Plant Molecular Biology 34: 759–770, 1997. 759c 1997 Kluwer Academic Publishers. Printed in Belgium.

Melon ascorbate oxidase: cloning of a multigene family, induction duringfruit development and repression by wounding

George Diallinas1;2;�, Irene Pateraki1;�, Maite Sanmartin1;2, Angela Scossa1;2,Eugenia Stilianou1, Nickolas J. Panopoulos2 and Angelos K. Kanellis1;2;+

1Institute of Viticulture, Vegetable Crops & Floriculture, National Agriculture Research Foundation,PO Box 1841, GR-711 10 Heraklion, Crete, Greece; 2Institute of Molecular Biology and Biotechnology,Foundation for Research and Technology-Hellas, PO Box 1527, GR-711 10 Heraklion, Crete, Greece (+authorfor correspondence; �the two first authors contributed equally)

Received 24 June 1996; accepted 23 April 1997

Key words: ascorbate oxidase, ethylene, fruit ripening, melon (Cucumis melo L.), repression, wounding

Abstract

A small family of at least four genes encoding melon ascorbate oxidase (AO) has been identified and three membersof it have been cloned. Preliminary DNA sequence determination suggested that melon AO genes code for enzymeshomologous to ascorbate oxidases from other plants and similar to other multicopper oxidases. We describe detailedmolecular studies addressing melon AO expression during organ specific differentiation, fruit development andripening, and in response to wounding. In particular, AO transcript accumulation was induced in ovaries and theouter mesocarp of mature preclimacteric melon fruits, before the expression of genes encoding the necessaryenzymatic activities for ethylene biosynthesis. On the other hand, AO was not expressed in late stages of fruitripening and was repressed in wounded fruits. The role of ethylene in transcriptional regulation of AO is discussed.

Introduction

Ascorbate oxidase (AO) is a copper containing metal-loenzyme found in higher plants. It catalyses thereversible oxidation of ascorbate (vitamin C) to 2-dehydro-ascorbate with the concomitant reduction ofmolecular oxygen to water. This enzyme is of specialinterest not only for its involvement in vitamin C meta-bolism [for reviews, see 11, 34, 40] in higher organ-isms, but also, for its complex and dynamic structureas well as its unknown function in plant metabolism[5, 9, 12, 13, 30–32, 36–41].

The most abundant natural sources of AO are mem-bers of the Cucurbitaceae (cucumber, zucchini, pump-kin, squash, melon, etc.), in which AO biochemistryand expression has been studied extensively [9, 20,22, 31, 41, 42]. Genes coding for AO have been isol-ated and sequenced [21, 27, 44–46]. AO is a cell wallenzyme [20, 32]; recent sequence analyses showed thatAO mRNA codes for a leader signal sequence typicalof extracellular proteins [21, 44]. AO transcript accu-

mulation, protein content and AO activity are highestin actively growing tissues and in the epidermis ofimmature fruits of Cucurbitaceae [2, 20, 21, 32, 41,44]. These and other observations led to the hypothes-is that AO is involved in cell wall loosening and cellwall reorganisation during the fast growing phase ofcucurbit fruits [20, 32, 41, 44].

AO genes have been sequenced from cucumber,pumpkin and tobacco [21, 27, 44–46]. The corres-ponding AO amino acid sequences from these plantsare highly conserved (75–82% identical amino acidresidues), and show similarity (28–30% identicalamino acid residues) to laccases and human ceruloplas-min, a group of blue multicopper oxidases possessingthree spectroscopically distinct copper centres [21, 25,37–40, 44]. In most cases, the role of these enzymesfound in organisms as distinct as bacteria, fungi, plantsand mammals is not well established.Biophysical char-acterisation of AO from Cucurbitaceae plants showedthat the active enzyme is a glycosylated homodimerof 136–140 000 Mr containing eight copper ions, rep-

GR: 201001975, Pips nr. 140348 BIO2KAP

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resenting one type I (blue copper centre), one type II(normal copper centre), and two type III (binuclearcopper centre) copper atoms per subunit [37–38]. Thecrystal structure of the resting form of AO from zuc-chini has been solved and refined at 1.9 A resolution.It showed that the two subunits, each consisting ofthree distinct domains, are arranged as tetramers withD2 symmetry, and the folding of all three domains isof a similar �-barrel type and related to plastocyaninand azurin [37–40]. The amino acid residues that bindeach copper atom, as well as an asparagine involvedin the attachment of the two N-linked sugar moieties,have been identified. The probable binding site for thereducing substrate has been proposed to be close to thetype I copper atom [39].

