Peach-gum: A promising alternative for retarding the ripening and senescence in postharvest peach fruit

Postharvest Biology and Technology 161 (2020) 111088

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Peach-gum: A promising alternative for retarding the ripening and senescence in postharvest peach fruit

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Li Zhang, Xiyi Kou, Xue Huang, Guohuai Li, Junwei Liu*, Junli Ye* Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, Wuhan 430070, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Peach-gum Coating Postharvest Retard Peach Ripening Transcriptome

To evaluate the potential of peach-gum coating in retarding postharvest fruit ripening and softening, the effects of peach-gum treatment on storage performance and transcriptomes of peach fruit were studied during cold storage. Compared to the controls (CK), treatment with all tested concentrations (1 %, 5 %, and 10 %, v/v) of peach-gum repressed ethylene production and fruit softening, and to some extent, prevented weight loss. Peachgum treatment did not alter malic acid, citric acid, quinic acid, glucose, fructose, or sucrose content, but it repressed the reduction in sorbitol. Transcriptomic analysis revealed that the expression of numerous genes related to fruit softening and cell wall degradation were repressed by peach-gum treatment, in accordance with the delayed softening observed. Meanwhile, the expression of senescence-associated genes, chitinase genes, and pathogenesis-related genes that were up-regulated during cold storage, were also inhibited by peach-gum treatment. Among the genes differentially expressed between peach-gum-treated and control fruit, genes involved in indole-3-acetic acid (IAA) transport and auxin response were relatively statistically overrepresented. A total of 90 transcription factors belonging to 26 families were differentially expressed. 21 of 23 zinc finger proteins from the four TF families, C2H2, C3H, CO, and Dof, were up-regulated in peach-gum-treated fruit. Additionally, abscisic acid and IAA content were markedly lower in peach-gum-treated fruit than in control fruit. Taken together, our study demonstrated that peach-gum can potentially serve as a new edible coating to preserve peach fruit. These results establish the basis for the future development of improved peach-gum-based edible coatings, by incorporating other effective compounds, and provide valuable information for further investigation of the regulatory mechanisms underlying fruit ripening and senescence in peaches.

1. Introduction Fruit are highly perishable, as 80–90 % of their weight is water, which evaporates quickly during postharvest storage, leading to poor product shelf life (Dhall, 2013). Fruit spoilage often occurs from the skin and increases the possibility of biochemical deteriorations including browning, off-flavor, texture breakdown and pathogenic microorganism infection. Consumers around the world demand chemicalfree fresh fruit and vegetables of high quality, nutritional value, and

extended shelf life. Therefore, attempts to reduce crop losses and maintain the quality of the postharvest fruit is a priority for all food producers (Velickova et al., 2013). Extending the postharvest life of food products requires a reduction in desiccation, maturation, senescence, and the onset and rate of microbial growth (Erbil and MuFtuGil, 1986). Some methods applied to extend the postharvest life of fruit are based on the principle of retarding or diminishing metabolic processes (Vishwasrao and Ananthanarayan, 2017). Edible coatings and films have become the

Abbreviations: ABA, abscisic acid; IAA, indole-3-acetic acid; SOD, superoxide dismutase; CAT, catalase; GC–MS, gas chromatography coupled to mass spectrometry; NIST, National Institute of Standards and Technology; JA, jasmonic acid; UFLC-ESI-MS/MS, ultra fast liquid chromatography-electrospray ionization tandem mass spectrometry; FPKM, Fragments Per Kilobase of transcript per Million fragments mapped; DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; SE, standard error; qRT-PCR, quantitative real time polymerase chain reaction; SAMS, S-adenosylmethionine synthase; ACS, 1aminocyclopropane-1-carboxylate synthase; ACO, 1-aminocyclopropane-1-carboxylate oxidase; PG, polygalacturonase; PLY, pectate lyase; PE, pectinesterase; EXP, expansin; Xyl, xylosidase; XTH, xyloglucan endo-transglucosylase; PMEI, pectin methylesterase inhibitor; ETR, ethylene receptor; EIN, ethylene-insensitive protein; EIL, EIN3-like protein; EBF, EIN3-binding F-box protein; ERF, ethylene-responsive factor; EREBP, ethylene-responsive element binding protein; SAG, senescence associated gene; PR, pathogenesis-related gene; TF, transcription factor; PYR, PYRABACTIN RESISTANCE; PYL, PYR: PYR1-LIKE ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Zhang), [email protected] (X. Kou), [email protected] (X. Huang), [email protected] (G. Li), [email protected] (J. Liu), [email protected] (J. Ye). https://doi.org/10.1016/j.postharvbio.2019.111088 Received 29 August 2019; Received in revised form 6 November 2019; Accepted 3 December 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.

