Identification of the Maize <i>PP2C</i> Gene Family and Functional Studies on the Role of <i>ZmPP2C15</i> in Drought Tolerance

The protein phosphatase PP2C plays an important role in plant responses to stress. Therefore, the identification of maize <i>PP2C</i> genes that respond to drought stress is particularly important for the improvement and creation of new drought-resistant assortments of maize. In this study, we identified 102 <i>ZmPP2C</i> genes in maize at the genome-wide level. We analyzed the physicochemical properties of 102 <i>ZmPP2Cs</i> and constructed a phylogenetic tree with <i>Arabidopsis</i>. By analyzing the gene structure, conserved protein motifs, and synteny, the <i>ZmPP2Cs</i> were found to be strongly conserved during evolution. Sixteen core genes involved in drought stress and rewatering were screened using gene co-expression network mapping and expression profiling. The qRT-PCR results showed 16 genes were induced by abscisic acid (ABA), drought, and NaCl treatments. Notably, <i>ZmPP2C15</i> exhibited a substantial expression difference. Through genetic transformation, we overexpressed <i>ZmPP2C15</i> and generated the CRISPR/Cas9 knockout maize mutant <i>zmpp2c15</i>. Overexpressing <i>ZmPP2C15</i> in <i>Arabidopsis</i> under drought stress enhanced growth and survival compared with WT plants. The leaves exhibited heightened superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) activities, elevated proline (Pro) content, and reduced malondialdehyde (MDA) content. Conversely, <i>zmpp2c15</i> mutant plants displayed severe leaf dryness, curling, and wilting under drought stress. Their leaf activities of SOD, POD, APX, and CAT were lower than those in B104, while MDA was higher. This suggests that <i>ZmPP2C15</i> positively regulates drought tolerance in maize by affecting the antioxidant enzyme activity and osmoregulatory substance content. Subcellular localization revealed that <i>ZmPP2C15</i> was localized in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments demonstrated <i>ZmPP2C15</i>’s interaction with ZmWIN1, ZmADT2, ZmsodC, Zmcab, and ZmLHC2. These findings establish a foundation for understanding maize <i>PP2C</i> gene functions, offering genetic resources and insights for molecular design breeding for drought tolerance.