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Effects of Nitrogen Sources on Glutamate Dehydrogenase and Glutamine Synthetase Activity in Suspension Cultures of Atropa belladonna L.
Biochem. Physiol. Pflanzen 188, 283-294 (1992) Gustav Fischer Verlag Jena
Effects of Nitrogen Sources on Glutamate Dehydrogenase and Glutamine Synthetase Activity in Suspension Cultures of Atropa belladonna L. MAIJA-LnSA SALONEN, RnTTA PARVIAINEN
and
LnsA KAARINA SIMOLA
Department of Botany, University of Helsinki, Helsinki, Finland Key Term Index: glutamate dehydrogenase, glutamine synthetase, ornithine, proline, suspension culture; Atropa belladonna
Summary Development of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activity, as well as growth, were studied in dark-grown suspension cultures of Atropa belladonna initiated from root callus. The nutrient media contained NaN0 3 (15 mM) or NH4 N0 3 (7.5 mM). In order to study the effect of early precursors of tropane alkaloids, the cultures were supplemented with proline or ornithine (2.5 mM) during the rapid growth phase on day 10. Growth was accelerated by NH4 N0 3 (days 0-13), but higher fresh and dry weights were obtained at the end of the growth period (day 30) with NaN0 3 . Ornithine temporarily retarded growth in NaN0 3 (days 13, 16). GDH and GS were assayed on days 9, 13 and 16. Considerable GS levels were found in all cultures. However, GS activity in NaN0 3 was markedly higher than in NH4N03 . In contrast, GDH activity was distinctly higher in NH4 N0 3 than in NaN0 3 . Proline and ornithine were effectively metabolized by the suspension cultures, and only transitional accumulation of these amino acids in the cells was observed after amino acid supplementation. GDH and GS were hardly affected by proline, whereas ornithine enhanced GDH activity in the NaN0 3-grown cultures and decreased GS in both nutrient media.
Introduction Atropa belladonna is a medicinal plant producing tropane alkaloids which originate from glutamate-derived amino acids (LIEBISCH and SCHUTTE 1967; LEETE 1990). When good conditions for growth and alkaloid production in tissue cultures of A. belladonna are desired, these amino acids may be of great value as early precursors of hyoscyamine and scopolamine. Ornithine has been found to stimulate the growth of callus cultures of A. belladonna when given together with glutamate, but ornithine alone is a poor nitrogen source. Glutamate or proline are used as the sole sources of nitrogen or together with low concentrations of N03 - or combinations of NH4 + and N0 3- (SALONEN and SIMOLA 1977; SALONEN 1980). The development of nitrate reductase in suspension cultures of A. belladonna is dependent on inorganic nitrogen sources and amino acids (SALONEN and SIMOLA 1989). Activity of this enzyme is distinctly enhanced by NH4N03 , as well as by proline alone or a Abbreviations: GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthetase; Tris, tris(hydroxymethyl)aminomethane 19*
BPP 188 (1992) 5
283
combination of proline and ornithine in the presence of NaN0 3 compared with that in cultures grown in medium containing only NaN0 3 . Increased activity of glutamate dehydrogenase (GDH) is often found in plant tissues e.g. in maize roots supplied with N~ + (OAKS and HIREL 1985; SRIVASTAVA and SINGH 1987). The enzyme GDH catalyzes the reversible conversion of glutamate and 2-oxoglutarate, providing a link between carbohydrate and amino acid metabolism. Correlation has been observed between the activity of GDH and the formation of organic nitrogen compounds (SINGH and SRIVASTAVA 1982), but strong evidence has recently been obtained with carrot cell cultures that the primary role of GDH is the oxidation of glutamate (ROBINSON et aI. 1991). According to current knowledge the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle is the major pathway for the incorporation of ammonia in higher plants (LEA et al. 1990). These enzymes are responsible for assimilation of ammonia at normal physiological concentrations (MIFLIN and LEA 1980). The high Km values obtained for ammonia of GDH enzymes (5-70 mM, STEWART et al. 1980) have, however, been criticized because they are influenced by substrate concentrations and pH (FURUHASHI and TAKAHASHI 1982; YAMAYA and OAKS 1987). The present study is directed at GDH and GS. The development of GDH and GS activity in suspension cultures of A. belladonna was determined in the presence of NaN0 3 or NH4N0 3 . The influence of precursor amino acids of the tropane skeleton, i.e. ornithine or proline, on GDH and GS activity, as well as on intracellular concentrations of these two amino acids, were also studied.
Material and Methods Culture The callus line of A. belladonna L. was derived from roots of intact plants (seed strain 16) (SIMOLA et al. 1988). During these experiments the age of the callus was about two years (27th passage), and the growing methods of the callus and suspension cultures were similar to those in the earlier studies (SALONEN and SIMOLA 1989). Slightly aggregated stock suspensions were used for experimental cultures which were grown in the dark in media (50 ml in 200-ml Erlenmeyer flasks) containing 15 mM NaN03 or 7.5 mM N~N03 at an initial pH of 5.2. On day 10, the media were supplemented with L-amino acids: 2.5 mM proline or ornithine. Suspension cultures (4-6 replicates) were harvested on days 9, 13, 16 and 30 by vacuum filtration and washed with distilled water. Both fresh and dry (after Iyophilisation) weights were determined (SALONEN 1980). Sterility of the cultures in each flask was tested by inoculating a sample of medium and tissue on enriched media containing tryptone, yeast extract and glucose (Orion Diagnostica). The pH of the medium filtrates was measured immediately, and samples of media stored at -20°C for analysis of N~ +, proline and ornithine. The chemicals used were analytical grade and purchased from Merck, Fluka, Sigma or BDH. Enzyme extraction The extraction buffer contained 100 mM Tris-HCI (PH 7.5), 10 mM MgClz and 10 mM ~ mercaptoethanol. Triton X-loo at 0.1 % (w/v) was added to the extraction buffer to facilitate organelle disruption (GOODCHILD and GIVAN 1990). The tissue was homogenized (4.0 ml buffer g-l fr.wt.) by grinding in a mortar and pestle with quartz sand for 10 min at +4°C. The homogenate was first centrifuged for 10 min at 700 x g, and then for 30 min at 14000 X g 284
BPP 188 (1992) 5
(SANCHEZ DE JIMENEZ and FERNANDEZ 1983). This extraction method was very good both for GS and GDH. The supernatant was filtered through a small column of Sephadex G-25 (1.5 X 5 cm; PD10, Pharmacia) to remove small-molecular-weight compounds using extraction buffer without Triton X-100 for elution. This partially purified filtrate was used for the enzyme assays. GS was assayed within one day, whereas GDH was determined from deep-frozen extracts which could be stored at -20°C for several months. Four different cultures were analyzed on days 9, 13 and 16. Assay of enzymes GDH: GDH activity was measured in the direction of amination by the modified method of YOUNG (1973) and TAKAHASHI and FURUHASHI (1980). The reaction mixture (3 rnI) contained at the optimal pH 7.5: 100 mM Tris-HCI, 3.3 mM 2-oxoglutarate, 100 mM N~CI, 0.1 mM CaCh (according to FURUHASHI and TAKAHASHI 1982), 0.1 mM NADH, and 0.05 or 0.1 rnl of the enzyme extract. Stock solutions of all reagents had been adjusted to pH 7.5. The reaction mixture without NADH was first stabilized at 25°C for 3 min. Then the reaction was initiated by adding NADH, and the oxidation of pyridine nucleotide was followed at 340 nm for about 10 min using a Shimadzu UV-visible recording spectrophotometer. The two blanks consisted of the complete reaction mixtures without 2-oxoglutarate or NH4Cl. GS: The GS activity assay was based on the procedure of WOOLFOLK et al. (1966) for the determination of transferase activity as modified by MOHANTY and FLETCHER (1980). The reaction mixture (2 rnl) contained at pH 6.5: 45 mM glutamine, 20 mM sodium arsenate, 4.5 mM MnCh, 26 mM hydroxylamine, 30 mM imidazole-HCI buffer, and 0.29 mM ADP. Stock solutions of all reagents had been adjusted to pH 6.5. After stabilization at 30°C the reaction was initiated by the addition of 0.05,0.1 or 0.2 ml enzyme extract, and the reaction mixture was incubated for 10 min. The end concentration of hydroxamate was determined according to MOHANTY and FLETCHER (1980). The blank was without glutamine. GOGAT: The NADH-GOGAT activity assay was based on the procedure of MOHANTY and FLETCHER (1980). Other methods Soluble proteins from the enzyme extracts were measured according to the modified method of BRADFORD (1976) using bovine serum albumin as a standard. However, 3 ml of the protein reagent was used instead of the 5 rnl proposed by BRADFORD. For the analysis of free proline or ornithine, the sample (1 or 1.5 g fr.wt.) was homogenized in 10 ml3 % (w/v) sulfosalicylic acid for 5 min at +4°C. The homogenate was centrifuged at 4200 X g for 10 min. Concentrations of these two amino acids in the supernatant were determined according to the method of BATES et al. (1973). Concentrations of NH4 +, proline and ornithine in the media were analyzed as described by SALONEN and SIMOLA (1989). The methods of HARTMANN et al. (1986) were applied for the tropane alkaloids.
