Figure 1.
Immunoblotting detection of LhcSR and CP47 on C. reinhardtii thylakoids with specific antibodies.
(A) LhcSR on thylakoids from WT, stt7 mutant, and cbs3 mutant cells, HL and LL acclimated. Two sample dilutions are loaded (100% = 0.8 µg of Chl). Arrows indicate three cross-reacting bands; the lowest is poorly represented. (B) LhcSR on WT and stt7 HL thylakoids untreated or treated with calf intestine alkaline phosphatase (CIP). Three sample dilutions were loaded (100% = 0.8 µg of Chl).
Figure 2.
NPQ analysis of C. reinhardtii cells (WT, stt7, and cbs3 genotypes), HL or LL long-acclimated.
Fv/Fm for each sample shown in left inset.
Figure 3.
Native Deriphat-PAGE and Western blotting.
(A) Native Deriphat-PAGE electrophoresis and Western blotting of HL C. reinhardtii (C.r.) cell membranes showing anti-LhcSR immuno-reacting bands (asterisks) at monomeric antenna protein MW and between monomeric and trimeric antennae MW. Note how, in native electrophoresis, band resolution is always much worse than in SDS-PAGE electrophoresis, and background signal is higher. A. thaliana (A.t.) membranes are for comparison. (B) Native Deriphat-PAGE separation of both HL and LL native complexes and Western blotting with anti-LhcSR, showing how phosphatase treatment does not influence LhcSR aggregation state.
Figure 4.
Sequence alignment of LhcSR1, LhcSR3, and A. thaliana CP29 and Lhcb1.
Chl- and neoxanthin-coordinating residues are shown. Note conservation of all these residues in both LhcSR isoforms, with exception of residues coordinating B3 and B6, with respect to CP29 and Lhcb1. At, A. thaliana; Cr, C. reinhardtii.
Figure 5.
Spectroscopic analysis of LhcSR3.
(A) Absorption spectra in visible region of LhcSR3 (red line) and CP29 (Z. mays, black line); maximum absorption peak in Qy region is also shown. (B) LhcSR3 fluorescence emission at 300K with excitation at 440 nm (black line, Chl a), 475 nm (red line, Chl b), and 500 nm (blue line, Cars). (C) CD spectra in visible region of LhcSR3 (red line) and CP29 (Z. mays, black line); Chl a (666 and 683 nm) major peaks are shown. (D) Fluorescence emission spectra at 77K (630 to 720 nm) of LhcSR3 (red line) and CP29 (Z. mays, black line) upon excitation of Chl a (440 nm); peak values are shown. (E) Deconvolution of LhcSR3 absorption spectrum in Qy spectral region with Chl spectral forms in protein environment. Several spectral forms are marked A (Chl a) and B (Chl b), with peak absorptions reported in the box insert. (F) Number of Chls represented by spectral forms in (E): data are normalized for six total Chls. (G) Deconvolution of LhcSR3 absorption spectrum in Qy spectral region with Chl spectral forms in protein environment and a single Gaussian with 16 half-height bandwidths peaking at 685.7 nm; spectral forms are marked A (Chl a), B (Chl b), and G (Gaussian function). (H) Number of Chls represented by spectral forms of (G): data are normalized for six total Chls.
Table 1.
Pigment content in picomoles of refolded LhcSR3 complexes in absence or presence of Zea (LV and LVZ, respectively) and with or without Chl b.
Figure 6.
Time-resolved fluorescence analysis.
Fluorescence emission kinetics of CP29 and LhcSR3 lutein/violaxanthin (LhcSR3 LV) or lutein/violaxanthin/Zea (LhcSR3 LVZ) binding was recorded at 685 nm (A). Fluorescence emission kinetics of LhcSR3 lutein/violaxanthin (LhcSR3 LV) or lutein/violaxanthin/Zea (LhcSR3 LVZ) was recorded at pH 7.5 or pH 5.5 (B). Decay curves were fitted with two or three exponential functions for CP29 and LhcSR3. Fitted curves are shown with dashed lines.
Table 2.
Lifetimes and relative fluorescence quantum yields of CP29 and LhcSR3.
Figure 7.
(A and B) TA traces from LhcSR3 LV and LhcSR3 LVZ at 940 nm (A) and 980 nm (B). (C and D) TA traces at 940 nm from LhcSR3 LV (C) and LhcSR3 LVZ (D) at pH 7.5 versus pH 5.5. (E and F) LhcSR3 LV (E) and LhcSR3 LVZ (F) TA spectra with fixed 15-ps time.
Figure 8.
In vivo effect of DCCD and Zea on NPQ.
(A) measurement of NPQ in HL acclimated C. reinhardtii cells with or without incubation with 20 µM DCCD. (B) NPQ analysis of C. reinhardtii cells (WT and npq1 genotypes) HL long acclimated (500 µmol m−2 s−1). Note that WT strain used in the experiment shown in (B) is CC425 for comparison with npq1mutant in the same genetic background [70]; WT in (A) is cw15.
Figure 9.
(A) SDS-PAGE separation of proteins labeled with 14C-DCCD. (B) Autoradiography of gel shown in (A). (C) Densitometric analysis of bands in (B) after normalization to Coomassie staining (A).
Figure 10.
Model showing Chl and xanthophyll chromophores bound to different sites in LhcSR3.
Model was built by homology based on crystal structure of LHCII by [46].