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Plant Physiol. (1998) 116: 37-43
Antioxidant Defenses in the Peripheral Cell Layers of Legume Root
Nodules1
David A. Dalton*,
Shannon L. Joyner,
Manuel Becana,
Iñaki Iturbe-Ormaetxe, and
J. Mark Chatfield
Biology Department, Reed College, Portland, Oregon 97202 (D.A.D.,
S.L.J.); Departamento de Nutrición Vegetal, Estación
Experimental de Aula Dei, Consejo Superior de Investigaciones
Científicas, Apdo. 202, 50080 Zaragoza, Spain (M.B.,
I.I.-O.); and Biology Department, West Virginia State College,
Institute, West Virginia 25112 (J.M.C.)
 |
ABSTRACT |
Ascorbate peroxidase (AP) is a key
enzyme that scavenges potentially harmful H2O2
and thus prevents oxidative damage in plants, especially in
N2-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago
sativa) root nodules contains elevated levels of AP protein, as
well as the corresponding mRNA transcript and substrate (ascorbate).
Enhanced AP protein levels were also found in cells immediately
peripheral to the infected region of soybean (Glycine
max), pea (Pisum sativum), clover
(Trifolium pratense), and common bean (Phaseolus
vulgaris) nodules. Regeneration of ascorbate was achieved by
(homo)glutathione and associated enzymes of the ascorbate-glutathione
pathway, which were present at high levels. The presence of high levels
of antioxidants suggests that respiratory consumption of O2
in the endodermis or nodule parenchyma may be an essential component of
the O2-diffusion barrier that regulates the entry of
O2 into the central region of nodules and ensures optimal
functioning of nitrogenase.
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INTRODUCTION |
All N2-fixing organisms face a critical
dilemma. Nitrogenase, the enzyme that catalyzes the conversion of
N2 into NH3, is quickly and
irreversibly inactivated by O2, yet
N2 fixation is an extremely energy-demanding
process, with at least 16 mol of ATP being consumed per mol of
N2 fixed (Ljones and Burris, 1972 ). Therefore,
relying on aerobic respiration to supply this energy puts the organism
at risk of inactivating nitrogenase. This "O2 problem" in N2-fixing organisms is handled by a
variety of mechanisms, including anaerobic metabolism
(Clostridium spp.); consumption of O2
by extremely rapid rates of respiration (Azotobacter spp.); physical compartmentalization of nitrogenase (heterocysts of
cyanobacteria and vesicles of Frankia spp., the symbiont in
roots of alder and other nonlegumes); and temporal separation of
photosynthesis and N2 fixation (nonheterocystous
cyanobacteria).
In legume root nodules, the O2 problem is dealt
with by three mechanisms (Appleby, 1984; Witty et al., 1986; Hunt et
al., 1987; Denison, 1992): an abundant amount of the
O2-binding protein leghemoglobin to facilitate
the flux of O2 to symbiotic bacteria (Rhizobium or Bradyrhizobium), while maintaining
an extremely low, nontoxic concentration of free
O2; a high rate of respiratory O2 consumption; and a variable diffusion barrier
that controls the entry of O2 into the central,
infected regions. The principles by which this barrier operates are not
clear, but they are responsible for the physiological control of the
size, distribution, and content of intercellular spaces. The diffusion
of O2 into the nodule interior can thus be
regulated by alterations in relative amounts of air, liquid, or
occluding glycoproteins within these intercellular spaces (James et
al., 1991; Van Cauwenberghe et al., 1993). The effectiveness of the
control of O2 levels in nodules has been unequivocally established with measurements by O2
microelectrodes that show that the O2
concentration rapidly decreases from atmospheric levels in the nodule
outer cortex to nearly anaerobic levels in the nodule central region
(Tjepkema and Yocum, 1974; Witty et al., 1987).
