Antioxidant metabolism in stem and calyx end tissues in relation to flesh browning development during storage of 1-methylcyclopropene treated ‘Empire’ apples

Postharvest Biology and Technology 149 (2019) 66–73

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Antioxidant metabolism in stem and calyx end tissues in relation to flesh browning development during storage of 1-methylcyclopropene treated ‘Empire’ apples Jinwook Leea,b, Lailiang Chenga, David R. Rudellc, Jacqueline F. Nocka, Christopher B. Watkinsa,

T

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a

Horticulture Section, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853, USA Department of Integrative Plant Science, Chung Ang University, Anseong, Gyeonggi-do 17546, Republic of Korea c USDA-ARS, Tree Fruit Research Laboratory, 1104 North Western Avenue, Wenatchee, WA 98801, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Malus x domestica 1-MCP Antioxidants Chilling injury Flesh browning Storage

Development of firm flesh browning, a physiological disorder, in ‘Empire’ apples during long term controlled atmosphere (CA) storage, can be enhanced by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. The disorder develops earlier in stem-end tissues than in calyx-end tissues. The antioxidant scavenging systems in the tissue zones of fruit stored under CA conditions (2 kPa O2/2 kPa CO2) at 3 °C at 6 and 10 months were investigated. Flesh tissue browning as indicated by lightness and hue angle was greater in 1-MCP treated than in untreated fruit, and in stem-end tissues than in calyx-end tissues. 1-MCP treatment decreased superoxide production as indicated by nitroblue tetrazolium (NBT) reducing activity but increased H2O2 concentrations, while treatment effects on malondialdehyde concentrations were inconsistent. Ascorbic acid (AsA) and glutathione (GSH) concentrations declined during storage, regardless of 1-MCP treatment, but were lower in stemend tissue than in calyx-end tissue. While ascorbate peroxidase (APX) activity was not affected by 1-MCP treatment, its activity in untreated fruit was lower in stem-end tissues than in calyx-end tissues. The activities of superoxide dismutase (SOD) and copper/zinc-superoxide dismutase (Cu/Zn-SOD) increased during storage. The activities of catalase (CAT) and peroxidase (POX) decreased in 1-MCP treated fruit but effects on activities of other enzymes were inconsistent. Overall, higher browning may be associated with lower AsA and GSH concentrations in stem-end tissues, but the enhanced browning resulting from 1-MCP treatment does not appear to be directly related to antioxidant metabolism.

1. Introduction Firm flesh browning is a chilling-related physiological storage disorder of apple fruit (Snowdon, 1990). ‘Empire’ apple is susceptible to developing this disorder during long term controlled atmosphere (CA) storage, and incidence of the disorder is greater as temperatures approach 0 °C (Jung et al., 2010; Watkins and Liu, 2010). Consequently, current storage recommendations are to store the fruit at 2 °C as a compromise between flesh browning development at lower temperatures and more rapid softening at 3 °C or higher with 2 kPa O2/ 2 kPa CO2 of CA storage system. However, flesh browning symptoms that cannot be distinguished from chilling injury can occur when fruit are treated with the ethylene action inhibitor 1-methylcyclopropene (1MCP) and stored at temperatures of 2 °C or higher (Watkins, 2008). Higher browning in 1-MCP treated fruit is associated with treatment of

more mature fruit (Doerflinger et al., 2015). Risk of flesh browning development is therefore a serious limiting factor for the long term CA storage of the cultivar. The disorder, sometimes called firm flesh browning, is illustrated in James et al., (2010). A number of aspects of 1-MCP-exacerbated flesh browning have been investigated, including antioxidant metabolism (Lee et al., 2012a) and metabolomic changes (Lee et al., 2012b). It has been hypothesized by Jung and Watkins (2011) that flesh browning may result from inhibition of ethylene production by the fruit, either at low temperatures (0.5 °C) with and without 1-MCP, where chilling injury limits ethylene production, or at higher temperatures (3–4 °C), when fruit have been treated with 1-MCP. Therefore, fruit under these conditions are unable to produce ethylene required for normal metabolism. In general, flesh browning in plant tissues is thought to be associated with enzymatic oxidative reaction of phenolic compounds catalyzed by polyphenol

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Corresponding author. E-mail addresses: [email protected] (J. Lee), [email protected] (L. Cheng), [email protected] (D.R. Rudell), [email protected] (J.F. Nock), [email protected] (C.B. Watkins). https://doi.org/10.1016/j.postharvbio.2018.11.015 Received 11 May 2018; Received in revised form 4 October 2018; Accepted 20 November 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.

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At harvest, and at 6 and 10 months, five fruit per treatment were sampled immediately on removal from storage, each fruit being processed individually without warming. Fruit were cut horizontally across the stem- and calyx-end regions and the tissues flash frozen in liquid nitrogen and stored at -80 °C until analysis. Peel tissues were removed while frozen, and the flesh tissues cryogenically milled to a fine powder using an IKA® A11 basic rotary mill (IKA® Works, Inc. Wilmington, NC, USA) and stored at -80 °C. The other side of each cut surface was used to assess color variables as described below.

