Suppression of the MADS-box gene SlMBP8 accelerates fruit ripening of tomato (Solanum lycopersicum)

Accepted Manuscript Suppression of the MADS-box gene SlMBP8 accelerates fruit ripening of tomato (Solanum lycopersicum) Wencheng Yin, Zongli Hu, Baolu Cui, Xuhu Guo, Jingtao Hu, Zhiguo Zhu, Guoping Chen PII:

S0981-9428(17)30210-3

DOI:

10.1016/j.plaphy.2017.06.019

Reference:

PLAPHY 4913

To appear in:

Plant Physiology and Biochemistry

Received Date: 17 January 2017 Revised Date:

13 June 2017

Accepted Date: 14 June 2017

Please cite this article as: W. Yin, Z. Hu, B. Cui, X. Guo, J. Hu, Z. Zhu, G. Chen, Suppression of the MADS-box gene SlMBP8 accelerates fruit ripening of tomato (Solanum lycopersicum), Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.06.019. 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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Title: Suppression of the MADS-box gene SlMBP8 accelerates fruit ripening of tomato (Solanum lycopersicum)

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Running Title: Silencing of SlMBP8 accelerates fruit ripening in tomato

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Wencheng Yin, Zongli Hu, Baolu Cui, Xuhu Guo, Jingtao Hu, Zhiguo Zhu, Guoping Chen *

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4 Laboratory of molecular biology of tomato, Bioengineering College, Chongqing University, Chongqing 400044,

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People’s Republic of China

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* Guoping Chen (Author for Correspondence): Room 523, Bioengineering College, Chongqing University,

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Campus B, 174 Shapingba Main Street, Chongqing 400030, P.R. China; Tel: 00862365112674; Fax:

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00862365112674; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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MADS-box genes encode important transcription factors that are involved in many biological processes of plants,

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including fruit ripening. In our research, a MADS-box gene, SlMBP8, was identified, and its tissue-specific

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expression profiles were analysed. SlMBP8 was highly expressed in fruits of the B+4 stage, in senescent leaves and in

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sepals. To further characterize its function, an RNA interference (RNAi) expression vector of SlMBP8 was

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constructed and transferred into tomato. In the transgenic plants, the ripening of fruits was shortened by 2-4 days

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compared to that of wild type. At the same time, carotenoids accumulated to higher levels and the expression of

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phytone synthase 1 (PSY1), phytoene desaturase (PDS) and ς-carotene desaturase (ZDS) was enhanced in RNAi fruits.

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The transgenic fruits and seedlings showed more ethylene production compared with that of the wild type.

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Furthermore, SlMBP8-silenced seedlings displayed shorter hypocotyls due to higher endogenous ethylene levels,

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suggesting that SlMBP8 may modulates the ethylene triple response negatively. A yeast two-hybrid assay indicated

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that SlMBP8 could interact with SlMADS-RIN. Besides, the expression of ethylene-related genes, including ACO1,

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ACO3, ACS2, ERF1, E4 and E8, was simultaneously up-regulated in transgenic plants. In addition,SlMBP8-silenced

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fruits showed higher ethylene production, suggesting that suppressed expression of SlMBP8 promotes carotenoid and

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ethylene biosynthesis. In addition, the fruits of transgenic plants displayed more rapid water loss and decreased

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storability compared to wild type, which was due to the significantly induced expressions of cell wall metabolism

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genes such as PG, EXP, HEX, TBG4, XTH5 and XYL. These results suggest that SlMBP8 plays an important role in

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fruit ripening and softening.

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Keywords: MADS-box gene; fruit ripening; carotenoid accumulation; storage; tomato (Solanum lycopersicum)

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Introduction Fruit ripening involves obvious complex biochemical and physiological changes, such as pigment accumulation

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for bright colouring, cell wall degradation for fruit softening and accumulation of volatiles for aromas. In climacteric

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fruits, ripening is accompanied by a peak in respiration and a concomitant burst of ethylene biosynthesis. ACS

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(1-Aminocyclopropane-1-carboxylate synthase) and ACO (1-Aminocyclopropane-1-carboxylate oxidase) are two key

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biosynthetic enzymes (Adams and Yang, 1979, Yang and Hoffman, 1984). The expression levels of both SlACO1 and

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SlACO3 are significantly increased at the onset of tomato fruit ripening (Barry et al., 1996). It has been revealed that

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the expression of SlACO3 is transiently induced at the breaker stage, while SlACO1 is sustained during ripening

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(Barry et al., 1996). Previous studies also have indicated that SlACS2 is an important factor in the transition between

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ethylene synthesis system 1 to ethylene synthesis system 2, and RNA interference (RNAi) inhibition of SlACS2

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strongly inhibits ethylene biosynthesis and fruit ripening (Barry et al., 2000). In addition to ethylene synthesis, the

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ability to perceive and respond to ethylene is necessary for fruit ripening. E4 and E8 are two classical

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ethylene-response genes that are involved in fruit ripening (Lincoln and Fischer, 1988, Kesanakurti et al., 2012).

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Fruit softening is type of fruit quality, but excessive softening limits fruit shelf-life and storage . The expression

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of cell wall-modifying genes has been used to investigate the role of particular activities of fruit softening during

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ripening and in the manufacture of processed fruit products (Brummell and Harpster, 2001, Vicente et al., 2007).

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Ripening-specific or postharvest-softening-specific genes encoding cell wall-modifying enzymes have been

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characterized and cloned. Polygalacturonase (PG), which is transcriptionally activated during fruit ripening, is a

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major cell wall polyuronide-degrading enzyme involved in the fruit softening process (Montgomery et al., 1993).

