Content uploaded by Richard M Higashi
Author content
All content in this area was uploaded by Richard M Higashi on Jan 19, 2015
Content may be subject to copyright.
Journal
of
Experimental Botany, Vol. 48, No. 314,
pp.
1655-1666, September 1997
Journal of
Experimental
Botany
Anaerobic nitrate and ammonium metabolism
in
flood-tolerant rice coleoptiles
Teresa W-M.
Fan15,
Richard M. Higashi2, Thomas A. Frenkiel3 and Andrew N. Lane4
1
Department
of
Land,
Air
and Water Resources, University
of
California, Davis, CA 95616,
USA
2
Crocker Nuclear Laboratory, University
of
California, Davis, CA 95616,
USA
3
MRC Biomedical NMR Centre, National Institute
for
Medical Research, The Ridgeway, Mill
Hill,
London
NW71AA,
UK
4
Division
of
Molecular Structure, National Institute
for
Medical Research, The Ridgeway, Mill
Hill,
London
NW71AA,
UK
Received
12
February 1997; Accepted
14 May
1997
Abstract
The tolerance
of
germinating rice seedlings
to
anaero-
biosis cannot
be
fully accounted
for by
ethanolic
fer-
mentation alone. Nitrate metabolism (nitrate reduction
to
NH4+
plus
its
subsequent assimilation)
may
provide
an additional sink mechanism
for
excess protons
and
NADH produced during anaerobiosis.
To
follow the fate
of nitrate, 15N-labelled nitrate
and
ammonium incorp-
oration
in
aerobic
and
anaerobic rice coleoptiles
was
examined using 18N-edited
1H NMR and gas
chromato-
graphy-mass spectrometry methods. After
22
h of
treatments, protein-free
Ala, Glu, Gin,
and
y-amino-
butyrate were
the
main 1SN-Iabelled products
for
both
nitrate
and
ammonium-treated anaerobic rice coleop-
tiles,
with
Gin, Glu, and Ala
being
the
most enriched.
The total amount
of 15N
label incorporation into
Ala
and GAB increased significantly
in
response
to
anaero-
biosis.
The
1SN-Iabelling pattern
of
Glu and
Gin
sug-
gests that
the
GS/GOGAT system
was
primarily
involved
in
ammonium assimilation whereas Glu dehy-
drogenase
may
play
a
role
in
nitrate assimilation.
1SN
incorporation into protein-derived amino acids
was
also significant and
was
more substantial
in
anaerobic
than
in
aerobic rice coleoptiles, which indicate that
protein biosynthesis remained active
in
anaerobic rice
coleoptiles. Thus, anaerobic assimilation
of
inorganic
N into amino acids, particularly
Ala and
Glu/GAB,
may serve
to
supplement ethanolic fermentation
in
sustaining glycolysis
and
energy production
in
rice
coleoptiles.
Key words:
15N
tracer, nitrate metabolism, ammonium
assimilation, anaerobiosis, rice.
Introduction
When aerobic organisms are deprived of O2, mitochon-
drial oxidative phosphorylation is inhibited while anaer-
obic glycolysis to fermentation pathways become
activated, leading to the accumulation of products such
as ethanol, lactate, Ala, succinate, y-aminobutyrate
(GAB),
glycerol, and malate (Crawford, 1978; Davies,
1980).
The concerted activity of glycolysis and fermenta-
tion allows limited ATP synthesis to continue by sub-
strate-level phosphorylation while regenerating NAD4"
and removing excess protons. The importance of ethanolic
fermentation is that it is more efficient on a molar basis
than lactic fermentation in regenerating NAD+ and con-
suming protons. Hence, organisms capable of ethanolic
fermentation (e.g. plants, microbes, invertebrates, some
fishes) are more tolerant of prolonged O2 deficiency than
those dependent on lactic fermentation (e.g. mammals).
Nevertheless, ethanolic fermentation alone is inadequate
to meet the normal metabolic demands of aerobic organ-
isms.
Eventually, NADH build-up, cytoplasmic acidosis,
sTo whom correspondence should
be
addressed. Fax:
+1
916 752 1552. E-mail: twfan©ucdavis.edu.
Abbreviations:
GAB,
y-aminobutyrate; GC-MS,
gas
chromatography-mass spectrometry;
PCA,
perchloric
acid;
MTBSTFA, AV-methyl-N-[tert-
butyldimethylsilylltnfluoroacetamide; TOCSY, total shift correlation spectroscopy; HSQC, heteronuclear single quantum coherence;
DSS,
2-2'-
dimethylsilapentane-5-sulphonate; GS/GOGAT, glutamine synthetase/glutamate synthase; GDH, glutamate dehydrogenase.
© Oxford University Press 1997
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
1656 Fan et al.
and ATP depletion lead to decreased metabolic rates and
cell injury (Hochachka and Mommsen, 1983; Garlick
et al., 1979).
Rice seedlings are among the few terrestrial plants that
are able to survive under prolonged O2 deficiency resulting
from flooding. In particular, rice coleoptiles have the
unusual ability to expand under anaerobic conditions
(Fan et al., 1992; Opik, 1973). Several fermentation
reactions are operative in anaerobic rice coleoptiles,
resulting in the accumulation of ethanol, Ala, succinate,
GAB,
and lactate (Menegus et al., 1988, 1989; Fan et al.,
1986a). Clearly, these reactions account for an important
part of the anaerobic tolerance of rice coleoptiles.
However, this picture of the anaerobic response in rice is
by no means complete. For example, externally supplied
nitrate promotes rice seed germination and seedling emer-
gence (Hagiwara and Imura, 1991) as well as improves
hypoxic growth of rice and several other seedlings (Prioul
and Guyot, 1985; Trought and Drew, 1981; Malavolta,
1954;
Arnon, 1937). In addition, nitrate is stored in
appreciable amounts in rice seeds, and mobilized and
reduced in the coleoptiles during anaerobic germination
(Reggiani et al., 1993a).
Despite the interesting effects of nitrate on the germina-
tion and development of anaerobic rice seeds, the physio-
logical and biochemical basis of these effects is still
unclear. It has been recently demonstrated that 15N-
nitrate was incorporated into protein-free amino acids
in the coleoptile of anaerobically germinating seeds
(Reggiani et al., 1995), which suggests that reductive
nitrate assimilation pathways are operative in rice seeds
under anaerobiosis. Nitrate reduction to ammonium con-
sumes four NAD(P)H (8 electrons) and six protons per
reaction cycle (Kamin and Privalle, 1987; Guerrero et al.,
1981),
which is much more efficient in regenerating
NAD+
and consuming protons than other known fer-
mentation reactions, including ethanol production. If
sufficiently active, anaerobic nitrate metabolism can facil-
itate energy production and alleviate cytoplasmic acidosis.
However, whether the extent of nitrate metabolism does
contribute significantly to the overall fermentation capa-
city requires a detailed characterization of the nitrate
assimilation pathway.
Here, 15N-edited 'H-detected NMR spectroscopy and
gas chromatography-mass spectrometry (GC-MS) was
used to follow the metabolic fate of l5N-nitrate as com-
pared to that of 15N-ammonium ion in rice coleoptiles
under both aerobic and anaerobic conditions.
Incorporation of 15N into both free and protein-derived
amino acids was measured to estimate the amount of
total label originated from 15N-nitrate or -ammonium.
This,
in turn, allowed the contribution of nitrate or
ammonium metabolism to fermentation capacity in anaer-
obic rice coleoptiles to be assessed.
