Abstract Background Pepper (Capsicum annuum L.) is a vegetable crop of significant economic importance, but its yield and quality are severely affected by the combined stress of low temperature and low light (LL), particularly in greenhouse environments. Despite this, the physiological and molecular mechanisms underlying pepper’s response to LL stress remain poorly understood. In this study, we conducted physiological and transcriptomic analyses on two pepper genotypes: Y2, a LL-sensitive genotype, and Y425, a LL-tolerant genotype. These genotypes were subjected to LL stress conditions (10 °C/5°C, 100 µmol m⁻²s⁻¹) and control (CK) conditions (28 °C/18°C, 300 µmol m⁻²s⁻¹). Results Three days after treatment, the phenotypes of the two pepper genotypes began to show clear distinctions, with Y425 seedlings exhibiting greater root length, shoot fresh weight, and root fresh weight compared to Y2. Additionally, comparative transcriptome analysis of leaf samples from both genotypes identified a total of 13,190 differentially expressed genes (DEGs). Gene Ontology (GO) enrichment analysis revealed that genes associated with photosynthesis, osmotic stress response, reactive oxygen species response, and other GO terms potentially contribute to LL tolerance. Moreover, three key pathways involved in the response to LL stress were identified: photosynthesis-antenna proteins, zeatin biosynthesis, and circadian rhythm pathways. The key DEGs in these pathways were expressed at higher levels in Y425 as compared with Y2. Furthermore, physiological indicators such as chlorophyll fluorescence parameters, chlorophyll content, osmoregulatory substances, and antioxidant enzyme activities decreased under LL stress; however, the reduction was significantly greater in Y2 compared to Y425, further validating the molecular findings from the transcriptome analysis. Conclusion This study identified significant physiological and transcriptomic differences in two pepper genotypes under LL stress. It highlighted key pathways and provide novel insights into the molecular and physiological mechanisms of pepper’s LL tolerance. These results emphasize the importance of optimizing greenhouse conditions for better crop productivity. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-025-06169-7. Keywords: Chilies, Light stress, Cold stress, Temperature stress, Photosynthesis Introduction Temperature and light are critical environmental factors that influence plant growth and development. They not only provide the energy necessary for plant growth but also ensure proper spatiotemporal development of plants [[34]1, [35]2]. However, fluctuations in temperature and light conditions can transform these essential factors into significant abiotic stresses, adversely affecting plant growth and development. In nature, plants rarely face isolated abiotic stresses; instead, multiple stresses often occur simultaneously, compounding their impact on growth [[36]3]. In practical production, light intensity and temperature often exhibit a correlation [[37]4]. Particularly during the autumn and winter seasons, diminished light levels are typically concomitant with reduced temperatures. Consequently, low-temperature stress frequently co-occurs with low-light stress, posing a severe threat to plant growth [[38]5, [39]6]. In recent years, China’s greenhouse vegetable industry has expanded rapidly, with greenhouse cultivation covering 2.6 million hectares, representing 33% of the country’s vegetable production by 2022 [[40]7]. However, in North China, plastic greenhouses dominate the landscape, offering limited control over temperature and light. As a result, these facilities often fail to provide the optimal conditions required for vegetable growth and development [[41]8]. Consequently, the combined stress of low temperature and low light (LL stress) has emerged as a major challenge to off-season vegetable production in greenhouse environments. Pepper (Capsicum annuum L.) is an important horticultural crop belonging to the Solanaceae family, originates from South America [[42]9]. Due to its high nutritional and economic importance, it has become the most extensively cultivated vegetable crop in China, covering over 2.1 million hectares annually [[43]10]. Peppers are predominantly grown in greenhouses, owing to their thermophilic and heliophilic nature, which makes them particularly vulnerable to LL stress [[44]11, [45]12]. Research indicates that LL stress can severely impair photosynthesis by disrupting the carbon and oxygen cycles, reducing CO₂ availability, and hindering the synthesis of photosynthetic pigments [[46]13]. Chlorophyll fluorescence parameters, which are closely linked to photosynthetic efficiency, are also dynamically altered under abiotic stress, especially when the photosynthetic machinery is compromised [[47]14]. For example, LL stress significantly affects the morphological characteristics of pepper roots, including reduced root length, surface area, and volume [[48]15]. Additionally, LL stress negatively impacts the development of palisade and spongy tissues in pepper leaves [[49]16]. This stress induces excessive production of reactive oxygen species (ROS) in plant cells, leading to oxidative damage. ROS interact with unsaturated fatty acids in cell membranes, forming malondialdehyde (MDA), which disrupts membrane protein structures and alters antioxidant enzyme activity [[50]16, [51]17]. Another critical adaptive mechanism involves the accumulation of osmoregulatory substances, such as proline, soluble sugars, and soluble proteins. These compounds rapidly increase in response to abiotic stress and play essential roles in maintaining cellular osmotic balance and enhancing stress tolerance [[52]18–[53]20]. At the molecular level, plants respond to abiotic stress through intricate signaling pathway interactions and differential gene expression [[54]5]. The photosynthesis pathway and photosynthesis-antenna proteins pathway are significantly enriched in pepper under LL stress [[55]8]. Additionally, carotenoids play critical roles in photoprotection, light harvesting, and antioxidant activities [[56]21]. Genes involved in the carotenoid metabolic pathway, such as ZDS, CA2, and NCED3, were found to be down-regulated under LL stress, whereas CA1 was upregulated in pepper [[57]8]. Zhang et al. revealed that the Ca²⁺ signaling pathway and MAPK signaling pathway are activated in pepper under cold stress, with several key genes identified. For instance, genes encoding CML, CIPK, and CDPK-related kinase (CRK) were upregulated, while the novel gene (Newgene_9060) encoding MEKK3 was significantly downregulated. Conversely, the genes MKK4/5 (LOC107861622) and MPK3 (LOC107873437) showed opposite expression patterns [[58]22]. Similarly, the photosynthesis-antenna proteins pathway was activated under low-light stress, with Lhca4 (Capan01g000647) and Lhcb1 (Capan00g002800) exhibiting significantly higher expression levels in cotyledons compared to hypocotyls [[59]23]. Hormone metabolism also plays a pivotal role in plant responses to abiotic stress [[60]24]. Previous studies indicated that the brassinolide signal transduction pathway is significantly enriched after low-light stress in pepper. Key components of this pathway, including Bri1 (Capana12g001867), Bki1 (Capana12g000698), Bin2 (Capana00g000724), Tch4 (Capana07g000060), and Cycd3 (Capana03g002253), were significantly upregulated in hypocotyls [[61]23]. Additionally, Gao et al. showed that several differentially expressed genes (DEGs) were enriched in the auxin hormone signaling pathway during cold stress in pepper, with genes such as Aux1, GH3, and SaUR upregulated during the early stages of cold stress [[62]25]. Combined stresses often have synergistic effects on plants, with simultaneous exposure to multiple stresses causing significantly greater damage compared to individual stresses. The physiological and molecular response mechanisms involved in coping with such combined stresses are inherently more complex [[63]26]. While numerous studies have explored the physiological and molecular changes in pepper under either low-temperature or low-light stress, research on the combined effects of these stresses is still limited. Furthermore, existing studies have predominantly focused on the physiological mechanisms of LL stress in pepper [[64]27–[65]29]. Consequently, there remains a lack of comprehensive information regarding the molecular mechanisms underlying LL stress tolerance in pepper. In a previous germplasm screening study, we identified two pepper varieties with contrasting levels of tolerance to low-temperature and low-light (LL) stress: Yangjiao NO.2, an LL-sensitive genotype, and Yanjiao 465, an LL-tolerant genotype. In this study, we conducted comparative physiological and transcriptomic analyses of these two genotypes under LL stress and control conditions. Our aim was to identify and characterize the key genes and regulatory pathways contributing to LL tolerance. These findings provide valuable insights into the molecular mechanisms of LL tolerance and lay a theoretical foundation for the development of LL-tolerant germplasm resources in pepper. Materials and methods Plant materials and growth conditions Two pepper varieties, Yangjiao NO.2 (an LL-sensitive genotype) and Yanjiao 425 (an LL-tolerant genotype), referred to as Y2 and Y425 respectively, were used in this study. These varieties were provided by the Jiangsu Lixiahe Institute of Agricultural Sciences. To prepare the seedlings, the seeds were surface-sterilized by soaking in a 10% sodium hypochlorite solution for 10 min, followed by thorough rinsing with distilled water. The sterilized seeds were then placed in petri dishes lined with moist filter paper and incubated in an artificial climate chamber (LISK, Nanjing, China) at 28 °C for germination. Once germinated, healthy seedlings were transplanted into 7-cm-wide plastic pots filled with a substrate mixture of peat, vermiculite, and perlite in a 3:1:1 ratio. The seedlings were placed in the artificial climate chamber for incubation under the specific environmental conditions described in Table [66]1 for the CK treatments. Uniform water and fertilizer management practices were applied to ensure consistent growth across all seedlings. Table 1. LL and CK treatment conditions Treatment Temperature/°C Photoperiod/h PPFD/µmol m^− 2s^− 1 Relative humidity/% CK 28/18 12 300 70 LL 10/5 12 100 70 [67]Open in a new tab PPFD: Photosynthetic photon flux density Stress treatments At the six true-leaf stage, seedlings of uniform size and growth status were selected for low-temperature and low-light (LL) stress and control check (CK) treatments. These conditions were based on, and slightly modified from, the method described by Li et al. [[68]28] as shown in Table [69]1 Functional leaves (3rd–4th fully expanded leaves) were randomly collected from the seedlings at 0, 1, 3, 5, and 7 days post-treatment. The collected leaves were immediately snap-frozen in liquid nitrogen and stored at -80 °C for subsequent analyses. Each treatment was conducted with three biological replicates, and each replicate consisted of 30 pepper seedlings. Determination of morphological parameters The plant height, stem diameter, leaf area, and fresh weight of pepper seedlings were measured at 0, 1, 3, 5, and 7 days after treatment. Seedlings were also photographed at each time point to document morphological changes. Plant height (measured as the distance from the base of the plant to the growing point) and root length (entire root system after washing) were measured using a tape measure. Stem diameter (measured at the base of the stem below the cotyledons) was determined using a vernier caliper. Leaf area was calculated using ImageJ software (Bethesda, Maryland, USA). For fresh weight measurements, the washed seedlings were separated into shoots and roots and weighed individually using an electronic balance (METTLER TOLEDO, Zurich, Switzerland). Determination of physiological and biochemical indices Chlorophyll content was determined following the method described by Xie et al. [[70]30]. Briefly, 100 mg of fresh leaf tissue was immersed in 15 mL of extraction solution until the leaves were completely decolorized. The samples were then shaken under dark conditions at 50 rpm for 24 h. The absorbance of the extract was measured at wavelengths of 665 nm and 649 nm using a UV-Vis spectrophotometer (METASH, Shanghai, China). Chlorophyll fluorescence parameters were measured using a chlorophyll fluorescence imaging system (BLT, Guangzhou, China) (Table [71]2). Before measurement, each pepper plant was dark-adapted for 30 min to ensure accurate parameter detection. Table 2. Chlorophyll fluorescence parameters used in this study Parameter Description Fv/Fm Maximum quantum yield of PSII photochemistry Y(PSII) Effective quantum yield of photochemical energy conversion in PSII qP Photochemical quenching of PSII [72]Open in a new tab The activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), along with the contents of malondialdehyde (MDA), superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), soluble protein, soluble sugar, and proline (Pro), were measured spectrophotometrically. For each assay, approximately 100 mg of fresh samples were used, and all measurements were performed in triplicate. Antioxidant enzyme activities (POD, SOD, and CAT) and reactive oxygen species (ROS, including O₂⁻ and H₂O₂) were determined using commercial kits (Solarbio, Beijing, China), with absorbance measurements at 470 nm, 560 nm, 240 nm, 530 nm, and 415 nm, respectively, following the manufacturer’s protocols. The detection of ROS was further supported by nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) staining. Malondialdehyde (MDA) content, an indicator of lipid peroxidation, was quantified using the thiobarbituric acid (TBA) method as described by Song et al. [[73]31]. Osmolyte contents, including soluble protein, soluble sugar, and proline, were measured using commercial kits (Comin, Suzhou, China), with absorbance readings at 620 nm (for both protein and sugar) and 520 nm (for proline), in accordance with the manufacturer’s instructions. RNA extraction and cDNA library construction Frozen pepper leaf samples were thoroughly ground in a mortar with liquid nitrogen, and total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The extracted RNA samples were then sent to Biomarker Technologies Co., Ltd. (Beijing, China) for library construction and sequencing. Briefly, mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. First-strand cDNA synthesis was carried out, followed by second-strand cDNA synthesis. The remaining overhangs were converted into blunt ends through nucleic acid exonuclease and polymerase activities. After adenylation of the 3’ ends of the DNA fragments, NEBNext Adapters with a hairpin loop structure were ligated to the DNA, preparing them for hybridization. