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Plant Cell Physiol. 43(7): 697–705 (2002) JSPP © 2002 The Use of Multiple Transcription Starts Causes the Dual Targeting of Arabidopsis Putative Monodehydroascorbate Reductase to Both Mitochondria and Chloroplasts Keisuke Obara 1, 3, Kazuyoshi Sumi 1 and Hiroo Fukuda 1, 2 1 2 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan ; region. Because mitochondria and chloroplasts share some overlapping functions, such as DNA replication, transcription, translation and protection from oxidative stress (Small et al. 1998), some enzymes are present in both organelles. Although it is generally accepted that one gene encodes one isozyme, there are some exceptions in which a single gene encodes multiple isoforms that carry out the same function in different organelles. Several tRNA synthetases, biosynthetic proteins and oxidoreductases coded by a single gene are dual-targeted to both mitochondria and chloroplasts (reviewed in Small et al. 1998, Peeters and Small 2001). These examples help us understand the mechanism of protein transport to mitochondria and chloroplasts, but the precise mechanism is still not fully understood. To further elucidate this mechanism, we used the transport mechanism of the protein involved in the antioxidant system as a model because some of the antioxidant molecules are present in both organelles, where they work to remove excess reactive oxygen species (ROS). During respiratory and photosynthetic electron transport, copious quantities of ROS are produced at very high rates, even under optimal conditions (Noctor and Foyer 1998). Recent studies indicated that plants utilize ROS, especially H2O2, as signal molecules in cases such as ABA signaling (Pei et al. 2000, Murata et al. 2001), and responses to cellular and environmental stress (Prasad et al. 1994, Wagner 1995). ROS are also used as a weapon against invading pathogens in the oxidative burst (Lamb and Dixon 1997, Somssich and Hahlbrock 1998). However, it is generally thought that ROS are toxic to the cell because of their ability to cause damage to proteins, lipids and DNA. Therefore, ROS production and removal must be strictly controlled. One efficient way to avoid the damage caused by excess accumulation of ROS is to remove them with antioxidant molecules. Ascorbate is a major primary antioxidant (Nijs and Kelley 1991) and is ubiquitous in animals and plants. Ascorbate is also involved in the plant antioxidant system as an electron donor for ascorbate peroxidase that scavenges H2O2 and produces H2O. Oxidation of ascorbate in this process generates the monodehydroascorbate radical. The monodehydroascorbate radical is then in turn reduced by Monodehydroascorbate reductase (MDAR) isoforms exist in mitochondria, chloroplasts, cytosol and microbodies. Two putative MDAR sequences with an extended Nterminal region are found in Arabidopsis. They differ in the length of the extension by 21 bp. We have shown that these two isoforms arise from a single gene by the use of multiple transcription starts. Green fluorescent protein was fused to each extension, revealing that the longer and shorter fusion proteins were imported into mitochondria and chloroplasts, respectively. These results demonstrate that putative MDAR is a dual-targeting protein transported into both mitochondria and chloroplasts. Although there have been several reports of dual targeting of proteins to mitochondria and chloroplasts, this is the first example in which the dual targeting of the protein to mitochondria and chloroplasts is achieved by the use of multiple transcription initiation sites. Keywords: Arabidopsis — Chloroplast — Dual targeting — Mitochondria — Monodehydroascorbate reductase — Multiple transcription initiation sites. Abbreviations: GFP, green fluorescent protein; MDAR, monodehydroascorbate reductase; PMDAR-L, putative monodehydroascorbate reductase-long; PMDAR-S, putative monodehydroascorbate reductase-short; ROS, reactive oxygen species; 5¢-UTR, 5¢-untranslated region. Introduction Plants acquired chloroplasts in addition to mitochondria by endosymbiosis. Following the endosymbiosis, many of the genes of the endosymbionts were transferred to the nucleus, and therefore most mitochondrial and chloroplast proteins are encoded by nuclear genes and are synthesized in the cytosol. These proteins are usually translated as precursor proteins that have mitochondrial or chloroplast targeting sequences (presequence and transit peptide, respectively) at the N-terminal 3 Corresponding author: E-mail, [email protected]; Fax, +81-3-5841-4462. 