钙离子（Ca²⁺）作为细胞内重要的第二信使，参与调控多种信号转导过程。细胞感受外界刺激后，会引发胞外或胞内钙库释放Ca²⁺，Ca²⁺传感器解码胞质中的Ca²⁺信号，将其信息转化为下游细胞反应。高渗促钙内流蛋白（hyperosmolality-induced [Ca²⁺]_i increase，OSCA）是目前发现的唯一能够直接感受外界渗透胁迫，并被激活的Ca²⁺机械通道。高渗刺激可以改变质膜上OSCA离子通道的亚基构型，促使离子通道的开放，实现Ca²⁺内流。拟南芥中共有15个OSCA蛋白，参与调控植物生长发育、气孔运动以及渗透胁迫应答等重要过程。其中OSCA1.1研究较为清楚，但关于OSCA1.5和OSCA1.6的功能研究较少，因此深入探究OSCA1.5/1.6调控拟南芥Ca²⁺和胁迫应答的分子机理，对挖掘抗性基因和培育改良新品种具有重要意义。

本研究构建了osca1.5/1.6双突变体作为实验材料，通过细胞生物学，分子生物学等手段明确了OSCA1.5/1.6参与调控拟南芥Ca²⁺和胁迫应答；研究发现磷脂酸（Phosphatidic acid，PA）能够与OSCA1.5/1.6相互作用，并验证了PA增强OSCA1的离子通道活性。主要研究结果如下：

1.外源添加脱落酸（Abscisic acid，ABA）、过氧化氢（H₂O₂）和Ca²⁺，观察其对osca1.5/1.6气孔运动的影响，发现与野生型（Wild type，WT）相比，osca1.5/1.6的气孔关闭受到抑制，表现为不敏感表型。在根部伸长和种子萌发阶段osca1.5/1.6也表现为ABA不敏感表型。渗透和热胁迫处理后，与WT相比，osca1.5/1.6根部伸长受阻更明显，其幼苗死亡率显著升高。以上生理实验验证了OSCA1.5/1.6在拟南芥的多种非生物胁迫过程中发挥调控作用。

2.通过染色观察osca1.5/1.6子叶气孔中的Ca²⁺和ROS水平，结果显示，突变体中的Ca²⁺、H₂O₂和ROS水平均显著低于WT，ABA处理诱导WT子叶气孔中的Ca²⁺、H₂O₂和ROS水平升高，导致染色程度及荧光亮度加深；在osca1.5/1.6突变体中这种诱导被显著抑制。这表明OSCA1.5/1.6参与调控拟南芥钙信号及ROS水平。

3.构建了OSCA1.5-GFP和OSCA1.6-GFP表达载体，侵染拟南芥获得转基因遗传材料，激光共聚焦显微镜观察其亚细胞定位。发现OSCA1.5和OSCA1.6均定位于拟南芥根尖细胞的细胞核中。

4.osca1.5/1.6突变体对PA介导的气孔关闭和根部伸长不敏感。通过脂-蛋白结合和ELISA实验证实了OSCA1.5和OSCA1.6的C端蛋白可以与PA特异性结合，且该结合具有浓度依赖性。突变OSCA1.5的741和742以及OSCA1.6的742和743位置的赖氨酸和精氨酸后，OSCA1.5^KRGG和OSCA1.6^KRGG与PA的结合均显著减弱。

5.利用非洲爪蟾卵母细胞体系验证OSCA1.1的钙离子通道活性。在正常渗透条件下，表达OSCA1.1的卵母细胞无Ca²⁺转运活性，高渗处理可以激活OCSA1.1的Ca²⁺转运能力；外源PA处理可以增加OSCA1.1的Ca²⁺转运能力。关键氨基酸位点突变后的OSCA1.1^KRGG丧失Ca²⁺转运活性。

综上所述，本研究初步揭示了OSCA1.5/1.6调控拟南芥钙信号和胁迫响应的分子机理；证实了OSCA1.5/1.6与PA相互结合并鉴定到两个关键的氨基酸结合位点；明确了PA对OSCA1钙离子通道活性的增强作用。本研究丰富了人们对OSCA基因家族的认知，为后续对OSCA1.5和OSCA1.6的生物学功能研究奠定重要的理论基础。

