Genetically Modified Arabidopsis thaliana Cells ... - Oxford Journals

Genetically Modified Arabidopsis thaliana Cells Reveal the Involvement of the Mitochondrial Fatty Acid Composition in Membrane Basal and Uncoupling Protein-Mediated Proton Leaks Regular Paper

Cécile Hourton-Cabassa1,∗, Ana Rita Matos1,2, Joao Arrabaça2, Chantal Demandre1, Alain Zachowski1 and François Moreau1 1Université

Pierre et Marie Curie (Paris 6), CNRS UR 5, 4 place Jussieu, 75005 Paris, France de Biologia Vegetal, Bio FIG, Faculdade de Ciências da Universidade de Lisboa, Centro de Engenharia Biológica, Campo Grande, 1749-016 Lisboa, Portugal 2Departamento

We investigated the role of membrane fatty acids in basal proton leaks and uncoupling protein (UCP)-dependent proton conductance in Arabidopsis mitochondria. Using wild-type cells, cold-sensitive fad2 mutant cells, deficient in ω-6-oleate desaturase, and cold-tolerant FAD3+ transformant cells, overexpressing ω-3-linoleate desaturase, we showed that basal proton leak in the non-phosphorylating state was dependent on lipid composition. The extent of membrane proton leak was drastically reduced in the fad2 mutant, containing low amounts of polyunsaturated fatty acids. Conversely, this proton leak was higher in FAD3+ mitochondria that exhibit a higher polyunsaturated fatty acid content and high protein to lipid ratio. The dependency of membrane leaks upon membrane potential was higher in FAD3+ and lower in fad2. UCP content was higher in both the fad2 mutant and FAD3+ transgenic lines compared with wildtype cells and so was the UCP activity, assayed by the reduction of phosphorylation yield (ADP/O) triggered by palmitate as UCP activator. This UCP assay was validated by measurements of UCP–proton leak in the non-phosphorylating state (flux–force relationships between proton flux and membrane potential). The potential uncoupling capacity of the UCP was high enough to allow the loss of respiratory control in the three genotypes. Taken together, the data reported here suggest that the cold tolerance of FAD3+ cells and the cold sensitivity of fad2 cells are associated with changes in their mitochondrial membrane basal proton leaks, whereas differences in functional expression of UCP are not simply related to cold adaptation in Arabidopsis cells. ∗Corresponding

Keywords: Arabidopsis thaliana • Cold sensitivity • Lipid desaturase mutants • Membrane and fatty acid composition • Membrane conductance • Uncoupling protein. Abbreviations: AOX, alternative oxidase; BSA, bovine serum albumin; DBI, double bond index; TPP+, tetraphenylphosphonium; UCP, uncoupling protein; WT, wild type.

Introduction Oxidative phosphorylation is affected by lipid composition and fatty acid unsaturation of the inner mitochondrial membrane (Daum 1985, De Santis et al. 1999, Matos et al. 2009). In plants, the membrane phospholipid composition is temperature dependent, and unsaturation of fatty acids influences the membrane physical state and functional properties (Goubern et al. 1990, Fontaine et al. 1996, Caiveau et al. 2001, Vaultier et al. 2006), although the relationships between fatty acid composition and functional properties of membranes are far from being understood. Nevertheless, progress can be made by using an approach based on genetic modifications of membrane lipid composition of plant cells. Plants synthesize fatty acids de novo and desaturate them by two independent pathways (Wallis and Browse 2002): the prokaryotic pathway, located in plastids, and the eukaryotic pathway, associated with the endoplasmic reticulum (responsible for the fatty acid desaturation in extraplastidial membranes, including mitochondria). In previous works we used the low temperature-sensitive fad2 mutant cells of Arabidopsis thaliana, deficient in oleate

author: E-mail: [email protected]; Fax: +33-1-44-27-61-51.

Plant Cell Physiol. 50(12): 2084–2091 (2009) doi:10.1093/pcp/pcp144, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

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Plant Cell Physiol. 50(12): 2084–2091 (2009) doi:10.1093/pcp/pcp144 © The Author 2009.

