Preharvest bagging and postharvest calcium treatment affects superficial scald incidence and calcium nutrition during storage of ‘Chili’ pear (Pyrus bretschneideri) fruit

Postharvest Biology and Technology 163 (2020) 111149

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Preharvest bagging and postharvest calcium treatment affects superficial scald incidence and calcium nutrition during storage of ‘Chili’ pear (Pyrus bretschneideri) fruit

T

Qian Li1, Chen-xia Cheng1, Xin-fu Zhang, Cai-hong Wang, Shao-lan Yang* College of Horticulture, Qingdao Agricultural University, No. 700 Changcheng Road, Chengyang, Qingdao City, 266109, Shandong Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Superficial scald Disorders Calcium

Superficial scald, which appears as black or brown necrotic patches on the skin, is a serious postharvest physiological disorder of pear fruit. The occurrence of scald in ‘Chili’ pear (Pyrus bretschneideri) fruit during storage at 2 °C in response to preharvest polyethylene (PE) bagging and non-woven fabric bagging has been investigated. The non-woven fabric bagging treatment of fruit prevented scald development, while the PE bagged fruit had a higher incidence of scald than the untreated fruit. Fruit from the PE bagging treatment had lower Ca content, less distribution and lower flux rate of Ca2+ than no bagging and non-woven fabric bagging fruit. The treatment also was associated with greater expression of genes encoding calmodulin-like (CML) proteins, such as PbCML19, PbCML5, PbCML38, PbCML42-1, and PbCML42-2. In contrast, non-woven fabric bagging did not affect the expression of these genes in the fruit. In addition, CaCl2 treatment reduced scald development in PE bagged pear fruit during storage. Our results suggest that Ca2+ may play a role in regulating the occurrence of superficial scald in pear fruit.

1. Introduction Superficial scald is one of the most common physiological disorders that occurs in apple [Malus pumila (L.) Mill], pear [European (Pyrus communis L.), and Asian pear (Pyrus serotine Redh.)] fruit, during or after low temperature storage (Lurie and Watkins, 2012). Symptoms are manifested as a browning or black appearance of the exocarp accompanied by necrosis of the epidermis and hypodermal cortical cells (Lurie and Watkins, 2012; Busatto et al., 2018; Watkins et al., 1995; Tsantili et al., 2007). Scald is believed to result from the production of α-farnesene, and its oxidation into conjugate trienols (CTols), leading to the production of ketone 6-methyl-5-hepten-2-one (MHO) (Rowan et al., 1995; Zhao et al., 2016). 1-Methycyclopropene (1- MCP) can prevent the scald development (Argenta et al., 2007; Rupasinghe et al., 2000; Gago et al., 2015). Several physiological disorders are related to calcium deficiency, including bitter pit in apple fruit (Ferguson and Watkins, 1992), blossom-end rot in tomato and pepper (de Freitas et al., 2012; Marcelis and Ho, 1999), and hard-end disorder in pear fruit (Lu et al., 2015; Wang et al., 2017). During the development of pear fruit, Ca2+ influx into the calyx end tissues, as well as free Ca2+ and storage Ca2+, was

lower in fruit with hard-end disorder than in normal fruit, although the calcium content was higher in hard-end fruit than normal fruit at harvest (Wang et al., 2017). CaCl2 treatment inhibited hard-end incidence from 81 % to 39 % in ‘Whangkeumbae’ pear fruit (Wang et al., 2017). An application of 4 % CaCl2 and 1% lecithin reduced the incidence of bitter pit from 35 % to 2 % in ‘Cox’s Orange Pippin’ apple fruit (Watkins et al., 1982). Preharvest and/or postharvest application of calcium alleviated scald incidence in ‘Golden Delicious’ apples (Raese and Drake, 2002; Gago et al., 2016). However, the relationship between calcium nutrition and superficial scald is still not completely understood. Preharvest bagging of fruit in the orchard during fruit development protects fruit from biotic and abiotic stress-related injuries, improving the microenvironment for fruit development, and increasing fruit quality (Sharma et al., 2014; Xu et al., 2010; Amarante et al., 2002). In China, Polyethylene (PE) bags are widely used in orchards to cover both pear and apple fruit due to their low cost and effective light transmission. The use of non-woven fabric bags has also increased in recent years as they are waterproof, have good air permeability, biodegradable, and can be recycled (Wang et al., 2017). Jakhar and Pathak (2016) found that pre-harvest bagging treatment improved mango fruit quality by increasing sugar content, reducing black spots and weight

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Corresponding author. E-mail address: [email protected] (S.-l. Yang). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.postharvbio.2020.111149 Received 22 November 2019; Received in revised form 10 February 2020; Accepted 10 February 2020 0925-5214/ © 2020 Elsevier B.V. All rights reserved.

