NH/ UPTAKE BY THE UNICELLULAR ALGA CYANIDIUM CALDARIUM POSSIBLE CONTROL MECHANISMS DEPENDENT ON NITROGEN STATUS

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1 New PhytoL {\9S7) 107, 5075\2 * :r t NH/ UPTAKE BY THE UNICELLULAR ALGA CYANIDIUM CALDARIUM POSSIBLE CONTROL MECHANISMS DEPENDENT ON NITROGEN STATUS BY VITTORIA DI MARTINO RIGANO, VINCENZA VONA, LUIGI MANZO AND CARMELO RIGANO Dipartimento di Biologia Vegetale, Universita di Napoli, Via Foria 223, Napoli, Italy {Accepted 13 May 1987) SUMMARY Measurements of NH4"^ uptake by Cyanidium caldarium (Tilden) Geitler, an acidophilic thermophilic nonvacuolate unicellular red alga, were made with cells grown either in batch culture with excess ammonium or in continuous culture with nitrogen limitation. Batchgrown cells absorbed NH^"^ at a lower rate than chemostatgrown cells, and uptake was greatly inhibited in darkness (83 %) and by COg deprivation (74%). In chemostatgrown cells, in contrast, darkness inhibited uptake by only 20 to 30%. In chemostatgrown cells subjected, after the addition of ammonium, to alternate light/dark cycles over 7 h of incubation, the rate of NH^^ uptake in the successive dark periods progressively decreased and fell to only 15 % of the initial rate after 5 h incubation. In the successive light periods, in contrast, there was a 15 % decrease in rate from one light period to the next, and after 7 h the rate of uptake was still 50 % of the initial value. At this time, transfer of the cells from light to darkness resulted in 85 % inhibition of uptake. It is suggested that in C. caldarium the overall process of ammonium assimilation is controlled, at the level of ammonium entry into the cell, either through the involvement of two independently controlled uptake systems or through multiple interconvertible forms of a single system, sharing different regulatory properties. Key words: Regulation of ammonium uptake, ammonium uptake, Cyanidium caldarium, algal nitrogen metabolism, nitrogen assimilation. INTRODUCTION It is now established that in microbial cells, algae and fungi (Kleiner, 1981; Boussiba, Resch & Gibson, 1984; Rai, Rowell & Stewart, 1984), the uptake of ammonium, like the uptake of other nutritive ions (Tamai, Tohe & Oshima, 1985; Krueger et al., 1986), is mediated by specific permease systems, and that control mechanisms operate on these systems. In photosynthetic organisms, the uptake of ammonium can be inhibited by darkness or by deprivation of COg. In the case of the unicellular alga Chlamydomonas (Thacker & Syrett, 1972), however, it was observed that inhibition was dependent on whether cells grown with excess nitrogen were directly used for experiments of ammonium uptake, or whether the cells were previously kept under conditions of nitrogen starvation. Inhibition of uptake by darkness was great in the first case but not so severe in the second. In the case of Phaeodactylum (Syrett et al., 1986), the ability of the cells to absorb ammonium and other nitrogen compounds can be developed or increased manifoldly during several hours of nitrogen starvation /87/ $03.00/ The New Phytologist

V. D I MARTINO RIGANO et al. Cyanidium caldarium is an acidophilic thermophilic nonvacuolate unicellular red alga. In previous papers (Di Martino Rigano et al.

