Plant Cell Reports (1994) 13:361-366 Plant Cell Reports �9 Springer-Verlag 1994

Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots

Hiroyuki I t o 1, Fusao Kimizuka 2, Akira Ohbayashi 2, H i r o k a z u M a t s u i 1, Mamoru Honma 1, A t s u h i k o S h i n m y o 3, Y u k o O h a s h i 4, A l a n B . C a p l a n 5, and Raymond L. Rodriguez 6

1 Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan 2 Bioproducts Development Center, Takara Shuzo Co., Ltd., Seta 3-4-1, Otsu, Shiga 520-21, Japan 3 Department of Biotechnology, Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan 4 Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba Science City, Ibaraki 305, Japan s Laboratorium voor Genetica, Rijksuniversiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium 6 Department of Genetics, University of California, Davis, CA 95616, USA

Received 30 June 1993/Revised version received 2 February 1994 - Communicated by A. Komamine

Abstract. PCR with oligonucleotide primers that corresponded to two highly homologous regions, in terms of amino acid sequence, of plant peroxidases was used to amplify a specific DNA fragment from a mixture of rice (Oryza sativa L.) cDNAs. We then screened a e D N A library prepared from mRNAs of rice shoots utilizing the product of PCR as probe. Two eDNA clones, prxRPA and prxRPN, were isolated. They encode dist inct i sozymes of peroxidase. Sequence analysis indicated that the clones encode mature proteins of approximately 32 kDa, both of which possess a putative signal peptide. Comparison of the amino acid sequences of the two rice peroxidases showed that they are about 70% similar to each other but are only 40% to 50% similar to other plant peroxidases. RNA blot hybridization revealed that mRNAs that corresponded to prxRPA and prxRPN cDNAs accumulate at high levels in roots but only at low levels in stems and leaves. In various tissues of rice plants, levels of both mRNAs were stimulated by wounding and by ethephon. These results indicate that at least two isozymes of peroxidase are expressed not only in shoots but also in roots of rice plants, and that the expression of these genes is influenced by ethylene which is the simplest plant hormone.

Introduction

Plant peroxidases [EC 1.11.1.7] are oxidoreductases that catalyze the oxidation of various proton donors by It202. Each has a protoheme IX moiety as a prosthetic group. Some molecular aspects of plant peroxidases have been reviewed by van Huystee (1987). Most higher plants possess a number of different isozymes a n d / o r i s o f o r m s o f p e r o x i d a s e . These i s o z y m e s can be distinguished by their isoelectric points (pI) or elution profiles during ion-exchange column chromatography and can be divided into three subgroups: anionic, neutral, and cationic peroxidases (van H u y s t e e 1987, S h i n m y o 1988). Many c D N A s and chromosomal DNAs encoding peroxidase isozymes have been isolated and cloned from dicotyledonous plants (Lagrimini et al. 1987, Roberts et al. 1988, Fuj iyama et al. 1988, Roberts and Kolattukudy 1989, Intapruk et al. 1990, Fujiyama et al. 1990, Buffard et al. 1990). In the case of monocotyledns, two genes for peroxidases were reported in barley (Rasmussen et al. 1991) and wheat (Hertig et al. 1991, Rebman et al. 1991). Analysis of the structures of these genes revealed that plant peroxidases consist of about 300 amino acid residues and that there are several highly conserved regions , which inc lude two heme-b ind ing , His- containing regions.

Peroxidases in plants are involved in several physiological functions, for example, wound-healing (Lagrimini and Rothstein 1987, Bowles 1990), b iosynthes is of cell wall (Negre l and

Correspondence to: H. Ito

Lherminier 1987) including lignification and suberization of the cell wall (Espelie et al. 1986, Lagrimini et al. 1987), organogenesis (Kay and Basile 1987), growth regulation (Zheng and van Huystee 1992), auxin catabolism (Hinnman and Lang 1965), and senescence (Abeles et al. 1988). Some of the isozymes are inducible by wounding (Svalheim and Robertsen 1990, Mohan et al. 1993), treatment with ethylene (Morgens et al. 1990) and some pathogens (Sheen and Diachun 1978, Hammerschmidt et al. 1982). Some o f t h e m a r e a l s o i m p l i c a t e d in t he o x i d a t i o n o f 1- aminocyclopropane-l-carboxylic acid (ACC)(Acosta et al. 1992), which is an intermediate in the biosynthesis of ethylene. However, the relationships between the isozymes and their physiological roles, as well as the cellular localization of each isozyme, are not fully understood. Cloning of peroxidase genes and construction of new transgenic plants with cloned peroxidase genes or antisense genes might help us to characterize the functions of peroxidase isozymes.

We previously reported the fractionation and isolation of peroxidase isozymes from rice (Oryza sativa L.) shoots and the existence of almost 25 isozymes/isoforms in the shoots (Ito et al. 1990). Recently, Mittal and Dubey (1991) reported the bchaviour of isoforms of rice peroxidase in relation to salt tolerance. In this report, we describe the cloning of two cDNAs that encode putative peroxidases from rice shoots, using a probe synthesized by amplification by PCR of a DNA fragment whose sequence was based on two highly conserved regions of other plant peroxidases. The amino acid sequences, deduced from the two eDNA sequences, were compared with those of other peroxidases. We also show that levels of the mRNAs for the two peroxidases increased in plants after exposure to stress.

Materials and Methods

Plant materials and stress treatments. Rice (Oryza sativa L. cv. Nipponbare) seedlings were grown for 25 days after sowing at 25 ~ in a temperature-controlled greenhouse. Harvested shoots were stored at -80~ until they were used for construction of a eDNA library and for extraction of total DNA. A line of suspension-cultured cells was derived from rice embryos of the same cultivar and cultured in A.A. medium ( M u l l e r and Grafe 1978) that con ta ined I mg /L of 2,4- dichlorophenoxyacetie acid, 0.2 mg/L of 6-furferylaminopurine, and 0.1 mg/L of gibberellin A3. The cells were grown in 50 ml of medium in a 200-ml Erlenmeyer flask at 25~ with 16 h of light per day and they were maintained by transfer of 5 ml of a 7-day-old culture to 50 ml of fresh medium. Cells were harvested 7 and 14 days after transfer and stored at -80*(2 until use.

