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Title: In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr-78 site

item Kim, Sang Yeol
item Harvey, Christopher
item GIESE, JONAS - Muenster University
item LASSOWSKAT, INES - Muenster University
item SINGH, VIJAYATA - University Of Illinois
item CAVANAGH, AMANDA - University Of Illinois
item SPALDING, MARTIN - Iowa State University
item FINKMEIER, IRIS - Muenster University
item Ort, Donald
item Huber, Steven

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2019
Publication Date: 8/26/2019
Citation: Kim, S., Harvey, C.M., Giese, J., Lassowskat, I., Singh, V., Cavanagh, A., Spalding, M.H., Finkmeier, I., Ort, D.R., Huber, S.C. 2019. In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr-78 site. Proceedings of the National Academy of Sciences.

Interpretive Summary: Rubisco activase (Rca) is central to the control of Rubisco activation state, the carboxylating enzyme of photosynthesis. Regulation of Rca by redox status of cysteine residues in species such as Arabidopsis is well recognized. Recently phosphorylation of Rca at threonine-78 has been identified and we now show the regulatory role of phosphorylation in vivo. Surprisingly, we also observed that the conservative substitution of serine for threonine-78 results in impaired functionality of Rca in vivo that is associated with retention of phosphorylation well into the light period and reduced plant growth. This likely reflects in part the specificity of the requisite protein kinase(s) for serine versus threonine and may explain the absence of serine at this position in terrestrial plants.

Technical Abstract: Arabidopsis Rubisco activase (Rca) is phosphorylated at the threonine-78 (Thr78) site at low light and in the dark suggesting a potential regulatory role in photosynthesis but this has not been directly tested. To do so, we transformed an rca-knockdown mutant with either wild type Rca-ß (control), or the phospho-null Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutants. Interestingly, the T78S mutant was hyper-phosphorylated at the serine-78 site relative to the Thr78 wild type controls as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II ('PSII) and reduced growth relative to the control transgenic plants expressing wild type Rca-ß (control plants) under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased 'PSII relative to control transgenic plants. While expression of the wild type Rca-ß or the T78A mutant fully rescued the slow growth phenotype of the rca-knockdown mutant grown in a square wave light regime, the T78A mutants grew faster than control plants at low light (30 µmol photons m-2 s-1) or in a fluctuating low light/high light environment. Collectively, these results suggest that phosphorylation of threonine-78 (or serine-78 in the T78S mutant) plays a negative regulatory role in vivo, and provides an explanation for the absence of serine at position 78 in terrestrial species.