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Tomato (Solanum lycopersicum) is generally considered to be a model plant for studying climacteric fruit

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ripening and the mechanism of tomato fruit ripening has been uncovered through a wide range of studies. Molecular

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and biochemical studies on tomato ripening mutants such as ripening inhibitor (rin), never ripe (Nr), nonripening 3

ACCEPTED MANUSCRIPT (nor) and color nonripening (cnr) have been conductted (Wilkinson et al., 1995, Vrebalov et al., 2002). In terms

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of floral organ development, a considerable number of MADS-box genes have been identified in Arabidopsis

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thaliana and Antirrhinum majus (Causier et al., 2003, Urbanus et al., 2009, Becker and Ehlers, 2016). In addition,

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regulation of fruit development, embryogenesis and vegetative organ development have been successively revealed in

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various species, which suggests a diverse role of these MADS-box transcriptional factors (Yanofsky et al., 1990,

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Sommer et al., 1990). SlMADS-RIN is a classical MADS-box protein involved in tomato fruit ripening, this protein

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is the essential positive regulator. FRUITFULL(FUL) plays an important role in dry and fleshy fruit development by

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regulating colour development and anthocyanin-related gene expression. The TM4 gene (Tomato MADS-box 4),

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which is homologous to Arabidopsis FUL, is repressed in the rin, cnr and nor mutants, indicating that the function of

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TM4 is associated with fruit development (Seymour et al., 2002, Fujisawa et al., 2012). It was revealed that FUL1

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and FUL2, which are highly similar to FRUITFULL in Arabidopsis, bind to the same genomic sites as RIN (Fujisawa

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et al., 2011, Giovannoni, 2007). Different from other identified ripening regulators, FUL1 and FUL2 generally

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regulate fruit ripening in an ethylene-independent manner (Shima et al., 2013). In addition, SlMADS1 negatively

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regulates fruit ripening, and overexpression of the novel MADS-box gene SlFYFL delays fruit ripening (Dong et al.,

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2013, Xie et al., 2014). Moreover, SlMADS1 interacts with RIN, similar to the behaviour of TOMATO

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AGAMOUS-LIKE1 (TAGL1), TAGL11 and FUL2. In addition, there may be many other unidentified MADS-box

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genes that play important roles in tomato fruit ripening.

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Previous reports have indicated that SlMBP8 transcripts increase during the process of fruit development and

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ripening, which suggests that the function of SlMBP8 might be related to fruit ripening(Hileman et al., 2006). To

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further reveal its regulatory function in fruit, RNA interference of SlMBP8 (GenBank accession XM_004252664)

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was performed, and darker red fruits were observed on transgenic plants, together with earlier ripening and more

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carotenoid accumulations, which supports our hypothesis that SlMBP8 plays an important role in regulating fruit 4

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ripening.

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2.1. Plant material and growth conditions

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Materials and methods

Tomato plants (Solanum lycopersicum Mill. cv. Ailsa Craig AC++) were used as the wild type (WT) in our experiments. Transgenic and wild-type plants were grown in greenhouse conditions (16-h-day/8-h-night cycle, 25/18℃

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day/night temperature, 80% humidity, and 250µmol m-2 s-1 light intensity). There were two generations used in the

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experiments: T0 originated from tissue culture, and T1 originated from seedlings. Flowers were tagged at anthesis, and

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the fruits were harvested according to the number of days post-anthesis (dpa) and fruit colour. In wild type, we

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defined MG (Mature green) as fruits of 35 dpa, B (Breaker stage) as fruits of 38 dpa in which the colour changed

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from green to yellow, and B+4 and B+7 as fruits of 4 days after break and 7 days after break , respectively. All

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samples were immediately frozen in liquid nitrogen and then stored at –80°C for further analyses.

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2.2. Isolation and sequence analysis of SlMBP8

Total RNA was extracted from all plant tissues for three biological repeats with the Trizol reagent (Invitrogen,

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USA). Then, a 1 µg aliquot of RNA treated with RNAiso Plus (TaKaRa) was used for synthesis of first-strand cDNA

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by reverse transcription polymerase chain reaction (M-MLV reverse transcriptase, TaKaRa, China) with tailed oligo

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d(T)18 primers (5’ GCT GTC AAC GAT ACG CTA CGT AAC GGC ATG ACA GTG TTT TTT TTT TTT TTT

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TTT 3’). An amount of 1-2 µl of cDNA was used to clone the complete sequence of the coding region of the SlMBP8

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gene with the primers SlMBP8-F (5’ CAA CAA TGG GAG AAC TGC TAC A 3’) and dT-R (5’GCT GTC AAC

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GAT ACG CTA CGT AAC G 3’) through high-fidelity PCR (Prime STARTM HS DNA polymerase, TaKaRa). The

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PCR procedure was performed at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for

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30 s, and a final extension at 72°C for 10 min. In addition, the amplified products were then subcloned into the

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pMD18-T vector (TaKaRa) and confirmed by sequencing (Invitrogen). Multiple sequence alignment of SlMBP8 with

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other MADS-box proteins was conducted using the DNAMAN 5.2.2 programs.

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2.3. Construction of the SlMBP8 RNAi vector and plant transformation

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TO repress the expression of the SlMBP8 gene, a vector (pBIN19RNAi) was constructed (Supplementary Fig.

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S2). The 423-bp SlMBP8-specific DNA fragment used in the hairpin was amplified using the primers SlMBP8-F

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(5’CAA CAA TGG GAG AAC TGC TAC A 3’) and SlMBP8-R (5’AGA AAC AAG AAC AAG GAT GAA TA 3’)

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with Hind III, Kpn I, EcoR I and Xba I restriction sites at the 5’ end. The resultant construct was used to transform

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tomato cotyledon explants with Agrobacterium tumefaciens (strain LBA4404) (Zhu et al., 2014). Transformed lines

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were selected for kanamycin (80 mg l-1) resistance and then analysed by PCR with the primers NPTII-F (5ˊGAC

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AAT CGG CTG CTC TGA 3ˊ) and NPTII-R (5ˊAAC TCC AGC ATG AGA TCC 3ˊ) to determine the presence of

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T-DNA.

