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First published online March 5, 2008; 10.1104/pp.108.117218 Plant Physiology 147:206-215 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Ammonia Triggers Photodamage of Photosystem II in the Cyanobacterium Synechocystis sp. Strain PCC 68031,[OA]Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, D–35392 Giessen, Germany (M.D., N.K., K.F.); Fachbereich Biologie-Molekulare Pflanzenphysiologie, Philipps-Universität Marburg, D–35032 Marburg, Germany (A.B.); Institut für Biochemie, Universität zu Köln, D–50674 Cologne, Germany (K.M., J.N.); and Lehrstuhl für Mikrobiologie/Organismische Interaktionen, Eberhard-Karls-Universität Tübingen, D–72076 Tuebingen, Germany (K.F.)
Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.
Ammonium (in this communication, the term ammonium denotes both NH3 and NH4+) is a molecule of fundamental importance to autotrophic organisms because the assimilation of nitrogen into organic material occurs through ammonium-dependent reactions. Ammonium is either directly taken up from exogenous sources or the organisms employ reaction pathways that generate ammonium from other nitrogenous compounds. On the other hand, ammonium is known to be toxic for many organisms, in particular to plants and oxygenic photosynthetic microorganisms. A variety of herbicides (such as glufosinate) act as inhibitors of the key enzyme of ammonium assimilation, Gln synthetase, thereby leading to the accumulation of toxic concentrations of ammonium. It is widely believed that ammonium toxicity is caused by the uncoupling of photophosphorylation; however, this assumption could be proven to be wrong for whole organisms (Zhu et al., 2000  ; Britto and Kronzucker, 2002). More recent investigations in higher plants suggested that futile transmembrane NH4+ cycling might be involved in ammonium sensitivity (Britto et al., 2001). Furthermore, ammonium toxicity is more pronounced under high light conditions, an observation that has not been explained so far (Zhu et al., 2000; Britto and Kronzucker, 2002). In contrast to plants, heterotrophic bacteria seem to be highly resistant to ammonium because they were shown to tolerate ammonium concentrations until they suffer osmotic stress (Müller et al., 2006). Cyanobacteria, a domain of bacteria that performs oxygenic photosynthesis, display metabolic properties similar to plants. Although, to our knowledge, ammonium sensitivity has so far not been systematically investigated in cyanobacteria, higher concentrations of ammonium are toxic to many strains.
The cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter called Synechocystis) is widely used to study fundamental processes of photosynthetic metabolism. As for many cyanobacteria, ammonium is the preferred nitrogen source for Synechocystis. The uptake of ammonium is affected by the pH-dependent equilibrium between NH3 and NH4+. Whereas ammonia (NH3), which prevails under alkaline conditions, can diffuse through membranes, the protonated ion (NH4+) cannot (Kleiner, 1981
The D1 protein of PSII itself is sensitive to light-induced damage, leading to loss of PSII function. The mechanistic basis of PSII photoinhibition has been an issue of controversial discussion. Recent evidence strongly suggests that photooxidation of the OEC is the initial reaction, which together with subsequent reactions leads to photodamage. The absorption of short wavelength blue and UV light by the Mn cluster causes Mn release from OEC and thereby its inactivation (Hakala et al., 2005
The ftsH2 (slr0228) Gene Is Required for Ammonium Tolerance of Synechocystis
According to the participation of FtsH2 in PSII repair, the ftsH2– mutant is sensitive to higher photon flux densities (above 40 µmol photons m–2 s–1) in standard BG-11 medium (nitrate supplemented) and is affected in pigmentation as well as in photosynthetic electron transport. Therefore, under standard growth conditions (liquid cultures in carbonate-buffered BG-11 medium, containing 17.6 mM NaNO3), the ftsH2– mutant cells already display a lower photosynthetic activity than wild-type cells (Mann et al., 2000
