Postharvest Biology and Technology 23 (2001) 197–208 www.elsevier.com/locate/postharvbio

Changes in chemical wax composition of three differentapple (Malus domestica Borkh.) cultivars during storage

Els A. Veraverbeke *, Jeroen Lammertyn, Stijn Saevels, Bart M. NicolaıFlanders Centre/Laboratory of Posthar�est Technology, Katholieke Uni�ersiteit Leu�en, W. de Croylaan 42,

3001 He�erlee, Belgium

Received 18 December 2000; accepted 16 May 2001

Abstract

The effects of year, picking date and storage conditions on the chemical composition of the wax layer of three applecultivars (‘Jonagold’, ‘Jonagored’ and ‘Elstar’) were investigated by means of GCMS and multivariate statisticaltechniques. Wax of apples with different surface characteristics also differed in chemical composition. Controlledatmosphere (CA) storage and subsequent shelf life affected wax properties and caused changes in the chemicalcomposition, especially during shelf life. The changes in wax composition of ‘Elstar’, however, were smaller thanthose in ‘Jonagold’ and ‘Jonagored’, possibly associated with lower diffusion resistance and more limited structuralchanges of ‘Elstar’ wax. Longer CA storage periods accelerated the changes in wax composition during subsequentshelf life. The components responsible for the changes were mainly the alkane and ester fractions. The secondaryalcohol nonacosan-10-ol was particularly important in explaining the changes in wax composition of ‘Jonagold’during shelf life and its presence could be linked with greater development of greasiness. © 2001 Elsevier Science B.V.All rights reserved.

Keywords: Wax; Chemical composition; PCA; Apple

1. Introduction

Like most aerial parts of plants, apples arecovered with a continuous extracellular cuticle.This cuticle consists of a triglyceride matrix orcutin layer and an epicuticular wax layer. Thelatter is deposited on and in the cutin matrix andforms the natural interface between the plant and

its environment. The wax layer primarily providesexcellent protection against water loss (Araus etal., 1991; Schreiber and Riederer, 1996). Besidesthat, it has other physiological and ecologicalfunctions such as limiting surface permeability(Baur et al., 1996), reduction of transpiration(Blanke and Holthe, 1997) and protection againstinvasion of micro-organisms and insects (Mark-stadter et al., 2000). Typical wax characteristicscan vary greatly among different apple cultivarsand the wax can also change when subjected toenvironmental stresses such as rain (Rinallo andMori, 1996), UV-B (Gordon et al., 1998), wind

* Corresponding author. Tel.: +32-16-322668.E-mail address: els.veraverbeke@agr.kuleuven.ac.be (E.A.

Veraverbeke).

0925-5214/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0925 -5214 (01 )00128 -4

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(Gardingen et al., 1991) and temperature (Roy etal., 1994).

For apples, this protective function of the waxis not only important on the tree, but also duringlong-term controlled atmosphere (CA) storage,especially in limiting water and hence weight loss.In storage, water loss is across the epidermis andthe wax layer is the most important transportbarrier (Jenks et al., 1994). During storage, how-ever, wax characteristics change and the effects ofenvironment and storage conditions have beeninvestigated in the past (Morice and Shorland,1973). For most plants, the role of the wax struc-ture is seen as more important as a protectivefunction, and so the wax layer is seen as a physi-cal rather than a chemical barrier (Riederer andSchneider, 1990; Schreiber and Riederer, 1996).The presence of cracks in the wax surface and thespecific melting of the wax at higher temperaturesto cover the cracks, have been found to be veryimportant in controlling water loss (Lurie et al.,1996; Maguire et al., 1999; Veraverbeke et al.,2001). Structure and composition both changeduring storage, however, and can not be consid-ered independently.

Most wax constituents originate from the elon-gation of C16 and C18 free fatty acids to longchain aliphatic acids with an even number of Catoms, which can be reduced to aldehydes orprimary alcohols. Alkanes arise from the decar-boxylation of these long chain fatty acids or thedecarbonylation of the fatty aldehydes, and theycan then be oxidised to ketones or secondaryalcohols (Hannoufa et al., 1993). Eventually, bythe combination of the alcohols and free fattyacids, a wide range of ester homologues results. Inmany waxes, including those of apple fruit, theC29 homologues were found to be the most abun-dant components (Morice and Shorland, 1973;Jenks et al., 1996; Belding et al., 1998). Little isknown, however, about wax composition differ-ences and the changes that occur during storagein different cultivars and in relation to specificfruit surface characteristics.

