L-ascorbic acid metabolism in an ascorbate-rich ...

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Accepted Manuscript L-ascorbic acid metabolism in an ascorbate-rich kiwifruit (Actinidia. Eriantha Benth.) cv. ‘White’ during postharvest Zhen-Ye Jiang, Yu Zhong, Jian Zheng, Maratab Ali, Guo-Dong Liu, Xiao-Lin Zheng PII:

S0981-9428(18)30005-6

DOI:

10.1016/j.plaphy.2018.01.005

Reference:

PLAPHY 5109

To appear in:

Plant Physiology and Biochemistry

Received Date: 19 October 2017 Revised Date:

3 January 2018

Accepted Date: 5 January 2018

Please cite this article as: Z.-Y. Jiang, Y. Zhong, J. Zheng, M. Ali, G.-D. Liu, X.-L. Zheng, L-ascorbic acid metabolism in an ascorbate-rich kiwifruit (Actinidia. Eriantha Benth.) cv. ‘White’ during postharvest, Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2018.01.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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L-ascorbic acid metabolism in an ascorbate-rich kiwifruit (Actinidia.

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Eriantha Benth.) cv. ‘White’ during postharvest

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Zhen-Ye Jiang a, Yu Zhong a, Jian Zheng a, Maratab Ali a, Guo-Dong Liu b, Xiao-Lin Zheng a,*

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a

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310018, PR China

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b Horticultural Sciences Department, IFAS, University of Florida, Gainesville, FL 32611-0690,

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USA

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College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou

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ABSTRACT

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Kiwifruit (Actinidia eriantha Benth.) ‘White’, a novel cultivar with higher L-ascorbic acid

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(AsA) level, is registered in China. Changes in AsA, related metabolites, enzymatic activity, and

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gene expression associated with AsA biosynthesis and recycling process were investigated in this

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paper. The results indicated that AsA biosynthesis through L-galactose pathway supplemented

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by D-galacturonic acid pathway and AsA recycling collectively contributed to accumulating and

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remaining higher AsA level in kiwifruit cv. ‘White’ during postharvest. Moreover, L-galactose

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*

Corresponding author. Tel.: +86 571 28008958; fax: +86 571 88053832.

E-mail addresses: [email protected] (X. Zheng)

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dehydrogenase (GalDH) activity and relative expressions of the genes encoding GDP-D-

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mannose pyrophosphorylase (GMP), L-galactose-1-P phosphatase (GPP), GDP-L-galactose

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phosphorylase (GGP), GalDH and D-galacturonate reductase (GalUR) were important for

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regulation of AsA biosynthesis, and the activity and expression of dehydroascorbate reductase

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(DHAR) were primarily responsible for regulation of AsA recycling in kiwifruit ‘White’ during

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postharvest.

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Key words: Ascorbic acid; Biosynthesis; Kiwifruit (Actinidia eriantha); Postharvest; Recycling

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1. Introduction L-ascorbic acid (AsA; i.e., vitamin C) is an essential dietary nutrient for human beings due

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to their inability to synthesize it. Also, AsA plays important roles in human body with a wide

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range of health benefits, such as reducing plasma cholesterol, enhancing collagen formation,

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minimizing nitrosoamine production, increasing iron absorption, boosting immune system,

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scavenging free radicals, and reducing the risk of cancer, etc (Blaschke et al, 2013; Cimmino et

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al., 2017; Schlueter and Johnston, 2011). However, humans are entirely reliant on the dietetic

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sources due to lack of the ability to synthesize AsA, and more than 90% AsA is derived from

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fruit and vegetables (Davey et al., 2000). Thus, AsA content acts as a preferential factor for

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evaluating the quality of most fruit, and increasing efforts have been devoted to AsA

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improvement in different fruit (Lee and Kader, 2000; Liu et al., 2015; Mellidou et al., 2012;

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Tsaniklidis et al., 2014).