Activated forms of O2, such as
H2O2 and the superoxide
radical, constitute another aspect of the O2
problem in legume nodules. Nodules have a high capacity to produce
these damaging chemical species because of the high rates of
respiration, the strong reducing conditions required to reduce
N2, the tendency of leghemoglobin to autoxidize,
and the likely ability of nitrogenase to directly reduce
O2 (Dalton, 1995). A major defense against
activated O2 in nodules is provided by AP (EC
1.11.1.11), a hemoprotein that uses the reducing power of ascorbate to
scavenge H2O2. Although AP
may be regarded as a nearly universal "housekeeper" in the cytosol
and chloroplasts of plant cells, it is especially abundant in the
cytosol of N2-fixing root nodules, where it makes
up almost 1% of the total soluble protein.
There is considerable variation between species in the structure of the
peripheral cell layers in nodules (Dakora and Atkins, 1989; Parsons and
Day, 1990; Hirsch, 1992; Brown and Walsh, 1996). This report follows
the terminology of Hirsch (1992), in which the organization (from
exterior toward the center) is referred to as the nodule cortex, nodule
endodermis, nodule parenchyma, and the N2-fixing
(infected) zone. Vascular bundles are present in the nodule parenchyma
layer. One advantage of this system is that the same terms apply to
both determinate and indeterminate nodules, although there are some
structural differences, such as the presence of a scleroid layer in the
nodule parenchyma of some determinate nodules.
In this report we have used various techniques of microscopy to define
the location of antioxidants, especially AP, in the peripheral cell
layers of nodules from several legume species. We hypothesize that
antioxidants may be involved in a type of "respiratory protection"
that limits the entry of O2 into the nodule
interior.
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MATERIALS AND METHODS |
Plant Culture
The following plant species and rhizobial symbionts were used:
soybean (Glycine max cv Williams) with Bradyrhizobium
japonicum 122DES; alfalfa (Medicago sativa cv Ladak)
with Rhizobium meliloti 104A14; clover (Trifolium
pratense cv Kenland) with Rhizobium leguminosarum bv
trifolii 162P28; pea (Pisum sativum cv Lincoln) with R. leguminosarum bv viceae NLV8; cowpea
(Vigna unguiculata cv California blackeye no. 5) with
Bradyrhizobium sp. 32H1; and bean (Phaseolus
vulgaris cv Contender) with R. leguminosarum bv phaseoli 3622. Plant growth conditions were as described by
Dalton et al. (1986).
Northern-Blot Analysis
RNA was isolated by the guanidinium-thiocyanate method using
RNAzolB (Tel-Test, Friendswood, TX). Electrophoresis, blotting, and
detection were as described by (Sambrook et al. 1989) with a
33P-labeled probe synthesized using the
EcoRI-EcoRI full-length cDNA insert of pSOYAP75
(Chatfield and Dalton, 1993) as a template. Each lane was loaded with
an equal amount (10 µg/lane) of total RNA based on
A265.
Immunodetection
Root nodules of various legume species, including alfalfa,
soybean, pea, clover, and common bean, were embedded in London White
resin and cut into 1-µm sections. Nodules were between 28 and 40 d old when harvested. At least five nodules from five separate plants
were examined for each of the observations reported here, but in most
cases the number examined was much higher. Immunodetection was
performed with a primary rabbit polyclonal antibody raised against
purified soybean AP (Dalton et al., 1993). The primary antibody was
used at a dilution of 1:50 for a 2-h incubation at room temperature.
For light-microscopic immunogold-Ag staining, the secondary antibody
consisted of affinity-purified goat anti-rabbit antibodies conjugated
to colloidal gold (Auroprobe LM, Amersham) and was used according to
the manufacturer's recommended procedures. For light-microscopic
immunofluorescence staining, the secondary antibody consisted of
affinity-purified goat anti-rabbit antibodies conjugated to a Cy3
fluorophore (excitation wavelength, 550 nm; emission wavelength, 570 nm; Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution
of 1:300 for 30 min. Sections were viewed with a microscope (Laborlux
S, Leitz, Wetzlar, Germany) equipped with a rhodamine filter. Negative
controls for both detection procedures were performed using rabbit
normal serum. Additional negative controls were performed by
preincubating 150 µL of diluted (1:50) primary antibody with 1 µg
of purified recombinant AP (Dalton et al., 1996) for 2 h with
gentle shaking at room temperature.