Although AO has been studied extensively at thelevel of expression and enzyme structure, its preciserole in higher plants remains unknown. AO activityshowed a significant increase during a specific periodof melon fruit development, suggesting that it mightplay an important role in cell wall metabolism duringfruit ripening [41]. Here, we identify a small mul-tigene family encoding melon AO and describe thecloning and initial characterisation of three AO genes.In addition, we performed detailed molecular studiesaddressing melon AO gene function relative to cell walllocalisation, organ specific differentiation, fruit devel-opment and ripening, and wounding. Our results estab-lish that AO expression is differentially regulated, atthe level of transcript accumulation, during fruit devel-opment and in response to wounding.

Materials and methods

Plant material

Melon seeds (Cucumis melo L., variety reticulatus,F1 alpha) were provided by Tezier Breeding Insti-tute, Velence, France. Melon plants were grown undergreenhouse conditions at 25/15 �C day/night temper-ature. Fruit were harvested at various developmentalstages (ovaries, 10-, 20-, 25-, 30-, 32-, 34-, 36-, 38-,and 40-day old after anthesis). For wounding experi-ments the melon mesocarp from 20- and 34-day oldfruit was separated from seeds and epidermis, slicedvery finely in uniform strips of 2 mm thickness usinga blade, placed in wet sterile Whatman paper in Petridishes and incubated at room temperature for 6, 12,and 24 h. For experiments studying spatial expres-sion of AO, ACS and ACO, inner, middle and outer

areas of mature (30-, 34-, 36-, 38-, and 40-day old)fruit mesocarp were separated using a blade. Samplesfrom 0-day represent ovaries while fruits 10- and 20-day old were whole mesocarp. Fruits 25-day old weredivided into two portions, inner and outer. In all casesdescribed above, tissue from three different fruit at thesame developmental stage were collected, immediatelyfrozen in liquid nitrogen, and stored at �80 �C. Mel-on tissue from seed, root, shoot, fast growing shootapex of mature plants, flower, and mature leaf was alsocollected and frozen at �80 �C.

Melon genomic DNA isolation

Total genomic DNA was isolated from melon ovarieswith a novel, simple and rapid method; frozen groundtissue (2–3 g) was homogenised in 15 ml of 50 mMTris HCl, pH 7.5, 20 mM EDTA, 0.7% sodium dodecylsulfate (SDS) using a Polytron apparatus and the mix-ture was incubated for 30 min at 65 �C. 4.5 ml of3 M potassium acetate, pH 4.8 were, then, added tothe mixture, left on ice for 30 min and then centri-fuged for 10 min at 10 000 g at 4 �C. The supernatantwas filtered through Miracloth (Calbiochem) (or blu-tex) and precipitated with 2 volumes of ethanol. Thismethod gave 50–100�g of good quality genomic DNAwhich was used for restriction, Southern analyses andPCR amplification reactions.

Standard techniques of bacteria and plasmidmanipulation

Plasmid preparations and manipulations were madeas described in Sambrook et al. [50]. Plasmid forsequencing was purified either by selective precipit-ation from 13% polyethylene glycol [50] or with theuse of QIAGEN purification columns. Plasmids wereintroduced in E. coli by transformation of competentcells as described in Sambrook et al. [50].

Construction of an AO probe

Based on comparative analyses of all AO DNAsequences available in databases, the followingoligonucleotides were synthesised: ‘sense primer’,50-TGGCATTTGCA(CT)GGCCATG-30, ‘antisenseprimer’, 50-GCAAATGAGGTTCAATATGGC-30.These primers, which correspond to highly conservedregions located close to the C-terminal region of AOgenes [21, 44], were used to amplify melon DNAsequences by PCR (94 �C for 30 s, 55 �C for 45 s, 72 �C

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for 1 min with 30 cycles and then 10 min extension at72 �C). The amplified DNA fragment was analysed byagarose gel electrophoresis and Southern blot analys-is with a cucumber AO cDNA. We cloned the ampli-fied DNA into the pMOSBlue T-vector (Amersham) bystandard procedures, and used vector-specific primersto determine its sequence. This PCR product was sub-sequently radiolabeled with 32P by random priming(Multiprime DNA labelling system, Amersham) andused as a melon AO-specific probe in various experi-ments described in this work.

Melon RNA isolation

Total RNA isolation from various frozen melon tissueswas performed as previously described [35].

Cloning melon AO genes

A melon genomic DNA library, constructed byCLONTECH in EMBL-3 phage vector, was platedin 20 large (150 mm) Petri dishes (a total of 106

recombinant clones), transferred onto Hybond filters(Amersham) in duplicate, and hybridised at 65 �Cwith the melon AO-specific PCR product describedabove. Four positive clones were initially collectedand subsequently re-screened twice at higher platingdilution. Phage DNA was isolated from these clones byQIAGEN-500 columns and analysed by digestion withrestriction endonucleases and Southern blotting. Threeout of the four clones hybridised with the melon AOprobe and showed distinct, non-overlapping, physicalmaps. Following standard techniques, we have sub-sequently subcloned AO-hybridising and neighbour-ing sequences from all three clones into appropriatevectors (pT3T7lac and pBluescript (+) KS) for fur-ther restriction analysis and sequencing by the dideoxychain termination method [50] using a Sequenase DNAsequencing Kit (USB).