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The metabolic pathways including the de novo biosynthesis, degradation and recycling of

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AsA collectively contribute to the regulation of AsA levle in plants (Fig. 1). Also, in fruit, AsA is

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biosynthesized from glucose through these four biosynthetic pathways, including L-galactose, D-

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galacturonate, L-gulose, and myo-inositol pathway, but the predominating AsA biosynthetic

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pathway is varied among fruit species, developmental stages or the fruit tissues (Bulley et al.,

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2009; Cruz-Rus et al., 2010; Davey et al., 2000). Moreover, AsA can be oxidized to

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monodehydroascorbate (MDHA) by ascorbate oxidase (AO) and ascorbate peroxidase (APX)

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respectively, and then MDHA can be either reduced to AsA by monodehydroascorbate reductase

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(MDHAR) or disproportionated to dehydroascorbate (DHA) and AsA, and DHA can also be

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reduced to AsA by dehydroascorbate reductase (DHAR) before being subject to hydrolysis

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(Huang et al., 2014).

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Kiwifruit varieties encompass a wide spectrum of varieties, among which most

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commercially significant varieties are A. deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson, A.

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chinensis Planch., and A. arguta (Sieb. et Zucc.) Planch. ex Miq (Crowhurst et al., 2008).

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Recently, a novel cultivar A. eriantha Benth ‘White’ has been recognized and entitled by the

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Horticulture Institute, Zhejiang Academy of Agricultural Sciences and then registered in China

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under code of PVR No. CNA20050673.0 (Wu et al., 2009). The kiwifruit cv. ‘White’ has

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attracted vital consideration as a new commercial fruit with large size, good flovor and nutrition,

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and easy peel like banana fruit, especially by its much higher AsA level (5.69 -11.37 g kg -1 FW)

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as compared to the most common kiwifruit cultivars and many other fruit crops (Wu et al., 2009;

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Zhang et al., 2011). The fruit morphology of kiwifruit cv. ‘White’ was showed in Fig. 2. To date,

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a range of investigations on the variation and regulation of AsA level in kiwifruit, especially, in

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the varieties of A. deliciosa and A. chinensis has been conducted during fruit development

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(Bulley et al., 2009; Li et al., 2014; Li et al., 2010). Our previous work has reported the mainly

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physiological and quality changes in ‘White’ fruit during storage at room temperature (Zhang et

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al., 2011), but information about the AsA metabolism associated with regulation of higher AsA

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level in kiwifruit cv. ‘White’ during development and postharvest is scarce. For understanding

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the AsA accumulation in kiwifruit cv. ‘White’ during postharvest, changes in contents of AsA

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and total-AsA (T-AsA) and related metabolites, activity of enzymes and expression profiles of

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genes involved in AsA biosynthesis and recycling process were investigated in this work. The

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results would be of benefit to improving targets for fruit crops breeding strategies, especially for

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kiwifruit breeding, with the purpose of increasing AsA level.

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2. Materials and methods

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2.1. Material

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Kiwifruit (A. eriantha Benth) cv. ‘White’ selected for uniformity of size and maturity, as

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well as without blemish and decay were harvested at 170 days after full bloom at a commercial

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orchard in Taizhou, China, when the soluble solid content (SSC) had reached about to 11.0%,

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and then were immediately transported to a laboratory in Hangzhou by air-conditioned car

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(approx.3 h). Each thirty fruit without physical injuries were placed in a clean plastic box with

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fruit touching, and each box was enclosed in a 0.05 mm thick polyethylene bag, and total 270

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fruit (nine boxes) were held in chambers (MIR-554, Sanyo, Oizumi, Japan) at 20 ± 0.5 ◦C for 21

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d., Triplicate of flesh samples were collected from the middle part of 27 fruits being removed

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peels and cores (9 fruits without decay each replicate from the 9 boxes) on the first day and at 3-

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day intervals thereafter during storage, and were rapidly frozen in liquid nitrogen and stored at -

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80 ◦C until use. Analysis in the triplicate of flesh samples was undertaken for measurements of

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the following parameters.

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2. 2 Assays for AsA and T-AsA

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One gram of sample was homogenized in 10 ml of 5% (w/v) trichloroacetic acid (TCA).