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to cell wall metabolism, fruit senescence and stress response, and phytohormone signal transduction were mainly investigated, with the intention of elucidating the response of peach fruits under peach-gum treatment.

focus of much research in recent years, as they can improve food appearance and quality due to their environmentally friendly nature. They provide an alternative to modifying the atmosphere by reducing changes in quality and helping prevent product loss (Dhall, 2013; Petersen et al., 1999). Edible coatings provide a barrier to moisture, oxygen, and solute movement (Bourtoom, 2008) and also provide protection from microbial contamination (Petersen et al., 1999). Recently, the development and application of bio-based polymers made from agricultural products, including starches, cellulose derivatives, chitosan/chitin, gums, proteins, and lipids, have drawn considerable attention from researchers in the field (Vásconez et al., 2009). Growing evidence from different plant species, including papaya, strawberry, tomato, banana, etc., indicate that chitosan (Ali et al., 2011), methyl cellulose (Nadim et al., 2015), starch (Oz and Ulukanli, 2012), pectin (Moalemiyan et al., 2012) and gum arabic (Ali et al., 2010; Maqbool et al., 2011) are effective for maintaining the freshness of fruit and vegetables due to their film-forming, antimicrobial, biodegradable and biochemical properties. They have been shown to delay the changes in weight, firmness, respiration rate, ethylene evolution, titratable acidity, soluble solids concentration, ascorbic acid content, and color development, induce the activities of superoxide dismutase (SOD) and catalase (CAT), and inhibit superoxide free radical production. Meanwhile, several studies on different fruit species showed that defense response related enzymes/genes, including peroxidase, polyphenol oxidase, phenylalanine ammonia-lyse, β-1,3-glucanase and chitinase, were induced under chitosan treatment, thereby could reduce the severity of postharvest anthracnose (Ali et al., 2014; Edirisinghe et al., 2014; Zahid et al., 2015). Our knowledge of edible coatings for preserving fruit and vegetables has increased substantially. However, most of these studies were mainly focused on changes in physiological indicators or expression of enzymes/genes related to stress response caused by coatings. There remains much to learn about the molecular response to edible coating treatment, especially for the global transcriptional changes, with the purpose of better understanding the molecular mechanism of coating responsive regulation and providing a theoretical basis for the development of new edible coatings. Peach-gum exudates are mainly produced on the trunks and fruit of peach trees and their production is caused by pathogenic fungi, like Botryosphaeria dothidea, B. rhodina, B. obtuse, Lasiodiplodia theobromae, and Diplodia seriata (Wang et al., 2011; Gao et al., 2016). Chemical analyses have shown that peach gum polysaccharides are acidic arabinogalactans, mainly composed of arabinose, xylose, galactose, uronic acids, and smaller amounts of rhamnose and mannose (Qian et al., 2011; Simas et al., 2008). Their structural variation is based on the proportion of monosaccharides and glycosidic linkages. Peach-gumderived oligosaccharides have been shown to have hypoglycemic activity (Wang et al., 2017), antioxidant activity, and high antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli (Yao et al., 2013). In cherry tomatoes, peach-gum polysaccharidebased coating has been proved to effectively inhibit the tissue softening, weight loss, and respiration rate during storage and simultaneously delays the changes in total acidity, ascorbic acid, and sugar content (Li et al., 2017), thus showing a promising alternative technique for preserving fruit. The peach (Prunus persica L.), a fleshy fruit from the Rosaceae family, is highly perishable due to its high water content and respiration rate during postharvest storage. To our knowledge, in addition to Aloe vera gel (Ahmed et al., 2009; Hazrati et al., 2017), there are limited published data on the application of edible coatings on peach fruit. This study aimed to investigate the potential application of peach-gum as an edible coating on postharvest peaches. The effects of peach-gum coating on the physiological indexes including firmness, weight loss, ethylene production rate, main sugar and acids content and hormones content were evaluated. To provide a preliminary description of the underlying mechanism, the global transcriptional response to peach-gum treatment was subsequently studied. The transcriptional changes of genes related

