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Melatonin treatment reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and phenolic metabolism
Accepted Manuscript Melatonin treatment reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and phenolic metabolism Hui Gao, ZeMian Lu, Yue Yang, DanNa Wang, Ting Yang, MaoMao Cao, Wei Cao PII: DOI: Reference:
S0308-8146(17)31637-0 https://doi.org/10.1016/j.foodchem.2017.10.008 FOCH 21831
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
8 June 2017 4 September 2017 3 October 2017
Please cite this article as: Gao, H., Lu, Z., Yang, Y., Wang, D., Yang, T., Cao, M., Cao, W., Melatonin treatment reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and phenolic metabolism, Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.10.008
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Melatonin treatment reduces chilling injury in peach fruit through its regulation of
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membrane fatty acid contents and phenolic metabolism
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Running title: Effects of MT treatment on chilling injury of peach fruit
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Hui Gao*, ZeMian Lu, Yue Yang, DanNa Wang, Ting Yang, MaoMao Cao, Wei Cao
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Department of Food Science and Engineering, Northwest University, Xi’an 710069, P. R. China
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*
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Email address: [email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
Corresponding author: Tel: +86-29-88302213; Fax: +86-29-88302213.
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ABSTRACT
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Effects of 0.1 mM melatonin (MT) on chilling injury (CI), membrane fatty acid content and
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phenolic metabolism in peach fruit were studied during storage at 1°C for 28 days. MT treatment
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delayed the development of CI in peach fruit, as was illustrated by MT-treated fruit showing lower
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CI incidence, CI index and firmness loss than the control. MT treatment prevented membrane lipid
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peroxidation and contributed to maintaining a higher ratio of unsaturated to saturated fatty acids in
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peach fruit. MT treatment also stimulated the activities of glucose-6-phosphate dehydrogenase,
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shikimate dehydrogenase and phenylalanine ammonia lyase, but inhibited the activities of
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polyphenol oxidase and peroxidase. This would help in activating the accumulation of total
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phenolic and endogenous salicylic acid that might have a direct function in alleviation of CI.
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These results indicate that MT treatment can be an effective technique to reduce postharvest CI
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during low temperature storage of peach fruit.
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Keywords: peach fruit, chilling injury, melatonin, fatty acid, phenolic metabolism
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1. Introduction
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Chilling injury (CI) is a physiological disorder commonly occurring in a large collection of
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peach cultivars during low temperature storage, especially when storing at a potential risk zone of
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temperature between 2.2 and 7.6 °C (Lurie and Crisosto, 2005). The disorder is characterized
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mainly by flesh browning and/or mealiness, abnormal ripening and higher sensitivity to decay
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(Lurie and Crisosto, 2005), and is considered as a primary limitation of application of low
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temperature to preserve peach fruit. Many hypotheses have been put forward for a CI mechanism
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in plants, such as involvement of imbalances in metabolism, accumulation of toxic compounds,
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decreased water dynamic state and increased permeability (Lyons, 1973; Naruke, Oshita, Kuroki,
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Seo, Kawagoe & Walton, 2003; Purwanto et al., 2013). To deal with this problem, researchers
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have been devoting a great deal of attention to finding economical, convenient and highly
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effective techniques to reduce CI in peach fruit.
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Melatonin (MT) is an indoleamine hormone ubiquitous in nature. In plants, MT has been
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identified in nearly all organs and tissues, and is shown to be a signaling molecule involved in
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numerous physiological processes such as differentiation, growth, ripening and senescence of
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plant and the protective effect against various forms of environmental stress (Reiter, Tan, Zhou,
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Cruz, Fuentes-Broto & Galano, 2015; Zhang et al., 2015). Exogenous MT has been demonstrated
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to be effective in alleviating chilling stress-induced damage in plants through different
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mechanisms. For example, Posmyk, Balabusta, Wieczorek, Sliwinska & Janas (2009) revealed
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that MT treatment significantly counteracts the adverse effect of chilling stress on cucumber seeds
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by influencing the structure and function of cellular membrane. Likewise, in another study,
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researchers found that application of MT contributes to reducing the risk of ultrastructural damage
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in meristematic cells of Vigna radiata roots due to chilling exposure (Szafrańska, Glińska & Janas,
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2013). Their data also documented that MT-medicated Vigna radiata root acclimation to chilling
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stress is associated with its ability to activate the pathway of phenolic (Szafrańska, Szewczyk &
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Janas, 2014). It was also shown that MT has a positive regulation in the expression of
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C-repeat-binding factors and reactive oxygen species (ROS)-related antioxidant genes, which
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helps Arabidopsis thaliana induce resistance to chilling stress (Bajwa, Shukla, Sherif, Murch &
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Saxena, 2014). However, more recently, attention has been partially directed toward the effect of
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exogenous MT application on oxidative stress-induced senescence and CI in postharvest fruit. Sun
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et al. (2016) found that MT plays a crucial role in the regulation of senescence in tomato fruit. Gao,
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Zhang, Lv, Cheng, Peng & Cao (2016) putted forward a similar result that MT at a concentration
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of 0.1 mM leads to a clear delay of senescence in peach fruit during ambient storage, through
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antioxidative mechanism. Aghdam and Fard (2017) reported that MT is conductive to attenuating
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postharvest decay in strawberry fruit by mechanisms that trigger the accumulation of hydrogen
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peroxide and phenolic compounds and induces the activity of γ-aminobutyric acid shunt. Cao,
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Song, Shao, Bian, Chen & Yang (2016) proved that MT ensures better prevention of CI in peach
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fruit during low temperature storage, and such effect has been attributed in part to MT-induced
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promotion of polyamine, γ-aminobutyric acid and proline. These findings undoubtedly reveal new
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insights into the physiological roles of MT in plants. However, further work along these lines
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would be valuable, since there is only a limited understanding at present.
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The purpose of this paper is to advance our understanding of the possible underlying
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mechanisms of how MT induces a defence response of peach fruit to avoid CI. In peach fruit,
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previous studies have reported that both biosynthesis of phenolic compounds (Jin, Zheng, Tang,
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Rui & Wang, 2009; Gao et al., 2016) and maintenance of a higher ratio of unsaturated to saturated
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fatty acids (Jin, Zhu, Wang, Shan, & Zheng, 2014) may account for the enhancement of chilling
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tolerance. Therefore, the focus of the present study was on whether the MT-induced changes in
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membrane fatty acid contents and phenolic metabolism are linked to the enhanced tolerance to CI
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in peach fruit during low temperature storage.
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2. Material and methods
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2.1. Plant material and treatments
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Mature pre-climacteric fruit of peach (Prunus persica Batsch cv ‘Chuanzhongdao’) were
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hand-collected from a well-managed commercial orchard in Xi’an, China. Fruit were chosen for
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uniformity without any defects, and then separated into 2 lots at random. Fruit of the first lot were
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dipped in a solution of 0.1 mM MT for 10 min in the low light to prevent light-induced MT
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degradation (Gao et al., 2016). Under the same condition, fruit of the second lot were soaked in
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distilled water and designed as the control. Afterwards, all fruit were dried in the air and stored at
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1 °C with a relative humidity of 85-90% for 28 days. Fruit were removed from each lot after 0, 7,
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14, 21 and 28 d of low temperature storage to evaluate CI incidence, CI index and firmness loss.
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Meanwhile, flesh tissue samples derived from 10 fruit were collected and stored at -80 °C for
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subsequent measurements. For each lot, three replicates were performed.
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2.2. CI incidence, CI index and firmness
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CI incidence was defined as the ratio of CI-fruit to total fruit and expressed as %.
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CI index was assessed visually based on the percentage of the cut surface of peach slices that
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exhibited browning with a scale from 0 to 4: 0 (none), 1 (<25%), 2 (25−50%), 3 (50−75%), 4
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(>75%), according to Wang, Chen, Kong, Li, & Archbold (2006). CI index was obtained from the
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formula: CI index = ∑ (CI scale × number of fruit in each scale) / (4 × total number of fruit). Firmness was determined with a fruit firmness tester (GY-3, Aidebao Instrument Co., Ltd,
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Leqing, China) fitted with an 8 mm diameter probe and expressed as N.
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2.3. Malondialdehyde content
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Malondialdehyde (MDA) content was tested using a modified method described by Dhindsa,
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Pulmb-Dhindsa & Thorpe (1981). Flesh tissue (2 g) was homogenized with 10 ml of 10%
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trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The mixture was boiled at 100 °C
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for 10 min, cooled quickly thereafter, and centrifuged at 5000 g for 15 min. Absorbance of the
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supernatant was measured at 450, 532 and 600 nm. MDA content was expressed on a fresh weight
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basis as µmol g−1.
