Plant Molecular Biology 14: 715-725, 1990. © 1990 Kluwer Academic Publishers. Printed in Belgium. 715

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

Peter H. Morgens*, Ann M. Callahan**, Linda J. Dunn and Fred B. Abeles United States Department of Agriculture, Agricultural Research Service, Appalachian Fruit Research Station, 45 Wiltshire Road, Kearneysville, WV 25430, USA (* author for correspondence)

Received 17 July 1989; accepted in revised form 3 January 1990

Key words: cDNA, Cucumis sativus L., DNA sequencing, ethylene, peroxidase

Abstract

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1-5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55 ~o sequence identity), a horseradish (539/o), a turnip (45 ~o ), and a potato (419/o ) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative per- oxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.

Introduction

Ethylene has been shown to increase peroxidase activity in sweet potato [4,7], cucumber [1, 20, 24], pea [23], tomato [8], tobacco [26], and mung bean [2]. Molecular aspects of plant peroxidases have recently been reviewed by van Huystee [25]. However, the physiological functions of both endogenous and hormonally regulated peroxidases are not fully understood. Evidence has accumulated which shows that per-

oxidase plays an important role in cell wall lignifi- cation [ 17, 23]. Evidence both for [2] and against [8, 24] the concept that peroxidase is used in the defense of plants against microbes has also been presented. While increased peroxidase activity in cucumber has been associated with senescence and chlorophyll degradation in vitro, it has not been established with any certainty that per- oxidase plays a role in chlorosis or other degrada- tive aspects of senescence [ 1 ].

We have previously shown that a 33 kDa per-

The nucleotide sequence data reported will appear in the EMBL, Genbank, and DDBJ Nucleotide Sequence data bases under the accession number M32742. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U S D A and does not imply its approval to the exclusion of other products or vendors that may also be suitable. * Co-senior authors.

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oxidase (pI = 8.6) and a 60kDa peroxidase (pI = 4.0) are induced during ethylene-enhanced senescence of cucumber cotyledons [ 1 ]. This paper describes the cloning and nucleotide sequencing of cDNA clones encoding putative cucumber peroxidases. These clones have enabled us to compare the sequences of these putative peroxidases with a number of other plant per- oxidases and to measure the accumulation of their mRNA following ethephon and ethylene treat- ment. These cloned RNA sequences are impor- tant because they should help elucidate the function or functions of peroxidases in plants and lead to the further understanding of ethylene- induced gene activity.

Materials and methods

Plant material

Cucumber (Cucumis sativus L. cv. Poinsett 76) plants were grown in a greenhouse and treated with 10 mM 2-chloroethylphosphonic acid (ethephon) as an ethylene generator or with 100 mg/1 ethylene in a 40 liter Plexiglass box as described earlier [ 1 ]. Controls were treated with air in a 40 liter Plexiglass box.

Nucleic acid labeling

Oligonucleotides were 5' end labeled in a reaction catalyzed by polynucleotide kinase [ 11 ]. Plasmid fragments for Maxam-Gilbert sequencing were 3' end labeled in a reaction catalyzed by reverse transcriptase [13]. Plasmid DNA used for RNA blot analyses was labeled by nick translation [ 12]. DNA fragments used for RNA blot analysis were labeled with a Random Primer DNA Labeling System (Bethesda Research Laboratories, Gaithersburg, MD) according to the recom- mendations of the manufacturer.

RNA isolation and blotting

Total RNA was extracted from cucumber cotyledons as previously described [5] with the modification that the extraction buffer included 1 mM ascorbic acid and that the initial proteinase K digestion was at 65 °C. Poly(A)÷-enriched RNA was isolated from total RNA after one pass over an oligo(dT) cellulose column [11 ].

