Genome-wide identification and functional dissection of the BES1 family reveals key regulators of alkali stress response in hemp

Plants frequently encounter diverse abiotic stresses, which require precise transcriptional regulation to maintain basic survival plasticity. The BES1 transcription factor family, a key component of brassinosteroid (BR) signaling, plays critical roles in plant development and stress responses. However, the genomic characterization, evolutionary characteristics, and biological functions of the BES1 family in industrial hemp (Cannabis sativa L.) remain unclear. In this study, we conducted a comprehensive genome-wide analysis of the BES1-like gene family in hemp and classified the 7 identified CsBES1 genes into 5 branches based on their structural and phylogenetic association analysis. Gene promoter analysis revealed that cis-regulatory elements related to development, hormone signaling, and environmental stress (mainly BOX4, G-box, ARE, and ABRE) exhibited a dense distribution, indicating that they are involved in a complex regulatory network. Integrated RNA-sequencing data and gene expression profiling through quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that CsBES1-2 and CsBES1-3 genes had a good response towards alkaline stress. Subsequently, the subcellular localization of these two genes showed the cytoplasmic and nuclear distributions are indicative of a phosphorylation-dependent regulatory mechanism. The functional analysis through Yeast Two-Hybrid (Y2H) assays confirmed the activation of transcriptional activity of CsBES1-2 and CsBES1-3 genes and revealed enhanced alkali tolerance in yeast by showing the potential gene expression. In crux, this study provides the first systematic genomic characterization of the BES1 family in hemp and identifies CsBES1-2 and CsBES1-3 as key candidates for improving alkali stress resilience, offering valuable genetic resources for hemp breeding and crop improvement.