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Plant Physiol. (1999) 119: 297-304
Arginase Is Inoperative in Developing Soybean
Embryos1
Ariel Goldraij and
Joe C. Polacco*
Department of Biochemistry and Interdisciplinary Plant Group, 117 Schweitzer Hall, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
Arginase (EC 3.5.3.1) transcript
level and activity were measured in soybean (Glycine max
L.) embryos from the reserve deposition stage to postgermination. Using
a cDNA probe for a small soybean arginase gene family, no transcript
was detected in developing embryos. However, arginase transcripts
increased sharply on germination, reaching a maximum at 3 to 5 d
after germination. There was low but measurable in vitro arginase
specific activity in developing embryos (less than 6% of seedling
maximum). During germination arginase specific activity increased in
parallel with the sharply increasing arginase transcript level.
Seedling arginase activity was largely localized in cotyledons.
Arginase activity was assayed in vivo by measuring urea accumulation in
a urease-deficient mutant. No urea was detected in developing embryos,
whereas accumulated urea paralleled arginase specific activity and
transcript level in germinating seedlings. As in planta embryos,
cultured cotyledons did not accumulate urea when arginine (Arg) was
provided with other amino acids in a "mock" seed-coat exudate. Arg
as the sole nitrogen source was converted to urea but did not support
cotyledon growth. There appeared to be a lack of recruitment of the
low-level arginase activity to hydrolyze free Arg in developing
embryos, thus avoiding a futile urea cycle.
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INTRODUCTION |
Arg, a nitrogen-rich compound, is one of the predominant amino
acids in the seed and storage organs of numerous plant species (Van
Etten et al., 1967 ; Polacco and Holland, 1993) and represents a major
form of reserve nitrogen. In developing soybean (Glycine max) cotyledons, Arg is both actively synthesized (Micallef and Shelp, 1989b) and supplied from the seed-coat exudate (Rainbird et al.,
1984). Arg is incorporated into protein, where it accounts for 18% of
the total seed-protein nitrogen, or it remains in the free amino acid
pool, constituting more than 60% of the free amino acid nitrogen in
developing cotyledons (Micallef and Shelp, 1989a).
During early seedling growth, storage proteins are mobilized to provide
amino acids for proteins synthesized in the expanding axes. As part of
the overall reconfiguration of free and storage-protein-bound amino
acid profiles to that of seedling protein (Thompson, 1980; Polacco and
Holland, 1993), Arg breakdown to urea and Orn occurs by the action
of arginase (L-Arg amidinohydrolase, EC 3.5.3.1). Arginase activity increases sharply during germination in several species, including pumpkin (Splittstoesser, 1969), broad bean (Kollöffel and Van Dijke, 1975), soybean (Matsubara and Suzuki, 1984; Kang and Cho, 1990), Arabidopsis (Zonia et al., 1995), and loblolly pine (King and Gifford, 1997). After arginase action, Arg
nitrogen contained in urea is recycled to ammonia by the action of
urease (Polacco and Holland, 1993).
There have been contradictory reports on arginase activity in
developing cotyledons in contrast to those on arginase in seedlings. Micallef and Shelp (1989c) suggested that during soybean embryo development, arginase metabolizes a portion of the abundant free Arg.
In developing pea cotyledons, however, no Arg-derived urea was
detected, although in vitro arginase activity was present at an early
stage of cotyledon development (De Ruiter and Kollöffel, 1983).
In the present study we used the cDNA clone AG1, which
encodes a soybean seedling axis arginase (Goldraij et al., 1998), and the pleiotropic urease-deficient mutant eu3-e1/eu3-e1
(Meyer-Bothling et al., 1987) to investigate the expression and in vivo
activity, respectively, of soybean embryo arginase from the reserve
deposition stage to postgermination. In addition, functional tests of
arginase were performed in in vitro-cultured cotyledons in which Arg
was tested as a nitrogen source and as a precursor of urea. Our results indicate that the increase of arginase activity upon germination was
due to an increase in arginase transcript, and are consistent with the
absence of an arginase-catalyzed reaction in developing embryos during
the reserve deposition stage.
