The Role of IP3 in NO-Enhanced Chilling Tolerance in Peach Fruit

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Agricultural and Environmental Chemistry

The role of IP3 in NO-enhanced chilling tolerance in peach fruit Caifeng Jiao, and Yuquan Duan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02871 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 16, 2019

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Journal of Agricultural and Food Chemistry

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The role of IP3 in NO-enhanced chilling tolerance in peach fruit

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Caifeng Jiao, Yuquan Duan *

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Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/ Key

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Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry

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of Agriculture and Rural Affairs, Beijing 100193, People’s Republic of China

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ACS Paragon Plus Environment


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ABSTRACT: The role of inositol 1,4,5-trisphosphate (IP3) in nitric oxide (NO)-reduced chilling

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injury (CI) in peach fruit was investigated. The fruit were immersed in sodium nitroprusside (SNP)

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(NO donor) and neomycin (IP3 inhibitor). Results showed that chilling tolerance was enhanced

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upon exogenous SNP in postharvest peach fruit. Further, GABA accumulationit was stimulated by

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SNP. The increase in protein expression and activity for enzymes in GABA biosynthesis, including

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glutamate decarboxylase (GAD), polyamine oxidase (PAO) and amino aldehyde dehydrogenase

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(AMADH)

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△1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine d-aminotransferase (OAT) and down

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regulation of proline dehydrogenase (PDH) were induced by SNP treatment, thereby accelating

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proline production. Additionally, SNP treatment elevated protein expression and activity of

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alternative oxidase (AOX). The above effects induced upon SNP were partly weakened by

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neomycin. Therefore, IP3 mediated NO-activated GABA and proline accumulation as well as AOX,

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thus inducing chilling tolerance in postharvest peach fruit.

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KEY WORDS: SNP; IP3; GABA; proline; AOX; peach fruit

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INTRODUCTION

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Cold storage is an effective technology used to migitate decay and to maintain quality of

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postharvest fruit.1 However, peach (Prunus persica) fruit, as one of the typical tropical fruit, is

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sensitive to chilling injury (CI).2, 3 Classic symptoms of CI in peach fruit are internal browning and

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flesh mealiness. These changes impose limitations for postharvest life and consumer acceptability.4

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Therefore, the exploration of potential methods to reduce CI in postharvest fruit is imperative.

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A previous study of our group has shown that application of nitric oxide (NO) donor, sodium

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nitroprusside (SNP), inhibited levels of lipoxygenase and phospholipase D and elevated levels of

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antioxidant enzymes and small ubiquitin-like modifier, therefore reducing CI in peach fruit.5 The

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delay of CI in postharvest fruit involves the regulation by signalling compounds.5 It is of a great

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importance to explore the possible downstream signal molecule mediating NO-migitated CI in

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peach fruit. Our previous work has also shown that inositol 1,4,5-trisphosphate (IP3) mediated

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NO-reduced CI in peach fruit.5 Nevertheless, more underlying physiological and molecular

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mechanism of the IP3-mediated inducton of chilling tolerance upon NO treatment remains to be

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done.

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γ-Aminobutyric acid (GABA) in plant usually acted as a vital signal mediating lots of

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physiological responses,6 such as accelerating pollen tube growth,7 promoting photosynthesis and

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facilitating the uptake of nitrogen.8 Especially, exogenous GABA treatment up regulated proline

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content and migitated CI in postharvest peach fruit.9 Moreover, GABA biosynthesis was stimulated

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by lots of exogenous stimulus in plants.10 Accordingly, exogenous melatonin treatment enhanced

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GABA production, and migitated CI in peach fruit.2 However, little is reported regarding the

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induction of GABA synthesis by NO treatment and its role in alleviation of CI in postharvest fruit.10

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It has been shown that edelfosine, as the antagonist of phospholipase C (PLC, a key enzyme in IP3

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production), blocked the increase in spontaneous GABA release induced by ethanol,11 suggesting

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that the PLC/IP3 is essential for ethanol-induced GABA release in animal. Little is reported

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regarding the induction of GABA synthesis by IP3 under NO treatment in postharvest peach fruit.

