Download Natural abundance of 15N in amino acids and

Journal of Experimental Botany, Vol. 49, No. 320, pp. 521–526, March 1998
Natural abundance of 15N in amino acids and polyamines
from leguminous nodules: unique 15N enrichment in
homospermidine
T. Yoneyama1,3, S. Fujihara1 and K. Yagi2
1 Plant Nutrition Diagnosis Laboratory, National Agriculture Research Center (NARC),
Kannondai 3–1-1,Tsukuba, Ibaraki 305, Japan
2 National Institute of Agro-Environmental Sciences, Kannondai 2–1-1, Tsukuba, Ibaraki 305, Japan
Received 30 May 1997; Accepted 5 November 1997
Abstract
The natural 15N abundance (d15N value) in acetylpropyl
derivatives of amino acids and in ethyloxycarbonyl
derivatives of polyamines was determined using a
gas chromatography/combustion/mass spectrometer
(GC/C/MS). d15N values determined for 12 amino acids
and five polyamines by GC/C/MS were identical to
those obtained by a direct combustion method using
an automatic nitrogen and carbon analysis (ANCA)
mass spectrometer, the difference being less than
±1.0‰ in most cases. The GC/C/MS method was used
to analyse d15N values in the amino acids and polyamines from root nodules of pea and faba bean and from
stem nodules of Sesbania rostrata. The analysis of
d15N values revealed that homospermidine had high
d15N values, as much as +40‰, while the amino
acids investigated had d15N values between −3 and
+6‰, putrescine between +2 and +8‰, cadaverine
between +1 and +7‰, spermidine between −2 and
+4‰, and spermine between 0 and +6‰. The
mechanism of 15N enrichment in homospermidine is
discussed.
Key words: Amino acid, legumes, natural abundance of
15N (d15N), nodules, polyamines.
Introduction
Leguminous plants are able to fix atmospheric nitrogen
by symbiosis with rhizobia (Rhizobium, Bradyrhizobium
and Azorhizobium) located in root or stem nodules.
Isotopic fractionation of 15N/14N during nitrogen fixation
by leguminous plants was small (−0.2 to −2‰) when
whole plant N was considered ( Yoneyama et al., 1986).
However, the analysis of natural 15N abundance in the
different tissues of leguminous plants has revealed that
nodules, in some instances, had very high d15N values,
while other plant tissues only showed differences of about
2‰ (Shearer et al., 1982; Yoneyama et al., 1986; Unkovich
et al., 1994). This 15N enrichment in nodules was first
recognized in legumes which export fixed N as ureides
from the nodules to host plants (Shearer et al., 1982).
However, further investigation showed that also in some
amide-exporting legumes the nodules are enriched in
15N compared to other plant tissues ( Yoneyama, 1988;
Unkovich et al., 1994). In the ureide-exporting nodules,
the bacteroids were the most 15N-enriched N fraction,
while the cytosol fraction was more 15N-enriched
than the bacteroids in the amide-exporting nodules
( Yoneyama, 1988; Yoneyama et al., 1991a). Of the chemical fractions investigated, the polyamine-containing fraction, which was eluted by 2–6 N HCl solution from a
cation exchange column (Dowex 50 W, H+ form), was
enriched in 15N ( Yoneyama, 1988).
The polyamine content in nodules from both amideand ureide-exporting species has been investigated. In
both types of nodules, the major polyamines found were
put, cad, spd, homospd, and spm (Fujihara et al., 1994).
Homospd is the major polyamine found in cultured
rhizobia (Fujihara and Yoneyama, 1993). Since the roots
of legumes do not contain homospd, homospd in the
3 To whom correspondence should be addressed. Fax: +81 298 38 8837. E-mail: [email protected]
Abbreviations: AP, acetylpropyl; EOC, ethyloxycarbonyl; Agm, agmatine; Put, putrescine; Cad, cadaverine; Spd, spermidine; Spm, spermine; Homospd,
sym-homospermidine; GC/C/MS, gas chromatography/combustion/mass spectrometry; ANCA, automatic nitrogen and carbon analysis.
© Oxford University Press 1998
522
Yoneyama et al.
nodules may be produced in the bacteroids, in which
homospd synthase activity was detected (Abe, 1994). In
this communication, d15N values of individual amino
acids and polyamines in the nodules from four legumes
as determined by GC/C/MS are presented. Among them,
homospd was specifically enriched in 15N.
