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| Plant Physiol. 2008 May; 147(1): 206–215. doi: 10.1104/pp.108.117218. | PMCID: PMC2330311 |
Copyright © 2008, American Society of Plant Biologists Ammonia Triggers Photodamage of Photosystem II in the Cyanobacterium Synechocystis sp. Strain PCC 6803 1[OA]Miriam Drath, Nicole Kloft, Alfred Batschauer, Kay Marin, Jens Novak, and Karl Forchhammer* 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.) Received February 1, 2008; Accepted February 25, 2008. |
Abstract 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 NH 3 and NH 4+) 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 NH 4+ 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 NH 3 and NH 4+. Whereas ammonia (NH 3), which prevails under alkaline conditions, can diffuse through membranes, the protonated ion (NH 4+) cannot ( Kleiner, 1981). Ammonium at low concentrations is taken up by high affinity transporters of the Amt family, whose activity is regulated by the nitrogen state of the cells ( von Wiren and Merrick, 2004). Energy metabolism of Synechocystis depends on oxygenic photosynthetic electron transport, involving two types of reaction centers: PSI and PSII. The oxidation of water takes place in the oxygen-evolving complex (OEC) of PSII ( Debus, 1992; Diner, 2001; Ferreira et al., 2004). The PSII structure of the cyanobacterium Thermosynechococcus elongatus revealed that the OEC consists of a cubane-like Mn 3CaO 4 cluster and a fourth manganese (Mn) atom linked to the complex by one of its oxygen atoms. The Ca 2+ and Cl − ions, which are associated with the OEC, are participating in deprotonation of the substrate water molecules and in stabilization of the intermediates ( Ferreira et al., 2004; Iwata and Barber, 2004; Barber, 2006). The metal ions of the OEC are tightly liganded to residues of the D1 protein of PSII; the C-terminal domain of D1 is involved in stabilization of the OEC and important for the first steps of the assembly of the Mn 4Ca cluster ( Diner et al., 1991; Cohen et al., 2007). 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; Ohnishi et al., 2005). When the reaction center chlorophyll a (chl a; donor P680) becomes photooxidized, the highly reactive P680 + cannot be reduced from water oxidation and damages the D1 protein ( Hakala et al., 2005, 2006; Ohnishi et al., 2005; Murata et al., 2007). Reactivation of PSII involves an efficient PSII repair cycle; the damaged D1 protein is triggered for removal and degradation by a membrane-bound ATP- and Zn 2+-dependent FtsH protease, which is a member of the AAA protease family. Following removal of inactive D1, newly synthesized D1 protein is assembled in PSII, ensuring proper photosynthetic electron transport ( Aro et al., 1993; Komenda and Barber, 1995; Nixon et al., 2005). Interestingly, the genome of Synechocystis encodes four putative FtsH homologs ( Kaneko et al., 1996). Out of these, the ftsH2 (slr0228) gene was shown to encode the protease responsible for D1 degradation ( Silva et al., 2003; Komenda et al., 2006). Apart from its role in D1 turnover, FtsH2 was also shown to be involved in degradation of various soluble enzymes in Synechocystis ( Stirnberg et al., 2007). This study was initiated to investigate the role of FtsH2 in protein turnover under changing nitrogen regimes. In the course of this investigation, FtsH2 turned out to be essential for acclimation of Synechocystis toward ammonium. Subsequent investigations revealed a novel mechanism of ammonium toxicity in photosynthetic organisms, namely, photosensitizing PSII. |
RESULTS AND DISCUSSION 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 m m NaNO 3), the ftsH2− mutant cells already display a lower photosynthetic activity than wild-type cells ( Mann et al., 2000; Silva et al., 2003). Initial experiments indicated that the ftsH2− mutant is highly sensitive toward ammonium chloride. When exposed to 5 m m NH 4Cl at a photon flux density of 20 μmol photons m −2 s −1, the cells rapidly lost photosynthetic activity (see below) and viability. After 24 h in the presence of NH 4Cl, the culture of the ftsH2− mutant was completely bleached (data not shown) and the cells were unable to reinitiate growth on standard BG-11 plates with nitrate as nitrogen source, whereas cells cultured in parallel in BG-11 in the absence of NH 4Cl were unaffected ( Fig. 1A). Further, in contrast to the wild type, the ftsH2− mutant was unable to grow on BG-11 NH 4Cl-supplemented plates ( Fig. 1B). To study the effect of NH 4Cl on photosynthetic activity, PSII activity was measured by two independent methods ( Fig. 2): (1) by measuring light-saturated rates of oxygen evolution using a Clark electrode; and (2) by using the modulated chlorophyll fluorescence method in a PAM chlorophyll fluorometer, which allows determination of the electron transport rate (ETR) through PSII (for details, see “Materials and Methods”). Following the addition of 5 m m NH 4Cl to the BG-11-grown cultures, a rapid decline in PSII activity reaching zero level within several hours was measured in the ftsH2− mutant by both methods, whereas in nontreated control cultures, PSII activity remained unaffected. The wild type also displayed an initial drop in PSII activity following ammonium chloride addition; however, its PSII activity gradually recovered after 2 to 4 h. Because both PSII measurements reported a similar response of activity, in all subsequent experiments, PSII activity was recorded only by the more convenient PAM measurements.  | Figure 1.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, (more ...) |
 | Figure 2.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 (more ...) |
To distinguish between ammonia (NH 3) or protonated ammonium ions (NH 4+) 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 NH 3 and NH 4+ (pK s of 9.2). When the medium was buffered with 20 m m 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.  | Table I. Effect of pH conditions on PSII activity in NH4Cl-treated ftsH2− mutant Synechocystis cells |
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 NH 4Cl 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 m m NH 4Cl. Higher concentrations led to a gradual decrease of PSII activity, but even at a concentration of 60 m m NH 4Cl, 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 m m NH 4Cl, they lost 40% of their photosynthetic activity and at 5 m m NH 4Cl, 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 m m NH 4Cl at a pH of 8.8, which was used in the subsequent studies.  | Figure 3.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 (more ...) |
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 NH 4Cl 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 m m NH 4Cl 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 NH 4Cl 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 NH 4Cl. 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. Ammonia Tolerance in Wild-Type Cells Depends on a Functional PSII Repair Cycle 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; Nixon et al., 2005). To investigate whether the ammonia sensitivity in the FtsH2-deficient mutant is indeed due to impaired PSII repair, protein synthesis in wild-type and mutant cells, treated with or without NH 4Cl, was inhibited by chloramphenicol. In the absence of de novo protein synthesis, no PSII repair cycle occurs. When protein synthesis was inhibited in NH 4Cl-treated wild-type cells, they rapidly lost PSII activity and became almost as sensitive toward light as the ftsH2− mutant ( Fig. 4A). In the NH 4Cl-exposed ftsH2− mutant, inhibition of protein synthesis did not further accelerate the loss of PSII activity compared to cells in which protein synthesis was not inhibited (compare Figs. 3B and 4A). In contrast, when at the same light intensity NaNO 3 was present instead of NH 4Cl, only a moderate inhibition of PSII was observed in the wild type and in ftsH2− mutant cells ( Fig. 4B).  | Figure 4.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 (more ...) |
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 NH 4Cl, whereas it was much more stable in the presence of NaNO 3 ( 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 NH 4Cl 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.  | Figure 5.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 (more ...) |
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 m m NH 4Cl. 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 NH 4Cl. 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 m m NH 4Cl lead to the cessation of photosynthetic oxygen evolution in less than 1 min (not shown). To assess whether NH 4Cl could affect the repair of photodamaged PSII, cells were first exposed to photoinhibitory conditions for 12 min in the presence of 5 m m NH 4Cl, 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 m m NH 4Cl. As shown in Figure 6B, recovery from photoinhibition was not affected by the presence of NH 4Cl, 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 NH 4Cl has no detrimental effect on the recovery of PSII from photodamage.  | Figure 6.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 (more ...) |
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 NH 4Cl 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 NH 4Cl and remained elevated after 1 h.  | Figure 7.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 (more ...) |
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 NH 4Cl, 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 NH 4Cl-treated ftsH2− mutant. Red light (680 nm, 700 nm, absorbed by chl a) 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 NH 4Cl, nor was the ftsH2− mutant affected, when illuminated in the presence of nitrate ( Fig. 8).  | Figure 8.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 (more ...) |
