J Sci Food Agric 1997, 73, 455È463

Developmental and Ripening-Related Effects onthe Cell Wall of Pepino muricatum)(SolanumFruit*

Erin M O’Donoghue,” Sheryl D Somerüeld, Leigh A de Vre� and Julian A Heyes

Crop & Food Research Ltd, Private Bag 4005, Levin, New Zealand

(Received 20 November 1995 ; revised version received 3 May 1996 ; accepted 2 October 1996)

Abstract : Several cell wall components in ripening pepino fruit have been quant-itatively and qualitatively characterised, with the aim of identifying their contri-butions to the loss of tissue Ðrmness. Pepinos were graded into nine groups basedon progressive, characteristic skin colour changes, previously shown to corre-spond with decreasing fruit Ðrmness. While fruit softening began when thepepinos were still green but with newly acquired purple stripes, the Ðrst signiÐ-cant quantitative signs of cell wall modiÐcation (total pectin and hemicellulosecontent declining and CDTA-soluble pectin content increasing, on a fresh weightbasis) were detectable later in ripening, when the fruit began to acquire yellowskin pigmentation. Gel fractionation studies demonstrated that there wereincreased levels of low-molecular-weight pectin and xyloglucan during pepinoripening. The change in molecular weight distribution of CDTA-soluble pectinoccurred as fruit started to acquire yellow pigmentation, while xyloglucan poly-mers were modiÐed at an earlier stage that coincided with the initial loss ofÐrmness.

Key words : pepino, pectin, hemicelluloses, xyloglucan, cell wall, molecularweight.

INTRODUCTION

Textural change associated with fruit ripening is strong-ly inÑuenced by modiÐcations to cell wall structure andcomposition. While there is a degree of conservationamong fruits regarding the outcome of changes to cellwall components during ripening, eg loss of neutralsugars, solubilisation and fragmentation of polymers(Fischer and Bennett 1991), the route to achieving thesemodiÐcations often di†ers between species, and can bedependent on the starting wall composition as well asthe capacity of the fruit to maintain wall synthesis, pro-duction of hydrolytic enzymes and ionic/pH conditionsin the apoplast. The e†ect of wall modiÐcations on wallstability in ripening fruit has been shown by electronmicroscopy, and the dissolution of the middle lamella

* Portions of this paper were presented at the New ZealandSociety of Plant Physiologists Annual Meeting, PalmerstonNorth, 1994.” To whom correspondence should be addressed.

region and general disarray of remaining wall materialis clear (eg Dallman et al 1989 ; Hallet et al 1992).

The solubilisation and hydrolysis of pectins in themiddle lamella is thought to be highly inÑuential in thesoftening of ripening fruit. Enzymes that inÑuence thestructure of pectin (eg polygalacturonase (PG), pectin-methylesterase, b-galactosidase) may have profounde†ects on the cohesiveness of the wall during ripening.It is now generally accepted that textural change duringripening is a composite e†ect of wall hydrolases actingin concert, but the individual e†ects of cell wall hydro-lases have been clariÐed by recent studies of transgenicfruit from plants that have been transformed by insert-ing a gene coding for a cell wall hydrolase in the anti-sense orientation. Polygalacturonase activity isresponsible for pectin hydrolysis and appears to beinvolved in pectin solubilisation (Carrington et al 1993),while pectinmethylesterase may regulate the binding ofcationsÈparticularly calciumÈin the cell wall and maymodulate the ensuing action of other cell wall hydro-lases (Tieman and Handa 1994).

455J Sci Food Agric 0022-5142/97/$09.00 1997 SCI. Printed in Great Britain(

456 E M OÏDonoghue et al

While changes in the structure of pectins have beengiven attention due to their location in the disinte-grating middle lamella of ripening fruit, other cell wallconstituents also inÑuence fruit texture. Cellulose micro-Ðbrils form a strong network around each cell and areinterlocked with hemicelluloses (Carpita and Gibeaut1993) and this network is susceptible to ripening-relatedmodiÐcation. The remarkably high levels of Cx-cellulase in avocado have been implicated in a changein cellulose Ðbril organisation occurring during ripening(OÏDonoghue et al 1994), while xyloglucan molecularweight also decreases in a number of fruits during(Mr)the ripening period (eg Huber and Lee 1986 ;OÏDonoghue and Huber 1992 ; Cutillas-Iturralde et al1994).

