Plant Physiol. (1999) 119: 399-408
The Site of Oxygen Limitation in Soybean Nodules1
Monika M. Kuzma,
Heike Winter,
Paul Storer,
Ivan Oresnik,
Craig A. Atkins, and
David B. Layzell*
Biology Department, Biosciences Complex, Queen's University,
Kingston, Ontario, Canada K7L 3N6 (M.M.K., D.B.L.); Department of
Botany, North Carolina State University, Raleigh, North Carolina 27607 (H.W.); Botany Department, University of Western Australia, Nedlands,
WA 6907, Australia (P.S., C.A.A.); and Department of Biological
Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
(I.O.)
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ABSTRACT |
In legume nodules the
[O2] in the infected cells limits respiration and
nitrogenase activity, becoming more severe if nodules are exposed to
subambient O2 levels. To identify the site of
O2 limitation, adenylate pools were measured in soybean
(Glycine max) nodules that were frozen in liquid
N2 before being ground, lyophilized, sonicated, and
separated on density gradients of nonaqueous solvents
(heptane/tetrachloroethylene) to yield fractions enriched in bacteroid
or plant components. In nodules maintained in air, the adenylate energy
charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was lower in
the plant compartment (0.65 ± 0.04) than in the bacteroids
(0.76 ± 0.095), but did not change when the nodulated root system
was exposed to 10% O2. In contrast, 10% O2
decreased the bacteroid AEC to 0.56 ± 0.06, leading to the
conclusion that they are the primary site of O2 limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitochondria are localized in
O2-enriched microenvironments adjacent to intercellular
spaces, we propose that steep adenylate gradients may exist between the site of ATP synthesis (and ADP use) in the mitochondria and the extra-mitochondrial sites of ATP use (and ADP production) throughout the large, infected cells.
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INTRODUCTION |
Root nodules are the site of a beneficial symbiotic association
between legume plants and certain soil bacteria of the
Rhizobium or Bradyrhizobium genera. The plant
supplies the bacteria with an energy source (e.g. malate or succinate)
and, in turn, the bacteria reduce (fix) the atmospheric
N2 gas to
NH4+, providing it to the plant
for assimilation into amino acids, protein, and other essential
nitrogenous compounds. The nitrogenase enzyme responsible for
N2 fixation is O2 labile,
and, presumably to protect nitrogenase from damage, legume nodules have
evolved mechanisms to regulate their permeability to
O2 (Hunt et al., 1987
; Witty et al., 1987; Hunt
and Layzell, 1993). O2 in the infected cell is
maintained at an extremely low concentration (5-50
nM) compared with that in cells in equilibrium
with air (approximately 250 µM) (King and
Layzell, 1991; Denison et al., 1992; Kuzma et al., 1993). Hunt et al.
(1989) have shown that gradual increases in the external partial
pressure of O2 result in small (2%-20%) but
significant stimulations in nodule respiration and nitrogenase activity. Moreover, in nodules in which nitrogenase is inhibited due to
photosynthate deprivation, nitrate fertilization, or
Ar:O2 exposure, much greater stimulations
(50%-300%) in their metabolism can be achieved by increasing the
external partial pressure of O2 (Hartwig et al.,
1987; Vessey et al., 1988; Denison et al., 1992; de Lima et al., 1994).
Thus, the metabolism of legume nodules is thought to be limited by the
O2 supply at all times; by controlling permeability to O2 diffusion, nodules are able to
reduce further the supply of O2 to the infected
cells and thereby down-regulate metabolism.
For more than 30 years the predominant view has been that in vivo,
bacteroids are limited by O2 availability,
presumably by restricting aerobic respiration and thereby ATP supply to
nitrogenase (Bergersen, 1962, 1984; Dilworth, 1974; McDermott et al.,
1989; Werner, 1992). This view is supported by studies that show
a positive correlation between ATP pools and nitrogenase activity in
free-living, anaerobically grown diazotrophic bacteria, in isolated
bacteroids, or in bacteria induced to fix N2
under O2-limiting conditions (Upchurch and
Mortenson, 1980; Ching et al., 1981; Privalle and Burris,
1983).
However, the Km (O2)
for the terminal oxidases of nodule mitochondria (50-100
nM; Rawsthorne and LaRue, 1986; Millar et al., 1995) is greater than that for bacteroids (5-20
nM; Bergersen and Turner, 1993), suggesting that
at the low [O2] in the infected cells (5-50
nM), O2 would limit
mitochondrial respiration rather than bacteroid respiration. However,
the mitochondria appear to be localized adjacent to intercellular
spaces and therefore may experience higher [O2]
than the bacteria that are found farther away from the spaces
(Bergersen, 1994; Thumfort et al., 1994).
