Physiol. Mol. Biol. Plants (2007), 13(2) : 163-167.
Short Communication
Nitrate-Induction of Nitrate Reductase and its Inhibition by Nitrite and Ammonium Ions in Spirulina platensis ★ Pamela Jha 1, Ahmad Ali 1 and Nandula Raghuram1,2★ of Life Sciences, University of Mumbai, Vidyanagari, Mumbai – 400 098, India 2 School of Biotechnology, GGS Indraprastha University, Kashmiri Gate, Delhi – 110 006, India 1Department
Nitrate assimilation in Spirulina platensis is not well characterized, despite its direct link with its high protein content. In other non-nitrogen fixing cyanobacteria, the reported role of nitrate, nitrite and ammonium ions in the regulation of nir operon and nitrate reductase (NR) activity varies considerably. We report here that NR activity in Spirulina is diminished upon withdrawal of nitrate from the medium, and is fully restored within two hours of resupplying nitrate, indicating substrate induction. Nitrite and ammonium, the downstream metabolites of nitrate assimilation, significantly inhibited nitrate-induced NR activity in a concentration dependent manner. These results in Spirulina indicate the need to reassess the role of nitrate in the expression of NR in this and other non-N 2-fixing, nitrate-assimilating cyanobacteria.
INTRODUCTION Spirulina platensis is a filamentous, nitrate-utilizing, non-nitrogen fixing, photosynthetic cyanobacterium with tremendous importance in nutritional, industrial and environmental biotechnology (Vonshak, 1997). It is best known for its high protein content (60-70 % by dry wt.), but it is not clear how the organism steers its nitrogen metabolism to produce so much protein. Nitrate assimilation is the major process of nitrogen acquisition in cyanobacteria (Guerrero et al. 1981). It is transported into the cells by an active transport system and reduced to ammonium by the sequential action of nitrate reductase (NR) and nitrite reductase (NiR) prior to fixation into amino acids through the glutamine synthetase (GS) pathway. The process of nitrate assimilation and its regulation has been widely studied in higher plants and fungi (Raghuram et al., 2006, Lochab et al., 2007), algae (Berges, 1997), bacteria (Lin and Stewart, 1998) and to a lesser extent in cyanobacteria (Hererro et al., 2001). Different responses to nitrate have been reported in literature between the non-nitrogen fixing (nitrate utilizing) and N 2 fixing cyanobacteria. In non-nitrogen fixers, high levels of NR have been reported in media lacking combined nitrogen (Palod et al., 1990), suggesting constitutive expression, whereas in N 2
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fixers, nitrate assimilation is known to be a substrateinducible system (Flores and Herrero, 1994). However, positive regulation of nitrate assimilation (nir operon) by nitrite has been reported in nitrate-limited conditions in unicellular, non-N 2 fixing Synechococcus sp., strain PCC 7942 (Kikuchi et al., 1996, Aichi et al., 2004). There is a paucity of information regarding the role of nitrate and nitrite in other non-N 2 fixing cyanobacteria in general and Spirulina in particular. Ammonium, the end product of the nitrate reduction pathway, acts as a very effective antagonist of nitrate assimilation and different inhibitory effects of this ion have been shown on nitrate uptake, NR synthesis and activity (Losada and Guerrero, 1979; Muro-pastor et al., 2005). The presence of NH 4+ in the culture media effectively prevents nitrate utilization. For example, in Synechococcus sp., it has been reported that NH 4 + ion acts as an effective antagonist to nitrate utilization causing repression of a 48-kDa nitrate transport element binding protein (Madueno et al., 1988), NR (Herrero et al., 1981), NiR (Herrero and Guerrero, 1986) and GS (Vega-Palas et al., 1990). Recently, Kobayashi et al. (2005) have reported that NR and NiR activities were insensitive to ammonium inhibition in Synechocystis sp. strain PCC 6803. Earlier, they reported that the ABC-type nitrate and nitrite bispecific transporter (encoded by the nrtABCD genes) and NR were completely inhibited by ammonium ions in Synechococcus elongatus strain PCC 7942. (Kobayashi et al., 1997). These reports indicate that different nitrateutilising cyanobacteria may respond differently to ammonium ions. Further, there is no information on
