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. 2020 Apr 26;15(6):1752534. doi: 10.1080/15592324.2020.1752534

Comparative transcriptome analysis of differentially expressed genes between the fruit peel and flesh of the purple tomato cultivar ‘Indigo Rose’

Xiaoxi Liu a, Yinggemei Huang b, Zhengkun Qiu b, Hao Gong a,✉
PMCID: PMC8570723  PMID: 32338177

ABSTRACT

Anthocyanins are considered health-promoting phytonutrients; however, anthocyanins strictly occurr in the fruit peel of purple tomato cultivars, making the total anthocyanin content limited per tomato fruit. In this study, we performed a transcriptome analysis between the fruit peel and flesh of a purple tomato cultivar ‘Indigo Rose’ at both the mature green stage and breaking stage. In total, 1,945 differently expressed genes, including 165 transcription factors, were detected between the fruit peel and flesh, both at and after the mature green stage. We further analyzed the transcription of anthocyanin biosynthesis genes and the regulatory genes composing the MYB-bHLH-WD40 (MBW) complex between the fruit peel and flesh at both development stages. In addition, several light-sensing genes and other transcription factor genes, including BBX family genes and WRKY genes, showed different expression patterns between the fruit peel and flesh. These findings deepen our understanding of anthocyanin biosynthesis in tomato fruit peels and facilitate the identification of genes limiting the anthocyanin biosynthesis in tomato fruit flesh.

KEYWORDS: Tomato cultivar ‘Indigo Rose’, anthocyanin, transcriptome analysis, R2R3-MYB, transcription factor

1. Introduction

Anthocyanin, a class of flavonoids, are widely present in fruits and vegetables.1 In addition to providing attractive colors (purple, blue, and red) in leaves, flowers, and fruits, increasing evidence has shown that anthocyanins play key roles in the adaption of plants to biotic and abiotic stressors.2-5 Due to their ubiquitous present in vegetables and fruits, anthocyanins are commonly consumed as part of the human diet and are considered to have health-promoting properties because they have high levels of antioxidants.6-8

Tomato (Solanum lycopersicum) is the one of the most important vegetables worldwide, but unlike other Solanum crops, including eggplants and pepper, anthocyanins are generally undetectable in cultivated tomato.9 In the last decade, attempts have been made to increase the anthocyanin content in tomato fruit.4,10-14 For example, a previous study introgressed Anthocyanin fruit (Aft) and atroviolacium (atv) loci from the wild species S. chilense and S. cheesmaniae, respectively, into the cultivated tomato.13 This led to tomato plants with purple fruit with a high anthocyanin content.13 ATV encodes an R3-MYB transcription factor, SlMYBATV, and acts as a repressor in anthocyanin biosynthesis.15,16 More recently, the gene underlying Aft was found to encode an R2R3-MYB protein, SlAN2-like, which positively regulates anthocyanin production in tomato fruit.17 Moreover, the previous study further revealed that Aft cooperates with ATV to form a feedback loop to fine-tune anthocyanin biosynthesis.17 In addition to Aft and atv, the Aubergine (abg) locus from S. lycopersicoides could also regulate anthocyanin accumulation in tomato fruit.18 Abg was mapped to chromosome 10, the same arm as Aft.18 However, it is still unclear whether Abg is an allele of Aft.