A common indicator of hypoxic metabolism in biological systems is the
relative size of the adenylate pools. In hypoxic cells the
concentration of ATP is typically lower, whereas ADP and AMP pools are
higher than those found in aerobic cells (Pradet and Raymond, 1983).
Consequently, values for the AEC (ATP + 1/2 ADP)/(ATP + ADP + AMP) are
0.80 or greater in aerobic cells but less than 0.75 in hypoxic cells.
Previous studies have shown that under optimal conditions, soybean
nodules have AEC values of 0.65 to 0.75 (de Lima et al., 1994; Oresnik
and Layzell, 1994), which decrease further by approximately 0.12 when
nodule metabolism is limited after stem girdling, nitrate
fertilization, or exposure to 10% O2 (de Lima et
al., 1994). In contrast, exposure to high O2
inhibited nitrogenase activity and increased the AEC by approximately 0.11 (de Lima et al., 1994).
To determine the site of hypoxic metabolism, nodules of soybean
(Glycine max) were frozen rapidly in liquid
N2, ground to a fine powder while frozen,
lyophilized, sonicated in nonaqueous solvents, and separated into plant
and bacteroid fractions using a nonaqueous density-gradient technique
(Gerhardt and Heldt, 1984; Riens et al., 1991) adapted for use with
soybean nodules. Under these conditions the distribution of metabolites
was not altered during the isolation procedure, a problem inherent in
all traditional aqueous methods for organelle separation (Farnden and
Robertson, 1980; Reibach et al., 1981). Adenylate concentrations were
assessed in the plant and bacteroid compartments of control nodules
maintained at an external [O2] of 21% (v/v)
and in nodules inhibited by exposure to 10% O2
for 3 min.
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MATERIALS AND METHODS |
Plant Culture
Soybean (Glycine max L. Merr. cv Maple Arrow) plants
were grown in gas-exchange pots in a plant growth chamber (model PGV36, Conviron, Winnipeg, Manitoba, Canada) as described previously (Kuzma
and Layzell, 1994). Plants were inoculated with Bradyrhizobium japonicum USDA 16 at planting time and were used for experiments 5 weeks after planting.
Experimental Treatments and Tissue Harvest
Four populations of soybean were used and each was divided into
two groups of 15 plants per group. The nodulated roots of the control
group were maintained at ambient conditions (21%
O2) before sampling, whereas the nodulated roots
of treated plants were switched from 21% O2 to
10% O2 in N2 for 3 min
before being frozen and sampled. The 10% O2
treatment is known to inhibit nitrogenase activity by increasing
O2 limitation (Hunt et al., 1987). Sampling involved rapidly uprooting the nodulated roots from the sand media and
immediately plunging them into liquid N2 (Oresnik
and Layzell, 1994). Nodules from all 15 plants within a group were
removed from the root system while frozen and ground in liquid
N2 using a mortar and a pestle until a very fine
powder was obtained (approximately 1 h). Two subsamples
(approximately 0.12 g each) of the homogenized nodule tissue were
removed for metabolite and protein analysis, respectively, whereas the
remaining tissue was lyophilized for 3 d under a vacuum (
100
kPa) in a flask maintained on an ice/methanol mixture (15°C) for
the first 24 h, then at room temperature for the remaining 2 d. A trap between the sample and the vacuum pump was maintained in
liquid N2 throughout this time. The dried tissue powder was then used for nonaqueous fractionation.
Nonaqueous Fractionation of the Nodule Tissue
A nonaqueous-fractionation method was developed for nodules as a
modification of a method designed for leaf tissue (Gerhardt and Heldt,
1984; Riens et al., 1991). The following steps were carried out in a
cold room (4°C): Lyophilized nodule tissue (approximately 0.6 g) was
sonicated using a microprobe (model 450 sonicator, Branson Ultrasonics,
Danbury, CT) in a dry-ice/heptane bath in 25 mL of dry (dried and
stored over Molecular Sieve 4A, Fisher Scientific) heptane and
tetrachloroethylene mixed at a ratio of 43:57 (v/v) to give 1.23 g
mL
1 density. The density was checked at 4°C
using calibrated hydrometers (Fisher Scientific). Sonication was for 12 min, during which time the material was exposed to four cycles, each
including 2 min of 5 s on, 10 s off and 1 min of 15 s
off, 2 s on. The sonication step, which was similar to that
carried out with leaf tissue in previous nonaqueous studies (Gerhardt
and Heldt, 1984; Stitt et al., 1989), was necessary to break the
electrostatic and other physical forces that tended to hold the dried
0.3- to 1.0-µ particles together within the nonaqueous solvents.
Through sonication a uniform suspension of the dried tissue particles
was obtained, thus making it possible to filter the samples and,
subsequently, to separate the particles by density-gradient
centrifugation.
