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Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family during Apple Fruit Ripening and Softening Zongying Zhang, Nan Wang, Shenghui Jiang, Haifeng Xu, Yicheng Wang, Chuanzeng Wang, Min Li, Jingxuan Liu, Changzhi Qu, Wen Liu, Shujing Wu, Xiaoliu Chen, and Xuesen Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04536 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Analysis of the Xyloglucan Endotransglucosylase/Hydrolase

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Gene Family during Apple Fruit Ripening and Softening

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Zongying Zhang†‡, Nan Wang†‡, Shenghui Jiang†, Haifeng Xu†, Yicheng Wang†,

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Chuanzeng Wang§, Min Li†, Jingxuan Liu†, Changzhi Qu†, Wen Liu#, Shujing Wu†,

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Xiaoliu Chen†, Xuesen Chen†*

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Engineering, Shandong Agricultural University, Tai’an, Shandong, 271018, China

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§

Shandong Institute of Pomology, Tai’an, Shandong, 271000, China

9

#

College of Life Science, Linyi University, Linyi, Shandong, 276005, China

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Co-first author

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*

Corresponding author

13

Tel: +86-538-8249338. E-mail: [email protected]

State Key Laboratory of Crop Biology, College of Horticulture Science and

10

14 15

Notes

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The authors declare no competing financial interest.

17 18 19 20 21 22 23 24 25 1

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Abstract: Ethylene and Xyloglucan endotransglucosylase/hydrolase (XTH) genes

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were important for fruit ripening and softening in ‘Taishanzaoxia’ apple. In this study,

28

we found it was ACS1-1/-1 homozygotes in ‘Taishanzaoxia’ apple, which determined

29

the higher transcription activity of ACS1. XTH1, XTH3, XTH4, XTH5 and XTH9 were

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mainly involved in the early fruit softening independent on ethylene, while XTH2,

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XTH6, XTH7, XTH8, XTH10 and XTH11 predominantly involved in the late fruit

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softening dependent on ethylene. Overexpression of XTH2 and XTH10 in tomato

33

resulted in the elevated expression of genes involved in ethylene biosynthesis (ACS2,

34

ACO1), signal transduction (ERF2) and fruit softening (XTHs, PG2A, Cel2 and

35

TBG4). In summary, the burst of ethylene in ‘Taishanzaoxia’ apple was predominantly

36

determined by ACS1-1/-1 genotype, and the differential expression of XTH genes

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dependent and independent on ethylene played critical roles in the fruit ripening and

38

softening. XTH2 and XTH10 may act as a signal switch in the feedback regulation of

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ethylene signaling and fruit softening.

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Keywords: apple; fruit softening; ethylene; XTH; gene expression

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Introduction

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The plant hormone ethylene plays a key role inthe ripening and softening of many

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climacteric fruits and in its absence the process fails to proceed to completion 1.

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However, once initiated, fruit ripening and softening is a one-way process and the

55

beneficial aspects of ethylene for generating a high-quality product can soon be

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outweighed by its propensity to stimulate fruit softening and decay 2. Fleshy fruits

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soften mainly as a consequence of changes in cell wall structures and the disassembly

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of different cell wall components

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cellulose, hemicellulose, pectin, and structural proteins. The solubilization of pectin

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and depolymerization of hemicelluloses are common features of fruit softening, which

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is an elaborated biochemical process involving coordinated actions of cell wall

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modifying enzymes and

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polygalacturonase (PG), β-galactosidase (β-Gal), expansin (EXP), and xyloglucan

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endotransglucosylase (XET) 5,6.

3,4

. The plant primary cell wall is composed of

proteins,

including pectin methylesterase (PME),

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PG and PME are two major enzymes associated with the solubilization of pectin.

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The demethylation of pectin by PME makes the cell walls susceptible to further

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degradation by PG through hydrolytic cleavage of α-(1-4) galacturonan linkages

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Differences in cortical microstructures and cell adhesion are responsible for the

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textural differences between ‘Scifresh’ and ‘Royal Gala’ apples. These differences are

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closely related to PME and PG activities 9. Previous studies determined that PG

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causes the most prominent changes during softening

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tomatoes indicated that the degradation of polyuronides in cell walls by PG is

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insufficient for fruit ripening and softening 13.

7,8

.