The physical and chemical properties of thewax also determine the apple’s appearance (Glennet al., 1990), one of the most important qualitycomponents determining consumer demand for

the fruit. During long term storage and shelf life,excessive water loss may occur, causing shrivel(Hatfield and Knee, 1988). The wax layer mayprevent such water loss by changes in its composi-tion and distribution, or by covering cracks andstomata. Therefore, changes in wax properties canhave economic consequences, especially whengreasiness develops. Optimal storage conditionsshould, therefore, include an appropriate balancebetween minimal moisture loss and optimal exter-nal appearance of the fruit. This requires moreinformation on wax properties and better statisti-cal analysis of the differences in wax characteris-tics of a large number of fruit of differentcultivars.

Our objective was to evaluate changes in chem-ical composition of the wax layer of three applecultivars with clearly different macroscopic sur-face characteristics, during long-term CA storageand shelf life. After chemical identification of thewax layer components, evaluation of the waxcharacteristics was carried out by comparison ofsimple gas chromatographic profiles of the purewax of 1200 apples by means of multivariatestatistical techniques.

2. Materials and methods

2.1. Experimental design

Apples used in this experiment were randomlypicked from 10 trees per cultivar at the NationaleProeftuin voor Grootfruit (Velm, Belgium). Sam-ples were taken over a period of 2 years from1998 to 2000. A complete factorial design wasused, consisting of three cultivars (‘Elstar’, ‘Jon-agold’ and ‘Jonagored’), two or three pickingdates, three CA storage periods and three shelflife periods. ‘Elstar’ was picked on two differentpicking dates (early, 11 September 1998 and 2September 1999; commercial, 18 September 1998and 11 September 1999), and ‘Jonagold’ and ‘Jon-agored’ on three different picking dates (early, 23September 1998 and 22 September 1999; commer-cial, 30 September 1998 and 29 September 1999;late, 8 October 1998 and 7 October 1999). In thesecond year, the mutant ‘Elshof’ was picked in-

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stead of ‘Elstar’. On each picking date, 90 ran-domly selected apples were harvested percultivar: 30 apples were analysed immediately afterharvest (0 months of CA storage), 60 werestored under commercial CA conditions until anal-ysis after 4 or 8 months. For ‘Elstar’ and ‘Elshof’the CA conditions were 2% O2 and �1% CO2 at1 °C and 95% RH, and for ‘Jonagold’ and‘Jonagored’ 1% O2 and 2.5% CO2 at 1 °C and95%RH.

At every CA storage period (0, 4 and 8 months),samples of 30 apples were selected for analysis: 10apples were evaluated immediately and 10 eachafter 1 and 2 weeks of shelf life at 20 °C. Eachevaluation included measuring apple weight andequatorial diameter and isolation and subsequentchemical analysis of the wax.

2.2. Chemical analysis

The total wax of each apple was extracted byimmersing the fruit in hexane for 45 s (Tulloch andBergter, 1981; Tulloch, 1987; Jenks et al., 1995;Smith et al., 1996; Rashotte et al., 1997). The timefor extraction was determined by checking thecompleteness of extraction with confocal micro-scopic imaging before and after extraction (Ver-averbeke et al., 2001). After extraction the solventwas removed under vacuum at 60 °C and theremaining wax transferred in 1 ml hexane to apre-weighed vial. These wax samples could bestored at −20 °C until GC analysis. Before GCanalysis all samples were dried under nitrogen(Jenks et al., 1995) for at least 72 h until completedryness and the weight of the total wax was thendetermined with a microbalance (Sartorius BPmaximum 160 g, Germany).

For GC analysis (GC Top 8000 series, CEInstruments, Interscience, Belgium) solutions ofthe total wax of one apple in 1 ml hexane wereprepared and heated at 60 °C until completesolubilisation of the wax. Then 1 �l of this solutionwas injected into a 32 m RTX-1 capillary column(0.32 mm ×0.25 �m) by means of split injectionat a temperature of 220 °C and a split ratio of 1/20.A standard FID detector was used at 320 °C. Thetemperature program was 180–290 °C with a 10 °Cper min increase and finally an isothermal period

of 21 min at 290 °C. Helium was used as carrier gasat a flow rate of 1 ml/min.

