The atroviolacea Gene Encodes an R3-MYB Protein Repressing Anthocyanin Synthesis in Tomato Plants

Abstract

The anthocyanin biosynthetic pathway is well characterized in plants. However, in tomato (Solanum lycopersicum L.) an exhaustive knowledge of its regulation is still lacking. Tomato mutants showing higher levels of anthocyanins in fruits or vegetative tissues, such as Anthocyanin fruit (Aft) or atroviolacea (atv), have been extensively exploited in the attempt to clarify the process. Nevertheless, only candidate genes have been proposed as responsible for such phenotypes. The recessive atv mutation likely represents an allelic variant of a gene introgressed in tomato from wild Solanum species. We performed genome sequencing of atv/atv plants followed by candidate gene analysis, and identified a mutated gene encoding an R3-MYB protein. When overexpressed, this protein abolished anthocyanin production in tomato seedlings and plants, by silencing key regulators and biosynthetic genes of the pathway. The functional analysis of the protein clearly showed that it can negatively interfere with the activation of the anthocyanin biosynthetic pathway mediated by the endogenous MYB-bHLH-WDR (MBW) complexes. In particular, this R3-MYB protein can directly bind the bHLH factors which are part of the MBW complexes, therefore acting as a competitive inhibitor. The R3-MYB protein here described is therefore involved in a feedback mechanism that dampens the production of anthocyanins once activated by endogenous or exogenous stimuli. The atv mutation causes the production of a truncated version of the R3-MYB factor that cannot retain the full potential to inhibit the MBW complexes, thus leading to a constitutively higher production of anthocyanins.

Anthocyanin Fruit encodes an R2R3‐MYB transcription factor, SlAN2‐like, activating the transcription of SlMYBATV to fine‐tune anthocyanin content in tomato fruit

“…The CRISPR/Cas9‐mediated SlMYBATV mutants have a much higher anthocyanin content, which is consistent with the higher expression levels of anthocyanin biosynthesis genes (except CHI‐like ) in fruit than that in LA1996 (Figs c,e, S10). The results herein were consistent with the repressive function of SlMYBATV in anthocyanin biosynthesis in tomato (Cao et al , ; Colanero et al , ). Notably, the expression level of SlMYBATV was much higher in the ATV ‐M1 mutant than that in LA1996 (Fig.…”

“…SlMYBATV was previously predicted to compete with the R2R3‐MYB regulator to bind bHLH proteins to inhibit the formation of the MBW complexes (Cao et al , ; Colanero et al , ). Thus, Y2H assays were used to investigate whether SlMYBATV (transcripts of SlMYBATV‐X1, ‐X2 and ‐X3) could bind to the bHLH regulator SlAN1 or SlJAF13.…”

“…In addition to transcriptional regulators, negative regulators, such as R2R3‐MYB and R3‐MYB proteins, have been characterized (Ma & Constabel, ). Negative regulators can disrupt the activity of the MBW complex by directly sequestering specific transcription factors and indirectly suppressing the transcription of genes encoding components of the MBW complex (Zhu et al , ; Albert et al , ; Colanero et al , ; Zhou et al , ). For example, the R3‐MYB repressors PtMYBx in petunia and CPC in Arabidopsis are thought to compete with R2R3‐MYB activators for bHLH binding sites and/or anthocyanin biosynthetic genes (Zhu et al , ; Albert et al , ).…”

“…For example, the tomato cultivar (cv) Indigo Rose contains the Aft and atv loci, which have been introgressed from Solanum chilense and S. cheesemanii , respectively (Kendrick et al , ; Jones, ); this cultivar bears purple fruit with a high anthocyanin content (Cao et al , ). Recently, a gene at the atv locus related to the repression of anthocyanin synthesis, SlMYBATV ( Solyc07g052490 ), was identified on the long arm of chromosome 7 and encodes an R3‐MYB protein (Cao et al , ; Colanero et al , ).…”

Optimizing shelf life conditions for anthocyanin-rich tomatoes

“…lycopersicum . Whereas the Aft gene likely encodes an R2R3 MYB activator of the anthocyanin biosynthetic pathway, whose precise identity is still under debate [ 17 , 18 ], the atv gene has been recently identified and encodes for an R3 MYB repressor of the pathway [ 19 , 20 ]. A further cross between the Aft and atv lines led to the double Aft/Aft atv/atv genotype (commercially known as “Indigo™ Rose” or “Sun Black™”) characterized by a highly pigmented fruit phenotype [ 13 , 21 – 23 ].…”