Transcriptome analysis of fresh-cut kiwifruit treated with slightly acidic electrolyzed water (SAEW) and 1-methylcyclopropene (1-MCP)

Fresh-cut kiwifruit is difficult to store due to the characteristics of thin and juicy rind, which results in the great limitation of development and utilization. The aim of the study was to evaluate the effect of slightly acidic electrolyzed water (SAEW) combined with 1-methylcyclopropene (1-MCP) on shelf life quality of fresh-cut kiwifruit by transcriptome technology. The results showed that SAEW combined with 1-MCP treatment suppressed ethylene production, respiratory rate, soluble solid content (SSC), polyphenol oxidase (PPO) and improved titratable acid content (TAC), total phenols content (TPC), total chlorophyll content (TCC), peroxidase (POD) and phenylalanine ammonia lyase (PAL) activities comparing with the untreated samples. There were 2754 differentially expressed genes (DEGs) between the combined treatment and the control on day 6. GO function annotation results showed that the related genes involved in cell membrane composition in samples might be involved in protein phosphorylation under the action of protein kinase genes. KEGG enrichment analysis showed that 715 DEGs were significantly enriched in 111 metabolic pathways on day 6 after combined treatment. In ethylene signaling pathway, expression levels of ETR and ERF1 were down-regulated, which proved that 1-MCP inhibited ethylene reaction by binding to ethylene receptors. Genes expression levels of SnRK2, MPK3/6, and BAP31 related to MAPK signaling pathway and protein processing in endoplasmic reticulum pathway were up-regulated, and the combined treatment had a positive regulatory effect on fruit defense during storage. Hence, the combination treatment of SAEW and 1-MCP may be a proven and effective technology to retain higher quality and nutritive properties of fresh-cut kiwifruit.


Introduction
As a kind of medicine and food homologous plant with rich nutrition and diverse biological active ingredients, fresh-cut kiwifruit is a popular fruit product in fruit market.It is difficult to store due to the characteristics of thin and juicy rind, which results in the great limitation of development and utilization of fresh-cut kiwifruit (Fu et al. 2018;Sun et al. 2021).Nevertheless, the physiological and biochemical metabolism of fresh-cut fruit is accelerated due to the mechanical damage, leading to surface browning, bacterial infection, loss of nutrients and commercial value (Yu et al. 2015).Therefore, it is urgent to ensure the quality and prolong the shelf life of fresh-cut kiwifruit.
As an ethylene receptor competitive inhibitor, 1-methylcyclopropene (1-MCP) interacts with ethylene receptor ETR1 and ERS1 proteins, thereby blocking the normal binding of ethylene receptors to ethylene and suppressing ripening of fruits (Fu et al. 2017;Saba and Watkins 2020).In addition, 1-MCP is also concerned with the biosynthesis of ethylene (Xu et al. 2020a, b).For example, 1-MCP can inhibit the synthesis of endogenous ethylene and delay the occurrence time of respiratory peak in kiwifruit (Salazar et al. 2019), pear fruit (Chen et al. 2016), jujube fruit (Zhang et al. 2012), blueberry (Xu and Liu 2017), durian fruit (Thongkum et al. 2018) and apple fruit (Zhou et al. 1 3 2016).However, the regulatory mechanism of 1-MCP for ethylene biosynthesis in fresh-cut kiwifruit was not yet been fully elucidated so far.
Slightly acidic electrolyzed water (SAEW) has a near neutral pH, which is a promising disinfectant for the inactivation of foodborne pathogens (Feng et al. 2021).As a broad-spectrum fungicide, SAEW has been widely used in food disinfection and fruit vegetable preservation due to its advantages of being safe, non-toxic, low cost and corrosive (Xing et al. 2021).For instance, Chen et al. (2019a, b a) found that the synergistic properties of the combination of SAEW and chemical methods could be used in fruit industry as an effective sanitizer.Zhi et al. (2017) suggested that SAEW treatment has potential as a commercial cleaning process to prolong the shelf-life and suppress browning of peaches.Tango et al. (2017) also reported that SAEW combined with chemical and physical treatments effectively decontaminated bacteria on fruits.However, there were few researches on the preservation effect of SAEW on fresh-cut kiwifruit.
In recent years, transcriptome technology has been widely used to study the enhancement of postharvest fruit disease resistance by preservatives, and to reveal the regulation mechanism of postharvest fruit disease resistance from the perspective of gene changes (Xu et al. 2016;Chen et al. 2017;Tang and Zheng 2019).However, the changes of differentially expressed genes (DEGs) in fresh-cut kiwifruit treated with SAEW combined with 1-MCP has not been reported so far.The study aims to evaluate the effectiveness of SAEW combined with 1-MCP on shelf life quality of fresh-cut kiwifruit by transcriptome analysis.The meaning of the study is to explore the application prospect of this method in maintaining the storage quality of fresh-cut kiwifruit.

