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Effect of Sulfur Deficiency on Protein Synthesis and Amino Acid Accumulation in Cell Suspension Cultures of Nicotiana tabacum
Effect of Sulfur Deficiency on Protein Synthesis and Amino Acid Accumulation in Cell Suspension Cultures of Nicotiana tabacum S. KLAPHECK, W.
GROSSE
and L. BERGMANN
Botanisches Institut der Universitat zu Kaln, Gyrhofstra~e 15, D-5000 Kaln 41 Received August 3,1982 . Accepted September 3,1982
Summary Sulfur starvation in photo heterotrophic and heterotrophic cell suspension cultures of Nico· tiana tabacum var. Samsun affects growth, protein synthesis and the accumulation of soluble nitrogen compounds. After depletion of the sulfate supply in the medium no further net-protein synthesis was observed and large amounts of soluble nitrogen accumulated. In photoheterotrophically grown cells this accumulation was mainly due to an intense increase in arginine and glutamine which accounted for 70-85 % of the free amino acid nitrogen. In heterotrophically grown cells only a small amount of arginine was formed and glutamine was the predominant soluble nitrogen compound accumulated. In contrast to the changes in the free amino acid fraction, the protein content of the cultures remained constant during the early period of sulfur deficiency and the amino acid composition of the bulk protein did not change appreciably. Therefore it appears that the amino acids accumulating during sulfur deficiency have been produced by de novo synthesis. Under sulfur starvation conditions photo heterotrophically grown cells contained citrulline and ornithine which were not present in detectable amounts in suspensions grown on sulfur-rich media. The observed twofold increase in arginine production under sulfur starvation also indicates a stimulation of arginine synthesis by these conditions.
Key words: Nicotiana tabacum, sulfur deficiency, arginine, cell suspension cultures.
Introduction Sulfur deficiency in higher plants results in a decrease in the rate of protein synthesis and an increase in the amount of free amino acids, amides and inorganic nitrogen in the cells (d. Hewitt, 1963; Thomas, 1958). The accumulation of soluble nitrogen compounds observed in sulfur starved plants probably reflects the degree to which the uptake and assimilation of inorganic nitrogen is sustained under these conditions. Analysis of the changes in the pattern of nitrogen metabolism caused by sulfur starvation should provide some information about the interpathway control of nitrogen and sulfur assimilation by which the rate of nitrogen and sulfur assimilation must be coordinated. This control becomes very difficult to be studied, if protein Abbreviations: TCA = trichloroacetic acid; D.W.
=
dry weight.
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S. KLAPHECK, W. GROSSE and L. BERGMANN
degradation, nitrogen uptake, assimilation, and storage occur in different plant organs and are blurred by transport processes. Therefore, we have analysed the effect of sulfur deficiency on nitrogen metabolism in cell suspension cultures which represent a more homogeneous material than whole plants and lack the difficulties arising from transport processes. In these cultures sulfur deficiency can easily be induced and its effects can be studied (Reuveny and Filner, 1977; Bergmann et aI., 1980). In the present paper we report on the influence of sulfur deficiency on protein synthesis and amino acid accumulation in cells of Nieo· tiana tabaeum grown under photo heterotrophic and heterotrophic conditions and supplied with different inorganic nitrogen sources.
Material and Methods Suspension cultures of Nicotiana tabacum var. Samsun have been cultured as described by Bergmann et aL (1976) in a modified Murashige-Skoog medium at 25°C under continuous illumination (3000 Ix) or in darkness on a rotary shaker (100 r.p.m.). In suspensions grown with nitrate as sole nitrogen source NtLN0 3 was replaced by equimolar amounts of KN0 3 • Sulfur deficiency was induced by growing the cells with 0.6 (OA) mM sulfate instead of 1.73 mM as in the controls. Cells were harvested after different periods of growth, washed twice with fresh sterile medium, and lyophilized. Total nitrogen was determined with the mikro-Kjeldahl method (Humphries, 1956), protein bound and TCA-soluble nitrogen after extraction of the cells with 5 % TCA. Amino acid nitrogen was calculated from the content of free amino acids. The free amino acids were extracted from lyophilized cells with picric acid in a lithiumcitrate buffer and analysed by an automated amino acid analyser as previously described in detail (Bergmann et aI., 1976). The TCA-insoluble protein of the cells was hydrolysed with 6 N HCI at 110°C for 24 h in a sealed test tube. The hydrolysate was evaporated in vacuo to dryness and for amino acid analysis dissolved in lithium-citrate buffer. Sulfate in the medium was determined turbidometrically (Rennenberg and Bergmann, 1979), nitrate was measured colorimetrically after Cataldo et aL (1975). The ammonium content of the medium was analysed by the enzymological method of Kun and Kearny (1970).
Results Sulfur deficiency in cell suspension cultures of Nieotiana tabaeum grown in batch cultures with limiting amounts of sulfur results in a decline of net-protein synthesis which comes to a complete stop after the sulfur supply in the medium has been exhausted (Fig. 1). As shown in Fig. 2 the amount of protein formed per culture is proportional to the amount of sulfur offered to the cultures. This indicates that protein synthesis is limited by the available sulfur most likely caused by an insufficient cysteine and methionine synthesis. During the early stage of sulfur limited growth the assimilation of nitrogen and the increase in dry weight continue. However, the percentage of protein in the biomass decreases whereas the amount of TeA-soluble nitrogen increases. A comparison of the nitrogen content of cells from cultures before and after the media became
z. Pjlanzenphysiol. Bd. 108. S. 235-245. 1982.
Sulfur deficiency in cell suspension cultures
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B.
12
16
20
12
days
16
20
days
Fig. 1: Levels of inorganic nitrogen (e), ammonium (_), nitrate (.A.), and sulfate (*) in the culture medium (A) and cell nitrogen increments (B) in Nicotiana tabacum cell suspensions grown in batch cultures under photoheterotrophical conditions. Sol-N: TCA soluble nitrogen; ProtN: TCA insoluble nitrogen; AS-N: nitrogen in amino acids and amides; Org-N: sum of Prot-N and AS-N; Total-N: sum of Sol-N and Prot-No depleted in sulfur shows the remarkable changes in protein nitrogen and TCA-soluble nitrogen under sulfur starvation conditions (Tab. 1). Cells cultivated with ammonium plus nitrate exhibit protein contents between 23 and 25 % of the dry matter during exponential growth, but show protein contents below 10 % after the sulfur supply of the medium has been exhausted. Culture with nitrate as sole nitrogen source results in a relatively low protein (13 % of the dry weight) and soluble nitrogen content, while culture with ammonium plus nitrate not only results in a higher protein content, but also in a high accumulation of soluble nitrogen. Despite of these differences in the nitrogen composition of the cells an increase in soluble nitrogen and a decrease in protein nitrogen is observed under sulfur starvation conditions in both cases. Table 1: Effect of sulfur deficiency on protein-N and soluble N of cell suspension cultures of Nicotiana tabacum. Nitrogen content of cells cultivated with S (mg N g-l Dry weight)
Light, NH. + and N0 3Light, N0 3Dark, NH. + and N03-
Nitrogen content of cells cultivated without S (mg N g-l Dry weight)
Soluble N
Protein N
lotal N
Soluble N
Protein N
Total N
21.6 7.5 14.0
37.9 20.9 43.8
59.5 28.4 57.8
38.3 16.2 29.9
15.9 11.6 15.3
54.2 27.7 45.2
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0
300
0
~
~
:; u
c 200 QI
2
Q. CJl
E
100
50
100
150
200
!lmol SO~- / culture
Fig. 2: Relationship between sulfur supply and maximal amount of protein formed in suspension cultures of Nicotiana tabacum grown photo heterotrophically (0) and heterotrophically (.).