Materials and methods
Nodules and rhizobia
Two cultivars (Nimura and Shokusen) of pea (Pisum sativum
L.) and one cultivar (Issun) of faba bean (Vicia faba L.) were
grown in a field at NARC, Tsukuba, Japan and their root
nodules were harvested, washed with distilled water and stored
in a freezer until used as described by Yoneyama et al. (1991a).
Sesbania rostrata was grown in a paddy field of NARC as
described by Yoneyama et al. (1991b), and the stem nodules
were harvested for analysis. Bradyrhizobium japonicum A1017
and Rhizobium fredii P220 were cultured as described by
Fujihara and Yoneyama (1993), and collected by centrifugation
for use in the experiments.
Isolation of polyamines from nodules and their derivatization
Homogenization of 2 g frozen nodules in 10 ml of 0.5 M HClO
4
for 10 min was followed by centrifugation at 10 000 rpm for
15 min at temperatures below 4 °C. To the supernatant 10 M
KOH was added until it reached pH 7. KClO was removed by
4
centrifugation. The supernatant thus obtained was put on a
cation exchange resin column (Dowex 50 W, 200–400 mesh,
H+ form). The eluate with 0.5 N HCl was discarded, and the
subsequent eluate with 6 N HCl was identified as the polyamine
fraction. Analysis with GC showed that put, cad, spd, homospd,
and spm were included in this fraction. The polyamine fraction
was evaporated to dryness in a rotary evaporator at 50 °C in
vacuo, and the residue was dissolved in distilled water to a
volume of 1 ml. The polyamines were made volatile by
conversion to EOC derivatives by reaction with ethyl chloroformate as described by Yamamoto et al. (1982). To the
supernatant solution, containing 100–200 nmol of each polyamine, 0.5 ml of 100 g l−1 NaOH and 0.2 ml ethyl chloroformate
were added, and it was shaken for 30 min at room temperature.
Addition of 3 ml diethyl ether caused the polyamine-derivatives
to move to the ethereal layer, and this was repeated three times.
The combined extracts were evaporated to dryness at 50 °C
under a N flow. After it had completely dried the residue was
2
dissolved in 60 ml ethyl acetate with addition of anhydrous
Na SO to remove contaminating water.
2 4
Isolation of free amino acids from nodules and their derivatization
Homogenization of 2 g frozen nodules in 10 ml of 80% (w/v)
ethanol for 10 min was followed by centrifugation at 10 000 rpm
for 15 min at temperatures below 4 °C. Ethanol in the
supernatant was evaporated in a rotary evaporator at 40 °C,
and the residue was put on a cation exchange resin (Dowex
50 W, 200–400 mesh, H+ form) column. The column was
washed with distilled water, and then eluted with 0.5 N HCl.
That this eluate contained the amino acids was confirmed by
GC analysis. The amino acids were made volatile by conversion
to AP derivatives by reaction with n-propanol and acetyl
chloride as described by Merritt and Hayes (1994). 0.3 ml
acidified n-propanol (produced by dropping acetyl chloride (5
vols) into n-propanol (20 vols) on ice) was added to the 0.5 N
HCl eluate containing approximately 200 nmol of each amino
acid. After cooling on ice, the acidified n-propanol was
evaporated at 110 °C under a N stream, and the residue was
2
again cooled on ice. One ml of acetyl chloride/dichloromethane
(151, v/v) was added, heated at 90 °C for 30 min, and thereafter
cooled on ice. By heating at 90 °C under N , the remaining
2
acetyl chloride and dichloromethane were removed. The residue
(derivatives) was dissolved in 60 ml ethyl acetate.
Isolation of amino acids and polyamines from rhizobia and their
derivatization
After centrifugation of each of the two rhizobial cultures, 4%
(w/v) HClO was added to the pellet and left overnight at 4 °C.
4
The supernatant obtained by centrifugation was neutralized by
addition of 10 M KOH, and centrifuged again. The neutralized
supernatant was put on a Dowex 50 W column, and washed by
distilled water. By elution with 0.5 N HCl, the amino acid
fraction was obtained, and by further elution with 6 N HCl,
the polyamine fraction was obtained. The derivatization of
polyamines and amino acids were conducted as described above
for nodules samples.
