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Circadian Profiling of the Arabidopsis Proteome Using 2D-DIGE.

Choudhary MK, Nomura Y, Shi H, Nakagami H, Somers DE - Front Plant Sci (2016)

Bottom Line: Clock-generated biological rhythms provide an adaptive advantage to an organism, resulting in increased fitness and survival.The phasing of maximum expression for the cyclic proteins was similar for both datasets, with a nearly even distribution of peak phases across the time series.Taken together, this study provides new insights into the importance of post-transcriptional circadian control of plant physiology and metabolism.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology Pohang, South Korea.

ABSTRACT
Clock-generated biological rhythms provide an adaptive advantage to an organism, resulting in increased fitness and survival. To better elucidate the plant response to the circadian system, we surveyed protein oscillations in Arabidopsis seedlings under constant light. Using large-scale two-dimensional difference in gel electrophoresis (2D-DIGE) the abundance of more than 1000 proteins spots was reproducibly resolved quantified and profiled across a circadian time series. A comparison between phenol-extracted samples and RuBisCO-depleted extracts identified 71 and 40 rhythmically-expressed proteins, respectively, and between 30 and 40% of these derive from non-rhythmic transcripts. These included proteins influencing transcriptional regulation, translation, metabolism, photosynthesis, protein chaperones, and stress-mediated responses. The phasing of maximum expression for the cyclic proteins was similar for both datasets, with a nearly even distribution of peak phases across the time series. STRING clustering analysis identified two interaction networks with a notable number of oscillating proteins: plastid-based and cytosolic chaperones and 10 proteins involved in photosynthesis. The oscillation of the ABA receptor, PYR1/RCAR11, with peak expression near dusk adds to a growing body of evidence that intimately ties ABA signaling to the circadian system. Taken together, this study provides new insights into the importance of post-transcriptional circadian control of plant physiology and metabolism.

No MeSH data available.


Related in: MedlinePlus

Two-dimensional difference gel electrophoresis (2-D DIGE) analysis of circadian regulated proteins. (A) Total protein isolated through a phenol extraction or (B) RuBisCO-depletion method were compared across the six time points by 2-D DIGE using 24 cm pH 4–7 (left to right) IPG strips and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein spots of interest are numbered. Panel (A) is an overlap image of gel no. 1 (Table S1) with the LL25 sample labeled with Cy3 and the LL37 sample labeled with Cy5. Panel (B) is an overlap image of gel no. 4 (Table S1) with the LL29 sample labeled with Cy3 and the LL33 sample labeled with Cy5.
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Figure 1: Two-dimensional difference gel electrophoresis (2-D DIGE) analysis of circadian regulated proteins. (A) Total protein isolated through a phenol extraction or (B) RuBisCO-depletion method were compared across the six time points by 2-D DIGE using 24 cm pH 4–7 (left to right) IPG strips and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein spots of interest are numbered. Panel (A) is an overlap image of gel no. 1 (Table S1) with the LL25 sample labeled with Cy3 and the LL37 sample labeled with Cy5. Panel (B) is an overlap image of gel no. 4 (Table S1) with the LL29 sample labeled with Cy3 and the LL33 sample labeled with Cy5.

Mentions: In both approaches four biological replicates were obtained for each time point (12 gels for each extraction method; Table S1). We first analyzed protein samples prepared in parallel from the same tissue samples on 1-D SDS-PAGE gels (Figure S1) and then separated the extracts on high resolution 24 cm 2D gels using the CyDye system (Table S1). The phenol–methanol and RuBisCO-depletion methods consistently yielded highly resolution gels with typically ~1350 protein spots resolved in each gel (Figure 1, Figure S2). Gel image analysis and protein spots were quantified using Progenesis same spot software and spots that reproducibly changed in abundance between two samples within the time series with p < 0.05 (n = 4) were selected for further examination (77 for the phenol-extracted samples and 59 for the RuBisCO-depleted samples; summaries of peptides matching each protein are in Table S2). A substantial set of changes are apparent by the colored overlay of the fluorescence images. Spots of interest were picked manually from post-electrophoretically silver stained preparative gels and in-gel trypsin-digested. Protein identities were analyzed by LC-MS/MS (Figures 1A,B indicated by arrows). MASCOT software (ver. 2.3.02) was used to simultaneously identify proteins.


