Plant Physiol. (1984) 75, 1104-1 1 10
0032-0889/84/75/1 104/07/$0 1.00/0
Purine Synthesis and Catabolism in Soybean Seedlings'
THE BIOGENESIS OF UREIDES
Received for publication February 27, 1984 and in revised form May 1, 1984
DEBORAH A. POLAYES2 AND KAREL R. SCHUBERT*
Department of Biology, Washington University, St. Louis, Missouri 63130
ABSTRACr
The ureides, allantoin and allantoic acid, are the major nitoge us
substances transported within the xylem of Nrfixing soybeans (Glycine
max L. Meff. cv Amsoy 71). The ureides accumulated in the cotyledons,
roots and shoots of soybean seedlings inoculated with Rhizobiwm or
grown in the presence of 10 millimolar nitrate. The patterns of activity
for uricase and allantoinase, enzymes involved in ureide synthesis, were
positively correlated with the accumulation of ureides in the roots and
cotyledons. Allopurinol and azaserine inhibited ureide production in 3day-old cotyledons while no inhibition was observed in the roots. Incubation of 4day-old seedlings with 4qClserine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of
ureides in both 3- and 4day-old roots was probably the cotyledons. The
inhibition of ureide accumulation by allopurinol but not azaserine in 8day-old cotyledons suggested that ureides in these older cotyledons arose
via nucleotide breakdown. Incubation of 8-day-old plants with 1'4Ciserine
suggested that the roots had acquired the capability to synthesize ureides
via de novo synthesis of purines. These data indicate that both de novo
purine synthesis and nucleotide breakdown are involved in the production
of ureides in young soybean seedlings.
The ureides, allantoin and allantoic acid, are the major xylary
nitrogenous substances of soybeans grown in association with
the bacterium, Rhizobium japonicum (17, 20, 25). The presence
of ureides in the xylem stream has been linked to the process of
symbiotic N2 fixation (20, 21). The production of ureides occurs
in the nodule and the ureides apparently arise from the de novo
synthesis and subsequent degradation of purines (3-6, 9-11, 31).
The concentration of ureides in young soybean cotyledons has
been found to increase 2 to 5 d after planting ( 19). The presence
of ureides in young seedlings suggests that soybeans are capable
of producing ureides without the aid or presence of the microsymbiont. The sites and pathway of ureide biosynthesis within
the developing soybean seedling are unknown. If purines play a
major role in ureide production within the seedling as in the
nodule, then there are two possible explanations for the observed
increase in production of ureides. The increase in ureide concen-
tration may result from (a) de novo synthesis of purines with
subsequent metabolism to ureides, or (b) the degradation of
preexisting nucleotides to form ureides. In either case the activity
of both uricase and allantoinase, two enzymes involved in ureide
synthesis via purine degradation, should be observed. The site of
ureide synthesis can be determined by examining the patterns of
activity of uricase and allantoinase.
The pathway or ureide synthesis can be investigated by using
the inhibitors azaserine and allopurinol. Azaserine blocks the
transfer of the amide-N of glutamine to formylglycinamide ribonucleotide (30). Allopurinol inhibits the enzyme xanthine
dehydrogenase (8). Thus, azaserine is an inhibitor of de novo
purine synthesis while allopurinol blocks purine degradation.
Fujihara and Yamaguchi (15) found no difference in the amount
of ureides in the roots of 3-d-old soybeans after treatment with
azaserine. They concluded that de novo synthesis of purines is
not involved in ureide production in young seedlings. However,
no data were presented for the effects of azaserine on the levels
of ureides in the cotyledons.
In this paper we examine the pattern of ureide accumulation
in the developing soybean seedling. The role of de novo synthesis
of purines in the production of ureides is addressed by examining
the effect of inhibitors of purine metabolism on ureide synthesis
and by measuring the incorporation of label from ['4C]serine
into purines, allantoin and allantoic acid.
MATERIALS AND METHODS
Growth of Plants. Soybean seeds (Glycine max L. Merr. cv
Amsoy 71) were washed with 1% (v/v) bleach, rinsed and soaked
for 1 h in dH20.3 After imbibition, seeds were placed on moist
paper towels on trays and germinated overnight in the dark.
Seeds were planted in 20-cm plastic pots (10 seeds per pot)
containing Perlite. Plants were grown in a greenhouse under
supplemental fluorescent lighting at an energy fluence rate of
120 w-m-2 with a 12-h photoperiod. On d 2 after imbibition,
half the plants were maintained on N-free nutrient solution (12)
while the remaining received nutrient solution supplemented
with 10 mM KNO3.
Ureide Determination. Plants were harvested daily during the
period 2 to 10 d after imbibition and every other day thereafter.
Data for d 0 represent seeds which were imbibed for 1 h. Plant
material was divided into cotyledons and axes (roots, shoots, and
leaves), weighed and frozen in liquid N2. The tissue (1 g) was
homogenized in 4 ml 0.25 N HC104 with a mortar and pestle
(1). The homogenate was centrifuged at 10,000g for 15 min. The
supernatant fluid was neutralized by the addition of 2 N KOH
and centrifuged to remove the potassium perchlorate precipitate.
The supernatant fluid was analyzed for ureides using the differential analysis of Vogels and Van der Drift (29).
' Supported by a grant to K. R. S. from the United States Department
of Agriculture Science and Education Administration Competitive Research Grants Office (Grant 79-CRCR-1-0388). D. A. P. was supported
by a Jessie Smith Noyes Foundation Fellowship. This research was
conducted by D. A. P. in partial fulfillment of the doctoral degree
requirement of the Department of Biochemistry, Michigan State University.
