Studies on the nitrogen supply of endosymbiotic chlorellae in greem paramecium bursaria

Plant Science Letters, 25 (1982) 85--90 Elsevier/North-Holland ScientificPublishersLtd.

85

STUDIES ON THE NITROGEN SUPPLY OF ENDOSYMBIOTIC CHLORELLAE IN GREEN P A R A M E C I U M B U R S A R I A

D. ALBERS, W. REISSER and W. WIESSNER

Pflanzenphysiologisches Institut der Univensit#t, Abt. Exp. Phykol., Untere Karspi~le 2, D-3400 G6ttingen (F.R.G.) (Received August 14th, 1981) (Revision received October 20th, 1981) (Accepted October 20th, 1981)

SUMMARY

Uptake of nitrate as well as uptake and excretion of ammonia by green and by algae-free Paramecium bursaria and by the symbiotic Chlorella spec. isolated from green ciliates were measured. Specific enzyme activities of nitrate reductase, NADH-dependent glutamate dehydrogenase and glutamine synthetase were assayed in the chlorellae within the symbiotic unit and in the isolated symbiotic algae, which were grown in mass cultures and supplied with different nitrogen sources (nitrate, ammonia, urea, glutamic acid, glutamine), or were cultivated under conditions of nitrogen deficiency. Neither green nor algae-free Paramecium bursaria take up nitrate. The ammonia metabolism of the ciliates shows a symbiosis specific feature: ammonia is excreted by algae-free Paramecium bursaria, but is taken up from the culture medium by green ciliates. These observations and enzymatic studies suggest that the nitrogen sources of the specialised chlorellae within the endosymbiotic unit are not nitrate but probably ammonia and glutamine.

INTRODUC~ON

Paramecium bursaria and a special strain of Chlorella spec. form an endosymbiotic unit which is called green Paramecium. Contrary to our knowledge of carbon metabolism of the association [1--4] only few data are known about its nitrogen metabolism. So far it has only been shown, that the Paramecium supplies the symbiotic Chlorella with some essential, yet unidentified nitrogen compounds [5]. Abbreviations: NAR, nitrate reductase; GDH/NADH, NADH-dependent glutamate dehydrogenase; GIS, glutamine synthetase. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.

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This paper reports on the nitrogen source of the endosymbiotic Chlorella spec. within the ciliate and on the specific activities of three key enzymes of its nitrogen metabolism, NAR, GDH/NADH, GIS, when the alga is cultivated either in mass culture or lives in symbiotic relationship with the Paramecium. METHODS

Green and algae-free P. bursaria as well as the isolated symbiotic Chlorella spec. were cultivated according to Reisser and coworker [6,7]. Before each experiment the paramecia were kept in an inorganic medium [ 8] for at least 24 h with a light/dark change of 14/10. When potassium nitrate was either omitted as nitrogen source of the isolated algae or was replaced by equimolar amounts of ammonia, urea, glutamic acid or glutamine, the potassium was added to the medium as potassium chloride in equimolar concentration. Green paramecia were separated into an algal and a Paramecium fraction according to Reisser [ 2]. Cell-free-extracts of the freshly isolated algal fraction and of algae harvested from mass cultures were prepared according to Reisser and Benseler [ 7]. NAR (EC 1.6.6.1)-activity was assayed according to Liu and Hellebust [ 9], modified by Tischner [10]. Reaction mixture (conc. in mM): MgSO4, 9; NADH, 0.2; KNO3, 1.8; potassium.phosphate-buffer, 0.1 M (pH 7.4). The reaction was started by addition of the algal extract. Final volume was 2 ml. After 5 min, 0.2 ml of the reaction mixture were added to 1.4 ml of sulfanilamide (1% in 1.5 N HC1) and 1.4 ml of N-(1-naphtyl)ethylendiamindihydrochloride (0.02%) in order to stop the reaction. The developing colour was measured at 540 nm. The specific activity was expressed as ~mol NO~ produced per min and mg protein (unit). GDH/NADH (EC 1.4.1.2)-activity was assayed according to Schmidt [11]. Reaction mixture (conc. in mM): NADH, 3.3; EDTA, 1.3; a-ketoglutarate, 6; ammoniumacetate, 300; potassium-phosphate-buffer, 0.1 M (pH 8.0). The reaction was started by the algal cell-free extract. Final volume was 2 ml. The specific enzyme activity was expressed as ~mol NADH oxidised per milligram protein (unit). GIS (EC 6.3.1.2)-activity was assayed according to Akimova et al. [12], modified by Tischner and Lorenzen [ 13]. Reaction mixture (conc. in raM): NH~OH • HC1, 5; ATP, 5; MgSO4, 10; Tricin buffer, 0.1 M (pH 7.8); extract equivalent to 400 ~g protein. Incubation at 37°C for 30 min. The reaction was stopped by the addition of 1 ml of the following mixture (conc. in mol): FeC1, 0.37 in HC1, 0.67 and trichloracetic acid, 0.2. Extinction was measured at 540 nm after centrifugation. Specific activity was expressed as ~mol ~-glutamylhydroxamate produced per min and mg protein (unit). Uptake of nitrate by green paramecia and isolated algae was measured with an ion selective electrode [14] (2 mV, 21°C, 6 ~mol NO~/1). For measurements of the ammonia uptake or excretion by the isolated algae and by green and algae-free paramecia, the organisms were washed

