DNA methylation, transcriptomic, and metabolomic analyses provide new mechanistic insights into cold shock-induced chilling tolerance in cucumber

Abstract

Cucumber is cold-sensitive; improper storage induces chilling injury. To investigate how 3 h 0 °C cold shock (CS) alleviates chilling injury in ‘Baijinyu’ cucumbers at 4 °C, we performed integrated DNA methylation, transcriptomic, and metabolomic analyses. CS alleviated chilling injury, delayed quality decline (soluble solids, firmness, color), and altered accumulation of lipids, keto acids/esters, amino acids, and organic acids. CS increased methylation of peroxidase, caffeic acid 3‑O‑methyltransferase, and cinnamyl‑alcohol dehydrogenase, upregulating peroxidase but downregulating the latter two, accompanied by accumulation of (E)-ferulic acid, L‑tyrosine, and trans‑5‑O‑(p‑coumaroyl)shikimate. CS induced hypomethylation of CG‑type distal intergenic region of NCED and CHH‑type promoter region of abscisic acid receptor PYL, leading to downregulation of NCED and upregulation of PYL, thereby suppressing ABA biosynthesis and modulating ABA signaling. Moreover, CS maintained membrane lipid stability. Overall, CS enhances chilling tolerance by DNA methylation-mediated regulation of transcriptional and metabolic networks, supporting its potential as a green postharvest treatment.

Introduction

Cucumber (Cucumis sativus L.) is a tropical and subtropical crop belonging to the family Cucurbitaceae. It is widely cultivated worldwide and represents one of the most important protected-cultivation vegetables in China, with the largest planting area and broad geographic distribution¹. The ‘Baijinyu’ cucumber, also referred to as mini or baby cucumber, is approximately half the length of conventional cucumbers, typically measuring 4–5 cm. It is characterized by a light green peel, smooth skin, crisp texture, and pronounced aroma. Owing to its superior sensory attributes, the economic value of ‘Baijinyu’ cucumber is approximately two to three times higher than that of common cucumber cultivars, leading to its increasing popularity in recent years. In addition, ‘Baijinyu’ cucumber is rich in water, vitamins, minerals, and antioxidant compounds². However, due to its thin peel and delicate tissue structure, ‘Baijinyu’ cucumber is highly susceptible to mechanical damage after harvest and rapidly undergoes wilting, shriveling, discoloration, and overall quality deterioration at ambient temperature³. Consequently, refrigerated storage has become the predominant commercial preservation method. Nevertheless, inappropriate low-temperature storage readily induces chilling injury (CI), which manifests as surface pitting, water-soaked lesions, discoloration, off-flavors, tissue breakdown, and internal decay⁴. Therefore, elucidating the molecular mechanisms underlying chilling injury in cucumber and developing effective mitigation strategies are of great practical importance for reducing postharvest losses, extending storage life, and maintaining the commercial quality of high-value cultivars such as ‘Baijinyu’ cucumber.

CI in cucumber triggers a cascade of physiological, biochemical, and molecular alterations, including pronounced changes in metabolite profiles and gene expression⁵. CI-induced cellular damage is often associated with membrane lipid disruption, which represents one of the most severe forms of injury⁶. With the occurrence of cold damage, lipid metabolism in cucumbers will be enhanced⁷. Previous studies have demonstrated that an elevated phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio promotes membrane phase separation and destabilization in cucumber fruit⁸. In addition, polyamines-such as putrescine, spermidine, and spermine are essential functional molecules involved in plant stress responses. These compounds contribute to improved membrane stability, reduced lipid peroxidation, and enhanced tolerance to low-temperature stress⁹. Recent studies in tomato have further suggested that DNA methylation participates in chilling responses by regulating key genes involved in hormone signaling pathways, providing new insights into the epigenetic regulation of CI and highlighting potential mechanisms relevant to cucumber fruit¹⁰. Currently, postharvest preservation strategies for cucumber include edible coatings, heat treatments, modified atmosphere packaging (MAP), and the application of bioactive compounds such as salicylic acid, melatonin, jasmonic acid, betaine, and polyamines¹¹. Although these approaches have proven effective to varying degrees, many require chemical inputs or complex handling procedures, underscoring the need for a simple, efficient, and practical physical preservation technology to alleviate CI during low-temperature storage.

Cold shock (CS) treatment involves the short-term exposure of produce to sufficiently low temperatures to induce cold acclimation without causing irreversible injury¹². CS treatment has been reported to extend the shelf life of various fruits and vegetables, including peach, sweet cherry, and bell pepper, by maintaining firmness, visual quality, and cold tolerance^13–15. In cucumbers, previous studies have shown that CS treatment can reduce weight loss and firmness decline during storage, enhance antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), and modulate respiration rate¹⁶. In particular, CS treatments at 0 °C for 40 min and at 3 °C for 20, 40, or 60 min have exhibited optimal preservation effects¹⁷.

Despite these advances, existing studies on CS treatment in cucumber have focused primarily on physiological responses, with limited insight into the underlying gene regulatory and epigenetic mechanisms. Therefore, the present study integrated DNA methylation profiling, transcriptomic and metabolomic analyses, and physiological assessments to systematically elucidate the molecular mechanisms by which CS treatment enhances chilling tolerance in ‘Baijinyu’ cucumbers. Furthermore, a regulatory network describing CS-mediated alleviation of chilling injury was constructed. The findings of this study provide a theoretical basis for the commercial application of CS technology and offer new perspectives for postharvest preservation of other thermophilic fruits and vegetables.

