Claudia Massa
email: massa@sissa.it

Mads H Clausen

Jure Stojan

Doriano Lamba

Cristiana Campa

Study of the Mode of Action of a Polygalacturonase from the Phytopathogen Burkholderia cepacia

Keywords: Burkholderia cepacia, glycoside hydrolase, pectin degradation, HPAEC-PAD, capillary electrophoresis, kinetic model

We have recently isolated and heterologously expressed an endo-polygalacturonase from the phytopathogen Burkholderia cepacia (BcPeh28A). Endo-polygalacturonases belong to the glycoside hydrolase family 28 and are responsible for the hydrolysis of non-esterified region of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain length ranging from 2 to 8. By inspection of products progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products.

Moreover, the investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized, based on hydrolysis of hexagalacturonides with different methylation patterns.

Introduction

Primary cell walls are important structures of plant cells that are involved in a variety of physiological processes [START_REF] Somerville | Toward a Systems Approach to Understanding Plant Cell Walls[END_REF]. These structures are composed mainly of polysaccharides such as cellulose, hemicellulose and pectin. Cellulose microfibrils, composed of 30 to 36 chains of β-1,4-linked glucose, together with hemicelluloses form a cohesive net work that is embedded in a soluble matrix of polysaccharides, glycoproteins and lowmolecular weight compounds. Pectic substances are the most abundant components within this matrix. On the basis of backbone structure, pectic substances can be divided into three classes: homogalacturonan (HGA), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II). HGA is a linear homopolymer composed of 1,4-linked α-D-galacturonic acid (GalA) residues, where several GalA residues can be methyl es terified or acetylated depending on the plant source. RG-I has a backbone of alternating rhamnose and galacturonic acid residues, where several rhamnose residues can be sub stituted at C-4 with neutral and acidic oligosaccharide side chains. RG-II is composed of at least seven 1,4-linked α-D-GalA residues, to which branched side chains are attached [START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF].

The modification of such a complex substrate is required in the course of plant physiological processes, like cell wall turnover and remodelling [START_REF] Ridley | Pectins: structure, biosynthesis and oligogalacturonide-related signaling[END_REF], but it also occurs as a consequence of a pathogen attack [START_REF] Prade | Pectins, pectinases and plant-microbe interactions[END_REF][START_REF] Collmer | The role of pectic enzymes in plant pathogenesis[END_REF]. Cell wall degrading enzymes isolated from pathogenic organisms include exo-and endo-polygalacturonases (endoPGs), pectin and pectate lyases, rhamnogalacturonases and rhamnogalacturonan lyases. Moreover other accessory enzymes are needed to degrade the branched side groups, like acetyl and methyl esterases [START_REF] De Vries | Aspergillus enzymes involved in degradation of plant cell wall polysaccharides[END_REF]. Interestingly, many pathogenic organisms produce multiple isoen zymes, suggesting that each isoform harbors a specific catalytic and thus physiological function [START_REF] Herron | Structure and function of pectic enzymes: virulence factors of plant pathogens[END_REF].

Among the pectin-degrading enzymes, a large number of endoPGs have been isolated from pathogenic fungi like Aspergillus niger [START_REF] Bussink | The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes[END_REF] and Botris cinerea [START_REF] Wubben | Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea[END_REF] and their specific hydrolytic activities have been investigated. EndoPGs are secretory enzymes responsible for the hydrolysis of the α-1,4 glycosidic bond in HGA. On the basis of their amino acid sequence similarities, these enzymes have been grouped into glycoside hydrolase family 28 that also includes exo-polygalacturonases, rhamnogalacturonases and xylogalacturonan hydrolases. The catalytic action of endoPGs proceeds according to an acid/base mechanism: a conserved aspartate residue (D201 in A. niger endoPGII, see Figure 1) acts as proton donor, while a water molecule, structurally conserved in two of the known endoPGs crystal structures [START_REF] Pickersgill | Crystal Structure of Polygalacturonase from Erwinia carotovora ssp. Carotovora[END_REF][START_REF] Van Santen | 1.68 Å crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis[END_REF], is the nucleophile. The activation of this water molecule is ascribed to two aspartic acid residues (D180 and D202 in A. niger endoPGII, see Figure 1) that act as general bases. These three aspartate residues are strictly conserved among the endoPGs [12].

In order to define the endoPGs mode of action, the most commonly used experimental approach consists of the detection of the products that are released upon hydrolysis of a substrate chain [13 -18]. On the basis of the hydrolysis product progression profile, endoPGs have been classified as single attack or multiple attack enzymes. Accordingly, in the case of single attack enzymes substrate molecules are cleaved only once after the formation of the enzyme-substrate (ES) complex, resulting in the release of oligomers of different chain lengths. The multiple-attack enzymes, also named processive enzymes, instead cleave the same substrate molecule several times, and small oligomers, or even monomers, are released in the initial period of the reaction. The available information on the mode of action of several fungal endoPGs is reported in Table 1. In particular, a comparative study of A. niger endoPGI and endoPGII modes of action has been carried out, combining the information taken from the amino acid sequence alignment, biochemical and structural studies. These two enzymes, despite their high degree of amino acid sequence identity (71.4%) and similarity (74.2%), exhibit a different mode of action on polygalacturonate. The two structures are very similar with a relative r.m.s.d of 0.8 Å, on 335 corresponding C α atoms [START_REF] Van Santen | 1.68 Å crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis[END_REF][START_REF] Van Pouderoyen | Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger[END_REF]. Both enzymes adopt a right-handed β-helix fold that has been observed in all the known structures of endoPGs [START_REF] Van Santen | 1.68 Å crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis[END_REF]12,[START_REF] Federici | Structural requirements of endopolygalacturonase for the interaction with endoPGIP (polygalacturonase-inhibiting protein)[END_REF][START_REF] Cho | The X-ray structure of Aspergillus aculeatus polygalacturonase and a modeled structure of the polygalacturonaseoctagalacturonate complex[END_REF][START_REF] Shimizu | Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution[END_REF]. The structure of A. niger endoPGII that includes 10 rungs, each consisting of three β-strands separated by turn regions, is depicted in Figure 1. Site directed mutagenesis studies on A. niger endoPGI and endoPGII demonstrated that the switch between a processive and a non-processive mode of action can be ascribed to the presence of an arginine or a serine residue, respectively, in a conserved segment belonging to rung 2 of the β-helix [START_REF] Pages | Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II[END_REF].

Little information is available on the mode of action of bacterial endoPGs and our current understanding has been gathered from data on enzymes isolated from Agrobacterium vitis, Ralstonia solanacearum and Erwinia carotovora that have been previously classified as endo-acting enzymes. By analyzing their product progression profile, the former enzyme has been classified as a dimer-releasing enzyme and both the latter two as trimer-releasing [START_REF] Herlache | Characterization of the Agrobacterium vitis PehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum[END_REF]. The three dimensional crystal structure of E. carotovora endoPG has been elucidated [START_REF] Van Santen | 1.68 Å crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis[END_REF], but no structural-functional relationship studies have been carried out so far.

Other studies aimed at characterizing the active site of endoPGs have been carried out by analyzing the products released following the hydrolysis of pure oligogalacturonides (OGAs) that differ in their degree of polymerization (DP) and in their methylesterification patterns [13][14][START_REF] Parenicova | Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties[END_REF][START_REF] Parenicova | endoPGaA and endoPGaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger[END_REF][START_REF] Bonnin | Study of the mode of action of endopolygalacturonase from Fusarium moniliforme[END_REF][START_REF] Kars | Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris[END_REF][START_REF] Kester | Performance of selected microbial pectinases on synthetic monomethyl-esterified di-and trigalacturonates[END_REF].

The active site of depolymerising enzymes includes several saccharide unit binding sites, referred to as subsites, that have been labelled, by convention, from -n to +n, where -n represents the non-reducing end and +n the reducing end of the sugar moieties, respectively [START_REF] Davies | Nomenclature for sugarbinding subsites in glycosyl hydrolases[END_REF]. The cleavage occurs between the subsites -1 and +1. The latter subsites have been most extensively studied, since they include strictly conserved amino acid residues that are involved in catalysis (Figure 1) [START_REF] Armand | The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis[END_REF]. The other subsites are mainly involved in substrate recognition and have been less well characterized since no significant conservation in the amino acid residues is detectable at these positions.

Structure-based site directed mutagenesis studies of the A. endoPGII unveiled a total of seven subsites, from -4 to + 3, allowing for several amino acid residues to be assigned to each of them (Figure 1), [START_REF] Pages | Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II[END_REF][START_REF] Pages | Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis[END_REF].

To the best of our knowledge, no characterization of the structural determinants responsible for the catalytic activity of bacterial endoPGs has been reported.

We have recently heterologously expressed and purified to homogeneity an endoPG secreted by the plant pathogen Burkholderia cepacia, strain ATCC 25416, referred to also as pectic hydrolase enzyme A, BcPeh28A [START_REF] Massa | Isolation, heterologous expression and characterization of an endopolygalacturonase produced by the phytopathogen Burkholderia cepacia[END_REF]. BcPeh28A does not exhibit a significant amino acid sequence identity with respect to fungal and other bacterial endoPGs: A. niger endoPGI (ID%: 21.5), A. niger endoPGII (ID%: 21.3), E. carotovora (ID%: 23.8), R. solanacaerum (ID%: 22.8) and A. vitis (ID%: 22.9) endoPGs.

With the aim of characterizing the active site of BcPeh28A, the evident lack of sequence similarity notwithstanding, we now for the first time report on the mode of action of a bacterial endoPG on polygalacturonate and on shorter substrate molecules that differ in their overall length and in their methylation patterns.

Experimental 1. Production and purification of BcPeh28A

The endoPG of B. cepacia (BcPeh28A) was heterologously expressed and purified as reported elsewhere [START_REF] Massa | Isolation, heterologous expression and characterization of an endopolygalacturonase produced by the phytopathogen Burkholderia cepacia[END_REF].

