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• W2034227623 abstract "Alternative splicing events in the 3′-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leafin vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE. Alternative splicing events in the 3′-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leafin vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE. Chloroplasts are potentially a powerful source of oxidants, and sites within the cells are at greatest risk of photooxidative damage (1Asada K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 601-639Google Scholar, 2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Google Scholar). Even under optimal conditions, the process of chloroplastic electron transport produces active oxygen species (AOS) 1The abbreviations used are: AOS, active oxygen species; APX, ascorbate peroxidase; chlAPX, chloroplastic (stromal and thylakoid-bound) APX; sAPX, stromal APX; tAPX, thylakoid-bound APX; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; RT, reverse transcriptase; FW, fresh weight; nt, nucleotide(s); RACE, rapid amplification of cDNA ends; CaMV, cauliflower mosaic virus; SRE, splicing regulatory cis element; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; SR protein, serine/arginine-rich protein. such as superoxide radical, H2O2, and hydroxyl radical. Ascorbate peroxidase (APX, EC 1.11.1.11), which plays an important role in scavenging H2O2, exists as stromal soluble (sAPX) and thylakoid membrane-bound (tAPX) forms in chloroplasts of higher plants (1Asada K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 601-639Google Scholar, 3Shigeoka S. Ishikawa T. Tamoi M. Miyagawa Y. Takeda T. Yabuta Y. Yoshimura K. J. Exp. Bot. 2002; 53: 1305-1319Google Scholar). We have previously demonstrated that, in spinach sAPX, the N-terminal 364 amino acids encoding the transit peptide and the catalytic domains are completely identical with those of tAPX, except for the C-terminal 50 amino acids, and that chloroplastic APX (chlAPX) isoenzymes, which are encoded by a single gene (ApxII), are produced by the alternative splicing of the 3′-terminal region of ApxII pre-mRNA in spinach (4Ishikawa T. Sakai K. Yoshimura K. Takeda T. Shigeoka S. FEBS Lett. 1996; 384: 289-293Google Scholar, 5Ishikawa T. Yoshimura K. Tamoi M. Takeda T. Shigeoka S. Biochem. J. 1997; 328: 795-800Google Scholar). The ApxII contained 13 exons split by 12 introns. The penultimate exon 12 consisted of one codon (Asp-365) for sAPX mRNAs before the termination codon and the entire 3′-untranslated region, including a potential polyadenylation signal. The final exon 13 consisted of the corresponding coding sequence of the thylakoid membrane-spanning domain for tAPX mRNA before the termination codon and the entire 3′-untranslated region, including a potential polyadenylation signal. Similar observations have also been made in pumpkin (6Mano S. Yamaguchi K. Hayashi M. Nishimura M. FEBS Lett. 1997; 413: 21-26Google Scholar) and Mesemnbryanthemum crystallinum(GenBankTM accession numbers AF069315 for tAPX and AF069316for sAPX). At the present time, only Arabidopsis thaliana has been found to contain two different genes for chlAPX isoenzymes (7Jespersen H.M. Kjaersgard I.V.H. Ostergaard L. Welinder K.G. Biochem. J. 1997; 326: 305-310Google Scholar). Alternative splicing is a common mechanism of regulation of gene expression at the post-transcriptional stage in eukaryotic organisms (8McKeown M. Annu. Rev. Cell Biol. 1992; 8: 133-155Google Scholar, 9Simpson G.G. Filipowicz W. Plant Mol. Biol. 1996; 32: 1-41Google Scholar). Some alternative splicing events are constitutive, with similar ratios of mRNA variants in different cells, whereas others are subject to tissue-specific or developmental regulation (8McKeown M. Annu. Rev. Cell Biol. 1992; 8: 133-155Google Scholar). In a few cases in insects and vertebrates, the mechanism underlying alternative splicing events has been elucidated, and cis elements andtrans-acting regulators have been characterized (8McKeown M. Annu. Rev. Cell Biol. 1992; 8: 133-155Google Scholar, 10Moore M.J. Query C.C. Sharp P.A. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-358Google Scholar, 11Valcarel J. Singh R. Green M.R. Pre-mRNA Processing. R. G. Landes Publishers, Georgetown, TX1995: 97-112Google Scholar). In higher plants, a number of cases of alternative splicing have been reported, although the biological significance of those produced isoforms is unknown (9Simpson G.G. Filipowicz W. Plant Mol. Biol. 1996; 32: 1-41Google Scholar). It is likely that a few alternative splicing events are regulated by unique factors specific to plants. The usage of alternative 5′ splice sites in Rubisco activase pre-mRNA produced two protein isoforms that differ only at the C terminus (12Rundle S.J. Zielinski R.E. J. Biol. Chem. 1991; 266: 14802-14807Google Scholar). Although both isoforms can activate Rubisco, only the larger one is redox-regulated. The alternative splicing of the hydroxypyruvate reductase gene in pumpkin, which produces two proteins, peroxisome and cytosol types, is regulated by light (13Mano S. Hayashi M. Nishimura M. Plant J. 1999; 17: 309-320Google Scholar). However, the regulatory mechanisms of alternative splicing are not clear in any of those cases in higher plants. RT-PCR analysis using polyadenylated RNA and polysomal RNA from spinach leaves revealed that four types of chlAPX mRNA variants, one form (tAPX-I) encoding tAPX and three forms (sAPX-I, sAPX-II, and sAPX-III) encoding sAPX, are produced by alternative splicing events, including alternative selection of polyadenylation sites and alternative splicing of introns 11 and 12, of the 3′-terminal region of chlAPX pre-mRNA (see Fig. 1) (5Ishikawa T. Yoshimura K. Tamoi M. Takeda T. Shigeoka S. Biochem. J. 1997; 328: 795-800Google Scholar, 14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). In the mature spinach leaf, the estimated expression ratios of mRNA variants sAPX-I, sAPX-II, sAPX-III, and tAPX-I based on by S1 nuclease analysis were 21:5:32:42 (14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). The levels of enzymatic activity of tAPX and sAPX were almost equal, which was roughly in agreement with the ratio of expression of total sAPX mRNAs to tAPX mRNA (15Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Plant Physiol. 2000; 123: 223-233Google Scholar). However, it seems likely that the relative levels of expression of tAPX and sAPX change according to the plant species, leaf age, and various environmental conditions (15Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Plant Physiol. 2000; 123: 223-233Google Scholar, 16Miyagawa Y. Tamoi M. Shigeoka S. Plant Cell Physiol. 2000; 41: 311-320Google Scholar). Mano et al. (6Mano S. Yamaguchi K. Hayashi M. Nishimura M. FEBS Lett. 1997; 413: 21-26Google Scholar) have reported that the accumulation of sAPX and tAPX isoenzymes in pumpkin cotyledons is differently regulated during germination and the subsequent greening. These observations suggest that alternative splicing events of chlAPX pre-mRNA may be essential for changing the ratios and the amounts of chlAPX isoenzymes. Therefore, a detailed analysis of the regulatory mechanism of the alternative splicing of pre-mRNA of chlAPX isoenzymes should help to elucidate their respective functions. In this study, we analyzed the ratio of expression of chlAPX isoenzymes in several tissues of mature spinach plants and the possible existence of a cis element involved as a regulatory factor in alternative splicing. Spinach seedlings (Spinacia oleracea) were grown in a climate chamber under the following conditions: an 8-h photoperiod, illumination of 300 μE m−2s−1, and 15 ± 2.5 °C for 4 weeks. Tobacco seedlings (Nicotiana tabacum cv. Xanthi) were grown in a climate chamber under the following conditions: a 12-h photoperiod, illumination of 300 μE m−2 s−1, and 25/20 °C (day/night) for 8 weeks. Spinach and tobacco mature plants were collected, frozen immediately in liquid N2, and stored at −80 °C until use in the analyses described in the text. All other chemicals were of the highest purity grade commercially available. Total RNA (30 μg each) was isolated from leaves, stems, and roots (1 g, FW each) of spinach plants as previously described (15Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Plant Physiol. 