Results

Effects of cold shock on chilling injury index, color, firmness, and soluble solids content of postharvest cucumbers

As shown in Fig. 1A, cucumbers stored at 4 °C for 8 days followed by 2 days (CK-10 d) at 20 °C developed typical chilling injury symptoms, including surface pitting, water-soaked lesions, and discoloration. In contrast, A 3 h cold shock treatment at 0 °C on cucumbers, followed by storage at 4 °C for 8 days and then at 20 °C for two days (CS-10 d), markedly suppressed CI development and helped maintain external fruit quality. After 10 days, the CI index of the CK group reached 0.70, whereas that of CS-treated cucumbers was only 0.23, representing a 66.67% reduction in CI severity compared with CK (Fig. 1B). At day 0 (CK-0 d), the soluble solids content (SSC) of cucumbers was 4.20%. After 10 days of storage, SSC significantly declined to 3.57% in CK fruit, whereas CS-treated fruit retained a higher SSC of 3.90%. Compared with CK-10 d, CS treatment significantly alleviated the reduction in SSC (Fig. 1C). As chilling injury progressed, fruit texture also deteriorated. Both groups exhibited significant firmness loss after 10 days compared with day 0; however, the magnitude of decline differed markedly between treatments. Firmness decreased by 7.56 N in CK fruit but by only 2.88 N in CS-treated fruit. Consequently, firmness in CS-10 d cucumbers was approximately 21.18% higher than that in CK-10 d fruit (Fig. 1D). Color parameters also changed during storage. The a^* value, reflecting greenness, increased significantly in CK-10 d fruit but was markedly suppressed in CS-10 d fruit, indicating that CS treatment delayed chlorophyll degradation and better maintained green coloration during storage (Fig. 1E). As shown in Fig. 1F, the L^* value increased with storage time. No significant difference was observed between CK-10 d and CK-0 d; however, CS treatment resulted in a significantly higher L^* value, suggesting improved brightness in CS-treated fruit. This might be due to light, oxidation and respiration, causing the cucumber to change from its natural green color to white¹⁸.

Fig. 1. Effects of cold shock on chilling injury index, color, firmness, and soluble solids content of postharvest cucumbers.

A Fruit appearance. B Chilling injury index. C Firmness. D soluble solids content. E a* value (red-green value, as the value of an increases, the color changes from green to red). F L* value (lightness).

Effects of cold shock on metabolite profiles of postharvest cucumbers

To elucidate the metabolic regulation underlying CS-induced chilling tolerance, widely targeted metabolomic analysis was conducted on CK-0 d, CK-10 d, and CS-10 d samples. Principal component analysis (PCA), including quality control (QC) samples, revealed clear separation among the three groups and tight clustering within each group, indicating high data reliability and repeatability (Supplementary Fig. S10). In total, 2,822 metabolites were detected, with lipids (15.4%), ketones–aldehydes–esters (14.1%), terpenoids (12.8%), sugars (10.3%), and organic acids (9.0%) representing the five most abundant categories (Fig. 2A).

Fig. 2. Overview of metabolomic, transcriptomic, and DNA methylation analyses in cucumber under cold shock treatment.

A Metabolite classification diagram. B The number of three groups of DAMs. C Up-regulation and down-regulation ratio of different types of metabolites in two groups of cucumbers. D The expression of TFs in CK-10 d vs. CS-10 d. E The number and changes of DEGs in the comparison group CK-0 d vs. CK-10 d. F The number and changes of DEGs in the comparison group CK-0 d vs. CS-10 d. G The number and changes of DEGs in the comparison group CK-10 d vs. CS-10 d. H Three sets of DEGs Venn diagrams. I Several differentially methylated regions (DMRs) were identified in the CK-0 d vs. CK-10 d, CK- 10 d vs. CS- 10 d comparisons. J Map of methylation levels of functional elements in the genome between groups. K Annotation of DMRs and their distribution in the CK-10 d vs. CS-10 d comparison group. L Methylation level and proportion of mC in different sequence contexts (CG, CHG, and CHH).

In CK-0 d vs CK-10 d, 1541 DAMs were detected, comprising 1043 increased and 498 decreased metabolites (Fig. 2B). The major DAM categories included ketones–aldehydes–esters (16.5%), lipids (16.2%), terpenoids (10.1%), organic acids (8.8%), sugars (8.6%), and amino acids (7.4%). Approximately 62% of lipid- and ketone–aldehyde–ester-related DAMs increased, while more than 70% of terpenoids, organic acids, sugars, and amino acids showed increased accumulation during storage (Fig. 2C). KEGG pathway enrichment analysis revealed that, relative to CK-0 d, DAMs in CK-10 d were mainly enriched in C5-branched dibasic acid metabolism, betalain biosynthesis, biotin metabolism, arginine biosynthesis, and D-amino acid metabolism (Supplementary Fig. S11). These pathways involved 26 DAMs, of which 17 were upregulated and 9 were downregulated. Metabolites showing marked increases included N-acetylornithine, pimelic acid, trans-aconitic acid, aconitic acid, and (Z)-aconitic acid, whereas 2-ketoglutaric acid, L-aspartic acid, L-glutamic acid, and related compounds exhibited significant decreases (Supplementary Table S1).

In CK-10 d vs CS-10 d, 1010 DAMs were identified, including 502 increased and 508 decreased metabolites (Fig. 2B). After applying thresholds of p < 0.05 and |log₂ (fold change) | ≥ 1262 DAMs remained, comprising 79 upregulated and 183 downregulated metabolites (Supplementary Table S2). These DAMs were mainly classified as lipids (19.8%), ketones–aldehydes–esters (17.1%), terpenoids (10.3%), sugars (6.4%), organic acids (6.4%), and amino acids (5.7%), with the majority of metabolites showing decreased levels following CS treatment (Fig. 2C). Notably, polyphenols exhibited an increasing trend after CS treatment. Several saturated fatty acids, including azelaic acid, 9-hydroxypentadecanoic acid, and 2-hydroxypalmitic acid, were significantly reduced in CS-10 d fruit (Fig. 5B). The reduction in saturated fatty acids suggests a shift toward a higher unsaturated-to-saturated fatty acid ratio, which may contribute to enhanced membrane fluidity under low-temperature stress. KEGG enrichment analysis of CK-10 d vs CS-10 d DAMs indicated significant enrichment in betalain biosynthesis, sulfur relay system, zeatin biosynthesis, pantothenate and CoA biosynthesis, tyrosine metabolism, and efferocytosis (Supplementary Fig. S12).

