Anaerobic Metabolism in Soybean Root Nodules
Angela von Richter Guse
A thesis submitted to the Department of Biology
in confomity with the requirements for
the d e p e of Master of Science
Queen's University
Kingston, Ontario. Canada
August, 1997
Copyright O Angela von Richter Guse, 1997
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A bstract
Although soybean nodules have the enzymes for ethanol synthesis. and although their CO,
evolution and nitrogenase activity Vary in the degree to which they are O, lirnited, Little is
known about the relative importance of anaerobic metabolism in these organs. This study
examined the role of anaerobic metabolism in nodules by cornparing measured and
theoretical aerobic values for respiratory quotient (RQ= CO, production 1 O2uptake) and
by measunng rates of ethanol production in nodules that Vary in the degree to which
respiration and nitmgenase activity are Limited by 0, supply. In control nodules which
have only minor levels of O2limitation. the measured RQ (1.18 to 1.26) was much greater
than theoretical (1.06 to 1.08), whereas in severely O, lirnited nodules caused by nitrate
fertilization or detopping. the measured RQ (1.12 to 1.23 and 1-06to 1.14, respectively)
was in better agreement with theoreticai. Therefore, if anaerobic metabolism does occur in
nodules, it does not seems to be associated with 0, limitation. Treatment of nodules with
0% 0,for 10 min demonstrated that nodules had the ability to produce large amounts of
ethanol, however under more physiological rates of 0 supply, ethanol production by
nodules was not able account for their high RQ values. It was concluded that net
anaerobic metabolism plays a minor role in nodule metabolism. and that either the
subsuates or the products of nodule metabolism were different fiom those that were
assurned to exist within the theoretical calculations for RQ.
Acknowledgrnents
There are a number of people I would like to thank for their help in completing this degree.
Firstly. 1 would Wre to thank my supervisor Dr.Dave Layzell for the opponunity to do this
research, as weU for his encouragement, advice and financial support dong the way. This
is also forwarded to the rnembers of my supervisory cornmittee, Dr. D.Canvin and Dr. W.
Plaxton, for their advice and assistance. Furthemore, 1 would like to thank Nick Dowiing
for his technical expertise. Th&
also to the members of the Layzell lab namely, Jennifer
( ~ e n nWillms,
~)
Monika Kuzma, ~ w e e Neo,
'
Steve Hunt. Kirby Duffy and Jill Besse, for
advice and fnendship.
To al1 my basketball friends thanks for your interest in the progress of my work. 1
am indebted to my friends Beth and Donnie Wartman who gave me a home in Kingston
this past summer. Thanks for providing al1 those delicious meals and for your
encouragement.
Finally, 1 would like to express loving thanks to my mom and dad von Richter and
mom and dad Guse for their love and unconditional support through out this project. Many
thanks to Dr. J. Mercier who believed in me. Lastly, a special thanks to Paul, my husband
mentor and friend.
1 also acknowledge the fuiancial help of the School of Graduate Studies at Queen's
University.
.
2.3.1 CER.OER ANA and RQ in nodules .................................................. 33
2.3.2 Relationship between respiratory gas exchange and nitrogenase activity in nodules
from control. nitrate inhibited and detopped plants..........................................35
2.4 Discussion ...................................................................................... 39
2.4.1 Methods for RQ measurement: Past and present ..................................... 39
2.4.2 Theoretical RQ vs. Measured RQ....................................................... 41
2.4.3 What does RQ tell us about the presence of anaerobic rnerabolism in nodules?..-45
2.5 Conclusions..................................................................................... 45
Chapter 3. Ethanol and 0,.Limi ted Metabolism .............................46
3.1 Introduction ................................................................................... -46
3.2 Materials md Methods........................................................................- 4 8
3.2.1 Plant Culture.............................................................................-48
3.2.2 Tissue Harvest and Ethanol Analysis.................................................. 49
3.2.3 Measurement of Ethanol Loss to the Gas Phase ..................................... 51
3.2.4 Treaunents ................................................................................ 51
3.2.5 Ethanol Movement within a Whole Plant ............................................ - 5 2
3.3 Results .........................................................................................-53
3.3.1 Nodule Excision ........................................................................-53
3.3.2 10% 0,Treatment ....................................................................... 54
3.3.3 0% O?Treatment ........................................................................-56
3.3.4 Whole Plant Analysis................................................................... - 5 6
3.4 Discussion..................................................................................... -56
3.4.1 Ethanoi Production in Nodules under 0% 4.........................................57
3.4.2 Ethanol Production in Nodules in 2 1% and 10% 0, ............................... - 5 8
3.4.3 The Important of Ethanol Production in Nodule Metabolism ....................... 60
3.5 Conchsion ..................................................................................... 61
Chapter 4 . Conc1usions .......................................................6 2
4.1 Future Research ............................................................................... - 6 3
Literature Cited .................................................................6 5
Vita
.............................................................................-74
List of Figures
1.1 A summary of key pathways for C.N. and O metabolisrn in legume nodules ..........7
1.2 The effect of infected ceil concentration on TNA and C O evolution ................... 1 1
1.3 Possible e' utilization sites in legume nodules.............................................-20
2.1 A schematic diagram of the RQ analyser ................................................... 24
2.2 Ureide synthesis from sucrose and ammonia .............................................. 32
2.3 Gas exchange of excised soybean nodules ................................................. 34
2.4 Respiratory gas exchange. TNA and R Q of conuol excised soybean nodules......... 36
2.5 Respiratory gas exchange TNA and RQ of nitrate excised soybean nodules.......... 37
2.6 Respiratory gas exchange. R I A and RQ of detopped excised soybean nodules ......38
2.7 The predicted relationship between RQ and TNA for nodules at different levels of
anaerobic metabolism ......................................................................... 42
3.1 Method for ethanol measusernent in nodule tissue. ........................................ 50
3.2 Method used to measure ethanol volatilization from nodules ............................52
3.3 Ethanol accumulation and volatiiization rate from excised nodules...................... 54
3.4 Ethanol accumulation as a result of 10% 0 exposure to intact plants .................. 55
3.5 Ethanol accumulation as a result of 0% O exposure to intact plants .................... 57
3.6 Carbon flow through glycolysis. TCA cycle and anaerobic metabolisrn ............... 60
.
.
A b breviations Used
AAR
ALLA
ALLN
ANA
AS
ASN
ATP
CA
CER
DW
e-
EAC
E.T.C.
w
GLN
GLU
GOGAT
GS
IMP
RGA
Lb
MDH
MFC
nod
OAA
OD
OER
PBM
PA
PD
PEP
PHB
P.P.P.
PYR
RQ
S1
TCA
TNA
m
v1 &V2
ammonia assimilation rate;
allantoic acid
allantoin
apparent nitrogenase activity (H?evolution in air)
asparagine synthe tase
asparagine
adenosine triphosphate
CO?analyzer
C O exchange rate
dry weight
electron (2e-= electron pair)
electron allocation coefficient of niuogenase
electron transport chah
fresh weight
glutamine
-olutamate
glutamate synthase
olutamine synthetase
:nosine monophosphate
infra-red gas analyser (CO?)
leghemoglobin
malate dehydrogenase
m a s flow controls
nodule
oxaioacetic acid
differential O, analyzer
0 exchange iate
peribacteroid rnem brane
aunospheric pressure
differential pressure between the reference and anal@cal Cas streams
phosphoenolpyruvate
pol y-B- hydroxyb y acid
pentose phosphate pathway
pyruvate
respiratory quotient
solenoid valve used to switch between reference and sample gas strearns
Tricarboxylic Acid Cycle
totai nitrogenase activity (H,
evolution in &O2)
temperature of the block that houses the differential O sensor
needle valves used to calibrate the O, analyzer
Symbols Used in Equations
equals 0.5 and is the number of CO, evolved in the complete aerobic
catabolism of sucrose per 2e- produced
the proportion of O E RArmbrc
~ o , , ,which
,
does not occur due to anaerobic
respiration.
equals 0.75 and is the moles of O, uptake which are not balanced by CO,
production per NH,' assimilated h o ureides from sucrose
CO, concentration in Pa caiculated from CA'
voltage output of the C o andyzer
fractional C O concentration in the major calibration gis
fractional C O concentration in minor calibration gas
measured CO, concentration differentiai in Pa between reference and
sample gas stiearn
the flow rate of the sample gas sueam entenng the cuvette
concentration of H,..in Pa
the m a s of the sample
the Pa of N2consurned by niuogenase in the sample gas Stream
measured 0 concentration differential in Pa between the reference and
sample gas Stream
the Pa of O2exchange in the sample gas stream after dilution correction
O, concentration in the reference gas (OA sensor, units of kPa)
fractionai 0 concentration in the major calibration gas
fractional O2concentration in the minor calibration $as
Arrobic
''%ormicd
theoretical 0 exchange in aerobic nodules
differential O, concentration in Pa
atmospheric pressure (PA sensor, units of kPa)
pressure differential generated (PD sensor. units of Pa)
the gas constant (8.3 145 1 Pa m' K1mol")
Apparent RQ value associated with the calibration gas
Aem bic
R eïïteoretieal
theoretical RQ value for nodules having a detined nitrogenase activity and
fuiiy aerobic metabolism
Chapter 1. Introduction and Literature Review
1.1 GeneraI Introduction
Nitrogen is the major limiting numient for plant growth. especially in agnculiurai systems
(Atkins 1994). Acquisition and assimilation of nitrogen is second in importance only to
photosyndietic carbon assimilation for enhancing plant growth and development. and the
production of high-quality, protein-rich food is extremely dependent on availability of
suffcient nitrogen. The crucial role that nitrogen plays in supporting optimal plant
growth and development requires that we understand the physiological, biochemical. and
molecular events that regulate the rate of nitrogen assimilation.
Plants normaiiy acquire niuogen from the soi1 in inorganic form, as either nitrate
or ammonium. In the absence of an adequate supply of available soi1 nitrogen, most
legumes are capable of forming symbiotic associations with certain soi1 micro-organisms.
In these symbiosis, the plant provides the micro-organisms with a 'home' in the form of a
root nodule and a source of reduced carbon. In retum, die micro-organisms (bactena of
the genus Rhizobium or Bradvrhizobium in legumes) reduces (fuces) nitrogen gas to
arnmonia and provides this to the plant In this way the plant is able to obtain part or d l
of the nitrogen required for plant growîh from its symbiotic partner.
It is weil known that legume nodules possess the ability to regulate the O?
concentration within the bacteria-infected cells located in the central zone of the nodules.
Since the nitrogenase enzyme (the bacterial enzyme that fixes N2to NHr*)is extrernely
0, labile, the 0, concentration maintained in the infected cells is very low (4 to 70 nM
O?). equivalent to that of water in equilibrium with an atmosphere containing 2.5 xlo"%
O,- (air is 20.7%). This regulation is achieved, in part by the ability of legurne nodules to
regulate their permeability to 4 diffusion from the rhizosphere to the bacteria-infected
cells. This O, concentration is so low that it limits the rate of respiratory o2consumption
and nodule metabolism even under optimal conditions for nitrogen fixation (Layzell and
Hunt. 1990). Given the O,-limited conditions that are known to exist in legume nodules,
various workers have proposed that at l e m a portion of nodule metabolism may be
anaerobic (Tajima and LaRue 1982; Sugunama et ai., 1987; higoyen et al., 1990).
Respiratory quotients (RQ = CO7evolution + O uptake) have been reported to be
greater than 1 at a sub-ambient PO, for attached nodules of Alnus rrtbra (Winship and
Tjepkema, 1985) and for detached soybean nodules (Pankhurst and Sprent, 1975;
Bergersen, 1982). This increase in RQ was coupled with the stimulation of ethanol
production (Serraj et al., 1994).and has also been attributed to increased rates of
anaerobic CO2 production (Sprent and Gallacher, 1976; Witty et ai., 1983).
Since studies indicate a pathway for anaerobic metabolism (Sprent and Gallacher,
1976; Tajima and LaRue, 1982; Tajima et al., 1982: Smith, 1985) which have been
related to increases in respiratory quotients (Sprent and Gallacher, 1976; Witty et al.,
1983), this thesis investigated the change of RQ values under various levels of O2
limitation using a new means of measuring O?, CO, and H2 simultaneously. As well.
ethanol was measured to determine the potential for anaerobic metabolism in soybean
root nodules and to provide supporting evidence for increasing RQ values. However,
before presenting the expenmental results, the following literature review will consider
nodule O2 regulation, nitrogen fixation and other evidence which has been published
conceming anaerobic metabolisrn in legume nodules.
1.2 Nodulation
Soi1 bacteria, referred to as rhizobia belonging to the genera Rhizobium, and
Brudyrhizobium have the unique ability to induce nitrogen-fixing nodules on the roots or
stems of leguminous plants. The steps of nodule development and differentiation of
bacteria has been descnbed in several recent reviews (Schultze et al.. 1994; Mylona et al.,
1995; van Rhijn and Vanderleyden 1995). Interaction of rhizobia and legumes begins
with signal exchange and recognition of the symbiotic partners. The bacteria show
chemotaxis toward many substances exuded by the plant root, including amino acids and
carbohydrates, as well as flavonoids that serve as chemoattractants as well as nod gene
inducers (Peters and Verma. 1990). This initial signahg event confers specificiry on the
interaction, such that a given rhizobia species interacts with a limited range of plant
species. Foilowing this recognition, the rhizobia attach to the plant root hairs. The root
hair defoms, resulting in the formation of 'shepherd's crooks' which is followed by the
infection of root hairs by bacteria through invagination of the root ce11 wall whose
continued growth leads to the formation of infection threads (Brewin 1991). These tubes
peneuate the ce11 layers toward the nodule primordium by growing and branching and
meanwhile transporting the multiplying bacteria. Simultaneously, cortical cells are
mitotically activated, giving rise to the nodule pnmordium. Infection threads grow
toward the primordium. and the bacteria are then released into the cytoplasm of the host
cells, surrounded by a plant-derived peribacteroid membrane (PBM). The nodule
prirnordium thereupon develops into a matiire nodule; outgrowth of the emerging nodule
begins. The bacteria during this time, differentiate into their endosymbiotic form, which
is known as the bacteroid. Bacteroids. together with the surrounding PBMs. are called
symbiosomes. At this stage, bacteria synthesize nitrogenase, which catalyzes the
reduction of nitrogen. The product of niuogen fixation, arnmonia, is then exported to the
plant fraction where it is assimilated into oqanic nitrogenous compounds.
