Peter Meszaros

Karin Klappe

Ina Hummel

Dick Hoekstra

Jan Willem Kok

Function of MRP1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

Keywords: ATP-binding cassette, CDC, methyl-β-cyclodextrin/cholesterol, CFDA, 5-carboxyfluorescein diacetate, CO, cholesterol oxidase, DRM, detergent resistant membrane, LO, lovastatin, LTC 4 , cysteinyl leukotriene

Synopsis

MRP1 ‡ has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. Here we show for the first time that nearly complete oxidation of free cholesterol in the plasma membrane of BHK-MRP1 cells did not affect MRP1 localization in lipid rafts or its efflux function, using 5-carboxyfluorescein diacetate as substrate. Inhibition of cholesterol biosynthesis, using lovastatin in combination with RO 48-8071, resulted in a shift of Mrp1 out of lipid raft fractions, but did not affect Mrp1-mediated efflux in Neuro-2a cells. Short-term methyl-β-cyclodextrin treatment was equally effective in removing free cholesterol from Neuro-2a and BHK-MRP1 cells, but affected MRP1 function only in the latter. The kinetics of loss of both MRP1/Mrp1 efflux function and lipid raft association during long-term methyl-β-cyclodextrin treatment did not match the kinetics of free cholesterol removal in both cell lines. Moreover, MRP1 activity was measured in vesicles consisting of membranes isolated from BHK-MRP1 cells using the substrate cysteinyl leukotriene C 4 and was not changed when the free cholesterol level of these membranes was either decreased or increased. In conclusion, MRP1/Mrp1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts.

Page heading title: MRP1 ‡ function is independent of cholesterol Supplementary Key Words: methyl-β-cyclodextrin, lovastatin, RO 48-8071, cholesterol oxidase, ATPase activity; LTC 4 transport assay

Introduction

One of the best characterized multidrug resistance mechanisms is the energy-dependent drug efflux by proteins belonging to the ATP-binding cassette (ABC) transporter protein superfamily. Multidrug-resistance-related protein 1 (MRP1) and P-glycoprotein are the most widely studied ABC transporters and are known to depend on their direct lipid environment for optimal functioning [START_REF] Dudeja | Reversal of multidrug resistance phenotype by surfactants: relationship to membrane lipid fluidity[END_REF][START_REF] Sinicrope | Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles[END_REF]. Lavie et al. [START_REF] Lavie | Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells[END_REF] have shown for the first time the association of an ABC transporter with a membrane domain. They found that a substantial fraction of Pglycoprotein was located in Cav-1 containing Triton X-100-based detergent resistant membranes (DRMs) in P-glycoprotein overexpressing cells. Later studies showed localization of both P-glycoprotein and MRP1 in non-caveolar DRMs. Both ABC transporters were more strongly enriched in Lubrol-based or Brij-based DRMs compared to Triton X-100-based DRMs [START_REF] Hinrichs | ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells[END_REF][START_REF] Radeva | P-Glycoprotein is localized in intermediatedensity membrane microdomains distinct from classical lipid rafts and caveolar domains[END_REF].

Given their localization in DRMs, the function of ABC transporters may well be dependent on cholesterol, which is known to be enriched in DRMs. Modulation of Pglycoprotein function by cholesterol and involvement of DRMs in this process are widely studied [START_REF] Orlowksi | P-glycoprotein and 'lipid rafts': some ambiguous mutual relationships (floating on them, building them or meeting them by chance?)[END_REF]. For example, cholesterol depletion resulted in a shift of P-glycoprotein out of DRM fractions and P-glycoprotein-mediated drug transport was also affected [START_REF] Troost | Modulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells[END_REF]. In Caco-2 cell monolayers, cholesterol depletion significantly impaired the efflux activity of Pglycoprotein [START_REF] Yunomae | Involvement of cholesterol in the inhibitory effect of dimethyl-ß-cyclodextrin on P-glycoprotein and MRP2 function in Caco-2 cells[END_REF]. Concerning modulation of MRP1 function by cholesterol, information is scarce. In one study, cholesterol was reported to modulate MRP1 function and this was related to the presence of MRP1 in DRMs [START_REF] Marbeuf-Gueye | Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells-modification of ABCC1 (MRP1) localization and functionality[END_REF]. It is important to rigorously establish whether cholesterol affects MRP1 function, as this could be the underlying mechanism for the appearance of MRP1 in lipid rafts and a potential target for manipulation of MRP1 activity in the context of tumour cell sensitization to cytostatics.

In this study we rigorously investigated the impact of cholesterol and the localization of MRP1/Mrp1 in cholesterol-dependent lipid rafts on efflux function of the ABC transporter in two cell lines. We used Neuro-2a (neuroblastoma) cells, which express endogenous murine Mrp1/Abcc1 and BHK-MRP1 (fibroblast) cells, which highly express stable transfected human MRP1/ABCC1 [START_REF] Chang | ATPase activity of purified multidrug resistance-associated protein[END_REF]. In addition, we studied ABCC1 function in vesicles consisting of membranes isolated from cholesterol-modulated BHK-MRP1 cells. In view of potential drawbacks using detergent-based protocols for lipid raft isolation [START_REF] Munro | Lipid rafts: Elusive or illusive[END_REF][START_REF] Heerklotz | Triton promotes domain formation in lipid raft mixtures[END_REF], we employed the detergent-free lipid raft isolation procedure recently developed by Macdonald and Pike [START_REF] Macdonald | A simplified method for the preparation of detergent-free lipid rafts[END_REF]. Various strategies were employed to manipulate cellular cholesterol levels, including 1) shortand long-term methyl-β-cyclodextrin (M-β-CD) treatment, which physically removes cholesterol from the plasma membranes of cells, 2) cholesterol oxidase treatment, which chemically converts cholesterol into cholestenon and 3) combined treatment with lovastatin, an inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase and RO 48-8071, an inhibitor of oxidosqualene cyclase. Abcc1/ABCC1 activity did not correlate with the variable levels of cholesterol, either in intact cells or isolated membranes. Neither did Abcc1/ABCC1 activity correlate with the protein's variable extent of lipid raft localization resulting from cholesterol modulation. We conclude that Abcc1/ABCC1 function is not dependent on cholesterol or cholesterol-dependent lipid raft localization, while the observed effect of (longterm) M-β-CD treatment in intact cells, likely relates to reduced membrane integrity.

Materials and methods

Materials

MK571 was a gift from Prof. A.W. Ford-Hutchinson (Merck-Frosst, Inc., Kirkland, Canada). All cell culture plastic was from Costar (Cambridge, MA, USA). Cell culture media, Hank's balanced salt solution (HBSS), antibiotics, L-glutamine, sodium pyruvate and trypsin were purchased from Gibco (Invitrogen, Paisley, UK). Fetal calf serum (FCS) was from Bodinco (Alkmaar, The Netherlands). RO 48-8071 was from Enzo Life Sciences AG (Farmingdale, NY, USA). Cholesterol oxidase was purchased from Calbiochem (Merck KgaA, Darmstadt, Germany). 5-Carboxyfluorescein diacetate (CFDA), cysteinyl leukotriene C 4 (LTC 4 ), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), lovastatin, and methyl-βcyclodextrin (M-β-CD) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[ 3 H]LTC 4 was purchased from PerkinElmer (Waltham, MA, USA). Cholesterol was from Avanti Polar Lipids (Alabaster, AL, USA). The rat monoclonal anti-ABCC1 (MRPr1) antibody was obtained from Alexis (Dedham, MD, USA). GF/C short drop filterplates were from Whatman (Kent, UK). OptiPrep was from Axis-Shield PoC AS (Dundee, Scotland).

Cell culture and incubation conditions

The murine neuroblastoma cell line Neuro-2a was purchased from the ATCC (Manassas, VA, USA). These cells were grown as adherent monolayer cultures in Dulbecco's modified Eagle medium supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate, under standard incubator conditions (humidified atmosphere, 5% CO 2 , 37°C). The hamster BHK cell line stably expressing the human ABCC1 gene, named BHK-MRP1, was a gift from Dr. Riordan (Mayo Clinic Arizona, S.C. Johnson Medical Research Center, Scottsdale, AZ, USA; ref . 10). These cells were grown as adherent monolayer cultures in Dulbecco's modified Eagle medium/NUT mix F-12 (1:1) supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine, under standard incubator conditions (humidified atmosphere, 5% CO 2 , 37 o C). The cells were kept under selective pressure by growing them in the presence of 100 µM methotrexate. In order to deplete cholesterol, cells were incubated in the presence of 10 mM M-β-CD for various time intervals in serum-free medium. Alternatively, Neuro-2a cells were incubated for 20 h in the presence of both 1 µM RO 48-8071, an inhibitor of oxidosqualene cyclase, and 1 µg/ml lovastatin, an inhibitor of 3-hydroxy-3methylglutarylcoenzyme A reductase, in serum-free medium. As a control in this case, cells were grown in serum-free medium for 20 h with vehicle. In order to chemically convert cholesterol to cholestenon, cells were incubated in the presence of 1 U/ml cholesterol oxidase for 1 h. For cell membrane preparations cells were grown in roller bottles, harvested by trypsinization, and then subjected to cholesterol oxidase or M-β-CD treatment for various time intervals to deplete plasma membrane cholesterol. For cholesterol loading of the plasma membrane, M-β-CD filled with cholesterol (M-β-CD/cholesterol) was used for different incubation times in serum-free medium at 37 °C (10 mM concentration). To prepare M-β-CD/cholesterol, 100 mg M-β-CD was dissolved in 2 ml water and 3 mg cholesterol (solution in ethanol) was added slowly while stirring at 60-70 °C. This was dried and used later as described above. After treatment cells were centrifuged (3000 rpm, 3 min), frozen in liquid nitrogen and stored at -80 °C until membrane isolation. Control cells were treated similarly but in the absence of cholesterol modulators, i.e. solvent controlled when applicable.

