Gene, 118 (1992) 261-266 0 1992 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

06567

localization

Structure and chromosomal peroxidase BP 2A (Hordeum

v&gave; nucleotide

Bodil Theilade

sequence;

by J.K.C.

signal peptide;

C-terminal

of the gene encoding

barley seed

processing;

acid)

RFLP;

intron;

abscisic

DK-4000

Roskilde, Denmark

and %-en K. Rasmussen

Plant Biology Section. Environmental

Received

261

0378-l 119/92/$05.00

Knowles:

Science

16 December

and Technology Department,

1991; Revised/Accepted:

Rim National Laboratory.

12 March

1992; Received

at publishers:

16 April 1992

SUMMARY

A clone, APrxb. I, coding for a barley seed peroxidase (BP; EC 1.11.1.7), was isolated from a genomic library using a cDNA coding for the barley seed peroxidase, BP 1, as a probe. The nucleotide sequence coded for a BP showing 73% amino acid (aa) sequence identity with BP 1 and less than 50% similarity with other sequenced plant peroxidases. The aa composition is 92% identical to that determined for BP 2 purified from mature barley grains, and therefore the gene product is named BP 2A. The alignment suggests that the coding region is interrupted by a 76-bp intron having the consensuses GT and AG, at the 5’ and 3’ ends, respectively. Alignment with BP 1 suggests that BP 2A has a leader peptide of 36 aa and the mature protein is 3 19 aa. Alanine and leucine account for 50 y0 of the residues of the leader peptide. Of the codons used 90% have a C or G in the third position. The promoter shows a putative abscisic acid-response element, 5’-GTACGTGTC, 115 bp upstream from the start codon. The BP 2A-encoding gene was RFLP-mapped on barley chromosome 3, and we suggest for this peroxidase locus the name Prx6.

INTRODUCTION

Barley peroxidases (BP; EC 1.11.1.7) are coded for by genes in at least five loci. Isozyme technique has been used to map one locus to chromosome 5 (Ainsworth et al., 1984), two on chromosome 2 (Brown and Munday, 1982; Benito et al., 1988), and a seed peroxidase locus on chromosome 1 (Nielsen and Hejgaard, 1987). The structural gene cod-

Correspondence

to: Dr. B. Theilade

or Dr. S.K. Rasmussen,

ogy Section, Environmental Science and Technology National Laboratory, DK-4000 Roskilde, Denmark. Tel. (+45)42371212; Abbreviations:

Plant Biol-

Department,

Rise

Fax (+45)46323383.

aa, amino

acid(s);

ABA, abscisic

acid; ABRE,

abscisic

acid response element; BP, barley peroxidase(s); bp, base pair(s); HRP, horse radish peroxidase(s); kb, kilobase or 1000 bp; nt, nucleotide(s); IPrx6.1,

clone carrying

Prx6 encoding

PAGE, polyacrylamide-gel POX, wheat peroxidase(s); fragment

length polymorphism;

mato peroxidase(s);

BP 2A; ORF, open reading frame;

electrophoresis; POP, potato peroxidase(s); Pm, gene encoding BP; RFLP, restrictionSDS, sodium

TP, turnip peroxidase(s).

dodecyl

sulfate; TOP, to-

ing for the seed peroxidase BP 1 has recently been RFLPmapped to chromosome 3 (Johansson et al., 1992). Barley peroxidases are studied at the biochemical, physiological, genetical and molecular level to delineate the biological function of the individual enzymes. So far, BP 1 is the best characterized. It has been purified from barley seeds and its cDNA cloned. It was found that BP 1 is tissue specifically as well as developmentally expressed in the endosperm showing maximum of expression 15 days after flowering (Rasmussen et al., 1991a). BP 1 is low in carbohydrate content, and one third of the enzyme is nonglycosylated (Johansson et al., 1992). BP 1 has a low specific activity for classical peroxidase substrates including substrates for lignification (Andersen et al., 1991), however, the in planta substrate remains to be found. Six peroxidases from barley leaves have been purified and partially characterized (Saeki et al., 1986). Kerby and Somerville (1989) found that two isoforms could be induced by inoculating barley leaves with a powdery mildew fungus. Recently, two cDNAs for leaf peroxidases, showing less than 50% aa sequence identity, were isolated (Rasmussen et al., 1991b;

