Urea metabolism in plants

Plant Science 180 (2011) 431–438

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Review

Urea metabolism in plants Claus-Peter Witte ∗ Department of Plant Biochemistry, Dahlem Centre of Plant Sciences, Freie Universität Berlin, Königin-Luise-Str. 12-16, 14195 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 4 October 2010 Received in revised form 17 November 2010 Accepted 22 November 2010 Available online 1 December 2010 Keywords: Nitrogen remobilization Urea transport Urease activation Arginase Ureide degradation Urea fertilization

a b s t r a c t Urea is a plant metabolite derived either from root uptake or from catabolism of arginine by arginase. In agriculture, urea is intensively used as a nitrogen fertilizer. Urea nitrogen enters the plant either directly, or in the form of ammonium or nitrate after urea degradation by soil microbes. In recent years various molecular players of plant urea metabolism have been investigated: active and passive urea transporters, the nickel metalloenzyme urease catalyzing the hydrolysis of urea, and three urease accessory proteins involved in the complex activation of urease. The degradation of ureides derived from purine breakdown has long been discussed as a possible additional metabolic source for urea, but an enzymatic route for the complete hydrolysis of ureides without a urea intermediate has recently been described for Arabidopsis thaliana. This review focuses on the proteins involved in plant urea metabolism and the metabolic sources of urea but also addresses open questions regarding plant urea metabolism in a physiological and agricultural context. The contribution of plant urea uptake and metabolism to fertilizer urea usage in crop production is still not investigated although globally more than half of all nitrogen fertilizer is applied to crops in the form of urea. Nitrogen use efficiency in crop production is generally well below 50% resulting in economical losses and creating ecological problems like groundwater pollution and emission of nitric oxides that can damage the ozone layer and function as greenhouse gasses. Biotechnological approaches to improve fertilizer urea usage bear the potential to increase crop nitrogen use efficiency. © 2010 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3. 4. 5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Urea transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. High affinity active transport of urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Passive transport of urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Urease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Urease accessory proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic sources of urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Ureides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological and agricultural aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Nitrogen limits plant growth in most terrestrial and aquatic ecosystems [1] exerting a constant selective pressure on plants for efficient nitrogen usage. This ecophysiological selection even led to a reduction of the nitrogen content of plant proteins [2]. Plants

∗ Corresponding author. Tel.: +49 30 83854787; fax: +49 30 83853372. E-mail address: [email protected] 0168-9452/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2010.11.010

431 432 432 432 432 433 434 434 434 435 436 436

possess a whole range of proteins involved in nitrogen uptake and partitioning, not only for nitrate and ammonium but also for amino acids, peptides, nucleotides and their degradation products, and urea [3–5]. Even entire microbes and whole proteins are used by plants as nitrogen sources [6,7]. However, the quantitative contribution of organic nitrogen sources in the soil to plant nitrogen nutrition is still under debate [8]. Not only uptake but also reallocation of nitrogen resources during development is important for optimal nitrogen usage. These processes are especially prominent during germination and senescence when nitrogen source

432

C.-P. Witte / Plant Science 180 (2011) 431–438

tissues (seed, senescing leaves) provide nitrogen for metabolic sinks (seedling, flowers, developing seeds, and storage organs). Urea plays a role as primary nitrogen source taken up actively by plants from the soil solution but is also an intermediate of plant arginine catabolism involved in nitrogen remobilization from source tissues. Urea is the most widely used nitrogen fertilizer in agriculture on a global scale (http://faostat.fao.org). About half of all nitrogen used for crop production is applied as urea. Its nitrogen is only accessible for assimilation after hydrolysis to ammonia and carbon dioxide. A widespread opinion is that plants mainly take up ammonium and nitrate generated by microbial conversion of urea in the soil and that direct urea uptake and internal hydrolysis by the plant are not significant. This view is now being challenged by the observation that plants possess dedicated urea transporters, hydrolyse urea very efficiently and can use urea as sole nitrogen source. A better understanding of plant urea metabolism involving uptake, storage, internal transport, hydrolysis and assimilation of urea nitrogen will be required to assess and possibly improve direct usage of urea by plants (without prior soil conversion) in agricultural settings employing urea fertilization. 2. Urea transport 2.1. High affinity active transport of urea Plants possess a high affinity secondary active urea transporter (DUR3) that is involved in taking up environmental urea but may also mediate internal urea transport. DUR3 of Arabidopsis thaliana (At5g45380) was identified by its similarity to the urea transporter of Saccharomyces cerevisiae (ScDUR3 [9]). Orthologs to AtDUR3 are present throughout the green lineage from algae to higher plants, mostly encoded by single copy genes [10]. Several lines of evidence suggest that AtDUR3 co-transports urea along with protons driving secondary active transport even against a urea concentration gradient [9]. With an apparent affinity constant of 3–4 ␮M for urea and saturation transport kinetics, AtDUR3 classifies as high affinity transporter [9,11]. Corresponding loss-of-function plants show reduced urea uptake that does not follow a saturation kinetic anymore [11]. The protein is expressed at the plasma membrane of root epidermal cells especially in nitrogen starved plants and the promoter is responsive to urea in the absence of other nitrogen sources which is consistent with its role as urea transporter [11,12]. A role in internal urea transport is indicated by the expression of AtDUR3 near the root xylem and in the shoot [9,11]. Transport of urea from root to shoot was observed in nickel-deprived ureasenegative plants grown on urea. These accumulate high amounts of urea in the shoot [13,14]. 2.2. Passive transport of urea Passive urea transport is mediated by major intrinsic proteins (MIPs) also called aquaporins. These proteins conduct selected low molecular solutes along a concentration gradient through a channel. Arabidopsis contains 35 MIPs grouped into 4 subclasses: the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nodulin 26-like membrane intrinsic proteins (NIPs), and small, basic membrane intrinsic proteins (SIPs) [15]. Four Arabidopsis TIPs (AtTIP1;1: At2g36830; AtTIP1;2: At3g26520, AtTIP2;1: At3g16240, and AtTIP4;1: At2g25810) were shown to mediate urea transport by complementing a yeast DUR3 mutant in a screen of an Arabidopsis seedling cDNA library [16]. Members of the plant PIP and NIP subfamilies also facilitate urea transport [17–20]. Aquaporins associate as tetramers with each monomer forming a hydrophobic pore through the membrane. Channel selectivity is difficult to predict, but selection by size appears to be a major determinant for urea conductivity [21]. PIPs have a rather

