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INTRODUCTION |
Unsaturation degree of fatty acids plays a crucial role in lipid
dynamics and lipid-protein interactions in the lipid matrix of
mitochondrial membranes (1, 2). However, the exact relationships between chemical, physical, and biological properties of these membranes are far from being elucidated (2-4). It is well established that the composition in fatty acids of plant membrane phospholipids is
temperature-dependent, fatty acids being more unsaturated
in chilling-resistant than in sensitive plants (2-5). Furthermore, it
has been shown that the unsaturation degree of fatty acids influences
the physical state of the membrane, microviscosity being higher in
membranes containing a low level of polyunsaturated fatty acids
(6-8).
In mitochondrial membranes, the oxidative phosphorylation is likely to
be affected by changes in fatty acid unsaturation of the inner membrane
phospholipids. Indeed, catalytic properties of carriers and redox
components of the respiratory chain (1, 9), and the proton-selective
leaks (10, 11) are sensitive to the unsaturation index. However,
contradictory results have been reported. In plant systems, a decrease
of unsaturation of mitochondrial membranes triggered by hypoxia had no
significant effect on the activation energy of respiration (12). In
mammalian mitochondria, the electron transport in respiratory chain did not absolutely behave as a lipid diffusion-limited process (13). Similarly, the role of lipids and unsaturated fatty acids in proton permeability of mitochondrial membranes is still debated, because the
mechanism of proton leak is not really known (14, 15).
To get further insight in the relationships between fatty acid
composition and dynamic and functional properties of plant mitochondrial membranes, we used an approach based on genetic modifications of membrane lipid composition. Experiments were carried
out with the fad2 mutant of Arabidopsis thaliana,
which belongs to a family of monogenic mutants deficient in fatty acid desaturase activities (16). Plants, contrary to animals, de novo synthesize their fatty acids and desaturate them by two
independent pathways (3, 4). The prokaryotic pathway, located in
plastidial membranes, is responsible for the desaturation of fatty
acids found in this compartment. The eukaryotic pathway is associated to endoplasmic reticulum and is responsible for the desaturation of
fatty acids present in extraplastidial membranes. As a
consequence of the existence of these two pathways,
Arabidopsis mutants affected in desaturase activities
associated to one pathway are viable. In peculiar, the fad2
mutant, deficient in oleate desaturase associated to endoplasmic
reticulum, grows well except if plants are exposed at low temperature
(5, 17). Such a mutant is a good model to analyze the importance of
polyunsaturated fatty acids
(PUFA)1 in extraplastidial
membrane functions. In addition, compared with animal mitochondria,
plant mitochondrial membranes exhibit a simple fatty acid composition
where only three unsaturated fatty acid species are found: one
monounsaturated, oleic acid (18:1), and two PUFA, linoleic
(18:2) and linolenic (18:3) acids.
Composition of mitochondrial membranes isolated from cell culture of
the fad2 mutant was analyzed, and the dynamic properties of
membrane lipids were investigated by measuring lipid microviscosity and
lateral diffusion. Oxidative phosphorylation parameters, such as
oxidation rates and activation energy of electron transport, were
analyzed. The flux-force relationships were established, and the
metabolic control analysis of the oxidative phosphorylation process was
carried out to estimate the impact of fad2 mutation on
proton leaks.
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MATERIALS AND METHODS |
Plant Material--
Cell suspension culture of wild type
A. thaliana L. (Heynh) ecotype Columbia was provided by Dr.
M. Axelos (CNRS, Toulouse). Cell suspension culture of the
fad2-2 genotype was established in the laboratory. The
culture medium (Gamborg B5) was bought from Duchefa, France. Every
fifth day, 25-ml aliquots of cell suspension were transferred into
500-ml Erlenmeyer flasks containing 200 ml of fresh medium. Cells were
grown under continuous light (100 µmol m
2
s
1) at 22 °C, with rotating agitation (140 rpm).