Isolation of detergent-free lipid rafts

Detergent-free lipid rafts were isolated as described [START_REF] Macdonald | A simplified method for the preparation of detergent-free lipid rafts[END_REF][START_REF] Klappe | Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1[END_REF].

Immunoblot analysis

Protein from equal volumes of the gradient fractions was processed as described [START_REF] Klappe | Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1[END_REF].

Cholesterol determination

To quantify free cholesterol, a lipid extraction [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] on cells or pooled detergent-free lipid fractions was performed after a protein determination [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF]. The cholesterol concentration was determined spectrophotometrically by a cholesterol oxidase/peroxidase assay [START_REF] Gamble | Procedure for determination of free and total cholesterol in micro-or nanogram amounts suitable for studies with cultured cells[END_REF]. The amount of cholesterol was expressed relative to the protein content.

Sphingolipid analysis by liquid chromatography-electrospray ionization tandem mass spectrometry

Sphingolipids were extracted and analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) as described previously [START_REF] Klappe | Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1[END_REF]. The amounts of individual sphingolipid species were added to obtain the total sphingolipid pool. Protein content of pooled OptiPrep gradient fractions was determined as described by Smith et al. [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF].

Detection of Abcc1/ABCC1-mediated efflux by flow cytometric analysis

Neuro-2a or BHK-MRP1 cells were plated to confluence in 25 cm 2 flasks one day prior to the experiment. Abcc1/ABCC1-mediated efflux of the substrate 5-carboxyfluorescein diacetate was performed by flow cytometric analysis as described [START_REF] Hummel | MRP1 function and localization depend on cortical actin[END_REF].

Measurement of cellular sensitivity to cytotoxic drugs (MTT assay)

One thousand cells/well were plated in microtiter plates. For depletion of cholesterol, cells were washed 24 h after plating with serum-free medium and incubated in the presence of 10 mM M-β-CD in serum-free medium for 1h at 37°C. Subsequently, cells were washed with serum-free medium and incubated for 48h in serum-containing medium. Viable cells were determined 72h after plating as previously described [START_REF] Klappe | Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1[END_REF].

Isolation of membrane vesicles from BHK-MRP1 cells

BHK-MRP1 cells were washed with HBSS, trypsinized, harvested and treated, then centrifuged (3000 rpm, 3 min), frozen in liquid nitrogen and stored in -80 °C. Pellets of ~ 8x10 8 frozen cells were resuspended in hypotonic solution (5 mM sodium phosphate, pH 7.4, containing protease inhibitors and 1 mM EDTA) by stirring for 90 min on ice. After centrifugation (100000 x g, 45 min, 4 °C) cell material was resuspended in isotonic buffer (10 mM Tris, pH 7.4, 250 mM sucrose, containing protease inhibitors and 1 mM EDTA) and homogenized using a Dounce tissue grinder. The material was carefully layered on top of a 38% sucrose solution and centrifuged (280000 x g, 2 h, 4 °C). The membrane interface was collected and diluted in isotonic buffer and centrifuged again (100000 x g, 45 min, 4 °C). The membrane pellet was finally resuspended in isotonic buffer at a concentration of 5 mg/ml of protein [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF], frozen in liquid nitrogen and stored at -80 °C.

Measurement of ABCC1-mediated ATPase and transport activities

Vanadate sensitive ATPase activites were measured as described earlier [START_REF] Sarkadi | Expression of the Human Multidrug Resistancec DNA in Insect Cells Generates a High Activity Drug-stimulated Membrane ATPase[END_REF][START_REF] Glavinas | ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions[END_REF]. Briefly, isolated membranes (8 µg/well) were incubated for 11 min in 50 µl ATPase assay mix (40 mM MOPS-Tris pH 7.0, containing 10 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol, 0.1 mM EGTA, 4 mM Na-azide, and 5 mM ATP). The reaction was stopped with 100 µl malachite green mix [START_REF] Hess | Assay of Inorganic and Organic Phosphorus in the 0.1-5 Nanomole Range[END_REF][START_REF] Veldhoven | Inorganic and Organic Phosphate Measurements in the Nanomolar Range[END_REF][START_REF] D'angelo | Rapid, Sensitive, Microscale Determination of Phosphate in Water and Soil[END_REF][START_REF] Carter | Inorganic phosphate assay with malachite green: An improvement and evaluation[END_REF][START_REF] Rumsfeld | High-Throughput Assay for Inorganic Pyrophosphatases Using the Cytosolic Enzymes of Saccharomyces cerevisiae and Human as an Example[END_REF], as modified in our lab (5.6 mM malachite green, 9.4 mM ammoniummolybdate, 2 % citric acid, 4.6 % ethanol, and 1.16 N HCl, final concentration). After two min 100 ul of citric acid was added and incubated at 37 °C for 15-20 min. The absorbance values were measured using a spectrophotometer (Biotek uQuant) at a wavelength of 630 nm. For transport of cysteinyl leukotriene C 4 (LTC 4 ) into membrane vesicles, vesicles were incubated as described before [START_REF] Pál | Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2[END_REF] in 40 mM MOPS-Tris pH 7.0, 180 mM sucrose, containing 10 mM MgCl 2 , 20 mM KCl, 47.8 nM LTC 4 and with or without 4 mM ATP, for various time intervals at 37 °C. LTC 4 at a concentration of 47 nM was mixed with 3 H-LTC 4 (specific activity of 190 Ci/mmol) at a concentration of 0.8 nM. The reaction was stopped with ice cold washing buffer (10 mM Tris pH 7.0, 180 mM sucrose, 20 mM KCl). The reaction mix was rapidly filtered in glass fibre filter plates (Whatman GF/C short drop), washed with 5x200 µl ice cold washing buffer, and dried. Radioactivity was measured by liquid scintillation counting (Packard Topcount microplate scintillation counter). ATPase activity was expressed as pmol Pi/mg/min and transport into membrane vesicles as pmol LTC 4 /mg/min. For graphic representation, values were normalized to maximal activity in control conditions (=100%).

Measurement inside-out vesicle content

Determination of the inside-out vesicle ratio was based on 5'-nucleotidase activity and performed as described earlier [START_REF] Grinstein | Measurement of Sidedness of Isolated Plasma-Membrane Vesicles: Quantitation of Actin Exposure by DNase I Inactivation[END_REF] with the following changes: 25-50 µg of membrane vesicles was incubated (30 min, 37 °C) in 50 mM Tris, pH 7.4, containing 4 mM MgCl 2 , and with or without 3 mM AMP and/or 0.3 % Triton X-100. The inorganic phosphate released by the 5'-nucleotidase was measured with the malachite green system as described above.

Results

Efficient depletion or uploading of cholesterol

First we established the efficacy of cholesterol modulation, using three different strategies. 1) M-β-CD was used to physically deplete cholesterol from cell membranes. 2) Lovastatin, an inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase, plus RO 48-8071, an inhibitor of oxidosqualene cyclase, were used to inhibit the biosynthesis of cholesterol. 3) Cholesterol oxidase was used to chemically convert cholesterol to cholestenon.

The results are summarized in Table 1. In Neuro-2a cells a 1 h treatment with 10 mM M-β-CD decreased the cholesterol level by 71.6% in whole cells. In detergent free lipid raft fractions from these cells, cholesterol was even more strongly depleted (87.0%). In BHK-MRP1 cells cholesterol depletion by M-β-CD was 64.1%. The M-β-CD treatment slightly affected Neuro-2a cell viability, as determined by a MTT assay. Neuro-2a cell viability was 81.4% ± 18.6% (n=6) for M-β-CD-treated cells (1 h) compared to control cells (=100%).

Conditions for inhibition of cholesterol biosynthesis in Neuro-2a cells were optimized in terms of concentrations and time of incubation (data not shown). Cells incubated for 20 h in the presence of both 1 µg/ml lovastatin and 1 µM RO 48-8071 showed a 52.9% decrease of the cholesterol level. Finally, when 1 U/ml cholesterol oxidase was used for 1 h in Neuro-2a cells, the cholesterol content was reduced by 29.0%. In BHK-MRP1 cells, the enzyme was much more efficient, reducing cholesterol by 96.3%.

The cholesterol levels in cell membranes isolated from cholesterol-modulated BHK-MRP1 cells are summarized in Table 2. Cholesterol oxidase treatment of intact BHK-MRP1 cells resulted in a maximal decrease in cholesterol level of ~50% in the isolated cell membranes. This was less efficient compared to previous experiments (Table 1), possibly due to the use of large scale cell cultures needed for the subsequent isolation of membranes. We also treated isolated membrane vesicles with cholesterol oxidase, but this resulted in degradation of ABCC1 (Supplementary Figure S1 at http://www.BiochemJ.org/bj/) and these preparations were therefore not suitable for analysis of ABCC1 function. ABCC1 was not degraded in membrane preparations obtained from cholesterol-modulated BHK-MRP1 cells, as compared to control membrane preparations (Supplementary Figure S1 at http://www.BiochemJ.org/bj/).

Cholesterol depletion by M-β-CD treatment reduced cholesterol by ~57% in isolated membranes, while cholesterol uploading with M-β-CD/cholesterol increased the cholesterol level by ~58%. The cholesterol content of control membrane vesicles was 240 ± 30 nmol cholesterol/mg protein (n=9).