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2.4. Measurement of composition of fatty acid
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The extraction and quantification of fatty acids was performed according to the method
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described by Jin et al. (2014). Flesh tissue (20 g) was homogenized with chloroform/methanol (2:1)
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solution and the mixture was acidified with 0.1 N HCl. After centrifugation at 4000g for 10 min,
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the organic layer was collected and taken to dryness. Total fatty acids were methylated by adding
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trifluoride 14% methanolic solution and methylated fatty acids were extracted with hexane. After
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that, the solvent was dried and the methylated fatty acids were redissolved in 0.2 ml chloroform
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and analyzed for fatty acid composition by a Hitachi 663-30 Gas Chromatograph with a flame
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ionization detector. Fatty acids were identified based on the retention time of the internal standard
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free fatty acid C17:0. The ratio of unsaturated to saturated fatty acid was calculated from the
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formula: [oleic acid (18:1) + linoleic acid (18:2) + linolenic acid (18:3)] / [palmitic acid (16:0) +
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stearic acid (18:0)].
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2.5. Total phenolic and endogenous salicylic acid contents
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Flesh tissue (2 g) was homogenized in 5 ml of methanol. After centrifugation at 12,000× g for
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15 min, the supernatant was collected and used to measure the content of total phenolic. In brief,
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0.5 ml of supernatant was gently mixed with 1.0 ml of Folin-Ciocalteu reagent and 3 ml of 1 M
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sodium carbonate and the total volume of the mixture were adjusted to 10 ml with distilled water.
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After the mixture had been kept at room temperature for 1 h, the absorbance was read at 760 nm
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(Hinneburg, Dorman, & Hiltunen, 2006). Results were expressed as the mass of gallic acid
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equivalents on a fresh weight basis in mg g-1.
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Endogenous salicylic acid content was measured by an enzyme-linked immunosorbent assay
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(ELISA). A plant salicylic acid ELISA kit was used following the instructions of the manufacturer
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(Nanjing Senbeijia Biological Technology Co., Ltd, Nanjing, China). In brief, flesh tissue (1 g)
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was homogenized in 5 ml of methanol and the supernatant collected. To test the supernatant 10 µl
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was diluted with 50 µl sample dilution in a 96-well microplate. The mixtures were incubated in the
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water bath at 37 °C for 30 min. After washing 5 repeats, HRP-Conjugate reagent and chromogen
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solution were added and incubated for 15 min at 37 °C in the dark. The reaction was stopped by
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sulfuric acid and the changes in colour were noted at 450 nm. The content of endogenous salicylic
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acid was calculated following the standard curve.
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2.6. Measurement of enzymes activities associated with phenolic metabolism
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For determination of lipoxygenase (LOX), shikimate dehydrogenase (SKDH), polyphenol
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oxidase (PPO) and peroxidase (POD), flesh tissue (2 g) was homogenized in 5 ml of 50 mM
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potassium-phosphate buffer, pH 6.8. And then sample was centrifuged at 12,000 g for 15 min at
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4 °C. For determination of glucose-6-phosphate dehydrogenase (G6PDH), flesh tissue (2 g) was
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homogenized in 10 ml of potassium-phosphate buffer, pH 7, containing 1 mM EDTA, 3 mM of
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magnesium sulfate and 1 mM polyvinylpyrrolidone. The sample was then centrifuged at 12,000 g
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for 15 min at 4 °C. For determination of phenylalanine ammonia-lyase (PAL), flesh tissue (2 g)
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was homogenized with 5 ml of 0.2 M borate buffer, pH 8.8, containing 6 g of
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polyvinylpyrrolidone. The sample was then centrifuged at 12,000 g for 15 min at 4 °C. The
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supernatants were then used for the enzyme activity.
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LOX activity was assayed according to the method of Surrey (1963). Reaction mixture
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consisted 2.775 ml 100 mM sodium acetate buffer, pH 5.5, 25µl linoleic acid and 0.2 ml of
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supernatant. The absorbance was measured by an increase in absorbance at 234nm. LOX activity
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was expressed on a fresh weight basis as U g−1, where U = 0.01 ∆A234 nm per min.
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G6PDH activity was determined using the method described in Debnam and Emes (1999). 0.2
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ml of supernatant was mixed with 1.8 ml of 50 mM Hepes-NaOH phosphate buffer, pH 8, 5 mM
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glucose-6-phosphate disodium salt, 0.8 mM NADP+ and 2 mM magnesium chloride. The
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absorbance was measured by an increase in absorbance at 340 nm. G6PDH activity was expressed
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on a fresh weight basis as U g−1, where U = 0.01 ∆A340 nm per min.
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SKDH activity was measured in a mixture consisting of 1.9 ml of 100 mM Tris-HCl, pH 9.0,
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1.45 ml of 2 mM shikimic acid and 1.45 ml of 0.5 mM NADP and 0.2 ml of supernatant
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(Sánchez-Rodríguez, Moreno, Ferreres, Rubio-Wilhelmi & Ruiz, 2011). The absorbance was read
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by the reduction of NADP at 340 nm. SKDH activity was expressed on a fresh weight basis as U
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g−1, where U = 0.01 ∆A340 nm per min.
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PAL activity was determined using the method described by Sánchez-Rodríguez et al. (2011).
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The reaction mixture consisted of 2 ml of 0.2 M borate buffer, pH 8.8, 1.0 ml of 0.6 mM
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L-phenylalanine
and 0.2 ml of supernatant for 1 h at 37 °C. An increase in absorbance at 290 nm
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caused by the formation of trans-cinnamate was recorded. PAL activity was expressed on a fresh
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weight basis as U g−1, where U = 0.01 ∆A290 nm per min.
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PPO activity was assayed in a mixture consisting of 2.8 ml of 100 mM catechol and 0.2 ml of
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supernatant (Murr and Morris1974). The increase in absorbance at 420 nm was recorded. PPO
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activity was expressed on a fresh weight basis as U g−1, where U = 0.01 ∆A420 nm per min.
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POD activity was measured according to the method of Kochba, Lavee & Spiege (1997). 0.4
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ml of the supernatant was mixed with 2.1 ml of 50mM phosphate buffer, pH 6.8, 0.25 ml of 0.16
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M guaiacol and 0.25 ml of 0.88 M H2O2. The absorbance was measured by an increase in
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absorbance at 470 nm. POD activity was expressed on a fresh weight basis as U g−1, where U =
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0.01 ∆A470 nm per min.
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2.7. Statistical analysis
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The experiments were performed in a completely randomized design. All experiments were
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conducted in triplicate and average values with standard errors are reported. Analysis of variance
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was carried out, and means were compared using Tukey’s multiple range tests at a significance
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level of 0.05 with Origin (Version 7.5).
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3. Results
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3.1. Effects of MT treatment on chilling injury and fruit firmness
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Flesh browning is a notable CI symptom in this study, and it was first observed in control fruit
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after 7 day storage at low temperature, and more intensive incidence and severity of flesh
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browning were exhibited thereafter. MT treatment has a good control effect for chilling-induced
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flesh browning as reflected by reduced CI incidence and CI index, as shown in Fig. 1A and B. By
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the end of storage, 46% and 63% decreases in CI incidence and CI index were respectively
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recorded in comparison to the control fruit.
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Accelerated loss of firmness is indicative of chilling-induced injury. Firmness loss in control
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fruit occurred mostly in the first 7 days of storage, and declined by 88% over the initial value. MT
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treatment inhibited the loss of firmness. After 7 days of storage, firmness in MT-treated fruit was
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about 3 times higher than that in control fruit (Fig. 1C).
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3.2. Effects of MT treatment on MDA content and LOX activity
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A similar change pattern was obtained for MDA content where MT-treated fruit displayed an
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obvious decrease. MT treatment caused 31% and 46% deceases of MDA content on day 14 and
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day 28, respectively (Fig. 2A).
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LOX activity in control fruit increased at first, and arrived at the maximum value of 209.92 U
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g-1 on day 21, and then decreased slightly. MT treatment inhibited the activity of LOX. On day 21,
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LOX activity in MT-treated fruit was 88.29 U g-1, which was about 2.4 times lower than that in
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control fruit (Fig. 2B).
