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. 2020 Aug 19;58(6):2246–2257. doi: 10.1007/s13197-020-04735-5

Transcriptional and metabolite analysis reveal a shift in fruit quality in response to calcium chloride treatment on "Kyoho" grapevine

Weihong Fu 1, Mengwei Zhang 1, Peian Zhang 1, Zhongjie Liu 1, Tianyu Dong 1, Saihang Zhang 1, Yanhua Ren 1, Haifeng Jia 1,✉, Jinggui Fang 1
PMCID: PMC8076366  PMID: 33967321

Abstract

‘Kyoho’ grapevine (Vitis vinifera) treated by calcium ions solution has been proved as an effective treatment to extend grape quality during storage to reduce disease, but its molecular mechanism was not clear yet. In the current work, grape berries were treated with different concentration of Calcium chloride (CaCl2) solution, and their effects on antioxidant enzyme activity and transcriptome and metabolome in fruit were investigated. CaCl2 treatments reduced weight loss and inhibited the decrement of flesh firmness. 80 mM CaCl2 significantly increased the activity of antioxidant enzymes POD, SOD and CAT, which was the optimum experimental concentration. The study showed that the expression level of heat shock transcription factor and UBX which involved in endoplasmic reticulum stress and degradation pathway increased significantly. Moreover, the corresponding metabolites, such as heat shock protein and organic acid, also increased significantly. The misfolded proteins are transported to the cytosol for degradation, so that the preservation ability of grape is improved.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04735-5) contains supplementary material, which is available to authorized users.

Keywords: Grapevine, Calcium chloride, Transcriptome, Metabolome, Fruit preservation

Introduction

Calcium plays a significant role in plant growth and development. Calcium ion is now firmly established as a second messenger molecule involved in regulation of diverse cellular functions.

It conveys a wide range of extracellular signals and environmental stimuli such as abiotic and biotic stress and light factors to appropriate physiological responses, which are termed as calcium signatures. Calcium signal can be decoded to affect gene transcription and participate in growth of root hair, the fertilization event, pollen tube growth, stomatal closure, and responding to environmental stress (Reddy and Day 2011). Calcium protects the cell wall of plant from the degradation of enzymes by binding pectins (Ranjbar et al. 2018), slows down mushroom browning by improving membrane integrity, and delays membrane lipid catabolic processes to delay the decline of apple quality (Picchioni et al. 1998).

Fruits lacking of Ca2+ are more susceptible to decay processes and begin with water soaked tissue, followed by damage to tissue integrity and dehydration, which eventually causes dark brown depression on fruit surface. These symptoms can be alleviated by exogenous calcium application. Calcium can control pathogenic bateria on jujube fruit by improving the biocontrol efficiency of yeast (Guo et al. 2016). Calcium treatment helps strawberry resist fungal pathogen Botrytis cinerea in vitro and in vivo growth (Langer et al. 2019). Calcium can also improve cell integrity as a secondary messenger in hormone signaling pathway so as to decrease the rising of rot index (Correia et al. 2019). CaCl2 is the main inorganic Ca salt that promotes fresh-keeping and storage of fruits after harvest due to its high efficiency (Youryon et al. 2018).

Grape (Vitis vinifera) is a popular fruit with high economic value all over the world, it can be consumed as table grape, raisins, fruit juice, wine and so on. During storage, fruits produce off-flavor in response to surface blackening, rotting, microbial attack, and it can even be linked to postharvest shelf life and economic value (Lu et al. 2018). In general, rapid postharvest deterioration of table grapes is partly due to the nutrient leakage from the tissues and water activities which make them susceptible to fungal decay (Lichter et al. 2002). Besides, table grape is a non-climacteric fruit which is prone to postharvest rotting because of irreversible processes such as ethylene biosynthesis, mechanical damage and oxidative damage (Wang et al. 2019). A series of externally applied procedures and explorations have been taken by means of refrigeration, controlled atmospheres (CA), modified atmosphere packaging (MAP), and Micro-perforated film packing (MPFP) to extend the storage life of grapes (Lu et al. 2018). ‘Kyoho’ grape is rot-prone during storage and transportation because of its soft and juicy flesh as well as loose clusters. Therefore, it is urgent to regulate grape berry mature process and improve its postharvest quality.

