Figure 1.
Expression of flv genes in Synechocystis WT.
(A) Accumulation of flv transcripts under HC (white) and LC (grey) conditions analyzed by RT-Q-RT-PCR. The transcript abundances were calculated relative to the reference gene (rnpB) expression level. Results are shown as a mean value of three to four independent experiments (bars indicate SE). (B) Protein abundances of Flv2, Flv3 and Flv4 under HC and LC analyzed by Western blot. Total cell extract (30 µg proteins) was applied to SDS-PAGE and probed with Flv2, Flv3 and Flv4 antibodies. The corresponding Δflv mutants (Δ) were used to indicate the specificity of the antibodies, and served as negative controls. (C) Induction of flv2 (red) and flv4 (blue) transcript accumulation upon shift of Synechocystis WT cells from HC to LC in response to different light intensity. Relative abundance of transcripts indicates a fold change in the amount of transcripts upon a shift of cells from HC to LC.
Figure 2.
Localization of Flv proteins in different cellular fractions.
Protein samples (30 µg) of the total cell extract, membrane and soluble fractions of WT cells grown at LC were applied to SDS-PAGE and probed with Flv2, Flv3 and Flv4 antibodies. The antibodies against D1 and Rubisco large subunit were used to indicate the purity of the fractions. The corresponding Δflv mutants (Δ) served as negative controls. The experimental molecular weight of each Flv was estimated by comparing the migration of the respective proteins with the molecular weight markers in SDS-PAGE.
Figure 3.
Expression of flv genes in WT and Δflv mutants under HC and LC conditions.
(A) Accumulation of flv transcripts analyzed by RT-Q-RT-PCR. The transcript level of rnpB is used as a reference. Bars represent the ratio of expression of individual flv genes at LC to that at HC±SE for three independent experiments. (B) Expression of Flv proteins. Immunoblotting was performed by loading 40 µg of total cell extract in each well, and probed with Flv2, Flv3 and Flv4 antibodies.
Figure 4.
Thylakoid protein content of WT and Δflv mutant cells grown at HC and LC.
(A) Membrane proteins (25 µg in each well) were separated by SDS-PAGE and immunoblotting was performed using D1, D2, AtpA/B, Cytf, PsaA/B, NdhJ and NdhD3 specific antibodies. (B) Relative amounts of the D1 and D2 proteins in WT and the Δflv mutant cells grown under HC and LC conditions. Protein amounts are indicated as a percentage of those measured in the WT cells grown at HC (100%). Values are the mean±SE from 3 independent experiments.
Figure 5.
PSII fluorescence and the phenotype of the Δflv mutants.
Cells were grown at HC and 50 µmol photons m−2 s−1 or at LC under 15, 50, 120 or 200 µmol photons m−2 s−1. (A) The amplitude of the flash-induced variable Chl fluorescence of the WT and Flv mutant cells was measured in the presence of 20 µM DCMU and at a Chl concentration of 5 µg Chl/ml. The results are shown as a percentage of the amplitude of the flash-induced variable Chl fluorescence in the WT-cells grown at HC (set as 100%). ±SD for three independent experiments. (B) Photographs of cell cultures grown under HC and 50 µmol photons m−2 s−1, (C) under LC and 50 µmol photons m−2 s−1, (D) under LC and 200 µmol photons m−2 s−1.
Table 1.
Net photosynthesis and PSII activities of WT and flv mutants.
Figure 6.
D1 turnover rates of WT and Δflv mutants under LC conditions.
Synechocystis cells were grown under LC and 50 µmol photons m−2 s−1. A radioactive pulse was given to the cell suspension for 5, 10, 20, and 30 min under 150 µmol photons m−2 s−1 illumination. Chase experiments were performed under similar conditions for 0.5, 1, 2, and 3 h. (A) Autoradiogram of the membrane proteins separated by SDS-PAGE. The bands corresponding to the D1 and pre-D1 proteins are indicated by arrows. (B) Quantification of radioactivity in the D1 protein during pulse. (C) Quantification of radioactivity in the D1 protein during chase. Values are the mean of two independent experiments.