Amino acid sequence alignments

The pair wise alignments have been performed, with default parameters, by algorithms available within the EMBOSS bio-informatics suite accessible at the following url: http://www.ebi.ac.uk/emboss/align/.

Substrates

Polygalacturonic acid

Polygalacturonic acid sodium salt (PGA) from citrus fruit was purchased from Sigma Aldrich. According to the manufacturer, the raw material was de-esterified with NaOH and the final level of esters was about 5%. The product has been purified from highgrade pectin with a molecular weight distribution of 50,000-150,000 Da.

Oligogalacturonides

A sample containing a mixture of OGAs with DP ranging from 1 to 8 was kindly provided by Dr. G. Salvi, Dipartimento di Biologia Vegetale, Università "La Sapienza", Roma, Italy. It has been used as standard in the analysis by capillary electrophoresis (CE) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The mixture was obtained as described in [START_REF] Bellicampi | Oligogalacturonides inhibit the formation of roots on tobacco explants[END_REF].

Decagalacturonide

A fully unmethylated decagalacturonide, purified from a pectin digest as described in [START_REF] Guillaumie | Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides[END_REF], was a kind gift from Prof. K. J. Jensen, Department of Natural Sciences, University of Copenhagen, Denmark.

Hexagalacturonides

The hexagalacturonides (1-4) (Figure 7) that differ in their methylation patterns were synthesized as reported in [START_REF] Clausen | Synthesis of Hexasaccharide Fragments of Pectin[END_REF].

Time course experiment

BcPeh28A (0.5 µg/mL) was incubated at 20 °C in 2.0 mL of 50 mM sodium acetate, pH 3.5, in the presence of 0.25% g/L PGA as substrate. Aliquots of 50 µl were taken at regular intervals and the reaction was stopped by adding an equal volume of derivatization solution, as described below. The samples were then boiled at 95 °C for 20 minutes. Capillary electrophoresis, as described below, was used for identifying and assessing the molar number distribution of the OGAs present in the mixture, following the enzymatic reaction.

Kinetic model

The detailed kinetic characterization of BcPeh28A, using PGA as substrate, was done in several steps. At first, direct information on the reaction path was gathered by inspecting the products progression curves. Then, a family of independent and likely reaction schemes was constructed and for each of them the corresponding sets of differential equations were derived. Each reaction scheme was then tested by simultaneous fitting to the progress curves. A non linear regression analysis was performed by a computer programme that fitted the parameters of a set of stiff differential equations to multiple experimental curves [33]. In a system of differential equations that describes the time course of six products more than twelve parameters should be evaluated. In most, if not all cases, it is necessary to make rational assumptions in order to reduce the number of unknowns, due to the limitations in the experimental data. Hence, the bimolecular rate constants for the enzyme-substrate binding were assumed to be close to the diffusion rate limit either for the PGA or for the oligomers (k

1 = k 3 = k 5 = k 7 = 5x10 8 [M -1 sec -1 ]
); the catalytic constants for OGAs with a DP 4-6 were assumed to be equal (k 4 = k 6 = k 8 ); the dissociation constant of the E-PGA complex, k -1 , always converging to very small values, was set equal to zero; the initial enzyme concentration was set to 2.0x10 -8 M (see Experimental section 4). Table 2 reports the imposed and derived kinetic constants in detail. The basic criteria for the goodness of the fit were obeyed throughout the analysis [START_REF] Cleland | Statistical Analysis of Enzyme Kinetic Data[END_REF].

Mode of action on OGAs

Hydrolysis of decagalacturonide

BcPeh28A (0.075 µg/mL) was incubated at 37 °C with decagalacturonide (50 µM) in 50 mM acetate buffer, pH 3.5. Aliquots (100 µL) of the reaction mixture were taken at regular time intervals and the reaction stopped by the addition of an equal volume of 2.0 mM Tris-hydrochloride, 50 mM sodium hydroxide, which resulted in a final pH of 8.3-8.5. In order to avoid the spontaneous breakdown of the obtained OGAs, reported to occur at high pH [13], the samples were immediately analysed by HPAEC-PAD, as described below. The identification of the hydrolysis products was done in accordance to the retention time of a standard containing a mixture of OGAs with a DP 1-8.

Hydrolysis of hexagalacturonides

BcPeh28A (1.0 µg/mL) was incubated at 37 °C with 25 µM of hexagalacturonides in 50 mM acetate buffer pH 3.5, for different incubation times. The enzymatic digest was analysed by electrospray mass spectrometry with a time-of-flight analyser (ESI-TOF-MS), as described below.

Analytical techniques

Capillary electrophoresis (CE)

Capillary electrophoresis experiments were carried out using a Hewlett-Packard HP3D Capillary electrophoresis system, equipped with a diode array UV detector. Fused capil laries were from Agilent Technologies (total length: 104 cm; effective length: 95.5 cm; i.d. 50 µm; extended light path). Before sample injection, a 4-min conditioning of the capillary with the running buffer was necessary, preceded by a 2-min wash with 0.1 M sodium hydroxide (pressure equal to 950 mbar). Signals were acquired by continuously monitoring UV absorbance at 195 nm and 285 nm. All analyses were done at 25 °C.

The separation potential was equal to 27 kV. Samples were injected at 50 mbar for 6 sec. Monomer, dimer and trimer of galacturonic acid (Sigma Aldrich) as well as a sample containing a mixture of OGAs with DP 1-8 were used as standards. To allow a sensitive UV detection, the saccharides were derivatized by reaction with 4-aminoben zonitrile (4-ABN) in the presence of sodium cyanoborohydride (NaCNBH 3 ) [START_REF] Schwaiger | Capillary zone electrophoresis and micellar electrokinetic chromatography of 4aminobenzonitrile carbohydrate derivatives[END_REF]. The derivatization solution was composed of 0.16 M NaCNBH 3 and 0.5 M 4-ABN in meth anol/acetic acid (95/5 v/v). Only one 4-ABN molecule chromophore is attached to each oligomer, leading to a UV-response independent of the length of OGAs. This represents an advantage over chromatographic techniques such as HPAEC-PAD where the electro chemical response must be determined for each oligomer [START_REF] Campa | Determination of average degree of polymerisation and distribution of oligosaccharides in a partially acid-hydrolysed homopolysaccharide: a comparison of four experimental methods applied to mannuronan[END_REF]. The derivatization solu tion was diluted 1:1 with an aqueous solution of the uronic acid(s) and the derivatization mixture was heated for 20 minutes at 95 °C. Prior to injection into the CE system, samples were spun down for 1 minute at 13000 g. The analysis was performed by mi cellar electrokinetic capillary chromatography (MEKC) as described in [START_REF] Campa | Determination of average degree of polymerisation and distribution of oligosaccharides in a partially acid-hydrolysed homopolysaccharide: a comparison of four experimental methods applied to mannuronan[END_REF]. The buffer used was 600 mM boric acid, containing 75 mM SDS (pH 8.0). The peak areas divided by migration time (A/t) were used for quantitative purposes [START_REF] Baker | Capillary Electrophoresis[END_REF]. To obtain a calibration curve, electrophoregrams for different amounts of galacturonic acid were recorded and their corresponding values of A/t were plotted as a function of nanomoles of galacturon ic acid. Calibration was linear in the concentration range comprised between 1.0 and 14.0 nanomoles.

High-performance anion-exchange chromatography with pulsed amperometric de tection (HPAEC-PAD)

The analysis of oligomers released upon hydrolysis of decagalacturonide by BcPeh28A was carried out with a HPAEC-PAD system (Dionex Corporation), equipped with a GP50 gradient pump, an ED50 electrochemical detector, and a LC25 chromatography oven. The separation was performed using a CarboPac PA-200 Analytical anion exchange column (3x250 mm) with Guard column (3x50 mm). The volume of the injection loop was 25 µL, and the column temperature was 35°C. The flow-through cell of the pulsed amperometric detector consisted of an Au working electrode (1.0-mm diameter) and a pH-Ag/AgCl combination reference electrode; the titanium body of the cell served as the counterelectrode. The sequence of potentials applied to the electrode was set as recommended by the manufacturer for carbohydrate detection, as follows: t = 0 s, E = 0.10 V; t = 0.20 s, E = 0.10 V (integration begin); t = 0.40 s, E = 0.10 V (integration end); t = 0.4 s, E = -2.00 V; t = 0.4 s, E = -2.00 V; t = 0.43 s, E = 0.60 V; t = 0.4 s, E = -0.10 V; t = 0.50 s, E = -0.10 V. The software Chromeleon, version 6.60 (Dionex Corporation) was used for the acquisition and processing of the chromatograms. The column was equilibrated with 0.1 M sodium hydroxide, 0.2 M sodium acetate. The samples were eluted applying a linear gradient of 0.2-0.8 M sodium acetate in 0.1 M sodium hydroxide at 0.5 mL/min for 60 minutes. The identification of the hydrolysis products was done by correlating with the retention times of a standard containing a mixture of OGAs with DP 1-8. was performed on a microTOF Focus system (Bruker Daltonics), equipped with a nitrogen generator N2LCMS1(Claind). The samples were diluted five times with a mixture of methanol/water/formic acid (49/49/2) and injected with a syringe pump in the mass spectrometer. The injection flow rate was 180 µL/h; the capillary voltage was 4500 V and the end plate offset was 500V (positive mode); the dry temperature was 180°C, the dry gas flow was 4 L/min and the nebulizer pressure was 0.4 bar.

Results and Discussion

Time course experiment

The mode of action of BcPeh28A on a polymeric substrate was investigated in a time course experiment that monitored the products released upon PGA hydrolysis by collecting electropherograms. The suitability of MEKC with UV detection for the separation of uronic acids [START_REF] Campa | Determination of average degree of polymerisation and distribution of oligosaccharides in a partially acid-hydrolysed homopolysaccharide: a comparison of four experimental methods applied to mannuronan[END_REF] was tested on a standard sample containing a mixture of OGAs with DP 1-8, as shown in Figure 2A.

