The SCL6-AGL80-GAPC2 module regulates tolerance to aluminum toxicity via malate efflux and the reactive oxygen...

Aluminum (Al) toxicity severely limits agricultural productivity in acidic soils worldwide. Although Al tolerance mechanisms have been extensively studied in crops, the molecular basis in forage crops remains poorly understood. Here, we investigated the physiological and transcriptomic responses of white clover (Trifolium repens L.) seedlings to Al toxicity. Al exposure triggered reactive oxygen species (ROS) accumulation and significantly inhibited plant growth and development. RNA-seq revealed a MADS-box transcription factor gene, TrAGL80, which exhibited strong induction under Al toxicity. Interference and silencing of TrAGL80 led to increased ROS levels and reduced malate secretion; conversely, heterologous expression of TrAGL80 in alfalfa (Medicago sativa) improved Al tolerance. We found that TrAGL80 interacts with TrGAPC2 in the nucleus and cytoplasm and that silencing TrGAPC2 reduces white clover Al tolerance. Additionally, we identified TrSCL6 as a negative regulator of TrAGL80 expression, and TrSCL6 negatively regulates Al tolerance by inhibiting malate secretion in white clover. Taken together, our findings unveil a TrSCL6-TrAGL80-TrGAPC2 regulatory module that plays a critical role in mediating Al tolerance in white clover, providing valuable genetic targets for breeding improved Al-resistant forage crops.