Small-scale column chromatography was carriedout using a Pasteur pipette filled with silica gelearlier activated at 120 °C. The total wax of oneapple dissolved in 1 ml hexane was loaded onto thecolumn and successively eluted with different 10 mlfractions of increasing polarity by combining hex-ane, diethylether (DEE) and acetone in differentconcentrations (100% hexane, 50% hexane 50%DEE, 100% DEE, 50% DEE 50% acetone, 100%acetone). Preparative TLC of the total wax of oneapple was carried out on silica gel plates (Uniplate,Analtech, Belgium) with a solvent system of hex-ane acetic acid (100:1) for 12 cm followed byhexane diethylether acetic acid (75:25:1) for 10 cm(Knowles et al., 1996). For detection of the differ-ent component classes, the plates were sprayedwith a 10% solution of phosphomolybdic acid inethanol. GC-MS was carried out at DCMS(Bierges, Belgium) on a Finnigan Trace MS withsplit injection and as ionisation mode an electronimpact of 70 eV. A full scan from m/z=41 to 500was acquired in 0.5 s. The GC conditions were thesame as for the regular GC analysis.

With GC, typical and reproducible chro-matograms were obtained for each apple cultivarand the different peaks identified by means of acombination of column chromatography, prepara-tive thin layer chromatography (TLC), GCMS andH-NMR (Veraverbeke, unpublished data). Aftercomparison of the different gas chromatograms of‘Jonagold’, ‘Jonagored’ and ‘Elstar’, a selectionwas made of 34 peaks that were always present inevery chromatogram (Table 1). The relative con-centrations of these components, calculated bytaking the ratio of the integrated peak areas andthe total area of all 34 integrated peaks, werefollowed during storage. Since not all of the 34selected peaks could be attributed to every cultivar,the missing peaks were assigned a zero area.

2.3. Statistical analysis

Large data sets often contain a considerableamount of information, which is partially hiddenbecause the data are too complex to be easilyinterpreted. PCA (principle component analysis)

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is a multivariate statistical technique used to re-duce the number of variables and facilitate inter-pretation of the data based on data visualisationtechniques. PCA is used to explain which vari-ables are really important to describe the varia-tion or information in the data matrix. Thevariance–covariance structure of the data matrixis described by means of a few linear combina-tions of the original variables called principalcomponents (PCs). The first PC explains most ofthe variance in the data and each subsequent PC

will explain less variance than the earlier one.Much of the variability will usually be accountedfor by only a small number of PCs.

Once the amount of useful information as op-posed to noise or meaningless variation containedin the data structure has been quantified, loadingplots and score plots are constructed to visualisethe data. It is then possible to find out in whatrespect one sample is different from another,which variables contribute most to this differenti-ation, and whether those variables contribute in

Table 1Peak number, identity and average concentration of wax components based on 450 fruits for ‘Jonagold’ and ‘Jonagored’ and 300fruits for ‘Elstar’

ComponentNr Average relative concentration (%)

‘Jonagold’ ‘Jonagored’ ‘Elstar’

�-farnesene1 2.35 2.97 0Heptadecanoic acid2 0.28 0.36 0

01.030.863 Linoleic acid0.19 0.294 0Oleic acid

Stearic acid 0.105 0.16 06 00.910.90FFA

0.320.23 0FFA78 Linoleic acid ethyl ester 0.51 0.60 1.90

Pentacosane9 0.13 0.14 0Unidentified 0.4210 0.48 0Unidentified 0.1011 0.17 0Unidentified 0.0812 0.08 0

13 00.040.06UnidentifiedHexacosane 0.0614 0.07 0

15 00.090.08Unidentified0.200.22 0Unidentified16

17 Heptacosane 0.598.847.92Unidentified18 0.18 0.11 0

19 23.831.010.95Octacosane0.160.15 1.59Tridecanal200.740.92 1.35Unidentified21

56.21 62.9654.84Nonacosane220.22Unidentified 00.61230.260.25Triacontane24 0.24

Unidentified25 0.420.41 0.3926 1.1800Unidentified

027 0Unidentified 1.9628 Untriacontane 0.10 0.19 029 Nonacosan-10-ol 20.85 18.43 0

0.250.510.6130 Ester131 0.63Ester2 0.54 0.16

Ester3 0.5432 0.34 00.652.512.5633 Ester X1

Ester4 0.51 0.51 0.3334

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the same way, i.e. are correlated or independentof each other. PCA analysis of the data set onwax composition was carried out by means of thesoftware ‘The Unscrambler’ version 6.11 (CAMO,ASA, Norway).