AU of the steps of nodule development involve the expression of nodule-specific
plant genes, the so-called nodulin genes (van Karnmen, 1984). The early nodulin genes
encode products that are expressed before the onset of nitropn fixation and are involved
in infection and nodule developrnent. The products of the late nodulin genes are involved
in the interaction with the endosymbiont and in the metabolic specialization of the nodule
(Nap and Bisseling, 1990).
In a mature soybean nodule the central tissue consists of a rnatrix of infected and
uninfected celis (e.g., Bergersen, 1982). to which O?is supplied via an interconnected net
work of $as-filled intercellular spaces (Witty et ai.. 1987). Access of O2from the extemal
atmosphere is considered to be resvicred by a layer of cells in the inner nodule cortex
(Witty and Minchin 1990). Consequently, the high respiratory demand of the infected
cells generates very low concentrations of O?within hem, thus protecùng nitrogenase in
the bacteroids from the deleterious effects of excess O2 (Robson and Postgate 1980).
Other mechanisms of 0 regulation will be discussed later in this chapter.
1.3 Nitrogenase Reaction and Ammonia Assimilation
Dunng biological N2 fixation, H' and N2 are simultaneously reduced by the bacterial
enzyme niuogenase, which is cornposed of two dissociating protein cornponents. The Fe
protein (dinitrogenase reductase), interacts with ATP and ~ g in" a hydrolytic reaction
which resu1t.s in a transfer of elecuons from an electron donor (ferredoxin or flavodoxin)
and passes them to the second cornponent, the MoFe protein, which transfers the
electrons to substrates. The reduction of the MoFe protein requires the hydrolysis of 2
ATP molecules per electron transferred (Dean and Jacobson 1992). The physiological
products of nitrogenase are Hz and NH,'. but other substrates besides N2 and H' c m be
reduced (including; acetylene, cyanide, azide, nitrous oxide, and hydrazine). Under
normal atmosphenc conditions, nitrogenase simultaneously reduces N2 to ammonium:
N fixation
7
N ? + ~ H +1+2 ~ ~ ~ + 6 e ' - - - - > 2 12ADP+
~,'+
12Pi
H,H , ~ r ~ d ~ ~ t i o n
2H
' + 4 ATP + 2 e- ---->
H, + 4ADP
+ 4Pi
Eqn. 1.la
Eqn. l.lb
full Reaction
N?
+ 1 0 ~ ' +16 ATP+
8e'---->2 N H , ' + H ~ + 16ADp+ 16 Pi
Eqn. l.lc
H, is normally produced during N2fixation , with at lest 1 HLproduced for every Nz
fixed (Simpson and Bums, 1984) as shown in Eqn. 1.lc. Therefore, in the presence of Nt
only a proportion of the electron tiux is used in the production of H2, thus measurements
of H, evolution represent only apparent nitrogenase activity (ANA, Hunt et al., 1987).
However, if nodules are exposed to an atmosphere lacking N, (ie. Ar:OZatmosphere), al1
electron flow through nitrogenase is used to reduce protons to Htas shown in Eqn. 1.2,
and the rate of HZevolution from the nodules represents a measure of total niuogenase
activity (TNA, Hunt et al., 1987).
16 ATP + 8e' + 8 ~ '---->4 HZ+ 16 ADP + 16Pi (H,production in Ar:O,)
Eqn. 1.2
A stoichiometry showing the production of 4H, has been chosen here so that the total
electron flow through the enzyme (8e' or 4 electron pairs) is similar to ihat in Eqn. 1.1~.
In many NZ fixing systems. Hzproduction and N, fixation can be variable, and the
terrn electron allocation coefficient (EAC = - (ANA + TNA)) is used to describe the
proportion of total electron fiow through nitrogenase which is ailocated to N, fixation
rather than H, production (Edie and Phillips, 1983). Thus. EAC is a measure of the
efficiency of N2 fixation (Moloney and Layzell, 1993). Since at least one H, is produced
per N2reduced. and & fixation consumes three electron pairs (i.e. 6e-)per N, reduced,
and Hzproduction requires only one electron pair, under optimal conditions the
maximum EAC is 0.75 (Layzell, 1990). However, it has been reported that under
ambient conditions the EAC is less that the theoretical maximum, usually between 0.60
to 0.72 (Moloney and Layzell, 1993).
1.32 Ammonium Assimilation
The ammonia generated by the bacteroids diffuses across the bacteroid and peribacteroid
membranes into the host plant cytosol where initially. it is incorporated into glutamine by
olutarnine synthetase
a
(GS) which is located in the cytosol of the infected cell. then
glutamate synthase (GOGAT)transfers amide N to an alpha-amino group. GOGAT is
located within the plastids of these celis. The assimilation of MI, requires the presence
of reductant (NADPH) in the plastids and ATP in the cytosol. This GS:GOGAT system
results in the net production of glutamate at a cost of one ATP and one NADPH per NH,
assimilated. The metabolism of this glutamate depends on the species of legume, for
present purposes the biosynthesis of only ureides and asparagine will be examined. The
metabolic pathway by which these compounds are formed is illustrated in Fig. 1.1 with
sucrose and ammonia as starting materials.
Ureides form the main N-compound transported from soybean nodules through
the xylem to other parts of the piant, nodules export up to 90% of the fixed nitrogen in
form of ureides. Their metabolism involves the transfer of an amino group from
glutamate to oxaloacetate to form aspartate and senne. the formation of glycine. and de
novo purine synthesis in plastids. Oxidation of purine nucleotides leads to the formation
of xanthine monophosphate and which is incorporated into xanthine in plastids located in
the cytosol of infected cells. The final steps including uric acid formation. oxidation of
uric acid to allantoin in peroxisomes and hydrolysis of allantoin to allantoic acid in the
smooth endoplasmic reticulum. are believed to occur in neighbounng uninfected cells of
the nodule (Meilor and Werner. 1990). Uricase catalyzes the oxidative cleavage of unc
acid to allantoin plus CO, (Eqn. 1.3).
Unc acid + 0, + HzO--s
allantoin + H,O, + CO,
Eqn. 1.3
Ureides are of particular significance in legumes as transporters of fixed nitrogen
as they have a sucrose:nitrogen ratio of 0.09 which is substantially lower ratio compared
to other export products of nitmgen futation (e.g. 0.24 for asparagine). Ureides therefore,
conserve carbon in the nodule and transfer the cost of N assimilation to the leaves where
energy may be more readily available (Pate and Layzell, 1990; Schubert and Boland,
1990).
Although ureides are the major export product in soybeans the concentration of
asparagine in nodules is several fold higher than the concentration of ureides, and
Fig. 1.1. A summary of key pathways of C, NTand O metabolism and their location in
the infected and uninfected ce11 of a legume nodule. Key reactions or processes are
depicted as Items 1. PEP carboxylase: 2, malate dehydrogenase (MDH); 3. glutamine
synthetase (GS);4, glutamate synthase (GOGAT); 5, asparagine synthetase (AS); 6,
xanthine oxidoreductase; 7, uncase; 8, aiiantoinase; 9, ATPase; 10, pyruvate kinase; 11,
bacteroid terminal oxidase; 12, mitochondriai terminal oxidase; 13, leghemoglobin
equilibrium reaction; 14, variable cortical diffusion banier. ALLA, allantoic acid;
ALLN, aliantoin; ASN. asparagine; ATP, adenosine triphosphate; e', reductant; E.T.C.,
electron transport chain; GLN, glutamine; GLU.glutamate; IMP, inosine
monophosphate; Lb, leghemoglobin; OAA. oxaloacetic acid; PEP. phosphoenolpyruvate;
PHB,poly-ll-hydroxybutync acid; P.P.P., pentose phosphate pathway; PYR,pyruvate;
TCA, Tricarboxylic Acid Cycle (Layzell and Atkins, 1997).
C, N & O METABOLISM IN NODULES.
SUCROSE
Q
PHB
E.T.C.
detectable amounts of asparagine are exported from the nodulated root systern especially
during early nodule development Asparagine may represent a N-compound exported by
soybean nodules as well as ureides. Its biosynthesis is catalyzed by aspartate
aminotransferase and asparagine synthetase from glutamine via glutamate and aspartate.
Aspartate fotmed in plastids of infected cells serves as a subsuate for asparagine
synthetase in a cytosol-localized reaction and can be regarded as an immediate precursor
of asparagine (Robinson et al., 1994). The end product of the reaction is one molecule of
asparagine and one of glutamic acid.
1.4 Carbon Metabolism in Legume Nodules
Photosynthate is transported to the nodule in the form of sucrose (Gordon et al.. 1985;
Streeter. 1987) and is stored in the plastids of the plant fraction of nodules as siarch
(Walsh et ai., 1987). Nodule starch has a slow turnover rate, and these reserves are
metabolized only when extemal. particularly shoot, reserves are depleted (Waish et al.,
1987). The sucrose provided to the nodules is not only a source of carbon for growth, but
provides carbon for the deposition of starch reserves in plastids of uninfected cells, for
poly-B-hydroxybutyrate (PHB) storage in bacteroids. and for the carbon skeletons
required in the assimilation of NH,' and the synthesis of ureides. In addition, sucrose is
the source of oxidizable substrates needed for plant and bacteroid respiration.
Ca-dicarboxylic acids, principally malate, are synthesized in the cytosol of the
infected ce11 and transported across the plant symbiosome membrane. via proton-ATPase
(reviewed by, Jording et ai., 1994), and the bacterial plasma membrane where they are
metabolized in a bacterial TCA cycle (Suganurna and LaRue, 1993). PEP carboxylase is
a key cytosolic enzyme in the infected cell. Together with malate dehydrogenase it
generates the malate for the bacteria. Bacteroids absorb C,-dicarboxylic acids via a
H+/dicarboxylateCO-transportsystem in the cytoplasmic membrane (Li et al.. 1990).
With both bacteroid and plant fraction conuibuting to the proton gradient across the
bacteroid cytoplasmic membrane treatrnenis affecting ATP genention in rnitochondria or
bacteroids (e.g. O2supply) may perturb these H+gradients and have rapid and dramatic
effects on nitrogenase activity (Bergersen, 1994; Sueeter, 1995).
Although Ca-dicarboxylicacids support bacteroid respiration and nitrogen
fixation. their support of the latter is not always simple or direct. For example, when "cmalate, or 'J~-succinateare supplied to suspensions of bacteroids prepared from soybean
nodules, in excess of immediate requirements. '"Cis stored as PHB,and nitrogen fixation
may be depressed to lower rares (Bergersen and Turner, 1990). If exogenous Csubstrates are then withdrawn, stored PHB is utilized, ' " ~is0evolved,
~
and enhanced
nitrogen fixation occurs (Bergersen and Tuner, 1990). Thus, delivery of substrates to the
bacteroids mus1 be suictly regulated, and possibly transport across the membranes
restricts this. Bergersen and Turner, (1989) also observed that this utilization of PHB did
not occur until= 80 minutes after exposure to C-limiting ueatments. That is. once
exposed to such a treatment 'C continued to accumulate in PHB until it accounted for =
90% of bacteroid "C.
Proline may also be an important carbon source for B. japonicum bacteroids based
on the stimulation of acetylene reduction activity following supply of proline to
nodulated roots (Zhu et al., 1992). However in different studies proline was considered
to be an unimportant carbon source as indicated by the failure of proline to be transported
into bacteroids (Udvardi et al., 1990). Even with these conflicting results proline has
been suggested to be an important carbon source for bacteroids in environmentally
stressed nodules (Kohl et al., 1994).
1.5 Oxygen Metabolism in Legume Nodules
The large ATP demands of nitrogenase are provided by bacteroid oxidative
phosphorylation, whereas mitochondriai oxidative phosphorylation generates most of the
ATP needed for NH, assimilation by the plant fraction, and for maintaining the proton
gradient across the symbiosome membrane as well as the needs for basic ce11 growth and
maintenance. To maintain the high rates of oxidative phosphorylation, the bacteria and
mitochondria have the capacity for high rates of O consumption. However, since the 0,
concentration in the infected celi must be stringentiy regulated to protect nitrogenase
from ineversible inhibition, it is maintained at a level(=25nM, equivalent to 104 of
atrnospheric) that limits the rate of oxidative phosphox-ylation. and therefore the
availability of ATP in support of cellular processes. Layzell and Hunt, (1990) and Witty
and Minchin, (1990) have shown that the low 0, concentration is maintained by a
variable physical barrier to the diffusion of gas from external pO, concentrations, since a
sharp decline in PO, (Tjepkema and Yocum. 1974) and a sharp increase in pHZ(Witty,
1991) have been rneasured in this region when electrodes were inserted in to nodules.
