Jasmonic acid and ERF family genes are involved in chilling sensitivity and seed browning of pepper fruit after harvest

Pepper (Capsicum annuum L.) fruit is sensitive to temperatures below 10 °C, which severely diminish fruit quality during cold chain distribution. Seed browning was a major chilling symptom in 36 genotypes of C. annuum fruit screened after storage at 2 °C for 3 weeks. Among them, pepper fruits of chilling-insensitive ‘UZB-GJG-1999–51’ and -sensitive ‘C00562’ were treated at 2 °C for 0 or 24 h, respectively. Analyses of integrated transcriptome-metabolome and relative gene expression in seeds obtained from the two genotypes were conducted to identify key factors involved in the seed browning induced by chilling. The relative contents of branched-chain amino acids such as leucine, isoleucine, and valine were significantly increased after chilling. Transcriptome identification showed 3,140 differentially expressed genes (log twofold change > 1.0 and FDR-corrected p value < 0.05) affected by chilling between the two genotypes. Particularly, genes related to jasmonic acid synthesis and signaling were differentially expressed. A regulatory network of jasmonic acid synthesis and signaling, and regulation of ERF family genes might contribute to chilling response in pepper fruit. The results of this study may help facilitate further studies to develop chilling-insensitive peppers and could be a basis for improving postharvest fruit quality.


Results
Seed browning rates of 36 pepper genotypes. Seed browning symptoms were the major chilling injury in the 36 genotypes of C. annuum screened in this study (Supplementary Table S1). Other chilling symptoms such as pitting on the surface and discoloration of the calyx were negligible during cold storage at 2 °C for 3 weeks. Seed browning was also observed as a major chilling symptom of C. annuum 'Cheongyang' fruit treated at 2 °C for 3 weeks 5 . We sorted 36 pepper genotypes according to seed browning rates from 0.0% (chillinginsensitive peppers) to over 60.0% (chilling-sensitive peppers) (Supplementary Table S1).
Differentially accumulated metabolites. A total 51 of untargeted and targeted metabolites in pepper seeds were quantified in this study. To confirm the relationships among identified metabolites, partial least squares-discriminant analysis (PLS-DA) was performed (Fig. 1B,C). The first and second principle components (PCs) accounted for 47.6% (PC 1, 34.6%; PC 2, 13.0%) of the variance in the dataset. The PLS-DA loading plot (Fig. 1C) was used to identify the causative metabolites of the PLS-DA score plot. We were able to confirm that pyruvic acid, lactic acid, oxalic acid, palmitic acid, and methionine were the major metabolites separating the two pepper genotypes (chilling-insensitive 'UZB-GJG-1999-51' and chilling-sensitive 'C00562'). In addition, among amino acids, isoleucine, valine, and leucine were the major metabolites contributing to the distribution between '0 h' and '24 h' pepper groups, with separation due to the chilling treatment at 2 °C for 24 h. Figure 2A shows the heat-map analysis of metabolites. Most organic acids, except for malic acid and citric acid, showed substantially higher contents in chilling-sensitive 'C00562' than in chilling-insensitive 'UZB-GJG-1999-51′. Also, leucine, valine, and isoleucine contents were clearly increased in the two pepper genotypes by chilling treatment; however, chilling-insensitive 'UZB-GJG-1999-51′ had substantially higher contents of leucine, valine, and isoleucine than chilling-sensitive 'C00562' .
The relative content levels of 10 selected metabolites from organic acids, amino acids, and fatty acids are compared in Fig. 2B. The levels of pyruvic acid, succinic acid, and fumaric acid were substantially higher in chilling-sensitive 'C00562' , regardless of chilling treatment, at 2 °C for 24 h. The pyruvic acid level was significantly increased in 'C00562' only after chilling treatment. In the cases of leucine, isoleucine, and valine, which are branched-chain amino acids (BCAA), there were no differences in their contents between the two pepper genotypes, and they showed similar levels to each other at 0 h, before chilling treatment. However, after chilling treatment (24 h), the three amino acid levels were significantly increased in the two pepper genotypes.
