Plants' Received for Publication September 17, 1984 and in Revised Form November 20, 1984

Plant Physiol. (1985) 77, 779-783 0032-0889/85/77/0779/05/$0 1.00/0

Ureide Metabolism in Leaves of Nitrogen-Fixing Soybean Plants' Received for publication September 17, 1984 and in revised form November 20, 1984

BARRY J. SHELP* AND ROBERT J. IRELAND Department ofHorticultural Science, University ofGuelph, Guelph, Ontario, Canada NJG 2WI (B.J.S.),

and Department ofBiology, Mount Allison University, Sackville, New Brunswick, Canada EOA 3CO Downloaded from https://academic.oup.com/plphys/article/77/3/779/6079839 by guest on 28 September 2021 (R.J.I.)

ABSTRACT natural lighting in vermiculite and fertilized with a nitrogen-free Hoagland-type nutrient solution. During the winter months, In leaf pieces from nodulated soybean (Glycine max ILI Meff cv Maple daylength was extended to 12 h by supplemental lighting of 5000 Arrow) plants, '4Cqurea-dependent NH3 and "CO2 production in the lux. dark showed an approximately 2:1 stoichiometry and was decreased to Chemicals and Radiolabel. Organic compounds and enzymes less than 11% of the control (12-19 micromoles NH3 per gram fresh were obtained from Sigma and Dowex AG 1-X8 (formate form) weight per hour) in the presence of 50 millimolar acetohydroxamate, a and AG 50W-X8 (hydrogen form) from Bio-Lab Laboratories. urease inhibitor. NH3 and CO2 production from the utilization of 12-'4Cj L-[2-'4C]Uric acid (60 gCi umol-') and ['4C]urea (250 sCi allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar ,umol-') were obtained from Amersham. [2-'4C]Allantoin was extent by the presence of acetohydroxamate with a concomitant accu- prepared by incubating [2-'4C]uric acid with uricase (10) followed mulation of urea which entirely accounted for the loss in NH3 production. by purification using ion-exchange chromatography (2). The almost complete sensitivity of NH3 and CO2 production from allan- Metabolism of Urea. In Vivo Studies. Freshly harvested, toin and urea metabolism to acetohydroxamate, together with the ob- chilled second and third trifoliate leaves from 4 to 5 week-old served stoichiometry, indicated a path of ureide assimilation (2.0 micro- symbiotic plants were deveined and cut into 2 x 2 mm pieces. moles per gram leaffresh weight per hour) via allantoate, ureidoglycolate, Leaf samples (0.2 g tissue to 4 ml of incubation liquid) were and glyoxylate with the production of two urea molecules yielding, in added to 34 ml serum vials containing 50 mm K-phosphate (pH turn, four molecules of NH3 and two molecules of CO2. 7.5) with 5% n-propanol (4) and 250 mM '4C-labeled (103 dpm Amol-') or unlabeled urea in the presence and absence of acetohydroxamate. The tissue was twice vacuum infiltrated and the vials sealed and incubated in the dark for varying periods in a shaking water bath at 30°C. To measure NH3 production, tissue was ground in the incubation liquid using sand and a chilled The ureides, allantoin and allantoate, constitute 60 to 80% of mortar and pestle. After filtration through four layers of cheese- xylem-borne nitrogen in nitrogen-fixing tropical legumes (1, 3, cloth, the homogenate was centrifuged (12,000g, 20 min, 4°C), 8, 9, 14, 16) and apparently provide most of the nitrogen used the supernatant fraction collected and NH3 content determined in amino acid and protein synthesis during plant growth (1, 2, in triplicate by microdiffusion plus nesslerization as described 16). Although the pathway of ureide assimilation in plant tissues previously (11). In experiments to test acetohydroxamate inhi- has not been critically examined, five metabolic sequences, each bition as a function of concentration, the incubation liquid only giving rise to glyoxylate, NH3, and CO2 are possible based on was sampled for NH3 content. Where the production of 14CO2 bacterial metabolism (Fig. 1; adapted from Refs. 1, 16, 18). from '4C-labeled substrates was measured, a small tube contain- Recently, Atkins et al. (2) demonstrated the transfer of '4C from ing 0.5 ml of0.1 M KOH was inserted before the vial was closed. [2-'4C]allantoin to allantoate, urea, and CO2 but it was not clear The reactions were terminated by adding 0.5 ml 2 N H2SO4 whether allantoate metabolism is achieved by paths involving through the serum cap and the closed vials were held at 4°C for either allantoicase (EC 3.5.3.4) or allantoate amidohydrolase (EC Table I. Predicted Production ofNH3 and '4C02, and Sensitivity to 3.5.3.9). Acetohydroxamate during the Metabolism of[2-'4C]Allantoin The present investigation determines the stoichiometry ofNH3 and CO2 production from urea and allantoin, and uses a specific Inhibition by 50 mM urease inhibitor, acetohydroxamate, to reduce their production Total NH3andCo2 Acetohydroxamate thereby causing the accumulation of urea (Table I). This infor- Routea Produced 14co2 NH3 mation indicates the exact route of ureide metabolism in leaves Production Production of nitrogen-fixing legumes. 1 3(4)NH3 + 14C02 + '2C02 50 66 (50)c MATERIALS AND METHODS 2 4NH3 + 14C02 + 12CO2 100 100 Plant Material. Soybean (Glycine max [L.] Merr. cv Maple 3 4NH3 + 14C02 + 12CO2 50 50 Arrow) plants, effectively nodulated with Rhizobium sp (North 4 2(3)NH3 + 14C02 + 12C02 0 0 American Plant Breeders, L) were grown in the greenhouse under 5 3(4)NH3 + 14C02 + 12C02 0 0 a See Figure 1. b It is assumed that one urea of allantoate is not 'Supported by grants from the Natural Sciences and Engineering preferentially cleaved. c Number in parentheses indicates the results Research Council of Canada to B.J.S. and R.J.I. if oxamate is hydrolyzed to oxalate liberating NH3. 779 780 SHELP AND IRELAND Plant Physiol. Vol. 77, 1985

