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Potassium deficiency causes severe losses in yield and quality in crops. Mepiquat chloride, a plant growth regulator, can increase K+ uptake in cotton (Gossypium hirsutum), but the underlying physiological mechanisms remain unclear. In this study, we used a non-invasive micro-test technique to measure K+ and H+ fluxes in the root apex with or without inhibitors of K+ channels, K+ transporters, non-selective cation channels, and plasma membrane H+-ATPases. We found that soaking seeds in mepiquat chloride solution increased the K+ influx mediated by K+ channels and reduced the K+ efflux mediated by non-selective cation channels in cotton seedlings. Mepiquat chloride also increased negative membrane potential (E m) and the activity of plasma membrane H+-ATPases in roots, due to higher levels of gene expression and protein accumulation of plasma membrane H+-ATPases as well as phosphorylation of H+-ATPase 11 (GhAHA11). Thus, plasma membrane hyperpolarization mediated by H+-ATPases was able to stimulate the activity of K+ channels in roots treated with mepiquat chloride. In addition, reduced K+ efflux under mepiquat chloride treatment was associated with reduced accumulation of H2O2 in roots. Our results provide important insights into the mechanisms of mepiquat chloride-induced K+ uptake in cotton and hence have the potential to help in improving K nutrition for enhancing cotton production. [ABSTRACT FROM AUTHOR]