Ca2+ Requirement for Aerobic Nitrogen Fixation by - Plant Physiology

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For Anabaena sp.,the culture medium of Arnon et al. (4), modified to contain 4 mM K2HPO4, was used. Anabaena. ATCC 33047 was grown on the medium ...
Plant Physiol. (1990) 92, 886-890 0032-0889/90/92/0886/05/$01 .00/0

Received for publication August 7, 1989 and in revised form October 20, 1989

Ca2+ Requirement for Aerobic Nitrogen Fixation by Heterocystous Blue-Green Algae1 Herminia Rodriguez, Joaquin Rivas*, Miguel G. Guerrero, and Manuel Losada Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla-Consejo Superior de Investigaciones Cientificas, Facultad de Biologia, Apartado 1113, 41080 Sevilla, Spain Among them, there is a thick multilayered envelope, which probably acts as an 02 barrier (7, 8, 26). From observations of the effect of Ca2+-chelating agents upon nitrogenase activity in the presence and absence of 02, Gallon and Hamadi (10, 12) have proposed that Ca2+ is required for aerobic N2 fixation in Gleothece, a unicellular blue-green alga which does not possess heterocysts, and suggested that the role of Ca2+ may be linked to respiratory protection of nitrogenase. Smith et al. (21, 22) have recently proposed a Ca2+ mediated regulation of heterocyst frequency in Nostoc PCC 6720 (Anabaenopsis circularis). The present report deals with a strict Ca2' requirement for aerobic growth and nitrogen fixation in two strains of the heterocystous blue-green alga Anabaena. This Ca2' requirement seems to be related to nitrogenase protection from 02-

ABSTRACT The requirement of Ca2+ for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N2 under microaerobic conditions, Ca2+ was not required for growth, at least in concentrations greater than traces. In contrast, Ca2+ was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca2+ to an aerobic culture without Ca2+ promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca2+ promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca2+ or low oxygen tension was dependent on de novo protein synthesis. The role of Ca2+ seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.

MATERIALS AND METHODS Biological Material and Culture Conditions Anabaena sp. is a phycoerythrin-rich strain isolated from Albufera de Valencia (Spain) (20). Anabaena ATCC 33047 was obtained from the American Type Culture Collection (Rockville, MD). For Anabaena sp., the culture medium of Arnon et al. (4), modified to contain 4 mM K2HPO4, was used. Anabaena ATCC 33047 was grown on the medium described by Moreno et al. (18), containing 86 mM NaCl, 50 mM NaHCO3, 8 mM KCI, 1 mm K2HPO4, 0.5 mM MgSO4, 0.35 mM CaCl2, as well as a supply of essential micronutrients and Fe-EDTA (4). When a combined source of nitrogen was utilized, the medium was supplemented with 20 mM NaNO3 or KNO3 (nitrate medium) or with 5 mm NH4C1 (ammonium medium). When Anabaena sp. was grown with NH4C1 as the nitrogen source, the culture medium contained 10 mm NaHCO3, in order to maintain the pH near the optimum for this strain (pH 7.3). Ammonium concentration in the medium was kept at a constant level by adding once a day an amount equivalent to that being consumed by the cells. For the experiments performed without added Ca2+ or when the concentration of added Ca2+ (CaCl2) was lower than that of the standard medium, the cells used as inoculum were washed two to four times with Ca2+-free medium. Cells were grown autotrophically, under either aerobic or microaerobic conditions, as indicated. For aerobic growth, cultures were bubbled with air (100 L L-' h-1) containing 0.4% (v/v) C02, whereas for microaerobic growth they were bubbled with N2 (100 L L' h-') containing 0.4% (v/v) CO2.

