Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in...

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Journal of Experimental Botany, Page 1 of 12doi:10.1093/jxb/ers090

RESEARCH PAPER

Developmental effects on ureide levels are mediated bytissue-specific regulation of allantoinase in Phaseolusvulgaris L.

Juan Luis Dıaz-Leal, Gregorio Galvez-Valdivieso, Javier Fernandez, Manuel Pineda and Josefa M. Alamillo*

Departamento de Botanica, Ecologıa y Fisiologıa Vegetal, Grupo de Fisiologıa Molecular y Biotecnologıa de Plantas, Campus deExcelencia Internacional Agroalimentario, CEIA3. Campus de Rabanales, Edif. Severo Ochoa, 1a planta, Universidad de Cordoba,14071, Cordoba, Spain

* To whom correspondence should be addressed. E-mail: bv1munaj@uco.es

Received 10 August 2011; Revised 29 February 2012; Accepted 1 March 2012

Abstract

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes.

In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source,

ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but

they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of

allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent withdevelopmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis

catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants

fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower

leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N2-fixing plants

and in the significant amounts of ureides observed in non-nodulated plants.

Key words: Allantoate, allantoin, allantoinase, development, gene expression, nitrogen fixation, ureides.

Introduction

The ureides allantoin and allantoate are major forms of

nitrogen transported from root nodules to shoots in tropicallegumes. In these plants, nitrogen fixed by the rhizobia is used

for purine synthesis. Through a series of enzymatic steps,

purines are oxidized to allantoin and allantoate (Supplemen-

tary Fig. S1 available at JXB online). Ureides synthesized in

the nodules are transported to the shoot where they should be

degraded and their N content re-assimilated. De novo purine

synthesis is the main route for ureide formation in nodules.

However, purines involved in the biogenesis of ureides mayalso arise by turnover of nucleic acids (Zrenner et al., 2006).

Whatever the biosynthetic route, degradation of ureides

starts with hydrolysis of the internal amide bond of allantoin,

giving rise to allantoate, in a reaction catalysed by allantoin

amidohydrolase [allantoinase (ALN); EC 3.5.2.5], which has

been characterized in plants (Webb and Lindell, 1993; Yang

and Han, 2004; Raso et al., 2007b). The pathway for

degradation of allantoate into its end-products, glyoxylateand ammonia, is still under debate, since enzymatic activities

have only been recently characterized in cell-free extracts,

physiological studies are controversial, and the occurrence of

several pathways for the degradation of both allantoate and

ureidoglycolate has been reported (Todd et al., 2006; Munoz

et al., 2011). Nevertheless, most recent reports suggest that

plants degrade allantoate to ureidoglycolate via allantoate

amidohydrolase (AAH; EC 3.5.3.9) and ureidoglycineaminohydrolase (EC 3.5.3.–) (Todd and Polacco, 2004, 2006;

Todd et al., 2006; Raso et al., 2007a; Werner et al., 2008,

2010, 2011; Serventi et al., 2010).

In soybean and other ureidic plants relying upon N2

fixation as the sole nitrogen source, ureides may comprise

up to 86% of the N in the xylem sap, whereas amino acids,

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amides and nitrate are the major forms of nitrogen exported

from the roots when plants are fertilized with nitrate

(McClure and Israel, 1979; McClure et al., 1980). In these

plants, it is assumed that ureides reach high concentrations

only in nodulated, nitrogen-fixing plants, and determination

of stem or petiole ureide levels has been established as an

easy method to determine nitrogen fixation rates (McClure

et al., 1980; Pate et al., 1980; Herridge, 1982; Patterson andLaRue, 1983; Herridge et al., 1990). Several reports have

shown that plant development strongly influences the level

of ureides in xylem sap and in leaves of ureidic plants

(Schubert, 1981; Herridge and Peoples, 1990; Aveline,

1995).

Changes in ureide levels upon plant development have been

considered an important constraint for the use of the ureide

assay as a convenient method to determine nitrogen fixationrates. The utilization of two different calibration curves for

vegetative and reproductive growth phases has been proposed

for soybean (Herridge and Peoples, 1990). Several reports

have shown that nitrogen fertilization has negative effects on

nodulation, nitrogen fixation, and the levels of ureides. This

has led to the idea that high ureide concentrations are the

direct consequence of nitrogen fixation (McClure et al., 1980;

Atkins et al., 1982; Tajima et al., 2004). In contrast, uponnitrogen fertilization, ureidic plants would use the amides

asparagine and glutamine, instead of ureides, as their storage

and exportable nitrogen (Pate et al., 1980; Streeter, 1985; Leidi

and Rodriguez-Navarro, 2000).

Ureide levels have been shown to rise under water stress

conditions, and it has been suggested that the accumulation

of ureides may be responsible for the feedback inhibition of

nitrogen fixation in these adverse situations (Serraj et al.,1999; Serraj, 2003; King and Purcell, 2005). Recent work

has shown that ureide levels increase considerably in non-

nodulated common bean plants suffering water stress.

Recycling of nitrogen from proteins or nucleic acids in

tissues undergoing drought-induced senescence was consid-

ered the possible source of these ureides (Alamillo et al.,

2010). Nevertheless, the actual source of ureides and the

molecular signalling leading to ureide accumulation underthese conditions need to be clarified further.