180

cgtcgcccgcctcgccctgacgccgtctgttcccagccacgcatgcagtggaggagccct / gcgtcgcccgcccgtccctgattaagctcggcactgcccatcgtcgtgtgcgcgttcccc / -

480 540 600 660

aggtgcggtaacggatttcgatgagtttcagtaatagattccgattagttctacggcagt

gcgtctcctcgcgacgaggtttgactgatttacgcattgttttatggggctcgtacgcac

cattttcaatcaaaactgtaaacaccttgtttttttacgggtttgatcattttaaggaat

ctattacagatgctctatggaagtgaatggagagtatcttgtgctggtgctggctgtgtc ABRE atgcacgatggtacctacagtacgtgtcaaagacaagcgtcgtctataaaagcaaggctc

1320

1380

1440

1500

GTCACGGCGCCGTCGTCTCCTGCTCCGACATCCTGGCGCTCGCGCGCGACTCCGTCGTCG CysHisGlyAlaValValSerCysSerAspIleLeuAlaLeuAlaArgAspSerValVal

CCACGGGCGGCCCGGACTACTGCGTGCCCCTCGGCCGCCGCGACAGCGCACGGTTCGCCA AlaThrGlyGlyProAspTyrCysValProLeuGlyArgArgAspSerAlaArgPheAla

CACGGGACGCAGTGGGCTCCGGCCTCCCGCGGCCGTCCTCCAACGTGACAACCCTCCTCG ThrArgAspAlaValGlySerGlyLeuProArgProSerSerAsnValThrThrLeuLeu

ACGTGTTCCGCAAGCTCGGCCTCGAAGCCACCGACCTCGTCGCGCTCTCCGGCGGCCACA AspValPheArgLysLeuGlyLeuGluAlaThrAspLeuValAlaLeuSerGlyGlyHis

tggttcatgcatgaacgcagccgctcatgattcatgaatgcatgcagGGGTGCGACGCGT GlyCysAspAla

1260

1140

CGCCTCCACTTCCACGACTGCTTCGTCCAGgtacctaccttgccttgccactcatgattt ArgLeuHisPheHisAspCysPheValGln

CGCTCCGCCCCTCCGCCTTGAAGGCCATCGACAACATCCGCGACCAGCTGGAGCACCACT ThrLeuArgProSerAlaLeuLysAlaIleAspAsnIleArgAspGlnLeuGluHisHis

1080

1200

1020

GCGGAGTCCATCGTGCGTAAGTTCGTCCAGGACGCCGTCCGCAAGGACAAAGGCCTCCTC AlaGluSerIleValArgLysPheValGlnAspAlaValArgLysAspLysGlyLeuLeu

CGGTGCTCCTGCACGGGTCCGCCGCCGAGCCAGGGGAACAGCAGGCGCCGCCCAACCTCA SerValLeuLeuHisGlySerAlaAlaGluProGlyGluGlnGlnAlaProProAsnLeu

960

AGGCAGCCTCCGATTACTCGCGGCCTGTCGTTCGACTTCTACCATGGGAGGTGCCCCGGG ArgGlnProProIleThrArgGlyLeuSerPheAspPheTyrHisGlyArgCysProGly

tgctagtttactatgtcgac

tatgtcaacttgtcatattttacatacaactttgccagaaaaatattttggttggccgtt

2640

2580

2520

tatagattttgtcaactacgctgtaaatccacaccgtgtttgacaaattattgttgtctt gtagcaattttcattattaatgatataaacgcatgtcatagatttggttgccaatttaaa

2460

agactacgagacacttagtagccacattaattaacctagttgtcaactggcaactgtgtg

2400

2340

acgatccgttttcatcgttagctttctcgcaatgagatctcaaaactagtcccatcatcg acacgatcaacaacttttttttatcgatarttrccagattactattagttagttgttgcc

2280

2220

cggagtcttttaatgttgaattttcaaaaagttttatcttttgaactataaattcaattg

ggcgggcgggatgcgatccgagcaggcatcgtacgccctgtgttagcatatgctgtgcga

2160

ggttatgtttctttgttggaaaaatctaagtaactacgtcgtacctcttccttcccgtgc 900

2100

atcgaccaagcggtgtaactacatcacacgtacggtgaccagttagtttgatcaatcgat

840

2040

1980

1920

1860

1800

1740

1680

1620

1560

ggagtggtagctgttaaccatgcatgtcg~~aactgagcaggcaagccatg

CTCGTGCAGACCGTCGTtgagctgccgcggagaacctcgggttctagaatctatactagt ProArgAlaAspArgArgEnd