There are many reports on the involvement of Ca2" as a macronutrient in a large variety of physiological processes in higher plants. Nevertheless, little is known about the molecular mechanisms by which Ca2" exerts its effects (17). A requirement of Ca2" for growth has also been shown for several microalgae (green and blue-green algae and diatoms), but the concentration required appears to vary greatly among the different species (3, 5, 11, 14, 17, 19). Among the processes which have been reported to be Ca2"-dependent in blue-green algae are stimulation of sheat formation (9), PSII activity (6), phosphate uptake ( 15), activity of proteases involved in heterocyst differentiation (13), and assimilation of inorganic nitrogen (1-3, 21). Nitrogen fixation is an anaerobic process, with nitrogenase being rapidly and irreversibly inactivated by oxygen. The nitrogen-fixing, blue-green algae are the only organisms that have solved the special problem of protecting nitrogenase during simultaneous photosynthetic 02 evolution. Many nitrogen-fixing cyanobacteria show specialized cells for nitrogen fixation, namely the heterocysts, which provide an appropriate environment for nitrogenase, maintaining a low internal concentration of oxygen by a variety of strategies (24, 25). Research supported by Comisi6n Interministerial Ciencia y Tecnologia (BI088-02 18). H. R. is a recipient of a fellowship from Junta de Andalucia.

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Ca2" REQUIREMENT FOR AEROBIC NITROGEN FIXATION When nitrate was the nitrogen source, the cultures of Anabaena sp. were bubbled with air (100 L L' h-') containing 1% C02, in order to maintain the pH of the medium around 7.3. Cells were grown at 350C in 5-cm wide glass containers of 1 -L capacity, which were laterally illuminated with fluorescent lamps at an irradiance of 40 W m-2. Growth was either in batch culture with continuous illumination or in semicontinuous culture subjected to 10 h light/14 h dark cycles, as indicated. Semicontinuous cultures were partially renewed with the corresponding fresh medium to a cell density of about 6 mg (Chl) L' at the beginning of the light period. Specific growth rates were calculated from the increase in dry weight measured from samples at the beginning and end of a 24 h period.

Analytical Methods For dry weight determinations, 50-mL aliquots of cell suspensions were filtered through previously weighted GF/C Whatman glass microfiber filters, washed twice with distilled water, and dried at 80'C for 24 h. Chl a was determined spectrophotometrically in methanolic extracts employing the extinction coefficient given by Mackinney (16). Nitrogenase activity was measured in whole cells by the acetylene reduction technique (23). Assays were carried out in 17-mL conical flasks sealed with rubber serum stoppers and containing either air (aerobic assays) or N2 (microaerobic assays) supplemented with 0.4% (v/v) CO2 to which 2 mL of acetylene were added. For microaerobic assays, aliquots of the cell suspension were withdrawn from the cultures with a syringe and immediately injected into the sealed flasks in order to avoid contact with air. The flasks were shaken in a bath at 40°C and illuminated from below (100 W m-2, white light). After 10 to 15 min, l-mL aliquots of the gas phase were removed, and the ethylene formed was determined by gas chromatography. Heterocyst frequencies were determined by counting at least 1000 cells under a light microscope.

Table I. Effect of Ca2+ on Growth of Anabaena sp. in Media with Different Nitrogen Sources under Aerobic and Microaerobic Conditions Cells were grown in semicontinuous culture with and without Ca2+ (1 0-4 M) added to the medium. Data are mean values ± SD of three independent determinations. Nitrogen Source and Growth Conditions

Specific Growth Rate Ca2+-free medium Ca2"-supplemented medium

0.39 ± 0.02 Nitrate, aerobic Ammonium, aerobic 0.35 ± 0.05 NGa N2, aerobic 0.25 ± 0.01 N2, microaerobic a No growth detected.

