Sequence analysis, transcriptional and posttranscriptional regulation of the rice vdac family

Biochimica et Biophysica Acta 1625 (2003) 43 – 51 www.bba-direct.com

Sequence analysis, transcriptional and posttranscriptional regulation $ of the rice vdac family Fawaz Al Bitar a,1, Nancy Roosens b,1,2, Mathias Smeyers a, Marc Vauterin b, Jos Van Boxtel c, Michel Jacobs b, Fabrice Homble´ a,* a

Laboratoire de Physiologie Ve´ge´tale, Universite´ Libre de Bruxelles, Campus Plaine (CP 206/2), B-1050 Brussels, Belgium b Laboratorium voor Plantengenetica, Vrije Universiteit Brussel, Paardenstraat, 65, B-1640 Sint Genesius Rode, Belgium c CIRAD-AMIS/biotrop, Avenue Agropolis, F-34398 Montpellier cedex 5, France Received 18 April 2002; received in revised form 25 September 2002; accepted 16 October 2002

Abstract The voltage-dependent anion-selective channel (VDAC) is a mitochondrial outer membrane ion channel. Different isoforms exist in plants but information about their specific role remains to be established. Our purpose is to find out the structural features common to three rice VDAC isoforms and to investigate their (post)transcriptional regulation in response to an osmotic stress. Two new cDNAs encoding mitochondrial VDAC from rice (Oryza sativa) were isolated, sequenced and characterized: a phylogenetic reconstruction permitted identification of orthologues in Poaceae and computer-based analyses predicted 18 transmembrane h-strands, one amphipathic a-helix and two different phosphorylation motifs. The expression of three rice vdac genes was investigated. Northern blot analyses indicated that they were expressed in all plant tissues. There was a differential expression of osvdac1 and osvdac3, whereas osvdac2 was homogeneously expressed in all tissues. No change in vdac expression was observed under an osmotic stress. However, a fast-enhanced expression of vdac was observed in roots during the recovery period after stress release. This enhanced expression is not correlated to the amount of VDAC protein detected in roots suggesting a posttranscriptional regulation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Voltage-dependent anion-selective channel; Rice; Mitochondria; Channel; Osmotic stress; Stress recovery

1. Introduction Mitochondria contain a voltage-dependent anion-selective channel (VDAC) that is the major component of the mitochondrial outer membrane [1]. This channel is probably the main pathway for ion diffusion through the outer membrane. Moreover, VDAC could be involved in several other functions such as compartmentation [2], apoptosis [3], enzyme binding [4] and cytoskeleton binding [5].

$ The nucleotide sequences reported in this paper have been submitted to GenBank under the accession number Y18104 (OSVDAC1), AJ251562 (OSVDAC2), AJ251563 (OSVDAC3). * Corresponding author. Tel.: +32-2-6505383; fax: +32-2-6505382. E-mail address: [email protected] (F. Homble´). 1 Both authors equally contributed to the work. 2 Present address: Laboratoire de Physiologie et Ge´ne´tique Mole´culaire des Plantes, Universite´ Libre de Bruxelles, Campus de la Plaine CP 242, B-1050 Bruxelles, Belgium.

VDACs from protozoa [6], mammals [7], fungi [8] and higher plants [9– 11] have similar channel properties. At voltages close to 0 mV, the channel exists mainly in its fully open state ( f 4 nS in 1 M KCl). Upon application of voltages larger than F 20 mV, it switches to subconducting states. In these subconducting states, the VDAC has not only a reduced permeability, but also a reversed selectivity. For instance, the channel allows the diffusion of negatively charged solutes like succinate, malate and ATP in the fully open state, but not after switching to a subconducting state that is cation selective. Therefore, it has been assumed that VDAC could regulate mitochondrial functions. VDACs purified from different organisms also have similar structure. Both electron-microscopic and spectroscopic (CD and FTIR) investigations indicate that it forms a h-barrel [12,13]. It is now well established that plant VDACs are encoded by a small multigene family [14 –17]. Two isoforms have recently been purified from cotyledons of bean seeds [11].

