The experiment was carried out in Jianji Village, Laojunshan Town, Jianchuan County, Dali Bai Autonomous Prefecture, Yunnan Province (E 99°33′, N 26°31′, altitude 2,565 m). The experimental material was the flue-cured tobacco variety Honghua Dajinyuan, cultivated using floating seedling technology. Transplanting of the seedlings under the film was conducted in the year 2021, with a row spacing of 120 cm × 60 cm. Topping was conducted on June 10, leaving 15–16 leaves after topping. Harvesting and curing of the lower leaves began on July 3 and ended on September 7. The soil type used in the experiment was loam, with a pH of 6.47, organic matter content of 56.19 g/kg, total nitrogen of 2.76 g/kg, total phosphorus of 1.11 g/kg, total potassium of 17.64 g/kg, water-soluble nitrogen of 210.8 mg/kg, available phosphorus of 91.3 mg/kg, and available potassium of 285.5 mg/kg.

The experiment was divided into two treatments: Treatment 1 was inside a plastic greenhouse (CK), where a random plot of tobacco leaves measuring 10 m in length and 5 m in width was selected, and a steel-frame greenhouse was constructed. The top was covered with polyvinyl chloride and polyethylene plastic for insulation, rain protection, and light transmission. The sides of the greenhouse were enclosed with two to three layers of shading net to achieve insulation and wind protection. Protective rows were established around the greenhouse. Treatment 2 was conducted under natural field conditions (Treat), where a plot of the same area as in Treatment 1 was randomly selected, with protective rows set up around it. Both treatments were conducted within the same field block, with 60 flue-cured tobacco plants in each treatment. The greenhouse was installed when cold stress was imminent, ensuring that the growth conditions inside and outside the greenhouse were identical before the cold stress.

A TH12R-EX temperature and humidity recorder (Shenzhen Huahanwei Science and Technology Co., Ltd., Shenzhen, China) was placed in the experimental field to record daily temperature and humidity changes. Additionally, a WH-2310 wireless weather station (Jiaxing Misu Electronic Co., Ltd., Zhejiang, China) was used to record daily information on temperature, precipitation, and wind speed. Irrigation of tobacco plants inside the greenhouse was conducted as necessary, based on the natural precipitation outside the greenhouse.

The experiment personnel observed meteorological data and tobacco leaf phenotypes. Once the temperature dropped and the tobacco leaves exhibited phenotypic changes, samples were taken. All tobacco samples were collected from the upper leaves (the fifth to sixth leaves from the top).

A TH12R-EX temperature and humidity recorder was placed in the experimental field to record daily temperature and humidity changes, while a WH-2310 wireless weather station was used to record daily information on temperature, precipitation, and wind speed. Meteorological data from July to August were collected comprehensively using the weather station, temperature and humidity recorder, and local meteorological bureau, specifically including temperature, rainfall, and wind speed, with a focus on analyzing the meteorological data when cold injury appeared in the field tobacco leaves.

Each treatment had three biological replicates. For each replicate, flue-cured tobacco plants with consistent growth were selected, and the samples were immediately stored in liquid nitrogen after sampling for the determination of physiological indicators and plastid pigment content.

Superoxide dismutase (SOD) activity was measured using the nitro blue tetrazolium (NBT) reduction method (Sadak et al., 2020), and peroxidase (POD) and catalase (CAT) activities were determined using the guaiacol method (Rácz et al., 2018) and ultraviolet spectrophotometry (Ekinci et al., 2020), respectively. The malondialdehyde (MDA) content was detected using the thiobarbituric acid (TBA) method (Chen et al., 2022), and the hydrogen peroxide (H₂O₂) content was determined using the titanium sulfate colorimetric method (Brennan and Frenkel, 1977).

The mean and standard deviation were calculated using IBM SPSS Statistics 27.0 software, and an independent samples t-test was used to analyze the significance of differences.