SofteningÈor loss of ÐrmnessÈin ripening fruit hasoften been measured by an objective compressive test,such as with a penetrometer or an Instron UniversalTesting Machine (Instron Corp, Canton, MA, USA).Compressive tests record the resistance to movement ofa probe through a Ðxed distance into the fruit Ñesh.Recently Heyes et al (1994) investigated both compres-sive and tensile properties of ripening pepino Ñesh anddescribed a range of textural parameters. This approachhas enabled some deductions to be made about whichfactors (cell wall strength, cell-to-cell adhesion, turgorand tissue anatomy (Holt and Schoorl 1985)), areimportant during pepino ripening. Heyes et al (1994)concluded that softening of pepino fruit is due to steadydecreases in the absolute cell wall strength and cell-to-cell adhesion, which begin very early in ripening.Tensile tests showed that there was a change from cellsrupturing as their walls broke under strain (in unripefruit) to cells separating when a tensile stress wasapplied (in ripe fruit). The work presented here, wherewe have examined quantitative and qualitative changesin pepino cell wall polymers during ripening, wasundertaken to deÐne ripening-associated wall modiÐ-cations in this ethylene-insensitive fruit, and to linkthese to the structural changes associated with soften-ing.

MATERIALS AND METHODS

Plant material

Pepino fruit (cultivar “El CaminoÏ) were propagatedfrom cuttings and grown in the glasshouse in individualpolythene bags. Fruiting was restricted to two mainstems per plant. Glasshouse conditions were asdescribed by Heyes et al (1994). Fruit selections werereplicated by two separate crops, which were harvestedin November 1993 and January 1994.

Pepino fruits were picked at nine developmental andripening classes based on days from Ñowering ([2, [1

TABLE 1Developmental and ripening class descriptions for pepino

fruit, including average days from fruit set (DFS)

Class DFS Description

[2 38 Green fruit[1 44 Green fruit

0 48 Green fruit1 51 Green fruit, Ðrst appearance of purple stripes2 55 Green fruit, with established purple stripes3 60 Loss of green colour, background pale cream4 65 \25% background pale yellow5 69 25È50% dull yellow orange6 71 [75% skin deep golden orange

and 0) and skin colour changes (1È6). Class criteria andaverage days from fruit set are listed in Table 1. Theripening classes 1È6 correspond to those texturallyanalysed by Heyes et al (1994). There were 4È6 fruitspooled in each class of each harvest replicate.

Firmness of fruit in each class was measured by com-pression using an Instron Universal Testing Machine.Probe diameter was 8 mm, crosshead speed was150 mm min~1 and penetration of the probe was 7 mm.Firmness was measured on peeled Ñesh from oppositesides of each whole fruit. Responses to compressionwere averaged within each class and reported as yieldload (Newtons). Immediately following compressiontests, fruits were peeled, the placenta and seeds removedand the pericarp chopped, frozen in liquid thenN2stored at [80¡C.

Cell wall preparations

Four ethanol-insoluble residue (EIR) preparations weremade from all ripeness classes in each harvest replicate.Approximately 10 g of frozen tissue was used for eachpreparation, following the method to inactivate wallenzymes with tris(hydroxymethyl)aminomethane (Tris)-bu†ered phenol (Huber 1992), with the exception thattissue/solvent homogenates were centrifuged (2000] g,5 min) rather than Ðltered between washes. Walls weredried overnight, ground and stored at [15¡C untilused.

