Characterization of the Nt4CL gene family in Tobacco (Nicotiana tabacum) and reveals Nt4CL9 response to cold stress

4-coumarate:CoA ligases (4CLs) are key enzymes in the phenylpropanoid pathway, which plays a central role in plant adaptation to diverse environmental stresses. Despite being a model Solanaceae species, tobacco’s 4CL gene family composition and functions remain largely unknown. Here, we identified 24 Nt4CL family genes in Nicotiana tabacum and comprehensively analyzed their phylogenetic relationships, conserved motifs, gene structures, and promoter cis -acting elements. The Nt4CL family clustered into three clades, all containing conserved ANL domains and catalytic motifs. Promoter analyses revealed abundant elements associated with light, hormone, and stress responses. Expression profiling under abiotic stresses revealed that different Nt4CL members participate in distinct stress responses, among which Nt4CL9 was strongly induced by cold stress. Nt4CL9 localized to the plasma membrane and cytoplasm. CRISPR/Cas9-mediated knockout of Nt4CL9 ( Nt4cl9 ) increased flavonoid accumulation, particularly rutin. Nt4cl9 markedly enhanced cold tolerance, as evidenced by reduced malondialdehyde accumulation, elevated antioxidant enzyme activities, and up-regulation of cold-responsive genes. Collectively, these findings demonstrate that Nt4CL9 not only modulates flavonoid metabolism but also functions as a negative regulator of cold tolerance in tobacco. This observation not only provides novel insights into the molecular mechanisms underlying phenylpropanoid-mediated stress adaptation but also identifies Nt4CL9 as a potential genetic target for enhancing cold resilience in tobacco and other Solanaceae family crops.