Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp

Abstract

We report here that osmotic effects and ionic effects are both involved in the NaCl-induced inactivation of the photosynthetic machinery in the cyanobacterium Synechococcus sp. PCC 7942. Incubation of the cyanobacterial cells in 0.5 M NaCl induced a rapid and reversible decline and subsequent slow and irreversible loss of the oxygen-evolving activity of photosystem (PS) II and the electron transport activity of PSI. An Na(+)-channel blocker protected both PSII and PSI against the slow, but not the rapid, inactivation. The rapid decline resembled the effect of 1.0 M sorbitol. The presence of both an Na(+)-channel blocker and a water-channel blocker protected PSI and PSII against the short- and long-term effects of NaCl. Salt stress also decreased cytoplasmic volume and this effect was enhanced by the Na(+)-channel blocker. Our observations suggested that NaCl had both osmotic and ionic effects. The osmotic effect decreased the amount of water in the cytosol, rapidly increasing the intracellular concentration of salts. The ionic effect was caused by an influx of Na(+) ions through potassium/Na(+) channels that also increased concentrations of salts in the cytosol and irreversibly inactivated PSI and PSII.

Figure 1

Changes in the photosynthetic oxygen-evolving activity of PSII in intact cells during incubation with NaCl or sorbitol. Cells were incubated in the presence of 0.5 NaCl or 1.0 m sorbitol at 32°C. At designated times, aliquots were withdrawn and oxygen evolution was measured at 32°C after addition of 1.0 mm BQ. The activity that corresponded to 100% was 594 ± 38 μmol O₂ mg⁻¹ Chl h⁻¹. ▵, Control (no addition); ○, 0.5 m NaCl; ▿, 1.0 m sorbitol. Each point and bar represent the average ± se of results from four independent experiments.

Figure 2

Reversibility of the effects of NaCl on the photosynthetic oxygen-evolving activity of PSII in intact cells. Cells were incubated for 0.5, 1, and 5 h with 0.5 m NaCl at 32°C. Aliquots were withdrawn at times indicated by arrows and cells were washed twice with fresh BG-11 medium. The washed cells were incubated at 32°C in BG-11 medium. A small aliquot of each suspension was withdrawn and oxygen evolution was measured at 32°C after addition of 1.0 mm BQ. The activity that corresponded to 100% was 612 ± 48 μmol O₂ mg⁻¹ Chl h⁻¹. ○, Incubation with 0.5 m NaCl; ▴, incubation without NaCl after washing. Each point and bar represent the average ± se of results from four independent experiments.

Figure 3

Effects of a Na⁺-channel blocker (phenytoin) and a water-channel blocker (p-chloromercuriphenyl-sulfonic acid) on the NaCl-induced inactivation of the oxygen-evolving machinery in intact cells. Cells were incubated with 0.5 m NaCl at 32°C in the presence of 100 μm phenytoin, or of 100 μm phenytoin plus 100 μm p-chloromercuriphenyl-sulfonic acid, or in their absence. At designated times, aliquots were withdrawn and oxygen evolution was measured at 32°C after addition of 1.0 mm BQ. The activity that corresponded to 100% was 572 ± 37 μmol O₂ mg⁻¹ Chl h⁻¹. ○, Cells in the presence of 0.5 m NaCl; □, cells in the presence of 0.5 m NaCl and 100 μm phenytoin; ▵, cells in the presence of 0.5 m NaCl and 100 μm p-chloromercuriphenyl-sulfonic acid; ⋄, cells in the presence of 0.5 m NaCl, 100 μm phenytoin, and 100 μm p-chloromercuriphenyl-sulfonic acid. Black symbols correspond to white symbols of the same shapes but in the absence of added NaCl. Each point and bar represent the average ± se of results from five independent experiments.

Figure 4

Changes in PSII-mediated electron transport activity in isolated thylakoid membranes during incubation with NaCl. Thylakoid membranes (10 μg Chl mL⁻¹) were incubated at 32°C in the presence of 0.5 m NaCl and in its absence. At designated times, aliquots were withdrawn and PSII-mediated electron transport activity from water to DCIP (○) and from DPC to DCIP (▿) was measured at 25°C by monitoring the light-induced reduction of DCIP after addition of 0.1 mm DCIP or 0.1 mm DCIP plus 0.5 mm DPC. Solid line, In the presence of 0.5 m NaCl; dashed line, in the absence of NaCl. Each point and bar represent the average ± se of results from five independent experiments.

