Ureide catabolism in nitrogen-fixing legumes

TIBS 13-March 1988 Symp. Quant. Biol. 51,515-523 12 Ginsburg, D., Handin, R. L, Bonthron, D. T. and Orkin, S. H. (1985) Science228,1401-1406 13 Fay, P. J., Kawai, Y., Wagner, D. D., Ginsburg, D., Bonthron, D., OhissonWilhehn, B. M., Chavin, S. l., Abraham, G. N., Handin, R. I., Orkin, S. H., Montgomery, R. R. and Marder, V. J. (1986) Sc/ence232, 995-998 14 Bonthron, D. T., On', E. C., Mitsock, L. M., Ginsburg, D., Handin, R. l. and Orkin, S. H. (1986) NudeicAcids Res. 14, 7125--7127 15 Bonthron, D. T., Handin, R. I., Kaufman, R. J., Wasley, L.C., On', E. C., Mitsock, L. M., Ewenstein, B., Loscalzo, J., Ginsburg, D. and Orkin, S. H. (1986) Nature 324, 270-273 16 Verweiji, C. L., deVries, C. J. M., Distel, B., van Zonneveld, A-J., van Kessel, A. G., van

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Mourik, J. A. and Pannekoek, H. (1985) Nucleic Acids Res. 13, 4699--4717 Verweiji, C. L., Diergaade, P. J., Hart, M. and Pannekoek, H. (1986)EMBOJ. 5,1839--1847 Montgomery, R. R. and Zimmerman, T. S. (1978) J. Clin. Invest. 61,1498-1507 Verweij, C. L., Hart, M. and Pannekoek, H. (1987) EMBO J. 6, 2885-2890 Federici, A. B., Elder, J. H., De Marco, L., Rugged, Z. M. and Zimmerman, T. S. (1984)

J. Clin.Invest.74, 2049-2055 21 Fujimura,Y., Titani, K., Holland,L. Z., Russell,S. R., Roberts,J. R., Elder,J. H. Rugged, Z. M. and Zimmerman,T. S. (1986)J. Biol. Chem. 261,381-385 22 Girma, J-P., Kalafafis, M., Pietu, G., Lavergne, J-M., Chopek, M. W., Edgington, T. S. and Meyer, D. (1986) Blood 67, 1356--

1366 23 Plow, E. F., Pierschbacher, M. D., Rouslahti, E., Marguerie, G. A. and Ginsberg, M. H. (1985) Proc. NatiAcad. ~i. USA 82, 8057-8061 24 Haverstick, D. M., Cowan, J. F., Yamada, K. M. and Santoro, S. A. (1985) Blood66, 946952 25 Pared, F. I., Fujimura, Y., Dent, J. A., Holland, L. Z., Zimme.~man, T. S. and Rugged, Z. M. (1986)J. Biol. Chem. 261, 15310-15315 26 Fujimura, Y., Titani, K., Holland, L. Z., Roberts, J. R., Kostel, P., Rugged, Z. M. and Zimmerman, T. S. (1987)J. Biol. Chem. 262, 1734-1739 27 Foster, P. A., Fulcher, C. A~, Marti, T., Titani, K. and Zimmerman, T. S. (~987)J. Biol. Chem. 262, 8443-8446

supplied directly to the developing embryo, but are instead metabo|ized in the seed coats and pod waU6,s to the amides, glutamine and asparagine, which then supply nitrogen to the developing embryo. Ureides are a poor Rodney G. Winider, Dale G. Bl~evins,Joseph C. Polacco and nitrogen source for growth of developing Douglas D. Randall cotyledons in culture, i.e. cultured cotyledons grown on allantoin accumulate nitrogen at 21% the rate of those grown on glutamine 9. The seed coat The ureides, allantoinand allantoate, are the major nitrogen transportcompounds in certain (maternal tissue) may play a critical role N2.~ legtcne& A novel pathway of agantoate catabolism ~ua releases 4NHh 2C0 z and in the contr.o! of seed development by its glyoxylatefrom aUantoate, independent of urease action, has beenproposedfor soybeans. control of nitrogen metabofism 6,8-~°.

