Acclimation of Chlamydomonas reinhardtii to different growth irradiances

Abstract

We report on the changes the photosynthetic apparatus of Chlamydomonas reinhardtii undergoes upon acclimation to different light intensity. When grown in high light, cells had a faster growth rate and higher biomass production compared with low and control light conditions. However, cells acclimated to low light intensity are indeed able to produce more biomass per photon available as compared with high light-acclimated cells, which dissipate as heat a large part of light absorbed, thus reducing their photosynthetic efficiency. This dissipative state is strictly dependent on the accumulation of LhcSR3, a protein related to light-harvesting complexes, responsible for nonphotochemical quenching in microalgae. Other changes induced in the composition of the photosynthetic apparatus upon high light acclimation consist of an increase of carotenoid content on a chlorophyll basis, particularly zeaxanthin, and a major down-regulation of light absorption capacity by decreasing the chlorophyll content per cell. Surprisingly, the antenna size of both photosystem I and II is not modulated by acclimation; rather, the regulation affects the PSI/PSII ratio. Major effects of the acclimation to low light consist of increased activity of state 1 and 2 transitions and increased contributions of cyclic electron flow.

FIGURE 1.

Growth curves of acclimated cells. Cells acclimated to HL (triangles), CL (stars), and LL (circles) were grown at different light intensity (20–60-400 μmol m⁻² s⁻¹, respectively) for at least 10 generations. The number of cells of the different acclimated cultures from early exponential to saturation phase is reported in logarithmic scale.

FIGURE 2.

Western blot quantification of light phase protein complexes. Photosystems I and II, Cyt b₆f, and ATP synthase content were estimated by Western blot analysis using antibody probes against specific subunits as follows: PsaA and D1 for PSI and PSII, respectively; Cyt f for Cyt b₆f ATPase δ subunit for the ATP synthase supercomplex. Different amounts of chlorophyll were loaded in the SDS-polyacrylamide gel to provide linearity in the quantification. A, Western blot reactions are reported, with the indication of the amount of micrograms of chlorophyll loaded for each lane. B and C, amounts of the different subunits are reported normalized on chlorophyll (B) or cell (C) basis. a.u., arbitrary units.

FIGURE 3.

Western blot quantification of Rubisco content in acclimated cells. A, Calvin cycle enzymes and in particular Rubisco content in the different acclimated cells were estimated by Western blot analysis against the large subunit of Rubisco. The micrograms of chlorophylls loaded on SDS-polyacrylamide gel are reported in the figure. B, amount of the Rubisco large subunit is reported normalized on chlorophyll or cell basis. a.u., arbitrary units.

FIGURE 4.

Western blot quantification of Lhcb antenna protein in acclimated cells. Lhcb antenna protein content was analyzed by Western blot using specific antibodies. A, immunoblot assays of PSII antenna proteins CP26, CP29, and LHCII compared with D1, used as reference for PSII core complex amount. Each sample was loaded in three dilutions to prevent saturation; in the figure the micrograms of chlorophylls loaded are reported. B and C, amount of the different Lhcb subunits is reported normalized to chlorophyll content (B) or D1 amount (C), to determine in the latter case the content of each Lhcb protein per PSII reaction center. The average content of Lhcb subunits per chlorophyll or D1 is also reported. avg, average; a.u., arbitrary units.

FIGURE 5.

Western blot quantification of LHCI antenna protein in acclimated cells. LHCI antenna protein content was analyzed by Western blot using specific antibodies. A, immunoblot assays of PSI antenna proteins Lhca1–9 compared with PsaA, used as reference for PSI core complex amount. Each sample was loaded in three dilutions to prevent saturation; in the figure the amount of chlorophylls loaded (in micrograms) is reported. B and C, amount of the different LHCI subunits is reported as normalized to chlorophyll content (B) or PsaA amount (C), to determine in the latter case the content of each LHCI protein per PSI reaction center. The average content of LHCI subunits per chlorophyll or PsaA is also reported. Antibodies directed against Lhca8 and Lhca9 partially recognize also the comigrating subunits Lhca1 and Lhca5, respectively. avg, average; a.u., arbitrary units.

FIGURE 6.

