Inactivation of photosystems I and II in response to osmotic stress in Synechococcus. Contribution of water channels

Abstract

The effects of osmotic stress due to sorbitol on the photosynthetic machinery were investigated in the cyanobacterium Synechococcus R-2. Incubation of cells in 1.0 M sorbitol inactivated photosystems I and II and decreased the intracellular solute space by 50%. These effects of sorbitol were reversible: Photosynthetic activity and cytoplasmic volume returned to the original values after removal of the osmotic stress. A blocker of water channels prevented the osmotic-stress-induced inactivation and shrinkage of the intracellular space. It also prevented the recovery of photosynthetic activity and cytoplasmic volume when applied just before release from osmotic stress. Inhibition of protein synthesis by lincomycin had no significant effects on the inactivation and recovery processes, an observation that suggests that protein synthesis was not involved in these processes. Our results suggest that osmotic stress decreased the amount of water in the cytoplasm via the efflux of water through water channels (aquaporins), with resultant increases in intracellular concentrations of ions and a decrease in photosynthetic activity.

Figure 1

Effects of sorbitol and a water-channel blocker on the photosynthetic electron-transport activities of PSII and PSI in intact cells. Cells were incubated in the presence of 1.0 m sorbitol and 100 μm p-chloromercuriphenyl-sulfonic acid (the water-channel blocker) or in their absence. At designated times, a portion of each cell suspension was withdrawn and the activities of PSII and PSI were determined. A, The oxygen-evolving activity of PSII was examined after the addition of 1.0 mm BQ to the suspension. The activity that corresponded to 100% was 533 ± 38 μmol O₂ mg⁻¹ Chl h⁻¹. B, The electron-transport activity of PSI was determined by monitoring the uptake of oxygen at 32°C after the addition of 15 μm DCMU, 0.1 mm DCIP, 5 mm ascorbate, and 0.1 mm MV to the suspension. The rate of oxygen uptake that corresponded to 100% was 353 ± 52 μmol O₂ mg⁻¹ Chl h⁻¹ in the absence of sorbitol and p-chloromercuriphenyl-sulfonic acid (○); in the presence of 1.0 m sorbitol (●); and in the presence of 1.0 m sorbitol and 100 μm p-chloromercuriphenyl-sulfonic acid (▴). Each point represents the average with se of results from four independent experiments.

Figure 2

Recovery of PSII and PSI in intact cells after removal of sorbitol. After cells had been incubated for 2 h with 1.0 m sorbitol, they were washed twice with fresh BG-11 medium and incubated in the presence of either lincomycin or p-chloromercuriphenyl-sulfonic acid and in the absence of these chemicals. At designated times, a portion of each cell suspension was withdrawn and the activities of PSII and PSI were determined. A, The oxygen-evolving activity of PSII was examined after the addition of 1.0 mm BQ to the suspension. The activity that corresponded to 100% was 544 ± 38 μmol O₂ mg⁻¹ Chl h⁻¹. B, The activity of PSI was determined by monitoring the uptake of oxygen after the addition of 15 μm DCMU, 0.1 mm DCIP, 5 mm ascorbate, and 0.1 mm MV to the suspension. The rate of oxygen uptake that corresponded to 100% was 364 ± 52 μmol O₂ mg⁻¹ Chl h⁻¹ in the absence of both chemicals (○); in the presence of 100 μm p-chloromercuriphenyl-sulfonic acid (▵); in the presence of 100 μg lincomycin mL⁻¹ (□). Each point represents the average with se of results from four independent experiments.

Figure 3

Changes in the yield of Chl fluorescence in intact cells during incubation with 1.0 m sorbitol. At designated times, a portion of each cell suspension was withdrawn and kept in darkness at 32°C for 15 min; the maximum fluorescence of Chl (F_max) was then determined at 25°C before and after the addition of 1 mg mL⁻¹ dithionite. ●, No dithionite; ▴, after addition of dithionite. Each point represents the average with se of results from three independent experiments.

Figure 4

Changes in cell volume during incubation in the presence of sorbitol. Cells were incubated with 1.0 m sorbitol in the presence of 100 μm p-chloromercuriphenyl-sulfonic acid and in its absence. At designated times, a portion of each cell suspension was withdrawn and the cell volume was determined from measurements of EPR. The cell volume that corresponded to 100% was 0.75 ± 0.05 fL per cell in the absence of p-chloromercuriphenyl-sulfonic acid (●); in the presence of 100 μm p-chloromercuriphenyl-sulfonic acid (▴). Each point represents the average with se of results from four independent experiments.

Figure 5

Recovery of cell volume after removal of sorbitol. After cells had been incubated for 2 h with 1.0 m sorbitol, they were washed twice with fresh BG-11 medium and then incubated in the presence and absence of 100 μm p-chloromercuriphenyl-sulfonic acid. At designated times, a portion of each cell suspension was withdrawn, and the cell volume was determined as described in the text in the absence of p-chloromercuri-phenyl-sulfonic acid (○); in the presence of 100 μm p-chloromercuriphenyl-sulfonic acid (▵). Each point represents the average with se of results from four independent experiments.

Figure 6

Changes in electron-transport activities in isolated thylakoid membranes during incubation in the presence and absence of sorbitol. Thylakoid membranes at a Chl concentration of 10 μg mL⁻¹ were incubated at 32°C in darkness in the presence of 1.0 m sorbitol and in its absence. At designated times, a portion of each suspension of membranes was withdrawn and the activities of PSII and PSI were determined. A, The transport of electrons by PSII from water to DCIP (○, ●) and from DPC to DCIP (▵, ▴) was monitored by following the light-induced reduction of DCIP after the addition of 0.1 mm DCIP and of 0.1 mm DCIP plus 0.5 mm DPC, respectively. B, The transport of electrons by PSI from the reduced form of DCIP to MV was monitored by following the light-induced uptake of oxygen after addition of 15 μm DCMU, 0.1 mm DCIP, 5 mm ascorbate, and 0.1 mm MV to the suspension. Black symbols, In the presence of 1.0 m sorbitol; white symbols, in the absence of sorbitol. Each point represents the average with se of results from three independent experiments.

Figure 7

Effects of a water-channel blocker on the uptake of sorbitol by Synechococcus R-2 cells. Cells at 100 μg Chl mL⁻¹ were incubated with 1.0 m sorbitol plus [U-¹⁴C]sorbitol (18.5 kBq mL⁻¹, 1.7 nm) in the presence of 100 μm p-chloromercuriphenyl-sulfonic acid (●) or in its absence (○). At designated times a portion of cell suspension was withdrawn and the uptake of radioactivity in cells was determined as described in “Materials and Methods.” Uptake of radioactivity is expressed as the radioactivity remaining on the glass-fiber filter divided by the radioactivity supplied as a percentage. Each value represents the average with se of results from three independent experiments.

Figure 8

A hypothetical schematic explanation of the osmotic stress-induced inactivation of PSI and PSII in cyanobacterial cells. ●, Three extrinsic proteins, namely, the 33-kD protein, Cyt c₅₅₀, and PsbU, of the oxygen-evolving machinery of PSII; ▪, plastocyanin or Cyt c₅₅₃ associated with PSI. PM, Plasma membrane; TM, thylakoid membrane; I, PSI complex; II, PSII complex.