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This is to certify that the
thesis entitled
DARK CO2 FIXATION AND AMINO ACID METABOLISM IN
SYMBIOTIC NZ-FIXING SYSTEMS
presented by
George T. Coker III
has been accepted towards fulfillment
of the requirements for
Ph. IL degreein Biochemistry
w QM
Date ngl J)
U (7' [52,
Major professor
0-7639
MSU
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DARK CO2 FIXATION AND AMINO ACID METABOLISM IN SYMBIOTIC N -FIXING SYSTEMS.
14C and 13N-Labeled Tracers
2
Labeling Studies with
By
George T. Coker III
A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Biochemistry
1982
6 [WHY/0
ABSTRACT
DARK C02 FIXATION AND AMINO ACID METABOLISM IN SYMBIOTIC N -FIXING SYSTEMS.
2
Labeling Studies with 14C and 13N-Labeled Tracers
By
George T. Coker III
Amino acids constitute a greater proportion of the organic
nitrogen transported from the roots of soybean (Glycine max) plants
treated with N03 or NH: and from the nodulated roots of alder (Algg§_
glutinosa) than they do from the nodulated roots of soybean plants
dependent solely on N2. This suggests that the metabolism of amino
acids is different in these tissues. Amino acid metabolism was
examined by monitoring the amino acids labeled with [14C] incorporated
during dark C02 fixation and with [13N] incorporated from 13NHZ,
13No; or [13N]N2.
Label from 14CO2 was directly incorporated in soybean roots and
the NZ-fixing root nodules of soybeans and alders. The rate of incor-
poration depended on the age and the nitrogen source for the plants.
The products of dark C02 fixation were primarily amino and organic
acids. The distribution of label incorporated from 14C02 into amino
acids depended on the plant species and the nitrogen source. The major
labeled amino acids in roots and nodules of soybean plants dependent on
N2 were aspartate and glutamate; in alder nodules, citrulline; in roots
of soybean plants treated with N03, asparagine; and in roots of soybean
plants treated with NHZ, asparagine and glutamine.
Asparagine was the major amino acid transported out of the soybean
George T. Coker III
root system. The pathway of asparagine synthesis was examined by
T4
exposing roots either to C0 followed by a chase with 12CO2 or to
2
[2,3-3H]aspartate and [4-]4C]aspartate. The results from these two exp-
eriments indicated that asparagine was synthesized directly from aspartate.
14
After exposure to C02, the specific activity of glutamine was
consistently higher than that of glutamate in soybean nodules and
roots of plants treated with N03. This was taken as evidence that
there were two pools of glutamate, only one of which was associated
with glutamine synthesis.
Alder and soybean nodules and roots were incubated with
13 I3
N—labeled tracers. Those tissues incubated with NHZ had a higher
ratio of [13N]glutamine to [13N]glutamate than similar tissues exposed
to 13N03 or [13NjN2. An explanation for these results based on the
relative rates of glutamine and glutamate synthesis is discussed.
To My Family
To my brother and mother who tan read this and to my father who cannot
"See dad, it only took 2 more years."
ACKNOWLEDGEMENTS
I wish to thank my major adviser, Dr. K. R; Schubert, and the
members of my guidance committee, Drs. Tolbert, Nadler, Averill and
Ferguson-Miller. I also wish to thank the people that I entered
graduate school with for their willingness to help me over the rough
_ spots. I would also like to thank the members of the laboratory -
Alan Christensen, Peter McClure and Debra Polayes, My special
thanks goes to Mary Tierney who stood by me.
I acknowledge the financial support of the American Cancer
Society, the National Institute of Health (GM lO9l), the National
Science Foundation (PCM 77-24683, PCM 80-ll736 and the National
Needs Training Grant), the Michigan Agricultural Experiment Station,
and the Department of Biochemistry at Michigan State University.
iii
TABLE OF CONTENTS
Pagi
LIST OF FIGURES ........................ vii'
LIST OF TABLES ......................... xi
LIST OF ABBREVIATIONS ..................... xi'
INTRODUCTION ........................... 1
LITERATURE REVIEW ....................... 4
A. Dark CO2 Fixation .................... 4
B. Role of Dark CO2 Fixation ................ 7
C. Amino Acid Metabolism ............. _ ..... 9
1. Enzymes of NH+ Assimilation .............. 9
2. Pathway of NH4 Assimilation .............. ll
3. Asparagine Synthesis ................. l4
4. Citrulline Synthesis ................. l7
5. Allantoin and Allantoate Synthesis .......... 17
D. Effect of N03 and NHZ+on N2 Fixation ........... 18
E. Effects of NO' and NH4 on Nitrogen Transport in Soybeans . 23
F. Effects of NH4 on Amino Acid Synthesis .......... 24
MATERIALS AND METHODS ..................... 26
A. Growth of Plants ..................... 26
B. Measurement of the Rate of CZHZ Reduction ........ 27
C. Measurement of Allantoate, Allantoin, N03 and Total
Amino Acids ....................... 28
D. Measurement of the Rate of O2 Consumption ........ 29
E. Production of 14CO2 ................... 29
F. Exposure of Plant Tissue to 14CO2 ............ 30
6. Measurement Of the Specific Activity of 14co2 .. ..... 30
H. Extraction of the Products of 14C02 Fixation ....... 3l
I. Separation of the Products of 14CO2 Fixation ....... 32
1. High Performance Liquid Chromatography (HPLC) ..... 32
2. Ion-Exchange Chromatography .............. 33
iv
. Isolation and Chromatography of
3. Thin-Layer Chromatography ...............
4. High-Voltage Electrophoresis . . . . . . .......
5. Gas-Liquid Chromatography ...............
14c-Labe1ed 2-Oxoacids
from Soybean Nodules ...................
Incorporation of [2,3-3H]Aspartate and [4-14C]Aspartate
into Asparagine .....................
. Distribution of Label Within Aspartate and Glutamate in
14
Soybean Nodules Exposed to CO2 .............
. Production of [IBM] ...................
. Exposure of Plant Tissue to [13N] ............
. Extraction of the Labeled Products after the Assimilation
of [13N] . . . ......................
. Distribution of Label in Glutamine after the Assimilation
l3 +
of NH4 .........................
CHAPTER l The Rate of Dark CO2 Fixation during the Development
of the Soybean Nz-Fixing System ...........
RESULTS ...........................
A.
B.
CO2 Fixation Assay ....................
Effect of Detachment of Nodules and Concentration of CO2
on the Rate of Dark C02 Fixation . - ...........
. Effect of CO2 Concentration on 02 Consumption and
CZHZ Reduction ......................
. Dark CO2 Fixation, CZHZ Reduction and Concentration of
Organic Nitrogen in the Xylem Exudate during the
Development of Soybean Plants ..............
DISCUSSION ..........................
Page
35
36
36
37
38
4O
42
44
44
45
46
47
47
48
49
52
59
L_______—---IIIlllIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Page
CHAPTER 2 The Products of Dark 602 Fixation in Soybean Nodules 64
RESULTS ........................... 64
A. Labeling of Organic Acids ................ 64
B. Labeling of Amino Acids ................. 66
C 4C02—Pulse 12COZ-Chase Experiment ............ 66
D. Position of [14C] within Aspartate and Glutamate ..... 68_
E. Labeling of Asparagine and Glutamine ........... 7l
DISCUSSION .......................... 74
A. Initial Step of Dark CO2 Fixation in Nodules ....... 74
B. Loss of Recently-Fixed C02 from Nodules ......... 77
C. Amino Acid Metabolism .................. 80
+
CHAPTER 3 The Effects of N05 and NH4 on Dark C02 Fixation and
Amino Acid Metabolism in Soybean Nodules and Roots . 88
RESULTS ........................... 88
A. Effect of Inorganic Nitrogen on C2H2 Reduction and
Distribution of Nitrogen in the Xylem Exudate ...... 88
B. Effects of N05 and NH: on Amino Acid Metabolism in Roots
of l3-day—old Plants ................... 95
l. Concentrations of Amino Acids ............. 95
2. Products of Dark CO2 Fixation ............. 97
C. Effects of N0; and NH: on Amino Acid Metabolism in Roots
of 29-day-old Plants ................... 99
l. Concentrations of Amino Acids and Organic Acids . . . . 99
2. Rates of Dark CO2 Fixation .............. 99
3. Products of Dark C02 Fixation ............. l02
4. Specific Activities of Amino Acids in Roots Exposed to
4co2 . . ' 102
0. Effects of N03 and NH: on Amino Acid Metabolism in Nodules 109
E. Rates of Incorporation of Carbon Derived from Dark CO2
Fixation into Amino Acids in Roots and Nodules ...... lll
vi
¢
F. Pathway of Asparagine Synthesis in Roots .........
l. Labeling of Asparagine from 14CO2 Incorporated in Roots
2. Synthesis of Asparagine from [2, 3- 3H]Aspartate and
[4 -] 4C]Aspartate ...................
DISCUSSION ..........................
A. Time Course of the Effect of NO3 and NH4 .........
B. Effects of NO3 and NH+ on Nodule Metabolism .......
C. Effects of NO3 and NH4 on Root Metabolism ........
1. Dark CO2 Fixation ...................
2. Synthesis of Amino Acids ...............
3. Specific Activities of Glutamate and Glutamine
D. Asparagine Synthesis ...................
CHAPTER 4 Dark CO2 Fixation in Alder Nodules .........
RESULTS ...........................
DISCUSSION ..........................
CHAPTER 5 The Incorporation of 13N-Labeled Tracers in Soybean
and Alder Nodules and Soybean Roots .........
RESULTS ...........................
DISCUSSION ..........................
SUMMARY ............................
REFERENCES ...........................
vii
Page
113
113
114
123
123
124
126
126
126
128
130
132
132
139
142
142
152
157
161
10.
11.
12.
13.
14.
LIST OF FIGURES
Metabolism of N2, NO3 and NH: in Nodulated Soybean Roots . .
Proposed Sources of the Atoms in Allantoin and Synthesis
of Tetrahydrofolate Derivatives ..............
Effect of the Concentration of CO2 on the Rates of Dark
CO2 Fixation in Roots and Nodules of Intact Soybean Plants .
Rates of Dark CO2 Fixation and C2H2 Reduction and the
Composition of the Xylem Exudate during the Development
of Soybean Plants .....................
Correlation of the Concentration of Amino Acids in Xylem
Exudate with the Rate of Dark CO2 Fixation in Nodules
Incorporation of Label into Organic Acids and Neutral Compounds
14
in Nodules Exposed to CO
2 ................
Time Course of the Distribution of Label in Organic Products
in Nodules Exposed to 14CO2 ................
Distribution of Label in Organic Compounds in Nodules during
a 14coz-Pu1se 12coz-cnase .- ................
Time Course of the Specific Activities of the Amino Acids
Labeled in Nodules Exposed to 14CO2 ............
Proposed Pathways for the Exchange of Label into Organic and
Amino Acids of Nodules Exposed to 14co2 ..........
Proposed Pathway for the Exchange of Label from 14CO2
Incorporated in Nodules into C-1 and C-4 of Oxalacetate
Proposed Pathway for the Exchange of Label into Serine and
Glycine in Nodules Exposed to 14CO2 ............
Proposed Pathway for the Exchange of Label into Alanine
in Nodules Exposed to 14CO2 ................
Time Course of the Effect of NO- on the Distribution of
3
Nitrogen in the Xylem Exudate and the Rate of C2H2 Reduction
viii
Page
20
51
57
58
65
67
7O
72
76
78
82
86
9O
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
_ Page
Time Course of the Effects of N03 and NH: on the
Distribution of Nitrogen in the Xylem Exudate ....... 94
Effect of N03 and NH: on the Distribution of Label in
Amino Acids in Roots Exposed to 14CO2 . . . . . ...... l05
Effect of N03 and NH: on the Specific Activities of Several
Amino Acids in Roots Exposed to 14CO2 . .‘ ......... 108
Effects of N05 and NH: on the Amounts of Several Amino Acids
in Nodules ......................... 110
Time Course of the Distribution of Label in Organic
Compounds in NOS-Treated Roots Exposed to a 14COZ-Pulse
~12C02-Chase ..................... V . . . 115
Proposed Pathways for the Incorporation of Label from
[2.3-3H]Aspartate and [4-]4C1A5partate into Asparagine . . . 118
Incorporation of Label and the 3WHO Ratio in Organic Products
in Roots Incubated with [2,3-3H]Aspartate and [4-]4CJAspartate 122
Distribution of Amino Acids in Xylem Exudate and Extracts of
Nodules of Alders ..................... 133
Time Course of the Distribution of Label in Organic Compounds
in Alder Nodules Exposed to 14C02 ............. 135
Incorporation of Label into Organic Acids and Neutral Compounds
in Alder Nodules Exposed to 14C02. ............. l36
Time Course of the Distribution of Label in Organic Acids in
Alder Nodules Exposed to 14co2 ............... 138
Proposed Pathways for the Incorporation of Label into Citrulline
in Alder Nodules Exposed to 14CO2 ............. 140
Incorporation of Label into Organic Products in Alder Nodules
Incubated with 13NH; .................... 144
Time Course of Incorporation of Label into Glutamine and
Glutamate in Soybean Roots and Alder Nodules Incubated with
13NH+ 146
4 OOOOOOOOOOOOOOOOOOOOOOOOOOO
ix
29.
30.
Page
Incorporation of Label into Organic Products in Soybean
Roots Incubated with 13N03 or 13NH: ............ l48
Incorporation of Label into Organic Products in Soybean
13 +
150
Nodules Exposed to [BNJN2 or NH4 ............
10.
11.
12.
13.
14.
LIST OF TABLES
HPLC Elution Gradient for Amino Acid Analysis .......
Effect of the Concentration of CO2 on the Rate of Dark CO
Fixation in Attached and Detached Nodules .........
Effect of the Concentration of CO2 on Nodule Respiration . .
Effect of the Concentration of CO2 on the Rate of C2H2
Reduction ..........................
Distribution of Label and Estimated Rates of Incorporation
of Carbon Derived from Dark CO2 Fixation into Amino Acids
in Nodules Exposed to 14CO2 ................
Effects of N03 and NH: on the Concentration of Amino Acids
in Roots and Nodules of Young Plants ............
Effects of NO3 and NH4 on the Distribution of Label
Incorporated from 14CO2 in Roots and Nodules of Young Plants
Effects of N05 and NH: on the Concentration of Amino Acids
in Roots of 29-day-old Plants ...............
Effect of KNO3 on Concentration of Several Organic Acids . .
Effects of N03 and NH: on the Rate of Dark CO2 Fixation in
Roots and Nodules .....................
Estimated Rates of Incorporation of Carbon Derived from
Dark CO2 Fixation into Several Amino Acids in Roots and
Nodules - ..........................
3H/MC Ratio in Aspartate and Asparagine after Incubation
of Roots with [2. 3-3H]Aspartate and [4-]4C]Aspartate .
Effect of NH4 and Aminooxyacetate on Production of [ 3H]H2 0
from [2, 3- 3H]Aspartate and 14CO from [4- 4C]Aspartate . . .
2
Incorporation of Label into Organic Products in Soybean
Roots Incubated with 13NHZor 13N03 ............
xi
Page
34
50
53
54
83
96
98
100
101
103
112
119
121
149
EZ‘I
aln
asn
asp
cit
citl
FH
fum
GDH
gln
glu
91y
GOGAT
GS
HPLC
mal
MSX
N-free
o.a. '1'
neu.
OPA
PEPC
POPOP
PPO
LIST OF ABBREVIATIONS
allantoate
asparagine
aspartate
citrate
citrulline
tetrahydrofolate
fumarate
glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase
[deaminating] EC l.4.l.3)
Iglutamine
-glutamate
glycine
glutamate synthase (L-glutamatezoxidoreductase [transaminating]
cofactor NAD+ EC 1.4.1.14; NADP+ EC 1.4.1.13; ferredoxin EC 1.4.7.1)
glutamine synthetase (L-glutamatezammonia ligase [ADP] EC l.4.l.3)
high performance liquid chromatography
malate
methionine sulfoximine
free of nitrogenexcept for N2
organic acids plus neutral compounds
orphthalaldehyde
phosphoenolpyruvate carboxylase (orthophosphate:oxalacetate
carboxyl-lyase [phosphorylating] EC 4.l.l.3l)
l,4-bis|2-(4-methyl-5-phenyloxazolyl)]-benzene
2,5-diphenyloxazole
xii
SEY‘
SUC
UV
serine
succinate
ultraviolet
INTRODUCTION
One of the problems the governments of the world are facing and
will continue to face is how can the people of the world be fed. Up
until recently, the efforts in the area of agricultural have concentrated
on developing new varieties of plants that respond to increased amounts
of fertilizer nitrogen (1). However, because of the increasing costs
of fertilizer nitrogen that is produced in chemical factories (2), the
developing countries can no longer afford to purchase the nitrogen that
makes these fertilizer-intensive plants more productive than other
varieties. In the future, biological N2 fixation is expected to
partially replace chemical factories as the source of nitrogen for
agricultural purposes. The amount of nitrogen that biological N2 fixation
, contributes may be dramatically increased by understanding the processes
by which N2 is reduced and is converted into usable forms of organic
nitrogen.
The synthesis of amino acids, in particular those most closely
associated with the tricarboxylic acid cycle, is especially important in
biological N2 fixation because of its role in the initial steps of
converting inorganic nitrogen to organic nitrogen. The enzymes that
catalyze the synthesis of these amino acids have, for some systems, been
isolated and partially characterized (3,4). Recent studies using
labeled tracers have partially identified the pathways by which nitrogen
is incorporated into organic compounds (5,6). Further work is needed to
confirm the pathways of amino acid synthesis and to determine what factors
regulate amino acid metabolism in biological systems that fix N2. My
research was directed towards the further understanding of the metabolism
of amino acids in the soybean -Rhizobium japonicum and alder-Frankia sp.
1
systems.
Previous investigators have proposed that dark CO2 fixation
in lupin (Lupinus angustifolius) (7) and broad bean (Vicia faba)
nodules (8) provides carbon skeletons for the synthesis of amino
acids. The synthesis of amino acids, and in particular asparagine,
is especially important in the nodules of these plants because amino
acids may represent up to 100% of the organic nitrogen that is
transported from the root to the shoot (9,10). Organic nitrogen is
transported from the root system of alders primarily as the amino acid
citrulline (ll) and from soybeans that are dependent on N03
primarily as asparagine (12). Therefore, carbon that is incorporated
during dark CO2 fixation in these systems might be expected to be
incorporated into amino acids. In contrast, soybeans that are dependent
on N2 that is fixed in the nodule synthesize and transport the
ureides, allantoin and allantoate, instead of amino acids (12). The
dependence of soybean nodules on dark CO2 fixation might be
expected to be minimal when compared to alder nodules or the root
systems of soybeans that are dependent on N03.
In this thesis I will address the following questions:
l) Does dark CO2 fixation occur in soybean and alder nodules and
soybean roots?
2) Is label incorporated into amino acids in soybean and alder nodules
and soybean roots exposed to 14C02?
3) What are the effects of N03 or NH: on amino acid metabolism in
alder and soybean nodules and soybean roots?
I initially characterized dark CO2 fixation in soybean
nodules (question l) and then determined the fate of the label that
was incorporated after exposure of soybean and alder nodules to
14CO2 (question 2). The effect of N03 or NH: on amino acid
metabolism in soybean roots and nodules was examined by monitoring
the labeling of amino acids after exposure of tissues to 14CO2
and by determining the concentrations of a number of amino acids
(question 3). In other experiments, [13
examine the metabolism of N03, NHZ, and N2 in nodulated soybean
N] was used as a probe to
roots and NH: in alder nodules (question 3).
I have arranged the results and discussion part of this thesis
into the following chapters:
Chapter 1 - The Rate of Dark CO2 Fixation during the Development
of the Soybean NZ-Fixing System
Chapter 2 - The Products of Dark CO2 Fixation in Soybean Nodules
Chapter 3 - The Effects of N0; and NH: on Dark CO2 Fixation
and Amino Acid Metabolism in Soybean Nodules and Roots
Chapter 4 - Dark CO2 Fixation in Alder Nodules
Chapter 5 - The Incorporation of 13N-Labeled Tracers in
Soybean and Alder Nodules and Soybean Roots
The work that is presented in Chapter 4 was done in collaboration with
Dr. P. McClure and that in Chapter 5 with Dr. K.R. Schubert.