To distinguish between ammonia (NH3) or protonated ammonium ions (NH4+) as causative agents of PSII activity loss in the ftsH2– mutant, the experiment was performed in the presence of different buffer and pH conditions (see Table I ), making use of the pH-dependent equilibrium between NH3 and NH4+ (pKs of 9.2). When the medium was buffered with 20 mM HEPES to a pH of 8.2, the mutant cells maintained 63% of their initial PSII activity after 5-h incubation. More alkaline conditions led to a stronger decrease of PSII activity, and at pH 8.8, PSII activity was completely lost after 5-h incubation. Controls carried out under the same illumination conditions showed that the PSII activity in the ftsH2– mutant was unaffected in the presence of nitrate or nitrite under any pH conditions tested (Table I shows the most alkaline condition tested). Apparently, the loss of PSII activity that was observed in the ftsH2– mutant correlates with the free ammonia concentration (Table I). When ammonium is present in the protonated form, its uptake is accomplished by ammonium transporters of the Amt family, whose activity is regulated by the nitrogen state of the cells. Ammonium-rich conditions cause inhibition of Amt activity (Montesinos et al., 1998 ), which avoids the accumulation of excess intracellular ammonium. However, when ammonia freely diffuses through membranes, the cells are not able to control its uptake. Under those conditions, ammonia causes damage to PSII in the ftsH2– mutant.
To determine the concentration of ammonia that can be tolerated by wild-type and mutant cells, experiments carried out as outlined above were performed in the presence of different concentrations of NH4Cl at a constant pH of 8.8, where a substantial amount of ammonium is present as nonprotonated ammonia. Figure 3A shows that the PSII activity in Synechocystis wild-type cells was not affected by up to 10 mM NH4Cl. Higher concentrations led to a gradual decrease of PSII activity, but even at a concentration of 60 mM NH4Cl, the cells maintained an appreciable PSII activity of more than 20% compared to untreated cells. By contrast, the mutant cells were highly susceptible, because already in the presence of 1 mM NH4Cl, they lost 40% of their photosynthetic activity and at 5 mM NH4Cl, no more variable chlorophyll fluorescence could be measured. Therefore, the optimal conditions to study the effect of ammonia on the FtsH2 mutant turned out to be 5 mM NH4Cl at a pH of 8.8, which was used in the subsequent studies.
Ammonia-Mediated Damage of PSII in the FtsH2-Deficient Mutant Is Light Dependent To unravel whether the observed loss of PSII function in the presence of NH4Cl in the ftsH2– mutant is a light-dependent process, the time course of PSII inhibition was investigated at three different photon fluence rates and in the dark (Fig. 3B). During exposure of the ftsH2– mutant to 5 mM NH4Cl in the presence of 10 µmol photons m–2 s–1 of white light, the PSII activity dropped to 40% after 60 min and was almost completely lost after 220 min. In the presence of 20 µmol photons m–2 s–1, the cells already lost 70% of their initial ETR during the first 40 min, and after 180 min the PSII activity was completely lost. In the presence of 40 µmol photons m–2 s–1, loss of PSII activity was even faster. However, when the mutant cells were exposed to NH4Cl in the absence of light, the cells were almost not affected. In contrast to the mutant, the wild-type cells were only transiently impaired in PSII activity following the addition of NH4Cl. After an initial drop in the first 60 min by 30% to 50% compared to the untreated control, the cells overcame the stress and recovered photosynthetic activity nearly completely after 2 to 3 h. The initial response suggests that ammonia damages PSII in the wild type as well; however, in the presence of functional FtsH2, PSII activity can be efficiently reactivated. Furthermore, the damaging effect of ammonia is clearly enhanced by increasing light intensity, suggesting that photodamage and FtsH2-dependent PSII repair is responsible for the response of PSII activity toward ammonia.