narrow and water selective pore allowing at best slow urea passage. In contrast, all TIP and NIP channels likely conduct sufficient urea to support growth of the yeast DUR3 mutant on urea as sole nitrogen source [17]. As indicated by the name, several AtTIPs are indeed located in the tonoplast membrane [16,22] but the exact membrane location of individual MIPs will require experimental confirmation and cannot rely only on sequence-based prediction. The tonoplast location of urea-conducting TIPs may contribute to the higher urea permeability of the tonoplast membrane compared to the plasmamembrane [23]. Urea in the vacuole is not accessible for hydrolysis and assimilation (see below), potentially slowing down urea assimilation rates. Urea was observed consistently to persist for over 36 h in potato leaves after foliar urea spray [24] despite the presence of sufficient leaf urease activity to potentially degrade this urea in only a few hours (see below). Such temporal urea storage in the vacuole may also occur under natural conditions after animal urine excretion leading to a sudden rise of urea in the soil to high millimolar concentrations and probably massive urea import into roots. Whether there is a physiological role of aquaporins in conducting urea or whether urea is just not excluded from many aquaporin channels remains to be shown. Maybe the export of urea generated during mitochondrial arginine degradation (see below) is facilitated by an aquaporin. TIP1;5 of Arabidopsis, expressed in pollen tube and located in the mitochondria, was suggested to play such a role [25]. Interestingly, two NIPs (At2g29870 and At2g34390) are among the few genes that are specifically up-regulated by urea in roots and shoots of Arabidopsis [12]. Consistently, urea uptake was increased in plants that were grown exclusively on urea but only when no additional nitrogen source was present [12]. 3. Urease Most ureolytic organisms employ the nickel metalloenzyme urease to hydrolyse urea. Only a few algae, fungi, and bacteria use an alternative urease-independent pathway comprising two enzymatic reactions. In these organisms urea is first carboxylated to allophanate in an ATP-dependent reaction by urea carboxylase and then allophanate is hydrolysed to ammonia and carbon dioxide by allophanate hydrolase [26–28]. The one-step enzymatic hydrolysis of urea by urease yields ammonia and carbamate [29]. The latter rapidly decays non-enzymatically forming a second molecule of ammonia and carbon dioxide [30] (Fig. 1A). Urease activity is found in many, if not all plants [31–33] and is also ubiquitously present in all plant tissues, for example in potato and soybean [34–36]. In Arabidopsis, the urease gene is transcribed in all tissues (https://www.genevestigator.com). The enzyme is located in the cytosol of jackbean seeds [37]. Urease is generally assumed to be cytosolic because the protein does not contain an apparent subcellular targeting peptide and because several proteins required for urease activation (see below) appear to be cytosolic as well. Recently, a partial cell wall and membrane localisation has been claimed in bromeliad species [38]. Interestingly, in certain lichens a polygalactosylated urease acts as cell wall-associated algal receptor for the recognition of adequate fungal partners by binding a fungal lectin that is related to arginase [39]. The KM constants of most plant ureases range from 0.15 to 3 mM but many possess a KM around 0.5 mM [33,40–44] which is similar to the range of urea concentrations (ca. 0.2–0.9 mM) measured in Arabidopsis and rice [12,40]. Leaves from potato contained about 100 mU urease per g fresh weight at 30 ◦ C. Activities in several other plants ranged from 50 to 180 mU per gram fresh weight [31]. Internal urea concentrations of up to 20 ␮mol/g fresh weight were measured after spraying potato leaves with a 330 mM (2% w/v) urea solution, which theoretically could be degraded by the leaf urease activity in less than 3.5 h. In reality, urea concentrations required 36–48 h to return to physi-


O

A

O

urease

NH2

H 2N

spontaneous

HO H2 O

urea

B

+

+

NH2 H2 O

NH 3

CO2 NH 3

apo-urease

urease

Lys

Lys C Ni O O Ni

UreD CO 2

Ni Ni

UreF

UreG UreF GTP

UreD

UreG GDP + Pi

Fig. 1. Urease reaction and model of urease activation. (A) Enzymatic hydrolysis of urea to carbamate catalysed by urease and subsequent non-enzymatic decay of carbamate. Two mol ammonia and one mol carbon dioxide are generated from urea by these reactions. (B) Hypothetical model of plant urease activation involving the binding of the three urease accessory proteins (UreD, UreF and UreG) to apo-urease, covalent modification of an active site lysine by nitrogen carboxylation, and specific incorporation of two nickel ions per active site. The accessory proteins dissociate from urease after activation. Activation may require GTP hydrolysis mediated by UreG.

ological level [24] indicating that urea is either compartmentalized in the leaf (for example to the vacuole, see above) and consequently not accessible for urease or alternatively urease activities in vivo are substantially lower than estimated from in vitro measurements. All ureases form a basic trimeric structure. In most bacteria, each unit of this trimer is itself a heterotrimer (of UreA, UreB and UreC subunits) harboring one active site ((UreABC)3 -structure) [30]. Plant (eukaryotic) ureases are highly similar to bacterial ureases in sequence and structure [45–47], but in plant enzymes the UreA, UreB and UreC subunits of bacterial ureases are fused in a collinear fashion to make up a single polypeptide chain of about 90 kDa. Plant ureases form trimers and two trimeric units can also associate to a hexameric structure [45,48]. Plant urease was extensively investigated in soybean. Soybean has two ureases, a highly expressed embryo-specific urease encoded by the Eu1 gene (Glyma05g27840 [49]) and an ubiquitous urease found in all tissues encoded by Eu4 (Glyma1 1g37250 [50]). Additionally, a third uncharacterized urease-like gene (Glyma08g10850) is present in the soybean genome. This paralog is probably not functional because of accumulated mutations (exchange of functionally essential amino acids, partial sequence deletions and a premature stop codon). Interestingly, some urease activity was retained even in an Eu1/Eu4 double mutant, which was explained by the presence of urease-positive bacteria living on the plant [51]. Alternatively, complementation between the defective Eu4 gene and the urease-like gene on chromosome 8 may cause the residual activity. Interallelic complementation between two independent defective Eu4 alleles, each carrying a different missense mutation has been described [52]. Interestingly, the highly active seed specific urease of soybean and other legumes has no assimilatory role but was proposed to be involved in plant defense [53]. High levels of seed urease activity also occur in some Curcubitaceae [54]. Urease or urease-like proteins (canatoxin) or peptides derived from these proteins exert toxic effects on fungi and certain insects even independent of urease activity [55,56]. Maybe the soybean ureaselike gene described above (Glyma08g10850) also plays a role in defense. In contrast to soybean, several solanaceous species and also A. thaliana possess only a single urease gene [47]. A single gene is also found in all sequenced plant genomes except

