Plant Physiol. (1998) 117: 385-395
Effects of Oxygen on Nodule Physiology and Expression of Nodulins
in Alfalfa1
Keith L. Wycoff2,
Stephen Hunt,
Michael B. Gonzales,
Kathryn A. VandenBosch,
David B. Layzell, and
Ann M. Hirsch*
Department of Molecular, Cell and Developmental Biology (K.L.W.,
A.M.H.), and Molecular Biology Institute (A.M.H.), University of
California, 405 Hilgard Avenue, Los Angeles, California 90095-1606; University of
California, 405 Hilgard Avenue, Los Angeles, California 90095-1606Department of Biology, Queen's University, Kingston, Ontario, Canada
K7L 3N6 (S.H., D.B.L.); and Department of Biology, Texas A&M
University, College Station, Texas 77843-3258 (M.B.G., K.A.V.)
 |
ABSTRACT |
Early
nodulin 2 (ENOD2) transcripts and protein are specifically found in the
inner cortex of legume nodules, a location that coincides with the site
of a barrier to O2 diffusion. The extracellular glycoprotein that binds the monoclonal antibody MAC236 has also been
localized to this site. Thus, it has been proposed that these proteins
function in the regulation of nodule permeability to O2
diffusion. It would then be expected that the levels of ENOD2 mRNA/protein and MAC236 antigen would differ in nodules with different permeabilities to O2. We examined the expression of ENOD2
and other nodule-expressed genes in Rhizobium
meliloti-induced alfalfa nodules grown under 8, 20, or 50%
O2. Although there was a change in the amount of MAC236
glycoprotein, the levels of ENOD2 mRNA and protein did not differ
significantly among nodules grown at the different [O2],
suggesting that neither ENOD2 transcription nor synthesis is involved
in the long-term regulation of nodule permeability. Moreover, although
nodules from all treatments reduced their permeability to
O2 as the partial pressure of O2
(pO2) was increased to 100%, the levels of extractable
ENOD2 and MAC236 proteins did not differ from those measured at the
growth pO2, further suggesting that if these proteins are
involved in a short-term regulation of the diffusion barrier, they must
be involved in a way that does not require increased transcription or
protein synthesis.
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INTRODUCTION |
Symbiotic N2 fixation is dependent upon
nitrogenase, a very O2-labile enzyme. To protect
nitrogenase from inactivation by O2, the
[O2] in the infected cells of functional legume
nodules is maintained at a very low level. Through leghemoglobin
oximetry, infected cell [O2] has been measured
at 30 to 50 nM in active N2-fixing
nodules (Kuzma et al., 1993
), whereas the [O2]
in soil water in equilibrium with the atmosphere is about 260 µM. The low [O2] in the infected
cells is maintained by a combination of a high rate of bacteroid and
mitochondrial respiration as well as a barrier to
O2 diffusion thought to be located in the nodule inner cortex (also known as nodule parenchyma; van de Wiel, 1990b) (for
review, see Hunt and Layzell, 1993). We will use the terms "nodule
inner cortex" and "nodule parenchyma" interchangeably in this
paper.
The nodule diffusion barrier was first measured directly by Tjepkema
and Yocum (1974), who inserted an O2
microelectrode into soybean nodules and found that the
[O2] dropped sharply near the region of the
common endodermis. O2 electrodes have since been used to confirm the presence of a barrier to O2
diffusion near the interface between the nodule cortex and central
infected zone in pea, French bean, and alfalfa nodules (Witty et al.,
1987; Masepohl et al., 1993). This barrier consists of layers of
parenchyma cells, some of which (i.e. the boundary layer), have
radially aligned cell walls and very few, small intercellular spaces
(Parsons and Day, 1990). Because the diffusion coefficient of
O2 in air is about 10,000 times greater than in
water, and because O2 solubility in water is only
0.03 of that in air, models of gas exchange in nodules predict that a
water-filled barrier of about the same thickness as the boundary layer
would be sufficient to produce the observed diffusion barrier (Sinclair
and Goudriaan, 1981; Hunt et al., 1988).
Nodule permeability to O2 diffusion is controlled
by short-term physiological mechanisms and long-term developmental
adaptations (Hunt and Layzell, 1993). The latter include changes in
nodule anatomy as well as changes in the types or abundance of proteins synthesized in the region functioning as the nodule diffusion barrier.
When legume roots are grown at subambient pO2,
intercellular spaces in the nodule inner cortex are larger and more
abundant. Also, this zone increases or decreases in cell number in
proportion to the rhizosphere pO2 present during
growth in many plants (Dakora and Atkins, 1989, 1990a, 1990b, 1990c,
1991; Parsons and Day, 1990; James et al., 1991; Arrese-Igor et al.,
1993; Atkins et al., 1993).
Short-term physiological regulation of nodule permeability to
O2 diffusion occurs within a few minutes
following changes in rhizosphere pO2 (King et
al., 1988) or other physiological treatments (Hunt and Layzell, 1993).
Such a rapid adjustment cannot be accounted for by structural changes
or by changes in gene expression. The mechanism responsible for
short-term diffusion control remains a topic of conjecture (Thumfort et
al., 1994).
We are interested in the molecular mechanisms underlying the
establishment of the diffusion barrier and its ability to adjust during
development to external pO2. At least three
proteins have been localized specifically to the nodule inner cortex,
which suggests that they may play a role in the regulation of the
diffusion barrier. One of these proteins is a glycoprotein that has
been identified by its reaction to the monoclonal antibody MAC236 and related antibodies (VandenBosch et al., 1989). The MAC236
cross-reacting glycoprotein has been immunolocalized primarily in the
cell wall and intercellular spaces of the nodule parenchyma in pea and
soybean nodules (VandenBosch et al., 1989). The amount of this
glycoprotein is related to the rhizosphere pO2
under which the nodules are grown: compared with controls, more of the
MAC236 cross-reacting glycoprotein was found occluding intercellular
spaces in nodules grown at higher [O2]
(40-50%), whereas less was found in nodules grown at lower
[O2] (10%) (James et al., 1991; Iannetta et
al., 1993b). Exposing lupin nodules to 20 mM
KNO3 for 2, 4, or 6 d decreased nodule
permeability to O2 and also increased the
abundance of the MAC236 cross-reacting glycoprotein within the
intercellular spaces of the nodule inner cortex (Iannetta et al.,
1993a).