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MATERIALS AND METHODS |
Plant Material and Germination/Growth Conditions
Wild-type Eu3/Eu3 soybean (Glycine max L. Merrill cv Williams 82) plants and an otherwise isogenic
Eu3/eu3-e1 heterozygote were grown in a
controlled-environment chamber at 27°C with a 12-h/12-h light/dark
regime at 180 µmol m 2
s1. The eu3-e1 mutation has been
characterized previously (Meyer-Bothling et al., 1987). The homozygous
eu3-e1/eu3-e1 mutant lacks the activities of all soybean
ureases. Eu3/eu3-e1 heterozygous plants (Stebbins et al.,
1991; Stebbins and Polacco, 1995) have urease levels similar to
wild-type Eu3/Eu3 plants. All urease-negative
eu3-e1/eu3-e1 seeds used in this study were derived from
selfed Eu3/eu3-e1 plants. Urease-positive and -negative
seeds were distinguished by a seed-chip urease assay (Meyer-Bothling
and Polacco, 1987). After removal from pods, immature embryos were
separated from the testa, frozen in liquid nitrogen, lyophilized, and
stored at  70°C. The relative water content of embryos (milligrams
of water per milligram of embryo fresh weight × 100) was taken as
the criterion for the physiological state of the embryo.
For seedling studies, seeds were germinated in the dark at 28°C in
rolls of germination paper (Anchor Paper, St. Paul, MN) moistened with
deionized water. Seeds routinely germinated 1 d after imbibition.
Whole etiolated seedlings were frozen immediately in liquid nitrogen
and stored at 70°C until use.
Arginase Activity Assay
Embryos or seedlings (1.5-2.0 g fresh weight) were ground and
powdered in a mortar with liquid nitrogen. The fine powder was resuspended to a final volume of 4 mL in cold 0.1 M
Tris-maleate, pH 7.0, containing 1 mM EDTA and 0.1 mM PMSF. The suspension was maintained on ice and disrupted
(three 15-s cycles) in a homogenizer (Ultra Turrax, Tekmar, Cincinnati,
OH) and centrifuged at 13,000g for 10 min at 4°C. The
supernatant was filtered through Miracloth (Calbiochem), brought to 5 mM in MnCl2, and left for
10 min at room temperature to activate arginase.
Arginase activity was assayed by measuring the Arg-dependent production
of urea. One milliliter of standard assay medium contained 160 mM L-Arg (adjusted to pH 9.7 with KOH), 33 µM phenyl phosphorodiamidate (a urease inhibitor, Liao
and Raines [1985]), and about 0.1 to 0.3 mg of extract protein. The
reaction mixture was incubated for 30 min at 30°C. Aliquots (400 µL) were removed and the reaction was stopped by adding 1 N H2SO4 to 600 µL. Urea released was determined colorimetrically (Schimke, 1970).
Arginase activity was expressed as nanomoles of urea released per
minute per milligram of protein. Protein was determined by the method
of Lowry et al. (1951).
Tissue Extraction and Urea Analysis
Lyophilized embryos or whole seedlings were frozen in liquid
nitrogen and ground in a mortar to a fine powder. Weighed portions (40-70 mg) were extracted three times with 250 µL of 3% (w/v) HClO4. Combined supernatants were neutralized by
adding 15 µL of 2.5 M
K2CO3, and 1 mM
EDTA per 100 µL of extract. Samples were placed on ice for 10 min,
and the potassium perchlorate pellet was removed by centrifugation.
Urea was measured by adding urease to the supernatant and determining
the ammonia liberated by the phenol-hypochlorite method (Weatherburn,
1967), basically as described by Zonia et al. (1995), except that
control reactions were also performed without urease.
Nucleic Acid Techniques
Genomic DNA was isolated from soybean leaves according to the
method of Dellaporta et al. (1983). For DNA analysis 8 µg of DNA was
digested overnight with EcoRl, BamHl,
XbaI, or EcoRV at 37°C, and then separated on a
1% agarose gel. DNA was blotted to a nitrocellulose membrane and
hybridized at high stringency in 50% (v/v) formamide, 100 µg
mL1 sonicated salmon-sperm DNA, 100 µg
mL1 yeast RNA, 5× Denhardt's solution (1×
Denhardt's is 0.02% Ficoll, 0.02% PVP, and 0.02% BSA), 50 mM NaPO4, pH 6.5, 5× SSC
(1× SSC is 0.15 M NaCl and 0.015 M sodium citrate), and 0.2% SDS at 42°C for 14 to 16 h. The filter was washed in 2× SSC, 0.2% SDS at room temperature for 15 min, followed by 0.2× SSC, 0.2% SDS at 65°C for
30 min. Low-stringency hybridization conditions were identical to
high-stringency conditions except that formamide was reduced to 25%.