The products progression profile (Figure 3A) indicates that during the first minutes of reaction, OGAs with a DP ranging from 3 to 8 were produced. This confirms that BcPeh28A is an endo-acting enzyme. An electropherogram of the reaction mixture is shown in Figure 2B that provides the molar product distribution present in the enzymatic digest. The occurrence of OGAs with different chain lengths clearly shows that BcPeh28A can be classified as single-attack enzyme. The minimum length of the released OGAs varies among pectin-degrading enzymes and provides further information on the active site architecture of the enzyme. For the PGA hydrolysis catalyzed by BcPeh28A, the end products of the reaction are monomers, dimers and trimers, with a clear excess of trimers (Figure 3B). To verify whether BcPeh28A is capable of further degrading the trimers, a commercial sample of trigalacturonic acid was incubated with the enzyme. No cleaved products were observed (data not shown).

Hence, the substrate binding cleft of BcPeh28A is likely to span at least four susbsites.

This finding allows us to classify BcPeh28A as a trimer-releasing enzyme, in keeping with other bacterial endoPGs [START_REF] Herlache | Characterization of the Agrobacterium vitis PehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum[END_REF].

In order to gain a more detailed insights into the BcPeh28A catalytic site, its amino acid sequence was pair-wise and structure-based aligned to that of the well-studied nonprocessive enzyme A. niger endoPGII. It appears (Figures 1 and4) that the catalytic amino acids forming subsites -1 and +1, belonging to the rungs 5, 6, 7 and 8, are very well conserved, while the amino acid residues associated with the distal subsites are more variable (M150/E192, N186/G234, E252/D310, D282/Q342, Q288/T347, Y326/E417). It is worthy of note that a serine residue, S104, is present in the putative rung 2 of BcPeh28A, thus supporting the previous established criteria according to which a single amino acid residue can determine the processive or non-processive behaviour of endoPGs [START_REF] Pages | Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II[END_REF].

Kinetic model

A kinetic model that describes the BcPeh28A mode of action on PGA has been con structed as reported in the Experimental section 5. The reaction scheme has been ra tionalized as illustrated in Figure 5A. By inspecting the product progress curves (Figure 3), it is possible to assume that OGAs with a DP 3-6 are produced rapidly after mixing PGA with the enzyme, while monomers and dimers emerge only later as second-genera tion products. Additionally, as previously mentioned monomers, dimers and trimers ac cumulate with time, while OGAs with a DP 4-6 appear at the beginning of the reaction and are cleaved later to yield shorter ones. Thus, trimer formation is the result of a rapid release from either PGA or DP 4-6 reprocessing. The percentage of uncleaved PGA at the end of the reaction has been evaluated to be 99%, confirming that the time course experiment has been performed in a large excess of substrate. The excellent agreement between the experimental progression curves shown in figure 3B and those obtained as the result of the kinetic model based on a numerical integration method, is shown in Figure 5B.

A number of conclusions can be drawn from kinetic constants derived from the model (see Table 2). It has been assumed that the dissociation rate constant of the E-PGA com plex, k -1 , can be approximated to zero. As a consequence, the k cat /K m value for PGA ap proaches the diffusion-controlled rate limits, i.e. the specificity constant of BcPeh28A for PGA approaches its maximum. This means that catalytic velocity on PGA is only re stricted by the rate at which BcPeh28A encounters the polymer in solution. Hence, every encounter between the enzyme and the substrate is productive. Moreover, the k cat value for PGA is higher if compared to the k cat values for oligomers, suggesting that BcPeh28A degrades long polymeric chain faster than the short OGAs.

Concerning the OGAs hydrolysis, it has been assumed that, once BcPeh28A binds to an oligomer with a DP 4-6, the catalysis takes place at the same rate, independent of the length of the substrate bound (k 4 = k 6 = k 8 ). This implies that the rate-determining step of the enzymatic reaction on OGAs is the formation of a stable and productive ES com plex. At this point, it is worthy of note that the only kinetic parameters that enable dif ferentiation of oligomers from one another are the dissociation constants, whose values decrease with increasing chain length of the substrates (k -3 > k -5 > k -7 ). In order to ex plain this trend, it is convenient to introduce the concept of binding modes. They can be referred to as the multiple putative modes of interaction that may occur between the en zyme subsites and the saccharide units of the substrate chain. From a catalytic point of view, only the binding modes that involve binding at the subsites -1 and +1 can lead to a product release, therefore allowing for discriminating between productive and non-pro ductive binding modes.

Longer substrate molecules, like DP 6, that can interact with a large number of enzyme subsites, exhibit a higher overall binding affinity when compared to those of shorter OGAs, like DP 4 and DP5. Binding of shorter OGAs is unfavourable, which is con firmed by their higher dissociation constants. Such a trend of dissociation constants con sequently determines the decreasing trend of K m values at the increase of the length of the substrate chain. It should be emphasized that since the binding of oligomers like DP 4 and DP 5 can occur according to more than one way (Figure 5C), one would expect different affinities for each binding mode. The kinetic model indirectly allowed the identification of the favoured binding modes by introducing a partition coefficient for the distribution of the end products. The resulting distribution led us to conclude that the subsites at positions -3, -2, -1 and +1 display the highest binding affinities, being those that are most likely to be involved in the formation of the favoured productive ES com plexes (Figure 5C). Similar conclusions have been reported by studying the active site architectures of endo-(1→5)-α-L-arabinanases from A. niger and A. aculeatus [START_REF] Pitson | Action patterns and mapping of the substrate-binding regions of endo-(1→5)-α-L-arabinanases from Aspergillus niger and Aspergillus aculeatus[END_REF] and of the endo-β-(1→4)-xylanase from Penicillium simplicissimum [START_REF] Schmidt | Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant[END_REF]. The P. simplicis simum xylanase crystallographic studies and biochemical data on the hydrolysis of xy looligosaccharides with a DP 2-6 revealed that four is the minimum substrate length for cleavage. This suggsts that the two catalytic residues divide the binding cleft into a "substrate recognition area", from the active site toward the non-reducing end of a bound xylan chain, with strong and specific binding for the first three xylose sugar units, and a "product release area" with considerably weaker and less specific binding.

Mode of action on OGAs

Hydrolysis of decagalacturonide

To investigate if BcPeh28A displays a preferential cleavage mode on shorter substrate chains, its mode of action on a pure decagalacturonide was studied. The hydrolysis products were analysed by the highly sensitive HPAEC-PAD technique, since the available amount of the initial substrate was under the sensitivity limit for OGA detection by MEKC-UV. The purity of the decagalacturonide, prior to the enzymatic incubation, was checked by HPAEC-PAD. Small amounts of octa-and nonagalacturonides were detected (Figure 6A). Following the incubation with the enzyme (Figure 6B), the decagalacturonide sample was hydrolyzed, and the formation of OGAs with DP 3-7 was detected. The reaction was stopped as previously described, by adding of 2.0 mM Tris-HCl, 50 mM sodium hydroxide. This buffer produced a large peak that masked the signal of monomer and dimer, making their detection impossible.

HPAEC-PAD analysis of the decagalacturonide digestion products pointed out that

BcPeh28A is an endo-acting enzyme that displays a non-processive behaviour also on shorter substrates. As previously observed for the PGA hydrolysis, the main end product of decagalacturonide hydrolysis was identified to be the trimer.

Hydrolysis of hexagalacturonides with different methylation patterns

In order to obtain a more thorough understanding of the enzyme-substrate topology interaction, the mode of action of BcPeh28A on hexagalacturonides (1-4) that differ in the methylation patterns (Figure 7A and 7B) was investigated. Namely, the hydrolysis of compound 1 and compound 2 that exhibit an asymmetric methylation pattern, allowed us to define the tolerance towards methylated saccharide unit of the individual subsites. The definition of the substrate location has been previously established by biochemical studies for endoPGII of A. niger [14] and further confirmed by crystallographic studies of the unbound endoPG from Stereum purpureum and of its binary complex with one D-galacturonate (a β-D-galactopyranouronate was found in the subsite +1) and of its ternary complex with two D-galacturonates (an additional β-Dgalactofuranuronate was found at the subsite -1) [START_REF] Shimizu | Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution[END_REF]. Thus the non reducing end of the substrate points towards the N-terminus of the enzyme, as illustrated in Figure 1.

The end products of the hexagalacturonides (1-4) hydrolysis were analysed by HPAEC-PAD and ESI-TOF-MS. As reported elsewhere [START_REF] Williams | A novel enzyme activity involving the demethylation of specific partially methylated oligogalacturonides[END_REF][START_REF] Van Alebeek | Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger[END_REF], the use of the HPAEC-PAD technique is not suitable for characterizing the hydrolytic products, since the methylated OGAs undergo spontaneous de-methylation at the pH values reached during the analysis. Consequently, although typical chromatograms showed peaks attributable to both methylated and de-methylated oligomers, it was not possible to quantify the amounts of the individual hydrolysis products.Nevertheless, HPAEC-PAD was used to determine the optimal relative concentrations of enzyme and substrate at which the hydrolysis could occur. Indeed, since the methylated hexagalacturonides (1-4) are not good substrates for BcPeh28A, a higher enzyme to substrates ratios were chosen with respect to previous experiments. HPAEC-PAD revealed that BcPeh28A does not hydrolyze compounds 2 and 4 (data not shown). It is possible to hypothesize that BcPeh28A cannot bind modified substrate molecules, such as extensively methyl-or acetyl-esterified OGAs. In fact, such chemical modifications affect the net charge distribution and the steric hindrance of the OGAs and interfere with the ionic interactions, hydrogen bonding network and hydrophobic interactions that are the driving forces in the formation and stabilization of the ES complexes. On the contrary, compounds 1 and 3 are substrates of BcPeh28A, since methylated hydrolysis products could be identified in the chromatograms (data not shown). The identity of the hydrolysis products was assessed by ESI-TOF-MS. Figure 8 (panels A and B) shows the mass spectra of the hydrolysis products originating from the enzymatic degradation of compound 1, after 1 hour of incubation at 37 °C. The identification of a peak corresponding to compound 1 (peak 1: m/z = 1139.25,

[M+Na] + ) suggested that it was not completely hydrolyzed. The monomethylated dimer (peak 1a: m/z = 407.08, [M+Na] + ) and the dimethylated tetramer (peak 1b: m/z = 773.16, [M+Na] + ) were identified as the hydrolysis products.