The sonicated homogenate was filtered through 82-µm nylon mesh to
remove any large debris, pelleted by centrifugation at 3000 rpm in a
swing-out rotor (clinical centrifuge, International Equipment, Boston,
MA) for 10 min, and then resuspended in a dry solvent mixture (density
of 1.23 g mL1) to a final volume of 8.5 mL.
The homogenate was set aside in a closed tube to prevent condensation
of water vapor in the solvents, and an exponential density gradient of
dry heptane and tetrachloroethylene was prepared using a custom-made
glass gradient maker similar to that described previously (Soboll et
al., 1979). Thirty milliliters of a 41:59 (v/v)
heptane:tetrachloroethylene mixture (density of 1.24 g
mL1) was placed in the open reservoir of the
gradient maker; 25 mL of a 25:75 (v/v) mixture of the same solvents
(density of 1.4 g mL1) was placed in the
sealed mixing reservoir of the gradient maker. The gradient was created
by allowing the contents of the open reservoir to flow through the
sealed chamber, which was mixed rapidly using a magnetic stirrer. The
gradient was poured into 1- × 3.5-inch nitrocellulose centrifuge tubes
(Beckman) containing a 4-mL cushion of pure tetrachloroethylene
(density of 1.64 g mL1). The density of
the poured gradient ranged exponentially from 1.29 to 1.4 g
mL1, as shown in Figure
1B. Four milliliters (containing
67.1 ± 3.6 mg of protein) of the homogenate (density of 1.23 g mL1) was gently placed on top of each
gradient and two gradients were run at the same time for each nodule
harvest.
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| Figure 1.
A, Photograph of a nonaqueous
(heptane:tetrachloroethylene) gradient of lyophilized particles from
soybean nodules. The approximate location of each of the 10 fractions
is shown to the right of the photograph. The red color is due to Lb. B,
The approximate density of the liquid associated with each fraction of
the gradient that is shown in A. The gradient was poured onto a 4-mL
cushion of tetrachloroethylene having a density of 1.64 g
mL1, and the sample was loaded in a
heptane:tetrachloroethylene mixture having a density of 1.23 g
mL1.
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The gradients were centrifuged at 12,000 rpm for 2.5 h using a
rotor and an ultracentrifuge (models L8-55M and SW28, respectively, Beckman) maintained at 4°C. This resulted in equilibrium distribution of the components in the gradient according to their density. From each
gradient, 10 fractions (2-4.5 mL each) were collected, starting at the
top and using a single 5-mL glass pipette per gradient to minimize
total losses. The approximate locations of these fractions are shown in
Figure 1A.
As soon as each fraction was collected, its density was lowered by the
addition of approximately 3 to 6 mL of dry heptane before the tissue
was pelleted and then resuspended in 1 mL of pure dry heptane. Each
fraction was split between two Eppendorf tubes (0.5 mL/tube) containing
acid-washed sand. One tube was subsequently used for metabolite
analysis and the other for marker enzymes and protein analysis. The
subfractions were dried overnight at room temperature under a vacuum
(60 kPa) in a dessiccator with silica gel and candle wax.
Tissue Extraction and Metabolite and Marker Enzyme Analysis
Dried subfraction samples were extracted for metabolites in 1 mL
of ice-cold 10% HClO4 containing 2.5 mM EGTA by vortexing each tube for four cycles of 30 s
of agitation and 10 s on ice (to cool the samples between each
agitation). Resuspended subfractions were left on ice for 15 min before
pelleting by centrifugation (14,000 rpm) for 10 min and neutralizing
the supernatant with KOH. Adenylates (ATP, ADP, and AMP) were measured
in each fraction using enzyme-coupled assays, as described by Oresnik
and Layzell (1994).
The second set of dried subfractions was extracted for marker enzyme
and protein analysis in 0.5 mL of 0.25 mM phosphate buffer (pH 7.5, containing 0.5 mM EDTA, 0.5 mM DTT, 2 mM PMSF, 15% glycerol [v/v], and 0.1% Triton-X) by
vortexing the samples on acid-washed sand, as described for metabolite
extraction. Samples were held on ice for 10 min before adding 0.5 mL of
a 100 mM phosphate buffer (pH 7.5, containing 0.5 mM EDTA, 0.5 mM DTT, 2 mM PMSF,
15% glycerol [v/v], and 0.1% Triton X-100).
