor mediating hyperosmolality induced [Ca2+]i increases (OSCA) in Arabidopsis. Based on the evolution and expression analysis of the OSCA family in rice, we have studied the the mechanism of OsOSCA1.4 mediating osmotic stress signaling pathway induced by hyperosmolality and high salinity. The detail results are as follows:

1. Using ratiometric imaging to measure hyperosmolality induced [Ca2+]i mediated by OsOSCAs in HEK293 cells, we found that one of ten OsOSCAs, OsOSCA1.4, mediates both of the osmotic stress-induced cytosolic [Ca2+] increase (OICIcyt) and the salt stress-induced cytosolic [Ca2+] increase (SICIcyt) in HEK293 cells. Then, patch clamp technique for detecting the ion permeability of OsOSCA1.4 in HEK293 cells showed that OsOSCA1.4 was a calcium-permeable non-selective cation channel, which generated an inward wholl-cell current induced by hyperosmolality and high salt. We also found that OsOSCA1.4-YFP was localized in the plasma membrane in HEK293.

2. Using calcium indicator Cameleon YC3.6 to detect cytosolic [Ca2+] changes in rice root cells, we found that the OICIcyt and SICIcyt were happened in wild-type rice ZH11. However, OICIcyt, but not SICIcyt, was disrupted in T-DNA mutant line ososca1.4. We further showed that OsOSCA1.4 was localized on the plasma membrane in Arabidopsis leaf mesophyll protoplasts. These results indicate that the membrane-localized OsOSCA1.4 mediates rice root cells perceiving hyperosmolality stress and induced [Ca2+]i increases. However, SICIcyt is still mediated by other uncharacerized mechnism in plants.

3. To analyze the OsOSCA1.4 regulated differentially expressed genes in response to hyperosmolality stress or high-salinity stress, wild-type rice ZH11 and mutant rice ososca1.4 were treated with 125 mM sorbitol and 75 mM NaCl respectively. We collected the whole plants of five-leaf rice seedling before treatment for control (WT for wild type ZH11 and os1_4 for rice mutant ososca1.4), as well as 1 hour and 36 hours post-sorbitol treatment (WTS1 and WTS36, os1_4S1 and os1_4S36), and 1 hour and 36 hours post-sodium chloride treatment (WTN1 and WTN36, 124 os1_4N1 and os1_4N36). There were totally 10 samples for RNA sequencing. The RNA sequencing results indicate that there are 65 OsOSCA1.4-mediated DEGs at 1 h post-treatment and 206 OsOSCA1.4-mediated DEGs at 36 h post-treatment which respond to both of sorbitol stress and sodium chloride stress; the amount of OsOSCA1.4-mediated specific-stress-responsive DEGs are 28 for S1 (at 1 h post sorbitol treatment), 294 for N1 (at 1 h post NaCl treatment), 253 for S36 (at 36 h post sorbitol treatment) and 241 for N36 (at 36 h post NaCl treatment).

4. Considering that the differences existed between the responsive phenotype of hyperosmolality and high-salinity stress, GO enrichment analysis was performed with the OsOSCA1.4-mediated specific-stress-responsive DEGs at 36 h post stress treatment. The results reveals that OsOSCA1.4-mediated specific-stress-responsive genes at 36 h post sorbitol treatment are enriched in 8 crucial ‘molecular function’ pathway terms, such as ‘kinase activity’, ‘DNA binding transcriptional factor activity’, ‘nucleotide phosphate binding’ and so on. OsOSCA1.4-mediated specific-stress-responsive genes at 36 h post NaCl treatment are only enriched in two pathways, including ‘catalytic activity’ and ‘hydrolase activity’. Promoter motif analysis indicates that OsOSCA1.4-mediated stress responsive genes enriched in the same pathway contain some consensus motifs, such as promoter motifs of 11 up-regulated genes in the pathway ‘DNA binding transcriptional factor activity’ containing sequence ‘G/CGG/CCCCCACCA/C’ and promoter motifs of 10 up-regulated genes in this pathway containing sequence ‘GCTTCCTCCCCC’. According to these results, we can speculate that loss function of OsOSCA1.4 protein affects downstream genes expression in some significant patnways, which finally represses the timely and effective response of rice plants to hyperosmotic stress, such as the closing process of stomata. The high-saltinity stress response genes mediated by OsOSCA1.4 may not play an important role in the process of high-salinity stress response to the whole plant, so lack of function of OsOSCA1.4 does not impede the perception of high salt stress signals in rice.

In summary, research results about hyperosmolality and high salt stress perception in animal cells indicate that OsOSCA1.4 gene encodes a calcium permeable non-selective cation channel which perceives hyperosmotic stress signal induced by hyperosmolality and high salt, inducing a calcium ion influx and a quick [Ca2+]i increases. Studies on the perception mechanism of hyperosmolality and high salt stress in rice root cells show that plasma membrane localized OsOSCA1.4 mediates rice root cells perceiving hyperosmolality stress and induces [Ca2+]i increases; but functional redundancy exists in the process of perceiving high-salinity stress except OsOSCA1.4. According to GO enrichment results in "molecular function" pathway, it’s also possible to speculate that OsOSCA1.4 mediated stress response genes participate in more important hyperosmotic stress response processes, and the role of OsOSCA1.4 mediated stress response genes in the process of high-salinity stress response is not the most critical. In addition to the OsOSCA 1.4 gene, another 10 members of OsOSCAs gene family may also function significantly in the process of stress signal perceiving. The functional research of OsOSCAs gene family has laid a solid foundation for analyzing signal transduction pathways of hyperosmolality and high-salinity stress in plant cells.