In Situ Hybridization
Digoxigenin-labeled antisense RNA probes for AP transcript were
made using pSOYAP75 (Chatfield and Dalton, 1993) digested with
BamHI as the template. The synthesis reaction
contained T7 polymerase and the components of the RNA-labeling kit
(Genius, Boehringer Mannheim). The sense probe (negative control) was
constructed using XhoI to linearize the plasmid and T3
polymerase. Details of the hybridization protocol were described by Li
et al. (1993). After an initial hybridization period of 15 h at
42°C, samples were incubated with rabbit anti-digoxin, washed, and
then incubated with protein-A gold (15 nm, Amersham) at a 1:100
dilution for 90 min. Specimens were photographed with dark-field
microscopy, because this accentuates the label and minimizes problems
of resolution that arise because the label and underlying tissue are in
slightly different planes of focus.
Histochemical Procedures
Localization of ascorbate was performed by incubating thin,
hand-cut sections of alfalfa nodules in 4% (w/v)
AgNO3 in 100 mm sodium acetate, pH
4.5, for 24 h at room temperature (Chinoy, 1984). For
(homo)glutathione localization, fresh cowpea or bean nodules were
halved with a sharp razor blade. To one-half, 20 µL of 50 mm K2-PO4, pH
7.0 (control), was immediately added, and the other half received the
same buffer containing 4 mm monochlorobimane (Thiolyte MC,
Calbiochem; Sanchez-Fernández et al., 1997). This compound
conjugates specifically with (homo)- glutathione, yielding a
fluorochrome (maximum excitation, 380 nm; maximum emission, 480 nm).
After incubation for 1 to 2 min, both halves were washed three times
with the same buffer, and the turquoise blue fluorescence was viewed
(excitation filter, 355-425 nm; suppression filter, 460 nm) and
photographed with a fluorescence microscope (Ortholux II, Leitz). To
visualize respiratory activity, intact alfalfa nodules were submerged
in 3 mm 2,3,5-triphenyltetrazolium chloride in 10 mm sodium phosphate buffer, pH 7.0, in the dark for 2 h, sectioned with a razor blade, and viewed without further staining.
Enzyme and Metabolite Assays
Nodules of cowpea and bean were dissected into cortex (including
nodule parenchyma) and infected tissue under a binocular dissecting
microscope. These species were chosen because of the relatively large
size of their nodules and, thus, the ease of separating tissue layers.
Extracts were prepared and assayed for activities of AP,
monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase
(EC 1.8.5.1), glutathione reductase (EC 1.6.4.2), and catalase (EC
1.11.1.6), as well as for concentrations of ascorbate and
(homo)glutathione, using procedures described by Gogorcena et al.
(1995). Samples containing similar amounts of whole nodules were run in
parallel and used as controls.
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RESULTS AND DISCUSSION |
Abundance of AP Transcript
Blots of total RNA from nodules, roots, shoots, and leaves of
soybean showed that the transcript level of AP was strongly enhanced in
nodules (Fig. 1). The transcript level in
nodules was 10-fold higher than in uninfected roots, 2.4-fold higher
than in shoots, and 1.5-fold higher than in leaves when quantified with
NIH Image software. These observations are consistent with earlier
results (Dalton et al., 1987) in which we estimated that the large
amount of AP protein in nodules corresponds to about 0.9% of the total
soluble protein.
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| Figure 1.
Northern-blot analysis showing abundance of AP
mRNA in soybean nodules (lane 1), roots (lane 2), shoots (lane 3), and
leaves (lane 4). Each lane contained 10 µg of total RNA.
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The slightly higher position of nodule AP mRNA (Fig. 1, lane 1) as
opposed to AP bands from other tissue types may be a gel artifact
caused by excess target, but the possibility of a slightly larger-molecular-weight form in nodules cannot be excluded.
Immunolocalization of AP
Immunostaining with rabbit polyclonal antibodies raised against
soybean AP indicated that the level of this protein was strongly enhanced in the nodule endodermis of alfalfa nodules (Fig.
