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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

Phosphorylation of RCA at the Thr78 site is light/dark regulated in vivo. (A) Plants sampled at different times of the normal diurnal light/dark cycle. (B) Transfer of plants at midday to low light and darkness for 1 h. (C) Time course of a light-dark-light transfer of plants at midday. The experiment started 5 h after the beginning of the photoperiod (L5 sample). 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.
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Figure 1: Phosphorylation of RCA at the Thr78 site is light/dark regulated in vivo. (A) Plants sampled at different times of the normal diurnal light/dark cycle. (B) Transfer of plants at midday to low light and darkness for 1 h. (C) Time course of a light-dark-light transfer of plants at midday. The experiment started 5 h after the beginning of the photoperiod (L5 sample). 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.

Mentions: Arabidopsis leaves contain roughly equal amounts of the RCA α- and β-isoforms and their abundance is relatively constant over a diurnal day/night cycle (Figure 1A). In contrast, phosphorylation of RCA at the Thr-78 site, as detected by immunoblotting with sequence- and modification-specific antibodies (anti-pT78 antibodies; Figure 1A), was only observed at night and both isoforms were equally phosphorylated. Full blots showing the specificity of these custom antibodies are presented in Supplementary Figure S1; apart from the α- and β-isoforms of RCA, only the anti-pT78 antibodies reacted with an additional off-target band (designated by the asterisk) that provides a useful loading control. Phosphorylation of Thr-78 was also triggered by exposure to low light, which resulted in partial phosphorylation compared to that observed after 1 h of complete darkness (Figure 1B). Thus, the light signal controlling RCA phosphorylation seems to not operate as an on–off switch, but rather is also responding to low irradiance with partial phosphorylation. In addition, the experiment presented in Figure 1B was performed in the middle of the day whereas the experiment in Figure 1A was performed where the dark period coincided with the normal night cycle. Thus is it is also clear that RCA phosphorylation is responding to instantaneous light conditions. The time course of phosphorylation and dephosphorylation at the Thr-78 phosphosite is presented in Figure 1C for plants transferred to darkness after 5 h of illumination. As shown, phosphorylation occurred rather slowly to reach a maximum value after 1 h of darkness and both RCA isoforms responded in similar fashion. In contrast, dephosphorylation upon transfer of plants to the light occurred rapidly and was nearly complete within 5 min.


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)

Phosphorylation of RCA at the Thr78 site is light/dark regulated in vivo. (A) Plants sampled at different times of the normal diurnal light/dark cycle. (B) Transfer of plants at midday to low light and darkness for 1 h. (C) Time course of a light-dark-light transfer of plants at midday. The experiment started 5 h after the beginning of the photoperiod (L5 sample). 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Phosphorylation of RCA at the Thr78 site is light/dark regulated in vivo. (A) Plants sampled at different times of the normal diurnal light/dark cycle. (B) Transfer of plants at midday to low light and darkness for 1 h. (C) Time course of a light-dark-light transfer of plants at midday. The experiment started 5 h after the beginning of the photoperiod (L5 sample). 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.
Mentions: Arabidopsis leaves contain roughly equal amounts of the RCA α- and β-isoforms and their abundance is relatively constant over a diurnal day/night cycle (Figure 1A). In contrast, phosphorylation of RCA at the Thr-78 site, as detected by immunoblotting with sequence- and modification-specific antibodies (anti-pT78 antibodies; Figure 1A), was only observed at night and both isoforms were equally phosphorylated. Full blots showing the specificity of these custom antibodies are presented in Supplementary Figure S1; apart from the α- and β-isoforms of RCA, only the anti-pT78 antibodies reacted with an additional off-target band (designated by the asterisk) that provides a useful loading control. Phosphorylation of Thr-78 was also triggered by exposure to low light, which resulted in partial phosphorylation compared to that observed after 1 h of complete darkness (Figure 1B). Thus, the light signal controlling RCA phosphorylation seems to not operate as an on–off switch, but rather is also responding to low irradiance with partial phosphorylation. In addition, the experiment presented in Figure 1B was performed in the middle of the day whereas the experiment in Figure 1A was performed where the dark period coincided with the normal night cycle. Thus is it is also clear that RCA phosphorylation is responding to instantaneous light conditions. The time course of phosphorylation and dephosphorylation at the Thr-78 phosphosite is presented in Figure 1C for plants transferred to darkness after 5 h of illumination. As shown, phosphorylation occurred rather slowly to reach a maximum value after 1 h of darkness and both RCA isoforms responded in similar fashion. In contrast, dephosphorylation upon transfer of plants to the light occurred rapidly and was nearly complete within 5 min.

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