oxidase (PPO) (Tomás-Barberán and Espin, 2001). In ‘Empire’ apples, PPO activity was usually higher in flesh tissues of 1-MCP treated fruit than in untreated fruit (Jung and Watkins, 2011). However, the role of phenolics and activity of enzymes, such as PPO, in development of physiological flesh browning disorders of apples and pears remains inconclusive (de Castro et al., 2008; Ma et al., 2015; Veltman et al., 1999). The concentrations of reactive oxygen species (ROS) are tightly controlled either by their production or removal by enzymatic and nonenzymatic antioxidant scavenging systems, such as AsA and glutathione (GSH) (Mittler, 2002). Lee et al. (2012a) found concentrations of various antioxidant and related enzymes of cortical flesh tissues were impacted by 1-MCP and storage temperature. 1-MCP treatment reduced superoxide concentrations, but enhanced those of H2O2 in the flesh of fruit stored at 3 °C. Lipid peroxidation, ostensibly driven by oxidative stress, increased in 1-MCP treated fruit at 0.5 °C, but not at 3 °C. However, 1-MCP treatment effects on the antioxidant metabolic scavenging system of whole fruit cortical tissues were greater at 3 °C than at 0.5 °C (Lee et al., 2012a). AsA and GSH concentrations were reduced by 1-MCP treatment later in storage, as was oxidized glutathione (GSSG), but not dehydroascorbate (DHA) concentrations. 1MCP treatment enhanced monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) enzymatic activities but not those of superoxide dismutase (SOD), Cu/ Zn-SOD, or ascorbate peroxidase (APX). Analysis of firm flesh browning development in ‘Empire’ has revealed that the disorder originates from the stem-end of the fruit (Lee et al., 2012a). Also, in ‘Cripps Pink’ apples, more flesh browning associated with CO2 injury was found in the stem-end than in other zones (de Castro et al., 2008), particularly radial flesh browning but not diffuse flesh browning (James and Jobling, 2009). In apples, calyx-end tissues have higher densities than stem-end tissues (James and Jobling, 2009), which is why the stem-end of fruit typically face up when floated on water. In pear fruit, gas diffusion properties were much higher in the vertical axis than in equatorial radius axis direction (Ho et al., 2006a, b). Ascorbic acid (AsA) concentrations were higher in calyx-end tissues than in stem-end tissues (de Castro et al., 2008). However, surprisingly little information is available about metabolic differences in different tissue zones of apple fruit. The primary objective of this work was to determine how 1-MCP treatment differentially impacts the enzymatic and non-enzymatic antioxidant scavenging systems of ‘Empire’ calyx and stem-end flesh tissues during CA storage at 3 °C. Because flesh browning occurs after 6 months as was reported in our previous work (Lee et al., 2012a), we focused in this study on differences between 6 and 10 months of storage.

2.2. Fruit color assessment Flesh color variables were measured at six sites within the calyx-end region (1.5 cm from the equator), equator, and stem-end region (1.5 cm from equator) using a Minolta chromameter (Model CR-300, Osaka, Japan). Color measurements were expressed as lightness (L*, dark to light on a scale of 0 –100) and hue angle (actual color, ho = 0–360) (McGuire, 1992). 2.3. Determination of superoxide anion radical, hydrogen peroxide, and lipid peroxidation, and antioxidant metabolite assays All methods followed in this work as exactly as described by Lee et al. (2012a) using the methods of Doke (1983) to assay superoxide anion radical production, Patterson et al. (1984) for H2O2 concentrations, and Hodges et al., (1999) for measuring malondialdehyde (MDA) concentrations. AsA and DHA concentrations were estimated according to Logan et al. (1998). GSH and GSSG were determined spectrophotometrically, using an enzymatic cycling method described by Griffith (1980). All concentrations are presented as units per kg on a fresh weight basis. 2.4. Antioxidant enzyme activities Enzyme extraction was carried out as described by Lee et al. (2012a). SOD activity was assayed by monitoring the inhibition of the photochemical reduction of NBT according to Giannopolitis and Ries (1977). Cu/Zn-SOD activity was also determined by monitoring the inhibition of the photochemical reduction of NBT (Frei et al., 2010). APX activity was determined using the methods of Nakano and Asada (1981), and Grace and Logan (1996). CAT activity was determined using the method described by Aebi (1984). MDHAR activity was assayed by monitoring the decrease in absorbance at 340 nm over 3 min due to NADH oxidation (Miyake and Asada, 1992). DHAR activity was measured by the method of Nakano and Asada (1981). GR activity was determined by the method described by Grace and Logan (1996). POX activity was measured by the method described by Hammerschmidt et al. (1982) using guaiacol as the hydrogen donor. Protein concentrations were determined using the Bradford assay (Bradford, 1976) according to the manufacturer’s micro-assay protocol (Sigma-Aldrich Co., St. Louis, MO, USA). All enzyme activities were expressed on a protein basis.

2. Materials and methods 2.1. Fruit source, treatments, storage conditions, and fruit sampling ‘Empire’ apple (Malus domestica Borkh) fruit used in this experiment were harvested from mature trees at the Cornell University orchard at Lansing, NY, USA. Uniform sized (6.9 cm in length and 8.2 cm in diameter) fruit were randomly sorted into experimental units of 5 fruit. Mean values (n = 10) for fruit maturity and quality attributes at harvest, carried out as previously described (Doerflinger et al., 2015), were internal ethylene concentration: 0.916 μL L−1; flesh firmness: 65.8 N; starch pattern index: 6.2; soluble solids concentration: 12.4%; titratable acidity:3.96 g kg−1. Fruit were then pre-cooled overnight at 3 °C and then, either treated or left untreated with 1 μL L−1 1-MCP (SmartFresh tablets, 0.36% a.i., AgroFresh Co., Spring House, PA, USA) for 24 h in a sealed 4 m3 plastic tent. Fruit were stored in 2 kPa O2/2 kPa CO2 balanced with N2 at 3 °C in 0.9 m3 stainless steel chambers (Storage Control Systems, Inc., Spartan, MI, USA) as described by Lee et al. (2012a). After sampling of fruit, atmospheres were restored within 6 h.