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Apart from GABA, proline production in postharvest fruit has been shown to be an adaptive

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mechanism to CI.12 Accordingly, proline production was induced by exogenous SNP,13 GA3,14

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glycine betaine

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postharvest fruit. Both of proline accumulation and IP3 activation are the results of the plants

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defense reactions. However, little is reported regarding the induction of proline accumulation by

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IP3 under SNP treatment in postharvest peach fruit.

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and melatonin

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treatments, playing an important role in migitating CI in

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Also, alternative oxidase (AOX), has been shown to be induced by methyl jasmonate (MeJA)

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and methyl salicylate (MeSA) treatments, playing a vital role in alleviating CI in tomato and sweet

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pepper.16 Accordingly, AOX reduced superoxide level, and thereby scavenged reactive oxygen

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species (ROS) in tobacco leaf.17 Thus, AOX might enhance chilling tolerance through ROS

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avoidance. Additionally, NO has been confirmed to induce AOX in tobacco plants.18 Little is

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available about the induction of AOX by NO in postharvest peach fruit. Furthermore, the mediation

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of AOX activation by IP3 under NO treatment remains to be revealed.

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Based on the above, the aim of our work was to investigate the modulation of the CI index and

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GABA and proline biosynthesis as well as AOX activation by IP3 upon NO application and to

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reveal the related mechanisms in peach fruit.

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MATERIALS AND METHODS

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Fruit Materials and Postharvest Treatments. Peach fruit (Prunus persica Batsch cv.

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Jinqiuhongmi) were harvested at commercial maturity from a local orchard in Beijing, China. The

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fruit were chosen for uniformity without any damage, and randomly divided into three groups:

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(1) Control group (CK): The fruit were immersed using sterile deionized water.

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(2) SNP (NO donor): The fruit were immersed using 15 μM SNP.

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(3) SNP+neomycin (IP3 inhibitor): The fruit were immersed using 15 μM SNP plus 10 μM

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neomycin.

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Treatment procedure and optimal concentrations of SNP and neomycin were based on our

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previous literature.5 The fruit used for each group were treated for approximately 10 min, followed

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by being dried in air for 40 min at room temperature. All fruit were then stored for 28 d at 4 °C and

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80 % relative humidity. During the whole storage, 20 fruit of each group were sampled at 7 day

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intervals for assays, and cut into small pieces. Each assay was repeated three times. The samples

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were kept at -80 °C for determination.

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CI Index Assay. CI index was calculated accordind to visual surface pitting: 0 =no damage, 1

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= superficial damage (damage 50%). The CI index was obtained using

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the following formula: CI index =Σ [(CI scale) × number of fruit at that CI)] / (5 × total number of

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fruit in each sample) × 100 %.

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GABA Content Analysis. Small pieces were lyophilized with a Labconco freeze-dryer. one

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gram of dried sampled peach from was extracted using 7% acetic acid. Then the mixture were

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centrifuged at 10,000 × g for 15 min. GABA contents were assayed by high performance liquid

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chromatography (HPLC) according to Yang et al.19 The contents were expressed to be micrograms

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of GABA per gram of DW.

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Activity of GAD, PAO and AMADH Determination. For the assay of GAD activity, one

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gram of sampled peach from small pieces was ground using 70 mM potassium phosphate buffer

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(pH 5.8) containing 2 mM β-mercaptoethanol, 2 mM ethylenediamine tetraacetic acid (EDTA), 0.2

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mM pyridoxal phosphate (PLP) and 10 % (w/v) glycerinum, and centrifuged at 4 °C at 10,000 × g

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for 20 min afterwards. GAD activity was determined according to Bai et al.20 One unit of GAD

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activity was defined as the amount of enzyme causing the 1 μg GABA production per hour. The

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results were expressed to be U/mg FW.