The aim was to provide a method of freshness preservation of ‘Kyoho’ grape. In our study, 20, 80 and 500 mM CaCl2 solutions were used as Ca donors to testify the effects of different concentrations of Ca on the storage quality of grapes. Our objectives were to (1) verify the hypothesis that calcium chloride can delay the senescence of grape berry, (2) find out the best calcium chloride concentration for grape storage, (3) shed light on the metabolic pathways and candidate genes underlying the postharvest storage, (4) profile the transcriptome and metabolome changes in grape during various storage time to investigate the maturation mechanism.

Methods

Plant materials and quality determination

‘Kyoho’ grape berries with 90% maturity were randomly divided into 4 groups, each group contained 20 berries of uniform size without mechanical damage. CaCl2 treatment groups (CT) were soaked in CaCl2 solution (mixed with 0.1% Tween 20) with 20 mM, 80 mM and 500 mM, respectively, while CK soaked in pure water for 10 s. All samples were sprayed with Botrytis cinerea then stored in greenhouse under 25 °C, samples were taken once every 2 days. After flushing the surface of grape with plenty of ddH2O and dividing the peel and pulp, they were frozen with liquid nitrogen quickly and stored at − 80 °C for subsequent tests.

Hardness was determined by fruit hardness tester (Edinburgh, GY-4), total soluble solids–TSS (%) was determined by direct reading with pocket refractometer (ATOGO, PAL-1); titratable acidity–TA was measured by titration with 0.1 M NaOH and TSS/TA ratio. The data were analyzed of variance and regression by DPS 9.5 software and the means were compared by the Tukey test (p < 0.05).

Scanning electron microscopy

Samples stored for 2 days were fixed at room temperature with glutaraldehyde of 3–4% volume fraction for 4–6 h, washed with phosphate buffer (PBS, pH 6.8) for 4–6 times at intervals of 20–30 min, then dehydrated gradient with series of acetone (volume fraction 30%, 50%, 70%, 80%, 90%, 95%, and 100%) at intervals of 30 min, of which 100% acetone dehydrated three times and pureed by acetic acid. Isoamyl ester was replaced twice, 30 min each time. The critical point of CO2 was dry, sticky and coated. The samples were observed and photographed under JSM-6360LV scanning electron microscope (Soylu et al. 2005).

Determination of antioxidant enzyme activity

The activity of SOD was determined by nitrous blue tetrazole (NBT) method. To obtain homogenate, 1.6 mL extract was added to 0.2 g tissue. The activity of SOD was calculated by the activity unit (U) of 50% NBT photoreductase at 560 nm. Total SOD activity was expressed by fresh weight enzyme unit per gram (U/g) (Peng et al. 2018).

The activity of POD was determined by Solarbio's POD activity detection kit (BC0090). The homogenization (0.1 g tissue was added 1 mL extract) was centrifuged at 4 °C for 10 min under 8,000 g, the supernatant was used to determine the activity of enzymes. The activity of POD was calculated with 0.01 change of A470 per min per g tissue in each mL reaction system as a unit of U. Units were expressed by U/g.

The activity of CAT was determined by using Solarbio's CAT activity detection kit (BC0200). The extraction method was the same as POD above, and the supernatant was used to determine enzyme activity. The degradation of 1 nmol H2O2 per g of tissue per min in the reaction system was defined as a unit of enzyme activity. Units were expressed by U/g.

RNA preparation, cDNA library construction and Illumina sequencing

Peeled pulp was ground into powder in liquid nitrogen, total RNA was extracted by Hexadecyl trimethyl ammonium Bromide (CTAB) method (Zhihua et al. 2004). RNA degradation and contamination were monitored on 1% agarose gels. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). Three biological replicates from each treatment were used for RNA-sequencing. The libraries were sequenced on the Illumina HiSeq platform and 125 bp/150 bp paired-end reads were generated (Novogene). Illumina sequencing was performed at novogene (www.novogene.cn). The clean reads were aligned to the reference genome of Vitis vinifera (https://genomes.cribi.unipd.it/DATA/V2/) by HISAT2 v2.0.4. HTSeq v0.9.1 was used to count the reads numbers mapped to each gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, was used for estimating gene expression levels. The genomic localization results of all reads data were assembled using Cufflinks (Ghosh and Chan 2016). Details of Illumina sequencing and assembly were showed in Table 1.