10-12

. Analyses of transgenic

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Xyloglucan is the predominant hemicellulose of cell walls, and acts as a tether

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between microfibrils through the formation of hydrogen bonds with cellulose 3

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microfibrils

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previously named xyloglucan endotransglucosylase (XET), is believed to be critical

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for fruit ripening and softening because of its role in disassembling xyloglucans and

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loosening the cell wall in preparation for further modifications by other cell

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wall-associated enzymes 16. The XTH enzymes exhibit XET activity, resulting in the

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integration of newly secreted xyloglucan chains into an existing wall-bound

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xyloglucan and restructuring of existing cell wall materials. The XTH enzymes also

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function as a hydrolase to hydrolyze xyloglucan molecules in the absence (or at low

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concentrations) of xyloglucan oligosaccharides 3.

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. Xyloglucan endotransglucosylase/hydrolase (XTH), which was

The characterization of apple XTHs has focused on changes in enzyme activity and 10,16,17

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gene expression levels during fruit ripening and softening

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transcripts in ripe apples are those of two XTH genes (i.e., MdXTH2 and MdXTH10) 16.

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The increase in XET activity in ethylene-treated apples may be due to the elevated

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expression of XTH10, which is highly expressed during ripening, and can be induced

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by ethylene 3. A previous study revealed that the differential expression of XTH genes

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may result in fruit textural differences in progenies of a cross between ‘Fuji’ and

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Malus sieversii f. niedzwetzkyana apples18. Depending on the fruit species, different

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modifications may occur and to different extents, so the roles of individual cell

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wall-modifying enzymes during fruit ripening and softening may differ between

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species.

96

. The most abundant

‘Taishanzaoxia’ is an early-ripening apple cultivar with excellent fruit appearance 19

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and quality characteristics

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accompanied by a burst of ethylene production during fruit late development period

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lowers its freshness and shelf life

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. Unfortunately, a sharp decrease in fruit firmness

20

. Nevertheless, this characteristic makes the

cultivar ideal for investigating the ethylene-dependent mechanisms regulating fruit 4

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ripening and softening. Previously, we isolated two XTH genes (i.e., XTH2 and

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XTH10) from suppression subtractive hybridization (SSH) libraries generated for

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‘Taishanzaoxia’ apples harvested around the climacteric stage. The up-regulation of

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XTH2 and XTH10 expression levels was significantly correlated with fruit firmness

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and ethylene production, suggesting XTH genes may be important for fruit softening

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in ‘Taishanzaoxia’ apples

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ripening and softening in ‘Taishanzaoxia’ apples were unclear. In this study, we

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investigated the effects of ethylene on the regulation of XTH expression levels, fruit

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ripening and softening. Our findings may help characterize the molecular mechanisms

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responsible for apple ripening and softening, which is critical for optimizing fruit

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quality during the breeding of new apple cultivars.

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. However, the roles of other XTH genes during fruit

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Materials and methods

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Plant materials

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‘Taishanzaoxia’ apples were harvested from the Shandong Agricultural University

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fruit breeding orchard (36°26′N, 117°29′E) in Tai’an, China. Fruits were collected at

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six developmental stages [i.e., 40, 50, 60, 65, 70, and 75 days after full bloom

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(DAFB)] and immediately transferred to our laboratory for the determination of

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ethylene production and fruit firmness. In post-harvest 1-MCP treatment, fruits

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harvested 70 DAFB were treated with 1 µL L−1 1-methylcyclopropene (1-MCP) and

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stored at 24 °C. Fruit firmness and ethylene levels were measured at 1 day intervals (0,

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1, 2, 3, 4 and 5 days after harvest). The apples were then cut into approximately 1 cm2

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pieces, frozen in liquid nitrogen, and stored at −80 °C after the determination.

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Determination of fruit firmness and ethylene levels 5

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Fruit firmness was measured using a TA.XT plus Texture Analyzer (Stable

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Microsystems, Godalming, UK) with a P/2 columnar probe (2 mm diameter). The

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pre-test, test, and post-test speeds were 2 mm s−1, 1 mm s−1, and 10 mm s−1,

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respectively. The depth of penetration was 10 mm, with a trigger force of 10 g. The

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firmness was automatically calculated using Texture Exponent 32. Each fruit was

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punctured twice near the equator, and six replicates were used to test fruit firmness.

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Two fruits were sealed in a 1.5-L glass jar and incubated at 24 °C for 6 h.