Cholesterol does not affect efflux function of Abcc1/ABCC1

Having established the extent of cholesterol modulation with three different strategies, we next measured the effects on Abcc1/ABCC1-mediated CFDA efflux. Efflux activity was the same in cholesterol oxidase-treated and control cells, regarding both Neuro-2a (Fig. 1A) and BHK-MRP1 (Fig. 1B) cells. Also in lovastatin/RO 48-8071-treated Neuro-2a cells, efflux activity was similar to control (Fig. 1C). MK571 was used as a positive control for inhibition of Abcc1/ABCC1-mediated efflux (Fig. 1A-C). With M-β-CD differential effects on Abcc1/ABCC1-mediated efflux kinetics were observed in the two cell types. In Neuro-2a cells, efflux activity was normal up to 10 min of M-β-CD treatment, but was reduced after 30 min and maximally affected after 45 min treatment (Fig. 2A). In BHK-MRP1 cells, on the other hand, ABCC1-mediated efflux activity was already reduced after 10 min of M-β-CD treatment, and subsequently kept fluctuating around this level (Fig. 2B). It was therefore important to measure in parallel the kinetics of cholesterol depletion in both cell types, which turned out to be very similar in Neuro-2a and BHK-MRP1 cells (Supplementary Figure S2 at http://www.BiochemJ.org/bj/). Cholesterol levels were equally decreased at t = 10 min in both cell types (Supplementary Figure S2 at http://www.BiochemJ.org/bj/). Interestingly, upon longer M-β-CD treatment, both cell types became sensitive to MK571, as indicated by loss of Abcc1/ABCC1 substrate likely due to membrane leakage. This occurred between 45 and 60 min for Neuro-2a (Fig. 2C) and between 15 and 30 min for BHK-MRP1 cells (Fig. 2D).

Abcc1/ABCC1 efflux function is not correlated with its localization in lipid rafts

We used a detergent-free method for the isolation of lipid rafts and first characterized the gradient fractions in terms of cholesterol and sphingolipid enrichment. For this purpose, fractions 1-2 were pooled, as well as 3-4, 5-6 and 7-9. Fractions 1-2 were most strongly enriched in both cholesterol and sphingolipids (Supplementary Figure S3 at http://www.BiochemJ.org/bj/), and to a lesser extent also fractions 3-4. This indicates that fractions 1-2, with the lowest buoyant density, optimally fulfil the criteria for lipid rafts. In accordance with the absence of an effect of cholesterol oxidation on Abcc1 efflux function, there was no effect on detergent-free lipid raft localization of the ABC transporter, as indicated by a similar gradient profile of Abcc1 compared to control Neuro-2a cells (Fig. 3A). Cholesterol oxidase was much more effective in reducing cholesterol content in BHK-MRP1 cells than in Neuro-2a cells. Therefore, we also rigorously tested lipid raft association of ABCC1 under cholesterol oxidase conditions in BHK-MRP1 cells and this turned out to be equal to control cells, as confirmed by quantification of the lipid raft-associated pools (Fig. 3B).

On the other hand, lovastatin/RO 48-8071 treatment did result in a clear shift of Abcc1 out of detergent-free lipid raft fractions in Neuro-2a cells, as confirmed by quantification of the relative amount of Abcc1 in lipid raft fractions (Fig. 3C). Upon M-β-CD treatment, detergent-free lipid raft association of Abcc1 was gradually reduced in Neuro-2a cells and this was significant after 60 min (Fig. 4A). In BHK-MRP1 cells, ABCC1 showed a tendency to shift out of lipid raft gradient fractions only after 60 min M-β-CD treatment, but this did not reach significance due to large variation (Fig. 4B).

ABCC1 ATPase and transport activities are not correlated with cholesterol levels in isolated cell membranes

The absence of effects of cholesterol modulation on Abcc1/ABCC1 function could have been due to confounding factors in the complex background of the intact cell. To overcome the complexity of the cell system, plasma membrane vesicles were isolated from cholesterol modulated BHK-MRP1 cells. To make sure that our procedures to modulate cholesterol in cells did not affect the inside-out ratio of ABCC1 in the isolated membrane vesicles, we measured this ratio. Under all conditions, the inside-out vesicle ratio was found to be invariable (Supplementary Figure S4 at http://www.BiochemJ.org/bj/). Moreover, ABCC1 integrity was not changed by our cholesterol modulation procedures, as shown by Western blot (Supplementary Figure S1 at http://www.BiochemJ.org/bj/). In this simplified system for ABCC1 function analysis, we measured ATPase activity of ABCC1 as well as transport of LTC 4 into membrane vesicles by ABCC1 (Fig. 5). The average vanadate sensitive ATPase activity and transport of LTC 4 into isolated control plasma membranes were 34 ±9 pmol Pi/mg/min (n=27) and 74 ±39 pmol/mg/min (n=27), respectively. There were no significant differences between cholesterol depleted (cholesterol oxidase or M-β-CD treated) or cholesterol uploaded (M-β-CD/cholesterol treated) membranes on the one hand and control membranes on the other (Fig. 5 and Supplementary Table S1 at http://www.BiochemJ.org/bj/). The average V max and K m values of the transport of LTC 4 into membrane vesicles were 661 ±171 pmol/mg/min and 942 ±220 nM, respectively. These kinetic parameters of the LTC 4 transport did not show any statistical differences between cholesterol-modulated and control membranes (data not shown).

Discussion

With regard to cholesterol-modulated ABC transporter activity, most studies have focused on P-glycoprotein. These studies have generated much information, but unfortunately not a coherent picture of whether and how cholesterol affects P-glycoprotein function [START_REF] Orlowksi | P-glycoprotein and 'lipid rafts': some ambiguous mutual relationships (floating on them, building them or meeting them by chance?)[END_REF][START_REF] Kok | Are lipid rafts involved in ABC transporter-mediated drug resistance of tumor cells? Trends Glycosci[END_REF]. Concerning modulation of Abcc1/ABCC1 by cholesterol, very little information is available. One study shows that when the cellular cholesterol level is lowered, ABCC1 function is reduced and concomitantly ABCC1 shifts out of lipid rafts in GLC4 multidrug-resistant lung cancer cells [START_REF] Marbeuf-Gueye | Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells-modification of ABCC1 (MRP1) localization and functionality[END_REF]. This conclusion was based on results obtained solely with M-β-CD treatment. In our study, we obtained similar effects of M-β-CD treatment. However, we did not attribute these effects to cholesterol, in view of the results obtained with the additional procedures for cholesterol modulation, i.e. cholesterol oxidase and lovastatin plus RO 48-8071. Abcc1/ABCC1-mediated efflux was not affected in these two treatment protocols, while the cholesterol level was strongly decreased, especially in cholesterol oxidase-treated BHK-MRP1 cells. Moreover, with M-β-CD treatment efflux was dissociated from the cholesterol level, since short-term treatment did affect efflux in BHK-MRP1, but not in Neuro-2a cells, while cholesterol levels were equally decreased in the two cell types. Long-term M-β-CD treatment in Neuro-2a cells decreased both cholesterol levels and Abcc1-mediated efflux. However, long-term M-β-CD treatment has been shown to result in side effects, such as removal of other lipids and potentially even proteins from membranes [START_REF] Yunomae | Involvement of cholesterol in the inhibitory effect of dimethyl-ß-cyclodextrin on P-glycoprotein and MRP2 function in Caco-2 cells[END_REF][START_REF] Arima | Contribution of cholesterol and phospholipids to inhibitory effect of dimethyl-betacyclodextrin on efflux function of P-glycoprotein and multidrug resistance-associated protein 2 in vinblastine-resistant Caco-2 cell monolayers[END_REF]. Thus, interpretation of such studies should be done with care. The data obtained with MK571 as inhibitor of Abcc1/ABCC1 efflux show that after short-term M-β-CD treatment the residual cell associated substrate is high, as expected. However, after long-term M-β-CD treatment substrate is lost from MK571-treated cells, suggesting that MK571 becomes toxic in combination with M-β-CD. This would indicate that membranes gradually destabilize with M-β-CD treatment. It is noteworthy that the M-β-CD effect becomes apparent at shorter incubation times in BHK-MRP1 cells, as compared to Neuro-2a cells. This could be related to the fact that BHK-MRP1 cells were subjected to forced expression of human ABCC1, possibly resulting in less stable integration of ABCC1 in the plasma membrane. As a consequence, ABCC1 would become more prone to the destabilizing effects of M-β-CD. On the other hand, when the M-β-CD effect on Neuro-2a cells sets in, it is more pronounced compared to that in BHK-MRP1 cells.

The studies on intact cells were extended to isolated membrane vesicles to overcome potential confounding effects in the complex system of the intact cell. The modulation of cholesterol performed on the cells did not change the integrity and inside-out ratio of ABCC1 in the vesicles consisting of isolated membranes from these cells, allowing us to properly measure potential effects on ABCC1 function. Data on ABCC1 function in these vesicles with decreased cholesterol levels confirm the conclusion that ABCC1 function does not depend on cholesterol. In addition, uploading of cholesterol was ineffective with regard to the function of ABCC1. Moreover, this shows that our results are not specific for the Abcc1/ABCC1 substrate CFDA, since in this assay for ABCC1 function in vesicles from isolated membranes a completely different substrate (LTC 4 ) was used.

Next we explored whether Abcc1/ABCC1-mediated efflux activity was related to localization of Abcc1/ABCC1 in cholesterol-stabilized lipid rafts. Comparison of results revealed that all 4 combinations of "effect/no effect" on these two parameters occurred. 1) After cholesterol oxidase treatment, both Abcc1/ABCC1-mediated efflux and its localization in lipid rafts were unaffected. possible combinations of "effect/no effect" on these 2 parameters occurred, we can conclude that Abcc1/ABCC1-mediated efflux activity and its localization in cholesterol-stabilized lipid rafts were not correlated. In a recent study we showed that Abcc1/ABCC1 efflux function was correlated with its localization in cortical actin-stabilized lipid rafts [START_REF] Hummel | MRP1 function and localization depend on cortical actin[END_REF]. Therefore, we should not only consider that Abcc1/ABCC1 (and other ABC transporters as well) may behave differently between various cell types in terms of (lipid raft) localization-function coupling. In addition, it may well be that within a certain cell type, different types of lipid rafts exist, which are sensitive to either cholesterol or cortical actin, while only the latter is relevant for ABCC1 function.

