Photosynthesis and state transitions in mitochondrial mutants of Chlamydomonas reinhardtii affected in respiration

Abstract

Photosynthetic activities were analyzed in Chlamydomonas reinhardtii mitochondrial mutants affected in different complexes (I, III, IV, I + III, and I + IV) of the respiratory chain. Oxygen evolution curves showed a positive relationship between the apparent yield of photosynthetic linear electron transport and the number of active proton-pumping sites in mitochondria. Although no significant alterations of the quantitative relationships between major photosynthetic complexes were found in the mutants, 77 K fluorescence spectra showed a preferential excitation of photosystem I (PSI) compared with wild type, which was indicative of a shift toward state 2. This effect was correlated with high levels of phosphorylation of light-harvesting complex II polypeptides, indicating the preferential association of light-harvesting complex II with PSI. The transition to state 1 occurred in untreated wild-type cells exposed to PSI light or in 3-(3,4-dichlorophenyl)-1,1-dimethylureatreated cells exposed to white light. In mutants of the cytochrome pathway and in double mutants, this transition was only observed in white light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. This suggests higher rates of nonphotochemical plastoquinone reduction through the chlororespiratory pathway, which was confirmed by measurements of the complementary area above the fluorescence induction curve in dark-adapted cells. Photo-acoustic measurements of energy storage by PSI showed a stimulation of PSI-driven cyclic electron flow in the most affected mutants. The present results demonstrate that in C. reinhardtii mutants, permanent defects in the mitochondrial electron transport chain stabilize state 2, which favors cyclic over linear electron transport in the chloroplast.

Figure 1.

Light saturation curves of oxygen evolution in cells of wild-type and dum19/25 double mutant (lacking complexes I and IV). A and B, Two scales of photosynthetic photon flux densities.

Figure 2.

Relationship between the rate of dark respiration and the apparent quantum yield of oxygen evolution (estimated from the initial slope of the light saturation curves and expressed in percentage of the wild-type control).

Figure 3.

Cellular ATP concentrations of mitochondrial mutants, expressed in percentage of the wild-type control. Cells were fixed under illumination with white light of 70 μmol m^-2 s^-1, as used during growth.

Figure 4.

Analysis of state transitions by 77 K fluorescence spectroscopy. A, Fluorescence emission spectra (77 K) of the wild-type control and of the dum19/25 double mutant (lacking complexes I and IV). Different pretreatments were applied to cell suspensions before freezing: 1, control under white light of 70 μmol m^-2 s^-1; 2, illumination by PSI light (λ > 705 nm) of 60 W m^-2 during 15 min; 3, illumination by white light of 70 μmol m-2 s^-1 in the presence of 10 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); and 4, dark incubation under nitrogen atmosphere during 20 min. B, F715 to F685 ratios measured in the same conditions for the wild type and for the indicated mutants.

Figure 5.

Immunoblot detection of phosphorylated LHCs of the wild-type control and of the dum17, dum1, and dum19/25 mutants lacking complex I, complex III, and complexes I and IV, respectively. Immunoblotting was performed on 10 μg of membrane proteins using an antibody against phospho-Thr. Different pretreatments were applied to cell suspensions before membrane isolation: 1, control under white light of 70 μmol m^-2 s^-1; 2, illumination by PSI light (λ > 705 nm) of 60 W m^-2 during 15 min; 3, illumination by white light of 70 μmol m^-2 s^-1 in the presence of 10 μm DCMU; and 4, dark incubation under nitrogen atmosphere during 20 min.

Figure 6.

Analysis of the redox state of PQs from fast fluorescence induction curves recorded in the absence or in the presence of DCMU (10 μm) after 5 min of dark adaptation. A and B, Variable fluorescence traces (averages of four recordings) of cell suspensions of the wild-type control and of the dum19/25 double mutant lacking complexes I and IV (only the variable part of the signal, from F_o to F_m, is shown here after normalization to F_m). C, F_v/F_m ratios in the absence of DCMU. D. Apparent numbers of oxidized PQ per PSII reaction center, calculated from the complementary areas over the fluorescence induction curves.

Figure 7.

Slow changes in room temperature fluorescence quantum yield associated with state 1 transition during illumination by white light in the presence of 10 μm DCMU. A, Recordings of the modulated fluorescence signal in wild-type and dum19/25 cells. Cells are illuminated by 10 μmol m^-2 s^-1, and 1-s saturating pulses of 100 μmol m^-2 s^-1 are given every 1 min to record F_m. After approximately 5 min, the photosynthetic photon flux density (PPFD) is kept continuously at 100 μmol m^-2 s^-1. B, Initial rate of F_m increase under 10 μmol m^-2 s^-1 in wild-type and mutant cells, expressed in percentage of the wild-type control.

Figure 8.

PSI ES measured from the photo-acoustic photothermal signal measured under modulated PSI light (λ > 715 nm). A, ES as a function of the modulation frequency of excitation light in cells of the wild-type control and of the dum19/25 mutant lacking complexes I and IV. B, ES values of cells of the wild type and of the indicated mutants.