Another protein postulated to be involved in regulation of the
diffusion barrier is ENOD2 (Nap and Bisseling, 1990).
GmENOD2 transcripts accumulate from early stages of soybean
nodule development, specifically in the cells of the nodule parenchyma
(Franssen et al., 1987; van de Wiel et al., 1990b). Transcripts for
ENOD2 homologs are found in nodules of pea, alfalfa, lupin,
cowpea, common bean, and Sesbania rostrata (Dickstein et
al., 1988; Szczyglowski and Legocki, 1990; van de Wiel et al., 1990a,
1990b; Padilla et al., 1991; Trese and Pueppke, 1991; Dehio and de
Bruijn, 1992), at a location comparable to that described for pea (van
de Wiel et al., 1990a, 1990b; Allen et al., 1991) except for lupin
nodules (W.M. Karlowski, personal communication). Antibodies raised
against a synthetic ENOD2 peptide have since been used to
immunolocalize ENOD2 to intercellular spaces of inner cortical cells of
pea and soybean nodules (D.J. Sherrier, G.S. Taylor, and K.A.
VandenBosch, unpublished results). Proteins that are immunoreactive
with MAC236 and the putative ENOD2 protein run at different apparent
Mrs, indicating that the two are independent
proteins even though they colocalize in the nodule parenchyma. As
additional support for their distinctiveness, immunoaffinity-purified
ENOD2 does not cross-react with MAC236. Moreover, the 236 protein
localizes to infection threads as well as intercellular spaces in
contrast to ENOD2, which is found only in intercellular spaces (D.J.
Sherrier, G.S. Taylor, and K.A. VandenBosch, unpublished results).
A third protein, peanut lectin, has been localized to the intercellular
spaces of peanut nodule parenchyma cells, but not to those of boundary
layer cells (VandenBosch et al., 1994). This lectin was also detected
in the vacuoles of the nodule parenchyma cells and to a lesser extent
within the symbiosome lumen of infected cells (VandenBosch et al.,
1994).
In the present study we examined the ability of alfalfa nodules to
adapt to changes in pO2 and tested whether
adaptation to high or low pO2 is correlated with
changes in nodulin gene expression. We hypothesized that the expression
of proteins such as ENOD2 or the MAC236 cross-reacting glycoprotein
might vary with the rhizosphere pO2 that was
present during nodule development.
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MATERIALS AND METHODS |
Plant Growth and Nodulation Conditions
Alfalfa (Medicago sativa cv Iroquois) seeds were
planted in plastic growth pots (volume equals 750 mL) that could be
sealed for open-circuit gas-exchange measurements. Each pot contained two to four plants grown in silica sand. Plants were grown at a
constant temperature of 20°C, and a photon flux density of 400 µmol
quanta m
2 s1 during a
16-h photoperiod. Pots were flushed twice daily with a modified
Hoagland solution containing 5 mM potassium nitrate to
inhibit spurious nodule formation. After 4 weeks of growth, the pots
were flushed with water and inoculated with a broth culture of
Rhizobium meliloti strain 1021. An acrylic lid was sealed
onto the pots using a flexible sealant (Qubitac, Qubit Systems, Inc., Kingston, Ontario, Canada). The lid had a slit to accommodate the
stems, and the stems were sealed to the lid with sealant. The lid was
fitted with a subseal through which nutrient solution could be
injected. Each pot received 10 mL of N2-free
nutrient solution twice daily. The lid was also fitted with a gas port through which 8, 20, or 50% O2 in
N2 was supplied continuously at about 50 cm3 min1. Gases were
humidified before entering the pots to prevent drying of the roots.
Gases were vented through a gas exit port at the base of the pot and
this port also served as a drain for excess nutrient solution.
[O2] in the effluent gas was monitored
periodically to ensure that a stable pO2 was
supplied to each treatment. Plants were used in gas-exchange
experiments 20 d after exposure to each O2
regime.
Gas-Exchange Measurements
Measurements of gas exchange from nodulated roots were made using
a computer-controlled, open-flow gas-exchange system as described
previously (Hunt et al., 1989). On initial attachment to the system,
plants were exposed to a gas mixture containing N2:O2 at the
pO2 at which they were grown without exposure
to atmospheric pO2.
When H2 evolution in
N2:O2 was stable, the gas
mixture was switched to Ar:O2 while maintaining a
constant pO2. The peak rate of
H2 evolution in Ar:O2 was
taken as a measure of TNA (Hunt et al., 1987). The rate of
CO2 evolution was taken as a measurement of
respiration rate. Immediately after TNA was measured, the
pO2 in the Ar:O2 mixture
was programmed to double every 20 min until 100%
O2 was reached. For example, in the 8%
O2 treatment, pO2 was
increased from 8 to 16%, 16 to 32%, and 32 to 64% in linear ramps,
each of 20-min duration, and then increased from 64 to 100% in 11.25 min. In the 20% O2 treatment,
pO2 was increased from 20 to 40% and from 40 to
80% in two linear ramps, each of 20-min duration, and then from 80 to
100% in 5 min. In the 50% O2 treatment,
pO2 was increased from 50 to 100% in a single
20-min ramp. These changes in pO2 ensured that
the nodulated roots were exposed to the same relative change in
O2 gradient between the rhizosphere and nodule
interior during elevation of pO2.
CO2 and H2 evolution were
monitored continuously during the increase in pO2, and the maximum rate of
H2 evolution was taken as a measurement of PNA
(Diaz del Castillo et al., 1992).
After the gas-exchange measurements the nodulated roots were removed
from their growth pots, rinsed rapidly to remove growth media, quickly
blotted dry, and weighed. The roots and nodules were immersed together
in liquid N2, and the nodules were picked frozen
and subsequently stored at 
80°C until extracted for protein and RNA
analysis.
