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Research ArticleReceived: 15 September 2008 Revised: 16 April 2009 Accepted: 16 April 2009 Published online in Wiley Interscience: 8 June 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3651

Discrimination of climactericand non-climacteric melon fruit at harvestor at the senescence stage by quality traitsJavier M Obando-Ulloa,a Mohammad-Mahdi Jowkar,b Eduard Moreno,c

M Kazem Souri,d Juan A Martınez,e Marıa C Bueso,f Antonio J Monfortec†

and J Pablo Fernandez-Trujilloa∗

Abstract

BACKGROUND: This paper characterizes the quality traits at harvest and the changes associated with fruit senescence basedon fruit physiological behaviour (climacteric or non-climacteric) found in a collection of near-isogenic lines (NILs) of melon(Cucumis melo L.). Data from both stages of postharvest development were analyzed by univariate and multivariate statisticalanalysis.

RESULTS: The principal components and random forest analyses of the fruit quality traits allowed the best classification of theNILs by time (harvest, senescence), or by climacteric behaviour at harvest, but not at the senescent stage. The overall qualityprofile of the non-climacteric senescent melons was, in general, very different from that of the climacteric ones, and was inaccord with a longer storage life. Most of the taste quality traits (individual sugars or sucrose equivalents, titratable acidity andthe citric, oxalacetic, glutamic and succinic acids) and the traits related to skin, flesh and juice colour parameters (chroma, hueangle) helped to distinguish the climacteric NILs from the non-climacteric ones independently of the time considered.

CONCLUSIONS: The time had a stronger effect on quality than the physiological behaviour. The discrimination by climactericor non-climacteric behaviour was usually better at harvest than at the senescent stage irrespective of the methodology used.Principal component analysis was the best multivariate method to discriminate by time and physiological behaviour followedby random forest and linear discriminant analysis.c© 2009 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: near-isogenic lines; fruit composition; texture; sugars; organic acids; fruit over-ripening

INTRODUCTIONThe timing of fruit ripening and susceptibility to over-ripening isgenetically determined1,2 and controlled by the degradation of atleast two ethylene receptors.3 Climacteric fruit reach senescence,the last developmental process at the end of fruit ripening,sooner than non-climacteric ones.1,2 Ethylene biosynthesis andperception regulates the expression of downstream genes whichproduces the typical ripening events before senescence.4 – 9

These changes reduce fruit shelf-life and affects different fruitquality traits.9 – 13

Senescent fruit can be assessed by overall decay in freshness,appearance and flavour. Typical senescence symptoms monitoredinstrumentally are chlorophyll degradation and external colouryellowing, excessive flesh softening or soluble solids, somewhatwater-soaked in appearance, skin greasiness, tissue disintegrationsometimes associated with vitreous flesh at fruit blossomend, wateriness of the seed cavity, loss of sweetness andacidity and sometimes aromatic flavour, fermentation (alcoholic)and/or off-flavour, abnormal increase in respiration rate andethylene production, and increased susceptibility to decay.1,2,11 – 15

∗ Correspondence to: J Pablo Fernandez-Trujillo, Department of Agricultural andFood Engineering, Technical University of Cartagena (UPCT), Campus AlfonsoXIII, Paseo de Alfonso XIII, 48, ETSIA, E-30203 Cartagena, Murcia, Spain.E-mail: juanp.fdez@upct.es or jf68es@terra.es

† Current address: Instituto de Biologıa Molecular y Celular de Plantas (CSIC-UPV),C/Ingeniero Fausto Elio, s/n.CPI ‘‘Ciudad Politecnica de la Innovacion’’-Edificio8E, 46022 Valencia, Spain.

a Department of Agricultural and Food Engineering, Technical University ofCartagena (UPCT), Campus Alfonso XIII, Paseo de Alfonso XIII, 48, ETSIA andInstitute of Plant Biotechnology E-30203 Cartagena, Murcia, Spain

b Islamic Azad University, Science and Research Branch, Tehran, I.R., Iran

c IRTA, Centre de Recerca en Agrigenomica, Ctra de Cabrils, Km 2 E-08348 Cabrils,Barcelona, Spain

d Tarbiat Modares University, Department of Horticultural Sciences, P.O. Box14115-336, Tehran, Iran

e Department of Plant Production, UPCT, Campus Paseo Alfonso XIII, Spain

f Department of Applied Mathematics and Statistics, UPCT, Campus Muralla delMar, Doctor Fleming s/n, ETSII, E-30202 Cartagena, Murcia, Spain

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Table 1. Means of the fruit quality traits evaluated in senescent fruit of the near isogenic lines (NILs) and the parental line ‘Piel de sapo’ (PS)

NILs and classification of climacteric behaviour ANOVA significance

Fruit quality atthe senescence

SC3-5(LC)

5M5(LC)

5M6(LC)

5M9(LC)

5M3(MC)

5M4(MC)

5M8(MC)

5M10(MC)

5M2(NC)

5M7(NC)

PS(NC) Pedigree Time Pxt

Flesh firmness (N) 2.7∗ 2.7 2.6 2.7 2.8∗ 2.7 2.7 2.7 2.6 2.7 2.6 ∗ ∗∗∗∗ NS

Extractable juice(g·kg−1)

601 370∗ 613 461 662 627 615 472 436 577 611 ∗∗∗∗ ∗∗∗∗ ∗

L∗ skin (u) 53.3 50.3 45.4 61.9∗ 52.8 57.4∗ 53.6 60.2∗ 48 45.8 44.5 ∗∗∗ NS ∗∗

C∗ skin (u) 48.9∗ 46.5 40.4 64.2∗ 47.6 56.1∗ 50.6∗ 60.9∗ 39.2 31.8 34.3 ∗∗∗∗ NS NS

