Wave-like SnRK1 Activation and Tre6P–Sucrose Imbalance Shape Early Salt Stress Response in Growing Alfalfa Leaves

Abstract Alfalfa (Medicago sativa L.) is a key forage crop valued for its adaptability and nutritional quality, yet salinity significantly limits its productivity, particularly in arid regions. Understanding early stress responses is crucial for improving resilience. Salt stress impairs leaf growth and photosynthesis, triggering complex, time-dependent signaling. Sucrose non-fermenting kinase 1 (SnRK1), a central metabolic sensor, regulates metabolism and growth under stress. We investigated the dynamics of SnRK1, sucrose, and trehalose-6- phosphate (Tre6P) during leaf expansion in a salt-tolerant alfalfa cultivar. Plants were hydroponically grown and exposed to 200 mM NaCl. Stress induced transient declines in chloroplast performance (Fv/Fm, performance index). SnRK1 activity peaked within 1 hour post-treatment (hpt), likely initiating metabolic shifts. By 3 hpt, sugar metabolism shifted, with increased catabolism, TCA cycle modulation, and glucose-6-phosphate accumulation. SnRK1 and sucrose showed opposing wave-like patterns, with sucrose peaking at 1 day post-treatment (dpt) as Tre6P declined, indicating a disrupted regulatory link. A second SnRK1 peak at 3 dpt correlated with reduced leaf growth. Exogenous sucrose inhibited SnRK1, while NaCl enhanced it. This is the first report of wave-like SnRK1 activation and Tre6P–sucrose uncoupling in alfalfa, highlighting early SnRK1 activation as key to salt stress adaptation. Highlights Early SnRK1 activation is a key determinant of salt stress response in alfalfa, linking early biochemical shifts to downstream metabolic alteration. First evidence of a wave-like SnRK1 activation pattern and disruption of the Tre6P– Sucrose nexus reveals novel dynamics in alfalfa’s stress signaling. Sucrose inhibits SnRK1 activity in source leaves with or without NaCl Competing Interest Statement The authors have declared no competing interest.