Molecular and functional characterization of a PEX14 cDNA from rice

Plant Science 166 (2004) 123–130

Molecular and functional characterization of a PEX14 cDNA from rice Jung Ro Lee1 , Kyun Oh Lee1 , Jin Ho Park, Ji Young Yoo, Jae Sook Kang, Hye Sook Jeon, Sun Young Kim, Young Mi Lee, Sun Tae Kim, Chae Oh Lim, Jeong Dong Bahk, Moo Je Cho, Sang Yeol Lee∗ Division of Applied Life Sciences (BK21 Program), PMBBRC, Gyeongsang National University, Chinju 660-701, South Korea Received 1 July 2003; received in revised form 20 August 2003; accepted 31 August 2003

Abstract In contrast to the translocation mechanisms determined in yeasts and mammalian cells, there is little information on the functions of plant peroxisomal proteins or their genomic structures. To understand the role that PEX14 plays in diverse plant peroxisomal functions and how peroxisomal translocation is mediated in plant cells, we cloned a 1827 bp cDNA encoding the peroxisomal membrane protein OsPex14p from a rice leaf cDNA library. The 54 kDa OsPex14p, which has a theoretical pI value of 6.06, contains a highly conserved N-terminal domain and a short putative transmembrane domain. The OsPEX14 gene in the rice genome exists as a single-copy gene, consists of eleven exons interrupted by ten introns, and spans about 5 kb of chromosome 5. The 5 -flanking region contains putative cis-acting light-responsive elements, and the transcription initiation site maps 114 bp upstream of the translation start codon. OsPEX14 mRNA is highly expressed in leaf tissues and is induced by exposure to several stresses. Heterologous expression of OsPex14p suppresses the defect in targeting of peroxisomal matrix proteins in a pex14 null mutant of Saccharomyces cerevisiae. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: OsPex14p; Peroxisome; PTS1; Rice; Targeting

1. Introduction Peroxisomes are respiratory organelles with a single membrane that serve a wide variety of functions in eukaryotic cells, including long-chain fatty acid ␤-oxidation, glyoxylate cycle metabolism, photorespiration, and hydrogen peroxide, superoxide radical and nitric oxide generation [1–3]. Peroxisomal matrix proteins are synthesized on free polyribosomes in the cytoplasm and translocated into peroxisomes [4]. Two evolutionarily conserved peroxisomal targeting signal (PTS) sequences, designated PTS1 and PTS2, are required for transport. PTS1, located at the C-terminal end of matrix proteins, contains the tripeptide SKL or a conservative variant [5], whereas PTS2, positioned Abbreviations: PTS, peroxisomal targeting signal; Os, Oryza sativa; OsPex14p, rice Pex14 protein; Sc, Saccharomyces cerevisiae; GFP, green fluorescent protein; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; UTR, untranslated region ∗ Corresponding author. Tel.: +82-55-751-5958; fax: +82-55-759-9363. E-mail address: [email protected] (S.Y. Lee). 1 These two authors contributed equally to this work.

at the N-terminus, contains a 9-amino acid signal sequence, namely, (R/K)-(L/V/I)-X5 -(H/Q)-(L/A/F). The PTS2 but not the PTS1 signal sequence is removed after transport [6,7]. The translocation of peroxisomal matrix proteins containing PTS1 or PTS2 requires specific recognition by receptor proteins, such as Pex5p for PTS1 [8,9] or Pex7p for PTS2 [10]. Once the translocated protein is loaded onto its receptor, the receptor–cargo complex binds to the peroxisomal integral membrane protein Pex14p, which directs it to the peroxisomal compartment [11]. Pex14p has been demonstrated to interact with Pex5p, Pex7p, Pex13p and Pex17p to form a docking complex that permits the binding of PTS1 and PTS2 receptor proteins to the peroxisomal membrane [12,13,16]. To date, five cDNAs encoding Pex14 proteins have been identified from yeasts [12–14], mice, humans [15] and Arabidopsis thaliana [2]. A peroxisome-deficient A. thaliana pex14 mutant exhibits significant defects in fatty acid ␤-oxidation and photorespiration, a reduction in the number of peroxisomal matrix enzymes, and a requirement for exogenous sucrose for post-germination development [2]. These observations suggest that in plant cells, Pex14p functions are critical for peroxisome biogenesis,

