Aug 30, 1983 - have a much greater enzymic potential (asparaginase) for direct deamidation ofasparagine (8). Deamidation of asparagne by asparaginase in ...
Plant Physiol. (1984) 74, 822-826 0032-0889/84/74/0822/05/$0 1.00/0
Amino Acid Metabolism in Pea Leaves' UTILIZATION OF NITROGEN FROM AMIDE AND AMINO GROUPS OF ['5N]ASPARAGINE Received for publication August 30, 1983
TRUNG CHANH TA, KENNETH W. JOY*, AND ROBERT J. IRELAND Department ofBiology and Institute of Biochemistry, Carleton University, Ottawa,
Ontario KIS 5B6, Canada ABSTRACT The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with '5N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination. Nitrogen from the amide group is found predominantly in 2-hydroxysuccinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation. The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.
using '5N or 14C labeling are consistent with the operation of these pathways (2-4, 15, 18). However, no distinction was made between the amide or amino group of asparagine as the source of nitrogen, and a complete picture of the individual nitrogen flow has not been realized. In this paper, we describe the utilization of asparagine, '5N-labeled individually in the amide or amino position, supplied through xylem to young pea leaves.
MATERIALS AND METHODS Pea seedlings (Pisum sativum cv Little Marvel) were grown for 3 weeks without nodulation in nutrient solution containing nitrate as the source of nitrogen, with a 12-h photoperiod (3). Plants selected for experiments had four fully expanded leaves, and the fifth leaf was not quite half-expanded (stage 4, Ref. 18). Samples of the terminal growing leaves (leaf 5 plus apex) were obtained from several shoots by cutting under water, and their cut ends were placed in labeled 5 mm asparagine solutions, pH 6.4. At intervals from 6 to 60 min, three or four leaf samples were rinsed, frozen in liquid N2, and extracted overnight in the cold after homogenization in 80% ethanol. After concentration and partition against chloroform, components of the aqueous fraction were analyzed. Separation of Nitrogenous Components. Amino acid levels were determined using a Beckman 1 l9BL amino acid analyzer, using lithium buffers. To obtain individual nitrogen groups from the amides for '5N analysis, a more complex method was required, modified from the procedure used by Fentem et al. (6). Free ammonia was first distilled from the plant extract, adjusted In a number of legumes, asparagine is a major nitrogenous to pH 10, using a stream ofacid-washed air at 40C and collecting transport compound for growth of young tissues and seed devel- in a small volume of 0.1 N HCI. The extract was adjusted to pH opment (2, 13, 14). Enzymic studies have shown the potential 6.5, and acidic amino acids were removed from the extract by for direct utilization of asparagine through both deamidation passage over Dowex 1 (acetate) from which glutamate was then and transamination (1, 8, 14-17) but the relative importance of eluted with 0.2 N acetic acid, and aspartate with 0.5 N acetic these pathways in vivo is not certain. In half-expanded pea leaves, acid. The amide nitrogen of glutamine in the Dowex 1-treated use of inhibitors suggested that a considerable proportion of extract was released by addition of 0.5 unit of glutaminase asparagine was transaminated, with subsequent deamidation of (Sigma, grade V, containing no detectable asparaginase) at pH the transamination products; however, younger leaves appear to 5, 2 h at 30°C. The resulting ammonia was collected by distillahave a much greater enzymic potential (asparaginase) for direct tion, as above. Asparagine-amide was then released with aspardeamidation of asparagine (8). aginase (0.5 unit, Sigma, grade V), pH 8, 2 h at 30C, and Deamidation of asparagne by asparaginase in the cytosol (10) released ammonia was again removed. The glutamate and asparyields aspartate and ammonia. Transamination of the 2-amino tate produced by deamidation were then separated on Dowex 1 nitrogen from asparagine is a peroxisomal reaction (10), cata- as