Despite the great relevance of ureides, especially in

legumes, there is little information on the regulation of

genes and enzyme activities of ureide metabolism. More-

over, the scarce genomic information in ureidic legumes has

precluded the development of a broad-range expression

analysis that could shed light on the regulation of these

pathways. The recent availability of the fully sequencedgenome of soybean (http://www.phytozome.net/soybean), as

well as the ongoing projects for the sequencing of bean

genomes, will help to compensate this lack of information,

but, to date, there are only a few reports, focusing on

a limited number of experimental conditions, in which

regulation of ureide metabolism gene expression has been

investigated in a ureidic plant (Werner et al., 2008; Charlson

et al., 2009; Alamillo et al., 2010; Yang et al., 2010).In this work, an in-depth physiological and molecular

analysis of ureide metabolism during development of

nodulated and nitrate-fertilized, non-nodulated, P. vulgaris

plants is presented. Under both regimes, ureide levels,

nitrogen fixation rates, and amino acid and nitrate concen-

trations were measured, and the changes in the expression

of genes and the activity of their encoded enzymes involved

in ureide degradation during plant development were

analysed.

The results presented here suggest that an increase inureides at the beginning of the reproductive stage of

development in Phaseolus vulgaris plants is mediated by the

induction of ALN, and that a significant proportion of

these ureides do not depend on nodule nitrogen fixation but

instead originate from remobilization of nitrogen in the

oldest vegetative tissues.

Materials and methods

Biological material and growth conditions

Phaseolus vulgaris L. cv. Great Northern seeds were surfacesterilized by sequential dipping in ethanol (30 s) and 0.2% (w/v)sodium hypochlorite (5 min), and washed thoroughly with distilledwater. Soaked seeds were allowed to germinate on wet paper understerile conditions. After germination, 3–4 seedlings were sown oneach pot (16 cm diameter, 18 cm height) filled with an artificialsubstrate composed of vermiculite/perlite (2/1, w/w) and inoculatedwith 1 ml per plant of a fresh suspension of Rhizobiumleguminosarum bv. phaseoli strain ISP 14, which had been culturedovernight at 28 �C to 0.8–1.0 (OD600 nm), corresponding to ;108

cells ml�1. Plants were cultured in a growth chamber under a 16 hlight, 8 h dark photoperiod, with 200 lE m�2 s�1 lighting, 70%relative humidity, and 26–21 �C day–night temperatures. Inocu-lated plants were first watered with 5 mM KNO3 as a single,starter nutrition, and then three times a week with nitrogen-freenutrient solution (Rigaud and Puppo, 1975) during the whole ofplant development, whereas the nitrate-fed, uninoculated, plantswere watered with nutrient solution containing 10 mM KNO3.Plant material collected at the indicated times after sowing wasfrozen with liquid nitrogen and stored at –80 �C until furtheranalysis. Routinely, tissue samples were obtained as follows: thefourth trifoliate leaves; shoot tissue portions including the basal,middle, and apical stem after removal of leaves; and whole rootsafter careful removal of nodules were collected. In some experi-ments, the primary leaves and the uppermost, unfolded, leaveswere also collected, and stored as independent leaf samples.Nodules collected from each individual plant were weighed andphotographed, as a visual control of similar nitrogen fixationcapacity among the different plants and experiments. Samples werecollected from three independent experiments.

Nitrogen fixation

Nitrogenase (EC 1.7.9.92) activity was measured as the represen-tative H2 evolution in an open-flow system (Witty and Minchin,1998) using an electrochemical H2 sensor (Qubit System Inc.,Canada). For nitrogenase activity measurements, nodulated rootsfrom individual plants were sealed in 0.5 litre cylinders and H2

production was recorded according to the manufacturer’s instruc-tions. The apparent nitrogenase activity (ANA; rate of H2

production in air) was determined under N2:O2 (80%:20%) usinga total flow of 0.4 l min�1. After reaching steady-state conditions,total nitrogenase activity (TNA) was determined under Ar:O2

(79%:21%) at a flow of 0.4 l min�1. The electron allocationcoefficient (EAC) of nitrogenase activity was calculated as1–(ANA/TNA). Standards of high-purity H2 were used to

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calibrate the detector. At least five replicates were measured ineach experiment (biological replicates) for each condition.

Gene expression analysis

Total RNA was isolated from different tissues using the TRIREAGENT� RNA–DNA/Protein Isolation Reagent (MolecularResearch Center, Inc., Cincinnati, OH, USA). Prior to reversetranscription-PCR (RT-PCR), the RNA was treated with RNase-free DNase I (Promega, Madison, WI, USA) at 37 �C for 30 min.Lack of PCR amplification of the 18S rRNA was used to check thesuccessful removal of DNA. First-strand cDNA synthesis wascarried out using 2 lg of DNase-treated RNA using IScript�reverse transcriptase (Bio-Rad, Hercules, CA, USA) following themanufacturer’s instructions. Quantitative RT-PCR (qRT-PCR)was carried out with the iCycler iQ System using the iQ SYBR-Green Supermix (Bio-Rad) and the gene-specific primers listed inSupplementary Table S1 at JXB online. The PCR program was aspreviously reported (Alamillo et al., 2010) and the amplificationefficiency of the primers, calculated by serial dilutions of cDNA,was >90%. The ureide metabolism transcript levels were normal-ized with Actin-2 transcript expression as the reference gene, andwere analysed using the method of Livak and Schmittgen (2001).All the reactions were set up in triplicate (three technical replicates)using three RNA preparations with plant tissues from threeindependent experiments (biological replicates).