H∗ skin (◦) 101.9 105.4 98.6 92.2 99.9 96.5 99.5 94.2 104.6 96.8 100.4 ∗ ∗∗∗∗ NS

L∗ flesh (u) 66.2 72.9 64.6 72.3 52.3 61.4 58.6 71.5 50.6 68.9 67.7 ND ND ND

C∗ flesh (u) 19 21.2 14 17.5 15.8 24 17 15.5 14.3 16.7 18.4 ∗∗∗∗ NS ∗∗∗∗

H∗ flesh (◦) 97.2 99.8 101 102.9 94.3 86.8 98.1 102.9 102.6 103.1 102.1 ND ND ND

L∗ juice (u) 28.4 28.6 27.8 27.9 28.3 29.1 28.2 28 28.2 28.3 28 ∗∗ NS NS

C∗ juice (u) 1.9 2 1.3 1.8 1.8 3 1.7 1.4 1.8 1.3 1.8 ∗∗∗ NS ∗∗

H∗ juice (◦) 110.8 109.2 105.8 108 104.8 107.6 104.6 104.8 107.2 107.9 109 NS ∗∗∗∗ ∗∗

pH 6 5.7 5.8 5.7 6.1 6.1 5.9 5.8 6.1 5.7 5.8 ∗∗ ∗∗∗∗ ∗∗∗

Juice density (kg·m−3) 1048.2 1042.6 1053.3 1051.9 1043.2 1050.3 1044.5 1052.4 1063.8 1045.5 1051.4 ∗∗∗∗ ∗∗∗∗ NS

Maturity index (◦Brix/%malic acid)

119.5 89.9 104.9 85.1 122.5 137 122.5 110.1 119.9 85.9 98 ND ND ND

Weight loss after12 days (% w/w)

2.8∗ 3.4 2.4∗ 3.4∗ 3.3∗ 3.6 3.2∗ 2.6∗ 3.1∗ 3.0∗ 3.9 – – –

Storage life (days)z 12 18 12 18 18 13 16 18 20 16 21 – – –

z Time at which at least 50% of the fruit were sound.NIL means within rows highlighted with ∗ showed statistical differences from PS data according to a Dunnett’s test at P = 0.05. Maturity index was theratio between soluble solids content and titratable acidity. The fruit physiological behaviour was classified as slight, moderate or non-climacteric (LC,MC or NC, respectively) as reported in the text. The significant interaction and mean effects of the two-way analysis of variance (ANOVA) consideringdata at senescence and at harvest (Table 2) are highlighted according to the probability level.∗ P ≤ 0.05∗∗ P ≤ 0.01∗∗∗ P ≤ 0.001∗∗∗∗ P ≤ 0.0001

However, the climacteric or non-climacteric pattern in melons9 iscritical when developing a reliable definition of the senescentphenotype for marketing and genetic research purposes.

Obando-Ulloa et al.16 found that aroma profile has beenrevealed as an efficient tool to discriminate climacteric from non-climacteric near-isogenic lines. In their study, ten NILs and theparental line ‘Piel de sapo’ (PS) were studied. The NILs 5M2, 5M7and PS behaved as non-climacteric fruit while two main groups ofclimacteric intensity were detected in the rest of the NILs. The goalof the present study is to use the differences in climacteric or non-climacteric behaviour of these NILs to characterize the associationbetween physiological behaviour and changes in fruit quality traitsfrom harvest to senescent stage. Our second goal was to establishthe best multivariate analysis for future studies with NILs withdifferences in climacteric behaviour or other quality traits.16

MATERIALS AND METHODSPlant materialThe fruit at harvest and at the senescent stage were obtained fromPS and ten near-isogenic lines (NILs), as follows: the climactericNIL SC3-5 and nine NILs 5Mx (x = 2 to 10).2,16 The nineNILs contained shorter introgressions than SC3-5 of the Koreanaccession ‘Shongwan Charmi’ PI 161375 of the Conomon Group(SC) into the linkage group III of the parental line ‘Piel de sapo’genetic background.2,17

Experimental design

The crop management, harvesting practices and harvesting indiceshave been previously described.2,16,18 The plantlets at the twotrue leaf stage were established in an open field 36 days aftersowing at the beginning of March. Soil preparation, fertigation,plant protection, weed control, and other growing practices werethose commonly used for melon cultivation in the Mediterraneanconditions in Torre Pacheco (Murcia, Spain). The soil of the plot wasclassified as Haplic Calcisols.19 Each replicate consisted in threeplants separated 1.5 m in the rows. The distance among replicateswas 3 m. The separation between rows was 2 m. The plantationwas surrounded by a border line of the cultivar Nicolas.

