BACKGROUND: Tobacco is an important economic crop and a model plant for molecular biology research. It exists in various cultivars and is processed using different curing methods. Fatty acids play a crucial role in the quality and flavor of tobacco leaves. However, there is limited information on the fatty acid composition across different cultivars, developmental stages, and curing methods. This study employed targeted metabolomics and transcriptomics to investigate the fatty acids and related pathway genes in tobacco leaves from different cultivars, developmental stages, and curing methods. RESULTS: This study focused on four tobacco cultivars: K326, Basma, Samsun, and Cuba1, and investigated fatty acid differences in the leaves at four developmental stages (seedling, transplanting, budding, and topping) under two curing methods (air-curing and flue-curing). K326 was used as the main cultivar for comparison with the other three. The analysis included short-chain fatty acids (C2-C6), free fatty acids (C8-C24), and gene expression differences. The fatty acid metabolic profile of different tissue types in K326 at the budding stage was also examined. The results showed significant differences in fatty acid content among the different tissues of K326 at the budding stage, with the highest levels of short-chain fatty acids found in flower buds and upper leaves. At the seedling stage, there were marked variations in short-chain fatty acid content across different periods. Three key genes Nta01g31980, Nta08g22780, and Nta23g11140 were identified as major differential genes in fatty acid-related pathways in K326 compared to the other three cultivars during this stage. Regarding the four cultivars, the total short-chain fatty acid content at the budding stage was ranked as Basma >
Samsun >
Cuba1 >
K326 before topping, but the order was reversed after topping. At the budding stage, 35 fatty acid pathway-related genes showed similar expression levels in Basma and Samsun, differing from K326 and Cuba1. Among the two curing methods, air-curing resulted in higher short-chain fatty acid content than flue-curing. Under air-curing, Samsun and Basma showed more downregulation of differential fatty acids compared to K326, while the opposite was observed under flue-curing. CONCLUSION: This study expands our understanding of fatty acids in tobacco across different cultivars and developmental stages, providing a molecular basis for the study of fatty acids and genes related to their biosynthesis and metabolism.