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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

Quantitative analysis of nuclear proteome duringtomato fruit ripening. Nuclear proteins were extracted fromtomato fruits at mature green (MG), breaker (Br), orange (Or), and redripe (RR) ripening stages, and subjected to isobaric tags for relative andabsolute quantification (iTRAQ) labeling coupled with NanoLC-MS/MS. The136 differentially expressed proteins were classified into sevenfunctional categories and the expression patterns within each werehierarchically clustered. Expression ratios were calculated using theearlier ripening stage, that is, MG, as denominator, and plotted in a heatmap on a log2 scale. Each row in the color heat mapindicates a single protein, and the gene identifiers (Solyc numbers) andfunctional annotations are shown. The green and red colors indicate down-and upregulation, respectively, in an indicated ripening stage relative tothe MG stage. Black represents no significant expression change. Data frombiologically repeated samples are averaged and the detailed information onproteins is listed in Additional file 2.
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Fig2: Quantitative analysis of nuclear proteome duringtomato fruit ripening. Nuclear proteins were extracted fromtomato fruits at mature green (MG), breaker (Br), orange (Or), and redripe (RR) ripening stages, and subjected to isobaric tags for relative andabsolute quantification (iTRAQ) labeling coupled with NanoLC-MS/MS. The136 differentially expressed proteins were classified into sevenfunctional categories and the expression patterns within each werehierarchically clustered. Expression ratios were calculated using theearlier ripening stage, that is, MG, as denominator, and plotted in a heatmap on a log2 scale. Each row in the color heat mapindicates a single protein, and the gene identifiers (Solyc numbers) andfunctional annotations are shown. The green and red colors indicate down-and upregulation, respectively, in an indicated ripening stage relative tothe MG stage. Black represents no significant expression change. Data frombiologically repeated samples are averaged and the detailed information onproteins is listed in Additional file 2.

Mentions: An iTRAQ-based quantitative proteomic analysis was utilized to gaina global view of nuclear proteome alteration during tomato fruit ripening. Anoverview of the iTRAQ experimental design and the workflow is depicted inAdditional file 1. The nuclear proteinswere isolated from tomatoes in four stages of ripening, that is, mature green,breaker, orange, and red ripe stages. Simultaneous comparison of nuclear proteinexpression across these samples was achieved using four-plex iTRAQ isobaric tagswith NanoLC-MS/MS. Two independent biological replicates for each sample were usedfor labeling. Using the Solanum lycopersicumprotein database, a total of 1,279 and 1,303 proteins were identified with aglobal false discovery rate (FDR) below 1% in the two biological replicates,respectively. These identified proteins were filtered to calculate the meaningfulcutoff using a population statistics applied to the biological replicates asproposed by Gan et al. [37]. The cutoff values were then used to verifywhether the changes in protein abundance are significant. A total of 136 proteinswere finally screened as significantly altered at one or more ripening stages.Additional file 2 shows thesedifferentially expressed proteins along with all relevant identificationinformation and the ratio of iTRAQ reporter ion intensities. According to theFunctional Catalogue (FunCat) annotation scheme [38] and the UniProt Knowledgebase (UniProtKB) [39], these proteins were classified into sevenfunctional categories, namely signaling and gene regulation, chromatin remodeling,protein degradation, cell defense and protein folding, ribosomal proteins andtranslation, metabolism, and uncharacterized. To identify the proteins showingsimilar expression profiles, hierarchical clustering [40] was applied within each functional category(FigureĀ 2).Figure 2


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)

Quantitative analysis of nuclear proteome duringtomato fruit ripening. Nuclear proteins were extracted fromtomato fruits at mature green (MG), breaker (Br), orange (Or), and redripe (RR) ripening stages, and subjected to isobaric tags for relative andabsolute quantification (iTRAQ) labeling coupled with NanoLC-MS/MS. The136 differentially expressed proteins were classified into sevenfunctional categories and the expression patterns within each werehierarchically clustered. Expression ratios were calculated using theearlier ripening stage, that is, MG, as denominator, and plotted in a heatmap on a log2 scale. Each row in the color heat mapindicates a single protein, and the gene identifiers (Solyc numbers) andfunctional annotations are shown. The green and red colors indicate down-and upregulation, respectively, in an indicated ripening stage relative tothe MG stage. Black represents no significant expression change. Data frombiologically repeated samples are averaged and the detailed information onproteins is listed in Additional file 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Quantitative analysis of nuclear proteome duringtomato fruit ripening. Nuclear proteins were extracted fromtomato fruits at mature green (MG), breaker (Br), orange (Or), and redripe (RR) ripening stages, and subjected to isobaric tags for relative andabsolute quantification (iTRAQ) labeling coupled with NanoLC-MS/MS. The136 differentially expressed proteins were classified into sevenfunctional categories and the expression patterns within each werehierarchically clustered. Expression ratios were calculated using theearlier ripening stage, that is, MG, as denominator, and plotted in a heatmap on a log2 scale. Each row in the color heat mapindicates a single protein, and the gene identifiers (Solyc numbers) andfunctional annotations are shown. The green and red colors indicate down-and upregulation, respectively, in an indicated ripening stage relative tothe MG stage. Black represents no significant expression change. Data frombiologically repeated samples are averaged and the detailed information onproteins is listed in Additional file 2.
Mentions: An iTRAQ-based quantitative proteomic analysis was utilized to gaina global view of nuclear proteome alteration during tomato fruit ripening. Anoverview of the iTRAQ experimental design and the workflow is depicted inAdditional file 1. The nuclear proteinswere isolated from tomatoes in four stages of ripening, that is, mature green,breaker, orange, and red ripe stages. Simultaneous comparison of nuclear proteinexpression across these samples was achieved using four-plex iTRAQ isobaric tagswith NanoLC-MS/MS. Two independent biological replicates for each sample were usedfor labeling. Using the Solanum lycopersicumprotein database, a total of 1,279 and 1,303 proteins were identified with aglobal false discovery rate (FDR) below 1% in the two biological replicates,respectively. These identified proteins were filtered to calculate the meaningfulcutoff using a population statistics applied to the biological replicates asproposed by Gan et al. [37]. The cutoff values were then used to verifywhether the changes in protein abundance are significant. A total of 136 proteinswere finally screened as significantly altered at one or more ripening stages.Additional file 2 shows thesedifferentially expressed proteins along with all relevant identificationinformation and the ratio of iTRAQ reporter ion intensities. According to theFunctional Catalogue (FunCat) annotation scheme [38] and the UniProt Knowledgebase (UniProtKB) [39], these proteins were classified into sevenfunctional categories, namely signaling and gene regulation, chromatin remodeling,protein degradation, cell defense and protein folding, ribosomal proteins andtranslation, metabolism, and uncharacterized. To identify the proteins showingsimilar expression profiles, hierarchical clustering [40] was applied within each functional category(FigureĀ 2).Figure 2

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