It has also been shown that O, limits and regulates nitrogenase activity and
respiration rate in legume nodules even under normal conditions (Fig. 1.2). this would
allow for some control over Oi in the low n~ concentration range. Physiological data
showing that srnall increases in extemal PO, stimulate respiration and nitrogenase activity
in legumes (Hunt and Layzeil 1993). and rneasurements of the nodule adenylate energy
charge (AEC = [ATP + 112 ADP] 1 [ATP + ADP + AMP]) provide evidence for O, and
ATP-limited metaboiism in control nodules. In aerobic tissues, AEC values are typicaiiy
0.80 or greater (Pradet and Raymond, 1983, 1984). whereas the central zone of legume
nodules have AEC values (0.60 to 0.72) (Oresnik and Layzell, 1994) typical of hypoxic
metabolism. Moreover. treaünents which cause a greater O2 limitation of nodule
metabolism and nitrogen ftxation generally reduce the AEC,whereas exposure to high
p 0 increases AEC (deLima et al., 1994). The site of 0 limitation of nodule rnetabolism
is not known,
An 0 Limitation within the bacteroid could easily account for the decline in
nitrogenase activity as the infected cell O2 concentration is reduced. However, the
K,(02) for the bacteroid terminal oxidase is very low (5-20 nM 02)
compared with that in
Fig. 1.2. The effect of infected ceiï O?concentration on (13)TNA (H, evolution in &/O,)
and (O) nodule respiration rate (CO2evolution) in single soybean nodules during a
gradual increase in external p 0 2 after the Ar-induced decline. Low Oi vaiues of < 16 nM
are indicative of inhibited nodules and Oi vaiues of 16 - 55 nM are indicative of control
plants (Adapted from Kuzma et ai., 1993).
--
P
;y<>%
o
:>y*.*
>,pz.:
g&
.*:/
g$$
~2
=mm mm a 0 mmom
o
-
o
-
000
-
O
MM Kinetics [Ks(O,)= 18 nM]
m
..
CO,Evolution
O
100
150
Infected CeIl [O,] (nM)
0.00 1 of the
ceiiuiar [O,] i l
equilibr&
the mitochondria (50-100 nM O?).mitochondrial respiration is therefore likely to be more
O?-limited than bacteroid respiration at the 0, concentration measured within infected
ceils (4-70 nM O z ) If this is the case. the lower nitropnase activity in more O?-Limited
nodules may be associated with the availability of ATP needed to maintain the proton
gradient across the symbiosome membrane that is required for malate transport to the
bacteroids. Perhaps then, an O,-limitation within the mitochondria may impose a
carbohydrate Limitation within the bacteroids.
1.5.1 Regulation of O, diffusion
Changes in the nodule environment. such as flooding or fluctuations in soi1 temperature.
c m alter the availability of 0 to the nodule or the rate of it's consumption in respiration.
thereby disrupting the balance between O2 concentration and flux. To compensate for
this. it is thought that the diffusion banier is able to vary it's resistance to O, entry (Witty
and Minchin, 1990; LayzelI and Hunt, 1993).
Studies with an O2 micro-electrode inserted into nodules have shown that the O2
concentration declines sharply in the region of the cortical diffusion banier (Tjepkema
and Yocum, 1974; Witty et ai., 1991). whereas measurement of oxygenation of
leghemoglobin (Lb) and in vivo spectroscopy has been used to predict free Oz
concentrations of about 5 to 60 nM in the infected cells (Layzrll et al.. 1990: Kuzma et
al., 1993).
Further evidence supporting the presence of a variable diffusion bamier in legume
nodules was provided by Hunt et al., (1987) and Weisz and Sinclair (1987a, b) who
showed that step changes in extemal p 0 rapidly inhibited nitrogenase activity and
respiration, but that after 30 minutes to 4 hours the initial rates of both processes were
recovered under the new PO,. During these ueaunents nodule permeability increased or
decreased in response to a decrease or an increase. respectively. in extemal PO, (Weisz
and Sinclair, 1987b). It was concluded that nodules can regulate their resistance to O,
diffusion to maintain constant rates of respiration and nitrogenase activity.
The diffusion barrier c m be considered in the context of Fick's tirst law of
diffusion. the following relationship may be derived to solve for the infected~cellO2
concentration (Oi)(Witty et al., 1986).
Oi = O, - (F x R)
Where F is O flux into die nodule (mole m-%l),
ce11 O, concenuarion (mole
Eqn. 1.4
Oeand Oi are the extemal and infected
respectively and R is resistance (s m-l). Since
nitrogenase is inhibited by even small levels of Oi. rhe right-hand side of Eqn. 1.4 must
remain zero. Therefore, a change in O, needs to be matched by changes in F and/or R.
So that F can respond to increases in O,.nodules must be o-limited under normal
conditions and thereby "buffer" changes in Oi. In effect, the O?-limitation of nodule
metabolism provides the nodule with a form of "feedback conuol" over Oi (Layzell and
Hunt 1990). Thus, legume nodules are able to regulate their resistance in response to
short-term. (minutes to days) changes in environmentai and physiological conditions such
as those described below. This time scale would not permit changes in the ce11 structure
of the nodule cortex similar to those occumng with longer O2 treatments (Hunt et ai.,
1989; Diaz del Castillo et al., 1992). In addition, Dakora and Atkins (1990, 1991)
showed that nodules grown at high PO, had smaller intercellular spaces in the cenual
zone than nodules grown at low p 0 2 , a result that shows that not ail the conuol of O2
diffusion is Iocalized in the nodule cortex.
Control mechanisms that have been proposed inclode changes in intra- and
extracellular solute or ion concentrations with consequent reversible flooding of gas
spaces (Hunt et al.. 1988. 1990; Streeter, 1992; Purceil and Sinclair, 1993). changes in the
shape or size of cells around the Sas spaces (Iannetta et al., 1993). or occlusion of the
spaces with an extracellular glycoprotein (James et al.. 1991; Iannetta et al., 1995). Al1
these mechanisms are based on the fact that solubility and diffusion of O2in an aqueous
liquid phase are much lower than in a gas phase.
Recent studies have provided evidence that the infected cells themselves rnay
have the ability to exercise some level of control over their own intercellular O?
concentration. For exarnple, Thumfort et ai., ( 1994) have shown that under certain
conditions a zone may occur in the infected ce11 adjacent to the air space in which
leghemoglobin (Lb) is saturated with Oz. Since LbO, cm only diffuse down a
concentration gradient, a zone of saturation would result in the collapse of Lb-facilitated
diffusion close to the air spaces. This collapse provides an intrinsic mechanism for
maintaining a low average Ozconcentration in the infected ce11 despite substantial
variation in the Q concentration in the space. In addition. it has been suggested that the
mitochondria are clustered around the gas spaces, providing a form of respiratory
protection by consuming much of the 0, before it cm diffuse to the symbiosomes within
the center of the infected cells (Millar et al., 1995). Both mechanisms would serve to
stabilize Oi in situations when there may be relatively large variations in the cenual zone
0, concentration. However, these infected ce11 mechanisms require increases in the rate
of O2 consumption to deal with higher space O2concentrations, and many of the shortterm, physiologically-regulated mechanisms (stem girdling, etc.) cause decreases in
respiration when nodule resistance increases. Hence, these mechanisms cannot account
for al1 short term regulatory responses, and there is still a need for direct control over
nodule resistance such as that which could be provided by changes in the spaces of the
nodule cortex.
1.6 Nitrogenase Activity under Changing Environmental Conditions
In the natural environment, factors affecting O2 supply to the nodules are constantly
changing, thus nodule O2consumption and nitrogenase activity must Vary to maintain
optimal OZsupply and O concentration. There are many examples of treatments that are
known to cause a change in O? concentration and nodule resistance (Hunt and Layzell,
1993):
In many symbioses, nodule exposure to O, concentrations of 25 kPa or more
(ambient air is Ca. 20.7 Wa), causes a rapid increase in Oi, and an inhibition of
nodule respiration an nitrogenase activity. However, within 5 to 10 minutes Oi
returns to iis 'normal' range, and over the subsequent 30 minutes nitrogenase activity
and respiration recover even though the external O2remains at the high level. This is
because R increases to compensate for the greater O2 differential (King e t al., 1988;
Kuzma et ai., 1993).
Exposing nodules to decreased O, concentrations to 10% causes a decrease in
nitrogenase activity within 4 minutes. This inhibition is followed by a steady
increase in niuogenase activity to 70% of initial within 45 minutes. ATP pool sizes
follow the sarne trend as nitrogenase recovering to initial by 45 minutes (de Lima et
ai,, 1994).
Nodule excision causes a rapid decline in H2production within 5 minutes, between 6
and 15 minutes there is a partial recovery followed by a second decline to a low
steady state at about 40 minutes after detachment (Sung et ai., 1991). Ralston and
Imsande (1982) found that nitrogenase activity in these inhibited nodules could be
partially recovered by increasing 0,.
Changes in nodule permeability on Oi are dso associated with cuning off phloem Sap
supply to nodules (e.g. by stem girdling or shoot removal), causing a reduction in
nodule respiration (Le. F in Eqn 1.4) and nitrogenase activity. Following such
ueatments, legume nodules decrease their permeability to Oz, causing a decrease in
Oi and a reduction in nodule metabolism through a greater O, limitation of
nitrogenase-linked respiration (Hartwig et ai., 1987; Walsh et al., 1987; Vessey et al.,
1988b). hcreases in 0, can recover up to 70% of the initial nodule activity.
providing evidence that the increased resistance had down-regulated metabolism by
making it more O,-limited.
Niirate inhibition of N2 fixation involves a drcreased O2 supply for respiration in the
nodule central zone (Vessey and Waterer. 1992; Hunt and Layzell. 1993). This
evidence includes a partial dleviation of nitrate inhibition by elevated O2(Vessey et
al., 1988).
1.7 Anaerobic Metabolism in Plants
Hypoxic metabolism is characterized by the concurrent activity of both limited
respiration (aerobic metabolism) and some degree of fermentation (anaerobic
metabolism). Under hypoxic conditions. the metabolism of an organ may be
heterogeneous since the outer layers receive more oxygen, and are therefore less hypoxic.
than the core of the tissue. Plant mitochondna cm survive anaerobic conditions for
several days, however, in the absence of molecular oxygen as a terminal electron
acceptor there c m be no mitochondrial respiration. Thus. anaerobiosis induces a shift
from normal respiration to the fermentative production of ethanol and lactic acid.
Although glycolysis can function well without 02.funher oxidation of pyruvate
and NADH by mitochondria requires oxygen. Therefore. when O? is limiting, NADH
and pymvate begin to accumulate. Under this condition plants carry out fermentation
(anaerobic metabolism), forming either ethanol or lactic acid (usually ethanol), as shown
in Eqn. 1.5a and 1Sb.
-pyruvate
-
alcohol
dthydrogenase
pyruvic a c i pyruvau
d ' l b acetaldehyde - ~ - y ethanol
w
Eqn 1.5a
decarboxylase
ÀTP
ADP
NADH
AD+
pynivate
Ny=riactic
pymvic acid
lactic acid
PEP,?~
a d
Eqn 1.5b
dehydrogenase
ADP
ATP
The reâction for ethanol production consisü of a decarboxylation of pyruvic acid to form
16
acetaldehyde. then rapid reduction of acelaldehyde by NADH to brm ethanol. These
reactions are catal yzed by pymvic acid decarboxylase and alcoho1 de hydrogenase. Some
cells contain Iactic acid dehydrogenase, which uses NADH to reduce pymvic acid to
lactic acid. Ethano1 or lactic acid, or both, are fermentation products, depending on the
activities of each of the enzymes present. In each case NADH is the reductant, but only
under anaerobic conditions is NADH abundant enough to cause reduction. Furthemore,
in some plants NADH is used to enable accumulation of other compounds when 4 is
limihg, especially malic acid and glycerol (Crawford, 1982; Davies. 1980a).
It c m be predicted that the supply of ATP is Iimited because the elecuontransport system and Krebs cycle cannot function without oxygen. Furthemore, the
products of fermentation, especiaiiy ethanol (in most species), lactic acid, malic acid, and
rarely glycerol, accumulate to some extent. However, fermentation products usually do
not become toxic because they leak out and diffuse away from roots.
Louis Pasteur discovered a surpnsing effect of hypoxic and anoxic conditions in
his studies of wine production by yeast over a century ago. When the O?concentration
around yeast and most plant cells is decreased gradually below the atmosphexic 20.9%,
CO?production from respiration decreases until a minimum is reached. but lower O,
concentrations cause a rapid nse in CO2 production. Under anaerobic conditions cells
grew slower but used more sugar and produced more CO, and ethanol. The Pasteur
effect is believed to occur so that the ce11 can meet its requirements for ATP despite the
rnuch lower efficiency of ATP production by fermentation. The increase in glycolytic
flux during hypoxia is accompanied by the accumulation of a number of glycolytic end
products including ethanol, lactate, alanine, and various organic acids and amino acids
(Roberts et al., 1992)
1.7.1 Ethanol in Nodules
Sprent and Gallacher, ( 1975) reported the production of ethanol from waterlogged
soybean nodules. As well Smith and ap Rees (1979) and ap Rees (1980) show that
nodules have a high capacity for ethanolic fermentation and a radier Iower capacity for
lactic fermentation. Uninhibited nodules accumulate 95 to 180 nmol ethanol ~ ' D w ,
which indicates that ethanol and acetaldehyde are normal metabolites of nodules, and not
formed solely in response to prolonged stress (Tajirna and LaRue, 1982).
Tracer experiments show that ethanol and acetaldehyde arise from pymvate. The
enzymes for ethanol formation were localized in the nodule cytosol, showing that ethanol
formation is a function of the plant fraction. Pymvate decarboxylase and alcohol
dehydrogenase are present throughout the development of the nodule (Tajima and LaRue,
1982). These enzymes are stimulated by increased stress conditions in intact nodulated
roots (Irigoyen et al., 1992).