0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.08.016

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peroxisomal differentiation, cell growth, and the conversion of fatty acids to sucrose. Much information has been obtained for yeasts and mammalian cells concerning the mechanism of translocation of peroxisomal proteins, but little experimental evidence has been reported for higher plants. Indeed, the A. thaliana PEX14 cDNA is the only member of this family cloned from plants [2]. To understand the role PEX14 plays in diverse plant peroxisomal functions and to assess how peroxisomal translocation is mediated in plants, it is of interest to clone and analyze additional PEX14 genes from various plant sources. For this purpose, we undertook to isolate PEX14 homologues from a rice cDNA library by using a 450 bp partial fragment of the A. thaliana PEX14 cDNA, which encodes the first 150 amino acids of the AtPex14p, as a probe. Here, we describe the cloning of the rice gene OsPEX14 (Oryza sativa PEX14) and the functional characterization of its gene product, as assessed by the suppression of the pex14 null mutation of Saccharomyces cerevisiae [13]. To investigate the genomic structure and transcriptional regulation of OsPEX14, we have also isolated and characterized an OsPEX14 genomic clone.

2. Materials and methods 2.1. Materials Surface-sterilized rice (O. sativa L. cv., Dong-jin) seeds were imbibed at 4 ◦ C for 48 h and transferred to 0.8% agar bottles or sterile soil. Seedlings were grown in a chamber maintained at 28 ◦ C with a 16 h photoperiod for 20– 40 days. S. cerevisiae wild type (CB80: MATα, ura3-52, leu2-1, trp1-63, his3-200) and pex14 mutant (CB81: MATα, ura3-52, leu2-1, trp1-63, his3-200, PEX14::KanMX4) strains were used to assess the peroxisomal targeting of the model PTS1 protein GFP-SKL [11,13]. To construct YCplac22-ScPEX14, the BamHI/SalI ScPEX14 insert from YCpPEX14 [13] was subcloned into YCplac22. To construct YCplac22-OsPEX14, PCR was performed with pBluescript II SK(−)-OsPEX14 as a template with the primers 14fn (5 -AGTTCGCTTCGCTTCGATCATATGGC-3 ) and 14rs (5 -GTCGACATGTAATGTAATGTAATGTATCGG-3 ). The promoter region of ScPEX14 was amplified from yeast genomic DNA by PCR using the primers Sc14Pf (5 -TTACCGAAGCGGCCGCTGTCA-3 ) and Sc14Pr (5 GGCCCACGTCACTCATATGTTATTCACC-3 ). The resulting PCR products were subcloned into the vector pGEM-T (Promega). The NotI/NdeI-digested insert containing the ScPEX14 promoter region and the NotI/SalI-digested coding region of the OsPEX14 were subcloned into YCplac22 containing a modified multiple cloning site. To construct pYES2-GFP-SKL, the C-terminus of the green fluorescent protein (GFP) gene was modified by PCR using the primers GFPf (5 -GATGGATCCATGAGTAAAGGAGAAGAACTTT-3 ) and GFPr (5 -CTCGAGTTATAGCT-

