Drought stress severely limits the growth, development, and productivity of crops, and therefore understanding the mechanisms by which plants respond to drought is crucial. In this study, we cloned a maize NAC transcription factor, ZmNAC49, and identified its function in response to drought stress. We found that ZmNAC49 is localized in the nucleus and has transcriptional activation activity. ZmNAC49 expression is rapidly and strongly induced by drought stress, and overexpression enhances stress tolerance in maize. Overexpression also significant decreases the transpiration rate, stomatal conductance, and stomatal density in maize. Detailed study showed that ZmNAC49 overexpression affects the expression of genes related to stomatal development, namely ZmTMM, ZmSDD1, ZmMUTE, and ZmFAMA. In addition, we found that ZmNAC49 can directly bind to the promoter of ZmMUTE and suppress its expression. Taken together, our results show that the transcription factor ZmNAC49 represses ZmMUTE expression, reduces stomatal density, and thereby enhances drought tolerance in maize.

Mitogen-activated protein kinase (MAPK) cascades are primary signaling pathways involved in various signaling pathways triggered by abiotic and biotic stresses in plants. The downstream substrate proteins of MAPKs in maize, however, are still limited. Here, we screened a WRKY IIa transcription factor (TF) in maize (Zeamays L.), ZmWRKY104, and found that it is a substrate of ZmMPK6. ZmWRKY104 physically interacts with ZmMPK6 in vitro and in vivo. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis results showed that threonine-59 (Thr-59, T59) was the major phosphorylation site of ZmWRKY104 by ZmMPK6. Subcellular localization analysis suggested that ZmWRKY104 acts in the nucleus and that ZmMPK6 acts in the nucleus and cytoplasmic membrane in the cytosol. Functional analysis revealed that the role of ZmWRKY104 in ABA-induced antioxidant defense depends on ZmMPK6. Moreover, overexpression of ZmWRKY104 in maize can enhance drought tolerance and relieve drought-induced oxidative damage in transgenic lines. The above results help define the mechanism of the function of ZmWRKY104 phosphorylated by ZmMPK6 in ABA-induced antioxidant defense and drought tolerance in maize.

Glycogen synthase kinase 3 (GSK3)–like kinases play important roles in stress responses in plants. However, the mechanism of GSK3-like kinases in drought-induced antioxidant defense is not clear. In this study, we discovered that the GSK3-like kinase SHAGGY-like kinase 1 (ZmSK1) negatively regulates drought tolerance by inhibiting antioxidant defense in maize (Zea mays). Then, we determined that cysteine-rich polycomb-like protein 2 (ZmCPP2) interacts with ZmSK1 and enhances maize drought tolerance by inducing antioxidant defense. ZmCPP2 is phosphorylated at Ser-250 by ZmSK1, which is dependent on ZmSK1 kinase activity and attenuates maize drought tolerance. Furthermore, ZmCPP2 directly binds to the promoter of the superoxide dismutase (SOD) gene ZmSOD4, encoding an antioxidant defense enzyme, and activates its expression. ZmSK1 phosphorylating ZmCPP2 at Ser-250 represses the binding of ZmCPP2 to the ZmSOD4 promoter. Taken together, our results indicate that the phosphorylation of ZmCPP2 by ZmSK1 results in decreased SOD activity and thus reduces drought tolerance in maize. These findings reveal a mechanism of GSK3-like kinases regulating antioxidant defense in the drought stress response.