RNA Isolation and Northern Hybridization
Total RNA was isolated from nodules 21 d postinoculation
using RNA STAT-60 (Tel-Test "B", Inc., Friendswood, TX). The RNA was size-fractionated on formaldehyde agarose gels (Sambrook et al.,
1989) with 5 µg of RNA loaded per lane. After transfer to Nytran
membranes (Schleicher & Schuell), blots were hybridized with
-[32P]dCTP-labeled DNA probes. The probe for
ENOD2 transcripts was a 298-bp EcoRI fragment
from the plasmid A2ENOD2 (Dickstein et al., 1988). The probe for
ENOD40 mRNAs was a 580-bp PstI fragment from the
plasmid MsENOD40-2 (Asad et al., 1994). The probe for leghemoglobin
mRNA was a 1.0-kb fragment from the plasmid MsLb3 (Löbler and
Hirsch, 1992), and the probe for R. meliloti nifHDK transcripts was a 4-kb fragment from the plasmid pRmR2 (Ruvkun and
Ausubel, 1981). Blots were also hybridized with Msc27
(Kapros et al., 1992) as an internal control to standardize loading.
Protein Extraction and Western Blotting
For extracts of total soluble protein, nodules were ground in
ice-cold homogenization buffer (50 mM Tris, pH 7.5, 0.5 M Suc, 10 mM DTT, and 5 mM PMSF) in
1.5-mL microcentrifuge tubes using a plastic pestle (Kontes, Hayward,
CA) attached to a portable electric drill. The nodules were ground with
5 to 10 µL of buffer and 0.3 mg of polyvinylpolypyrolidone per
milligram of nodule (wet weight). The tubes were centrifuged at 10,000 rpm at 4°C for 10 min and the supernatants were removed to new tubes.
The extracts were stored frozen at 20°C. After aqueous extraction, some nodules were subjected to harsher extraction procedures (Fry, 1988). The protein concentrations of the supernatants were determined by the Bradford assay (Bio-Rad) using BSA as a standard. For
competitive ELISA, a different extraction buffer consisting of 40 mM Tris (pH 8.0), 3 mM EDTA, and 1 mM PMSF was used because DTT interfered with antibody
binding in the competition assays.
Extracts containing 10 to 20 µg of soluble protein were subjected to
SDS-PAGE (Laemmli, 1970) using 12.5% acrylamide gels, then transferred
to nitrocellulose in a Transblot apparatus (Hoefer Scientific Products,
San Francisco, CA) containing transfer buffer (25 mM Tris,
192 mM Gly, and 20% methanol, pH 8.3) at 100 mA overnight at 4°C. Blots were stained with Ponceau S (Sigma) to locate molecular mass markers, and then incubated with TBS (20 mM Tris, pH
7.5, and 180 mM NaCl) plus 1% BSA for 1 h prior to
probing with antibodies. Blots were sealed in plastic pouches with 10 mL of antibodies diluted in TBS plus 1% BSA. Affinity-purified
antibody against ENOD2 (D.J. Sherrier, G.S. Taylor, and K.A.
VandenBosch, unpublished results) was diluted to 1 µg/mL. MAC236
(gift of N. Brewin, John Innes Institute, Norwich, UK) was diluted
1:100 from hybridoma culture supernatant. Anti-leghemoglobin (gift of
C. Vance, University of Minnesota, St. Paul) was diluted 1:1000, and
cross-reacting antibodies were first removed by incubating the diluted
antiserum with a nitrocellulose filter blotted with an alfalfa root
extract. A monoclonal antibody against ribosomal protein P0 (Uchiumi et al., 1990) (gift of T. Uchiumi, Niigata, Japan) was diluted 1:200.
The filters were incubated with antibody solution at 4°C with gentle
shaking overnight, and then washed once with TBS, three times with TBS
plus 0.05% Nonidet P-40, and once with TBS (5 min each wash). The
filters were then incubated with the appropriate secondary antibody
diluted 1:1000 in TBS plus 1% BSA. Goat anti-rabbit IgG alkaline
phosphatase conjugate was used to detect anti-ENOD2 and
anti-leghemoglobin. Goat anti-rat IgG alkaline phosphatase conjugate
was used to detect MAC236, and goat anti-mouse IgG alkaline phosphatase
conjugate was used to detect anti-ribosomal protein P0. All secondary
antibodies were affinity purified (Sigma). The filters were incubated
with secondary antibody for 4 to 6 h at 4°C, washed as before,
and then incubated in color development buffer until color was
apparent. Color development buffer consisted of 50 mM
Tris (pH 9.0), 50 mM NaCl, 2.5 mM
MgCl2, 330 µg/mL nitroblue tetrazolium, and 165 µg/mL
5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt. The
filters were rinsed with water and allowed to dry before being
photographed.
Quantitative Competitive ELISA
For quantitative competitive ELISA, nodule extract was diluted in
PBS to 5 µg protein/mL. Aliquots of 50 µL were loaded into wells of
microtiter plates (Immulon IV, Dynex, Chantilly, VA), which were stored
overnight at 4°C. Unbound antigen was removed and plates were
incubated for 1 h at 25°C with 100 µL per well of blocking
solution (PBS plus 1% BSA). Blocking solution was removed and 25 µL
of competitor solution was added followed by 25 µL of antibody
solution. Nodule extracts to be tested were usually used at 2 µg
protein/mL when testing for ENOD2 and 10 µg protein/mL when testing
for the MAC236 glycoprotein. The anti-ENOD2 antibody was used at a
final concentration of 0.25 µg/mL, and the MAC236 antibody was used
at a final dilution of 1:100. These antibody concentrations were
previously determined to give about one-half saturation binding.
To make the ELISA quantitative, a standard curve of competitor was used
on each plate. Competitor for ENOD2 was a 21-amino acid synthetic ENOD2
peptide (POHEKPOHENTPOEYQPOHEK, where O = Hyp; D.J. Sherrier, G.S.
