Cumulative effect of Aox1 inactivation and N source modulation on the cellular proteome of Chlamydomonas reinhardtii - New insights in the metabolic interconnexion between respiration and photosynthesis

In Chlamydomonas reinhardtii, the Aox1 gene encoding the mitochondrial alternative oxidase (AOX) is tightly regulated by the N source: its transcription is stimulated by nitrate and repressed by ammonium. AOX has been proposed to play a key role in regards to the N source by acting as a trigger factor for the TCA cycle to promote the production of reduced cofactors which, together with photo-generated reductant, could contribute to support nitrate assimilation. In the present work, the cellular proteomes of wild-type and AOX-deficient strains grown either on nitrate or ammonium in the presence of acetate have been compared using two dimensional-differential in-gel electrophoresis (2D-DIGE) coupled to hierarchical clustering and ANOVA-2 statistical analyses in order to further investigate the physiological role(s) of AOX, particularly in regards to the N source. The dramatic up-regulation of chloroplastic components of the carbon-concentrating mechanism (CCM) in response to Aox1 inactivation importantly suggests that AOX plays a key role in the CCM in both N sources by allowing the production of CO2 through the TCA cycle. The concomitant down-regulation of photorespiratory enzymes in cells lacking AOX indicates that such an induction of the CCM could actually be sufficient to over-compensate the effects of AOX deficiency on CO2 availability in the local environment of Rubisco and maintain high rates of inorganic carbon fixation despite lower TCA cycle-mediated CO2 production. The global overexpression of diverse photosynthetic chain and Calvin cycle components in cells lacking AOX further evidences that a tight functional relationship exists between mitochondrial and chloroplastic processes of energy transduction, and that there could be a global redirection of bioenergetics towards photosynthesis to limit the harmful consequences (particularly oxidative stress) of mitochondrial deficiency. The observation that numerous N-responsive proteomic modifications are strongly attenuated (or even abolished) in response to Aox1 inactivation supports that AOX also exerts a more specific role as a central effector of the metabolic plasticity induced by modulating the N source in the growth medium. Our proteomic results tend notably to confirm that respiration (through AOX activity) and photosynthesis collaborate to provide nitrate reduction with electrons in mixotrophic condition and that acetate oxidation could be promoted to support AOX involvement in this context.