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. 2011;6(12):e28389.
doi: 10.1371/journal.pone.0028389. Epub 2011 Dec 22.

Double mutation in photosystem II reaction centers and elevated CO2 grant thermotolerance to mesophilic cyanobacterium

Jorge Dinamarca  1 , Oksana Shlyk-Kerner, David Kaftan, Eran Goldberg, Alexander Dulebo, Manuel Gidekel, Ana Gutierrez, Avigdor Scherz
Affiliations

Double mutation in photosystem II reaction centers and elevated CO2 grant thermotolerance to mesophilic cyanobacterium

Jorge Dinamarca et al. PLoS One. 2011.

Abstract

Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43 °C but only at elevated CO(2) (1%). Under the same conditions, the Synechocystis control strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated Q(A)(-)→Q(B) electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO(2) concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant.

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Conflict of interest statement

Competing Interests: A.S. is the incumbent of the Robert and Yaddele Sklare Professorial Chair in Biochemistry. J.D. was supported by a fellowship from the Programa Bicentenario-Banco Mundial, Conicyt, Chile. This program has no commercial interest. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Thermotolerance of Synechocystis sp. PCC 6803 AC mutant.
ΔKS and AC cells (circles and squares, respectively) were grown in liquid medium for 7 days at 30, 38, 40 and 43°C under 1% CO2 aeration and 40 µmol photons m−2 s−1. Growth was monitored by measuring the optical density at 730 nm (OD730). The values represent the mean ± SD of three independent experiments.
Figure 2
Figure 2. Growth of Synechocystis control and AC mutant.
Growth was estimated by measuring dry weight (A), OD730 (B and C) and chlorophyll content (D). A. ΔKS and AC cells (circles and squares, respectively) that were grown in liquid medium for 7 days at 30° or 43°C (open and filled symbols, respectively) under 1% CO2. B. ΔKS and AC cultured in liquid medium under stirring and normal air. The strains were incubated at 43°C for 5 days (filled symbols) and then transferred to 30°C (open symbols) for 5 days to test their viability. C. Wild-type (triangles), ΔKS (circles) and AC cells (squares) were grown at 43°C under 1% CO2. D. Chlorophyll content in wild type, ΔKS and AC cells (triangles, circles and squares, respectively) that have been transferred to 43°C and 1% CO2 after 3–4 days incubation at 30°C and 1% CO2. The values represent the mean ± SD of three independent experiments.
Figure 3
Figure 3. Changes in the D1 and Rubisco large subunit protein content.
ΔKS and AC cells were grown in liquid medium at 43°C under 1% CO2. Cells were collected at indicated times for isolation of proteins. Samples containing 1 µg of chlorophyll were immunoblotted using antibodies specific against D1 and Rubisco (see Materials and methods).
Figure 4
Figure 4. Activity of the PSIIRC in control and AC mutant.
Cells were grown for three days at 30° or 43°C (as indicated). A. The rate of oxygen evolution was measured at 30°C (white bars) and 43°C (gray bars) after 10 min incubation at the measuring temperature. The values represent the mean ± SD of three independent experiments. B. Temperature dependence of the QA −→QB ET rate constant for ΔKS (circles) and AC (squares) grown at 30°C (closed symbols) and 43°C (open symbols). The corresponding curves for T. elongatus grown at 43°C are denoted by empty triangles. C. The values from B were used to construct the corresponding Eyring plots. The bold lines represent the linear fits of the various curves from which ΔH ‡ (slope) and ΔS ‡ (intercept with the Y axis) were derived. For more detailed conditions, see Materials and methods. For B & C the values represent the mean of 10–12 independent measurements, the error bars are not shown here for clarity.
Figure 5
Figure 5. The effect of high irradiance and elevated temperature on D1 protein content and PSII oxygen evolution.
The control ΔKS (circles) and AC (squares) cells were incubated at 43°C and illuminated with 500 µmol photons m−2 s−1 in the absence (open symbols) or presence (closed symbols) of lincomycin. Aliquots of the suspensions were taken at the indicated times. Samples were used for Western blot analysis and to measure the oxygen evolving activity as described in Materials and methods. A. D1 protein content. Thylakoid membrane samples were analyzed by SDS-PAGE and immunoblotting using D1-specific antibody. The insert shows the contribution of the repair mechanisms, calculated as the difference between the content of D1 protein in the absence and presence of lincomycin. The data are shown after normalization to the value at the 0 time point. B. Oxygen evolution. The oxygen evolving activity was assayed in whole cells. For A & B the values represent the mean ± SD of three independent experiments.
Figure 6
Figure 6. Binding interactions between D1 and D2 proteins.
The proposed conformations for the ground (GS, conf 1) and transition (TS, conf2) states of QA −→QB in the in silico mutated D1-AC209/212SS (A) and the resolved wild-type structure of T. elongatus (B) representing the AC mutant structure.
Figure 7
Figure 7. Molecular dynamics simulation of D helix conformations and interactions of the D1 and D2 proteins.
A–D. Following energy minimization, the Ca atoms of the D helices of Synechocystis sp. PCC6803 and T. elongatus exhibit almost identical structures at the start of the simulation (A). Significant deviation is seen mostly on the periphery of the helices at the end of the simulated 20 ns dynamics (B). Close up of the central part of the helices shows additional details of the hydrogen bond network in T. elongatus (C) and Synechocystis sp. PCC6803 (D). D1 and D2 helices are respectively shown in blue and red for Synechocystis sp. PCC6803 and in yellow and cyan for T. elongatus.
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