7 Enzymology of Nitrogen Assimilation in Mycorrhiza

Enzymology of Nitrogen Assimilation in Mycorrhiza IFTIKHAR AHMAD and JOHAN A. HELLEBUST Centre for Plant Biotechnology, Department of Botany, University of Toronto, Toronto, Ontario M5S 3B2, Canada

........................................

I. Introduction .

181 rrhizal fungi for studies of nitrogen metabolism ................ ....................... 184 A. Fungal cultures ............................................... 184 B. Nitrogen utilization ...................................

11. Pure culture

A . Isolation .................................................

IV. V. VI. VII.

Pathways of ammonium assimilation ......................................... Induction of anabolic and catabolic pathways Extracellular enzymes ............................................... Discussion .............................................. References ... ...............................................

I.

194 198 200

Introduction

It has long been recognized that the widespread occurrence of mycorrhiza, in both natural and managed fields is a symbiotic relationship based on the exchange of nutrients between fungal mycelia and host roots. However, the conventional view of mycorrhizal associations as an extension of the root system to increase the absorption of free nutrients in the soil appears to be an oversimplification. Read et al. (1989) have recently pointed out that most of the biomass of the fungus in some forms of mycorrhizal associations is located within or close to the root; a formation not extremely efficient in increasing the absorption area. It is becoming evident that the fungal symbiont may be playing an important METHODS IN MICRORIOI.OGY VOLUME 23 ISBN O-L?-5215?3-I

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role in mobilizing resources that are not directly accessible to the host root. Ectomycorrhizal fungi have long been known to be efficient utilizers of amino acids (Melin and Nilsson, 1953; Carrodus, 1966; Abuzinadah and Read, 1988), and a number of recent studies have demonstrated their release of proteinases (protein degrading enzymes) to free amino acid from the soil litter (El-Badaoui and Botton, 1989; Leake and Read, 1989). These features would render mycorrhizal fungi in direct competition with their saprotrophic counterparts and it has been speculated that the supply of carbohydrates by the host plant gives an advantage to the mycorrhizal fungi in this competition (Abuzinadah et af., 1986). This raises many important questions about the relationship between carbon balance and nitrogen metabolism in mycorrhizal systems. First of all, there is a need for the characterization of various fungal symbionts for their ability to utilize different nitrogen sources. Such data may be obtained for intact mycorrhiza and where possible, also for pure cultures of the fungal symbiont. The utilization of nitrogen sources by the fungal symbiont should be characterized in terms of uptake, assimilation, and the release of assimilated or waste products to the external medium. The question arises as to the regulation of enzymes of nitrogen metabolism in the fungal symbiont. The pathway of ammonium assimilation in higher plants is well studied, and in the root tissue of mycorrhiza the incorporation of ammonium into amino acids is expected to be carried out primarily via the combined action of glutamine synthetase (GS) and glutamate synthase (GOGAT) (Miflin and Lea, 1980; Oak and Hirel, 1985). The operation of a GS-GOGAT cycle in mycorrhizal fungi is still under scrutiny however, and so far no clear evidence has been presented for the presence of GOGAT in these micro-organisms (see Ahmad et af., 1990). There is evidence that the incorporation of ammonium in mycorrhizal fungi is at least in part carried out via the alternative pathway involving the aminating activity of glutamate dehydrogenase (GDH) (Martin er af., 1986; Dell er af., 1989). Deaminating activities of G D H enzymes, on the other hand, are known to play a major catabolic role generating 2-oxoglutarate from glutamate for carbohydrate metabolism. Thus GDH enzymes may play a key role in nitrogen metabolism of the fungal symbiont and may also act at branch points between nitrogen and carbon metabolism. Coupled with these pathways, the presence of highly active aminotransferases in mycorrhizal fungi (Khalid er af., 1988; Ahmad et af., 1990) is indicative of an efficient metabolic machinery present in these symbionts for the utilization of diverse nitrogen sources. Figure 1 presents a summary diagram of major pathways of nitrogen metabolism in mycorrhizal fungi. Fungi

Fig. 1. Summary diagram of major pathways of nitrogen metabolism. The scheme presented here emphasises the role of both the GS-GOGAT cycle and the NADPH-GDH pathway in inorganic nitrogen assimilation and that of the NADH-GDH in amino acid catabolism. The TCA cycle is shown to be linked to both ammonium assimilation and intermediary nitrogen metabolism. The scheme also signifies the role of membrane transport in the exchange of small molecular weight metabolites and the excretion of proteinase activities. The hydrolysis of soil proteins by excreted proteinases contributes to the extracellular amino acid pool.

are able to adapt to changing nutritional conditions by invoking diverse anabolic and catabolic pathways, which, because of subcellular localization of enzyme activities, are compartmentalized in cell organelles. So far little attention has been paid to the subcellular organization of metabolic pathways in mycorrhizal fungi. There is also a need for a better understanding of the synthesis and the regulatory properties of proteolytic enzymes released by fungal symbionts. All of these studies are essential for elucidating nitrogen metabolism of symbiotic fungi and would lead to a better understanding of the basis of the plant-fungus mutualism in mycorrhizal associations.