RNA samples were fractionated on 1.4~o agarose gels containing 5 mM methyl mercuric hydroxide [3] and transferred to Nytran mem- brane according to the manufacturer's recom- mended procedure (Schleicher and Schuell, Keane, NH). Prehybridization and hybridization conditions for the oligonucleotide probes were 4 0 ~ formamide, 5 x Denhardt's solution, 8x SSC, 0 .1~ SDS, 50mM Tris pH7.8 at 37 °C. Final wash conditions were 2x SSC, 0.1 ~o SDS, 25 °C. Prehybridization and hybridi- zation conditions for either whole plasmid or purified DNA fragment probes were 509/0 forma- mide, 3 x Denhardt's solution, 5 x SSC, 0.19/o SDS, 50 mM Tris 7.8, 45 °C. Final wash condi- tions were 0.1x SSC, 60 °C. Filters were exposed to Kodak XAR-5 X-ray film for auto- radiography. Radioactive bands were cut out and counted in a Packard Tri-Carb 1500 scintillation counter.

cDNA library construction

Poly(A) + RNA was isolated from two-week-old cotyledons, 24 h after treatment with 10mM ethephon. First-strand synthesis of cDNA was done as described by Maniatis et al. [ 11 ]. Second strand synthesis was performed as described by Gubler and Hoffman [9] using RNase H and DNA polymerase I. The ends were filled in by incubating with reverse transcriptase (Life Sciences Inc., St. Petersburg, FL) and Klenow polymerase (Bethesda Research Laboratories) for 30 min at 37 °C. Phosphorylated EcoRI linkers (New England Biolabs, Beverly, MA) were added by ligation and cleaved with an excess of EcoRI. The cDNA was fractionated by chromatography

over Sepharose CL-4B [11] to remove excess linkers and cDNAs smaller than 400 bp in length. The double stranded cDNA was then ligated at a 1 : 80 ratio by weight to lambda Zap vector DNA (Stratagene, San Diego, CA) that had been digested with EcoRI and dephosphorylated. The resulting DNA was packaged using Gigapack Gold extracts as directed by the supplier (Stratagene). The library was amplified according to Maniatis et al. [ 11 ]. Aliquots were frozen in a final concentration of 7.5~o v/v DMSO at - 8 0 °C.

Screening by plaque hybridization

Duplicate nitrocellulose filters prepared as described [11] were prehybridized in 3 × Den- hardt's solution, 8 × SSC, 50mM Tris pH 8, 0.1~o SDS, and denatured salmon testes DNA at 100/ag/ml at 25 ° C for 2 h. Each filter was hybrid- ized with 1-2 x 10 6 cpm/ml peroxidase mixed oligonucleotide probe in the prehybridization buf- fer minus salmon DNA at 25 °C for 16 h. Final washes were done in 2× SSC, 0 .1~ SDS at temperatures described in the text.

DNA extractions, restriction digests and subcloning into Bluescript plasmid and phage

Phage were grown by inoculating one liter of media (NZYM) as described by Parkinson and Houts [16] and then concentrated by PEG precipitation and CsC1 banding [11 ]. The phage DNA was extracted using formamide as described by Davis et al. [6]. The lambda Zap phage were converted into pBluescript phagemids according to manufacturers instructions (Strata- gene). Briefly, lambda-infected cells were super- infected with fl phage allowing the lambda inserts and sequences required for phagemids to replicate and circularize. Fresh cells were infected with the phagemid and plasmid-containing cells were selected by ampicillin resistance. Several trans- formed colonies were grown and plasmid DNA was isolated and restricted to verify faithful con-

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version of the lambda Zap insert. Phagemid DNA was extracted by the alkaline lysis method [11 ] from 5 ml L-broth cultures. The DNA was digested with restriction enzymes according to the suppliers' directions (Bethesda Research Labora- tories, New England Biolabs). Bluescript phage were induced by superinfection of the phagemid carrying strains with fl phage according to the Stratagene directions. Single-stranded DNA from Bluescript phage was isolated following the directions supplied by Stratagene.

Sequencing

DNA sequences were determined by a combi- nation of the chemical method of Maxam and Gilbert [13] and the dideoxy chain termination method of Sanger et aL [22]. Ends of inserts were labeled at the vector AvaI sites and digested with appropriate second enzymes for DNA sequence analysis by the chemical method. The dideoxy sequencing used a combination of single-stranded DNA isolated from phage and double-stranded DNA from pBluescript plasmid DNA that had been eluted through an EluDtip (Schleicher and Schuell). The sequencing procedure used with Sequenase (USB, Cleveland, OH) was performed as indicated by the manufacturer's instructions. Initially KS and SK primers (Stratagene) were used in the sequencing protocols and then primers obtained from Research Genetics (Huntsville, AL) that were complementary to the sequenced DNA were used to continue the sequencing. Just prior to gel electrophoresis all the sequencing reactions were heated to 80 °C for 3 min and samples were loaded on an 8 M urea-6~ poly- acrylamide gel and electrophoresed for varying amounts of time. The gels were dried onto Whatman 3MM paper on a gel dryer and exposed directly to X-ray film at room temperature.