Analysis of d15N in derivatives by GC/C/MS
For measurement of d15N values of polyamines and amino
acids, a GC/C/MS method was employed, essentially as
described by Merritt and Hayes (1994) for determination of
d15N values in amino acids. The separation of individual
derivatives of polyamines and amino acids was performed using
a Hewlett Packard 5890 gas chromatograph with capillary
columns of Hewlett Packard Ultra 1 (cross-linked methyl
silicone gum, 25 m×0.32 mm×0.17 mm film thickness) or
Shimadzu CB1-S25 (Methylsilicone 25 m×0.32 mm×0.50 mm).
Both were non-polar and gave similar separation of derivatives.
Injection temperature was 285 °C. The temperature of the
capillary column was raised from 140 to 280 °C at the rate of
8 °C min−1 for EOC-derivatives (polyamines) and from 100 to
260 °C at the rate of 6 °C min−1 for AP-derivatives (amino
acids). Helium gas was used as carrier gas (1 ml min−1) at
10 psi as the head pressure. The combustion was done by an
oxidation column of NiO/Pt/CuO at 960 °C and the reduction
column of Cu was kept at 600 °C Standard N gas (−4.3‰)
2
was injected at the start, the end and also occasionally between
peaks. A Finnigan Mat 252 mass spectrometer was used to
monitor the intensity of mass 28, 29, and 30, and the outputs
of the mass 28 peak and the ratio of 29/28 were obtained.
Analysis of d15N by ANCA mass spectrometer
The d15N values of 12 authentic amino acids and five authentic
polyamines, and amino acids and polyamine fractions from
nodules were analysed with an on-line ANCA-SL mass
spectrometer (Europa Scientific, Crewe, UK ). All the d15N
values of the samples were expressed as permil (‰) deviation
from that of the atmospheric dinitrogen (standard ):
d15N=[(15N/14N )
/(15N/14N )
−1]×103 .
sample
standard
Results and discussion
d15N determination of authentic amino acids and
polyamines
The d15N values of 12 amino acids and five polyamines
determined by GC/C/MS were compared to those measured by on-line ANCA-SL mass spectrometer ( Table 1).
d15N of polyamines from nodules 523
Table 1. The d15N values of amino acids and polyamines
determined by ANCA-SL and GC/C/MS
Amino acid/polyamine
Glycine
Serine
-Valine
Leucine
Isoleucine
Threonine
Proline
Aspartic acid
Glutamic acid
-Arginine
Histidine
-Lysine
Putrescine
Cadaverine
Spermidine
Homospermidine
Spermine
d15N (‰)
ANCA-SL
GC/C/MS
+1.9±0.1
+8.2±0.2
+9.5±0.2
+0.7±0.3
−1.6±0.5
−3.5±0.1
−5.1±0.1
−1.9±0.1
−3.8±0.1
−2.3±0.2
−3.0±0.1
−0.5±0.1
−8.7±0.3
−3.4±0.2
−0.7±0.3
−5.0±0.2
+0.5±0.2
+1.3±0.8
+9.2±0.7
+10.2±1.5
+0.8±0.7
−0.6±0.5
−3.5±0.3
−4.8±0.8
−1.8±0.7
−3.5
−4.6
−2.6
−1.5
−8.9±0.8
−3.3±0.9
−1.3±0.5
−4.7±0.8
−1.6±0.6
Data are means ±SD of 2–3 analyses, except for the GC/C/MS data
of glutamic acid, -arginine, histidine and -lysine which were
single analyses.
The differences in d15N values between the two methods
were within 1‰, except for -arginine and spermine,
whose d15N values measured by GC/C/MS were 2‰
lower than those by ANCA-SL. Previous analysis of d15N
values in nine amino acids (alanine, glycine, leucine,
norleucine, serine, proline, aspartic acid, hydroxyproline,
and phenylalanine) by GC/C/MS also gave identical
results as those by conventional ratio mass spectrometer
(Merritt and Hayes, 1994). The variations of d15N values
in amino acids and polyamines from biological samples
reported by Gaebler et al. (1963) and Minagawa et al.