Sample preparation and treatments
Kiwifruits were purchased from the local supermarket of Jilin, China.Scatheless samples of the same maturity (80%), size (120 ± 10 g) and colour were selected and immediately transported to the laboratory for treatment.Samples were divided into four groups of 2000 g each for further treatment.
1-MCP was purchased from Sinopharm Chemical Reagent Co., Ltd., Shenyang, China in a ready to use form.SAEW was prepared by an acid oxidation potential water generator (HRW-1500, Burning Man Jingchuang Medical Equipment Co., Ltd, China).According to preliminary experimental results, 0.9 μl L -1 was selected as 1-MCP treatment concentration, and the fumigation time was 22 h.SAEW with available chlorine concentration (ACC) of 50 mg L -1 and pH of 2.5 for 20 min had the best preservation effect on fresh-cut kiwifruit.

Measurement of physiological indexes
A 3 L glass chamber with samples (approx.100 g) was enclosed for 1 h, and 1 mL of gas from the chamber was collected for each test.Afterwards, the samples were injected into a gas chromatograph (7890B, Agilent, USA) equipped with a flame ionization detector (FID) and a column (1 m × 0.4 mm, Agilent, USA).The parameters were shown below: column temperature 50 ℃, detector temperature 240 ℃, and current velocity of N 2 4.0 mL min -1 .The results of ethylene production were expressed in ng kg −1 s −1 (Xu et al. 2018).
Respiration rate was detected as CO 2 production.A 1 L glass jars with samples (approx.100 g) was enclosed for 30 min prior to CO 2 sampling, and the gas was detected with an infrared gas analyzer (IR400, Yokogawa Co., Ltd., China), and the results of respiration rate were expressed in ng kg −1 s −1 (Xu et al. 2018).
The samples (10 g) were ground with 0.3 g of quartz sand and diluted with 30 mL of distilled water to a triangular flask, which was incubated in the 90 ℃ water for 30 min.Then the mixture was cooled down and filtered, the filtrate (10 mL) was titrated with 0.1 M NaOH.Titratable acid content (TAC) was calculated with the formula below (Xu et al. 2019): The samples (50 g) were homogenized, and then diluted with 50 mL of deionized water for 5 min, which was centrifuged at 4000 × g for 10 min.Subsequently, the supernatant was used to measure soluble solid content (SSC) with a refractometer (MASTER-53S, ATAGO, Japan) (Xu et al. 2021).
Total phenols content (TPC) was determined according to the method of Singelton et al. (1999).Homogenized samples (2 g) were added with Folin-Ciocalteu reagent (0.2 mL) and mixed completely.Then 15% Na 2 CO 3 (1 mL) was added after 5 min and the solution was stationary for Titratable acid content(%) 2 h.Whereafter, the absorbance at 760 nm was determined with a spectrophotometer (UV-4802, Unico, China).Total chlorophyll content (TPC) was determined according to the method of Xu et al. (2019).The samples (5 g) were ground by a pestle in the ice-bath with 0.2 g of quartz sand.Then 100 mL of absolute ethyl alcohol and 80% acetone (1:1) were added to the above thick liquid to extract for 24 h.The mixture was centrifuged at 4,000 × g for 10 min, whereafter, absorbances of supernatant at 663 and 645 nm were measured by a spectrophotometer (UV-4802, Unico, China).