2 Present address: Department of Biology B022, University of Califor3Abbreviations: dH2M, distilled H20; XMP, xanthosine 5'-monophosphate.
nia at San Diego, La Jolla, CA 92093.
1104
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.
UREIDE BIOGENESIS IN SOYBEAN SEEDLINGS
Enzyme Assays. Plant material was divided into leaves, roots,
and cotyledons. The tissi'c (1 g) was homogenized in 4 ml 10
mm K-phosphate buffer (pH 7.6), 10% (w/w) sorbitol, and 10%
(w/w) PVP with a mortar and pestle. The homogenate was
filtered through four layers of cheesecloth and centrifuged at
5OOg for 10 min. The supernatant fluid was analyzed for uricase
and allantoinase activity.
Uricase activity was assayed in a reaction mixture containing
20 Ml 0.15 M 2-(N-cyclohexylamino)ethanesulfonic acid (pH 9.5),
10 Ml 0.59 mM [2-'4C]uric acid (57-60 mCi/mmol; Amersham)
in 0.15 mM Ches (pH 9.5) and 10 Ml enzyme extract (13, 14).
Immediately after the addition of enzyme extract, 5 Ml of reaction
mixture was removed and spotted on PEI-cellulose plates (Brinkman MN-polygram cel 300 PEI) which had been predeveloped
in dH20 overnight (13). The reaction mixture was incubated at
30C and 5 Ml aliquots were removed and spotted on plates at 5,
10, and 15 min after the addition of enzyme extract. Onedimensional chromatography was performed using a solvent
system consisting of 0.5 M NaCl:95% ethanol (4:1). Chromatograms were developed to a distance of 15 cm. The plates were
air dried and uric acid was visualized under short wavelength
UV. Allantoin and allantoic acid were identified using Erlich's
reagent (13). The following relative mobilities were observed:
uric acid (0.34), allantoic acid (0.43), and allantoin (0.68). The
appropriate areas were cut out of chromatograms and radioactivity was determined by liquid scintillation spectrometry.
Allantoinase activity was determined as described by Hanks et
al. (16). Enzyme extract (5-20 Ml) was incubated with 1 ml 20
mM allantoin in 20 mm Tricine (pH 7.8) for 20 min at 25C. The
reaction was stopped by the addition of 1 ml 0.15 N HCI and the
amount of allantoic acid produced was determined (29). Protein
concentration of extracts was determined by a modified Lowry
procedure (7).
Inhibition Studies. Azaserine Treatment. Plants grown on Nfree nutrient solution were harvested at 3, 8, and 12 d after
imbibition. Seedlings were washed with dH20 and placed in 125ml Erlenmeyer flasks (4 seedlings per flask) containing 10 mM
K-phosphate buffer, pH 7.0 (buffer A) with or without 0.5 mM
azaserine (15). The flasks were entirely covered with aluminum
foil and placed on a rotary shaker (28C) in the dark for 24 h.
After the incubation, seedlings were washed with dH20 and
separated into shoots and leaves, roots, and cotyledons. Extraction of tissue and determination of ureide content within the
tissues were as described above.
Allopurinol Treatment. Plants grown on N-free nutrient solution were harvested as described for azaserine treatment. Four
seedlings were placed in flasks containing buffer A with or
without 0.5 mm allopurinol (15). The base of the flasks were
wrapped in aluminum foil. Seedlings were incubated in flasks
under fluorescent lights (12-h photoperiod) for 24 h with continuous aeration. After incubation, seedlings were harvested and
ureide determinations made as described above.
The amount of xanthine and allopurinol in the extracts was
determined by ion-suppression, reverse phase HPLC with a
Whatman Partisil 1O-ODS 2 column. All solvents were prepared
with deionized H20 and filtered daily through a 0.45 MAm nitrocellulose filter. Samples were filtered through 0.2 Mm nitrocellulose filters by centrifugation at 5,000g for 5 min. Filtered extract
(20 Ml) was injected onto the column which was equilibrated with
20 mm ammonium phosphate buffer (pH 7.5). Samples were
eluted from the column as described by Atkins et al. (6). Purine
bases and nucleosides were detected at 260 nm. The following
retention times (min) were observed: xanthosine (5.9), xanthine
(10.8), hypoxanthine (13.2), allopurinol (15.8), adenosine (22.8),
and adenine (24.3).
Labeling Studies. Incubation Conditions. Soybean seedlings
grown on N-free nutrient solution were harvested at 4 and 8 d
1105
after imbibition. Four-d-old seedlings were placed in 10-ml beakers (I seedling per beaker) containing 1 ml buffer A, 0.5 mM
allopurinol or 0.5 mM azaserine, and 20 AI [U-'4C]serine (140
mCi/mmol; ICN Radiochemicals). The control plants were incubated in buffer A and [U-'4C]serine without either of the
inhibitors. The incubation conditions were similar to those described above under Inhibitor Studies. After 2 h, the plants
treated with allopurinol received an additional 2 ml of buffer A
containing 0.5 mM allopurinol. After 8 h, 1.5 ml of buffer A with
0.5 mM allopurinol and 10 Ml [U-'4Cjserine were added to the
beakers containing the seedlings.
Eight-d-old seedlings were placed in 10-ml test tubes containing 7 ml buffer A, 0.5 mm allopurinol, and 50 Ml [U-_4C]serine.
