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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

Specific E2 genes are involved in the regulation offruit ripening. Virus-induced gene silencing assay in tomatoreveals the effect of SlUBC32 andSlUBC41 on fruit ripening. Imagesshow the ripe fruit of plants infected with vectors containing no insert(Ev), specific PHYTOENE DESATURASEsequence (PDS), specific SlUBC32 sequence, or specific SlUBC41 sequence.
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Fig8: Specific E2 genes are involved in the regulation offruit ripening. Virus-induced gene silencing assay in tomatoreveals the effect of SlUBC32 andSlUBC41 on fruit ripening. Imagesshow the ripe fruit of plants infected with vectors containing no insert(Ev), specific PHYTOENE DESATURASEsequence (PDS), specific SlUBC32 sequence, or specific SlUBC41 sequence.

Mentions: To examine whether E2 genes participate in the regulation of fruitripening, a virus-induced gene silencing (VIGS) assay was performed. All genesthat were demonstrated to be directly regulated by RIN, namely SlUBC6, 8, 24, 32, 41, and 42, wereanalyzed by this system. A specific cDNA fragment of these genes was cloned andinserted into the pTRV2 vector, respectively. The inflorescence peduncles at thepreanthesis stage were used for infiltrated and the fruits were visually inspecteddaily. Obvious phenotype was detected in plants silenced for SlUBC32 or SlUBC41cDNA. As shown in FigureĀ 8, control fruitinoculated with pTRV2 alone (empty vector) showed a homogenous orange at theorange stage. By contrast, the color of fruits infected with pTRV2 carrying a 362bp fragment of the SlUBC32 gene was patchy withsectors of different shades of yellow and orange. Similar result was observed onfruits infected with the virus vector pTRV2 carrying a 477 bp fragment of theSlUBC41 gene. The mRNA levels of SlUBC32 and SlUBC41was measured by quantitative RT-PCR in the fruit pericarps of plants infiltratedwith pTRV2-SlUBC32 and pTRV2-SlUBC41. The results showed that the mRNA levels ofSlUBC32 and SlUBC41 in the yellow areas were reduced by approximately 70% and60%, respectively, when compared with the orange tissues. Inversely, the level ofthe TRV capsid protein mRNA was significantly higher in the yellow tissues (datanot shown). These data demonstrated that SlUBC32 and SlUBC41 weresuccessfully silenced and both genes play an important role in the regulation oftomato fruit ripening.Figure 8


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)

Specific E2 genes are involved in the regulation offruit ripening. Virus-induced gene silencing assay in tomatoreveals the effect of SlUBC32 andSlUBC41 on fruit ripening. Imagesshow the ripe fruit of plants infected with vectors containing no insert(Ev), specific PHYTOENE DESATURASEsequence (PDS), specific SlUBC32 sequence, or specific SlUBC41 sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Specific E2 genes are involved in the regulation offruit ripening. Virus-induced gene silencing assay in tomatoreveals the effect of SlUBC32 andSlUBC41 on fruit ripening. Imagesshow the ripe fruit of plants infected with vectors containing no insert(Ev), specific PHYTOENE DESATURASEsequence (PDS), specific SlUBC32 sequence, or specific SlUBC41 sequence.
Mentions: To examine whether E2 genes participate in the regulation of fruitripening, a virus-induced gene silencing (VIGS) assay was performed. All genesthat were demonstrated to be directly regulated by RIN, namely SlUBC6, 8, 24, 32, 41, and 42, wereanalyzed by this system. A specific cDNA fragment of these genes was cloned andinserted into the pTRV2 vector, respectively. The inflorescence peduncles at thepreanthesis stage were used for infiltrated and the fruits were visually inspecteddaily. Obvious phenotype was detected in plants silenced for SlUBC32 or SlUBC41cDNA. As shown in FigureĀ 8, control fruitinoculated with pTRV2 alone (empty vector) showed a homogenous orange at theorange stage. By contrast, the color of fruits infected with pTRV2 carrying a 362bp fragment of the SlUBC32 gene was patchy withsectors of different shades of yellow and orange. Similar result was observed onfruits infected with the virus vector pTRV2 carrying a 477 bp fragment of theSlUBC41 gene. The mRNA levels of SlUBC32 and SlUBC41was measured by quantitative RT-PCR in the fruit pericarps of plants infiltratedwith pTRV2-SlUBC32 and pTRV2-SlUBC41. The results showed that the mRNA levels ofSlUBC32 and SlUBC41 in the yellow areas were reduced by approximately 70% and60%, respectively, when compared with the orange tissues. Inversely, the level ofthe TRV capsid protein mRNA was significantly higher in the yellow tissues (datanot shown). These data demonstrated that SlUBC32 and SlUBC41 weresuccessfully silenced and both genes play an important role in the regulation oftomato fruit ripening.Figure 8

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