Mitochondrial fatty acid composition and H+ leaks

desaturase of the eukaryotic pathway, to analyze the physical and functional properties of mitochondrial membranes (Caiveau et al. 2001, Vaultier et al. 2006). We also used FAD3+ suspension-cultured cell lines overexpressing ω-3-linoleate desaturase to investigate the impact of membrane fatty acid composition on cell growth and mitochondrial respiration, with special attention to the alternative oxidase (AOX) (Matos et al. 2007). Changes in membrane composition were accompanied by modifications in both the membrane physical state and the rate of the cytochrome electron transport pathway. Cell growth rates under cold stress were also affected. As a consequence, FAD3+ cells exhibited a relatively better cold tolerance compared with the wild type (WT), whereas the fad2 genotype was cold sensitive and behaved like cells adapted to high temperature. Here, we focused on membrane basal proton leaks and uncoupling protein (UCP)-dependent proton leaks. In Arabidopsis, UCP belongs to a multigenic family (HourtonCabassa et al. 2004, Borecky et al. 2006). Unlike their mammalian homolog UCP1, responsible for thermogenesis in brown adipose tissue, the activities and physiological roles of plant UCPs are poorly understood. Increasing evidence suggests that UCP1 homologs are involved in preventing formation of reactive oxygen species by the respiratory chain under different stress conditions (Brandalise et al. 2003a, Brandalise et al. 2003b, Considine et al. 2003, Goglia et al. 2003). Recently, it has been shown that plant UCP1 plays a crucial role in photorespiration metabolism (Sweetlove et al. 2006). In various isolated mitochondria, free fatty acid-dependent uncoupling processes, resulting from increased membrane proton leaks, have been ascribed to UCP. These leaks were generally not sensitive to nucleotides, putative UCP inhibitors (Borecky et al. 2001, Hourton-Cabassa et al. 2002). More recently, it was shown that UCP activation led to a nucleotide-sensitive decrease in ATP synthesis measured by the ADP/O ratio (Navet et al. 2005). In this work, we report on the impact of membrane fatty acid composition on membrane basal or UCP-dependent proton leaks using mitochondria isolated from cell cultures of Arabidopsis Col 0 (WT), fad2 and FAD3+ genotypes. We assessed UCP activity in the phosphorylating state by measuring the reduction of the phosphorylation yield triggered by exogenous free fatty acid to activate UCP fully, and we established proton conductance curves by varying membrane potential in the non-phosphorylating state. Our results show that the opposite changes in polyunsaturated fatty acid content of mitochondrial phospholipids occurring in the fad2 and FAD3+ mutants affect the basal proton leaks whereas the extent of UCP-dependent leaks is not directly related to either changes in lipid membrane composition or the adaptation to cold stress, but rather to UCP content.

Results Respiratory parameters Table 1 shows the differences of membrane fatty acid composition found in mitochondria isolated from fad2 and FAD3+ genotypes represented by their double bond index (DBI), calculated as detailed in the Materials and Methods and according to previous data (Matos et al. 2007). Compared with the WT, the fad2 mitochondrial DBI was reduced by 35%, due to the high 18 : 1 content (50% of the total fatty acids), whereas that of FAD3+ mitochondria was slightly increased (8%), as a consequence of the linoleate desaturase overexpression in this genotype. Oxidation rates in the phosphorylating state (state 3) were slightly higher in FAD3+ compared with the WT and fad2. In the non-phosphorylating state (state 4), the oxidation rate was significantly lower in fad2 mitochondria compared with the WT and FAD3+ (by 40 and 50%, respectively), whereas the membrane potential was slightly lower in the WT. Thus, the respiratory control was higher in fad2 (3.0 ± 0.3) than in WT (2.0 ± 0.1) and FAD3+ mitochondria (1.8 ± 0.2). Moreover, fad2 mitochondria exhibited the highest values of the ADP/O ratio.

