{Accepted 28 March 1985) SUMMARY

Transcription

1 NOT f/iy/o/(1985) 100, 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 Biochemistry, Waite Agricultural Research Institute and Botany*, University of Adelaide, Adelaide, South Australia 5000 {Accepted 28 March 1985) SUMMARY Growth of Pezizella ericae Read in solution culture with ammonium (NHJ") as the primary nitrogen source was accompanied by decrease in ph and amtnonium concentration. Both glutamate dehydrogenase (GDH) and glutamine synthetase (GS) were involved in ammonium assimilation. Activities of these enzymes changed during the time course of the experitnent (28 d). Maximum GDH activity [035 /imox NAD(P)H oxidized min"' mg protein"'] occurred when external NHJ concentration was high (5 to 10 mm). Maximum GS activity (0-2 fimo\ of y-glutamyl hydroxamate min"' mg protein"') occurred when external NHj concentration was low (1 mm). L-Methionine DL sulphoximine (MSX), a specific inhibitor of GS, inhibited uptake of '^NHjCl in 13-d.-old mycelium of P. ericae. The nitrogen nutrition of the fungus and the host in the soil environment is discussed. Key words: Ericaceous mycorrhiza, N assimilation, '^N uptake, glutamine synthetase, glutamate dehydrogenase. INTRODUCTION Ericaceous plants, e.g. Calluna and Vaccinium, often grow on acid soils where soil nitrogen is likely to be present as ammoniuna. Mycorrhizal infection results in increased plant growth and higher nitrogen content of host plants (Read & Stribley, 1973; Stribley & Read, 1974). Pearson & Read (1975) showed that Pezizella ericae Read, a mycorrhizal fungus of ericaceous plants, could use ammonium as a primary source of nitrogen when grown in axenic culture. Stribley & Read (1975) showed that Vaccinium plants became enriched with ^^N when [^^N]glutamine was supplied to the associated P. ericae. Thus the ability of the fungus to use amnnonium and to translocate ^^N-labelled compounds is potentially important in the nitrogen nutrition of the host. However, the mechanisms by which ammonium is absorbed and metabolized by the fungus have not been studied. The enzymes of NH+ assimilation likely to be important in the fungus are glutamate dehydrogenase (GDH) and glutamine synthetase (GS). In Neurospora crassa, some yeasts and bacteria, GDH is though to be the most im^portant enzyme of NH J assimilation when external concentrations of ammonium are high (Brown, Macdonald-Brown & Meers, 1974; Mora et al., 1980). The GS/GOGAT pathway is considered to be more important in NH^ assimilation in plants when external concentrations of NHJ are low (Miflin, Lea & Wallsgrove, 1980). In this paper we report results of experiments in which the growth of P. ericae, the activities of GS and GDH and the uptake of I'NHjCl were investigated X/85/ $03,00/ The New Phytologist

2 580 B. J. ST. JOHN et al. MATERIALS AND METHODS Culture Pezizella ericae isolated from Calluna vulgaris (isolate 1) and kindly supplied by Dr V. Gianinazzi-Pearson, was maintained on malt extract agar at 22 to 25 C. Mycelium and agar were cut from 28-d-old cultures and macerated in sterile distilled water (08 cm- colony + agar ml"'). One millilitre of this macerate was transferred into each 100 ml Erlenmeyer flask, containing approximately 35 ml of ammonium medium at ph The culture medium contained, in one litre: glucose 10 g; NH,H,PO, 2 g; KH.PO, 04 g; MgSO,. 7H,O 0 5 g; CaCl,. 2H,O 55-5 mg; yeast extract 50 mg; ferric citrate 5 mg; MnSO4.4H2O 5mg; ZnSOj. 7H2O 4 4 mg (Pearson & Read, 1975). To avoid precipitation of calcium phosphate, the medium was adjusted to ph 58, autoclaved, and prior to inoculation re-adjusted to ph Flasks were incubated at 22 C and mycelial mats were harvested at intervals. Three replicate flasks were harvested for fresh-weight and dry-weight determinations on the same day as samples used for enzyme determinations. Samples for dry weight were oven-dried at 80 C overnight. Measurements of ph and ammonium concentration were made in filtrates of culture media from every flask. Enzymes Mycelial mats were harvested and washed with ice-cold extraction buffer (0-05 M 3-[N-morpholino] propane sulphonic acid (MOPS), 5 mm EDTA, 3 mm MnClj. 4H20,1 mm dithiothreitol (DTT), ph 7 25) and ground in a chilled mortar (extraction buffer: g fresb mycelium 5:1). Extracts were centrifuged at g for 20 min, and the supernatant decanted and stored on ice. All enzyme assays were performed using this crude extract. GDH activity was measured spectrophotometrically by following the oxidation of NADH and NADPH at 340 nm. The 3 ml reaction mixture contained 100 mm potassium phosphate buffer ph 8; 133 mm (NHJ^SO^; 80//M C^^Cl^. llip; Q-ll mm NAD(P)H; and 30//I crude extract. This mixture was equilibrated for 2 min and the reaction was started by addition of the substrate, 7 5 mm a-oxoglutarate (after the method of Bhandari & Nicholas, 1981). GS activity was assayed by tbe transferase method (Shapiro & Stadtman, 1970). Reaction mixture (total volume 1 ml) contained 50 mm MOPS; 20 mm Na2AsO4.7H2O; 3 mm MnCl2.4H2O; 250 mm glutamine; 250 mm hydroxylammomum chloride; 5 mm ADP ph 6 5 at 30 C. Control tubes without glutamine were always included. The reaction was started by the addition of extract (50 fi\) and terminated after 15 min by the addition of 1 ml of 3-3 % (w/v) FeClg, 8 % (w/v) TCA in 2 N HCl. The absorbance of y-glutamyl hydroxamate at 540 nm was measured spectrophotometrically. GDH activity is expressed as //mol NAD(P)H oxidized min~> mg protein"' in the crude extract and GS activity as/fmol y-glutamyl hydroxamate produced min"^ mg protein "^ in the crude extract. Protein content was assayed by microbiuret method (Itzhaki & Gill, 1964) and ammonium concentration was measured using an Orion NH, electrode. At early harvests, fungal mats from several flasks were pooled to obtain sufficient material. At later harvests, replicate determinations were performed on extracts of mycelium from three separate flasks. Means and standard errors of means where given are for differences between flasks. The effect of L-methionine DL sulphoximine (MSX), a specific inhibitor of GS,