Table 1.

The significantly enriched pathways for DEGs in grape berry

Pathway Pathway ID Input number Background number P value Corrected P-value
Protein processing in endoplasmic reticulum vvi04141 74 222 1.62E-05 0.001943762
Ribosome biogenesis in eukaryotes vvi03008 33 87 0.000601702 0.036102134
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Differential expression genes (DEG) analysis

Differential expression analysis between CT and CK was performed using the DEGSeq R package (1.18.0). The resulting P-values were adjusted using the Benjamini and Hochberg’ s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed. The FPKM value > 1 was used as a threshold for judging whether or not the gene was expressed.

Gene ontology and Kyoto encyclopedia of genes and genomes analysis

Gene Ontology (GO) enrichment analysis was performed by GOseq R package, in which gene length bias was corrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) can identify the most important biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes through Pathway significant enrichment. Pathway enrichment analysis was performed using KOBAS.

Quantitative real-time PCR (qRT-PCR)

The cDNA was synthesized using the Prime Script RT reagent Kit (Perfect Real Time) (TaKaRa). The reaction mixture (20 μL) contained 10 μL SYBR Premix Mixture, 0.4 μM each of the forward and reverse primers, 1 μL of template cDNA and made up to 20 μL with ddH2O. PCR amplification was performed under the following conditions: 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 20 s and 72 °C for 30 s. All primers used in this study were listed in Table S1. The test was set up for 3 repetitions, and the fluorescence value change curve and the melting curve were analyzed after the reaction completed, and the test data was analyzed by the 2−ΔΔCT method. Primer sequence used for qRT-PCR was shown in Table S3.

Metabolites extraction and metabonomic profile of grape pulp

LC–MS-based metabonomic profile was performed with extracts of grape pulp (Novogene, Beijing, China). The frozen pulp was ground using a bead beater, 100 mg powder samples were placed into 2 mL EP tubes and then 500 μL of 80% methanol solution containing 0.1% formic acid was added, vortexed and shaked, incubated at 4 °C for 5 min, centrifuge for 15 min at 15 °C, 12,000 g. The supernatant was diluted with mass spectrometry water to a methanol content of 53%, the mixture was centrifuged for 15 min at 4 °C, 12,000 g. Supernatant was transferred into a fresh 2 ml LC/MS glass vial for LC–MS analysis.

The scan range was selected as m/z 70–1050; ESI source conditions were set as follows: Sheath gas flow rate 35arb, Aux Gas flow rate 10arb, source temperature 320 °C, Spray Voltage 3.2 kV. Polarity was in positive or negative modes, respectively. Data-related scan was the second. Raw date was imported, the retention time and mass/load ratio were screened, and then peak comparison was conducted for different samples according to the retention time deviation of 0.2 min and mass deviation of 5 ppm. Peak extraction was performed after setting parameters. The blank samples were used to remove the background ions, and the quantitative results were normalized to obtain the qualitative and quantitative results.

The metabonomic profiles between groups were evaluated with independent component analysis. PCA, all metabolites identified were recombined into a new set of comprehensive variables, which showed a trend of separation between CT and CK. The smaller the QC sample difference, the closer the distribution of QC samples in the PCA analysis chart was, the better the stability of the whole method, the higher the data quality was. The QC samples were shown to be very close, and the CT and the CK could be well distinguished (Fig S2). These showed that the results of the metabolome analysis were quite reliable.

The comprehensive analysis of metabolites and transcriptome included the correlation analysis of differential metabolites and differential genes and KEGG analysis. For correlation analysis, the data needed to be standardized to eliminate the influence of the order of magnitude. The relevant icons were created aiming at the first 50 differential metabolites and the first 100 differential genes. KEGG analysis was mainly to capture the common pathway of the differential metabolites and genes. Python 2.7.6 and R-3.4.3 were used for data processing and chart making.