AM (day-') 0.42 ± 0.31 ± 0.39 ± 0.43 ±

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RESULTS Table I shows the effect of Ca2+ on the growth in semicontinuous culture of Anabaena sp. in media with different nitrogen sources. With a combined source of nitrogen, either nitrate or ammonium, Ca2+ was not required for aerobic growth, the specific growth rate being very similar in the absence and in the presence of l0-4 M Ca2+ in the growth medium. On the contrary, with molecular nitrogen as the sole nitrogen source, a strict Ca2' requirement for growth was evident under aerobic conditions. In the absence of added Ca2' and with molecular nitrogen as the sole nitrogen source, cells could grow under low 02 tension (microaerobic conditions), although at a slower rate than in a Ca2+-supplemented medium. Analogous results were obtained for Anabaena ATCC 33047 (data not shown). As shown in Figure 1, virtually no growth of Anabaena sp. was detected with molecular nitrogen under aerobic conditions if the concentration of Ca2' added to the medium was 1 Mm or lower. At 10 AM Ca2 , significant growth occurred,

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Figure 1. Effect of Ca2+ concentration on growth (@) and nitrogenase activity (0) of Anabaena sp. under aerobic conditions. Cells were grown in semicontinuous culture with air as the sole nitrogen source. Nitrogenase activity was estimated under aerobic conditions. Data are mean values of at least four determinations.

maximal growth rate being attained at 100 MM, and inhibition of growth being observed when Ca2` concentration was increased to 1 mm. An analogous trend in response to the concentration of Ca2+ in the medium was also evident with regard to the nitrogenase activity level of the cells (Fig. 1). With molecular nitrogen as the sole nitrogen source, the cellular nitrogenase level was very low under aerobic conditions in the absence of added Ca2+. Notwithstanding, this level increased significantly when the cells grew microaerobically, amounting to about one-third of those in Ca2+-supplemented cultures, either microaerobic or aerobic (Table II). The data in Table II show moreover that, whereas Ca2+ supply and 02 tension clearly influenced the nitrogenase activity level of

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Table II. Effect of Ca2" on Nitrogenase Activity and Heterocyst Frequency of Anabaena sp. under Aerobic and Microaerobic Conditions Cells were grown in semicontinuous culture with and without Ca2+ (10-4 M) added to the medium. Nitrogenase assays were performed at about the middle of the light periods under microaerobic conditions. Data are mean values ± SD of three independent determinations. Culture Conditions

Nitrogenase Activity

Heterocyst Frequency

MmoI C2H4 mg Chl-1

%

10.3 ± 1.4 21.5 ± 1.7

9.7 ± 0.2 8.9 ± 0.6

60.3 ± 3.4 65.3 ± 1.8

8.4 ± 1.0 9.3 ± 1.0

Ca2+-free medium Aerobic Microaerobic

Ca2+-supplemented medium Aerobic Microaerobic

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TIME (h) Figure 2. Ca2-promoted development of nitrogenase activity in an aerobic batch culture of Anabaena sp. At t = 0 a suspension of Anabaena sp. filaments in Ca2"-free medium under aerobic conditions (0) was divided into two halves, to one of which (e) Ca2` was added to a final concentration of 10-4 M. Nitrogenase activity assays were carried out under aerobic conditions.

Anabaena sp. cells, these factors did not practically affect the heterocyst frequency in the filaments. Analogous results were also obtained for Anabaena ATCC 33047. Figure 2 shows that an aerobic batch culture of Anabaena sp. on a medium without added Ca2+ could attain a high nitrogenase activity level in response to the addition of l0-4 M Ca2 . It is worthy to mention that it took about 3 h until this Ca2+-promoted development of nitrogenase activity was initiated, maximal nitrogenase activity being reached 5.5 h

0

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TIME (h) Figure 3. Air-promoted decay of nitrogenase activity in a Ca2+-free batch culture of Anabaena sp. and further recovery under microaerobic conditions. At t = 0 N2 was replaced by air (0) in the gas mixture sparged through a culture of Anabaena sp. filaments. After 1 h (arrow) the suspension was divided into two halves, one of which was made again microaerobic (0), whereas the other remained aerobic. Nitrogenase activity assays were carried out under microaerobic conditions.

after the addition of Ca2+ to the medium. This evolvement by Ca2+ of nitrogenase activity was prevented by chloramphenicol, thus indicating a requirement for de novo protein synthesis. Again, analogous results were obtained for Anabaena ATCC 33047, except that the time required for full development of nitrogenase activity was longer (about 10 h) than for Anabaena sp.