2). Quantification of AP as Ag-grain
deposition per cell area indicated that levels of AP were 3.5- or
2.6-fold more abundant in the endodermis region than in the nodule
cortex or in the infected zone, respectively. The antibodies used in
these studies recognized a single band in sensitive chemiluminescent
western immunoblots of crude extracts of total protein from nodules of
soybean, alfalfa, clover, common bean, and red alder (Alnus
rubra; not shown). They also recognized recombinant soybean AP
expressed in Escherichia coli (Dalton et al., 1996).
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| Figure 2.
Immunolocalization of AP in alfalfa nodules.
A, Typical section showing Ag particles (AP protein)
concentrated in the infected region (INF) and in the nodule endodermis
(NE) between the nodule cortex (NC) and the nodule parenchyma (NP). B,
Negative control indicating the absence of labeling with Ag when normal
rabbit serum is used in place of antibody raised against AP. C, Similar to A, except that detection is based on Cy3 fluorescence. D, Negative control with a fluorescent probe in which the primary antibody was
blocked by preincubation with purified recombinant AP. Each bar
indicates 100 µm.
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Enhanced immunostaining of AP was consistently observed in the
endodermis of indeterminate nodules from several species (alfalfa, Fig.
2, A and C; pea and clover, not shown) with detection procedures based
on either Ag precipitation or fluorescence. A different labeling
pattern was observed in determinate nodules (bean, Fig. 3A; soybean, not shown). In these species
the labeling was most intense at the exterior edge of the nodule
parenchyma (just interior to the scleroid layer of soybean) and
diminished inwardly before sharply intensifying in the infected region.
This region with enhanced AP expression corresponds to the "boundary
layer" referred to in other studies (e.g. Parsons and Day, 1990).
Enhanced labeling was especially apparent in the parenchyma immediately
surrounding vascular bundles (Fig. 3A). As with indeterminate nodules,
controls with normal rabbit serum showed essentially no labeling of
bean (Fig. 3B) or soybean (not shown) nodules.
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| Figure 3.
Immunolocalization of AP in bean nodules (A) and
negative control (B) using normal rabbit serum. Micrographs were taken
using dark-field illumination, which makes Ag grains (AP) appear as bright dots. V, Vascular bundle. Other abbreviations are as in Figure
2. Each bar indicates 100 µm.
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Ag immunolabeling of AP could be seen with both bright-field (Fig. 2A)
and dark-field microscopy (Fig. 3A); however, this labeling was more
clearly seen with a dark field. Although Ag precipitation is a proven
and stable technique for localization of both proteins and transcripts,
the resultant micrographs suffer from limited resolution because the Ag
grains and the target tissue are in different planes of focus.
Dark-field microscopy maximizes the visibility of the Ag particles by
allowing them to appear as bright spots of light on an in-focus
background.
Cell wall artifacts are sometimes observed with immunostaining of plant
tissues. Several lines of evidence strongly suggest that our
observations were not attributable to such artifacts. Electron-microscopic studies (Dalton et al., 1993; also repeated here
but not shown) indicate clearly that the labeling is present almost
exclusively in the cytoplasm. Sections of alfalfa nodules treated with
normal rabbit serum instead of antiserum to AP showed no labeling in
the endodermis and very little in the infected regions, even if the
serum was used at an undiluted strength (Fig. 2B). Furthermore,
pretreating of primary antibody by the addition of purified,
homogeneous recombinant AP (Dalton et al., 1996) resulted in a complete
absence of labeling, with amounts of blocking antigen ranging from 1 µg to 1 ng in 150 µL of antibody solution (Fig. 2D). These controls
also eliminated the possibility of misleading results due to
autofluorescence.
In Situ Hybridization
The use of digoxigenin-labeled RNA antisense probes confirmed that
the endodermis region of alfalfa nodules contained high levels of
transcript for AP relative to adjacent regions of the cortex (Fig.
4A) and that central, infected cells had
high levels relative to adjacent, uninfected cells (Fig. 4B). Controls
performed identically with sense RNA probes showed only very light,
nonlocalized labeling. The apparent labeling of the epidermis in Figure
4A was an artifact that was also seen in controls.
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| Figure 4.