The base of the test tube was wrapped with aluminum foil and
plants were placed under fluorescent lamps for 24 h with continuous aeration of the nutrient solution.
Seedlings were harvested after incubation and extracts prepared as described for ureide determinations. Centrifugation was
performed for 5 min using an Eppendorf microfuge. The acidinsoluble precipitate was resuspended in 1 N NaOH and mcubated at 37°C for 16 h (2). Aliquots of neutralized and NaOHsolubilized samples were placed in vials containing toluene:Triton X 100:PPO:POPOP (66:33:5:0.01, v/v/w/w) and radioactivity was determined. Neutralized extracts were stored at
-20°C until analysis by HPLC and TLC.
Analysis of'4C-Labeled Products. Products of labeling studies
were analyzed by HPLC and TLC. Bases, nuclosides, and nucleotides were separated using ion-paired, reverse phase HPLC
with a Zorbax-ODS (7 Mm) column. The column was equilibrated with 5 mM tetrabutyl ammonium hydroxide (pH 6.5) and
samples (20 Ml) were eluted from column as described by Atkins
et al. (6). Purines were detected at 260 nm. Radioactivity was
determined with a Radioactive Flow Detector (ROMAC. FLOONE Model HS). The efficiency of the flow cell was determined
using [U-'4C]serine and [2-'4C]uric acid. The delay time between
the UV detector and the flow cell was 1.1 min as determined
using [2-'4C]uric acid. The following retention times (min) were
observed: serine, allantoin and allantoic acid (2.6), aminoimidazole carboxamide (4.0), guanine (7.0), hypoxanthine (8.0),
xanthine (8.9), allopurinol (12.0), guanosine (12.6), uric acid
(13.6), adenine (17.0), adenosine (18.1), xanthosine (20.2), aminoimidazole carboxamide ribonucleotide (27.4), IMP (28.6),
AMP (31.5), XMP (39.2), ADP (40.0), and ATP (42.0).
Serine, allantoin and allantoic acid were separated by TLC
(22). Neutralized extracts (25 Ml) were spotted on PEI-cellulose
F plates (E. Merck Reagents, Germany). One-dimensional ascending chromatography was performed as described by Obendorf and Marcus (22). Sample lanes were scanned for radioactivity with a gas-flow scanner (Berthold) and the appropriate areas
were cut out of the plates for determination of radioactivity by
liquid scintillation spectrometry. The following relative mobilities were observed: xanthine (0.32), uric acid (0.36), hypoxanthine (0.46), xanthosine (0.52), XMP (0.62), allantoin (0.68),
allantoic acid (0.78), and serine (0.94).
RESULTS
Patterns of Ureide Accumulation. The level of total ureides
present in the cotyledons and in the embryonic axis, that tissue
which develops into the roots and shoots of the soybean, was
determined during the period 0 to 18 d after imbibition (Fig. 1).
Allantoic acid accounted for approximately 80% of the total
ureides present in all tissues. The amount of total ureides in the
cotyledons increased until d 6, after which time a gradual decrease in the level of ureides was observed (Fig. 1). Nitrate
appeared to have no effect on the pattern or level of ureides in
the cotyledons. The level of ureides in the cotyledons was greater
than in the axes until d 5. After this time, the level of ureides in
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.
1106
POLAYES AND SCHUBERT
A
*
Plant Physiol. Vol. 75, 1984
Nitrate, root S leaves
5050
15
'5
E
1012
O
Doys after Imbi bi tion
FIG. 1. Total ureide concentrations in developing soybean seedlings.
Plants were grown on N-free nutrient solution (open symbols) or nutrient
solution containing I10 mm KNO3 (closed symbols). Plants were harvested, separated into cotykldons (O, *) and axes (A, A), and ureide
levels were determined. The values presented for cotyledons are for a
pair of cotyledons (part). Each value is the mean of two experiments
SE. Each experiment included triplicate samples.
18
o
12
18
Days after knbibition
FIG. 2. Total uricase activity in the cotyledonsand roots ofdeveloping
soybean seedlings. Plants were grown on N-free nutrient solution (open
symbols) or nutrient solution containing 10 mM KNO3 (closed symbols).
Plants were harvested and separated into cotyledons, panel A (0, 0) and
roots, panel B (A, A). For cotyledons a part is a pair of cotyledons. Each
value is the mean of two experiments ± SE. Each experiment included
triplicate samples. The means of the two experiments were averagd.
.c
Table I. A Comparison of Two Methods for Determining Uricase
Activity
Extracts of tissue were, prepared as descibed in "Materials and Mlethods". Assay 1: Uricase activity was measured by monitoring the change
in A at 293 nm as described by Hanks et al. (I16). Assay 2: Uricase activity
was determined by measuring the conversion of '4Clabeled uric acid
into allantoin as described in "Materials and Methods." For Assay 1, 20
to 50 #1 aliquots of nodule extract were assayed. For Assay 2, nodule
extract was diluted 100-fold. An aliquot (10#1u) of diluted extract was
°0.
:r
@ o
o!
0.6
0,4
Z
E 02
0,4
assayed.
Uricase Activity
Assay I
Assay 2
nmol mg'
protein * min172
155
Nodule?'
NDC
1-2
Cotyledonse
NDc
Roots"
16-16.5
' Nodules were removed from 48-d-old soybeans.
b Cotyledons and roots were from 7-d-old plants.
c ND, not detectable.