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qRT-PCR was performed using the SYBR® Premix Ex Taq II kit (TaKaRa, China) in a 10-µl total sample

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volume (5.0 µl of 2× SYBR Premix Ex Taq, 1.0 µl of primers, 1.0 µl of cDNA, 3.0 µl of ddH2O), and all reactions

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were performed using the CFX96

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min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. To verify that the PCR master mixes were free of

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contamination and to remove the template from the environment, a no-template control (NTC) and a

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no-reverse-transcription control (NRT) were included with each assay. In addition, three technical replicates and

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three biological replicates for each sample were used. The tomato clathrin adaptor complexes medium subunit ( CAC )

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Real-Time System (Bio-Rad) under the following conditions: 95°C for 3

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gene was selected as an internal standard (Exposito-Rodriguez et al., 2008). The expression levels of SlMBP8 in WT,

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Nr, rin and transgenic lines were determined with SlMBP8 (RT)-F and SlMBP8 (RT)-R primers (Supplementary

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Table S1). The relative gene expression levels were detected using the 2

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2001).

method (Livak and Schmittgen,

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Furthermore, the mRNA expression levels of other genes, including fruit ripening-related genes (E4, E8, PG,

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Pti4, LOXA and LOXB), carotenoid biosynthesis genes (PSY1, PDS and ZDS), ethylene biosynthesis pathway genes

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(ACO1, ACO3, ACS2 and ERF1) and cell-wall-modifying genes (PE, CEL2, EXP, HEX, MAN, TBG4, XTH5 and

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XYL), were determined simultaneously, their primers are shown in Table S1.

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2.5. Measurement of carotenoid contents

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One-gram samples of each line were cut and removed from the pericarp in a 5-mm-wide strip around the equator of

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38-dpa, 42-dpa and 45-dpa fruits-. Then, 10 ml of 60:40 (v/v) hexane-acetone was added to extract total carotenoids.

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The extract was centrifuged (5 min at 4000 g), and the absorbance of the supernatant was determined at 450 nm. The

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carotenoid content was calculated as follows: total carotenoids (mg ml-1) = (OD450)/0.25 (Forth and Pyke, 2006).

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Three technical replicates and three biological replicates were measured for each sample.

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2.6. Ethylene triple response assay

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The seeds of wild-type plants and transgenic plants were surface sterilized and cold treated at 4°C for 3- 4 days

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in the dark. The wild-type seeds were sown on MS medium supplemented with 0, 1.0, 2.0, 5.0, 10.0 and 20.0 µM

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ethylene precursor 1-aminocyclopropane-1-carboxylate synthase (ACC). Transgenic lines seeds (T1) were sown on

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MS medium fortified with 0 µM or 5.0 µM ACC. Seedlings were grown at 25°C in the dark for 5 days, after which

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the hypocotyl length and root length were measured, with each culture including 30 seedlings. To further explore 7

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changes in the expression of genes related to the ethylene response pathways in transgenic plants, the expression of

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ACO1, ACS1A, ACS2 and ACS6 in the WT and RNAi lines was detected. The expression level of SlMBP8 was also

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detected in WT seedlings treated with various concentrations of ACC (Supplementary Fig. S4).

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Fruits of B (breaker stage), B+4, B+7, and B+14 were harvested and placed in open jars for 4 h. As in the

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experiment of the ethylene triple response assay, seedlings were grown at 25°C in the dark for 5 days and then placed

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into 25-mL jars. The ias were sealed and incubated at room temperature (24 h). The ethylene contents were then

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detected using Hewlett-Packard 5890 series gas chromatograph following the methods of (Chung et al., 2010).

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2.8. Yeast two-hybrid assay

A yeast two-hybrid assay was performed according to the methods of Dong, T in 2013. The ORF of SlMBP8

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was amplified by PCR with the primer pair SlMBP8(Y)-F (5'GGCCATATGGGGCTTTTAATCGGCGAAAAA3')

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and SlMBP8(Y)-R (5'CGCGGATCCCGACGAATACGA CGATAATCA 3’).

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2.9. Postharvest storage test All

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under standard greenhouse conditions. Water loss from transgenic and wild-type fruit was assessed by measuring

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the weight reduction over the 30-day period in fruits removed from the vine at the red ripe stage (Fray and Grierson,

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1993).

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2.10. Statistical analysis 8

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The mean values of the data were measured from three replicates, and the ‘Standard Error’ of the means was

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calculated. The data were analysed using Origin 8.0 software, and the t test was used for assessing significant

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differences among the means.

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3.

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3.1. Isolation and expression pattern analyses of SlMBP8 in tomato

Results

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Based on the sequence in GenBank (GenBank accession XM_004252664), we designed specific primers and

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cloned the SlMBP8 gene from tomato fruit tissue. The nucleotide sequence analysis showed that SlMBP8 contains an

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open reading frame ( ORF ) of 597 nucleotides that encodes 198 amino acids. Amino acid homology analysis showed

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that SlMBP8 contains a M–I–K–C protein structure, suggesting that SlMBP8 is a typical MADS-box transcription

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factor (Supplementary Fig. S1).

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To extend our understanding of the role of SlMBP8, we examined its expression pattern in various tomato

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organs by quantitative real-time PCR. The maximum expression of SlMBP8 was observed in senescent leaves, and

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moderate expression was observed in sepals, young leaves, mature leaves and fruits at the B (Breaker), B+4 (4 days

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after Breaker) and B+7 (7 days after Breaker) stages. At the same time, very low levels of transcripts accumulated in

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stems, flowers and roots (Fig. 1A). In addition, during fruit enlargement and ripening, an increasing trend was

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observed from the MG (immature green) to the B+4 stage, but the expression decreased at the B+7 stage. A similar

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increasing trend was found for SlMBP8 expression in fruits of Nr and rin mutants (Fig. 1B). Fig. 1C shows that

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SlMBP8 mRNA highly accumulated in flower sepals and increased with the development of sepals, which indicated

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that SlMBP8 may participate in the sepal development process. Thus, these results suggest that SlMBP8 may be

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correlated with leaf and sepal development and fruit ripening.