Previous studies have revealed that anthocyanin production is controlled by the MYB-bHLH-WD40 (MBW) complex of R2R3-MYB, basic helix-loop-helix (bHLH), and WD-repeat proteins.19 Aside from Aft, three other R2R3-MYB transcription factors, SlAN2, SlANT1, and SlANT1-like were identified in tomato.11 SlAN2, SlANT1, and SlANT1-like are located near Aft on chromosome 10.11,20 Overexpression of SlAN2 and SlANT1 could significantly increase anthocyanin production in tomato fruit.11,12,21 The bHLH factor SlAN1, which underlies the AH locus, positively controls anthocyanin biosynthesis in tomato.4 SlAN1-overexpressed tomato bears purple fruit with high anthocyanin content.4 However, anthocyanins only occurred in the fruit peel even in the overexpression lines of R2R3-MYB genes (SlAN2, SlANT1 or Aft) and the SlAN1-overexpressed line. Because the fruit peel accounts for only about 5% of the total fruit mass, the total anthocyanin content was 300 μg per g fresh weight.14 A purple tomato with high anthocyanin content in the flesh was produced by the ectopic expression of two selected transcription factors (R2R3 MYB: Ros1; bHLH: Del1) from snapdragon (Antirrhinum majus).14 The anthocyanin content could reach approximately 3 mg per g fresh weight, with anthocyanins produced in both the fruit peel and flesh, in the Del1/Ros1 transgenic lines.14 The striking phenotype of Del1/Ros1 fruit was due to the high expression levels of the anthocyanin biosynthesis genes, including phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and flavonoid 3ʹ5’-hydroxylase (F3ʹ5’H).14,22

Tomato cultivar (cv.) ‘Indigo Rose’ contains the Aft and atv loci, bearing purple fruit with a high anthocyanin content in the fruit peel.16 However, it is unclear why anthocyanins only accumulate in the fruit peel, not the flesh. Therefore, in this study, we used RNA sequencing (RNA-seq) of the fruit peel and flesh from the cv. ‘Indigo Rose’ at the mature green and breaking stages to obtain a better understanding of anthocyanin accumulation. A comparative analysis of their transcriptomes demonstrated that most of the anthocyanin biosynthesis genes, especially the later biosynthesis genes, were strongly downregulated in fruit flesh compared to the fruit peel. We further observed the differential transcription of tens of other anthocyanin-related genes between the fruit peel and flesh and found that several regulators showed different expression patterns in the fruit peel and flesh at both the mature green stage and the breaking stage. These findings will facilitate the identification of genes limiting anthocyanin accumulation in tomato fruit flesh.

2. Materials and methods

2.1. Plant material and growth conditions

The seeds of the tomato cultivar ‘Indigo Rose’ were obtained from Johnny’s Selected Seeds (https://www.johnnyseeds.com/). ‘Indigo Rose’ contains the Anthocyanin fruit (Aft) and atroviolacea (atv) loci from the wild species S. chilense and S. cheesemanii, respectively. It bears purple fruit with a high anthocyanin content in fruit peel at and after the mature green stage. The plants were grown in a plastic greenhouse from September to December of 2018 (Guangzhou, Guangdong, China).

2.2. Anthocyanin extraction and quantification

The fruit peel and flesh at the mature green stage and breaking stage were used for anthocyanin quantification. Anthocyanin extraction and quantification were performed as described in a previous study.23 Briefly, fruit peel or flesh were ground with liquid nitrogen into powder. 2 g of the powder was extracted with 1 volume HCl 1% (v/v) in methanol with the addition of two-thirds volume of distilled water. Extracts were recovered, and 1 volume of chloroform was added to remove chlorophylls through mixing and centrifugation (1 min at 14,000 g). The amount of anthocyanins was determined spectrophotometrically (A535-A650) and expressed as mg of petunidin-3-(p-coumaroyl rutinoside)-5-glucoside per g, based on an extinction coefficient of 17,000 and a molecular weight of 934. Mean values were obtained from three independent replicates. Each replicate was collected from five fruits collected from different plants.