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Subsequently, PCR amplification was performed using 3 µL of USER Enzyme (NEB, USA) with junction-ligated cDNAs. The reaction was conducted at 37 °C for 15 min and 95 °C for 5 min. Further amplification was carried out using Phusion High-Fidelity DNA Polymerase, Universal PCR Primers, and Index (X) Primers. The PCR products were then purified again using the AMPure XP system. Finally, the quality of the library was evaluated using the Agilent Bioanalyzer 2100 system. Determination of differentially expressed genes (DEGs) DESeq2 (v1.20.0) was used to perform differential expression analysis on all biological replicate samples, including the treatment and reference groups. Genes with ∣log₂(Fold Change)∣ ≥ 1 and a false discovery rate (FDR) < 0.01, as identified by DESeq2, were considered differentially expressed genes (DEGs) [[74]32]. Gene expression levels were quantified using fragments per kilobase of transcript per million mapped reads (FPKM) [[75]33]. Functional enrichment analysis of DEGs The functions of the DEGs were annotated using the Gene Ontology (GO; [76]http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; [77]http://www.kegg.jp/kegg/) databases. Enrichment analyses of the DEGs were conducted across the whole genome using ClusterProfiler (v4.4.4) and the hypergeometric test. A corrected p-value < 0.05 was considered indicative of a significantly enriched GO term or pathway. Quantitative real-time PCR (qRT-PCR) validation To validate the RNA-seq results, eight DEGs were randomly selected for verification using quantitative real-time PCR (qRT-PCR). Primer sequences for qRT-PCR were designed using Primer Premier 5 and synthesized by Tsingke Biotechnology (Beijing, China). Complementary DNA (cDNA) was synthesized from total RNA using HiScript Reverse Transcriptase (Vazyme, Nanjing, China). qRT-PCR analysis was conducted on an iQ™ 5 Multicolor Real-Time PCR Detection System (Bio-RAD, USA). The relative expression levels of the selected genes were calculated using the 2⁻∆∆CT method [[78]34]. Statistical analysis Statistical analyses were performed using Microsoft Excel (v16.72) and SPSS (v26.0). Significant differences were evaluated using one-way ANOVA followed by Duncan’s multiple range test (p < 0.05). GraphPad Prism (v10.0) was used for data visualization, and the results were presented as mean ± standard deviation (SD) based on at least three biological replicates. Results Different morphological changes in two peppers under LL treatment Morphological changes in the two pepper genotypes were observed at 0, 1, 3, 5, and 7 days after LL treatment to assess their tolerances to LL stress. As shown in Fig. [79]1A, the LL-tolerant variety Y425 maintained relatively normal growth throughout the treatment period, whereas the LL-sensitive variety Y2 exhibited delayed growth, with leaves drooping noticeably from day 3 onwards. Additionally, root morphology observations indicated more severe root damage in Y2 compared to Y425 three days after treatment. Fig. 1. [80]Fig. 1 [81]Open in a new tab Phenotypic changes of pepper seedlings exposed to LL stress at different time periods. (A) The phenotype of pepper plants and roots at different time periods under different treatments. (B) The plant height, (C) steam diameter, (D) leaf area, (E) root length, (F) root fresh weight, and (G) shoot fresh weight. Y2LL: Y2 in LL conditions, Y425LL: Y425 in LL conditions, Y2CK: Y2 in CK conditions, Y425CK: Y425 in CK conditions. The same as below. Bars are presented as means ± SD (n = 3), and different lowercase letters indicate significant differences among the means at p < 0.05 by Duncan’s multiple range tests. ns: not significant Measurements of plant height, stem diameter, leaf area, root length, shoot fresh weight, and root fresh weight were taken at each time point following LL treatment. As illustrated in Fig. [82]1B-G, no significant phenotypic differences were observed between the genotypes during the early stages under both CK and LL conditions. However, significant differences emerged by day 3 in root length, shoot fresh weight, and root fresh weight; by day 5 in leaf area; and by day 7 in plant height and stem diameter. These differences became more pronounced with prolonged LL treatment. Notably, the adverse effects of LL stress were significantly less pronounced in Y425 than in Y2. Based on phenotypic and observational data, day 3 appears to be a critical turning point for LL tolerance in peppers. RNA-seq analysis reveals DEGs in response to LL stress in pepper To gain a comprehensive understanding of the molecular mechanisms underlying pepper’s response to LL stress, we performed transcriptomic analyses of Y425 and Y2, integrating these with phenotypic data. Samples were collected under both CK conditions and LL treatment (three days post-treatment). A total of 12 samples (three biological replicates per group) were sequenced, yielding 77.24 Gb of clean data after filtering low-quality reads. The Q20 and Q30 bases exceeded 97.96% and 94.1%, respectively, while the GC content ranged between 42.25% and 42.79% (Table [83]S1). Clean reads were mapped to the specified pepper reference genome ([84]http://peppersequence.genomics.cn/page/species/index.jsp), with mapping rates ranging from 84.23 to 95.64% and unique mapping rates between 80.80% and 91.40% (Table [85]S1). The correlation heatmap demonstrated a strong correlation among replicate samples (Fig. [86]2A), while PCA revealed that biological replicates from the same group clustered together, with clear separation between different groups (Fig. [87]2B). To validate the transcriptomic data, eight DEGs were randomly selected for qRT-PCR analysis (Table [88]S7). The expression trends of these genes were consistent with the RNA-seq data (Fig. [89]S1), confirming the reliability of the transcriptomic dataset for downstream bioinformatics analyses. Fig. 2. [90]Fig. 2 [91]Open in a new tab RNA-seq analysis reveals DEGs in response to LL stress in pepper. (A) Sample correlation clustering heatmap. (B) PCA of transcriptomes. (C) Numbers of up- and downregulated genes in the four comparison groups. (D) Venn diagram of all DEGs in the four comparisons To identify DEGs associated with LL stress, we established four comparison groups: Y2CK vs. Y2LL, Y425CK vs. Y425LL, Y425CK vs. Y2CK, and Y425LL vs. Y2LL. Genes with ∣log₂(Fold Change)∣ ≥ 1 and FDR < 0.01 were considered DEGs. In total, 13,190 DEGs were identified across the four comparison groups (Table [92]S2). Specifically, in Y2, 2,212 genes were upregulated and 2,531 were downregulated under LL stress (Y2CK vs. Y2LL). In Y425, 1,698 genes were upregulated and 1,686 were downregulated under LL stress (Y425CK vs. Y425LL) (Fig. [93]2C). Furthermore, under CK conditions (Y425CK vs. Y2CK), 835 genes were upregulated and 1,203 were downregulated, while under LL stress (Y425LL vs. Y2LL), 1,211 genes were upregulated and 1,814 were downregulated (Fig. [94]2C). These results indicate that LL stress activated more genes compared to CK