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Novel R2R3 MYB transcription factors regulate anthocyanin synthesis in Aubergine tomato plants

Abstract

Background

A high content in anthocyanins, for their health beneficial properties, represents an added value for fruits and vegetables. Tomato (Solanum lycopersicum) is one of the most consumed vegetables worldwide and is rich in vitamins and carotenoids. In recent years, purple-skinned tomatoes, enriched of anthocyanins, were produced recovering allelic variants from wild Solanum species. The molecular basis of the Anthocyanin fruit (Aft) locus, exploited by breeders to activate the anthocyanin synthesis in tomato epicarp, has been recently identified in the correct splicing of the R2R3 MYB gene AN2like. Aubergine (Abg) is a tomato accession which introgressed from Solanum lycopersicoides a locus activating the synthesis of anthocyanins in the fruit. The Abg locus was mapped in the region of chromosome 10 containing Aft and the possibility that Abg and Aft represented alleles of the same gene was hypothesized.

Results

We dissected the R2R3 MYB gene cluster located in the Abg genomic introgression and demonstrated that AN2like is correctly spliced in Abg plants and is expressed in the fruit epicarp. Moreover, its silencing specifically inhibits the anthocyanin synthesis. The Abg allele of AN2like undergoes alternative splicing and produces two proteins with different activities. Furthermore, in Abg the master regulator of the anthocyanin synthesis in tomato vegetative tissues, AN2, is very poorly expressed. Finally, a novel R2R3 MYB gene was identified: it encodes another positive regulator of the pathway, whose activity was lost in tomato and in its closest relatives.

Conclusion

In this study, we propose that AN2like is responsible of the anthocyanin production in Abg fruits. Unlike wild type tomato, the Abg allele of AN2like is active and able to regulate its targets. Furthermore, in Abg alternative splicing leads to two forms of AN2like with different activities, likely representing a novel type of regulation of anthocyanin synthesis in tomato.

Background

The large chemical diversity of the secondary metabolites existing in plants is made possible by a set of enzymes acting in their biosynthetic pathways and encoded by structural genes transcriptionally regulated in response to environmental or developmental cues. The transcription factors (TFs) modulating these processes are encoded by regulatory genes often belonging to multigene families and arranged in genomes as tandem repeats, deriving from duplication events. In higher plants, indeed, gene duplication represented an important mechanism in the diversification of the secondary metabolism [1, 2].

The R2R3 MYB constitutes one of the plant largest groups of TFs: they belong to the family of MYB (MYeloBlastosis) proteins, ubiquitous in the eukaryotic organisms, and exhibit the conserved MYB DNA-binding domain consisting of two imperfect repeats (R2 and R3) of about 50 amino acids. These TFs have been associated with a multiplicity of different processes, including regulation of cell identity and fate, control of primary and secondary metabolism, and response to biotic and abiotic stresses [3, 4].

One of the best characterized biosynthetic pathways regulated by R2R3 MYBs is the synthesis of anthocyanins, a class of secondary metabolites playing essential roles in defense mechanisms and flower and fruit colors [5]. Anthocyanins are soluble polyphenolic pigments belonging to the group of flavonoids, synthesized through a specific branch of the phenylpropanoid pathway and accumulated in vacuoles of plant cells [6]. The structural genes of this pathway are conventionally divided in early biosynthetic genes and late biosynthetic genes (LBGs), being the early genes involved in the first common enzymatic steps of the flavonoid synthesis and acting the LBGs in the specific final ramifications of the pathway, leading to anthocyanins or proanthocyanidins [7].

In the same species multiple R2R3 MYB TFs can finely regulate the anthocyanin synthesis in different tissues and at different times, acting either in positive or negative ways, and both singularly and taking part to multiprotein MBW complexes where the MYB TF physically interacts with a basic Helix-Loop-Helix (bHLH) factor and a WD40-repeat (WDR) protein [8]. The R2R3 MYB TFs are conventionally divided in subfamilies, according to the presence of specific amino acidic signatures conferring them peculiar properties [3]. In the anthocyanin pathway the positive R2R3 MYB regulators usually belong to the subgroup 6, whereas the negative regulators to the subgroup 4 [9]. When part of a MBW complex, the MYB factor confers target gene specificity to the activity of the complex, and its production is strictly regulated by specific developmental or environmental stimuli. On the other hand, bHLH and WDR partners can be less specific, often regulating more than one biological process [10].

The R2R3 MYB proteins involved in anthocyanin synthesis can be encoded by paralogous genes which often lie in tandem on the same genomic region. In Arabidopsis thaliana, for example, four R2R3 MYB TFs (PAP1, PAP2, MYB113 and MYB114), showing a high sequence similarity, can induce anthocyanin synthesis, and three of the relative genes (PAP2, MYB113 and MYB114) are tightly linked on chromosome 1 [11]. In petunia (Petunia x hybrida), four R2R3 MYB genes were identified as positive regulators of anthocyanin production in different tissues: AN2, AN4, DPL, and PHZ [12, 13]. Except for AN2, the other three genes cluster together in the same genomic scaffold. Recently, other three MYB genes, ASR1, ASR2 and ASR3, have been identified in two petunia wild species (P. axillaris and P. inflate) arranged in a second cluster, representing a genomic duplication of the first one [14].

Clusters of R2R3 MYB genes modulating anthocyanin production are also present in the genome of some cultivated Solanaceae. In tomato (Solanum lycopersicum), four R2R3 MYB TF-encoding genes, namely ANT1, ANT1like, AN2 and AN2like, lie on the distal part of the long arm of chromosome 10 [15]. In the tomato accession Anthocyanin fruit (Aft) the same cluster of MYB genes was introgressed from Solanum chilense [16] and shows extensive variation in coding and promoter sequences compared to the tomato reference genome [17]. In eggplant (Solanum melongena), a major quantitative trait locus, responsible for most of the phenotypic variation in fruit and leaf anthocyanin pigmentation, is in chromosome 10 [18]. Similarly, in wild (Solanum commersonii) and cultivated (Solanum tuberosum) species of potato, R2R3 MYB paralogous genes are in tandem in the same genomic scaffold of chromosome 10 and show sequence variability and functional divergence [19]. Finally, a major region in chromosome 10 containing 12 genes related to the accumulation of anthocyanins in fruits was recently found in pepper [20]. The presence of these clusters of R2R3 MYB genes in the same chromosome, often organized in syntenic blocks, indicates a relatively recent divergence of these species from a common ancestor, where the R2R3 MYB genes likely expanded by duplication and subsequent diversification.

In tomato the R2R3 MYB genes belonging to the cluster in chromosome 10 show highly conserved sequences but also functional divergence [15, 16]. For three of them, ANT1, ANT1like, and AN2, a promoting activity on anthocyanin biosynthesis was demonstrated, with a major role exerted by AN2 in vivo in vegetative tissues [15, 16]. Furthermore, the presence of a splicing mutation in AN2like leads to the production of a non-functional TF, preventing the induction of anthocyanin pigmentation in fruits, where this gene is expressed [16, 21]. On the contrary, in Aft plants the allele of AN2like, derived from S. chilense, is functional [16, 21, 22] and allows the production of anthocyanin spots in the fruit peel [23].