Materials and methods
Plant
growth
and
treatments
Rice (Oryza sativa M201) seeds were obtained from the
University of California Rice Research Station, Biggs,
California. Sodium 15N-nitrate and 15N-ammonium sulphate
(99%
enriched) were purchased from Cambridge Isotopes
Laboratory (Andover, MA). Rice seeds were sterilized, imbibed,
and germinated according to our previous procedure (Fan
et al., 1992). Apical 3-5 mm shoots were excised from 3-d-old
dark-grown rice seedlings and
1.7-3.5
g tissues (wet weight)
were incubated in the dark at 25 °C in 200 ml each of the
appropriate treatment buffer aerated with 0.2 /xm-filtered CO2-
free air or He for 22-28 h. Treatment buffers contained 0.5 mM
CaSO4,
5mM glucose, 5 mM NaHCO3, 10 mM MES
(2-[JV-
morpholinojethanesulphonate), and either 5 mM Na15NO3 or
(15NH4)2SO4 at pH 6.0, which was sterilized by passing through
0.22 (iin cellulosic filters. After incubation, aerobic and anaer-
obic tissues were rinsed extensively with oxygenated and
He-purged deionized water, respectively, before lyophilization.
Each treatment was repeated separately at least twice using
similar conditions with treatment durations of 22-28 h.
PCA and
protein extractions
The lyophilized tissues were pulverized in a micro ball mill (B.
Braun Instruments, Melsungen, Germany) to <3/^m particles
and extracted with 5% ice-cold perchloric acid (PCA) as
described previously (Fan et al., 1986ft). An aliquot of the PCA
extract was lyophilized and derivatized with N-methy\-N-[tert-
butyldimethylsilyl]trifluoroacetamide (MTBSTFA, Regis
Technologies, Morton Grove, IL) for analysis by GC-flame
ionization detection (FID) (Fan el al., 1993a) and GC-MS,
and the rest was lyophilized for analysis by NMR. The same
tissues were also extracted for total proteins with a buffer
containing 62.5 mM TRIS-HC1, 10% glycerol, 2% SDS, 5%
2-mercaptoethanol, and 12.5 ppm bromophenol blue, pH 6.8
(BioRad sample buffer) and boiled for lOmin. After centrifu-
gation, the protein extract was dialysed extensively against
deionized water using a Spectrapor
1
kDa molecular weight
cutoff membrane (Spectrum Medical Industries, Inc., Los
Angeles, CA). An aliquot of the dialysed protein extract was
then lyophilized, weighed (approximately
1
mg each), and
digested in 6 N HO plus 10 mM phenol and 2.5% thioacetic
acid at 110°C under vacuum for 24 h (Moore, 1972). The final
digest was lyophilized and redissolved in 0.9 M HC1 in 10%
D2O for NMR analysis, followed by lyophilization and
derivatization of an aliquot with MTBSTFA for GC-MS
analysis. The digestion conditions were optimized based on the
recovery of individual amino acids from a digestion time-course
(12,
24, and 36 h) of one of the protein extracts. The digestion
conditions chosen yielded some free Gin which is usually
reported to be lost from the digest (Moore, 1972). However,
no Asn was observed, which is expected to be lost through
digestion.
NMR and
GC-MS
analysis
Lyophilized tissue extracts were first dissolved in 100% D2O for
analysis by 'H NMR including two-dimensional (2-D) TOCSY
(total shift-correlation spectroscopy) for metabolite identifica-
tion as described previously (Fan et al., 1993ft).
Tissue extracts were then re-lyophilized and redissolved in
0.9 M HC1 in D2O: H2O (1:9) for analysis of the exchangeable
protons. The protein digests were prepared similarly. Under
these conditions, the exchange of ammonium protons was
sufficiently slow to be observed with water suppression (Preece
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
and Cerdan, 1993). NMR spectra were recorded at 11.75 T and
14.1 T on Varian (Palo Alto, CA) UnityPlus and Unity NMR
spectrometers, respectively. Simple one-dimensional (1-D) spec-
tra were recorded using the pulsed gradient (Watergate) method
for water suppression (Piotto et al., 1992) to avoid effects of
saturation transfer from the water protons. The acquisition
time was 2 s and the relaxation delay was
3
s.
For selectively observing and identifying I5N-labelled meta-
bolites, both 1-D and 2-D 'H-15N HSQC (heteronuclear single
quantum coherence) and HSQC-TOCSY spectra were recorded
on either spectrometer using inverse detection of 15N, with and
without "N decoupling at
10 °C
to further reduce the NH
exchange rate. In all cases, 15N decoupling was produced using
a GARP sequence (Shaka et al., 1985) while the spin-lock field
was generated with a MLEV-17 sequence (Bax and Davis,
1985) with a typical strength of
5
kHz and mixing times of
36-58 ms. With 15N decoupling, the acquisition time was
restricted by hardware to 0.15 s which limited the spectral
resolution. However, with the 15N-coupled spectra, the acquisi-
tion time was 2 s, which resolved the fine splitting due to 1H-1H
coupling and provided additional information on the identity
of NH (see below). The recycle time for 1-D experiments was
4 s. For 2-D HSQC-TOCSY (Bax and Pochapsky, 1992; Wider
and Wuthrich, 1993), phase-sensitive data were recorded using
the method of States et
al.
(1982) with Watergate (Piotto et al.,
1992) for water suppression. Proton chemical shifts were
referenced to the C-1H of glucose at 5.24 ppm (as relative to
that of DSS (2,2'-dimethylsilapentane-5-sulphonate)). Nitrogen
chemical shifts were referenced indirectly to liquid ammonia
using the proton shifts and the ratio of the gyromagnetic ratios
ofN and H (Live et al., 1984).
Derivatized tissue extracts were analysed on a Varian 3400
gas chromatograph fitted with a 0.18 mm i.d. x40m open-
tubular column of 0.25 fim DB-1 stationary phase (permethyl-
siloxane) coat (J&W Scientific, Rancho Cordova, CA), that
was fed through a line-of-site interface to a Finnegan (San
Jose,
CA, USA) ITD-806 mass spectrometer. GC temperatures
were: injector =
260
°C,
interface =
290
°C,
column =
60 °C
initial,
held for 2.0 min, ramped to 150°C at 20°Cmin~\ then to
290°C at 6°Cmin~l, splitless valve closed for 1.5 min, then
open for the duration of run. Hydrogen carrier gas, at
40 cm s"1, was used in conjuction with the El function of the
ITD-806; this resulted in classical electron ionization mass
spectra with hydrogen gas. MS parameters were: manifold
temperature =
220
°C,
electron energy =
70
eV, filament cur-
rent =
10 ;*A
with beam restrictor removed, automatic gain
control =
99
amu, scan range = m/z 150-500, 4 full scans s"1
averaged into 1 spectrum s"1, mass calibration and tune
performed using perfluorobutylamine. A mass defect of
1.0 mmu/amu was applied to all spectra.
Isotope enrichment calculation
NMR
analysis:
The 1-D water-suppressed 'H NMR spectra of
tissue extracts (Fig. 3A) and protein digests in 0.9 M HC1 were
obtained for estimating the %"N of GAB and NH4+. This was
achieved by comparing the peak area of the 15N coupled proton
satellites with that of the 14N coupled proton resonances. The
ratio of the area of the satellites to that of the total NH
resonance (15N + 14N) represents the % enrichment in 15N.
