Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms

Journal name:
Nature
Volume:
524,
Pages:
366–369
Date published:
DOI:
doi:10.1038/nature14599
Received
Accepted
Published online
Corrected online

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth’s climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere1. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests2. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes3, 4, 5, 6. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

At a glance

Figures

  1. ECS measures the PMF in P. tricornutum.
    Figure 1: ECS measures the PMF in P. tricornutum.

    a, Deconvolution of the experimental ECS spectrum (black) into linear (blue) and quadratic (red) spectral components (see Methods); r.u., relative units. b, Schematic representation of polar (blue) and polarizable (red) pigments, and their associated linear (blue) and quadratic (red) ECS responses to the electric field. Black: thylakoid lipid bilayer. Green ‘+’ and ‘−’ symbols: ΔΨ. Red arrows: pigment polarization induced by ΔΨ. c, d, Relationship between quadratic and linear ECS in control (c) and in cells treated with uncoupler (8 µM FCCP, black squares), anaerobiosis (red circles) and respiratory inhibitors (AA, 5 µM, and SHAM, 1 mM; blue circles) (d). Green arrow: extent of the dark electric field (ΔΨd). Representative of five (c) and three (d) independent biological samples. e, Schematic representation of the energetic interactions between plastid (left) and mitochondria (right) in the dark. Red arrows: respiratory electron flows. Green dashed line: putative ATP/ADP exchange pathway between the organelles. ATPase, ATPase/synthase; b6f, cytochrome b6f; I/III/IV, respiratory complexes I, III and IV.

  2. Energetic interactions between mitochondria and plastid in P. tricornutum.
    Figure 2: Energetic interactions between mitochondria and plastid in P. tricornutum.

    a, Relationship between CEF capacity and total electron flow (TEF, mean ± s.d. from data in Extended Data Fig. 3a, c). b, Relationship between oxygen uptake (U0) and gross photosynthesis (E0) as measured by membrane-inlet mass spectrometry (mean ± s.e.m. from data in Extended Data Fig. 3b, d). c, Dependency of photosynthetic activity (ETRPS II) on respiration rates (as the percentage of control, mean ± s.d. from data in Extended Data Fig. 4). Closed circles: SHAM + AA; open circles: SHAM + myxothiazol treatments (see Methods). d, Schematic representation of possible plastid–mitochondria metabolic interactions in the light. Continuous and dashed blue arrows: photosynthetic linear and cyclic flows, respectively. Yellow arrow: exchange of reducing equivalents. For other symbols, see text and Fig. 1e.

  3. Phenotypic traits of AOX knockdown lines of P. tricornutum.
    Figure 3: Phenotypic traits of AOX knockdown lines of P. tricornutum.

    a, Relative sensitivity of photosynthesis (ETRPS II) to addition of respiratory inhibitors: AA (magenta), SHAM (dark yellow) and AA + SHAM (black) (n = 3 ± S.D), or to knockdown of AOX (n = 5 ± s.d.). Blue and red: kd-c5 and kd-c9, respectively. b, Western blot analysis of photosynthetic and respiratory complexes. c, Growth rates of the wild-type and AOX lines (n = 7 ± s.d.). d, In vivo light dependency of NADPH redox state in wild-type and AOX lines. Data are normalized to the maximum value in the light. e, In vivo 31P-NMR evaluation of the NTP content in wild-type and AOX knockdown lines, in the dark or in low light, with or without AA (data normalized to the dark values). d, e, Three independent biological samples.

  4. ATP transfer from mitochondria to plastid in representative diatoms.
    Figure 4: ATP transfer from mitochondria to plastid in representative diatoms.

    a, Spectra of the linear (blue) and quadratic (red) ECS probes in four diatoms. Blue and red vertical dashed lines represent the wavelengths used for linear and quadratic ECS, respectively. Spectra are normalized to 1 at the maximum value of the linear ECS. b, Relationship between the quadratic and the linear ECS in control conditions (open symbols) and in AA + SHAM conditions (filled symbols). ΔΨd is represented as a horizontal arrow. Data are representative of three independent biological samples.