Similar changes in the nitrogen composition of plant cells have repeatedly been reported as typical symptoms of sulfur deficiency in higher plants (d. Hewitt, 1963; Thomas, 1958) and it has been assumed by some authors that these changes are caused by proteolysis (Eaton, 1941, 1942; Coleman, 1957). The present results clearly show that the protein content of the cultures stays almost constant during a considerable period after net-protein synthesis has been curtailed. It therefore can be excluded that the observed decrease in percentage of cell protein in sulfur starved tobacco cells is due to protein degradation. Analysis of the composition of the soluble nitrogen fractions from sulfur starved, photo heterotrophic cells reveals that 60-90 % of the nitrogen in these fractions is present in form of amino acids and amides. This clearly demonstrates that nitrogen is assimilated during sulfur starvation. In case of nitrate grown cells the origin of the nitrogen assimilated is exclusively nitrate. Also in ammonium-nitrate cultures a substantial proportion of the nitrogen assimilated during sulfur deficiency originates from nitrate as the increase in organic nitrogen inside the cells is higher than the decrease in ammonium in the culture medium (Fig. 1). On the 14th day of culture the cells have taken up nearly all of the ammonium supplied in the medium (6 mmol). The protein and the amino acids at this time contain 10.7 mmol nitrogen, so that 4.7 mmol nitrate must have been reduced and incorporated into organic compounds. Two days later, on the 16th day of culture, the ammonium-nitrate cells contain 11.3 mmol nitrogen so that 5.3 mmol have to originate from nitrate reduction. In Z. Pjlanzenphysiol. Ed. 108. S. 235-245. 1982.
Sulfur deficiency in cell suspension cultures
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Fig. 3: Increments of cell nitrogen in Nicotiana ta.bacum cell suspensions grown in batch cultures under photoheterotrophical condition with nitrate as sole nitrogen source. Legends as in Fig. 1.
contrast, nitrate grown cells contain only 2.6 mmol of organic nitrogen at the 14th day of culture and 2.95 mmol on the 16th day. From the increase in organic nitrogen in nitrate grown cells a rough calculation of the rate of nitrate reduction can be performed. This calculation shows that the utilisation of nitrate in the cells declines during sulfur starvation from 170 p.mol g-I D.W. day-I to 70 p.mol g-I D.W. day-I, compaired to 400 p.mol g-I D.W. day-I in exponentially growing cells. The increase in amino acid nitrogen during sulfur deficiency is caused by an accumulation of only few amino acids as revealed by the quantitative determination of the amino acids and amides in this fraction. In cells grown photo heterotrophically and supplied with ammonium and nitrate these are arginine and glutamine which account for 84-87 % of the nitrogen accumulated in amino acids. The cells double their glutamine content during sulfur starvation and store 30 times more arginine than the controls in which glutamine represents the bulk of amino acid nitrogen (Tab. 2 A). An intense accumulation of arginine characterizes sulfur deficiency also in photoheterotrophic cells grown with nitrate as sole nitrogen source. In these cells arginine Z. Pjlanzenphysiol. Bd. 108. S. 235-245. 1982.
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s. KLAPHECK, W. GROSSE and L. BERGMANN
Table 2 A: Content of free amino acids and amides (~mol g-I dry weight) of photoheterotrophically grown cell suspensions of Nicotiana tabacum cultured on M +S Medium with normal (1.73 mM) or limited (0.6 mM) sulfur supply. Days of culture Growth Conditions
10
8 NI-LN0 3
N03-
NI-LN0 3
+S
-S +
+S
-S +
-S
Asparagine 12.6 26.3 Glutamic acid 13.1 25.8 Glutamine 258.3 423.3 Alanine 14.1 43.6 4-Aminobutyric acid 25.4 21.0 Citrulline + Ornithine + Arginine 4.0 6.8 Remaining amino acids 30.6 43.7
Days of culture Growth Conditions
12 N0 3-
-S
19.5 47.2 13.8 12.3 294.4 398.4 18.8 37.3 28.9 18.6 10.2 3.1 6.4 107.7
17.3 5.6 17.2 10.1
+ + + 13.4
34.2
14
N0 3-
+S
-S
-S
19.6 46.3 15.3 18.6 303.5 422.4 26.3 38.0 30.5 13.1 17.0 4.5 9.6 139.5
14.3 5.9 21.9 16.0
+ + +
56.9
NI-LN0 3
14.9
36.4
16
50.9
2.1 13.2 15.3 43.7 29.1 2.0
+ 2.1 25.6
18
NI-LN0 3
N0 3-
NI-LN03
N03-
NI-LN03
N03-
+S
-S
+S
-S
+S
-S
4.8 17.2 28.7 46.5 24.0 2.4 3.0 33.1
23.0 38.8 15.8 16.8 309.7 452.7 40.8 25.7 24.0 13.8 16.4 8.4 8.4 202.2
4.4 15.5 32.7 50.9 31.4 3.0 3.6 52.3
30.1 41.6 13.0 14.4 291.4 357.8 40.6 37.4 20.8 39.4 9.2 7.3 9.3 190.8
-S
Asparagine 20.6 44.5 Glutamic acid 16.9 19.4 Glutamine 311.3 623.8 Alanine 36.4 32.3 4-Aminobutyric acid 26.7 13.1 Citrulline 22.0 Ornithine 9.2 Arginine 8.1 203.5 Remaining amino acids 38.8 64.5
33.7
-S
29.4
53.8
36.1
27.1
-S
54.4
3.7 15.2 29.0 49.8 33.6 1.8 3.5 73.6 42.0
Table 2 B: Content of free amino acids and amides (~mol g-I dry weight) of heterotrophically grown cell suspensions of Nicotiana tabacum cultured on M +S Medium with normal and limited sulfur supply. Days of culture
8
Growth Conditions
+S
-S
+S
-S
+S
-S
+S
-S
8.4 41.5 295.0 37.3 57.9
8.7 43.9 296.0 34.9 42.7
10.4 27.9 286.0 20.2 44.6
32.5 34.6 490.0 16.9 18.8
20.6 17.0 338.0 9.7 38.4
80.8 23.7 556.0 8.4 19.0
61.0 29.2 307.1 13.3 13.5
130.8 18.1 527.0 9.0 10.0
3.7 45.4
2.4
4.7 48.4
76.1
121.6
120.9
Asparagine Glutamic acid Glutamine Alanine 4-Aminobutyric acid Citrulline Ornithine Arginine Remaining amino acids
11
13 Ammonium Nitrate
1.5 14.6 73.5
67.3
73.5
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63.0
87.5
15
Sulfur deficiency in cell suspension cultures