Results and Discussion Effect of nitrogen source on growth, pH and protein level A combination of NH4 + and N0 3 - (7.5 mM NH4N0 3 ) gave better growth of the suspension cultures of A. belladonna (initiated from root callus) than NaN0 3 (15 mM) during the early stages of growth (days 0-13) (Table 1). This confirms our earlier finding with suspension cultures derived from stem callus (SALONEN and SIMOLA 1989). However, higher fresh and dry weights were obtained with NaN0 3 than with NH4 N0 3 at the end of the experiment. The water content of the cells in each inorganic medium was almost the same (91 - 96 % ). BPP 188 (1992) 5
285
N
00
VI
~
~
-
~
...gg
t:O
0\
mM
+ Om 2.5
2.5 mM
+ Om
d .wt.
2.89 ±0. 16
1.59 ±0.06
0.24 ±0.02
0.16 ±0.01
3.66 ±0.25
4.17 ±0.07
4.40 ±0.16
2.64* ±0.22
3.99 ±0.34
3.38 ±0.19
0.30 ±0.01
0.29 ±O.OO
0.30 ±0.01
0.24 ±0.02
0.33* ±0.01
0.28 ±0.02
3.73 ±0.51
5.96* ±0.23
4 .77 ±0.22
3.55* ±0.35
4 .83 ±0.48
4 .96 ±0.33
fLwt.
d .wt.
6.85 ±0.1l
9.43 ±0.39
9.56* ±0.20
8.43 ±0.44
0.27 ±0.02
6. 11 ±0.81
0 .35*** 7.30 ±O.OO ±0.31
0 .30 ±O.OO
0.33 ±0.03
0.44 ±0.03
0 .37 ±0.02
fr.wt.
d.wt.
0.25 ±0.02
0 .29 ±0.Q1
0.27 ±O.OO
0.37 ±0.01
0.41 ** ±0.Q1
0.36 ±0.01
4 .69 ±0. 11
3.40 ±0.07
fr.wt .
d.wt.
3.40* ±0.22
3.63 ±0.43
4 .20 ±0.09
2.19* ±0.07
2.81 * ±0.05
2.54 ±0.08
2.40 ±0.11
2.49 ±0.07
2.48 ±0.16
4.36 ±0.12
3.90 ±0.45
3.80 ±0.33
16
fr.wt.
13
9
30
13
9
16
Soluble protein mg ' g-l fro wt. Day
Fresh and dry weight g Day
1) Significance of differences between controls: fr. wt. on day 9 *** , day 13 *, day 30 *; d. wt. on day 9 * * day , 16 * ,day 30 ** *; protein on day 9 and 13 * * * , day 16 *.
2.5 mM
+ Pro
NH4N0 3 7.5 mMl)
mM
+ Pro 2.5
NaN0 3 15 mM1)
Nitrogen source
Table 1. Effect of NaN0 3, NH~03 and their combinations with proline or ornithine on the growth and accumulation of soluble protein in suspension cultures of Atropa belladonna. Addition of proline or ornithine on day 10. Initial fresh weight and dry weight approximately 0.60 g and 0.04 g, respectively. Soluble protein was determined using Coomassie brilliant blue G-250. Significance of differences from corresponding controls in NaN0 3 or NH4N0 3 (days 13, 16,30) assessed by Student's t-test are shown by asterisks at the 0.1 % (***), 1 % (**), and 5 % (*) level. Mean ± SE, n = 3-6.
Growth of suspension cultures of A. belladonna was scarcely affected by proline (2.5 mM) , whereas ornithine (2.5 mM) clearly retarded growth in NaN0 3 media at the beginning of the growth period (Table 1). Together with high inorganic nitrogen (60 mM), ornithine (1 mM) has no effect on growth of tobacco cell cultures (KOIWAI et al. 1970), whereas proline (2 mM) causes 54 % inhibition in suspension cultures of Distichlis spicata (RODRIGUEZ and HEYSER 1988). On the other hand, growth of cultured Ipomoea spp. cells is not affected by proline (1 mM) combined with inorganic nitrogen (20 mM) (ZINK 1982). Table 2. Changes in the concentration of medium NHt and medium pH in suspension cultures of A. belladonna grown with NaN0 3 or NH,lV03 supplemented with proline or ornithine on day 10. Concentration of NHt was determined using the phenol-hypochlorite reaction. Initial pH in the medium 5.2. Significance of differences (medium NHt) as in Table 1. Mean ± SE, n = 3-5, - = not detected. pH
Nitrogen source Medium NHt mM Day Nt4N0 3 7.5mM
+
Pro 2.5 mM
+ Om 2.5mM NaN0 3 15mM1) + Pro 2.5mM
+ Om2.5mM
9
13
16
30
0.5 ±0.1
0.1 ±O.O
0.3 ±O.O
1.5 3.9 ±O.O ±O.O 1.9** ±O.l 2.7*** ±O.l 0.1 4.6 ±o.o ±0.1 0.2 ±0.1 0.1 ±0.1
0.3 ±o.o 0.6*** 1.3* ±O.O ±0.2
9
13
16
30
3 .7 ±0.1 3.7 ±O.l 3.6 ±0.1 4.9 ±0.2 4.6 ±0.1 4.5 ±0.2
3.6 ±O.O 3.8 ±O.l
5.6 ±O.O 5.8 ±O.O 4.9 ±0.3
3.8 ±o.o 5.7 ±o.o 5.7 ±O.O 5.6 ±O.O
5.5 ±0.4 5.9 ±0.1 5.9 ±0.1
I) Slight positive phenol-hypochlorite reaction (cf. SALONEN 1984) was also obtained in the presence of NaN0 3 at the end of the growth period.
The pH of the suspension cultures of A. belladonna dropped distinctly during 16 days of growth, but it approached 5.5-6.0 at the end of the growth period (Table 2). Plant tissues supplied with NH4 + acidify the surrounding medium, whereas those supplied with N0 3 - make it alkaline (e .g. MENGEL et al. 1983). Rather high concentrations of NH4 + were found in the media of NH4NOr grown cultures of A. belladonna (Table 2: day 30). This NH4 + might come from the degradation of proteins in the cells and from damaged cells. The soluble protein level of our cultures was very little affected by the nutrient medium (Table 1). However, a low concentration of N~ + (0.9 mM) in the presence of N0 3 - (24 mM) significantly increased the level of total protein in suspension cultures of rose as compared with that in cultures grown in medium containing only N0 3 (MOHANTY and FLETCHER 1980). BPP 188 (1992) 5
287
Effect of inorganic nitrogen source on GDH and GS GDH activity in NH4N0 3 was distinctly higher on day 13 (g-l fr.wt.) and 16 (g-l fr.wt. and mg- 1 protein) compared with that of NaN0 3-grown cultures of A. belladonna (Table 3). The specific activity in NH4N0 3-grown cultures increased during days 9-16 (at Table 3. Changes in glutamate dehydrogenase (GDH) activity in suspension cultures of A. belladonna grown with NaN0 3 or NH~03 supplemented with proline or ornithine on day 10. Aminating GDH activity was determined following the oxidation of NADH at 340 nm for 10 min (25°C). Results were expressed as nanokatals (nkat, kat = mol· S-I) g-I fresh weight or mg- I protein ± SE, n = 4. Significance of differences as in Table 1. Nitrogen source
GDH activity nkat
g-Ifr. wt.
mg- I
g-Ifr.wt.