  5. Deconvolution of the quadratic and linear ECS in P. tricornutum.
    Extended Data Fig. 1: Deconvolution of the quadratic and linear ECS in P. tricornutum.

    a, Absorption difference (ΔI/I) kinetics followed at different wavelengths in P. tricornutum, after a series of six saturating laser flashes, in anaerobic conditions. Solid lines correspond to the global fit of the experimental data with a sum of two exponential decays, with time constants τ and 2τ, respectively, as expected for linear and quadratic dependencies (see Methods). b, ΔI/I spectra are shown at different times during ECS relaxation. All spectra were normalized to 1 at 520 nm for better comparison. The observation that the blue and green parts of the spectrum are homothetic during relaxation, while changes are seen in the red most part of it, reflects the presence of the two ECS components, having different relaxation kinetics.

  6. Separation of c-type cytochrome signals from linear and quadratic ECS signals in P. tricornutum.
    Extended Data Fig. 2: Separation of c-type cytochrome signals from linear and quadratic ECS signals in P. tricornutum.

    a, Kinetics of ΔI/I changes at 520, 554 and 566 nm during an ~10 ms pulse of saturating red light (4,500 µmol quanta m−2 s−1) and the subsequent dark relaxation (top: control conditions; bottom: AA + SHAM). b, Kinetics of ecslin, ecsquad changes and c-type cytochrome redox state, from kinetics in a, after deconvolution as explained in Methods. c, d, Relationship between the quadratic and the linear ECS, before (ecslin, ecsquad, c) and after (ECSlin, ECSquad, d) correction for the dark electric field (see Methods). Dark yellow and magenta symbols correspond to control and AA + SHAM conditions, respectively. The green arrow indicates the value of ΔΨd in control conditions. Data are representative of five independent biological samples. The black boxes in a and b indicate periods of darkness.

  7. Cyclic electron flow and water-to-water cycles in P. tricornutum.
    Extended Data Fig. 3: Cyclic electron flow and water-to-water cycles in P. tricornutum.

    a, Representative traces of changes in ECSlin (normalized as explained in Methods, namely expressed in charge separation per PS) to evaluate linear and cyclic electron flow. Cells were illuminated with 1,870 µmol quanta m−2 s−1 of red light, in absence (filled circles) and presence (open circles) of DCMU and then transferred to the dark; r.u., relative units. b, Representative traces of the 16O2 and 18O2 concentrations at the offset of a 280 µmol quanta m−2 s−1 blue light; a.u., arbitrary units. In a and b, light and dark periods are represented by white and black boxes, respectively. c, Photochemical rate corresponding to total electron flow (TEF, dark symbols, data from four independent biological samples) and CEF (red symbols, n = 8 ± s.d.) at different irradiances. TEF and CEF were estimated from the initial slope of the ECS decay, as (SDSL control) and (SDSL DCMU), respectively (see Methods). d, Light-dependencies of oxygen uptake (U0, half-filled symbols) and gross photosynthesis (E0, open symbols) in control conditions (dark) and in the presence of DCMU (red). Data from two independent biological samples (squares and circles).

  8. [Dgr][PSgr]d and photosynthesis under respiratory inhibition in P. tricornutum.
    Extended Data Fig. 4: ΔΨd and photosynthesis under respiratory inhibition in P. tricornutum.

    ac, Dependency of the ETRPS II (a), ΔΨd (b) and dark respiration (c) upon inhibition of the cyanide-sensitive respiratory pathway with different concentrations of antimycin A, in the presence of saturating SHAM (1 mM). Data from two independent biological samples. Experimental data were fitted with a mono-exponential decay function. d, e, ETRPS II (d), ΔΨd and dark respiration (e), expressed as percentage of control, in the presence of saturating AA (5 µM), SHAM (1 mM) or AA + SHAM (four independent experiments ± s.d.). f, Relationship between ΔΨd and mitochondrial respiration in samples treated with increasing concentrations of AA in the presence of 1 mM SHAM (mean value ± s.e.m. from b and c).

