The interaction complexes of TsHKT1 splicing variants enhance salt tolerance of Thellungiella salsuginea by decreasing Na+ uptake

Abstract

HKT1 is a membrane-localized Na⁺/K⁺ cotransporter that enables plants adaptation to abiotic stresses. Extreme abiotic stresses can induce extensive alternative splicing (AS) of various genes in plants. However, the mechanisms by how splice variants contribute to stress tolerance in Thellungiella salsuginea remain unclear. Here, we demonstrated that high salinity triggers widespread AS events in T. salsuginea, with three HKT1 genes (TsHKT1;1, TsHKT1;2, and TsHKT1;3) producing six splice variants. These proteins encoded by splice variants form interaction complexes in the plasma membranes of root epidermal cells, which are capable of reducing Na⁺ uptake. Specifically, Heterologous expressed TsHKT1s in yeast indicated that TsHKT1;1, TsHKT1;2b, and TsHKT1;3 preferentially transport Na⁺ with limited K⁺ uptake, whereas the interaction complexes exhibit enhanced K⁺ uptake capacity. Ectopically expressed TsHKT1;1, TsHKT1;2a, TsHKT1;2b and TsHKT1;3 in Arabidopsis conferred salt sensitivity, while co-expression of TsHKT1;1 with TsHKT1;2b or TsHKT1;3 improved salt tolerance in Arabidopsis. Electrophysiological analyses revealed that roots of transgenic Arabidopsis coexpressing TsHKT1;1-TsHKT1;2b or TsHKT1;1-TsHKT1;3 exhibited reduced Na⁺ influx, particularly under low K⁺ and high salt conditions. These findings demonstrate that T. salsuginea employs AS to generate novel protein interaction complexes that mitigate ion toxicity, which suggests this mechanism is widely utilized by halophytes to survive in harsh environments.