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Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism

Nature volume 605, pages 366–371 (2022)Cite this article

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Abstract

Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The high efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO2 fixation2. Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified3,4, how microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains unknown4,5,6. Here we show that in the green alga Chlamydomonas reinhardtii, the combined action of cyclic electron flow and O2 photoreduction—which depend on PGRL1 and flavodiiron proteins, respectively—generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO2. We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity.

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Fig. 1: Deletion of PGRL1 and FLVB impairs photosynthetic affinity for Ci.
Fig. 2: Growth of pgrl1 flvB double mutants is impaired at low CO2 in the presence of CCM components.
Fig. 3: CEF, PCEF and CMEF contribute energy to the CCM.
Fig. 4: Proposed mechanism of the CCM-energization network in algal cells.

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Data availability

Genes studied in this Article can be found on https://phytozome-next.jgi.doe.gov/ under the loci Cre12.g531900 (FLVA), Cre16.g691800 (FLVB), Cre07.g340200 (PGRL1), Cre16.g662600 (BST1), Cre16.g663400 (BST2) and Cre16.g663450 (BST3).

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Acknowledgements

This work was supported by the A*MIDEX (ANR-11-IDEX-0001-02) project and by the ANR OTOLHYD. A.B. acknowledges support from the Carnegie Institution for Science. O.D. is the recipient of a PhD grant awarded to Y.L-.B. We thank A. Grossman for use of the JTS-100; G. Peers for stimulating discussion; K. K. Niyogi and M. Iwai for critical reading of the manuscript; J. V. Moroney for the bsti-1 mutant; L. Mackinder for the BST3 antibody; and H. Fukuzawa for the LCIA and LCI1 antibodies. We gratefully acknowledge the contributions of S. Moulin for artistic drawings in Fig. 4, S. Blangy for LCIC antibody preparation, E. Calikanzaros and V. Epting for technical assistance, and A. Gosset for performing genetic crosses of flvB and pgrl1 mutants. The authors acknowledge the European Union Regional Developing Fund, the Region Provence Alpes Côte d’Azur, the French Ministry of Research, and the CEA for funding the HelioBiotec platform.

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  1. Aix Marseille Univ, CEA, CNRS, Institut de Biosciences et Biotechnologies Aix-Marseille, CEA Cadarache, Saint-Paul-lez-Durance, France

    Adrien Burlacot, Ousmane Dao, Pascaline Auroy, Stephan Cuiné, Yonghua Li-Beisson & Gilles Peltier

  2. Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA

    Adrien Burlacot

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Contributions

A.B. and G.P. designed the research; A.B., O.D., P.A., S.C. and G.P. performed research; A.B. and G.P. contributed new reagents and analytic tools; A.B. and G.P. analysed data; A.B. and G.P. wrote the paper with input from Y.L.-B.

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Correspondence to Gilles Peltier.

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Extended data figures and tables

Extended Data Fig. 1 Obtaining and characterizing of pgrl1 flvB double mutants.

a, Upon crossing of pgrl1 and flvB-21 opposite mating types, progenies were screened for FLVB deficiency based on the chlorophyll fluorescence pattern, and for insertion in the PGRL1 locus by PCR. Five independent double mutants and control strains were randomly selected among the pool of double mutants and wild-types for further analysis. b, c, Chlorophyll fluorescence patterns of parental strains (pgrl1 and flvB-21) and of their respective control strains (137AH and CC-4533 respectively) in b, and of progenies of the crossing (pgrl1 flvB-1, 2, 3, 4 and 5; WT-1, 2, 3, 4) in C. Chlorophyll fluorescence measurements were performed using a PAM fluorimeter in the dark and under red actinic light (100 µmol photon m−2 s−1, from t = 22 s to t = 110 s) at a Ci concentration of 1 mM. Data are normalized to initial FM and slightly shifted on the time axis for clarity. d, PCR amplification targeting the PGRL1 locus in the different strains. Shown is representative of n = 3 independent experiments.

Extended Data Fig. 2 Maximum net O2 evolution and K1/2 values measured in pgrl1, flvB, pgrl1 flvB and their respective controls when grown under Low CO2 or High CO2.

Net O2 production rates were measured as described in Fig. 1 in low CO2 (a, b) or high CO2 (c, d) grown cells. a, c, Maximal net O2 production rates. Shown are mean values and replicates (n = 3 biologically independent sample). b, d, K1/2 values determined for each strain from hyperbolic fits. Shown are mean values ±SD of the fit of data shown in Fig. 1a–f. Letters (a–d) above bars in (b) represent significant differences (p < 0.05) between strains based on one-way ANOVA analysis.

Extended Data Fig. 3 Growth of pgrl1, flvB, pgrl1 flvB mutants and their control strains.

