Silencing of NtFAR3 disrupts membrane lipid homeostasis and increases cold stress sensitivity in Nicotiana tabacum

Although fatty acyl-CoA reductase 3 (FAR3) plays essential roles in plant growth and development, its function in cold stress responses remains poorly understood. Here, we characterized NtFAR3 in tobacco. Phylogenetic analysis revealed that NtFAR3 shares the highest homology with its ortholog from Nicotiana sylvestris . Expression profiling showed that NtFAR3 is predominantly expressed in flowers and glandular trichomes, with peak levels at the seedling stage. Subcellular localization confirmed that NtFAR3 resides in the endoplasmic reticulum. Lipidomic analysis of NtFAR3 -silenced lines identified 218 differentially accumulated lipids compared to wild-type (WT), including 194 upregulated and 24 downregulated species. The downregulated lipids were primarily phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), with 15 lipids reduced by 59.7–100%. Specifically, FFA (17:0) and FFA (20:0) increased by 1.68- and 1.56-fold, respectively, while FFA (22:1) and FFA (28:0) specifically accumulated at 0.12 μg·g⁻¹ and 1.25 μg·g⁻¹ . Moreover, the unsaturation levels of PG, PC, and PE decreased by 70%, 52.51%, and 62.35%, respectively. Physiologically, silenced lines exhibited reduced peroxidase (POD) and catalase (CAT) activities and enhanced cold sensitivity. The cold-induced expression of key regulatory genes ( NtICE1 , NtCBF2 , NtCBF3 , NtLEA14 and NtGOLS1 ) was also attenuated. Collectively, our results demonstrate that NtFAR3 is crucial for maintaining membrane lipid homeostasis and cold stress adaptation in tobacco.