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Excel 2010 software were used for data analysis (Qu et al., 2016).

loss. In the present study, the incidence of superficial scald and calcium nutrition in Chili’ pear (Pyrus bretschneideri) fruit during cold storage was investigated in response to different preharvest bagging treatments (polyethylene vs. non-woven fabric bags).

2.6. Calcium ion flux An approximately 1 cm2 portion of peel tissue with a thickness of about 0.3 mm was removed from the equatorial portion of the pears. Peel tissues were rinsed with distilled water and equilibrated in the measuring solution (10−4 mol L−1 CaCl2, 10−4mol L−1 KCl2, 3 × 10−4 mol L−1 MES, PH 6.0) for 30 min in preparation for measuring calcium ion flux. The peel tissues were transferred and fixed in a petri dish containing 8 ml of measuring solution. The calcium ion flux at the peel was measured using a Non-invasive Micro-test Technology [NMT100 Series, Younger USA LLC, Amherst, MA 01002, USA; Xuyue (Beijing) Sci. &Tech. Co., Ltd., Beijing, China]. The NMT measurement rationale was according to Zhang et al. (2009). Data and image acquisition of the microscope stage were performed with ASET software. Three fruit of each treatment were used for measurement and 3 equatorial portions were taken from each fruit.

2. Materials and methods 2.1. Preharvest, fruit bagging treatments, postharvest treatments, and sampling protocol Fifteen-year-old ‘Chili’ pear trees in an orchard located in Laiyang City, China were used in the study. On June 17, 2017, 200 fruit were covered with PE bags, 200 fruit with non-woven fabric bags, and 200 fruit received no bagging treatment. Water and fertilizer management was consistent throughout the fruit growth process. Fruit were harvested on September 28, 2017, 180 d after anthesis and the bags were removed. Fruit maturity were determined at harvest. Ten fruit were used to assess firmness and soluble solids content (SSC). Firmness was measured on the opposite sides of each fruit using a texture analyzer (CT3-4500, Brookfield, USA) equipped with a 2 mm diameter probe, and the expressed juice was used for SSC measurement with a refractometer (Atago PR101, Atago Co., Japan). Fruit without disease, insect pest, or physical damage were immediately transported to the laboratory and were placed in storage at 2 ± 0.5 °C. Fruit samples were taken every 15 days. Pear peel and flesh tissues were separately collected, frozen in liquid nitrogen, and stored at -80 °C. In the year of 2018, PE bags and non-woven fabric bags were covered on June 16, and the fruit was harvested on September 28, 2018. The PE bagged fruit were separated as two groups, one group were dipped in 2 % CaCl2 for 15 min and another group were subject without CaCl2 in the air. After that, all fruit were stored at 2 ± 0.5 °C.

2.7. Quantitative real-time PCR (qRT-PCR)

Scald incidence was assessed on all fruit of each treatment at 0, 15, 30, 45, 60, 75 and 90 d after harvest, and calculated as percentages.

Samples of ‘Chili’ pear peel tissue from each of treatments sampled at 0 d, 30 d, and 60 d after harvest were selected for qRT-PCR. RNA was extracted with a RNA prep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (TIANGEN, China), and was then reverse transcribed into cDNA using the Prime Script™ RT reagent Kit (Takara, Japan). Gene-specific primers used for the qRT-PCR analysis and actin gene, which was used for normalization, were designed using Primer Premier 5. The primer sequences are listed in Table S1. qRT-PCR was carried on each sample in a total volume of 20 μL which included 1 μL of cDNA, 0.8 μL of each primer, and 10 μL of 2 × chamQ SYBR Color qPCR Master Mix (Vazyme, China), and was performed on a Light Cycler® 480 instrument (Roche, Switzerland). Transcript levels were determined using the 2−ΔΔCt method (Livak and Schmittgen, 2000). A negative control without template was included for each primer pair in each run. Three replicates were performed for each gene.