The level of palmitic acid was not significantly changed in chilling-insensitive 'UZB-GJG-1999-51' by chilling treatment, but it was significantly increased in chilling-sensitive 'C00562' . Citric acid and malic acid levels were not significantly different. Linoleic acid content was slightly higher in chilling-insensitive 'UZB-GJG-1999-51' Scientific Reports | (2020) 10:17949 | https://doi.org/10.1038/s41598-020-75055-z www.nature.com/scientificreports/ than in chilling-sensitive 'C00562' but there were no significant differences according to chilling sensitivity of genotypes or according to the chilling treatment at 2 °C for 24 h. To reveal the biological pathways involved in a chilling-induced seed browning in peppers by chilling treatment at 2 °C for 24 h, metabolite enrichment was analyzed (Fig. 3A). As a result, galactose metabolism; valine, leucine, and isoleucine biosynthesis; phenylalanine, tyrosine, and tryptophan biosynthesis; and starch and sucrose metabolism pathways in peppers were determined to be significantly affected by the chilling treatment at 2 °C for 24 h.
Differentially expressed genes (DEGs). Through the transcriptome analysis of seeds obtained from chilling-insensitive 'UZB-GJG-1999-51' and chilling-sensitive 'C00562' before or after chilling treatment, an average of 23.9 million reads ranging from 15.1 to 36.8 million reads among 12 samples (two genotypes + before and after chilling + 3 biological replicates) were obtained. On average, 82.7% of total reads were mapped to the transcript library. Furthermore, the percentages of Q30 base in all samples were higher than 96.50% and the percentages of Q20 were higher than 98.03% (Supplementary Table S3).
In total, 84,627 contigs were assembled with a reference genome (Pepper_Zunla_1_Ref_v1.0_rna.fna, accession number: GCF_000710875.1). The average contig length was 1010.6 bp, and the N50 was 1518 bp. To confirm the relationships among each sample and replicate, PCA and PLS-DA were performed using the changes  Supplementary Fig. S2). In the PCA score plot and PLS-DA score plot, the sum of first and second principal components accounted for 69.5% and 68.9% of the variance in the dataset, respectively. Also, the results of hierarchical analysis showed a clear distinction between the two pepper genotypes and the chilling treatment ( Supplementary Fig. S3).
To minimize false positives, DEGs were selected using a threshold of ≥ twofold upregulated or downregulated genes with an FDR < 0.05. As a result, a total of 6493 contigs were defined as DEGs of chilling-insensitive (Ins) 'UZB-GJG-1999-51' and chilling-sensitive (Sen) 'C00562' (Fig. 3B). Gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed using 3,140 DEGs to identify the differences in specific gene expressions induced by the chilling treatment at 2 °C for 24 h. As an exception, 3353 contigs belong to only 'Ins-0 h vs. Sen-0 h' (787 contigs), and only in an intersection between 'Ins-0 h vs. Sen-0 h' and 'Ins-24 h vs. Sen-24 h' (2566 contigs).
Upregulated or downregulated biological functions by the application of chilling treatment at 2 °C for 24 h were investigated with GO-based enrichment analysis (Fig. 3C)

Protein-protein interaction (PPI) network.
DEGs were used to construct the PPI network which was composed of 760 nodes and 228 edges ( Supplementary Fig. S4). Among them, 11 of the JA-related genes including LOX, AOC, JAR1, JAZ1, and JAZ3 were highlighted by a colored circle in Supplementary Fig. S4A. Also, except for PKT3, all of the JA-related genes were involved in abiotic, stress-related gene networks (Supplementary Fig. S4B). Therefore, it could be inferred that JA signaling is activated in the two genotypes; chilling-insensitive 'UZB-GJG-1999-51' and sensitive 'C00562' .

Heat-map of DEGs.
