Potato Virus X-induced LeHB-1 Silencing Delays Tomato Fruit Ripening

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  • 1 Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China

Tomato (Solanum lycopersicum) fruit ripening is a complex genetic trait correlating with notable fruit phenotypic, physiologic, and biochemical changes. Transcription factors (TFs) play crucial roles during this process. LeHB-1, an HD-zip homeobox protein, is a ripening-related TF and acts as an important regulator of fruit ripening. However, the detailed biochemical and molecular basis of LeHB-1 on tomato fruit ripening is unclear. In the current study, the biologic functions of LeHB-1 were determined by a potato virus X (PVX)-mediated gene-silencing approach. The results indicate that PVX-induced LeHB-1 silencing in tomato could decrease pigment accumulation and delay fruit ripening. Compared with controls, nonripening flesh retains a greater pH value and a lesser anthocyanin content. By evaluating expression levels of genes related to tomato fruit ripening, we inferred that LeHB-1 located at the downstream of LeMADS-RIN-mediated regulatory network. In addition, LeHB-1 silencing mainly disturbed phytoene desaturation and isomerization, and led to a decrease in trans-lycopene accumulation, but did not influence flavonoid biosynthesis directly in tomato fruit. The findings provide a theoretical foundation for illustrating the biologic functions of LeHB-1 in tomato fruit ripening and quality.

Abstract

Tomato (Solanum lycopersicum) fruit ripening is a complex genetic trait correlating with notable fruit phenotypic, physiologic, and biochemical changes. Transcription factors (TFs) play crucial roles during this process. LeHB-1, an HD-zip homeobox protein, is a ripening-related TF and acts as an important regulator of fruit ripening. However, the detailed biochemical and molecular basis of LeHB-1 on tomato fruit ripening is unclear. In the current study, the biologic functions of LeHB-1 were determined by a potato virus X (PVX)-mediated gene-silencing approach. The results indicate that PVX-induced LeHB-1 silencing in tomato could decrease pigment accumulation and delay fruit ripening. Compared with controls, nonripening flesh retains a greater pH value and a lesser anthocyanin content. By evaluating expression levels of genes related to tomato fruit ripening, we inferred that LeHB-1 located at the downstream of LeMADS-RIN-mediated regulatory network. In addition, LeHB-1 silencing mainly disturbed phytoene desaturation and isomerization, and led to a decrease in trans-lycopene accumulation, but did not influence flavonoid biosynthesis directly in tomato fruit. The findings provide a theoretical foundation for illustrating the biologic functions of LeHB-1 in tomato fruit ripening and quality.

Fruit ripening is an important stage for plants bearing fleshy fruit. During the process of ripening, there are noticeable changes in biophysical and biochemical attributes—such as pigmentation, flesh texture, aroma, and nutrient components—that determine the quality and commodity of fruit (Gapper et al., 2013). Currently, tomato has been an excellent model system for fruit ripening studies as a result of their ease of cultivation, short life cycle, dramatic ripening process, various ripening mutants, efficient gene editing approaches, and fully sequenced genome (Kudo et al., 2017; Tomato Genome Consortium, 2012). Accumulating evidence indicates that the internal determinants of regulating tomato fruit ripening include phytohormone signaling pathways, epigenetic changes, and TF networks (Shinozaki et al., 2018).

As a typical climacteric fruit, perception and signal transduction of ethylene in tomato has been well characterized (Bapat et al., 2010; Cara and Giovannoni, 2008; Wang et al., 2016). Also, it has been well accepted that abscisic acid and indole-3-acetic acid play crucial roles in controlling fruit ripening by a complicated network of feedback and crosstalk among phytohormones (McAtee et al., 2013; Mou et al., 2016; Weng et al., 2015). Some reports suggest that epigenetic modification plays important roles in fruit-ripening progress (Kanazawa et al., 2011; Kasai and Harada, 2015; Manning, et al., 2006; Zhong et al., 2013). Liu et al. (2015) defined a direct cause-and-effect relationship between active DNA demethylation and induction of gene expression to explain the correlation between genomic DNA demethylation and fruit ripening.

In addition, a wide range of studies in fruit-specific transcriptional control of ripening in tomato has received considerable attention (Rohrmann et al., 2012). Several TFs, such as LeMADS-RIN, SlFUL1/SlFUL2, SlTAGL1, SlMADS1, SlAP2a, and SlNAC4, have been shown to play central regulatory roles by both ethylene-dependent and -independent pathways. Mutations of these TF genes result in phenotypes with changes in all aspects of fruit ripening, including pigment biosynthesis, softening, size and shape, chloroplast degradation, and chromoplast development (Dong et al., 2013; Fujisawa et al., 2013, 2014; Karlova et al., 2011; Vrebalov et al., 2009; Zhu et al., 2014). In intricate transcriptional regulatory networks, LeHB-1 (GenBank accession no. CAP16664) encodes a 285-aa protein with a conserved homeobox domain (aa 49–125) and a leucine zipper (aa 121–163) (Tomato Genome Consortium, 2012). The protein with a helix-loop-helix-turn-helix structure can bind the promoter of a 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene LeACO1. LeHB-1 silencing can result in a reduced LeACO1 transcript, and thus a considerable delay in tomato fruit ripening. In addition, ectopic overexpression of LeHB-1 in developing tomato plants can produce multiple flowers within on sepal whorl, which leads to fusion of sepals and petals, and convert sepals into fruit (Lin et al., 2008). However, as a key TF, the detailed biochemical and molecular basis of LeHB-1 on tomato fruit ripening is unclear. Effects of LeHB-1 on the biosynthesis of ripening-associated secondary metabolites are also rarely reported.

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for the study of gene function in plants. Upon viral infection, plants use their innate RNAi-mediated defense machinery to target the viral genome explicitly (Senthil-Kumar and Mysore, 2011). PVX, a single-strand RNA virus, is a member of the potexvirus group (Lico et al., 2006). The PVX-mediated VIGS has been used successfully for gene function analysis in tomato fruit (Chen et al., 2015; Lin et al., 2008; Manning et al., 2006; Zhou et al., 2012). Although PVX-mediated VIGS cannot provide uniform efficiency and a stable effect, its advantages are obvious: 1) for keeping away from constructing transgenic lines, PVX-mediated VIGS in tomato fruit is efficient and rapid; 2) the induced ripening-related phenotype is distinct and easily identifiable; 3) it can largely simulate targeted gene mutant in transcriptional level; 4) the intensity of PVX-mediated VIGS was various which can be used to assess the function of genes whose mutation is lethal; and 5) partial sequencing information of ripening-related genes is sufficient to silence genes with multiple copies or multiple family members.