In experiments to examine the developmental and tissue-specific

362

accumulation of transcripts of peroxidase genes, rice plants were grown in a growth chamber at 25"C with a 16 h of light per day. Seven, 14, and 21 days after germination, shoots, leaves, stems, and roots were harvested separately.

In experiments to examine the induction of peroxidase mRNAs in various tissues, 21-day-old plants were treated by spraying of leaves and soil with 0.01% HzC h, 50 mM salicylic acid, or 1 mM 2-ehloroethyl- phosphonic acid (ethephon). They were also wounded by rubbing the leaves with sea sand. Various tissues were harvested 2 days (48 h) after such treatment.

Enzymes and chemicals. Restriction endonueleascs, modification enzymes, and 7-deaza-sequeneing, kilo-sequence deletion, Megalabel, random primer labeling, BcaBEST labeling, and ligation kits were products of Takara Shuzo (Kyoto, Japan) and were used in accordance with the manufacturer's instructions. [~.32p] dCTP (14.7 and 110 TBq/mmol) was from Amersbam Japan (Tokyo, Japan). All other chemicals were of reagent grade.

Construction of a eDNA library. Rice shoots (30 g) were powdered with a homogenizer in liquid nitrogen. Total RNA was isolated from the powdered shoots by guanidinium/cesium chloride centrifugation (Sambrook et al. 1989). Poly(A)-enriched RNA was prepared from total RNA using OligoTex TM-dT30 (Takara). Doable-stranded eDNA was synthesized from poly(A)-enriched RNA by an oligo(dT) priming method (Gubler and Hoffman 1983). First-strand DNA was synthesized using an oligo(dT)12.a8 primer and avian myeloblastosis virus reverse lrangcriptase XL. Second-strand synthesis was performed using RNasc H and DNA polymerase I ofEscherichia coli. Blunt-ended eDNA was generated by treatment with T4 DNA polymerase. The double-stranded eDNA was ligated with EcoRI-SmaI adaptors (5'-ouAATI'CCCGGG-3' and 5'-pCCCGGG-3') using a ligation kit and it was phosphorylated by T4 polynucleotide kinase, dsDNA containing EcoRI cohesive termini was inserted into the unique EcoRI site of the ~,gtll phage vector (Stratagene Cloning Systems, CA, USA). The recombinant DNAs were packaged into bacteriophage particles using packaging extract (Stratagene) and grown on E. coli Y1090,

Polymerase chain reaction (PER). There are at least four highly conserved regions in the amino acid sequences of plant mature peroxidases (Fig. 3, boxed). The His residue in region I (Fig. 3) is predicted to be involved in acid/base catalysis as an active site (AS), while the His residue in region III (Fig. 3) is predicted to be the 5th ligand of heme (heine-binding site, HBS). Two primers, named AS and HBS; were synthesized using a DNA synthesizer (380B DNA Synthesizer; Applied Biosystem Ine, CA, USA). The AS primer [5" CA(T/C)TT CCAff/C)G ACTGC TT(T/C)GT IAAT-3'] and the HBS primer (3'-CCTCI IGTGT GCTAG CCTGC-5') corresponded to the overlining indicated by a and b, respectively, in Figure. 1. Amplification by PCR was performed in a DNA Thermal Cycler (Perkin Elmer Cetus Inc., CT, USA). The amplification reaction mixture of 100 IXl consisted of 0.5 lag eDNA, 20 pmol of the AS primer, 20 pmol of the HBS primer, 0.02% gelatin, I mM each dNTP, and 2.5 U AmpliTaqTM polymerase in Taq buffer [10 mM Tris-HC1 buffer (pH 8.3) containing 50 mM KCI and 1.5 mM MgClz]. The DNA-denaturation step was set at 94"C for 1 min, the primer-annealing step at 42"C for 2 min, and the primer-extension step at 72"C for 2 min. The reaction was allowed to run for 30 cycles. The product of PCR was fraetionated on a 1% Seakem GTG agarose gel (FMC BioProducts, ME, USA) and desired products were purified from the gel. Blunt-ended products were generated using a blunting kit and subeloned into the Hinell site of the pUC18 vector. A probe (the AS-HBS probe) for screening was prepared from the recombinant plasmids.

Screening by plaque hybridization. The eDNA library in ~gtll was screened with the AS-HBS probe. The AS-HBS probe was labeled with 1.85 MBt] of [ct-~P]dCTP using a random primer labeling kit. About 2.5 x 10 plaques of the library were transferred onto duplicate nylon membranes (HybondN +; Amersham). The membranes were treated with the 32p-labeled AS-HBS probe in hybridization buffer [6x SSC

solution (lx SSC: 0.15 M NaC1 and 0.015 M sodium citrate), 5x Denhardt 's solution (Ix Denhardt's: 0.02% Fieoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin), 0.1% SDS, and 0.1 mg/ml salmon testes DNA] at 55"C. They were rinsed with 2x SSC and 0.1% SDS at 55"C for 30 min and then with 0.1x SSC and 0.5% SDS at 55"C for 30 min. These membranes were exposed to X-Omat AR film (Kodak, CT, USA) for aatoradiography with an enhancer screen (Lighting Plus; Dupont, DE, USA).

Analysis of nucleotide sequences, Nucleotides sequencing was performed by the dideoxy chain-termination method (Sanger et al. 1977), but 7-deaza-dGTP was used instead of dGTP. Various fragments (0.2-0.4 kb) of the putative genes for pemxidase were prepared from the subeloned plasmids and cloned into appropriate sites of the vectors M13mp18 or mp19 to provide templates. Alternatively, deletion derivatives were generated from the subelones using a kilo-sequence deletion kit. The sequences were deduced from the results for both strands. The DNA sequences and the predicted amino acid sequences were analyzed using the Gene~otein sequence database of a DNASIS computer (Hitachi, Japan). The complete nueleotide sequences of the eDNAs for the rice peroxidases, prxRPA and prxRPN, have been submitted to the DDBJ, EMBL and GenBank data bases under the accession numbers D14481 and D14482, respectively.