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3.3. Effects of MT treatment on fatty acid composition
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Five fatty acids were analyzed in flesh tissue of peach fruit. Among the quantified fatty acids,
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palmitic and stearic acids are saturated fatty acid, whereas oleic, linoleic and linolenic acids
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belong to unsaturated fatty acid. During the whole low temperature storage, an increasing trend
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was found for the contents of palmitic, stearic and oleic acids (Fig. 3A, B and C); however,
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contents of linoleic and linolenic acids and the ratio of unsaturated to saturated fatty acids
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presented an opposite change pattern, namely, decreased continuously (Fig. 3D, E and F). MT
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treatment has a good control effect for the enhancement of palmitic, stearic and oleic acids and the
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reduction of linoleic and linolenic acids, which resulted in a higher ratio of unsaturated to
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saturated fatty acids in MT-treated fruit.
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3.4. Effects of MT treatment on total phenolic and endogenous salicylic acid contents
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Control fruit showed a gradual decrease in total phenolic content from 0.34 mg g-1 (at initial)
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to 0.15 mg g-1 (at the end of storage). However, MT treatment suppressed the decline (Fig. 4A).
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During the whole low temperature storage, MT treatment helped to reduce the loss of total
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phenolic in peach fruit by up to 82% as compared to the control.
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Change in endogenous salicylic acid content was similar to that of total phenolic. As shown in
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Fig. 4B, MT treatment delayed the loss of endogenous salicylic acid as well, showing about 29%
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decline over the initial value by the end of storage.
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3.5. Effects of MT treatment on G6PDH, SKDH and PAL activity
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As shown in Fig. 5A, G6PDH activity in control fruit decreased at first, and then increased
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and declined again by the end of storage. MT treatment enhanced G6PDH activity, and the activity
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of G6PDH was more than 2.3 times higher in MT-treated fruit than in control on day 28. SKDH
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activity in MT-treated fruit increased before 7 day storage under low temperature and fluctuated
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thereafter between 0.96 and 0.76 U g-1, and was higher than that in control fruit during the whole
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storage (Fig. 5B). A similar trend was observed for PAL activity where MT-treated fruit displayed
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an obvious increase. MT treatment resulted in 36% enhancement of PAL activity after 28 day
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storage under low temperature, as shown in Fig. 5C.
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3.6. Effects of MT treatment on PPO and POD activity
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PPO activity in MT-treated fruit increased before 15 days and decreased thereafter. Higher
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activity of PPO was observed in control fruit than MT-treated fruit during the whole low
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temperature storage (Fig. 6A). POD activity in control fruit decreased between day 0 and day 7,
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and then increased thereafter (Fig. 6B). MT treatment inactivated POD activity and the activity of
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POD in MT-treated fruit was 1.31 U g-1, which was about 2.1 times lower than that in control
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fruit.
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4. Discussion
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CI is the principal limiting factor for application of low temperature to preserve peach fruit.
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Therefore, seeking effective techniques for alleviating CI in peach fruit has always been a centre
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of attention. Previous studies have demonstrated that MT fulfills a crucial role in protection
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against CI in plants (Posmyk et al., 2009; Szafrańska et al., 2013; Bajwa et al., 2014; Szafrańska et
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al., 2014; Turk, Erdal, Genisel, Atici, Demir & Yanmis, 2014; Turk and Erdal, 2015; Ding, Liu &
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Zhang, 2017); however, there are few studies on the effect of MT on CI in postharvest fruit. In a
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recent study, Cao et al. (2016) concluded that exogenous MT application leads to a decreased
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incidence of CI in ‘Jinghu’ peach cultivar, providing the first evidence that MT treatment
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contributes to enhancing chilling resistance in postharvest fruit. In this study it was found that MT
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also alleviates CI in ‘Chuanzhongdao’ peach cultivar, as was illustrated by which MT-treated fruit
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showed lower CI incidence, CI index and firmness loss (an indicative of CI) than that control (Fig.
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1), consistent with the findings of Cao et al. (2016).
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It is well known that destabilization of the cell membrane is the primary cause of CI in plants
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(Lyons, 1973). Increase of membrane lipid desaturation during chilling acclimatisation promotes
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the stability of cell membrane, and as a result, acts as an important determinant of plant tolerance
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to chilling stress (Graham and Patterson, 1982). In accordance with this, previous studies have
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shown that membrane fatty acids from chilling-sensitive plants presented a lower proportion of
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unsaturated fatty acids than did chilling-resistant plants (Boonsiri, Ketsa & van Doom, 2007;
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Wongsheree, Ketsa & van Doom, 2009). Maintenance of a high level of membrane lipid
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desaturation has also been shown to be propitious to control CI in postharvest fruit. For example,
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‘Nanguo’ pear fruit fumigated with 1- methylcyclopropene resulted in a higher ratio of unsaturated
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to saturated fatty acids, which attenuated the impact of CI (Cheng, Wei, Zhou, Tian & Ji, 2015).
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Treatment of peach fruit with oxalic acid improved resistance to CI and was accompanied by an
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elevated ratio of unsaturated to saturated fatty acids (Jin et al. 2014). The results presented in our
272
study showed that the development of CI in peach fruit was associated with the increase in
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contents of palmitic and stearic acids and the decrease in contents of linoleic and linolenic acids.
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However, MT treatment maintained higher contents of the two unsaturated fatty acids, which
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inclined the MT-treated fruit to exhibit an advanced ratio of unsaturated to saturated fatty acids
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(Fig. 3). These results suggested that MT may help the cell membrane avoid destabilization by
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desaturating fatty acids of membrane lipids, giving rise to the chilling resistance in peach fruit
278
during low temperature storage. Aghdam and Fard (2017) have come to a similar conclusion
279
focusing on the effect of MT on membrane lipid desaturation in strawberry fruit during ambient
280
temperature storage.
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On the other hand, under chilling stress, cell membranes can become destabilized because of
282
lipid peroxidation. The peroxidation of membrane lipids can be triggered by the enzyme LOX
283
which catalyzes the reaction of molecular oxygen with polyunsaturated fatty acids containing cis,
284
cis-1, 4-pentadiene structures to produce hydroperoxy fatty acids (Shewfelt and del Rosario, 2000).
285
Eventually, MDA is formed and has been utilized very often as a reliable estimator for lipid
286
peroxidation (Dhindsa, Plumb-Dhindsa, & Thorpe, 1981). Higher LOX activity and MDA content
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have been found to contribute to the development of CI in peach fruit during low temperature
288
storage (Cao, Hu, Zheng & Lu, 2010; Gao et al., 2016). In the current study, the activation of LOX
289
and the formation of MDA in peach fruit due to chilling stress were inhibited by MT treatment
290
(Fig. 2). This indicated that MT treatment has facilitated the reduction of membrane lipid
291
peroxidation and cell membrane destabilization, which could, in turn, promote the ability of peach
292
fruit to resist CI. Similar alleviation of CI by suppression of membrane lipid peroxidation due to
293
MT treatment has been reported in cucumber seeds (Posmyk et al., 2009), Vigna radiata root
294
(Szafrańska et al., 2013; Szafrańska et al., 2014), wheat and maize seedlings (Turk et al., 2014;
295
Turk et al., 2015) and tomato plants (Ding et al., 2017).
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During low temperature storage of postharvest peach fruit, the increased accumulation of total
297
phenolics appears to confer increased resistance to CI (Jin et al., 2009; Gao et al., 2016).
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Undoubtedly that is partially because phenolic compounds can protect membrane lipid against
299
peroxidation by preventing the initiation and propagation of oxidizing chain reactions
300
(Pennycooke, Cox & Stushnoff, 2005). In this study, the result of the correlation analysis showed
301
that total phenolic content was negatively related to LOX activity (r = -0.94) in control fruit, while
302
the loss in the content of total phenolic was delayed by MT treatment during the whole low
303
temperature storage (Fig. 4A), which indicated that MT might lead to accumulation of phenolic
304
compounds and thus provide peach fruit with direct and sufficient chain-breaking activity. This
305
observation is further supported by two types of observations, namely, endogenous salicylic acid
306
content is higher in MT-treated fruit (Fig. 4B), and there is a strong inverse relationship between
307
endogenous salicylic acid content and LOX (r = -0.95). Salicylic acid has been reported to be
308
effective in reducing lipid peroxidation in peach fruit during low temperature storage (Wang et al.,
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309
2006), and as a consequence, contributing to the activation of the chilling tolerance. Taken
310
together, it is therefore believed that phenolic compounds are implicated in the MT-induced
311
chilling tolerance in peach fruit, and it can improve the antioxidant potential of peach fruit under
312
chilling stress.
313
G6PDH, SKDH and PAL are three essential enzymes for phenolic anabolism. Among them,
314
G6PDH is the first rate-limiting enzyme of the pentose phosphate pathway, and its main function
315
is to supply the substrate erythrose-4-phosphate for shikimate pathway (Debnam and Emes, 1999).