0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 5 9 0 - 0

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However, little is known about the regulation and function of these various isoforms. The different VDAC isoforms studied have similar basic channel properties and structure [11,13,18]. This suggests that the differences between isoforms should be dealing with their regulation and/or their differential expression. Elkeles et al. [15] have shown that the three wheat vdac genes were differentially expressed in flowers, where the expression of two out of them was significantly higher in anthers than in other parts. Information about the factors that could regulate the expression of vdac genes is very scarce. The expression of vdac-like cDNA in Arabidopsis thaliana is increased in the hypersensitive response [19]. Environmental stress factors that are known to affect the tissue respiration could also have an effect on vdac gene expression. Moreover, the identification of the factors inducing the expression of the various genes will permit to understand the function of the different isoforms. Here, we report on the molecular identification of two new rice vdac genes. Analysis of the primary structure is done to predict the secondary structure and the membrane topology of the VDAC. We investigate the expression pattern of the three rice vdac genes with respect to plant development, to tissue specificity and to abiotic stress. Particular attention is given to the effect of osmotic stress recovery that is poorly investigated until now.

MS solution supplemented with either 360 mM mannitol (final osmolality of 0.645 osmol/kg) or an isoosmotic solution of 200 mM NaCl. For drought stress, the plantlets were allowed to desiccate for 2 h under the laminar flow. For stress recovery, the 3-day osmotic stressed plantlets or the 2 h drought stressed plantlets were put back in nonstressing MS medium for 6– 24 h. For organ specific expression studies, seeds were germinated and grown on a water saturated soil (1/5 compost, 4/5 sand (w/w)) in the greenhouse (12 h light/12 h dark cycle). mRNA was extracted from different parts of the plants before pollination (stage 61 in the BBCH scale): the root, the first leaf, the internode below the first leaf (internode1), the third leaf, the internode below the third leaf (internode3), the panicular leaf, the internode between the panicular leaf and the panicle (panicular internode) and the flower. Moreover, 2 weeks after pollination (stage 73 in the BBCH scale), mRNA was extracted from the pre-mature seed at the milky stage (flower after pollination), the panicular leaf and the panicular internode. 2.2. DNA sequencing

2. Materials and methods

Plasmids were extracted from a midi scale culture according to the alkaline lysis procedure described by Maniatis et al. [22]. Their sequences were determined using the dideoxynucleotide chain termination method with an automatic laser fluorescence (ALF Pharmacia) detection.

2.1. Plant growth and stress treatments

2.3. Sequence analysis and phylogenetic reconstruction

Oryza sativa indica seeds (cultivar CR203) were germinated and grown in vitro on solid MS medium [20] in a culture room (24 jC, 16 h illumination at 26 AE m 2 s 1 intensity, 8 h dark cycle). CR203 is an elite breeding line selected by the Institute of Plant Protection (Hanoi, Vietnam) from IR-8423-132-622 of the International Rice Research Institute (Los Ban˜os, Philippines). One-day-old, three-day-old, five-day-old and ten-day-old plantlets were used for developmental expression studies. These developmental stages, respectively, correspond to stages 03, 10, 11 and 12 in the BBCH scale [21]. For stress expression studies, 10-day-old plantlets were transferred in liquid MS medium during 1 day and were then placed for 1 –3 days in

Analysis of the nucleotide sequences and phylogenic prediction were done using the GeneCompar program (Applied Maths, Kortrijk, Belgium). The Neighbour Joining method [23] was used to reconstruct the phylogenetic tree of the fully sequenced VDAC proteins. The reliability of the internal branches was tested by bootstrapping with 500 resamples [24]. 2.4. DNA probes Twenty-five to fifty nanograms of template were labeled with (g-32P)dCTP (7.4 MBq) using the rediprime II random prime labelling system (Amersham Pharmacia, UK).