Results

Quality index of control and CaCl2-treatment grape berries

Effect of CaCl2 treatment on grape preservation was shown in Fig. 1. The fruit epidermis gradually wrinkled, dehydrated and the pulp became soft with the increase of storage time. Control group began to rot on the 12th day. However, the CaCl2-treatment group was spoiled later than the control group, and the grape preservation period was extended. On the 20th day, the grape berries in the 80 mM group still maintained a relatively complete appearance.

Fig. 1.

Fig. 1

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Appearance of grape fruit in the control group and CaCl2 treatment group within 20 days

In the CaCl2-treatment group mycelial growth of Botrytis cinerea was disordered, most of them shrivelled, the overall growth rate slowed down or even no longer grew. By contrast, in control group the Botrytis cinerea grew well and mycelia were slender, straight, uniform in thickness and smooth in line. The surface of mycelia was smooth, and the growth point was thin and well extended (Fig. 2). These showed that CaCl2 inhibited the Botrytis cinerea grew healthy on the grape.

Fig. 2.

Fig. 2

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SEM of Botrytis cinerea on grape skin. Samples were labeled as: A. control, B. 20 mM CaCl2, C. 80 mM CaCl2 and D. 500 mM CaCl2

Grape is a kind of non-climacteric fruits with low physiological activity. The loss of water in the fruit will lead to softening and shrinking, and further fruit stalks will wither, which will make the grape lose commercial value. The change of grape quality was shown in Fig. 3. During the storage, in the control group the weight loss rate was about 1.5% per day, and in the CaCl2-treatment group the weight loss rate was lower than the control group, especially the 80 mM of CaCl2 (Fig. 3c). Meantime, the CaCl2-treatment group maintained firmer than the control, especially the 80 mM CaCl2, remaining firmer until the 20th day (Fig. 3b). However, the application of CaCl2 did not influence total soluble solids content (TSS) and titratable acidity (TA) of grapes during storage (Fig. 3a, d, e).

Fig. 3.

Fig. 3

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Weight loss rate (a), Total Soluble Solids (b), Hardness(c), Titratable acidity (D) and TSS/TA ratio (E) of ‘Kyoho’ grapes during storage (25 °C) treated with CaCl2 and the control group. The error bars represent the standard error (n = 3). Different letters indicate statistically significant differences at p < 0.05 as determined by Student’s t-test

Changes of antioxidant enzyme activities in grape berries after CaCl2 treatment

Compared with control, the activities of CAT, POD, SOD were increased after CaCl2 treatment at different concentrations. The most significant increase of enzyme activities was observed in 80 mM of CaCl2 treatment group (Fig. 4a). However, the enzyme activities dropped when the concentration of CaCl2 reached 500 mM. After 48 h of CaCl2 treatment, most of the enzyme activities decreased slightly, but were still higher than the control. Based on the above data, the samples of 24 h/80 mM CaCl2-treatment and 24 h/CK were selected for transcriptome sequencing.

Fig. 4.

Fig. 4

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a, Antioxidant enzyme activities of grape berries changed after 24 h and 48 h treatment with different concentrations of CaCl2. The error bars represent the standard error (n = 3). b, Heat Cluster of all expressed genes of CT and CK. The horizontal axis represented the fold change of differential gene expression in each sample. The vertical axis represented the statistical significance of the difference gene expression levels. Red and green dots represented the up-regulated and down-regulated genes, respectively, and blue ones showed those without significance. c, The GO classification of 3,693 DEGs. GO terms were summarized in three main categories of biological process (BP), cellular component (CC) and molecular function (MF)

Global analysis of transcriptomic response and qRT-PCR validation

The RNA-seq of the three replicates in control group yielded 7.85, 8.37, and 6.76G clean bases after removing impurities and linker sequence, respectively, while the CaCl2-treatment group obtained 6.4, 6.71, 6.27 G clean bases, respectively (Table S1). The Q30 of all samples were greater than 89%, and the lowest reads sequence of 67.85% could be used for comparison with reference genome. The reliability of the experiment was assessed by the correlation of gene expression levels between samples, then abnormal samples were screened out, which was reflected in Pearson correlation coefficient (Fig S1). The closer the correlation coefficient between two duplicate samples was to 1, the higher the correlation was. The correlation analysis showed extremely significant correlation between samples in each group (CT and CK). In summary, the clean reads were good enough for subsequent analysis.