described above, to yield the amino groups of glutamine and lyzed by the same enzyme responsible for serine-glyoxylate trans- asparagine. amination (9). Asparagine transamination produces 2-oxosuccinSeparation of some remaining amino acids were carried out amate but this compound appears to be rapidly converted to using the amino acid analyzer, and collecting the effluent after HSA, which accumulates in peas (15). Results of in vivo studies reaction with ninhydrin. Nitrogen was released from the ninhydrin complex in individual peaks by addition of 0.2 ml H2S04 ' Supported by grants from the Natural Sciences and Engineering (7), and the resulting ammonia was recovered by distillation at Research Council, Canada, to K. W. J. and R. J. I. IOOC following addition of 10 N NaOH. In some cases, the total 2 Abbreviations: HSA, 2-hydroxysuccinamic acid; AOA, aminooxy- amino nitrogen of residual extracts (with ammonia, Glu, Asp, acetate; Hse, homoserine; MSO, methionine sulfoximine. Gln, and Asn already removed) were recovered for '5N analysis 822
UTILIZATION OF ASPARAGINE NITROGEN by reaction of the extract with ninhydrin, and recovery of ammonia after decomposition with H2SO4, as above. Organic acids were recovered from a separate aliquot of extract, by passage through Dowex 50 (H+) and then absorption on Dowex 1 (chloride); the fraction containing HSA was eluted with 0.2 N HC1. Further separation of HSA was carried out using HPLC on an aminopropyl column, eluted with 0.1 M KH2PO4 buffer, pH 3.4, and detected by measuring A at 214 nm. Labeling Experiments. ['5N-amide]Asparagine (99% atom excess) was obtained from Isotope Labeling Corp (Whippany, NJ) and ['5N-amino]asparagine (95% atom excess) was from Merck, Sharp and Dohm, Montreal, Canada. [U-'4C]Asparagine was from Amersham. 'sN was determined by emission spectrometry (Jasco NIA-1) after conversion of ammonia or amino acid samples to N2 gas in a discharge tube, using the Dumas procedure, modified from the method of Kano et al. (12). 14C was measured using liquid scintillation counting.
RESULTS AND DISCUSSION Leaf tissue chosen for this work was 1 d younger than used in earlier inhibitor experiments (8). The tissue had higher asparaginase activity (8), but inhibitor experiments (not shown) indicated that transamination was still of considerable importance in asparagine utilization. In '5N-feeding experiments, label was supplied directly through the cut stem to the detached apex. Although phloem transport from older leaves is disrupted when a growing apex is excised, the normal supply of material through the xylem can be maintained, and the results confirm that this provides a useful and 'physiological' method for introduction of experimental substrate. Typical values for some nitrogenous components of growing leaves, during a feeding experiment, are in Table I, and show little change over the experimental period. The supply of asparagine, provided at a concentration similar to that normally found in xylem sap (A.A. Urquhart, unpublished) caused no significant alteration to amino acid levels. Asparagine and HSA were the major components, followed by glutamate, aspartate, glutamine, alanine, and homoserine. Treatment with the glutamine synthetase inhibitor MSO decreased the level of glutamine, and inTable I. Changes in Levels of the Major Soluble Amino Acids and Other Nitrogen Metabolites in Detached Expanding Pea Leaves during Experimental Feeding with Asparagine MSO (1 mM) and AOA (4 mM) were prefed for 60 min before supplying asparagine. Metabolite Concentration Asn only Asn + MSO Asn + AOA (60 min) 0 min 12 min 30 min 60 min (60 min) ;tmolg`' fresh wt 20.20 Asn 22.10 27.50 26.90 24.10 23.45 2.84 Gln 2.83 0.64 1.48 2.85 2.99 3.60 4.18 9.11 3.56 3.62 4.46 Asp 4.82 4.98 7.46 8.22 Glu 5.10 5.06 Thr 0.32 0.27 0.45 0.40 NAa NA 0.92 0.94 NA Serb 0.81 0.95 NA 1.47 Hse 1.25 1.13 1.64 NA 0.18 0.21 0.18 0.17 Gly Ala 1.61 NA 1.51 1.60 1.83 0.67 0.68 0.74 0.78 2.42 NH3 HSA 15.25 15.30 15.70 15.90 15.80 'NA, not analyzed. bValues for serine are approximate due to poor resolution unknown amino compound.