2.3. RNA isolation and RNA-seq analysis

The fruit peel or flesh from cv. ‘Indigo Rose’ at the mature green stage and breaking stage were used for an RNA-seq analysis. The RNA-seq analysis was performed as described in a previous study.24 Briefly, total RNA was extracted using an Eastep Super RNA extraction isolation kit (Cat. No LS1040, Promega) and used for the development of an RNA-seq library. The RNA-seq library was generated using an NEBNext UltraTM RNA Library Prep kit for Illumina (NEB) following the manufacturer’s protocols; index codes were added to attribute sequences to each sample. Three biological replicates were performed for RNA-seq analyzes. STAR software25 was used to align the sequencing reads to the tomato reference genome (SL3.0), FeatureCounts26 was then used to analyze the transcript levels of the annotated genes, and DEGseq27 was used to calculated the P-values. P < .01 was used as a threshold for the identification of the DEGs. GO enrichment analysis for biological process was performed in GENEONTOLOGY with the default parameters.28

2.4. cDNA synthesis and quantitative reverse transcription PCR (qRT-PCR) analysis

cDNA was synthesized from 1 μg of total RNA using the GoScriptTM Reverse Transcription System (cat. No. A5001, Promega). qRT-PCR was performed following our previously.16 A tomato ACTIN (Solyc03g078400) gene was used as the reference. All analyses were performed with three technical replicates. The 2−Δct method was used to calculate the relative expression of each gene.29 Primers used for qRT-PCR are listed in Table S1.

3. Results

3.1. Phenotype and transcriptome analysis of the fruit peel and flesh in cv. ‘Indigo Rose’

Cv. ‘Indigo Rose’ bears purple/black fruit at and after the mature green stage (Figure 1a).16 A quantitative analysis of anthocyanin content revealed that anthocyanin content reached ~200 mg per 100 g fresh weight in the fruit peel at the mature green or breaking stages (Figure 1a). Very few anthocyanins were detected in the fruit flesh both at the mature green and breaking stages (Figure 1a).

Figure 1.

Figure 1.

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Transcriptome analysis of differentially expressed genes (DEGs) between the fruit peel and fruit flesh of cv. ‘Indigo Rose.’ (a) The phenotype and a quantitative analysis of anthocyanin content in the fruit peel and flesh of cv. ‘Indigo Rose.’ Data are means of three biological replicates ± standard error. (b) The Venn diagram represents the DEGs between the fruit peel and flesh of cv. ‘Indigo Rose’ at the mature green and breaking stages. (c) The enriched GO terms in biological process of the 1,945 DEGs shared between the mature green stage and breaking stage. MG means mature green stage and BR means breaking stage.

To further identify the candidate genes related to the anthocyaninless phenotype in fruit flesh, transcriptome analyses were performed on the fruit peel and flesh of cv. Indigo Rose at the mature green and breaking stages. A total of approximately 154.8 million clean reads were generated and ~95.5% of the clean reads could be uniquely mapped to the tomato reference genome (SL3.0). Gene expression was normalized using fragments per kilobase of transcript per million mapped reads (FPKM).

From the transcriptome data at the two developmental stages, a total of 3,207 and 3,524 differentially expressed genes (DEGs) were detected between the fruit peel and fruit flesh of cv. ‘Indigo Rose’ at the mature green and breaking stages, respectively (Table S2 and Table S3). In the mature green and breaking stages, 1,945 DEGs, including 165 TFs, were shared (Figure 1b, Table S4). Among the 165 TFs, 104 TFs had a higher level of expression in the fruit peel than flesh; 58 TFs had a lower level of expression in the fruit peel than flesh (Figure S1). Three TFs showed the opposite expression pattern between the mature green stage and breaking stage (Figure S1). A Gene Ontology (GO) analysis of biological process revealed that the 1,945 shared-DEGs were significantly enriched in ‘primary and secondary metabolic process,’ ‘response to auxin,’ ‘photosynthesis, light harvesting,’ and ‘regulation of transcription’ (Figure 1c).

