ZmSK4 activates ZmSLAC1 through relief of repression by ZmGCT1 and ZmPP2Cs in ABA signaling

Guard cell movement is orchestrated by complex internal and environmental signals, with ZmGCT1 and ZmGCT2 playing critical roles in maintaining guard cell turgor in maize. Here, we demonstrate that ZmGCT1 interacts with and inhibits ZmSK4, a GSK3-like kinase. Under drought stress, ABA-activated ZmSnRK2s phosphorylate ZmGCT1, relieving its partial inhibition of ZmSK4, which, together with the relief of ZmPP2Cs-mediated inhibition, leads to ZmSK4 activation. ZmSK4 directly phosphorylates ZmSLAC1 and activates its anion currents in Xenopus oocytes, which ultimately promotes stomatal closure under drought stress. Consistent with this mechanism, mutations in ZmSK4 and its homolog ZmSK3 not only confer drought sensitivity but also suppress the constitutively closed stomata phenotype of the Zmgct1 mutant. Notably, in contrast to its BIN2 homologs, which do not regulate stomatal movement in Arabidopsis but stomatal development, ZmSK4 and ZmSK3 do not influence stomatal development in maize. Our findings thus delineate a complete phosphorylation relay within the ABA signaling pathway that dynamically regulates stomatal movement under drought stress in maize.