The filter was washed in 2× SSC, 0.2% SDS at room temperature for 15 min, followed by a second wash in the same solution at 42°C for 15 min. The dried blot was exposed for 3 to 5 d to radiographic film
(X-Omat, Kodak) with intensifying screens at 70°C.
Total RNA was isolated from embryos or seedlings according to the
method of Murfett et al. (1994). For RNA analysis 8 µg of RNA was
separated in a 16.2% formaldehyde and 1% agarose gel and then
transferred to a nylon membrane. Before hybridization the membrane was
stained with 0.3 M sodium acetate, pH 5.2, containing 0.03% methylene blue to reveal RNA. Hybridization conditions and washes were identical to the high-stringency conditions used for DNA
analysis. The RNA blot was exposed to radiographic film as indicated
above.
Cotyledon Culture
Developing urease-positive (Eu3/Eu3 and
Eu3/eu3-e1) and -negative (eu3-e1/eu3-e1)
cotyledons (80-100 mg fresh weight), derived from selfed
Eu3/eu3-e1, were collected and their phenotype was identified by a seed-chip assay performed on excised embryonic axes
(Meyer-Bothling and Polacco, 1987). The testa and axes were aseptically
removed and each embryo was halved into separate cotyledons. Cotyledons
derived from the same embryo were used to compare different nitrogen
sources. Cotyledons were cultivated in 3 mL of culture medium as
described by Thompson et al. (1977), except that vitamins were at
half-strength, Gly was omitted, and 5.9 mM
K2SO4 was used as the
sulfur source.
A mock seed-coat exudate was made up according to the reported five
main amino acids in the in vivo soybean seed coat exudate and used to
provide amino acids to the cotyledon (Rainbird et al., 1984). The amino
acid mixture was 12.6 mM Gln, 4.5 mM Asn, 1.1 mM Ser, 0.7 mM His, and 0.6 mM Arg
(N = 40 mEq L1). Arg was provided as
the sole nitrogen source at either 10 mM (N = 40 mEq L1) or at 0.6 mM. In both cases the results with respect to
urea evolution, growth, and total protein after 6 d of culture
were identical. For cotyledon protein determination, the 3%
HClO4 pellet (see above) was boiled for 15 min in
1 mL of 1.5 N NaOH. The insoluble material was
removed by centrifugation and the solubilized protein was determined in
the supernatant by the Lowry method (Lowry et al., 1951).
In experiments with radiolabeled Arg, 1.1 × 106 dpm
L-[guanido-14C]Arg (51.5 Ci/mol,
DuPont NEN) were added to the culture medium. Treatment with Dowex 50 W-X8 columns separated [14C]urea in cotyledon
extracts and in the culture medium from unreacted L-[guanido-14C]Arg, as indicated by
Daghigh et al. (1994), and then measured by liquid scintillation
counting. The eluate was confirmed to be urea by its conversion to
14CO2 with added urease.
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RESULTS |
Arginase Sequence and Copy Number
Using a full-length Arabidopsis arginase cDNA (Krumpelman et
al., 1995) as a probe, we have previously reported (Goldraij et al.,
1998) the cloning of a 1324-bp cDNA fragment (AG1) encoding a soybean seedling arginase. Figure 1
shows a comparison of the deduced amino acid sequences of the two
enzymes. They are similar in size (36.5 and 38.6 kD for Arabidopsis and
soybean, respectively) and are 78% identical. The most notable
differences are in the N-terminal region. Considering that all plant
arginases reported so far are mitochondrial enzymes (Polacco and
Holland, 1993), this region constitutes a putative transit peptide. In
addition, both sequences have characteristics common to N-terminal
transit peptides, such as abundance of hydroxylated and positively
charged residues (Hartl et al., 1989).
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| Figure 1.