DNA blot hybridization analysis. Total DNA was extracted from 25-day-old rice shoots by the method of Murray and Thompson (1980). Samples of DNA (5 ~tg) were digested with various restriction endonucleases, fraetionated by electrophoresis on a 0.7% agarose gel, and blotted onto a nylon membrane which was then probed with [c~- 3zP]dCTP-labeled eDNAs for rice peroxidase, prxRPA and prxRPN.

32. These cDNAs were labeled with 1.85 MBq of let- P]dCTP using BeaBest and random primer labeling kits. Each blot was washed twice with 0.1x SSC and 0.5% SDS at 65 or 70"C for 1 h. The membranes were exposed to film for autoradiography with an enhancer screen for a short (3 h) time or a longer time (1411) at -80"C.

RNA blot hybridization analysis. Total RNA was isolated from various tissues by the phenol/SDS method (Ausubel et al. 1987), fractionated on a 1% formaldehyde agarose gel (Sambrook et al. 1989) and Iransferred to a nylon membrane. The blot was probed with 32p-labeled peroxidase cDNAs, prxRPA and prxRPN, and washed twice with 0.1x SSC and 0.5% SDS at 65"C for 0.5 h. The 32p-labeled probes were prepared with 1.85 MBq of [ct-32P]dCTP using a BeaBest labeling kit. The filters were subjected to autoradingraphy for 12 h or 48 h with intensifying screens at -80"C. The relative intensity of the bands on autoradiograms was determined densitometrically with a dual-wavelength scanner (CS- 9000; Shimadzu, Kyoto, Japan).

Results and Discussion

Isolation of putative eDNA clones for peroxidase. D NA fragments of about 400 bp were amplified from a mixture of rice shoot eDNA by PCR with the AS and HBS primers. The products of PCR were heterogeneous, as expected since there should be several different cDNAs that encode rice peroxidases with highly homologous sequences. The amplified fragments were subeloned into the pUC18 vector and one subclone was selected and analyzed. From the nucleotide and deduced amino acid sequences, we concluded that the inserted fragment corresponded to regions I and III of rice peroxidase (data not shown). Therefore, this subcloned fragment was used as a probe to screen the eDNA library; it was named the AS-HBS probe.

A eDNA library consisting of approximately 250,000 recombinant phages was screened with the 32p-labeled AS-HBS probe and 12 positive clones were selected. These clones were plaque-purified and phage DNAs were purified for subeloning and analysis of inserted fragments. From the restriction maps, the positive clones were appeared to consist of members of two groups, designated ~.I and ~.II: 11 of 12 positive clones were in

363

the kI group and only one clone was in the ~.II group.

Nucleotide sequence analysis. The cDNAs inserted in one member each of the ~.I and ~.II groups, designated prxRPA and prxRPN cDNAs, were subcloned into pUC18 and M13mpl8 vectors and the nucleotides were sequenced as described in "Materials and Methods". The complete nucleotide sequences of prxRPA eDNA (1,204 bp) and prxRPN eDNA (1,348 bp) are shown in Figure. 1. The sequence of the AS-HBS probe corresponded exactly to the regions overlined and marked a and b in prxRPA eDNA. The amino acid sequences of this region are more than 50% homologous among 10 plant peroxidases, as shown in Figure. 3. mRNAs corresponding to the two clones both have one of the typical polyadenylation signals of eukaryotic mRNA, AGUAAA. This sequence is located 174 nucleotides before the addition of poly(A) in prxRPA mRNA and 35 nucleotides before the po ly(A) in prxRPN mRNA (Fig. 1, double underlined sequences), on the basis of the results of Sheets et al. (1990). We assume that the different distances between the signal and poly(A) are involved in the differential regulation of the gene expression and turnover of the mRNAs.

Figure 2 shows the deduced amino acid sequences of the open reading frames (ORFs) of both eDNAs. It is predicted from these sequences that the proteins (designated RPA and RPN) are synthesized as preproteins of Mr 35,611 (326 amino acids) and 35,149 (335 amino acids), with hydrophobic putative signal sequences of 27 and 33 residues, respectively. The predicted amino acid sequences of RPA and RPN are 70% homologous to one another. The theoretical pI Values of mature RPA and RPN are 4.7 and 5.8, respectively. The numbers of acidic (Glu and Asp) and basic amino acids (His, Lys, and Arg) are 40 and 28 for mature RPA and 28 and 29 for mature RPN. These results indicate that RPA is an acidic and RPN is a neutral isozyme. Although there are at least 25 peroxidase isozymes and/or isoforms in rice shoots (Ito et al. 1990), we isolated only two cDNAs in this study. Both predicted proteins are found in the pattern of rice shoot isozyme as minor bands (data not shown), as judged from their theoretical pI values. However, their actual pI values might differ from the theoretical values because of glycosidic side chains.

Therefore, we cannot accurately identify the deduced proteins in dee shoots.

Putative sites of N-glycosylation were found at positions 154, 236, and 307 of RPA and at positions 101, 160, 188, 215, and 316 of RPN (Fig. 2, Asn residues marked with asterisks). From the different numbers and positions of the sites, it seems likely that RPA might differ from RPN in both cellular location and physiological function.