LITERATURE REVIEW
Soybean plants with the bacteria Rhizobium japonicum (13)
and alder plants with the bacteria Frankia sp. (14) are able to
form symbioses that can use N2 as their sole source of nitrogen
(15,16). During the development of these symbioses, the growth
and metabolism of both the plant and the bacteria are altered. Upon
infection by the bacteria, the root tissue develops into root
nodules, the organ of N2 fixation. The bacteria differentiate into
a form called endophytes (17,18) which are enclosed in vesicles
contained within the plant cell (19). These endophytes in the
R. japonicum - soybean symbiosis are called bacteroids (18,20).
Both plant and bacteria are necessary for the efficient
fixation of N2 in these symbioses. The plant provides the environment
and the source of energy necessary for N2 fixation. The endophytes
synthesize nitrogenase and nitrogenase reductase, the enzyme complex
which reduces N2 to NH: (21,22). Ammonium is released from the
bacteroids (23,24) and enters the host cell where it is assimilated (25).
Based on both the spatial and metabolic organization of the nodule,
the endophyte may be thought of as a plant cell organelle that reduces
NZ to NHZ. A skematic of some of the metabolic processes occuring in
soybean nodules is shown in Figure 1.
A. DARK COO FIXATION
Various tissues (26—30) including legume root nodules (7,8,31,32)
directly incorporate CO2 in a process referred to as dark CO2 fixation.
Phosphoenolpyruvate carboxylase (PEPC), which catalyzes the synthesis
of oxalacetate, was proposed to be the enzyme that is primarily
4
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The enzyme isolated from pea leaves has Km values for glutamate,
+
4
Glutamine synthetase in prokaryotes is regulated by an elaborate
NH and MgATP of 8 mM, 0.019 mM and 0.5 mM, respectively (65).
mechanism involving the adenylylation and deadenylylation of the
enzyme (66). This mechanism, however is apparently not involved in
the regulation of GS in higher plants (67,68). Glutamine synthetase
in plants does appear to be regulated by energy charge (3,69) and
light (70). The effect of light on the activity of GS in
Chlorella sorokiniana was examined by growing cells on a 7 h light:
5 h dark photoperiod and monitoring GS activity (70). The specific
activity of GS from cells grown in the dark for 5 h followed by l h
of light was 7 times the activity from cells examined immediately
after th 5 h dark period. This increase in specific activity of GS
in the light was not dependent on ge_ngyg_synthesis of GS. Rhodes
et a1. (71) showed that the activity of GS purified from Lemna minor
is reversibly altered by the titration of four thiol groups of the
11
enzyme with thiol reagents. The regulation of GS in_yjyg_by
altering thiol groups suggests that GS activity might be modulated
by the LEM (light effect modulators) system as proposed by
Anderson and Avron (72) or a similar system proposed by Buchanon (73).
Glutamine synthetase has recently been found to exist as
several different isozymes (74-77). One of the isozymes in rice
plants is localized in the chloroplasts, a second in the cytosol of
leaves and a third in the roots (74). Although the kinetic analysis
is preliminary, several investigators (75,76) have proposed that the
isozymes have different roles in nitrogen assimilation.
Glutamate synthase catalyzes the following reaction:
2-oxoglutarate + L-glutamine + NAD(P)H + H+-—-—«-2 L-glutamate +
NAD(P)+
Several tissues including pea leaves (60) contain a GOGAT enzyme
that uses reduced ferredoxin instead of reduced pyridine nucleotides
(58). The enzyme isolated from lupin nodules has Km values for
2-oxoglutarate, glutamine, and NADH of 39 uM, 400 uM and 1.3 uM,
respectively (4). The regulation of GOGAT jn_yjyg_is not completely
understood; however, product inhibition may be involved. For the
enzyme from lupin nodules, NAD+ is a competitive inhibitor (Ki=0.l mM)
with respect to NADH and glutamate is a competitive inhibitor (Ki:
0.7 mM) with respect to 2-oxoglutarate (4).
2. Pathway of NH: Assimilation
The proposal that GS/GOGAT is the primary pathway for NH:
assimilation in plants is supported by two lines of evidence:
1) tracer studies with labeled nitrogen, either [13N] or [ISN] and
2) measurements of enzyme levels. If GS and GOGAT are the enzymes
‘- I
12
responsible for the assimilation of NH: then glutamine would be
expected to be the initial organic compound formed from NH: and
glutamate the second. Although Kennedy (6,52) indicated that glutamate
and glutamine were the primary amino compounds synthesized in Nz-fixing
nodules of serradella plants (Ornithopus sativus) exposed to [15N1N2,
he concluded that glutamate is the primary amino acid synthesized
from NH: produced during N2 fixation. The use of the radioactive
isotope of nitrogen,[13N] (half-life = 10 min),has allowed other
investigators to determine the products of NH: assimilation after
shorter labeling periods. Glutamine accounted for 82% of the
total label in the organic compounds in soybean nodules that were
exposed to [13N]N2 for 20 sec, while glutamate accounted for the
remaining 18% (5). The percentage of label in glutamine declined
and the percentage in glutamate increased after longer periods of
exposure. Similar results were obtained using tobacco cells
that were incubated with 13NH: or 13N03 (78) and rice roots incubated
with 15NH: (79) or 15N03 (80). The results of these studies are
consistent with NH: being assimilated into glutamine and glutamate
by GS and GOGAT.
The pathway of NH: assimilation has been further studied
using inhibitors of GS and GOGAT. Methionine sulfoximine (MSX),
an inhibitor of GS, when added to a number of tissues has been shown
to inhibit the incorporation of labeled-NH4+ into glutamine and
glutamate (78,81,82), suggesting that GS is the enzyme primarily
responsible for the initial assimilation of NH2. In several of the
experiments (78,81), MSX inhibited the synthesis of glutamine more
than glutamate. However,if GS is the sole enzyme that is responsible
13
for NH: assimilation, then inhibiting GS should inhibit the
incorporation of labeled-NH: into glutamine and glutamate to the
same extent. To explain the paradox between this hypothesis and
the observed results, Skokut gt al.(78) proposed that one of two
events is occurring. One, either a small portion of the 13NH: is
assimilated by GDH or two, the GS activity that is not eliminated by
MSX synthesizes glutamine which is preferentially used for the synthesis
of glutamate. Azaserine, an inhibitor of GOGAT, inhibited the incor-
poration of 13
NH: into glutamine and glutamate by 56 and 99%,
respectively (78), which is consistent with GOGAT being the enzyme
primarily responsible for glutamate synthesis.
The second line of evidence for GS/GOGAT being the primary route
for NH: assimilation comes from the measurement of enzyme levels
‘jgnyitrg. The levels of GS in nodules of 11 different species of
legumes were an average of l6-times higher than the levels of GDH
when both enzymes were measured with saturating amounts of substrates
(83). When the relative Km values for each of the enzymes and the
estimated concentration of NH: in the nodules are considered, GS
would be more than 90% saturated with NH; whereas GDH would operate
at about 10% of its maximum velocity (83). On this basis, Boland
gt 31. (83) concluded that GS and GOGAT are responsible for the primary
assimilation of newly fixed NH: produced by the bacteroid. Based
on similar considerations as those above, the activity of GDH in
Lemna minor can be estimated to be between 0.4 and 4.0% the activity
of GS (71,84) suggesting that GS is also responsible for the
assimilation of NH: in this organism as well. In summary, it is
generally concluded that GS/GOGAT is the primary pathway or NH:
14
assimilation (85,86). However, the possibility that GDH or some
other enzyme assimilates a portion of the NH: has not been excluded.
Once NH: is assimilated into glutamine and glutamate, these
amino acids are used in the synthesis of other nitrogenous compounds
such as aspartate, asparagine, allantoate and citrulline. Aspartate
is synthesized by aspartate aminotransferase which catalyzes the
following reaction:
oxalacetate + L-glutamate 4—————_'> L-aspartate + 2-oxoglutarate
Several different isozymes of aspartate aminotransferases are found
in soybean (87) and lupin nodules (88). The activity of a specific
isozyme of aspartate aminotransferase increased during the development
of lupin nodules (88). Reynolds and Farnden (88) proposed that this
isozyme is specifically associated with N2 fixation.
3. Asparagine Synthesis
Asparagine is one of the most prominent amino acids in many
plants. It is used for the transport of nitrogen within plants
(for a recent review see ref. 89) and for the storage of nitrogen in
a form which is less toxic than free NH: (90). Asparagine in soybeans
grown on N05 (12) and in lupins (9) accounts for up to 70% of the
organic nitrogen translocated from the roots to the shoot. In the
shoot the asparagine is degraded and the nitrogen and carbon are used
to synthesize other compounds (91,92).
Asparagine accumulates during seed germination (93-96). The
carbon skeletons of the storage protein are oxidized to provide
energy for the developing plant. The NH: that remains after the
protein is degraded is used to synthesize asparagine. With the
15
development of the photosynthetic apparatus of the developing plant
and the ability to fix carbon, the accumulated asparagine is
degraded and the nitrogen is used for protein synthesis (for a
review see ref. 96).
Three different pathways have been proposed for asparagine
synthesis: 1) direct amidation of aspartate (97-100), 2) an indirect
pathway via succinate and/or fumarate (101) and 3) hydrolysis of
cyanoalanine (102-104).
The evidence for the first pathway includes enzymological data
and labeling studies. An asparagine synthetase catalyzing the following
reaction has been detected in a number of tissues (98,100,103):
L-aspartate + MgATP + L-glutamine ——- L-asparagine + L-glutamate +
MgAMP + PPi
The asparagine synthetase in lupin seedlings has Km values for aspartate,
MgATP and glutamine of 1.3 mM, 0.14 mM and 0.16 mM, respectively (100).
Label from [14C]aspartate vacuum infiltrated into soybean leaves was
incorporated into asparagine. This labeling of asparagine was
inhibited by MSX and stimulated by glutamine (99). Stewart (99)
concluded that asparagine is synthesized jn_yjyg_by a glutamine-
dependent asparagine synthetase. Because uniformly labeled aspartate
was used, the results do not exclude the possibility that succinate
and/or fumarate are intermediates.
Exogenous aspartate does not appear to be the direct precursor
of asparagine in pea roots (101). On the basis of isotope-competition
experiments, Mitchell and Bidwell (101) concluded that succinate and/
or fumarate are more direct precursors for asparagine synthesis than
16
exogenous aspartate. They further proposed that asparagine synthesis
is compartmentalized. Lever and Butler (105) reached similar
conclusions and further suggested that aspartate was an intermediate
in the synthesis of asparagine from fumarate. If this proposal is
true, then the first two pathways listed above are actually the result
of the relative distribution of aspartate between two pools: one
that is used for asparagine synthesis, and a second that is in
equilibrium with succinate and/or fumarate. Streeter (106) using
soybean cotyledons concluded that a fraction of the asparagine is
synthesized via succinate but that another fraction is synthesized
directly from aspartate.
Cyanoalanine, formed by the condensation of HCN and cysteine,
can be hydrolyzed to form asparagine (102-104). While the
enzymes catalyzing this reaction have been isolated, the extent
to which this pathway contributes to the synthesis of asparagine
appears to be limited jn_ijg_because the amount of CN' that is
available to the plant appears to be minimal (89).
The status of asparagine synthesis in soybean nodules is not
clear. Although Streeter (106) reported that he was unable to
detect a glutamine-dependent asparagine synthetase in soybean
nodules, two preliminary communications (107,108) have reported
measuring asparagine synthetase activity in cell-free extracts of
soybean nodules. The results of experiments attempting to incorporate
label into asparagine in intact nodules are mixed. Asparagine accounted
for 34 and 86% of the total label incorporated in soybean nodules
15 +
that were vacuum infiltrated with NH4 and [amide-15N]glutamine,
respectively (109). In contrast, asparagine accounted for less than
17
1% of the label incorporated in soybean nodules that were exposed to
[13N]N2. Based on the incorporation of label from 15NH: and
[amide-lsNJQlutamine into asparagine and the recent reports of the
presence of an asparagine synthetase in soybean nodules,
asparagine can be synthesized in these nodules. Whether asparagine
is synthesized in the nodule from nitrogen derived from N2 is not
clear.
4. Citrulline Synthesis
Citrulline is the primary product of N2 fixation in alder
root nodules (110). It also accounts for a large percentage of
the nitrogen transported from the root to the shoot in several
species of plants including alders (A130; sp.) and persimmons
(Diospyros kaki) (11). The proposed pathway for citrulline synthesis
in higher plants is : l) N-acylation of glutamate, 2) conversion of
N-acylglutamate to N-acyl ornithine, 3) removal of the acyl group
to form ornithine, 4) synthesis of carbamyl phosphate and 5) synthesis
of citrulline from ornithine and carbamyl phosphate (for a review
see ref. 111).
5. Allantoin and Allantoate Synthesis
The ureides, allantoin and allantoate, account for up to 98% of
the nitrogen that is transported to the shoot from the root
nodules of several species of grain legumes, including soybeans
(12), cowpeas (Vigna unguiculata; 112) and dwarf french beans
(Phaseolus vulgaris; 31). Allantoate is proposed to be synthesized
from purines which are synthesized gg_novo in the nodule (113,114;
for a review see ref. 115,116). According to this proposal, the
, W'fi
I
I
18
initial step is the synthesis of phosphoribosylpyrophosphate.
Then in a series of reactions, the amide nitrogens of two glutamines,
the amino group of aspartate, glycine, carbon from bicarbonate and
two carbons from derivatives of tetrahydrofolate are added
resulting in the synthesis of inosine monophosphate. This compound
in another series of reactions is metabolized to form allantoin and
allantoate. Figure 2 illustrates the origins of the atoms of
allantoate.
+
4
D. EFFECT OF NO; AND NH ON NO FIXATION
The fixation of N is both inhibited (12,117-121; for
2
reviews see ref. 122,123) and stimulated (121,124) by N05 and
NH1. Whether N03 or NH: inhibits or stimulates depends on the
timing and the amount of nitrogen that is applied. As stimulants,
N03 and NH: are suggested to alleviate nitrogen stress, especially
when they are added during the early development of the plant
(124). The rate of N2 fixation of 21 to 28-day-old soybean plants
that had been treated with 7 mM KNO for the first 14 days after
3
they had been planted was three times that of plants that had
3 3
act to inhibit initiation and subsequent nodule development
not been treated with KNO (124). As inhibitors N0 and NH;
(for a review see ref. 125). The inhibitory effects of N0; and
NH: on N2 fixation are also observed when the nitrogen is added
to the plants after N2 fixation is established. The rate of
C2H2 reduction ( an assay for nitrogenase activity) in 78-day-old
soybean plants that had been treated with 7 mM KNO3 for the
prior 7 days was 30% the rate of plants that had not been treated
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21
with KNO3 (12).
The time necessary for the first effects of N03 or NH:
on N2 fixation to be noticeable is of the order of l to 2 days
after the initial application. Two days after the addition of
7 mM KNO3 to 24-day-old soybeans, the rate of C2H2 reduction of
the KNO -treated plants was 80% of that of the control plants (124).
3
Similar observations have been made using peas (120) and soybeans
treated with NH4C1 (124).
Two mechansims have been proposed to explain the inhibitory
effects of N03 on N2 fixation. The first is that the uptake
and utilization of N03 influences the distribution of photosynthate
to the various organs of the plant (124,126,127). The second
mechanism is that N03 or one of the products of its reduction has a
direct effect on N2 fixation (124,128,129).
The fixation of N2 is closely linked to the availability
of photosynthate. Manipulations that result in increased rates
of N2 fixation such as removing other sinks for photosynthate
(130) or increasing the rate of photosynthesis (131,132) are
said to have acted by increasing the supply of photosynthate to
the nodules, thereby increasing the energy available for N2 fixation.
Manipulations such as removing the shoot of a plant (133) or
decreasing the light intensity (134) result in a reduced rate
of N2 fixation presumably by reducing the flow of photosynthate
to the nodules. The proponents of this mechanism argue that
upon the application of N03 the sites of N03 reduction, the leaves
and roots, retain the photosynthate that was previously destined
to be used by the nodule for N2 fixation. The nodule, therefore,
22
has a reduced rate of N2 fixation because of the reduced supply
of energy that is available.
This proposed mechanism is based, in part, on the effect
of N05 on the distribution of photosynthate. Small and Leonard
(126) treated nodulated pea and clover plants with 14 mM KNO3 for
5 days, at which time the plants were exposed to 14
CO2 for 15 min
during the photosynthetic period. Nodules from plants that
were treated with KNO3 contained less label than nodules from
plants that had not been treated with KN03. In contrast, the
roots of plants that were treated with KN03 had higher levels of label
than roots from control plants. Similar experiments (127,134) with
soybeans have yielded similar results.
The second proposed mechaism to explain the effect of N03
on N2 fixation is that N03 or one of the products of its reduction
directly affects nodule metabolism (124,128,129). Based on the
effects of tungsten on the inhibition of N2 fixation by hog,
Harper and Nicholas (135) concluded that N03 must be metabolized
to affect N2 fixation. This suggests that N03 does not directly
affect N2 fixation, but does not eliminate the possibility that
one of the metabolites of N03 reduction such as NOE, NHZ,
glutamine or glutamate does so. Houwaard (l36) concluded that
NH: must be assimilated to inhibit nitrogenase activity. The
rate of C2H2 reduction in nodules that were treated with NH401
for 4 h was 40% the value for control nodules. If MSX was
.4.
4
acids and NH: can inhibit N2 fixation by affecting the synthesis of
included, NH had no effect on the rate of 02H2 reduction (136). Amino
nitrogenase (137) but they do not appear to inhibit purified
23
nitrogenase (138).
Nitrite, the initial product of N03 reduction, was suggested
to mediate the inhibition of N2 fixation by N03 because of the
low concentrations of N05 (0.1 to 0.8 mM) needed to inhibit C2H2
reduction in isolated bacteroids (139) and by purified nitrogenase
(140). The N03 reductase in bacteroids was thought to be responsible
for the synthesis of N05 that inhibits N2 fixation; however,
Gibson and Pagan (141) and Manhart and Wong (142) concluded on
the basis of their work with N03
that the bacteroid N03 reductase is not involved in the inhibition
reductase mutants of rhizobia,
of N2 fixation by N03. Streeter (129) in a recent preliminary
report suggested that the N03 reductase in the plant portion of
the nodule produces NOE which inhibits N2 fixation. Direct
evidence linking N03 reduction in the nodule to the inhibition
of N2 fixation is not yet available.
E. EFFECTS OF NO3
AND NH: ON NITROGEN TRANSPORT IN SOYBEANS
Nitrate and ammonium affect the amount of amino acids that
are transported from the soybean root to the shoot, indicating that
N03 and NH: affect amino acid metabolism in the root system
(12,143). Amino acids account for 40% of the total nitrogen in the
3
as compared to 20% or less in the xylem exudate of plants dependent
xylem exudate of plants that are dependent on NO for nitrogen
on N2 fixation (12). The synthesis of asparagine, in particular,
appears to be stimulated in the roots of plants treated with
N03 (12,144).
The effect of NH: on the transport of nitrogen in soybeans
has not been well documented. In one study (143), 5-day-old
24
soybeans were treated with 10 mM NH4C1 or 10 mM KNO3 for 8 days,
at which time the xylem exudate was collected and analyzed. The
+.
4
contained 6-times the amount of glutamine and 2-times the amount
xylem exudate of the soybeans that had been treated with NH
of asparagine as found in the xylem exudate of the NOé-treated
plants.
F. EFFECTS OF NH} ON AMINO ACID SYNTHESIS
Ammonium may affect the synthesis of amino acids either as
a substrate or as a modulator of enzyme activity. In response
to the addition of high levels of NHZ, plants will synthesize'
large amounts of the amides, asparagine and glutamine (145).
This increased synthesis is apparently because of the increased
concentration of substrates, NH; for GS and glutamine for
asparagine synthetase.
Ammonium has been suggested to affect amino acid synthesis
by directly altering the activities of certain enzymes. Several
investigators (146,147), based on the levels of metabolites in
Chlorella pyrenoidosa or alfalfa (Medicago sativa) leaves after
the addition of NH2, proposed that NH: stimulates pyruvate kinase.
