NUCLEAR MAGNETIC RESONANCE STUDY OF AMMONIUM ION ASSIMILATION BY LEMNA GIBBA L.

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$BY EDNA BEN-IZHAK MONSELISE\ DANIEL KOST^ DAN PORATH* AND MOSHE ^Department of Biology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel and ^ Department of Chemistry, Ben Gurion$

1 X/87/ S03.00/ The New Phytologist Pftyto/. (1987) 107, v^ 34I NUCLEAR MAGNETIC RESONANCE STUDY OF AMMONIUM ION ASSIMILATION BY LEMNA GIBBA L. BY EDNA BEN-IZHAK MONSELISE\ DANIEL KOST^ DAN PORATH* AND MOSHE ^Department of Biology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel and ^ Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel {Accepted 31 March 1987) SUMMARY ^*N Nuclear magnetic resonance (NMR) spectroscopy was used to follow nitrogen assimilation and amino acid production in Lemna gibba L. exposed to 0 4 mm ^^NH^Cl solution for 24 h. NMR analysis of the "N content of the treated plant tissues (aqueous extract) revealed "N incorporation into glutamine (N-amide plus amino-n or amide-n only) and glutamic acid and no detectable free ammonium ion. Methionine sulphoximine (MSO), an inhibitor of glutamine synthetase, at 10 mm inhibited completely the incorporation of ^*N. In the presence of 05 mm azaserine (AZA), a glutamate synthase inhibitor, the incorporation of ^*N was detected only in the amide group of glutamine. The results confirm the involvement of the glutamine synthetase/glutamate synthase (GS/ GOGAT) pathway in the assimilation of ammonium ions. Key words: Lemna gibba, GS/GOGAT pathway, ammonium ion assimilation; ^*N-NMR, MSO and AZA. INTRODUCTION The most common source of plant nitrogen is nitrate. However, ammonium ions can also be used as a source of nitrogen by some higher plants in which they are taken up at similar or even at higher rates than nitrate. For many years the assimilation of ammonia in plants was thought to be mediated by the GDH (glutamate dehydrogenase) pathway (Folkes & Yemm, 1958; Singh & Srivastava, 1982; Quetz, Tischner & Lorenzen, 1982; Cammaerts & Jacobs, 1985), but more recently an alternative GS/GOGAT pathway has been established (Lea & Miflin, 1974; Miflin & Lea, 1980; Woo & Osmond, 1982; Berger et al., 1986). The exact pathway(s) of ammonia assimilation has not been elucidated. Both the GS/GOGAT (involving glutamine as the primary product) pathway and the GDH pathway of NH4^ assimilation, are potentially operative in higher plant cells (Martin et al., 1986). The relative importance of these two alternative pathways is still a matter of debate. One approach to a solution of this problem is by examination of the uptake of "N-labelled precursors by the plant and their conversion to labelled products. The incorporation of ^^N can be followed by nuclear magnetic resonance (NMR) spectroscopy (Moor, Ratcliffe & Williams, 1983), and there is an increasing interest in applying this tool to plant tissues (Loughman & Ratcliffe, 1984; Belton, Lee & Ratcliffe, 1985; Martin et al., 1986). The work reported here investigates the pathway of ammonium ion assimilation in Lemna gibba, a floating aquatic angiosperm. The assimilation of ^^N-labelled

