Song et al BMC Genomics (2020) 21:390 https://doi.org/10.1186/s12864-020-06797-3 RESEARCH ARTICLE Open Access iTRAQ-based quantitative proteomics analysis of cantaloupe (Cucumis melo var saccharinus) after cold storage Wen Song, Fengxian Tang, Wenchao Cai, Qin Zhang, Fake Zhou, Ming Ning, Huan Tian and Chunhui Shan* Abstract Background: Cantaloupe is susceptible to cold stress when it is stored at low temperatures, resulting in the loss of edible and commercial quality To ascertain the molecular mechanisms of low temperatures resistance in cantaloupe, a cold-sensitive cultivar, Golden Empress-308 (GE) and a cold-tolerant cultivar, Jia Shi-310 (JS), were selected in parallel for iTRAQ quantitative proteomic analysis Results: The two kinds of commercial cultivars were exposed to a temperature of 0.5 °C for 0, 12 and 24 days We found that the cold-sensitive cultivar (GE) suffered more severe damage as the length of the cold treatment increased Proteomic analysis of both cultivars indicated that the number of differentially expressed proteins (DEPs) changed remarkably during the chilly treatment JS expressed cold-responsive proteins more rapidly and mobilized more groups of proteins than GE Furthermore, metabolic analysis revealed that more amino acids were up-regulated in JS during the early phases of low temperatures stress The DEPs we found were mainly related to carbohydrate and energy metabolism, structural proteins, reactive oxygen species scavenging, amino acids metabolism and signal transduction The consequences of phenotype assays, metabolic analysis and q-PCR validation confirm the findings of the iTRAQ analysis Conclusion: We found that the prompt response and mobilization of proteins in JS allowed it to maintain a higher level of cold tolerance than GE, and that the slower cold responses in GE may be a vital reason for the severe chilling injury commonly found in this cultivar The candidate proteins we identified will form the basis of future studies and may improve our understanding of the mechanisms of cold tolerance in cantaloupe Keywords: Cantaloupe, Cold storage, Proteomics, iTRAQ Background The cantaloupe (Cucumis melo var saccharinus) is rich in various nutrients and is one of the main economic crops of Xinjiang, China, where it plays an important role in promoting local economic development [1] Cantaloupe has a high sugar content and is susceptible to infection by pathogenic microorganisms [2], therefore, refrigerated storage is considered to be the most effective method for preserving the good quality of cantaloupe * Correspondence: 972338194@qq.com College of Food, Shihezi University, Xinjiang 832000, China during long-distance transport [3] However, cantaloupe is susceptible to cold damage during refrigeration, resulting in peel pitting and softening [4] Use of coldsensitive cultivars, longer storage times and lower temperatures are the major factors that contribute to the chilly injury As a major environmental stress, chilly stress affects plant growth and triggers a series of changes in many physiological and molecular processes [5] It results in electrolyte leakage, accumulation of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) and malondialdehyde (MDA), as well as changes in the levels © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Song et al BMC Genomics (2020) 21:390 of endogenous abscisic acid [6, 7], ethylene [8, 9] and soluble sugars [10] in plants The accumulation of ROS may result in oxidative stress, which damages the plant plasma membrane, decreases enzyme activity, and inhibits the rates of photosynthesis and protein processing [11, 12] Plant has evolved complex regulatory mechanisms to cope with cold stress Stress-responsive signaling pathways regulate the