Fruit were harvested by two people in 2 weeks during morninghours (6 : 30 to 10 am). The harvest season lasted less than 10 daysin every NIL. Minimum harvest indices were the presence of a wellformed and defect-free fruit, firm, well healed and dry epidermiswith lignified netting, high density according to the experienceof the harvester, skin colour, withering of the stem and leafcloser to the fruit peduncle suberisation, and yellowing of theground spot.2 The most common harvest indices for the non-climacteric NILs and PS were the development of skin netting andsometimes development of annular ring or yellow colour arounda partly suberised peduncle. For the climacteric NILs (dehiscent atdifferent extent), the harvest indices were the development andfruit dehiscence (about 1/2 to 3/4 slip for harvest), whole fruittexture and stylar-end texture, yellowing of the skin (including the

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Table 2. Means of the fruit quality traits evaluated at harvest in the near-isogenic lines (NILs) and the parental line ‘Piel de Sapo’ (PS) melon fruit

NILs and classification of climacteric behaviour

Fruit quality trait at harvestSC3-5(LC)

5M5(LC)

5M6(LC)

5M9(LC)

5M3(MC)

5M4(MC)

5M8(MC)

5M10(MC)

5M2(NC)

5M7(NC)

PS(NC)

Flesh firmness (N) 6.7 4.1∗ 5.2∗ 4.3∗ 3.7∗ 4.2∗ 4.3∗ 5.2∗ 4.2∗ 7.3 8.1

Extractable juice (g kg−1) 424∗ 269∗ 422∗ 296∗ 477 479 429∗ 303∗ 341∗ 411∗ 499

L∗ skin (u) 50.0∗ 50.6 47.1 52.2∗ 47.7 52.3∗ 46.4 52.6∗ 41.6 45.3 44.9

C∗ skin (u) 44.9∗ 50.7∗ 37.1 49.5∗ 43.6∗ 47.9∗ 37.5 49.5∗ 30.6 37.1 31.5

H∗ skin (◦) 105.1 104.4 108.3 105.6 103.8 106.5 109.9∗ 105.1 109.1 105.3 105.6

L∗ flesh (u) 72.6∗ 72.0∗ 68.1 72.4∗ 73.5∗ 71 69.3 72.8∗ 67.5 72.2∗ 69.2

C∗ flesh (u) 15.9 18.8∗ 16.8∗ 17.4∗ 17.7∗ 18.0∗ 19.9∗ 16.9∗ 16.4∗ 16.4∗ 14.7

H∗ flesh (◦) 101.2∗ 104.6 105.1 103.6∗ 102.4∗ 99.2 104.8 104.8 106.1 106.4 105.8

L∗ juice (u) 28.9 28.4 27.9 27.7 28.2 28.4 27.8 27.2∗ 27.2 28.2 28.4

C∗ juice (u) 1.7∗ 1.9∗ 1.6∗ 1.7∗ 1.5 1.9∗ 1.8∗ 1.6∗ 1.6∗ 1.4 1.3

H∗ juice (◦) 120.5 118.3∗ 117.4∗ 112.3∗ 119.1 118.4∗ 119.1 115.4∗ 120.4 126 125.1

pH 5.8 5.6 5.7 5.9∗ 5.6 5.7 5.5∗ 5.8 5.7 5.5∗ 5.7

Juice density (kg·m−3) 1056 1058 1060 1066∗ 1052 1065∗ 1061∗ 1067∗ 1054 1049∗ 1055

Maturity index (◦Brix/% malic acid) 82.7 65.7∗ 88.8 84.5 73.6 86.9 65.1∗ 92.6 86.2 66.6∗ 83.3

Means highlighted with ∗ showed statistical differences within rows from PS data according to a Dunnett’s test at P = 0.05. Maturity index was theratio between soluble solids content and titratable acidity. The fruit physiological behaviour was classified as slight, moderate or non-climacteric (LC,MC or NC, respectively) as reported in the text.

ground spot), the cracking or history of cracking of each NIL, andvolatile emission detected by human nose.1,2,16,18

Two fruits per replicate were assessed at harvest (n = 9 for NILs5M2, 5M9 and 5M10; n = 20 for the parental line PS; n = 7 for therest of the NILs).

Other fruit were stored at 21 ± 1 ◦C and 66 ± 6% relativehumidity (mean ± SD) until senescence (n = 5–6 replicates ofindividual fruit). To ensure environment with ethylene levels below0.01 µL L−1, continuous air renewal was established throughoutthe experiment. To ensure senescent fruit, the NILs were analyzedand sampled after 3 weeks, except the NILs SC3-5, 5M4 and 5M6(2 weeks, earlier senescence), and PS (26 days). After senescence,quality traits were evaluated using the methodology previouslyreported for these NILs.2,16

Major fruit quality traits evaluationFruit quality traits evaluated according to Obando et al.18 wereflesh firmness, soluble solids content (SSC), titratable acidity(TA, in mmol L−1 of H+ and in percentage of malic acid), thematurity index (the ratio between TSS and TA, in◦Brix divided bypercentage of malic acid), pH, skin, flesh and juice colour (recordedin tristimulus coordinates lightness, chroma and hue angle or L∗,C∗ and H∗; illuminant C), extractable juice and juice density, drymatter, sugar and organic acid content. Dry matter, soluble solidscontent, TA and, individual sugars and organic acid content wereconsidered as fruit taste traits while the rest of the traits wereconsidered as major quality traits.

The fruit weight loss (% on a fresh weight basis) was evalu-ated after 12 days of storage, and the storage life was calculatedas the time at which at least 50% of the fruit were freefrom any symptom of initial external decay or soft stylar-endcracking (fruit not commercial). When the fruit became senes-cent, they were photographed according to Fernandez-Trujilloet al.1,20

The physiological behaviour of the NILs has been previouslyreported.2,16 The NIL 5M2, 5M7 and PS showed non-climacteric

behaviour, with a trend to decrease respiration and ethyleneproduction rates during ripening, and without ethylene peak.The rest of the NILs showed two kinds of climacteric intensityat 21 ◦C after 4–5 days of postharvest: slight (SC3-5, 5M5, 5M6and 5M9) or moderate (5M3, 5M4, 5M8 and 5M10).2,16 The NILsclassified as slightly climacteric showed respiration rate levelsof 100–300 nmol kg−1 s−1 CO2 and ethylene production levelsof 40–125 pmol kg−1 s−1 C2H4. The moderately climactericNILs showed respiration rate levels of 200–800 nmol kg−1

s−1 CO2 and ethylene production levels of 80–200 pmol kg−1

s−1 C2H4.