Overlap of the NH resonance of Ala, Glu, and Gin precluded
the use of this method for these amino acids. However, the
three-bond coupling from 13N to the methyl protons of Ala in
the tissue extract spectra was sufficient to apply a similar
procedure to measure the %I5N enrichment of Ala in D2O
(data not shown). The relative amount of "N label incorpora-
Rice 16N anaerobic metabolism 1657
tion into Ala and GAB was calculated based on their NH peak
areas in the 1-D HSQC spectra (Fig. 3B, C). The amount of
15N label in ammonium ion of
the
protein digests was estimated
by multiplying the %15N enrichment by the content. The
ammonium content ([NH4]) of the digests was derived from
the peak intensities of ammonium ion and Ala in the water-
suppressed spectra:
[NH4] =
[Ala]
x 0.25 x I(NH4)/r(AIa) x 3 (1)
where I(NH4) is the sum of the intensities of the ammonium
resonances, and f(Ala) is the intensity of the alanine methyl
resonance and Ala content
([Ala])
was obtained from GC-MS
analysis.
GC-MS
analysis:
15N enrichment analyses by GC-MS were
performed as follows, using instrument conditions identical to
that described above, and 20 scans were averaged in each case
to obtain the mass spectrum. First, natural abundance isotope
ratios were obtained for the 'pseudomolecular ion' (pM) cluster
of a given analyte, using authentic standards derivatized by
MTBSTFA. This pM is the M-57 ion, representing the highly-
favoured loss of the tert-buty\ group from the molecular ion
(cf. Mawhinney and Madsen, 1982; Anderson et al., 1987).
Thus,
in all cases, a high yield ofpM was obtained, which
contains the entire target analyte. Since at natural abundance
the predominant isotope of N is 14N (99.63%), this pM cluster
effectively represents the 14N amino acid. Then, samples from
the 15N-enriched experiments were analysed in identical fashion.
Subtraction of natural abundance (standard) from enriched
(sample) pM cluster (each normalized to the pM) yielded a
spectrum representing only the 15N-labelled amino acid (Fig. 4).
From these two spectra, %15N enrichment was then:
%liN=height of
pM*/(height
ofpM+height ofpM*)
where pM= pseudomolecular ion from the 14N spectrum,
pM*
=
pseudomolecular ion from the 15N (difference) spectrum.
For double-"N-labelled compounds such as Gin**, the above
process was repeated on isotope peaks of one mass unit larger.
Linearity of isotope ratio response was verified by successively
diluting the sample containing the highest 15N enrichment (that
from the "NH^"/anaerobic experiment) with natural abundance
standards, followed by analysis in identical fashion (data not
shown). 15N label incorporation in ftmolg"1 dry wt was
calculated from the product of %15N and content of a given
amino acid (with the multiplification factor of 2 for doubly
labelled Gin and Asn). The total %15N enrichment was
calculated from the ratio of total 15N label incorporation and
content of total N in amino acids from both PCA and protein
fractions.
For the determination of %!5N enrichment at the N-2 (a-
amino) and N-5 (S-amido) positions of Gin, the m/z 198 ion
cluster which was fragmented from the tri-silylated Gin was
also analysed. This cluster exhibited an enrichment in the m/z
199 ion, but not in the m/z 200 ion, in both singly and doubly
15N labelled Gin, which showed that only one of either N-2 or
N-5 nitrogens were contained in this fragment ion. More
importantly, 15N labelled Glu also resulted in the enrichment
of the m/z 199 ion (data not shown), which is consistent with
the m/z 198 ion cluster containing the N-2 instead of the N-5
nitrogen. Additionally, non-labelled tri-silylated Gin gave m/z
198-200 ion ratios very similar to the theoretical ion ratios of
the expected fragment ion structure (C10H21NOSi) containing
only the N-2 nitrogen (data not shown). Since the ion trap MS
instrument gave very stable ion intensities and ratios under full-
scan conditions for the fragments of the tri-silylated Gin, the
m/z 198 ion cluster was employed instead of the more typically
used fragment ions from the tetra-silylated Gin and selected
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
1658 Fan et al.
ion monitoring (Anderson et al., 1987; Gibbs, 1992; Williams
and Wolfe, 1994).
The m/z 198 ion cluster was analysed in identical manner
described above for the pM cluster. The %Gln* at the N-2
(%Gln*^2) and the N-5 (%Gln+N-5) positions was computed
by (Gibbs, 1992):
%Gln*N"2 = %Glntotil N"2-%Gln**
%Gln*N"5 = %Gln*-%Gln*N-2
%Glnt0U" N"5 =%Gln*N"5 + %Gln**
where %Gln* is the % singly-labelled Gin in the N-2 plus
the N-5 positions, %Gln** is the % doubly-labelled Gin,
%Glnlotal N'2 is the %Gln* + Gln** at the N-2 position, and
%Glnloul N"5 is the %Gln* + Gln** at the N-5 position.
Results
Effect of anaerobiosis and N treatments on selected
metabolite content
The effect of anaerobiosis on the content of major rice
coleoptile metabolites was measured in the presence of
either nitrate or ammonium ions. These metabolites were
identified using the 'H TOCSY spectra and quantified by
GC-FID as described previously (Fan
et
al.,
1993, 19866).
Under both nitrate and ammonium treatments, ethanol,
Ala, succinate, GAB, and lactate accumulated in anaero-
bic rice coleoptiles (Table 1), with the first four being the
major products. A large quantity of ethanol was also
observed in the medium, which was presumably excreted
from the tissues. These results were qualitatively similar
to those obtained in the absence of exogenous nitrogen
sources (Menegus et ah, 1988, 1989; Fan et al., 1986a).
The anaerobic accumulation of
ethanol,
GAB, and especi-
ally Ala was greater under ammonium than under nitrate
treatments. In addition, the levels of Gin and Asn were
substantially higher with ammonium than with nitrate
treatments under both aerobic and anaerobic conditions
(Table 1). This enhancement has also been reported in
other studies (Pilbeam and Kirkby, 1992). Similar results
were observed in three separate experiments (data not
shown).
The amino acid content of
rice
coleoptile protein digests
was also analysed for both nitrate and ammonium treat-
ments as shown in Table
2.
A similar content of major
protein-derived amino acids were observed for the two N
treatments under both aerobic and anaerobic conditions.
Interestingly, anaerobiosis induced a comparable increase
(at least 2-fold) in the content of protein-derived amino
acids for both N treatments. These results were in contrast
with the differences in free amino acid content (Table 1).
Identification of 1SN-Iabelled metabolites by NMR and
GC-MS
To determine the fate of exogenous nitrate and ammo-
nium in rice coleoptiles, we supplied the tissues with
either 15N-labelled nitrate or ammonium and analysed
the tissue extracts and protein digests for 15N-labelled
products by NMR and GC-MS. Instead of the conven-
tional, insensitive 15N-detected NMR method (Menegus
et al., 1993), the 15N-edited HSQC method was used,
Table 1. Effects of anaerobiosis on the content of free amino acids and fermentation products in rice coleoptiles under two N treatments
Treatment conditions were as described in Materials and methods. Tissue metabolite content
(/imolg"1
dry wt) were determined by GC as
described previously (Fan et al., 1993a), nd denotes not-detected; values in parenthesis represent ethanol in the medium Similar results were
obtained from three separate experiments.
CompoundTreatments
Air/15N-nitrateHe/13N-nitrateAir/"NH4+He/"NH4+
Fermentation products
ethanol
lactate
succinate
Ala
GAB
Glu
Other metabolites
Asn
Asp
Gin
Gly
He
Leu
Met
Phe
Pro
Ser
Thr
Val
nd (nd)
11 7
6.8
52.1
0 3
41.5
67
150
13.7
5.3
32
7.1
11.7
2.4
4.5
16.2
5.6
69
126.6 (
17.5
134.3
166.5
36.1
48.8
160
11.7
9.0
15.1
10.7
15.2
18.7
6.5
10.0
21.4
22.5
21.8
10321.7)nd (nd)
78
3.4
66 9
1.2
19.5
999
26.5
85.1
5.2
2.0
8.8
13.4
2.8
4.6
15.2
17.4
12.9
189.0(15728.6)
12.5
111.0
491.7
70.8
16.0
30.5
11 3
32.8
17.4
8.6
127
20.3
5.5
8.7
26.2
6.4
24.2
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
Rice 15N anaerobic metabolism 1659
Table 2. Effects of anaerobiosis on the content of protein-derived amino acids in rice coleoptiles under two N treatments
Treatment conditions and protein digestion were as descnbed in Materials and methods. Amino acid content (^mol g~' dry wt) of protein digests
was determined by GC-MS as described in Materials and methods; NH,+ content was estimated from NMR analysis.
