Metabolomic and Transcriptome Analysis of Phytohormone Metabolism in Hami Melon during Low-Temperature Storage

ZHOU Fake, TANG Fengxian, ZHANG Qin, SONG Wen, NING Ming, CAI Wenchao, SHAN Chunhui

(School of Food Science and Technology, Shihezi University, Shihezi 832003, China)

Abstract: Hami melon is vulnerable to chilling stress during postharvest storage. Phytohormones play an important role in regulating crop responses to chilling stress. In order to explore the molecular mechanism of endogenous phytohormone metabolism in Hami melon under chilling stress, the effects of different storage temperatures (3 and 21 ℃) on storage quality, endogenous phytohormone metabolism and gene expression levels in Hami melon were investigated by targeted metabolomics and transcriptomics. The results showed that low-temperature storage kept fruit mass and hardness at higher levels, delayed decay, and inhibited the increase of malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents, but also caused chilling injury to a certain extent. In addition, metabolomics analysis showed that low-temperature storage inhibited the accumulation of abscisic acid (ABA) in Hami melon fruit, but promoted the accumulation of indole-3-acetic acid (IAA) and salicylic acid (SA). Transcriptome analysis showed that low temperature significantly affected the expression levels of key genes (such as PYR/PYLs, IAAs, TIRs, GH3, NPRs, and TGAs) involved in the phytohormone signal transduction pathway, and the expression levels of some of the key genes (bHLH13, IAA9-like, IAA27, MYC2-like, and PYL4) were validated by real-time quantitative polymerase chain reaction (qPCR). These findings provide a theoretical basis for understanding the molecular mechanism of phytohormone metabolism during low-temperature storage of Hami melon.

Keywords: Hami melon; plant hormone; transcriptome; metabolomics; low-temperature storage

The Hami melon (Cucumis melovar. Saccharinus)is an economically important crop in Xinjiang province in northwestern China[1]. To prolong the shelf life and maintain the sensory quality of Hami melons, we selected the appropriate cold storage temperature according to the varieties of Hami melon. Previous studies have shown that the optimal temperature of Jiashi Hami melon was 3 ℃[2].However, since the Hami melon is sensitive to cold, the risk of chilling injury (CI) increases greatly during cold storage[3].Hami melons often show various cold-damage symptoms,such as spotting on the surface during cold storage, pitting of the pericarp, softening, and even rotting[4]. These injuries affect the appearance of Hami melons and impact consumer acceptance, resulting in huge economic losses. Hence, there is a practical and urgent need to understand the basis of these physiological effects and the molecular mechanisms involved in cold damage to increase melon resistance to chilling stress.

Plant hormones play a key role in regulating plant growth and development and responding to abiotic stresses.Complex hormone signal networks and crosstalk mechanisms make them ideal messengers to mediate defense responses[5-8].Several naturally occurring plant hormones, such as abscisic acid (ABA), indole-3-acetic (IAA), salicylic acid (SA), have been suggested to be the key players in the response of fruit to abiotic stress. A study has showed that exogenous ABA improved the resistance of peach fruit to low-temperatureinduced oxidative stress during cold storage[9]. In addition, the accumulation of endogenous ABA was helpful in reducing water loss and CI in zucchini fruit during cold storage[10].Research has shown that the overexpression of ABA receptors,PtPYRL1andPtPYRL5, significantly enhanced the resistance of poplar to low-temperature stress by positively regulating ABA signaling[11]. Exogenous application of IAA can improve the drought resistance of white clover by increasing the content of endogenous hormone ABA and up regulating auxin response genes (GH3.1,GH3.9,IAA8,etc.)[12]. A previous study has revealed that chilling stress up-regulated IAA production in cucumber seedlings,indicating that endogenous IAA was involved in the response to chilling stress. Postharvest application of SA can significantly extend the cold-storage period of “Kinnow”citrus fruit and reduce fruit mass loss, rotting, and loss of firmness[13]. Additionally, transcriptome analysis showed that cold treatment increased the expression of SA pathway marker genes,PR2andPR5, as well as the accumulation of hydrogen peroxide inArabidopsis thaliana[6]. However, the dynamic changes in the endogenous hormone levels and the mechanisms of hormone signal transduction in Hami melons under different storage temperatures are poorly understood.