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For the assay of PAO activity, one gram of sampled peach from small pieces was ground in 70

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mM potassium phosphate buffer (pH 6.5) containing 10 % (w/v) glycerinum, and centrifuged at 4

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°C at 10,000 × g for 20 min afterwards. PAO activity was determined as described by Yang et al.21

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One unit of PAO activity was defined as a change of 0.01 of absorbance per minute. The results

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were expressed to be U/mg FW.

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For the assay of AMADH activity, one gram of sampled peach from small pieces was

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extracted by 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM

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dithiothreitol (DTT) and 10 % (w/v) sucrose, and then centrifuged at 4 °C at 12,000 × g for 20 min.

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AMADH activity was detected according to Yin et al.22 One unit of AMADH activity was defined

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as a change of 0.01 of absorbance per minute. The results were expressed to be U/mg FW.

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Proline Content Determination. 0.5 g peach fruit from small pieces was ground with 3 %

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(v/v) sulfosalicylic acid at 100 °C for 10 min. The collected supernatant was mixed with an equal

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volume of glacial acetic acid and acid ninhydrin reagent, and the mixture was boiled for 30 min.

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After cooled, the mixture was partitioned against toluene. The determintion of proline content was

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conducted according to Shang et al.9 The results were expressed as micrograms of proline per gram

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of FW.

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Activity of P5CS, OAT and PDH Determination. For P5CS and PDH activity, 2.0 g peach

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fruit from small pieces was ground in 50 mM Tris-HCl buffer (pH 7.4) containing 7 mM MgCl2,

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0.6 M KCl, 3 mM EDTA, 1 mM DTT, and 5 % (w/v) insoluble polyvinylpyrrolidone, and

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subsequentgly centrifuged at 4 °C at 12,000 × g for 20 min. The analysis of P5CS and PDH activity

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was carried out according to Shang et al.9 One unit of the two enzymes activity was defined as the

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amount of enzyme resulting in a 0.001 reduction of absorbance per minute. The results were

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expressed to be U/g FW.

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For OAT activity, 2.0 g peach fruit from small pieces was ground in 100 mM potassium

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phosphate buffer (pH 7.9) with 1 mM EDTA, 15 % (v/v) glycerol, and 10 mM 2-mercaptoethano,

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and then centrifuged at 4 °C at 12,000 × g for 20 min. The analysis of OAT activity was carried out

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according to Shang et al.9 One unit of OAT activity was defined as the amount of enzyme resulting

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in a 0.001 reduction of absorbance per minute. The results were expressed to be U/g FW.

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Activity of AOX Determination. 0.5 g peach fruit from small pieces was ground in 10 mL of

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10 mM phosphate buffer (pH 7.2) at 4 °C. Peach samples were centrifuged at 10,000g at 4 °C for 15

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min. Then, AOX activity was assayed using assay Kit (Nanjing Jiancheng Bioengineering Institute,

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Jiangsu, China). The results were compared with a standard curve and expressed to be U/mg FW.

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Western Blot. The peach fruit sampled from small pieces after 28 d of storage were extracted

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with RIPA lysis buffer containing cocktail (a protease inhibitor). Then, the homogenate was

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centrifuged to obtain the tissue lysates. Western blot analysis was carried out according to Jiao et

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al.23

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Statistical Analyses. All the data in this work were expressed as the mean ± standard

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deviation (SD). The SPSS 21.0 software (SPSS Inc., Chicago, IL, USA) was used to conduct the

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assay of significant difference at p < 0.05. All the data were compared using ANOVA procedure

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and Duncan analysis.

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RESULTS

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Effects of Exogenous SNP and Neomycin on CI Index in Peach Fruit. CI symptoms in

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peach fruit were visible after 7 d of cold storage. During storage, CI index increased. CI in peach

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fruit was delayed by exogenous SNP treatment. After 28 d of cold storage, CI index upon SNP

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treatment was reduced by 33% compared with the control. More interestingly, the reduction of CI

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index under SNP treatment were weakened by neomycin (IP3 inhibitor) treatment. After 28 d of

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storage, CI index upon SNP plus neomycin treatment increased by 33% compared with that under

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SNP treatment (Fig. 1).