Photoinhibition was previously suggested to be mediated by two different photosensitizers: in addition to a contribution of chl a, 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 chl a would lead in a second step to the generation of long-lived P 680+ 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 chl a 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. |
CONCLUSION 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). The absence of such a motif suggests that the increased expression of ftsH2 upon ammonium addition is not a response mediated by NtcA but is probably a consequence of increased photoinhibition caused by ammonium. The increased expression of ftsH2 may enhance the capacity of the repair cycle and contribute to ammonium tolerance. The mechanism of ammonia-induced ftsH2 gene expression is intriguing and deserves further investigation. 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), implying that a photoinhibitory process is involved. Second, ammonia was shown in vitro to be a potent inhibitor of the water-splitting reaction in PSII preparations from plants by binding to the Mn cluster ( Britt et al., 2004; Evans et al., 2005). These findings are in perfect agreement with the photosensitizing effect of ammonia described here for Synechocystis and suggest that ammonium toxicity could operate in many oxygenic photosynthetic organisms by photosensitizing PSII. Further biochemical studies on isolated PSII particles are required to clearly elucidate the molecular details of the coaction of ammonia with the blue light/UV absorbing photosensitizer that mediates PSII photodamage. Eventually, such investigations could provide further insights into the various steps involved in PSII photodamage and may help to clarify the role of the OEC in this important biological process. |
MATERIALS AND METHODS 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  Km R; Stirnberg et al., 2007) were used in this study. All cells were grown photoautotrophically in liquid BG-11 medium containing 17.6 m m NaNO 3 ( Rippka, 1988) supplemented with 5 m m NaHCO 3 at 28°C and under continuous light of 10 μmol photons m −2 s −1 from white fluorescent tubes (LUMILUX de Luxe Daylight; Osram). They were constantly shaken at 140 rpm. The mutant was maintained with kanamycin (80 μg mL −1). 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. Assay of Survival in the Presence of Ammonia 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. Measurements of Chlorophyll Concentration The cells (1 mL) were sedimented by centrifugation (20,000 g, 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 (20,000 g, 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). Oxygen Evolution Measurement 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. Measurement of Photosynthetic Activity by PAM Chlorophyll Fluorescence 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; Schreiber et al., 1995). The relative ETR was calculated from the quantum yield (Y) as described previously ( Sauer et al., 2001). Cultures were diluted 1:10 in BG-11 medium before the measurements in a final volume of 2 mL. Monochromatic Light Treatments 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. Gel Electrophoresis and Immunoblot Analysis 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). ftsH2 Transcript Quantification Experiments For analysis of ftsH2 mRNA abundances under different nitrogen sources, Synechocystis wild-type cells grown in BG-11 medium with 17.6 m m NaNO 3 were treated with or without 5 m m NH 4Cl for 60 min. Thereafter, 50 mL of the cultures were harvested on ice by centrifugation (10,000 g, 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). RNA probes were denatured by treatment with formamide. The samples were loaded and separated in a 1.2% (w/v) agarose gel containing formaldehyde and transferred to nylon membrane (Roti-Nylon plus, Roth). DNA probes internal to ftsH2 and rnpB (RNase P subunit B) were obtained by PCR amplification of chromosomal template DNA of Synechocystis using the following primer sets: slr0228 (5′-ATGAAATTTTCCTGGAGAACTG-3′ forward primer and 5′-AAGCTCTAATACGACTCACTATAGGGTGTTGCTGGAACGGCGGAAG-3′ reverse primer) and rnpB (5′-GTGAGGACAGTGCCACAGAA-3′ forward primer and 5′-GGCAGGAAAAAGACCAACCT-3′ reverse primer). The DNA probes were labeled with [ 32P]dCTP (Amersham-Pharmacia) by using the Megaprime DNA labeling kit (Amersham-Pharmacia). The hybridization signals were visualized by exposing the membrane to a phosphorimager screen (K-Imaging Screen; Bio-Rad) and the data were recorded in a phosphorimager (Molecular Imager FX; Bio-Rad). 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). |
Notes |
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