1.7.2 increased CO, evolution during O,-lirnited conditions
Pankhurst and Sprent, (1975) reported enhanced CO, and ethanol production in detached
soybean nodules and the effects of water stress, this was also seen later by Sprent and
Gallacher, (1976) in drought stessed soybean. Respiratory quotients (RQ) above 1.O at
arnbient predict anaerobic metabolism (Serrajet al., 1994), since they suggest that the O2
limitation results in less complete oxidative pathways being followed, so that CO,
evolved is in excess of 0 taken up, and thus reflect anaerobic fermentation by nodules.
Values of nodule RQ 2 1 have been measured a sub-ambient p 0 for nodules attached to
the roots of Alnus rubra (Winship and Tjepkema, 1985), this was also the case for
detached nodules exposed to a range of 20 to 80% oxygen (Pankhurst and Sprent, 1975).
Possible sites for electron sinks are shown in Fig 1.3. Theoretically, when sugar
provided to the nodule in the phloem is fully oxidized to C O and -0,there would be an
equal number of CO2produced and 4 consumed and the R Q would be 1.0. The RQ in
symbiotic legume nodules is higher as elecuons are also used in the reduc tion of N, to
MI,'. However, under net anaerobic metabolism, the RQ would be expected to nse
above that which would be expected in aerobic nodules fixing a given amount of N, gas.
As the electrons would be used not only for purposes of
H1production, N, fixation and
nitrate reduction, but they would dso used in the synthesis of reduced organic molecules
(e.g. ethanol, acetddehyde) produced from fennentative pathways. Thus increases in RQ
may indicate net ethanol accumulation within soybean nodules exposed to minor and
adverse O2limitations.
In the present thesis two methods were used to quantify anaerobic metabolism.
The Fust rnethod involved using a novel. highly sensitive differential O2 sensor to
measure values for RQ, where RQ values greater than predicted would indicate anaerobic
metabolism. WhiIe the second method measured the rates of ethanol accumulation and
volatilization in soybean nodules exposed to conditions that would result in greater or
lesser degrees of O,-limited meiabolism.
Fig. 1.3. Possible sites where electrons are used (sinks)within a nitrogen fixing le,Oume
nodule (- - -). An RQ of I would occur if the CO, evolution equaled the 0, uptake is the
ratio. In nodules there are other potential processes which will change the RQ, narnely
nitrogenase activity (TNA, ANA. NIfixation), arnmonia assimilation (GOGAT), PHE!
biosynthesis (reductive biosynthesis) and fermentation. GLU, glutamate; C,H,,O,,
hexose.
Sucrose
Ethanol
Acetaldehyde
etc.
2 NH,
2 GLU
Chapter 2. Respiratory Quotients and Fermentative Metabolism in O,Limited Soybean Nodules
2.1 Introduction
Soybean root nodules fix N, gas to ammonia via the bacterial enzyme, nitrogenase.
Although nitrogenase requires large amounts of ATP synthesis (> 16 ATP per N, fixed)
provided by bacterial oxidative phosphorylation, the enzyme is irreversibly darnaged by
0 (Robson and Postgate 1980). Therefore. the 0, concentration in the infected cells (5-
60 nM Oz, Kuzma et al., 1993) is maintained at a level equivalent to about 104 of the O?
concentration in cells that are in equilibrium with air (ca. 260 ph4 O?, Hunt and Layzeii,
1991). To do this, legume nodules regulate their pemeability to O? diffusion from the
rhizosphere to the bacteria infected cells so as to limit the high rate of respiratory O?
consumption that occurs within the rnitochondria or bacteroids (Hunt et al., 1990; Layzeii
et al., 1990; Witty and Minchin, 1990; Hunt and Layzell, 1991).
A variety of evidence supports the conclusion that the metabolism of legume
nodules is Oz-limited. For example, increasing the PO,around N, fixing nodules
typically stimulates nodule C O evolution and nitrogenase activity. and the magnitude of
the stimulation increases in plants exposed to treatments that inhibit nitrogenase activity
such as nitrate fertilization (Minchin et al., 1986; Vessey et al., 1988a, 1988b). stem
girdling (Vessey et al.. 1988b, Walsh et ai., 1987). defoliation (Hartwig et al., 1987),
GH, or Ar:O, exposure (Witty et al., 1984; Hunt et al., 1987; Rosendahl and Jakobsen,
1988; King and Layzeli, 1991). or nodule excision (Ralston and Imsande, 1982; Hunt et
al.. 1989; Sung et ai., 1991). These same treatments have been shown to reduce the
infected ce11 O, concentration from levels of about 20 to 60 nM 0 in untreated plants, to
3 to 15 nM O2in those that are inhibited. These low 4 concentrations would be
expected to lirnit respiratory 0 consumption since the Km(02)for the terminal oxidase
of nodule mitochondna is only 50 to 100 nM O2 ( Rawsthorne and LaRue. 1986; Day et
al.. 1988; Millar et al., 1995) while that of bacteroids is about 20 nM 0 (Bergersen and
Turner, 1990, MUar et al., 1994, 1995)
The adenylate energy charge (AEC = (ATP + 0.5 ADP)c(ATP + ADP + AMP))
of actively fixing nodules is indicative of hypoxic metabolism (AEC 10.75; Oresnik and
Layzell, 1994), rather than fully aerobic metaboIism (AEC 2 0.80; Pradet and Raymond,
1983. 1984) and in inhibited nodules the. AEC declines by an additional 0.1 uni&
(DeLima et al., 1994).
Finally, anaerobic end-products such as acetaldehyde and ethanol have been
measured in soybean nodules (Sprent and Callacher, 1976; Tajima and LaRue. 1982).
and the enzymes involved in their synthesis (pymvic decarboxylase and alcohol
dehydrogenase) have been found in the plant cytosol (Tajima et al.. 1982; Smith, 1985;
Thym and Werner, 1996).
Clearly, there is strong evidence that nodule metaboiism is 0 limited (hypoxic),
and there is also evidence that this hypoxic metabolism is critical to the maintenance of
the biochemicd pathways supporting N2 fixation (Kuzma et al., 1993) and to the control
of the 0 concentration in the bacteria-infected cells (Denison, 1992; Bergersen, 1994;
Thumfon et al., 1995; Bergersen, 1996). However, various workers have proposed that
at least a portion of nodule metabolism may be anaerobic (Peterson and LaRue, 1981;
Tajima and LaRue, 1982; Smith, 1985; Irigoyen et al., 1992, Serraj et al., 1994),
especially when the plants are stressed (plant disturbance, stem girdling, nitrate
fertilization or nodule excision) and the nodule metabolism is more severely 0,limited.
To test whether soybean root nodules exhibit anaerobic metabolism, the present
study used a novel differential
gas analyzer (Layzell et al.. 1996; Willms et al., 1997)
in combination with a CO, and H, anaiyzer to obtain accurate, simultaneous
measurements of the respiratory quotient (RQ = CO, evolution + O?uptake) and
nitrogenase activity of nodules. These ernpirical values were compared with RQ values
that would be expected from a theoretical analysis of CO, and H2 exchange in nodules
having various proportions of aerobic and anaerobic metabolism. It was hypothesized
that the measured RQ should be higher than the theoretical RQ for aerobic nodules,
especially in treatments which result in a more severe 4 limitation.
2.2 Methods
2.2.1 Plant Culture
Seeds of soybean (Glycine mar L. Men cv Maple Arrow) were inoculated at planting
with Brudyrhizobiumjnponicum USDA 16, a strain lacking uptake hydrogenase activity
(Layzell et al.. 1984). AU pots (ca. 9 cm diameter X 15 cm high) were provided with
small Stones to a depth of five centimeters before being filled to the top with silica sand
(Grade 16) (Hunt et al., 1987). Plants were grown in a growth chamber (Mode1 PGV36,
Conviron Environments Ltd., Winnipeg. MB) at a constant temperature of 25°C and 80%
relative humidity. with a photon flux density of 800 pmol m-2 s-1 photosynthetically
active radiation (PAR) and a 16 hour photo penod. The plants were irrigated twice daily
with a rnodified Hoagland's nutrient solution (Walsh et al.. 1987) containing 0.5 m M
KNO, until one week af'ter germination and then with the same solution lacking nitrate
for the remainder of the growth period. Plants were used in experiments at 27-35 days
after inoculation.
2.2.2 Experimental Treatments
Nodule Excision - Nodules (ca. 0.16 gDW) were removed from a 28 day plant and
placed in a 3-ml syringe b m l which acted as a gas exchange cuvette. Within 1 to 2
minutes of detachment. the cuvette was incorporated into a gas exchange system capable
of measuring CO,, O2and $ exchange simultaneously in an open flow sysiem as shown
in Fig. 2.1 and descnbed in more detail below. Gas exchange was monitored
Fig. 2.1. A schematic diagram of the open-flow Respiratory Quotient (RQ) Anaiyzer,
which aiiowed for continuous. sequential gas exchange measurements. This system
contained a HZanalyzer, a differential O analyzer (OD) and a C O anaiyzer (CA). Two
mass flow controllers (MFC) were used to prepare Cases (N2:OJ for the sample.
reference and the major calibration gas streams. The minor calibration $as Stream
originated from a tank of compressed air containing 19.8% CO, and lacking in O,. The
differential 0 analyzer contained a number of environmental sensors, which monitored
changes in temperature (TO), atmospheric pressure (PA), and differential pressure (PD)
between the reference and analytical gas streams. Humidity was avoided by drying all of
the gas streams thoroughly using magnesium perchlorate before analysis. Using the
needle valves (V1 and V2) the differential O2analyzer could be pressure calibrated. A
solenoid valve (S 1) aiiowed the user to switch between reference and sample gas sueams.
Cuvette
I
I
Magnesiurn Perchlorate
Drying Columns
1
Mixing
Volume
!
1
1
Differential 0,Analvzer
1
continuously for 40 minutes as the nitrogenase activity declined. Nodule frrsh weights
were obtained at the completion of each expenment and then the nodules were dried at
85'C for 72 h before obtaining dry weiphts.
Nitrate Inhibition and Nodule Excision - At 28 days after inoculation, plants were
irrigated with the growth nutrient solution supplemented with either 10 m M KNO,
(nitrate-treated plants) or 5 m M K2S04(conuol plants). The nutrient solution was
provided twice daily for 2 days. and 48 hours after €ist treatment. approximately 10
nodules were picked from the root systems and placed into a sealed cuvette connected to
the gas exchange system. Four replicated measurements were made for each of the
nitrate treated and control plants.
Detopped and Nodule Excision - Soybean plants (28 days old) were detopped by
excising the shoot just above the point of cotyledon attachment, thereby stopping phloem
Sap supply to nodules. After 3 h of the detopping ueatment. the nodulated roois were
removed from the pots and approximately 10 nodules were picked and placed
immediately into a Cas exchange cuvette within the gas analysis treatment CO2,0, and
Hzexchange were monitored for 40 min.
At the end of al1 experiments, al1 nodule and
root materials were washed free of silica sand, dried separately at 85'C for 72 h and
weighed.
2.2.3 Measurements of CO,0, and H,Exchange
A gas exchange system was set up as shown in Fip. 2.1 in which N,:O gas composition
was provided by mass flow controllers (MFC)(mode1 FMA- 100 series, Omega
Engineering, Stamford, CT)to three gas strearns: a reference gas, a sample gas and the
major calibration gas. Each of the gas strearns were dried thoroughly by passing through
magnesium perchlorate columns before entering the Respiratory Quotient (RQ) Analyzer.
The reference gas Stream had a flow rate of ca. 50 to 60 mL min-' while the sample gas
was provided to the cuvette at a flow rate of ca. 30.0 mL min-'. The final calibration gas
was a mixture of two gas strearns: a major gas (ca. 2 L min'') havin; a composition
identicai to the reference gas, and a minor calibration (variable tlow of O to 5 mL min-')
with a composition of 19.8% C O in N2 which was provided by tank air lacking O?.
Either the sample or the calibration gas Stream, user selectable. was provided to a H,
- gas
sensor (Layzell et al., 1984) which then connects to the sample bypass port of the
Respiratory Quotient (RQ) analyzer (Layzell et al., 1996; Willms et al., 1997) while the
reference strearn was connected direcdy to the reference bypass port of the R Q analyzer.
The RQ Analyzer was designed and built to measure srnaIl differences (typically
10 to 500 ppm) in both O and CO,concentration between a reference and sample gas
stream even when the background O, concentration is air (ca. 209.500 ppm). Key to the
operation of this analyzer is a new design for a differential 0,sensor, and the
incorporation within the instrument of other environmental sensors that are used to
calibrate the instrument, and to minimize drift due to variations in environmental
conditions.
Although the detailed operation of this instrument has been described previously
(Layzell et al., 1996; Willms et al., 1997). it will be summarized here. A pump within the
R Q Analyzer pulled gas (eu. 20 mL min-') simulüineously through two gas sueams: one
that flushes the reference side of a differential O2sensor (OD), and a second gas stream
that flushes the analytical side of the same sensor, as well as the analytical ce11 in an
infra-red COz analyzer (mode1 225, Mark III, ADC.,Hoddesdon, U.K.).A three-way
solenoid vaive (S 1) was included upstrearn of the analytical side of the OD sensor so that
the gas provided to that stream could be selected from either the reference or the sample
gas streams provided to the RQ Analyzer. Needle valves (VIand V2) were located in
each gas strearn so that the differential pressure could be precisely controlleZ across the
OD sensor. The analyzer also contained sensors for the temperature of the OD block
(TO), atmosphenc pressure (PA) and a sensor (PD) that monitored the differential
pressure between the sample and reference side of the OD sensor.