Fig. 1. Characterisation of the rice VDAC gene family. (A) Comparison of the deduced amino acid sequence of the rice OSVDAC1 [17] with the one of OSVDAC2 and OSVDAC3. Asterisks indicate OSVDAC2 and OSVDAC3 identical amino acids to the OSVDAC1 amino acids. (B) Structural model of VDAC insertion in a lipid bilayer membrane. A cylinder is used for the a-helix and wide arrows for h-strands. Putative phosphorylation sites for casein kinase II (CK2) and protein kinase C (PKC) are indicated by dark grey and light grey ovals, respectively. Bold circles show the putative phosphorylated residues. (C) Unrooted phylogenetic tree for fully sequenced VDACs. The tree contains all the published VDAC sequences. Among the plant kingdom: Wheat VDAC1-3 [15], Maize VDAC1 – 2 [16], Maize VDAC3 [39], Rice VDAC1 [17], Potato VDAC1 – 2 [14], Pea VDAC [39], Arabidopsis VDAC [19]. Among the animal kingdom: Mouse VDAC1 – 3 [40], Rat VDAC1,3 [41], Rat VDAC2 [42], Human VDAC1 – 2 [4], Human VDAC3 [43], Bovine VDAC [44], Dogfish VDAC [45], Fruitfly VDAC [46]. Among the fungi kingdom: Yeast VDAC1 [47], Yeast VDAC2 [48], Neurospora VDAC [49], Dictyostelium VDAC [50]. The rice VDAC4 was deduced from the full genomic sequence (BAC clone B1131B07) and both VDAC2 and VDAC3 are given in (A).

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2.5. Total RNA extraction and Northern blot hybridization Total RNA was isolated from 1 g of rice plantlets according to the method described by Rerie et al. [25]. The RNA concentration was determined by measures of absorbance at 260 nm. Then, 30 Ag of RNA samples containing ethidium bromide (0.03 Ag/Al) were submitted to electrophoresis in a 1% agarose gel containing 6% formaldehyde (37%). The equal amount of RNA loaded was verified by the fluorescent signal of the RNA – ethidium bromide complex under UV radiation. Total RNA was transferred by gravity blotting onto positively charged nylon membranes (Boehringer Mannheim). The equal amount of transferred RNA on the membranes was checked by methylene blue coloration. The full-length O. sativa vdac1, vdac2 and vdac3 cDNAs were, respectively, used as radioactive probes. Hybridizations were carried out at 62 jC in a solution containing 7% SDS, 0.25 M Na2HPO4, 1% (w/v) Bovine Serum Albumin, 0.2% (v/v) H3PO4 (85% orthophosphoric acid). The membranes were first washed in 2 SSC (sodium chloride/sodium citrate: 0.3 M NaCl; 0.03 M Na3citrate.2H2O) at room temperature for 20 min, and then in 0.5  SSC at 42 jC for 40 min. Membranes were subsequently exposed to X-ray films (Du Pont) for 24 h. Each RNA extraction and Northern blot was repeated at least three time. The same membrane was hybridized with a rice vdac probe and with the full-length salT cDNA probe [26]. SalT radioactive signal can easily be distinguished from the vdac radioactive signal, as their mRNA sizes are different. 2.6. Southern blot hybridization The pBLUESCRIPT plasmid containing an osvdac isoform (10 ng) were digested with SalI and NotI restriction enzymes and subjected to 1% agarose gel eletrophoresis. The DNA was transferred by gravity blotting onto a positively charged nylon membrane (Boehringer Mannheim). The membrane was hybridized with one of the osvdac probe. The hybridizations were carried out as for Northern blot hybridization.

fuged 20 min at 14,000  g (SA300, Sorvall). The pellet was resuspended in 200 Al of PBS solution [22]. The protein concentration was about 1 Ag/Al. It was determined with the BCA-kit (Pierce) using BSA (Sigma) as standard. 2.9. Western blot analysis Five micrograms of protein samples were separated by SDS-PAGE (12%) and transferred to nitrocellulose membrane (Schleicher and Schuell, Germany) by mini Trans-Blot (Bio-Rad, USA). The membrane was blocked by incubation in PBS and 0.1% Tween 20 (PBS-T) plus 1% (w/v) BSA for 2 h at room temperature. The membrane was then incubated in the presence of the maize porin antibody [28] diluted 1:500 in PBS-T plus 1% (w/v) BSA at 4 jC overnight. The membrane was washed three times in PBS-T and incubated 1 h with peroxidase-conjugated anti-mouse lgG HRP (Chemicon) diluted 1:2000 in PBS-T. After four washes in PBS-T, labeling was detected by chemiluminescence (Amersham Pharmacia) and 30 s of exposure to Kodak BioMax film at room temperature. The protein extractions and the Western blot analyses were repeated three times. 2.10. Structural analysis The Prof method was used to predict the secondary structures [29]. The secondary structure prediction was first done on rice VDACs and then compared to that of other VDAC sequences obtained from yeast (P04840, P40478), Neursopora (P07144), human (P21796, P45880, Q9Y277) and Arabidopsis (CAA10363). A secondary structure was considered as acceptable if it was detected in all VDAC sequences. This prediction was then compared to that obtained with both PHD method [30,31] and the neural network-based method [32] to check for

