Changes in fruit firmness, cell wall composition and transcriptional

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Changes in fruit firmness, cell wall composition and transcriptional profile in the yellow fruited tomato 1 (yft1) mutant Ling Li, Weihua Zhao, Xuechao Feng, Lulu Chen, Lida Zhang, and Lingxia Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04611 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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Journal of Agricultural and Food Chemistry

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TITLE PAGE

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Changes in fruit firmness, cell wall composition and transcriptional profile in the

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yellow fruited tomato 1 (yft1) mutant

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Ling Li,† Weihua Zhao,†,

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Zhao,†, ‡, *

‡

Xuechao Feng,† Lulu Chen,†,

‡

Lida Zhang, †,* Lingxia

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*Corresponding authors:

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Lida Zhang (e-mail: [email protected]); Lingxia Zhao (e-mail: [email protected],

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phone: 0086-21-34205775).

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† Department of Plant Science, School of Agriculture and Biology, Shanghai Jiao

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Tong University, Shanghai 200240, China

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‡Joint

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Tong University, Shanghai 200240, China

Tomato Research Institute, School of Agriculture and Biology, Shanghai Jiao

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1 ACS Paragon Plus Environment


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ABSTRACT

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Fruit firmness is an important trait in tomato (Solanum lycopersicum), associated with

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shelf life and economic value; however, the precise mechanism determining fruit

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softening remains elusive. A yellow fruit tomato 1(yft1) mutant, harbors a genetic

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lesion in the YFT1 gene, and has significantly firmer fruit than those of the cv. M82

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wild type at a red ripe stage, 54 dpa (days post anthesis). When softening was further

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dissected, it was found that the yft1 firm fruit phenotype correlated with a difference

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in cellulose, hemicellulose and pectin deposition in the primary cell wall (PCW)

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compared to cv.M82. Alterations in the structure of the pericarp cells, chemical

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components, hydrolase activities and expression of genes encoding these hydrolases,

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were all hypothesized to be a result of the loss of YFT1 function. This was further

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affirmed by RNA-seq analysis, where a total of 183 differentially expressed genes

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(DEGs, 50/133 down/up-regulated) were identified between yft1 and cv.M82. These

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DEGs were mainly annotated as participating in ethylene and auxin related signal

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transduction, sugar metabolism and photosynthesis. This study provides new insights

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into mechanism underlying the control of fruit softening.

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KEYWORDS: yellow fruited tomato 1 mutant, fruit firmness, softening, cell

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structure, transcriptomics, primary cell wall


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INTRODUCTION

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Tomato (Solanum lycopersicum) is a major vegetable crop worldwide, with 177

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million tons produced in 2016 representing a value of $ 95.7 billion.1 It is frequently

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used as a model plant to investigate fundamental biological questions,2-6 and it is a

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source of nutrients and vitamins in the human diet.1,7 Tomato is also beneficial to

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human health in preventing risk of skin cancer, lung cancer, prostate cancer, and some

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cardio-cerebrovascular diseases, due to the high levels of carotenoids, which act as

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antioxidants. 8,9

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Firmness is an important fruit attribute as it is associated with shelf life and the

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capacity for long distance transport.

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that is influenced by fruit development, cell wall disintegration, 12,13 quality decline 10,

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as well as with synthesis of ethylene gas in tomato.14 Fruit firmness is dependent on

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polysaccharides deposited in the cell wall, cell-to-cell adhesion and the water status

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inside the cells.

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cross-links to form the main structural component of the cell wall, while pectin

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polymers and structural proteins provide additional components of the cell wall

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network. In addition to polysaccharides in the cell wall, soluble sugars such as sucrose,

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fructose and glucose accumulating inside the cells are also associated fruit firmness

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through their effect on cellular turgor and swelling. 16

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During tomato fruit ripening, the cell wall undergoes substantial disassembly, which

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is mediated by a number of cell wall proteins.11,

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(PG) and pectin methylesterase (PME) modify the pectic homogalacturonan structure,

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11,18

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rhamnogalacturonan I (RGI) .