Calibration of the OD and CA sensors was cariied out by two caiibration systems
as described previously (Willms et ai.. 1997). The OD sensor was caiibrated by (a)
providing both sides of the sensor with the same reference gas, (b) adjusting needle valve
V2 to generate lower or higher gas pressures at the analytical side of the OD sensor, and
(c) at each differentiai pressure setting, record the pressure differential generated (PD
sensor, units of Pa), the atrnospheric pressure (PA sensor. units of kPa), the O2 pressure in
the reference Cas (OA sensor, units of kPa) and the voltage generated by the OD sensor.
The differential O2 concentration in Pa (OD")associated with each volta,oe was
calculated as:
Eqn. 2.1
~ PA* are the relevant sensor value in units of Pa, kPa and kPa.
where PD", O A and
respectively. The relationship between O D and
~ OD sensor voltage was innately linear
across the entire range of the sensor ( p a t e r than -2000 to +2000 Pa O? differentiai)
(Willms et al., 1997).
The RQ was cdibrated by simuitaneously exposing both C O analyzer and the
OD sensor to a final calibration gas in which there was a fixed relationship between the
Pa of CO, e ~ c h m e nand
t the Pa of O, depletion Fig. 2.1. When the major and minor
calibration gases (Fig. 2.1) are mixed, the resultant gas Stream (i.e. finai calibrafion gas)
is e ~ c h e in
d CO2and depleted in 0 by a constant ratio ( R Q ~ ;), regardles of the
relative flow rates of the initial two gas streams. That ratio cari be calculated as:
Eqn. 2.2
where O A ~and
,
a
:
,
,are the fractional O2and CO, concentrations, respectively, in
concentrations. respectively, in the major calibration gas. Therefore, if the major
calibration gas is CO- free air at 20.95% 0,. and the minor calibration _osis 19.84 COZ
in Nt, the final calibration gas will be equivalent to the effluent gas from a biological
system having an R Q of~ 0.945 (Willms et al.. 1997).
Therefore. to calibrate the CA analyzer, the minor calibration gas was bled into
the major calibration gas stream at various flow rates and measurements were made of
the differential O2concentration in Pa (oD")and the voltage output of the CA anaiyzer
(CA'). The C O concentration in Pa CA^) associated with each CA' value was
caiculated from the OD":
Eqn. 2.3
2.2.4 Caiculation of Gas Exchange Rates
The H, Cas sensors provided a measure of the concentration of H, in Pa (d[H,]")
produced by the nodules in the sample air stream, and this value was used to calculate the
apparent niuogenase acùvity (ANA, units of pmoles HIg l DWad hfl)using the standard
equation:
.
,=[
where FR,
1
FR,, x 6 O x ~ [ H , ] "
R x 273.15
+
Eqn. 2.4
is the flow rate of the sarnple gas stream entenng the cuvette (mL min'' at
S V ) , R is the gas constant (8.3 1451 Pa m3 K" mol-'), 273.15 is the temperature in 'K at
STP,M is the mass of the sarnple. and 60 is a conversion factor to change minutes to
hours. Parallel studies (data not shown) reveaied that the nodules had an electron
allocation coefficient (EAC) of 0.65, a value consistent with previous studies with this
symbiosis (Moloney et al.. 1994; Moloney and Layzell, 1993). Therefore, total
nitrogenase activity (TNA, units of pmoles 2e' g-'DWnd hi') W
~ calculated
S
from
ANA,
and assuming this EAC value using the following equation:
ANA
]
TIVA=[I - E A C
Eqn. 2.5
The CO, exchange rate (CER, units of pmolzs CO1 $DW,
hr") was determined
from the difference in the Pa of CO2(A[co~]")
between the inflow and effluent gas
streams of the cuvette using an calculation identical to that in Eqn. 2.4, except A[CO,]"
replaced A[H~]'~.However, a similar approach could not be used to determine the O2
exchange rate (OER, units of pmoles O2g-'DW,, hi1)from the measured O2
concentration differential (A[o,]") between the inflow and effluent gas streams of the
curette. This was because the high background O, concentration in air (ca. 20,994 Pa)
would be affected by any imbalance in the gases that are produced and consumed by the
plant material in the cuvette. For example, if C O production is greater than O2
consumption or if Hzproduction is greater than Nz fixation, more molecules of gas will
leave the cuvette than will enter it (Le. the flow rate out of the cuvette will be slightly
higher than that entering) and therefore the O2concentration in the effluent gas will be
diluted. This will appear as a greater O2 differential than that which occuned solely as a
result of the respiratory Q consumption of the tissue. The following equation was used
to provide a value for the O2concentration differential that was associated only with the
biological processes of O? exchange (A[o,]"
- COV.) (Willms et al. 1997):
(A[o]"
~[q]:,.
= "[oJPa
+
+A[O~]"+A[H,]~~+A[N,]~~)
X
Eqn. 2.6
, OA*~
PA f f A
where OA- and PA^ are Ihe OZconcentration in kPa and the atmospheric pressure in
is the Pa of N, consumed by nitrogenase. This was
P a , respectively, and A[N, lR
calculated from the measured A[H,
1" values as:
Eqn. 2.7
where the EAC was assumed to be 0.65, and the '3' is the number of electron pairs (H,
equivalents) of the TNA required to fix each N,.
Finally. the OER (units of pmoles 0: g" DW h-') was calculated using a
modification of Eqn. 2.4. where ( A[O~];_,) replaced A[HJ~'.
2.2.5 Calculation of the Theoretical Respiratory Quotient
To calculate a theoretical RQ (RQ,)
value for a nodule, measured values were
accepted for CER and nitropnase activity and fram these, a theoretical rate was
calculated for 0, exchange. These calculations were carried out using the following
assumptions:
1. Sucrose or starch were assumed to be the sole C source for metabolism, and in a
nodules having no anaerobic metabolism, CO?, water. starch. cellulose and ureides
(allantoin and allanroic acid) were the only end-products of metabolism (Layzell and
LaRue, 1981). Therefore, CO2 production would equal O, consumption in al1 metabolic
processes within the nodule except for those which generate the reducing power to meet
the electron demands for nitrogenase (TNA, units of pmoles 2e- g-1 DW h-l) or the
enegy, carbon and 0 demands for ammonia assimilation into ureides (see item 3,
below). Hence the theoretical O, exchange ( O E ~aerobic
Q m I i c dthese
) i n aerobic nodules :
O E R ~ =: -[CER
~ ~ ~ (A
~ x TNA)- ( B x AAR)]
Eqn. 2.8
where 'A' = 0.5 and is the number of C O evolved per 2e- produced in the complete
oxidation of glucose to CO, and water, and 'B' is the moles of 0 consumption which are
not balanced by CO, production per NH,' assimilated into ureides (see item 3, below).
2. The rate of arnmonia assimilation (AAR (units of kmoles NH,' g " ~ ~ h - ' )was
),
calculated from the TNA. assuming an EAC of 0.65:
Eqn. 2.9
where the value '3' is the nurnber of 2e- required to reduce N, to MI,' and the value '2'
represents the 2e- consurned by glutamate synthase per N2 (Le. two NH,') fixed.
3. To caiculate coefficient 'B'in Eqn. 2.8. ureides were assumed to be the only product
of Nz fixation. and were produced from sucrose and NH,' through purine biosynthesis
and breakdown as described previously (Layzell et al., 1988a. Pate and Layzell, 1990).
An analysis of the biochemical pathways revealed that to assimilate 1 NH,+into ureide,
0.094 sucrose and 0.375 0 would be consumed while 0.125 C O would be evolved (Fig.
2.2). To achieve this, one pair of 2e- were produced whereas there was a dernand for 1.94
ATP. The O2 and CO, exchange associated with reducing this energy balance to zero
were calculated by assuming (a) reductant use via a respiratory electron transport chah
(P:O = 3) to meet the demands for ureide synthesis and (b) any excess reductant would
decrease the need for sucrose metabolism through glycolysis and TCA cycle. Therefore,
the incorporation of each mole of NH,+ into a ureide molecule would consume 0.080
moles sucrose, and result in the uptake of 0.034 mole C O and of 0.7 16 mole O,. The O,
uptake which is not baianced by C o production (coefficient 'B.in Eqn 8) would be 0.75 mole per NH,'.
4. The theoretical RQ (
~ ~ & y ~wast :calculated
~ ~ ) as:
-
-1
1
-
CER
R Q =~ ~ L
Eqn. 2.10
OER~omticd
5. To calculate the theoretical O2exchange rate ( 0 ~ ~ ~ ~ rnay
~ ~ exist
) t inh nodules
a t
having various percentages of their respiration being carried out anaerobically (AR'). the
following equation was used:
O E ~ ; , = [(i - AR%)x O E C ; ~ ~ ~ ]
where
Eqn. 2.1 1
OER",
is the value for aerobic nodules using Eqn. 2.8, and AR%is the
proportion (0.05 to 0.40) of the O E RAerobic
,eorcticai which was assumed not to occur due to
anaerobic metabolism.
Fig. 2.2. Ureide synthesis from sucrose and ammonia. The basic C O and 0 exchange is
shown on the left. To set the energy balance to zero, elecuons were assumed to be
consumed by an electron transport chain (ETC)and any excess reductant decreased the
need for sucrose metabolism (dotted lines).
The net reaction is shown on the right. 'B' represents the moles of O, consumption
which is not balance by C O production per unit ammonia assimilated from sucrose and
MI, into ureides which is used in Eqn. 2.8. Negative signs indicate consumption.
I
Basic Pathways
1
I
To set energy
balance to zero
1
Net Reaction
Sucrose
t
0.25 Allantoin
2.04
ATP
v
0.25 Allantoin
2.3 Results
2.3.1 CER,OER, ANA and RQ in nodules
The CER, OER and ANA of excised soybean nodules was measured sirnultaneously in an
open flow gas analysis system, yielding a CO2enrichment or O1 depletion in the range 25
to 70 Pa (ca. 250 to 700 ppm), and a H2 enrichment of 3 to 10 Pa. An exarnple of a
typical run is shown in Fig. 2.3A and B. Assuming an EAC of 0.65, the Pa of N,
depletion was calculated (Eqn. 2.7) for each rneasurement of H2measurement. as shown
in Fie. 2.3B.
The nodules modified the composition of the gas Stream entering the cuvette to
the greatest extent in freshly excised nodules. but over the subsequent 40 min penod, the
CO?. O2 and HIdifferentials declined to 47.7%. 44.8% and 35.6%.respectively, of chat
which was observed within the 3 minutes of excision.
When the C O e ~ c h m e nwas
t divided by the corresponding value for O2
depletion, apparent RQ (RQmmmJvalues were obtained that ranged from 1-16 to 1.2 1 in
the example data shown in Fie. 2.3A. To calculate the tme RQ, the observed differential
O2 concentration was corrected using Eqns. 6 and 7 to reflect only that O depletion that
was associated directly with biological O2 exchange. The corrected values for O2
depletion ( A[o~]~,.)
were consistently lower than the measured 0 depletion ( A [ O ~ ) ~ )
(Fïg. 2.3A). resulting in m e R Q values which were consistently higher than the
corresponding values for RQ,
In the example daca of Fig. 2.3A, m e R Q values
ranged from 1.23 to 1.28. The RQmamid values caiculated via Eqn. 2.8 to 2.10. were
typically the lowest, ranping from 1.06 to 1.07 (Fig.2.3C).
Fig. 2.3. Gas exchange of excised soybean nodules (0.69 g FW) immediately following
excision. A. Measured concentration differentials for C O (A[COJ~.thick h e ) and 4
(A[o,]",
thin line) between the inflow and effluent gas stream provided to nodules widiin
a cuvette. The 0 concentration differentid associated only with biological O, exchange
(A[02lPa,symbol), was calculated from Eqns. 2.6 and 2.7. B. Measured concentration
W, thick line) between the inflow and effluent gas stream
differentials for HI(A[HJ
provided to nodules within a cuvette and the calculated (Eqn. 2.7) NI differential (A[H,]~~,
thin line) that would have existed assuming an EAC of 0.65. C. The apparent (
RQ,,),
true (RQ) and theoreticai (RQnme",)
data presented in panels A and B.
values for respiratory quotient caiculated from the
O
10
20
30
40
Time after nodule excision (min)
50
2.3.2 Relationship between respiratory gas exchange and nitrogenase activity in
nodules from control, nitrate inhibited and detopped plants.
In freshly excised nodules from control plants. TNA was estimated to be 103.3 f9.0
pmol2e' g-' DW,, h'' and it declined to 60.4 k 7.0 pmol 2e- g-' DW,
h" within 40 min.
of excision (Fig. 2.4). CER and OER declined dong with the TNA, from maximal values
of 3 11.0 f 13 p o l CO, g*' DW,
to minimal rates of 198.9
h-' and 248.0 f 1 1 p m o l 0 5-'DW,, h-', respectively,
+ 15 pmol CO2g-' DW,,
h'' and 162.5 f 1 1 pmol O g1DW,,
h" ,respectively (Fig. 2.4A). Average RQ values ranged from 1.18 to 1.26. Theoretical
values for OER that assumed aerobic respiration ( OER$'~~,~~~ ) were higher than
measured OER, and therefore the RQnmrdd values were consistently less than the
measured values ranging from 1.056 to 1.O6 1 (Fig. 2.4B).
