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.

Analyzing the transcriptome of maize leaves under drought stress and rewatering conditions revealed that transcription factors were involved in this process, among which ZmbZIP33 of the ABSCISIC ACID-INSENSITIVE 5-like protein 5 family was induced to significantly up-regulated. The functional mechanism of ZmbZIP33 in Abscisic acd (ABA) signaling pathway and its response to drought stress and rewatering has not been studied yet. The present study found that ZmbZIP33 contains a DNA-binding and dimerization domain, has transcriptional activation activity, and is highly homologous to SbABI1,SitbZIP68 and OsABA1. The expression of ZmbZIP33 is strongly up-regulated by drought, high salt, high temperature, and ABA treatments. Overexpression of ZmbZIP33 remarkably increased chlorophyll content and root length after drought stress and rewatering, and, moreover, cause an accumulation of ABA content, thereby improving drought resistance and recovery ability in Arabidopsis. However, silencing the expression of ZmbZIP33 (BMV-ZmbZIP33) remarkably decreased chlorophyll content, ABA content, superoxide dismutase and peroxidase activities, and increased stomatal opening and water loss rate compared with BMV (control). It showed that silencing ZmbZIP33 lead to reduced drought resistance and recovery ability of maize. ABA sensitivity analysis found that 0.5 and 1 μmol/L treatments severely inhibited the root development of overexpression ZmbZIP33 transgenic Arabidopsis. However, the root growth of BMV was greatly inhibited for 1 and 5μmol/L ABA treatments, but not for BMV-ZmbZIP33. Subcellular localization, yeast two-hybrid and BIFC further confirmed that the core components of ABA signaling pathways ZmPYL10 and ZmPP2C7 interacted in nucleus, ZmPP2C7 and ZmSRK2E as well as ZmSRK2E and ZmbZIP33 interacted in the plasma membrane. We also found that expression levels of ZmPYL10 and ZmSRK2E in the BMV-ZmbZIP33 mutant were lower than those of BMV, while ZmPP2C7 was the opposite under drought stress and rewatering. However, expression of ZmPYL10 and ZmSRK2E in normal maize leaves were significantly up-regulated by 3–4 folds after drought and ABA treatments for 24 h, while ZmPP2C7 was down-regulated. The NCED and ZEP encoding key enzymes in ABA biosynthesis are up-regulated in overexpression ZmbZIP33 transgenic line under drought stress and rewatering conditions, but down-regulated in BMV-ZmbZIP33 mutants. Together, these findings demonstrate that ZmbZIP33 played roles in ABA biosynthesis and regulation of drought response and rewatering in Arabidopsis and maize thought an ABA-dependent signaling pathway.

Maize is an important food and feed crop. Under limited arable land area, the cultivation of high-density-tolerance crops is a key factor in promoting yield improvement. Leaf width and stalk strength are important influences on density tolerance in maize. However, no comprehensive transcriptomic analysis has focused on maize’s leaf width and stalk strength formation mechanisms. In this study, comparative transcriptomic analyses demonstrated that significant transcriptome changes occurred regarding leaf width and stalk strength of narrow-leaved and wide-leaved maize inbred lines, with a total of 5001 differentially expressed genes (DEGs) identified. Enrichment analysis showed that phenylpropanoid biosynthesis, starch and sucrose metabolism, phytohormone signaling, amino acid metabolism, and brassinosteroid biosynthesis were significantly correlated with the formation of maize leaf shape and stalk strength and that the genes in these pathways were primarily involved in cell wall formation. Weighted gene co-expression network analysis identified 2 modules significantly correlated with leaf width and stalk strength, from which 11 hub genes were further identified. The 11 leaf and stem development genes in different pathways were validated using qRT-PCR. These findings can provide a theoretical basis for the mechanism of narrow-leaf and high-strength stalk formation in high-density-tolerance maize and contribute to the proposal of a breeding strategy for yield improvement.