. As shown in Extended Data Fig. 1a, an ECS signal is visible in P.tricornutum, the characteristics of which depend on the physiological conditions. Deconvolution of the ECS decay-associated spectra (DAS) (see Supplementary Information and Extended Data Fig. 1b,c) explains these observations by revealing the existence of two ECS components (Fig. 1a), respectively characterized by linear and quadratic responses to the ΔΨ (Fig. 1b). The existence of a quadratic ECS, predicted by theory 7 but observed to date only in green algae mutants (Fig. 1c), but was also suppressed by anaerobiosis or by pharmacological inhibition of mitochondrial activity (Fig. 1d, e). This suggests that the PMF is generated in the dark by the plastidial ATPase, which hydrolyses ATP of mitochondrial origin, as previously suggested in green algae 9 .In Viridiplantae (including green algae and higher plants), the AEPs generating additional ATP in the light comprise cyclic electron flow (CEF) around PS1 10 and the water-to-water cycles (WWC). uptake (U 0 ) increased with light, being ~2.5-fold higher at saturating light intensities than in the dark (Extended Data Fig. 2b, d). We further found that the light-stimulated consumption of oxygen was blocked by DCMU (Extended Data Fig. 2c, d), indicating that it was fed by electrons derived from PS2.Moreover, U 0 linearly increased with O 2 evolution, in agreement with earlier findings in the diatom
Thalassiosira pseudonana
15, with a slope indicating that ~10% of the electron flow from PSII participate in WWC, regardless of light intensity (Fig 2b). These results indicate that WWC produces a constant extra ATP per photosynthetically-generated NADPH. This is expected for an AEP that contributes to optimizing CO 2 assimilation at any light intensity, and is not the case for CEF, which is completely insensitive to changes in the photosynthetic flux (LEF, Fig 2a).If this WWC is due to the export of photosynthetic products towards the mitochondrial oxidases, then any mitochondrial dysfunction should negatively affect photosynthetic electron transfer rates (ETR PSII ) and light-dependent growth. Mitochondrial respiration comprises a cyanidesensitive pathway (involving Complex III) and an insensitive pathway involving the alternative oxidase (AOX). We therefore modulated mitochondrial activity by adding increasing amounts of Antimycin A (AA) or myxothiazol (Mx), inhibitors of Complex III, in conditions where the AOX was inhibited by SHAM (see legend to Fig. 2d). Both the ΔΨ d and ETR PSII followed respiration linearly (Fig. 2c, d and Extended Data Fig. 3). The almost complete shut-down of respiration resulted in a decrease of photosynthesis which was independent of light intensity (Fig. 3b).Overall we found that in the dark a PMF is generated in the plastid by hydrolysis of ATP produced in mitochondria (Fig 1d,e and Fig. 2c). Conversely, in the light, respiration increases linearly with photosynthesis (Fig. 2b), and vice versa (Fig. 2d). This tight energetic coupling is likely instrumental for adjusting ...