12.1 ±0.4
3.6 ±0.1
14.3 ±1.1
3.1 ±O.3
+ Pro 2.5mM + Om 2.5mM
+ Om 2.5mM
mg- I
g-Ifr. wt.
mg- I protein
10.2 ±0.5 10.4 ±0.9 13.6** ±0.2 14.0 ±O.7 14.2 ±0.4 15.5 ±1.5
2.7 ±0.2 2.7 ±0.1 3.1 ±0.1 5.7 ±O.2 5.7 ±0.3 6.5 ±0.7
protein
protein
+ Pro 2.5 mM
Day 16
Day 13
Day 9
9.7 ±O.5 9.6 ±0.8 11.6 ± 1.0 13.6 ±O.5 11.7* ±0.2 14.8 ±0.7
3.8 ±0.3 3.4 ±0.2 5.3* ±0.3 3.2 ±0.1 3.3 ±0.3 4.4 ±0.4
1) Significance of differences between controls: on day 13 **/g fr. wt.; on day 16 **/g fr. wt. and
***/mg protein.
0.1 % level). Between those days GDH activity decreased in NaN0 3 (at 5 % level) even though the cultures were actively growing (Table 3). GS activity in NH~03 was markedly lower than in NaN0 3 both at fresh weight and on a protein basis on day 9 and 13 (Table 4). As in A. belladonna cultures, higher GDH activity and lower GS activity in the presence of NH4 + have been obtained in rose cells (MOHANTY and FLETCHER 1980), as well as in roots and leaves of Arabidopsis thaliana (CAMMAERTS and JACOBS 1985). In contrast, the GS activity in suspension cultures of Bouvardia ternifolia (MURILLO and SANCHEZ DE JIMENEZ 1984) and in tobacco and sunflower cells (LENEE and CHUPEAU 1989) responds more weakly than GDH to the source of nitrogen employed. On the other hand, in Ipomoea spp. cells activity of GDH increases with increasing amounts of inorganic nitrogen in the medium (NH4 + or N0 3-, 2.5-20 mM) (ZINK 1989). The N0 3- was effectively reduced in suspension cultures of A. belladonna, but the cell level ofNH4+ remained low (about 0.4 !-lmol g-l fr.wt.) in cultures grown in NaN0 3 (15 mM) media (SALONEN and SIMOLA 1989). The nutrient NH4 + was also rapidly 288
BPP 188 (1992) 5
• Table 4. Changes in glutamine synthetase (GS) activity in suspension cultures of A. belladonna grown with NaN0 3 or NH,I{03 supplemented with proline or ornithine on day 10. The formation of y-glutamylhydroxamate in the transferase reaction was followed at 30°C for 10 min. Results were expressed as nanokatals (nkat, kat = mol, S-I) g-I fresh weight or mg- I protein ± SE, n =4. Significance of differences as in Table 1. Nitrogen source
GS activity nkat
NaN0 3 15 mM!)
g-Ifr.wt.
mg- I protein
g-Ifr. wt.
mg- I protein
g-'fr. wt.
mg- I protein
186.5 ±2.1
54.9 ±0.6
159.6 ±7.0
34.1 ± 1.8
171.8 ±5.3 145.8 ± 10.5 114.0*** ±6.9 142.0 ±9.2 151.0 ±2.9 115.2 ±2.2
67.9 ±3.9 51.8* ±3.4 52.2* ±2.4 33.8 ±2.0 43.5 ±5.4 34.4 ±2.7
172.4 ±21.1 160.8 ± 17.3 111.3 ±4.5 119.7 ±6.7 132.8 ±3.0 74.0** ±7.9
45.4 ±4.1 41.9 ±3.9 25.5* ±0.7 48.4 ±0.7 53.4* ±1.5 30.9* ±3.1
+ Pro 2.5mM + Om 2.5mM NH4 N0 3 7.5 mMI)
+ Pro 2.5mM + Om 2.5mM
Day 16
Day 13
Day 9
I) Significance of differences between controls: on day 9 **/g fro wt., ***/mg protein; on day 13 *1 g fro wt. and ***/mg protein.
removed from the medium of our cultures: on day 9 more than 90 % of the NH4 + had disappeared (Table 2). Quite high cell amounts ofN~ + (2.6-2.9 !-lmol g-l fr.wt.) were previously detected in cultures grown in NH4N0 3 (7.5 mM) (SALONEN and SIMOLA 1989). These concentrations could be suitable for both GDH and GS. A good correlation between GDH activity and intracellular NH4 + is observed in suspension cultures of B. ternifolia (MURILLO and SANCHEZ DE JIMENEZ 1984) but not in Acer pseudoplatanus cells (GOODCHILD and GIVAN 1990). The levels of GDH in A. belladonna (Table 3) were up to ten times those measured in rose cells (MOHANTY and FLETCHER 1980), and rather similar to those in Ipomoea spp. cells (ZINK 1989). Two- or threefold GDH activities have been obtained in carrot cells during the exponential growth phase (ROBINSON et al. 1991) compared with those measured in our suspension cultures. The highest GS activities in A. belladonna cells (Table 4) were about fourfold those reported for rose cells (MOHANTY and FLETCHER 1980) when a similar assay method was used. However, the rate of GS activity obtained in the transferase assay is generally several times higher than those obtained in synthetase assays (STEWART et al. 1980). In B. ternifolia cells the increase in GDH activity precedes the exponential phase of growth, suggesting an anabolic role for this enzyme (MURILLO and SANCHEZ DE JIMENEZ 1984). Our results for NH4NOr grown cultures were not in agreement with this (Tables 1, BPP 188 (1992) 5
289
3) . A similar trend for a late m~imum in GDH as in A. belladonna has been observed in sunflower (LENEE and CHUPEAU 1989) and carrot cells (ROBINSON et al. 1991). During natural senescence GDH activity also increases in leaves (CAMMAERTS and JACOBS 1985; TIRADO et al. 1990). Evidence of a catabolic role for GDH has been obtained from callus cultures of Nicotiana plumbaginifolia (MAESTRI et al. 1991). GDH appears to catalyze the oxidation of glutamate in response to a deficiency of carbon in carrot cells as shown by ROBINSON et al. (1991) using nuclear magnetic resonance spectroscopy and mass spectrometry. In our experiments the sucrose concentration was still half the initial level (2 % w/v) in the medium of cultures during days 9-16 when GDH and GS activity was determined (data not shown). This indicates that carbon was not a limiting factor in the suspension cultures of A. belladonna. The GOOAT activity in suspension cultures of A. belladonna was either very low (below 1/10 of that obtained with GDH) or not measurable at all (data not shown). This was probably due to the general instability of this enzyme (STEWART et al. 1980).
Effect of proline and ornithine on GDH and GS Ornithine (2.5 mM) could increase GDH by 30-40% in NaN0 3-grown cultures of A. belladonna (Table 3) but proline (2.5 mM) had no effect on this enzyme. GS activity was decreased with 25-35 % by ornithine both in NaN03 and NIL.N03 (Table 4) . Several studies on GDH and GS are based on relatively short treatments of amino acids used in quite high concentrations . In maize roots, GDH (aminating) is inhibited with 37-44%, e.g. by proline, arginine and phenylalanine (5 mM) (SINGH and SRIVASTAVA 1983), whereas proline or ornithine (25 mM) in the reaction mixture has no effect on rice leaf GS (YUAN and Hou 1989). Medium proline and ornithine were effectively utilized by the suspension cultures of A. belladonna. Proline was almost completely exhausted from the medium by day 13 and on day 16, about 90% of the ornithine in the medium had also disappeared (Table 5) . The levels of both proline and ornithine in the cells increased following supplementation compared to the control cultures grown with inorganic nitrogen (Table 5). Proline is a common amino acid in the free amino acid pools of suspension cultures of A. belladonna, as well as in normal roots, whereas ornithine is below the detection limit (SALONEN and SIMOLA 1986) as usually in plant tissues (e.g. Datura stramonium, LEWIS 1975). The accumulation of free proline is characteristic of many types of stress (e.g. RHODES et al. 1986), but only transitional accumulation of proline was observed in the suspension cultures of A. belladonna (Table 5). Ornithine (0.25 mM) is known to stimulate carbamoyl phosphate synthetase, and this amino acid is a precursor of arginine (pea shoots, O'NEAL and NAYLOR 1976). Both ornithine and arginine are needed for the synthesis of polyamines (TIBURCIo et al. 1990). Of these, putrescine is also an intermediate in the biosynthesis of tropane alkaloids (LEETE 1990). Our suspension cultures did not synthesize tropane alkaloids and the biosynthetic pathway was not induced by the amino acids tested (data not shown). A lack of feed-in induction by tropane alkaloid precursors has been reported earlier in suspension cultures 290
BPP 188 (1992) 5
Table 5. Concentrations of proline and ornithine in the medium, and the effect of these two amino
acids on accumulation of cell proline or ornithine in suspension cultures of A. belladonna grown with NaN03 or NH~03' Addition of proline or ornithine on day 10. Proline and ornithine were
assayed using the acid-ninhydrin method. Significance of differences (cell proline or ornithine) as in Table 1. Mean ± SE, n = 3 - 5, - = below the detection level of the assay. Nitrogen source Day
Medium proline or ornithine!) mM 13
16
NaN03 15mM
30
Cell proline or ornithine2) !lmol g-I fro wt.