3. Results and discussion

3.1. Chemical composition of the wax of‘Jonagold’, ‘Jonagored’ and ‘Elstar’

For a first exploration of the data obtainedfrom the GC analysis, an overview of the 34selected peaks with their identification and meanrelative concentration was created as shown inTable 1. From this table, the alkane nonacosane(C29H60) could be assigned as the most abundantcomponent for all three cultivars with the highest(relative) concentration for ‘Elstar’. Components1 (�-farnesene), 17 (heptacosane) and 29 (nona-cosan-10-ol) are typical for ‘Jonagored’ and ‘Jon-agold’ while 19 (octacosane) and two unidentifiedpeaks at positions 26 and 27 are almost exclu-sively present for ‘Elstar’. The most obvious dif-ference among the wax composition of the threecultivars was the complete absence of nonacosan-10-ol in the wax of ‘Elstar’ while it was one of themost important components for ‘Jonagold’ and‘Jonagored’, with a slightly higher relative concen-tration for ‘Jonagold’. If nonacosan-10-ol is thecompound determining the main differences insurface characteristics among the three cultivarsthen conditions that influence the presence of thiscomponent may control the surface properties ofthe fruit. Based on these differences in wax com-position, a first diversification between the threecultivars was possible.

PCA analysis performed on the total data set of1200 apples with 34 variables revealed that 95% ofthe variance could be explained with the first twoprinciple components (PCs) (PC1: 88%; PC2: 7%).The third PC only added another 2% of explainedvariance. The score plot of PC3 versus PC1 pro-vided the best visualisation of the separation be-tween the three cultivars (Fig. 1a). ‘Elstar’ couldbe clearly separated from ‘Jonagold’ and ‘Jon-agored’ along the PC1 axis. The cultivars ‘Jon-

agold’ and ‘Jonagored’ were only slightlyseparated along PC3. This result could be ex-pected as ‘Elstar’ was chosen as a cultivar withvery different surface characteristics from theother cultivars. ‘Elstar’ is a cultivar with a drysurface, a limited sensitivity to greasiness buthighly prone to water and hence weight loss,during storage. ‘Jonagold’ and ‘Jonagored’ on theother hand are cultivars with a smoother surface,a more prominent wax but less water loss duringstorage. In this respect ‘Jonagored’ was cultivatedas a strain of ‘Jonagold’ in order to obtain anapple with the same wax quality as ‘Jonagold’ butless stickiness development. The fact that ‘Jon-agold’ and ‘Jonagored’ are related cultivars ex-plains why their wax characteristics are so similarand why these groups of data were not clearlyseparated.

The loading plot of PC3 versus PC1 is shown inFig. 1b. As PC1 mainly defines the separationbetween ‘Elstar’ on the one hand and ‘Jonagored’and ‘Jonagold’ on the other, the variables or waxcomponents �-farnesene, heptacosane, octa-cosane, nonacosane, nonacosan-10-ol and esterX1 can be considered mainly responsible for theseparation of the cultivars. Further, by compari-son of both the score and the loading plot, itcould be concluded again that �-farnesene, hepta-cosane, nonacosan-10-ol and ester X1 were con-tributing in a negative way (thus, more specific for‘Jonagold’ and ‘Jonagored’) while octacosane andnonacosane were contributing positively (thus,more specific for ‘Elstar’ wax).

In order to explain the separation between ‘Jon-agold’ and ‘Jonagored’, with respect to the waxcomposition, the contribution of the different waxcomponents on PC3 was considered as well. Sincethe ‘Jonagored’ data in the score plot (Fig. 1a)were shifted a little downwards along the PC3axis compared with those of ‘Jonagold’, a nega-tive contribution of a chemical constituent alongPC3 in the corresponding loading plot (Fig. 1b),indicated a constituent characteristic for the waxof ‘Jonagored’ whereas a positive loading indi-cated a component more specific for ‘Jonagold’.Again, nonacosan-10-ol was the determining com-ponent here with a higher concentration for ‘Jon-agold’ than for ‘Jonagored’. The presence of

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Fig. 1. Score (a) and loading (b) plots for PC3 versus PC1 corresponding to a PCA analysis of the total data set of 34 variables and1200 apple samples. Cultivar ‘Elstar’ is clearly differentiated from cultivars ‘Jonagold’ and ‘Jonagored’ along the axis of PC1. In theloading plot (b) the components responsible for this separation are shown.

nonacosan-10-ol is, therefore, important in ex-plaining the differences in wax composition andsurface characteristics among all three cultivars.