This stimulation presumably results in an increase in the amount of
ketoacids for amino acid synthesis. The jn_vitro activity of
pyruvate kinase, which requires a monovalent cation for activity
+
4
not sufficient to saturate the enzyme. Although similar results
(148), can be increased by NH but only if levels of K+ are
were observed in Anabaena cylindrica (149) as in C, pyrenoidosa
after NH: was added (146), the in vitro activity of pyruvate kinase
25
from A, cylindrica is not stimulated by NHZ. These results
suggest that NH: increases the flow of carbon through pyruvate
kinase by some mechanism other than direct stimulation of the
enzyme.
Ammonium may also affect amino acid metabolism by regulating
the synthesis or degradation of enzymes that assimilate nitrogen.
The levels of GDH increase in several plants when high levels of
NH: are present (62-64, 150,151). In Chlorella sorokiniana,
NH: appears to regulate the activity of the NADP+-dependent
isozyme of GDH by reducing the rate at which this isozyme
is inactiviated (152). In contrast, the increase in GDH activity
in Lemna minor (64) and oat (Avena sativa) leaves (62) after the
addition of NH: appears to be the result of gg_ngy9_synthesis.
Rhodes et_al,(64) suggested that glutamine, a product of NH:
assimilation, rather than NH: regulates the synthesis of GDH.
Although it is apparent that NH: increases the rate of synthesis
of amino acids, the mechanisms by which it does so are not completely
understood.
MATERIALS AND METHODS
A. GROWTH OF PLANTS
Soybean seeds (Glycine max L. Merr. cv. Amsoy 71) were
inoculated with broth cultures of Rhizobium japonicum USDA strain
3Ilb 110 (obtained as a gift from D. Weber, USDA, Beltsville,
MD) and planted in 20-cm plastic pots filled with Perlite. All of
the plants were irrigated with N-free nutrient solution (153)
until 15 days after they were planted unless otherwise noted. The
plants were then divided into four groups. One group continued
to receive N-free nutrient solution while the other three groups
of plants were irrigated daily with 1 liter of N-free nutrient
solution that was supplemented with KNO3 to a final concentration
of 3 or 10 mM or (NH4)ZSO4 to a final concentration of 1.5 mM.
Plants, nodules and roots of plants that were treated with (NH4)ZSO4
will be referred to as NHZ-treated plants, MHZ-treated nodules and
NHZ-treated roots. A similar format will be used for plants
treated with KNO3. Unless otherwise specified NOé-treated will
refer only to those plants treated with 10 mM KN03. Plants, nodules
and roots of plants that were maintained solely on N-free nutrient
solution will be referred to as control plants, control nodules
and control roots.
Young alder seedlings (Alnus glutinosa) either collected
from the Kellogg Forest, Kalamazoo M1, or grown from seed
(obtained as a gift from 0. Hanover, MSU, E. Lansing, MI) were planted
in plastic pots filled with a commercial peat-vermiculite mixture.
Stationary phase broth cultures of Frankia Avc 11 or Cp 1 (obtained
26
27
as gifts from D. Baker, Middlebury College, Middlebury, VT) or
suspensions of nodules (10 g in 50 ml of H20) obtained from
field-grown plants were used to inoculate the roots of 21 to 28-day-old
alder plants grown from seed. Both soybean and alder plants were
grown in the greenhouse under daylight extended to a photoperiod of
-1
16 h with fluorescent lighting (55 pEom'gsec at pot height).
8. MEASUREMENT OF THE RATE OF C H2 REDUCTION
2
Besides reducing N2, nitrogenase is capable of reducing
C2H2 to C2H4,thereby forming the basis for a convenient and
sensitive assay for nitrogenase activity (154). The rate of
C2H2 reduction was measured on nodules that were still attached
to the roots of either intact plants or roots from which the shoots
were excised or on detached alder and soybean nodules. Intact
soybean plants were removed from the Perlite and each root system
was placed into either a 20-ml single arm Warburg flask or a 50-ml
round bottom flask. A split rubber stopper was then placed around
the stem of the plant and the stopper was inserted into the top
of the flask. Plasticine was used to seal around the plant, stopper
and flask. Alternatively, intact plants were removed from the
Perlite, the shoots were excised and each root system was placed into
a 250-ml Erlenmeyer flask. The flask was sealed with a serum
stopper. For other experiments, soybean or alder nodules were
removed from the plant and were placed in a 10 or 50-m1 Erlenmeyer
flask and the flask was sealed with a serum stopper.
The assay was started by removing 0.1 volume of air and
replacing it with the same volume of C2H2 which was produced
from CaC (Sargent Welch) and water. Gas samples were removed
28
from each flask at various times up to l h and were injected into
a gas chromatograph (Varian 3700). Ethylene was separated from
C2H2 on a Porapak N column (2 m x 2 mm) with either He or N2
as a carrier gas and was detected with a flame ionization detector.
The assay was ended by removing the plant tissue and placing it
in a 70 C oven for 24 h.
C. MEASUREMENT OF ALLANTOATE, ALLANTOIN, NO; AND TOTAL AMINO ACIDS
Allantoin and allantoate were degraded to glyoxylate which
was measured colorimetrically after its reaction with phenylhydra-
zine and K4Fe(CN)6 (155). The assay was linear in the range that
was used, 2 to 100 nmol.
Nitrate was assayed either colorimetrically after its
reaction with salicylic acid (156) or by its absorption of UV
light after analysis by high performance liquid chromatography
(HPLC) (157). The colorimetric assay was linear over the range
that was used, 2 to 150 nmol, as was the HPLC method from 0.1 to
20 nmol.
Total amino acids were assayed colorimetrically after
their reaction with ninhydrin (158) or fluorometrically after
their reaction with g¢phthalaldehyde (OPA) in a procedure adapted
from Hill gt_al, (159). The colorimetric assay was linear over the
range that was used, 10 to 200 nmol. In the adapted procedure,
the GPA solution consisted of methanol (590 pl), saturated sodium
borate (210 pl), ethanethiol (Aldrich) (50 p1), and GPA (Sigma)
(30 mg). One microliter of sample, which contained 1 to 20 nmol of
amino acids was added to 8 pl of OPA solution. One minute later,
2 ml of methanol were added and the solution was vortexed.’ The
29
fluorescence of the derivatized amino acids was measured with
fluorometer fitted with a Corning 7-54 primary filter and Wratten
2A secondary filter. The fluorescent OPA-amino acid adduct
has two excitation maxima, one at 230 nm and one at 330 nm, and
one emission maximum at 460 nm.
D. MEASUREMENT OF THE RATE OF 0 CONSUMPTION
2
The rate of 02 consumption of detached nodules and nodules
of intact plants was measured amperometrically with a Clark
oxygen electrode (YSI 4004, Yellow Springs, OH) (160). An
intact plant was removed from the Perlite and the roots were
trimmed so that they would fit into a 7-ml Lucite chamber in
which the electrode was fitted. A split rubber stopper was placed
around the stem of the plant and the stopper was inserted into the
top of the chamber. Plasticine was used to seal around the stopper
and the plant. Alternatively, nodules were removed from the
plant and placed in the chamber which was then sealed with a
rubber stopper. The system was calibrated by injecting into
the chamber known volumes of O2 (Matheson Gas Products). The
assay was ended by removing the plant tissue and drying it in a 70 C
oven for 24 h.
E. PRODUCTION OF 14CO2
Labeled CO2 was generated by adding [14C]NaHCO3 (59 mCi mmo1“;
Amersham) and 1 ml of 36 N H2504 to a 10-ml flask. The flask was
sealed with a serum stopper and was gently stirred and heated until
the solution was boiling. The 14CO2 that was produced was removed from
the flask with a l or 3-ml syringe.
30
F. EXPOSURE OF PLANT TISSUE T0 14CO
2
Roots and nodules were sealed into flask in a fashion
analogous to that presented in section B. The assay for the
rate of dark CO2 fixation was started by injecting 5 to 20 OCT
14
of C02 into the sealed vessel containing the plant material. For
12
some experiments, CO2 (Matheson Gas Products) was added just
14
prior to the addition of CO2 to increase the CO2 concentration
in the flask. In those experiments in which the products of
14
dark C02 fixation were to be examined 10 to 100 pCi of CO
2
were added.
One minute before an assay was ended, two 0.5 or l-ml gas
samples were removed from the flask so that the specific activity
of the 14
C02 could be determined (section G). The assay for the
rate of dark C02 fixation was ended for soybean nodules and roots
after a 10 min exposure and for alder nodules after 20 min. All
14CO2 fixation assays were ended by removing the plant tissue and
freezing it in liquid nitrogen.
14
G. MEASUREMENT OF THE SPECIFIC ACTIVITY OF CO2
One of the two gas samples that had been removed from each
incubation flask was injected into a glass scintillation vial that
was sealed with a serum stopper. The vial contained 1 ml of
methylbenzethonium (Sigma). One hour after the sample was
injected, 10 ml of scintillation fluid (6 g PPO in 1 liter of
toluene) was added and the radioactivity from the 14C02 which
was absorbed by the methylbenzethonium (161) was determined by
scintillation spectrometry. The CD2 of the second sample was
31
separated on a Porapak N column (2 mex 2 mm) using He as a
carrier gas and was measured with a thermal conductivity detector.
An analyzed gas mixture (COzzozzNz; 0.04: 1.0: 98.96, v/v/v) that
was purchased from Matheson Gas Products was used as a standard.
H. EXTRACTION OF THE PRODUCTS OF 14CO FIXATION
2
The plant material that had been frozen after exposure to
14CO2 was lyophilized. Nodules and roots were separated from each
other, weighed and 10 to 50 mg added to a 15-ml polypropylene
centrifuge tube. The dried tissue was ground with a glass stirring rod
and was extracted three times with 3 ml of ethanoleZO (80:20,v/v).
The slurry was vortexed and then clarified either by centrifugation
at 10,000 g for 10 min or filtration through 0.45 pm Millipore
filters. The clarified solutions from each extraction were combined
and dried jn_yagug at 50 C either to complete dryness or to the
point where the odor of ethanol was not detectable. The remaining
H20 was removed by lyophilization to prevent labile compounds such as
glutamine and allantoate from being destroyed by exposure to 50 C
for longer than necessary. The dried material was redissolved in
0.05 to 1.0 ml of H20 or ethanoleZO (80:20,v/v). The radioactivity
of an aliquot of the redissolved material was measured by scintillation
spectrometry after Triton X-100 scintillation fluid (666 m1 toluene,
333 m1 Triton X-100 (Research Products Int. Corp., Mount Prospect,
IL), 5 g PPO, 0.1 g POPOP) was added.
32
I. SEPARATION OF THE PRODUCTS OF 14CD FIXATION
2
1. High Performance Liquid Chromatography (HPLC)
Amino acids were separated and analyzed by a procedure
adapted from Hill gt al, (159). The amino acids were derivatized
with OPA and ethanethiol to form a fluorescent product. The amino
acid adducts were then separated by reverse-phase liquid chromatography.
The amino acids in plant extracts were derivatized by adding 10 pl
of a nodule or root extract (representing about 3 mg dry weight) to
25 pl methanol, 10 pl of saturated sodium borate and 5 pl of OPA
solution. The OPA solution contained 0.9 m1 methanol, 0.1 m1
saturated sodium borate, 50 pl of ethanethiol and 60 mg of OPA.
Approximately 1 min after the OPA solution was added to the sample,
the solution containing the aminoeacids was added to a microfilter
system (BioAnalytical Systems, W. Lafayette, IN) which was centrifuged
at 8000 g for 2 min to remove particulate material. Twenty microliters
of the resulting filtrate was injected onto the HPLC column
The HPLC system consisted of two pumps, a microprocessor,
a stirring chamber, an injection valve fitted with a 20 p1 sample
loop, either a 5 pm Ultrasphere or a 10 pm Ultrasil C18 reverse-
phase column (Beckman), a fluorometer and a fraction collector. The
fluorometer was fitted with a 70 pl flow cell, a Corning 7-54 primary
filter and a Wratten 2A secondary filter. The column eluate passed
through the fluorometer to a fraction collector where 0.3 to
1.0 ml fractions were collected in scintillation vials. The
radioactivity collected in the scintillation vials was measured by
scintillation spectrometry after the addtion of Triton X-100
33
scintillation fluid. The amino acids in the plant extract were
identified on the basis of the retention times of their OPA
derivatives and those of standard amino acids. The standard amino
acids that were used routinely were aspartate, glutamate, asparagine,
glutamine, serine, glycine and alanine. Occasionally arginine, leucine,
isoleucine, valine, phenylalanine, ornithine, lysine, NH: and
citrulline were used as standards. The concentration of the
standard amino acids in the solution that was injected onto the
HPLC column varied from 0.025 to 0.50 mM per amino acid. The OPA
derivatives of the amino acids were detected with a fluorometer and
were quantified by an electronic integrator. To minimize the time
necessary to analyze each sample, the elution gradient was modified
from that described by Hill 25.91. (159) to that listed in Table l.
2. Ion-Exchange Chromatography
The organic acids were separated from amino acids by
ion-exchange chromatography using a procedure adapted from
Stumpf and Burris (162). Dowex resins were prepared according
to Atkins and Canvin (163). A plant extract was applied to a
disposable pipet that contained 1 ml of Dowex 50-X8 (hydrogen,
200-400 mesh). The organic acids and neutral compounds were
eluted with 2 to 3 m1 of H20. The amino acids were eluted from the
Dowex 50 column with 3 to 6 m1 of 14.5 N NH4OH. The organic acid
and neutral compound fraction obtained from the Dowex 50 column was
_either lyophilized or applied to a 1 ml column of Dowex l-X8
(formate, 200-400 mesh). The neutral compounds were eluted from the
Dowex 1 column with 2 to 3 m1 of H20 and the organic acids were eluted
with 2 to 3 m1 of 16 N formic acid. All fractions were lyophilized
34
Table 1 HPLC Elution Gradient for Amino Acid Analysis
Time
(min)
CH3CN concentration = 12%
Flow rate = 1.5 ml-min'1
0 Sample injection
0 CH3CN concentration increased to 26% at
9 Flow rate increased to 2.0.ml-min'1 at
15 CH3CN concentration increased to 100% at
25 CH3CN concentration decreased to 12% at
28 Flow rate decreased to 1.0 ml-min"1 at
1 at
37 Flow rate decreased to 0 ml-min'
Plant extracts, xylem exudate and standard amino acids were
Comments
Initial conditions
Initial conditions
Rate
Rate
Rate
Rate
Rate
Rate
l%-min'1
0.1 ml-min'
1
1
10.6%fmin'
l4.7%-min-
1.0 m1~min'
1.0 ml-min'
derivatized with o-phthalaldehyde and ethanethiol and injected
onto a C18 reverse-phase column. The amino acid adducts were
eluted with a CH3CNzNaH2PO4 (12.5 mM; pH=7.2) gradient formed
by two microprocessor controlled pumps.
1
1
1
35
before they were analyzed further.
The organic acids from alder nodules were separated from
one another by ion-exchange chromatography. A mixed bed of Dowex l
(chloride, acetate) resin was prepared according to Berl §t_al,(164).
The advantage to not converting the resin completely to the acetate
or chloride form is that the organic acids, which would not be retained
if the column was in the chloride form, were retained by the mixed-bed
column and were eluted from this column with more dilute solutions
of eluant than that required if the column was completely in the
acetate form. A 170-ml gradient of 0 to 1.2 N formic acid was used to
elute the organic acids. The 1-ml fractions collected from the column
were dried in a 100 C sand bath to remove the formic acid. Water
(0.2 ml) and Trition X-lOO scintillation fluid (2 ml) were added
to the dried vials and the radioactivity was measured by scintillation
spectrometry.
3. Thin-Layer Chromatography
Plant extracts were spotted on either a 5 x 20 cm, 0.1 mm
thick or 20 x 20 cm, 0.25 mm thick, thin-layer cellulose plates
(Brinkmann). The extracts were Chromatographed using a solvent system
of either phenoleZO (80:20,v/v) or n-butanolzacetic acidzHZO
(120:30:50, v/v/v). The phenol solvent system was used to separate amino
acids from one another, and the n-butanol system was used to separate
organic acids from one another. About 1 pg of allantoin and allantoate
and 0.05 pCi of [14C]malate (15 mCiommol'I; Cal Atomic, Los Angeles, CA),
[14C]glutamate (180 mCi-mmol-1; Cal Atomic, Los Angeles, CA) and
[14C]aspartate (180 mCi-mmol'1; ICN) were applied to each plate and
used as standards. The ureides, allantoate and allantoin, were detected
36
by spraying the plates with a solution containing 1 g diamino-
benzaldehyde in 3 m1 of 12 N HCl and 12 ml of n-butanol (165).
Amino acids were made visible either with UV light after spraying the
plates with OPA (166) or by heating the plates after spraying them
with 0.25% ninhydrin in n-butanol.
4. High-Voltage Electrophoresis
The high-voltage electrophoresis procedure developed by
Wolk gt 31, (167) was used primarily for the analysis of the products
of [13N] assimilation. Samples were spotted on a 5 x 20 cm, 0.1 mm
thick, thin-layer cellulose plate (Brinkmann). The plate was
sprayed with 70 mM sodium borate (pH=9.2) and was subjected to
electrophoresis for 6 to 12 min at 1.5 to 3 kV. Amino acids were
localized by spraying the plate with ninhydrin (167) or with OPA (166).
The radioactivity on the plates was detected with a gas-flow scanner
(Berthold). About 5 pg of aspartate, glutamate, asparagine, glutamine,
alanine and allantoate were spotted on the plates and used as
standards. Allantoate was localized by spraying the plate with
diaminobenzaldehyde (165).
5. Gas-Liquid Chromatography of Organic Acids
The organic acids from nodule and root extracts were purified
on Dowex l (formate) resins as described in section 1.2 (paragraph 1).
The organic acids were converted to their trimethylsilyl derivatives
using a procedure adapted from Stumpf and Burris (162). Thirty microliters
of redistilled, dry pyridine and 15 pl of N,O-bis-(trimethylsi1y1)-
trifluroacetamide (BSTFA; Pierce Chemical Co.) were added to the dried
organic acids. The reaction was carried out at room temperature for
37
10 min in 2-ml capped vials. The derivatized organic acids (0.3 to
40 nmol per organic acid) were separated by gas-liquid chromatography
on a 2 m x 2 mm, 3% SE-30 column (Supelco, Inc., Bellefonte, PA)
using N2 as a carrier gas and were measured with a flame ionization
detector. The temperature program was 80 to 185 C at 5 C'min'].
The organic acids were identified on the basis of their retention
times and quantified on the basis of the detector response as compared
to the standards malonate, succinate, fumarate and malate.
14
J. ISOLATION AND CHROMATOGRAPHY OF C-LABELED 2-OXOACIDS FROM
SOYBEAN NODULES
The oxoacids in nodules were isolated as the 2,4-dinitrophenyl-
hydrazone derivatives, prepared using a procedure adapted from
Isherwood (168). About 1 g (fresh weight) of soybean nodules were
14
exposed to CO2 for 5 or 60 min, at which time the nodules were
removed from the vessel containing the 14
C02 and frozen in liquid
nitrogen. The frozen nodules were ground, and 5 ml of 0.6 M (HPO3)n
(meta-phosphoric acid) and 0.5 ml of 1% (w/v) 2,4-dinitrophenylhydrazine
in 5 N H2S04 were added. The mixture was vortexed and then incubated
at 37 C. After incubation for l h, the mixture was extracted four times
with 2 m1 of diethyl ether to removed the 2,4-dinitropheny1hydrzone
derivatives of the oxoacids. The ether extracts were combined and
extracted twice with 5 ml of saturated NaHCO3 to separate the derivatized
oxoacids from the other ether soluble compounds. The NaHCO3 extracts
were combined and the pH adjusted to 2 with 3 N H2S04. The acidified
solutions were extracted three times with 0.5 volumes of chloroformzether
(85:15,v/v). The extracts were combined and dried with a stream of air
38
to a final volume of 100 pl. One to five microliters of the
chloroformzether extract was spotted on a thin-layer cellulose
plate. The derivatives were separated by chromatography using
n-butanolzethanolezo (70:10:20, v/v/v) as the solvent system
(169). The plates were scanned for radioactivity with a gas-flow
scanner (Berthold). The standards glyoxylate, pyruvate, 2-oxog1utarate,
and acetone were detected by their yellow color.
