Gene, 134(1993)217-221 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/SO6.00 217

GENE 07454

Short Communications

Isolation and characterization of a gene involved in ethylene biosynthesis from Arabidopsis thaliana

(Amino-cyclopropane-carboxylic acid (ACC) oxidase; cDNA; cloning; ethylene forming enzyme)

Miguel Angel Gbmez-Lim, Victor ValdCs-Lbpez*, And& Cruz-Hernandez

and Luis Jorge Saucedo-Arias

Department of Genetic Engineering, CINVESTAV, Apurtado Postul629. Irapuatu, GTO, Mexico

Received by F. Bolivar: 13 January 1993; Revised~Accepted: 14 June/16 June 1993; Received at publishers: 27 July I993

SUMMARY

The ethylene forming enzyme (EFE) is a key factor in ethylene biosynthesis. To understand better the regulation of ethylene biosynthesis in vegetative tissues, we set out to isolate and characterize a complementary DNA (cDNA) encoding the EFE from Arabidopsis thaliana. An A. thaliana cDNA library was screened with pTOM 13, a tomato cDNA coding for the EFE. A cDNA clone (pEAT1) was isolated. The cDNA is 1200 nucleotides (nt) in length and predicts a protein of M, 36663. The insert includes the complete open reading frame of 972 bp and shows strong homology with several reported sequences, both at the nt and amino acid level, In whole seedlings, expression of pEAT1 was enhanced by wounding, ethrel, Fe’+, and 1-amino-cyclopropane-carboxylic acid (ACC) treatments. In contrast, heat shock had no effect on the expression.

INTRODUCTION

Ethylene is an endogenous regulator of many develop- mental processes, from seed germination to fruit ripening and leaf abscission in plants (Abeles, 1973). The biosyn- thetic pathway of ethylene has been established:

Correspondence to: Dr. M.A. Gomez-Lim. Department of Genetic

Engineering, CINVESTAV, Apartado Postal 629, Irapuato, GTO,

Mexico. Tel. (52-462) 5-16-00; Fax (52-462) S-12-82.

*Present address: Department of Biology, Faculty of Sciences and

Department of Plant Molecular Riology, Institute of Biotechnology,

UNAM, Ap. Postal 62 271, Cuernavaca, Morelos, Mexico. Tel.

(52-73) 13-99-88.

Abbreviations: A., A~~b~~opsjs; aa, amino acid(s); ACC, l-amino-

cyclopropane-carboxylic acid; bp, base pair(s); EFE, ethylene-forming

enzyme; EFE, gene (DNA) encoding EFE; kb, kilobase or 1000 bp;

nt, nucleotide(s); ORF, open reading frame: SAM, S-adenosyl-

methionine; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/O.OlS M

Na,citrate pH 7.6: SSPE, 0.18 M NaC1 10mM NaH,PO,

pH 7.4/l mM EDTA.

Methionine is converted to ethylene with S-adenosyl- methionine (SAM) and l-aminocyclopropane-l-carboxy- lit acid (ACC) as intermediates (Kende, 1989). These two reactions are catalyzed by ACC synthase and the ethylene forming enzyme f EFE), respectively. The occur- rence of both reactions is known to increase dramatically during some developmental processes of plant growth like fruit ripening and petal wilting resulting in increased ethylene production.

The key regulatory step in ethylene biosynthesis is the formation of ACC, its conversion into ethylene being mainly a constitutive reaction (Yang and Hoffman, 1984; Theologis, 1992). The cloning of the genes coding for ACC synthase and EFE is a prerequisite for the study of the regulation of ethylene biosynthesis at the molecular level. ACC synthase has been cloned from various sources and its expression analyzed at the molecular level (re- viewed by Theologis, 1992). EFE has also been cloned and studied at the molecular level (Hamilton et al., 1991; McGarvey et al., 1991; Callahan et al., 1992; Pua et al.,

218

1992; Ross et al., 1992). Unlike ACC synthase, EFE is

constitutively expressed in most vegetative tissues and is

induced during fruit ripening and by several stimuli

(Yang and Hoffman, 1984).

The system most intensively studied in this respect is

the production of ethylene by fruits during ripening. To

help understand the regulation of this enzyme in vegeta-

tive tissues, we set out to isolate and characterize the

EFE gene from A. thaliuna and study its expression under

different experimental conditions.

