Light-independent regulation of algal photoprotection by CO2 availability

Abstract

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO₂ conditions. When light energy exceeds CO₂ fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO₂ Concentrating Mechanism (CCM). How light and CO₂ signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO₂ concentrations and that depletion of CO₂ drives these responses, even in total darkness. High CO₂ levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO₂ and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO₂ levels.

Fig. 1. Acetate needs to be metabolized to inhibit LHCSR3 accumulation.

WT, icl, icl-C and dum11 strains were acclimated for 16 h in LL (15 µmol photons m⁻² s⁻¹) in HSM; sparged with air (labelled as “air”); sparged with air and supplemented with 10 mM sodium acetate (labelled as “acet”); sparged with air enriched with 5% CO₂ (labelled as “CO₂”). After sampling for the LL conditions, light intensity was increased to 600 µmol photons m⁻² s⁻¹ (HL); samples were taken 1 h (RNA) or 4 h (protein) after exposure to HL. a, c. Accumulation of LHCSR3 mRNA at the indicated conditions normalized to WT LL ctrl (n = 3 biological samples, mean ± s.d.). The p-values for the comparisons of acetate and CO₂ conditions to air are based on ANOVA Dunnett’s multiple comparisons test of log10 transformed mRNA data as indicated in the graphs (*P < 0.005, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant). Exact p-values can be found at the Source Data file. b, d. Immunoblot analyses of LHCSR3 and ATPB (loading control) under the indicated conditions. Representative datasets of experiments repeated three times.

Fig. 2. LHCSR3 inhibition is driven by CO₂ derived from the metabolism of acetate.

Experiment at LL: a, b mRNA accumulation of LHCSR3 and RHP1 and c concentration of sodium acetate in the growth medium in WT and icl strains. Cells were acclimated overnight at LL (15 µmol photons m⁻² s⁻¹) in HSM sparged with air. At t = 0 cells either continued being sparged with air (labelled “air”); or sparged with air and supplemented with 10 mM sodium acetate (labelled “acet”); or sparged with air enriched with 5% CO₂ (labelled “CO₂”). The addition of acetate or CO₂ is indicated with a green mark on the x-axis. Samples were taken at t = 0, 1 h, 4 h and 8 h. Experiment at HL: d, e mRNA accumulation of LHCSR3 and RHP1 and f concentration of sodium acetate in the growth medium in WT and icl strains. Cells were acclimated overnight at LL (15 µmol photons m⁻² s⁻¹) in HSM sparged with air; at t = 0 light intensity was increased to 600 µmol photons m⁻² s⁻¹. At t = 1 h cells either continued being sparged with air (labelled “air”); or sparged with air and supplemented with 10 mM sodium acetate (labelled “acet”); or bubbled with air enriched with 5% CO₂ (labelled “CO₂”), always at 600 µmol photons m⁻² s⁻¹. The time of addition of acetate or CO₂ is highlighted in green on the x-axis. Samples were taken at t = 0, 1 h, 2 h, 5 h and 9 h. (n = 3 biological samples, mean ± s.d.). The p values for the comparisons of acetate and CO₂ conditions to air (LL; t = 1, 4, 8 h, HL; t = 2, 5, 9 h) are based on ANOVA Dunnett’s multiple comparisons test of log10 transformed mRNA data as indicated in the graphs (*P < 0.005, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant), following the color-code of the datasets. Exact p-values can be found at the Source Data file. Please note that in some cases the error bars are smaller than the data point symbols.

Fig. 3. Cross-talk of responses to HL and low-CO₂.

a CC-125 WT, cia5 and cia5-c strains were acclimated for 16 h in LL (15 µmol photons m⁻² s⁻¹) in HSM bubbled with air (labelled as “LL”); after sampling for the LL conditions, light intensity was increased to 600 µmol photons m⁻² s⁻¹ (HL); samples were taken after 1 h (RNA) and 4 h (protein and photosynthesis measurements). Accumulation of mRNA of genes at the indicated conditions were normalized to WT LL ctrl. (n = 3 biological samples, mean ± s.d.). The p-values for the comparisons of WT with cia5 and cia5 with cia5-C are based on ANOVA Dunnett’s multiple comparisons test of log10 transformed mRNA data as indicated in the graphs. b Immunoblot analyses of LHCSR3, LHCSR1 and ATPB (loading control) under the indicated conditions; PSBS was non-detectable at these experimental conditions. Representative dataset of experiment repeated three times. c qE of WT, cia5 and cia5-C under LL and HL conditions (n = 3 biological samples, mean ± s.d.). The statistical analyses (two-way ANOVA Tukey’s multiple comparison test) are shown in the graph. Exact p-values can be found at the Source Data file. Raw fluorescence and NPQ curves can be seen in Supplementary Fig. 4.