In the current study, PVX-induced LeHB-1 silencing was performed in tomato fruit. Physiologic and biochemical attributes of ripening and nonripening sectors in inoculated fruit were determined first. Subsequently, the expression changes of ripening-associated genes were assessed. Research findings indicated that LeHB-1 silencing led to a decrease in pigment accumulation and a delay of fruit ripening. In addition, LeHB-1 could locate at the downstream of LeMADS-RIN-mediated regulatory network and mainly influence carotenoid biosynthesis. These results will provide useful information to explore the functional mechanism of LeHB-1.

Materials and Methods

Plant materials and growth conditions.

Tomato cultivar Ailsa Craig (AC) and tobacco (Nicotiana benthamiana) were cultured normally in a growth chamber at 25 °C with a photoperiod of 16/8 h day/night. Flowers were tagged at anthesis. Developmental and ripening stages of fruit were recorded as days postanthesis (DPA) and days postbreaker (DPB).

Detection of LeHB-1 expression.

Total RNA was extracted from roots, stems, leaves, and fruit (15 DPA, 30 DPA; 5 DPB, 10 DPB) using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand complementary DNA (cDNA) was produced using the Fast Quant RT Kit (Tiangen, Beijing, China) according to the product manual. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using 2× Ultra SYBR mixture (CWBIO, Beijing, China) in a PCR detection system (CFX96; Bio-Rad Laboratories, Hercules, CA). The PCR program was as follows: 95 °C (10 min), followed by 40 cycles at 95 °C for 15 s, 58 °C for 15 s, and 72 °C for 20 s. The intensity change in SYBR Green fluorescence and the threshold cycle (Ct) over the background was calculated for each reaction. Samples were normalized using 18S rRNA, and the relative expression levels were measured using the 2(–ΔCt) analysis method.

Construction of PVX/mLeHB-1 vector.

A nonsense mutant LeHB-1 gene (mLeHB-1) was designed in which a start codon was replaced by a stop codon. mLeHB-1 was amplified using DNA polymerase (PrimeSTAR; Takara, Kusatsu, Japan) from fruit cDNA (15 DPA) in a thermal cycler (S1000; Bio-Rad Laboratories). The PCR products were digested by restriction enzymes Cla I and Eag I (New England BioLabs, Ipswich, MA) and cloned into the PVX vector to generate PVX/mLeHB-1. The construct was confirmed by nucleotide sequencing using a pair of PVX sequencing primers. The sequence of primer pairs for vector construction is shown in Table 1.

Table 1.

Primer pairs used for vector construction and gene expression analysis.

Table 1.

In vitro transcription and virus-induced LeHB-1 silencing.

The PVX or PVX/mLeHB-1 vector was linearized using the restriction enzyme Spe I (New England BioLabs), purified with a High Pure PCR Product Purification Kit (Roche, Rotkreuz, Switzerland), and then suspended in RNase-free water with 1 unit·μL–1 RNasin Ribonuclease Inhibitor (Promega, Fitchburg, WI). The Riboprobe In Vitro Transcription System (Promega) was used to synthesize single-strand PVX or PVX/mLeHB-1 RNA transcripts. Afterward, RNA transcripts were inoculated mechanically on young leaves of N. benthamiana. After 10 d of inoculation, systemically infected leaves were collected and freeze-dried in a freeze-drying machine (FreeZone 2.5 Plus; Labconco, Kansas City, MO). Subsequently, ≈0.1 g leaf tissue was ground in 2 mL TE buffer (pH 7.5) and needle-injected into the carpopodium of immature tomato fruit (about 15 DPA). At 5 DPB, fruit with obvious nonripening sectors were photographed, harvested, and stored at –80 °C. The total RNA extraction and first-strand cDNA synthesis of the red zone and green zone of harvested fruit were described as above. The transcripts of PVX/mLeHB-1 were validated by PCR amplification using a pair of PVX sequencing primers and electrophoresis analysis on a 1% agarose gel (Fig. 1C).

Fig. 1.
Fig. 1.

(A) Developmental expression of LeHB-1 in tomato cultivar Ailsa Craig and (B–D) construction of potato virus X (PVX)/mLeHB-1 vector. Fruit development and ripening stages were recorded as days postanthesis (DPA) and days postbreaker (DPB). (B) Analysis of linearized plasmids and in vitro transcription RNA products for PVX and PVX/mLeHB-1. (C) Detection of PVX/mLeHB-1 in tomato fruit at 5 DPB with a biologic repeat by reverse transcription–polymerase chain reaction (RT-PCR) assays. An equal amount of total RNA was used for RT-PCR. PVX/mLeHB-1G and PVX/mLeHB-1R, respectively, represent green zone and red zone in injected tomato fruit. (D) The phenotype of Nicotiana benthamiana inoculated with TE buffer, PVX, or PVX/mLeHB-1 through mechanical inoculation. Photographs were taken at 10 d postinoculation. Analysis of variance was used to compare more than two means, and Duncan’s multiple range tests were used for means separations. Bars represent sd of the means. Lowercase letters indicate significant differences at P < 0.05.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04497-18

Determination of physiologic and biochemical parameters.

Soluble solids content was determined using a refractometer (LB20T; Suwei, Shenzhen, China). The pH value was evaluated using a pH meter (3520; Jenway, Stone, UK) in 15 mL diluted juice from 5 g fruit flesh. Anthocyanin was extracted using the method reported by Zhang et al. (2014). Briefly, the sample (5 g) was ground in liquid nitrogen and diluted with methanol supplemented with 0.1 mol·L–1 hydrochloric acid to 30 mL. The suspensions were placed in the dark for 24 h and centrifuged to remove the precipitate. Subsequently, absorbance of the supernatant was measured using a spectrophotometer (SmartSprec Plus, Bio-Rad Laboratories) at 525 nm, and the relative content of anthocyanin was represented by absorbance per gram fresh weight. Chlorophyll content was evaluated as described by Wang et al. (2009). The 5-g sample was ground in liquid nitrogen and diluted with 80% acetone to 30 mL. After adequate mixing and centrifugation, the acetone phase was collected, and absorbance at 645 nm (OD645) and at 663 nm (OD663) were measured for determination of chlorophyll a and chlorophyll b. Chlorophyll content was calculated using the formula Total chlorophyll content = 8.33 × (8.02 × OD663 + 20.20 × OD645), measured in micrograms per gram.

qRT-PCR analysis.

