Tree Physiology 12,93-100 0 1993 Heron Publishing-Victoria, Canada Effect of ammonium on glutamine synthetase activity in ectomycorrhizal fungi, and in mycorrhizal and non-mycorrhizal Scats pine seedlings TYTTI SARJALA The Finnish Forest Research Institute, Parkano Research Station, SF-39700 Parkano, Finland Received December 3, 1991 Summary The influence of ammonium on glutamine synthetase activity (GS, EC 6.3.1.2) was studied in three species of ectomycorrhizal fungi, Paxillus involutus (Batsch:Fr) Fr, Piloderma c~~o-oc eum Erikss. and Hjortst. and Suillus variegatus (Fr) 0 Kuntze growing in pure culture, as well as in the roots and needles of nursery-grown, non-mycorrhizal and mycorrhizal Scats pine (Pinus sylvestris L.) seedlings inoculated with Paxillus involutus or Piloderma croceum as the symbiont. In response to increasing concentrations of ammonium in the nutrient solution, GS activity (expressed on a dry weight basis) increased slightly in Suillus variegatus but not in the other fungi. Glutamine synthetase activity increased in the roots and decreased in the needles of non-mycorrhizal seedlings as the ammonium concentration in the nutrient solution was increased from 0 to 1 mm, but no response was noted with further increases from 1 to 12 mm. Interspecies differences in GS activity were noted among the fungi growing in pure culture, but no significant interspecies differences were observed among the same fungi in the mycorrhizal state. Keywords: mycorrhizae, nitrate, nitrogen assimilation, Paxillus involutus, Piloderma croceum, Pinus sylvestris, Suillus variegatus, free-living fungi. Introduction Conifers preferentially use reduced nitrogen as a nitrogen source, although nitrate is also utilized by some species (Bigg and Daniel 1978, Ingestad 1979, Sarjala et al. 1987, Scheromm and Plassard 1988, Peuke and Tischner 1991). The uptake of nitrogen is enhanced by mycorrhizae, and mycorrhizal effects are more noticeable for ammonium uptake than for nitrate uptake (Rygiewicz et al. 1984a, 1984b). In angiosperms, the importance of ectomycorrhizal symbiosis in the mineral nutrition of the host plant has been extensively studied, but information about nitrogen assimilation of conifers and ectomycorrhizae is restricted to a few species (Finlay et al. 1988, Chalot et a1.1991, Vezina et al. 1988, Vezina et al. 1989, Finlay et al. 1992). Vezina et al. (1989) suggested that, in some conifer species, changes in the enzyme activities involved in nitrogen metabolism of mycorrhizae depend on the fungus. On the other hand, Botton et al. (1989) found that repression of aspartate aminotransferase and glutamate dehydrogenase in mycorrhizae was due to the host plant. However, the mechanism of the repression remains unknown. In angiosperms, glutamine synthetase (GS, EC 6.3.1.2) is the main enzyme involved in ammonium assimilation (Haynes and Goh 1978, Oaks and Hire1 1985, Joy 1988), but both GS and glutamate dehydrogenase are involved in ammonium assimilation in mycorrhizal fungi (Genetet et al. 1984, Kershaw and Stewart 1989, Ahmad et al. 1990). Corresponding information on coniferous species is lacking.
94 SARJALA However, studies on nitrate reductase (Sarjala 1990, 1991) indicate that the response of the enzyme in mycorrhizae to increasing ammonium concentrations differs from the response of the enzyme in the free-living fungus, suggesting that information obtained from fungus symbionts grown in pure culture may not be applicable to the mycorrhizal state. This study was undertaken to compare enzyme activities associated with ammonium assimilation in mycorrhizae and in free-living fungi. Glutamine synthetase was selected as the target enzyme because it is the main enzyme involved in ammonium assimilation. Specifically, the responses of GS to increasing ammonium concentration in the nutrient solution were studied in (1) three ectomycorrhizal fungi growing in pure culture, (2) mycorrhizal roots of Scats pine seedlings, and (3) in the roots and needles of one-year-old Scats pine seedlings. Materials Fungal material and methods Mycelia of Paxillus involutus (Batsch:Fr) Fr, Piloderma croceum Erikss. and Hjortst. and Suillus variegatus (Fr) 0 Kuntze were cultured on modified Melin Norkrans medium (MMN) (Marx 1969). The origin of the fungal isolates has been described (Sarjala 1990). For the GS assays, the mycelia were grown floating on liquid MMN in petri dishes. One day before the enzyme assays, the nutrient medium was removed with a Pasteur pipette and replaced with 10 ml of fresh MMN containing 0, 1, 6, or 12 mm (NHd)zHPOd (corresponding to 0,2, 12,24 mm NH;) as the nitrogen source. Vegetative mycelia of Paxillus involutus and