  9. Dark respiration, PMF and growth in AOX knockdown lines of P. tricornutum.
    Extended Data Fig. 5: Dark respiration, PMF and growth in AOX knockdown lines of P. tricornutum.

    a, Respiratory activity of wild-type and AOX knockdown lines. Total respiration rate (red bars) and the contribution of the AOX capacity (white bars, see Methods) were normalized to wild-type values (n = 5 ± s.d.). b, ECS-based measurements of ΔΨd in wild-type (n = 3 ± s.d.) and AOX knockdown lines (n = 2 ± s.e.m.), in control conditions (green), in the presence of AA (dark green) and in the presence of AA + SHAM (white). c, Growth curves of wild-type and AOX knockdown lines in the presence/absence of AA (2 µM). Three independent growth curves are shown for each strain/condition. AA was added every day and cells were grown in continuous light to prevent them from dying in the dark because of lack of respiration.

  10. Subcellular localization of AOX in P. tricornutum and plastid-mitochondria interaction in P. tricornutum wild-type cells.
    Extended Data Fig. 6: Subcellular localization of AOX in P. tricornutum and plastid–mitochondria interaction in P. tricornutum wild-type cells.

    a, Subcellular localization of AOX. Cells were treated with an anti-AOX antibody and then with a secondary Alexa Fluor 488 antibody (see Methods). Positions of plastid and nuclei are indicated by chlorophyll a autofluorescence (red) and DAPI staining (blue), respectively. The pattern of AOX localization is highly similar to that observed with the mitochondria-specific mito-tracker probe (data not shown). Images are representative of 60 cells from three independent biological samples. b, Electron micrographs of the plastid–mitochondria juxtaposition in P. tricornutum. Arrows indicate possible physical contacts between the plastid and mitochondrial membranes. Image is representative of 51 images from seven independent biological samples.

  11. Cytochrome b6f turnover in P. tricornutum wild-type and AOX knockdown lines.
    Extended Data Fig. 7: Cytochrome b6f turnover in P. tricornutum wild-type and AOX knockdown lines.

    a, Schematic representation of the electron-flow reaction steps in the cytochrome b6f complex, which can be evaluated by spectroscopic measurements. b, Slow phase of ECSlin indicating cytochrome b6 activity (top) and time-resolved redox changes of cytochromes c6/f (bottom) in wild-type and AOX knockdown lines (kd-c5 and kd-c9). P. tricornutum cells were exposed to saturating single-turnover laser flashes given 10 s apart. Data were normalized to the amplitude of the fast phase of the ECSlin signal. Cytochrome c and ECSlin were deconvoluted as explained in Methods. Three independent biological samples are shown in red, blue and green colours. Cell concentration was 2 × 107 cells per millilitre. Note that both the slow phase of the ECSlin and the reduction of cytochromes c6/f were completely abolished by the plastoquinone competitive inhibitor DBMIB (10 µM; black arrow).

  12. In vivo changes in the NADPH redox state and ATP in wild-type and AOX knockdown lines.
    Extended Data Fig. 8: In vivo changes in the NADPH redox state and ATP in wild-type and AOX knockdown lines.

    a, Changes in NADPH at different light intensities. Light and dark periods are represented by white and black boxes, respectively. Light intensities were 50, 100, 200 and 400 µmol quanta m−2 s−1 (green, blue, red, and black traces, respectively). Chlorophyll concentration was ~5 µg ml−1. b, Spectra from cells of wild-type (left) and AOX knockdown lines c5 (middle) and c9 (right) in the dark (red), light (green) and light + AA (blue) conditions are shown, with normalization to the internal standard (methylenediphosphonate; pH 8.9). The positions of the α-, β- and γ-phosphates of NTPs are shown. a, b, Representative of three independent biological samples.

