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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

RuBisCO depletion from total protein extracts. Silver stained SDS-PAGE (12%) gel showing RuBisCO depletion after immunoaffinity purification using a Seppro IgY-RuBisCO spin column. Large (LSU) and small (SSU) subunit of RuBisCO are marked. M, Molecular weight marker; T, Total protein; U, Unbound protein fraction; SB, Specifically bound proteins.
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Figure 3: RuBisCO depletion from total protein extracts. Silver stained SDS-PAGE (12%) gel showing RuBisCO depletion after immunoaffinity purification using a Seppro IgY-RuBisCO spin column. Large (LSU) and small (SSU) subunit of RuBisCO are marked. M, Molecular weight marker; T, Total protein; U, Unbound protein fraction; SB, Specifically bound proteins.

Mentions: RuBisCO-depletion spin columns (Seppro® IgY affinity purification; SIGMA) effectively removed the large and small subunit of RuBisCO (Figure 3; compare boxed areas in Figures S2A,B). One indication of the effectiveness of RuBisCO depletion is the many fewer number of RuBisCO peptides identified in the depleted samples (10) compared to the phenol-extracted samples (45; Table S7). Surprisingly, only four proteins were found common to both data sets as the top hit in the respective spots: cold, circadian rhythm RNA-binding 2 (CCR2/ATGRP7; spot P69 and S59), phosphoglycerate kinase 1 (PGK1; spot P42 and S50), RuBisCO activase (RCA; spot P06, P12 and spot S11), and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (RBCS; spot P73 and S49; Tables S3, S4; Figure 4). CCR2/ATGRP7 is an RNA-binding protein that is part of a molecular slave oscillator associated with the Arabidopsis circadian system (Schmal et al., 2013). Both extraction methods showed the same late-day peak phase of CCR2 protein (Heintzen et al., 1997), similar to the transcript, but the amplitude of oscillation was more robust using the RuBisCO-depletion extraction method (Figures 4A–C).


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

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

RuBisCO depletion from total protein extracts. Silver stained SDS-PAGE (12%) gel showing RuBisCO depletion after immunoaffinity purification using a Seppro IgY-RuBisCO spin column. Large (LSU) and small (SSU) subunit of RuBisCO are marked. M, Molecular weight marker; T, Total protein; U, Unbound protein fraction; SB, Specifically bound proteins.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: RuBisCO depletion from total protein extracts. Silver stained SDS-PAGE (12%) gel showing RuBisCO depletion after immunoaffinity purification using a Seppro IgY-RuBisCO spin column. Large (LSU) and small (SSU) subunit of RuBisCO are marked. M, Molecular weight marker; T, Total protein; U, Unbound protein fraction; SB, Specifically bound proteins.
Mentions: RuBisCO-depletion spin columns (Seppro® IgY affinity purification; SIGMA) effectively removed the large and small subunit of RuBisCO (Figure 3; compare boxed areas in Figures S2A,B). One indication of the effectiveness of RuBisCO depletion is the many fewer number of RuBisCO peptides identified in the depleted samples (10) compared to the phenol-extracted samples (45; Table S7). Surprisingly, only four proteins were found common to both data sets as the top hit in the respective spots: cold, circadian rhythm RNA-binding 2 (CCR2/ATGRP7; spot P69 and S59), phosphoglycerate kinase 1 (PGK1; spot P42 and S50), RuBisCO activase (RCA; spot P06, P12 and spot S11), and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (RBCS; spot P73 and S49; Tables S3, S4; Figure 4). CCR2/ATGRP7 is an RNA-binding protein that is part of a molecular slave oscillator associated with the Arabidopsis circadian system (Schmal et al., 2013). Both extraction methods showed the same late-day peak phase of CCR2 protein (Heintzen et al., 1997), similar to the transcript, but the amplitude of oscillation was more robust using the RuBisCO-depletion extraction method (Figures 4A–C).

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