Taylor, and K.A. VandenBosch, unpublished results) at 5 to 50 ng/mL.
This peptide was synthesized based on the deduced amino acid sequence
of a PsENOD2 clone (van de Wiel et al., 1990b), with a predicted
pattern of posttranslational hydroxylation based on known Pro-rich
proteins (Kieliszesky and Lamport, 1994). The repetitive motifs PPHEK
and PPEYQ are highly conserved among predicted amino acid sequences of
ENOD2 genes in legumes, including alfalfa (Dickstein et al., 1988). A
competitor for the MAC236 glycoprotein was an extract from nodules
grown at 8% O2, at 5 to 40 µg protein/mL.
Controls included wells without competitors. Plates containing
competitor/antibody mixtures were incubated overnight at 4°C. Unbound
antibody was removed and the plate was washed three times with PBS.
Secondary antibody solution, consisting of affinity-purified goat
anti-rabbit (for anti-ENOD2) or anti-rat (for MAC236) IgG alkaline
phosphatase conjugate (Sigma) diluted 1:1000 in PBS plus 1% BSA, was
added (50 µL/well) and the plate was incubated for 4 to 6 h at
4°C. The secondary antibody solution was removed and the plate was
washed four times with TBS. The substrate solution was then added and
the plates were incubated at 37°C until a yellow color developed. The
substrate solution consisted of 104 phosphatase substrate
(p-nitrophenyl phosphate, disodium, and hexahydrate; Sigma)
at 1 mg/mL in 1 M diethanolamine, pH 9.8, 0.5 mM MgCl2. The reaction was stopped by
the addition of 100 µL of 1 M NaOH.
A405 was assayed on a plate reader.
Straight line standard curves were generated by a logit-log
transformation of the data (Signorella and Hymer, 1984). The logit Y was calculated as:
![<UP>logit </UP><IT>Y</IT><UP> = ln(</UP><IT>Y</IT><UP>/[1 − </UP><IT>Y</IT><UP>])</UP>](/content/vol117/issue2/fulltext/385/img001.gif)
|
(1)
|
where Y = B/B0 (B
is the absorbance at some competitor concentration, and B0
is the mean absorbance of the uncompeted controls). The linear
competitor concentration standard curve is described by the equation:
|
(2)
|
where X is the concentration of competing antigen, and
m and b are constants determined by a
least-squared linear regression of the logit-log transformed data. The
concentration of competing antigen in the ENOD2 assay was expressed in
terms of nanograms of ENOD2 peptide equivalents per milliliter. The
concentration of competing antigen in the MAC236 assay was expressed in
terms of arbitrary units based on the standard curve of 8% nodule
extract.
Differences between measurements of ENOD2 and MAC236 were analyzed for
significance using a one-sided Student's t test.
Light-Microscopic Analysis of Nodules
Nodules were harvested from plants grown under different
O2 regimes as described above, placed in buffered
fixative, and subjected to a slight vacuum to facilitate infiltration
of fixative. The fixative contained 4% paraformaldehyde and 1%
glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.0. The nodules were fixed overnight on ice at ambient pressure. They were
then dehydrated in an ethanol series and embedded in London Resin White
as previously described (Sherrier and VandenBosch, 1994). Semithin
sectioning, immunolabeling, and silver enhancement were also conducted
as previously described (Sherrier and VandenBosch, 1994), using
affinity-purified anti-ENOD2 IgGs at a concentration of 5 mg/mL as a
primary antibody. Labeled sections were counterstained with 1% (w/v)
aqueous basic fuchsin to impart contrast to the embedded tissue.
Nodules were also fixed in formaldehyde-acetic acid-alcohol and
embedded in paraffin as previously described (McKhann and Hirsch,
1993). Slides were stained for starch with the periodic acid Schiff's
reaction (Jensen, 1962). Photographs were taken on a Zeiss Axiophot
microscope using Ektachrome Tungsten 160 film, and prepared using Adobe
Photoshop.
| |
RESULTS |
Gas-Exchange Measurements
TNA and respiration rates were not significantly different in
nodulated roots grown at 8, 20, and 50% O2 when
assayed at their respective growth pO2 (Table
I). Also, the PNA was not significantly different among the three treatments. PNA represents the nitrogenase activity attainable when nitrogenase-linked respiration is not limited
by O2. Thus, the degree of
O2 limitation of nitrogenase activity in a
nodulated root can be indexed by the ratio of TNA to PNA. This ratio is
termed OLCN (Diaz del Castillo et al., 1992), and
the lower the value, the greater the degree of O2
limitation. All three treatments had OLCN values
close to 0.9 (Table I), indicating that despite being grown at
different pO2, a similar, small degree of
O2 limitation of nitrogenase activity occurred in
each.
Although PNA values were similar in nodulated roots grown at different
pO2, nitrogenase sensitivity to elevated
pO2 differed among treatments. At all three
pO2, nitrogenase activity declined during the
initial few minutes of the O2 ramp (Fig.
1A). This was attributed to an Ar-induced
inhibition of nitrogenase activity. After the initial Ar-induced
decline in nitrogenase activity, activity in all three treatments
increased above initial levels. PNA values were attained at 60, 80, and
90% O2 in the nodulated roots grown at 8, 20, and 50% O2, respectively. In the 8%
O2 treatment, PNA was followed by a decline in
nitrogenase activity at pO2 above 60%. In the
other two treatments, there were no significant differences between the
PNA values and the nitrogenase activities measured at 100%
O2.
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| Figure 1.
Nitrogenase activities (A) and respiration rates
(B) of nodulated roots of alfalfa exposed to gradual increases in
pO2 in a balance of Ar. Increases in pO2 were
from initial growth pO2 of 8% ( ), 20% ( ), and 50%
( ). Each point represents the mean of four replicates ± representative SEs. Absolute initial values for TNA and
respiration rates are shown in Table I.