(2 - )- Allantoin a Allantoicase b Allantoate amidohydrolas. t c U ease NH2 NH2

"CO COOH CO

- NH-CH-NH

Allantoate Oxo-acid Amino Acid

Ureidoglycine Oxalurate I I NH3 * C02 Oxamate NHU3r T 'b* NH3

Ureidoglycolate Downloaded from https://academic.oup.com/plphys/article/77/3/779/6079839 by guest on 28 September 2021 Carbamoyl - P 'bn Oxalate

t Glyoxylate Glyoxylate Urea NH3 + CO2 c I 2NH3+ C02 2 Urea c NH3+ C02 4 NH3+ 2C°2 2 NH3 + C02 FIG. 1. Possible metabolic pathways for the utilization of allantoin in ureide-producing legumes (adapted from Refs. 2, 24, 27). Oxamate metabolism (----) has not been demonstrated in organisms known to metabolize ureides.

- 100

0 80 0 A

0 60 * + Urea > 20 0 ~~~~~~~~~~~~~~0 E

2 0 40 0W

10 D

20 -Urea j I z M-_n __ ---l_ 0 60 120 180 40 80 12(' 200 Incubation time (min) [Acetohydroxamate] mM FIG. 3. Production ofNH3 (A, A, 0) and "CO2 (0, 0) from soybean FIG. 2. Effect ofacetohydroxamate concentration on urea-dependent leafpieces incubated in the dark with ["C]urea. Tissues from symbiotic NH3 production from leaf pieces incubated in the dark. Leafpieces from plants were incubated either in the presence of 250 mm ["C]urea (103 symbiotic plants were incubated in the presence (0) and absence (0) of dpm Mmol-') with (0, A) and without (0, A, E) 50 mm acetohydroxa- 250 mm urea and the NH3 content ofthe incubation medium determined mate, or in the absence of urea (El). The results are from two separate after 2 h. Results are pooled from two separate experiments; rates in the experiments; each point is the average of duplicate samples within each absence ofacetohydroxamate were 16.5 and 18.5 ,umol NH3 g9' fresh wt experiment, and is corrected for NH3 and '4CO2 present at zero time. h-'. The data are corrected for NH3 present at time zero. Metabolism of Allantoin in Leaf Pieces. Metabolism ofallan- a further 2 h before the "C content of the KOH was determined toin (suspension equivalent to 100 mM) and to by liquid scintillation spectrometry (2). All data are corrected for [2-'4C]allantoin form NH3 and "CCO2, respectively, were assayed as above. Allan- nonenzymic production of NH3 and "CO2 in the absence of leaf tissue. toin suspensions (13) were required to approach saturation of In Vitro Studies. Ureolytic activity was also measured in cell- NH3 production. When urea content was determined, 0.5 g leaf free extracts prepared using 5 volumes of 50 mm K-phosphate tissue was added to 10 ml of incubation liquid. After the desired (pH 7.5). The homogenate was filtered and the supernatant incubation period, the liquid was removed and centrifuged to collected after centrifugation (1 2,000g, 20 min) and used as the remove excess allantoin; the tissue was ground in 10 ml of 10 source of enzyme. The reaction mixture contained 40 mM K- mM Tris-acetate (pH 6.5). The supernatants were combined and phosphate (pH 7.5), 250 mm urea, and 1.0 ml of plant extract in used for NH3 determination, and assayed for urea by reaction a total volume of 3 ml, contained in a closed 34 ml serum vial. with a-isonitrosopropiophenone after deproteinization and ion- The NH3 content of the incubation liquid was determined after exchange chromatography to remove interfering compounds I and 2 h and corrected for NH3 present at time zero. such as ureidoglycolate, allantoate, and allantoin (1 1). All data UREIDE METABOLISM IN LEGUMES 781