9
13
16
2.7 ±0.1
0.5 ±0.1
0.3 ±0.1
2.4 ±0.7
0.7 ±0.2 2.1 * ±0.4
0.1 ±O.O 0.1 ±O.O
0.5** ±O.O
0.1* ±O.O
1.0 ±0.1
0.6* ±0.1
0.1 ±O.O 0.1 ±O.O
+ Pro 2.5mM + Om 2.5mM
0.6 ±0.1 0.1 ±O.O
N14N037.5mM
+ Pro 2.5mM + Om 2.5mM
0.7 ±O.O
0.2 ±0.1
5.1 *** ±0.3 0.1 ±O.O
30
!) The detection limit of the assay 0.02 !lmol ml- I . 2) The detection limit of the assay 0.10 !lmol g -I fr. wt.
of A. belladonna (SIMOLA et al. 1990), but the total alkaloid content is increased by ornithine (3-6 mM) in shoot cultures of this plant (BENJAMIN et al. 1987).
Conclusions The two ammonia-assimilating enzymes, GDH and GS, were active in suspension cultures of A. belladonna cultivated in nutrient media containing different nitrogen sources. The high activity of GS in the cells grown in NaN0 3 suggested a primary role for this enzyme in these cultures. In NaN03-grown cells, ornithine decreased GS activity and increased GDH, even though it temporarily retarded growth. Proline and ornithine at a concentration of 2.5 mM were rapidly metabolized by NaN0 3- and N~N03-grown cultures. The substantial level of GDH found in the presence of NH4N0 3 suggested that under high ammonia availability, GDH could also playa role in the nitrogen metabolism of A. belladonna. GDH from NH4 N0 3-grown cultures of A. belladonna was not affected by the amino acids tested, but GS was decreased by ornithine. The inhibitory effects of ornithine suggest that relatively high concentrations of the precursor amino acids (2.5 mM) of tropane alkaloids in nutrient medium could lead to changes in nitrogen metabolism. The results obtained with suspension cultures of A. belladonna based on GDH and GS activity indicate the contributions of these enzymes in the nitrogen metabolism of this medicinal plant, but confirmatory evidence on the flow of nitrogen requires 15N-Iabeling studies. BPP 188 (1992) 5
291
Acknowledgements Financial support for this work came from the LEO and REGINA WAINSTEIN Foundation (M.L.S.) and the University of Helsinki (L.K.S.). The English was revised by Mrs. CAROL NORRIS (Ph.D.) and Mr. JOHN DEROME.
References BATES, L. S., WALDREN, R. P., and TEARE, I. D.: Rapid determination of free proline for waterstress studies. Plant & Soil 39, 205-207 (1973). BENJAMIN, B. D., ROJA, P. C., HEBLE, M. R., and CHADHA, M. S.: Multiple shoot cultures of Atropa belladonna: Effect of physico-chemical factors on growth and alkaloid formation. J. Plant Physiol. 129, 129-135 (1987). BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976). CAMMAERTS, D., and JACOBS, M.: A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana. Planta 163, 517-526 (1985). FURUHASHI, K., and TAKAHASHI, Y.: Control of glutamate dehydrogenase from green tobacco callus mitochondria by Ca2 + and pH. Plant Cell Physiol. 23, 179-184 (1982). GOODCHILD, J. A., and GIVAN, C. V.: Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus. Physiol. Plant. 78,29-37 (1990). HARTMANN, T., WITTE, L., OPRACH, F., and TOPPEL, G.: Reinvestigation of the alkaloid composition of Atropa belladonna plants, root cultures, and cell suspension cultures. Planta Med. 52, 390-395 (1986). KOlWAI, A., TANNO, Y., and NOGUCHI, M.: The effects of amino acids on the growth and glutamic acid decarboxylase activity of tobacco cell cultures. Agr. BioI. Chern. 34, 1754-l756 (1970). LEA, P. J., ROBINSON, S. A., and STEWART, G. R.: The enzymology and metabolism of glutamine, glutamate, and asparagine. In: The Biochemistry of Plants, Vol. 16 (Eds. MIFLIN, B. J., and LEA, P. J.), pp. 121-159. Academic Press, San Diego 1990. LEETE, E.: Recent developments in the biosynthesis of the tropane alkaloids. Planta Med. 56, 339-352 (1990). LENEE, P., and CHUPEAU, Y.: Development of nitrogen assimilating enzymes during growth of cells derived from protoplasts of sunflower and tobacco. Plant Sci. 59, 109-117 (1989). LEWIS, O. A. M.: An 15N_ 14C study of the role of the leaf in the nitrogen nutrition of the seed of Datura stramonium L. J. Exp. Bot. 26, 361-366 (1975). LIEBISCH, H.-W., und SCHUTTE, H. R.: Zur Biosynthese der Tropanalkaloide. VIII. Vorstufen des Pyrrolidinringes. Z. Pflanzenphysiol. 57,434-439 (1967). MAESTRI, E., RESTIVO, F. M., GULLI, M., and TASSI, F.: Glutamate dehydrogenase regulation in callus cultures of Nicotiana plumbaginifolia: effect of glucose feeding and carbon source starvation on the isoenzymatic pattern. Plant Cell Environm. 14, 613-618 (1991). MENGEL, K., ROBIN, P., and SALSAC, L.: Nitrate reductase activity in shoots and roots of maize seedlings as affected by the form of nitrogen nutrition and the pH of the nutrient solution. Plant Physiol. 71, 618-622 (1983). MIFLIN, B. J., and LEA, P. J.: Ammonia assimilation. In: The Biochemistry of Plants, Vol. 5 (Ed. MIFLIN, B. J.), pp. 169-202. Academic Press, New York 1980. MOHANTY, B., and FLETCHER, J. S.: Ammonium influence on nitrogen assimilating enzymes and protein accumulation in suspension cultures of Paul's Scarlet rose. Physiol. Plant. 48, 453-459 (1980). MURILLO, E., and SANCHEZ DE JIMENEZ, E.: Alternative pathways for ammonium assimilation in Bouvardia ternifolia cell suspension cultures. J. Plant. Physiol. 117,57-68 (1984). OAKS, A., and HIREL, B.: Nitrogen metabolism in roots. Annu. Rev. Plant Physiol. 36, 345-365 (1985).