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11. Pure culture manipulation of mycorrhizal fungi for studies of nitrogen metabolism

A. Fungal cultures

Many ectomycorrhizal fungi grow readily in pure cultures (Molina and Palmer, 1982). Over the years a number of artificial media have been formulated for propagating these symbiotic fungi under defined conditions (Melin and Rama Das, 1954; Palmer and Hacskaylo, 1970; Martin et al., 1983, 1988). Surprisingly, all of these so-called defined media omit at least one major nutrient, calcium, and even when supplemented with trace elements as suggested by Molina and Palmer (1982), do not contain a full complement of micronutrients and vitamins. The use of these incomplete nutrient 'media can hamper the study of nitrogen metabolism seriously since enzymes such as glutamate dehydrogenase and nitrate reductase are known to be affected adversely by the lack of calcium and molybdenum, respectively. We have recently formulated a buffered medium (Ahmad et al., 1990) that contains all major mineral nutrients and is enriched with micronutrients and vitamins. The medium can be prepared from the following solutions. Macronutrients and buffer stock solution. 65 mM KCl + 20 mM MgS04 + 3 mM CaC12 + 15 mM NaCl + 20 mM 2-(N-morpholino) ethanesulphonic acid (MES) adjusted to pH 5.5 with NaOH. Micronutrient stock solution. 2 mM NaH2P04 + 1 mM boric acid + 1 8 p M MnCI2 1 6 p M ZnS04 + 0 . 6 p M Na2Mo04 + 1 p M CoCI2 + 0.8 p M CuS04 + 230 p M sodium iron salt of EDTA + 200 p g litre-' thiamine hydrochloride + 10 pg litre-' biotin 10 p g litre-' vitamin B12.

+

+

Carbon stock solution. 500 mM glucose. Inorganic nitrogen stock solution. 50 mM NaN03 (nitrate media) or 50 mM NH4CI (ammonium media). The stock solutions are kept under refrigeration. The medium is prepared by adding 100ml each of the four stock solutions to a final volume of 1 litre. In experiments where amino acids are used as nitrogen sources, the inorganic nitrogen stock solution is substituted by solid additions of amino acids. Glucose addition can also be substituted or omitted according to experimental conditions. Media are made ready for inoculation by autoclaving 100 ml fractions in 500 ml wide-mouth

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conical flasks except for solutions of heat-labile nitrogen sources such as glutamine, which are filter-sterilized separately.

B. Nitrogen utilization Most ectomycorrhizal fungi are able to utilize both inorganic and organic nitrogen sources. Growth rates and final biomass production give an overall estimate of the fungal efficiency in assimilating a given nitrogen source. The disappearance of nutrients from the growth media as a function of growth rate ( p ) has been used for estimating the long-term uptake of compounds in microbial cultures. The most conventional way of measuring nutrient uptake by micro-organisms is by determining the radioactivity of filtered cells after a period of incubation in the presence of a radiolabelled nutrient source (Hellebust and Lin, 1978). The uptake of I4C- and "-labelled compounds can be quantified conveniently by scintillation counting. In laboratories with facilities for heavy isotope analysis, the incorporation of "N-labelled compounds allows a direct and more specific measure of nitrogen utilization (Rhodes et a)., 1981; Genetet et al., 1984). 111. Isolation and characterization of enzyme activities

A.

Isolation

Most nitrogen assimilatory enzymes are soluble proteins and thus are readily extracted by any of the standard tissue homogenization techniques. Grinding with acid-washed sand has been successfully used for extracting various mycorrhizal fungal enzymes (Khalid et af., 1988; Ahmad et al., 1990). Mycorrhiza and non-mycorrhizal roots may be ground in the presence of polyvinylpyrrolidone (Dell er af., 1989) to protect enzymes against phenolic compounds released during tissue maceration. The macerate is usually clarified by centrifugation. These crude enzyme preparations can be purified partially either by ammonium sulphate fractionation and/or by one of the standard chromatographic techniques (Martin et af., 1983; Dell et af., 1989; Ahmad et af., 1990). Glutamine synthetase, glutamate dehydrogenase and aminotransferases show good resolution on anion-exchange columns, and chromatographic separations have been improved considerably by the introduction of fast protein liquid chromatography. The stability of enzyme activities during purification and the level of purification required for assay procedures are the two important criteria for selecting appropriate

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extraction and purification protocols. These issues are addressed in the following discussion of the characterization of various enzyme activities. B.

Enzyme characterization and assay procedures

1. Nitrate reductase Nitrate reductase (NR) is a labile enzyme and for this reason a number of protectants are added to the extraction media (Wray and Fido, 1990). These protectants usually include nitrate, which is an NR substrate; FAD, which is a prosthetic group present in the protease-sensitive region of NR; EDTA, which chelates toxic metals released during cell breakage; and sulphydryl compounds such as dithiothreitol and mercaptoethanol, which prevent the oxidation of essential SH-groups of NR. Protease inhibitors such as phenylmethylsulphonyl fluoride are usually added to inhibit proteolysis during extraction. Exogenous proteins such as bovine serum albumin and casein are routinely used to enhance the stability of NR. A detailed study of in vitro measurement of NR in the basidiomycete Hebeloma cylindrosporum has been carried out by Plassard et al. (1984a). Nitrate reductase can be assayed directly in crude cell-free preparations. The overall physiological reaction of NR is usually determined by the reduction of nitrate in the presence of NAD(P)H followed by the colorimetric measurement of the nitrite produced: Nitrate