2 342 E. B.-I. MoNSELiSE e^ a/. t ammonium ion and the eflfect of the inhibitors, methionine sulphoximine (MSO) and azaserine (AZA), on this process were studied by applying the NMR t e c h n i q u e, i '-" ''J'--'^';;" "' ' - - ' ''-"--'^ '. : '. MATERIALS AND METHODS Axenic cultures of L. gibba L. G-3 (Cleland & Briggs, 1968) and L. gibba L. Hurfeish (Porath, Efrat & Arzee, 1980) were used. The plants were grown at 24±1 C in 250 ml Erlenmeyer flasks containing 100 ml Hutner's medium supplemented with 1 5 x 10"^ M sucrose, under continuous light (about IOW m~^) supplied by cool white fluorescent tubes. Ammonium ion uptake was studied by adding to 100 ml Erlenmeyer flasks, 50 ml sterilized distilled water, filter-sterilized (Sartorius Membrane filter 0*45 ju,) ammonium ions (0*4 mm ^^NH4Cl-99 Atom % Amersham International pic, Amersham, UK) and methionine sulphoximine (MSO 1 mm; Sigma, USA) or azaserine (0-5 mm O-diazoacetyl-L-serine; Sigma, USA) as mentioned. About 0-1 % (fresh weight) duckweed biomass was added to each flask and incubated for 24 h in continuous light as mentioned above. Ammonium ions remaining in the medium were determined with a Klett-Summerson spectrocolorimeter using Nessler reagent according to Rand, Greenberg & Tarns (1975) and with Na/K tartrate (2%) as a stabilizer. The plants (1-0 g) were removed for NMR analysis. They were washed with distilled water sonicated (10 min) with 10 cc distilled water and centrifuged at 5000^ for 10 min to obtain a 5 CC supernatant which was taken for analysis. 15^ NMR measurements were performed on a Bruker WP-200 SY Fourier transform NMR spectrometer operating at MHz. Broad-band protondecoupled ^^N NMR spectra were obtained with the following spectrometer conditions: 30/AS pulse duration, 7-2 s recycle time, to accumulations, and 16 K Fourier data transform. Spectra were recorded in 10% DgO to provide a lock signal. Chemical shifts were reported relative to ^^NH^"*" at 0 ppm. Temperature was maintained at 27 C. Measurements were carried out at ph 5-7. RESULTS Uptake of ammonium ions In medium without addition of inhibitors, clones G-3 and Hurfeish removed and %, respectively, of the added ammonium ions. In both clones, MSO, a GS inhibitor, completely blocked the ammonium uptake, while AZA, a GOGAT inhibitor, caused only partial inbibition of the uptake ( % of the added ammonia in G-3 and % in Hurfeish). (The percentages are averages of 10 replicates+ SE.) studies The NMR method allowed the rapid determination of intracellular enrichment of the ^^N-labelled amino acids. Under (normal) optimal uptake conditions, aqueous extracts from L. gibba clones showed, after exposure to ^^NH^"*" for 24 h three peaks: a strong one at 90-6 ppm due to the glutamine amide- N, another strong peak at 19-7 ppm assigned to the amino-n of glutamine and glutamic acid, and a weaker resonance at 11-6 ppm due to y-aminobutyrate (according to Martin, 1985). Ammonium ion at 0 ppm could not be detected (Fig. 1).

3 Assimilation of ammonium by Lemna gibba 343 I I I I I r I I 1 I 1 I I I I I I (b) _L (ppm) Fig. 1. (a) ^'NMR spectra (2O-26 MHz) of aqueous extract of Lemna gibba Hurfeish exposed to "NH^Cl (99 Atom %) for 24 h. (b) N NMR spectra of free amino acids at ph 5-7. Spectral conditions were as given in 'Materials and Methods'. Chemical frequencies (mgppm): "NH/, 0; 11-6, y-aminobutyrate; Gin N^, 19-7; Gin N^, In the presence of AZA, a GOGAT inhibitor, a distinct change in the ^^N NMR spectra was observed. This inhibitor caused an increase in the signal at 90*6 ppm due to glutamine amide-n and a dramatic decrease in the signal at 19*7 ppm due to the amino-n group of glutamine and glutamic acid (Fig. 2). DISCUSSION Our results are consistent with the GS/GOGAT pathway being the major route of NH4'^ assimilation in L. gibba, as was reported previously for L. minor (Stewart & Rhodes, 1976; Rhodes, Sims & Folkes, 1980). Ammonium ion uptake was drastically inhibited by the presence of GS/GOGAT inhibitors. GDH activity was not able to sustain NH^"*^ assimilation when GS was inhibited by MSO. It appears that despite the high ammonium ion concentration used in the present study, the GDH pathway did not play a substantial role in the assimilation of this ion in L. gibba. From the incorporation into amino acids (Fig. 1), it is indicated that the major recipients of newly assimilated ^^N during ^^NH^^ feeding were glutamine, glutamic acid and y-aminobutyrate (non-protein amino acid) Azaserine

4 344 E. B.-L MoNSELiSE et ah (ppm) Fig. 2. *'N NMR spectra (20-26 MHz) of aqueous extracts of Lemna gibba Hurfeish clone exposed to "NH«C1 (99 Atom %) + AZA (GOGAT-inhibitor) for 24 h. Spectral conditions were as given in 'Materials and Methods'. Chemical shifts (ppm):"nh/, 0; Gin N., 197; Gin N^, caused a sharp decrease in the ^*N labelling of glutamic acid and glutamine amino- N, and an increase in the labelling of glutamine amide-n. These changes are consistent with the direct incorporation of newly absorbed ammonium nitrogen into the N-amide position of glutamine (showing the strong signal at 90 6 ppm) with the N-amino of this amino acid being derived from originally unlabelled glutamic acid and showing a weaker resonance at 19*7 ppm (Fig. 2). In conclusion, use of ^*N-NMR allowed the rapid examination of nitrogen assimilation and amino acid biosynthesis. This paper appears to be the first to report on ^*N-NMR analysis of an aqueous extract of a higher plant. By using this technique, we conflrmed the results of other authors (Lea, 1982), emphasizing the importance of the action of the two enzymes GS/GOGAT in ammonium ion assimilation in higher plants. However, as stated by Stewart & Rhodes (1977), care should be taken in extrapolating results from relatively simple systems, such as Lemna or tissue culture cells in an aquatic environment, to land plants in which transport of metabolites away from their cellular subcellular sites of synthesis occurs rapidly and which never flnd themselves surrounded completely by a medium rich in NH4''". ACKNOWLEDGEMENTS We wish to thank Professor A. Lapidot for invaluable suggestions and Mrs A. Levkoviz, Mrs R. Massil and Mr G. Razial for kind and skilled co-operation. REFERENCES BELTON, P. S., LEE, R. B. & RATCLIFFE, R. G. (1985). A "N nuclear magnetic resonance study of inorganic nitrogen metabolism in barley, maize and pea roots. Journal of Experimental Botany, 36 (163), BERGER, M. G., SPRENGART, M. L., KUSNAN, M. & FOCK, H. P. (1986). Ammonia fixation via glutamine