expression levels of several downstream stress-related genes in response to low temperature stress [13] Alongside this, non-enzymatic and enzymatic antioxidant systems participate in ROS scavenging to protect plant cells from oxidative damage [14] To understand how plant copes with abiotic stresses, previous studies have employed physiological and transcriptomics approaches [4, 15] Although transcriptome sequencing is a powerful method for identifying novel transcripts and analyzing gene expression at the transcriptional level, the changes in mRNA levels determined by transcriptomics not always correlate with corresponding proteins changes [16] In addition, the proteome of cantaloupe in response to cold remains largely unknown In our study, we applied proteomics technology to directly visualize the proteins being expressed in cantaloupe To better investigate cold response mechanisms in cantaloupe over a period of time, we used iTRAQ coupled to liquid chromatography-tandem mass A Page of 15 spectrometry (LC-MS/MS) to study two different cultivars (Golden Empress-308, GE; Jia Shi-310, JS) in parallel during cold treatments at 0.5 °C We found that the cold damage began to appear at 12 days, and, by 24 days it was more obvious and severe The findings of this study will lay a new foundation for the further investigation of cold tolerance mechanisms in cantaloupe, which may inform breeding programs and improve the commercial storage of this fruit Results Phenotypic changes in GE and JS under cold treatment After 12 days of cold storage, we observed several phenotypic changes in GE, but not in JS After 24 days, GE fruits were suffering more severe chilling injuries including rotting and bacterial infections, while JS fruits retained a higher quality with fewer injuries (Fig 1a) Cold stress resulted in increases in relative electrolyte leakage (REL) in both cultivars during 12 days of storage, which was higher in GE than in JS, but the RELs of both cultivars decreased after 24 days of storage (Fig 1b) Similar tendencies were also identified among the changes in malondialdeobhyde (MDA) and H2O2 levels (Fig 1b) Overall, our analysis confirmed that coldsensitive GE suffered a more severe cold damage, with higher levels of electrolyte leakage, lipid peroxidation, and H2O2 than JS during the early phase of cold storage B GE 90 a a JS b c b c REL/% 60 30 0d 12 d 24 d 60 a 50 a -1 40 b b b c 30 H 2O 20 10 0d 12 d 24 d 22 a 20 MDA Content/nmol.g-1 18 16 14 a 12 10 b b b c 0d 12 d 24 d Fig The phenotypic changes of GE and JS under cold treatment at different time points a, Representative pictures of untreated GE and JS fruits, and fruits exposed to 12 or 24 d of cold treatment; b, Comparison of REL, MDA content and H2O2 content in GE and JS fruits after cold treatment with the control The different letters represent that they are significantly different based on ANOVA (P < 0.05) Song et al BMC Genomics (2020) 21:390 Page of 15 down-regulated (Fig 2a) Similarly, we found 331 DEPs in JS after 24 days, of which 138 were up-regulated and 193 were down-regulated (Fig 2a) A higher number of DEPs were identified in JS than in GE at the early phase of cold treatment, but during the later phase of treatment, the number of DEPs in GE increased (Fig 2a) According to these observations, we assumed that the cold-tolerant cultivar JS responded faster than GE in terms of expressing cold-responsive proteins Rapid upregulation of proteins that regulate the response to the chilling stress and protect the plant cells from damage induced by ROS is important for cold tolerance Therefore, the delayed cold response in GE may be a critical reason for the severe chilling injury [17] Meanwhile, among the 807 DEPs in GE, 181 (22.42%) and 268 (33.20%) were