Preparation of Mitochondria and Submitochondrial
Particles--
Mitochondria were isolated from 6-day-old suspension
cultures according to Davy de Virville et al. (18). Purified
mitochondria were immediately used for functional measurements or kept
at
20 °C for further analysis. Based on galactolipid content (used
as a marker of plastidial membranes), the purity of the mitochondrial fraction was higher than 95%. The outer membrane integrity of fresh
mitochondria was higher than 90%, as deduced from cytochrome c oxidase activity measurements (19). To prepare
submitochondrial particles, intact mitochondria (1.5 mg of protein/ml)
in 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 20 mM Mops (pH 7.2) medium were sonicated six times for 10 s in an MSE sonicator. After
centrifugation at 100,000 × g for 60 min, the membrane
pellet was resuspended in a minimal volume of the same medium and used
for protein and lipid phosphorus determinations.
Lipid Analysis and Liposome Preparation--
Lipids were
extracted and separated on silica gel plates (Merck) by two-dimensional
thin layer chromatography as previously described (20). Fatty acids
were analyzed after transmethylation (18) using methylheptadecanoate
(C17) as internal standard. Large unilamellar liposomes of total
mitochondrial lipids were prepared by extrusion (Liposofast, Avestin
Inc., Ottawa, Canada) in 100 mM KCl, 5 mM
MgCl2, 1 mM EDTA, and 20 mM Mops
(pH 7.2) medium.
Oxygen Uptake--
Oxygen consumption was measured between 5 and
35 °C using a Clark-type oxygen electrode (19). Air-saturated
electrode medium contained 0.3 M mannitol, 10 mM phosphate buffer, pH 7.2, 10 mM KCl, 5 mM MgCl2, 1 g/liter fatty acid-free bovine
serum albumin (Sigma). Mitochondria (0.1-0.4 mg of protein/ml) were
incubated with 200 µM ATP and 100 µM propyl
gallate to inhibit the alternative oxidase. Oxidation was triggered by
the addition of succinate (10 mM) and then of ADP (from 25 to 100 µM).
Membrane Potential Measurements--
The membrane potential was
measured continuously and simultaneously with oxygen consumption
using a tetraphenylphosphonium (TPP+)-sensitive electrode
in the presence of 2 µM TPP+ (21). The values
of membrane potential were calculated using the equation of Kamo
et al. (21), without corrections for cation binding and
assuming a mitochondrial volume of 1 µl/mg of protein.
Determinations of Proton Conductance and Overall Control
Coefficients over Proton Fluxes--
Since in plant mitochondria the
protonmotive force (
p) is totally expressed as membrane
potential (
) (22, 23) and assuming both an entirely delocalized
protonmotive force (22) and no slip reactions of the proton pumps
(i.e. a constant H+/O stoichiometry of 6 for
succinate oxidation (24)), the proton conductance (CmH+),
expressed in nmol of
H+·min
1·mg
1
(protein)·mV
1, was estimated under
nonphosphorylating conditions by a ratio between 6 times the value of
the oxidation rate (Jo expressed in nmol of atomic
oxygen·min
1·mg
1
protein) and membrane potential. The flux control coefficients exerted
by each of the three subsystems involved in oxidative phosphorylation
(namely, the respiratory chain (c), the proton leaks
(l), and the block of phosphorylation (p)) over
the overall proton flux through the respiratory chain were determined
according to the top down approach of metabolic control analysis (25) using the protonmotive force (assimilated to membrane potential) as
common intermediate. The flux control coefficients exerted by each
block were deduced from oxidation rate measurements
(Jo) from which proton flux for each subsystem
(Jc, Jl, and
Jp) were derived using the relation
JH+ = Jo·H+/O
and from the determination of the overall elasticity coefficient,
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(Eq. 1)
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of the three subsystems, utilizing the equations of Hafner
et al. (25). In the nonphosphorylating state, control
coefficients were given by the relations,
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(Eq. 2)
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and
|
(Eq. 3)
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for the respiratory chain and the proton leaks, respectively. In
the phosphorylating state, control coefficients were given by the
equations,
|
(Eq. 4)
|
and
|
(Eq. 5)
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for the control exerted by the respiratory chain, the proton
leaks, and the block of phosphorylation. Experimentally, all determinations were carried out according to Kesseler et al.
(23). The elasticity coefficient of the respiratory chain to
p (
pc)
was determined by measuring succinate oxidation at several
p values obtained in the presence of various amounts of
hexokinase with ATP and glucose (500 µM each) in excess.
