Characterization of Cuticle Composition after Cold Storage of âCeleste

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Characterization of Cuticle Composition after Cold Storage of “Celeste” and “Somerset” Sweet Cherry Fruit Burcu Belge,†,‡ Montserrat Llovera,§ Eva Comabella,†,‡ Ferran Gatius,† Pere Guillén,† Jordi Graell,‡,∥ and Isabel Lara*,†,‡ †

Departament de Química, ‡Unitat de Postcollita-XaRTA, §Serveis Científico-Tècnics, ∥Departament de Tecnologia d’Aliments, Universitat de Lleida, Alcalde Rovira Roure 191, 25198 Lleida, Spain ABSTRACT: Cuticle composition and structure may be relevant factors affecting the storage potential of fruits, but very few studies have analyzed fruit cuticle composition from a postharvest perspective. In this work, the chemical composition of waxes and cutin (major cuticular components) was analyzed in cuticle samples isolated from “Celeste” and “Somerset” cherries (Prunus avium L.) after cold storage at 0 °C. Total cuticle amounts per surface unit (μg cm−2) increased along with cold storage. The triterpene ursolic acid, the alkane nonacosane, linoleic acid, and β-sitosterol were the most abundant components of cuticular waxes, whereas cutin composition was dominated by C18-type monomers. In spite of being comprised of similar chemical families, cultivar-related differences were found regarding the abundance and the evolution of some compound families during cold storage. To the best of our knowledge, this is the first report on changes in cuticle composition of sweet cherry during postharvest storage. KEYWORDS: cold storage, cuticle, cutin, Prunus avium L., postharvest, waxes

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“Navelate” oranges15 suggest that the biosynthesis of cuticular waxes in these fruit species might be under ethylene control. Sweet cherry (Prunus avium L.) fruit is very perishable, owing to high respiration and water loss rates which favor rapid postharvest deterioration due to weight loss, detrimental effects on appearance, and increased susceptibility to infections and mechanical injuries, which notably restrict storage and shelf life potential. Postharvest quality of fruit produce is greatly impacted by cuticle composition, structure, and properties (reviewed in ref 2). Cuticles of sweet cherry fruit have been studied at commercial maturity16 and during on-tree development regarding their chemical composition,6 mechanical properties,17 and frequency and distribution of microcracks.18 However, no study has addressed the compositional changes in sweet cherry cuticles after harvest or during the refrigerated storage commonly used for its preservation. Therefore, the purpose of this work was to assess the evolution of the chemical constituents of the cuticle of two important cherry cultivars during cold storage.

INTRODUCTION The plant cuticle is a lipidic layer synthesized by the epidermis, which surrounds aerial, nonlignified organs, including fruits. Cuticles are composed mainly by cutin, a polyester polymer matrix rich in hydroxylated and epoxy-hydroxylated C16 and C18 fatty acids, embedded with amorphous waxes and a minor fraction of phenolics. The outer side of the cuticle is covered by epicuticular waxes, while in its inner side the cutin matrix mixes with polysaccharides from the epidermal cell walls.1 Because the cuticle covers the epidermis of the fruit, it represents the first barrier against the abiotic and biotic conditions in which it develops. The composition and structure of this surface layer have a noticeable influence on the postharvest storage potential of fruits, inasmuch as it protects against transpirational water loss, mechanical injuries and microbial infections, and provides mechanical support.2 In spite of this relevance, there are few reports on cuticle modifications taking place along the life span of a fruit, which have often focused on physiological or morphological rather than on biochemical aspects. Some studies, though, have reported the evolution of the chemical composition of the cuticle during ontree development of fruit,3−9 and some genes putatively involved in the biosynthesis of cuticular wax and cutin components during fruit development have been identified.10,11 Even more limited research attention has been apparently paid to the changes in fruit cuticle composition along its postharvest life. For apple, there seems to exist considerable cultivar-associated variability as to the progress of total wax amounts, properties, or chemical components during long-term cold storage8,12 or in response to controlled atmosphere storage and subsequent shelf life.13 The observations that 1-methylcyclopropene attenuated composition changes in cuticular waxes during storage of “Autumn Gold” and “Royal Gala” apples14 and that exogenous ethylene induced structural and compositional changes in surface waxes of © XXXX American Chemical Society