2 V. D I MARTINO RIGANO et al. Cyanidium caldarium is an acidophilic thermophilic nonvacuolate unicellular red alga. In previous papers (Di Martino Rigano et al., 1986, 1987), it was shown that the pattern of ammonium utilization by C. caldarium grown in excess ammonium differed from that of Nlimited cells. It was suggested that NH4+ uptake system(s) with different biochemical and regulatory properties could be present in the two types of cells, which enable the cells to regulate ammonium uptake. Further observations with Cyanidium which support this contention are reported in the present paper. M A T E R I A L S AND M E T H O D S C. caldarium (Tilden) Geitler, strain 0206, was supplied by Professor T. D. Brock who isolated it from a thermal spring in Yellowstone Park (USA). The alga was grown at 42 C in continuous light in batch cultures with excess ammonium, or in chemostat cultures under conditions of nitrate limitation. The continuous culture apparatus and composition of the culture medium was as previously described (Rigano et al., 1981). The dilution rate was 025 d~\ Cells collected by lowspeed centrifugation were resuspended in nitrogenfree medium, adjusted to ph 19 with sulphuric acid, at a density of 2 to 4 mg cell dry weight m r ^ Cell density was estimated by centrifuging a known volume in a haematocrit tube and converting the cellular volume so obtained to dry weight by use of a calibration curve. The cell suspensions were then kept at 40 C in light or in darkness, with or without COg, as described in the text. NH4+ was added as required. Ammonium disappearance from the external medium was monitored with an ammonia electrode. The two channels of a peristaltic pump continuously sampled the cell suspension and NaOH solution which were then pumped through a single capillary tygon tube into a reaction chamber containing the electrode. This was connected to an Ionalyzer (Orion model 901) adjusted in the concentration mode, which, in turn, was connected to a data printer and recorded ammonia concentration at 1 min intervals. The recording apparatus is described elsewhere (Di Martino Rigano et al., 1986). RESULTS Experiments on ammonium uptake by suspensions of cells grown in batch culture Cells grown with excecs ammonium (20 mm) were collected during exponential growth by lowspeed centrifugation, resuspended in Nfree medium (ph 19) at a density of 35 mg cell dry weight ml"^ and kept at 40 C. The suspension was sufflated with air containing 5 % COg and, at zero time, ammonium (final concentration 7 mm) was added. The suspension was submitted alternately to light/dark periods and to a period of COg deprivation. Rate of uptake was measured by following the disappearance of N H / from the external medium. As shown in Figure 1, in light with COg, the cells took up ammonium linearly with time at a rate of 032 //mol (mg cell dry weight)^ h ' ^ Upon darkening the suspension, rate of uptake decreased almost immediately by 83 %; the rate was fully restored by illumination. Uptake of ammonium, in light, was also decreased by 74% following removal of CO2 and was again restored when COg was added. Ammonium uptake was almost completely suppressed in cell suspensions sufflated with pure nitrogen to create anaerobic conditions (results not shown).

3 uptake by Cyanidium Time (min) Fig. 1. Ammonium uptake by a cell suspension of Cyanidium caldarium grown in batch culture with excess ammonium (20 mm). The cell suspension (35 mg cell dry weight ml"^) was kept in light at 40 C, suffiated with air containing 5 % COg and ammonium added at zero time to give a final concentration of 7 mm. At the time indicated by the first arrow, the cell suspension was transferred to darkness and successively again to light (second arrow). At the time indicated by the third arrow, COj was removed from the sufflating air (the suspension remained in the light) and then CO2 again added (as indicated by the fourth arrow). To remove COj, air was passed through 10 M NaOH before it reached the cell suspension. The initial rate of uptake was 032 //mol NH4"^ h~^ (mg cell dry weight)"^ The numbers in parentheses indicate the uptake rates under the different conditions of incubation, as percentages of the initial rate. The duration of the dark period is shown on the abscissa. Experiments with cells grown in a nitrogenlimited chemostat Cells were collected and resuspended into Nfree medium at a density of 27 mg cell dry weight mr\ as indicated above. The cell suspension was kept at 40 C and sufflated with air containing 5 % COg throughout the experiment (about 7 h in all). At zero time, ammonium (final concentration 8 mm) was added and the suspension subjected to light/dark periods (each of about 1 h duration) and ammonium disappearance followed. As shown in Figure 2, in the initial light period ammonium was taken up at a rate of 061 //mol mg cell dry weight h^^ In the following period of darkness, the rate of NH4+ assimilation abruptly decreased to 65 % of the initial rate in light. Illumination in the second light period resulted in stimulation of the rate of uptake over the preceding rate in darkness, but the rate attained was only 85 % of that in the initial light period. During the second dark period, the rate decreased further to only 30 % of the initial rate in light. In the third light period, almost 65 % of the initial activity was restored. In a third period of darkness, the rate further decreased to only 15 % of the initial value but increased to 50 % of the initial rate during the final light period. The results can be summarized as follows: in the dark periods, the rate of ammonium uptake decreased progressively to reach only 15 % of the initial activity after 5 h of incubation. In light, in contrast, there was a 15 % decrease in rate from one light period to the next, and after 7 h and three intervening periods of darkness, the rate in light was still 50 % of the initial value. However, at this time, transfer of the cells from light to darkness resulted in an 85 % decrease in rate of NH4^ uptake. In two similar cell suspensions freshly prepared from chemostatgrown