The GC contents of coding regions are 54% for prxRPA

1 ) RPA b~YSYSYRFM LVCSVLV LCLNTRGARCQLSDDFYDYICPDVYTV 44

2) RPN ---ATRGDRTASCLSFL-NIV-LLG-AAAAGSG--T--y---C--Q--RI 50

I) RPA VQQBA"ZAAI~%TEMRMGASLLRLHFHDCFVNGCDGSILLDGDDGEKFALPN 94

2) RPN -RSR-A---KA ...................... A ...... TNS .... A-- 100

i) RPA KNSVRGFEVIDAI~DLENICPEVVSCADIVALAAGYGVLFSGGPYYDVL 144

2) RPN N ..... Y ....... A---GA--G ............ K .... L .... D .... 150

i) RPA LGRRDGLVANQSGADNG LPSPFEPIKSIIQKFNDVGL~GGH 193

2) RPN ........... T---SN ..... DS-SV-TAR-K .... NA ........ A- 199

i) RPA TIGRARCTLFSNRL STTSSSAI~TLDATMAANLQSLCAGGDGNETTV 240

2) RPN .... S--L ...... ANF-A-N-V ..... SSL-SS--QV-R--ADQLAA 247

I) RPA LDITSAYVFDNRYYQNLLNQKGLLSSDQGLFSS DDGI ANTKELVETYS 288

2) RPN --VN--DA---H ...... AN .... A ..... V--SG-PAV-A--A--QA-- 297

i) RPA ADAHKFFWDFGRSMVKMGNISPLTGDDGQIRKNCRVV~ 326

2) RPN -NGQR-SC---N ............. SA ........ A-- 335

Fig. 2. Amino acid sequences predicted from the nucleotide sequences of the two cDNAs. The sequences have been aligned to maximize sequence similarity. Potential sites of N-glycosylation are marked with asterisks. The putative N-terminus is indicated by an arrow. 1) and 2) show the amino acid sequences of RPA and RPN. The bars indicate the same amino acid residues as those in RPA and only the residues of RPN different from those of RPA are shown. The gaps have been introduced to maximize alignment.

i) prXRPA 2) prxRPN

1) prxRPA 2) prXRPN

1) prxRPA 2) prxRPN

1) prxRPA 2) prxRPN

1) prxRPA 2) prXRPN

1) pEXRPA 2) prxRPN

1) prxRPA 2) prXRPN

1) prxRPA 2) prxRPN

1) prxRPA 2) prxRPN

1) pEXRPA 2) prxRPN

1) prxRPA 2} prXRPN

2) prxRPN

tgtgagagATGGAGTACTCTTACAGCTACAGGTTCATG CTTGTATGCTCTGTTCTTGTA CTGTGCCTTAATACTCGGGGTGCGAGATGCCAGTTATCCGACG 102 gccATGGAGTACGCTACTCGTGGAGATCGCACGGCTAGCTGCCTGAGTTTCCTCTGCAATATCGTCGTTCTGCTGGGCCTCGCCGCCGCGGCGGGCAGCGGGCAGCTGACGGACG 115

(a)

ATTTcTACGACTACATATGTCCTGATGTGTACACCGTTGTCCAGCAGCATGTTTATGCTGCCATGAGGACTGAGATGAGGATGGGTGCCTCCCTCCTAAGGCTCCATTTCCATGACTGCT 222 ACTACTACGACTATTGCTGcCCCCAGGTTTACCGCATCGTCCGGTCCCGCGTGGCCGCCGCGATGAAGGCCGAGATGCGCATGGGCGCCTCCCTGCTCAGGCTTCACTTCCACGACTGCT 235

TTGTCAATGGGTGTGACGGTTCCATCCTTCTGGACGGTGACGACGGCGAGAAATTTGCACTTCCCAACAAGACCTCTGTCAGAGGGTTCGAAGTCATTGACGCGATAAAGGAAGATCTCG 342 TCGTCAATGGCTGTGACGCGTCCATCCTCCTTGACGGCACAAACAGCGAGAAGTTTGCACTTCCCAACAAGAACTCGGTGAGAGGGTACGAAGTCATCGATGCGATAAAGGCCGACCTCG 355

AGAACATCTGCCCTGAAGTTGTTTCCTGCGCCGACATTGTAGCCCTTGCAGCTGGCTATGGAGTACTATTTAGTGGAGGCCCTTACTATGACGTTCTTCTCGGTAGAAGGGATGGTCTTG 462 AGGGCGCCTGCCCGGGAGTCGTCTCCTGCGCCGACATAGTAGCCCTTGCAGCTAAATACGGAGTACTACTTAGTGGAGGACCTGATTATGATGTCCTCCTGGGAAGAAGAGATGGTCTGG 475

cD TCGCAAATCAATCAGGAGCTGACAACGGC CTCCCTTCACCGTTCGAACCCATCAAATCGATCATACAGAAGTTCAATGATGTCGGCCTCGACACAACCGATGTTGTCGTCCTATCAG 579 TGGCAAATCAGACGGGGGCG AACAGTAACTTGCCTAGCCCTTTCGATTCGATCAGCGTTATCACTGCGAGGTTCAAGGATGTCGGTCTCAACGCAACCGATGTTGTGGTCTTATCAG 592

GAGGGCACACGATCGGACGAGCCCGGTGCACGCTGTTCAGCAACCGGTTG TCGACCACCTCAAGCTCAGCCGACCCGACGCTGGACGCCACCATGGCCGCCAACCTCCAGA 690 GGGCGCACACGATCGGGCGATCTCGCTGCCTGCTGTTCAGCAACCGGCTGGCGAACTTCTCGGCGACCAACTCCGTC GACCCGACGCTGGACTCGTCGCTGGCGTCCAGCCTGCAGC 709

GCCTCTGTGCCGGTGGAGACGGCAACGAGACCACCGTGCTGGACATCACCTCCGCCTACGTTTTCGACAACCGCTACTACCAGAACCTCCTCAATCAGAAAGGCCTCCTGTCCTCCGACC 810 AGGTGTGCCGCGGCGGCGCTGACCAGCTGGCGGCG CTGGACGTCAACTCCGCCGACGCGTTCGACAACCACTACTACCAGAACCTGCTGGCCAACAAGGGCCTCCTCGCCTCCGACC 826

AGGGCCTCTTCTCCAGCGAC GACGGCATC GCCAACACCAAGGAGCTGGTGGAGACTTACAGCGCAGATGCCCACAAGTTCTTCTGGGATTTTGGCAGATCCATGGTGAAGATGG 924 AGGGCCTCGTCTCCAGCTCCGGCGACCCCGCCGTCGCCGCCACCAAGGCGCTGGTGCAGGCCTACAGCGCCAATGGCCAGCGCTTCTCCTGCGACTTCGGCAACTCCATGGTCAAGATGG 946

