Possible mechanisms of warming effects for amelioration of superficial scald development on ‘Fuji’ apples

Possible mechanisms of warming effects for amelioration of superficial scald development on ‘Fuji’ apples

Postharvest Biology and Technology 62 (2011) 43–49 Contents lists available at ScienceDirect Postharvest Biology and Technology journal homepage: ww...

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Postharvest Biology and Technology 62 (2011) 43–49

Contents lists available at ScienceDirect

Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio

Possible mechanisms of warming effects for amelioration of superficial scald development on ‘Fuji’ apples Xingang Lu, Xinghua Liu ∗ , Shunfeng Li, Xiaojiao Wang, Lihua Zhang College of Food Science and Engineering, Northwest A&F University, No. 28, Xinong Road, Yangling 712100, Shaanxi, China

a r t i c l e

i n f o

Article history: Received 24 January 2011 Accepted 24 April 2011 Keywords: Superficial scald Warming treatment Ethylene Antioxidant Conjugated trienols ‘Fuji’

a b s t r a c t Warming of fruit during storage has been shown to decrease scald development, but the mechanisms involved in this effect are poorly understood. The effects of a single warming of 5 days at 20 ◦ C after 2, 4, 6 and 8 weeks storage at 0 ◦ C on development of superficial scald of ‘Fuji’ apples in relation to ethylene, afarnesene and conjugated trienol (CTol) concentrations have been studied. Malondialdehyde (MDA) and hydrogen peroxide (H2 O2 ) concentrations, catalase (CAT) and peroxidase (POX) activities, total phenolic contents and total antioxidant activity were measured in order to assess the effects of the treatments on membrane damage and oxidant and antioxidant activity. Warming after 4 weeks storage reduced scald to the lowest level among all treatments. Warming greatly stimulated internal ethylene concentrations (IECs) and in turn, increased ␣-farnesene and CTol accumulation. Scald resistance, indicated by CAT and POX activities, total phenolic contents and total antioxidant activity, was higher in fruit in early than in late storage. The warming treatment after 4 weeks of storage resulted in higher concentrations of CTols and H2 O2 , as well as MDA, compared with the control fruit when kept at 20 ◦ C after 6 and 12 weeks of storage, but lower than after 20 and 28 weeks. These results suggest that warming could inhibit scald development by modifying CTol accumulation as well as by affecting generation of accompanying active oxygen species (AOS), and reducing oxidative damage. These changes may cause a shift from a scald-sensitive metabolism to a resistant stage during storage. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Superficial scald is a commercially important physiological disorder that occurs in apples and pears during storage. Smith (1959) demonstrated that a single warming treatment of 5 days at 16 and 20 weeks during storage inhibited the subsequent scald development in ‘Bramley’s Seedling’ apples. Watkins et al. (1995) found similar effects for ‘Granny Smith’ after 1–4 weeks at 0 ◦ C with a warming at 20 ◦ C for 3 or more days. However, Alwan and Watkins (1999) found that although multiple warming treatments actually reduced scald in ‘Cortland’, ‘Delicious’ and ‘Law Rome’ fruit, the magnitude of the effects varied among cultivars. In addition, responses to intermittent warming and pre-conditioning differ among cultivars, regions, and years (Watkins et al., 2000). The reasons for the effect that a warming treatment has, whether effective or not, on scald inhibition, are not known. A possible hypothesis is that resistance of fruit tissue to damage increases as a result of the effect of warming on ␣-farnesene production and categories and ratios of its conjugated trienol (CTol) oxidation products, as well as on ethylene-induced ripening (Watkins

