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International audience; Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H$^+$ /K$^+$ antiporter KEA3 in the model diatom $Phaeodactylum\ tricornutum$. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca$^{2+}$ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

In eukaryotic organisms, oxygenic photosynthesis is catalyzed by 4 multiprotein complexes present on the thylakoid membranes inside the chloroplasts: Photosystem (PS) II, Cytochrome b6f (Cyt b6f), PSI and chloroplast ATP synthase (ATPase). The three former mediate a Linear Electron Flow (LEF) from H2O to NADP+ to synthetize NADPH. LEF also generate a proton gradient across the thylakoid membrane, supporting ATP synthesis. NADPH and ATP are exploited by Calvin Benson cycle to fix inorganic CO2 in sugars available for the metabolism. Photosynthetic process is accompanied by the formation of unstable molecules that can generate reactive oxygen species (ROS) harmful for the cell. Particular regulatory mechanisms prevent the formation of these molecules. Among these, the Alternative Electron Flow (AEF) divert electrons from photosynthetic processes preventing over-reduction of electron transporters and ROS production. AEF mechanisms can be divided in Cyclic Electron Flow (CEF) and Pseudo-CEF (PCEF) according to the acceptors of the pathways, respectively the plastoquinone and O2. Two major CEF are known and mediated by PGR5/PGRL1 or by NDH-1 complex. PCEF is instead mediated by Flavodiiron proteins (FLV). In this thesis I focused on the early land plant Physcomitrella patens, this moss presents all the main AEF identified in algae and vascular plants and is thus a suitable model to directly compared their activity in the same organisms. FLV proteins were lost in Angiosperms, one of the plausible explanations to this loss is that another AEF mechanism replaces their function, making FLV dispensable. FLV were shown to be fundamental to respond to light fluctuation that occurs in the natural environments, a function that in angiosperms was replaced by PGR5/PGRL1. To evaluate the hypothesis that PGRL1/PGR5 CEF mediated pathway can replace FLV, in chapter 3 we isolated a P. patens mutant defective for both FLV and PGRL1. Our experiments highlighted a strong synergy between the 2 pathways especially when plants were exposed to stressful light regimes, suggesting a functional overlap and the possible replacement of one pathway over the other during evolution. Further analysis allowed us to establish that FLV and PGRL1 are particularly important for protection of PSI from over-reduction, thus preventing photodamage. In chapter 4, we characterized NDH-1 complex in P. patens isolating a line defective for NDH-1 function. NDH-1 defective mutants showed a small defect in PSI functionality in the first moment after light exposure. Reiterating cycle of dark and light, the defect observed increased significantly in NDH-1 defective plants suggesting that this complex may operates in first seconds after light exposure to avoid PSI over-reduction. However, this defect did not impact the plant growth not even in fluctuating light conditions, probably because of the compensation from other mechanisms present in the chloroplast. In order to entangle this possible compensation in chapter 5 NDH-1 was depleted in plants knock out for both PGRL1 and FLV, by isolating a double mutant lacking both CEF pathways (pgrl1-ndhm) and a triple mutant defective for both CEF and PCEF (flva-pgrl1-ndhm). Both exhibited a drastic decrease in growth and photosynthetic performances showing that NDH-1 depletion has a much larger effect if other mechanisms for AEF are also absent, eliminating compensating effects. The triple mutant showed growth defects even in non-stressing conditions suggesting these mechanisms are important also for steady state photosynthesis. Spectroscopic and biochemical characterization detected extensive damage to PSI, correlated with the growth defects.