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the effect of ammonium ions on nitrate assimilation in Spirulina to the best of our knowledge. Therefore, we have examined the effect of nitrate and its downstream metabolites on NR activity in Spirulina platensis as a basis for further characterization of nitrate assimilation in this organism. MATERIALS AND METHODS Growth conditions Pure cultures of Spirulina platensis (strain ARM 729) were grown in shake flasks in Zarouk’s medium at pH 10 with the following composition: Macroelements (g/ l) consisted of NaCl: 1.0, CaCl 2 : 0.04, NaNO 3 : 2.5, FeSO 4 .7H 2 0: 0.01, EDTA(Na): 0.08, K 2 SO 4 : 1.0, MgSO 4.7H 2O: 0.2, NaHCO3: 18, K 2HPO4: 0.5 (added in the same order). Microelements (mg/l) consisted of H 3BO 3 : 2.86, Co(NO 3) 2 : 0.049, CuSO 4: 0.079, MnCl 2: 1.81, NaMoO 4 .2H 2 0: 0.39, ZnSO 4 .7H 2 0: 0.22. The cultures were grown on a shaker at 30±2 o C under continuous white light illumination (4 Klux) for 8-9 days, till they reached the exponential phase. Treatments All treatments were done using exponentially growing cultures (9 day old). 100 ml of culture was taken for each treatment and control. The cultures were washed twice with ice-cold sodium bicarbonate buffer (10 mM, pH 10.5). The pellets were resuspended in a treatment medium containing appropriate amounts of NaNO 3 , NaNO2, NH 4Cl or combinations of NaNO3 with NaNO 2 or NH 4Cl in Zarouk’s medium. The control media had either no nitrogen source or optimum nitrate concentration. The controls and treated samples were kept on a shaker under continuous white light illumination (4 Klux) at 30 °C for an appropriate period of time. At the end of the specified time interval, 50 ml of cultures were taken from each flask, washed twice with bicarbonate buffer (20 mM, pH-10.5) and crude extract was prepared as described below. Preparation of crude extract for enzyme assay 50 ml of exponentially growing culture was centrifuged at 15,000 RPM at 25 oC for 10 min. The pellet was washed twice with 50 ml of ice-cold sodium bicarbonate buffer (10 mM, pH 10.5) and centrifuged at 15,000 RPM for 10 minutes at 25 oC. The pellet was weighed and resuspended in an ice-cold extraction buffer containing sodium bicarbonate buffer (100 mM, pH 10.5), glycerol (20 %), magnesium chloride (1 mM) and DTT (1 mM). The tissue to buffer ratio was 1:5 (w/v). The suspension was sonicated at 4 o C for 5 minutes (10 sec on, 30 sec off) at 18 KHz. using MSE Soniprep 150. The sonicated sample was centrifuged at 14,000 rpm for 15 minutes at 4 °C. The supernatant was transferred into a pre-cooled microfuge tube and used for enzyme assays.
Nitrate reductase assay Spirulina NR activity was measured using dithionitereduced methyl viologen as an artifical electron donor (Manzano et al. 1976). The reaction mixture consisted of sodium carbonate/bicarbonate buffer (100 mM, pH 10.5), 20 mM KNO 3, 4 mM methyl viologen, 10 mM sodium dithionite freshly prepared in 0.3 mM sodium bicarbonate, and 50 µg of crude extract protein in a total reaction volume of 0.4 ml. The reaction mixture was incubated for 10 minutes at 25 o C. The blanks contained all the assay components except methyl viologen. The reaction was stopped by adding 0.6 ml of 1:1 (v/v) mixture of sulfanilamide (1 % w/v in 3 N HCl) and NED (0.1 % w/v). The pink color developed was measured spectrophotometrically at 540 nm. The amount of nitrite formed was calculated from a standard curve plotted using the A 540 values obtained from known amounts of nitrite. NR activity was defined as nmoles of nitrite produced per ml extract per hour. The enzyme assay was set up in triplicates and the mean data from at least two independent experiments was plotted along with standard errors as shown in the results. Protein estimation was carried out using Bradford’s method for calculation of specific activities. RESULTS AND DISCUSSION Regulation of nitrate reduction by changes in the activities of the enzymes envolved is extremely important in the control of the overall process of nitrate assimilation, in order to prevent accumulation of downstream metabolites, which could be toxic to the cell. However, as compared to higher plants, the emphasis in cyanobacteria has traditionally been on N 2-fixation and hence nitrate assimilation has received relatively lesser attention. Nitrate is known to act as an inducer of nitrate uptake and assimilation in higher plants, algae, fungi and bacteria, including N 2 -fixing cyanobacteria (Raghuram et al., 2006; Lochab et al., 2007; Berges, 1997; Lin and Stewart, 1998). But in nonN 2 fixing, nitrate-utilising cyanobacteria, the role of nitrate as an inducer seems to vary significantly in the few organisms in which it has been studied (Herrero et al., 2001; Kikuchi et al., 1996). There are no studies on the roles of nitrate and its downstream metabolites (nitrite and ammonium ions) in the regulation of nitrate assimilation in Spirulina, hence this report on S. platensis. Nitrate induction Dose-dependent response is a typical feature of regulation. In this study, experiments were conducted to determine the optimum nitrate concentration required for maximum NR activity using NaNO 3 in the range of 0-60 mM as described in Materials and Methods. The data presented in Fig. 1 shows that 30
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Figure 1 : Effects of Nitrate Concentration on NR Activity in Spirulina. Exponential phase Spirulina culture grown for 9-days was harvested, washed and resuspended in media containing different concentrations of NaNO 3 (mM) as indicated. Samples were collected after 8 hrs of treatment and processed for extraction and NR assay as described in Materials and Methods. The mean data from 3 different experiments are shown along with std. error bars.