For aqueous cell wall preparations, approximately100 g of frozen pepino tissue was homogenised in cold40 mM HEPES (pH 7É0) using a Waring blender(Waring, New Hartford, CO, USA) and a KinematicaAG polytron (Kinematica, Littau, Switzerland), then Ðl-tered through two layers of organza cloth. The cell wallswere washed slowly with 4 litres of ice-cold deionisedwater at 4¡C, then frozen, freeze-dried, ground andstored at [15¡C. One cell wall preparation was madefor each ripening class in both harvest replicates.

Developmental and ripening-related e†ects on the cell wall of pepino fruit 457

Pectin quantiÐcation

Total pectin in EIR preparations was measured usingthe hydrolysis method of Ahmed and Labavitch (1977)and the uronic acid detection assay of Blumenkrantzand Asboe-Hansen (1973). Chelator-soluble pectinswere released from 15 mg of EIR with 4 ml of 50 mM

trans -1 , 2 - cyclohexanediamine -N ,N ,N@ ,N@ - tetraaceticacid (CDTA) in 50 mM Na-acetate (pH 6É0) by shakingfor 4 h at room temperature. The extracted pectin wasÐltered through Whatman GF/A paper and the solidresidue washed with 2 ml of the extracting bu†er.Uronic acids released were quantiÐed as above. Levelsof methyl esteriÐcation in pectin from pepino pericarpof each developmental and ripening class were assessedin 5 mg portions of EIR by saponiÐcation followed bymeasurement of the released methanol using the pro-cedure of Wood and Siddiqui (1971). Total pectin,CDTA-soluble pectin and methyl esteriÐcation assayswere repeated four times for the ripening classes of eachharvest replicate.

Cellulose quantiÐcation

Samples (5 mg) of aqueous cell wall preparations fromeach developmental and ripening stage were analysedfor cellulose content using a micro-version of themethod of Updegra† (1969) followed by quantitation ofcellulose-derived hexoses (Dische 1953). Assays wererepeated four times for the ripening classes of eachharvest replicate.

Hemicellulose quantiÐcation

Samples (20 mg) of aqueous cell wall preparations fromeach developmental and ripeness class were shaken in3 ml of 6 M NaOH including 26 mM for 4 h atNaBH4 ,room temperature. Samples were centrifuged (20 min,1250 ] g, 4¡C) and the supernatant collected and neu-tralised with glacial acetic acid. This extract was kept at1¡C overnight, while the wall residue was suspended in2 ml of deionised water and incubated overnight at37¡C to further extract hemicelluloses (Edelmann andFry 1992). Following centrifugation as above, this waterextract was added to the previous alkali extract and thepH re-adjusted to neutral. Hemicellulose extracts weredialysed in 2000 cut-o† tubing using the procedureMrof de Vetten and Huber (1990), followed by freeze-drying. To remove co-extracted acidic polymers(primarily pectins), spin-columns of DEAE-Sephadex(Sigma, St Louis, MO, USA) were prepared using theconcept introduced by Neal and Florini (1973) with thefollowing modiÐcations. Plastic syringe barrels (5 ml),partially plugged with glass wool, were Ðlled with aslurry of DEAE-Sephadex equilibrated in 10 mM

sodium phosphate, 20 mM NaCl, pH 6É8, placed inside

50 ml centrifuge tubes and centrifuged very slowly (c200 ] g for 2 min). Final column volumes were approx-imately 3 ml. Freeze-dried hemicellulose extracts weretaken up in 3 ml of the column operating bu†er andcentrifuged through the column for 2 min. The eluentwas circulated through the columns a total of threetimes, then retained and the column washed with 2 mlof the phosphate bu†er. This procedure resulted in afast, e†ective removal of uronic acids from the hemi-cellulose extracts and has been found to remove allpolygalacturonic acid from a 5 ml sample of dextranand polygalacturonic acid at 200 kg ml~1 each.

The depectinated hemicellulose extracts were freeze-dried, then dissolved in 1 ml of 50 mM Na-acetate con-taining 10 mM NaCl (pH 5É0), and desalted on asimilarly prepared Sephadex G-25 spin column. Thecarbohydrate levels in the hemicellulose extracts wereassayed using the phenol-sulphuric acid assay of Duboiset al (1956). Hemicellulose extractions were duplicatedfor the ripening classes of each harvest replicate.