433

soybean [40] accessible via the Phytozome (version 5) database (http://www.phytozome.net). The presence of several paralogous urease genes as in soybean appears to be the exception and not the rule. 4. Urease accessory proteins Urease is the only enzyme known to contain nickel in plants [57]. In the bacterium Klebsiella aerogenes, four urease accessory proteins (UreD, UreF, UreG and UreE) are required for urease activation involving the carboxylation of an active site lysine and the incorporation of two nickel ions per active site that are bridged by the carboxyl group of the modified lysine. The precise role of the urease accessory proteins in metallocentre assembly is not yet clearly understood [30]. Apo-urease (encoded by ureA, ureB and ureC on the urease operon of K. aerogenes) forms a complex with UreD, the U–UreD (urease–UreD) complex can in turn bind UreF, and the U–UreDF complex is competent to bind UreG, forming a stoichiometric U–UreDFG complex that can be isolated from cells grown in the absence of nickel [58]. UreE is a nickel-binding protein that is thought to join the U–UreDFG complex delivering the nickel [59]. Experimental evidence for the existence of a bacterial U–UreDFGE complex has been obtained using a mutant variant of urease [60]. After assembly of the U–UreDFGE complex, GTP hydrolysis carried out by UreG is required for urease activation [61] and the complex dissociates into its components releasing active urease. A UreDFG complex could be isolated from bacteria lacking urease and UreE [62] but it is unclear whether a preformed UreDFG complex contributes in vivo to apo-urease activation. Using microbial accessory protein sequences, putative plant orthologs for UreD, UreF, and UreG were identified and the corresponding cDNAs cloned [34,63,64]. Plants seem not to possess a gene with similarity to bacterial UreE [65]. It was hypothesized that the function of the bacterial metallochaperone UreE may have been taken over in plants by the UreG protein. In comparison to bacterial UreG proteins, plant UreGs contain an extended nitrogen terminus rich in His and Asp residues that binds nickel [34,64]. The Eu3 mutant of soybean was defective in ureG (Glyma02g20690) resulting in the absence of both embryo-specific and ubiquitous urease activities [64] and even lacked any further background activities that were proposed to be of bacterial origin [49]. The identification of the Eu3 protein was the first functional evidence for the presence of a urease accessory protein in plants. A complete set of plant urease accessory proteins was identified in Arabidopsis. The analysis of Arabidopsis knockout mutants for UreD (At2g35035), UreF (At1g21840) and UreG (At2g34470) as well as urease (At1g67550) demonstrated the requirement of each of these proteins for plant urease activity, because mutants lacking any one of them were unable to grow on urea as sole nitrogen source and lacked urease activity [65]. Evidence that the three accessory proteins are not only necessary but probably also sufficient for activating plant urease was obtained in the same study by simultaneously co-expressing Arabidopsis urease and the three accessory proteins in (ureasenegative) Escherichia coli. Urease activity was only generated in these bacteria when all four plant proteins were present but not when any of them was missing (Fig. 1B) [65]. Functional proof for the orthologous set of accessory proteins from rice was obtained by transiently reconstituting the rice urease activation machinery in Nicotiana benthamiana [40]. The urease-negative Eu2 mutant in soybean, long believed to affect a different component of the activation process not identical with UreD, F or G [63], is defective in one of the two ureF paralogs (Glyma02g44440; J.C. Polacco, personal communication). Interestingly, the other paralog (Glyma14g04380), which was previously shown to complement Schizosaccharomyces pombe defective in ureF [63] is almost inactive in planta despite being transcribed (J.C. Polacco, personal communication). A possi-

434


ble explanation may come from a structural analysis of plant ureF genes [40]. The mRNA corresponding to this ureF paralog contains an upstream out-of-frame AUG codon in the 5 leader sequence, probably blocking efficient translation from the genuine translation start codon located further downstream. Plant and bacterial UreF and UreD only share about 20% sequence identity, but secondary structure prediction in silico revealed that plant and bacterial UreF and in part also UreD are probably conserved on structural level [40]. Not only the ureases but also the urease activation machinery appears to be similar in plants and bacteria. This notion is strengthened by the observation that (i) potato UreG could partially complement a bacterial (K. aerogenes) urease operon with a ureG deletion [34] and that (ii) rice UreD interacts directly with rice urease [40] as do the corresponding proteins in K. aerogenes [58]. The structural differences between plant and bacterial UreD may account for different binding requirements when interacting with the plant homomeric versus the bacterial heterotrimeric urease. UreF was hypothesized to be a GTPase-activating protein, maybe acting on UreG [66], which resembles a small G protein. It appears that UreF and UreG form the structurally conserved catalytic core needed for urease activation, while UreD functions as an adapter protein for attachment of this core to the respective urease (Fig. 1B).

protein degradation

MITOCHONDRION

+

NH3 H 2N

-

NH

H 2N

COO

+

arginine NADH + H H2O

arginase

NAD

NH2

H 2N urea

H3N

-

+

COO

ornithine

2 glutamate

urea transporter 2 glutamate 2 glutamine

2 NH3 urease

5. Metabolic sources of urea

The catabolism of arginine by arginase in the mitochondrial matrix generates ornithine and urea. Mitochondrial ornithine metabolism converts this compound to glutamate [67]. Urea is exported to the cytosol possibly by an aquaporin [25], then hydrolysed by urease, and the ammonium is re-assimilated by (cytosolic) glutamine synthetase using glutamate (from ornithine catabolism) as substrate. All nitrogen of arginine is incorporated into glutamine by these reactions while urease is required to mobilize half of the nitrogen stored in arginine (Fig. 2). In fact, this is the only firmly established role of urease in plant metabolism (apart from hydrolyzing root-imported urea) and arginase is the only enzyme in plants known to generate urea in vivo. Arginine catabolism is central to the mobilization of nitrogen from source tissues. In trees, arginine is an important amino acid for nitrogen storage and transport [68]. High arginine concentrations also occur in underground storage organs of several plants [69,70]. Arginine is the most important single metabolite for nitrogen storage in many plant seeds [71]. Many legume seeds are rich in canavanine, an amino acid structurally related to arginine and involved in plant defense [72]. This compound can also be degraded by arginase to canaline and urea [73]. The biosynthesis of arginine during seed/embryo development is located in the plastids [74]. In soybean, a futile urea cycle with simultaneous arginine biosynthesis and degradation during embryogenesis is not observed, despite detectable arginase activity and arginine import capacity of embryonic mitochondria. One explanation may be that the seed arginine reserve is deposited out of reach for mitochondrial import [75,76]. Upon germination, arginase activity rises [40,76,77] and arginine is degraded in the mitochondria [53,75]. The importance of urease for recycling arginine nitrogen during germination is highlighted by the fact that aged Arabidopsis seeds failed to germinate when urease was chemically inhibited but could be rescued by an external nitrogen source [78]. Arginine is subject to net degradation also during senescence. In nickel-deprived plants which consequently do not contain an active urease, urea accumulates from arginine turnover, especially in senescing tissues [13,79].