The degradation of the D1 protein by the FtsH2 protease is a key process in the PSII repair cycle, which mediates recovery from photoinhibition (Aro et al., 1993
To directly reveal the effect of ammonia on the degradation of D1, western-blot analysis with antibodies against PsbA (D1 protein) were performed in chloramphenicol-treated wild-type and mutant cells. In wild-type cells, the D1 protein was completely degraded in the presence of NH4Cl, whereas it was much more stable in the presence of NaNO3 (Fig. 5 ). This feature indicates that ammonia indeed accelerates D1 destruction, as visualized by the complete D1 removal in wild-type cells. As expected, the FtsH2-deficient mutant was not able to degrade the D1 protein, and therefore the protein levels remained high in both nitrogen regimes (Fig. 5), confirming that the rapid disappearance of D1 in the wild type in the presence of NH4Cl was due to FtsH2-mediated activity. Altogether, this analysis shows that ammonia indeed accelerates PSII photodamage even at low light intensity (20 µmol photons m–2 s–1), an effect that becomes evident only when the PSII repair cycle is inhibited, either by inhibiting de novo protein synthesis or by deleting the FtsH2 protease.
Additional evidence that ammonia enhances photoinhibition by stimulating photodamage and not by impairing the repair of photodamage was obtained by a set of experiments performed with Synechocystis wild-type cells. Cells were exposed to photoinhibitory light conditions (1,500 photons m–2 s–1) in the absence or in the presence of 5 mM NH4Cl. Samples were taken after different time points to determine the quantum yield of PSII (Y) by PAM chlorophyll fluorescence, which is a reliable indicator of photoinhibition. As shown in Figure 6A , photoinhibition (as revealed by the decrease in PSII quantum yield) is clearly accelerated in the presence of NH4Cl. A similar result was obtained by measuring oxygen evolution under photoinhibitory high light conditions; under light conditions in which oxygen evolution decayed within 15 min, the addition of 5 mM NH4Cl lead to the cessation of photosynthetic oxygen evolution in less than 1 min (not shown). To assess whether NH4Cl could affect the repair of photodamaged PSII, cells were first exposed to photoinhibitory conditions for 12 min in the presence of 5 mM NH4Cl, where the PSII quantum yield had dropped to zero. Then, the cells were transferred to fresh medium and allowed to recover from photoinhibition at low light intensities (20 µmol photons m–2 s–1) in the presence or absence of 5 mM NH4Cl. As shown in Figure 6B, recovery from photoinhibition was not affected by the presence of NH4Cl, whereas the addition of chloramphenicol completely inhibited the recovery from photodamage, indicating that recovery required the synthesis of novel protein. In conclusion, this experiment demonstrates that NH4Cl has no detrimental effect on the recovery of PSII from photodamage.
Because the FtsH2 protease seems to be crucial for ammonia tolerance of Synechocystis wild-type cells, the expression of the ftsH2 gene in response to NH4Cl treatment was investigated. The abundance of ftsH2 mRNA was analyzed by northern-blot DNA-RNA and slot-blot RNA-RNA hybridization, revealing a significantly increased level of the ftsH2 (slr0228) transcript in ammonium-treated cells in contrast to cells grown on nitrate (Fig. 7 ). The ftsH2 transcript abundance was increased approximately 2.5-fold (densitometrically estimated from RNA-RNA hybridization signals) after the addition of NH4Cl and remained elevated after 1 h.
The Primary Target of Ammonia-Induced PSII Photoinhibition May Be the OEC To clarify the mechanism of coaction of light and ammonia in PSII destruction, a rough action spectrum of ammonia-induced photoinhibition of PSII was taken. Mutant cells, and wild-type cells as control, were illuminated with monochromatic light of different wavelengths from 360 to 700 nm in the presence of NH4Cl, and the photosynthetic activity of PSII was monitored after 240 min of irradiation. As a further control, the ftsH2– mutant illuminated in the presence of nitrate was used. As shown in Figure 6, irradiation with light from 440 to 700 nm led to only a slight decrease of PSII activity (4%–25% inhibition) in the NH4Cl-treated ftsH2– mutant. Red light (680 nm, 700 nm, absorbed by chla) showed no effect on PSII destruction, whereas orange light of 621 nm, absorbed by phycobiliproteins, caused a slight activity loss of 25% in the ftsH2– mutant. The strongest effects, however, were observed with short wavelength blue and near UV (UV-A) irradiation. Illumination with light at 421 nm, 393 nm, and 360 nm caused significantly increasing damage of PSII, with PSII inhibition rates of 30%, 60%, and 85%, respectively (Fig. 8 ). As expected from their ammonium-tolerance, PSII activities in the wild-type cells were not severely affected by the addition of NH4Cl, nor was the ftsH2– mutant affected, when illuminated in the presence of nitrate (Fig. 8).