Molecular weight analysis

The distributions of chelator-soluble pectin andMrhemicellulose polymers from pepino fruits wereanalysed by low pressure gel Ðltration chromatographyusing a 1 ] 30 cm Superose 6HR column (Pharmacia,LKB, Uppsala, Sweden). The column elution proÐlewas calibrated with dextran standards [5000 kDa,500 kDa, 73 kDa, 40 kDa, 9É5 kDa and glucose, at aÑow rate of 0É5 ml min~1.

For chelator-soluble pectins of each developmental/ripeness class from each harvest replicate, approx-imately 300 kg of uronic acid equivalents were appliedto the column in a total volume of 200 kl. The columnwas operated in a bu†er of 30 mM Na-acetate with20 mM NaCl and 10 mM EDTA, pH 6É5. Fractions of0É5 ml were collected and assayed for uronic acids usingthe method of Blumenkrantz and Asboe-Hansen (1973).

For distribution analysis of hemicelluloses fromMreach developmental and ripeness class from eachharvest replicate, approximately 800 kg of glucoseequivalents were applied to the column in a Ðnalvolume of 200 kl. The elution bu†er was 50 mM Na-acetate with 10 mM NaCl, pH 5É0. Fractions (0É5 ml)were assayed for total carbohydrates (Dubois et al 1956)and xyloglucan (Kooiman 1960).

Statistical analysis

Results from compositional assays were examined usingthe analysis of variation (ANOVA) test with SAS(Version 6). Averages and LSD values (a \ 0É05) arepresented for each assay in Table 2. In addition, thecontrast procedure was applied to compositional datato improve the detection of ripening-related trends. For

458 E M OÏDonoghue et al

TABLE 2Compositional analysis of pepino fruit at each developmental/ripening stage (mg g~1 fresh weight ;

esteriÐcation is a % of total pectin)a

Cellulose Hemicellulose T otal pectin EsteriÐcation CDT A-Solublepectin

A [2 2É84 0É78 1É55 8É1 0É21[1 2É53 0É63 1É80 5É4 0É23

0 2É83 0É75 1É70 6É9 0É24

B 1 2É70 0É63 1É42 5É6 0É252 2É51 0É71 1É68 6É9 0É333 2É65 0É64 1É58 6É1 0É38

C 4 2É39 0É51 1É58 6É1 0É715 2É35 0É47 1É20 5É2 0É556 1É86 0É38 1É22 4É1 0É76

LSD a \ 0É05 0É46 0É27 0É57 3É8 0É21

ProbabilitiesaA/B 0É36 0É40 0É41 0É52 0É11B/C 0É01* 0É02* 0É16 0É29 \0É01**A/C \0É01** \0É01** 0É04* 0É11 \0É01**

a Probabilities refer to the statistical contrasts between groupings A (late development), B (early ripening)and C (late ripening). Probabilities test the null hypothesis that there is no di†erence between the con-trasts. * signiÐcantly di†erent ; ** highly signiÐcantly di†erent.

the contrast procedure, the nine developmental/ripeningstages were re-grouped into three main classes based onexternal features at harvest :

(A) “developmentalÏ (fruits are green all over, includesstages [2, [1 and 0) ;

(B) “early ripeningÏ, (fruit has purple stripes but noyellow colouration, includes stages 1, 2 and 3) ;and

(C) “late ripeningÏ (fruit has increasing yellow skinpigmentation, includes stages 4, 5 and 6).

Group mean values for wall composition were con-trasted and results are presented in Table 2 as probabil-ity values (ie the probability that there is no di†erencebetween the comparisons).