Determination of enzymatic activities

All procedures for crude extract preparation were carried out at0–4 �C. Frozen plant material was ground to a fine powder withliquid nitrogen. Plant extracts were obtained by adding 4 ml ofextraction buffer per gram of tissue. The extraction buffer foreach enzyme was the same as used in each of the assays,according to published reports. After 5–10 min of incubation,the resulting homogenate was centrifuged at 15 000 g for 5 min at4 �C and the resulting supernatant was used as the crude extract.Uricase (UO) activity was measured by the decrease in absor-bance at 292 nm due to the aerobic oxidation of urate ina reaction mixture containing 0.1 mM uric acid, in 0.1 M TRIS-HCl, pH 8.5, and an appropriate amount of crude extract(Pineda et al., 1984). ALN activity was determined basically bythe procedure described in Raso et al. (2007b). Briefly, enzymaticproduction of allantoate was determined in a reaction mixtureconsisting of 50 mM TRIS-HCl, pH 7.8, 1 mM MnSO4, 12 mMallantoin, and plant extract. The reaction was carried out at 35�C and aliquots were taken at several time points to determinethe allantoate concentration. Allantoate-degrading activity wasdetermined following the production of ureidoglycolate usinga slight modification of the protocol described by Raso et al.(2007a). The reaction mixture was 50 mM triethanolamine-NaOH, pH 7.0, 1 mM MnSO4, 6 mM potassium allantoate, 0.70mM phenylhydrazine-HCl, and an appropriate amount of plantextract. The reaction was carried out at 35 �C and aliquots weretaken at several time points to determine the ureidoglycolateconcentration. Controls were systematically used to account forthe non-enzymatic decay of substrates. One unit (U) of enzy-matic activity is the amount of enzyme that catalyses thetransformation of 1 lmol of substrate per minute. Results ofenzymatic activities are given as their specific activity (mU mg�1

protein).The results are expressed as means of the values from at least

three independent experiments. Enzymatic assays and analyticaldeterminations from each biological experiment were carried outin duplicate (technical replicate).

Xylem sap collection

Xylem sap was collected at 1 week intervals from 3- to 7-week-oldnitrogen-fixing or nitrate-supplied plants and analysed for ureide,

nitrate and amino acid contents. For xylem sap harvesting, theshoots were cut with a razor blade, just above the cotyledonarynode (McClure and Israel, 1979). To avoid contamination, the cutsurface was rinsed with sterile water, and the sap was collected fora period of 20 min per plant. Sap collection from all the plantswithin a sampling lasted ;2 h, and it was always carried outbefore midday. Xylem sap was kept on ice during the collectionprocess and then immediately frozen until analysis.

Analytical determination

The concentration of ureides was determined by the colorimetricassay of glyoxylate derivatives as described by Vogels and Van derDrift (1970). In this assay, allantoin and allantoate are indepen-dently determined after their chemical transformation to glyox-ylate. The values of total ureides in crude extracts are the sum ofallantoin plus allantoate.Individual free amino acids of the sap were separated and

quantified by reverse-phase HPLC of their OPA (o-phthaldialde-hyde) and FMOC (9-fluorenylmethoxycarbonyl chloride) deriva-tives using a Zorbax Eclipse AAA (4.6 mm3150 mm35 lm)chromatography column and an Agilent 1100 series HPLC system(Agilent Technologies Inc., Santa Clara, CA, USA). Thechromatographic separation was carried out at 40 �C using a flowrate of 2 ml min�1 according to the method described byHenderson et al. (2000). For detection, a diode array detector at338 nm was used for OPA-amino acids and 262 nm for FMOC-amino acids; and a fluorescence detector (340 nm excitation and450 nm emission from 0 min to 15 min, and excitation at 266 nmand emission at 305 nm from 15 min to 26 min). Peakidentification was performed using the 24 amino acids standardmix as described by Henderson et al. (2000).Nitrate in the xylem sap was determined according to Cataldo

et al. (1975).Soluble protein concentration was determined by the method of

Bradford (1976), using bovine serum albumin as a proteinstandard.The results are expressed as means of the values from at least

three independent experiments. Determinations from each biolog-ical experiment were carried out in duplicate (technical replicate).

Results

Developmental stage regulates ureide levels in commonbean tissues

To study the effects of development on the regulation of

ureide metabolism, the levels of ureides were first

determined in tissues of P. vulgaris depending on N2

fixation as the sole nitrogen source or in plants fed with

nitrate, during both the vegetative and reproductive phasesof development. For that, plants were cultured under either

condition, as described in the Materials and methods, and

tissues were collected at 21, 28, 35, 42, and 49 days after

sowing (DAS). At these times, plants were at the vegetative

stage at 21 d and 28 d, and most of them have already

flowered at 35 d, whereas samples at 42 d and 49 d

correspond to pod development and early seed-filling stages

(Table 1).The ureides allantoin and allantoate were determined in

roots, shoots, and leaves from the tissues collected at the

different times after sowing. The ureide concentration

increased during development in roots, shoots, and leaves

from nitrogen-fixing plants and it was higher in most tissues

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from nitrogen-fixing plants than in the samples from the

plants fertilized with nitrate (Fig. 1). It was noticeable that

accumulation of ureides in nodulated plants was mostly in

the form of allantoate in leaves and stems, but it wasequally distributed between allantoin and allantoate in

roots. In shoots, similar amounts of both ureides were

found at 21 d, but allantoate was more abundant than

allantoin from 28 d on, at it reached its maximum levels at

the reproductive stage of development (35, 42, or 49 DAS)

(Fig. 1). In roots, the concentration of ureides increased

;2.0- to 2.5-fold from 21 to 28 and 35 DAS. This increase

in ureide levels was only observed in the nitrogen-fixingplants, whereas in the nitrate-fed plants the lowest level of

ureides was found in roots collected at 35 DAS (Fig. 1A). In

contrast to the moderate (2- to 3-fold) increase in ureide

levels found in roots, stems and leaves from nitrogen-fixing

plants showed a sharp increase in the level of ureides at

35 DAS. The concentration of ureides was maintained at

a high level from 35 d to 49 d in shoots (Fig. 1B), whereas

in leaves the ureide level showed a clear peak at 35 d anda rapid decline in subsequent weeks (Fig. 1C). As for roots,

stems and leaves from nitrate-fed plants showed the lowest

level of ureides at 35 d, but contained significant ureide

concentrations, at both early (21 d) and late developmental

stages (49 d). Interestingly, the level of ureides at 21 d was

slightly higher in all tissues from nitrate fed-plants than in

the equivalent tissues from plants relying on nitrogen

fixation as the sole nitrogen source (Fig. 1).