P5CDH

δOAT

+

NH3

+

urea

5.1. Arginine

+

α-ketoglutarate

O

+

GS1

Fig. 2. Arginine catabolism. Arginine is hydrolysed in the mitochondria by arginase to urea and ornithine. Urea leaves the mitochondria is hydrolysed by cytosolic urease and the released ammonia re-assimilated by cytosolic glutamine synthetase (GS1). Mitochondrial ␦-ornithine aminotransferase (␦OAT) transfers the side chain amino group of ornithine to ␣-ketoglutarate generating one molecule of glutamate and pyrroline-5-carboxylate which is oxidized to a second molecule of glutamate by pyrroline-5-carboxylate dehydrogenase (P5CDH). Glutamate can be exported from the mitochondria and serve as substrate for the cytosolic GS1-reacation. In total, all four nitrogen atoms of arginine are incorporated into glutamine. Urease is required to mobilize half of the arginine nitrogen.

5.2. Ureides The ureides allantoate and ureidoglycolate are intermediates of purine degradation. Fungal enzymes that catabolize these compounds have been cloned [80] and the corresponding sequences reported [81,82]. In S. cerevisiae allantoate is degraded by an allantoate amidinohydrolase (allantoicase; ScDAL2) releasing urea and S-ureidoglycolate, which in turn is further catabolized by an Sureidoglycolate lyase (ScDAL3) generating urea and glyoxylate (Fig. 3). Several ureidoglycolate lyases from different organisms have been cloned, but only glyoxylate (and not urea) was generally measured to demonstrate the corresponding activities. Direct proof that urea is indeed the product of DAL3-type ureidoglycolate lyases was only recently presented for such an enzyme from E. coli (AllA [83]). The exact route of allantoate degradation in plants is still a matter of debate (for earlier discussion see [53,84,85]). The generation of urea from allantoate has been reported in soybean and tentatively assigned to ureidoglycolate lyase activity [86]. However, physiological or enzymatic studies of ureide degradation are always complicated by the fact that these compounds (in particular ureidoglycine and ureidoglycolate) are unstable and rapidly generate urea, especially when exposed to acidic or basic pH [83,87,88]. Putative ureidoglycolate lyases have been purified from legumes [89,90] but the corresponding genes have not yet been identified. Proteins of diverse evolutionary origin possess ureidoglycolate lyase activity although ureidoglycolate may not be their natural substrate. For example, a protein from Burkholderia cepacia belonging to the family of fumarylacetoacetate hydrolases was found to have ureidoglycolate lyase activity [91]. However, the genome of


purine degradation

-

COO O

O H 2N

NH NH allantoate

H2O NH 3 CO2

NH2

AAH

-

pathway in Arabidopsis

COO O ALC

H 2N

NH

NH2

yeast DAL2

H S-ureidoglycine H2O

UGlyAH

NH 3

-

-

COO O

COO O HO

NH NH2 H S-ureidoglycolate H2O

2 NH 3 CO2

UAH

-

O

+

HO

NH NH2 H2N NH2 H S-ureidoglycolate urea yeast DAL3

UGL

-

COO

COO

O glyoxylate

O glyoxylate

O

+ NH2

H 2N

435

allantoate amidohydrolase releasing ammonia (and not urea) from allantoate has been cloned and characterized from Arabidopsis and soybean (At4g20070, Glyma15g16870 [100,101]). Additionally, genes coding for two enzymes acting downstream of allantoate amidohydrolase were cloned from Arabidopsis and the enzymes biochemically characterized: (i) S-ureidoglycine aminohydrolase (At4g17050), converting the product of the allantoate amidohydrolase reaction (S-ureidoglycine) to S-ureidoglycolate and ammonia [83,102], and (ii) ureidoglycolate amidohydrolase (At5g43600) degrading this compound to glyoxylate, ammonia and carbon dioxide [83] (Fig. 3). That these enzymes form a pathway for plant ureide degradation is indicated by the following facts: (i) common subcellular location in the endoplasmic reticulum (for allantoate amidohydrolase and ureidoglycine aminohydrolase: [83,102]; for ureidoglycolate amidohydrolase: I.A. Sparkes, C.-P. Witte, unpublished); (ii) KM values in the low micromolar range similar to the ureide concentrations found in plants [83,101]; (iii) compatible stereochemical specificity of ureidoglycine aminohydrolase for the reaction product of allantoate amidohydrolase and of ureidoglycolate amidohydrolase for the reaction product of ureidoglycine aminohydrolase; (iv) presence of orthologous, mostly single-copy genes coding for these three enzymes in all plants for which complete genome sequence information is available; (v) growth reduction on allantoin as sole nitrogen source and ureidoglycolate accumulation in a ureidoglycolate amidohydrolase mutant of Arabidopsis and complementation of these phenotypes by a ureidoglycolate amidohydrolase transgene (A.K. Werner, C.-P. Witte, unpublished). It appears that plants in general have the enzymatic capacity to degrade ureides without generating a urea intermediate, leaving arginine catabolism as the only confirmed source for metabolic urea. However, the in-vivo relevance of this pathway still needs to be investigated in more detail. 6. Physiological and agricultural aspects

urea

Fig. 3. Comparison of ureide degradation in Arabidopsis and Saccharomyces. Plants possess enzymes that can degrade allantoate derived from purine degradation without generating a urea intermediate. The enzymes involved are allantoate amidohydrolase (AAH), ureidoglycine aminohydrolase (UGlyAH) and ureidoglycolate amidohydrolase (UAH). Urease is not required. In contrast, yeast degrades allantoate and ureidoglycolate by enzymes that generate urea (allantoate amidinohydrolase or allantoicase, DAL2; and ureidoglycolate lyase, DAL3).