Sequence data was analyzed using PC/GENE (Intelligenetics, Mountain View, CA) and DNA Inspector (Textco, West Lebanon, NH) computer software.

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Southern analysis

Southern analysis was done using an Oncor Probe Tech I apparatus, Oncor membranes and Oncor reagents according to manufacturers direc- tions (Oncor, Gaithersburg, MD). Total DNA used for the Southern analysis was isolated from cucumber cotyledons [15]. A 530bp HindlII fragment (bases 60-590) of pCuPer2 was hybrid- ized with PstI, HindlII, HinclI, and Sau3A digests of total cucumber. AvaI digested pCuPer2 was also probed in amounts equivalent to 0.3 copies, 1.5 copies, and 7 copies per haploid genome [ 18]. The blot was washed with a low stringency wash of 2 x S SC at 25 ° C and exposed to film. A duplicate blot was then washed with a higher stringency of 0.1 × SSC at 52 °C and exposed to film. The first blot was then washed progressively more stringently (37 °C, 2 x SSC followed by 45 ° C, 2 × SSC) and exposed to film.

Results

Determination of the most efficient oligonucleotide probe by RNA blot screening

Four groups of mixed oligonucleotides, each containing 256 different sequences, were synthe- sized to correspond to the antisense strand of the previously determined [1] partial peroxidase amino acid sequence GAHTFG(S) (see Table 1). The terminal serine residue was misread in the

amino acid sequence, so in fact only 18 bases out of 20 bases were appropriate. Each of the four oligonucleotide groups was used to probe RNA blots of control and ethylene-treated poly(A) + cotyledon RNA. Only probe 2 hybridized preferentially with the RNA from the ethylene- treated cotyledons. We then proceeded to isolate putative peroxidase cDNA clones that hybridized with probe 2.

Construction of a cDNA library from ethephon- induced cucumber RNA and isolation of putative peroxidase eDNA clones

Double-stranded cDNA was synthesized from poly(A) + RNA that had been extracted from cotyledons treated with 10mM ethephon for 24 h. A cDNA library of 115000 primary clones was constructed in lambda Zap from 8 ng of double-stranded cDNA. A total of 16 000 plaques were screened with probe 2 as described in Mate- rials and methods. After a 37 °C wash all the plaques exhibited an equal hybridization signal. After a 45 °C wash two plaques showed a preferential hybridization. After a 55 °C wash, none of the plaques gave a signal above background. The two putative plaques from the 45 °C wash were purified and shown by plaque hybridization to preferentially hybridize with eDNA probes made from ethephon-treated poly(A) + RNA versus control poly(A) ÷ RNA (data not shown).

Table 1. Sequence of four groups of oligonucleotide probes constructed corresponding to the antisense strand of the amino acid sequence GAHTFGS.

Probe No Sequence

GANCCA/GAAT/GGTATGNGCNCC

GANCCA/GAAT/GGTGTGNGCNCC

GANCCA/GAAC/AGTATGNGCNCC

GANCCA/GAA~AGTGTGNGCNCC

N = all four nucleotides at this location.

Restriction mapping of lambda and Bluescript clones

DNA from the two clones (lambda CuPerl and lambda CuPer2) that hybridized with probe 2 was prepared and subjected to restriction analysis. Insert size of the two clones was measured using the vector flanking PvuII and AvaI sites since the junction EcoRI sites were apparently lost during cloning. The cDNA insert of clone lambda CuPerl was 850 base pairs and that of clone lambda CuPer2 was 1150 base pairs. The inserts were converted into phagemids. DNAs prepared

from small plasmid cultures were digested with AvaI and PvulI to confirm the fidelity of the plas- mid conversion. A restriction map was then con- structed to determine useful sites for sequencing and to determine the orientation and similarity of the two clones (see Fig. 1). Restriction analysis of the two clones suggested that they were identical except that clone pCuPer2 had 300 more bases of the 5' end of the gene than did clone pCuPerl.