GCAACATCAGCCCACTCACCGGTGACGACGGCCAGATTCGCAAGAACTGCAGGGTTGTTAATTAActgagcttcagtgtgttgaaaagataqtaaatttcttgtacttttcacaaggcga 1044 GCAACATCAGCCCTCTCACCGGCTCTGCCGGCCAGATTCGCAAGAACTGCAGGGCCGTCAACTGAtgagcaaaaaggcaaagattttttgca-~ccatgaccccatcttggatttgcc 1066

ttgagacgatgtgtgttatgttttgatgttacatgaatgcttgaagaagcagaagtaataacgatgtgattgtgactagtttgtgaactgatcgtcaacaaagatgtgatccattggaaa 1164 agaagctatatcgtcttcttggactcaagtgtgaatctgtcgtttttaatgtgttgtggagcgctacatgtttcgttttgttcaagctatctaggattctctCtccaatgcacgagtaga 1186

aaaagaactgaatgtatgtgccttaaaaaaaaaaaaaaaa 1204 ataagcaattagcatgcaaagttgctcgtcctacacgaatctgcagctgcattttcagtgggtgtatcaccaattattagagcacagacaaacgcgctgtgtctatcaga~taaaagaaa 1306

gaatatgcaactgccatatggtttaaaaaaaaaaaaaaaaaa 1348

Fig. 1. Nucleotide sequences of two cDNAs. 1) and 2) indicate the sequences of the prxRPA and prxRPN cDNAs, respectively. The nueleotide sequences are aligned to maximize sequence similarity. Capital letters indicate the coding regions. Gaps have been introduced to maximize alignment. The double underlining indicates putative polyadenylation sequences. The overlining marked (a) and (b) indicates the sequences that correspond to the AS and HBS primers, respectively.

364

eDNA and 62% for prxRPN eDNA. The third nucleotide in all codons is G or C in 66% and 81% of cases in prxRPA and prxRPN eDNA, respectively. The bias of codon usage in prxRPN eDNA agrees well with the results obtained from many genes from mon0cots (Murray et al. 1989).

Comparison of peroxidases. The amino acid sequences of the two rice peroxidases were compared with those of peroxidases from other plants, namely, tobacco (Lagrimini et al. 1987), horseradish (Fujiyama et ah 1988, Fujiyama et al. 1990), turnip (Mazza and Welinder 1980), potato (Roberts et al. 1988), Arabidopsis (Intapruk et al. 1990), peanut (Buffard et al. 1990), wheat (Rebman et al. 1991), and barley (Theilade and Rasmussen 1992) (Fig. 3). Mature RPA and RPN were about 50% homologous to the tobacco peroxidase and PNC1 from peanut, and they were 41% to 47% homologous to other peroxidases. Four conserved regions were found, with 80-95% homology in region I, 56-91% in region II, 57-86% in region III, and 44-88% in region IV (Fig. 3, boxed).

Two histidine and eight cysteine residues are conserved in all the peroxidases shown in Figure. 3. Both histidine residues have been predicted to be involved in catalysis. From an analysis of the protein sequences from horseradish (Welinder 1979) and turnip peroxidases (Mazza and Welinder 1980), we can predict the presence of four intramolecular disulfide linkages in plant peroxidases (cystine residues cl /c4, c2/e3, c5/c8, and c6/c7 in Fig. 3).

Recently, cloning of genes for peroxidases from wheat (Rebman et al. 1991) and barley (Theilade and Rasmussen 1992) has been reported. However, the putative rice peroxidases exhibit higher homology to tobacco peroxidase than to wheat and barley peroxidases. The results of amino acids comparisons indicate

that there is no clear distinction between mature peroxidases from dieots and monoeots.

Genomic DNA blot analysis. Southern hybridization of rice genomic DNA was performed using prxRPA and prxRPN eDNAs as probes (Fig. 4). A different set of fragments hybridized to each of the two probes without cross-hybridization between the two clones. With the prxRPA eDNA probe, identical patterns were obtained after short and long exposures (Fig. 4A and B). Only a single signal was detected after EcoRI, HindlII, and Xba I digestions. With prxRPN cDNA probe, the only one or two strong signals were found after short and long exposures but more hybridizing bands were detected after the long exposure (Fig. 4C and D). This result suggests that the genes on the rice genome that correspond to the prxRPN eDNA might form a multigenefamily. However, given the high GC content (about 70%) of prxRPN eDNA, it is possible that extensions are interrupted by secondary structures and that many short fragments that are generated anneal with non-specific regions of rice genomic DNA when this eDNA, as probe, is labeled with Klenow fragment and random primers (6-mers). Therefore, we labeled the eDNA with random 9-meric primers and Bca polymerase, whose the optimum temperature is 65"C (Ishino et al. submitted). The same pattern as shown in Figure. 4C was obtained after short and long exposures (data not shown). These results indicate that the genes that correspond to both prxRPA and prxRPN are present as single copies in the rice genome.

Expression of rice peroxidase mRNAs. We examined the levels of expression of the two peroxidase mRNAs in various tissues of plants at different stages by Northern hybridization analysis, using prxRPA and prxRPN cDNAs as probes (Fig. 5). With rice

Rice RPA

Rice RPN

Tobacco

Horseradish Cla Turnip TP7

Potato

Arabidopsis Ca Peanut PNCl

Wheat POX1 Barley BP1

II

I

el h c2 c 3 QLHDDFYDYICP~AAMRTEMRMGASLL RLHFHDCFV~GCDGSILLDG

--.--.--.-.--.,-..-.---. ......... ............. A ......

---AT---TT--N-TS I-RGVMDQRQ- -DA-A--K I I ...................

--TPT-- -NS--N-SNI-RDTIVNEL-SDP-IA--I .............. A .....

--TTN--STT--NLLST-KSG-KS -VS SQP ..... I- --F ................ I

--TP EA- -FSA-RGV-DS -IDA-T ..... I .......... D----G .... !

--TPT---TS--T-TNI-RDTIVNEL-SDP-IAG-I .............. A ..... N

---SN--ATK--NALSTIKSA-NSCVAK-A .............. -Q---A-V---0

-- -ST-- -TS--RALVAIKSG-A--VSSDP ...... ........... A-V--T-

AEPPVAPG--F---RRT--RAES I-R~F -QE-V-KDIGLA-G-- ........ -Q---A-V~--

IVALAAGYGVLFSGGPYYDVLLGRRD

...... K .... L .... D ......... -,-.-..,.-----..--.--.-

LLTI --QQS-TLA---SWR-P .....