In order to analyze the complex changes of gene expressions and hormone levels in Hami melon fruits under low temperature stress, this article studied the influence of different storage temperatures (3 and 21 ℃) on the storage quality of Hami melons. Targeted metabonomic analysis was used to detect the dynamic changes of plant hormones at different storage temperatures, and the transcriptome analysis method was used to identify the key genes of plant hormone signal transduction pathways that respond to low temperature stress.

1 Materials and Methods

1.1 Materials and reagents

Jiashi Hami melons were selected from Shihezi 121 farm(44°N, 86°E) in Xinjiang. The picking requirements were as follows: maturity 80% (soluble solid content (12.0 ± 0.5)%,firmness (152 ± 1) g), uniform size, clear reticulation, no disease,pest and mechanical damage. These melons were transported to the modified atmosphere storage of School of Food Science and Technology, Shihezi University, within 12 h.

UNlQ-10 Column TRIzol Total RNA Extraction Kit Sangon Bioengineering (Shanghai) Co., Ltd.; Fast SYBR Green Master Mix Applied Biosystems USA; Plant metabolite standards Sigma-Aldrich Inc. USA; RevertAid Premium reverse transcriptase Solarbio Science &Technology Co., Ltd. Beijing, China.

1.2 Instrumental and equipment

Texture analyzer Stable Micro Systems, UK; WD-2102A Multi-function microplate reader American Porton Instruments Co., Ltd.; 5500 QTRAP Mass spectrometer (MS)AB SCIEX Pte. Ltd. USA; LightCycler480 II Quantitative real-time polymerase chain reaction (qPCR) Roche,Rotkreuz, Switzerland; Acquity UPLC HSS T3 column(2.1 mm × 100 mm, 1.7 μm) Waters Corporation USA.

1.3 Methods

1.3.1 Sample preparation

Jiashi Hami melon was stored at 21 ℃ (control group)and 3 ℃ with relative humidity between 75% and 85%.Three parallel groups were made for each temperature, with 70 melons in each group. Among them, 35 melons were used to detect the basic storage indexes (rotting rate, mass loss rate, CI index), and then put back to the original place;the other 35 melons were used for sampling: we took about 5 mm×5 mm subcutaneous tissue from the equatorial region of Hami melon every 6 days, and 3 melons were taken as a parallel group each time to determine firmness, MDA content and H2O2content, and the rest samples were frozen in liquid nitrogen and then was stored in the refrigerator at –80 ℃for subsequent analysis (i.e. RNA sequencing, qPCR and metabonomics analysis, etc.).

1.3.2 Determination of physiological characteristics

The CI of Hami melon was graded according to the area of dent and spot on the surface[14]. The specific grading standards of CI are as follows: grade 0: no CI symptoms;grade 1: CI area ratio ≤ 25%; grade 2: CI area ratio between 25%–50%; grade 3: CI area ratio between 50%–75%; grade 4:CI area ratio ≥ 75%. The CI index was calculated using the equation (1).

wherenis the highest CI grade;Nis the total number of fruits.

The firmness of Hami melon pulp was determined using a previously described method[4]. The Hami melon was cut longitudinally, and then cross-cut into slices with a thickness of about 1.5 cm, then use a 1.6 cm diameter puncher to take a cylindrical pulp with a height of about 1 cm, and its firmness was measured by P/2 stigma on the texture analyzer.The parameters were set as follows: preloading speed was 5.00 mm/s, descending speed was 2.00 mm/s, ascending speed was 2.00 mm/s after puncture, pressing distance was 8.000 mm, trigger force was 10.0 g, each melon was measured five times, and the average value was calculated.