TTGATTTGTATAGTTCATC-3 ), and the product was cloned into the vector pGEM-T (Promega). The BamHI/ XhoI-digested insert was subcloned into the vector pYES2. All constructs were confirmed by DNA sequencing. 2.2. Screening of a rice cDNA library and DNA sequencing To clone full-length PEX14 cDNA from rice, a 450 bp partial fragment of the A. thaliana PEX14 cDNA that encodes the first 150 amino acids of the AtPex14p was used as a probe. The fragment was then labeled with [␣-32 P]ATP to a specific activity of ca. 5×108 cpm/␮g by the random-primer labeling method. Approximately 5 × 106 clones from a phagemid-based rice leaf cDNA library were screened with this probe. The library was plated on E. coli XL1-Blue MRF (Stratagene) to a density of approximately 3 × 104 plaques per 15 cm agar plate and replicated to a nylon membrane (Amersham). The membranes were hybridized and washed under proper stringency conditions as previously described [17]. After three rounds of screening, five positive clones were isolated, all of which were shown by sequencing to represent a single gene. Nucleotide sequences of both strands were determined by the dideoxy chain-termination method with an automated sequencer (Applied Biosystems model 377) using a BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems). 2.3. Southern and Northern blot analyses Ten micrograms of rice genomic DNA was digested with EcoRI, HindIII or XbaI, separated on a 0.8% agarose gel, and transferred to a nylon membrane (Amersham). Hybridization was performed using the [␣-32 P]ATP-labeled OsPEX14 cDNA as a probe and the membrane was washed twice in 0.5 × SSC, 0.1% SDS at 42 ◦ C for 20 min. The filter was dried and exposed to an X-ray film for the appropriate time at −70 ◦ C. For Northern blot analysis, total RNA was extracted from rice seeds, seedlings and suspension culture cells exposed to various stresses as a previously described [18]. Twenty micrograms of total RNA was separated on a 1.2% formaldehyde agarose gel and transferred to a nylon membrane (Amersham). The blots were hybridized with [␣-32 P]ATP-labeled specific cDNA probes. 2.4. Cloning of the 5 -flanking region of the OsPEX14 gene and primer extension analysis The 5 -flanking region of the OsPEX14 gene was amplified by PCR using the primers 14Gf (5 -CCTTGGATTCCACTAGCCTAGCAAGTCCC-3 ) and 14Gr (5 -GAATCGAAGCGAAGCGAACGTACGT-3 ) and rice genomic DNA as a template. The amplified fragment was subcloned into the vector pGEM-T (Promega) and used as a template to derive the DNA sequence ladder used in primer extension analysis. For primer extension, the 14Gr primer, which

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is complementary to the OsPEX14 5 -untranslated region (UTR), was radio-labeled by T4 polynucleotide kinase (Roche) with [␥-32 P]ATP (5000 Ci/mmol, Amersham Pharmacia). Five micrograms rice leaf mRNA was denatured at 65 ◦ C and incubated in a reaction buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2 , 20 mM dithiothreitol) containing 0.5 ng of the labeled oligonucleotide, Moloney murine leukemia virus reverse transcriptase (Roche) and deoxynucleotide triphosphates (1 mM) at 37 ◦ C for 1 h. The synthesized cDNA was denatured at 65 ◦ C and analyzed on a 6% acrylamide gel. The same primer was used to generate the DNA sequence ladder. 2.5. Targeting analysis of the PTS1-containing protein GFP-SKL by fluorescence microscopy The wild type (CB80) and pex14 knockout (CB81) yeast strains and the yeast vector YCpPEX14 expressing ScPex14p [13] were kindly provided by Dr. Andreas Hartig. All plasmids were purified by ultracentrifugation on a CsCl gradient. The YCplac22-ScPEX14 and YCplac22-OsPEX14 constructs were introduced into CB80 and CB81, respectively, which were previously transformed with pYES2-GFP-SKL. All transformations were performed by the LiAc method. Co-transformed cells were selected on synthetic complete media without uracil and tryptophan, inoculated into Sabouraud-raffinose 4% (w/v) media without uracil and tryptophan and cultured at 30 ◦ C for about 48 h. Galactose was added when the OD600 of the cultures reached 2.0, and samples were periodically harvested by centrifugation (5000 × g for 5 min). GFP-SKL expression was monitored at various time points after galactose induction, and images were captured with a cooled charge-coupled device camera using a Zeiss Axioplan fluorescence microscope. The XF116 (exciter, 474AF20; dichroic, 500DRLP; emitter, 510AF23) filter sets were used for GFP detection.

3. Results and discussion 3.1. Molecular cloning of OsPEX14 from a rice leaf cDNA library and sequence analysis To identify Pex14p homologues that play a key role in the translocation of peroxisomal matrix proteins in plants, we screened a rice leaf cDNA library by using a fragment of the A. thaliana PEX14 cDNA as a probe. We found a full-length cDNA clone, OsPEX14, which contained a 1503 bp open reading frame with a 102 bp 5 -UTR and a 222 bp 3 -UTR including a poly-A tail. This sequence was deposited in the GenBank database under accession number AY262026. The deduced amino acid sequence of OsPex14p consists of 501 amino acids with a predicted molecular mass of 53,836 Da and a theoretical pI value of 6.06. GenBank database searches using the NCBI/BLAST program revealed that OsPex14p shares the highest homology with the A.