U~~~~~ -Aceohdrxaat 20

I .

-6

I z .5 a / 10 / Downloaded from https://academic.oup.com/plphys/article/77/3/779/6079839 by guest on 28 September 2021 /

+ Ace tohydroxamate FIG. 4. Production of NH3 and urea from soybean .* (symbiotic plants) leafpieces incubated in the dark with * - - J allantoin in the absence (0, E) and presence (0, *) of 0 50 mm acetohydroxamate. The experiment was done twice and results shown are from a single typical experiment; each time point shows duplicate samples.

I-I.

Ot 10

.. E-

Incubation time (min)

are corrected for nonenzymic NH3, CO2, and urea production in the absence of leaf tissue. RESULTS To minimize the influence of photorespiratory NH3 production, ureide breakdown was studied in the dark using leaf pieces; 13.6 A addition of MSO2 did not significantly stimulate NH3 accumulation and was therefore omitted in the remainder of the experiments. Under these circumstances, NH3 production should orig- E 4 inate chiefly from ureide metabolism and, depending on the pathway involved, could be reduced by the presence of a specific 14 urease (EC 3.5.1.5) inhibitor, acetohydroxamate (6, 7). Figure 2 2 Co shows the effect of increasing concentrations of acetohydroxamate on urea-dependent NH3 production; 50 mm acetohydroxamate caused 89% inhibition and was used in the remaining 0 60 120 180 experiments. Incubation time (min) Leaf pieces incubated with ['4C]urea exhibited a linear pro- FIG. 5. Production of NH3 (A, A) and '4CO2 (0, *) from soybean duction of NH3 and '4CO2 with rates of 12.7 and 5.9 ,mol g-' leaf pieces incubated in the dark with [2-"4C]allantoin. Tissues from fresh wt h-', respectively, giving an NH3:CO2 stoichiometry of symbiotic plants were incubated with 25 mm [2-'4CJallantoin (5 x 103 2.2:1 (Fig. 3). Acetohydroxamate completely prevented NH3 and dpm Mmol-') with (0, A) and without acetohydroxamate (0, A). The "'CO2 production after 1 h. Figure 4 shows that leaf pieces results are from two separate experiments; each point is the average of incubated in a suspension of allantoin also produced NH3 in an duplicate determinations within each experiment. Each value has been corrected for NH3 and "4CO2 present at time zero. 2Abbreviation: MSO, 1-methionine sulfoximine. 782782SHELP AND IRELAND Plant Physiol. Vol. 77, 1985 approximately linear fashion at a rate ofabout,mol6.7g-' fresh g-' fresh wt h-', respectively). It is noteworthy that leaf pieces wt h-'. The urea content initially increased, followed by a de- fed allantoin showed a decrease in urea concentration from 1 to crease over the next 2 h. In contrast, the presence of acetohy- 3 h (Fig. 4), suggesting that allantoin was either no longer droxamate inhibited any further production of NH3 together available in the free space or not being taken up by the tissue to with a concomitant increase in tissue urea, which did not exhibit replenish the internal poo1. This is supported by the fact that a decrease during the study period. Urea accumulation, calcu- allantoin suspensions gave rates of NH3 production which ex- lated as the difference between the two treatments was equivalent ceeded those at solubility limits (compare Figs. 4 and 5); saturat- to,umol3.2 g-' fresh wt h-' and was about 48% of NH3 ing rates were about 8.0 umol g'1 freshh-'wt (Shelp and Ireland, production in the absence of the inhibitor (i.e. NH3 produc- unpublished). Although vacuum infiltration and n-propanol were tion:urea accumulation = 2.1:1). Metabolism[2-'4C]allantoinof used, it is suggested that this technique is probably not adequate produced NH3 and "4CO2 in a 4.1:1 ratio (Fig. 5) indicating for periods longer than 3 h. Despite this difficulty, the metabo- (since only 50% of the CO2 will be labeled) a 2.1:1 stoichiometry lism of allantoin and urea in the dark suggests that ureide between NH3 and CO2 (see Fig. 