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Headspace samples (1 mL) were collected and analyzed with a GC-9A gas

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chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector.

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The temperatures of the separation column and detector were 70 °C and 120 °C,

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respectively. Nitrogen and hydrogen were used as the carrier gases at 20 mL min−1

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and 50 mL min−1, respectively. The rate of ethylene production was calculated based

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on peak area quantification. The average ethylene concentration from three jars was

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calculated and used in subsequent analyses.

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Xyloglucan endotransglucosylase activity assay

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Enzyme extraction. Fruit tissue (1 g) was ground into a fine powder in liquid

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nitrogen, and then added 800 µL 10 mM citrate-phosphate buffer (pH 7.0). The

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extracts were centrifuged at 12000 rpm for 20 min (4 °C). The sediment was washed

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with 10 mM citrate-phosphate buffer (pH 7.0) twice, and then centrifuged at

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12000 rpm for 10 min (4 °C). The sediment was resuspended with 10 mM

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citrate-phosphate buffer (containing 1 M NaCl, pH 6.0), and then placed at 4 °C for

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24 h. The resuspending was centrifuged at 12000 rpm for 10 min (4 °C), and the

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supernatant was the enzyme mixture used for the determination of XET activity.

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Determination of XET activity. XET activity was assayed with a colorimetric method 6

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developed by Sulova et al 22. Briefly, the reaction mixture contained: 50 µL tamarind

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xyloglucan solution (2 mg mL−1; Megazyme, Bray, Ireland), xyloglucan-derived

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oligosaccharide solution (0.5 mg mL−1; Megazyme), 10 mM sodium phosphate (pH

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6.0), and an enzyme mixture, respectively. The blank sample lacked xyloglucan and

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the control sample lacked the XET enzyme mixture. The mixtures were incubated at

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37 °C for 30 min before the reaction was stopped with the addition of 100 µL 1 M

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HCl. We added 0.2 ml iodine-potassium iodide solution and 0.8 mL 20% (w/v)

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Na2SO4 to the samples, which were then incubated in darkness for 30 min. The optical

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density of the samples was measured at 620 nm. The XET activity rate was calculated

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as the percentage of xyloglucan degradation catalyzed by 1 g fresh weight.

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Determination of ACS1 genotype

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Genomic DNA was isolated from leaves of three apple cultivars, ‘Fuji’, ‘Golden

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Delicious’ and ‘Taishanzaoxia’ using plant genomic DNA kit (TIANGEN, Beijing,

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China). PCR was carried out to identify the ACS1 allelic forms in apple cultivars. The

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reaction mixture (25 µL) contained 25 ng of template genomic DNA, 1 µL forward

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and reverse primer (10 µM), 2.5 µL 10×buffer, 2 µL dNTP (2.5 mM) and 0.3 µL Taq

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DNA polymerase (5 U µL-1). The amplification program consisted of 94 °C for 3 min,

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followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 50 s, with a

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final 3 min extension step at 72 °C. The PCR products were examined on a 1%

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agarose gel.

172 173

Quantitative real-time PCR analysis

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Total RNA was isolated from (2 g) leaves, fruit peels, and fruit flesh that had been

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quickly ground into a fine powder in liquid nitrogen. A 1 µg aliquot of RNA was 7

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analyzed by 1% agarose gel electrophoresis as a quality check. Total RNA

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concentration (ng µL−1) and quality (optical density ratio: 260 nm/280 nm) were

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determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham,

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MA, USA). First-strand cDNA was synthesized from 1 µg total RNA using the

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TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgene,

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Beijing, China). The quantitative reverse transcription polymerase chain reaction

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(qRT-PCR) assay was conducted using a CFX96 Real-Time PCR Detection System

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(Bio-Rad, Hercules, CA, USA). The reaction samples consisted of 1 µL cDNA (5-fold

184

dilution), 10 µL TransStart Top Green qPCR SuperMix, 1 µL forward and reverse

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primer (10 µM), and 7 µL double-distilled H2O. The qRT-PCR program was as

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follows: 94 °C for 30 s and then 40 cycles of 94 °C for 5 s, 58 °C for 15 s, and 72 °C

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for 10 s. The qRT-PCR assay was completed in triplicate. The Actin gene (for apple)

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and 18S gene (for tomato) served as an internal control, and the relative quantities of

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specific transcripts were determined using the 2−∆∆Ct method

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designed using Primer Premier 6 software.

