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

Key message 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. Abstract 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.