Rotting and mass loss rates were calculated using the equation (2) and (3), respectively.

The content of MDA was determined by thiobarbituric acid (TBA) method[15]. The absorbance was recorded at 532, 600 and 450 nm respectively. Results were expressed as nmol/g of fresh mass.

H2O2content was assayed as [15]described, with some modifications. Hami melon exocarp was ground in liquid nitrogen, then added 5.0 mL acetone, and centrifuged at 10 000 r/min for 15 min at 4 ℃. 1.0 mL sample supernatant was collected and mixed with 0.1 mL TiCl4solution and 0.2 mL concentrated ammonia for 5 min. After centrifugation at 4 ℃ 10 000 r/min for 15 min, the precipitated titanium dioxide complex was dissolved in 4 mL of 2 mol/L sulfuric acid. After dissolution, the absorbance was measured at 415 nm,and the results were expressed as μmol/g of fresh mass.

1.3.3 Targeted metabolomic detection of endogenous plant hormones

The contents of endogenous ABA, IAA, and SA in Hami melons stored at low temperature (3 ℃) and room temperature (21 ℃) for 0, 6, 12, 18, 24, and 30 days were absolute quantitatively detected. This experiment was mainly carried out by Shanghai Bioprofile Technology Company Ltd. (Shanghai, China) using the following methods.

1.3.3.1 Sample preparation and extraction

Samples were weighed before the extraction of metabolites, and dried lyophilized samples were ground in 2 mL Eppendorf tube containing a 5 mm tungsten bead for 1 min at 65 Hz in a grinding mill. Metabolites were extracted using 1 mL of a precooled mixture of methanol, acetonitrile, and water (2:2:1,V/V), then placed for 1 h in an ice bath with ultrasonic shaking. Subsequently, the mixture was kept at–20 ℃ for 1 h and centrifuged at 14 000 ×gfor 20 min at 4 ℃. The supernatants were recovered and concentrated by drying in a vacuum.

A 100 μL sample was thoroughly mixed with 400 μL of cold methanol acetonitrile (1:1,V/V) via vortexing. Then,mixtures were processed with sonication for 1 h in an ice bath, incubated at –20 ℃ for 1 h, and centrifuged at 4 ℃ for 20 min at 14 000 ×g. The supernatants were then harvested and dried in a vacuum for liquid chromatography-mass chromatography (LC-MS) analysis.

The metabolites were extracted from cell residue with 1 mL precooled methanol/acetonitrile/water (2:2:1,V/V) under sonication for 1 h in an ice bath. The mixture was incubated at –20 ℃ for 1 h followed by centrifugation at 14 000 ×g,then placed at 4 ℃ for 20 min and transferred to the sampling vial for LC-MS analysis.

1.3.3.2 UHPLC-MS analysis

The LC-MS portion of the platform was based on a Shimadzu Nexera X2 LC-30AD system equipped with an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.7 μm)and a triple-quadruple mass spectrometer. Metabolites were detected in electrospray negative-ionization and positive-ionization modes. The 2 μL?samples were injected sequentially with an LC autosampler. The ACQUITY UPLC HSS T3 column was heated to 45 ℃ under a flow rate of 300 μL/min. The gradient used to separate the compounds consisted of 10 mmol/L ammonium acetate at pH 8.0 (solvent A) and 100% acetonitrile (solvent B). The gradient started with 5% solvent A for 0.5 min and linearly increased to 65%solvent A over 6 min, then linearly increased to 98% solvent A over 2.5 min, followed by a 2 min hold before returning to the starting mixture over 0.1 min and re-equilibrating for 2 min. Quality contrl (QC) samples were injected every six or eight samples during acquisition.

The MS conditions were set as follows: source temperature: 550 ℃; ion source gas 1 (GAS1): 40; ion source gas 2 (GAS2): 50; curtain gas (CUR): 35; ion spray voltage floating (ISVF): 5 500 V for positive and –4 500 V for negative. The mass spectrometer was operated with a dwell time of 200 ms.