292
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O'NEAL, T. D., and NAYLOR, A. W.: Some regulatory properties of pea leaf carbamoyl phosphate synthetase. Plant Physiol. 57, 23-28 (1976). RHODES, D., HANDA, S., and BRESSAN, R. A.: Metabolic changes associated with adaptation of plant cells to water stress. Plant Physiol. 82, 890-903 (1986). ROBINSON, S. A., SLADE, A. P., Fox, G. G., PHILLIPS, R., RATCLIFFE, R. G., and STEWART, G. R.: The role of glutamate dehydrogenase in plant nitrogen metabolism. Plant Physiol. 95,
509-516 (1991). RODRIGUEZ, M. M., and HEYSER, J. W.: Growth inhibition by exogenous proline and its metabolism in saltgrass (Distichlis spicata) suspension cultures. Plant Cell Rep. 7, 305-308
(1988). SALONEN, M.-L.: Glutamate- and aspartate-derived amino acids as nitrogen sources for the callus of Atropa belladonna L. Ann. Bot. Fennici 17, 357-378 (1980). SALONEN, M.-L.: Influence of ammonium and nitrate nutrition on levels of ammonium, nitrate and total nitrogen in the callus of Atropa belladonna (Solanaceae). Ann. Bot. Fennici 21,367-381
(1984). SALONEN, M.-L., and SIMOLA, L. K.: Dipeptides and amino acids as nitrogen sources for the callus of Atropa belladonna. Physiol. Plant. 41, 55-58 (1977). SALONEN, M.-L., and SIMOLA, L. K.: Nitrogen metabolism of callus and suspension cultures of Atropa belladonna. In: Proc. VI International Congress of Plant Tissue and Cell Culture (Eds. SOMERS, D. A., GENGENBACH, B. G., BIESBOER, D. D., HACKETT, W. P., and GREEN, C. E.). Abstracts p. 355. August 3-8, Minneapolis 1986. SALONEN, M.-L., and SIMOLA, L. K.: Effect of nitrate, ammonium and some amino acids on growth and nitrate reductase activity in suspension cultures of Atropa belladonna. Plant Cell Physiol. 30,1177-1181 (1989). SANCHEZ DE JIMENEZ, E., and FERNANDEZ, L.: Biochemical parameters to assess cell differentiation of Bouvardia ternifolia Schlecht callus. Planta 158, 377-383 (1983). SIMOLA, L. K., NIEMINEN, S., HUHTIKANGAS, A., YLINEN, M., NAARANLAHTI, T., and LOUNASMAA, M.: Tropane alkaloids from Atropa belladonna, Part II. Interaction of origin, age, and environment in alkaloid production of callus cultures. J. Nat. Prod. 51, 234-242 (1988). SIMOLA, L. K., PARVIAINEN, R., MARTINSEN, A., HUHTIKANGAS, A., JOKELA, R., and LOUNASMAA, M.: Feeding experiments using suspension cultures of Atropa belladonna; limiting steps in the biosynthesis of tropane alkaloids. Phytochem. 29, 3517-3523 (1990). SINGH, R. P., and SRIVASTAVA, H. S.: Glutamate dehydrogenase activity and assimilation of inorganic nitrogen in maize seedlings. Biochem. Physiol. Pflanzen 177, 633-642 (1982). SINGH, R. P., and SRIVASTAVA, H. S.: Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings. Physiol. Plant. 57, 549-554 (1983). SRIVASTAVA, H. S., and SINGH, R. P.: Role and regulation ofL-glutamate dehydrogenase activity in higher plants. Phytochem. 26, 597-610 (1987). STEWART, G. R., MANN, A. F., and FENTEM, P. A.: Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. In: The Biochemistry of Plants, Vol. 5 (Ed. MIFLIN, B. J.), pp. 271-327. Academic Press, New York 1980. TAKAHASHI, Y., and FURUHASHI, K.: Properties of glutamate dehydrogenase purified from green tobacco callus tissue. Plant Cell Physiol. 21, 1067-1075 (1980). TIBURCIO, A. F., KAUR-SAWHNEY, R., and GALSTON, A. W.: Polyamine metabolism. In: The Biochemistry of Plants, Vol. 16 (Eds. MIFLIN, B. J., and LEA, P. J.), pp. 283-325. Academic Press, San Diego 1990. TIRADO, J.-L., CONEJERO, G., et ROBIN, P.: Comparaison des activites glutamine synthetase et glutamate deshydrogenase dans la feuille de Glycine max. Distribution dans la plante et effet de l'ammonium et du nitrate. Plant Physiol. Biochem. 28, 239-250 (1990). WOOLFOLK, C. A., SHAPIRO, B., and STADTMAN, E. R.: Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch. Biochem. Biophys. 116, 177-192 (1966). BPP 188 (1992) 5
293
YAMAYA, T., and OAKS, A.: Synthesis of glutamate by mitochondria - An anaplerotic function for glutamate dehydrogenase. Physiol. Plant. 70, 749-756 (1987). YOUNG, M.: Studies on the growth in culture of plant cells. XVI. Nitrogen assimilation during nitrogen-limited growth of Acer pseudoplatanus L. cells in chemostat culture. 1. Exp. Bot. 24,
1172-1185 (1973). YUAN, H.-F., and Hou, C.-R.: Regulatory properties of rice leaf glutamine synthetase by.amino acids and nucleotides. Bot. Bull. Acad. Sinica 30, 71-89 (1989). ZINK, M. W.: Regulation of nitrate reductase by various nitrogen sources in cultured Ipomoea sp. Can. 1. Bot. 60, 386-396 (1982). ZINK, M. W.: Regulation of ammonia-assimilating enzymes by various nitrogen sources in cultured Ipomoea spp. Can. 1. Bot. 67, 3127-3133 (1989).
Received February 21, 1992; revised/orm accepted May 15, 1992 Authors' address: Phil. Lic. M.-L. SALONEN, M. Sc. R. PARVIAINEN and Prof. Dr. L. K. SIMOLA, Department of Botany, University of Helsinki, Unioninkatu 44, SF -00170 Helsinki, Finland.
294
BPP 188 (1992) 5
Effects of Nitrogen Sources on Glutamate Dehydrogenase and Glutamine Synthetase Activity in Suspension Cultures of Atropa belladonna L. MAIJA-LnSA SALONEN, RnTTA PARVIAINEN
and
LnsA KAARINA SIMOLA
Department of Botany, University of Helsinki, Helsinki, Finland Key Term Index: glutamate dehydrogenase, glutamine synthetase, ornithine, proline, suspension culture; Atropa belladonna
Summary Development of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activity, as well as growth, were studied in dark-grown suspension cultures of Atropa belladonna initiated from root callus. The nutrient media contained NaN0 3 (15 mM) or NH4 N0 3 (7.5 mM). In order to study the effect of early precursors of tropane alkaloids, the cultures were supplemented with proline or ornithine (2.5 mM) during the rapid growth phase on day 10. Growth was accelerated by NH4 N0 3 (days 0-13), but higher fresh and dry weights were obtained at the end of the growth period (day 30) with NaN0 3 . Ornithine temporarily retarded growth in NaN0 3 (days 13, 16). GDH and GS were assayed on days 9, 13 and 16. Considerable GS levels were found in all cultures. However, GS activity in NaN0 3 was markedly higher than in NH4N03 . In contrast, GDH activity was distinctly higher in NH4 N0 3 than in NaN0 3 . Proline and ornithine were effectively metabolized by the suspension cultures, and only transitional accumulation of these amino acids in the cells was observed after amino acid supplementation. GDH and GS were hardly affected by proline, whereas ornithine enhanced GDH activity in the NaN0 3-grown cultures and decreased GS in both nutrient media.
Introduction Atropa belladonna is a medicinal plant producing tropane alkaloids which originate from glutamate-derived amino acids (LIEBISCH and SCHUTTE 1967; LEETE 1990). When good conditions for growth and alkaloid production in tissue cultures of A. belladonna are desired, these amino acids may be of great value as early precursors of hyoscyamine and scopolamine. Ornithine has been found to stimulate the growth of callus cultures of A. belladonna when given together with glutamate, but ornithine alone is a poor nitrogen source. Glutamate or proline are used as the sole sources of nitrogen or together with low concentrations of N03 - or combinations of NH4 + and N0 3- (SALONEN and SIMOLA 1977; SALONEN 1980). The development of nitrate reductase in suspension cultures of A. belladonna is dependent on inorganic nitrogen sources and amino acids (SALONEN and SIMOLA 1989). Activity of this enzyme is distinctly enhanced by NH4N03 , as well as by proline alone or a Abbreviations: GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthetase; Tris, tris(hydroxymethyl)aminomethane 19*
BPP 188 (1992) 5
283
combination of proline and ornithine in the presence of NaN0 3 compared with that in cultures grown in medium containing only NaN0 3 . Increased activity of glutamate dehydrogenase (GDH) is often found in plant tissues e.g. in maize roots supplied with N~ + (OAKS and HIREL 1985; SRIVASTAVA and SINGH 1987). The enzyme GDH catalyzes the reversible conversion of glutamate and 2-oxoglutarate, providing a link between carbohydrate and amino acid metabolism. Correlation has been observed between the activity of GDH and the formation of organic nitrogen compounds (SINGH and SRIVASTAVA 1982), but strong evidence has recently been obtained with carrot cell cultures that the primary role of GDH is the oxidation of glutamate (ROBINSON et aI. 1991). According to current knowledge the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle is the major pathway for the incorporation of ammonia in higher plants (LEA et al. 1990). These enzymes are responsible for assimilation of ammonia at normal physiological concentrations (MIFLIN and LEA 1980). The high Km values obtained for ammonia of GDH enzymes (5-70 mM, STEWART et al. 1980) have, however, been criticized because they are influenced by substrate concentrations and pH (FURUHASHI and TAKAHASHI 1982; YAMAYA and OAKS 1987). The present study is directed at GDH and GS. The development of GDH and GS activity in suspension cultures of A. belladonna was determined in the presence of NaN0 3 or NH4N0 3 . The influence of precursor amino acids of the tropane skeleton, i.e. ornithine or proline, on GDH and GS activity, as well as on intracellular concentrations of these two amino acids, were also studied.