Measurement of enzyme activities
The samples (5 g) were extracted for 10 min with 3 mL of 0.05 M sodium phosphate buffer (pH 7.8) containing some quartz sand at 4 ℃.The extract solution was centrifuged for 30 min at 10,000 × g, and the supernatant was collected as enzyme solution for the measurement of enzyme activity (Xu and Liu 2017;Xu et al. 2020a, b).
Polyphenol oxidase (PPO, EC 1.10.3.1) was determined according to the method of Xu et al. (2020a, b). 1 mL of phosphate buffer (pH 7.6), 1 mL of catechol (0.2 M) and 0.5 mL of enzyme solution were added in sequence to the quartz cell.The absorbance at 410 nm was determined with a spectrophotometer (UV-4802, Unico, China).
Peroxidase (POD, EC 1.11.1.7)was measured according to the method of Kochba et al. (1977), which use guaiacol as donor and H 2 O 2 as a substrate.One unit of POD activity (U g −1 ) was defined as an increase of 0.01 in absorbance per minute at 460 nm under the assay conditions.
Phenylalanine ammonia lyase (PAL, EC 4.3.1.5)was measured by the methods of Zucker (1965).One milliliter of enzyme solution was added into 4 mL of 0.05 M borate saline buffer (pH 8.8) containing 10 mM phenylalanine.After the reaction solutions were incubated for 30 min at 25 ℃ in a thermostated water bath, the absorbance was measured with a spectrophotometer (UV-4802, Unico, China) at 290 nm.One unit of PAL activity (U g −1 ) was defined as the amount of enzyme that causes the increase in absorbance of 0.01 at 290 nm in 60 min under the specified conditions.

Library preparation and transcriptome sequencing
Six samples were randomly selected on day 6 for transcriptome sequencing, representing three independent biological replicates of the untreated and combined treated samples (named CK1, CK2, CK3, SY1, SY2 and SY3, respectively).Total RNA was isolated using the Trizol Reagent.Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA).Illumina HiSeq 4000 sequencing technology platform was used for sequencing that was delegated to Shanghai Personalbio Technology Co., Ltd.

Differential genes screening
DESeq was used to screen for differentially expressed genes with the conditions of expression difference multiple |log 2 Fold Change|> 1 and significant p-value < 0.05.

Differential enrichment analysis
The Gene ontology (GO) database established by Gene Onotology Consortium was used to annotate the GO classification of the differential genes.At the same time, KEGG pathway functional enrichment analysis of different genes was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Quantitative real-time PCR (qRT-PCR) analysis
DEGs were verified by qRT-PCR (CFX96 Touch, Bio-Rad, Hercules, USA) according to the methods of Jiang et al. (2022).The extraction of total RNA was as described above.cDNA was synthesized using the Primer Script™ RT reagent Kit with gDNA Eraser (Takara, Japan), subsequently diluted tenfold and used as templates.All PCR reactions were performed in a final volume of 20 μL, containing 1 μL of diluted cDNA, 10 μL of 2 × SYBR green reaction mix (Takara, Japan), 1 μL (5 pmol) of forward and reverse primers and 8 μL ddH 2 O.The following steps were used: initial denaturation at 95 °C for 2 min, followed by 40 cycles of 94 °C for 10 s, 57 °C for 10 s, and 72 °C for 40 s.The fluorescence signal was measured once every 1 °C.The non-cDNA template negative PCR control was run to detect possible contamination in every PCR analysis.The actin gene was used as an internal control in qRT-PCR and fold change was calculated by using 2 −ΔΔCT .The primers were designed and synthesized by Tiangen Biotechnology Co. Ltd. (Table 1).

Statistical analysis
All experiments were performed in triplicate.The results were analyzed by SPSS 21.0 software.Significant differences were analyzed by Duncan's multiple range tests at the 5% level.All experimental results are showed as the mean ± SE.

