Plasma membrane-localized H⁺-ATPase OsAHA3 functions in saline-alkaline stress tolerance in rice

A novel function of plasma membrane-localized H⁺-ATPase, OsAHA3, was identified in rice, which is involved in saline-alkaline tolerance and specifically responds to high pH during saline-alkaline stress. Saline-alkaline stress causes serious damage to crop production on irrigated land. Plants suffer more severe damage under saline-alkaline stress than under salinity stress alone. Plasma membrane-localized proton (H⁺) pump (H⁺-ATPase) is an important enzyme that controls plant growth and development by catalyzing H⁺ efflux and enabling effective charge balance. Many studies about the role of plasma membrane H⁺-ATPases in saline-alkaline stress tolerance have been reported in Arabidopsis, especially on the AtAHA2 (Arabidopsis thaliana H⁺-ATPase 2) gene; however, whether and how plasma membrane H⁺-ATPases play a role in saline-alkaline stress tolerance in rice remain unknown. Here, using the activation-tagged rice mutant pool, we found that the plasma membrane-localized H⁺-ATPase OsAHA3 (Oryza sativa autoinhibited H⁺-ATPase 3) is involved in saline-alkaline stress tolerance. Activation-tagged line 29 (AC29) was identified as a loss-of-function mutant of OsAHA3 and showed more severe growth retardation under saline-alkaline stress with high pH than under salinity stress. Moreover, osaha3 loss-of-function mutants generated by CRISPR/Cas9 system exhibited saline-alkaline stress sensitive phenotypes; staining of leaves with nitrotetrazolium blue chloride (NBT) and diaminobenzidine (DAB) revealed more reactive oxygen species (ROS) accumulation in osaha3 mutants. OsAHA3-overexpressing plants showed increased saline-alkaline stress tolerance than wild-type plants. Tissue-specific expression analysis revealed high expression level of OsAHA3 in leaf, sheath, glume, and panicle. Overall, our results revealed a novel function of plasma membrane-localized H⁺-ATPase, OsAHA3, which is involved in saline-alkaline stress tolerance and specifically responds to high pH.