  13. Cyclic electron flow in representative diatoms.
    Extended Data Fig. 9: Cyclic electron flow in representative diatoms.

    a, Total electron flow (filled symbols) was measured at different light irradiances, as illustrated in Extended Data Fig. 3, in T. weissflogii (black, n = 3 independent biological samples), T. pseudonana (blue, n = 2) and F. pinnata (red, n = 4). Cyclic electron flow capacity was also measured for every species (open circles, five independent biological samples ± s.d.). b, CEF capacity was plotted against TEF. CEF and TEF are presented as mean values ± s.d. from a. The green line corresponds to CEF = 5% of the maximal total electron flow.

  14. [Dgr][PSgr]d and photosynthesis under conditions of respiratory inhibition in representative diatoms.
    Extended Data Fig. 10: ΔΨd and photosynthesis under conditions of respiratory inhibition in representative diatoms.

    Dark respiration (ad), ΔΨd (eh) and ETRPS II (il) in untreated cells and after treatment with inhibitors of respiration antimycin A, and/or SHAM at saturating concentrations. a, e, i, T. weissflogii (black). b, f, j, T. pseudonana (blue). c, g, k, F. pinnata (red). d, h, l, D. brightwellii (green). The data represent the mean value ± s.d. of three (l), four (d), five (f, h, k), six (a, c, e, j), seven (b, i) or eight (g) independent experiments. All data were normalized to the control value.

Change history

Corrected online 19 August 2015
Affiliation number 4 was corrected.