Sequence alignment of plant arginases. Deduced
amino acid sequence of soybean seedling arginase (Goldraij et al.,
1998) was compared with that of Arabidopsis arginase
(Krumpelman et al., 1995). Amino acids conserved between the two
proteins are highlighted. The N-terminal region, which exhibits the
weakest identity, is a putative transit peptide. Dashes are gaps for
optimization of alignments.
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To examine the copy number of the soybean arginase gene and the
presence of related genes, we used AG1 as a probe to perform DNA analysis. Under high-stringency conditions, genomic DNA digested with XbaI and EcoRI showed two and three bands,
respectively, whereas BamHI and EcoRV digestions
showed four or more bands (Fig. 2A). The
cloned AG1 had only one restriction site for
BamHI and EcoRV, indicating the presence of more
than one arginase gene and/or the presence of intron restriction sites
for the enzymes used in the analysis. Additional bands appeared in all
DNA digestions when the hybridization was performed at low stringency
(Fig. 2B), indicating the existence of more distantly related genes in
the soybean genome.
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| Figure 2.
DNA analysis of arginase genes in the soybean
genome. Autoradiogram of blot containing soybean genomic DNA (8 µg)
digested with XbaI (lanes X), BamHI
(lanes B), EcoRV (lanes EV), and EcoRI
(lanes EI). Hybridization was carried out under high- (A) and low- (B)
stringency conditions using radiolabeled AG1 cDNA as a
probe. Asterisks indicate additional bands not present under
high-stringency conditions. Numbers at left indicate marker sizes.
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Arginase Expression in Developing Embryos and in Germinating Seeds
Arginase expression was studied at the transcript level in
developing embryos and in germinating soybean seeds. Total RNA isolated
from immature embryos, quiescent seeds, and whole 0- to 6-DAG seedlings
was subjected to blot analysis and probed with AG1 (Fig.
3). Hybridization revealed a single band
migrating slightly faster than the 1.38-kb standard, which is
consistent with the size of the arginase cDNA. The transcript appeared
at 1 DAG and stayed at the same level at 2 DAG. It accumulated to
higher levels in the 3- to 5-DAG interval and then decreased slightly
by 6 DAG. In contrast, no transcript was seen in developing embryos or
in quiescent seeds, although the amount of RNA analyzed was nearly four
times greater. A nearly identical pattern was obtained for arginase
specific activity of the same samples used in the RNA blot experiment
(Fig. 4). Arginase activity was barely
detectable in developing embryos and in quiescent seed, but a sharp and
constant increase was seen from 2 DAG until reaching a maximum at 5 DAG. Therefore, the increase of arginase activity is consistent with an
increase in the transcript level, indicating that expression of the
arginase gene(s) is a developmentally controlled process coincident
with germination and seedling development.
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| Figure 3.
Soybean arginase expression in developing embryos
and in germinating seeds. A, Autoradiogram of blot containing total RNA
isolated from developing embryos (30 µg) and whole seedlings (8 µg). An embryo water content of 8% corresponds to quiescent seed.
Hybridization was carried out using radiolabeled AG1
cDNA as a probe. B, Uniformity of RNA loading and transfer was
confirmed by nylon membrane staining with methylene blue. Numbers at
left indicate marker sizes.
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| Figure 4.
Soybean arginase specific activity in developing
embryos and in germinating seeds. Arginase specific activity was
measured in developing embryos at the reserve deposition stage and in
germinating seeds 0 to 6 DAG. The values are averages of duplicate
reactions and are representative of two separate experiments.
Replicates were within 5% of the average values shown.
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The organ distribution of arginase expression and activity was
investigated in 3- to 4-DAG seedlings. The results showed that the
highest arginase specific activity was in the cotyledon, followed by
the hypocotyl and radicle, with only about 30% and 15% of the cotyledon specific activity, respectively (Fig.
5A). The arginase activity levels of the
different seedling organs roughly corresponded to their arginase
transcript levels (Fig. 5B). Because the total activity in the
cotyledon was about 50 times higher than in the radicle or hypocotyl,
we concluded that arginase was localized mainly in the cotyledons.
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| Figure 5.