1 and Table I). Allantoin metabolism is not linked to photosynthetic generation of ATP utilization was also extremely sensitive to acetohydroxamate, and reductant (1, 16) and is consistent with the view that urea Downloaded from https://academic.oup.com/plphys/article/77/3/779/6079839 by guest on 28 September 2021 which decreased the production Of CO2 and NH3 to less than breakdown does not proceed either by the allophanate pathway 10% of control values. or by NADP+ (NAD+) urea dehydrogenase, but rather by urease (5). DISCUSSION In conclusion, the data presented provides indirect evidence in ureide-producing legumes for a pathway of allantoin utiliza- Metabolism of urea and allantoin produced NH3 andCO2 in tion via allantoate, ureidoglycolate, and glyoxylate with the pro- an approximately 2:1 stoichiometry (Figs. 3 and 5) suggesting duction of urea yielding NH3 and CO2. Rates of urea accumu- that these products are derived solely from urea breakdown. This lation andNH3 production from allantoin indicate that ureides is supported by the fact that acetohydroxamate inhibition of are metabolized in leaf tissue at about 1.6 to,umol2.0 g-' fresh ureolytic activity (Figs. 2 and 3) decreased to a similar extent wth-'. Although these experiments were performed in the dark (1 1% or less of the control; Figs. 2-5) NH3 andC02 production to minimize complications arising from photorespiratory metab- from both substrates. Moreover, the acetohydroxamate-induced olism, it is expected that ureides are utilized at similar rates in inhibition was accompanied by an accumulation of urea from the light. If so, it is tempting to speculate that ureide-derived allantoin (Fig. 4) in the amount expected from the loss in NH3 NH3 can be reassimilated via glutamine synthetase 1,( 19) and production (urea = 2NH3), indicating that the path of ureide that ureide-derived glyoxylate can feed into the photorespiratory metabolism was still operating under these circumstances. IfNH3 pathway (19). In the latter case, 0.20 to 0.25 Amol NH3g ' fresh formation did not result from urea breakdown, acetohydroxa- wt would be in the conversion of two molecules 66% (paths1 h-' produced of mate inhibition would not be expected to exceed glycine to serine. Currently, studies are underway to examine and 3), the stoichiometry of NH3 andCO2 production would not these hypotheses and to determine the carbon flow through the be 2:1 (path 4), and urea would not accumulate in the presence suspected intermediates in an attempt to identify rate-limiting of acetohydroxamate (paths 4 and 5) (Fig. 1; TableI). Thus, in ureide metabolism. except from the metabolism of urea, it seems unlikely that an steps additional source of NH3 and CO2 was available during ureide Acknowledgments-The authors wish to thank Professor K. W. Joy for helpful assimilation. The stoichiometry studies, together with the almost discussion and use of equipment in his laboratory and Joelle Marmonier for complete sensitivity ofNH3 andCO2 production to acetohydrox- technical assistance. amate, are consistent only with the operation of route 2 (Table where allantoate is metabolized via allantoicase to ureidogly' LITERATURE CITED colateI) and then to with the formation oftwo molecules glyoxylate 1. ATKINS CA 1982 Ureide metabolism and the significance of ureides in legumes. of urea (Fig. 1). In NS Subba Rao, ed, Advances in Agricultural Microbiology. Oxford and Studies of symbiotic tropical legumes have shown that the IBH, New Delhi, pp 25-51 ureides, allantoin and allantoate, are the major nitrogenous 2. ATKINS CA, JS PATE, A RITCHIE, MB PEOPLES 1982 Metabolism and translo- (1, 16). cation ofallantoin in ureide-producinggrain legumes. Plant Physiol 70:476- compounds travelling in xylem to developing organs In 482 cowpea plants, Atkins et al. (2) have recovered"1C from [2-"4C] 