Nitrogen fixation rates during the development ofP. vulgaris

Nitrogen fixation was measured as the ANA and TNA, and

the EAC was also estimated (Witty and Minchin, 1998).

The maximum nitrogen fixation estimated during common

bean development was found at 28 DAS (Fig. 2A;

Supplementary Table S2 at JXB online). Moreover, nodule

fresh weight (Fig. 2B), uricase activity (UO), and ureide

content were measured in nodules from P. vulgaris roots

collected at different times during plant development. Asexpected, uricase, a late nodulin with a key activity involved

in ureide synthesis (Sanchez et al., 1987), showed a pattern

of activity similar to that exhibited by nitrogen fixation

(Fig. 2C), and similar kinetics were also obtained for the

ureide content in these nodules (Fig. 2D). In contrast,

maximum nodule fresh weight was slightly delayed with

respect to nitrogen fixation (Fig. 2B), probably because at

the times of highest nodule number (35–42 d) a significant

part of these nodules might be already entering intosenescence.

Developmental stage regulates allantoinase transcriptlevels

To get a better understanding of how changes in the ureide

concentration in P. vulgaris tissues are regulated by the

developmental stage, the expression level of genes involved in

either allantoate synthesis or degradation was determined.

For that, qRT-PCR was carried out using gene-specific

primers for PvALN and PvAAH, and cDNAs derived from

RNAs isolated from roots, stems, and leaves collected atseveral different times during plant development.

The expression level of ureide metabolism genes was

normalized with respect to expression of Actin-2, used as

internal control, and the cDNA from root samples at 21 DAS

was used as the reference in each condition. A single-copy

gene codes for AAH in P. vulgaris (Dıaz-Leal et al., personal

communication; EF650088), whereas two highly similar

sequences coding for possible ALN genes have been found inthe P. vulgaris genome (Duran and Todd, 2012; this study).

The full-length clones have been obtained and their sequences

have been deposited in the NCBI data bank (JQ282796 and

JQ277455). However, only one of these two genes (PvALN 1)

showed significant expression in P. vulgaris tissues analysed in

this work, whereas basal levels of expression were found for

the second ALN-coding gene (PvALN 2). The expression

pattern shown in Fig. 3A does, therefore, resemble theexpression of PvALN 1, although it was obtained with

a primer pair potentially able to determine the expression of

both genes (Supplementary Fig. S2 at JXB online). A

comparison of expression of both ALN genes showing the

low levels of PvALN 2 mRNA is presented in Supplementary

Fig. S3.

Expression of PvALN was higher in the stems than in the

roots or leaves from N2-fixing plants. Moreover, a stronginduction of PvALN transcript with growth time was shown

in shoots from nitrogen-fixing plants. In contrast, PvALN

mRNA did not show any significant changes in roots and it

was only slightly induced in the leaves, with an expression

pattern with time that resembles the nitrogen fixation

Table 1. Growth parameters of P. vulgaris plants grown under N2 fixation or fertilized with nitrate

Number of nodes (V), percentage of plants with flowers, and the presence or absence of fruits, at each sampling time in nodulated plantsgrown under nitrogen fixation conditions or in uninoculated plants fertilized with nitrate.

Days N2 fixation Nitrate

Growth (nodes) Flowers (%) Fruits Growth (nodes) Flowers (%) Fruits

21 V562 0 – V562 0 –

28 V662 15 – V762 0 –

35 V763 84 + V964 42 +

42 V863 100 + V963 100 +

49 V862 100 + V962 100 +

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activity of these plants (Fig. 3A). Surprisingly, the expres-sion of ALN was similar or even higher in roots and leaves

from plants fed with nitrate compared with the expression

in nitrogen-fixing plants, although it did not show any large

changes during vegetative development. Unlike roots and

leaves, ALN expression was lower in stems from nitrate-

fertilized plants than in the nitrogen-fixing plants, and it

was induced only at the late developmental stage (42 DAS

samples) (Fig. 3A).In contrast to PvALN, PvAAH transcript expression

(Fig. 3B) was higher in the leaves than in roots or shoots

and, in general, its expression increased slightly along with the

nitrogen fixation rates in shoots and leaves from nodulated

plants. PvAAH expression in roots from nitrogen-fixing

plants did not change significantly, except for a moderate

induction at late development stages. As already mentioned

for PvALN transcript expression, the PvAAH expression levelwas similar, or even higher, in samples from nitrate-fed plants

than in the same tissue from nitrogen-fixing plants; in roots

and stems, there were only small differences between both

conditions, except for the significantly lower expression found

at 35 d in shoots from nitrate fed-plants compared with the

nodulated plants, while in leaves PvAAH reached even higher

expression levels in the samples from nitrate-fed plants than

in those from the nodulated plants (Fig. 3B).It was remarkable that the changes in the expression level

of PvAAH during development were quantitatively lower

(<2-fold induction) than those of the ALN gene in shoots

from nitrogen-fixing plants, that reached values ;10 times

higher in the latest growth stages than early in development

(Fig. 3A, B). Besides the small changes in its expression

levels, relative to the expression in 21-day-old roots, the

expression of PvAAH, normalized according to the internalcontrol Actin-2, was in general several fold lower than the

Fig. 1. Ureide concentration during plant development in

P. vulgaris tissues. Crude extracts were obtained from frozen

tissues collected at several time points during common bean

development. Concentrations of allantoin (grey bars) and allantoate

(black bars) in roots (A), stems (B), and trifoliate leaves (C) are

shown. Results are the mean 6SD of four independent

experiments.

Fig. 2. Developmental effects in N2 fixation, nodule mass, uricase

(UO) activity, and ureide contents in nodules from P. vulgaris.