A fruit phenotype similar to Aft is shown by the Aubergine (Abg) tomato accession, which carries a genomic introgression from the wild nightshade (Solanum lycopersicoides) [24]. This phenotype cosegregates as a monogenic dominant trait with a RAPD marker in the long arm of chromosome 10: this raises the possibility that Aft and Abg may represent alleles of the same gene [24, 25]. However, the variegated anthocyanin pigmentation shown by Abg fruit peel, which can appear spotted, blotched or fully homogeneous even in different fruits of the same plant, is peculiar [24], and the degree of anthocyanin accumulation can be stronger than in Aft [26]. In Abg, as in Aft, the fruit pigmentation is light-dependent, a trait reminiscent of the light-induced strong anthocyanin accumulation shown by the fruits of the wild nightshade, which is native of the Andes region between Peru and Chile, where the high-elevation climate is characterized by very high light intensities [27]. Under adequate light exposition, also Abg fruits can show a very intense coloration, resembling the black peel of the “aubergine” (S. melongena) fruits, from which the accession took the name [24].

Because of the mutation identified in the gene AN2like, the plants of domesticated tomato cannot produce anthocyanin-pigmented fruits. In recent years, however, the growing interest in the dietary effects of anthocyanins in human health [5] led to the production by genetic engineering or breeding of purple tomatoes, which can synthesize and accumulate anthocyanins in addition to carotenoids [28]. Breeding exploited the possibility to cross S. lycopersicum with some wild Solanum species which produce anthocyanins in their fruit peel, as the species S. chilense and S. lycopersicoides above mentioned [29].

The strong interest in the anthocyanin-enriched tomatoes and the necessity to further improve their qualities prompted us to carry out the molecular characterization of the Abg accession, known for a long time but never studied in detail, with the aim to ascertain the genetic basis of its phenotype and to possibly find out new sources of genetic variability for tomato breeding.

Results

The Abg plants show a peculiar anthocyanin phenotype

The Abg tomato line was originally selected for its fruit phenotype [24]: when fruits are light-exposed, they can indeed synthesize anthocyanins in the upper epicarp, accumulating them in diverse patterns, from spotted to totally flushed purple motifs, from the mature green stage on, and anthocyanins persist in the red ripe fruit peel (Fig. 1a-c).

Fig. 1
figure 1

Phenotype of Abg fruits. a Tomatoes at the mature green stage from cv. Ailsa Craig (wild type, WT) (left), and Abg heterozygous genotype (Abg/+) (right). b Anthocyanin content measured in the fruit epicarp of WT and Abg/+ tomatoes at the mature green stage. Anthocyanins are expressed in mg petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside g− 1 fresh weight (FW). Data are means of six biological replicates. Unpaired t-test was carried out and **** asterisks indicate significant difference (P ≤ 0.0001). c Different patterns of anthocyanin accumulation in Abg fruits at the mature green and red stages. d Fruit morphology of Abg/Abg and Abg/+ plants. e Anthocyanin content measured in the fruit peel of Abg/Abg and Abg/+ tomatoes at the mature green stage. Anthocyanins are expressed in mg petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside g− 1 FW. Data are means of six biological replicates. Unpaired t-test (P ≤ 0.05) was carried out. Photographs are from the authors

The Abg phenotype is dominant and the anthocyanin pigmentation in fruits is similar in homozygous (Abg/Abg) and heterozygous (Abg/+) plants (Fig. 1d, e). Morphology, instead, is very different, with the homozygous fruits which expand less, reaching final sizes lower than those of the heterozygous fruits, and are seedless and often malformed (Fig. 1d).

The growth habits of the two genotypes are also quite different: both types of plants grow and develop normally in the first weeks after germination, but, later, the homozygous plants start to yellow, while producing flowers and fruits (Fig. 2a). The yellow leaves tend to roll and curl, starting from the oldest and moving to the youngest. When are still green, the homozygous plants show a very pale colour in their leaves, stems, and leaf veins compared to the heterozygotes (Fig. 2b, c); this is confirmed by the low levels of anthocyanins measured in these tissues (Fig. 2d). Whereas leaves can slowly accumulate anthocyanins over time, leaf veins and stems remain pale green (Fig. 2b).

Fig. 2
figure 2

Anthocyanin phenotype of Abg plants. a Growth habits of 50-day-old Abg heterozygous (Abg/+) plants (left), 50-day-old Abg homozygous (Abg/Abg) plants (centre), and three-month-old Abg/Abg plants (right). Leaf abaxial side, magnification of the leaf veins in the abaxial side, and internode of the stem of an Abg/Abg (b) and an Abg/+ (c) plant. d Anthocyanin content measured in the leaf, leaf veins and stem of 50-day-old Abg/Abg and Abg/+ plants. Anthocyanins are expressed in mg petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside g− 1 fresh weight (FW). Data are means of six biological replicates. Unpaired t-test was carried out and **** asterisks indicate significant difference (P ≤ 0.0001). Photographs are from the authors

Analysis of Abg R2R3 MYB genes involved in anthocyanin synthesis

Given the peculiar phenotype in fruits and vegetative tissues, each of the R2R3 MYB genes located in the distal part of the long arm of chromosome 10 (where the genomic introgression from S. lycopersicoides was mapped [24, 25]) and known to be involved in regulation of the anthocyanin biosynthetic pathway in tomato [15, 16] was cloned and sequenced in Abg homozygous plants. The corresponding wild type (WT) tomato sequences [17], and the genomic sequences recently assembled for S. lycopersicoides [30] were used for comparison. Four genes orthologous to the four R2R3 MYB genes of tomato were identified (Fig. S1S4). They showed several polynucleotide and single nucleotide polymorphisms (SNPs) compared to WT, and most of them, as expected, were also present in the wild nightshade corresponding genomic sequences (Fig. S5-S8).

Unexpectedly, a fifth and novel gene, very similar to the previous four, was additionally identified in Abg, and its presence was confirmed in S. lycopersicoides chromosome 10, contiguous to the other four R2R3 MYB genes (Fig. S9). Due to its similarity with the gene MYB113 present in other Solanaceae (Fig. S10-S11), we called it MYB113Abg. A Blast search in the tomato genome, using MYB113Abg as a query, revealed the existence of a similar sequence in the very distal part of chromosome 10, 8.2 kb far from the genomic sequence of AN2like (Solyc10g086290) (Fig. S12a, c). We cloned and sequenced this tomato genomic region: it resulted shorter than the 1340 nucleotide-MYB113Abg open reading frame, matching its first 457 and last 540 nucleotides (Fig. S12b-S13). The first part of the tomato sequence (in forward orientation) well aligned with MYB113Abg, but the corresponding putative transcript contains a premature stop codon (Fig. S12b). This sequence is not annotated as a transcript in the tomato genomic database. On the contrary, from the final part of the genomic sequence of tomato aligning with MYB113Abg, in reverse orientation a putative cDNA, corresponding to the Solyc10g086300.1.1 transcript, is annotated as “Unknown protein” (Fig. S12d, e).