1.3.4 RNA sequencing analysis

RNA sequencing (RNA-Seq) was performed by BGI Shenzhen (Shenzhen, China) using Illumina HiSeq 500 platform. Total RNA from the pulp of Hami melons stored at 3 ℃ and 21 ℃ for 0, 12, and 24 days was used for cDNA library construction. Clean reads were mapped to reference genes and genomes using Software Bowtie 2[16]and HISAT[17]. The read numbers were transformed into the fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) value for gene expression quantification. The Possion distribution method[18]was used to screen differentially expressed genes (DEGs). TheP-value threshold was determined by controlling the false discovery rate (FDR). In this study, the criteria of |log2ratio| ≥ 1 and FDR ≤ 0.001 were set to search DEGs. Three biological replicates were used for each sample.

1.3.5 qPCR analysis

qPCR was used to test the expression ofbHLH13,IAA9-like,IAA27,MYC2-like,PYL4,PYL4-like,PYR1-like, andSTS14in Hami melon fruits. Total RNA was extracted by TRIzol total RNA Extraction Kit, and cDNA was synthesized using reverse transcriptase. The RNA sequence data were verified by qPCR analysis. Primer pairs for each gene(Table 1) were designed and synthesized by Sangon Biotech(Shanghai). qPCR was conducted using Fast SYBR Green Master Mix on LightCycler480 II qPCR instrument. The PCR conditions were as follows: 95 ℃ for 3 min, denaturation at 95 ℃ for 7 s, annealing at 60 ℃ for 10 s, elongation at 72 ℃ for 12 s, for a total of 45 cycles.GAPDHwas used to calculate the relative expression level of of each target gene through the formula of 2–ΔΔCt[19]. Three biological replicates were taken for all qPCR experiments.

Table 1 Sequences of specific primers used for q-PCR analysis

1.4 Statistical analysis

SPSS 25 software was used to analyze the data and evaluate?the?differences?between?the?treatment?groups.?Oneway?analysis?of?variance?(ANOVA)?was?used?for?significance?analysis.?Significant?difference?was?calculated?at?the?0.05?level.?Data?were?expressed?as?the?mean?value?±?standard?deviation.?Origin?2017?software?was?used?to?draw?bar?graphs?and?line?graphs.

2 Results and Analysis

2.1 Changes in phenotype and physiology under low- and room temperature storage conditions

At the beginning of storage (0 d), the appearance of the Hami melons was good and the surfaces were full and smooth (Fig. 1A). After 30 days of storage, Hami melons stored at low temperature (3 ℃) showed typical CI symptoms(pitting and browning) (Fig. 1B). Hami melons stored at room temperature (21 ℃) showed no chilling injuries, but the level of decay was much worse than in melons stored at low temperature (Fig. 1C).

Fig. 1 Appearance of Jashi-grown Hami melons stored for 0 and 30 days at different temperatures

CI index is often used in studying the cold tolerance of postharvest fruits. In the storage experiment, we observed that CI began to appear on Hami melons after 12 days of storage at 3 ℃, then increased in the subsequent storage period(Fig. 2A). Compared with room temperature storage, the low temperature effectively delayed and reduced the decay of Hami melons (Fig. 2B). In addition, low temperature storage maintained the mass of the fruit. The mass loss rate of the melons stored at 3 ℃ was significantly lower than that of the melons stored at 21 ℃ (Fig. 2C). Firmness of Hami melon pulp decreased during storage at both temperatures, but the firmness in the low temperature was higher than that in room temperature conditions (Fig. 2D). As shown in Fig. 2E,MDA content was higher in room-temperature-stored than cold-stored Hami melons. The results showed that H2O2content of the Hami melons initially increased, then decreased, reaching the peak on the 18th day at both storage temperatures. H2O2content of the melons was much higher at 21 ℃ than at 3 ℃ storage (Fig. 2F). The results indicated that low temperature would inevitably cause CI of Hami melon, but low temperature storage could also maintain the quality of Hami melon by inhibiting the content of MDA and H2O2, making the fruit firmness and mass higher than that of room temperature storage, thus delaying the decay of Hami melons.