Pattern of (A) apparent nitrogenase activity (ANA), (B) total nodule

mass, (C) UO activity, and (D) ureide content in nodules from

P. vulgaris sampled during plant development.

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expression of PvALN 1 (results not shown). In the nodules,

the relative expression of PvALN 1, PvALN 2, PvAAH, and

the nodulin uricase (PvUO) presented a similar trend (see

Supplementary Fig. S4 at JXB online). The four genes

showed the pattern of expression expected according to the

kinetics of nitrogen fixation, with their highest expression

levels at 21–28 d, thus preceding the maximum of nitrogen

fixation activity.

Developmental effects on the activities of the enzymesof ureide metabolism

Enzymatic activities of ALN and AAH were measured

in the plant tissues used previously to determine gene

expression and ureide levels (Fig. 4). In stems and leaves,

the specific ALN activity was ;10 times higher than the

AAH activity, whereas in roots ALN activity was only 2–3

times higher than that of AAH (Fig. 4).

ALN activity was higher in tissues from nitrogen-fixing

plants than in plants grown on nitrate, except for samples at

early times of development (21 d) in which lower ALN

activity was found in roots and shoots from nitrogen-fixingplants compared with the plants grown on nitrate. The

highest ALN activity was found in stems, with ;3- to 5-fold

the activity found in roots or leaves. Noticeably, the

temporal evolution of activity in all analysed tissues was

similar to the pattern of the ureide levels, although the only

tissue showing strong increases in ALN-specific activity was

the shoot (Fig. 4A).

In contrast to the effect of plant growth on ALN,developmental stage did not exert any significant effect on

AAH activity, apart from a slight increase in activity at late

times of development in roots. The highest level of specific

1500 N2-FIX

21 28 35 42 49 21 28 35 42 49 21 28 35 42 49

LR t St

LeavesRoots Stems

Days after sowing

150 N2-FIX

21 28 35 42 49 21 28 35 42 49 21 28 35 42 49

LeavesRoots Stems

Days after sowing

Fig. 4. Activity of ureide metabolism enzymes in roots, stems, and

leaves during development of P. vulgaris. Crude extracts were

prepared from frozen tissues from nodulated plants grown under

nitrogen fixation conditions (white bars) and non-nodulated,

nitrate-fertilized plants (black bars). (A) Allantoinase activity.

(B) Allantoate amidohydrolase activity. Tissue samples were

collected at 21, 28, 35, 42, and 49 days after sowing (DAS). The

enzymatic activities are represented as mU per mg of total soluble

protein. Results are the mean 6SD of three independent

experiments and each assay was performed in duplicate (technical

replicate).

Fig. 3. Analysis of relative transcript expression of allantoinase

(ALN) and allantoate amidohydrolase (AAH) during development of

Phaseolus vulgaris plants grown under nitrogen fixation or nitrate-

fertilized conditions. Relative transcript expression of ureide

metabolism genes analysed by qRT-PCR during plant

development in tissues from common bean cultivated either under

nitrogen fixation conditions or fertilized with nitrate. Data were

normalized to the expression of the Actin-2 gene and are

expressed relative to the expression of the corresponding gene in

roots at 21 d, using the 2–DDCT formula (Livak and Schmittgen,

2001). (A) Relative transcript expression of PvALN in roots, stems,

and leaves at 21, 28, 35, and 42 days after sowing (DAS).

(B) Relative PvAAH transcript in roots, stems, and leaves at 21, 28,

35, and 42 DAS.

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activity was found in roots, it was ;3 times lower in shoots,

and the lowest level of activity was found in leaves (Fig. 4B).

The high specific AAH activity in roots was most probably

due to the low protein level of this tissue, since low activity

levels were found for all the tissues analysed when expressed

as activity per fresh tissue weight. Moreover, there were no

clear differences between samples from nitrogen-fixing plants

and those from nitrate-fed plants (Fig. 4B).

Young developing leaves accumulate large amounts ofureides, even in the absence of nitrogen fixation

The results presented in Fig. 1 showed that plant age

strongly influenced ureide levels in trifoliate leaves. To

check whether development exerts the same effect in old

and young leaves co-existing within a plant, ureide content

and ALN activity were determined in the oldest, primary

leaves, and in the uppermost, still unfolded youngest leaves,from plants grown either under nitrogen fixation conditions

or with nitrate as the main nitrogen source (Fig. 5). In the

primary leaves, low concentrations of ureides were found in

samples collected at 21–42 DAS, both in plants relying on

nitrogen fixation and in nitrate-fed plants (Fig. 5A, B). In

contrast, in the uppermost, youngest leaves, the level of

ureides showed a sharp peak at 35 DAS in nitrogen-fixing

plants (Fig. 5A), and a gradual increase upon developmentin the plants fed with nitrate (Fig. 5B). Moreover, the

concentration of ureides was higher in the uppermost,

unfolded leaves than in the mature trifoliate leaves analysed

in Fig. 1, although they showed similar patterns during

plant development. Surprisingly, in the youngest leaves

from nitrate-grown plants, the ureides reached concentra-

tions as high as those in the equivalent leaves from

nodulated, nitrogen-fixing plants (Fig. 5A, B).As well as the ureides, the ALN activity measured in the

same samples was lower in the primary leaves, at any

developmental stage, than in the uppermost, youngest,

leaves. In general, similar levels of enzyme activity were

found in the nodulated and in the non-nodulated plants

(Fig. 5C, D). ALN activity showed a gradual, but not very

high, increment with plant age in the primary leaves from

plants grown under nitrogen fixation and it was maintainedat constant levels in the plants fertilized with nitrate. In

contrast, in the youngest leaves, ALN activity exhibited

a similar pattern in both sets of plants. As previously stated,

the activity was ;2–3 times higher in the uppermost,

youngest, leaves than in oldest, primary leaves (Fig. 5C, D).