Burkholderia species also contains a DAL3-type ureidoglycolate lyase gene that is more likely to carry out ureidoglycolate hydrolysis in vivo, because it is genetically linked to other genes coding for enzymes involved in purine degradation. Another protein with ureidoglycolate lyase activity was purified from rat liver mitochondria [92] and ureidoglycolate lyase activity was also demonstrated for the peptidyl-␣-hydroxyglycine ␣-amidating lyase domain of peptidylglycine ␣-amidating monooxygenase involved in C-terminal peptide amidation in rat [93]. These enzymes are definitely not involved in ureidoglycolate hydrolysis in vivo because this metabolite does not occur in rats. In the light of these reports it appears possible that the enzymes with ureidoglycolate lyase activity purified from legumes might not be involved in ureidoglycolate catabolism in vivo. The recent findings that these enzymes require phenylhydrazine at millimolar concentrations as co-substrate for ureidoglycolyl group transfer strengthens this notion because phenylhydrazine is not a plant metabolite [94]. In excellent studies it was demonstrated that soybean plants can degrade allantoate without a urea intermediate [85,95–97]. Consistently, soybean cell cultures lacking urease activity were not growth restricted when allantoin was used as sole nitrogen source [98] and urease negative soybean plants did not grow more slowly or accumulated less seed nitrogen than the wild type [99]. An

Urea can serve as sole nitrogen source for plant growth. This was demonstrated in laboratory settings avoiding any non-plant nitrogen conversions like microbial urea hydrolysis or nitrification. However, nitrogen nutrition only based on urea leads to reduced growth and in some cases to symptoms of nitrogen starvation compared to nitrogen nutrition with nitrate or ammonium nitrate [12–14,40,79]. The molecular reasons for the inefficient usage of urea as sole nitrogen source by plants are not understood but explanations like altered osmotic homeostasis [13] or inefficient distribution of nitrogen [40] have been put forward. When plants grow exclusively with urea, the concentrations of the nitrogen storage and transport amino acids, in particular glutamine but often also asparagine, are increased resulting in higher total amino acid concentrations [12–14,40]. These high levels of amide amino acids may contribute to the observed growth depression, perhaps because these amino acids indicate nitrogen abundance to the plant resulting in shut down of further nitrogen uptake and distribution. Similar changes in amino acid profiles were also observed when Arabidopsis was grown with ammonium sulfate as sole nitrogen source [12]. Ammonium nutrition often results in growth depression as well [103]. Interestingly, growth deficits are not observed when urea is supplied together with nitrate [12,104]. In wheat, nitrate boosts urea uptake and urea nitrogen assimilation [105]. In crop production relying on urea fertilization, it is a widespread opinion that urea hardly ever reaches the plants but is degraded by soil microbes to ammonia and nitrate which are then taken up by plants. This view is based on the observation that (i) some soils contain high urease activity, rapidly converting urea to ammonia [106,107] and that (ii) high losses of urea fertilizer mainly by ammonia volatilization can occur in some soils and under certain conditions [108]. Such losses would probably

436


not occur if plants rapidly absorbed all urea. Additionally, it was long unknown that plants are capable of actively importing urea from the soil solution. It has now been demonstrated that plants possess a dedicated active urea transporter and facilitated passive urea transport was observed as well (see above). Internal urea concentrations hardly differ in plants grown on urea versus mineral nitrogen, indicating that plants possess sufficient urease activity to process soil imported urea even at higher external concentrations [12,14,40]. Although urea is certainly converted in part to ammonium and nitrate in the soil, it is possible that the direct urea import of urea fertilizer and its hydrolysis by the plant is generally underestimated. Further research is required to understand the adverse effects of urea nutrition on plants better and to assess the role of plant urea uptake and metabolism for urea fertilizer usage. For example, it would be interesting to compare wild type plants and urease negative mutants fertilized with urea in field experiments to assess the contribution of plant metabolism to urea fertilizer usage. If the plant urea metabolism only plays a minor role in fertilizer urea conversion then urease negative plants will grow as well with urea as with other nitrogen sources in such an experiment. More than half of the nitrogen fertilizer applied in agriculture is not absorbed by crops resulting in pollution of the ground water with nitrate and of the atmosphere with ammonium and nitric oxides [109]. An improvement of plant urea uptake and metabolism bears the potential of reducing nitrogen losses from agricultural systems. For example, it may be possible to boost urea uptake or urea partitioning in the plant by (inducible) overexpression of urea transporters. However, plant urea metabolism including uptake, storage, internal transport, hydrolysis and assimilation of urea nitrogen still needs to be investigated better to develop strategies for knowledge-based crop improvement. Acknowledgements This work was supported by (i) the German Academic Exchange Service (DAAD) from funds of the Federal Ministry for Education and Research (BMBF), program German-Chinese Research Groups and (ii) by the Deutsche Forschungsgemeinschaft (DFG), Grant WI3411/1-2. References [1] P.M. Vitousek, R.W. Howarth, Nitrogen limitation on land and in the sea – how can it occur, Biogeochemistry 13 (1991) 87–115. [2] J.J. Elser, W.F. Fagan, S. Subramanian, et al., Signatures of ecological resource availability in the animal and plant proteomes, Mol. Biol. Evol. 23 (2006) 1946–1951. [3] T. Mohlmann, C. Bernard, S. Hach, et al., Nucleoside transport and associated metabolism, Plant Biol. 12 (2010) 26–34. [4] D. Rentsch, S. Schmidt, M. Tegeder, Transporters for uptake and allocation of organic nitrogen compounds in plants, FEBS Lett. 581 (2007) 2281–2289. [5] S. Kojima, A. Bohner, N. von Wiren, Molecular mechanisms of urea transport in plants, J. Membr. Biol. 212 (2006) 83–91. [6] C. Paungfoo-Lonhienne, D. Rentsch, S. Robatzek, et al., Turning the table: plants consume microbes as a source of nutrients, PLoS One 5 (2010) e11915. [7] C. Paungfoo-Lonhienne, T.G.A. Lonhienne, D. Rentsch, et al., Plants can use protein as a nitrogen source without assistance from other organisms, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 4524–4529. [8] T. Nasholm, K. Kielland, U. Ganeteg, Uptake of organic nitrogen by plants, New Phytol. 182 (2009) 31–48. [9] L.H. Liu, U. Ludewig, W.B. Frommer, et al., AtDUR3 encodes anew type of high-affinity urea/H+ symporter in Arabidopsis, Plant Cell 15 (2003) 790–800. [10] W.H. Wang, B. Kohler, F.Q. Cao, et al., Molecular and physiological aspects of urea transport in higher plants, Plant Sci. 175 (2008) 467–477. [11] S. Kojima, A. Bohner, B. Gassert, et al., AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots, Plant J. 52 (2007) 30–40. [12] P. Merigout, M. Lelandais, F. Bitton, et al., Physiological and transcriptomic aspects of urea uptake and assimilation in Arabidopsis plants, Plant Physiol. 147 (2008) 1225–1238.

