Figure 1.
Chlorophyll a fluorescence quenching in R. salina.
Cells were dark adapted for 20 minutes before and after irradiation. NPQ was induced by 100 s of orange actinic light (622 nm, 600 µmol m−2 s−1; white bar). Fluorescence induction curve (black line) represents a typical curve. The extent of NPQ (grey symbols, top part of the figure) was calculated as quenching of maximal fluorescence (FM′-FM)/FM′ for every saturating flash (n = 3); the maximal fluorescence measured after light period (FM″) reflects a fast recovery part of the FM quenching. The value of maximal PSII efficiency calculated in dark (FV/FM) and on light (Genty parameter - φPSII) was 0.79 and 0.1 respectively.
Figure 2.
Light dependence of NPQ and the efficiency of PSII (Genty parameter) in R. salina.
Fresh sample was used for each measurement and values were recorded always after 40 s of irradiation by orange light (622 nm). Data represent average and standard deviation for n = 3.
Figure 3.
Effect of various inhibitors on NPQ in R. salina.
Cells were dark adapted for 20 minutes and then the NPQ was induced by 100 s exposure to orange light (622 nm, 600 µmol m−2 s−1; see white bar). A) Effect of ΔpH uncouplers nigericin and NH4Cl. The maximal efficiency of PSII photochemistry (FV/FM) in the presence of uncouplers was 0.75 for control, 0.62 for nigericin and 0.68 for NH4Cl. B) Effect of inhibitors of linear and cyclic electron transport DCMU, antimycin and rotenone. All data represent typical curves aligned to the same Fo level.
Table 1.
Relative pigment content in intact cells of R. salina and in their chlorophyll a/c2 antennae (CAC antennae).
Figure 4.
Fluorescence emission spectra (Panel A) and spectral dependence of NPQ (Panel B) of R. salina cells.
All curves were measured using green light (520 nm, 500 µmol m−2 s−1) absorbed by phycoerythrin or blue light (465 nm, 1100 µmol m−2 s−1) absorbed by chlorophyll a. A) The black FM spectrum induced by saturating flash (200 ms, 1100 µmol m−2 s−1) shows the fluorescence emission spectra of dark adapted cells for green light excitation at 520 nm; the grey FM′ spectrum induced by saturating flash (200 ms, 1100 µmol m−2 s−1) was recorded after 120 s of continuous irradiation by green light. Similar measurements using blue light excitation at 465 nm are presented in the insert. B) Fluorescence emission spectra described in A were used for calculation of spectral dependence of NPQ(λ) based on Stern-Volmer formalism, green light excitation at 520 nm – grey line, blue light excitation at 465 nm – black line. Data represent typical curves, characteristic maxima are marked.
Figure 5.
Fluorescence quenching in isolated CAC antennae.
A) CAC proteins in a native state were isolated by ultracentrifugation in a sucrose gradient as a ‘free’ antennae (Fr. 1) or in a supercomplex with PSII (Fr. 2). See also Figures S2 and S4 for the complete figure of gradient and the spectroscopic analysis. B) Protein samples were diluted 10-fold to decrease concentration of dodecyl-β-maltoside – sample addition to buffer (20 mM HEPES, pH 8.0) is visible as chlorophyll fluorescence appearance (arrow ‘Sample’) that slowly decrease. After 70 s of incubation the pH in the sample was lowered from 8.0 to 5.5 (see arrow pH = 5.5) causing a fluorescence quenching. Reversibility of quenching has been confirmed by addition of 200 µM DCCD.
Figure 6.
The 2D eletrophoresis of membrane protein complexes of R. salina and spectral characteristic of isolated bands.
A) Membrane proteins were solubilised by dodecyl-β-maltoside and separated in a first dimension by clear-native electrophoresis (CN-PAGE). The protein complexes resolved on the CN-PAGE were further separated in the second dimension by denaturing gel (SDS-PAGE) and stained by Coomassie Blue. Position of protein complexes separated by CN-PAGE are marked as follows: CAC[1] – CAC monomers; CAC[c] – CAC oligomer; PSI[1] and PSII[1] - PSI and PSII monomers; PSI sc. and PSII sc. – supercomplexes of PSI and PSII. Proteins further resolved by SDS-PAGE are marked: CP47, CP43, D1, D2 - PSII core subunits; PsbA/B - PSI core subunits; CAC –chlorophyll a/c antenna. B) Absorbance spectra of Band I, II and III separated by CN-PAGE; positions of all three bands at the CN-PAGE are marked. C) Fluorescence emission spectra of the Band III (CAC oligomer) after excitation at 435 nm; positions of particular maxima are highlighted.
Table 2.
Effect of different DCCD (N,N′-dicyclohexyl-carbodiimide) concentrations on the maximal efficiency of PSII photochemistry (FV/FM) and on NPQ.