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Tomato nuclear proteome reveals the involvement of specific E2 ubiquitin-conjugating enzymes in fruit ripening.

Wang Y, Wang W, Cai J, Zhang Y, Qin G, Tian S - Genome Biol. (2014)

Bottom Line: Virus-induced gene silencing assays show that two E2s are involved in the regulation of fruit ripening.Our results uncover a novel function of protein ubiquitination, identifying specific E2s as regulators of fruit ripening.These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Fruits are unique to flowering plants and play a central role in seed maturation and dispersal. Molecular dissection of fruit ripening has received considerable interest because of the biological and dietary significance of fruit. To better understand the regulatory mechanisms underlying fruit ripening, we report here the first comprehensive analysis of the nuclear proteome in tomato fruits.

Results: Nuclear proteins were isolated from tomatoes in different stages of ripening, and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. We show that the proteins whose abundances change during ripening stages are involved in various cellular processes. We additionally evaluate changes in the nuclear proteome in the ripening-deficient mutant, ripening-inhibitor (rin), carrying a mutation in the transcription factor RIN. A set of proteins were identified and particular attention was paid to SlUBC32 and PSMD2, the components of ubiquitin-proteasome pathway. Through chromatin immunoprecipitation and gel mobility shift assays, we provide evidence that RIN directly binds to the promoters of SlUBC32 and PSMD2. Moreover, loss of RIN function affects protein ubiquitination in nuclei. SlUBC32 encodes an E2 ubiquitin-conjugating enzyme and a genome-wide survey of the E2 gene family in tomatoes identified five more E2s as direct targets of RIN. Virus-induced gene silencing assays show that two E2s are involved in the regulation of fruit ripening.

Conclusions: Our results uncover a novel function of protein ubiquitination, identifying specific E2s as regulators of fruit ripening. These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.

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Related in: MedlinePlus

Electrophoretic mobility shift assay of RIN bindingto the regulatory regions of target genes. (a) SDS-PAGE gelshows the affinity purification of the recombinant RIN protein used forthe electrophoretic mobility shift assay. (b) RIN binds specifically to the promoters of target genescontaining CArG box elements. The promoter structures of the target genesare presented. Blue boxes indicate CArG box elements in the promoterregion and numbers represent the position of these motifs relative to thetranslational start site. The probe sequences for each target gene areshown, with red letters representing the CArG box. The mutated bases inthe probes are represented by blue letters. wt, probe with intact CArG boxelement; mt, probe with mutated CArG box element. As competitors,1,000-fold excess amounts of unlabeled probes were added to the bindingreaction. The retarded bands and the free probes are indicated byarrowheads.
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Fig7: Electrophoretic mobility shift assay of RIN bindingto the regulatory regions of target genes. (a) SDS-PAGE gelshows the affinity purification of the recombinant RIN protein used forthe electrophoretic mobility shift assay. (b) RIN binds specifically to the promoters of target genescontaining CArG box elements. The promoter structures of the target genesare presented. Blue boxes indicate CArG box elements in the promoterregion and numbers represent the position of these motifs relative to thetranslational start site. The probe sequences for each target gene areshown, with red letters representing the CArG box. The mutated bases inthe probes are represented by blue letters. wt, probe with intact CArG boxelement; mt, probe with mutated CArG box element. As competitors,1,000-fold excess amounts of unlabeled probes were added to the bindingreaction. The retarded bands and the free probes are indicated byarrowheads.

Mentions: To confirm that RIN interacts with the promoters of E2 genesidentified in the ChIP assay, we carried out an EMSA with purified recombinant RINprotein (Figure 7a). For each gene, adouble-stranded and biotin-labeled probe (26-mer oligonucleotide) (Additional file11) containing the CArG-box elementwas made, and its binding by the RIN protein was analyzed. A shift band wasobserved for each gene when the recombinant RIN protein was mixed with thebiotin-labeled probe, indicating that RIN protein bound well to the biotin-labeledpromoter fragments (Figure 7b). Thebinding of RIN protein to these fragments was effectively competed by addition ofan excessive amount of the corresponding unlabeled probe with intact CArG boxelement, but not by the probe with mutated CArG box element. These resultsindicated that RIN binds specifically to the biotin-labeled probe. Furthermore, weobserved different extents of competition by the unlabeled DNA fragment. Thissuggests that RIN has differential binding ability to the promoters of thesegenes. Together, our data suggest that RIN binds directly to the promoters ofSlUBC6, 8,24, 41,and 42. Considering SlUBC32, the direct RIN target identified on basis of thecomparative analysis of nuclear proteome, a total of six E2 genes were identifiedas the direct targets of RIN.Figure 7


Tomato nuclear proteome reveals the involvement of specific E2 ubiquitin-conjugating enzymes in fruit ripening.

