A putative R3 MYB repressor is the candidate gene underlying atroviolacium, a locus for anthocyanin pigmentation in tomato fruit

Abstract

Anthocyanins are potential health-promoting compounds in the human diet. The atv (atroviolacium) locus, derived from the wild tomato species Solanum cheesmaniae, has been shown to enhance anthocyanin pigmentation in tomato fruit when it co-exists with either the Aft (Anthocyanin fruit) or the Abg (Aubergine) locus. In the present study, the atv locus was fine-mapped to an approximately 5.0-kb interval on chromosome 7. A putative R3 MYB repressor was identified in this interval and is hereby designated as SlMYBATV. The allele of SlMYBATV underlying the atv locus harbored a 4-bp insertion in its coding region, which is predicted to result in a frame-shift and premature protein truncation. The other candidate R3 MYB and R2R3 MYB repressors of anthocyanin biosynthesis were also identified in tomato via a genome-wide search. Transcriptional analysis showed that most of the structural genes and several regulatory genes of anthocyanin biosynthesis were up-regulated in the tomato SlMYBATV mutant lines. These findings may facilitate the elucidation of the molecular mechanisms underlying anthocyanin pigmentation in tomato fruit and help in the marker-assisted selection of anthocyanin-enriched tomato cultivars.

Keywords: Anthocyanin; MYB repressor; SlMYBATV; atroviolacium; fine-mapping; tomato; transcriptional analysis.

Fig. 1.

Anthocyanin accumulation in the fruit of different tomato genotypes. (A) Photographs of fruit at the mature green (MG) and fully ripened (FR) stages. (B) Scanned images of tomato peel taken from the fruit at the mature green and fully ripened stages. (C) Anthocyanin content in peel collected from fruit at the mature green and fully ripened stages. Heinz1706 is the wild-type, Indigo Rose is a variety containing both the Aft and atv loci. AftAft ATVATV, AftAft ATVatv, and AftAft atvatv are three genotypes of a F₃ population that was derived from a cross between Heinz1706 and Indigo Rose. Scale bars in (A, B) indicate 1 cm.

Fig. 2.

Fine-mapping of the atv locus. (A) The genotype and fruit color of the recombinants in the F₃ populations. N indicates the number of recombinants. (B) Genotypes of three recombinants and the annotated gene according to ITAG release 2.40 at the atv locus. RI indicates the recombinant identification number. (C) The exon–intron structures of the SlMYBATV gene based on its three transcripts. Only the 4-bp insertion at the beginning of the second exon of the SlMYBATV mutant gene is shown. The other sequence polymorphisms are shown in Supplementary Figs. S1 and S2.

Fig. 3.

Multiple alignment of predicted protein sequences of SlMYBATV with AtCPC and PhMYBx. Entire protein sequences were aligned by using Clustal X ver. 2 (Larkin et al., 2007) with default settings, and the conserved amino acids were shaded by using GeneDoc (2.6) (Nicholas et al., 1997). The R3/bHLH-binding domain is marked above the alignment. ‘SlMYBATV-mutant’ indicates the deduced protein sequence from the transcripts of the purple-fruit tomato variety Indigo Rose.

Fig. 4.

Phylogenetic analysis of SlMYBATV and the other MYB repressors. The phylogenetic tree was constructed by using MEGA6 (Tamura et al., 2013) with 1000 bootstrap replicates, which was based on the N-terminal protein sequences comparing the R2R3 domain or only the R3 domain for the small MYB proteins. Numbers on the tree indicate the percentage of consensus support.

Fig. 5.

Transcriptional analysis of the structural genes of the anthocyanin biosynthetic pathway in the peel of tomato fruit at the mature green stage. (A) The anthocyanin biosynthetic pathway (modified from Povero et al., 2011). (B) Analysis of the transcriptional expression of structural genes of the anthocyanin biosynthetic pathway was determined by qRT-PCR in the fruit peel of Aft/Aft ATV/ATV (abbreviated as Aft/Aft) and Aft/Aft atv/atv in a F₅ population, and the two parent lines Heinz1706 (genotype, AFT/AFT ATV/ATV) and Indigo Rose (Aft/Aft atv/atv). A tomato ACTIN (Solyc03g078400) gene was used as the reference: relative expression levels are shown. Different letters indicate statistically significant differences among groups (Tukey’s honestly significant difference test, P<0.01). PAL, phenyl alanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; CHI-LIKE, chalcone isomerase-like; F3H, flavanone 3-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, leucoanthocyanidin dioxygenase; 3-GT, 3-O-glucosyltransferase; RT, rhamnosyl transferase; AAC, anthocyanin acyltransferase; 5-GT, 5-O-glucosyltransferase; PAT, putative anthocyanin transporter; GST, glutathione S-transferase; F3′H, flavonoid 3′-hydroxilase; FLS, flavonol synthase.

Fig. 6.

Transcriptional analysis of the candidate regulators of the anthocyanin biosynthetic pathway in the peel of tomato fruit. (A) The candidate members of the MBW activation complex. (B) The candidate MYB repressors of the anthocyanin biosynthetic pathway. A tomato ACTIN (Solyc03g078400) gene was used as reference: relative expression levels are shown. Different letters indicate statistically significant differences among groups (Tukey’s honestly significant difference test, P<0.01).

Fig. 7.

A putative model of the anthocyanin gene regulation network in the peel of tomato fruit. This model is modified from fig. 8 in the previously published paper [Albert et al. (2014). A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. The Plant Cell 26, 962–980. Republished with permission (www.plantcell.org), ‘Copyright American Society of Plant Biologists’] according to the expression patterns and putative functions of the corresponding genes that we determined in the present study. In the fruit peel of Aft/Aft (abbreviation of Aft/Aft ATV/ATV) plants (1) anthocyanin biosynthesis is initiated by activating the expression of the AFT gene under high light or low temperature. (2) The AFT protein (MYB activator, most likely SlAN2 or SlAN2-like) interacts with SlJAF13 and SlAN11 to form a MBW complex that activates the expression of SlAN1 (3). (4) SlAN1 interacts with AFT and SlAN11 to form a core MBW activation complex, and the complex activates the expression of the SlAN1 gene and most of the structural genes (5), and enhances the anthocyanin pigmentation. (6) The MBW complex also activates the expression of R2R3 MYB repressors. (7) The R2R3 MYB repressor, most likely SlMYB76, might intrude into the MBW complex and repress the anthocyanin pigmentation. (8) The MBW complex also activates the expression of R3 MYB repressors. (9) The R2R3 MYB repressor, e.g. SlMYBATV, competes with AFT to bind SlAN1 to inhibit the formation of new MBW complexes. This feedback inhibition can prevent the production of too much anthocyanin.