2. Materials and methods 2.1. Preparation of peach-gum solutions Peach-gum exudates were sampled from the experimental base of peach at Huazhong Agricultural University, in Wuhan, China. Peachgum exudates that were newly exuded, transparent, and uncontaminated by clutter were chosen for follow-up experiments. They were first dried to a constant weight in an oven at 40 °C and then ground into a powder. The peach-gum powder was dissolved in boiling water at a ratio of 1:100 (w/v), according to a previous method reported by our group (Wang et al., 2017). The supernatant solution obtained by filtration was regarded as the original solution (concentration = 1 %). Peach-gum solutions with concentrations of 5 % and 10 % were obtained by concentrating to a 1/5 and 1/10 vol, respectively, using a rotary evaporator. The solutions were stored at 4 °C for approximately 3 d until used for the coating treatment. 2.2. Plant material and treatments Mature pre-climacteric peach (Prunus persica L. cv ‘Jinxiu’) fruit were hand-collected from a well-managed commercial orchard named ‘Shiwaitaoyuan’ in Yingcheng, HuBei Province, China. Fruit were chosen for uniformity and lack of defects and then separated into 4 lots at random. Fruit of the three lots were dipped in peach-gum solutions at concentrations of 1 %, 5 %, and 10 % for 10 min. Under the same conditions, fruit of the fourth lot were soaked in distilled water and were designated as the control group (CK). All fruit were then air dried and stored at 8 °C for 25 d. Fruit of each lot were removed after 1d, 5d, 9d, 13d, 17d, 21d, and 25 d of cold storage to evaluate firmness, ethylene production, and weight loss rate. Meanwhile, 15 fruit were sampled and randomly divided into three biological repliates with five fruit in each replicate. Flesh tissue samples were collected along the equator, frozen in liquid nitrogen and subsequently, stored at −80 °C for follow-up measurements. The storage experiment was performed twice at year in 2015 and 2016. 2.3. Measurement of ethylene production Five fruit samples were put into a 2.6 L preservation box (LOCK & LOCK, Seoul, Korea) with a rubber plug on the top of lid for two hours. Then, 1 mL of gas was extracted from the airtight box using a gas-tight syringe. Ethylene was detected using a gas chromatograph (7890A; Agilent, Snata Clara, USA), equipped with a DB-624 column and a flame ionization detector (FID), as described by (Gao et al., 2016). The temperature of the injection port, FID, and chromatographic column were 250 °C, 250 °C, and 40 °C, respectively. The carrier gas was pure nitrogen (N2), with a flow rate of 16 mL min−1. An external standard method was used for the determination of ethylene production. A standard ethylene sample (2 %, N2), from Newradar Special GAS Co., Ltd. (Wuhan, China), was used to create a standard curve. The rate of ethylene production was expressed as ng kg−1 s−1 and presented as the mean ± SE of three independent biological replicates. 2.4. Measurement of firmness Fruit firmness was determined using a texture analyzer (TA-XT2i Plus; Stable Micro System Ltd., Surrey, UK) fitted with a 7.9-mm-diameter head. The rate of penetration was 1 mm s−1 with a final penetration depth of 10 mm. The firmness of 9 fruit samples was measured and every fruit was evaluated at 6 points along the equator. Fruit 2

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firmness was expressed as ‘N’ and presented as the mean ± SE of the 9 fruit samples.

2.9. Verification of RNA-seq by qRT-PCR To test the reliability of RNA-seq data, 15 genes that were differently expressed between the CK group and peach-gum-treated fruit at day 13, were selected for qRT-PCR analysis. Specific primers were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA) and synthesized by Tsingke (Beijing, China). PpeIF-1A was used as a reference gene, based on our previous study (Kou et al., 2017). Primer sequences are listed in Table S1. Total RNA was extracted from the selected samples using RNA extraction kit SK8661 from Sangon. cDNA was then synthesized from 1 μg of total RNA using HiScript ®ⅡReverse Transcriptase (Vazyme, Nanjing, China). qRT-PCR amplification was performed on an ABI Prism 7900 H T system (Applied Biosystems) with the following cycling conditions: 1 cycle of 5 min at 95 °C and 40 cycles of 10 s at 95 °C, 20 s at 60 °C, and 20 s at 72 °C. Relative gene expression was calculated using the 2−△△Ct method.

2.5. Determination of weight loss Eight fruit of each treatment were individually labeled. They were weighed and then put back in everytime of sampling. The weight loss rate was calculated and expressed as ‘%’ and presented as the mean ± SE of the 8 fruit samples. 2.6. Measurement of the main sugars and acids The content of main sugars and acids was evaluated using GC–MS (gas chromatography coupled to mass spectrometry). Non-targeted metabolite profiling was performed by GC–MS using a method described by (Yun et al., 2013), with some modifications. A total of 0.15 g of ground flesh samples was extracted in 2.7 mL of methanol and an adonitol solution (300 μL, 0.2 g L−1) was added as an internal standard. Samples were centrifuged, dried, and derivatized. GC–MS analysis was performed using a Thermo Trace GC Ultra, coupled with a Thermo Fisher DSQ II mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Metabolites were identified using an available chromatogram library NIST (National Institute of Standards and Technology). Values are expressed as g kg−1 adonitol equivalent fresh weight (FW) of three independent biological replicates.

3. Results and discussion 3.1. Effects of peach-gum treatment on ethylene production, fruit firmness, and weight loss Ethylene release and fruit softening are typical characteristics of fruit ripening. Ethylene production by fruit in the CK group and the 1 % peach-gum treatment group increased with time and peaked at day 13, while ethylene was produced at low levels throughout the storage period in the 5 % and 10 % peach-gum treatment groups (Fig. 1A). The firmness of CK fruit and 1 % peach-gum-treated fruit decreased rapidly from day 1 to day 13 and reached its lowest value (approximately 1 N) at day 13, after which it was stable until to day 25. The firmness of 5 % peach-gum-treated fruit decreased significantly from day 5 to day 9 and remined comparatively high (approximately 2.3 N) from day 9 to day 25. The firmness of 10 % peach-gum-treated fruit showed a slight decrease throughout the storage period. The rate of fruit softening was negatively correlated with the concentration of peach-gum (Fig. 1B). The rate of weight loss in the 10 % peach-gum treatment group was lower than the other three groups, which did not exhibit obvious differences between each other (Fig. 1C). In summary, higher concentrations of peach-gum treatment resulted in lower production of ethylene and accordingly, a lower rate of softening. Peach-gum treatment suppressed the rate of weight loss to some extent. The data from a 2015 study also support the conclusion that peah-gum treatment represses ethylene production and fruit softening (Fig. S1). Considering that both the 5 % and 10 % treatments significantly retarded peach fruit ripening, we chose fruit from the CK and 5 % treatment groups for further investigations, to avoid potential physiological disorders caused by an overdose of peach-gum coating.