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|
In general, the ramps in [O2] affected the
respiration rates of nodulated roots in a fashion similar to the effect
on nitrogenase activities in all three pO2
treatments (Fig. 1B). In each case, an initial decline in respiration
rate was followed by a recovery as the O2 ramps
progressed. However, only in the case of the 8% O2 treatment did the respiration rate increase
significantly above initial levels. Also, this treatment was the only
one to show a decline in respiration rate at pO2
values greater than that at which the maximum respiration rate was
reached.
Expression of Nodulin Genes
Total RNA was extracted from frozen alfalfa nodules that developed
under conditions of 8, 20, or 50% O2. There was
little difference in the amount of total RNA per gram fresh weight
recovered from the three treatments (1.4, 1.32, and 1.23 mg/g fresh
weight for 8, 20, or 50% O2-grown nodules,
respectively). Northern-blot analysis (Fig.
2A) revealed that steady-state levels of
ENOD2 mRNA remained constant between 8 and 50%
pO2. Levels of leghemoglobin and
ENOD40 mRNAs were also not affected by
[O2], nor was the expression of the R. meliloti nifHDK genes (Fig. 2B).
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| Figure 2.
RNA blot of total RNA from alfalfa nodules grown
under 8, 20, or 50% O2. A, Blot probed for
MsENOD2 and Msc27 mRNAs. B, Blots probed
for MsENOD40, MsLb3, and R. meliloti nifHDK mRNAs. Five micrograms of total nodule RNA
was loaded per lane.
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|
Immunological Assays of Nodulins
Because there was no apparent effect of O2
level on the level of accumulation of nodulin transcripts, we assessed
the abundance of leghemoglobin, ENOD2, and the MAC236 glycoprotein in
nodule extracts using immunological techniques. Although total soluble protein content as a fraction of nodule fresh weight (in milligrams per
gram) was highest in the control nodules (25.7 versus 19.6% for
nodules developed under conditions of 8% O2 and
17.4% for 50% O2-grown nodules), all
immunological comparisons were based on using the same amount of
protein from each extract. Western blots of total soluble protein from
nodules grown in 8, 20, or 50% O2 were probed
with rabbit anti-leghemoglobin antiserum, rabbit anti-ENOD2 antiserum,
and the rat monoclonal antibody MAC236. Anti-leghemoglobin antiserum
showed significant cross-reaction to proteins other than leghemoglobin
(data not shown). Cross-adsorption of this antiserum to alfalfa root
proteins removed much of the cross-reaction, and when
subsequently used, it was clear that the quantity of leghemoglobin was
similar under all pO2s tested (Fig.
3A).
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| Figure 3.
Immunological detection of different proteins on
western blots. A, Leghemoglobin. Blot probed with anti-leghemoglobin
antiserum that was previously exposed to alfalfa root proteins to
remove cross-reacting antibodies. The leghemoglobin band is indicated by the arrow at 15 kD. Twenty micrograms of total protein was loaded
per lane. B, ENOD2. Extracts from nodules grown at 8, 20, and 50%
O2 were subjected to electrophoresis on a 7.5% acrylamide gel and blotted onto nitrocellulose. The blot was probed with anti-ENOD2 antibody at 1 µg/mL. The major ENOD2 protein band is indicated by the arrow. C, MAC236 glycoprotein. Blot probed with MAC236
at 1:100 and monoclonal anti-ribosomal protein P0 at 1:200 dilution.
The MAC236 glycoprotein is indicated by the black arrow, and the P0
protein (which served as a loading control) is indicated by the white
arrow.
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Western blots of nodule extracts were probed with the anti-ENOD2
antiserum. The antibody reacted primarily to a broad band at about 155 kD and one or two smaller bands at about 140 and 120 kD (Fig. 3B). In
some extracts, a fainter band could be seen at about 70 kD (data not
shown). There was little difference in the intensity or pattern of
bands between nodules grown at 8 and 50%; a slightly higher amount of
ENOD2 antigen was detected in the 20%-grown nodules (Fig. 3B).
Nodule extracts were also assayed for ENOD2 protein by an indirect
competitive ELISA with anti-ENOD2 antibodies (Fig.
4A), using a synthetic 20-amino acid
peptide as a standard. Comparison of the competition by nodule extracts
with the standard curve allowed us to measure the quantity of ENOD2 in
units of "peptide equivalents." This method more accurately
quantifies relative antigen concentrations than the minimal dilution
method employed by Iannetta et al. (1993b). There was no significant
difference in the relative quantity of extractable ENOD2 protein
between nodules grown at 8 and 20% pO2, or
between those grown at 8 and 50% pO2. However,
there was approximately 25% less ENOD2 in extracts of nodules grown at
50% pO2, and this was significant at the 99% confidence level determined by using a one-sided Student's
t test.
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| Figure 4.
Quantification of MAC236 glycoprotein and ENOD2.
A, Relative quantity of ENOD2 in nodules. B, Relative quantity of
MAC236 glycoprotein in nodules. Bars represent the mean of three to
four assays on each of six individual nodules. Error bars are
SD for the six nodules.
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Western blots of nodule extracts were probed with the monoclonal
antibody MAC236 together with a monoclonal antibody against ribosomal
protein P0 (used as a loading control). A glycoprotein recognized by
MAC236 was present in alfalfa nodules and increased in quantity with
increasing pO2 (Fig. 3C). It should be noted that
this, and similar antibodies, also react with protein in infection
threads (VandenBosch et al., 1989). Our study did not determine whether
the increase in alfalfa nodules was in nodule parenchyma cells,
infection thread-containing cells, or both. The apparent molecular mass
of the MAC236 glycoprotein in alfalfa nodules was 195 kD. This was
somewhat smaller than the size of 240 kD reported for lupin nodules (de
Lorenzo et al., 1993), but larger than the 95 kD reported for pea
nodules (VandenBosch et al., 1989). After aqueous extraction of the
nodules, the remaining cell wall debris were subjected to harsher
extraction procedures, but only a small amount of additional MAC236
glycoprotein was recovered (data not shown).