4 5IO V. Di MARTINO RIGANO et al. 7 6 i 5 \ (100) \ 1 c 1 : ) /.0 80 Time (min) Time (min) I Fig. 2. Ammonium uptake by a cell suspension of Cyanidium caldarium grown in chemostat under conditions of N limitation. The cell suspension (2 7 mg cell dry weight ml"^) was kept at 40 C and sufflated with air containing 5 % COj. The experiment started with the addition of NH^"'" to give a concentration of 8 mm. The cells were kept initially in light and were then subjected to alternate darklight periods as indicated by the arrows and the black bars (darkness) on the abscissa. The numbers in parentheses above the trace line indicate the rate of uptake of NH^* as a percentage of the initial rate. The initial rate measured in the first light period was 061 //mol NH^* h~^ (mg cell dry weight)"^. The inset shows the ammonium uptake of cells collected at the end of the experiment (7 h) and resuspended in a medium containing 3 mm cultures, the rate of ammonium uptake was almost 20 % higher in light than darkness over 100 min (result not shown). In cell suspensions sufflated with pure nitrogen the uptake of ammonium was stopped completely. DISCUSSION Cells of C. caldarium grown in chemostat culture with nitrogen limitation took up ammonium faster than cells grown in batch culture with excess ammonium. This finding agrees with reports in the literature which emphasize that in bacteria (Jayakumar et al., 1986) and algae (Syrett et al., 1986) the ability to assimilate ammonium is fully developed only under conditions of nitrogen limitation or nitrogen starvation. In cells grown with excess ammonium, the uptake rate was not only lower, but it was drastically, and almost immediately, inhibited by darkness and by CO2 deprivation. Nitrogenlimited cells, by contrast, assimilated ammonium at sustained rates both in light and in darkness, and, as shown previously (Di Martino Rigano et al., 1987), the inhibition by COg deprivation was not immediate but behaved like a timedependent phenomenon. Thus, in

5 uptake by Cyanidium 511 C. caldarium, ammonium assimilation does not necessarily depend directly on photosynthesis; ATP and carbon skeletons generated by dark metabolism are apparently suitable for this purpose. This conclusion suggests that the inability of Nsufficient cells (see above) to assimilate ammonium in darkness, or when CO2 is removed in light, does not necessarily indicate a strict dependence of ammonium assimilation on photosynthesis but rather that in Nsufficient cells the unavailability of an exogenous carbon source or darkness may act as regulatory signals that trigger an inhibitory mechanism operating on the entry of ammonium into the cell. It is noteworthy that chemostatgrown cells assimilated ammonium in light 20 to 30% faster than did cells in darkness. This finding suggests that a similar inhibitory phenomenon occurring at the level of ammonium entry into the cell is responsible for the partial (in chemostatgrown cells) or almost total (in batchgrown cells) inhibition of ammonium utilization by darkness. Ammonium entry into the cell is mediated by a specific permease system. Perhaps, in C. caldarium, a permease for ammonium can exist in multiple interconvertible forms, or, more probably, two distinct permease systems are present, which differ in their regulatory properties, as deduced from the observation that the uptake is sometimes inhibited by darkness greatly and rapidly, or by COg deprivation, and sometimes is unaffected either by darkness or, in the short term, by COg deprivation. If two permease systems exist, one of theni may be formed constitutively and may thus occur both in Nsufficient cells and in Nhmited cells. The other permease, in contrast, may be repressed in Nsufficient cells and derepressed by nitrogen limitation and may thus occur only in Nlimited cells. This hypothesis, which accounts not only for difterent rates of NH4+ uptake but also for a different pattern of ammonium assimilation dependent on the nitrogen status of the Cyanidium cells, is supported by data from the literature. The ammonium uptake system, in fact, can be repressed by high ammonium concentrations in several organisms (Sharak Genthner & Wall, 1985; Jayakumar et al., 1986). Two distinct permease systems with different regulatory and biochemical properties are known for other nutritive ions (Fuggi et al 1984Tamai et al., 1985). The existence of a regulatory site at the level of ammonium entry in C. caldarium is supported by the experiment of Figure 2, where assimilation of ammonium by chemostatgrown cells was followed over 7 h of alternate light/ dark cycles. In the first part of the experiment (zero time to 2 h), the transfer of the cells from light to darkness decreased N H / assimilation by only 35 %. In the second part, by contrast (from 4 to 7 h), the transfer from light to darkness inhibited by 8 5 %. This finding supports the view that chemostatgrown cells possess two distinct uptake activities, one of which is inhibited by darkness and the other not, and moreover, that addition (and assimilation) of ammonium causes a timedependent loss of the latter activity but not of the former. This hypothesis can explain why, at the end of the experiment, the rate of ammonium uptake was inhibited by 85 % in darkness. On the other hand, the failure to assimilate NH^^ in the dark cannot be attributed to a loss of the capacity of the cell to incorporate ammonium into organic matter since, in the light, this capacity is preserved, nor can it be attributed to a depletion of organic compounds, since these compounds are largely replenished during the light periods. Thus, the most plausible explanation is that, after a certain time from ammonium addition, the cells lose that uptake activity which can absorb ammonium in the dark. On the basis of results discussed above, and of results in previous papers