(1992) and in this report were larger than the variation
caused by GC/C/MS analysis. Therefore, it is considered
that GC/C/MS is very valuable for the analysis of d15N
values in individual N-containing compounds, using volatile derivatives.
d15N values of amino acid and polyamine fractions
The d15N values in amino acid and polyamine fractions
from nodules are shown in Table 2. The d15N values in
the amino acid fraction were close to the reference (atmo-
spheric N ), while those in the polyamine fraction were
2
higher than the reference value. In particular, the amounts
of polyamine N in the nodules from pea cv. Nimura and
in faba bean cv. Issun were small, but their d15N values
were extremely high.
Previous analysis ( Yoneyama et al., 1991a, b) showed
that the total N contents of nodules were 5–7 mg g−1
FW, and their d15N values were −0.5‰ in pea cv.
Nimura, +1.0‰ in pea cv. Shokusen, +6.1‰ in faba
bean cv. Issun, and +8.1‰ in Sesbania rostrata used in
the present investigation. When the nodules were fractionated into the plant cytosol, bacteroids, and nodule residue,
the plant cytosol (in particular its soluble N ) showed
slight enrichment in 15N in cv. Nimura and cv. Shokusen
nodules, and the bacteroids and plant cytosol (in particular its soluble N ) had high d15N values in faba bean and
Sesbania rostrata nodules. The proportion of the N from
whole nodules found in the polyamine fraction was: 1.7%
in pea cv. Nimura, 2.9% in pea cv. Shokusen, 1.2% in
faba bean cv. Issun, and 4.2% in Sesbania rostrata. The
contribution of d15N by the polyamine fractions was
around 0.2‰ in the whole nodules, indicating that other
nitrogenous compounds contribute significantly to the
increase in d15N of nodules like in those from faba bean
and Sesbania rostrata in this investigation.
d15N values of amino acids
In Table 3, the d15N values of major N peaks of amino
2
acids from nodules and cultured rhizobia on GC/C/MS
are shown. In nodules, aspartic acid (including aspartic
acid from asparagine) was the most abundant amino acid
followed by glutamic acid (including glutamic acid from
glutamine) ( Kouchi and Yoneyama, 1986). In contrast,
glutamic acid was present in the largest amount in the
rhizobial cells (Fujihara and Yoneyama, 1993). The variation in d15N values among amino acids in nodules was
between −3 and +6‰ and that in rhizobia was between
−2 and +8‰. The relatively high d15N values in arginine
from pea and faba bean nodules may suggest 15N enrichment of the guanidino in this molecule as reported by
Medina and Schmidt (1982). Separation of some amino
acids was not so clearcut as that of polyamines, due to
the fact that many amino acids were present in small
Table 2. The N contents and d15N values of amino acid and polyamine fractions from nodules
Nodules
Pea (Pisum sativum) cv. Nimura
Pea (Pisum sativum) cv. Shokusen
Faba bean (Vicia faba) cv. Issun
Sesbania rostrataa
aStem nodules.
Amino acids
Polyamines
N
( mg g−1 FW )
d15N
(‰)
N
( mg g−1 FW )
d15N
(‰)
983
620
520
233
−1.1
−1.5
+0.7
+0.2
125
209
69
270
+14.1
+3.7
+10.2
+5.7
524
Yoneyama et al.
Table 3. The d15N values of amino acids from nodules of four legume species and two strains of rhizobia
d15N (‰)
Amino acid
Pea
cv. Nimura
Pea
cv. Shokusen
−2.7±0.1
+4.4
Glycine, alanine, serine
Valine
c-Amino butyric acid
Threonine
Proline
Aspartic acida
Glutamic acidb
Arginine
−3.2±0.6
+4.0±2.0
−3.1±2.0
+5.0
+0.9±0.1
−1.6±0.8
+2.6±0.9
−1.2±0.3
Faba bean
cv. Issun
+2.5
+3.2
+1.6
−1.8
+2.1±0.6
+0.6±0.2
+6.1
S. rostrata
+1.9±0.1
+1.6±1.3
−2.1±0.9
−2.0
+0.8
B. japonicum
A1017
R. fredii
P220
+5.3±0.9
+2.0
+2.0
+5.8±0.8
+5.8±2.3
+8.1±0.6
+4.9±1.3
−0.9±0.2
+5.7±0.5
−1.7±0.2
+2.3
+5.5±1.2
+5.0±1.6
+6.9±1.2
+4.0±0.6
Data are means ±SD of 3–4 analyses.
aAspartic acid from aspartic acid and asparagine.
bGlutamic acid from glutamic acid and glutamine.
quantities in the nodules and their peak heights were
lower, and more variable than those of neighboring amino
acids present in larger amounts. The largest peaks
(aspartic acid in nodules and glutamic acid in rhizobia)
gave consistent d15N values in repeated analysis. However,
further separation of the less abundant amino acids is
required.