2.7. Determination of ABA, IAA, and JA The content of ABA, IAA, and JA (Jasmonic acid) were determined as described by (Liu et al., 2012). Briefly, 0.1 g of ground sample was added to 750 μL of extraction buffer, containing methanol:H2O:acetic acid (80:19:1, v/v/v) in a 2 mL tube. After shaking at 140 g overnight at 4 °C, the samples were centrifuged at 12,000g for 15 min. The upper phase was then transferred to a new 2 mL tube. A total of 400 μL of extraction buffer was added to the remainder of the precipitate, followed by shaking for 4 h at 4 °C and centrifugation at 12,000g for 15 min. The two supernatants were combined and filtered using a nylon filter with a pore size of 0.22 μm. The filtrate was dried by evaporation under a flow of nitrogen gas for approximately 4 h at room temperature and then dissolved in 200 μL of methanol. The extracted solution (10 μL) was analyzed by UFLC-ESI-MS/MS for ABA, IAA, and JA measurements. 2.8. RNA isolation, library preparation, and sequencing RNA was extracted from the day 1 and day 13 samples of the CK group and the group treated with a 5 % peach-gum solution. RNA was extacted from three biological replicates, using a plant total RNA kit, according to the manufacturer's instructions (SK8661; Sangon Biotech, Shanghai, China). RNA was quantified using an Agilent RNA Nano 6000 Assay Kit and RNA purity and integrity were assessed using a NanoDrop 2000 ultramicrospectrophotometer (Thermo Scientific) and 1 % agarose gel electrophoresis, respectively. Differentially expressed gene (DEG) library construction and sequencing on an Illumina instrument were performed at Annoroad Gene Technology (Beijing, China). Paired-end reads (150 bp) were generated and aligned to the Prunus persica genome assembly v2.0 (https://www.rosaceae.org/species/ prunus_persica/genome_v2.0.a1). Gene expression was quantified as fragments per kilobase of transcript per million mapped reads (FPKM). Fold change (|log2FC| ≥ 1) and q-value (q < 0.05) were used as statistical significance indices. DEGs were annotated using Gene Ontology (GO, http://www.geneontology.org), Kyoto Encyclopedia of Genes and Genomes (www.genome.jp/kegg/), and Pfam (http://pfam.xfam.org/) online resources.

3.2. Effects of peach-gum treatment on the transcriptomes of cold-stored peaches To identify molecular mechanisms triggered by peach-gum treatment, high-throughput RNA sequencing analysis was performed on CK and 5 % peach-gum treatment groups at day 1 (designated “CK1” and “T1”) and day 13 (designated “CK13” and “T13”) of cold storage. Each of the cDNA libraries yielded 44.4–47.9 million clean reads and 90 % to 93 % of them were mapped to the peach genome (https://www. rosaceae.org/organism/Prunus/persica). Pair-wise comparisons between different storage stages and different groups were performed to gain a better understanding of the effects of peach-gum treatment on cold-stored peaches. Gene expression levels were calculated based on FPKM values. In total, 20346 genes were found to be expressed in the 12 samples analyzed. Fold change (|log2FC| ≥ 1) and p-value (p ≤ 0.01) were used as statistical significance indices. There were 4400 DEGs (1956 up-regulated genes and 2444 down-regulated genes) detected between day 13 and day 1 in CK fruit, while 3459 DEGs (1340 3

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Fig. 2. Summary of differently expressed genes. “T” indicates fruit treated with 5 % peach-gum. Fold change (|log2FC| ≥ 1) and q-value (q < 0.05) were used as statistical significance indices for DEGs (differentially expressed genes).

stimulus”, “auxin-activated signaling pathway”, “response to auxin”, “response to abscisic acid”, and “fruit ripening”. Few DEGs in the cellular-component category were enriched, while the remarkable terms within the molecular-function category were “oxidoreductase activity”, “chitinase activity”, “hydrolase activity, hydrolyzing O-glycosyl compounds”, and “hydrolase activity, acting on glycosyl bonds”. The GO terms assigned to the DEGs between CK13 and T13 suggested that peach-gum treatment influenced ripening and senescence, possiblly by affecting processes related to stress responses and hormone-mediated signaling pathways represented by auxin.