RESULTS AND DISCUSSION

General comments

In this report, we describe the composition of the cellwall of pepino fruit during late development through tocomplete ripening. The progression of Ðrmness lossduring pepino fruit ripening is shown in Fig 1 and is theaverage of the two harvest replicates. Values for yieldload remained high until the appearance of purplestripes. There was a marked decrease in Ðrmnessbetween stages 2 and 4 (the onset of yellowpigmentation), with the intermediate stage 3 (where the

background appears almost white) notable for highÐrmness variation among the fruits. Fruits in the laterripeness stages 4, 5 and 6 softened steadily, but thesechanges were comparatively small compared to thosethat occurred during early ripening. Fruit from the Ðrstreplicate crop was Ðrmer than the counterparts of thesecond replicate until stage 4 was reached. We noticed adistinct replicate e†ect when compositional data fromboth harvest replicates were analysed by ANOVA eventhough the same skin-colour assessments were used rig-orously, and this replicate e†ect is probably due to the

Fig 1. Firmness (yield load) of pepino fruit at eachdevelopmental/ripening stage. Values are averages of two

harvest replicates. Bars are ^1 SE.

Developmental and ripening-related e†ects on the cell wall of pepino fruit 459

Ðrmness variation between fruits of the two crops. Skincolour-based ripening classiÐcations have been suc-cessfully compared to objective assessments of pepinocolour using a colorimeter, and generally correlate wellwith Ðrmness-based assessments using an Instron(Heyes et al 1994). The pepino cultivar “El CaminoÏ is aclonal selection and there should be no variation due toinherent plant di†erences when plants are propagatedfrom cuttings. It is difficult to reconcile these crop di†er-ences, but they may well be a function of growing tem-perature and glasshouse conditions. It is likely that theexternal indicators of purple stripes and disappearanceof chlorophyll may not be as tightly coordinated withinternal changes in individual fruits as Ðrst thought,while the increase in yellow pigmentation reliably sig-niÐes the end of the initial intense softening phase.

The variability between harvest replicates oftenresulted in high LSD values, and difficulties in assigningclear ripening-related trends to the compositional data.The contrast procedure for comparing di†erent groupsof means is especially valuable in this situation, allow-ing broad changes related to the later stages of fruitdevelopment and early ripening to be identiÐed, whiledi†erences in individual developmental/ripening stagescan be distinguished with the standard data analysis.

The amounts of aqueous-prepared cell walls andethanol-insoluble wall material recovered from pepinofruit are maintained until the late stages of fruit ripeningwhen there is a slight decline in yield (data not shown).Since pepino fruits ripen abnormally when removedfrom the plant, it has not been possible to record indi-vidual fruit weight changes during ripening, but it hasbeen observed that the expansion phase is completedprior to the onset of ripening, and ripening is notaccompanied by fruit swelling (Burge G, pers comm).This observation would suggest that the late stages ofripening involve a genuine loss of wall components.

Cellulose and hemicelluloses

Using the contrast procedure to compare groups ofmeans (Table 2), cellulose content decreased in the lateripening stage compared to late development or earlyripening groups. Looking at individual stages, cellulosecontent decreases markedly when pepino fruit reach thefully ripe state (stage 6, Table 2), but it seems likely thatthe earlier, intense ripening-related Ðrmness loss (stages2È4) is not reliant on a change in absolute levels of cel-lulose. Levels of hemicellulose (on a fresh weight basis)were maintained during late development and earlyripening (stages [2 to 3), but began to decrease aspepino fruit entered the late ripening stages (Table 2), apattern similar to cellulose changes. Unlike cellulosecontent, however, there were no signiÐcant di†erences inhemicellulose content between the late ripening stages 4,5 and 6 (Table 2). Quantitation of alkali-extracted hemi-

celluloses gives an overall indication of changes in thisdisparate group of polymers, and values given here forripe fruit are similar to those reported by Redgwell andTurner (1986) in their composite analysis of ripepepinos.

Qualitative analyses of hemicellulose compositionshow that modiÐcations in the proportion of partici-pating sugar residues (McCollum et al 1989 ; Redgwellet al 1991) and alterations in the nature of bond types(Tong and Gross 1988) may accompany changes incontent. Another qualitative property, the of alkali-Mrsoluble pepino hemicelluloses, was examined to investi-gate the relationship between polymer size and the stateof softening in pepino fruit. Elution proÐles in Fig 2 arethe average of each harvest replicate. Two size classeswere distinguishable. One peak eluted close to the voidvolume of the column and the bell shape of this peakindicates that it contains only a small proportion ofpolymers that are completely excluded from thecolumn. The second peak is of lower and containsMrthe majority of hemicellulosic polymers.