Assimilation of ammonium by Lemna gibba 345 synthetase and glutamate synthase in the CAM plant Cissus quadrangular is L. Plant Physiologv 81 356-360. J' BJ',, CAMMAERTS, D. & JACOBS, M. (1985).

5 Assimilation of ammonium by Lemna gibba 345 synthetase and glutamate synthase in the CAM plant Cissus quadrangular is L. Plant Physiologv J' BJ',, CAMMAERTS, D. & JACOBS, M. (1985). A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana. Planta, 163, CLELAND, C. F. & BRIGGS, W. K. (1968). EfFect of low intensity red and far red light and high intensity white light on the flowering response of the long day plant L. gibba G-3. Plant Physiology FOLKES, B. F. & YEMM, E. W. (1958). The respiration of barley plants. X. Respiration and the metabolism of amino acids and proteins in germinating grain. New Phytologist, 57, LEA, P. J. & MIFLIN, B. B. (1974). An alternative route for nitrogen assimilation in higher plants. Nature, 251, LEA, P. J. (1982). The assimilation of ammonia. In: Techniques in Bioproductivity Photosynthesis (Ed bv J. Coombs & D. O. Hall), pp Pergamon, Oxford. LOUGHMAN, B. C. & RATCLIFFE, R. G. (1984). Nuclear magnetic resonance and the study of plants In: Advances in Plant Nutrition, vol. 1 (Ed. by A. Lauchi & P. B. Tinker), pp Praeger New York. ' MARTIN, F. (1985). "N-NMR studies of nitrogen assimilation and amino acid biosynthesis in the ectomycorrizal fungus Cenococcum grartiforme. FEBS Letters, 182, MARTIN, F., STEWART, G. R., GENETET, I. & LETACON, F. (1986). Assimilation of "NH/ by Beech {Fagus sylvatica L.) eetomycorrhizas. New Phytologist, 102, MIFLIN, B. J. & LEA, P. J. (1980). Ammonia assimilation. In: The Biochemistry of Plants vol 5 (Ed bv B. J. Miflin), pp Academic Press, New York. * ^ ' MOOR, G. R., RATCLIFFE, R. G. & WILLIAMS, R. J. P. (1983). NMR and the biochemist Essavs in Biochemistry, 19, " PoRATH, D., EFRAT, Y. & ARZEE, T. (1980). Morphological patterns and heterogeneity in populations of duckweeds. Aquatic Botany, 9, QuETZ, P. C, TiscHNER, T. & LoRENZEN, H. (1982). Changes in the activity of enzymes involved in nitrogen metabolism in maize seedlings dependent on diflterent nitrogen sources. Biochemie und Phvsioloeie der Pflanzen, 177, RAND, H. C, GREENBERG, A. E. & TARNS, M. J. (1975). Standard Methods for the Examination of Water and Waste water, 14th Edn, pp American Public Health Association, Washington D.C. RHODES, D., SIMS, A. P. & FOLKES, B. F. (1980). Pathway of ammonia assimilation in illuminated Lemna minor. Phytochemistry, 19, SINGH, R. P. & SRIVASTAVA, H. S. (1982). Glutamate dehydrogenase activity and assimilation of inorganic nitrogen in maize seedlings. Biochemie und Physiologie der Pflanzen, 177, STEWART, G. R. & RHODES, D. (1976). Evidence for the assimilation of ammonia via the glutamine pathway in nitrate-grown Lemna minor L. FEBS Letters, 46, STEWART, G. R. & RHODES, D. (1977). Control of enzyme levels in the regulation of nitrogen assimilation. In: Regulation of Enzyme Synthesis and Activity (Ed. by H. Smith). London, Academic Press. Woo, K. C. & OSMOND, C. B. (1982). Stimulation of ammonia 2-oxo-glutarate-dependent Oj evolution in isolated chloroplasts by dicarboxylate and the role of the chloroplasts in photorespiratory nitrogen recycling. Plant Physiology, 69,

{Accepted 28 March 1985) SUMMARY

{Accepted 28 March 1985) SUMMARY NOT f/iy/o/(1985) 100, 579-584 5 7 9 ENZYMES OF AMMONIUM ASSIMILATION IN THE MYCORRHIZAL FUNGUS PEZIZELLA ERICAE READ BY B. J. ST. JOHN, S. E. SMITH, D. J. D. NICHOLAS AND F. A. SMITH* Departments of Agricultural