Piloderma croceum grown on MMN solidified with agar were used as inocula for the Scats pine seedlings. Mycorrhizal seedlings Scats pine seeds (Pinus sylvestris L.) from the Kainuu district of eastern Finland were washed with tap water and shaken in water with a few drops of Tween 20 and surface sterilized for 1 h with 30% hydrogen peroxide (Trappe 1961). The seeds were then germinated in sterilized test tubes (30 x 200 mm) containing 15 ml of nutrient solution (Ingestad 1962) (1.8 mm NbN03 as a nitrogen source) solidified with 1.5% agar and closed by a cotton plug. The agar absorbed the 15 ml of nutrient solution that was added to each test tube during the growth period. The seedlings were grown under controlled environment conditions that provided a day/night temperatures of 20/18 C and a 16-h photoperiod from two 400-W high pressure sodium lamps (Philips SGR232) giving a photon flux density of 800 pmol rnp2 s-. Two months after germination, half of the seedlings were inoculated with pieces of agar containing mycelia of Paxillus involutus or Piloderma croceum. Both mycorrhizal and non-mycorrhizal seedlings were grown for a further 4 months under the controlled environment conditions. The roots of the six-month-old mycorrhizal and non-mycorrhizal seedlings were washed and the seedlings placed in Erlenmeyer flasks with the roots immersed in 100 ml of nutrient solution (Ingestad 1962)
GLUTAMINE SYNTHETASE IN ECTOMYCORRHIZAL FUNGI AND TREES 95 containing 1 mm (NH&HP04 (which contains 2 mm NH$) as the nitrogen source. This treatment was designed to provide the seedlings with a full supply of ammonium nitrogen before the assays for GS were performed. After 24 h, the fresh weights of the roots and needles were determined, and mycorrhizal roots from the inoculated seedlings and non-mycorrhizal roots from the control seedlings were collected for the GS assays. One-year-old nursery seedlings Scats pine seeds were sown in June 1989 in an open area. Fertilization and irrigation of the seedlings during the growing season followed the usual practice at the nursery in western Finland (61 40 N, 21 42 E). On May 17, 1990, the seedlings were transplanted to clay pots (18 cm) filled with the peat in which they had been growing and placed for two weeks in the same environmental conditions as the mycorrhizal seedlings (day/night temperatures of 20/18 C and a 16-h photoperiod at a photon flux density of 800 pmol mm2 s-l). The roots were then washed with water and each seedling was placed in an Erlenmeyer flask with roots immersed in 100 ml of nutrient solution (Ingestad 1962) without any nitrogen. The next day the medium was replaced with fresh nutrient solution (Ingestad 1962) with 0, I,6 or 12 mm (NH&HP04 (which contains 0,2, 12 or 24 mm NHd+) as the nitrogen source. After 24 h, the seedlings were removed and the needles and roots collected for assay of GS. In all seedlings, part of the root system was colonized by unknown mycorrhizal symbionts of nursery origin. Glutamine synthetase assay Glutamine synthetase (GS, EC 6.3.1.2) activity was determined in crude tissue extracts by the transferase reaction (Shapiro and Stadtman 1970). A crude extract of the mycelium was prepared by grinding the fungal tissue in a chilled mortar in 3.0 ml of buffer solution (ph 7.25) containing 50 mm 3-(N- morfolino) propanesulphonic acid (MOPS), 3 mm MnCl*, 5 mm EDTA, and 1 mm dithiothreitol (DTT) (St. John et al. 1985). The homogenate was clarified by centrifugation at 17,000 g for 30 min and the supernatant used for the GS assay and protein determination. The GS assay reaction mixture contained 50 pl of enzyme solution in 1.5 ml of assay mixture ph 6.5 (Smith et al. 1985), containing 50 mm MOPS, 3 mm MnC12, 20 mm Na2As0.7H20, 200 mm glutamine, 25 mm hydroxylamine hydrochloride and 0.5 mm ADP. Samples without glutamine were used as references. After a 15-min incubation at 30 C, the reaction was terminated by adding 1.5 ml of 3.3% (w/v) FeCls and 8% (w/v) trichloroacetic acid (TCA) in 2 M HCl. The production of y-glutamyl hydroxamate in the reaction mixture was measured spectrophotometritally (Hitachi 101) at 540 nm. Crude extracts of roots and needles were prepared and assayed in the same way as the fungal tissue extracts except that the extraction buffer, ph 7.6, contained 50 mm Tris-HCl, 0.5 mm DTT, 5 mm glutamate, 10 mm MgS04.7H20, 10% (v/v) glycerol and 1% Nonidet P-40 (Vezina et al. 1988); and the reaction buffer, ph 6.8, contained 50 mm Tris-HCl, 100 mm glutamine, 3 mm MnC12, 25 mm hydroxylamine-hcl, 20