316
The reaction that SKDH catalyzes is the third and fourth steps of the shikimate pathway,
317
converting 3-dehydroquinate into shikimate. Shikimate itself is a precursor to those aromatic
318
amino acids like l-phenylalanine (Díaz and Merino 1997). PAL is the principal enzyme
319
responsible for transformation of L-phenyalanine into trans-cinnamic acid at the entry point of the
320
phenylpropanoid
321
(Sánchez-Rodríguez et al., 2011). There is empirical evidence that enzymes involved in phenolic
322
compounds mobilization have been relevant to plant tolerance to chilling stress. In cucumber
323
seedlings, for example, Arbuscular mycorrhizal fungi inoculation resulted in higher activities of
324
G6PDH, SKDH and PAL as well as their corresponding gene expression, along with the induction
325
of chilling adaptive response (Chen et al., 2014). It was also reported that 24-epibrassinolide
326
immersion induced tolerance of peach fruit to chilling stress, which has been partially attributed to
327
the elevated activities of SKDH and PAL (Gao et al., 2016). We reported herein that MT induced
328
increases in activities of G6PDH, SKDH and PAL, and stimulated the accumulation of total
329
phenolic as well as endogenous salicylic acid. Furthermore, MT-treated fruit showed a lower
330
occurrence of CI than control fruit during the whole low temperature storage. These findings
pathway
driving
towards
15
the
generation
of
phenolic
compounds
331
revealed that the ability of MT treatment protects peach fruit from CI may be due to activation of
332
phenolic compounds biosynthesis to some extent. This is in agreement with a formerly reported
333
result by Szafrańska et al. (2014), who proposed that one of the functional routes of MT activity
334
may be to prevent Vigna radiate root from CI, by stimulating the biosynthesis of phenolic
335
compounds. Molecular evidence also indicates that MT can up-regulate the expression of genes
336
pertaining to salicylic acid pathway in Arabidopsis plants (Weeda, Zhang, Zhao, Ndip, Guo, &
337
Buck, 2014). In strawberry fruit, Aghdam and Fard (2017) proposed that the phenolic anabolism
338
due to MT treatment is beneficial to attenuating fungal decay.
339
In the case of CI, PPO and POD are deemed to have an antagonistic effect on enzymes
340
affecting phenolic anabolism. These two enzymes work together to catalyze the oxidation of
341
phenolic compounds to quinines and result in flesh browning (Zhang et al., 2015), which has been
342
reported to be a notable CI symptom in peach fruit (Lurie and Crisosto, 2005). Therefore, for
343
example, inhibited activities of PPO and POD were observed in peach fruit treated by hot air in
344
combination with methyl jasmonate vapour, this being less sensitive to CI after treatment (Jin et
345
al., 2009). Also, Gao et al. (2016) found that 24-epibrassinolide alleviated CI of peach fruit, and
346
this process was accompanied by lower activities of PPO and POD. The results presented in our
347
study showed that the application of MT treatment led to a decrease in activities of PPO and POD
348
during the whole low temperature storage compared to control fruit, for which increase of these
349
two enzymes was observed (Fig. 6). In this sense, the decrease in activities of PPO and POD could
350
be a defence mechanism against chilling stress, and could be account for the lower flesh browning
351
found in MT-treated peach fruit.
352
In conclusion, treatment with 0.1 mM MT alleviated CI in peach fruit and the enhancement of
16
353
resistance against CI by MT treatment may be derived from its capacity to maintain a higher ratio
354
of unsaturated to saturated fatty acids, inhibit membrane lipid peroxidation as well as stimulate
355
phenolic mobilization. Given that MT has minimal toxicity to human beings over a very wide dose
356
range (Galano et al., 2011), these results suggest that the application of MT treatment to peach
357
fruit could be considered as an effective postharvest technique with good results in terms of
358
reducing CI damage.
359
Acknowledgements
360
This work was supported by the National Natural Science Foundation of China (grant number
361
31401550) and the National Innovation Experiment Project for College Students of China (grant
362
number 201510697022).
363
Conflict of interest
364
We declare that we have no conflict of interest.
365
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473 474 475 476 477 478 479 480 481 482 483 484 485 22
486 487
Figure Captions
488
Fig. 1 Effects of MT treatment on CI occurrence (A), CI index (B) and firmness (C) of peach fruit
489
during low temperature storage. Vertical bars represent the standard errors of the means of
490
triplicate assays. Values with different letters for each day were significantly different at P < 0.05.
491
Fig. 2 Effects of MT treatment on MDA content (A) and LOX activity (B) of peach fruit during
492
low temperature storage. Vertical bars represent the standard errors of the means of triplicate
493
assays. Values with different letters for each day were significantly different at P < 0.05.
494
Fig. 3 Effects of MT treatment on palmitic acid (A), stearic acid (B), oleic acid (C), linoleic acid
495
(D) and linolenic acid (E) contents and the ratio of unsaturated to saturated fatty acids (F) of peach
496
fruit during low temperature storage.
497
Fig. 4 Effects of MT treatment on total phenolic (A) and endogenous salicylic acid (B) contents of
498
peach fruit during low temperature storage. Vertical bars represent the standard errors of the
499
means of triplicate assays. Values with different letters for each day were significantly different at
500
P < 0.05.
501
Fig. 5 Effects of MT treatment on G6PDH (A), SKDH (B) and PAL (C) activities of peach fruit
502
during low temperature storage. Vertical bars represent the standard errors of the means of
503
triplicate assays. Values with different letters for each day were significantly different at P < 0.05.
504
Fig. 6 Effects of MT treatment on PPO (A) and POD (B) activities of peach fruit during low
505
temperature storage. Vertical bars represent the standard errors of the means of triplicate assays.
506
Values with different letters for each day were significantly different at P < 0.05.
507
23
80
508 CI incidence (%)
Control MT
60 40 20 0 0
7
14
21
28
14
21
28
0.5 Control
CI index
0.4
MT
0.3 0.2 0.1 0 0
7
120 Control 100 Firmness (N)
509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549
MT
80 60 40 20 0 0
7
14
21
Storage time (days)
24
28
MDA content (µmol g -1)
3.5 Control 2.8
MT
2.1 1.4 0.7 0 0
7
14
21
28
14
21
28
250 Control LOX activity (U g -1)
550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
200
MT
150 100 50 0 0
7
Storage time (days)
25
40
3.6
30
2.7
20
1.8 Control
Control
MT
MT
0.9
0
0 0
7
14
21
28
0
7
14
21
28
21
35
18
28
15 12
21
9
14
6 3
Control
Control
MT
MT
Linoleic acid (%)
Oleic acid (%)
Stearic acid (%)
4.5
7
0
0 0
7
14
21
28
0
7
14
21
28
40
4
32
3
24 2 16 8 0 0
1
Control
Control
MT
MT
7
14
21
28
0
Storage time (days)
7
14
21
Storage time (days)
26
0 28
Unsaturated to saturated fatty acid ratio
Palmitic acid (%)
50
10
Linolenic acid (%)
591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634
Total phenolics (mg g-1)
0.5 0.4 0.3 0.2 Control
0.1
MT 0 0
7
14
21
21
28
Salicylic acid content (µg g-1)
1.5
18 Oleic acid (%)
635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
15 12 9 6
Control
3
MT
0
1.2 0.9 0.6 0.3
Control MT
0
0
7
14
21
28
0
7
14
21
Storage time (days)
27
28
G6PDH activity (U g -1)
10 Control 8
MT
6 4 2 0 0
7
14
21
28
28+3
SKDH activity (U g -1)
1.2 0.9 0.6 Control
0.3
MT 0 0
7
14
21
28
14
21
28
1.4 1.2 PAL activity (U g -1)
675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710
1 0.8 0.6 0.4
Control
0.2
MT
0 0
7
Storage time (days)
28
25 PPO activity (U g -1)
Control 20
MT
15 10 5 0 0
7
14
21
28
21
28
3 POD activity (U g -1)
711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743
Control 2
MT
1
0 0
7
14 Storage time (days)
744
29
745
Highlights
746
►MT delayed the development of chilling injury in peach fruit.
747
►MT maintained higher ratio of unsaturated to saturated fatty acids in peach fruit.
748
►MT stimulated phenolic anabolism and prevented lipid peroxidation in peach fruit.
749
►MT inhibited PPO and POD activities leading to reduced flesh browning in peach fruit.
