Preharvest Application of Exogenous 2,4-Epibrassinolide and Melatonin Enhances the Maturity and Flue-Cured Quality of Tobacco Leaves

Tobacco (<i>Nicotiana tabacum</i>) is a globally cultivated crop, with its quality closely associated with the color and chemical composition of cured tobacco leaves. In this experiment, the effects of spraying exogenous 2, 4-epibrassinolide (EBR) and melatonin (MT) on the development of tobacco leaves at maturity stage and the quality after curing were investigated. Both EBR and MT treatments significantly enhanced the appearance quality of tobacco leaves at the stem-drying stage. Following preharvest applications, the sugar-to-alkali ratio and potassium content increased, while the contents of starch, total alkaloids, and proteins decreased. The levels of conventional chemical components were improved, enhancing the overall coordination of the tobacco. Transcriptome analysis revealed that EBR treatment down-regulated the chlorophyll biosynthetic genes <i>hemA</i>, <i>MgPEC</i>, and <i>ChlD</i>, while up-regulating the chlorophyll degradation genes <i>CHL2</i>, <i>SGR</i>, and <i>PAOs</i>. Similarly, MT treatment down-regulated the chlorophyll biosynthetic genes <i>FC2</i> and <i>MgPEC</i> and up-regulated the degradation genes <i>CHL2</i> and <i>SGR</i>, thus promoting chlorophyll degradation. Furthermore, in the downstream carotenoid biosynthetic pathway, both EBR and MT treatments regulated abscisic acid-related genes, with <i>NCEDs</i> being up-regulated and <i>CYP707A1s</i> down-regulated, thereby promoting the leaf ripening. Metabolomics analysis indicated that EBR treatment primarily regulated alkaloids, terpenoids, and flavonoids, while MT treatment mainly affected flavonoids. Both treatments also reduced the accumulation of the harmful substance aristolochic acid B. Comprehensive evaluations of appearance quality, physiological parameters, transcriptome, and metabolomics analyses demonstrated that exogenous spraying of EBR and MT treatments improved the maturity and quality of cured tobacco leaves, with EBR treatment exhibiting a greater effect than MT treatment.