Metabolic modelling identifies mitochondrial Pi uptake and pyruvate efflux as key aspects of daytime metabolism and proton homeostasis in crassulacean acid metabolism leaves

Crassulacean acid metabolism (CAM) leaves are characterized by nocturnal acidification anddiurnal deacidification processes related with the timed actions of phosphoenolpyruvate carbox-ylase and Rubisco, respectively. How CAM leaves manage cytosolic proton homeostasis, particu-larly when facing massive diurnal proton effluxes from the vacuole, remains unclear. A 12-phase flux balance analysis (FBA) model was constructed for a mature malicenzyme-type CAM mesophyll cell in order to predict diel kinetics of intracellular proton fluxes. The charge- and proton-balanced FBA model identified the mitochondrial phosphate carrier(PiC, Pi/H+symport), which provides Pi to the matrix to sustain ATP biosynthesis, as a majorconsumer of cytosolic protons during daytime (>50%). The delivery of Pi to the mitochon-drion, co-transported with protons, is required for oxidative phosphorylation and allows suffi-cient ATP to be synthesized to meet the high energy demand during CAM Phase III.Additionally, the model predicts that mitochondrial pyruvate originating from decarboxylationof malate is exclusively exported to the cytosol, probably via a pyruvate channel mechanism,to fuel gluconeogenesis. In this biochemical cycle, glyceraldehyde 3-phosphate dehydrogen-ase (GAPDH) acts as another important cytosolic proton consumer. Overall, our findings emphasize the importance of mitochondria in CAM and uncover ahitherto unappreciated role in metabolic proton homeostasis. ; Funding was provided by the Research Fund KU Leuven. Figures were created with BioRender.com.