+ NAD(P)H NR, Nitrite + NAD(P)

In higher plants, nitrate reductase is NADH-specific, whereas in fungi the enzyme shows a preference for NADPH (Beevers and Hageman, 1980). In Hebeloma cylindrosporum the activity of NR is shown to be strictly dependent on NADPH as the electron donor (Plassard et al., 1984a). The following assay procedure is a modification of Scholl et al. (1974). The reaction mixture contains 25 mM potassium phosphate (pH 75), 10 mM potassium nitrate, 0.5 mM NADH or NADPH and up to 0.25 ml enzyme in a final volume of 0.5 ml. The reaction is carried out for 20-30 min at 25 "C and stopped by adding 0.25 ml of a 200 mM zinc acetate solution. The precipitated material is cleared by centrifugation. The residual pyridine nucleotide in the reaction mixture is oxidized by adding 0.25 ml of a freshly prepared 50 p M phenazine methosulphate solution and leaving the mixture at room temperature for about 20 min. The colour development is initiated by adding 1 ml each of the diazocoupling reagents 1% sulphanilamide solution in 3 N HCI and 0.02%

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N - ( 1-naphthy1)-ethylenediamine dihydrochloride solution in 0.1 N HCl. After 20 min the absorbance of the pink diazo dye is read at 540 nm and the amount of nitrite is determine from a standard curve for 10-100 nmol nitrite. The K , for nitrate and NADPH of the fungal nitrate reductase range from 60-200 p M and 9-60 kM , respectively (Beevers and Hageman, 1980). The K , values for nitrate and NADH are similarly low for the plant enzyme. Nitrate reductase is a substrate-induced enzyme and is usually found only in the presence of nitrate. In fungi, the presence of ammonium in the culture medium completely abolishes the nitrate-mediated induction of nitrate reductase (Lewis and Fincham, 1970). A similar inhibition of nitrate reductase induction has been shown in plant roots (Smith and Thompson, 1971; Radin, 1975).

2. Nitrite reductase One of the major differences between the plant and fungal nitrite reductase (NiR) is in their specificity for electron donors. The plant enzyme accepts electrons from reduced ferredoxin whereas the fungal enzyme utilizes pyridine nucleotides as the electron donor and generally shows a preference for NADPH. The reaction catalysed by NADPH-NR is considered to involve a sequential transfer of electrons: NADPH + FAD + siroheme + nitrite NADPH-NR extracted from Hebeloma cylindrosporum shows a rapid loss of activity but can be reactivated by the addition of sodium dithionite and methylviologen as the electron donor (Plassard et al., 1984b). These authors have suggested that the loss of NADPH-NR activity in the fungal extract was associated with the step involved in the transfer of electrons from NADPH to FAD, and the reduction of siroheme by methylviologen bypassed the need for electrons from NADPH. The overall reaction of NiR can be summarized as: Nitrite

+ reduced ferredoxin or NAD(P)H NIR\ NH; + oxidized ferredoxin or NAD(P)

The activity of nitrite reductase is usually measured by determining the disappearance of nitrite in the assay system. For pyridine nucleotidedependent assay, the following modification of Garret (1972) may be used. The reaction mixture contains 50 mM potassium phosphate (pH 7 . 9 , 1 mM potassium nitrite, 0.05 mM FAD, 0.5 mM NADPH or NADH and enzyme in a final volume of 0.5 ml. After 15 or 20 min incubation at 25 "C, the reaction is stopped and the amount of nitrite in the sample is

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determined according to the procedure described for nitrate reductase assay. For assaying ferredoxin-dependent nitrite reductase activity, the above reaction mixture is modified to substitute NAD(P)H by 0.5 mM ferredoxin and 12 mM sodium dithionite. The reaction is started by the addition of dithionite and stopped by vigorous shaking. Residual nitrite is measured as above. As with nitrate reductase, nitrite reductase is a high affinity enzyme and shows K , values for nitrite in the micromolar range. The fungal and plant NiR differ considerably in their molecular size. In higher plants NiR has a molecular mass of about 63 kDa (Wray and Fido, 1990) which is four times lower than the molecular weight of 290 kDa reported for NiR isolated from Neurospora crassa (Nason er al., 1954). The molecular mass of the purified NiR is usually determined by gel chromatography. 3. Glutamate dehydrogenase The in vitro activity of glutamate dehydrogenase (GDH) enzymes can be assayed both in the aminating (assimilatory) and deaminating (catabolic) directions. NH:

+ 2-oxoglutarate + NAD(P)H

- Glutamate + NAD(P)

GEH

The root tissue of higher plants usually contains a mitochondria1 NADH-linked GDH, which functions primarily in the deaminating direction. Many fungi possess both the NADH-linked catabolic enzyme and an NADPH-linked assimilatory GDH which can be separated in a one step anion-exchange chromatographic procedure (Fig. 2). The usual assay system of GDH activities is based on spectrophotometric monitoring of the oxidation of NAD(P)H, which gives a stoichiometric measure of the amount of glutamate produced. The following is a modification of Ahmad and Hellebust (1984). The reaction mixture of 1 ml contains (final concentration) 50 m M potassium phosphate (pH 7.5), 100 mM ammonium chloride, 20 mM 2-oxoglutarate, 0.2 mM NADH or NADPH and 0.2ml enzyme. The oxidation of NAD(P)H is monitored at 340 nm using an extinction coefficient ( E ) of 6.2. The assay must be corrected for non-specific oxidase activities present in the enzyme extract. These contaminating activities can be determined by omitting 2-oxoglutarate from the reaction mixture. Membrane-bound oxidases can be removed either by 20% ammonium sulphate precipitation followed by centrifugation at 10000 g for 10 min (Ahmad and Hellebust, 1986) or by high speed (30000 g ) centrifugation for 30 min (Ahmad et al., 1990).