Figure 7.
Photoinhibition of PSII in WT, Δflv2 and Δflv4 under HC and LC.
The cells were grown under HC and LC conditions at 50 µmol photons m−2 s−1, and then subjected to high light illumination in a photobioreactor at 1500 µmol photons m−2 s−1 for 90 min with continuous bubbling of the cell cultures with 3% (HC) and air level (LC) of CO2, respectively. Samples were withdrawn during the high light treatment, and the PSII activity was monitored by steady-state oxygen evolution measurements in the presence of 2 mM DMBQ as an artificial electron acceptor. The results are shown as a percentage of oxygen evolution measured before the high light treatment, which is set as 100% (for absolute O2 evolution activities of the controls from HC and LC conditions, see Table 1). Mean±SD for three independent experiments.
Figure 8.
Phylogenetic analysis of flavodiiron proteins in anaerobes, facultative aerobes and oxygenic photosynthetic organisms.
Amino acid sequences of cyanobacterial proteins were obtained from CyanoBase (http://bacteria.kazusa.or.jp/cyanobase/), Selaginella amino acid sequences from JGI (http://genome.jgi-psf.org/Selmo1/Selmo1.home.html) and the other amino acid sequences from NCBI (http://www.ncbi.nlm.nih.gov/). Ana, Anabaena sp. PCC 7120 (Flv1a: All0177, Flv1b: All3891, Flv2: All4444, Flv3a: All0178, Flv3b: All3895, Flv4: All4446); Ama, Acaryochloris marina MBIC11017 (Flv1: Am1_1384, Flv3: Am1_1386); Cya, Cyanothece sp. ATCC 51142 (Flv1: Cce_2580, Flv2: Cce_3835, Flv3: Cce_3635, Flv4: Cce_3833); Cre, Chlamydomonas reinhardtii (FlvA: XP_001692916, FlvB: XP_001699345); Dgi, Desulfovibrio gigas (ROO: AAG34792); Eco, Escherichia coli (FlRd: Q46877); Gvi, Gloeobacter violaceus PCC 7421 (Flv1: Glr1776, Flv3: Glr1775); Mae, Microcystis aeruginosa NIES-843 (Flv1: MAE61610, Flv2: MAE50820, Flv3: MAE01310, Flv4: MAE50840); Mth, Moorella thermoacetica (FDP: AAG00802); Npu, Nostoc punctiforme ATCC 29133 (Flv1a: Npun_F4867, Flv1b: Npun_F5838, Flv2: Npun_R0591, Flv3a: Npun_F4866, Flv3b: Npun_F5837); PMM, Prochlorococcus marinus MED4 (FlvA: PMM0042, FlvB: PMM0043); PMT, Prochlorococcus marinus MIT9313 (FlvA: PMT2165, FlvB: PMT2164); Ppa, Physcomitrella patens subsp. patens (FlvA: XP_001759251, FlvB: XP_001756079); Pro, Prochlorococcus marinus SS120 (FlvA: Pro0044, FlvB: Pro0045); Smo, Selaginella moellendorffii (FlvA: estExt_Genewise1.C_61201, FlvB: estExt_fgenesh2_pg.C_1160033); Sel, Synechococcus elongatus PCC 6301 (Flv1: Syc2283, Flv3: Syc2284); Syc, Synechococcus sp. CC 9902 (FlvA: Syncc9902_2183, FlvB: Syncc9902_2180); Syn, Synechocystis sp. PCC 6803 (Flv1: Sll1521, Flv2: Sll0219, Flv3: Sll0550, Flv4: Sll0217), Tel, Thermosynechococcus elongatus BP-1 (Flv2: Tll1373, Flv4: Tlr1088); Ter, Trichodesmium erythraeum IMS 101 (Flv1: Tery_0770, Flv3: Tery_0302).
Table 2.
Oligonucleotide sequences used to perform RT-Q-RT-PCR.