The elasticity of proton leaks to
p
(
pl) was determined in
the nonphosphorylating state by progressively inhibiting succinate
oxidation with increasing amounts of malonate (0-5 mM).
The elasticity of combined
p consumers (namely block of
phosphorylation and proton leaks) was determined in the presence of 1 mM ADP (maximal phosphorylating state) by progressively
inhibiting succinate oxidation with malonate. The elasticity
coefficient of the phosphorylation subsystem
(
pc) was determined by
an appropriated replot against
p (23).
Fluorescence Measurements--
The lateral diffusion of
1-pyrenedodecanoic acid (Molecular Probes, Inc., Eugene, OR), an
excimer-forming probe, was determined at different temperatures (from 5 to 35 °C) in purified mitochondrial membranes or in liposomes
according to Galla and Luisetti (26). Aliquots of membranes (100 nmol
of phospholipids) were suspended in 1.2 ml of 20 mM Mops, 1 mM EDTA, 5 mM MgCl2, 100 mM KCl (pH 7.2) medium. Successive additions of the probe
were made to give a molar ratio (probe/lipids) varying from 0.001 to
0.13. Microviscosity was assayed by measuring fluorescence
depolarization of 2-, 3-, 6-, 7-, 9-, or 12-(9-anthroyloxy) stearic
acid or 16-(9-anthroyloxy) palmitic acid (Molecular Probes) embedded in
mitochondrial membrane or liposomes. The probe/phospholipid molar ratio
was 0.02. The samples were incubated in the presence of probe at
20 °C for 20 min, and the measurements were carried out between 5 and 35 °C. Measurements were performed on an LS-5 Perkin-Elmer
spectrofluorimeter equipped with a thermostated device.
Cytochrome Content--
Cytochrome concentration was determined
by differential absorption spectrophotometry (reduced state minus
oxidized state) at room temperature, using an Aminco DW 2A
spectrophotometer. The specific absorption coefficients of cytochromes
a + a3 (602-630 nm), cytochromes
b (560-575 nm), and cytochrome c (550-540 nm) were 16, 20, and 19 mM
1·cm
1, respectively.
Phosphorus and Protein Analysis--
Lipid phosphorus content
was assayed according to Rouser et al. (27). Protein content
was determined according to Lowry et al. (28) using bovine
serum albumin as a standard.
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RESULTS |
Lipid Analysis and Cytochrome Content Determination--
As shown
in Fig. 1A, a drastic
reduction in the amounts of PUFA, linoleic acid (18:2), and linolenic
acid (18:3) was observed in fad2 mitochondria. In addition,
the amounts of palmitic acid (16:0) and stearic acid (18:0) were also
greatly lowered. As a consequence, the amount of oleic acid (18:1) was
considerably enhanced (~10 times), since it represented about 75% of
total fatty acids. Consequently, the PUFA/monounsaturated acid ratio was extremely reduced (about 40 times), and the double bond index dropped from 3.04 to 2.18. By contrast, lipid class analysis showed that the distribution of the phospholipids present in mitochondrial membranes (i.e. phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylinositol
(PI), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG)) was only slightly modified in
fad2 mitochondria (Fig. 1B); the
phosphatidylcholine/phosphatidylethanolamine ratio decreased from 1.5 to 1.1, and the amount of phosphatidylinositol was slightly reduced.
Fatty acid composition of individual lipids showed that each class of
lipid was differently affected by the fad2 mutation (Table
I). The main phospholipids,
phosphatidylcholine and phosphatidylethanolamine, possessed an
extremely high amount of 18:1 (
75%) and low amounts of PUFA as well
as of saturated fatty acids. DPG, which was the more unsaturated
phospholipid in wild type mitochondria, was altered in a comparable
way; the level of 18:1 increased to 70% and the amount of 18:2 dropped to less than 2%, but the level of 18:3 fatty acid still accounted for
~20%. In phosphatidylinositol and phosphatidylglycerol, changes induced by the mutation were characterized by a quasiabsence of PUFA,
by a decrease in the level of 18:0, and by an expected enhancement in
18:1 content. As a consequence, the double bond index of each phospholipid class was severely reduced in membranes from the mutant.