Membrane basal proton leaks Using data reported in Table 1 (i.e. the oxidation rate and membrane potential in state 4), maximal values of membrane proton leaks were calculated (see the Materials and Methods). The highest proton leaks were found in FAD3+ mitochondria [5.60 ± 0.5 nmol H+ min−1 mg (protein)−1 mV−1] while fad2 mitochondria exhibited lower proton leaks [2.80 ± 0.2 nmol H+ min−1 mg (protein)−1 mV−1] than those of the WT [4.9 ± 0.5 nmol H+ min−1 mg (protein)−1 mV−1]. To determine membrane basal proton leaks at different values of the membrane potential, the flux–force relationships between the oxidation rate and membrane potential were established in non-phosphorylation states as describe in the Materials and Methods (Fig. 1). In the WT, fad2 or FAD3+ mitochondria, typical non-linear (non-ohmic) relationships were obtained. Taking into account that CmH+ can be directly calculated from these relationships {CmH+ = [(rate of oxygen uptake) (H+/O)]/∆Ψ}, these data indicate an increase in membrane proton conductance for membrane potential ranging between state 3 and state 4 values (Fig. 1 and Table 1). At membrane potential values close to state 3 (different for each genotype: between −185 and −200 mV for the WT, between −200 and −215 mV for fad2 and between −195 and −210 mV for FAD3+), the nonohmic character of the curve was more pronounced in FAD3+ than in the WT, and less accentuated in fad2.

UCP expression and phosphorylation yield Immunodetection analysis presented in Fig. 2A indicates that UCP was more abundant in fad2 and FAD3+ mitochondria


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Table 1 Membrane features and functional parameters of mitochondria isolated from WT, fad2 and FAD3+ cells WT

DBIa

3.5

fad2

FAD3+

2.2

3.8

Oxidation rate nmol O2 min–1 mg (protein)–1

Parameter

150

Oxidation rateb State 3

178 ± 7

161 ± 25

190 ± 20

State 4

87 ± 5

53 ± 6

103 ± 12

Respiratory control

2.0 ± 0.1

3.0 ± 0.3

1.8 ± 0,2

ADP/O

1.15 ± 0.05

1.3 ± 0.05

1.10 ± 0.05

∆Ψstate 4c

210 ± 6

225 ± 7

220 ± 8

∆Ψstate 3d

190 ± 5

205 ± 5

200 ± 5

Oxidation rates were measured in state 3 and state 4 using a combination of succinate plus NADH as respiratory substrates, and respiratory controls were calculated from these values. Data are the average ± SD of ≥3 independent mitochondrial preparations. aDouble bond index calculated according to the formula: DBI = 2 (1[18 : 1] + 2[18 : 2] + 3[18 : 3])/100. bIn nmol. O min−1 mg (protein)−1. 2 cMembrane potential in state 4 expressed in mV. dMembrane potential in state 3 expressed in mV.

A

WT

fad2

FAD3+ 100 WT

fad 2 50

0

B

O2 ADP 1

32 kDa

∆ψ

b

Pal

a

C ADP

2

100 ADP/O (%/contorl)

220

Fig. 1 Dependence of membrane proton leaks on membrane potential in wild-type (triangles), fad2 (circles) and FAD3+ (squares) Arabidopsis mitochondria. The relationships between the oxidation rate and membrane potential were established by progressive inhibition of state 4 respiration by KCN using NADH as substrate. Data are the average from three independent experiments.

FAD3+

UCP

160 180 200 Membrane potential (mV)

140

90 b

WT

80

GTP Pal

70 FAD3+ 60

a

3

ADP

fad2 50 b 4 6 8 0 2 PAL / prot. ratio (x10–2 mg mg–1)

25 µM O2

a 1min

Fig. 2 UCP expression and ADP/O ratio in Arabidopsis mitochondria treated by palmitate. (A) Immunodetection of UCP proteins was performed on mitochondria isolated from 6-day-old wild-type, fad2 and FAD3+ cells cultured at 22°C. In each lane, 40 µg of total mitochondrial proteins were separated by SDS–PAGE before hybridization with specific antibodies as described in the Materials and Methods. (B) Effect of palmitate and GTP on the state 3/state 4 transitions triggered by addition of a limiting amount of ADP (50 µM) on mitochondria from the wild-type genotype treated or not by palmitate or palmitate plus (1 mM) GTP, using a palmitate/protein ratio of 7.1 × 10−2 (mg mg−1). Oxidation rates and membrane potential were measured simultaneously as described in the Materials and Methods. (C) Effect of increasing palmitate concentrations, expressed as the palmitate to protein ratio (mg mg−1), on the ADP/O ratio in wild-type (triangles), fad2 (circles) and FAD3+ (squares) mitochondria.