References

  1. Falkowski, P. G. The evolution of modern eukaryotic phytoplankton. Science 305, 354360 (2004)
  2. Field, C. B., Behrenfeld, M. J., Randerson, J. T. & Falkowski, P. G. Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281, 237240 (1998)
  3. Shikanai, T. Cyclic electron transport around photosystem I: genetic approaches. Annu. Rev. Plant Biol. 58, 199217 (2007)
  4. Asada, K. The water–water cycle as alternative photon and electron sinks. Phil. Trans. R. Soc. Lond. B 355, 14191431 (2000)
  5. Cardol, P. et al. An original adaptation of photosynthesis in the marine green alga Ostreococcus. Proc. Natl Acad. Sci. USA 105, 78817886 (2008)
  6. Ort, D. R. & Baker, N. R. A photoprotective role of O2 as an alternative electron sink in photosynthesis? Curr. Opin. Plant Biol. 5, 193198 (2002)
  7. Petersen, J., Förster, K., Turina, P. & Gräber, P. Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast. Proc. Natl Acad. Sci. USA 109, 1115011155 (2012)
  8. Allen, J. F. Photosynthesis of ATP-electrons, proton pumps, rotors, and poise. Cell 110, 273276 (2002)
  9. Lucker, B. & Kramer, D. M. Regulation of cyclic electron flow in Chlamydomonas reinhardtii under fluctuating carbon availability. Photosynth. Res. 117, 449459 (2013)
  10. Allen, J. F. Oxygen reduction and optimum production of ATP in photosynthesis. Nature 256, 599600 (1975)
  11. Radmer, R. J. & Kok, B. Photoreduction of O2 primes and replaces CO2 assimilation. Plant Physiol. 58, 336340 (1976)
  12. Badger, M. R. Photosynthetic oxygen exchange. Annu. Rev. Plant Physiol. 36, 2753 (1985)
  13. Prihoda, J. et al. Chloroplast-mitochondria cross-talk in diatoms. J. Exp. Bot. 63, 15431557 (2012)
  14. Bowler, C. et al. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456, 239244 (2008)
  15. Grouneva, I., Rokka, A. & Aro, E.-M. The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery. J. Proteome Res. 10, 53385353 (2011)
  16. Witt, H. T. Energy conversion in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods. The central role of the electric field. Biochim. Biophys. Acta 505, 355427 (1979)
  17. Joliot, P. & Joliot, A. Characterization of linear and quadratic electrochromic probes in Chlorella sorokiniana and Chlamydomonas reinhardtii. Biochim. Biophys. Acta 975, 355360 (1989)
  18. Diner, B. & Joliot, P. Effect of the transmembrane electric field on the photochemical and quenching properties of photosystem II in vivo. Biochim. Biophys. Acta 423, 479498 (1976)
  19. Finazzi, G. & Rappaport, F. In vivo characterization of the electrochemical proton gradient generated in darkness in green algae and its kinetics effects on cytochrome b6f turnover. Biochemistry 37, 999910005 (1998)
  20. Waring, J., Klenell, M., Bechtold, U., Underwood, G. J. C. & Baker, N. R. Light-induced responses of oxygen photo-reduction, reactive oxygen species production and scavenging in two diatom species. J. Phycol. 46, 12061217 (2010)
  21. Kinoshita, H. et al. The chloroplastic 2-oxoglutarate/malate transporter has dual function as the malate valve and in carbon/nitrogen metabolism. Plant J. 65, 1526 (2011)
  22. Lemaire, C., Wollman, F. A. & Bennoun, P. Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: an example of mitochondria-dependent photosynthesis. Proc. Natl Acad. Sci. USA 85, 13441348 (1988)
  23. Cardol, P. et al. Impaired respiration discloses the physiological significance of state transitions in Chlamydomonas. Proc. Natl Acad. Sci. USA 106, 1597915984 (2009)
  24. Dang, K. V. et al. Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii. Plant Cell 26, 30363050 (2014)
  25. Vartanian, M., Desclés, J., Quinet, M., Douady, S. & Lopez, P. J. Plasticity and robustness of pattern formation in the model diatom Phaeodactylum tricornutum. New Phytol. 182, 429442 (2009)
  26. Guillard, R. R. L. in Culture of Marine Invertebrate Animals (eds Smith W. L. & Chanley M. H.) 2660 (Plenum, 1975)
  27. Joët, T., Cournac, L., Horvath, E. M., Medgyesy, P. & Peltier, G. Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I. Plant Physiol. 125, 19191929 (2001)
  28. Wishnick, M. & Lane, M. D. Inhibition of ribulose diphosphate carboxylase by cyanide. Inactive ternary complex of enzyme, ribulose diphosphate, and cyanide. J. Biol. Chem. 244, 5559 (1969)
  29. Nakano, Y. & Asada, K. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 28, 131140 (1987)
  30. Asada, K., Takahashi, M. A. & Nagate, M. Assay and inhibitors of spinach superoxide dismutase. Agric. Biol. Chem. 38, 471473 (1974)
  31. Joliot, P. & Delosme, R. Flash induced 529 nm absorption change in green algae. Biochim. Biophys. Acta 357, 267284 (1974)
  32. Santabarbara, S., Redding, K. E. & Rappaport, F. Temperature dependence of the reduction of p-700+ by tightly bound plastocyanin in vivo. Biochemistry 48, 1045710466 (2009)
  33. Melis, A. Kinetic analysis of P-700 photoconversion: effect of secondary electron donation and plastocyanin inhibition. Arch. Biochem. Biophys. 217, 536545 (1982)
  34. Bailleul, B., Cardol, P., Breyton, C. & Finazzi, G. Electrochromism: a useful probe to study algal photosynthesis. Photosynth. Res. 106, 179189 (2010)
  35. Johnson, X. et al. A new setup for in vivo fluorescence imaging of photosynthetic activity. Photosynth. Res. 102, 8593 (2009)
  36. Genty, B., Briantais, J. M. & Baker, N. R. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 8792 (1989)
  37. Bilger, W. & Björkman, O. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth. Res. 25, 173186 (1990)
  38. Peltier, G. & Thibault, P. O2 uptake in the light in Chlamydomonas. Plant Physiol. 79, 225230 (1985)
  39. Kana, T. M. et al. A membrane inlet mass spectrometer for rapid and high-precision determination of N2, O2, and Ar in environmental water samples. Anal. Chem. 66, 41664170 (1994)
  40. Rivasseau, C. et al. Accumulation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate in illuminated plant leaves at supraoptimal temperatures reveals a bottleneck of the prokaryotic methylerythritol 4-phosphate pathway of isoprenoid biosynthesis. Plant Cell Environ. 32, 8292 (2009)
  41. Bligny, R. & Douce, R. NMR and plant metabolism. Curr. Opin. Plant Biol. 4, 191196 (2001)
  42. Van de Meene, A. M. L. & Pickett-Heaps, J. D. Valve morphogenesis in the centric diatom Rhizosolenia setigera (Bacillariophyceae, Centrales) and its taxonomic implications. Eur. J. Phycol. 39, 93104 (2004)
  43. De Riso, V. et al. Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37, e96 (2009)
  44. Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C. & Bowler, C. Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. (NY) 1, 239251 (1999)