There was a slight up-shift in the distribution of bothpeaks of the hemicelluloses from developmental stages[2 to 0, a situation also noted by Cutillas-Iturralde etal (1994) in the very early stages of ripening of per-simmon fruit. Since the overall levels of hemicellulosedo not change during this phase, it is likely that newbonds are forming within the hemicelluloses alreadylaid down in the wall. From stages 0 to 4 there was asequential change in the distribution, initially in theMrlarge-sized polymers then within the smaller group. Bystage 3 the distribution of both groups of polymers hadbroadened and Ñattened, although the average ofMronly the larger group of polymers had decreased. Atstage 4 the average of the smaller polymers had alsoMrundergone a downshift. This same distributionremained for polymers up to stage 6 (fully ripe fruit),although there is some loss of very large polymers evenat this point.

The general distribution of hemicelluloses fromMrpepino fruit is similar to those reported for tomato(Huber 1983 ; Huber and Lee 1986 ; Tong and Gross1988), strawberry (Huber 1984) and muskmelon(McCollum et al 1989). There does appear to be a muchsharper resolution between the two size peaks forpepino, however, which may be due to the gel per-meation column packing used here, and the method formore extensive extraction of alkali-soluble polymers.Additionally, the proportion of smaller hemicellulosepolymers does not increase to the same extent duringpepino ripening as is seen for these other fruits. Varia-tions in hemicellulose distributions have been reportedfor ripening kiwifruit (three size classes ; Redgwell et al1991), and for avocados (one peak, OÏDonoghue andHuber 1992), while 4 M KOH-soluble hemicellulosesfrom unripe persimmon fruit also elute as one peak butsplit into two as ripening progresses, eventually ending

460 E M OÏDonoghue et al

Fig 2. Molecular size distribution of 6 M NaOH-soluble hemi-celluloses extracted from cell wall preparations of pepino fruitat maturity stages [2 to 6. Total carbohydrates were frac-tionated on Superose 6HR and detected at 490 nm using themethod of Dubois et al (1956). Developmental and ripeningstages are indicated on each graph. Tick marks at the top ofeach graph indicate elution positions of standard dextrans :from left to right, 5 ] 103 kDa (exclusion), 500 kDa, 73 kDa,

40 kDa, 9É5 kDa and glucose.

up in one size classiÐcation of lower average Mr(Cutillas-Iturralde et al 1994).

The distribution of pepino xyloglucan is shown inMrFig 3. In fruit at the late development stages ([2, [1,0) the majority of xyloglucan was of large size, elutingclose to the exclusion volume of the column. Ripeningtrends in xyloglucan size distribution were very similarto that of the general hemicellulose distribution, withthe main feature being the adjustment in fractionationoccurring from stage 2 onwards. Polymers of large sizebecame more broadly distributed, with a slightly

Fig 3. Molecular size distribution of xyloglucan in 6 M

NaOH-soluble hemicellulose extracts from cell wall prep-arations of pepino fruit at maturity stages [2 to 6. Xyloglu-can was fractionated on Superose 6HR and detected at640 nm using the method of Kooiman (1960). Developmentaland ripening stages are indicated on each graph. Molecular

weight markers are as for Fig 2.

decreased average and there was a sharpening ofMr ,distribution of smaller-sized polymers. There was alsoan increase in the proportion of smaller-sized xyloglu-can from stage 2 to 6.

Ripening-related xyloglucan size distributions havebeen reported for tomato (Huber and Lee 1986), per-simmon (Cutillas-Iturralde et al 1994) and avocado fruit(OÏDonoghue and Huber 1992). In all these cases, thexyloglucan distribution tends to follow that for thegeneral hemicelluloses. Xyloglucan is a comparativelyminor constituent of the lower hemicellulose poly-Mrmers in ripening pepino fruit, but probably contributessigniÐcantly to the pool of hemicelluloses of large size.