23

. All primers were

191 192

Transformation of tomato

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The XTH2 and XTH10 genes were individually incorporated into the pBI121 vector

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at the BamHI and SalI sites. The constructs were inserted into Agrobacterium

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tumefaciens strain LBA4404 cells using a thermal stimulation method (i.e., incubation

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in an ice bath for 5 min, liquid nitrogen for 5 min, and at 37 °C for 5 min). The A.

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tumefaciens cells were then used to transform tomato cotyledons using a dipping

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method. Transformed materials were cultured three times on Murashige and Skoog

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medium supplemented with 100 mg L−1 kanamycin to isolate the transgenic material.

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The plants were grown in the illumination incubator, with a photoperiod of 16 h light 8

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(27 °C), 8 h dark (19 °C). Leafs of the transgenic tomato were used for gene

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expression analysis. Three lines were tested for each gene.

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RESULTS AND DISCUSSION

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ACS1 Genotype, ethylene and fruit softening

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In previous study, we found fruit firmness was on the decline during the fruit

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development period in ‘Taishanzaoxia’ apple, accompanying with a burst of ethylene

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production. The decline of fruit firmness was significantly correlated with ethylene

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production (r=-0.851*), suggesting fruit softening in ‘Taishanzaoxia’ apple was

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dependent on ethylene21. In this study, we found 1-MCP delayed the burst of ethylene

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production and the decline of fruit firmness during 0-3 DAH. Accompanying by the

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increase of ethylene production, fruit firmness declined quickly during 2-5 DAH

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(Figure 1). The results further confirmed that ethylene is required for normal ripening

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and softening of many climacteric fruits2.

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ACS and ACO are two key enzymes in the ethylene biosynthesis 2. In apple, ACS1 is

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specifically expressed in ripening fruits and closely related with ethylene production

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and fruit softening24. Two allelic forms of ACS1 (i.e. ACS1-1, ACS1-2) were isolated

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from a genomic library of ‘Golden Delicious’ 25. ACS1-2 is linked with the low level

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of ethylene production, as the insertion of a short interspersed nuclear element (SINE)

220

into the promoter region results in the sharp decline of the transcription activity of

221

ACS1-2

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combinations, ACS1-1/-1, ACS1-2/-2 and ACS1-1/-2 generally confer high, low and

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medium ethylene production, respectively24,26. Consistent with the results, we found it

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was ACS1-1/-1 homozygote in ‘Taishanzaoxia’ apple as there was one band of 489bp

225

in length (Figure 2A). The transcription levels of ACS1 in ‘Taishanzaoxia’ apple were

25-27

. ACS1 is inherited in a Mendelian fashion, and the three allelic

9

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elevated during the fruit development period (Figure 2B) and significantly correlated

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with ethylene production (rACS1=0.949**). In previous study, we found that the

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up-regulation of ACO1 was also correlated with ethylene production 21. Therefore, the

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burst of ethylene production in ‘Taishanzaoxia’ apple was closely associated with

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higher transcription activity of ACS1 determined by ACS1-1/-1 genotype, and ACO1

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also played important roles in ethylene production.

232 233

Roles of XTH Genes in Fruit Ripening and Softening

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The XET activity level increased throughout the fruit development period in

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‘Taishanzaoxia’ apple, with a distinct surge during the rapid fruit softening stage

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(Figure 3). The XET activity was significantly correlated with ethylene production

237

(r=0.852*) and fruit firmness (r=-0.888*), suggesting that the elevated XET activity

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dependent on ethylene played important roles in fruit ripening and softening.