697 698 Dual targeting of Arabidopsis putative MDAR Fig. 1 Alignment of the deduced amino acid sequences of Arabidopsis MDAR-like sequences. Amino acid residues conserved in all sequences were highlighted. Gray boxes indicate conserved residues in more than four sequences. Note that only D84417 and NP_564818 have an N-terminal extension. monodehydroascorbate reductase (MDAR) using NAD(P)H as the electron donor to become ascorbate (Hossain et al. 1984). Therefore, MDAR plays an important role in the plant antioxidant system through its ability to regenerate ascorbate to maintain the ascorbate pool in the cell. Recently, MDAR was shown to be also capable of reducing phenoxyl radicals to their respective parental phenols (Sakihama et al. 2000), which are also thought to be potent antioxidants (Rice-Evans et al. 1997). In plants, isoforms of MDAR exist in chloroplasts, mitochondria, microbodies and in the cytosol, and cDNA of MDAR has been cloned from cucumber (Sano and Asada 1994), garden pea (Murthy and Zilinskas 1994), tomato (Grantz et al. 1995), rice (Accession No. D85764), Arabidopsis (Accession No. D84417) and leaf mustard (Accession No. AF109695). Because mitochondria and chloroplasts are major sources of ROS in the cell, MDAR activity in these organelles might be essential for cell function (Puntarulo et al. 1991, Asada 1999, Møller 2001). Our preliminary search of the Arabidopsis genome and cDNA databases indicated the presence of two MDAR-like sequences that possess putative mitochondrial/ chloroplast targeting sequences. Interestingly, the longer one contained the entire sequence of the shorter one. Therefore, we tried to elucidate the targeting compartment of the two MDARlike proteins and the mechanism by which the two sequences are made. Our results demonstrate that alternative transcription of a single gene generates two different proteins, of which one is transported into mitochondria and the other into chloroplasts. Results A single putative MDAR gene generates two mRNAs A search of the Arabidopsis thaliana genome and cDNA databases revealed the presence of at least seven MDAR-like sequences. There were eight hits in Arabidopsis, but the two of them, D84417 and AAG52455, overlapped and contained a one base mismatch, which may have been caused by polymorphism or misreading of the sequence. Therefore, the two sequences were represented by D84417 in this paper. Fig. 1 shows their deduced amino acid sequences, of which two (D84417 and NP_564818) had an extended region at the Nterminus. The results of several computer programs predicting protein localization or signal sequences suggested that the two putative MDARs with the extended N-terminal region were targeted to two organelles at high frequency; the longer one to mitochondria and the shorter one to chloroplasts. The others were not predicted to be targeted to either mitochondria or chloroplasts. Interestingly, the Arabidopsis genome database indicated that the two putative MDARs arise from the same ORF on chromosome 1. Taken together, it is suggested that one putative MDAR gene generates two mRNAs that differ in length by 21 b and that their products are targeted to different organelles. We designated the longer one as PMDAR-L (putative monodehydroascorbate reductase – long) and the shorter one as PMDAR-S (putative monodehydroascorbate reductase – short). There was no difference between the PMDAR-L and PMDAR-S sequences except that PMDAR-L had the extended seven amino acid residues from the first Met of PMDAR-S Dual targeting of Arabidopsis putative MDAR 699 Fig. 2 RT-PCR experiments showing the existence of two mRNAs originating from a PMDAR gene. (A) Genome sequence of the 5¢ region corresponding to PMDAR-L and PMDAR-S mRNAs. Protein-coding sequences are underlined and their corresponding amino acid sequences are described below them. Start codons, ATGs, are boxed. An intron (gray letters) ranging approximately 90 bp exists between the two start codons, and conserved consensus sequences of the initiation and termination of intron, GT and AG, are dotted. Hooked arrows represent the forward primers used in the RT-PCR analysis. The reverse primer was designed from the stop codon of PMDAR (see B). Note that primers S-2 to S-6 were designed within the first intron of PMDAR-L, so that they can amplify only PMDAR-S specifically. In contrast, the primer S-1 can amplify the fragment from both PMDAR-L and PMDAR-S. (B) The primer sequences used. (C) PCR amplification of PMDAR-L and PMDAR-S