3 Ammonium assimilation by Pezizella ericae on uptake of ^^NH^Cl by fungal mats was investigated. Replicate fungal mats were incubated with 0 5 mm MSX for 1 h, washed with 100 ml sterile NHJ-free culture medium (ph 6-6) and incubated with 1 mm "NH^Cl (30 atom % excess) for 1 h. In control experiments fungal mats were subjected to the above procedure but not exposed to MSX. After incubation with ^"NH^Cl the mats were washed with 100 nml NH^-free medium, oven dried (80 C overnight) and dry weights were recorded. Nitrogen contents of dried mats were determined by micro-kjeldahl analysis. The '^N enrichments were determined in a VG micromass 602 E mass spectrometer (Isomass, Middlewich, Cheshire, UK) by the method of Brownell & Nicholas (1967). RESULTS AND DISCUSSION The results in Figure 1 show that increases in dry weight of the fungus P. ericae were associated with decreases in both ammonium concentration and ph of the culture medium. Uptake and assimilation of ammonium results in the extrusion of approximately one proton per ammonium ion (Raven & Smith, 1976) so that acidification of the culture solution occurs r E < 28 Fig. 1. Changes in ammonium concentration ( - )andph(< -#) in the culture medium associated with dry weight increase (O O) of Pezizella ericae. Means and standard errors of means where given are for three replicate cultures. The data in Figure 2 illustrate changes in the specific activities of GS and GDH at different stages of growth of the fungus. At early harvests (up to day 16) when NH^ concentration was high, GDH was more active than GS, but GS activity, while initially low, increased as the concentration of NH^ decreased in the culture medium. Maximum GDH activity of 035/^mol NAD(P)H oxidized min"^ mg protein"! was recorded at an ammonium concentration of 5 to 10 mm. Maximum

4 582 B. J. ST. JOHN et al i: 12 g < 4 1 c 'a> proi E - T ' mil - 1 GS activity O-l - - i < 1 \ Jr^ Yd I \, E O-l I o Time(d) Fig. 2. Changes in GS { ) and GDH (D D) activity during ammonium utilization by Pezizella ericae. Means and standard errors of means where given are for three replicate cultures. Amnrionium data taken from Figure Fig. 3. Changes in relative activities of NADH- (A A) and NADPH- (A ) dependent GDH during ammonium utilization by Pezizella ericae. Means and standard errors of means where given are for three replicate cultures. GS activity of 0-2 /*mol y-glutamyl hydroxamate produced min ' nig protein ' occurred at 1 mm ammonium. Figure 3 shows changes in the activities of NADH- and NADPH-dependent GDH. Biosynthetic activity (NADPH-GDH) contributed up to 85% (0-3/tmol min"^ mg protein"^) of the total GDH activity. As ammonium was utilized, biosynthetic activity decreased, indicating that the enzyme is most active under conditions of high NH+ availability. Catabolic GDH activity (NADH-GDH) remained comparatively constant despite large changes in NH^ concentration. High turnover of NH via this enzyme did not occur during the course of this experiment. Data from later harvests were variable. However, GS activity remained approximately constant from day 16 onwards while GDH activity declined. The

5 Ammonium assimilation by Pezizella ericae 583 decrease in GDH activity occurred in parallel with the decrease in the external ammonium concentration and in ph (Figure 1). As external ph decreases towards three, NH^ influx is likely to be inhihited because the membrane transport system is directly sensitive to low ph (e.g. Smith & Walker, 1978). Alternatively, decreased ph might decrease the electrical potential difference across the plasma membrane, as in other organisms, thereby decreasing the driving force for NH^ uptake (e.g. Smith & Walker, 1978). Ammonium is therefore 'functionally' less available and uptake of NHJ would proceed via GS. An experiment to ascertain the effect of MSX on '^N uptake was performed on 13-d-old fungal cultures. In mycelia of this age, GS activity had become a significant proportion of the total (GS plus GDH) enzyme activity (Figure 2). Exposure to MSX, a specific inhibitor of GS, resulted in a 53 % inhibition of'^nh^ uptake from 0253 to 0-118//.g N mg d. wt"^* h~^ This demonstrates the relative importance of glutamine synthetase in the uptake of ammonium from the culture medium at this stage of growth. We have shown that both GS and GDH are present in this fungus - however, the contribution of either enzyme to ammonium uptake from the soil solution is likely to depend on the concentration of ammonium ions in the root environment. Fungal GS able to operate in conditions of low ammonium availability is likely to be important in nitrogen acquisition by a mycorrhizal host plant. Moreover, fungal hyphae can penetrate soil outside the depletion zone and exploit nutrient sources otherwise unavailable to the host. In situations of nutrient abundance, GDH would gain relative importance. In soils of neutral or alkaline ph, soil nitrogen can exist as nitrate. Further experiments are needed to investigate the nitrate-assimilating capabilities of these fungi. As it is possible to culture ericaceous mycorrhizal fungi axenically, the opportunity exists to study, both separately and in symbiosis, the biochemistry of nitrogen assimilation of the fungal and plant partners. ACKNOWLEDGEMENTS We would like to thank Mr D. Hein and Mr M. Jurgs for skilled technical assistance. The financial support of the Australian Research Grants Scheme is gratefully acknowledged. REFERENCES BHANDARI, B. & NICHOLAS, D. J. D. (1981). Some properties of glutamine synthetase from the nitrifying bacterium Nitrosomonas europaea. Australian Journal of Biological Science, 34, BROWN, C. M., MACDONALD-BROWN, D. S. & MEERS, J. L. (1974). Physiological aspects of microbial inorganic nitrogen metabolism. Advances in Microbial Physiology, 11, BKOWNELI., P. F. & NICHOLAS, D. J. D. (1967). Some effects of sodium on nitrate assimilation and Nj fixation in Anabaena cylindrica. Plant Physiology, 42, ITZHAKI, R. F. & GILL, D. M. (1964). A microbiuret method for estimating proteins. Analytical Biochemistry, 9, MiFLiN, B. J., LEA, P. J. & WALLSGROVE, R. M. (1980). The role of glutamine in ammonia assimilation and reassimilation in plants. In: Glutamine : Metabolism, Enzymology and Regulation (Ed. by J. Mora & R. Palacios), pp Academic Press, New York. MORA, J., DXVILA, G., ESPIN, G., GONZALEZ, A., GUZMAN, J., HERNANDEZ, G., HUMMELT, G., LARA, M., MARTINEZ, E., MORA, Y. & ROMERO, D. (1980). Glutamine metabolism in Neurospora crassa. In: Glutamine: Metabolism, Enzymology and Regulation (Ed. by J. Mora & R. Palacios), pp Academic Press, New York.

6 584 B. J. ST. JOHN e/a/. PEARSON, V. & READ, D. J. (1975). The physiology of the myeorrhizal endophyte of Calluna vulgaris. Transactions of the British Mycological Society, 64, 1-7. RAVEN, J. A. & SMITH, F. A. (1976). Nitrogen assimilation and transport in vascular land plants in relation to intracellular ph regulation. The New Phytologist, 76, READ, D. J. & STRIBLEY, D. P. (1973). Effect of mycorrhizal infection on nitrogen and phosphorus nutrition of ericaceous plants. Nature, Neu: Biology, 244, SHAPIRO. B. M. & STADTMAN, E, R. (1970). Glutamine synthetase (Escherichia coli). Methods in Enzymology, 17 A, SMITH, F. A. & WALKER, N. A. (1978). Entry ofmethylammonium and ammonium ions in CAara internodal cells. Journal of Experimental Botany, 29, STHIBLEY, D. P. & RE.\D, D. J. (1974). The biology of mycorrhiza in the Ericaceae. IV, The effect of mycorrhizal infection on uptake of "N from labelled soil by Vaccinium viacrocarpon (Ait). The New Phytologist, 73, STRIBLEY, D. P. & READ, D. J. (1975). Some nutritional aspects of the biology of ericaceous myccrrhizas. In: Endomycorrhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker), pp Xcademic Press, New York.

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