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The supernatants obtained after a 20 min centrifugation at 4 ◦C at 12,000 g were diluted 40 times

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for measuring AsA. Moreover, each 1.0 ml supernatant was mixed with 0.5 ml 60 mM

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dithiothreitol-alcohol and then added NaH2PO4-Na2HPO4 buffer to pH 7~8. The mixed solution

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was incubated for 10 min at 30 ºC for reduction of oxidised ascorbate to AsA, and then added 0.5

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ml 20 % (w/v) TCA for assaying T-AsA.

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The contents of AsA and T-AsA content were assayed according to a method described by

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Cao et al. (2013) with some modifications. A reaction mixture (containing 1 ml of the diluent, 1

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ml absolute ethyl alcohol, 0.5 ml 0.4% H3PO4-ethyl alcohol, 1 ml 1% (w/v)

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bathophenanthroline-ethyl alcohol and 0.5 ml 0.3% (w/v) FeCl3-ethyl alcohol) were analyzed at

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534 nm using an ultraviolet-1800 spectrophotometer after in water baths at 60 ◦C for 30 min.

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AsA and T-AsA were expressed on a fresh weight as g kg-1 FW.

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DHA content was calculated by T-AsA minus AsA in terms of as g kg-1 FW.

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2.3 HPLC assay of metabolites Ten grams of flesh samples was grounded with 5 ml distilled water, and then centrifuged at

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8 000 g and 4 ◦C for 30 min. The supernatant was filtered through 0.22 µm filter paper, and the

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filtrate was used to assay glucose and fructose. The determinations were conducted with some

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modifications of the method described by Andrés et al. (2015). Chromatographic separation was

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performed on an XBridge BEH Amide Column (3.0 × 150 mm). The mobile phase was 80%

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(w/v) acetonitrile in distilled water with a flow rate of 0.4 ml per min at 30 ◦C。

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Two grams of flesh samples was grounded with 2 ml 0.2% HPO3, and then centrifuged at

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12 000 g and 4 ◦C for 30 min. Centrifugation was repeated for two times. The combined

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supernatant was filtered through 0.22 µm filter paper, and the filtrate was used to assay oxalic

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acid and tartaric acid. The determinations were conducted with some modifications of the

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method described by Ma et al. (2015). Chromatographic separation was performed on a Vennusil

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MP C18 (250 mm × 4.6 mm × 5 µm). The mobile phase was 5% (v/v) acetonitrile in 0.1 mM

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K2HPO3(pH 2.05)with a flow rate of 0.8 ml per min at 30 ◦C。

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2.4 Determination of related enzymes in AsA metabolism

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Two grams of flesh were homogenized with 5 ml of 0.1 M potassium phosphate buffer (pH

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7.5) containing 0.1 mM phenylmethanesulfonyl fluoride, 0.5% (v/v) Triton X-100, 0.2% (v/v) 2-

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mercaptoethanol, and 2% (w/v) PVP, and then centrifuged at 12,000×g at 2 ◦C for 20 min. The

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supernatants were collected to assay L-galactose dehydrogenase (GalDH). GalDH activity was

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assayed according to the method of Gatzek et al. (2002).

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Two grams of flesh were homogenized with 8 ml of 0.1 M potassium phosphate buffer (pH

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7.4) containing 0.4 M sucrose, 30 mM mercaptoethanol, 10% (v/v) glycerol, 1% (w/v)

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polyvinylpyrrolidone (PVP) and 1 mM Ethylenediaminetetraacetic acid (EDTA) and centrifuged

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at 500 g and 4 ◦C for 15 min to remove chloroplast and cell debris. The supernatant was

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centrifuged again at 12,000×g for 20 min at 4 ◦C to obtain the pellet. The pellet was suspended in

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3 ml of 50 mM Tris-HCl (pH 8.5) containing 5 mM glutathione and 10% (v/v) glycerol. This

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suspended solution was again centrifuged at 2,000×g for 10 min at 4 ◦C, and the supernatant was

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used to determine L-galactono-1, 4-lactone dehydrogenase (GalLDH). GalLDH activity was

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assayed following Ôba et al. (1995).

