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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: [email protected]
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,
ª The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]
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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
ALN
1000
1500 N2-FIX
NO3
-
g-1 p
ro
t)
A
21 28 35 42 49 21 28 35 42 49 21 28 35 42 49
0
500
LR t St
AL
N (m
U.m
g
LeavesRoots Stems
Days after sowing
AAH
150 N2-FIX
NO3
-
pro
t)
B
21 28 35 42 49 21 28 35 42 49 21 28 35 42 49
0
50
100
AA
H (m
U.m
g-1 p
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
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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
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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.
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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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