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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

Genes involved in ubiquitin-proteasome pathway areidentified as direct RIN targets. (a) Expression analysis ofSlUBC32 and PSMD2 at protein and mRNA levels. The protein expression inwild-type (WT) and rin mutant wasassessed by quantitative proteome analysis at breaker (Br) and orange (Or)ripening stages. The mRNA expression was examined by quantitative RT-PCR.The gene transcript levels are normalized against the actin gene. Values are means ± SD of threeindependent experiments. (b) ChIP-qPCRassays show that RIN direct binds to the promoter regions of SlUBC32 and PSMD2. The promoter structures of the target genes arepresented. Blue boxes represent CArG box elements and numbers indicate theposition of these motifs relative to the translational start site. Greenfragments with upper-case letters represent the regions used forChIP-qPCR. Values are the percentage of DNA fragments thatco-immunoprecipitated with anti-RIN antibodies (black bars) ornon-specific antibodies (preimmune rabbit IgG; grey bars) relative to theinput DNAs. Error bars represent the SD of three independent experiments.(c) Gel mobility shift assays revealthe direct binding of RIN to CArG box elements in the promoter regions ofSlUBC32 and PSMD2. The probe sequences corresponding to the SlUBC32 and PSMD2 promoters are shown, with red letters representing theCArG box. The mutated bases in the probes are represented by blue letters.wt, probe with intact CArG box element; mt, probe with mutated CArG boxelement. As competitors, 1,000-fold excess amounts of unlabeled probeswere added to the binding reaction. The retarded bands and the free probesare indicated by arrowheads.
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Fig4: Genes involved in ubiquitin-proteasome pathway areidentified as direct RIN targets. (a) Expression analysis ofSlUBC32 and PSMD2 at protein and mRNA levels. The protein expression inwild-type (WT) and rin mutant wasassessed by quantitative proteome analysis at breaker (Br) and orange (Or)ripening stages. The mRNA expression was examined by quantitative RT-PCR.The gene transcript levels are normalized against the actin gene. Values are means ± SD of threeindependent experiments. (b) ChIP-qPCRassays show that RIN direct binds to the promoter regions of SlUBC32 and PSMD2. The promoter structures of the target genes arepresented. Blue boxes represent CArG box elements and numbers indicate theposition of these motifs relative to the translational start site. Greenfragments with upper-case letters represent the regions used forChIP-qPCR. Values are the percentage of DNA fragments thatco-immunoprecipitated with anti-RIN antibodies (black bars) ornon-specific antibodies (preimmune rabbit IgG; grey bars) relative to theinput DNAs. Error bars represent the SD of three independent experiments.(c) Gel mobility shift assays revealthe direct binding of RIN to CArG box elements in the promoter regions ofSlUBC32 and PSMD2. The probe sequences corresponding to the SlUBC32 and PSMD2 promoters are shown, with red letters representing theCArG box. The mutated bases in the probes are represented by blue letters.wt, probe with intact CArG box element; mt, probe with mutated CArG boxelement. As competitors, 1,000-fold excess amounts of unlabeled probeswere added to the binding reaction. The retarded bands and the free probesare indicated by arrowheads.

Mentions: In the quantitative analysis of nuclear proteome between wild-typeand rin mutant, it is noticeable that 14proteins showing similar expression patterns were identified as proteins involvedin the ubiquitin-proteasome system. Two of these proteins, ubiquitin-conjugatingenzyme E2 N (SlUBC32; Solyc07g062570) and 26S proteasome regulatory subunit(PSMD2; Solyc07g053650), were subjected to further characterization. SlUBC32 wasthe only E2 enzyme identified in the iTRAQ analysis, and PSMD2 represented theprotein showing the highest changes in protein abundance (0.58 at breaker stageand 0.48 at orange stage) among the 26S proteasome regulatory subunits identified.SlUBC32 and PSMD2 were downregulated in the rinmutant at both breaker and orange stages. To examine whether the proteinexpression patterns were also present at the transcript level, quantitativereverse transcription polymerase chain reaction (RT-PCR) was performed. Theresults showed that the transcript alterations in the rin mutant were in agreement with the protein expression variationsfor both genes (Figure 4a).Figure 4


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)