3.2. Analysis of the anthocyanin biosynthesis genes

Anthocyanin biosynthesis genes can be divided into two groups (Figure 2). One group is the early biosynthetic genes (EBGs), including phenylalanine ammonia lyase (PAL), 4-coumaryl:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3 H). The other group is the late biosynthetic genes (LBGs), including flavonoid 3ʹ5’-hydroxylase (F3ʹ5’H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) rhamnosyl transferase (RT), anthocyanin acyltransferase (AAC), flavonol-5-glucosyltransferase (5-GT), putative anthocyanin transporter (PAT), and glutathione-S-transferase (GST). In order to characterize the key biosynthetic genes related to anthocyanin accumulation, we observed the transcription levels of all genes involved in anthocyanin biosynthesis in the fruit peel and flesh of cv. ‘Indigo Rose.’ Consistent with the anthocyanin content, the transcription levels of both the EBGs and LBGs were significantly lower in the fruit flesh than that in the peel (Figure 2). The expression of PAL was dramatically deceased in the fruit flesh at the breaking stage compared to the mature green stage (Figure 2).

Figure 2.

Figure 2.

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Transcription analysis of anthocyanin biosynthesis genes in the fruit peel and flesh of cv. ‘Indigo Rose.’ EBG indicates early biosynthetic genes; LBG indicates late biosynthetic genes. PAL, phenylalanine ammonia lyase; 4CL, 4-coumaryl:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3 H, flavanone 3-hydroxylase; F3ʹ5’H, flavonoid 3ʹ5’-hydroxylase; DFR, dihydroflavonol 4-reductase; AN2, anthocyanidin synthase; RT, rhamnosyl transferase; AAC, anthocyanin acyltransferase; 5-GT, flavonol-5-glucosyltransferase; PAT, putative anthocyanin transporter; GST, glutathione-S-transferase. Two CHS (CHS1, up; CHS2, down) and CHI (CHI, up; CHI-like, down) genes were previously found in tomato. MG and BR mean mature green stage and breaking stage, respectively. *P < .05 and ** P < .01 indicate statistically significant differences between the fruit peel and fruit flesh of cv. ‘Indigo Rose.’.

To validate the RNA-seq expression data, we performed qRT-PCR to analyze the transcription levels of eight anthocyanin-related genes and found the qRT-PCR results were consistent with those obtained from the RNA-seq experiment (Figure S2).

3.3. Analysis of the anthocyanin regulatory genes of the MBW activation complex

Anthocyanin biosynthesis genes are transcriptionally regulated by the MBW complex. Four R2R3-MYB TFs (SlAN2, SlANT1, SlANT1-like, and Aft), two bHLH TFs (SlAN1 and SlJAF13), and one WDR (SlAN11) were previously identified.16 Among the four R2R3-MYB TFs, SlAN2, SlANT1, and SlANT1-like showed limited expression in both the fruit peel and flesh (Figure 3a). Aft had a much higher expression level in the fruit peel than in fruit flesh at both the mature green and breaking stages (Figure 3a). However, Aft was still highly expressed in the fruit flesh at the mature green stage (FPKM = 75.1 ± 6.5, Figure 2a). SlAN1 was highly expressed in the fruit peel, but showed very low expression levels in the fruit flesh at both development stages (Figure 3b). Unlike SlAN1, no significant differences of SlJAF13 transcription were detected between the fruit peel and flesh (Figure 3b). The transcription levels of SlAN11 were higher in the fruit peel than in the fruit flesh (Figure 3b). However, SlAN11 was still moderately expressed in the fruit flesh (Figure 3b).

Figure 3.

Figure 3.

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Transcription analysis of positive regulators in the fruit peel and flesh of cv. ‘Indigo Rose.’ (a) Transcription levels of positive R2R3-MYB factors and (b) bHLH and WDR factors. Data are means of FPKM of three biological replicates±standard error. Different letters indicate statistically significant differences among groups (Tukey’s honest significant difference test, P < .01). MG and BR indicate the mature green stage and breaking stage, respectively.

