BackgroundOxytropis ochrocephala, one of locoweeds, is widely distributed in arid regions of China. Previous studies have primarily focused on understanding its toxicity to livestock, with limited attention paid to its stress tolerant mechanism. The objective of this study is to elucidate the molecular basis of drought resistance in O. ochrocephala by deciphering the regulatory mechanisms of its internal signaling pathways under drought stress.ResultsIn this study, we obtained 105 downstream genes for the O. ochrocephala transcription factor OoAREB1 by DNA affinity purification sequencing (DAP-seq). These genes were mostly involved in ABA response and drought adaptation. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays further demonstrated that OoAREB1 directly binds to and activates the promoters of Ultraviolet-B receptor 8 (OoUVR8), Macro domain-containing protein (OoMacroD), Aspartic proteinase nepenthesin-1 (OoAP), HVA22-like protein (OoHVA22), U-box domain-containing protein 19 (OoPUB19) and Late embryogenesis abundant 3 (OoLEA3). Notably, since OoLEA3 is well recognized as a key stress-responsive gene, it was selected as the focus for subsequent functional characterization. Heterologous expression of OoLEA3 in Escherichia coli significantly enhanced the bacterium's tolerance to osmotic stress, while in vitro experiments further demonstrated that OoLEA3 exerts a protective effect against desiccation-induced inactivation of lactate dehydrogenase (LDH). In Arabidopsis, overexpression of OoLEA3 improved drought tolerance and ABA sensitivity.ConclusionsOur findings demonstrate that heterologous overexpression of OoLEA3 - a target gene of the O. ochrocephala transcription factor OoAREB1 - enhances drought resistance in Arabidopsis. This study provides a foundation for future research into the environmental acclimation mechanisms of locoweeds.