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MATERIALS AND METHODS

Plant Material, Assessment of Standard Quality, and Postharvest Handling. Cherries (Prunus avium L. cv. “Celeste” and “Somerset”) were hand-collected in 2011 (May 23rd and June 7th, respectively) from the same commercial orchard located in Corbins, in the area of Lleida (NE Spain), at marketable maturity according to the usual indices (size and color) in the producing area. Uniform, defect-free samples were selected and transported directly to the laboratory. Standard quality parameters were determined immediately thereafter in order to check adequate maturity of samples. Firmness was measured with a Durofel DFT 100 durometer (Agro-Technologie, Forges Les Received: June 3, 2014 Revised: August 1, 2014 Accepted: August 4, 2014

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dx.doi.org/10.1021/jf502650t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Eaux, France) fitted with a 5.64 mm tip, on two opposite faces on the cheek region of 30 fruit, and results were expressed as Durofel units (1, no resistance, to 100, maximum resistance). For the assessment of juiciness, three replicate samples (10 fruit each) were stoned in each case and squeezed until no more juice was released. After filtration, the volume of juice recovered was measured and expressed as mL 100 g−1 fresh weight. Soluble solids content (SSC) and titratable acidity (TA) were assessed in juice obtained as described above. SSC was determined with a hand-held refractometer (Atago, Tokyo, Japan), and results expressed as °Brix. For TA determination, 10 mL of juice were diluted in 10 mL of distilled water, and titrated with 0.1 M NaOH to pH 8.1; results were given as g malic acid L−1. Skin color was determined at two opposite equatorial points of 30 fruit using a portable tristimulus colorimeter (Chroma Meter CR-200, Minolta Corp., Osaka, Japan), with CIE D65 illuminant and 8 mm aperture diameter. Lightness (L*) values were recorded, and hue angle was calculated from a* and b* parameters. After quality assessments, fruits were stored at 0 °C and 92% relative humidity during 14 days followed by 3 days at 20 °C to simulate commercial shelf life. Skin samples were taken at harvest, upon removal from cold storage, and 3 days thereafter, and cuticles isolated and analyzed in triplicate as described below. Cuticle Isolation. Exocarp segments were excised from the cheek region of 15 fruit using a cork borer (13 mm, i.d.). Four skin disks were so obtained from each individual fruit (5.31 cm2 per fruit). Cuticular membranes (CM) were isolated enzymatically by incubation at 37 °C in 1.6 U mL−1 cellulase and 100 U mL−1 pectinase in 50 mM citrate buffer (pH 4.0) until no more material was released, in the presence of 1 mM NaN3 to prevent microbial growth. After isolation, CM were washed in citrate buffer (50 mM, pH 4.0) at 37 °C until no material was left in suspension, thoroughly rinsed in distilled water in order to remove materials sorbed during isolation,19 dried at 40 °C, and kept in hermetically capped vials until use. CM mass was determined gravimetrically and expressed per unit of fruit surface area (μg cm−2) as well as on total fruit surface basis (mg fruit−1). The reagents and standards used for the analysis of cuticular components were supplied by Sigma-Aldrich (Steinheim, Germany). All standards used were of analytical grade. Extraction and Analysis of Cuticular Wax Compounds. Waxes were recovered from the 60 dry CM disks (a total of 79.64 cm2 per cultivar and analysis date) obtained in each case. CM samples were dewaxed three times by shaking in CHCl3 (1 mg sample mL−1) 24 h at room temperature, followed by incubation (15 min) in an ultrasonic bath. After filtration, the chloroform extracts were pooled and concentrated using a rotary evaporator at 50 °C and the waxes transferred to a preweighed vial, dried under N2 until complete dryness, and weighed in a microbalance for calculation of total wax yield (μg cm−2). Free hydroxyl and carboxyl groups were converted respectively into their trimethylsilyl (TMSi) ethers and esters by derivatizing with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in pyridine (2:1, v/ v) for 15 min at 100 °C. After derivatization, wax samples were added eicosane (C20) and dotriacontane (C32) as internal standards and injected (1 μL) in oncolumn mode into a gas chromatography−mass spectrometry (GC-MS) system for compound identification. A GC equipment (Agilent 7890A) coupled with a quadrupole mass selective detector (Agilent 5973N) was used for these analyses, equipped with a capillary column (BPX5, 30 m × 0.25 mm, 0.25 μm; SGE Europe Ltd., Milton Keynes, UK). Wax compounds were identified by comparing their retention indices with those of standards and by matching their electron ionization-mass spectra (EI-MS) (70 eV, m/z 50−700) with those from the NIST 08 MS library. The oven was set at 50 °C (2 min), and the temperature was raised initially by 40 °C min−1 to 200 °C, kept at 200 °C for 2 min, raised again by 3 °C min−1 to 310 °C, and then kept constant at 310 °C for 30 min. Helium was used as the carrier gas (2 mL min−1). Quantitative determination of wax compounds was carried out under the same chromatographic conditions using a GC system equipped with a flame ionization detector (FID). Data were expressed as relative %. Extraction and Analysis of Cutin Monomers. Dewaxed cuticular membranes (DCM) were hydrolyzed in 3 mL of 1 M HCl in 100%