2.3. Weight loss

2.8. Statistical analyses

The weight was of nine fruit from the different bagging treatments and the control was recorded at 0, 30, 60, and 90 d after harvest and percent weight loss was calculated as follows: Weight loss (%) = (Weight 0 d −Weight 30/60/90 d) / Weight 0 d × 100 %. Three replicates were performed on each treatment.

Figures were generated and significant differences were analyzed using GraphPad Prism software version 7.0 for Mac (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS software (version 19.0, IBM, USA), respectively.

2.2. Incidence of superficial scald

3. Results 2.4. Determination of Ca, K, Mg, Na, P, S, B, Fe, Mn and Zn contents 3.1. Harvest maturity Ten grams of peel samples collected at 0 and 90 d after harvest were used to determine the mineral elements as described by Wang et al. (2018). Peel samples were dried at 105 °C for 30 min and then maintained at 80 °C in the oven to a constant weight. The level of Ca, K, Mg, Na, P, S, B, Fe, Mn, and Zn were analyzed using an anatomic absorption spectrophotometer (AAnalyst100, PerkinElmer Inc., USA). Three replicates were performed on each treatment. 2.5. Ca

2+

On the harvest day, fruit flesh firmness of pear fruit were about 3 N in 2017, and were from 3.32 N to 3.59 N in 2018. SSC of PE bagging fruit were lower than non-woven fabric bagging fruit and no bagging fruit both in 2017 and 2018. SSC increased in non-woven fabric bagging fruit and control fruit harvest in 2018, but it was similar of the PE bagging fruit in 2018 with fruit in 2017 (Table 1). 3.2. The incidence of superficial scald and weight loss in pear fruit

localization

‘Chili’ pear exhibited significantly different appearance in response to the different bagging treatments. At harvest day (0 d), the fruit from the PE bags were more green relative to the control and had large lenticels. In contrast, the peel tissues from pears that been in the nonwoven fabric bags were either bright yellow and green with sparsely distributed, small lenticels (Fig. 1A). Pear fruit from the PE bags began to develop scald at 15 d after harvest, and continued to increase during cold storage. In some cases, the entire fruit had scald. In contrast, fruit obtained from the non-woven fabric bags did not develop scald during

Observations of calcium ion fluorescent staining was conducted as described by Qu et al. (2012) with some modifications. Five fruit from each bagging treatment and the control sampled at 0 and 90 d after harvest were sliced along the equator and immersed in a 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPE) buffer. After staining with 10−5 mol L−1 fluo-4/AM fluorescent dye, the samples were placed at 4 °C in the dark for 2 h. Then, samples were placed on slides and observed at 10 X under a BP500-550 scanning laser microscope (TCSSP5 Ⅱ, Leica, Germany). Image-pro Plus software and Microsoft 2

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loss of fruit during postharvest cold storage while non-woven fabric bags did not significantly influence this process, compared to nonbagged control fruit.

Table 1 SSC and firmness of pear fruit on the harvest day. Treatment

Control PE bagging Non-woven fabric bagging

2017

2018

SSC (%)

Firmness(N)

SSC (%)

Firmness(N)

11.90 ± 0.31 a 10.40 ± 0.68 b 11.70 ± 0.41 a

3.00 ± 0.57 a 3.00 ± 0.56 a 3.01 ± 0.48 a

12.78 ± 0.17 a 10.58 ± 0.34 b 12.83 ± 0.63 a

3.32 ± 0.54 a

3.3. Mineral element contents The Ca content in fruit obtained from PE or non-woven fabric bags at 0 d was higher than that in control fruit. Contents in peel tissues of fruit from PE bags was lower than that in control fruit, while the Ca content of peel tissues of fruit obtained from non-woven fabric bags was significantly higher than that in control fruit at 90 d after harvest. In addition, P, K, Na, Fe and Zn content in peel tissues of fruit obtained from PE bags was significantly lower than that in control fruit. No differences in the Mg, B and Mn content of peel tissues was observed between fruit obtained from the PE bagging treatment and the control. Similar to that of Ca, the contents of K, Mg, and Mn in peel tissues of fruit from the non-woven fabric bagging treatment were higher than that in the control fruit. No difference in the Na content of peel tissues was observed in fruit obtained from the non-woven fabric bagging treatment and peel tissues of control fruit. Based on these results, we speculate that the occurrence of superficial scald may be caused by an imbalance in mineral elements (Fig. 2).