Calcium ions (Ca²⁺) act as essential second messengers regulating diverse cellular signaling processes. Upon external stimuli, Ca²⁺ is released from extracellular or intracellular stores, with cytosolic sensors translating these signals into downstream responses. The hyperosmolality-induced [Ca²⁺]i increase (OSCA) channels represent the sole identified Ca²⁺-permeable mechanosensors directly activated by osmotic stress through plasma membrane subunit reconfiguration, enabling Ca²⁺ influx. Arabidopsis thaliana contains 15 OSCA members implicated in growth, stomatal dynamics, and osmotic adaptation. Although OSCA1.1 has been well characterized, OSCA1.5/1.6 remain functionally obscure. Elucidating their molecular mechanisms in Ca²⁺ signaling and stress regulation holds significance for stress-resistance gene discovery and crop improvement.

This study used an osca1.5/1.6 double mutant. Through cell and molecular biology methods, it clarified that OSCA1.5/1.6 regulates Ca²⁺ and stress responses in Arabidopsis. It found phosphatidic acid (PA) interacts with OSCA1.5/1.6 and verified PA enhances OSCA1 ion channel activity. Main results are as follows:

1.Exogenous abscisic acid (ABA), hydrogen peroxide (H₂O₂), and Ca²⁺ were added to observe their impacts on osca1.5/1.6 stomatal movement. Results showed that, unlike the wild type (WT), stomatal closure of osca1.5/1.6 was inhibited, presenting an insensitive phenotype. During root elongation and seed germination, osca1.5/1.6 was also insensitive to ABA. After osmotic and heat stress, osca1.5/1.6 root elongation was more inhibited and seedling mortality increased significantly compared to the WT. These physiological experiments confirmed that OSCA1.5/1.6 regulates various abiotic stress processes in Arabidopsis.

2.Staining was used to observe Ca²⁺ and ROS levels in osca1.5/1.6 cotyledon stomata. The mutant had significantly lower Ca²⁺, H₂O₂, and ROS levels than the WT. ABA treatment induced an increase in Ca²⁺, H₂O₂, and ROS in WT cotyledon stomata, with deeper staining and brighter fluorescence, which was notably inhibited in the osca1.5/1.6 mutant. This suggests that OSCA1.5/1.6 regulates calcium signals and ROS levels in Arabidopsis.

3.OSCA1.5-GFP and OSCA1.6-GFP expression vectors were constructed, and Arabidopsis was infected to obtain transgenic genetic materials. Laser confocal microscopy was used to observe their subcellular localization. It was found that both OSCA1.5 and OSCA1.6 were localized in the nuclei of the root tip cells of Arabidopsis.

4.The osca1.5/1.6 mutant was insensitive to PA-mediated stomatal closure and root elongation. Lipid-protein binding and ELISA experiments confirmed that the C-terminal proteins of OSCA1.5 and OSCA1.6 could specifically bind to PA, and this binding was concentration-dependent. After mutating the lysine and arginine at positions 741 and 742 of OSCA1.5 and positions 742 and 743 of OSCA1.6, the binding of both OSCA1.5^KRGG and OSCA1.6^KRGG to PA was significantly weakened.

5.The calcium ion channel activity of OSCA1.1 was verified using the Xenopus laevis oocyte system. Under normal osmotic conditions, the oocytes expressing OSCA1.1 had no Ca²⁺ transport activity. Hyperosmotic treatment could activate the Ca²⁺ transport ability of OCSA1.1, and exogenous PA treatment could increase the Ca²⁺ transport ability of OSCA1.1. OSCA1.1^KRGG with mutated key amino acid sites lost its Ca²⁺ transport activity.

Overall, this study uncovers molecular mechanisms of how OSCA1.5/1.6 regulates calcium signals and stress responses in Arabidopsis. It validates binding between OSCA1.5/1.6 and PA, pinpoints two key binding sites, and clarifies PA’s enhancing effect on OSCA1 calcium channel activity. This work enriches understanding of the OSCA gene family and paves the way for further research on OSCA1.5/1.6 biological functions.