Ca2+ REQUIREMENT FOR AEROBIC NITROGEN FIXATION

As mentioned above, the nitrogenase activity level of Anabaena sp. is very high in batch culture without added Ca" under low 02 tension (microaerobic conditions). However, the data in Figure 3 show that, when N2 was replaced by air in the gas mixture sparged through the culture, the nitrogenase activity level dropped almost instantaneously to a very low level. Original nitrogenase activity could be recovered by placing again the cells under a microaerobic atmosphere but not if the aerobic conditions prevailed. It is important to underline that recovery of nitrogenase activity was completed in about 2 h, without any lag phase, in contrast with the Ca"promoted development of nitrogenase activity (Fig. 2). Restoration of the initial nitrogenase activity level of a culture without added Ca>2 under microaerobic conditions was also inhibited by chloramphenicol. For comparative purposes, when N2 was replaced by air in a Ca2+-supplemented culture, cellular nitrogenase activity dropped also instantaneously to a low level, but it recovered rapidly even under aerobic conditions (data not shown).

DISCUSSION The dispensability of Ca>2 for inorganic nitrogen assimilation by heterocystous blue-green algae (1-3, 21) still remains a matter of controversy. Whereas it has been reported that Ca> is essential for growth with either nitrate or molecular nitrogen (1, 21), it has otherwise been suggested that Ca>2 is required with molecular nitrogen only (2). Our results with two strains of Anabaena show that Ca>2 is not required, at least in concentrations greater than traces, for growth in the presence of combined nitrogen. In contrast, an absolute Ca>2 requirement as a macronutrient (10'O M) is observed with air as the nitrogen source but not with N2 under microaerobic conditions (Table I). However, Ca2+ is not essential for nitrogen fixation itself, as indicated by the high level of nitrogenase activity in the absence of added Ca>2 at low oxygen tension (Table II; Fig. 3). Introduction of air promotes a drastic drop in nitrogenase activity, which rapidly recovers its initial level upon restoration of microaerobic conditions in a process which is dependent on de novo protein synthesis (Fig. 3). This is evidence against the suggestion of a catalytic role of Ca>2 in nitrogen fixation (2). Under aerobic conditions, Ca>2 is required for nitrogen fixation (Figs. 1 and 2). It seems, therefore, that Ca2+ exerts its effect by protecting nitrogenase against inactivation by oxygen. Enhancement of nitrogen fixation capacity under air in response to Ca2' addition is not immediate but exhibits a lag of several hours (Fig. 2). Ca2+ might act by conferring to preexisting heterocysts a resistance to oxygen, rather than by influencing heterocyst frequency as proposed by Smith et al. (21, 22) for Anabaenopsis. In fact, a preliminary light and electron microscopic study of Anabaena sp. and Anabaena ATCC 33047 filaments grown in the absence of added Ca>2 revealed the presence of abnormally shaped heterocysts with morphological alterations in their envelope, which might be indicative of a defective barrier against oxygen. ACKNOWLEDGMENTS We thank A. Friend and M. J. Perez de Le6n for skillful secretarial

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22. Smith RJ, Hobson S, Ellis IR (1987) Evidence for calciummediated regulation of heterocyst frequency and nitrogenase activity in Nosloc 6720. New Phytol 105: 531-542 23. Stewart WDP, Fitzgerald GP, Burris RH (1967) In situ studies on N2 fixation using the acetylene reduction technique. Proc Natl Acad Sci USA 58: 2071-2078 24. Van Baalen C (1987) Nitrogen fixation. In P Fay, C Van Baalen,

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eds, The Cyanobacteria. Elsevier Science Publishers, Amsterdam, pp 187-198 25. Wolk CP (1980) Cyanobacteria (blue-green algae). In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol I. Academic Press, New York, pp 659-686 26. Wolk CP (1982) Heterocysts. In NG Carr, BA Whitton, eds. The Biology of Cyanobacteria. Blackwell, Oxford, pp 359-386