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Palmitate-dependent proton flux and UCP-dependent leak To correlate the decrease of phosphorylation yield triggered by palmitate to membrane proton leak induced by UCP, we established the flux–force relationships in the presence

24

FAD3+ + PAL

A

WT + PAL Proton flux

18

fad2 + PAL

FAD3+ 12 [x102 nmol H+ min–1 mg (protein)–1]

UCP- dependent proton flux

than in the WT, the highest level being found in fad2. This is mainly due to UCP1 gene expression in all genotypes (data not shown). In vitro assay of UCP activity requires activation by exogenous free fatty acids. Here we used palmitate as previously reported (Hourton-Cabassa et al. 2002, Navet et al. 2005). Since nucleotides (GTP or GDP) should inhibit UCP-dependent proton leaks activated by exogenous fatty acids in phosphorylating states, we used a UCP assay revealing this specific feature, namely the effect of palmitate on the ADP/O ratio, as suggested recently (Navet et al. 2005). In Fig. 2B, the state 3/state 4 transitions triggered by addition of a limiting amount of ADP (50 µM) to WT mitochondria treated or not by palmitate (palmitate/protein ratio of 7.1 × 10−2 mg mg−1) or palmitate plus 1 mM GTP were followed by simultaneous measurements of (i) the oxidation rate and (ii) the membrane potential. Under conditions of palmitate-dependent mild uncoupling, the extent of the depolarization triggered by ADP addition was reduced and the time course of state 3 was enhanced (traces 1 and 2). The resulting respiratory control was close to 1 and the ADP/O ratio was lower by approximately 25%. In the presence of GTP plus palmitate, the time course of state 3 was significantly reduced and the value of the ADP/O ratio was restored to around 80% (trace 3) that of the control (trace 1). Variations in the time course of depolarization occurring in state 3 respiration (upon addition of a limiting amount of ADP) fitted with the changes in oxygen consumption. Based on these data, the mitochondrial uncoupling induced by palmitate can reasonably be ascribed to UCP-dependent proton leak, as previously reported in potato tuber mitochondria (Navet et al. 2005). It can also be mentioned that, as expected (Hourton-Cabassa et al. 2002, Navet et al. 2005), during the state 4 respiration that followed ADP exhaustion, GTP was unable to restore the membrane potential and oxidation rate prevailing in control mitochondria. When the ADP/O ratio (expressed as a percentage of control) was determined at an increasing palmitate/protein ratio (2.0 × 10−2−8 × 10−2 mg mg−1), its reduction was more pronounced in fad2 and FAD3+ than in WT mitochondria (Fig. 2C). For a given palmitate/protein ratio (5.0 × 10−2 mg mg−1), the reduction of the phosphorylation yield was 15, 28 and 40% in the WT, FAD3+ and fad2, respectively. Comparison between Fig. 2A and C showed a correlation between the relative UCP content and palmitate-induced reduction of phosphorylation yield.

WT fad2

6

0 FAD3+

B 18

fad2 WT 12

6

0 140

160 180 200 Membrane potential (mV)

220

Fig. 3 Relationships between proton fluxes and membrane potential in wild-type (triangles), fad2 (circles) and FAD3+ (squares) mitochondria treated or not with palmitate. (A) Curves were established in progressively inhibiting state 4 respiration by KCN using NADH as substrate in the absence or in the presence of palmitate. The palmitate to protein ratio was adjusted to provide a similar reduction in the ADP/O ratio in wild-type (triangles), fad2 (circles) and FAD3+ (squares) mitochondria: 7.1 × 10−2, 2.6 × 10−2 and 4.5 × 10−2 mg mg (protein)−1, respectively. (B) Relationships between UCP-dependent proton fluxes and membrane potential in wild-type (triangles), fad2 (circles) and FAD3+ (squares) mitochondria calculated from the data obtained in A.

