In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site

Sang Yeol Kim, Christopher M. Harvey, Jonas Giese, Ines Lassowskat, Vijayata Singh, Amanda P. Cavanagh, Martin H. Spalding, Iris Finkemeier, Donald Richard Ort, Steven C. Huber

Research output: Contribution to journalArticle

Abstract

Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine- 78 (Thr78) in 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 largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, 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 (ΦPSII) and reduced growth relative to control plants expressing wildtype Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ΦPSII relative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of the rcaknockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 μmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Original languageEnglish (US)
Pages (from-to)18723-18731
Number of pages9
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number37
DOIs
StatePublished - Sep 10 2019

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Ribulose-Bisphosphate Carboxylase
Tissue Plasminogen Activator
Threonine
Phosphorylation
Light
Photosystem II Protein Complex
Photosynthesis
Mutation
Growth
Photons
Immunoblotting
Oxidation-Reduction
Arabidopsis Rca protein
Mass Spectrometry
Phenotype
Gene Expression
Antibodies

Keywords

  • Phosphorylation
  • Photosynthesis
  • Rubisco activase

ASJC Scopus subject areas

  • General

Cite this

In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site. / Kim, Sang Yeol; Harvey, Christopher M.; Giese, Jonas; Lassowskat, Ines; Singh, Vijayata; Cavanagh, Amanda P.; Spalding, Martin H.; Finkemeier, Iris; Ort, Donald Richard; Huber, Steven C.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 37, 10.09.2019, p. 18723-18731.

Research output: Contribution to journalArticle

Kim, SY, Harvey, CM, Giese, J, Lassowskat, I, Singh, V, Cavanagh, AP, Spalding, MH, Finkemeier, I, Ort, DR & Huber, SC 2019, 'In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site', Proceedings of the National Academy of Sciences of the United States of America, vol. 116, no. 37, pp. 18723-18731. https://doi.org/10.1073/pnas.1812916116
Kim, Sang Yeol ; Harvey, Christopher M. ; Giese, Jonas ; Lassowskat, Ines ; Singh, Vijayata ; Cavanagh, Amanda P. ; Spalding, Martin H. ; Finkemeier, Iris ; Ort, Donald Richard ; Huber, Steven C. / In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site. In: Proceedings of the National Academy of Sciences of the United States of America. 2019 ; Vol. 116, No. 37. pp. 18723-18731.
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abstract = "Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine- 78 (Thr78) in 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 largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, 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 (ΦPSII) and reduced growth relative to control plants expressing wildtype Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ΦPSII relative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of the rcaknockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 μmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.",
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T1 - In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site

AU - Kim, Sang Yeol

AU - Harvey, Christopher M.

AU - Giese, Jonas

AU - Lassowskat, Ines

AU - Singh, Vijayata

AU - Cavanagh, Amanda P.

AU - Spalding, Martin H.

AU - Finkemeier, Iris

AU - Ort, Donald Richard

AU - Huber, Steven C.

PY - 2019/9/10

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N2 - Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine- 78 (Thr78) in 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 largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, 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 (ΦPSII) and reduced growth relative to control plants expressing wildtype Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ΦPSII relative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of the rcaknockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 μmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

AB - Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine- 78 (Thr78) in 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 largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, 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 (ΦPSII) and reduced growth relative to control plants expressing wildtype Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ΦPSII relative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of the rcaknockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 μmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

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