CompoundTreatments
Air/1!N-nitrateHe/15N-nitrateAir/15NH;He/"NH4+
Ala
Asp
Gin
Glu
Gly
He
Leu
Pro
Ser
Tyr
Val
NH;
71.1
46.6
6.6
12.3
71.4
29.4
178.0
31.3
40.7
2.1
48.0
126.7
140 6
104.3
20.7
43.5
136.1
60.7
421 6
68.8
80.2
7.1
91.6
262.1
88.6
55 2
8.2
17.5
94.1
36.7
209.5
37.4
45.3
2.8
56.7
151.8
167.1
133.7
29.3
69.6
162.9
71.6
389.7
80.7
95.9
8.5
110.7
280.0
which selected only those protons directly bonded to 15N
and provides up to a 1000-fold enhancement in sensitivity.
Also,
when combined with lH TOCSY (Bodenhausen
and Ruben, 1980), this method allows structure identi-
fication of both known and unknown 15N-labelled com-
pounds directly in crude extracts.
Figure
1
shows a representative 2-D 15N-'H HSQC-
TOCSY spectrum obtained from a crude extract of 15N-
ammonium-treated aerobic rice coleoptiles. Also shown
in Fig.
1
is the corresponding 1-D 15N-XH HSQC-TOCSY
spectrum. One major cross-peak at 7.62 ppm (k, in the
'H dimension) of the extract spectrum was connected to
three other prominent cross-peaks at 3.15 (f), 2.62 (d),
and 2.06 ppm (b), which by comparison with the standard
spectrum corresponded to the y-NH, y-CH2, a-CH2, and
j3-CH2 of GAB, respectively. Similarly, the three cross-
peaks at 8.19 (1), 4.25 (h), and 1.60 (a) ppm were assigned
to the a-NH, a-CH, and j3-CH3 of Ala, respectively while
the four cross-peaks at 8.29 (n), 4.18 (g), 2.58 (e), and
2.24 (c) ppm were assigned to the a-NH, a-CH, y-CH2,
and j9-CH2 of Gin (the y-NH signal of Gin was not
detected). Moreover, two more peaks at 7.16 (j) and
8.24 ppm (m) were noted. Peak j was assigned to ammo-
nium based on the 'H chemical shift (22) and lack of 'H-
'H correlations, and the other (peak m), to the a-NH of
Glu based on the 'H chemical shift (Fig. 1). The lack of
'H connectivity of the m proton to the rest of the Glu
protons was presumably due to its fast exchange in water.
Additional confirmation of these assignments was
obtained from the 15N chemical shifts (Martin, 1985),
one-bond NH coupling constants, and fine splitting struc-
ture from proton-proton coupling (Fig. 3A). For
example, the doublet of the NH resonance of Ala is
consistent with its coupling to the a-CH whereas the
triplet of the NH resonance of GAB represents coupling
to the y-CH2. Except for the ammonium ion, the identity
of these 1SN-Iabelled metabolites was confirmed by
GC-MS analyses of the same extract samples.
o
7 S S 4 3 2
'H Chemical Shift (ppm)
Fig. 1. 2-D 15N-'H HSQC-TOCSY spectrum of the PCA extract of
15N-ammonium-treated rice coleoptiles. Both 1-D and 2-D HSQC-
TOCSY spectra was recorded at 10°C and at 11.75 T using a spin-lock
field strength of 5.3 kHz for a duration of 58 ms as described in
Materials and methods. Additional parameters for the 2-D spectrum
included a spectral width of 2 kHz in the N dimension and 6 kHz in
the H dimension, 128 increments each with 96 acquisitions, and a
relaxation delay of 3 s. The final data matrix was zero-filled to 8192 by
1024 complex points, and apodized using a mild Gaussian function in
both dimensions Respectively, peaks a, h, and
1
denote /3-CH3, a-CH,
and a-NHj* of Ala; b, d, f, and k, j3-CH2, a-CH2, y-CH2, and y-NH3+
of GAB; c, e, g, and n. 0-CH2, y-CH2, a-CH, and a-NH3+ of Gin; m,
a-NHj of Glu ;
j,
NH4+; i, residual H2O.
The protein digests were analysed by 2-D 15N-'H
HSQC spectroscopy for 15N labelled amino acids and a
representative spectrum for the ammonium/air treatment
is shown in Fig. 2. A total often cross peaks were resolved
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
1660
Fan
etal.
a 28^
3 :
nlcal Shift
o ^
44^
o-NhVGly
\
@ ^>, o-NHIlB
t-HWLy
1
8.48.0 7.8 7.8
1H Chemical Shift (ppm)
Fig. 2. 2-D 15N-'H HSQC spectrum of protein digest from "N-
ammonium-treated aerobic rice coleoptiles The spectrum was recorded
at H.7T and 10°C using Watergate (Piotto el ai, 1992) for water
suppression, and GARP for 15N decoupling during the proton
acquisition. The spectral widths were 5000 Hz in F2 and 2500 Hz in
Fl.
128 complex points were collected in tl and the acquisition time in
12
was 0.147 s. The data matrix was zero-filled to 4096 by 2048 complex
points, and apodized using a Gaussian function in both dimensions,
prior to Fourier transformation.
in the spectrum, which were assigned to NH4+, t-NH of
Lys,
and a-NH of Gly, Leu, He, Val, Ala, Glu, Ser, and
Tyr, based on their chemical shifts, 15N-1H connectivity
as shown, and ^-'H TOCSY connectivity (data not
shown). NH4+ was presumably the hydrolytic product of
Asn and Gin during acid digestion. Except for Lys, the
presence of 15N label in all of the amino acids were
confirmed by GC-MS analysis. Lys yielded no discernible
GC-MS peaks, possibly due to a poor derivatization
under the conditions used.
Quantification of 15N-enrichment in amino acids of rice
coleoptiles
Figure 3A illustrates two examples of 1-D water-
suppressed 'H NMR spectra of anaerobic rice coleoptile
PCA extracts, which were acquired without 15N editing.
Consequently, all 15N and 14N-coupled protons were
observed. The two relatively intense resonances at
7.62 ppm and 8.19 ppm were assigned to GAB and Ala,
respectively (see above). These resonances consisted of a
broad central component and two satellites spaced by
74 Hz due to the one-bond 15N-'H coupling. The areas
of these satellites compared with the total peak integral
provided an estimate of the %15N-enrichment (see
Materials and methods).
In comparison, the 15N-edited 1-D HSQC spectra
(Fig. 3B, C) were greatly simplifed because only those
protons that are bonded to 15N were observed. Using this
method, the pattern of 15N-labelled amino acids in 15N-
ammonium- and -nitrate-treated rice coleoptiles was com-
pared. By acquiring spectra at 10°C with a long recycle
8.6 8.4 8.2
3 "N-Alo
HN-Glu
\
"N-GIn /
i
i
1
i i i i
1
i
8.0 7.8
Chemical
1
1
I
1111
<
11111111111111 11111
7.« 7.4 7.2 7.0 6.8
Shift (ppm)
GAB
PCH,
aCH,-C00-
(He/15NH4+
l»N-GAB
^ . 1.