∗ Corresponding author. Tel.: +86 29 8709 2452; fax: +86 29 8709 2821. E-mail address: [email protected] (X. Liu). 0925-5214/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2011.04.008

et al., 1995; Alwan and Watkins, 1999). Differences in response to warming among cultivars and growing regions may be related to factors such as skin permeability and wax composition that could influence concentrations of volatiles or other materials in the cuticle (Watkins et al., 2000). However, discrepancies exist in ethylene responses to altered temperature conditions among cultivars and a potential role in scald development. It has been shown that ␣-farnesene concentration increases with the ethylene climacteric during ripening (Meigh and Filmer, 1969; Du and Bramlage, 1994; Watkins et al., 1993, 1995; Ju and Curry, 2000; Pechous et al., 2005). In addition, treatments, that block ethylene action, such as diazocyclopentadiene and 1methylcyclopropene (1-MCP), decrease scald development (Gong and Tian, 1998; Fan et al., 1999; Rupasinghe et al., 2000; Watkins et al., 2000; Shaham et al., 2003). Ethylene production can be stimulated by chilling in apple cultivars such as ‘Lady Williams’, ‘Granny Smith’ and ‘Fuji’, which are chilling-sensitive and can be induced to ripen by exposure to 0 ◦ C in air (Jobling et al., 1991, 2003; Jobling and McGlasson, 1995; Lara and Vendrell, 2003). Low temperatures appear to hasten homogeneous ripening and induce the competency to synthesize autocatalytic ethylene through enhancement of both ACC synthase (ACS) and ACC oxidase (ACO) activities in apples (Jobling et al., 2003; Lara and Vendrell, 2003). Upon removal of fruit from cold storage,

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ethylene production is strongly hastened and the amount of ethylene depends on the chilling period (Lara and Vendrell, 2003). On the basis of these findings, it is possible that stimulated ethylene production and physiological changes after chilling are related to the effects of intermittent warming on inhibiting scald development. To test this hypothesis, we have examined the warming effects on ‘Fuji’ apples for amelioration of scald during and after storage from two aspects: (1) influences of warming of fruit after various chilling periods on internal ethylene concentrations (IECs), and accumulation of ␣-farnesene and CTols, and (2) effects of warming treatments on membrane damage as assessed by malondialdehyde (MDA) concentrations, as well as on selected oxidant and antioxidant species. 2. Materials and methods 2.1. Plant materials ‘Fuji’ apples were harvested on 2 October 2008 (Experiment 1), and 15 October 2009 (Experiment 2), from an orchard in Shaanxi, China. Apples were transported immediately after harvest to the laboratory and divided into groups of three replicates for each experimental treatment. Each replicate contained 310 fruit in 2008 or 620 fruit in 2009. 2.1.1. Experiment 1. Effects of warming treatments on superficial scald and IECs, ˛-farnesene and CTols The apples were stored in a chamber at 0 ◦ C with a relative humidity of about 90% for 2, 4, 6 and 8 weeks. A control sample was kept at 20 ◦ C. Following each period of chilling, 30 fruit were transferred to 20 ◦ C for analysis of internal concentrations of ethylene (IECs) after 1, 5, 10, 15 and 20 days at 20 ◦ C. After 2, 4, 6 and 8 weeks at 0 ◦ C, fruit were kept at 20 ◦ C for 5 days and then returned to 0 ◦ C. Control fruit were held continuously at 0 ◦ C. All fruit were kept for a total 28 weeks at 0 ◦ C. For each treatment, there were 16 fruit per replicate for measurement of IECs, ␣-farnesene and CTol concentrations at each interval of storage, plus 100 fruit for evaluation of scald after 28 weeks storage plus 7 days at 20 ◦ C. 2.1.2. Experiment 2. Relationships between oxidants and antioxidants during warming treatments After storage at 0 ◦ C for 4 weeks, apples were transferred to 20 ◦ C for 5 days and then returned to 0 ◦ C for a further 24 weeks. The control sample was held continuously at 0 ◦ C. After 6, 12, 20, and 28 weeks storage at 0 ◦ C, physiological parameters were analysed with 20 fruit per replicate, including catalase (CAT) and peroxidase (POX) activities, total phenolic concentrations and total antioxidant activity, and 60 fruit per treatment per replicate were transferred to 20 ◦ C and CTol, hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA) contents were measured at 1, 3, 5, 7, and 9 days. Replicates of 100 fruit were assessed for scald after 7 days at 20 ◦ C after 12, 20 and 28 weeks storage from harvest. 2.2. Measurement of IECs IECs were measured on a gas sample withdrawn via a syringe and hypodermic needle inserted in the calyx end of the fruit. Ethylene was determined with a GowMac Model 580 gas chromatograph fitted with an activated alumina column (2 m × 2 mm ID, stainless steel) and flame ionization detector with nitrogen carrier gas at 40 mL min−1 , hydrogen 35 mL min−1 , air 350 mL min−1 , and oven temperature at 150 ◦ C. One milliter of gas samples were used and the lower detection limit for ethylene was 0.01 mL L−1 .