Figure 5

Changes in the photosynthetic electron transport activity of PSI in intact cells during incubation with NaCl or sorbitol. Cells were incubated in the presence of 0.5 NaCl or 1.0 m sorbitol or in their absence at 32°C. At designated times, aliquots were withdrawn and activity of the PSI complex was monitored at 32°C by measuring the uptake of oxygen after addition of 15 μm DCMU, 0.1 mm DCIP, 5 mm sodium ascorbate, and 0.1 mm MV. The oxygen-uptake activity that corresponded to 100% was 312 ± 46 μmol O₂ mg⁻¹ Chl h⁻¹. ▵, Control (no addition); ○, 0.5 m NaCl; ▿, 1.0 m sorbitol. Each point and bar represent the average ± se of results from four independent experiments.

Figure 6

Effects of a Na⁺-channel blocker (phenytoin) and a water-channel blocker (p-chloromercuriphenyl-sulfonic acid) on the NaCl-induced inactivation of the PSI complex in intact cells. Cells were incubated in the presence of 0.5 m NaCl at 32°C with 100 μm phenytoin or with 100 μm phenytoin plus 100 μm p-chloromercuriphenyl-sulfonic acid, or in their absence. At designated times, aliquots were withdrawn and the activity of PSI was monitored by measuring the uptake of oxygen at 32°C after addition of 15 μm DCMU, 0.1 mm DCIP, 5 mm sodium ascorbate, and 0.1 mm MV. The oxygen uptake activity that corresponded to 100% was 337 ± 45 μmol O₂ mg⁻¹ Chl h⁻¹. ○, Cells in the presence of 0.5 m NaCl; □, cells in the presence of 0.5 m NaCl and 100 μm phenytoin; ⋄, cells in the presence of 0.5 m NaCl, 100 μm phenytoin, and 100 μm p-chloromercuriphenyl-sulfonic acid. Each point and bar represent the average ± se of results from five independent experiments.

Figure 7

Changes in PSI-mediated electron transport activity in isolated thylakoid membranes during incubation with NaCl. Thylakoid membranes (10 μg Chl mL⁻¹) were incubated at 32°C in the presence of 0.5 m NaCl and in its absence. At designated times, aliquots were withdrawn and transport of electrons by PSI from reduced DCIP to MV was measured at 25°C by monitoring the light-induced uptake of oxygen after addition of 15 μm DCMU, 0.1 mm DCIP, 5 mm sodium ascorbate, and 0.1 mm MV. Solid line, In the presence of 0.5 m NaCl; dashed line, in the absence of NaCl. Each point and bar represent the average ± se of results from four independent experiments.

Figure 8

Effects of a Na⁺-channel blocker (phenytoin) and a water-channel blocker (p-chloromercuriphenyl-sulfonic acid) on cytoplasmic volume during incubation with NaCl. Cells were incubated with 0.5 m NaCl at 32°C in the presence of phenytoin or of p-chloromercuriphenyl-sulfonic acid or in their absence. At designated times, aliquots were withdrawn and cytoplasmic volume was determined. The cytoplasmic volume that corresponded to 100% was 0.75 ± 0.05 fL. ○, In the presence of 0.5 m NaCl; □, in the presence of 0.5 m NaCl and 100 μm phenytoin; ⋄, in the presence of 0.5 m NaCl, 100 μm phenytoin, and 100 μm p-chloromercuriphenyl-sulfonic acid. Each point and bar represent the average ± se of results from five independent experiments.

Figure 9

Changes in Na⁺/H⁺ antiport activity during incubation with NaCl or sorbitol. Cells (200 μg Chl mL⁻¹) were incubated in the presence of 0.5 m NaCl or of 1.0 m sorbitol or in their absence. At designated times, 20-μL aliquots were withdrawn and diluted 100-fold with Na⁺-free medium that contained 5 μm acridine orange. Then the Na⁺/H⁺ antiport activity was measured as described previously (Allakhverdiev et al., 1999). The Na⁺/H⁺ antiport activity was calculated from the initial rate of recovery of fluorescence quenching upon addition of NaCl, divided by the difference between the level of fluorescence before the addition of NaCl and the steady-state level of fluorescence 1 min after addition of Triton X-100. ▵, Control (no addition); ▿, in the presence of 1.0 m sorbitol; ○, in the presence of 0.5 m NaCl. Each point and bar represent the average ± se of results from four independent experiments.

Figure 10

A hypothetical model of the NaCl-induced inactivation of PSI and PSII in cyanobacterial cells. ●, Extrinsic proteins of the oxygen-evolving machinery of PSII, namely, the 33-kD protein, cytochrome c₅₅₀, and PsbU; ▴, a protein associated with the PSI complex, namely, plastocyanin or cytochrome c₅₅₃. I, PSI complex; II, PSII complex.