cDNA. Onefiftieth of the PCR products were run on a 1% agarose gel and stained with ethidium bromide. The primer sets used are shown above each lane. The positions of 1700 bp and 1159 bp are given on the left. Lane 1, l DNA digested with PstI was used as a molecular marker. Lanes 2–4, PCR products amplified with the forward primers designed at 5¢-UTR of PMDAR-L and the reverse primer. Lane 5, PCR products amplified with the forward primer S-1 and the reverse primer. Lanes 6–10, PCR products amplified with the primer sets that are specific to PMDAR-S. (D) The Nterminal sequence of the fragments L-1 and S-6 obtained by RT-PCR with the forward primers L-1 and S-6, respectively. Translation start codons were boxed. The protein coding region was underlined. Note that the fragment S-6 contained a part of the intron sequence of fragment L-1 (gray letters) at its 5¢-UTR. This region was spliced out in the fragment L-1. (data not shown). To elucidate how a single PMDAR gene can generate two mRNAs, we first analyzed the genomic structure of the gene. Fig. 2A shows the genome sequence of the 5¢ region of the gene. The extended region of the PMDAR-L sequence contained an intron ranging approximately 90 bp. This intron had conserved consensus sequences, GT and AG, which are usually seen at the initiation and termination sites of the intron, respectively. The start codon, ATG, for the PMDARS sequence lies in the second exon of the PMDAR-L. Although the two start codons for PMDAR-L and PMDAR-S sequences are in-frame even through the intron, there are two stop codons, in this frame, in the intron. Next, to examine the existence of two mRNAs corresponding to PMDAR-L and PMDAR-S, RTPCR was performed with the oligonucleotides shown in Fig. 2B as forward primers and cDNA copied from Poly(A)+ RNA as a template. Each of the primers used amplified a single band of the expected size (Fig. 2C), of which the two fragments amplified with L-1-Reverse and S-6-Reverse set (hereafter referred to as fragment L-1 and S-6, respectively) were cloned and sequenced. As predicted, the fragment L-1 had a sequence 700 Dual targeting of Arabidopsis putative MDAR Fig. 3 Determination of two transcription initiation sites of the Arabidopsis PMDAR. (A) The positions of primers designed to amplify the fragments containing the transcription initiation site of PMDAR-L and PMDAR-S. The first PCR was performed with the primer sets of 1RC (see B) and R-1. For the nested-PCR, 2RC primer (see B) was used as a forward primer, and R-2 or R-3 as reverse primers. (B) The primer sequences used to determine the transcription starts. 1RC and 2RC are the forward primers designed at the oligonucleotides that were copied from oligoribonucleotides artificially attached to replace the 5¢ cap structure (see Materials and Methods). (C) The amplified products were run on a 4% GTG Nusieve Agarose gel and stained with ethidium bromide. The primer sets used are shown above each lane. The positions of 500 bp and 100 bp are given at left. Lane 1, a molecular marker of 100-bp ladder. Lane 2, the first PCR product. Lanes 3–7, nested PCR products amplified with primer sets, 2RC/R-2, 2RC/R-3, only 2RC, only R-2, and only R-3, respectively. In Lane 2, no band was detected. In Lanes 3 and 4, several bands were detected. Of these bands, those that were not amplified in negative control experiments (Lanes 5–7) are indicated by arrowheads. The fragments corresponding to these two bands were cloned and sequenced. Note that R-3 primer was designed inside the first intron of PMDAR-L so that the fragment specific to PMDAR-S was expected to be amplified. (D) Transcription starts for PMDAR-L and PMDAR-S (TS-L and TS-S, respectively) shown by arrowheads. A putative TATA box (double underline) was found upstream of TS-S. Start codons, ATGs, are boxed. The gray sequence indicates the first intron of PMDAR-L, and the donor and acceptor site of the intron are dotted. Protein coding regions are underlined and the amino acid residues corresponding to each triplet are shown below. Note that TS-S lies inside the first intron of PMDAR-L. (E) Schematic representation of how the two isoforms are produced from a single PMDAR gene. in which the region between the GT and AG was spliced out, and the fragment S-6 contained, in its 5¢-untranslated region (5¢-UTR), a part of the spliced-out sequence in the fragment L1 (Fig. 2D). It was confirmed that these PCR products had not been originated from contaminated genomic DNAs because they did not contain intron sequences that they have within their genomic