Fraction number

Fig. 2. Elution profiles for the fractionation by anion-exchange chromatography of glutamate dehydrogenase (NADH-GDH, closed symbols; NADPH-GDH open symbols); glutamine synthetase (transferase activity, closed symbols; synthetase activity, open symbols) and alanine aminotransferase. A 4 ml extract prepared by grinding 1 ml packed volume of Laccaria bicolor was clarified by centrifugation (30000 g, 30min) and a 2ml fraction loaded onto a Mono-Q column attached to a fast protein liquid chromatography assembly. The composition of the elution media was as described by Ahmad and Hellebust (1987).

The pH optima of the amination reactions of both NADH-GDH and NADPH-GDH from various sources fall in the pH range 7-8, whereas the p H optima of the deaminating reactions are usually 1.0-1.5 pH units higher. These differences in pH optima appear to have broad implications for the functional relations of these enzymes. NADH-GDH from various sources have a high K , for ammonium ( 1 0 - 8 0 m ~ ) . The K , for ammonium of NADPH-GDH from higher plants and many fungi is also high. However, NADPH-GDH from some ectomycorrhizal fungi show biphasic kinetics with different K , values for ammonium (Martin et af., 1983; Ahmad et af., 1990). The lower K , value for these NADPH-GDH enzymes is in the range of 2 - 5 m ~ ammonium. The

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NADH-linked GDH of higher plants is a metalloprotein and requires added calcium to prevent inactivation by EDTA or other chelating agents. No calcium requirement has been reported for the fungal G D H enzymes. 4.

Glutamine synthetase

Two separate forms of glutamine synthetase (GS) exist in higher plants; one located in the cell cytosol and the other in plastids. GS activity in plant roots is shown to be predominantly cytosolic (Emes and Fowler 1979; Suzuki et al., 1981). The anion-exchange study of GS in the ectomycorrhizal fungus, Laccaria bicolor, shows the presence of a single molecular form (Fig. 2) with an elution profile on a sodium chloride gradient similar to that observed for cytosolic enzymes from many plants and algal sources (McNally er al., 1983; Casselton er al., 1986). The plastid enzyme requires the presence of sulphydryl reagents to prevent inactivation during isolation. However, these reagents are shown to suppress the activity of cytosolic GS from some sources (Wallsgrove et al., 1983; Ahmad and Hellebust, 1987). It may therefore be necessary to isolate the two GS forms by separate extraction procedures with appropriate sulphydryl composition of the isolation media. The two GS isoforms are also reported to differ in their thermal stability with the cytosolic form being considerably less labile (Mann et al., 1979; Ahmad et al., 1982). GS catalyses the formation of glutamine from ammonium and glutamate at the expense of ATP hydrolysis; the overall reaction can be summarized as: Glutamate

+ NH; + ATP

Glutamine

+ ADP + Pi + H 2 0

This reaction can be determined by a coupled spectrophotometric assay procedure where the ADP produced in GS reaction is linked to the conversion of phosphoenolpyruvate by pyruvate kinase to pyruvate, which in turn is linked to the oxidation of NADH by lactate dehydrogenase. The following is a modification of Stewart and Rhodes (1977a). The reaction mixture of 1 ml is prepared with 50 mM sodium phosphate buffer (pH 7.5) containing 100mM sodium glutamate, 10mM ATP, 10 mM ammonium chloride, 20 mM magnesium sulphate, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 5 units pyruvate kinase (PK), 2 units lactate dehydrogenase (LDH) and 200 p1 enzyme extract. The oxidation of NADH monitored at 340nm gives a stoichiometric estimate of the amount of glutamine produced by the GS reaction. A colorimetric procedure has been developed by substituting hydroxylamine (NH20H) for ammonium and determining the amount of

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y-glutamylhydroxamate produced spectrophotometrically following the development of a brown colour with ferric chloride. Glutamate

+ NHzOH + ATP GS,

y-Glutamylhydroxamate

+ ADP + Pi + H 2 0

This alternative assay gives a measure of GS reaction in the biosynthetic direction and is usually termed the synthetase assay. The V,,, and K , values determined by this method are usually similar to those obtained by a coupled assay system. The reaction is started in a 1 ml mixture prepared with 100mM Tris-HC1 buffer (pH 7.6) and containing 80mM glutamate, 20 mM hydroxylamine, 20 mM ATP, 50 mM magnesium sulphate and 200 pl enzyme extract (Ahmad and Hellebust, 1984). After a desired period of incubation (20-30 min) at 25 "C, the reaction is stopped by the addition of 1 ml of acidified ferric chloride solution (26 g of ferric chloride and 40 g of TCA in one litre of 1 N HCI). It is essential to run zero time controls by stopping the reaction immediately after the addition of enzyme extract to the reaction mixture. Where necessary, the precipitated material is removed by sedimentation in a bench-top centrifuge before reading the aborbance at 540 nm. A standard plot using a commercial glutamylhydroxamate preparation is established as a reference for GS activity. A third assay has been developed on the basis of the ability of GS to catalyze the y-glutamyl transfer reaction that results in the formation of y-glutamylhydroxamate from glutamine and hydroxylamine: Glutamine