Download references

Author information

Affiliations

  1. Génétique et Physiologie des Microalgues, Département des Sciences de la vie and PhytoSYSTEMS, Université de Liège, B-4000 Liège, Belgium

    • Benjamin Bailleul,
    • Nicolas Berne &
    • Pierre Cardol
  2. Environmental Biophysics and Molecular Ecology Program, Departments of Marine and Coastal Sciences and of Earth and Planetary Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA

    • Benjamin Bailleul &
    • Paul G. Falkowski
  3. Institut de Biologie Physico-Chimique (IBPC), UMR 7141, Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie, 13 Rue Pierre et Marie Curie, F-75005 Paris, France

    • Benjamin Bailleul,
    • Fabrice Rappaport &
    • Pierre Joliot
  4. Ecole Normale Supérieure, PSL Research University, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, 46 rue d’Ulm, F-75005 Paris, France

    • Benjamin Bailleul,
    • Omer Murik,
    • Judit Prihoda,
    • Atsuko Tanaka,
    • Leila Tirichine &
    • Chris Bowler
  5. Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, Centre National de la Recherche Scientifique (CNRS), Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), Université Grenoble Alpes, Institut National Recherche Agronomique (INRA), Institut de Recherche en Sciences et Technologies pour le Vivant (iRTSV), CEA Grenoble, F-38054 Grenoble cedex 9, France

    • Dimitris Petroutsos,
    • Richard Bligny,
    • Serena Flori,
    • Denis Falconet &
    • Giovanni Finazzi
  6. Fermentalg SA, F-33500 Libourne, France

    • Valeria Villanova
  7. Institute for Integrative Biology of the Cell (I2BC), Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, Institut de Biologie et de Technologie de Saclay, F-91191 Gif-sur-Yvette cedex, France

    • Anja Krieger-Liszkay
  8. Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via Celoria 26, I-20133 Milan, Italy

    • Stefano Santabarbara

Contributions

B.B., L.T., C.B. and G.F. designed the study. B.B., N.B., O.M., D.P., J.P., A.T., V.V., R.B., S.F., D.F., A.K-L, F.R., P.J., L.T., P.C. and G.F. performed experiments. B.B., N.B., O.M., D.P., R.B., A.K.-L., S.S., F.R., P.J., L.T., P.F., P.C., C.B. and G.F. analysed the data. B.B., C.B. and G.F. wrote the manuscript, and all authors revised and approved it.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Deconvolution of the quadratic and linear ECS in P. tricornutum. (183 KB)

    a, Absorption difference (ΔI/I) kinetics followed at different wavelengths in P. tricornutum, after a series of six saturating laser flashes, in anaerobic conditions. Solid lines correspond to the global fit of the experimental data with a sum of two exponential decays, with time constants τ and 2τ, respectively, as expected for linear and quadratic dependencies (see Methods). b, ΔI/I spectra are shown at different times during ECS relaxation. All spectra were normalized to 1 at 520 nm for better comparison. The observation that the blue and green parts of the spectrum are homothetic during relaxation, while changes are seen in the red most part of it, reflects the presence of the two ECS components, having different relaxation kinetics.