conditions, with a higher proportion of downregulated genes across all comparisons, except for Y425CK vs. Y425LL. Venn diagram analysis revealed 1,855 and 1,302 unique responsive genes in the Y2CK vs. Y2LL and Y425CK vs. Y425LL comparisons, respectively. Additionally, 641 and 597 unique responsive genes were identified in the Y425LL vs. Y2LL and Y425CK vs. Y2CK comparisons, respectively. A total of 68 genes were shared across all four comparison groups, potentially providing critical insights into the molecular mechanisms underlying pepper’s response to LL stress (Fig. [95]2D). GO enrichment analysis of DEGs To explore the potential functions of DEGs involved in the response of pepper to LL stress, GO enrichment analysis was performed (Fig. [96]3). Bubble diagrams were used to visualize the top 30 significantly enriched GO terms for each comparison group. GO annotations were categorized into three main branches: biological process (BP), cellular component (CC), and molecular function (MF). In the Y2CK vs. Y2LL comparison group, 3,433 DEGs were mapped to the GO database, accounting for 79.01% of the total DEGs in the group. In BP, DEGs were significantly enriched in ‘photosynthesis, light harvesting’ (GO:0009765), ‘photosynthesis’ (GO:0015979), and ‘chlorophyll biosynthetic process’ (GO:0015995). In CC, DEGs were significantly enriched in ‘chloroplast thylakoid membrane’ (GO:0009535), ‘photosystem II’ (GO:0009523), and ‘photosystem I’ (GO:0009522), with the highest number of DEGs involved in the term ‘chloroplast’ (GO:0009507). In MF, DEGs were significantly enriched in ‘chlorophyll binding’ (GO:0016168), with the largest number of DEGs associated with ‘monooxygenase activity’ (GO:0004497). These results suggest that DEGs associated with photosynthesis are activated in response to LL stress. Fig. 3. [97]Fig. 3 [98]Open in a new tab GO enrichment analysis of the DEGs identified in the four comparison groups. (A) Y2CK vs. Y2LL comparison group. (B) Y425CK vs. Y425LL comparison group. (C) Y425CK vs. Y2CK comparison group. (D) Y425LL vs. Y2LL comparison group. The horizontal coordinate is the proportion of DEGs enriched in this term to all DEGs, and the vertical coordinate is the GO annotation term In the Y425CK vs. Y425LL comparison group, 2,364 DEGs were mapped to the GO database, representing 78.70% of the total DEGs in this group. In BP, DEGs were significantly enriched in ‘photosynthesis, light harvesting’ (GO:0009765) and were also prominently involved in ‘response to temperature stimulus’ (GO:0009266), ‘response to osmotic stress’ (GO:0006970), ‘response to antibiotic’ (GO:0046677), and ‘response to reactive oxygen species’ (GO:0000302). In CC, DEGs were enriched in ‘photosystem II’ (GO:0009523) and ‘photosystem I’ (GO:0009522). In MF, the majority of DEGs were associated with ‘DNA binding’ (GO:0003677). These results suggest that DEGs involved in osmotic stress resistance and ROS response are induced by LL stress. In the Y425CK vs. Y2CK comparison group, 1,175 DEGs were mapped to the GO database, accounting for 73.21% of the DEGs in this group. In BP, DEGs were significantly enriched in ‘defense response’ (GO:0006952), which had the highest number of DEGs. In CC, DEGs were primarily enriched in ‘cell wall’ (GO:0005618), ‘integral component of membrane’ (GO:0016021), ‘extracellular region’ (GO:0005576), and ‘plant-type cell wall’ (GO:0009505). In MF, the highest number of DEGs were associated with ‘heme binding’ (GO:0020037), with additional enrichment in ‘monooxygenase activity’ (GO:0004497) and ‘endopeptidase inhibitor activity’ (GO:0004866). These results suggest that DEGs involved in cell wall composition contribute to defense responses under LL stress. In the Y425LL vs. Y2LL comparison group, 2,010 DEGs were mapped to the GO database, representing 76.86% of the DEGs in this group. In BP, DEGs were significantly enriched in ‘photosynthesis’ (GO:0015979), ‘plant-type cell wall organization’ (GO:0009664), and ‘chlorophyll biosynthetic process’ (GO:0015995). In CC, DEGs were most significantly enriched in ‘chloroplast thylakoid membrane’ (GO:0009535), which had the highest number of DEGs. In MF, DEGs were prominently associated with ‘chlorophyll binding’ (GO:0016168), ‘oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen’ (GO:0016705), and ‘protein disulfide oxidoreductase activity’ (GO:0015035). These findings further highlight the activation of DEGs related to photosynthesis and chlorophyll biosynthesis in response to LL stress. KEGG pathway enrichment analysis of DEGs To further investigate the metabolic pathways involved in pepper’s response to LL stress, KEGG pathway enrichment analysis was performed on the DEGs identified in the four comparison groups (Fig. [99]4; Table [100]S3). In the Y2CK vs. Y2LL comparison group, 2,870 DEGs were mapped to the KEGG database, accounting for 66.05% of the DEGs in this group. Among these, the pathways ‘Photosynthesis-antenna proteins’ (ko00196), ‘Circadian rhythm-plant’ (ko04712), and ‘Carbon fixation in photosynthetic organisms’ (ko00710) were significantly enriched, with 32, 40, and 36 DEGs, respectively. In the Y425CK vs. Y425LL comparison group, 2,002 DEGs were mapped to the KEGG database, representing 66.64% of the DEGs in the group. Key enriched pathways included ‘Circadian rhythm-plant’ (ko04712), ‘Photosynthesis-antenna proteins’ (ko00196), and ‘Plant hormone signal transduction’ (ko04075), with 36, 15, and 88 DEGs, respectively. Notably, the ‘Plant-pathogen interaction’ pathway (ko04626) had the highest number of enriched DEGs, with 157. Fig. 4. [101]Fig. 4 [102]Open in a new tab KEGG pathway enrichment analysis of the DEGs identified in the four comparison groups (the top 20 KEGG pathways). (A) Y2CK vs. Y2LL comparison group. (B) Y425CK vs. Y425LL comparison group. (C) Y425CK vs. Y2CK comparison group. (D) Y425LL vs. Y2LL comparison group. The horizontal coordinate is the number of DEGs enriched in the pathway, and the vertical coordinate is the enriched pathway For the Y425CK vs. Y2CK comparison group, 975 DEGs were mapped to the KEGG database, accounting for 60.75% of the DEGs in this group. Pathways enriched at higher levels included ‘Zeatin biosynthesis’ (ko00908), ‘Anthocyanin biosynthesis’ (ko00942), and ‘Taurine and hypotaurine metabolism’ (ko00430), with 18, 6, and 5 DEGs, respectively. In the Y425LL vs. Y2LL comparison group, 1,655 DEGs were mapped to the KEGG database, representing 63.33% of the DEGs in this group. Prominently enriched pathways included ‘Photosynthesis-antenna proteins’ (ko00196), ‘Carbon metabolism’ (ko01200), and ‘Carbon fixation in photosynthetic organisms’ (ko00710), with 21, 71, and 32 DEGs, respectively. Interestingly, among the top 20 pathways, only one, ‘Circadian rhythm-plant’ (ko04712), was shared across all four comparison groups. This finding underscores its potential role as a common regulatory mechanism in pepper’s response to LL stress. Analysis of DEGs in photosynthesis-antenna proteins pathway Based on the gene function enrichment analysis, photosynthesis-related pathways were significantly enriched in response to LL stress, with the photosynthesis-antenna proteins pathway being particularly prominent (only the Y425CK vs. Y2CK group did not show enrichment among the four comparison groups). This highlights that LL stress activates key DEGs in