Sequencing of clones

Initially the ends of the two clones were sequenced by the Maxam-Gilbert technique from the pBluescribe vector A va I ends. This confirmed the identity of the 3' ends by the presence of a poly(A) + tail and provided information on the internal sequence by sequencing of the 5' end of the smaller pCuPerl clone. Both single and double stranded DNA was then used as substrate for the dideoxy method of sequencing as described in Materials and methods. Figure 1 schematically presents the sequencing strategy.

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The 1126 base nucleic acid sequence and predicted amino acid sequence from the insert of CuPer2 is shown in Fig. 2. The 819 base nucleic acid sequence of CuPerl differs at two positions from the CuPer2 sequence. A C to T difference at position 470 of the CuPer2 sequence does not affect the predicted amino acid sequence and a C to T difference at position 691 changes a proline to a leucine. The CuPer2 clone has one long open reading frame of 876 bases followed by a TAA stop codon that would encode a protein of Mr 32 576 with a predicted pI of 6.07. There is a potential polyadenylation signal AATTAAA starting at 19 bases before the poly(A) + tail. The probe sequence used to isolate the peroxidase clones is found starting at base number 484. The homology of one of the 256 oligonucleotides in probe 2 is perfect except for the serine encoding residues. There are, however, discrepancies between the partial amino acid sequence previ- ously determined from the 33 kDa peroxidase ( D L V S L S G A H T F G R S R N R F F ) [1] and that predicted from the cDNA sequence (DLVA LSGAHTFGKSRCQ FF).

D-SK . d D-P4

M -G D-P6

D-KS i l l ,

M-G

Hindlll Pstl Bglll Hindlll H~dlll EcoRV

I , ', I[ I I I I I I I I I

0 100 300 400 500 600 700 800 900

"~ S'P3

200

d l

D-KS S-P4

4 D-P5

D-P6

I I 1000 1126

S'SK

~ID M-G T

Fig. 1. Restriction map and sequencing strategy of the cucumber putative peroxidase clones pCuPerl and 2. The sequence corresponding to the coding region is indicated by the hatched box. The direction of the arrows indicate which strand was sequenced: the arrowhead to the right is the sense strand and the arrowhead to the lett is the antisense strand. Arrows below the line represent data derived from the pCuPer2 clone while the arrows above the line represent data derived from the pCuPer 1 clone. M-G indicates sequences obtained by the Maxam-Gilbert sequencing method, S- and D- indicates sequences obtained by using the Sanger dideoxy technique on single-stranded and double-stranded DNA, respectively, and -KS, -SK, -P3, -P4, -P5,

and -P6 indicate which primer was used.

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T F Y D E S C P D V S N I V R R V V Q Q