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Mediation by IP3 of GABA Biosynthesis and Activity and Protein Expression of GAD,

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PAO and AMADH under SNP Treatment. During cold storage, GABA production accumulated

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continuously in peach fruit under control and exogenous SNP treatments. GABA accumulation was

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induced by exogenous SNP. After 28 d of cold storage, the GABA content after SNP treatment was

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1.6 times of the control. Interestingly, the induction of GABA production by SNP treatment was

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blocked by neomycin. After 28 d of storage, the GABA synthesis under SNP plus neomycin

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treatment was weakened by 9% compared with that under SNP treatment (Fig. 2A).

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During cold storage, the GAD activity increased continuously in peach fruit under control and

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exogenous SNP treatments. The GAD activity was enhanced by exogenous SNP. After 28 d of

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storage, GAD activity after SNP treatment was 1.9 times of the control. During storage, the activity

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of PAO and AMADH increased initially, followed by a reduction under control and SNP treatments.

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The activity of PAO and AMADH was up regulated by SNP. The activity of PAO and AMADH

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under SNP treatment reached a maximum on 21 d, which was 1.5 and 2.1 times of the control,

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respectively. After 28 d of storage, there was no statistical difference in PAO activity under

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between control and SNP treatments, and the activity of AMADH under SNP treatment was 2.5

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times of the control. Moreover, during storage, the increment of activity of GAD, PAO and

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AMADH by SNP treatment was blocked by neomycin. After 28 d of storage, compared with that

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under SNP treatment, the activity of GAD, PAO and AMADH after SNP plus neomycin application

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was weakened by 23%, 20% and 33% (Fig. 2B-D).

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After 28 d of storage, protein expression of GAD, PAO and AMADH after SNP application in

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peach fruit was 1.4, 6.6 and 2.0 times of the control. Moreover, neomycin inhibited the SNP

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treatment-induced protein expression of GAD, PAO and AMADH. After 28 d of storage, compared

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with that under SNP treatment, the protein expression of GAD, PAO and AMADH decreased by

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14%, 12% and 73% after SNP plus neomycin application (Fig. 3).

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Mediation by IP3 of Proline Content and Activity and Protein Expression of P5CS, OAT

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and PDH under SNP Treatment. During cold storage, the proline production increased at the

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early stages, followed by a decrease in peach fruit under control and exogenous SNP treatments.

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Exogenous SNP treatment enhanced the proline production. The proline content under SNP

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treatment reached a maximum on 14 d, which was 2.3 times of the control. After 28 d of storage,

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the proline content after SNP application was 1.8 times of the control. More interestingly, during

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storage, neomycin inhibited proline accumulation under SNP treatment. After 28 d of storage,

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compared with that under SNP treatment, the proline content decreased by 28% after SNP plus

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neomycin application (Fig. 4A).

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During storage, the activity of P5CS and OAT increased at the early stages, followed by a

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reduction in peach fruit under control and SNP treatments. The activity of P5CS and OAT was

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elevated by SNP treatment. Both of the activity of P5CS and OAT after SNP application reached a

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maximum on 21 d, which was 1.6 and 2.0 times of the control, respectively. After 28 d of storage,

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the activity of P5CS and OAT after SNP application was 1.7 and 1.9 times of the control. Moreover,

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during storage, neomycin weakened the increase in the activity of P5CS and OAT by SNP treatment.

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After 28 d of storage, neomycin inhibited the P5CS and OAT activity by 26% and 33% in peach

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fruit under SNP treatment (Fig. 4B and 4C). During storage, the PDH activity continuously

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decreased in peach fruit under control and exogenous SNP treatments. The PDH activity was

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weakened by exogenous SNP treatment. Moreover, during storage, neomycin weakened the

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reduction in the PDH activity induced by SNP treatment. After 28 d of storage, there were no

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statistical differences in PDH activity in peach fruit under among the control, SNP and SNP pius

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neomycin treatments (Fig. 4D).