3.3. Effects of peach-gum treatment on ethylene biosynthesis and fruit softening-related genes In accordance with the production of ethylene, the expression of ethylene biosynthetic genes were repressed by peach-gum treatment. SAMS1, ACS1, and ACO1 were down-regulated by 0.56-, 3.57-, and 1.57-fold, respectively, at day13 (Fig. 4). Genes encoding enzymes involved in cell wall remodeling showed lower transcript abundance in peach-gum treated fruit, with several, including polygalacturonase (PG), pectate lyase (PLY), pectinesterase (PE), α-galactosidase, β-galactosidase, expansin (EXP), xylosidase (XYL), xyloglucan endo-transglucosylase (XTH), and endoglucanase mRNAs, being 1.09- to 3.36-fold lower. Simultaneously, the pectin methylesterase inhibitor (PMEI) gene displayed the opposite trend, being 5.85-fold higher in peach-gumtreated fruit than in CK fruit. Thus, peach-gum treatment repressed the biosynthesis and release of ethylene and also the flesh-softening process that depends on ethylene. Ethylene is known to regulate fruit ripening through a series of genes in the ethylene signaling pathway (Fig. S3). ETRs (Ethylene receptors) are negative regulators, while EIN2 (Ethylene-insensitive protein 2) and the downstream EIN3/EILs are transcription factors that positively regulate ethylene signaling. EIN3 and EIL1 (EIN3-like protein 1) regulate the vast majority of downstream target genes; however, they are degraded by two critical F-box proteins, EBF1 (EIN3-binding F-box protein 1) and EBF2 (Zhang et al., 2018). EIN3 regulates the expression of ERF1 (Ethylene-responsive transcription factor 1) and other target genes by binding directly to their promoters and subsequently, ERF1 and other EREBPs (ethylene-responsive element binding proteins) bind to the GCC box in the promoter of target genes and activate downstream ethylene responses (Solano et al., 1998). Contrary to the common recognition that EIN3 positively regulates the ethylene-response pathway, ETR1, ETR2, EIN2, EIN3-1, EIN3-2, and EIL were all

Fig. 1. Effect of peach-gum treatment on ethylene production (A), fruit firmness (B), and weight loss rate (C) of peaches. The rate of ethylene production was expressed as ng kg−1 s−1 and presented as the mean ± SE of three independent biological replicates. Fruit firmness was expressed as ‘N’ and presented as the mean ± SE of 9 fruit samples. The weight loss rate was expressed as ‘%’ and presented as the mean ± SE of 8 fruit samples. The significance of difference was analysed according to a Duncan’s multiple range test at p = 0.05.

up-regulated genes and 2119 down-regulated genes) were detected in peach-gum-treated fruit (Fig. 2). Moreover, there were 622 genes and 1807 genes (948 were induced by peach-gum treatment and 859 were repressed) differently expressed between the two groups at day 1 and day 13, respectively. Fifteen genes that were differently expressed in CK and peach-gum-treated fruit at day 13 were selected for qRT-PCR analysis. All 15 genes exhibited similar patterns of expression as observed in the RNA-seq data, although the degree of change was not completely consistent (Fig. S2). As the number of DEGs at day 1 was small, more attention was paid to the DEGs identified at day 13. GO terms were assigned to the annotated DEGs identified in the present study (Fig. 3). These were classified into three GO categories: “biological process”, “cellular component”, and “molecular function”. Within the biological-process category, some of the remarkable and significantly enriched terms were “response to stimulus”, “response to chemical”, “response to stress”, “chitin metabolic process”, “response to oxidative stress”, “response to reactive oxygen species”, “signal transduction”, “response to hormone”, “hormone-mediated signaling pathway”, “cellular response to auxin 4

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Fig. 3. q value distribution of enriched Gene Ontology terms in different comparison groups. “T” indicates fruit treated with 5 % peach-gum. q < 0.05 was taken as the threshold for GO enrichment analysis.

3.4. Effects of peach-gum treatment on main sugars and acids

upregulated in peach-gum-treated fruit, while EBF1 and EBF2 were downregulated compared to CK (Fig. S3). Obviously, there was an inconsistency between the transcription of genes involved in ethylene signal transduction pathway and upstream biosynthetic genes and downstream flesh-softening-related genes. This phenomenon may be explained by a previously reported negative-feedback mechanism (Potuschak et al., 2003), in which EIN3 can accelerate its own degradation by inducing EBF2 transcription after triggering ethylene-responsive gene expression when ethylene synthesis is at normal level. Considering the potential targets of EIN3/EILs, 11 differently expressed ERFs that are potentially associated with genes involved in the metabolism of cell walls or other pathways were identified (Fig. S3). To determine whether genes involved in cell wall metabolism were regulated by ethylene, their promoter regions (1.5 kb) were scanned for ERF-binding sites, ethylene response elements, and EIN3-binding sites (Fig. S4). As expected, EIN3-binding sites (EBSs) were identified in the promoters of all genes analyzed, while ethylene response elements (EREs) were identified in 8 genes and ERF-binding sites were identified in 11 genes. The enrichment of EIN3-binding sites in the promoters of genes involved in cell wall metabolism suggested that they are more likely to be regulated by EIN3.

