Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery

Abstract

Absorption of excess light energy by the photosynthetic machinery results in the generation of reactive oxygen species (ROS), such as H2O2. We investigated the effects in vivo of ROS to clarify the nature of the damage caused by such excess light energy to the photosynthetic machinery in the cyanobacterium Synechocystis sp. PCC 6803. Treatments of cyanobacterial cells that supposedly increased intracellular concentrations of ROS apparently stimulated the photodamage to photosystem II by inhibiting the repair of the damage to photosystem II and not by accelerating the photodamage directly. This conclusion was confirmed by the effects of the mutation of genes for H2O2-scavenging enzymes on the recovery of photosystem II. Pulse labeling experiments revealed that ROS inhibited the synthesis of proteins de novo. In particular, ROS inhibited synthesis of the D1 protein, a component of the reaction center of photosystem II. Northern and western blot analyses suggested that ROS might influence the outcome of photodamage primarily via inhibition of translation of the psbA gene, which encodes the precursor to D1 protein.

Fig. 1. Light-induced inactivation of PSII in the presence of reagents that accelerate the production of ROS in wild-type Synechocystis. Cells were exposed to light at 1.5 mmol photons/m²/s with standard aeration. (A) In the presence of methyl viologen at 2 µM (open triangles) and at 5 µM (open squares) and in its absence (open circles); filled circles: in the presence of 5 µM methyl viologen in darkness. (B) The same as (A) but in the presence of 200 µg/ml chloramphenicol. (C) In the presence of H₂O₂ at 0.5 mM (open triangles) and at 2 mM (open squares) and in its absence (open circles); filled circles: in the presence of 2 mM H₂O₂ in darkness. (D) The same as (C) but in the presence of 200 µg/ml chloramphenicol. PSII activity was monitored in terms of the photosynthetic evolution of oxygen in the presence of 1 mM 1,4-benzoquinone as the electron acceptor. The activity taken as 100% was 527 ± 46 µmol O₂/mg chlorophyll/h. Values are means ± SD (bars) of results from four independent experiments.

Fig. 2. Light-induced inactivation of PSII in wild-type Synechocystis and in katG^–/tpx^– mutant cells. Cells were exposed to light at 1.5 mmol photons/m²/s in the presence of 200 µg/ml chloramphenicol and in its absence. The other experimental conditions were the same as described in the legend to Figure 1. (A) Wild-type cells. (B) katG^–/tpx^– cells. Open triangles and circles: in the presence and absence of chloram phenicol, respectively. PSII activity was monitored in terms of the photosynthetic evolution of oxygen. The oxygen-evolving activity of katG^–/tpx^– cells that was taken as 100% was 560 ± 44 µmol O₂/mg chlorophyll/h. Values are means ± SD (bars) of results from four independent experiments.

Fig. 3. Repair of photodamage to PSII and the effects of H₂O₂ after light-induced inactivation in wild-type Synechocystis and katG^–/tpx^– cells. Cells were exposed to light at 3 mmol photons/m²/s for 60 min (wild type) or 40 min (katG^–/tpx^–) without aeration to induce ∼80% inactivation of PSII. Cells were then incubated in light at 70 µmol photons/m²/s with standard aeration in the presence of 0.5 mM H₂O₂ (open triangles), 2 mM H₂O₂ (open squares) or 200 µg/ml chloramphenicol (filled triangles), and in the absence of these reagents (open circles). (A) Wild-type cells. (B) katG^–/tpx^– cells. Diamond symbols indicate repair after 0.1 µM catalase had been added, at the time indicated by the vertical arrow, to a suspension of cells that had been incubated in the presence of 0.5 mM H₂O₂. Values are means ± SD (bars) of results from three independent experiments.

Fig. 4. Changes in the level of the D1 protein during the light-induced inactivation of PSII in wild-type cells. (A) Results of western blot analysis. (B) Quantitation of the results shown in (A). Cells were exposed to light at 1.5 mmol photons/m²/s with standard aeration in the absence (lane 1; open circles) and in the presence (lane 2; open triangles) of 5 µM methyl viologen. Cells were also exposed to light in the presence of 200 µg/ml chloramphenicol (lane 3; filled circles) and in the presence of 200 µg/ml chloramphenicol plus 5 µM methyl viologen (lane 4; filled triangles). Thylakoid membranes were isolated from cells at the indicated times. Values are means ± SD (bars) of results from three independent experiments.

Fig. 5. The synthesis of the D1 protein de novo in the presence of ROS in wild-type Synechocystis and in katG^–/tpx^– cells, as monitored in terms of the incorporation of radioactive [³⁵S]methionine into proteins of thylakoid membranes. (A) Lanes 1–3, wild-type cells were labeled for 20 min in the absence of added reagents (lane 1), in the presence of 5 µM methyl viologen (lane 2) and in the presence of 2 mM H₂O₂ (lane 3). Lane 4, katG^–/tpx^– cells were labeled in the absence of any reagent. The arrow indicates the D1 protein. (B) Changes in the level of labeled D1 protein that had been incorporated into thylakoid membranes in wild-type cells in the absence of any added reagents (open circles), in the presence of 5 µM methyl viologen (open triangles), and in the presence of 0.5 mM H₂O₂ (open squares); filled circles indicate labeled D1 protein in katG^–/tpx^– cells in the absence of any added reagents. Values are means ± SD (bars) of results from three independent experiments.

Fig. 6. Effects of ROS on the light-induced expression of psbA genes in wild-type Synechocystis and katG^–/tpx^– cells. (A) Results of northern blot analysis. (B) Quantitation of the results in (A). Prior to exposure to light, cells were incubated at 30°C in darkness for 60 min. Wild-type cells were exposed to light at 1.5 mmol photons/m²/s in the absence of any added reagents (control; open circles) and in the presence of 5 µM methyl viologen (MV; open triangles), 0.5 mM H₂O₂ (open squares) and 200 µg/ml chloramphenicol (Cm; filled triangles); filled circles indicate induction of the expression of psbA genes in katG^–/tpx^– cells in the absence of any added reagents. Total RNA (5 µg) was loaded in each lane of the gel. Values are means ± SD (bars) of results from three independent experiments.

Fig. 7. Changes in the level of the pre-D1 protein during the light-induced inactivation of PSII in wild-type cells. Cells were exposed to light at 1.5 mmol photons/m²/s in the absence of H₂O₂ (open circles), in the presence of 0.5 mM H₂O₂ (open squares), and in the presence of 200 µg/ml chloramphenicol (open triangles). Thylakoid membranes were isolated from cells at the indicated times. Values are means ± SD (bars) of results from three independent experiments.

Fig. 8. Effects of H₂O₂ on the distribution of psbA mRNAs that are free or are associated with polysomes in wild-type cells. Cells were incubated at 30°C in darkness for 60 min and then exposed to light at 1.5 mmol photons/m²/s for 20 min in the presence of 0.5 mM H₂O₂ and in its absence. Cells were disrupted and membrane-bound polysomes (M), cytosolic polysomes (C) and polysome-free RNA (F) were prepared as described in the text. RNA was isolated from each fraction and subjected to northern blotting with a labeled fragment of the psbA2 gene as the probe. The amount of RNA in each lane corresponded to that from each individual fraction, and each fraction was derived from cells equivalent to 5 µg chlorophyll. (A) Gel-electrophoretic pattern. The results shown are representative of the results of four independent experiments, each of which gave similar results. (B) Quantified results. Values are means ± SD (bars) of results from four independent experiments. T represents the total of the three fractions of psbA mRNA.