|Kim, Sang Yeol|
|Bender, Kyle - University Of Illinois|
|Zielinski, Michael - University Of Illinois|
|Spalding, Martin - Iowa State University|
Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/2016
Publication Date: 3/31/2016
Citation: Kim, S., Bender, K.W., Walker, B.J., Salvucci, M.E., Zielinski, M.E., Spalding, M.H., Ort, D.R., Huber, S.C. 2016. The plastid casein kinase 2 phosphorylates Rubisco activase at the Thr-78 site but is not essential for regulation of Rubisco activation state. Frontiers in Plant Science. doi: 10.3389/fpls.2016.00404.
Interpretive Summary: Rubisco is the enzyme that catalyzes the fixation of atmospheric carbon dioxide in photosynthesis, and its activity is dependent on the continual action of its helper protein, Rubisco activase. The activity of Rubisco activase is known to be regulated by reversible formation of a disulfide bond between two cysteine residues located near the carboxy terminus of the protein. The disulfide is formed at low light and in darkness, which reduces Rubisco activase activity and as a result Rubisco activity as well. Phosphorylation of Rubisco activase was recently identified and was also shown to occur in a light/dark-regulated manner and thus could contribute along with the redox mechanism to control Rubisco activity. However, studies with recombinant Rubisco activase in vitro did not identify an effect of mimicking the phosphorylation event by substitution of an acidic residue for the phosphorylated threonine. In addition, preventing phosphorylation or Rubisco activase in vivo by genetically removing the requisite protein kinase, which was determined to be plastid casein kinase 2, did not affect the induction kinetics of leaf photosynthesis in response to changes in light. Collectively, the results suggest that phosphorylation of Rubisco activase occurs in the dark but is not essential for down regulation of Rubisco activity, at least under optimal growth conditions.
Technical Abstract: Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is encoded by a single gene (At2g39730) that is alternatively spliced to form a large alpha-RCA and small beta-RCA isoform. 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 alpha-RCA subunit. Additionally, phosphorylation of RCA threonine-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. However, details of RCA phosphorylation and its functional role are lacking. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and non-reducing SDS-PAGE to monitor the redox status of the alpha-RCA subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark. Feeding DTT to leaf segments prevented the dark-induced oxidation of the alpha-RCA isoform and strongly inhibited phosphorylation of both isoforms suggesting that stromal redox was the dark signal. A nearly similar inhibition of phosphorylation at the Thr-78 site was observed when transgenic plants (rwt43) expressing only the beta-RCA isoform were provided exogenous DTT, indicating that redox was affecting phosphorylation via activation of the protein kinase and/or inhibition of the protein phosphatase. The plastid-targeted casein kinase 2 (cpCK2) was identified as the major protein kinase involved in phosphorylation of Thr-78 based on studies with an Arabidopsis cpCK2 knockout mutant and confirmed with in vitro phosphorylation studies with recombinant proteins. Characterization of a phosphomimetic mutant of beta-RCA (T78D/E) suggested that phosphorylation had no effect on ATPase activity in the absence or presence of ADP. Moreover, the cpCK2 knockout mutant was similar to the wild type in terms of activation kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that phosphorylation of RCA Thr-78 does not act in concert with redox regulation to control Rubisco activation state under normal conditions. We conclude that the phosphorylation of both isoforms of RCA at the Thr-78 site is regulated by redox status of the chloroplast stroma, but does not impact RCA activity in a similar manner to redox regulation of the alpha-RCA isoform.