2000; 123: 223-233Google Scholar), subjected to electrophoresis on 1.2% agarose gel containing 2.2 m formaldehyde, and transferred to a Hybond N membrane (Amersham Biosciences, Buckinghamshire, UK). Prehybridization was carried out at 55 °C for 6 h in a hybridization buffer containing 5× SSC, 5× Denhardt's solution, 1% SDS, and 100 μg ml−1 denatured salmon sperm DNA. The membrane was hybridized at 55 °C for 12 h in the presence of [α-32P]CTP random-primed cDNA probe. As a result of alternative splicing events, four mRNA variants are generated (see Fig. 1). sAPX-II and sAPX-III mRNAs contained a sequence derived from exon 13, including the coding sequence of the membrane-anchoring segment of tAPX as a untranslated region. Furthermore, nucleotide lengths of sAPX-I, -II, -III, and tAPX-I were 1634, 1961, 1860, and 1691 nt, respectively. Therefore, we found it difficult to determine the respective transcript levels of sAPX and tAPX isoenzymes by the Northern blot analysis and detected as the transcript level of chlAPX added together with those of tAPX and sAPX using a tAPX-I cDNA as a probe (15Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Plant Physiol. 2000; 123: 223-233Google Scholar). The membrane was washed twice in 2× SSC and 0.1% SDS for 10 min each at room temperature and in 0.1× SSC and 0.1% SDS at 60 °C for 60 min. The relative levels of chlAPX transcripts were estimated using a Mac BAS 2000 image scanner (Fuji Photofilm, Tokyo, Japan) and were expressed as the mean values from three independent experiments. RNase protection analysis was carried out according to the instruction manual of the RPAII kit (Ambion, Austin, TX). The 3′-terminal region of sAPX-III cDNA was amplified by PCR using a set of primers (sense, 5′-TATGCAGCTGACCAAGAAGCA-3′; antisense, 5′-GGCTTATTAGGGCTACCTCC-3′) and originally cloned into pT7 Blue-T vector (Novagen, Madison, WI). The plasmid DNA was linearized with HindIII. The radioactive antisense RNA probe was synthesized with [α-32P]CTP (Amersham Biosciences) using T7 RNA polymerase (Takara, Kyoto, Japan) and purified by electrophoresis on a 6% polyacrylamide/8 m urea gel. Total RNA was isolated from various tissues of spinach plants as in Northern blot analysis. The RNA probe (about 1 × 105 cpm) was hybridized to 20 μg of spinach total RNA for 12 h. The hybridization mixtures were incubated at 80 °C for 10 min, gradually cooled to 42 °C, and then incubated for 16 h. The mixtures were treated with 0.1 unit of RNase A and 100 units of RNase T1 at 37 °C for 30 min. The protected fragments were separated by electrophoresis on a 3.5% (w/v) polyacrylamide/8 m urea gel. The radioactivity of each protected APX mRNA was estimated using a Mac BAS 2000 image scanner (Fuji Photofilm), and the expression ratios of chlAPX mRNA variants were expressed as the mean values from three independent experiments. Various tissues (0.1 g each, FW) of spinach plants were homogenized with SDS-loading buffer (150 mm Tris-HCl, pH 6.8, 4% SDS, and 10% 2-mercaptoethanol) as previously described (17Leammli U.K. Nature. 1970; 227: 680-685Google Scholar). The homogenates were boiled for 5 min and centrifuged at 10,000 × g for 10 min. The supernatants were analyzed to determine their protein contents and subjected to SDS-PAGE and immunoblotting. SDS-PAGE was performed in 12.5% slab gels according to the method of Laemmli (17Leammli U.K. Nature. 