Two Brassica rapa cultivars with contrasting cold tolerance, the cold-tolerant ‘Longyou 7’ and the cold-sensitive ‘Lenox’, were used as experimental materials in this study (Xu et al., 2024; Fahim et al., 2025). Following germination, seedlings were transplanted into nursery pots and grown in a growth chamber under a 14-hour light (25 °C)/10-hour dark (20 °C) cycle until they reached the seventh leaf stage. Plants were then subjected to a low-temperature treatment of 0 °C for 6, 12, and 24 hours, with three biological replicates per treatment time point. Leaf samples were collected from both control (ambient temperature) and treated plants for subsequent RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) analysis.

The genome and annotation files for Arabidopsis thaliana, Brassica rapa subsp. pekinensis, Brassica juncea, and Brassica napus were retrieved from the Ensembl Plants (http://plants.ensembl.org/index.html) database (Bolser et al., 2016). Genomic data for Brassica rapa were provided by our research group. TIL proteins from these five species were identified using HMMER v3.0 with the TIL domain (PF08212) Hidden Markov Model (HMM) profile from the PFAM (http://pfam.xfam.org/) database (Mistry et al., 2021; Yang et al., 2024), using a default E-value threshold. The physicochemical properties of the identified TIL proteins were analyzed using the ExPASy ProtParam tool (https://web.expasy.org/protparam/) (Zhang et al., 2025), and their subcellular localizations were predicted using the WoLF PSORT (https://psort.hgc.jp/) online tool (Liu et al., 2023).

A phylogenetic tree of the identified TIL genes was constructed using MEGA11 software and visualized using the iTOL (Letunic and Bork, 2021; Tamura et al., 2021). The protein motifs and conserved domains of the TIL proteins were analyzed using MEME software and the NCBI’s Batch CD-Search tool (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), respectively. The cis-acting elements in the promoter regions of the TIL genes were identified using the Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database (Chen et al., 2023). The chromosomal locations of the TIL genes were mapped using TB tools software. Tandem and segmental duplication events were investigated using the MCScanX module in TB tools with default parameters (Wang et al., 2013). The non-synonymous to synonymous substitution rate ratios (Ka/Ks) for the duplicated gene pairs were calculated using the Ka/Ks Calculator tool in TB tools (Chen et al., 2020). Finally, interspecies synteny relationships were analyzed using the Amazing Super Circos tool in TBtools-II. All visualizations were generated using TBtools-II software.

RNA-seq expression data for five tissues (root, stem, leaf, flower, and silique) of Brassica napus, Brassica rapa, and Brassica juncea were obtained from the BnIR (https://yanglab.hzau.edu.cn/), BRAD (http://brassicadb.cn/#/), and BjuIR (https://yanglab.hzau.edu.cn/BjuIR) databases, respectively, and subsequently analyzed (Chen et al., 2022; Yang et al., 2023; Zhang et al., 2024). For low-temperature stress expression analysis, transcriptome data (generated in our laboratory) from Brassica rapa and Brassica napus were used. All results were visualized using TBtools-II software (Chen et al., 2020).

Total RNA was extracted using an RNA Pure Plant Kit (Tiangen, Beijing, China). Complementary DNA (cDNA) was synthesized from the extracted RNA using a reverse transcription kit (Tiangen). Quantitative real-time PCR was performed using the FastReal qPCR PreMix (SYBR Green) kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Actin (gene ID: 103850356) was used as the reference gene for normalization, and all primers used are listed in Supplementary Table 1 (Tao et al., 2023). Relative gene expression levels were calculated using the 2^−ΔΔCT method.

The BrTIL1 protein sequence was submitted to the STRING database (http://string-db.org) (Szklarczyk et al., 2023) for protein-protein interaction (PPI) network, using Brassica rapa as the reference model with a Confidence threshold of 0.4. The resulting network was visualized and refined using Cytoscape software (v3.10.3), wherein isolated nodes (those without any interactions) were removed (Ono et al., 2025).