87 several times in an inorganic medium [8], in which Ca(NO3)2 was replaced by an equimolar a m o u n t o f CaC12. The organisms were then transferred for 7 h into an ammonia-containing medium consisting of CaCl2, 1.2 • 10-3; NH4CI, 1.9.10-6;MgSO4, 1 . 6 . 1 0 - 4 ; K2HPO4, 1 . 1 5 . 1 0 - 4 ; NaC1, 2 . 0 2 . 10-2; Fe(SO4)3, 2.03 • 10 -6 (all conc. in tool/l). The ammonia concentration was measured at the beginning and at the end of each experiment by the phenylhypochlorite-method of Solorzano [15]. During the experiments both algae and green paramecia were illuminated with 14.9 W . m -2. The protein c o n t e n t of cell-free extracts was determined according to Lowry et al. [16]. Thin-layer chromatography of amino acids in supematants of algal mass cultures was done according to Myhill and Jackson [ 17]. RESULTS

Neither green nor algae-freeP. bursaria take up nitrate from the culture m e d i u m (Table I). A m m o n i a is assimilated by green paramecia and excreted by algal-freeciliates.Mass cultures of the isolated symbiotic chlorellae use both, nitrate and ammonia, as nitrogen sources (Table I). The results on nitrate uptake are supported by measurements of the activity of N A R , which is lacking in the algal fractions of green paramecia as well as in the Paramecium fraction of green ciliatesand in algae-free paramecia (Table If).However enzyme activity is present in symbiotic chlorellae which are cultivated separately in mass cultures with nitrate as nitrogen source. Because ammonia can be u.sed as a nitrogen source only by'green paramecia, the activities of key enzymes of the ammonia metabolism, the GDH/ NADH and GIS, were assayed in mass cultures of symbiotic algae under different culture conditions. Their activities were compared with those measured in the algal fraction of green paramecia (Table II). The GDH/ NADH activity is higher in separately grown algae than in algae living in TABLE I UPTAKE AND EXCRETION OF AMMONIA AND UPTAKE OF NITRATE BY GREEN AND ALGAE-FREE P. B U R S A R I A AND THE ISOLATED SYMBIOTIG C H L O R E L L A SPEC.

+-

uptake;-

= excretion

Isolated symbiotic

Green P. bursaria

Algae-free

Chloreila spec.

NO~ NH4+

P. bursaria nrnol/h • 10 3 paramecia

nmol/h mg

nmol/h • mg

Chlorophyll

chlorophyll

nmol/h • 10 3 paramecia

+ (6.75 ± 0.37) • 102 +3.76 ± 0.18

0 +0.21 _+0 . 0 1

0 0 +(0.61 + 0.03) • 10 -4 -2.41 ± 0.13

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TABLE II SPECIFIC ENZYME ACTIVITIES OF THE ALGAL FRACTION OF GREEN P. BURSARIA AND OF THE SYMBIOTIC CHLORELLA SPEC. CULTIVATED IN