Being not known the exact dimension of the introgressed region of S. lycopersicoides in Abg, we cloned another R2R3 MYB gene, THM27(Solyc10g055410), located in the long arm of chromosome 10 not far from the other five R2R3 MYB genes above described (Fig. S14a). Indeed, the THM27WT protein shows some structural features which would indicate a possible involvement in the flavonoid metabolism as a negative regulator: a bHLH interaction motif in the R3 domain and an “ethylene-responsive element binding factor-associated amphiphilic repression” (EAR) motif in the C-terminal region (Fig. S14b). Again, THM27Abg shared with the corresponding sequence of S. lycopersicoides most of the nucleotide polymorphisms towards the THM27WT gene (Fig. S15), indicating its origin from the wild parental species.

To easily assess the genotype of the Abg plants, which were phenotypically very similar in the early stages of development, a Cleaved Amplified Polymorphic Sequence marker for the gene AN2like, allowing discrimination among wild type (+/+), Abg/+ and Abg/Abg genotypes, was designed (Fig. S17).

Alternative splicing and splicing mutations can crucially affect the activity of R2R3 MYB TFs involved in the regulation of anthocyanin synthesis [16, 21]. For this reason, we first inferred the cds of the Abg MYB genes identified in chromosome 10 from the relative gene sequences and predicted the corresponding polypeptide sequences, without finding out major mutations expected to affect their activity (Fig. S16). Then we cloned the transcripts of the novel Abg alleles of AN2like and MYB113 from fruits and leaves, respectively, of Abg homozygous plants and compared them with the cds bioinformatically predicted. Whereas the cds of MYB113Abg was identical with what expected from the canonical splicing of its gene (Fig. S9a), two different AN2like transcripts were isolated from the fruit epicarp. One, deriving from the combination of three exons (Fig. S19a, b), corresponded to the canonically spliced cds and well aligned with the AN2like mRNA expressed in Aft fruits (Fig. 3a; Fig. S19c) [16, 21]. The other transcript, shorter for the presence of an 81-nt deletion in the third exon, which was spliced as an additional intron, maintained the frameshift in the downstream sequence, thus producing a putative polypeptide 27 amino acids shorter than the one translated from the longer transcript (Fig. 3a; Fig. S19b, d).

Fig. 3
figure 3

Alternative splicing of AN2likeAbg and cellular localization of the novel Abg R2R3 MYB proteins. a Structure of the gene AN2like of Abg plants, showing the start and stop codons, exons represented by boxes and introns represented by lines, and of the long and the short transcripts produced from the same gene with the positions of R2 and R3 MYB domains and of the transactivation domain. The different parts of the transcripts are proportionally scaled. b Cellular localization of total AN2likeAbg-GFP, short AN2likeAbg-GFP, long AN2likeAbg-GFP and MYB113Abg-GFP fusion proteins, co-expressed with the Nuclear-Localization Signal (NLS)-mCherry protein in tomato protoplasts. Bright field, GFP, NLS-mCherry, and merged GFP + NLS-mCherry or bright field + GFP + NLS-mCherry images of protoplasts are shown

Functional analysis of the Abg R2R3 MYB proteins

To firstly ascertain their possible activities, we analysed the subcellular localization of AN2likeAbg and MYB113Abg proteins. To do that, their genomic sequences fused with the GFP marker were co-transfected with the Nuclear Localization Signal-mCherry sequence in tomato protoplasts. As shown in Fig. 3b, both AN2likeAbg and MYB113Abg proteins resulted clearly localized in the cell nuclei. With the same strategy, we checked the cellular localization of the two different cds produced from AN2likeAbg, finding that both resulted nucleus-localized (Fig. 3b).

Then, in order to verify whether the R2R3 MYB proteins from the Abg locus were functional as TFs, a transactivation assay was carried out. The promoter of the Dihydroflavonol 4-Reductase (DFR) LBG, encoding the enzyme catalysing the first committed step in plant anthocyanin synthesis [6], driving the firefly luciferase reporter gene, was transfected in tomato protoplasts. At the same time, each single AN2Abg, ANT1Abg, ANT1likeAbg, AN2likeAbg and MYB113Abg gene sequence, in combination with AN1, encoding the bHLH partner taking part to the MBW complex of tomato [31], were co-expressed as effectors in the protoplasts. The corresponding WT and Aft R2R3 MYB alleles [16] were used for comparison.

We found that all the Abg MYB proteins in the presence of the bHLH partner could transactivate the DFR promoter. Moreover, whereas the AN2Abg, ANT1Abg, and ANT1likeAbg sequences showed an efficiency very similar to the orthologous WT and Aft alleles (Fig. S18a-c), AN2likeAbg transactivated the DFR promoter with a little lower efficiency than AN2likeAft (Fig. 4a). On the contrary, and as already known [16], the AN2likeWT allele did not produce a TF able to activate the DFR promoter (Fig. 4a).

Fig. 4
figure 4

Transactivation activity of the novel Abg R2R3 MYB proteins in tomato protoplasts. DFR promoter driving firefly luciferase gene was transactivated by effector plasmids containing: the AN2like genomic sequences from wild type (WT), Abg or Aft plants a; the MYB113 genomic sequences from WT and Abg plants and the MYB113 cds from Abg b; the THM27 genomic sequences from WT and Abg plants singularly and in combination with the AN2like genomic sequence from Abg, and the single AN2like genomic sequence from Abg c; and the AN2like cds from WT, Abg (both the short and the long transcripts) or Aft plants d. In all the transactivation assays, the effector R2R3 MYB plasmids were co-expressed with the effector plasmid containing the bHLH factor AN1. Data are expressed as relative luciferase activity (RLU) (Firefly_Luc/Renilla_Luc) with the value of the DFR promoter basal level set to 1 and are means of four biological replicates. One-way ANOVA with Tukey’s HSD post hoc test was performed. Different letters indicate significant differences at P ≤ 0.05

MYB113Abg, differently to the corresponding WT protein, resulted functional in the transactivation assay, giving consistency to a putative role of R2R3 MYB TF able to induce anthocyanin synthesis (Fig. 4b).

Finally, THM27Abg, expressed in protoplasts, behaved as an anthocyanin negative regulatory protein because it could repress the activation of the DFR promoter induced by the co-expression of AN2likeAbg and AN1 (Fig. 4c). In this case, however, also the THM27WT allele was functional, and the relative protein repressed the activation of the DFR promoter with a similar efficiency (Fig. 4c).

Interestingly, in the transactivation assay with the DFR promoter, both the AN2likeAbg transcripts resulted functional when co-expressed with AN1 (Fig. 4d), but the short transcript was less efficient than the long one (Fig. 4d). Thus, to verify if the deletion in the C-terminal part of the polypeptide, due to the splicing of the 81-nt additional intron adjacent to some conserved functional domains (Fig. 5a), was responsible of the lower efficiency, a corresponding 81-nt deletion was produced by mutagenesis in the AN2likeAft cds. The transactivation assay with the DFR promoter was then carried out, and, as observed with the Abg transcripts, the Aft shorter cds resulted a less efficient effector than the longer one, and the Abg and Aft short AN2like cds resulted quite similar in their transactivation efficiency (Fig. 5b).