MeOH and esterified in the same solvent for 2 h at 80 °C. After cooling, 2 mL of saturated NaCl was added to the methanolysate. The cutin monomers were extracted three times in 2 mL of hexane for 10 min, followed by thorough mixing and centrifugation at 20 °C. The monomer extracts were combined and evaporated to dryness in a N2 steam. Cutin yields were determined gravimetrically as μg cm−2. Dry cutin samples were derivatized with BSTFA for 15 min at 100 °C, then added heptadecanoate (C17) and tricosanoate (C23) as internal standards, and injected (1 μL) to GC-FID for quantitative determination. Cutin compounds were identified by comparison with standards, and from their EI-MS spectra (70 eV, m/z 50−700) after GC-MS analysis using the same GC-MS system as described in the previous section on wax analysis. The oven was set and held at 50 °C for 2 min, and then the temperature was raised to 250 °C by 10 °C min−1, kept at 250 °C for 1 min, raised again by 3 °C min−1 to 310 °C, and kept at that temperature for 20 min. The same chromatographic conditions were used for quantitative determination of cutin compounds by GC-FID. Results were expressed as relative %. Statistical Analysis. A multifactorial design with storage and shelf life periods as factors was used to statistically analyze the results within each cultivar. All data were tested by analysis of variance (ANOVA) with the Minitab 16 program package, and means were separated by the Fisher’s LSD test at P ≤ 0.05.

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RESULTS AND DISCUSSION The usual indicators of commercial quality show that samples were picked at marketable maturity according to the standards in the producing area (Table 1). Average weight and diameter at Table 1. Maturity and Quality Indicators of “Celeste” and “Somerset” Sweet Cherry Fruit at Commercial Harvest parametera

‘Celeste’

‘Somerset’

weight (g) diameter (mm)b firmness (Durofel units) juiciness (mL 100 g−1 FW) SSC (°Brix) TA (g L−1) hue (°) lightness (L*)

9.02 b 27.95 b 77.67 a 61.07 b 16.07 b 6.70 b 21.88 a 33.51 a

10.49 a 30.73 a 80.43 a 65.37 a 19.43 a 7.32 a 16.97 b 27.01 b

a

Values represent means of 30 or three (SSC, TA, juiciness) replicates. Mean values followed by a different lower-case letter within the same row are significantly different at P ≤ 0.05 (LSD test). bDiameter is given as the average between the wider and the narrower sides of each fruit.

harvest were higher for “Somerset” than for “Celeste” fruit (Table 1). “Somerset” cherries were also firmer, juicier, and displayed higher SSC and TA values. Skin color of “Celeste” fruit was less red, but lighter, than that of “Somerset”, as indicated by higher hue and lightness values. In addition to these differences in the usual quality indicators, both cultivars also differed in relation to cuticle amount recovered from fruit skin (Table 2). Cuticle yields (μg cm−2) obtained from 60 skin disks were noticeably lower in “Celeste” than in “Somerset” fruit (100 vs 206 at harvest, respectively). Higher cuticle amounts per surface area might be indicative of thicker cuticles, but no measurements of cuticle thickness were undertaken in this study. Incidentally, weight loss and firmness changes after 3 days at 20 °C were more pronounced in “Celeste” than in “Somerset” samples (Table 3), showing a somewhat better conservation potential for “Somerset” fruit. However, published studies on the relevance of cuticle thickness for postharvest weight loss have reported contradictory results B