Total RNA extraction and first-strand cDNA synthesis of samples (AC fruit at 30 DPA, AC fruit at 5DPB, the red zone and green zone of AC fruit injected with PVX/mLeHB-1 at 5 DPB) were described as above. Expression of genes involved in ripening-associated TFs, ethylene biosynthesis, and carotenoid and flavonoid biosynthesis was determined by qRT-PCR using specific primer pairs as described by Lai et al. (2015), Pandey et al. (2015), and Su et al. (2015). The sequence of all primer pairs for qRT-PCR analysis is shown in Table 1.

Statistical analysis.

Data were collected from at least three independent experiments, and statistical analyses were performed using SPSS (version 20.0; IBM Corp., Armonk, NY). Analysis of variance was used to compare more than two means. Mean separations were analyzed using Duncan’s multiple range test. Differences at P < 0.05 were considered to be significant.

Results

Expression profile of LeHB-1.

LeHB-1 can be detected in roots, stems, leaves, flowers, and fruit at different development stages. Of these plant tissues, it has the greatest expressional level in flowers followed by leaves and stems, and little amount in roots. In fruit, the expression level of LeHB-1 decreased gradually from immature green stage to mature green stage and was followed by a rapid decline at the breaker stage and red ripening stage. The LeHB-1 transcripts were maintained at a relatively lower level during the fully ripening stage (Fig. 1A).

Construction and ectopic expression of PVX/mLeHB-1.

The coding region for mLeHB-1 was amplified and cloned into the PVX-based vector. RNA transcripts were produced by in vitro transcription (Fig. 1B) and inoculated onto the leaves of N. benthamiana. About 10 d after inoculation, virus-inoculated leaves developed obvious chlorotic lesions. In addition, curling and mosaic symptoms were observed on systemically infected leaves (Fig. 1D). The phenotype was consistent with typical symptoms of wild-type PVX infection (Lico et al., 2006). After reverse transcription–polymerase chain reaction (RT-PCR) detection and direct sequencing, the recombinant viral RNAs containing a complete mLeHB-1 sequence were confirmed in infected N. benthamiana leaves.

PVX-induced LeHB-1 silencing.

Immature green fruit were needle-injected with leaf sap containing PVX/empty or PVX/mLeHB-1 RNA transcripts. Under standard greenhouse condition, development of control fruit and inoculated fruit all proceeded normally. About 38 DPA, almost all fruit showed a break in color from green to tannish yellow, pink, or red on no more than 10% of the tomato fruit surface. At 5 DPB, more than 95% of the surface shown red color in control fruit and fruit inoculated with PVX/empty RNA transcripts. However, about 10% of the fruit inoculated with PVX/mLeHB-1 RNA transcripts shown obvious green or orange sectors that occupied about 20% to 45% of the fruit surface. The maturity of the sarcocarp was heterogeneous as well, as observed in cross-section (Fig. 2). The effectiveness of gene silencing was analyzed at the transcript level by monitoring LeHB-1 messenger RNA accumulation. The expression of LeHB-1 in green sectors was downregulated about 30% to 50% than that in red sectors.

Fig. 2.
Fig. 2.

Ripening phenotypes of (A) wild-type tomato cultivar Ailsa Craig (AC) fruit, (B) AC fruit injected with potato virus X (PVX), and (C, D) AC fruit injected with PVX/mLeHB-1 5 d after breaker. Fruit are bidissected to show internal ripe and/or nonripening tissues.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04497-18

Fig. 3.
Fig. 3.

Effect of potato virus X (PVX)-induced LeHB-1 silencing on (A) soluble solids content, (B) pH, (C) anthocyanin content, and (D) chlorophyll content in tomato cultivar Ailsa Craig (AC) fruit at 5 d postbreaker. mLeHB-1R and mLeHB-1G represent the red zone and green zone, respectively, of AC fruit injected with PVX/mLeHB-1. Analysis of variance was used to compare more than two means, and Duncan’s multiple range tests were used for means separations. Lowercase letters indicate significant differences at P < 0.05; bars represent sd.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04497-18

Fig. 4.
Fig. 4.

Expression of genes related to (A) ripening-associated transcription factors, (B) ethylene biosynthesis, (C) carotenoid biosynthesis, and (D) flavonoid biosynthesis in tomato cultivar Ailsa Craig (AC) fruit injected with potato virus X (PVX)/mLeHB-1. The 5 DPB and 30 DPA represent fruit at 5 d postbreaker and fruit at 30 d postanthesis, respectively. mLeHB-1R and mLeHB-1G represent the red zone and green zone of AC fruit, respectively, injected with PVX/mLeHB-1 at 5 DPB. Analysis of variance was used to compare more than two means, and Duncan’s multiple range tests were used for means separations. Different lowercase letters represent significant differences at P < 0.05 in the expression of indicated genes among four groups; bars represent sd. ACS, 1-aminocyclopropane-1-carboxylate (ACC) synthase; ACO, ACC oxidase; PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, zeta-carotene isomerase; ZDS, zeta-carotene desaturase; CRTISO, carotenoid isomerase; β-CYC, β-cyclase; β-LCY, lycopene β-cyclase; ε-LCY, lycopene ε-cyclase; β-CRTR, β-carotene hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavanol 4-reductase; ANS, anthocyanidine synthase; GT, glucosyltransferase; RT, rhamnosyltransferase.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04497-18

Effects of PVX-mediated LeHB-1 silencing on fruit quality parameters.

Generally, control fruit and fruit inoculated with PVX/empty RNA transcripts possessed similar ripening attributes, which indicates that injection of PVX/empty has no significant influence on fruit ripening. Through PVX-mediated LeHB-1 silencing, normal ripening (red sector) and ripening delay (green sector) could be inspected visually in one fruit. Soluble solids and anthocyanin contents of the red and green sectors were almost same (Fig. 3A and C). However, pH and chlorophyll content in green sectors were significantly greater than those in the red sectors (Fig. 3B and D). These results mean that PVX-induced LeHB-1 silencing was effective and that fruit color correlates positively with physiologic and biochemical changes of fruit during ripening.

Effects of PVX-mediated LeHB-1 silencing on expression of genes related to fruit ripening.

For most examined genes, their differential expression trends between the green zone and the red zone of LeHB-1 silenced fruit were similar with those between fruit at 30 DPA and fruit at 5 DPB. For other genes, different or contradictory results may be the result of the different silencing efficiency of LeHB-1 and individual kinetic trends of genes in developing fruit (Fig. 4).