The coding sequence (CDS) of BrTIL1 was cloned and ligated into the pSuper1300-GFP vector to generate a pSuper1300-BrTIL1-GFP fusion construct. This construct, along with a plasma membrane marker and the empty pSuper1300-GFP vector as controls, was transiently expressed in tobacco (Nicotiana benthamiana) leaves via Agrobacterium tumefaciens-mediated transformation. After 2–3 days, GFP fluorescence was observed using confocal laser scanning microscopy. To confirm membrane localization, the leaf tissues were subsequently treated with a 50% sucrose solution for 20 minutes to induce plasmolysis and then re-examined under the confocal microscope.

The full-length cDNA of BrTIL1 was amplified by PCR and cloned into the pDONR vector using a BP recombination reaction. The entry clone was then transferred into the pEarlyGate101 destination vector via an LR recombination reaction to generate an expression construct for plant transformation. The resulting plasmid was introduced into Agrobacterium tumefaciens strain GV3101 for floral dip transformation of Arabidopsis thaliana. Homozygous T3 generation plants were selected using 0.01% Basta (Marques and Gallazzini, 2024). These transgenic plants, along with wild-type controls, were subjected to cold treatment at -4 °C for 0, 3, 6, 12, and 24 hours. Following treatment, a subset of plants was used for RNA extraction and measurement of physiological parameters, including soluble protein (SP) concentration and antioxidant enzyme activities (SOD, POD, CAT). Another subset was returned to normal growth conditions for a one-week recovery period before phenotypic observation and survival rate analysis were conducted. The experiment included three biological replicates.

To validate protein-protein interactions for BrTIL1 in yeast, the yeast two-hybrid assay was performed. The constructed plasmids include the positive control (pGBKT7-53 + pGADT7-T), negative control (pGBKT7-Lam + pGADT7-T), and the experimental group (pGBKT7-BrTIL1 + pGADT7) were individually transformed into the Saccharomyces cerevisiae Y2HGold strain. The transformed yeast cells were plated on double-dropout (DDO) medium (SD/-Leu/-Trp) containing X-α-Gal and incubated at 30 °C for 3–5 days. Subsequently, single colonies were picked and spotted onto quadruple-dropout (QDO) medium (SD/-Ade/-His/-Leu/-Trp) supplemented with X-α-Gal and Aureobasidin A. After incubation at 30 °C for 3–5 days, the results indicated that the pGBKT7-BrTIL1 bait construct did not exhibited self-activation. Positive blue colonies from the screening were selected for one-to-one interaction validation.

A total of 23 TIL genes were identified across the five Brassicaceae species: one in Arabidopsis thaliana (AtTIL), three in Brassica rapa (BrTIL1–3), five in Brassica rapa subsp. pekinensis (BraTIL1–5), six in Brassica juncea (BjTIL1–6), and eight in Brassica napus (BnTIL1–8). The genes were named systematically according to their chromosomal locations. Physicochemical analysis showed that these TIL proteins ranged from 137 (BraTIL5) to 346 amino acids (BraTIL4, BnTIL4, BjTIL3). Their molecular weights ranged from 15.7 kDa (BraTIL5) to 39.0 kDa (BnTIL4). All proteins exhibited an instability index greater than 31.72, and their theoretical pI values ranged from 4.9 (BnTIL1) to 9.25 (BraTIL5). Subcellular localization predictions indicated that the TIL proteins from Arabidopsis thaliana and Brassica rapa were localized to the cytoplasm. For the other species, cytoplasmic localization was predicted for three (60%) BraTIL proteins, four (66.7%) BjTIL proteins, and six (75%) BnTIL proteins (Table 1). These results suggest that this gene family primarily functioned in the cytoplasm.

To elucidate the evolutionary relationships of the TIL gene family, a phylogenetic tree was constructed using 23 TIL proteins from five Brassicaceae species, along with three OsTIL proteins from rice (Oryza sativa) and three SlTIL proteins from tomato (Solanum lycopersicum L.). The tree was divided into four major clades, designated I–IV (Figure 1, Supplementary Table 2). Clade I contained the most members (20), followed by Clade III (5), while Clades II and IV contained the fewest members, with 2 members each. The phylogenetic analysis revealed that Clade III contained TIL proteins from both monocots (rice) and dicots (tomato, Brassica rapa subsp. pekinensis, Brassica juncea, Brassica napus). The TIL proteins from the five Brassicaceae species (dicots) were primarily clustered in Clade I, which exhibited a greater genetic distance from Clade IV (monocots) but showed a closer phylogenetic relationship with Clade II (also dicots). These findings indicated that TIL genes existed prior to the divergence of monocots and dicots. The conserved presence of TIL genes in Clade III across both plant groups suggested that their functions were highly conserved. Furthermore, the predominant clustering of Brassicaceae TIL proteins in Clade I implied potential functional conservation within this family.