Competitive ELISA with MAC236 (Fig. 4B) revealed that the MAC236
glycoprotein increased 2.4-fold in nodules grown at 20% compared with
those grown at 8% O2 (significant at the 99%
confidence level). There was no significant difference in the abundance
of the MAC236 glycoprotein between nodules grown at 20 and 50%
O2 based on the large SEs. However,
as shown in the western-blot analysis (Fig. 3C), there was a trend
toward increased MAC236 protein levels as the
[O2] was raised.
All of the nodules from the experiments described above were from
plants harvested after O2 ramping. In a separate
gas-exchange experiment, nodules taken from plants harvested before
O2 ramping were compared with nodules from plants
harvested after O2 ramping. MAC236
glycoprotein was assayed in nodules that developed under 20%
O2, and ENOD2 was assayed in nodules that
developed under 8 and 50% O2. In no case was
there a significant difference in the amount of extractable antigen
between nodules harvested before and after O2
ramping (Table II).
Localization of Nodulins in Nodules Grown under Different
[O2]
Longitudinal sections of nodules were compared to discern whether
the ENOD2 protein was distributed differently in nodules grown under
different [O2]. An antibody against
distinctive, conserved motifs of ENOD2 was used in an immunolabeling
silver-enhancement protocol. The distribution of immunoreactive protein
in alfalfa nodules from all three growing conditions resembled that
seen previously in pea nodules (D.J. Sherrier, G.S. Taylor, and K.A. VandenBosch, unpublished results). Labeled protein was abundant in
intercellular spaces in the nodule parenchyma and was adjacent to the
nodule endodermis starting in interzone II-III (data not shown) and
continuing into zone III (Fig. 5, A-C).
Interzone II-III lies between the prefixation zone II and the
N2-fixation zone III (Vasse et al., 1990). Label
was heaviest at the cell corners and was sometimes observed lining the
nodule parenchyma cells. No qualitative or obvious quantitative
differences in labeling patterns were seen among the different
O2 treatments. In all cases, label was found in
intercellular spaces, in two to four cell layers of the nodule
parenchyma. ENOD2 protein was occasionally detected in the
intercellular spaces of the central infected zone (Fig. 5C), but it
accumulated to much lower levels and occurred at a much lower frequency
than what was observed in the nodule parenchyma. Omission of primary
antibody or incubation in preimmune serum resulted in a lack of
specific labeling (data not shown).
View larger version (69K):
[in this window]
[in a new window]
| Figure 5.
Immunolabeling of ENOD2 protein in alfalfa nodules
grown under different pO2s. Following silver enhancement,
immunogold labeling appeared as an opaque black deposit on
intercellular spaces in the nodule parenchyma (arrowheads). The regions
of the nodules depicted were comparable and are all in upper zone III.
C, Outer cortex; E, nodule endodermis; Inf, infected cells in zone III. All views are magnified ×315. Bar, 50 µm. A, Labeled section of a
nodule grown in 8% O2. B, Labeled section of a nodule
grown in 20% O2. C, Labeled section of a nodule grown in
50% O2.
|
|
In contrast to the immunolabeling results, a dramatic difference in the
patterns of starch accumulation was observed for the different
treatments. Vasse et al. (1990) noted that starch deposition in
amyloplasts occurs in a clearly demarcated cell layer (interzone II-III). In the present study, we utilized periodic acid Schiff's staining to visualize the abundant, plate-like amyloplasts in interzone
II-III in nodules of plants grown in 20% O2.
Small, round amyloplasts were present in uninfected cells of the
central region and also in the boundary layer cells (arrow, Fig.
6B). There was a gradual decrease in
starch abundance from interzone II-III toward the base of the nodule
grown in 20% O2 (data not shown).
View larger version (92K):
[in this window]
[in a new window]
| Figure 6.
Periodic acid Schiff's staining of
paraffin-embedded alfalfa nodules that had been grown under different
[O2]. Zones I, II, and the periphery of zone III are
shown. A, Longitudinal section of a nodule grown in 8% O2.
The arrow points to small starch grains in the nodule cortex. B,
Longitudinal section of a nodule grown in 20% O2. The
arrow points to starch grains in the boundary layer. C, Longitudinal
section of a nodule grown in 50% O2.
|
|
Starch was even more abundant in nodules grown in 8%
O2. Amyloplasts were found in the uninfected
cells of interzone II-III, zone II, and along the periphery of zone I
(the meristem), as well as in the cells of the nodule cortex (Fig. 6A).
Moreover, amyloplasts were evident throughout the entire length of the
nodule (data not shown). There was a copious accumulation of both
plate-like amyloplasts in the infected cells surrounding the central
region and small, round amyloplasts in the boundary layer. In contrast, starch grains were sparse in nodules grown in 50%
pO2 (Fig. 6C). Very few amyloplasts were observed
in the nodule, except in interzone II-III, and in both infected and
uninfected cells. Few periodic acid Schiff's positive staining bodies
were observed in either zone I or zone II in these nodules, and an
insignificant number of amyloplasts were observed in the more mature
regions of the nodule, with the exception of the senescent zone.
| |
DISCUSSION |
Gas Exchange
Although O2 supply to the central infected
zone of legume nodules often limits nitrogenase activity under normal
and adverse environmental conditions (Hunt et al., 1987, 1989; Hunt and
Layzell, 1993), nitrogenase activity did not differ among nodules that were grown and assayed at 8, 20, and 50% O2.
Also, respiration rates and the degree of O2
limitation of nitrogenase activity were similar in all three
O2 treatments. Taken together, these results
indicate that [O2] in the central zone of
nodules from the three treatments was similar, despite large
differences in the O2 gradient between the
rhizosphere and the central zone. This could only be achieved if the
nodules from the three treatments had very different permeabilities to
O2. Using a simplification of Fick's Law (Sheehy
et al., 1983):
|
(3)
|
in which F represents nodule O2
consumption (assuming equivalence with CO2
production), P represents nodule permeability to
O2, Oe represents
rhizosphere [O2], and
Oi represents the
[O2] in the nodule central zone, maintaining
F and Oi constant would require
that nodule permeability would change in inverse proportion with
increases in Oe. Therefore, the nodules
grown at 8% O2 would have had permeabilities to
O2 diffusion approximately 2.5 times greater than
those grown at 20% O2 and 6.3 times greater than those grown at 50% O2.