2.3. Extraction and analysis of ˛-farnesene and CTols Extraction and analysis of ␣-farnesene and CTols were performed as described by Isidoro and Almeida (2006). Peel was removed from the equatorial region on opposite sides of 10 fruit. After carefully scraping off all cortical tissue, a disk 10 mm in diameter was excised from the peel. Two disks excised from each fruit were immersed in 10 mL of hexane and incubated for 10 min at 22 ◦ C with agitation. After incubation the solvent was filtered through a cellulose paper filter and the final volume adjusted to 10 mL. Immediately after filtration, absorbance of the extract was measured at 232 nm and in the range 281–290 nm with a UV–visible spectrophotometer (UV-1700 Pharmaspec, Shimadzu, Tokyo, Japan). ␣-Farnesene and CTols concentrations were calculated using molar extinction coefficients ε232 nm = 27,740 (Anet, 1969) for ␣-farnesene and ␧281–290 nm = 25,000 for the conjugated trienes (Anet, 1972) and expressed as mg kg−1 on a fresh weight basis. 2.4. CAT and POX assays CAT and POX were extracted from 1 g FW of frozen peel tissue using 10 mL of ice-cold 50 mM potassium phosphate buffer (pH 7.0), containing 1 mM ascorbate, 1 mM EDTA, and 5% (w/v) PVPP at 4 ◦ C. The homogenate was centrifuged twice at 20,000 × g for 15 min at 4 ◦ C. POX activity was determined using the method described by Rao et al. (1996). One unit of CAT was defined as the amount of enzyme which consumes 1 ␮mol H2 O2 min−1 at 25 ◦ C. One unit of POX was defined as the amount of enzyme responsible for the changes of optical density (OD) at 470 nm mg−1 protein. CAT activity was determined by the method of Klapheck et al. (1990). Enzyme activities were all based on total protein. Protein content was determined according to the method described by Bradford (1976) using bovine serum albumin as a standard. 2.5. Determination of total phenolic concentrations and total antioxidant activity The total phenolic concentration of apple peel was measured using a modified Folin–Ciocalteu colorimetric method (Singleton et al., 1999). Absorbance was measured at 750 nm versus a blank after 90 min at room temperature. The results were expressed as gallic acid equivalents on a fresh weight basis, g kg−1 . The total antioxidant activity was determined using the 1-diphenyl-2-picrylhydrazyl (DPHH)-radical-scavenging activity assay (Kondo et al., 2002). A test sample (20 mL) was added to 980 mL of 0.1 M DPPH in ethanol, and the combination was mixed and kept for 20 min at room temperature in the dark. The decrease in absorbance at 516 nm was monitored, and IC50 based on 50% inhibition of reaction in the solution of the sample and DPPH. The IC50 value represented the concentration of the sample in 1 L reaction mixture. Distilled water was used instead of the sample solution as a control. 2.6. Determination of H2 O2 and MDA concentrations The H2 O2 concentration was determined according to Loreto and Velikova (2001). Two grams of frozen peel were homogenized at 4 ◦ C in 0.1% (w/v) trichloroacetic acid. The homogenate was centrifuged at 12,000 × g for 15 min and 1 mL of the supernatant was mixed with 1 mL of 10 mM potassium phosphate buffer (pH 7.0) and 2 mL of 1 M KI. The H2 O2 content of the supernatant was evaluated by comparing its absorbance at 390 nm with a standard calibration curve and expressed as mmol kg−1 fresh weight. MDA content was determined by the thiobarbituric acid (TBA) reaction according to Imahori et al. (2008). Frozen peel tissues