  2. Extended Data Figure 2: Separation of c-type cytochrome signals from linear and quadratic ECS signals in P. tricornutum. (234 KB)

    a, Kinetics of ΔI/I changes at 520, 554 and 566 nm during an ~10 ms pulse of saturating red light (4,500 µmol quanta m−2 s−1) and the subsequent dark relaxation (top: control conditions; bottom: AA + SHAM). b, Kinetics of ecslin, ecsquad changes and c-type cytochrome redox state, from kinetics in a, after deconvolution as explained in Methods. c, d, Relationship between the quadratic and the linear ECS, before (ecslin, ecsquad, c) and after (ECSlin, ECSquad, d) correction for the dark electric field (see Methods). Dark yellow and magenta symbols correspond to control and AA + SHAM conditions, respectively. The green arrow indicates the value of ΔΨd in control conditions. Data are representative of five independent biological samples. The black boxes in a and b indicate periods of darkness.

  3. Extended Data Figure 3: Cyclic electron flow and water-to-water cycles in P. tricornutum. (232 KB)

    a, Representative traces of changes in ECSlin (normalized as explained in Methods, namely expressed in charge separation per PS) to evaluate linear and cyclic electron flow. Cells were illuminated with 1,870 µmol quanta m−2 s−1 of red light, in absence (filled circles) and presence (open circles) of DCMU and then transferred to the dark; r.u., relative units. b, Representative traces of the 16O2 and 18O2 concentrations at the offset of a 280 µmol quanta m−2 s−1 blue light; a.u., arbitrary units. In a and b, light and dark periods are represented by white and black boxes, respectively. c, Photochemical rate corresponding to total electron flow (TEF, dark symbols, data from four independent biological samples) and CEF (red symbols, n = 8 ± s.d.) at different irradiances. TEF and CEF were estimated from the initial slope of the ECS decay, as (SDSL control) and (SDSL DCMU), respectively (see Methods). d, Light-dependencies of oxygen uptake (U0, half-filled symbols) and gross photosynthesis (E0, open symbols) in control conditions (dark) and in the presence of DCMU (red). Data from two independent biological samples (squares and circles).

  4. Extended Data Figure 4: ΔΨd and photosynthesis under respiratory inhibition in P. tricornutum. (309 KB)

    ac, Dependency of the ETRPS II (a), ΔΨd (b) and dark respiration (c) upon inhibition of the cyanide-sensitive respiratory pathway with different concentrations of antimycin A, in the presence of saturating SHAM (1 mM). Data from two independent biological samples. Experimental data were fitted with a mono-exponential decay function. d, e, ETRPS II (d), ΔΨd and dark respiration (e), expressed as percentage of control, in the presence of saturating AA (5 µM), SHAM (1 mM) or AA + SHAM (four independent experiments ± s.d.). f, Relationship between ΔΨd and mitochondrial respiration in samples treated with increasing concentrations of AA in the presence of 1 mM SHAM (mean value ± s.e.m. from b and c).

  5. Extended Data Figure 5: Dark respiration, PMF and growth in AOX knockdown lines of P. tricornutum. (210 KB)

    a, Respiratory activity of wild-type and AOX knockdown lines. Total respiration rate (red bars) and the contribution of the AOX capacity (white bars, see Methods) were normalized to wild-type values (n = 5 ± s.d.). b, ECS-based measurements of ΔΨd in wild-type (n = 3 ± s.d.) and AOX knockdown lines (n = 2 ± s.e.m.), in control conditions (green), in the presence of AA (dark green) and in the presence of AA + SHAM (white). c, Growth curves of wild-type and AOX knockdown lines in the presence/absence of AA (2 µM). Three independent growth curves are shown for each strain/condition. AA was added every day and cells were grown in continuous light to prevent them from dying in the dark because of lack of respiration.