na
0.85 0.20 0.97 NA na
from an
823
creased ammonia, glutamate, and aspartate. Addition of the transamination inhibitor AOA decreased the glutamine, homoserine, and alanine levels but increased the accumulation of glutamate. Amide Utilization. When ['5N-amide]asparagine was supplied to the detached shoot apices, there was a linear accumulation of label in the tissue (Table II), and an increasing flow of label into metabolic products over the 1-h experimental period. The heaviest incorporation was into the amide groups of glutamine and HSA, followed by glutamate. There was a considerable flow into the total pool of other amino compounds, although labeling was not high in any individual amino acids. Threonine, serine, homoserine, glycine, and alanine were also analyzed separately, but had relatively low levels of '5N incorporation. The flow of amide nitrogen to HSA confirms that this compound results from transamination of asparagine (via the nonaccumulating intermediate 2-oxosuccinamate) and complements the evidence derived from '4C-labeling (15) and enzymological studies (8, 9). The heavy labeling of glutamine amide could result from a direct transamidation, or from assimilation of ammonia, released by deamidation, through glutamine synthetase. When MSO, a glutamine synthetase inhibitor, was provided with the ['5N-amidelasparagine, the total flow of '5N from asparagine to glutamine was substantially reduced, by about 86% at 60 min (Table II), while a similar amount of '5N accumulated in the pool of free ammonia. The decrease in flow of '5N resulted from a considerable inhibition of glutamine synthesis (Table I), with relatively little effect on the atom % labeling of the small amount of glutamine that was produced (Table II). In a parallel series of experiments (not shown), the flow of total '5N from ('5NH4)2SO4 to glutamine was comparably reduced (by 81-84%) by a similar concentration of MSO. These results suggest that the major flow of nitrogen from asparagine to glutamine is MSO-sensitive, and through a small, actively turning over pool of ammonia, rather than through any direct transamidation. In an attempt to determine whether asparagine, or the transamination products 2oxosuccinamate or 2-hydroxysuccinamate, were the substrates for deamidation reactions producing ammonia, the transaminase inhibitor AOA was added together with MSO (Table II). This decreased the labeling of HSA by over 90%, and also had a great effect on the transfer of label to ammonia (almost 75% inhibition). This indicates that a major proportion of the asparagine is transaminated before the amide group is released. The labeling of a number of other compounds, including aspartate and glutamate, was also decreased in the presence of AOA. Glutamate, the amino group of glutamine, and other amino compounds also received label from the amide group of asparagine, as would be expected ifthe glutamine synthetase/glutamate synthase cycle, plus transamination, were in operation. Administration of the glutamate synthase inhibitor azaserine (2 mM) together with ['5N-amide]asparagne resulted in an 80% fall in '5N labeling of glutamate, with an increased labeling of glutamine-amide, adding further support to this conclusion (results not shown). Amino Utilization. When '5N was used to label the amino group of asparagine (Table III), accumulation of label and metabolism were linear and at a similar level to that found for amide-labeled experiments. Aspartate, glutamate, alanine, and homoserine were the main recipients of asparagine amino nitrogen, and there was a considerable input into the general pool of other amino compounds. Aspartate can be formed directly from asparagine by deamidation, whereas other amino compounds may be formed through transamination of this aspartate (e.g. to glutamate), or direct transamination of asparagine (e.g. to alanine; Ref. 9). This is borne out by supply of AOA, which had little effect on aspartate labeling, but approximately halved the total flow of amino nitrogen.
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Plant Physiol. Vol. 74, 1984
TA ET AL.