Organ distribution of arginase expression and
specific activity in etiolated 3- to 4-DAG soybean seedlings. A,
Arginase specific activity. Results are means ± SD of two
separate experiments. B, Autoradiogram of blot containing total RNA (8 µg) isolated from the three organs. Hybridization was similar to that
in Figure 3. C, Uniformity of RNA loading and transfer was confirmed by
nylon membrane staining with methylene blue. Numbers at left indicate
marker sizes.
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The Arginase Reaction Is Inoperative in Developing Embryos
The arginase expression patterns (Figs. 3 and 4) suggest that the
role of arginase is largely confined to germination and seedling
development. We tested this suggestion by an in-vivo approach
exploiting mutant eu3-e1/eu3-e1, which lacks all urease activity (Meyer-Bothling et al., 1987). The quantity of urea
accumulated by the mutant is a useful indicator of in vivo arginase
activity (Stebbins and Polacco, 1995). Virtually identical, low levels of urea were found in developing embryos of the mutant and its urease-positive siblings (all progeny of selfed
Eu3/eu3-e1 plants) (Fig. 6).
The lack of urea accumulation in the mutant suggests that the
arginase-catalyzed reaction is inoperative in developing embryos.
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| Figure 6.
Urea accumulation in developing embryos and in
germinating seeds. Urea was measured in urease-positive
(Eu3/Eu3 or Eu3/eu3-e1) ( ) and
urease-negative (eu3-e1/eu3-e1) ( ) developing embryos
at the reserve deposition stage and in germinating seeds 0 to 6 DAG.
Each value represents the mean ± SD of two separate
experiments. Bars are shown only when they are bigger than the
symbol.
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In contrast, there was a sharp increase in urea upon germination of
urease-negative seeds, reaching a peak at 3 DAG. This pattern is
roughly in agreement with the pattern for arginase expression and
arginase specific activity, albeit with an earlier peak. Based on
studies using labeled precursors and an inhibitor (allopurinol) of
purine conversion to ureides (Stebbins and Polacco, 1995), we
demonstrated previously that the vast majority of urea generated in
soybean seedlings comes from Arg rather than from ureides. As expected,
much less urea accumulated in urease-positive seedlings, although the
levels were somewhat higher than previously reported.
Arginase Is Conditionally Operative in in Vitro-Cultured
Cotyledons
Developing soybean cotyledons were cultured in vitro in defined
media to evaluate Arg as a nitrogen source and to test for a functional
arginase using the criterion of urea accumulation in the
urease-deficient mutant eu3-e1/eu3-e1. We previously determined that Arg uptake was not altered in the urease-deficient mutant compared with the wild type (results not shown). Arg was supplied to immature cotyledons as the sole nitrogen source or included
in an amino acid mixture approximating the seed-coat exudate (Rainbird
et al., 1984) that nourishes the developing embryo (see ``Materials and Methods'').
In both the urease-positive seedlings and in the isogenic
urease-deficient sibling, the growth and total protein in
cotyledons cultured with Arg as the sole nitrogen source were almost
identical to values obtained in cotyledons cultured without nitrogen
(Fig. 7A), even though urea derived from
Arg accumulated in the urease-deficient mutant (Fig. 7B). Arg is the
precursor of urea when it is the sole nitrogen source, as confirmed by
the conversion of
L-[guanido-14C]Arg to
[14C]urea (Table
I). Therefore, the products derived from
Arg breakdown (urea and Orn) did not support growth and protein
deposition beyond developing cotyledons cultured without nitrogen.
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| Figure 7.
In vitro culture of urease-positive
(Eu3/Eu3 or Eu3/eu3-e1) and
urease-negative (eu3-e1/eu3-e1) immature cotyledons.
After detaching the two cotyledons from a given embryo, one was
cultivated on mock exudate (ME) or nitrogen-free medium (No N), whereas
the other cotyledon was cultivated on Arg. Fresh weight, total protein,
and total urea were determined in each cotyledon after 6 d of
culture. A, Fresh weight increase (gray bars) and total protein (white
bars) relative to values of paired cotyledon cultured on Arg,
established as 100%. B, Total urea accumulation in cotyledon and
culture medium. Results are means ± SD of three separate
experiments.
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Table I.
Total urea accumulation in cotyledons cultivated
with L-[guanido-14C]Arg
Results are averages ± SD of two different
experiments.