EXPERIMENTAL AND DISCUSSION

(a) Isolation and identification of a ethylene-biosynthesis-

related cDNA

We screened a hGEM-2 cDNA library prepared from

whole Arabidopsis plants (Columbia strain) according to

Huynh et al. (198.5) with the insert of pTOM13 (encoding

the EFE gene from tomato) labeled by random priming

(Feinberg and Vogelstein, 1984) to a specific activity of

6 x lo* dpm/ug. Hybridization was carried out for 18 h

at 60°C in 5 x SSPE (20 x SSPE is 3.6 M NaC1/200 mM

NaH,PO, pH 7.4/20 mM EDTA)/S x Denhardt’s

solution/O.1 % SDS/100 mg denatured salmon sperm

DNA/l x lo6 dpm probe (per ml). Filters were washed

with increasing stringency with the final wash being

0.1 x SSPE/O. 1% SDS at 55°C. Three positives were iden-

tified from an initial screen of approximately 7.5 x 10”

recombinant phage and the one containing the largest

insert (PEAT) was selected for further studies. The cDNA

insert was subcloned into pBluescript as an XhaI-EcoRI

fragment.

(b) Structure of the pEAT1 cDNA

The 1200-bp cDNA sequence (Fig. 1) contains a 323-aa

ORF which begins with the ATG codon nearest to the

5’ end. This ORF predicts a M, 36663 protein, which is

comparable to that of the tomato EFE protein (33 512)

(Holdsworth et al., 1987). pEAT1 contains a poly(A) se-

quence of 16 nt and several potential polyadenylation sig-

nals at the 3’ end, including the animal consensus

sequence AATAAA and the most conserved plant polya-

denylation signal AATAAT. This is identical to the

tomato EFE gene (Holdsworth et al., 1987). A search in

the GenBank database revealed no homology to other

sequences except to those belonging to the superfamily

of Fe’+/ascorbate oxidases to which EFE belongs

(McGarvey et al., 1992).

(c) Genomic organization of EFE-related sequences

A. thuliana genomic DNA was cut with different re-

striction enzymes and fractionated on a 0.7% agarosc gel

before being transferred to a nylon membrane. The result-

ing filter was hybridized to the labelled pEAT1 cDNA

insert. This analysis revealed one strongly hybridizing

band for for each restriction enzyme (Fig. 2). These data

suggest that the corresponding sequence is probably rep-

resented once in the Arabidopsis genome. In addition,

other faint bands hybridizing to the probe were detecta-

ble at low stringency (data not shown) which suggests

that other partially related sequences to pEAT1 are

present.

This result was unexpected as it is known that in

tomato, apple and peach, EFE is a member of a small

multigene family (Holdsworth et al., 1987; Callahan et al.,

1992; Ross et al., 1992). Since EFE seems to be induced

by different stimuli, one functional gene in Aruhidopsis

may imply several promoter sequences responding to

different signals.

(d) Expression of pEATI-homologous mRNA

To study the expression of pEAT1, Arabidopsis seeds

were germinated and grown for 3 weeks and the resultant

plants subjected to different treatments (wounding, ACC,

ethrel, heat shock and Fe). After each treatment, the seed-

lings were frozen in liquid nitrogen and total RNA ex-

tracted and hybridized to pEAT1 in a Northern-type

experiment. Fig. 3 shows the results of these experiments.

The gene seems to be expressed in control plants, i.e.,

without any treatment, albeit at a low level, which con-

firms previous results and suggests that this gene is ex-

pressed constitutively in vegetative tissues. This result is

consistent with previous findings indicating that this

enzyme is expressed constitutively in most vegetative tis-

sues (Yang and Hoffman, 1984; Theologis, 1992). Heat-

shock treatment does not seem to affect significantly the

level of EFE mRNA. It has been shown that high temper-

atures inhibit ethylene production and that the inhibition

occurs at the EFE step (Steed and Harrison, 1993).

Recovery is relatively rapid and is followed by increased

ethylene production. Our results seem to indicate that

this inhibition might not be regulated at the transcrip-

tional level, but at a later step.