-L- I--RDS-VQL---NWN-KV ....

-L- I --RDS -AKL- -QT-N-A- --S -

MLTI --QQS _Try.- -SWK-P- ....

-L-V--RDS -VAL--ASWN .......

-LTV--RDS -VAL--SWT-P .....

-L .... RDS-V%r----D-R-P ....

GL VANQSGAD~LPSPFEPIKSIIQKFNDVGLD TTDV

...... T--NSN ..... DS-SV-TAR-K----N A---

S- T--R---NSDI ..... TLAVM-PQ-TNK-M- L--L

S- Q-FLDL -NAN - -A- -FTLPQLKDS-RN- --NRS S -I

AK T-S-AA-NSNI-A-SMSLSQL-SS-SA---S -R-)

AR T--FT--LTQ--A--DNLTVQ- ..... KICFT LRE~

S- Q-FLDL-NAN--A--FTLPQLKAN-KN .... RPS-L

ST T-SL-S-NSD--A--FNLSGL-SA-SNK-FT -KEL

ST T-SA-L-NSD--G-SSSRSQLEAA-LKKN-N -V-~

SRSF -STQDVLSD- -G-SSNVQ-LLALLGRL--- A--I

!II

h c6 VVLSC-GHTIGRARC

..... A .... -S--

-A---A--F .....

-A ...... F-~0- -A---A- ---Q~ --

-A-A-A--V-F---

-A---A--F-KNQ-

-T---A----Q-Q-

-A~-A .... K-Q-

-TI ...... -C-H-

IV

c4 DDG EKFALPNKN SVRGFEVIDAI ~EDLENICPEV

TNS .... A--N ..... Y ....... A---GA--G-

-GTQT --13- -ANV GAG--DIV-D--TA---V--G-

TT SFRT--D-FG-A- -A---P---RM-AAV-SA--RT

TS SFTG-QN-G--R- -A---T--ND--SAV-KA--G-

IN-TFTG-QNSP--A- -A--Y---AQA-QSVIDT--NIS

TT SFRT--D--G-A- -A---P---RM-AAV-RA--RT

TS NFTG--T-G--A- -I ...... -T--SQV-SL--G-

M-QN-G--VG -L- --G- - -N --TQ--SV-KQT

SA TGPG -QQ-P --LTL RP SA-KAVlqD -RDP - -RE -I~F=A~

VSC~D

c7 TLFSNRL STTS SSADPTLDATMAANLQSLC AGGDGNETTV

L ...... ANF-A-N-V .... SSL-SS--QV- R-- ADQLAA

GT-EQ--FNFNG-GNP -L-V---FLQT--GI-FQ G-NN--TF-N

RFIRD--~I~F-N-GLP .... NT-YLQT-RG--PL N-N LSALVD

VN-RA-VI~/ETN IN -AF -TLR-RS -PRA--AG-ANLAP

--VCT-GNVNP --Q--CN- S -TLTDSDLQQ

RFIMD--YNF-N-GLP .... NT-YLQT-RGQ-PR N-N QSVLVD

-A-RT-I YRESNZ-P-Y-KS--AN-PS V---T-L SP

SN-RT-I YC~-TNINTAF-TS-KAN-PQ S--NT-L AN

SS-ED-- FPRP---ISP-FL~R-KRT- PVKQTDRR--

c8 LDITSAYVFDNRYYQNLLNQKGLLSSDQGLFSS DDGI ANTKELVETYSADAKKFFWDFGRS MVKM~NISPLTGDDGQIRKNCRVVN

--VN--DA--~H ...... AN .... A ..... V--SG-PAV-A--A--4~A-- -NGQ~-SC---N ............. SA ........ A- -

---STPND---D-FT--Q6NQ- --QT--E- --TSG SA- IAI -NR-AGSQfD~ --D- -VS- -I-L ........ TN .... TD-KR--

F-LRTPTI---K--V--EE .... IQ---E .... PNATD- IP --RSFANS TQT - -NA-VEA -DR .... T .... TQ----L ......

---.--.---,-.--.-.---.---.-- NGG S-DSI-RG--NSPSS-NS--AAA -I---D- ..... SS-E---V-GKT-

--T- PTM--KV--D--N-NQ-IR~---V- TG- -T-AGF-TD--N-VSV-LG--AAA -,------..-.,..--.-.--

F-LRTPL--~K--V--KE .... IQ---E .... PNATD-IP--RA-ADGTQT--NA-VEA -NR--~T-T--TQ----L ......

.-.-..---.--,--.--.----.--.- N-VSTDSQ -TAuNN-AT-NT---NA --IM----L .... TS .... T---KT-

--TMTP/IA---AMT---S .... H~-V--NNETTDN TVRN FASN-AA-SSA-TTA -I---~A .... TQ--~LS-SK--

--VRTPH .... K--ID-V-RE~FV~-D--TN - I -RPI --RFARSQQD --EQ--V- IG---QMR%~-S -Q-EV-R--S -R-

(299) (302) 71% (302) 52%

SNSLLHDMVEVVDFVSSM (323) 46% (296) 47%

PTSVASM (290) 45% SNSLLEIDVVDIVDFVSSM (323) 46% (292) 51%

S (289) 46% PGPGADALQWPSLVQTIVDEAKC~SIG (331) 41%

Fig. 3. Amino acid homology among ten mature peroxidases. The maximum alignment of the amino acid sequences of the two rice peroxidases (mature RPA and RPN) with peroxidases from tobacco, horseradish (Cla), turnip (TP7), potato, Arabidopsis (Ca), peanut (PNC1), wheat (POX1), and barley (BP1) is shown. Highly homologous regions are boxed and labeled I to IV. el to c8 and h represent consensus cysteine and histidine residues, respectively. The bars indicate the same amino acid residues as those in RPA. Gaps have been introduced to maximize alignment. The numbering of amino acids is shown in parentheses. The % similarity was calculated relative to RPA.