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thaliana Pex14 protein, AtPex14p [2], although the extent of sequence identity (28%) was very low. However, significant sequence conservation was found at the N-terminal peptide domain of OsPex14p from residues 42 R to 83 A, which is conserved in most Pex14 proteins and is considered to be the recognition motif for the PTS1 receptor protein, Pex5p. Besides this motif, two amino acids of OsPex14p, 143 W and 170 P, are perfectly conserved in all Pex14 proteins. The deduced amino acid sequence of OsPex14p is aligned with that of other Pex14 proteins cloned to date from various organisms in Fig. 1A. To explore the evolutionary relationship of OsPex14p with other Pex14 proteins, we generated a phylogenetic tree by using the Clustal package in the MegAlign program (Fig. 1B). This analysis confirmed that OsPex14p is most closely related to AtPex14p and more distantly related to animal and yeast homologues. Since Pex14 proteins are reported to be membrane-bound [2,12–15], we examined OsPex14p by using the TMHMM database (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and found that the peptide from 146 A to 164 V is highly likely to be in a transmembrane domain, as shown in Fig. 1C. Coincidently, this putative transmembrane domain exists between the two highly conserved amino acids 143 W and 170 P. 3.2. Isolation of the genomic clone of OsPEX14 and its structural characteristics To obtain a genomic clone of OsPEX14 containing the promoter, we carried out a BLAST search (http://www. ncbi.nlm.nih.gov/BLAST/) using the OsPEX14 cDNA sequence as a query and identified a 156,772 bp fragment of a rice chromosome five PAC clone, P0036D10 (http://genome.sinica.edu.tw/, accession number AC073405), which contains the complete OsPEX14 genomic sequence. Alignment of the cDNA and genomic sequences allowed us to identify the exons and introns in the genomic sequence. The structure of the OsPEX14 gene, its mRNA transcript and predicted translational product are summarized in Fig. 2A. The genomic OsPEX14 comprises eleven exons interrupted by ten introns and spans about 5 kb of the clone. The exons vary in size from 32 kb (exon 7) to 658 bp (exon 11). The first exon contains the translational start codon and the last exon contains the translational stop codon, TAG. A comparison of the primary structure of the protein with the genomic sequence shows that the conserved N-terminal domain and putative transmembrane domain of OsPex14p are translated from exon 2 and exon 4, respectively. To obtain additional information concerning the genomic organization of OsPEX14, Southern blot analysis was performed using the full-length cDNA as a probe (Fig. 2B). Under high stringency hybridization and washing conditions, one to four hybridizing bands were observed on a blot of genomic DNA digested with EcoRI (lane 1), HindIII (lane 2) and XbaI (lane 3). The hybridizing bands (Fig. 2B) coincide exactly in number and size with the fragments that

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Fig. 1. Amino acid sequence analysis of OsPex14p. (A) Comparison of the deduced amino acid sequence of OsPex14p with that of other Pex14 proteins identified to date. Open reading frames of the corresponding cDNA clones were translated and compared after alignment by using the Clustal method in the MegAlign program. The GenBank accession numbers of the Pex14 proteins are: AtPex14p (BAB17668), HsPex14p (BAA36837), MmPex14p (NP 062755), HpPex14p (P78723) and ScPex14p (P53112). The highly conserved peptide positioned from 42 R to 83 A of OsPex14p is boxed and the putative transmembrane domain is indicated by a bold line above the sequence. Asterisks mark positions of perfectly conserved amino acid residues and dots mark well conserved positions. Amino acid numbers are indicated at right and amino acid sequence identities with OsPex14p are shown in the last column (%). (B) Phylogenetic analysis of OsPex14p compared with other Pex14 proteins constructed using the Clustal method. The bottom scale indicates relative distances between the sequences. (C) The OsPex14p transmembrane domain predicted with the use of the TMHMM program. The Y-axis represents the probability scores for a transmembrane helical region and the X-axis shows the amino acid position of OsPex14p.