02
Tissue
6
6
12
18
Days after Imbibition
FIG. 3. Total allantoinAse activity in cotyledons and roots of developing soybean seedlings. Plants were grown on N-free nutrient solution
(open symbols) or nutrient solution supplemented with 10 mm KNO3
(closed symbols). Plants were separated into cotyledons (0, 0) and roots
(A, A). For cotyledons a part is a pair of cotyledons. Each value is the
mean of two experiments + SE. Each experiment included triplicate
samples. The means of the two experiments were averaged.
the axes was substantially greater than that observed in the 14). The use of ['4C]uric acid increased the sensitivity 100-fold,
cotyledons. At the peak of ureide accumulation for both tissues, thus enabling the detection of uricase activity in the roots and
ureide-N represented 0.7% and 6% of the total N of the cotyle- cotyledons of the seedling (Table I). Even with this incsed
dons and axes, respectively. Nitrate appeared to have no effect sensitivity, no uricase activity was detected in the developing first
on the level of ureides present in the axes prior to d 10. After d
leaves of the seedling.
10 the amount of ureides in the axes of plants grown on nitrate
Uricase activity was measured during the period 0 to 18 d after
was greater than that of plants grown on N-free nutrient solution. imbibition (Fig. 2). Uricase activity was determined for both the
Patterns of Uricase and Allantoinase Actti during Seedliag dry seed and a seed after 1 h imbibition. A value of 2.5 + 0.1
Development. Uricase activity was not detected in the seedlings nmol-part'- min-' was observed in both samples. Nitrate did
using the standard assay in which a decrease in the concentration not affect the level of uricase activity in the cotyledons and roots.
of uric acid is monitored at 293 nm (Table I). Uricase activity of Uricase activity peaked in the cotyledons 2 to 4 d after imbibition
the soybean nodule was easily detected with this procedure. The (Fig. 2A). The level of activity was higher in the cotyledons
assay finally employed entailed the incubation of enzyme extract during this early period than in the roots. Uricase activity in the
with ['4C]uric acid and the separation of products by TLC (13, roots increased gradually until d 6 and then declined (Fig. 2B).
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.
UREIDE BIOGENESIS IN SOYBEAN SEEDLINGS
1107
Table II. Effecl ofAllopurinol and Azaserine on Total Ureide
Accumulation
Plants were treated for 24 h with either 0.5 mm azaserine (Az) or 0.5
mM allopurinol (Al). Controls are plants treated for 24 h with only buffer
(10 mm K-phosphate buffer, pH 7.0). The levels of ureides (nmol-g-'
tissue) in the control plants were as follows: for cotyledons, 600 ± 50, d
3; 857 ± 50, d 8; 535 ± 30, d 12; for roots, 1904 ± 100, d 3, 1799 ± 120,
d 8, 1189 ± 70, d 12; and for shoots, 4734 ± 80, d 8; 3628 ± 100, d 12.
Each value is the mean of three experiments ± SE.
Total Ureide Accumulation
Roots
Plant Age
Shoots
Cotyledons
Az
Al
Al
% of control
0
59±6 72±2 94 ±4 93±5
0
92±8 80± 1 80± 1 78±6 79± 1 79±5
92±2 64± 1 64±2 68±5 72±3 68±4
Az
d
3
8
12
Al
Az
Table III. Accumulation ofXanthine in Plants Treated with Allopurinol
The cotyledons contained measurable amounts of xanthine in the
absence of treatment with allopurinol. The following amounts of xanthine were found in untreated cotyledons: 158 ± 39, d 3; 80 ± 16, d 8;
144 ± 46, d 12. This amount was subtracted from the amount of xanthine
in the cotyledons of plants treated with allopurinol. Each value is the
mean of two experiments ± SE.
Xanthine Content
Plant Age
Cotyledons
Roots"
Shoots'
nmol.g' fresh wt
3
162 ± 22
268 ± 4
8
157±9
165±9
266± 18
12
129±26
70±4
205±21
The levels of xanthine in roots and shoots of plants grown in the
absence of allopurinol were not measurable.
d
Table IV. Decrease in Total Ureides for Plants Treated with
Allopurinol
The derease in total ureides due to treatment with allopurinol was
calculated as follows: Decrease = total ureides control - total ureides
allopurinol treatment. Each value is the mean of three experiments ± SE.
Each analysis included three replicates.
Total Ureides
Plant Age
Roots
Shoots
Cotyledons
d
nmol-g' fresh wt
3
171 ± 20
260 + 40
177 ± 4
8
325 ± 56
920 ± 15
12
187±26
407 ±60
1222± 100
Nitrate did not affect allantoinase activity in the roots or
cotyledons of soybean seedlings (Fig. 3). The pattern of allantoinase activity in the cotyledons was similar to that observed for
uricase activity. The level of allantoinase activity in the cotyledons on d 4 was 10-fold greater than that measured in the roots.
Allantoinase activity in the roots did not vary with time, in
contrast to the developmental pattern observed for uricase activity in the roots. The amount of allantoinase activity in either
tissue was substantially greater than that determined for uncase
activity. Leaf allantoinase activity was assayed from d 8 to 18.
The level of allantoinase activity in the leaves was relatively
constant (70 gmol.part'Imin-').