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tomato involved in tethering cellulose microfibrils, and its de-polymerization, a

7,15

10,11

However, it is a complex horticultural trait

Cellulose is connected by hydrogen bonded hemicellulose

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For example, polygalacturonase

while β-galactosidase (β-Gal) acts on the branched galactan sidechains of 19

Xyloglucan is the predominant hemicellulose in



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notable feature of tomato fruit softening, may be catalyzed by enzymes such as

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xyloglucan endo-transglucosylases (XET),

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20,21

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involved in sucrose biosynthesis and degradation. INV cleaves sucrose into glucose

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and fructose22, SS converts it into fructose and uridine diphosphate glucose (UDPG)23

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and SPS reversibly catalyzes the synthesis of sucrose phosphate (S-6-P) from UDPG

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and 6-phosphate fructose (F-6-P).24

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Alterations of the physiological and biochemical properties of tomato fruit during

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ripening are regulated by genes associated with cell wall degradation and the

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metabolism of soluble sugars. PG2 down-regulation was reported to inhibit aspects of

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pectin degradation

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leaves and mature green tomato fruits.26 Suppression of TBG4 (TOMATO

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BETA-GALACTOSIDASE 4) expression caused lower β-galactosidase levels and

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enhanced firmness in processing tomato.27 It was reported that EL2 (EGase2)

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contributes to tomato fruit softening associated cellulose decomposition, and its

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expression is known to be repressed in the ripening-inhibitor (rin) mutant despite the

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presence of ethylene.28 SLXTH8 (Xyloglucan Endo-transglycosylase/Hydrolase 8), a

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member of the glycosyl hydrolase family 16 (GH16), has both XET and xyloglucan

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endo-Hydrolases (XEH) activity, and is highly expressed during fruit ripening, where

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it catalyzes xyloglucan hydrolysis and affects cell wall mechanical properties .29

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Finally, SINENSIS SUCROSE SYNTHASE 1 (SUS1) and SPS control sucrose

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synthesis and degradation via regulation of SS and SPS in Citrus.30

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Several tomato mutants, such as non-ripening (nor),13 rin,31 never ripe (Nr)

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green-ripe (gr) 33 have enhanced fruit firmness due to ripening suppression; however

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the taste and nutritional value of these mutants are not as good as wild type fruit.34 An

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and endo-β-1,4-glucanases (EGases).

Invertase (INV), sucrose synthase (SS) and sucrose phosphate synthase (SPS) are

7,25,

while silencing of PME1 reduced the PME activity in both


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and

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ethylene receptor mutant, sletr1-2, also has enhanced firmness, but again this

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mutation is associated with undesirable characteristics.35 Suppressed expression of PG

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or PME resulted in fruits with some desirable characteristics, but does not prevent

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softening.18,36 An approach to extend storage time and shelf life, without altering gene

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expression, is to suppress ethylene synthesis by treating postharvest tomatoes with

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aminoethoxyvinyl glycine (AVG).37 It has also been proposed that an alternative

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strategy for extending fruit shelf life would be to control the deposition of cell wall

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components rather than the hydrolytic enzymes.11 However, importantly, the precise

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mechanisms involved in fruit softening remain elusive. 7

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The yellow fruit tomato 1 (yft1) mutant was created from S. lycopersicum (cv. M82)

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by exposure to fast neutrons. The yft1 target was isolated by map-based cloning in our

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laboratory and found to be a genetic lesion occurring in the 5′ UTR of YFT1.38

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Besides the yellow fruited phenotype, the enhanced fruit firmness of yft1 provided an

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opportunity to better understand processes associated with fruit softening. In this

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study, we investigated the mechanisms involved in tomato fruit textural changes and

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dissected the contribution of YFT1 to fruit softening.

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MATERIALS AND METHODS

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Plant materials

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cv. M82 and yft1 (n3122) seeds were kindly provided by Prof. Dani Zamir at the

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Hebrew University of Jerusalem (http://zamir.sgn.cornell.edu/mutateds). Seeds were

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sown in a 60 cell breeding plug tray (Taizhou Sophia Import & Export Co., Ltd,

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Zhejiang, China) with humid peat pellets to germinate at 26/20°C(day/night) in an

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intelligent illuminating incubator (Shanzhi precision instrument technology Co., Ltd,

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Shenzhen, China), and seedlings with four fully expanded true-leaves were planted in



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a polycarbonate standard greenhouse with natural light at the Pujiang experiment farm

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in the School of Agriculture and Biology at the Shanghai Jiao Tong University

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(Shanghai city, China).