K. INCORPORATION OF [2,3-3HJASPARTATE AND [4-‘4CJASPARTATE INTO
ASPARAGINE
The pathway of asparagine synthesis in soybean roots was
examined by measuring the incorporation of label from [2,3-3H]aspartate
and [4-14C]aspartate into asparagine. Thirty-nine-day-Old plants
that had received 10 mM KNO3 for 24 days prior to the experiment were
removed from the pots in which they had been planted. About 4 g of
secondary roots were harvested and cut into pieces approximately 1 cm
long. About 1 g of the roots was placed into each of four 20-m1 glass
scintillation vials and one of four different solutions was added to
each of the vials:
0.5 mM L-aspartate
0.5 mM KCl
2.0 mM aminooxyacetate pH=7.0
1+
or
0.5 mM L-aspartate
0.5 mM NH4C1
2.0 mM aminooxyacetate pH=7.0
l+
In addition to one of the above solutions, each vial contained 26 pCi
of L-[2,3-3H]aspartate (20 Ci mmoi“; ICN) and 1.7 pCi of
39
L-[4-14CJaspartate (50 mCiommol'1
; Amersham). The vials were
sealed with a serum cap and incubated at 25 C. After 1 h, the
roots were removed, rinsed well with H20, patted dry and frozen.
The roots were lyophilized and prepared for analysis according to
the procedures discussed in section H.
Just prior to Opening the scintillation vial to remove the
roots, 14CO2 formed from [4-14C]aspartate, was assayed by removing
3 m1 of the gas from each vial and injecting this sample through
a serum cap into a separate scintillation vial containing 1 m1 of
methylbenzethonium hydroxide 90 min after the gas sample was added
to the vial. The vials were then stored at -20 C for 2 h to allow
the fluorescence, which is sometimes observed in this system, to
decay. The radioactivity due to the 14
CO2 which was absorbed by the
methylbenzethonium hydroxide was measured by scintillation spectrometry.
The solution in which the roots had been incubated was assayed
for 3
H20, produced from [2,3—3H]aspartate, by the microdistillation
technique of Varner and Burton (170). The labeled amino acids contained
in this solution were derivatized and separated by HPLC and analyzed
by flurometry and scintillation spectrometry as discussed in section
1.1 and below.
The amino acids extracted from the plant roots were derivatized
as described in section 1.1 except that 0.2 ml of H20, 0.2 ml of
saturated sodium borate, 0.5 ml of methanol and 0.1 ml of OPA solution
were added to the dried extract of each root. Because of the low amount
of label that was recovered from the roots in asparagine (about 3700 cpm
of [14C]og fresh weight-10f roots), the entire solution containing the
derivatized amino acids was injected onto the HPLCcolumn in multiple
40
injections. The CH3CN concentration in the mobile phase was zero
(i.e. the mobile phase was 12.5 mM NaH2P04, pH=7.2) during the
injection procedure so that the amino acids would not be prematurely
eluted from the column. Once the entire solution had been injected,
the CH3CN concentration was increased to 12% and the elution gradient
started (Table l). The disadvantage of injecting large amounts of
material onto the column is that the separation between amino acids
is not as good as when smaller amounts of material are injected.
The eluate from the HPLC column was collected in fractions
and the radioactivity was determined by scintillation spectrometry
after the addition Of Triton X-100 scintillation fluid. The
radioactivity due to [3H] and to [140] was determined using two different
channels of a liquid scintillation counter (Program number 4 of a
Beckman LS 7000). To ensure that the CH3CNzNaH2PO4 buffer did not affect
the relative counting efficiences of either isotope, several HPLC runs
using unlabeled aspartate and asparagine were made. Standard [3H]aspartate
and [14CJaspartate were added to the vials containing the unlabeled
aspartate and asparagine eluted from the column. The counting efficiencies
obtained for these standards were used to calculate the 3H/MC ratio in
aspartate and asparagine that was present in the root extracts and
the solution in which the roots had been incubated.
L. DISTRIBUTION OF LABEL WITHIN ASPARTATE AND GLUTAMATE IN SOYBEAN
14
NODULES EXPOSED TO CO,
L
Root systems of soybean plants were exposed to 14
CO2 for 15
and 30 min. The amino acids were extracted and prepared as discussed in
section H. Aspartate and glutamate were purified from one another
41
and the other amino acids by ion-exchange chromatography on Dowex 1
(chloride, acetate) according to the procedure of Berl et_al, (164).
The fractions containing aspartate were combined and lyophilized
as were the fractions that contained glutamate. Aspartate and
glutamate accounted for 98% of the total amino acids in the purified
aspartate and glutamate fractions, respectively, as determined by
HPLC (section 1.1).
The position of [14C] within aspartate and glutamate was
examined by selectively removing one or more of the carbons. The
carboxyl groups of aspartate and C-1 of glutamate were removed with
ninhydrin by a procedure adapted from Sobel gt al. (171). Sixty
microliters of ninhydrin (0.3 g.m1"1 of 95% ethanol) and 0.12 ml of
l M citrate (pH=2.2) were added to 0.3 m1 of sample (about 5 nmoloml'1
of the appropriate amino acid) in a glass scintillation vial. A
glass marble was placed on the top of the vial, and the vial was
placed into a boiling H20 bath. After 10 min in the H20 bath, the
vials were removed and 30 pl of 5 N HCl were added. The vials
were then placed in a heated (70 C) vacuum desiccator and dried under
reduced pressure to remove traces of [14C]HC03 produced by the
decarboxylation of aspartate and glutamate. The vials were removed from
the desiccator and 0.7 m1 of H20 and 7 ml of Triton X-100 scintillation
fluid was added after the vials had cooled. The radioactivity which
had not been lost during the treatment with ninhydrin represented the
label in C-2,3 of aspartate and C-2 to 5 of glutamate. The standards
were L-[4-14C]aspartate, L-[u-14C]aspartate, L-[U—14C]glutamate and
L-[l-14C]ornithine. Ornithine labeled in C-l was used to determine if
ninhydrin completely removed C-1 of an amino acid.
42
The amount Of label in the C-4 position of aspartate was
determined by a procedure adapted from Luehr and Schuster (172).
Seventy microliters of purified aspartate (5 nmol'ml'1) were added
to 30 p1 of 3.8 M sodium acetate (pH=5.4) and 100 p1 of pyridoxal-H01
(Sigma) (120 mg-ml'1) and A13(SO4)2 (5 mg-ml") in a glass
scintillation vial. The vials were placed in a 70 C H20 bath. After
30 min, the vials were removed and 100 p1 of 5 N HCl were added.
The vials were placed in a heated (70 C) desiccator and dried under
reduced pressure to remove traces of [14C]HCO§ produced by the
decarboxylation of aspartate. Water (0.7 m1) and Triton X-100
scintillation fluid (7.0 ml) were added to each vial, and the
radioactivity determined by scintillation spectrometry. The radioactivity
remaining after the treatment with pyridoxal represented the
radioactivity in C-1,2,2 of aspartate. The standards used were
[U-14C]aspartate and [4-14C]aspartate.
M. PRODUCTION OF [13N]
Nitrogen-13 was generated by irradiation of a target for 10 min
with a 12 MeV proton beam produced at the Michigan State University
Heavy Ion Laboratory. The target consisted of either 18.6 mg of
amorphous carbon, 97 atom % of [13C] (173) (Monsanto Research Corp.,
Mound Laboratory, Miamisburg, OH) or 1 ml of H20 (174). Bombardment
of the [130] with protons resulted in the production of a neutron and
a loss of a proton to form [13N] according to the nuclear reaction
13C(p,n)13N. Bombardment of [16O]H20 with protons resulted in the
production of [13N] according to the reaction 16O(p,ot)13N.
After bombardment of the target, the particular chemical form
of [13N] that was desired was purified according to the schemes
43
developed in the laboratories of Tiedje (174) and Wolk (167,173,175).
The [13C] target was converted to [BNJN2 by the Dumas combustion
procedure of Austin gt al. (173). After bombardment, the [13C]
target was mixed with 0.6 mg of fine CuO powder and 0.18 mg
of KNO3 in a quartz tube. The sample was then heated to 800 C.
A stream of CO2 was used to sweep the combustion products through
post heater tubes filled with CuO and metallic Cu, where the nitrous
oxides produced during combustion were reduced to N2. The products
were then passed through a liquid nitrogen trap, to remove C02 and
unreacted oxides of nitrogen , to a previously evacuated Toeppler
pump.
After bombardment, the [160]H20 target contained varying
13 13NH+ 13 13
amounts of N03, NH4 and N05. The N03 was purified by adding
2 to 3 drops of 10 N NH4OH to the water target which was then flash
13N H+
evaporated to remove the 4(174). One ml of H20 was added to
the dried material and the solution was neutralized with 2 or 3 drops
of 10 N HC1. The purity of the ‘3
N03 was determined by separating
the radioactive constituents of a 10 p1 sample by HPLC using a Partisil-
10 SAX anion-exchange column (Whatman) (174). The elution buffer
was 50 mM KH2P04 (pH=3.0). The 13NHZ, 13N03 13N05 eluted
separately and their radioactivity was measured with a sodium
and
iodide coincidence detector.
13
The NH4 was purified from the H20 target as follows. After
bombardment, the H20 target was placed in a flask and about 100 mg
of Devardas alloy and 10 m1 of 10 N NaOH were added to reduce the
13 13N H+ (176). The flask was fitted to a steam distillation
13
N03 to
apparatus (167,177) and the NH4 was transferred from the flask by
44
steam distillation under vacuum into a side arm test tube
immersed in liquid nitrogen. Up to 10 mCi of 13NH: was recovered.
N. EXPOSURE OF PLANT TISSUE T0 [‘3N1
Detached nodules or segments of nodulated roots were exposed
to [BNJN2 in l or 3-m1 vials. The vials were briefly evacuated
before the [BNJN2 (about 0.2 ml) was transferred to the sealed
vial using a Toeppler pump. The pressure inside the vessel was
adjusted to atmospheric pressure with a mixture of Ar:C02:O2
(80:1:10, v/v/v). Root systems of intact plants were trimmed of
excess roots and placed in a test tube containing 2 to 10 mCi of
13NH4 or 13
of the labeled compounds.
N0; in H20. Detached nodules were dipped in a solution
0. EXTRACTION OF THE LABELED PRODUCTS AFTER THE ASSIMILATION OF [13N]
After the tissues had been exposed to [13N], they were ground
and extracted twice with 1 m1 of methanoleZO (80:20,v/v). The
resulting slurry was clarified by centrifugation at 10,000 g for
3 to 5 min. The supernatant fluids were combined and dried
jn_vacuo at 50 C. MethanoleZO (20 to 100 p1) and amino acid standards
1 per amino acid) were
(5 to 20 p1 of solution containing 1 mg-ml'
added to the dried extract. The standard amino acids included
aspartate, glutamate, asparagine, glutamine, alanine and arginine.
For certain experiments, allantoate was also added as a standard.
The labeled compounds were separated by high-voltage electrophoresis
as described in section 1.3.
45
P. DISTRIBUTION OF LABEL IN GLUTAMINE AFTER THE ASSIMILATION OF 13NH:
Alder nodules were incubated in a solution containing 13NH:
for 2,5 and 10 min. The nodules were rinsed with H20 and labeled
compounds were extracted with methanoleZO as described above.
Two to three drops Of 14 N NH4OH were added to ensure that any
13NH: remaining from the incubation solution was removed during the
drying of the extract. One ml of 10 mM sodium acetate (pH=4.9)
containing 25 units of glutaminase (Sigma, Grade V) was added to the
dried extract. After a 10 or 20 min incubation, the solution was
added to a 2-ml Dowex 1 (acetate) column. The 13NH: released from the
amide position of glutamine was eluted by washing the column with
3 ml of H20. The[]3N]glutamate from the [13N]glutamate that was
originally present and hydrolyzed [13nglutamate was eluted from
the column with 3 m1 of 2 N acetic acid (178). The radioactivity
of each fraction was measured by scintillation spectrometry after
Triton X-100 scintillation fluid was added. The procedure was
1
standardized with [14C1g1utamine (50 mCi-mmol' ; ICN).
CHAPTER 1
THE RATE OF DARK CO FIXATION DURING THE DEVELOPMENT OF THE SOYBEAN
2
N -FIXING SYSTEM
2
Christeller 93.21-(7) proposed that dark C02 fixation
was involved in the synthesis of asparagine in lupin nodules.
In support of this proposal, these investigators reported that
the rate of dark CO2 fixation was correlated with the rate of
N2 reduction during the development of the NZ-fixing system of
lupin (Lupinus angustifolius) and Rhizobium lupinus. The major
form of organic nitrogen transported from nodulated lupin plants
is asparagine (9), whereas allantoate is the major form transported
from soybean nodules (12). Allantoate, based on the pathways
proposed for its synthesis (115,116), is not expected to contain
carbon that enters the metabolism of the nodule via dark CO2 fixation.
Therefore, the relationship between the rate of dark CO2 fixation
and N2 fixation is expected to be different in soybeans from what it
is in lupins. _
In this chapter I report and discuss the following: 1) the
mechanics of the assay for dark CO2 fixation, 2) the effect of
several parameters on the rate of dark CO2 fixation in soybean
nodules and roots and 3) changes in the rate of dark CO2 fixation
and N2 reduction during the development of soybean nodules and
roots.
46
RESULTS
A. CO2 FIXATION ASSAY
Previous workers have used [14C] in the form of 14002 and
[14CJHC05 to measure the rate and/or products of CO2 fixation in
14
plants. After exposure of plant tissue to [14C]HC03 or CO
23
the labeled products were usually extracted with either an acidic
solution (8,179,180) or an organic solvent such as methanol or
ethanol (28,31,181). The extracts were dried, thereby removing any
14
residual CD2 or [14C]HCOS. In this study, the nodules and roots were
extracted with ethanol rather than acidified solutions because some
of the products of 14
C02 fixation, most notably asparagine and
glutamine, are labile in acidic solutions.
The efficiency of extracting labeled compounds from nodule and
root tissue with ethanol was examined by extracting several tissue
sample (40 mg dry weight per sample) that had been allowed to incorporate
label from 14
C02, six times with 3 m1 of ethanoleZO (80:20,v/v). The
first three extractions, combined, accounted for 95 to 99% Of the
total radioactivity that was extracted; therefore, three extractions
were used routinely.
If [14C] that had not been fixed, i.e. [14C]Hco§, was present
in the dried extracts, then the calculated rates of CO2 fixation would
be artificially high. An extract was prepared from nodules that had
been allowed to incorporate label from 14
C02. Fifty microliters of
6 N HCl or glacial acetic acid were added to a 200 pl aliquot of this
extract. The acidified extract and an aliquot of the same extract
without added acid were dried jn_yagug_at 55 C. The amounts of the
radioactivity in the extracts with and without acid were the same,
47
48
14
demonstrating that CD2 or [14C]HCO§ was not present in the dried
extracts.
Prior to ending a C02 fixation assay, two gas samples were
routinely removed from the container in which the plant tissue
14
was exposed to C02. Based on the amount of radioactivity
measured in one sample and the total concentration of CO2 measured
1
in the other sample, the specific activity of the 4CO2 was
calculated. This value together with the amount of [14C] that
was recovered from the tissue was used to calculate the rate of
dark C02 fixation. For different experiments the specific activity
14
of the CO2 varied from 0.5 to 25 x dpm-pmol'l.
14
The incorporation of label from CO in attached and detached
2
nodules was linear during the time period that was examined, 2 to 12
min. The external concentration of CO2 did not affect the linearity
of the assay.
8. EFFECT OF DETACHMENT OF NODULES AND CONCENTRATION OF CO2 ON THE
RATE OF DARK CO FIXATION
2
Based on other investigators' work (7,182,183) at least
two factors needed to be considered in measuring the rate of
dark CO2 fixation in soybean nodules. First, since the rate of
C2H2 reduction in detached nodules is less than in intact systems
(182,183), detachment of nodules might have a similar effect on the
rate of dark C02 fixation. The second fact is that the rate of dark
CO2 fixation in lupin nodules was affected by the concentration of
CO that surrounded the nodules with high levels of CO2 resulting
2
in high rates of dark CO2 fixation (7)
49
The rates of dark C02 fixation in attached and detached
soybean nodules was measured when both high and low concentrations
of C02 were present. The rates of dark CO2 fixation in detached
nodules appeared to be less than the rates measured in attached
nodules regardless of the concentration of CO2 that was present
(Table 2). Higher rates of dark CO2 fixation in nodules were measured
when higher concentrations of C02 were present (Table 2). The
maximal rate of dark CO2 fixation in attached nodules was attained
when the external CO2 concentration was 1.5% (v/v) or greater
(Figure 3). A similar saturation of the rate of dark CO2 fixation
with high concentrations of CO2 was observed in detached nodules. The
rate of dark CO2 fixation in roots was not affected by increasing the
concentrations of CO2 around the roots from 1.0 to 3% (v/v) (Figure 3).
C. EFFECT OF CO2 CONCENTRATION ON 09 CONSUMPTION AND C2H2 REDUCTION
Mahon (184) reported that concentrations of CO2 between 0.1
and 3% (v/v) inhibited the respiration of nodulated pea roots. If
a similar event occurs in soybean nodules, then the rate of dark CO2
fixation observed at high concentrations of 002 may not be reflective
of the rate of dark CO2 fixation in nodules. Rather, the rates measured
under high concentrations of CO2 may be the result of an increase in
Specific activity of 14
14
CO2 within the nodule because of the reduced
dilution of the ‘2
CO2 by C02 produced during respiration.
The effect of high concentrations of C02 on the respiration of
nodulated soybeans was examined. The rate of oxygen consumption of
attached and detached nodules was measured amperometrically in the
presence of high or low concentrations of 002. The initial addition
of both high and low concentrations of CO2 to separate samples was
50
Table 2 Effect of the Concentration of C02 on the Rate of Dark CO2
Fixation in Attached and Detached Nodules
Treatment CO2 Concentration Rate of Dark CO2 Fixation
% (v/v) nmol mg dry wt of nodules'Jh.1
Attached 0.2 to 0.6 10 t 2 (9)
1.5 to 3.5 36 i 5 (9)
Detached 0.2 to 0.6 4 i l (6)
1.5 to 3.5 19 i 3 (6)
Nodules that were removed from the root (detached) or the entire
nodulated root system of 26-day-old plants were placed into a flask.
The tissue was exposed to 5 to 20 pCi of 14CO2 and either low
(0.2 to 0.6%, v/v) or high (1.5 to 3.5%) concentrations of CO2 for
10 min, at which time the tissue was removed and frozen in liquid
nitrogen. The values reported are t SE and the number of replicates
for each determination are in parentheses. The rates of dark CO2
fixation in attached nodules in low and high concentrations of CO2
were significantly different (P5_0.10) from one another as
determined by the Student's t-test. The rates for detached nodules
in high and low concentrations of CO2 were also significantly different
(P§O.10) from one another.
51
60- °-
nodule 0
o
e 1
,2 .f
1 . .
S ,_ 4O
.5 3 0°C 0
u. «3‘
N ‘0 00
(3 GD
0 E o
1. 1: 2K3 ’ '
a E A A root A
a G A AA . ‘ A
l/z””"45"—'——u—f A A
A ' A
0 L I L l L
0.5 1.0 20 3.0
CO: concentration (7.1
Figure 3 Effect of the Concentration of CO2 on the Rates of Dark C0
Fixation in Roots and Nodules of Intact Soybean Plants
2
Nodulated roots of intact 19-day-old soybean plants were incubated with
5 to 10 pCi of 14CO2 and various concentrations of 12CO2 for 10 min, at
which time the tissue was removed and frozen in liquid nitrogen. The
rate of'dark CO2 fixation in nodules (O) and in roots (A) was
calculated by dividing the amount of label incorporated during the 10 min
exposure by the specific activity of the 14CO2 that the samples were
exposed to. The lines that are presented were fitted by eye.
52
to ensure that the order Of addition did not affect the results. There
appeared to be no significant effect of the concentrations of CO2
on the oxygen consumption of attached nodules (Table 3). If
detached nodules were initially exposed to high concentrations of C02,
then the rate of oxygen consumption was greater under high
concentrations of CO2 than under low concentrations.