Inhibition Studies. The amount of ureides in the various tissues
of plants treated with the inhibitors, allopurinol and azaserine,
was examined (Table II). Azaserine and allopurinol inhibited
FIG. 4. The effect of allopurinol treatment on the distribution of label
from ['4C]serine within 4-d-old cotyledons. Plants were incubated with
10 mm K-phosphate buffer (pH 7.0) containing (U-'4C)serine (140 mCi/
mmol; 0.5 1uCi/ml) and with 0.5 mM allopurinol, panel A, or with buffer
plus ['4C]serine, panel B. An aliquot (20 ;d) of acid-soluble extract was
eluted from a Zorbax-ODS column as described. Absorbance was monitored at 260 nm and radioactivity by a flow-counter. The retention
times for the radioactivity data have been corrected to reflect the delay
time (1.1) between absorbance detection and radioactivity detection.
Table V. Distribution of Total Radioactivity in the Soluble Fraction of
4-DayOld Cotyledons Incubated with f'4CJSerine
Whole soybean seedlings (4-d-old) were incubated with [U-'4C]serine
(140 mCi/mmol, 0.5 ,Ci/ml). The incubation mixture contained 10 mM
K-phosphate buffer (pH 7.0) alone (C), or buffer plus 0.5 mm allopurinol
(AL) or 0.5 mM azaserine (Az). Total soluble radioactivity for all treatments was 20 ± 2 x l0 dpm-g-' fresh wt. Each value is the mean of
three experiments. The SE was less than 5% of the observed value.
% Total Acid-Soluble
Radioactivity
Compounds
AL
C
Az
Xanthine
4
15
0
Uric acid
6
0
0
Allantoin
2
1
0
Allantoic acid
40
13
0
Serine
42
58
100
ureide production in the cotyledons of 3-d-old soybeans; no
inhibition was observed in the roots. Azaserine did not affect
ureide accumulation in the cotyledons of 8- and 12-d-old seedlings, while a substantial inhibition was observed with allopurinol. A comparable decrease in ureide content in the roots was
found in the presence of both inhibitors during this period. The
inhibition of ureide accumulation in the shoots was similar to
that observed in the roots.
The level of xanthine in extracts of tissues treated with allopurinol was examined by HPLC. The extracts of tissues treated
with 0.5 mm allopurinol were found to contain substantial quantities of xanthine (Table III). The cotyledons were the only tissue
that contained measurable amounts of xanthine in the absence
of treatment with allopurinol. The cotyledons accumulated approximately 160 nmol xanthine.g' fresh weight at all harvests.
The cotyledons and shoots accumulated approximately 300 nmol
allopurinol -g' fresh weight.
The decrease in ureides for the cotyledons (Table IV) was
comparable to the increase in xanthine observed (Table III). On
d 3 after imbibition, the decrease in the amount of ureides within
the roots was equal to the amount of xanthine accumulated. As
the plants aged, the decrease in the level of ureides was greater
than the amount of xanthine accumulated within the roots. The
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.
1108POLAYES AND SCHUBERT
1108
A.
0
E
0.4
UA
1~~~~~~~~~
6
:ul t X ~~*4 .4\
40
A^
.1'2^
30
20
Retention Time (min)
0
FIG. 5. The effect of allopurinol treatment on the distribution of label
from ['4C]serine within 8-d-old roots. Plants were incubated with buffer
A containing [U-'4C]serine (140 mCi/mmol; 0.5 uCi/ml) and 0.5 mM
allopurinol, panel A, or buffer A plus ['4C]serine, panel B. Acid-soluble
extract (20 ul) was injected onto a Zorbax-ODS column and eluted as
described. Absorbance was monitored at 260 nm and radioactivity by a
flow-counter. The retention times for the radioactivity data have been
corrected to reflect the delay time (1.1) between absorbance detection
and radioactivity detection.
Table VI. Distribution of Total Radioactivity in the Soluble Fraction of
8-Day-Old Seedlings Incubated with f4CJSerine
Soybeans (8-d-old) were incubated with [U-'4C]serine (140 mCi/
mmol, 0.5 MCi/ml) as described in "Materials and Methods". The incubation mixture contained 10 mm K-phosphate buffer (pH 7.0) alone (C),
or buffer plus 0.5 mM allopurinol (AL). Each value is the mean of three
experiments. SE was less than 5% of the observed value. Xanthine and
uric acid were separated by TLC and HPLC. Allantoin, allantoic acid,
and serine were separated by TLC. For cotyledons and shoots, the level
of radioactivity was not sufficient to determine percentage radioactivity
in allantoin, allantoic acid, and seine separated by TLC.
% Total Acid-Soluble
Radioactivity
Compound
Roots
C
0
39
AL
37
0
1
7
Cotyledons
C
AL
53
0
Shoots
C
3
33
AL
50
3
Xanthine
Uric acid
7
Allantoin
40a
22
40 }
Allantoic acid
40a
26
28
Seine
a % Radioactivity in allantoin, allontoic acid and serine
greatest decrease in the amount of ureides was found in the
shoots (Table IV). This decrease was substantially greater than
the level of xanthine accumulated in the shoots (Table III).
Labeling Studies. The role of de novo synthesis of purines in
0
44
ureide production was examined by incubating whole seedlings
with [U-'4C]serine and analyzing the distribution of radioactivity
in the acid-soluble products. The total acid-soluble radioactivity
recovered was 20 + 2 x I05 dpm .g' fresh weight for the
cotyledons and 7 ± 1 x I05 dpm .g' fresh weight for the roots
of 4-d-old plants. Both the cotyledons and roots incorporated
approximately the same amount of label (16 ± 4 x I05 dpm *g-'
fresh weight) in the acid-insoluble fraction (proteins, nucleic
acids, etc.) at this time.