Table II. Flow of'5N from ['5N-AmidelAsparagine Supplied to Expanding Pea Leaves in the Presence and Absence of MSO (1 mM), or MSO Plus Aminooxyacetate (4 mM) Inhibitors were supplied for I h before addition of labeled asparagine. '5N Content Atom % excess
With Asn only Asn-amide Gln-amide Gln-amino Asp Glu Other amino compounds NH3 HSA
+MSO Asn-amide Gln-amide Gln-amino Asp Glu Other amino compounds NH3 HSA
+MSO + AOA Asn-amide Gln-amide Gln-amino Asp Gln Other amino compounds NH3 HSA
6 min
12 min 20 min 30 min ng '5N.g' fresh wt
60 min
at 60 min
2,395 25
4,235 66 12 15 42 63 5 86
18,382 615 83 58 209 845 31 579
5.60 1.55 0.22 0.09 0.32 0.19 0.28 0.26
4 4 21 21 2 43
5,880 139 23 20
10,576 254 36 35
64
91 409 13 264
149 8 153
15 min
30 min
60 min
Atom % excess at 60 min
4,045
8,005
17 2
44 5
11 39 108 99 108
33 98 239 247 239
14,865 87 8 89 188 531 848 531
5.30 1.01 0.12 0.08 0.20 0.14 2.50 0.24
15 min
30 min
60 min
Atom % excess at 60 min
3,421 16
6,848 35 3
13,406 66 6 34 116 260 223 39
5.25 0.81 0.08 0.05 0.12 0.08 1.60 0.02
1
8 24 72 43 9
The AOA had a variable effect on labeling of different amino acids. Transamination of asparagine (as indicated by flow of amide nitrogen to HSA; Table II and Ref. 8) and labeling of alanine and homoserine (Table III) were almost completely inhibited. In contrast, there was much less effect on labeling of "other amino compounds" (Table III); this group would include threonine, lysine, and methionine, which can be derived directly from aspartate. Labeling of glutamate was approximately halved by treatment with AOA; however, the total pool size was almost doubled. It is possible that there is a differential sensitivity of transaminases to this inhibitor, or that it does not penetrate equally to all compartments in the cell, allowing the flow of '5N from aspartate. The overall pattern of distribution of nitrogen from asparagne is summarized in Table IV. The total recovery of 'sN metabolized from amino-labeled asparagine is somewhat lower than for amide-labeling. This may be due to experimental variation; it is also likely that it is more difficult to recover absolutely '5N label present at low concentration in many compounds, after widespread distribution by transamination, in comparison with higher levels of labeling found in fewer compounds receiving 'IN from the amide group. The values in Table IV are not absolute rates, but represent the actual accumulation of '5N (100 atom %) during the 1-h experiment. Since the actual precursor pool of asparagine reached a maximum of 5.6 atom % 'IN (Table II), a 17.9-fold correction
14 54
135 102 19
gives a value closer to the real rate of asparagine metabolism. A further correction is necessary, to adjust for the steady rise in labeling of the asparagine pool, from 0 at the start of the experiment. As the rise was quite linear, and the pool size was relatively unchanged, a 2-fold correction is a reasonable approximation. Thus, the corrected rate of asparagine metabolism, derived from this study, is 169 nmol/h x 17.9 x 2 x 24 (h) = 145.2 umol/d/g fresh weight or 4.06 mg N/d/g fresh weight. Between developmental stages 4 and 5, an expanding leaf increases by 1.1 mg N/d, or 5.5 mg N/d/g fresh weight at the midpoint stage ( 18); thus, the data obtained here (4.06 mg N/d) are consistent with the role of asparagine as the principal nitrogen source for the developing leaf. The values also confirm that growth and metabolism can continue for a while at a high level in detached apices, even though the supply ofnutrients in phloem has been disrupted. The results for homoserine raise some interesting questions. The accepted