Juice sampling for organic acid and sugar compositionanalysesJuice samples were obtained according to Obando et al.18 andstored at −60 ◦C until their HPLC analyses to determine the sugarcomposition according to Fernandez-Trujillo et al.1,19 Organicacids were analyzed in 10 µL aliquots of 1 : 10 juice solution witha non-polar derivative Lichrospher column (RP-Select B, 5 µm;Merck, Darmstadt, Germany). The mobile phase (0.3 mL min−1)was a 99 : 1 v/v combination of deionised water and methanol witha buffer of 50 mmol L−1 phosphate di-hydrogen potassium withH2SO4 (pH = 3) and degassed with an ultrasonic system. Sampleswere read at 210 nm with a UV–visible detector (L-7400; Hitachi,Tokyo, Japan) for 45 min at 30 ◦C. Results for sugars and organicacid concentrations were expressed in µmol g−1 of fresh weightusing juice density and juiciness measurements. Sweetness wascalculated as sucrose equivalents (SEq) according to the formula:SEq = 1× [sucrose] +0.74× [glucose] +1.73× [fructose].21

For quantification, external standards were used to spike juicesamples and to prepare calibration curves for each organic acid andindividual sugars over the range of 0.03–7.8 mg mL−1, yieldingcorrelation coefficient values greater than. The standards werepurchased from Sigma Aldrich (Steinheim, Germany), exceptglutamic acid, which was from Merck.

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Table 3. Means of the fruit taste quality traits (including sugars and organic acids in µmol g−1) in full senescent fruit in the near isogenic lines (NILs)and the parental line ‘Piel de sapo’ (PS) melons stored at 21 ◦C

NILs and classification of climacteric behaviour ANOVA significance

Fruit quality traitat the senescence

SC3-5(LC)

5M5(LC)

5M6(LC)

5M9(LC)

5M3(MC)

5M4(MC)

5M8(MC)

5M10(MC)

5M2(NC)

5M7(NC)

PS(NC) Pedigree Time Pxt

Dry matter (g·kg−1) 114.7 97.4∗ 118.7 116.6 107 113.6 102.6 117.5 105.1 110.8 126.1 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗

Soluble solidscontent (◦Brix)

10.5 9.0∗ 11.1 10.8 8.7∗ 9.8 8.9∗ 12.1 10 10.2 11.8 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗

Fructose (µmol ·g−1)

20.9 11.1 12.4 6.7 12.3 28.4 25.7 16.6 12.6 20.5 29.6 ND ND ND

Glucose (µmol ·g−1)

18.3 11.5 9.4 2.64 11.6 32.4 20.5 11.8 15.1 25.1 32.1 ND ND ND

Sucrose (µmol ·g−1)

33.1 22.5 37.1 16.7 12.7 32.6 44.9 25.2 19 43.3 39.6 NS NS NS

Sucroseequivalents(µmol · g−1)

82.9 50.3 65.5 30.3 42.6 105.7 81 80 52 97.4 104.5 ND ND ND

Titrable acidity [H+(mmol·L−1)]

14 15.2 15.8 19.4 10.8∗ 11.3∗ 12.1∗ 17.6 12.5∗ 17.9 18.3 ND ND ND

Glutamic (µmol ·g−1)

0.864 1.314 0.363 0.8 0.078 ND 1.091 0.922 0.309 0.21 0.27 ND ND ND

Oxalacetic (µmol ·g−1)

0.173 0.166 0.142 0.064 0.119 ND 0.201 0.1 0.099 0.288 0.281 ∗∗∗∗ ∗∗∗ NS

Quinic (µmol · g−1) 0.043 0.07 0.05 0.027 0.026 0.049 0.035 0.066 0.031 0.063 0.052 NS ∗∗∗∗ NS

Isocitric (µmol ·g−1)

0.235 0.165 0.244 0.1 0.196 ND 0.266 0.138 0.157 0.309 0.265 ∗∗ ∗ ∗∗

Ascorbic (µmol ·g−1)

ND 0.019 0.038 0.014 0.004 0.053 0.039 0.033 0.016 0.054 0.062 ∗∗∗∗ ND ND

Citric (µmol · g−1) 1.938 1.731 2.202 0.955 0.735 3.191 2.833 1.667 1.304 3.226 3.209 ∗ ∗∗∗∗ ∗

Succinic (µmol ·g−1)

2.242 3.83 2.9 0.945 0.515 1.87 3.04 2.802 1.82 2.382 3.33 NS ∗∗∗∗ NS

NIL means within rows highlighted with ∗ showed statistical differences from PS data according to a Dunnett’s test at P = 0.05. In the cases of notdetectable (ND) levels in PS, the means not connected by the same letter are significantly different among them, according to a Tukey-Kramer HSDtest at P = 0.05. The fruit physiological behaviour was classified as slight, moderate or non-climacteric (LC, MC or NC, respectively) as reported in thetext. The significant interaction and mean effects of the two-way analysis of variance (ANOVA) considering data at senescence and at harvest (Table 4)are highlighted according to the probability level.∗ P ≤ 0.05∗∗ P ≤ 0.01∗∗∗ P ≤ 0.001∗∗∗∗ P ≤ 0.0001