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Two grams of flesh were homogenized with 50 mM sodium phosphate buffer (pH 7.2,

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containing 2 mM EDTA, 2 mM DTT, 20% glycerol and 2% PVP). The supernatant obtained

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after a 30 min centrifugation at 4 ◦C at 6,000×g and used to assay D-galacturonate reductase

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(GalUR). GalUR activity was determined as described by Agius et al. (2003).

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Two grams flesh was homogenized in 6 ml potassium phosphate buffer (50 mM/L, pH 7.5)

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containing 2 ml potassium phosphate buffer (100 mM, pH6.8), 0.1 mM ethylenediami-

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netetraacetic acid (EDTA), 0.3% (w/v) Triton X-100, 4% (w/v) insoluble polyvinyl-

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polypyrrolidone (PVPP), and 0.1 mM ascorbic acid, and then centrifuged at 1, 6000×g for 15

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min. The supernatant was used immediately to assay for APX, AO, MDHAR, and DHAR. APX

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and AO activities were assayed following the previously protocol by Nakano and Asada (1981),

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and Yoshimura et al. (2000), respectively, while DHAR and MDHAR activities following the

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method of Ma and Cheng (2003).

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One unit of GalDH, GalLDH, GalUR, APX, and AO activity was defined as the amount of

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enzyme causing an increase of 0.01 in the absorbance per min at 340, 550, 340, 290, and 265 nm

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at 25 ◦C respectively, and one unit of MDHAR or DHAR activity was defined as the substance

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produced or consumed per minute per unit of total protein.

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2.5 Quantitative real-time PCR analysis for expression of genes involved in AsA metabolism

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Two grams of flesh samples were homogenized in liquid N2, and total RNA was extracted

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from the frozen flesh using a commercial RNA extraction kit (Tiangen, Beijing, China).

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Extraction was conducted according to the manufacturer's instructions. Using SuperScript™ III

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First-Strand Synthesis SuperMix, the first strand cDNA was synthesized by reverse transcription

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following the manufacturer’s protocols. The primers used for the quantitative real-time PCR

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(qPCR) were identified by Primer 6 software Primer Premier 6.0 and Beacon designer 7.8 (Table

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1). Transcript levels of the genes involved in the metabolic pathways of AsA, including GMP,

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GME, GGP, GPP, GalDH, GalLDH, GalUR, APX, AO, MDHAR, and DHAR, were monitored

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via qPCR using the Power SYBR® Green Master Mix Kit (Toyobo, Osaka, Japan) following

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manufacturer's guidelines on a CFX384 Touch Real time PCR Detection System (Bio-Rad,

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USA).

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The qPCR program was carried out as follows: 1 min at 95 ◦C; 40 cycles of 15 s at 95 ◦C,

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and 25 s at 63 ◦C and followed by an automatic melting curve analysis. Three independent

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biological replicates were determined for each sample. The relative expression level for each

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gene was calculated using the comparative Ct method (2

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repeated three times using biological replicates.

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2.6 Statistical analysis

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method). All analyses were

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Data were expressed as mean values ± SD and were subjected to one-way analysis of

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variance (ANOVA) using SPSS 20.0 software (SPSS Inc., USA). The differences at P ≤ 0.05

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were considered as significant.

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3. Results

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3.1 Changes in AsA, DHA, T-AsA, and AsA/DHA ratio in the fruit during storage

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AsA content of kiwifruit showed a considerably increasing trend from the initial day to 17 d of storage and then decline slightly, which indicating that AsA content was 7.20 g kg

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FW at

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first day of storage, and increased continuously to the highest AsA contents of 8.30 g kg -1 FW at

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17 d, and then declined to 7.98 g kg -1 FW at 21 d of storage as shown in Fig 3A. Overall, DHA content of kiwifruit ‘white’ showed a continuously decreasing trend during

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whole storage of 21 d. At 5 d and 17 d of storage, a little increasing trend was observed (Fig.

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3B).

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In the case of T-AsA, the level slightly declined to 5 d of storage, after which it increased

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and then remained stable to 21 d of storage (Fig. 3C).

The AsA/DHA ratio showed an increasing trend during whole period as shown in Fig. 3D.