specifically identified at the 12 and 24 day time points, respectively A further 179 (22.18%) DEPs were shared by both time points (Fig 2b) However, in JS, only 114 out of 722 DEPs (15.79%) were common to both time points, while 277 (38.37%) and 217 (30.06%) were specifically identified after 12 or 24 days, Identification and quantitation of DEPs by iTRAQ We compared protein levels in JS and GE before and after the cold treatment to identify differentially expressed proteins (DEPs) Using iTRAQ labeling LCMS/MS analysis, 5450 proteins were specifically identified from 107,101 LC-MS/MS spectra and 30,927 peptides in GE, and 5291 proteins were identified from 107, 048 LC-MS/MS spectra and 29,829 peptides in JS (Additional file 1: Table S1) We used ratio fold changes of > 1.200 or < 0.833 in expression (P < 0.05) as the cut-off points for upregulated and downregulated proteins, respectively, and found a total of 807 DEPs (12 days: 360; 24 days: 447) in GE, and 722 DEPs (12 days: 391; 24 days: 331) in JS, after cold treatment (Additional file 2: Table S2) After 12 days of cold treatment, we identified 360 DEPs in GE, 142 of which were up-regulated and 218 of which were downregulated (Fig 2a) In JS, there were 391 DEPs, of which 251 were up-regulated and 140 were down-regulated (Fig 2a) After 24 days, there were 447 DEPs in GE, 160 of which were up-regulated and 287 of which were A B C D d d 24 12 JS VS JS 0.50 K A GE 12d 0.00 G R O C JS X 12d W V J -0.50 -1.00 -1.50 P F 1.00 D T E M contrib I N B U H S JS 24d Q Cluster GE 24d 0.0 2.5 5.0 Dim1 (60.1%) 0d Cluster Cluster Cluster Cluster Cluster Cluster Cluster JS JS 0d VS VS 0d E 0d G G E VS G G E E 12 24 d d XP_008465603.2 (N) L 1.50 Dim2 (25.4%) XP_008462188.1 (I) XP_008437058.1 (S) XP_008455767.1 (U) XP_008444340.1 (W) XP_008448277.1 (X) XP_008458563.1 (C) XP_008463154.1 (V) XP_008447193.1 (K) XP_008445559.1 (P) XP_008464962.1 (G) XP_008449885.1 (O) XP_008454243.1 (B) XP_008459050.1 (M) XP_008448943.1 (E) XP_008455252.1 (D) NP_001284457.1 (T) XP_008446744.1 (J) XP_008446239.1 (R) XP_008462390.1 (F) XP_008454470.1 (A) XP_008460196.1 (L) XP_008458953.1 (Q) YP_004841791.1 (H) Fig Differentially expressed proteins (DEPs) in GE and JS after 0, 12 and 24 days of cold treatment a, The number of DEPs in GE and JS at different time points; b, Venn diagram of common and specific identified DEPs for GE and JS under cold treatment at different time points; c, Hierarchical clustering analysis of common expressed DEPs from GE and JS; d, Principal component analysis of common expressed DEPs from GE and JS Song et al BMC Genomics (2020) 21:390 respectively (Fig 2b) These findings demonstrate that much more different groups of proteins were mobilized in cold-tolerant JS under low temperature treatment [12] In order to identify the proteins that are most likely to be related to the acquisition of cold tolerance in cantaloupe, a careful analysis of common expressed DEPs was carried out The selected proteins with differential expression patterns were commonly expressed during the whole treatment period in both cultivars A hierarchical cluster analysis (HCL) was performed to analyze the correlations of common expressed DEPs in GE and JS after cold treatment Notably, the changes among 24 common DEPs were statistically significant and their abundance can be illustrated as seven clusters, revealing that two cultivars mobilized numerous proteins and differentially regulated their abundance to cope with cold stress (Fig 2c; Additional file 2: Table S2) Furthermore, the principal component analysis (PCA) we performed on the expression data above indicated that, in all conditions, the two cultivars presented different protein expression patterns The changes in protein expression