TFs are required for the initiation and promotion of fruit ripening. In LeHB-1 silenced fruit, expression levels of SlMADS1, SlMYB12, and SlAP2a were greater in the green sectors than in the red. No statistically significant changes were observed in the expression levels of SlTAGL1, LeMADS-RIN, LeTDR4, LeSPL-CNR, and LeNAC-NOR (Fig. 4A).

As a typical respiration climacteric fruit, ethylene biosynthesis is an important parameter for tomato fruit ripening. Comparing red sectors, the expression levels of LeACS1, LeACS4, LeACO1, and LeACO3 increased significantly; the expressions of LeACS6 decreased significantly in the green sectors of LeHB-1 silenced fruit. No statistically significant changes were observed in the expression levels of LeACS2, LeACS3, LeACO2, and LeACO4 (Fig. 4B).

The color change from green to red is a very important indicator of tomato ripening and can be measured easily by chromametry. This change is associated with the degradation of chlorophylls and the composition of carotenoid. The levels of transcripts coding for carotenoid biosynthesis enzymes were monitored in LeHB-1 silenced fruit. Results indicate that, compared with the red sectors, the expression of SlZISO, SlZDS, CRTISO, and β-CRTR was downregulated in the green sectors. The expression of other genes related to carotenoid biosynthesis in the current study were not influenced by LeHB-1 silencing (Fig. 4C).

The flavonoid group as an important secondary metabolite plays crucial roles in the tomato development and ripening process. The expressions of genes involved in flavonoid synthesis in LeHB-1 silenced fruit was determined as well. The data indicate that almost all genes showed greater expression levels in green sectors than in red (Fig. 4D).

Discussion

In this study, PVX-mediated LeHB-1 silencing was performed to estimate the function of LeHB-1 on tomato fruit ripening. When the PVX vector that harbored a host-derived target gene sequence (mLeHB-1) infected immature fruit, a double-strand RNA copy of the insert was produced and induced the activation of the host plant’s RNA silencing pathway, resulting in the subsequent degradation of the endogenous LeHB-1 transcript (Becker and Lange, 2010; Senthil-Kumar and Mysore, 2011). Hence, fruit failed to ripen normally and generated a distinct green or orange sector. Compared with mature parts, the nonripening parts had a greater pH value and chlorophyll content, which corresponds to the immature phenotype. The soluble solids content and anthocyanin content were not influenced by LeHB-1 silencing.

Transcriptional profiling studies indicate that amounts of TFs show expression alterations during fruit development and ripening consistent with functions (Arhondakis et al., 2016). Mutations of TF genes, including LeMADS-RIN, LeSPL-CNR, and LeNAC-NOR, can abolish the normal ripening process. In addition, SlTAGL1, TDR4, SlAP2a, SlMADS1, and SlMYB12 have been found to play significant roles in tomato fruit ripening. Among them, LeMADS-RIN was considered to be a master regulator because its target genes are involved in ethylene synthesis and signaling, cell wall modification, carotenoid accumulation, aroma formation, and transcriptional regulation of ripening-related TF genes (Fujisawa et al., 2013). Meanwhile, LeMADS-RIN, SlFUL1/SlFUL2, and SlTAGL1 could form a higher order tetramer that is mainly responsible for ripening regulation (Fujisawa et al., 2014; Shima et al., 2013). Several studies using a chromatin immunoprecipitating approach revealed that LeMADS-RIN interacted directly with the promoters of SlAP2a, LeTDR4, LeNAC-NOR, LeSPL-CNR, and LeHB-1 (Fujisawa et al., 2012, 2013; Ito et al., 2008; Qin et al., 2012). LeHB-1 was the only reported negatively regulated target (Martel et al., 2011). In the current study, expression levels of these TFs were not influenced in LeHB-1 silenced fruit, which is consistent with reports that the promoter of LeHB-1 was the target of LeMADS-RIN, and also indicates that LeHB-1 is located downstream from the LeMADS-RIN-mediated regulatory network. SlMADS1 belongs to the MADS-box gene family and plays an important role in fruit ripening as a repressive modulator (Dong et al., 2013). Its transcripts decreased significantly in accordance with fruit ripening. As a result of ripening delay in the LeHB-1 silencing sector, the greater expression level of SlMADS1 in green sectors was reasonable.

In climacteric fruit, ethylene is present throughout the various development stages and is essential for the onset and completion of the ripening process (Bapat et al., 2010). Two systems of ethylene biosynthesis have been studied extensively in plants. System 1, with an autoinhibitory manner, represents basal ethylene in vegetable tissues and immature fruit. System 2, with an autocatalytic manner, stands for a significant increase in ethylene production involved in flower senescence and fruit ripening. The main rate-limiting step in the ethylene biosynthetic pathway is the production of ACC. The reaction is catalyzed by ACC synthase (ACS), which is followed by the conversion of ACC to ethylene by ACC oxidase (ACO). System 2 regulated mainly by LeACS2, LeACS4, and LeACS6. LeACS2 and LeACS4 are controlled in a positive feedback manner, whereas LeACS6 is regulated by a negative feedback system. Expression of LeACS2 and LeACS4 peak and LeACS6 sustains a lower level at around breaker stage. Five genes encoding the ACO enzyme have been defined in tomato and three of them—LeACO1, LeACO3, and LeACO4—have been shown to be expressed differentially in fruit. The expression levels of LeACO1 and LeACO4 increase dramatically at the onset of climacteric burst and ripening. The expression of LeACO3 is reduced but transitory at the breaker stage (Cara and Giovannoni, 2008). In the current study, the expression of LeACS2, LeACS4, LeACO1, LeACO3, and LeACO4 is upregulated in the green sector, whereas the expression of LeACS6 is downregulated. Compared with the red sector, the green sector was at around the breaker stage, and the respiration climacteric in the green sector was only beginning. The ripening of tomato was delayed considerably by mLeHB-1 silencing.

The red color of ripening tomato fruit is mainly a result of carotenoid (in flesh) and flavonoid (in peel) pigments (Ballester et al., 2010). The carotenoid biosynthetic pathway in tomato has been well described by Giuliano (2014). Briefly, two molecules of geranylgeranyl diphosphate are first condensed to 15-cis-phytoene by phytoene synthases. The 15-cis-phytoene is then transformed into all-trans-lycopene through a series of reactions of phytoene desaturase (PDS), zeta-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), and zeta-carotene isomerase (ZISO). Subsequently, the conversion of lycopene to γ-carotene and δ-carotene are catalyzed by lycopene β-cyclases (β-CYC) and ε-cyclase (ε-LCY). The β-carotene and α-carotene are further synthesized by β-CYC. Finally, the carotenes are transformed into violaxanthin and lutein by β-carotene hydroxylases (β-CRTR) (Su et al., 2015). In this study, expressional intensities of PSY1, PDS, ε-LCY, β-LCY, and β-CYC had no significant differences between the red and green sectors of tomato fruit. However, the expression of ZISO, ZDS, and CRTISO was downregulated in nonripening zones. This indicates the desaturation and isomerization of phytoene were disturbed by LeHB-1 silencing, which then led to a decrease in all-trans-lycopene accumulation.