2.5. Statistical analysis All data were subjected to analysis of variance (ANOVA) using the general linear model (Proc GLM) to determine main effects and interactions. Data for 1-MCP treatment and storage duration were treated as independent. Storage treatment was not replicated as is common in postharvest research because of limitations of cold rooms and CA chambers, and therefore were treated as pseudo-replication. Means were compared using Fisher’s least significant difference (LSD) test, P = 0.05. Experimental data are presented as means of five individual fruit replicates. Pearson correlation coefficient analysis was performed to determine relationships between color variables and antioxidant metabolites by using SAS version 9.3 (SAS Institute, Cary, NC, USA). 67

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fruit by 10 months. In the calyx tissues, H2O2 concentrations in untreated fruit declined, while remaining similar to concentrations at harvest in 1-MCP treated fruit by 6 months. However, by 10 months, the H2O2 concentrations increased in tissues of both untreated and 1MCP treated fruit except for the untreated stem-end tissue, although concentrations tended to be higher in 1-MCP than in untreated fruit. Lipid peroxidation as indicated by MDA accumulation was different between tissues of untreated and 1-MCP treated fruit over time (Table 1). In control tissues, lipid peroxidation decreased by 6 months before increasing in 10 months, while opposite changes occurred in 1MCP treated tissues, except at 6 months.

LSMEANS statements were used in PROC GLM from SAS (version 9.3, SAS Institute Inc., Cary, NC, USA) to derive means for the untreated and 1-MCP treated fruit. The CONTRAST option was used to test comparisons depending on treatment and tissue localization effects at 6 or 10 months. Antioxidant metabolite data were analyzed using partial leastsquares discriminant analysis (PLS-DA) (Pérez-Enciso and Tenenhaus, 2003). The antioxidant metabolites were considered to be predictor variables, whereas treatment factors (untreated or 1-MCP-treated), storage duration (duration, 0–10 month), and lightness values (flesh tissue color) were considered to be response variables. Treatments factors were introduced as separate categorical variables (reading either -1 or 1), whereas storage duration and lightness values were included as continuous variables. Both X- and Y-data were mean centered and scaled to unit variance to give all variables an equal chance to influence the model. PLS-DA was performed using The Unscrambler (version 10.0, Camo A/S, Trondheim, Norway).

3.3. AsA and GSH AsA concentrations were lower in stem-end than calyx-end tissues, with a decreasing trend from harvest to 6 and 10 months, respectively (Table 2). AsA concentrations were not impacted by 1-MCP treatment, but they were influenced by storage duration and tissue location. By contrast, DHA concentrations were affected only by an interaction between storage duration and 1-MCP treatment; concentrations were similar in both tissue types of untreated and 1-MCP treated fruit until 6 months, but then increased in tissues of untreated fruit and declined in 1-MCP treated fruit (Table 2). The AsA/(AsA + DHA) ratio decreased during storage, but was also affected by tissue location. GSH concentrations tended to be lower in stem-end tissue than in calyx-end tissues but were not affected by 1-MCP treatment (Table 2). However, stem- and calyx-end GSH concentrations changed differentially over time with GSH declining gradually during storage in the calyx-end, while increasing in stem-end tissues until 6 months and then declining by 10 months. No other interactions among treatments were detected. GSSG concentrations were affected by an interaction among storage duration, 1-MCP treatment, and tissue type (Table 2). GSSG levels were higher in stem-end than in calyx-end tissues at harvest, and increased consistently in calyx-end tissues, but changes during storage were not consistent in stem-end tissues. Changes of the GSH/(GSH + GSSG) ratio were not consistent in stem-end tissues but the ratio decreased in calyx-end tissues during storage in control and 1-MCP treated fruit (Table 2).

3. Results 3.1. Flesh tissue color Color responses of stem-end and calyx-end flesh were assessed using the L* and ho parameters (Table 1). The L* values were relatively stable for 6 months, but then declined in the stem-end tissues of untreated fruit. 1-MCP treatment reduced L* values of stem-end tissues more than in control fruit, while the L* values changed little in the calyx-end tissues of untreated fruit. A similar pattern of change of ho values was found, except that a gradual decline of values occurred in all tissues over time, before decreasing more rapidly at 10 months in 1-MCP treated stem-end tissues. 3.2. Superoxide anion generation, H2O2 concentrations and lipid peroxidation Superoxide anion production as indicated by NBT reducing activity was mostly lower in stem-end tissues than in calyx-end tissues (Table 1). NBT reducing activity increased during storage, but to a greater extent in calyx-end tissues than in stem-end tissues of untreated fruit. Activity was higher in stem-end and especially calyx-end tissues of untreated fruit, compared with those of 1-MCP treated fruit. H2O2 accumulation was affected by an interaction among storage duration, 1-MCP treatment, and tissue location (Table 1). In stem-end tissues, H2O2 concentrations of both control and 1-MCP treated fruit decreased by 6 months, but then increased in tissues of 1-MCP treated

