The basic leucine zipper (bZIP) family of transcription factors (TFs) regulate diverse phenomena during plant growth and development and are involved in stress responses and hormone signaling. However, only a few bZIPs have been functionally characterized. In this paper, 54 maize bZIP genes were screened from previously published drought and rewatering transcriptomes. These genes were divided into nine groups in a phylogenetic analysis, supported by motif and intron/exon analyses. The 54 genes were unevenly distributed on 10 chromosomes and contained 18 segmental duplications, suggesting that segmental duplication events have contributed to the expansion of the maize bZIP family. Spatio-temporal expression analyses showed that bZIP genes are widely expressed during maize development. We identified 10 core ZmbZIPs involved in protein transport, transcriptional regulation, and cellular metabolism by principal component analysis, gene co-expression network analysis, and Gene Ontology enrichment analysis. In addition, 15 potential stress-responsive ZmbZIPs were identified by expression analyses. Localization analyses showed that ZmbZIP17, -33, -42, and -45 are nuclear proteins. These results provide the basis for future functional genomic studies on bZIP TFs in maize and identify candidate genes with potential applications in breeding/genetic engineering for increased stress resistance. These data represent a high-quality molecular resource for selecting resistant breeding materials.

OSCAs are hyperosmolality-gated calcium-permeable channel proteins. In this study, two co-expression modules, which are strongly associated with maize proline content, were screened by weighted correlation network analysis, including three ZmOSCA family members. Phylogenetic and protein domain analyses revealed that 12 ZmOSCA members were classified into four classes, which all contained DUF221 domain. The promoter region contained multiple core elements responsive to abiotic stresses and hormones. Colinear analysis revealed that ZmOSCAs had diversified prior to maize divergence. Most ZmOSCAs responded positively to ABA, PEG, and NaCl treatments. ZmOSCA2.3 and ZmOSCA2.4 were up-regulated by more than 200-fold under the three stresses, and showed significant positive correlations with proline content. Yeast two-hybrid and bimolecular fluorescence complementation indicated that ZmOSCA2.3 and ZmOSCA2.4 proteins interacted with ZmEREB198. Over-expression of ZmOSCA2.4 in Arabidopsis remarkably improved drought resistance. Moreover, over-expression of ZmOSCA2.4 enhanced the expression of drought tolerance-associated genes and reduced the expression of senescence-associated genes. We also found that perhaps ZmOSCA2.4 was regulated by miR5054.The results provide a high-quality molecular resource for selecting resistant breeding, and lay a foundation for elucidating regulatory mechanism of ZmOSCA under abiotic stresses.

Nuclear factor Y (NF-Y) genes play important roles in many biological processes, such as leaf growth, nitrogen nutrition, and drought resistance. However, the biological functions of these transcription factor family members have not been systematically analyzed in maize. In the present study, a total of 52ZmNF-Ygenes were identified and classified into three groups in the maize genome. An analysis of the evolutionary relationship, gene structure, and conserved motifs of these genes supports the evolutionary conservation of NF-Y family genes in maize. The tissue expression profiles based on RNA-seq data showed that all genes apart fromZmNF-Y16,ZmNF-YC15, andZmNF-YC17were expressed in different maize tissues. A weighted correlation network analysis was conducted and a gene co expression network method was used to analyze the transcriptome sequencing results; six core genes responding to drought and rewatering were identified. A real time fluorescence quantitative analysis showed that these six genes responded to high temperature, drought, high salt, and abscisic acid (ABA) treatments, and subsequent restoration to normal levels.ZmNF-YC12was highly induced by drought and rewatering treatments. The ZmNF-YC12 protein was localized in the nucleus, and the Gal4-LexA/UAS system and a transactivation analysis demonstrated thatZmNF-YC12in maize (Zea mays L.) is a transcriptional activator that regulates drought resistance and recovery ability. SilencingZmNF-YC12reduced net photosynthesis, chlorophyll content, antioxidant (superoxide dismutase, catalase, peroxidase and ascorbate peroxidase) system activation, and soluble protein and proline contents; it increased the malondialdehyde content, the relative water content, and the water loss rate, which weakened drought resistance and the recoverability of maize. These results provide insights into understanding the evolution of ZmNF-Y family genes in maize and their potential roles in genetic improvement. Our work provides a foundation for subsequent functional studies of the NF-Y gene family and provides deep insights into the role of theZmNF-YC12regulatory network in controlling drought resistance and the recoverability of maize.

The protein phosphatase PP2C plays an important role in plant responses to stress. Therefore, the identification of maize PP2C 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 ZmPP2C genes in maize at the genome-wide level. We analyzed the physicochemical properties of 102 ZmPP2Cs and constructed a phylogenetic tree with Arabidopsis. By analyzing the gene structure, conserved protein motifs, and synteny, the ZmPP2Cs 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, ZmPP2C15 exhibited a substantial expression difference. Through genetic transformation, we overexpressed ZmPP2C15 and generated the CRISPR/Cas9 knockout maize mutant zmpp2c15. Overexpressing ZmPP2C15 in Arabidopsis 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, zmpp2c15 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 ZmPP2C15 positively regulates drought tolerance in maize by affecting the antioxidant enzyme activity and osmoregulatory substance content. Subcellular localization revealed that ZmPP2C15 was localized in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments demonstrated ZmPP2C15’s interaction with ZmWIN1, ZmADT2, ZmsodC, Zmcab, and ZmLHC2. These findings establish a foundation for understanding maize PP2C gene functions, offering genetic resources and insights for molecular design breeding for drought tolerance.