2.7. Proline content Proline was extracted from 0.3 g fresh weight of rice plantlets and quantified according to the method of Bates [27]. A unit of proline concentration represents 1 Amol of proline per gram fresh weight. Each analysis is the mean of four independent measures. 2.8. Total protein extraction Root samples (1 g FW) were ground in liquid nitrogen, resuspended in extraction medium (0.3 M mannitol, 0.6% PVP, 1 mM EDTA, 25 mM MOPS, pH 7.5) and centrifuged 20 min at 1500  g (SA300, Sorvall). The supernatant was filtered through Miracloth (Calbiochem, USA) and centri-

Fig. 2. Specificity of the different Osvdac probes. Ten nanograms of each pBLUESCRIPT plasmid was digested with SalI and NotI restriction enzymes and blotted on three nylon membranes. Lane 1 was loaded with osvdac1 (clone Y18104), lane 2 was loaded with osvdac2 (clone AJ251562) and lane 3 was loaded with Osvdac3 (clone AJ251563). Blots were hybridized with the 32P-labelled full-length cDNAs of (a) osvdac1, (b) osvdac2 and (c) osvdac3.

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Fig. 3. Northern blot analysis of total RNA isolated from rice shoots 1, 3, 5 and 10 days old. Blots were hybridized with the 32P-labelled full-length cDNAs of osvdac1, osvdac2 and osvdac3.

consistency. A Prosite search was done to find consensus motifs [33].

3. Results 3.1. Gene analysis To identify rice cDNAs encoding VDAC isoforms, we used the previously identified nucleotide sequence of osvdac1 [17] to screen the EST database of GenBank. Several ESTs of rice VDACs were identified. Multiple sequence analysis and inspection of overlapping cDNA regions resulted in the identification of cDNA clones corresponding to new rice vdac transcripts, respectively named osvdac2 and osvdac3. One clone was selected for each vdac (respectively, C12629 and E10458 from the MAFF DNA Bank) and their full sequence was determined. The cDNA inserts have a size of 1189 and 1148 bp, respectively. For both of them, we identified the putative first ATG and the poly-A tail. They contain minimum open reading frames of 840 and 825 nucleotides, encoding putative proteins of 280 and 275 amino acids, respectively.

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The predicted molecular weight of OSVDAC2 and OSVDAC3 is, respectively, 29.6 and 29.9 kDa and their theoretical isoelectric points (pI) are 8.56 and 7.24, respectively. The nucleotide sequences of osvdac2 and osvdac3 share, respectively, 79.6% and 78.9% identity to the nucleotide sequence of osvdac1. Alignment of the deduced amino acid sequences of OSVDAC1, OSVDAC2 and OSVDAC3 (Fig. 1A) indicates high levels of identities (70%) and similarities (81%) that are evenly distributed along the sequence. A fourth putative VDAC isoform has been recently identified on the rice chromosome I (BAC clone B1131B07). It displays between 64% and 69% of identity with the three other isoforms. Several algorithms were used to predict the secondary structure of the rice VDACs from their primary structure. The VDAC sequences were analysed with secondary structure prediction algorithms such as the Prof, PHD and neural network methods. In the VDACs, all the h-strands should be transmembrane segments [13]. Therefore, only the hstrand predictions consisting of at least seven residues were considered to be correctly assigned. We found a number of 18 membrane spanning h-strands and a a-helix of about 14 residues at the amino terminal end in all the sequences tested (see Materials and methods). When plotted on a helical wheel, this a-helix displays an amphipathic character, suggesting that it could be partly embedded in the membrane lying parallel to the surface. The putative membrane topology of OSVDAC2 based on secondary structure prediction is shown in Fig. 1B. Similar predictions were obtained for the other VDACs. Two motifs, one for the protein kinase C (PKC) and the other for the casein kinase II (CK2) were identified. PKC was found in all the VDACs tested, whereas CK2 was found mainly in the plant VDACs.