A total of 32,272 DEGs were identified between CT and CK groups. Out of these DEGs, 3,693 genes were assigned as differentially expressed levels with the threshold of P-adjust < 0.05, including 1,983 up-regulated and 1,710 down-regulated genes (Fig. 4b). GO analysis showed that the identified DEGs were enriched into 6, 14, and 10 terms, respectively, which were annotated in biological processes, cellular components and molecular functions. (Fig. 4c, Table S2).

Among these clusters, 11 Go terms had extreme significant difference (P<0.001). The terms of “carbohydrate derivative metabolic process” (GO:1,901,135), “nitrogen compound metabolic process” (GO:0,006,807), “fatty acid metabolic process” (GO:0,006,631), “monocarboxylic acid metabolic process” (GO:0,032,787) were the dominant groups in the biological process; “cellular component” (GO:0,005,575), “membrane” (GO:0,016,020), “intracellular” (GO:0,005,622), “intracellular part” (GO:0,044,424), “ribonucleoprotein complex” (GO:0,030,529), “cell” (GO:0,005,623), “cell part” (GO:0,044,464), “membrane part” (GO:0,044,425) were the representative groups in the cellular component; While among the molecular function, a great number of DEGs were focused on categories of “unfolded protein binding” (GO:0,051,082).

The biological pathway analysis for CT and CK was performed using KEGG database to determine pathways that were significantly enriched in those 3,693 DEGs. Q-value < 0.05 indicated that the DEGs were significantly enriched. Among 120 of the enriched KEGG pathways, only 2 significantly enriched KEGG pathways were identified with P value < 0.05 (Table 1). As shown in Fig. 5a, DEGs in the Endoplasmic reticulum-associated degradation (ERAD) were closely related to Ubiquitin Regulatory X (Ubx), Heat shock protein 90 (Hsp90) and Derlin. Ubx, Hsp90 and Derlin had significantly higher expression and were highlighted on the Protein processing in endoplasmic reticulum (ER).

Fig. 5.

Fig. 5

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a, Model of CaCl2 in maintaining grape fruit quality. External stimulation caused Ca2+ homeostasis imbalance of ER. On the one hand, the accumulation of misfolded protein promoted chaperones GRP78 and Hsp40, triggering ERS. Caspase 3 activated as the executioners of apoptosis, which led to apoptosis through the hydrolysis of caspase target protein. Bip recognized proteins that bound to misfolded proteins, and guided terminally misfolding of abnormal proteins through ER-degradation enhancing alpha-mannosidase like proteins (EDEM). Derlin-1 played an important role in substrate recognition. They could combine with ERAD specific ubiquitin ligase Hrd1 to form a protein dislocon channel, which could induce them to move from ER to cytosol. Hsps (Hsp40, Hsp70, Hsp90) co-operated with many different proteins, bound to different substrates and performed changes in the structure of proteins. Then in cytosol, the protein was ubiquitinated and RAD23 provided an additional layer of specificity in substrate targeting to govern Ub chain extension or timely processing of the substrate. b, Expression level validation of DEGs using qRT-PCR in comparation with corresponding data detected in RNA-Seq. Relative expression ratio of each DEG was presented in a log2 value of post-PCF vs. pre-PCF libraries. The error bars represent the standard error (n = 3). c, Metabolites with significant changes between CT and CK

To validate the RNA-Seq data, qRT-PCR was used to determine the expression of DEGs with the same RNA samples. Nine up-regulated and seven down-regulated DEGs related to grape berry maturation was tested, the qRT-PCR results were highly consistent with the RNA-Seq results (Fig. 5b). It confirmed the reliability of the RNA-Seq results.

Global analysis of metabolomic response

After quality validation, 1,137 metabolites were identified across broad chemical classes from the spectra. 101 metabolites were significantly upregulated and 98 were significantly down-regulated between CaCl2 treatment and control groups (Table S4). The identified metabolites were divided into several groups including alcohols, organic acids, amino acids, phospholipids, and miscellaneous compounds. Several representative metabolites were emphatically analyzed as ripening associated metabolites.

