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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

Preparation of tomato nuclei for proteomic analysis.(a) Micrographs showing representative nuclear fractions fromtomato fruits after 4′,6-diamidino-2-phenylindole (DAPI) staining. Thephase-contrast micrograph and the fluorescence micrograph of the nucleiare presented. Scale bar, 25 μm. (b)Western blot analysis of the different purification fractions withantibodies directed against histone H3, UDP-glucose pyrophosphorylase(UGPase), and photosystem II reaction centre protein D1 (PsbA). T, totalprotein extract; S1, supernatant fraction from centrifugation at3,000 × g; S2, supernatant fractionafter 1% Triton X-100 treatment and centrifugation; S3, supernatantfraction from sucrose density centrifugation; N, nuclear proteinextract.
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Fig1: Preparation of tomato nuclei for proteomic analysis.(a) Micrographs showing representative nuclear fractions fromtomato fruits after 4′,6-diamidino-2-phenylindole (DAPI) staining. Thephase-contrast micrograph and the fluorescence micrograph of the nucleiare presented. Scale bar, 25 μm. (b)Western blot analysis of the different purification fractions withantibodies directed against histone H3, UDP-glucose pyrophosphorylase(UGPase), and photosystem II reaction centre protein D1 (PsbA). T, totalprotein extract; S1, supernatant fraction from centrifugation at3,000 × g; S2, supernatant fractionafter 1% Triton X-100 treatment and centrifugation; S3, supernatantfraction from sucrose density centrifugation; N, nuclear proteinextract.

Mentions: Purification of nuclei away from other cellular contaminants isvital to nuclear subproteome analysis. We isolated intact nuclei from tomato fruitusing differential centrifugation and sucrose density enrichment. The integrity ofthe isolated nuclei was assessed using 4′,6-diamidino-2-phenylindole (DAPI)staining and examined by fluorescence microscopy (Figure 1a). The nuclei were uniform spheres with an average diameter ofapproximately 10 μm. To further evaluate the enrichment and purity of the nuclei,western blotting was performed with antibodies against organelle specificproteins. The nuclear protein histone H3 was detected in the nuclear fraction, butnot in the cytoplasmic fraction. By comparison, the cytoplasmic proteinUDP-glucose pyrophosphorylase (UDPase) and the chloroplast protein photosystem IIreaction center protein D1 (PsbA), which are absent from the nuclei, were notfound in the purified nuclear fraction (Figure 1b). These data suggest that the nuclei were successfullyenriched and there was no appreciable level of contamination by chloroplast orcytoplasm in the isolated nuclei. Nuclear proteins were prepared from thenuclei-enriched fraction using a phenol-based method to avoid contamination bynucleic acids.Figure 1


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)

Preparation of tomato nuclei for proteomic analysis.(a) Micrographs showing representative nuclear fractions fromtomato fruits after 4′,6-diamidino-2-phenylindole (DAPI) staining. Thephase-contrast micrograph and the fluorescence micrograph of the nucleiare presented. Scale bar, 25 μm. (b)Western blot analysis of the different purification fractions withantibodies directed against histone H3, UDP-glucose pyrophosphorylase(UGPase), and photosystem II reaction centre protein D1 (PsbA). T, totalprotein extract; S1, supernatant fraction from centrifugation at3,000 × g; S2, supernatant fractionafter 1% Triton X-100 treatment and centrifugation; S3, supernatantfraction from sucrose density centrifugation; N, nuclear proteinextract.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Preparation of tomato nuclei for proteomic analysis.(a) Micrographs showing representative nuclear fractions fromtomato fruits after 4′,6-diamidino-2-phenylindole (DAPI) staining. Thephase-contrast micrograph and the fluorescence micrograph of the nucleiare presented. Scale bar, 25 μm. (b)Western blot analysis of the different purification fractions withantibodies directed against histone H3, UDP-glucose pyrophosphorylase(UGPase), and photosystem II reaction centre protein D1 (PsbA). T, totalprotein extract; S1, supernatant fraction from centrifugation at3,000 × g; S2, supernatant fractionafter 1% Triton X-100 treatment and centrifugation; S3, supernatantfraction from sucrose density centrifugation; N, nuclear proteinextract.
Mentions: Purification of nuclei away from other cellular contaminants isvital to nuclear subproteome analysis. We isolated intact nuclei from tomato fruitusing differential centrifugation and sucrose density enrichment. The integrity ofthe isolated nuclei was assessed using 4′,6-diamidino-2-phenylindole (DAPI)staining and examined by fluorescence microscopy (Figure 1a). The nuclei were uniform spheres with an average diameter ofapproximately 10 μm. To further evaluate the enrichment and purity of the nuclei,western blotting was performed with antibodies against organelle specificproteins. The nuclear protein histone H3 was detected in the nuclear fraction, butnot in the cytoplasmic fraction. By comparison, the cytoplasmic proteinUDP-glucose pyrophosphorylase (UDPase) and the chloroplast protein photosystem IIreaction center protein D1 (PsbA), which are absent from the nuclei, were notfound in the purified nuclear fraction (Figure 1b). These data suggest that the nuclei were successfullyenriched and there was no appreciable level of contamination by chloroplast orcytoplasm in the isolated nuclei. Nuclear proteins were prepared from thenuclei-enriched fraction using a phenol-based method to avoid contamination bynucleic acids.Figure 1

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