The effect of the concentration of C02 on metabolic processes
in nodules was examined by measuring the rates of C2H2 reduction under
high and low concentrations of C02. The rates of C2H2 reduction in
attached nodules of intact plants or of plants that had had the
shoot excised were not affected by the presence of high concentrations
of C02. In contrast, the rate of C2H2 reduction in detached nodules
was higher in the presence of high concentrations of CO2 than the
rate measured in low concentrations (Table 4).
D. DARK CO2 FIXATION, C.,H2 REDUCTION AND CONCENTRATION OF ORGANIC NITROGEN
IN THE XYLEM EXUDATE DURING THE DEVELOPMENT OF SOYBEAN PLANTS
Previously, Streeter (185) noted a decline in the amount of
amino acids and an increase in ureides in the xylem exudate of
developing soybeans. If dark CO2 fixation in soybean nodules provides
carbon for the synthesis of amino acids translocated to the shoot
as suggested for lupin nodules (7), then the rate of dark C02 fixation
may be related to the transport of amino acids in soybeans. The
concentration of amino acids and the ureides,allantoin and allantoate,
in the xylem exudate and the rates of C02 fixation and 02H2 reduction
were measured in developing soybeans.
The concentrations of amino acids was greater than that of ureides
in the xylem exudate of plants younger than 18 days (Figure 4, Panel B).
53
Table 3 Effect of the Concentration of CO2 on Nodule Respiration
Rate of O2 Consumption in High COZ/Rate of O2 Consumption
in Low CO2
Treatment Attached Nodules Detached Nodules
ratio
Ambient 002: 31.5% 0.92 i 0.04 (7) 0.91 i 0.03 (7)
31.5%: Ambient CO 1.11 i 0.06 (7) 1.25 i 0.03 (11)
2
The oxygen consumption of tissues incubated either in ambient or 1.5%
(v/v) or greater concentrations of CO2 were measured for 10 to 15 min.
To the sample originally incubated in ambient levels of C02, CO2 was
added to increase the concentrations of CO2 (Ambient C02: 31.5%); the
chamber of those sample that were initially incubated in high
concentrations of CO2 was flushed to lower the concentration of CO2
(31.5%:Ambient 002). The oxygen consumption for each sample was
measured again. The measured rates of O2 consumption werelbetween
and 48 to 60
for attached and detached nodules,
126 and 180 nmol O2 consumedrmg dry weight of nodules-1 h
nmolomg dry weight of nodules'L h"1
respectively. The values reported are t SE and the number of replicates
for each determination are in parentheses.
aThese values are significantly different (P§_0.05) from one another
as determined by the Student's t-test.
54
Table 4 Effect of the Concentration of C02 on the Rate of C H
2 2
Reduction
Treatment CO2 Concentration Rate of CZH2 Reduction
% (v/v) nmol-mg dry wt of nodules-Jh']
Attached Nodules 0.2 to 0.6 37 s 6 (7)
on intact plants 1.5 to 3.5 37 i 6 (7)
Attached Nodules 0.2 to 0.6 48 i 4 (8)
w1th shoots excised 1.5 to 3.5 53 i 6 (7)
Detached Nodules 0.2 to 0.6 18 i 1 (7)a
1.5 to 3.5 24 s 1 (7)a
Nodules, either on intact plants (attached) or removed from the
roots of 26-day-old plants (detached), or attached nodules from
36-day-Old plants that had had the shoot excised just prior to the
assay were placed in separate flasks and exposed to low (0.2 to 0.6%,
v/v) or high (1.5 to 3.5%) concentrations of C02 and to C2H2 (10%, v/v).
Periodically, gas samples from the flasks were removed and assayed
for C H
2 4'
for each determination are in parentheses.
The values reported are t SE and the number of replicates
aThese values were significantly different from one another (P50.10)
as determined by the Student's t-test.
55
After the onset of N2 fixation, the ureides became the major form
nitrogen in the xylem exudate while the concentration of amino
acids slowly declined.
The maximal observed rate of dark CO2 fixation in secondary
roots was measured on day 8 (Figure 4, Panel A). Seven days later
1-h"1.
the rate was 10 nmol~mg dry weight of roots' Nodules
’were first observed on day 12 and noticeable rates of dark CO2
fixation and 02H2 reduction in nodules were first measured on
day 15. The maximal observed values were 117 nmol CO2 fixedo
mg dry weight of nodules-1 h'1 and 138 nmol 02H2 reducedomg dry
weight of nodules-1 h'1. The specific activity of C2H2 reduction
remained relatively constant until day 29. In contrast, the
specific activity of nodule C02 fixation declined after day 18 reaching
a value of 20 nmol-mg dry weight-1 h'1 on day 35.
The decline in the rate of dark CO2 fixation observed for
nodules corresponded to a decrease of the concentration of amino
acids in the xylem exudate. The values for the concentrations of
amino acids in the xylem exudate and the rates of dark CO2 fixation
for the plants from the last four sampling dates were correlated
with a coefficient of 0.96 (Figure 5).
56
Figure 4 Rates of Dark CO2 Fixation, C2H2 Reduction and the
Composition of the Xylem Exudate during the Development
of Soybean Plants
After intact nodulated roots of soybean plants that had been
maintained on N-free nutrient solution were sealed in to a flask, they
were exposed to either C2H2 (10%, v/v) or 5 to 10 pCi of 14C02 and
saturating levels of 12CO2 (3f1.5%, v/v). The root systems that were
exposed to 14CO2 were removed from the flask after 10 min and frozen
in liquid nitrogen. The reported values of the rates of dark CO2
fixation in roots ([3 , Panel A) are averages of 15 to 20 replicates
and in nodules (C) , Panel A) 3 to 10 replicates. The reported values
Of the rates of C2H2 reduction ([3 , Panel A) are averages of 5 replicates.
Bars are presented about the rate of C2H2 reduction and the rate of
dark C02 fixation in nodules and roots where the SE is greater than the
size of the symbOl. The xylem exudate of plants that were left in
their pots was collected for 30 to 45 min and was assayed for amino
acids (0, Panel B) and the ureides, allantoin and allantoate (A ,
Panel B). The ratio of nmoles Of amino acids to nmoles of ureides
(. ------ , Panel A) in the xylem exudate is also presented.
Activity
(nmohmg'dny wt"oh"1
Composition of Exudate"
'1
(nmol-ml
160
120
m
o .
A
O
57
anthosis
.. 4
(:sz reduction “3
i-z
\ cozfixation “I
é\¢ (nodule)
\ ~‘fi-..---.T\2
.:.a—g-
0402 fixation (root). 0'
ureides
\- A
\
x‘ /
O\ A
_ \
\
b- - - --o
l 1 g L L 1
16 20 24 28 32 36
Plant Age (days)
Figure 4
J lnxylom exudate
[amino .a'ciOJIEiroido
58
O
I: EI" ‘
o
g . ..
b
E
u _ 5. .
s E
0 _:. I- 0 q
1, 6
“5,5 4 ~
4! so - I
o
.5 - .
3 0
2.9L '4 p .
15 30 45 60
(nmol-mg dry wi'lh")
002 Fixation Rate
Figure 5 Correlation of the Concentration of Amino Acids in Xylem
Exudate with the Rate of Dark CO Fixation in Nodules
The values reported in Figure 4 for the concentration of amino acids in
the xylem exudate and the rate of dark CO2 fixation in nodules of plants
21-days or older are presented.
The line was fitted by least squares
analysis and had a correlation coefficient of 0.96.
DISCUSSION
The rates of dark CO2 fixation in this chapter were calculated
based on the label recovered from the nodules and roots exposed to
14CO2 for 10 min. These rates, however, may not represent the
rate at which net fixation of carbon occurs in the nodule, but may
instead primarily reflect the rate at which carbon is exchanged
through the system. Net fixation results in an increase in the
amount of carbon in the system, whereas exchange results in
carbon incorporated during dark C02 fixation being lost as CO2
via the respiratory activities of the nodule. The extent to
which dark CO2 fixation results in net fixation or exchange
cannot be determined from the data presented in this chapter.
14
The specific activity of the C02 in the gas phase
surrounding the tissue was used in calculating the rate of dark
CO2 fixation. The use of this specific activity may result
in a calculated rate of dark CO2 fixation that does not accurately
reflect the actual rate of dark C02 fixation. The specific activity
that should be used in the calculations is the specific activity
at the site of dark CO2 fixation within the nodule. The two
specific activities are probably not the same because the
12
respiratory activities of the nodule releases CO diluting the
14
2
CO2 within the nodule. I was unable to experimentally determine
the 14CO2 specific activity at the site of dark CO2 fixation;
therefore, I used the value measured for the gas phase just
prior to ending the incubation. During a 10 min incubation, the
concentration of CO2 within a flask increased because of the
respiratory activities of the plant tissue. This increase had
59
60
a minor effect (less than 10%) on the specific activity of 002,
and hence the calculated rate of dark CO2 fixation, when
saturating concentrations of CO2 were present.
Christeller gt 31. (7) noted that for lupin nodules, high
concentrations of C02 were necessary to Obtain maximal rates of
dark CO2 fixation. The internal specific activity of the
‘14C02 was assumed to approach that outside the nodule only at
very high external concentrations of 002. Christeller gt a1.(7)
concluded that the rate of dark CO2 fixation measured at saturating
concentrations of CO2 is the best estimate of the "true“ rate
of dark CO2 fixation jn_yjyg, The response of the rate of dark
CO2 fixation to high concentrations of CO2 was similar in soybean
and lupin nodules; the rate of dark CO2 fixation in soybean roots
was not affected by the concentration of C02. Unless otherwise
indicated the rate of dark CO2 fixation presented represents the
rate Observed at saturating concentrations of C02.
High concentrations of CO2 did not inhibit respiration of
nodulated soybean roots, in contrast to the inhibition of respiration
noted in pea roots (184). The reason for the difference of the
pea and soybean root system is not clear. Other than possible
inherent differences between peas and soybeans, the method by which
respiration was measured may have affected the results. I measured
respiration as oxygen consumption, whereas Mahon (184) measured
respiration as the efflux of 002. The addition of high concentrations
of CO2 may have affected the meaurement of CO2 efflux but not
oxygen consumption. Mahon (184) also performed his measurements
jn_situ, whereas I removed plants from their pots and placed them
61
into a small vessel prior to the assay.
The presence of high concentrations of CO2 did appear to
affect the metabolism of detached nodules. The rate of C2H2 reduction
in the presence of high concentrations of CO2 was significantly
higher (Table 4) than the rate in the presence of low concentrations
of 002. The respiration of detached nodules was altered by exposure
to high concentrations of CO2 prior to exposure to ambient levels of
CO2 versus the reverse treatment (Table 3). The results of this
experiment do not indicate whether oxygen consumption was inhibited
or stimulated by exposure to high concentrations of C02. Oxygen
consumption may have been stimulated when high concentrations of
CO2 were initially present or inhibited in low concentrations of
CO2 after a exposure to high concentrations of C02.
The differences in the rates of dark CO2 fixation, C2H2 reduction
and oxygen consumption between attached and detached nodules demonstrates
that detachment of the nodules even for short periods of time affects
the metabolism of the nodule. Similar declines in the rates of
CZH2 reduction (182,183) have been observed in nodules and nodulated
root systems removed from the rest of the plants. For this reason,
conclusions based on the results with detached systems should be
applied with caution to the intact plant.
The rate of dark CO2 fixation in attached soybean nodules
1
varied between 4 and 24 pmol-g fresh weight' oh"1 (Figure 2; assumes
5 g dry weight = 1 g fresh weight) during the development of the
soybean nodule. Layzell et_al,(181) reported that the rate of
dark CO2 fixation in detached cowpea and lupin nodules was 18 and
1
0.8 pmo CO2 fixed-g fresh weight of nodules-Lih' , respectively.
62
The values reported for cowpea and lupin nodules probably represent
low estimates of the activity of attached nodules. The concentrations
Of CO2 used in the assay for dark CO2 fixation (181) were not
reported and may have been below the concentrations necessary to
obtain maximal rates. Based on the rates of dark CO2 fixation
(Figure 4) and respiration (Table 3) an estimated 10 to 13% of the
carbon respired by the nodule could be refixed by in 1119 dark CO2
fixation in both lupin (181) and soybean nodules .
Several investigators have proposed that dark CO2 fixation
catalyzed by PEPC in lupin (7) and broad bean nodules (8) provides
carbon for the synthesis of asparagine, the major product synthesized
from N2 reduced in the nodules from these plants. Christeller
et al. (7) noted that the rates of CZHZ reduction and dark 002
fixation were correlated during the development Of the lupin nodule.
The major product of N2 fixation in soybeans, allantoate, does not
appear to require carbon incorporated by dark CO2 fixation catalyzed
by PEPC for its synthesis (115,116); therefore, a correlation
between the rates of N2 fixation and C02 fixation might not be
expected and was not observed in soybeans (Figure 4). On the other
hand, the parallel decline of the rate of dark CO2 fixation in the
nodule and the concentration of amino acids in the xylem exudate
(Figure 5) suggests that there may be a relationship between dark
CO2 fixation and amino acid synthesis in soybean nodules. The
results, however, do not indicate whether the parallel decline was
because the reduced rates of dark CO2 fixation were responsible for
the reduced Concentrations of amino acids, or vice versa, or
whether the decline in both measurements is caused by a third factor
63
such as the development of the nodulated root.
The next chapter is concerned with the pathway and products
of dark CO2 fixation in soybean nodules.
CHAPTER 2
THE PRODUCTS OF DARK CO2 FIXATION IN SOYBEAN NODULES
A portion of the label incorporated in broad bean (8) and
dwarf french bean (31) nodules was recovered in amino acids. The
amino acids accounting for the highest percentages of label in
roots and nodules exposed to 14
CO2 are generally those most closely
associated with the tricarboxylic acid cycle; aspartate, glutamate,
asparagine and glutamine (8,31,186). However,the relative incorporation
of label from 14
CO2 into these amino acids as well as other products
depends on the species of plant that is examined. For example,
asparagine is the major labeled amino acid in broad bean nodules (8),
whereas citrulline is in alder nodules (186). In chapter 1, I
demonstrated that CO2 was taken up by soybean nodules. In this
Chapter, I examine the products of dark C02 fixation in soybean nodules.
RESULTS
A. LABELING OF ORGANIC ACIDS
The incorporation of label into organic acids in nodules
that were exposed to 14CO2 was examined. The fraction containing
the organic acids and neutral compounds accounted for 43 to 72% of
14
the total radioactivity extracted from nodules exposed to CO
2
for 2 to 60 min. The label in this fraction was distributed among
malate, which accounted for 29% of the total radioactivity extracted
from nodules that had been exposed to 14
CO2 for 60 min, succinate
(5%), fumarate (4%), citrate (7%), allantoate (5%) and several
unidentified components (Figure 6).
64
65
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t
“-
I
sue tum
Radioactivity (apm)
(J 1!) 23¢)
Distance (cm)
Figure 6 Incorporation of Label into organic Acids and Neutral
Compounds in Nodules Exposed to 14CO
2
Soybean plants (29-days old) were uprooted and the shoots were excised.
The root systems were placed into a flask where they were exposed to
14CO2 for 60 min. The nodulated roots were removed from the flask and
frozen in liquid nitrogen. The labeled products were extracted from
the nodules with ethanoleZO (80:20, v/v). The extract was separated
into two fractions, one containing the organic acids and neutral compounds,
and a second containing amino acids. An aliquot of the former fraction
was spotted on a thin-layer cellulose plate at the origin and
Chromatographed with butanol:glacial acetic acidzHZO (120:30z50, v/v/v)
as a solvent system. Malate (mal), citrate (cit) and allantoate (aln)
were identified on the basis of co-migration with standards. Succinate
(sac) and fumarate (fum) were identified on the basis of published
Rf values (206). Radioactivity was detected with a gas-flow scanner.
66
B. LABELING OF AMINO ACIDS
The amino acids accounted for 28 to 57% of the total
14
radioactivity extracted from nodules exposed to CO2 (Figure 7).
Aspartate and glutamate accounted for the highest percentage
of the label in the amino acid fraction. After a 2 min exposure
14
to C02, the percentage of the total label in aspartate was 7%,
increased to 16% after a 5 min exposure, and remained at about
that value for the remainder Of the experiment (Figure 7, Panel A).
The percentage of label in glutamate increased until it represented
22% of the total radioactivity after a 60 min exposure of the
nodules to 14
002.
Lesser amounts of label were recovered in several other
amino acids (Figure 7; Panels A and B). Asparagine, glutamine,
alanine, serine and glycine accounted for approximately 7,1,3,3 and
2% of the total radioactivity recovered from nodules. A small
'percentages (§_5%) did not elute with the amino acids presented
in Figure 7 but occasionally eluted with several unidentified
amino acids.
14 12
CO -CHASE EXPERIMENT
C. 2
COz-PULSE
Lawrie and Wheeler (8) noted that a large proportion of
14
C0 in broad bean nodules was lost rapidly
2
from the nodules. The retention of recently-fixed 14CO2 in
the recently-fixed
soybean nodules was examined. The Shoots of nodulated soybeans
were excised, the nodulated root systems were placed in a 250-ml
flask, the flask was sealed and 14CO2 was added to the flask.
67
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After a 3 min exposure to 14C02, the root systems were removed
from the flask, flushed with a stream of air for 30 sec, and
placed into another 250-m1 flask that was left open. The root
systems were removed from the open flask at intervals and were
frozen in liquid nitrogen.
The major portion of the recently-fixed 14
12
CO2 was lost
from the nodule during the chase with C02. After a 60 min
chase, only 28% of the label incorporated during the 3 min
exposure to 14CO2 was still present (Figure 8, Panel A). The
percentage of label in aspartate remained relatively constant
throughout the chase; the percentage in the organic acid plus
neutral fraction declined slightly; the percentage in glutamate
increased during the chase (Figure 8, Panel B). Labeled asparagine
and glutamine were not detected in the extracts of the nodule because
of the low amounts of radioactivity incorporated during Short
exposure to 14CO2 and the low percentage of the total label
incorporated into these amino acids.
D. POSITION OF [14C] WITHIN ASPARTATE AND GLUTAMATE
Previous investigators have proposed that dark CO2 fixation
in nodules was primarily catalyzed by PEPC (7,8). The position
of [14C] within the molecules of aspartate and glutamate that were
isolated from nodules that had been exposed to 14
CO2 was determined
to provide further evidence for this proposal. Oxalacetate that
is produced from phosphoenolpyruvate and [14CJHC03 in the reaction
catalyzed by PEPC should be labeled solely in C-4. Aspartate
that is derived from this oxalacetate should, therefore also be
labeled solely in C-4. .Labeled glutamate synthesized from labeled
69
Figure 8 Distribution of Label in Organic Compounds in Nodules
14coz-Puise 12COZ-Chase
Soybean plants (29-days old) were uprooted and the shoots were excised.
The root systems were placed into a flask where they were
exposed to 14CO2 for 3 min at which time the nodulated roots were
14C02 and placed into a
separate flask. At various times, the root systems were removed from
the second flask and frozen in liquid nitrogen. The labeled products
were analyzed by HPLC. Radioactivity was measured by scintillation
spectrometry. Panel A, total radioactivity remaining after pulse as
per cent of that present immediately after the pulse ended. Panel B,
the distribution of radioactivity in organic acids and neutral compounds
(D), aspartate ( A) and glutamate (C).
during a
removed from the flask containing the
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71
oxalacetate via the tricarboxylic acid cycle should be labeled
solely in C-l (Figure 10 and 11).
Aspartate and glutamate were isolated by ion-exchange
chromatography from nodules that had been exposed to 14CO2 for
15 and 30 min. Ninety-five percent of the label in glutamate
was in C-l. The C-4 and C-1 of aspartate contained 75 and 25%
of the label in aspartate, respectively.
E. LABELING OF ASPARAGINE AND GLUTAMINE
Asparagine and glutamine are two important amino acid amides in
soybeans (5,12) yet these amides account for relatively low
percentages of the total radioactivity incorporated in nodules
exposed to 14
CO2 when compared to their respective acids,
aspartate and glutamate (Figure 7). This differenCe
in the labeling of these acids and amides may exist for two
reasons. Either the amides were labeled at a slower rate than the
acids or that the rates of labeling were similar but there was
a large difference in the amounts of the acids and amides. For
example, glutamate and glutamine may be labeled to the same
extent, i.e. both have the same specific activity, yet if the
amount of glutamate was larger than glutamine, glutamate would
account for a larger percentage of the label than glutamine. The
specific activities and amounts of the amino acids in nodules
exposed to 14
CO2 were measured to determine if the rate of labeling
or the amounts of amino acids could account for the low percentage
of label in asparagine and glutamine.
