Limits...
The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Kim SY, Bender KW, Walker BJ, Zielinski RE, Spalding MH, Ort DR, Huber SC - Front Plant Sci (2016)

Bottom Line: Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco.In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit.Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements.

View Article: PubMed Central - PubMed

Affiliation: Global Change and Photosynthesis Research Unit, United States Department of Agriculture - Agricultural Research Service, UrbanaIL, USA; Plant Biology, University of Illinois at Champaign-Urbana, UrbanaIL, USA.

ABSTRACT
Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

No MeSH data available.


Related in: MedlinePlus

Feeding DTT to leaves inhibits the dark-induced phosphorylation of RCA at the Thr-78 site. Leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT followed by 1 h in the light or dark. Extracts were subjected to non-reducing or reducing SDS-PAGE as indicated and blots were probed with anti-RCA or anti-pT78 antibodies. (A)Arabidopsis Col wild type plants with both RCA isoforms, subjected to SDS-PAGE under reducing or non-reducing conditions as indicated. (B) Transgenic rwt43 plants expressing only the RCAβ isoform, electrophoresed under reducing conditions only. In each blot, the bands corresponding to the α- and β-isoforms are indicated. The red asterisk in the anti-pT78 blots indicates an off-target signal that serves as a loading control, and the black asterisk in (A) identifies the RbcL protein that shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814456&req=5

Figure 3: Feeding DTT to leaves inhibits the dark-induced phosphorylation of RCA at the Thr-78 site. Leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT followed by 1 h in the light or dark. Extracts were subjected to non-reducing or reducing SDS-PAGE as indicated and blots were probed with anti-RCA or anti-pT78 antibodies. (A)Arabidopsis Col wild type plants with both RCA isoforms, subjected to SDS-PAGE under reducing or non-reducing conditions as indicated. (B) Transgenic rwt43 plants expressing only the RCAβ isoform, electrophoresed under reducing conditions only. In each blot, the bands corresponding to the α- and β-isoforms are indicated. The red asterisk in the anti-pT78 blots indicates an off-target signal that serves as a loading control, and the black asterisk in (A) identifies the RbcL protein that shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.

Mentions: To determine if stromal oxidation is the factor that triggers phosphorylation of RCA, wild type Arabidopsis or transgenic plants expressing just the non-redox regulated 43-kDa RCAβ isoform were examined for responses to exogenous DTT. In the experiment presented in Figure 3, leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT prior to incubation for 1 h in the light or dark as indicated. Leaves were harvested at the end of light or dark treatment, and extracts were prepared and fractionated by non-reducing or reducing SDS-PAGE as indicated prior to immunoblot analysis. In wild type Arabidopsis leaf extracts analyzed by non-reducing SDS-PAGE, three bands of RCA protein were detected, including two corresponding to the reduced and oxidized forms of RCAα and one band corresponding to RCAβ (Figure 3A, middle). Incubation of leaves in the light with increasing concentrations of the reductant DTT promoted reduction of the intramolecular disulfide bond in RCAα such that at 10 mM DTT only a single RCAα band was observed that corresponds to the slower migrating reduced isoform. In the dark, control leaves (0 mM DTT) had a single α-isoform band corresponding to the oxidized, faster migrating form, but in the presence of DTT the reduced RCAα subunit was restored (Figure 3A). It is known that DTT can reduce oxidized thioredoxins (Buchanan et al., 1979) and that redox-regulated chloroplast enzymes can be reduced in the dark by DTT treatment of leaves (Kolbe et al., 2006); because RCAα is reductively activated by thioredoxin-f (Zhang and Portis, 1999), the results are indeed expected and confirm that the DTT treatment has altered the redox status of the chloroplast stroma. Analysis of the same samples on reducing SDS-PAGE detected only the single (reduced) RCAα and RCAβ bands (Figure 3B). Also as expected, phosphorylation of RCA Thr-78 was observed only in the dark in the control (-DTT) leaf sample where phosphorylation of both α- and β-isoforms was apparent. The most important result is that treatment of leaves with DTT in the dark largely prevented the phosphorylation, suggesting that oxidation of the stromal thioredoxin(s) was the trigger for phosphorylation of RCA (Figure 3A, bottom). The changes in RCA Thr-78 phosphorylation were also apparent when proteins were electrophoresed on non-reducing SDS-PAGE (not shown). In order to determine whether stromal redox was affecting phosphorylation of RCA indirectly as a result of reversible disulfide bond formation in the C-terminal extension of RCAα, we tested the effect of DTT treatment on leaves expressing only the non-redox regulated RCAβ isoform (Figure 3B). Because these plants only contain the non-redox regulated RCA isoform, electrophoresis was performed only under reducing conditions, where bands are sharper and non-specific antibody reactions are less pronounced (e.g., reaction of the antibodies with the RbcL band). As shown, phosphorylation of RCAβ at the Thr-78 site occurred in the darkened control leaf sample and was greatly reduced by 10 mM DTT. These results suggest that it is not the redox status of RCA itself that affects phosphorylation of the protein, but rather other steps in the process.