In situ localization of mRNA transcript for AP (A
and B) and histochemical localization of ascorbate (C and D) in alfalfa nodules. A, In situ hybridization with antisense RNA
probe for AP showing enrichment in the nodule endodermis when viewed by dark-field illumination. B, Similar in situ hybridization showing high levels of mRNA labeling in an infected cell (I) and lower
levels in an adjacent, uninfected cell (U). The label is evident as
small bright dots. Other features include numerous bacteroids (nearly
continuous bumps in I) and starch grains in uninfected cells. C, Ag
particles precipitated from AgNO3, indicating the presence
of ascorbate in high levels in the endodermis region. D, Negative
control for C in which the tissue was preincubated in pH 9.0 buffer to
oxidize the ascorbate before exposure to AgNO3. Other
abbreviations are as in Figure 2. Bar indicates 100 µm in A, C, and
D; 10 µm in B.
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As was the case with Ag immunolabeling, labeling of AP transcript with
Ag could be seen with both bright-field (not shown) and dark-field
microscopy, with the latter producing much clearer images.
Histochemistry
Staining with AgNO3 indicated a distinct
band of highly elevated ascorbate concentrations in the periphery of
alfalfa nodules (Fig. 4C). This band was apparently located at the
periphery of the nodule parenchyma, slightly interior to the
endodermis, although this distinction is difficult to make in fresh,
hand-cut sections. It may be that high utilization of ascorbate by AP
locally depletes the ascorbate in the endodermis region. This would
account for the absence of Ag deposition in the cell layers immediately
exterior to the region of highest ascorbate concentration (Fig. 4C). If this interpretation is correct, then ascorbate could be resupplied to
the endodermis through the abundant adjacent supply in the nodule
parenchyma. The procedure for ascorbate localization was based on the
specific reduction of Ag+ to dark crystals of
elemental Ag by ascorbate (Chinoy, 1984). No labeling was present in
control sections in which ascorbate was first destroyed by oxidation
with either high pH or copper sulfate before exposure to
AgNO3 (Fig. 4D). Additional evidence for elevated
ascorbate levels was provided by staining of fresh sections with
2,6-dichloro-indophenol, in which case there was a region of
clearing (indicating reduction of the dye by ascorbate) in the same
peripheral band in which Ag deposition had indicated high ascorbate
levels (data not shown).
Histochemical techniques indicated a high concentration of
(homo)glutathione in the nodule parenchyma of cowpea (Fig.
5) and bean nodules (not shown).
Glutathione ( Glu-Cys-Gly) is a major antioxidant in many
organisms. Curiously, legumes partially rely on a homolog,
homoglutathione (Glu-Cys- Ala), with antioxidant properties
presumably similar to glutathione found in other plant families
(Klapheck, 1988). Because glutathione and homoglutathione are not
distinguishable by the techniques used here, this report uses the term
(homo)glutathione to indicate both compounds. In plants one of
(homo)glutathione's chief roles involves a coupled series of redox
reactions by which ascorbate is regenerated for continuing
H2O2 scavenging (Dalton,
1995).
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| Figure 5.
Histochemical localization of (homo)glutathione in
cowpea nodules. A, Turquoise blue fluorescence in the
nodule parenchyma emitted by the adduct formed between
(homo)glutathione and monochlorobimane. B, Negative
control treated similarly except for the omission of monochlorobimane.
Abbreviations are as in Figure 2.
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Enzyme Activities and Metabolite Concentrations
Additional evidence for the enhanced antioxidant properties of the
peripheral regions of nodules was obtained by microdissection of
nodules into cortical and infected tissue. The corresponding extracts
were analyzed for concentrations of antioxidant metabolites and
activities of antioxidant enzymes (Table
I). This technique did not allow a
fine-enough separation to distinguish between discrete cell layers,
such as the endodermis versus other peripheral layers, but it did
provide a clear separation of peripheral versus central infected
tissue. Ascorbate contents were 44 and 67% higher in the peripheral
layers of bean and cowpea nodules, respectively, whereas
(homo)glutathione was 44 and 120% higher (Table I). Considering that
85% of the nodules is water, the average concentration of ascorbate in
the periphery can be estimated as 1 to 1.5 mm and that of
(homo)glutathione as 0.5 mm.
