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. 2009 Dec;5(12):e1000777.
doi: 10.1371/journal.pgen.1000777. Epub 2009 Dec 18.

Fruit-surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-regulated Transcriptional Network

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Fruit-surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-regulated Transcriptional Network

Avital Adato et al. PLoS Genet. .
Free PMC article

Abstract

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB-type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The y mutant phenotype and its cuticular properties.
(A) Enzymatically isolated cuticles (n = 20) of wt and y mutant at five stages of fruit development. (B) Biomechanical tests of isolated fruit cuticles (n = 7) at the red stage of development reveal significant differences in the elastic phase between wt and y mutant cuticles.
Figure 2
Figure 2. Gene-expression alterations in the y mutant fruit as revealed by array analysis.
(A) Functional category distribution among differentially expressed wt and y mutant transcripts at the three latest stages of fruit development. (B) The expression profile (obtained by array analysis) of genes belonging to cluster 14 (total 38 members) in the peel tissue of y mutant and wt fruit. In the y mutant, array analysis was carried out on three of the five stages of fruit development examined in the wt fruit.
Figure 3
Figure 3. Differences between metabolic profiles of wt and y mutant peel and flesh tissues detected by principal component analysis (PCA) of GC-MS and LC-QTOF-MS data sets.
(A) PCA of metabolic profiles obtained by GC-MS analysis, with samples of wt and y peel and flesh tissues at five stages of fruit development (n = 3). (B) PCA of metabolic profiles obtained by UPLC-QTOF-MS analysis, with samples of wt and y peel and flesh tissues in the last three stages of fruit development (n = 3). Distinct metabolic profiles that correspond to particular stages of y and wt fruit development are encircled in (A,B).
Figure 4
Figure 4. GC-MS analyses detected 27 out of 56 assigned polar metabolites (Mintz-Oron et al. [6]) as having significantly different levels between wt and y mutant peel and/or flesh at at least one tested stage of fruit development.
Indicated by asterisks are significant differences as analyzed by two-way ANOVA and post-hoc analyses, see Materials and Methods (n = 3 for each sample). Y axis indicates metabolites' relative quantification by normalization of their response values to the Ribitol internal standard (IS) (see also Mintz-Oron et al. [6]).
Figure 5
Figure 5. Alterations in metabolite and gene-expression levels in the phenylpropanoid pathway as detected in the y mutant fruit peel tissue.
(A) Changes in gene expression (detected using array and/or real-time PCR analyses) and metabolite levels (detected by UPLC-QTOF-MS and GC-MS analyses) in tomato fruit peel. Red and blue colors represent up- and down-regulation, respectively. (B) Real-time PCR relative expression analyses of selected transcripts from the phenylpropanoid pathway in wt and y mutant tomato peels at the breaker stage of fruit development. Indicated by asterisks are significant differences analyzed by Student's t-test (n = 3; P<0.05; bars indicate standard errors). Gene identifiers and RT–PCR primers are listed in Table S3. #, caffeoylquinic acid derivatives that include: 4-caffeoylquinic acid, 5-caffeoylquinic acid, dicaffeoylquinic acid I/II/III, tricaffeoylquinic acid and 3-dicaffeoylquinic acid, detected but not altered in both peel and flesh of the y mutant. Metabolites marked by * or ** may be NarCh or Nar derivatives.
Figure 6
Figure 6. Composition of the y mutant and wt fruit cuticular waxes was tested at three stages of fruit development, analyzed by GC-MS and GC-FID (n = 5; P<0.05; bars indicate standard errors).
(A) Total cuticular wax load expressed as mg/cm2. (B) Amounts (%) relative to total wax coverage of individual compounds that were significantly altered at the breaker stage of fruit development, (C) at the orange stage of fruit development, and (D) at the ripe red stage of fruit development. 29an, C29 alkane; 30an, C30 alkane; iso31an, C31 iso alkane; 24ic, tetracosanoic acid.
Figure 7
Figure 7. Changes in metabolite and gene-expression levels in the isoprenoid pathway detected in y mutant fruit tissues.
(A) HPLC-PDA analysis detected six isoprenoids that differ significantly between the wt and y mutant fruit tissues at the orange stage of fruit development. Indicated by asterisks are significant differences analyzed by Student's t-test (n = 3; P<0.05; bars indicate standard errors). (B) Changes in isoprenoid-related gene expression (detected by array analysis) and metabolite levels (detected by HPLC-PDA) in tomato fruit peel and flesh tissues. Red and blue colors represent up- and down-regulation, respectively. Significant down-regulation of SlCRTR-B2 and SlNCED transcript levels in the y mutant peel was confirmed by RT–PCR analyses (Student's t-test, n = 3; P<0.05; bars indicate standard errors). Primers, transcript identifiers/accessions and expression data are listed in Table S4.