Beside these drastic changes, fad2 mitochondria exhibited a
significant increase of the lipid/protein ratio (Table
II), which was about 40% higher than in
the wild type, both in total mitochondria and submitochondrial
particles. This suggested that membrane proteins represented the same
fraction of total mitochondrial protein (about 30%). Consequently, the
fad2 mutation induced a significant decrease of the
mitochondrial membrane protein density. To further analyze this effect,
the cytochrome concentrations were determined by differential
absorption spectrophotometry. The total amounts of cytochromes
expressed per mg of protein were barely significantly different between
both genotypes (Table III). As a
consequence, the lipid/cytochrome ratio was enhanced in the fad2 mutant, indicating a specific reduction in redox
complex density that was consistent with the reduction in overall
protein density of these membranes.

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Fig. 1.
Fatty acid (A) and
glycerolipid (B) composition of mitochondrial
membranes isolated from wild type (open
columns) and fad2 (closed
columns) cells of A. thaliana. Separation
of the phospholipid classes was performed by thin layer chromatography
as indicated under "Materials and Methods." Fatty acids were
analyzed by gas chromatography after lipid saponification and
transmethylation. Values are the mean of two determinations on two
independent preparations ± the interval between the two
experimental values.
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Table I
Fatty acid composition of the various phospholipid classes found in
mitochondrial membranes from wild type (WT) and fad2 genotypes of
A. thaliana
Data correspond to the mean of values obtained in two independent
preparations. Differences between values of every couple were less than
10%. Separation of phospholipids from a total lipid extract was
performed by thin layer chromatography as indicated under "Materials
and Methods." Fatty acid content was assayed by capillary gas
chromatography after saponification and transmethylation. DBI, double
bond index = 2 × [(18:1) + (18:2 × 2) + (18:3 × 3)]/100 except for DPG: 4 × [(18:1) + (18:2 × 2) + (18:3 × 3)]/100. ND, not detectable. PC,
phosphatidylcholine; PE, phosphatidylethanolamine; DPG,
diphosphatidylglycerol; PI, phosphatidylinositol; PG,
phosphatidylglycerol.
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Table II
Lipid/protein ratio (w/w) in mitochondria and submitochondrial
particles (SMP) from wild type and fad2 mutant
SMP were obtained after sonication of intact mitochondria as described
under "Materials and Methods." Lipid content was assayed on a lipid
extract. Data correspond to the mean ± S.D. of five different
experiments.
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Table III
Total cytochrome content of mitochondria from wild type and fad2
genotypes of A. thaliana
Cytochrome content was determined from absorption of the -bands of
each cytochrome using the molar extinction coefficients given under
"Materials and Methods." The technique applied for these
determinations was differential absorption spectroscopy between the
reduced state (by dithionite) and the oxidized state of cytochromes.
Values are the mean of values obtained in two independent
preparations ± the interval between the two experimental values.
Lipid/cytochrome ratios were calculated using values of lipid/protein
ratios given in Table II and lipid content expressed in mol of
phosphate.
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This lower protein density could be a direct consequence of the
peculiar lipid composition of the fad2 membrane.
Alternatively, it could represent an adaptative tendency of the plant,
which could be considered as tropical (from its lipid unsaturation) to
a moderate temperature. To test these hypothesis, we isolated mitochondria from cells grown for 12 days at 12 °C. Contrary to what
was found at 22 °C, where lipid/protein ratios were different by
40%, the two ratios found at 12 °C fell in the range
0.40-0.45.2 This shows that
the reduction in this ratio was higher in fad2 membranes
than in wild type ones, suggesting that the lipid/protein ratio was not
solely controlled by the polyunsaturated fatty acid content.
Lipid Dynamics in Membranes and Liposomes--
Effects of
fad2 mutation on the dynamic properties of membrane lipids
were determined by measuring local microviscosity and lipid lateral
diffusion in mitochondrial membranes and liposomes prepared from
mitochondrial lipids. Microviscosity at different depths along the acyl
chains was studied measuring fluorescence anisotropy of
anthroyloxy-labeled fatty acids at 20 °C. In membranes from
fad2 mitochondria (Fig.