Cells were spotted on plates containing minimal medium at pH 7.2 (a, c, e) or pH 8.2 (b, d, f) and grown under continuous high light (100 µmol photon m−2 s−1, a, b), medium light (60 µmol photon m−2 s−1, c, d) or low light (30 µmol photon m−2 s−1, e, f). For each pH and light conditions, cells were grown either at 100 ppm CO2 (Very low CO2), 400 ppm CO2 (Low CO2) or 3% of CO2 (High CO2). Growth was assessed in pgrl1, flvB, and their respective control strains (137AH and CC-4533) (left panels) and on double mutants (pgrl1 flvB-1 to -5) and their control strains (WT-1 to -4) (right panels); the CCM1 mutant cia5 was introduced as a CCM-deficient control together with its reference strain CC-125. Shown are representative spot tests of n = 10 independent experiments.

Extended Data Fig. 4 Photosynthesis and CCM components assessment.

a, Maximum PSII efficiency (FV/FM) of double mutants (pgrl1 flvB-1 to -5) and their control strains (WT-1 to -4), measured using a PAM fluorimeter after 15 min dark adaptation. b, Immunodetection of the PSII subunit PsbD, the PSI subunit PsaC, the cytochrome b6f complex subunit PetB, the large Rubisco subunit RbcL, the type II NADH dehydroagenase NDA2, the mitochondrial cytochrome aa3 oxidase subunit CoxIIB, of the alternative oxidase AOX1, and of the Fe superoxide dismutase FeSOD, in two independent pgrl1 flvB double mutants and controls grown under Low CO2 or High CO2. c, Immunodetection of LCI1, BST3, RbcL and LHCSR3 in pgrl1, flvB, and their respective control strains (137AH and CC-4533). d, Immunodetection of FLVA, FLVB, PGRL1, LCI1, and BST3 in bsti-1 and its control strain (D66) at low density. e, Immunodetection of LHCSR3 in bsti-1 and its control strain (D66) cultivated at low (LD, 6 µg chlorophyll. mL−1) or high (LD, 12 µg chlorophyll. mL−1) cell density. When cultivating algae at different cell densities, which affects light and CO2 availability (two factors known to modulate LHCSR3 accumulation), we observed that bsti-1 accumulates lower LHCSR3 levels as compared to D66 at LD, but similar levels at HD. Data shown in b–e have been reproduced with n = 2 independent experiments. f, Carbonic anhydrase (CA) activity was determined in vivo by following the unlabelling of 18O-enriched CO2 in the same strains and conditions as in Fig. 2b. Shown are mean values and replicates (n = 3 biologically independent sample).

Extended Data Fig. 5 Electrochromic carotenoid absorbance changes measurements and pmf size and partitioning between ΔpH and ΔΨ.

ECS signal was measured upon light to dark transition after 2 min red light illumination (500 µmol photons m2 s−1) in the presence of 1mM NaHCO3. a, Typical ECS traces are shown for single mutants (pgrl1, flvB and bsti-1), their control strains (137AH, CC-4533 and D66 respectively), for double mutants (pgrl1flvB-1 to -5) and their controls (WT-1 to -4). Light was turned off at t = 0. b, relative pmf size was determined for each strain. c. Proportion of ΔpH in the pmf was determined for each strain. Shown are means and replicate values (n = 3 biologically independent sample). pgrl1 flvB double mutants showed a significantly lower pmf and ΔpH as compared to simple mutants and control strains (p < 0.05, one way ANOVA).

Extended Data Fig. 6 Chlorophyll fluorescence, NPQ and Ci measured during dark-light-dark transients in pgrl1, flvB, pgrl1 flvB and their respective controls.

a–f, Chlorophyll fluorescence patterns (upper panels), NPQ and Ci measurements (lower panels). Shown are representative experiments of three biological replicates. g, h, CO2-dependent NPQ was determined by subtracting the NPQ value measured after Ci injection from the NPQ value measured upon the initial Ci depletion as indicated by dotted lines (a–f). Immunodetection of LHCSR3 is shown on top of each corresponding strain. Shown are mean values and replicates (n = 3 biologically independent sample). All strains were grown as in Fig. 1 under low CO2. Red arrows indicate addition of bicarbonate. i, j, pgrl1 flvB double mutants have maintained a full qE potential. Chlorophyll fluorescence and NPQ were measured in pgrl1 flvB-1 and pgrl1 flvB-2 following the addition of acetic acid (13 mM) decreasing the pH to 5.5 and further addition of KOH (13 mM) according to the experimental protocol described in ref. 34 of the main text. Shown are representative experiments of three biological replicates.