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Table 1 Effect of warming treatments on scald incidence and severity in ‘Fuji’ apples stored for 28 weeks in 2008 and 20 or 28 weeks in 2009. Evaluation time

Time of warming treatment

Superficial scald

Incidence (%)

Severity (1–4)

2008 (28 weeks)

None 2 weeks 4 weeks 6 weeks 8 weeks

71a 66a 11d 25c 57b

2.1a 1.8b 0.8d 1.1c 1.6b

2009 (20 weeks)

None 4 weeks

24b 2d

1.1b 0.1d

2009 (28 weeks)

None 4 weeks

60a 8c

1.8a 0.7c

Different letters indicated significance difference within the same column at p < 0.05.

(2 g) were homogenized in a cooled mortar and pestle with 10 mL of ice-cold 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 20,000 × g for 10 min at 4 ◦ C. A 1 mL aliquot of the supernatant was mixed with 4 mL of 20% TCA containing 0.5% TBA. The mixture was incubated at 95 ◦ C for 30 min and then quickly cooled in an ice-bath. After centrifugation at 20,000 × g for 10 min at 4 ◦ C, the absorbance of the supernatant at 532 nm was recorded and corrected for non-specific absorption at 600 nm using a spectrophotometer (UV-1700 Pharmaspec, Shimadzu, Tokyo, Japan). The MDA concentration was calculated using the extinction coefficient 155 mM cm−1 . 2.7. Assessment of superficial scald Scald was defined by incidence and severity. Incidence was evaluated as all fruit with symptoms. Scald severity was evaluated visually using the percentage of the fruit surface area affected, where none = 0, 1–10% = 1, 11–33% = 2, 34–66% = 3 and 67–100% = 4, and divided by the number of affected fruit.

Fig. 1. Internal ethylene concentrations (IECs) in fruit kept continuously at 20 ◦ C for 20 days after 0 (), 2 (), 4 (), 6 (♦) or 8 () weeks storage at 0 ◦ C (A) and in those without warming () or warmed for 5 days at 20 ◦ C before being transferred to 0 ◦ C after 2 (), 4 (), 6 (♦), or 8 () weeks storage at 0 ◦ C (B). Vertical bars represent LSD at the 5% level.

2.8. Statistical analysis The experimental design was a completely randomized one with three replications. Data were subjected to analysis of variance (ANOVA), with treatment and storage time as sources of variation. Mean comparisons at p = 0.05 were performed using the least significant difference (LSD) method. Pearson correlations were used to quantify the relationships among factors assessed. 3. Results 3.1. Experiment 1 Scald incidence and severity were reduced by warming treatments with the maximum reduction occurring in fruit treated after 4 weeks of storage. Warming after 2 weeks did not influence scald incidence significantly, but it reduced its severity (Table 1). Generally, the effect of warming declined with prolonged storage time before warming. Cold storage of fruit stimulated ethylene production, and advanced the onset of the autocatalytic ethylene rise. During 20 days in air at 20 ◦ C, the IECs of fruit stored for 2 weeks were higher than those in fruit kept continuously at 20 ◦ C, with fruit of neither treatment reaching maximum concentrations. IECs of fruit stored for 4, 6, 8 weeks peaked at 15, 8, 3 days, respectively (Fig. 1A). The longer the cold exposure, the shorter the lag period required to achieve the greatest ethylene production upon warming. However,