Wounding induces the expression of this gene several-

fold, similarly to what occurs in other systems like tomato

(Holdsworth et al.. 1987) and apple (Ross ct al.. 1992).

With ACC treatment, an increase in gene expression was

also observed, which is again consistent with previous

findings (Cameron et al., 1979). Application of ACC is

not as effective in preclimacteric fruits and flowers, which

have a very limited capacity not only to convert SAM to

ACC, but also to convert ACC to ethylene (Yang and

Hoffman, 1984) as in climacteric fruits. Fe’+ treatment

caused a detectable increase in the accumulation of EFE

mRNA. Fe’+ has been shown to be an essential factor

219

CGTTGCTGTC GAAGTTAGGC E K L N G E E AGAAGCTTAA TGGAGAAGAG VNH GISL

GTGAACCATG GGATTTCACT RF K E S I

GAGATTCAAG GAATCGATTA F Y L K xi L P

TCTACCTCAA GCACCTTCCC FAG KIEK

TTCGCCGGAA AGATAGAGAA KKV F Y G

AMAAAGGTG TTTTACGGGT LVK G L R A TAGTCAAGGG TCTCCGAGCC L L K D G E W

CTTCTTAAAG ACGGCGAGTG I TN G K Y

GATAACCAAT GGGAAGTACA S F Y N P G S CATTCTATAA TCCGGGAAGC EN Y P R F V

GAGAACTATC CGAGATTTGT E AM KAM

TGAAGCCATG AAAGCTATGG TAATAAATAT ATATATATAT GTTCATGTTG TTGTATGTTT

M E S F P I I N L 9 aa

CAAGAAACCC ATTTAAAAAA AAAGAGAGAG AGATGGAGAG TTTCCCGATC ATCAATCTCG 80 nt

R A’ I T M E K I K D A C E N W G F FEC 36 aa AGAGCAATCA CTATGGAGAA GATCAAAGAC GCTTGTGAAA ACTGGGGCTT CTTTGAGTGT 160 nt

E L L D K V E K M T K E H Y K K C M E E 63 aa CGAGCTTTTG GACAAAGTGG AGAAGATGAC CAAGGAACAT TACAAGAAGT GCATGGAAGA 240 nt K N R G L D S L R S E V N D V D W E S T 89 aa AGAACAGAGG TCTTGACTCT CTTCGCTCTG AAGTCAACGA CGTTGACTGG GAATCCACTT 320 nt

V S N I S D V P D L D D D Y R T L M K D 116 aa GTCTCTAATA TCTCCGATGT CCCTGATCTC GACGACGATT ACAGAACGTT AATGAAAGAC 400 nt

L S E E L L D L L C E N L G L E K G Y L 143 aa GTTGTCGGAG GAGCTACTGG ATCTGCTGTG CGAGAATCTC GGTTTAGAGA AGGGTTATTT 480 nt S K R P T F G T K V S N Y P P c P N P D 169 aa CGAAAAGACC GACTTTTGGA ACCAAAGTCA GCAATTATCC ACCTTGTCCT AATCCGGACC 560 nt

H T D A G G I I L L F Q D D K V S G L Q 196 aa CACACCGACG CCGGCGGCAT CATCCTCCTC TTCCAAGACG ACAAAGTCAG TGGACTTCAG 640 nt

V D V P P v K H S I VVN L G D Q L E V 223 aa GGTCGATGTT CCTCCGGTTA AGCATTCAAT CGTCGTTAAT CTCGGCGATC AACTTGAGGT 720 nt KS V E Ii RV L S Q T D G E G R M S I A 249 aa AGAGTGTGGA ACATAGAGTG CTATCTCAGA CAGACGGAGA AGGAAGAATG TCGATCGCAT 800 nt

D S V I F P V P E L I G K E A E K E K K 276 aa GACTCTGTTA TTTTTCCGGT GCCGGAGCTG ATCGGAAAAG AAGCAGAGAA GGAGAAGAAA 880 nt

F E D Y M K L Y S A V K F Q A K E P R F 303 aa GTTTGAAGAT TACATGAAAC TCTACTCTGC TGTCAAGTTT CAGGCCAAGG AACCAAGGTT 960 nt ET T V ANN V G P LATA * 323 aa AGACAACTGT GGCCAACAAT GTTGGACCAT TGGCCACTGC GTGAATGATA TGTAACTGGT 1040 nt ATATATATAG TCTTTATATA ATGTCTTAGA AACTTGATTA TTCACTATAC GAATAATTTT 1120 nt AAGTGGTGAA TGTGTTATAT ATGGGAATTA ATGTTTTCTG TTCGAAAAAA AAAAAAAAAA 1200 nt

Fig. 1. Complete nt sequence of the cDNA insert of pEAT1 and the deduced aa sequence. Three putative polyadenylation signals are underlined.