To better understand the molecular responses of a chilling-induced seed browning of pepper fruit, a heat-map analysis was conducted using 29 DEGs consisting of 20 kinds of AP2/ERF family genes and 9 kinds of JA-related genes ( Fig. 4 and Supplementary Table S4). Heat-map analysis clearly showed the differences in expression levels of 29 DEGs between chilling-insensitive 'UZB-GJG-1999-51' and chillingsensitive 'C00562' before and after chilling treatment at 2 °C for 0 h and 24 h (Fig. 4). Supplementary Fig. S5 shows the correlation and correlation network analysis of 51 metabolites and 29 DEGs of seeds obtained from 'UZB-GJG-1999-51' and 'C00562' peppers.
The expression level of CaJAR1, a JA-Ile biosynthesis gene, was up-regulated by chilling treatment in the two pepper genotypes, 'UZB-GJG-1999-51' and 'C00562' (Fig. 4). A higher CaJAR1 expression level was observed in chilling-insensitive 'UZB-GJG-1999-51' than in chilling-sensitive 'C00562' . Overall expression trends of CaDREB3 and CaERF11 were opposite to the overall expression trends of CaJAR1, CaERF1, CaERF3_1, CaERF10, and CaERF5. The levels of CaDREB3 and CaERF11 were more highly expressed in chilling-sensitive 'C00562' than in chilling-insensitive 'UZB-GJG-1999-51' and the expression levels of CaDREB3 and CaERF11 were downregulated by the chilling treatment at 2 °C in both genotypes.
The expression levels of transcription factors CaERF1, CaERF3_1, CaERF5, and CaERF10 were significantly upregulated by chilling treatment in the 6 pepper genotypes (Fig. 5). At 0 h, before the chilling treatment, their expression levels were similar regardless of the genotypes. However, at 24 h, after chilling treatment, their expression levels were substantially higher in the chilling-insensitive genotypes of 'UZB-GJG-1999-51' , 'Takanotsume' , and 'Hungarian Wax' than in the chilling-sensitive genotypes of 'C00562' , 'chili bangi' , and 'Gyeonggiyangpyeong' .
Scientific Reports | (2020) 10:17949 | https://doi.org/10.1038/s41598-020-75055-z www.nature.com/scientificreports/ At 0 h, the relative expression levels of CaERF11, CaDREB3, and CaLOX2.1 were significantly higher in the chilling-sensitive genotypes than in the chilling-insensitive genotypes (Fig. 5). However, their expression levels were substantially down-regulated in the 6 pepper genotypes after chilling treatment for 24 h. Chilling-insensitive genotypes showed substantially lower expression levels of CaERF11, CaDREB3, and CaLOX2.1 than chillingsensitive genotypes. Overall, their expression trends were very similar to each other, and the responses of their cellular mechanisms to chilling might be closely connected to each other in pepper fruit. We could assume that CaERF11 and CaDREB3 transcription factors act as negative regulators in the chilling response of pepper fruit.
CaLOX1.5_1 expression levels were not different, however. The expression levels of CaLOX1.5_2 were significantly upregulated in the chilling-insensitive and chilling-sensitive genotypes after chilling treatment (Fig. 5). The expression of CaLOX1.5_2 was more highly upregulated in chilling-sensitive genotypes than in insensitive genotypes. The expression levels of CaLOX1.5_2 showed similar levels in all peppers at 0 h.
The CaJAR1 expressions were opposite to the CaERF11 and CaDREB3 expressions (Fig. 5). CaJAR1 expressions were significantly upregulated in chilling-insensitive genotypes by the chilling treatment, and were slightly upregulated in chilling-sensitive genotypes. Chilling-insensitive groups originally had higher CaJAR1 expressions than chilling-sensitive groups.
