A salt-signaling network involving ethylene, extracellular ATP, hydrogen peroxide, and calcium mediates K⁺ /Na⁺ homeostasis in Arabidopsis

This work aimed at investigating the interactive effects of salt-signaling molecules, i.e., ethylene, extracellular ATP (eATP), H₂ O₂, and cytosolic Ca²⁺ ([Ca²⁺ ]_cyt), on the regulation of K⁺ /Na⁺ homeostasis in Arabidopsis thaliana. The presence of eATP shortened Col-0 hypocotyl length under no-salt conditions. Moreover, eATP decreased relative electrolyte leakage and lengthened root length significantly in salt-treated Col-0 plants but had no obvious effects on the ethylene-insensitive mutants etr1-1 and ein3-1eil1-1. Steady-state ionic flux kinetics showed that exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) and eATP-Na₂ (an eATP donor) significantly increased Na⁺ extrusion and suppressed K⁺ loss during short-term NaCl treatment. Moreover, ACC remarkably raised the fluorescence intensity of salt-elicited H₂ O₂ and cytosolic Ca²⁺. Our qPCR data revealed that during 12 h of NaCl stress, application of ACC increased the expression of AtSOS1 and AtAHA1, which encode the plasma membrane (PM) Na⁺ /H⁺ antiporters (SOS1) and H⁺-ATPase (H⁺ pumps), respectively. In addition, eATP markedly increased the transcription of AtEIN3, AtEIL1, and AtETR1, and ACC treatment of Col-0 roots under NaCl stress conditions caused upregulation of AtRbohF and AtSOS2/3, which directly contribute to the H₂ O₂ and Ca²⁺ signaling pathways, respectively. Briefly, ethylene was triggered by eATP, a novel upstream signaling component, which then activated and strengthened the H₂ O₂ and Ca²⁺ signaling pathways to maintain K⁺ /Na⁺ homeostasis under salinity.