The nodules grown at each pO2 were able to
regulate permeability in response to changing rhizosphere
pO2, evidenced by maintenance of high nitrogenase
activities as pO2 was increased to 100%.
Respiration rate changed little with elevated
pO2. Therefore, increased respiratory O2 consumption would have had a very little role
in maintaining a central zone [O2] conducive to
nitrogenase activity. Applying the Fick's Law analog in Equation 3
above, these results suggest that between the initial growth
pO2 and the pO2 at which
PNA was reached, permeabilities of nodules grown at 8, 20, and 50%
O2 decreased to approximately 13, 25, and 50% of
initial values, respectively. Therefore, any developmental changes in
alfalfa nodules that allow them to adapt to long-term changes in
rhizosphere pO2 do not change the ability of the
nodules to regulate diffusion resistance physiologically during
short-term changes in pO2. A similar conclusion
was reached in earlier studies with soybean (Atkins et al., 1993).
Nodule Structure
In a previous study, alfalfa plants nodulated under conditions of
1% pO2 had macroscopically observable outgrowths
of loosely packed cells that resembled aerenchyma on both roots and
nodules (Arrese-Igor et al., 1993). The presence of loosely packed
cells is likely to affect gas permeability. However, we did not observe aerenchymatous tissue on nodules or roots in our experiments. A
concentration of 8% pO2 is apparently not low
enough to induce this kind of modification. Nevertheless, the nodule
tissues did appear less compact in the hypoxically grown nodules,
whereas tissues in the 50% pO2-grown nodules
appeared more compact. Even zone I, the nodule meristem, appeared to
consist of smaller, more densely packed cells in nodules grown in 50%
pO2 (compare Fig. 6C). Parsons and Day (1990)
observed a similar increase in tissue compactness for soybean nodules
formed under high O2.
A major anatomical difference among the different treatments was the
level of starch grain abundance. Like Arrese-Igor et al. (1993), we
found that nodules developed under low-O2
conditions amassed numerous starch granules. These authors suggested
that the accumulation of starch in indeterminate nodules such as
alfalfa might be related to stress. However, on the basis of
nitrogenase activity and respiration measurements, there was no
evidence that the nodules at 8% O2 were exposed
to stressful conditions. Moreover, nodules grown under 50%
O2 accumulated very little starch, and these
nodules had metabolic activity similar to those at 8 and 20%
O2. One interpretation for the presence of excess
starch is that the available carbon exceeded the respiratory demand of
the tissue and accordingly accumulated as starch. In any case, starch accumulation is a relatively long-term response and does not explain the short-term regulation of the nodule to changes in rhizosphere pO2, although Layzell et al. (1990) proposed that
carbohydrates may be involved in osmotic regulation of the diffusion
barrier.
Protein Levels and Nodulin Expression
The amount of protein per nodule fresh weight was higher in the
control nodules (20% pO2) than in the nodules
grown at 8 or 50% pO2. This is in agreement with
measurements of soluble protein in soybean nodules grown at 10, 21, and
40% pO2 (James et al., 1991). It cannot
presently be determined whether this represents a difference in the
abundance of one protein or many. Leghemoglobin, the most abundant
protein in nodules, facilitates the diffusion of
O2 across the gradient that exists between the
infected cell surface and the bacteroids (Appleby, 1984). We found no
apparent difference in the abundance of leghemoglobin, when visualized on western blots, among the different pO2
treatments. This is consistent with measurements of leghemoglobin in
other N2-fixing symbioses (Dakora et al., 1991).
Because there was no significant difference in the respiration rate of
the nodules grown at all three pO2, it is not
surprising that leghemoglobin levels were similar among the treatments.
It has been shown previously that nodules of soybean and cowpea grown
at sub- and supra-ambient pO2 maintain different
permeabilities by a change in thickness of the cortical diffusion
barrier and by a change in the proportion of the nodule parenchyma that
is composed of open (unoccluded) intercellular spaces (Dakora and Atkins, 1990c, 1991; Parsons and Day, 1990). Other studies at higher
growth [O2] demonstrated that the occlusion of
intercellular spaces is correlated with the deposition of a
glycoprotein recognized by the monoclonal antibody MAC236 (James et
al., 1991; Iannetta et al., 1993b, 1995). We found that a glycoprotein
recognized by MAC236 was also present in alfalfa nodules, and that its
abundance increased as pO2 increased from 8 to
20% O2 during development. Although there was no
significant difference between the abundance of MAC236 glycoprotein in
nodules grown at 20 and 50% O2, there was a
trend toward increasing amounts of MAC236 protein in the nodules
exposed to higher [O2] (compare Fig. 3C).
Iannetta et al. (1995) found that the MAC236 antibody cross-reacted
with increasing amounts of material in the intercellular spaces of
white lupin nodules exposed for 15 or 30 min to 50%
O2, whereas control nodules showed little or no
labeling of the cell walls. They proposed that an MAC236-type
glycoprotein could be involved in rapidly regulating permeability in
lupin nodules. However, MAC236 also cross-reacts with glycoproteins
from infection threads and infection droplets (VandenBosch et al.,
1989), and as pointed out by James et al. (1997), indeterminate nodules
such as those on alfalfa have a large number of infection threads in
contrast to white lupin nodules, which have few.