The amount of glutamine in nodules was 1 nmol-mg dry weight-1
versus 22 nmol-mg dry weight.1 for glutamate. The amounts of
72
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aspartate and asparagine were about the same, 11 and 8 nmol-mg
dry weight-1, respectively.
The concentration of these amino acids within the nodule
can be estimated by making several assumptions: 1) the amino
acids are evenly spread throughout the nodule, 2) 1 mg dry weight
of nodule is equal to 5 mg fresh weight and 3) l g of tissue
is equal to 1 ml of volume. The concentration of glutamine,
glutamate, aspartate and asparagine are estimated to be
0.2 mM, 4.4 mM, 2.2 mM and 1.6 mM.
The specific activity of glutamine was consistently higher
than that of glutamate (Figure 9). After a 2 min exposure of
14
nodules to C02, the specific activities of glutamine and
glutamate were 136 and 6 cpm-nmol'], respectively. The specific
activity of glutamate increased with time reaching 80 cpm-nmol'1
after a 60 min exposure, whereas, the specific activity of glutamine
slowly declined during the period from 2 to 28 min at which time
it increased. The reasons for this decline and rise in the
Specific activity of glutamine is not known.
The specific activity of aspartate was consistently higher
than that of asparagine. The specific activity of aspartate
increased with increasing exposure times up to 28 min when
a maximum of 123 cpmonmol'1 was reached. The maximum observed
specific activity of asparagine, 51 cpm-nmol'], was attained
14
after a 60 min expoure to CO
2.
DISCUSSION
A. INITIAL STEP OF DARK C09 FIXATION IN NODULES
I presented evidence in chapter 1 which demonstrated that
1 vivo dark CO2 fixation occurred in soybean nodules and roots and
which suggested that dark CO2 fixation may be related to amino acid
metabolism. In this chapter, I demonstrated that organic acids
and amino acids were labeled in nodules exposed to 14
C02.
The initial product(s) or step(s) of dark CO2 fixation in
nodules is not known; the results from the labeling studies
presented in this chapter are generally consistent with PEPC
being the enzyme primarily responsible for dark CO2 fixation in
soybean nodules (Figure 10). The product of the reaction catalyzed
by PEPC is oxalacetate. Oxalacetate is difficult to identify and
isolate because it is susceptible to spontaneous decarboxylation.
I attempted to isolate oxalacetate as its 2,4-dinitropheny1-
14
hydrazone derivative from nodules exposed to 002. Less than
1000 cpmig fresh weight'1 were recovered from nodules that were
exposed to 14
CO2 for l h as derivatives of 2,4-dinitrophenyl-
hydrazine. Because of the low levels of radioactivity that were
recovered as these derivatives,the identity of the labeled
derivative(s) was not established.
Oxalacetate, in most tissues, is rapidlyconverted to
one of three compounds, malate, aspartate or citrate. In soybean
14
nodules exposed to C02, these three compounds accounted for 42%
of the total radioactivity incorporated, which is consistent
14
with CO2 being fixed primarily by PEPC.
74
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Table 5 Distribution of Label and Estimated Rates of Incorporation
of Carbon Derived from Dark C02 Fixation into Amino Acids
l4
in Nodules Exposed to 002
Amino Acid Radioactivity Rate of Incorporation
% of Total nmol of carbon-mg dry wt
1
of nodules'. h-1
Aspartate l5.8 4.8
Glutamate 22.0 6.6
Asparagine 7.0 2.2
Glutamine 1.3 0.4
Serine 3.3 0.9
Glycine 0.9 0.3
Alanine 3.0 0.9
Soybean plants (29—days old) were uprooted and the shoots were
excised. The root systems were placed into a flask where they were
exposed to 14002. After 60 min, the nodulated roots were removed
from the flask and frozen. The amino acids were analyzed by HPLC.
Radioactivity was measured by scintillation spectrometry. The rates
of incorporation were estimated by multiplying the percentage of the
total radioactivity that each amino acid represented (column 1)
times the rate of dark C02 fixation (30 nmol CO2 incorporatedomg dry
wt of nodules-1 h'I).
84
probably better estimates for those amino acids that are metabolic
end products such as asparagine than they are for those amino acids
that are used as substrates in other reactions, such as aspartate and
glycine, because the percentage of radioactivity in each amino acid
only reflects the carbon that has accumulated in that particular amino
acid. It does not account for the total amount of carbon that has
passed through each amino acid.
An estimate of the carbon incorporated during dark C02 fixation
that passed through glycine and serine can be made if it is assumed that
the majority of glycine and serine synthesized in the nodules is used
in the synthesis of allantoate. The percentage of label incorporated
from 14
CO2 in allantoate , 5%, added to the percentages of label in
glycine and serine, 0.9 and 3.3%, respectively (Table 5) represents the
major portion of the label that could have passed through glycine and
serine. If these values are used, the estimated rates at which carbon
derived from dark C02 fixation are incorporated into glycine and serine
are 1.8 and 2.4 nmol of carbon-mg dry weight of nodules-1 '1
h , respectively.
The total rate of incorporation of carbon into glycine and serine
can be made based on an estimated rate of allantoate synthesis. For
this calculation several assumptions have to be made:
l) The rate of N2 fixation equals the rate of CZHZ reduction
divided by four (l90).
2) Eighty per cent of the N2 that is reduced in soybean nodules
is used in the synthesis of allantoate (l2).
3) Two moles of glycine and serine are synthesized for each
mole of allantoate synthesized (llS, ll6; Figure 2).
Based on these assumptions and the rate of 02H2 reduction for nodules
from 29—day-old soybean plants (44 nmol1mg dry weight"1 h71), the
85
estimated rate of synthesis for both glycine and serine is
8.8 nmol-mg dry weighth-h'1. Expressed on the amount of carbon that
is required, the synthesis of glycine requires 17.6 nmol of carbon
mg-dry weight'l h'1 and the serine requires 25.4 nmol of carbon-mg dry
weight-1 h']. These values are about l0-times higher than the rates
estimated above for the rates of incorporation of carbon incorporated
via dark C02 fixation. If the rates that are presented are accurate,
then the contribution of dark C02 fixation to the synthesis of glycine
and serine, and hence allantoate is minimal.
The labeling of alanine during dark €02 fixation can occur by
at least two different pathways (Figure l3). Aspartate that is labeled
in C-l can be decarboxylated forming alanine directly (lQl).
Alternatively, pyruvate formed by the decarboxylation of labeled
malate can be transaminated to form labeled alanine. There has been no
published report of an enzyme catalyzing the B-decarboxylation of aspartate
in nodules. Malic enzyme, which catalyzes the decarboxylation of
malate, is known to occur in nodules (8); therefore, the latter pathway
is the more likely route for the synthesis of labeled alanine.
Glutamine is synthesized from glutamate and NH: in a reaction
catalyzed by GS. The label from 14
C02 is proposed to enter into
glutamine as follows:
14002 --> oxalacetate -- 2-oxoglutarate -I- glutamate ---glutamine
Based on this series of reactions, the specific activity of glutamate,
the direct precursor of glutamine, is expected to be greater than or
equal to the specific activity of glutamine. Contrary to the expected
results, the specific activity of glutamine was higher than that of
86
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4.
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Figure l3 Proposed Pathways for the Exchange of Label into Alanine
in Nodules Exposed to 14(:02
The asterisks refer to the carbons that are expected to be labeled when
14002 is incorporated. Both carboxyl groups of oxalacetate are labeled
because of the conversion of oxalacetate to fumarate and fumarate back to
oxalacetate (Figure ll). The number in parentheses near each reaction
refer to the enzyme that catalyzes the indicated reaction:
(1) phospoenolpyruvate carboxylase
(2) malate dehydrogenase
(3) malic enzyme
(4) pyruvate aminotransferase
(5) aspartate aminotransferase'
. (6) aspartate beta-decarboxylase
87
glutamate in nodules exposed to 14
CO2 (Figure 9). To explain
this discrepancy, I propose that there are at least two different
pools of glutamate. One of these proposed pools of glutamate is
assumed to be rapidly labeled with [140] from 14
002 attaining
a high specific activity, while a second pool is at best only
slowly labeled. It is the former pool of glutamate which is
presumed to be used for the synthesis of glutamine. The differential
labeling of glutamate and glutamine will be more fully discussed in
chapter 3.
88
CHAPTER 3
3 AND NH: ON DARK coz FIXATION AND AMINO ACID
METABOLISM IN SOYBEAN NODULES AND ROOTS
THE EFFECTS OF NO
For a number of years N03 and NH: have been known to affect
the process of N2 fixation in nodulated legumes (l22,123). The
addition of N03 also appears to increase asparagine synthesis in
the root system of soybeans (12). The work presented in this
chapter is concerned with two areas: the effect of N07 and NH+
3 4
on amino acid metabolism in soybean nodules and roots, and the
pathway of asparagine synthesis in the soybean roots.
RESULTS
£h_EFFECTS OF INORGANIC NITROGEN ON__C2H9 REDUCTION AND DISTRIBUTION OF
NITROGEN IN THE XYLEM EXUDATE
I initially examined the effect of inorganic nitrogen on
amino acid metabolism by determining the time necessary for N03
to have an observable effect on N2 fixation and the distribution of
nitrogen in the xylem exudate. One group of nodulated plants was
treated daily with N-free nutrient solution while another group
\was treated with nutrient solution containing 10 mM KNO3 for 8 days
prior to the experiment. When the plants were 2l-days old, some of
the plants that had been grown on N-free nutrient solution were now
provided daily with nutrient solution containing l0 mM KNO3 until the
end of the experiment. These plants are referred to as "shifted"
.P7a nts. The control plants continued to receive N-free nutrient
soltxtion throughtout the experiment.
After 2 days of treatment with NO', the"shifted" plants had
89
Figure 14 Time Course of the Effect of N03 on the Distribution of
Nitrogen in the Xylem Exudate and the Rate of CZHZ Reduction
Soybean plants were irrigated with N-free nutrient solution (C)) or
with nutrient solution containing 10 mM KNO3 starting when the plants
were l3-days old (A) or 2l-days. old ([3 ). On the indicated days,
the shoots were excised and the xylem exudate was collected for 45 min.
Amino acids and N03 were analyzed by HPLC. Organic N is the sum of the
nitrogen in ureides, allantoin and allantoate, and in the amino acids
(12). The values reported for the concentration of N03, the ureide
content and the rate of CZHZ reduction are averages for 3 plants.
The average asparagine content was determined by chromatographing one
sample that consisted of equal volumes of xylem exudate collected
from 3 plants. Bars are presented in Panels A, B and C when the SD
is greater than the size of the symbol.
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91
a higher rate of CZHZ reduction than control plants, 3.2 versus
1 h-l
1.2 nmol plant- ; after 6 days, the rate of C2H2 reduction
of the “shifted" plants was the same as that measured for Nog-treated
plants, both of which were considerably lower than that of
control plants (Figure 14, Panel A).
The concentration of N05 in the xylem exudate of the "shifted"
plants, within 2 days, increased to 10 11mol~m1"1 (Figure 14, Panel B).
The comparable values for Nog-treated and control plants were
14 and 0.2 umoloml'1,respectively.
The ureide content (sum of allantoate and allantoin) of
the xylem exudate expressed as the percentage of the organic nitrogen
rose in control plants from 50 to 80%, the value that it remained
at for the next 12 days (Figure 14, Panel C). In contrast, the
ureide content of the "shifted" plants initially increased and
then decline. The relative ureide contents of the xylem exudate
of "shifted" plants after a 12-day treatment and the NOS-treated
plants were similar.
While asparagine is the major amino acid in the xylem
exudate of plants that are dependent on N03 and N2, it makes up
a larger percentage of the organic nitrogen in the xylem exudate
of plants that are dependent on N03 than those dependent solely on
N2 (l2). The major products of N2 fixation in soybean are the
ureides, allantoin and allantoate. A decline in the ureide
content and an increase in the asparagine content (the percentage
of organic nitrogen in the xylem exudate as asparagine), therefore,
indicates a shift of metabolism away from N2 fixation to N0;
utilization. After 6 days of treatment, asparagine accounted for
92
13% of the organic nitrogen. This value was 5-times more than the levels
in control plants but only 60% the levels observed in NOS-treated
plants (Figure 14, Panel 0). After 12 days, the relative asparagine
contents of the "shifted" and NOS-treated plants were similar.
The drop in the relative asparagine content that was observed
in the xylem exudate of control plants has been observed previously
(114) and appears to be related to developmental process.
The effect of NH: and N03 on the distribution of nitrogen
in the xylem exudate was next examined. Plants (ZS-days old) that
had been grown on N-free nutrient solution were irrigated with 1
liter of 1.5 mM (NH
SO4 or 10 mM KNO3 at 10:00 a.m. and 12, 24
- +
3 or NH4
the plants there was a small diference in the relative ureide
4)2
and 48 h later. Twelve hours after NO was first added to
contents in the xylem exudate of both the treated plants and
the control plants (Figure 15, Panel A). Fourty-eight hours after the
original treatment, the ureides in NH: and Nog-treate plants accounted
for 80 and 68% of the organic nitrogen in the xylem exudate, respectively
versus 90% in control plants. The percentage of organic nitrogen
as asparagine in Nog-treated plants and asparagine and glutamine
in NHZ-treated plants increased at the same time the ureide
content decreased (Figure 15, Panels C and D). The concentration
of N03
the first 12 h until it was l4-times higher than that of the
control and NHZ-treated plants (Figure 15, Panel B). These results
in the xylem exudate of NOE-treated plants increased within
suggest that within 12 h and certainly within 24, N03 and NHZ
affected plant metabolism. The following experiments were concerned
with amino acid metabolism and dark €02 fixation in plants that
93
Figure 15 Time Course of the Effects of N03 and NH: on the
Distribution of Nitrogen in the Xylem Exudate
Twenty-five-day-old soybean plants that had been previously irrigated
with N-free nutrient solution were irrigated either with N-free
nutrient solution (0) or nutrient solution containing 10 mM KNO3
( [5) or 1.5 mM (NH4)ZSO4 ([3 ) at 10:00 a.m. and 12, 24 and 48 h
later. At each time, the shoots of 3 plants from each group were
excised and the xylem exudate was collected for 45 to 60 min. Amino
acids and N05 were analyzed by HPLC. Organic N is the sum of
nitrogen in the ureides, allantoin and allantoate, and in the amino
acids (12). The values reported for the ureide content and concentrations
of N03 are averages of values for 3 plants. The asparagine and glutamine
content was determined by chromatographing one sample that consisted
of equal volumes of xylem exudate collected from 3 plants per
treatment. Bars are presented in Panel A when the SD is greater than
the size of the symbol.
94
. a J q . u . . m d J 4
AGL
H
48
24
,1 H
H .4 /H
/H. Ho
<:_____
H B
L - n p - n n \l
«I. m 9 6 3 O M H
:1.6~.o.=3 5:32.230 mo: 056339 no 2 2.330 8 m m
.. . . u
(d d d J 4 1 a d J a d 8 o.” g
.. 10114.. 1.01.. - n. A Mo .4 H
H 0/ AH: u
m\&11 SUA/
. r AM, . b p 1 c n /-O J o
O O O 9 6 O
9 7 5
.0295 no 2 2.3302. 2.30.2.3. no 2 2.3958.
95
- +
were treated with N03 or NH4 for 5 to 14 days.
B. EFFECTS OF NOEAND NH: ON AMINO ACID METABOLISM IN ROOTS OF
13-DAY-OLD PLANTS
1. Concentrations of Amino Acids
The effect of treatments with N05 or NH: for 5 days or longer on
the concentrations of amino acids and the incorporation of label from
14CO2 into amino acids was first examined in roots of l3-day-old plants
that had been treated with 10 mM KNO or 1.5 mM (NH4)ZSO4 for 5 days.
3
The N03 and MHZ-treated roots had, respectively, 2.2 and 2.7-times the
concentration of amino acids as the 13-day-old control roots did (Table 6).
Most of this difference was accounted for by differences in the amount
of asparagine. Roots of NOS-treated plants had 2.4-times the level of
asparagine as l3-day-old control roots. The NHZ-treated roots had
higher levels of asparagine (3.2-times) and glutamine (4.3-times) than
the l3-day-old control roots. For comparision, roots of 6-day-old plants
were also analyzed. They contained 7.5-times as much glutamine and 5.3-
times the total amount of amino acids as the 13-day-old control roots
when the values are expressed on a dry weight basis.
Nodules were present on the roots of 13-day-old control
plants, but were not observed on the plants that had been treated
with either NO' or NHZ. When split open, the nodules were white
3
indicating that leghemoglobin was not present and that the nodules were
not fixing N2 (25). The distribution of amino acids in nodules
and control roots was similar except for the amount of asparagine
and glutamate that was present (Table 6). In nodules the
concentration of glutamate was substantially higher than that
96
ocp How mospo> och
poppcoo How oopcomopo mozpo> as»
ocpso popop
.mpoEom Loo mpcopo N cppz mopoaom N Lop momoco>o opo mozmmpp Hospo
.oposom Loo mpcopo N sppz mmposom o Ho Amm Av momopo>o ogo mpooc oco mopzoo:
.oopoopoo mopoo ocpso ogp ppm Ho coppoeszm osp op mcopog mopoo
.ompoogpxo mopoo oopso ogp oco opo mxoo1mp co m opoz mogp cos: oopmo>pog ogoz mpcopo
asp .oomNflozzv 25 m.p co mozx zs op mcpcpopcoo coppzpom pcopgpzc .w hon co mopppopm Ho Apocpcoov
pcospcoaxm ogp pzogmsoggp coppopom pcopgpzc mogp1z gmcppo sppz zppoo oopompppp ogmz mpcopo coonaom.
m A No m.o A m.p o A mp m._ A a.mp m.m mp pocpcoo
mopzooz
HHH H.m «NH m.N N.N mp omNquzH
opp o.N No o.m m.m mp mso.
N A mm m.o A m._ p A mm m.o A o.N N.o A m.N mp poppcoo
mom w.¢p NAN 0.3 o.o o pocpcoo
mpoom
1p: xco as.posc
mopo< ocps< om<
popoh ocpEopzpw ocpmogoom< oposopzpm opoppoom< pcopa pcospooph
mpcopa mcso>
Ho mo—zooz oco mpoom op mopo< oops< mo coppocpcoocoo ocp co wzz oco moz Ho mpoowwm m opoop
97
measured in roots , 15 versus 2.4 nmolsmg dry weight-1. The
reverse was true for the concentration of asparagine. Roots had a higher
concentration of asparagine than nodules, 39 versus 13 nmol-mg
dry weight'].
2. Products of Dark CO2 Fixation
14
The roots of 6 and 13-day-old plants were exposed to CO
2
for 5 to 28 min. The labeled compounds in these roots were then
analyzed. Only the values that were obtained after a 28 min
exposure to 14
CO2 are presented (Table 7), because the distribution
of label did not appear. to be affected by the length of exposure.
Between 70 and 86% of the total label at all times and for all
tissues was recovered in the fraction containing the organic acids
and neutral compounds. A larger percentage of the total label was
in the amino acids of NOS-treated (30%) or MHZ-treated (23%) roots
than was in the amino acids of the l3-day-old control roots (14%).
In N03 and in NHZ-treated roots, asparagine accounted for 5 and 6%
of the total label, whereas in control roots only 1% of the
14
total label was in asparagine. The percentage of label from C0
2
in glutamine in MHZ-treated roots was the same as in the other roots
even though the concentration of glutamine in NHZ-treated roots
was higher than in the other roots of the same age. Glutamate
accounted for a higher percentage of the total label recovered from
nodules (10%) than it did in roots (3 to 5%).