Fig. 5
figure 5

Conserved domains in the AN2likeAbg transcripts and analysis of the activity of the corresponding proteins. a Structure of AN2likeAbg showing the three exons and the two introns in the pre-mRNA giving origin to the long transcript (up), and the three introns and the four exons in the pre-mRNA giving origin to short transcript (down). ATG and TAA start and stop codons, S6A, S6B and S6C domains [47] are shown. The different parts of the transcripts are proportionally scaled. b Transactivation of the DFR promoter driving firefly luciferase in protoplasts by effector plasmids containing the long and short transcripts of AN2like produced from the genes of Abg or Anthocyanin fruit (Aft) plants. The short Aft transcript was produced artificially

To complete the functional characterization with an in vivo assay, Nicotiana benthamiana leaves were agroinfiltrated with vectors expressing the AN2likeAbg long or short transcripts or the MYB113Abg cds compared with the corresponding Aft and WT counterparts. The bHLH AN1 was co-expressed with the MYB TFs to produce a partner for the formation of the MBW complex or alone as a negative control. Ectopic anthocyanin synthesis was effectively produced in all the leaves where both AN2like (either Abg or Aft) and AN1 were expressed, with a higher pigment accumulation in correspondence of the longer transcripts (Fig. 6a), as also demonstrated by the quantification of the anthocyanins isolated from the relative spots on Nicotiana leaves (Fig. 6b).

Fig. 6
figure 6

Functional in vivo analyses of AN2likeAbg and MYB113Abg. a Anthocyanin accumulation in leaves of Nicotiana benthamiana agroinfiltrated with plasmids containing the AN2like long and short transcript sequences of Abg or Aft co-expressed with the effector plasmid containing the bHLH factor AN1, also used alone as negative control. White dotted circles indicate the agroinfiltrated areas. b Quantification of the anthocyanins produced in the different areas of the leaves agroinfiltrated as described in a. c Anthocyanin accumulation in N. benthamiana leaves agroinfiltrated with plasmids containing the MYB113 genomic sequences of Abg or wild type (WT) plants co-expressed with the effector plasmid containing the bHLH factor AN1, also used alone as negative control. As positive control, the AN2likeAbg long transcript was co-expressed with AN1. White dotted circles indicate the agroinfiltrated areas. d Quantification of the anthocyanins produced in the different areas of the leaves agroinfiltrated as described in c. In b and d anthocyanins are expressed in mg petunidin-3-(p-coumaroyl rutinoside)-5-glucoside g− 1 fresh weight (FW). Data are means of ten biological replicates. One-way ANOVA with Tukey’s HSD post hoc test was performed. Different letters indicate significant differences at P ≤ 0.05. Photographs are from the authors

Anthocyanin production was also induced in leaves where MYB113Abg and AN1 were co-expressed, but not when the MYB113WT sequence was infiltrated with AN1 (Fig. 6c, d).

Expression studies of the R2R3 MYB genes in Abg plants

To characterize the process of anthocyanin synthesis in planta, an expression analysis of the main genes involved in the regulation of the pathway, including the novel Abg R2R3 MYB alleles, was carried out in the main tissues of Abg plants. For the analysis, the vegetative tissues were sampled from young plants, when the differences in terms of anthocyanin pigmentation between homozygous and heterozygous genotypes were more visible. Peel was sampled from fruits at the mature green stage, at the beginning of the anthocyanin synthesis.

By examining the expression levels of the R2R3 MYB genes in the vegetative tissues of the homozygous plants, all of them appeared transcribed at barely detectable levels, with MYB113Abg showing a little bit higher expression in the stems (Fig. 7a). Furthermore, AN2Abg, ANT1Abg, ANT1likeAbg and MYB113Abg resulted almost not transcribed in the fruit peel, where, on the contrary, AN2likeAbg resulted well expressed (Fig. 7a). The analysis of the other regulatory genes of the pathway showed that the bHLH JAF13 and the WDR AN11, considered constitutively expressed in tomato [16], were expressed at similar good levels everywhere, whereas the bHLH AN1 and the structural LBGs DFR and ANS, all regulated by the MBW complex and thus generally correlated with the accumulation of anthocyanins [16], resulted significantly expressed only in the fruit peel (Fig. 7a).

Fig. 7
figure 7

Expression analysis of regulatory and structural genes involved in anthocyanin synthesis in Abg plants. Expression levels of the regulatory R2R3 MYB (AN2, AN2like, ANT1, ANT1like, MYB113, THM27), bHLH (JAF13, AN1), WDR (AN11), R3 MYB (MYBATV), and of the structural late biosynthetic genes DFR and ANS were analysed by qPCR in leaves, leaf veins, stems and fruit peel from Abg homozygous plants (Abg/Abg) a and Abg heterozygous plants (Abg/+) b. Data are means of six biological replicates

In the heterozygous Abg plants, the Abg alleles of the R2R3 MYB genes followed the same trend shown in the Abg/Abg plants: all of them resulted poorly transcribed in both vegetative tissues and fruits, with the only exception of AN2like, whose expression in the fruit epicarp was considerable (Fig. 7b). Also in these plants, JAF13 and AN11 were highly expressed in all the tissues, whereas AN1, DFR and ANS were transcribed not only in the fruit peel, as in the homozygotes, but also in the vegetative tissues (Fig. 7b). On the other hand, in the Abg/+ plants anthocyanins were accumulated also in leaves and stems (Fig. 2). Noteworthy, the expression of the AN2WT allele in the heterozygous plants was significantly higher than the corresponding Abg allele in all the vegetative organs (Fig. S20a).

In both genotypes, the anthocyanin R3 MYB repressor MYB-ATV, known to be transcriptionally regulated by the MBW complex [31], basically followed the expression pattern of AN1 and of the LBGs, whereas the novel R2R3 MYB repressor THM27 showed a sustained expression in both the fruit peel and the vegetative tissues (Fig. 7). Remarkably, in the heterozygous plants, no big differences were observed in the expression levels of the two alleles of THM27, whereas the AN2likeWT allele was expressed in the fruit epicarp much less than the Abg allele (Fig. S20b, c).

Since AN2likeAbg resulted well expressed in the fruit epicarp, the expression levels of its two different transcripts were analysed and compared: the two isoforms were well expressed in the peel of the homozygous and heterozygous fruits, but in both cases the shorter transcript much less than the longer one (Fig. S21).

Virus Induced Gene silencing of AN2like Abg inhibited pigmentation in Abg fruit peel

To prove that the expression of AN2likeAbg in the fruit epicarp was indeed responsible of the accumulation of anthocyanins, a VIGS experiment was carried out. Immature green Abg fruits were bagged with a light-impermeable cover for two weeks, after which they were agroinfiltrated with a genetic construct designed to silence the two transcripts of AN2likeAbg. Four days after the treatment, fruits were uncovered and left on the plant. Within the mature green stage, anthocyanin spots started to appear on the peel of the fruits infiltrated with empty vectors, representing the negative controls, but not in those infiltrated with the gene-silencing genetic cassette (Fig. 8a). Anthocyanin content was then measured on the fruit peel sampled from the negative controls and from the silenced fruits at mature green stage, and qPCR analysis for the genes involved in the anthocyanin pathway was performed, confirming the silencing of AN2likeAbg (but not that of the other R2R3 MYB genes – Fig. S22) and of the LBGs analysed, as well as the strong reduction in the anthocyanin content, in the silenced fruits (Fig. 8b, c).