3.2. Influence of year and picking date on waxcomposition

Comparison of the chemical wax layer compo-sition of all three cultivars over both years

through PCA showed a reasonable year influencefor ‘Elstar’; this was smaller for ‘Jonagored’ and‘Jonagold’. For ‘Elstar’ the first three PCs ex-plained 98% of the variance in the dataset (PC1:86%, PC2: 9%, PC3: 3%). The score plot of thefirst and second PC gave the best representationof the differences in wax characteristics over 2years by a shift of the data to the right along thePC1 axis (Fig. 2). From the loading plot (not

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shown) this shift could be attributed to the twocharacteristic alkanes of ‘Elstar’, namely octa-cosane (more specific for 1998) and nonacosane(more specific for 1999). Wax component 27(unidentified) and the ester X1, on the other hand,seemed to be not related at all to the differencesbetween 2 years, while they accounted for some ofthe variance in the total data set.

For ‘Jonagold’ the changes in wax characteris-tics between the 2 years were smaller and the firstthree PCs explained 89% of the variance (PC1:57%, PC2: 24%, PC3: 8%); (data not shown). Theinfluence of �-farnesene, heptacosane and ester X1

was the largest, and these components were re-sponsible for most of the variance between 2years. Nonacosane and its secondary alcohol onthe other hand, could not explain the differencesin wax composition over the 2 years. The resultsfor ‘Jonagored’ were similar to those of ‘Jon-agold’ (data not shown). For none of the cultivarsfor either of the 2 years was there an importantinfluence of picking date on the wax composition.

3.3. Influence of storage time

PCA analysis of the data of each cultivar re-vealed a slight change in wax composition duringCA storage that was only dependent on the culti-var and not on the year or picking date. For‘Elstar’ this change was most clearly seen in the

score plot of PC2 and PC3 along a directionrunning from the bottom right to the top left,which was aligned with the data from 0 to 8months of storage (Fig. 3). Ester X1 was the mostimportant component influencing this shift. Othercomponents that were highly correlated with esterX1 were the components at positions 30 (ester)and 34 (ester). It could be concluded from theloading plot (not shown) that the concentration ofthese specific wax constituents decreased duringstorage. This could also be confirmed from thenumerical data of the GC analysis (Table 2). Allof these peaks belonged to a group of estersconsisting of an alcohol and a C16 or C18 fattyacid (Veraverbeke, unpublished data). A loss inrelative concentration can then possibly be at-tributed to the hydrolysis of these esters duringstorage. Nonacosane and octacosane, on the otherhand, were not correlated with the earlier peaksand didn’t change enough to be responsible forany changes in wax characteristics after long-termCA storage.

For ‘Jonagold’ and ‘Jonagored’ the relativeconcentration of ester X1 declined during storagewhile nonacosane and nonacosan-10-ol increasedin relative concentration. This was a remarkableresult, as nonacosane was not prevalent in ‘Elstar’wax and nonacosan-10-ol is an important compo-nent in the changes in composition of ‘Jonagold’and ‘Jonagored’. �-Farnesene and heptacosane

Fig. 2. Year influence from 1998 to 1999 on the chemical composition of ‘Elstar’ wax.

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Fig. 3. Influence of the duration of CA storage on the chemical composition of ‘Elstar’ wax. From 0 to 8 months of CA storagea shift in the data from the bottom right to the top left (indicated by the arrow) in the score plot occurs.

did not change during storage. Heptacosane(‘Jonagold’ and ‘Jonagored’) and octacosane (‘El-star’) were very characteristic for the specific culti-vars but also did not change appreciably.