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The cotyledons used in the experiment shown in Figure 7 received 10 mM Arg. Identical results, including urea accumulation (data not shown), were obtained when Arg was reduced to 0.6 mM. With a mock seed-coat exudate (containing 0.6 mM Arg, see ``Materials and Methods'') as the nitrogen
source, growth and protein yield were increased about 40% to 50% in
both genotypes with respect to cotyledons cultured with Arg (Fig. 7A),
whereas no evolution of urea was detected in the urease-deficient
mutant (Fig. 7B). As expected, no urea accumulation was seen in
urease-positive cotyledons in any of the three treatments assayed. In
preliminary experiments, when L-[guanido-14C]Arg was provided in
a mock exudate, the label was rapidly incorporated into protein, as
previously reported (Micallef and Shelp, 1989c). Thus, in agreement
with the lack of Arg degradation by developing embryos in vivo (Fig.
6), there was no arginase-catalyzed degradation of Arg by cultured
cotyledons provided with a mock seed-coat exudate (Fig. 7).
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DISCUSSION |
We investigated the expression and functioning of soybean arginase
in developing embryos and in germinating seeds. We used the cDNA clone
AG1, which encodes a soybean seedling arginase (Goldraij et
al., 1998) that is 78% identical to that of Arabidopsis (Fig. 1), the
only other plant arginase whose sequence is currently available.
High-stringency genomic hybridization indicated that arginase may be a
member of a small gene family, whereas low-stringency conditions
indicated the presence of more distantly related homologs in soybean
(Fig. 2).
Arginase activity increased 20-fold upon germination to a maximum at 5 DAG, whereas RNA blots probed with AG1 revealed a transcript peak at 3 to 5 DAG. In contrast, arginase activity in developing embryos was basal and no transcript signal was detected at any time
during the reserve deposition stage. Therefore, the increased arginase
activity during germination and seedling development was most likely to
be due to de novo enzyme biosynthesis and not to activation of
pre-existing enzyme.
Germination as the signal for the synthesis of arginase is in agreement
with a catabolic role. Its substrate, Arg, is a major nitrogenous
storage compound in plants. For 379 species analyzed, Arg averaged 7.7 mol % of seed amino acids and 21.1% of total amino acid nitrogen, the
highest contribution of any amino acid (Van Etten et al., 1967; Polacco
and Holland, 1993). In soybean, Arg contains 18% of seed protein-bound
nitrogen (Micallef and Shelp, 1989a). Arg degradation by arginase
during germination is consistent with the sharp increase in urea in
urease-deficient mutant seedlings (Fig. 6; Stebbins and Polacco, 1995).
Urease has been proposed to function coordinately with arginase in the utilization of seed protein reserves during germination (Thompson, 1980). In Arabidopsis, urease functions to recycle urea nitrogen derived from Arg breakdown during seedling development (Zonia et al.,
1995).
The preponderance of arginase activity in seedling cotyledons (Fig. 5)
indicates that Arg degradation might occur mainly in the cotyledon,
prior to the delivery of its nitrogen to the growing points. This is in
agreement with observations in pumpkin cotyledons (Chou and
Splittstoesser, 1972), in which Arg constitutes 30% of amino acid
nitrogen but is only a minor component of the amino acid nitrogen
(1.7%) transported out of the cotyledon.
In contrast to the abundant arginase activity and urea reaction product
in developing seedlings, the enzyme does not appear to be active in
developing embryos. This conclusion is based mainly on the absence of
urea accumulation in urease-deficient developing embryos (Fig. 6). At
this stage arginase activity was extremely low but detectable. The
absence of any transcript detectable by the seedling axis clone
AG1 is consistent with either a lack of arginase transcript
in the developing embryo or with a poorly cross-hybridizing minor
arginase mRNA.
In vitro cotyledon culture was used to evaluate the capacity of Arg to
support growth and protein synthesis in immature soybean cotyledons. A
mixture of amino acids containing Arg and resembling the seed-coat
exudate (mock exudate) stimulated an increase in fresh weight and
protein deposition similar to that obtained with Gln alone (2.5 times
the initial fresh weight and 95 mg of protein per cotyledon after
6 d culture), which was previously shown to be the best nitrogen
source (Thompson et al., 1977). Increases in fresh weight and protein
supported by the mock exudate were not, however, accompanied by Arg
breakdown caused by arginase, because no urea accumulated in
urease-deficient cotyledons (Fig. 7).