The relative amounts of the three main acids and four main sugars in peach fruit were determined (Fig. 5). Malic acid and citric acid decreased with storage in both the CK and 5 % peach-gum treatment groups, while quinic acid did not show any obvious change. Peach-gum treatment had little effect on the amount of these acids (p ≤ 0.05). The ratio of malic acid, citric acid, and quinic acid content in the 5 % treatment group relative to the CK group was approximately 0.76–0.92, 0.88–1.08, and 0.87–1.14, respectively. In both groups, glucose and fructose increased with time, while sucrose decreased. Peach-gum treatment had no obvious effect on these sugars (p ≤ 0.05). The ratio of glucose, fructose, and sucrose content in the 5 % treatment group relative to the CK group was approximately 0.94–1.17, 0.92–1.12, and 1.00–1.09, respectively. In the CK group, sorbitol decreased from day 13 to day 25, while it was stable in peach-gum-treated fruit. The ratio of sorbitol content in the 5 % treatment group relative to the CK group reached 1.74 at day 25. Thus, peach-gum treatment had little effect on malic acid, citric acid, quinic acid, glucose, fructose, or sucrose levels, while it repressed the reduction in sorbitol at the later stage of storage. Sorbitol accounts for 60–80 % of the total end products of photosynthesis in the Rosaceae family and serves as the major transport carbohydrate (Meng et al., 2018). It is synthesized from glucose by aldose-6-phosphate reductase and sorbitol-6-phosphate phosphatase in 5

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Fig. 4. Expression profiles of genes related to ethylene biosynthesis and cell wall metabolism. The expression level (FPKM) of each gene was collected from the transcriptomic data and was expressed as mean ± SE of three independent biological replicates. Abbreviations used are as follows: SAMS, S-adenosylmethionine synthase, Prupe.1G107000; ACS, 1-aminocyclopropane-1-carboxylate synthase, Prupe.2G176900; ACO, 1-aminocyclopropane-1-carboxylate oxidase, Prupe.3G209900; PG, polygalacturonase, Prupe.4G262200, Prupe.4G261900; PLY, pectin lyase, Prupe.4G116600, Prupe.3G025600; PE, pectinesterase, Prupe.7G192800, Prupe.1G529500, Prupe.8G263900; α-galactosidase, Prupe.1G247000, Prupe.1G352200; β-galactosidase, Prupe.7G163100; Expansin, Prupe.6G075100, Prupe.6G042000, Prupe.2G237000; XYL, xylosidase, Prupe.1G123100; XTH, xyloglucan endotransglucosylase, Prupe.8G201000; Endoglucanase, Prupe.1G367400; PMEI, pectin methylesterase inhibitor, Prupe.1G113800.

Fig. 5. Effect of peach-gum treatment on content of main sugars and acids. The relative content of each metabolite was calculated against the quantified internal standard, adonitol. Values are expressed as g kg−1 adonitol equivalent fresh weight (FW) of three independent biological replicates. 5 %/CK represents the ratio of the metabolite content in the 5 % peach-gum-treated fruit to the metabolite content in control fruit. The values with different letters are significantly different according to a t-test at *p < 0.05 and **p < 0.01.

2007; Zhang et al., 2015). Here, no matter gene expression data or concentration data, sorbitol biosynthesis and accumulation is not induced in cold-stored peaches. In our previous study, sorbitol displayed steady levels during the early stage of cold storage, followed by a significant decrease in the later stage (data not shown). Meanwhile, the

source organs, and is metabolized to fructose by sorbitol dehydrogenase after being transported to sink organs (Meng et al., 2018). Sorbitol is also implicated in multiple stress response processes, such as osmotic, drought, salt, and cold stress. Its accumulation increased under stress conditions (Busatto et al., 2018; Li et al., 2011; Pommerrenig et al., 6

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Here, many pathogenesis-related genes, including PR1, PR4, PR5, PR10, and chitinase, exhibited lower expression levels in peach-gum-treated fruit than CK fruit at day 13 (Fig. 6). This may suggest that senescence or stress was relieved by peach-gum treatment.

expression of two genes encoding D-sorbitol-6-phosphate dehydrogenase (Prupe.1G339000 and Prupe.1G245400) and a gene encoding sorbitol dehydrogenase (Prupe.2G288800) were up-regulated in the later stage of storage, suggesting the existence of sorbitol catabolism. Concerning the accumulation of glucose and fructose in coldstored peaches, the catabolism of sorbitol may have occurred to meet the need for energy sources. In this study, sorbitol in the CK group exhibited a trend similar to the previous study, while its decreased levels in the later stage of storage was inhibited by peach-gum treatment, implying that the deterioration of fruit quality was retarded by peachgum treatment.