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From 1 d to 13 d of study, a slight increase regarding AsA/DHA ratio was observed. After that a

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sharp increase level was observed from the onward days of 13 d to 17 d. After 17 d, AsA/DHA

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ratio tended to a decreasing level continuously until 21 d. Overall, from 1 d to 21 d of storage, a

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significantly increasing trend was found.

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3.2 Changes in activities of enzymes involved in AsA metabolism in the fruit during storage

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At the first day, 10 U g

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activity of GalDH was observed which tended to increase

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significantly to the highest activity (38.33 U g -1) at 17 d of storage, and then decreased to 25.33

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Ug

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AsA content (r2 = 0.841 *) (Fig. 4A). GalLDH activity maintained a relatively steady level for

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the first 17 d of storage, and then decreased a lowest level at 21 d of storage (Fig.4B). GalUR

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at 21 d. Correlation analysis showed that GalDH activity was positively correlated with

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activity remained at stable levels during whole storage period, and a minor change was observed

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in GalUR activity as compared to GalDH and GalLDH (Fig. 4C). AO activity gradually decreased to a minimum level at 17 d, and then sharp increased to a

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level similar to that at 5 d (Fig.4D). APX activity slightly decreased for the first 17 d and then

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sharp reduced to a minimum level at 21 d (Fig.4E). MDHAR activity was tending towards steady

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levels except it showed a peak at 17 d (Fig. 4F), while DHAR activity enhanced rapidly for the

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first 5 d and then slightly increased for the remainder of storage time (Fig. 4G).

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3.3 Changes in the relative expression of AsA-related genes in the fruit during storage

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Changes in expression levels of the 11 AsA-related genes in fruit during postharvest were

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showed in Fig.5. The genes including GMP, GME, GGP, GPP, GalDH, and GalLDH involve in

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L-galactose pathway. In kiwifruit cv. ‘White’, the expression of GMP and GME remained steady

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at the first 5 d and 13 d repectively, and then GMP gradually increased until 21 d, while GME

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significantly decreased during the remainder days in storage (Fig. 5A and B). The expression of

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GGP increased to a peak value at 17 d, which was over 2 times higher than that of the first day,

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and then sharply reduced to the initial level (Fig. 5C). The relative expression of GPP increased

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signifiantly during storage (Fig. 5D). The expression of GalDH increased sharply during the first

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9 d, and then gradually increased to about 4 times higher level compared to the initial level

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whereas, GalLDH was stably expressed with a slight fluctuation during storage (Fig. 5E and F).

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The relative expression of GalUR that involves in the D-galacturonate pathway increased sharply to a peak at 13 d, and then gradually decreased continuously (Fig. 5G). AO, APX, MDHA and DHAR involve in the ascorbate-glutathione cycle. In kiwifruit cv.

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White, the expression of AO and APX gradually decreased, while the DHAR increased during the

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storage, but the expression of MDHA remained nearly unchanged level except increasing to

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double its original level at 17 d during storage (Fig. 5H, I, J and K).

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3.4 Change in contents of AsA-related metabolites in the fruit during storage

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The contents of glucose and fructose in the fruit increased rapidly in the first 5 d, and then

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fluctuated slightly during the remainder of storage time (Fig. 6A and B). The tartaric acid content

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gradually increased until 9 d and then slightly decreased, while the oxalic acid content sharply

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decreased in the first 5 d and almost varied slightly up to 17 d, and then increased to the similar

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level on the first day (Fig 4C and D).

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4. Discussion

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4.1 Accumulation of AsA in kiwifruit ‘White’ during postharvest

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Generally, AsA content in fruit decreases during postharvest. For example, Lee and Kader

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(2000) have reported that AsA losses in fruit are boosted due to the extended storage period,

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higher temperature and low relative humidity. Kalt (2005) has also reported that AsA content

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reduces simultaneously with the deprivation of fleshy fruit tissues (e.g. strawberry) after over

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ripening. In kiwifruit, AsA content in the fruit of A. deliciosa and A. chinensis decreases during

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postharvest, even the loses of AsA are up to 50~70 % while fruit ripened fully (Sharma et al.,

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2015). Our results showed an increasing trend of AsA content in kiwifruit cv. ‘White’ during

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storage at room temperature for 21 d, while T-AsA maintained steady accompanied with higher

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AsA/DHA ratio. The pattern of AsA/DHA followed that of T-AsA remained reduction status in

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the fruit even over ripening. Thus, this comprehensive evidence suggested that a higher

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efficiency of AsA metabolism, especially in AsA biosynthesis or /and AsA regeneration, must

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take place for remaining higher level of AsA in kiwifruit cv. ‘White’ during postharvest.