The distribution of label within the acid-soluble fraction of 4d-old cotyledons was examined by HPLC (Fig. 4). The cotyledons
Plant Physiol. Vol. 75, 1984
from soybeans were treated with allopurinol (Fig. 4A) and the
distribution of label was compared to that for cotyledons incubated in the absence of inhibitor (Fig. 4B). Most of the radioactivity for the untreated cotyledons and the cotyledons incubated
with allopurinol was recovered in the first peak. This peak
corresponded to allantoin, allantoic acid and serine, which were
not resolved on this column. The amount of label recovered in
the peak at 13.6 min was different between allopurinol-treated
and untreated cotyledons. The retention time of this peak corresponded to that of a uric acid standard. The identity of this
peak was confirmed by separation on TLC plates. Label was
incorporated into uric acid in the cotyledons of untreated plants
(Fig. 4B). Treatment with allopurinol led to the disappearance
of the uric acid peak while the amount of label in xanthine (RT
= 8.9 min) increased (Fig. 4A). Some other peaks ofradioactivity
were observed in the allopurinol-treated plants that were not
found in the untreated plants. The identity of these peaks was
not determined. The peak at approximately 39 min was tentatively identified as XMP.
Allantoin, allantoic acid, and serine were separated by TLC
and the per cent of total radioactivity in these compounds was
determined (Table V). In untreated plants, the ureides represented 42% of the total radioactivity in the cotyledons. Some
radioactivity was found in uric acid and xanthine with the
remainder being in the serine fraction. Treatment with allopurinol led to the disappearance of label in uric acid and a decrease
in the percentage of label in the ureides. The majority of the
label was present in serine. Treatment with azaserine resulted in
the label remaining in serine. Examination of the labeling pattern
in 4-d-old roots was difficult since the amount of radioactivity
was very low (approximately one-third of the radioactivity found
in the cotyledons). The only measurable peak of radioactivity
corresponded to serine.
The distribution of total radioactivity in 8-d-old plants was
also examined. The majority of the label was recovered in the
roots (64 ± 3 x I05 dpm .g' fresh weight in the acid-insoluble
fraction and 42 ± 3 x I0O dpm .g' fresh weight in the acidsoluble fraction). Little incorporation of label was observed in
the cotyledons. An equal distribution of label (5.6 ± 0.3 x 105
dpm .g' fresh weight) between the soluble and insoluble fractions was observed for the shoots. The distribution of label within
the acid-soluble fraction of 8-d-old roots is shown in Figure 5.
The roots of plants incubated without allopurinol contained two
peaks of radioactivity (Fig. SB). One corresponded to the allantoin, allantoic acid, and serine peak while the other was uric
acid. Treatment with allopurinol led to the complete disappearance of radioactivity in uric acid (Fig. 4A). Instead, a large
amount of radioactivity was recovered in xanthine. The distribution of label in the shoots and cotyledons was similar to that
found in the roots (Table VI). The per cent of label in uric acid
for 8-d-old cotyledons was substantially greater than that found
in 4-d-old cotyledons.
Separation of allantoin, allantoic acid, and serine was performed on root extracts. Analysis of shoot and cotyledon extracts
by TLC was not possible. These samples contained a very low
amount of radioactivity (20 cpm/,ul, cotyledons; 50 cpm/,l,
shoots). The loading of large volumes (>50 ,ul) onto the TLC
plates generally resulted in a distortion of relative mobilities.
Attempts to concentrate samples led to variable losses in radioactivity. In the roots, ureides represented 29% of the total radioactivity in plants incubated without allopurinol. Treatment with
allopurinol resulted in a decrease in the percentage of label in
the ureides.
DISCUSSION
The pattern of ureide accumulation within the cotyledons was
similar to that reported by Matsumoto et al. (19). Examination
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.
UREIDE BIOGENESIS IN SOYBEAN SEEDLINGS
of ureide levels in the roots and shoots, however, revealed that
these tissues also accumulated ureides. The cotyledons appeared
to accumulate ureides prior to the appearance of measurable
levels of ureides in the developing axes, suggesting that early
synthesis of ureides was carried out by the cotyledons.
The effect of nitrate on ureide production was investigated to
examine the role of ureides in the N-economy of the seedling.
The application of nitrate to symbiotically-grown soybeans leads
to a decrease in N2 fixation and a decrease in the level of ureides
in the xylem stream (20). The mechanism of inhibition has not
been defined. The growth of seedlings on nitrate had no effect
on ureide accumulation in the cotyledons and led to an increase
in ureide levels in the roots and shoots after d 10. The effect of
nitrate on ureide accumulation in the roots and shoots after d
10 may reflect either an increase in ureide synthesis due to higher
N availability or a decrease in the utilization of ureides due to
the availability of another N source.
The patterns of activity for uricase and allantoinase were
examined to determine the site of ureide synthesis and to investigate the effect of nitrate on ureide synthesis. Comparison between the enzyme activities of plants grown on nitrate or on Nfree nutrient solution suggested that ureide synthesis was not
altered by nitrate. Treatment with nitrate did not change the
level or developmental pattern of uricase or allantoinase activities.
Differences in the patterns of uricase activity for roots and
cotyledons, however, were observed. Total uricase activity in the
cotyledons was higher at early harvests than the activity of the
root enzyme. The pattern of activities was positively correlated
with the accumulation of ureides observed for both tissues. A
high level of uricase activity was found in the roots at a time
when activity in the cotyledon was declining, suggesting that
roots were capable of synthesizing ureides. Uricase activity was
not detected in the leaves; therefore, the ureides in this tissue
presumably arose via transport from other plant tissues.- The
pattern of allantoinase activity in the cotyledons was similar to
the pattern of uricase activity observed.