Freshly excised nodules from nitrate inhibited plants had similar M A values to
those nodules from control plants, but the CER and OER were only 76.8 and 78.7 %
+
(238.9 25 pmol CO, g" DW,
h-' and 195.3 f 22 pmol Ozg-' DWd h". respectively)
of that in the control treatment, As in the control treatment, TNA, CER and OER
declined with time after excision (Fig. 2SA), but the average RQ rernained relatively
stable between 1.12 and 1.23 (Fig. 2.5B). In comparison, RQbe,,,,
values were lower
than measured and ranging between 1.O9 and 1.08.
Within 3 minutes of uprooting and excising nodules from previously detopped
plants, TNA was estimated to be only 42.6 % (44 f 2.0 pmol2e- g" DW,, h-l) of that in
the control plants, whereas the CER and OER (136.9 t 4.6 pmol CO2 g" DW,, h-l, and
120.9 pmol O, g'l DW,
h", respectively) were only 44.0 and 48.8 8 of that in nodules
from control. As in the control ueatrnent, TNA, CER and OER declined with time after
excision (Fig. 2.6A), but the average RQ remained relatively stable between 1.06 and
1.14 (Fig. 2.6B). In comparison, theoreticai R Q values were predicted to be lower, in the
range of 1.O6 to 1.07.
Fig. 2.4. The relationship between the respiratory gas exchange of nodules frorn control
plants, and the total niuogenase activity of those nodules as al1 gas exchanges declined
following excision. A. Measured rates of CO, (CER) and 0 (OER) exchange in nodules
plotted against the TNA (caiculated frorn measured ANA values, Eqn. 2.5) at various
times after excision. Theoretical O2exchange rates assuming fully aerobic metabolism
Aemhc
(OERniconIICa1)
were calculated according to Eqns. 2.8 and 2.9. Each data point represents
the mean of 6 replicate measurements. A typical standard error (SE) for the measurement
of each parameter is shown on the figure. B. The rneasured me (RQ, solid squares) and
Aerobic
caiculated theoretical (RQ,,,,,
open circles) values for respiratory quotient of nodules
at each measured value for TNA. Values are presented as mean f standard error of 6
replicate measurements.
Respiratory Quotient
CO2and 0 2 Exchange Rates
(pmole g-lDW,,,, h-l)
3P
G O
a
C
CI.
Fig. 2.5. The relationship between the respiratory gas exchange of nodules from nitrate
treated plants, and the total nitrogenase activity of those nodules as al1 gas exchanges
declined following excision. A. Measured rates of CO, (CER) and O2 (OER) exctange in
nodules plotted against the TNA (calculated from measured ANA values. Eqn. 2.5) at
various times after excision. Theoretical O2exchange rates assuming fully aerobic
Aembic
metabolism (OER,,,,,)
were calculated according to Eqns. 2.8 and 2.9. Each data
point represents the mean of 4 replicate measurements. A typical standard error (SE) for
the measurement of each parameter is shown on the figure. B. The measured m e (RQ,
solid squares) and calculated theoretical ( R Q ~ ~open
~ circles)
~ ~ ,values
,
for respiratory
quotient of nodules at each measured value for TNA. Values are presented as mean f
standard error of 4 repiicate measurernents.
Nitrate & Excision
p CER
11SE
More O2 Lirnited
H
40
60
80
100
Total Nitrogenase Activity
Fig. 2.6. The relationship between the respiratory sas exchange of nodules from
detopped plants, and the total nitrogenase activity of those nodules as al1 gas exchanges
declined following excision. A. Measured rates of CO2 (CER) and 0 (OER) exchange in
nodules plotted against the TNA (calculated from measured ANA values, Eqn. 2.5) at
various times after excision. Theoretical 0, exchange rates assuming fully aerobic
Aerobic
metabolism ( OER,,,,)
were caiculated according to Eqns. 2.8 and 2.9. Each data
point represents the mean of 4 replicate measurements. A typical standard error (SE) for
the measurernent of each parameter is shown on the figure. B. The measured m e (RQ,
solid squares) and calculated theoretical (,,QR
Acmbic
open circles) values for respiratory
quotient of nodules at each measured value for TNA. Values are presented as mean f
standard error of 4 replicate measuremenü.
Detopped & Excision
More O2Limited
1
Total Nitrogenase Activity
(pmole g-1DW,, h-1)
H
2.4 Discussion
2.4.1 Methods for RQ measurement: Past and present
This study is one of the few studies with plants that have attempted to measure RQ values
in an open flow gas exchange system. These R Q measurements were made possible by
the development of a highly sensitive differential oxygen analyzer (Layzell et al., 1996).
Accurate values for RQ in plant tissue have been difficult to obtain in the past because of
the low rates of O exchange that occur in plants against a high background O2
concentration of air (20.78 or 207,000 pUL O,). Most previous studies (Pankhurst and
Sprent, 1975; Dixon et ai., 1981) used closed gas exchange systems to generate larger
differentials in 0 concentration that could be measured by an 0, electrode. However
closed system methods have a number of drawbacks, including; (a) the conditions inside
the sample cuvette change over time, resulting in aunospheres with elevated levels of
CO, and HZand depleted levels of O?, environmental conditions which have al1 been
reported to alter nodule metabolism and nitrogenase activity (Bethenod et ai., 1984; Hunt
et ai., 1987; Moloney and Layzeli, 1993). (b) removal of gas sarnple for analysis can
inuoduce artifacts, by changùig the pressure and p 0 2 in the cuvette, and by increasing the
probability of leaks, and (c) in most studies, C O is measured first in an open system and
then 0 exchange is measured in a closed system (Lambers et al., 1980;de Visser, 1985;
Serraj et ai., 1994; Serraj et al., 1995; Serraj and Sinclair, 1996; Vadez et al., 1996).
Depending how the studies were done, the nodules may become more inhibited with
tirne, resulting in an underestimate of O2uptake relative to CO, evolution.
Although open flow gas exchange systems have many advantages over closed gas
exchange systems, reliable and accurate measurements of O uptake depend on the
careful monitoring or control of the gas pressure and temperature and the need to
incorporate appropriate volumetric corrections in the calculation of O2 exchange.
Oxygen is a difficult gas to measure because it is highly affected by environmentai
conditions. Very small pressure differentials can have a large effect on the measurernent
of differential O?. For example. a differential of about 1.0 cm water column is equivalent
to about 20 Pa (200 ppm). At higher PO,. the same differential would have a
proportionately greater effect on the measured O2differential. The methods described in
this chapter were the fust to incorporate sensors which monitor changes in atmospheric
pressure and differential pressure changes between reference and analytical gas streams.
In the field of respiratory physiology it is well known that volumeuic comections
are needed to account for the volumetric effects on O2concentration that C O has when
its production does not precisely balance O?uptake (Haidane, 1912). Most of the
previous RQ studies with plants have not accounted for the volumetric dilution or
concentration of O?,
despite the fact that errors cm be very significant in plants. For
example, if CO, and Hzproduction in nodules is greater than O and NZ uptake, there
would be a dilution of the O concentration in air such that the measured O, differentials
would overestimate actual biological O2exchange. The scmbbinp of CO, in al1 gas
streams before measurement would partially solve this problem but would not correct for
the dilution effect caused by H,. In most legumes Hzproduction is 1.5 to 2.0 times the
rate of N, fixation (Moloney and Layzell, 1993), and in Ar02 environments, N2 fixation
is zero, H, production increases 3-4 fold (Hunt et al., 1987). Even HUP' symbioses,
which do not produce HZgas, will also require a volumetric correction since they still fix
N, and therefore, there would be a tendency to concentrate O? in the gas phase, which in
turn would cause an underestimate of O?uptake. The importance of this correction cm
be seen in Fig. 2.3A where the correction of OER resulted in lower A[O,]:~.
values (by
5.6% from the apparent OER)resulting in an increase in the estimate of RQ (Fig. 2 . K ) .
2.4.2 Theoretical RQ vs. Measured RQ
In the control and nitrate treatments, the measured RQ vaiues were higher than the
RQ,i
vaiues that were calculated from Our knowledge of the biochemical pathways
that are thought to operate within legume nodules. These calculations assumed that
carbohydrate (sucrose and starch) was the C substrate, and ureides were the only end-
product of N, fixation. Three reasons may be proposed to account for the discrepancy
between measured and theoreticai RQ.
a) The presence of anaerobic metabolism.
Fig. 2.7 shows the theoretical relationship between RQ and TNA in soybean
nodules with various capacities for anaerobic metabolism. Given the relationship
between CER and TNA in Fig. 2.3, the theoreticai RQ in a fully aerobic nodule
(R
Q,,,)Acrobic
at high TNA would be expected to be about 1.O62 in nodules fixing NZ and
synthesizing ureides (Fig. 2.4A). At al1 percentages of anaerobic metabolisrn, the RQ
increased. Therefore, the RQ was expected to increase if anaerobic metabolism were
present.
If the theoretical estimates are valid, the measured RQ values of 1.18 to 1.26
found in excised nodules would predict that 15% of the CO, evolution coupled to the
E.T.C.in an aerobic nodule (oxidative CO, production) would be coupled to anaerobic
endproduct production (Fig. 2.4B). Treatments of nitrate excision predicted that > 10%
of oxidative CO, production would be coupled to anaerobic end-produce production at a
TNA of 100 pmole g-'DW,h"
(RQ = 1.23) and dropping to < 5% at TNA 40 pmole g-
'DW,, h-' (RQ = 1.12). While detopped excision ueatrnents appear to have < 5%
oxidative CO, production coupled to anaerobic end-product production (Fig. 2.4B) in the
RQ ranges foond ( 1.14 to 1.06). It was interesting that the highest RQ values, which
would be indicative of the highest rates of anaerobic metabolism were found in the
inhibitory treatment which resulted in the least inhibition (excision only). Treatments
with intact plants under detopped and nitrate conditions result in inhibition of nitrogenase
activity and respiration rate, which are accompanied by a decline in Oi (Minchin et al.,
1986; Hartwig et al., 1987; Vessey et al., 1988a. 1988b). Increases in O, can recover the
decreased permeability resulting in a stimulation of O2uptake and nitrogenase activity,
however the maximum activity attained in the inhibited plant is less than that in the
Fig. 2.7. The predicted relationship between respiratory quotient ( R Q ~ : ~ ~ ~and
, , TNA
)
in soybean nodules having various capacities for anaerobic metabolism. The thick Iine
depicts the RQnmetid for a nodule having only aerobic respiration, as caiculated from
Eqns. 2.8 to 2.10 using data from a linear regression of the CER and TNA values in Fig.
2.3A (CER= 2.72 X TNA + 17.2 1). The other curves use the same data but the
theoretical OER values are modified by Eqn 11 to provide estimates of RQ[:;~,,
anaerobic rnetabolism at a TNA of zero accounts for 5% to 40% of the CER not
associated with electron flow to nitrogenase or AAR (see text).
where
Total Nitrogenase Activity(pmole g-ID~,,h-1)
control representing a decrease in apparent Vmax (Layzell et al.. 1990; Sun2 et al., 1991).
In this study excised nodules from nitrate and detopped plants exhibited lower R Q values
than excision alone, indicating that the decrease in RQ may be associated with the
availability of C reserves.
b) Other carbon substrates were used during Nz fixation.
Acrohc
In the calculation of OER,,,,,
, an assumption was made that the nodule's
primary substrate was sucrose or starch since these represent the principal C substrates
either supplied to nodules in phloem (sucrose) or stored in nodules (starch, especially in
cortical cells). However. nodule excision would have stopped al1 phloem supply to the
nodules, and if the starch pools were inaccessible, the nodule may have begun to
breakdown other C substrates. Since the measured RQ was greater than the theoreUcal
RQ for fhis explmation to account for the experimental results, the C supply for
respiration would need to have been a highly oxidized subsrnte (per C present) such as
malate or succinate. There is no evidence on the change of succinate or malate pools
with excision, however, increases in PO,result in an increase in malate pools at a rate of
= 5 vmol g ' ' ~ ~ h(Suganuma
-'
and LaRue. 1995). If nodule excision caused a depletion
of malate by the same rate (5 pmol g ' D ~ h ' ' )this would indicate that .- 20 pmol gelDWh'
I
of the C O evolved in nodules was associated with malate utilization. However this
utilization would only increase the theoretical R Q in fully aerobic nodules by 0.99%
3) Other products were made dunng N, fixation.
Armbic
The RQnmmi, values were calculated based on the OERThomticd
of nodules
synthesizing ureides (Eqn. 2.8) using a 'B' coefficient of - 0.75 (Fig. 2.2). It is possible
that other products were formed in the nodules which significandy altered C O and O2
exchange. One candidate is poly-8-hydroxybutyrate (PB),a storage compound within
bacteroids that has been reported to increase in concentration in nodules that are stressed
(Vasseileva and Ignatov, 1996). However when Eqn. 2.8 was changed to include PHB
synthesis dong with ureide synthesis the RQ,,,,
was only slightly increased (by
0.08% data not shown). On the other hand. if the nodules were synthrsizing asparagine
(ASN) rather than ureides. the 'B'coefficient in Eqn. 2.8 would be - 0.5 instead of - 0.75.
Consequently, the R QAemhc values would be higher, (e.g. 1.104 for ASN vs 1.O62 for
T ~ C RCUI
O ~ ~
ureides). With these theoretical values the measured RQ for excised and nitrate-excised
ueatments would result in a prediction of = 1 0 1 of the oxidative C O production was
coupled to anaerobic end-product production, while in the detopped plants, the RQ values
would be consistent with a fully aerobic nodule.