3.4. Antioxidant enzyme activities APX activity in both the stem-end and calyx-end flesh remained similar until 6 months (Table 3). Activity increased in stem-end tissues of 1-MCP treated fruit but was inconsistent in untreated fruit, while in calyx-end tissues, activity increased in untreated fruit and remained

Table 1 Lightness (L*), hue angle (h°), nitroblue tetrazolium (NBT) reducing activity, and the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in stem-end and calyx-end localized tissues of ‘Empire’ apple fruit untreated, or treated with 1 μL L−1 1-MCP at harvest, and then stored at 2 kPa O2/2 kPa CO2 for 6 and 10 months at 3 °C. Means are for five individual fruit replicates. The least significant difference (LSD) represents P = 0.05. Storage duration (months)

0 6 10

Treatment

Control Control 1-MCP Control 1-MCP

LSD0.05 Storage duration (D) Treatment (T) Tissue location (L) D×T D×L T×L D×T×L

ho (0-360)

L* (0-100)

NBT activity (OD580 kg−1 s−1)

H2O2 (μmol kg−1)

MDA (μmol kg−1)

Stem

Calyx

Stem

Calyx

Stem

Calyx

Stem

Calyx

Stem

Calyx

81.6 81.6 81.1 79.7 77.2

81.6 81.7 81.4 82.3 79.2

105.4 101.1 101.8 95.6 91.6

106.5 103.0 103.0 100.2 96.1

0.017 0.094 0.056 0.222 0.183

0.028 0.164 0.175 0.272 0.178

129.5 101.8 96.9 100.0 147.8

119.6 95.6 114.3 149.2 162.2

6.8 5.2 7.4 6.2 4.2

5.9 3.9 8.8 6.8 4.4

1.94 0.0029 0.0033 0.0589 0.0264 0.0695 0.8554 0.7165

2.82 < 0.0001 0.0264 0.0016 0.0092 0.8351 0.1173 0.8831

0.051 < 0.0001 < 0.0001 0.0649 0.9235 0.2232 0.0532 0.8989

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16.60 < 0.0001 0.0007 0.0262 0.0268 0.0038 0.5841 0.0067

1.36 0.0093 0.0667 0.9166 < 0.0001 0.6907 0.0972 0.0294

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Table 2 Ascorbic acid (AsA), dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) concentrations on a fresh weight basis, and ratios of AsA to (AsA + DHA) and GSH to (GSH + GSSG) in stem-end and calyx-end localized tissues of ‘Empire’ apple fruit untreated, or treated with 1 μL L−1 1-MCP at harvest, and then stored at 2 kPa O2/2 kPa CO2 for 6 and 10 months at 3 °C. Means are for five individual fruit replicates. The least significant difference (LSD) represents P = 0.05. Storage duration (months)

0 6 10

Treatment

Control Control 1-MCP Control 1-MCP

LSD0.05 Storage duration (D) Treatment (T) Tissue location (L) D×T D×L T×L D×T×L

AsA (μmol kg−1)

DHA (μmol kg−1)

AsA/ (AsA + DHA)

GSH (μmol kg−1)

GSSG (μmol kg−1)

GSH/ (GSH + GSSG)

Stem

Calyx

Stem

Stem

Calyx

Stem

Calyx

Stem

Calyx

Stem

162.4 136.2 148.6 125.1 96.8

200.1 172.1 172.5 131.0 119.5

40.1 38.2 40.0 42.3 31.4

0.79 0.78 0.79 0.75 0.76

0.84 0.84 0.83 0.72 0.79

21.3 25.5 23.9 20.3 17.8

32.8 29.8 26.1 23.5 21.1

3.5 3.5 2.0 2.4 3.2

1.8 2.6 3.0 3.4 3.9

0.86 0.88 0.92 0.89 0.84

28.82 < 0.0001 0.4402 0.0020 0.1353 0.4920 0.8909 0.4097

Calyx 38.4 34.1 35.2 47.7 32.8 12.74 0.8486 0.0592 0.6651 0.0214 0.4782 0.6518 0.7240

0.07 0.0029 0.3074 0.0411 0.2944 0.3490 0.4970 0.2919

4.59 0.0011 0.0933 0.0004 0.9507 0.1168 0.7137 0.7164

1.01 0.1654 0.8045 0.9287 0.0285 0.0098 0.1190 0.0365

Calyx 0.95 0.92 0.89 0.87 0.84 0.05 0.0045 0.3086 0.1304 0.0905 0.0509 0.3518 0.1182

SOD activity only changed with storage duration. Cu/Zn-SOD activity was neither altered by tissue location nor by 1-MCP treatment (Table 3). CAT activity was affected by storage duration, declining slightly by 6 months before increasing at 10 months (Table 3). POX activity was not affected by tissue location, but increased during storage (Table 3). However, POX activity increased more in untreated flesh, regardless of tissue type.

unchanged in 1-MCP treated fruit. MDHAR activity was lower in stemend than in calyx-end tissues at harvest, increasing from 6 months to 10 months in stem-end but not in calyx-end tissues (Table 3). DHAR activity increased between 6 and 10 months, regardless of 1MCP treatment and tissue location (Table 3). GR activity increased over time (Table 3). However, activity was not impacted by 1-MCP treatment in calyx-end tissues.