Southern and northern blot analyses

Genomic DNA was digested with different restrictionenzymes, fractionated in 0.8% agarose-TAE-ethidiumbromide gels, transferred onto Hybond-N filters andhybridised with radiolabelled 32P DNA probes, asdescribed in Church and Gilbert [10]. Total RNAfrom various melon organs was fractionated in form-aldehyde denaturing agarose gels, transferred ontoHybond-N filters and hybridised to radiolabelled 32PDNA probes, basically as described for Southern

Figure 1. Melon AO is encoded by a multigene family. Genom-ic DNA (� 5 �g) from ovaries was digested with EcoRI (R) andBamHI (B), fractionated in 0.8% agarose gel electrophoresis, trans-ferred to Hybond-N-membranes and hybridised at 58 �C with a spe-cific, 32P-radiolabelled, 0.9 kb EcoRV-EcoRI restriction fragmentfrom melon clone CM-AO1. Three strong-hybridising and one weak-hybridising bands (indicated with arrows in (B), a longer exposureof the same autoradiogram shown in (A)) were detected in bothdigests. 32P-radiolabelled DNA molecular weight markers (M) wereco-electrophoresed, co-transferred and shown here.

blot analysis. Quantification of RNA was carried outby spectroscopic measurements at 260/280 nm andby fractionation in native 2% agarose-TAE-ethidiumbromide gels. Equilibrated loading was followed bymethylene blue staining of denatured RNA transferredin Hybond-N filters [50], and by hybridisation with anactin radiolabelled probe [52]. For re-utilisation, filterswere washed for 10 min in 0.1% SDS at 100 �C.

Computer-based analyses of DNA sequences

DNA sequence analysis (translation, open readingframe and restriction maps, etc.) as well as comparis-ons of DNA and protein sequences (FASTA, BLAST,BESTFIT, GAP, etc.) were made using GCG facilities[14].

AO specific activity measurements

AO enzymatic activities were performed according to amodified version of the method of Moser and Kanellis

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[41]. Melon tissue was extracted in buffer A (20 mMpotassium phosphate, pH 7.4, 0.5 mM PMSF, 1.5%PVPP). The mixture was homogenised with a Polytronand allowed to stand on ice for 20 min with occa-sional vortexing every 2 min. After a centrifugationat 15 000 g for 15 min at 4 �C, the supernatant (sol-uble fraction) was filtered through Miracloth (Calbio-chem) and assayed directly for AO activity by follow-ing spectrophotometrically the oxidation of ascorbicacid at 265 nm [41]. The pellet (particulate fraction)was washed twice in buffer A and dissolved in bufferB (20 mM potassium phosphate, pH 7.4, 1 M calciumchloride, 0.5 mM PMSF) and vortexed for 20 min at4 �C. It was found that the use of calcium chloride,instead of monovalent ions, gave excellent results forthe recovery of cell wall associated AO. The mixturewas finally centrifuged again at 15 000 g for 15 min at4 �C and the supernatant containing cell wall proteinsassayed directly for AO activity as described above.Protein content was quantified according to Bradford[8] and one unit of AO activity was defined as theoxidation of 1 �M ascorbic acid per min at 25 �C con-sidering an extinction coefficient for ascorbic acid of9246 M�1 cm�1 at 265 nm.

Results

Melon ascorbate oxidase is coded by a family of fourgenes

We have previously reported that purified melon AO iscomposed of two subunits of molecular weight 68 000,and forms at least six isoenzymes with isoelectricpoints in the range of pH 7.7 to 8.3 [41]. This observa-tion suggested the presence of more than one AO gene.However no molecular studies have, so far, addressedthe question whether AO is encoded by a single or mul-tiple genes, as is often the case in plants. The primarystructure of AO was only deduced from the cDNAs ofcucumber, pumpkin and tobacco clones [21, 27, 44].The three sequences were highly similar (approxim-ately 80% identity along the entire DNA sequencesdetermined), strongly suggesting that AO genes of thethree species are homologous. Moreover, comparisonof AO sequences with all available DNA sequencesin databases showed that multicopper oxidases sharehighly conserved short sequences corresponding tothe copper binding regions of the proteins. Basedon such comparisons we identified AO DNA regionswhich would be expected to be highly conserved in all

AO genes, and designed degenerate oligonucleotidesto amplify by the polymerase chain reaction (PCR)a 206 bp DNA region from genomic DNA isolatedfrom melon ovaries (for experimental details and oli-gonucleotide sequences see Materials and methods).The resulting PCR product cross-hybridised stronglywith an AO clone from cucumber (results not shown).Both the PCR product and the cucumber clone gaveidentical results in northern blot studies of melon AOgene expression (as described later). The melon PCRDNA product was cloned into an appropriate vector(pMOSBlueT-vector, Amersham) and sequenced bythe dideoxy chain-termination method (see Materialsand methods). The 206 bp DNA sequence obtainedcorresponded to a putative amino acid sequence highlysimilar (approximately 93% identity) to the expectedconserved region of cucumber and pumpkin AO (seeFigure 3). This melon PCR DNA product was used asa probe for detecting melon AO genes.