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We previously isolated XTH2 and XTH10 from an SSH library for the

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‘Taishanzaoxia’ apple. Their elevated expression levels suggested they played

241

important roles during fruit ripening and softening

242

differences in the XTHs expression patterns (Figure 3 and 4). The decreased XTH1,

243

XTH5 and XTH9 expression levels during the fruit development period were

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significantly correlated with fruit firmness (positive), suggesting they may play a role

245

in the early period of fruit softening, which was consistent with results in the

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soft/crisp strains of Malus sieversii f. niedzwetzkyana18. It was similar with the roles

247

of AdXTH7 in kiwifruit and LeEXT1 in tomato16,28, which may play a role in early

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softening of fruits allowing access for other cell wall hydrolases during the rapid

249

softening phase. Accompanied by a sharp increase in ethylene levels, XTH2, XTH7,

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XTH10 and XTH11 were highly expressed during the rapid fruit softening stage and

21

. In this study, we observed

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significantly correlated with fruit firmness (negative) and ethylene production

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(positive), suggesting they may be involved in fruit ripening and softening via an

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ethylene-regulated pathway, which was consistent with the results of a study

254

involving ethylene-treated apples 3. This was also supported by the fact that fruit

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softening was delayed in fruits treated with 1-MCP and XTH2, XTH7, XTH10, and

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XTH11 expression levels were suppressed 0-2 DAH, but were up-regulated 2-5 DAH

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accompanying with the accumulation of ethylene (Figure 4). Accompanying the

258

accumulation of ethylene, the expression of XTH3 was up-regulated (Figure 3 and 4)

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and it was significantly correlated with ethylene (negatively), suggesting it may be

260

negatively regulated by ethylene. XTH4 mainly expressed during early fruit

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development (Figure 3), and 1-MCP had little effect on the expression (Figure 4),

262

suggesting the expression of XTH4 may be independent of ethylene. XTH6 and XTH8

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had two expression peaks during early and late fruit development respectively (Figure

264

3), suggesting they may play a role in both early and late fruit development. The

265

expression of XTH6 and XTH8 was evidently suppressed by 1-MCP during late fruit

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development (Figure 4), suggesting the late fruit softening was dependent on ethylene.

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Thus, to conclude, the differential expression of XTH genes was critical for the fruit

268

ripening and softening in ‘Taishanzaoxia’ apple. The early fruit softening mainly

269

resulted from the expression of XTH1, XTH3, XTH4, XTH5 and XTH9 was

270

independent on ethylene. The late fruit softening was dependent on ethylene, which

271

was associated with up-regulation of XTH2, XTH6, XTH7, XTH8, XTH10 and XTH11.

272

The role of XTHs was further verified in transgenic tomatoes overexpressing XTH2

273

and XTH10. Interestingly, not only the expression of XTH2 and XTH10 was

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significantly up-regulated in transgenic tomatoes, but also the expression of fruit

275

softening-related genes (PG2A, XTHs, Cel2 and TBG4) was up-regulated (Figure 5). 11

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Fruit softening is a genetically programmed development process, involving changes

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in the structure and composition of the fruit cell wall, which are resulted from the

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co-ordinated and interdependent action of a range of hydrolytic enzymes including

279

PG, β-Gal and XTH

280

softening by loosening the cell wall in preparation for further modification by other

281

cell wall-associated enzymes and through disassembly of xyloglucan16. Therefore,

282

XTH2 and XTH10 may act as a switch in fruit softening through activating the

283

expression of softening-related genes. Additionally, the expression of ethylene

284

biosynthesis and signaling pathway genes (ACS2, ACO1 and ERF2) was also

285

up-regulated in transgenic tomato, suggesting there may be feedback regulation

286

mechanism between ethylene biosynthesis and fruit softening. To conclude, XTH2 and

287

XTH10 may act as a signal switch triggered off the increase of ethylene production

288

and initiated the process of fruit softening. Therefore, it will be our focus to further

289

identify the function mechanism of XTH2 and XTH10 in the process, which is critical

290

for a more comprehensive characterization of the mechanisms regulating fruit quality

291

and breeding programs aimed at optimizing fruit quality.

4,29

. XTH enzymes are thought to play a key role in fruit

292 293

Other regulatory factors in fruit ripening and softening

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Fruit softening is a one-way process and once initiated, the process can not be

295

completely inhibited by inhibitors of ethylene action (e.g. 1-MCP) 2. This implies

296

there may be regulatory factors upstream of ethylene or other regulatory factors that

297

are part of ethylene-independent pathways. In tomato, RIN, which is a member of the

298

MADS-box family of transcription regulators, is essential for fruit ripening. Its

299

encoded protein acts as an upstream regulator of ethylene, and interacts with the

300

promoters of genes involved in fruit softening, including ACS, PG2A, and EXP1 30,31. 12

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In apple, specific MADS2.1 alleles are significantly associated with qualitative

302

assessments of fruit texture

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and ethylene-modulated ripening traits, are inhibited in MADS8/9-suppressed apples

304

33

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associated with fruit ripening and softening and may act as a negative regulator

306

Similar to RIN, the MADS genes are important for regulating flesh formation and

307

softening of fleshy fruits, potentially through the activation of ethylene biosynthesis

308

genes. Therefore, confirming the role of MADS genes during the regulation of

309

‘Taishanzaoxia’ apple ripening and softening, and determining whether their functions

310

depend on ethylene may be of value.