As indicated in Figure 1A , the temperature and rainfall at the Jianji Village experimental site in Laojunshan Town, Jianchuan County, exhibited considerable fluctuations in August. The daily maximum temperature varied from 14.6°C to 30.8°C, the daily minimum temperature ranged from 8.6°C to 16.6°C, and the daily rainfall ranged from 0 mm to 35.8 mm. On August 26, the greatest diurnal temperature variation was recorded at 22.2°C, but without any rainfall. On August 18, the smallest diurnal temperature variation was 3.1°C, accompanied by 35.8 mm of rainfall. According to the local survey of field chilling stress, a significant temperature drop occurred on August 18, accompanied by heavy rainfall and a small amount of hail. In the following 4–5 days, large-scale chilling stress symptoms appeared in flue-cured tobacco, with the most severe symptoms in the middle and upper leaves. The leaf surface color gradually changed, as shown in Figure 1B : the normal tobacco leaf surface was green; after 1 day of chilling stress, the color changed to light pink; after 1–2 days of chilling stress, it changed to dark green; after 3–4 days, the leaf surface turned purple-black.

On August 19, samples of normal tobacco leaves (CK) and chilling-stressed tobacco leaves (Treat) were collected from the experimental site ( Figure 2C ), with the sampling locations being the fifth to sixth leaves from the bottom to the top. As seen in Figure 2C , the normal tobacco leaves on the right showed no obvious symptoms of chilling stress, while the chilling-stressed tobacco leaves on the left clearly showed large pink spots on the leaves, indicating that chilling stress has occurred.

MDA, as one of the final products of lipid peroxidation in membranes, can effectively reflect the degree of cell membrane damage. Figure 2A shows that the MDA levels in tobacco leaves significantly increase after chilling stress. The transmission of plant stress signals is influenced by changes in H₂O₂ content. As shown in Figure 2B , the H₂O₂ content in tobacco leaves under chilling stress is significantly higher than that in normal tobacco leaves (p < 0.05). Chilling stress also led to a significant decrease in SOD and POD activities in tobacco leaves (p < 0.05) ( Figures 2C, D ), whereas CAT activity significantly increased (p < 0.05) ( Figure 2E ). Additionally, we observed that under chilling stress, the SPAD, chlorophyll a, chlorophyll b, lutein, and β-carotene values in tobacco leaves were all significantly reduced (p < 0.05) ( Figure 2 ).

Figure 3A presents the results of the metabolite data identified using PCA, with the first principal component (PC1) scoring 36.43% and the second principal component (PC2) scoring 16.1%. The figure demonstrates that the CK-treated tobacco leaves and the chilling-stressed tobacco leaves were clearly separated, with replicate samples from different treatments closely clustered together, indicating good data reproducibility. Additionally, cluster analysis of differential metabolites was conducted for each of the 12 sample comparison groups in this study. The results, as shown in Figures 2B, C , indicate that the biological replicates of the experimental samples are relatively consistent and stable. The selection of differential metabolites was mainly based on three parameters—VIP, FC, and p-value—with thresholds set at VIP > 1.0, FC > 2.0 or FC < 0.5, and p-value <0.05. The differential metabolites selected are shown in Figure 3D : a total of 1,035 metabolites were identified in the metabolomics analysis, with 240 differential metabolites identified, of which 172 were upregulated and 68 were downregulated.

As shown in Figure 3E , the KEGG enrichment results indicate that the differential metabolites are significantly enriched in three metabolic pathways: Tyrosine metabolism (map00350), Isoquinoline alkaloid biosynthesis (map00950), and Stilbenoid, diarylheptanoid, and gingerol biosynthesis (map00945).

Further analysis revealed that chilling stress significantly upregulated several key metabolites in the Tyrosine metabolism (map00350), Isoquinoline alkaloid biosynthesis (map00950), and Stilbenoid, diarylheptanoid, and gingerol biosynthesis (map00945) pathways in tobacco leaves, including l-Dopa, Tyramine, Gentisic acid, Rosmarinic acid, 6-Gingerol, Resveratrol, and Piceatannol. In addition, we found that in stress resistance-related metabolic pathways, Saccharopine in the Lysine degradation pathway (map00310), Spermidine in the ABC transporter pathway (map02010), and Palmitic acid and ACAA1 protein in the Fatty acid degradation pathway (map01212) were all significantly upregulated, while Biotin was downregulated in the ABC transporter pathway ( Figure 3F ).