Effects of different treatments on ethylene production and respiratory rate
As shown in Fig. 1A, ethylene production of fresh-cut kiwifruit increased sharply from day 0 to day 6, then decreased until day 8, which showed a positive correlation (r = 0.981, p = 0.05) with respiration rate (Fig. 1B).Fresh-cut kiwifruit treated with combination of SAEW and 1-MCP had the lowest values of ethylene production and respiration rate with the reduction of 41.7% and 36.0%compared with the control on day 6 of storage, respectively.So, SAEW or 1-MCP treatment restrained the ethylene release and respiratory action of fresh-cut kiwifruit.Similar results were reported on phyllanthus emblica fruit and longan fruit by Jiang et al. (2021) and Chen et al. (2020a, b), who considered that SAEW inhibited fruit ripening that was caused by the promotion of respiratory action.Besides, Dong et al. (2019) also reported that the amount of CO 2 in apples treated with 1-MCP was inhibited, which was in agreement with the current results.

Effects of different treatments on TAC and SSC
TAC and SSC are important indexes of quality and flavor in fruit during shelf life (Shan and Xu 2015).According to the results of Fig. 1C, TAC in samples went down gradually and SAEW or 1-MCP treatment delayed the decrease of TAC.At the beginning of shelf life, SAEW or 1-MCP had little effect on the maintenance of TAC in fresh-cut kiwifruit.The effect of SAEW or 1-MCP on TAC was more and more prominent with the extension of storage time.TAC in samples treated with SAEW plus 1-MCP maintained high compared to other groups throughout the storage period.The reason for the results may be that the combined treatment inhibited ripening and delayed the decline of TAC in fresh-cut kiwifruit.
As shown in Fig. 1D, SSC in untreated fresh-cut kiwifruit increasingly went up and SAEW or 1-MCP treatment postponed the accumulation of SSC, especially from day 4 to day 8 (p < 0.05).This was in agreement with the results of mango fruit treated with 1-MCP reported by Bai and Zhu (2010).However, the combination treatment of SAEW and 1-MCP had the most obvious effect on delaying the increase of SSC in comparison with the control group.In particular, SSC in fresh-cut kiwifruit treated with the combination was 7.5% on day 8 of storage, which was reduced by 21.1% comparing with the control.The results indicated that the combined treatment had the best efficacy on maintaining TAC and delaying the accumulation of SSC.The present results were in line with the results of TAC, but different from the conclusions of SSC in fresh-cut Hami melon (Yu et al. 2015), which showed that SAEW effectively reduced the consumption of TAC and SSC.

Effects of different treatments on TPC and TCC
Phenol compounds are secondary metabolites, which participate in and regulate the growth and development of plants, giving them the functions of antioxidation and antiviral action (Chen et al. 2020a, b).Cell structure of fruit is destroyed by mechanical cutting, which enhances the membrane permeability and results in the outflow of phenol compounds in the membrane (Zhi et al. 2017).
As shown in Fig. 1E, TPC in fresh-cut kiwifruit showed a descending trend with the extension of shelf life, and similar patterns were found in TCC in samples (Fig. 1F).Application of SAEW or 1-MCP dramaticlly reduced the consumption of TPC and TCC during the whole storage time to some extent.Samples treated with SAEW plus 1-MCP had the highest values of TPC and TCC with improvements of 26.6% and 54.5% compared to the control on day 8 of storage, respectively.The data demonstrated that SAEW or 1-MCP efficaciously maintained the TCC in fresh-cut kiwifruit, which was comparable with the results of broccoli and kiwifruit reported by Wang et al. (2013) and Xu et al. (2019).Zhi et al. (2017) also considered SAEW kept higher TPC in peach fruit in comparison with the control group.However, diverse outcome of TPC in broccoli was found, and the researchers considered that SAEW reduced the accumulation of TPC because the accumulation of TPC in broccoli may be related to the increase of PAL enzyme activity in the tissue (Wang et al. 2013).Zhou et al. (2012) also reported that SAEW inhibited the increase of TPC in pulp of harvested juicy peach, which was not in agreement with the current results.