It had been reported that the contents of organic acids such as malic acid (MA), fumaric acid (FA), tartaric acid (TA) and citric acid (CA) gradually decreased during the development of grape fruits due to respiration (Sabir et al. 2010). FA treatment could remove pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus from plant leaves which could cause plant decay. The combined treatment of Ultrasonic and organic acid (CA and MA) could markedly inactivate E. coli ATCC 25,922 in the juice owing to their synergistic action (Salleh-Mack and Roberts 2007). MAP treatments alone or with MeJA caused significant changes in specific organic acid (CA, MA, FA) content stating that the MAP technique reduced the loss of organic acids that occurred during the storage period (Ozturk et al. 2019). All the above studies had confirmed that organic acids could help plants resist pathogenic bacteria infection. In this study, the content of FA and MA increased significantly after CaCl2 treatment compared with CK, which proved that CaCl2 could reduce the loss of organic acids in grape fruits and make them more resistant to diseases (Fig. 5c).

75 pathways involving at least one annotated metabolite were screened as potential primary target pathways of interest relating to the CaCl2treatment. Among these, two pathways had lower P values (< 0.05) of phenylalanine metabolism and biosynthesis of plant hormones, which were related to phytosphingosine and resveratrol metabolism (Table S5).

A total of 15 DEGs could be classified as phenylalanine metabolism through KEGG analysis (Table S6). Caffeoyl-CoA O-methyltransferase (CCOAMT) was essential in lignin and coumarin biosynthetic process in plants (Zhong et al. 2000). In plant, phenolic compounds lignin precursors acted as potential antioxidant that might work as ROS-scavenging compounds (Olga et al. 2003). Coumarin was one of the phenolic derivatives in plants which possess a significant antioxidant ability scavenging peroxyl radicals. 4-coumarate–CoA ligase 2 (4CL2) was also involved in the synthesis of several phenylpropanoid-derived compounds, including anthocyanins, flavonoids, coumarins, lignin, suberin and wall-bound phenolics. This suggested that a series of processes might result in accumulation of certain metabolites.

Comprehensive analysis of metabolites and genes expression

At the transcriptional level, change of genes that associated with the plant irritability response were firstly observed (Fig. S3). The prominent genes enriched in up-regulated treatment-responsive transcripts were stress-associated proteins (SAPs) (VIT_213s0064g01220). These proteins were composed of special types (A20/AN1) of zinc finger protein (ZFP) domains which were pivotal for conferring tolerance to multiple stress in plants (Aghdam et al. 2019). In addition, heat shock proteins (Hsps) were also enriched in CaCl2 treatment, indicating the defense system was activated. Heat shock protein 12 (Hsp12, VIT_213s0019g02740) and Heat stress transcription factor B-2a (HFB2A, VIT_216s0100g00720) both participated in plant stress response. As a transcription factor, HFB2A up-regulated the transcription of various genes associated with thermotolerance by activating downstream genes and thereby improve heat resistance (Gong et al. 2014). Hsp12 was a highly hydrophilic protein, which broke the H-bond and ion interaction between adjacent glucan chains of cell wall and played an important role in the flexibility of cell wall (Precious et al. 2004).

The accumulation of metabolites related to transcription factors such as L-Glutathione (Com_308_pos) and Indole-3-butyric acid (Com_358_pos) were identified. L-Glutathione was involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates. Glucosinolates were a heterogeneous family of compounds derived from a variety of protein and nonprotein amino acids, that converted these amino acids to secondary metabolites to ensure plants survival under different stresses. Most of them existed as inactively glycosylated forms which offered an efficient mechanism of regulating the homeostasis of secondary metabolites (Matthew et al. 2003). Indole-3-butyric acid-response5 (IBR5) was an active phosphatase that was able to inactivate the activated form of Mitogen-activated protein kinases (MAPKs), MAPK-based signal transduction modules regulate plants response to environmental stresses and phytohormones (Lee et al. 2009).