2A), the microviscosity was
higher at all of the positions, and the mobility gradient along the
chain was different. The "plateau" region extended down to the 9th
carbon in the mutant (instead of the 6th in the wild type), and then the motion became easier toward the center of the bilayer but with a
different profile. It is interesting to note that anisotropy values did
not vary significantly with the temperature in the range of
5-35 °C. Different results were found with liposomes prepared from
the whole mitochondrial lipids (Fig. 2B). Compared with
native membranes, the liposomes from both genotypes exhibited a higher
microviscosity at the 3rd carbon level, and then, down to the 12th
carbon, the microviscosity was not different in liposomal or
mitochondrial membranes. At the 16th carbon level, the microviscosity values in liposomes were lower than in native membranes. Moreover, the
difference was more pronounced in liposomes prepared with lipids from
wild type mitochondria. Nevertheless, in absolute value,
fad2 liposomes were more rigid than wild type liposomes.

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Fig. 2.
Local microviscosity at different depths in
mitochondrial membranes (A) and liposomes
(B) from wild type (open
symbols) and fad2 (closed
symbols) genotypes of A. thaliana. Liposomes were obtained by extrusion of a
suspension prepared from a total mitochondrial lipid extract. Local
microviscosity was assayed by measuring the fluorescence anisotropy of
anthroyloxy fatty acid derivatives with the fluorophore attached on
different carbon of the acyl chain. The probe/lipid molar ratio was
0.02. Data are the mean of four measurements made on a single
preparation (liposomes) or eight measurements made on two independent
preparations (mitochondria).
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The lateral diffusion of lipids within the bilayer was probed using a
pyrene-labeled fatty acid at different temperatures between 5 and
35 °C. The lipid diffusion coefficient was always higher in
fad2 (from 1.4-5.5 µm2
s
1) than in wild type (0.5-1.2
µm2 s
1) membranes (Fig.
3A). The activation energy of
lipid diffusion was 33 ± 3 kJ mol
1 and
18 ± 4 kJ mol
1 for fad2 and
wild type mitochondrial membranes, respectively (Fig. 3B),
indicating that the thermal dependence of lateral diffusion in
fad2 mitochondria was higher. By contrast, lateral diffusion rates in fad2 (from 2.5 to 6.7 µm2
s
1) or wild type (from 1.6 to 4.6 µm2 s
1) liposomes were much
less different, and the thermal dependence of the motion was the same
in both genotypes (about 37 ± 5 kJ mol
1). Interestingly, the activation energies
found in membranes and liposomes were very close for the mutant line,
whereas in wild type, these values differed by a factor of 2.

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Fig. 3.
Lipid lateral diffusion in mitochondrial
membranes (circles) and liposomes
(triangles) from wild type (open
symbols) and fad2 (closed
symbols) genotypes of A. thaliana. Lipid lateral diffusion was determined by
measuring the collision rate of 1-pyrenedodecanoic acid (see
"Materials and Methods"). A, diffusion rate as a
function of temperature. B, Arrhenius plots of exchange
frequencies (in MHz). Data are the mean of values obtained with two
independent preparations ± the interval between the two
experimental values.
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Functional Analysis--
To characterize the consequence of the
fad2 mutation on functional properties of
Arabidopsis mitochondria, various parameters of oxidative
phosphorylation such as oxidation rates, respiratory control, and
ADP/atomic oxygen ratio were determined. Under either nonphosphorylating (state 4) or phosphorylating (state 3) conditions, the oxidation rates (expressed per mg of protein) measured at 20 °C
were significantly lower (by about 20%) in fad2
mitochondria. However, if expressed versus cytochrome
content, no significant differences could be found between the two
systems (not shown). The yield of oxidative phosphorylation probed by
ADP/atomic oxygen ratio (1.4 ± 0.2) and respiratory control
(1.8 ± 0.2) were identical between both genotypes (average of
four independent determinations). Temperature differently affected the
oxidation rates in the two genotypes (Fig.