Proteomics analysis was performed on normal and chilling-stressed tobacco leaves. As shown in Figure 4A , the reproducibility of the biological replicate sample data was good. A total of 246,819 spectra were obtained, with 68,496 spectra matched, 27,350 peptides identified, and 5,950 proteins identified ( Figure 4B ). On the basis of the entire proteome, the protein data obtained from the biological replicates were combined, and the commonly expressed proteins were used for differential protein screening. A total of 410 commonly differentially expressed proteins were identified between the chilling-stressed and normal tobacco leaves in the comparison group, with 176 upregulated (p ≤ 0.05, fold change >1.5) and 234 downregulated (p ≤ 0.05, fold change <0.67) ( Figure 4C ).

Gene Ontology (GO) functional annotation analysis was performed on the differentially expressed proteins in different groups, focusing on biological processes, cellular components, and molecular functions. Figure 4D shows the GO annotation results for the differentially expressed proteins in chilling-stressed and normal tobacco leaves, with the top 20 GO enrichments including eight biological processes, nine cellular components, and three molecular functions. The biological processes were mainly enriched in translation, ribosome biogenesis, cellular biopolymer biosynthetic process, biopolymer biosynthetic process, ribonucleoprotein complex biogenesis, and macromolecule biosynthetic process. The cellular components were mainly enriched in ribosomal subunit, ribosome, large ribosomal subunit, cytosolic large ribosomal subunit, and cytosolic ribosome. The molecular functions were mainly enriched in the structural constituent of ribosome, structural molecule activity, and RNA binding ( Figure 4D ).

KEGG pathway enrichment analysis revealed that the differential proteins were significantly enriched in pathways such as Ribosome (map03010), Starch and sucrose metabolism (map00500), and Glycolysis/Gluconeogenesis (map00010). The upregulated proteins were significantly enriched in metabolic pathways such as Starch and sucrose metabolism (map00500) and Amino sugar and nucleotide sugar metabolism (map00520). The downregulated proteins were significantly enriched in pathways such as Ribosome (map03010) and Photosynthesis (map00195) ( Figure 4E ).

To validate the abundance differences of proteins related to chilling-stressed and normal tobacco leaves, 18 important proteins were randomly selected for PRM verification. The comparison of PRM quantification and isobaric tag for relative and absolute quantitation (iTRAQ) for the protein expression differences in the Treatcomparison group is shown in Figure 5 among them, 16 proteins showed consistent levels of expression changes, while two proteins showed opposite trends. Overall, the results are largely consistent with our iTRAQ-based proteomics quantification results ( Figure 5 ).

In summary, the physiological, metabolic, and protein-level responses of tobacco leaves under cold stress form a highly coordinated process, with interactions between these layers working together to ensure the plant’s adaptability to adverse environments. The results of this study reveal the multi-level response mechanisms of tobacco leaves to cold stress, from physiological to metabolic to molecular levels. The measured altitude of the experimental site in this study was 2,565 m, significantly higher than the main tobacco-growing areas in Yunnan. The temperature fluctuations during the curing period were exceptionally intense, with August temperatures ranging from 14.6°C to 30.8°C, making field chilling injury highly likely. The investigation found that on the day of chilling stress (August 18), the temperature difference at the experimental site was 25.4°C. Over the next 4 days, the appearance of the middle and upper tobacco leaves changed from green to light pink, then to dark green, and finally to purple-black. This color change is a visual indicator of the plant’s progression from mild to severe damage due to chilling, reflecting the complex physiological and biochemical changes within the leaves. Research indicates that low-temperature stress causes numerous visible changes in plants, particularly phenotypic alterations, representing the most direct expression of environmental interference. With prolonged exposure to low-temperature stress, plants show visible symptoms such as wilting, slowed growth, and leaf chlorosis (Cheng et al., 2021). Under continued chilling stress, cells may undergo irreversible damage leading to cell death. The rupture of cell membranes and lipid peroxidation reactions are likely the primary causes of the tobacco leaves turning dark green or purple-black (Zhang et al., 2020; Bilska-Kos et al., 2017; Stefanowska et al., 1999).