sequences (16 introns for PMDAR-L and 15 introns for PMDAR-S). Therefore, we concluded that two mRNAs encoding PMDAR-L (481 a.a. from 17 exons) and PMDAR-S (474 a.a. from 16 exons) are produced in vivo. We then determined the transcription initiation site for PMDAR-L and PMDAR-S using the cap site hunting method (Maruyama and Sugano 1994). Fragments containing each transcription start were amplified from cap site cDNA by Dual targeting of Arabidopsis putative MDAR 701 Fig. 4 Transport of green fluorescent proteins (GFPs) fused to the N-terminal extension of PMDAR-L and PMDAR-S. (A) Schematic representation of the fusion proteins. The linker sequence, a three-times repetition of GlyGlyGlySer, was designed between the N-terminal extension and GFP. These constructs and a control construct (35S-GFP-nosT) were introduced into Arabidopsis epidermal and vascular bundle sheath cells by particle bombardment. (B–F) Fluorescence images of cells having GFP proteins. (B) A cell transformed by the control construct (35S-GFP-nosT), showing green fluorescence in the nucleus and cytoplasm. An arrowhead indicates a gold particle shot into the nucleus. (C) A cell with Signal-LGFP showing green fluorescence in mitochondria. (D) A cell with Signal-S-GFP showing green fluorescence in chloroplasts. (E) Autofluorescence of chloroplasts. (F) A merged image of E and F. Note that the fluorescence of GFP overlapped with that of chloroplasts. The bar indicates 20 mm. nested-PCR with primer sets listed in Fig. 3B. The first PCR was performed with the primer set of R-1 and 1RC that was designed inside the oligonucleotides copied from oligoribonucleotides that were artificially fused to replace the 5¢ cap structure. The PCR product was subjected to nested-PCR with R-2 and 2RC that was also designed inside the oligonucleotides. Although this primer set was expected to amplify two fragments that contained the transcription start of PMDAR-L and PMDAR-S, only a specific fragment that was not seen in the negative control experiment was amplified (Fig. 3C, compare Lanes 3 and 5). Cloning and sequencing of this fragment revealed the transcription start of PMDAR-L. The fragment with the transcription start of PMDAR-S was not amplified with this primer set, probably because of competition for R-2 between PMDAR-L and PMDAR-S cDNA. Therefore, to amplify the fragment with the transcription start of PMDAR-S specifically, we designed the reverse primer (R-3) inside the first intron of the PMDAR-L. This primer amplified a fragment containing the transcription start of PMDAR-S (Fig. 3C, Lane 4). Cloning and sequencing of this fragment revealed that the transcription initiation site of PMDAR-S lies inside the first intron of PMDAR-L, further confirming the existence of the multiple transcripts. In conclusion, two PMDAR mRNAs are produced from a single gene by alternative transcription (Fig. 3E). The N-terminal extension of PMDAR-L and PMDAR-S proteins can transport green fluorescent protein into mitochondria and chloroplast, respectively Next, we examined the subcellular localization of the PMDAR-L and PMDAR-S proteins. We constructed fusion proteins (Fig. 4A) in which the extended N-terminal regions of PMDAR-L and PMDAR-S (Signal-L and Signal-S, respectively) were fused to the green fluorescent protein (GFP) sequence. Constructs for the GFP fusion proteins were introduced into Arabidopsis leaves, and the fluorescence was observed (Fig. 4B–F). Cells expressing Signal-L-GFP exhibited green fluorescence from many small moving compartments (Fig. 4C). The shape and number of the compartments was characteristic of mitochondria. We have confirmed that a 702 Dual targeting of Arabidopsis putative MDAR Fig. 5 A structure of Signal-L. (A) An amino acid sequence of Signal-L. Basic amino acid residues are colored red. Non-polar and polar residues are in green and blue, respectively. The putative mitochondrial presequence cleavage site and a relatively conserved motif are dotted and underlined, respectively. (B) Helical wheel representation of amino acid residues 1–8 of Signal-L. Hydrophobic residues are clustered on one face of the helix whereas basic and polar residues are on the other face. similar fluorescence pattern was obtained by MitoTracker Orange (a mitochondrial-staining dye) staining (data not shown). No green fluorescence was detected from chloroplasts in these cells, suggesting that Signal-L is not an ambiguous targeting sequence that can direct the proteins to both mitochondria and chloroplasts, as is often seen (Creissen et al. 1995, Chow