Based on phylogenetic relationships, gene structure and conserved motif analyses were conducted for the 23 TIL genes from five Brassicaceae species. The phylogenetic tree demonstrated close homology among the TIL proteins (Figure 2A). Conserved motif analysis revealed that all TIL members contained Motif 1, Motif 2, and Motif 5. However, certain motifs were exclusive to specific clades; for instance, Motif 3 was only identified in Clade I (Figure 2B). Further analysis showed that the Clade III members (BraTIL4, BjTIL3, and BnTIL4) possessed the lipocalin_FABP superfamily and lipocalin_CHL conserved domains. In contrast, the remaining 20 TIL proteins contained either Lipocalin-2 or lipocalin_Blc-like conserved domains (Figure 2C), suggesting potential functional divergence between these clades. Gene structure analysis indicated that Clade I members contained fewer introns (0–2), while Clade III members possessed a higher number (4) (Figure 2D), reflecting increased structural diversity that arose during evolutionary divergence. These results were consistent with the phylogenetic classification and suggested that TIL proteins within the same clade performed similar biological functions.

A comprehensive analysis of cis-acting elements was conducted in the promoter regions (2,000 bp upstream of the transcription start site) of all 23 TIL genes to investigate their potential functions. The analysis identified 26 distinct types of response elements (Figure 3A, Supplementary Table 3), which were categorized into four main groups: light-responsive, hormone-responsive, stress-responsive, and development-related elements. This finding indicated that the expression of TIL gene family members is coordinately regulated by multiple cis-acting elements. Further analysis revealed that light-responsive elements, ABA-responsive elements, and MeJA-responsive elements were widely distributed across all 23 genes. Notably, all three TIL genes from Brassica rapa contained low-temperature responsive elements, suggesting a potential association with cold acclimation during species evolution. This discovery indicated that TIL genes might contribute to this species’ capacity for low-temperature tolerance (Figure 3B). Furthermore, every TIL gene was found to contain at least one stress-responsive element (Figure 3C). Collectively, these results demonstrated that the expression of TIL genes is regulated by a diverse set of cis-acting elements, underscoring the extensive involvement of this gene family in plant responses to environmental stresses and hormonal signals.

The chromosomal distribution of TIL genes was analyzed across five Brassicaceae species. Arabidopsis thaliana contained a single AtTIL gene on chromosome 5 (Figure 4A), Brassica rapa harbored three BrTIL genes on chromosomes A02, A03, and A10 (Figure 4B), while its subspecies, Brassica rapa subsp. pekinensis, possessed five BraTIL genes on chromosomes A02, A03, A06, and A10 (Figure 4C). In Brassica juncea, six BjTIL genes were located on chromosomes A02, A03, A06, B02, B05, and B08 (Figure 4D), and Brassica napus contained eight BnTIL genes distributed across chromosomes A02, A03, A06, A10, C02, and C09 (Figure 4E). To understand the mechanisms of gene family expansion, duplication events were investigated. The analysis identified one tandem-duplicated gene pair in Brassica rapa subsp. pekinensis and two in Brassica napus (Supplementary Table 4). No segmental duplications were detected, indicating that tandem duplication was a major evolutionary force driving the expansion of the TIL gene family in these species.