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4.2 The enzymes and genes involved in AsA biosynthesis contributed to AsA accumulation in

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kiwifruit ‘White’ during postharvest

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Recently, it has been concluded that the activity of some key enzymes and expression of

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some genes involved in AsA biosynthesis collectively regulate the variation of AsA content in

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fruit, in a parallel way to AsA accumulation (Huang et al., 2014; Li et al., 2010; Ioannidi et al.

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2009; Tsaniklidis et al., 2014). AsA biosynthesis in fruit through L-galactose pathway is well

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established due to all of the involved enzymes and genes being identified, where the last two

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steps are oxidation of L-Gal to L-GalL by GalDH, and then oxidation of L-GalL to AsA by

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GalLDH (Laing et al., 2004). During fruit development, the GalDH activity and GalDH

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expression in kiwifruit are associated with AsA level, but GalLDH is post-transcriptionally

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controlled and is not a main factor to regulate AsA concentration (Li et al., 2010). Also, no clear

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relationship between GalLDH expression and AsA synthesis has been found by Bulley et al.

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(2009) and Ioannidi et al. (2009). Our results indicated that GalDH activity and GalDH

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expression in kiwifruit cv. ‘White’ increased and maintained higher levels, which were

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significantly positive correlations to AsA content. Although GalLDH activity maintained higher

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levels, the GalLDH expression almost unchanged during postharvest. Thus, it was suggested that

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GalDH and up-regulated expression of GalDH might play important role in AsA synthesis, and

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GalLDH might impart its role in AsA synthesis with respective to AsA accumulation in kiwifruit

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‘White’ during postharvest.

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The expression of the other genes involved in L-galactose pathway including GMP, GME,

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GGP and GPP also contributes to regulation of AsA accumulation in fruit. For example, Huang

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et al. (2014) have reported that GMP, GME, GalDH, GalLDH and GGP are up- regulated when

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the AsA accumulation rate rapidly increases in chestnut rose fruit. A greater AsA concentration

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may be connected to a greater expression of GPP in fruit pulp of Citrus (Yang et al., 2011).

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Ioannidi et al. (2009) have also stated that GPP expression is correlated with AsA concentration

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and imparts a vital part in maintaining pool size of AsA during the maturing period of tomato

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fruit. Conversely, Mellidou et al. (2012) have revealed that only the expression of one orthologue

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of GGP associates with a peak level of AsA accumulation in tomato fruit. Moreover, in various

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genotypes of kiwifruit, the level of GGP expression is a chief regulator as its transcriptions is

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interrelated with AsA strength during growth of fruit, while variations in GPP expression are

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correlated with changes in AsA concentration rate. Li et al. (2010) have observed a strong

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association between the expression levels of GMP and GPP and AsA accumulation in kiwifruit

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development. Interestingly, in the kiwifruit of A. eriantha, the GPP expression is greater as

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compared with GGP and GME, where the GGP expression is still higher than that of A. deliciosa

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and A. chinensis (Bulley et al., 2009). Data here showed the expressions for GMP, GPP and

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GGP were up-regulated in kiwifruit ‘White’ along with it’s AsA accumulation during

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postharvest. Thus, these genes might also play important role for regulating AsA biosynthesis in

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relation to AsA accumulation in kiwifruit ‘White’ during postharvest.

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GalUR is a key enzyme involved in D-galacturonate patyway of AsA biosynthesis, and

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changes in its activity or/and gene expression also affect AsA level in some fruit. For example,

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previous works have showed that GalUR expression is correlated to AsA content and

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accumulation in strawberry (Agius et al., 2003) and in chestnut rose fruit (Huang et al., 2014)

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during the period of development, as well as in grape berries during growth and ripening (Cruz-

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Rus et al., 2010). Our experimental results revealed that GalUR activity in kiwifruit ‘White’

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remained at a steadily low level compared to the activities of GalDH and GalLDH during

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posthavest, but the GalUR expression was up regulated, which indicated that GalUR might also

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play a role on regarding AsA accumulation in kiwifruit ‘White’ during postharvest.