et al. 1997, reviewed in Peeters and Small 2001). On the other hand, green fluorescence of Signal-S-GFP was detected from chloroplasts (Fig. 4D). This result was confirmed by merging the red autofluorescence of chloroplasts (Fig. 4E, F). Cells in which a control construct, 35S-GFP, was introduced, exhibited green fluorescence from the nucleus and cytoplasm (Fig. 4B). These results strongly suggest that two PMDAR isoforms produced by alternative transcription are targeted to different organelles: PMDAR-L to mitochondria and PMDAR-S to chloroplasts. It is also suggested that the first 50 or 43 amino acid residues are sufficient to target this protein to the mitochondria or to the chloroplasts, respectively. Discussion The use of multiple transcription starts causes dual targeting of PMDAR proteins to both mitochondria and chloroplasts Our results show that PMDAR is a dual-targeted protein to mitochondria and chloroplasts, which results from the use of multiple transcription starts. Although most proteins playing the same role in different compartments are encoded by different genes, many exceptions have been reported in all of the eukaryotic systems that have been thoroughly investigated (reviewed in Danpure 1995, Small et al. 1998). These exceptions include the examples of dual targeting of proteins to mitochondria and chloroplasts. A glutathione reductase of pea was reported to be targeted to mitochondria and chloroplasts (Creissen et al. 1995). An Arabidopsis ferrochelatase-I is another example of a protein shown to be imported into both mitochondria and chloroplasts, although this was demonstrated in vitro (Chow et al. 1997). Dual targeting of these proteins is achieved by an ambiguous targeting signal that can be used for targeting both mitochondria and chloroplasts. A spinach protoporphyrinogen oxidase II (Che et al. 2000) was also imported into both mitochondria and chloroplasts (Watanabe et al. 2001). A single mRNA of this enzyme generated two isoforms that differed in length by alternative use of translation initiation codons. These are examples of dual targeting of proteins that arise from a single mRNA. We have shown in this research that at least two mRNAs were generated from a single PMDAR gene by alternative transcription. Therefore, the mechanism of dual targeting of Arabidopsis PMDAR is clearly different from that of the examples enumerated above in that it is regulated, in the case of PMDAR, at the transcriptional level rather than at the translational or post-translational level. The use of multiple transcription initiation sites itself is seen in many genes (reviewed in Danpure 1995). In plants, several genes with multiple ATG at the N terminal region have been reported to use multiple transcription starts (Cheng et al. 1994, Lumbreras et al. 1995, Cunillera et al. 1997, Luo et al. 1997). However, in every case, the relationship between the multiple transcripts and the localization of their products has not been demonstrated clearly. Moreover, none of these is an example of dual targeting of the proteins to mitochondria and chloroplasts. Therefore, to our knowledge, this is the first example of dual targeting to mitochondria and chloroplast that is achieved by the use of multiple transcription starts of a single gene. In Arabidopsis, there was no sequence that is similar to known MDAR other than the seven sequences listed in Fig. 1. Of the seven sequences, the only two sequences, PMDAR-L and PMDAR-S, had the extended N-terminal region and were predicted to be targeted to mitochondria or chloroplasts. Indeed, Signal-L and Signal-S could transport the fused GFP into mitochondria and chloroplasts, respectively (Fig. 4). All the other sequences were expected by the several computer predictions to localize in compartments other than mitochondria and chloroplasts. Therefore, it is strongly suggested that PMDAR-L and PMDAR-S proteins are responsible for the MDAR activities existing in mitochondria and chloroplasts, respectively. However, further analysis will be needed to verify this. PMDAR-S cDNAs were more amplified in the RT-PCR experiment than PMDAR-L. However, this RT-PCR was not Dual targeting of Arabidopsis putative MDAR performed under quantitative conditions, and therefore the result may not reflect the amount of mRNA expressed in vivo. It will be interesting to determine the expression level of PMDAR-L and PMDAR-S in different tissues by the quantitative RT-PCR method and to compare the ratio of PMDAR-L to PMDAR-S between the photosynthetic and non-photosynthetic tissues, because