ACGTTTTACGACGAATCATGCCCCGACGTA TCCAACATTGTGCGCCGTGTGGTACAACAA 60

A L V S D E R A G A R L I R L H F H D C GCTTTGGTCTCTGACGAACGTGCTGGTGCC AGACTCATTCGACTTCATTTCCACGACTGC 120

F V N G C D G S V L L E D Q P G V V S E TTCGTAAATGGGTGTGATGGGTCGGTTTTA CTAGAGGACCAACCTGGTGTGGTGAGTGAG 180

L A A P G N A N I T G F N I V N N I K A CTTGCCGCTCCTGGAAATGCCAATATTACA GGGTTTAACATTGTTAATAACATCAAAGCT 240

A V E K A C P G V V S C A D I L A I A S GCAGTCGAAAAGGCTTGCCCTGGTGTTGTT TCATGTGCTGACATCTTAGCTATAGCTTCC 300

V G S V N L A G G P C W E V Q L G R R D

GTCGGATCAGTTAATTTGGCAGGAGGACCG TGTTGGGAGGTACAACTAGGAAGAAGAGAC 360

S R R A N L Q G A I D G L P S P F E N V AGTAGAAGGGCAAATTTACAAGGCGCAATA GATGGCCTTCCAAGTCCTTTCGAAAATGTT 420

T Q L K R K F D R V D L D S T D L V A L

ACACAACTTAAACGCAAGTTCGATAGAGTA GATCTTGATTCTACTGATCTCGTTGCCTTA 480

S G A H T F G K S R C Q F F D R R L N V TCTGGTGCACACACATTCGGGAAATCGAGA TGTCAATTTTTCGACCGACGTTTAAACGTT 540

S N P D S T L N P R Y A Q Q L R Q A C S TCAAATCCAGATAGCACGCTGAATCCCAGG TATGCACAACAGCTCCGTCAAGCTTGCAGT 600

S G R D T F V N L D P T T P N K F D K N AGCGGCCGAGACACGTTTGTGAATCTGGAC CCCACGACTCCCAACAAGTTCGACAAGAAC 660

Y Y T N L Q S N T G P L T S D Q V L H S

TACTACACAAACCTTCAGTCCAACACCGGC CCTCTCACCAGTGACCAAGTCCTTCATTCC 720

T P G E D T V K I V N L F A A S Q N Q F ACTCCCGGCGAAGACACCGTCAAAATCGTC AACCTCTTCGCTGCCAGCCAGAACCAGTTC 780

F E $ F G Q S M I N M G N I Q P L T G N

TTCGAAAGCTTCGGCCAGTCCATGATCAAC ATGGGAAATATCCAACCGTTGACTGGTAAC 840

Q G E I R S N C R R L N CAAGGAGAAATTAGATCCAATTGCCGAAGG CTGAACTAAAATTATATATATGATATCATA 900

TATGGTGGTTCCGTTACGGTCAAACTCAAT TGTATTATAATAATCATATCTTGTTTTTGT 960 GGTTGCTTTTTATTGTTCGAGGGGAAAAAG TGGGTTTTGATCAATTTTCTCTTGTGGCTT 1020 AAAATGAGAGAGTGCATGGGGATGCCAAAC ATCAAAGCTCAAAAACATATAGTGTCTTTT 1080 GTTCTATATATGATCAATAAAAGTGGTGTC TTTCTATTTTAAAAAA 1126

Fig. 2. Nucleofide sequence of cucumber peroxidase cDNA CuPer2 and the pre~cted amino acid sequence. The translated amino acid sequence is above the DNA sequence. The 5' end of CuPerl is underlined ~ base 307. The C's at positions 470 and 691 th~ ~e Ts m the CuPerl sequence are shown in outline. The sequence corresponding to We probe 2 sequence is underlined

base 483. The long open reading ~ame ends ~ the underlined stop codon TAA ~ base 877. There is a potenti~ polkA) sign~ site underlined at base 1096.

Comparisons with known plant peroxidase se- quences

The CuPer2 nucleic acid sequence was compared to the coding regions of published potato [ 21] and tobacco [ 10] peroxidase nucleic acid sequences. The tobacco sequence had 5 8 ~ identity, with 10~o o f the mismatches being in the third base

resulting in no change in amino acid. The potato sequence had 51 ~ identity, with 7 ~/o of the mis- matches being in the third base resulting in no change in amino acid.

Figure 3 shows the comparison of the CuPer2 predicted amino acid sequence and published peroxidase sequences from tobacco [ 10], horse- radish [27], potato [21], and turnip [14]. Based

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Cucumber

Tobacco

Horserad

Turnip

Potato

Cucumber

Tobacco

Horserad

Turnip

Potato

Cucumber

Tobacco Horserad

Turnip

Potato

Cucumber

Tobacco

Horserad

Turnip

Potato

Cucumber

Tobacco

Horserad

Turnip

Potato

Cucumber

Tobacco

Horserad

Turnip

Potato

Cucumber

Tobacco

Horserad Turnip

Potato

c* h'c* c*

.... TFYDES CPDVSNIVR- RWQQALVSD ERAGARLIRL HFHDCFVNGC

QLSA .... TT ..~.TS...- G.MD.RQRT. A .... KI .............

ZLTP .... N. ..~ ...... - DTIVNE.R.. P.IA.S.L ............

ZLTTN..ST. ..NLLST.KS G.KSAVSSQP A.M..S.L.. F .........

QLTP .... .A .VFSA--..- G..DS,IDAE T.M.,S ........... D..

c* i00

DGSVLLED-Q PGVVSELAAP GNA~--ITGF NIVN-NIKAA VEKACP-GVV

...I..D--T D.TQT.KD.. --..VGAG.. D..D-D..T. L.NV..-...

