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. 2016 Jul;171(3):1821-36.
doi: 10.1104/pp.16.00282. Epub 2016 May 6.

Characterization of a New Pink-Fruited Tomato Mutant Results in the Identification of a Null Allele of the SlMYB12 Transcription Factor

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Characterization of a New Pink-Fruited Tomato Mutant Results in the Identification of a Null Allele of the SlMYB12 Transcription Factor

Josefina-Patricia Fernandez-Moreno et al. Plant Physiol. .
Free PMC article

Abstract

The identification and characterization of new tomato (Solanum lycopersicum) mutants affected in fruit pigmentation and nutritional content can provide valuable insights into the underlying biology, as well as a source of new alleles for breeding programs. To date, all characterized pink-pigmented tomato fruit mutants appear to result from low SlMYB12 transcript levels in the fruit skin. Two new mutant lines displaying a pink fruit phenotype (pf1 and pf2) were characterized in this study. In the pf mutants, SlMYB12 transcripts accumulated to wild-type levels but exhibited the same truncation, which resulted in the absence of the essential MYB activation domain coding region. Allelism and complementation tests revealed that both pf mutants were allelic to the y locus and showed the same recessive null allele in homozygosis: Δy A set of molecular and metabolic effects, reminiscent of those observed in the Arabidopsis (Arabidopsis thaliana) myb11 myb12 myb111 triple mutant, were found in the tomato Δy mutants. To our knowledge, these have not been described previously, and our data support the idea of their being null mutants, in contrast to previously described transcriptional hypomorphic pink fruit lines. We detected a reduction in the expression of several flavonol glycosides and some associated glycosyl transferases. Transcriptome analysis further revealed that the effects of the pf mutations extended beyond the flavonoid pathway into the interface between primary and secondary metabolism. Finally, screening for Myb-binding sites in the candidate gene promoter sequences revealed that 141 of the 152 co-down-regulated genes may be direct targets of SlMYB12 regulation.