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contributes nearly 60 % to the amino acid nitrogen. Also the glutamine content of the cells increased under sulfur starvation, but reached only a low concentration (30 /Lmol g-l D.W.) and accounted for just about 15 % of the amino acid nitrogen. Furthermore, an increase in alanine can be observed in the nitrate grown cells, accumulating at about 50/Lmolg- 1 D.W. or 10% of the amino acid nitrogen in the sulfur starved cells. Similar levels of alanine are found in cells from ammonium-nitrate cultures. However, because of the intensive accumulation of glutamine and arginine, alanine accumulation contributes only to a small extent to the pool of soluble amino acid nitrogen. This limited accumulation of alanine may suggest a restriction of alanine synthesis by feedback inhibition. Cells from both ammonium-nitrate and nitrate grown cultures display an increase in citrulline and ornithine with increasing sulfur deficiency. Both intermediates were not present in sulfur-rich cultures in detectable amounts. The observed increases under sulfur starvation probably indicate changes in the pool sizes of ornithine and citrulline which could be linked with the intensive arginine synthesis observed under these conditions. Sulfur deficiency in heterotrophically grown tobacco cells, however, is characterized by an intensive accumulation of glutamine alone which accounts for almost 80 % of the soluble amino acid nitrogen in sulfur starved cells (Tab. 2 B). Beside glutamine, asparagine also increases whereas arginine accumulation in heterotrophical Table 3: Protein amino acids of sulfur-starved cell suspensions of Nicotiana tabacum grown under photo heterotrophic conditions. The values are given as a percentage of the content (mol) of each individual amino acid of the total amino acids detected. Amino acid
Protein amino acids of sulfur starved tobacco cells after 4 days 8 days 15 days 11 days 13 days
Aspartic 9.7 Threonine 5.4 Serine 6.7 Glutamic 10.4 Proline 5.4 Glycine 9.0 Alanine 8.5 Valine 7.5 Methionine 1.2 Isoleucine 5.2 Leucine 9.1 Tyrosine 2.8 Phenylalanine 4.3 Lysine 7.5 Histidine 2.3 Cysteine 0.49 Arginine 4.7
9.8 5.5 6.7 10.5 5.6 8.9 8.6 7.8 1.6 4.3 9.0 2.9 4.2 7.3 2.3 0.61 4.5
9.7 5.5 7.1 10.3 5.7 8.7 8.6 7.8 1.3 5.4 8.8 2.9 4.4 6.9 2.3 0.42 4.2
9.7 5.3 6.9 10.4 6.0 9.3 9.0 7.9 1.3 5.0 8.8 3.0 4.3 6.6 2.1 4.4
9.3 5.3 6.9 10.3 5.8 8.5 8.4 7.7 1.4 5.1 8.7 3.2 4.1 7.8 2.5 0.74 4.2
17 days
21 days
11.0 6.0 8.1 10.6 6.2 8.2 8.2 7.5 0.8 4.5 8.4 3.5 2.7 7.3 2.4 0.59 4.0
10.8 5.8 7.5 10.4 6.1 8.3 8.1 7.5 1.2 4.9 8.4 3.2 4.0 7.1 2.5 0.52 3.9
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and L.
BERGMANN
cells is small compaired with photoheterotrophical cells grown under sulfur starvation. The amino acid composition of the TeA-insoluble fraction (bulkprotein) of heterotrophically grown tobacco cells does not change appreciably during sulfur deficiency indicating that the synthesis of different proteins is not modified by sulfur deficiency to a great extent (Tab. 3). Monitoring the arginine content of the bulk protein as well as the concentration of free arginine in the cells, a comparison of the rate of arginine synthesis during growth on media with different sulfur supplies can be made. As the result of such an experiment (Fig. 4) there is a remarkable increase in arginine production during the early period of sulfur deficiency followed by a gradual decrease in the later period. 48 h after sulfur deficiency becomes evident by the decline of net-protein synthesis, 75 % more arginine is found in the sulfur starved cultures than in the controls (Tab. 4). This difference increased with time, and 96 h after the beginning of sulfur deficiency nearly twice the amount of arginine has been synthesized in the sulfur starved cultures than in the controls. The difference in arginine accumulation between normal
---
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10
4
6
8
10
12
14
16
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Fig. 4: Comparison of arginine content of photoheterotrophic cell suspension cultures of Nico· tiana tabacum grown with different amounts of sulfur. Control, 1.73 mM sol-: proteine arginine (0), proteine and free arginine (.). Sulfur deficiency, 0.6 mM SO/-: proteine arginine (0), proteine and free arginine (.). Z. Pf/anzenphysiol. Bd. 108. S. 235-245. 1982.
Sulfur deficiency in cell suspension cultures
243
and sulfur deficient cultures decreases subsequently when the deficiency becomes severe and the metabolism of the sulfur starved cells deteriorates. The difference in arginine synthesis between sulfur starved cells and normal cells is also reflected by the different amount of arginine produced per unit dry weight. As shown in table 4 cells grown with an adaequate sulfur supply contain about 881lmol arginine g- I D.W., whereas an amount of 250-2751lmol arginine g-I D.W. during sulfur starvation demonstrates the higher rate of arginine synthesis under these conditions. Table 4: Effect of sulfur deficiency on the amount of proteine arginine and free arginine in cell suspension cultures of Nicotiana tabacum grown in batch cultures under photo heterotrophic co~d~tions with (0.52 mmol) and without (0.18 mmol) sulfate. Values are given as Ilmol argmme. Cultures without Sulfate
Cultures with Sulfate Culture Proteine- Soluble Arginine Arginine ARG per ARG per per Time per (Days) Culture Culture Culture gD.W.
Proteine- Soluble Arginine Arginine ARG per ARG per per per Culture Culture Culture gD.W.