Material and Methods Culture The callus line of A. belladonna L. was derived from roots of intact plants (seed strain 16) (SIMOLA et al. 1988). During these experiments the age of the callus was about two years (27th passage), and the growing methods of the callus and suspension cultures were similar to those in the earlier studies (SALONEN and SIMOLA 1989). Slightly aggregated stock suspensions were used for experimental cultures which were grown in the dark in media (50 ml in 200-ml Erlenmeyer flasks) containing 15 mM NaN03 or 7.5 mM N~N03 at an initial pH of 5.2. On day 10, the media were supplemented with L-amino acids: 2.5 mM proline or ornithine. Suspension cultures (4-6 replicates) were harvested on days 9, 13, 16 and 30 by vacuum filtration and washed with distilled water. Both fresh and dry (after Iyophilisation) weights were determined (SALONEN 1980). Sterility of the cultures in each flask was tested by inoculating a sample of medium and tissue on enriched media containing tryptone, yeast extract and glucose (Orion Diagnostica). The pH of the medium filtrates was measured immediately, and samples of media stored at -20°C for analysis of N~ +, proline and ornithine. The chemicals used were analytical grade and purchased from Merck, Fluka, Sigma or BDH. Enzyme extraction The extraction buffer contained 100 mM Tris-HCI (PH 7.5), 10 mM MgClz and 10 mM ~ mercaptoethanol. Triton X-loo at 0.1 % (w/v) was added to the extraction buffer to facilitate organelle disruption (GOODCHILD and GIVAN 1990). The tissue was homogenized (4.0 ml buffer g-l fr.wt.) by grinding in a mortar and pestle with quartz sand for 10 min at +4°C. The homogenate was first centrifuged for 10 min at 700 x g, and then for 30 min at 14000 X g 284
BPP 188 (1992) 5
(SANCHEZ DE JIMENEZ and FERNANDEZ 1983). This extraction method was very good both for GS and GDH. The supernatant was filtered through a small column of Sephadex G-25 (1.5 X 5 cm; PD10, Pharmacia) to remove small-molecular-weight compounds using extraction buffer without Triton X-100 for elution. This partially purified filtrate was used for the enzyme assays. GS was assayed within one day, whereas GDH was determined from deep-frozen extracts which could be stored at -20°C for several months. Four different cultures were analyzed on days 9, 13 and 16. Assay of enzymes GDH: GDH activity was measured in the direction of amination by the modified method of YOUNG (1973) and TAKAHASHI and FURUHASHI (1980). The reaction mixture (3 rnI) contained at the optimal pH 7.5: 100 mM Tris-HCI, 3.3 mM 2-oxoglutarate, 100 mM N~CI, 0.1 mM CaCh (according to FURUHASHI and TAKAHASHI 1982), 0.1 mM NADH, and 0.05 or 0.1 rnl of the enzyme extract. Stock solutions of all reagents had been adjusted to pH 7.5. The reaction mixture without NADH was first stabilized at 25°C for 3 min. Then the reaction was initiated by adding NADH, and the oxidation of pyridine nucleotide was followed at 340 nm for about 10 min using a Shimadzu UV-visible recording spectrophotometer. The two blanks consisted of the complete reaction mixtures without 2-oxoglutarate or NH4Cl. GS: The GS activity assay was based on the procedure of WOOLFOLK et al. (1966) for the determination of transferase activity as modified by MOHANTY and FLETCHER (1980). The reaction mixture (2 rnl) contained at pH 6.5: 45 mM glutamine, 20 mM sodium arsenate, 4.5 mM MnCh, 26 mM hydroxylamine, 30 mM imidazole-HCI buffer, and 0.29 mM ADP. Stock solutions of all reagents had been adjusted to pH 6.5. After stabilization at 30°C the reaction was initiated by the addition of 0.05,0.1 or 0.2 ml enzyme extract, and the reaction mixture was incubated for 10 min. The end concentration of hydroxamate was determined according to MOHANTY and FLETCHER (1980). The blank was without glutamine. GOGAT: The NADH-GOGAT activity assay was based on the procedure of MOHANTY and FLETCHER (1980). Other methods Soluble proteins from the enzyme extracts were measured according to the modified method of BRADFORD (1976) using bovine serum albumin as a standard. However, 3 ml of the protein reagent was used instead of the 5 rnl proposed by BRADFORD. For the analysis of free proline or ornithine, the sample (1 or 1.5 g fr.wt.) was homogenized in 10 ml3 % (w/v) sulfosalicylic acid for 5 min at +4°C. The homogenate was centrifuged at 4200 X g for 10 min. Concentrations of these two amino acids in the supernatant were determined according to the method of BATES et al. (1973). Concentrations of NH4 +, proline and ornithine in the media were analyzed as described by SALONEN and SIMOLA (1989). The methods of HARTMANN et al. (1986) were applied for the tropane alkaloids.
Results and Discussion Effect of nitrogen source on growth, pH and protein level A combination of NH4 + and N0 3 - (7.5 mM NH4N0 3 ) gave better growth of the suspension cultures of A. belladonna (initiated from root callus) than NaN0 3 (15 mM) during the early stages of growth (days 0-13) (Table 1). This confirms our earlier finding with suspension cultures derived from stem callus (SALONEN and SIMOLA 1989). However, higher fresh and dry weights were obtained with NaN0 3 than with NH4 N0 3 at the end of the experiment. The water content of the cells in each inorganic medium was almost the same (91 - 96 % ). BPP 188 (1992) 5
285
N
00
VI
~
~
-
~
...gg
t:O
0\
mM
+ Om 2.5
2.5 mM
+ Om
d .wt.
2.89 ±0. 16
1.59 ±0.06
0.24 ±0.02
0.16 ±0.01
3.66 ±0.25
4.17 ±0.07
4.40 ±0.16
2.64* ±0.22
3.99 ±0.34
3.38 ±0.19
0.30 ±0.01
0.29 ±O.OO
0.30 ±0.01
0.24 ±0.02
0.33* ±0.01
0.28 ±0.02
3.73 ±0.51
5.96* ±0.23
4 .77 ±0.22
3.55* ±0.35
4 .83 ±0.48
4 .96 ±0.33
fLwt.
d .wt.
6.85 ±0.1l
9.43 ±0.39
9.56* ±0.20
8.43 ±0.44
0.27 ±0.02
6. 11 ±0.81
0 .35*** 7.30 ±O.OO ±0.31
0 .30 ±O.OO
0.33 ±0.03
0.44 ±0.03
0 .37 ±0.02
fr.wt.
d.wt.
0.25 ±0.02
0 .29 ±0.Q1
0.27 ±O.OO
0.37 ±0.01
0.41 ** ±0.Q1
0.36 ±0.01
4 .69 ±0. 11
3.40 ±0.07
fr.wt .
d.wt.
3.40* ±0.22
3.63 ±0.43
4 .20 ±0.09
2.19* ±0.07
2.81 * ±0.05
2.54 ±0.08
2.40 ±0.11
2.49 ±0.07
2.48 ±0.16
4.36 ±0.12
3.90 ±0.45
3.80 ±0.33
16
fr.wt.
13
9
30
13
9
16
Soluble protein mg ' g-l fro wt. Day
Fresh and dry weight g Day
1) Significance of differences between controls: fr. wt. on day 9 *** , day 13 *, day 30 *; d. wt. on day 9 * * day , 16 * ,day 30 ** *; protein on day 9 and 13 * * * , day 16 *.