Developmental and ripening-related e†ects on the cell wall of pepino fruit 461

It is clear that changes in distribution of hemi-Mrcellulosic polymers (and xyloglucan speciÐcally) doaccompany the main decreases in pepino fruit Ðrmnessduring ripening, and at the point when Ðrmness changeshave slowed (stages 4È6), the size of these polymers isstable. The data indicate that at least a correlativerelationship exists between hemicellulose and Ðrm-Mrness loss. It could be argued that the softening betweenstages 2 and 4 would appear to be more dramatic thanthe changes in hemicellulose distribution, and thenetwork/matrix interaction of the wall may be able tobu†er the occasional bond breakage. Nevertheless,while these modiÐed polymers appear to be still largeenough to maintain some degree of strength within thewall and there is no appearance of oligomeric hemi-cellulose, it is conceivable that the continued cleavage ofmatrix polymers may reduce cell-to-cell adhesion andhence lead to movement between cells under an appliedcompressive stress.

The mediation of hemicellulose molecular weightchanges in ripening fruit has not yet been clariÐed.Undoubtedly there is some involvement of hydrolyticenzymes as seen in the changes found when hemi-Mrcellulose isolates are incubated with crude enzymeextracts from ripe fruit (OÏDonoghue and Huber 1992)and there is evidence of continuing synthesis of hemi-celluloses during the ripening of some fruit (Tong andGross 1988) although this has not been proven to resultin a change in polymer length. Ripening pepino fruitdoes not display cellulase-type activity as measured byreductometric assays with carboxymethylcellulose(Heyes and Blaikie, unpublished data), and the xyloglu-can changes occurring during ripening support theMrconcept that a separate, speciÐc b-1,4-glucanase (or“xyloglucanaseÏ) hydrolyses this polymer (Maclachlanand Brady 1992, 1994 ; OÏDonoghue and Huber 1992).

Pectin modiÐcation

There are marked quantitative and qualitative changesin the pectic component of ripening pepino fruit cellwalls. There is a slight but steady decline in total pectincontent during ripening (Table 2). Statistically signiÐ-cant di†erences appear when the late development andlate ripening groups are compared by contrast pro-cedures (Table 2). The values for total pectin in ripefruit are higher than those reported by Redgwell andTurner (1986). The level of esteriÐed pectin, expressed asa percentage of total pectin, changes signiÐcantly onlybetween stages [2 and 6 (Table 2) and when groups ofmeans are contrasted (Table 2), it is clear that pectinesteriÐcation is not a†ected by the ripening process. Theextent of esteriÐcation is quite limited in pepino fruitand is of the order of that reported for strawberry(Wade 1964)Èother fruits examined have more than10-fold this amount (Knee 1978 ; Redgwell et al 1988 ;

Dawson et al 1992). Since total pectin declines duringripening without a change in the esteriÐcation of theremaining pectin it is likely that esteriÐed and non-esteriÐed pectin are both targeted for loss as pepinoundergo Ðnal development and ripening, but the contri-bution to the solubilised pectin would be slight. Pecti-nmethylesterase activity increases during pepinoripening (Heyes et al 1994), but does not appear to havea large impact on the esteriÐcation of wall pectins.

The quantity of pectin solubilised by the chelatingagent CDTA increases markedly in the late ripeningstages (4, 5 and 6) when contrasted with levels in latedevelopment or early ripening fruit (Table 2). A keypoint in the change in solubility properties appears tobe at stage 4, although there are indications that theincrease is initiated earlier, when purple stripingappears on the skin (stages 1 and 2, Table 2). Theapproximately 2É5-fold increase in CDTA-soluble pectinas fruit enter the late ripening phase may be due to thede novo synthesis of smaller-sized pectic molecules, orcould be due to de-esteriÐcation of pectin polymers inthe cell wall. We have no evidence to support or dis-claim the de novo synthesis scenario. There is a steadyincrease in pectinmethylesterase activity (Heyes et al1994) and a slight but steady decrease in levels of ester-iÐed pectin during pepino fruit ripening which does notcorrelate with the stage 4-speciÐc increase in CDTA-soluble pectin. It seems unlikely that CDTA-solublepectin increases at stage 4 simply by de-esteriÐcation.