15N enrichment in polyamines
This study indicates that the alkaline fraction, eluted by
6 N HCl, had much higher d15N values than the amino
acid fraction ( Table 2). Separation into individual polyamines ( Table 4) revealed that homospd was especially
15N-enriched, with d15N values as high as 40‰. The d15N
values of put were between +2 and 8‰, those in cad
were between +1 and +7‰, those in spd were between
−2 and +4‰ and those in spm were between 0 to +6‰.
However, homospd from rhizobia was not much enriched
in15N, compared to put ( Table 4) and amino acids
( Table 3) from rhizobia.
Homospd is produced in the bacteroids by homospermidine synthase encoded by the rhizobia (Abe, 1994;
Fujihara et al., 1995). It is very likely that homospd
produced in the bacteroids is highly enriched in d15N and
that this homospd is largely retained in the bacteroids in
soybean (Glycine max), adzuki bean (Vigna angularis),
Sesbania rostrata and faba bean, while the homospd
produced in the bacteroids of pea may easily leak out of
the bacteroids. This may result in 15N enrichment of the
bacteroids in the former nodules and of the soluble N in
the latter nodules ( Yoneyama et al. 1991a, b). In fact,
the polyamine fractions in the cytosol were slightly
enriched, while those fractions from the bacteroids in
adzuki bean and soybean nodules were highly enriched
in 15N ( Yoneyama, 1988). The 15N enrichment in the
soluble N of the cytosol in pea and faba bean nodules
was more obvious in mature and senescent nodules than
in young nodules ( Yoneyama et al., 1991b). 15N-enriched
homospd might be more released to the plant cytosol in
aged nodules.
Possible processes resulting in 15N enrichment of homospd
Put is synthesized from ornithine by ornithine decarboxylase or from arginine by three enzymes, arginine
decarboxylase, agm iminohydrolase and N-carbomoylputrescine amidohydrolase ( Tiburcio et al., 1990). Cad is
synthesized from lysine decarboxylase. Spd is synthesized
from put and decarboxylated S-adenosylmethionine by
spd synthase, while homospd is synthesized from two
molecules of put by homospd synthase with the reduction
of NAD ( Tait, 1985). The catalytic reaction of homospd
synthase consists of the following three processes: (1)
oxidation of put to form 4-aminobutyraldehyde (a deamination reaction), (2) Schiff base formation between put
Table 4. The d15N values in polyamines from nodules of four legume species and two strains of rhizobia
Polyamine
Putrescine
Cadaverine
Spermidine
Homospermidine
Spermine
d15N (‰)
Pea
cv. Nimura
Pea
cv. Shokusen
Faba bean
cv. Issun
S. rostrata
B. japonicum
A1017
R. fredii
P220
+2.4±0.5
+1.4±0.3
−2.4±0.2
+39.8±2.7
+2.0±1.7
+4.8±2.2
+3.6±1.3
−2.4±1.3
+40.1±2.5
+0.4±0.4
+8.2+0.9
+9.3±3.1
+4.4±0.5
+39.1±2.6
+6.1±2.4
+3.8±1.1
+1.9±1.1
−2.4±1.4
+38.9±2.9
−0.1±0.7
+5.6±1.3
+7.8±2.2
+8.4±1.9
+7.0±1.6
Data are means ±SD of 3–4 analyses.
d15N of polyamines from nodules 525
and 4-aminobutyraldehyde, and (3) reduction of the
intermediate to form the final product homospd using
the NADH produced by process (1).