I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' I I I I I I I I I T 1 I I
8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8
"N-Glu
HN-QInV
Chemical Shift (ppm)
"N-QAB
He/Na15NO,
Alr/Na15NO,
1 i '
8.4
1 I '
8.2
1 I '
8.0
1 I '
7.8
1 I '
7.8
Chemical Shift (ppm)
Fig. 3. 1-D water-suppressed 'H and HSQC NMR spectra of PCA
extracts of rice coleoptiles grown on different nitrogen and O2 supplies.
All spectra were recorded at 10 X in 90% HCl/10% D2O at 14.1 T with
a spectral width of 6 kHz, using Watergate for water suppression
(Piotto el ul., 1992). Additional parameters for the HSQC spectra
included a delay of \J (6.7 ms), acquisition time of 0.15 s, and
relaxation delay of 2.5 s. The free induction decays were apodized using
a 0.5 Hz line-broadening exponential function. (A) Water-suppressed
spectra (without 15N-editing) from anaerobic "N-ammonium and
nitrate treatments. (B) "N-decoupled HSQC spectra from 15N-
ammonium/aerobic (lower) and "N-ammonium/anaerobic (upper)
treatments. (C) "N-decoupled HSQC spectra from 15N-nitrate/aerobic
(lower) and 15N-mtrate/anaerobic (upper) treatments, Y-scale expanded
10-fold relative to that in (B). Spectra (B) and (C) were directly
comparable by normalizing to spectral parameters and tissue mass.
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
time (4 s) and using the Watergate method for water
suppression (Piotto et al., 1992), signal attenuation due
to incomplete relaxation and saturation transfer from
water to the NH resonances was minimized. Thus, the
signal intensity for Ala and GAB in the 1-D HSQC
spectra (Fig. 3B, C) was proportional to their total 15N
label, i.e. the product of %15N enrichment and content.
However, for Gin and Glu, their a-NH signal intensities
were distorted somewhat due to a faster exchange. In
addition, the y-NH of Gin was presumably in such a fast
exchange at low pH that the signal was not observed. It
should be noted that a significant fraction of the GAB
peak from aerobic nitrate treatment in Fig. 3C was attrib-
uted to the natural abundance of 15N (0.37%) since
negligible selective enrichment was detected using the
GC-MS method (cf. Fig. 5).
Figure 3B also compares the 15N decoupled-HSQC
spectra for aerobic and anaerobic rice coleoptiles after
22 h of ammonium treatments. In both cases, signals
arising from Ala, Glu, and GAB dominated the spectra,
indicating a substantial incorporation of 15N label into
these amino acids. The presence of 15NH4+ signal was
expected and indicates the uptake of ammonium ion from
the medium. Moreover, the three amino acid signals
increased in response to anaerobiosis. In contrast, with
nitrate as the N source, the aerobic incorporation of 15N
into Ala, Glu, and GAB was low (Fig. 3C). However,
under anaerobic conditions, the amount of 15N-enrich-
ment in these amino acids increased substantially. The
presence of 15NH,J" signal indicates that I5NO^~ was taken
up and reduced to ammonium ion in rice coleoptiles.
A similar set of 1-D *H spectra were also acquired for
the protein digests from all treatments (data not shown).
Due to a significant overlap, it was difficult to discern the
a-NH signals arising from individual amino acids.
However, regardless of the oxygen status, the signal
intensities of the 1-D HSQC spectra were generally greater
•
for the ammonium than for the nitrate treatments, indicat-
ing a higher amount of 15N label incorporation into
proteins for the former.
Except for the ammonium ion, quantification of 15N-
label (%"N and total 15N) in free and protein-derived
amino acids was obtained from the GC-MS data, where
all component peaks were well-resolved. A typical
GC-MS chromatogram of the PC A extract of anaerobic
rice coleoptiles is shown in Fig. 4A. Also illustrated
(Fig. 4B) are the mass regions of the pseudo molecular
ion of Ala with natural abundance isotope distribution
and enriched in 15N; the %l5N-enrichment was calculated
from such a data set. The %l5N-enrichment and ratio of
total 15N label for Ala and GAB in the PCA extracts
were independently verified by the analysis of 1-D water-
suppressed (Fig. 3A) and HSQC NMR spectra (Fig. 3B,
C) since the pertinent NMR signals were adequately
resolved.
Rice 15W anaerobic metabolism 1661
Figures 5A and B summarize the %15N enrichment in
various N-metabolites from 15N-ammonium and -nitrate
treated coleoptiles, respectively. For the free amino acids,
Gin and Glu were among the most labelled (up to 80%)
under all treatments. Ala, GAB, Asp, and Pro were also
significantly enriched, particularly for Ala and GAB in
air/ammonium-treated tissues (c. 70-80%) and for Pro in
the nitrate-treated tissues (up to 70%). The %15N-enrich-
ment patterns responded differently to anaerobiosis
between nitrate and ammonium treatments. For example,
the % doubly labelled Gin (Gin**), Glu*, Asn*, Asn**,
and Asp* increased in nitrate-treated tissues but decreased
in ammonium-treated tissues. Similar results were
obtained for Gly, He, and Ser. The %15N-enrichment of
amino acids was generally lower in nitrate (Fig. 5B) than
ammonium-treated tissues (Fig. 5A). Interestingly, while
Gin was present mainly in the Gin** form in ammonium-
treated tissues, the singly-labelled species (Gin*) became
much more significant in anaerobic nitrate-treated tissues
(up to 57% enriched). Except for the aerobic nitrate
treatment, the 15N label of Gin* was largely in the N-2
(60-100% of the Gin* label) instead of the N-5 position.
These enrichment results were confirmed with the NMR
data wherever feasible (Fig. 3) and were consistent in
three separate experiments (data not shown).
In comparison, the %15N enrichment was generally
lower in protein-derived than in free amino acids for all
treatments. The enrichment pattern in the free amino
acids was not reflected in that of the protein-derived
amino acids, most notably for Gin**, Gly*, Val*, Ser*,
and Pro* under the nitrate treatment. Oxygen deficiency
caused a decrease in the %15N enrichment of most protein-
derived amino acids with the ammonium treatment.
However, with the nitrate treatment, the %Gln*, Asp*,
and Pro* increased in response to anaerobiosis. Thus, the
distribution of %15N enrichment in protein-derived amino
acids also differed between the two N treatments, as for
the case of the free amino acids (see above).
By summing all of the 15N-labelled products normalized
against the total amino acid content, the total %15N
enrichment for the tissues and the two amino acid frac-
tions of all treatments were estimated as shown in Table 3.
It is clear that anaerobiosis led to a substantial decrease
in the overall %I5N enrichment in free amino acids from
the ammonium treatment while a small increase was
observed for the nitrate treatment. In addition, the overall
%15N in protein-derived amino acids decreased in
response to anaerobiosis under both N treatments.
Table 3 also lists the total amount of 15N label in tissues
and the two amino acid fractions. Sixty-four to 72% of
the label resided in the free amino acid fraction under the
ammonium treatment, while that under the nitrate treat-
ment was lower (17-49%). A substantial increase in the
total amount of label in tissues and both amino acid
fractions was observed for the nitrate treatment in
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
1662 Fan et al.
AlaGAB
«I \l
3
o
o
5
o
B
Hu
Ii ..