1970; 227: 680-685Google Scholar). The proteins on the gel were stained with Coomassie Brilliant Blue R-250. For immunoblotting, the gel was transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA) using a semidry electroblotting system (model 200/2.0, Bio-Rad) at 15 V for 1 h. The membrane was incubated with monoclonal antibody (chl-mAb6) raised against spinach sAPX, which reacts with both sAPX and tAPX proteins (18Yoshimura K. ishikawa T. Wada K. Takeda T. Kamata Y. Tada T. Nishimura K. Nakano Y. Shigeoka S. Biochim. Biophys. Acta. 2001; 1526: 168-174Google Scholar). The immunocomplexes on the membrane were visualized with alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad). The ratio of expression of sAPX to tAPX proteins was quantified densitometrically and expressed as the mean value from three independent experiments. To amplify a chlAPX-specific partial cDNA fragment from tobacco, degenerate oligonucleotide primers (sense, 5′-TGGCA(C/T)GATGC(C/T)GG(A/T)ACTTA-3′; antisense, 5′-(C/T)TCAT(A/C/G/T)GAATC(C/T)GA(A/C/T)AGCTC-3′) were designed from the common region of the amino acid sequences of tAPX and sAPX from higher plants described previously (4Ishikawa T. Sakai K. Yoshimura K. Takeda T. Shigeoka S. FEBS Lett. 1996; 384: 289-293Google Scholar, 13Mano S. Hayashi M. Nishimura M. Plant J. 1999; 17: 309-320Google Scholar). RT-PCR was carried out with poly(A)+ RNA isolated from tobacco leaves as described previously (14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). 5′ and 3′RACE to amplify the full-length cDNAs encoding tAPX and sAPX were carried out using sets of primers corresponding to 5′-upstream (antisense, 5′-TTGTCTTTGATTGGCTGGAG-3′) and 3′-downstream (sense, 5′-AAACTGAGCAACCTTGGAGC-3′) regions of partial cDNA fragments according to the instruction manuals of commercial kits (5′-RACE System for Rapid Amplification of cDNA Ends, Version 2.0 and 3′-RACE System for Rapid Amplification of cDNA Ends, Invitrogen, Rockville, MD). DNA sequencing was performed by the dideoxy chain primer method with an automatic DNA sequencer (model 310, PerkinElmer Life Sciences/Applied Biosystems, Chiba, Japan). Genomic DNA was isolated from tobacco leaves as described previously (5Ishikawa T. Yoshimura K. Tamoi M. Takeda T. Shigeoka S. Biochem. J. 1997; 328: 795-800Google Scholar). PCR of genomic DNA for isolation of the chlAPX gene was carried out using a set of primers (sense, 5′-CCAGAGAAGTTTGTGGCAGC-3′; antisense, 5′-CTCAGTTTCCAAGCAAAGAT-3′) according to the method of Ishikawa et al. (5Ishikawa T. Yoshimura K. Tamoi M. Takeda T. Shigeoka S. Biochem. J. 1997; 328: 795-800Google Scholar). The constructs for transformation into cultured tobacco cells were based on pBI121 vector derived from Ti plasmid (Toyobo, Osaka, Japan). The 3′-terminal region (encoding exons 10–13) ofApxII, which was involved in the alternative splicing events, was amplified by PCR and was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in a sense orientation. Transformation of tobacco plants by Agrobacterium tumefaciens strain LBA4404 was performed according to the method of Shikanai et al. (19Shikanai T. Takeda T. Yamauchi H. Sano S. Tomizawa K. Yokota A. Shigeoka S. FEBS Lett. 1998; 428: 47-51Google Scholar). To detect the mRNA variants derived from the transgene in extracts from leaves of transgenic tobacco plants, RT-PCR analysis was carried out with poly(A)+ RNA isolated from the leaves of transgenic tobacco as described previously (14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). First-strand cDNA was synthesized using ReverTra Ace (reverse transcriptase, Toyobo) with an oligo(dT) primer. The PCR reactions were performed using sets of primers (for detection the exogenous chlAPX mRNAs/sense, P-1: 5′-TATGCAGCTGACCAAGAAGC-3′ (located in exon 11); antisense, P-2: 5′-CACAAGAAAATAGCTTCATCTTGC (located in exon 12) or P-3: 5′-ACTGCCAAAACTCCAATCACAATC-3′ (located in exon 13); for detection the endogenous chlAPX mRNAs/sense, P-4: 5′-AAACTGAGCAACCTTGGAGC-3′ (located in exon 11); antisense, P-5: 5′-CTCCTTCATTAGGCTACCAA-3′ (located in exon 12) or P-6: 5′- CTCAGTTTCCAAGCAAAGATGTC-3′ (located in exon 13)) under the following conditions: 27 cycles of 1 min at 94 °C, 1 min at 55 °C, and 30 s at 72 °C. The extraction of protein from nuclei was carried out according to Robinson and Hinz (20Robinson D.G. Hinz G. Plant Cell Biology. 