ACTGCGCCGTGCCCAACCCCGGCACCGTTGATGGCGACGACGCCTTGAGTGGCCGTCTGC AsnCysAlaValProAsnProGlyThrValAspGlyAspAspAlaLeuSerGlyArgLeu

TGTCCATTGGTGAAGATGGCCAGATAAGGGTGCTCACCGGCGACCAGGGCCAAGTCCGCA ValSerIleGlyGluAspGlyGlnIleArgValLeuThrGlyAspGlnGlyGlnValArg

CCCAGCCCATCGTCGAGAGCTTCGCGCGCAGCCAAGGCGACTTCTTCGACCAGTTCGGCG ThrGlnProIleValGluSerPheAlaArgSerGlnGlyAspPhePheAspGlnPheGly

ACCTGGTGAACCAGGAGGGGCTCTTCGTGTCCGACCAGGATCTCTACACCAACGCCATCA AsnLeuValAsnGlnGluGlyLeuPheValSerAspGlnAspLeuTyrThrAsnAlaIle

GACGGCCAGCCGCGTTGGACGTGCGCACCACCAACGTGTTCGACAACAAATACTTCGTGA GlyArgProAlaAlaLeuAspValArgThrThrAsnValPheAspAsnLysTyrPheVal

CCATGAGCCCTTCCTTCGTCGCCAGGCTGAAGCGGACGTGCCCG.~CCATGGGCACCGACG ThrMetSerProSerPheValAlaArgLeuLysArgThrCysProT~rMetGlyThrAsp

CCATCGGGCTGGGACACTGCAACTCCTTCGAGAAGCGGCTCTTCCCGCTCCCGGACACCA ThrIleGlyLeuGlyHisCysAsnSerPheGluLysArgLeuPheProLeuProAspThr

780

GTGCTGGCCCTTGGTTCTGCCGTGGCGTATGCGGCGGCAGAGCATGAGGAGCTGAGGTCG ValLeuAlaLeuGlySerAlaValAlaTyrAlaAlaAlaGluHisGluGluLeuArgSer

tacgttgtagagcagagcagagcacagtaccactgtggcatacacgaactgtgagacgat --Y-Y cgagtgctgagatcgATGGCTC~CTC\CTCTCGCAGC*GC*GTAGCACTGCTG MetAlaProSerSerProLeuLeuAlaAlaAlaValAlaLeuLeu

420

tcaagccggctgcaattcgatccagctacggcaaattttgacttatggcgacttcatccg

720

360

gtcgtggccgcgcgctcccccatcaccgccccgctcttctccggtgtctgccccgttccg -7 attcgggccgccggtggcctagtccgccctgcctttgacggccttgctcggcagtggagg

300

240

120

ttttcctcccgtcccgacgatccatctctggccacggtgtatctcgctctggcgcctcgg

-7

60

gtcgacactgaagactccgtaaatgaaactgtatctcattatagctcatttctttttctt

z

IJ

263 Thordal-Christensen,

1992). Increasing

amounts

of tran-

codes a mature

script for each of the two cDNAs were detected 4 h after inoculating leaves with a powdery mildew fungus. Thus, at least two peroxidases must take part in the plant response to pathogenic attack. We are interested in the structurefunction relationship of the many isozymes and in eluci-

protein

of 3 19 aa, with an aa composition

92% identical to that determined for BP 2 which has been purified from resting barley grains (Hejgaard et al., 1991). The M, can be calculated to be 34 500 which is close to the 36500 Da determined for BP 2 by SDS-PAGE (Hejgaard et al., 1991) taking into account that BP 2 might be N-glycosylated. We therefore conclude that iPrx6.1 gene codes for a BP 2 isozyme, and hence we designate this

dating the tissue and developmental regulation of gene expression. Here we report on the nt sequence of the first cloned BP-encoding gene and the chromosomal localiza-

peroxidase

BP 2A.

tion of this gene.