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4.3 The key enzymes and genes involved in AsA recycling contributed to AsA accumulation in

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kiwifruit ‘White’ during postharvest

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AsA regeneration through ascorbate-glutathione cycle commonly contributes to AsA

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accumulation in fruit, where the enzymes such as AO, APX, MDHAR and DHAR involve in

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(Huang et al., 2014; Liu et al., 2015). Data here showed that the activities and the relative

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expression of AO and APX in the fruit dramatically decreased during postharvest, which

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indicated that AsA oxidation was relatively lower level. Moreover, numerous works have

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revealed that the contribution of MDHAR or DHAR to AsA level varies among fruit species. For

284

instance, AsA level in tomato fruit and strawberry fruit is associated well with MDHAR rather

285

than with DHAR (Cruz-Rus et al., 2011; Stevens et al., 2008). By contrast, the AsA content, and

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the activity and expression of DHAR in kiwifruit reduce with fruit ripening, but the activity and

287

expression of DHAR is positive correlations with AsA content (Li et al., 2010). Similar results

288

have been reported in chestnut rose fruit, where DHAR, not MDHA, correlates strongly with

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variations in reduced AsA concentration in both gene expression and enzyme activity (Huang et

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al., 2014). Our data showed that the activity and expression of MDHAR almost remained steady

291

throughout storage except at 17 d, but the expression and activity of DHAR increased

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dramatically and corrected positively with AsA content during postharvest. Therefore, it was

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suggested that DHAR might be as one of the most crucial enzymes for AsA regeneration and in

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turn contributed to AsA accumulation in kiwifruit ‘White’ during postharvest.

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5. Conclusion

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Final AsA level in fruit during postharvest depends on it’s biosynthesis, catabolism, and

298

recycling. Our data indicated that, in the flesh of kiwifruit ‘white’, AsA catabolism seemed to

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affect AsA content minimally, as the metabolite contents such as oxalic acid and tartaric acid in

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the fruit was relatively low during postharvest. AsA biosynthesis through L-galactose pathway

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supplemented by D-galacturonic acid pathway and AsA recycling might collectively contribute

302

to accumulating and remaining higher AsA level in the flesh of kiwifruit cv. ‘White’ during

303

postharvest. Also, the GalDH activity and the gene expressions including GMP, GPP, GGP,

304

GalDH and GalUR might play important roles in regulating on AsA biosynthesis, and the

305

expression and activity of DHAR might be primarily responsible for regulation of AsA recycling

306

in the flesh of kiwifruit ‘White’ during postharvest, which would be suggested as potentially

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good candidates for breeding and selection new cultivars with higher AsA content during

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postharvest storage up to over-ripening.

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Acknowledgements

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This work was supported by National Natural Science Foundation of China (No. 31371848) and National Key R&D Program of China (No.2016YFD0400901).

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involved in sugar metabolism of apple fruit in controlled atmosphere storage. Food Chem.

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141, 3323-3328.

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Table 1 The sequences of specific primers used for qRT-PCR analysis.

407

Fig. 1 Proposed pathways for biosynthesis, degradation and recycling of AsA in plants

408

The enzymes catalyzing the reactions are: GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3′–5′-

409

epimerase (GME), GDP-L-galactose transferase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-

410

galactose dehydro- genase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), D-galacturonic acid

411

reductase

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monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), ascorbate oxidase (AO),

413

ascorbate peroxidase (APX). Adapted from Smirnoff (2011).

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Fig. 2 The fruit morphology of kiwifruit cv. ‘White’

415

Fig. 3 Changes in AsA (A), DHA (B), T-AsA (C), and AsA/DHA ratio (D) in the fruit during

416

storage. Data were the means of three replicates ± SD. 