pathway for synthesis (5) involves aspartate as the starting compound, and the nitrogen and carbon flow together, via aspartic semialdehyde, into homoserine. Several lines of evidence suggest that in peas (which accumulate high quantities of homoserine, unlike most other plants) transamination may also play a role in synthesis of this amino acid. Aminooxyacetate reduced the flow of amino nitrogen from asparagine to homoseine by about 90%, although the labeling of aspartate was unchanged (Table III). In a dual labeling experiment with [14C]-
825
UTILIZATION OF ASPARAGINE NITROGEN Table III. Flow of'5Nfrom /5N-AminoJAsparagine Supplied to Expanding Pea Leaves, in the Presence and Absence of AOA (4 mM) '5N Content Atom % excess
-AOA Asn-amino Gln-amide Gln-amino Asp Glu Gly Ala Hse Other amino compoundsb
6 min
12 min 20 min 30 min ng '5N.g-' fresh wt
60 min
at 60 min
2,362 NDa 3
4,024
18,030 28 99 281 270 7 128 97 462
5.20 0.07 0.25 0.48 0.40 0.31 0.51 0.42 0.12
29 28 ND 10 8 22
4 8 45 50 ND 22 13
23 15 min
5,611 8 23 65 75 2 31 21 74
30 min
+AOA Asn-amino 4,398 8,125 1 2 GIn-amide 3 12 Gln-amino 44 115 Asp 23 69 Glu ND ND Gly 2 6 Ala 2 6 Hse 55 151 Other amino compoundsb a ND, not detectable. b Total amino nitrogen, other than amino acids listed above. Table IV. Metabolism of Nitrogen Groups ofAsparagine by Developing Pea Leaves during a 60-Minute Feeding Period Values have been adjusted to account for the small differences in atom % labeling of the two sources of asparagine. Flow of Nitrogen
'5N-amide '5N-amino nmol '5N.h-'- g' fresh wt Residual Asn Gin-amide Gin-amino Glu Asp Hse Ala Other amino acids NH3 HSA Total recovery of metabolized Asn
1337
1426
44 6 15 4
2 8 20 21 8
NDa
11
ND 56 2 42
34 ND ND
169
104
ND, not detectable.
and ['5N-amino]asparagine, the labeling ratios of several amino acids were compared (Table V). For alanine, the labeling ratio was low, indicating a flow of nitrogen (by transamination) without any corresponding carbon flow. The high ratio for aspartate indicates that more carbon than nitrogen was retained in the compound: this can be explained by an equilibration of aspartate amino-N through transamination (11), and possibly an additional flow of carbon to aspartate via oxosuccinamate/hydroxysuccinamate (15). Homosenne showed an intermediate level of
9,820 11 44 113 132 3 54 37
250
60 min
Atom % excess at 60 min
16,915
5.15 0.03 0.11 0.45 0.12 0.04 0.09 0.08 0.07
6 23 280 138 1
12 10 310
Table V. A Comparison of 4C and '5N Flow from Asparagine to Homoserine and Other Amino Acids Expanding pea leaves were supplied with "1C- and '5N-amino-labeled asparagine, and the `'C/'5N ratio (cpm/ng '5N) was determined at times up to 60 min.
14C/15N
Time min 12 20 30 60
Asn
Asp
40.4 40.2 44.8 42.7
106.7 208.7 210.1 242.2
Glu cpm/ng
Ala
Hse
51.8
5.5 7.3 7.2 4.3
26.0 34.7 50.3 41.9
84.1 83.6 107.4
labeling, very different from the main pool of aspartate, which therefore does not appear to be its precursor. This is consistent with synthesis of homoserine by transamination, using a ketoacid derived from the general carbon pool (which also supplies carbon to glutamate). An alternate possibility would be homoserine synthesis in a compartment where the labeling ratio for aspartate (produced from asparagine) was very different from the main aspartate pool. This is unlikely, as asparaginase activity is not present in major organelles (10) and in any case active transaminases (which would equilibrate the labeling of aspartate) are associated with the major organelles. Active homoserine transamination has been demonstrated in pea leaf extracts (K. W. Joy and C. Pearson, unpublished).