the maximal concentration showed a negative correlation with the chilling time. After return of fruit to cold storage, the IECs were maintained at higher levels, the exception being a rise of IECs in control fruit between 9 and 12 weeks. The IECs of fruit warmed after 4 and 6 weeks in storage after transfer to the cold were 2–3 times above that of unwarmed fruit (Fig. 1B). Fruit warmed after 4 and 6 weeks at 0 ◦ C had the highest ␣-farnesene accumulation (approximately 92 and 84 nmol cm−2 , respectively) for the first half storage period than control samples (16 weeks, 75 nmol cm−2 ). Also the peak of accumulation occurred at 11 and 13 weeks, respectively, compared with 16 weeks in the control fruit. However, few other effects were detected (Fig. 2A). A warming treatment after 2 weeks storage at 0 ◦ C did not affect CTol production significantly. The increase of CTol accumulation was more rapid in fruit warmed after 4, 6 and 8 weeks, than in control fruit. Also, the earlier the warming, the higher the CTol accumulation and the sooner the maximum values occurred. However, despite being lower at the start of storage, CTol accumulation in control fruit reached higher levels than those in fruit warmed, after 4, 6 and 8 weeks (Fig. 2B). 3.2. Experiment 2 Scald did not occur in fruit of either treatment after 12 weeks cold storage plus 7 days at 20 ◦ C. With longer storage time, scald

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Fig. 2. Concentrations of ␣-farnesene (A) and conjugated trienols (B) in fruit without warming () or warmed for 5 days at 20 ◦ C before being transferred to 0 ◦ C after 2 (), 4 (), 6 (♦), or 8 () weeks storage at 0 ◦ C. Vertical bars represent LSD at the 5% level.

incidence and severity increased, and were higher after 28 weeks than after 20 weeks in both control and warmed fruit (Table 1). However, as shown in Experiment 1, scald development was greatly inhibited by the warming treatment. CAT activities were higher in warmed than control fruit throughout storage, but the patterns of change were similar, the highest activities occurring after 12 weeks of storage and decreasing to low levels by the end of storage (Fig. 3A). POX activities of fruit from both treatments were significantly higher (p < 0.05) at the early stage of storage, compared with those after 20 and 28 weeks storage (Fig. 3B). No significant differences were observed between treatments at any storage time. Phenolic concentrations and antioxidant activity of warmed fruit were higher than those of the control fruit at each assessment time, and after 12 and 20 weeks storage, respectively. Total phenolic concentrations and antioxidant activity were both higher in fruit after 6 and 12 weeks than those after 20 and 28 weeks of storage (Fig. 4). Total phenolic concentrations were positively associated with total antioxidant activity (r = 0.7096, p < 0.05), suggesting that phenolics make a significant contribution to the total antioxidant capacity in apple skin. After transfer of fruit to 20 ◦ C, at any storage period, the differences of CTol, H2 O2 and MDA accumulation between treatments followed the same pattern. Warmed fruit had higher CTol, H2 O2 and MDA concentrations than the controls after 6 and 12 weeks at 0 ◦ C, whereas they were lower after 20 and 28 weeks (Fig. 5). CTol concentrations of both treatments increased at all stages (Fig. 5A). No clear patterns were observed in changes of H2 O2 and MDA con-

Fig. 3. CAT (A) and POX (B) activities in fruit without warming or warmed for 5 days at 20 ◦ C after 4 weeks storage at 0 ◦ C and measured after 6 ( ), 12 ( ), 20 ( ), 28 ( ) weeks storage at 0 ◦ C.

tents after 6 weeks, however, they both decreased after 12 weeks and increased after 20 and 28 weeks when fruit of both treatments were kept at 20 ◦ C (Fig. 5B and C). 4. Discussion This study demonstrated the positive effects of warming treatments for amelioration of scald development of ‘Fuji’ apples, which were similar to results obtained with ‘Bramley’s Seedling’ (Smith, 1959), and ‘Granny Smith’ (Watkins et al., 1995, 2000) fruit. However, basic issues raised by comparing these results are why the effects of warming are different among cultivars as well as diverse chilling periods and what factors are crucial to determine whether warming after a certain chilling time is effective or not on inhibition of scald. Previous studies focused on ␣-farnesene production and its conjugated trienol (CTol) oxidation products found no evidence of a direct role of warming on their accumulation and no certain mechanisms was described (Watkins et al., 1995; Alwan and Watkins, 1999). Fruit showed varying degrees of IECs at 20 ◦ C after different periods of chilling, which was consistent with effects shown earlier for ‘Fuji’ (Jobling and McGlasson, 1995; Jobling et al., 2003), ‘Granny Smith’ (Jobling et al., 1991; Johnston et al., 2002), ‘Lady Williams’ (Jobling and McGlasson, 1995) and ‘Braeburn’ (Tian et al., 2002) fruit. In addition, higher IECs were also maintained after return to storage (Fig. 1B), suggesting that the warming period has long term effects on ethylene-dependent metabolism. Such effects could be important factors affecting responses of ‘Fuji’ apples to storage disorders during storage after warming.