3.6. Effects of peach-gum treatment on JA, ABA, and IAA content and genes related to phytohormone signal transduction The JA, ABA, and IAA content of CK and 5 % peach-gum-treated fruit were evaluated at day 1, day 13 and day 25 (Fig. 7). JA did not exhibit significant difference (p ≤ 0.05) between CK and peach-gumtreated fruit, even though a slight decrease was observed during storage. ABA content in both groups was relatively stable with increased storage time, while peach-gum treatment significantly reduced ABA content in peach fruit. IAA content in CK fruit increased with time, but decreased from day 13 to day 25 in peach-gum treated fruit. Furthermore, IAA content in peach-gum-treated fruit was significantly lower than in CK fruit at day 1 and day 25. According to the transcriptomic comparison between CK and peachgum-treated fruit at day13, DEGs involved in auxin-mediated signaling pathways were predominant (Fig. 3). 25 auxin-related genes were shown to be differentially expressed (Fig. 8). Compared to CK fruits, three genes related to IAA transport were up-regulated, while 22 genes encoding auxin-responsive proteins and auxin-induced proteins were down-regulated at day 13 in peach-gum-treated fruit. These data revealed the important role of IAA in regulating peach ripening and softening. It can be speculated that peach-gum treatment had a greater effect on IAA transport and response than synthesis in the earlier stage (day 1 to day 13) of cold storage. However, the processes of IAA synthesis, transport and response were all extensively repressed by peach-gum treatment in the later stage (day 13 to day 25) according to the dramatic difference in IAA content at day 25. IAA has been reported to have some crosstalk with ethylene during peach ripening and a high concentration of IAA is required to generate a large amount of system-2

3.5. Genes involved in senescence and stress responses Senescence-associated genes (SAGs) are genes that execute the senescence process and take part in macromolecule breakdown, detoxification of oxygen reactive species, induction of defense-related metabolism, and signaling and regulatory events (Gepstein et al., 2003). Some SAGs may protect the senescing cell from stress, since the process of senescence is thought to stress the cell (Weaver et al., 1998). Two SAGs, SAG29 and SAG12, were up-regulated with storage time in both CK and peach-gum-treated fruit, while their expression levels were lower in peach-gum-treated fruit at day 13 (Fig. 6). Our results suggest that these two SAGs could be considered as further good markers of fruit senescence process in peach. Pathogenesis-related proteins (PRs) are widely induced by pathogens and stressful situations (Edreva, 2005). They are considered to play important roles in conferring tolerance to fungal pathogen and abiotic stress in the various postharvest practices, such as salicylic acid, nitric oxide, UV-C and heat treatment (Horváth et al., 2007; Hu et al., 2014; Li et al., 2010; Pavoncello et al., 2001). Some PRs are confirmed to be senescence-associated genes (Hanfrey et al., 1996) and are used widely as marker genes to study the plant self-defense mechanism responding to stress situations (Jwa et al., 2006; Kosova et al., 2014).

Fig. 6. Expression profiles of genes related to senescence, pathogenesis, and chitinase. The expression level (FPKM) of each gene was collected from the transcriptomic data and was expressed as mean ± SE of three independent biological replicates. SAG29 (Prupe.1G220700), SAG12 (Prupe.4G059600), PR1 (Prupe.8G153800, Prupe.8G153700), PR4 (Prupe.6G141100), PR5 (Prupe.3G144000, Prupe.3G148300, Prupe.5G094200), PR10 (Prupe.1G127800, Prupe.1G128700, Prupe.1G127300, Prupe.1G128200, Prupe.1G127600) and chitinase (Prupe.7G259000, Prupe.1G205600, Prupe.7G258500, Prupe.1G205400, Prupe.1G391300). 7

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Fig. 7. Effect of peach-gum treatment on JA, ABA, and IAA content. The final results are expressed as μg kg−1 FW and presented as the mean ± SE of three independent biological replicates. The values with different letters are significantly different according to a t-test at *p < 0.05 and **p < 0.01.

Fig. 8. Influence of peach-gum treatment on genes related to auxin signaling. The expression level (FPKM) of each gene was collected from the transcriptomic data and was expressed as mean ± SE of three independent biological replicates. 8

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Table 1 Differently expressed zinc finger proteins from four TF families. TF ID

TF family

Description

log2(Fold change)

Prupe.8G142400 Prupe.6G060000 Prupe.6G127700 Prupe.1G336500 Prupe.6G238500 Prupe.1G381800 Prupe.5G239100 Prupe.6G323100 Prupe.7G125800 Prupe.7G216200 Prupe.1G416500 Prupe.1G219800 Prupe.1G093900 Prupe.1G398700 Prupe.3G245100 Prupe.8G022200 Prupe.6G079500 Prupe.5G126800 Prupe.2G314800 Prupe.6G092600 Prupe.4G042500 Prupe.7G140700 Prupe.6G253300

C2H2 C2H2 C2H2 C2H2 C2H2 C2H2 C2H2 C2H2 C2H2 C3H C3H CO-like CO-like CO-like CO-like Dof Dof Dof Dof Dof Dof Dof Dof

Zinc finger protein ZAT10

−1.01 1.00 1.06 1.20 1.20 1.29 1.71 1.81 1.89 2.68 2.72 1.05 1.43 1.85 2.05 −1.29 1.14 1.24 1.27 1.78 1.87 1.50 1.39