750 751 752
30
S0308-8146(17)31637-0 https://doi.org/10.1016/j.foodchem.2017.10.008 FOCH 21831
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
8 June 2017 4 September 2017 3 October 2017
Please cite this article as: Gao, H., Lu, Z., Yang, Y., Wang, D., Yang, T., Cao, M., Cao, W., Melatonin treatment reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and phenolic metabolism, Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.10.008
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1
Melatonin treatment reduces chilling injury in peach fruit through its regulation of
2
membrane fatty acid contents and phenolic metabolism
3
Running title: Effects of MT treatment on chilling injury of peach fruit
4
Hui Gao*, ZeMian Lu, Yue Yang, DanNa Wang, Ting Yang, MaoMao Cao, Wei Cao
5
Department of Food Science and Engineering, Northwest University, Xi’an 710069, P. R. China
6
*
7
Email address: [email protected]
8
[email protected]
9
[email protected]
10
[email protected]
11
[email protected]
12
[email protected]
13
[email protected]
Corresponding author: Tel: +86-29-88302213; Fax: +86-29-88302213.
14 15 16 17 18 19 20 21 22
1
23
ABSTRACT
24
Effects of 0.1 mM melatonin (MT) on chilling injury (CI), membrane fatty acid content and
25
phenolic metabolism in peach fruit were studied during storage at 1°C for 28 days. MT treatment
26
delayed the development of CI in peach fruit, as was illustrated by MT-treated fruit showing lower
27
CI incidence, CI index and firmness loss than the control. MT treatment prevented membrane lipid
28
peroxidation and contributed to maintaining a higher ratio of unsaturated to saturated fatty acids in
29
peach fruit. MT treatment also stimulated the activities of glucose-6-phosphate dehydrogenase,
30
shikimate dehydrogenase and phenylalanine ammonia lyase, but inhibited the activities of
31
polyphenol oxidase and peroxidase. This would help in activating the accumulation of total
32
phenolic and endogenous salicylic acid that might have a direct function in alleviation of CI.
33
These results indicate that MT treatment can be an effective technique to reduce postharvest CI
34
during low temperature storage of peach fruit.
35
Keywords: peach fruit, chilling injury, melatonin, fatty acid, phenolic metabolism
36 37 38 39 40 41 42 43 44
2
45
1. Introduction
46
Chilling injury (CI) is a physiological disorder commonly occurring in a large collection of
47
peach cultivars during low temperature storage, especially when storing at a potential risk zone of
48
temperature between 2.2 and 7.6 °C (Lurie and Crisosto, 2005). The disorder is characterized
49
mainly by flesh browning and/or mealiness, abnormal ripening and higher sensitivity to decay
50
(Lurie and Crisosto, 2005), and is considered as a primary limitation of application of low
51
temperature to preserve peach fruit. Many hypotheses have been put forward for a CI mechanism
52
in plants, such as involvement of imbalances in metabolism, accumulation of toxic compounds,
53
decreased water dynamic state and increased permeability (Lyons, 1973; Naruke, Oshita, Kuroki,
54
Seo, Kawagoe & Walton, 2003; Purwanto et al., 2013). To deal with this problem, researchers
55
have been devoting a great deal of attention to finding economical, convenient and highly
56
effective techniques to reduce CI in peach fruit.
57
Melatonin (MT) is an indoleamine hormone ubiquitous in nature. In plants, MT has been
58
identified in nearly all organs and tissues, and is shown to be a signaling molecule involved in
59
numerous physiological processes such as differentiation, growth, ripening and senescence of
60
plant and the protective effect against various forms of environmental stress (Reiter, Tan, Zhou,
61
Cruz, Fuentes-Broto & Galano, 2015; Zhang et al., 2015). Exogenous MT has been demonstrated
62
to be effective in alleviating chilling stress-induced damage in plants through different
63
mechanisms. For example, Posmyk, Balabusta, Wieczorek, Sliwinska & Janas (2009) revealed
64
that MT treatment significantly counteracts the adverse effect of chilling stress on cucumber seeds
65
by influencing the structure and function of cellular membrane. Likewise, in another study,
66
researchers found that application of MT contributes to reducing the risk of ultrastructural damage
3
67
in meristematic cells of Vigna radiata roots due to chilling exposure (Szafrańska, Glińska & Janas,
68
2013). Their data also documented that MT-medicated Vigna radiata root acclimation to chilling
69
stress is associated with its ability to activate the pathway of phenolic (Szafrańska, Szewczyk &
70
Janas, 2014). It was also shown that MT has a positive regulation in the expression of
71
C-repeat-binding factors and reactive oxygen species (ROS)-related antioxidant genes, which
72
helps Arabidopsis thaliana induce resistance to chilling stress (Bajwa, Shukla, Sherif, Murch &
73
Saxena, 2014). However, more recently, attention has been partially directed toward the effect of
74
exogenous MT application on oxidative stress-induced senescence and CI in postharvest fruit. Sun
75
et al. (2016) found that MT plays a crucial role in the regulation of senescence in tomato fruit. Gao,
76
Zhang, Lv, Cheng, Peng & Cao (2016) putted forward a similar result that MT at a concentration
77
of 0.1 mM leads to a clear delay of senescence in peach fruit during ambient storage, through
78
antioxidative mechanism. Aghdam and Fard (2017) reported that MT is conductive to attenuating
79
postharvest decay in strawberry fruit by mechanisms that trigger the accumulation of hydrogen
80
peroxide and phenolic compounds and induces the activity of γ-aminobutyric acid shunt. Cao,
81
Song, Shao, Bian, Chen & Yang (2016) proved that MT ensures better prevention of CI in peach
82
fruit during low temperature storage, and such effect has been attributed in part to MT-induced
83
promotion of polyamine, γ-aminobutyric acid and proline. These findings undoubtedly reveal new
84
insights into the physiological roles of MT in plants. However, further work along these lines
85
would be valuable, since there is only a limited understanding at present.
86
The purpose of this paper is to advance our understanding of the possible underlying
87
mechanisms of how MT induces a defence response of peach fruit to avoid CI. In peach fruit,
88
previous studies have reported that both biosynthesis of phenolic compounds (Jin, Zheng, Tang,
4
89
Rui & Wang, 2009; Gao et al., 2016) and maintenance of a higher ratio of unsaturated to saturated
90
fatty acids (Jin, Zhu, Wang, Shan, & Zheng, 2014) may account for the enhancement of chilling
91
tolerance. Therefore, the focus of the present study was on whether the MT-induced changes in
92
membrane fatty acid contents and phenolic metabolism are linked to the enhanced tolerance to CI
93
in peach fruit during low temperature storage.
94
2. Material and methods
95
2.1. Plant material and treatments
96
Mature pre-climacteric fruit of peach (Prunus persica Batsch cv ‘Chuanzhongdao’) were
97
hand-collected from a well-managed commercial orchard in Xi’an, China. Fruit were chosen for
98
uniformity without any defects, and then separated into 2 lots at random. Fruit of the first lot were
99
dipped in a solution of 0.1 mM MT for 10 min in the low light to prevent light-induced MT
100
degradation (Gao et al., 2016). Under the same condition, fruit of the second lot were soaked in
101
distilled water and designed as the control. Afterwards, all fruit were dried in the air and stored at
102
1 °C with a relative humidity of 85-90% for 28 days. Fruit were removed from each lot after 0, 7,
103
14, 21 and 28 d of low temperature storage to evaluate CI incidence, CI index and firmness loss.
104
Meanwhile, flesh tissue samples derived from 10 fruit were collected and stored at -80 °C for
105
subsequent measurements. For each lot, three replicates were performed.
106
2.2. CI incidence, CI index and firmness
107
CI incidence was defined as the ratio of CI-fruit to total fruit and expressed as %.
108
CI index was assessed visually based on the percentage of the cut surface of peach slices that
109
exhibited browning with a scale from 0 to 4: 0 (none), 1 (<25%), 2 (25−50%), 3 (50−75%), 4
110
(>75%), according to Wang, Chen, Kong, Li, & Archbold (2006). CI index was obtained from the
5
111 112
formula: CI index = ∑ (CI scale × number of fruit in each scale) / (4 × total number of fruit). Firmness was determined with a fruit firmness tester (GY-3, Aidebao Instrument Co., Ltd,
113
Leqing, China) fitted with an 8 mm diameter probe and expressed as N.
114
2.3. Malondialdehyde content
115
Malondialdehyde (MDA) content was tested using a modified method described by Dhindsa,
116
Pulmb-Dhindsa & Thorpe (1981). Flesh tissue (2 g) was homogenized with 10 ml of 10%
117
trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The mixture was boiled at 100 °C
118
for 10 min, cooled quickly thereafter, and centrifuged at 5000 g for 15 min. Absorbance of the
119
supernatant was measured at 450, 532 and 600 nm. MDA content was expressed on a fresh weight
120
basis as µmol g−1.
