The aquaporin MePIP2;7 improves MeMGT9-mediated Mg^{2 +} acquisition in cassava

Aquaporins are important transmembrane water transport proteins which transport water and several neutral molecules. However, how aquaporins are involved in the synergistic transport of Mg²⁺ and water remains poorly understood. Here, we found that the cassava aquaporin MePIP2;7 was involved in Mg²⁺ transport through interaction with MeMGT9, a lower affinity magnesium transporter protein. Knockdown of MePIP2;7 in cassava led to magnesium deficiency in basal mature leaves with chlorosis and necrotic spots on their edges and starch over-accumulation. Mg²⁺ content was significantly decreased in leaves and roots of MePIP2;7-RNA interference (PIP-Ri) plants grown in both field and Mg²⁺ -free hydroponic solution. Xenopus oocyte injection analysis verified that MePIP2;7 possessed the ability to transport water only and MeMGT9 was responsible for Mg²⁺ efflux. More importantly, MePIP2;7 improved the transportability of Mg²⁺ via MeMGT9 as verified using the CM66 mutant complementation assay and Xenopus oocytes expressing system. Yeast two-hybrid, bimolecular fluorescence complementation, co-localization, and co-immunoprecipitation assays demonstrated the direct protein-protein interaction between MePIP2;7 and MeMGT9 in vivo. Mg²⁺ flux was significantly elevated in MePIP2;7-overexpressing lines in hydroponic solution through non-invasive micro-test technique analysis. Under Mg²⁺ -free condition, the retarded growth of PIP-Ri transgenic plants could be recovered with Mg²⁺ supplementation. Taken together, our results demonstrated the synergistic effect of the MePIP2;7 and MeMGT9 interaction in regulating water and Mg²⁺ absorption and transport in cassava.