Plant Physiol. (1998) 117: 1083-1093
Analysis of Respiratory Chain Regulation in
Roots of Soybean
Seedlings1
A. Harvey Millar,
Owen K. Atkin,
R. Ian Menz,
Beverley Henry,
Graham Farquhar, and
David A. Day*
Division of Biochemistry and Molecular Biology, Faculty of Science
(A.H.M., R.I.M., D.A.D.), and Environmental Biology Group, Research
School of Biological Sciences (O.K.A., B.H., G.F.), The Australian
National University, Canberra 0200, Australia
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ABSTRACT |
Changes in the respiratory rate and
the contribution of the cytochrome (Cyt) c oxidase and
alternative oxidase (COX and AOX, respectively) were investigated in
soybean (Glycine max L. cv Stevens) root seedlings using
the 18O-discrimination method. In 4-d-old roots respiration
proceeded almost entirely via COX, but by d 17 more than 50% of the
flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate
all decreased substantially. In extracts from whole roots of different
ages, the ubiquinone pool was maintained at 50% to 60% reduction,
whereas pyruvate content fluctuated without a consistent trend. In
whole-root immunoblots, AOX protein was largely in the reduced, active
form at 7 and 17 d but was partially oxidized at 4 d. In
isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas
both AOX capacity and protein abundance remained unchanged. The amount
of mitochondrial protein on a dry-mass basis did not vary significantly
with root age. It is concluded that decreases in whole-root respiration
during growth of soybean seedlings can be largely explained by
decreases in maximal rates of electron transport via COX. Flux via AOX
is increased so that the ubiquinone pool is maintained in a moderately
reduced state.
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INTRODUCTION |
The rate of plant respiration is linked to the rate of metabolism
and growth due to requirements for ATP, reductant, and carbon skeletons
during cell maintenance, division, and expansion (Hunt and Loomis,
1979
; Lambers et al., 1983). For example, respiration rates are often
lower in species with intrinsically slower growth rates (Poorter et
al., 1991). Moreover, respiration is rapid in tissues with high energy
demands, such as thermogenic floral spadices (Meeuse, 1975), and in
rapidly growing tissues, such as the elongation zone of roots (Lambers
et al., 1996). Plant respiration can also increase rapidly in response
to both biotic and abiotic stress (for a recent review, see Lambers et
al., 1996). Conversely, decreases in respiratory rate often occur as
plant tissues age (Azcon-Bieto et al., 1983; McDonnell and Farrar,
1993; Atkin and Cummins, 1994; Winkler et al., 1994). Various factors
may be responsible for these changes, including substrate availability,
enzyme activation, specific protein degradation or de novo protein
synthesis, and alterations in mitochondrial numbers.
The extent to which such changes in respiration rate alter the rate of
oxidative phosphorylation also depends on the partitioning of electron
flux between the Cyt and the alternative pathways of electron
transport. The Cyt pathway (terminating at COX) couples the reduction
of O2 to water with the translocation of protons across the inner mitochondrial membrane, thereby building a
proton-motive force that drives ATP synthesis. The alternative pathway
branches directly from Q and reduces O2 to water
without further proton translocation. This pathway appears to consist
of a single-subunit cyanide-resistant quinol oxidase, AOX. Electron
flow via AOX in plants can allow carbon flux through the TCA cycle when
ADP is limiting, thereby providing carbon skeletons for other cellular processes (Lambers and Steingröver, 1978). This pathway may also protect against harmful reactive O2 generation
when the Q pool is highly reduced (Purvis and Shewfelt, 1993; Wagner
and Krab, 1995), allow respiration to proceed in the presence of nitric oxide (Millar and Day, 1996), and help avoid the production of fermentation products when pyruvate accumulates (Vanlerberghe et al.,
1995).
Partitioning between COX and AOX can be dramatically affected by
factors that influence the AOX activation state (Hoefnagel et al.,
1995; Ribas-Carbo et al., 1995a, 1997). AOX exists as a dimer in
plants, and sulfhydryl linkages between paired subunits must be reduced
for maximal AOX activity (Umbach and Siedow, 1993). A variety of 2-oxo
acids, notably pyruvate, have been shown to specifically and reversibly
stimulate AOX activity at micromolar concentrations (Millar et al.,
1993, 1996). These activators apparently increase the
Vmax of AOX and may prevent inhibition by
oxidized Q (Hoefnagel et al., 1997). Because of these regulatory
features, the previous use of inhibitor titrations to estimate the
partitioning between respiratory pathways in vitro and in vivo has been
largely discredited (Millar et al., 1995; Day et al., 1996). A
noninvasive technique using differences in discrimination against
18O has now been developed to measure
partitioning between the Cyt and alternative pathways in whole-plant
tissues and isolated mitochondria (Guy et al., 1992; Robinson et al.,
1992; Ribas-Carbo et al., 1995a).
In this report ontogenetic changes in whole-root respiration and the
partitioning between COX and AOX are investigated using the
18O-discrimination technique. Respiratory changes
of whole roots were measured in conjunction with the redox poise of the
Q pool and of the AOX protein in whole-root extracts. These were then compared with the kinetics of Q-oxidizing and -reducing pathways and
the abundance of terminal oxidase proteins in isolated mitochondria. This information is used to identify factors influencing root respiration during growth and differentiation.