121
2.4. Measurement of composition of fatty acid
122
The extraction and quantification of fatty acids was performed according to the method
123
described by Jin et al. (2014). Flesh tissue (20 g) was homogenized with chloroform/methanol (2:1)
124
solution and the mixture was acidified with 0.1 N HCl. After centrifugation at 4000g for 10 min,
125
the organic layer was collected and taken to dryness. Total fatty acids were methylated by adding
126
trifluoride 14% methanolic solution and methylated fatty acids were extracted with hexane. After
127
that, the solvent was dried and the methylated fatty acids were redissolved in 0.2 ml chloroform
128
and analyzed for fatty acid composition by a Hitachi 663-30 Gas Chromatograph with a flame
129
ionization detector. Fatty acids were identified based on the retention time of the internal standard
130
free fatty acid C17:0. The ratio of unsaturated to saturated fatty acid was calculated from the
131
formula: [oleic acid (18:1) + linoleic acid (18:2) + linolenic acid (18:3)] / [palmitic acid (16:0) +
132
stearic acid (18:0)].
6
133
2.5. Total phenolic and endogenous salicylic acid contents
134
Flesh tissue (2 g) was homogenized in 5 ml of methanol. After centrifugation at 12,000× g for
135
15 min, the supernatant was collected and used to measure the content of total phenolic. In brief,
136
0.5 ml of supernatant was gently mixed with 1.0 ml of Folin-Ciocalteu reagent and 3 ml of 1 M
137
sodium carbonate and the total volume of the mixture were adjusted to 10 ml with distilled water.
138
After the mixture had been kept at room temperature for 1 h, the absorbance was read at 760 nm
139
(Hinneburg, Dorman, & Hiltunen, 2006). Results were expressed as the mass of gallic acid
140
equivalents on a fresh weight basis in mg g-1.
141
Endogenous salicylic acid content was measured by an enzyme-linked immunosorbent assay
142
(ELISA). A plant salicylic acid ELISA kit was used following the instructions of the manufacturer
143
(Nanjing Senbeijia Biological Technology Co., Ltd, Nanjing, China). In brief, flesh tissue (1 g)
144
was homogenized in 5 ml of methanol and the supernatant collected. To test the supernatant 10 µl
145
was diluted with 50 µl sample dilution in a 96-well microplate. The mixtures were incubated in the
146
water bath at 37 °C for 30 min. After washing 5 repeats, HRP-Conjugate reagent and chromogen
147
solution were added and incubated for 15 min at 37 °C in the dark. The reaction was stopped by
148
sulfuric acid and the changes in colour were noted at 450 nm. The content of endogenous salicylic
149
acid was calculated following the standard curve.
150
2.6. Measurement of enzymes activities associated with phenolic metabolism
151
For determination of lipoxygenase (LOX), shikimate dehydrogenase (SKDH), polyphenol
152
oxidase (PPO) and peroxidase (POD), flesh tissue (2 g) was homogenized in 5 ml of 50 mM
153
potassium-phosphate buffer, pH 6.8. And then sample was centrifuged at 12,000 g for 15 min at
154
4 °C. For determination of glucose-6-phosphate dehydrogenase (G6PDH), flesh tissue (2 g) was
7
155
homogenized in 10 ml of potassium-phosphate buffer, pH 7, containing 1 mM EDTA, 3 mM of
156
magnesium sulfate and 1 mM polyvinylpyrrolidone. The sample was then centrifuged at 12,000 g
157
for 15 min at 4 °C. For determination of phenylalanine ammonia-lyase (PAL), flesh tissue (2 g)
158
was homogenized with 5 ml of 0.2 M borate buffer, pH 8.8, containing 6 g of
159
polyvinylpyrrolidone. The sample was then centrifuged at 12,000 g for 15 min at 4 °C. The
160
supernatants were then used for the enzyme activity.
161
LOX activity was assayed according to the method of Surrey (1963). Reaction mixture
162
consisted 2.775 ml 100 mM sodium acetate buffer, pH 5.5, 25µl linoleic acid and 0.2 ml of
163
supernatant. The absorbance was measured by an increase in absorbance at 234nm. LOX activity
164
was expressed on a fresh weight basis as U g−1, where U = 0.01 ∆A234 nm per min.
165
G6PDH activity was determined using the method described in Debnam and Emes (1999). 0.2
166
ml of supernatant was mixed with 1.8 ml of 50 mM Hepes-NaOH phosphate buffer, pH 8, 5 mM
167
glucose-6-phosphate disodium salt, 0.8 mM NADP+ and 2 mM magnesium chloride. The
168
absorbance was measured by an increase in absorbance at 340 nm. G6PDH activity was expressed
169
on a fresh weight basis as U g−1, where U = 0.01 ∆A340 nm per min.
170
SKDH activity was measured in a mixture consisting of 1.9 ml of 100 mM Tris-HCl, pH 9.0,
171
1.45 ml of 2 mM shikimic acid and 1.45 ml of 0.5 mM NADP and 0.2 ml of supernatant
172
(Sánchez-Rodríguez, Moreno, Ferreres, Rubio-Wilhelmi & Ruiz, 2011). The absorbance was read
173
by the reduction of NADP at 340 nm. SKDH activity was expressed on a fresh weight basis as U
174
g−1, where U = 0.01 ∆A340 nm per min.
175
PAL activity was determined using the method described by Sánchez-Rodríguez et al. (2011).
176
The reaction mixture consisted of 2 ml of 0.2 M borate buffer, pH 8.8, 1.0 ml of 0.6 mM
8
177
L-phenylalanine
and 0.2 ml of supernatant for 1 h at 37 °C. An increase in absorbance at 290 nm
178
caused by the formation of trans-cinnamate was recorded. PAL activity was expressed on a fresh
179
weight basis as U g−1, where U = 0.01 ∆A290 nm per min.
180
PPO activity was assayed in a mixture consisting of 2.8 ml of 100 mM catechol and 0.2 ml of
181
supernatant (Murr and Morris1974). The increase in absorbance at 420 nm was recorded. PPO
182
activity was expressed on a fresh weight basis as U g−1, where U = 0.01 ∆A420 nm per min.
183
POD activity was measured according to the method of Kochba, Lavee & Spiege (1997). 0.4
184
ml of the supernatant was mixed with 2.1 ml of 50mM phosphate buffer, pH 6.8, 0.25 ml of 0.16
185
M guaiacol and 0.25 ml of 0.88 M H2O2. The absorbance was measured by an increase in
186
absorbance at 470 nm. POD activity was expressed on a fresh weight basis as U g−1, where U =
187
0.01 ∆A470 nm per min.
188
2.7. Statistical analysis
189
The experiments were performed in a completely randomized design. All experiments were
190
conducted in triplicate and average values with standard errors are reported. Analysis of variance
191
was carried out, and means were compared using Tukey’s multiple range tests at a significance
192
level of 0.05 with Origin (Version 7.5).
193
3. Results
194
3.1. Effects of MT treatment on chilling injury and fruit firmness
195
Flesh browning is a notable CI symptom in this study, and it was first observed in control fruit
196
after 7 day storage at low temperature, and more intensive incidence and severity of flesh
197
browning were exhibited thereafter. MT treatment has a good control effect for chilling-induced
198
flesh browning as reflected by reduced CI incidence and CI index, as shown in Fig. 1A and B. By
9
199
the end of storage, 46% and 63% decreases in CI incidence and CI index were respectively
200
recorded in comparison to the control fruit.
201
Accelerated loss of firmness is indicative of chilling-induced injury. Firmness loss in control
202
fruit occurred mostly in the first 7 days of storage, and declined by 88% over the initial value. MT
203
treatment inhibited the loss of firmness. After 7 days of storage, firmness in MT-treated fruit was
204
about 3 times higher than that in control fruit (Fig. 1C).
205
3.2. Effects of MT treatment on MDA content and LOX activity
206
A similar change pattern was obtained for MDA content where MT-treated fruit displayed an
207
obvious decrease. MT treatment caused 31% and 46% deceases of MDA content on day 14 and
208
day 28, respectively (Fig. 2A).
209
LOX activity in control fruit increased at first, and arrived at the maximum value of 209.92 U
210
g-1 on day 21, and then decreased slightly. MT treatment inhibited the activity of LOX. On day 21,
211
LOX activity in MT-treated fruit was 88.29 U g-1, which was about 2.4 times lower than that in
212
control fruit (Fig. 2B).