Hence, the identification of these two hydrolysis products strongly indicates that the only productive ES complex would involve binding of the hexagalacturonate 1 with the un-methylated saccharide units at the subsites -2, -1 and +1 respectively (Figure 9.1).

The structural studies of the ternary complex of S. purpureum endoPG with two Dgalacturonates [START_REF] Shimizu | Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution[END_REF] revealed that in fact the carboxylate of a galacturonic acid residue located at position -1 is engaged in electrostatic interactions with several conserved, positively charged amino acids (e.g., the arginine and lysine residues belonging to the RIK segment present in the rung 8, see Figures 1 and4). These interactions significantly contribute to the stability of the ES complex. In addition, as observed in the crystal structure of the ternary complex of S. purpureum endoPG [START_REF] Shimizu | Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution[END_REF], the binding at subsite +1 may provide adequate free energy to permit distortion of the conformation of the sugar residue at subsite -1. The distorted half-chair conformation of the sugar ring bound at site -1 is thought to be involved in the stabilization of the oxocarbenium cation-like transition state [START_REF] Rye | Glycosidase mechanisms[END_REF]. Therefore, the binding of a methylated saccharide unit at the subsite -1 prevents the formation of a productive ES complex.

The mass spectra of the products released upon the enzymatic hydrolysis of compound were identified as end products. The identification of these two hydrolysis products strongly suggests that two productive ES complexes can occur (Figures 9.2 and 9.3). In either case, BcPeh28A shall accommodate three consecutive un-methylated saccharide units at the subsites -2, -1 and + 1.

The ESI-TOF-MS analyses showed that compound 1 was not fully hydrolysed by BcPeh28A, after 1 hour of incubation at 37 °C (Figure 8B), in contrast to compound 3 that, under the same conditions, was completely degraded after 20 minutes. This finding suggests that compound 3 is a preferred substrate compared to compound 1. Their different susceptibilities to the enzymatic hydrolysis can be ascribed to the two versus the only one productive binding mode that likely exist for compound 3 and 1, respectively (Figure 9). However, it should be emphasized that these findings are of a qualitative nature. The ionisation efficiency of methylated OGAs is influenced by their chain length and methylation pattern. Namely, the methylation degree of hexagalacturonides 1 and 3 may affect changes in their volatility and hydrophobicity [START_REF] Zaia | Mass spectrometry of oligosaccharides[END_REF]. Therefore, ESI-TOF-MS was reliable only for a qualitative assessment of the mixtures analysed.

Overall, the observed action patterns of BcPeh28A on compounds 1 and 3 and its inability to hydrolyse compounds 2 and 4 indicate that in order to form a productive ES complex, the substrates should at least contain three consecutive un-methylated saccharide units. In addition, among the six subsites identified, those referred to as -2, -1 and +1 can be supposed to be responsible for crucial ES contacts. Indeed, methylated saccharide units cannot be accommodated at these subsites, thus excluding the formation of a productive ES complex as it is observed for compounds 2 and 4. The subsites -4, -3, +2, +3 and +4, if all of them exist, are likely less critical for the formation of a productive ES complex, since they can accommodate a methylated saccharide unit (Figure 9). This finding is in agreement with results obtained from the digestion of pectins with a different degree of methyl-esterification by A. niger and E. carotovora endoPGs [START_REF] Mort | Separation of 8-aminonaphthalene-1,3,6trisulfonate labeled oligomers containing galacturonic acid by capillary electrophoresis: application to determining the substrate specificity of endopolygalacturonases[END_REF].

In perspective, further studies are needed to fully elucidate the structural determinants responsible for the binding affinities of each subsite by using pure unmethylated and ad hoc methylesterified OGAs with DP 5-8, in concert to a site directed mutagenesis approach aimed at determining the exact amino acid residue topology at each subsite.

7. 3 E

 3 lectrospray mass spectrometry with a time-of-flight analyser ( ESI-TOF-MS) Mass spectrometry with electrospray ionization and a time-of-flight analyser (ESI-TOF)

3

  are shown in Figure 8, panels C and D. No peak attributable to compound 3 was found after an incubation time of 20 minutes at 37 °C. The monomethylated dimer (peak 3a: m/z = 407.08, [M+Na] + ) and monomethylated trimer (peak 3b: m/z = 583.11, [M+Na] + )

family 28 :

 28 sequence-structural features, specificities and evolution. Protein Eng. 14, 615-631. 13 Parenicova, L., Benen, J. A. E., Kester, H.C.M. and Visser, J. (1998) pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. Eur. J. Biochem. 251, 72-78.

Figure 1 .

 1 Figure 1. The three dimensional structure of Aspergillus niger endoPGII. The β-helix encompasses 10 complete rungs (numbered from 1 to 10). The amino acid residues associated with subsites -3 to +3 are reported in the scheme[START_REF] Pages | Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis[END_REF]. The aspartate residues D180, D201, D202 are involved in catalysis. The residues at subsites -1 and +1 shown in the table are strictly conserved among fungal endoPGs[12]. The figure was prepared with PyMOL[START_REF] Delano | The PyMOL Molecular Graphics System[END_REF].

Figure 2 .

 2 Figure 2. Electropherogram of a sample containing a mixture of OGAs with DP 1-8 (A). Electropherogram of the reaction mixture after 30 minutes of incubation at 37 °C on PGA 0.25 % g/L (B). The details are given in the Experimental section 7.1.

Figure 3 .RUNG 1

 31 Figure 3. The progression profile curves of released products upon incubation of PGA (0.25 g/L) with BcPeh28A at 20 °C over 10 minutes (A) and over 1.5 hours (B). Δ, DP 1; ♦, DP 2; ▲, DP 3; ■, DP 4; □, DP 5; ▼, DP 6; ×, DP 7; ,٭ DP 8. The details are given in the Experimental sections 4 and 7.1.

Figure 4 .

 4 Figure 4. Structure based amino acid sequence alignment of Aspergillus niger endoPGII (PDB code: 1CZF) and BcPeh28A. The amino acid residues of A. niger endoPGII shown in Figure1are in bold and those that are conserved in BcPeh28A are also underlined. The serine residues S91 (A. niger endoPGII ) and S104 (BcPeh28A) are underlined. They belong to the rung 2 and are responsible for the non-processive enzymatic behaviour. The "*", ":" and "." symbols indicate: identical residues, conserved substitutions and semi-conserved substitutions, respectively.

Figure 5 .

 5 Figure 5. Reaction scheme constructed on the basis of the product progression curves reported in Figure3B (A). The progression curves of OGAs with a DP 1-6, derived from the experimental data (straight lines: ) and reconstructed according to the kinetic model (dashed lines: ----) (B). A graphical view of the productive binding modes between the enzyme and the substrates (oligomers with a DP 4, DP 5 and DP 6, respectively). The reducing end of OGAs is indicated by the symbol Ø while an arrow indicates the cleavage sites. The percentage of occurrence of each binding mode and the associated standard deviations are also reported (C).

Figure 6 .

 6 Figure 6. HPAEC-PAD analysis of the hydrolysis of the decagalacturonide, prior to (A) and subsequent to the incubation for 5 minutes at 37 °C with BcPeh28A (B). The details are given in the Experimental sections 6.1 and 7.2.

Figure 7 .

 7 Figure 7. Partially methyl-esterified hexagalacturonates (A). Methylation patterns of the hexagalacturonides (B). The reducing end is indicated by the symbol Ø.

Figure 8 .

 8 Figure 8. Mass spectra of the hydrolytic products released after the enzymatic hydrolysis by BcPeh28A of compound 1 (after 1 hour of incubation at 37 °C, panels A and B) and compound 3 (after 20 minutes of incubation at 37 °C, panels C and D). The molecular masses indicated by the arrows have been assigned as follows: peak 1: m/z = 1139.25, [M+Na] + , hexagalacturonide 1; peaks 1a and 3a: m/z = 407.08, [M+Na] + , monomethylated dimer; peak 1b: m/z = 773.16, [M+Na] + , dimethylated tetramer; peak 3b: m/z = 583.11, [M+Na] + , monomethylated trimer. The details are given in the Experimental sections 6.2 and 7.3.

Figure 9 . 6 k 7 =

 967 Figure 9. The BcPeh28A binding modes of compounds 1 (Fig. 9.1) and 3 (Fig. 9.2 and 9.3). The reducing end is indicated by the symbol Ø while an arrow indicates the cleavage sites.

Table 2 .

 2 The BcPeh28A kinetic parameters for different substrates. For each derived ki netic constant the associated standard deviation is also reported.

Biochemical Journal Immediate Publication. Published on 13 Jul 2007 as manuscript BJ20061833 © 2007 Biochemical Society

Acknowledgements

We gratefully acknowledge Prof. William G. T. Willats (Department of Molecular Biology, University of Copenhagen, Denmark) and Prof. K. J. Jensen (Department of Natural Sciences, University of Copenhagen, Denmark) for their suggestions and critical reading of the manuscript.