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MATERIALS AND METHODS |
Reagents
Percoll was purchased from Pharmacia and Folin and Ciocalteau's
reagents were purchased from BDH Chemicals (Melbourne, Australia). All
other reagents were purchased from Sigma.
Plant Culture and Organelle Isolation
Roots were harvested from soybean (Glycine max L. cv
Stevens) seedlings propagated in trays of vermiculite in a growth
cabinet at 28/25°C with a 16-h light/8-h dark cycle. At d 4 the
cotyledons and hypocotyls were greening and the root system
(approximately 150 mg fresh mass/seedling) consisted of a single
taproot without branches. At d 7 cotyledons were green and beginning to
open, and the primary leaf was expanding. The primary root
(approximately 300 mg fresh mass/seedling) had developed branches at
the base in a classic taproot structure. At d 17 cotyledons were fully open and slightly yellowing, primary leaves were fully expanded, and
the first trifoliate leaf was expanding. The root system (approximately 600 mg fresh mass/seedling) was a network of first- and second-order branches. Published methods were used to isolate mitochondria from
roots of 4-, 7-, and 17-d-old seedlings (Day et al., 1985).
Mitochondrial Assays
O2 consumption was measured at 25°C using
an electrode (Rank Brothers, Cambridge, UK). A standard reaction medium
(0.3 M Suc, 10 mM TES
(N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid) buffer, [pH 7.2], 5 mM
KH2PO4, 10 mM
NaCl, 2 mM MgSO4, 0.1% [w/v] BSA)
was assumed to contain an air-saturated O2
concentration of 240 µM. Assays containing succinate also
included 0.1 mM ATP to activate succinate dehydrogenase.
The redox state of Q was measured voltametrically with glassy carbon
and platinum electrodes according to the method of Moore et al. (1988).
COX activity was measured as Cyt c-dependent
O2 consumption sensitive to 0.5 mM KCN in the presence of 0.05% (w/v) Triton X-100 and 5 mM
ascorbate. Whole tissues were frozen in liquid
N2, ground to a fine powder with sand, and
resuspended in mitochondria-grinding buffer (Day et al., 1985)
supplemented with 0.05% (w/v) Triton X-100, and the filtrate was used
for COX assays. Significant ascorbate-dependent O2 consumption was observed in whole-tissue
extracts in the absence of added Cyt c, but this activity
was not affected by 0.5 mM KCN. This value was subtracted
from that in the presence of Cyt c to provide the rate of
COX activity. In isolated mitochondria endogenous ascorbate-dependent
O2 consumption was negligible. Protein content was determined by the method of Lowry et al. (1951). NAD-ME activities were assayed as NADH production at 340 nm, according to the method of
Day et al. (1984), in a reaction medium consisting of 2 mM NAD+, 2 mM
MnCl2, 4 mM DTT, 0.02% (v/v) Triton
TX-100, 1 µM antimycin A, 50 µM
n-propyl gallate, and 50 mM
Mes/1,3-bis[Tris(hydroxymethyl)-methylamino]propane, pH 6.5.
Whole-Tissue Pyruvate Content
Root samples (1-1.5 g) were snap frozen in liquid
N2, ground to a powder, thoroughly mixed with 6 mL of 6% perchloric acid, and kept in ice for 10 min. Extracts were
then centrifuged for 5 min at 9500g and the supernatant
filtered and neutralized with KOH. After removal of precipitate,
aliquots were assayed for lactate dehydrogenase (10 units)-dependent
NADH oxidation at 340 nm in a solution of 0.1 mM NADH, 50 mM Hepes, pH 7.5.
On-Line 
18O Measurements on Intact Tissues
Whole root systems (5-10 g fresh weight) were placed in a
water-jacketed, 50-mL, adjustable-volume (but note that the volume was
constant throughout the experiment) closed cuvette maintained under
darkness at 25°C, either directly after harvest or after prior
treatment with inhibitors. A constant supply of gaseous HCN was formed
in the chamber from a 1 M swab of KCN; SHAM was administered by soaking tissue in 20 mM SHAM for 20 min and
lightly patting dry before placing in the cuvette. Treatment with both inhibitors together decreased O2 consumption to
10% to 15% of the control values. The cuvette was connected through a
two-way valve (Valco, Sydney, Austalia) to a 125-µL sample loop and
to a 5-mL syringe to facilitate gas mixing before sampling. In one position, the two-way valve connected the cuvette and the sample loop;
in the other position, the valve connected the sample loop to a
pressure-regulated He (ultra-high purity) carrier gas line. The time
between withdrawal of successive samples from the reaction chamber was
270 s.
The sample gas stream flowed through a water/CO2
trap to the GC column of a NA 1500 elemental analyzer (Strumentazione,
Milan, Italy) and through an open split to an Isochrom-EA mass
spectrometer (Micromass UK, Ltd., Manchester, UK).