.A.I..DN-T TSFRT.KD.F .... -SAR.. PVID-RM ..... S...-RT.

...I..D.-T SSFTG.QN.G P.R.-SAR.. TVI.-D..S ....... -...

..GI..D.IN GTFTG.QNS. P...-SAR.Y EVIAQAKQSV IDT-..NIS.

c* 150

SCADILAIAS VGSVNLAGGP CWEVQLGRRD SRRANLQGAI DGLPSPFENV

....... L.. EIG.V..K.. S.Q.LF..K. .LT..RS..N SDI ..... TL

.... L.T..A QQ..T ..... S.R.P ...... LQ.F.DL,N AN..A..FTL

......... A RD..VQL... N.N.KV .... AKT.SQAA.N SNI.A.SMSL

......... A RD.,AKL..Q TYN.A...S. A.T..FT..L TQ..A..D~L

h* c* 200

TQLKRKFDRV DLD-STDLVA LSGAHTFGKS RCQFFDR-RL ~VS .... NPD

AVMIPQ.TNK GM.-L ............. RA ..GT.EQ-.. FNF~GSG...

P...DS.RN. G.NR.S ....... G ..... N Q.R-.IMD.. YNFSNTGL..

S..ISS.SA. G.STR-.M ........ I,Q. ,.VN.-.A,V YNETN .....

.VQIQ..NDK NFT-LREM.. .A .... V,FA ..STVC ........ TSG.--

c* 250

STLNPRYAQQ LRQ-AC .... S-SGRDTFVN LDPTTPNKFD KNYYTNLQSN

L.VDATFL.T .-.GI.FQG- G-NNGA..T. ..IS...D.. ND.F ...... P,,~TT.L.T ,,-GL.PLN- G-NLSALVDF -.LR..TI., NK..V,.EEQ

--I.AAF.-T ...RS.PRAA G-..DANLAP ..INSATS.. NS.FK..MAQ

--V..--.A. .-.C~.---- .ATLT.SDLQ QLD...TM.. .V..D..NN.

3OO

TGPLTSDQVL HSTPGED-TV KIVNLFAASQ NQFFESFGQS MINMGNIQPL

Q.L.QT..E. F..S.SA-.I A...RY.G.. T...DD.VS. ..KL...S.,

K.LIQ...E. F.S.~ATD.I PL.RS..~.T QT,.NA.VEA .DR .... T..

R.L.H ..... F-NG.STDS- -..RGYSN.P .S.NSD.AAA ..K..D.S..

Q.IMF ..... TGDATTAGF. ---TDYSNDV SV.LGD.AAA ..K..DLP.S

c* 320

TGNQGEIRSN CRRLN

..TN.Q..TD .K.V.

..T..Q..L. ..VV.SNS

..SS .... KV .GKT. A.A.L...DV .S.V~PTSVA SM

Fig. 3. Mature peroxidase protein sequence comparisons. The predicted peroxidase protein sequences encoded by the cucumber pCuPer2 clone (this report), the tobacco [10] and the potato [21] are shown with the amino acid sequences of horseradish (horserad) [27] and turnip [14]. Only the cucumber sequence is shown in full; other sequences are shown by a dot for homology or where they differ from the top sequence. Dashes represent a gap introduced in the sequence to maximize homology, h* represent consensus histidine residues potentially involved in the heine binding sites, c* represent consensus cysteine residues potentially involved in formation of disulfide bridges. The horseradish peroxidase has four such bridges at amino acid residues 11-95, 45-50, 102-311, and 182-216. Underlined N's represent potential carbohydrate attachment sites based on those found

in horseradish peroxidase.

on the other plant peroxidase amino acid se- quences , the predicted CuPer2 amino acid se- quence starts 4 amino acids from the amino- terminal end o f the expected mature protein. The percentages o f identity o f CuPer2 amino acid residues to the peroxidases are: t obacco 55~o;

horseradish 5 3 ~ ; turnip 45~o ; and potato 41~o. The single amino acid change for the predicted C u P e r l amino acid sequence would change the proline at posi t ion 253 to a c o n s e n s u s leucine. Us ing information from the horseradish sequence [27] , the locat ions o f 8 cysteine residues involved

722

in disulfide bridges and two histidine residues involved in heme binding are conserved in all the sequences (indicated in Fig. 3). However, there were 8 glycosylation sites specified by Asn-X- Thr/Ser amino acid sequences and identified in the horseradish information that do not appear to be conserved.