Genomic DNA isolated from melon ovaries wasrestriction digested with various enzymes, fractionatedby agarose electrophoresis, transferred onto nitrocel-lulose filters, and hybridised, at 58 �C, initially withthe AO-specific PCR product described above, andsubsequently with a 0.9 kb EcoRV-EcoRI restrictionfragment from melon clone CM-AO1 isolated in thiswork (see below). Two of several restriction digestsare shown in Figure 1. EcoRI restriction digest gavethree strong hybridising bands of approximate sizes9.2, 6.3 and 3.0 kb. BamHI also gave three stronghybridising bands of approximate sizes around 20–25 kb. A fourth hybridising band of approximate size4.8 kb in the EcoRI digest and 4.0 kb in the BamHIdigest was evident only after long exposures of themembranes (Figure 1). Three strong hybridising and asingle weak hybridising band appeared in all differentrestriction digests performed in the course of this work.The presence of four AO-hybridising melon genomicDNA sequences gained further evidence from the sub-sequent cloning of a number of putative genomic AOclones whose pattern of restriction analysis correspon-ded to the Southern blot analysis presented in Figure 1(see below). Thus, our results suggest that melon AO iscoded by a small multigene family consisting of at leastfour members. The isolation of a single putative AO-specific PCR product can be explained by the fact thatsubsequent DNA sequencing analysis of three putativeAO clones showed that only one (CM-AO1) had 100%identity with both oligonucleotides designed for thePCR reaction.

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Figure 2. Physical characterisation of three melon AO genomic clones. Restriction enzyme analysis was performed for three genomic melonclones which included AO-specific sequences. The region of AO homologous sequences in all clones is shown with grey box. The approximateposition of the region corresponding to the PCR product used as a probe to identify these clones is also shown with black box. The direction oftranscription is also indicated with arrows. R, EcoRI; RV, EcoRV; X, XbaI; S, SalI; B, BamHI; H, HincII.

Cloning of melon AO multigene family

In order to clone melon AO genes, we screened a gen-omic library using as a radiolabelled probe the 206 bp,PCR-generated, AO-specific DNA fragment. Afterthree rounds of screening, four AO-positive cloneswere isolated. Subsequent restriction and Southernblot analysis showed that three out of the four includeAO-specific sequences (results not shown). Detailedrestriction maps were constructed and showed to cor-respond to three different clones, designated CM-AO1,-2, -3 (Figure 2). Furthermore, restriction analysisof these clones showed that they contain sequencesidentical in size to AO-specific genomic DNA restric-tion fragments detected previously in Southern blots.The assignment of the isolated clones to specific AOgenomic restriction fragments was confirmed by rep-robing the Southern blot of Figure 1 to specific probesisolated from CM-AO1, CM-AO2 or CM-AO3 (data notshown). Under high stringency conditions of hybridisa-tion at 65 �C, CM-AO1 sequences hybridised stronglyonly to the 6.3 kb, CM-AO2 to the 4.8 kb, and CM-AO3to the 9.2 kb EcoRI restriction fragments, respectively(see Figure 1, lane R). None of the probe hybridisedto the 3.0 kb EcoRI restriction fragment. Thus, ourresults strongly suggested that we have cloned threeout of four putative members of melon AO multigenefamily previously detected in Southern blot analysis.

Positively hybridising and neighbouring DNAregions from CM-AO1, -2 and -3 were subcloned inpBluescript KS(+) or pT3T7 lac vectors for moredetailed restriction analysis and sequencing. Appropri-ate subclones were sequenced with the dideoxy chaintermination method [50]. DNA sequences were ana-lysed, computer translated and compared to existingDNA and protein sequences in all data bases. All threeclones contain sequences highly similar to pumpkin,cucumber and tobacco AO DNA sequences and less,but significantly, similar to other multicopper oxidases[21, 25, 27, 44–46 and SwissProt Data Base] and otherrelated proteins [3] (Figure 3). The similarity amongall putative AO sequences, including the three mel-on AO sequences reported herein, extends beyond thehighly conserved active site region found in all multi-copper oxidases. Similarity scores of melon AO withAO sequences from other plants range at 68–82% atthe DNA level and at 75–86% at the protein level.These scores strongly suggest that all plant AO pro-teins analysed, so far, are homologous enzymes con-served throughout evolution. At present, DNA cor-responding to 60–90% (340–500 amino acids of thepredicted protein sequences) of exon AO sequenceshas been sequenced. In the three melon AO genes,putative exon-intron positions are highly conserved,and seem to correspond to the exon-intron splice junc-tion as determined by comparative analysis of genomic