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After 28 d of storage, protein expression of P5CS and OAT in peach fruit after SNP

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application was 1.4 and 1.5 times of the control. Moreover, neomycin blocked the SNP

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treatment-induced protein expression of P5CS and OAT. After 28 d of storage, compared with that

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under SNP treatment, the protein expression of P5CS and OAT was reduced by 86% and 33% in

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peach fruit under SNP plus neomycin treatment. In addition, after 28 d of storage, the protein

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expression of PDH after SNP application decreased by 48% in comparison with control. Moreover,

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neomycin blocked the SNP treatment-reduced protein expression of PDH. After 28 d of storage,

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compared with that under SNP treatment, the protein expression of PDH increased by 114% after

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SNP plus neomycin application (Fig. 5).

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Mediation by IP3 of Activity and Protein Expression of AOX under SNP Treatment.

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During cold storage, the AOX activity increased at the early stages, followed by a decrease in peach

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fruit under control and exogenous SNP treatments. Exogenous SNP treatment enhanced the AOX

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activity. The AOX activity under SNP treatment reached a maximum on 14 d, which was 1.8 times

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of the control. After 28 d of storage, the AOX activity after SNP application was 2.0 times of the

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control. More importantly, during storage, neomycin inhibited AOX activity under SNP treatment.

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After 28 d of storage, compared with that under SNP treatment, the AOX activity decreased by 15%

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after SNP plus neomycin application (Fig. 6).

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After 28 d of storage, the protein expression of AOX after SNP application was 2.3 times of

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the control. Furthermore, neomycin blocked the SNP treatment-induced protein expression of AOX.

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After 28 d of storage, compared with that under SNP treatment, protein expression of AOX was

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reduced by 51% in peach fruit under SNP plus neomycin treatment (Fig. 7).

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DISCUSSION

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Our work showed that during cold storage, SNP treatment delayed CI (Fig. 1), suggesting that immersing in SNP might be employed as a potential method to alleviate CI in peach fruit.

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During storage, GABA content in peach fruit increased after SNP application (Fig. 2). This

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resulted from the increase in protein expression and activity of GAD, PAO and AMADH (Fig. 3).

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In plants, GABA produced from glutamate catalyzed by GAD, a critical enzyme in the GABA

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shunt pathway.24 Also, GABA could be biosynthesized from polyamine degradation pathway.10

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PAO is the key enzyme in polyamine catabolism, and AMADH is involved in in polyamine

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degradation pathway.25,

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treatment-induced GABA production and protein expression and activity of of GAD, PAO and

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What’s more, neomycin (IP3 inhibitor) weakened the SNP

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AMADH (Fig. 2 and 3). The data indicated that the SNP-induced GABA production in peach fruit

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was mediated by IP3. GABA, as one of the important secondary metabolites in plants, was verfied

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as an adaptive mechanism to CI in postharvest fruit.2, 27 However, SNP application does not directly

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stimulate the physiological reactions, instead of triggering some signalling compounds, e.g. IP3.5

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Subsequently, the activation of signal compounds modulated the enzymes involved in secondary

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metabolism synthesis, thereby enhancing target secondary metabolites production.28 Ca2+ was

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possibly involved in the GABA accumulation enhancement by IP3. Accordingly, IP3 regulated

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endomembrane Ca2+ release channels in plants.29 This suggested that IP3 acts as a regulator of Ca2+

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in plant. Moreover, calcium facilitated sprout growth, and induced GABA accumulation by

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elevating the expression and activity of diamine oxidase (DAO) in soybean sprouts,30 and GAD is a

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Ca2+-CaM dependent enzyme in plants.31 Thus, we could deduce that the stimulation of GABA

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biosynthesis by IP3 was possibly mediated by calcium.