EXPERIMENTAL

(c) The aa sequence of BP 2A From the alignment with BP 1 (Fig. 2) it is evident that

AND DISCUSSION

BP 2A has 36 aa residues preceding the N-terminal Gln (nt 796 to 904 in Fig. l), which forms a typical signal peptide with many hydrophobic residues in the middle region (von Heijne, 1990). Ala and Leu constitute 50% of the residues in the signal peptide as noted for the a-amylase inhibitors expressed during barley grain filling (Rasmussen and Johansson, 1992). The alignment with other plant peroxidases suggests that BP 2A might have an 18-residue C-terminal extension from nt 1884 to 1937 (Fig. 1). In fact, it has been shown by comparing the aa sequence deduced from cDNA with that obtained by aa sequence analysis of the mature protein that seed BP 1 (Rasmussen et al., 1991a) and HRP C (Fujiyama et al., 1988) both are processed at the C terminus. The cellular localization of BP 1, BP 2A, and HRP C is unknown and it remains to be shown whether these peroxidases are actually targetted to the vacuoles. The proposed C-terminal extension is, however, unusual by having two Arg residues at the terminus (Fig. 1). A cDNA, pcD 13 11, coding for a barley leaf peroxidase also have Asp-Arg-Arg as the C-terminal sequence (Rasmussen et al., 1991b). The BP 2A contains two sites for possible N-glycosylation (Asn-Xaa-Ser/Thr), Asn” and Asn14’ (Fig. 2). The seed BP 1 is low in glycosylation (Rasmussen et al., 1991a) compared, e.g., to HRP C where eight out of nine possible sites are glycosylated. From the predicted aa sequence, it is evident that BP 2A also belongs to the peroxidases of low glycosylation.

(a) Barley peroxidase genomic clones A barley genomic library in phage 1 was screened at low stringency, 5 x SSPE (20 x SSPE: 3 M NaC1/0.2 M NaH,P0,/0.02 M EDTA), using a seed-specific BP 1 cDNA clone, pcR7 (Rasmussen et al., 1991a) as a probe. Upon purification two clones, APrx6. I and W66 11, showed hybridization to pcR7 at high stringency, 0.1 x SSPE. A restriction analysis of the phage DNA of APrx6.1 followed by Southern blotting and hybridization to pcR7 showed that the gene could be located to a 7.8-kb BumHI restriction fragment, which was then subcloned in pUC13. Further analysis showed that the entire coding region was located on a 2.7-kb Sal1 fragment, which was subcloned in pUC13 and subsequently characterized by nt sequence analysis. There was no indication of a tandem repeat of the gene as found in the case for the horseradish neutral prxCla and prxClb (Fujiyama et al., 1988) and tomato anionic peroxidase genes (Roberts and Kolattukudy, 1989). (b) The IPrx6.2 clone codes for a BP 2A isozyme The 2660-bp Sal1 fragment of APrx6.1 contains an ORF from nt 796 to 1940. Alignment with known plant peroxidase sequences suggests this is interrupted by a 76-bp intron from 105 1 to 1127 (Fig. 1). Comparison with the aa sequence of BP 1 (Johansson et al., 1992) showed that APrx6.1 codes for a BP 73% identical with BP 1 (Fig. 2). The alignment led us to suggest that Gln at nt 904 is theNterminal residue. BP 1 is the only open to Edman degradation (Johansson et al., 1992), whereas all other sequenced peroxidases have a pyroglutamate as the N terminus. These alignments allow us to suggest that APrx6.1 enFig.

(d) Similarity of plant peroxidases The aa sequence of BP 2A is aligned with three well characterized peroxidases, BP 1 (Johansson et al., 1992) TP 7 (Mazza and Welinder, 1980) and HRP C (Welinder, 1985). Furthermore, the sequence from two cloned wheat

I. Nucleotide sequence and deduced aa sequence of IPrx6. I, A barley genomic library constructed

Rhode, Cologne, was screened using the seed BP cDNA, using pcR7 (Rasmussen

and kindly provided

by Dr. Wolfgang

et al., 1991a) as a probe. A central 2.7-kb Sal1 fragment

in /IEMBL4

from the positive

clone 1.Pr.v6.1 was characterized by nt sequence analysis (Tabor and Richardson, 1987). ClaI fragments were cloned in the pBluescript KS plasmid, SmuI and XbaI fragments were cloned in pUC13. HpaII, Sau3A and TuqI restriction fragments were cloned into the replicative form of Ml3mp8. quencing

primers were synthesized

in the light of known

sequences

to fill in gaps and to complete

sequencing

of both strands.

