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 CO 2 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.