Multiple attack enzymes

Table 1. Fungal endoPGs whose mode of action has been elucidated. The enzymes have been grouped into two subgroups according to their mode of action on PGA. For each of the entries the source, the Swiss Prot (a) or EMBL/Genbank (b) database accession numbers, the PDB ID code and the relevant bibliographic references have been includ ed.


Nicolas Arnaud

Karl Ravet

Andrea Borlotti

Brigitte Touraine

Jossia Boucherez

Cécile Fizames

Jean-François Briat

Françoise Cellier

Frédéric Gaymard
email: gaymard@supagro.inra.fr

The iron-responsive

Keywords: ACO, aconitase, IRE, iron responsive element, IRP, iron regulatory protein, cytosolic aconitase, iron, Arabidopsis, ferritin

Introduction

As the major cofactor of proteins involved in essential processes like photosynthesis, respiration, DNA replication or nitrogen fixation, iron is an essential element for life. Nonetheless, in the free ionic form, iron is toxic as it can catalyze the formation of reactive oxygen species (ROS) through the Fenton reaction. These ROS damage the cell membranes, DNA and proteins [START_REF] Guerinot | Iron: Nutritious, noxious, and not readily available[END_REF][START_REF] Noctor | Ascorbate and glutathione: Keeping active oxygen under control[END_REF]. Thus, iron homeostasis has to be tightly regulated, to avoid starvation that impairs the metabolism, and to avoid excess that may lead to cell death. Iron homeostasis is strongly dependent on ferritins, which are iron-storage proteins, found in bacteria, animals and plants. Plant and animal ferritin structures are very similar, and are formed by 24 subunits arranged to form an hollow sphere able to sequester iron in a non-toxic and bioavailable form [START_REF] Harrison | The ferritins: Molecular properties, iron storage function and cellular regulation[END_REF].

A major regulatory pathway of iron homeostasis in pluricellular eukaryotic organisms has been documented from studies with animal systems [START_REF] Harrison | The ferritins: Molecular properties, iron storage function and cellular regulation[END_REF][START_REF] Hentze | Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress[END_REF]. In these systems, the balance between iron uptake and iron storage is mainly achieved by the post-transcriptional regulation of ferritin and transferrin receptor synthesis by the IRE/ IRP1-cytosolic aconitase iron switch. In the 5'untranslated region of ferritin mRNA and in the 3'-untranslated region of transferrin receptor transcript are located specific sequences, named IREs (Iron-Responsive-Elements [START_REF] Klausner | Regulating the fate of mRNA: The control of cellular iron metabolism[END_REF]). The IREs function as binding sites for two related trans-acting factors, namely the iron regulatory proteins IRP1 and IRP2. When bound to the IRE of the ferritin mRNA, the IRP inhibits translation of the transcript [START_REF] Hentze | Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress[END_REF]. Conversely, the binding of IRP to the IREs found in the 3'-untranslated sequence of the transferrin receptor mRNA stabilizes the transcript, thus leading to the synthesis of the protein.

IRP1 is a bifunctional protein that, when iron is abundant, possesses a 4Fe-4S cluster and acts as a cytosolic aconitase. When iron level is low, the 4Fe-4S cluster disassembles and the apoprotein acquires IRP activity, thus repressing ferritin translation. High levels of iron lead to the 4Fe-4S cluster reconstitution and therefore to the protein aconitase activity. Exposure to nitric oxide (NO • ) and ROS was shown to convert the aconitase into an IRP by releasing the cluster, consequently leading to the IRE binding, and finally modulating the expression of ferritins [START_REF] Hirling | Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase[END_REF][START_REF] Gardner | Superoxide radical and iron modulate aconitase activity in mammalian cells[END_REF].

IRP2 is an IRP1 homologue which does not contain a Fe-S cluster and thus has no aconitase activity. Its activity is primarily regulated by iron-dependent degradation [START_REF] Rouault | The role of iron regulatory proteins in mammalian iron homeostasis and disease[END_REF].

Several reports suggested that the IRE/ IRP1-cytosolic aconitase could also operate in plants [START_REF] Peyret | Structure, genomic organization, and expression of the Arabidopsis thaliana aconitase gene. Plant aconitase show significant homology with mammalian iron-responsive element-binding protein[END_REF][10][11], making it a universal regulatory pathway in eukaryotic pluricellular organisms. Indeed, several lines of evidence suggest a putative relation between ferritins and aconitase activity. NO and oxidative stress have been shown to modulate the expression of ferritins in plants [11][START_REF] Petit | Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family[END_REF][START_REF] Arnaud | An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopis AtFer1 ferritin gene expression[END_REF], and to inactivate the aconitase [10,[START_REF] Verniquet | Rapid inactivation of plant aconitase by hydrogen peroxide[END_REF], and thus potentially converting it into an IRP. However, indirect evidences are not in favor of an IRE/ IRP1-cytosolic aconitase regulatory switch in plants. Indeed, the synthesis of plant ferritins and iron transporters has been shown to be regulated at the transcriptional level in response to the iron status, and not at the mRNA stability or translational levels as described above for animal cells [START_REF] Petit | Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family[END_REF][START_REF] Lescure | Ferritin gene transcription is regulated by iron in soybean cell cultures[END_REF]. IRE sequences are not found in the plant ferritin genes. No IRP activity is detectable in plant cell extracts by RNA shift experiments using animal IRE as a probe [START_REF] Rothenberger | The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution[END_REF], or in a translational assay using animal ferritin mRNA and wheat germ extract [START_REF] Brown | Requirements for the translational repression of ferritin transcripts in wheat germ extracts by a 90-kda protein from rabbit liver[END_REF]. However, it must be pointed out that the bacterial aconitase does have a RNA binding activity, but does not recognize a canonical IRE sequence [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF][START_REF] Tang | Escherichia coli aconitases and oxidative stress: Post-transcriptional regulation of sodA expression[END_REF]. The potential RNA binding activity of plant aconitase was recently enforced by showing that an Arabidopsis aconitase protein is able to bind to the 5'-UTR of the superoxide dismutase CDS2 gene [20].

In order to directly address the involvement of a putative IRP1-cytosolic aconitase regulatory switch in the regulation of ferritin expression in plants, we searched for IRP homologues in the Arabidopsis genome, identified three genes encoding putative aconitase, and isolated the corresponding loss-of function mutants. In the three mutants, the expression of genes involved in iron homeostasis was not altered, demonstrating that cytosolic aconitase, in contrast to animals and bacteria, are not involved in the regulation of iron metabolism in plants.

EXPERIMENTAL Isolation of aco knock-out mutants

Mutants in the three aconitase genes ACO1 (At4g35830), ACO2 (At4g26970), ACO3 (At2g05710) were identified in the collections from the Salk Institute [START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF] for ACO2 (line Salk-054196), and ACO3 (line Salk-013368) and from the GABI-Kat [START_REF] Rosso | An Arabidopsis thaliana T-DNA mutagenized population (gabi-kat) for flanking sequence tag-based reverse genetics[END_REF] for ACO1 (line 138A08).

Plants were back-crossed and homozygous mutant plants were identified by PCR on genomic DNA.

The two T-DNA / gene junctions were amplified and sequenced to determinate the location of the T-DNA insertion.

Plant Culture

Plants were grown on soil (Humin substrate N2 Neuhaus, Klasmann-Deilmann, Geeste, Germany) in a greenhouse at 23°C with a light intensity maintained above 300 E.s -1 per m 2 and a day/night regime of 16 h/ 8 h. For determining the expression pattern of ACO genes, wild type (Col ecotype) plants were grown in hydroponic conditions for 5 weeks as described [START_REF] Cellier | Characterization of AtCHX17, a member of the cation/H + exchangers, CHX family, from Arabidopsis thaliana suggests a role in K + homeostasis[END_REF]. The other studies were performed on 10 days old plantlets grown in liquid medium in sterile conditions [START_REF] Arnaud | An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopis AtFer1 ferritin gene expression[END_REF].

For the iron-starvation treatment, plants were transferred in the same medium, except that Fe(III)-EDTA was omitted. For iron excess treatment, a mix of FeSO 4 / Na 3 -citrate was added (final concentration in the medium: 300 M FeSO 4 / 600 M Na 3 -citrate).

RNA preparation and analysis

Total RNA were extracted, and northern blot experiments were performed as indicated in Lobréaux et al. [24]. The EF1 , AtFer1 and IRT1 specific probes were obtained as described [START_REF] Arnaud | An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopis AtFer1 ferritin gene expression[END_REF][START_REF] Vert | IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth[END_REF]. The AtFer3 specific probe consists in a chimeric fragment containing the 5'-and 3'-UTR regions of the AtFer3 cDNA. The 5'-UTR region was amplified with thermostable Pfu DNA p o l y m e r a s e ( P r o m e g a , M a d i s o n , U S A ) u s i n g t h e p r i m e r s 5 ' -GGTACCTATATTCCCTCCTCCGCCACCACCAATC -3' a n d 5 ' -GAATTCAAACCGGAGACGAAATGGAGG -3' introducing a Kpn I and an EcoR I sites at the 5' and 3' end of the amplified fragment respectively. The 3'-UTR region was amplified using the primers 5'-TGGGAATTCTGCTTAATCTGAG -3' and 5'-GGGTACTAGTAAATATCAGTG -3' introducing an EcoR I and a Spe I sites at the 5' and 3' ends of the amplified fragment respectively.

The fragment was cloned at the corresponding sites in pBluescript and sequenced. The specific probe (462 bp) was obtained by digesting the resulting construct by Kpn I and Spe I. The aconitase specific probes were amplified by PCR on genomic DNA as described above for AtFer3, cloned in pBluescript at the Eco RV site, and sequenced. The ACO1 probe, located in the 3'-UTR, was amplified with the primers 5'-GAGTTGGCTTATTTCGATCAGG-3' and 5'-GACTCGACAGTGATAATGTTTCC-3'. The two other ACO probes, located in the 5'-UTR, were

a m p l i f i e d w i t h 5 ' -C T T G A C G A A A T C T C T T C T C C G T C T C -3 ' a n d 5 ' - G A G C A A A C T C G A A A G G A G C G G A G A G C C -3 ' f o r A C O 2
, and 5'-CGCAGCCCTAGGCATTTTCG-3' and 5'-GGGGAAATGGTACCAAAAGATCTACG-3' for ACO3. 25S RNA and specific probes were labeled, and hybridization were performed as described [START_REF] Arnaud | An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopis AtFer1 ferritin gene expression[END_REF].