The sequencing was performed by the dideoxy chain-terminaton procedure using [35S]dATP and a double-stranded template procedure provided in

the Sequenase kit (US Biochemical, Cleveland, OH, USA). The entire insert was sequenced on both strands. The nt sequence data reported in this

paper have been submitted to the EMBL, GenBank and DDBJ nt sequence databases and have been assigned the accession number X66719.

nt ABCDEF

l-

Fig. 2. Southern blot analysis of genomic DNA from A. thuliuna. Total

DNA from A. hdianu leaves was digested with Xhal (lane A), EcoRI

(lane B) or Smal (lane C) and hybridized to the insert of pEAT1.

Methods: Genomic DNAs (10 ug) prepared from green tissues of

A. thalianu (Leutwiler et al., 1984) were digested to completion with

the corresponding restriction enzyme and separated by electrophoresis

on a 0.7% agarose gel. The fragments were transferred to Hybond-N

membranes (Amersham) and probed with a “P-labeled random-primed

EAT insert at 65°C (5 x SSC/O.OZ% SDS/5 x Denhardt’s solution

100 mg denatured salmon sperm DNA with 1 x lo6 dpm probe per ml),

washed in 0.1 x SSC/O.l% SDS at 65‘C and subjected to autoradiogra-

phy at -80°C with Cronex Lightning Plus intensifying screens. The

probe was prepared by oligo labeling (Feinberg and Vogelstein, 1983)

using [n-32P]dCTP.

Fig. 3. Northern blot analysis of pEATl-related sequences during sev-

eral experimental conditions. The analysis was performed in control

(untreated) plants (lane A); plants treated with 10 mM ACC for 4 h

(lane B); plants kept at 42°C for 3 h (lane C); plants treated with 1 mM

ethrel for 4 h (lane D); plants treated with 0.2 mM Fe for 4 h (lane E);

plants mechanically wounded and incubated for 3 h (lane F). The

number on the left indicates the approximate size of the message, deter-

mined using RNA standards (0.24-9.5 kb RNA ladder, Gibco BRL,

Gaithersburg, MD, USA). Methods: Seeds of the Columbia strain of

A. thaliana were grown in liquid medium ( Murashige-Skoog) with shak-

ing in constant light at 25-C. After 3 weeks the seedlings were subjected

to the different experimental treatments. When needed, the chemicals

were added directly to the incubation medium. Total RNA was subse-

quently extracted from whole plants (Lopez-Gomez and Gomez-Lim,

1992) and 20 ug were subjected to 2.2 M formaldehyde-l % agarose gel

electrophoresis and transferred to Hybond-N (Amersham). After UV

crosslinking, following the manufacturer’s instructions, the blot was

probed with randomly labeled pEAT1. Hybridization and autoradiog-

raphy were carried out as for Southern blots (see Fig. 3).

for the conversion of ACC into ethylene and to enhance

ethylene production (Lau and Yang, 1976; Bouzayen

et al., 1991). It is possible that the stimulation of ethylene

production by exogenous Fe’+ occurs at the transcrip-

tional level. Treatment of seedlings with ethrel (an ethy-

lene-releasing compound) also caused an increase in the

220

EFE mRNA level. This is in agreement with previous

results obtained with green tissues which showed an

increased capacity to convert ACC into ethylene after

ethylene treatment (Riov and Yang, 1982; Chalutz et al.,

1984). The effect of exogenous ethylene on plants is a

complex one. Exogenous ethylene can promote (autoca-

talysis) or inhibit (autoinhibition) its own synthesis de-

pending on the tissue and the time of application in

several fruits and vegetables (Mattoo and White, 1991).

