Knockout of NtMSI4c enhances cold tolerance in tobacco through metabolic reprogramming of flavonoid biosynthesis

Nicotiana tabacum ( N. tabacum ) is a warm-season but cold-sensitive crop of significant economic importance. Cold stress frequently leads to reductions in both yield and quality of tobacco, causing substantial economic losses. The molecular mechanisms underlying cold tolerance in tobacco remain incompletely understood. Here, we identified NtMSI4c , a putative homolog of Arabidopsis thaliana ( A. thaliana ) FLOWERING LOCUS VE/ MULTICOPY SUPRESSOR OF IRA 4 ( FVE/MSI4 ), as a negative regulator of cold tolerance through its modulation of flavonoid biosynthesis. Consistent with its role as a negative regulator, NtMSI4c expression was significantly suppressed by cold stress. Subcellular localization assays confirmed that NtMSI4c is localized to the nucleus. Under cold stress, the msi4c mutant exhibited enhanced cold tolerance, reduced reactive oxygen species (ROS) accumulation, and elevated flavonoid content compared to wild-type (WT) plants. Metabolic analysis demonstrated that knockout of NtMSI4c led to extensive reprogramming of the metabolome, particularly enhancing flavonoid accumulation. RNA-sequencing (RNA-seq) analysis further confirmed that upregulation of key flavonoid biosynthesis-related genes in the msi4c mutant contributed to the flavonoid accumulation, thereby enhancing cold tolerance. Collectively, these results establish NtMSI4c as a key negative regulator of cold adaptation in tobacco and elucidate a mechanism by which its disruption enhances flavonoid-mediated ROS scavenging and cold tolerance. • Cold stress suppresses the expression of NtMSI4c. • Knockout of NtMSI4c elevates flavonoid accumulation. • NtMSI4c enhances cold tolerance via flavonoid-mediated ROS scavenging.