Genes involved in ubiquitin-proteasome pathway areidentified as direct RIN targets. (a) Expression analysis ofSlUBC32 and PSMD2 at protein and mRNA levels. The protein expression inwild-type (WT) and rin mutant wasassessed by quantitative proteome analysis at breaker (Br) and orange (Or)ripening stages. The mRNA expression was examined by quantitative RT-PCR.The gene transcript levels are normalized against the actin gene. Values are means ± SD of threeindependent experiments. (b) ChIP-qPCRassays show that RIN direct binds to the promoter regions of SlUBC32 and PSMD2. The promoter structures of the target genes arepresented. Blue boxes represent CArG box elements and numbers indicate theposition of these motifs relative to the translational start site. Greenfragments with upper-case letters represent the regions used forChIP-qPCR. Values are the percentage of DNA fragments thatco-immunoprecipitated with anti-RIN antibodies (black bars) ornon-specific antibodies (preimmune rabbit IgG; grey bars) relative to theinput DNAs. Error bars represent the SD of three independent experiments.(c) Gel mobility shift assays revealthe direct binding of RIN to CArG box elements in the promoter regions ofSlUBC32 and PSMD2. The probe sequences corresponding to the SlUBC32 and PSMD2 promoters are shown, with red letters representing theCArG box. The mutated bases in the probes are represented by blue letters.wt, probe with intact CArG box element; mt, probe with mutated CArG boxelement. As competitors, 1,000-fold excess amounts of unlabeled probeswere added to the binding reaction. The retarded bands and the free probesare indicated by arrowheads.
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Related In: Results  -  Collection

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Fig4: Genes involved in ubiquitin-proteasome pathway areidentified as direct RIN targets. (a) Expression analysis ofSlUBC32 and PSMD2 at protein and mRNA levels. The protein expression inwild-type (WT) and rin mutant wasassessed by quantitative proteome analysis at breaker (Br) and orange (Or)ripening stages. The mRNA expression was examined by quantitative RT-PCR.The gene transcript levels are normalized against the actin gene. Values are means ± SD of threeindependent experiments. (b) ChIP-qPCRassays show that RIN direct binds to the promoter regions of SlUBC32 and PSMD2. The promoter structures of the target genes arepresented. Blue boxes represent CArG box elements and numbers indicate theposition of these motifs relative to the translational start site. Greenfragments with upper-case letters represent the regions used forChIP-qPCR. Values are the percentage of DNA fragments thatco-immunoprecipitated with anti-RIN antibodies (black bars) ornon-specific antibodies (preimmune rabbit IgG; grey bars) relative to theinput DNAs. Error bars represent the SD of three independent experiments.(c) Gel mobility shift assays revealthe direct binding of RIN to CArG box elements in the promoter regions ofSlUBC32 and PSMD2. The probe sequences corresponding to the SlUBC32 and PSMD2 promoters are shown, with red letters representing theCArG box. The mutated bases in the probes are represented by blue letters.wt, probe with intact CArG box element; mt, probe with mutated CArG boxelement. As competitors, 1,000-fold excess amounts of unlabeled probeswere added to the binding reaction. The retarded bands and the free probesare indicated by arrowheads.
Mentions: In the quantitative analysis of nuclear proteome between wild-typeand rin mutant, it is noticeable that 14proteins showing similar expression patterns were identified as proteins involvedin the ubiquitin-proteasome system. Two of these proteins, ubiquitin-conjugatingenzyme E2 N (SlUBC32; Solyc07g062570) and 26S proteasome regulatory subunit(PSMD2; Solyc07g053650), were subjected to further characterization. SlUBC32 wasthe only E2 enzyme identified in the iTRAQ analysis, and PSMD2 represented theprotein showing the highest changes in protein abundance (0.58 at breaker stageand 0.48 at orange stage) among the 26S proteasome regulatory subunits identified.SlUBC32 and PSMD2 were downregulated in the rinmutant at both breaker and orange stages. To examine whether the proteinexpression patterns were also present at the transcript level, quantitativereverse transcription polymerase chain reaction (RT-PCR) was performed. Theresults showed that the transcript alterations in the rin mutant were in agreement with the protein expression variationsfor both genes (Figure 4a).Figure 4

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