365

Fig. 4. Southern blot analysis of rice DNA using prxRPA and prxRPN cDNAs as probes. Total DNA (5 Ilg o was digested with EcoRl/HindIII (lane 1), EcoRI (lane 2), Hindlll (lane 3), SphI (lane 4), and Xbal (lane 5), fractionated on a 0.7% agarose gel, transferred to a nylon membrane and probed with 32P-labeled prxRPA (A and B) or prxRPN (C and D) eDNA. EcoT14l-digested ~. DNA was used as a source of size markers. X-ray films were exposed for 3 h (A and C) and 14 h (B and D) at -70"C.

ribosomal RNAs as markers, the size of the prxRPA and prxRPN mRNAs was estimated to be about 1,300 bases (Figs. 5 and 6). When Southern hybridizations were carried out under the same conditions as for RNA hybridization, patterns identical to those shown in Figure. 4 were obtained (data not shown). Therefore, we concluded that our Northern hybridization conditions could distinguish between the transcripts that corresponded to our two el ones.

No accumulat ion of prxRPA and prxRPN mRNA was observed in seeds or suspension-cultured cells (Fig. 5A and B; lanes 1 to 3). Since peroxidase activity is found in suspension- cultured cells (data not shown), it is suggested that peroxidase gene(s) different from those represented by prxRPA and prxRPN are active in the cultured cells. In leaves and stems, prxRPA mRNA became detectable within 14 days after germination, whereas prxRPN mRNA was clearly detected only in 21-day-old stems. It is puzzling that the size of transcripts of prxRPA in 14-day-old leaves and stems (Fig. 5A; lanes 5 and 7) was greater than 1.3 kb. We cannot explain this phenomenon at present. Expression of both mRNAs was greatly enhanced in roots during development of seedling (Fig. 5A and B; lanes 9 to 11). These findings demonstrate that the pattern of expression of genes for peroxidase isozymes is developmentally regulated.

Messenger RNAs hybridizing to the prxRPA and prxRPN cDNAs accumulated to high levels in roots, but they were only barely detectable in stems and leaves (Fig. 5C and D). The relative levels of expression in leaves, stems and roots of prxRPA and prxRPN transcripts were assessed densitometrically to be 1:4:12 and 1:7:80, respectively. These results suggest that the genes corresponding to prxRPA and prxRPN cDNAs, which were isolated from rice shoots, were activated to a greater extent in roots than in shoots. On the basis of the duration of autoradiography, we estimated that levels of prxRPA mRNA in each tissue were at least 4-fold higher than those of prxRPN mRNA. This difference in levels of expression probably explains why most of the positive eDNA clones isolated in this study were in the ~.I group that contained the prxRPA eDNA.

Induction of rice peroxidase mRNAs. To investigate whether the expression of rice genes for peroxidases is stimulated by wounding or treatment with H202, salicylic acid or ethephon, levels of peroxidase mRNAs were monitored by Northern hybridization with prxRPA and prxRPN cDNAs as probes (Fig. 6). The levels of prxRPA mRNA in leaves increased about 10-fold, as compared to those in healthy (21-old-day) leaves, when plants

Fig. 5. Accumulation of prxRPA mRNA and prxRPN mRNA in various rice tissues and suspension-cultured cells. Total RNA in each lane was isolated from tissues as follows. Lane 1, 15 Ixg of total seed RNA; lanes 2 and 3, 15 ttg of total RNA from 7- and 14-day-old suspension-cultured cells, respectively; lane 4, 15 lag of total RNA from 7-day old-shoots; lanes 5 and 6, 15 ~tg of total RNA from 14- and 21-day-old leaves, respectively; lanes 7 and 8, 15 ttg of total RNA from 14- and 21-day-old stems, respectively; lanes 9, 10, andll, 6 Ixg of total RNA from 7-, 14-, and 21-day-old roots, respectively; lanes 12, 13, and 14, 15 ~tg of total RNA from leaves, stems, and roots, respectively, of 21-day-old plants. The transcript was approximately 1.3 kb long. The filters were probed with prxRPA eDNA (A and C) and prxRPN eDNA (B and D). The autoradiograms were exposed for 12 h (A and C) or 48 h (13 and D) at -80"C.

Fig. 6. Accumulation of prxRPA mRNA and prxRPN mRNA in leaves (A and D), stems (B and E), and roots (C and F) following various treatments. Lanes WL, HL, SL, and EL were loaded with 15 ~tg of total RNA isolated t~om leaves after wounding or treatment with H202, salicylic acid, and ethephon, respectively. Lanes WS, HS, SS, and ES ~were loaded with 15 t~g of total RNA from stems after the aforementioned treatments. Lanes WR, HR, SR, and ER were loaded with 2.5 ~tg of total RNA from roots after the aforementioned treatments. Lanes CL, CS, and CR were loaded with 15, 15, and 2.5 ~tg of total RNA from leaves, stems, and roots, respectively, of 21-day-old plants as controls. The transcript was about 1.3 kb long. The autoradiograms were exposed for 12 h (A, B, and C) or 48h (D, E, and F) at -80"C.

366

were wounded or treated with ethephon (Fig. 6A). In healthy leaves prxRPN mRNA was hardly detectable. Wounding and ethephon, however, each caused at least a 10-fold enhancement of the level of prxRPN mRNA (Fig. 6D). In stems and roots levels of prxRPA transcripts were enhanced about 2- to 4-fold by each treatment (Fig. 6B and C). Levels of mRNAs that hybridized to the prxRPN eDNA were increased about 2- to 3-fold by wounding or treatment with I-I202 or ethephon in stems (Fig. 6E) and were enhanced approximately 10-fold by all treatments in roots (Fig. 6F). We also found that levels of both mRNAs were increased in leaves and stems by UV irradiation (data not shown). These results suggest that both rnRNAs were most strongly inducible by wounding and ethephon in leaves and that regulation of levels of both enzyme in response to environmental stress occurred at the level of transcription. The present data suggest that one function of these genes may be involved in recovery from wounding.