the photosynthesis-antenna proteins pathway to mitigate stress. A total of eight DEGs associated with this pathway were shared across three comparison groups (Fig. [103]5B-C; Table [104]S4). Among these, seven DEGs were downregulated by LL stress. These genes encode chlorophyll a-b binding protein 3 C (Capana00g000031 and Capana04g002820), chlorophyll a-b binding protein 1B (Capana00g002800), chlorophyll a-b binding protein P4 (Capana01g000647), chlorophyll a-b binding protein 6 A (Capana05g002549 and Capana05g002550), and chlorophyll a-b binding protein CP24 10a (Capana08g002012). Interestingly, only one gene, Capana00g002794, encoding chlorophyll a-b binding protein 21, was upregulated under LL stress. This gene showed particularly high expression levels in the LL-tolerant genotype Y425, suggesting that its elevated expression may play a critical role in conferring LL tolerance in Y425. Chlorophyll a-b binding proteins (CAB) are localized on the chloroplast thylakoid membrane, where they bind to chlorophyll to form the light-harvesting chlorophyll protein complex (LHC). These proteins are essential for capturing and transferring light energy [[105]35] (Fig. [106]5A). In summary, LL stress induces the expression of CAB genes, which contribute to enhancing LL tolerance in pepper. Fig. 5. [107]Fig. 5 [108]Open in a new tab Analysis of shared DEGs in photosynthesis-antenna proteins pathway. (A) Light-harvesting chlorophyll protein complex in the KEGG pathway database. (B) Venn diagram of shared DEGs in photosynthesis-antenna proteins pathway from Y2CK vs. Y2LL, Y425CK vs. Y425LL and Y425LL vs. Y2LL comparison groups. (C) Heatmap analysis of the shared DEGs expressions. A yellow rectangle indicates that the transcription level of the gene was upregulated, and a blue rectangle indicates that the transcription level of the gene was downregulated Analysis of DEGs in zeatin biosynthesis pathway Zeatin plays a crucial role in regulating plant defense responses to abiotic stresses and is essential for plant growth and development [[109]36]. In this study, the zeatin biosynthesis pathway was significantly enriched in both the Y425CK vs. Y2CK and Y425LL vs. Y2LL comparison groups. A total of six DEGs were shared between these groups (Fig. [110]6; Table [111]S5). Among them, one DEG encoding zeatin O-glucosyltransferase (Capana00g004282), one encoding cytokinin hydroxylase (Capana02g002776), and another encoding a hypothetical protein FXO38_29178 (NewGene_6641) were upregulated in Y425 and downregulated in Y2. Conversely, the DEG encoding an uncharacterized mitochondrial protein AtMg00860-like (NewGene_23445) exhibited the opposite expression pattern, indicating that the differential expression of these genes may collectively influence the phenotypes of the two genotypes. Fig. 6. [112]Fig. 6 [113]Open in a new tab Analysis of shared DEGs in zeatin biosynthesis pathway. (A) Venn diagram of shared DEGs in zeatin biosynthesis pathway from Y425CK vs. Y2CK and Y425LL vs. Y2LL comparison groups. (B) Heatmap analysis of the shared DEGs expressions Interestingly, another DEG encoding zeatin O-glucosyltransferase (Capana05g000785) and one encoding UDP-glycosyltransferase (Capana10g001519) displayed similar expression patterns, being upregulated in Y2 under CK conditions and in Y425 under LL stress. Notably, zeatin O-glucosyltransferase (ZOG) genes were the most abundant in this cluster and were consistently upregulated in response to LL stress, suggesting that they may serve as key genes contributing to LL stress tolerance. Analysis of DEGs in circadian rhythm-plant pathway Plant defense responses to abiotic stresses are significantly influenced by the circadian rhythm-plant signaling pathway [[114]37]. In the KEGG enrichment analysis, the circadian rhythm-plant pathway was found to be enriched in all four comparison groups, with numerous DEGs identified across these groups. A total of 23 DEGs were shared between the Y2CK vs. Y2LL and Y425CK vs. Y425LL groups, while 7 DEGs were shared between the Y425CK vs. Y2CK and Y425LL vs. Y2LL groups (Fig. [115]7A-B; Table [116]S6). This indicates that genetic differences between genotypes play an important role in coping with LL stress. Among the 23 shared DEGs in the Y2CK vs. Y2LL and Y425CK vs. Y425LL groups (Fig. [117]7C), five genes encoding zinc finger proteins (ZFPs; Capana00g004030, Capana02g003199, Capana07g000030, Capana08g002625, and Capana12g000414) exhibited similar expression trends. Notably, Capana07g000030 was downregulated in both genotypes after LL stress, while the remaining four genes were upregulated. Four genes encoding REVEILLE proteins (RVEs; Capana02g000058, Capana02g002266, Capana08g000974, and Capana10g001790) were consistently upregulated after LL stress, with significantly higher expression levels in Y425 than in Y2. Similarly, four genes encoding cyclic dof factors (CDFs; Capana02g000842, Capana02g003361, Capana04g002144, and Capana05g001141) were upregulated in both genotypes, with stronger upregulation in Y425. Fig. 7. [118]Fig. 7 [119]Open in a new tab Analysis of shared DEGs in circadian rhythm-plant pathway. (A) Venn diagram of shared DEGs in circadian rhythm-plant pathway from Y2CK vs. Y2LL and Y425CK vs. Y425LL comparison groups. (B) Venn diagram of shared DEGs in circadian rhythm-plant pathway from Y425CK vs. Y2CK and Y425LL vs. Y2LL comparison groups. (C) Heatmap analysis of the shared DEGs expressions from Y2CK vs. Y2LL and Y425CK vs. Y425LL comparison groups. (D) Heatmap analysis of the shared DEGs expressions from Y425CK vs. Y2CK and Y425LL vs. Y2LL comparison groups Three genes encoding TCP transcription factors (Capana02g003665, Capana03g000409, and Capana08g000047) showed differential expression patterns: Capana02g003665 and Capana03g000409 were upregulated in both genotypes, with higher levels in Y425, while Capana08g000047 was downregulated. One gene encoding a bHLH84 transcription factor (Capana04g000660) was downregulated in both genotypes under LL stress. Two genes encoding two-component response regulators (Capana03g000812 and Capana04g000899) were upregulated in both genotypes, with higher expression in Y425. Additionally, a gene encoding chalcone synthase (Capana05g002274), a SPA1-related protein (Capana07g002184), and a poly(A) polymerase (Capana12g000508) were upregulated in both genotypes after LL stress. Interestingly, a FLOWERING LOCUS T (FT) gene (Capana12g002498) was upregulated only in Y2 after LL stress, with no change observed in Y425. Among the seven shared DEGs in the Y425CK vs. Y2CK and Y425LL vs. Y2LL groups (Fig. [120]7D), two genes encoding HEADING DATE proteins (Capana05g002506 and Capana12g002497) showed a similar pattern of being downregulated in Y425 and upregulated in Y2 under LL stress. Two genes encoding chalcone synthase (Capana12g000350 and NewGene_7121) were downregulated in both genotypes. The FT gene (Capana12g002553) showed higher expression levels in Y2 than in Y425, particularly after LL stress. A gene encoding a two-component response regulator (NewGene_6965) exhibited significantly higher expression in Y425 than in Y2, although it was slightly downregulated in Y425 under LL stress. Notably, one CDF gene (Capana05g001141) was shared across both gene clusters and appears to positively regulate LL tolerance in pepper based on its expression