Statistical analysisData were subjected to a two-way ANOVA using general linearmodel procedures, with storage time (harvest or end of storage)and pedigree as factors in JMP v5.1.2 for Windows (SAS InstituteInc., NC). If significant differences were found by ANOVA usingpedigree, the differences between NILs and PS were evaluated byDunnett’s test at P = 0.05 using JMP according to Obando-Ulloaet al.18 In those cases in which quality trait data were not detectedin PS, the NILs were compared by Tukey’s test at P = 0.05in JMP. The ANOVA also allows identifying the compoundsthat discriminate the NILs according to their climacteric ornon-climacteric behaviour.16

Fruit and taste quality trait data were also subjected tomultivariate analyses as follows: principal components analysis(PCA), partial least square-discriminant analysis (PLS-DA), lineardiscriminant analysis (LDA) and random forest (RF).22

The PCA was conducted using JMP. The PC values of theNILs of all the traits were analyzed by ANOVA with time andpedigree as factors in JMP. The PLS-DA was performed withThe Unscrambler (version 9.6; CAMO A/S, Trondheim, Norway),according to Obando-Ulloa et al.18 Because this software only

accepts a matrix of 20 levels (NILs at harvest and at the senescentstage together) it was not possible to classify the NILs according tothe time tested. LDA and RF were conducted with the MASS library(version 7.2–40) and the randomForest package (version 4.5–23)in R language.22 – 25 Random forest results were represented usingmulti-dimensional scaling plots.

RESULTSShelf-life and weight loss in the NILsThe average shelf-life at 21 ◦C ranged from 12 to 18 days in theNILs and 21 days in PS, which was in agreement with the lowerstorage time from harvest to senescence and consequently lowerweight loss (Table 1).

Quality traits of the PS parental line and the NILs in senescentfruit versus harvest fruit (univariate analysis)At harvest, most of the NILs showed significant differences insome of the quality traits analyzed (firmness, extractable juiceand skin, flesh and fruit juice colour coordinates) compared to

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Table 4. Means of the fruit taste quality traits (including sugars and organic acids) at harvest in the near isogenic lines (NILs) and the parental line‘Piel de sapo’ (PS) melons stored at 21 ◦C

NILs and classification of climacteric behavior

Fruit quality trait at harvestSC3-5(LC)

5M5(LC)

5M6(LC)

5M9(LC)

5M3(MC)

5M4(MC)

5M8(MC)

5M10(MC)

5M2(NC)

5M7(NC)

PS(NC)

Dry matter (g·kg −1) 134.1 130.8 145.8∗ 149.2∗ 130.5 148.6∗ 140.1 147.9∗ 128.2 107.6∗ 128.4

Soluble solids content (◦Brix) 11.3 11 11.9∗ 12.6∗ 10.4 12.7∗ 11 13.1∗ 10.5 8.2∗ 10.6

Fructose (µmol · g−1) 11.04 8.09 12.17 6.32 13.4 9.84 14.92 6.01∗ 8.83 13.35 11.41

Glucose (µmol · g−1) 9.44 5.85∗ 10.84 4.41∗ 10.84 7.46 13.97 4.02∗ 9.01 13.32 11.91

Sucrose (µmol · g−1) 19.04 15.6 23.45 21.66 19.3 24.64 22.65 13.74 15.95 15.48 17.41

Sucrose equivalents (µmol · g−1) 50.97 39.41 57.6 47.05 52.66 53.66 58.78 32.76∗ 43.09 50 50.34

Titrable acidity or TA [H+ (mmol·L−1)] 21.3 24.2∗ 20.2 22.8∗ 21.1 21.7 24.4∗ 21.2 18.7 19.1 19.5

Glutamic (µmol · g−1) 1.83∗ 1.47 1.3 1.61∗ 1.24 1.61∗ 1.72∗ 1.22 0.89 0.88 0.71

Oxalacetic (µmol · g−1) 0.17 0.02∗ 0.06∗ 0.04∗ 0.2 0.07∗ 0.03∗ 0.04∗ 0.04∗ 0.08∗ 0.21

Quinic (µmol · g−1) 0.09 0.02 0.08 0.06 0.05 0.10 0.02 0.05 0.03 0.11 0.07

Isocitric (µmol · g−1) 0.44 0.15 0.44 0.23 0.34 0.41 0.10∗ 0.3 0.23 0.29 0.38

Ascorbic (µmol · g−1) 0.04 0.28∗ 0.25∗ 0.17 0.06 0.28∗ 0.43∗ 0.1 0.19 0.24∗ 0.04

Citric (µmol · g−1) 2.32 0.43∗ 1.27∗ 0.83∗ 2.52 1.11∗ 0.02∗ 1.13∗ 0.38∗ 2.26 2.57

Succinic (µmol · g−1) 6.74 4.60∗ 7.12 6.05 3.95∗ 7.3 5.94 5.94 3.09∗ 3.34∗ 8.52

NIL means wit hin rows highlighted with ∗ showed statistical differences from PS data according to a Dunnett’s test at P = 0.05. The fruit physiologicalbehaviour was classified as slight, moderate or non-climacteric (LC, MC or NC, respectively) as reported in the text.

PS (Table 2). However, in the traits pH, SSC, TA, sugars or sucroseequivalents, or organic acids, only a few differences between NILsand PS were found. None of the quality traits analyzed at harvestor at the senescence stage showed significant differences for theentire set of climacteric NILs compared to PS (Tables 1–4, andSupplementary Table 1).

