Enhancing Rice Salinity Tolerance with Lignite Fulvic Acid: Structural Determinants of Molecular Weight Fractionations and Regulation Mechanisms of Plasma Membrane H+-ATPase/SOS1

Fulvic acid (FA) has been widely used as a plant growth regulator to alleviate salt stress. However, the mechanism by which it enhances salt tolerance in rice remains unclear. This study systematically investigated the chemical structure of FA and its role in enhancing rice salt tolerance through the regulation of plasma membrane (PM) H + -ATPase. The results demonstrated that the increased molecular weight (MW) of FA companied by progressive aromaticity enhancement and functional group depletion. Principal component analysis revealed that low-MW FA fractions (< 3 kDa), enriched in carboxylic-C, phenolic-C, and lignin-like substances, exhibited a strong positive correlation with root growth, membrane potential (MP) hyperpolarization, and Na + efflux under 100 mmol/L NaCl stress. Mechanistically, FA directly activated PM H + -ATPase (activity increased by 30.3%‒86.2%), generating a proton gradient that energized SOS1 to mediate Na + efflux, leading to salt tolerance in rice. Genetic validation using osa1 knockout mutants confirmed the indispensable role of PM H + -ATPase in FA-induced responses, as both MP and Na + efflux remained unaltered upon FA1‒FA5 treatment. This research found that FA fractions with high functional group density and low aromaticity can serve as optimal plant biostimulants for salt stress mitigation, providing a new strategy for sustainable lignite valorization in precision agriculture.