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Examination of fruit firmness and pericarp thickness

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Tomato fruits were sampled at 35 days post anthesis (dpa), 47dpa and 54 dpa,

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corresponding to the MG (mature green), BR (breaker) and RR (red ripe)

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developmental stages of cv.M82. Tomato fruit firmness (TFF) was examined using a

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Texture Analyzer (Model TA.XT Plus, SMSTA, UK) fitted with a 6 mm cylindrical

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plunger (Model SMS P/5). The firmness was measured by Texture Profile Analysis

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(TPA) and each fruit was compressed to 11mm at a speed of 100mm/min with a

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trigger force of 0.3 N. The firmness value was the maximum force developed during

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the test. Each tomato fruit was analyzed at five points: the top, pedicle, and three

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points with 120° intervals around the equatorial zone (n=5).

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Pericarp thickness was measured at three points with 120° intervals around the

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equatorial zone using an electronic vernier caliper (CD-15CPX, Mitutoyo, Japan)

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(n=5).

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Pericarp tissue microstructure

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The tomato pericarps were sampled at 35 dpa, 47dpa and 54 dpa, cut into small pieces

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(1-2 mm2) and then immediately immersed in the FAA (formalin-acetic acid) fixative

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(100mL FAA contains 5mL 37% formaldehyde, 5mL acetic acid, 63mL anhydrous

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ethanol and 27mL water). The FAA fixative was replaced with fresh FAA after

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vacuum infiltration for 30 min and then stored at 4°C. The samples were dehydrated

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using an ethanol series (70%, 80% and 95%) for 30 min each and 100% ethanol for 2

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hours, before being immersed in mixture of anhydrous ethanol and Basic liquid

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Technovit 7100

(v:v = 1:1) for 2 hours. Finally, the samples were transferred into


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permeation solution (1g hardener I dissolved in 100 mL Basic liquid Technovit 7100,

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Kulzer and Co., Wehrheim, Germany) overnight. Embedding was performed by

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transferring the samples to embedding solution (1 mL hardener II dissolved in 15mL

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permeation solution) and incubating at 60°C for 48h. Samples were sectioned using a

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slicer (LEICA RM2265) with a slice thickness of 2-3μm. Finally, the sections were

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stained with periodic acid-schiff (PAS), and the microstructure of tomato pericarp

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cells was observed with a microscope (Nikon Eclipse 80i, USA).

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Extraction and determination of carbohydrates

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cv.M82 and yft1 tomato pericarps (35dpa, 47dap and 54 dpa) were cut into small

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pieces and ground into a fine powder in liquid nitrogen (n=3). Cellulose was extracted

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and detected using the sulphuric acid-potassium bichromate oxidation method,39 with

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some modification. Pericarp powder (2g) was fully mixed with 10 mL of extraction

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buffer containing acetic acid and nitric acid (v:v=1:1), boiled in a water bath for

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30min, an then diluted into 45mL with dd H2O. The homogenate was centrifuged at

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12,000 g for 15 min and the pellet was washed twice with dd H2O, and then dried at

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105°C overnight. The dried residue was mixed well with 10mL sulphuric

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acid-potassium bichromate (10% sulphuric acid with 0.1 potassium bichromate)

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5mL 20% KI and titrated until the appearance of blue color with 0.2 M sodium

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hyposulfite. Hemicelluloses were extracted by mixing pericarp powder (2g) with

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10mL of 80% calcium nitrate solution (w:v), and adding ddH2O to 45 mL. The

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samples were boiled in a water bath for 5min, and then centrifuged at 12,000 g for

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15min. The supernatant was discarded and the residues washed twice with 45 mL

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ddH2O before drying into a constant weight at 105°C. Ten mL of HCl was added and

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the samples boiled in a water bath for 45min, after which a drop of 1%

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phenolphthalein indicator (w:v) was added, and titration performed until the


and


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appearance of a pink color with 2 N NaOH. Finally, the samples were diluted to 50

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mL with ddH2O. Pectin was extracted and measured as previously described.40

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Soluble sugars were extracted and detected using the ethanol and anthrone sulfuric