The exceedingly high enrichment of d15N in homospd
molecules separated from nodules and the contrasting
small enrichment of those from rhizobia ( Table 4) suggest
the possibility that reactions to form homospd in nodule
bacteroids may include the processes resulting in higher
15N enrichment in homospd than in rhizobia growing in
the synthetic liquid media. Both put in rhizobia and in
bacteroids may be synthesized via the ornithine pathway
and/or from arginine by the pathway as has been reported
in most bacteria ( Tabor and Tabor, 1985). Although the
arginine pathways includes two reactions liberating
ammonia, which may result in 15N enrichment in the
precursor substances, the guanidino N does not remain
in the put molecule. As the source of ornithine in the
bacteroids, glutamic acid transferred from the cytosol
( Kouchi et al., 1991) is possible. In the bacteroids, a part
of the glutamic acid may be deaminated (releasing ammonia), to form a-keto glutaric acid as the source of respiration. The remaining glutamic acid (source of ornithine)
may be enriched in d15N. However, the d15N values of
glutamic acid were not high, when whole nodules were
analysed ( Table 3). Separate analysis of d15N of glutamic
acid from the cytosol and bacteroids is necessary.
Homospd synthesis from put includes ammonia liberation
to form 4-aminobutyraldehyde; This process may result
in 15N enrichment of the remaining put. In the nodules,
the d15N values of put ( Table 4) were higher than those
of glutamic acid, a possible precursor for ornithine and
then put ( Table 3). Put degradation by amine oxidase,
which releases ammonia ( Tiburcio et al., 1990; Ozawa
et al., 1997) is another step leading to 15N enrichment in
the remaining put. These branched reactions may increase
the 15N content in the final substrate (put) to synthesize
homospd. d15N values of put were relatively higher than
those of plant-produced polyamines (cad, spd, and spm),
but not as much as those of homospd in the nodules
( Table 4). Put is produced both in plant cytosol and
bacteroids; Separate analysis of d15N of this molecule is
also necessary. Predominant formation of homospd in
bacteroids (Abe, 1994) from the put thus enriched in 15N
may result in 15N enrichment. Synthesis and retention of
homospd in rhizobia last a short period, whereas those
in bacteroids may take a long period. Under such conditions, homospd in the nodules might be metabolized
further releasing ammonia like in oxidative deamination
of spd (Smith, 1985). Recently homospd metabolites were
identified in various legume nodules (Fujihara et al.,
1996). The homospd metabolite, which contains unsaturated bonds, was not detected in cultured rhizobia. It is
not yet known whether homospd is deaminated in nodules, although activity of spd oxidase was found in
soybean nodules (Ozawa et al., 1997). The deamination
of homospd may result in further 15N enrichment of the
remaining homospd. To verify which of the above mentioned processes is the key step resulting in 15N enrichment
of bacteroid homospd, it is necessary to clarify the
biosynthetic and biodegradative pathways of polyamines
in rhizobia and in nodule bacteroids together with a
detailed analysis of amino acids and polyamines isolated
separately from the plant cytosol and bacteroids.
Acknowledgements
We thank Mr M Ohori of Finnigan Mat Instrument Ink
(Japan) for his guidance of operation of GC/C/MS, Mr M
Okamura for his help in the preparation of rhizobial samples,
Dr W Engelaar for his careful reading of the manuscript and
Ms J Terakado for typing the manuscript. This study was
supported by a research grant from the Bio-Media Program of
the Ministry of Agriculture, Forestry and Fisheries of Japan
(BMP 97-V-1–3-9).
References
Abe H. 1994. Polyamines in leguminous nodules with special
reference to homospermidine synthesis. Master’s thesis,
University of Tsukuba, Ibaraki, Japan.
Fujihara S, Abe H, Minagawa Y, Akao S, Yoneyama T. 1994.
Polyamines in nodules from various plant-microbe symbiotic
associations. Plant and Cell Physiology 35, 1127–34.
Fujihara S, Abe H, Yoneyama T. 1995. A new polyamine
4-aminobutylcadaverine. Occurrence and its biosynthesis in
root nodules of adzuki bean plant Vigna angularis. Journal
of Biological Chemistry 270, 9932–8.
Fujihara S, Takenaka M, Yoneyama T. 1996. Occurrence of
unsaturated polyamines in legume root nodules. International
Symposium on Polyamines (Tokyo), Abstracts 122–3.
Fujihara S, Yoneyama T. 1993. Effect of pH and osmotic stress
on cellular polyamine contents in the soybean rhizobia
Rhizobium fredii P220 and Bradyrhizobium japonicum A1017.
Applied and Environmental Microbiology 59, 1104–9.