Photoinhibition was previously suggested to be mediated by two different photosensitizers: in addition to a contribution of chla, a photosensitizer that absorbs blue/UV light is responsible for the high sensitivity of PSII toward UV-A and UV-B light (Greenberg et al., 1989 ; Ohnishi et al., 2005). The blue light/UV photosensitizer was originally suggested to be the plastosemiquinone anion radical (Greenberg et al., 1989). However, the blue light/UV sensitivity of PSII in isolated thylakoids disappeared following the removal of the OEC together with its Mn ions, despite that plastosemiquinone was still present (Hakala et al., 2005). More recently, it was suggested that blue light/UV absorption by the OEC causes release of Mn ions and subsequent photoinhibition by destruction of D1, in agreement with the similarity of the absorption spectrum of a Mn(III/IV) model compound and the action spectrum of photoinhibition (Hakala et al., 2005, 2006; Ohnishi et al., 2005). Following the destruction of the OEC, excitation of chla would lead in a second step to the generation of long-lived P680+ radicals that ultimately damages D1. Apparently, the coaction of ammonia with light is confined to the blue/UV light-absorbing photosensitizer, whereas the lack of response to red light (680 and 700 nm) rules out a direct coaction of ammonia with the reaction center chla molecules. If the blue/UV light photosensitizer is indeed the Mn cluster in the OEC as suggested by Hakala et al. (2005) or Ohnishi et al. (2005), then the direct target of ammonia is the OEC. This suggestion is in accordance of the action spectrum of ammonia-triggered photodestruction with the action spectrum of OEC photodamage (Hakala et al., 2005; Ohnishi et al., 2005). Furthermore, biochemical studies on isolated PSII particles showed previously that ammonia is a competitive inhibitor of the water-splitting reaction. Ammonia is a known inhibitor of water oxidation because it binds to the OEC as structural analog of the substrate water (Evans et al., 2005; Fang et al., 2005), and it was proposed that ammonia is incorporated into the Mn complex as a bridging ligand (Britt et al., 2004). These biochemical studies, however, did not consider physiological consequences of ammonia binding to the OEC. Our observation that ammonia causes enhanced sensitivity of PSII toward light that is absorbed by the Mn cluster leads us to suggest that ammonia binding to the Mn complex could be the reason for ammonia-enhanced photodamage in PSII.
This investigation revealed the importance of the PSII repair cycle in allowing phototrophic growth in the presence of millimolar concentrations of ammonia under alkaline conditions in the cyanobacterium Synechocystis. In general, cyanobacteria are alkaliphilic and grow readily in media up to pH values of 10. Alkaline conditions with elevated ammonia concentrations might arise in natural environments, e.g. from urea decomposition in the presence of urease-positive bacteria; in the natural habitat of Synechocystis, such conditions might occur temporarily. Because free ammonia diffuses through biological membranes, its uptake cannot be controlled by the global nitrogen regulatory system, and, consequently, ammonia can reach intracellular concentrations, which are potentially harmful to cells. In the absence of the FtsH2 protease, the cells are highly susceptible toward these ammonia concentrations in a light-dependent manner, whereas the FtsH2-bearing wild-type cells are able to tolerate appreciable concentrations of ammonia in the light. Clear evidence is presented that shows the PSII activity in Synechocystis is affected by ammonia through accelerated photodamage and not through impaired PSII repair. Whereas the wild type is able to compensate the increased damage by elevating the expression of ftsH2 and by carrying out an efficient PSII repair cycle, the FtsH2-deficient mutant is unable to do so and, therefore, dies upon prolonged incubation with ammonium in the light. In agreement, prevention of the repair cycle by inhibition of de novo protein synthesis causes similar ammonium sensitivity in the wild type as in the ftsH2– mutant.