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Figure 3. Alignment of partial amino acid sequences from the three cloned melon AOs with all known AO amino acid sequences from otherplants and two other selected homologous sequences. The alignment was created using the GCG/PILEUP program [14]. Identical amino acidsare shown in bold letters. Dots indicate gaps introduced by the program to maximise similarities. The protein sequences shown are the following:Mao1, Mao2, Mao3, melon AOs; Pao, pumpkin AO [21]; Cao, cucumber AO [44]; Tao, tobacco AO [27]; Bp10, a pollen specific protein relatedto ascorbate oxidase from Brassica napus [3]; Lac1, laccase from Neurospora crassa [25]. The region compared corresponds to a part of thefourth exon of AOs including the highly conserved copper binding sites (underlined). Similarity scores of melon AO with AO sequences fromother plants are 75–86% at the protein level and when compared to laccase and Bp10 50% and 38%, respectively.

and cDNA AO sequences in cucumber and pumpkin[21, 44–46]. Taken together, these observations fur-ther suggest that the genes isolated encode putativeAO enzymes.

AO specific activity is high in ovaries and outermesocarp of mature preclimacteric fruits

In previous studies we found that AO specific activitywas very high in melon ovaries but declined dramatic-ally in young developing fruits (5–20 days after anthes-is), increased transiently in 30- and 35-day-old maturefruits, to decrease again in ripe 36-, 38- and 40-day-oldfruits [41]. In these studies, parallel measurements of

fruit ethylene production have shown that AO activitywas expressed prior to ethylene production and fruitripening. Here, we extended our measurements in dif-ferent melon tissues and performed a more detailedanalysis of AO specific activity in cell wall and sol-uble fractions from crude extracts of the inner, middleand outer parts of fruit mesocarp. AO specific activityin melon fruits was associated (85% of total specificactivity) with cell wall fractions (results not shown).Figure 4 shows that highest specific activities wererecorded in ovaries and the outer part of mature pre-climacteric fruits, 34–36-day-old after anthesis. No,or low, activities were found in younger (10–32-day-old) or over-ripe (38–40-day-old) fruits. Protein blot

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analysis has also demonstrated that AO protein con-tent is high in ovaries and appreciable in 34-day oldmature fruits, but undetectable or very low in fruit ofother developmental stages [16, 41; A. Al Madhoonand A. K. Kanellis, in preparation].

AO expression is regulated at the level of transcriptaccumulation and precedes the expression of genesinvolved in ethylene biosynthesis

We addressed the question whether differentialincrease in AO specific activity and accumulation ofAO protein content are due to organ-specific or devel-opmental regulation of AO message (mRNA) accumu-lation. We performed RNA blot analyses using as aspecific probe, initially a homologous AO cDNA fromcucumber, and subsequently, a melon-specific AO gen-omic DNA (the 0.9 kb EcoRV-EcoRI restriction frag-ment from clone CM-AO1, see Figure 2). Both probesgave identical results (data not shown). AO transcriptaccumulation was detected in total RNA extracted fromdifferent melon tissues and fruits in defined develop-mental stages. For mature pre- and post-climactericfruits, RNA was extracted specifically from the outer,middle and inner parts of mesocarp tissue. In parallel,we followed the accumulation of 1-aminocyclopropanecarboxylate (ACC) synthase (ACS) and ACC oxidase(ACO) transcripts encoding the enzymes necessary forethylene biosynthesis [1, 6, 49], to relate specific-ally AO expression to the molecular events underlyingethylene production and fruit ripening.

Figure 5 shows that AO expression parallels that ofAO specific activity. This was found for all organs ofmelon tested, throughout fruit development,and withinthe different mesocarp areas of a given fruit. Maxim-um AO message accumulation was found in ovariesand the outer mesocarp areas of mature preclimactericfruits. No or very low AO message accumulation wasdetected in young or overripe fruits and in other melonorgans. When compared to the expression of the ethyl-ene biosynthetic genes, AO is expressed prior to theexpression of both ACS and ACO. In addition, and incontrast to the preferential spatial expression of AO inouter mesocarp areas, ACS and ACO transcripts werefound to accumulate equally well throughout inner,middle and outer mesocarp areas of the fruit.

Figure 4. AO specific activity from particulate crude extracts frominner (stars), middle (circles) and outer (triangles) parts of fruitmesocarp during fruit development. Squares represent the averagevalues of specific activities of inner, middle and outer parts at eachsampling period. Vertical bars represent standard deviation derivedfrom the specific activities from three different fruit.