[13] J. Gerendas, B. Sattelmacher, Significance of N source (urea vs. NH4 NO3 ) and Ni supply for growth, urease activity and nitrogen metabolism of zucchini (Cucurbita pepo convar. giromontiina), Plant Soil 196 (1997) 217–222. [14] J Gerendas, Z. Zhu, B. Sattelmacher, Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.), J. Exp. Bot. 49 (1998) 1545–1554. [15] K.L. Forrest, M. Bhave, Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype, Funct. Integr. Genomics 7 (2007) 263–289. [16] L.H. Liu, U. Ludewig, B. Gassert, et al., Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis, Plant Physiol. 133 (2003) 1220–1228. [17] M. Dynowski, M. Mayer, O. Moran, et al., Molecular determinants of ammonia and urea conductance in plant aquaporin homologs, FEBS Lett. 582 (2008) 2458–2462. [18] F. Klebl, M. Wolf, N. Sauer, A defect in the yeast plasma membrane urea transporter Dur3p is complemented by CpNIP1, aNod26-like protein from zucchini (Cucurbita pepo L.), and by Arabidopsis thaliana delta-TIP or gamma-TIP, FEBS Lett. 547 (2003) 69–74. [19] M Gaspar, A. Bousser, I. Sissoeff, et al., Cloning and characterization of ZmPIP15b, an aquaporin transporting water and urea, Plant Sci. 165 (2003) 21–31. [20] M. Eckert, A. Biela, F. Siefritz, et al., New aspects of plant aquaporin regulation and specificity, J. Exp. Bot. 50 (1999) 1541–1545. [21] U. Ludewig, M. Dynowski, Plant aquaporin selectivity: where transport assays, computer simulations and physiology meet, Cell Mol. Life Sci. 66 (2009) 3161–3175. [22] S. Gattolin, M. Sorieul, P.R. Hunter, et al., In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots, BMC Plant Biol. 9 (2009) 133. [23] P. Gerbeau, J. Guclu, P. Ripoche, et al., Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes, Plant J. 18 (1999) 577–587. [24] C.P. Witte, S.A. Tiller, M.A. Taylor, et al., Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of 15 N after foliar urea application in wild-type and urease-antisense transgenics, Plant Physiol. 128 (2002) 1129–1136. [25] G. Soto, R. Fox, N. Ayub, et al., TIP5;1 is an aquaporin specifically targeted to pollen mitochondria and is likely involved in nitrogen remobilization in Arabidopsis thaliana, Plant J. (2010), ahead of print. [26] T. Kanamori, N. Kanou, H. Atomi, et al., Enzymatic characterization of a prokaryotic urea carboxylase, J. Bacteriol. 186 (2004) 2532–2539. [27] G.S. Maitz, E.M. Haas, P.A. Castric, Purification and properties of the allophanate hydrolase from Chlamydomonas reinhardii, Biochim. Biophys. Acta 714 (1982) 486–491. [28] R.J. Roon, B. Levenberg, Urea amidolyase. I. Properties of enzyme from Candida utilis, J. Biol. Chem. 247 (1972) 4107–4113. [29] R.L. Blakeley, J.A. Hinds, H.E. Kunze, et al., Jack bean urease (Ec 3.5.1.5). Demonstration of a carbamoyl-transfer reaction and inhibition by nydroxamic acids, Biochemistry-US 8 (1969) 1991–2000. [30] E.L. Carter, N. Flugga, J.L. Boer, et al., Interplay of metal ions and urease, Metallomics 1 (2009) 207–221. [31] C.P. Witte, N. Medina-Escobar, In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction, Anal. Biochem. 290 (2001) 102–107. [32] M.E. Hogan, I.E. Swift, J. Done, Urease assay and ammonia release from leaf tissues, Phytochemistry 22 (1983) 663–667. [33] W.T. Frankenberger, M.A. Tabatabai, Amidase and urease activities in plants, Plant Soil 64 (1982) 153–166. [34] C.P. Witte, E. Isidore, S.A. Tiller, et al., Functional characterization of urease accessory protein G (ureG) from potato, Plant Mol. Biol. 45 (2001) 169–179. [35] R.S. Torisky, J.C. Polacco, Soybean roots retain the seed urease isozyme synthesized during embryo development, Plant Physiol. 94 (1990) 681–689. [36] J.C. Polacco, R.G. Winkler, Soybean leaf urease – a seed enzyme, Plant Physiol. 74 (1984) 800–803. [37] L. Faye, J.S. Greenwood, M.J. Chrispeels, Urease in jack-bean (Canavaliaensiformis (L) Dc) seeds is a cytosolic protein, Planta 168 (1986) 579–585. [38] C.A. Cambui, M. Gaspar, H. Mercier, Detection of urease in the cell wall and membranes from leaf tissues of bromeliad species, Physiol. Plant 136 (2009) 86–93. [39] M. Vivas, M. Sacristan, M.E. Legaz, et al., The cell recognition model in chlorolichens involving a fungal lectin binding to an algal ligand can be extended to cyanolichens, Plant Biol. 12 (2010) 615–621. [40] F.Q. Cao, A.K. Werner, K. Dahncke, et al., Identification and characterization of proteins involved in rice urea and arginine catabolism, Plant Physiol. 154 (2010) 98–108. [41] A. Menegassi, G.E. Wassermann, D. Olivera-Severo, et al., Urease from cotton (Gossypium hirsutum) seeds: isolation, physicochemical characterization, and antifungal properties of the protein, J. Agric. Food Chem. 56 (2008) 4399–4405. [42] N. Das, A.M. Kayastha, P.K. Srivastava, Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L.) seeds, Phytochemistry 61 (2002) 513–521. [43] C. Hirayama, M. Sugimura, H. Saito, et al., Purification and properties of urease from the leaf of mulberry, Morus alba, Phytochemistry 53 (2000) 325–330. [44] H.M. Davies, L.M. Shih, Urease from leaves of Glycine max and Zea mays, Phytochemistry 23 (1984) 2741–2745.