The levels of CaDREB2, CaAOC, CaJAZ3, and CaAFP3 expressions were significantly higher in the 6 pepper genotypes at 0 h, before the chilling treatment (Fig. 5). However, they were clearly downregulated after the chilling treatment. Contrary to the expression levels of CaDREB2, CaAOC, CaJAZ3, and CaAFP3, the expression level of CaAOS3 was significantly upregulated by the chilling treatment but there were no differences according to the genotypes. The chilling treatment upregulated the expression level of CaJAZ1 only in chilling-sensitive groups, but not in chilling-insensitive groups. www.nature.com/scientificreports/ Figure 6 shows the putative chilling response pathway predicted in C. annuum exposed to chilling conditions. Chilling-insensitive pepper fruit synthesizes more JA-Ile by higher upregulation of JAR1, and by higher rates of increasing isoleucine content than chilling-sensitive pepper fruit under chilling conditions. The transcription factors of CaERF1, CaERF3_1, CaERF5, and CaERF10 might act as positive regulators to increase the chilling tolerance by showing higher expression levels in chilling-insensitive pepper fruit than in chilling-sensitive pepper fruit. However, CaDREB3 and CaERF11 transcription factors might act as negative regulators by showing highly upregulated expression levels in sensitive pepper fruit before chilling as well as by showing downregulated expression levels after chilling.

Discussion
We analyzed a total of 51 targeted and untargeted metabolites of seeds obtained from chilling-insensitive 'UZB-GJG-1999-51' and chilling-sensitive 'C00562' peppers exposed to 2 °C for 24 h, and confirmed the changes of various metabolite levels ( Fig. 1 and Fig. 2A). Our PLS-DA analysis indicated that pepper fruit are apparently separated according to their genotypes (PC 1); however, they are not clearly separated according to the chilling exposure time of 24 h (PC 2) (Fig. 1B). Metabolites are the end products of cell biology regulation processes and are closely related to phenotypes. Their levels can be regarded as the response of plant development to genetic and environmental changes 25,26 . Our results indicate that chilling-insensitive and sensitive pepper genotypes show differentially expressed metabolites, and that their levels are affected by chilling. We found that chilling exposure for 24 h was not enough to induce significant changes in all metabolites that were directly associated with the phenotype of seed browning induced by chilling.
The major differentially-expressed metabolites in the pepper fruit seeds were the organic acids; pyruvic acid, succinic acid, and fumaric acid. Their levels were significantly higher in chilling-sensitive 'C00562' peppers than in chilling-insensitive 'UZB-GJG-1999-51' peppers. Particularly, the pyruvic acid content was only significantly increased in chilling-sensitive 'C00562' peppers by the chilling treatment ( Fig. 2A,B). According to a previous study, pyruvic acid was highly accumulated in cucumber and eggplant which were exposed to a chilling   28 . Increased AOX gene expressions and AOX protein content then scavenges reactive oxygen species (ROS) generated under chilling conditions, resulting in higher chilling tolerance in the plant 4 . We speculate that the increased level of pyruvic acid in chilling-sensitive 'C00562' peppers after chilling treatment is probably due to the faster and more sensitive response to chilling stress compared to chilling-insensitive 'UZB-GJG-1999-51' peppers. Increased pyruvic acid has been known to accelerate amino acid synthesis 29 . Among amino acids, BCAAs such as isoleucine, leucine, and valine are known to control abiotic stress responses induced by chilling 30 through the regulation of osmotic pressure 31 . BCAA levels of leucine, isoleucine, and valine in rice seedlings increased to 6.8, 2.6, and 4.5 times higher than the initial level, respectively, after a chilling treatment at 4 °C for 24 h 32 . In this study, BCAA levels were significantly increased in seeds of pepper fruit treated with a chilling temperature of 2 °C at 24 h, regardless of the pepper genotype, and the increased levels were the same in both pepper genotypes. A difference in BCAA levels according to the chilling sensitivity of pepper fruit was not observed.