Table 3 Ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), superoxide dismutase (SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and guaiacol peroxidase (POX) activities on a fresh weight basis in stem-end and calyx-end localized tissues of ‘Empire’ apple fruit untreated, or treated with 1 μL L−1 1-MCP at harvest, and then stored at 2 kPa O2/2 kPa CO2 for 6 and 10 months at 3 °C. Means are for five individual fruit replicates. The least significant difference (LSD) represents P = 0.05. Storage duration (months)

0 6 10

Treatment

Control Control 1-MCP Control 1-MCP

APX (mmol s−1 kg−1)

0 6 10

Treatment

Control Control 1-MCP Control 1-MCP

LSD0.05 Storage duration (D) Treatment (T) Tissue location (L) D×T D×L T×L D×T×L

DHAR (mmol s−1 kg−1)

GR (mmol s−1 kg−1)

Stem

Calyx

Stem

Calyx

Stem

Calyx

Stem

Calyx

2.63 3.01 2.86 2.49 3.39

2.88 2.94 3.19 4.56 3.06

0.61 0.83 0.69 0.92 0.94

0.76 0.86 0.97 0.89 0.89

1.04 0.92 1.03 1.27 1.40

1.12 0.86 1.16 1.19 1.17

0.17 0.29 0.19 0.28 0.39

0.23 0.28 0.32 0.33 0.33

LSD0.05 Storage duration (D) Treatment (T) Tissue location (L) D×T D×L T×L D×T×L Storage duration (months)

MDHAR (mmol s−1 kg−1)

0.69 0.0140 0.4841 0.0387 0.3263 0.0346 0.0075 0.0003

0.14 0.0003 0.9202 0.0133 0.7696 0.0192 0.1692 0.1015

0.26 0.0013 0.0654 0.7259 0.2834 0.2779 0.9303 0.2049

SOD (1000 Unit kg−1)

Cu/Zn-SOD (1000 Unit kg−1)

CAT (mmol s−1 kg−1)

0.08 < 0.0001 0.6164 0.0544 0.0415 0.1510 0.6676 0.0049 POX (mmol s−1 kg−1)

Stem

Calyx

Stem

Calyx

Stem

Calyx

Stem

Calyx

1.54 0.92 1.17 2.04 1.73

1.23 0.82 0.93 1.61 1.63

0.63 0.45 0.80 1.12 0.71

0.47 0.46 0.62 0.60 0.66

0.07 0.04 0.04 0.07 0.06

0.07 0.05 0.05 0.07 0.06

0.37 1.27 1.97 6.77 4.34

0.49 1.38 2.60 8.09 4.06

0.52 < 0.0001 0.8909 0.0703 0.2450 0.9277 0.7250 0.3893

0.41 0.1749 0.7120 0.1057 0.0614 0.6163 0.5388 0.1332

69

0.01 < 0.0001 0.1142 0.4572 0.1475 0.3264 0.3087 0.7209

1.67 < 0.0001 0.0132 0.4341 < 0.0001 0.8421 0.5364 0.2278

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Fig. 1. Pearson correlation coefficient (r) matrices generated using changes of color variables, antioxidant metabolites and enzyme activities in stem-end tissue or calyx-end localized tissues of ‘Empire’ apple fruit untreated or treated with 1 μL L−1 1-MCP at harvest, and then stored at 2 kPa O2/2 kPa CO2 for up to 10 months at 3 °C. Red and blue colors indicated positive and negative correlation coefficients between variables. Data are means of five individual fruit replicates (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

(r=-0.637*) but positively with AsA (r = 0.650**) and GSH/(GSH + GSSG) (r = 0.515*) (Fig. 1,. lower right). Overall, correlations among antioxidant enzyme activities had higher correlation coefficients, not only in 1-MCP treatment than in control, but also in stem-end tissues than in calyx-end tissues.

3.5. Pearson correlations between L* and antioxidant metabolism Correlation coefficients were assessed for stem- and calyx-end tissues of individual fruit between 6 and 10 months (Fig. 1). The L* and ho values were correlated positively with AsA concentrations (r = 0.696* and r = 0.744*, respectively) in 1-MCP treated (Fig. 1, upper right) but not control flesh (Fig. 1. upper left). MDA and AsA concentrations were correlated positively with the L* and ho values (r = 0.598* and r = 0.641* for MDA, and r = 0.592* and r = 0.734** for AsA, respectively) only in stem-end flesh. In addition, the ho values correlated negatively with the activities of MDHAR, DHAR, GR, and POX activities (r=-0.620*, r=-0.618*, r=-0.676**, and r=-0.655**, respectively). AsA concentrations also correlated negatively with MDHAR, DHAR, and GR (r=-0.594*, r=-0.658**, and r=-0.634*, respectively), and GSH concentrations correlated negatively with DHAR and GR (r=0.673** and r=-0.519*, respectively; Fig. 1. lower left). In calyx-end tissues, the L* values correlated negatively with H2O2 concentration (r=-0.574*) and positively with DHA concentration (r = 0.573*). The ho values also correlated negatively with NBT activity (r=-0.618*), H2O2 (r=-0.812***), GSSG (r=-0.519*), MDHAR (r=-0.606*), and GR

3.6. The association of antioxidant metabolites with response variables PLS loading plots revealed associations among antioxidant metabolites and L*, storage duration, and 1-MCP treatment (Fig. 2). LV 1 and 2 from the loading plot for stem-end tissues accounted for 46% and 20% of the total X variance and 57% and 18% of the total Y variance, respectively (Fig. 2A). LV 1 and 2 for calyx-end tissues accounted for 47% and 26% of the total X variance and 46% and 32% of the total Y variance, respectively (Fig. 2B). The 1-MCP treatment variable was less impacted than L* and storage duration. AsA concentrations were closely linked with the L* variable in stemend tissues but not in calyx-end tissues. GSSG concentrations and GR activity were tightly associated with the storage duration in calyx-end tissues, but not consistently in stem-end tissues. However, 1-MCP was 70

