Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

Abstract

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.

Figure 1.

Activities of PSII, hydrogenases, and CBB cycle upon a shift from dark anoxia (1 h) to light (250 µmol photons m^–2 s^–1) in the wild type (wt; A), pgrl1 (B), hydg-2 (C), and pgrl1 hydg-2 (D). Dark circles indicate ETR_PSII (e^– s^–1 PSII^–1); gray squares indicate J_H2 (e^– s^–1 PSI^–1); and a white triangle indicates electron flow toward carbon fixation (J_CO2, e^– s^–1 PSI^–1), calculated as follows: J_CO2 = ETR_PSII – J_H2. See text for further information. All measurements were performed at least in triplicate (n ≥ 3), and data are presented as means ± sd.

Figure 2.

Activities of PSII, hydrogenases, and CBB cycle upon a shift from dark anoxia (1 h) to light (250 µmol photons m^–2 s^–1) in the wild type (wt; A and B), hydg-2 (C and D), and pgrl1 (E and F) in conditions of inhibition of the CBB cycle. Glycolaldehyde (10 mm; A, C, and E) or CCCP (20 µm; B, D, and F) were added prior to illumination. Dark circles indicate ETR_PSII (e^– s^–1 PSII^–1); gray squares indicate J_H2 (e^– s^–1 PSI^–1); and a white triangle indicates electron flow toward carbon fixation (J_CO2, e^– s^–1 PSI^–1), calculated as follows J_CO2 = ETR_PSII – J_H2. See text for further information. All measurements were performed at least in triplicate (n ≥ 3), and data are presented as means ± sd.

Figure 3.

J_CEF upon a shift from dark anoxia (1 h) to light in the wild type (wt; A and E), pgrl1 (B and F), hydg-2 (C and G), and pgrl1 hydg-2 (D and H). Dark circles (A–H) indicate ETR_PSII (e^– s^–1 PSII^–1); white circles (A–D) indicate ETR_PSI (e^– s^–1 PSI^–1); gray circles (E–H) indicate R_ph (e^– s^–1 PS^–1); gray diamonds (A–D) indicate J_CEF (e^– s^–1 PSI^–1), calculated as follows: J_CEF = ETR_PSI – ETR_PSII; and gray diamonds (E–H) indicate J_CEF (e^– s^–1 PSI^–1), calculated as follows: J_CEF = 2(R_ph – ETR_PSII). See text for further information. All measurements were performed at least in triplicate (n ≥ 3), and data are presented as means ± sd.

Figure 4.

Schematic model of photosynthetic electron transfers in C. reinhardtii upon a shift from dark anoxia (1 h) to light in the wild type (A), hydg-2 (B), pgrl1 (C), and pgrl1 hydg-2 (D). PSI-CEF (J_CEF), J_H2, and electron transport rate toward CO2 fixation (J_CO2) refer to electron rates (e^– s^–1 PSI^–1) taken from Figures 1 and 3. PC, Plastocyanin; Cyt b₆f, cytochrome b₆f complex; PQ/PQH₂, plastoquinone pool; CF₁F₀, chloroplastic ATP synthase.

Figure 5.

ETR_PSII (e^– s^–1 PSII^–1) in the wild type (wt), pgrl1, hydg-2, and pgrl1 hydg-2 upon a shift from dark anoxia (10 min) to light (250 µmol photons m^–2 s^–1; A), upon a shift from dark anoxia (16 h) to light (250 µmol photons m^–2 s^–1; B), upon a shift from dark anoxia (1 h) to light (120 µmol photons m^–2 s^–1; C), and upon a shift from dark anoxia (1 h) to light (1,000 µmol photons m^–2 s^–1; D). All measurements were performed at least in triplicate (n ≥ 3), and data are presented as means ± sd.

Figure 6.

In vivo hydrogenase activity. A and B, Concomitant measurements of J_H2 (e^– s^–1 PSI^–1; gray squares) and dissolved oxygen concentration (µm O₂, dark triangles) in the wild type (wt; A) and pgrl1 (B) upon a shift from dark anoxia (1 h) to light. Anoxia was reached by bubbling with nitrogen for 5 min prior to incubation in the dark for 1 h. C, J_H2 (e^– s^–1 PSI^–1) upon a shift from dark anoxia (1 h) to light. The arrow indicates when glycolaldehyde (10 mm, dark squares) was added to inhibit CBB cycle activity. After 2 min of incubation in the dark, light is switched on for at least an extra 6 min. All measurements were performed at least in triplicate (n ≥ 3), and data are presented as means ± sd.

Figure 7.

Growth in anoxic conditions. A, Specific growth rate (µ, d^–1) of the wild type (wt) and mutants in 3-h-dark/3-h-light cycles in TMP liquid medium. B, Proportion of pgrl1 hydg-2 mutant within a coculture of pgrl1 hydg-2 and wild-type cells in a 3-h-dark/3-h-light cycle in TAP liquid medium (for further details, see “Materials and Methods”). Dark squares indicate an aerated culture, and gray squares indicate an anoxic sealed details in triplicate (n ≥ 3), and data are presented as means ± sd.