3.4. Analysis of the candidate MYB repressors related to flavonoid biosynthesis

Three candidate R3-MYB repressors (SlMYBATV, SlMYBATV-like, and SlTRY) and four candidate R2R3-MYB repressors (SlMYB3, SlMYB7, SlMYB32, and SlMYB76) related to anthocyanin biosynthesis were previously identified.16 Herein, we found that the transcription levels of Slmybatv and SlMYBATV-like were much higher in the fruit peel than in fruit flesh at the both mature green and breaking stages (Figure 4a). Extremely low transcription levels of SlTRY were detected in both fruit peel and flesh (Figure 4a). The expression pattern of R2R3-MYB repressors differed from R3-MYB expression, except for SlMYB76, which shown higher expression levels in the fruit peel than in the fruit flesh (Figure 4b). The expression level of SlMYB3, SlMYB7, and SlMYB32 was significantly higher in the fruit peel than that in the fruit flesh at the breaking stage, while no significant difference was detected at the mature green stage (Figure 4b).

Figure 4.

Figure 4.

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Transcription analysis of negative regulators in the fruit peel and flesh of cv. ‘Indigo Rose.’ (a) Transcription levels of candidate R3-MYB repressors and (b) candidate R2R3-MYB repressors. Data are means of FPKM of three biological replicates±standard error. Different letters indicate statistically significant differences among groups (Tukey’s honest significant difference test, P < .01).

3.5. Analysis of the other candidate anthocyanin-related regulators

Anthocyanin accumulation in most plant species requires light stimulation.30 To characterize whether the absence of anthocyanin in fruit flesh may be due to shading by the peel, we compared the transcription levels of the light-sensing genes, such as the red- and far-red light-sensing phytochrome genes (SlPHYA, SlPHYB1, and SlPHYB2), the blue/ultraviolet (UV)-A-perceiving cryptochrome (CRYs) genes (SlCRY1a, SlCRY1b, SlCRY2, and SlCRY-DASH), and the UV-B-sensing photoreceptor gene (SlUVR8), between the fruit peel and flesh in cv. ‘Indigo Rose’ at both the mature green and breaking stages. We found that none of these genes were significant differently expressed between the fruit peel and flesh at both the mature green and breaking stages (Figure 5). SlCRY1a, SlPHYB1, and SlPHYB2 had higher expression levels in the fruit flesh relative to the fruit peel at the mature green stage, while no significant differences were observed at the breaking stage (Figure 5). The expression level of SlCRY1b was much lower in fruit flesh than in the peel at the breaking stage, while no difference was detected at the mature green stage (Figure 5). No significant difference of the expression levels of SlCRY2, SlPHYA, and SlUVR8 were detected between the fruit peel and flesh at both the mature green and breaking stages (Figure 5). HY5 played a positive role in mediating light-activated anthocyanin production. SlHY5 showed upregulation in the fruit peel compared to the fruit flesh only at the mature green stage. No significant difference of SlHY5 expression was detected between the fruit peel and flesh at the breaking stage.

Figure 5.

Figure 5.

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Transcription analysis of other candidate anthocyanin-related regulators in the fruit peel and flesh of cv. ‘Indigo Rose.’ *P < .05 and ** P < .01 indicate statistically significant differences between the fruit peel and fruit flesh of cv. ‘Indigo Rose;’ – indicates no statistically significant differences between the fruit peel and fruit flesh of cv. ‘Indigo Rose.’.