Examination of the upstream region of the ftsH2 gene failed to reveal a consensus-binding motif for NtcA, the global nitrogen control transcription factor in cyanobacteria. All genes regulated by NtcA (whereby NtcA can act as inducer or repressor depending on the promoter architecture) display a characteristic DNA sequence motif (Herrero et al., 2004
Although this study reveals a novel target for ammonium toxicity, it remains to be elucidated whether ammonium-triggered PSII photodestruction can be generalized to all oxygenic photosynthetic organisms. Two so-far unconnected observations argue in favor of a general significance. First, the toxicity of ammonia in higher plants is frequently more pronounced at high light intensity (Britto and Kronzucker, 2002
Growth of Cyanobacteria
The transformable wild-type strain of Synechocystis sp. strain PCC 6803 (hereafter referred to as Synechocystis) and the derived ftsH2– mutant (slr0228::KmR; Stirnberg et al., 2007 For conditioning with ammonium, the mid-exponential-phase cultures were treated with 5 to 60 mM NH4Cl and illuminated with 10 to 40 µmol photons m–2 s–1 or kept in the dark, as indicated. BG-11 medium was buffered with 20 mM HEPES or 20 mM MES to a final pH of 8.2 to 8.8, as indicted. For protein synthesis inhibitor studies, chloramphenicol (30 µg mL–1) was added to the cell culture. Growth of the cultures was monitored by measuring the optical density (OD) at 750 nm.
To assay the ability of the cells to survive in the presence of ammonia for various time periods, 10 µL of the ammonium-treated cell culture (OD750 of 0.4) were dropped on a BG-11 agar plate, containing 17.6 mM NaNO3, 0.3% sodium thiosulfate, and supplemented with 5 mM NaHCO3 (for the mutant the agar plates were supplemented with kanamycin [50 µg mL–1]). The plates were incubated at 28°C and under continuous light of 20 µmol photons m–2 s–1 from white fluorescent tubes (LUMILUX de Luxe Daylight; Osram). After 7 d of incubation, the plates were photographed. To assay the ability to grow in the presence of ammonia, BG-11 nitrate agar plates were prepared that were supplemented with 5 mM ammonium chloride. Ten microliters of mid-exponential-phase cell culture at the OD750 of 0.4 were dropped on the agar plate and incubated as described above.
The cells (1 mL) were sedimented by centrifugation (20,000g, 3 min, room temperature) and resuspended in 1 mL 99% methanol. The suspension was incubated for 30 min on ice and in the darkness. Afterward the cell debris was sedimented (20,000g, 1 min, room temperature) and the clear supernatant was used for absorption measurements (665 nm). The concentration of chlorophyll was calculated from absorbance of the supernatant at 665 nm according to Mackinney (1941)
Photosynthetic oxygen evolution was measured in vivo using a Clark-type oxygen electrode (Hansatech DW1). Light was provided from a high-intensity white-light source (Hansatech L2). Oxygen evolution was measured at room temperature of 2 mL of mid-exponential-phase cell culture at the OD750 of 0.4 using the Hill reaction. The culture was illuminated with 3,000 µmol photons m–2 s–1 in the presence of an artificial electron acceptor system of 1 mM 2,5-dimethyl-p-benzoquinone and 1 mM potassium ferricyanide.