Qlu
. JL
Qln
900 1000 1100 1200 1300 1400 1S00 1600 1700
Scan # 1800
slty
c
a
j:
CD
latl
CD
IX
zoo
, ||| , ,|
261274
I I I I I 1
278
274
260280
m/z270280 240250260 270 280
m/z
Fig. 4. GC-MS chromatogrdm of silylated extract of rice coleoptiles and mass spectra of silylated natural abundance and 15N-enriched Ala. The
tissue extraction and GC-MS conditions were as described in Materials and methods. The chromatogram shown in (A) was acquired from
anaerobic rice coleoptiles under 15N-ammonium treatments and illustrates the N sinks discussed in this paper. (B) shows mass spectra of silylated
Ala standard (natural abundance) (left), and of the Ala peak from the chromatogram above (right).
response to anaerobiosis while the ammonium treatment
exhibited an opposite trend. The elevated content
of protein-derived amino acids (Table2) primarily
accounted for the increase in the total amount of I5N
label in the protein fraction of nitrate-treated tissues,
despite the reduction in the overall %15N enrichment.
Discussion
As with previous findings (Menegus et al., 1988, 1989;
Fan et al., 1986a; Gibbs, 1992), the large accumulation
of free ethanol, Ala, succinate, and GAB reported here
(Table 1) indicated that these are the major fermentation
products in anaerobic rice coleoptiles, regardless of the
exogenous N source. However, the extent of anaerobic
accumulation of ethanol, Ala. and GAB was greater in
ammonium- than in nitrate-treated tissues. This could
simply reflect a higher glycolytic rate for energy produc-
tion in ammonium- than in nitrate-treated tissues during
anaerobiosis. On the other hand, nitrate reduction in
nitrate-treated tissues should have consumed some of the
NADH and protons generated by glycolysis, thereby
reducing the demand for the synthesis of fermentation
products.
Protein-free Gin, Glu, and Ala were the most enriched
amino acids (in terms of the %15N) (Fig. 5), which is
consistent with their role as the primary N assimilation
products, regardless of the oxygen status (Gibbs, 1992;
Lea et al., 1990; Rhodes et al., 1989). GAB was also
among the most enriched metabolites under the aerobic/
ammonium treatment, which suggests a channeled path-
way from ammonium to GAB in rice coleoptiles. A
similar conclusion can be made for Pro under the aerobic/
nitrate treatment. Although the free Glu content was
higher with the nitrate than ammonium treatment, its
%15N enrichment showed the opposite, which indicates
that endogenous N sources (e.g. protein degradation)
may have contributed more to its production under nitrate
treatments (see below).
The %15N enrichment pattern in Glu and Gin suggests
that the enzymes involved in their synthesis (Fig. 6)
exhibited different activities between the nitrate and
ammonium treatments, particularly under anaerobic con-
ditions. Under ammonium treatments, the much higher
%Gln** over %Gln* (Fig. 5) indicates that Gin was
primarily synthesized via the Gin synthetase/Glu synthase
(GS/GOGAT) system (Lea et al., 1990; Rhodes et al.,
1989) by utilizing exogenous 15NH4+ (Fig. 6) and that
Glu* was readily available for Gin** synthesis. This is
also consistent with the observation that the %Gln** and
Glu* were similar (Fig. 5).
Under the anaerobic/nitrate treatment, the large
increase in %Gln*, the dominance of 15N label in the N-2
(a-amino) position of Gin*, and the maintenance of high
%Glu* (Fig. 5) requires a different interpretation. One
possibility is that catabolic deamination (e.g. from protein
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
Rice 15W anaerobic metabolism 1663
15N-Ammonium Treatment
u
I
ao
70
60-
60-
40-
30-
20-
10-
li
Dtt/lmu
BHMMU
0 Air/protein
u
S
Q.
B15N-Nitrate Treatment
Fig. 5. %1!N-enrichment of free and protein-derived amino acids in rice
coleoptiles under different N and oxygenation treatments. Treatments
and 15N isotope analysis of the GC-MS data were as described in
Materials and methods. Total 15N
(^molg"1
dry wt) is expressed as
the product of %15N and absolute concentration of a given amino acid
from Table 1.
*
and ** indicate singly and doubly 15N-labclled,
respectively. The Gin content for the aerobic/nitrate treatment was low,
which made
it
difficult to determine its %13N enrichment. (A) "N-
ammomum treatments. (B) 15N-nitrate treatments.
degradation) (Menegus et al., 1993) added a significant
amount of endogenous N (14N) to the internal ammonium
pool, in addition to that derived from the reduction of
exogenous 15NO^". This would have led to a lower %15N
in this pool, which would in turn favour Gin* over Gin**
production, as observed. However, the label of Gin*
would have been at the N-5 (8-amido) position, if Gin*
were synthesized via the GS/GOGAT system. The domin-
ance of the label at the N-2 position of
Gin*
suggests the
existence of a distinct pool of Glu* which is synthesized
by the Glu dehydrogenase (GDH) reaction (Rhodes
et al., 1989) (Fig. 6), followed by Gin* (at N-2 position)
synthesis from Glu* and unlabelled ammonium ion. In
addition, this Glu* pool is not accessible to the plastid
GS/GOGAT system, which is consistent with the occur-
rence of mitochondrial GDH isolated from plastid GS
(Yamaya et al., 1986) and an enhancement of the GDH
activity in anaerobic rice coleoptiles (Reggiani et al.,
1993ft).
The overall pattern of %15N enrichment (Fig. 5) is
consistent with the transfer of the label from the primary
products to other amino acids, which were then incorpor-
ated into proteins. The presence of 15N label in proteins
is evidence for de
novo
protein synthesis utilizing extern-
ally supplied nitrate or ammonium ion. In fact, protein
synthesis appeared to remain active under anaerobic
conditions in rice coleoptiles (Tables 2, 3), which is in
contrast
to
the reduced activity observed for flood-
sensitive tissues such as maize root (Sachs et al., 1980).
Maintenance of
a
high protein turnover rate was reported
in anaerobic rice embryos previously (Mocquot et al.,
1981).
However,
a
significant fraction of the protein
synthesis in anaerobic rice coleoptiles utilized endogenous
amino acid sources since the %15N in all protein-derived
amino acids was lower than that in free amino acids
(Fig. 5). Whether the observed incorporation of exogen-
ous nitrogen into proteins is relatively specific to a subset
of proteins required for anaerobic metabolism remains to
be determined.
For the ammonium treatment, the overall lower % and
total 15N enrichment in response to anaerobiosis (Fig. 5A;
Table 3) is to be expected since ammonium assimilation
into amino acids and proteins requires ATP, which is in
short supply. A similar argument should also apply to
the nitrate treatment. However, the nitrate treatment
resulted in the increase in %15N of several free and
protein-derived amino acids in response to anaerobiosis
(Fig. 5B). It is possible that the pathways from nitrate to
Table 3. Effects of anaerobiosis on total % i5N enrichment and amount of
15JV
label in rice coleoptiles under two
N
treatments
Treatment"
Air/15N-nitrateHe/15N-nitrateAir/"NH4+He/15NH4+
% "N
Free AA's
Protein AA's
Total
"N content
Free AA's
Protein AA's
Total
15.9
4.7
54
7.1
33.6
40.7
19.3
33
5.6
48.9
51.8
100.7
68.6
13.5
32.7
318.8
117.3
436 1
46.1
5.6
13.0
178.8
98.0
276.8
" "N label content is in ^mol g
'
dry wt, the total amount of "N label was summed from that of individual components including doubly
labelled Gin and Asn.