The situation with Arg as the sole nitrogen source (at 0.6 or 10 mM) was exactly reversed. Arg did not support growth or
protein deposition in urease-positive or -negative cotyledons, but was actively broken down by arginase, as evidenced by urea accumulation in
the mutant. We observed no induction of arginase by Arg. The in vitro
arginase specific activity of cultured cotyledons (<11 nmol urea
min1 mg1 protein) was
lower than the specific activity observed in embryos in planta. Because
the other amino acids in the mock exudate appear to prevent arginase
action, they could prevent uptake of Arg into the cotyledon or into the
mitochondrion, the site of all or most plant arginases (Polacco and
Holland, 1993). Inhibition of uptake into the cotyledon appears
unlikely, because we observed no differences in Arg uptake whether it
was provided alone or included in the mock exudate. In the latter case,
Arg was abundantly incorporated (35% of total Arg uptake) into
perchloric-acid-precipitable material (A. Goldraij and J. Polacco,
unpublished results).
Other studies have examined the role of arginase in developing
cotyledons of pea (De Ruiter and Kollöfel, 1983) and soybean (Micallef and Shelp, 1989b, 1989c). In both species Arg was reported to
be actively incorporated into protein and was also the major constituent of the free amino acid pool. After injection of
L-[guanido-14C]Arg into excised
cotyledons, little urea accumulation or
14CO2 release was detected
in pea despite the in vitro arginase activity detected in developing
seeds. Contrary to our results, Micallef and Shelp (1989c) found that
approximately 20% of injected Arg was routed to urea and Orn in
soybean. Perhaps this activity was the result of arginase being
released from mitochondria by mechanical disruption caused by Arg
injection.
We propose that arginase is not operative in vivo in developing soybean
embryos. Although very low, in vitro arginase activity could be
detected and was sufficient to generate large amounts of urea when Arg
was the sole nitrogen source in cultured cotyledons. The presence of
arginase and a large free Arg pool without a reaction taking place
indicates that a regulatory mechanism impedes the reaction. Considering
that all plant arginases reported so far are mitochondrial or
particulate enzymes (for review, see Polacco and Holland, 1993), this
regulation may be achieved by different intracellular locations of
substrate and enzyme. A similar mechanism has been suggested for pea
(De Ruiter and Kollöfel, 1983).
The inoperative arginase in developing soybean embryos containing
abundant free Arg avoids a futile, energy-wasting urea cycle. Unimpeded
action of arginase in the developing embryo would result in a
functional urea cycle with a wasteful conversion of ammonia and
 -amino acid nitrogen to urea in the overall reaction of the Arg/urea
cycle:
Urea is then reassimilated by urease, which is active in
developing cotyledons:
The combined action of urease and an Arg/urea cycle would lead to
a very expensive deamination of -amino acids:
Nature uses a variety of strategies to avoid arginase engaging in
a futile cycle with Arg biosynthesis in tissues/organisms lacking a
functional urea cycle (Vissers et al., 1986; Jenkinson et al., 1996).
In developing embryos of soybean, we propose that the low activity of
arginase, its mitochondrial location, or both, keeps biosynthetic and
catabolic phases of the urea cycle spatially and/or temporally
separated. The identification of the putative Arg transporter in
mitochondrial membrane and the time in which it becomes active may be
the key toward a more thorough understanding of Arg metabolism in
soybean seeds.
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FOOTNOTES |
1
This work was supported by the Missouri
Agricultural Experimental Station and by the U.S. Department of
Agriculture (grant no. 97-35 305-4629 to J.C.P.). A.G. was supported by
a postdoctoral fellowship from the Interdisciplinary Plant Group. This
is journal contribution no. 12,804 from the Missouri Agricultural
Experimental Station.
*
Corresponding author; e-mail polaccoj{at}missouri.edu; fax
1-573-882-5635.
Received July 20, 1998;
accepted October 12, 1998.
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ABBREVIATIONS |
Abbreviation:
DAG, days after germination.
| |
ACKNOWLEDGMENTS |
We thank Dale Blevins and Krystyna Lukaszewska for critical
reading of the manuscript.
| |
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