+ NH20H

y-Glutamylhydroxamate

+ NH:

This reaction -termed transferase assay -gives rates several times higher than those obtained by the above biosynthetic reactions. After establishing the transferasesynthetase ratio of a given GS enzyme, this method can be used as a sensitive indicator of its activities at different stages of purification protocols. The transferase assay is based on the colorimetric determination of glutamylhydroxamate produced. The reaction is started in a 1 ml mixture with 100 mM Tris-acetate buffer (pH 6.4) containing 100 mM glutamine, 30 mM hydroxylamine, 30 mM sodium arsenate, 1.5 mM MnC12, 0.2 mM ADP and 200 p1 enzyme (Ahmad and Hellebust, 1984). The reaction is stopped after 30 min by the addition of acidified ferric chloride and GS activity is quantified as described for the synthetase assay. The kinetics of GS have been extensively studied. Various purified preparations have been shown to have high affinity for ammonium, but a considerably lower affinity for glutamate (Stewart et af., 1980). The ATP-dependent reaction of glutamine synthetase has a specific requirement for magnesium. The enzyme shows complex kinetics with respect

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to concentrations of ATP and magnesium and pH of the reaction system (Stewart et al., 1980).

5. Glutamate synthase Glutamine produced via the glutamine synthetase pathway is utilized by glutamate synthase (GOGAT), which catalyses the transfer of amido nitrogen to 2-oxoglutarate, resulting in the formation of two molecules of glutamate. The enzyme from green plants is specific for ferredoxin or ferredoxin-like proteins (Miflin and Lea, 1980; Suzuki et al., 1985) as the electron donor Glutamine

+ 2-oxoglutarate + reduced ferredoxin

2 Glutamate

G a T

+ oxidized ferredoxin

whereas GOGAT from various non-green micro-organisms is found to be NAD(P)H-specific (Stewart et al., 1980): Glutamine

+ 2-oxoglutarate + NAD(P)H

GoGqT

2 Glutamate

+ NAD(P)

The net result of the combined action of GS and GOGAT is the synthesis of glutamate from ammonium and 2-oxoglutarate; this combined action is frequently referred to as the GS-GOGAT cycle or simply as the glutamate synthase pathway. GOGAT can be measured directly in crude extracts. The enzyme is usually extracted in 50 mM phosphate buffer (pH 7.5) containing protectants such as sulphydryl reagents, mercaptoethanol and DTT, substrate 2-oxoglutarate and protease inhibitor PMSF (Marquez et al., 1988; Wallsgrove et a f . , 1977). In some cases it is necessary to solubilize membrane-associated ferredoxin-dependent GOGAT from plant tissue by the addition of 0.05% to 0.5% Triton X-100 to the extraction media. GOGAT activity is usually measured by the quantitative measurement of glutamate produced. For assaying in vitro ferredoxin-dependent GOGAT activity the electron donor can be replaced by reduced methyl viologen without significant change in activity (Marquez et a[., 1988). The enzyme extract is pre-incubated for 20 min in a 0.5 ml mixture containing 100 mM phosphate buffer, pH 7.5, 10 mM glutamine, 10 mM 2-oxoglutarate and 15 mM methyl viologen. The reaction is started by adding 100 pl of freshly prepared dithionite reductant mixture (235 mg sodium dithionite, 250 mg sodium bicarbonate in 5 ml water). In NAD(P)H-dependent GOGAT assays, methyl viologen in the reaction mixture and its reduction by dithionite mixture are omitted, and the reaction is started by the addition of 0.5 mM NADH or NADPH. After

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20 min at 25 "C, the reaction is stopped either by boiling or by the addition of 1 ml ethanol. Glutamate produced can be separated from glutamine and quantified by one of the chromatographic procedures employing paper chromatography (Wallsgrove et al., 1977), thin layer chromatography (see Lea et a f . , 1990), high-performance liquid chromatography (Martin et al., 1982), anion-exchange column chromatography (Hecht et al., 1988) or paper electrophoresis (Chen and Cullimore, 1988). Ninhydrin-based colorimetric determination of glutamate is routinely applied in these studies. The use of radiolabelled 14C glutamine in the reaction mixture allows a more sensitive determination of glutamate by scintillation counting (Wallsgrove et a f . , 1982). Pyridine nucleotidedependent GOGAT activity can be measured spectrophotometrically by following the oxidation of NAD(P)H provided the enzyme preparation does not contain non-specific NAD(P)H oxidase activities. The composition of the reaction mixture for spectrophotometric measurements is the same as that described above for NAD(P)H GOGAT activity except for the concentration of NAD(P)H, which is lowered to 0.25 mM to give an initial absorbance reading of less than 2. 6 . Aminotransferases