Fig. 5. Membrane lipid metabolism.

A Expression of genes related to membrane lipid metabolism pathway. B The content of metabolites related to membrane lipid metabolism. C Differential methylation of genes related to membrane lipid metabolism. Phosphatidylinositol (PI), Phosphatidylcholine (PC), Free fatty acids (FFAs), Phosphatidic acid (PA), Unsaturated fatty acids (USFAs), Saturated fatty acids (SFAs), Lysophosphatidic acid (LPA), diacylglycerol (DAG).

Overall, chilling injury induced pronounced disruptions in amino acid metabolism, organic acid metabolism, membrane lipid composition, and energy metabolism. CS treatment mitigated these alterations by reducing saturated fatty acid accumulation, promoting polyphenol enrichment, and modulating amino acid and organic acid metabolic balance, thereby alleviating cold-induced physiological damage in postharvest cucumbers.

Effects of cold shock on gene expression in postharvest cucumbers

To characterize the transcriptional regulatory effects of CS treatment during postharvest storage, RNA sequencing was performed on three sample groups: CK-0 d, CK-10 d, and CS-10 d. A total of nine libraries were constructed and sequenced, generating 52.68 Gb of clean data. Each sample yielded at least 5.71 Gb of clean reads, with Q30 values ≥ 94.16%. More than 97.00% of the clean reads were successfully mapped to the cucumber reference genome, indicating high sequencing quality and mapping efficiency (Supplementary Table S3).

Differential expression analysis revealed extensive transcriptional reprogramming during storage and in response to CS treatment. In CK-0 d vs CK-10 d, 4335 differentially expressed genes (DEGs) were identified, including 1651 upregulated and 2684 downregulated genes (Fig. 2E). In CK-0 d vs CS-10 d, 4,185 DEGs were detected, comprising 1470 upregulated and 2715 downregulated genes (Fig. 2F). By contrast, only 864 DEGs were identified in CK-10 d vs CS-10 d, including 340 upregulated and 524 downregulated genes (Fig. 2G), indicating that CS treatment markedly attenuated transcriptional changes induced by chilling stress. Venn diagram analysis showed that 342 DEGs were shared among all three comparisons (Fig. 2H), whereas 711, 667, and 116 DEGs were uniquely identified in CK-0 d vs CK-10 d, CK-0 d vs CS-10 d, and CK-10 d vs CS-10 d, respectively. KEGG pathway enrichment analysis revealed distinct transcriptional response patterns among the comparisons. In CK-0 d vs CK-10 d, the top enriched pathways included plant hormone signal transduction, photosynthesis, phenylpropanoid biosynthesis, MAPK signaling pathway–plant, and linoleic acid metabolism (Supplementary Fig. S1). In CK-0 d vs CS-10 d, enrichment was observed in largely overlapping pathways, namely plant hormone signal transduction, photosynthesis, phenylpropanoid biosynthesis, MAPK signaling pathway–plant, and phenylalanine metabolism (Supplementary Fig. S2). In CK-10 d vs CS-10 d, the most significantly enriched pathways were phenylpropanoid biosynthesis, plant hormone signal transduction, glutathione metabolism, α-linolenic acid metabolism, and DNA replication (Supplementary Fig. S3). At day 10, CS treatment significantly upregulated genes involved in α-linolenic acid metabolism and plant hormone signal transduction (Supplementary Fig. S13), while suppressing genes associated with phenylpropanoid biosynthesis, glutathione metabolism, and DNA replication (Supplementary Fig. S14).

Given that chilling injury is closely associated with texture deterioration (Fig. 1D), genes related to cell wall modification and firmness were further examined (Supplementary Table S4). GDSL esterase/lipase proteins (GELPs) are known to function as acyltransferases or hydrolases involved in cutin and wax biosynthesis, thereby contributing to water retention and mechanical resistance. In contrast, pectinesterase (PE), polygalacturonase (PG), β-glucosidase (β-Glu), and β-galactosidase (β-Gal) are typical pectin-degrading enzymes that promote fruit softening. During chilling injury development (CK-0 d vs CK-10 d), GELP genes were predominantly downregulated, whereas PE, PG, β-Glu, and β-Gal were upregulated. In CK-10 d vs CS-10 d, CS treatment markedly induced GELP expression while suppressing PE, PG, β-Glu, and β-Gal, indicating that CS enhanced tissue resistance and delayed softening by activating cuticle- and wax-related genes while inhibiting cell wall degradation.

A substantial proportion of DEGs encoded transcription factors (TFs), which play key roles in regulating downstream gene expression at both transcriptional and post-transcriptional levels. In CK-0 d vs CK-10 d, 26 unique TFs were identified, belonging mainly to the bHLH, ERF/AP2, WRKY, HSF, bZIP, MYB, and NAC families (Supplementary Table S5). In CK-0 d vs CS-10 d, 19 unique TFs were detected, including members of the WRKY, MYB, bHLH, HSF, and bZIP families (Supplementary Table S5). These differences indicate that CS treatment substantially altered TF expression profiles during low-temperature storage. Notably, 28 TFs were co-expressed in CK-10 d vs CS-10 d, encompassing ERF/AP2, MYB, WRKY, bHLH, Trihelix, MADS-box, GATA, and TCP families (Fig. 2D). Compared with CK-10 d, TFs from the bHLH, MYB, WRKY, GATA, and TCP families were predominantly upregulated by CS treatment, suggesting their potential involvement in CS-mediated alleviation of chilling injury in postharvest cucumbers.

