NtHHL5 modulates lipid metabolism and negatively regulates cold tolerance in Nicotiana tabacum

NtHHL5 is a 3-hydroxyisobutyryl-CoA hydrolase-like protein and a potential important member of this family, whose biological functions in plants remain largely unexplored, especially its involvement in stress responses in tobacco ( Nicotiana tabacum L. ). Cold stress is a major abiotic constraint that severely impairs tobacco growth and productivity, yet the specific role of NtHHL5 in this process remains to be elucidated. Phylogenetic analysis revealed that NtHHL5 is closely related to homologs from solanaceous species and contains a conserved ECH_2 domain involved in fatty acid metabolism. Expression profiling showed that NtHHL5 is preferentially expressed in trichomes and strongly induced by low temperature. Subcellular localization indicated that NtHHL5‑GFP fusion protein targets both the nucleus and endoplasmic reticulum. Lipidomic analysis of NtHHL5 RNAi lines revealed significant remodeling of the leaf lipidome, with elevated levels of specific galactolipids, phospholipids, and lysophospholipids, as well as increased unsaturation of multiple lipid classes, which likely enhance membrane stability under cold stress. Phenotypically, NtHHL5 RNAi lines exhibited superior cold tolerance, accompanied by reduced ROS accumulation, lower MDA and H 2 O 2 contents, and higher antioxidant enzyme activities (POD, SOD, CAT) compared with WT plants. Moreover, NtHHL5 silencing upregulated key cold‑responsive genes, including NtICE1 , NtCBF1-2 , and NtCOR413 , under cold stress. NtHHL5 is characterized as a negative regulator of cold tolerance in Nicotiana tabacum . Our findings demonstrate that NtHHL5 negatively modulates cold defense by disrupting membrane lipid homeostasis, compromising antioxidant defense systems, and suppressing the CBF signaling. This study identifies NtHHL5 as a promising genetic target for improving cold tolerance in tobacco and other crops.