Some quality traits showed a pedigree × time interaction(Tables 1 and 3). The interaction was explained, with someexceptions, in terms of the lower changes in fruit showing non-climacteric behaviour compared with the climacteric ones. Forexample, TA was reduced by 6% in senescent 5M7 and PS fruitcompared with harvest levels, but TA diminished by 15–50% in theclimacteric NILs. The mean pedigree was significant in 16 qualitytraits, while the time effect, which evaluated the net changefrom harvest to senescence, was usually significant in most ofthe traits (Supplementary Table 1). In general, the time effect wasvery important, as in the case of organic acids or maturity index.The maturity index of all senescent NILs increased by 1–88%compared with harvest levels (Tables 1 and 2, and SupplementaryTable 1). As regards organic acids, NIL 5M4 was the most affectedby senescence with concentrations of four organic acids below orclose to the detection level (Tables 3 and 4).

Grouping the NILs using pedigree and time as factorsby multivariate analysis of fruit quality data at harvestand senescent stageThe PLS-DA did not completely separate the NILs at harvest orat the senescent stage but 80% of total variability was explained(Supplementary Fig. 1), and consequently only PCA results areshown.

The first three PCA components (PCiq, i = 1–3) explained 20, 17and 12% of total variance, respectively (Fig. 1 and SupplementaryFig. 2). The PC1q discriminated the climacteric NIL 5M9 at thesenescent stage from the rest. This axis was mostly affected bytaste traits (the content of fructose, glucose, sucrose, sucroseequivalents and oxalacetic acid) and, to a lesser extent, textural

traits (juiciness) and maturity index on the positive side, and by TAand others on the negative side (Fig. 1B).

PC2q allowed the discrimination of the non-climacteric NILs(5M7 and PS) at harvest from the rest towards the negativeside, and allowed senescent PS fruit to be separated from therest of the NILs (Fig. 1A). The PC2q was mostly affected by theflesh and juice colour (mainly H∗) and flesh firmness on thenegative side and by other major quality traits (mainly skinL∗ and C∗, juice C∗ and TA) (Fig. 1B). In fact, the time andpedigree effects were significant in the ANOVA of PC1q andPC2q (P < 0.001). The interaction of pedigree × time (Tables 1and 3) also allowed senescent PS to be separated from the rest ofthe NILs by PC2q.

Grouping the NILs using climacteric or non-climactericbehaviour and time as factors by multivariate analysisof quality traits data (PCA, LDA and random forest analysis)The PCA partly allowed the classification of the NILs by climactericbehaviour and time into the four groups expected (Fig. 2). The non-climacteric NILs at harvest were separated from the climactericones at harvest or senescence and the senescent non-climactericones due to PC1q (Fig. 2A), which was previously defined (Fig. 1B).However, PC2q (also defined before) allowed the NILs to beseparated according to the time effect (Fig. 2A), and this separationwas also possible by PC3q (Fig. 2B and Supplementary Fig. 2). Themost significant discrimination between the non-climacteric NILsat both times was obtained in Fig. 2A, because the ellipses at 95%did not intersect.

LDA allowed discrimination between groups, obtaining a meanclassification error of 7% (Fig. 3). The random forest analysis of thequality traits allowed the NILs to be separated by the climactericbehaviour at harvest, but not at the senescent stage. Thus, atharvest the classification error in non-climacteric as climactericNILs was of 8% and 3%, vice versa. The classification error in fruitat harvest classified as senescent was of 0% (SupplementaryFig. 3A). Using the importance measure based on the mean

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Figure 1. Biplot of centroids after principal component analysis of all the fruit quality traits of near-isogenic lines (NILs) of melon (at harvest, aftersenescence; no subscript or subscript (s), respectively). The traits were identified in senescent fruit of the near-isogenic lines and ‘Piel de sapo’ (PS) storedat 21 ◦C. The percentage of variance explanation is beside each axe. (A) Plot of the NILs in the first two axes. (B) Plot of the variables in the first and secondaxes.

decrease in accuracy, clustering of the NILs was based on thevalues of C∗ of the skin, H∗ of the juice, flesh firmness and drymatter (Supplementary Fig. 3B). It was not possible to clusterthe NILs by their climacteric behaviour at the senescent stageusing fruit quality traits due to a classification error above 40% inboth cases.

Classification of the NILs by univariate or multivariate analysisof the quality traits of senescent fruitDiscriminant power of all the quality traits of senescent fruitby univariate analysisOnly flesh firmness, L∗ and C∗ skin, SSC, TA and dry mattermoderately discriminated climacteric from the non-climactericNILs.

Multivariate analysis of the quality traits of senescent fruitThe RF analysis of all quality traits did not clearly cluster theNILs according to their physiological behaviour and consequentlydata are not reported. In this case, LDA allowed the NILs to be

discriminated by their climacteric behaviour without classificationerror (data not shown). Also, the loading values of the tastetraits (sugars, sucrose equivalents and isocitric and oxalacetic acidcontents) were higher than for other quality traits in LDA (datanot shown). A PCA and PLS-DA multivariate analysis were alsoconducted.

As regards PCA of quality traits of senescent fruit, the first threePCs explained 30, 16 and 13% of total variance, respectively (Fig. 4).PC1qs was mostly affected by taste traits (the content of fructose,glucose, sucrose, sucrose equivalents, quinic, oxalacetic, isocitricand citric acids) and allowed the non-climacteric NIL 5M7 to beseparated from the rest. PC2qs was mostly affected by TA, fleshfirmness, the true colour of the skin, flesh and juice of the fruit(i.e. H∗) on the negative side, while the positive side of PC2qs wasinfluenced by extractable juice, pH, maturity index, skin C∗ andL∗. PC2qs confirmed the separation of NIL 5M7 and also allowed5M4 to be separated from the rest (Fig. 4A). On the other hand,PC3qs was associated with the most abundant and relevant tastetraits in fruit composition apart from water (SSC, dry matter andjuice density) on the negative side, while the coordinates of juice

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Figure 2. Plot of centroids and ellipses after principal component analysis of fruit quality trait of melon fruit (n = 5), using climacteric behaviour andtime (harvest and senescence) as factors. The percentage of variance explanation by the first three PCs is beside each axe. (A) Plot of the NILs in PC1q andPC2q. (B) Plot of the NILs in PC1q and PC3q.

