Non-enzymatic acetylation of lysine residues affects the stability of Rubisco activase

Post-translational modifications of non-histone proteins are critical for plant adaptation to fluctuating environmental conditions. Rubisco activase (RCA), a chloroplastic ATPase, plays a crucial role in CO₂ fixation by activating ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In this study, we determined that RCA isolated from tobacco (Nicotiana tabacum) leaves is acetylated at multiple lysine residues, with K126 and K164 being key acetylation sites. The acetylation level of RCA varies under different light regimes (day vs. night) and heat-stress conditions. RCA undergoes non-enzymatic acetylation in vitro through direct interactions with various metabolites, particularly acetyl-CoA (Ac-CoA). Using deuterium-labeled Ac-CoA, we confirmed that lysine residues K126 and K164 are acetylated; this acetylation event leads to a significant reduction in the ATPase activity of RCA, with only a minor impact on Rubisco activation. Additionally, higher acetylation levels of RCA accelerate its degradation in chloroplasts, a process independent of ubiquitination. Our findings uncover a previously unknown function of protein acetylation in regulating RCA stability, which is essential for modulating carbon assimilation efficiency in plants under varying energy conditions.