Besides carotenoids, flavonoids are essential for determining the color of tomato fruit peel (Ferreyra et al., 2012). They are rarely accumulated in the fruit flesh because of a lack of expression of biosynthetic genes. The first committed step of flavonoid biosynthesis is the condensation of one molecule of coumaroyl CoA and three molecules of malonyl CoA to form naringenin–chalcone in a reaction catalyzed by chalcone synthase. Naringenin is then generated by chalcone isomerase. Subsequently, naringenin is hydroxylated to the precursor of delphinidin, pelargonidin, or cyanidin by flavonoid 3-hydroxylase, flavonoid 3′-hydroxylase, and/or flavonoid 3′5′-hydroxylase (F3′5′H). Finally, under catalyzing of dihydroflavonol reductase and anthocyanidine synthase, the anthocyanin pigments are produced; under catalyzing of flavonol synthase, myricetin, kaempferol, and quercetin are produced. In addition, quercetin can be transformed to rutin in a series of actions catalyzed by glucosyltransferase and rhamnosyltransferase (Koes et al., 2005). The genes of these key enzymes almost show the same pattern of expression in tomato peel. Levels of transcripts increase with fruit development, peak at the breaker or turning stage, and decline gradually in red-stage fruit (Pandey et al., 2015). In the current study, all the examined genes related to flvonoid biosynthesis were upregulated in the green sector 5 DPB, which is similar to the expression trend of TF gene LeMYB12. The result is consistent with a report that LeMYB12 plays an important role in regulating the flavonoid pathway (Ballester et al., 2010). The upregulation of primary genes in flavonoid biosynthesis pathway may be due to the delayed respiratory climacteric induced by LeHB-1 silencing. Therefore, we speculate that LeHB-1 does not affect flavonoid biosynthesis in a direct way.

In conclusion, PVX-induced LeHB-1 silencing in tomato can decrease pigment accumulation and delay fruit ripening. Through qRT-PCR analysis, we inferred that LeHB-1 is located downstream from the LeMADS-RIN-mediated regulatory network and it did not influence flavonoid biosynthesis directly. In addition, LeHB-1 can influence carotenoid biosynthesis by regulating the desaturation and isomerization of phytoene. Further exploration is needed to illuminate the exact mechanism of LeHB-1 in regulating tomato fruit ripening.

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  • Fujisawa, M., Nakano, T., Shima, Y. & Yasuhiro, I. 2013 A larger-scale identification of direct targets of the tomato MADS Box transcription factor ripening inhibitor reveals the regulation of fruit ripening Plant Cell 25 371 386

    • Search Google Scholar
    • Export Citation
  • 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 6 1107 1122

    • Search Google Scholar
    • Export Citation
  • Fujisawa, M., Shima, Y., Nakagawa, H., Kitagawa, M., Kimbara, J., Nakano, T., Kasumi, T. & Ito, Y. 2014 Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS Box proteins Plant Cell 26 89 101

    • Search Google Scholar
    • Export Citation
  • Gapper, N.E., McQuinn, R.P. & Giovannoni, J.J. 2013 Molecular and genetic regulation of fruit ripening Plant Mol. Biol. 82 575 591

  • Giuliano, G. 2014 Plant carotenoids: Genomics meets multi-gen engineering Curr. Opin. Plant Biol. 19 111 117

  • Ito, Y., Kitagawa, M., Ihashi, N., Yabe, K., Kimbara, J., Yasuda, J., Ito, H., Inakuma, T., Hiroi, S. & Kasumi, T. 2008 DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN Plant J. 55 212 223

    • Search Google Scholar
    • Export Citation
  • Kanazawa, A., Inaba, J., Shimura, H., Otagaki, S., Tsukahara, S., Matsuzawa, A., Kim, B.M., Goto, K. & Masuta, C. 2011 Virus-mediated efficient of epigenetic modifications of endogenous genes with phenotypic changes in plants Plant J. 65 156 168

    • Search Google Scholar
    • Export Citation
  • Karlova, R., Rosin, F.M., Busscher-Lange, J., Parapunova, V., Do, P.T., Fernie, A.R., Fraser, P.D., Baxter, C., Angenent, G.C. & de Maagd, R.A. 2011 Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening Plant Cell 23 923 941

    • Search Google Scholar
    • Export Citation
  • Kasai, A. & Harada, T. 2015 Epimutant induction as a new plant breeding technology Jpn. Agr. Res. Qrtly. 49 301 305

  • Koes, R., Verweij, W. & Quattrocchio, F. 2005 Flavonoids: A colorful model for the regulation and evolution of biochemical pathways Trends Plant Sci. 10 236 242

    • Search Google Scholar
    • Export Citation
  • Kudo, T., Kobayashi, M., Terashima, S., Katayama, M., Ozaki, S., Kanno, M., Saito, M., Yokoyama, K., Ohyanagi, H., Aoki, K., Kubo, Y. & Yano, K. 2017 Tomatomics: A web database for integrated omics information in tomato Plant Cell Physiol. 58 1 12

    • Search Google Scholar
    • Export Citation
  • Lai, T.F., Wang, Y., Zhou, T., Mei, F.L., Zhang, P.C., Zhou, Y.Y., Shi, N.N. & Hong, Y.G. 2015 Virus-induced LeSPL-CNR silencing inhibits fruit ripening in tomato J. Agr. Sci. 7 184 195

    • Search Google Scholar
    • Export Citation
  • Lico, C., Capuano, F., Renzone, G., Donini, M., Marusic, C., Scaloni, A. & Baschieri, S. 2006 Peptide display on potato virus X: Molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles J. Gen. Virol. 87 3103 3112

    • Search Google Scholar
    • Export Citation
  • Lin, Z.F., Hong, Y.G. & Yin, M.G. 2008 A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening Plant J. 55 301 310