In addition to the light-sensing genes, we also analyzed the transcription of other candidate anthocyanin-related genes in the fruit of cv. ‘Indigo Rose,’ including two bHLH TF genes (PIF3 and PIF4), three B-box (BBX) genes (SlBBX20, SlBBX24 and SlBBX22), one Broad Complex/Tramtrack/Bric-a-Brac (BTB) gene (SlBT2), and two WRKY TF genes (SlWRKY53 and SlMRKY54). Among them, only SlBT2 was both significant highly expressed in fruit flesh compared to the fruit peel at the mature green and breaking stages. Eight candidate TFs (Solyc03g120620, Solyc10g084380, Solyc04g081000, Solyc10g083450, Solyc12g007070, Solyc01g100510, SlAN1, and SlMYBATV) that may regulate anthocyanin biosynthesis in an HY5-independent manner were previously identified.24 Four (Solyc03g120620, Solyc10g084380, Solyc04g081000, and Solyc10g083450) out of the six regulators had higher expression levels in the fruit peel than in the fruit flesh at both developmental stages. The expression of Solyc12g007070 was only significantly higher in the fruit peel than in the flesh at the breaking stage. No significant differences were detected for Solyc01g100510 expression between the fruit peel and flesh at both development stages. The two anthocyanin-related WRKY genes (SlWRKY53 and SlWRKY54) were only significantly differentially expressed between the fruit peel and flesh at the mature green stage.

4. Discussion

Anthocyanins are health-promoting compounds, but unfortunately, they are not usually accumulated in cultivated tomato.9 Some tomatoes, such as cv. ‘Indigo Rose,’ which contains the Aft and atv loci, bear purple or black tomato fruits at and after the mature green stage.16 However, anthocyanins in cv. ‘Indigo Rose’ only occur in the fruit peel, limiting the total anthocyanin content per tomato fruit. Previous studies have indicated that a major limitation in the flavonoid production in tomato fruit was the lack of expression of the CHI gene.31 PAL and F3ʹ5’H was subsequently reported as other genes limiting flavonoid biosynthesis in tomato fruit.14,22 However, in this study, though the expression of CHI and PAL were significant higher in the fruit feel than in fruit flesh, they were still moderately expressed in fruit flesh at and after the mature green stage (Figure 2). Another study also showed similar results.23 CHI was highly expressed in the peel of the non-anthocyanin-producing fruit peel of cv. ‘Ailsa Craig.23ʹ We found that several EBGs (CHS1, CHS2, CHI-like, and F3 H) and LBGs (all, except 5-GT) were highly expressed in the fruit peel, but showed limited expression in the fruit flesh at and after the mature green stage (Figure 2), suggesting that these genes limit anthocyanin production in tomato fruit.

Anthocyanin biosynthesis genes are transcriptionally regulated by the MBW complex.32,33 In apple, R2R3-MYB TFs MdMYB10 and MdMYB110a play important roles in generating type 1 and type 2 red fleshed fruit, respectively.34-36 The high expression levels of MdMYB10 and MdMYB110a lead to the high anthocyanin content in apple fruit flesh.37 Very recently, the gene underlying the Aft locus was identified.17 Aft encodes an R2R3-MYB protein, SlAN2-like, which interacts with SlJAF13 and SlAN11 to form an MBW complex that induces the transcription of SlAN1 and SlAN11.17 SlAN2-like then interacted with SlAN1 and SlAN11 to form a new MBW complex to active the transcription of anthocyanin biosynthesis genes.17 As reported herein, SlAN2-like was highly expressed in the fruit peel relative to the fruit flesh at and after the mature green stage (Figure 3a). Notably, SlAN2-like showed a low level of expression in the fruit flesh at the mature green stage (Figure 3a). Combined with the previous study that no anthocyanins were produced in SlAN2-like-overexpressed tomato lines,10 we speculated that there are some other limiting regulatory factors (e.g., bHLH TFs) related to the anthocyanin biosynthesis in tomato fruit flesh. Indeed, the bHLH gene SlAN1 showed very low levels of expression in fruit, while it was strongly expressed in the fruit peel (Figure 3). SlAN1 plays an important role in anthocyanin biosynthesis in the whole tomato plant.4 Overexpression of SlAN1 in tomato could lead to high anthocyanin content in the fruit peel.4