between JS chilled at 12 and 24 days were smaller than those observed in GE, clustering close together with little separation in either axes (Fig 2d; Additional file 2: Table S2) In contrast, the changes in protein expression in GE between 12 and 24 days clustered away from each other Consequently, we speculated that, compared with JS, a longer duration of cold stress had a greater impact on the expression of proteins in GE, which may explain a more severe damage to GE during cold treatment [18] Primary functional classification of DEPs From the Clusters of Orthologous Groups (COG) database, we found that the largest group of DEPs are involved in ‘posttranslational modification, protein turnover, chaperones’ (119 DEPs), followed by ‘general function prediction only’ (94 DEPs) and ‘translation, ribosomal structure and biogenesis’ (83 DEPs; Fig 3a; Additional files 3: Table S3) The further analysis will be discussed below DEPs in response to the early phase of cold stress Using gene ontology (GO) analysis, the DEPs were classified into three categories: cellular components (CC), molecular function (MF) and biological processes (BP) [19] During the early phase of cold stress in both cultivars, the most highly represented categories were ‘cell’, ‘cell part’, ‘intracellular’, ‘intracellular part’ and ‘cytoplasm’ in CC (Fig 3b); ‘catalytic activity’, ‘binding’ and ‘heterocyclic compound binding’ in MF (Fig 3b); and ‘metabolic process’, ‘organic substance metabolic process’ and ‘cellular process’ in BP (Fig 3b) The results indicated that the majority of DEPs were involved in Page of 15 metabolic processes, cellular processes, cell and catalytic activities, suggesting the cold treatment mainly affected physiological metabolism and cell differentiation in cantaloupe (Additional files 4: Table S4) More intriguingly, all three categories of proteins were expressed at higher levels in JS compared with GE, revealing that, the proteome of cold-tolerant JS responds more rapidly to cold stress than that of cold-sensitive GE To further identify the roles of the DEPs, we performed KEGG pathway analysis Only significantly enriched categories with P-values < 0.05 were selected We found that cold stress affected ribosome, phagosome and phenylpropanoid biosynthesis in both cultivars Proteins involved with protein processing in the endoplasmic reticulum, plant-pathogen interactions and photosynthesis-antennae were highly enriched in GE compared with JS On the other hand, proteins involved in photosynthesis, galactose metabolism and cyanoamino acid metabolism, were considerably enriched in JS (Table 1; Fig 4a, c; Additional files 6: Table S5) More interestingly, there were conspicuous protein-protein interactions among ribosome and other functions (P < 0.05) (Fig 5a, c) Thus we speculate that ribosome related DEPs may play a significant role in regulating the metabolic mechanisms in cantaloupe at the early phases of cold stress DEPs in response to the later phase of cold stress As above, during the later phase of the cold stress, proteins were characterized by ‘cell’, ‘cell part’, ‘intracellular’, ‘intracellular part’ and ‘cytoplasm’ in CC (Fig 3c); ‘catalytic activity’, ‘binding’ and ‘heterocyclic compound binding’ in MF (Fig 3c); and ‘metabolic process’, ‘cellular process’ and ‘organic substance metabolic process’ in BP (Fig 3c) After 12 days of cold storage, all three categories were dramatically higher in GE compared with JS, indicating that GE experienced greater levels of cold stress as the length of storage increased (Additional files 4: Table S4) The tardiness of the cold response in GE may be a critical reason for its severe cold damage KEGG pathway analysis indicated that protein processing in the endoplasmic reticulum