Effects of different treatments on enzyme activities
The change of fruit color is mainly caused by enzymatic browning (Li et al. 2017).Figure 2A showed that PPO activity in untreated samples gradually increased because cutting destroyed the cellular space of the fruit tissue (Li et al. 2017).However, PPO activity in SAEW-treated or 1-MCP-treated samples suppressed to some extent, which suggested that SAEW or 1-MCP contributed to the delay of browning rate, as reported for fresh-cut celery and lotus root by Massolo et al. (2019) and Li et al. (2017), respectively.Besides, the combined treatment had the remarkable efficacy (p < 0.05) on inhibiting PPO activity and browning rate of fresh-cut kiwifruit.On the other hand, Saba & Watkins (2020) reported that 1-MCP increased PPO activity of apple in comparison with the control group, which was not in agreement with the current results.
A series of defense enzymes in plants undergo extensive pathological changes when they are infected by pathogens, which contributes to the expression of disease resistance (Massolo et al. 2019).POD and PAL activities in untreated samples increased gradually from day 0 to day 6 due to the production of free radicals as a result of mechanical damage, and then decreased during the later stage of storage (Fig. 2B  and C).The decrease might be caused by fruit senescence with the extension of shelf life.Obviously, SAEW or 1-MCP treatment enhanced the POD and PAL activities of fresh-cut kiwifruit to varying degree.However, the combined treatment of the two methods was the most effective and the improvements of POD and PAL activity were 41.3% and 42.9% respectively on day 6 compared to the control group.Xu et al. (2020a, b) reported that SAEW or 1-MCP increased PAL activity of broccoli sprouts and Huangguan pears compare with the control group.Chen et al. (2019a, b) also found that acidic electrolyzed oxidizing water improved the storage capacity of blueberry by enhancing activities of SOD, CAT and APX.The above results were consistent with the current results on fresh-cut kiwifruit.Therefore, SAEW and 1-MCP may be useful to enhance defense enzyme activity, thereby improving quality and extending shelf life of freshcut kiwifruit.

Differential gene expression
Each dot in the volcano plots represented a gene.The abscissa represented the log value of the multiple difference between the expression levels of a gene in the two samples, and the greater the absolute value was, the greater the multiple difference between the expression levels of a gene in the two samples was.The ordinate represented the negative logarithm of the significance level of the variation of gene expression levels, and the larger the value was, the more significant the differential expression was, and the more reliable the differential genes obtained by screening were (Yang et al. 2021).
Figure 3 showed that there were 2754 DEGs (1705 upregulated and 1049 down-regulated) between the untreated (CK) and combined treated samples (SY) on day 6.Cluster analysis of the six samples was used to compare the expression patterns between samples (Fig. 4).The color gradient (blue to red) corresponded to the gene expression levels (low

GO enrichment analysis of the DEGs
The three categories of GO function include molecular biological process and cellular component (Yang et al. 2021).As shown in Fig. 5, the samples on day 6 could be annotated into 20 functional groups in the GO database.There were 5 and 12 types of genes annotated to molecular function and biological process, among which the functional group with the largest number of genes involved were protein kinase activity and protein phosphorylation, respectively.Besides, there were 3 types of genes annotated to component of membrane, but the number of genes involved was similar.The results indicated that the related genes involved in cell membrane composition in fresh-cut kiwifruit might be involved in protein phosphorylation under the action of protein kinase genes, thus inhibiting fruit decay.