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families, both types of cuticular components showed cultivarrelated differences in the specific composition and in the abundance of particular compounds. Noticeable differences in the total content of cuticular waxes and cutin were also observed between both cherry cultivars. Total wax yields of fruit at harvest were around 33 (“Celeste”) and 48 (“Somerset”) μg cm−2, corresponding to 33 and 23% of total cuticle material recovered, respectively. Triterpenes were the most abundant class of wax components in both cherry cultivars considered (Table 4), with ursolic acid as the dominating compound (49% and 47% of total waxes at harvest for “Celeste” and “Somerset”, respectively). This is in accordance with an early report on “Bing” cherries.16 The prevalence of ursolic acid agrees as well with a more recent investigation6 in which cuticular wax composition was analyzed in four cherry cultivars (“Hedelfinger”, “Kordia”, “Sam”, and “Van”) during ontree fruit development, although in that study the amount of triterpenes was found to account for up to 76% of total wax, which is higher than the levels observed in this work at harvest (55−57%). This difference was due to greater contents of ursolic acid (around 60%) because the relative amounts of oleanoic acid, the second triterpene in quantitative terms, were equivalent to those found in this study (7.5%). Alkanes were the second most important class of wax components in quantitative terms, among which n-nonacosane (C29) was the most abundant compound, followed by nheptacosane (C27) (Table 4), which is also consistent with previous observations.6,16 However, “Somerset” cuticles were richer in alkanes than those obtained from “Celeste” fruit (roughly 15 vs 10% at harvest, correspondingly), particularly in those with longer chains (C27 to C31), and also contained higher percentages of phytosterols. In contrast, “Celeste” cuticles were richer in fatty acids than those of “Somerset” fruit, although in both cultivars linolenic acid predominated quantitatively among this type of wax components. Cuticular waxes of “Bing” cherries were also characterized to be rich in palmitic, stearic, oleic, and linoleic acids and to contain significant amounts of sitosterol.16 None of these fatty acids, and only trace amounts of sitosterol, which are characteristic for membrane lipids, were found in a subsequent study,6 leading to the suggestion that their presence in the previous report was due to the use of pressed, dehydrated fruit skins as the source of wax. Nevertheless, we wish to point out that we used enzymatically isolated cuticles herein as the source material for wax extraction. Therefore, presence of these compounds in the wax fraction is not likely to be artifactual but rather to be reflecting actual cultivar-related differences in cuticular wax composition. Similarly to the observations for waxes, total cutin content was also higher in “Somerset” than in “Celeste” samples, expressed per unit surface area (85 vs 45 μg cm−2, respectively) as well as on a whole fruit basis (2.5 vs 1.1 mg per fruit), even though it was a bit lower when expressed as a percentage over total cuticle yield (41 vs 45%) (Table 2). The analysis of cutin monomer composition revealed discrepancies with previous reports that the cutin fraction of mature “Kordia” fruit consisted mainly of C16 (69.5%) and, to a much lesser extent, C18 (19.4%) monomers.6 In this work, in contrast, we found that the cutin fraction of “Celeste” and “Somerset” cherry fruit at harvest maturity was composed mainly of C18 monomers (65.3%), comprising αmonocarboxylic, α,ω-dicarboxylic, and ω-hydroxylated acids (Table 5). The most abundant of these C18 constituents were octadecane-1,18-dioic acid, 18-hydroxy-linoleic acid, 18-hydroxy-oleic acid, and oleic acid, whereas hexadecane-1,16-dioic

Table 2. Total Amount of Cuticle Recovered, and Wax and Cutin Yields Obtained from “Celeste” and “Somerset” Sweet Cherries at Harvest and after Cold Storagea days at 0 °C + days at 20 °C 0+0 cuticle amount (mg)b cuticle yield (μg cm−2) cuticle yield (mg/fruit) wax yield (μg cm−2) wax yield (mg/fruit) cutin yield (μg cm−2) cutin yield (mg/fruit) wax (%) cutin (%) cuticle amount (mg)b cuticle yield (μg cm−2) cuticle yield (mg/fruit) wax yield (μg cm−2) wax yield (mg/fruit) cutin yield (μg cm−2) cutin yield (mg/fruit) wax (%) cutin (%)

0+3

‘Celeste’ 8.0 13.6 100.45 c 170.77 b 2.47 c 4.19 b 32.74 b 37.13 a 0.80 b 0.91 a 45.14 c 38.29 d 1.11 c 0.94 c 32.59 a 21.74 b 44.94 b 22.42 c ‘Somerset’ 16.4 19.1 205.93 b 239.83 b 6.11 b 4.83 c 47.51 b 57.46 a 1.41 a 1.16 b 84.53 c 140.08 ab 2.51 bc 2.82 b 23.07 a 23.96 a 41.05 b 58.41 a

14 + 0

14 + 3

17.9 224.76 a 5.48 a 24.66 c 0.60 c 122.90 a 3.00 a 10.97 c 54.68 ab

10.6 133.10 bc 2.30 c 24.90 c 0.43 d 82.45 b 1.42 b 18.71 bc 61.95 a

26.4 331.49 a 7.47 a 33.91 c 0.76 c 162.90 a 3.67 a 10.23 c 49.14 ab

17.1 214.72 b 4.33 c 34.91 c 0.70 c 105.86 bc 2.14 c 16.26 b 49.30 ab

a

Yields and percentages represent means of three replicates. Mean values followed by a different lower-case letter within the same row are significantly different at P ≤ 0.05 (LSD test). bRecovered from 60 skin discs (4 disks × 15 fruit), corresponding in total to 79.64 cm2.