  6. Extended Data Figure 6: Subcellular localization of AOX in P. tricornutum and plastid–mitochondria interaction in P. tricornutum wild-type cells. (678 KB)

    a, Subcellular localization of AOX. Cells were treated with an anti-AOX antibody and then with a secondary Alexa Fluor 488 antibody (see Methods). Positions of plastid and nuclei are indicated by chlorophyll a autofluorescence (red) and DAPI staining (blue), respectively. The pattern of AOX localization is highly similar to that observed with the mitochondria-specific mito-tracker probe (data not shown). Images are representative of 60 cells from three independent biological samples. b, Electron micrographs of the plastid–mitochondria juxtaposition in P. tricornutum. Arrows indicate possible physical contacts between the plastid and mitochondrial membranes. Image is representative of 51 images from seven independent biological samples.

  7. Extended Data Figure 7: Cytochrome b6f turnover in P. tricornutum wild-type and AOX knockdown lines. (146 KB)

    a, Schematic representation of the electron-flow reaction steps in the cytochrome b6f complex, which can be evaluated by spectroscopic measurements. b, Slow phase of ECSlin indicating cytochrome b6 activity (top) and time-resolved redox changes of cytochromes c6/f (bottom) in wild-type and AOX knockdown lines (kd-c5 and kd-c9). P. tricornutum cells were exposed to saturating single-turnover laser flashes given 10 s apart. Data were normalized to the amplitude of the fast phase of the ECSlin signal. Cytochrome c and ECSlin were deconvoluted as explained in Methods. Three independent biological samples are shown in red, blue and green colours. Cell concentration was 2 × 107 cells per millilitre. Note that both the slow phase of the ECSlin and the reduction of cytochromes c6/f were completely abolished by the plastoquinone competitive inhibitor DBMIB (10 µM; black arrow).

  8. Extended Data Figure 8: In vivo changes in the NADPH redox state and ATP in wild-type and AOX knockdown lines. (390 KB)

    a, Changes in NADPH at different light intensities. Light and dark periods are represented by white and black boxes, respectively. Light intensities were 50, 100, 200 and 400 µmol quanta m−2 s−1 (green, blue, red, and black traces, respectively). Chlorophyll concentration was ~5 µg ml−1. b, Spectra from cells of wild-type (left) and AOX knockdown lines c5 (middle) and c9 (right) in the dark (red), light (green) and light + AA (blue) conditions are shown, with normalization to the internal standard (methylenediphosphonate; pH 8.9). The positions of the α-, β- and γ-phosphates of NTPs are shown. a, b, Representative of three independent biological samples.

  9. Extended Data Figure 9: Cyclic electron flow in representative diatoms. (243 KB)

    a, Total electron flow (filled symbols) was measured at different light irradiances, as illustrated in Extended Data Fig. 3, in T. weissflogii (black, n = 3 independent biological samples), T. pseudonana (blue, n = 2) and F. pinnata (red, n = 4). Cyclic electron flow capacity was also measured for every species (open circles, five independent biological samples ± s.d.). b, CEF capacity was plotted against TEF. CEF and TEF are presented as mean values ± s.d. from a. The green line corresponds to CEF = 5% of the maximal total electron flow.

  10. Extended Data Figure 10: ΔΨd and photosynthesis under conditions of respiratory inhibition in representative diatoms. (180 KB)

    Dark respiration (ad), ΔΨd (eh) and ETRPS II (il) in untreated cells and after treatment with inhibitors of respiration antimycin A, and/or SHAM at saturating concentrations. a, e, i, T. weissflogii (black). b, f, j, T. pseudonana (blue). c, g, k, F. pinnata (red). d, h, l, D. brightwellii (green). The data represent the mean value ± s.d. of three (l), four (d), five (f, h, k), six (a, c, e, j), seven (b, i) or eight (g) independent experiments. All data were normalized to the control value.

Additional data