bZIP‐Mediated Activation of a Late Embryogenesis Abundant Protein MsLEA2 Enhances Aluminium Tolerance in Medicago sativa

The phytohormone abscisic acid (ABA) is rapidly induced in roots exposed to aluminium (Al) stress, yet the mechanisms by which it modulates Al-detoxification genes remain elusive. Here we uncover a hierarchical ABA-responsive regulatory network that orchestrates a Late Embryogenesis Abundant (LEA)_2 protein MsLEA2, which modulates Al tolerance in alfalfa (Medicago sativa L.). Using yeast one-hybrid (Y1H) assays, luciferase complementation imaging (LCI), mutagenesis, and electrophoretic mobility shift assays (EMSA), we show that three antagonistic bZIP transcription factors-MsABF2 (activator), MsDPBF3 (hyper-activator) and MsGBF1 (repressor)-dynamically regulate MsLEA2 expression through spatially distinct ABRE motifs, enabling precise transcriptional control. Functional analyses reveal that MsLEA2 acts as a multifunctional hub, that promotes (i) oxalate exudation via interactions with aquaporins (MsPIP2;1, MsPIP2;2, MsTIP1;1), (ii) reactive oxygen species (ROS) scavenging, and (iii) cell wall remodelling through binding to UDP-xylose synthase (MsUXS1). Golgi-targeted MsLEA2 facilitates apoplastic Al exclusion, a mechanism distinct from its cytoplasmic homologue AtLEA14, underscoring the importance of subcellular compartmentalisation in Al detoxification. Our findings establish LEA_2 proteins as pivotal components of secretory-pathway-mediated Al tolerance and provide actionable targets for engineering crops resilient to acidic soils.