(L∗, C∗ and H∗) and flesh C∗ coordinates were on the positiveside (Fig. 4B). This axis confirmed the separation of the climactericsenescent NIL 5M4 from the rest.

The PLS-DA of senescent fruit using all the fruit quality traitstogether did not establish a clear differentiation among the NILs(Supplementary Fig. 4), although 82% of total variability wasexplained. Consequently, two additional PLS-DAs were performedon selected quality traits of senescent fruit.

In the first sub-analysis, the PLS-DA conducted using major fruitquality traits of senescent fruit explained 86% of the total variability(67% for PLS1qs and 19% for PLS2qs). The PLS1qs was defined byflesh L∗ oriented on the positive side and maturity index on theopposite side. The high values of the quality trait on the positiveside reported for NILs 5M5 (with internal disorder problems such asvitreous texture and mealiness, data not shown) and 5M9 allowedthem to be distinguished from the rest (Table 1 and Fig. 5A). Theclimacteric NIL 5M4 at senescent stage was located on the left sidein the middle of the graph, due to its high maturity index (Table 1and Fig. 5A). On the other hand, the PLS2qs was defined by skinH∗ on the positive side, while the negative side was influencedby skin L∗ and C∗. The highest values in skin L∗ and C∗ allowedthe NILs 5M9 and 5M10 to be separated from the rest (Table 1

and Fig. 5A). The NILs 5M2, 5M7 and PS were separated by skinH∗ (visually observed as dark green) and were placed in the uppermiddle area of the graph (Table 1 and Fig. 5A).

In the second sub-analysis, the PLS-DA conducted using tastetraits of senescent fruit explained 78% of total variance (51%for PLS1tts and 27% for PLS2tts). According to this analysis, thenon-climacteric NIL 5M7 was separated from the rest due toPLS1tts, which was influenced by the content of fructose, glucose,sucrose equivalents, oxalacetic and isocitric acids (Table 3 andFig. 5B). On the other hand, NILs 5M6, 5M9, 5M10 and PS wereplaced in the upper area of the graph, according to PLS2ttsbecause of the high values of SSC, dry matter and TA (Table 1 andFig. 5B).

DISCUSSIONThe lack of a specific correlation between climacteric behaviourand the individual quality traits analyzed at harvest or aftersenescence (Tables 1 and 2) indicated at least a partial ethylene-independent control of most of the quality traits studied, inagreement with Flores et al.5 Some effects of sample variabilitycannot be discarded.

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Figure 3. Linear discriminant analysis (LDA) score plots of all the fruit quality traits of near-isogenic lines (NILs) of melon using climacteric or non-climactericbehavior and time (at harvest and after senescence) as factors. The traits were identified in senescent fruit of the near-isogenic lines and ‘Piel de sapo’(PS) stored at 21 ◦C. Climacteric at harvest (�); non-climacteric at harvest (�); climacteric at the senescence stage (�); non-climacteric at the senescencestage (�).

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Figure 4. Plot of centroids after principal component analysis of the quality traits of near-isogenic lines (NILs) of melon and ‘Piel de sapo’ (PS) aftersenescence. The percentage of variance explanation by the first three PCs is beside each axe. (A) Plot of the NILs in PC1qs and PC2qs. (B) Plot of the NILsin PC1qs and PC3qs.

The relationship between climacteric behaviour and fruit qualitytraits did not support a variation in these traits caused bypleiotropic effects of the gene or genes that induce the climactericbehaviour in SC3-5. This variation could be due, at least in part, togenes other than those controlling the switch from non-climactericto climacteric ripening in melons.9

The climacteric and senescence factors affected sugar andorganic acid metabolism (including glutamic acid metabolism),as well as the carotenoid biosynthesis and skin chlorophylldegradation (because of colour traits; see Fig. 4 and Tables 1–4).All these compounds have a high impact on consumer perceptionand preferences.10,26 Individually, either sugars or organic acids

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Figure 5. Plots of centroids after partial least square analysis of quality traits (n = 5) in senescent melon fruit of ‘Piel de sapo’ (PS) and near-isogenic lines(NILs) stored at 21 ◦C. The variability percentage explained by the latent variables (x and y axes) is shown on the bottom of each graph. Ellipses representr2 = 50 and 100% explained by the model. (A) Major quality traits. The first and second latent components (PLS1qs and PLS2qs, respectively) werelocated on x and y axes. (B) Fruit taste traits. The first and second latent components (PLS1tts and PLS2tts, respectively) were located on x and y axes.

were unable to discriminate climacteric from non-climacteric NILs,which was confirmed by several statistical techniques. However,the trend toward higher levels of glutamic acid in the climactericNILs than in PS at harvest (see above) and after senescence(Tables 3 and 4) correlated well with a reduced shelf-life (Table 1),as observed in tomato.27