mM NaNO 3 was the optimum nitrate concentration required for maximum NR activity. This dosedependent response also indicated the possible role of nitrate as an inducer. Nitrate induction studies were conducted using cultures grown on Zarouk’s medium containing 30 mM nitrate, washed and resuspended in fresh media without any form of nitrogen. Upon nitrate removal, NR activity dropped to below 38 % of the control level by 8 hrs (Fig. 2). Upon re-addition of nitrate (30 mM NaNO 3) into the medium, NR activity was restored to normal levels within 2 hrs, indicating that nitrate could induce NR activity in Spirulina.
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Figure 2 : Nitrate Induction of NR Activity in Spirulina. Exponential phase culture was washed and resuspended under sterile conditions in media containing either no N-source (Minus N) or 30 mM NaNO 3 (Control) . Samples were collected after every 2 hrs under sterile conditions and NR activity (NRA) assayed as described in Materials and Methods. After 8 hrs, 30 mM KNO 3 was added to the ‘Minus N’ culture (see arrow) and NRA assayed every 2 hrs. The mean data from 3 different experiments are shown along with std. error bars.
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This is the first report of nitrate-induction of NR activity in Spirulina, and also perhaps in any nonnitrogen fixing, nitrate-utilizing cyanobacterium. Kikuchi et al. (1996) reported that NR transcription increased in the presence of nitrate and nitrite in Synechococcus sp. strain PCC 7942, but attributed it to nitrite rather than nitrate, based on comparison with a NR-deficient mutant. However, these experiments were conducted in the presence of MSX to prevent NH 4 + assimilation and the resultant repression of the nir operon. They emhasize the derepression of the NiR operon as a prerequisite to induction by nitrite, whereas we observed straightforward response to nitrate as the sole source of N. Moreover, our finding that nitrite inhibits nitrate - induction of NR activity (Fig. 3) argues against the possibilty of nitrite being an inducer of NR activity in Spirulina. According to Herrero et al. (1984), though nitrate does not seem to be required as an inducer in Synechococcus sp. PCC 6301, the stability of preformed NR was higher in the presence of nitrate or ammonium than in the absence of combined nitrogen. While the effect of nitrate on the stability of NR in Spirulina needs to be assessed, the slow decline in NR activity upon nitrate removal and quick recovery upon re-addition (Fig. 2) seem to argue against such post-translational effects, which normally follow the opposite kinetic pattern. However, it is also possible that the slow decline is due to residual nitrate inside the cells. Regulation by downstream metabolites An intriguing feature of NR regulation is that all the products of nitrate reduction, i.e., nitrite and ammonium ions, as well as glutamine and asparagine affect NR mRNA levels or NR activity. But the precise role of nitrite and ammonium ions and their mechanism of NR regulation is not well understood even in higher plants, as their mode of action and degree of influence on NR seems to vary depending on the experimental systems/conditions (Raghuram & Sopory, 1999; Ali et al., 2007; Lochab et al., 2007). In N 2 -fixing cyanobacteria, nitrite and nitrate are known to be inducers of the nir operon, though this was not considered to be the case with non-N 2 -fixing cyanobacteria (Herrero et al., 1981), till it was reported that nitrite could be an inducer in Synechococcus sp., strain PCC 7942 (Kikuchi et al., 1996, Aichi et al., 2004). However, our results on the effect of exogenous nitrite supply on NR activity in Spirulina in this study clearly indicate that nitrite is inhibitory to nitrateinduced NR activity in a dose dependent manner in the concentration range 0.1-1 mM (Fig. 3). The level of inhibition increased from 18.79 % in 0.2 mM NaNO 2 to 35.29 % in 1 mM NaNO2. The differences between our results and those of Kikuchi et al. (1996) and Aichi et al. (2004) can be attributed to differences between
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Figures 3 & 4 : Effects of Nitrite and Ammonium on NR Activity in Spirulina. Exponential phase Spirulina culture grown for 9-days was harvested, washed and resuspended in media containing 30 mM NaNO 3 (NO 3 ) and different concentrations of NaNO 2 (NO 2) as shown in Fig 3 on the left and NH 4 Cl (NH 4) Fig 4 on the right as indicated. The controls consisted of Zarouk’s medium with (control) or without 30 mM NaNO 3 (Minus N). Samples were collected after 8 hrs of treatment and processed for extraction and NR assay as described in Materials and Methods. The mean data from 3 different experiments are shown along with std. error bars.