The HBD was assayed as described previously (Farnden and Robertson,
1980) and used as the marker enzyme for bacteroids. Preliminary studies
(data not shown) indicated that the distribution of HBD activity among
fractions was similar to that of Ala dehydrogenase, another bacterial
enzyme (Wiame et al., 1965), and to the reaction of a polyclonal
antibody made against the Fe component of Klebsiella pneumoniae nitrogenase (Oresnik, 1995). To identify an appropriate marker for the plant compartment, the following enzyme activities of
each fraction were assayed on a spectrophotometer (Milton Roy Spectronic 3000, Spectronic Instruments, Rochester, NY) using standard enzyme-coupled assays for PEPC (Gerhardt and Heldt, 1984) and
xanthine dehydrogenase (Atkins et al., 1980) as cytosolic markers, and
GDH as a mitochondrial marker (Yamaya et al., 1984). In
addition, Lb was assayed as a possible cytosolic marker using the
pyridine hemochrome method for heme (Appleby and Bergersen, 1980) and
an ELISA assay (Engvall and Perlmann, 1971) incorporating antibodies
against the soybean Lb apoprotein. The protein content of each fraction
was determined using a Bio-Rad protein assay kit.
Determination of Adenylate Contents of the Plant versus Bacteroid
Fractions
Because the distribution of marker enzymes was similar in the
fractions from the replicate gradients within each of the four nodule
populations, the proportions of each adenylate and marker enzyme were
averaged from the two gradients for each of the 10 fractions (numbered
from the top of the gradient). Therefore, four replicates were
available for subsequent calculations of the distribution of adenylates
in the bacteroid and plant compartments. These were carried out
individually for each of the four replicate gradients.
The proportion of adenylates (to the total nodule content) that could
be attributed to the bacteroid and plant compartments of the nodule was
determined using a computer program developed by Riens et al. (1991).
This program tests all of the possible combinations of metabolite
proportions found associated with "marker A" and "marker B" at
1% intervals (i.e. marker A = 1%, marker B = 99% ... marker A = 99%, marker B = 1%) against experimental data,
in this case the proportion of the metabolite distribution to the
marker enzymes in all of the fractions. This method assumes that the
metabolites and marker enzymes from the same subcellular compartment
segregate together into the various fractions of the gradient. The
best-fitting metabolite proportions between the bacteroid marker and
the plant cell marker with the experimental data, as predicted by the
computer program, were used to calculate the metabolite content of the
bacteroids and the plant cells' compartment of the nodule.
To calculate the percent recovery of each metabolite, the total
recovered from the gradient (in nanomoles per milligram of protein) was
divided by the amount (in nanomoles per milligram of protein) of that
metabolite measured in the rapidly frozen fresh nodule tissue. To
correct for losses during fractionation, the proportion of each
metabolite ascribed to the bacteroids or plant cells was multiplied by
the total content (in nanomoles per milligram of protein) of the
metabolite determined in the frozen nodule tissue that was not
lyophilized. This calculation assumes that any losses in metabolites
were not differentially associated with either the plant or the
bacteroid compartments.
Electron Microscopy of Bacteroid and Plant-Enriched Fractions
Aliquots of fractions 4 and 8 were prepared for electron
microscopy by fixing the dried material in aqueous 4% glutaraldehyde and then in 2% osmium tetroxide before dehydration in an acetone series and embedding in Spurr's resin (Spurr, 1969). Sections (0.1 µm) were stained with uranyl acetate and lead citrate and the
electron micrographs were obtained using a transmission electron microscope (model JEM 2000FXII, Jeol). Although rehydration of the
material in the glutaraldehyde solution may have modified disrupted
membranes, it was found to be necessary. In preliminary studies fixing
and substituting the dry material directly in 2% osmium tetroxide in
pure acetone at 80°C resulted in most of the material falling out
of the thin sections (data not shown).
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RESULTS |
Metabolite Content and AEC of Whole Nodules under Ambient and Low
O2
In rapidly frozen soybean nodules assayed directly for adenylates,
no significant differences were measured in adenylate pools between
nodules from plants maintained in air (control plants) and those in
which the nodulated roots were exposed to 10% O2 for 3 min (treated plants) (Table I). However, mean AEC
values calculated from individual samples were significantly different, being greater in the control (0.70 ± 0.01) than in the treated (0.61 ± 0.04) nodules (Table 1).
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Table I.
The effect of reducing the rhizosphere
[O2] from air to 10% O2 (3-min exposure) on
the pool sizes of adenylates in rapidly frozen soybean nodules that
were subsequently used for nonaqueous fractionation
Values are presented as means ± SE (n = 4).
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Identification of Marker Enzymes
When the frozen tissue had been ground, lyophilized, sonicated,
and fractionated on a nonaqueous gradient, the resultant profile showed
distinct bands or zones of red- and white-colored material (Fig. 1).
The gradients were separated into 10 fractions, as shown in Figure 1,
and aliquots of each were taken for analysis of potential marker
enzymes. Initially, HBD, Ala dehydrogenase, and the Fe protein of
nitrogenase were assayed as potential markers for bacteroids. HBD was
chosen because its separation was similar to that of the other enzymes
(data not shown), its activity was relatively high, and the assay was
simple.
A number of potential markers for the plant compartments were also
examined. All of these fractionated differently from HBD (Fig.