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Fig. 2. Genomic organization of OsPEX14. (A) Schematic representations of the OsPEX14 genomic clone, its mRNA transcript and putative protein structure including restriction enzyme sites (E, EcoRI; H, HindIII; X, XbaI). The 5’ flanking region (gray boxes), exons (black boxes) and introns (white boxes) of the OsPEX14 gene were assigned by comparison and alignment of the cDNA and genomic DNA sequences. The OsPEX14 mRNA along with the exon numbers is shown. The highly conserved N-terminal peptide positioned between 42 R and 83 A of the OsPex14p, and a putative OsPex14p transmembrane domain are represented by black and gray boxes, respectively. (B) Genomic Southern blot analysis of OsPEX14. Rice genomic DNA (10 ␮g per lane) was digested with EcoRI (lane 1), HindIII (lane 2), or XbaI (lane 3) and hybridized with an OsPEX14 cDNA probe as described in Section 2. DNA size markers are indicated to the left.

are predicted to be yielded by digestion with these enzymes. This indicates that OsPEX14 is a single copy gene. 3.3. Analysis of the OsPEX14 promoter and regulation of gene expression The 5 -flanking region of OsPEX14 was amplified by PCR using the specific primers 14Gf and 14Gr (see Section 2) and the reaction product was completely sequenced. Analysis of the 5 -flanking sequence of OsPEX14 by referring to the PlantCARE database revealed the presence of a G-box, a GT-1 motif, a CAAT box, an ATCT motif, a GA-motif, a GAG motif, and a TATA box (Fig. 3A). Interestingly, most of the elements in the OsPEX14 promoter have been previously identified as cis-acting regulatory elements that are involved in plant cell light responsiveness [19,20]. To determine the transcriptional start site of OsPEX14, we carried out

primer extension analysis using mRNA isolated from young rice leaves and the 14Gr primer labeled with [␥-32 P]ATP (Fig. 3B). The DNA fragment containing the 5 -flanking region of OsPEX14 was sequenced using the same primer. The reaction products obtained from both the primer extension and sequencing reactions were separated on a 6% polyacrylamide gel and visualized by autoradiography. A guanine (G) residue located 114 bp upstream of the translational start codon appears to be the major transcriptional initiation site of the OsPEX14 gene. A putative TATA box identified by the database search is present in the 26 bp region upstream of the transcriptional initiation site. We examined the expression of OsPEX14 mRNA in different tissues under diverse stress conditions by Northern analysis using the [␣-32 P]ATP-labeled full-length OsPEX14 cDNA as a probe (Fig. 3C and D). OsPEX14 was highly expressed in leaf tissues, and a low transcript level was detected

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Fig. 3. Analysis of the OsPEX14 promoter sequence and regulation of expression. (A) The 5 flanking region of OsPEX14 was analyzed with the use of the PlantCARE database (http://sphinx.rug.ac.be:8080/PlantCARE/cgi/index.html). Several putative cis-elements including the TATA box are boxed. +1 indicates the transcription initiation site. Sequences complementary to the underlined nucleotides correspond to the oligonucleotide primer (14Gr) that was used for the primer extension assay. (B) Primer extension analysis of the OsPEX14 gene. The arrowhead indicates the major band detected in the experiment and the nucleotide sequences around the band are shown to the right. The nucleotide marked in bold (G) represents the mapped transcription initiation site. (C) Tissue-specific expression of OsPEX14 in rice tissues. Total leaf (L) and root (R) RNA was prepared from 20-day-old rice seedlings, and seeds (S) were obtained from fully-grown (about 6-month-old) rice plants and subjected to Northern analysis with OsPEX14 (upper panel) and R1C-Prx (lower panel) probes. (D) Stress-induced expression of OsPEX14 mRNA. Embryo-derived rice suspension culture cells were treated with Magnaporthe grisea spores (5 × 105 per ml) (P), fungal elicitor (50 ␮g of glucose equivalents per ml) (E), 100 ␮M methyl viologen (M), 200 mM NaCl (S) or 5 mM hydrogen peroxide (H) for 12 h. Untreated calli (C) were used as a control. The blot was hybridized with the OsPEX14 (upper panel) and ␤-tubulin (lower panel) cDNA probes.