KEGG enrichment analysis of the DEGs
As shown in Table S1, 715 DEGs were significantly enriched in 111 metabolic pathways on day 6 after combined Fig. 3 Volcano plots of the differentially expressed genes (DEGs).The red dots indicate up-regulated genes with significant differences, the blue dots indicate down-regulated genes with significant differences, and the gray dots indicate genes that did not significantly differ treatment.The combined treatment of SAEW plus 1-MCP resulted in changes of gene expression in multiple pathways, however the current study focused on the pathways with the largest number of DEGs such as plant hormone signal transduction, MAPK signaling pathway-plant, plantpathogen interaction and protein processing in endoplasmic reticulum (Fig. 6, Table 2).
Genes expression levels of ETR and ERF1 related to ethylene signaling pathway were down-regulated after combined treatment on day 6, which indicated that 1-MCP binded to ethylene receptors competitively thereby inhibiting fruit ripening (Xu et al. 2019).These findings were consistent with the current results of ethylene production (Fig. 1A).Xie et al. (2014) andDal Cin et al. (2006) also reported that 1-MCP reduced the expression levels of ETR1 and ETR2 in pears and apples.The up-regulation was mainly enriched with genes involved into MAPK signaling pathway, such as SnRK2 and MPK3/6.Therefore, combined treatment had a positive regulatory effect on fruit defense during storage.Genes expression levels of MKK45 related to plant-pathogen  interaction pathway were down-regulated, which proved that the production of reactive oxygen species (ROS) was suppressed by combined treatment, consequently improving disease resistance of fruits.BAP31 is mainly involved in B cell activation and vesicle transport that has anti-apoptotic function (Zhou et al. 2019).Genes expression level of BAP31 related to protein processing in endoplasmic reticulum pathway was up-regulated after combined treatment on day 6.The defense response of fresh-cut kiwifruit was induced by the DEGs in multiple energy metabolism pathways, such as amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism.

qRT-PCR validation
Among these DEGs, four DEGs were screened for qRT-PCR validation, including ETR and ERF1 involved in ethylene signaling pathway, SnRK2 and MPK3/6 involved in MAPK signaling pathway.As shown in Fig. 7, relative expression levels of ETR and ERF1 in the combined treatment samples were prominently (p < 0.05) higher than that in control.However, relative expression levels of SnRK2 and MPK3/6 in the combined treatment samples were prominently (p < 0.05) lower than that in control.This was consistent with the results of transcriptomics.

Conclusion
The effect of SAEW combined with 1-MCP on shelf life quality through transcriptome analysis in fresh-cut kiwifruit compared to control was investigated.Treatment of SAEW or 1-MCP alone controlled ethylene production, respiratory rate, SSC, PPO and enhanced TAC, TPC, TCC, POD and PAL activities compared to the untreated samples to varying degree.Moreover, the combined effectiveness of SAEW plus 1-MCP was more remarkable than the treatment alone.Transcriptome data indicated that 2754 differentially expressed genes (DEGs) were found between the combined and the untreated samples on day 6.The samples on day 6 could be annotated into 20 functional groups in the GO database and the related genes involved in cell membrane composition in samples might be also involved in protein phosphorylation under the action of protein kinase genes.Besides, 715 DEGs were significantly enriched in 111 metabolic pathways on day 6 after combined treatment.In ethylene signaling pathway, expression levels of ETR and ERF1 were down-regulated, which proved that 1-MCP inhibited ethylene reaction by binding to ethylene receptors.Genes expression levels of SnRK2, MPK3/6, and BAP31 related to MAPK signaling pathway and protein processing in endoplasmic reticulum pathway were up-regulated, and the combined treatment had a positive regulatory effect on fruit defense during storage.
In plant-pathogen interaction pathway, genes expression levels of MKK45 were down-regulated, which proved that the production of ROS was suppressed by combined treatment.The findings revealed that combined application of SAEW and 1-MCP had a certain potential in delaying the quality degradation of fresh-cut kiwifruit, which revealed the preservation mechanism of SAEW plus 1-MCP on fresh-cut kiwifruit from the transcriptome level and provided reference for the breakthrough of fruit storage and preservation technology.Deeper researches are required to explore the action mechanism of SAEW and 1-MCP treatment in suppressing ethylene receptor genes expression of fresh-cut kiwifruit by genomics techniques.

Fig. 2
Fig. 2 Effects of SAEW, 1-MCP, and their combination on PPO activity (A), POD activity (B) and PAL activity (C) of fresh-cut kiwifruit during storage.Each value is a mean of three replications ± SE.Bars represent SE (p < 0.05)

Fig. 4
Fig.4Cluster analysis of the total expressed genes.The log 10 values of the genes were used for gene expression normalization and hierarchical cluster analysis.The color gradient (blue to red) corresponds to the gene expression levels (low to high)

Table 1
Sequences of primers for qRT-PCR analysis