O2 and N2 were separated by
a 2-m × 6-mm o.d., 4-mm i.d. molecular sieve MS 5-A column (Alltech, Sydney, Australia) maintained at 40°C. The
O2 and N2 were detected
using a thermal conductivity detector and integrated using OS/2.2.1
Isochrom-EA software (Micromass UK, Ltd.). The isotope ratio
18O/16O was measured as the
ratio of masses 34 to 32. D values were determined from slopes of
ln(R/Ro) versus
ln(f) plots, according to the method of
Guy et al. (1989). The reproducibility of measurement of the isotope
ratio of O2 in the empty chamber was ± 0.01 to 0.1
. The cuvette was tested for leaks by filling with He, closing the cuvette, and sampling; no leaks were detected. Leaks were also
tested for during an experiment by plotting
ln(R/Ro) against ln(f) and looking at the slope; in the
presence of a leak, one would expect to see a flattening of the curve
as the contribution of air, and hence 16O,
leaking into the cuvette became more significant as the tissue respiration depleted the O2. We did not observe
this, and the linear regressions for these fits, from 7 to 12 points
per curve, had r2 values of 0.94 to 0.99 even when the O2 concentration was very low
toward the end of an experiment.
Analysis of Q-Pool Reduction
The extraction of Q from intact root material was conducted
according to a modified version of a procedure described by Wagner and
Wagner (1995). Approximately 1 g of whole soybean root systems was
immersed in liquid N2 and crushed to a fine
powder with a mortar and pestle. Extracts were freeze dried overnight
under a vacuum. This step removed the root aqueous phase and decreased the possibility of Q oxidation during organic extraction. Dried samples
were vortexed for 3 min in a mixture of 1.5 mL of methanol (containing
0.2 M perchloric acid) and 1.5 mL of petroleum ether (35-50°C boiling point, D = 0.64). After centrifugation at
1000g for 3 min to separate the phases, the upper phase was
evaporated to dryness under N2. Additional
petroleum ether was added to the lower phase, the earlier steps
repeated, and the upper phases combined. The extraction and drying was
performed under safe lights (Ilford S902, Kodak GBX-2) in a darkroom to
prevent light-dependent formation of semiquinone species. Dried samples
were dissolved in 150 to 300 µL of methanol containing 1 mM HCl, purged with N2, and passed
through a 0.22-µm filter before injection onto an HPLC column. A 250- × 4.6-mm reverse-phase C18 Alltima column (Alltech, Sydney, Australia) was used with an LKB-Pharmacia system with
an isocratic mobile phase of ethanol:methanol (7:3, v/v) under
N2 at a flow rate of 1 mL
min
1. The effluent from the column was
monitored continuously at 275 and 290 nm. Retention times of reduced
and oxidized Q9 and Q10 (Sigma) standards were determined for comparison with root extracts. The retention times were 8.5 and 13.9 min for reduced and oxidized Q9, and 10.9 and 18 min for reduced and oxidized
Q10. Both Q homologs were reduced to their
respective QH2 according to the method of Rich
(1978). The extinction coefficients for reduced and oxidized Q homologs
at 275 and 290 nm in standard solutions were determined spectrally,
according to the method of Crane and Barr (1971), and used to determine
the ratio of reduced and oxidized Q in root extracts.
Electrophoresis and Immunological Probing
For purified mitochondria, aliquots containing 40 µg of protein
were solubilized in sample buffer (2% [w/v] SDS, 62.5 mM
Tris-HCl [pH 6.8], 10% [v/v] glycerol, 0.002% [w/v] bromphenol
blue, and 50 mM DTT) and boiled for 1 to 2 min. For
whole-tissue extracts, approximately 250 mg fresh weight of soybean
roots was snap frozen in liquid N2, glass beads
(80 mesh) were added, and the sample was crushed to a fine powder with
a mortar and pestle. Samples were then solubilized in 400 µL of
standard sample buffer, boiled for 5 min, and centrifuged at
10,000g for 5 min; 20 µL of the supernatant was
loaded per lane for SDS-PAGE. For oxidation of samples, 5 mg of diamide
(azobis-dimethyl formamide) in 200 µL of 50 mM Tris-HCl,
pH 7.0, was added to powdered root samples, and the mixture was allowed
to thaw and incubate at room temperature for 5 min before addition of
sample buffer. Whole-root extracts were supplemented with 1 mM PMSF, 1 mM paminobenzamidine,
and 5 µM
trans-epoxysuccinyl-L-leucylamide(4-guanidino)-butane
to inhibit proteases.
Proteins were separated by SDS-PAGE as described by Kearns et al.
(1992). A modified version of the method of Towbin et al. (1979) was
used for immunoblotting. The probes were the AOA monoclonal antibody
raised against AOX proteins from Voodoo lily (Sauromatum guttatum; generously supplied by T.E. Elthon, University of
Nebraska, Lincoln, and by L. McIntosh, Michigan State University, East
Lansing) and a monoclonal antibody raised against human COX subunit I
(Molecular Probes, Eugene, OR). Immunoreactive proteins were visualized
using a chemiluminescence system (Boehringer Mannheim) for isolated mitochondria blots, and the Super-Signal Ultra chemiluminescence system
(Pierce) for whole-tissue extract blots.
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RESULTS |
Respiration and Growth of Soybean Roots
The rate of O2 consumption by whole soybean
root systems declined by 60% on a dry-mass basis from d 4 to d 17 of
seedling development (Table I). The
18O fractionation of respiration increased from
16.4 at d 4 to 20.5 at d 17, whereas the
18O fractionation via COX (plus SHAM) or AOX
(plus KCN) operating alone remained unchanged at the three root ages
tested (Table I). This indicates that a change in electron partitioning
away from the Cyt pathway and toward the alternative pathway of
mitochondrial O2 consumption occurred during root
growth. Calculation of the contribution of each pathway to total
respiration, using the D values in Table I, shows that the percentage
of respiration via AOX increased from 5% on d 4 to 35% on d 7, and
finally reached 55% on d 17. Presentation of this data on a dry-mass
basis shows that part of the change in percentage respiration via AOX
was caused by a decline in the respiration rate via COX, although AOX
activity per se also increased (Fig. 1A).