PRESENCE OF DIFFERENT NITROGEN SOURCES Spec. act., GDH/NADH, 0.0832 units ~ 100%; GIS, 0.0185 units ~- 100%; NAR, 3.46 units ~ 100% Specific activities NAR

GDH/NADH

GIS (%)

0

100

100

100 0

217 257

Nitrogen deficiency

0

3 24

Urea Glutamine Glutamic acid

0 0

217 109 no growth

56 39 158 56 98

(%) Algal fraction of green paramecia Symbiotic ChloreUa

(%)

s p e c . grown in mass

cultures on Nitrate Ammonia

the symbiotic unit. Only when glutamine is present in the medium, similar activities can be detected in both cases. The activity of the GIS is always lower in the chlorellae when cultivated in pure mass culture, except in the case of nitrogen deficiency or when glutamine has been given as nitrogen source (Table II). In the P a r a m e c i u m fractions of green paramecia neither NAR nor GDH/ NADH b u t a significant GIS activity could be detected, which was up to 130% of the GIS activity f o u n d within the chloreUae in the symbiotic unit. As the Chlorella spec. did n o t grow with glutamic acid and algal mass cultures grown on glutamine get acid very rapidly the s u p e m a t a n t of the giutamine grown cultures was tested for glutamic acid. Experiments show that glutamic acid is excreted by algae grown on glutamine as nitrogen source. DISCUSSION The experimental results demonstrate the ability of the isolated symbiotic Chlorella spec. to reduce nitrate and to use it as nitrogen source. But when

living within the endosymbiotic unit the algae appear unable to use nitrate as is shown by the lack of detectable NAR-activity. P. bursaria which has the animal-type of metabolism is unable to take up nitrate and to supply the chlorellae with this nitrogen source.

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The release of ammonia by algae-free P. bursaria is in agreement with former investigations on aposymbiotic Paramecium species, in which an excretion but not an uptake of ammonia is reported for P. eaudatum_ [ 18] and for P. aurelia [19]. As in contrast to algae-free P. bursaria green cfliates take up ammonia (Table I) this behaviour seems to be a symbiotic feature and due to the nitrogen requirement of the symbiotic algae. A similar observation could be made with zooxanthellae-bearing reef corals: reef corals containing endosymbionts take up more ammonia than corals without endosymbionts [20]. The uptake of ammonia by green paramecia suggests that this compound is the nitrogen source of the endosymbiotic Chlorella spec. But the question arises, whether this ammonia is available to the algae in inorganic form or bound to an organic transport metabolite. Different activities of the key enzymes of ammonia metabolism in algae living within the symbiotic unit and in algae grown in mass cultures with ammonia as nitrogen source indicate that ammonia could probably not be the only nitrogen source of the endosymbiotic algae in the paramecia. Correspondence between enzymatic patterns in the isolated symbiotic algae grown on glutamine and the chloreUae within the symbiotic unit suggest glutamine as a possible additional nitrogen source of the algae. Glutamine can serve as a non-poisonous transport metabolite of ammonia in ammonothelic animals, to which also the genus Paramecium belongs [ 18]. Since the isolated symbiotic chlorellae excrete glutamic acid when grown on glutamine, a glutamine-glutamic acid shuttle system for ammonia supply of the symbiotic algae is conceivable. What is more, the Paramecium fraction of green ciliates shows a significant GIS-activity. Probably because of its poisonous character for their metabolism, ammonia is excreted by aposymbiotic Paramecium species [18,19] and algae-free P. bursaria. In the green P. bursaria the symbiotic chlorellae probably detoxicate the host metabolism from ammonia and also prevent a loss of nitrogen from the symbiotic unit. Even more, ammonia uptake from the culture medium by green paramecia leads to the assumption that the endosymbiotic chlorellae are able to withdraw nitrogen compounds from the Paramecium, so forcing the ciliate to take up an external nitrogen compound, which normally is a waste product of its metabolism. ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft. We wish to thank Dr. R. Tischner for his advice in measurements of nitrate uptake. REFERENCES 1 J.A. Brown and P.J. Nielsen, J. Protozool., 21 (1974) 569. 2 W. Reiuer, Arch. Mikrobiol., 111 (1976) 161.

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