Table 3. Some Physical Properties of “Celeste” and “Somerset” Sweet Cherries at Harvest and after Cold Storagea days at 0 °C + days at 20 °C 0+0 firmness (Durofel units) weight loss (%) diameter (mm)b fruit surface (cm2/fruit)c fruit volume (cm3/fruit)c firmness (Durofel units) weight loss (%) diameter (mm)b fruit surface (cm2/fruit)c fruit volume (cm3/fruit)c

‘Celeste’ 77.7 a − 27.95 a 24.54 a 11.43 a ‘Somerset’ 80.4 b − 30.73 a 29.67 a 15.19 a

0+3

14 + 0

14 + 3

72.1 ab 9.6 c 27.94 a 24.52 a 11.42 a

68.5 b 15.8 b 27.87 a 24.40 a 11.33 a

69.9 b 22.4 a 23.43 b 17.25 b 6.73 b

77.2 b 2.4 c 25.31 b 20.12 c 8.49 c

86.5 a 7.2 b 26.79 b 22.55 b 10.07 b

82.1 ab 15.4 a 25.35 b 20.19 c 8.53 c

a

Values are the means of 30 replicates. Mean values followed by a different lower-case letter within the same row are significantly different at P ≤ 0.05 (LSD test). bDiameter is given as the average between the wider and the narrower sides of each fruit. cTotal surface and volume were estimated from the average diameter of each fruit assuming a spherical shape.

(reviewed in ref 2), and such a relationship has been actually shown in only a few instances.20 The composition and/or structure of the fruit surface may be of more relevance for postharvest performance-related physiological traits. Cuticular Wax and Cutin Composition at Harvest. Cutin and cuticular waxes were extracted from cuticles obtained enzymatically from fruit samples and analyzed chromatographically. In spite of being comprised of similar chemical C



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Table 4. Wax Constituents (Relative %) Identified in Cuticles of “Celeste” and “Somerset” Sweet Cherries at Harvest and after Cold Storagea days at 0 °C + days at 20 °C 0+0

0+3

14 + 0

14 + 3

67.3 a 9.68 a 57.62 a 12.27 a 0.01c nd c nd b 0.01 b 0.03 b 0.02 c 0.14 b 0.13 b 1.35 b 0.72 b 8.51 a 0.52 a 0.83 a 3.43 c 0.18 b nd c 0.76 b 0.18 b 2.31 c 0.00 b 2.24 a 0.76 ab 1.48 a 0.87 a 0.34 a 0.19 a 0.05 b 0.10 a 0.19 a 86.08 a 13.92 b

69.22 a 9.71 a 59.51 a 12.33 a 0.01 c nd c nd b 0.01 b nd c 0.05 c 0.16 b 0.19 b 1.49 a 0.79 a 8.32 a 0.53 a 0.78 b 2.56 c 0.11 b nd c 0.89 b 0.15 b 1.41 c 0.00 b 1.65 c 0.72 b 0.93 b 0.74 ab 0.25 ab 0.17 a 0.04 b 0.11 a 0.17 b 86.49 a 13.51 b

51.15 b 8.21 a 42.94 b 18.406 a 0.06 ab 0.02 ab 0.02 a 0.13 a 0.31 a 0.58 a 0.75 a 2.24 a 1.47 a 10.68 a 0.97 a 1.23 a 5.79 b 0.20 b 0.02 b 1.18 b 0.21 b

53.85 a 7.68 b 46.17 a 15.25 b 0.06 ab 0.01 b nd b 0.05 ab 0.12 b 0.27 b 0.34 b 1.99 b 1.08 b 9.67 bc 0.71 b 0.95 b 7.73 a 0.46 a 0.05 a 1.70 a 0.36 a

‘Celeste’ triterpenes oleanolic acid ursolic acid alkanes decane (C10) undecane (C11) dodecane (C12) docosane (C22) tricosane (C23) tetracosane (C24) pentacosane (C25) hexacosane (C26) heptacosane (C27) octacosane (C28) nonacosane (C29) triacontane (C30) hentriacontane (C31) fatty acids C16:0 C17:0 C18:0 C18:1 (9) C18:2 (9, 12) trans-C18:2 (9,12) phytosterols campesterol β-sitosterol fatty alcohols tricosanol (C23) tetracosanol (C24) hexacosanol (C26) octacosanol (C28) triacontanol (C30) identified wax unidentified