While cotyledons and roots appear to have the capacity for
purine degradation, direct evidence for ureide production via de
novo purine synthesis has not been reported previously. The
pathway of ureide synthesis was examined by treatment with
azaserine and allopurinol, inhibitors of purine metabolism. Allopurinol and azaserine inhibited ureide production in cotyledons, but not in the roots of 3-d-old plants, which suggests that
early synthesis of ureides was carried out in the cotyledons. The
inhibition by azaserine indicated that the production of ureides
was due, at least in part, to de novo purine synthesis. Fujihara
and Yamaguchi (15) performed similar experiments and also
observed no difference in ureide levels of 3-d-old roots after
treatment with azaserine. They concluded that de novo purine
synthesis was not involved in ureide production. However, the
effect of azaserine on the level of ureides within the cotyledons
was not examined.
The inhibition by allopurinol and the lack of inhibition by
azaserine in 8- and 1 2-d-old cotyledons suggested that the ureides
in these older cotyledons may be arising via nucleotide breakdown. The inhibition of ureide production by both allopurinol
and azaserine in the roots of 8- and 12-d-old plants correlated
with the pattern of uricase activity observed in the roots. Roots
appeared to acquire the ability to form ureides via de novo
synthesis of purines with time. Azaserine and allopurinol inhibited ureide accumulation in the leaves. This inhibition would
suggest that either the leaves were capable of ureide synthesis or
that the ureides are synthesized in other tissues and transported
to the leaves. The lack of uricase activity in this tissue would
indicate that the decrease in ureide levels may reflect the inhibition of ureide synthesis in the roots resulting in a decrease in the
1109
transport of ureides to the leaves.
The accumulation of xanthine in tissues treated with allopurinol instead of hypoxanthine has been reported for other plant
systems (23, 26, 27). In the cotyledons, the accumulation of
xanthine corresponded to the decrease in the level of ureides
present suggesting that no utilization of ureides had occurred. A
similar result was observed in 3-d-old roots. However, on d 8
and 12, the decrease in ureide accumulation in the roots was
greater than the amount of xanthine accumulated in this tissue.
This corresponded to the period of leaf emergence. Therefore,
the difference between the decrease in ureides and the accumulation of xanthine probably reflected the increased transport of
ureides from the roots to the shoots.
The data obtained from the azaserine inhibition studies suggested that ureide production via de novo synthesis of purines
may occur within the cotyledons or roots at various developmental stages. To confirm the role of de novo synthesis of purines in
the production of ureides, plants were incubated with ['4C]serine.
Incubation of 4-d-old seedlings with labeled serine resulted in
42% of the total radioactivity in the acid-soluble fraction from
cotyledons being in the ureides. Treatment with allopurinol
resulted in the expected decrease in the incorporation of label
into ureides and uric acid. The data for 4-d-old roots suggested
that ureide production via de novo synthesis of purines was not
occurring. For 8-d-old plants little radioactivity was recovered in
the cotyledons. However, analysis of the acid-soluble fraction
from the cotyledons revealed that 40% of the label was present
as uric acid. This accumulation of label in uric acid corresponded
with the decreased level of uricase activity in 8-d-old cotyledons.
In contrast to reports from other investigators (15, 24), these
data indicate that de novo purine synthesis is involved in the
production of ureides in young soybean seedlings. In 4-d-old
cotyledons, ureides arise via de novo synthesis of purines, as
shown by the inhibition of ureide synthesis by azaserine and the
incorporation of label from ['4C]serine into allantoin and allantoic acid. At later harvests, the ureides may result from the
breakdown of preexisting nucleotides within the cotyledons. The
source of ureides in 4-d-old roots was probably the cotyledons,
since the roots were found to have low levels of uricase activity;
azaserine and allopurinol did not alter the levels of ureides in
this tissue; and the label from ['4C]serine was not incorporated
into ureides. As the seedling developed the level ofuricase activity
in the roots increased, and the accumulation of ureides in this
tissue was inhibited by both azaserine and allopurinol. Also, label
from ['4C]serine was incorporated into allantoin and allantoic
acid.
These results suggest that there was a shift in the site of ureide
biogenesis from the cotyledons to the roots during seedling
development. Ureides were formed in the cotyledons from purines synthesized de novo initially. As the cotyledons began to
senesce, formation of ureides from preexisting nucleic acids
increased and de novo purine synthesis declined. At the same
time, purine biosynthetic activity increased in roots producing
purines for ureide biogenesis and transport to the developing
leaves.
Acknowledgment-We would like to thank Bonnie Williams for diligently typing
this manuscript.
LITERATURE CITED
1. ANDERSON JD 1977 Adenylate metabolism of embryonic axes from deteriorated soybean seeds. Plant Physiol 59: 610-614
2. ANDERSON JD 1979 Purine nucleotide metabolism of germinating soybean
embryonic axes. Plant Physiol 63: 100-104
3. ATKINS CA 1981 Metabolism of purine nucleotides to form ureides in nitrogenfixing nodules of cowpea ( Vigna unguiculata L. Walp.). FEBS Lett 125: 8389
4. ATKINS CA 1981 The legume-Rhizobium symbiosis: Ureide biosynthesis. In
AH Gibson, WE Newton, eds. Current Perspectives in Nitrogen Fixation.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.
l110
POLAYES AND SCHUBERT
Australian Academy of Science, Canberra, pp 27 1-272
5. ATKINS CA, RM RAINBIRD, JS PATE 1980 Evidence for a purine pathway of
ureide synthesis in Nrfixing nodulesofcowpea( Vigna ungukulata L. Walp).