Fig. 4. Kinetic resolution of photoprotective gene and protein expression at different light and CO₂ availabilities.

Cells were acclimated overnight at LL (15 µmol photons m⁻² s⁻¹) bubbled with air (labelled “air”). At t = 0 the light intensity was raised to 600 µmol photons m⁻² s⁻¹ under air bubbling or bubbling with 5% CO₂ and mRNA and protein were followed for 25 h. a LHCSR1, LHCSR3 and PSBS mRNA accumulation. (n = 3 biological samples, mean ± s.d.). The p-values for the comparisons of CO₂ conditions to air for t = 1, 4, 8, 24 and 25 h are based on two-way ANOVA Šídák’s multiple comparisons test of log10 transformed mRNA data as indicated in the graphs (*P < 0.005, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant). Exact p-values can be found at the Source Data file. b Immunoblot analyses of LHCSR1, LHCSR3 and ATPB (loading control). Representative dataset of experiment repeated three times.

Fig. 5. Low CO₂ levels can trigger qE and CCM genes in the absence of light.

WT, cia5 and cia5-C cells were bubbled with air overnight in darkness; next day air bubbling was either maintained or replaced by CO₂-limited-air bubbling in the darkness or in the presence of 600 µmol photons m⁻² s⁻¹ light. Sampling was performed after 1 h (RNA) or 4 h (protein). a mRNA accumulation of LHCSR3.1 (qE gene) and CAH4, LCIA, LCI1 (CCM genes) in WT, cia5 and cia5-C. Data were normalized to WT air dark; (n = 3 biological samples, mean ± s.d.). The p-values for the comparisons of WT with cia5 and cia5 with cia5-C are based on ANOVA Dunnett’s multiple comparisons test of log10 transformed mRNA data as indicated in the graphs (*P < 0.005, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant). Exact p-values can be found at the Source Data file. b Immunoblot analyses of LHCSR3 and ATPB (loading control) under the indicated conditions. Representative dataset of experiment repeated three times. c Immunoblot analyses of LHCSR3 and ATPB (loading control) of WT samples presented in b. Above the immunoblot shown are the amount of protein loaded per lane and the quantification of LHCSR3 protein accumulation (calculated as LHCSR3 /ATPB ratio) normalized to the air dark conditions. Representative dataset of experiment repeated three times.

Fig. 6. Photosynthetic electron transfer draws down the intracellular CO₂ concentration, relieving inhibition of LHCSR3 transcription.

a CO₂ concentration measured in the air stream coming out of the headspace of a column filled with 50 mL of HSM, sparged with air under HL. The two arrows in the graph indicate the addition of cells or DCMU. b WT cells were acclimated to LL HSM overnight shaken in flasks; the next day they were exposed to 300 µmol photons m⁻² s⁻¹ light in the presence or absence of 40 µM DCMU, shaken in flasks without or with sparging with VLCO₂. Samples were taken after 1 h. Presented are mRNA accumulation of LHCSR3, CAH4, LCIA. Data were normalized to LL (shown as dotted line in graph); n = 2 biological samples.

Fig. 7. CO₂- and light-dependent signals converge to regulate photoprotection and CCM in Chlamydomonas.

The intracellular levels of CO₂, defined by the equilibrium between CO₂ fixation in chloroplasts and the generation of CO₂ by mitochondrial metabolism (e.g. acetate assimilation) is the key determinant of the regulation of gene expression controlling two major processes of photosynthetic organisms: CCM and photoprotection. Changes in light availability have a direct impact on intracellular CO₂ levels; exposure to HL increases CO₂ fixation rates leading to depletion of CO₂ and to activation of not only photoprotection- but also CCM-related genes. Conversely, depletion of CO₂ is sufficient to drive high expression levels of CCM genes and LHCSR3 even in complete darkness (indicated by the black arrows). High CO₂ levels, either exogenously supplied by sparging or metabolically produced via acetate metabolism or by inhibiting photosynthetic electron flow using DCMU, repress LHCSR3 and CCM genes while at the same time they stabilize LHCSR1 protein levels. The close interconnection of photoprotection and CCM is further corroborated by the fact that CIA5, the regulator of expression of genes associated with the CCM, also exerts control over LHCSR3 and to a lesser extent over PSBS mRNA levels and acts as repressor of LHCSR1 protein accumulation. Independent of CIA5, light strongly impacts expression of all of these photoprotective genes (yellow arrows). This impact can be the consequence of both photoperception (e.g. phototropin) and the production of reactive oxygen species.