Wang Y, Wang W, Cai J, Zhang Y, Qin G, Tian S - Genome Biol. (2014)

Electrophoretic mobility shift assay of RIN bindingto the regulatory regions of target genes. (a) SDS-PAGE gelshows the affinity purification of the recombinant RIN protein used forthe electrophoretic mobility shift assay. (b) RIN binds specifically to the promoters of target genescontaining CArG box elements. The promoter structures of the target genesare presented. Blue boxes indicate CArG box elements in the promoterregion and numbers represent the position of these motifs relative to thetranslational start site. The probe sequences for each target gene areshown, with red letters representing the CArG box. The mutated bases inthe probes are represented by blue letters. wt, probe with intact CArG boxelement; mt, probe with mutated CArG box element. As competitors,1,000-fold excess amounts of unlabeled probes were added to the bindingreaction. The retarded bands and the free probes are indicated byarrowheads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269173&req=5

Fig7: Electrophoretic mobility shift assay of RIN bindingto the regulatory regions of target genes. (a) SDS-PAGE gelshows the affinity purification of the recombinant RIN protein used forthe electrophoretic mobility shift assay. (b) RIN binds specifically to the promoters of target genescontaining CArG box elements. The promoter structures of the target genesare presented. Blue boxes indicate CArG box elements in the promoterregion and numbers represent the position of these motifs relative to thetranslational start site. The probe sequences for each target gene areshown, with red letters representing the CArG box. The mutated bases inthe probes are represented by blue letters. wt, probe with intact CArG boxelement; mt, probe with mutated CArG box element. As competitors,1,000-fold excess amounts of unlabeled probes were added to the bindingreaction. The retarded bands and the free probes are indicated byarrowheads.
Mentions: To confirm that RIN interacts with the promoters of E2 genesidentified in the ChIP assay, we carried out an EMSA with purified recombinant RINprotein (Figure 7a). For each gene, adouble-stranded and biotin-labeled probe (26-mer oligonucleotide) (Additional file11) containing the CArG-box elementwas made, and its binding by the RIN protein was analyzed. A shift band wasobserved for each gene when the recombinant RIN protein was mixed with thebiotin-labeled probe, indicating that RIN protein bound well to the biotin-labeledpromoter fragments (Figure 7b). Thebinding of RIN protein to these fragments was effectively competed by addition ofan excessive amount of the corresponding unlabeled probe with intact CArG boxelement, but not by the probe with mutated CArG box element. These resultsindicated that RIN binds specifically to the biotin-labeled probe. Furthermore, weobserved different extents of competition by the unlabeled DNA fragment. Thissuggests that RIN has differential binding ability to the promoters of thesegenes. Together, our data suggest that RIN binds directly to the promoters ofSlUBC6, 8,24, 41,and 42. Considering SlUBC32, the direct RIN target identified on basis of thecomparative analysis of nuclear proteome, a total of six E2 genes were identifiedas the direct targets of RIN.Figure 7

Bottom Line: Virus-induced gene silencing assays show that two E2s are involved in the regulation of fruit ripening.Our results uncover a novel function of protein ubiquitination, identifying specific E2s as regulators of fruit ripening.These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Fruits are unique to flowering plants and play a central role in seed maturation and dispersal. Molecular dissection of fruit ripening has received considerable interest because of the biological and dietary significance of fruit. To better understand the regulatory mechanisms underlying fruit ripening, we report here the first comprehensive analysis of the nuclear proteome in tomato fruits.

Results: Nuclear proteins were isolated from tomatoes in different stages of ripening, and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. We show that the proteins whose abundances change during ripening stages are involved in various cellular processes. We additionally evaluate changes in the nuclear proteome in the ripening-deficient mutant, ripening-inhibitor (rin), carrying a mutation in the transcription factor RIN. A set of proteins were identified and particular attention was paid to SlUBC32 and PSMD2, the components of ubiquitin-proteasome pathway. Through chromatin immunoprecipitation and gel mobility shift assays, we provide evidence that RIN directly binds to the promoters of SlUBC32 and PSMD2. Moreover, loss of RIN function affects protein ubiquitination in nuclei. SlUBC32 encodes an E2 ubiquitin-conjugating enzyme and a genome-wide survey of the E2 gene family in tomatoes identified five more E2s as direct targets of RIN. Virus-induced gene silencing assays show that two E2s are involved in the regulation of fruit ripening.

Conclusions: Our results uncover a novel function of protein ubiquitination, identifying specific E2s as regulators of fruit ripening. These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.

Show MeSH
Related in: MedlinePlus