organisms or nitrate-replete vs. nitrate-limited experimental conditions. Considering that NO 2 accumulation is toxic to the cell under nitrate-replete conditons, its levels can be controlled either by inhibiting NR activity and/or nitrate transport, or by enhancing nitrite reductase (NiR) activity. Since all three are a part of the same nir operon in cyanobacteria, differential regulation is only possible at the post translational level. The downregulation of NR activity observed in this study needs further characterization in this regard. Ammonium ions are well known to repress the expression of the cyanobacterial genes involved in nitrate uptake and assimilation (Herrero, 2001, Muropastor et al., 2005), though Kobayashi et al. (2005) have reported recently that NR and NiR activities were insensitive to ammonium inhibition in Synechocystis sp. strain PCC 6803. In the present study, addition of NH 4 + to exponential Spirulina cultures growing in the presence of nitrate led to a decrease NR activity in a concentration dependent manner (Fig. 4). While 1 mM NH 4 Cl did not have any significant effect on NR activity, the % inhibition was 26 % in 5 mM NH4 Cl and 45 % in 10 mM NH 4 Cl. When a higher concentration range (10-40 mM) was tested, the cells became yellow within a few hours of treatment and were therefore not processed further. Whether the inhibitory effect of ammonium ions is due to nitrate uptake, NR downregulation, or uncoupling of photosynthesis or due to a derivative like glutamine (Flores et al., 2005; Muro-pastor et al., 2005) remains to be assessed. In conclusion, this study reports nitrate-induction of NR activity and its inhibition by nitrite and
ammonium ions in Spirulina for the first time. Further characterization of these preliminary findings may reveal the biochemical and molecular basis of nitrate assimilation and high protein content in this intriguing organism. ACKNOWLEDGMENTS We thank Sunila Lochab and Ravi Ramesh Pathak for their help in finalising the manuscript. This work was supported in part by research grants awarded to NR from UGC, New Delhi, University of Mumbai and Bharat Serums and Vaccines, Mumbai, India. LITERATURE CITED Ali, A., Sivakami, S. and Raghuram N. (2007). Effect of nitrate, nitrite, ammonium, glutamate, glutamine and 2oxoglutarate on the RNA levels and enzyme activities of nitrate reductase and nitrite reductase in rice. Physiol. Mol. Biol. Plants 13 (1): 17-25. Aichi M, Maeda S, Ichikawa K, Omata T. (2004) Nitriteresponsive activation of the nitrate assimilation operon in cyanobacteria plays an essential role in up-regulation of nitrate assimilation activities under nitrate-limited growth conditions. J. Bacteriol 186: 3224–3229. Archondeguy, T., Jack, R. and Merrick , M. (2001). PII Signal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control. Microbiol. Mol. Biol Rev. 65(1): 80–105. Berges, J.A. (1997). Algal nitrate reductases. Eur. J. Phycol., 32: 3-8. Flores, E., Frias, J.E., Rubio, L.M. and Herrero, A. (2005). Photosynthetic nitrate assimilation in cyanobacteria. Photosyn Res. 83: 117–133 Flores, E. and Herrero, A. (1994). Assimilatory nitrogen metabolism and its regulation. In: The molecular biology of cyanobacteria, ed. Bryant, D.A., Kluwer Academic, Dordrecht, The Netherlands. pp: 487-517. Guerrero, M.G., Vega, J.M. and Losada, M. (1981). The assimilatory nitratereducing system and its regulation. Annu. Rev. Plant Physiol. 32: 169–204.
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