Taken together, we thoroughly investigated potential effects of cholesterol on Abcc1/ABCC1 function in two different cell lines and in vesicles from isolated membranes. Moreover, in order not to rely completely on M-β-CD treatment we used 3 different cholesterol modulation procedures. Based in this comprehensive approach, we show for the first time that Abcc1/ABCC1 function can not be categorically linked to cholesterol levels or cholesterol-stabilized lipid raft localization in two different cell lines. A previous study did show an effect on ABCC1 function and localization by lowering the level of cholesterol in yet another cell line [START_REF] Marbeuf-Gueye | Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells-modification of ABCC1 (MRP1) localization and functionality[END_REF]. Therefore, studies in additional Abcc1/ABCC1 (over)expressing cell types have to be performed before more general conclusion can be drawn. In any case, cholesterol is not essential for ABCC1 function and therefore does not seem to take part in the mechanism underlying functional association of ABCC1 with lipid rafts, as has been observed for cortical actin [START_REF] Hummel | MRP1 function and localization depend on cortical actin[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  5'nucleotidase activity was measured in 4 conditions; A: with AMP and Triton X-100, B: with AMP, without Triton X-100, C: without AMP, with Triton X-100 and D: without AMP, without Triton X-100. A-C yields the total activity of the enzyme, B-D the activity of the right side-out vesicles only. The percentage of inside out vesicles was calculated as [(A-C)-(B-D)]x100/(A-C). Biochemical Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

2 )

 2 Upon long-term M-β-CD treatment both efflux and lipid raft localization of Abcc1/ABCC1 were reduced. 3) On the other hand, with short-term M-β-CD treatment in BHK-MRP1 cells, efflux was affected, while raft localization was normal. 4) The opposite occurred after lovastatin plus RO 48-8071-treatment, which resulted in a shift of Abcc1 out of lipid raft fractions without any effect on efflux activity. Given the fact that all Biochemical Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Figure 1 .

 1 Figure 1. Cholesterol modulation does not affect Abcc1/ABCC1-mediated efflux in Neuro-2a and BHK-MRP1 cells A,B: Neuro-2a (A) or BHK-MRP1 cells (B) were untreated (control) or treated with 1 U/ml cholesterol oxidase for 1 h (CO). C: Neuro-2a cells were untreated (control: 20 h in serumfree medium) or treated with 1 µg/ml lovastatin + 1 µM RO 48-8071 for 20 h in serum-free medium (LO/RO). Subsequently, cells were loaded with CFDA (0.5 µM) and allowed to efflux at 37°C during several time intervals. The remaining cell-associated fluorescence was determined by cytometric analysis and expressed as % of the 0 min value. Control: open squares; CO or LO/RO: closed squares. MK571 (20 µM) was used as a positive control for Abcc1/ABCC1 efflux inhibition. Control + MK571: open triangles; CO + MK571 or LO/RO + MK571: closed triangles. Data represent the mean ± SD (n=3).

Figure 2 .

 2 Figure 2. Effects of M-β-CD treatment on Abcc1/ABCC1-mediated efflux in Neuro-2a and BHK-MRP1 cells Neuro-2a (A,C) or BHK-MRP1 cells (B,D) were treated with 10 mM M-β-CD for various time intervals. Subsequently, cells were loaded with CFDA (0.5 µM) and allowed to efflux at 37°C for 5 min. The remaining cell-associated fluorescence was determined by cytometric analysis and expressed as % of the 0 min value. MK571 (20 µM) was used as a control for Abcc1/ABCC1 efflux inhibition (C,D). Data represent the mean + SD (n=3).

Figure 3 .

 3 Figure 3. Detergent-free lipid raft localization of Abcc1 is affected by lovastatin/RO 48-8071 but not by cholesterol oxidase in Neuro-2a cells (and BHK-MRP1 cells) Cells were untreated (CON) or treated with either 1 U/ml cholesterol oxidase for 1 h (CO) or with 1 µg/ml lovastatin + 1 µM RO 48-8071 for 20 h in serum-free medium (LO/RO). A: Lipid raft association of Abcc1 in CO-treated Neuro-2a cells compared to control. B: Lipid raft association of ABCC1 in CO-treated BHK-MRP1 cells compared to control. C: Lipid raft association of Abcc1 in LO/RO-treated Neuro-2a cells compared to control. The numbers indicate the percentage of Abcc1/ABCC1 found in the pooled gradient fractions, relative to total Abcc1/ABCC1 in the entire gradient (9 fractions). Data represent the mean + SD (n=3). Asterisks indicate values that are significantly different from CON (P < 0.05, as determined by Student's t-test). The right panels show representative blots from three experiments concerning Abcc1/ABCC1 distribution along the gradient. Gradient samples were applied on SDS-PAGE based on equal volume. Lipid raft fractions are indicated.

Figure 4 .

 4 Figure 4. Effects of M-β-CD treatment on detergent-free lipid raft localization of Abcc1/ABCC1 in Neuro-2a and BHK-MRP1 cells Neuro-2a (A) or BHK-MRP1 cells (B) were untreated (0 min) or treated with 10 mM M-β-CD for various time intervals (5, 30, 60 min) and lipid raft association of Abcc1/ABCC1 was determined under these conditions. The numbers indicate the percentage of Abcc1/ABCC1 found in the gradient fractions, relative to total Abcc1/ABCC1 in the entire gradient (9 fractions). Data represent the mean + SD (n=3). Asterisks indicate values that are significantly different from CON (P < 0.05, as determined by Student's t-test). The right panels show representative blots from three experiments concerning Abcc1/ABCC1 distribution along the gradient. Gradient samples were applied on SDS-PAGE based on equal volume. Lipid raft fractions are indicated.

Figure 5 .

 5 Figure 5. Vanadate sensitive ATPase activity and transport of LTC 4 into membrane vesicles by ABCC1 in cholesterol modified membrane vesicles BHK-MRP1 cells were treated with cholesterol oxidase (CO; 1 U/ml; A,B), M-β-CD (CD; 10 mM; C,D), or M-β-CD/cholesterol (CDC; 10 mM; E,F). Cell membranes were isolated from these cells and control cells. The vanadate sensitive ATPase activity (A,C,E) or the transport of LTC 4 into membrane vesicles (B,D,F) by ABCC1 were measured. For each treatment, three independent membrane preparations were generated. For each membrane preparation, the measurements were performed in triplicate. Each data point in a curve represents the average ± SD of three independent membrane preparations. (a) represents membranes from control cells and (b), (c) and (d) membranes from cells treated for various time intervals with CO, CD or CDC, respectively. In case of CO, (b), (c) and (d) refer to 30, 60 and 90 min treatment, respectively. In case of CD and CDC, this is 10, 20 and 30 min. (a) ♦ solid line; (b) ■ dotted line; (c)▲dashed line; (d) • dashed dotted line. ATPase activity was expressed as pmol Pi/mg/min and LTC 4 transport as pmol LTC 4 /mg/min. For graphic representation, values were normalized to maximal activity in control conditions (=100%).

  Figure 1

  Figure 2

  Figure 3

  Figure 4

  Figure 5

  Biochemical Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110427	A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Table 1 : Cholesterol depeletion methods and efficiency in intact cells

 1 Various procedures were performed to manipulate cholesterol levels, including M-β-CD treatment (10 mM, 1 h), which physically removes cholesterol from the plasma membranes of cells, cholesterol oxidase treatment (1 U/ml, 1 h), which chemically converts cholesterol into cholestenon and combined treatment with lovastatin (1 µg/ml), an inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase and RO 48-8071 (1 µM), an inhibitor of oxidosqualene cyclase. Cholesterol content was measured and normalized to protein. These values were normalized to control (=100%). Data indicate the mean ± SD of three independent measurements.

	Biochemical Journal Immediate Publication. Published on 24 May 2011 as manuscript BJ20110427
	TABLES
	Cell type	Fraction	Method	Cholesterol content
				(control = 100%)
	Neuro-2a	whole cells	M-β-CD	28.4% ± 12.3
	Neuro-2a	detergent-free lipid rafts	M-β-CD	13.0% ± 7.5
	Neuro-2a	whole cells	lovastatin + RO 48-8071	47.1% ± 7.8
	Neuro-2a	whole cells	cholesterol oxidase	71.0% ± 5.0
	BHK-MRP1	whole cells	M-β-CD	35.9% ± 3.0
	BHK-MRP1	whole cells	cholesterol oxidase	3.7% ± 2.1

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Cholesterol depletion with Cholesterol Oxidase (CO)

Cholesterol depletion with M-β-Cyclodextrin (CD)

Cholesterol upload with M-β-CD/cholesterol (CDC)


Elizabeth Allen

Annick Moing

Jonathan Ad Wattis

Tony Larson

Mickael Maucourt

Ian A Graham

Dominique Rolin

Mark A Hooks
email: m.a.hooks@bangor.ac.uk

Jonathan A D Wattis

Mickaël Maucourt

Evidence that ACETATE NON-UTILIZING 1 prevents carbon leakage from peroxisomes during lipid mobilization in Arabidopsis seedlings

Keywords: acetyl-CoA, Arabidopsis, glyoxylate cycle, metabolism, modelling, substrate cycle

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The process of seed germination has been delineated into three steps, water absorption by the seed coat, the initiation of metabolic processes within the outer cellular layers (the endosperm of oilseeds) and the establishment of metabolic processes within the embryo leading to radical emergence from the seed coat [START_REF] Bewley | Seeds: Physiology of Development and Germination[END_REF]. In the oilseed Arabidopsis, the cotyledons of the embryo contain about 90% of the total triacylglycerol (TAG) for carbon nutrition. The endosperm contains about 10% of the total seed TAG, degradation of which is essential to kick-start embryo metabolism and permit normal development of the seedling [2]. Prior to seedling emergence the degradation of TAG begins in earnest in order to feed the rapidly developing seedling and they are completely consumed by the time the cotyledons have spread and greened completely. Fatty acids released from TAGs are transported to glyoxysomes where they are activated to their acyl-CoA esters to enter -oxidation [START_REF] Graham | Seed storage oil mobilization[END_REF]. The -oxidation spiral releases acetyl-CoA, which enters the glyoxylate cycle for conversion to organic acids that can be exported to mitochondria for subsequent gluconeogenesis [START_REF] Beevers | Metabolic production of sucrose from fat[END_REF] or to fuel respiration [START_REF] Raymond | Peroxisomal Beta-Oxidation of Fatty-Acids and Citrate Formation by a Particulate Fraction from Early Germinating Sunflower Seeds[END_REF][START_REF] Eastmond | Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle[END_REF]. However, only recent work on Arabidopsis mutants has revealed the enzymes critical for linking -oxidation to primary carbon metabolism. At least one glyoxysomal isozyme of citrate synthase and malate dehydrogenase are essential for lipid catabolism and seedling establishment, whereas isocitrate lyase and malate synthase are not [START_REF] Pracharoenwattana | When is a peroxisome not a peroxisome?[END_REF].

