Photosynthesis redox regulation in microalgae revealed by in-cell real-time Nuclear Magnetic Resonance – PhotOxIN

CO2 assimilation in the Calvin-Benson-Basham (CBB) cycle is dependent on light and the availability of ATP and NADPH, produced upon the photochemical phase of photosynthesis. It is inhibited in the dark by the oxidation of thiol groups of some of CBB enzymes, by pH shift, and by proteins-proteins interactions. This project aims at understanding the molecular basis of CBB enzyme regulation, taking into account new elements of the redox network that have been recently described as well as the multiphasic chloroplastic medium. In particular, the glyceraldehyde phosphate dehydrogenase (GAPDH) and the phosphoribulokinase (PRK) are inhibited in the dark by the formation a ternary complex with a small protein redox mediator, CP12. In the light, CP12 is disordered, the complex dissociates, and other functions have been proposed for CP12 on the basis of in vivo studies. Although purified CBB enzymes are highly soluble in water, few in vivo studies localised them in the vicinity of thylakoid membranes. It could influence spatial confinement and molecular crowding but has not been considered until now to investigate the molecular basis of GAPDH and PRK regulation by CP12. In order to take into account this physiological and physico-chemical environment, we propose to study the structural properties and transitions of CP12 in the cellular context by in-cell NMR using the model microalga Chlamydomonas reinhardtii. We will couple and correlate the structural transitions of CP12 with the function of the CBB cycle by quantifying specific CBB metabolites using high resolution magic angle spinning NMR (HR-MAS NMR). This structure/metabolism tandem will be studied in the light, in the dark, and upon dark-to-light and light-to-dark transition using ultra-fast NMR mehods.