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
1664
Fan
etal.
coo-
Fig. 6. 15NO3 and "NH4 assimilation pathways in anaerobic rice
coleoptiles. The proposed pathways are based on both the present
findings and the literature. Process (1) converts 15NO^~ to 15NH^ via
nitrate and nitrite reductases (Reggiani et al., 1993); processes (2) and
(3) assimilate 15NH4+ into Gin and Glu via GS/GOGAT and GDH
(Lea et al., 1990; Rhodes et al., 1989), respectively; process (4)
incorporates 15NH^ into Ala via Glu:pyruvate aminotransferase
(Menegus e; a/, 1993); process (5) produces GAB via Glu decarboxylase
(Narayan and Nair, 1990); process (6) generates Ala via Ala
dehydrogenase (Brunhuber and Blanchard, 1994). Also shown is the
synthesis of Gin* at N-2 position from Glu* and unlabelled ammonium
ion (e.g. derived from catabolism) The reversed N represents 15N while
* and ** denote singly and doubly 15N-labelled species, respectively. It
should be noted that the GDH reaction may occur in a distinct
compartment (such as the mitochondria) separate from the
GS/GOGAT system.
these amino acids are selectively activated and that their
incorporation into proteins is favoured in anaerobic rice
coleoptiles.
Under the anaerobic/nitrate treatment, the major
labelled products, Ala, GAB, and Glu which also accumu-
lated to a high content, may be significant in several
aspects. Besides the 6 electrons and 8 protons required in
nitrate reduction, Glu synthesis via GDH additionally
consumes two electrons and one proton. The synthesis of
GAB from Glu, via the action of Glu decarboxylase
(GDC, Streeter and Thompson, 1972; Narayan and Nair,
1990;
Carroll et al., 1994) removes one more proton.
Thus,
the nitrate-Glu-GAB pathway may contribute
towards the ability of anaerobic rice coleoptiles in main-
taining the cytoplasmic pH (Fan et al., 1992). Although
consuming fewer protons and electrons, the ammonium-
Glu-GAB pathway can also serve the same function. A
similar argument can be made for the anaerobic nitrate
or ammonium to Ala pathway. Ala synthesis can involve
Ala aminotransferase (Menegus et al., 1993) or dehydro-
genase (Magalhaes, 1991; Brunhuber and Blanchard,
1994).
The latter reaction is analogous to the GDH
reaction in consuming additional electrons and protons.
The additional sink for electrons and protons provided
by nitrate reduction may have, in part, contributed to the
reduced production of ethanol by rice coleoptiles under
anaerobic conditions as compared with the ammonium
treatments (Table 1). In addition, the large accumulation
of Ala and GAB may help maintain the turgor pressure
of the coleoptile (Menegus et al., 1993). Moreover, GAB
accumulation has been associated with the induction of
mammalian hibernation (Nilsson and Lutz, 1993) which
shares a common trait of low oxygen status as anaero-
biosis. It is plausible that GAB may also be involved in
some form of metabolic stasis in anaerobic rice coleop-
tiles.
The wide occurrence of GAB in plants (Robinson,
1980),
particularly in response to stresses (Narayan and
Nair, 1990; Mayer et al., 1990; Crawford et al., 1994;
Thompson et al., 1966; Wallace et al., 1984) warrants
further investigations on the physiological role(s) of this
amino acid.
In conclusion, the reductive assimilation of nitrate into
Ala, GAB, Glu, and other amino acids may have import-
ant implications for the flood tolerance of germinating
rice seedlings, and perhaps plants in general, as a means
for sustaining glycolysis and energy production while
minimizing cytoplasmic acidosis and conserving N.
Acknowledgements
The authors wish to thank Mr Jim Webster of UC Davis, for
the generous gift of rice seeds and Dr Hank Greenway for his
critical comments. This research was supported, in part, by the
California Rice Research Board (project no. RB-5). The NMR
instrumentation was made available by the Biomedical NMR
Centre of the Medical Research Council, UK and the GC-MS
instrumentation was supported in part by US EPA (grant No.
R819658) Center for Ecological Health Research at UC Davis.
Although the information in this document has been funded
partly by the United States Environmental Protection Agency,
it may not necessarily reflect the views of the Agency and no
official endorsement should be inferred.
References
Anderson LW, Zaharevitz DW, Strong JM. 1987. Glutamine
and glutamate: automated quantification and isotopic enrich-
ments by gas chromatography/mass spectrometry. Analytical
Biochemistry 163, 358-68.
Amon DI. 1937. Ammonium and nitrate nitrogen nutrition of
barley and rice at different seasons in relation to hydrogen-
ion concentrations, manganese, copper, and oxygen supplied.
Soil Science 44,
91-121.
Bax A, Davis DG. 1985. MLEV-17-based two-dimensional
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
homonuclear magnetisation transfer spectroscopy. Journal of
Magnetic Resonance 65, 355-60.
Bax A, Pochapsky SS. 1992. Optimized recording of heteronu-
clear multidimensional NMR spectra using pulsed field
gradients. Journal of Magnetic Resondnce 99, 638—43.
Bodenhausen G, Ruben DJ. 1980. Natural abundance
nitrogen-15 NMR by enhanced heteronuclear spectroscopy.
Chemistry and Physics Letters 69, 185-9.
Brunhuber NMW, Blanchard JS. 1994 The biochemistry and
enzymology of amino-acid dehydrogenases. Critical Review
in Biochemistry and Molecular Biology 29, 415—67.
Carroll AD, Fox GG, Laurie S, Phillips R, Ratcliffe RG, Stewart
GR. 1994, Ammonium assimilation and the role of y-
aminobutyric acid in pH homeostasis in carrot cell suspension.
Plant Physiology 106, 513-20.
Crawford RMM. 1978. Metabolic adaptations to anoxia. In:
Hook DD, Crawford RMM, eds. Plant life in anaerobic
environments. Ann Arbor: Ann Arbor Science, 119-36.
Crawford LA, Bown AW, Breitkreuz KE, Guinel FC. 1994. The
synthesis of y-aminobutyric acid in response to treatments
reducing cytosolic pH. Plant Physiology 104,
865-71.
Davies DD. 1980. Anaerobic metabolism and the production of
organic acids. In: Davies DD, ed. The biochemistry of plants,
Vol. 2. New York: Academic Press, 581-611.
Fan TW-M, Higashi RM, Lane AN. 1986a. Monitoring of
hypoxic metabolism in superfused plant tissues by in vivo
'H NMR. Archives of Biochemistry and Biophysics 251,
674-87.
Fan TW-M, Higashi RM, Lane AN, Jardetzky O. 19866.
Combined use of 'H NMR and GC-MS for monitoring
metabolites and in vivo 'H NMR. Biochimica et Biophysica
ActaSS2, 154-67.
Fan TW-M, Lane AN, Higashi RM. 1992. Hypoxia does not
affect rate of ATP synthesis and energy metabolism in rice
shoot tips as measured by 31P NMR in vivo. Archives of
Biochemistry and Biophysics 294, 314-18.
Fan TW-M, Colraer TD, Lane AN, Higashi RM. 1993a.
Determination of metabolites by 'H NMR and GC: Analysis
for organic osmolytes in crude tissue extracts. Analytical
Biochemistry 214,
260-71.
Fan TW-M, Lane AN, Higashi RM. 19936. Energy and
fermentation metabolism in hypoxic rice coleoptiles—a
multinuclear NMR approach. In: Jackson MB, Black CR,
eds.
Interacting stresses on plants in a changing climate, NATO
ASI Series, Vol. 16, Berlin: Springer-Verlag, 333-52.
Garlick PB, Radda GK, Seeley PJ. 1979. Studies of acidosis in
the ischaemic heart by phosphorus nuclear magnetic reson-
ance.
Biochemical Journal 184, 547-54.
Gibbs DJ. 1992. Alanine accumulation in anaerobic rice
coleoptiles. PhD thesis, the University of Western Australia.
Guerrero MG, Vega JM, Losada M. 1981. The assimilatory
nitrate-reducing system and its regulation. Annual Review of
Plant Physiology 32, 169-204.