Effects of cold shock on DNA methylation in cucumbers

Whole-genome bisulfite sequencing was performed on CK-0 d, CK-10 d, and CS-10 d samples to characterize DNA methylation changes associated with CI development and CS treatment. Quantification of methylated cytosines under CG, CHG, and CHH contexts revealed the global distribution of 5-methylcytosine (5mC) across the cucumber genome. The numbers of detected methylated cytosine sites were 8047635.667 in CK-0 d, 7766990 in CK-10 d, and 8417731.333 in CS-10 d, respectively (Supplementary Fig. S16). Analysis of average methylation levels in different genomic regions showed that, under the CG context, methylation was relatively higher in upstream regions and internal introns. Under CHG and CHH contexts, higher methylation levels were observed in both upstream and downstream regions (Fig. 2J).

In the CK-0d, CK-10d, and CS-10d samples, methylation levels were highest in the CG context, followed by CHG and CHH. Compared with CK-0d, the overall methylation level increased slightly in CK-10d, and further increased in CS-10d, with the most pronounced increase observed in the CHG context. Analysis of the proportion of methylated cytosines in each context revealed that the CHH context accounted for the highest proportion (43.27% to 44.73%), followed by CG (35.93% to 37.09%) and CHG (19.34% to 19.72%). Inter-treatment comparison showed that the proportion of methylated cytosines in the CHH context was higher in the CS-10d group than in the CK groups, while the proportions in the CG and CHG contexts slightly decreased (Fig. 2L). In CK-0 d vs CK-10 d, 34,359 CG-, 33,090 CHG-, and 173,386 CHH-type differentially methylated regions (DMRs) were identified. In CK-10 d vs CS-10 d, 44,419 CG-, 36,860 CHG-, and 180,916 CHH-type DMRs were detected (Fig. 2I). Given their numerical dominance, subsequent analyses focused primarily on CHH-type DMRs. In both CK-0 d vs CK-10 d and CK-10 d vs CS-10 d comparisons, hypomethylated DMRs outnumbered hypermethylated DMRs. Most DMRs were distributed within gene Body and Intergenic regions (Fig. 2K, Supplementary Fig. S15). We found that the occurrence of chilling injury primarily relates to nucleocytoplasmic transport, plant hormone signal transduction, and carbon metabolism, while CS treatment inhibits chilling injury mainly by regulating the DNA methylation levels of pathways such as the citrate cycle (TCA cycle), ribosome biogenesis in eukaryotes, and plant hormone signal transduction (Supplementary Fig. S4, 5).

Multi-omics integration reveals mechanisms underlying cold shock–induced chilling tolerance in cucumbers

Integration of DNA methylation and transcriptomic data

Integrated analysis of DNA methylation and transcriptomic datasets was performed to elucidate the regulatory relationship between methylation changes and gene expression. In CK-0 d vs CK-10 d, a total of 1702 DEGs exhibited significant methylation alterations, among which 838 showed positive correlations and 864 showed negative correlations between methylation levels and gene expression (Fig. 3A). KEGG enrichment analysis indicated that these genes were mainly associated with plant hormone signal transduction, phenylpropanoid biosynthesis, and MAPK signaling pathway–plant (Supplementary Fig. S6). In CK-10 d vs CS-10 d, 354 DEGs displayed differential methylation (Fig. 3B), including 138 with positive and 216 with negative methylation–expression correlations. These DEGs were primarily enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and α-linolenic acid metabolism (Supplementary Fig. S7).

Fig. 3. Arginine metabolism and ethylene and abscisic acid signaling pathways.

A Correlation between methylation levels and the expression level of DEGs (CK-0 d vs. CK-10 d, |coefficient | > 0.80 and p value < 0.05). B Correlation between methylation levels and the expression level of DEGs (CK-10 d vs. CS-10 d, |coefficient | > 0.80 and p value < 0.05). C Expression of genes related to arginine and proline metabolism. D Content of metabolites related to arginine and proline metabolism. E Ethylene biosynthesis pathway. F Abscisic acid biosynthesis and signal transduction pathways.

Integration of transcriptomic and metabolomic data

Joint analysis of transcriptomic and metabolomic datasets further revealed coordinated regulation of gene expression and metabolite accumulation. In CK-10 d vs CS-10 d, DEGs and DAMs were most frequently co-enriched in plant hormone signal transduction, linoleic acid metabolism, and phenylpropanoid biosynthesis (Supplementary Fig. S8). In CK-0 d vs CK-10 d, co-enrichment was most pronounced in plant hormone signal transduction, phenylpropanoid biosynthesis, glycerophospholipid metabolism, and biosynthesis of amino acids (Supplementary Fig. S9). Across transcriptomic, metabolomic, and methylomic layers, phenylpropanoid biosynthesis, arginine and proline metabolism, plant hormone signal transduction, and membrane lipid–related pathways were consistently implicated in CS-mediated alleviation of chilling injury.

Phenylpropanoid biosynthesis

Within the phenylpropanoid biosynthesis pathway, 11 DEGs and 4 DAMs were identified (Fig. 4A). At day 10 of storage, CS treatment significantly downregulated genes encoding caffeic acid 3-O-methyltransferase (COMT), phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), shikimate O-hydroxycinnamoyltransferase (HCT), and cinnamyl-alcohol dehydrogenase (CAD), while upregulating peroxidase (POD), and caffeoylshikimate esterase (CSE). Correspondingly, metabolites including (E)-ferulic acid, L-tyrosine, and trans-5-O-(p-coumaroyl)shikimate increased under CS treatment, whereas 4-vinylphenol decreased (Fig. 4B). The methylation levels of POD, COMT, and CAD were significantly increased after CS treatment (Fig. 4C). A total of 5 DMRs were associated with DEGs in this pathway, including 2 CG-, 1 CHG-, and 2 CHH-type DMRs (Fig. 4C). Among these, 2 DMRs were located in promoter regions.