3.68 ± 0.46 a 3.59 ± 0.34 a

Different letters represent significant differences (P < 0.05) between different bagging.

cold storage. In the case of the fruit that was not bagged (control), a low level of scald was evident at 90 d after harvest (Fig. 1A). There was a greater incidence of scald in fruit from the PE bag treatment compared with unbagged fruit and fruit from the non-woven fabric bags (Fig. 1B). The incidence of superficial scald in the fruit bagged with PE bags was 0, 42 %, 55 %, 63 %, 73 %, 92 % and 97 % at 0, 15, 30, 45, 60, 75, and 90 d after harvest during cold storage, respectively. On the other hand, the incidence of superficial scald was 20.0 % and 0.0 % in fruit that was not bagged or had been placed in non-woven fabric bag, respectively at 90 d after harvest. These results indicate that preharvest PE bagging of fruit increases the incidence of superficial scald in subsequent postharvest storage at low temperatures while non-woven fabric bags prevent superficial scald. The weight loss rate of fruit obtained from the different bagging treatments was measured at 0, 30, 60, and 90 days of cold storage. Results indicated that fruit obtained from PE bags experienced a greater loss in weight (24.9 % at 90 d after harvest) than control fruit or fruit obtained from non-woven fabric bags (12.2 % and 11.9 %, respectively). This indicates that PE bagging significantly increases weight

3.4. Ca2+ location in fruit tissues To further verify whether the occurrence of scald is related to calcium imbalance, the localization of free Ca2+ in pear flesh was observed using fluo-4/AM. Low levels of free Ca2+ were observed in the flesh of fruit from the non-woven fabric bagging treatment at 0 d after harvest, but not in fruit obtained from the PE bagging treatment or control fruit (Fig. 3). High levels of free Ca2+ were observed in the flesh of pear fruit obtained from the non-woven fabric treatment and control fruit at 90 d after harvest, whereas no free Ca2+ was observed in the flesh of pear fruit obtained from the PE bagging treatment. These results Fig. 1. Superficial scald development and weight loss of cold-stored ‘Chili’ pear fruit A. The appearance of fruit from the different bagging treatments. Photos are representative of no bagging, PE-bagged (bagged 65 days after anthesis), and non-woven fabricbagged (bagged 65 days after anthesis) ‘Chili’ pear fruit at 0 d, 30 d, 60 d, and 90 d after harvest. B. Percent incidence of superficial scald incidence in pear fruit from the different bagging treatments. C. The weight loss of pear fruit during postharvest storage. Data of weight loss represent the mean ± SD (standard deviation) of three independent biological replicates. Different letters represent significant differences (P < 0.05) between the different bagging treatments.

3

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Fig. 2. Minerals content in pear fruit obtained from the different bagging treatments over the course of postharvest storage at low temperature. Ca: calcium; K: potassium; Mg: magnesium; Fe: ferrum; B: boron; P: phosphorus; Na: sodium; Zn: zinc; Mn: manganese; S: sulfur. The data represent the mean ± SD of three biological replicates. Different letters represent significant differences (P < 0.05) between the different bagging treatments.

indicate that the absence of free Ca2+ in fruit obtained from the PE bagging treatment may represent an important factor responsible for increasing the incidence of superficial scald.