photosynthesis generates a copious amount of ROS in chloroplasts. The N-terminal extended region of PMDAR-L possesses the features of both mitochondrial and chloroplast targeting sequences Signal-L could import the fused GFP into mitochondria whereas Signal-S, shorter by only seven amino acid residues, could import it into chloroplasts. How is it possible to sort the protein into different organelles with a seven amino acid residue extension? Fig. 5A shows the whole amino acid sequence of Signal-L. Signal-L has the features characteristic of a mitochondrial presequence (von Heijne et al. 1989), namely, the absence of acidic residues and enrichment in basic, hydroxylated, and hydrophobic residues. It contains the sequence of RIAS (amino acid residues 45–48) that fits the most commonly reported mitochondrial targeting peptide cleavage motif RX/XS (where X represents any amino acid) (von Heijne et al. 1989, Sjöling and Glase 1998). Moreover, it contains, at amino acid residues 1–8, a potent positively charged amphiphilic ahelix in which hydrophobic residues are clustered on one side of the helix whereas the basic and polar residues lie at the other side (Fig. 5B). Amphiphilic a-helixes have been suggested to be important for the function of mitochondrial targeting sequences. On the other hand, Signal-S has some features of chloroplast transit peptides. It contains three distinct regions that are often seen in stromal-targeting transit peptides (Bruce 2000, Bruce 2001). These are: (1) an uncharged N-terminal domain with Ala following the initial Met; (2) a central domain lacking acidic residues but enriched in hydroxylated residues, Ser and Thr in particular; and (3) a C-terminal domain enriched in Arg. Moreover, Signal-S was predicted to display a random coil structure almost throughout the sequence in an aqueous environment (data not shown). A random coil structure in an aqueous environment is also a typical feature of chloroplast transit peptides (Schmidt et al. 1979, von Heijne and Nishikawa 1991). Therefore, Signal-L possesses some features of both mitochondrial and chloroplast targeting sequences within its amino acid sequence. By fusing the mitochondrial and chloroplast targeting sequences in tandem, Silva Filho et al. (1996) reported that the targeting signal at the more extreme N-terminal position is important in determining the direction of the transport. The amphiphilic a-helix of Signal-L is, therefore, likely to play an essential role in mitochondrial targeting of the protein by having a dominant influence against the downstream chloroplast transit peptide. In the case of spinach protoporphyrinogen oxidase II, the product with the longer extended region was imported into chloroplasts, whereas the 703 shorter isoform was imported into mitochondria (Watanabe et al. 2001). A typical chloroplast transit peptide was found at the most N-terminal region of the longer isoform. Similarly, an Arabidopsis THI1 gene encoding thiamine biosynthetic enzyme generates two isoforms by alternative translation, and the longer isoform, which possesses a chloroplast transit peptide upstream of the mitochondrial presequence, was imported into chloroplasts. The shorter one without the most N-terminal chloroplast transit peptide was targeted to mitochondria (Chabregas et al. 2001). On the basis of our results and these reports, it may be rather common that the most N-terminal targeting sequence exerts a larger influence in the determination of the final location of the protein. Besides Arabidopsis, some MDAR sequences from several plant species are found in the databases. Of these sequences, only one MDAR protein from spinach has the second Met at amino acid residue 8. Interestingly, it also contains the third Met at amino acid residue 11. However, the relationship between the three Met residues and the intracellular localization of the protein is unknown. The other MDAR sequences did not contain the second Met at or around amino acid residue 8. Further accumulation of information about MDAR in various plants will help us understand the evolution of dual targeting of MDAR. Materials and Methods Cloning of PMADR-L and PMDAR-S cDNA cDNA of PMDAR-L and PMDAR-S was cloned by RT-PCR methods. Total RNA was extracted from the above-ground parts of Arabidopsis ecotype Columbia, at 4 weeks after sowing. Poly(A)+ RNA was purified from the total RNA using oligotex-dT30 (JSR, Tokyo). The purified Poly(A)+ RNA was primed with oligo(dT) and then copied into cDNA using reverse transcriptase. The cDNA was subjected to