Fig. 4. Phenylpropanoid biosynthesis.

A Expression of genes related to phenylpropanoid biosynthesis. B Differential methylation of genes related to phenylpropanoid biosynthesis. C The content of metabolites related to phenylpropanoid biosynthesis.

Arginine and proline metabolism

Three DEGs (aldehyde dehydrogenase (NAD + ) (ALDH), polyamine oxidase 2 (PAO2), and glutamate dehydrogenase 2 (GDH2)) and four DAMs related to arginine metabolism exhibited significant changes. Compared with CK-10 d, GDH2 expression was reduced by 1.12-fold in CS-10 d, whereas ALDH and PAO2 were upregulated by 1.48- and 1.01-fold, respectively (Fig. 3C). Metabolite analysis showed that L-citrulline and N-acetyl-L-glutamic acid decreased by 0.33- and 1.40-fold, respectively, while L-arginine and S-adenosylmethionine increased by 0.58- and 0.47-fold (Fig. 3D). Among them, PAO2 was associated with 3 DMRs (2 CG-type and 1 CHG-type). After CS treatment, the expression of PAO2 was upregulated, while its methylation level decreased, showing a negative correlation between the two. These results indicate that CS activates arginine metabolism by inhibiting DNA methylation of key regulatory genes and promoting gene expression and metabolite accumulation (Supplementary Table S6).

Ethylene and abscisic acid signaling pathways

In the ethylene biosynthesis pathway (Fig. 3E), ACO and SAM were downregulated in CS-10 d compared with CK-10 d. Following CS treatment, SAM exhibited promoter hypomethylation in the CG context, and methylation positively regulates the transcriptional expression of SAM (Supplementary Table S7). CS treatment also suppressed abscisic acid (ABA) biosynthesis, as indicated by a 1.12-fold downregulation of NCED (Fig. 3F). One CG-type DMR was detected in the distal intergenic regions of NCED, which was hypomethylated after CS treatment (Supplementary Table S7). In ABA signaling, bscisic acid receptor PYL4 (PYL) was upregulated, whereas protein phosphatase 2 C 51 (PP2C) was downregulated (Fig. 3F). A CHH-type DMR associated with the promoter region was detected in PYL, which showed hypomethylation and increased expression under CS treatment (Supplementary Table S7).

Furthermore, in the jasmonic acid signaling pathway, compared to CK-10d, the expression of JAZ was upregulated. The content of its upstream metabolite, jasmonoyl-L-isoleucine, was decreased.

Membrane lipid metabolism

Chilling injury caused pronounced disruption of membrane lipid metabolism (Fig. 5A). Relative to CK-0 d, phosphatidate phosphatase (PAP) expression decreased 2.98-fold in CK-10 d, whereas CS treatment alleviated this reduction. In CS-10 d, phospholipid:diacylglycerol acyltransferase (LPAAT) expression was higher than in CK-10 d, while Glycerol-3-phosphate acyltransferase (GAPT) expression was suppressed by CS. Acetylesterase (AES) expression declined during CI development, and this suppression was mitigated by CS treatment. Genes encoding membrane lipid–degrading enzymes, including phospholipase A (PLA), and phospholipase D (PLD), were inhibited by CS, whereas Lipoxygenase (LOX), Phospholipase C (PLC), and Diacylglycerol kinase (DGK), which were strongly induced during CI development, were markedly downregulated by CS. In addition, CS treatment significantly upregulated Fatty acid desaturase (FAD), supporting enhanced fatty acid desaturation and membrane stabilization. Metabolite profiling showed that CS reduced the accumulation of saturated fatty acids and their derivatives (including hydroxy-, di-carboxylic acids and esters), including 2-hydroxypalmitic acid, azelaic acid, docosanoic acid, ethyl 16-fluorohexadecanoate, and 9-hydroxypentadecanoic acid (Fig. 5B). The differentially methylated regions associated with membrane lipid-related genes were primarily of the CHH type (4 DMRs) and CG type (4 DMRs) (Fig. 5C). In the CHH context, the distal intergenic region of the PAP gene exhibited hypomethylation after CS treatment, which was associated with its transcriptional upregulation. CHH hypomethylation in the PLD promoter may have contributed to its downregulation. For the PLA gene, hypermethylation predominantly occurred in the CG context. The hypomethylation of the PLA gene showed a negative correlation with its decreased expression level. CS treatment led to increased methylation in the distal intergenic region of PLC in the CHH context. The methylation contexts of LOX were all of the CHH type, and significant hypomethylation occurred after CS treatment. Additionally, CS treatment caused hypomethylation in the exon region of the FAD gene, which was negatively correlated with its gene expression.

Overall, CS treatment modulated membrane lipid homeostasis through DNA methylation–mediated regulation, suppressing lipid degradation, promoting fatty acid desaturation and lipid biosynthesis, and change fatty acid composition to enhance membrane stability under chilling stress.