2.5 mM
+ Pro
NH4N0 3 7.5 mMl)
mM
+ Pro 2.5
NaN0 3 15 mM1)
Nitrogen source
Table 1. Effect of NaN0 3, NH~03 and their combinations with proline or ornithine on the growth and accumulation of soluble protein in suspension cultures of Atropa belladonna. Addition of proline or ornithine on day 10. Initial fresh weight and dry weight approximately 0.60 g and 0.04 g, respectively. Soluble protein was determined using Coomassie brilliant blue G-250. Significance of differences from corresponding controls in NaN0 3 or NH4N0 3 (days 13, 16,30) assessed by Student's t-test are shown by asterisks at the 0.1 % (***), 1 % (**), and 5 % (*) level. Mean ± SE, n = 3-6.
Growth of suspension cultures of A. belladonna was scarcely affected by proline (2.5 mM) , whereas ornithine (2.5 mM) clearly retarded growth in NaN0 3 media at the beginning of the growth period (Table 1). Together with high inorganic nitrogen (60 mM), ornithine (1 mM) has no effect on growth of tobacco cell cultures (KOIWAI et al. 1970), whereas proline (2 mM) causes 54 % inhibition in suspension cultures of Distichlis spicata (RODRIGUEZ and HEYSER 1988). On the other hand, growth of cultured Ipomoea spp. cells is not affected by proline (1 mM) combined with inorganic nitrogen (20 mM) (ZINK 1982). Table 2. Changes in the concentration of medium NHt and medium pH in suspension cultures of A. belladonna grown with NaN0 3 or NH,lV03 supplemented with proline or ornithine on day 10. Concentration of NHt was determined using the phenol-hypochlorite reaction. Initial pH in the medium 5.2. Significance of differences (medium NHt) as in Table 1. Mean ± SE, n = 3-5, - = not detected. pH
Nitrogen source Medium NHt mM Day Nt4N0 3 7.5mM
+
Pro 2.5 mM
+ Om 2.5mM NaN0 3 15mM1) + Pro 2.5mM
+ Om2.5mM
9
13
16
30
0.5 ±0.1
0.1 ±O.O
0.3 ±O.O
1.5 3.9 ±O.O ±O.O 1.9** ±O.l 2.7*** ±O.l 0.1 4.6 ±o.o ±0.1 0.2 ±0.1 0.1 ±0.1
0.3 ±o.o 0.6*** 1.3* ±O.O ±0.2
9
13
16
30
3 .7 ±0.1 3.7 ±O.l 3.6 ±0.1 4.9 ±0.2 4.6 ±0.1 4.5 ±0.2
3.6 ±O.O 3.8 ±O.l
5.6 ±O.O 5.8 ±O.O 4.9 ±0.3
3.8 ±o.o 5.7 ±o.o 5.7 ±O.O 5.6 ±O.O
5.5 ±0.4 5.9 ±0.1 5.9 ±0.1
I) Slight positive phenol-hypochlorite reaction (cf. SALONEN 1984) was also obtained in the presence of NaN0 3 at the end of the growth period.
The pH of the suspension cultures of A. belladonna dropped distinctly during 16 days of growth, but it approached 5.5-6.0 at the end of the growth period (Table 2). Plant tissues supplied with NH4 + acidify the surrounding medium, whereas those supplied with N0 3 - make it alkaline (e .g. MENGEL et al. 1983). Rather high concentrations of NH4 + were found in the media of NH4NOr grown cultures of A. belladonna (Table 2: day 30). This NH4 + might come from the degradation of proteins in the cells and from damaged cells. The soluble protein level of our cultures was very little affected by the nutrient medium (Table 1). However, a low concentration of N~ + (0.9 mM) in the presence of N0 3 - (24 mM) significantly increased the level of total protein in suspension cultures of rose as compared with that in cultures grown in medium containing only N0 3 (MOHANTY and FLETCHER 1980). BPP 188 (1992) 5
287
Effect of inorganic nitrogen source on GDH and GS GDH activity in NH4N0 3 was distinctly higher on day 13 (g-l fr.wt.) and 16 (g-l fr.wt. and mg- 1 protein) compared with that of NaN0 3-grown cultures of A. belladonna (Table 3). The specific activity in NH4N0 3-grown cultures increased during days 9-16 (at Table 3. Changes in glutamate dehydrogenase (GDH) activity in suspension cultures of A. belladonna grown with NaN0 3 or NH~03 supplemented with proline or ornithine on day 10. Aminating GDH activity was determined following the oxidation of NADH at 340 nm for 10 min (25°C). Results were expressed as nanokatals (nkat, kat = mol· S-I) g-I fresh weight or mg- I protein ± SE, n = 4. Significance of differences as in Table 1. Nitrogen source
GDH activity nkat
g-Ifr. wt.
mg- I
g-Ifr.wt.
12.1 ±0.4
3.6 ±0.1
14.3 ±1.1
3.1 ±O.3
+ Pro 2.5mM + Om 2.5mM
+ Om 2.5mM
mg- I
g-Ifr. wt.
mg- I protein
10.2 ±0.5 10.4 ±0.9 13.6** ±0.2 14.0 ±O.7 14.2 ±0.4 15.5 ±1.5
2.7 ±0.2 2.7 ±0.1 3.1 ±0.1 5.7 ±O.2 5.7 ±0.3 6.5 ±0.7
protein
protein
+ Pro 2.5 mM
Day 16
Day 13
Day 9
9.7 ±O.5 9.6 ±0.8 11.6 ± 1.0 13.6 ±O.5 11.7* ±0.2 14.8 ±0.7
3.8 ±0.3 3.4 ±0.2 5.3* ±0.3 3.2 ±0.1 3.3 ±0.3 4.4 ±0.4
1) Significance of differences between controls: on day 13 **/g fr. wt.; on day 16 **/g fr. wt. and
***/mg protein.
0.1 % level). Between those days GDH activity decreased in NaN0 3 (at 5 % level) even though the cultures were actively growing (Table 3). GS activity in NH~03 was markedly lower than in NaN0 3 both at fresh weight and on a protein basis on day 9 and 13 (Table 4). As in A. belladonna cultures, higher GDH activity and lower GS activity in the presence of NH4 + have been obtained in rose cells (MOHANTY and FLETCHER 1980), as well as in roots and leaves of Arabidopsis thaliana (CAMMAERTS and JACOBS 1985). In contrast, the GS activity in suspension cultures of Bouvardia ternifolia (MURILLO and SANCHEZ DE JIMENEZ 1984) and in tobacco and sunflower cells (LENEE and CHUPEAU 1989) responds more weakly than GDH to the source of nitrogen employed. On the other hand, in Ipomoea spp. cells activity of GDH increases with increasing amounts of inorganic nitrogen in the medium (NH4 + or N0 3-, 2.5-20 mM) (ZINK 1989). The N0 3- was effectively reduced in suspension cultures of A. belladonna, but the cell level ofNH4+ remained low (about 0.4 !-lmol g-l fr.wt.) in cultures grown in NaN0 3 (15 mM) media (SALONEN and SIMOLA 1989). The nutrient NH4 + was also rapidly 288
BPP 188 (1992) 5
• Table 4. Changes in glutamine synthetase (GS) activity in suspension cultures of A. belladonna grown with NaN0 3 or NH,I{03 supplemented with proline or ornithine on day 10. The formation of y-glutamylhydroxamate in the transferase reaction was followed at 30°C for 10 min. Results were expressed as nanokatals (nkat, kat = mol, S-I) g-I fresh weight or mg- I protein ± SE, n =4. Significance of differences as in Table 1. Nitrogen source
GS activity nkat
NaN0 3 15 mM!)
g-Ifr.wt.
mg- I protein
g-Ifr. wt.
mg- I protein
g-'fr. wt.
mg- I protein
186.5 ±2.1
54.9 ±0.6
159.6 ±7.0
34.1 ± 1.8
171.8 ±5.3 145.8 ± 10.5 114.0*** ±6.9 142.0 ±9.2 151.0 ±2.9 115.2 ±2.2
67.9 ±3.9 51.8* ±3.4 52.2* ±2.4 33.8 ±2.0 43.5 ±5.4 34.4 ±2.7
172.4 ±21.1 160.8 ± 17.3 111.3 ±4.5 119.7 ±6.7 132.8 ±3.0 74.0** ±7.9
45.4 ±4.1 41.9 ±3.9 25.5* ±0.7 48.4 ±0.7 53.4* ±1.5 30.9* ±3.1
+ Pro 2.5mM + Om 2.5mM NH4 N0 3 7.5 mMI)
+ Pro 2.5mM + Om 2.5mM
Day 16
Day 13
Day 9
I) Significance of differences between controls: on day 9 **/g fro wt., ***/mg protein; on day 13 *1 g fro wt. and ***/mg protein.