Similar to the result of the pyruvic acid analysis, palmitic acid, a major saturated fatty acid in pepper seeds, was significantly higher in chilling-sensitive 'C00562' than in chilling-insensitive 'UZB-GJG-1999-51' , regardless of the chilling treatment (Fig. 2B). Also, it was only significantly increased in chilling-sensitive 'C00562' after the chilling treatment, but not in chilling-insensitive 'UZB-GJG-1999-51' . Under chilling conditions, unsaturated fatty acids in cell membranes are usually converted to saturated fatty acids through the lipid peroxidation process by ROS 33 . In a comparison of pepper genotypes, chilling-insensitive 'UZB-GJG-1999-51' peppers are thought to be less prone to lipid peroxidation in chilling conditions because the palmitic acid level did not change even after the chilling treatment (Fig. 2B). On the other hand, chilling-sensitive 'C00562' has a rapid lipid peroxidation process as shown by a higher level of palmitic acid after chilling than chilling-insensitive 'UZB-GJG-1999-51' . The difference in the degree of lipid peroxidation between the two genotypes might be a major factor in determining the chilling sensitivity of the two genotypes. In addition to the metabolite analysis, transcriptome analysis was performed to confirm a specific chilling response mechanism in pepper seeds.
Through transcriptome analysis of seeds obtained from chilling-insensitive and sensitive peppers treated at 2 °C for 24 h, a total of 84,627 contigs were identified. Our results of multivariate analysis using all contigs (Supplementary Fig. S2) and hierarchical analysis (Supplementary Fig. S3) show clear separations by pepper genotypes as well as by the chilling treatment. The effect of chilling treatment could be confirmed more reliably through transcriptome analysis than by metabolite analysis.
JA is involved in chilling responses by controlling ROS through ERF transcription factors 34 . However, the period of the chilling exposure for the activation of the JA synthesis pathway varies by plant. In Arabidopsis, JA content increased by 3 times after 1.5 h of chilling treatment at 4 °C and JA biosynthesis genes such as LOX1_4, AOS, and AOC1_3 were significantly upregulated after 1.5 h of the chilling treatment 8 . Conversely, some crops require at least 24 h of chilling treatment to activate the JA synthesis pathway. For example, JA content in rice seedlings was increased by only 1.1 times after 1 day and increased by 2.0 times after 3 days of chilling treatment at 4 °C 35 . In addition, under a chilling treatment of 72 h or more, the expression levels of LOX, AOS, and AOC were significantly increased in Camellia japonica 'Nuccio's Bella Rossa' compared to the other cultivars 36 . www.nature.com/scientificreports/ In pepper fruit stored at 0 °C, the expression levels of CaLOX, CaAOS, and CaAOC were not different until 7 days of storage, but they were significantly different compared to the control after 14 days 37 . Similarly, in an experiment with C. annuum 'Cheongyang' stored at 2 °C for 25 days, CaLOX and CaAOC expression levels were not changed until 12 h, but the CaAOC expression level was significantly increased after 10 days during cold storage 5 . In this study, the expression levels of CaLOX1.5_2 and CaAOS3 were significantly increased after 24 h of the chilling treatment at 2 °C in both pepper genotypes (chilling-sensitive and insensitive peppers) (Fig. 5); however, the expression levels of CaLOX2.1 and CaAOC were significantly decreased after chilling. These results might imply that the JA synthesis pathway in peppers was not perfectly activated in our experiment condition of chilling at 2 °C for 24 h.
The expression level of CaJAR1 synthesizing JA-Ile, an active form of JA, was significantly increased in chilling-insensitive peppers but slightly increased in chilling-sensitive peppers (Fig. 5). Similarly, in Arabidopsis, the expression level of JAR1 was increased by 8.1 times after chilling treatment at 4 °C for 24 h compared to before the treatment 8 . JAR1 was more rapidly upregulated than LOX1 and AOC in Artemisia annua treated with chilling at 4 °C 38 . In summary, we could assume that expression levels of CaJAR1 in chilling-insensitive and sensitive peppers are very similar before chilling treatment but once pepper fruits are exposed to chilling, chilling-insensitive peppers increase the synthesis of JA-Ile, an active form of JA, by up-regulating CaJAR1 in response to chilling stress.