    • Search Google Scholar
    • Export Citation
  • Liu, R.E., How-Kit, A., Stammitti, L., Teyssier, E., Rolin, D., Mortain-Bertrand, A., Halle, S., Liu, M.C., Kong, J.H., Wu, C.Q., Degraeve-Guibault, C., Chapman, N.H., Maucourt, M., Hodgman, T.C., Tost, J., Bouzayen, M., Hong, Y.G., Seymour, G.B., Giovannoni, J.J. & Gallusci, P. 2015 A DEMETER-like DNA demethylase governs tomato fruit ripening Proc. Natl. Acad. Sci. USA 112 10804 10809

    • Search Google Scholar
    • Export Citation
  • Manning, K., Tör, M., Poole, M., Hong, Y.G., Thompson, A.J., King, G.J., Giovannoni, J.J. & Seymour, G.B. 2006 A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening Nat. Genet. 38 948 952

    • Search Google Scholar
    • Export Citation
  • Martel, C., Vrebalov, J., Tfelmeyer, P. & Giovannoni, J.J. 2011 The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPRNING-dependent manner Plant Physiol. 157 1568 1579

    • Search Google Scholar
    • Export Citation
  • McAtee, P., Karim, S., Schaffer, R. & David, K. 2013 A dynamic interplay between phytohormones is required for fruit development, maturation, and ripening Front. Plant Sci. 4 79

    • Search Google Scholar
    • Export Citation
  • Mou, W.S., Li, D.D., Bu, J.W., Jiang, Y.Y., Khan, Z.U., Luo, Z.S., Mao, L.C. & Ying, T.J. 2016 Comprehensive analysis of ABA effects on ethylene biosynthesis and signaling during tomato fruit ripening PLoS One 11 e0154072

    • Search Google Scholar
    • Export Citation
  • Pandey, A., Misra, P., Choudhary, D., Yadav, R., Goel, R., Bhambhani, S., Sanyal, I., Trivedi, R. & Trivedi, P.K. 2015 AtMYB12 expression in tomato leads to large scale differential modulation in transcriptome and flavonoid content in leaf and fruit tissues Scientific Rpt. 5 12412

    • Search Google Scholar
    • Export Citation
  • Qin, G.Z., Wang, Y.Y., Cao, B.H., Wang, W.H. & Tian, S.P. 2012 Unraveling the regulatory network of the MADS box transcription factor RIN in fruit ripening Plant J. 70 243 255

    • Search Google Scholar
    • Export Citation
  • Rohrmann, J., McQuinn, R., Giovannoni, J.J., Fernie, A.R. & Tohge, T. 2012 Tissue specificity and differential expression of transcription factors in tomato provide hints of unique regulatory networks during fruit ripening Plant Signal. Behav. 7 1639 1647

    • Search Google Scholar
    • Export Citation
  • Senthil-Kumar, M. & Mysore, K.S. 2011 New dimension for VIGS in plant functional genomics Trends Plant Sci. 16 656 665

  • 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. 82 427 438

    • Search Google Scholar
    • Export Citation
  • Shinozaki, Y., Nicolas, P., Fernandez-Pozo, N., Ma, Q., Evanich, D.J., Shi, Y., Xu, Y., Zheng, Y., Snyder, S.I., Martin, L.B.B., Ruiz-May, E., Thannhauser, T.W., Chen, K., Domozych, D.S., Catalá, C., Fei, Z., Mueller, L.A., Giovannoni, J.J. & Rose, J.K.C. 2018 High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening Nat. Commun. 9 364

    • Search Google Scholar
    • Export Citation
  • Su, L., Diretto, G., Purgatto, E., Danoun, S., Zouine, M., Li, Z., Roustan, J., Bouzayen, M., Giuliano, G. & Chervin, C. 2015 Carotenoid accumulation during tomato fruit ripening is modulated by the auxin–ethylene balance BMC Plant Biol. 15 114

    • Search Google Scholar
    • Export Citation
  • Tomato Genome Consortium 2012 The tomato genome sequence provides insights into fleshy fruit evolution Nature 485 635 641

  • Vrebalov, J., Pan, I.L., Arroyo, A.J.M., McQuinn, R., Chung, M., Poole, M., Rose, J., Seymour, G., Grandillo, S., Giovannoni, J. & Irish, V.F. 2009 Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1 Plant Cell 21 3041 3062

    • Search Google Scholar
    • Export Citation
  • Wang, Q., Lai, T.F., Qin, G.Z. & Tian, S.P. 2009 Response of jujube fruits to exogenous oxalic acid treatment based on proteomic analysis Plant Cell Physiol. 50 230 242

    • Search Google Scholar
    • Export Citation
  • Wang, R.H., Yuan, X.Y., Meng, L.H., Zhu, B.Z., Zhu, H.L., Luo, Y.B. & Fu, D.Q. 2016 Transcriptome analysis provides a preliminary regulation route of the ethylene signal transduction component, SlEIN2, during tomato ripening PLoS One 11 e0168287

    • Search Google Scholar
    • Export Citation
  • Weng, L., Zhao, F.F., Li, R., Xu, C.J., Chen, K.S. & Xiao, H. 2015 The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato Plant Physiol. 167 931 949

    • Search Google Scholar
    • Export Citation
  • Zhang, H.B., Jordheim, M., Lewis, D.H. & Arathoon, S. 2014 Anthocyanins and their differential accumulation in the floral and vegetative tissues of a shrub species (Rhabdothamnus solandri A. Cunn) Scientia Hort. 165 29 35

    • Search Google Scholar
    • Export Citation
  • Zhong, S., Fei, Z.J., Chen, Y.R., Zheng, Y., Huang, M.Y., Vrebalov, J., McQuinn, R., Gapper, N., Liu, B., Xiang, J., Shao, Y. & Giovannoni, J.J. 2013 Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening Nat. Biotechnol. 31 154 161

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    • Export Citation
  • Zhou, T., Zhang, H., Lai, T.F., Qin, C., Shi, N.N., Wang, H.Z., Jin, M.F., Zhong, S.L., Fan, Z.F., Liu, Y.L., Wu, Z.R., Jackson, S., Giovannoni, J.J., Rolin, D., Gallusci, P. & Hong, Y.G. 2012 Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening Scientific Rpt. 2 836

    • Search Google Scholar
    • Export Citation
  • Zhu, M.K., Chen, G.P., Zhou, S., Tu, Y., Wang, Y., Dong, T.T. & Hu, Z.L. 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. 55 119 135

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Contributor Notes

This study was supported by the Zhejiang Provincial Natural Science Foundation (LY18C150009) and the National Natural Science Foundation of China (NSFC31401926).

These authors equally contributed to this work.

Corresponding author. E-mail: laitongfei@163.com.