2), providing evidence that the gradient
was able to separate plant- and bacterial-enriched fractions. As a
cytosolic marker, PEPC was determined to be better than the heme of Lb,
because the heme of Lb and the apoprotein of Lb fractionated
differently from one another (Fig. 2A). Proportionately, more of the
heme of Lb than the apoprotein of Lb was associated with HBD,
suggesting that a small but significant amount of the heme in the
nodule may be in bacteroids, compromising the assumption that the heme of Lb marked the plant cytosol fraction. The apoprotein of Lb distribution resembled that of PEPC.
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| Figure 2.
The distribution of potential marker enzymes or
proteins for bacteroid or plant cell compartments obtained in each of
the 10 fractions that comprised a nonaqueous gradient of lyophilized
nodule tissue. A, A gradient in which assays were carried out for HBD,
the heme group of Lb (Lbheme), the apoprotein of Lb
(Lbprotein), PEPC, and xanthine dehydrogenase (XDH). B, A
gradient in which assays were carried out for PEPC and GDH.
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PEPC fractionated in much the same way as xanthine dehydrogenase (Fig.
2A) and GDH (Fig. 2B), showing that the nonaqueous gradient could
distinguish between bacteroid- and plant-enriched fractions, but could
not separate the plant subcellular components. Therefore, we used PEPC
as the marker enzyme for the entire plant fraction. Previous studies
(Robinson et al., 1996) have shown that PEPC is present in the cytosol
of both infected and noninfected cells, as well as cortical cells,
although to a lesser degree.
Recovery of Marker Enzymes and Metabolites from the Nonaqueous
Gradients
The recoveries of marker enzymes and adenylates from the
nonaqueous gradients were expressed as a percentage of the amount measured directly in extracts from the rapidly frozen nodules (Table
II). The HBD activity recovered from the gradient was
154% (treatment) and 220% (control) of that in fresh nodule tissue, indicating that the lyophilization, sonication, and exposure to nonaqueous solvents were able to elicit greater HBD activity than a
simple aqueous buffer extraction of fresh tissue. This finding is
consistent with the observation that maximum HBD activity from fresh
tissue required two or more passages through a French press at 16,000 p.s.i. units (Oresnik, 1995). All other marker enzymes and metabolites
that were assayed showed recoveries that varied from 85% to 129% of
that measured in the fresh tissue, and no mean values were
significantly different from 100% (Table II).
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Table II.
Recovery of marker enzyme activity and adenylates
from the nonaqueous gradients from the same population of plants
Values are presented as means ± SE (n = 4).
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Distribution of Marker Enzymes and Adenylates within the
Gradients
The distribution of HBD, PEPC, and protein was very similar in the
gradient fractions that were used to separate the lyophilized particulate components from the four control and in the four treated populations of the nodules (Fig. 3). The
HBD activity was highest in fraction 4 (Fig. 3A), whereas PEPC activity
peaked in fractions 8 and 9, with a lesser peak in fraction 4 (Fig.
3B). These activities correlated with the regions of intense red color
in the gradients, although a large amount of HBD activity was
associated with white-colored particulate material in fraction 3 (Fig. 1A).
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| Figure 3.
The proportional distribution of HBD (A), PEPC
(B), and protein (C) within the fractions taken from a nonaqeuous
gradient of lyophilized particles from soybean nodules of control
plants ( ) and of nodulated roots exposed to 10% O2 for
3 min ( ). Values are presented as the mean (±SE) of
four replicates, each replicate being the mean of two gradients that
were run simultaneously on nodule material harvested from a single
population of plants. Values within a fraction that were significantly
different (P = 0.05) between control and treated nodules are
marked with an asterisk.
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No significant differences were observed in the proportional
distribution of HBD activity between the control and treatment gradients (Fig. 3A). Similarly, PEPC and total protein distribution were not significantly different between treatments, except for the
protein fraction 7 (Fig. 3, B and C). These results were in contrast to
those for the proportional distribution of adenylates, where
significant differences were observed between control and treated
nodules in a number of fractions, particularly nos. 3, 5, 6, and 7 (Fig. 4). Although these differences were
subtle, they demonstrated that the 10% O2
treatment altered the adenylate pools in the nodule.
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| Figure 4.
The proportional distribution of ATP (A), ADP (B),
and AMP (C) within the fractions taken from a nonaqeuous gradient of
lyophilized particles from soybean nodules of control plants () and
from nodules of nodulated roots exposed to 10% O2 for 3 min (). Values are presented as the mean (±SE) of four
replicates. Values within a fraction that were significantly different
(P = 0.05) between control and treated nodules are marked with
asterisks.