The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Kim SY, Bender KW, Walker BJ, Zielinski RE, Spalding MH, Ort DR, Huber SC - Front Plant Sci (2016)

Feeding DTT to leaves inhibits the dark-induced phosphorylation of RCA at the Thr-78 site. Leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT followed by 1 h in the light or dark. Extracts were subjected to non-reducing or reducing SDS-PAGE as indicated and blots were probed with anti-RCA or anti-pT78 antibodies. (A)Arabidopsis Col wild type plants with both RCA isoforms, subjected to SDS-PAGE under reducing or non-reducing conditions as indicated. (B) Transgenic rwt43 plants expressing only the RCAβ isoform, electrophoresed under reducing conditions only. In each blot, the bands corresponding to the α- and β-isoforms are indicated. The red asterisk in the anti-pT78 blots indicates an off-target signal that serves as a loading control, and the black asterisk in (A) identifies the RbcL protein that shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814456&req=5

Figure 3: Feeding DTT to leaves inhibits the dark-induced phosphorylation of RCA at the Thr-78 site. Leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT followed by 1 h in the light or dark. Extracts were subjected to non-reducing or reducing SDS-PAGE as indicated and blots were probed with anti-RCA or anti-pT78 antibodies. (A)Arabidopsis Col wild type plants with both RCA isoforms, subjected to SDS-PAGE under reducing or non-reducing conditions as indicated. (B) Transgenic rwt43 plants expressing only the RCAβ isoform, electrophoresed under reducing conditions only. In each blot, the bands corresponding to the α- and β-isoforms are indicated. The red asterisk in the anti-pT78 blots indicates an off-target signal that serves as a loading control, and the black asterisk in (A) identifies the RbcL protein that shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.
Mentions: To determine if stromal oxidation is the factor that triggers phosphorylation of RCA, wild type Arabidopsis or transgenic plants expressing just the non-redox regulated 43-kDa RCAβ isoform were examined for responses to exogenous DTT. In the experiment presented in Figure 3, leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT prior to incubation for 1 h in the light or dark as indicated. Leaves were harvested at the end of light or dark treatment, and extracts were prepared and fractionated by non-reducing or reducing SDS-PAGE as indicated prior to immunoblot analysis. In wild type Arabidopsis leaf extracts analyzed by non-reducing SDS-PAGE, three bands of RCA protein were detected, including two corresponding to the reduced and oxidized forms of RCAα and one band corresponding to RCAβ (Figure 3A, middle). Incubation of leaves in the light with increasing concentrations of the reductant DTT promoted reduction of the intramolecular disulfide bond in RCAα such that at 10 mM DTT only a single RCAα band was observed that corresponds to the slower migrating reduced isoform. In the dark, control leaves (0 mM DTT) had a single α-isoform band corresponding to the oxidized, faster migrating form, but in the presence of DTT the reduced RCAα subunit was restored (Figure 3A). It is known that DTT can reduce oxidized thioredoxins (Buchanan et al., 1979) and that redox-regulated chloroplast enzymes can be reduced in the dark by DTT treatment of leaves (Kolbe et al., 2006); because RCAα is reductively activated by thioredoxin-f (Zhang and Portis, 1999), the results are indeed expected and confirm that the DTT treatment has altered the redox status of the chloroplast stroma. Analysis of the same samples on reducing SDS-PAGE detected only the single (reduced) RCAα and RCAβ bands (Figure 3B). Also as expected, phosphorylation of RCA Thr-78 was observed only in the dark in the control (-DTT) leaf sample where phosphorylation of both α- and β-isoforms was apparent. The most important result is that treatment of leaves with DTT in the dark largely prevented the phosphorylation, suggesting that oxidation of the stromal thioredoxin(s) was the trigger for phosphorylation of RCA (Figure 3A, bottom). The changes in RCA Thr-78 phosphorylation were also apparent when proteins were electrophoresed on non-reducing SDS-PAGE (not shown). In order to determine whether stromal redox was affecting phosphorylation of RCA indirectly as a result of reversible disulfide bond formation in the C-terminal extension of RCAα, we tested the effect of DTT treatment on leaves expressing only the non-redox regulated RCAβ isoform (Figure 3B). Because these plants only contain the non-redox regulated RCA isoform, electrophoresis was performed only under reducing conditions, where bands are sharper and non-specific antibody reactions are less pronounced (e.g., reaction of the antibodies with the RbcL band). As shown, phosphorylation of RCAβ at the Thr-78 site occurred in the darkened control leaf sample and was greatly reduced by 10 mM DTT. These results suggest that it is not the redox status of RCA itself that affects phosphorylation of the protein, but rather other steps in the process.

Bottom Line: Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco.In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit.Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements.

View Article: PubMed Central - PubMed

Affiliation: Global Change and Photosynthesis Research Unit, United States Department of Agriculture - Agricultural Research Service, UrbanaIL, USA; Plant Biology, University of Illinois at Champaign-Urbana, UrbanaIL, USA.

ABSTRACT
Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

No MeSH data available.


Related in: MedlinePlus