3.4. Effect of shelf life

From the different score plots of the wax dataof 1998 and 1999 there was only a very slighttrend visible in the data on wax composition of‘Elstar’ fruit during 0–2 weeks of shelf life (datanot shown). Changes in the wax compositioncould be mainly attributed to a decrease in rela-tive concentrations of nonacosane and octa-cosane. The effect of shelf life on the wax of‘Jonagold’ was more obvious. A clear shift oc-curred in the score plot of the wax data of both1998 and 1999 with PC1 and PC2. In Fig. 4, thescore plots for 0 and 4 months of CA storage in1999 are shown with PC1 explaining 67% of thevariance and PC2 22%. From these plots, theinfluence of CA storage on the subsequent shelflife could also be established. Immediately afterharvest (0 months CA, upper plot in Fig. 4), theeffect of 1 week of shelf life on the wax composi-tion was still very small, evident from a verylimited shift between the data of 0 and 1 weekshelf life. After 2 weeks of shelf life, a much more

prominent shift occurred. After 4 (lower part ofFig. 4) and 8 months (not shown) of CA storagethis clear shift of the data already occurred after 1week of shelf life and this time the differencebetween 1 and 2 weeks of shelf life was much

Table 2Influence of CA storage (0 and 8 months) on the relativeconcentration of some components of ‘Elstar’ fruit in 1999

Component Relative concentration (%)

0 months CA 8 months CAstoragestorage

Linoleic acid *1.50 1.79ethyl ester

*19.16 22.36Octacosane*1.59Tridecanal 1.22*0.70 0.9321

59.40Nonacosane 68.59 **0.6523 0.20

0.43Triacontane 0.16 *0.50 *26 0.651.4527 1.81 *

30 (ester) 0.18 0.03 *31 (ester) 0.26 *0.39Ester X1 1.16 0.53 *

0.29 0.1934 (ester) 0.01

* Means significant difference between 0 and 8 months ofCA storage at P=0.05.

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Fig. 4. Changes in chemical wax composition for ‘Jonagold’ in 1999–2000 at picking date 2 after 0 and 4 months of CA storageand 0, 1 and 2 weeks of shelf life at 20 °C. The shift in data caused by shelf life occurs after 2 weeks before CA storage and alreadyafter 1 week at 4 months of CA storage.

smaller. Thus, the longer the apples are stored,the more sensitive they become to the negativeeffects of subsequent ambient conditions.

From the corresponding loading plots (not

shown) the contribution of all wax components tothese changes could be deduced. This time �-far-nesene, ester X1, nonacosane and nonacosan-10-olwere found to influence the changes while hepta-

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cosane seemed less important. Here again nona-cosane-10-ol seemed to be associated with theadverse effect of shelf life on the wax quality.

Similar effects were found with ‘Jonagored’only this time there was a greater overlap betweenthe different groups of data for 0, 1 and 2 weeksof shelf. This could be due to the fact that ‘Jon-agored’ is a strain less predisposed to greasiness,one of the major problems during shelf life stor-age. On the other hand, the most important com-ponents involved in the wax changes during shelflife were not that much different from ‘Jonagold’,although, the effect of nonacosane was found tobe smaller from the loading plots.

4. Conclusions

PCA coupled with a simple and quick chro-matographic analysis allowed separation of thethree apple cultivars based on their wax composi-tion and the way they changed in storage. ‘Elstar’with a rough surface and considerable susceptibil-ity to water loss during storage was distinct from‘Jonagold’ and ‘Jonagored’, which have smoothersurfaces and lower susceptibility to water loss.The differentiation between ‘Jonagold’ and ‘Jon-agored’ was less obvious. This was expected as‘Jonagored’ is a strain of ‘Jonagold’. From thetotal analysis, a secondary alcohol of nonacosane(nonacosan-10-ol) is likely to be the most impor-tant component in explaining the differences insurface characteristics among the three cultivars.Although in our analysis, this compound was notfound in ‘Elstar’ wax, its was earlier reported forother apple cultivars (Morice and Shorland, 1973;Belding et al., 1998). Further, it can be seen in theGC data of Table 1 that the relative nonacosaneconcentration was higher in ‘Elstar’ than in theother cultivars and also that the relative octa-cosane concentration in ‘Elstar’ and the nona-cosan-10-ol concentration in ‘Jonagold’ and‘Jonagored’ were both around 20%. The presenceof an alcohol instead of an alkane can also help inexplaining the wax glossiness, typical for ‘Jon-agold’ and ‘Jonagored’. Glossiness has alreadybeen correlated with the presence of alcoholswhile alkanes correlate with a more glaucous ap-

pearance (Baker, 1974). For many species, thepresence of nonacosan-10-ol has been associatedwith the formation of typical tube-like crystals(Franich et al., 1978; Hannoufa et al., 1993; Gulz,1994). This, however, was not seen in the micro-scopic images of ‘Jonagold’ and ‘Jonagored’ slicesthat show a surface with more platelet-shapedcrystals (Veraverbeke et al., 2001). If nonacosan-10-ol is responsible for the development of greasi-ness then the transformation of the wax to a moreamorphous smooth layer as a reaction to highertemperatures and lower relative humidities,should also be related to the typical crystal forma-tion behaviour of nonacosan-10-ol.