Steady-state levels of the mRNA after ethephon and ethylene treatment

To measure the rate of accumulation of CuPer2 mRNA in intact cucumber seedlings treated with ethephon, total RNA was isolated at 0, 1, 3, 5, 8, 15, 18, 24, 36, and 48 hours from ethephon- treated seedlings. The RNA populations were size-fractionated and hybridized with radio- actively labeled pCuPer2 DNA as described in Materials and methods (Fig. 4). The transcript size was estimated to be 1300 bases or approxi- mately 170 bases larger than the insert size of pCuPer2. Increased levels of CuPer2 mRNA were detectable by 3 hours after ethephon treat- ment and plateaued by 15 hours. There is a 10-20-fold increase in the steady-state level of the

Fig. 4. Expression of peroxidase RNA following ethephon induction in cucumber cotyledons. Autoradiograph of north- ern blot of RNA extracted at various amounts of time after exposure to ethephon and hybridized with labeled pCuPer2 DNA. The X-ray exposure was at room temperature with no intensifying screens for two hours. The transcript size of 1300 bases was determined from Hpa II digested T7 DNA resolved

on the same gel.

mRNA based on the amount of radioactivity hybridized at each time point (Fig. 5).

We wanted to test (1)whether induction with ethylene gas was similar to induction with ethephon, and (2)whether a related coding sequence with a distinct 3' untranslated region might be the mRNA that is actually induced rather than our cloned RNA. Hence northern analyses with RNAs from air-treated and ethylene-treated cotyledons were hybridized with a coding region probe (bases 60-590 of Fig. 2) and a 3' untranslated region probe (bases 895-1126). Quantitation of those northern analy-

z 1 0 0 ~ O

80 W

x 60 w

40

x < 20

0 0 8 1 6 2 4 3 2 4 0 4 8

ETHEPHON

CuPer2 probe

ETHYLENE coding probe

--_ noncoding probe

AIR " ~ O ' - coding probe

noncoding probe

HOURS OF TREATMENT

Fig. 5. mRNA accumulation after ethephon, ethylene, and air treatment. Radioactive bands were excised from northern blots and quantitated in a scintillation counter. The counts were normalized to 100 % for the peak expression detected with each probe. The X's represent the data from Fig. 4. The filled symbols were probed with the coding sequence (coding probe) and the open symbols were probed with the 3' end untranslated sequence (non-coding probe). The pattern of accumulation of CuPer2 related mRNA is essentially identical following ethephon or ethylene treatment and essentially identical when detected with coding or

3' end probe. Increased accumulation of peroxidase RNA is seen by 3 hours and plateaus by 15 hours.

ses (Fig. 5) indicates that the accumulation after ethylene treatment is indistinguishable from the ethephon treatment and that the apparent mRNA accumulation patterns do not differ when meas- ured with a 3' untranslated part of the clone versus a probe entirely within the coding sequence. The small increase in mRNA accumu- lation seen in the air controls is presumably due to the stress from being placed in the Plexiglass chamber.

Determination of number of genes

Southern analysis using the coding region probe described above was done to determine the num- ber of genes in the genome. Different stringencies of washes (as described in Materials and methods) were done to see if there were additional related genes. Only the specific bands shown in Fig. 6 were detected in all the washes. The lower- stringency washes showed smeared backgrounds. The low number of bands that hybridize indicate

Fig. 6. Southern analysis ofcucumberDNAwith the coding region probe ofpCuPer2. Total cucumber DNA was digested with Pstl (P), HindlII (H3), HinclI (H2), and Sau3A (S). Eight #g of undigested DNA(U) and 0.3, 1.5, and 7 gene copy equivalents (8 pg of a 1140 bp is equivalent to 1 gene copy/haploid genome/6 #g of total DNA) were resolved along with 6 #g of the digested DNAs on a 0.7% agarose gel and transferred to a nylon membrane. One sequence environ- ment is detected. Copy number appears to be 1-5 copies per

haploid genome.