3.5. Analysis of the Ca

2+

flux in peel tissues of pear fruit during storage

Ca2+ fluxes were measured in fruit peel tissues using NMT technology (Fig. 4C). Results revealed that all three treatments exhibited differences in Ca2+ influx in peel tissues at 0 d vs. 90 d (Fig. 4A and B). Ca2+ influx in peel tissues of control fruit decreased with prolonged Fig. 3. The distribution of calcium ions in cells of pear flesh as observed by fluorescence staining and with a scanning laser microscope. The figure contains bright field, fluorescent field (fluo-4/AM), and merged images in which the amount and intensity of green fluorescence indicates the level of calcium ions in flesh cells of fruit obtained from the different bagging treatments. The fluorescent dye used to stain calcium ions was fluo-4 /AM at a working concentration of 10−5 mol L−1. Scale bar =150 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

4

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Fig. 4. Changes in Ca2+ flux between 0 and 300 s at the peel cells. Samples were obtained from the different bagging treatments. A. Time course of Ca2+ flux in samples of pear fruit at 0 d after harvest measured using Non-invasive Micro-test Technology (NMT). B. Time course of Ca2+ flux in samples of pear fruit at 90 d after harvest measured using NMT. C. The image shows the detection of Ca2+ flux in fruit peel tissues. D. The mean value of Ca2+ flux between 0 and 300 s in samples of pear fruit obtained from the different bagging treatments. The data represent the mean ± SD from at least three biological replicates of 3 fruit for each treatment Different letters represent significant differences (P < 0.05) between different bagging treatments.

fruit obtained from PE bags was monitored over the course of storage. Results indicated that the CaCl2 treatment decreased the incidence of superficial scald in fruit obtained from PE bags (Fig. 6). The incidence of superficial scald in CaCl2-treated fruit was lower from 15 d to 75 d after harvest, compared to untreated fruit obtained from PE bags.

storage time, while the same trend was observed in fruit peel tissues of fruit obtained from the PE or non-woven fabric bags. Moreover, the PE bagging treatment induced a strong and steady Ca2+ influx, which measured 5.48 mmol m−2 s−1 at 0 d and 1.44 mmol m−2 s−1 at 90 d after harvest. Ca2+ influx in peel tissues of fruit obtained from the PE bagging treatment was significantly higher than in peel tissues of unbagged (control) fruit and fruit obtained from the non-woven bagging treatment (Fig. 4D).

4. Discussion Superficial scald development in the skin of pear is an important storage problem, and it is different among varieties and maturities (Zoffoli et al., 1998). Here, the incidence of scald was higher in PE bagging fruit which has lower SSC level on the harvest day, which might in agreement with the literatures generally regarded superficial scald as a maturity-related disorder more prevalent on immature than on mature fruit (Ginsburg et al., 1969; Truter et al., 1994). Ca2+, as a ubiquitous signaling messenger, plays a key role in plant tissue growth and development, cell wall structure, and stress responses by relaying endogenous and exogenous signals to appropriate cellular responses (Clapham, 2007; Dodd et al., 2010; Kudla et al., 2010; Reddy et al., 2011). Ca2+ homeostasis is responsible for maintaining normal plant growth (Conn et al., 2011; Cybulska et al., 2011; Freitas et al., 2012), and it has been reported that localized deficiencies in calcium results in cell wall and membrane degradation, and causes several disorders in developing fruit, such as blossom end rot (Freitas et al., 2012). A high incidence of scald was found in the fruit that had been covered in PE bags during fruit development. The study also documented that when fruit obtained from PE bags was immersed in a 2 % CaCl2 solution after harvest, the incidence of physiological scald during storage could be reduced or delayed. Actually, preharvest bagging affect the Ca uptake through stem, while postharvest Ca dipping affect the Ca uptake might through stem or fruit surface. About the mineral nutrition analysis, fruit tissue from PE bagged pear fruit with visible superficial scald symptoms also had lower levels of Ca content in peel tissues than healthy fruit or fruit that had been covered with non-woven fabric bags. Which was consistent with the results of Sharples (1979) that low calcium could cause the occurrence of superficial scald in apples. In addition to calcium, the content or ratio of other mineral elements is also the factors that causes fruit physiological disorders