and galactose metabolism may be affected in both cultivars after cold stress Proteins involved in ribosomes, carbon fixation in photosynthetic organisms, plant-pathogen interactions, one carbon pool by folate, and phenylpropanoid biosynthesis were highly enriched in GE while proteins involved in photosynthesis-antennae, phagosomes, amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, pentose and glucuronate interconversions, and linoleic acid metabolism were enriched in JS (Table 1; Fig 4b, d; Additional files 5: Table S5) Like the hallmark of the protein-protein interaction at 12 days, there was still a significant interaction among in tr ac ga n cy cytmem elle t o o s b ce p o r ll no in lasmlic ane np t m em pl race ribic part as ll o a b tid ul so rt ribran ar m t i h e in on e-b tr uc o ntrayla par 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mrom e s i s p ac ol onllul e to muess ro ec s ar s lu m ul p e t p tr s ox olece bi met ho o st rocess re id u os ab to im es sp at le yn o sy u s on io m th lic nt lu se n-r eta et pr hes s to ed bo ic p oc is ch uc lic ro es em tio pr ce s ica n p oc ss l s ro ess tim ce ul ss us ell ul ar or Number of proteins cy t op oso li r cy otei rib c pa t o n o s rt pl co om as m e pl as mic plex tid pl p pa as in thy last rt tid tr la id ac k o th yla or ellu id k ga la in oid ne r t ch racemem clle in llu br ell ph loro tr la a ot ac os plas memr p ne ell a y t in ul tr th nth p thy bra rt ar ac yl et la la n no i e a s l k c t k e n- m m e lula oid memid poid em m r m a br bra org embra rt an b ne an ne e r e- - e bo nc cy lle ane un lo to pa s p r or ded ed l las t no ga or um m nne ga e m lle ne n em l l br c um le an th th ell en yla y pa ein o b ko lak rt tr rg int ou ac an nd i o ell ell cel ed cd paid ul e s lu o yt rt ar ub lar rg os rib co o r a n o l on m ga ell p n e uc c leo hl ch artmelle pr oro lor e str ot pl op nt uc e a tu or in c st plast ga om a r l nu mo nell ple t l strhet c e lei cu e pa x uc ero t u cy ca c ac le a rt cl ta id cti l c ic lyt bi vi o n co ic nd ty sin st m a in gl e o s ituepou b ctiv g o nt nd in ity rg ing o r rg a a l o b di ga n ni ic s nisme or f ribind ng cs u i g ub bst b me ani oso ng m st an io ta sm e re anc ce msyntboli pr e sp b e he c p oc on io ta ti r es se sy bo c p oce s to nth lic ro ss a e p ce re bio tic proc ss m sp tic r es ac o s o r r es cel nse stim ces po lu to u s m om ac ol ns lar s lus ro ecu e t p t re m le m ph o ro s o ole b e o st ce s re xid cul iosytabotosy imu ss sp at e m nt li nt lu on ion e he c p he s se -r ta tic ro sis to ed bo p ce ch uc lic roc ss em tio pr es ica n p oce s l s roc ss tim es ul s us rib on uc le Number of proteins Song et al BMC Genomics (2020) 21:390 A U V T S R M 160 200 Page of 15 70 60 50 40 30 20 10 -10 -20 -30 A L B C N A: RNA processing and modification O K B: Chromatin structure and dynamics D C: Energy production and conversion E: Amino acid transport and metabolism P D: Cell cycle control, cell division, chromosome partitioning E Q F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism F H: Coenzyme transport and metabolism G H I J GE 12d GE 24d JS 12d JS 24d Cellular Component Cellular Component ribosome and other functions in GE, while dramatic changes happened in JS (Fig 5b, d) This further demonstrates that various proteins were mobilized in JS during the cold treatment and that there was a positive relationship between the diversity of proteins and cold tolerance I: Lipid transport and metabolism J: Translation, ribosomal structure and biogenesis K: Transcription M: Cell wall/membrane/envelope biogenesis L: Replication, recombination and repair N: Posttranslational modification, protein turnover, chaperones O: Inorganic ion transport and