Table 6_Analysis of the TIL gene family in Brassicaceae species and functional study of BrTIL1 in cold tolerance.xlsx

Temperature-induced lipocalins (TILs) are a class of thermoregulated lipid-transporting proteins crucial for plant stress responses. However, systematic research on the TIL gene family remains relatively limited. In the present study, we conducted a comparative analysis of the TIL gene family in five Brassicaceae species (Arabidopsis thaliana, Brassica rapa L., Brassica rapa subsp. pekinensis, Brassica juncea L., and Brassica napus L.), identifying a total of 23 TIL genes. Analyses of their gene structures, evolutionary relationships, conserved motifs, and cis-acting elements showed extensive collinearity, close homology, and functional conservation, implying they may possess similar biological functions across different Brassicaceae species. The Brassica rapa TIL1 (BrTIL1) gene was significantly upregulated under low-temperature stress. Functional validation showed that Arabidopsis thaliana plants overexpressing BrTIL1 exhibited higher survival rates, soluble protein levels, and peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities under low-temperature conditions, confirming that BrTIL1 positively regulates cold tolerance. The BrTIL1 protein was localized to the cell membrane. A yeast two-hybrid screen identified six proteins interacting with BrTIL1. The genes encoding these interacting proteins exhibited differential expression under low-temperature stress, suggesting they may affect the functional activity of BrTIL1. In summary, this study provides a systematic analysis of the TIL gene family in five Brassicaceae species, elucidates the role of BrTIL1 in cold tolerance, and establishes a foundation for deciphering the molecular mechanisms of the cold stress response in Brassicaceae species.