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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

The cpck2 mutant is generally similar to wild type Arabidopsis suggesting that phosphorylation of RCA at the Thr-78 site is not playing an essential role in vivo. Induction kinetics of photosynthesis following pretreatment in (A) low light or (B) darkness. (C) Rubisco activation state after exposure of leaves for 60 min to low or high light as in the experiment in (A). (D) Relative plant growth rate in short days determined based on leaf area expansion. (E) Chloroplast structure showing normal granal development and accumulation of starch granules at the end of the day.
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Figure 8: The cpck2 mutant is generally similar to wild type Arabidopsis suggesting that phosphorylation of RCA at the Thr-78 site is not playing an essential role in vivo. Induction kinetics of photosynthesis following pretreatment in (A) low light or (B) darkness. (C) Rubisco activation state after exposure of leaves for 60 min to low or high light as in the experiment in (A). (D) Relative plant growth rate in short days determined based on leaf area expansion. (E) Chloroplast structure showing normal granal development and accumulation of starch granules at the end of the day.

Mentions: As one approach to study the functional role of phosphorylation in vivo, we examined the induction of leaf photosynthesis by gas exchange following changes in irradiance with wild type Arabidopsis and the cpck2 knockout mutant that has strongly reduced phosphorylation at the Thr-78 site (Figure 5A). It is generally accepted that RCA activity controls the rate of induction of photosynthesis following transfer from low to high light (Mott and Woodrow, 2000; Carmo-Silva and Salvucci, 2013). Therefore, if phosphorylation of Thr-78 is important in regulating RCA activity during light transitions, preventing phosphorylation of RCA by genetic removal of the requisite protein kinase, cpCK2α should impact induction kinetics of photosynthesis. Nearly identical photosynthetic induction kinetics were observed for wild type and the cpck2 knockout mutant when plants were transferred from low light (Figure 8A) or darkness (Figure 8B) to high light. Additionally, in vitro Rubisco activity assays measured under similar conditions revealed deactivation of Rubisco in leaves of wild type plants and the cpck2 knockout mutant at low light compared to high light and the differences between the genotypes under each light condition were not statistically significant based on the t-test (Figure 8C). These results strongly suggest that at least in wild type Arabidopsis plants expressing both RCA isoforms, phosphorylation of RCA does not play an essential and non-redundant role relative to redox regulation in deactivation of RCA activity at low light.


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)

The cpck2 mutant is generally similar to wild type Arabidopsis suggesting that phosphorylation of RCA at the Thr-78 site is not playing an essential role in vivo. Induction kinetics of photosynthesis following pretreatment in (A) low light or (B) darkness. (C) Rubisco activation state after exposure of leaves for 60 min to low or high light as in the experiment in (A). (D) Relative plant growth rate in short days determined based on leaf area expansion. (E) Chloroplast structure showing normal granal development and accumulation of starch granules at the end of the day.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: The cpck2 mutant is generally similar to wild type Arabidopsis suggesting that phosphorylation of RCA at the Thr-78 site is not playing an essential role in vivo. Induction kinetics of photosynthesis following pretreatment in (A) low light or (B) darkness. (C) Rubisco activation state after exposure of leaves for 60 min to low or high light as in the experiment in (A). (D) Relative plant growth rate in short days determined based on leaf area expansion. (E) Chloroplast structure showing normal granal development and accumulation of starch granules at the end of the day.
Mentions: As one approach to study the functional role of phosphorylation in vivo, we examined the induction of leaf photosynthesis by gas exchange following changes in irradiance with wild type Arabidopsis and the cpck2 knockout mutant that has strongly reduced phosphorylation at the Thr-78 site (Figure 5A). It is generally accepted that RCA activity controls the rate of induction of photosynthesis following transfer from low to high light (Mott and Woodrow, 2000; Carmo-Silva and Salvucci, 2013). Therefore, if phosphorylation of Thr-78 is important in regulating RCA activity during light transitions, preventing phosphorylation of RCA by genetic removal of the requisite protein kinase, cpCK2α should impact induction kinetics of photosynthesis. Nearly identical photosynthetic induction kinetics were observed for wild type and the cpck2 knockout mutant when plants were transferred from low light (Figure 8A) or darkness (Figure 8B) to high light. Additionally, in vitro Rubisco activity assays measured under similar conditions revealed deactivation of Rubisco in leaves of wild type plants and the cpck2 knockout mutant at low light compared to high light and the differences between the genotypes under each light condition were not statistically significant based on the t-test (Figure 8C). These results strongly suggest that at least in wild type Arabidopsis plants expressing both RCA isoforms, phosphorylation of RCA does not play an essential and non-redundant role relative to redox regulation in deactivation of RCA activity at low light.

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