Preparation of cytosolic and mitochondrial matrix extracts

All procedures were carried out at 4°C. Plantlets (50 g) were cut with a razor blade and ground in a mortar containing extraction buffer (330 mM sorbitol, 30 mM MOPS-BTP, 2 mM PMSF, 2 mM 2 ß-mercaptoethanol, 2 mM Na 3 -citrate, 2 mM ascorbic acid, 1 mM EDTA, 10 µg.ml -1 leupeptin, 1% PVP, pH 7.5) in ratio 1/3 w/v. The homogenate was filtered through two layers of Miracloth (Calbiochem, Merck Biosciences, Darmstadt, Germany), and centrifuged at 280 g for 10 min to remove the cell debris and the chloroplasts. The supernatant was centrifuged at 10800 g for 30 min.

The resulting pellet contained mitochondria, while the supernatant corresponded to the cytosol and the membranes fractions. This supernatant was centrifuged at 100000 g for 45 min to pellet the membranes. Glycerol (final concentration 10%) was added to the resulting supernatant (cytosolic fraction), and samples were stored in liquid nitrogen until use. The pellet containing mitochondria was resuspended in 2 ml of lysis buffer (10 mM Tris-HCl, 2 mM 2 ß-mercaptoethanol, 2 mM Na 3citrate, 1 mM PMSF, 1 mM EDTA, 10 µg.ml -1 leupeptin, pH 7.2). Mitochondria were broken by vortexing and freezing three times the samples in liquid nitrogen. After a centrifugation at 100000 g for 45 min, the supernatant containing matrix proteins was recovered. Glycerol (final concentration 10%) was added, and samples were stored in liquid nitrogen. Protein concentration was determined according to Schaffner and Weissmann [START_REF] Schaffner | A rapid, sensitive, and specific method for the determination of protein in dilute solution[END_REF] using BSA as standard.

Enzymatic activities

Aconitase activity was assayed at 20 ºC in 100 mM Tris-HCl, 50 mM DL-isocitric acid (trisodium salt), pH 7.4, by measuring the production of cis-aconitate. The extinction coefficient used for cis-aconitate was of 3.6 mM -1 . cm -1 at 240 nm. The reaction was started by adding from 75 g to 150 g of protein in 1 ml final volume. Before measuring the citrate synthase activity, mitochondrial and cytosolic extracts were purified in a BioSpin 6 column (Bio-Rad, Hercules, USA) following the manufacturer's instructions to discard 2 ß-mercaptoethanol that negatively influences the activity. Citrate synthase activity was determined in 100 mM Tris-HCl, 0.1 mM 5,5'dithiobis(2-nitrobenzoic acid) (DTNB), 0.2 mM acetyl-CoA, pH 8.0, from 50 g of protein extract in 1 ml final volume. After 5 min of incubation at 30ºC, the reaction was initiated by adding oxaloacetic acid (1 mM final concentration) at room temperature. The reduction of DTNB was measured by detection of 2-nitro-5-thiobenzoic acid reduced form (TNB-SH). The extinction coefficient used for TNB-SH was of 13,8 mM -1 . cm -1 at 412 nm.

Immunodetection of ferritins

Total protein extracts were prepared from 1 g of each sample as described [27]. Proteins were subjected to electrophoresis on a 13% polyacrylamide / 0.1% SDS gel according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF].

After electroblotting onto Hybond-P membrane (Amersham Biosciences), immunodetection of ferritin was performed using a rabbit polyclonal antiserum raised against purified AtFer1 protein [START_REF] Dellagi | Siderophore-mediated upregulation of Arabidopsis ferritin expression in response to Erwinia chrysanthemi infection[END_REF] and the Aurora Western blotting kit (MP Biomedicals, Irvine, USA) following the manufacturer's recommendations.

Bioinformatics

IRE motif searches were performed on Arabidopsis thaliana 5'-and 3'-UTR sequences (available at the TAIR website http://www.arabidopsis.org/) using a Perl script specially written for this purpose, and using the sequence CXXXXXCAGUGNYYYYY as consensus of the IRE loop.

The constraints imposed in the script were as follow: N designs any of the 4 bases. The five bases noted X and Y are involved in the stem formation, and must be complementary (A/U, C/G) or form a stable weak interaction (U/G). For the putative bacterial acn binding site, the two paired motifs, ACACAAUGC and CAUUUU, as defined by Tang and Guest [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF] were searched. To determine the probability for the motifs to occur by chance in the sequences, the SMILE software was used [30].

This algorithm allows dealing with structured motifs eventually associated by some distance constraints.

RESULTS

Identification of IRP-like proteins in Arabidopsis

In order to determine whether the animal IRE/ IRP regulatory system may have a counterpart in plants, we first try to identify IRP1 and IRP2 homologues in Arabidopsis genome. We identified three genes encoding aconitate hydratase, named ACO1 (At4g35830), ACO2 (At4g26970) and ACO3 (At2g05710). ACO1 corresponds to the gene previously identified by Peyret et al. [START_REF] Peyret | Structure, genomic organization, and expression of the Arabidopsis thaliana aconitase gene. Plant aconitase show significant homology with mammalian iron-responsive element-binding protein[END_REF]. The two others are identical to the genes recently identified by Moeder et al. [20]. The three proteins are closely related, and share more than 90% similarity, and have about 80% of similarity with IRP1 and IRP2 (Figure 1). The three cysteine residues (indicated by squares on Figure 1) in the IRP1 sequence, involved in the Fe-S cluster formation, are completely conserved in the three plant ACO proteins, suggesting that they could associate a cluster, and could have an aconitase activity. The crystal structure of the IRP1:ferritin H IRE complex allowed to identify amino acids directly interacting with the RNA loop ( [START_REF] Walden | Structure of dual function Iron Regulatory Protein 1 complexed with ferritin IRE-RNA[END_REF]; indicated by stars on Figure 1). Interestingly, most of these residues are conserved in the three plant ACO proteins (Figure 1), raising the hypothesis that plant ACO could bind IRE sequences.

Isolation of aco loss-of-function mutants

To investigate the involvement of Arabidopsis aconitase genes in ferritin regulation, we have isolated knock-out mutants in the three corresponding genes from the Salk Institute [START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF] (for ACO2: line Salk054196, and for ACO3: line Salk013368) and the GABI-Kat [START_REF] Rosso | An Arabidopsis thaliana T-DNA mutagenized population (gabi-kat) for flanking sequence tag-based reverse genetics[END_REF] (for ACO1: line 138A08) collections. In the mutants, the two T-DNA flanking sequences were determined. The three mutants were named aco1-1, aco2-1 and aco3-1. In the three mutant lines, the T-DNA were found to be inserted in inverted tandem (Figure 2A). In aco1-1, the insertion is located in the 14th exon of the gene, and led to a small deletion of 5 bp, and to the insertion of 12 and 8 bp extra sequences at the 5'-and 3'-junctions respectively. In aco2-1, the insertion is located in the 6th exon, and led to a 413 bp deletion, and to the insertion of 12 bp extra sequence at the 3'-junction. In aco3-1 mutant, the insertion was found to be located in the 5th exon, and led to a deletion of 8 bp, and to the addition of 5 bp extra sequence at the 3'-junction. The aco3-1 line has been previously described by Moeder et al. and was named KO-661 [20]. Lines homozygous for the disruption were isolated, and used for further studies. When the mutants were grown in greenhouse, or in hydroponic cultures under controlled conditions, they displayed no macroscopic phenotype. Furthermore, changes in environmental parameters, such as light intensity or iron concentration in the culture solution, did not lead to any obvious phenotype (data not shown).

The expression of the three ACO genes was investigated in the aco mutant backgrounds by northern blot with specific probes corresponding to the three genes (Figure 2B). In each mutant, the transcript corresponding to the mutated gene was undetectable, indicating that the mutants isolated are true knock-out lines. The relative mRNA accumulation of the two remaining genes was not affected in any of the three mutants (Figure 2B). This result indicates that the disruption of an ACO gene does not alter the expression of the two other genes.

ACO genes encode aconitase, a key enzyme in the Krebs cycle located in the mitochondria. To determine whether these genes could encode cytosolic aconitase, both cytosolic and mitochondrial aconitase activities were measured from 10 days old plants grown in liquid medium. Mitochondrial matrix and cytosolic extracts were prepared, and the aconitase activity was measured in each extracts. The contamination of the cytosolic extract with mitochondrial proteins was determined by measuring the activity of the matrix-specific enzyme citrate synthase. In all experiments, the contamination of the cytosolic extracts by matrix mitochondria was estimated to be less than 5%

(data not shown). In aco1-1 mutant, the cytosolic and mitochondrial aconitase activities were reduced of about 75% and 20% respectively when compared to wild-type plants. In aco3-1, the reduction was around 25% for the cytosolic, and 55% for the mitochondrial activities. The cytosolic aconitase activity was not affected in the aco2-1 background, whereas the mitochondrial activity was reduced of 20%. (Figure 2C). The mutations affected the ratio between mitochondrial and cytosolic activities. This indicates that, in our experimental conditions, ACO1 is mainly involved in the cytosolic activity, and ACO3 is responsible of the largest part of the mitochondrial activity.