57.28 b 7.76 b 49.54 b 10.28 b 0.06 a 0.03 a 0.09 a 0.03 b 0.08 a 0.22 a 0.33 a 0.33 a 1.18 c 0.76 ab 5.96 c 0.52 a 0.66 d 13.03 a 1.06 a 0.09 a 1.27 a 0.31 a 10.27 a 0.03 a 1.78 bc 0.83 a 0.95 b 0.89 a 0.34 a 0.14 a 0.14 a 0.11 a 0.16 bc 83.23 b 16.77 a

triterpenes oleanolic acid ursolic acid alkanes decane (C10) undecane (C11) docosane (C22) tricosane (C23) tetracosane (C24) pentacosane (C25) hexacosane (C26) heptacosane (C27) octacosane (C28) nonacosane (C29) triacontane (C30) hentriacontane (C31) fatty acids C16:0 C17:0 C18:0 C18:1 (9)

54.62 a 7.47 b 47.15 a 14.80 b 0.07 a 0.03 a 0.01 ab 0.08 ab 0.16 b 0.31 b 0.46 b 1.73 c 1.15 b 8.94 c 0.81 ab 1.05 b 7.58 a 0.41 a 0.06 a 1.20 b 0.25 b

65.73 a 9.19 a 56.55 a 12.91 a 0.04 b 0.01 b nd b 1.84 a 0.03 b 0.13 b 0.18 b 0.19 b 1.22 c 0.72 b 7.34 b 0.49 b 0.72 c 7.29 b 0.46 b 0.06 b 0.89 b 0.21 b 5.66 b 0.01 b 2.11 ab 0.71 b 1.40 a 0.53 b 0.22 b 0.04 b 0.05 b 0.07 b 0.15 c 86.40 a 13.60 b ‘Somerset’ 55.19 a 7.39 b 47.80 a 14.93 b 0.05 b 0.01 b 0.01 ab 0.06 b 0.11 b 0.21 b 0.30 b 1.75 c 0.98 b 9.77 b 0.69 b 0.99 b 7.01 ab 0.33 ab 0.06 a 1.13 b 0.24 b

D



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Table 4. continued days at 0 °C + days at 20 °C 0+0

0+3

14 + 0

14 + 3

5.25 a 3.01 b 1.07 b 1.94 b 0.96 b 0.23 b 0.17 b 0.17 a 0.15 ab 0.24 b 81.10 b 18.90 a

4.18 b 4.82 a 1.19 a 3.63 a 1.01 0.28 b 0.06 c 0.12 c 0.16 a 0.39 a 81.24 ab 18.76 ab

5.16 a 2.91 b 1.12 ab 1.79 b 1.07 0.46 a 0.06 c 0.12 c 0.14 b 0.29 b 80.83 b 19.17 a

‘Somerset’ C18:2 (9, 12) phytosterols campesterol β-sitosterol fatty alcohols tricosanol (C23) tetracosanol (C24) hexacosanol (C26) octacosanol (C28) triacontanol (C30) identified wax unidentified

5.66 a 3.61 ab 0.94 c 2.67 ab 1.05 a 0.29 b 0.21 a 0.15 b 0.16 a 0.24 b 81.66 a 18.34 b

a Values represent means of three replicates (nd, nondetectable). Mean values followed by a different lower-case letter within the same row are significantly different at P ≤ 0.05 (LSD test).

