Species-specific functional divergence of MAH1-L genes enhances wax biosynthesis and drought tolerance in cotton (Gossypium hirsutum L.).

Drought remains a major limitation in Cotton (Gossypium spp.) productivity, with high-yielding Gossypium hirsutum being stress-sensitive, whereas Gossypium arboreum exhibits inherent drought resilience. The superior drought tolerance of G. arboreum is closely associated with its higher epicuticular wax content, yet the mechanistic basis underlying this trait is poorly understood. Here, we revealed species-specific divergence of Midchain Alkane Hydroxylase-Like (MAH1-L) genes between the two species and evaluated their potential to enhance drought tolerance in G. hirsutum. Introduction of GaMAH1-L into G. hirsutum resulted in significant increase in drought tolerance compared to native GhMAH1-L overexpression and non-transgenic control plants. Improved stress resilience was underpinned by elevated epicuticular wax accumulation, particularly secondary alcohols and ketones, and enhanced physiological performance under water deficit conditions. Moreover, leaf surface analysis revealed substantially dense wax deposition in GaMAH1-L-expressing plants. Regulatory analysis identified the unique presence of drought- and ABA-responsive cis-elements in GaMAH1-L promoter, while structural comparison indicated stronger substrate-binding affinity of GaMAH1-L protein compared to GhMAH1-L. Furthermore, upon drought stress, GaMAH1-L expression was more strongly induced compared to GhMAH1-L, demonstrating enhanced transcriptional responsiveness. This integrated analysis of functional, regulatory, and structural features establishes GaMAH1-L as a key determinant of wax-mediated drought tolerance and provides a mechanistic framework for the development of drought-tolerant cotton cultivars through targeted breeding and genome editing.