Several genes related to ribosomal protein (RP) were also significantly up-regulated, such as VIT_213s0019g03260, VIT_214s0066g01110, and VIT_209s0002g00610. The main function of RP was in the composition of the ribosome and regulation of the translational process of protein biosynthesis. Some ribosomal proteins also played a role in plant disease defense and were related to some signaling pathways associated with defense responses.

A number of genes and metabolites were induced in grape to participate in stress response after CaCl2-treatment. The results suggested that CaCl2 could initiate some basic defense by induction of a defense-oriented recombination of the transcriptome and metabolome.

Discussion

CaCl2 treatment improved ‘Kyoho’ grape berry quality

Grapevine are non-climacteric plants. During storage, the weight of the fruit was usually reduced via respiration. As the storage time increased, the probability of infection by external bacteria increased. In this study, compared with the control group, ‘Kyoho’ grape treated with CaCl2 had smaller weight loss, higher firmness maintenance, better shape and longer storage time for 4–8 days under the storage conditions of 25 °C. This was consistent with the studies that CaCl2 could improve the postharvest storage time of loquat fruit (Akhtar et al. 2010). CaCl2 treatment improved free radical scavenging ability and prolonged storage time of fruits and enhanced the resistance of strawberry cell wall to fungi by reducing pectin hydrolase activity. Studies had reflected that CaCl2 could enhance the disease resistance of climacteric fruits (pear, kiwifruit, mango) by regulating the ethylene release process and the content of polyphenol (Shao et al. 2019). These experimental results suggested that CaCl2 treatment could effectively prolong the preservation time both of non-climacteric and climacteric fruits.

The optimum concentration of CaCl2 was identified by the activity of antioxidant enzymes determination. Several reactive oxygen species (ROS) including O2−, • OH and H2O2 were continuously generated during the aerobic metabolism (Yanqun et al. 2019). Whereas plants were surfeited with mechanisms to combat increased ROS levels using various antioxidant enzymes, such as SOD, POD and CAT. Continuous generation of ROS in plants could cause oxidative damages, initiating a variety of ROS-related disorders or cellular toxicity (Mou et al. 2015). At present, CaCl2 had been extensively confirmed to improve the antioxidant capacity of fruits (goji, tomato and apple). In this study, after treated with different concentrations of CaCl2, the activity of antioxidant enzymes in grape berry was detected at different time after treatment. It was found that the effect of 80 mM CaCl2 treatment on grape quality improvement was the best. 80 mM CaCl2 was the optimal concentration for grape preservation.

Enrichment pathways related to grape preservation

Carboxylic acids were involved in the synthesis of aromatic substances or directly contributed to adversity resistance. Studies had proved that carboxylic acids were closely related to fruit aroma and flavor. L-malic acid and tartaric acid, as the most two important organic acid in grape, increased significantly under abiotic stress to perform antioxidant function which confirmed the important role of organic acids in fruit storage and preservation (Estrada et al. 2013). Compared with CK, the synthesis pathway of these substances was up-regulated in CaCl2 treatment. The acceleration of the synthesis and metabolism of acid substances was conducive to maintaining the flavor of the fruit.

Sumoylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, played an important role in regulating the immune function of eukaryotic organisms. Studies showed that sumoylation of NPR1 by SUMO3 activated defense gene expression by transforming the association between NPR1 and transcription repressors into transcription activators (Saleh et al. 2015). The rapid and reversible formation of SUMO1/2 conjugationed following exposure of plants to stress pointed to sumoylation as a key modulator of the stress response. In this study, the expression of SUMO-specific protease activity pathway was significantly enriched to better adapt to external stress and prolong shelf life.

Under biotic and abiotic stresses, the NADPH oxidase (Nicotinamide adenine dinucleotide phosphate oxidase) was activated which was responsible for superoxide anion generation and H2O2 biosynthetic process. The accumulation of H2O2 was crucial for attenuating stresses by triggering stomatal closure and programmed cell death process (Aghdam et al. 2018). The oxidation–reduction triggered specific molecular function which mainly involved in electron carrier associated activity such as ion transport (GO:0,006,811), oxidoreductase activity (GO:0,016,616), and acting on NAD binding (GO:0,051,287) as acceptor. The enzyme production was majorly associated with cellular component (GO:0,005,575) such as ribonucleoprotein complex (GO:0,030,529), membrane protein complex (GO:0,098,796) and ribosome (GO:0,005,840). Any disruption to cell homeostasis could result in an accumulation in unfolded proteins, thus causing endoplasmic reticulum stress response (Bennett et al. 2019). Together, this demonstrated that cell defense reactions occurred during grape storage.