set”). The main difference between cultivars, though, was found for cutin yields, which decreased in both absolute and relative terms in “Celeste” fruit, while the opposite was observed for “Somerset” cherries (Table 2). Detailed analysis of cuticle composition revealed different evolution of particular compounds after harvest. For example, in the case of “Celeste”, the percentage of triterpenes and alkanes was higher after 3 days at 20 °C, while that of free fatty acids and alcohols decreased after the same period (Table 4). The decreased content of fatty acids could be related to increases in alkanes, which are believed to arise from fatty acid decarboxylation or fatty aldehyde decarbonylation (reviewed in ref 31). On the contrary, no significant differences in the percentage of the main wax compound families were found for “Somerset” cuticles (Table 4). However, triterpenes must have been also actively synthesized in this cultivar after harvest in order to keep pace with increased cuticle yields, as shown by clearly augmented amounts in quantitative terms (Figure 1). Some increase was also observed for alkane content in spite of unchanged alkane percentages over total waxes (Table 4). Little information has been published to date on postharvest changes in cuticular waxes, but epicuticular waxes increased in ripe “Navelate” oranges (Citrus sinensis L. Osbeck) kept at 22 °C for 14 days, this increase being accentuated in ethylene-treated fruit.15 Formation of new waxes was suggested to serve protective purposes, covering stomata, cracks or areas lacking wax, and improving physical barriers against pathogen penetration.15,32 Significant increases in the amount per surface unit of different classes of wax compounds have also been reported for “Jesca” peach fruit, likewise cherry a Prunus species,33 accounting for augmented cuticle yields after harvest and suggestive of up-regulation of the biosynthetic pathways involved in cuticle biosynthesis. In contrast to waxes, the percentages of particular cutin monomer types in fruit cuticles showed limited changes after shelf life at 20 °C, the only significant variations observed being restricted to some decline in monocarboxylic acids, particularly in “Somerset” samples (Table 5). Yet, some differences in the evolution of these compound families were found between both cultivars. For “Celeste”, the content of hydroxy acids declined also in absolute terms (Figure 2A). In contrast, the observation of increased contents of dicarboxylic and hydroxy acids in cuticles of “Somerset” fruit after harvest (Figure 2B) suggests net biosynthesis taking place thereafter and accounting, at least

acid predominated among the C16-type monomers. Only minor quantitative differences in cutin composition were found between both cultivars considered at harvest, the main constituent compounds being the same. Because the procedures for cuticle isolation and cutin depolymerization were similar, the discrepancies with that report6 may illustrate cultivar-related differences which should be explored in relation with their potential relevance for distinctive agronomic, physiological, and quality traits of each particular genotype, such as keeping potential, water loss rates or predisposition to cracking. For example, the prevalence of monohydroxylated monomers (Table 5) instead of di- and trihydroxy acids6 may have consequences for the cross-linking of the cutin network. Although currently available information on the specific composition of fruit cuticles is limited, significant differences have been found among different cultivars of apple,5,8,12,13,21 grape,22−24 pepper,25,26 persimmon,27 or tomato mutants.28−30 At least for apple, pepper, persimmon, and tomato, these differences appeared to be connected to relevant physiological traits related to cuticle functionality such as water loss rates or mechanical properties. Changes in Cuticular Wax and Cutin Composition after Harvest. After harvest, fruit remained 3 days at 20 °C, after which the amount of cuticle per unit surface area and per fruit increased in “Celeste” (Table 2). While this could have arisen from decreased surface area leading to apparently higher cuticle yields per surface unit, data show that fruit diameter, surface, and volume were similar to those at harvest (Table 3). These data thus suggest that some deposition of cuticle material took place during that period. The physiological significance of this postharvest cuticle formation is unclear. Although cuticle deposition in sweet cherry is known to cease early during fruit development,10 postharvest modifications in the regulation of cuticle-related metabolic pathways cannot be ruled out, as no postharvest studies on sweet cherry cuticles have been published to date. In contrast, CM mass per surface area did not change significantly in “Somerset” samples, and it was actually found to decrease when expressed on a whole fruit basis (Table 2), which suggests the existence of cultivar-related differences in cuticle dynamics. Significantly higher wax yields in absolute terms (μg cm−2) were observed in both cases. However, due to augmented cuticle amounts during the shelf life period, the percentage of cuticular waxes decreased (“Celeste”) or remained unchanged (“SomerE



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Table 5. Cutin Constituents (Relative %) Identified in Cuticles of “Celeste” and “Somerset” Sweet Cherries at Harvest and after Cold Storagea days at 0 °C + days at 20 °C 0+0 α-monocarboxylic acids C15:0 C16:0 C16:1 (9) C18:0 C18:1 (9) C18:2 (9, 12) C22 α,ω-dicarboxylic acids 1,16-dioic C16 1,18-dioic C18:1 1,18-dioic C18:2 hydroxyacids 2-OH C16:0 16-OH C16:0 18-OH C18:1 18-OH C18:2 identified cutin unidentified α-monocarboxylic acids C16:0 C16:1 (9) C18:0 C18:1 (9) C18:2 (9, 12) C20:0 C22:0 C28:0 C30:0 α,ω-dicarboxylic acids 1,16-dioic C16 1,18-dioic C18:1 1,18-dioic C18:2 hydroxyacids 2-OH C16:0 16-OH C16:0 18-OH C18:1 18-OH C18:2 identified cutin unidentified