Changes in amino acid, ureide, and nitrate levels duringdevelopment in xylem sap from nitrogen-fixing andnitrate-fed plants

Amino acids, ureide, and nitrate contents in the xylem sapfrom common bean plants were analysed in samples

collected at several different times during plant develop-

ment, both in nodulated and in non-nodulated, nitrate-fed

plants. Xylem sap collection was carried out during the

morning to avoid diurnal effects on transpiration, and each

sample comprised the xylem saps collected during 20 min

from four plants in each condition. Sample aliquots were

used to measure the ureide, amino acid, and nitrate content

within the same sample. The individual amino acid concen-

tration for up to 18 amino acids was determined by HPLC

analysis after their derivatization and calibration with

appropriate standard curves. A summary of the results,

showing total amino acid, ureide, and nitrate content incommon bean xylem saps measured at 21, 28, 35, 42, and 49

DAS, is given in Table 2, whereas the concentration of the

four most prominent amino acids, as well as contents of

allantoin and allantoate, is depicted in Fig. 6. The profile

for total soluble amino acids is also presented in Supple-

mentary Fig. S5 at JXB online.

Under N2 fixation, the amides asparagine and glutamine

were the most abundant amino acids, followed by aspartateand glutamate. Aspartate and asparagine followed a similar

pattern, with a higher level at 21 d, lower at 28 d and 35 d,

and a sharp increase at 42 d, whereas the levels of glutamate

and glutamine showed a steady increase during develop-

ment, also peaking at 42 d (Fig. 6A). Interestingly, levels of

glutamine (Fig. 6A) closely resembled the pattern of ureide

accumulation (Fig. 6C). In contrast, increments in ureide

levels at 28 and 35 DAS (Fig. 6C) coincided with the lowestlevels of asparagine in these plants (Fig. 6A). In plants

fertilized with nitrate (Fig. 6B), asparagine was the most

abundant organic nitrogen compound, followed by aspar-

tate, glutamate, and glutamine. In these plants, the aspara-

gine levels increased during development, showing a peak at

42 d, reaching concentrations that were ;8 times the

concentration of glutamine, glutamate, or even aspartate.

In N2-fixing plants, the increment in xylem ureidecorrelates with increases in nodule fresh weight and

matched the nitrogen fixation rates (Fig. 2) during the

vegetative growth phase, but sharply increased after flower-

ing (35–42 d) (Fig. 6C). The contents of allantoin and

allantoate followed a similar pattern during plant growth,

although allantoic acid was the major component of the

ureide fraction at any of the developmental stages.

Interestingly, significant levels of ureides were also found inthe xylem from nitrate-fed plants, in which, at 42 d, ureides

reached values that were ;40% of the concentration found

in the N2-fixing plants (Fig. 6C). Levels of both amino acids

and ureides were lower at 49 DAS than at 42 DAS,

suggesting that sources of organic nitrogen, either by

fixation or assimilation, begin to be less operative.

As expected, the nitrate concentration was maintained at

low levels in xylem sap collected from nodulated plants, butit reached concentrations that were 2–4 times higher than

the 10 mM used in the nutrient solution in the plants

fertilized with nitrate (Table 2).

Ureide, amino acid, and nitrate contents were used to

determine the relative ureide nitrogen (RUN) content

during development in common bean plants, according to

Herridge (1982). RUN reached 91–93% at the times of the

highest nitrogen fixation rates (28 d and 35 d) in thenodulated plants, and slightly decreased at later develop-

mental stages. In contrast, in nitrate-fertilized plants, RUN

nloaded from

was ;10% during vegetative development, but rose to 16–

17% at the late, reproductive phase of development(Table 2).

Discussion

Many reports have shown a direct correlation between ureide

levels and nitrogen fixation, although large amounts of

ureide have also been observed in uninoculated plants and in

non-nitrogen-fixing species, challenging the frequently held

idea that significant ureide levels are only produced by

nitrogen fixation in ureidic plants (Thomas et al., 1979,

1980; Brychkova et al., 2008; Santos et al., 2009).The results presented in this work demonstrate that in

P. vulgaris the concentration of ureides only shows a good

correlation with nitrogen fixation until the onset of

flowering. Afterwards, the amount of ureides in leaves and

stems increases more than nitrogen fixation (Figs 1, 2).

Moreover, leaves and stems from uninoculated, nitrate-

fertilized plants also show significant ureide levels during

their late development (Figs 1, 5; Table 2). The amount ofureides in uninoculated plants is comparable with the

apparent excess of ureides in relation to fixation rates found

Fig. 5. Effect of developmental stage on ureide content and allantoinase activity in the primary and the uppermost leaves from nodulated

and nitrate-fed plants. Ureide contents in the oldest, primary leaves, and in the youngest, unfolded trifoliate (uppermost) leaves during the

development of P. vulgaris plants grown under nitrogen fixation conditions (A) or of nitrate-fertilized P. vulgaris plants (B). Allantoinase

activity in the primary and the uppermost leaves at several time points during the development of nitrogen-fixing plants (C) and plants

grown with nitrate as the main nitrogen source (D). Results are the mean of four independent experiments.

Table 2. Distribution of nitrogen compounds during development in the xylem sap from P. vulgaris plants grown under N2 fixation or

fertilized with nitrate

Ureide represents the sum of allantoin plus allantoate. Amino acids are the sum of soluble amino acids detected after o-phthaldialdehyde (OPA)and 9-fluorenylmethoxycarbonyl chloride (FMOC) derivatization. Relative ureide nitrogen (RUN) was estimated according to the formula ofHerridge (1982).