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3.2. Silencing of SlMBP8 accelerates fruit ripening SlMBP8 transcripts were markedly reduced in 6 independent SlMBP8-RNAi lines. Among these transgenic lines,

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the accumulation of SlMBP8 transcripts in lines 01 and 22 were markedly reduced to approximately 10% of control

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levels, while the transcript accumulations were not significantly reduced in the other lines (Fig. 2A). The expression

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of SlMBP15, the most homologous gene of SlMBP8, didn’t change (Supplementary Fig. S3). Thus, lines 01 and 22

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were selected for further investigation. During the process of fruit development, the time from pollination to the

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breaker stage was measured. The colour of transgenic fruits changed 2 to 4 days earlier than wild type (Fig. 2B)

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(Table 1). As the characteristic red pigmentation of tomato fruits is determined by accumulation of the carotenoid

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pigment lycopene, the carotenoid accumulation of transgenic and wild-type fruit at different fruit ripening stages was

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measured in this study. As shown in Fig. 3A, the carotenoid contents in transgenic lines increased approximately 2-

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to 3-fold compared with those of wild-type fruits at 38 dpa (days after anthesis), 42 dpa and 45 dpa. Even at the same

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stage, the accumulation of carotenoids in transgenic lines was higher than that in the wild-type (Fig. 3B). Thus,

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SlMBP8 may play an important role in inhibiting biosynthesis of carotenoids during fruit developments. In this study,

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the transcripts of carotenoid biosynthetic genes were detected in the wild-type and transgenic lines by quantitative

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RT-PCR. Compared with the wild-type, the expression levels of phytone desaturase ( PDS ) and carotene desaturase

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( ZDS ) were up-regulated by 15–40% at 38 dpa and 42 dpa in transgenic lines, respectively, whereas, the expression

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levels of

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repressed expression of SlMBP8 may facilitate carotenoid biosynthesis, leading to the darker red fruit observed in

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transgenic plants.

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phytone synthase1 (PSY1) were not significantly different (Fig. 3C, D, E). These results indicate that

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3.3. Ethylene- and ripening-related genes are significantly up-regulated in SlMBP8-silenced fruits The expression of ethylene- and ripening-related genes in fruits was measured to characterize the molecular 10

ACCEPTED MANUSCRIPT regulation mechanism of SlMBP8 in fruit ripening. Two ethylene biosynthesis genes (Centeno et al., 2011), ACO1

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and ACS2, were significantly up-regulated in the B+4 stage of transgenic fruits (Fig. 4A, C). In addition, the

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transcript level of ACO3 increased markedly in the SlMBP8-RNAi fruits at all stages (Fig. 4B). Two ripening-related

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genes (Fraser et al., 1994), E4 and E8, were also significantly up-regulated in the SlMBP8-RNAi fruits at the B+4

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stage (Fig. 4D, E). In addition, two ethylene-responsive genes (Barry and Giovannoni, 2007), ethylene-responsive

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element binding factor 1 (ERF1) and pto-interacting protein (Pti4), were up-regulated approximately 45% at the MG

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stage (Fig. 4F, I). Fig. 4G and H show that the transcripts of two ethylene-inducible lipoxygenase (LOX) genes

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(Lincoln et al., 1987), LOXA and LOXB, were significantly increased in SlMBP8-silenced fruits at the B/B+4 stage.

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All of these results suggested that SlMBP8 might inhibit ripening by directly or indirectly regulating ethylene

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biosynthesis and ethylene -related gene expression.

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3.4. More ethylene is produced by SlMBP8-silenced lines

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Ethylene production during fruit development and ripening was measured. As shown in Fig. 5F, there was a

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rapid increase in ethylene production at the B+4 stage, and more ethylene was produced during fruit ripening in

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transgenic fruits than in wild type and remained at high levels until the B+14 stage. SlMBP8-silenced seedlings

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showed 2-fold increase in ethylene production compared with those of wild type (Fig. 5G).

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The relationship between SlMBP8 and ethylene in non-fruit tissues needs to be further investigated, thus, an

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ethylene triple response assay was performed in our study. Seeds of wild-type and transgenic lines were germinated

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on MS medium fortified with 0 µΜ or 5 µΜ ACC. The length of the hypocotyl of transgenic lines was markedly

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shorter both in the absence (0 µΜ) and presence (5.0 µΜ) of ACC 5 days after sowing, however, the root length in 11

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the presence 5.0µΜ ACC did not show obvious differences (Fig. 5A, B and C). The expression levels of ACS1A, ACS2, ACS6, and ACO1 were measured in transgenic lines (0 µΜ ACC). The

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transcripts of ACS1A and ACO1 were up-regulated dramatically, while the expression of ACS2 and ACS6 was only

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slightly increased in transgenic lines (Fig. 5D), suggesting that the expression of ethylene biosynthesis genes could be

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activated by silencing of SlMBP8. In addition, the expression level of SlMBP8 in WT seedlings decreased

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significantly after the treatment of ACC, and there was a slow declining trend with increasing concentration of ACC

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(Fig. 5E), which indicated that ACC and ethylene could promote the expression of SlMBP8.

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3.6. SlMBP8 interacts with SlMADS-RIN

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We conducted a yeast two-hybrid assay for SlMBP8 and for the ripening regulator protein RIN. The ORF of

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SlMBP8 was amplified and cloned into pGBKT7 as the bait, and the ORF of SlMADS-RIN was amplified and

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cloned into pGADT7 as the prey. Self-activation of the pGBKT7-MBP8 tested negative (Fig. 6). An empty prey and

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bait vector was used as a negative control. The yeast grew and turned blue on QDO/X indicator plates, indicating that

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SlMBP8 could interact with SlMADS-RIN in vivo (Fig. 6).

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3.7. Metabolism of the cell wall is promoted in transgenic lines

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To investigate the regulatory mechanism of SlMBP8 in pericarp development, the RNA expression from a set of

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cell wall-modifying genes was measured in the fruits of transgenic plants and wild type. Among them, six genes,

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polygalacturonase

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endotransglucosylase/hydrolase 5 (XTH5) and xylosidase (XYL), were markedly up-regulated in SlMBP8-silenced

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fruit at the B+4 stage, whereas no obvious change was detected in the expression of polyethylene (PE) and

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mannanase (MAN) (Fig. 7A). The results indicate that SlMBP8 may inhibit the metabolism of cell walls.