Fig. 2 Changes in CI index (A), rotting rate (B), mass loss rate (C),firmness (D), MDA content (E), and H2O2 content (F) of Hami melons stored at 3 or 21 ℃

2.2 Changes in the endogenous plant hormone concentrations in Hami melons under low-temperature and room temperature storage

ABA content of Hami melons stored at low temperature was always lower than in those kept at room temperature during the entire storage period. However, it is worth noting that ABA accumulated in Hami melons stored at 3 ℃ with the increase of storage time (Fig. 3A). The pattern of change in IAA content was similar for the two storage temperatures: both groups showed a trend of an initial increase in IAA content and then a gradual decrease with storage time. The content of IAA peaked on the 12th day at both storage temperatures and then gradually decreased.However, unlike ABA, the IAA content in the 3 ℃-storage group was significantly higher than that in the control group.The results show that low temperature significantly promoted the synthesis of IAA (Fig. 3B). As shown in Fig. 3C, the SA content of Hami melons stored at 3 ℃ was lower than for those stored at 21 ℃ before 18th day. However, after 18 days,the SA content increased significantly as the 3 ℃ storage time increased and peaked on day 30. And on day 30, the SA content in 3 ℃ group was even higher in the control group.

Fig. 3 Contents of ABA (A), IAA (B), and SA (C) in Hami melons stored at 3 or 21 ℃

2.3 Analysis of DEGs involved in plant hormone signal transduction pathway in Hami melons response to postharvest cold storage

2.3.1 ABA signal transduction pathway

Because the contents of several endogenous hormones,including ABA, IAA, and SA, changed significantly in the Hami melons during postharvest low-temperature stress, we screened the RNA-seq data for key genes involved in plant hormone signal transduction pathways.

In the ABA signal transduction pathway, 27 genes displayed different expression patterns at 3 and 21 ℃(Table 2). Seven of these genes were ABA receptors (PYR/PYLs), most of which were significantly up-regulated at 3 ℃compared with the control. Five were protein phosphatase 2C (PP2C) genes that were up-regulated at 3 ℃ but downregulated at 21 ℃. Four were serine/threonine-protein kinase(SRK) genes, most of which were down-regulated at 3 ℃compared with the control. However, one gene (SRK2I)showed a different trend and was up-regulated at 3 ℃.Three genes encoding ABA-insensitive 5-like proteins(103495243, 103487883, 103491314) were down-regulated during the entire storage period compared with the control group. Transcription factorRF2b(103496745) was upregulated, while transcription factorVIP1(103500087) was down-regulated at 3 ℃ compared with the control group.

Table 2 DEGs involved in plant hormone signal transduction pathway

Table 2 (continued)

2.3.2 Auxin signaling transduction pathway

The expression of 45 genes showed different patterns at 3 and 21 ℃ in the auxin signal transduction pathway(Table 2). Among these genes, 11 were auxin-responsive protein IAAs, four genes of which (IAA14,IAA16,IAA26,and 103488319) were up-regulated at 3 ℃, while the rest(IAA6,IAA9,IAA13,IAA27(103501033, 103500598),IAA28,and 103487427) were down-regulated at 3 ℃ compared with the control group. The other three auxin-responsive protein genesSAURs(103497676, 103496295, 103498869) were down-regulated at 3 ℃ compared with the 21 ℃ group. Two transport inhibitor response 1 (TIR1)genes (103503672,103503674) were down regulated during the entire storage period compared with the control group. There were 12 auxin response factor (ARF) genes that down-regulated at 3 ℃ exceptARF19-like(103492260), which was up-regulated compared with the control group. Three were indole-3-acetic acid-amido synthetase genes,GH3.1(103484816),GH3.5(103485860), andGH3.6(103490488), which were downregulated at 3 ℃ compared with the 21 ℃ group. In addition,one gene (103484554) encodingCucumis meloprotein auxin signaling F-box 2-likewas significantly up-regulated at 3 ℃compared with the control group. Four genes (103501432,103495810, 103486311, 103503441) encoding auxin transporter-like proteins were down-regulated at 3 ℃ at 12 d compared with 21 ℃ at 12 d.