32

. Ripening characteristics, such as starch degradation

. We previously identified a MADS gene in an SSH library, which was closely 21

.

311 312 313

Supporting information

314

S1 Table. (A) Analysis of correlation between XTH expression levels and fruit

315

firmness, ethylene production.

316

S2 Figure 1. Differential expression of XTH genes in the leaf, peel and flesh of

317

‘Taishanzaoxia’ apple

(B) Primers used in the study

318 319

Funding

320

This research was funded by the Special Fund for Agro-scientific Research in the

321

Public Interest of China (to XSC, grant no.201303093, http://www.most.gov.cn/),

322

Natural

323

http://www.nsfc.gov.cn/), and National Key Basic Research Program of China (to

324

XSC, grant no.2011CB100606, http://www.most.gov.cn/) of the Ministry of Science

325

and Technology and Agriculture of the People’s Republic of China.

Science

Foundation

of

China

(to

XSC,

13

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no.31171932,

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Table of Contents Figure 1. Changes of fruit firmness and ethylene production in ‘Taishanzaoxia’ apple treated with 1-MCP. A: Ethylene production rate B: Fruit firmness. Capital letters mean the significant level of 1% and lowercase letters mean the significant level of 5%. Figure 2. Band patterns of ACS1 genotypes (A) and the transcription levels of ACS1 \during fruit development period in ‘Taishanzaoxia’ apple (B). (A) Lane 1: ‘Taishanzaoxia’, ACS1-1/-1 homozygote (one band of 489 bp in length); Lane 2: ‘Golden Delicious’, ACS1-1/-2 heterozygote (two bands of 489bp, 655 bp in length); Lane 3: ‘Fuji’, ACS1-2/-2 homozygote (one band of 655 bp in length); M: 1kb plus marker. Figure 3. Changes of the XET activity and transcription levels of XTH genes during fruit development period in ‘Taishanzaoxia’ apple Figure 4. Effect of 1-MCP on the expression of XTH genes in ‘Taishanzaoxia’ apple. ** means the significant level of 1% and * means the significant level of 5%. Figure 5. Characterization of the expression of genes involved in fruit ripening and softening in tomatoes overexpressing XTH2 and XTH10. ** means the significant level of 1% and * means the significant level of 5%.

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Figure 1. Changes of fruit firmness and ethylene production in ‘Taishanzaoxia’ apple treated with 1-MCP. A: Ethylene production rate B: Fruit firmness. Capital letters mean the significant level of 1% and lowercase letters mean the significant level of 5%. 96x43mm (300 x 300 DPI)

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Figure 2. Band patterns of ACS1 genotypes (A) and the transcription levels of ACS1 \during fruit development period in ‘Taishanzaoxia’ apple (B). (A) Lane 1: ‘Taishanzaoxia’, ACS1-1/-1 homozygote (one band of 489 bp in length); Lane 2: ‘Golden Delicious’, ACS1-1/-2 heterozygote (two bands of 489bp, 655 bp in length); Lane 3: ‘Fuji’, ACS1-2/-2 homozygote (one band of 655 bp in length); M: 1kb plus marker. 84x29mm (300 x 300 DPI)

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Figure 3. Changes of the XET activity and transcription levels of XTH genes during fruit development period in ‘Taishanzaoxia’ apple 109x38mm (300 x 300 DPI)

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Figure 4. Effect of 1-MCP on the expression of XTH genes in ‘Taishanzaoxia’ apple. ** means the significant level of 1% and * means the significant level of 5%. 90x45mm (300 x 300 DPI)

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Figure 5. Characterization of the expression of genes involved in fruit ripening and softening in tomatoes overexpressing XTH2 and XTH10. ** means the significant level of 1% and * means the significant level of 5%. 101x44mm (300 x 300 DPI)

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For TOC only TOC graphic 85x47mm (300 x 300 DPI)

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