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

Abstract

<i>Arabidopsis</i> 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 <i>rca</i>-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 (ϕ_PSII) and reduced growth relative to control plants expressing wild-type 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 ϕ_PSII 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 <i>rca-</i>knockdown 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.