Gain-of-function mutations of AtNHX1 suppress sos1 salt sensitivity and improve salt tolerance in Arabidopsis

Soil salinity severely hampers agricultural productivity.Under salt stress,excess Na+accumulation causes cellular damage and plant growth retardation,and membrane Na+transporters play central roles in Na+uptake and exclusion to mitigate these adverse effects.In this study,we performed sos1 suppressor mutant(named sup)screening to uncover potential genetic interactors of SOS1 and additional salt tolerance mechanisms.Map-based cloning and sequencing identified a group of mutants harboring dominant gain-of-function mutations in the vacuolar Na+/H+antiporter gene AtNHX1.The gain-of-function variants of AtNHX1 showed enhanced transporter activities in yeast cells and increased salt tolerance in Arabidopsis wild type plants.Ion content measurements indicated that at the cellular level,these gain-of-function mutations resulted in increased cellular Na+accumulation likely due to enhanced vacuolar Na+sequestration.However,the gain-of-function suppressor mutants showed reduced shoot Na+but increased root Na+accumulation under salt stress,indicating a role of AtNHX1 in limiting Na+translocation from root to shoot.We also identified another group of sos1 suppressors with loss-of-function mutations in the Na+transporter gene AtHKT1.Loss-of-function mutations in AtHKT1 and gain-of-function mutations in AtNHX1 additively suppressed sos1 salt sensitivity,which indicates that the three transporters,SOS1,AtNHX1 and AtHKT1 function independently but coordinately in controlling Na+homeostasis and salt tolerance in Arabidopsis.Our findings provide valuable information about the target amino acids in NHX1 for gene editing to improve salt tolerance in crops.