Comprehensive evolutionary, differential expression and VIGS analyses reveal the function of GhNST1 in regulating drought tolerance and early maturity in upland cotton.

NAC secondary wall thickening promoting factor 1 (NST1) is crucial for abiotic stress responses and plant growth and development. However, the study of the function of GhNST1 in upland cotton is rather limited. Our study revealed that its promoter contains multiple cis-elements linked to hormones, stress, and development. Among various abiotic stresses (drought, heat, NaCl, and cold), GhNST1 presented the strongest response to drought. The transcriptional level of GhNST1 was higher in early-maturing varieties at the three-leaf stage compared to those of late-maturing varieties. VIGS experiments confirmed that GhNST1 was essential for drought tolerance in cotton. The TRV:GhNST1 presented a lower relative water content (RWC) and faster water loss rate (WLR) in isolated leaves than the TRV:00 plants. Under drought stress, the TRV:GhNST1 plants presented more wilting leaves than did the control plants. The chlorophyll content, activities of superoxide dismutase (SOD) and peroxidase (POD) were lower, the contents of malondialdehyde (MDA), lignin and proline (Pro) were higher, 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining was more intense, while the transcript levels of stress-responsive genes (GhDREB2A, GhRD29A, and GhCBL3) were significantly downregulated. Furthermore, the budding, flowering and boll-opening of the CLCrV:GhNST1 plants occurred significantly later than those of the empty vector plants. In the CLCrV:GhNST1 plants, the GhAP1, GhFT and GhSOC1 expression levels were lower than those in the controls, whereas the GhSVP expression level was elevated. These findings provide critical information for further investigations into how GhNST1 regulates both the early maturity and drought stress response in cotton.