in capital letters and the first nt in the start codon is No. I in the nt sequence. Direct and inverted repeats are marked tative promoter sequences, CAAT, TATA and ABRE are underlined. The two polyadenylation signals 5’-AATAAA peptidc

is shown in hold-face

letters. The sequence

is reported

to GenBank

under accession

No. M83671.

and Se-

The coding region is shown

with horizontal arrows. The puare boxed. The proposed signal

264 FY

L BP

2A

BP

1

A

CP

RLHFHDCFV

GCD

S &

E

E

AN

QPPITRGLSEDFYHGRCPGAESIVRKFVQDAVRKD----KGLLRLHFHDCFVQ~CDASVLLHGS-AAEPGEQQAPPNLTLRPSALKAIDNIRDQ~EH F~EPPVAPGLsFDFYRRTCPIlnESIVREFVeEAVRKDIGLAAGLLRLHFH~CFVQGCDASYLLDGS-ATGPEEQQAPPNLTLRPSAFKAVNDI.RDRLER

pPox3t-n

QLSPTFYDT~CPRALAIIKsGVMAAVSSDPRMGASLLRLHIEA

POX1

QLSSTFYDTsCPRALVAIKSGVAAAVSSDPRMGASLLRLHES ZLTTNFYST~CPNLLSTVK~GVKSAVSSQ~RMGASILRLFFHDCFVNGC~GSILLDDT-S~FTGEQNAGP~RN-SARGFTVINDIKSAVEK

TP

7

HRP

c

ZLRPDFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLLiFHDCFVNGCDASILLDNT-TSFRTEKDAFGNAN-SARGFPV~DRMKAAVES

POP

A

QLTPE----AC--VFSAVRGVVDSAIDAETRMGASLIRLHFHDCFVDGCDGGILL~DINGTFTGEQNSPPNAN-SA~GYEVIAQAKQSVID

TOP

A

QLSATFYDT~CPNVTSIVRGMDQRQRTD~~GAKIIRL~FHDCFVNGCDGSILLDTD-GT-QTEKDAPAN~G--AGGFDIVDDIKTALEN

COIWIIO~

BP

2A

BP

1

c

vsc

DI&

&

sy

GGP

VG1151

A

F

llP

D v LSG E

ECRGAVVSCSDILALAARDSVVtrSGGPDYRVP~GRRDSRSFA~TQDVLSDLP~PSSNVQSLL~LLGRLGLD-A~DLVTISGGH~IGLAHCSSF~DRLFICNQ-TVSCADILTVAA_RDSVVALGGPSWTVPLGRRDSTD-ANEAAANSDLPGFTSSRSDLELAFRNKGLL-TIDMVALSGAHTIGQAQCGTFKDRIY-

POX1

VCKQ-TVSCADILTVAARDSVVALGGPSWTVPLGRRDSTT-ASASLANSDLPGPSSSRSQLEAAFLKKNLN-TVDMVALSGAHTIGKAQCSNFRTRIY-

TP

7

ACPG-VVSCADILAIAARDSV~QLGGPNWNVKVGRRDAKT-ASQAAANSNIPAPSMSLSQLISSFSAVGLS-TRDMVALSGAHTIGQSRCVNFRARVY-

HRP

c

ACPR-TVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQ-AFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYN

POP

A

TCPNISVSCADILAIAARDSVAKLG~QTY~ALGR~DART-ANFTG~LTQLPAPFD~LTVQIQKFN~KNFT-LREMV~LAGAHTVGF~RCSTV------

TOP

A

VCPG-VVSCADILALASEIGVVLAKGPSWQVLFGRKDSLT-ANRSGANSDIPSPFETLAVMIPQFTNKGMD-LTDLVALSGAHTFGRARCGTFEQ~LFN

pcLll311

TLISSFANRSLD-VADLVSLSGAHTFGVAHCPAFEDRSS-

BP 2A 1

pPox381 POX1 TP

7

HRP

C

POP

A

TOP

A

pcD1311

G

TG

G

E

C

N

BP 2A

DGQIRVLTGDQGQVR-NCAVPNPG

BP

MGQMRVIJTSDQGEVRRNCSVRNPGPG

1

pPox381

MGNIAPKTGTQGQIRLSCSRVNS

POX1

MGNLAPLTGTQGQIRLSCSKVNS



TVDGDDALSGRLPRADRR



t319>

ADALQWPSLVQTIVDEAAGSIG



73%

t290>

44%

t289>

45%

TP

7

MGDISELTGSSGEIRI&VCGKTN

HRP

c

MGNITPLTGTQGQIRLNCRWNSNS

POP

A

MGDLPPSAGAQLEIRDVCSRVNPTSVASfj

TOP

A

P&1311

C

HCHGAVVsCSDILALA-RDSVVATGGPDYCVPLGRRDSARFATRDAVGSGLPRPSSNVTTLLDVFRKLGLE-ATDLVALSG~HTIGLGHCN~FEKRLF-

pPox381

BP

G

t296>

40%



LGNISPLTGTNGQIRTDCKRN



MSNMDILTGTKGEIRNNCAVPN



LLHDMVEVVDFVSSM





39% 32% 40%

RRVRTSRPPSPARGDRR



39%

R

265 peroxidases,

pPOX381

(Rebman

et al., 1991) and POX1

(Hertig et al., 1991) from POP A (Roberts and Kolattukudy, 1989) and TOP A (Lagrimini et al., 1987) is included in the alignment. The partial cDNA pcD 13 11, coding for leaf BP is also included (Rasmussen et al., 1991b). The regions around the proximal His”l and distal His44 are highly conserved among plant peroxidases (Welinder, 1985). Eight of the nine Cys in BP 2A are at identical positions to those forming the four highly conserved disulfide bridges in HRP C. The overall identity to the monocot and dicot peroxidases is less than 50 %, except for 73 y0 identity with BP 1. This supports the idea that the cationic BP from barley grain have a distinct biological function.

TABLE

I

Segregation

of Est4, Pr.u5 and Prx6 Combined

Loci” Allele

SV

AL

V

43P 4

2OP

A d The loci are: esterase, h Combined

Nielsen,

segregation

1

V

A

44P 0

24~

in locus Esr4 for allele Sv and At (Hvid

allele V and A (p = parent

from homozygous

1

Est4; BP 1, Prx5; and BP 2A, Pm6.

1977) in locus Pm.5 for allele V and A (Johansson

and Pm6

(e) Codon usage The overall G+C content in the coding region of APrx6.1 is 65 %, and 907; of the codons have a C or G in the third position as is the case for BP 1. This bias in codon usage has been observed for many genes expressed in the barley seeds, probably reflecting a high translation rate (Fincher, 1989).

PIT5

Est4

Prx6

DNA

segregation’

genotype).

lines derived

Southern

and

et al., 1992) blots of barley

from chromosome-doubled

hap-

loids (Doll et al., 1989) were digested with AIuI and probed with the KpnI fragment

(nt 673- 1053) from IPrx6.1.

is under regulation by ABA. Two polyadenylation signals 5’-AATAAA found in many plant genes are located 70 and 74 nt downstream from the TGA stop codon. (g) Chromosome localization The structural gene for BP 2A was mapped using the 380-bp KpnI fragment covering the N-terminal part of the gene to detect RFLP. This KpnI fragment does not crossreact with the BP 1 cDNA. Linkage was found to the isozyme loci Est4 showing 8.7% & 3.39 recombination and to the BP 1 locus Prx5 showing 1.45% k 1.44 recombination (Table I). The Est4 has been assigned to barley chromosome 3 (Hvid and Nielsen, 1977). Hence the structural gene for BP 2A is located on barley chromosome 3 and tightly linked to PrxS. We suggest that this new locus is named Prx6, and hereby a total of seven BP loci on four different chromosomes are known.