(PG),

expansin

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(HEX),

β-galactosidase

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xyloglucan

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3.8. Silencing of SlMBP8 reduces the storability of fruits Fruits of wild-type and transgenic lines were harvested at the B+4 stage and kept at room temperature for 32

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days. After fourteen days, transgenic fruits became soft and dark red, yet wild-type fruits remained hard and lighter in

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colour. After thirty-two days, transgenic fruits were soft, dehydrated and mouldy, while wild-type fruits were just

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begining to soften (Fig. 7B). To test the hypothesis, the weight of the detached fruits was monitored. Fig. 7C shows

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that the transgenic fruits lost 15% of their weight compared with 8% in the wild-type fruits during the first 15 days,

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and only the transgenic fruits had obvious water loss in the following 15 days, concluding that the transgenic fruit

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softened faster than the wild-type fruit.

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Discussion

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Fruit ripening in tomato requires the regulation of ethylene. Although the role of ethylene in mediating

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climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the

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involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato MADS-box

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transcription factor gene SlMBP8 regulates fruit ripening via regulation of ethylene biosynthesis and signalling.

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4.1. SlMBP8 influences carotenoid accumulation during fruit ripening

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It is reported that most ripening-deficient mutant fruits have defects in carotenoid biosynthesis (Barry and

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Giovannoni, 2007). Reduced expression of SlMBP8 resulted in a substantially increased total carotenoid content (Fig.

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3A), which may be attributed to the earlier ripening and darker red colour compared with wild-type fruits. In addition,

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the expression of three genes PSY1, PDS and ZDS, which encode major regulators of metabolic flux towards

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downstream carotenoids, was simultaneously increased in response to reduced SlMBP8 (Fig. 3C, D and E). It has

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been shown that the relative ratio of lycopene and β-carotene in ripening tomato fruit is mediated by up-regulation of 13

ACCEPTED MANUSCRIPT PSY1. Similarly, (Dong et al., 2013) showed that the MADS-box transcription factor MADS1 negatively regulates

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ethylene synthesis, and repression of MADS1 resulted in increased ethylene production and the accumulation of

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carotenoids in ripening fruit. According to these results, we speculate that SlMBP8 functions as a key regulator

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during the carotenoid pathway flux towards β-carotene, possibly through regulation of ethylene synthesis and

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signalling.

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4.2. SlMBP8 inhibits ethylene biosynthesis and fruit ripening

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In our study, shorter ripening time was observed in SlMBP8-RNAi fruits (Fig. 2). Both in the absence and

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presence of ACC, transgenic lines showed shorter hypocotyls compared with wild type, suggesting that the SlMBP8

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negatively modulates the ethylene tiple response (Fig. 5A and B). Nine ACS genes and five ACO genes, which act as

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rate-limiting enzymes in ethylene biosynthesis, have been identified in tomato (Nakatsuka et al., 1998, Sell and Hehl,

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2005). SlACS1A and SlACS6 participate in system 1 and are expressed before the onset of ripening . SlACS2-RNAi

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fruits cannot ripen normally (Oeller et al., 1991). SlACO1 and SlACO3 contribute to the triggering of fruit ripening

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(Barry and Giovannoni, 2007). Therefore, consistent with the phenotypes of shorter ripening time and hypersensitive

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response, the expression levels of ACS2, ACS1A, ACS6, ACO1 and ACO3 in RNAi fruits and seedlings were

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noticeably higher compared to those of the wild type (Fig. 4A, B, C and 5D), and the transgenic fruits produced more

17

ethylene (Fig. 5F). These results indicated that SlMBP8 probably plays an important role in tomato fruit ripening

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through interactions with the promoter region of ethylene biosynthesis genes in the ethylene pathway, but whether

19

these interactions are direct or not remains to be defined. Recently, it was reported that the MADS-box transcription

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factor TAGL1 can bind to the promoter region of ACO1 and ACS2 (Itkin et al., 2009), which provides an effective

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basis that ethylene biosynthesis genes can be directly regulated by MADS-box transcriptional factors in the tomato

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fruit ripening process.

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ACCEPTED MANUSCRIPT E8 and E4 are two important ethylene-responsive genes during fruit ripening. The expression levels of E8 and E4

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were significantly up-regulated in the SlMBP8-RNAi fruits compared to the wild-type fruits (Fig. 4D, E). In addition,

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the transcript levels of two ethylene-responsive genes, ERF1 and Pti4, were markedly induced in SlMBP8-RNAi fruit

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(Fig. 4F, I). LOXA and LOXB, associated with flavour, aroma and nutrition, also increased dramatically (Fig. 4G and

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H). Interestingly, our investigation showed that SlMBP8 expression was much higher in the rin mutant (Fig. 1B), and

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RIN mRNA accumulation was up-regulated in SlMBP8-RNAi fruit (Fig. 4J). Moreover, the interaction between

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SlMBP8 and SlMADS-RIN was verified by the yeast two-hybrid assay (Fig. 6). All of these results suggested that

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SlMBP8 probably binds to SlMADS-RIN and might depress its activity, subsequently regulating the expression of

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ethylene biosynthesis and response genes, reducing the production of ethylene, and ultimately inhibiting fruit

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ripening. Therefore, SlMBP8 adds a new component to the emerging network that regulate tomato fruit ripening.

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However, whether or not other MADS-box domain family members, whose functions are not clear, are involved in

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tomato fruit ripening needs further research.

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4.3. SlMBP8 enhances fruit shelf-life

The gene expression analysis showed that SlMBP8-RNAi fruits exhibited increased abundance of mRNA of

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genes involved in cell wall modification, including PG, EXP, HEX, TBG4, XTH5 and XYL (Fig. 7A), which are

17

associated with cell wall structure and impact fruit softening. In addition to altered cell wall metabolism and the other

18

ripening-related changes described above, we observed that the transgenic postharvest fruits became softened earlier

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and showed faster water loss rates than the wild-type fruits (Fig. 7B, C), indicating that SlMBP8 negatively regulates

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tomato fruit ripening, which results in extended shelf-life.