Role of ERAD during extending shelf life in grape berry

ER was the membrane system of cytoplasm which promoted the synthesis, folding and maturation of transmembrane proteins, serving as site of lipid and sterol biosynthesis and stores calcium. Normal ER calcium homeostasis could be destroyed by a variety of mechanisms, such as excessive accumulation of misfolded or unfolded proteins, imbalance of sterol and lipid levels, which triggered ERS thus promoting the upregulation of UPR species including the transcription factor XPB-1 and chaperone proteins GRP78 and Hsp40. If endoplasmic reticulum function continued to disrupt, then cells will eventually initiated apoptotic processes (Bennett et al. 2019). Misfolded proteins were identified, ubiquitized, and deliverred to 26S proteasomes for degradation in the cytosol. These events were collectively referred to as ERAD (Needham et al. 2019). Some toxins caused ERS by interfering with protein folding, the signaling pathways that ERS activated came to be known as the unfolded protein response (UPR).

ATPase p97 provided the force needed for retrotranslocation in ERAD and served as a processing station for the substrate once in the cytosol. Proteins containing ubiquitin regulatory X (Ubx) ubiquitin-like domains bound directly to the amino terminus of the ATPase, acting as the adapter of p97, enhancing the activity of p97 to specific substrate proteins, thus mediating the decomposition of protein complexes (Lalonde and Anthony 2011). Molecular chaperones (also known as Hsps) were multi-domain proteins involved in protein folding, unfolding and remodeling, such as Hsp40, − 70, − 90 and Hsc70. To achieve their goals to control the quality of ER, Hsps functioned in different ways by co-operating with many different proteins, binding to different substrates and performing changes in the structure of proteins (Niforou et al. 2014). Derlins were a family of membrane proteins involved in ERAD. Derlin-1 and Derlin-2 played an important role in substrate recognition. They were capable of binding with the ERAD-specific ubiquitin ligase HRD1 to form a protein-conducting channel, which induced their dislocation from the ER to the cytosol, where they were degraded by the proteasome. In addition, Derlin-1 interacted with its ERAD substrates even when their ER luminal domain had been exposed to the cytosol, indicating that Derlin-1 interacted with the ERAD substrates throughout the dislocation process (Huang et al. 2013).

The results showed that CaCl2 up-regulated the expression of Ubx, Hsps and Derlin in grape berry, indicating that grape berry was subjected to external stress, which stimulated ERAD to break down misfolded proteins, thus reducing fruit damage and extending shelf life.

Conclusion

In this study, combination of transcriptomics and metabolomics was used a to explore the molecular mechanism of which CaCl2 treatment maintained the postharvest quality of grape berry. The transcriptome analysis showed that CaCl2 was involved in stress resistance, such as resistance protein, antioxidant enzyme system, ER protein processing and degradation pathway. According to metabonomic data, CaCl2 treated berries accumulated higher secondary metabolites during storage, such as lignin and coumarin, which increased the antioxidant and antifungal activities of grape berries. The purpose of this study was to develop a suitable concentration of CaCl2 treatment which helps to maintain the appearance and quality of fresh grapes, and prolong the storage time of table grapes for the further economic benefit.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 775 kb) (775.4KB, docx)

Acknowledgments

We would like to express our gratitude to Jiangsu Academy of Agricultural Sciences for providing the grape material. We also thank Shaoyan Lin and Ruiping Tian from the State Key Laboratory of Crop Genetics & Germplasm Enhancement for helping me analyze UPLC and GC-MS data. This work was supported by The national key research and development program (2018YFD1000200), the Fundamental Research Funds for the Central Universities (KYXJ202003), China National Natural Science Fund (31872938, 31872047), Jiangsu Excellent Youth Fund (BK20180076).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Footnotes

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