At least in our system, ethrel seems to induce the level of

EFE mRNA severalfold over background.

It is well established that all plant tissues are capable

of producing ethylene, although the production rate is

normally low (Abeles, 1973; Yang and Hoffman, 1984).

So far, ethylene production has been predominantly

studied during fruit ripening. EFE-related sequences have

been cloned from three different fruits and two flowers.

Only in two cases has an EFE sequence been isolated

from vegetative tissues, that from Brussica junceu and

from A. thaliana. The availability of pEAT1 will permit

the evaluation of ethylene production by the different

organs of A. thaliana at the molecular level. A genomic

clone for pEAT1 has been recently isolated and the pro-

moter sequences are being currently investigated

(M.A.G.-L., L.J.S.-A., G.B. Xoconostle-Cazares and P.

Guzman-Villate, unpublished results). Current studies of

transformation of A. thaliana with pEAT1 will allow a

closer examination of the regulation of pEAT1 at the

molecular level.

(e) Homology to other EFE-related aa sequences

The derived aa sequence of pEAT1 was compared with

the tomato (Holdsworth et al., 1987) avocado

(McGarvey et al., 1992), carnation (Wang and Woodson,

1991) apple (Ross et al., 1992), Brassica juncea (Pua

et al., 1992) peach (Callahan et al., 1992) and petunia

(Wang and Woodson, 1992) sequences using the

GeneWorks program, version 2.2. The Arabidopsis pro-

tein exhibited a high degree of conservation with over

70% aa sequence identity with tomato, avocado, peach

and carnation. However, the identity between Arabidopsis

and apple and Brassica juncea is 59 and 50%,

respectively.

Sequences of several EFE-related proteins were aligned

with pEAT1, introducing gaps in the sequences to allow

the best fit (Fig. 4). The similarities between the different

proteins are apparent throughout the entire coding

region. Several regions of high sequence conservation are

obvious in the alignment.

(e) Conclusions

(I ) The EFE from A. thaliana is highly homologous

to the EFE from tomato (74% at the aa level). The sim-

Fig. 4. A comparison of the predicted aa sequence between

A. tkalianrr clone and related clones. Avocado (pAVOe3) is a clone

isolated from a ripe fruit cDNA library (McGarvey et al.. 1992). Peach

(pPch3 13) is a clone expressed during peach softening (Callahan et al.,

1992). Tomato (pTOM13) codes for EFE (Holdsworth et al., 1987).

Carnation (pSR120) represents a clone expressed during carnation

flower senescence (Wang and Woodson, 1991). A. thuhna (pEAT1) is

a clone expressed constitutively in vegetative tissues. Apple (pAP4) is

a clone expressed during apple fruit ripening (Ross et al., 1992). Petunia

(pPHEFE) is a clone expressed in senescing corolla tissue and polli-

nated pistil concomitant with ethylene production (Wang and

Woodson, 1992). Brassicu (pMEFE) is a clone isolated from vegetative

tissues from B. junea (Pua et al., 1992). Dashes represent gaps in the

sequences and dots represent residues similar to those of Arahidopsis.

The different sequences were analyzed and compared using the

GeneWorks program, version 2.2 (IntelliGenetics, Mountain View. CA).

ilarities in the hydropathy plot of various EFE-related

proteins suggest common structural features and a re-

lated function.

(2) By Southern analysis in stringent conditions only

one band is detectable in the A. thaliana genome, suggest-

ing one gene copy. Since EFE is induced by various stim-

uli, one functional gene in A. thulium may imply several

promoter sequences responding to different signals.

(3) EFE was expressed constitutively in vegetative tis-

sues from A. thaliana. Wounding, ACC, Fe’+ and ethrel

can induce EFE expression severalfold over the control

but heat shock did not have any detectable effect on the

EFE mRNA level.

221

ACKNOWLEDGEMENTS

We are grateful to Dr. L. Herrera-Estrella for providing

the A. thaliana cDNA library and to Prof. D. Grierson

for providing the plasmid pTOM 13. Our thanks to

B. Jimenez and to M. Gidekel for help with the sequen-

cing and the computer work respectively. Thanks are also

due to Dr. P. Guzman for helpful discussions and help

with the A. thaliana work and to Dr. J. Simpson for kindly

correcting the English version of the manuscript.