4 6 8 9 10 11 12 13 14 15 16
23.5 39.3 86.1 70.4 84.4 100.0 99.0 91.0 127.0 128.0 125.5
21.4 38.9 75.4
1.2 2.7
21.4 40.1 78.1
88 82.1 89.9
140.0
6.2
146.2
93.9
218.5
15.9
234.4
94.1
438.2
53.8
492.0
87.6
651.4
76.1
727.4
77.5
0.5 1.7 6.9 99.8 171.6 266.0 356.0 564.0 636.0 739.0 773.5
24 41 93 170 256 366 455 655 764 867 899
100 76 88 133 161 172 179 274 245 237 222
Discussion
The main symptom of sulfur deficiency in tobacco cell suspensions is a tremendous accumulation of soluble nitrogen compounds accompanied by a constant protein content. As observed by other investigators (d. Hewitt, 1963; Thomas, 1958) the accumulation of soluble nitrogen is mainly due to an accumulation of arginine and glutamine which account for 84-87 % of the soluble nitrogen in sulfur deficient photoheterotrophic cells. In heterotrophic cells the soluble nitrogen that has been accumulated during sulfur starvation consists predominantly of glutamine. The arginine content in heterotrophic cells rises to 40llmolg-1 D.W. only, whereas 200ilmoi arginine g-I D.W. were found in photoheterotrophic cells. The reason for the different effect of sulfur starvation on the composition of soluble nitrogen in photoheterotrophic and heterotrophic cells is not yet clear. Steward et al. (1959) reported that the composition of soluble nitrogen in Mentha piperita L. is highly influenced by the length of day, probably pointing to an participation Z. Pjlanzenphysiol. Ed. 108. S. 235-245. 1982.
244
S. KLAPHECK, W. GROSSE and L. BERGMANN
of the phytochrom system. In these experiments it was shown that sulfur deficiency in MENTHA grown under long day condition is characterized by an intense accumulation of arginine in the leaves whereas glutamine and asparagine were accumulated under short day conditions. On the other hand, the difference in arginine accumulation in photoheterotrophic, chloroplast-containing tobacco cells and heterotrophic, chloroplast-free cells may indicate an participation of the chloroplast in arginine biosynthesis. Two enzymes involved in arginine synthesis, carbamoylphosphate synthetase and ornithine carbamoyltransferase, have recently been shown by Shargool et al. (1978) to be associated with a plastid fraction isolated from soy bean cells. On the basis of this information it appears possible that the photosynthetic capacity of the chloroplasts could be used for an intensified arginine synthesis. Work is in progress to examine this possibility which is consistant with the increasing evidence that plastids in higher plants play an important role in amino acid synthesis (Miflin and Lea, 1977). The accumulation of the soluble nitrogen compounds in the cells starts after netprotein synthesis is arrested and the protein content of the cultures remains constant. As there is no interference by translocation and redistribution in cell cultures, the accumulation of soluble nitrogen compounds must result from a continuous synthesis of these compounds, primarily arginine and glutamine. Our results clearly show that the amount of arginine formed during sulfur starvation is much higher than the amount synthesized under normal conditions and mainly used for protein synthesis. The high amount of arginine formed in the sulfur starved cells is consistent with the observed increase in the ornithine and citrulline pool in these cells and suggests that arginine synthesis is stimulated by sulfur deficiency. Since arginine synthesis in plant cells is regulated by feedback inhibition (Dougall and Fulton, 1967; Morris and Thompson, 1977) and is reduced by 98 % when protein synthesis is inhibited by cycloheximid in suspension cultures of Paul's Scarlet rose (Fletcher and Beevers, 1971; Fletcher, 1975), the accumulation of arginine during sulfur starvation can take place only if the arginine is removed from the sites of synthesis and stored in vacuoles. Work with protoplasts and intact vacuoles from tobacco mesophyll cells has already shown that arginine is a constituent of vacuoles (Bergmann, 1981). It remains to be shown whether arginine accumulates in the vacuoles during sulfur starvation and which factors are responsible for the transport into the vacuoles under these conditions. Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
References BERGMANN, L.; Aspects of S- and N-metabolism in tissue cultures. In: BOTHE, H. and A.TREBST (Eds.), Biology of inorganic nitrogen and sulfur. Springer, Berlin, pp. 341-351 (1981). BERGMANN, L., W. GROSSE, und P. KOTH; EinfluB von Ammonium und Nitrat auf Stickstoffme-
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tabolismus, Malatanhaufung und Malatenzym-Aktivitat in Suspensionskulturen von Nicotiana tabacum var. «SAMSUN». Z. f. Pflanzenphysiol. 80, 60-70 (1976). BERGMANN, L., J. D. SCHWENN, and H. URLAUB: Adenosine triphosphate sulfurylase and o-acetylserin sulfhydrylase in photo heterotrophically and heterotrophically cultured tobacco cells. Z. Naturforsch. 35c, 952-957 (1980). CATALDO, D. A., M. HAROON, L. E. SCHRADER, and V. L. YOUNGS: Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Science and plant analysis 6, 71-80 (1975). COLEMAN, R. G.: The effect of sulfur deficiency on the free amino acids of some plants. Austr. J. BioI. Sci. 10, 50-56 (1957). DOUGALL, D. K. and M. M. FULTON: The biosynthesis of protein amino acids in plant tissue cultures III. Studies on biosynthesis of arginine. Plant Physiol. 42, 387-390 (1967). EATON, S. V.: Influence of sulfur deficiency on metabolism of the sunflower. Bot. Gaz. 102, 536-556 (1941).
- Influence of sulfur deficiency on metabolism of black mustard. Bot. Gaz. 104, 306-315 (1942).
FLETCHER,
J.
S.: Control of amino acid synthesis in tissue culture cells. Plant Physiol. 56,
450-451 (1975).
FLETCHER, J. S. and H. BEEVERS: Influence of cycloheximide on the synthesis and utilisation of amino acids in suspension cultures. Plant Physiol. 48, 261-264 (1971). HEWITT, E. J.: The essential nutrient elements, requirements and interactions in plants. In: F. C. STEWARD (Ed.), Plant Physiology, Vol. III, pp. 137-362. Academic Press, 1963. HUMPHRIES, E. c.: Mineral components and ash analysis. In: K. PEACH and M. V. TRACEY (Eds.), Modern Methods of Plant Analysis, Vol. I, Springer, Berlin, pp. 468-502 (1956). KOIWAI, A., M. NOGUCHI, and E. TAMAKI: Changes in the amino acid composition of tobacco cells in suspension cultures. Phytochemistry 10,561-566 (1971). KUN, E. and T. B. KEARNEY: Ammoniak. In: H. U. BERGMEYER (Ed.), Methoden der enzymatischen Analyse, 2. Aufl., pp. 1748-1752. Verlag Chemie, Weinheim, 1970. MIFLIN, B. J. and P. T. LEA: Amino acid metabolism. Ann. Rev. Plant Physiol. 28, 299-329 (1977).
MORRIS, C. J. and J. F. THOMPSON: Formation of N-acetylglutamate by extracts of higher plants. Plant Physiol. 59, 684-687 (1977). RENNENBERG, H. und L. BERGMANN: EinfluB von Ammonium und Sulfat auf die Glutathionproduktion in Suspensionskulturen von Nicotiana tabacum. Z. f. Pflanzenphysiol. 92, 133-142 (1979).