Among the ERF family genes regulated by JA signaling, the CaERF1, CaERF3_1, CaERF5, and CaERF10 expression levels were significantly increased by chilling treatment and found to be more significantly increased in chilling-insensitive genotypes. On the contrary, the expression levels of CaERF11 and CaDREB3 were significantly decreased in both genotypes by the chilling treatment (Fig. 5). ERF1, ERF3, and ERF5 have been known to increase their expression levels under chilling stress in Arabidopsis 39 and cotton 40 . In banana, MaERF10 positively regulates chilling tolerance 41 . DREB3 is also known to increase chilling tolerance in tomato 42,43 , but in this study we obtained the opposite result for CaDREB3. Therefore, further studies are needed to confirm whether the expression pattern of CaDREB3 is specific to pepper fruit.
The involvement of ERF11 in chilling responses has not been well reported. ERF11 has been known to promote internode growth by activating gibberellin 44 or to inhibit a stress response mechanism by antagonistically regulating ERF6 in the plant 45 . CaERF11 could be expected to play a role in suppressing the chilling tolerance of pepper fruit (Fig. 6).
In conclusion, the harvested pepper fruit in our study showed seed browning, which is a typical chilling symptom during cold storage or market distribution. Seed browning rates varied greatly according to pepper genotypes from chilling-insensitive (0.0%) to chilling-sensitive (over 60.0%). Integrating transcriptomic and metabolomics analysis clearly revealed differential metabolite accumulations and gene expressions between the chilling-insensitive and sensitive pepper genotypes. CaERF1, CaERF3_1, CaERF5, and CaERF10 were found to be the key genes positively contributing to the chilling tolerance of insensitive pepper genotypes by showing significantly increased expressions under chilling conditions. However, CaDREB3 and CaERF11 always showed significantly higher expressions and were therefore found to be the key genes negatively contributing to the chilling tolerance of chilling-sensitive pepper genotypes. To the best of our knowledge, this study is the first to report on the molecular mechanisms regulating seed browning occurrence in C. annuum pepper fruit. These results can be used as a foundation for breeding pepper cultivars with high postharvest quality that show resistance to chilling stress. However, further research is required to confirm whether these ERE genes play a regulatory role in the chilling sensitivity of peppers, by up-or downregulating key genes.

Materials and methods
Plant materials and chilling treatment. We obtained seeds from 36 genotypes of C. annuum from the National Agrobiodiversity Center, Jeonju, Korea (Supplementary Table S1). Pepper seeds were planted in a greenhouse in Suwon, Korea. The plants were managed according to standard practices and fruits were harvested at maturity, approximately 45-50 days after full bloom, depending on the genotype. Pepper fruits of uniform size and color were harvested by hand for the experiment. They were immediately transported to the laboratory and then precooled at 18 °C for 8 h. After precooling, the fruits were transferred to storage chambers for cold storage at 2 °C for 3 weeks under 90% relative humidity and dark conditions.
Chilling injury symptoms in each genotype were carefully observed and photographed after cutting each fruit in half, lengthwise. This was done weekly during cold storage at 2 °C for 3 weeks. The seed browning rate of each fruit was calculated by the following equation and an average rate of seed browning for each genotype was determined. Twenty fruits were used as biological replicates in each pepper genotype.
For transcriptome and metabolomics studies, we selected two different pepper genotypes to find key genes involved in a chilling-induced seed browning based on our screening data of seed browning rates (Supplementary Table S1). The variety 'UZB-GJG-1999-51' was selected as a chilling-insensitive pepper (seed browning rate 0.00%) and 'C00562' as a chilling-sensitive pepper (seed browning rate 63.86%). Both 'UZB-GJG-1999-51' and 'C00562' fruits were exposed at 2 °C for 24 h without cold storage for 3 weeks. Pepper fruits were sampled before chilling treatment at 2 °C (0 h) and after chilling treatment for 24 h at 2 °C (24 h). All seeds obtained were collected without placenta, then immediately frozen in liquid nitrogen and kept at − 80 °C for all experiments.