Expression of ACO genes in Arabidopsis

Biochemical Journal Immediate Publication. Published on 16 Apr 2007 as manuscript BJ20061874

The expression pattern of the three ACO genes in planta was investigated. Total RNA extracted from seeds, roots, leaves, stem, caulinary leaves and flowers was analyzed by northern hybridization using the ACO specific probes (Figure 3). The three genes present a very similar expression pattern, being expressed in all organs, except in the seed, where ACO1 mRNA accumulation was undetectable, and ACO2 and ACO3 genes were weakly expressed. ACO1 mRNA was more abundant in roots, stems and caulinary leaves. The highest level of expression of ACO2 was detected in roots, caulinary leaves and flowers.

To gain further insight regarding the potential IRP activity of the proteins encoded by the ACO genes, we have first investigated the expression of the aconitase genes in standard, iron-excess and iron-starved conditions (Figure 4). Under iron-excess condition, the abundance of the AtFer1 transcript increased rapidly. By contrast, the expression of the three aconitase genes was not affected by this treatment (Figure 4A). Under iron-deficient condition, the mRNA corresponding to the iron-starvation inducible gene IRT1 [START_REF] Vert | IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth[END_REF][START_REF] Eide | A novel iron-regulated metal transporter from plants identified by functional expression in yeast[END_REF] accumulated two days after application of the treatment. Under such condition, the mRNA abundance of ACO3 was not significantly affected comparatively to standard iron nutrition condition, whereas those corresponding to ACO1 and ACO2 were slightly decreased after three days of starvation (Figure 4B).

In animal cells, the switch between cytosolic aconitase and IRP activity is associated with a decrease of the cytosolic aconitase activity. To determine whether such a switch may occur in plants, we investigated whether iron addition or depletion may lead to a modulation of the cytosolic aconitase activity. Aconitase activity was measured on ten days old plants grown in liquid medium, in control conditions (standard medium), 3 hours after the addition of iron-citrate (final concentration 300 µM), and after one day of growth in an iron-depleted medium. Cytosolic and mitochondrial aconitase activities were measured in wild-type and in the three aco mutants (Figure 4C). In wild-type plants, the iron treatment led to a small increase of the cytosolic aconitase activity of about 25%. The iron starvation did not significantly modulate the activity. In the three aco mutants, the effects of iron addition and depletion on the cytosolic aconitase activity were similar to those observed in wild-type plant. Both iron starvation and addition led to a slight decrease of mitochondrial aconitase activity in all the genetic backgrounds.

Taken together, our results indicate that the modulation of the plant iron status did not strongly affect the expression of ACO genes in Arabidopsis. Moreover, cytosolic aconitase activity was not greatly affected by iron addition or depletion. In the three loss-of-function mutants, the modulation of the cytosolic aconitase activity in response to the iron status was the same than in wild-type suggesting that neither ACO1, nor ACO2, nor ACO3 activity is strongly affected by iron.

Ferritin gene expression in aco mutants

The involvement of Arabidopsis aconitase in the regulation of iron-responsive genes expression was finally investigated by analyzing the expression of two ferritin genes in the different aco genetic backgrounds. The AtFer1 and AtFer3 genes were chosen since the corresponding mRNA and protein quickly accumulate in response to iron addition [START_REF] Petit | Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family[END_REF][START_REF] Arnaud | An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopis AtFer1 ferritin gene expression[END_REF][START_REF] Gaymard | Characterization of a ferritin mRNA from Arabidopsis thaliana accumulated in response to iron through an oxidative pathway independent of abscisic acid[END_REF]. Moreover, in animal cells, ferritin genes are one of the primary targets of the IRE/IRP regulation. We first determined the kinetic of AtFer1 and AtFer3 mRNA accumulation in the wild type and in the three aco mutants in response to excess iron (Figure 5A). In none of the mutants the accumulation of the two ferritin mRNA was affected. The ferritin protein accumulation was also investigated in the three genetic backgrounds (Figure 5B). Total protein extracts were prepared before, 24 and 48 hours after iron addition. The same level of ferritin accumulated in the three mutants compared to the wild-type.

These results show that ACO1, ACO2 and ACO3 are not involved in the regulation of plant ferritin expression in response to iron, neither at the transcriptional, nor at the post-transcriptional level.

Functional redundancy between the cytosolic aconitase proteins

Our results indicated that ACO2 protein does not have cytosolic aconitase activity, at least in our experimental conditions (Figure 4C). On the other hand, in aco1-1 and aco3-1 mutants, the cytosolic aconitase activity is reduced of about 75% and 25% respectively in comparison to the wild-type (Figure 2 and 4C), indicating that the corresponding proteins represent the two major sources of plant cytosolic aconitase activity. The expression pattern of ACO1 and ACO3 genes are very similar (Figure 3), suggesting that the two proteins may, at least partially, be redundant. Thus, it can not be excluded that the absence of modulation of ferritin gene expression in the single aco1-1 and aco3-1 mutants (Figure 5) was due to a compensation mechanism between ACO1 and ACO3.

In the way to isolate and characterize a double aco1-aco3 mutant, the progenies of plants homozygous for one mutation and heterozygous for the other (namely aco1aco1-ACO3aco3 and ACO1aco1-aco3aco3) were sawed, and the genotype of the progeny was analyzed by PCR to detect the wild-type and mutant alleles. More than 100 plants from the two progenies were analyzed, but we failed to identify a double aco1aco1-aco3aco3 mutant, suggesting that a mutation in both ACO1 and ACO3 genes is lethal. Moreover, when examining the seeds in the siliques of aco1aco1-ACO3aco3 and ACO1aco1-aco3aco3 plants, some aborted seeds were systematically observed (Figure 6A). The proportion of aborted seeds in the progeny of the two parental lines was close to 25%, a value expected for a lethal double mutation (Figure 6B). development of the seed, and as a consequence unable us to study ferritin expression in the double aco1-aco3 mutant.

Identification of IRE and potential bacterial aconitase binding sites in the Arabidopsis genome

Our results (Figure 5) show clearly that Arabidopsis ACO are not involved in the transcriptional activation of AtFer1 and AtFer3 ferritin genes. However, it must be pointed out that, most of the residues known to be essential for the RNA binding activity of rabbit IRP1 are conserved in Arabidopsis ACO proteins (Figure 1). Such an important aminoacid conservation is intriguing, and indicates that a nucleotide binding activity of these proteins has to be considered. These essential residues for the IRP activity are also conserved in the bacterial aconitase protein that has been shown to bind to motifs unrelated to IRE [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF]. The exact recognized motif (or sequence) by bacterial aconitase is still unknown, but some conserved elements have been identified in the 3'-UTR of acnA and acnB genes [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF]. The high conservation of residues among animal, plant and bacterial proteins suggests that plant aconitase may also have a RNA binding activity. This hypothesis is supported by the recent work of Moeder et al. [20] which shows that Arabidopsis ACO1 protein is able to bind to the 5'-UTR of CSD2 superoxide dismutase transcript.

To further study the potential involvement of plant aconitase as a nucleotide binding protein, we took advantage of the availability of the whole Arabidopsis genome sequence to look for potential binding sites in the 5'-and 3'-UTR of all the transcripts. For this bioinformatic search, we used the animal IRE, and the sequences identified in E. coli acnA and acnB genes [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF]. In animals, IREs are stem-loops made of 30 nucleotides with a bulge. The loop has a conserved sequence, 5' CAGUGN 3', N usually being a pyrimidine [34]. We performed a search with the loop consensus sequence surrounded by a stem consisting of at least 5 bases in 5' complementary to the 5 bases located in 3'.

Using this rule, we found only 4 genes containing a putative IRE, one gene with the sequence in the 5'-UTR, and the three others in the 3'-UTR (Table 1). The binding site of the bacterial aconitase is still unknown, but from the sequence homologies between the 3'-UTRs of the two target genes acnA and acnB, Yang and Guest [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF] have suggested that two paired motifs ACACAAUGC and CAUUUU may, at least partly, be involved in the formation of aconitase binding sites. We searched for these two paired motifs in the Arabidopsis genome, and found them in the 5'-UTR of two genes and in the 3'-UTR of 4 others (Table 1). Because the probability to find them in UTRs sequence is likely more than 99% due to chance, as indicated by the SMILE software [30], such sequences are unlikely to be functional.

DISCUSSION

To directly address the question of a potential link between plant cytosolic aconitase and the regulation of ferritin expression, we took advantage of the availability of the Arabidopsis thaliana genome sequence. We searched for IRP1 and IRP2 homologues, and three proteins (ACO1 to -3) were identified. These IRP-like proteins were found to encode mitochondrial aconitase (Figures 2 and4), a result consistent with studies which found the 3 ACO proteins in the mitochondrial proteome [35]. In animals, mitochondrial and cytosolic aconitases are encoded by distinct genes [36,[START_REF] Kennedy | Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein[END_REF]. It is not the case in S. cerevisae, in which a unique gene encode both mitochondrial and cytosolic aconitase [START_REF] Gangloff | Molecular cloning of the yeast mitochondrial aconitase gene (aco1) and evidence of a synergistic regulation of expression by glucose plus glutamate[END_REF][START_REF] Regev-Rudzki | Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing[END_REF]. In plants, several reports indicate that, like in yeast, both mitochondrial and cytosolic aconitase are encoded by the same genes, therefore suggesting a dual targeting of the proteins [START_REF] Brouquisse | Characterization of a cytosolic aconitase in higher plant cells[END_REF][START_REF] De Bellis | Purification and characterization of aconitase isoforms from etiolated pumpkin cotyledons[END_REF][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]. Therefore, the question we had to address was to determine whether or not the three identified aconitases were targeted to the cytosol. The contribution of each ACO protein to the cytosolic aconitase activity was estimated from the analysis of the aco knock-out mutants (Figure 2), and suggests that ACO1 and ACO3 are the two major proteins displaying a cytosolic aconitase activity. ACO2, which is present in the mitochondria [35, [START_REF] Kruft | Proteomic approach to identify novel mitochondrial proteins in Arabidopsis[END_REF][START_REF] Millar | Analysis of the Arabidopsis mitochondrial proteome[END_REF] Our major goal was to determine whether or not cytosolic aconitase is involved in the regulation of ferritin expression in plants. To address this question, we first studied the effect of the plant iron status on ACO gene expression (Figure 4). At the mRNA level, neither iron excess, nor iron depletion modulated the expression of aconitase genes. Moreover, when measuring the effect of iron treatments on the cytosolic aconitase activity, no strong variations were observed in wild-type, as well as in the mutants (Figure 4). Consistent with an IRP1-cytosolic aconitase switch, a variation of the plant iron status should have led to a modulation of the cytosolic aconitase activity by affecting the Fe-S assembly of the protein. Finally, the direct evidence that Arabidopsis ACO are not involved in the regulation of ferritin expression was given by studying the expression of two ferritin genes at the mRNA and protein levels in the aco mutant genetic backgrounds. In the three aco mutants, both the level of AtFer1 and AtFer3 transcript accumulated and the amount of ferritin subunit were identical to those detected in the wild-type. A potential functional redundancy between ACO1 and ACO3 could explain the lack of effect of the mutations on ferritin expression. However, we did not observe any compensation between the three aconitase genes (Figure 2A), since the mutation in one gene did not affect the expression of the two others. The remaining activity in aco1-1 mutant (probably due to ACO3 activity) could be sufficient to explain the absence of modulation of ferritin expression. We therefore intend to isolate a double aco1-1 aco3-1 mutant, but failed to, as the presence of the two mutations leads to the abortion of seeds. This result indicates that the cytosolic aconitase activity is essential for the proper formation of the embryo or for the development of the seed.