Ureide catabolism in nitrogenfixing legumes

and allantoate (reviewed in Refs 2 and 3). In contrast, most other leguminous plants studied to date transport asparagine under N2-fixing conditions and are designated amide transporters. In soybean, the proportion of total xylem nitrogen in ureides is highly correlated to the level of N2-fixation4. When soybean receives N as nitrate (NO3-), N2-fixation is inhibited and both NO 3and asparagine are transported to the shoot. Although much of the nitrogen released from ureides is ultimately stored in seed storage proteins, the primary site of ureide ca~bofism 3 is the leaf. There is little apparent storage of ureides in the leafs. The nitrogen released is allocated to leaf growth and maintenance, and to the synthesis of amino acids which are transported to the developing fruit or pods6. Ureides account for only 33% of the nitrogen delivered to the developing cowpea fruit via both xylem (from roots) and phioem (from leaves) in the first half of fi-ait development, and relatively less during R. G. Winkler, D. G. Blevins, 1. C Polacco and the second half of fruit development 6. D. D. Randall are at the Interdisciplinary Plant Some of the ureides delivered to the Physiology and Biochemistry Group, University of developing fruits ~ the phloem may be Missouri, 117 Schweitzer Hall, Columbia, MO derived from direct xylem-to-phloem 65211, USA. R. G. Winkler ~spresently at the Departtransfer in the |ear 7. Ureides are not ment of Genetics, UC ~3erkeley, USA.

The availability of nitrogen frequently limits plant growth and the application of nitrogenous fertilizers derived from fossil fuels, in modem agricultural production is expensive. Leguminous plants and rhizobial bacteria have evolved a symbiotic relationship which allows them to reduce and assimilate atmospheric N 2 and thereby meet all nitrogen needs for plant growth and reproduction. Thus, legumes are likely to become relatively more important in agriculture as the supply of fossil fuels becomes limited. The ureides, allantoin and allantoate (Fig. 1, compounds I and H), have a one to one C: N ratio and are the major nitrogen storage and transport compounds in a number of plant familiesL The recent resurgence of interest in ureide metabolism follows the observation that certain leguminous plants (e.g~ soybean, winged bean, mung bean, snap bean and cowpea) transport the majority of their fixed n~trogen to the shoot as allantoin

Ureide synthesis and economy

Ureides are the oxidation products of purines synthesized de novo in the root nodule (see Ref. 2 and TIBS 7,366-368). The estimated 'metabolic cost" of ureide synf,hesis in ATP equivalents is less than 50% that of asparagine synthesis per N atom z. In theory, this allows the plant to invest relatively less energy in the synthesis of nitrogen-transport compounds in the nodule and may provide a selective; advantage to ureide-transporting plants. In contrast to amide-transporting legumes, u~'eide-transporting legumes are genera~ly of tropical or subtropical origin and have a determinate nodule structure, which is expected to allow them a greater flux of transp','~'ational water through the nodulen. It was hypothesized that this higher flux of water was necessary to transport ureides because ureides were supposedly less soluble than asparagine n. Although this hypothesis has been widely accepted, we find that the potassium salt of allantoate is, in fact, more soluble than asparagine. The previous investigators us~:dall~ntoic acid instead of the potassium salt. However, at cellular pHs, which are normally at least 3 units above the p K a Of ~dlantoate (pH 2.4), the potassium salt of allantoate will predominate. C~1988,ElsevierPublicationsCambridge 0376-5067/88/$02.00

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Ni2+, indicating that allantoin can be at least partially catabolized independently "c" coo" o 0 I = , of urease. I IK 15 II 8 1N--C=--N--C--NH= ~ N--C--N--C~NH= Winkler et al. 16 studied allantoin H H H 7 H H H catabolism in intact leaf tissue and proI ALLANTOIN 2 / II ALLANTOATE posed that it was degraded to 4NH 3, 2CO 2 and glyoxylate without the involvement of a urea intermediate. [2,714C]Allantoin (urea carbons labeled) I o coo -I ,9 coo'o II I .I I II released 14CO2 at linear rates; 14CO2 H z N - - C - - N - - C - - N H ; I . NHs +COl H,,N--C--NH. • HOC--N--C--NH., H H release was not affected by the irreversH H -J ible urease inhibitor, phenylphosphorffl UREIDOGLYCINE In_ UREA UREIDOGLYCOLATE diamidate at concentrations eliminating all urease activity. Additionally, neither 14C-urea nor any other 14C-metabolite other than allantoate could be detect0 CO0" CO0" 0 II I I II HaN-C--N--C--OH + NH= /C ÷ H=N--C--NH= ed in leaf tissue. In contrast, leaf .'"b discs released 14CO2, [14C]glyoxylate, H H [14C]glycine and [14C]sedne when incuR7 UREIDOGLYCOLATE GLYOXYLATE UREA bated with [4,5-14C]allantoin (acetate ~4 Fig. 1. Catabolic pathways of allantoin assimilation. (1) carbons labeled). These results are conCO0" Allantoin amidohydrolase; (2) Allantoate amidohydrolase; sistent with the action of allantoate amidoI (3) Ureidoglycine amidohydrolase; (4) Ureidoglycolate hydrolase and a second amidohydrolase C* aNH, * COa amidohydrolase: (5) Allantoate amidinohydrolase: (6) Ureido- activity (ureidoglycolate amidohydroH / \'O glycolate urea.lyase. Soybeans have activities, 1, 2, 3 and 4. At lase), that degrades the proposed interpresent, there is only indirect evidence for ureidoglycine aminomediate, ureidoglycolate (Fig. 1) and the hydrolase. GLYOXYLATE subsequent metabolism of glyoxylate through the photorespiratory pathway. Recent studies of allantoin catabolism In addition to the hypothesized advan- with the endoplasmic reticulum in soy- in soybean tissue culture (R. Stahlhut, tage of ureides in terms of carbon use bean nodules 14 and is membrane-bound PhD dissertation, 1987. University of Illinois, Urbana, IL, USA) have also efficiency (C:N ratio), ureides may pro- in leaf extracts is. Most published discussions on ureide indicated that allantoin can be comvide the plant with greater control over nitrogen transport and metabolism. In catabolism state that allantoate is pletely catabolized without the involvelupine (an amide transporter) certain catabolized via allantoate amidino- ment of a urea intermediate. Phenylamino acids can be selectively removed hydrolase and ureidoglycolate urea- phosphordiamidate eliminated urease from the xylem stream, while others are lyase to glyoxylate and urea (Reactions activity and growth on urea but did not selectively transferred to the phloem 12. 5 and 6, Fig. 1). This assertion is due affect the release of 14CO2 nor cause an accumulation of [14C]urea when the cells Thus the plant may control nitrogen partly to allantoate amidinohydrolase metabolism by controlling the form of being the only aUantoate degrading were fed [2J4C]allantoin. These results N metabolites. While asparagine is in- activity known in earlier studies an, to confirm the Ni2+ starvation experiments volved in many aspects of metabolism, reports of urea accumulation in allan- of Polacco et al. 17 As found with leaf ureides appear to serve only in nitrogen toin-fed plants (see, for example, Refs 1 discs, [4,5-14C]allantoin was metabolized transport and storage. This 'specializa- and 6). However, the analytical by soybean tissue culture to [14C]glyoxytion' of metabolites may give the techniques themselves could have gener- late, [14C]glycine and [14C]serine. Howplant greater control of itg nitrogen ated some or all of the urea detected; ever, the major 14C-labeled product metabolism. e.g. corrections for non-enzymic degra- detected by ion exclusion HPLC analysis dation of allantoate were not made (see, was an unidentified acidic compound. Pathway of ureide catabolism for discussion, Ref. 16). We know of no This is apparently a product of glyoxylate Allantoinase (allantoin amidohydro- reports that clearly establish in vitro metabolism since cell cultures incubated lase, Fig. 1), which hydrolyses allantoin ureide-dependent urea formation in with [U-14C]glyoxylate yield the same to allantoate, has been described in a plants. compound. These results are consistnumber of plants including non-ureide ent with the presence of allantoate transporting species. In soybeans, allan- Studies of ureide catabolism in intact amidohydrolase and ureidoglycolate toin amidohydrolase is found in nodules, tissue amidohydrolase (Fig. 1), but suggest that leaves, developing fruits and mature Recent studies with intact tissue have glyoxylate is metabolized predominantly seeds at levels 80 times those of allan- provided the dearest information on through an undescribed, non-photorestoate degradation activities (see Ref. 13 ureide catabolism. Polacco eta/. 17 took piratory pathway. This is not surprising and F. Rice MS Thesis, 1987. University advantage of the Ni2+ requirement of since there is little glycine decarboxylase of Missouri, Columbia, MO, USA). The urease to study the metabolism of urea activity in non-green tissue. Allantoinnoduie activity would appear to be most and allanto~n in soybean cell culture. dependent urea accumulation in soycritical to ureide metabolism since most Ni2+-deficient soybean cell cultures bean leaf discs in the presence of analyses of xylem sap have found allan- could not grow when urea was the sole acetohydroxamate (a urease inhibitor) toate to be the predominant ureide a. nitrogen source, but growth on allantoin has been reported but this was not Allant.oin amidohydrolase is associated was not affected by the availability of observed in cell cultures. However, H

O. aN

0

\ \ / X II 2C C 4

O

NH=

°l

99

TIBS 1 3 - March 1988

acetohydroxamate has been found to inhibit a number of enzymatic activities in addition to urease, including allantoate, a amidohydrolase and glyoxylate reductase is.19 and thus is of very limited utility in ureide studies. Catabolic studies in cell-free systems There are very few reported isolations of allantoate-degrading enzymes from plants. Although some reports claim that allantoate amidinohydrolase is found in plants, we know of no report that dearly establishes this activity. Winkler et al. 2° have proposed that allantoate is degraded in soybean seed coat extracts by an enzyme complex that contains allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Allantoate is degraded by a Mn2+dependent activity to CO 2and glyoxylate in a ratio of 2.3 to 1 and phenylphosphordiamidate neither inhibits 14CO2release from [2,7-14C]allantoate, nor causes an accumulation of [14C]urea. Urea would accumulate if allantoate amidinohydrolase or ureidoglycolate urea-lyase were involved in allantoate degradation. That glyoxylate is enzymaticallyreleased from allantoate without detectable urea production indicates that two amidohydrolase reactions act to degrade allantoate to 4NH3, 2CO2 and glyoxylate (see Fig. 1). [Ureido-14C]ureidoglycolate was also degraded to 14CO2under conditions in which urease was completely inhibited. Equimolar CO2 and glyoxylate were released from ureidoglycolate, consistent with ureidoglycolate amidohydrolase actMty. The observation that the ratio of CO2 to glyoxylate released from allantoin is greater than two to one is consistent with the formation of a glyoxylate derivative. The only intermediate found by ion exclusion chromatography (which separates many organic acids and polar neutral compounds) was 2-hydroxyethylthio, 2'ureido, acetic acid [E2NCOHNCH(CO2H)SCH2CH2OH] (Ref. 18) which is approximately 20% of the final produ~ (in terms of C-4 and C-5 of allantoate) and is apparently a reaction product between 2-mercaptoethanol and a reactive intermediate (perhaps enzymebound). Ureidog~)cine, a presumed intermediate between allantoate and ureidoglycolate, is not available commercially and no synthesis procedure has been established. We know of no naturally-occurring or chemically-synthesized primary .q-amino add in which C-2 is bonded to a second N atom. This a~ay reflect the lack of suitable compounds

from which to synthesize this type of amino acid or possibly an inherent instability of this structure. The second presumed intermediate, ureidoglycolate, is relatively unstable, especially at alkaline or acidic pH. Both of these possible intermediates either do not accumulate or accumulate only at very low levels in vitro. Ureidoglycolate does not accumulate in vivo ~6. Several of our observations suggest that allantoate amidohydrolase and ureidoglycolate amidohydrolase are part of an enzyme complex. The rate of 14CO2 production from [2,7-14C]ailantoate (urea labeled) was unaffected by increasing the reaction volume suggesting that 14CO2 production is not dependent upon the accumulation of free intermediates. Furthermore, 14CO2 production from [2,7-14C]allantoate was not proportionally diluted by unlabeled exogenously added ureidog!ycolate indicating that ureidoglycolate does not accumulate as a free intermediate. Thus, there is now very strong evidence that allantoate can be degraded directly by the enzymes allantoate amidoh~drolase and ureidoglycolate amidohydrolase (Fig. 1), rather than by enzymes which release urea as previously assumed. Regulation of m'eide catabolism by nitrogen nutrHon The regulation of ureide catabolism is not well defined but some elements of control have been elucidated. Allantoin amidohydrolase is constitutive in leaves, fruits and nodules and does not appear to be regulated by the level of tissue ureides or the flux of ureides into the tissue (Refs 22, 23 and F. Rice, op. dr.). In both soybean leaves and cowpea fruits, allantoin amidohydrolase varies more with the developmental stage of the plant than with nitrogen source. The high levels of allantoin amidohydrolase which accumulate in developing embryos may be more important for degradation of ureides that result from nucleic acid catabolism during germination, as it is unlikely that the developing embryo receives ureides from the seed coat6.8. Extractable allantoate amidohydrolase activity is low in the leaves of young seedlings, but increases as N2-fixation levels become significant at about three to four weeks. There is a slight peak in leaf allantoate amidohydrolase activity during flowering (F. Rice, op. cit.). Seed coats have an extractable allantoate anfidohydiolase at levels sigrfificantly higher than in embryos and pods, con-

sistent with the proposed role of the seed coat in ureide catabolism TM. Extractable allantoate amidohydrolase activity is affected by nitrogen source. Allantoate amidohydrolase is approximately 50% lower and ureide transport is 80% lower in NO3--fed relative to N2-fixing soybeans (Ref. 22 and F. Rice, op. cit.). In N2-fixing soybean plants treated with 20 mM NO3-, allantoate amidohydrolase levels are reduced by 44% on the third day of treatment. Several investigators have observed that tissue levels of ureides are not related to the level of ureide transport or N2-fixation (Refs 22-24, and F. Rice, op. cit.). In fact, leaf ureide levels are elevated by 150% after three days of NO 3- treatment (F. Rice, op. cit.). Urease-independent 14CO2release by leaf discs from [2,7-14C]allantoin does not vary over a day/night cycle. Likewise the levels of ure'des in the tissue are constant, even though no import i,to the tissue could be detected in the dark (F. Rice, op. cit.). These observations suggest that leaf ureides may be sequestered, or intercellular transport of ureides may be regulated. IntraceUular compartmentation has been dearly shown to be critical for short-term regulation of nitrogen (including ureide) metabolism in yeast25. Role of urea, Niz+ and urease in ureide metabolism Urease was previously considered essential to ureide catabolism. However, the observation that ureides can be completely catabolized in Ne-fixing soybeans without the involvement of urease raises questions about the role of urease. Polacco and co-workers26,2"t have recovered a series of soybean urease-negafive mutants that will help to define the role of urea, Ni2+, and urease in soybean metabolism. That mutants lacking one or both urease isozymes, viz., the embryo-specific and ubiquitous ureases, complete their life cycle with no phenotypic effects other than some leaftip necrosis in mutants lacking the ubiquitous urease, strongly suggests that urea is not a primary nitrogen metabolite. Future prospects Purification and characterization of allantoate amidohydrolase and ureidoglycolate amidohydrolase will be necessary to understand fully the propo~d pathway of ureide catabolism. Some key questions will address how ureide transport aad flax are factors in regulation and enzyme induction, whether allan-

100

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toate amidohydrolase and ureidoglycolate amidohydrolase constitute the only pathway for ureide utilization and the subceUnlar compartmentalization of the pathway. The urease mutants will be crucial in defining the role of urease in nitrogen metabolism. Acknowledgement Our research is supported by the Missouri Agricultural Research Station and USDA, SEAJARS Grant 85-CRCR-11638. This is report 10358 from the MoAES. References I Mothes, K. (1961) Can. J. Bot. 39,1785-1807 2 Schubert, K. (1986)Annu. Rev. Plant Physiol. 37, 539-574 3 Pate, J. S. and Atkins, C. A. (1983) in Nitrogen Ftralion Vol. 3 (Broughton, W. J., ed.), pp. 245-298, Oxford University Press 4 McClure, P. R. and Israel, D. W. (1979) Plant Physiol. 64,411-416 50hyama, T. (1984)Soil Sci. Plant Nutrit. 30,

219-229 6 Peoples, M. B., Atkins, C. A., Page, J. S. and Murray, D. R. (1985) Plant Physiol. 77, 382388 7 Atkins, C. A., Pate, J. S., Richie, A. and Peoples, M. B. (1982) Plant Physiol. 70, 471482 8 Rainbird, M. B., Thome, J. H. and Hardy, R. W. F. (1984) Plant Physiol. 74, 329-334 9 Coker, G. T. IlI and Schaeffer, J. (1985) Plant Physiol. 77,129-135 10 Murray, D. R. (1979) Plant Physiol. 64, 763-769 11 Sprent, J. I. (1980) Plant Cell Environ. 3, 35--43 12 McNeil, D. L., Atkins, C. A. and Pate, J. S. (1979) Plant Physiol. 63,1076-1081 13 Thomas, R. J. and Schader, L. E. (1981) Plant Physiol. 67,973-976 14 Hanks, J. F., Tolbert, N. E. and Schubert, K. R. (1981) Plant Physiol. 68, 65-69 15 Thomas, R. J., Meyers, S. P. and Schrader, L. E. (1983) Phytochemistry 22,1117-1120 16 Winlder, R. G., Blevins, D. B., Polacco, J. C. and Randall, D. D. (1987) Plant Physiol. 83, 585-591 17 Polacco, J. C., Thomas, A. L. and Bledsoe, P. J. (1982)PlantPhysiol, 69,1233-1240

18 Winkler, R. G., Polacco, J. C., Blel~ins,D. B. and Randall, D. D. (1985) Plant Physiol. 79, 787-793 19 Kleczkowski, L. A., Randall, D. D. and Blevins, D. G. (1987) Plant Phy~'ioL 84, 619623 20 Winkler, R. G., Blevins, D. G. and Randall, D. D. Plant Physiol. (in press) 21 Schuiler, K. A., Day, D. A., Gibson, A. H. and Gresshoff, P. M. (1986) Plant Physiol. 80, 646-650 22 Rice, C. F., Walker, S., Lukaszewski, K. M., Blevins, D. G. and Randall, D. D. (1986) Plant Physiol. bO, S-66 23 Pate, J. S., Atkins, C. A., White, S. T., Rainbird, R. M. and Woo, K. C. (1980) P/ant Physiol. 65,961-965 24 Yoneyama, T., Karasuyama, M., KouchL H. and Ishizuka, J. (1985)SoilSci. PlantNulrit. 31, 133-140 25 Cooper, T. G. (1984) Adv. Enzymol. 56, 91139 26 Meyer-Bothling, L. E. and Polacco, J. C. (!q87) Mol. Gen. Genet. 209, 439--444 27 Meyer-Bothling, L. E., Polacco, J. C. and Ciat~zio, S. R. (1987) MoL Gcn. Genet. 209, 432--438

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