The levels of ENOD2 and MAC236-extractable protein did not change pre-
versus post-ramp in the ramping experiments. Given that the duration of
the ramped increases in pO2 were only 71, 45, and
20 min for nodules grown at 8, 20, and 50% O2,
respectively, it is unlikely that the short-term decreases in nodule
permeability could be caused by increased synthesis of either MAC236
glycoprotein or ENOD2 protein. However, it is possible that the MAC236
or ENOD2 proteins play a role in the short-term regulation of nodule
permeability by altering the gas:water content of intercellular spaces
in response to changes in [O2]. For example,
one could envision a mechanism whereby a cell wall matrix glycoprotein
was rapidly cross-linked in response to changes in
O2, thereby creating a gel or adhesive that would
hold water and displace gas within intercellular spaces. If this were
to occur in the innermost layers of the inner cortex or in the
outermost layers of the central infected zone, then the result could be
a wider, more convoluted path of O2 diffusion and
a rapid, physiologically induced change in nodule permeability to
O2 diffusion. Nevertheless, this hypothesis is
challenged in that Van Cauwenberghe et al. (1994), using a cryoplaning
technique, did not observe any measurable change in water content of
the intercellular spaces in the inner cortex of soybean nodules.
The question now is whether either ENOD2 or MAC236 (or both) is
cross-linked in response to O2. Although the
immunohistological study did not show a significant change in ENOD2
levels in nodules grown at the different [O2],
a slight decrease in ENOD2 levels was detected using western-blot and
ELISA analyses. This decrease may reflect a change in extractable
protein due to cross-linking, and thus may not necessarily exclude a
role for ENOD2 in the diffusion barrier. The deduced amino acid
sequence of ENOD2 is similar to the sequence of SbPRP2 from soybean, a
protein that is rapidly insolubilized in cell walls following wounding
or pathogen infection (Bradley et al., 1992). These authors also
reported cross-linking of the MAC265 antigen upon elicitor treatment.
MAC265 and MAC236 colocalize to the same tissues in legumes. The two
monoclonals recognize either neutral (MAC265) or acidic (MAC236)
components of a 95-kD antigen (VandenBosch et al., 1989). Thus, it is
reasonable to suggest that ENOD2 acts in concert with other cell wall
components, such as the MAC236 antigen.
Based on our results, changes in ENOD2 gene expression or protein
synthesis do not appear to be involved in the regulation of
O2 diffusion either in the short term or the long
term. The data for the MAC236 glycoprotein, on the other hand,
are inconclusive. The larger question now is whether ENOD2 is
absolutely required for nodule development and/or the proper
functioning of the diffusion barrier. To this end, we have generated
ENOD2-antisense alfalfa plants and have found that when
grown at different [O2], they nodulate
normally, suggesting that full expression of ENOD2 is not
required for a functioning nodule (K.L. Wycoff, S. Hunt, D.B. Layzell,
and A.M. Hirsch, unpublished results). Moreover, although some of the antisense lines appear to lack sense transcripts, several
express ENOD2 protein, albeit at lower levels based on western-blot
analysis (K.L. Wycoff, S. Hunt, D.B. Layzell, and A.M.
Hirsch, unpublished results). However, a conundrum is that ENOD2 is
also expressed in mycorrhizal roots (van Rhijn et al., 1997), which a
priori would not be expected to have an O2
diffusion barrier. ENOD2 expression in these roots may be
reflective of a change in endogenous hormone levels brought about by
colonization by mycorrhizal fungi. Indeed, expression of the
ENOD2 gene in uninoculated alfalfa roots is up-regulated by
cytokinin treatment (van Rhijn et al., 1997). Thus, the major role
for ENOD2 in nodule development is still obscure and must await either
the isolation of an ENOD2 mutant plant or the discovery of a nodulating
legume that lacks the ENOD2 gene.
| |
FOOTNOTES |
1
This research was supported by the U.S.
Department of Agriculture-National Research Initiative Competitive
Grants Program (grant no. 92-37305-7717 to K.L.W. and grant nos.
92-37305-7815 and 95-37305-2366 to K.V.B.), the Natural Sciences and
Engineering Research Council (Canada) (research grant to D.B.L.), and
the National Science Foundation (grant no. 90-23888 to A.M.H.).
2
Present address: Planet Biotechnology, Inc.,
2462 Wyandotte St., Mountain View, CA 94043.
*
Corresponding author; e-mail ahirsch{at}ucla.edu; fax
1-310-206-5413.
Received November 12, 1997;
accepted February 24, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ENOD2, early nodulin 2.
ENOD40, early nodulin
40.
OLCN, O2-limitation coefficient of
nitrogenase.
pO2, partial pressure of O2.
PNA, potential nitrogenase activities.
TNA, total nitrogenase activities.
| |
ACKNOWLEDGMENTS |
The authors wish to thank Bruce Brand, Jennifer Kuo, and
Pieternel van Rhijn. We thank W.M. Karlowski for his helpful comments on the manuscript. Margaret Kowalczyk is acknowledged for her help with
the figures.
| |
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Legocki AB
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Isolation and nucleotide sequence of cDNA clone encoding nodule-specific (hydroxy)proline-rich protein LENOD2 from yellow lupin.
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Layzell DB
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A re-evaluation of the role of the infected cell in the control of O2 diffusion in legume nodules.
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[Abstract]
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[CrossRef][Web of Science]
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Kominami R
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Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.
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[Abstract/Free Full Text]
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Layzell DB
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A peanut nodule lectin in infected cells and in vacuoles and the extracellular matrix of nodule parenchyma.
Plant Physiol
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[Abstract]
van de Wiel C,
Norris JH,
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Hirsch AM
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Nodulin gene expression and ENOD2 localization in effective, nitrogen-fixing and ineffective, bacteria-free nodules of alfalfa.
Plant Cell
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1009-1017
[Abstract/Free Full Text]
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Scheres B,
Franssen H,
van Lierop MJ,
van Lammeren A,
van Kammen A,
Bisseling T
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The early nodulin transcript ENOD2 is located in the nodule parenchyma (inner cortex) of pea and soybean root nodules.
EMBO J
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1-7
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Fang Y,
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Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.
Proc Natl Acad Sci USA
94:
5467-5472
[Abstract/Free Full Text]
Vasse J,
deBilly F,
Camut S,
Truchet G
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Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules.
J Bacteriol
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[Abstract/Free Full Text]
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Direct evidence for changes in the resistance of legume root nodules to O2 diffusion.
J Exp Bot
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1129-1140
[Abstract/Free Full Text]
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Abstract
Introduction