Hagiwara M, Imura M. 1991. Promotion of seedling emergence
of patty rice from flooded soil by coating seed with potassium
nitrate. Japanese Journal of Crop Science 60, 441-6.
Hochachka PW, Mommsen TP. 1983. Protons and anaerobiosis.
Science 219, 1391-7.
Karain H, Privalle LS. 1987. Nitrite reductase. In: Ullrich WR,
Aparicio PJ, Syrett PJ, Castillo F, eds. Inorganic nitrogen
metabolism. Berlin: Springer Verlag, 112-17.
Lea PJ, Robinson SA, Stewart GR. 1990. In: Miflin BJ, Lea PJ,
eds.
The biochemistry of plants, Vol. 16. Ch. 4. San Diego:
Academic Press.
Live DH, Davis DG, Agosta WC, Cowburn D. 1984. Long range
Rice 15W anaerobic metabolism 1665
hydrogen bond mediated effects in peptides: 15N NMR study
of Gramicidin S in water and organic solvents. Journal of
American Chemical Society 106,
1939-41.
Magalhaes JR. 1991. Kinetics of (NH4+)-N-15 assimilation in
tomato plants—evidence for (NH^)-N-15 assimilation via
GDH in tomato roots. Journal of Plant Nutrition 14, 1341-53.
Malavolta E. 1954. Studies on the nitrogenous nutrition of rice.
Plant Physiology 29, 98-9.
Martin F. 1985. "N-NMR studies of nitrogen assimilation and
amino acid biosynthesis in the ectomycorrhizal fungus
Cenococcum graniforme. FEBS Letters 182, 350—4.
Mawhinney TP, Madsen MA. 1982. JV-methyl-./V-(rm-butyldime-
thylsilyl)trifluoroacetamide and related A'-rerf-butyldimethyl-
silylamides as protective silyl donors. Journal of Organic
Chemistry 47, 3336-9.
Mayer RR, Cherry JH, Rhodes D. 1990. Effects of heat shock
on amino acid metabolism of cowpea cells. Plant Physiology
94,
796-810.
Menegus F, Cattaruzza L, Chersi A, Fronza G. 1989. Differences
in the anaerobic lactate-succinate produciton and in the
changes of cell sap for plants with high and low resistance to
anoxia. Plant Physiology 90, 29-32.
Menegus F, Cattaruzza L, Chersi A, Serva A, Fronza G. 1988.
Production and organ distribution of succinate in rice
seedlings during anoxia. Physiologia Plantarium 74, 444-9.
Menegus F, Cattaruzza L, Molinari H, Ragg E. 1993. Rice and
wheat seedlings as plant models of high and low tolerance to
anoxia. In: Surviving hypoxia: mechanisms of adaptation and
control. Boca Raton: CRC Press, 53-64.
Mocquot B, Prat C, Mouches C, Pradet A. 1981. Effect of
anoxia on energy charge and protein synthesis in rice embryo.
Plant Physiology 68, 636-40.
Moore S. 1972. The precision and sensitivity of amino acid
analysis. In: Chemistry and biology of peptides. Ann Arbor
Science Publishers, Ann Arbor, 629-53.
Narayan VS, Nair PM. 1990. Metabolism, enzymology and
possible roles of 4-aminobutyrate in higher plants.
Phytochemistry 29, 367-75.
Nilsson GE, Lutz PL. 1993. Role of GABA in hypoxia tolerance,
metabolic depression and hibernation—possible links to
neurotransmitter evolution. Comparative Biochemistry and
Physiology 105C, 329-36.
Opik H. 1973. Effect of anaerobiosis on respiratory rate,
cytochrome oxidase activity, and mitochondrial structures in
coleoptiles of rice (Orvza sativa L). Journal of Cell Sciience
12,
725-59.
Pilbeara DJ, Kirkby EA. 1992. Some aspects of the utilization
of nitrate and ammonium by plants. In: Mengel K, Pilbeam
DJ, eds. Nitrogen metabolism of plants, Proceedings of the
Phytochemical Society of Europe, Vol. 33, Oxford: Clarendon
Press,
55-70.
Piotto M, Saudek V, Sklenar V. 1992. Gradient-tailored
excitation for single-quantum NMR spectroscopy of aqueous
solutions. Journal of Biomolecular NMR 2, 661-5.
Preece NE, Cerdan S. 1993. Determining 15N to 14N ratios in
biofluids by single-pulse 'H Nuclear Magnetic Resonance.
Analytical Biochemistry 215,
180-3.
Prioul J.-L, Guyot C. 1985. Role of oxygen transport and
nitrate metabolism in the adaptation of wheat plants to root
anaerobiosis. Physiologie Vegetate 23, 175-85.
Reggiani R, Mattana M, Aurisano N, Bertani A. 1993a.
Utilization of stored nitrate during the anaerobic germination
of rice seeds. Plant Cell Physiology 34, 379-83.
Reggiani R, Mattana M, Aurisano N, Bertani A. 19936. The
rice coleoptile-an example of anaerobic nitrate assimilation.
Physiologia Plantarum 89,
640-3.
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
1666
Fan
etal.
Reggiani R, Bertini F, Mattana M. 1995. Incorporation of
nitrate nitrogen into amino acids during the anaerobic
germination of nee. Amino Acids 9, 385-90.
Rhodes D, Bnink DG, Magalhaes JR. 1989. Assimilation of
ammonia by glutamate dehydrogenase. In: Poulton JE,
Romeo JT, Conn EE, eds. Plant nitrogen metabolism—recent
advances in phvtochemistry series, Vol. 23. New York: Plenum
Press,
191-226.
Robinson T. 1980. The organic constituents of higher plants.
North Amherst: Cordus Press, 230.
Sachs MM, Freeling M, Okimoto R. 1980. The anaerobic
proteins of maize. Cell 20, 761-7.
Shaka AJ, Barker PB, Freeman R. 1985. Computer-optimized
decoupling scheme for wideband applications and low-level
operation. Journal of Magnetic Resonance 64, 547-52.
States DJ, Haberkorn RA, Ruben DJ. 1982. A two-dimensional
nuclear Overhauser experiment with pure absorption phase
in four quadrants. Journal of Magnetic Resonance 48, 286-92.
Streeter JG, Thompson JF. 1972. Anaerobic accumulation of y-
amino butyric acid and alanine in radish leaves (Raphanus
sativus L.). Plant Physiology 49, 572-8.
Thompson JF, Stewart CR, Morris CJ. 1966. Changes in amino
acid content of excised leaves during incubation. I. The effect
of water content of leaves and atmospheric oxygen level.
Plant Physiology 41, 1578-4.
Trought MCT, Drew MC. 1981. Alleviation of injury to young
wheat plants in anaerobic solution cultures in relation to the
supply of nitrate and other inorganic nutrients. Journal of
Experimental Botany 32, 509-22.
Wallace W, Secor J, Schrader L. 1984. Rapid accumulation of
y-aminobutyric acid and alanine in soybean leaves in response
to an abrupt transfer to lower temperature, darkness, or
mechanical manipulation. Plant Physiology 75, 170-5.
Wider G, WDthrich K. 1993. A simple experimental scheme
using pulsed field gradients for coherence-pathway rejection
and solvent suppression in phase-sensitive heteronuclear
correlation spectra. Journal of Magnetic Resonance 102,
239-41.
Williams BD, Wolfe RR. 1994. Determination of amino-N-15
and amide-N-15 glutamine enrichment with tertiary butyldi-
methylsilyl derivatives. Biological Mass Spectrometry 23,
682-8.
Yamaya T, Oaks A, Rodes D, Matsumoto H. 1986.
Mitochondrial GDH? Plant Physiology 81, 754-7.
by guest on July 6, 2011jxb.oxfordjournals.orgDownloaded from