4). Between 5 and 30 °C, oxidation
rates (in nmol of O2·min
1·mg of
protein
1) varied from 20 to 155 in wild type and from 15 to 155 in fad2 mitochondria in state 4 (Fig. 4A)
and from 50 to 200 in wild type and from 30 to 170 in fad2
mitochondria in state 3 (Fig. 4B) (error on any value was
less than 10%). For both genotypes and in states 3 or 4, Arrhenius
plots were linear at least up to 25 °C (Fig. 4). This clearly
indicated that, although fad2 membranes were deprived from
polyunsaturated chains, no discontinuity occurred at low temperature
(i.e. below 15 °C). Moreover, this showed that the
activation energy was significantly higher in fad2
mitochondria under both energetic states: 66 ± 3 kJ·mol
1 instead of 57 ± 1 kJ·mol
1 in state 4 and 53 ± 2 kJ·mol
1 instead of 42 ± 2 kJ·mol
1 in state 3.

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Fig. 4.
Arrhenius plots of oxidation rates in
mitochondria from wild type (open symbols)
and fad2 (closed symbols)
genotypes of A. thaliana under nonphosphorylating
(A) and phosphorylating (B)
conditions. Respiration was measured with succinate as substrate
(see "Materials and Methods") and expressed in nmol of
O2·min 1·mg of protein 1.
Values are the mean of measurement made on two independent
preparations ± the interval between the two experimental
values.
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To further analyze the consequence of fad2 mutation on
bioenergetic properties of Arabidopsis mitochondria, the
proton conductance of the inner mitochondrial membrane was determined.
For this purpose, the flux-force relationship was established in
nonphosphorylating state by simultaneously measuring membrane potential
and oxidation rate with succinate as substrate and in the presence of
increasing amounts of malonate (23). Fig.
5A shows that a typical
nonlinear (nonohmic) relationship was obtained at 20 °C with
mitochondria from the wild type. However, the nonohmic character was
less pronounced in fad2 mitochondria (Fig. 5B).
Indeed, at the highest membrane potential, the oxidation rate (and
corresponding proton flux) was considerably reduced in fad2
mutant. As a consequence, the proton conductance was lower in membrane
from fad2 mitochondria (3.0 ± 0.3 nmol of
H+·min
1·mg of
protein
1·mV
1)
than in membrane from wild type mitochondria (5.0 ± 0.5 nmol of
H+·min
1·mg of
protein
1·mV
1).
Proton conductance in fad2 mitochondria was always lower
than in wild type ones between 5 and 30 °C (data not shown). In
addition, a higher activation energy of the proton conductance was
found in fad2 mitochondria (72 ± 7 kJ·mol
1) than in wild type ones (60 ± 6 kJ·mol
1).

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Fig. 5.
Plots of respiration rates versus membrane
potential under different incubation conditions to measure relative
elasticities of mitochondrial respiratory chain, proton leaks and block
of phosphorylation. Experiments were carried out with wild type
(A) and fad2 (B) genotypes of A. thaliana. The closed circles represent a
malonate titration of succinate oxidation during state 4 respiration
carried out in the presence of propyl gallate (100 µM) to
inhibit the alternative pathway and represent the dependence of
the proton leak on p. The open
circles correspond to steady states with different levels of
hexokinase (intermediate and noninhibited phosphorylating states) and
represent the dependence of respiratory chain rate on p.
The open triangles are a malonate titration of
respiratory rate and p in the presence of an excess of
ADP (inhibited phosphorylating state). The elasticity coefficients were
calculated as follows for each block of reactions,
|
(Eq. 6)
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Data correspond to a representative experiment carried out at
20 °C. Error was 10% for oxygen consumption and 5% for membrane
potential measurements.
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Since the fad2 mutation was responsible for changes in
metabolic and bioenergetic properties of Arabidopsis
mitochondria, we tried to get more insights in the oxidative
phosphorylation process using metabolic control analysis. The controls
exerted by substrate oxidation, by ATP turnover, and by proton leak on the respiration were investigated. For this purpose, in addition to the
proton leak titration reported above, the proton fluxes through the
respiratory chain and the phosphorylating system were measured. For
each system, the elasticity (i.e. the sensitivity of the
block to a small variation of membrane potential) was determined as
described under "Materials and Methods." An example of each of the
three titration curves is presented for wild type (Fig. 5A)
and fad2 (Fig. 5B) genotypes at 20 °C. The
control coefficients exerted by either the respiratory chain (substrate
oxidation), the block of phosphorylation (ATP turnover), or the proton
leaks over proton flux through the respiratory chain (oxidation rate) (Table IV) were derived from elasticity
measurements (25). Under nonphosphorylating conditions, the control was
shared only between the proton leaks and the respiratory chain. This
latter was predominant (almost 70%) in wild type mitochondria. In
contrast, respiration of fad2 mitochondria was mainly
controlled by proton leaks (70%). Under phosphorylating conditions and
in both genotypes, the control was mainly exerted by the respiratory
chain (55%) and the phosphorylating subsystem (30%), the control
exerted by the proton leaks being low (15%).
