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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

Changes in nuclear proteome inrinmutant reveal the potential downstream targets ofRIN. (a) Nuclear proteins were isolated from wild-type (WT)and rin mutant fruits at breaker (Br)and orange (Or) ripening stages, and subjected to isobaric tags forrelative and absolute quantification (iTRAQ) labeling coupled withNanoLC-MS/MS. A total of 127 proteins showing differential expression inthe rin mutant relative to thewild-type at Br or Or stage were identified and classified into sevenfunctional categories. The expression patterns of the proteins within eachfunctional category were hierarchically clustered based on the expressionratio as a log2 scale. Each row in the color heatmap indicates a single protein. The gene identifiers (Solyc numbers) andthe functional annotations are shown. The green and red colors indicatedown- and upregulation, respectively, in the rin mutant relative to the wild-type. Black represents nosignificant expression change. Data from biologically repeated samples areaveraged and the detailed information on proteins is listed in Additionalfile 4. (b) Venn diagram showing the overlap of proteins thatchanged abundance in the process of fruit ripening and those that changedabundance in the rin mutant at the sameripening stage (Br or Or).
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Fig3: Changes in nuclear proteome inrinmutant reveal the potential downstream targets ofRIN. (a) Nuclear proteins were isolated from wild-type (WT)and rin mutant fruits at breaker (Br)and orange (Or) ripening stages, and subjected to isobaric tags forrelative and absolute quantification (iTRAQ) labeling coupled withNanoLC-MS/MS. A total of 127 proteins showing differential expression inthe rin mutant relative to thewild-type at Br or Or stage were identified and classified into sevenfunctional categories. The expression patterns of the proteins within eachfunctional category were hierarchically clustered based on the expressionratio as a log2 scale. Each row in the color heatmap indicates a single protein. The gene identifiers (Solyc numbers) andthe functional annotations are shown. The green and red colors indicatedown- and upregulation, respectively, in the rin mutant relative to the wild-type. Black represents nosignificant expression change. Data from biologically repeated samples areaveraged and the detailed information on proteins is listed in Additionalfile 4. (b) Venn diagram showing the overlap of proteins thatchanged abundance in the process of fruit ripening and those that changedabundance in the rin mutant at the sameripening stage (Br or Or).

Mentions: Transcription factor RIN represents a global developmentalregulator of fruit ripening. To further dissect the complex networks ofripening-related pathways, nuclear protein extracts from wild-type and rin mutant at breaker as well as orange ripening stageswere analyzed in iTRAQ experiments with two independent biological replicates(Additional file 3). In total, 1,379 and1,339 proteins were identified in the two biological replicates, respectively. Themeaningful cutoff was calculated following the method of Gan et al. [37] to assess whether the changes in protein abundance aresignificant. A total of 127 proteins were found to experience significant up- ordownregulation in the rin mutant at oneripening stage or both (Additional file 4). These proteins were classified into seven functionalcategories as described above, and the differential expression patterns withineach were hierarchically clustered (FigureĀ 3a).Figure 3


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)

Changes in nuclear proteome inrinmutant reveal the potential downstream targets ofRIN. (a) Nuclear proteins were isolated from wild-type (WT)and rin mutant fruits at breaker (Br)and orange (Or) ripening stages, and subjected to isobaric tags forrelative and absolute quantification (iTRAQ) labeling coupled withNanoLC-MS/MS. A total of 127 proteins showing differential expression inthe rin mutant relative to thewild-type at Br or Or stage were identified and classified into sevenfunctional categories. The expression patterns of the proteins within eachfunctional category were hierarchically clustered based on the expressionratio as a log2 scale. Each row in the color heatmap indicates a single protein. The gene identifiers (Solyc numbers) andthe functional annotations are shown. The green and red colors indicatedown- and upregulation, respectively, in the rin mutant relative to the wild-type. Black represents nosignificant expression change. Data from biologically repeated samples areaveraged and the detailed information on proteins is listed in Additionalfile 4. (b) Venn diagram showing the overlap of proteins thatchanged abundance in the process of fruit ripening and those that changedabundance in the rin mutant at the sameripening stage (Br or Or).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Changes in nuclear proteome inrinmutant reveal the potential downstream targets ofRIN. (a) Nuclear proteins were isolated from wild-type (WT)and rin mutant fruits at breaker (Br)and orange (Or) ripening stages, and subjected to isobaric tags forrelative and absolute quantification (iTRAQ) labeling coupled withNanoLC-MS/MS. A total of 127 proteins showing differential expression inthe rin mutant relative to thewild-type at Br or Or stage were identified and classified into sevenfunctional categories. The expression patterns of the proteins within eachfunctional category were hierarchically clustered based on the expressionratio as a log2 scale. Each row in the color heatmap indicates a single protein. The gene identifiers (Solyc numbers) andthe functional annotations are shown. The green and red colors indicatedown- and upregulation, respectively, in the rin mutant relative to the wild-type. Black represents nosignificant expression change. Data from biologically repeated samples areaveraged and the detailed information on proteins is listed in Additionalfile 4. (b) Venn diagram showing the overlap of proteins thatchanged abundance in the process of fruit ripening and those that changedabundance in the rin mutant at the sameripening stage (Br or Or).
Mentions: Transcription factor RIN represents a global developmentalregulator of fruit ripening. To further dissect the complex networks ofripening-related pathways, nuclear protein extracts from wild-type and rin mutant at breaker as well as orange ripening stageswere analyzed in iTRAQ experiments with two independent biological replicates(Additional file 3). In total, 1,379 and1,339 proteins were identified in the two biological replicates, respectively. Themeaningful cutoff was calculated following the method of Gan et al. [37] to assess whether the changes in protein abundance aresignificant. A total of 127 proteins were found to experience significant up- ordownregulation in the rin mutant at oneripening stage or both (Additional file 4). These proteins were classified into seven functionalcategories as described above, and the differential expression patterns withineach were hierarchically clustered (FigureĀ 3a).Figure 3

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