Research indicates that the level of photosynthetic pigments is closely related to photosynthetic function (Wang et al., 2022; Yin et al., 2022), and it is generally believed that the content of photosynthetic pigments decreases when plants are subjected to stress (Jaleel et al., 2008; Anjum et al., 2017). In this study, after chilling stress, the chlorophyll and carotenoid contents in tobacco leaves significantly decreased compared to the control, indicating that chilling stress directly inhibited the photosynthetic capacity of the tobacco leaves. The reduction in photosynthetic pigments not only affects the efficiency of light absorption and conversion but may also lead to structural damage to the photosynthetic apparatus, which corresponds to the color changes observed in tobacco leaves after chilling stress.

Abiotic stresses such as drought, low temperature, salt stress, and heavy metal pollution significantly inhibit plant growth and development. These adverse conditions can damage or disrupt various metabolic processes within plant cells, leading to a substantial accumulation of reactive oxygen species (ROS) (Choudhury et al., 2017). H₂O₂, as a primary form of ROS, can indicate the extent of damage in plants (Hossain et al., 2015). When plants are subjected to stress, lipid peroxidation of cell membranes occurs, leading to increased membrane permeability. MDA, a byproduct of lipid peroxidation, increases in content, reflecting the degree of peroxidation and the intensity of the stress faced by the plant (Orts et al., 2024). Previous studies have found a positive correlation between decreasing temperature and increasing MDA content (Gao et al., 2021). In this study, chilling stress significantly increased the levels of H₂O₂ and MDA in tobacco leaves, indicating that the tobacco leaf cells suffered severe oxidative damage, with the structure and function of cell membranes impaired. Although plants typically eliminate excess ROS through their antioxidant system, under the chilling stress in this study, the activities of key antioxidant enzymes such as SOD and POD were significantly reduced, with only CAT activated in an attempt to remove the excess ROS (He et al., 2017). This is contrary to previous studies on the response of antioxidant enzymes in tobacco leaves to chilling stress (Gu et al., 2021), possibly because, under natural conditions, the large and sudden temperature drop caused the tobacco leaves’ antioxidant system to be unable to adapt to the rapidly changing environment in a short time, thereby weakening its ability to eliminate ROS.

Changes at the physiological level are often accompanied by the reorganization of metabolic networks to enhance the plant’s resistance to stress. In this study, chilling stress significantly upregulated several key metabolites in the tyrosine metabolism, isoquinoline alkaloid biosynthesis, and stilbenoid, diarylheptanoid, and gingerol biosynthesis pathways in tobacco leaves, including l-Dopa, Tyramine, Gentisic acid, Rosmarinic acid, 6-Gingerol, Resveratrol, and Piceatannol. The upregulation of these metabolites may reflect a physiological response in tobacco to enhance its resistance to chilling stress by strengthening antioxidant and defense mechanisms. l-Dopa and Tyramine play important roles in the tyrosine metabolism and isoquinoline alkaloid biosynthesis pathways, and their increase may promote the accumulation of antioxidant compounds and defense-related secondary metabolites, enhancing the plant’s ability to cope with oxidative stress (Schenck and Maeda, 2018; Cherubino Ribeiro et al., 2023). Additionally, Gentisic acid and Rosmarinic acid, as phenolic compounds with strong antioxidant capacities, may be upregulated to further protect cells from oxidative damage caused by chilling stress (Cao et al., 2005; Li et al., 2022). The upregulation of polyphenolic compounds such as 6-Gingerol, Resveratrol, and Piceatannol suggests that the plant may mitigate the damage to cell structure and function caused by chilling by enhancing its antioxidant capacity (Stagos, 2019).

Additionally, we found that in stress resistance-related metabolic pathways, Saccharopine in the Lysine degradation pathway, Spermidine in the ABC transporter pathway, and Palmitic acid and ACAA1 protein in the Fatty acid degradation pathway were significantly upregulated, while Biotin was downregulated in the ABC transporter pathway. These changes suggest that flue-cured tobacco enhances its stress adaptation through various metabolic regulatory strategies under chilling stress. The accumulation of Saccharopine may be related to the carbon skeleton supply and NADH production in the lysine degradation pathway, providing energy support for stress response (Fujioka, 1975). Spermidine, as a polyamine, may help stabilize cell membranes and enhance antioxidant defense when upregulated (Saleethong et al., 2016), while the downregulation of Biotin may be related to the plant’s metabolic reprioritization under low-temperature conditions. Additionally, the upregulation of Palmitic acid and ACAA1 protein suggests accelerated fatty acid degradation, providing more energy and metabolic intermediates to support the plant’s physiological needs under chilling stress (Guan and Liu, 2020). These results indicate that chilling stress enhances the accumulation of various stress resistance-related metabolites in tobacco leaves by regulating multiple metabolic pathways, thereby improving the plant’s adaptation to low-temperature environments.