6 512 V. D I MARTINO RIGANO et al. (Di Martino Rigano et al., 1986, 1987), it can be concluded that the control of ammonium assimilation in C. caldarium involves the participation of two uptake systems for ammonium (or, less probably, of multiple interconvertible forms of a single system), sharing different regulatory properties which allow the cell to modulate ammonium uptake according to the overall nutritional conditions. One system appears to be primarily controlled by the nitrogenous state of the cell and is developed only under conditions of nitrogen limitation when it is advantageous if the cell can scavenge trace amounts of ammonium from the external medium. This system works independently of whether an exogenous carbon source or light is immediately available, and uptake and assimilation of ammonium can then occur at the expense of ATP and carbon compounds generated by dark metabolism. The cell does not develop this system when it has a high nitrogen content and the absence of this system prevents unnecessary further ammonium uptake. In such cells, only the other uptake system is present which, apparently, is dependent on other nutritional factors (carbon availability, for instance, or light). This constitutive system thus functions only when all the nutritional requirements for growth are largely present. Studies are in progress to elucidate the molecular basis of these differing regulatory patterns. ACKNOWLEDGMENTS This work was aided by a grant from 'Consiglio Nazionale delle Ricerche' and 'Ministero della Pubblica Istruzione' of Italy. We wish to thank Professor T. D. Brock, University of Wisconsin, for his generous gift of a strain, 0206, of Cyanidium caldarium. REFERENCES BoussiBA, S., RESCH, C. M. & GIBSON, J. (1984). Ammonia uptake and retention in some cyanobacteria. Archives of Microbiology, 138, D I MARTINO RIGANO, V., VONA, V., Di MARTINO, C. & RIGANO, C. (1986). Effect of darkness and COg starvation on NH^^ and NOg"^ assimilation in the unicellular alga Cyanidium caldarium. Physiologia Plantarum, 68, Di MARTINO RIGANO, V., VONA, V., Di MARTINO, C. & RIGANO, C. (1987). Regulatory aspects of NH^* utilization in the acidophilic thermophilic unicellular red alga Cyanidium caldarium. New Phytologist, 105, FuGGi, A., VONA, V., Di MARTINO RIGANO, V., Di MARTINO, C, MARTELLO, A. & RIGANO, C. (1984). Evidence for two transport systems for nitrate in the acidophilic thermophilic alga Cyanidium caldarium. Archives of Microbiology, 137, JAYAKUMAR, A., SCHULMAN, I., MACNE'L, D. & BARNES, JR, E. M. (1986). Role of the Escherichia coli glnalg operon in regulation of ammonium transport. Journal of Bacteriology, 166, KLEINER, D. (1981). The transport of NHg and NH^^ across biological membranes. Biochimica et Biophysica Acta, 639, KRUEGER, R. D., HARPER, S. H., CAMPBELL, J. W. & FAHRNEY, D. E. (1986). Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphatelimited continuous culture of Methanobacterium thermoautotrophicum. Journal of Bacteriology, 167, RAI, A. N., ROWELL, P. & STEWART, W. D. P. (1984). Evidence for an ammonium transport system in freeliving and symbiotic cyanobacteria. Archives of Microbiology, 137, RIGANO, C, D I MARTINO RIGANO, V., VONA, V. & FUGGI, A. (1981). Nitrate reductase and glutamine synthetase activities, nitrate and ammonia assimilation, in the unicellular alga Cyanidium caldarium. Archives of Microbiology, 129, SHARAK GENTHNER, B. R. & WALL, J. D. (1985). Ammonium uptake in Rhodopseudomonas capsulata. Archives of Microbiology, 141, SYRETT, P. J., FLYNN, K. J., MOLLOY, C. J., DION, G. K., PEPLINSKA, A. M. & CRESSWELL, R. C. (1986). Effects of nitrogen deprivation on rates of uptake of nitrogenous compounds by the diatom, Phaeodactylum tricornutum Bohlin. New Phytologist, 102, 3 9 ^ 4. TAMAI, Y., TOHE, A. & OsHlMA, Y. (1985). Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae. Journal of Bacteriology, 164, & SYRETT, P. J. (1972). The assimilation of nitrate and ammonium by Chlamydomonas reinhardi. New Phytologist, 71, THACKER, A.

Uptake and assimilation of nitrite in the acidophilic red alga Cyanidium caldarium Geitler

New Phytol. (1993), 125, 3.S1-360 Uptake and assimilation of nitrite in the acidophilic red alga Cyanidium caldarium Geitler BY AMODIO FUGGI Dipartimento di Agrochimica ed Agrobiologia, Facolta di Agraria,