Differences in wax composition between thetwo considered harvest years (1998–1999 and1999–2000) were small for ‘Jonagold’ or ‘Jon-agored’ but were more striking for ‘Elstar’. This,however, could also be attributed to the fact thatin the first year ‘Elstar’ was used while in thesecond year the related mutant ‘Elshof’ waspicked. The year differences mostly correlatedwith changes in the alkane fraction. Apples werealso picked at different picking dates each year.The wax composition, however, did not changemuch with picking date.

The most obvious visual changes of the surfaceoccur during storage. The wax changes its struc-ture by external stresses that occur during storageand in some cases becomes sticky or shows abloom. The most obvious chemical changes alsooccur at this stage. Storage of apples commonlyconsists of long-term CA storage and short-termshelf life. Due to the stable conditions of CAstorage, the changes immediately after storageand up to 8 months were not so great. They wererelated mostly to hydrolysis of the ester fraction,with an increase in relative concentration of nona-cosan-10-ol for ‘Jonagold’ and ‘Jonagored’. Thismay explain the increased glossiness and greasi-ness of these cultivars during storage. The hydrol-ysis of the esters would mean an increase in thefree fatty acid fraction especially the C16 and C18

fatty acids, since they are the components onwhich the biosynthesis of the wax is based (Han-noufa et al., 1993; Jenks et al., 1995, 1996). Thisincrease in free fatty acid concentration duringcold storage of apples was earlier shown by

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Morice and Shorland (1973). In addition, thepresence of C16 and C18 free fatty acids in the waxof the apple cultivars in this study could be shownafter methylation and silylation of the wax tofacilitate the detection of free fatty acids with GCanalysis.

Acknowledgements

The authors wish to thank the Flemish Govern-ment and the Ministry of SME and Agriculture(project S5901) for their financial support. ElsVeraverbeke is a doctoral fellow of the IWT andJeroen Lammertyn is a Research Assistant of theFund for Scientific Research-Flanders (Belgium)(FWO-Vlaanderen). The financial support of bothInstitutes is acknowledged with gratitude.

References

Araus, J.L., Febrero, A., Vendrell, P., 1991. Epidermal con-ductance in different parts of durum wheat grown underMediterranean conditions: the role of epicuticular waxesand stomata. Plant Cell Environ. 14, 545–558.

Baker, E.A., 1974. The influence of environment on leaf waxdevelopment in Brassica oleracea var. gemmifera. NewPhytol. 73, 955–966.

Baur, P., Marzouk, H., Schonherr, J., Bauer, H., 1996. Mobil-ities of organic compounds in plant cuticles as affected bystructure and molar volumes of chemicals and plant spe-cies. Planta 199, 404–412.

Belding, R.D., Blankenship, S.M., Young, E., Leidy, R.B.,1998. Composition and variability of epicuticular waxes inapple cultivars. J. Am. Soc. Hortic. Sci. 123, 348–356.

Blanke, M.M., Holthe, P.A., 1997. Bioenergetics, main-tainance, respiration and transpiration of pepper fruits. J.Plant Physiol. 150, 247–250.

Franich, R.A., Wells, L.G., Holland, P.T., 1978. Epicuticularwax of Pinus radiata needles. Phytochemistry 17, 1617–1623.

Gardingen van, P.R., Grace, J., Jeffree, C.E., 1991. Abrasivedamage by wind to the needle surfaces of Picea sitchensis(Bong.) Carr. and Pinus syl�estris L. Plant Cell Environ.14, 185–193.

Glenn, G.M., Rom, C.R., Rasmussen, H.P., Poovaiah, B.W.,1990. Influence of cuticular structure on the appearance ofartificially waxed ‘Delicious’ apple fruit. Sci. Hortic. 42,289–297.

Gordon, D.C., Percy, K.E., Riding, R.T., 1998. Effects ofuv-B radiation on epicuticular wax production and chemi-cal composition of four Picea species. New Phytol. 138,441–449.

Gulz, P.-G., 1994. Epicuticular leaf waxes in the evolution ofthe plant kingdom. Plant Physiol. 143, 453–464.