Glutamate synthesized either via the GDH pathway or the GS-GOGAT cycle is the primary amino donor for the synthesis of most other amino acids. Several enzymes catalysing the transfer of amino groups from glutamate to different keto acids have been identified in plants and fungi. The most active of these aminotransferases are aspartate aminotransferase (AsAT) and alanine aminotransferase (AlAT). The reactions of both aminotransferases are reversible: 2T + oxaloacetate A.Aspartate + 2-oxoglutarate AET Glutamate + pyruvate .- Alanine + 2-oxoglutarate

Glutamate

These enzymes are routinely measured spectrophotometrically by coupling the production of keto acids to the oxidation of NAD(P)H catalyzed by an auxiliary dehydrogenase enzyme. A typical reaction mixture for AsAT assay contains in a final volume of 1 ml, 50 mM sodium phosphate buffer (pH 7.5), 100 mM sodium aspartate, 10 mM 2-oxoglutarate, 0.25 mM NADH, 2 units malate dehydrogenase and purified enzyme extract (Ahmad and Hellebust, 1989). The reaction mixture for AlAT assay is similar in its composition to the reaction mixture for AsAT except for aspartate and for malate dehydrogenase, which are replaced by 1 0 0 m ~alanine and 2 units of lactate dehydrogenase, respectively. When the coupled reaction proceeds linearly, the oxidation of NADH gives a stoichiometric measure of

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aminotransferase activity. The spectrophotometric procedure for measuring aminotransferase activities using glutamate as the amino donor has been described elsewhere (Ahmad and Hellebust, 1989). Enzyme preparations clarified by high speed centrifugation (30 000 g , 30 min) and/or ammonium sulphate precipitation are usually adequate for the coupled enzyme assays. The reactions catalysed by aminotransferases require pyridoxal 5'-phosphate as a coenzyme, but in plants and fungi it usually remains tightly bound to the enzyme during different stages of purification and therefore its addition to the reaction mixture is generally not needed. Several isoforms of both alanine and aspartate aminotransferases have been identified from various plant and microbial sources. Figure 2 shows the elution profile by anion-exchange chromatography of alanine aminotransferase from the ectomycorrhizal fungus, Laccaria bicolor. The fungal extract is fractionated into two peaks of AlAT activity; eluting first a cationic minor isoform followed by a major anionic isoform. Cationic aminotransferases are considered mitochondrial in origin whereas anionic isoforms are thought to be cytosolic enzymes (Givan, 1980). IV. Pathways of ammonium assimilation Prior to the demonstration that GOGAT occurs in higher plants (Lea and Miflin, 1974), G D H was considered to be the primary enzyme of ammonium assimilation in both plants and micro-organisms. There is now considerable evidence that in vascular plants ammonium assimilation occurs almost exclusively via the GS-GOGAT cycle (see Miflin and Lea, 1980 for review). Incubating plants with methionine sulphoximine -a specific inhibitor of GS -almost invariably results in a complete inhibition of nitrogen assimilation in both root and shoot tissue. Feeding plants with "N-labelled nitrate or ammonium and following the enrichment of amino acid pools by gas chromatography/mass spectrometry ( G C N S ) or by nuclear magnetic resonance spectroscopy shows a rapid labelling of amido nitrogen of the glutamine pool that is indicative of the entry of inorganic nitrogen to organic molecules primarily via GS activity. These results exclude GDH from playing a role in ammonium assimilation by higher plants. The relative importance of the G D H pathway and GS-GOGAT cycle for ammonium assimilation in mycorrhizal fungi is, however, not clear. It may be noted that a large group of fungi lack GOGAT activities. Furthermore, "N labelling studies have produced convincing evidence for the assimilation of ammonium via the G D H pathway in the fungus

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Candida urilis (Folkes and Sims, 1974). There are, however, conflicting reports about the presence of GOGAT in mycorrhizal fungi. Recently, Vezina et al. (1989) reported an active NADH-dependent GOGAT in Laccaria bicolor. However, the levels of GOGAT activity measured by Vezina et al. (1989) appear extremely low compared with the activity of GS and other enzymes of nitrogen assimilation present in the same 'N labelling of Cenococcum fungal extract. Using a combination of ' geophilum and the inhibition of its GS activity by methionine sulphoximine, the work of Martin et al. (1988) has excluded a role of GOGAT in the synthesis of glutamate by this ectomycorrhizal ascomycete. The incorporation of nitrogen into amino acids in this fungus probably occurs via both the reductive amination reaction of G D H producing glutamate and the amination of glutamate by GS producing glutamine. A concurrent role of GS and NADPH-linked GDH pathways was also evident in our study of L. bicolor (Ahmad et al., 1990) which showed that the activity of these enzymes reached maximum levels in rapidly growing mycelia and declined rapidly during the onset of the stationary growth phase. A highly active NADPH-GDH has also been reported in Heheloma spp. (Dell et a f . , 1989). These basidiomycetes and possibly other mycorrhizal fungi with similar enzymic characteristics provide useful material for elucidating the relative importance of the two potential pathways in the assimilation of nitrogen by fungal symbionts. It will be rewarding to adopt the isotopic procedures described by Martin and his co-workers (Martin, 1985; Martin et a f . , 1986) for C. geophilum in monitoring "N labelling patterns of other fungal symbionts. The use of the GS inhibitor, methionine sulphoximine, has afforded information regarding the contribution of the GS pathway in the nitrogen assimilation of higher plants and green algae (Fentem et a[., 1983; Ahmad and Hellebust, 1985, 1986), and one expects that it can be applied equally successfully in mycorrhizal studies. Selection of GS and G D H fungal mutants has surprisingly not been undertaken so far in mycorrhizal research. Intraspecific variability of the NADPH-GDH from Hebeloma has been studied by Wagner et al. (1989). V.