2nd Ed. Oxford University Press, New York2001: 295-324Google Scholar) with some modifications. All procedures were carried out at 4 °C. Each tissue (30 g FW) of spinach and tobacco was frozen in liquid N2 and homogenized using a mortar and pestle with 25 mm MES-KOH, pH 6.0, containing 250 mm sucrose, 5 mm EDTA, 10 mm KCl, 0.5 mm DTT, 0.5 mm spermidine, 0.5 mm PMSF, and 0.3% Triton X-100 (buffer 1). The resulting suspensions were filtered through double layers of a nylon filter (50 μm, Advantec, Tokyo, Japan). The filtrates were centrifuged at 3,300 × gfor 10 min, and the pellets (crude nuclear fractions) were resuspended with 30 ml of buffer 1 without Triton X-100. The suspensions were centrifuged at 3,300 × g for 10 min. The pellets, crude nuclear fractions, were resuspended with 25 mmTris-HCl, pH 7.9, containing 5 mm MgCl2, 0.1 mm EDTA, 25% glycerol, 2 mm DTT, and 1 mm PMSF; NaCl was added to a final concentration of 0.5m, and the mixtures were mixed gently for 30 min. After the mixtures were centrifuged at 25,000 × g for 30 min, the supernatants were dialyzed against 3 liters of 20 mmHEPES-KOH, pH 7.9, containing 100 mm KCl, 12.5 mm MgCl2, 0.2 mm EDTA, 20% glycerol, 1 mm PMSF, and 2 mm DTT for 12 h. The dialyzed nuclear protein extracts were then frozen in liquid N2 and stored at −80 °C. The RNA mobility-shift analysis was performed essentially according to Guo et al. (21Guo W. Mulligan G.J. Wormsley S. Helfman D.M. Genes Dev. 1991; 5: 2096-2107Google Scholar) with some modifications. The 162-bp fragment, including the SRE region, and the 184-bp fragment, including the acceptor site of intron 11 in the chlAPX gene of spinach, were amplified by PCR using the following sets of primers (sense, 5′-TTTTAATAAGCAAGATGAAG-3′; antisense, 5′-GGGCTACCTCCAAAACCTTC-3′; and sense, 5′-CCATGACATTGTTAGATTAT-3′; antisense, 5′-TTTTAGATGTGACTGACTAC-3′; respectively) and originally cloned into pT7 Blue-T vector (Novagen). The plasmid DNAs were linearized with EcoRI. [α-32P]CTP-labeled RNA probes were synthesized using T7 RNA polymerase (Takara) and purified by electrophoresis on a 6% polyacrylamide/8 murea gel. RNA-protein binding reactions were assembled by mixing the extract of nuclear protein (5 μg each) prepared from each tissue in a 50-μl final volume with 32P-labeled RNA (about 1 × 105 cpm), 17 mm HEPES-KOH, pH 7.9, 60 mm KCl, 7.5 mm MgCl2, 0.12 mm EDTA, 17% glycerol, 0.6 mm PMSF, 1.2 mm DTT, 10 units of RNasin, and 5 μg of yeast tRNA. The reaction mixtures were incubated for 30 min and then electrophoresed on 3.5% (w/v) polyacrylamide gels. The RNAs in the gels were visualized with a Mac BAS 2000 image scanner (Fuji Photofilm). Mutation of the SRE region was carried out according to the instruction manual of the QuikChange site-directed mutagenesis kit (Stratagene, CA). The mutagenic PCR was performed using PfuTurbo TM DNA polymerase, the mutagenic primer sets (M1, 5′-GTGTTGTGTTGCTATAAAGTTAAAGCAG-3′/5′-CTGCTTTAACTTTATAGCAACACAACAC-3′; M2, 5′-AAACGAGTGTGTA-GAGAAGCATATTTGAATTTGCAG-3′/5′-CTGCAAATTCAAATATGCTTCTCTACACA-CTCGTTT-3′; and M3, 5′-ACGAGTGTGTTGT-GAAGCAATAAAGAATTTGCAG-3′/5′-CTGCAAATTCTTTATTGCTTCACAACACACTCGT-3′), and the 3′-terminal region of ApxII was cloned into pT7 Blue-T vector (Novagen) as a template. The PCR conditions were as follows: 18 cycles of 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 7 min. The amplified fragment was transformed intoEscherichia coli strain DH5α cells, and a clone of the candidate mutant obtained thereby was isolated and sequenced. To determine whether the transcription level of ApxII varied in several tissues, we analyzed the steady-state transcript