417

Fig. 4 Changes in activity of enzymes involved in AsA biosynthesis and recycling in the fruit

418

during storage. Data were the means of three replicates ± SD. 


419

Fig. 5 Changes in the relative expression of genes involved in AsA biosynthesis and recycling in

420

the fruit during storage. Data were the means of three replicates ± SD.

421

Fig.6 Changes in the contents of metabolites related to AsA biosynthesis and catabolism in the

422

fruit during storage. Data were the means of three replicates ± SD.

myo-inositol

oxygenase

(MIOX),

L-gulono-1,4-lactone

oxidase

(GLOase),

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Table 1 The sequences of specific primers used for qRT-PCR analysis

Actinidia GME

GU339037.1

Actinidia GGP

GU339036.1

Actinidia GPP

AY787585.1

Actinidia GalDH

EU525847.1

Actinidia GalLDH

GU339039.1

Actinidia GalUR1

GU339035.1

Actinidia APX

JQ011279.1

Actinidia MDHAR

GU339041.1

Actinidia DHAR

GU339034.1

Actinidia AO

FG523033.1 JX236280.1

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Actinidia 18S

GCTGAACCCATCTGTTCTCGAT GTAATCCCTTGGCTGTCCAATG

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FJ643600.1

Product (bp)

Reverse primer (5' to 3')

GAGAAAGCCCCTGCTGCATTC

CAATGAAGGTGAAAGATCGGGTT GTGTGGGAAATCAGCGGACAT

GCCTCCAAGCATTTCCTTCAGA CTGCATTTCCCTGACCACAAGT

GTTGTCCCATCAAGAGGATCAACA

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Forward primer (5' to 3')

CGCCTCGGCATCAACTTCTT

CCACACTTGGTCGACACAATGTA

CCACTACAGGCGTTTGACTCAAA

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CCTCAATCTTGGCCCAATGTTC

CCAGTCAGATTGACCCAACACGT CTCCATGCCTTCCCAGACTGT

GGTGCCACAAGGAGCGTTCT GAAGGCGGAATCAGCGAGAA

CTCGGCTTTTCACAGCTGGTATA

CCAACAGCCACAGTTCCCTTGA

GCACACGGACCATACGTTAATGA

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CACATCAAGGTGGAACAGCTTTG GCGATTGGGAATCACAAGAT

AATAGCTCTCGCCGGAGTAAA GCCCTATCAACTTTCGATGGTAGGA

CCTTGGATGTGGTAGCCGTTTCTCA

100

79

120

128

74

93

152

91

81 100 113

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Fig. 1 Proposed pathways for biosynthesis, degradation and recycling of AsA in plants.

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The enzymes catalyzing the reactions are: GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3′–5′epimerase (GME), GDP-L-galactose transferase (GGP), L-galactose-1-phosphate phosphatase (GPP), Lgalactose dehydro- genase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), D-galacturonic acid reductase (GalUR), myo-inositol oxygenase (MIOX), L-gulono-1,4-lactone oxidase (GLOase), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), ascorbate oxidase (AO), ascorbate peroxidase (APX). Adapted from Smirnoff (2011).

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b 8.0

c d

d

7.0 6.5

a a

a

a

a

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1

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c

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a

a

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MDHAR activity (U g-1 FW)

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GalUR activity (U g-1 FW)

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APX activity (U g-1 FW)

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G

Fig.4

DHAR activity (U g -1 FW)

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B

A 1.5

GMP

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a b

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Tartaric acid content ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights: 1. AsA content increased in kiwifruit ‘White’ during postharvest. 2. AsA biosynthesis and recycling contributed to AsA accumulation in ‘White’.

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3. AsA biosynthesis is mainly through L-galactose pathway in kiwifruit ‘White’.

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4. DHAR was primarily responsible for AsA recycling in kiwifruit ‘White’

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Contributions

Conceived and designed the experiments: XLZ, ZYJ YZ. Performed the experiments: ZYJ YZ JZ. Analyzed the data: YZ ZYJ JZ. Contributed reagents/materials/analysis tools: XLZ. Wrote the paper:

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ZYJ MA. Revised the paper: GDL XLZ. All authors have read and approved the final manuscript.