Functional antenna size of photosystem I and II. Fluorescence emission kinetics of PSII from dark-adapted acclimated cells were treated with DCMU (A). The time required for reaching ⅔ of the maximum is inversely proportional to PSII antenna size (B). Kinetics of P700 oxidation were measured as absorption differences (ΔAbs) at 705 nm from dark-adapted acclimated cells treated with DCMU to inhibit electron transport from PSII; in these measurements methylviologen is used as final electron acceptor (C). The time required for reaching ⅔ of the maximum is inversely proportional to PSII antenna size (D). a.u., arbitrary units.

FIGURE 7.

State transition in acclimated cells. A, Western blot analysis of STT7 accumulation in acclimated cells; micrograms of chlorophyll loaded for each lane is reported in the figure. B and C, level of STT7 kinase accumulation in acclimated cells quantified by Western blot (A) and normalized to chlorophyll (B) or cell (C) content. D, maximal amplitude of state transitions induced in acclimated cells measured as percentage of PSII fluorescence difference between cells in states I and II, as induced by adding DCMU and NaN₃, respectively, to dark-adapted cells as described previously (39). E–G, low temperature (77 K) fluorescence emission spectra of whole cells dark-adapted (black lines) or light-treated at the irradiance used for growth for 2 h (dotted lines for LL, CL and HL cells, E–G, respectively): fluorescence emission spectra were normalized to CP47 emission peak at 694 nm. a.u., arbitrary units.

FIGURE 8.

Photosynthetic properties of acclimated cells. A, oxygen production of LL (⊕), CL (*), and HL (△) cells normalized to chlorophyll content (milligrams)/h, measured at different actinic light intensities. B, electron transport rate (ETR) at the level of PSII of the different acclimated cells. C, photochemical quenching of acclimated cells at different light intensities. D, estimation of total protonmotive force (ΔPMF) in acclimated cells upon exposure to 5 min of illumination at different light intensities, measured as difference in absorption at 520 nm, because of electrochromic shift of carotenoid absorption spectrum (ECS). E, extrapolation of trans-thylakoid ΔpH formation in acclimated cells on the basis of results reported in D; ΔpH is calculated as the portion of PMF relaxing in 20 s upon dark exposure, as described previously (38). F, extrapolation of total ΔΨ formation in acclimated cells on the base of results reported in D and F; ΔΨ is calculated as the difference between ΔPMF and ΔpH as described previously (38).

FIGURE 9.

Nonphotochemical quenching and LhcSR3 accumulation in acclimated cells. A, NPQ induction kinetics of the different acclimated cells measured with very strong actinic light (1850 μmol m⁻² s⁻¹) followed by dark NPQ relaxation. B, maximum NPQ values measured at different light intensities. C, Western blot analysis on LhcSR protein accumulation in acclimated cells. The antibody used recognizes both LhcSR3 and LhcSR1 (29). In particular LhcSR3 appears on the filter as a double band, the higher molecular weight one corresponding to LhcSR3 in phosphorylated form. In the text the amount of chlorophyll loaded for each lane is reported in micrograms. D, LhcSR3 level (sum of phosphorylated and unphosphorylated) per PSII (normalization to D1 content) in the acclimated cells quantified by Western blot analysis. E, linear correlation between LhcSR3/D1 ratios (D) and qE maximum induction in the different acclimated cells; qE amplitude was calculated by subtracting the level of NPQ induced upon illumination with 1850 μmol m⁻² s⁻¹ not yet relaxed after 500 s in the dark to NPQ maximal value.

FIGURE 10.

Alternative electron flows in acclimated cells. A, dependence of protonmotive force (PMF) from photosynthetic oxygen evolution in acclimated cells. Increased activation of CEF results in high PMF at lower oxygen production; both ΔPMF and oxygen evolution are normalized to chlorophyll content. B, dependence of trans-thylakoid ΔpH formation from photosynthetic oxygen evolution in acclimated cells. Increased activation of CEF results in high ΔpH at lower oxygen production·oxygen; both ΔPMF and oxygen evolution are normalized to chlorophyll content. C, PTOX protein level in acclimated cells quantified by Western blot analysis and normalized to chlorophyll or cell content. D, oxygen production in LL, CL, and HL cells normalized to chlorophyll content (milligram)/h, measured at 650 μmol m⁻² s⁻¹ in presence (+) or absence (−) of PTOX inhibitor PGAL.