Functional characterization of GhNAC2 promoter conferring hormone- and stress-induced expression: a potential tool to improve growth and stress tolerance in cotton

The GhNAC2 transcription factor identified from G. herbaceum improves root growth and drought tolerance through transcriptional reprogramming of phytohormone signaling. The promoter of such a versatile gene could serve as an important genetic engineering tool for biotechnological application. In this study, we identified and characterized the promoter of GhNAC2 to understand its regulatory mechanism. GhNAC2 transcription factor increased in root tissues in response to GA, ethylene, auxin, ABA, mannitol, and NaCl. In silico analysis revealed an overrepresentation of cis-regulatory elements associated with hormone signaling, stress responses and root-, pollen-, and seed-specific promoter activity. To validate their role in GhNAC2 function/regulation, an 870-bp upstream regulatory sequence was fused with the GUS reporter gene (uidA) and expressed in Arabidopsis and cotton hairy roots for in planta characterization. Histochemical GUS staining indicated localized expression in root tips, root elongation zone, root primordia, and reproductive tissues under optimal growth conditions. Mannitol, NaCl, auxin, GA, and ABA, induced the promoter-driven GUS expression in all tissues while ethylene suppressed the promoter activity. The results show that the 870 nt fragment of the GhNAC2 promoter drives root-preferential expression and responds to phytohormonal and stress signals. In corroboration with promoter regulation, GA and ethylene pathways differentially regulated root growth in GhNAC2-expressing Arabidopsis. The findings suggest that differential promoter activity governs the expression of GhNAC2 in root growth and stress-related functions independently through specific promoter elements. This multifarious promoter can be utilized to develop yield and climate resilience in cotton by expanding the options to control gene regulation.