This is consistent with the age-dependent decrease in SHAM-insensitive
respiration rate in whole roots (Table I).
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Table I.
Respiration rates and 18O-discrimination
values of whole soybean root respiration, first in the absence of
inhibitors and then in the presence of KCN or SHAM in 4-, 7-, and
17-d-old roots
Data are means ± SE (n = 3).
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| Figure 1.
A, O2 consumption via COX ( ) and
AOX ( ) in intact roots from 4- to 17-d-old soybean seedlings based
on the 18O- discrimination values of Table I. B, The
theoretical ATP yield via oxidative phosphorylation ( ) and the
relative growth rate ( ) of soybean roots of the same ages. DW, Dry
weight.
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Calculation of the theoretical ATP yield of mitochondrial oxidative
phosphorylation showed a nearly 4-fold decline in the rate of ATP
synthesis in roots from 4- to 17-d-old seedlings (Fig. 1B) Several
assumptions underlie these estimations. We did not know the precise
mixture of the substrates oxidized in vivo but assumed that matrix NADH
was the main substrate and set an ADP/O2 ratio of 2.5 via
COX, and 1 via AOX, based on ADP/O2 values determined for
isolated soybean cotyledon mitochondria (Day et al., 1988). Whatever
the real values, it is obvious that the ATP yield will decline as the
respiration rate declines with age, and Figure 1B serves to illustrate
this trend. The relative growth rate of the soybean root system
declined nearly 7-fold during development, from 0.67 g
g1 d1 in 2-d-old
seedlings to 0.095 g g1
d1 in 17-d-old seedlings (Fig. 1B).
Redox Poise of the Q Pool in Vivo
Organic extraction and reverse-phase HPLC separation were used to
determine the redox poise of the Q pool in snap-frozen soybean roots.
Comparison of root extracts with commercial Q samples revealed that the
Q10 homolog was the predominant form of Q present
in soybean roots (Fig. 2), in agreement
with studies on isolated soybean root mitochondria (Ribas-Carbo et al.,
1995b; Millar et al., 1997). The oxidized and reduced forms of
Q10 were differentiated by retention time and
their extinction coefficients at 275 and 290 nm.
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| Figure 2.
Typical HPLC separation chromatographs of standard
Q homologs (top) and organic extracts of whole soybean roots
(bottom).
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The Q pools of control extracts from 4-, 7-, and 17-d-old roots were
53% to 62% reduced (Table II). Addition
of KCN and SHAM to inhibit respiratory oxidases increased Q-pool
reduction to 76% to 79% in extracts from 4- and 7-d-old roots. For
reasons unknown, we were unable to avoid oxidation of the extracted Q from 17-d-old roots to which KCN and SHAM had been added. Repeated freeze thawing of root tissue decreased Q-pool reduction to 6 to 10%
in roots of all ages, as did heating of roots to 65°C for 5 min (data
not shown). We found it very difficult to protect the Q complement of
roots from chemical oxidation during sample handling, especially in
older roots, with some samples showing only 0% to 2% reduced Q,
despite attempts to remove O2 and maintain aqueous phases at an acid pH. These samples were omitted from the
results presented. In all other samples, the Qr/Qt ratio was found to
be in the range 50% to 65%, which agrees with the data of Wagner and
Wagner (1995) and with measurements on isolated mitochondria (see
below). We assume, therefore, that these values are reasonable
estimations of the status of the Q pool in vivo.
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Table II.
Redox poise of extracted Q from soybean roots of
various ages (4, 7, or 17 d old), untreated or treated with respiratory
inhibitors (1 mM KCN + 20 mM SHAM) or by
freeze-thawing
Numbers are means ± SE (n = 3-6).
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Respiration by Isolated Root Mitochondria from Different-Aged
Seedlings
The respiratory characteristics of roots were further investigated
by comparing the relationship between Q redox poise (Qr/Qt) and the
respiratory rate in mitochondria purified from 4-, 7-, and 17-d-old
soybean root systems. Using succinate as a substrate, O2 consumption rates were titrated with the
specific succinate dehydrogenase inhibitor malonate (Fig.
3). Cyt pathway rates were measured in
the presence and absence of ADP. AOX rates were measured in the
presence of myxothiazol, with or without added pyruvate.
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| Figure 3.
Succinate-dependent O2 consumption
rate as a function of Qr/Qt in root mitochondria isolated from 4-, 7-, and 17-d-old soybean seedlings. Data points are in the presence
(squares) and in the absence (triangles) of ADP, and in the presence of
myxothiazol (5 µM) with (circles) or without (diamonds)
added pyruvate (1 mM). Filled symbols denote data in the
absence of malonate; unfilled symbols are data from malonate titration
of succinate oxidation. prot, Protein.