PSII activity was analyzed in vivo in a WATER-PAM chlorophyll fluorometer (Walz GmbH). The chlorophyll fluorescence (F0) of dark-adapted cells was determined and the effective quantum yield (Y) of PSII photochemistry with actinic light of 655 nm (55 µmol photons m–2 s–1) was determined with the saturation pulse method (Genty et al., 1989
Five milliliters of mid-exponential-phase cell cultures were illuminated in petri dishes with monochromatic light applied through double interference filters (Schott). The half bandwidth of all filters was 14 nm or less. The fluence rate during monochromatic light treatments was set to 5 µmol m–2 s–1. Energy fluence rates were determined using a P-2000 optometer combined with a calibrated PD-9306-2 detector (Gigahertz) and photon fluence rates calculated from these measurements.
Cell extracts were prepared by vortexting the cells with glass beads (110-µm diameter) followed by centrifugation (3,500g, 5 min, 4°C). Protein composition was assessed by electrophoresis in a denaturing 12.5% SDS-PAGE containing 6 M urea. Then 7 µg of total protein per lane was loaded. After electrophoresis, proteins were blotted onto nitrocellulose membranes. The membrane was incubated with chicken anti-PsbA antibody (AgriSera) followed by incubation with rabbit anti-chicken IgY antibody (AgriSera) and the bands visualized by colorimetric reaction using LumiLight for western blotting (Roche).
For analysis of ftsH2 mRNA abundances under different nitrogen sources, Synechocystis wild-type cells grown in BG-11 medium with 17.6 mM NaNO3 were treated with or without 5 mM NH4Cl for 60 min. Thereafter, 50 mL of the cultures were harvested on ice by centrifugation (10,000g, 10 min, 4°C) and used for RNA extraction. RNA for northern-blot analysis was isolated by using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Then 15 µg of total RNA was used for northern-blot analysis according to the method as described (Sambrook et al., 1989 For slot-blot RNA-RNA hybridization analysis, RNA was isolated from samples treated as above except that one sample was removed form NH4Cl cultures already after 30 min. Cells were broken by phenol chloroform treatment and RNA extracted by subsequent use of the NucleoSpin RNA II kit (Macherey und Nagel) according to the manufacturer's instructions. Two micrograms of total RNA was denatured by treatment with formamide, loaded onto a nitrocellulose membrane, and covalently bound by UV cross-linking. Probes were obtained by PCR amplification of DNA probes internal to ftsH2 and rnpB with chromosomal DNA of Synechocystis as template and ftsH2-specific and rnpB-specific primers, with the 3' primers containing the T7 promoter sequence that was used for in vitro transcription using the DIG RNA Labeling kit SP6/T7 (Roche): rnpB_5', 5'-GTGAGGACAGTGCCACAGAA-3'; rnpB_3', 5'-GGGCCCTAATACGACTCACTATAGGGGGCAGGAAAAAGACCAACCT-3'; ftsH2_5', 5'-ATGAAATTTTCCTGGAGAACTG-3'; and ftsH2_3', 5'-GGGCCCTAATACGACTCACTATAGGGTGTTGCTGGAACGGCGGAAG-3'. The RNA-RNA hybridization was performed for 16 h at 52°C and the signals were visualized by using Anti-Digoxigenin-AP FAB fragments and CSPD as substrate (Roche) and recorded by the LAS-1000 imager (Fuji). Signal intensity was estimated by using the PC-BAS program (Raytest). To exclude the unspecific binding of the ftsH2 probe to other ftsH mRNAs, we performed a control experiment using plasmid DNA containing each of the four ftsH genes. One microgram of plasmid DNA was blotted onto nitrocellulose membrane and used for hybridization with the ftsH2 probe. Only in the case of the plasmid with the cloned ftsH2 gene was a hybridization signal obtained, indicating the strict specificity of the used RNA probe (data not shown). Received February 1, 2008; accepted February 25, 2008; published March 5, 2008.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. Fo195/6–1 and Fo195/4).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Karl Forchhammer (karl.forchhammer{at}uni-tuebingen.de).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.117218 * Corresponding author; e-mail karl.forchhammer{at}uni-tuebingen.de.
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ABSTRACT
RESULTS AND DISCUSSION