Extended Data Fig. 7 Ci-dependent NPQ variations are independent of state transition and linked with trans-thylakoidal ΔpH.

a, b, 77K chlorophyll fluorescence spectra were measured during Ci depletion in the control strain WT3. Samples were taken at 22 µM Ci (blue arrow, a) and 0 µM Ci (orange arrow, a) and 77K chlorophyll fluorescence spectra were recorded (b). Addition of carbonyl cyanide trifluoromethoxy phenylhydrazone (FCCP, 5 µM) was used as a control to induce state 2 (pink curve). Shown are representative experiments of n = 3 biologically independent sample. a, c–g, Chlorophyll fluorescence patterns (upper panels), NPQ and Ci measurements (lower panels) in control strains, the state transition mutant stt7-9 and bsti-1 mutant during Ci depletion in the light followed either by a dark period (a, c) or subsequent addition of the uncoupler nigericin (10 µM, purple arrow) (d–g). Shown are representative experiments of n = 3 biologically independent sample. (H, I) CO2-dependent NPQ was determined as described in Extended Data Fig. 5 (black bar, c–f). Nigericin-dependent NPQ was determined from NPQ values measured before and after nigericin addition (red bars, d–f). All strains were grown as in Fig. 1 under low CO2. Red arrows indicate addition of bicarbonate.

Extended Data Fig. 8 Ci dependency of O2 exchange rates measured by using MIMS in the presence of 18O2-enriched O2 in pgrl1, flvB and their respective controls.

Strains were grown under low CO2 as in Fig. 1. a, b, O2 exchange rates in pgrl1 and its control strain 137AH. c, Difference in O2 uptake rates between 137AH and pgrl1. d, Difference in O2 uptake rates as determined in (c) normalized to the net O2 production of pgrl1 is used to determine the CEF contribution (e, f) O2 exchange rates in flvB and its control strain (CC-4533). g, Difference in O2 uptake rates between CC-4533 and flvB. h, Light-induced O2 uptake in flvB mutant. i, The difference in O2 uptake rates as determined in (g) normalized to the net O2 production in flvB is used to determine the contribution of PCEF. j, the difference in O2 uptake rates as determined in (h) normalized to the net O2 production in flvB is used to determine the contribution of CMEF. k, Effect of the mitochondrial respiration inhibitors myxothiazol and SHAM on the light-induced O2 uptake rate measured in flvB mutant at low Ci after 3 min of illumination. Shown are mean values and replicates (n = 3 biologically independent sample). l, Effect of myxothiazol and SHAM on O2 exchange rates in pgrl1 flvB-1 double mutant. Shown are representative exchange rates from 3 independent replicates. The full inhibition of photosynthetic O2 exchange observed in these conditions reflects the requirement of CO2 fixation for additional ATP supply by CEF, PCEF and CMEF.

Extended Data Fig. 9 Effect of mitochondrial respiration inhibitors on the Ci affinity of net O2 photosynthesis.

Photosynthetic net O2 production was measured as in Fig. 1 in low CO2 (a, b, c, g, h, k) or high CO2 (d, e, f, i, j, l) grown cells in the absence or presence of the two mitochondrial respiration inhibitors myxothiazol (Myxo, 2.5 µM) and salicylhydroxamic acid (SHAM, 400 µM). a–j, For each replicate, net O2 production was measured at four different concentrations of Ci and normalized to the maximum photosynthetic net O2 production. Shown are three replicates for each strain (dots) and hyperbolic fit with variability (plain lines, dotted lines). k, l, Maximum net O2 production rates. Shown are mean values and replicates (n = 3 Shown are mean values and replicates (n = 3 biologically independent sample). m, K1/2 Ci values of high CO2 grown cells in the absence or presence of myxothiazol and SHAM. Shown are mean values ±SD of the fit of data shown in Extended Data Fig. 9 d–f, i, j.

Extended Data Fig. 10 Effects of respiratory inhibitors myxothiazol and SHAM added separately or simultaneously on the Ci affinity of net O2 photosynthesis.

Photosynthetic net O2 production was measured as in Fig. 1 in WT-3 low CO2 grown cells in the absence or in the presence of respiratory inhibitors. a, In the absence of inhibitors. b, in the presence of 400 µM SHAM. c, in the presence of 2.5 µM myxothiazol. d, in the presence of both 2.5 µM myxothiazol and 400 µM SHAM. Data shown on a–d are from n = 3 biologically independent sample (dots) and hyperbolic fit with variability (plain lines, dotted lines). e, K1/2 values were determined from hyperbolic fits. Shown are mean ± SD of the fit of experimental data shown in a–d. Asterisk represent significant difference (p < 0.05, multiple t-test).

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Burlacot, A., Dao, O., Auroy, P. et al. Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism. Nature 605, 366–371 (2022). https://doi.org/10.1038/s41586-022-04662-9

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