Shi Fu, Li Li, Wang Yaqiong, Guan Yanbin, Wu Ya'Nan, Zhang Qian, Chang Junli, Chen Mingjie, Wang Yuesheng, Li Sanhe, Zhang Xuebin, He Guangyuan, Li Yin, Yang Guangxiao
The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, the Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science & Technology, Wuhan 430074, China.
Key Laboratory of Crop Molecular Breeding, Ministry of Agriculture and Rural Affairs, Hubei Key Laboratory of Food Crop Germplasm and Genetic Improvement, Food Crops Institute, Hubei Academy of Agricultural Sciences, Wuhan 430068, China.
J Adv Res. 2025 Jun 5. doi: 10.1016/j.jare.2025.06.004.
Drought poses a significant environmental challenge, disrupting plant growth and reducing crop productivity. As sensors and effectors in calcium signaling, calcium-dependent protein kinases (CPKs) regulate plant development and environmental adaptation. However, the specific roles and molecular networks of many OsCPKs in rice adaptation to abiotic stress remain to be elucidated.
Drought-tolerant rice holds significant potential for enhancing yield stability and global food security. The study aimed to isolate drought-responsive OsCPKs and explore their molecular mechanisms.
To elucidate the mechanisms underlying OsCPK9-mediated drought tolerance, we integrated assays of Co-immunoprecipitation, yeast two-hybrid, GST pull-down and bimolecular fluorescence complementation to validate the OsCPK9-interacting proteins. In vitro protein phosphorylation analysis was conducted to identify phosphorylated substrates and sites. Rice yield performance was evaluated through three-year field trials.
OsCPK9 overexpressing plants enhanced drought tolerance in rice, accompanied by lower HO content and higher catalase activity than wild-type (WT) plants. Conversely, OsCPK9 knockout plants exhibited sensitive to drought stress compared with WT plants. Myristoylation and palmitoylation contributed to plasma membrane localization of OsCPK9, which is required for drought tolerance. OsCPK9 interacts with and phosphorylates Catalase C (OsCATC) at the plasma membrane. Phosphorylation of OsCATC at the highly conserved Thr105 enhanced its self-interaction, promoting oligomer formation and thereby increasing catalytic activity. OsCATC overexpressing plants showed higher catalase activity and improved drought tolerance compared to WT plants. Moreover, OsCPK9 overexpressing plants mitigated a 17.6% average yield loss per plant relative to WT plants.
This study reveals that myristoylation and palmitoylation of OsCPK9 contribute to its plasma membrane localization and drought tolerance. The post-translational regulatory mechanism mediated by OsCPK9 enhances environmental adaptability and provides a promising strategy to improve rice grain yield under drought stress conditions.
干旱构成了重大的环境挑战，扰乱植物生长并降低作物生产力。作为钙信号传导中的传感器和效应器，钙依赖性蛋白激酶（CPK）调节植物发育和环境适应性。然而，许多水稻CPK（OsCPK）在水稻适应非生物胁迫中的具体作用和分子网络仍有待阐明。
耐旱水稻在提高产量稳定性和全球粮食安全方面具有巨大潜力。本研究旨在分离干旱响应型OsCPK并探索其分子机制。
为阐明OsCPK9介导的耐旱机制，我们整合了免疫共沉淀、酵母双杂交、GST下拉和双分子荧光互补分析，以验证与OsCPK9相互作用的蛋白。进行了体外蛋白质磷酸化分析以鉴定磷酸化底物和位点。通过为期三年的田间试验评估水稻产量表现。
过表达OsCPK9的植株增强了水稻的耐旱性，与野生型（WT）植株相比，其过氧化氢含量更低，过氧化氢酶活性更高。相反，与WT植株相比，敲除OsCPK9的植株对干旱胁迫敏感。肉豆蔻酰化和棕榈酰化有助于OsCPK9定位于质膜，这是耐旱所必需的。OsCPK9在质膜上与过氧化氢酶C（OsCATC）相互作用并使其磷酸化。OsCATC在高度保守的苏氨酸105处的磷酸化增强了其自身相互作用，促进寡聚体形成，从而增加催化活性。与WT植株相比，过表达OsCATC的植株表现出更高的过氧化氢酶活性和更好的耐旱性。此外，与WT植株相比，过表达OsCPK9的植株平均单株产量损失减轻了17.6%。
本研究表明，OsCPK9的肉豆蔻酰化和棕榈酰化有助于其质膜定位和耐旱性。由OsCPK9介导的翻译后调控机制增强了环境适应性，并为在干旱胁迫条件下提高水稻籽粒产量提供了一种有前景的策略。
