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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

RIN directly binds to the promoter regions of targetgenes as revealed by chromatin immunoprecipitation. (a)Western blot revealed the specificity of the affinity-purified RINpolyclonal antibodies used for chromatin immunoprecipitation (ChIP) assay.Nuclear proteins were isolated from wild-type and rin mutant fruit at the orange ripening stage and hybridizedwith the RIN polyclonal antibodies. (b)ChIP-qPCR shows the binding of RIN to the promoter regions of five E2genes. The promoter structures of the target genes are presented. Blueboxes represent CArG box elements and numbers indicate the position ofthese motifs relative to the translational start site. Green fragmentswith upper-case letters represent the regions used for ChIP-qPCR. Valuesare the percentage of DNA fragments that co-immunoprecipitated withanti-RIN antibodies (black bars) or non-specific antibodies (preimmunerabbit IgG; grey bars) relative to the input DNAs. Error bars representthe SD of three independent experiments.
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Fig6: RIN directly binds to the promoter regions of targetgenes as revealed by chromatin immunoprecipitation. (a)Western blot revealed the specificity of the affinity-purified RINpolyclonal antibodies used for chromatin immunoprecipitation (ChIP) assay.Nuclear proteins were isolated from wild-type and rin mutant fruit at the orange ripening stage and hybridizedwith the RIN polyclonal antibodies. (b)ChIP-qPCR shows the binding of RIN to the promoter regions of five E2genes. The promoter structures of the target genes are presented. Blueboxes represent CArG box elements and numbers indicate the position ofthese motifs relative to the translational start site. Green fragmentswith upper-case letters represent the regions used for ChIP-qPCR. Valuesare the percentage of DNA fragments that co-immunoprecipitated withanti-RIN antibodies (black bars) or non-specific antibodies (preimmunerabbit IgG; grey bars) relative to the input DNAs. Error bars representthe SD of three independent experiments.

Mentions: Gene expression analysis showed that RIN affected the expression of15 E2 genes at two or more stages of ripening (Figure 5b). Among these genes, SlUBC32 has been identified as the direct target of RIN(Figure 4). To investigate whether RINregulates the other 14 E2 genes by directly binding to their promoters, the ChIPassay was performed to probe DNA-protein interactions within the naturalchromatin. Sequence analysis indicated that, except one (SlUBC38), 13 E2 genes contain CArG box binding motifs in theirpromoters (Additional file 5). For theChIP assay, the affinity-purified anti-RIN polyclonal antibody(Figure 6a) was used toimmunoprecipitate the cross-linked DNA-protein complexes. The enriched DNA waspurified and then submitted to real-time quantitative PCR analysis. Primer setswere designed for those 13 genes that contain CArG box binding motifs in theirpromoters (Additional file 6). Ourresults indicate that RIN binds to the promoters of SlUBC6, 8, 24, 41, and42 (Figure 6b). Notably, RIN shows differential binding ability to thepromoter fragments of these genes. The relative amounts of precipitated promoterfragments of SlUBC6, 24, and 42 were low (<0.2%).By contrast, the relative amounts of precipitated promoter fragments of SlUBC8 and 41 weremuch higher. The highest enrichments (>1%) was found for SlUBC41 promoter fragments. Very low enrichments wereobserved for all fragments when cross-linked DNA-protein complexes wereimmunoprecipitated with pre-immune rabbit IgG, the non-specific antibody. This isconsidered as non-specific background enrichment.Figure 6


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)

RIN directly binds to the promoter regions of targetgenes as revealed by chromatin immunoprecipitation. (a)Western blot revealed the specificity of the affinity-purified RINpolyclonal antibodies used for chromatin immunoprecipitation (ChIP) assay.Nuclear proteins were isolated from wild-type and rin mutant fruit at the orange ripening stage and hybridizedwith the RIN polyclonal antibodies. (b)ChIP-qPCR shows the binding of RIN to the promoter regions of five E2genes. The promoter structures of the target genes are presented. Blueboxes represent CArG box elements and numbers indicate the position ofthese motifs relative to the translational start site. Green fragmentswith upper-case letters represent the regions used for ChIP-qPCR. Valuesare the percentage of DNA fragments that co-immunoprecipitated withanti-RIN antibodies (black bars) or non-specific antibodies (preimmunerabbit IgG; grey bars) relative to the input DNAs. Error bars representthe SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: RIN directly binds to the promoter regions of targetgenes as revealed by chromatin immunoprecipitation. (a)Western blot revealed the specificity of the affinity-purified RINpolyclonal antibodies used for chromatin immunoprecipitation (ChIP) assay.Nuclear proteins were isolated from wild-type and rin mutant fruit at the orange ripening stage and hybridizedwith the RIN polyclonal antibodies. (b)ChIP-qPCR shows the binding of RIN to the promoter regions of five E2genes. The promoter structures of the target genes are presented. Blueboxes represent CArG box elements and numbers indicate the position ofthese motifs relative to the translational start site. Green fragmentswith upper-case letters represent the regions used for ChIP-qPCR. Valuesare the percentage of DNA fragments that co-immunoprecipitated withanti-RIN antibodies (black bars) or non-specific antibodies (preimmunerabbit IgG; grey bars) relative to the input DNAs. Error bars representthe SD of three independent experiments.
Mentions: Gene expression analysis showed that RIN affected the expression of15 E2 genes at two or more stages of ripening (Figure 5b). Among these genes, SlUBC32 has been identified as the direct target of RIN(Figure 4). To investigate whether RINregulates the other 14 E2 genes by directly binding to their promoters, the ChIPassay was performed to probe DNA-protein interactions within the naturalchromatin. Sequence analysis indicated that, except one (SlUBC38), 13 E2 genes contain CArG box binding motifs in theirpromoters (Additional file 5). For theChIP assay, the affinity-purified anti-RIN polyclonal antibody(Figure 6a) was used toimmunoprecipitate the cross-linked DNA-protein complexes. The enriched DNA waspurified and then submitted to real-time quantitative PCR analysis. Primer setswere designed for those 13 genes that contain CArG box binding motifs in theirpromoters (Additional file 6). Ourresults indicate that RIN binds to the promoters of SlUBC6, 8, 24, 41, and42 (Figure 6b). Notably, RIN shows differential binding ability to thepromoter fragments of these genes. The relative amounts of precipitated promoterfragments of SlUBC6, 24, and 42 were low (<0.2%).By contrast, the relative amounts of precipitated promoter fragments of SlUBC8 and 41 weremuch higher. The highest enrichments (>1%) was found for SlUBC41 promoter fragments. Very low enrichments wereobserved for all fragments when cross-linked DNA-protein complexes wereimmunoprecipitated with pre-immune rabbit IgG, the non-specific antibody. This isconsidered as non-specific background enrichment.Figure 6

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