CONCLUSIONS Feeding with [ '5N]asparagine has confirmed that both nitrogen groups of asparagine are readily utilized as a nitrogen source by expanding pea leaves. Metabolism can occur by two distinct routes: deamidation to aspartate and ammonia, or transamina-
826
TA ET AL.
tion to produce oxosuccinamate, which is rapidly converted to HSA, followed by deamidation of one or both of these products. Although young leaves have considerable amounts of asparagnase, transamination appears to provide the major pathway for primary metabolism of asparagine. Since the transamination products of asparagine can themselves be deamidated, and the aspartate resulting from asparaginase activity is readily transaminated, there is little difference overall resulting from the operation of the two pathways. Ammonia is released and reassimilated through the glutamine synthetase/glutamate synthase cycle, and a range of transamination products are formed. The significance may lie in the localization and regulation of the enzymic systems involved, and the potential for subsequent metabolism of HSA, which is formed in the tissue. Acknowledgment-We thank C. Shay for assistance with amino acid analysis. LITERATURE CITED 1. ATKINS CA, JS PATE, MB PEOPLES, KW Joy 1983 Amino acid transport and metabolism in relation to the nitrogen economy of a legume leaf. Plant Physiol 71: 841-848 2. ATKINS CA, JS PATE, PJ SHARKEY 1975 Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds. Plant Physiol 56: 807-812 3. BAUER A, AA URQUHART, KW JoY 1977 Amino acid metabolism of pea leaves: diurnal changes and amino acid synthesis from "5N-nitrate. Plant Physiol 59: 915-919 4. BAUER A, KW Joy, AA URQUHART 1977 Amino acid metabolism of pea leaves: labeling studies on utilization of amides. Plant Physiol 59: 920-924 5. BRYAN JK 1980 Synthesis of the aspartate family and branched-chain amino acids. In BJ Miflin, ed, The Biochemistry of Plants, Vol 5. Academic Press,
Plant Physiol. Vol. 74, 1984
New York, pp 403-452 6. FENTEM PA, PJ LEA, GR STEWART 1983 Ammonia assimilation in the roots of nitrate- and ammonia-grown Hordeum vulgare (cv Golden Promise). Plant Physiol 71: 496-501 7. FUJIHARA S, M YAMAGUCHI 1981 Assimilation of "NH3 by root nodules detached from soybean plants. Plant Cell Physiol 22: 797-806 8. IRELAND RJ, KW JoY 1981 Two routes of asparagine metabolism in Pisum sativum L. Planta 151: 289-292 9. IRELAND RJ, KW Joy 1983 Purification and properties of an asparagne aminotransferase from Pisum sativum leaves. Arch Biochem Biophys 223: 29 1-296 10. IRELAND RJ, KW Joy 1983 The subcellular localization of asparaginase and asparagine aminotransferase in Pisum sativum leaves. Plant Physiol 72: 1127-1129 11. Joy KW, RJ IRELAND, PJ LEA 1983 Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor. Plant Physiol 73: 165168 12. KANO H, T YONEYAMA, K KUMAZAWA 1975 Emission spectrometric '"N analysis of the amino acids and amides in plant tissues separated by thinlayer chromatography. Anal Biochem 67: 327-331 13. LEA PJ, L FOWDEN 1975 Asparagine metabolism in higher plants. Biochem Physiol Pflanz 168: 3-14 14. LEA PJ, BJ MIFLIN 1980 Transport and metabolism of asparagine and other nitrogen compounds within the plant. In BJ Miflin, ed, The Biochemistry of Plants, Vol 5. Academic Press, New York, pp 569-607 15. LLOYD NDH, KW Joy 1978 2-Hydroxysuccinamic acid: a product of asparagine metabolism in plants. Biochem Biophys Res Commun 81: 186-192 16. SODEK L, PJ LEA, BJ MIFLIN 1980 Distribution and properties of a potassiumdependent asparaginase isolated from developing seeds of Pisum sativum and other plants. Plant Physiol 65: 22-26 17. STREETER J 1977 Asparaginase and asparagine transaminase in soybean leaves and root nodules. Plant Physiol 60: 235-239 18. URQUHART AA, KW Joy 1982 Transport, metabolism, and redistribution of xylem-borne amino acids in developing pea shoots. Plant Physiol 69: 12261232