of palmitate, in amounts leading to a 25% reduction in the ADP/O ratio [corresponding to 8.0 × 10−2, 3.0 × 10−2 and 4.5 × 10−2 mg palmitate mg (protein)−1 for the WT, fad2 and FAD3+, respectively] as shown in Fig. 2C. Relationships between the total proton flux obtained in the presence of palmitate and the membrane potential (Fig. 3) were not linear (non-ohmic), especially when the membrane potential was comprised between state 3 and state 4 values. For membrane potentials between −170 and −200 mV, proton fluxes were the highest in the WT, whereas at maximal


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membrane potential the highest proton fluxes occurred in FAD3+ (Fig. 3A). Comparing palmitate-dependent depolarization at constant proton fluxes (ordinate) in the three genotypes (Fig. 3A), the highest UCP contribution to total depolarization (abscissa) was found in fad2 and the lowest in the WT. The UCP-dependent proton conductance curves (i.e. UCP-dependent proton fluxes vs. membrane potential) were established after subtracting the basal proton fluxes from the total proton fluxes determined with palmitate. The same relationship was obtained in the three genotypes (Fig. 3B). Taking into account the prevailing conditions of this assay in which similar reductions in the ADP/O ratio were induced in the three genotypes, the similar UCP-dependent proton leaks measured in Fig. 3B demonstrate a complete correlation between reduction of phosphorylation yield and UCP-dependent proton leaks, thus validating the ADP/O reduction assay as a good measure of UCP activity and, in fact, of amounts of activated UCP. The respective contribution of UCP-dependent proton leaks and membrane leaks in state 4 were also calculated using data reported in Figs. 1 and 3. UCP-dependent proton leaks [6.1 ± 0.1, 6.6 ± 0.2, 9.1 ± 0.5 nmol H+ min−1 mg (protein)−1 mV−1 in the WT, fad2 and FAD3+, respectively] always exceeded membrane basal proton leaks [4.9 ± 0.5, 2.8 ± 0.2, 5.6 ± 0.5 nmol H+ min−1 mg (protein)−1 mV−1, respectively].

Discussion This work was undertaken to investigate the role of membrane fatty acid composition in basal proton leaks and the UCP-dependent proton conductance in Arabidopsis mitochondria. We used a FAD3+ cell line overexpressing the ω-3-linoleate desaturase which displays highly unsaturated fatty acids in membrane lipids, and an fad2 mutant cell line, deficient in oleate desaturase, with a low level of polyunsaturated fatty acids in its membranes (Caiveau et al. 2001, Matos et al. 2007). In addition, the membranes of fad2 mitochondria exhibited a lower protein to lipid ratio and higher membrane rigidity potential than FAD3+ mitochondria, this latest point being important for the cold sensitivity observed for fad2 and the cold tolerance observed for FAD3+ (Matos et al. 2007).

Membrane basal proton leak We measured the flux–force relationships (proton flux vs. membrane potential) to analyze proton leaks in fad2 and FAD3+ mitochondria; our results show that the membrane basal protons leaks are dependent upon the lipid membrane composition. In fad2 mitochondria, the low values of membrane proton fluxes in the non-phosphorylating state can be ascribed to a low membrane proton permeability and are

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responsible, at least in part, for the cold sensitivity of this genotype. In contrast, FAD3+ membrane proton leak was higher at maximal membrane potential, but was dramatically reduced at lower membrane potentials, such as those prevailing in phosphorylating states, indicating that proton leaks were more dependent upon membrane potential in this genotype. This complex behavior could contribute to the cold tolerance of this FAD3+-overexpressing line. Indeed, in the non-phosphorylating state, high proton leaks in FAD3+ allow high rates of respiration compatible with potentially important carbon fluxes at low temperatures. In addition, the high respiration rate of FAD3+ observed in phosphorylating state 3 allows higher rates of ATP synthesis than in the WT and fad2 at 22°C and is compatible with previous results showing that the extent of reduction of the phosphorylating state 3 respiration rate under cold stress is important in the cold-sensitive fad2 cells but does not occur in the coldtolerant FAD3+ cells (Matos et al. 2007).