Light plays a central role in anthocyanin biosynthesis.30 Numerous light-sensing genes have been identified to induce the expression of genes of the MBW complex.38-41 Mutations in these light-sensing genes lead to an anthocyaninless phenotype.39,42,43 In our study, among the eight light-sensing genes (SlCRY1a, SlCRY1b, SlCRY2, SlCRY-DASH, SlPHYA, SlPHYB1, SlPHYB2, and SlUVR8), none were significantly expressed between the fruit peel and flesh at both the mature green and breaking stages (Figure 5). Thus, a conclusion cannot be made about whether shading caused by the fruit flesh of cv. ‘Indigo Rose’ caused the anthocyaninless phenotype. HY5 acts as a central regulator in anthocyanin biosynthesis under both visible and UV-B light.24,38 The expression levels of SlHY5 were obviously higher in the fruit peel than that in flesh at the mature green stage (Figure 5). In addition to regulating anthocyanin biosynthesis, SlHY5 was previously revealed to control carotenoid production in tomato.44 Thus, it is not surprising that SlHY5 was strongly expressed in both the fruit peel and flesh at the breaking stage (Figure 5). Notably, except for SlAN1 and SlMYBATV, four (Solyc03g120620, Solyc10g084380, Solyc04g081000, and Solyc10g083450) out of the six HY5-independent TFs were strongly induced in the fruit peel compared to the fruit flesh at both stages, suggesting that they would play roles in anthocyanin biosynthesis in tomato (Figure 5). Indeed, Solyc03g120620, Solyc10g084380, and Solyc10g083450 encode an HD-ZIP TF, a WRKY TF, and a NAC TF, respectively.24 The homologies of these three genes in Arabidopsis are GLABRA 2, TTG2, and ANAC032, respectively.24 GLABRA 2 and ANAC032 are positively related in anthocyanin biosynthesis, whereas TTG2 is negatively regulated in anthocyanin biosynthesis.45-48 However, further study about the function of these HY5-independent factors in tomato are still needed.

The BBX family proteins act as cofactors to HY5 to mediate light-regulated developmental responses.49,50 An increasing number of anthocyanin-related BBX genes were identified, for example, MdBBX22 and MdBBX37 in apple, PpBBX16, PpBBX18, and PpBBX21 in peach.51-53 MdBBX22, PpBBX16, and PpBBX18 positively regulated in anthocyanin accumulation,51-53 while MdBBX37 and PpBBX21 are negatively regulated in anthocyanin accumulation.53,54 In tomato, SlBBX22 is homologous to MdBBX22/PpBBX16, SlBBX20 is homologous to PpBBX18, and SlBBX24 is homologous to PpBBX21. These homologous genes in tomato were not significant differentially expressed between the fruit peel and flesh at and after the mature green stage (Figure 5). In tomato, SlBT2 is homologous to MdBT2; the expression of SlBT2 was significantly higher in fruit flesh than that in the peel at and after the mature green stage (Figure 5). In apple, MdBT2 was confirmed to be a negative regulator of the UV-B-induced anthocyanin accumulation by degrading the MdBBX22 protein through the 26 S proteasome pathway.51 Further studies are needed to determine whether SlBT2 acts as a repressor resulting in the anthocyaninless tomato fruit phenotype.

Overall, we performed transcriptome profile analysis of the DEGs between fruit peel and flesh of a purple tomato cv. ‘Indigo Rose’ at the mature green stage and breaking stage. We specially observed the transcription levels of anthocyanin biosynthesis genes, transcription factors of MBW complex, R3- and R2R3-MYB repressors, and light-sensing genes and several other type regulator genes between fruit peel and fresh. These findings will facilitate the identification of genes limiting anthocyanin accumulation in tomato fruit flesh.

Supplementary Material

Supplemental Material
Click here for additional data file. (3.2MB, zip)

Funding Statement

This research was funded by Guangdong Science and Technology Project (2019A050507003) and Guangzhou Municipal Science and Technology Project (201807010033).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

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