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Apart from GABA production, proline biosynthesis also increased in response to SNP

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treatment in postharvest peach fruit (Fig. 4). This resulted from the elevation of protein expression

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and activity of P5CS and OAT and the reduction of protein expression and activity of PDH (Fig. 4

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and 5). Proline production in plant could scavenge free-radical, regulate osmotic pressure, stabilize

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protein, and inhibit lipid peroxidation.2, 32 Especially, proline has been shown to enhance chilling

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tolerance in postharvest fruit.9,

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ornithine catalyzed by OAT, while proline production was also modulated by degradation via

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PDH.33, 34 In addition, exogenous GABA treatment has been shown to enhance the P5CS and OAT

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activity and reduce the PDH activity, and thereby induce proline production in postharvest peach

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fruit.9 Hence, we could speculate that the proline accumulation enhancement (Fig. 4) in response to

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Proline produces from glutamate catalyzed by P5CS or

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SNP treatment partly resulted from GABA biosynthesis (Fig. 2). Interestingly, neomycin (IP3

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inhibitor) inhibited the SNP treatment-regulated proline production and protein expression and

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activity of of P5CS, OAT and PDH (Fig. 4 and 5). The data indiated that IP3 was involved in SNP

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application-triggered proline biosynthesis. Accordingly, proline-rich extensin-like receptor kinase 4

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has been shown to be a modulator of Ca2+ in Arabidopsis thaliana,35 from which we could speculate

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proline production might facilitate to the induction of Ca2+ level afterwards. Moreover, a previous

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report has confirmed that the firmness increased by exogenous application of CaCl2 in postharvest

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strawberry fruit.36 Thus, the alleviation of CI by proline possibly involves the activation of Ca2+,

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the downstream messenger.

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Also, the protein expression and activity of AOX increased by SNP treatment (Fig. 6 and 7).

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AOX, a non-energy conserving terminal oxidase in plant, has been shown to regulate lots of signal

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compounds.37 Excess ROS-resulted in oxidative damage is a critical reaction to CI in postharvest

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fruit.15 Accordingly, AOX down regulated superoxide level, thereby reducing reactive oxygen

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species (ROS) generation in tobacco leaf.17 Thus, AOX might migitate CI by scavenging ROS.

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Furthermore, neomycin (IP3 inhibitor) inhibited the SNP treatment-enhanced protein expression

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and activity of AOX (Fig. 6 and 7). These indiated that IP3 was involved in SNP

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application-triggered AOX activation. It has been shown that EGTA (calcium chelator) weakened

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alternative respiratory pathway in chilling-stressed Arabidopsis callus,38 indicating that Ca2+ was

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essential to the induction of alternative respiratory pathway during cold storage. Thus, Ca2+ might

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mediate IP3-induced AOX activation in peach fruit.

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Exogenous SNP treatment protected peach fruit against CI. SNP also enhanced the protein

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expression and activity of GAD, PAO and AMADH, thus inducing GABA biosynthesis.

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Additionally, the elevation of protein expression and activity of P5CS and OAT and the reduction

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of protein expression and activity of PDH were stimulated by SNP application, leading to the

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enhancement of proline accumulation. Moreover, the protein expression and activity of AOX

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increased uopn SNP application. However, neomycin, a IP3 inhibitor, blocked the above

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SNP-triggered impacts. Overall, IP3 was involved in the stimulation of chilling tolerance under

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SNP treatment in postharvest peach fruit.

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AUTHOR INFORMATION

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*Corresponding Author

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Tel/Fax: 86-10-62815971

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E-mail: [email protected]

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FUNDING SOURCES

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The work was funded by the National Natural Science Foundation of China (31871862).

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388 389 390 391 392 393 394 395 396 397

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398 399

Figure captions

400

Fig. 1. Effect of neomycin on CI under SNP treatment in peach fruit. Values are the mean ± SE

401

(standard error). Values not sharing the same letter are significantly different at p < 0.05. The below

402

is same.

403

Fig. 2. Effects of neomycin on GABA content (A) and activity (B, C and D) of GAD, DAO and

404

AMADH under SNP treatment in peach fruit.

405

Fig. 3. Effects of neomycin on protein expression of GAD, DAO and AMADH under SNP

406

treatment in peach fruit. Panels show representative bands (A). Histograms represent relative

407

protein expression of GAD (B), DAO (C) and AMADH (D) normalized to the corresponding

408

Fig. 4. Effects of neomycin on proline content (A) and activity (B, C and D) of P5CS, OAT and

409

PDH under SNP treatment in peach fruit.