in roots and seeds (Fig. 3C, upper panel). When the same blot was hybridized with an [␣-32 P]ATP-labeled R1C-Prx probe as a control, seed-specific expression was observed as reported [17] (Fig. 3C, lower panel). In addition, using rice embryo-derived suspension culture cells, we examined the regulatory factors that affect gene expression under various

conditions, such as pathogen (Magnaporthe grisea), elicitor, methyl viologen, NaCl and hydrogen peroxide stresses. Overall, OsPEX14 mRNA expression generally increased as a result of these treatments (Fig. 3D, upper panel), whereas the control rice ␤-tubulin transcript levels did not change (Fig. 3D, lower panel). From these results, we conclude

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that OsPEX14 expression is induced by a variety of external stresses, including light stimuli, which is consistent with the results of the promoter analysis (Fig. 3A). 3.4. Suppression of the yeast PEX14 mutation by heterologous expression of OsPEX14 Although OsPex14p contains several highly conserved domains that are present in most Pex14 proteins, it shares very low amino acid sequence homology (28%) with these proteins, including AtPex14p (Fig. 1A). Therefore, we asked whether the OsPEX14 gene is a functional rice counterpart of the S. cerevisiae PEX14 gene by expressing the protein in a yeast pex14 knockout mutant (CB81), which is defective in the peroxisomal translocation of PTS1 proteins [13]. When a GFP-SKL construct containing the PTS1-specific tripeptide SKL at the GFP C-terminus was expressed under the control of the GAL1 promoter in a wild type strain (CB80), peroxisomal GFP-SKL fluorescence defined a punctate staining pattern as expected (Fig. 4A). However, in CB81 harboring the GFP-SKL construct, the fluorescence was exclusively localized in the cytosol (Fig. 4B), suggesting that GFP-SKL could not be transported into the peroxisome. In contrast, when the pYES2-GFP-SKL vector was co-transformed with YCplac22-ScPEX14 into CB81, GFP-SKL was correctly targeted to the peroxisome (Fig. 4C) and the same pattern of punctate staining was observed as in CB80 cells expressing GFP-SKL (Fig. 4A). Moreover, co-transformation of pYES2-GFP-SKL and YCplac22-OsPEX14 into CB81 cells resulted in a pattern of fluorescence similar to that of cells expressing ScPEX14 (Fig. 4D), indicating that OsPex14p can promote the targeting of GFP-SKL to peroxisomes in the mutant yeast strain. These results strongly suggest that the heterologous expression of OsPex14p suppresses the per-

Fig. 4. Functional suppression of the yeast pex14 mutation by heterologous expression of OsPex14p. GFP-SKL was expressed in wild type (CB80; A) and pex14 null (CB81; B) strains under the control of the GAL1 promoter. ScPex14p (C) and OsPex14p (D) were co-expressed with GFP-SKL under the control of the ScPEX14 promoter in pex14 cells (CB81). Cells were grown on selective media and shifted to induction media containing galactose. After 12–16 h of induction, cells were harvested and viewed by fluorescence microscopy (400× magnification).

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oxisomal targeting defect of a pex14 knock-out yeast strain with an efficiency comparable to the endogenous protein, ScPex14p. From these data, we conclude that OsPex14p is a functional homologue of yeast ScPex14p and that organisms ranging from yeast to plants use similar or even identical peroxisomal translocation systems. In summary, we have cloned a rice PEX14 gene and analyzed its genomic structure, patterns of expression and functional characteristics. This clone will be helpful in achieving a better understanding of the transport processes that occur in plant peroxisomes.

Acknowledgements We thank Dr. Kyu Young Kang (Gyeongsang National University, Korea) for generously supplying the rice leaf cDNA library and Dr. Andreas Hartig (University of Vienna, Austria) for kindly providing control plasmids and yeast cells. This research was supported by a National Laboratory Program grant (project #2000-N-NL-01-C-236), by the Crop Functional Genomic Center of the Frontier Program (project #CG1511 and #1512), by the Brain Korea21 project in 2003, and by the Kyongnam High-Tech Foundation (2000) supported by Kyongsang Namdo, Korea.

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