removed from the medium of our cultures: on day 9 more than 90 % of the NH4 + had disappeared (Table 2). Quite high cell amounts ofN~ + (2.6-2.9 !-lmol g-l fr.wt.) were previously detected in cultures grown in NH4N0 3 (7.5 mM) (SALONEN and SIMOLA 1989). These concentrations could be suitable for both GDH and GS. A good correlation between GDH activity and intracellular NH4 + is observed in suspension cultures of B. ternifolia (MURILLO and SANCHEZ DE JIMENEZ 1984) but not in Acer pseudoplatanus cells (GOODCHILD and GIVAN 1990). The levels of GDH in A. belladonna (Table 3) were up to ten times those measured in rose cells (MOHANTY and FLETCHER 1980), and rather similar to those in Ipomoea spp. cells (ZINK 1989). Two- or threefold GDH activities have been obtained in carrot cells during the exponential growth phase (ROBINSON et al. 1991) compared with those measured in our suspension cultures. The highest GS activities in A. belladonna cells (Table 4) were about fourfold those reported for rose cells (MOHANTY and FLETCHER 1980) when a similar assay method was used. However, the rate of GS activity obtained in the transferase assay is generally several times higher than those obtained in synthetase assays (STEWART et al. 1980). In B. ternifolia cells the increase in GDH activity precedes the exponential phase of growth, suggesting an anabolic role for this enzyme (MURILLO and SANCHEZ DE JIMENEZ 1984). Our results for NH4NOr grown cultures were not in agreement with this (Tables 1, BPP 188 (1992) 5
289
3) . A similar trend for a late m~imum in GDH as in A. belladonna has been observed in sunflower (LENEE and CHUPEAU 1989) and carrot cells (ROBINSON et al. 1991). During natural senescence GDH activity also increases in leaves (CAMMAERTS and JACOBS 1985; TIRADO et al. 1990). Evidence of a catabolic role for GDH has been obtained from callus cultures of Nicotiana plumbaginifolia (MAESTRI et al. 1991). GDH appears to catalyze the oxidation of glutamate in response to a deficiency of carbon in carrot cells as shown by ROBINSON et al. (1991) using nuclear magnetic resonance spectroscopy and mass spectrometry. In our experiments the sucrose concentration was still half the initial level (2 % w/v) in the medium of cultures during days 9-16 when GDH and GS activity was determined (data not shown). This indicates that carbon was not a limiting factor in the suspension cultures of A. belladonna. The GOOAT activity in suspension cultures of A. belladonna was either very low (below 1/10 of that obtained with GDH) or not measurable at all (data not shown). This was probably due to the general instability of this enzyme (STEWART et al. 1980).
Effect of proline and ornithine on GDH and GS Ornithine (2.5 mM) could increase GDH by 30-40% in NaN0 3-grown cultures of A. belladonna (Table 3) but proline (2.5 mM) had no effect on this enzyme. GS activity was decreased with 25-35 % by ornithine both in NaN03 and NIL.N03 (Table 4) . Several studies on GDH and GS are based on relatively short treatments of amino acids used in quite high concentrations . In maize roots, GDH (aminating) is inhibited with 37-44%, e.g. by proline, arginine and phenylalanine (5 mM) (SINGH and SRIVASTAVA 1983), whereas proline or ornithine (25 mM) in the reaction mixture has no effect on rice leaf GS (YUAN and Hou 1989). Medium proline and ornithine were effectively utilized by the suspension cultures of A. belladonna. Proline was almost completely exhausted from the medium by day 13 and on day 16, about 90% of the ornithine in the medium had also disappeared (Table 5) . The levels of both proline and ornithine in the cells increased following supplementation compared to the control cultures grown with inorganic nitrogen (Table 5). Proline is a common amino acid in the free amino acid pools of suspension cultures of A. belladonna, as well as in normal roots, whereas ornithine is below the detection limit (SALONEN and SIMOLA 1986) as usually in plant tissues (e.g. Datura stramonium, LEWIS 1975). The accumulation of free proline is characteristic of many types of stress (e.g. RHODES et al. 1986), but only transitional accumulation of proline was observed in the suspension cultures of A. belladonna (Table 5). Ornithine (0.25 mM) is known to stimulate carbamoyl phosphate synthetase, and this amino acid is a precursor of arginine (pea shoots, O'NEAL and NAYLOR 1976). Both ornithine and arginine are needed for the synthesis of polyamines (TIBURCIo et al. 1990). Of these, putrescine is also an intermediate in the biosynthesis of tropane alkaloids (LEETE 1990). Our suspension cultures did not synthesize tropane alkaloids and the biosynthetic pathway was not induced by the amino acids tested (data not shown). A lack of feed-in induction by tropane alkaloid precursors has been reported earlier in suspension cultures 290
BPP 188 (1992) 5
Table 5. Concentrations of proline and ornithine in the medium, and the effect of these two amino
acids on accumulation of cell proline or ornithine in suspension cultures of A. belladonna grown with NaN03 or NH~03' Addition of proline or ornithine on day 10. Proline and ornithine were
assayed using the acid-ninhydrin method. Significance of differences (cell proline or ornithine) as in Table 1. Mean ± SE, n = 3 - 5, - = below the detection level of the assay. Nitrogen source Day
Medium proline or ornithine!) mM 13
16
NaN03 15mM
30
Cell proline or ornithine2) !lmol g-I fro wt.
9
13
16
2.7 ±0.1
0.5 ±0.1
0.3 ±0.1
2.4 ±0.7
0.7 ±0.2 2.1 * ±0.4
0.1 ±O.O 0.1 ±O.O
0.5** ±O.O
0.1* ±O.O
1.0 ±0.1
0.6* ±0.1
0.1 ±O.O 0.1 ±O.O
+ Pro 2.5mM + Om 2.5mM
0.6 ±0.1 0.1 ±O.O
N14N037.5mM
+ Pro 2.5mM + Om 2.5mM
0.7 ±O.O
0.2 ±0.1
5.1 *** ±0.3 0.1 ±O.O
30
!) The detection limit of the assay 0.02 !lmol ml- I . 2) The detection limit of the assay 0.10 !lmol g -I fr. wt.
of A. belladonna (SIMOLA et al. 1990), but the total alkaloid content is increased by ornithine (3-6 mM) in shoot cultures of this plant (BENJAMIN et al. 1987).
Conclusions The two ammonia-assimilating enzymes, GDH and GS, were active in suspension cultures of A. belladonna cultivated in nutrient media containing different nitrogen sources. The high activity of GS in the cells grown in NaN0 3 suggested a primary role for this enzyme in these cultures. In NaN03-grown cells, ornithine decreased GS activity and increased GDH, even though it temporarily retarded growth. Proline and ornithine at a concentration of 2.5 mM were rapidly metabolized by NaN0 3- and N~N03-grown cultures. The substantial level of GDH found in the presence of NH4N0 3 suggested that under high ammonia availability, GDH could also playa role in the nitrogen metabolism of A. belladonna. GDH from NH4 N0 3-grown cultures of A. belladonna was not affected by the amino acids tested, but GS was decreased by ornithine. The inhibitory effects of ornithine suggest that relatively high concentrations of the precursor amino acids (2.5 mM) of tropane alkaloids in nutrient medium could lead to changes in nitrogen metabolism. The results obtained with suspension cultures of A. belladonna based on GDH and GS activity indicate the contributions of these enzymes in the nitrogen metabolism of this medicinal plant, but confirmatory evidence on the flow of nitrogen requires 15N-Iabeling studies. BPP 188 (1992) 5
291
Acknowledgements Financial support for this work came from the LEO and REGINA WAINSTEIN Foundation (M.L.S.) and the University of Helsinki (L.K.S.). The English was revised by Mrs. CAROL NORRIS (Ph.D.) and Mr. JOHN DEROME.
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Received February 21, 1992; revised/orm accepted May 15, 1992 Authors' address: Phil. Lic. M.-L. SALONEN, M. Sc. R. PARVIAINEN and Prof. Dr. L. K. SIMOLA, Department of Botany, University of Helsinki, Unioninkatu 44, SF -00170 Helsinki, Finland.
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