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Fig. 4. Total phenolic concentrations (A) and total antioxidant activity (B) in fruit without warming or warmed for 5 days at 20 ◦ C after 4 weeks storage at 0 ◦ C and measured after 6 ( ), 12 ( ), 20 ( ), and 28 ( ) weeks storage at 0 ◦ C.

Ethylene regulates ␣-farnesene production (Ju and Curry, 2000; Whitaker et al., 2000; Pechous et al., 2005). Our results also support a close link between changes in IECs and ␣-farnesene synthesis. Not only did warming after 4 and 6 weeks at 0 ◦ C increase IECs, but also brought the climacteric forward, which led to higher levels and advanced peaks of ␣-farnesene accumulation compared with control and warming treatments at other times (Fig. 2B and 3A). Watkins et al. (1995) also found that warming stimulated ␣farnesene accumulation even in treatments that inhibited scald. Though levels of ␣-farnesene were evidently different in the early storage stage, no significant correlation was found between ␣farnesene and scald development late in storage. Therefore, just as the previous studies (Du and Bramlage, 1994; Rao et al., 1998; Whitaker et al., 2000; Jemric et al., 2006) indicate, ␣-farnesene is not the main factor for scald occurrence, but rather its oxidation product CTols are. Despite its indirect role on scald development, higher levels of ␣-farnesene accumulation indicated more CTol production (Fig. 2). The relationship between ␣-farnesene and CTols in our experiments was in accordance with reports that rapid ␣-farnesene synthesis early in storage typically results in the relatively early accumulation of CTols (Whitaker et al., 1997; Whitaker, 1998; Watkins et al., 2000). Warming also reduced CTol concentrations later during storage for treatments after 4, 6 and 8 weeks and they developed slightly less scald (Fig. 2B). Correlation coefficients of 0.728** and 0.742** were found in the present study for CTols and scald incidence, and severity, respectively. Although these positive correlations are supported by other workers (e.g. Anet and Coggiola, 1974; Meir and Bramlage, 1988; Rowan et al., 2001), contrary reports (e.g. Rao et al., 1998; Whitaker et al., 2000; Jemric et al.,