The glyoxylate cycle and its association with -oxidation were elucidated almost 50 years ago through feeding studies using radiolabelled acetate [START_REF] Canvin | Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway[END_REF]. For exogenous acetate to be assimilated through the glyoxylate cycle, it requires transport into the peroxisome and activation to acetyl-CoA. important means by which acetate is recycled in Arabidopsis. However, ACS activity also resides within the cytosol, which is greater than either plastid or peroxisome associated activities [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF]. Several objectives toward ascertaining the importance of ACN1/AAE7 in developing seedlings were to determine the effects of eliminating this step on metabolite labelling patterns, metabolite levels and gene expression profiles using the acn1-2 mutant. We observed changes to the labelling of the sink metabolite Gln and in the levels of both metabolites and transcripts. This suggests an important metabolic role of ACN1/AAE7 during lipid mobilization, which may subsequently alter transcriptional responses. These results highlight the still unknown complexity of carbon utilization and partitioning and its regulation during seedling establishment. We discuss our data as a foundation of mathematical modelling efforts to explain the metabolic differences between acn1-2 and Col-7, which suggest a role of ACN1/AAE7 to prevent carbon loss from peroxisomes during lipid mobilization [START_REF] Hooks | Modelling the peroxisomal carbon leak during lipid mobilization in Arabidopsis[END_REF].

EXPERIMENTAL Plant material and growth conditions

All seeds were surface sterilized and imbibed in the dark at 4 o C for 3 days before sowing onto agar plates. For all experimental conditions, seeds were germinated at 20 o C at 70 mol of photons/m 2 /s constant illumination. Standard agar media plates contained 0.8% agar, 1/2-strength MS salts [START_REF] Murashige | A revised medium for rapid growth and bioassay with tobacco tissue cultures[END_REF]. The media, prior to addition of agar and subsequent autoclaving, was adjust to pH 5.7 with 0.1 N KOH. In order to obtain conclusive results regarding the behaviour of mutant compared to wild-type seedlings from the metabolite and transcript profiling, it was necessary to obtain equally developed seedlings. Due to the more rapid development of acn1-2 [START_REF] Hooks | Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings[END_REF] (6 to 8 h before Col-7 to attain Principal Growth Stage (PGS) 0.7 [START_REF] Boyes | Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants[END_REF]) we selected batches of seedlings with approximately the same average fresh weight in addition to visually staging seedling development. The average fresh weights of 50 seedlings from plates used for RNA extraction were 8.8 ± 1.2 (n=8) and 8.9 ± 1.3 (n=8) for Col-7 and acn1-2, respectively. All seedling batches used for metabolite quantification and feeding studies were selected to fall within these weights.

[ 13 C]Acetate and [ 13 C]Gln quantification

Seedlings were washed from standard agar media plates using distilled water, rinsed well, and ~1 g of seedlings was bubbled with air in a sterile solution of 1/2-strength MS salts, 20 mM sucrose, and 50 mM MES, pH 5.7. After 1 hour, [2- 13 C]-labelled sodium acetate was added to a concentration of 4 mM and the seedlings incubated for designated lengths of time. Chase experiments were performed by transferring seedlings to an air-bubbled solution of 4 mM unlabelled acetate two hours after adding the [2- 13 C]acetate, and continuing the incubations for either 2 or 4 h. At each sampling time, the seedlings were removed from the solution, rinsed thoroughly with distilled H 2 O, and frozen in liquid nitrogen. Metabolites were extracted according to Weckwerth et al. [20] and lyophilised overnight. The residue was resuspended in 550 l of an aqueous solution containing 18% (v/v) D 2 O, 1.75 g/ml streptomycin, 3.5 g/ml NaN 3 , and 8 mM 13 C-formate, which served as an internal standard. The pH was not adjusted. Standard broadband, proton-decoupled, Fourier transform NMR 13 C spectra were acquired on a Bruker AC250 spectrometer operating at 62.9 MHz The interval between 90 o pulses was 2 s, and the spectral width and data points were 16000 Hz and 8K, respectively.

Metabolite quantification

Approximately 0.6 g of seedlings was washed from the surface of the agar petri dishes with distilled sterile water into a filtration unit. Once the water had passed through the seedlings were washed in 10 ml more water, weighed and immediately frozen in liquid nitrogen. The time from opening the petri dish to freezing the sample was approximately 4.5 min. Metabolites were A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

extracted according to Weckwerth et al. [20] and metabolite quantification by 1 H-NMR was conducted as described in Moing et al. [21]. Briefly, the dried extracts were resuspended in 400 mM phosphate buffer pH 6.0 in D 2 O and analyzed at 500.162 MHz on a Bruker spectrometer (Bruker Biospin Avance). Special care was taken to allow absolute quantification of the individual metabolites through addition of ethylene diamine tetraacetic acid sodium salt solution (5 mM final concentration in the NMR tube) to improve the resolution and quantification of organic acids such as malate and citrate, adequate choice of the NMR acquisition parameters (pulse angle 90°, relaxation delay 10 s) and use of an electronic reference calibrated with glucose, fructose, Gln and Glu sodium salt solutions as described previously [21]. Individual metabolites were identified using published data [21], acquisition of NMR spectra of reference compounds under exact solvent conditions, and spiking extracts with reference compounds. They were quantified using the metabolite mode of AMIX software (Bruker Biospin v. 3.5.6) based on the number of protons comprising the corresponding resonance.

Samples for fatty acid measurements were sown onto agar plates and tissue samples harvested after the same length of time under the growth conditions specified above. Samples of 50 seeds or 25 seedlings were collected by forceps, weighed in screw-cap tubes, and immediately frozen in liquid nitrogen. Fatty acids were measured using the method of Browse et al. [22]. Acetyl-CoA levels were determined by HPLC according to Larson et al. [START_REF] Larson | Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds[END_REF]. Acetyl-CoA extraction efficiency and relative quantification was confirmed using the extraction protocol of Tumaney et al. [START_REF] Tumaney | Acetyl coenzyme A concentrations in plant tissues[END_REF].

Transcript profiling and data analysis

Total RNA was extracted from approximately 0.5 g of PGS 0.7 seedlings. The RNA was sent to the GARNet transcript profiling service at Nottingham University for quality analysis, dye labelling and hybridisation to the Affymetrix ATH1 GeneChip TM arrays. Twelve arrays were produced with one target hybridised to each chip. Three replicates of two sample types were prepared of Col-7 and acn1-2. The data pre-processing was carried out at the GARNet transcript profiling service where poor hybridisation events were noted and quantification of hybridisation intensity was performed. Further data mining was carried out at UWB using GeneSight TM v4.1 (Biodiscovery Inc., San Mateo, USA). The raw expression profiling data is available from the GARNet transcript profiling service (affymetrix.arabidopsis.info). Microarray expression data and all associated experimental metadata were presented to comply with MIAME guidelines [START_REF] Brazma | Minimum information about a microarray experiment (MIAME)-toward standards for microarray data[END_REF].

Identification of differentially expressed genes between acn1-2 and Col-7 was conducted using GeneSight TM v 4.1 (BioDiscovery Inc.). Unless stated otherwise, data normalisation was performed using the pre-set "Affy" data transformation sequence. A dataset was created in GeneSight in order to identify gene expression ratios between Col-7 and acn1-2 that were consistent throughout the microarrays. First, three groups were created that gave gene expression ratios for each acn1-2 replicate compared to one of the three Col-7 replicates. For example, group 1 comprised expression ratios calculated for every pair between acn1-2 (1) and Col-7 (1), acn1-2 (2) and Col-7 (1), and acn1-2 (3) and Col-7 (1). To do this, signal intensities in this dataset were transformed, except that replicates were not combined as the final step of the pre-set 'Affy' transformation process. This kept the data within two categories each containing the minimum three replicates needed in order to conduct the ANOVA (P < 0.05) to establish the set of consistent ratios between acn1-2 and Col-7 (GeneSight Users Manual v. 4.1, 2003). This set of 14,256 genes represented the core set of genes common to each group from which differentially expressed genes were determined. We adopted a combined approach of using a discretionary 'fold' cut-off level in conjunction with further statistical tests to determine the group of DE genes. A value at which genes showed DE was first identified based on reiterative hierarchical clustering of genes at various fold-change thresholds. The determined fold cut-off level was used in conjunction with a t-test (P < 0.05) to identify a statistically confident group of genes that showed DE between acn1-2 and Col-7. For each group, the log 2 differences in gene expression of the 14,256 genes were calculated between acn1-2 and Col-7 replicate. DE genes were identified in each group using a bootstrapping algorithm [START_REF] Kerr | Bootstrapping cluster analysis: Assessing the reliability of conclusions from microarray experiments[END_REF]. Through a reiterative process of decreasing the fold cut-off value while maintaining 95% confidence limits we obtained a set of 201 genes showing DE between acn1-2 and Col-7 at a cut-off threshold of 1.4-fold. Twenty of twenty times, hierarchal clustering of this set of genes resulted in complete separation of acn1-2 from Col-7. The hierarchical clustering was employed to determine the optimal number of windows to create 2-D self-organising maps to explore the structure of the data.