REUVENY, Z. and P. FILNER: Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. J. BioI. Chern. 252, 1858-1864 (1977). SHARGOOL, P. D., T. STEEVENS, M. WEAVER, and M. RUSSELL: The localization within plant cells of enzymes involved in arginine biosynthesis. Can. J. Biochem. 56, 273-279 (1978). STEWARD, F. c., F. CRANE, K. MILLAR, R. M. ZACHARIUS, R. RABSON, and D. MARGOLIS: Nutritional and environmental effects on the nitrogen metabolism of plants. Symp. Soc. Exp. BioI. Nr. XIII, Utilization of nitrogen and its compounds by plants. Cambridge, 1959. THOMAS, M. D.: Assimilation of sulfur and physiology of essential S-compounds. Encyclopedia of Plant Physiol. IX, 37-63 (1958) Ed. W. RUHLAND.
z. Pjlanzenphysiol. Bd. 108. S. 235-245. 1982.
GROSSE
and L. BERGMANN
Botanisches Institut der Universitat zu Kaln, Gyrhofstra~e 15, D-5000 Kaln 41 Received August 3,1982 . Accepted September 3,1982
Summary Sulfur starvation in photo heterotrophic and heterotrophic cell suspension cultures of Nico· tiana tabacum var. Samsun affects growth, protein synthesis and the accumulation of soluble nitrogen compounds. After depletion of the sulfate supply in the medium no further net-protein synthesis was observed and large amounts of soluble nitrogen accumulated. In photoheterotrophically grown cells this accumulation was mainly due to an intense increase in arginine and glutamine which accounted for 70-85 % of the free amino acid nitrogen. In heterotrophically grown cells only a small amount of arginine was formed and glutamine was the predominant soluble nitrogen compound accumulated. In contrast to the changes in the free amino acid fraction, the protein content of the cultures remained constant during the early period of sulfur deficiency and the amino acid composition of the bulk protein did not change appreciably. Therefore it appears that the amino acids accumulating during sulfur deficiency have been produced by de novo synthesis. Under sulfur starvation conditions photo heterotrophically grown cells contained citrulline and ornithine which were not present in detectable amounts in suspensions grown on sulfur-rich media. The observed twofold increase in arginine production under sulfur starvation also indicates a stimulation of arginine synthesis by these conditions.
Key words: Nicotiana tabacum, sulfur deficiency, arginine, cell suspension cultures.
Introduction Sulfur deficiency in higher plants results in a decrease in the rate of protein synthesis and an increase in the amount of free amino acids, amides and inorganic nitrogen in the cells (d. Hewitt, 1963; Thomas, 1958). The accumulation of soluble nitrogen compounds observed in sulfur starved plants probably reflects the degree to which the uptake and assimilation of inorganic nitrogen is sustained under these conditions. Analysis of the changes in the pattern of nitrogen metabolism caused by sulfur starvation should provide some information about the interpathway control of nitrogen and sulfur assimilation by which the rate of nitrogen and sulfur assimilation must be coordinated. This control becomes very difficult to be studied, if protein Abbreviations: TCA = trichloroacetic acid; D.W.
=
dry weight.
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degradation, nitrogen uptake, assimilation, and storage occur in different plant organs and are blurred by transport processes. Therefore, we have analysed the effect of sulfur deficiency on nitrogen metabolism in cell suspension cultures which represent a more homogeneous material than whole plants and lack the difficulties arising from transport processes. In these cultures sulfur deficiency can easily be induced and its effects can be studied (Reuveny and Filner, 1977; Bergmann et aI., 1980). In the present paper we report on the influence of sulfur deficiency on protein synthesis and amino acid accumulation in cells of Nieo· tiana tabaeum grown under photo heterotrophic and heterotrophic conditions and supplied with different inorganic nitrogen sources.
Material and Methods Suspension cultures of Nicotiana tabacum var. Samsun have been cultured as described by Bergmann et aL (1976) in a modified Murashige-Skoog medium at 25°C under continuous illumination (3000 Ix) or in darkness on a rotary shaker (100 r.p.m.). In suspensions grown with nitrate as sole nitrogen source NtLN0 3 was replaced by equimolar amounts of KN0 3 • Sulfur deficiency was induced by growing the cells with 0.6 (OA) mM sulfate instead of 1.73 mM as in the controls. Cells were harvested after different periods of growth, washed twice with fresh sterile medium, and lyophilized. Total nitrogen was determined with the mikro-Kjeldahl method (Humphries, 1956), protein bound and TCA-soluble nitrogen after extraction of the cells with 5 % TCA. Amino acid nitrogen was calculated from the content of free amino acids. The free amino acids were extracted from lyophilized cells with picric acid in a lithiumcitrate buffer and analysed by an automated amino acid analyser as previously described in detail (Bergmann et aI., 1976). The TCA-insoluble protein of the cells was hydrolysed with 6 N HCI at 110°C for 24 h in a sealed test tube. The hydrolysate was evaporated in vacuo to dryness and for amino acid analysis dissolved in lithium-citrate buffer. Sulfate in the medium was determined turbidometrically (Rennenberg and Bergmann, 1979), nitrate was measured colorimetrically after Cataldo et aL (1975). The ammonium content of the medium was analysed by the enzymological method of Kun and Kearny (1970).
Results Sulfur deficiency in cell suspension cultures of Nieotiana tabaeum grown in batch cultures with limiting amounts of sulfur results in a decline of net-protein synthesis which comes to a complete stop after the sulfur supply in the medium has been exhausted (Fig. 1). As shown in Fig. 2 the amount of protein formed per culture is proportional to the amount of sulfur offered to the cultures. This indicates that protein synthesis is limited by the available sulfur most likely caused by an insufficient cysteine and methionine synthesis. During the early stage of sulfur limited growth the assimilation of nitrogen and the increase in dry weight continue. However, the percentage of protein in the biomass decreases whereas the amount of TeA-soluble nitrogen increases. A comparison of the nitrogen content of cells from cultures before and after the media became
z. Pjlanzenphysiol. Bd. 108. S. 235-245. 1982.
Sulfur deficiency in cell suspension cultures
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B.
12
16
20
12
days
16
20
days
Fig. 1: Levels of inorganic nitrogen (e), ammonium (_), nitrate (.A.), and sulfate (*) in the culture medium (A) and cell nitrogen increments (B) in Nicotiana tabacum cell suspensions grown in batch cultures under photoheterotrophical conditions. Sol-N: TCA soluble nitrogen; ProtN: TCA insoluble nitrogen; AS-N: nitrogen in amino acids and amides; Org-N: sum of Prot-N and AS-N; Total-N: sum of Sol-N and Prot-No depleted in sulfur shows the remarkable changes in protein nitrogen and TCA-soluble nitrogen under sulfur starvation conditions (Tab. 1). Cells cultivated with ammonium plus nitrate exhibit protein contents between 23 and 25 % of the dry matter during exponential growth, but show protein contents below 10 % after the sulfur supply of the medium has been exhausted. Culture with nitrate as sole nitrogen source results in a relatively low protein (13 % of the dry weight) and soluble nitrogen content, while culture with ammonium plus nitrate not only results in a higher protein content, but also in a high accumulation of soluble nitrogen. Despite of these differences in the nitrogen composition of the cells an increase in soluble nitrogen and a decrease in protein nitrogen is observed under sulfur starvation conditions in both cases. Table 1: Effect of sulfur deficiency on protein-N and soluble N of cell suspension cultures of Nicotiana tabacum. Nitrogen content of cells cultivated with S (mg N g-l Dry weight)
Light, NH. + and N0 3Light, N0 3Dark, NH. + and N03-
Nitrogen content of cells cultivated without S (mg N g-l Dry weight)
Soluble N
Protein N
lotal N
Soluble N
Protein N
Total N
21.6 7.5 14.0
37.9 20.9 43.8
59.5 28.4 57.8
38.3 16.2 29.9
15.9 11.6 15.3
54.2 27.7 45.2
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0
300
0
~
~
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c 200 QI
2
Q. CJl
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100
50
100
150
200
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Fig. 2: Relationship between sulfur supply and maximal amount of protein formed in suspension cultures of Nicotiana tabacum grown photo heterotrophically (0) and heterotrophically (.).