2.3.3 SA signaling transduction pathway

In this study, 13 genes showed differential expression levels associated with SA (Table 2). Of these, four were regulatory proteinNPRgenes, which were significantly upregulated at 3 ℃ at 24 d compared with at 0 d. TheSTS14gene (103497402) andTGAtranscription factors (TGA2,TGA6, andTGA7) were down-regulated at 3 ℃, while others,including transcription factorHBP-1b(c38)(103488155),TGA1(103494398),PSL11(103488443), and pathogenesisrelated genePRB1-2-like(103495329), were up-regulated at 3 ℃ compared with the control. Overall, these results indicate that SA signal transduction pathways were activated in response to low-temperature stress.

2.4 RNA-Seq expression validation by qPCR

In order to verify the accuracy of the RNA sequence data, we randomly selected eight genes involved in the plant hormone signal transduction pathway and used qPCR to detect their transcription levels. Among these genes, the expression levels ofIAA9(103488149),IAA27(103501033),STS14(103497402), andPYR1-like(103490785) were higher at 3 ℃ than at 21 ℃ (Fig. 4). The results show that the gene expression levels identified by RNA-Seq were basically consistent with those indicated by qPCR, confirming the reliability of the transcriptome results in this study.

Fig. 4 Relative expression levels of eight selected genes involved in plant hormone signal transduction

3 Discussion

As a simple and effective method of maintaining the quality of food, cold temperatures are often used to extend the storage period of postharvest fruits, such as Hami melons[3-4], apples[20], blueberries[21], peaches[22], and citrus fruit[23], However, low-temperature storage inevitably causes CI to the fruits, which greatly spoils the appearance and reduces storability and consumer acceptance[2,24-25]. In the present study, the fruit quality of ‘Jiashi’ Hami melons gradually changed after long-term cold storage. The CI of Hami melon stored at low temperature began to appear on the 18th day (Fig. 2A). Compared with the control group,low temperature storage reduced the rotting rate (Fig. 2B),mass loss rate (Fig. 2C) and maintained higher fruit firmness(Fig. 2D).The contents of MDA and H2O2gradually accumulated during fruit ripening. Membrane lipid peroxidation would damage cell membrane, and then lead to fruit decay[21]. In this study, MDA content of the room temperature-stored Hami melons increased quickly and was always higher than that of the cold-stored group, indicating that low temperature storage could effectively inhibit the increase of MDA and H2O2content, thus delaying the ripening and decay of fruit. These manifestations are consistent with the findings of previous studies[4].

ABA is a key regulator of the cold stress response during the postharvest cold-storage of fruit. Recent studies have shown that the ABA content of zucchinis increased after low-temperature storage[26]. However, low-temperature storage was found to reduce the concentration of ABA in citrus fruits[27]. This disparity may be a result of different regulatory mechanisms employed by the two species. In the present study, the ABA content of Hami melons stored at a low temperature was lower than in those stored at room temperature (Fig. 3A), which indicates that the low temperature inhibited the accumulation of ABA in the fruit.This result is consistent with the regulation mechanisms of Hami melons at room temperature. In addition, research has shown that ABA levels are significantly and negatively correlated with mass loss rate and CI rate but positively correlated with firmness[26]. However, we found ABA is significantly and positively correlated with the rotting and mass loss rates but negatively correlated with firmness(Date was not attached). ABA signal transduction modulates the cold stress response of ABA via the “PYR/PYLs-PP2Cs-SnRK2s-AREB/ABFs” pathway[28]. By studying the transcriptional changes of ABA response-related genes,we revealed aspects of the low-temperature perception and ABA signal transduction of Hami melons. The results show that 3 ℃ storage increased the expression of the receptorsPYL2andPYL12. A previous study showed that the heterotopic expression ofPYL3in rice enhanced the cold tolerance of transgenicArabidopsis thaliana[29]. In addition,the constitutive expression ofPtPYRL1andPtPYRL5significantly improved the cold resistance of poplar by actively regulating ABA signaling[11].