  • View in gallery

    (A) Developmental expression of LeHB-1 in tomato cultivar Ailsa Craig and (B–D) construction of potato virus X (PVX)/mLeHB-1 vector. Fruit development and ripening stages were recorded as days postanthesis (DPA) and days postbreaker (DPB). (B) Analysis of linearized plasmids and in vitro transcription RNA products for PVX and PVX/mLeHB-1. (C) Detection of PVX/mLeHB-1 in tomato fruit at 5 DPB with a biologic repeat by reverse transcription–polymerase chain reaction (RT-PCR) assays. An equal amount of total RNA was used for RT-PCR. PVX/mLeHB-1G and PVX/mLeHB-1R, respectively, represent green zone and red zone in injected tomato fruit. (D) The phenotype of Nicotiana benthamiana inoculated with TE buffer, PVX, or PVX/mLeHB-1 through mechanical inoculation. Photographs were taken at 10 d postinoculation. Analysis of variance was used to compare more than two means, and Duncan’s multiple range tests were used for means separations. Bars represent sd of the means. Lowercase letters indicate significant differences at P < 0.05.

  • View in gallery

    Ripening phenotypes of (A) wild-type tomato cultivar Ailsa Craig (AC) fruit, (B) AC fruit injected with potato virus X (PVX), and (C, D) AC fruit injected with PVX/mLeHB-1 5 d after breaker. Fruit are bidissected to show internal ripe and/or nonripening tissues.

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    Effect of potato virus X (PVX)-induced LeHB-1 silencing on (A) soluble solids content, (B) pH, (C) anthocyanin content, and (D) chlorophyll content in tomato cultivar Ailsa Craig (AC) fruit at 5 d postbreaker. mLeHB-1R and mLeHB-1G represent the red zone and green zone, respectively, of AC fruit injected with PVX/mLeHB-1. Analysis of variance was used to compare more than two means, and Duncan’s multiple range tests were used for means separations. Lowercase letters indicate significant differences at P < 0.05; bars represent sd.

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    Expression of genes related to (A) ripening-associated transcription factors, (B) ethylene biosynthesis, (C) carotenoid biosynthesis, and (D) flavonoid biosynthesis in tomato cultivar Ailsa Craig (AC) fruit injected with potato virus X (PVX)/mLeHB-1. The 5 DPB and 30 DPA represent fruit at 5 d postbreaker and fruit at 30 d postanthesis, respectively. mLeHB-1R and mLeHB-1G represent the red zone and green zone of AC fruit, respectively, injected with PVX/mLeHB-1 at 5 DPB. Analysis of variance was used to compare more than two means, and Duncan’s multiple range tests were used for means separations. Different lowercase letters represent significant differences at P < 0.05 in the expression of indicated genes among four groups; bars represent sd. ACS, 1-aminocyclopropane-1-carboxylate (ACC) synthase; ACO, ACC oxidase; PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, zeta-carotene isomerase; ZDS, zeta-carotene desaturase; CRTISO, carotenoid isomerase; β-CYC, β-cyclase; β-LCY, lycopene β-cyclase; ε-LCY, lycopene ε-cyclase; β-CRTR, β-carotene hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavanol 4-reductase; ANS, anthocyanidine synthase; GT, glucosyltransferase; RT, rhamnosyltransferase.

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  • Fujisawa, M., Nakano, T., Shima, Y. & Yasuhiro, I. 2013 A larger-scale identification of direct targets of the tomato MADS Box transcription factor ripening inhibitor reveals the regulation of fruit ripening Plant Cell 25 371 386

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    • Export Citation
  • 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 6 1107 1122

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  • Fujisawa, M., Shima, Y., Nakagawa, H., Kitagawa, M., Kimbara, J., Nakano, T., Kasumi, T. & Ito, Y. 2014 Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS Box proteins Plant Cell 26 89 101

    • Search Google Scholar
    • Export Citation
  • Gapper, N.E., McQuinn, R.P. & Giovannoni, J.J. 2013 Molecular and genetic regulation of fruit ripening Plant Mol. Biol. 82 575 591

  • Giuliano, G. 2014 Plant carotenoids: Genomics meets multi-gen engineering Curr. Opin. Plant Biol. 19 111 117

  • Ito, Y., Kitagawa, M., Ihashi, N., Yabe, K., Kimbara, J., Yasuda, J., Ito, H., Inakuma, T., Hiroi, S. & Kasumi, T. 2008 DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN Plant J. 55 212 223

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  • Kanazawa, A., Inaba, J., Shimura, H., Otagaki, S., Tsukahara, S., Matsuzawa, A., Kim, B.M., Goto, K. & Masuta, C. 2011 Virus-mediated efficient of epigenetic modifications of endogenous genes with phenotypic changes in plants Plant J. 65 156 168

    • Search Google Scholar
    • Export Citation
  • Karlova, R., Rosin, F.M., Busscher-Lange, J., Parapunova, V., Do, P.T., Fernie, A.R., Fraser, P.D., Baxter, C., Angenent, G.C. & de Maagd, R.A. 2011 Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening Plant Cell 23 923 941

    • Search Google Scholar
    • Export Citation
  • Kasai, A. & Harada, T. 2015 Epimutant induction as a new plant breeding technology Jpn. Agr. Res. Qrtly. 49 301 305

  • Koes, R., Verweij, W. & Quattrocchio, F. 2005 Flavonoids: A colorful model for the regulation and evolution of biochemical pathways Trends Plant Sci. 10 236 242

    • Search Google Scholar
    • Export Citation
  • Kudo, T., Kobayashi, M., Terashima, S., Katayama, M., Ozaki, S., Kanno, M., Saito, M., Yokoyama, K., Ohyanagi, H., Aoki, K., Kubo, Y. & Yano, K. 2017 Tomatomics: A web database for integrated omics information in tomato Plant Cell Physiol. 58 1 12

    • Search Google Scholar
    • Export Citation
  • Lai, T.F., Wang, Y., Zhou, T., Mei, F.L., Zhang, P.C., Zhou, Y.Y., Shi, N.N. & Hong, Y.G. 2015 Virus-induced LeSPL-CNR silencing inhibits fruit ripening in tomato J. Agr. Sci. 7 184 195

    • Search Google Scholar
    • Export Citation
  • Lico, C., Capuano, F., Renzone, G., Donini, M., Marusic, C., Scaloni, A. & Baschieri, S. 2006 Peptide display on potato virus X: Molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles J. Gen. Virol. 87 3103 3112

    • Search Google Scholar
    • Export Citation
  • Lin, Z.F., Hong, Y.G. & Yin, M.G. 2008 A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening Plant J. 55 301 310