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The control respiration rate (i.e. without myxothiazol) in the presence
of ADP declined more than 2-fold on a mitochondrial protein basis with
increasing root age, but the Qr/Qt ratio in the absence of malonate
(i.e. at maximum O2 uptake rate) remained relatively stable at 0.2 to 0.3 (Fig. 3). When ADP was withheld, O2 consumption was lower at each age of a root,
and under these conditions the Qr/Qt ratio in the absence of malonate
was 0.7 to 0.8. Malonate-titration kinetics in the absence of
myxothiazol reflect flux via the Cyt pathway alone. This is because
without added pyruvate AOX did not significantly contribute to
respiratory flux below a Qr/Qt ratio of 0.8 to 0.9 (Fig. 3; see also
Millar et al., 1997). Data taken from malonate titrations of
respiration (in the presence of ADP) versus Qr/Qt in mitochondria from
different-aged roots were only able to be fitted on a single kinetic
curve by decreasing the apparent Vmax of
the Q-oxidizing pathway (not shown). Because electron transport under
these conditions is predominantly via COX, the capacity of the Cyt
pathway is less at any given Qr/Qt in mitochondria from older roots
compared with those from younger roots.
Upon addition of pyruvate, respiration via AOX in the absence of
malonate increased 3- to 4-fold at all root ages, to a rate of 100 to
120 nmol O2 min1
mg1 protein. Data from malonate titrations of
AOX activity in the presence of pyruvate could be fitted to a single
sigmoidal curve without modification, regardless of the age of roots,
suggesting that the amount of functional AOX on a protein basis
remained unchanged in mitochondria isolated from different-aged roots.
The last data point in each curve of Figure 3 (i.e. the value obtained
in the absence of malonate, shown as filled symbols) represents the
point of intersection between the COX and AOX kinetic curves, and the
kinetics of the Q-reducing pathway (in this case, succinate
dehydrogenase). A curve fitted through these points represents the
activity of succinate dehydrogenase in the face of an increasingly
reduced Q pool (Fig. 3, broken lines); these curves are mirror images
of Q-oxidizing-pathway kinetics. When these data points (obtained with
mitochondria from roots of different ages) were plotted on a separate
graph versus root age, they would fit on a single curve only when the
apparent Vmax of the Q-reducing pathway was
decreased (not shown), suggesting that succinate dehydrogenase activity
also decreased with root age.
Steady-state Qr/Qt values reflect the balance between Q-reducing and
Q-oxidizing pathways (Van den Bergen et al., 1994). A coordinated
decrease in both Q-oxidizing and Q-reducing pathways would, therefore,
explain why the steady-state Qr/Qt values in root mitochondria in
different metabolic states were constant with increasing root age,
despite the large decrease in absolute respiratory rate (Fig. 3).
Presumably, a decrease in succinate dehydrogenase activity also
contributes to the rather constant endogenous Qr/Qt ratio in intact
roots (Fig. 3). Total Q content of the mitochondria did not change
significantly over the time period studied (not shown).
COX Activity in Mitochondria and Whole Roots
Measurement of cyanide-sensitive Cyt c-dependent
O2 consumption (COX activity) provides a basis
for comparison of isolated mitochondria and whole-tissue respiratory
measurements. In isolated mitochondria from 4-, 7-, and 17-d-old roots,
COX activity declined with age on a protein basis (Table
III). Whole-tissue measurements showed a
similar decline in Cyt c-dependent O2
consumption with root age on a dry-mass basis. The ratio of the two
measurements provides an estimate of the amount of mitochondria in
soybean roots in milligrams of mitochondrial protein per gram of root dry mass. This parameter did not change during the period of growth measured.
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Table III.
COX activity in whole-tissue extracts and isolated
mitochondria from soybean roots of various ages (4, 7, and 17 d old)
Numbers are means ± SE (n = 4).
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The data in Table III also allow a comparison between the measured rate
of respiration and the capacity of COX in intact roots. Control
respiratory rates from Table I were only 14% to 20% of the COX
capacity in whole roots from Table III. Furthermore, the rates of
succinate-dependent respiration in the presence of ADP in isolated
mitochondria (Fig. 3) was only 30% to 38% of the COX capacity in
solubilized mitochondrial samples (Table III). This suggests that root
mitochondria were not strictly limited in vivo by the capacity of COX,
even though this decreased more than 2-fold during the time of the
experiment, but rather are limited by the combined effect of decreasing
dehydrogenase and COX activities. Respiration in vivo may also be
restricted by adenylates and/or substrate supply to the mitochondria.
COX and AOX Protein Abundance in Isolated Mitochondria
To determine whether the observed changes in AOX and COX
activities were correlated with changes in de novo synthesis or
degradation of oxidase proteins, antibodies against both oxidases were
reacted with mitochondrial proteins isolated from 4-, 7-, and 17-d-old plants on immunoblots (Fig. 4). A
monoclonal antibody specific for AOX cross-reacted with a single 36-kD
polypeptide among mitochondrial proteins from all three ages of soybean
roots and no significant difference was observed in the intensity of
the reactions. A monoclonal antibody specific for COX subunit I reacted
with a 52-kD protein in soybean root mitochondria, but in this case the
intensity of the reaction decreased noticeably with root age (Fig. 4).
This suggests that turnover of key COX subunits may be largely
responsible for the decreased total COX activity in mitochondria
isolated from older roots (Table III).
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| Figure 4.
Immunoblots of AOX and COX subunit I proteins in
root mitochondria isolated from 4-, 7-, and 17-d-old soybean seedlings.