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By analysing the phenotype and gene expression, it is proved that SlMBP8 is involved in the negative regulation

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of fruit ripening, carotenoid accumulation and cell wall metabolism. In summary, SlMBP8 plays a role in fruit 15

ACCEPTED MANUSCRIPT ripening as a repressive modulator by interacting with SlMADS-RIN and adds a new component to the emerging

2

mechanisms regulating fleshy fruit ripening. However, more information about the developmental regulatory cascade

3

of SlMBP8 needs to be discovered, such as the identification of the direct or indirect targets and the interaction

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relationship of these regulatory factors, which will likely contribute to further mapping of the regulatory network of

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tomato fruit ripening.

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ACCEPTED MANUSCRIPT Abbreviations Used

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ACC, 1-aminocyclopropane-1-carboxylic acid; WT, Wild-type; B, breaker; IMG, immature green; MG, mature green;

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RNAi, RNA interference; RT-PCR, reverse transcription-polymerase chain reaction.

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Acknowledgments

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This work was supported by National Natural Science Foundation of China (no. 31572129), and the Natural Science

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Foundation of Chongqing of China (cstc2015jcyjA80026), and the Fundamental Research Funds for the Central

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Universities (No. 106112015CDJZR235504).

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Conflict of interest

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The authors declare that they have no conflict of interest.

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Author contribution

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G. Chen and Z. Hu designed and managed the research work and improved the manuscript. W. Yin, B. Cui, J. Hu, X.

15

Guo and Z. Zhu performed the experiments. W. Yin wrote the manuscript. All authors read and approved the final

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manuscript.

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ACCEPTED MANUSCRIPT

ADAMS, D. O. & YANG, S. F. 1979. Ethylene Biosynthesis - Identification of 1-Aminocyclopropane-1-Carboxylic Acid as an Intermediate in the Conversion of Methionine to Ethylene. Proceedings of the National Academy of Sciences of the United States of America, 76, 170-174. BARRY, C. S., BLUME, B., BOUZAYEN, M., COOPER, W., HAMILTON, A. J. & GRIERSON, D. 1996. Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato. Plant Journal, 9, 525-535.

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BARRY, C. S. & GIOVANNONI, J. J. 2007. Ethylene and fruit ripening. Journal of Plant Growth Regulation, 26, 143-159. BARRY, C. S., LLOP-TOUS, M. I. & GRIERSON, D. 2000. The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. Plant Physiology, 123, 979-986.

BECKER, A. & EHLERS, K. 2016. Arabidopsis flower development-of protein complexes, targets, and transport. Protoplasma, 253, 219-230. in transgenic plants. Plant Molecular Biology, 47, 311-340.

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BRUMMELL, D. A. & HARPSTER, M. H. 2001. Cell wall metabolism in fruit softening and quality and its manipulation CAUSIER, B., COOK, H. & DAVIES, B. 2003. An antirrhinum ternary complex factor specifically interacts with

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C-function and SEPALLATA-like MADS-box factors. Plant Mol Biol, 52, 1051-62.

CENTENO, D. C., OSORIO, S., NUNES-NESI, A., BERTOLO, A. L., CARNEIRO, R. T., ARAUJO, W. L., STEINHAUSER, M. C., MICHALSKA, J., ROHRMANN, J., GEIGENBERGER, P., OLIVER, S. N., STITT, M., CARRARI, F., ROSE, J. K. & FERNIE, A. R. 2011. Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening. Plant Cell, 23, 162-84.

CHUNG, M. Y., VREBALOV, J., ALBA, R., LEE, J., MCQUINN, R., CHUNG, J. D., KLEIN, P. & GIOVANNONI, J. 2010. A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. Plant

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Journal, 64, 936-947.

DONG, T. T., HU, Z. L., DENG, L., WANG, Y., ZHU, M. K., ZHANG, J. L. & CHEN, G. P. 2013. A Tomato MADS-Box Transcription Factor, SlMADS1, Acts as a Negative Regulator of Fruit Ripening. Plant Physiology, 163, 1026-1036.

EXPOSITO-RODRIGUEZ, M., BORGES, A. A., BORGES-PEREZ, A. & PEREZ, J. A. 2008. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol, 8, 131.

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References

FORTH, D. & PYKE, K. A. 2006. The suffulta mutation in tomato reveals a novel method of plastid replication during fruit ripening. J Exp Bot, 57, 1971-9. FRASER, P. D., TRUESDALE, M. R., BIRD, C. R., SCHUCH, W. & BRAMLEY, P. M. 1994. Carotenoid Biosynthesis during

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1

Tomato Fruit Development (Evidence for Tissue-Specific Gene Expression). Plant Physiol, 105, 405-413. FRAY, R. G. & GRIERSON, D. 1993. Identification and Genetic-Analysis of Normal and Mutant Phytoene Synthase Genes of Tomato by Sequencing, Complementation and Co-Suppression. Plant Mol Biol, 22, 589-602. FUJISAWA, M., NAKANO, T. & ITO, Y. 2011. Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation. BMC Plant Biol, 11, 26. FUJISAWA, M., SHIMA, Y., HIGUCHI, N., NAKANO, T., KOYAMA, Y., KASUMI, T. & ITO, Y. 2012. Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses. Planta, 235, 1107-1122. GIOVANNONI, J. J. 2007. Fruit ripening mutants yield insights into ripening control. Current Opinion in Plant Biology, 10, 283-289. HILEMAN, L. C., SUNDSTROM, J. F., LITT, A., CHEN, M., SHUMBA, T. & IRISH, V. F. 2006. Molecular and phylogenetic analyses of the MADS-box gene family in tomato. Mol Biol Evol, 23, 2245-58. 18

ACCEPTED MANUSCRIPT ITKIN, M., SEYBOLD, H., BREITEL, D., ROGACHEV, I., MEIR, S. & AHARONI, A. 2009. TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network. Plant Journal, 60, 1081-1095. KESANAKURTI, D., KOLATTUKUDY, P. E. & KIRTI, P. B. 2012. Fruit-specific overexpression of wound-induced tap1 under E8 promoter in tomato confers resistance to fungal pathogens at ripening stage. Physiologia Plantarum, 146, 136-148. LINCOLN, J. E., CORDES, S., READ, E. & FISCHER, R. L. 1987. Regulation of Gene-Expression by Ethylene during Lycopersicon-Esculentum (Tomato) Fruit-Development. Proceedings of the National Academy of Sciences of

RI PT

the United States of America, 84, 2793-2797.