Discussion

Although low-temperature storage is widely applied to preserve fruit quality by suppressing respiration and delaying nutrient depletion, cold-sensitive commodities are highly susceptible to CI^19,20. In this study, we systematically investigated the regulatory effects and underlying mechanisms of a 0 °C cold shock (CS) treatment for 3 h on CI development in ‘Baijinyu’ cucumbers. The results demonstrated that CS markedly alleviated CI symptoms, delayed textural softening, and effectively maintained soluble solids content (SSC) and color quality. A study by Chen et al.¹⁶ showed that cold shock treatment (3 °C cold water for 40 min) inhibited firmness decline, and that a cold shock temperature below the threshold of 3–6 °C avoided membrane lipid peroxidation. The 0 °C treatment used in our study is indeed below this threshold. Similarly, another study found that 0 °C could maintain cucumber quality during storage¹⁵. Previous research on cold shock treatment of cucumbers has mostly focused on physiological responses and cold shock intensity, while the understanding of the genetic regulation and epigenetic mechanisms underlying the mitigation of chilling injury by CS remains limited. In this study, integrated multi-omics analyses further revealed that CS alleviates CI through coordinated regulation at the epigenetic, transcriptional, and metabolic levels.

To explore the epigenetic basis of this CS-induced chilling tolerance, we next analyzed global DNA methylation patterns in response to CS treatment. Low-temperature stress is known to induce extensive DNA methylation reprogramming, thereby altering the expression of stress-responsive genes and modulating transposable element (TE) activity, which collectively influence fruit adaptive responses^21,22. In the present study, extensive CHH hypomethylation was observed in CS-treated cucumbers relative to CK-10 d fruit. Previous studies have shown that cold stress exerts pronounced genome-wide effects on small RNA–mediated DNA methylation (RdDM), particularly reducing CHH methylation associated with TEs²³. Consistently, CS treatment in this study primarily altered CG- and CHH-type methylation within gene bodies and intergenic regions, with CHH-type DMRs far outnumbering CG- and CHG-type DMRs. This pattern agrees with earlier findings that CHH methylation plays a dominant role in chilling stress responses in cucumber²⁴. Given that CHH methylation constitutes the major methylation context in cucumber genomes and is highly dynamic in response to environmental cues, it likely serves as a central epigenetic regulator of CI development²⁵.

Next performed integrated transcriptomic and metabolomic analyses to identify key pathways affected by CS treatment in response to chilling injury. Chilling injury profoundly disrupts key metabolic pathways in horticultural crops, including arginine metabolism, sugar metabolism, and the tricarboxylic acid (TCA) cycle^26,27. Integrated transcriptomic and metabolomic analyses revealed that differential expression of GDH2 was closely associated with altered L-arginine accumulation. L-Arginine has been reported to alleviate CI by enhancing antioxidant capacity, as demonstrated in pomegranate and cherry tomato^28,29. In addition, the concurrent upregulation of ALDH and PAO2 observed in CS-treated cucumbers suggests enhanced polyamine biosynthesis. Polyamines are well known to promote cold-regulated protein production, stabilize membranes, and improve chilling tolerance in various fruits, including peach, plum, and banana, supporting their protective role under CS treatment^30–32.

In addition to polyamines, plant hormones represent another key regulatory layer in chilling tolerance, which we next investigated. Phytohormones such as methyl jasmonate and salicylic acid enhance cold tolerance by activating antioxidant defenses, modulating secondary metabolism, and regulating membrane lipid and cell wall metabolism. In cucurbit crops, salicylic acid (SA), abscisic acid (ABA), and brassinosteroids (BRs) positively regulate cold responses, whereas ethylene accumulation exacerbates CI³³. In the present study, CS treatment suppressed the expression of ethylene biosynthesis genes SAM and ACO, consistent with reduced CI severity. Moreover, CS upregulated the ABA receptor PYL while downregulating the negative regulator PP2C, thereby enhancing ABA signaling. These changes were accompanied by increased accumulation of polyphenolic compounds, suggesting that CS enhances chilling tolerance through coordinated inhibition of ethylene biosynthesis and activation of ABA-mediated protective pathways³⁴.

Membrane integrity is a critical determinant of chilling tolerance. In CK fruit, CI development was accompanied by the induction of lipid-degrading genes (PLA, PLD, LOX, PLC, and DGK) and repression of fatty acid desaturase (FAD), a pattern consistent with observations in banana fruit³⁵. In cucumbers, CI severity has been positively correlated with increased activities of PLC, PLD, and LOX³⁶. CS treatment markedly suppressed the expression of PLA, PLD, LOX, PLC, and DGK, while preventing CI-induced accumulation of saturated fatty acids³⁷. In addition, CS alleviated the repression of lipid biosynthesis–related genes such as LPAAT, PAP, and FAD, thereby contributing to membrane stabilization³⁸. These findings are consistent with reports in peach and highlight membrane lipid homeostasis as a key target of CS-mediated CI alleviation.

Transcription factors (TFs) play central roles in orchestrating stress responses. In this study, CS treatment induced the upregulation of TF families including bHLH, MYB, WRKY, GATA, and TCP, while suppressing ERF/AP2, forming a core transcriptional regulatory network associated with CI mitigation. TFs such as GATA contribute to maintaining cellular energy homeostasis by regulating sucrose and energy metabolism³⁹, whereas WRKY activates stress-responsive pathways⁴⁰. Suppression of ERF/AP2, a major component of ethylene signaling, may further restrain CI progression³³. Previous studies have shown that bHLH TFs directly activate membrane lipid–related genes in banana, while MYB regulates fatty acid desaturation through targeting FAD⁴¹. In sweet orange, bHLH binds the POD promoter to enhance reactive oxygen species (ROS) scavenging⁴². Consistently, POD expression was elevated following CS treatment in this study, suggesting a conserved TF-mediated regulatory mechanism underlying chilling tolerance in cucumbers.