213
3.3. Effects of MT treatment on fatty acid composition
214
Five fatty acids were analyzed in flesh tissue of peach fruit. Among the quantified fatty acids,
215
palmitic and stearic acids are saturated fatty acid, whereas oleic, linoleic and linolenic acids
216
belong to unsaturated fatty acid. During the whole low temperature storage, an increasing trend
217
was found for the contents of palmitic, stearic and oleic acids (Fig. 3A, B and C); however,
218
contents of linoleic and linolenic acids and the ratio of unsaturated to saturated fatty acids
219
presented an opposite change pattern, namely, decreased continuously (Fig. 3D, E and F). MT
220
treatment has a good control effect for the enhancement of palmitic, stearic and oleic acids and the
10
221
reduction of linoleic and linolenic acids, which resulted in a higher ratio of unsaturated to
222
saturated fatty acids in MT-treated fruit.
223
3.4. Effects of MT treatment on total phenolic and endogenous salicylic acid contents
224
Control fruit showed a gradual decrease in total phenolic content from 0.34 mg g-1 (at initial)
225
to 0.15 mg g-1 (at the end of storage). However, MT treatment suppressed the decline (Fig. 4A).
226
During the whole low temperature storage, MT treatment helped to reduce the loss of total
227
phenolic in peach fruit by up to 82% as compared to the control.
228
Change in endogenous salicylic acid content was similar to that of total phenolic. As shown in
229
Fig. 4B, MT treatment delayed the loss of endogenous salicylic acid as well, showing about 29%
230
decline over the initial value by the end of storage.
231
3.5. Effects of MT treatment on G6PDH, SKDH and PAL activity
232
As shown in Fig. 5A, G6PDH activity in control fruit decreased at first, and then increased
233
and declined again by the end of storage. MT treatment enhanced G6PDH activity, and the activity
234
of G6PDH was more than 2.3 times higher in MT-treated fruit than in control on day 28. SKDH
235
activity in MT-treated fruit increased before 7 day storage under low temperature and fluctuated
236
thereafter between 0.96 and 0.76 U g-1, and was higher than that in control fruit during the whole
237
storage (Fig. 5B). A similar trend was observed for PAL activity where MT-treated fruit displayed
238
an obvious increase. MT treatment resulted in 36% enhancement of PAL activity after 28 day
239
storage under low temperature, as shown in Fig. 5C.
240
3.6. Effects of MT treatment on PPO and POD activity
241
PPO activity in MT-treated fruit increased before 15 days and decreased thereafter. Higher
242
activity of PPO was observed in control fruit than MT-treated fruit during the whole low
11
243
temperature storage (Fig. 6A). POD activity in control fruit decreased between day 0 and day 7,
244
and then increased thereafter (Fig. 6B). MT treatment inactivated POD activity and the activity of
245
POD in MT-treated fruit was 1.31 U g-1, which was about 2.1 times lower than that in control
246
fruit.
247
4. Discussion
248
CI is the principal limiting factor for application of low temperature to preserve peach fruit.
249
Therefore, seeking effective techniques for alleviating CI in peach fruit has always been a centre
250
of attention. Previous studies have demonstrated that MT fulfills a crucial role in protection
251
against CI in plants (Posmyk et al., 2009; Szafrańska et al., 2013; Bajwa et al., 2014; Szafrańska et
252
al., 2014; Turk, Erdal, Genisel, Atici, Demir & Yanmis, 2014; Turk and Erdal, 2015; Ding, Liu &
253
Zhang, 2017); however, there are few studies on the effect of MT on CI in postharvest fruit. In a
254
recent study, Cao et al. (2016) concluded that exogenous MT application leads to a decreased
255
incidence of CI in ‘Jinghu’ peach cultivar, providing the first evidence that MT treatment
256
contributes to enhancing chilling resistance in postharvest fruit. In this study it was found that MT
257
also alleviates CI in ‘Chuanzhongdao’ peach cultivar, as was illustrated by which MT-treated fruit
258
showed lower CI incidence, CI index and firmness loss (an indicative of CI) than that control (Fig.
259
1), consistent with the findings of Cao et al. (2016).
260
It is well known that destabilization of the cell membrane is the primary cause of CI in plants
261
(Lyons, 1973). Increase of membrane lipid desaturation during chilling acclimatisation promotes
262
the stability of cell membrane, and as a result, acts as an important determinant of plant tolerance
263
to chilling stress (Graham and Patterson, 1982). In accordance with this, previous studies have
264
shown that membrane fatty acids from chilling-sensitive plants presented a lower proportion of
12
265
unsaturated fatty acids than did chilling-resistant plants (Boonsiri, Ketsa & van Doom, 2007;
266
Wongsheree, Ketsa & van Doom, 2009). Maintenance of a high level of membrane lipid
267
desaturation has also been shown to be propitious to control CI in postharvest fruit. For example,
268
‘Nanguo’ pear fruit fumigated with 1- methylcyclopropene resulted in a higher ratio of unsaturated
269
to saturated fatty acids, which attenuated the impact of CI (Cheng, Wei, Zhou, Tian & Ji, 2015).
270
Treatment of peach fruit with oxalic acid improved resistance to CI and was accompanied by an
271
elevated ratio of unsaturated to saturated fatty acids (Jin et al. 2014). The results presented in our
272
study showed that the development of CI in peach fruit was associated with the increase in
273
contents of palmitic and stearic acids and the decrease in contents of linoleic and linolenic acids.
274
However, MT treatment maintained higher contents of the two unsaturated fatty acids, which
275
inclined the MT-treated fruit to exhibit an advanced ratio of unsaturated to saturated fatty acids
276
(Fig. 3). These results suggested that MT may help the cell membrane avoid destabilization by
277
desaturating fatty acids of membrane lipids, giving rise to the chilling resistance in peach fruit
278
during low temperature storage. Aghdam and Fard (2017) have come to a similar conclusion
279
focusing on the effect of MT on membrane lipid desaturation in strawberry fruit during ambient
280
temperature storage.
281
On the other hand, under chilling stress, cell membranes can become destabilized because of
282
lipid peroxidation. The peroxidation of membrane lipids can be triggered by the enzyme LOX
283
which catalyzes the reaction of molecular oxygen with polyunsaturated fatty acids containing cis,
284
cis-1, 4-pentadiene structures to produce hydroperoxy fatty acids (Shewfelt and del Rosario, 2000).
285
Eventually, MDA is formed and has been utilized very often as a reliable estimator for lipid
286
peroxidation (Dhindsa, Plumb-Dhindsa, & Thorpe, 1981). Higher LOX activity and MDA content
13
287
have been found to contribute to the development of CI in peach fruit during low temperature
288
storage (Cao, Hu, Zheng & Lu, 2010; Gao et al., 2016). In the current study, the activation of LOX
289
and the formation of MDA in peach fruit due to chilling stress were inhibited by MT treatment
290
(Fig. 2). This indicated that MT treatment has facilitated the reduction of membrane lipid
291
peroxidation and cell membrane destabilization, which could, in turn, promote the ability of peach
292
fruit to resist CI. Similar alleviation of CI by suppression of membrane lipid peroxidation due to
293
MT treatment has been reported in cucumber seeds (Posmyk et al., 2009), Vigna radiata root
294
(Szafrańska et al., 2013; Szafrańska et al., 2014), wheat and maize seedlings (Turk et al., 2014;
295
Turk et al., 2015) and tomato plants (Ding et al., 2017).
296
During low temperature storage of postharvest peach fruit, the increased accumulation of total
297
phenolics appears to confer increased resistance to CI (Jin et al., 2009; Gao et al., 2016).
298
Undoubtedly that is partially because phenolic compounds can protect membrane lipid against
299
peroxidation by preventing the initiation and propagation of oxidizing chain reactions
300
(Pennycooke, Cox & Stushnoff, 2005). In this study, the result of the correlation analysis showed
301
that total phenolic content was negatively related to LOX activity (r = -0.94) in control fruit, while
302
the loss in the content of total phenolic was delayed by MT treatment during the whole low
303
temperature storage (Fig. 4A), which indicated that MT might lead to accumulation of phenolic
304
compounds and thus provide peach fruit with direct and sufficient chain-breaking activity. This
305
observation is further supported by two types of observations, namely, endogenous salicylic acid
306
content is higher in MT-treated fruit (Fig. 4B), and there is a strong inverse relationship between
307
endogenous salicylic acid content and LOX (r = -0.95). Salicylic acid has been reported to be
308
effective in reducing lipid peroxidation in peach fruit during low temperature storage (Wang et al.,
14
309
2006), and as a consequence, contributing to the activation of the chilling tolerance. Taken
310
together, it is therefore believed that phenolic compounds are implicated in the MT-induced
311
chilling tolerance in peach fruit, and it can improve the antioxidant potential of peach fruit under
312
chilling stress.