The accumulation of metabolites depends on protein regulation, and changes in protein expression further modulate the plant’s stress resistance strategies. After tobacco leaves are subjected to chilling injury, energy metabolism plays a crucial role. Insufficient energy supply can lead to physiological imbalances in the tobacco leaves, thereby exacerbating the occurrence of chilling injury. In this study, under chilling stress, proteins in the glycolysis pathway, including hexokinase (HK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enolase (ENO), were significantly upregulated. Similarly, proteins in the Starch and sucrose metabolism pathway, such as hexokinase (HK) and glucan endo-1,3-beta-d-glucosidase (EGLC), were also significantly upregulated (Ruan, 2014; Poonam et al., 2016). Additionally, the Amino sugar and nucleotide sugar metabolism pathways play a crucial role in plant stress resistance through various mechanisms, including cell wall reinforcement, antioxidant defense, signal regulation, energy support, and immune enhancement (Ma et al., 2021; Ran et al., 2023). The study found that chilling stress activated the upregulation of proteins such as Chitinase (E3.2.1.14) and Hexokinase (HK) in the Amino sugar and nucleotide sugar metabolism pathways in tobacco leaves. This further indicates that the synergistic effects of multiple metabolic pathways help tobacco leaves maintain cell structure integrity and physiological function under chilling conditions, thereby enhancing the plant’s overall stress resistance.

However, under low-temperature stress, 61 proteins in the Ribosome (map03011) pathway, including Small subunit ribosomal protein S10 (RP-S10), Large subunit ribosomal protein L4e (RPL4), Large subunit ribosomal protein L2 (MRPL2), Large subunit ribosomal protein L14 (MRPL14), and Small subunit ribosomal protein S11 (rpsK), were downregulated, indicating that chilling injury caused ribosomal instability in tobacco leaves, thereby inhibiting protein synthesis. This is consistent with the findings of Lv et al. (2021) under low-temperature treatment where Volvariella volvacea primarily responds to stress by activating the VVO_00750 gene in the ribosome pathway. The ribosome is the central machinery for protein synthesis, and the reduction of ribosomal proteins directly weakens ribosome assembly and stability, thereby reducing overall translation efficiency (Rodnina et al., 2016). This implies that during the chilling process, new proteins cannot be effectively synthesized in tobacco leaves, which in turn hinders the cells’ ability to repair and respond to chilling stress damage.

Furthermore, through additional screening, this study identified differentially abundant proteins enriched in the Photosynthesis pathway in leaves under chilling stress, including psbC, psbF, and psbO of PSII; psaA and psaK of PSI; and petD, petA, and petF of the cytochrome b ₆ f complex, which is consistent with the findings of Cui et al. (2014) on the differential gene expression profile of tobacco seedlings under low-temperature stress. The downregulation of these proteins indicates that chilling stress inhibited the photosynthetic electron transport chain in tobacco leaves, leading to a decline in photosynthetic efficiency (Peredo and Cardon, 2020). This inhibition is closely related to the reduction in chlorophyll a and b contents. Chlorophyll a and b are key pigments in capturing light energy and are involved in energy transfer in photosystems I (PSI) and II (PSII). As these photosynthetic pigments decrease, the light energy capture ability declines, further leading to the downregulation of related proteins in PSII and PSI, ultimately weakening the entire photosynthesis process (Leister and Schneider, 2003; Allakhverdiev et al., 2008). In conclusion, at the protein level, chilling stress intensifies the physiological imbalance and damage in tobacco leaves by disrupting energy metabolism, inhibiting protein synthesis, and impairing photosynthesis in a synergistic manner.