C.-P. Witte / Plant Science 180 (2011) 431–438 [45] A. Balasubramanian, K. Ponnuraj, Crystal structure of the first plant urease from jack bean: 83 years of journey from its first crystal to molecular structure, J. Mol. Biol. 400 (2010) 274–283. [46] C. Follmer, Insights into the role and structure of plant ureases, Phytochemistry 69 (2008) 18–28. [47] C.P. Witte, S. Tiller, E. Isidore, et al., Analysis of two alleles of the urease gene from potato: polymorphisms, expression, and extensive alternative splicing of the corresponding mRNA, J. Exp. Bot. 56 (2005) 91–99. [48] J.C. Polacco, E.A. Havir, Comparisons of soybean urease isolated from seed and tissue culture, J. Biol. Chem. 254 (1979) 1707–1715. [49] L.E. Meyerbothling, J.C. Polacco, S.R. Cianzio, Pleiotropic soybean mutants defective in both urease isozymes, Mol. Gen. Genet. 209 (1987) 432–438. [50] R.S. Torisky, J.D. Griffin, R.L. Yenofsky, et al., A single gene (Eu4) encodes the tissue-ubiquitous urease of soybean, Mol. Gen. Genet. 242 (1994) 404–414. [51] M.A. Holland, J.C. Polacco, Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in 2 soybean mutants, Plant Physiol. 98 (1992) 942–948. [52] A. Goldraij, L.J. Beamer, J.C. Polacco, Interallelic complementation at the ubiquitous urease coding locus of soybean, Plant Physiol. 132 (2003) 1801– 1810. [53] J.C. Polacco, M.A. Holland, Roles of urease in plant cells, in: International Review of Cytology – A Survey of Cell Biology, vol. 145, 1993, pp. 65–103. [54] A.S. Fahmy, M.A. Mohamed, M.Y. Kamel, Ureases in the cucurbitaceae, distribution and properties, Phytochemistry 35 (1994) 151–154. [55] C.R Carlini, J.C. Polacco, Toxic properties of urease, Crop Sci. 48 (2008) 1665–1672. [56] C. Follmer, R. Real-Guerra, G.E. Wasserman, et al., Jackbean, soybean and Bacillus pasteurii ureases – biological effects unrelated to ureolytic activity, Eur. J. Biochem. 271 (2004) 1357–1363. [57] N.E. Dixon, C. Gazzola, R.L. Blakeley, et al., Jack bean urease (Ec 3.5.1.5) metalloenzyme – simple biological role for nickel, J. Am. Chem. Soc. 97 (1975) 4131–4133. [58] I.S. Park, R.P. Hausinger, Requirement of carbon dioxide for in-vitro assembly of the urease nickel metallocenter, Science 267 (1995) 1156–1158. [59] A. Soriano, G.J. Colpas, R.P. Hausinger, UreE stimulation of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein complex, Biochemistry-US 39 (2000) 12435–12440. [60] S. Quiroz-Valenzuela, S.C. Sukuru, R.P. Hausinger, et al., The structure of urease activation complexes examined by flexibility analysis, mutagenesis, and small-angle X-ray scattering, Arch. Biochem. Biophys. (2008). [61] A. Soriano, R.P. Hausinger, GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 11140–11144. [62] M.B.C. Moncrief, R.P. Hausinger, Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease, J. Bacteriol. 179 (1997) 4081–4086. [63] M. Bacanamwo, C.P. Witte, M.W. Lubbers, et al., Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean, Mol. Genet. Genomics 268 (2002) 525–534. [64] S.K. Freyermuth, M. Bacanamwo, J.C. Polacco, The soybean Eu3 gene encodes an Ni-binding protein necessary for urease activity, Plant J. 21 (2000) 53–60. [65] C.P. Witte, M.G. Rosso, T. Romeis, Identification of three urease accessory proteins that are required for urease activation in Arabidopsis, Plant Physiol. 139 (2005) 1155–1162. [66] M. Salomone-Stagni, B. Zambelli, F. Musiani, et al., A model-based proposal for the role of UreF as a GTPase-activating protein in the urease active site biosynthesis, Proteins 68 (2007) 749–761. [67] D. Funck, B. Stadelhofer, W. Koch, Ornithine-delta-aminotransferase is essential for arginine catabolism but not for proline biosynthesis, BMC Plant Biol. 8 (2008) 40. [68] H. Rennenberg, H. Wildhagen, B. Ehlting, Nitrogen nutrition of poplar trees, Plant Biol. 12 (2010) 275–291. [69] A. Ninomiya, Y. Murata, M. Tada, et al., Change in allantoin and arginine contents in Dioscorea opposita ‘Tsukuneimo’ during the growth, J. Jpn. Soc. Hortic. Sci. 73 (2004) 546–551. [70] U. Bausenwein, P. Millard, B. Thornton, et al., Seasonal nitrogen storage and remobilization in the forb Rumex acetosa, Funct. Ecol. 15 (2001) 370–377. [71] C.H. Vanetten, W.F. Kwolek, J.E. Peters, et al., Plant seeds as protein sources for food or feed. Evaluation based on amino acid composition of 379 species, J. Agric. Food Chem. 15 (1967) 1077–1089. [72] G.A. Rosenthal, l-canavanine: a higher plant insecticidal allelochemical, Amino Acids 21 (2001) 319–330. [73] S. Dabir, P. Dabir, B. Somvanshi, The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by l-proline and branched-chain amino acids, J. Enzyme Inhib. Med. Chem. 21 (2006) 727–731. [74] R.D. Slocum, Genes, enzymes and regulation of arginine biosynthesis in plants, Plant Physiol. Biochem. 43 (2005) 729–745. [75] A. Goldraij, J.C. Polacco, Arginine degradation by arginase in mitochondria of soybean seedling cotyledons, Planta 210 (2000) 652–658. [76] A. Goldraij, J.C. Polacco, Arginase is inoperative in developing soybean embryos, Plant Physiol. 119 (1999) 297–303.