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Electron Microscopy of the Gradient Fractions
Electron micrographs of the nonaqueous fraction that was enriched
in HBD (Fig. 5A) clearly showed the
presence of many bacteroids interspersed with amorphous material. In
contrast, micrographs of the plant-enriched fraction (Fig. 5B) showed
few bacteroids and appeared to contain large amounts of amorphous
material (presumably cytosol) and portions of organelles (presumably
mitochondria and plastid membranes). The bacteroids were better able to
maintain their structure than the plant organelles in spite of the
stresses to which the tissues were exposed during the nonaqueous
fractionation and preparation for microscopy.
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| Figure 5.
Electron micrographs of the nodule material from
fractions 4 (A) and 8 (B) of the nonaqueous gradient. Fraction 4 was
dominated by bacteroids, whereas in the material from fraction 8, it
was difficult to distinguish any particular cellular structures.
Bars = 2 µm (A) and 0.5 µm (inset and B).
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Adenylates and AEC in Plant and Bacteroids under Ambient and
Low-O2 Treatment
To obtain an estimate of how much of each metabolite was
associated with the bacteroid compared with the plant compartment, the
proportional distribution of the marker enzymes and metabolites from
gradients associated with individual plant populations were entered
into the computer program of Riens et al. (1991). The plant compartment
was estimated to contain 63% ± 3% and 56% ± 2% of the total
adenylates in the control group and in those treated with 10%
O2, respectively. These values were not
significantly different (P = 0.05) from one another and were
similar to those obtained in an earlier study (Oresnik and Layzell,
1994) that used an aqueous-fractionation technique to quantify the
distribution of total adenylates in fractions from soybean nodules.
The ATP, ADP, and AMP pool sizes calculated to exist in the bacteroid
and plant fractions are shown in Figure
6, A and B, respectively. Due to the
large variance, no significant differences were observed in any of the
pool sizes between control and treated plants. However, when AEC values
were calculated for plant and bacteroid compartments from each of the
four nodule populations, the AEC in bacteroids was found to be
significantly lower (P = 0.05) in the treated (0.56 ± 0.06)
than in the control (0.76 ± 0.05) nodules (Fig. 5C). In contrast,
the AEC in the plant fraction was not affected by the treatment and
remained at 0.65 ± 0.02. Therefore, in whole control nodules, the
relatively low AEC (0.70 ± 0.01) was a composite of a high
bacteroid AEC and a low plant AEC. The decrease in whole-nodule AEC
that occurred when nodules were exposed to 10%
O2 (Table I) was associated with a reduction in
the AEC of the bacteroid, not of the plant component of the nodule.
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| Figure 6.
The effect of exposing nodulated soybean roots to
10% O2 for 3 min on the pool sizes of ATP (), ADP
( ), and AMP ( ) in the bacteroid (A) and plant (B) compartments of
soybean nodules. These values were used to calculate the AEC ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) (C) for the bacteroid () and plant
( ) compartment of nodules and for whole nodules  . Values are
presented as the means (±SE) of four replicates, each of
which is the mean of two gradients that were run simultaneously using
nodule material harvested from a single population of plants.
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DISCUSSION |
The Nonaqueous Separation of Nodule Fractions
This study is the first report, to our knowledge, of the
nonaqueous fractionation of nodule tissue into its component parts to
identify in vivo pool sizes of key metabolites. By maintaining the
vacuum flask containing nodule tissue in an ice/methanol mixture (15°C) for the first 24 h of lyophilization, and by allowing a
full 3 d for lyophilization while maintaining a liquid
N2 trap in the vacuum line, it was possible to
avoid the large (about 60%) losses in the adenylate pools that
occurred in previous studies (Oresnik and Layzell, 1994). High
recoveries were also obtained for the marker enzyme PEPC (Table I). The
abnormally high (154%-220%) recovery for HBD was attributed to the
fact that high pressure is required to extract full HBD activity from
fresh bacteroids (Oresnik, 1995). Presumably, sonication of the
lyophilized tissue extracted a larger proportion of the enzyme activity
than the simple aqueous-buffer extraction of fresh, frozen nodules.
Assuming that there were no treatment effects on the ability of
sonication to extract HBD activity from bacteroids, the high percentage
of recovery should have no effects on the findings of this study.
Unlike aqueous-gradient fractionation of legume nodules (Farnden and
Robertson, 1980; Reibach et al., 1981), in which bacteroids are more
dense than most plant organelles, in the nonaqueous gradients developed
here, the bacteroid fractions were localized near the top of the
gradient and the plant fraction was recovered near the bottom. This was
confirmed not only by electron microscopy (Fig. 5), but by HBD activity
(Fig. 3A), Ala dehydrogenase activity, and localization of the Fe
protein of nitrogenase (data not shown). Presumably, grinding,
lyophilization, and sonication of frozen nodules resulted in dried
plant material that was more dense than that of the bacteroids.