In higher plants, ethylene is induced by various stresses, such as mechanical wounding, flooding, and chemicals such as metal ions, ozone, or fungal exudates (Yang and Hoffman 1984). Additionally, it is known that ethylene activates some of the genes for peroxidases, chitinases, 13-1,3-glucanases, phenylalanine ammonia lyases, and pathogenesis-related proteins. These enzymes are also defense-related proteins in higher plants (Bowles 1990). Therefore, it can be assumed that a gene activated by ethylene is also induced by various stresses that include wounding. This possibility is supported by the results of the present study. At least two factors are necessary to trigger expression of the genes that correspond to prxRPA and prxRPN in wound-healing tissue: one is a signal that elicits the synthesis of ethylene upon wounding; and the other is a signal for activation by ethylene of the genes that encode peroxidases, As yet, it is unknown whether ethylene acts directly as an inducer in the latter case.

As described above, we have isolated two cDNAs that encode putative peroxidase isozymes. Both corresponding mRNAs in various tissues were induced by ethephon. We are currently isolating and characterizing the genes that correspond to the cDNAs. We have already confirmed the existence of the genes by the PCR with two primers, based on the N- and C-terminal regions of proteins encoded by prxRPA or prxRPN cDNAs. Furthermore, we are also preparing rice plants into which we will introduce the gene for ACC deaminase (Sheehy et al. 1991) and in which production of ethylene can be controlled. We will examine the levels of peroxidase mRNAs in the new transgenic plants. Cloning of the genes and creation of new transgenic plants should help to clarify the functions of each peroxidase isozyme.

Acknowledgements. The authors thank Mrs. A. Sadaoka and Mrs. H. Shimato of Takara Shuzo Co., Ltd., for their technical assistance. This work was supported by the Agrobiological Gene Analysis Research Association (AGARA), Japan.

References

Abeles FB, Dunn L1, Morgens P, Callahan A, Dinterman RE, Schmidt J (1988) Plant Physiol. 87:609-615

Aeosta M, Arnao MB, Casas JL, De Rio JA, Vioque B, Fernandez- Maculet JC (1992) Biochemical, Molecular, and Physiological Aspects of Plant Peroxidases (Lobarzewski J, Greppin H, Penel C, Gaspar TH eds.), pp. 121-125, Imprimerie Nationale, Geneva

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current Protocols in Molecular Biology, Wiley, New York

Bowles DJ (1990) Annu. Rev. Biochem. 59:873-907 Buffard D, Breda C, van Huystee RB, Asemota O, Pierre M, Dang Ha

DB, Esnault R (1990) Proe. Natl. Aead. Sei. USA 87:8874-8878 Espelie KE, Francesehi VR, Kolattukudy PE (1986) Plant Physiol. 81:

487-492

Fujiyama K, Takemura H, Shibayama S, Kobayashi K, Choi J-K, Shinmyo A, Takano M, Yamada Y, Okada H (1988) Eur. J. Biochem. 173:681-687

Fujiyama K, Takemura H, Shinmyo A, Okada H, Takano M (1990) Gene 89:163-169

Gubler U, Hoff~an BJ (1983) Gene 25:263-269 Hammerschmidt R, Nuckles E, Kuc J (1982) Physiol. Plant Pathol. 20:

73-82 Hertig C, Rebmann G, Bull J, Maueh F, Dudler R (1991) Plant Mol.

Biol. 16:171-174 I-Iinnman RL, Lang J (1965) Biochemistry 4:144-154 lntapruk C, l-Iigashimura N, Yamamoto K, Okada N, Shinmyo A, Takano

M (1990) Gene 98:237-241 Ishino Y, Uemori T, Fujita K, Asada K, Kato I, J. Biol. Chem., submitted Ito H, Hiraoka N, Ohbayashi A, Ohashi Y (1991) Agric. Biol. Chem.

55:2445-2454 Kay LE, Basile DV (1987) Plant Physiol. 84:99-105 Lagrimini LM, Rothstein S (1987) Plant Physiol. 84:438-442 Lagrimini LM, Burkhart W, Moyer M, Rothstein S (1987) Proe. Natl.

Aead. Sci. USA 84:7542-7546 Mazza G, Welinder KG (1980) Eur. J. Bioehem. 108:481-489 Mittal R, Dubey S (1991) Plant Physiol. Biochem. 29:31-40 Mohan R, Bajar AM, Kolattukudy PE (1993) Plant Mol. Biol. 21:

341-354 Morgens PH, Callahan AM, Dunn LJ, Abeles FB (1990) Plant Mol.

Biol, 14:715-725 Muller A, Grafe R (1978) Mol. Gen. Genet. 161:67-76 Murray EE, Lotzer J, Eberle M (1989) Nuel. Acids Res. 17:477-498 Murray MG, Thompson WF (1980) Nucl. Acids Res. 8:4321-4325 Negrel J, Lherminier J, (1987) Planta 172:494-501 Rasmussen SK, Welinder KG, Hejgaard J (1991) Plant Mol. Biol. 16:

317-327 Rebman G, Hertig C, Bull J, Mauch F, Dudler R (1991) Plant Mol.

Biol. 16:329-331 Roberts E, Kutchan T, Kolattukudy PE (1988) Plant Mol. Biol. 11:

15-26 Roberts E, Kolattukudy PE (1989) Mol. Gen. Genet. 217:223-232 Sambrook J, Fritseh EF, Maniatis T (1989) Molecular Cloning: A

Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York

Sanger F, Nieklen S, Coulson AR (1977) Proc. Natl. Aead. Sci. USA 74:5463-5467

Sheehy RE, Honma M, Yamada M, Sasaki T, Martineau B, Hiatt WR (1991) J. Baeteriol. 173:5260-5265

Sheen SJ, Diachun S (1978) Aeta Phytopathologica Academiae Scientiaram Hungaricae 13:21-28

Sheets MD, Ogg SC, Wiekens MP (1990) Nuel. Acids Res. 18:5799-5805 Shinmyo A (1988) Cell Technology 7:588-590 (in Japanese) Svalheim O, Robertsen B (1990) Plant Physiol. 78:261-267 Theilade B, Rasmussen SK (1992) Gene 118:261-266 van Huystee RB (i987) Annal. Rev. Plant physiol. 38:205-219 Welinder KG (1979) Eur. J. Bioehem. 96:483-502 Yang SF, Hoffman NE (1984) Ann. Rev. Plant Physiol. 35:155-189 Zheng X, van Huystee RB (1992) Plant Science 81:47-56