Further intra-genomic collinearity analysis of TIL genesidentified 3, 3, 10, and 11 collinear gene pairs in Brassica rapa, Brassica rapa subsp. pekinensis, Brassica juncea, and Brassica napus, respectively, while no such events were detected in Arabidopsis thaliana (Figure 5A, Supplementary Table 5). These widely distributed collinear pairs further contributed to the family’s expansion. An examination of Ka/Ks ratios for these collinear pairs revealed that all values were less than 1 (Supplementary Table 6), indicating that the TIL genes had undergone purifying selection and possessed evolutionarily conserved functions. Interspecific collinearity analysis revealed the fewest collinear gene pairs (2) between Arabidopsis thaliana and Brassica rapa, and the most (20) between Brassica juncea and Brassica napus (Figure 5B, Supplementary Table 7), suggesting that many of these genes descended from shared ancestral genes. Furthermore, the number of collinear gene pairs increased from that in Arabidopsis thaliana to those in the more complex Brassica species, reflecting the accumulation of genomic duplication events during evolution. Given that AtTIL1 in Arabidopsis thaliana is known to confer abiotic stress tolerance (Charron et al., 2008). TIL genes in Brassica rapa, Brassica rapa subsp. pekinensi, Brassica juncea, and Brassica napus likely perform similar, conserved roles in abiotic stress tolerance.

To further investigate the TIL gene family, we analyzed the expression patterns of 17 TIL genes across various tissues of Brassica rapa, Brassica juncea, and Brassica napus. The results revealed distinct tissue-specific expression profiles. In Brassica rapa, BrTIL2 was highly expressed in flowers, while BrTIL3 showed high expression in leaves. In Brassica juncea, although all six BjTIL genes were expressed at high levels, BjTIL5 and BjTIL6 exhibited the highest expression in flowers and stems, respectively. In Brassica napus, BnTIL8 was highly expressed in flowers, whereas the expression of BnTIL1, BnTIL2, BnTIL3, and BnTIL6 was down-regulated in roots (Figure 6A, Supplementary Table 8). These tissue-specific patterns suggest that TIL genes may be functionally specialized for roles in development and environmental adaptation. Analysis of transcriptome data under cold stress showed that the expression of BrTIL1, BrTIL2, and BrTIL3 in Brassica rapa leaves is induced by low temperature (Figure 6B, Supplementary Table 9). To validate these findings, we performed RT-qPCR on two Brassica rapa cultivars with contrasting cold tolerance: the cold-tolerant ‘Longyou 7’ and the cold-sensitive ‘Lenox’. The results demonstrated that BrTIL1 expression was continuously up-regulated with prolonged cold treatment (0 °C), peaking at 24 hours in both cultivars (Figure 6C). This confirmed that BrTIL1 is a cold-responsive gene, with its expression directly modulated by the duration of low-temperature stress.

A protein-protein interaction network for BrTIL1 was predicted using the STRING database with Brassica rapa as the reference species. The analysis revealed that BrTIL1 potentially interacts with 10 proteins, including three transcription factors containing AP2 domains (M4D2F7, M4D5S6, and M4DMZ2), (Figure 7). Studies indicate that AP2 domain transcription factors serve as key regulators of low-temperature stress in Brassica rapa (Jaglo et al., 2001; Song et al., 2016; Mooney et al., 2019). Therefore, we hypothesize that BrTIL1 may participate in low-temperature stress signaling mechanisms by binding to AP2 domain transcription factors.

To investigate the biological function of BrTIL1, we generated homozygous BrTIL1-overexpressing Arabidopsis thaliana lines through Agrobacterium-mediated floral dip transformation (Supplementary Figures 1A–C). PCR analysis confirmed the successful integration of the transgene, indicating the presence of an approximately 400 bp target band in the transgenic lines (Supplementary Figure 1D). Phenotypic characterization revealed that while BrTIL1-overexpressing lines and wild-type plants were similar at the seedling stage, the transgenic lines exhibited significant developmental alterations post-floral transition. These changes included a dwarf stature and delayed flowering and fruiting times. A notable phenotypic difference was a significantly higher root-to-shoot ratio in the BrTIL1-overexpression lines compared to wild-type controls (Supplementary Figure 1E). These preliminary results suggest that BrTIL1 may enhance cold tolerance in transgenic Arabidopsis thaliana by modulating plant architecture and resource allocation, favoring root development.