Fig. 4. Northern blot analysis of total RNA isolated from different rice organs. Blots were hybridized with the 32P-labelled full-length cDNAs of osvdac1, osvdac2 and osvdac3. Total RNA was isolated from: the root, the first leaf (1st leaf), the internode below the first leaf (1st int.), the third leaf (3rd leaf), the internode below the third leaf (3rd int.), the panicular leaf before pollination (pan. leaf b.p.), the panicular internode before pollination (pan. int. b.p.), the flower before pollination (b.p.), the panicular leaf after pollination (pan. leaf a.p.), the panicular internode after pollination (pan. int. a.p.) and the flower after pollination (a.p.).

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The putative phosphorylation sites found in all rice VDACs are shown in Fig. 1B. The rice VDAC sequences together with all the full VDAC sequences currently available in the literature were used to construct a phylogenetic tree by the Neighbour Joining method [23]. Thus, the horizontal length of joining branches was a measure of the evolutionary distances. As shown in Fig. 1C, VDACs of plants, animals and fungi are located in three significantly separated branches of the tree. Among the branch representing the plant VDAC sequences, monocotyledonous and dicotyledonous sequences fall in separate groups. The monocotyledonous group splits in, at least, three different subgroups, separating the various VDAC isoforms. Among the animal VDAC sequences, here again significant clusters are formed for the various known isoforms. Furthermore, the clustering indicates that the BovineVDAC sequence is most probably a VDAC1 type sequence. Despite the significant divergence observed within the fungal VDAC sequences, they do cluster together in a single group. The evolutionary distance in between the fungal VDAC

sequences is less compared to the evolutionary distances in between the plant – fungi and animal – fungi VDAC sequences. The evolutionary distance between plants and animals is smaller than between plants and fungi or animals and fungi. 3.2. Differential gene expression Gene expression studies of the three rice vdac isoforms were performed by Northern blot hybridization with the fulllength 32P-cDNA of each isoform. The specificity of the different probes was tested by Southern blot analysis. As shown in Fig. 2, no cross-hybridization occurred between the three different vdac isoforms indicating that the probe was highly specific of its target. During the seedling development, osvdac1, osvdac2 and osvdac3 genes were similarly expressed in 1 day to 10-dayold plantlets (Fig. 3). Moreover, their transcript level was the highest just after the germination and decreased with time. In 3-month-old plants, the three rice vdac genes were expressed in all the organs (Fig. 4). The expression pattern

Fig. 5. Northern blot analysis of total RNA isolated from 10-day-old rice plantlets submitted to osmotic stress and during osmotic stress recovery. Total RNA was extracted from the shoots and from the roots. Blots were hybridized with the 32P-labelled full-length cDNAs of osvdac1, osvdac2, osvdac3 and salT. For salT hybridization, the membranes already hybridized with osvdac1 were reused. Proline (Pro) content is expressed in micromoles of proline per gram of fresh weight. Each value is the mean of four independent samples and vertical bars represent standard errors. Total RNA isolation and proline measurement were performed from 10-day-old plantlets after treatments (24 and 72 h) with 360 mM mannitol and upon recovery (6 and 24 h) in control medium after 3 days of this mannitol stress treatment. The control condition consisted in MS medium.

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of osvdac2 was different from both osvdac1 and osvdac3. The osvdac2 transcript level was relatively similar in all the analyzed organs except in the flower after pollination, where the gene expression level was very low. The osvdac1 and osvdac3 genes presented a similar expression pattern, showing a high variation in expression from one organ to another. Their transcript level was much higher in the root, in the different internodes and in the flower before pollination, compared to leaves and flower after pollination. In contrast to the osvdac1 transcript, the osvdac3 transcript level was the highest in the flower before pollination. For both osvdac1 and osvdac3, a similar level of expression was found before and after pollination in both the panicular leaf and the panicular internode. 3.3. Effects of abiotic stress and stress recovery To investigate the expression of rice vdacs in response to osmotic stress and recovery, rice plantlets were submitted to an osmotic stress of 360 mM mannitol for 1 –3 days and then put back in control medium for 6 –24 h of recovery. Mannitol was used because it gives rise to osmotic stress sensu stricto, lowering the water potential of the medium while the plant tissue does not absorb it [34]. In the presence of mannitol, the expression of each vdac transcript in both shoots and roots was similar to that observed in the control condition (Fig. 5). However, the expression of the three vdac genes was strongly induced during the stress recovery period in the root but not in the shoot. The expression of salT, an osmotic-stress induced gene, and the level of proline content were used as controls to check whether the plants did effectively respond to the applied stress [26,35]. As expected, in the presence of