Induction of anabolic and catabolic pathways

For the purpose of this chapter, anabolic pathways of nitrogen metabolism refer to activities associated with primary nitrogen assimilation leading to the biosynthesis of glutamate and the conversion of glutamate to other primary amino acids. Catabolic pathways refer to activities associated with the breakdown of amino acids for supplying carbon

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skeletons of amino acids to support energy production and growth. In this sense, the trophic condition of the organisms becomes the distinguishing mark for the expressed pathway. Fungi are usually able to utilize a variety of nitrogen sources including nitrate, ammonium, amino acids and proteins. In the fungal literature these nitrogen sources are often categorized as either inducers, repressors or neutrals according to their effects on the expression of a given pathway. The pathway leading to the utilization of a given nitrogen source may be either substrate inducible, product repressible or both. For example, in the ascomycetes, Emericella (Aspergillus) nidulans and Neurospora crassa , NR is induced by its substrate, nitrate, but is repressed by the product of nitrate reduction, ammonium, and also by the product of ammonium assimilation, glutamine (Tomsett, 1989). It may be noted, however, that the basidiomycete Hebeloma cylindrosporum has been reported to show similar levels of NR activity when incubated in the presence of either ammonium or nitrate as the sole nitrogen source (Scheromm et al., 1990~).Nitrogen sources such as urea and uric acid are considered neutral for NR as they neither induce nor repress NR activity. Substrate inducibility in E. nidulans and N . crassa is shown to be pathway specific. Their product-repression mechanisms, however, have been found to influence several pathways simultaneously. Thus a number of amino acid-catabolizing enzymes are completely repressed when these fungi are supplied either with ammonium or some primary amino acids such as glutamate and glutamine as nitrogen sources (Marzluf, 1981; Jennings, 1989). The non-filamentous ascomycete Saccharomyces cerevisiue has also been found to possess a similar control mechanism (Cooper, 1982). This control-termed nitrogen catabolite control-facilitates a preferred assimilation of ammonium, glutamate and glutamine by maintaining high levels of anabolic activities of GS and NADPH-GDH, and only after a complete utilization of these preferred nitrogen sources permits the synthesis and/or activation of enzymes and transport systems necessary for catabolizing other nitrogen sources. These observations, based on combined biochemical and genetic analysis, allow a clear understanding of how the various processes of anabolism and catabolism of nitrogen are regulated in these non-symbiotic ascomycetes. Little is known, however, about the regulation of nitrogen metabolism in mycorrhizal fungi. Ectomycorrhizal fungi are generally considered to be poor utilizers of nitrate nitrogen. Contrary to such assumptions, the growth of the Hebeloma cylindrosporum thalli is shown to be much faster in the presence of nitrate than in the presence of ammonium (Scheromm et ul., 1990b). A recent study showed that the ectomycor-

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rhizal basidiomycete, Laccaria bicolor is able to grow on both ammonium and nitrate as well as on several organic nitrogen sources (Ahmad et al., 1990). Of a large number of amino acids tested, glutamate was the most efficient nitrogen source for this fungus, and during exponential growth the utilization of glutamate was accompanied by high mycelial levels of GS, NADPH-GDH and AsAT activities. O n the other hand, mycelia growing under strict catabolic conditions, where media contained either arginine or alanine as sole carbon and nitrogen sources, contained high levels of NADH-GDH and AlAT activities and very low levels of GS, NADPH-GDH and AsAT activities. These results indicate that the regulation of nitrogen metabolism in the fungal symbiont is highly integrated and may be subject to elaborate transcriptional control, as has been observed in the well-studied non-symbiotic ascomycetes. Furthermore, the work of Wagner et al. (1988, 1989) suggests significant intraspecific genetic variations in the levels of nitrogenassimilating enzymes in fungal symbionts. There appears to be an urgent need for detailed biochemical and genetic analysis of the regulatory features of nitrogen metabolism in mycorrhizal fungi. Our knowledge of the regulatory features of nitrogen metabolism in plant roots is very limited at present. In vascular green plants, GSGOGAT constitutes an integral part of nitrogen anabolism in both roots and leaves (Miflin and Lea, 1980; Stewart et al., 1980), and is usually present in excess to ensure rapid ammonium assimilation (Blackwell et al., 1987). GS is, however, strongly inhibited by various amino acids, notably alanine and glycine. Glutamine, the product of GS reaction, has been identified as the key regulator of GS levels in plants (Rhodes et al., 1976; Stewart and Rhodes, 1977b). Glutamine has also been implicated in the control of NR levels in plants. However, Oak and Hire1 (1985) have argued that the substrate nitrate is the main regulator of both the synthesis of NR protein and its activity. Plants growing under autotrophic conditions are not dependent on exogenous carbon supplies. However, they usually possess a significant capacity for the degradation of a number of amino acids to TCA cycle intermediates. The activity of the mitochondria1 NADH-GDH in both leaves and roots is considered to be catabolic, and the enzyme is usually more active in the root tissue than in the leaves (Lee and Stewart, 1978). Some detailed studies of the regulatory diversity of nitrogen metabolism in the fungal symbiont and the plant root are clearly needed. However, any extrapolation of the regulatory mechanisms seen in pure cultures of the fungal symbiont and the host plant to the mycorrhizal tissue must be undertaken with caution and, wherever possible, attempts should be made to include the analysis of the mycorrhizal tissue in these

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studies. As the work on several mycorrhizal species (Martin, 1985; Genetet et al., 1984; Martin et al., 1986, 1988; Dell et al., 1989) suggests, overarching the endogenous control of nitrogen metabolism in the fungal symbiont there may be a control by the host root. The biochemical and genetic basis of such regulation of nitrogen metabolism by plant roots in symbiotic associations is not clear. It may be noted that a regulatory control of nitrogen metabolism exerted by plant roots is also found in legume-Rhizobium symbioses, where a number of nodulespecific proteins (nodulins) are encoded by specific plant genes (Verma et al., 1983). Interestingly, among these proteins is a GS isoform designated as GSN (Lara et al., 1983) that is synthesized by the plant root after nodulation where it functions together with the root-specific GS isoform (GSR) to ensure rapid ammonium assimilation during nitrogen fixation. Free living Rhizobium contain two GS isoforms; both are suppressed after nodulation (Ludwig, 1980). It must be added that any study of the fungus-plant interaction in mycorrhizal formations should consider the long-term nature of such symbiosis. This is an important consideration for monitoring the beneficial effects of the mycorrhizal association as short-term studies often fail to show any nutritional or growth difference between mycorrhizal and non-mycorrhizal plants (see Scheromm et al., 1990a).

VI. Extracellular enzymes

A special case of nitrogen catabolism in mycorrhiza is the utilization of soil proteins by some fungal symbionts. A number of ericoid and ectomycorrhizal fungi are able to grow on proteins as sole nitrogen sources (Read et al., 1989). These fungi-some of which have been termed “protein fungi” because of their preference for proteinaceous nitrogen sources (Abuzinadah and Read, 1986)-excrete acid proteinase enzymes when incubated in the presence of pure proteins such as bovine serum albumin (Leake and Read, 1989), or protein extracted from soil litter (El-Badaoui and Botton, 1989). Amino acids released by proteinase activity are taken up by the fungi and assimilated. For the induction of proteinase secretion the fungus is grown for several days in pure liquid cultures containing 0.5-1 glitre-’ protein. The appearence of proteinase activity can be monitored by determining the concentration of residual proteins in the culture filtrates at regular intervals. Protein can be quantified by the procedure described by Bradford (1976) or Lowry et al. (1951). A procedure allowing direct

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measurements of proteinase activities in the culture filtrate has been described by Twining (1984). This assay is based on the spectrofluorometric measurement of fluorescein isothiocyanate (F1TC)-labelled proteins. A modification of this procedure for mycorrhizal proteinase activities has been described by Leake and Read (1989). The kinetics of extracellular acid proteinases from mycorrhizal fungi are similar to those of acid and alkaline proteinases from other microbial sources (Read er al., 1989). The work of El-Badaoui and Botton (1989) suggests that ectomycorrhizal fungi are capable of releasing alkaline proteinases. These activities are highly stimulated when the fungus is incubated with proteins extracted from forest litter, and suppressed by low concentration of ammonium.

VII.

Discussion

The complexity of regulation of nitrogen assimilation in fungi has been clearly recognized in extensive studies of the three non-symbiotic fungi, Emericella (Aspergillus) nidulans , Neurospora crassa and Saccharomyces cerevisiae. These ascomycetes, in spite of sharing common pathways of nitrogen metabolism and displaying an overriding catabolite control, exhibit distinct patterns of regulation of nitrogen metabolism in response to changes in trophic conditions (Cooper, 1982; Tomsett, 1989). It has been realized that one of the key elements of the regulatory complexity in these fungi is the separation of certain enzymes in different cell or hyphal compartments. This allows simultaneous activation of several anabolic and catabolic processes, enabling the fungus to explore diverse nitrogen and carbon sources. In comparison with these studies of non-symbiotic fungi, work on the regulation of nitrogen metabolism in symbiotic fungi is still in its infancy. Recent developments in mycorrhizal research include the establishment of appropriate culturing and inoculating techniques, isotopic studies of inorganic and organic nitrogen utilization, and preliminary characterization of certain enzymes active in primary nitrogen assimilation. That litter proteins may play a major role in the nitrogen nutrition of mycorrhiza is being increasingly recognized lately. There is, therefore, a need to appreciate the importance of the regulatory diversity and compartmentation of various pathways of nitrogen metabolism in mycorrhizal fungi. Efforts are needed for the selection of appropriate mutants to evaluate the relative importance of various pathways in the nitrogen nutrition of mycorrhizal fungi. The roles of NADPH-GDH and GS in

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ammonium assimilation, for example, can be compared by using mutants specifically lacking one or other of these enzymes. Furthermore, many novel techniques to study the genetic control of nitrogen metabolism in plant-microbe association have been developed recently in the study of nodulated roots. These genetic approaches can be expected to provide great assistance in elucidating the precise biochemical mechanisms involved in nitrogen assimilation by mycorrhiza.

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