PCR using TaKaRa Extaq HS (TaKaRa, Tokyo) or KOD plus (TOYOBO, Tokyo) as a DNA polymerase, forward primer designed at 5¢-UTR of each mRNA, and the reverse primer designed at the stop codon. Forward primers of PMDAR-S were designed inside the first intron of PMDAR-L to amplify the cDNA of PMDAR-S specifically (see Fig. 2A, B). Amplified fragments were concentrated using Microcon PCR (Millipore, Bedford, MA, U.S.A.) and cloned into pGEM-T Easy vector (Promega, Madison, WI, U.S.A.). Determination of transcription initiation sites Determination of the transcription starts was performed following the methods described in the manual attached to Cap Site cDNA (NIPPON GENE, Tokyo). Cap Site cDNA is composed of cDNAs reverse transcribed from mRNAs 5¢ cap structure of which had been replaced by artificially synthesized oligoribonucleotides (Maruyama and Sugano 1994). Cap Site cDNA of Arabidopsis leaves was obtained from NIPPON GENE and subjected to nested-PCR using primer sets designed at the artificial oligonucleotide region and specific reverse primers (see Fig. 3A, B). TaKaRa Extaq HS (TaKaRa) was used as a DNA polymerase. Nested-PCR products were electrophoresed in 4% Nusieve GTG agarose (BioWhittaker Molecular Applications, Rockland, ME, U.S.A.) and stained with ethidium bromide. Specific fragments that were not amplified in negative control experiments were cloned into pGEM-T Easy vector (Promega) and sequenced. 704 Dual targeting of Arabidopsis putative MDAR Construction of GFP fusion proteins Signal-L and Signal-S were amplified from each cDNA by PCR using KOD plus polymerase (TOYOBO) and primer sets (forward primer for Signal-L: 5¢-GGGTCGACCATGTCTGCAGTTCGTAGAGTC-3¢; forward primer for Signal-S: 5¢-GGGTCGACATGGCGTTAGCATCAACC-3¢; reverse primer for both: 5¢-GGCCATGGACCCCCCCCCGGACCCCCCCCCGGACCCCCCCCCGCTTCTGGAAGCG ATTCTA-3¢) designed to have a linker sequence (three-times repetition of GlyGlyGlySer) and restriction enzyme sites (SalI and NcoI at 5¢ end and 3¢ end, respectively). Amplified products were inserted inframe into the corresponding site of the pTH-2 vector (kind gift from Dr. Niwa, Y., University of Shizuoka; Chiu et al. 1996, Niwa et al. 1999) that contains the CaMV 35S promoter, sGFP (S65T), SalI and NcoI sites between the promoter and sGFP, and the nos-terminal. Introduction of the genes into Arabidopsis The constructs were introduced into cells of Arabidopsis rosette leaves by the particle bombardment method. Rosette leaves at 4 weeks after sowing were cut off and placed gently on a MS plate with the abaxial side up. Gold particles (1.0 mm; Bio-Rad, Hercules, CA, U.S.A.) coated with the constructs were shot to the abaxial side using a particle gun (TANAKA, Hokkaido). Leaves were then incubated on a MS plate with the adaxial side up for one night at 22°C in the dark. Observation After the one-night incubation, leaves were observed under an inverted fluorescence microscope (model IX70, Olympus, Tokyo) equipped with a cooled CCD camera (MicroMAX-1300B, NIPPON ROPER, Chiba). A successful introduction of the plasmids was confirmed by the fluorescence of GFP from some epidermal and vascular bundle sheath cells. Vascular bundle sheath cells expressing fusion proteins were observed further because they have differentiated chloroplasts whereas epidermal cells often do not contain differentiated chloroplasts. GFP was excited using a mercury lamp, an excitation filter (BP470–490, Olympus) and a dichroic mirror (DM505, Olympus). Fluorescence was detected through the dichroic mirror and a band-pass filter (BA515–550, Olympus) allowing the recording of green fluorescence without detecting the red autofluorescence of chloroplasts. Autofluorescence of chloroplasts was detected using an excitation filter (S555/28x, Chroma Technology, Brattleboro, VT, U.S.A.), a dichroic mirror (86100BS, Chroma Technology) and an emission filter (S617/73m, Chroma Technology) to record the red autofluorescence without detecting the fluorescence of GFP. Images were acquired using MetaMorph software (UNIVERSAL IMAGING, Downingtown, PA, U.S.A.). Acknowledgments This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (14036205, 10219201 to H.F.), from the Japan Society for the Promotion of Science (13440236 to H.F. and Research Fellowship for Young Scientists to K.O.), and from the Ministry of Agriculture, Forestry and Fisheries (Gene discovery and elucidation of functions of useful genes in rice genome by gene-expression monitoring system to H.F.). References Asada, K. 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