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Table I.
Antioxidant metabolites and enzymes in the periphery
(cortex plus nodule parenchyma) and infected zone of bean and cowpea nodules
Metabolite contents are expressed as nmol mg 1 protein.
Activities are expressed as µmol substrate min1
mg1 protein (catalase and AP) or in nmol substrate or
product min1 mg1 protein (others). Values
are means ± se (n = 4-7). For each
species, means denoted by the same letter do not differ significantly
at P = 0.05 based on Duncan's multiple-range test.
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Enzymes assayed in the peripheral and infected tissue included AP and
the related enzymes of H2O2
scavenging: dehydroascorbate reductase, monodehydroascorbate reductase,
and glutathione reductase. These enzymes participate in the
ascorbate-glutathione pathway by which
H2O2 is scavenged in higher
plants (Dalton et al., 1986; Dalton, 1995). The specific activity of AP
was similar in the peripheral and infected regions of bean and cowpea
nodules. However, this does not contradict the immunolocalization data
indicating enhancement in the nodule parenchyma, because the peripheral
extracts are diluted by regions that immunolocalization studies
indicate contain very little of this enzyme, whereas the infected
regions contain a uniform, moderately high level.
In terms of specific activity, the peripheral cells of both legume
nodules contain 57% more monodehydroascorbate reductase activity than
the corresponding infected regions. Dehydroascorbate reductase activity
was 77% higher in the peripheral cells of bean nodules compared with
levels in the infected zone; however, no such elevation of
dehydroascorbate reductase was observed in the periphery of cowpea
nodules (Table I). These elevated activities may allow the functioning
of the ascorbate-glutathione pathway at high rates in the nodule
periphery by continuously regenerating ascorbate from its oxidized
forms. However, glutathione reductase activity was similar in the
periphery and infected zone of cowpea nodules, and 27% higher in the
infected zone of bean nodules. This observation suggests that this
enzyme may have additional roles in the infected tissue, unrelated to
the ascorbate-glutathione pathway, such as the maintenance of high
levels of (homo)glutathione to reduce ferryl leghemoglobin to the
ferric form (Puppo et al., 1993).
Catalase activity, another direct scavenger of
H2O2 in plants, was
markedly higher in the central zone than in the periphery of both
legume nodules (Table I), an observation consistent with the primary
role of catalase in the elimination of
H2O2 generated as a
consequence of ureide synthesis in the central zone. Ureides are the
primary form by which N is transported out of nodules in these species.
Despite its considerable reputation, catalase is not a primary means of
antioxidant defenses in the cytoplasm of plant cells because of its
very high Km for
H2O2 and its restricted location in peroxisomes (Dalton, 1995).
Implications for the O2-Diffusion Barrier
The elevated levels of antioxidants in the endodermis or nodule
parenchyma suggest that this region plays a critical role in the
O2 relations of nodules. In particular, we
suggest that O2 diffusion into the interior of
nodules is restricted by respiratory demand at the endodermis region
(indeterminate nodules) or boundary layer (determinate nodules).
Aerobic respiration invariably produces activated forms of
O2, primarily by leakage of electrons from dehydrogenases in the electron transport chain. Elevated levels of AP
and ascorbate are thus required to provide adequate antioxidant defenses. Staining with triphenyl tetrazolium dye (an indicator of
respiratory activity) indicated that the endodermis region of alfalfa
nodules does indeed have high rates of respiration, as opposed to the
rates in the nodule cortex (Fig. 6).
This procedure also revealed diminished staining in the nodule
parenchyma and the central infected regions that may be interpreted as
a consequence of limited diffusion of the dye or as an indication of
lesser respiratory activity.
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| Figure 6.
Localization of respiratory activity in the
periphery of alfalfa nodules. Dark-red staining in the
endodermis/nodule parenchyma region is caused by reduction of
tetrazolium by respiratory dehydrogenases. Abbreviations are as in
Figure 2. The bar indicates 100 µm.
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Because the phloem tissue in nodules lies directly adjacent to the
endodermis, there is likely to be an abundant supply of photosynthate
to support the high rates of respiration. The respiratory production of
H2O2 is centered in
mitochondria, whereas AP is primarily cytosolic. However, this
separation does not refute the above arguments because
H2O2 readily crosses
biological membranes.