RESULTS

Global acetate consumption appears normal

We had previously reported that the acn1-2 mutant appeared to be less tolerant of acetate during the prolonged exposure provided through germination and emergence on standard agar media plates containing acetate [START_REF] Hooks | Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings[END_REF]. We hypothesized that the lack of acetate assimilation resulted in accumulation that would lead to acidification of the cytosol and ultimately death of the seedlings. Accordingly, the relatively high tolerance of the mutant to butyrate compared to acetate reflected the greater importance of ACN1 in activating acetate, and that butyrate may be activated by another enzyme. In previous [2- 14 C]acetate feeding studies on similarly staged seedlings, substantial label appeared in non-carbohydrate compound classes at near wild-type levels, except for the ethanol insoluble and organic acid fractions [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF]. This indicated that acetate metabolism was not completely abolished, but it was unclear how much it was reduced in the mutant. The relatively higher level of label in the organic acid fraction seen in the mutant from that study may have come from higher levels of un-metabolised free acetate indicating reduced assimilation. In order to test this hypothesis, we compared the uptake and utilization of acetate by acn1-2 compared to Col-7. Using NMR, we quantified the resonance corresponding to the C2 carbon of [2- 13 C]acetate-fed seedlings. It was necessary to conduct the feeding experiment in the presence of sucrose for observable levels of acetate to be taken up. Not only was sucrose promoting uptake, it was also exerting a protective effect on the seedlings. Seedlings fed acetate alone had a barely detectable 13 C signature after even 8 h, and seedlings rapidly deteriorated as observed by their tendency to fall apart during harvest. This was not unexpected as the toxic nature of free acetate on Arabidopsis seedlings and plant cells had already been reported [START_REF] Graham | Induction of Malate Synthase Gene Expression in Senescent and Detached Organs of Cucumber[END_REF]. The degree of deterioration at any given time was comparable between mutant and Col-7 during incubation times up to 24 h. Having sucrose present permitted us to extract and estimate free acetate levels for up to 24 h after feeding started. There was no difference in labelled acetate levels between acn1-2 and Col-7 (Fig. 1). Isotopically labelled acetate increased from undetectable to maximum levels of 40-50 mol g -1 fwt within 2 h. Levels remained elevated for 24 h with Col-7 and acn1-2 having 29 ± 11 and 41 ± 18 mol g -1 fwt, respectively. This shows that labelling of the endogenous acetate pool reaches steady-state within 2 h, and that we were measuring the capacity of the entire seedling to take up acetate and not just a specific tissue. To test if acetate was being metabolised, samples were chased with isotopically normal acetate and the fate of labelled acetate followed. Acetate had decreased within two hours to about 10 mol g -1 fwt. The fall in labelled acetate was similar for both acn1-2 and Col-7. In the subsequent 2 h, only a small further decrease was observed.

Disruption of the flow of carbon from acetate into Gln

Under the nitrogen replete conditions that we used, the predominant sink for label was Gln, and it appeared in all three carbons C2, C3 and C4 in the NMR spectra. We determined the levels of Gln based on each of the three labelled carbon atoms in the same extracts used for acetate quantification in Col-7 and acn1-2 (Figs. 2A-C). In both Col-7 and acn1-2, label in C2 appeared to reach steady-state within 2 h. In Col-7, the amount of label in C3 and C4 continued to increase up to 4 h after feeding, but reached steady-state levels in acn1-2 by 2 h. At 4 h, labelling in C3 and C4 were maximal in Col-7 (data not shown). By 4 h, there was approximately a 50% reduction in the amount of label appearing in the C3 and C4 carbons in acn1-2. There was only a slight reduction in label at position C2 for acn1-2. The differences in the amount of total label in each carbon and the timing in reaching apparent steady state, suggested that the Gln pool size may be smaller in acn1-2 than in Col-7. Upon chasing with isotopically normal acetate, the levels of label in Gln remained level or rose in the wild-type over the following 2 h. However, for acn1-2, there was no change or a slight decrease in labelled Gln post-chase, which showed that the flow of label from acetate to Gln was affected.

Depression of major metabolite levels in acn1-2

The timing and degree of Gln labelling suggested that the pool size was less in acn1-2 than in Col-7. To confirm our conclusion, the levels of Gln and other metabolites were quantified by 1 H-NMR. We quantified 27 distinct resonances corresponding to a variety of known and unknown metabolites, including soluble carbohydrates, amino acids and organic acids (Table I). Acetyl-CoA levels were determined by HPLC [START_REF] Larson | Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds[END_REF]. In general, the absolute levels of amino acids quantified on a nmol g -1 fwt basis were of the same order as those observed in seeds of the Arabidopsis ecotype Wassilewskija [START_REF] Li | Overexpression of a bacterial branched-chain-keto acid dehydrogenase complex in Arabidopsis results in accumulation of branched-chain acyl-CoAs and alteration of free amino acid composition in seeds[END_REF] or in seed germination series for other species, such as the legumes soybean and lupin [START_REF] Martínez-Villaluenga | Kinetics of free protein amino acids, free non-protein amino acids and trigonelline in soybean (Glycine max L.) and lupin (Lupinus angustifolius L.) sprouts[END_REF].

On average, Gln was 70% lower in acn1-2 than in Col-7, which corresponds to the labelling data. Furthermore, there was a depression of primary metabolite levels in acn1-2, which extended to all classes of metabolites that were NMR visible and quantifiable (Table I). The change was significant at p<0.05 for every compound measured except fumarate. Individual amino acids in acn1-2 were lower by 67%, on average, to those in Col-7, and ranged from 80% less proline to 59% less leucine. Individual soluble sugars in acn1-2 were on average 63% lower than those in Col-7 with the largest decrease observed for rhamnose at 69%. When fumarate was excluded from the calculation, organic acids in acn1-2 were on average 58% lower to the levels of corresponding ones in Col-7, ranging from 70% less formate to 40% less citrate. Malate was 50% lower in acn1-2. The unknown compounds were affected similarly to the other classes with levels between 40 and 60% lower. Fumarate showed a small, but insignificant increase in the mutant. If one effect of the mutation is to reduce carbon flow through the glyoxylate cycle via isocitrate lyase, then a potential decrease in succinate levels could have a corresponding effect on fumarate levels. If succinate was not the primary organic acid transported into mitochondria, since ICL activity appears unnecessary in light grown seedlings [START_REF] Eastmond | Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle[END_REF], then fumarate may be a metabolite not affected by the anapleurotic/gluconeogenic functions of glyoxylate/TCA cycle couple. In contrast to the behaviour of primary metabolites, acetyl-CoA was found to be more than 2-fold greater in acn1-2 than in Col-7.

Measurements of fatty acid catabolism

One possibility for lower metabolite levels in acn1-2 may have been due to less carbon entering metabolism from lipid mobilization. We conducted a time-course analysis of the disappearance of eicosenoic acid (C20:1), which is a marker of TAG degradation [START_REF] Rylott | The gluconeogenic enzyme phosphoenolpyruvate carboxykinase in Arabidopsis is essential for seedling establishment[END_REF], at three different time points, imbibed seeds and 3 and 5 days post-imbibition (Fig 3). The amount of eicosenoic acid in seeds was similar suggesting that fatty acid and TAG synthesis and storage is not affected in developing seeds. During germination and seedling growth, virtually all eicosenoic acid was gone in both Col-7 and acn1-2 by day 5. For both genotypes, the decrease in eicosenoic acid was significant between successive time points and the difference in levels between acn1-2 and Col-7 was significant at day 3, despite the seedlings being at a slightly more advanced stage of growth. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Transcript profiling

A group of differentially expressed genes were identified in each dataset using an algorithm based on the bootstrapping algorithm developed by Kerr and Churchill [START_REF] Kerr | Bootstrapping cluster analysis: Assessing the reliability of conclusions from microarray experiments[END_REF]. Genes were assigned as being either differentially expressed or not, based on the intensity of the replicated spots. For a gene to be identified as differentially expressed between acn1-2 and Col-7, the gene expression values from all three replicated microarrays need to be identified as differentially expressed. We employed a reiterative process of decreasing the fold cut-off value while maintaining 95% confidence limits [START_REF] Kerr | Bootstrapping cluster analysis: Assessing the reliability of conclusions from microarray experiments[END_REF] for DE among replicates. In this manner we obtained a set of 201 genes showing DE between acn1-2 and Col-7 at a cut-off threshold of 1.4-fold, which represented a statistically confident group of DE genes. Hierarchal clustering of the DE genes showed consistent segregation of seedling samples between mutant and Col-7 (Supplemental Fig. 1) and that the optimal number of bins for visualisation of the data by 2D-SOM was 9 (Supplemental Fig. 2). Three (bins 7-9) and six (bins 1-6) of the nine bins contained genes that were up-and downregulated, respectively (Supplemental Fig. 3). Of the 201 DE genes, 71 were up-regulated in acn1-2 with an increase in expression from 1.8 to 27.7-fold, whereas 130 genes were down-regulated with a decrease in expression ranging from 3.8 to 52.87-fold. All the DE genes are listed according to bin number for which repetitive mapping gave the greatest proportion of instances a gene fell into a particular bin (Supplemental Table 1).

The up-and down-regulated sets of genes were analysed for ontological patterns according to the bins assigned by the 2D-SOM [START_REF] Berardini | Functional annotation of the Arabidopsis genome using controlled vocabularies[END_REF]. A variety of genes with assigned identity were found in each cluster. Of the genes with assigned biological function based on ontology, Cluster 1 was comprised of genes involved in developmental processes, protein metabolism, nucleic acid related and signal transduction. Cluster 2 contained genes for protein metabolism, signalling and transcription. Cluster 3 contained genes involved in responses to stress or external stimuli, development, signalling and transcription. Genes from clusters 4 and 5 were examined jointly as these genes clustered together intermittently upon repeated 2D-SOM clustering, but no enrichment of a particular type of gene was noted. Similarly, genes in cluster 6 did not show a bias towards any specific function or process. Analysis of cluster 7 showed that the majority of genes were involved in response to stress or external stimuli, signalling and nucleic acid related. The genes of cluster 8 did not show enrichment in any specific ontology. Analysis of cluster 9 showed the genes were mainly involved in transcription or development.