Hannoufa, A., McNevin, J., Lemieux, B., 1993. Epicuticularwaxes of Eceriferum mutants of Arabidopsis thaliana. Phy-tochemistry 33, 851–855.

Hatfield, S.G.S., Knee, M., 1988. Effects of water loss onapples in storage. Int. J. Food Sci. Technol. 23, 575–583.

Jenks, M.A., Joly, R.J., Peters, P.J., Rich, P.J., Axtell, J.D.,Ashworth, E.N., 1994. Chemically induced cuticle muta-tion affecting epidermal conductance to water vapor anddisease susceptibility in Sorghum bicolor (L.). Moench.Plant Physiol. 105, 1239–1245.

Jenks, M.A., Tuttle, H.A., Eigenbrode, S.D., Feldmann, K.A.,1995. Leaf Epicuticular Waxes of the Eceriferum Mutantsin Arabidopsis. Plant Physiol. 108, 369–377.

Jenks, M.A., Rashotte, A.M., Tuttle, H.A., Feldmann, K.A.,1996. Mutants in Arabidopsis thaliana altered in epicuticu-lar wax and leaf morphology. Plant Physiol. 110, 377–385.

Knowles, L.O., Knowles, R.N., Tewari, P.J., 1996. Aliphaticcomponents of the epicuticular wax of developing Saska-toon (Amelanchier alnifolia) fruit. Can. J. Bot. 74, 1260–1264.

Lurie, S., Fallik, E., Klein, J.D., 1996. The effect of heattreatment on apple epicuticular wax and calcium uptake.Postharvest Biol. Technol. 8, 271–277.

Maguire, K.M., Lang, A., Banks, N.H., Hall, A, Hopcroft,D., Bennett, R., 1999. Relationship between water vapourpermeance of apples and micro-cracking of the cuticle.Postharvest Biol. Technol. 17, 89–96.

Markstadter, C., Federle, W., Jetter, R., Riederer, M., Holl-dobler, B., 2000. Chemical composition of the slipperyepicuticular wax blooms on Macaranga (Euphorbiaceae)ant-plants. Chemoecology 10, 33–40.

Morice, I.M., Shorland, F.B., 1973. Composition of the sur-face waxes of apple fruits and changes during storage. J.Sci. Food Agr. 24, 1331–1339.

Rashotte, A.M., Jenks, M.A., Nguyen, T.D., Feldmann, K.A.,1997. Epicuticular wax variation in ecotypes of Arabidopsisthaliana. Phytochemistry 45, 251–255.

Riederer, M., Schneider, G., 1990. The effect of the environ-ment in the permeability and composition of Citrus leafcuticles II composition of soluble cuticular lipids and cor-relation with transport properties. Planta 180, 154–165.

Rinallo, C., Mori, B., 1996. Damage in apple (Malus domes-tica Borkh) fruit exposed to different levels of rain acidity.J. Hortic. Sci. 71, 17–23.

Roy, S., Watada, A.E., Conway, W.S., Erbe, E.F., Wergin,W.P., 1994. Low-temperature scanning electron mi-croscopy of frozen hydrated apple tissues and surfaceorganisms. HortScience 29, 305–309.

Schreiber, L., Riederer, M., 1996. Determination of diffusioncoefficients of octadecanoic acid in isolated cuticular waxesand their relationship to cuticular water permeabilities.Plant Cell Environ. 19, 1075–1082.

Smith, R.M., Marshall, J.A., Davey, M.R., Lowe, K.C.,Power, J.B., 1996. Comparison of volatiles and waxes inleaves of genetically engineered tomatoes. Phytochemistry43, 753–758.

E.A. Vera�erbeke et al. / Posthar�est Biology and Technology 23 (2001) 197–208208

Tulloch, A.P., Bergter, L., 1981. Epicuticular wax of Juniperusscopulorum. Phytochemistry 20, 2711–2716.

Tulloch, A.P., 1987. Epicuticular waxes of Abies balsamea andPicea glauca : occurence of long-chain methyl esters. Phyto-chemistry 26, 1041–1043.

Veraverbeke, E.A., Van Bruaene, N., Van Oostveldt, P., Nico-laı, B.M., 2000. Non-destructive analysis of the wax layerof apple (Malus domestica Borkh.) by means of confocallaser scanning microscopy. Planta (published online 14March 2001: DOI 10.1007/s004250100528).