The textural traits were the most affected by pedigree and timeand the interactions of both factors (Tables 1 and 3). The reductionof flesh firmness is partly ethylene-dependent5,15,28 and mighthave influenced the increase in extractable juice at the senescentstage (Tables 1 and 2, and Supplementary Table 1). However, thetrend followed by the textural traits depends on other factors,such as maturity at harvest, fruit morphology, internal structure,the development of internal disorders or rots.1,4,29 Particularly inmelon, flesh softening at the fruit equator is accompanied by thesoftening and later disruption of tissue close to the placenta andstylar-end. The reduced softening in non-climacteric lines 5M7 andPS was mostly influenced by greater firmness at harvest than thefirmness values found in the NILs (Tables 1 and 2). The best texture

in non-climacteric melons could be due to the maintenance ofcell wall integrity in the covalently bound pectin polymers, and/orto the abundance of cellulosic polysaccharides, as occurs in longshelf-life melons stored at 20 ◦C.30 The textural traits influencedthe grouping of NILs at harvest or senescence (Fig. 4), and alsoflesh firmness and, to a lesser extent, extractable juice alloweddiscrimination among climacteric and non-climacteric NILs.

Miccolis and Saltveit31 reported that the external and fleshcolour of melons from the Inodorus group did not markedlychange with storage at different temperatures. The higher fleshL∗ and C∗ and lower flesh H∗ values of the NILs compared withPS did not match climacteric or non-climacteric NIL behaviour atharvest (Table 2) or in senescent fruit (Table 1). This trend agreeswith the classification of flesh colour changes as an ethylene-independent process. Some exceptions were also found for skincolour, previously classified as an ethylene-dependent processin melon.5 However, the skin L∗ and C∗ of the senescent fruitallowed the classification of the climacteric NILs into differentgroups (Table 1 and Fig. 1A). Some of the lightest coloured

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NILs (i.e. 5M4) had the fastest ripening process in the fieldand some senescent fruit showed a light orange flesh (datanot shown). This may be explained by the fact that in otherclimacteric cultivars flesh colour changes are influenced by theharvest date.5,32

Dry matter in the main collection of melon NILs shows a highdegree of correlation with SSC18 and a low level in melon is usuallyan indicator of poor organoleptic properties.10 Compared withsenescent PS fruit, the dry matter was only significantly lowerin one NIL (5M5), which also had lower SSC (Table 3) and mealytexture, as revealed by a very low juice extractability (Tables 1and 2). The NIL 5M2 and others (5M9, 5M10) also presented lowlevels of extractable juice and mealiness to different extents, aspreviously reported2, but these NILs did not show lower SSC or drymatter levels than the PS (Tables 1 and 2). The ‘Piel de sapo’ typemelon fruit usually attains maximum SSC at an optimum stageof maturity accompanied by a peak in sucrose content which ishighly correlated with melon sweetness.10 The subsequent loss ofSSC was found in climacteric NILs and 5M2 (around 2–2.5◦Brix).This loss of SSC seems to be independent of the degree ofclimacteric behaviour (Tables 2 and 4, and Supplementary Table 1)in agreement with Flores et al.,5 in which it has also been found tobe independent of the storage conditions in commercial ‘Piel desapo’ type melons.33

The effect of senescent stage on fruit quality traits in the NILfruit could be partly explained from a physiological point of view.For example, the low sugar content reported in the senescentfruit of the NILs compared with harvest levels (Tables 3 and 4)could be due to the synthesis of the organic acids required forthe respiratory metabolism as it has been reported for senescent‘Tendral’ winter melon.14 However, some organic acids were notdetected in senescent fruit (Table 3), probably due to their functionas respiration precursors or their roles in the synthesis of aromacompounds, such as esters.12,13,29

In summary, the time had a stronger effect on quality thanthe physiological behaviour. The principal components andrandom forest analyses of the fruit quality traits allowed thebest classification of the NILs by time (harvest, senescence), orby climacteric behaviour at harvest, but not at the senescentstage. The overall quality profile of the non-climacteric senescentmelons was, in general, very different from that of climactericones and agreed with a longer storage life. Most of the tastequality traits (individual sugars or sucrose equivalents, TA andthe citric, oxalacetic, glutamic and succinic acids) and the traitsrelated to skin, flesh and juice colour parameters (C∗, H∗) helped todistinguish the climacteric NILs from the non-climacteric onesindependently of the time considered. Additionally, the bestmultivariate method from those used was the principal componentanalysis because it allowed a clear classification of NILs accordingto their physiological behaviour and time as it was expected, whilethe other techniques applied only separated the NILs accordingto the time considered.

Supporting informationSupporting information may be found in the online version of thisarticle.

ACKNOWLEDGEMENTSThis work was funded by grants 05676/PI/07 and 00620/PI/04(Fundacion Seneca de la Region de Murcia), BIO-AGR06/02-0011(Consejerıa de Educacion y Cultura de la Region de Murcia),

AGL2003-09175-C02-01 and AGL2003-09175-C02-02 (SpanishMinistry of Education and Science and European Fund for RegionalDevelopment, FEDER, European Union), and funds donated byJ.P. Fernandez-Trujillo supporting part of the predoctoral stay ofM.K. Souri. J.M. Obando-Ulloa acknowledges a fellowship fromSpanish Ministry of Foreign Affairs. Eduard Moreno was supportedby an AGAUR fellowship (Generalitat de Cataluna). Thanks toSemillas Fito S.A. (Barcelona, Spain) for providing the seeds of PS,to Claudia Miranda for sampling assistance, to Jeroen Lammertynfor a critical review of this manuscript, and to Placido Varo and histeam (CIFEA-Torre Pacheco, Consejerıa de Agricultura, Region deMurcia) for crop management.

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