437

[77] T. Flores, C.D. Todd, A. Tovar-Mendez, et al., Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development, Plant Physiol. 147 (2008) 1936–1946. [78] L.E. Zonia, N.E. Stebbins, J.C. Polacco, Essential role of urease in germination of nitrogen-limited Arabidopsis thaliana seeds, Plant Physiol. 107 (1995) 1097–1103. [79] J. Gerendas, B. Sattelmacher, Influence of Ni supply on growth and nitrogen metabolism of Brassica napus L. grown with NH4 NO3 or urea as N source, Ann. Bot. London 83 (1999) 65–71. [80] H.S. Yoo, F.S. Genbauffe, et al., Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae, Mol. Cell Biol. 5 (1985) 2279–2288. [81] H.S. Yoo, T.G. Cooper, The ureidoglycollate hydrolase (Dal3) gene in Saccharomyces cerevisiae, Yeast 7 (1991) 693–698. [82] H. Lee, Y.H. Fu, G.A. Marzluf, Nucleotide sequence and DNA recognition elements of Alc, the structural gene which encodes allantoicase, apurine catabolic enzyme of Neurospora crassa, Biochemistry 29 (1990) 8779–8787. [83] A.K Werner, T. Romeis, C.P. Witte, Ureide catabolism in Arabidopsis thaliana and Escherichia coli, Nat. Chem. Biol. 6 (2010) 19–21. [84] C.D. Todd, P.A. Tipton, D.G. Blevins, et al., Update on ureide degradation in legumes, J. Exp. Bot. 57 (2006) 5–12. [85] R.G. Winkler, D.G. Blevins, J.C. Polacco, et al., Ureide catabolism in nitrogenfixing legumes, Trends Biochem. Sci. 13 (1988) 97–100. [86] C.D. Todd, J.C. Polacco, Soybean cultivars ‘Williams 82’ and ‘Maple Arrow’ produce both urea and ammonia during ureide degradation, J. Exp. Bot. 55 (2004) 867–877. [87] E.J. Gravenmade, G.D. Vogels, C. Van der Drift, Hydrolysis, racemization and absolute configuration of ureidoglycolate, a substrate of allantoicase, Biochim. Biophys. Acta 198 (1970) 569–582. [88] C. van der Drift, F.E. de Windt, G.D. Vogels, Allantoate hydrolysis by allantoate amidohydrolase, Arch. Biochem. Biophys. 136 (1970) 273–279. [89] A. Munoz, M.J. Raso, M. Pineda, et al., Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urealyase, Planta 224 (2006) 175–184. [90] A. Munoz, P. Piedras, M. Aguilar, et al., Urea is a product of ureidoglycolate degradation in chickpea. Purification and characterization of the ureidoglycolate urea-lyase, Plant Physiol. 125 (2001) 828–834. [91] J.K. McIninch, J.D. McIninch, S.W. May, Catalysis, stereochemistry, and inhibition of ureidoglycolate lyase, J. Biol. Chem. 278 (2003) 50091–50100. [92] S. Fujiwara, T. Noguchi, Degradation of purines – only ureidoglycollate lyase out of 4 allantoin-degrading enzymes is present in mammals, Biochem. J. 312 (1995) 315–318. [93] M. De, J. Bell, N.J. Blackburn, et al., Role for an essential tyrosine in peptide amidation, J. Biol. Chem. 281 (2006) 20873–20882. ˜ [94] A. Munoz, G.L. Bannenberg, O. Montero, et al., An alternative pathway for ureide usage in legumes: enzymatic formation of a ureidoglycolate adduct in Cicer arietinum and Phaseolus vulgaris, J. Exp. Bot. (2010), ahead of print. [95] R.G. Winkler, D.G. Blevins, D.D. Randall, Ureide catabolism in soybeans. III. Ureidoglycolate amidohydrolase and allantoate amidohydrolase are activities of an allantoate degrading enzyme complex, Plant Physiol. 86 (1988) 1084–1088. [96] R.G. Winkler, D.G. Blevins, J.C. Polacco, et al., Ureide catabolism of soybeans. II. Pathway of catabolism in intact leaf tissue, Plant Physiol. 83 (1987) 585–591. [97] R.G. Winkler, J.C. Polacco, D.G. Blevins, et al., Enzymic degradation of allantoate in developing soybeans, Plant Physiol. 79 (1985) 787–793. [98] R.W. Stahlhut, J.M. Widholm, Ureide catabolism by soybean [Glycine-Max (L) Merrill] cell-suspension cultures. I. Urea is not an intermediate in allantoin degradation, J. Plant Physiol. 134 (1989) 85–89. [99] N.E. Stebbins, J.C. Polacco, Urease is not essential for ureide degradation in soybean, Plant Physiol. 109 (1995) 169–175. [100] C.D. Todd, J.C. Polacco, AtAAH encodes a protein with allantoate amidohydrolase activity from Arabidopsis thaliana, Planta 223 (2006) 1108– 1113. [101] A.K. Werner, I.A. Sparkes, T. Romeis, et al., Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean, Plant Physiol. 146 (2008) 418–430. [102] F Serventi, I. Ramazzina, I. Lamberto, et al., Chemical basis of nitrogen recovery through the ureide pathway: formation and hydrolysis of S-ureidoglycine in plants and bacteria, ACS Chem. Biol. 5 (2010) 203–214. [103] D.T. Britto, H.J. Kronzucker, NH4 + toxicity in higher plants: a critical review, J. Plant Physiol. 159 (2002) 567–584. [104] F. Houdusse, A.M. Zamarreno, M. Garnica, et al., The importance of nitrate in ameliorating the effects of ammonium and urea nutrition on plant development: the relationships with free polyamines and plant proline contents, Funct. Plant Biol. 32 (2005) 1057–1067. [105] M. Garnica, F. Houdusse, J.C. Yvin, et al., Nitrate modifies urea root uptake and assimilation in wheat seedlings, J. Sci. Food Agric. 89 (2009) 55–62. [106] K.H. Han, W.J. Choi, G.H. Han, et al., Urea-nitrogen transformation and compost-nitrogen mineralization in three different soils as affected by the interaction between both nitrogen inputs, Biol. Fert. Soils 39 (2004) 193–199. [107] V. Kumar, R.J. Wagenet, Urease activity and kinetics of urea transformation in soils, Soil Sci. 137 (1984) 263–269.

438


[108] C.J. Watson, H. Miller, P. Poland, et al., Soil properties and the ability of the urease inhibitor N-(N-butyl) thiophosphoric triamide (nbtpt) to reduce ammonia volatilization from surface-applied urea, Soil Biol. Biochem. 26 (1994) 1165–1171.

[109] B. Hirel, J. Le Gouis, B. Ney, et al., The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches, J. Exp. Bot. 58 (2007) 2369–2387.