Fig. 8
figure 8

Virus Induced Gene Silencing (VIGS) of AN2like in Abg fruits. a Representative picture of a homozygous Abg (Abg/Abg) fruit that underwent VIGS with the empty TRV2 vector (left) and of a Abg/Abg fruit that underwent VIGS with the TRV2 vector carrying the silencing construct for the gene AN2likeAbg (right). Both fruits were collected at the mature green stage. b Anthocyanin quantification in the fruit peel of unsilenced (VIGS + empty TRV2 vector) and silenced (VIGS + TRV2 vector silencing the gene AN2likeAbg) Abg/Abg fruits. Anthocyanins are expressed in mg petunidin-3-(p-coumaroyl rutinoside)-5-glucoside g− 1 fresh weight (FW). Data are means of six biological replicates. Unpaired t-test was carried out and **** asterisks indicate significant difference (P ≤ 0.0001). c qPCR analysis of the regulatory R2R3 MYB gene AN2like and of the structural late biosynthetic genes DFR and ANS in the peel of Abg/Abg unsilenced or silenced fruits. Data are means of six biological replicates. Unpaired t-test was carried out and *** asterisks indicate significant difference (P ≤ 0.001) and **** asterisks indicate significant difference (P ≤ 0.0001). Photographs are from the authors

MYB113 is mutated in several Solanum species

Finally, to better characterize the evolution of the novel MYB113 gene, functional in Abg plants but completely disrupted in tomato, sequences orthologous were identified and analysed in other Solanum species and accessions. As shown in Fig. S23a, the large deletion in the second intron of MYB113 identified in S. lycopersicum was also found in very close relatives, such as S. lycopersicum var. cerasiforme and S. pimpinellifolium, and in wild Solanum species located more distant in the evolutionary scale, such as S. chilense and S. pennellii. Furthermore, in all the species analysed, SNPs or short nucleotide deletions were present in the first or second exon leading to premature stop codons (Fig. S23b). MYB113Aft showed similar mutations (Fig. S23b). Only one species, Solanum sitiens, among those analysed, maintained an intact MYB113 genomic sequence as Abg and S. lycopersicoides (Fig. S23b, c).

Discussion

Domestication and selection, mainly focused on yield increase, strongly reduced over time the genetic diversity of many crops, often at the expense of important disease resistance and nutritional traits [32]. To counteract this trend of “genetic erosion”, modern plant breeding often makes use of wild species which maintained in nature a wide genetic variability from which new alleles or genes can be transferred into the cultivated plants.

Tomato is a horticultural species among the most important worldwide, both for fresh market and processing industry [33]. The global interest towards this plant has recently increased thanks to the recognition of the nutraceutical qualities of its fruits [5, 34]. However, domestication and breeding caused a strong genetic erosion in tomato [35]. Consequently, modern tomato was genetically improved through guided crosses with wild relatives to increase resistance against biotic and abiotic stresses and to recover fruit nutritional quality, taste, and flavour [36]. Colour, important to guide consumer’s choice, is object of great interest and tomato varieties characterized by novel fruit pigmentation patterns have recently appeared on the market.

Novel colours may have been recovered from old or heirloom cultivars, exploiting mutations in the chlorophyll, carotenoid, or flavonoid biosynthetic pathways [28, 37, 38], or may have been created ex novo. Purple-skinned tomatoes, enriched of anthocyanins in their epicarp, have been repeatedly obtained in recent years through hybridization with wild Solanum species which, differently from tomato, can synthesize these compounds in their fruits. Allelic variants in positive and negative regulator-encoding genes of the anthocyanin pathway were recently identified at the bases of these novel genotypes [28]. One of them is Aft, an allelic variant of the gene AN2like, which may trigger the anthocyanin synthesis in fruit peel under high light [25], and was introgressed from S. chilense, one of the species of the “tomato clade” (corresponding to the Section Lycopersicon of the genus Solanum), which are frequently used as breeding material because are genetically close to S. lycopersicum, show colinear and homologous genomes [27], and result interfertile among them [29].

Wild Solanum species belonging to more distantly related lineages may offer new traits of potential interest to breeders. S. lycopersicoides is a very interesting wild relative of tomato, characterized by extreme resistance to many biotic and abiotic stresses and production of fruits which can naturally accumulate very high amounts of anthocyanins [27, 39]. This species belongs to the Section Lycopersicoides of the genus Solanum, which is more distantly related to the cultivated tomato than the species of the tomato clade [27]: its hybridization with S. lycopersicum is naturally limited by the existence of strong reproductive barriers and the common sterility of the hybrids [40]. However, in recent years S. lycopersicoides was artificially hybridized with tomato, obtaining introgression lines potentially very useful to increase its genetic variability [41,42,43].

Is Abg allelic to Aft? Finally, the answer

The Abg tomato accession introgressed from S. lycopersicoides the genetic locus on chromosome 10 linked with the synthesis of anthocyanins in the fruit epicarp under high light (Fig. 1). The possibility that Abg and Aft represent alleles of the same gene was early hypothesized [24, 25], but never proved so far. We started our analysis trying to understand how many and which of the R2R3 MYB genes lying in the long arm of chromosome 10 and known for their involvement in anthocyanin synthesis were inherited in Abg from the wild nightshade parental donor.

First, the presence of the entire R2R3 MYB cluster was confirmed in Abg plants (Fig. S1-S4). These genes showed significant genetic variability compared with tomato and resulted very similar to the sequences contained in the corresponding S. lycopersicoides genomic scaffolds [30] (Fig. S5-S8). This was a genetic confirmation of the origin of the Abg introgression from the wild nightshade and indicated that the entire MYB cluster derived from this species.

All these Abg R2R3 MYB genes showed the typical amino acidic signatures of positive anthocyanin MYB regulators: the [DE]Lx2[RK]x3Lx6Lx3R motif for interaction with bHLHs [44], and the ANDV sequence of Element 3 for regulation of anthocyanin synthesis [45] (Fig. S16b). A transactivation assay in protoplasts proved their ability to interact with the AN1 protein (the main bHLH partner of the R2R3 MYB TFs activating the anthocyanin synthesis in tomato [28, 46]) and to activate the promoter of DFR, a typical marker gene for anthocyanin synthesis (Fig. 4). For AN2, ANT1 and ANT1like no differences were observed between Abg and WT proteins in terms of activity (Fig. S18). As far as AN2like is concerned, the sequence in Abg was different from both WT and Aft but shared with the allele of Aft the SNP at the 5’ splice site of the second intron that was shown to be necessary for a correct splicing of the pre-mRNA to produce a functional TF [21] (Fig. S8). AN2likeAbg resulted indeed functional like AN2likeAft in the transactivation assay of the promoter of DFR (Fig. 4a) and in the agroinfiltration assay in N. benthamiana leaves (Fig. 6a, b). We also found that AN2likeAbg was highly expressed in the fruit epicarp where anthocyanins were synthesized (Fig. 7), and, most importantly, its specific silencing via VIGS inhibited the anthocyanin pigmentation of the fruits (Fig. 8). All these results finally demonstrated that the “purple” phenotype of Abg and Aft fruits relies on the expression of functional alleles of the same gene, AN2like.