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2006) show that CTols are not essential for scald development. The role of CTols in scald development is not confirmed, therefore, and other analytical approaches are required to clarify this issue. Scald development is viewed as a two-stage event, where the induction events are separated from symptom development (Bramlage and Meir, 1990). In spite of the unclear role of CTols, it is generally accepted that active oxygen species (AOS) generated by oxidation of ␣-farnesene are a contributory cause of scald (Whitaker, 2004). Thus, CTols are still available indicators of scald development since their accumulation continued throughout the storage period. Thus we need to re-examine the relationship between CTols and scald during the whole event, especially at the damage-inducing stage. Anet (1972) reported that the time at which high conjugated triene levels occur is important to scald development. Barden and Bramlage (1994) also pointed out that sensitivity of cells to CTols probably changes with time. In this study, treatments resulting in high CTol levels early in storage and low in later stages greatly reduced scald development. Fruit warming after 4 weeks in storage developed the lowest scald incidence and severity, expressing this most obvious trend in CTol accumulation (Fig. 2B). From this point of view, fruit cells may be able to provide defence against CTols and AOS early in storage, but such defence becomes weaker during prolonged cold storage. Intermittent warming could affect scald by increasing resistance of the fruit tissue to the damaging effects of the ␣-farnesene oxidation products as well as accompanying AOS (Watkins et al., 1995; Alwan and Watkins, 1999). IECs, greatly modified by warming, contributed to advanced fruit ripening, which is associated with fruit resistance to scald (Barden and Bramlage, 1994). The increase of total phenolic concentrations and antioxidant activity in fruit could result from advanced ripening and they could play a greater role in inhibiting ␣-farnesene oxidation and scavenging AOS (Barden and Bramlage, 1994; Shaham et al., 2003). However, while total phenolic concentrations and antioxidant activity were higher in warmed fruit compared with the controls, warmed fruit accumulated relatively more CTols when kept continuously at 20 ◦ C after 6 and 12 weeks, in spite being lower after 20 and 28 weeks (Fig. 4B and 5A). These results suggest that effects of warming on improvement of antioxidant capacity do not eliminate the negative affects caused by more ␣-farnesene synthesis and oxidation early in storage. After 6 and 12 weeks at 0 ◦ C, although CTols increased with the prolonged time at 20 ◦ C, H2 O2 contents decreased and were kept at comparatively low levels (Fig. 5A and B). However, positive correlations were found for CTols and H2 O2 contents after 20 and 28 weeks (r = 0.912 and 0.892; p < 0.05, respectively). Low levels of H2 O2 after 6 and 12 weeks depend on high activities of POX and CAT, and H2 O2 accumulation after 20 and 28 weeks was due to reduced activities, which was in agreement with these reports that POX and CAT are involved with H2 O2 degradation thereby improving resistance of fruit to scald (Rao et al., 1998; Fernández-Trujillo et al., 2003; Kochhar et al., 2003). AOS are able to initiate lipid peroxidation reactions (Shewfelt and Purvis, 1995) and the increase in peroxidation products may have been associated with changes in cellular conditions that result in symptom development. MDA levels were extremely low in fruit after storage for 6 weeks and were reduced in fruit after 12 weeks when kept continuously at 20 ◦ C (Fig. 5C), which might be attributed to low concentrations of H2 O2 on the one hand, or resulting from high tolerance and resistance of fruit to damage early in storage on the other. However, high MDA concentrations and their increase after 20 and 28 weeks indicated a more distinct deterioration of membrane integrity and aggravation of lipid peroxidation in the fruit and severely damaged membranes during these periods. At the same time, continuous increase of MDA contents during storage made it clear that scald development was not only a rapid

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Fig. 5. Concentrations of conjugated trienols (A), H2 O2 (B) and MDA (C) in fruit without warming () or warmed for 5 days at 20 ◦ C after 4 weeks () and transferred to 20 ◦ C for 9 days after 6, 12, 20 or 28 weeks storage at 0 ◦ C. Vertical bars represent LSD at the 5% level.

change in symptoms but also a long-term accumulation in damage. Whatever antioxidant system, or CTols, H2 O2 and MDA, their changes were evidently different among storage periods and these variations typically depend on physiological conditions and mutual influence of these factors. We believe that warming treatments are effective in inhibiting scald development on ‘Fuji’ apples by the transfer of harmful factors contributing to scald development from a scald-sensitive stage to a fairly resistant period during storage to relatively reduce oxidative damage. In summary, warming changed ethylene, ␣-farnesene and CTol production and physiological resistance of fruit to scald development. Effects of warming on ethylene biosynthesis probably played a key role in this process and were probably the main reasons for metabolic changes of CTols and AOS, probable sources of damage to scald induction. By modifying the timing of CTol production and accompanying AOS generation, warming may be able to maintain the balance of fruit between susceptibility and resistance of fruit tissues to injury. A reduction of damage during the period of induction would delay or inhibit scald development. Whether a certain time for warming is effective or not depends on whether ethylene biosynthesis is appropriately modified and whether the appropriate balance is maintained. This study has examined only on ‘Fuji’ apples, and further research should be carried out with other apple cultivars, especially those having attributes different in ethylene production or perception to confirm these results and find the exact mechanisms for various responses of apples to warming treatments.

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