3. HERRIDGE DF. CA ATKINS, JS PATE, RM RAINBIRD 1978 Allantoin and allantoin as allantoate, urea, and CO2 indicating the involvement allantoic acid in the nitrogen economy of the cowpea ( Vigna unguiculata L. of allantoinase, urease, and possibly allantoicase and ureidogly- Walp). Plant Physiol 62: 495-498 colase allantoicase in higher plants has 4. HOGAN ME, IE SwIFT, J DONE 1983 Urease assay and ammonia release from (EC 4.3.2.3). Although leaf tissue. Phytochemistry 22: 663-667 been reported for peanut cotyledons (12), its existence, and that 5. KERR PS, DG BLEvINs, BJ RAPP, DD RANDALL 1983 Soybean leaf urease: of ureidoglycolase (converts ureidoglycolate to glyoxylate and comparison with seed urease. Physiol Plant 57: 339-345 urea) have not been shown unambigiously in leaves of ureide- 6. KOBASHI K, J HASE, K UEHARA 1962 Specific inhibition of urease by hydrox- the designed for micro- amic acids. Biochim Biophys Acta 65: 383-385 producing legumes (1, 16). Using assay 7. KoBASHi K, N TERASHIMA, S TAKEBE, J HASE 1978 A new method of organisms (17), we have, as reported by others (1, 16), not been determination of hydroxamic acid by its urease inhibition and application able to detect these enzyme activities in cell-free leaf extracts to biochemical studies. J Biochem 83: 287 despite the addition of PVP, glycerol, reducing agents (DTT, 8. MCCLURE PR, DW ISRAEL 1979 Transport of nitrogen in the xylem of soybean Mn and EDTA, and phen- plants. Plant Physiol 64: 411-416 GSH, j3-mercaptoethanol), Mg ions, 9. PATE JS, CA ATKINS, ST WHITE, RM RAINBIRD, KC Woo 1980 Nitrogen ylmethylsulfonyl fluoride (a protease inhibitor). On the other nutrition and xylem transport ofnitrogen in ureide-producinggrain legumes. hand, abundant levels of ureolytic and allantoinase activities Plant Physiol 65: 961-965 have been demonstrated (1, 2, 4, 5, 15, 16). Using the in vivo 10. RAINBIRD RM, JH THORNE, RWF HARDY 1984 Role of amides, amino acids, and ureides in the nutrition of developing soybean seeds. Plant Physiol 74: assay for ureolytic activity, high urea concentrations (greater 329-334 than 200 mM) are required (4,5; Shelp and Ireland, unpublished), 11. SHELP BJ, K SIECIECHOWIcz, RJ IRELAND, KW Joy 1984 Determination of presumably to expedite diffusion into the tissue producing linear urea and ammonia in leaf extracts: Application to ureide metabolism. Can rates of production of NH3 and CO2 when both the incubation J Bot. In press and tissue are 3 and In contrast to a 12. SINGH R 1968 Evidence for the presence ofallantoicase in germinating peanuts. liquid analyzed (Figs. 4). Phytochemistry 7: 1503-1508 previous study (4), similar rates of ureolytic activities were found 13. SPRENT JJ 1980 Root nodule anatomy, type ofexport product and evolutionary using either the in vivo or in vitro assay (15.4 versus 13.8 -mol origin in some Leguminosae. Plant Cell Environm 3: 35-43 UREIDE METABOLISM IN LEGUMES 783 14. STREETER JG 1979 Allantoin and allantoic acid in tissues and stem exudate 17. TRIJBELS F, GD VOGELS 1967 Allantoate and ureidoglycolate degradation by from field-grown soybean plants. Plant Physiol 63: 478480 Pseudomonas aeruginosa. Biochim Biophys Acta 132: 115-126 15. THOMAS RJ, LE SCHRADER 1981 The assimilation of ureides in shoot tissues 18. VOGEIs GD, C VAN DER DRWr 1976 Degradation of purines and pyrimidines of soybeans. I. Changes in allantoinase activity and ureide contents of leaves by microorganisms. Bact Rev 40: 403-468 and fruits. Plant Physiol 67: 973-976 16. THOMAS RJ, LE SCHRADER 1981 Ureide metabolism in higher plants. Phyto- 19. WALLSGROVE RM, AJ KEYs, PJ LEA, BJ MIFLIN 1983 Photosynthesis, photo- chemistry 20: 361-371 respiration and nitrogen metabolism. Plant Cell Environ 6: 301-309 Downloaded from https://academic.oup.com/plphys/article/77/3/779/6079839 by guest on 28 September 2021