Very interestingly, we found the existence of an alternative splicing of the AN2likeAbg allele with the production of a shorter mRNA in addition to the canonically spliced functional transcript similar to the Aft one (Fig. S19). This short AN2likeAbg mRNA is translated in a protein which is functional but less efficient in the induction of anthocyanins than the longer one, both in vitro and in vivo assays (Figs. 5b and 6a and b). This is due to the lack of a 27-amino acidic sequence in a region of the C-terminus adjacent to the “S6B” motif (Fig. 5a), a sequence which is highly conserved in the R2R3 MYB TFs inducing anthocyanin synthesis and probably involved in protein-protein interactions [47]. Its artificial deletion in the AN2likeAft TF produced a similar reduction in the capacity to induce anthocyanin synthesis (Figs. 5b and 6a and b), indicating that the sequence additionally spliced in the third exon of the gene did contain features important for enhancing the activity of the TF.

The long transcript appeared much more expressed than the short one in the mature green peel of the Abg fruits (Fig. S21). Alternative splicing strongly contributes to increase the diversity of the mRNAs expressed from a genome and the different isoforms produced from a single gene may show different biological functions [48]. It is therefore plausible that the alternative splicing of AN2likeAbg has a functional meaning and may contribute to the modulation of the production of anthocyanins in the fruit epicarp under environmental changes or at different times. This is the first evidence of the existence in tomato of mechanisms of alternative splicing possibly regulating the anthocyanin pigmentation of the fruits and further experiments are necessary to clarify this process.

The Abg locus contains an additional R2R3 MYB gene

In Abg a novel R2R3 MYB gene, named MYB113Abg for its similarity with other Solanaceae genes [49, 50], and encoding a protein carrying domains typical of MYB TFs involved in anthocyanin regulation, was identified (Fig. S9, S10). Its structural features, including the presence of the interaction domain with bHLHs and specific signatures conserved in activators of the anthocyanin pathway, are typical of the subgroup 6 of R2R3 MYBs [3]. MYB113Abg resulted more similar to the TFs of the group of tomato AN2 and AN2like than to the group of ANT1 and ANT1like (Fig. S11). The presence of this gene was confirmed in S. lycopersicoides genome (Fig. S9): thus, also this fifth R2R3 MYB TF was introgressed in Abg from the wild parent. In a recent resequencing project of S. lycopersicoides genome, this gene was called AN2-like2 for its similarity with AN2like [30].

In tomato a gene orthologous to MYB113Abg is not known. However, a similar sequence was found in chromosome 10, close to AN2like and in a more distal position: it has not been annotated so far probably because its cds is very short: compared with MYB113Abg, this tomato genomic sequence showed indeed a large nucleotide deletion in the second intron and a SNP at the beginning of the third exon which created a premature stop codon (Fig. S12; Fig. S13). Actually, it was not able to transactivate the DFR promoter in vitro (Fig. 4b) or to activate anthocyanin synthesis (Fig. 6c, d). We thus concluded that the orthologous of MYB113Abg was lost in tomato.

MYB113Abg represents a novel gene involved in anthocyanin synthesis and its activity was demonstrated in the transactivation assay of the DFR promoter (Fig. 4b) and confirmed in vivo through agroinfiltration of N. benthamiana leaves (Fig. 6c, d). However, its expression was found negligible in Abg fruit peel (Fig. 7), indicating that it was not involved in fruit pigmentation. It resulted transcribed in vegetative tissues, but, excluding the stem in Abg/Abg plants, its expression level was very low (Fig. 7) and thus a functional role cannot be assigned at the moment. It would be interesting to understand if MYB113 in S. lycopersicoides may be individually modulated according to different environmental or developmental factors. In eggplant, a TF with a sequence similar to MYB113Abg can increase anthocyanin synthesis under low temperatures through interaction with C-repeat binding factors, which act as central regulators in cold response [50]. Due to the natural habitat of S. lycopersicoides plants, characterized by high light intensities and low temperatures [27], a similar function for MYB113Abg might be hypothesized.

Summarising, the R2R3 MYB cluster in chromosome 10 contains five functional genes in Abg: this is distinctive from both WT tomato, where only three genes out of five can produce functional TFs (AN2, ANT1 and ANT1like) (Fig. S18), and Aft plants, where AN2like is correctly spliced as in Abg, but MYB113 is mutated and not functional as in tomato (Fig. S23c). Remarkably, significant rearrangements in MYB113 were identified in all the other species of the tomato clade whose genomic sequences were available (Fig. S23). MYB113 sequence resulted complete only in S. lycopersicoides and S. sitiens, both belonging to the Section Lycopersicoides [27]: this means that the loss of MYB113 activity probably occurred in the separation between the Sections Lycopersicoides and Lycopersicon of the genus Solanum, whereas the activity of AN2like, being it functional in both Abg and Aft, was maintained in the tomato clade, at least till the separation of the green-fruited species from the red ones [28].

In Abg/Abg plants a positive regulator of the anthocyanin pathway is absent in vegetative tissues

As far as we know, the Abg accession originated from a spontaneous cross between S. lycopersicum and S. lycopersicoides, but the original tomato background is not known. The line has been maintained in collection as heterozygous plants, being the homozygous unstable and not fertile [25]. We found, as expected, that Abg/Abg plants were sterile, and produced small malformed and seedless fruits (Fig. 2d), whereas the heterozygotes were fertile. The genetic aspects were not the focus of our work, but homozygous and heterozygous plants captured our attention for their peculiar phenotype in terms of anthocyanin pigmentation in vegetative tissues. Leaves, leaf veins and stems in young Abg/Abg plants were pale green due to a strong deficiency of anthocyanins and this phenotype tended to persist, even if leaves could increase their anthocyanin content over time (Fig. 2). Such a phenotype may be consequence of two different scenarios: the expression of a strong repressor of the anthocyanin biosynthetic pathway in the Abg/Abg plants, probably deriving from the wild parental donor being more active when in homozygosis, or the presence of an activator of the pathway in the Abg/+ plants, deriving from the tomato parental donor and absent in the homozygotes.

We looked for a putative negative regulator of the pathway among the genes located in the long arm of chromosome 10 and focused our attention on the protein encoded by the THM27 gene [51, 52], which shows the typical structural features of the R2R3 MYB repressors, included an EAR motif in the C-terminal domain (Fig. S14b), characteristics which are typical of subgroup 4 of R2R3 MYBs [3]. This gene in Abg was indeed introgressed from S. lycopersicoides (Fig. S15) and its protein could effectively repress the activation of the promoter of DFR in a transactivation assay, confirming its putative role of repressor (Fig. 4c). However, its activity did not result significantly different from that of the THM27WT protein (Fig. 4c), and the expression level of the gene in vegetative tissues was similar in homozygous and heterozygous plants (Fig. 7). Furthermore, the THM27Abg allele was expressed at levels similar to the THM27WT allele in the heterozygotes (Fig. S20c). These results, even if very interesting because confirming for the first time the role of THM27 as a negative regulator of the anthocyanin pathway in tomato, did not allow us to correlate its activity with the peculiar vegetative phenotype shown by Abg homozygous plants.