3.6. Expression of Ca-related genes In the current study, eight Ca-related genes with increased transcript abundance were identified in fruit obtained from the PE bagging treatment at harvest using high-throughput sequencing (Wang et al., 2017; Fig. 5A). These genes encode Ca2+ sensors and Ca2+ channel proteins. To further validate gene expression patterns, qRT-PCR was conducted at 0, 30, and 60 d after harvest. Results indicated that the expression of calcium-like (CML) genes, PbCML19, PbCML38, PbCML5, PbCML42-1, PbCML42-2, and PbCML23 significantly increased in transcript abundance in peel tissues of fruit obtained from the PE bagging treatment over the course of cold storage, relative to samples obtained from control fruit obtained from the non-woven fabric bagging treatment (Fig. 5C–H). PbCML11 transcript levels significantly increased at 0 and 30 d in peels of fruit obtained from the PE and non-woven fabric bagging treatments, and then decreased at 60 d after harvest (Fig. 5B). A Ca2+ channel (PbCNGC1, cyclic nucleotide-gated ion channel 1) gene was up-regulated in peels of fruit obtained from the PE and non-woven fabric bagging treatments at 0 d after harvest (Fig. 5I). At 90 d, however, its expression was significantly higher in fruit obtained from the PE bags than in fruit obtained from the no bagging control or the nonwoven fabric bags. These data indicate that increased transcript levels of Ca2+ sensors and Ca2+ channel genes in fruit obtained from the PE bagging treatment may contribute to the greater incidence of superficial scald through their impact on Ca2+ homeostasis. 3.7. Superficial scald incidence with and without postharvest calcium chloride (CaCl2) treatment The incidence of superficial scald in calcium treated and non-treated 5

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Fig. 5. The expression pattern of calcium-related genes in pear fruit from the different bagging treatments. A. Heat map: changes in gene expression are indicated by RPKM (RPKM = total exon reads / mapped reads (millions) × exon length (KB)) values. Different shades from blue to yellow denote the RPKM values. Orange indicates that the threshold of 100 was exceeded. CML, Calmodulin-like protein; CNCG1, Cyclic nucleotide-gated ion channel 1. B-I. The expression levels of calciumrelated genes in A were verified by qRT-PCR. The data indicate the mean ± SD from three biological replicates. Different letters represent significant differences (P < 0.05) between different bagging treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

observed in fruit obtained from non-woven fabric bags; indicating that the calcium deficiency may result from alterations in cellular Ca2+ partitioning and distribution. The NMT results also indicated that fruit obtained from PE bags had higher Ca2+ influx, which may indicate that it was more deficient in Ca2+. These results are consistent with previous studies which demonstrated that abnormal distribution of Ca2+ and the lack of Ca2+ influx in cells and tissues may be the cause of calcium deficiency disorders (Dong et al., 2015; Freitas et al., 2010;

(Wang et al., 2018). Here we also found that the content of Mg, Mn, B, P, K, and Fe was all lower in PE bagging fruit. Consistent with our results, tomato plants grown in a low calcium nutrient solution had a high incidence of blossom end rot (Coolong et al., 2014), suggesting that calcium deficiency may contribute to the occurrence of physiological disorders in fruit. The use of fluorescence imaging of calcium using a scanning laser microscope indicated that the distribution of calcium ions in PE bagged fruit was significantly lower than the levels 6

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during low-temperature storage was investigated; as well as the ability of calcium to modulate the incidence of superficial scald. Our results suggest that preharvest bagging of fruit in PE bags dramatically increases the incidence of superficial scald in pear fruit during low-temperature storage, which may be at least partially due to an impairment in Ca2+ homeostasis in fruit resulting in a Ca2+ imbalance during cold storage. Immersing fruit in a 2 % CaCl2 solution prior to storage at low temperature reduced the incidence of superficial scald in pear fruit obtained from the PE bags. Our data indicates that Ca2+ plays a crucial role in regulating the occurrence of superficial scald in pear fruit. Author statement We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We understand that the Corresponding Author is the sole contact for the Editorial process. He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. The authors contribution are as followings: Shaolan Yang and Chenxia Cheng conceived and designed the experiments, Qian Li and Caihong Wang performed the experiments and analyzed the data, Xinfu Zhang and Shaolan Yang wrote the manuscript. All authors read and approved the manuscript.

