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

Abstract

Summary Crassulacean acid metabolism (CAM) leaves are characterized by nocturnal acidification and diurnal deacidification processes related with the timed actions of phosphoenolpyruvate carboxylase and Rubisco, respectively. How CAM leaves manage cytosolic proton homeostasis, particularly when facing massive diurnal proton effluxes from the vacuole, remains unclear. A 12‐phase flux balance analysis (FBA) model was constructed for a mature malic enzyme‐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 major consumer of cytosolic protons during daytime (> 50%). The delivery of Pi to the mitochondrion, co‐transported with protons, is required for oxidative phosphorylation and allows sufficient ATP to be synthesized to meet the high energy demand during CAM Phase III. Additionally, the model predicts that mitochondrial pyruvate originating from decarboxylation of malate is exclusively exported to the cytosol, probably via a pyruvate channel mechanism, to fuel gluconeogenesis. In this biochemical cycle, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) acts as another important cytosolic proton consumer. Overall, our findings emphasize the importance of mitochondria in CAM and uncover a hitherto unappreciated role in metabolic proton homeostasis.