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acid methods,41 with some modifications. Pericarp powder (1g) was used to extract

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soluble sugars, and the supernatant collected after centrifugation at 12,000g for 15

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min. A glucose standard curve was drawn using the 3, 5-dinitrosalicylic acid (DNS)

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method. 42

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Cell wall hydrolase extraction and activity assay

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Crude PG, PEM and β-GAL extracts were made according to previously described

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methods.43 Estimated PG activity was determined using the DNS method,

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PME and β-GAL activities were assayed using a titrimetric method

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p-nitrophenyl

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CELLULASE was extracted from tomato pericarp with 80 % alcohol (v:v), and

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activity determined through detection of carboxymethyl cellulose (CMC) reduction by

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the DNS method.46 The XET preparation and assay is described as in Miedes et al.. 47

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Extraction of crude enzyme preparations for AI, NI, SS and SPS and detection of their

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activities are described in Hubbard et al..48

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Gene expression analysis by real time-quantitative PCR (RT-qPCR)

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Total RNA was extracted from tomato pericarp at 35dpa, 47dpa and 54dpa with the

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RNA prep pure plant kit (Tiangen, Beijing, China), and served as the template for

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cDNA synthesis using the PrimeScript ™ RTMaster Mix kit (Takara, Dalian, China).

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The first strand cDNA synthesized using 1 μg total RNA in a 20 μl reaction system

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was diluted 50-fold to fulfill RT-qPCR analysis. Twenty μL reaction volumes

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containing 2μL cDNA, 10μL Maxima SYBR Green (Takara, China), 0.6μL each

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forward and reverse primer with 10 μM, and 6.8μL RNase-free water were prepared.

β-D-galactopyranoside

(PNPG)

method,45


42

44

respectively.

while and a Crude

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The RT-qPCR procedure was performed at 94°C (initial denaturation) for 3 mins and

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then for 40 cycles of 94°C for 20 s, 55°C for 20 s, 72°C for 20 s. ACTIN (GenBank

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accession: BT013524) was used as the reference gene to normalize the expression of

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hydrolase genes. Gene specific primers are listed in Table S1.

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Transcriptome analysis

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Tomato pericarps were sampled from yft1 and cv.M82 at 35dpa, 47dpa and 54 dpa,

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respectively (three biological replicates, with each replicate sampled from three fruits).

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Extraction of total RNA was conducted using the RNA prep-pure Plant Kit according

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to the manufacturer’s instructions (Tiangen, Peking, China). The concentration and

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quality of the total RNA were estimated by NanoDrop 2000 (Thermo Scientific,

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Waltham, USA) and Agilent 2200 (Agilent Technologies, Santa Clara, USA). The

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RNA samples were used to construct libraries for paired-end RNA sequencing (2×100

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bp) on an Illumina HiSeq 2000 sequencing system at the Shanghai Majorbio

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Bio-pharm Biotechnology Co., Ltd. (Shanghai, China).

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The FASTX-Toolkit (http://hannonlab.cshl.edu/fastx toolkit) was adopted to remove

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the adaptor sequences and low-quality regions from the raw reads, and the remaining

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high-quality reads were mapped to the tomato reference genome (ITAG2.4,

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ftp://ftp.solgenomics.net/tomato_genome/annotation/ITAG2.4_release/)

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TopHat2. 49 The relative transcript abundance of each gene was determined using the

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fragments per kilo-base of transcript per million mapped reads (FPKM) method. The

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differentially expressed genes (DEGs) between yft1 and cv.M82 at the same

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developmental stage were identified using a significance analysis by Cuffdiff with a

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p-value < 0.05 and at least two-fold changes (either up- or down-regulation). Genes

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whose expression levels showed significant changes between yft1 and cv.M82 at all

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three developmental stages were used for further pathway analysis.


using


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For gene annotation, sequences were analyzed using the (Kyoto Encyclopedia of

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Genes and Genomes) database (http://www.genome.ad.jp/kegg/kegg2) with a cut-off

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E-value of ≤10-10.50 KEGG pathway enrichment analysis of the DEGs was performed

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using Fisher’s exact test implemented in a Perl script against a reference tomato

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genome dataset. The significance level in the pathway analysis was set to FDR

Changes in fruit firmness, cell wall composition and transcriptional

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