Consequences of omega -6-Oleate Desaturase Deficiency on Lipid Dynamics and Functional Properties of Mitochondrial Membranes of Arabidopsis thaliana*

Olivier Caiveau, Dominique Fortune, Catherine Cantrel, Alain Zachowski, and François MoreauDagger

From the Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes UMR 7632 CNRS, Université Pierre et Marie Curie, Paris, France

Received for publication, July 13, 2000, and in revised form, November 10, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We probed the role of the polyunsaturated fatty acids on the dynamic and functional properties of mitochondrial membranes using the fad2 mutant of Arabidopsis thaliana, deficient in omega -6-oleate desaturase. In mitochondria of this mutant, the oleic acid content exceeded 70% of the total fatty acids, and the lipid/protein ratio was greatly enhanced. As a consequence, local microviscosity, probed by anthroyloxy fatty acid derivatives, was increased by 30%, whereas the lipid lateral diffusion, assayed using 1-pyrenedodecanoic acid, was approximately 4 times increased. Functional parameters such as oxygen consumption rate under phosphorylating and nonphosphorylating conditions and proton permeability of the inner mitochondrial membrane were significantly reduced in fad2 mitochondrial membranes, while the thermal dependence of the respiration was enhanced. Moreover, metabolic control analysis of the respiration clearly showed an enhancement of the control exerted by the membrane proton leaks. Our data suggest that the loss of omega -6-oleate desaturase activity in Arabidopsis cells induced an enhancement of both microviscosity and lipid/protein ratio of mitochondrial membranes, which in turn were responsible for the change in lateral mobility of lipids and for bioenergetic parameter modifications.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta p) is totally expressed as membrane potential (Delta Psi ) (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,
ϵ<SUP>J<SUB><UP>o</UP></SUB></SUP><SUB>&Dgr;p</SUB>=(<UP>d</UP>J<SUB><UP>o</UP></SUB><UP>/d&Dgr;</UP>p)×(&Dgr;p/J<SUB><UP>o</UP></SUB>) (Eq. 1)
of the three subsystems, utilizing the equations of Hafner et al. (25). In the nonphosphorylating state, control coefficients were given by the relations,
C<SUP>J<SUB><UP>o</UP></SUB></SUP><SUB>c</SUB>=ϵ<SUP>l</SUP><SUB>&Dgr;p</SUB>/<FENCE>ϵ<SUP>l</SUP><SUB>&Dgr;p</SUB>−ϵ<SUP>c</SUP><SUB>&Dgr;p</SUB></FENCE> (Eq. 2)
and
C<SUP><UP>J</UP><SUB><UP>o</UP></SUB></SUP><SUB>l</SUB>=ϵ<SUP>c</SUP><SUB>&Dgr;p</SUB>/<FENCE>ϵ<SUP>c</SUP><SUB>&Dgr;p</SUB>−ϵ<SUP>l</SUP><SUB>&Dgr;p</SUB></FENCE> (Eq. 3)
for the respiratory chain and the proton leaks, respectively. In the phosphorylating state, control coefficients were given by the equations,
C<SUP>J<SUB><UP>c</UP></SUB></SUP><SUB>c</SUB>=<FENCE>ϵ<SUP>p</SUP><SUB>&Dgr;p</SUB>×J<SUB>p</SUB>+ϵ<SUP>l</SUP><SUB>&Dgr;p</SUB>×J<SUB>l</SUB></FENCE>/<FENCE>ϵ<SUP>p</SUP><SUB>&Dgr;p</SUB>×J<SUB>p</SUB>+ϵ<SUP>l</SUP><SUB>&Dgr;p</SUB>×J<SUB>l</SUB>−ϵ<SUP>c</SUP><SUB>&Dgr;p</SUB>×J<SUB>c</SUB></FENCE> (Eq. 4)
and
C<SUP>J<SUB>c</SUB></SUP><SUB>l</SUB>=J<SUB>l</SUB>×<FENCE>1−C<SUP>J<SUB>c</SUB></SUP><SUB>c</SUB></FENCE>/J<SUB>c</SUB> <UP>and</UP> C<SUP>J<SUB>c</SUB></SUP><SUB>p</SUB>=J<SUB>p</SUB>×<FENCE>1−C<SUP>J<SUB>c</SUB></SUP><SUB>c</SUB></FENCE>/J<SUB>c</SUB> (Eq. 5)
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 Delta p (epsilon Delta pc) was determined by measuring succinate oxidation at several Delta 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 Delta p (epsilon Delta pl) was determined in the nonphosphorylating state by progressively inhibiting succinate oxidation with increasing amounts of malonate (0-5 mM). The elasticity of combined Delta 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 (epsilon Delta pc) was determined by an appropriated replot against Delta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

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).

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.

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.

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 Delta 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 Delta p. The open triangles are a malonate titration of respiratory rate and Delta p in the presence of an excess of ADP (inhibited phosphorylating state). The elasticity coefficients were calculated as follows for each block of reactions,
ϵ<SUP>J<SUB>o</SUB></SUP><SUB>&Dgr;p</SUB>=(<UP>d</UP>J<SUB>o</SUB>/<UP>d</UP>&Dgr;p)×(&Dgr;p/J<SUB>o</SUB>) (Eq. 6)
Data correspond to a representative experiment carried out at 20 °C. Error was 10% for oxygen consumption and 5% for membrane potential measurements.

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 Delta p, the substrate oxidation system (respiratory chain) producing Delta p, and the two blocks that consume the Delta 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 Delta 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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes UMR 7632 CNRS, Université Pierre et Marie Curie, case 154, 4 place Jussieu, 75252 Paris cedex 05, France. Tel.: 33 1 44 27 59 20; Fax: 33 1 44 27 61 51; E-mail: fmoreau@ccr.jussieu.fr.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M006231200

2 O. Caiveau, D. Fortune, C. Cantrel, A. Zachowski, and F. Moreau, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: PUFA, polyunsaturated fatty acid(s); Mops, 4-morpholinepropanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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