AO transcript accumulation is repressed in responseto wounding

Apart of organ-specific and developmental regula-tion, the expression of a great number of plant genesis known to be highly controlled in response to arange of environmental stress factors such as flood-ing, mechanical wounding and pathogen infection[6, 15, 18, 19, 29, 33, 47]. We have previouslyshown that the expression of a number of genes cod-ing for enzymes involved in phenylpropanoid biosyn-thesis (phenylalanine ammonia lyase (PAL), chalconesynthase (CHS), chalcone isomerase (CHI)) in melonfruits is rapidly induced within 90 min after wound-ing, and reaches a maximum 10 to 20-fold overallincrease over unwounded fruits within 24 h [15]. Inlight of increasing indirect evidence that ascorbate andits metabolism might play an important role in variousplant defence systems [23, 24], we examined whetherAO gene expression is regulated in response to wound-ing. For that, we performed northern blot analysis ofRNAs extracted from young green (20-day old) andmature (34-day old) fruits, wounded for different timeperiods, with a melon AO-specific probe. The probeused was either the PCR DNA product described aboveor the 0.9 kb EcoRV-EcoRI restriction fragment fromclone CM-AO1. AO transcript accumulation was dra-matically repressed within 6 h of wounding, and per-sisted so for at least 24 h after wounding (Figure 6A),in both young unripe and ripe fruits. As a control for

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the effectiveness of wounding in our experiment, thesame blots were washed and rehybridised to a melonPAL-specific probe. This showed, as previously estab-lished, normal, wounding-specific induction of the PALmessage (Figure 6B).

Discussion

This is the first report of a small multigene familyencoding AO in plants. Southern blot analyses andthe subsequent isolation and physical mapping of AOclones from a melon genomic library, suggested thatmelon AO is coded by at least four genes. Three ofthem were cloned in this work while experiments arein progress to clone the fourth one. Preliminary DNAsequence determination of the three cloned AO genesrevealed that they code for putative enzymes homo-logous to ascorbate oxidases and other multicopperoxidases and related proteins [3, 21, 27, 44–46 andSwissProt Data Base]. All three melon AO sequencesreported here, including the most divergent encoded byCM-AO2, show highest similarities with all previouslyreported AO sequences. This similarity is not confinedin the highly conserved sequences which include theactive site (copper binding) of all multicopper oxidases.Finally, the relative exon-intron positions found in thegenes reported here correspond very well with those oftwo previously reported AO genes [44–46 and Swiss-Prot data bases]. All these observations strongly sug-gest that the genes isolated encode melon AO enzymes.We cannot exclude however, that one or more of thegenes isolated are pseudogenes since no correspondingcDNAs were isolated. The identification of at least fourmelon AO genes is in agreement with the presence ofmultiple isoforms, 12–14 in total, detected recently inmelon [2]. The existence of further melon AO genes,which might have escaped detection in our screeningprocedures due to more divergent DNA sequence com-position, should not be excluded. Finally, in contrast tomelon AO multigene family reported here, in tobacco,it has been proposed that AO is coded by a single gene[27].

In previous studies, we have detected high AO spe-cific activity and protein accumulation in ovaries andmature fruits before the onset of endogenous ethyleneproduction [16, 17, 41]. Here, we confirmed these res-ults and extended our studies by analysing cell wall andsoluble fractions in crude extracts from different mel-on tissues and different parts of fruit mesocarp. Previ-ous observations in zucchini squash [32] and pumpkin

fruits [20] have shown that AO protein accumulationand AO activity are high during anthesis, in the epi-dermis of very young fruits, and in young and growingleaves or stems. Lin and Varner [32] had proposed thatAO activity might be associated with cell wall loosen-ing during cell expansion in early fruit development.In tobacco, it has also been shown recently that AOis expressed in young and growing tissues, suggestingthat non-cucurbitaceous plants exhibit a similar expres-sion pattern [27]. The cell wall localisation of melonAO and its high activity in ovaries and very younggrowing fruits shown in this study is compatible toa similar function. On the other hand, we detectedonly low AO expression in melon leaves, roots or evengrowing shoot apex. As far as it concerns leaves androots, this might be due to the fact that we did not useyoung growing tissue. However, low AO expressionin growing shoot apex of melon clearly contrasts thegeneral proposed idea of AO as a cell wall looseningenzyme in plant tissues.

