ZmWRKY104 Transcription Factor Phosphorylated by ZmMPK6 Functioning in ABA-Induced Antioxidant Defense and Enhance Drought Tolerance in Maize

SIMPLE SUMMARY: Current knowledge about the downstream substrate proteins of MAPKs is still limited. Our study screened a new WRKY IIa transcription factor as the substrate protein of ZmMPK6, and its phosphorylation at Thr-59 is critical to the role of ZmWRKY104 in ABA-induced antioxidant defense. Moreover, overexpression ZmWRKY104 in maize enhances the drought tolerance of transgenic plants. These findings define a mechanism for the function of ZmWRKY104 phosphorylated by ZmMPK6 in ABA-induced antioxidant defense and drought tolerance. ABSTRACT: 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 (Zea mays 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.