Figure 8
Figure 8. Phylogeny and expression analyses of putative phenylpropanoid/flavonoid-related transcription factor genes.
(A) Phylogenetic analysis of the putative tomato regulators reported in our study and known phenylpropanoid/flavonoid-related transcription factors from other species. The ClustalX and NJplot softwares were used to compute the tree and its significance (bootstrap) values. (B) RT–PCR relative expression analyses of selected tomato transcription factors putatively related to the regulation of the phenylpropanoid/flavonoid pathway in wt and y mutant tissues at the breaker stage of fruit development. Indicated by asterisks are significant differences analyzed by Student's t-test (n = 3; P<0.05; bars indicate standard errors). Gene identifiers and primers are listed in Table S3. (C) Real Time RT–PCR relative expression analysis of SlMYB12 in wt fruit tissues through five developmental stages reveals a peel-associated expression pattern. IG, Immature Green; MG, Mature Green; Br, Breaker; Or, Orange; Re, Red, stages of fruit development (n = 3; P<0.05; bars indicate standard errors). (D) Structure of the SlMYB12 gene. Gray regions represent coding sequence and white represents UTRs. Arrows indicate the position of RT–PCR primers, red brackets indicate the positions of premature polyadenylation sites (in the y-1 mutant allele), and the black bracket indicates the position of the alternative wt polyadenylation site.
Figure 9
Figure 9. The transgenic amiR-SlMYB12 lines exhibit a y-like phenotype.
(A) Red box indicates the location of the amiR-SlMYB12 target sequence on the SlMYB12 gene, arrows indicate the position of RT–PCR primers, and the sequence alignment on the right demonstrates the specificity of this artificial microRNA. (B) Expression of the amiR-SlMYB12 precursor in samples extracted from leaves of 35S:amiR-SlMYB12-transgenic lines and non-transgenic controls. (C) Fruit of 35S:amiR-SlMYB12-transgenic lines display colorless peel. (D) RT–PCR relative expression analysis of phenylpropanoid/flavonoid-related regulators and structural genes in fruit peel of wt and 35S:amiR-SlMYB12-transgenic lines. Indicated by asterisks are significantly reduced levels analyzed by Student's t-test (n = 3; P<0.05; bars indicate standard errors). (E) PCA of metabolic profiles obtained by UPLC-QTOF-MS analysis carried out on peel samples of wt cv. AC and cv. MT, y mutant and an amiR-SlMYB12-transgenic line at the red stage of fruit development. Analysis was performed with the TMEV program using normalized and log-transformed data. (F) Total ion chromatograms (TICs) of wt (cv. MT) and 35S:amiR-SlMYB12 peels at the red stage of fruit development, acquired in the negative mode using UPLC-QTOF-MS (in relative intensity, 100% corresponds to 6.14×104 counts). The putative identity of the differential compounds is: 1 - quercetin-dihexose-deoxyhexose, 2 - quercetin-hexose-deoxyhexose-pentose, 3 - quercetin-rutinoside (rutin), 4 - phloretin-di-C-hexose, 5 - kaempferol-glucose-rhamnose, 6 - naringenin chalcone, 7 - dicaffeoylquinic acid III, 8 - tricaffeoylquinic acid. Red and blue numbers indicate metabolites that showed elevated or reduced levels in the transgene samples in comparison to those of their corresponding wt, respectively. (G) Relative levels of NarCh in cv. MT and 35S:amiR-SlMYB12, expressed as chromatographic peak areas, calculated for m/z 271.06 Da (n = 5).
Figure 10
Figure 10. Sectorial phenotype complementation in a transgenic y line constitutively expressing the SlMYB12 gene under the 35S CaMV promoter (35S:SlMYB12).
(A) Peels of red fruit from the 35S:SlMYB12 line on a y background, the y mutant and wt cv. AC. (B) UPLC-PDA analysis of red fruit peels reveals significantly different levels of flavanoids between regions of phenotype complementation in peels of the 35S:SlMYB12 line and those of the y mutant (n = 3; P<0.01; bars represent standard error). The UPLC instrument used in this analysis is equipped with an Acquity 2996 PDA detector. Sample preparation and LC conditions were as described for the UPLC-QTOF-MS analysis. Compounds peak areas were determined by Empower 2 software (Waters) at 370 nm for naringenin chalcone (NarCh) and at 256 nm for quercetin-hexose-deoxyhexose-pentose (Q-trisacch) and quercetin-rutinoside (rutin).

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