levels. These results indicate that the circadian rhythm-plant pathway is strongly activated in pepper under LL stress. The ZFP, RVE, and CDF genes enriched in this pathway showed significant upregulation in response to LL stress, playing crucial roles in mitigating the damage caused by these conditions. Different physiological responses in two peppers under LL treatment Through transcriptome analysis, we identified significant enrichment of GO terms and metabolic pathways associated with photosynthesis-antenna proteins, chlorophyll biosynthesis, reactive oxygen species (ROS) response, and osmotic stress after LL stress. To validate these findings and gain a more comprehensive understanding of the physiological response mechanisms in pepper under LL stress, we measured key physiological indices following CK and LL treatments. Chlorophyll fluorescence parameters (Fv/Fm, Y(PSII), qP), chlorophyll content, and osmoregulatory substance contents (soluble sugars, soluble proteins, and Pro) were analyzed to compare the physiological responses of the two genotypes to LL stress (Fig. [121]8). As shown in Fig. [122]8A, Fv/Fm values decreased in both genotypes only at the late stage of LL treatment. Notably, Y425 showed a decline only on day 7, while Y2 exhibited a decreasing trend starting from day 5. The Y(PSII) values displayed a significant decline from day 3, with Y2 showing a more rapid decrease compared to Y425. Similarly, the qP value in Y2 began to decline markedly from day 3, whereas Y425 exhibited only a slight reduction. Chlorophyll content in both Y2 and Y425 decreased progressively with prolonged LL treatment (0–7 days). The reduction was more pronounced in Y2, with its total chlorophyll content reduced by 37.08% compared to CK by day 7, whereas Y425 experienced a significantly smaller reduction of 8.12% compared to CK during the same period. Fig. 8. [123]Fig. 8 [124]Open in a new tab Chlorophyll fluorescence parameters, chlorophyll content and osmoregulatory substances content of pepper seedlings at different periods after LL stress. (A) Images of Fv/Fm, Y(PSII) and qP. False colors represent values of the parameter ranging from low (black) to high (purple). (B) Chlorophyll a content, (C) chlorophyll b content, (D) total chlorophyll content, (E) soluble protein content, (F) soluble sugar content and (G) Pro content. Bars are presented as means ± SD (n = 3), and different lowercase letters indicate significant differences among the means by Duncan’s multiple range tests (p < 0.05). ns: not significant LL stress also induced significant increases in osmoregulatory substances in both genotypes. In Y2, soluble sugar content increased consistently throughout the treatment period, while soluble protein and Pro contents increased initially and peaked on day 5, followed by a decline. At their peaks, soluble sugar, soluble protein, and Pro levels increased by 412.29%, 152.91%, and 806.79%, respectively, compared to CK. In Y425, soluble sugars, soluble proteins, and Pro levels all increased consistently with the duration of LL treatment, reaching their highest levels on day 3. At their peaks, soluble sugar, soluble protein, and Pro levels increased by 521.19%, 179.89%, and 905.66%, respectively, compared to CK. These results confirmed that under LL stress, Y425 mitigated osmotic stress damage more effectively than Y2 by accumulating higher levels of compatible organic solutes. Differences in oxidative damage and antioxidant levels between the two pepper varieties under LL stress were assessed by measuring the contents of MDA, H₂O₂, and superoxide anion (O₂⁻), as well as the activities of antioxidant enzymes (SOD, POD, CAT). These findings were further validated through DAB and NBT staining (Fig. [125]9). In Y2, H₂O₂ content increased initially, peaking at 3 days, and then decreased as LL stress continued. In contrast, MDA and O₂⁻ levels increased continuously throughout the treatment period and were significantly higher than those in Y425 at all stages. In Y425, O₂⁻ levels also increased but at a slower rate than in Y2, while MDA and H₂O₂ levels showed an initial increase followed by a decline. These levels were consistently lower in Y425 compared to Y2 at all time points. DAB and NBT staining of pepper leaves after 7 days of LL stress revealed darker coloration in Y2 compared to Y425, while leaves under CK conditions showed the lightest coloration in both genotypes (Fig. [126]9A-B). These results indicate that Y2 accumulated more ROS than Y425 under LL stress, leading to more severe oxidative damage in Y2. Fig. 9. [127]Fig. 9 [128]Open in a new tab Differences in oxidative damage and antioxidant enzyme activities of pepper under LL stress. (A) Superoxide anion (O[2]^−) contents were detected by NBT staining and (B) H[2]O[2] levels were detected by DAB staining. (C) MDA content, (D) H[2]O[2] content, (E) O[2]^− content, (F) SOD activity, (G) POD activity and (H) CAT activity. Bars are presented as means ± SD (n = 3), and different lowercase letters indicate significant differences among the means by Duncan’s multiple range tests (p < 0.05). ns: not significant As shown in Fig. [129]9F-H, LL stress induced a significant increase in antioxidant enzyme activities in both genotypes, though the magnitude of the increase varied between Y2 and Y425. In Y2, SOD and POD activities peaked at 3 days (increasing by 78.17% and 89.15%, respectively), while CAT activity peaked at 5 days (increasing by 366.64%). In Y425, all three enzyme activities reached their maximum levels at 3 days, with SOD increasing by 126.29%, POD by 272.38%, and CAT by 577.41%. Notably, antioxidant enzyme activities were higher in Y425 than in Y2 at all stages, indicating that Y425 had a stronger ROS-scavenging capacity under LL stress. Overall, the antioxidant enzyme activities in both genotypes followed a trend of increasing initially and then decreasing after 3 days. However, Y425 maintained higher enzyme activity levels than Y2 throughout the treatment period. These results suggest that prolonged LL stress may impair the antioxidant system in peppers, with 3 days representing a critical turning point in their physiological response to LL stress. Discussion LL stress is a significant abiotic stress affecting facility-grown vegetables, often leading to severe declines in both yield and quality. This is particularly problematic during the off-season, as non-heated greenhouses reliant on natural light are highly vulnerable to LL stress, resulting in substantial agro-economic losses [[130]29]. Pepper, a widely cultivated facility vegetable, is especially susceptible to LL stress [[131]28]. Under LL conditions, the yield and quality of pepper are drastically reduced. While foliar applications of glycine betaine [[132]29], zeaxanthin [[133]27], and melatonin [[134]15] have been shown to mitigate some of the adverse effects of LL stress, these measures fail to fully prevent the damage caused by such conditions. Therefore, uncovering the physiological and molecular mechanisms underlying pepper’s response to LL stress and developing LL-tolerant varieties remain critical strategies. This study aimed to investigate the physiological and transcriptomic changes in the LL-tolerant genotype Y425 and the LL-sensitive genotype Y2 under LL stress, with the goal of providing valuable insights for future