Similar changes in the nitrogen composition of plant cells have repeatedly been reported as typical symptoms of sulfur deficiency in higher plants (d. Hewitt, 1963; Thomas, 1958) and it has been assumed by some authors that these changes are caused by proteolysis (Eaton, 1941, 1942; Coleman, 1957). The present results clearly show that the protein content of the cultures stays almost constant during a considerable period after net-protein synthesis has been curtailed. It therefore can be excluded that the observed decrease in percentage of cell protein in sulfur starved tobacco cells is due to protein degradation. Analysis of the composition of the soluble nitrogen fractions from sulfur starved, photo heterotrophic cells reveals that 60-90 % of the nitrogen in these fractions is present in form of amino acids and amides. This clearly demonstrates that nitrogen is assimilated during sulfur starvation. In case of nitrate grown cells the origin of the nitrogen assimilated is exclusively nitrate. Also in ammonium-nitrate cultures a substantial proportion of the nitrogen assimilated during sulfur deficiency originates from nitrate as the increase in organic nitrogen inside the cells is higher than the decrease in ammonium in the culture medium (Fig. 1). On the 14th day of culture the cells have taken up nearly all of the ammonium supplied in the medium (6 mmol). The protein and the amino acids at this time contain 10.7 mmol nitrogen, so that 4.7 mmol nitrate must have been reduced and incorporated into organic compounds. Two days later, on the 16th day of culture, the ammonium-nitrate cells contain 11.3 mmol nitrogen so that 5.3 mmol have to originate from nitrate reduction. In Z. Pjlanzenphysiol. Ed. 108. S. 235-245. 1982.
Sulfur deficiency in cell suspension cultures
1
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Fig. 3: Increments of cell nitrogen in Nicotiana ta.bacum cell suspensions grown in batch cultures under photoheterotrophical condition with nitrate as sole nitrogen source. Legends as in Fig. 1.
contrast, nitrate grown cells contain only 2.6 mmol of organic nitrogen at the 14th day of culture and 2.95 mmol on the 16th day. From the increase in organic nitrogen in nitrate grown cells a rough calculation of the rate of nitrate reduction can be performed. This calculation shows that the utilisation of nitrate in the cells declines during sulfur starvation from 170 p.mol g-I D.W. day-I to 70 p.mol g-I D.W. day-I, compaired to 400 p.mol g-I D.W. day-I in exponentially growing cells. The increase in amino acid nitrogen during sulfur deficiency is caused by an accumulation of only few amino acids as revealed by the quantitative determination of the amino acids and amides in this fraction. In cells grown photo heterotrophically and supplied with ammonium and nitrate these are arginine and glutamine which account for 84-87 % of the nitrogen accumulated in amino acids. The cells double their glutamine content during sulfur starvation and store 30 times more arginine than the controls in which glutamine represents the bulk of amino acid nitrogen (Tab. 2 A). An intense accumulation of arginine characterizes sulfur deficiency also in photoheterotrophic cells grown with nitrate as sole nitrogen source. In these cells arginine Z. Pjlanzenphysiol. Bd. 108. S. 235-245. 1982.
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Table 2 A: Content of free amino acids and amides (~mol g-I dry weight) of photoheterotrophically grown cell suspensions of Nicotiana tabacum cultured on M +S Medium with normal (1.73 mM) or limited (0.6 mM) sulfur supply. Days of culture Growth Conditions
10
8 NI-LN0 3
N03-
NI-LN0 3
+S
-S +
+S
-S +
-S
Asparagine 12.6 26.3 Glutamic acid 13.1 25.8 Glutamine 258.3 423.3 Alanine 14.1 43.6 4-Aminobutyric acid 25.4 21.0 Citrulline + Ornithine + Arginine 4.0 6.8 Remaining amino acids 30.6 43.7
Days of culture Growth Conditions
12 N0 3-
-S
19.5 47.2 13.8 12.3 294.4 398.4 18.8 37.3 28.9 18.6 10.2 3.1 6.4 107.7
17.3 5.6 17.2 10.1
+ + + 13.4
34.2
14
N0 3-
+S
-S
-S
19.6 46.3 15.3 18.6 303.5 422.4 26.3 38.0 30.5 13.1 17.0 4.5 9.6 139.5
14.3 5.9 21.9 16.0
+ + +
56.9
NI-LN0 3
14.9
36.4
16
50.9
2.1 13.2 15.3 43.7 29.1 2.0
+ 2.1 25.6
18
NI-LN0 3
N0 3-
NI-LN03
N03-
NI-LN03
N03-
+S
-S
+S
-S
+S
-S
4.8 17.2 28.7 46.5 24.0 2.4 3.0 33.1
23.0 38.8 15.8 16.8 309.7 452.7 40.8 25.7 24.0 13.8 16.4 8.4 8.4 202.2
4.4 15.5 32.7 50.9 31.4 3.0 3.6 52.3
30.1 41.6 13.0 14.4 291.4 357.8 40.6 37.4 20.8 39.4 9.2 7.3 9.3 190.8
-S
Asparagine 20.6 44.5 Glutamic acid 16.9 19.4 Glutamine 311.3 623.8 Alanine 36.4 32.3 4-Aminobutyric acid 26.7 13.1 Citrulline 22.0 Ornithine 9.2 Arginine 8.1 203.5 Remaining amino acids 38.8 64.5
33.7
-S
29.4
53.8
36.1
27.1
-S
54.4
3.7 15.2 29.0 49.8 33.6 1.8 3.5 73.6 42.0
Table 2 B: Content of free amino acids and amides (~mol g-I dry weight) of heterotrophically grown cell suspensions of Nicotiana tabacum cultured on M +S Medium with normal and limited sulfur supply. Days of culture
8
Growth Conditions
+S
-S
+S
-S
+S
-S
+S
-S
8.4 41.5 295.0 37.3 57.9
8.7 43.9 296.0 34.9 42.7
10.4 27.9 286.0 20.2 44.6
32.5 34.6 490.0 16.9 18.8
20.6 17.0 338.0 9.7 38.4
80.8 23.7 556.0 8.4 19.0
61.0 29.2 307.1 13.3 13.5
130.8 18.1 527.0 9.0 10.0
3.7 45.4
2.4
4.7 48.4
76.1
121.6
120.9
Asparagine Glutamic acid Glutamine Alanine 4-Aminobutyric acid Citrulline Ornithine Arginine Remaining amino acids
11
13 Ammonium Nitrate
1.5 14.6 73.5
67.3
73.5
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87.5
15
Sulfur deficiency in cell suspension cultures