levels of total chlAPX mRNA, which were expressed as the sum of the four types of mRNA variants encoding sAPX and tAPX, in the leaves, stems, and roots of mature spinach by Northern blotting using tAPX cDNA as a probe. Interestingly, the chlAPX mRNA variants were expressed at almost equal levels in the leaves, stems, and roots of mature plants (see Fig. 2). Four types of chlAPX mRNA variants, tAPX-I, encoding tAPX, and sAPX-I, -II, and -III, encoding sAPX, were produced by alternative splicing of the 3′-terminal region of chlAPX pre-mRNA (Fig. 1) (14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). Although no difference in the total transcription levels of chlAPX in the various tissues was detected (Fig. 2), it is possible that the efficiency of alternative splicing could have been modulated in each tissue. To examine this possibility, we analyzed the relative levels of expression of four types of mRNA variants in the leaves, stems, and roots of mature spinach plants by RNase protection analysis (Fig. 3). The relative expression ratio of the three types of sAPX mRNAs combined (sAPX-I, -II, and -III) to tAPX mRNA were extremely different depending on the tissue. In leaf, the ratios of sAPX mRNAs compared with tAPX mRNA was about 1 (Fig. 3 C), which was in agreement with the previous result (14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). In contrast to leaf, those of sAPX mRNAs to tAPX mRNA in stem and root were strongly elevated. The elevation of those ratios in both tissues was caused by an increase in sAPX-III and the decrease in tAPX-I. The ratios of sAPX-I and sAPX-II were not altered. Immunoblotting analysis using the monoclonal antibody (chl-mAb6), which reacts specifically with chlAPX isoenzymes in higher plants (18Yoshimura K. ishikawa T. Wada K. Takeda T. Kamata Y. Tada T. Nishimura K. Nakano Y. Shigeoka S. Biochim. Biophys. Acta. 2001; 1526: 168-174Google Scholar), showed that the ratios of the expression of sAPX to tAPX in each tissue nearly correlated with the ratios of levels of the respective mRNA variants (Fig. 4). These results clearly indicate that the alternative splicing regulates the relative levels of expression of chlAPX isoenzymes in a tissue-specific manner. To analyze the regulatory mechanism of alternative splicing in higher plants, it is necessary to utilize transgenic technology for the in vivo assay system for splicing. Thus, we isolated the cDNAs and genomic DNA fragment encoding chlAPX isoenzymes from tobacco plants that are suitable for transgenic techniques. Four cDNA clones were obtained by RT-PCR analysis and the 5′/3′RACE technique. Comparison of the nucleotide sequences of these clones with the deduced amino acid sequences of spinach chlAPX clearly showed that one of them encoded tAPX and the others encoded the sAPX isoenzymes of tobacco (tAPX; AB022273, sAPX; AB022274) (data not shown). Partial genomic DNA fragments containing 3′-terminal regions of chlAPX isoenzymes were then amplified by genomic PCR and sequenced. By alignment with the previously isolated cDNAs for tobacco chlAPX isoenzymes, the fragment was found to contain three exons corresponding to the 3′-terminal region of chlAPX genes from spinach and pumpkin (Fig. 5). The first exon in the fragment encoding the common sequence of tAPX and sAPX corresponded to exon 11 of spinach ApxII. The second exon, corresponding to the penultimate exon 12 of spinach ApxII, consisted of one codon for Asp before the TAA termination codon and the entire 3′-untranslated region of the sAPX mRNA. The third exon contained the sequence encoding the C-terminal extension with a predicted transmembrane domain, the TGA termination codon, and the entire 3′-untranslated region for tAPX mRNA. These results show that chlAPX isoenzymes in tobacco are produced by the alternative splicing of the pre-mRNA, as are those of spinach ApxII, which is in agreement with the results found in spinach and pumpkin (5Ishikawa