Chemical reagents. All  Free amino acids analysis. Free amino acids were analyzed following a previously described method 5,46 with some modifications. First, frozen pepper seeds were completely ground into a fine powder using a mortar and pestle in liquid nitrogen. Then, 1.2 mL of 5% trichloroacetic acid was added to 100 mg of pepper seed powder and the mixture was sonicated at room temperature for 30 min. After reaching a concentration of 16,000×g at 4 °C, 1 mL of the supernatant was collected and filtered through a 0.45 μm polyvinylidene fluoride membrane filter. After mixing 5 μL of 0.4 N borate buffer (pH 10.2) and 1 μL of sample, 1 μL of o-phthalaldehyde, and 1 μL of fluorenylmethyloxycarbonyl were added for derivatization. Finally, 64 μL of distilled water was added and analyzed by HPLC. The column was equipped with a Zorbax eclipse AAA (4.6 × 150 mm, Agilent, Santa Clara, CA, USA) and the flow rate was set to 2 mL min −1 . Untargeted polar phase metabolite analysis. Polar phase compounds were extracted following a previously described method 5,48,49 with some modifications. Fifty-mg of ground frozen pepper seeds was mixed with 1.2 mL of methanol and shaken at 75 °C for 30 min; then centrifuged at 13,500×g for 10 min. Next, 0.7 mL of the supernatant was transferred to a 2 mL-microtube and mixed with 0.5 mL of chloroform and 20 μL of ribitol (internal standard). Immediately after adding 0.7 mL of distilled water, it was centrifuged at 2500×g for 10 min. Then 0.5 mL of the supernatant was concentrated using a nitrogen evaporator (MG-2200, Eyela, Japan). Methoxyamine hydrochloride (50 μL) was added and incubated at 37 °C for 2 h. After incubation, 40 μL of sample and 100 μL of N-methyl-N-trifluoroacetamide were mixed and incubated at 37 °C for 30 min. Then, 1 µL of the sample was injected into the GC-MS ISQ LT system (Thermo Fisher Scientific, Waltham, MA, USA) using an auto sampler. The DB-5-fused silica capillary column (0.25 mm × 30 m × 0.25 μm, Agilent, Santa Clara, CA, USA) was used and the oven temperature was set to increase from 50 °C to 310 °C at a rate of 5 °C min −1 . The injector was in the split-less mode at 250 °C. Helium was used as the carrier gas at a flow rate of 1 × 10 -3 L min −1 . The range of mass scan was from 35 to 550 m/z.
Total RNA isolation, cDNA library construction, and sequencing. Frozen seeds were ground into a fine powder using a mortar and pestle in liquid nitrogen and 100 mg of powder was used for total RNA extraction. Total RNA was extracted using Ribospin Seed/Fruit Kit (GeneAll, Seoul, Korea) following the manufacturer's instructions. Extracted total RNA was used for RNA-seq and cDNA synthesis. cDNA was synthesized using an amfiRivert Platinum cDNA Synthesis Master Mix Kit (GenDEPOT, Baker, TX, USA) following the manufacturer's instructions.
Transcriptome analysis by RNA-Seq. Total RNA from 'UZB-GJG-1999-51' and 'C00562' peppers treated with chilling for 0 h and 24 h were used for RNA-seq. RNA-seq was performed using the Illumina Hi-Seq 2500 system (Illumina, San Diego, CA, USA) with 151-bp paired-end at the National Instrumentation Center for Environmental Management (NICEM), Seoul National University, South Korea. As a pretreatment process, artifacts such as adaptor sequence, contaminant DNA, and PCR duplicates, were removed from raw reads. Then, preprocessed reads were assembled and mapped to Pepper Zunla 1 Ref_v1.0 reference genome (GCF_000710875.1, NCBI).

Identification of DEGs.
To identify DEGs between the two different genotypes, 'UZB-GJG-1999-51' and 'C00562' pepper fruits were treated with chilling for 0 h or 24 h, at 2 °C. The expression levels of each read were calculated according to the fragment per kilobase of transcript per million mapped reads (FPKM) value. DEGs were selected using a threshold of ≥ twofold upregulated or downregulated genes with an FDR < 0.05 and all processes were performed by EdgeR software.

GO term and KEGG enrichment analyses for DEGs. Functional-enrichment analysis including GO
and KEGG were performed using Blast2GO 5.2 50