The method described here achieved highly enriched plant fractions
(fractions 8 and 9) with little contamination from bacteroids, but was
less successful in achieving bacteroid-enriched fractions (fractions
4-6) that were free of plant material. This would be expected if some
dried symbiosome membranes and cytosolic material remained attached to
the bacteroids during fractionation. The fact that the cytosolic
apoprotein Lb marker (representing infected cells only) was
proportionately higher in the bacteroid-enriched fractions and lower in
the plant-enriched fractions than the cytosolic PEPC marker
(representing infected and noninfected cells) is consistent with
this proposition. Nevertheless, the separation that was achieved was
sufficient for the computer program to attribute reliably each
adenylate to the two cell compartments.
AEC and O2 Limitation in Control Nodules
The observation that active, control nodules of soybean had AEC
levels (0.70 ± 0.009; Table I) typical of hypoxic or anaerobic tissues (Pradet and Raymond, 1983) confirmed previous findings (de Lima
et al., 1994; Oresnik and Layzell, 1994). Typically, fully aerobic
tissues have AEC values that are 0.80 or greater (Pradet and Raymond,
1983). The low AEC in legume nodules has been provided as further
evidence that their respiration and nitrogenase activity is limited by
the availability of O2 (Hunt and Layzell, 1993).
Through nonaqueous fractionation, the low AEC of control nodules was
attributed primarily to the plant compartment (AEC = 0.65 ± 0.04), whereas the bacteroid compartment had an AEC (0.76 ± 0.05)
that was close to that found in fully aerobic organisms. Because the
nodule cortex contains less than 5% of the adenylates in whole nodules
(Oresnik and Layzell, 1994), and because 72% to 90% of the central
zone tissue is occupied by infected cells (Lin et al., 1988;
Dakora and Atkins, 1990), the adenylate pools measured in this
study probably reflected conditions in the bacteria-infected cells.
These findings are consistent with the observation that the terminal
oxidases in bacteroids have a higher affinity to
O2 (5-26 nM; Bergerson and Turner,
1990, 1993) than those in nodule mitochondria (50-100 nM;
Rawsthorne and LaRue, 1986; Millar et al., 1995). Thus, the relatively
low [O2] found within the infected cells (<60
nM; Kuzma et al., 1993) may be sufficient for bacteroid
metabolism but limiting for mitochondrial metabolism.
A major problem with this interpretation is the cytological evidence
that mitochondria are clustered around the gas-filled intercellular
spaces within the infected tissue zone of the nodule (Millar et al.,
1995), and mathematical models that predict large free-O2 gradients in this region of the cell
(Bergersen, 1994; Thumfort et al., 1994). Therefore, the mitochondria
within the infected cell may occupy a very different microenvironment
(100 to >1000 nM O2) than the
bacteroids (3-40 nM O2) with respect to O2 availability. If this is the case, it is
difficult to see how mitochondrial metabolism could be
O2 limited.
Adenylate Energy Charge and O2 Limitation in Nodules
Inhibited by 10% O2
If an O2 limitation within the plant
fraction is responsible for the low (i.e. <0.80) AEC in active,
N2-fixing nodules, then imposing a more severe
O2 limitation on the nodule should further reduce
the AEC of the plant fraction. This was not observed in the present
study. Three minutes of exposure to 10% O2
causes a rapid inhibition of nitrogenase activity and nodule
respiration (Hunt et al., 1987), a sharp reduction in infected cell
[O2] (King et al., 1988; Layzell et al., 1990),
and a decrease in the AEC of the whole nodule (Table I) (de Lima et
al., 1994). However, the AEC of the plant fraction was unaffected by
the 10% O2 treatment, whereas the AEC of
bacteroids declined sharply from 0.76 to 0.56 (Fig. 6).
These findings suggest that it is the bacteroids, not the plant
compartment of the nodule, that is the primary site of
O2-limited metabolism.
Adenylates and the Site of O2 Limitation in Soybean
Nodules
Low AEC values in plant tissues occur when the rate of ATP
synthesis does not keep up with the rate of ATP utilization. This may
or may not be due to an O2 limitation of nodule
metabolism.
For example, it is possible that the 10% O2
treatment decreased ATP demand within the plant compartment, thereby
offsetting any O2 limitation on mitochondrial
activity and accounting for the lack of a change in plant AEC. In the
infected cells the most important sink for ATP would be Gln synthetase,
an enzyme responsible for assimilating the
NH4+ produced by nitrogenase.