98
.csspoo cpm: ocp :o oocpopoc po: ocoz posp moczoosoo omogp op Howoc monsooeoo Poppaoc + mopo< upcomcoo
.oposom Hog mpcopo N sppz
moposom N so» momoHo>o pzomopoog oopcomogo mm=Po> osh .zgpoeocpooom coppoppppcpom >3 oocpEHopoo
mo; app>ppooopoom .upo: >o oo~>Poco ogoz mposoopo oopooop och .:omocpp: opzopp :p coNocp oco xmopp
ogp sogw oo>oeop ago; msmpmam poo; ogp .:ps mN copp< .Nou op oomooxo opmz amcp ogogz xmopw o opcp
ep
oooo—a osoz msmpmzm poo; onp oco .oompoxm msoz mpoogm ocp .ompoogo: ogoz xmgp .opo mxoo1mp go 0 ago;
mpcopo ocp cos: .eomNAozzv :5 m.~ Lo mozx ze op mcpcpopcoo coppzpom pcopgpzc .w zoo co mopppopm Ho
apogpcoov pcmsppooxm ocp paogmzossp coppopom pcopgpzc mogw1z Hosppo gppz oopompggp ago; mpcopo :oooxom
mm o o op N mp poppcoo
mopzooz
AH m m m 8 mp vomNHezzv
mm m p m m mp mozx
ow o o A pp 0 _ogpcoo
mpoom
app>ppooopooa Popop A maoo
m caooso o; so on
o+ mopow wpcwmcu oopsopzpw ocpmogoom< oposopzpo opopcoom< pond; pcmspooph
m
mpcopa mczo> wo mopzooz oco mpoom
soap oopmtoatoocp Foams Ho coppsnptpmpo asp go «I: use moz Ho mpompcu H opnap
N
:p coop + 1
99
- +
3 AND NH4
29-DAY-OLD PLANTS
C. EFFECTS OF NO ON AMINO ACID METABOLISM IN ROOTS OF
1. Concentrations of Amino Acids and Organic Acids
Starting when they were 15-days old some plants began receiving
nutrient solution containing 10 mM KNO3 or 1.5 mM (NH4)ZSO4. When
the plants were 29-days old, the roots were harvested and their amino
acid content was determined. Nitrate and ammonium had a similar effect
on the distribution of amino acids in roots of 29-day-old plants
(Table 8) as they did on the distribution in the roots of l3-day-old
plants. The concentrations of amino acids in Nog-treated and in
NHg-treated roots were 2.4 and 3.2-times the levels in control roots.
Most of the difference in the concentration of total amino acids was
accounted for by larger concentrations of asparagine in N03 and NH:-
treated roots and by a larger concentration of glutamine in NH:-
treated roots than in control roots.
The effect of N03 on the concentration of several organic acids
in roots was also determined. The presence of high levels of malonate
and malate in nodules and in control roots previously reported (162)
was confirmed (Table 9). Control roots, however, had higher concentra-
tions of malonate than either NOé-treated roots or control nodules.
Nitrate-treated roots had lower amounts of all the organic acids than
were measured in either nodules or control roots.
2. Rates of Dark CO2 Fixation
The rates of dark CO2 fixation in roots appeared to be
affected by the presence of NH2. The rate of dark CO2 fixation
+
in NH4-treated roots from 29-day-old plants was significantly
1OO
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.oapoapao mopoo ospEa asp Fpa Ho sopposszm asp op msaAas mopoa ospsa Papop .uss: xs oaprosa
asaz mopoa ospsa ash .oapoaspxa mopoa ospEa asp osa opo mxao1mN asaz zasp sasz oapma>sos agaz
mpsapo asp .oomNsvzzv zs m.~ so mozx :5 o_ newspapsoo coppspom psapspsc .m_ aoo so msppsapm
so apospsoov psaspsaoxa asp pzosmzossp soppzpom psapspas aasA1z sppz oapampssp asaz mpsapa saasAom
A A m__ a.p A o.HN N A so m.o A m.A N.o A m.m AomNHAIZH
e A NN m.o A p.N m A mm .m.o A N.m ¢.o A m.m mozx
m A om p.o A o.p N A pp —.o A m._ N.o A m.N pospsou
P1p: ago mE.PoEs
mopo< ospe< aspsapzpo aspmasaom< apasapzpw apapcaam< pcaspaasp
papop
mpcapa Apo-sao-am so mpoom =H mapo< ocps< Ho coppaspcaocoo asp so N12 oca moz so mpoaaaa a apnap
101
Table 9 Effect of KNO on the Concentration of Several Organic
3
Acids
Treatment Malonate Malate Succinate Fumarate
nmol~mg dry wt"1
Roots
control 123 i 22 45 i 8 11 i 3 14 i 5
KNO3 16 i 3 23 i 2 2 i 1 1 i 1
Nodules
control 33 i 5 30 i 3 12 i 2 16 i 3
Soybean plants were irrigated with N-free nutrient solution until
they were lS-days old, after which time a number of the plants
continued to receive N-free nutrient solution (control) while the
other plants were irrigated with nutrient solution containing 10
mM KNO3. When the plants were 29-days old, the indicated tissue
was extracted with ethanoleZO (80:20, v/v). The organic acids were
purified by ion-exchange chromatography. The organic acids were
dissolved in pyridine and derivatized with BSTFA. The trimethylsilyl
derivatives of the organic acids were separated by gas-liquid
chromatography on a 3%-SE 30 column with a temperature program of
85 to 185 C at 5 C-min']. Malonate, malate, succinate and fumarate
were used as standards. The values reported are averages ( i SE)
fbr 4 plants.
102
higher than the rates measured for the other roots of comparable
age (Table 10). The rate of dark CO2 fixation in roots of 21-day-old
plants for each of the treatments, however, were not significantly
different from one another.
3. Products of Dark CO2 Fixation
The presence of N03 or NH: had a dramatic effect on the
distribution of label in 29-day-old roots exposed to 14
C02. Amino
acids in control roots accounted for at most 22% of the total
recovered radioactivity, with aspartate and glutamate, the two
major labeled amino acids, accounting for 6 and 8% of the total
label, respectively (Figure 16, Panel A). In contrast, after only
a 3 min exposure, 33% of the total label recovered from MHZ-treated
roots was present in the form of amino acids (Figure 16, Panel B).
The percentage of label in amino acids in NHZ-treated roots continued
to increase until after a 28 min exposure, asparagine and glutamine,
the two major labeled amino acids accounted for 14 and 24% of the
total label, respectively. In NOé-treated roots, the percentage of
14
label in the amino acids after a short exposure to CO2 was
lower than that observed in MHZ-treated roots (Figure 16, Panels B
and C). With longer exposures, the percentage of label in asparagine
increased, representing 25% of the total label after a 60 min
exposure.
4. Specific Activities of Amino Acids in Roots Exposed to 14co
2
The specific activities of glutamate and glutamine in
14
nodules after exposure to CO2 could not be explained by the
simple conversion of labeled glutamate to glutamine (discussion in
103
Table 10 Effects of N03 and NH: on the Rate of Dark co2 Fixation
in Roots and Nodules
Roots Nodules
Treatment 21-days 29-days 21-days 29-days
nmolsmg dry wt']~h'1
Control 16 1 2a 16 i 3a 51 i 6a 30 i 2a
KNO3 (10 mM) 12 i la 14 i la 20 i 3c 10 i lb
KNO3 (3 mM) 15 i la 17 i 2a 41 : 3ab 12 i 1b
(NH4)ZSO4 17 i 2a 24 i lb 34 i 3bc 14 i 2b
(1.5 mM)
Soybean plants were irrigated with N-free nutrient solution until they
were lS-days old, after which time a number of the plants continued to
receive N-free nutrient solution (control) while the other plants were
irrigated with nutrient solution containing 3 or 10 mM KNO3 or 1.5 mM
(NH4)ZSO4. When the plants were 21 or 29—days old, the shoots were
excised and the nodulated root systems were placed into a flask where
they were exposed to 14
C02. After a 10 min exposure, the nodulated
roots were removed and frozen in liquid nitrogen. The amount of
radioactivity that was incorporated was determined by scintillation
spectrometry. The values are averages for 6 replicates (t SE). In
each column, the values that are followed by the same letter are not
significantly (P§_0.05) from one another according to Duncan's
multiple-range test.
104
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osa Noe» saazpas aasasaAwpo asp .Aspasospoasm soppappppspom As oaszmaas mo: App>ppoaopoam .uss: As
oaNAAosa asaz mpasooss oapasop asp .apssam gas mpsaps N sppz apsEam aso so mpmAAasa psamassas
sapsamaso maon> asp .Nouvp op oamosxa asaz Aasp asasz smapm a opsp oaoapa asaz mEapmAm
poo; asp osa .oampoxa asaz mpoosm asp .oapoosss asaz Aasp .spo mAao1mN asaz mpso—o asp sasz
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105
op assmHH
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106
chapter 2). The relationship between the specific activities of
14
glutamate and glutamine in roots exposed to CO2 was different
depended on what the source of nitrogen for the roots was. The
specific activities of glutamate and glutamine in control roots
were about equal, as they rapidly increased reaching maximum values
of 302 and 288 cpmmmoi'1
, respectively (Figure 17, Panel A). 'In
+-
4
were also about equal, but they increased at a slower rate than they
NH treated roots the specific activities of glutamate and glutamine
did in control roots (Figure 17, Panel B). In NOé-treated roots
the specific activity of glutamate (21 cpm'nmol'I) was initially
higher than that of glutamine (O cpm-nmol") (Figure 17, Panel C).
However, after a 5 min exposure, the specific activity of glutamine
was 105 cpmonmol'1
, ZO-times that of glutamate. The maximum
specific activities of glutamine and glutamate observed in NOE-treated
roots were 314 and 195 cpmonmol'1, respectively.
14
After a 3 min exposure to C02, the specific activity of
aspartate in control, N03 and NHZ-treated roots was 98, 36 and
1, respectively (Figure 17). The specific activity of
88 cpm «nmol-
aspartate increased in control and NOE—treated roots reaching the
maximum observed values after a 28 min exposure. In contrast, the
specific activity of aspartate in NHZ-treated roots remained
relatively constant. The specific activity of asparagine increased
in all tissues although at a slower rate in control and NHZ-treated
roots than it did in Nog-treated roots.
107
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asaz mopoa ospEa ash .Aspasospoasm soppop—ppspom As oassmaas ma; App>ppoaopoom .oss: As
oaNAPasa asaz mpozooss oapasap asp .apssom gas mpsaps N sppz aAsEam aso 4o mpmApasa psamassas
oapsamass maapa> asp .Nou op oamosxa asaz Aasp asasz swap» a opsp oaoapo asaz msapmAm
AH
poo; asp use .oampoxa asaz mpoosm asp .oapooss: asaz Aasp .opo mAao1mN asaz mpsaps asp sasz
.Ho paces .mozv mozx :2 o_ co Hm paces .szs Aommsezzv :5 m.p mepepepeoo coppspoA pechpse co
a< chmm «POLHCOUV :OwquPOm Hcmeflzc QQLWIZ 5&5) UmHMOwLLw wLm3 main—h 0;“ meH £U_.£3
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108
NH acsmpa
..:.... 2:;
cm on o 00 on o Om on o
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109
0. EFFECTS OF NO3 AND NH: ON AMINO ACID METABOLISM IN NODULES
The results in the previous section demonstrated that the
concentrationsof amino acids in roots were elevated when either
N03 or NH: was added to the plants. The effect of N03 and NH: on
amino acid metabolism in nodules was next examined. For 13 or 14
days prior to an experiment, plants were provided with either N-free
nutrient solution or nutrient solution that contained 3 mM KNO3 or
1.5 mM (NH4)ZSO4. The effect of N05 and NH: on the rate of CZHZ
reduction and the total weight of nodules per plant reported previously
(193) was confirmed. The rate of CZHZ reduction of the N03 and NH:-
treated nodules from 29-day-old plants was 36 and 66% of the rate
of control nodules, respectively. The total weight of the nodules
from N03 and NHZ-treated plants, was on a per plant basis, 52 and 75%
of the control plants, respectively.
The concentration of amino acids in NH: and NOS-treated nodules
was about 75% of the levels in control nodules (Figure 18). Aspartate
and glutamine in N03 and MHZ-treated nodules accounted for less than 40%
of the values they did in control nodules. There were, to a lesser
extent, differences between the concentrations of asparagine, serine and
glutamate in Nog-treated nodules and asparagine in MHZ-treated nodules
and the concentrations of those amino acids in control nodules.
Differences in the incorporation of [14C] in the nodules
14
exposed to CO2 were observed between control nodules and NH:
and NOS-treated nodules. Aspartate accounted for 11% of the label
in control nodules exposed to 14
C02 for 28 min, whereas, it accounted
for only 5% in NOS-treated nodules and 3% in NHZ-treated nodules.
Glutamate accounted for 18% of the label in control nodules but only
110
=5 ...
g 100- — ~
0
2‘.
3 ' 75- w_, —' -
.3. . 1__.1 .—
:(.1
/‘ . 1
At
0.1 l l J .l
.0 . 20 4.0
Time (min)
Figure 23 Time Course of the Distribution of Labe1 in Organic Compounds
in Aider Nodu1es Exposed to 14C02
Aider nodu1es were removed from the roots and were p1aced into a f1ask
where they were exposed to 14C02. At the indicated time, the nodu1es
were removed from the f1ask and frozen in 1iquid nitrogen. The
1abe1ed products were extracted with ethan01:H20 and were ana1yzed
by HPLC. Radioactivity was measured by scintiTIation spectrometry.
The o.a. + neu. refers to the fraction containing the organic acids
and neutra1 compounds. Cit1. is the abbreviation for citru11ine.
136
Radlaactlvity (cpm)
fltdl
755 - ' t -
' cit .
5.0- ..
origin euc tum
1 1 1 -
0 .L p I
(D ‘ ' l() 21)
Distance (cm)
Figure 24 IncorporatiOn of Labe1 into Organic Acids and Neutra1
Compounds in A1der Nodu1es Exposed to 14CO2
A1der nodu1es were removed from the roots and p1aced in a f1ask where
they were exposed to 14C02. After 20 min, the nodu1es were removed
and the 1abe1ed products were extracted with ethano1zH20 (80:20, v/v).
The extract was separated into two fractions, one containing the organic
acids and neutra1 compounds, and a second containing amino acids. An
a1iquot of the former fraction was spotted on a thin-1ayer ce11u1ose
p1ate at the origin and Chromatographed with butano1zg1acia1 acetic
acidzHZO (120:30:50, v/v/v) as a so1vent system. Ma1ate (ma1) and citrate
( cit ) were identified on the basis of co-migration with standards.
Succinate (suc) and fumarate (fum) were identified on the basis of
pub1ished Rf va1ues (206). Radioactivity was detected with a gas-
f1ow scanner.
137
Figure 25 Time Course of the Distribution of Label in Organic
Acids in Alder Nodu1es Exposed to 14CO2
A1der nodules were removed from the roots and placed in a flask
where theywere exposed to 14C02. At the indicated time, the
nodules were removed and frozen. The labeled products were separated
by ion-exchange chromatography into two fractions, one containing the
organic acids and neutral compounds and a second that contained the
amino acids. The former fraction was Chromatographed on Dowex 1 (chloride,
acetate) using gradient elution with water and 1.2 N formic acid.
Radioactivity was measured by scintillation spectrometry. The columns
were standardized with [14C]ma1ate and [14C1citrate. Succinate was
identified on the basis of its relative retention to malate and
previously published separations (204,205). Based on reports
in the literature (204) a portion of the label that eluted with citrate
may be radioactive fumarate. Less than 2% of the radioactivity eluted
in the void volume of the column.
36' Radioactivity
80
0t
0
A
O
20
138
O
O
citrate
. O .
succinate 0
e’.\‘ .* ~e
O 20 ' 40
Time (min)
Figure 25
DISCUSSION
Carbon that was incorporated during dark CO2 fixation in
alder nodules was incorporated into citru11ine. There are at least
two enzymes, PEPC and carbamylphosphate synthetase, that fix CO2
into products that are used as intermediates in the synthesis of
citrulline in other organism (111) (Figure 26). The presence of
label in glUtamate and aspartate is consistent with a part of the
14CO2 being incorporated via PEPC in alder nodules. The relationship
between the percentage of label in glutamate and citru11ine (Figure 23)
suggests that the [14C] was entering citrulline via glutamate. The
pathway from glutamate to citrulline in other systems has ornithine
as an intermediate. Labeled ornithine may not have been detected
either because the amount of label fell below the detection limits of
the system or because ornithine and thus citrulline may not be
synthesized from labeled glutamate. Carbamylphosphate synthetase,
which catalyzes the synthesis of carbamylphosphate from HCOS and
[14C] incorporated during dark
ATP, may be the enzyme by which the
CO2 fixation is incorporated into citrulline.
The change in the distribution of label in the fraction containing
the organic acids and neutral compounds (Figure 27) may be explained
if two pools of malate are present. MacLennan 33 31. (192) proposed
that different pools of several organic acids exist in plant cells.
One of the pools for each of the organic acids appears to be closely
associated with the respiratory centers, whereas the other(s) is
physically remote from these centers and is metabolically stable.
I propose that a similar separation of pools exist in the alder
nodule. The major portion of the citrate but only a minor portion
139
140
phoephoenol- glutamate ornithine citrulline
pyruvate
cozw . 'cozw ”00214 *00211
fi-oeoa Hz .L'lswzw-cw _‘Q... HzN-CH HzN-C'll-i
CH2 “f"z’z ' (9"212 (3) ‘fH2’3
+ COzH ‘ ‘ (.:Hz. NH
10 _ , l .
11cc3 Nl-iz 'c=o
l
4‘ hfliz
.f a ‘_ (4) qktb
2 ATP~ + NH4 + H003 ’C-OPO3H2
0' Hz"
glutamine
carbamylphosphate
Figure 26 Proposed Pathways for the Incorporation of Label into Citrulline
in A1der Nodules Exposed to 14C02
The asterisks refer to the carbons that are expected to be labeled when
14CO2 is incorporated. The numbers in parentheses refer to an enzyme or
series of enzymes that catalyze the indicated reactions:
(1) phosphoenolpyruvate carboxylase
citrate synthase
aconitase
isocitrate dehydrogenase
glutamate synthase
(2) carbamylphosphate synthetase
(3) glutamate acetyltransferase
acetylglutamate kinase
N-acetyl-y-glutamylphosphate reductase
acetylornithine deacetylase
(4) ornithine carbamyltransferase
141
of the malate is in a pool that is actively turning over; the
.major portion of the malate is sequestered in a metabolically
inactive pool. As label accumulates in the nodule, the percentage
of label in malate would increase because the metabolically inactive
pool of malate would account for a larger portion of the label.
CHAPTER 5
13
THE INCORPORATION OF N-LABELED TRACERS IN SOYBEAN AND ALDER NODULES
AND SOYBEAN ROOTS
The experiments that were described in the previous chapters
[14C] to explore amino acid metabolism. This
used the tracer
approach is limited, however, in that only the metabolism of the
carbon skeletons can be monitored. The use of the radioactive
istope of nitrogen, [13M], provides a way of tracing the path of
the nitrogen of amino.acids. The following experiments were
designed to follow the pathway of nitrogen assimilation in nodulated
roots of soybeans using radioactive N2, N03 and NH: and to
explore the assimilation of NH: in alder nodules using labeled NHZ.
RESULTS
The pathway of NH: assimilation in alder nodules was examined
initially by incubating detached nodules with ‘3
NHZ. At intervals
nodules were removed and were extracted with methanol. Labeled
products were separated by high-voltage electrophoresis. Glutamine
accounted for the highest percentage of label with lesser amounts
in glutamate, alanine, aspartate and asparagine (Figures 27 and 28).
The amide-group of glutamine accounted for 90% of the label in
glutamine. Alder nodules that were still attached to the roots
incorporated label from 13
NH: in a fashion similar to that of
detached alder nodules.
The relationship between the incorporation of [13N] into
glutamine and glutamate was examined. Alder nodules were incubated
142
143
with NH: for various lengths of time, at which time they were
‘either immediately extracted or incubated with 13NH: for 5 min,
washed with H20 for 3 min and then incubated for a further 10 min
14
in water containing 10 mM NH: before they were extracted.
Glutamine accounted for 90 to 80% of the total radioactivity
13 +
NH4
min (Figure 28). Similar results were obtained with the
incorporated after exposure of the nodules to for l to 15
pulse-labeling experiment, in which glutamine accounted for 70%
14NH+.
of the incorporated label even after the 10 min chase with 4
The role of GS and GOGAT in the assimilation of NH: in
alder nodules was examined using an inhibitor of GOGAT, azaserine,
and an inhibitor of GS, methionine sulfoximine (MSX). Nodules
were incubated in water containing 1 mM azaserine for 10 min,
at which time 13
NH: was added. Ten minutes later the nodules were
extracted with methanol. The presence of azaserine did not have
any effect on the distribution of label in glutamine and glutamate.