LL-tolerant pepper breeding programs. The results revealed significant differences between Y2 and Y425 at both physiological and molecular levels following LL stress. A series of DEGs were identified in key pathways, including the photosynthesis-antenna protein pathway, zeatin biosynthesis pathway, and circadian rhythm-plant pathway. These pathways and DEGs play critical roles in regulating the adaptive mechanisms of pepper in response to LL stress. Morphological and physiological response of pepper to LL stress The damage caused by LL stress in pepper involves a complex interplay of physiological responses and gene regulation, with the most noticeable changes being morphological. In this study, LL stress significantly reduced plant height, stem diameter, leaf area, root length, shoot fresh weight, and root fresh weight compared to CK, consistent with the findings of Ding et al. [[135]27]. These observations are also similar to the responses of snapdragon to LL stress [[136]5]. The reductions can primarily be attributed to LL stress inhibiting cell elongation and differentiation, thereby reducing biomass [[137]38, [138]39]. However, studies have shown that low light can promote root elongation and reduce root branching in wheat, indicating that the effect of low light on roots varies depending on light intensity and plant species [[139]40]. The physiological responses of pepper to LL stress are more intricate and diverse. Chlorophyll fluorescence parameters are widely used in abiotic stress studies as they provide acute insights into changes in plant photosynthetic activity [[140]41]. Parameters such as Fv/Fm, Y(PSII), and qP, commonly regarded as key photochemical quenching indicators, generally show a declining trend under abiotic stress. In this study, Fv/Fm exhibited a significant decrease only during the later stages of LL treatment, while Y(PSII) and qP began declining from day 3. These findings align with the response of chlorophyll fluorescence parameters under drought stress in lettuce, as reported by Shin et al. [[141]42]. The genotype Y2 showed a pronounced reduction in chlorophyll content after LL stress, likely due to ROS accumulation triggered by abiotic stress, leading to membrane lipid peroxidation and damage to the PS action center. This, in turn, disrupted chloroplast structure and compromised photosynthetic activity [[142]43, [143]44]. Conversely, plants accumulate osmoregulatory substances under LL stress, including sugars, amino acids, and lipids, which help protect cells from stress-induced damage. These substances maintain cellular osmotic pressure and preserve the integrity of functional proteins [[144]45, [145]46]. Soluble sugars, soluble proteins, and proline are key osmoregulatory substances that regulate osmotic pressure and stabilize cell membranes [[146]19]. In this study, both pepper genotypes exhibited significant increases in these substances following LL stress, with Y425 showing significantly higher levels than Y2, consistent with the findings of Zhang et al. [[147]22]. The LL-sensitive genotype Y2 was more prone to osmotic disturbances under prolonged stress, leading to greater growth retardation. During LL stress, the accumulation of ROS, such as O₂⁻ and H₂O₂, damages enzyme activity, membrane lipids, and nucleic acids in plant cells [[148]47]. MDA is a critical indicator of peroxidative damage to plant cell membranes [[149]48]. In this study, Y2 accumulated higher levels of ROS and MDA compared to Y425. Meanwhile, plants protect themselves from oxidative damage through the enzymatic antioxidant system, which scavenges ROS [[150]49]. SOD, POD, and CAT are key antioxidant enzymes that efficiently neutralize ROS, balancing H₂O₂ and O₂⁻ levels [[151]50]. Upon exposure to low-temperature stress, the tolerant tomato variety elevated CAT activity compared to sensitive counterparts, thereby sustaining normal oxidative metabolic levels [[152]24]. Notably, antioxidant enzyme activities in Y425 were significantly higher than those in Y2, highlighting Y425’s stronger ROS-scavenging ability. However, these enzyme activities declined after 3 days of LL stress, likely due to cumulative cellular damage weakening the antioxidant system. Nevertheless, Y425 maintained relatively stable growth, reflecting its superior tolerance to LL stress compared to Y2. Activation of photosynthesis-antenna protein metabolic pathway after LL stress in pepper Photosynthesis, a process essential for plant growth and high yields, is strongly influenced by light and temperature [[153]51]. Low-temperature stress degrades chlorophyll, which in turn impairs photosynthetic efficiency. As temperatures drop, the contents of chlorophyll a, chlorophyll b, and total chlorophyll decrease, as observed in L. angustifolia leaves, negatively affecting photosynthesis. This response is similar to the changes in chlorophyll content and chlorophyll fluorescence parameters observed in pepper under LL stress in this study. At the molecular level, these changes are primarily attributed to the downregulated expression of CHLH-1, CHLH-2, CHLH-3, and GUN-4, along with the upregulated expression of CHLG. These gene expression changes result in reduced chlorophyll content, diminished light energy capture by the antenna system, and ultimately decreased light energy utilization [[154]52]. Photosynthesis-antenna proteins are critical components of the photosynthetic machinery and play a key role in light capture and energy transfer [[155]53]. In this study, the photosynthesis-antenna protein pathway was significantly enriched in multiple comparison groups (Fig. [156]7), indicating its involvement in the response to LL stress. Furthermore, eight CAB genes were identified in this pathway. CAB proteins, essential pigmented proteins, are involved in light capture and transfer during photosynthesis and are known to play active roles in abiotic stress responses [[157]54]. For instance, CAB genes in Paeonia ostii positively regulate drought stress responses by maintaining chloroplast homeostasis, regulating chlorophyll content, promoting photosynthesis, and accumulating osmoregulatory substances [[158]55]. Similarly, CAB genes were significantly upregulated in ginseng in response to heat stress [[159]56]. In this study, LL stress caused the downregulation of most CAB genes, except for CAB21 (Capana00g002794), which was upregulated in the LL-tolerant genotype Y425 under LL stress (Fig. [160]8; Table [161]S3). These findings suggest that while most CAB genes are suppressed by LL stress, CAB21 functions as a positive regulator of LL tolerance in pepper. Therefore, CAB genes in pepper appear to adopt distinct mechanisms to respond to LL stress, with CAB21 playing a pivotal role in conferring tolerance. Activation of zeatin biosynthesis pathway after LL stress in pepper Zeatin, a cytokinin derivative originally isolated from corn kernels, is known to enhance plant resistance to abiotic stresses and regulate growth and development [[162]57, [163]58]. In bell pepper, high concentrations of zeatin in the root system have been shown to aid in the recovery of normal photosynthesis under salt stress [[164]59]. In maize, zeatin functions as an inhibitor of leaf senescence [[165]60], while in wheat, its content increases significantly during the early stages of cold stress to mitigate damage [[166]61]. In this study, the zeatin biosynthesis pathway was enriched in both the Y425CK vs. Y2CK and Y425LL vs. Y2LL comparison groups (Fig. [167]9; Table [168]S4), supporting the hypothesis that this pathway plays a role in pepper’s response to LL stress. Additionally, six shared DEGs were identified within the zeatin biosynthesis pathway, with the highest number being ZOG genes. Glycosylation is a critical process in cytokinin metabolism and is closely linked to the formation of these compounds [[169]62]. For instance, TaCisZOG1 has been reported to exhibit elevated expression levels, promoting early wheat grain development [[170]63]. In this study, both ZOG genes were upregulated in Y425 under LL stress, likely contributing to enhanced stress tolerance. However, the precise mechanism underlying this is yet to be further elucidated. Activation of circadian rhythm metabolic pathway after LL stress in pepper Circadian rhythms are well recognized for their critical role in regulating various physiological and developmental processes in plants, including photosynthesis, stomatal opening, odor release, metabolism, and defense mechanisms [[171]64]. These rhythms are governed by the cellular circadian oscillator, enabling plants to adapt to environmental fluctuations [[172]65]. Previous studies have demonstrated that certain defense-related genes and stomatal closure are regulated by circadian rhythms, thereby contributing to plant immunity through circadian clock regulation [[173]66]. Thus, circadian rhythms are vital for plants to enhance their resilience to abiotic stresses. In this study, the circadian pathway was enriched across all four comparison groups (Fig. [174]7), indicating its significant role in the response to LL stress. Earlier studies have suggested that circadian rhythms regulate photosynthetic rhythms by modulating chlorophyll levels to adapt to environmental changes [[175]67], aligning with the current findings on chlorophyll content changes in pepper leaves under LL stress (Fig. [176]2). Zinc finger proteins (ZFPs) are nucleic acid-binding proteins found abundantly in eukaryotic genomes, playing key roles in plant responses to adversity [[177]68]. For instance, in petunia, the TFIIA-type ZFP genes ZPT2-2 and ZPT2-3 respond to cold and drought stress, with overexpression of ZPT2-3 enhancing dehydration tolerance [[178]69]. In this study, five shared ZFP genes were identified within the circadian pathway and co-expressed in the Y2CK vs. Y2LL and Y425CK vs. Y425LL groups (Fig. [179]10; Table [180]S5). Among these, four ZFP genes (Capana00g004030, Capana02g003199, Capana08g002625, and Capana12g000414) were upregulated after LL stress, with higher expression levels in Y425 compared to Y2, suggesting a positive regulatory role in LL tolerance. Conversely, one ZFP gene (Capana07g000030) was downregulated following LL stress, implying a potential negative regulatory role. These findings highlight the diverse functional roles of ZFP genes in plant stress responses. Fig. 10. [181]Fig. 10 [182]Open in a new tab Physiological and molecular mechanisms underlying LL stress tolerance in pepper, highlighting key pathways, gene regulation, and contrasting responses between LL-tolerant and LL-sensitive genotypes REVEILLE (RVE) proteins are key components of the circadian pathway. RVE4, RVE6, and RVE8 are the primary transcriptional activators of the plant biological clock, enhancing circadian robustness and regulating biological rhythms to adapt to environmental changes [[183]70]. In Arabidopsis, RVE4 and RVE8 specifically regulate heat tolerance by modulating the expression of downstream transcription factors such as ethylene response factor 53 (ERF53) and ERF54, particularly around midday, underscoring their link to the circadian clock [[184]71]. In this study, four shared RVE genes (Capana02g000058, Capana02g002266, Capana08g000974, and Capana10g001790) were identified in the circadian pathway. All four RVE genes were upregulated following LL stress, with expression levels higher in Y425 than in Y2. This differential expression likely contributes to the contrasting physiological and phenotypic responses of LL-tolerant and LL-sensitive genotypes. Cyclic DOF factors (CDFs) are known to regulate plant flowering and are associated with abiotic stress tolerance. In Arabidopsis, overexpression of SlCDF1 and SlCDF3 enhances tolerance to drought and salt stress [[185]72]. Similarly, in potato, StCDF1 regulates water loss and improves drought tolerance by modulating stomatal dynamics [[186]73]. In this study, four shared CDF genes (Capana02g000842, Capana02g003361, Capana04g002144, and Capana05g001141) were identified within the circadian pathway. All these CDF genes were upregulated in response to LL stress, suggesting that they positively regulate LL tolerance in pepper. Based on our findings, these CDF genes could be further explored for their roles in enhancing LL tolerance mechanisms. Conclusion In summary, the present study revealed the physiological and molecular mechanisms under LL stress in two pepper genotypes with contrasting LL tolerance (Fig. [187]10). LL stress induces complex physiological and molecular responses in pepper, leading to varying levels of tolerance between LL-tolerant and LL-sensitive genotypes. LL-tolerant genotypes exhibit better maintenance of photosynthetic activity (F[v]/F[m], Y(PSII), qP) and chlorophyll content, coupled with lower oxidative damage (reduced ROS and MDA accumulation) and higher antioxidant enzyme activities (POD, SOD, CAT). These responses are supported by the upregulation of key molecular pathways, including CAB21, ZOG, ZFP, RVE, and CDF genes, which enhance photosynthetic efficiency, mitigate oxidative stress, and maintain osmotic balance through the accumulation of soluble sugars, proteins, and proline. Conversely, LL-sensitive genotypes show greater photosynthetic and oxidative damage, resulting in worse LL tolerance. These findings provide insights into the physiological and molecular mechanisms underpinning LL tolerance and highlight potential genetic targets for breeding stress-resilient pepper varieties. This work provides a solid foundation for future research on enhancing pepper resistance in exposed to complex and variable environments, and the next step should be to explore the specific molecular networks of pepper in response to LL stress. Electronic supplementary material Below is the link to the electronic supplementary material. [188]12870_2025_6169_MOESM1_ESM.zip^ (2.6MB, zip) Supplementary Material 1: Fig. 1 qRT-PCR expression validation for eight randomly selected genes. The left Y-axis is the qRT-PCR expression levels, and the right Y-axis is the FPKM values of RNA-seq. Bars are presented as means ± standard error (n=3). Table S1 RNA-seq data quality. Table S2 Information of all genes in the different comparison groups. Table S3 Statistics of KEGG pathways. Table S4 Common DEGs involved in photosynthesis-antenna protein pathway in Y2CK vs. Y2LL and Y425CK vs. Y425LL and Y425LL vs. Y425LL. Table S5 Common DEGs involved in zeatin biosynthesis pathway in Y425CK vs. Y2CK and Y425LL vs. Y2LL. Table S6 Common DEGs involved in circadian rhythm-plant pathway in Y2CK vs. Y2LL and Y425CK vs. Y425LL and Y425CK vs. Y2CK and Y425LL vs. Y2LL. Table S7 Primers used in this study. Acknowledgements