metabolism P: Secondary metabolites biosynthesis, transport and catabolism R: Function unknown Q: General function prediction only S: Signal transduction mechanisms T: Intracellular trafficking, secretion, and vesicular transport U: Cytoskeleton V: Defense mechanisms B Molecular Function Molecular Function Biological Process GE JS 120 80 40 C 160 Biological Process 120 80 40 Fig Function classification of the DEPs a, COG function classification of DEPs in GE and JS; b, GO annotation of DEPs in GE and JS during the early phase of cold treatment; c, GO annotation of DEPs in GE and JS in the later phase of cold treatment Functional distribution analysis of cold induced proteins in JS Based on GO analysis, functional distribution analysis were performed and the DEPs identified in cold-tolerant JS after 12 days cold treatment were selected as the cold induced proteins [20] (Additional files 4: Table S4; Song et al BMC Genomics (2020) 21:390 Page of 15 Table KEGG pathway analysis of proteins in GE and JS during chilling stress at different time period Pathway name Pathway ID Ribosome Number of proteins GE ko03010 JS 12 d 24 d 12 d 24 d 34 42 38 – Phenylpropanoid biosynthesis ko00940 18 11 13 – Protein processing in endoplasmic reticulum ko04141 25 32 – 21 Photosynthesis - antenna proteins ko00196 – – Plant-pathogen interaction ko04626 12 11 – – Galactose metabolism ko00052 – 10 10 Carbon fixation in photosynthetic organisms ko00710 – 11 – – One carbon pool by folate ko00670 – – – Photosynthesis ko00195 – – 11 – Phagosome ko04145 – 11 12 Cyanoamino acid metabolism ko00460 – – – Amino sugar and nucleotide sugar metabolism ko00520 – – – 11 Fructose and mannose metabolism ko00051 – – – Pentose and glucuronate interconversions ko00040 – – – Linoleic acid metabolism ko00591 – – – B A Ribosome Ribosome 10 10 20 20 30 Pval e 40 Pathway Pathway 30 Phenylpropanoid biosynthesis Pval e 0.005 0.04 0.004 Phenylpropanoid biosynthesis 0.003 0.03 0.002 0.02 0.001 0.01 Phagosome 0.2 0.4 0.150 0.6 0.175 0.200 0.225 0.250 D C Pval e Ribosome 10 Photosynthesis 0.03 20 0.02 Phagosome 30 Pval e Phagosome Pathway Pathway 0.01 Phenylpropanoid biosynthesis 0.020 0.015 0.010 10 0.005 15 20 0.18 0.21 0.24 0.2 0.4 0.6 Fig KEGG pathway enrichment a, KEGG pathway enrichment in GE after 12 days of cold treatment; b, KEGG pathway enrichment in GE after 24 days; c, KEGG pathway enrichment in JS after 12 days; d, KEGG pathway enrichment in JS after 24 days Song et al BMC Genomics A (2020) 21:390 Page of 15 B XP_008444852.1 XP_008450738.1 XP_008461617.1 XP_008461390.1 XP_008463526.1 XP_008452210.1 XP_008447742.1 XP_008447825.1 XP_008455742.1 XP_008461363.1 XP_008437779.1 XP_008442779.1 XP_008439326.1 XP_008436991.1 XP_008450608.1 XP_008450453.1 XP_008438796.1 XP_008456396.1 XP_008460657.1 XP_008461261.1 XP_008452633.1 XP_008446809.1 XP_008462084.1 XP_008441387.1 XP_008453374.1 XP_008462521.1 XP_008459494.1 XP_008450136.1 XP_008438176.1 XP_008446192.1 XP_008441387.1 XP_008463223.1 XP_008448659.1 XP_008461018.1 XP_008441351.1 XP_008444383.1 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XP_008451565.1 XP_008442648.1 XP_008461261.1 XP_008446578.1 XP_008465452.1 XP_008453374.1 XP_008446109.1 XP_008450536.1 XP_008443423.1 XP_008456139.1 XP_008457143.1 XP_008456170.1 XP_008450536.1 XP_008446732.1 XP_008446109.1 XP_008439282.1 XP_008449776.1 XP_008454987.1 XP_008461924.1 XP_008439282.1 XP_008445911.1 XP_008443471.1 XP_008447950.1 XP_008453813.1 XP_008443634.1 XP_008447950.1 XP_008459498.1 XP_008452205.1 XP_008466112.1 XP_008451468.1 XP_008443553.1 XP_008449773.1 XP_008445323.1 XP_008448860.1 XP_008449771.1 XP_008463154.1 XP_008456761.1 XP_008463154.1 XP_008448860.1 GAS1 XP_008452767.1 XP_008449500.1 XP_008452767.1 XP_008454987.1 XP_008452205.1 C D