Ca2+ sensor CBL2/3-CIPK3/9/26 phosphorylates MTP11 to modulate manganese efflux via degradation and Golgi retention

Abstract Manganese (Mn) is essential for plant growth and development, yet plants in acidic and waterlogged soils are vulnerable to Mn2+ toxicity due to increased bioavailability. Metal tolerance protein 11 (MTP11) is a key Mn transporter in Arabidopsis (Arabidopsis thaliana) mediating Golgi-dependent exocytosis under high Mn stress; however, its regulatory mechanisms are thus far unknown. Here, we determined that complexes composed of calcineurin B-like proteins (CBL2/3) and CBL-interacting protein kinases (CIPK3/9/26) interact with and phosphorylate MTP11, mainly at Ser194/201, both in vitro and in vivo. Phospho-null mutants MTP11S194A and MTP11S201A displayed high tolerance to Mn stress, while phospho-mimetic mutants MTP11S194D and MTP11S201D were sensitive. Furthermore, CBL2/3-CIPK3/9/26-mediated phosphorylation promoted MTP11 degradation via the 26S proteasome and restricted its trafficking to the plasma membrane, confining MTP11 to the endomembrane system. In summary, our findings establish that the CBL2/3-CIPK3/9/26 module fine-tunes Mn homeostasis by suppressing MTP11 function through degradation and spatial retention, preventing excessive efflux of cellular Mn. This work advances understanding of post-translational control mechanisms underlying plant adaptation to Mn toxicity.