410

Fig. 5. Effects of neomycin on the protein expression of P5CS, OAT and PDH under SNP treatment

411

in peach fruit. Panels show representative bands (A). Histograms represent relative protein

412

expression of P5CS (B), OAT (C) and PDH (D) normalized to the corresponding rubisco.

413

Fig. 6. Effects of neomycin on AOX activity under SNP treatment in peach fruit.

414

Fig. 7. Effects of neomycin on the protein expression of AOX under SNP treatment in peach fruit.

415

Panels show representative bands (A). Histograms represent relative protein expression of AOX (B)

416

normalized to the corresponding rubisco.

417 418 419

20


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420

Chilling injury index (%)

100

CK NO NO+neomycin

80

a b

60

c

40 20

f gf

de e f

ab c

d

0 0 421 422

7 14 21 Storage time (d)

Figure 1

423 424 425 426 427 428 429 430 431 432 433 434 435 436 21


28

GABA content (μg/g DW)


140 120 100

CK NO NO+neomycin

60 40

c d

80 hhh

f gg

a

b

ef

d e

b

(A)

d

20 0

GAD activity (U/mg FW)

100

a

80

b

60 40 20

(B)

iii

ggh h

e fg ef

c cd de de

PAO activity (U/mg FW)

0

0.06

a

0.05

b bc cde bc cdbc de e

0.04

de f f

0.03 0.02

(C)

ggg

0.01

AMADH activity (U/mg FW)

0.00 a

0.10 0.08

0.04

fff

438

cd e f

cd e

c

de

e f

0.02 0.00 0

437

b

b

0.06

(D)

7 14 21 Storage time (d)

28

Figure 2

22


Page 22 of 28

Page 23 of 28


(A) GAD PAO AMADH Rubisco

GAD/Rubisco

8

(B)

a b

6

c

4 2

b

b

0

PAO/Rubisco

(C)

a

8

b

6 4 2

c

b

0

AMADH/Rubisco

4

(D) (D)

a

3 2

b c

1 0 CK

440 441

NO NO+neomycin

Figure 3

23


Proline content (¦Ìg/g FW)


50 40

CK NO NO+neomycin

ee

20

(A)

a c

30

10

a

b

cd

d

d e

e

f ggg

P5CS activity (U/g FW)

0

600

a

500

bbc bc bc de de def ef

400 300

(B)

a

ggg

cd f

200 100

OAT activity (U/g FW)

0

(C)

a 60

b c d

40 20

hhh

c

c

d

d

e

g f

f

0

PDH activity (U/g FW)

200

(D)

aaa 150 b

100

d

c

50

bc de e

e ff

f ff

21

28

0 0

7

14

Storage time (d)

443 444

Figure 4

24


Page 24 of 28

Page 25 of 28


(A) P5CS OAT PDH Rubisco

P5CS/Rubisco

8 6

a

(B)

b

4 2

c

0

OAT/Rubisco

4

(C)

a

5

b

b

3 2 1 0

3.0

PDH/Rubisco

2.5

a

a

(D)

2.0 1.5

b

1.0 0.5 0.0 CK

446 447

NO NO+neomycin

Figure

5

25


AOX activity (U/mg FW)


60 50

CK NO NO+neomycin

bbc

40

bc d

30 20

a

f

bc e

c d

d f

ggg

10 0 0

14

21

Storage time (d)

448 449

7

Figure 6

450 451 452 453 454 455 456 457 458 459 460 461 462 463

26


28

Page 26 of 28

Page 27 of 28


(A)

AOX

Rubisco

5

(B)

a

AOX/Rubisco

4 3 2

b

b

1 0 CK

464 465

NO NO+neomycin

Figure 7

466 467 468 469 470 471 472 473 474 475 476

27



477

TOC graphic

478

28


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The Role of IP3 in NO-Enhanced Chilling Tolerance in Peach Fruit

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