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Fig. 2. PLS loading plots from models containing antioxidant metabolites and response variables in stem-end (A) or calyx-end (B) localized cortex tissues of ‘Empire’ apple fruit untreated, or treated with 1 μL L−1 1-MCP at harvest, and then stored at 2 kPa O2/2 kPa CO2 for up to 10 months at 3 °C.

with higher SOD activity but lower superoxide and H2O2 concentrations, and lower activities of GSH, GR and POX in stem-end tissues than in calyx-end tissues (Fig. 3B). In stem-end tissues, MDA and DHA concentrations were higher in 1-MCP treated fruit than in untreated ones (Fig. 3C). At 10 months, the contrast illustrates how superoxide and MDA concentrations, and POX and APX activities were lower in stem-end than in calyx-end tissues, while SOD and Cu/Zn-SOD activities were higher (Fig. 3D). However, 1-MCP treatment enhanced NBT reducing activity and Cu/Zn-SOD, POX, MDHAR, and GR activities in stem-end tissues but reduced H2O2 concentrations and GSSG activity (Fig. 3E). The other contrast focused on treatment factors to evaluate the effects only in stem-end tissues. 1-MCP treatment decreased NBT reducing activity, CAT activity, and GSH and DHA concentrations but increased MDHAR and DHAR activities in stem-end tissues (Fig. 3F).

relatively less tightly linked with any metabolite or enzyme activity rate in either the stem-end or calyx-end tissues. POX, NBT, APX, and DHAR activities were closely linked, regardless of treatment factors and tissue type. The loading values of SOD, CAT, GR, and MDHAR activities, and AsA concentrations were relatively unchanged in either plot, indicating that these antioxidant variables were less influenced by tissue type. 3.7. Overall antioxidant metabolic responses We focused on antioxidant metabolism at 6 and 10 months as the greatest changes in tissue browning occurred during these two time points (Fig. 3). At 6 months, MDA concentrations and the activities of APX and GR enzymes increased in stem-end tissues more than in calyxend tissues in untreated fruit (Fig. 3A). 1-MCP treatment was associated

Fig. 3. Schematic pathway of antioxidant metabolism (metabolites and enzyme activities) of both control and 1-MCP treated ‘Empire’ apple fruit stored in 2 kPa O2/ 2 kPa CO2 for 6 and 10 months at 3 °C. Data are means of five individual fruit replicates. A general linear model was generated to the significance of each contrast using SAS. Letters A and D indicate tests between stem- and calyx-end flesh of control fruit at 6 or 10 months, respectively; B and E indicate tests between stem- and calyx-end flesh of 1-MCP treated fruit at 6 or 10 months, respectively; C and F indicate tests between stem-end flesh of 1-MCP treated fruit and control at 6 or 10 months, respectively. AO, ascorbate oxidase; APX, ascorbate peroxidase; AsA, L-ascorbate; CAT, catalase; Cu/Zn-SOD, cupper/zinc-superoxide dismutase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; γ-EC, γ-glutamylcysteine; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced form of glutathione; GSSG, oxidized form of glutathione; GST, glutathione-S-transferase; MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate reductase; POX, peroxidase; SOD, superoxide dismutase. 71