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that only one sequence environment is hybridiz- ing. The strength of the signal compared to recon- struction experiments indicates that there are 1-5 copies of the sequence per haploid genome.

Discussion

We have constructed a cDNA library from ethephon-treated cotyledons and isolated two cDNA clones that hybridized to a synthetic probe that was based on a partial amino acid sequence of an ethylene-induced 33 kDa peroxidase. DNA sequencing analysis detects only two single base differences between the two clones, one of which leads to a single amino acid difference in the predicted amino acid sequence. Ethephon or ethylene treatment of cotyledons results in increased steady-state levels of a 1300 base mRNA by three hours that is highly homologous to both the coding regions and the 3' untranslated regions of the clones.

Our deduced amino acid sequence for these cDNA clones shows homology with the amino acid sequences of a horseradish cationic 15er- oxidase [27], a turnip cationic peroxidase [ 14], a potato anionic peroxidase [21], and a tobacco anionic peroxidase [ 10]. The amino acid sequence identity is 55~o to tobacco, 53 ~o to horseradish, 41 ~o to potato, and 45 ~ to turnip. These levels of identity strongly suggest that the clones encode peroxidases. There are three highly conserved regions, the first being from amino acid residues 37-57 with a predicted disulfide bridge in the middle and a potential heme binding site. The second is from amino acid residues 94-109, again with two cysteines that may form disulfide bridges. The third region is at amino acid residues 167-178, with a potential heme binding site in the middle. There are other smaller regions of homol- ogy, but these are not directly related to potential disulfide bridges or to potential glycosylation sites. The function of these conserved regions is not known but may play a role in the positioning of the heme moiety. The three Asn-X-Thr/Ser amino acid sequences identified in the cucumber sequence are normally associated with glycosyla-

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tion sites. In the cucumber putative peroxidases there is a region at amino acid residues 98-119 that could be predicted using the method of Rao and Argos [19] to be a membrane-spanning region. The horseradish and tobacco amino acid sequences, but not those of potato and turnip, also predict a transmembrane helix of at least 14 residues in the same region when the parameters are set for peak minimal value = 1.11, base line value = 1.05, and minimum length of transmem- brane helix = 14.

Discrepancies in the amino acid sequence determined from the protein and that predicted from the cDNA sequence suggest that the clone does not encode the 33 kDa peroxidase that we had described earlier. We consider the identifi- cation of our clones as peroxidases to be only putative because we do not have a purified protein with identical sequence that shows peroxidase activity. There is also a discrepancy between the observed isoelectric point of the protein (pI 8.4) and the isoelectric point predicted from the cDNA sequence (pI 6.07), but these differences could be due to post-translational modification. We might expect the synthetic probes to hybridize to related peroxidase sequences because they were made to correspond to a highly conserved amino acid sequence. Furthermore, only two cDNAs were isolated in our screen-this is not likely to be exhaustive. It is possible that the cloned sequences correspond to the 60 kDa per- oxidase (pI 4.0) that migrates as a 33 kD protein on a denaturing gel [ 1 ], but comparisons of the amino acid content of the cloned sequences with those of the 33 kDa and 60 kDa peroxidases were inconclusive for identification purposes (data not shown). Because the Southern analysis detects only one sequence environment and because the northern analyses indicate an accumulation of mRNA with both coding and non-coding region probes, we believe that a mRNA corresponding to one or both of these cDNA clones (rather than a related sequence) accumulates following exposure to ethylene. Our hybridization conditions could not distinguish between transcripts from our two highly related clones, however, and thus it is pos- sible that one or the other does not actually

accumulate. Although it is conceivable that the differences in the cDNA sequences are due to cloning artifacts, we suspect that the two sequences are due to the cucumber cultivar being heterozygous for that locus or that the population of seeds from which the cotyledons arose was not completely homogeneous.

The isolation of these putative peroxidase- encoding cDNAs should help decipher the role of this enzyme in ethylene-induced senescence as well as in other physiological responses (wounding, development, and lignification) in which peroxidases have been implicated. It should also lead directly to the isolation and characterization of genomic sequences that respond to ethylene treatment.

Acknowledgements

Advice and synthesis of the four original per- oxidase oligonucleotide probes was provided by Ann L. Abeles and Marilyn Powers of the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD.

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