If however, the nodules were considered to have stopped NH, assimilation, and
the energy, carbon and 0 demands for ammonia assimilation went down to zero the 'B'
coefficient in Eqn. 2.8 would be zero. Here the values of % oxidative C O production
coupled to anaerobic end-product production, would increase by 1 1.4% of initial. and R Q
values would need to be greater than about 1.20 to represent anaerobic metabolism. In
this situation, measured RQ values for excision represent only = 5% of the oxidative CO,
production was coupled to anaerobic end-product production, and nitrate-excised RQ
predict < 5%, while detopped-excised RQ values were l e s than predicted. However,
there is no evidence for MI, accumulation in excised nodules in the literature, thus it is
reasonable to expect continued ureide synthesis in nodules from detopped piants (Walsh
et ai., 1987).
2.4.3 What does RQ tell us about the presence of anaerobic metabolism in nodules?
It has been suggested that increases in RQ values found in sub-ambient p 4 could reflect
anaerobic fermentation by nodules (Sprent and Gallacher. 1976; Witty et al., 1983; Senaj
et al., 1994). The RQ values f0ur.d in this study indicate that anaerobic metabolism may
be occumng in nodules fixing N, and synthesizing ureides.
The RQ analyzer provided a relatively easy means of measuring accunte values
of respiratory quotients. The RQ values found in this study may indicate thai up to =
15%of the oxidative C O production was coupled to anaerobic end-product production.
that is not directly associated with N, fixation and NH,' assimilation. However it was
interesting to find that freshly excised nodules exhibited higher R Q values than those
from plants that were first detopped. then excised. Since this latter treatment has been
shown to reduce Oi in nodules, p a t e r . not lesser rates of anaerobic metabolism would
have been expected in the nodules from the detopped plants.
Further work is required io test whether O,-limited nodules produce anaerobic
end-products such as. ethanol, and to compare this rate of production to the prediction
that up to 15% of the oxidative CO' production was coupled to the production of
anaerobic end-products.
Chapter 3. Ethanol and O,-Limited Metabolism
3.1 Introduction
The fixation of nitrogen to ammonia is catalyzed by the bacterial enzyme nitrogenase.
This reaction is energeticaily expensive, requiring at least 16 ATP for each molecule of
N, converted into two molecules of ammonia. The energy demands of nitrogenase are
metabolized through oxidative phosphorylation in the bacteroid (Robson and Postgate
1980), and to support this there must be an adequate flux of O2 to the bacteroid terminal
oxidases. However, oxygen is a potent. irreversible inhibitor of nitrogenase. The nodule
must therefore exercise stringent control over its intemal O, concentrations to maintain
optimal conditions for both respiration and nitrogenase activity. Low concentrations of
free 0 (3-10 nM O?)c m be maintained within the infected zone (Bergersen 1980;
Bergersen 1982) by (a) physiological regdation of the resistance of a variable barrier to
O, diffusion (see Layzell and Hunt 1990; Witty and Minchin 1990 for references), and
(b) a high rate of respiratory consumption of O, which is facilitated by leghemoglobin
(Bergersen 1962).
The low concentration of O, which is maintained in the infected zone limits and
regulates respiration activity under normal conditions (Layzell and Hunt, 1990), and this
O, limitation becomes more severe under many adverse environmental conditions.
including plant disturbance. stem girdling, nitrate fertilization or nodule excision.
Following such treatrnents, legume nodules decrease their permeability to O?, causing a
decrease in Oiand a reduction in nodule metabolism through a greater O, limitation of
nitrogenase-linked respiration (Minchin et al., 1986; Hartwig et al., 1987; Walsh et al.,
1987; Vessey et al., 1988a. 1988b; Hunt et al., 1989; Sung et al.. 1991; Diaz del Castillo
et al.. 1992). In such nodules increases in rhizosphere PO, causes a large stimulation of
nodule respiration and nitrogenase activity (Vessey et al., 1988a; Denison et al., 1992;
Hunt and LayzelI, 1993).
Hypoxic or anaerobic metabolism may also be indicated by the tact that the
central zone of legume nodules have low values for adenylate energy charge*(AEC =
[ATP + 0.5 ADP] + [ATP+ADP+AMP]) in the range of 0.60 to 0.72 (Oresnik and
Layzell, 1994) compared to that typical of aerobic ceils (AEC 2 0.80 (Pradet and
Raymond, 1983, 1984)). In addition, the AEC is lower in nodules that are stressed by
stem girdling, nitrate inhibition, and &:O2 (deLima et ai., 1994). Since the Km (0,)for
bacteroid respiration is 5-20 nrnolar O? (Bergersen and Turner 1990, Millar et al., 1994,
1995). whereas that for plant respiration it is 50 to 100 nmolar O2 (Rawsthome and
LaRue 1986; Day et al., 1988; Millar et al., 1995) the O2limitation is more likely to be
associated with the plant mitochondna than with the bacteroids.
Evidence that nodules support anaerobic metabolism include the presence of
anaerobic end products acetaldehyde and ethanol in nodules (Sprent and Gallacher, 1976;
Tajima and LaRue, 1982) and high activilles of various anaerobic enzymes (Tajima and
La Rue, 1982, T h y m and Werner, 1996). De Vries and coworkers (1980) found that
alcohol dehydrogenase (ADH)activity in Pisum sativum nodules were higher than that in
roots. In nodules of aifalfa (Medicago sativa cv. Aragon) ADH activity was 100 times
greater in the plant fraction than that in the bacteroids (Irigoyen et al., 1990),and in
soybeans it was higher in uninfected cells and cortical tissues than in the infected cells
(Sugunama et al., 1987). These studies indicate that anaerobic metabolism may be
especiaily important in uninfected and cortical tissues.
In chapter 2, measured values for respiratory quotient (RQ = C o evolution + 0,
uptake) were found to be higher than theoretical RQ values, a condition which rnay be
indicative of anaerobic metabolism. This stimulation was highest in excised nodules of
control plants, rather than those from nitrate ueated or detopped plants.
Consequently, there is considerable support for the suggestion that legume
nodules not only have the capability for anaerobic metabolism, but that it may play a
significant role in the C metabolism of the legume nodule under 'normal* physiological
conditions. Since rthanol is the end product of anaerobic metabolism in most plants, the
purpose of this study was to quantify the rates of ethanol accumulation and volatilization
in soybean nodules exposed to conditions that would result in greater or lesser degrees of
O,-limited metabolism. It was hypothesized that the rate of ethanol production in
nodules will account for the differences in measured and theoretical RQ reported in
chapter 2.
3.2 Materials and Methods
3.2.1 Plant Culture
Seeds of soybean (Glycine mar L. M e n cv Maple Amow) were inoculated at planting
with Bradyrhizobiurnjaponicum USDA 16, a strain lacking uptake hydrogenase activity
(Layzell et al., 1984). AU pots contained small Stones to a depth of five centimeters, and
then filled to the top with silica sand (Grade 16) (Hunt et ai., 1987). Al1 plants were
grown in a growth chamber (Mode1 PGV, Conviron Environments Ltd., Winnipeg, MB)
at a constant temperature of 25'C and 80% relative humidity, with a photon flux density
of 800 p o l m"
S-' photo synthetically active radiation (PAR)
and a 16 hour photo
period. The plants were irrigated twice daily with a modified Hoagland's nutrient
solution (Walsh et al., 1987) containing 0.5 m M KNO, until on week after germination
and then with the sarne solution lacking nitrate for the remainder of the growth period.
Plants were used in experiments at 27-35 days after sowing.
3.2.2 Tissue Hanest and Ethanol Analysis
Plants were harvested, by rapidly uprooting the plant and immediately plunging the
nodulated root system into liquid niaogen. Nodules were then picked while frozen,
separated from roots and sand grains. The frozen nodule tissue was ground to a fine
powder in liquid nitrogen and then weighed to yield fresh weight (FW). The nodule
powder was then placed ont0 0.8 mL of frozen 10% (w/v) perchloric acid (0.8 mL per
200-300 mg FW nodule) in a glass, hand-held tissue hornogenizer. The tissuewas
homogenized on ice as the liquid nitrogen evaporated and the perchloric acid thawed.
The homogenate was decanted into 1.5- mL microfup tubes, and the homogenizer was
rinsed with another 0.2 rnL of 10% (w/v) perchlonc acid, which was added to the original
homogenate. A 1 mL sample of the homogenate was then injected into an evacuated 3
mL red top vacutainer tube (Becton Dickinson and Company, New Jersey), containing
0.3g of NaCl to minimize ethanol solubility. The pressure in each test tube were
equilibrated to 1 ATM, and then placed in a 27'C water bath for 10 minutes. After 10
minutes a 1 mL gas sarnple was sarnpled from each test tube and injected into a gaschromatograph (mode1GC-8A. Shimadzu) for ethanol measurement (Fig. 3.1).
Standards were made up in which a known amount of ethanol was added to the perchlonc
acid/NaCl mixture. Recovery of ethanol into the gas phase was assessed by mnning
standards in which a small volume of 95% ethanol was added to an othenvise empty vial,
heating it to volatilize the ethanol and measunng the ethanol in the _pasphase.
The gas-chromatograph was equipped with a flame ionization detector. and
separaùon was achieved with a 6 ft x 2 mm ID Glas column packed with 0.2%
~arbowax@1500 on ~ r a ~ h ~ a c801
@100.
- ~ The
~ , column temperature was held at 70°C
while the detector was at 115'C and the carrier gas was nitrogen at a flow rate of 20 mL
min".
Addition of NaCl to the perchloric acid extract increased the amount of ethanol in
the head-space by approximately 6 0 1 and resulted in a recovery of approximately 7 1%
of the ethanol from the nodule tissue. The results presented here have been corrected for
recovery.
Foilowing injection into the gas-chromatograph a number of other volatiles were
also present within the nodule extracts. The retention times were longer than that for
ethanol, but attempts to identify them by mass-spectrometry were not successful since the
mas-spectrometer was not sufficiently sensitive.
Fig. 3.1. Summary of the method used to analyze the concentration of ethanol and other
volatile compounds in soybean root nodules.
Roots fkozen in
Nodules ground
in mortar and pistil
under liquid N, then
ground in frozen
perchlonc acid A
sample of
homogenate injected
into evacuated 3ml
vacutainer containing
NaCl
3.2.3 Measurement of Ethanol Loss to the Cas Phase
To quantify the rate of ethanol loss to the gas phase. approximately 10 nodules were
harvested fresh and placed into a syringe throush which ethanol free air of 02:N7Cas was
passed (10 mL min-') (Fig. 3.2). The effluent gas frorn the nodule charnber was sarnpled
( 1 mL),and
injected into the gas-chromatograph for ethanol anaiysis at 3, 8, 13. 18, 24,
or 30 minutes after nodule excision.
3.2.4 Treatrnents
Pool sizes of ethanol in intact attached nodules were measured in control plants and in
plants exposed to stress treatments known to decrease the inkcted cell O, concentration
and induce a greater OZlimitation of nodule metabolism. These treatments included:
Nodule excision - Nodules were excised and left for 0.0.5,
l,
or 10 minutes before being
frozen in liquid nitrogen and used for ethanol analysis. Previous studies have shown that
excision causes O&nitation on nodule metabolism (Sung et al., 199 1).
10% 0,Treatment - The nodulated roots of intact plants were sealed in theu growth pots
and nushed with room air at 500 mL min" for 20 minutes to obtain stable steady-state
conditions within the pot. The plants were then exposed to 10% 0 at 2000 mL min-' for
0, 0.5, 1.0, 2.0, 3.0, or 10 minutes after which the nodulated root system was rapidly
frozen in liquid nitrogen and used for ethanol analysis.
0% O, Treatment - The nodulated roots of intact plants were sealed in their growth pots
and flushed with room air at 500 mL min" for 20 minutes before being exposed to 0% O2
for 0, 1.0, 3.0 or 10 minutes, after which the nodulated root system was rapidly piunged
into liquid nitrogen. The nodules were then harvested under liquid nitrogen and used for
ethanol analysis.
3.2.5 Ethanol Movement within a Whole PIant
To determine whether ethanol formed within nodules may be lost to the gas phase, or
transported to the shoot in the xylem, nodulated soybean plants were sealed in their
growth pots and connected to a gas exchange system. After being flushed with air for 20
Fig. 3.2. Summary of the method used to measure ethanol volatilization from soybean
nodules. The Cas was provided at 10 mL min" from the mass flow controllers, and a 1
mL sample of the output $as sueam was analyzed via gas-chromatography,
lm1 Gas Sarnple
Input Gas from Mass Flow
Controiler at 10 mL min-1
minutes the flow rate was dropped to 10 mL min" and the effluent gas sueam was
sampled (1 mL) periodicdly and injected into the gas chromatograph for analysis.
In the same plant, the shoot was sealed in a plastic bag to which a continuous air
flow of 6 L min" was provided. The transpiration water lost from the Leaves was trapped
in three erlenmeyer flasks in series which were held at 0°C.Ethanol was measured in the
transpired water using a variation of the ethanol anaiysis technique. Briefly, a 1 mL
sample of the transpired water was injected into an evacuated test tube containing 0.3g of
NaCl. The pressure in each test tube was equilibrated to 1 ATM, following this the test
tubes were placed in a 27'C water bath for 10 minutes. A 1 mL gas sample was remove
from each test tube and injected into the gas chromatograph for edianol measurement.
When al1 measurernents had been made, the nodules were rapidly frozen in liquid
N2 and analyzed for ethanol content by ethanol analysis as described above.
3.3 Results
3.3.1 Nodule Excision
In attached nodules that were rapidly frozen in liquid N, the ethanol pool size was 6 1.5
+
18.9 nmol ~"Dw,. Within the first 10 minutes following excision, no change was
observed in the tissue ethanol content (Fig. 3.3A).