    • Search Google Scholar
    • Export Citation
  • Liu, R.E., How-Kit, A., Stammitti, L., Teyssier, E., Rolin, D., Mortain-Bertrand, A., Halle, S., Liu, M.C., Kong, J.H., Wu, C.Q., Degraeve-Guibault, C., Chapman, N.H., Maucourt, M., Hodgman, T.C., Tost, J., Bouzayen, M., Hong, Y.G., Seymour, G.B., Giovannoni, J.J. & Gallusci, P. 2015 A DEMETER-like DNA demethylase governs tomato fruit ripening Proc. Natl. Acad. Sci. USA 112 10804 10809

    • Search Google Scholar
    • Export Citation
  • Manning, K., Tör, M., Poole, M., Hong, Y.G., Thompson, A.J., King, G.J., Giovannoni, J.J. & Seymour, G.B. 2006 A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening Nat. Genet. 38 948 952

    • Search Google Scholar
    • Export Citation
  • Martel, C., Vrebalov, J., Tfelmeyer, P. & Giovannoni, J.J. 2011 The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPRNING-dependent manner Plant Physiol. 157 1568 1579

    • Search Google Scholar
    • Export Citation
  • McAtee, P., Karim, S., Schaffer, R. & David, K. 2013 A dynamic interplay between phytohormones is required for fruit development, maturation, and ripening Front. Plant Sci. 4 79

    • Search Google Scholar
    • Export Citation
  • Mou, W.S., Li, D.D., Bu, J.W., Jiang, Y.Y., Khan, Z.U., Luo, Z.S., Mao, L.C. & Ying, T.J. 2016 Comprehensive analysis of ABA effects on ethylene biosynthesis and signaling during tomato fruit ripening PLoS One 11 e0154072

    • Search Google Scholar
    • Export Citation
  • Pandey, A., Misra, P., Choudhary, D., Yadav, R., Goel, R., Bhambhani, S., Sanyal, I., Trivedi, R. & Trivedi, P.K. 2015 AtMYB12 expression in tomato leads to large scale differential modulation in transcriptome and flavonoid content in leaf and fruit tissues Scientific Rpt. 5 12412

    • Search Google Scholar
    • Export Citation
  • Qin, G.Z., Wang, Y.Y., Cao, B.H., Wang, W.H. & Tian, S.P. 2012 Unraveling the regulatory network of the MADS box transcription factor RIN in fruit ripening Plant J. 70 243 255

    • Search Google Scholar
    • Export Citation
  • Rohrmann, J., McQuinn, R., Giovannoni, J.J., Fernie, A.R. & Tohge, T. 2012 Tissue specificity and differential expression of transcription factors in tomato provide hints of unique regulatory networks during fruit ripening Plant Signal. Behav. 7 1639 1647

    • Search Google Scholar
    • Export Citation
  • Senthil-Kumar, M. & Mysore, K.S. 2011 New dimension for VIGS in plant functional genomics Trends Plant Sci. 16 656 665

  • 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. 82 427 438

    • Search Google Scholar
    • Export Citation
  • Shinozaki, Y., Nicolas, P., Fernandez-Pozo, N., Ma, Q., Evanich, D.J., Shi, Y., Xu, Y., Zheng, Y., Snyder, S.I., Martin, L.B.B., Ruiz-May, E., Thannhauser, T.W., Chen, K., Domozych, D.S., Catalá, C., Fei, Z., Mueller, L.A., Giovannoni, J.J. & Rose, J.K.C. 2018 High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening Nat. Commun. 9 364

    • Search Google Scholar
    • Export Citation
  • Su, L., Diretto, G., Purgatto, E., Danoun, S., Zouine, M., Li, Z., Roustan, J., Bouzayen, M., Giuliano, G. & Chervin, C. 2015 Carotenoid accumulation during tomato fruit ripening is modulated by the auxin–ethylene balance BMC Plant Biol. 15 114

    • Search Google Scholar
    • Export Citation
  • Tomato Genome Consortium 2012 The tomato genome sequence provides insights into fleshy fruit evolution Nature 485 635 641

  • Vrebalov, J., Pan, I.L., Arroyo, A.J.M., McQuinn, R., Chung, M., Poole, M., Rose, J., Seymour, G., Grandillo, S., Giovannoni, J. & Irish, V.F. 2009 Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1 Plant Cell 21 3041 3062

    • Search Google Scholar
    • Export Citation
  • Wang, Q., Lai, T.F., Qin, G.Z. & Tian, S.P. 2009 Response of jujube fruits to exogenous oxalic acid treatment based on proteomic analysis Plant Cell Physiol. 50 230 242

    • Search Google Scholar
    • Export Citation
  • Wang, R.H., Yuan, X.Y., Meng, L.H., Zhu, B.Z., Zhu, H.L., Luo, Y.B. & Fu, D.Q. 2016 Transcriptome analysis provides a preliminary regulation route of the ethylene signal transduction component, SlEIN2, during tomato ripening PLoS One 11 e0168287

    • Search Google Scholar
    • Export Citation
  • Weng, L., Zhao, F.F., Li, R., Xu, C.J., Chen, K.S. & Xiao, H. 2015 The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato Plant Physiol. 167 931 949

    • Search Google Scholar
    • Export Citation
  • Zhang, H.B., Jordheim, M., Lewis, D.H. & Arathoon, S. 2014 Anthocyanins and their differential accumulation in the floral and vegetative tissues of a shrub species (Rhabdothamnus solandri A. Cunn) Scientia Hort. 165 29 35

    • Search Google Scholar
    • Export Citation
  • Zhong, S., Fei, Z.J., Chen, Y.R., Zheng, Y., Huang, M.Y., Vrebalov, J., McQuinn, R., Gapper, N., Liu, B., Xiang, J., Shao, Y. & Giovannoni, J.J. 2013 Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening Nat. Biotechnol. 31 154 161

    • Search Google Scholar
    • Export Citation
  • Zhou, T., Zhang, H., Lai, T.F., Qin, C., Shi, N.N., Wang, H.Z., Jin, M.F., Zhong, S.L., Fan, Z.F., Liu, Y.L., Wu, Z.R., Jackson, S., Giovannoni, J.J., Rolin, D., Gallusci, P. & Hong, Y.G. 2012 Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening Scientific Rpt. 2 836

    • Search Google Scholar
    • Export Citation
  • Zhu, M.K., Chen, G.P., Zhou, S., Tu, Y., Wang, Y., Dong, T.T. & Hu, Z.L. 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. 55 119 135

    • Search Google Scholar
    • Export Citation
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