Deciphering the cucumber ATG6 and its promoter to enhance abiotic stress tolerance and drive stress-responsive transgene expression in plants.

Developing climate-resilient plants with enhanced tolerance to abiotic stresses such as drought and salinity is essential for mitigating the impact of climate change on agriculture. One promising approach is the heterologous expression of stress-responsive genes under appropriate regulatory elements. In this study, we evaluated the potential of the CsATG6 gene from cucumber (Cucumis sativus) to improve abiotic stress tolerance through heterologous expression and examined its promoter's utility for driving transgene expression in dicots. Overexpression of CsATG6 in Arabidopsis enhanced seedling root growth under nitrogen deficiency, drought, and salt stress, and improved survival rates following high-salinity treatment, indicating its potential application in stress-tolerant crop development. The CsATG6 promoter was cloned and analyzed for cis-acting elements responsive to environmental stimuli, hormones, and developmental signals. A series of 5' deletion constructs were fused to a GUS reporter and tested in Arabidopsis, tobacco leaves, and cassava calli under various treatments, including salicylic acid (SA), abscisic acid (ABA), drought, salt, and nitrogen starvation. Among the tested constructs, pCsATG6::GUS-1027 and pCsATG6::GUS-512 showed strong and inducible expression in response to ABA, SA, and multiple abiotic stresses. Furthermore, an ABA-repressive element within the promoter was identified and functionally validated via site-directed mutagenesis. Together, these findings demonstrate that CsATG6 is a valuable candidate gene for enhancing stress tolerance through heterologous expression, and that its promoter fragments can serve as stress- and hormone-responsive regulatory elements for transgene expression in some dicots, including economically important crops like cassava.