UCP-dependent proton leak In animals, UCP activity plays a crucial role in modulating oxidative phosphorylation and dissipating energy as heat in response to cold (for a review, see Bouillaud et al. 2001). Similarly, plant UCP involvement in cold resistance has been hypothesized (for a review, see Borecky et al. 2006, HourtonCabassa and Moreau 2008). Here, we used the fad2 and FAD3+ Arabidopsis lines as a model of a cold-sensitive and a cold-resistant plant, respectively, to address this question. It appeared that UCP content is higher in both cell lines, suggesting that higher expression levels of UCP are not correlated to cold adaptation in these cells. Indeed, transcriptomic analyses suggested that expression of UCP mRNAs was not affected by cold stress in Arabidospsis (Hourton-Cabassa and Moreau 2008) and immunodetection analyses showed that, after a 4 d cold stress at 9°C, UCP protein content was unchanged in WT cells and lower in fad2 (data not shown). However, in the case of Arabidopsis leaves, higher UCP contents were recently found after long-term exposure to cold (5°C), indicating the possible involvement of UCP during cold acclimation (Armstrong et al. 2008). It is noteworthy that the AOX, another energy-dissipating system present in plant mitochondria, is repressed in the two Arabidopsis mutants under the same cell culture conditions (Matos et al. 2007). This suggests that the overexpression of UCP we see here and the repression of the AOX previously observed are indirect consequences of the genetic modifications of these cell lines. This situation is different from the thermogenic skunk cabbage in which a specific co-expression of SrUCPb and SrAOX protein was found in thermogenic tissue (Onda et al. 2008). The participation of UCP in the heat dissipation process in Arabidopsis cells cannot be excluded. Based on phosphorylation yield measurements, we first confirm that,



in Arabidopsis cells, UCP is involved in the palmitate-inducible proton leaks, although it is insensitive to GTP under nonphosphorylating conditions, as previously reported for native potato UCP (Navet et al. 2005). Then, we show that UCP activity, measured by the reduction of phosphorylation yields, is directly related to UCP content. In addition, data reported herein suggest that UCP-dependent leaks, as measured by palmitate-inducible proton flux, exceed membrane basal proton leaks in all genotypes. It is noteworthy that under the conditions used here, the UCP-dependent uncoupling levels reach a high rate of respiration comparable with state 3, thus indicating that the functional expression of UCP in all genotypes and its uncoupling capacity is high enough to allow a loss of respiratory control. Therefore, UCP involvement in heat dissipation and/or preventing reactive oxygen species production system is possible in the three genotypes despite their differences in UCP expression level. Additional regulation processes should be required in vivo to control UCP activity and make this possibility a reality. In particular, the real UCP activators in vivo are still under debate. Because the free fatty acid level in mitochondrial membranes found in vivo is extremely low, compared with the high palmitate amounts used in vitro, palmitate or other fatty acids are unlikely to be UCP activators in Arabidopsis. The peroxidized fatty acids generated under oxidizing stress could play this role, as suggested by Considine et al. (2003). In any case, a better understanding of the UCP activation and regulation mechanisms in cold-sensitive and cold-tolerant plants is required.

Materials and Methods Plant material The initial cell suspension culture of WT A. thaliana L. (Heynh) ecotype Columbia, cultured for >10 years in our laboratory was provided by Dr. M. Axelos (CNRS, Toulouse, France). The fad2 genotype was obtained from the lipid mutant library established by Lemieux et al. (1990), and the FAD3+ genotype (line 4-2) was constructed by Arondel et al. (1992). Cell suspension cultures of fad2 and FAD3+ genotypes were established in the laboratory. The culture conditions have been described in Matos et al. (2007).

Isolation of mitochondria Mitochondria were isolated from 6-day-old suspension cultures and purified on Percoll gradients according to Davy de Virville et al. (1994). Based on the galactolipid content (used as a marker of plastidial membranes), the purity of the mitochondrial fraction was >95%. The outer membrane integrity of fresh mitochondria was >90%, as deduced from cytochrome c oxidase activity measurements (Davy de Virville et al. 1994). Protein concentration was determined

according to Lowry et al. (1951) using bovine serum albumin (BSA) as a standard.