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Table IV
Control coefficients exerted by the proton leak, the respiratory chain
and the phosphorylation subsystem over respiration rate in wild type
and fad2 mitochondria of A. thaliana
According to the top-down metabolic control analysis, the oxidative
phosphorylation system can be divided into two (in state 4) or three
blocks (in state 3) of reactions linked by the common intermediate
p, the substrate oxidation system (respiratory chain)
producing p, and the two blocks that consume the
p, either without ATP synthesis (proton leaks) or coupled
to ATP synthesis (the block of phosphorylation). The analysis is based
on measuring the relative elasticities of the blocks to p
(cf. Fig. 5) from which the control coefficients can be
derived. The set of equations used to calculate the control
coefficients exerted by each block over respiration rate is given under
"Materials and Methods." , no phosphorylation occurred in state 4 as checked by insensitivity to oligomycin of oxygen consumption rate in
A. thaliana mitochondria. Data are the mean of values
obtained with two independent preparations ± the interval between
the two experimental values.
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 |
DISCUSSION |
The availability of Arabidopsis mutants with altered
desaturase functions provides a genetic approach to investigate the
role of PUFA in the structure and function of membranes (16). In this
study, we used the fad2 mutant of Arabidopsis,
deficient in reticulum oleate desaturase activity that is responsible
for production of PUFA through the eukaryotic pathway of lipid
synthesis (3), to reexamine the role of lipids unsaturation in
mitochondrial respiration. This mutant is killed by low temperature (5,
17), and authors have suggested that this sensitivity resulted from disfunction in specific processes in membrane metabolism rather than
from membrane disruption. We thus focused our attention on the
mitochondrial compartment that plays a crucial role in energy production.
As expected, the fatty acid composition of mitochondrial membranes was
drastically changed in fad2 cells. The content in saturated species, i.e. palmitic and stearic acids, was more than 2 times diminished. Conversely, unsaturated species increased and
distributed as can be expected after the quenching of the oleate
desaturase activity. Indeed, oleic acid accounted for more than 75% of
the fatty acid content, while PUFA were reduced at 12%. This clearly shows that mitochondria do not possess an autonomous desaturase system
besides those present in the endoplasmic reticulum or plastidial membranes. Surprisingly, the amount of 18:3 fatty acid still present in
fad2 mitochondrial membranes remained significant (~10%),
contrary to that of 18:2 species. An explanation would be that the
presence of a residual activity of oleate desaturase (29) provided some 18:2 fatty acid, immediately desaturated in 18:3 fatty acid by a fully
active linoleate desaturase. Linolenic acid, being the final product,
accumulated, but its content among the different phospholipids
decreased at least by 50% when compared with control membranes.
An unexpected consequence of the mutation was an increase of ~40% in
the lipid/protein mass ratio in the membranes. This lower protein
density in the bilayer was associated with a specific reduction
of redox complexes as measured by the cytochrome content.
The impact of the fad2 mutation on lipid dynamic depends on
the type of lipid motion. The microviscosity at different depths of the
monolayer, probed by fluorescence depolarization of anthroyloxy fatty
acid derivatives, was higher in membranes from fad2
mitochondria or liposomes made from their lipid extract than in the
wild type counterparts. This result was in good agreement with the
widely accepted concept of a decrease in the unsaturation of
phospholipid acyl chains going along with an increase of microviscosity
(30). However, the presence of proteins influenced the dynamic
characteristics of the membrane lipid layer; in the deeper part of the
bilayer, and not toward its surface, the microviscosity was higher in
membranes than in liposomes, suggesting that the presence of protein
hampered the lipid motions toward the bilayer center. This effect was
more pronounced in wild type than in fad2, which was
probably due to the difference in protein density. It can be noticed
that the activation energy of the local motions of fatty acyl chains
was extremely low, indicating that microviscosity was not very
dependent on temperature changes (30).
