The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1(BZR1) transcription factors play crucial roles in plant growth, development, and stress response. However, little is known about the function of maize’s BES1/BZR1s. In this study, the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes were cloned from maize’s inbred line, B73, and they were functionally evaluated by analyzing their expression pattern, subcellular localization, transcriptional activation activity, as well as their heterologous expression in Arabidopsis, respectively. The results of the qRT-PCR showed that the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes were predominantly expressed in the root, and their expression was significantly down-regulated by drought stress. The ZmBES1/BZR1-3 and ZmBES1/BZR1-9 proteins localized in the nucleus but showed no transcriptional activation activity as a monomer. Subsequently, it was found that the heterologous expression of the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes in Arabidopsis decreased drought tolerance, respectively. The transgenic lines showed a more serious wilting phenotype, shorter root length, lower fresh weight, and higher relative electrolyte leakage (REL) and malondialdehyde (MDA) content compared to the control under drought stress. The RNA-sequencing data showed that the 70.67% and 93.27% differentially expressed genes (DEGs) were significantly down-regulated in ZmBES1/BZR1-3 and ZmBES1/BZR1-9 transgenic Arabidopsis, respectively. The DEGs of ZmBES1/BZR1-3 gene’s expressing lines were mainly associated with oxidative stress response and amino acid metabolic process and enriched in phenylpropanoid biosynthesis and protein processing in the endoplasmic reticulum. But the DEGs of the ZmBES1/BZR1-9 gene’s expressing lines were predominantly annotated with water deprivation, extracellular stimuli, and jasmonic acid and enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. Moreover, ZmBES1/BZR1-9 increased stomatal aperture in transgenic Arabidopsis under drought stress. This study indicates that ZmBES1/BZR1-3 and ZmBES1/BZR1-9 negatively regulate drought tolerance via different pathways in transgenic Arabidopsis, and it provides insights into the underlying the function of BES1/BZR1s in crops.

Drought stress significantly limits agricultural productivity and threatens food security. The identification of drought tolerance genes and the dissection of their regulatory networks in crops are crucial for ensuring future food production. The BRI1-EMS1 suppressor (BES1)/brassinazole-resistant 1 (BZR1) family is a kind of plant-specific transcription factor that regulates growth, development, and stress responses in plants. Here, the maize ZmBES1/BZR1-7 gene was cloned and transformed into Arabidopsis and rice to characterize plant phenotypes under drought stress conditions. Sequence analysis revealed that the ZmBES1/BZR1-7 protein contains a conserved basic helix-loop-helix (bHLH) domain, localizes to the nucleus, and exhibits transcriptional activation activity. Expression profiling revealed that ZmBES1/BZR1-7 is induced by drought stress in maize. Overexpression of ZmBES1/BZR1-7 significantly enhanced drought tolerance in both transgenic Arabidopsis and rice. Under drought stress conditions, all transgenic lines showed higher survival rates and relative water content (RWC), increased root length, reduced relative electrolyte leakage (REL) and malondialdehyde (MDA) content, as well as higher yield of transgenic rice than the wild type. Integrated analyses of RNA-seq and qRT-PCR assays demonstrated that ZmBES1/BZR1-7 modulates transcription of stress-responsive genes to enhance drought tolerance. Collectively, the study provides insights into the molecular mechanisms by which ZmBES1/BZR1-7 mediated drought stress response in maize.