LINCOLN, J. E. & FISCHER, R. L. 1988. Diverse Mechanisms for the Regulation of Ethylene-Inducible Gene-Expression. Molecular & General Genetics, 212, 71-75.

LIVAK, K. J. & SCHMITTGEN, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-8.

MONTGOMERY, J., POLLARD, V., DEIKMAN, J. & FISCHER, R. L. 1993. Positive and Negative Regulatory Regions 1049-1062.

SC

Control the Spatial-Distribution of Polygalacturonase Transcription in Tomato Fruit Pericarp. Plant Cell, 5, NAKATSUKA, A., MURACHI, S., OKUNISHI, H., SHIOMI, S., NAKANO, R., KUBO, Y. & INABA, A. 1998. Differential and

internal

feedback

regulation

of

1-aminocyclopropane-1-carboxylate

M AN U

expression

synthase,

1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiol, 118, 1295-305.

OELLER, P. W., LU, M. W., TAYLOR, L. P., PIKE, D. A. & THEOLOGIS, A. 1991. Reversible inhibition of tomato fruit senescence by antisense RNA. Science, 254, 437-9.

SELL, S. & HEHL, R. 2005. A fifth member of the tomato 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene family harbours a leucine zipper and is anaerobically induced. DNA Sequence, 16, 80-82.

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SEYMOUR, G. B., MANNING, K., ERIKSSON, E. M., POPOVICH, A. H. & KING, G. J. 2002. Genetic identification and genomic organization of factors affecting fruit texture. Journal of Experimental Botany, 53, 2065-2071. SHIMA, Y., KITAGAWA, M., FUJISAWA, M., NAKANO, T., KATO, H., KIMBARA, J., KASUMI, T. & ITO, Y. 2013. Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN. Plant Mol Biol, 82, 427-38.

EP

SOMMER, H., BELTRAN, J. P., HUIJSER, P., PAPE, H., LONNIG, W. E., SAEDLER, H. & SCHWARZSOMMER, Z. 1990. Deficiens, a Homeotic Gene Involved in the Control of Flower Morphogenesis in Antirrhinum-Majus - the Protein Shows Homology to Transcription Factors. Embo Journal, 9, 605-613. URBANUS, S. L., DE FOLTER, S., SHCHENNIKOVA, A. V., KAUFMANN, K., IMMINK, R. G. H. & ANGENENT, G. C. 2009. In

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana. Bmc Plant Biology, 9.

VICENTE, A. R., SALADIE, M., ROSE, J. K. C. & LABAVITCH, J. M. 2007. The linkage between cell wall metabolism and fruit softening: looking to the future. Journal of the Science of Food and Agriculture, 87, 1435-1448. VREBALOV, J., RUEZINSKY, D., PADMANABHAN, V., WHITE, R., MEDRANO, D., DRAKE, R., SCHUCH, W. & GIOVANNONI, J. 2002. A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (Rin) locus. Science, 296, 343-346. WILKINSON, J. Q., LANAHAN, M. B., YEN, H. C., GIOVANNONI, J. J. & KLEE, H. J. 1995. An ethylene-inducible component of signal transduction encoded by never-ripe. Science, 270, 1807-9. XIE, Q., HU, Z., ZHU, Z., DONG, T., ZHAO, Z., CUI, B. & CHEN, G. 2014. Overexpression of a novel MADS-box gene SlFYFL delays senescence, fruit ripening and abscission in tomato. Sci Rep, 4, 4367. YANG, S. F. & HOFFMAN, N. E. 1984. Ethylene Biosynthesis and Its Regulation in Higher-Plants. Annual Review of 19

ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7

Plant Physiology and Plant Molecular Biology, 35, 155-189. YANOFSKY, M. F., MA, H., BOWMAN, J. L., DREWS, G. N., FELDMANN, K. A. & MEYEROWITZ, E. M. 1990. The Protein Encoded by the Arabidopsis Homeotic Gene Agamous Resembles Transcription Factors. Nature, 346, 35-39. ZHU, M., CHEN, G., ZHOU, S., TU, Y., WANG, Y., DONG, T. & HU, Z. 2014. A new tomato NAC (NAM/ATAF1/2/CUC2) transcription factor, SlNAC4, functions as a positive regulator of fruit ripening and carotenoid accumulation. Plant Cell Physiol, 55, 119-35.

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ACCEPTED MANUSCRIPT Table 1. Days from anthesis to breaker stage for control

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and SlMBP8-silenced lines

Wild type (WT)

38.0±0.50

RNAi01

34.6±0.46

RNAi05

36.6±0.48

RNAi22

35.4±0.53

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Days

Values represent means ± SD in days for at least 20 fruits of each line.

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Tomato Lines

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS

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Figure 1.

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Expression profile of SlMBP8 in tissues of the cultivar Ailsa Craig (WT) and non-ripening mutant fruits. (A)

4

Expression of SlMBP8 in WT as indicated. Rt, roots; St, stems; Se, sepal of flower in anthesis; Pe, petal; Sta, stamen;

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Ca, carpel; Yl, young leaves; Ml, mature leaves; Sl, senescent leaves; IMG, immature green fruits; MG, mature green

6

fruits; B, breaker-stage fruits; B+4, 4 days after breaker fruits; B+7, 7 days after breaker fruits. (B) Expression of

7

SlMBP8 in WT, Nr and rin fruits. (C) Expression of SlMBP8 in sepals of WT. BPS, sepals of flowers before

8

pollination; IPS, sepals of flowers in pollination; APS, sepals of flowers after pollination. The data represent the

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means of three replicates with three biological repeats. *, indicates P < 0.05 between the wild type and others. Error

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bars indicate SE. CAC was the internal standard gene.

