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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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Genome-wide screening and expression analysis ofgenes encoding E2 ubiquitin-conjugating enzymes in tomato.(a) Phylogenetic analysis of tomato E2s. Phylogenetic treewas produced using MEGA version 5.2. Bootstrap values from 1,000replications for each branch are shown. (b) Gene expression analysis of tomato E2s in wild-type (WT)and rin mutant during the period offruit ripening, as determined by quantitative RT-PCR. The actin gene was used as the internal control.The stages of fruit ripening include mature green (MG), breaker (Br),orange (Or) and red ripe (RR). Values are means ± SD of three independentexperiments. Asterisks indicate values that changed more than twofold inthe rin mutant at indicated ripeningstages.
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Fig5: Genome-wide screening and expression analysis ofgenes encoding E2 ubiquitin-conjugating enzymes in tomato.(a) Phylogenetic analysis of tomato E2s. Phylogenetic treewas produced using MEGA version 5.2. Bootstrap values from 1,000replications for each branch are shown. (b) Gene expression analysis of tomato E2s in wild-type (WT)and rin mutant during the period offruit ripening, as determined by quantitative RT-PCR. The actin gene was used as the internal control.The stages of fruit ripening include mature green (MG), breaker (Br),orange (Or) and red ripe (RR). Values are means ± SD of three independentexperiments. Asterisks indicate values that changed more than twofold inthe rin mutant at indicated ripeningstages.

Mentions: Protein ubiquitination is mediated through the action of threeenzymes known as ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme(E2), and ubiquitin ligase (E3). Substrate specificity is mainly determined by E2together with E3. In our quantitative analysis of nuclear proteome, we found thatthe expression of one ubiquitin E2 enzyme, SlUBC32, was downregulated in therin mutant fruits. However, due to thelimited sensitivity and resolution of proteomic technologies, the effect of RIN onthe expression of other E2 remains unclear. By screening the SGN Tomato database,we identified 52 non-redundant E2 genes. These E2 genes were named SlUBC1 to SlUBC52according to their location on the chromosomes (Additional file 8). All the E2 genes contain a highly conservedubiquitin-conjugating (UBC) domain with an active-site cysteine residue confirmedby ScanProsite (Additional file 9).Phylogenetic analysis revealed that tomato ubiquitin E2 enzymes can be dividedinto a dozen of subgroups based on >50% bootstrap support (Figure 5a). Many tomato E2 proteins shared high similaritywith another one (Additional file 10),suggesting gene duplications. The largest tomato E2 subgroup was formed by SlUBC2and eleven other tomato E2s: SlUBC6, 11, 12, 20, 24, 28, 33, 34, 36, 40, and 49.SlUBC11, 20, 24, 28, 34, 36, 40, and 49 are very similar to SlUBC2, with more thanor equal to 95% amino acid identity. SlUBC33 shows 91% identity to SlUBC2, whileSlUBC6 and 12 show 87% and 84% identity to SlUBC2, respectively (Additional file10). Gene expression of the 52 tomatoE2 genes was analyzed in wild-type and rinmutant during fruit ripening using quantitative RT-PCR. Figure 5b shows the expression patterns of these genes asrelated to their phylogenetic relationships. Generally, gene expression patternswere frequently similar within subgroups. For example, the expression patterns ofduplicated paralogs SlUBC42 and SlUBC43 showed high similarity. By contrast, theexpression patterns of some paralogs, for example, SlUBC20 and SlUBC40, were quitedifferent. This suggests that, after duplication, one daughter gene may retain theancestral function while the other acquires new function. We found that theexpression of 14 E2 genes (SlUBC7, 8, 12, 17, 18, 24, 30, 32, 38, and41 to 45)was downregulated, whereas one (SlUBC6) wasupregulated more than two-fold, in the rinmutant at two or more stages of ripening (Figure 5b).Figure 5


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)

Genome-wide screening and expression analysis ofgenes encoding E2 ubiquitin-conjugating enzymes in tomato.(a) Phylogenetic analysis of tomato E2s. Phylogenetic treewas produced using MEGA version 5.2. Bootstrap values from 1,000replications for each branch are shown. (b) Gene expression analysis of tomato E2s in wild-type (WT)and rin mutant during the period offruit ripening, as determined by quantitative RT-PCR. The actin gene was used as the internal control.The stages of fruit ripening include mature green (MG), breaker (Br),orange (Or) and red ripe (RR). Values are means ± SD of three independentexperiments. Asterisks indicate values that changed more than twofold inthe rin mutant at indicated ripeningstages.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269173&req=5

Fig5: Genome-wide screening and expression analysis ofgenes encoding E2 ubiquitin-conjugating enzymes in tomato.(a) Phylogenetic analysis of tomato E2s. Phylogenetic treewas produced using MEGA version 5.2. Bootstrap values from 1,000replications for each branch are shown. (b) Gene expression analysis of tomato E2s in wild-type (WT)and rin mutant during the period offruit ripening, as determined by quantitative RT-PCR. The actin gene was used as the internal control.The stages of fruit ripening include mature green (MG), breaker (Br),orange (Or) and red ripe (RR). Values are means ± SD of three independentexperiments. Asterisks indicate values that changed more than twofold inthe rin mutant at indicated ripeningstages.
Mentions: Protein ubiquitination is mediated through the action of threeenzymes known as ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme(E2), and ubiquitin ligase (E3). Substrate specificity is mainly determined by E2together with E3. In our quantitative analysis of nuclear proteome, we found thatthe expression of one ubiquitin E2 enzyme, SlUBC32, was downregulated in therin mutant fruits. However, due to thelimited sensitivity and resolution of proteomic technologies, the effect of RIN onthe expression of other E2 remains unclear. By screening the SGN Tomato database,we identified 52 non-redundant E2 genes. These E2 genes were named SlUBC1 to SlUBC52according to their location on the chromosomes (Additional file 8). All the E2 genes contain a highly conservedubiquitin-conjugating (UBC) domain with an active-site cysteine residue confirmedby ScanProsite (Additional file 9).Phylogenetic analysis revealed that tomato ubiquitin E2 enzymes can be dividedinto a dozen of subgroups based on >50% bootstrap support (Figure 5a). Many tomato E2 proteins shared high similaritywith another one (Additional file 10),suggesting gene duplications. The largest tomato E2 subgroup was formed by SlUBC2and eleven other tomato E2s: SlUBC6, 11, 12, 20, 24, 28, 33, 34, 36, 40, and 49.SlUBC11, 20, 24, 28, 34, 36, 40, and 49 are very similar to SlUBC2, with more thanor equal to 95% amino acid identity. SlUBC33 shows 91% identity to SlUBC2, whileSlUBC6 and 12 show 87% and 84% identity to SlUBC2, respectively (Additional file10). Gene expression of the 52 tomatoE2 genes was analyzed in wild-type and rinmutant during fruit ripening using quantitative RT-PCR. Figure 5b shows the expression patterns of these genes asrelated to their phylogenetic relationships. Generally, gene expression patternswere frequently similar within subgroups. For example, the expression patterns ofduplicated paralogs SlUBC42 and SlUBC43 showed high similarity. By contrast, theexpression patterns of some paralogs, for example, SlUBC20 and SlUBC40, were quitedifferent. This suggests that, after duplication, one daughter gene may retain theancestral function while the other acquires new function. We found that theexpression of 14 E2 genes (SlUBC7, 8, 12, 17, 18, 24, 30, 32, 38, and41 to 45)was downregulated, whereas one (SlUBC6) wasupregulated more than two-fold, in the rinmutant at two or more stages of ripening (Figure 5b).Figure 5

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