Abstract: Saline–alkali stress is one of the major abiotic factors limiting crop production and affecting the ecological environment. The plasma membrane (PM) H⁺-ATPases are involved in modulating the membrane potential in response to alkaline stress. The central loop (cytoplasmic domain) of the PM H⁺-ATPase AHA2, in contrast to its well-studied C-terminal regulatory domain, remains poorly understood in terms of its regulatory function. In this study, we found that CARK1 and CARK3 (cytosolic ABA receptor kinase 1 and 3) positively modulate saline–alkali stress tolerance in Arabidopsis. Using molecular biology and biochemistry approaches, we reveal that CARK1 and CARK3 interact with and phosphorylate AHA2 at Thr469 in the central loop domain. Molecular mechanism indicates that CARK1/3-mediated phosphorylation elevates AHA2 activity through two key actions: First, by increasing Thr947 phosphorylation and promoting binding to 14-3-3 protein, and second, by releasing autoinhibitory interaction between the C-terminus and the central loop of AHA2. Functional and genetic analyses reveal that the phosphorylation-mimicking mutation AHA2^T469D dramatically rescues hypersensitivity to alkali tolerance, H⁺ efflux, and cytosolic ROS accumulation in aha2 and cark1/3aha2 triple mutants. Collectively, our work reveals the central regulatory loop of AHA2 in response to alkali stress and reports that its activity is enhanced through Thr469 phosphorylation by CARK1/3.

Key words: alkaline stress, phosphorylation, PM H⁺-ATPase AHA2, the CARK family