In alternative, the lack of a WT dominant activator of the pathway might be responsible of the “anthocyanin-free” phenotype. Such an activator should be expressed in the heterozygous Abg plants because they maintain single copies of the WT alleles in the genomic region corresponding to the segment introgressed in chromosome 10. AN2 would be the favourite candidate, being the master anthocyanin positive regulator in tomato vegetative tissues [15]. Remarkably, whereas the capacity of the Abg allele of AN2 to transactivate the promoter of DFR was identical to the capacity of the WT allele (Fig. S18a), their expression levels in the vegetative tissues of the heterozygous plants (where both the alleles are transcribed) appeared very different, with AN2WT showing a good expression level and AN2Abg being very little expressed (Fig. 7; Fig. S20a). Very low levels of AN2Abg transcripts were measured also in the homozygous plants, where the WT allele is not present (Fig. 7). Thus, the lack of a significant expression of the gene AN2 in the vegetative tissues might be at the basis of the scarce capacity of the homozygous Abg plants to synthesise anthocyanins in those organs. Differences in the promoter regions of the WT and Abg alleles in terms of cis-acting elements might be responsible for such a significant diversity in their response to the same environmental and developmental cues. Other studies are necessary to understand if in S. lycopersicoides R2R3 MYB TFs different from AN2 and encoded by genes located outside of the genomic region introgressed in Abg, substitute it in its role of main activator of the anthocyanin pathway in vegetative tissues, or if AN2 is active in other developmental stages or environmental conditions.

Conclusion

This study allowed us to genetically and functionally demonstrate that the anthocyanin-rich fruit phenotype of the Abg accession was due to the expression in the fruit epicarp of a functional allele of AN2like, which can be transcribed in a pre-mRNA correctly spliced in a TF taking part to the MBW complex inducing the anthocyanin biosynthetic pathway in fruits. This clearly resembles what already found in the Aft accession [16, 21]. In Abg plants, however, a further modulation of the pathway has been observed, since the AN2likeAbg allele can be alternatively spliced in two transcripts characterized by different length and activity, and this is the first report of a mechanism of alternative splicing in a R2R3 MYB TF gene which may fine tune the synthesis and the accumulation of these pigments in the fruit epicarp. Furthermore, the strong deficiency of anthocyanins observed in the vegetative tissues of Abg homozygous plants was due to failed expression of all the R2R3 MYB genes encoding positive activators of the pathway, and in particular of the gene AN2, which is the most important one in tomato [15]. An additional TF, MYB113Abg, was finally identified in Abg as produced from a novel gene belonging to the R2R3 MYB cluster, potentially able to act as a positive regulator of the anthocyanin pathway and lost in tomato and in its closest relatives. This gene might work in specific developmental stages or environmental conditions, giving to Abg plants a trait which is not present anymore in tomato, and which is, for this reason, worthy of further studies.

Methods

Plant materials and growth conditions

Seeds of S. lycopersicum cv. Ailsa Craig (LA2838A) and Abg (LA3668) were provided by the Tomato Genetic Resource Center (https://tgrc.ucdavis.edu/). Seeds were germinated in rock-wool plugs (Grodan) soaked in a nutritive solution [53]. Two-week-old seedlings were transplanted in pots containing a 70:30 soil/expanded clay mixture and placed in a growth chamber with 23 °C/20°C day/night temperature, 12 h photoperiod, 150 µmol photons m− 2 s− 1, and 40% relative humidity.

Gene cloning and plasmid construction

Genes of Abg plants orthologous to tomato Solyc10g086250 (AN2), Solyc10g086260 (ANT1), Solyc10g086270 (ANT1like), Solyc10g086290 (AN2like), and Solyc10g055410 (THM27) genes (SOL Genomics Network, https://sgn.cornell.edu), as well as the sequence of MYB113Abg gene, were amplified by PCR starting from Abg +/+ genomic DNA using the “Phusion High-Fidelity DNA Polymerase” (Thermo Fisher Scientific) and the oligonucleotide primers reported in Table S1. Full length cds of Abg AN2like and MYB113 were amplified from RNA extracted from leaves or fruit peel using the “Spectrum Plant Total RNA Kit” (Merck), treated with DNase and reverse-transcribed with the SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) using the same primers reported in Table S1. The sequences were individually cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific) and sequenced (Eurofins Genomics). For mutagenesis of AN2likeAft transcript, the entry vector containing the sequence, prepared in a previous study [16], was amplified by overlap extension PCR using the pair of primers listed in Table S2, and the shorter AN2likeAft cds was cloned as described above. The entry clones were recombined with different destination vectors, as described below, via Invitrogen™ Gateway™ recombination cloning technology (Thermo Fisher Scientific). Multiple sequence alignments were performed using ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalo) sequence analysis software.

Cleaved amplified polymorphic sequences analysis

Amplified DNA fragments with specific oligonucleotide primers (Table S1), producing a 444 nt product from WT and a 439 nt product from Abg, were digested with the restriction endonuclease Spe I, whose site is present only in the AN2likeWT sequence, to display by gel electrophoresis different patterns according to genotypes.

Phylogenetic analysis

The analysis was performed on the Phylogeny.fr platform [54] using default programs and parameters. MUSCLE was used for multiple alignment and PhyML for phylogeny.

Anthocyanin quantification

Anthocyanins were extracted and quantified as described in [31], and finally expressed as mg petunidin-3-(p-coumaroyl rutinoside)-5-glucoside g− 1 fresh weight [15].

Transient transformation of tomato protoplasts

Leaf protoplasts were isolated following the protocol in [55] from 3-week-old Micro-Tom plants, grown as reported above. Polyethylene glycol-mediated protoplast transformation was carried out as in [56].

Transactivation assays in tomato protoplasts

Transactivation assays by dual-luciferase system were performed with the Renilla reniformis (Renilla) and Photinus pyralis (Firefly) luciferase (Luc) enzymes. The effector constructs 35S:AN2, 35S:ANT1, 35S:ANT1like, 35S:AN2like, 35S:MYB113, and 35S:THM27 were produced as reported in [31] with the R2R3 MYB sequences cloned from Abg and Ailsa Craig DNA. The effector constructs 35S:AN2like_long transcript and 35S:AN2like_short transcript were produced with the two transcripts of the AN2like allele isolated from Abg fruit peel. All the other effector plasmids containing Ailsa Craig or Aft MYB sequences, the effector construct 35S:AN1, and the promoter_DFR:Firefly_Luc reporter construct were produced in a previous study [31]. A 35S:Renilla_Luc vector was used to normalize luminescence values detected in protoplasts [57]. Effector and reporter plasmids were co-transfected in protoplasts (5 µg for each effector plasmid, 5 µg for the promoter_DFR:Firefly_Luc reporter construct and 2.5 µg for the 35S:Renilla_Luc vector) and luminescence relative levels were measured as described in [15]. In each assay, data were expressed as relative luciferase activity (Firefly_Luc/Renilla_Luc).

Agro-infiltration assay

Transient expression assay was performed using Nicotiana benthamiana plants cultivated in a growth chamber with 16 h daylight, 100 µmol photons m− 2 s− 1, 23 °C/20°C day/night temperature. Overexpression vectors were generated by recombining the entry clones containing the sequences of AN2like, MYB113, and AN1 with the Gateway™ compatible binary vector pK7WG2 [58]. Agrobacterium tumefaciens GV3101 (MP90) strains hosting the different constructs were infiltrated in Nicotiana leaves following the protocol of [59]. Each leaf was infiltrated in four or five points with different constructs. pK7WG2 vectors recombined with the sequence of AN1 were singularly used as negative controls. Anthocyanins were quantified in single portions sampled from leaves in relation to the infiltrated areas at five days after infiltration.