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4. Discussion

capability to scavenge ROS resulting from oxidative stress during storage, compared with calyx-end tissues. Moreover, Davey et al. (2004) reported that AsA and GSH concentrations gradually decreased from peel to core tissue in ‘Ontario’ apple fruit. de Castro et al. (2008) also found that AsA concentrations were different between stem-end tissues and calyx-end tissue. These results suggest that AsA and GSH concentrations are not evenly distributed within a fruit vertically or horizontally. AsA concentrations in cortical tissues decline during storage, with the decrease being greater in 1-MCP treated fruit than in control fruit (Lee et al., 2012a). 1-MCP treatment resulted in lower DHA concentrations by 10 months in both tissues although DHA concentrations in control tissues increased. AsA concentrations were more closely linked with storage duration than DHA concentrations in either tissue type (Fig. 2). GSH and GSSG concentrations were also inversely correlated with each other in calyx-end tissues but not in stem-end tissues. 1-MCP reduced superoxide production (Table 1) but did not impact the activities of SOD and Cu/Zn-SOD in either tissue during CA storage (Table 3). The contrast test was performed to evaluate the responses of those variables at a specific time or between tissues (Fig. 3). Superoxide production was higher in stem-end tissues at 10 months, compared with that at 6 months, but SOD activity was only higher at 6 months while Cu/Zn-SOD activity was only higher at 10 months in stem-end vs calyxend tissues in 1-MCP treated fruit. Brown tissues tended to have higher superoxide production, and SOD and Cu/Zn-SOD activities increased potentially detoxifying superoxide anion radical in both tissues. H2O2 accumulated further in brown tissues, which may also be associated with stress. Similarly, AsA and GSH concentrations decreased as they were mobilized to scavenge H2O2 alongside enhanced CAT and POX activities. APX activity was not associated with any particular tissue, treatment, or storage duration. APX plays a signaling role in the fine modulation of ROS but CAT activity has a direct scavenging role in the removal of excess ROS (Mittler, 2002). By contrast, 1-MCP enhanced H2O2 concentrations at 10 months of storage, while those of AsA and GSH remained low. POX activity was only reduced by 1-MCP treatment but CAT activity was slightly reduced in both tissues by 10 months. However, POX activity was closely linked with 1-MCP treatment in stem-end tissues but less in calyx-end tissues (Fig. 2). Similarly, 1-MCP treatment enhanced POX activity but did not impact CAT activity in ‘Golden Smoothee’ apple stored in air at 1 °C (Vilaplana et al., 2006). While 1-MCP treatment did not affect APX activity in either tissue, APX activity increased in control fruit by 10 months. MDHAR, DHAR, and GR activities were inconsistent. To investigate the effects of tissue location or 1-MCP treatment on antioxidant scavenging systems, we focused on 6 and 10 months data for contrast tests (Fig. 3). The stem-end tissue in untreated fruit had lower superoxide production, MDA concentrations, and POX activity than calyx-end tissue. However, 1-MCP treated stem-end tissue had higher POX and CAT activities, and AsA-GSH cycle metabolites, than either 1-MCP treated calyx-end or untreated stem-end tissues. MDHAR activity in 1-MCP treated stem-end tissues was higher, regardless of the other two factors for the contrast test. Superoxide production, and POX and CAT activities were inversely related with these factors, suggesting that untreated stem-end tissue had the highest ability to produce superoxide and hydrolyze H2O2 by POX and CAT activities, while 1-MCP treated calyx-end tissue had the lowest ability. Therefore, each factor was mutually exclusive with respect to required antioxidant scavenging components. 1-MCP treated stem-end tissues had higher POX, CAT, and GR activities, compared with 1-MCP treated calyx-end tissues, while it had lower POX and CAT activities, higher DHAR activity, and higher GSH concentrations, compared with untreated stem-end tissues. In conclusion, flesh browning occurs first in the stem-end flesh and can be enhanced by 1-MCP treatment. Reduced AsA and GSH concentrations are linked with ROS accumulation, and the lower AsA and GSH concentrations might be associated with higher MDA concentrations in stem-end tissues. Loss of enzymatic and non-enzymatic antioxidant capacity through the enhanced ROS levels results in the decline

The degrees of replication reported in the literature are variable, from individual to bulked samples of different numbers, e.g. replicates of three (Gapper et al., 2017), four (Chiu et al., 2015) and ten (Ma et al., 2015) fruit. Disorder incidence is a population based phenomenon, which can range from 0 to 100%; therefore in the example of 50% of a given disorder, there is a 50% chance of not detecting injuries. In the case of mineral relationships with a disorder, such as bitter pit, greater accuracy is obtained by bulking fruit samples (Ferguson and Watkins, 1989). However, for metabolite analyses, this approach can be problematic. Bulking samples averages metabolite changes and could result in missed detection of changes in a fruit population with low disorder incidence, whereas small fruit numbers could result in selection of fruit that will not develop the disorder. Here, as in earlier research (Lee et al., 2012a, b), we adopted an experimental design using individual fruit sampling and processing. In this study, flesh browning developed first in the stem-end of fruit and then progressed towards the calyx-end, as indicated by L* and ho values (Table 1). This pattern is similar to that observed earlier (Lee et al., 2012a, b; Ma et al., 2015), and in two other cultivars, “Pink Lady” (de Castro et al., 2008) and ‘Royal Gala’ (Lee et al., 2013, 2016). Flesh browning was further aggravated by 1-MCP treatment (Table 1; Lee et al., 2012a). Oxidative stress caused by CA storage combined with chilling storage temperatures may result in loss of cellular membrane integrity and stability, and subsequent browning (Hodges et al., 2004). Browning in stem-end tissues of “Pink Lady” was associated with higher electrolyte leakage than in calyx-end tissues (de Castro et al., 2008). Higher electrolyte leakage was strongly linked with the accumulation of H2O2 and MDA concentrations in pomegranate fruit as chilling injury developed (Babalar et al., 2018). Increased H2O2 concentrations were associated with higher electrolyte leakage and browning indices in white button mushrooms during cold storage (Dokhanieh and Aghdam, 2016). In this study, superoxide generation, and H2O2 and MDA concentrations were affected by storage duration (Table 1). In a previous report (Lee et al., 2012a), superoxide generation was decreased by 1-MCP treatment during CA storage but H2O2 and MDA concentrations were not consistently affected. MDA concentrations fluctuated for reasons that we cannot explain. Critically, the higher H2O2, MDA and NBT concentrations in calyx-end regions indicate a lack of association with greater browning in the stem-end tissues, compared with those in the calyxend. PLS loading plots (Fig. 2) indicated that superoxide generation was closely associated with storage duration in both tissues but only closely linked to the 1-MCP treatment variable in stem-end tissues. Superoxide production was less affected by 1-MCP treatment in stem-end tissue, but reduced by 1-MCP treatment in calyx-end tissue. On the other hand, H2O2 concentrations were strongly linked with storage duration in calyx-end tissues (Fig. 2B) but not with storage duration or 1-MCP treatment in stem-end tissues (Fig. 2A). H2O2 concentrations decreased steadily in stem-end tissues of control fruit but 1-MCP treatment enhanced the H2O2 concentration by the end of storage. In contrast, the H2O2 concentration increased in calyx-end tissues by the end of storage, irrespective of 1-MCP treatment (Table 1). The MDA concentration was not linked to the L*, 1-MCP, or with storage duration variables in either tissue (Fig. 2). The biochemical responses of ROS and their byproduct from different tissue regions may be associated with structural difference of apple fruit. These differences could affect gas transport properties and influence gas diffusivities in different parts of the fruit during CA storage (Ho et al., 2006a, b; Schotsmans et al., 2003), and might affect responses of different tissues to ROS and their byproducts. Antioxidant metabolites, including AsA, DHA, GSH, and GSSG, are involved in scavenging ROS (Apel and Hirt, 2004). AsA and GSH concentrations were lower in stem-end tissues than in calyx-end tissues at harvest. Also, GSSG concentrations were inversely related with those of GSH but not DHA at harvest. Stem-end tissues might have lower 72