Circadian Profiling of the Arabidopsis Proteome Using 2D-DIGE.

Choudhary MK, Nomura Y, Shi H, Nakagami H, Somers DE - Front Plant Sci (2016)

Two-dimensional difference gel electrophoresis (2-D DIGE) analysis of circadian regulated proteins. (A) Total protein isolated through a phenol extraction or (B) RuBisCO-depletion method were compared across the six time points by 2-D DIGE using 24 cm pH 4–7 (left to right) IPG strips and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein spots of interest are numbered. Panel (A) is an overlap image of gel no. 1 (Table S1) with the LL25 sample labeled with Cy3 and the LL37 sample labeled with Cy5. Panel (B) is an overlap image of gel no. 4 (Table S1) with the LL29 sample labeled with Cy3 and the LL33 sample labeled with Cy5.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940426&req=5

Figure 1: Two-dimensional difference gel electrophoresis (2-D DIGE) analysis of circadian regulated proteins. (A) Total protein isolated through a phenol extraction or (B) RuBisCO-depletion method were compared across the six time points by 2-D DIGE using 24 cm pH 4–7 (left to right) IPG strips and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein spots of interest are numbered. Panel (A) is an overlap image of gel no. 1 (Table S1) with the LL25 sample labeled with Cy3 and the LL37 sample labeled with Cy5. Panel (B) is an overlap image of gel no. 4 (Table S1) with the LL29 sample labeled with Cy3 and the LL33 sample labeled with Cy5.
Mentions: In both approaches four biological replicates were obtained for each time point (12 gels for each extraction method; Table S1). We first analyzed protein samples prepared in parallel from the same tissue samples on 1-D SDS-PAGE gels (Figure S1) and then separated the extracts on high resolution 24 cm 2D gels using the CyDye system (Table S1). The phenol–methanol and RuBisCO-depletion methods consistently yielded highly resolution gels with typically ~1350 protein spots resolved in each gel (Figure 1, Figure S2). Gel image analysis and protein spots were quantified using Progenesis same spot software and spots that reproducibly changed in abundance between two samples within the time series with p < 0.05 (n = 4) were selected for further examination (77 for the phenol-extracted samples and 59 for the RuBisCO-depleted samples; summaries of peptides matching each protein are in Table S2). A substantial set of changes are apparent by the colored overlay of the fluorescence images. Spots of interest were picked manually from post-electrophoretically silver stained preparative gels and in-gel trypsin-digested. Protein identities were analyzed by LC-MS/MS (Figures 1A,B indicated by arrows). MASCOT software (ver. 2.3.02) was used to simultaneously identify proteins.

Bottom Line: Clock-generated biological rhythms provide an adaptive advantage to an organism, resulting in increased fitness and survival.The phasing of maximum expression for the cyclic proteins was similar for both datasets, with a nearly even distribution of peak phases across the time series.Taken together, this study provides new insights into the importance of post-transcriptional circadian control of plant physiology and metabolism.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology Pohang, South Korea.

ABSTRACT
Clock-generated biological rhythms provide an adaptive advantage to an organism, resulting in increased fitness and survival. To better elucidate the plant response to the circadian system, we surveyed protein oscillations in Arabidopsis seedlings under constant light. Using large-scale two-dimensional difference in gel electrophoresis (2D-DIGE) the abundance of more than 1000 proteins spots was reproducibly resolved quantified and profiled across a circadian time series. A comparison between phenol-extracted samples and RuBisCO-depleted extracts identified 71 and 40 rhythmically-expressed proteins, respectively, and between 30 and 40% of these derive from non-rhythmic transcripts. These included proteins influencing transcriptional regulation, translation, metabolism, photosynthesis, protein chaperones, and stress-mediated responses. The phasing of maximum expression for the cyclic proteins was similar for both datasets, with a nearly even distribution of peak phases across the time series. STRING clustering analysis identified two interaction networks with a notable number of oscillating proteins: plastid-based and cytosolic chaperones and 10 proteins involved in photosynthesis. The oscillation of the ABA receptor, PYR1/RCAR11, with peak expression near dusk adds to a growing body of evidence that intimately ties ABA signaling to the circadian system. Taken together, this study provides new insights into the importance of post-transcriptional circadian control of plant physiology and metabolism.

No MeSH data available.


Related in: MedlinePlus