Mitochondrial protein equivalent to a 40-µg BSA standard was loaded
in each lane in the presence of DTT (50 mM).
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AOX Redox State in Whole Roots
The AOX protein exists in two forms: an inactive, covalently
linked dimer and an active, noncovalently linked dimer (Umbach and
Siedow, 1993). These two forms can be identified on SDS-PAGE under
nonreducing conditions: the reduced dimer is separated into monomers of
32 to 39 kD, whereas the oxidized dimers remain in pairs and migrate
with an apparent molecular mass of 70 to 80 kD. The redox status of AOX
in isolated mitochondria can be measured in this manner, but this ratio
does not necessarily reflect that in vivo because oxidation can occur
during the mitochondrial isolation process (Umbach and Siedow, 1997).
Using recent advances in chemiluminescence technology, the AOX protein
can now be immunodetected in whole-tissue extracts, even in
nonthermogenic tissues such as soybean, in which AOX is expressed at
relatively low levels. In Figure 5,
immunoblots of total root protein rapidly extracted under denaturing
conditions are shown after separation in the presence and absence of
chemical oxidants and reductants. Each blot was probed with the
monoclonal anti-AOX antibody. In root extracts from 7- and 17-d-old
seedlings, control lanes show that nearly all of the immunoreactive
protein is present at an apparent molecular mass of 36 kD. At both
ages, the addition of DTT had little effect, whereas incubation of root samples with diamide (a strong oxidant) yielded an immunoreactive protein with an apparent molecular mass of 75 kD. This suggests that
most, if not all, of the AOX protein in d-7 and -17 roots was present
in the reduced, active form in vivo. Extracts from d-4 roots were more
problematic, with high background-to-signal ratios observed in all
experiments. This was probably caused by the substantially higher
protein content in the young roots, which dilutes the mitochondrial
component. However, focusing only on the bands at 75 and 36 kD, which
we think is justified because these were the only bands observed in the
blots of roots from older seedlings, it can be seen that the addition
of DTT markedly increased the intensity of an immunoreactive band at 36 kD (Fig. 5). The relative intensities of the 75- and 36-kD
immunoreactive bands, representing oxidized and reduced AOX,
respectively, were 0.65 and 0.55 in the control and 0.35 and 1.0 in the
DTT-treated lanes, respectively, as determined by image analysis of the
x-ray film. We suggest, therefore, albeit tentatively, that AOX was partly oxidized in d-4 roots.
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| Figure 5.
Immunoblots of AOX in rapidly extracted
whole-root samples from 4-, 7-, and 17-d-old soybean seedlings. Samples
treated in the presence and absence of DTT (50 mM) and
diamide (5 mM) are presented. For each age, lanes are from
a single gel, and each lane shown was separated by two empty lanes to
avoid cross-contamination of redox chemicals. The intensity of the
reaction at 35 and 75 kD was measured using National Institutes of
Health imaging software. At a given root age, the 35-kD band in the DTT
lanes had the highest density of all bands present in the three lanes,
and this was arbitrarily set at 1.0; the densities of the other bands
in those lanes are presented as fractions of that highest-density
band.
|
|
Tissue Pyruvate Content and Mitochondrial NAD-ME Activities
The activity of AOX is reversibly stimulated from within the
mitochondrial matrix by short-chained 
-keto acids such as pyruvate. In vivo, intramitochondrial pyruvate may be supplied via the transport of cytosolic pyruvate from the cytosol or via the decarboxylation of
malate to pyruvate by NAD-ME in the matrix. Pyruvate content in total
soybean root tissue increased from d 4 to 7 and then decreased to the
former value by d 17 (Table IV). The
activity of NAD-ME in isolated mitochondria did not change
significantly during this time (Table IV) and was only 20% to 40% of
activity measured in soybean cotyledon, potato tuber, and Arum
maculatum spadix mitochondria (data not shown).
View this table:
[in this window]
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Table IV.
Pyruvate content of whole-root systems and NAD-ME
activities of mitochondria isolated from whole-root systems of soybean
seedlings of various ages (4, 7, and 17 d old)
Numbers are means ± SE (n = 3).
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|
| |
DISCUSSION |
Ontogenetic Changes in Total Respiration and Electron
Partitioning
This study demonstrates that total respiration, and the
contribution of COX to that respiration, decreases with age in whole roots of soybean seedlings. The amount of AOX protein and the capacity
of AOX did not change during this period, but electron flux through AOX
increased with age and at 17 d AOX activity equaled that of COX
(Fig. 1). This increase agrees with previous developmental work on
legumes (Azcon-Bieto et al., 1983; Obenland et al., 1990; Wen and
Liang, 1993; Lennon et al., 1995) and two Arctic species (Atkin and
Cummins, 1994).
The ontogenetic decline in total respiration and COX activity of our
soybean roots correlated with a decline in root relative growth rate
during aging, as reported by others (Azcön-Bieto et al., 1983;
McDonnell and Farrar 1993; Winkler et al., 1994; Lambers et al., 1996).
The decline in Cyt pathway activity with age may reflect a decline in
the demand for ATP associated with the slower growth rates, as has been
suggested previously (Amthor, 1989). Because the amount of COX I
protein also declined, there may be a feedback effect of ATP demand on
the synthesis of Cyt-chain components. This may not be a general
phenomenon, however, because a decline in Cyt-chain capacity was not
seen in developing pea leaves (Lennon et al., 1995).