XP_008451468.1 XP_008438517.1 XP_008444460.1 XP_008442483.1 XP_008464962.1 XP_008448041.1 XP_008452205.1 XP_008444814.1 XP_008454421.1 XP_008443553.1 XP_008443526.1 XP_008454491.1 XP_008437269.1 XP_008454421.1 XP_008464962.1 XP_008449885.1 aga2 XP_008458587.1 XP_008446334.1 XP_008453374.1 XP_008445497.1 XP_008442483.1 XP_008455603.1 XP_008454987.1 XP_008445559.1 XP_008448040.1 XP_008463154.1 XP_008461463.1 XP_008457837.1 XP_008465194.1 XP_008437664.1 XP_008447450.1 XP_008446192.1 XP_008467065.1 XP_008440392.1 XP_008452010.1 XP_008453835.1 XP_008449885.1 XP_008448943.1 XP_008456611.1 XP_008461453.1 XP_008459233.1 XP_008447584.1 XP_008440181.1 XP_008447511.1 XP_008456941.1 XP_008466451.1 XP_008459377.1 XP_008451826.1 XP_008450816.1 XP_008451369.1 XP_008454823.1 XP_008455742.1 XP_008467320.1 XP_008466710.1 XP_008462725.1 XP_008458496.1 XP_008448943.1 XP_008450536.1 XP_008441854.1 XP_008446013.1 XP_008449286.1XP_008463199.1 XP_008445491.1 XP_008450738.1 XP_008451956.1 XP_008439282.1 XP_008442676.1 XP_008437269.1 XP_008459498.1 XP_008461924.1 XP_008445559.1 XP_008452767.1 XP_008456347.1 XP_008462084.1 XP_008449241.1 XP_008452292.1 XP_008446109.1 XP_008453995.1 XP_008441527.1 XP_008450298.1XP_008453211.1 XP_008443426.1 XP_008450453.1 XP_008442676.1 Fig PPI analysis of DEPs based on KEGG pathway enrichment a, PPI in GE after 12 days (Different node colors show the types of enrichment according to P-value: ribosome in red, phenylpropanoid biosynthesis in blue, protein processing in endoplasmic reticulum in green, photosynthesis-antenna proteins in yellow, plant–pathogen interactions in pink, phagosome in dark green); b, PPI in GE at 24 days (Different node colors show the types of enrichment according to P-value: ribosome in red, protein processing in endoplasmic reticulum in blue, galactose metabolism in green, carbon fixation in photosynthetic organisms in yellow, plant–pathogen interactions in pink, one carbon pool by folate in dark green, phenylpropanoid biosynthesis in light blue); c, PPI in JS after 12 days (Different node colors show the types of enrichment according to P-value: ribosome in red, photosynthesis in blue, phagosome in green, phenylpropanoid biosynthesis in yellow, galactose metabolism in pink); d, PPI in JS after 24 days (Different node colors show the types of enrichment according to P-value: photosynthesis-antenna proteins in red, phagosome in blue, galactose metabolism in green, protein processing in endoplasmic reticulum in yellow, amino sugar and nucleotide sugar metabolism in pink, fructose and mannose metabolism in dark green, linoleic acid metabolism in light blue) STRING tool (http://string.embl.de/) was used to predict protein–protein interaction networks Additional files 6: Fig S1) In terms of cellular components, membrane, cell part, cell, and organelle proteins were significantly enriched in JS (P < 0.05) In the molecular function category, proteins with catalytic activity and binding were the most positively regulated (P < 0.05) In biological processes, proteins involved in cellular processes, metabolic processes and organic substance metabolic processes were the most highly enriched (P < 0.05) ... phase of cold treatment Functional distribution analysis of cold induced proteins in JS Based on GO analysis, functional distribution analysis were performed and the DEPs identified in cold- tolerant... (Fig 3c) After 12 days of cold storage, all three categories were dramatically higher in GE compared with JS, indicating that GE experienced greater levels of cold stress as the length of storage. .. the metabolic mechanisms in cantaloupe at the early phases of cold stress DEPs in response to the later phase of cold stress As above, during the later phase of the cold stress, proteins were