AO function has also been related to cell division.Earlier reports have shown that ascorbic acid medi-ates the biosynthesis of hydroxyproline-rich proteinsnecessary for the progression from G1 to S phase inthe cell cycle [4]. Blocks in ascorbic acid biosynthesisby chemical treatment leads to interrupted cell divi-sion in G1. This cell cycle arrest is reversed by theaddition of exogenous ascorbic acid [4]. More recentresults have shown that AO message accumulation andAO activity are high in specialised non-dividing cellsknown as the quiescent center (QC) [28]. QC consistsof non-dividing cells found in the most central part ofthe root apical meristem where they function as organ-ising centres for postembryonic morphogenesis [28].It has been proposed that AO function is to controlcell division and growth rates in the root apex by con-trolling the levels of ascorbic acid via its oxidation todehydroascorbic acid [28]. This model might be alsocompatible with high melon AO expression in ovariesfollowed by a dramatic drop in its expression duringearly stages of fruit development. Before fertilisation,ovary cells do not divide. Upon fertilisation, cell divi-sion is estimated to last for 3–5 days [48]. All sub-sequent fruit development is by cell expansion. Thus,high AO expression in ovaries might establish a G1arrest by keeping ascorbic acid levels low, while uponfertilisation, a dramatic drop in its expression mightallow cell division to occur for a period necessary forfruit development.

A novel aspect of our studies comes from the obser-vation that AO is also expressed specifically in the outer

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Figure 5. Regulation of AO, ACS and ACO transcript accumulation in response to organ specific differentiation, fruit development and ripening.Total RNAs (� 20 �g) from various melon organs (r, root; sh, shoot; t, shoot apex; l, leaf; ov, ovaries) and fruit at different developmental stages(10-, 20-, 25-, 30-, 34-, 36-, 38- and 40-day after anthesis) were fractionated in denaturing agarose gels, transferred to Hybond-N-membranesand hybridised with specific 32P-radiolabelled probes for melon AO (CM-AO1), melon ACO (pMEL-1) [6] and tomato ACS (ptACC2) [49]genes, as described previously [15]. c, indicates inner (central) fruit tissue, m, indicates middle fruit tissue, o, indicates outer fruit tissue. Equalloading, integrity and transfer was checked by washing out the AO, ACS and ACO probes and hybridising the blots with an actin-specific probefrom soybean [52] (results not shown). Autoradiograms shown were adjusted by differential exposure to mRNA loading according to bothtotal RNA and actin hybridisation signals. Here, and in previous studies, we have established that for all melon tissues studied, actin messageaccumulation levels were in total agreement with total RNA concentrations as estimated spectrophotometrically and by ethidium bromidestaining. Thus, as in many other biological systems, actin hybridisation signals represent a proper internal control in northern blot analysis. Thenorthern blot analysis shown represents one out of the three independent experiments performed. All experiments gave identical results.

Figure 6. Repression of AO expression in response to wounding.Total RNAs (� 10 �g) from 20- and 34-day old fruits, wounded for0, 6, 12 or 24 h were fractionated in denaturing agarose gels, trans-ferred to Hybond-N-membranes and hybridised with AO (CM-AO1)and PAL (pmPAL1, [15]) specific 32P-radiolabelled probes. Equalloading, integrity and transfer was controlled as described in thelegend to Figure 5. The RNA blot analysis shown represents one outof three independent experiments performed. All three experimentsgave identical results.

tissues of mature preclimacteric fruits, preceding thedramatic molecular and physiological changes associ-ated with ethylene production and ripening [1, 51, 53].This observation opens new possibilities concerningthe role of AO and ascorbic acid metabolism in fruitdevelopment. The fact that AO is not expressed signi-ficantly in mature zucchini [32] or pumpkin [20] fruitssuggests that transient expression in the outer tissues ofmature unripe melons might serve a function specificto the development of climacteric fruits.

We found that organ-specific and developmentalexpression of AO activity correlates well with AO mes-sage levels observed. Both AO activity and AO messageaccumulation are high in ovaries and the outer meso-carp areas of mature preclimacteric fruits. AO genewas also found to be expressed at low levels in otherorgans tested (roots, shoot, shoot apex, leaf, immaturefruit). This observation is also in agreement with otherresults showing that low levels of AO isoenzymes andAO specific activity are detected in the above organs[2; A. Al Madhoon and A. K. Kanellis, in preparation].Generally, the results presented here and in previouspublications [2, 16, 17, 20, 21, 44] imply first, thatthe increase in AO enzymatic activity is due to de

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novo synthesis of protein and second that the actualincrease in transcript levels is directly related to theaccumulation of active AO enzyme. Thus, it seemsthat regulation of AO expression occurs basically atthe transcriptional level. This contrasts results recentlyobtained in tobacco where AO activity and the amountof AO protein were not in accord with changes in themRNA [27].