Fig. 6. Northern blot analysis of total RNA isolated from 10-day-old rice plantlets submitted to salt and water stresses and during their recovery. Total RNA was extracted from the shoots and from the roots. Blots were hybridized with the 32P-labelled full-length cDNAs of osvdac1, osvdac2 and osvdac3. Total RNA was isolated from 10-day-old plantlets after treatments (24 and 72 h) with 200 mM NaCl, after drought stress condition (2 h) and upon recovery (6 and 24 h) in control medium after 3 days of 200 mM NaCl stress or 2 h of drought stress. The control condition consisted in the MS medium.

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Fig. 7. Western blot analysis. Proteins were detected with the maize porin monoclonal antibody (PM035). Ten-day-old plantlets were stressed 0, 24 or 72 h in 360 mM mannitol. After 3 days of stress, they were put back in the control medium for a recovery period of 24 or 48 h.

mannitol, there was an increase in salT expression and proline content in both shoots and roots. During the recovery period, the proline content decreased by about 30% in the root but not in the shoot. Similar vdac expressions were observed when mannitol was replaced by either an isoosmotic solution in NaCl (200 mM) or a 2 h dehydration treatment, followed by a recovery period (Fig. 6). Western blot analyses were carried out to detect a change in VDAC protein level in roots from plants grown in similar mannitol stress conditions. The recovery periods were 24 and 48 h in place of 6 and 24 h to take into account a possible delay between the transcription and the translation events. As shown in Fig. 7, the VDAC protein level did not change during both stress and stress recovery experiments.

4. Discussion To study the transcriptional regulation of the rice vdac family, two new clones were isolated and characterized. As the assignment of the plant VDAC sequence to the secondary structure is not known, a computer-based search for secondary structure was performed. It predicts 18 transmembrane h-strands and a large extra-membranar loop between residues 156 and 174. At the amino terminus, 14 residues are predicted to form an amphiphilic a-helix that would be embedded in the surface of the membrane with its hydrophobic and hydrophilic faces directed towards the interior of the lipid bilayer and the solvent, respectively. These predictions are in good agreement with those obtained for the yeast VDAC [36] and the human VDAC [37] using other methods and with spectroscopic studies done on purified plant VDAC proteins reconstituted in lipid bilayers [13]. Thus, despite the poor homology ( < 30%) between the primary structure of plant, yeast and animal VDACs, their structure should be relatively well conserved. Moreover, putative phosphorylation sites for two different kinases (PKC and CK2) were found in the primary sequence. They are located in loops between hstrands. The PKC motif was found in all VDACs, whereas the CK2 motif was mainly found in the plant VDACs. Both kinases could phosphorylate serine or threonine residues. The CK2 motif is located on opposite sides of the membrane, which is consistent with the enzyme localisation in both the