Lipid lateral diffusion in fad2 mitochondrial membranes was
3-5 times higher than in wild type ones. By contrast, lipid lateral diffusion measured in liposomes made from the mitochondrial lipids showed that (i) the difference between the two membranes was much smaller (50% only); (ii) there was no difference between liposomes and
mitochondrial membranes in fad2 system, indicating that the high lipid mobility found in mutant mitochondrial membranes was mainly
due to its low protein density. This hypothesis is in good agreement
with reports showing that lateral diffusion of lipids in complex
membranes is significantly reduced when proteins are incorporated in
the bilayer (31, 32). On the other hand, it is interesting to note that
the activation energy of lipid lateral diffusion was lower in wild type
mitochondrial membranes than in fad2 membranes or in
liposomes. An explanation would be that the high protein content of
wild type membranes generated defects that would attenuate the
temperature effect on the lipid diffusion. Thus, protein content
influenced in an independent manner the rate and the thermal dependence
of lipid diffusion.
fad2 mutation also affects mitochondrial bioenergetics.
Respiratory rates were reduced in the mutant if expressed per membrane protein amount but not significantly different if expressed per nmol of
cytochromes. However, the difference between the wild type and the
mutant mitochondrial respiration was not constant with the assay
temperature: a higher marked difference was found at low temperature,
as expected from the difference in fatty acid composition of the
membranes. However, although fad2 membrane contains 50% of
dioleoyl phospholipids, no discontinuities could be detected in the
response of respiration to temperature. This latter result is similar
to what was described with mitochondria from Sycomore cells whose fatty
acid composition was modified in vitro (12). The variation
with temperature of the difference between the two mitochondria was
reflected by a difference in the activation energies
(Ea) in state 4 as well as in state 3 of the
respiration; in each case, Ea was higher in
fad2 mitochondria. Thus, changes in fad2 membrane
have affected the respiration regulation by temperature, as they have
modified the diffusion properties of the lipid phase. Moreover,
Ea of respiration was higher than
Ea of diffusion, suggesting that quinone diffusion
(equivalent to the lipid diffusion) (33) was not a limiting step in
respiration, as previously reported for mammalian mitochondria
(13).
As a consequence of membrane changes, the proton permeability was lower
in fad2 than in wild type mitochondria, and the control exerted by the membrane proton leaks in state 4 was enhanced. But as
soon as another proton pathway, independent of the bilayer, was
involved (i.e. the ATP synthase in state 3), differences
were no longer apparent.
However, it is difficult to precisely determine which of the changes
consecutive to the mutation (fatty acid composition or lipid/protein
ratio) was the most important for the functional differences detected.
For instance, it has been reported that there is no correlation between
liposomal proton permeability and phospholipid fatty acid composition
and that the proton pathway through the bulk phospholipid bilayer
accounts for a small proportion of the total mitochondrial proton leak
(14, 15). Consequently, our results are better explained, assuming that
the low protein density in fad2 mitochondrial membranes was
mainly responsible for the reduction in proton leak. Similarly, the
lipid lateral diffusibility appeared to be more controlled by the
lipid/protein ratio than by the fatty acid content, since there was a
higher difference between mitochondrial membranes than between
liposomal membranes. In fact, only lipid microviscosity seemed to be
mainly influenced by the unsaturation degree of the bilayer. Thus, it appears that the enhancement in lipid/protein ratio noticed in fad2 mitochondrial membranes from cells grown at 22 °C
would be responsible for the major part of the functional or dynamic
modifications of the mitochondria.
It could be hypothesized that this high lipid/protein ratio is an
adaptative mechanism to growth at 22 °C of a plant seemingly adapted
to high temperature, because of its high content in monounsaturated acid, and unable to synthesize PUFA. However, growth at a lower temperature (12 °C) showed that fad2 membranes can be as
rich in protein as are wild type membranes. This suggests that the high
lipid/protein ratio is not a general feature of the mutant but a
characteristic of fad2 mitochondrial membranes at 22 °C and that other parameters are involved in the control of the membrane protein content at low temperature. Experiments are currently under way
to determine these parameters.