313
G6PDH, SKDH and PAL are three essential enzymes for phenolic anabolism. Among them,
314
G6PDH is the first rate-limiting enzyme of the pentose phosphate pathway, and its main function
315
is to supply the substrate erythrose-4-phosphate for shikimate pathway (Debnam and Emes, 1999).
316
The reaction that SKDH catalyzes is the third and fourth steps of the shikimate pathway,
317
converting 3-dehydroquinate into shikimate. Shikimate itself is a precursor to those aromatic
318
amino acids like l-phenylalanine (Díaz and Merino 1997). PAL is the principal enzyme
319
responsible for transformation of L-phenyalanine into trans-cinnamic acid at the entry point of the
320
phenylpropanoid
321
(Sánchez-Rodríguez et al., 2011). There is empirical evidence that enzymes involved in phenolic
322
compounds mobilization have been relevant to plant tolerance to chilling stress. In cucumber
323
seedlings, for example, Arbuscular mycorrhizal fungi inoculation resulted in higher activities of
324
G6PDH, SKDH and PAL as well as their corresponding gene expression, along with the induction
325
of chilling adaptive response (Chen et al., 2014). It was also reported that 24-epibrassinolide
326
immersion induced tolerance of peach fruit to chilling stress, which has been partially attributed to
327
the elevated activities of SKDH and PAL (Gao et al., 2016). We reported herein that MT induced
328
increases in activities of G6PDH, SKDH and PAL, and stimulated the accumulation of total
329
phenolic as well as endogenous salicylic acid. Furthermore, MT-treated fruit showed a lower
330
occurrence of CI than control fruit during the whole low temperature storage. These findings
pathway
driving
towards
15
the
generation
of
phenolic
compounds
331
revealed that the ability of MT treatment protects peach fruit from CI may be due to activation of
332
phenolic compounds biosynthesis to some extent. This is in agreement with a formerly reported
333
result by Szafrańska et al. (2014), who proposed that one of the functional routes of MT activity
334
may be to prevent Vigna radiate root from CI, by stimulating the biosynthesis of phenolic
335
compounds. Molecular evidence also indicates that MT can up-regulate the expression of genes
336
pertaining to salicylic acid pathway in Arabidopsis plants (Weeda, Zhang, Zhao, Ndip, Guo, &
337
Buck, 2014). In strawberry fruit, Aghdam and Fard (2017) proposed that the phenolic anabolism
338
due to MT treatment is beneficial to attenuating fungal decay.
339
In the case of CI, PPO and POD are deemed to have an antagonistic effect on enzymes
340
affecting phenolic anabolism. These two enzymes work together to catalyze the oxidation of
341
phenolic compounds to quinines and result in flesh browning (Zhang et al., 2015), which has been
342
reported to be a notable CI symptom in peach fruit (Lurie and Crisosto, 2005). Therefore, for
343
example, inhibited activities of PPO and POD were observed in peach fruit treated by hot air in
344
combination with methyl jasmonate vapour, this being less sensitive to CI after treatment (Jin et
345
al., 2009). Also, Gao et al. (2016) found that 24-epibrassinolide alleviated CI of peach fruit, and
346
this process was accompanied by lower activities of PPO and POD. The results presented in our
347
study showed that the application of MT treatment led to a decrease in activities of PPO and POD
348
during the whole low temperature storage compared to control fruit, for which increase of these
349
two enzymes was observed (Fig. 6). In this sense, the decrease in activities of PPO and POD could
350
be a defence mechanism against chilling stress, and could be account for the lower flesh browning
351
found in MT-treated peach fruit.
352
In conclusion, treatment with 0.1 mM MT alleviated CI in peach fruit and the enhancement of
16
353
resistance against CI by MT treatment may be derived from its capacity to maintain a higher ratio
354
of unsaturated to saturated fatty acids, inhibit membrane lipid peroxidation as well as stimulate
355
phenolic mobilization. Given that MT has minimal toxicity to human beings over a very wide dose
356
range (Galano et al., 2011), these results suggest that the application of MT treatment to peach
357
fruit could be considered as an effective postharvest technique with good results in terms of
358
reducing CI damage.
359
Acknowledgements
360
This work was supported by the National Natural Science Foundation of China (grant number
361
31401550) and the National Innovation Experiment Project for College Students of China (grant
362
number 201510697022).
363
Conflict of interest
364
We declare that we have no conflict of interest.
365
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366
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Figure Captions
488
Fig. 1 Effects of MT treatment on CI occurrence (A), CI index (B) and firmness (C) of peach fruit
489
during low temperature storage. Vertical bars represent the standard errors of the means of
490
triplicate assays. Values with different letters for each day were significantly different at P < 0.05.
491
Fig. 2 Effects of MT treatment on MDA content (A) and LOX activity (B) of peach fruit during
492
low temperature storage. Vertical bars represent the standard errors of the means of triplicate
493
assays. Values with different letters for each day were significantly different at P < 0.05.
494
Fig. 3 Effects of MT treatment on palmitic acid (A), stearic acid (B), oleic acid (C), linoleic acid
495
(D) and linolenic acid (E) contents and the ratio of unsaturated to saturated fatty acids (F) of peach
496
fruit during low temperature storage.
497
Fig. 4 Effects of MT treatment on total phenolic (A) and endogenous salicylic acid (B) contents of
498
peach fruit during low temperature storage. Vertical bars represent the standard errors of the
499
means of triplicate assays. Values with different letters for each day were significantly different at
500
P < 0.05.
501
Fig. 5 Effects of MT treatment on G6PDH (A), SKDH (B) and PAL (C) activities of peach fruit
502
during low temperature storage. Vertical bars represent the standard errors of the means of
503
triplicate assays. Values with different letters for each day were significantly different at P < 0.05.
504
Fig. 6 Effects of MT treatment on PPO (A) and POD (B) activities of peach fruit during low
505
temperature storage. Vertical bars represent the standard errors of the means of triplicate assays.
506
Values with different letters for each day were significantly different at P < 0.05.
507
23
80
508 CI incidence (%)
Control MT
60 40 20 0 0
7
14
21
28
14
21
28
0.5 Control
CI index
0.4
MT
0.3 0.2 0.1 0 0
7
120 Control 100 Firmness (N)
509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549
MT
80 60 40 20 0 0
7
14
21
Storage time (days)
24
28
MDA content (µmol g -1)
3.5 Control 2.8
MT
2.1 1.4 0.7 0 0
7
14
21
28
14
21
28
250 Control LOX activity (U g -1)
550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
200
MT
150 100 50 0 0
7
Storage time (days)
25
40
3.6
30
2.7
20
1.8 Control
Control
MT
MT
0.9
0
0 0
7
14
21
28
0
7
14
21
28
21
35
18
28
15 12
21
9
14
6 3
Control
Control
MT
MT
Linoleic acid (%)
Oleic acid (%)
Stearic acid (%)
4.5
7
0
0 0
7
14
21
28
0
7
14
21
28
40
4
32
3
24 2 16 8 0 0
1
Control
Control
MT
MT
7
14
21
28
0
Storage time (days)
7
14
21
Storage time (days)
26
0 28
Unsaturated to saturated fatty acid ratio
Palmitic acid (%)
50
10
Linolenic acid (%)
591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634
Total phenolics (mg g-1)
0.5 0.4 0.3 0.2 Control
0.1
MT 0 0
7
14
21
21
28
Salicylic acid content (µg g-1)
1.5
18 Oleic acid (%)
635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
15 12 9 6
Control
3
MT
0
1.2 0.9 0.6 0.3
Control MT
0
0
7
14
21
28
0
7
14
21
Storage time (days)
27
28
G6PDH activity (U g -1)
10 Control 8
MT
6 4 2 0 0
7
14
21
28
28+3
SKDH activity (U g -1)
1.2 0.9 0.6 Control
0.3
MT 0 0
7
14
21
28
14
21
28
1.4 1.2 PAL activity (U g -1)
675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710
1 0.8 0.6 0.4
Control
0.2
MT
0 0
7
Storage time (days)
28
25 PPO activity (U g -1)
Control 20
MT
15 10 5 0 0
7
14
21
28
21
28
3 POD activity (U g -1)
711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743
Control 2
MT
1
0 0
7
14 Storage time (days)
744
29
745
Highlights
746
►MT delayed the development of chilling injury in peach fruit.
747
►MT maintained higher ratio of unsaturated to saturated fatty acids in peach fruit.
748
►MT stimulated phenolic anabolism and prevented lipid peroxidation in peach fruit.
749
►MT inhibited PPO and POD activities leading to reduced flesh browning in peach fruit.
750 751 752
30