The results of this study indicate that CS treatment coordinately regulates phenylpropanoid metabolism, arginine/polyamine metabolism, ethylene/ABA biosynthesis and signaling, as well as membrane lipid metabolism under cold stress. Although these pathways are often discussed separately, recent advances highlight a regulatory hub centered on ethylene (ET), nitric oxide (NO), and polyamines (PAs), which are metabolically interconnected through their common precursors S‑adenosylmethionine (SAM) and L‑arginine⁴³. In this study, CS treatment led to the accumulation of L‑arginine (0.57‑fold) and SAM (0.47‑fold). Ethylene synthesis was not driven (ACO expression was downregulated); instead, the accumulated SAM was preferentially channeled into polyamine biosynthesis, as evidenced by the upregulation of the polyamine oxidase gene PAO2 and its hypomethylation⁴⁴. Elevated PAO2 activity generates H₂O₂, which in turn induces NO production via nitrate reductase (NR) or NOS‑like enzymes—a mechanism well documented in plants under low‑temperature stress^45,46. It is well established that nitric oxide (NO) inhibits ethylene biosynthesis via ACS (1-aminocyclopropane-1-carboxylic acid synthase) and ACO (ACC oxidase)^47,48. This finding is consistent with the reduced ACO expression observed in the CS treatment in this study. Therefore, under CS treatment, a regulatory network involving arginine, polyamines, NO, and ethylene may exist. Importantly, this regulatory network is also closely linked to membrane lipid metabolism and the phenylpropanoid pathway. The H₂O₂ derived from polyamine oxidation not only serves as a signal for NO production but also upregulates POD expression, leading to the accumulation of phenolic compounds such as ferulic acid and trans‑5‑O‑(p‑coumaroyl) shikimic acid, thereby contributing to ROS scavenging and cell wall stabilization⁴⁹. Meanwhile, the inhibition of ethylene and ABA biosynthesis (e.g., downregulation of NCED) reduces stress-accelerating signals, thereby enabling plants to engage in membrane protection. CS treatment also induces hypomethylation of FAD, PLD, and LOX in CG or CHH contexts, shifting metabolism from lipid degradation toward fatty acid desaturation and membrane stabilization. Based on the above analysis, a regulatory model was constructed in which CS treatment coordinately modulates pathways related to phenylpropanoid biosynthesis, arginine metabolism, ethylene/ABA biosynthesis/signaling, and membrane lipid metabolism, thereby delaying the onset of chilling injury (Fig. 6).

Fig. 6. Multilevel regulatory network model of CS treatment on chilling injury in ‘Baijinyu’ cucumber fruit.

Compared with CK-10 d, the red text or dots indicate upregulation of physiological indices, relative metabolite content, gene FPKM, and DNA methylation levels in CS-10 d, while the green text or dots indicate downregulation. Gray arrows represent connections between distinct metabolic pathways via common metabolites or signaling molecules (L-arginine, SAM, H₂O₂, NO, etc.). CS treatment leads to the accumulation of L-arginine and SAM, suppresses ethylene synthesis while promoting the polyamine pathway, and orchestrates phenylpropanoid metabolism, inhibition of ABA synthesis, and membrane lipid remodeling through H2O2/NO signaling, thereby ultimately enhancing plant chilling tolerance.

CS treatment effectively alleviated chilling injury in ‘Baijinyu’ cucumbers during low-temperature storage, reducing CI symptoms—including surface pitting, water-soaked lesions, and discoloration—by 66.67%. CS significantly delayed firmness loss, mitigated declines in soluble solids content, and restrained changes in a^* values, thereby preserving visual appearance, texture, and nutritional quality. CS enhanced the accumulation of ketones, aldehydes, esters, lipids, and terpenoids, while suppressing the accumulation of saturated fatty acids, contributing to improved stress resistance and reduced membrane lipid oxidation. At the molecular level, CS induced the upregulation of key transcription factor families (bHLH, MYB, WRKY, GATA, and TCP) while suppressing ERF/AP2. CS primarily reprogrammed CG- and CHH-type DNA methylation within gene body and intergenic regions. Integrated multi-omics analyses revealed that CS coordinately modulated DNA methylation, gene expression, and metabolite accumulation in pathways associated with phenylpropanoid biosynthesis, arginine metabolism, ethylene and ABA biosynthesis/signaling, and membrane lipid metabolism (e.g., PAL, COMT, NCED, PYL, and PLD), thereby delaying CI development. Overall, this study provides mechanistic insights into CS-induced chilling tolerance and supports the application of cold shock as an effective, green postharvest preservation strategy for cucumbers and other cold-sensitive horticultural crops. Future studies will conduct genetic functional validation and expand experimental conditions and materials, so as to further clarify the epigenetic regulatory network and promote the practical application of cold shock treatment in cucumber postharvest preservation.

Methods

Plant materials and cold shock treatment

‘Baijinyu’ cucumbers (Cucumis sativus L.) with uniform size and moderate maturity, free from pests, diseases, and mechanical damage, were harvested from a commercial vegetable production base in Shouguang, Weifang, Shandong Province, China, and immediately transported to the laboratory. A total of 96 fruits with uniform appearance and color were selected and gently cleaned using a cotton cloth. Given that household refrigerators, cold storage facilities, and cold-chain transportation systems typically operate at approximately 4 °C-well below the safe storage threshold for ‘Baijinyu’ cucumbers-this study selected 4 °C as the storage temperature to realistically simulate chilling conditions commonly encountered during postharvest handling. In preliminary experiment, we set three different treatment gradients, namely cold shock at 0 °C for 2, 3, and 4 h. Based on the changes in appearance at the end of storage, we ultimately determined that the 3 h cold shock treatment was the most effective. The fruits were randomly divided into two groups: Cold shock (CS) treatment group (n = 48): fruits were subjected to cold shock at 0 °C for 3 h, followed by storage at 4 °C with a relative humidity of 70% for 8 days. Control group (CK, n = 48): fruits were directly stored at 4 °C with a relative humidity of 70% for 8 days. After the 8-day cold storage period, fruits from both groups were transferred to 20 °C for an additional 2 days to evaluate chilling injury symptoms. For omics analyses, peel tissues from 15 fruits per group were sampled at day 0 and day 10, cut into small pieces, immediately frozen in liquid nitrogen, and stored at -80 °C until further analysis.