We examined more closely the top twenty up-and down-regulated genes (Table 2) following the assumption that the genes that demonstrate the largest change in expression levels between two samples are the pivotal genes around which the difference in biological basis revolves [32]. Of the twenty genes that showed the greatest increase in expression, 16 had a predicted biological function. These included a gene associated with the phytohormone auxin was induced (At5g13350) as well as a number of genes possibly involved in the regulation of translation or transcription (At3g49460, At1g49920, At5g19920, At2g46780, and At2g17920). Examining the top twenty down-regulated genes in acn1-2, the identities of 16 were assignable. Eight of which were involved with early seedling development, including genes encoding late embryogenesis abundant (LEA) proteins (At2g36640, At1g32560, At4g36600, At4g21020), seed storage protein (At5g39110) and a protein associated with embryonic development (At5g62210). Genes associated with hormone regulators of plant development At1g48660 (auxin) and At3g02480 (ABA) and a Ring-finger family transcription factor (At4g35480) were also repressed in acn1-2.

Transcript levels of genes associated with the assimilation of acetate

Since the sequential action of the glyoxylate cycle, TCA cycle and gluconeogenesis is important for assimilating both free acetate and acetyl-CoA from -oxidation, it was of particular interest to examine what happened with the expression of glyoxylate cycle, anapleurotic and gluconeogenic genes in acn1-2. None of the genes examined showed differential expression between acn1-2 and Col-7 when the 1.4-fold change in gene expression threshold was used in conjunction with a t-test (P > 0.05). However, certain genes showed DE when a simple threshold cut-off value of +/-1.4-fold was applied (Table 2). Two genes, a putative cytosolic malate dehydrogenase (At5g56720) and one phosphoenolpyruvate carboxykinase (PCK) showed an increase in transcript levels in acn1-2 of 2.9-and 2.0-fold, respectively. The putative PCK is not the one known to be developmentally important during early seedling establishment [START_REF] Rylott | The gluconeogenic enzyme phosphoenolpyruvate carboxykinase in Arabidopsis is essential for seedling establishment[END_REF]. The genes for which transcript levels were lower in acn1-2 were malate synthase (At5g03860) [33], isocitrate lyase (At3g21720) [START_REF] Eastmond | Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle[END_REF], a malate dehydrogenase-related protein (At3g53910), citrate synthase 3 (CSY3, At2g42790) [START_REF] Pracharoenwattana | Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination[END_REF], an aspartate aminotransferase (ASP1, At2g30970) [35] and ACN1/AAE7 (At3g16910) [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF] in the range of 5.5-to 1.4-fold. We have shown previously that a partial transcript for ACN1/AAE7 is detectable in the acn1-2 mutant [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF], therefore, its expression in the mutant could be determined.

DISCUSSION

We have employed a combination of metabolite and transcript profiling to investigate the effects on metabolism and gene expression caused by eliminating the peroxisomal acetyl-CoA synthetase step catalysed by ACN1/AAE7. Because other acetyl-CoA synthetase activities are present in Arabidopsis [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF][START_REF] Ke | The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds[END_REF], it was necessary to determine if the lack of ACN1/AAE7 affected the overall assimilation of acetate. It was evident from our previous studies that acetate metabolism is not greatly compromised in acn1-2. However, this component of acetate assimilation represents the gluconeogenic contribution as shown by the large decrease in [ 14 C]acetate appearing in soluble carbohydrate in the acn1-2 and comatose/acn2 mutants compared to wild-type [START_REF] Hooks | The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism[END_REF][START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF]. We conclude that the effects that we observe on metabolite levels and gene expression reflect a defect in a relatively narrow, but metabolically important, aspect of acetate assimilation.

One possibility is that it serves to recycle acetate in young seedlings. Several mechanisms for acetate production have been described [START_REF] Bao | Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue[END_REF]. For example, cysteine biosynthesis is likely to be a substantial producer of free acetate. In loblolly pine, free cysteine levels go from being essentially undetectable in dry seeds, in contrast to all other amino acids, to become the tenth most abundant free amino acid within several days of radical emergence [38]. Furthermore, the flux through cysteine would be high, since it serves as the route by which sulphur is assimilated into proteins and metabolic intermediates [39] and a variety of sulphur-containing defence compounds [40]. Therefore, it is highly likely that cysteine biosynthesis with contributions from protein deacetylation provides a steady and high flux of free acetate within developing seedlings. In our study, the amount of labelled acetate did not change dramatically during the second two hours of the chase, thereby suggesting that it continues to be produced. We have shown that plant cells have an enormous capacity to accumulate acetate if presented with an exogenous source, but that levels of free acetate in untreated seedlings are very low. Thus, it appears that Arabidopsis seedlings have a large capacity to turn-over free acetate. From the disappearance of label in acetate, the turnover of the acetate pool is estimated to be about 5.4 nmol s -1 g -1 fwt and the entire acetate pool could turn over in approximately 8 s. It must be noted that this rate is an estimate from sucrose-fed seedlings and the rate may be different in non-treated seedlings. However, we previously reported that most free acetate assimilation is ACN1 independent in seedlings not fed with sucrose, and a large cytosolic ACS activity in seedlings that may account for acetate activation [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF]. Therefore, we can conclude that re-assimilation of extra-peroxisomal acetate is not likely to be a major function of ACN1 in young seedlings. This function would not appear to fall onto ACS either, since acs1 does not appear to be affected by exogenous acetate in seedlings less than 10 days old [START_REF] Lin | The role of acetyl-coenzyme a synthetase in Arabidopsis[END_REF]. The key difference between mutant and wild-type that suggests an intra-peroxisomal function in acetate cycling is the difference in acetyl-CoA levels. Greater acetyl-CoA levels in acn1-2 appear counterintuitive for an enzyme that would convert acetate to acetyl-CoA. However, these results are consistent with a model of lipid mobilization where ACN1/AAE7 prevents the loss of carbon from peroxisomes as free acetate by reactivating it (Fig. 4). Acetyl-CoA cannot cross the peroxisomal membrane [START_REF] Van Roermund | The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions[END_REF], but there are a number of acyl-CoA thioesterases present in peroxisomes of Arabidopsis, some of which are active with short-chain substrates or the substrate specificity is currently unknown [START_REF] Tilton | Two families of acyl-CoA thioesterases in Arabidopsis[END_REF][START_REF] Reumann | In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes[END_REF]. This situation would be analogous to the increase in acetyl-CoA levels observed for the heterologous expression of the S. cerevisiae acetyl-CoA hydrolase in mitochondria of tobacco plants [START_REF] Bender-Machado | Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis[END_REF]. Their explanation was that free acetate is exported from mitochondria and reactivated in the cytosol. The lack of acetate reactivation in peroxisomes would not expect to have the severe consequences on plant growth as observed for the acetyl-CoA hydrolase expressing plants, because sufficient acetyl-CoA would be produced by -oxidation. Increased levels of cytosolic acetyl-CoA may even promote biosynthetic processes allowing faster development under ideal growing conditions. A similar conclusion was made about an aconitase isozyme of tomato that was hypothesized to be a factor affecting the distribution of carbon between the TCA cycle and sucrose biosynthesis [START_REF] Carrari | Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato[END_REF]. Elimination of this aconitase resulted in reduce levels of primary metabolites and an increase in various soluble sugars indicating a shift in carbon allocation from respiration to biosynthetic processes. The tomato aconitase mutant grew taller and produced greater fruit mass than the corresponding wild-type plant. The lack of ACN1/AAE7 may affect the rate at which acetyl-CoA enters the glyoxylate cycle from fatty acid degradation. The opposing activities of ACN1/AAE7 and a thioesterase, in conjunction with the ability of peroxisomes to import and export acetate, would constitute a true substrate cycle [START_REF] Fell | Understanding the Control of Metabolism[END_REF]. Metabolic rather than transcriptional control of lipid mobilization processes may explain why the expression of -oxidation and glyoxylate cycle genes is unaffected even in situations where lipid mobilization is severely diminished [START_REF] Pracharoenwattana | Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination[END_REF], and why we observed an effect on the expression of very few metabolic genes. It is known that germination and lipid mobilization are regulated independently [47]. Whereas the molecular mechanisms of germination are quite well defined, the regulation of metabolic processes remains poorly understood.

We conducted mathematic modelling to further explore the possibility that ACN1/AAE7 is necessary to prevent acetate leakage from peroxisomes [START_REF] Hooks | Modelling the peroxisomal carbon leak during lipid mobilization in Arabidopsis[END_REF]. The aim was to model the situation during lipid mobilization with acn1-2 containing higher levels of acetyl-CoA than Col-7. The assumptions placed into the model were: i) a steady-state production of acetyl-CoA from lipid mobilization, which is likely to be the case just after seedling emergence [START_REF] Cornah | Synthesis and function of glyoxylate cycle enzymes[END_REF], ii) a higher rate of acetyl-CoA production from fatty acid degradation than from extra-peroxisomal sources, iii) an increase in ACN1 activity during seedling growth, iii) an extra-peroxisomal source of acetyl-CoA activity and a peroxisomal thioesterase active with acetyl-CoA, iv) the pool of primary metabolites is fed by fatty acid degradation via steps of the glyoxylate cycle, v) acetyl-CoA can be derived from the metabolite pool. Through extensive varying of parameters to mimic various conditions of metabolic fluxes, we could not obtain the observed differences in acetyl-CoA levels by forming acetyl-CoA exclusively via input from the metabolite pool. In addition, it would not seem likely that a reduced metabolite pool would generate higher acetyl-CoA levels in the mutant. However, we could produce models fulfilling these observations by having acetate leave peroxisomes and activated by an external acetyl-CoA synthetase as long as this activity was greater than that of ACN1/AAE7. More extensive modelling of acn1-2 metabolism using with targeted label input, such as through labelled fatty acids, at different developmental stages will be required to solidify our conclusions.

