AbstractPlants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a posttranslational modification to directly modulate metabolic function. We generated a lysine acetylome of the mossPhyscomitrium patensand identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. InP. patensmMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected inArabidopsis thalianamMDH1. We assessed the kinetic behavior of recombinantA. thalianaandP. patensmMDH1 with site-specifically incorporated acetyl-lysines. Acetylation ofA. thalianamMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation ofP. patensmMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only inA. thalianamMDH1, where acetylation of K334 highly activated it. Comparative homology modelling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.Significance statementWe explore the role of lysine acetylation as a mechanism to directly modulate mitochondrial metabolism in land plants by generating the lysine acetylome of the mossPhyscomitrium patensand comparing with available angiosperm data. We found acetylation of evolutionarily conserved lysines as a strategy to fine-tune the activity of mitochondrial malate dehydrogenase in a species-dependent molecular context.