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cytoplasmic and the mitochondrial intermembrane space [38]. To further characterize the rice vdac gene family, a phylogenetic reconstruction of all the fully sequenced VDAC proteins was done. The result of Fig. 1C, showing the evolutionary distance between animals, plants and fungi, suggests that the rate of sequence change was greater in the fungal lineage than in the animal or plant lineage. The duplications that gave rise to the multigene family of vdacs probably occurred independently in animals and plants after the divergence between the two kingdoms. In plants, vdac duplications probably happened after the divergence between monocotyledons and dicotyledons. This conclusion does not agree with that of Elkeles et al. [15] who suggested that these duplications occurred before the divergence between monocotyledons and dicotyledons. This discrepancy arises mainly from the limited number of sequenced VDACs used by these authors to build their reconstruction. Among the Poaceae, the vdacs should have split into at least three paralogous genes before the divergence between the genera. Each of the paralogous vdac1 – 3 gene gave rise to different orthologous genes during speciation events. The position of rice VDAC4 suggests the presence of a fourth paralogous gene in Poaceae. Further identification of new vdac isoforms in the other genera is required to confirm this hypothesis. Paralogous vdac genes are also found in animals. These genes were probably duplicated before the divergence between the placental mammals. Transcription analysis showed specific expression of the rice vdac genes in the different organs: osvdac2 is constitutively expressed whereas osvdac1 and osvdac3 genes are differentially expressed in the plant. Although, osvdac1 and osvdac3 have a similar expression pattern, osvdac3 is much more expressed in flowers. This could be related to a high mitochondrial activity in these organs since VDAC is thought to regulate the transport of metabolites involved in respiration. For all vdacs, expression levels in flowers are significantly higher before pollination than after pollination. This is consistent with the results of Elkeles et al. [15], who showed that the vdac expression in flower organs decreases after anthesis. This decrease is correlated with the flower degenerescence, as it does not happen in other organs. Moreover, there is also a developmental regulation during the seedling development for all the rice vdac gene expressions. The developmental rice vdac regulation and the differential expression in function of the organs are most probably coordinated by transcriptional regulatory factors. Gene promoter analysis of the rice vdacs will probably provide more insights in these regulatory processes. Under the mannitol stress condition, no change in vdac expression was observed after 24 or 72 h of stress although both proline content and salT gene expression increased indicating that plantlets did effectively respond to the applied stress. In contrast, a fast-enhanced expression of vdac was observed during the recovery period after mannitol removal from the medium. Similar results were observed in the

presence of a salt stress or a drought stress. As these stresses also have an osmotic component, we can reasonably assume that the enhanced vdac expression observed during the recovery period arises from the same regulation process in the three conditions. The antibody used in this work reacts with the different VDAC isoforms [28]. Therefore, our Western blot experiment shows that the total amount of VDAC protein does not change during the stress recovery period. The lack of correlation between RNA expression and protein level indicates that there may be a posttranscriptional regulation of the vdac gene expression during the stress recovery period. Acknowledgements F. Al Bitar is a Research Assistant and F. Homble´ is a Research Director of the National Fund for Scientific Research (Belgium), N. Roosens was supported by a F.R.I.A. fellowship and she is a ‘‘Charge´ de Recherche’’ for the Belgian Fond National de la Recherche Scientifique. The authors wish to thank C. Bouton, M. De Kerpel and M. Claes for their technical assistance. The authors are grateful to MAFF DNA Bank, for the gift of the O. sativa EST clones (C12629 and E10458), to the Laboratory of Genetics of the University of Ghent for the gift of the salT cDNA and to J. Balk (University of Oxford) for the gift of maize porin antibody. This work was supported by a grant of the Communaute´ Franc¸aise de Belgique-Actions de Recherche Concerte´es. References [1] L.S. Zalman, H. Nikaido, Y. Kagawa, Mitochondrial outer membrane contains a protein producing nonspecific diffusion channels, J. Biol. Chem. 255 (1980) 1771 – 1774. [2] V. Adams, L. Griffin, J. Towbin, B. Gelb, K. Worley, E.R. McCabe, Porin interaction with hexokinase and glycerol kinase: metabolic microcompartmentation at the outer mitochondrial membrane, Biochem. Med. Metabol. Biol. 45 (1991) 271 – 291. [3] S. Shimizu, M. Narita, Y. Tsujimoto, Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC, Nature 399 (1999) 483 – 487. [4] E. Blachly-Dyson, E.B. Zambronicz, W.H. Yu, V. Adams, E.R. McCabe, J. Adelman, M. Colombini, M. Forte, Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel, J. Biol. Chem. 268 (1993) 1835 – 1841. [5] M. Linden, G. Karlsson, Identification of porin as a binding site for MAP2, Biochem. Biophys. Res. Commun. 218 (1996) 833 – 836. [6] S.J. Schein, M. Colombini, A. Finkelstein, Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria, J. Membr. Biol. 30 (1976) 99 – 120. [7] M. Colombini, A candidate for the permeability pathway of the outer mitochondrial membrane, Nature 279 (1979) 643 – 645. [8] M. Colombini, Structure and mode of action of a voltage dependent anion-selective channel (VDAC) located in the outer mitochondrial membrane, Ann. N.Y. Acad. Sci. 341 (1980) 552 – 563. [9] D.P. Smack, M. Colombini, Voltage-dependent channel found in the membrane fraction of corn mitochondria, Plant Physiol. 79 (1985) 1094 – 1097.

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