Heterologous expression of DobHLH25 from Dendrobium officinale enhances drought tolerance in Arabidopsis.

Drought stress severely constrains the growth, yield, and accumulation of bioactive compounds in Dendrobium officinale (D. officinale), a valuable medicinal orchid, and this challenge is exacerbated under simulated wild cultivation where plants are inevitably exposed to recurring water deficits. Basic helix-loop-helix (bHLH) transcription factors are well-established regulators of plant abiotic stress responses. However, the molecular mechanisms by which bHLH transcription factors respond to drought stress in this species remain largely unknown. In this study, a bHLH transcription factor gene, DobHLH25, was cloned from D. officinale. Phylogenetic analysis revealed that DobHLH25 shares the highest sequence identity with its ortholog in Dendrobium nobile. Additionally, subcellular localization analysis indicated that DobHLH25 is targeted to the nucleus and possesses a functional transcriptional activation domain. Expression pattern analysis showed that DobHLH25 is most abundantly expressed in old leaves, and its expression in roots, stems, and leaves is induced by polyethylene glycol treatments. Heterologous expression of DobHLH25 in Arabidopsis thaliana resulted in higher seed germination rates and longer root lengths under mannitol-induced osmotic stress compared to wild-type plants. Under drought stress, DobHLH25 heterologous expression lines exhibited higher survival rates, reduced leaf water loss, lower malondialdehyde accumulation, and increased proline content. Moreover, the activities of antioxidant enzymes such as superoxide dismutase and peroxidase were significantly enhanced, and the expression levels of multiple drought-responsive genes were markedly upregulated. Collectively, these findings suggest a correlation between DobHLH25 expression and plant drought tolerance, as evidenced by reduced oxidative damage, increased osmolyte accumulation, enhanced antioxidant enzyme activities, and upregulation of drought-responsive genes. Together, these results suggest that DobHLH25 plays a positive role in drought tolerance, and provides a basis for future dissection of its regulatory network in D. officinale.