96 SARJALA mm NazAs0.7H20, and 0.5 mm ADP. Proteins in the crude extracts of the fungus, roots and needles were precipitated with 10% TCA, dissolved in distilled water, and protein content determined by the method of Bradford (1976). Statistical analysis Linear regression analysis and analysis of variance were used to analyze the results. The Student s t-test (Steel and Torrie 1960) was used to assess the results for the mycorrhizal and control seedlings. Results and discussion Fungal cultures There were significant differences (P < 0.001) in GS activity (expressed on a dry weight basis, gnw) between Piloderma croceum cultures of different ages (Figure IA). Significant differences (P < 0.001) in GS activity per gdw were found among all three ectomycorrhizal fungi (Figure la), but when GS activity was expressed per mg protein significant differences (P < 0.001) were found only between Paxillus involutus and the other fungi (Figure IC). In response to increasing concentrations of ammonium in the nutrient solution, GS activity per gnw increased slightly, but significantly (P < O.Ol), in Suillus variegatus but not in the other fungi (Figure 1A). Increasing concentrations of ammonium had no effect on GS specific activity in any of the fungi (Figure 1C). Protein concentrations of the fungal extracts varied from 6 to 32 mg gnw- (Figure 1B). The greatest variation in protein concentrations among the fungi was observed in the presence of 1 and 6 mm (NH)zHPOd. All three fungi had similar protein contents when grown in the presence of 12 mm (NH&HPOd. Sen (1990) found differences in mycelial protein content between Suillus variegatus and 5. hovinus. Because protein extraction from fungal mycelia is often difficult, incomplete extraction may have resulted in underestimation of the protein content of some or all of the fungal extracts. Mycorrhiza Glutamine synthetase activity per gow in root and needles was similar in control seedlings and in seedlings inoculated with Paxillus involutus or Piloderma croceum (Figure 2), and the significant difference in GS activity found between Piloderma croceum and Paxillus involutus in pure culture was not detected when the same fungi were in the mycorrhizal state. Scats pine seedlings Glutamine synthetase activity per gow was higher in roots than in needles of Scats pine seedlings. Margolis et al. (1988) reported higher GS activity in needles than in roots of Pinus banksiana. The addition of up to 1 mm ammonium in the nutrient
GLUTAMINE SYNTHETASE IN ECTOMYCORRHIZAL FUNGI AND TREES 91 0 2 4 6 8 10 12 (NH4 l2 HP04, mm Figure 1. Influence of ammonium concentration in the nutrient medium on (A) GS activity per gow, (B) protein concentration, and (C) GS activity per mg protein in Puxillus involutus after 21 days of culture (A,), Piloderma croceum after 19 (0) and 22 days (A) of culture, and Suillus variegatus after 22 days of culture (0). Vertical bars represent SE (n = 3). medium increased GS activity per gnw in roots of one-year-old Scats pine seedlings 0, = 293.0 + 226.3x, F = 32.14, P = 0.001) but reduced it in needles (y = 172.5-7 1.0x, F = 8.99, P = 0.024) (Figure 3A). Further increases in ammonium concentration from 1 to 12 mm produced no response in GS activity in either the roots or needles. When expressed on a per mg protein basis, GS activity in the roots and needles of one-year-old seedlings showed no response to an increase in ammonium concentration in the nutrient medium (Figure 3B). Vezina et al. (1989) measured the activities of five enzymes involved in nitrogen metabolism in jack pine and concluded that the changes in the activities of the enzymes as a result of ectomycorrhizal symbiosis were dependent on the fungal
98 SARJALA ; 800, roots needles Figure 2. Glutamine synthetase activity per gow in the needles and root tips of control seedlings (open column), seedlings inoculated with Puxillus involutus (hatched column), and seedlings inoculated with Pilode~~u c r-oceutn (crosshatched columns). Vertical bars represent SE (n = 3). & 400 i. 5 5 200 0 0 2 4 6 8 10 12 (NH412 HP04. mm Figure 3. Influence of ammonium concentration in the nutrient medium on (A) GS activity per gow, and (B) GS activity per mg protein in the roots (dashed lines) and needles (solid lines) of one-year-old, nursery-grown Scats pine seedlings. Lines al and a2 represent regression function from 0 to 1 mm ammonium and lines bl and b2 represent regression function from 1 to 12 mm ammonium. associate. The results of the present study indicate that, in Scats pine, GS activity in a mycorrhizal symbiosis is not dependent on the fungal species, although the symbiotic relationship decreases GS activity in the root. Botton et al. (1989) concluded that repression of aspartate aminotransferase and glutamate dehydrogenase
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