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Figure 2.

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SlMBP8 repression phenotypes. (A) Expression of SlMBP8 in RNAi lines and wild type (WT). RNAs were extracted

14

for the qPCR assay from breaker-stage fruits of RNAi line 01 and the wild type. Three replicates for each sample

15

were performed. (B) Genotypes of fruit ripening in SlMBP8-RNAi lines. The colour of SlMBP8-silenced fruits

16

changed earlier than wild-type fruits. 20 days-50 days, statistical time starting from pollination. CAC was the internal

17

standard gene.

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Figure 3.

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Carotenoid accumulation and expression of PSY1, PDS and ZDS in SlMBP8-silenced and wild-type (WT) fruits. (A)

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Analysis of carotenoid accumulation in 38-dpa, 42-dpa and 45-dpa fruits of transgenic lines and wild type. (B) 22

ACCEPTED MANUSCRIPT Analysis of carotenoid accumulation of transgenic lines and wild-type fruits in the B, B+4 and B+7 stages. (C) to (E)

2

represent expression of PSY1, PDS and ZDS in the MG, B, B+4 and B+7 stages fruits of transgenic lines and wild

3

type. The data represent the means of three technical replicates and three biological replicates. *, indicates P < 0.05

4

between the wild type and others. Error bars indicate SE. CAC was the internal standard gene.

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Figure 4.

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Ripening- and ethylene-related gene expression in SlMBP8-silenced and wild-type (WT) fruits. (A) to (J) represent

8

the expression analysis of ACS2, ACO1, ACO3, E4, E8, ERF1, LOXA, LOXB, RIN and Pti4 in wild-type and

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SlMBP8-silenced fruits of different stages. RNAs were extracted for the qPCR assay from MG, B and B+4 fruits of

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RNAi lines and the wild type.

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(4 days after breaker fruits), 42 dpa. In transgenic lines: MG, 30 dpa; B, 34 dpa; B+4, 38 dpa. The data represent the

12

means of three technical replicates and three biological replicates. *, indicates P < 0.05 between the wild type and

13

others . Error bars indicate SE. CAC was the internal standard gene.

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In wild-type lines: MG (mature green), 35 dpa; B (breaker-stage fruits), 38 dpa; B+4

Figure 5.

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Ethylene production and ethylene triple response assay in WT and SlMBP8-silenced lines. (A) Seedling of wild-type

17

Ailsa Craig and an RNAi line (RNAi 01) treated with 0 or 5.0 µM ACC. (B) to (C) represent elongation of the

18

hypocotyl and root growth in different concentrations of ACC. (D) Expression of ACS1A, ACS2, ACS6 and ACO1 in

19

RNAi 01 and wild type. (E) Expression of SlMBP8 in seedlings of the wild type treated with 0 (A0), 1.0 (A1), 2.0

20

(A2), 5.0 (A5), 10.0 (A10) or 20.0 (A20) µM ACC. (F) Production of ethylene in WT and SlMBP8-silenced fruits.

21

Fresh fruits of B (breaker stage), B+4, B+7 and B+14 were sealed in airtight vials, and 1 mL of gas was sampled from

22

the headspace after 24 h. The values represent the means of at least three individual fruits. WT, wild type. (G)

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ACCEPTED MANUSCRIPT 1

Production of ethylene in WT and SlMBP8-silenced seedlings. *, indicates P < 0.05 between the wild type and others.

2

Error bars represent SE. CAC was the internal standard gene.

3

Figure 6.

5

Yeast two-hybrid assay for SlMBP8 and SlMADS-RIN proteins. SDO, SD medium without Trp; TDO, SD medium

6

without Trp, His, and adenine; QDO, SD medium without Trp, Leu, His, and adenine; QDO/X, SD medium without

7

Trp, Leu, His, and adenine with X-a-Gal. Numbered wedges are as follows: 1, pGBKT7-MBP8 and pGADT7-RIN

8

(interaction of SlMBP8 and SlMADS-RIN); 2, pGBKT7-53 and pGADT7-T (positive control); 3, pGBKT7-Lam and

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pGADT7-T (negative control); 4, pGBKT7-MBP8 (autoactivation assay); 5, pGBKT7 and pGADT7-RIN (empty bait

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vector); 6, pGBKT7-MBP8 and pGADT7 (empty prey vector).

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Figure 7.

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Fruit storability phenotype and expression of related genes in SlMBP8-silenced and wild-type (WT) fruits. (A)

14

Expression profiles of cell wall metabolism genes in the pericarp between transgenic lines and wild type. (B) Fruit

15

storability phenotype of transgenic lines and wild type after harvesting at the B+4 stage. 14 days and 32 days,

16

post-harvest storage time. (C) Water loss in in SlMBP8-silenced and wild-type fruits after harvesting at the B+4 stage.

17

The y-axis depicts the percentage of weight loss beginning on the day of harvesting. The data represent the means of

18

three technical replicates and three biological replicates. *, indicates P < 0.05 between the wild type and others. Error

19

bars indicate SE. CAC was the internal standard gene.

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Figure 7.

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ACCEPTED MANUSCRIPT Highlights 1. Gene SlMBP8 plays an important role in fruit ripening. 2. A yeast two-hybrid assay revealed a clear interaction between SlMBP8 and SlMADS-RIN.

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3. Silencing of SlMBP8 reduces the storability of fruits.

ACCEPTED MANUSCRIPT Author contribution G. Chen and Z. Hu designed and managed the research work and improved the manuscript. W. Yin, B. Cui, J. Hu, X. Guo and Z. Zhu performed the experiments. W. Yin wrote the manuscript.

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All authors read and approved the final manuscript.