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contributes nearly 60 % to the amino acid nitrogen. Also the glutamine content of the cells increased under sulfur starvation, but reached only a low concentration (30 /Lmol g-l D.W.) and accounted for just about 15 % of the amino acid nitrogen. Furthermore, an increase in alanine can be observed in the nitrate grown cells, accumulating at about 50/Lmolg- 1 D.W. or 10% of the amino acid nitrogen in the sulfur starved cells. Similar levels of alanine are found in cells from ammonium-nitrate cultures. However, because of the intensive accumulation of glutamine and arginine, alanine accumulation contributes only to a small extent to the pool of soluble amino acid nitrogen. This limited accumulation of alanine may suggest a restriction of alanine synthesis by feedback inhibition. Cells from both ammonium-nitrate and nitrate grown cultures display an increase in citrulline and ornithine with increasing sulfur deficiency. Both intermediates were not present in sulfur-rich cultures in detectable amounts. The observed increases under sulfur starvation probably indicate changes in the pool sizes of ornithine and citrulline which could be linked with the intensive arginine synthesis observed under these conditions. Sulfur deficiency in heterotrophically grown tobacco cells, however, is characterized by an intensive accumulation of glutamine alone which accounts for almost 80 % of the soluble amino acid nitrogen in sulfur starved cells (Tab. 2 B). Beside glutamine, asparagine also increases whereas arginine accumulation in heterotrophical Table 3: Protein amino acids of sulfur-starved cell suspensions of Nicotiana tabacum grown under photo heterotrophic conditions. The values are given as a percentage of the content (mol) of each individual amino acid of the total amino acids detected. Amino acid
Protein amino acids of sulfur starved tobacco cells after 4 days 8 days 15 days 11 days 13 days
Aspartic 9.7 Threonine 5.4 Serine 6.7 Glutamic 10.4 Proline 5.4 Glycine 9.0 Alanine 8.5 Valine 7.5 Methionine 1.2 Isoleucine 5.2 Leucine 9.1 Tyrosine 2.8 Phenylalanine 4.3 Lysine 7.5 Histidine 2.3 Cysteine 0.49 Arginine 4.7
9.8 5.5 6.7 10.5 5.6 8.9 8.6 7.8 1.6 4.3 9.0 2.9 4.2 7.3 2.3 0.61 4.5
9.7 5.5 7.1 10.3 5.7 8.7 8.6 7.8 1.3 5.4 8.8 2.9 4.4 6.9 2.3 0.42 4.2
9.7 5.3 6.9 10.4 6.0 9.3 9.0 7.9 1.3 5.0 8.8 3.0 4.3 6.6 2.1 4.4
9.3 5.3 6.9 10.3 5.8 8.5 8.4 7.7 1.4 5.1 8.7 3.2 4.1 7.8 2.5 0.74 4.2
17 days
21 days
11.0 6.0 8.1 10.6 6.2 8.2 8.2 7.5 0.8 4.5 8.4 3.5 2.7 7.3 2.4 0.59 4.0
10.8 5.8 7.5 10.4 6.1 8.3 8.1 7.5 1.2 4.9 8.4 3.2 4.0 7.1 2.5 0.52 3.9
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cells is small compaired with photoheterotrophical cells grown under sulfur starvation. The amino acid composition of the TeA-insoluble fraction (bulkprotein) of heterotrophically grown tobacco cells does not change appreciably during sulfur deficiency indicating that the synthesis of different proteins is not modified by sulfur deficiency to a great extent (Tab. 3). Monitoring the arginine content of the bulk protein as well as the concentration of free arginine in the cells, a comparison of the rate of arginine synthesis during growth on media with different sulfur supplies can be made. As the result of such an experiment (Fig. 4) there is a remarkable increase in arginine production during the early period of sulfur deficiency followed by a gradual decrease in the later period. 48 h after sulfur deficiency becomes evident by the decline of net-protein synthesis, 75 % more arginine is found in the sulfur starved cultures than in the controls (Tab. 4). This difference increased with time, and 96 h after the beginning of sulfur deficiency nearly twice the amount of arginine has been synthesized in the sulfur starved cultures than in the controls. The difference in arginine accumulation between normal
---
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8
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Fig. 4: Comparison of arginine content of photoheterotrophic cell suspension cultures of Nico· tiana tabacum grown with different amounts of sulfur. Control, 1.73 mM sol-: proteine arginine (0), proteine and free arginine (.). Sulfur deficiency, 0.6 mM SO/-: proteine arginine (0), proteine and free arginine (.). Z. Pf/anzenphysiol. Bd. 108. S. 235-245. 1982.
Sulfur deficiency in cell suspension cultures
243
and sulfur deficient cultures decreases subsequently when the deficiency becomes severe and the metabolism of the sulfur starved cells deteriorates. The difference in arginine synthesis between sulfur starved cells and normal cells is also reflected by the different amount of arginine produced per unit dry weight. As shown in table 4 cells grown with an adaequate sulfur supply contain about 881lmol arginine g- I D.W., whereas an amount of 250-2751lmol arginine g-I D.W. during sulfur starvation demonstrates the higher rate of arginine synthesis under these conditions. Table 4: Effect of sulfur deficiency on the amount of proteine arginine and free arginine in cell suspension cultures of Nicotiana tabacum grown in batch cultures under photo heterotrophic co~d~tions with (0.52 mmol) and without (0.18 mmol) sulfate. Values are given as Ilmol argmme. Cultures without Sulfate
Cultures with Sulfate Culture Proteine- Soluble Arginine Arginine ARG per ARG per per Time per (Days) Culture Culture Culture gD.W.
Proteine- Soluble Arginine Arginine ARG per ARG per per per Culture Culture Culture gD.W.