Fig. 6. Superficial scald incidence of ‘Chili’ pear fruit from the PE bagging treatment with and without postharvest treatment with 2 % CaCl2. A. The appearance of PE-bagged fruit from untreated control (no calcium dip) and the CaCl2 treatment. Photos are representative of PE-bagged (bagged 65 days after anthesis) fruit from the untreated control and 2% CaCl2 treatment at 0 d, 30 d, and 60 d after harvest. B. Percentage incidence of superficial scald in control (untreated) and 2 % CaCl2 treated pear fruit obtained from the PE bags. Eightyfour fruit were evaluated from each treatment to determine the percentage incidence of superficial scald.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements

Park et al., 2005; Tonetto de Freitas et al., 2011). Ca2+ signal transduction moves through activated Ca2+ channels, such as the CNGC, and through some Ca2+-binding proteins such as CaMs, CMLs, calcium-dependent protein kinases (CDPKs), and Calcineurin-B-like (CBL) proteins (White and Broadley, 2003). After binding Ca2+, these proteins can activate downstream pathways leading to specific cellular responses to different types of stimuli (White and Broadley, 2003). In our study, the expression pattern of PbCNGC and seven calmodulin genes were analyzed. Results indicated that PbCML19, PbCML38, PbCML5, PbCML42-1, PbCML42-2 and PbCML23, as well as PbCNGC1, were induced during the storage of pear fruit that had received the PE treatment. Calmodulin genes were also induced in pear fruit exhibiting superficial scald. These results are consistent with a study by Wang et al. (2017) of pear fruit with hard-end disorder, where the expression of PbCML4 was up-regulated in the crown of hardend fruit (Wang et al., 2018). Based on our results, we suggest that CMLs play a role in the high incidence of superficial scald in PE-bagged pear fruit. Many CML genes in Arabidopsis (Arabidopsis thaliana) are stress responsive (McCormack et al., 2015; Zhu et al., 2015). Up-regulation of AtCML9 occurs in plants exposed to salt, cold, or dehydration stress (Magnan et al., 2008). In our study, calmodulin genes were upregulated during low-temperature storage by the abnormal distribution of Ca2+ and the lack of free Ca2+ ions in cells; similar to the response to environmental stress in Arabidopsis. Based on the collective results of our study, we speculate that the occurrence of superficial scald is related to an imbalance in the distribution and levels of calcium.

This Science Science Modern

work was supported by the Project of Shandong Natural Foundation (ZR2017MC006), Project of National Natural Foundation of China (31201608), the Project of Shandong Fruit Technology Industry System (SDAIT-06-06).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2020. 111149. References Amarante, C., Banks, N.H., Max, S., 2002. Effect of preharvest bagging on fruit quality and postharvest physiology of pears (Pyrus communis). N. Z. J. Crop Hortic. Sci. 30, 99–107. https://doi.org/10.1080/01140671.2002.9514204. Argenta, L.C., Fan, X., Mattheis, J.P., 2007. Responses of’ Golden Delicious’ apples to 1mcp applied in air or water. Hortic. Sci. 42. https://doi.org/10.21273/hortsci.42.7. 1651. Busatto, N., Farneti, B., Commisso, M., Bianconi, M., Iadarola, B., Zago, E., Ruperti, B., Spinelli, F., Zanella, A., Velasco, R., Ferrarini, A., Chitarrini, G., Vrhovsek, U., Delledonne, M., Guzzo, F., Costa, G., Costa, F., 2018. Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes. Plant J. 93, 270–285. https://doi.org/10.1111/tpj.13774. Clapham, D.E., 2007. Calcium signaling. Cell 131, 1047–1048. https://doi.org/10.1016/ j.cell.2007.11.028. Conn, S.J., Gilliham, M., Athman, A., Schreiber, A.W., Baumann, U., Moller, I., Cheng, N.H., Stancombe, M.A., Hirschi, K.D., Webb, A.A., Burton, R., Kaiser, B.N., Tyerman, S.D., Leigh, R.A., 2011. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis. Plant Cell 23, 240–257. https://doi.org/10.1105/tpc.109.072769. Coolong, T., Mishra, S., Barickman, C., Sams, C., 2014. Impact of supplemental calcium chloride on yield, quality, nutrient status, and postharvest attributes of tomato. J. Plant Nutr. 37, 2316–2330. https://doi.org/10.1080/01904167.2014.890222. Cybulska, J., Zdunek, A., Konstankiewicz, K., 2011. Calcium effect on mechanical properties of model cell walls and apple tissue. J. Food Eng. 102, 217–223. https://doi.

5. Conclusions In the present study, the effect of different preharvest bagging treatments on the postharvest incidence of superficial scald in pear fruit 7

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