Days N2 fixation Nitrate

Ureide (mM) Amino acid (mM) Nitrate (mM) RUN (%) Ureide (mM) Amino acid (mM) Nitrate (mM) RUN (%)

21 0.6260.2 1.1060.3 0.9960.6 54.562.1 0.6160.1 2.2160.04 23.967.2 8.5860.5

28 2.7661.0 0.5660.2 0.4460.06 91.660.4 0.6260.06 2.3560.4 17.761.8 10.960.1

35 3.9261.7 0.7760.1 0.2960.03 93.660.6 0.7060.1 3.3060.5 16.763.1 12.563.3

42 5.5360.7 2.2660.8 0.6460.4 88.663.1 2.2160.01 5.8560.5 41.968.0 15.761.9

49 2.8560.1 1.7360.1 0.4560.1 84.160.7 1.4160.2 3.4660.1 23.165.8 17.761.4

nloaded from

after flowering in the nodulated, nitrogen-fixing plants,

suggesting that they probably originated from the same

source. The results presented in Fig. 5 strongly support thishypothesis, and point to remobilization of nitrogen from

the oldest leaves as the main source for ureide synthesis and

accumulation in shoots and developing tissues in the non-

nodulated plants, and for the sharp increase in ureides

during early pod filling in the nodulated plants when

nitrogen fixation starts to decline (Figs 1, 2).

Changes in the correlation between nitrogen fixation and

ureide levels with transition to reproductive development

have been clearly demonstrated (Herridge and Peoples,

1990). Furthermore, early nitrogen remobilization in sen-

escent tissues in an early maturing cultivar of soybean was

reported as a possible source of error in the estimation of

nitrogen fixation by the ureide assay method (Aveline, 1995).

In fact, it has recently been shown that, under conditions ofwater stress, P.vulgaris plants accumulate ureides even if they

lack root nodules (grown with nitrate), and major accumula-

tion of ureides occurs after drought has completely inhibited

the N2 fixation in nodulated plants (Alamillo et al., 2010).

Remobilization of nitrogen from drought-induced senescent

tissues was suggested as the most likely alternative source of

ureides under such conditions.

Fischinger et al. (2006) showed that nitrogen re-trans-location from senescing lower leaves to common bean root

nodules might be involved in the N-feedback regulation of

nitrogen fixation. Moreover, ureide accumulation upon

drought stress has been hypothesized to be responsible for

N2 fixation inhibition (Serraj et al., 1999). Therefore, it

would be interesting to analyse further the actual contribu-

tion of ureides originating from senescing lower leaves to

the decline in nodule activity shown at late developmentalstages (Fig. 2).

The present results resemble those from early studies

showing increases in ureide-N at flowering and early pod

formation in non-nodulated P. vulgaris L. (bushbean)

(Thomas et al., 1979, 1980). It has been shown that the

increase in ureides correlated with a peak in the specific

activity of ALN in soybean (Thomas and Schrader, 1981).

However, in these early reports, lack of genetic informationprecluded the study of the regulation of ureide metabolism

at the molecular level. Interestingly, when the ureide

concentration was determined in tissues from nodulated

N2-fixing plants, only the roots showed a pattern of ureide

accumulation resembling the nitrogen fixation kinetics, and

neither gene expression nor ureide metabolism enzymatic

activities changed significantly during plant development in

roots, suggesting that they make a limited contribution tothe generation of ureides upon the remobilization occurring

after flowering.

In the present study, allantoate was the most abundant

ureide in most tissues and under most conditions, except for

roots that contained similar allantoin and allantoate levels.

Except for roots, similar or even higher allantoin than

allantoate levels were found in samples during early

vegetative growth (Fig. 1). Remarkably, the presence ofallantoin coincided with low ALN activity (Fig. 4).

Induction of ALN activity, mainly in shoots, led to the

reduction of allantoin and to the steady accumulation of

allantoate, thus suggesting that AAH is limiting the rate of

ureide degradation. This was previously suggested by

Thomas and Schrader (1981), although neither gene expres-

sion analysis nor the assay of enzymatic activity able to

degrade allantoate was feasible at that time. The results inFigs 3 and 4 strongly suggest that the induction of expression

and activity of ALN, mostly in the stem, is responsible for

Fig. 6. Amino acid and ureide concentrations in xylem sap from

P. vulgaris plants grown under nitrogen fixation or fertilized with

nitrate. (A) Aspartate (ASP), asparagine (ASN), glutamine (GLN),

and glutamate (GLU) composition in xylem sap from nodulated,

nitrogen-fixing plants. (B) ASP, ASN, GLN, and GLU concentra-

tions in xylem sap from nitrate-fertilized plants. (C) Ureides,

allantoin, and allantoate in xylem from nodulated and nitrate-

fertilized plants. Xylem sap collection was carried out during

20 min, at about midday, from plants at 21, 28, 35, 42, and 49

days after sowing (DAS). Xylem sap from at least five plants was

pooled together in each sampling. Results are the mean from three

independent experiments.

nloaded from

the significant increase in allantoate. It was noticeable that

the ALN activity pattern did match the transcript levels.

Similarly, low levels of AAH activity match the lower

induction of PvAAH transcript expression, supporting the

transcriptional regulation of ureide metabolism, although

other levels of regulation could also be possible.

Despite the large number of reports studying ALN activity

in plants, mainly in legumes, analysis of ALN geneexpression is very limited. The results in Fig. 3A show that

ALN expression is highly induced upon development, mainly

in stems. Maximum ALN expression levels were reported in

bark/cambial zone from black locust (Robinia pseudoacacia)

shoots (Yang and Han, 2004), suggesting that preferential

expression in shoots is also a feature for ALN in this non

ureide-type legume. The nitrogen source has been shown to

regulate ALN expression in Arabidopsis (Werner et al., 2008),which apparently contradicts the present results showing

small differences in transcript abundance in tissues from

N2-fixing and nitrate-fed plants (Fig. 3). In Arabidopsis, the

lowest mRNA level was found in nitrate-treated plants, and

it increased in response to nitrogen starvation or allantoin

feeding. However, it is unclear whether these results corre-

spond to repression by nitrate or to induction by nitrogen

starvation in the other conditions.Whereas ALN activity agrees well with its transcript

abundance in all tissues examined, the highest relative AAH

gene expression was found in leaves, although they showed

the lowest AAH activity, thus suggesting that some post-

translational regulation of AAH activity could operate in this

tissue. Post-translational regulation of AAH has already

been suggested to explain the accumulation of allantoate

observed under drought conditions when AAH gene expres-sion was either maintained at constant levels (Charlson et al.,

2009) or even induced (Alamillo et al., 2010).