To determine whether ethanol was Leaving the nodule in the gas phase, excised
soybean nodules were placed in an open flow gas exchange system as shown in Fig. 3.2.
The ethanol content in the effluent gas rose rapidly from the time the nodules were put
into the cuvette (3 minutes after excision) and stabilized at 202.9 f 57.5 nmol ~ " D W , at
28 minutes after excision (Fig. 3.3B).
3.3.2 10% 4 Treatment
When nodulated roots were exposed to 10% O?, the O concentration in the pot declined
rapidly and stabilized at 10% within 30 seconds of the start of the treatment (Fig. 3.4A).
The initial ethanol pool size was 23 f 3.7 nmol ~'Dw,, rose to a high of 47.6 t 9.3
Fig. 3.3. Nodule excision as method of causing O2 limitation. A. The ethanol
concentration within nodules after 0.5, 1, or 10 minutes of excision. B. Ethanol lost to
the gas phase after 3. 8, 13, 18.24, or 30 minutes of excision. Mean values are presented,
the SE bars are presented where these exceed the dimensions of the symbols (n=3).
1
Excised Nodules
B
Ethaol loss to gas phase
10
20
Time after excision (min)
Fig. 3.4. Whole plants were exposed to 10% O2 for 0.5, 1,2,3. or 10 minutes. A. The
level of O2 concentration inside the pot with time. B. The response of ethmol over time
as a result of 10% O2exposure. Mean values for ethanol levels are presented, the SE bars
are presented where these exceed the dimensions of the symbols (n=9).
2
4
6
8
Time in 10% 0, (min)
10
12
nmol ~"DW,, h-' at 3 minutes. but declined back
CO 24.6
+ 9.0 nmol ~'Dw,,,h-' within
10 minutes (Fig. 3.4B).
3.3.3 0% O, Treatrnent
To determine the potential of nodules to produce ethanol, nodulated roots were exposed
to an environment lacking in 0:. The 0, concentration declined to less than 1% ( v h ) in
approximately 1 minute (Fig. 3.5A) and ethanol pool sizes remained low, between 20 f
5.2 and 143 f 43.6 nmol ~'Dw,,
for the first 3 minutes of exposure (Fig. 3.58).
However, by 10 minutes of exposure to 0% O?, the ethanol pool increased to 2179.8 f
359.4 nmol g*'DW,,, a nse equivalent to a rate of 18684 nmol ~*'DW,, h-'.
3.3.4 Whole Plant Analysis
Ethanol was rneasured in the water transpired from the leaves of nodulated plants. from
the gas phase surrounding the nodulated roots and within the nodule itself to determine
whether ethanol was exported out of the nodule. The results for this experiment are
presented in Figure 3.6. Under normal growth conditions ethanol was lost from the
leaves in the uanspired water at a rate of 258.1 k 42.1 nmol p"DW,,
loss from the gas phase around nodules was 16.7
accumulated by 105.7 t 37.2 nmol $DW,
h-', whereas ethanol
+ 2.3 nrnol ~ * ' D wh",,
while ethanol
within the nodule.
3.4 Discussion
3.4.1 Ethano1 Production in Nodules under 0% 0,
Legume nodules have the capacity to produce ethanol when exposed to severely O,Limiting conditions, under 0% O2 conditions fermentation has been reported to be
stimulated to a rate of 6640 nmol e thanol ~ " D w ,h-' (Serraj et al., 1994) and Heckmann
and Drevon, (1987) reported ethanol pool sizes of 5000 nmol ~ ' ' D wBoth
~ ~studies
~.
used detached nodules (100-200mg)which had been placed in closed test tube (150 mm x
20 mm) containing 0%0, and incubated at 20°C for Ihr (Witty et al., 1983). The
present study measured ethanol accumulation in intacted nodules under 0%O2
Fig. 3.5. Whole plants were exposed to 0%O for 0, 1,3. or 10 minutes. A. The level of
OZconcentraùon inside the pot with time. B. The response of ethanol over time as a
result of 0%0 exposure. Mean values for ethanol levels are presented, the SE bars are
presented where these exceed the dimensions of the symbols (n=6).
2
4
6
8
Time in 0% O, (min)
10
12
conditions resulting in pool sizes of 2179.8 nmol glDW,,which reprexnred a rate of
18684 nmol glDW,
h" production in the 7 minutes measured (Fig. 3.33). The higher
apparent rate of ethanol accumulation in this study may be attributed to the shoner
incubation time than that used in previous studies. However. nodules are rarely exposed
to such severe conditions of 0, limitation. Therefore, these studies show that ethanol
production is possible but provide no information on whether it occurs under 'normal'
physiological conditions.
3.4.2 Ethanol Production in Nodules in 21 % and 10% 0,
Although nodule excision is not an 'normal' physiological condition it has been shown to
give rise to a greater O, Limitation of nodule metabolism as observed in nitrate fertilized
plants or in plants exposed to long term dark treatments (Layzell et ai., 1990; Sung et ai.,
1991). Sung et al.. (1991) reported that nodule excision caused a rapid decline in
Hz
production of 46% of that in intact plants within 5 minutes. however. between 6 and 15
minutes after detachment nodules exhibited partial recovery of nitrogenase activity which
was followed by a second decline to a low of 20% of initial after 40 minutes of excision.
A similar trend was seen in ethanoi accumulation, 3 minutes after excision ethanol pool
sizes had increased by 1696 of the initial, by 10 minutes the pool sizes declined to 38.5%
of initial (Fig. 3.3A). However, ethano1 lost to the gas phase was observed to increase
and stabilize at 203 t 57.5 nmol ~ ' D w ,h-' indicating that excised nodules are
continuously producing ethanol and most of the ethanol is lost to the gas phase (Fig.
3.3B).
Treatrnents of 10% 0 are reported to cause a decline in ANA within 4 minutes by
30% of the initial rate which is followed by a steady, gradua1 increase in HI evoiution to
70% of initial within 45 minutes (de Lima et al., 1994). Ethanol accumulation responded
in a similar fashion, 3 minutes &ter exposure to 10% O?,the ethanol pool size had
increased by 50% of the initial which then dropped down to the level of initial (Fig. 3.4B)
after 10 minutes of exposure.
Exposure to 10% oxygen and nodule detachment represent minor to moderate
levels of 0, limitation, respectively. However. the net ethanol accumulation does not
appear to represent a significant carbon l o s for the nodule unless exposed to severe O2
limiting conditions (0% Cl2).
Tajima and LaRue, (1982) found that ethanol accumulated in freshIy harvested
nodules to a level of between 95 and 180 nmol g"DW,.
Their observations suggest that
anaerobic metabolism was a normal physiological activity of the soybean nodule and not
formed solely as a response to prolonged stress as reported by Sprent and Gdlacher
(1976). Non-stressed soybean nodules accumulated ethano1 to the level of 105 37.2
nmol ~"DW,, which is consistent with those of Tajima a d LaRue, (1982). and
represents a rate of 1.42 nmol g''~W,h-'. this rate was caicuiated using the assurnption
for relative growth rate of nodules (0.1 gDW d-') and grains of dry weight nodule (0.31
oDW).
L'
To determine if ethanol was exported from the nodules, ethanol was measured in
the trmspired water from leaves of uninhibited nodulated plants, from the gas phase
surrounding the nodulated roots and within the nodules themselves. The rate of the
ethanol accumulated within the nodule (1.42 nmol g - l ~ ~ , h ' ' ) was much lower than that
of ethanol lost in the transpired water (258.14 f 42.1 nmol g"DW,h-')
or that lost in the
gas phase surrounding the nodules (16.7 f 2.29 nmol g-'DW,, h*').
3.4.3 The Important of Ethanol Production in Nodule Metabolism
To assess the relative importance of ethanol production with respect to metabolism within
a nodule, a mode1 was developed in which it was assumed that (a) the whole nodule CO,
evolution rate was 300 jmol ~''DW, h'' (Sung et al., 1991). (b) two thirds of the nodule
respiration was from the bacteroids (Layzell et al., 1988) and (c) hexose was the substrate
used by nodules (Fip. 3.7). Therefore. the rate of hexose consumption would be 50 pmol
~"Dw,,h", resulting in the production of 100 p o 1 PEP ~ - ' D W , h", half of which is
L
Fig. 3.6. A cornparison between the carbon flow through glycolysis and TCA cycle. and
the carbon flow through anaerobic metabolism. The mode1 was derived based on a CO2
evolution rate of 200 m o l $'DW,
h-' and the assumption that hexose was the substrate
used by nodules. Ethanol values were denved from whole plant experimentation under
control conditions. Each ethanol value represents an average of 6. OAA, oxaioacetic
acid; PEP, phosphoenolpyruvate; TCA, Tricarboxylic Acid Cycle.
mlHexose
Transpired from
Leaves
-- .
I\
'
Metabolized ,
1 - Exported
in Shoots
-* -Ethanol-
dehydrogenase
py1 U VUCC
I
decarboxytase
ldehyde 1
NADH
NAD+>
.(?)
1
/
air /
mala te
dehydrogenase
I
l
-
NAD'
in room
NADH
.-.
.
v olatilizea
frorn Nodules
7
I
I
I
b
Accumulated
in Nodules
I
used to produce malate for bacteroid metabolisrn, and half to support plant metabolisrn.
On the b a i s of the results obtained in this study. ethanol production accounts for only
0.56 8 of the pyruvate used by the plant fraction of the nodule. Therefore, anaerobic
metabolism to ethanol represents only a small component of nodule membolism.
3.5 Conclusion
Ethanol accumulates in uninhibited nodules. and the potential for the pathway for
anaerobic metabolism was demonstrated in conditions of O 1 O?. Despite the fact that
legume nodules have the capacity to support very high rates of ethanol production, under
21% or 10% 02,net ethanol production accounts for l e s than 0.093 8 of the CO2
evolution within the nodule. This however, does not mean that anaerobic metabolism is
not significant within the nodule, as the methods for ethanol measurement could only
measure net ethanol production. It is possible that anaerobic metabolism is continuously
active in plant fraction of nodules and the ethanol produced is utilized by the bacteroids
as a carbon source, this utiiization of ethanol would also result in no net change in CO, or
O2 evolution, thus RQ values would not be a useful indicator.
Chapter 4.
Conclusions
This thesis used two approaches to study the role of net anaerobic metabolism in legume
nodules. RQ was measured and compared with theoreticai values on the assumption that
values greater than theoretical would be indicative of anaerobic respiration. Secondly. an
attempt was made to quantiCy ethanol production by nodules. The results of this study
showed that:
1) In excised control nodules, measured RQ values were higher than the RGae,,,
values
indicating up to = 15% of the nodule CO, production. not involved with N, reduction and
NH,' assimilation, may be associated with anaerobic respiration.
2) In excised-detopped nodules, the measured RQ was similar to the theoretical RQ values.
This was surprising, since previous studies (Sung et ai., 199 1; Hartwig et al., 1987)had
shown that detopping caused severe O limitation in nodules.
3) A theoretical analysis of CO, and 0 exchange in nodules predicted that the pathways of
C and N metabolism would have a major effect on the RQ of nodules. nierefore. when
using RQ to study anaerobic metabolism in nodules, it is crucial to know the other products
and su bstrates of nodule metabolism.
4) Although Ethanol is produced at very high rates in nodules exposed to 0% O, the rate of
net ethanol production under physiological levels of O, limitation does not appear to
represent a significant proportion of the respiration of soybean nodules.
Based on the data collected in this study, it c m be concluded that o d y a minor
proportion of the C O from nodules was associated with ethanol production, the high RQ
values observed in excised control nodules could be due ro changed C and N metabolism.
This does not indicate that anaerobic metabolism is not significant within the nodule
as the methods for RQ and ethanol measurement could only measure net ethanol
production. It is quite possible that ethanol is produced continuousty and utilized by the
bacteroids as a carbon source (Peterson and LaRue 1981. 1982). It is therefore, possible
that the pathway for ethanol production is highly active wirhin the nodules at dl times.
which is consistent with the low levels of O, found within nodules and the potential of the
anaerobic pathway under 0% O?.
4.1 Future Research
This study found that measured RQ values from excised control nodules were higher than
theoretical R Q values, and this increase was not linked to ethanol production. To resolve
this apparent discrepancy, intact plant R Q values need to be obtained as this would provide
a clearer picture on R Q change, intact plants are more stable and any treaunents on intact
plants would result in slower biologicai changes as compared to excision treaunents.
Ethanol rates and accumulation levels suggest that ethanol has a very srnail
contribution to total nodule metabolism. however it is possible that the ethanoi is utilized by
the bacteroid and only under extrerne conditions, which severely limit the bacteroids,
ethanol accumulates. To examine this possibility the turnover rate of the ethanol pool
would need to be exarnined. This would show if the ethanol pool was stagnant or if it was
in continuous use.
Possible Experiments;
1) Select for bacteria which can not utilize ethmol as a carbon source. These bacteria can
be used to inoculate the soybean root system. If nodules are formed, ethanol could be
measured under various O,-limited conditions to see if there is a higher level of
accumulation in these nodules, also under these conditions it cm be determined if the
nodules iack tolerance to O, limitation.
2) Exposing the leaves of a soybean plant to CO, labeled with I3C,the labeled CO2would
be incorporated into sugars which would be transported to the nodules. The level of
13c02
coming off the nodule and '%ethano1 measured within the nodule could be compared to
detemine the turnover rate, and thus. the flux through the pathway. If the nodule ethanol
pool size is npidly tuming over. the specific acljvity of C in the ethanol pool would be
similar to the specific activity of the CO, evolved by the nodule.
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