Figures

Figure 1.
Figure 1.
Schematic overview of the flavonoid biosynthetic pathway and regulation in tomato fruit. The amino acid l-Phe is transformed into naringenin chalcone by the action of PAL, C4H, 4CL, and CHS enzymes. Naringenin chalcone is the precursor of different flavonoid classes: chalcones, flavonones, flavones, isoflavones, dihydroflavonols, and flavonols. SlMYB12 TF is the main activator of the flavonol biosynthetic branch to act on PAL, 4CL, CHS, CHI, F3H, and FLS genes (Adato et al., 2009; Ballester et al., 2010). 3GT, 3-O-GLYCOSYL TRANSFERASE; RT, RHAMNOSYL TRANSFERASE.
Figure 2.
Figure 2.
The pink fruit phenotype in both the pf1 and pf2 mutants. A, The phenotype for tomato fruit, fruit skin, and methanolic extracts in both pf mutants and the wild type. Relative quantifications for both naringenin chalcone (Nc) and naringenin (N) metabolites (Metabol.) are also shown (see metabolites #8 and #14, respectively, in Supplemental Table S1). B, Gene expression level of SlMYB12 detected by RT-PCR analysis in both pf mutants and wild-type fruit skin. C, Allelism test of both pf mutants and the wild type. D, Complementation tests comparing pf1 and pf2 mutants, and both pf mutants and the y mutant (cv Ailsa Craig; AC-y). Alleles in C and D are based on molecular results (see about Δy further in the text). MeOH Extr., methanol extracts; ywt, wild-type allele for the y locus.
Figure 3.
Figure 3.
The Slmyb12-Δy allele in the pf mutants. A, SlMYB12 gene expression levels (FPKMs) in RNA-Seq analysis for both Δy mutants and the wild type. B, SlMYB12 genomic region and RNA-Seq read mapped gene expression profile. The two genes placed downstream of SlMYB12 in the genome are also shown. C, SlMYB12 PCR amplicons using genomic DNA (gDNA) templates from the wild type, Δy1, and Δy2 mutants. See both size and location of the PCR reactions in D. D, SlMYB12 genomic sequence (available in tomato database: http://www.solgenomics.net/) and the PCR/RT-PCR oligonucleotide distribution in the gene sequence. The location of the putative deletion in SlMYB12 is indicated by a red asterisk. MFR, Myb-like flavonol regulation motif; SG7, subgroup 7 with the flavonol regulation motif; SG7-2, subgroup 7-2 with the specific flavonol regulation motif.
Figure 4.
Figure 4.
Flavonol biosynthesis is blocked in the Δy mutants. Genes differentially expressed in the Δy mutants are indicated by blue (down-regulated) or red (up-regulated) arrows (thicker arrows represent a higher-fold change). The MBS motifs found in the promoter region of the down-regulated genes are also included (in colored boxes). Bar graphs show the metabolite levels for flavonols. The wild type is represented in yellow (left), the Δy1 mutant in dashed gray (middle), and the Δy2 mutant in dotted gray (right). Error bars are standard deviations (n = 3). Genes and metabolites differentially expressed also in the y mutant and in the other y lines are marked “a” and “b,” respectively, and those differentially expressed only in the Δy mutants are marked “c” (in red). Numbers in bar graphs correspond to metabolites in Supplemental Table S1. EBGs, early biosynthetic genes; N, naringenin; Nc, naringenin chalcone; HNc, hydroxylated naringenin chalcone (eriodictyol-chalcone); diHNc, dihydronaringenin chalcone (phloretin); HN, hydroxylated naringenin (eriodictyol); DK, dihydrokaempferol; DQ, dihydroquercetin; K, kaempferol; Q, quercetin; H, hexose; dxh, deoxyhexose; P, pentose; gl, Glc; rh, rhamnose; mh, malonyl-hexose; pCA, p-coumaric acid; Rut, rutin; a’, the ANS gene was down-regulated in the y mutant.
Figure 5.
Figure 5.
Genes differentially expressed in the Δy mutants detected by the RNA-Seq approach. Venn diagram including all genes differentially expressed in mutant Δy1 and Δy2 (dark-blue and light-blue circles, respectively). Those genes also detected as being differentially expressed in the y mutant (pink circle) by the Affymetrix microarray approach were also included. The genes shared between mutant Δy1 and Δy2, and between both the Δy mutants and the y mutant, were represented in a white circle. Shared up-regulated genes (↑) and shared down-regulated genes (↓) are also represented.
Figure 6.
Figure 6.
Genes up- and downstream of flavonol biosynthesis are also affected in the Δy mutants. The figure represents the next biosynthetic processes upstream of flavonoid biosynthesis: TCA cycle; shikimate, chorismate, and arogenate pathways; core phenylpropanoid pathway; phenolic acid and lignin pathways; resveratrol biosynthesis; and l-Phe catabolism. In addition, the flavonoid transport process is shown downstream of flavonoid biosynthesis. Those genes differentially down-regulated (blue) are shown by arrows (the thicker the arrow, the higher the fold change). MBS motifs found in their promoters were also included (in colored boxes). Genes in gray were not differentially expressed in the Δy mutants. Genes and metabolites differentially expressed both in the y mutant and in other y-lines are marked “a” and “b,” respectively; those differentially expressed only in the Δy mutants are marked “c” (in red). NAD+-ME, NAD+-MALIC ENZYME; PDT, PREPHRENATE DEHYDRATASE; COMT, CAFFEIC ACID O-METHYLTRANSFERASE; HCT, CINNAMOYL COA TRANSFERASE; UDP-GT, GLUCURONOSYL TRANSFERASE.
Figure 7.
Figure 7.
In silico MBS motifs found in potential target genes of SlMYB12. A, Venn diagram representing the MBSs (MBS1 and MBS2) recognized by AtMYB12 TF and found using FUZZNUC in 114 of the 152 genes in the down-regulated gene set of Δy mutants. Bar graphs represent the number of genes with promoters containing one or more than one MBS1 (in red), MBS2 (in blue), or both MBS (in gray) motifs. B, Consensus sequences for MBS1 and MBS2 FUZZNUC motifs detected in the 114 genes. MYB12-related MBS motifs detected by the Tomtom tool in both MEME and DREME motifs are also shown. These were AtMYB111 (primary motif) and AtMYB111_2 (secondary motif) MBSs. Finally, the consensus sequence for the six R2-R3-type MBSs detected in the analyses was also provided (see Supplemental Tables S5, A–D). C, Venn diagram showing the distribution of those down-regulated genes containing MBS motifs based on the different MBS search tools used.

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