Systematic genomic characterization of β-glucosidase genes in tobacco reveals NtBGLU50 as a multifunctional regulator of glycoside metabolism and defense-associated pathways

β-Glucosidases (BGLUs) are critical enzymes involved in plant stress responses and secondary metabolism. However, their genomic and functional roles in Nicotiana tabacum , a model species for specialized metabolism, remain poorly understood. In this study, we systematically identified 56 NtBGLU genes, which were classified into 10 distinct clades. Most of these genes exhibit strong conservation in gene structure, motif composition, and chromosomal distribution, with notable species-specific expansions in tobacco. Promoter analysis revealed abundant stress- and hormone-responsive elements, particularly for methyl jasmonate (MeJA) and abscisic acid (ABA), consistent with the known roles of BGLUs in stress signaling and metabolic regulation. Transcriptomic and qRT-PCR analyses revealed tissue-specific expression and differential responses to abiotic stresses and phytohormones. Among the identified genes, NtBGLU50 was selected for functional analysis due to its substantial upregulation under MeJA treatment. RNA interference (RNAi)-mediated knockdown of NtBGLU50 disrupted root development, altered phytohormone crosstalk, and impaired secondary metabolism, leading to reduced levels of nicotine and anabasine. These findings suggest that NtBGLU50 plays a role in integrating jasmonate (JA) signaling with glycoside metabolism and defense pathways, particularly in regulating nicotine biosynthesis. These results provide valuable insights into the functional roles of NtBGLU genes, particularly NtBGLU50 , in mediating the integration of JA signaling with secondary metabolism, which may contribute to tobacco's stress response and alkaloid biosynthesis. • Fifty-six NtBGLU genes were identified and systematically characterized in Nicotiana tabacum . • NtBGLUs exhibit tissue-specific expression and differential responses to abiotic stresses and phytohormones. • NtBGLU50 is highly induced by MeJA and expressed primarily in the root cortex and endodermis. • NtBGLU50 integrates jasmonate (JA) signaling with defense pathways, particularly in regulating nicotine biosynthesis.