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. 2010 Jan;152(1):71-84.
doi: 10.1104/pp.109.147322. Epub 2009 Nov 11.

Biochemical and Molecular Analysis of Pink Tomatoes: Deregulated Expression of the Gene Encoding Transcription Factor SlMYB12 Leads to Pink Tomato Fruit Color

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Biochemical and Molecular Analysis of Pink Tomatoes: Deregulated Expression of the Gene Encoding Transcription Factor SlMYB12 Leads to Pink Tomato Fruit Color

Ana-Rosa Ballester et al. Plant Physiol. .
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Abstract

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

Figures

Figure 1.
Figure 1.
Schematic overview of the flavonoid pathway in tomato. PAL, Phe ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; HCT, cinnamoyl-CoA shikimate/quinate transferase; C3H, p-coumaroyl ester 3-hydroxylase; HQT, hydroxycinnamoyl-CoA quinate transferase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; F3′H, flavonoid-3′-hydroxylase; F3′5′H, flavonoid-3′5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; 3GT, flavonoid-3-O-glucosyltransferase; RT, flavonoid 3-O-glucoside-rhamnosyltransferase; AAC, anthocyanin acyltransferase; 5GT, flavonoid-5-glucosyltransferase.
Figure 2.
Figure 2.
Phenotypic analysis of S. lycopersicum ‘Moneyberg’ (red tomato) and S. chmielewskii IL1b (pink tomato). Whole fruit (left and middle) and peel (right) of red (top) and pink (bottom) ripe tomato fruit are shown.
Figure 3.
Figure 3.
Schematic overview of tomato chromosome 1 showing the locations of 14 COSII markers (whose names begin with C2_At) and a simple sequence repeat (SSR) marker. Numbers near the marker names indicate the genetic distances in cM from the top of the chromosome, based on the tomato-EXPEN 2000 mapping population (http://sgn.cornell.edu/). At the right part of chromosome 1, the total genetic length is mentioned. The S. chmielewskii IL population comprised four lines with single homozygous introgressions on chromosome 1 (black bars). The borders of the bars are arbitrarily drawn midway between markers positive and negative for the introgressed S. chmielewskii segment. The genetically mapped interval of the locus underlying the pink fruit phenotype is indicated by inward-facing arrows.
Figure 4.
Figure 4.
Phenylpropanoid and flavonoid contents in peel and flesh of fruit from tomato cv Moneyberg (black squares) and IL1b (white circles) at four different stages of ripening. Values represent averages of three biological replicates. FW, Fresh weight.
Figure 5.
Figure 5.
Carotenoid contents in peel and flesh of fruit from tomato cv Moneyberg (black squares) and IL1b (white circles) at four different stages of ripening. Values represent averages of three biological replicates. FW, Fresh weight.
Figure 6.
Figure 6.
Relative expression of phenylpropanoid and flavonoid genes in peel of Moneyberg (black squares) and IL1b (white circles) tomatoes at different stages of ripening. Expression levels were determined by qRT-PCR and expressed relative to the expression of the ubiquitin gene. Values represent averages of three biological replicates, each with two technical replicates.
Figure 7.
Figure 7.
A, Relative expression of MYB12 in peel and flesh of red (black squares) and pink (white circles) tomatoes harvested at different stages of ripening, determined by qRT-PCR. Values represent means of three biological replicates ± sd. B, Correlation analysis of the structural phenylpropanoid and flavonoid gene expression profiles with the expression levels of the MYB12 gene.
Figure 8.
Figure 8.
A, Alignment of deduced amino acid sequences of SlMYB12 alleles, including sequences of tomato cv Moneyberg (accession no. CBG76063.1), IL1b (accession no. CBG76064.1), the published sequence EU419748, and four pink tomato cultivars (TJ1, TJ2, TJ3, and TJ4). Amino acids different in one of the three sequences are boxed in gray. B, Schematic overview of the genomic structures of the SlMYB12 gene in Moneyberg (MB) and S. chmielewskii (Sc). The MYB12 gene consists of three exons (large boxes, light gray) and three introns (small boxes, dark gray). The duplicated sequence (53 bp) in the second intron is shown as a hatched box. The yellow box represents the additional 72 nucleotides detected in the S. chmielewskii sequence. Primers used to map the gene are indicated as i628F and i909R.
Figure 9.
Figure 9.
Mapping of the MYB12 gene in the IL population derived from a cross between cultivated tomato S. lycopersicum ‘Moneyberg’ (MB) and the wild species S. chmielewskii (Sc). Ch., Chromosome. M, S. lycopersicum ‘Moneymaker’. Asterisks indicate pink tomatoes.
Figure 10.
Figure 10.
SlMYB12 gene silencing in the cultivated tomato Moneymaker (M). A, Peel sections of fruit agroinjected with pTRV1 + pTRV2-SlMYB12 (right) or pTRV1 alone as control (left). M_S, Silenced sectors showing a colorless phenotype; M_NS, nonsilenced sectors. B, Peel of fruit agroinjected with pTRV1 + pTRV2-SlMYB12, showing the colorless silenced sectors and the colored nonsilenced sectors (control). C, Effect of SlMYB12 gene silencing in Moneymaker tomato fruit. Contents of naringenin chalcone, rutin, and chlorogenic acid in peel of four independent silenced tomato fruit and in peel of six control fruit agroinjected with pTRV1 alone are shown. FW, Fresh weight.

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