Z Pflanzenphysiol 97: 249-260
6. ATKINS CA, A RfTCHIE, PB ROWE, E MCCAIRNS, D SAUER 1982 De novo
purine synthesis in N-fixing nodules of cowpea ( Vigna unguiculata L. Walp)
and soybean (Glycine max L. Merr). Plant Physiol 70: 55-60
7. BENSADOUN A, D WEINSTEIN 1976 Assay of proteins in the presence of
interfering materials. Anal Biochem 70: 241-250
8. BOLAND MJ 1981 NAD*:xanthine dehydrogenase from nodules of navy beans:
partial purification and properties. Biochem Int 2: 567-574
9. BOLAND MJ, IF HANKS, PHS REYNOLDs, DG BLEVINS, NE TOLBERT, KR
SCHUBERT 1982 Subcellular organization of ureide biogenesis from glycolytic
intermediates and ammonium in nitrogen-fixing soybean nodules Planta
155: 45-51
10. BOLAND MJ, KR SCHUBERT 1982 Purine biosynthesis and catabolism in
soybean root nodules. Incorporation of '4C from `CO2 into xanthine. Arch
Biochem Biophys 213: 486-491
11. BOLAND MJ, KR SCHUBERT 1983 Biosynthesis of purines by a proplastid
fiwtion from soybean nodules. Arch Biochem Biophys 220:179-187
12. FISHBECK K, HJ EVANS, LL BOERSMA 1973 Measurements of nitrogenase
activity in intact legume symbionts in situ using the acetylene reduction
assay. Agron J 65: 429-433
13. FRIEDMAN TB, CR MERRILL 1973 A microradiochemical assay for urate
oxidase. Anal Biochem 55: 292-296
14. FRIEDMAN TB, DH JOHNSON 1977 Temperature control of urate oxidase
activity in Drosophila: Evidence of an autonomous timer in malpighian
tubules. Science 192: 477-479
15. FUJIHARA S, M YAMAGUCHI 1978 Effects of allopurinol (4-hydroxpyrazolo
(3,4-d) pyrimidine) on the metabolism of allantoin in soybean plants. Plant
Physiol 62: 134-138
16. HANKS JF, NE TOLBERT, KR SCHUBERT 1981 Localization of enzymes of
ureide biosynthesis in peroxisomes and microsomes of nodules, Plant Physiol
68: 65-69
17. HERRIDGE DF, CA ATKINS, JS PATE, RM RAINBIRD 1978 Allantoin and
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Plant Physiol. Vol. 75, 1984
allantoic acid in the nitrogen economy of cowpea ( Vigna unguiculata (L)
Walp). Plant Physiol 62: 495-498
HONG Y-N, P SCHOPFER 1981 Control by phytochrome of urate oxidase and
allantoinase activities during peroxisome development in the cotyledons of
mustard (Sinapis alba L.) seedlings. Planta 152: 325-335
MATSUMOTO T, M YATAZAWA, Y YAMAMOTO 1977 Distribution and change
in the contents of allantoin and allantoic acid in developing nodulating and
non-nodulating soybean plants. Plant Cell Physiol 18: 353-359
MCCLURE PR, DW ISRAEL 1979 Transport of nitrogen in the xylem of soybean
plants. Plant Physiol 64: 411-416
MCCLURE PR, DW ISRAEL, RJ VOLK 1980 Evaluation of the relative ureide
content of xylem sap as an indicator of N2 fixation in soybeans. Greenhouse
studies Plant Physiol 66: 720-725
OBENDORF RL, A MARCUS 1974 Rapid increase in adenosine 5'-triphosphate
during early wheat embryo germination. Plant Physiol 53: 779-781
OGUTUGA DBA, DH NORTHCOTE 1970 Biosynthesis of caffeine in tea callus.
Biochem J 117: 715-720
PATE JS, CA ATKINS 1983 Nitrogen uptake, transport, and utilization. In WJ
Broughton, ed, Ecology of Nitrogen Fixation, Vol 3, Legumes. Oxford
University Press, Oxford, pp 245-298
STREETER JG 1979 Allantoin and allantoic acid in tissues and stem exudate
from field-grown soybean plants. Plant Physiol 63: 478-480
SUZUKI T 1973 Metabolism of methylamine in the tea plant (Thea sinensis
L.). Biochem J 132: 753-763
SUZUKI T, E TAKAHASHI 1975 Metabolism of xanthine and hypoxanthine in
the tea plant (Thea sinensis L.). Biochem I 146: 79-85
TRIPLETT EW, DO BLEVINs, DD RANDALL 1980 AHantoic acid synthesis in
soybean root nodule cytosol via xanthine dehydrogenase. Plant Physiol 65:
1203-1206
VOGEts GD, C VAN DERDRIFr 1970 Differential analysis of glyoxylate derivatives. Anal Biochem 33: 143-157
WEBB JL 1966 Enzyme and Metabolic Inhibitors, Vol 2. Academic Press, NY,
p 933
31. Woo KC, CA ATKINS, JS PATE 1980 Biosynthesis of ureides from purines in a
cell-free system from nodule extractsof cowpea( Vigna unguiculata L. Walp).
Plant Physiol 66: 735-739
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1984 American Society of Plant Biologists. All rights reserved.