In contrast, a pretreatment of alder nodules with l or 10 mM
MSX for 10 or 20 min before the nodules were exposed to 13NH:
increased the distribution of label into glutamate, aspartate and
alanine in relation to the amount of label in glutamate when only
13NH: was added. Labeled glutamate and aspartate accounted for
only 52% of the total radioactivity incorporated in MSX-treated
nodules versus less than 15% in nodules that were not treated with
MSX.
The incorporation of labeled nitrogen insoybean was first
examined by determining the incorporation of 13NH: and 13N03
into soybean roots. The roots of soybean plants that had been grown
144
I
hD
C)
1
(D
In.
(cpm x164.5 )(.....
9
an
°
ll) ' 155 12¢)
Distance (cm)
Radioactivity (cpm x '0-3 H 1
' (i
in
(3
«A;
Figure 27 Incorporation of Label into Organic Products in Alder Nodules
Incubated with 13NH:
Alder nodules were removed from the plant roots and placed into H20
containing 13NHZ. Eleven minutes later, the nodules were removed and
the labeled products were extracted with methanoleZO (80:20, v/v).
After standard amino acids were added to the extract, an aliquot was
spotted on a thin-layer cellulose plate. The plate was sprayed with
70 mM sodium borate (pH=9.2) and subjected to electrophoresis (3 kV).
The radioactivity on the plate was detected with a gas-flow scanner and
the standard amino acids were visualized by spraying the plate with
ninhydrin. The arrows indicate the location of the standard amino acids
after electrophoresis.
145
Figure 28 Time Course of Incorporation of Label into Glutamine
and G1utamate in Soybean Roots and Alder Nodules
Incubated with 13M”:
Soybean roots (. ,I) or alder nodu1es (O ,D) were incubated in
H20 containing 13NHZ‘for the indicated time. The organic products
were extracted with methanoleZO (80:20, v/v). After standard amino
acids were added to the extract, an aliquot was spotted on a
thin-layer cellulose p1ate.‘ The plate was subjected to electrophoresis
(3 kVO. The radioactivity on the plate was detected with a gas-flow
scanner and the standard amino acids were visualized by spraying the
plate with ninhydrin. The amount of label that co-migrated with
glutamine (., O) and glutamate (I ,D) was quantified by
integration of the peaks after scanning. Areas were corrected for
decay. The difference between 100% and the sum of the label in
glutamine and glutamate was accounted for by label in aspartate and
alanine.
146
100
80 '
6 . .4
3383.3: .28.:
15
Time (min)
Figure 28
147
on N-free nutrient solution actively incorporated label from
both 13NH: and 13N0; into organic products (Figure 29). The
distribution of label from 13
NH: after exposure of soybean roots
was similar to that observed in alder nodules (Figure 28) with
glutamine accounting for about 80% of the total label incorporated.
13NHZ, the percentage of label
In soybean roots incubated with
in glutamine versus glutamate was higher than that in roots incubated
with 13110; (Table 14). ' I
Meeks gt al.(5) examined the time course of incorporation
of label from [BNJN2 into the organic products of soybean nodules.
One product, which migrated between glutamate and asparagine, was
not identified. In an attempt to identify this compounds, we
exposed soybean nodules to 13NH: for 10 min, at which time the
labeled prOducts were extracted and subjected to electrophoresis.
Labeled NH: was used rather than [BNJN2 because larger amounts
13NH: versus [BNJN2 (preliminary
of label can be incorporated using
observations ). The previously unidentified compound was
identified as allantoate on the basis of its migration with
standard allantoate during electrophoresis. Allantoate accounted
for only a minor portion of the label (8%) in nodules incubated with
13NH: (Figure 30, Panel B) versus the 30% reported by Meeks EEJEL-
(5) for the unidentified compound in nodules exposed to [‘3NJN2.
The experiment performed by Meeks et_al. (5) was repeated to determine
if the distribution of label in nodules exposed to 13NH: or [13NJN2
was different. Our results with detached nodules exposed to
[13NJN2 (Figure 30, Panel A) were similar to those previously
reported (5). Allantoate accounted for 48% of the incorporated label
C)
I
)>
I!
C)
03
1
9
(I
I
(31:. ‘—‘* __2:
15- B '3NH1'
lC)- .
91’“ ale
5 -' asp can I -
Radioactivity (cpm 910's )
o
‘1
1’
11
l
o e ' . lO 15 20
Distance (cm)
Figure 29 Incorporation of Label into Organic Products in Soybean Roots
Incubated with 13NO; or 13NH:
Soybean roots were incubated in H20 containing N03 for 12 min (Panel A)
r 13NH: for 15 min (Panel B). At the indicated time, the roots were
removed from the solution and the labeled products were extracted with
methanoleZO (80:20, v/v). After standard amino acids were added to
the extract, an aliquot was spotted on a thin-layer cellulose plate. The
plate was sprayed with 70 mM sodium borate (pH=9.2) and subjected to
electrophoresis (3 kV). The radioactivity on the plate was detected
with a gas-flow scanner and the standard amino acids were visualized by
spraying the plate with ninhydrin. The arrows indicate the location of
the standard amino acids after electrophoresis.
13
149
Table 14 Incorporation of Label into Organic Compounds in Soybean
Roots Incubated with 13NH: or 13N03
Treatment Glutamine Glutamate Othera
% of Total Radioactivity
131m: (5) . 83 e 2 17 e 1 0
13N03 (3) 47 e 6 33 i 6 26 i 2
Soybean roots were incubated in water containing either 13NH:
or 13N03 for 7 to 12 min, at which time the roots were removed and
the labeled products were extracted with methanoleZO (80:20, v/v).
After standard amino acids were added to the extract, an aliquot
was spotted on a thin-layer cellulose plate. The plate was sprayed
with 70 mM sodium borate (pH=9.2) and subjected to electrophoresis
(3 kV). Radioactivity was detected with a gas-flow scanner and
the standard amino acids were visualized by spraying the plate
with ninhydrin. The amount of label that co-migrated with each
peak was determined by integration of the peaks after scanning. The
values that are reported are t SE. The number in parentheses
indicates the number of experiments.
aOther refers to those compounds that did not co-migrate with
glutamine or glutamate. Because of the number of experiments
and the poor separation of the labeled compounds,the identity
of these compounds was not established.
F3 I I '
A A N2 010 ’2‘
glu l g
1.5- as 1 gln -l.5 :
C“
1.0 '-
_e:
5.
(DJS ‘3
C)
Radioactivity lcpm 1: IO.3 N
1
.ppf 1....
l
(3 £5 ll) 125 1213
Dietanceicm)
Figure 30 Incorporation of Label into Organic Products in Soybean
Nodules Exposed to [13NJN2 or 13NH:
Soybean nodules were exposed to [13N]N2 (Panel A) or were incubated in
H20 containing 13NH: (Panel B). After a 10 min exposure, the nodules
were removed from the vessel containing the label and the labeled
products were extracted with methanoleZO (80:20, v/v). After standard
amino acids and allantoate were added to the extract, an aliquot was
spotted on a thin-layer cellulose plate. The plate was sprayed with
70 mM sodium borate (pH=9.2) and subjected to electrophoresis (3 kV).
The radioactivity on the plate was detected with a gas-flow scanner.
The standard amino acids were visualized by spraying the plate with
ninhydrin and allantoate by spraying with diaminobenzaldehyde. The arrows
indicate the location of the standard amino acids and allantoate (aln)
after electrophoresis.
151
with lesser amounts of label in aspartate, glutamate, glutamine
and alanine (Figure 30, Panel A).
DISCUSSION
Glutamine was the principal product of NH: assimilation in
alder nodules. This result plus the observation that the amide
position accounted for 90% of the label in glutamine indicates that
GS was the enzyme primarily responsible for the assimilation of
NH: in alder nodules. A number of investigators (78,83,85) concluded
that GS plays a similar role in a number of other systems including
soybean nodules. The question of whether other enzymes, such as GDH,
also assimilates NH: in nodules or in other tissues has not been
answered.
Methionine sulfoximine, a specific inhibitor of GS, inhibited
13
the incorporation of label from NH: into glutamine more than it did
into glutamate and the other amino acids labeled in alder nodules.
Similar results have been observed in tobacco cells (78) and rice
roots (81) that were incubated with labeled NHZ. It has been assumed
+
49
then MSX should inhibit the synthesis of glutamine and glutamate to the
that if GS/GOGAT is the only pathway for the assimilation of NH
same extent. To explain the discrepancy between this assumption and
the results obtained with tobacco cells, Skokut 33 31. (78) suggested
that either a small amount of the NH: was assimilated by GDH or
that the activity of GS that remained in the presence of MSX permitted
the synthesis of glutamine which could be preferentially used in
the synthesis of glutamate.
Although similar explanations may be put forth to explain
the effect of MSX on the assimilation of labeled NH: in alder nodules,
it is not necessary to invoke the assimilation of NH: by GDH. Instead
there may be enough residual activity of GS in both alder nodules
152
153
and tobacco cells in the presence of MSX to provide glutamine for
the activity of GOGAT to remain at a substantial percentage of its
activity in the absence of MSX. This assumes that the activity of
GS is greater than that of GOGAT in tobacco cells and alder nodules,
as is the case in a number of other systems that have been examined
(83,207,208). To illustrate, the relative enzyme rates may be assumed
to be similar to those in soybean nodules where the in vitro
1
activities of GS and GOGAT are 89 and 10 nmolomg protein'o min"1
(83). If the activity of GS is inhibited 95% by MSX, then the
residual activity could provide 4.5 nmol of glutamineomg protein-'Jmin"1
or enough glutamine for GOGAT to have 45% of the activity it had
in the absence of MSX. Methionine sulfoximine, therefore, could end
up inhibiting GS activity by 95%, but inhibit GOGAT activity by only
55%. The labeled glutamine would not accumulate in the presence of
MSX, but instead be used in the synthesis of glutamate. While the
differential inhibition of the incorporation of labeled NH: into
glutamine and glutamate by MSX can be explained in terms of differences
of jn_vitro enzyme activities, further experimentation is necessary
to determine if differences in enzyme activities occur jg_vivo and if
these differences are indeed responsible for the differences in the
labeling patterns. Further, based on the results that are now
available, the possibility that this differential labeling of glutamine
and glutamate is the result of GDH assimilating NH: cannot be excluded.
Differences in the distribution of label in glutamine and
glutamate are observed in tissues that incorporate NH: (tobacco cells,
(78); rice roots, (81) and soybean nodules and roots (Figures 29 and
30)) and the same tissues that incorporated [13N]N2 (soybean nodules,
154
Figure 30 and ref. 5) or labeled N05 (tobacco cells (78); rice roots (81);
and soybean roots (Figure 29)). More labeled glutamate than glutamine
was formed in tissues that were exposed to [13NJN2 or labeled N03,
13 +
NH4.
I propose that these differences may be the result of differences in
whereas the reverse was true in tissues that were exposed to
the relative rates of synthesis of labeled glutamine and glutamate from
labeled NO and NH+. I assume that the rate of labeled NH:
3’ "2 4
production in the tissues that were exposed to [BNJN2 and labeled N03,
and the rate of glutamate synthesis in those tissues exposed to exogenous
labeled NH: is lower than the rate at which exogenous labeled NH: is
incorporated. Although the data are not available for all the enzymes
in all the tissues, these assumptions hold for soybean nodules, if the
_i_n_ v_i_t;r_g activities of GS and GOGAT are reflective of their relative
activities in 1119. The estimated rate of N2 reduction is 12 nmol NH:
produced-g fresh weight of nodules"1-h"1 in soybeans. This value is
based on the maximum rate of CZHZ reduction that was presented in this
thesis (Figure 4), 124 nmol CZHZ reduced g dry weight"1 h", and
assumes that the rate of N2 reduction equals the rate of CZHZ reduction
divided by 4 and that l g dry weight is equal to 5 g fresh weight. The
estimated potential rate for glutamine and glutamate synthesis is 108
and 12 umolog fresh weight of nodules-1 h']. These values are based
on EM activities of GS and GOGAT (83).
. According to this proposal, exogenous labeled NH: is rapidly
incorporated in the nodules into glutamine by GS. This 1abe1ed
glutamine may then be used in the synthesis of labeled glutamate.
However, the synthesis of labeled glutamate is assumed to be much
slower than that of labeled glutamine because of the relative
'155
activities of GS and GOGAT. The result would be a high ratio of labeled
glutamine to 1abe1ed glutamate.
In experiments in which labeled NH: is provided exogenously,
the synthesis of glutamine is proposed to be limited by the activity
of GS. In contrast, the rate at which labeled glutamine can be
synthesized from [IBNJN2 is limited by the rate of N2 fixation. If
the rates of N2 fixation and glutamate synthesis are assumed to be
12 and 10 nmolog fresh weight of nodules’kvh’I, respectively, as
discussed above, then the rates at which labeled glutamine and
glutamate would be synthesized from [BNJN2 are the approximately
the same. The amount of labeled glutamine would be about the same
as that of labeled glutamate. The rate of glutamine synthesis in roots
treated with labeled N03 may be limited by the rate of N0; reduction
in a manner similar to that discussed above.
The differences in the labeling patterns when different sources
of radioactive nitrogen were used was not confined to differences
in the labeling of glutamine and glutamate, Allantoate accounted
for 48% of the radioactivity incorporated in soybean nodu1es that
had been exposed to [13N]Nz, but appeared to account for only 8%
13
of the label incorporated in nodules incubated with NH: (Figure 30).
These observations confirm the work of Ohayama and Kumazawa (144),
who demonstrated the preferential incorporation of label from
[15N1N2 into allantoate in soybean nodules, and Fujihara and Yamaguchi
15
(109), who demonstrated that NH: was primarily incorporated into
amino acids rather than allantoate.
The difference in the incorporation of label from 13NH: and
[13N]N2 into allantoate may be explained by differences in the
156
distribution of enzymes within the nodule. The distribution of
enzymes involved in the allantoate synthesis was examined in the
nodules of cowpeas (Vigna unguiculata) (209), another plant that
transports nitrogen from the nodules as allantoate. Those cells that
contain bacteroids and are in the center of the nodule have higher
levels of these enzymes than the cells that are on the outside of
the nodule (209). Thus, when nodules are incubated with 13NHZ,
e 13
th NH: may be assimilated in the cells on the outside of the
nodule and never reach the cells that contain the enzymes necessary
13
for allantoate synthesis. In contrast, NH: produced during the
reduction of [UNJN2 would be in close proximity to the site of
allantoate synthesis and therefore, would be incorporated into
allantoate.
In contrast to nodules, labeled allantoate was not detected in
roots incubated with 13NH: or 13
N03 indicating that the synthesis
of allantoate occurs primarily in the nodule. This statement is
consistent with previous results (12,144,193) demonstrating that
allantoate is the major form of nitrogen transported when plants
are dependent on the N2 that is reduced in the nodule.
SUMMARY
The major results presented in this thesis are summarized -
below:
1) Carbon-l4 was incorporated in soybean nodules and roots
and alder nodules exposed to 14C02.
2) Most of the label incorporated in soybean nodules during
exposure to 14CO2 was rapidly lost from the nodules.
3) The major labeled compounds in nodules and roots exposed
to 14CO2 were closely associated with the tricarboxylic acid
cycle.
4) The source of nitrogen of the plants affected the distribution
of the label incorporated in nodules and roots exposed to 14C02.
13 13
5) Plant tissue exposed to NOS, NH: or [BNJN2 had different
distributions of labeled products.
6) After exposure to 14C02. the specific activities of glutamine
was hiaher than that of glutamate in soybean nodules and in
roots of sovbean plants treated with N03.
7) After incubation of soybean roots with 3H- and 14c-iabeied
aspartate, most of the labeled asparagine was recovered from
the medium surrounding the roots; the remainder was recovered
from the roots. The 3WHO ratio of the asparagine recovered
from the medium was the same as that of the added aspartate3,
the asparagine recovered from the roots had a lower 3H/MC ratio
than the added aspartate.
Nodules and roots of soybean plants and nodules of alder plants
have a system by which CO2 can be directly taken up and enter the
157
158
metabolism of the nodules and roots. This system is referred to
as dark CO fixation. The rate of dark CO2 fixation in soybean
2
nodules varied from 30 to 120 nmol-mg dry weight of nodules-L h"1
and in roots from 10 to 120 nmol-mg dry weight of roots'1 h“.
The rate of dark CO2 fixation in alder nodules was similar to
O
that in soybean nodules.
Dark CO2 fixation in soybean nodules appeared to be involved
primarily in the exchange of carbon rather than the net fixation
of carbon. About 70% of the recently-fixed [14C] from 14CO2 was
12
lost from the nodules after a l h chase with C02. If this label
was lost via the respiratory activities of the nodules, then net
fixation of carbon via dark CO2 fixation is minimal.
The products of dark CO2 fixation were primarily the organic
acids and amino acids clbsely associated with the tricarboxylic acid .
cycle: malate, citrate, aspartate, glutamate, asparagine and glutamine
and in alder nodules, citrulline. This data together with other
evidence suggests that one of the major enzymes catalyzing dark CO2
fixation is phosphoenolpyruvate carboxylase. Other enzymes such as
malic enzyme or carbamylphosphate synthetase may also be involved.
The relative distribution of label from 14
CO2 into the above
products depended in part on the source of nitrogen of the plant.
The major labeled amino acids in soybean nodules and roots dependent
on N2 were aspartate and glutamate. The relatively low percentage
of label in asparagine in these nodules and roots is consistent
with asparagine adcounting for only a small proportion of the organic
nitrogen transported from the root systems of plants dependent solely
on N2. In contrast, asparagine was the major labeled amino acid in
159
roots of plants treated with No; as were asparagine and glutamine
in roots of plants treated with NHZ. The increased synthesis of
these amino acids, as indicated by the increased incorporation of
14C02, in roots of plants treated with N03 or with NH:
label from
was also reflected by asparagine and glutamine accounting for an
increased percentage of the organic nitrogen in the xylem exudate
of these plants.
While the addition of N03 or NH: appeared to increase the
synthesis of amino acids in roots, it reduced the rates of dark CO2
fixation and CZH2 redUction andithe concentration of amino acids
in nodules. These results are consistent with the nodules of plants
treated with N03 or NH: having been deprived of photosynthate and
thus energy to maintain high rates of metabolic activities.
The effect of N03 and NH: on amino.acid metabolism was also
13
explored using N-labeled tracers. Differences were observed in
the labeling of glutamine and glutamate inseveral different tissues
exposed to 13N-tracers. In those tissues that were exposed to
13NH: - alder nodules, soybean nodules and soybean roots- the ratio
of [13N]glutamine to [13N]glutamate was higher than in the tissue
exposed to 13NOS, soybean roots, and [13N]N2, spybean nodules. These
results were explained by assuming that the rates of glutamine and
glutamate synthesis depended on the source of nitrogen.
The metabolism of glutamine and glutamate was also explored by .
monitoring the incorporation of label from 14
14
CO2 into these amino acids.
'After exposure to C02, the specific activity of glutamine was higher
than that of glutamate in roots of soybean plants treated with NO}
and in soybean nodules. These results suggest that there were
160
two pools of glutamate, only one of which was used in the synthesis
of glutamine.
In another experiment, the pathway of asparagine synthesis
was explored by incubating soybean roots with [2.3-3H]aspartate
and [4-14CJaspartate. Over 90% of the labeled asparagine was
recovered from.the medium surrounding the roots, the remainder was
in the roots.‘ The asparagine in the medium had the same 3H/MC ratio
as that of the added aspartate whereas the asparagine in the roots
had a lower ratio. These results suggest that there may be two
sites of asparagine synthesis in roots, one where aspartate is
directly converted to asparagine that is transported out of the
roots and a second where aspartate is converted to asparagine that
inretained within the roots.
I have shown that dark CO2 fixation occurs in the nodules and
roots of soybean plants and the nodules of alder plants. The labeling
14
'of amino acids via the incorporation of [14C] from CO2 has provided
a means by which amino acid metabolism could be explored. The use
13
of N-labeled tracers has permitted-the nitrogen group of amino acids
14
to be monitored. thereby complimenting the use of CD as a probe.
2
to 00 \l 01
e e e e
14.
15.
16.
17.
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