Based on the above analysis, we selected BrTIL1 for functional validation. To investigate the function of the BrTIL1 gene under low-temperature stress, we subjected wild-type (WT) and BrTIL1-overexpressing Arabidopsis thaliana lines (OE) to -4 °C cold stress treatment at the seedling stage. Survival rates were recorded after one week of recovery at room temperature. The results showed no significant phenotypic differences between the two groups after 3 hours of cold treatment. However, after 24 hours, the leaves of WT plants exhibited severe yellowing and wilting, resulting in a low survival rate of only 27%. In contrast, OE lines maintained significantly better leaf integrity and exhibited a survival rate of 65% (Figures 8A, B), demonstrating that BrTIL1 overexpression markedly enhances freezing tolerance. Consistent with its role in cold adaptation, BrTIL1 expression in OE lines was strongly induced by low temperature, reaching an expression level 12.3-fold higher than in WT plants after 24 hours of cold stress treatment (Figure 8C). Physiological analysis revealed that antioxidant enzyme activities (SOD, POD, CAT) and soluble protein content were similar between WT and OE lines under normal conditions. However, upon cold stress, these parameters increased significantly in the OE lines. After 24 hours at -4 °C, the OE lines displayed peak POD, SOD, and CAT activities, which were 11.75%, 15.21%, and 40.00% higher than those in WT plants, respectively, alongside a 19.65% higher soluble protein content (Figures 8D–G). These results indicate that BrTIL1 is a cold-induced gene that positively regulates freezing tolerance. Its overexpression enhances the plant’s capacity to scavenge ROS by boosting key antioxidant enzyme activities and osmolyte accumulation, thereby mitigating cold-induced oxidative damage.

To determine the subcellular localization of BrTIL1, a pSuper1300-BrTIL1-GFP fusion construct was transiently expressed in Nicotiana benthamiana leaves. Confocal laser scanning microscopy showed that the green fluorescence signal of the BrTIL1-GFP fusion protein co-localized precisely with the red fluorescence of the plasma membrane marker. To confirm this localization, plasmolysis was induced by treating the leaf tissue with a 50% sucrose solution. The resulting separation of the plasma membrane from the cell wall demonstrated that the BrTIL1-GFP signal remained exclusively associated with the membrane (Figure 9). These results conclusively identifyed BrTIL1 as a plasma membrane-localized protein, supporting a potential role in membrane protection and stabilization, particularly under stress conditions.

To identify proteins that interact with BrTIL1 in Brassica rapa, a yeast two-hybrid (Y2H) cDNA library was constructed. Through self-activation validation (Supplementary Figure 2A), re-screening (Supplementary Figure 2B), and GO annotation with KEGG functional analysis (Supplementary Figures 2C, D), combined with molecular feature and signaling pathway analysis, six candidate proteins were selected for yeast single-hybrid validation with pGBKT7-BrTIL1. The results indicated that the BrTIL1 protein specifically interacted wit COP9, unnamed protein, the GNAT9, TMT2, TOPP1, and PPI1 in yeast cells (Figure 10A). Gene expression regulation is a fundamental component of the plant stress response. To investigate the relationship between BrTIL1 and its interacting partners, we analyzed the expression patterns of the six confirmed interacting protein genes under cold stress using RT-qPCR. The results revealed distinct expression profiles among the interactors (Figure 10B). Notably, the expression of COP9 and TMT2 closely mirrored that of BrTIL1, both showing significant upregulation in response to cold stress. This induction was more pronounced in the cold-tolerant cultivar ‘Longyou 7’ than in the cold-sensitive ‘Lenox’. Specifically, COP9 expression peaked at 12 hours, with 8.2-fold and 3.3-fold increases in ‘Longyou 7’ and ‘Lenox’, respectively. Similarly, TMT2 expression peaked at 24 hours, with 4.6-fold and 3.1-fold upregulation in the respective cultivars. In contrast, other interactors displayed divergent expression. The unnamed protein gene was downregulated in ‘Longyou 7’ but upregulated in ‘Lenox’. PPI1 was upregulated in both cultivars but showed overall higher expression in ‘Lenox’, potentially indicating a role in cold stress perception rather than tolerance. Meanwhile, GNAT9 expression was significantly suppressed, and TOPP1 exhibited fluctuating expression levels under cold stress. Collectively, these findings demonstrate that BrTIL1-interacting proteins display diverse and cultivar-specific expression patterns under low-temperature stress, implicating them in a complex regulatory network governing cold adaptation.