Lipid analysis Mitochondrial lipids were extracted according to Folch et al. (1957). Fatty acid methyl esters were determined according to Caiveau et al. (2001). The DBI was calculated according to the formula: DBI = 2(1[18 : 1] + 2[18 : 2] + 3[18 : 3])/100.

Oxygen uptake and ADP/O measurements Oxygen consumption was measured at 20°C using a Clarktype oxygen electrode. Air-saturated electrode medium contained 0.3 M mannitol, 10 mM phosphate buffer pH 7.2, 10 mM KCl, 5 mM MgCl2 and 0.1 g l−1 fatty-acid free BSA (Sigma). Mitochondria (0.1–0.4 mg protein ml−1) were incubated with 100 µM propyl gallate to inhibit the AOX. Oxidation was triggered by addition of 10 mM succinate plus 1 mM NADH, and, in the case of phosphorylation states, of ADP (25–100 µM). ADP/O ratio measurements were carried out on mitochondria treated or not with palmitate [2.0 × 10−2–810−2 mg mg (protein)−1] and GTP (1 mM).

Membrane potential and proton conductance Membrane potential was measured simultaneously with oxygen consumption using a tetraphenyl-phosphonium (TPP+)-sensitive electrode, in the presence of 2 µM TPP+, as described in Kamo et al. (1979). The values of the membrane potential were calculated without corrections for cation binding and assuming a mitochondrial matrix volume of 1 µl µg of protein. In plant mitochondria the protonmotive force (∆p) is totally expressed as membrane potential (∆Ψ) and it is assumed that the protonmotive force is entirely delocalized and that no slip reactions of the proton pumps occur (Kesseler et al. 1982, Brand et al. 1994). As a consequence, proton fluxes are directly correlated to oxidation rates by a constant H+/O stoichiometry (taken as 6 for succinate oxidation). Membrane proton conductance expressed in nmol H+ min−1 mg (protein)−1 mV−1 was calculated using the relationship CmH+ = [(rate of oxygen uptake) (H+/O)]/∆Ψ.

Flux–force relationships and determination of proton leaks Relationships between oxidation rate and membrane potential were determined in the non-phosphorylating state (state 4) in the absence (membrane basal proton leaks) or in the presence of exogenous free fatty acid (total proton leaks), at palmitate to protein mass ratios ranging between 2.6 and 7.7 × 10−2 (to activate UCP), using 1 mM exogenous NADH as substrate, 6 µM atractylate to inhibit ADP–ATP carrier, and increasing amounts of KCN up to 1 mM to inhibit respiration. After converting the oxidation rates into proton fluxes, the flux–force relationships were established and


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UCP-dependent proton leak was calculated by subtracting the membrane basal proton leak (in the absence of palmitate) from the total leaks obtained in the presence of fatty acid.

Western blot analysis of UCP content Mitochondrial protein analysis was performed as in Matos et al. (2007) and is the result of three independent experiments performed in duplicate. Proteins were separated by SDS–PAGE in 11% (v/v) polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad) in a Trans-Blot SemiDry Electrophoretic Transfer Cell (Bio-Rad) for 30 min at 135 mA using transfer buffer [25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3]. Equal loading was ensured by ‘Red Ponceau’ staining. Detection of Arabidopsis UCPs was performed with affinity-purified anti-UCP1 antibodies, referred to as 375.5 (Miroux et al. 1992), diluted 4,000-fold and horseradish peroxidase-linked anti-sheep IgGs as secondary antibodies, and revealed by chemiluminescence (ECL kit, from Perkin Elmer Life Sciences).

Funding The ‘Centre Nationale de la Recherche Scientifique’, France; the ‘Fundação para a Ciência e a Tecnologia’, Portugal.

Acknowledgments The authors wish to thank D. Cicek, C. Cantrel and W. Samson for their technical assistance, and Pierre Carol for his critical reading of the manuscript.

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(Received September 21, 2009; Accepted October 12, 2009)


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