RNA isolation, cDNA synthesis, and quantitative RT-PCR analysis (qPCR)

Total RNA, extracted from leaves, leaf veins, stems and fruit peel with the “Spectrum™ Plant Total RNA Kit” (Merck) was subjected to DNase treatment and then reverse transcribed into cDNA using the “Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase” (Thermo Fisher Scientific). Quantitative RT-PCR was performed with a QuantStudio 3 Real-Time PCR system (Applied Biosystems) using the “PowerUp™ SYBR® Green Master Mix” (Thermo Fisher Scientific) and the primers listed in Table S3 and in [15]. Expression levels relative to the reference gene Elongation Factor 1-alpha (EF1A) were quantified for each target gene.

Cellular localization of MYB proteins

The Abg AN2like and MYB113 entry vectors were recombined with the Gateway™ compatible destination vector p2FGW7 [58]. Protoplasts were isolated as described, transformed with 5 µg DNA for each plasmid, and incubated in the dark at 25 °C for 16 h before subsequent analysis. Fluorescence for GFP and RFP was imaged with a Nikon Eclipse Ti-5 video-confocal microscope (https://www.nikon.it) using suitable filters.

Virus Induced Gene silencing (VIGS)

TRV-based T-DNA binary vectors pTRV1, pTRV2 and pTRV2/GATEWAY are from [60]. A fragment of the cDNA of AN2likeAbg was amplified using the oligonucleotide primers listed in Table S1. The amplified sequence was cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific) and sequenced (Eurofins Genomics). The entry clone was then recombined into the Gateway compatible pTRV2 vector. Agrobacterium cultures were grown as described in [61], and cell concentration in the infiltration media was adjusted to an OD of 0.05. Abg fruit peel, from fruits bagged with a light-impermeable cover for two weeks starting from the immature green stage, was agroinfiltrated with a 1:1 (v/v) mixture of two A. tumefaciens GV3101 strains, containing the pTRV1 and the pTRV2 binary vectors with the silencing fragment (or the empty pTRV2 as control), respectively. Infiltrated fruits were kept in the dark for other 4 days and then grown under light to promote anthocyanins accumulation. Fruits were photographed and peel sampled at the mature green stage. The RNA was extracted from silenced and unsilenced fruits and the expression levels of regulatory and target genes were measured as described above.

Statistics

Statistical analyses were performed with GraphPad Prism 9.00 (www.graphpad.com/scientific-software/prism/). Data were analysed by t-test or one-way ANOVA with differences measured using the Tukey’s honest significant difference (HSD) multiple comparisons test.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request. The sequences of the Abg R2R3 MYB genes and transcripts are now being deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank): ANT1likeAbg gene (OP094090); ANT1Abg gene (OP094091); AN2likeAbg gene (OP094092); AN2Abg gene (OP094093); MYB113Abg gene (OP094094); THM27Abg gene (OP094098); ANT1Abg mRNA (OP094102); AN2likeAbg long form mRNA (OP094099); AN2likeAbg short form mRNA (OP094100); MYB113Abg mRNA (OP094101). The following sequences were also deposited: MYB113Aft gene (OP094096); Solanum chilense MYB113 gene (OP094097); MYB113WT gene (OP094095).

Abbreviations

Aft :

Anthocyanin fruit

Abg :

Aubergine

TF:

transcription factor

MYB:

MYeloBlastosis

MBW:

MYB-bHLH-WDR

bHLH:

basic Helix-Loop-Helix

WDR:

WD40-repeat

SNP:

single nucleotide polymorphism

nt:

nucleotide

VIGS:

Virus Induced Gene Silencing

WT:

wild type

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Acknowledgements

We thank Dr. Giacomo Novi for his expertise and assistance with plant growth.

Funding

This work was supported by Scuola Superiore Sant’Anna, Pisa, Italy. The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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S.G. and P.P. conceived and directed the research. J.M. performed the experiments and the overall data analysis. S.G. wrote the manuscript and J.M. prepared the figures. All authors revised the manuscript and approved its final version.

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Correspondence to Silvia Gonzali.

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Additional File 1: Fig. S1

. Sequence of the gene AN2 in Abg, S. lycopersicoides and WT plants. Fig. S2. Sequence of the gene ANT1 in Abg, S. lycopersicoides and WT plants. Fig. S3. Sequence of the gene ANT1like in Abg, S. lycopersicoides and WT plants. Fig. S4. Sequence of the gene AN2like in Abg, S. lycopersicoides, WT and Aft plants. Fig. S5. ClustalW alignment of the gene AN2 sequenced in Abg, S. lycopersicoides and WT plants. Fig. S6. ClustalW alignment of the gene ANT1 sequenced in Abg, S. lycopersicoides and WT plants. Fig. S7. ClustalW alignment of the gene ANT1like sequenced in Abg, S. lycopersicoides and WT plants. Fig. S8. ClustalW alignment of the gene AN2like sequenced in Abg, S. lycopersicoides, WT and Aft plants. Fig. S9. Sequence analysis of the gene MYB113 in Abg and S. lycopersicoides. Fig. S10. MUSCLE 3.8.31 alignment of the sequences of Solanaceae R2R3 MYB proteins similar to MYB113 identified in Abg. Fig. S11. Phylogenetic tree of MYB113Abg and other Solanaceae R2R3 MYB proteins. Fig. S12. Analysis of the gene MYB113 identified in Abg in WT plants. Fig. S13. ClustalW alignment of the gene MYB113 sequenced in Abg, S. lycopersicoides and WT plants. Fig. S14. Sequence analysis of the gene and protein THM27 in Abg, S. lycopersicoides and WT plants. Fig. S15. ClustalW alignment of the gene THM27 sequenced in Abg, S. lycopersicoides and WT plants. Fig. S16. cds and protein sequences of the R2R3 MYB transcription factors belonging to the cluster of chromosome 10 cloned in Abg plants. Fig. S17. CAPS marker for the gene AN2like. Fig. S18. Dual-Luc assay of the genes AN2, ANT1 and ANT1like. Fig. S19. AN2like transcripts and relative proteins in Abg plants. Fig. S20. Expression levels of the WT and Abg alleles of the regulatory R2R3 MYB AN2, AN2like, and THM27 genes in Abg heterozygous plants. Fig. S21. Expression levels of the total transcripts (long transcript + short transcript) and the short transcripts of the AN2likeAbg gene analysed by qPCR in the fruit peel of Abg homozygous and heterozygous plants. Fig. S22. qPCR analysis of the regulatory R2R3 MYB gene AN2, ANT1 and ANT1like in the peel of Abg/Abg unsilenced or silenced fruits. Fig. S23. Sequence analysis of the gene MYB113 from different tomato accessions and wild Solanum species.

Additional File 2: Table S1.

List of oligonucleotide primers used for gene cloning, CAPS analysis and as VIGS guides. Table S2. List of oligonucleotide primers used for mutagenesis of AN2likeAft. Table S3. List of oligonucleotide primers used for qPCR analysis.

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Menconi, J., Perata, P. & Gonzali, S. Novel R2R3 MYB transcription factors regulate anthocyanin synthesis in Aubergine tomato plants. BMC Plant Biol 23, 148 (2023). https://doi.org/10.1186/s12870-023-04153-7

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Keywords

  • Solanum lycopersicum
  • Tomato
  • Abg
  • Anthocyanins
  • R2R3 MYB
  • Alternative splicing