(f) Structure of the BP ZA-encoding gene The BP 2A-encoding gene consists of two exons and one intron. The 76-bp intron has the consensus GT and AG at 5’ and 3’ terminus and it is located between the codon Glns5 and Gly” close to the distal His. Precisely at the same position in the protein an intron is found in all genomic clones published so far, e.g., Arabidopsis prxCa and prxEa (Intapruk et al., 1991), horseradish prxC2 and prxC3 (Fujiyama et al., 1990) prxCla and prxClb (Fujiyama et al., 1988), tomato TAP 1 and TAP2 (Roberts and Kolattukudy, 1989) and wheat pseudogene POX1 (Hertig et al., 1991). In total, two introns are found in the peroxidase-encoding gene isolated from wheat and tomato, and three introns in the genes from horseradish and Arabidopsis thaliana. The size of introns at identical positions varies between genes as well as between plant species. In the 5’ noncoding region, the putative promoter sequence boxes, TATA and CAAT are found 91 bp and 249 bp upstream from the start codon, respectively. The se688 115 bp upstream is closely quence 680GTACGTGTC related to the ABA-responsive element 5’-GTACGTGGC in Rub16 from rice (Skriver et al., 1991) and 5’-ACACGTGGC in the Em promoter from wheat (Guiltinan et al., 1990). This could indicate that the BP 2A-encoding gene

(h) Conclusions This is the first report on the primary structure of a BP-encoding gene. The genomic clone 2Prx6. I has an ORF encoding a sequence of 355 aa. The coding region is interrupted by a 76-bp intron located near the distal His44. At exactly the same location an intron has been found in all peroxidase-encoding genes characterized so far (horseradish, tomato, Arabidopsis and in a pseudogene from wheat). The aa sequence is 73 y0 identical to seed BP 1 (Johansson et al., 1992). Alignment with BP 1 suggests that this gene has a putative signal peptide of 36 aa giving a mature

Fig. 2. Alignment of the tentative aa sequence of BP 2A with that of seed BP 1, the wheat peroxidases pPOX381 (Rebman ct al., 1991) and POX1 (Hertig et al., 1991) TP 7 (Mazza and Welinder, 1980), HRP C (Welinder, 1985) POP A (Roberts and Kolattukudy, 1989) TOP A (Lagrimini et al., 1987) and a partial sequence of barley leaf peroxidase shows invariable

residues

(underlined)

pcD 13 11 (Rasmussen

and aa conserved

et al., 199lb). Every tenth residue is underlined

in all except one. The C-terminal

extension

for each sequence.

The first lint (Common)

is shown for BP 1 and HRP

C and tentatively

for

BP 2A and pcDl311. The aa numbers for the mature protein are shown in brackets and for BP 1, BP 2, pcD1311 and HRPC also for the C-terminal extension. The ‘% similarity is calculated relative to BP 2A. Gaps are introduced to maximize alignment and calculated as nonidentical residues. The N-terminal

Q in BP 2A is tentative,

as is the case for pPOX381,

POX],

POPA

and TOPA.

266

protein of 319 aa. The aa composition of the deduced aa sequence shows 92”/, identity to that determined for BP 2 purified from resting grains (Hejgaard et al., 1991). We therefore conclude that 3,Prx6.1 encodes a BP 2 isozyme and designate this BP 2A. We have RFLP-mapped this gene to a new locus on barley chromosome 3 and suggest that this locus is named Prx6. In total, one gene and three cDNAs coding for four different BP have been characterized and seven Prs loci mapped to four barley chromosomes.

Aspects of Plant Pcroxidases. Hertig, C.. Rebmann, and tissue-specific

Intapruk,

C., Higashimura,

and

Takano,

encoding 241.

M.:

Johansson.

Lagrimini,

M.D.: The chromosomal

locations

E.A., Miller, T.E. and Gale.

of leaf peroxidaac

genes in hexap-

loid wheat, rye and barley. Theor. Appl. Genet. 69 (1984) 205-210. Andersen, M.B.. Johansson. T., Nyman. P.O. and Welinder, K.G.: Substrate specificity oxidases. (Eds.),

In: Lobarzewski, Biochemical,

Pcroxidases. Benito,

M.C..

Sanchez,

Brown,

stimulation

J., Grcppin,

Molecular

University

chromosome Genet.

and manganese

of plant and fungal per-

H., Gaspar,

and Physiological

T. and Pcnel, C. Aspects

of Plant

199 1, pp. 169- 173.

of Geneva,

M., Shin, J. and Blake, T.: A map of barlcl

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