In contrast to the changes in PvAAH presented here, the

expression of AAH in A. thaliana was similar in all tissues

and growth stages examined (Todd and Polacco, 2006), and

upon N starvation or nitrate feeding (Werner et al., 2008).

Whereas these studies point to constitutive AAH mRNA

expression, the present results show that PvAAH expressionis not affected by the nitrogen source, but it is regulated by

the plant developmental stage. These discrepancies might be

explained by the high sensitivity of the qRT-PCR used in

this study, whereas the semi-quantitative RT-PCR used in

previous reports may not be accurate enough to detect the

moderate changes in AAH expression shown here.

Low allantoate-degrading capacity has been considered as

the bottleneck for ureide metabolism (Thomas and Schrader,1981), and the reason for the accumulation of ureides under

drought stress in soybean (King and Purcell, 2005). However,

data in common bean show that accumulation of ureides is,

indeed, caused by the induction of allantoate synthesis

through the positive regulation of ALN (Alamillo et al., 2010;

this study). These results should be considered for future

strategies aiming to improve nitrogen fixation (i.e. limiting

ureide accumulation under drought stress) and suggest thatthey should focus on the control of ALN regulation, instead

of looking for improved allantoate degradation.

The distribution of the ureide, amino acid, and nitrate

content in xylem sap of P. vulgaris summarized in Table 2

and depicted in Fig. 6 strongly supported the generalized

idea that ureidic plants behave as amidic plants upon nitrate

fertilization (Pate et al., 1980). Furthermore, the concen-

trations of amino acids shown in Table 2 are similar to

previously reported levels in nitrate-fertilized Phaseolus

plants (Leidi and Rodriguez-Navarro, 2000). Nevertheless,although nitrate-fed plants transported more asparagine

and glutamine in their xylem than plants fixing nitrogen, the

significant levels of asparagine and glutamine measured

during the pod-filling stage in the nodulated plants is still

remarkable. Remobilization of nutrients in the oldest tissues

is the most likely source of both amides and ureides found

at late developmental stages.

Nitrogen remobilization has been reported to be preferen-tially partitioned to reproductive organs in pea and other

grain legumes (Salon et al., 2001; Schilzt et al., 2005),

although enhancement of nitrogen and CO2 fixation in pea

nodules was also suggested as a mechanism to supply the

high demands of early fruit development (Fischinger and

Schulze, 2010). However, the present data show that nitrogen

fixation in Phaseolus peaks several days before flowering,

and, therefore, mobilization of N stored in vegetative tissuesshould satisfy the pod’s development demands. The results

suggest that nitrogen mobilized from senescent tissues is

mainly channelled through synthesis of ureides, thus support-

ing the idea of ureide-N having a predominant role in pod

development. Ishizuka (1977) suggested that ureide-N, aris-

ing predominantly from N fixation, was used more efficiently

in seed protein production than N in the form of amino

acids, amides, and nitrate. According to this, ureides reachedtheir highest levels in the developing fruits from Phaseolus

(Raso et al., 2007b), also supporting a role for ureides in pod

development.

In summary, results in this report show that the

developmental stage influences ureide levels in P. vulgaris,

both under nitrogen-fixing conditions and in nitrate-fed

plants, and that changes in ureide levels during P. vulgaris

development are mediated by the tissue-specific regulationof ALN. In addition, they suggest that remobilized N from

lower leaves is involved in the sharp rise in ureides in shoots

and leaves during early pod filling in N2-fixing common

bean, and in the moderate, although significant, amounts of

ureides observed in non-nodulated plants

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Purine catabolism in plants.

Figure S2. Specificity of primers used to determine theexpression of PvALN genes.

Figure S3. Analysis of relative transcript expression of

allantoinase genes during development of Phaseolus vulgaris

plants grown under nitrogen fixation or nitrate-fertilized

conditions.

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Figure S4. Relative transcript expression of ureide

synthesis and degradation genes during development in

nodules from P. vulgaris plants.

Figure S5. Amino acid composition in the xylem sap from

nodulated, N2-fixing, and nitrate-fertilized P. vulgaris

plants.

Table S1. Primers used for quantification of mRNAs

from French bean tissues by qRT-PCR.Table S2. Evolution of the N2 fixation activity during the

development of common bean plants.

Acknowledgements

This work was supported by grants AGL2009-11290

(Ministerio de Educacion y Ciencia, Spain), AGR01283, and

P07-RNM-03307 from Consejerıa de Innovacion, Ciencia y

Empresa (Junta de Andalucia, Spain) and Plan Andaluz de

Investigacion (BIO-115; Junta de Andalucıa, Spain). JMA

and JLDL were supported by a post-doctoral contract anda fellowship from Consejerıa de Innovacion, Ciencia y

Empresa (Junta de Andalucia, Spain). The authors would

like to thank Professor C.D. Todd (Saskatchewan Univer-

sity, Canada) for sharing unpublished results. We are also

grateful to Professor A. de Ron and Dr P. Rodino (MBG,

Pontevedra, Spain) for their generous gift of the Phaseolus

vulgaris seeds, and to Dr Dulcenombre Rodrıguez (C.I.F.A.,

Sevilla, Spain) for the gift of Rhizobium leguminosarum ISP14, as well as to Professor C. Lluch and Dr F. Palma

(University of Granada, Spain) for their help in the

determination of nitrogen fixation. The excellent technical

assistance of Mrs Marta Robles is also acknowledged.

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