Gaebler OH, Choitz HC, Vitti TG, Vukmirovich R. 1963.
Significance of N15 excess in nitrogenous compounds of
biological origin. Canadian Journal of Biochemistry and
Physiology 41, 1089–97.
Kouchi H, Fukai K, Kihara A. 1991. Metabolism of glutamate
and aspartate in bacteroids isolated from soybean root
nodules. Journal of General Microbiology 137, 2901–10.
Kouchi H, Yoneyama T. 1986. Metabolism of [13C ]-labelled
photosynthate in plant cytosol and bacteroids of root nodules
of Glycine max. Physiologia Plantarum 68, 238–44.
Medina R, Schmidt H-L. 1982. Nitrogen isotope ratio variations
in biological material, indicator for metabolic correlations?
In: Schmidt H-L, Förstel H, Heinzinger K, eds. Stable
isotopes. Amsterdam, Elsevier Scientific Publishing
Company, 465–73.
Merritt DA, Hayes JM. 1994. Nitrogen isotopic analysis by
isotope-ratio-monitoring gas chromatography/mass spectrometry. Journal of American Society of Mass Spectrometry
5, 387–97.
Minagawa M, Egawa S, Kabaya Y, Karasawa-Tsuru K.1992.
Carbon and nitrogen isotope analysis for amino acids from
biological sample. Mass Spectroscopy 40, 47–56.
526
Yoneyama et al.
Ozawa T, Kato S, Sanai K, Sawamoto M. 1997. Polyamine as
a negative regulatory substance in defence responses of
soybean against the infection and nitrogen fixation by
Bradyrhizobium japonicum. In: Ando T, Fujita K, Mae T,
Matsumoto, Mori S, Sekiya J, eds. Plant nutrition—for
sustainable food production and environment. Tokyo: Kluwer
Academic Publishers, 711–12.
Shearer G, Feldman L, Bryan BA, Skeeters JL, Kohl DH,
Amarger N, Mariotti F, Mariotti A. 1982.15N abundance of
nodules as an indicator of N metabolism in N -fixing plants.
2
Plant Physiology 70, 465–8.
Smith TH. 1985. The di- and poly-amine oxidases of higher
plants. Biochemical Society Transactions 13, 319–22.
Tabor CW, Tabor H. 1985. Polyamines in microorganisms.
Microbial Reviews 49, 81–99.
Tait GH. 1985. Bacterial polyamines; structures and biosynthesis. Biochemical Society Transactions 13, 316–18.
Tiburcio AF, Kaur-Sawhney R, Galston AW. 1990. Polyamine
metabolism. In: Miflin BJ, Lea PJ, eds. Intermediary nitrogen
metabolism. New York: Academic Press, 283–325.
Unkovich MJ, Pate JS, Sanford P, Armstrong EL. 1994.
Potential precision of the d15N natural abundance method in
field estimates of nitrogen fixation by crop and pasture
legumes in south-west Australia. Australian Journal of
Agricultural Research 45, 119–32.
Yamamoto S, Itano H, Kataoka K, Makita M. 1982. Gas-liquid
chromatographic method for analysis of di- and polyamines
in foods. Journal of Agricultural and Food Chemistry 30, 435–9.
Yoneyama T. 1988. Natural abundance of 15N in root nodules
of pea and broad bean. Journal of Plant Physiology
132, 59–62.
Yoneyama T, Fujita K, Yoshida T, Matsumoto T, Kambayashi
I, Yazaki J. 1986. Variation in natural abundance of 15N
among plant parts and 15N/14N fractionation during N
2
fixation in the legume-rhizobia symbiotic system. Plant and
Cell Physiology 27, 791–9.
Yoneyama T, Uchiyama T, Sasakawa H, Gamo T, Ladha JK,
Watanabe I. 1991b. Nitrogen accumulation and changes in
natural 15N abundance in the tissues of legumes with emphasis
on N fixation by stem-nodulating plants in upland and
2
paddy fields. Soil Science and Plant Nutrition 37, 75–82.
Yoneyama T, Uchiyama T, Yazaki J. 1991a. Ontogenic change
of nitrogen accumulation and natural 15N abundance in pea
and faba bean with special reference to estimate of N
2
fixation and 15N enrichment of nodules. Mass Spectroscopy
39, 267–76.