The aquaporin MePIP2;7 improves MeMGT9-mediated Mg2+ 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 Me PIP2;7 was involved in Mg2+transport through interaction with Me MGT9, a lower affinity magnesium transporter protein. Knockdown of Me PIP2;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 Me PIP2;7-RNA interference(PIP-Ri) plants grown in both field and Mg2+-free hydroponic solution. Xenopus oocyte injection analysis verified that Me PIP2;7 possessed the ability to transport water only and Me MGT9 was responsible for Mg^(2+)effux.More importantly, Me PIP2;7 improved the transportability of Mg^(2+)via Me MGT9 as verified using the CM66 mutant complementation assay and Xenopus oocytes expressing system. Yeast twohybrid, bimolecular fuorescence complementation,co-localization, and co-immunoprecipitation assays demonstrated the direct protein–protein interaction between Me PIP2;7 and Me MGT9 in vivo. Mg2+fux was significantly elevated in Me PIP2;7-overexpressing lines in hydroponic solution through non-invasive micro-test technique analysis. Under Mg^(2+)-free condition, the retarded growth of PIP-Ri transgenic plants could be recovered with Mg^(2+)supplementation. Taken together, our results demonstrated the synergistic effect of the Me PIP2;7 and Me MGT9 interaction in regulating water and Mg2+absorption and transport in cassava.