Protein TRANSPARENT TESTA 1 Zinc finger protein 4 Zinc finger protein JACKDAW Zinc finger protein MAGPIE Protein SHOOT GRAVITROPISM 5 Zinc finger protein MAGPIE Zinc finger protein ZAT5 Zinc finger CCCH domain-containing protein 20 Zinc finger CCCH domain-containing protein 20 Zinc finger protein CONSTANS-LIKE 14 Zinc finger protein CONSTANS-LIKE 5 Zinc finger protein CONSTANS-LIKE 4 Zinc finger protein CONSTANS-LIKE 2 Dof zinc finger protein DOF1.2 Dof zinc finger protein DOF2.4 Dof zinc finger protein DOF4.6 Dof zinc finger protein DOF4.6 Dof zinc finger protein DOF2.1 Dof zinc finger protein PBF Dof zinc finger protein DOF5.3 Dof zinc finger protein DOF2.4

Fold change = FPKM (5 %) / FPKM (CK).

3.7. Regulation of transcription factors

ethylene (Tadiello et al., 2016; Tatsuki et al., 2013; Trainotti et al., 2007). During this process, IAA functions upstream of ethylene and therefore, which maybe explain why IAA content showed no difference in CK and peach-gum-treated fruit, while ethylene production was several times higher in CK fruit than peach-gum-treated fruit at day 13. The difference in IAA content between CK and peach-gum-treated fruit was evident in the later stage of cold storage, while large numbers of related DEGs were seen earlier, indicating that physiological changes lagged behind changes in gene expression. Since it has been reported that ethylene can inhibit IAA transport (Burg and Burg, 1967; Lewis et al., 2011; Suttle, 1988), the upregulation of genes related to IAA transport may have resulted from the inhibition of ethylene biosynthesis. Compared with the enrichment of auxin-related DEGs, the number of DEGs related to other phytohormones (abscisic acid, jasmonic acid, salicylic acid, gibberellin, and cytokinin) was relatively lower. Contrary to our expections, three DEGs (abscisic acid receptor PYL2, abscisic acid receptor PYL4 and ABA responsive element binding factor) related to ABA, the phytohormone best known for involvement in ripening and senescence, were downregulated during storage and displayed higher expression levels in peach-gum-treated fruit than CK fruit at day 13 (Fig. S5). ABA has been reported to be involved in response to multiple biotic and abiotic stress and serves as a chemical signal to trigger adaptation to the stress conditions (Lee and Luan, 2012). There exists a signaling dynamic adjustment that different levels of ABA in stress situations alters the perception by various PYR (PYRABACTIN RESISTANCE)/PYL (PYR1-LIKE) members. Six HvPYR/PYL genes in barley were reported to be down-regulated under prolonged stress, whereas it was again up-regulated when the ABA level reduced (Seiler et al., 2014). Hence, the down-regulation of PpPYL1 and PpPYL4 in coldstored peach and the relative up-regulation of them in peach-gum treated fruit were possibly caused by the signaling dynamic adjustment. Salicylic acid has been reported to modulate the plant response to abiotic stress (Asghari and Aghdam, 2010). Three salicylic acid-binding proteins were markedly induced in CK fruit during cold storage, but they exhibited lower expression levels in peach-gum-treated fruit than in CK fruit at day 13, thus indicating the relief of stress to the fruit.

A total of 90 transcription factors (TFs) belonging to 26 families were found to be differentially expressed in CK and peach-gum-treated fruit at day 13. The most abundant TF families were MYB, ERF, C2H2, Dof, and WRKY (Fig. S6). Interestingly, 21 of 23 zinc finger proteins from four TF families, consisting of C2H2, C3H, CO, and Dof, were upregulated in peach-gum-treated fruit (Table 1). Genes from the above four families are mostly reported to be involved in plant development, including floral organogenesis, leaf initiation, lateral shoot initiation, embryogenesis, and meristem and circadian clock modulation and in regulating genes that are involved in carbohydrate metabolism, seed germination, and gibberellin and auxin responses (Chrispeels et al., 2000; Griffiths et al., 2003; Li and Thomas, 1998; Lijavetzky et al., 2003). The higher transcriptional level of zinc finger proteins in peachgum-treated fruit may suggest a lower degree of ripeining and senescence and that they function in maintaining fruit freshness.

4. Conclusions Above results showed that peach-gum coating is a potentially alternative treatment to delay postharvest fruit ripening and maintain the functional properties of peaches. Peach-gum treatment delayed ethylene production and fruit softening; prevented weight loss, to some extent; and repressed the reduction in sorbitol at the later stage of cold storage. According to transcriptome data, a series of genes related to ethylene biosynthesis, fruit softening, senescence, and stress response were inhibited by peach-gum treatment. Meanwhile, numerous zinc finger proteins from four TF families that have been reported to play crucial roles in the plant development, exhibited higher expression levels in peach-gum-treated fruit than in CK fruit, implying their potential role in retaining fruit freshness. Moreover, our data suggested that auxin-mediated signaling pathways play an important role in the process of fruit ripening and senescence. These results provide the theoretical basis for further application of peach-gum in fruit preservation and a better understanding of the molecular mechanisms of ripening and senescence regulation in peach fruit.

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Funding sources

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