60-day-old Nicotiana tabacum L. seedlings were selected for hydroponic cultivation. Each treatment was composed of three replicates, and each replicate was cultured in 1 L Hoagland nutrient solution. Specifically, the treatments included separately adding 1 mL of DMSO (CK), 1 mL of 50 mM MeJA solution (MeJA, dissolved in DMSO), 1 mL of 500 mM SA solution (SA, dissolved in DMSO), and 1 mL of a combined mixture of 50 mM MeJA and 500 mM SA (SA+MeJA, dissolved in DMSO) to 1 L of Hoagland nutrient solution. After 24 hours of treatment, root samples were collected for transcriptome sequencing.