REFERENCES

Abeles, F.B.: Ethylene in Plant Biology. Academic Press, New York,

1973.

Bouzayen, M., Felix, G., Latche, A., Pech, J.C. and Boller, T.: Fe’+: an

essential cofactor for the conversion of l-aminocyclopropane-l-car-

boxylic acid to ethylene. Planta 184 (1991) 2444247.

Callahan, A.M., Morgens, P.H., Wright, P. and Nichols, K.E.:

Comparison of Pch313 (pTOM 13 homolog) RNA accumulation

during fruit softening and wounding of two phenotypically different

peach cultivars. Plant Physiol. 100 (1992) 482-488.

Cameron, A.C., Fenton, C.A.L, Yu, Y.B., Adams, D.O. and Yang, S.F.:

Increased production of ethylene by plant tissues treated with

1-aminocyclopropane-1-carboxylic acid. HortScience 14 (1979)

178180.

Feinberg, A.P. and Vogelstein, B.: A technique of radiolabeling DNA

restriction endonuclease fragments to high specific activity. Anal.

Biochem. 132 (1983) 6613.

Holdsworth, M.J., Bird, C.R., Ray, J., Schuch, W. and Grierson, D.:

Structure and expression of an ethylene-related mRNA from tomato.

Nucleic Acids Res. 15 (1987) 731-739.

Huynh, T.V., Young, R.A. and Davies, R.W.: Constructing and screen-

ing libraries in Xgt 10 and hgt 11. In: Clover, D.M. (Ed.), DNA

Cloning. A Practical Approach, Vol. I. IRL, Oxford. 1985, pp. 49978.

Kende, H.: Enzymes of ethylene biosynthesis. Plant Physiol. 91

(1989) 1-4.

Lau, O.L. and Yang, S.F.: Stimulation of ethylene production in the

mung bean hypocotyls by cupric ion. calcium ion and kinetin. Plant

Physiol. 57 (1976) 88.

Leutwiler. L.S., Hough-Evans, B.R. and Meyerowitz, E.M.: The DNA

of Arahidopsis thaliana. Mol. Gen. Genet. 194 (1984) 15523.

Lopez-Gomez, R. and Gomez-Lim, M.A.: A method for extracting

intact RNA from fruits rich in polysaccharides using ripe mango

mesocarp. HortScience 27 (1992) 440-442.

Matto, A.K. and White, W.B.: Regulation of ethylene biosynthesis. In:

Mattoo. A.K. and Suttle, J.C. (Eds.), The Plant Hormone Ethylene.

CRC Press, Boca Raton, FL. 1991, pp. 21-42.

McGarvey, D.J., Yu, H. and Christoffersen, R.E.: Nucleotide sequence

of a ripening-related cDNA from avocado fruit. Plant Mol. Biol. 15

(1991) 1655167.

McGarvey, D.J., Sirevag, R. and Christoffersen, R.E.: Ripening-related

gene from avocado fruit. Plant Physiol. 98 (1992) 5544559.

Pua, C.E., Sim, G.E. and Chye, M.L.: Isolation and sequence analysis

of a cDNA clone encoding ethylene-forming enzyme in Brussica

juncea (L.) Czern & Coss. Plant Mol. Biol. 19 (1992) 541-544.

Ross. G.S., Knighton, M.L. and Lay-Yee, M.: An ethylene-related

cDNA from ripening apples. Plant Mol. Biol. 19 (1992) 231-238.

Theologis, A.: One rotten apple spoils the whole bushel: the role of

ethylene in fruit ripening. Cell 70 ( 1992) 181-184.

Wang, H. and Woodson, W.R.: A flower senescence-related mRNA

from carnation shares sequence similarity with fruit ripening-related

mRNAs involved in ethylene biosynthesis, Plant Physiol. 96 ( 1991)

1000~1001.

Wang, H. and Woodson, W.R.: Nucleotide sequence of a cDNA encod-

ing the ethylene forming enzyme from Prtunia corollas. Plant

Physiol. 100 (1992) 535-536.

Yang, SF. and Hoffman, N.E.: Ethylene biosynthesis and its regulation

in higher plants. Annu. Rev. Plant Physiol. 35 (1984) 155-189.