NtFATB silencing impairs lipid homeostasis and enhances cold sensitivity associated with reduced the ICE-CBF-COR expression in Nicotiana tabacum.

Fatty acyl-acyl carrier protein thioesterases B (FATB) plays a critical role in regulating fatty acid chain length and saturation in plants. However, the function of FATB genes in tobacco remains poorly understood. In this study, we identified and characterized NtFATB, a FATB homolog in tobacco. Phylogenetic analysis revealed that NtFATB is closely related to FATB proteins from Nicotiana tomentosiformis and several Solanaceae species. Sequence alignment and domain prediction identified conserved catalytic residues and two characteristic acyl-ACP thioesterase domains. Expression analysis showed that NtFATB is predominantly expressed in trichomes and flowers, peaks at the seedling stage, and is strongly induced by cold stress. Subcellular localization demonstrated that NtFATB-GFP localizes to the endoplasmic reticulum. To investigate its biological function, we generated NtFATB RNAi lines and performed untargeted lipidomic profiling. Comparative analysis revealed that silencing NtFATB reduced the levels of 7 glycerophospholipids and 3 free fatty acids, and that triacylglycerols and ceramides were the lipid classes exhibiting the most robust changes. Moreover, the unsaturation level of several major membrane lipids, including DGDG, PA, PI, PG, PC, and LPA, was significantly decreased. Silencing of NtFATB caused lipid-class-specific and statistically significant changes in acyl chain length (ACL), with small increases observed in PA and PE, and small decreases in DGDG and MGDG. Functionally, NtFATB RNAi lines displayed heightened sensitivity to cold stress, as evidenced by earlier wilting, elevated reactive oxygen species (O2-) and malondialdehyde accumulation, and reduced activities of antioxidant enzymes (SOD, POD, and CAT). Consistently, the expression levels of core cold-responsive genes (NtICE1, NtCBF1-4, NtLEA14, NtCOR413, and NtGOLS1) were markedly suppressed in RNAi lines under cold stress. Collectively, our findings establish that NtFATB is essential for maintaining lipid homeostasis, and it positively regulates cold stress tolerance in association with enhanced antioxidant capacity and elevated expression of ICE-CBF-COR pathway genes in tobacco.