Accompanying the increasing amounts of chelator-soluble pectin is a change in their distribution.MrFigure 4 shows the distributions of CDTA-solubleMrpectins from stages [2 through to 6, averaged for eachharvest replicate. Soluble pectins from stages in the latedevelopment and early ripening of the pepino fruit(stages [2 to 3) elute from this column as one peakclose to the column void volume. Although thereappear to be di†erences in the sharpness of these peaks,no consistent trends are evident. As with the quantitat-ive measurements of CDTA-soluble pectin, the mainchange in elution proÐle occurs at stage 4 when there isa sudden decrease in the proportion of pectin largeenough to elute close to the void volume of the column,and an increase in fractions of intermediate and lowmolecular weight. By stage 6 the main peak in themolecular weight distribution elutes close to the inclu-sion volume of the column. The molecular weightmarkers used to calibrate the Superose 6HR column arelinear dextrans and can only be used as size indicators,not to determine the absolute molecular weight of thepectin, since this might be inÑuenced by the presence/absence of neutral residue side-chains. The change in

of chelator-soluble pectin is a consistent theme inMrripening fruit and has been documented extensively (egHuber and Lee 1986 ; Dawson et al 1992 ; Redgwell et al1992 ; Huber and OÏDonoghue 1993). Chelator-solublepectin from fruits that ripen in an ethylene-insensitive

462 E M OÏDonoghue et al

Fig 4. Molecular size distribution of CDTA-soluble pectinsisolated from ethanol-insoluble wall preparations of pepinofruit at maturity stages [2 to 6. Pectic polymers were frac-tionated on Superose 6HR and uronic acids were detected at520 nm using the method of Blumenkrantz and Asboe-Hansen (1973). Developmental and ripening stages are indi-cated on each graph. Molecular weight markers are as for

Fig 2.

manner, and do not possess readily measurable PGactivity, such as persimmon and muskmelon, also showsome size modiÐcations during ripening (McCollum etal 1989 ; Cutillas-Iturralde et al 1993).

Between stages 3 and 4 there is a dramatic release ofCDTA-soluble pectin and an associated increase inmuch smaller pectic fragments. PG activity increasesfrom stage 1 onwards (Heyes et al 1994), but there is noindication during early ripening that PG activity has animpact on CDTA-soluble pectin (either quantitativelyor qualitatively), although it is possible that other sol-vents may detect solubility changes. It may well be that

the ionic conditions in the apoplast become conducivefor PG to be active in vivo between stage 3 and 4, whenthe fruits start to develop yellow coloration, as pro-posed by Huber and OÏDonoghue (1993). Alternatively,PG may be active at an early stage (around stage 1 or 2)in some limited capacity, hydrolysing internal bondswithin extremely large pectic polymers (and these poly-mers are insoluble in CDTA).

CONCLUSIONS

We have deÐned ripening-associated changes in thestructure and composition of the cell walls of pepinofruit. We suggest that the progressive decline in cell wallstrength and cell-to-cell adhesion described by Heyes etal (1994) can be linked to qualitative changes in matrixpolysaccharides. The contribution of cell wall modiÐ-cation to softening can be separated into an initial earlyphase, which is related to changes in molecular weightof hemicellulose polymersÈparticularly xyloglucansÈand the initiation of pectin solubilisation, and a laterphase contributed to by changes in the amount and Mrof chelator-soluble pectin. The changes in pectin solu-bility may be inÑuenced by the degree of de-branchingand loss of neutral sugars, issues that have not yet beenaddressed.

ACKNOWLEDGEMENTS

The authors thank Sandy Wright and John Jowett fortheir advice on the statistics required for this research.

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