Measurement of Electron Partitioning between Respiratory
Pathways
In this study an 18O-discrimination method
was used to determine the partitioning of electrons between AOX and COX
(Guy et al., 1989). This method overcomes many of the problems
associated with the classic inhibitor-titration methods used previously
(Atkin et al., 1995; Millar et al., 1995; Day et al., 1996). Our
results generally agree with other recent work using this technique,
which has shown that 30% to 40% of respiratory flux occurs via AOX in whole soybean roots (Robinson et al., 1992, 1995). We observed consistent D values by the Cyt and alternative pathways measured in
soybean roots of various ages (Table I). Our determinations for AOX do
not vary much from others reported in the literature. Published values
for the Cyt pathway in soybean vary substantially between studies, from
17.2 (Robinson et al., 1995) to 20.3 (Ribas-Carbo et al., 1997); our
values fall closer to the former. The reason for the different values
may reflect differences in growth and measuring conditions in the
different experiments. Clearly, it is necessary to determine these
values for each set of experiments, even with the same species.
Control of AOX Activity in Vivo
To explain the observed changes in partitioning between
respiratory pathways, we attempted to determine factors known to affect AOX activity in isolated mitochondria and intact roots.
Intramitochondrial pyruvate is one such factor. Measurements of
mitochondrial pyruvate content in vivo are extremely difficult, but
measurements of whole-tissue pyruvate content have been made in the
hope that these might help elucidate the importance of this regulatory
factor in vivo. Wagner and Wagner (1995) showed that addition of the
uncoupler S13 to a petunia cell culture stimulated AOX activity without
increasing Q-pool reduction. They suggested that a concomitant increase
from 100 to 166 nmol pyruvate g1 fresh weight
in the cells on addition of the uncoupler may have been responsible for
this stimulation. Variation in pyruvate content from 30 to 60 nmol
g1 fresh weight in pea, spinach, and wheat
leaves also positively correlates with partitioning to AOX as judged by
inhibitor titration (Day and Lambers, 1983). In soybean roots, pyruvate
content did vary during development but showed no clear trend that
correlated with the in vivo respiratory flux via AOX (Table IV).
Because only very small amounts of intramitochondrial pyruvate are
needed to activate AOX in soybean (Millar et al., 1996), estimates of whole-tissue pyruvate contents are unlikely to answer the question of
whether AOX is fully activated by -keto acids in vivo. However, the
lack of significant change in mitochondrial ME activity (Table IV)
indicates that the potential for intramitochondrial pyruvate formation
in soybean roots does not change with seedling age. Furthermore, Q
redox titrations of mitochondria isolated from 17-d-old roots indicate
that pyruvate was required to obtain appreciable AOX activity at 60%
to 70% reduced Q (Fig. 3); because this was the value of Q reduction
in intact roots (Fig. 2) and because AOX was clearly active in these
roots (Fig. 1), it can be concluded that pyruvate levels in vivo were
sufficient to activate AOX. Because the same level of pyruvate was
observed in 4-d-old roots in which AOX was inactive (Table IV), and
Q-reduction levels were also similar (Fig. 2), it is obvious that
factors other than whole-tissue pyruvate content were involved.
The redox state of AOX protein is known to affect AOX activity (Umbach
and Siedow, 1993), and our results suggest that in roots of very young
soybean seedlings AOX oxidation may be one factor contributing to its
low activity (Fig. 5). In this context, a study of AOX in developing
pea leaves found that although AOX activity increased during the time
period studied, the amount of AOX protein in mitochondria remained
constant (Lennon et al., 1995). The increase in AOX activity was
correlated with a shift in the redox state of AOX protein toward the
more reduced state (Lennon et al., 1995).
Electron flow to AOX is also stimulated by the absence of ADP, and it
is likely that respiration in the roots under study here was adenylate
limited, particularly in the older roots, in which the relative growth
rates were relatively low. The observation that the ratio of
succinate-dependent respiratory rate (Fig. 3) to COX capacity (Table
III) for isolated root mitochondria in the absence of ADP is very
similar to the ratio observed between respiratory rate and COX activity
of whole tissues (Tables I and III) supports this prediction, as does
the relatively high whole-root Q-reduction levels (Fig. 2, compare with
Fig. 6).
View larger version (15K):
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| Figure 6.
Succinate-dependent O2 consumption and
Q-pool reduction by mitochondria isolated from 17-d-old soybean roots.
Ten millimolar succinate (succ), 2 µM myxothiazol (myxo),
and 1 mM pyruvate were added as indicated. Numbers on
traces indicate nmol O2 min1
mg1 protein and the steady-state Qr/Qt ratio (in
parentheses).
|
|
It is interesting to note that the KCN-insensitive respiration rate
measured in intact roots decreased during seedling growth from 7 to
17 d (Table I), whereas AOX capacity in isolated mitochondria did
not change (Fig. 3). This discrepancy could be the result of restricted
substrate supply to the mitochondria in the oldest roots; this could
have resulted from high cytosolic ATP/ADP ratios, which, together with
low ADP concentrations (Dry and Wiskich 1982), could also have
restricted electron transport-chain activity. However, it must also be
kept in mind that structural changes during root development will
underlie the changes in respiration seen on a whole-root basis. During
root development cortical cell tissue is replaced with vascular cell
tissue and the proportion of meristematic to nongrowing tissue in the
whole-root system also changes (Esau, 1977). The relative contributions
of the meristematic and nongrowing tissues to both in vivo respiration
rates and the isolated mitochondria remain unknown, and may influence
comparisons of the two.
