Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Acta Physiologiae Plantarum Aims and scope Submit manuscript

Abstract

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 via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

Abbreviations

AA:

Amino acids

N:

Nitrogen

NILs:

Near-isogenic lines

NH4 + :

Ammonium

NO3 − :

Nitrate

TSS:

Total soluble sugar

Agm:

Agmatine

Spm:

Spermine

Spd:

Spermidine

Put:

Putrescine

NR:

Nitrate reductase

UU:

Ubiquitous urease

ARGAH:

Arginase—arginine amidohydrolase

OTC:

Ornithine carbamoyltransferase or ornithine transcarbamylase

OAT:

Ornithine aminotransferase

P5CR:

Pyrroline-5-carboxylate-reductase

ODC:

Ornithine decarboxylase

ADC:

Arginine decarboxylase

References

  • 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

    PubMed  Google Scholar 

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

    CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  Google Scholar 

  • 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

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    CAS  PubMed  Google Scholar 

  • 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

    Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • Dixon NE, Riddles PW, Gazzola C, Blakeley RL, Zerner B (1980) Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds. Can J Biochem 58:1335–1344

    PubMed  CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    PubMed  PubMed Central  CAS  Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • 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

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

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

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

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

    PubMed  CAS  Google Scholar 

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

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

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

    PubMed  Google Scholar 

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

    PubMed  PubMed Central  CAS  Google Scholar 

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

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  CAS  Google Scholar 

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

    PubMed  Google Scholar 

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

    CAS  Google Scholar 

  • 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

    Google Scholar 

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

    CAS  Google Scholar 

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

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

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

Authors and Affiliations

  1. Department of Plant Biology, Institute of Biology, University of Campinas, PO Box 6109, Campinas, SP, 13083-970, Brazil

    Sarah Caroline Ribeiro de Souza, Ladaslav Sodek & Paulo Mazzafera

  2. Department of Botany, Federal University of São Carlos, PO Box 676, São Carlos, SP, 13565-905, Brazil

    Sarah Caroline Ribeiro de Souza

  3. Department of Biochemistry, Interdisciplinary Plant Group, University of Missouri, 117 Schweitzer Hall, Columbia, MO, 65211, USA

    Joe Carmine Polacco

  4. Department of Crop Science, College of Agriculture “Luiz de Queiroz”, University of São Paulo, CP 09, Piracicaba, SP, 13418-900, Brazil

    Paulo Mazzafera

Authors
  1. Sarah Caroline Ribeiro de Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ladaslav Sodek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joe Carmine Polacco
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paulo Mazzafera
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sarah Caroline Ribeiro de Souza.

Additional information

Communicated by P. Wojtaszek.

Publisher's Note

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

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

Check for updates. 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). https://doi.org/10.1007/s11738-020-3020-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11738-020-3020-9

Keywords

Search

Navigation