4 6 8 9 10 11 12 13 14 15 16
23.5 39.3 86.1 70.4 84.4 100.0 99.0 91.0 127.0 128.0 125.5
21.4 38.9 75.4
1.2 2.7
21.4 40.1 78.1
88 82.1 89.9
140.0
6.2
146.2
93.9
218.5
15.9
234.4
94.1
438.2
53.8
492.0
87.6
651.4
76.1
727.4
77.5
0.5 1.7 6.9 99.8 171.6 266.0 356.0 564.0 636.0 739.0 773.5
24 41 93 170 256 366 455 655 764 867 899
100 76 88 133 161 172 179 274 245 237 222
Discussion
The main symptom of sulfur deficiency in tobacco cell suspensions is a tremendous accumulation of soluble nitrogen compounds accompanied by a constant protein content. As observed by other investigators (d. Hewitt, 1963; Thomas, 1958) the accumulation of soluble nitrogen is mainly due to an accumulation of arginine and glutamine which account for 84-87 % of the soluble nitrogen in sulfur deficient photoheterotrophic cells. In heterotrophic cells the soluble nitrogen that has been accumulated during sulfur starvation consists predominantly of glutamine. The arginine content in heterotrophic cells rises to 40llmolg-1 D.W. only, whereas 200ilmoi arginine g-I D.W. were found in photoheterotrophic cells. The reason for the different effect of sulfur starvation on the composition of soluble nitrogen in photoheterotrophic and heterotrophic cells is not yet clear. Steward et al. (1959) reported that the composition of soluble nitrogen in Mentha piperita L. is highly influenced by the length of day, probably pointing to an participation Z. Pjlanzenphysiol. Ed. 108. S. 235-245. 1982.
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of the phytochrom system. In these experiments it was shown that sulfur deficiency in MENTHA grown under long day condition is characterized by an intense accumulation of arginine in the leaves whereas glutamine and asparagine were accumulated under short day conditions. On the other hand, the difference in arginine accumulation in photoheterotrophic, chloroplast-containing tobacco cells and heterotrophic, chloroplast-free cells may indicate an participation of the chloroplast in arginine biosynthesis. Two enzymes involved in arginine synthesis, carbamoylphosphate synthetase and ornithine carbamoyltransferase, have recently been shown by Shargool et al. (1978) to be associated with a plastid fraction isolated from soy bean cells. On the basis of this information it appears possible that the photosynthetic capacity of the chloroplasts could be used for an intensified arginine synthesis. Work is in progress to examine this possibility which is consistant with the increasing evidence that plastids in higher plants play an important role in amino acid synthesis (Miflin and Lea, 1977). The accumulation of the soluble nitrogen compounds in the cells starts after netprotein synthesis is arrested and the protein content of the cultures remains constant. As there is no interference by translocation and redistribution in cell cultures, the accumulation of soluble nitrogen compounds must result from a continuous synthesis of these compounds, primarily arginine and glutamine. Our results clearly show that the amount of arginine formed during sulfur starvation is much higher than the amount synthesized under normal conditions and mainly used for protein synthesis. The high amount of arginine formed in the sulfur starved cells is consistent with the observed increase in the ornithine and citrulline pool in these cells and suggests that arginine synthesis is stimulated by sulfur deficiency. Since arginine synthesis in plant cells is regulated by feedback inhibition (Dougall and Fulton, 1967; Morris and Thompson, 1977) and is reduced by 98 % when protein synthesis is inhibited by cycloheximid in suspension cultures of Paul's Scarlet rose (Fletcher and Beevers, 1971; Fletcher, 1975), the accumulation of arginine during sulfur starvation can take place only if the arginine is removed from the sites of synthesis and stored in vacuoles. Work with protoplasts and intact vacuoles from tobacco mesophyll cells has already shown that arginine is a constituent of vacuoles (Bergmann, 1981). It remains to be shown whether arginine accumulates in the vacuoles during sulfur starvation and which factors are responsible for the transport into the vacuoles under these conditions. Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
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tabolismus, Malatanhaufung und Malatenzym-Aktivitat in Suspensionskulturen von Nicotiana tabacum var. «SAMSUN». Z. f. Pflanzenphysiol. 80, 60-70 (1976). BERGMANN, L., J. D. SCHWENN, and H. URLAUB: Adenosine triphosphate sulfurylase and o-acetylserin sulfhydrylase in photo heterotrophically and heterotrophically cultured tobacco cells. Z. Naturforsch. 35c, 952-957 (1980). CATALDO, D. A., M. HAROON, L. E. SCHRADER, and V. L. YOUNGS: Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Science and plant analysis 6, 71-80 (1975). COLEMAN, R. G.: The effect of sulfur deficiency on the free amino acids of some plants. Austr. J. BioI. Sci. 10, 50-56 (1957). DOUGALL, D. K. and M. M. FULTON: The biosynthesis of protein amino acids in plant tissue cultures III. Studies on biosynthesis of arginine. Plant Physiol. 42, 387-390 (1967). EATON, S. V.: Influence of sulfur deficiency on metabolism of the sunflower. Bot. Gaz. 102, 536-556 (1941).
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FLETCHER, J. S. and H. BEEVERS: Influence of cycloheximide on the synthesis and utilisation of amino acids in suspension cultures. Plant Physiol. 48, 261-264 (1971). HEWITT, E. J.: The essential nutrient elements, requirements and interactions in plants. In: F. C. STEWARD (Ed.), Plant Physiology, Vol. III, pp. 137-362. Academic Press, 1963. HUMPHRIES, E. c.: Mineral components and ash analysis. In: K. PEACH and M. V. TRACEY (Eds.), Modern Methods of Plant Analysis, Vol. I, Springer, Berlin, pp. 468-502 (1956). KOIWAI, A., M. NOGUCHI, and E. TAMAKI: Changes in the amino acid composition of tobacco cells in suspension cultures. Phytochemistry 10,561-566 (1971). KUN, E. and T. B. KEARNEY: Ammoniak. In: H. U. BERGMEYER (Ed.), Methoden der enzymatischen Analyse, 2. Aufl., pp. 1748-1752. Verlag Chemie, Weinheim, 1970. MIFLIN, B. J. and P. T. LEA: Amino acid metabolism. Ann. Rev. Plant Physiol. 28, 299-329 (1977).
MORRIS, C. J. and J. F. THOMPSON: Formation of N-acetylglutamate by extracts of higher plants. Plant Physiol. 59, 684-687 (1977). RENNENBERG, H. und L. BERGMANN: EinfluB von Ammonium und Sulfat auf die Glutathionproduktion in Suspensionskulturen von Nicotiana tabacum. Z. f. Pflanzenphysiol. 92, 133-142 (1979).
REUVENY, Z. and P. FILNER: Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. J. BioI. Chern. 252, 1858-1864 (1977). SHARGOOL, P. D., T. STEEVENS, M. WEAVER, and M. RUSSELL: The localization within plant cells of enzymes involved in arginine biosynthesis. Can. J. Biochem. 56, 273-279 (1978). STEWARD, F. c., F. CRANE, K. MILLAR, R. M. ZACHARIUS, R. RABSON, and D. MARGOLIS: Nutritional and environmental effects on the nitrogen metabolism of plants. Symp. Soc. Exp. BioI. Nr. XIII, Utilization of nitrogen and its compounds by plants. Cambridge, 1959. THOMAS, M. D.: Assimilation of sulfur and physiology of essential S-compounds. Encyclopedia of Plant Physiol. IX, 37-63 (1958) Ed. W. RUHLAND.
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