No previous studies have approached the relation-ship of AO expression with endogenous ethylene pro-duction [1, 53] and fruit ripening [51]. When comparedto the expression of the ethylene biosynthetic genes,AO was expressed prior to both ACS and ACO. In addi-tion, and in contrast to the preferential spatial expres-sion of AO in outer mesocarp areas, ACS and ACOtranscripts accumulated equally well throughout inner,middle and outer mesocarp areas of fruit. These obser-vations strongly suggest that induction of AO geneexpression during melon fruit development is inde-pendent of ethylene biosynthesis. This, however, doesnot exclude, as we discuss later, a role of ethylene onAO transcription regulation.

Ascorbate and its metabolism might play an import-ant role in various plant defence systems [23, 24, 43].Oxidative damage is experienced by plants both in nat-ural abiotic stress throughout their life-time and dur-ing terminal developmental processes such as the laststages of fruit ripening [24, 26]. Senescence, whichaccompanies the final stage of ripening, is believedto be partially caused by a reduced efficiency in fruitdefence mechanisms against oxidation. Plant oxidativedefence mechanisms involve mainly the biosynthes-is of antioxidant compounds (such as ascorbate) andinduced expression of antioxidant enzymes (catalase,superoxide dismutase, ascorbate peroxidase) [24, 26].Ascorbate is a central component in plant defenceagainst oxidative damage because of its role as a freeradical scavenger and as a cofactor in many enzymat-ic reactions [26]. In addition, studies with potatotubers have shown that wounding leads to inductionof ascorbate biosynthesis and increased ascorbate con-tent [43]. The above observations led us to investig-ate whether AO gene expression is part of a defenceresponse leading to alterations in total ascorbate con-tent. We subjected melon fruit of different develop-mental stages to wounding, a typical stress conditioninducing most, if not all, plant defence molecular sys-tems [18, 19, 29, 47]. AO expression is rapidly anddramatically repressed by wounding. Parson and Mat-too [47] have previously shown that the expressionof pT53, a cDNA clone from tomato fruit, is weakly

repressed by wounding in the early-red and red stage.Also, Lincoln et al. [33] have reported that the expres-sion of LE-ACS2, an ACS gene in tomato fruit, is tran-siently repressed by wounding during the first 2 h andthen increases after 4–6 h. In both these cases wound-ing causes a partial and transient repression which isfollowed by induction of expression. Both these genes,pT53 and LE-ACS4, are known to be expressed in ripetomato fruit. In our case, AO expression is stronglyrepressed from 6 h till at least 24 h after wounding. Aninteresting observation is that AO is also not expressedin ripe melon fruit. In other words, AO is not expressedeither under stress conditions or in a specific devel-opmental stage which lead to massive ethylene pro-duction. Moreover, in transgenic melon fruit whichdo not produce ethylene, because ACO expression is‘blocked’ with antisense technology, AO expression isnot repressed by wounding [M. Guis, M. Sanmartin,I. Pateraki, G. Diallinas, A. K. Kanellis, J.-P. Pech,unpublished results]. It seems that in wild-type mel-on fruit, endogenous ethylene produced in response towounding might be the cause of AO repression. Thus,ethylene is a good candidate for being the molecularsignal repressing AO expression both in overripe orwounded fruits.

Our results suggest the presence of at least twoindependent transcriptional mechanisms underlyingAO expression in response to plant development andwounding. We can speculate from these results that,although AO gene expression seems to be specific-ally induced at particular stages during plant develop-ment and fruit ripening, plant response to wounding iscapable of overriding developmentally pre-establishedinduction and repressing totally AO transcription, atleast in mature preclimacteric fruit. Previous res-ults from studies with potato tubers have shown thatwounding leads to an increase in ascorbate content[43]. This and our results suggest that repression ofAO expression might occur in parallel to increasedascorbate biosynthesis [43] contributing to a gener-al increase in ascorbate content in wounded tissues.Increased ascorbate content in wounded tissues mightbe necessary to stimulate cell division and tissue heal-ing.

In conclusion, this work has led to the isolation andinitial characterisation of a multigene family encod-ing AO and established a number of important novelaspects concerning AO expression in melon. The isol-ation of an AO multigene family allows us to constructgene-specific probes [7] and further study the mechan-isms controlling AO expression in response to organ-

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specificity, organ development, fruit ripening and inresponse to stress signals. This should in turn per-mit us to construct appropriate transgenic plants andapproach the elucidation of the physiological functionof this enzyme, and in particular, understand its role inascorbic acid metabolism.

Acknowledgements

We thank C. Balague, J.-C. Pech and D. Griersonfor the ACO plasmid, A. Theologis for the ptACC2plasmid, A. Shimino for the cucumber AO plas-mid (pASO11) and R. Meagher for the actin plas-mid (pSAc3). This work was supported by the EU-BIOTECH-8102-CT93-0400 grant to A.K.K.. M.S.and A.S. were recipient of student fellowships bythe Mediterranean Agronomic Institute of Chania andConsiglio Nationale di Richerche, Italy, respectively.

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