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of AsA and GSH concentrations, and higher POX activity. Overall, higher flesh browning may be associated with lower AsA and GSH concentrations in stem-end tissues but the enhanced browning resulting from 1-MCP treatment does not appear to be directly related to antioxidant metabolism.

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Acknowledgements Jinwook Lee was supported by a Department of Horticulture Graduate Assistantship. We thank Jeanne Grace, and Drs. Rujira Deewatthanawong, Pengmin Li, and Li-Song Chen for their technical support, and Gerald Tangren at the Tree Fruit Research and Extension Center of Washington State University for the statistical assistance. Revision of this study was supported by the Chung-Ang University Research Grant (2017). This work was also supported by the USDA National Institute of Food and Agriculture, Hatch project 2013-14-483, Improving Quality and Reducing Losses in Specialty Fruit Crops through Storage Technologies (NE-1336). Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the National Institute of Food and Agriculture (NIFA) or the United States Department of Agriculture (USDA). References Aebi, H., 1984. Catalase in vitro. In: Lester, P. (Ed.), Methods Enzymol. Academic Press, pp. 121–126. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Physiol. Plant Mol. Biol. 55, 373–399. https://doi. org/10.1146/annurev.arplant.55.031903.141701. Babalar, M., Pirzad, F., Sarcheshmeh, M.A.A., Talaei, A., Lessani, H., 2018. Arginine treatment attenuates chilling injury of pomegranate fruit during cold storage by enhancing antioxidant system activity. Postharv. Biol. Technol. 137, 31–37. https:// doi.org/10.1016/j.postharvbio.2017.11.012. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. Chiu, G.Z., Shelp, B.J., Bowley, S.R., DeEll, J.R., Bozzo, G.G., 2015. Controlled atmosphere-related injury in ‘Honeycrisp’ apples is associated with ɤ-aminobutyrate accumulation. Can. J. Plant Sci. 95, 879–886. https://doi.org/10.4141/CJPS-2015-061. Davey, M.W., Franck, C., Keulemans, J., 2004. Distribution, developmental and stress responses of antioxidant metabolism in Malus. Plant Cell Environ. 27, 1309–1320. https://doi.org/10.1111/j.1365-3040.2004.01238.x. de Castro, E., Barrett, D.M., Jobling, J., Mitcham, E.J., 2008. Biochemical factors associated with a CO2-induced flesh browning disorder of Pink Lady apples. Postharv Biol. Technol. 48, 182–191. https://doi.org/10.1016/j.postharvbio.2007.09.027. Doerflinger, F.C., Miller, W.B., Nock, J.F., Watkins, C.B., 2015. Variations in zonal fruit starch concentrations of apples: a developmental phenomenon or an indication of ripening? Hortic. Res. 2, 15047. https://doi.org/10.1038/hortres.2015.47. Dokhanieh, A.Y., Aghdam, M.S., 2016. Postharvest browning alleviation of Agaricus bisporus using salicylic acid treatment. Sci. Hort. 207, 146–151. https://doi.org/10. 1016/j.scienta.2016.05.025. Ferguson, I.B., Watkins, C.B., 1989. Bitter pit in apple fruit. Hort. Rev. 11, 289–355. Frei, M., Wang, Y., Ismail, A.M., Wissuwa, M., 2010. Biochemical factors conferring shoot tolerance to oxidative stress in rice grown in low zinc soil. Funct. Plant Biol. 37, 74–84. https://doi.org/10.1071/FP09079. Gapper, N.E., Hertog, M.L.A.T.M., Lee, J., Buchanan, D.A., Leisso, R.S., Fei, Z., Qu, G., Giovannoni, J.J., Johnston, J.W., Schaffer, R.J., Nicolaï, B.M., Mattheis, J.P., Watkins, C.B., Rudell, D.R., 2017. Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis. BMC Plant Biol. 17, 77. https://doi.org/10.1186/s12870-017-1030-6. Giannopolitis, C.N., Ries, S.K., 1977. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol. 59, 309–314. https://doi.org/10.1104/pp.59.2.309. Grace, S.C., Logan, B.A., 1996. Acclimation of foliar antioxidant systems to growth irradiance in three broad-leaved evergreen species. Plant Physiol. 112, 1631–1640. https://doi.org/10.1104/pp.112.4.1631. Griffith, O.W., 1980. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207–212. https://doi. org/10.1016/0003-2697(80)90139-6. Hammerschmidt, R., Nuckles, E.M., Kuć, J., 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol. Plant Pathol. 20, 73–82. https://doi.org/10.1016/0048-4059(82)90025-X.

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