From gene expression profiles taken over the course of seedling establishment we have evidence that seedlings gradually attain photosynthetic competence after germination, and it is not complete until the first true leaves begin to appear [49]. This suggests that transcriptional programs promoting development are moderated by some mechanism, which may involve catabolite repression [50] and/or genetic factors. It has been hypothesized that genetic factors present in the embryo to regulate cotyledon expansion may continue to exert their effects well into establishment [START_REF] Penfield | Cold and light control seed germination through the bHLH transcription factor SPATULA[END_REF]. Our results show that both development and gene expression are affected by elimination of ACN1/AAE7. When global patterns of gene expression in acn1-2 seedlings are compared to Col-7 at the same stage of development, we see lower transcript levels of genes that are associated with embryogenesis in acn1-2. Subsequently, the down-regulation of genes, such as the LEA proteins, may promote seedling growth like we observe with acn1-2. Genetic mechanisms preventing unimpeded (or causing suboptimal) growth have been demonstrated for the microorganism B. subtilis [START_REF] Fischer | Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism[END_REF]. In general, transcript levels of acetate assimilation genes are also lower in acn1-2. This may reflect the lower rate at which fatty acids appear to be degraded in acn1-2 or could be the result of a diminished metabolic signal from a reduced primary metabolite pool. At this time it is not possible to conclude if altered gene expression is a cause or result of perturbed development, but it is evident that metabolic processes involving peroxisomal acetate/acetyl-CoA metabolism are important for normal seedling development. It is likely that ACN1/AAE7 begins to exert it influence on peroxisomal metabolism during imbibition. It is expressed in imbibed seeds and fluoroacetate, which is activated by ACN1/AAE7, greatly delays germination when seeds are imbibed in the presence of it [START_REF] Turner | Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle[END_REF]. In conclusion, our results support the findings of Bender-Machado et al. [START_REF] Bender-Machado | Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis[END_REF] and Oliver et al. [START_REF] Oliver | Acetyl-CoA-Life at the metabolic nexus[END_REF] that acetate and acetyl-CoA levels are tightly regulated, and disrupting peroxisomal acetyl-CoA metabolism during germination affects both metabolism and gene expression and appears to have knock-on consequences to germination and seedling development. ]acetate chase (arrow). The experimentation is briefly described in the legend to Figure 1. Panels A, B and C refer to the C2, C3 and C4 positions of Gln, respectively. The values represent the mean ± sd of 3 independent experiments. According to Student's t-tests, 2 h pre-chase values between genotypes for all C positions were not significant with P < 0.05. At 4 h, the pre-chase, betweengenotype values were 0.3, 0.06 and 0.07 for C2, C3 and C4, respectively. All post-chase, betweengenotype values were significant with P < 0.01, except for C2 at 4 h (P = 0.054).

Figure 3. Degradation of eicosenoic acid. Fatty acids were extracted from seeds at 4 days after imbibing in the cold (day 0) and 3 and 5 days after transfer to growth conditions. The values represent means ± sd of five independent samples. According to Student's t-tests, for both genotypes the levels were significantly different between days with P < 0.01, and only at day 3 were the levels of acn1-2 significantly different from those in Col-7 (P < 0.01). A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Biochemical Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. Acetate utilization by acn1-2. Symbols: ○, • Col-7; □, ■ acn1-2. Darkened symbols represent acetate levels after [2-12 C]acetate chase. Approximately 1 g samples of 3 day-old seedlings (PSG 0.7) seedlings were fed with 4 mM [2-13 C]acetate plus 20 mM sucrose in incubation media for the lengths of time specified. Chase experiments were performed by removing seedlings at 2 h and transferring them to a solution with 4 mM isotopically normal acetate plus 20 mM sucrose (arrow). Tissue samples were extracted with 80% ethanol. Acetate was quantified using NMR by comparison of the intensities of the 13 C resonances of acetate and the internal standard formate. Symbols and error bars represent the average ± sd, respectively, of values from 3 independent experiments. According to Student's T-tests, the differences between values pre-and post-chase at 4 h for both genotypes were significant with P < 0.01. There were no significant differences between 4 and 6 h for post-chase values for either genotype.

Figure 2 .

 2 Figure 2. Time course quantification of Gln by13 C-labelled carbon resonances. Symbols: ○, • Col-7; □, ■ acn1-2. Darkened symbols represent measured Gln levels after chasing with [2-12 C]acetate chase (arrow). The experimentation is briefly described in the legend to Figure1. Panels A, B and C refer to the C2, C3 and C4 positions of Gln, respectively. The values represent the mean ± sd of 3 independent experiments. According to Student's t-tests, 2 h pre-chase values between genotypes for all C positions were not significant with P < 0.05. At 4 h, the pre-chase, betweengenotype values were 0.3, 0.06 and 0.07 for C2, C3 and C4, respectively. All post-chase, betweengenotype values were significant with P < 0.01, except for C2 at 4 h (P = 0.054).

Figure 4 .

 4 Figure 4. Models to explain the role of ACN1 preventing the loss of carbon from peroxisomes. Abbreviations: Ac, unesterified (free) acetate; Ac-CoA, acetyl-CoA; Cit, citrate, Mal, malate; TE, thioesterase. (A) The wild-type situation showing the anapleurotic/gluconeogenic function of the glyoxylate cycle. The principle flow of carbon from fatty acids out of peroxisomes is through citrate and malate. (B) Removal of ACN1 permits the exit of acetate from peroxisomes due to the action of a thioesterase, where is it reactivated in the cytosol by an acetyl-CoA synthetase activity.

Figure 1 .Figure2

 1 Figure 1.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

  Biochemical Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764

Table II Comparative gene expression of Col-7 and acn1-2 from transcript profiling.

 II The 20 greatest up-and down-regulated genes and acetate assimilation genes with fold-DE greater than 1.4 are shown. The gene descriptions are those given in TAIR10 Genome Release. Fold-DE, fold differential expression (ratio of intensity values). Biochemical Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764 A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 11 May 2011 as manuscript BJ20101764 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101764

	At5g62210	Embryo-specific protein 3, (ATS3)	20.1
	At1g33265	Transmembrane proteins 14C	19.1
	At1g24090	RNase H family protein	17.0
	At4g35480	Encodes a putative RING-H2 finger protein RHA3b.	16.5
	Gene ID At3g45460 At4g36830	Gene Description IBR domain containing protein HOS3-1	16.0 Fold-DE 15.5
	At3g02480 At4g20800	Late embryogenesis abundant protein (LEA) family protein Up-regulated in acn1-2 FAD-binding Berberine family protein	15.1 14.9
	At4g31070 At2g32790	Tetratricopeptide repeat (TPR)-like superfamily protein Ubiquitin-conjugating enzyme family protein	27.7 14.6
	At3g49460 At5g39110	Unknown protein RmlC-like cupins superfamily protein	23.9 14.3
	At1g49920	MuDR family transposase	23.7
	At1g56680	Chitinase family protein Acetate Assimilation Genes	21.2
	At5g13350 At5g03860	Auxin-responsive GH3 family protein Encodes a protein with malate synthase activity.	19.6 5.5
	At1g22080 At3g21720	Cysteine proteinases superfamily protein Encodes a glyoxylate cycle enzyme isocitrate lyase (ICL).	16.7 4.3
	At1g27590 At3g53910	Protein of unknown function (DUF3453) malate dehydrogenase-related	16.0 3.5
	At5g19920 At2g42790	Transducin/WD40 repeat-like superfamily protein Peroxisomal citrate synthase	14.0 1.7
	At2g04620 At3g16910	Cation efflux family protein ACN1/AAE7	14.0 1.5
	At2g46780 At2g30970	RNA-binding (RRM/RBD/RNP motifs) family protein Aspartate aminotransferase 1	13.7 1.4
	At3g28510	P-loop containing NTP hydrolases superfamily protein	12.7
	At1g75230	DNA glycosylase superfamily protein	11.2
	At2g17920	Nucleic acid binding	10.2
	At1g66360	Calcium-dependent lipid-binding family protein	9.4
	At2g06480	Transposable element gene	9.0
	At3g19330	Protein of unknown function (DUF677)	9.0
	At5g24655	RESPONSE TO LOW SULFUR 4 (LSU4)	8.4
	At4g18780	Cellulose synthase family	8.1
	At5g25950	Protein of Unknown Function (DUF239)	7.5
	At4g05240	Ubiquitin-like superfamily protein	7.5
		Acetate Assimilation Genes
	At5g56720	Lactate/malate dehydrogenase family protein	2.9
	At1g08650	Phosphoenolpyruvate carboxylase kinase	2.0
		Down-regulated in acn1-2
	At2g36640	Late embryogenesis abundant (LEA) protein in group 3	52.8
	At1g33055	Unknown protein	45.0
	At1g72070	Chaperone DnaJ-domain superfamily protein	38.4
	At1g48660	Auxin-responsive GH3 family protein	34.4
	At1g32560	Late embryogenesis abundant protein, group 1 protein	30.6
	At4g36600	Late embryogenesis abundant (LEA) protein	28.5
	At2g33850	Unknown protein	24.9
	At4g21020	Late embryogenesis abundant protein (LEA) family protein	24.9
	At5g28630	Glycine-rich protein	23.1
	At1g10640	Pectin lyase-like superfamily protein	21.7

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited -17 -

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

We would like to thank Dr. Martine Dieuaide at INRA-Bordeaux for helping with the 13 C-NMR, the GARNet microarray facility at the University of Nottingham for carrying out the array hybridization, and Professor A. Deri Tomos for aiding in the supervision of Elizabeth Allen. We would also like to acknowledge the contribution of the following people to the modelling effort: Martin Berglund, Hilmar Castro, Anna Lovrics, Tal Pearson, Nikolas Popovich, Jamie Twycross, Maria Grazia Vigliotti and Minaya Villasania. The PhD studentship funding for Elizabeth Allen came from the Sir Williams Roberts trust provided to Bangor University. The work was funded by the UK Biotechnology and Biological Sciences Research Council [grant no. P19408].

The letters and numbers of unknown peaks represent the type of signal and the chemical shift relative to TSP: unk, unknown, D, doublet; M, multiplet; S, singlet. b The number of samples for Col-7 and acn1-2 were 5 and 6, respectively. c The probability statistic p is derived from Student's t-tests with 9 degrees of freedom.