Previous studies have indicated that IAA is involved in chilling tolerance in cucumber seedlings[30]. Low-temperature storage can accelerate auxin signal transduction, delay senescence, and maintain the fruit quality of Powell (Citrus sinensis) fruits[29]. In our experiment, we found that lowtemperature storage significantly increased the content of IAA in Hami melons (Fig. 3B), which is consistent with previous research, indicating that IAA plays a key role in the process of Hami melon cold-stress resistance. In addition,we also found that levels of IAA were significantly and negatively correlated with H2O2content. Research has shown that several genes related to auxin signal transduction,includingAux/IAA,GH3,SAUR,andARFgene family members, are differentially expressed under abiotic stress conditions, which suggests there is crosstalk between auxin and abiotic stress signals[31]. In the present study,IAA14andIAA26were dramatically up-regulated at 3 ℃ storage for 24 days compared with at 0 days, andGH3.5,GH3.6,SAUR36, andARF19were down-regulated in melons stored at 3 ℃. In addition, theOsGH3-2gene was reported to increase the drought and cold tolerance of rice by regulating the dynamic balance of endogenous IAA and ABA[32], which concurs with the results of this study. The crosstalk between ABA and IAA plays a crucial role in the developmental process and environmental responses through a complex signaling network[33]. Interestingly, we found that the contents of IAA and ABA showed diametrically opposite trends,indicating that ABA and IAA may have antagonistic effects during the cold storage of Hami melons, which provides a new perspective for understanding the interactions of plant hormones.

SA is also considered to be essential for the postharvest preservation of fruits and vegetables and in responses to adverse environmental stress conditions, especially in reducing chilling damage in fruit, such as peaches[22],mandarins[34], and longan fruit[35]. The accumulation of endogenous SA has been reported to increase the cold tolerance of cucumber (Cucumis sativusL.) seedlings[36]. In this study, we found a significant increase in the SA content of Hami melons with increasing cold storage time after 18 d(Fig. 3C), indicating that SA was involved in the response of Hami melon to low temperature stress, and the accumulation of SA content was helpful to improve the resistance of Hami melon to low temperature stress. A previous study found that cold treatment increased the expression of the SA pathway marker genes,PR2andPR5, inArabidopsis thaliana[37]. In our work, we found that SA pathway is activated by cold stress, and many genes that play an active role in defense response, such asNPR3andNPR5, transcription factorTGAs,have significantly different expression under different storage temperatures (Table 2, Fig. 4).

4 Conclusion

The results showed that low temperature storage kept Hami melon fruits with higher mass and firmness, delayed the decay, inhibited the increase of MDA and H2O2content,but also inevitably caused a certain degree of CI. In addition,metabonomic analysis showed that the content of ABA in Hami melon fruits at low temperature was significantly lower than that in the control group, while the contents of IAA and SA were significantly higher than those in the control group.These results indicated that ABA, IAA and SA play important roles in the response mechanism of Hami melon fruits to low temperature stress. Furthermore, transcriptome analysis showed that the expression levels of key genes in ABA, IAA and SA signal transduction pathways under low temperature storage were significantly different from those under room temperature storage, which may be the key genes in the response of Hami melon fruits to low temperature stress.These findings provided new insights for understanding physiological metabolism and molecular mechanism of the response to low temperature stress in Hami melon.