Beside the question of the involvement of cytosolic aconitase on the regulation of iron metabolism in plants, our work provides tools to study the impact of disturbing the glyoxylate and/or citric acid cycles on plant growth and metabolism. The relative contribution of each ACO isoform on the mitochondrial and cytosolic aconitase activities, on the metabolism [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], on the regulation of oxidative stress and on the establishment of defense against pathogens [20, 45] could be studied with the loss-of-function mutants.

As a complementary approach to determine whether the IRE/ IRP1-cytosolic aconitase switch may occur or not in plants, we performed a bioinformatic search for putative aconitase RNA binding sites in the 5'-and 3'-UTR of Arabidopsis transcripts. Only 10 genes contain one of these sequences (Table 1). Among them, 4 genes contained a putative IRE sequence, and the others 6 contained the two consensus sequences [START_REF] Tang | Direct evidence for mRNA binding and posttranscriptional regulation by Escherichia coli aconitases[END_REF] found in the bacterial aconitase regulated genes. The low number of genes containing these elements raise therefore the question of their significance.

Four genes encode unknown proteins, and among the others, none of them is obviously related to iron metabolism.

Recent results show that plant cytosolic aconitase protein may have a RNA binding activity.

Indeed, Arabidopsis ACO1 is able to bind to the 5'-UTR of the CDS2 superoxide dismutase transcript [20]. This result raises the interesting point that, like in animals, plant cytosolic aconitase may have a RNA binding activity. However, the CDS2 aconitase binding site is not an IRE nor a bacterial aconitase binding site [20]. Our bioinformatic analysis, which indicates that animal and bacterial cis-acting sequences are probably not functional in plants, is consistent with this result, since the CDS2 gene was not detected by our analysis. These data suggest that the potential RNA binding activity of plant aconitase proteins does not involve a canonical cis-acting sequence, but would be more likely related to the secondary structure of the RNA region. The identification of the nucleotides involved in the CDS2 ACO1-binding site, and the search for other transcripts able to bind ACO1 will be of primary importance in the future. Even more important will be the demonstration that this in vitro binding activity does exist in vivo, and participates to the regulation of so far unknown biological functions. aco3-1 and ACO1 aco1-1 aco3-1 aco3-1) were grown in green house, and seeds were collected.

LEGENDS TO FIGURES

The proportion of aborted seeds was estimated visually under binocular.

  may be not targeted to the cytosol. Alternatively, our experimental conditions may not reveal the cytosolic aconitase activity of this protein. Our results bring additional information to the work of Moeder et al [20] who determine the total (cytosolic + mitochondria matrix) aconitase activity in various aco mutant genetic backgrounds. It has also to be mentioned that the aco1-1 and aco2-1 mutants we have characterized in this work are different alleles than those used by Moeder et al [20].

Figure 1 .

 1 Figure 1. Sequence alignment of the deduced Arabidopsis aconitases with human IRP1 and IRP2.The human IRP1 (Genbank accession NP002188) and IRP2 (Genbank accession NP004127) was aligned with their Arabidopsis homologues At4g35830 (ACO1), At4g26970 (ACO2) and At2g05710 (ACO3). Identical and similar residues between at least three proteins are boxed in black and grey respectively. Gaps were introduced to maximize alignment. Aminoacids are numbered from the translational start methionine. Stars indicate aminoacids directly interacting with the RNA loop of the IRE[START_REF] Walden | Structure of dual function Iron Regulatory Protein 1 complexed with ferritin IRE-RNA[END_REF]. The three cysteine residues involved in the 4Fe-4S cluster formation in IRP1 are indicated by squares.

Figure 2 .

 2 Figure 2. Isolation of aco knock-out mutants. A. Characterization of the aco1-1, aco2-1 and aco3-1 mutant lines. The diagram illustrates the site of insertion on the T-DNA in the different genes. Boxes and lines represent exons and introns, respectively. The gene/ T-DNA junction sequences are indicated. The gene sequences are indicated in bold type, the T-DNA sequences are in upper case, and additional sequences found at the junctions are in lower case. LB: left border of the T-DNA. Numbering is given relative to the first codon of the translation initiation site. B. Expression of ACO genes in wild-type and aco mutants. Wild-type Arabidopsis (Col), aco1-1, aco2-1 and aco3-1 plantlets were grown for 10 days in liquid medium. RNA was analyzed by northern blotting and hybridized successively with the ACO1, ACO2, ACO3 and 25S probes. 25S RNA abundance was shown as loading control. C. Effect of the mutations on cytosolic and mitochondrial aconitase activities. Plants were grown as described in B. Cytosolic and matrix extracts were prepared, and the aconitase activities were measured. Results are presented as the percentage of the cytosolic (black histograms) and mitochondrial (white histograms) aconitase activities measured for Col plants. Bars correspond to the standard deviation from three independent experiments.

Figure 3 .

 3 Figure 3. Expression of aconitase genes in the different organs of Arabidopsis. Plants were grown in hydroponic conditions for 5 weeks. Total RNA was extracted from seeds (S), roots (R), rosette leaves (L), stem (St), caulinary leaves (CL) and flowers (F), and analyzed by northern blot as described in the legend to figure 2.

Figure 4 .

 4 Figure 4. Effect of iron nutrition on aconitase gene expression and aconitase activities. A. Effect of excess iron on ACO mRNA accumulation. Wild-type plantlets (Col) were grown as described in the legend to figure 2. Iron-citrate (final concentration 300 M) was added, and plantlets were collected

Figure 5 .

 5 Figure 5. Effect of aco mutations on ferritin expression. A. AtFer1 and AtFer3 mRNA abundance in aco mutants. Col, aco1-1, aco2-1 and aco3-1 plantlets were grown for 10 days in liquid medium.Iron-citrate was added (300 M final), and plantlets were collected before (0), 1, 3 and 6 h after the treatment. RNA was extracted, analyzed by northern blotting, and hybridized successively with the AtFer1, AtFer3, EF1 and 25S probes. EF1 mRNA and 25S RNA abundance were used as loading controls. B. Effect of the mutation on ferritin protein accumulation in response to iron. Col, aco1-1, aco2-1 and aco3-1 plantlets were grown for 10 days in liquid medium. Iron-citrate was added (300 M final), and plantlets were collected before (0), 24 and 48 h after the treatment. Total protein were extracted. Ten g protein extracts were loaded for each lane. A polyclonal serum (1/20000 dilution) raised against AtFer1 protein was used for immunodetection. A coomassie blue stained gel is shown as loading control.

Figure 6 .

 6 Figure 6. The double mutant aco1-1 aco3-1 is lethal. A. Aborted seeds present in siliques of a plant mutant in ACO1 gene and heterozygous at the ACO3 locus. Wild type (Col) and mutant (genotype aco1-1 aco1-1 ACO3 aco3-1) plants were grown in green house and siliques were collected just before dehiscence. Siliques were opened and seeds were examined under binocular. All the seeds were normal in Col plants (left), whereas some were aborted in the mutant (right, aborted seeds are indicated by arrows). B. Proportion of aborted seeds in the progeny of plants mutant at one locus, and heterozygous at the second. Wild-type (Col) and mutant plants (genotype aco1-1 aco1-1 ACO3

  UTR ACACAAUGC and CAUUUU At5g63780 zinc fingerFor each accession, the location (in the 5'-or 3'-UTR) of the putative element in the transcript is indicated. For IRE sequence, the corresponding sequence found in the database is given. The bases of the IRE consensus are indicated in bold type. The AGI number, and function of the corresponding genes are indicated.

Biochemical Journal Immediate Publication. Published on 16 Apr 2007 as manuscript BJ20061874

Our results indicate that ACO1 and ACO3 protein activities are required for the development of the embryo, or for the proper

Table 1 .

 1 Putative IRE and bacterial aconitase binding sites in the UTR of Arabidopsis thaliana genes.

Biochemical Journal Immediate Publication. Published on 16 Apr 2007 as manuscript BJ20061874 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgments

This work was funded by Institut National de la Recherche Agronomique and Centre National de la Recherche Scientifique, and by the Action Concertée Incitative "Biologie Cellulaire Moléculaire et Structurale" n°BCMS166 from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. NA and KR were supported by a thesis fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche.