Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Urease deficiency alters nitrogen metabolism and gene expression in urease-null soybean without affecting growth or productivity under nitrate supply


Urea is a product of arginine catabolism in plants and its Nitrogen is recycled into the plant metabolism as ammonium after hydrolysis by urease. The eu3-a soybean mutant is null for the Ni insertion protein (UreG) necessary for urease activity. No UreG protein nor any activity of the urease enzymes is detectable in these eu3-a mutants. In order to understand the mechanisms of nitrogen cycling in soybean and the possible physiological benefits to N metabolism, eu3-a (urease-null) and control soybean near-isogenic Eu3 plants were studied. They were grown to two different developmental stages (vegetative-V5 and reproductive-R5) with 15 mM nitrate as the sole source of nitrogen. Growth and biochemical parameters (such as amino acid, nitrate, and polyamine pools) were evaluated in leaves. Gene transcript levels were determined for some enzymes related to Arg catabolism, together with those of the DUR3 active urea transporter and the UreG Ni-insertion accessory protein, whose transcript was confirmed to be absent in eu3-a. The absence of urease activity in the eu3-a null plants did not affect growth or yield although there was a substantial and progressive accumulation of urea in the leaves. Metabolic changes occurred mainly in the pool of amino acids and in the expression of genes related to the pathway of Arg degradation. There are indications that the pathway may be diverted to form polyamines, but to a limited extent. Thus, considering both developmental stages, the degradation of Arg to urea and Orn remains the main path for nitrogen recycling from Arg, despite the progressive accumulation of urea and consequently immobilization of N.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Amino acids




Near-isogenic lines

NH4 + :


NO3 :



Total soluble sugar










Nitrate reductase


Ubiquitous urease


Arginase—arginine amidohydrolase


Ornithine carbamoyltransferase or ornithine transcarbamylase


Ornithine aminotransferase




Ornithine decarboxylase


Arginine decarboxylase


  1. Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF (2010) Polyamines: Molecules with regulatory functions in plant abiotic stress tolerance. Planta 231:1237–1249

  2. Bender RR, Haegele JW, Below FE (2015) Nutrient uptake, partitioning, and remobilization in modern soybean varieties. Agron J 107:563–573

  3. Bieleski RL, Turner NA (1966) Separation and estimation of amino acids in crude plant extracts by thin-layer electrophoresis and chromatography. Anal Biochem 17:278–293

  4. Bitrián M, Zarza X, Altabella T, Tiburcio AF, Alcázar R (2012) Polyamines under abiotic stress: metabolic crossroads and hormonal cross talks in plants. Metabolites 2:516–528

  5. Bohner A, Kojima S, Hajirezaei M, Melzer M, von Wirén N (2015) Urea retranslocation from senescing Arabidopsis leaves is promoted by DUR3-mediated urea retrieval from leaf apoplast. Plant J 81:377–387

  6. Bortolotti C, Cordeiro A, Alcázar R, Borrell A, Culiañez-Macià FA, Tiburcio AF, Altabella T (2004) Localization of arginine decarboxylase in tobacco plants. Physiol Plant 120:84–92

  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  8. Brien AO, Vega A, Krouk G, Gojon A, Coruzzi G, Gutie RA (2016) Nitrate transport, sensing, and responses in plants. Mol Plant 9:837–856

  9. Carter EL, Fluggaa N, Boer JL, Mulrooney SB, Hausinger RP (2009) Interplay of metal ions and urease. Metallomics 1:207–221

  10. Cataldo DA, Haroon M, Schrader L, Youngs VL (1975) Rapid colorimetric determination of nitrate in plant tissues by nitration of salicylic acid. Commun Soil Sci Plant Anal 6:71–80

  11. Cruchaga S, Lasa B, Jauregui I, González-Murua C, Aparicio-Tejo PM, Ariz I (2013) Inhibition of endogenous urease activity by NBPT application reveals differential N metabolism responses to ammonium or nitrate nutrition in pea plants: a physiological study. Plant Soil 373:813–827

  12. Dixon NE, Riddles PW, Gazzola C, Blakeley RL, Zerner B (1980) Jack bean urease (EC V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds. Can J Biochem 58:1335–1344

  13. Egli DB (1999) Variation in leaf starch and sink limitations during seed filling in soybean. Crop Sci 39:1361–1368

  14. Farrugia MA, Macomber L, Hausinger RP (2013) Biosynthesis of the urease metallocenter. J Biol Chem 288:13178–13185

  15. Fehr W, Caviness C, Burmood D, Pennington J (1971) Stage development descriptions for soybeans Glycine max (L) Merril. Crop Sci 11:929–931

  16. Felker P (1977) Microdetermination of nitrogen in seed protein extratcs, 49th edn. Analytical chemistry, Washington

  17. Freitas DS, Rodak BW, Reis AR, Reis F de B, Carvalho TS, Schulze J, Carneiro MAC, Guilherme LRG (2018) Hidden nickel deficiency? Nickel fertilization via soil improves nitrogen metabolism and grain yield in soybean genotypes. Front Plant Sci 9:1–16

  18. Freyermuth SK, Bacanamwo M, Polacco JC (2000) The soybean Eu3 gene encodes an Ni-binding protein necessary for urease activity. Plant J 21:53–60

  19. Funck D, Stadelhofer B, Koch W (2008) Ornithine-δ-aminotransferase is essential for arginine catabolism but not for proline biosynthesis. BMC Plant Biol 8:40

  20. Goldraij A, Polacco JC (2000) Arginine degradation by arginase in mitochondria of soybean seedling cotyledons. Planta 210:652–658

  21. Graham D, Smydzuk J (1965) Use of anthrone in the quantitative determination of hexose phosphates. Anal Biochem 11:246–255

  22. Hageman RH, Reed AJ (1980) [24] Nitrate reductase from higher plants. Methods Enzymol 69:270–280

  23. Hildebrandt TM, Nunes-Nesi A, Araújo WL, Braun H-PP (2015) Amino acid catabolism in plants. Mol Plant 8:1563–1579

  24. Hoagland DR, Arnon DI (1938) The water culture method for growing plants without soil. Soil Calif Agric Exp Sta Bull Circular 3

  25. Imsande J, Touraine B (1994) N demand and the regulation of nitrate uptake. Plant Physiol 105:3–7

  26. Islam M, Ishibashi Y, Nakagawa ACS, Tomita Y, Iwaya-inoue M, Arima S, Zheng S (2016) Nitrogen redistribution and its relationship with the expression of GmATG8c during seed filling in soybean. J Plant Physiol 192:71–74

  27. Jaworski EG (1971) Nitrate reductase assay in intact plant tissues. Biochem Biophys Res Commun 43:1274–1279

  28. Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59

  29. Kaiser WM, Huber SC (2001) Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. J Exp Bot 52:1981–1989

  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

  31. Majumdar R, Barchi B, Turlapati SA, Gagne M, Minocha R, Long S, Minocha SC (2016) Glutamate, ornithine, arginine, proline, and polyamine metabolic interactions: the pathway is regulated at the post-transcriptional level. Front Plant Sci 7:78

  32. Masclaux C, Valadier MH, Brugière N, Morot-Gaudry J-FF, Hirel B (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211:510–518

  33. Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A (2010) Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann Bot 105:1141–1157

  34. Mattoo AK, Minocha SC, Minocha R, Handa AK (2010) Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine. Amino Acids 38:405–413

  35. Mattoo AK, Fatima T, Upadhyay RK, Handa AK (2015) Polyamines in plants: biosynthesis from arginine, and metabolic, physiological and stress-response roles Polyamine biosynthesis in plants. CAB International, Wallingford, pp 177–194

  36. Mokochinski JB, Soares DX, Bruns RE, Mazzafera P, Sawaya ACHF (2013) Optimization of extraction conditions of free amino acids in plants by factorial design. In: Congresso Brasileiro de Espectrometria de Massas. Campinas, SP, Brazil, p 8075

  37. Nam KH, Lee SH, Lee JH (1997) Purification and characterization of arginine decarboxylase from soybean (Glycine max) hypocotyls. Plant Cell Physiol 38:1150–1155

  38. Nelson-Schreiber BM, Schweitzer LE (1986) Limitations on leaf nitrate reductase activity during flowering and podfill in soybean. Plant Physiol 80:454–458

  39. Patel J, Ariyaratne M, Ahmed S, Ge L, Phuntumart V, Kalinoski A, Morris PF (2017) Dual functioning of plant arginases provides a third route for putrescine synthesis. Plant Sci 262:62–73

  40. Pinton R, Tomasi N, Zanin L (2016) Molecular and physiological interactions of urea and nitrate uptake in plants. Plant Signal Behav 11:e1076603

  41. Polacco JC, Winkler RG (1984) Soybean Leaf Urease: A Seed Enzyme? Plant Physiol 74:800–803

  42. Polacco JC, Thomas AL, Bledsoe PJ (1982) A soybean seed urease-null produces urease in cell culture. Plant Physiol 69:1233–1240

  43. Polacco JC, Hyten DL, Medeiros-Silva M, Sleper D, Bilyeu KD (2011) Mutational analysis of the major soybean UreF paralogue involved in urease activation. J Exp Bot 62:3599–3608

  44. Polacco JC, Mazzafera P, Tezotto T (2013) Opinion: nickel and urease in plants: still many knowledge gaps. Plant Sci 199–200:79–90

  45. Puiatti M, Sodek L (1999) Waterlogging affects nitrogen transport in the xylem of soybean. Plant Physiol Biochem 37:767–773

  46. Rechenmacher C, Iebke-Strohm B, de Oliveira-Busatto LA, Polacco JC, Carlini CR, Bodanese-Zanettini MH (2017) Effect of soybean ureases on seed germination and plant development. Genet Mol Biol 40:1–8

  47. Shargool D, Jain JC, McKay G (1988) Ornithine biosynthesis, and arginine biosynthesis and degradation in plant cells. Phytochemistry 27:1571–1574

  48. Stebbins NE, Polacco JC (1995) Urease Is Not essential for ureide degradation in soybean. Plant Physiol 109:169–175

  49. Stebbins N, Holland MA, Cianzio SR, Polacco JC (1991) Genetic tests of the roles of the embryonic ureases of soybean. Plant Physiol 97:1004–1010

  50. Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97

  51. Tezotto T, Souza SCR, Mihail J, Favarin JL, Mazzafera P, Bilyeu K, Polacco JC (2016) Deletion of the single UreG urease activation gene in soybean NIL lines: characterization and pleiotropic effects. Theor Exp Plant Physiol 28:307–320

  52. Wang WH, Köhler B, Cao FQ, Liu LH (2008) Molecular and physiological aspects of urea transport in higher plants. Plant Sci 175:467–477

  53. Wiebke-strohm B, Ligabue-braun R, Rechenmacher C, Oliveira-busatto LA, Bodanese-zanettini MH (2016) Structural and transcriptional characterization of a novel member of the soybean urease gene family. Plant Physiol Biochem 101:96–104

  54. Williams L, Miller A (2001) Transporters responsible for the uptake and partitioning of Nitrogenous solutes. Annu Rev Plant Physiol Plant Mol Biol 52:659–688

  55. Wingler A, Masclaux-Daubresse C, Fischer AM (2009) Sugars, senescence, and ageing in plants and heterotrophic organisms. J Exp Bot 60:1063–1066

  56. Winter G, Todd CD, Trovato M, Forlani G, Funck D (2015) Physiological implications of arginine metabolism in plants. Front Plant Sci 6:1–14

  57. Witte C-PP (2011) Urea metabolism in plants. Plant Sci 180:431–438

  58. Witte C, Tiller S, Taylor M, Davies H (2002) Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of 15N after foliar urea application in wild-type and urease-antisense transgenics. Plant Physiol 128:1129–1136

Download references


SCRS, JCP, and PM thank the São Paulo Research Foundation—FAPESP, respectively, for a post-doctoral Grant (2013/25094-8), visiting professor Grant (2014/09730-4), and research support (Grant 2008/58035-6). PM and LS thank the National Council for Scientific and Technological Development-CNPq for research fellowships.

Author information

Correspondence to Sarah Caroline Ribeiro de Souza.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by P. Wojtaszek.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 14 kb)

Supplementary file2 (DOC 54 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

de Souza, S.C.R., Sodek, L., Polacco, J.C. et al. Urease deficiency alters nitrogen metabolism and gene expression in urease-null soybean without affecting growth or productivity under nitrate supply. Acta Physiol Plant 42, 34 (2020).

Download citation


  • Arginase
  • Arginine decarboxylase
  • Polyamines
  • Amino acids
  • DUR3
  • Nitrate reductase enzyme