T. Yoshimura K. Tamoi M. Takeda T. Shigeoka S. Biochem. J. 1997; 328: 795-800Google Scholar, 6Mano S. Yamaguchi K. Hayashi M. Nishimura M. FEBS Lett. 1997; 413: 21-26Google Scholar, 14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). Thus, it is clear that the tobacco plant becomes a good tool for the in vivoanalysis of the alternative splicing mechanism as described below. The significant structural differences among the four spliced mRNA variants of chlAPX are associated with the alternative splicing of the 3′-terminal region consisting of intron 11 to exon 13 of the gene (ApxII). This view suggests that an essential regulatory factor such as a cis element for alternative splicing is present within the 3′-terminal region. Fig. 5 shows a comparison of the nucleotide sequences of the 3′-terminal regions (exons 11 to 13) of the chlAPX genes in spinach (5Ishikawa T. Yoshimura K. Tamoi M. Takeda T. Shigeoka S. Biochem. J. 1997; 328: 795-800Google Scholar), tobacco (this study), and pumpkin (6Mano S. Yamaguchi K. Hayashi M. Nishimura M. FEBS Lett. 1997; 413: 21-26Google Scholar). Interestingly, we found a common sequence with a high homology in introns 11 and 12 among the three chlAPX genes. On the other hand, the other upstream introns (introns 1 through 10), which were spliced out constitutively, showed no significant homology (data not shown). Furthermore, the region immediately upstream of the acceptor site in intron 12 was highly conserved in the three genes. These facts suggest that the common region may be a ciselement involved in regulating the tissue-specific alternative splicing, and thus we designated this region as a putative splicing regulatory element (SRE, bold line in Fig. 5). It seemed likely that the SRE region acts as a cis element that regulates the alternative splicing of chlAPX isoenzymes. Therefore, the effect of SRE on the efficiency of the alternative splice site selection in vivo was investigated using transformed tobacco plants. The truncated 3′-terminal regions of spinach ApxIIwere placed under the control of the CaMV 35S promoter of pBI121 vector (pBI/chlAPX3′SRE, Fig. 6 A). The mutant constructs (pBI/chlAPX3′ΔSRE and pBI/chlAPX3′mutSRE), which deleted the middle 10 bases of the SRE sequence and partially substituted the SRE sequence, respectively, in pBI/chlAPX3′SRE, were also prepared. However, the sequence of the acceptor site of the intron 12 (-GAATTTGCAG/AGATCA-) remained in these mutants, because the SRE was close to this site. The transgenes were transformed into tobacco plants by the A. tumefaciens infection method (19Shikanai T. Takeda T. Yamauchi H. Sano S. Tomizawa K. Yokota A. Shigeoka S. FEBS Lett. 1998; 428: 47-51Google Scholar). The transgenic tobacco plants were selected, and the splicing efficiency of chlAPX pre-mRNA derived from the transgene in the leaf was analyzed by RT-PCR as described previously (14Yoshimura K. Yabuta Y. Tamoi M. Ishikawa T. Shigeoka S. Biochem. J. 1999; 338: 41-48Google Scholar). No amplified band was detected in tobacco transformed with the control vector (pBI/GUS, β-glucuronidase) (Fig. 6 B). On the other hand, many amplified bands, which were in agreement with the findings in spinach plants as a control, were detected in the leaf of pBI/chlAPX3′SRE-transformed tobacco plants. By the DNA sequencing analysis, these bands were found to correspond to the four mRNA variants (sAPX-I, -II, -III, and tAPX-I mRNAs) derived from the transgene, although large amounts of unspliced pre-mRNAs were also detected (Fig. 6 B). This result suggests that the processing of the truncated pre-mRNA by the mRNA processing system occurs, including the alternative" @default.
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• W2034227623 title "Identification of a cis Element for Tissue-specific Alternative Splicing of Chloroplast Ascorbate Peroxidase Pre-mRNA in Higher Plants" @default.
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