The high respiratory demand of nodules can be attributed in part to
bacteroid (as opposed to host plant cell) activity. AP is not present
in bacteroids (Dalton et al., 1986), and this would have no direct role
in bacteroid antioxidant defenses. However, nodule respiration occurs
in large part in the plant host cell, where AP is essential.
Nevertheless, the respiratory consumption of O2
cannot account for much of the well-known variability of the
O2-diffusion barrier. Numerous studies have
provided evidence that diffusion is regulated by physical changes in
the intercellular spaces of the nodule periphery (Dakora and Atkins,
1989; James et al., 1991; Atkins et al., 1993). These physical changes
alter the rate at which O2 diffuses into the
nodule interior such that the interior concentration of
O2 remains unaffected by changes in the external
concentration of O2.
In short-term studies (minutes to hours), this compensation does not
involve any changes in the observed rates of CO2
evolution, indicating that a variable rate of respiratory
O2 consumption does not account for differences
in rates of O2 entry into the nodule interior and
that there is no respiratory protection of nitrogenase (Sheehy et al.,
1983; Hunt et al., 1987; Weisz and Sinclair, 1987; Dakora and Atkins,
1989). However, the exposure of nodules to supra-ambient levels of
O2 does result in a 194% increase in the rate of
CO2 evolution after 5 d (Dalton et al., 1991). This long-term response also involves a concomitant increase of
81% in the activity of AP and a 53% increase in ascorbate
concentration. The observations presented here regarding enhanced
antioxidants in the nodule periphery further implicate a role of
respiration in controlling O2 entry into
nodules, and support the suggestion of Minchin et al. (1992)
that the diffusion barrier contains a respiration component that
reflects respiratory activity of nodule parenchyma and vascular
bundles.
Respiratory O2 consumption at the nodule
endodermis (indeterminate nodules) or nodule parenchyma (determinate
nodules) could be an important component of the diffusion barrier that
operates in conjunction with poorly understood changes in intercellular spaces to provide for maximum regulation of O2
entry into the nodule interior. Although protective respiration does
not appear to be involved in short-term variation in
O2 diffusion, respiration is almost certainly
important as a baseline defense and as a component of long-term
variability. Respiratory activity is high at the endodermis and nodule
parenchyma, where supplies of O2 and
photosynthate are maximal. Enhanced respiration requires enhanced
antioxidant defenses, as shown by the elevated levels of ascorbate and
AP. Therefore, AP and related antioxidants play a critical, supporting role in maintaining the microaerobic conditions required to prevent the
inactivation of nitrogenase in the infected, central region of nodules.
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FOOTNOTES |
1
Work done at Reed College and West Virginia
State College was supported by National Science Foundation grant no.
IBN-9507491. Additionally, partial support was provided by a grant from
the Howard Hughes Medical Institute to the Reed College Biology
Department under the 1991 Undergraduate Biological Sciences Initiative.
Work done at Consejo Superior de Investigaciones Científicas
(Zaragoza) was supported by grant no. PB95-0091 from Dirección
General de Enseñanza Superior (Ministry of Education and Culture,
Spain).
*
Corresponding author; e-mail david.dalton{at}reed.edu; fax
1-503-777-7773.
Received June 19, 1997;
accepted September 18, 1997.
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ABBREVIATIONS |
Abbreviation:
AP, ascorbate peroxidase.
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ACKNOWLEDGMENTS |
Technical support, assistance, and advice were provided by Dr.
Vince Franceschi and Chris Davitt, Electron Microscopy Center, Washington State University, Pullman, and Dr. Maria Herrero, Servicio de Investigación Agraria, Spain. We thank Dr. Frank Minchin for making valuable comments about the manuscript.
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LITERATURE CITED |
Appleby CA
(1984)
Leghemoglobin and Rhizobium respiration.
Annu Rev Plant Physiol
35:
443-478
[CrossRef][ISI]
Atkins CA,
Hunt S,
Layzell DB
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Top
Abstract
Introduction