Because nitrogenase activity would be inhibited by the 10%
O2 treatment, the ATP demands of Gln synthetase would decline with time. However, this is not likely to account for the
results of the present study, because the pools of
NH4+ are large in nodules and
are probably not depleted during the 3-min exposure to 10%
O2. Support for this conclusion comes from studies (King, 1989; Walsh et al., 1989) that have examined pool sizes
of NH4+, amino acids, and
ureides in nodules following exposure to Ar:O2, a
treatment that stops NH4+
production and is therefore more severe than the 50% reduction in
NH4+ production associated with
10% O2 exposure. Following
Ar:O2 treatment, nodule
NH4+ and amino acid pools were
still approximately 70% of their initial levels after 60 min (King,
1989), and ureide pools were maintained for 20 to 30 min (King, 1989)
before they declined, with a half-time of about 2 h (Walsh et al.,
1989). It seems unlikely that there would be a significant reduction in
the plant's demand for ATP in
NH4+ assimilation during the
initial 3 min of exposure to 10% O2.
The simplest explanation for the lack of a 10%
O2 effect on plant AEC is that the plant fraction
is not a site of O2-limited metabolism in
nodules. If this is the case, why was the plant AEC so low (0.65) and
typical of that found in tissues having hypoxic metabolism? This could
be explained as a characteristic of the infected cells.
Bacteria-infected cells are much larger (having radii of approximately
30 µm) than most other plant cells (having radii of approximately
5-10 µm). The sinks for ATP within the plant compartment (e.g.
symbiosome ATPases or Gln synthetase) would occur throughout the entire
cell, in many cases at long distances from the mitochondria that are
clustered near the gas-filled spaces. The translocation of ATP from the
mitochondria to extra mitochondrial sites within the cell and the
translocation of ADP and AMP back to and into the mitochondria may be
the major factor limiting the rate of ATP synthesis in these cells.
This proposal may explain why the 10% O2
treatment did not affect the AEC of the plant fraction. It would help
to resolve an apparent discrepancy between the results of the present
study and those of mathematical models that have predicted that the
mitochondria may be in a microenvironment where
[O2] greatly exceeds the
Km (O2) of the
terminal oxidase (Bergersen, 1994; Thumfort et al., 1994).
When the [O2] around the nodule was decreased
to 10%, the sharp decline in the AEC of the bacteroid fraction was
most readily explained as an O2 limitation of ATP
synthesis. This treatment results in inhibition of nitrogenase activity
to 30% of initial values within 2 to 3 min (Hunt et al., 1987; de Lima
et al., 1994), and a corresponding decline in the AEC of the whole
nodule (de Lima et al., 1994), as shown in Table I.
Researchers have long assumed that bacteroids were the site of
O2-limited metabolism in legume nodules
(Bergersen, 1962, 1984; Dilworth, 1974; McDermott et al., 1989;
Werner, 1992). However, to our knowledge, this study is the first to
provide evidence that illustrates this in intact, attached nodules.
Although the mechanism of nitrogenase inhibition is unknown, it could
be attributed to a shortage of ATP in meeting the metabolic needs of
nitrogenase or to a direct inhibition of the nitrogenase enzyme by
ADP (Miller et al., 1986).
Nonaqueous Fractionation and the Study of Nodule Metabolism
The nonaqueous method described here is potentially a powerful
tool for the study of C and N metabolism in legume nodules. In addition
to its use in studies of adenylates, it could monitor changes in the
pool sizes of metabolites such as malate, PEP, pyruvate, or Gln, after
perturbations in O2 supply, carbohydrate availability, or N fixation, and thereby provide insight into the
factors that regulate subcellular pools of metabolites.
Preliminary studies indicate that it may be possible (with some
modifications to the gradient) to obtain fractions that are enriched in
individual organelles such as mitochondria and plastids. For example,
if separate mitochondrial and cytosolic fractions could be recovered,
it may be possible to test the hypothesis proposed here, that the
mitochondria are fully aerobic in nodules and that the low AEC in the
plant compartment is a result of the long diffusion path from the
mitochondria to the site of ATP utilization.
| |
FOOTNOTES |
1
This research was supported by a Natural
Sciences and Engineering Research Council (Canada) research grant to
D.B.L., an Australian Research Council grant to C.A.A., and an
Alexander von Humboldt Foundation (Germany) grant to H.W.
*
Corresponding author; e-mail layzelld{at}biology.queensu.ca; fax
1-613-545-6617.
Received September 15, 1998;
accepted October 23, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AEC, adenylate energy charge.
GDH, glutamate
dehydrogenase.
HBD, hydroxybutyrate dehydrogenase.
Lb, leghemoglobin.
PEPC, PEP carboxylase.
| |
ACKNOWLEDGMENTS |
We would like to thank Drs. Cyril Appleby and David Goodchild
for their gift of antibodies to soybean Lb apoprotein, Drs. David Day
and Fraser Bergersen for useful criticisms of the manuscript, Robert
Campbell for construction of the gradient maker, Dr. Stephen Hunt for
photographing the gradient, and Natalie Fletcher for assistance with
the electron microscopy.
| |
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Abstract
Introduction