A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I

Abstract

In photosynthetic organisms like cyanobacteria and plants, the main engines of oxygenic photosynthesis are the pigment-protein complexes photosystem I (PSI) and photosystem II (PSII) located in the thylakoid membrane. In the cyanobacterium Synechocystis sp. PCC 6803, the slr1796 gene encodes a single cysteine thioredoxin-like protein, orthologs of which are found in multiple cyanobacterial strains as well as chloroplasts of higher plants. Targeted inactivation of slr1796 in Synechocystis 6803 resulted in compromised photoautotrophic growth. The mutant displayed decreased chlorophyll a content. These changes correlated with a decrease in the PSI titer of the mutant cells, whereas the PSII content was unaffected. In the mutant, the transcript levels of genes for PSI structural and accessory proteins remained unaffected, whereas the levels of PSI structural proteins were severely diminished, indicating that Slr1796 acts at a posttranscriptional level. Biochemical analysis indicated that Slr1796 is an integral thylakoid membrane protein. We conclude that Slr1796 is a novel regulatory factor that modulates PSI titer.

Keywords: cyanobacteria; photosynthesis; photosystem I biogenesis; photosystem II; protein complex; redox regulation.

FIGURE 1.

Slr1796 is a single cysteine thioredoxin-like protein in thylakoid membrane. A, schematic diagram showing the domains of the Slr1796 protein in Synechocystis 6803. Transmembrane sequence prediction was performed using the TMHMM program. The single cysteine thioredoxin-like fold was predicted by UniProt. The positions of cysteine residues are labeled. B, phylogenetic analysis of the evolutionary relationship between Slr1796 and its orthologs in selected cyanobacterial species. Red, thermophile; green, nitrogen-fixing; brown, strain lacking thylakoids. The land plant A. thaliana was used as an out-group. The scale bar indicates the number of amino acid substitutions per site.

FIGURE 2.

Genetic deletion and complementation of the slr1796 gene of Synechocystis 6803. A, scheme of slr1796 replacement by a Km^R cassette. B, PCR confirmation of complete segregation of the Δslr1796 mutant. Primer positions are indicated. Primer pair p3 + p4 is specific to the slr1796 gene (0.37-kb product) but not to Km^R. The expected size of the PCR products from primers p1 + p2 is 2.0 kb in WT and 2.6 kb in the mutant. M, DNA size standard. C, construction of the Comp strain. Slr1796 was fused at the C terminus with a His₆ tag and expressed under the P_cpc560 promoter in the Δslr1796 background.

FIGURE 3.

Phenotypic characterization of WT, the Δslr1796 mutant, and the complemented strain of Synechocystis 6803. A, growth curves of three strains grown under 40 μmol of photons m⁻²·s⁻¹. Error bars represent the standard deviation of three biological replicates. B, strains grown in liquid culture to the same OD_{730 nm} showing differences in the culture color. C, whole cell absorption spectra of three strains adjusted to equal cell numbers. Absorption peaks for chlorophyll a are at ∼440 and 680 nm and for phycobilins at ∼620 nm. D, low temperature (77 K) chlorophyll fluorescence emission spectra. The excitation wavelength was 435 nm. Peaks at 685 and 695 nm arise from PSII; the peak at 721 nm arises from PSI. For C and D, three or more independent replicates were performed, and representative data are presented. r.u., relative units.

FIGURE 4.

Kinetic spectroscopic characterization of WT (black line), the Δslr1796 mutant (blue), and the complemented strain (red). Cells were suspended in BG11 to equal cell numbers at which WT cells were at a chlorophyll concentration of 5 μg/ml. A dotted line was drawn to facilitate comparisons between strains. Data are presented as the average of three biological replicates. A, reoxidation kinetics of the reduced PSII primary electron acceptor Q_A⁻ in three strains. B, P700 redox kinetics in WT, Δslr1796 mutant, and Comp cells grown under the same conditions. After 10-s dark adaption, actinic light was turned on (up arrow) for 5 s and then off (down arrow) to allow the re-reduction of P700⁺ to P700. Inhibitors (Inh.; dotted lines), 10 μm DCMU and 10 μm 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone, were added to inhibit linear and cyclic electron flow. A negative slope indicates P700 oxidation. Rel., relative.

FIGURE 5.

Transcript levels of PSI and PSII genes in WT and Δslr1796 mutant cells. The constitutively expressed rnpB gene was used as a reference and to show that samples do not contain contamination from genomic DNA when no reverse transcriptase (RTase) was added. The primer pair used for the detection of slr1796 gene was p3 + p4 as in Fig. 2A. Optimal PCR cycles were determined for each primer pair to ensure that the amplifications were in the linear range. Three or more independent replicates were performed, and representative results are shown.

FIGURE 6.

Representative membrane protein profiles in WT, the Δslr1796 mutant, and the complemented strain. Immunoblotting analyses of photosynthetic membrane proteins are shown. Equal amounts of membrane proteins were loaded for the detection of each protein. A dilution series of WT protein (10, 25, 50, and 100%) were loaded. Antibodies were directed against PSI subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, and PsaK), PSI assembly factors (BtpA, Ycf3, and Ycf4), PSII core subunit D1, and a subunit of the cytochrome (Cyt) b₆f complex (Rieske FeS). The number at the right of each band is the percentage of protein in the Δslr1796 mutant relative to that of WT (set as 100%). Values represent the standard deviation of at least 10 measurements for each protein.

FIGURE 7.

Blue native PAGE of membrane protein complexes of WT and the Δslr1796 mutant. An equal protein amount of about 35 μg was loaded to each lane. The gel is shown as unstained (left) and stained with Coomassie Brilliant Blue (right). Molecular mass markers are indicated, and the assignments of PSI and PSII complexes are given.

FIGURE 8.

Subcellular localization of the Slr1796 protein. Protein fractions were prepared from the Comp strain. A, immunoblotting analysis of the cellular localization of the Slr1796-His protein using His tag antibodies. Lane 1, total cellular protein; lanes 2 and 3, pellet and soluble fractions after centrifugation; lanes 4 and 5, plasma and thylakoid membranes isolated by two-phase partitioning, respectively, that were validated by the detection of specific marker proteins NrtA and CP47, respectively. B, immunoblotting detection of Slr1796-His in pellet (P) and soluble (S) fractions of thylakoid membrane treated with the indicated solubilizing agents. D1 was used as a control integral membrane protein, and PsaE was used as a control extrinsic protein.

FIGURE 9.

Transmission electron micrographs of WT, the Δslr1796 mutant, and the complemented Synechocystis 6803 strains. A and B, WT; C and D, Δslr1796 mutant; E and F, Comp strain. Scale bars in A, C, and E represent 200 nm. The boxed region is enlarged in B, D, and F, respectively. The black lines in B, D, and F depict the measurements of the interthylakoidal space in each strain. Thylakoid membranes (T) are also labeled.