Maintenance of Q Redox State in Roots
Table II shows that intact roots maintained a relatively constant
Q redox poise despite a 3-fold decrease in respiratory rate (Table I)
and a large change in partitioning between respiratory pathways
(Fig. 1). In isolated mitochondria steady-state Qr/Qt ratios under
standard conditions in the absence of malonate also remained relatively
constant with root age (Fig. 3). However, although 95% of total
cellular Q is located within mitochondria (Swiezewska et al.,
1993), the Qr/Qt derived from Q-electrode measurements on isolated
mitochondria (Fig. 3) may not accurately represent the redox poise of Q
extracted from roots, because 10% to 30% of mitochondrial Q appears
to be redox inactive in isolated plant mitochondria (Van den Bergen et
al., 1994; Ribas-Carbo et al., 1995b). The residual reduced and
oxidized Q in the presence of KCN plus SHAM can be used as estimates of
nonreactive Q in vivo. On this basis, 50% to 60% of Q reduction in
vivo is equivalent to 0.6 to 0.7 redox active Qr/Qt in isolated
mitochondria. However, it is possible that the inactive Q pool found in
isolated mitochondria is an artifact caused by damage of a proportion
of mitochondria during isolation. In this case, the in vivo percentage
Q reduction could be compared directly with Qr/Qt in isolated
mitochondria.
Wagner and Wagner (1995) have reported that petunia cell cultures also
maintain a Q-pool reduction of approximately 60% for most of the
culture cycle, despite large changes in respiratory rate. These authors
suggested that electron partitioning between respiratory pathways is
varied in vivo to maintain a steady-state Q redox poise, rather than
vice versa.
The Role of AOX in Soybean Roots
This study shows that respiratory rates of soybean roots during
aging are largely dictated by changes in the activity of Cyt-pathway electron transport (probably mediated by changes in the content of
relevant enzymes, as well as by adenylate control) and changes in AOX
activation status. The decline in Cyt-chain activity is at least
partially offset by increased AOX activity to provide a relatively
stable steady-state Q-pool reduction in the mitochondrial inner
membrane. Possible changes in succinate dehydrogenase activity may also
play a role here. The changes in AOX activity may be important to
ensure a smooth transition through developmental changes in ATP demand.
It seems that although ATP demand decreases as roots age, some
provision for carbon flux through respiratory pathways is maintained (as proposed by Palmer, 1976), and the role of AOX here may be to
ensure that this occurs without overreduction of the Q pool with its
associated generation of destructive O2
intermediates (Wagner and Krab, 1995; Millar and Day, 1997). The
ability of AOX engagement to modulate the redox state of mitochondrial
Q is illustrated in Figure 6. This shows simultaneous recordings of
O2 consumption and Q reduction during succinate
oxidation by mitochondria isolated from 17-d-old roots. In these
mitochondria, AOX protein is largely reduced, but pyruvate must be
added to observe AOX activity (Day et al., 1994). Respiration was rapid and the level of Q reduction quite low (Qr/Qt = 0.4) in the
presence of ADP; upon depletion of ADP, respiration slowed and
Q-reduction level increased to 0.75. Subsequent addition of pyruvate to
activate AOX stimulated respiration almost 2-fold and decreased Qr/Qt
to 0.55 (Fig. 6). (It is interesting to note that this was the level of
Q reduction in intact roots of this age [Fig. 2].) Thus, AOX activation allows increased resting rates of respiration without large
increases in Q-reduction levels.
More work is needed to understand the mechanisms behind down-regulation
of Cyt-pathway activity during development of plant tissues, but it is
interesting that the decline in roots with age is accompanied by
decreases in COX protein levels, implying some coarse control of
respiratory pathways. COX deficiencies in mammals have also been
detected in whole tissues and in isolated mitochondria (Nicoletti et
al., 1995). Such deficiencies have been linked to aging and to a
variety of inflammatory and degenerative myopathies (Chariot et al.,
1996). Establishing a link between this mammalian work and the
ontogenetic- and stress-induced changes in plant respiration will be a
valuable step in furthering our understanding of the regulation of
mitochondrial respiration.
| |
FOOTNOTES |
1
This research was supported by a grant from the
Australian Research Council to D.A.D.
*
Corresponding author; e-mail david.day{at}anu.edu.au; fax
61-2-6249-0313.
Received November 13, 1997;
accepted April 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AOX, alternative oxidase.
COX, Cyt
c oxidase.
D, discrimination value for 18O
fractionation.
ME, malic enzyme.
Q, ubiquinone.
Qr/Qt, fraction of the
Q pool in the reduced state.
SHAM, salicylhydroxamic acid.
| |
ACKNOWLEDGMENTS |
Sue Young and Julie Styles are thanked for technical assistance.
David Greber and Agnieszka Dombek contributed to this work through
undergraduate research projects. The generous support of Micromass UK,
Ltd., to this project is acknowledged.
| |
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Top
Abstract
Introduction