Determination of chilling injury index, firmness, soluble solids content, and color

Nine fruits from each group were randomly selected for chilling injury assessment and subsequently used for measurements of firmness, soluble solids content (SSC), and color.

Chilling injury (CI) index was determined by the method of Madebo et al.⁵⁰. CI symptoms mainly included surface pitting and water-soaked lesions. Fruits were classified according to the proportion of affected surface area as follows: 0%, 0–25%, 25–50%, 50–75%, and 75–100%, corresponding to grades 0, 1, 2, 3, and 4, respectively. The CI index was calculated using the following equation:

CI index = ∑ (CI grade × number of fruits at that grade)/4 × total number of fruits

Firmness: When measuring firmness, the cucumber peel was retained. Firmness was measured at the equatorial region of the cucumber fruit using a texture analyzer equipped with a TA39 probe (2 mm diameter) at a penetration speed of 2 mm·s⁻¹ and a penetration depth of 10 mm.

Soluble solids content (SSC): SSC was determined using homogenized juice samples. Measurements were conducted in triplicate for each group (n = 9) using a handheld digital refractometer (PAL-1, Atago, Japan) and expressed as percentage of fresh weight.

Color: Fruit color was measured using a chromameter (CR-400, Konica Minolta, Tokyo, Japan). The color parameters L^* (lightness), a^* (red–green), and b^* (yellow–blue) were recorded. The instrument was calibrated with a standard white plate prior to measurements¹⁸. Three fruits per group were randomly selected, with three measurements taken at different positions on each fruit.

Metabolomic analysis

Freeze-dried cucumber samples (50 mg) were extracted with 1000 μL of extraction solvent (methanol: acetonitrile: water = 1:2:1, v/v/v). After vortexing for 30 s, steel beads were added, and samples were ground at 45 Hz for 10 min, followed by ultrasonication in an ice-water bath for 10 min. Samples were incubated at -20 °C for 1 h and centrifuged at 12,000 rpm for 15 min at 4 °C. A total of 300 μL of the supernatant was filtered through a 0.22 μm organic membrane into 2 mL vials. Equal volumes (10 μL) from each sample were pooled to generate quality control (QC) samples. The difference analysis was conducted according to the method of Bai et al.⁵¹. Differentially accumulated metabolites (DAMs) were identified based on variable importance in projection (VIP) values > 1 and |log₂ (fold change) | ≥ 1.0. VIP values were derived from the OPLS-DA model after log transformation and mean centering of the data. A 200-time permutation test was performed to evaluate model reliability and avoid overfitting. Metabolites were annotated using the KEGG compound database and mapped to metabolic pathways via the KEGG pathway database⁵².

Transcriptomic analysis

Total RNA was extracted using the RNAprep Pure Plant Kit according to the manufacturer’s instructions⁵³. RNA concentration was measured using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed with the RNA Nano 6000 Assay Kit on an Agilent Bioanalyzer 2100 system. For library construction, 1 μg of total RNA was used per sample. mRNA was enriched using oligo(dT) magnetic beads, fragmented, and reverse-transcribed into cDNA. After purification with AMPure XP beads, library quality was assessed using the Bioanalyzer 2100 system. Sequencing was performed on an Illumina NovaSeq platform to generate 150 bp paired-end reads. Raw sequencing data were processed on the BMKCloud platform. Differential gene expression analysis was conducted using DESeq2 based on a negative binomial distribution model. Genes with a false discovery rate (FDR) < 0.01 and |fold change | ≥ 2 were considered differentially expressed. Functional annotation was performed using the Nr, KEGG, and Gene Ontology (GO) databases.

DNA methylation analysis

Genomic DNA was extracted using a commercial plant DNA extraction kit. DNA integrity, purity, and concentration were assessed by 1% agarose gel electrophoresis, microvolume spectrophotometry, and fluorescence-based quantification, respectively. Bisulfite conversion was performed using the EZ DNA Methylation-Gold™ kit following the manufacturer’s protocol. Bisulfite-treated single-stranded DNA was amplified using KAPA HiFi HotStart Uracil+ ReadyMix and sequenced on the Illumina HiSeq platform. Raw sequencing data were subjected to quality control using FastQC, and low-quality reads were removed using Trimmomatic. All libraries achieved Q30 values above 96.54%. Differentially methylated regions (DMRs) were identified based on uniquely mapped reads and cytosine positions. DMRs were annotated to genomic features, including promoters, exons, introns, CpG islands (CGIs), CGI shores, transcription start sites (TSS), and transcription end sites (TES). Genes associated with DMRs were functionally annotated using BLAST searches against the KEGG and NR databases⁵⁴.

Statistical analysis

All statistical analyses were performed using SPSS version 25. Data were analyzed by one-way analysis of variance (ANOVA), and differences were considered statistically significant at p < 0.05. Results are presented as mean ± standard deviation (SD). Graphical representations were generated using Origin 2021 and Metware Cloud.

Author contributions

Yiting Ren: Data analysis, Methodology, Validation, Visualization, Data curation, Writing-original draft & editing. Jianlou Mu: Visualization, Writing-review & editing, Data curation. Yunxiang Wang: Methodology, Supervision. Chunmei Bai, Lili Ma, Jiejie Tao: Methodology, Data curation. Xinyi Feng, Tianyu Li, Xinhuan Yan: Conceptualization, Writing-review & editing, Supervision. Jinhua Zuo, Yanyan Zheng: Conceptualization, Writing-review & Editing, Supervision, Funding acquisition, Project administration.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to the fact that related research is still in progress but are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41538-026-00926-0.