Survival and growth of Synechocystis wild-type and ftsH2⁻ mutant in the presence of ammonium chloride. A, Cultures in mid-exponential growth phase (OD₇₅₀ of 0.4) were supplemented with 5 mm NH₄Cl and further incubated for 5 h or 24 h. As a control, cells were kept without ammonium supplementation for 5 and 24 h. Then, 10 μL of the cell culture was dropped on BG-11 nitrate agar plates and incubated for 7 d (for details, see “Materials and Methods”). B, Prior to ammonium addition, the mid-exponential-grown culture was dropped on BG-11 nitrate agar plate, containing 5 mm NH₄Cl and incubated as in A.

Ammonium tolerance of Synechocystis wild-type and ftsH2⁻ cells. A, NH₄Cl concentration-dependent impairment of PSII activity. Cultures were grown in standard BG-11 medium to an OD₇₅₀ of 0.5 under continuous irradiation with white light at a fluence rate of 10 μmol photons m⁻² s⁻¹. Ammonium chloride was then added to the cultures in different concentrations, and the cultures were further incubated for 5 h in presence of 20 μmol photons m⁻² s⁻¹. Samples were taken for measuring PSII activity (relative ETR, see “Materials and Methods”). B, Fluence rate-dependent damage of PSII in the presence of 5 mm ammonium chloride. Cultures of wild-type and ftsH2⁻ strains were grown in standard BG-11 medium (OD₇₅₀ of 0.5) under illumination with white light at a fluence rate of 10 μmol photons m⁻² s⁻¹. Ammonium chloride (5 mm) was added to the culture at time point zero and incubated with different light intensities (10 [squares], 20 [triangles], or 40 [diamonds] μmol photons m⁻² s⁻¹) or in darkness (circles). Samples were taken at the indicated time points for measuring relative ETR. The data in parts A and B are the means of three independent experiments, with error bars showing the sd.

Effect of ammonium on the degradation of PSII subunit D1. Immunoblot analysis of wild-type and ftsH2⁻ cultures during incubation with NH₄Cl or NaNO₃⁻ and treated with chloramphenicol for time periods as indicated. Cells were grown in standard BG-11 medium under white fluorescent light (10 μmol photons m⁻² s⁻¹) to an OD₇₅₀ of 0.5, then pre-incubated or not with 5 mm NH₄Cl, and 10 min thereafter chloramphenicol (30 μg mL⁻¹) was added (time point 0). The fluence rate of white light was 20 μmol photons m⁻² s⁻¹ during the following incubation period. D1 protein was detected in total protein extracts by immunoblot analysis from samples removed after the indicated time points.

Ammonium-dependent accumulation of ftsH2 transcript. A, Northern-blot analysis of ftsH2 transcript in NH₄Cl-treated Synechocystis wild-type cells. Samples were taken from cells grown in BG-11 medium containing 17.6 mm NaNO₃ as a control (C) or following the addition of 5 mm NH₄Cl for 1 h (NH₃) under illumination with white light (20 μmol photons m⁻² s⁻¹). RNA was isolated and northern blot was performed as described in “Materials and Methods.” The hybridization signals of ftsH2 and rnpB, which serves as a loading control, were visualized by phosphorimaging. B, RNA-RNA slot-blot analysis of ftsH2 transcript in NH₄Cl-treated Synechocystis wild-type cells. Cells were treated as above, except that one sample was already taken after 30 min. RNA was isolated and slot blot was performed as described in “Materials and Methods.” The hybridization signals of ftsH2 and rnpB, which serves as a loading control, were visualized by a chemiluminescence imager workstation.

Effect of ammonium addition on PSII activity. Cultures of wild type (white symbols) and ftsH2⁻ (black symbols) grown in standard BG-11 medium to mid-exponential phase of growth were treated with 5 mm NH₄Cl (squares) at time zero. As control, cells were grown in standard BG-11 medium without addition (circles). Cells were illuminated with 20 μmol m⁻² s⁻¹ of white light, and at the indicated time points, samples were removed for determination of PSII activity by tow different methods. A, Measurement of light-saturated rates of oxygen evolution. B, ETR through PSII by PAM chlorophyll fluorometry as described in “Materials and Methods.” The values are the averages of three independent experiments and the error bars indicate sd.

Inhibition of PSII repair by chloramphenicol. Cultures of wild type (white squares) and ftsH2⁻ mutant (black squares) were grown in standard BG-11 medium illuminated with 20 μmol photons m⁻² s⁻¹ of white light. A, At time point zero, 5 mm ammonium chloride was added or as control, B, no addition occurred. After 10 min of incubation, chloramphenicol (30 μg mL⁻¹) was added (arrow) to all cultures. Samples were taken at the indicated time points and relative ETR was determined. The values represent the mean of three independent experiments, with error bars showing the sd.

Ammonium-dependent PSII photodamage and repair in Synechocystis wild-type cells. A, Synechocystis wild-type cells were diluted in standard BG-11 nitrate medium to an OD₇₅₀ of 0.3. Three-milliliter aliquots were removed into glass test tubes, supplemented (white triangles) or not supplemented (black circles) with 5 mm NH₄Cl, and placed in the light beam of a halogen lamp at a fluence rate of 1,500 photons m⁻² s⁻¹. At indicated time points, 100-μL aliquots were withdrawn for assessment of PSII quantum yield. For this purpose, the 100-μL samples were diluted into 1.9 mL of BG-11 nitrate medium and transferred into the measuring cuvette of a WATER-PAM chlorophyll fluorometer. After 2 min in the dark measuring cell, the measuring beam was switched on for determination of F₀. Saturating light pulses were applied to determine F_m and to quantify quantum yield of PSII (Y) from the F₀−F_m/F_m ratio as described by Schreiber et al. (1995). B, The 8-mL samples of Synechocystis wild-type cells were photodamaged for 12 min in the presence of 5 mm NH₄Cl as described in A. The damaged cells were gently transferred to ammonium-free, nitrate supplemented BG-11 medium by 5 min centrifugation at 1,400g, twice washing the pellet with 8 mL ammonium-free BG-11 nitrate medium. After the last centrifugation, the cells were resuspended in 8 mL BG-11 nitrate medium. To one sample, chloramphenicol was added (30 μg/mL final concentration) to inhibit novel protein synthesis (black squares). The samples were incubated in Erlenmeyer flasks with a photon fluence rate of 20 μmol photons m⁻² s⁻¹. After different time points as indicated, 100-μL aliquots were withdrawn for determination of PSII quantum yield as described above. After 40 min, at the onset of recovery in the absence of chloramphenicol, 5 mm NH₄Cl was added to one sample (indicated by the arrow), and the samples were further incubated to record the recovery from photoinhibition in the absence (black circles) or presence (white triangles) of NH₄Cl. Triplicate experiments yield almost identical results (sd < 5%). All experimental steps were performed at 25°C.

Wavelength-dependent inhibition of PSII activity in the presence or absence of ammonium. Exponentially grown cultures of ftsH2⁻ were treated with 5 mm NH₄Cl (black circles). For controls, exponentially grown cultures of wild-type cells were treated with 5 mm NH₄Cl (black squares) and ftsH2⁻ culture was grown in standard BG-11 medium containing 17.6 mm NaNO₃ (white circles). Cells were illuminated with the indicated wavebands at 5 μmol m⁻² s⁻¹ for 240 min before relative ETR was determined. The values are the means of three independent experiments with error bars showing the sd. The 100% values correspond to the values determined at 462 nm.