0+3

‘Celeste’ 21.38 ab 15.52 bc nd b nd b 3.72 ab 2.70 b nd b nd b 0.99 a 0.66 b 11.53 a 8.28 ab 3.27 b 2.68 b 1.88 a 1.21 b 26.94 a 26.34 a 7.67 a 7.08 ab 18.05 a 18.14 a 1.23 a 1.13 ab 35.90 a 37.38 a 0.70 ab 0.50 b 4.98 a 4.89 a 14.23 a 14.92 a 15.98 a 17.07 a 84.22 a 79.24 ab 15.78 c 20.76 bc ‘Somerset’ 22.38 b 19.24 c 3.64 bc 3.06 c 0.21 b 0.39 a 1.12 c 0.99 c 13.30 b 11.11 b 2.80 b 2.19 b nd c nd c 1.31 b 1.50 c nd nd nd b nd b 23.87 a 24.10 a 6.27 ab 6.72 a 16.30 a 16.12 a 1.30 a 1.26 a 27.29 a 26.80 a 0.87 a 0.73 ab 3.31 a 3.02 a 12.39 a 11.94 a 10.72 a 11.10 a 74.31 a 70.55 ab 25.69 b 29.45 ab

14 + 0

14 + 3

24.99 a 0.41 a 6.11 a 0.50 a 1.03 a 10.42 a 5.08 a 1.42 ab 20.57 b 5.54 b 14.06 c 0.96 b 28.30 c 1.26 a 3.69 c 10.79 c 12.56 c 75.32 b 24.68 b

11.88 c nd b 2.17 b 0.16 b 0.84 ab 4.73 b 2.20 b 1.78 a 25.35 a 8.10 a 15.98 b 1.28 a 31.24 b 0.21 b 4.29 b 12.68 b 14.06 b 68.47 c 31.53 a

27.64 a 4.25 b 0.35 a 1.36 b 16.45 a 3.49 b 0.36 b 0.94 c nd 0.43 a 17.02 b 4.57 c 11.51 b 0.95 b 15.74 b 1.31 a 1.66 b 6.35 b 6.43 b 66.47 b 33.53 a

26.91 a 5.31 a 0.35 a 1.72 a 12.01 b 6.23 a 0.43 a 0.86 c nd nd b 23.21 a 5.65 b 16.39 a 1.17 a 26.45 nd b 3.37 a 12.52 a 10.55 a 77.63 a 22.37 b

Figure 1. Amount (μg cm−2) of the main classes of wax constituents identified in cuticles of “Celeste” (A) and “Somerset” (B) sweet cherries at harvest and after cold storage. For each class of wax constituents, values bearing different letters are significantly different at P ≤ 0.05 (LSD test).

a

Values represent means of three replicates (nd, nondetectable). Mean values followed by a different lower-case letter within the same row are significantly different at P ≤ 0.05 (LSD test).

Figure 2. Amount (μg cm−2) of the main types of cutin monomers identified in cuticles of “Celeste” (A) and “Somerset” (B) sweet cherries at harvest and after cold storage. For each type of cutin monomers, values bearing different letters are significantly different at P ≤ 0.05 (LSD test).

partially, for the augment in cutin percentage over total cuticle material (Table 2). Differences in the postharvest evolution of particular types of cutin monomers also seem to exist between melting- and nonmelting-type peach genotypes.33 Changes in Cuticular Wax and Cutin Composition after Cold Storage. For both cultivars, the amount of cuticle per unit surface area and per total area increased after storage of fruit at 0 °C for 2 weeks, but decreased again during the subsequent 3 days of shelf life at 20 °C (Table 2), in parallel to lowered fruit surface and volume (Table 3). In the case of “Celeste”, augmented cuticle yields after cold storage were detectable as soon as after

only 7 days (data not shown). Total wax yield and wax percentage in both cultivars decreased significantly after keeping fruit at 0 °C for 14 days. In contrast, although cutin contents were higher in absolute terms than those at harvest (per surface unit as well as per fruit), the percentage over total cuticle amount remained the same owing to augmented cuticle yields per unit surface area (Table 2). F



Article

were not sufficient to keep pace with enhanced cuticle deposition (Table 2). This must have had consequences for cuticle structure and properties, as α,ω-diacid compounds are thought to be important for cross-linking of the cutin polymer.35 In turn, the ωhydroxyl group in ω-hydroxyacids is also necessary for the formation of the polymer.36 Similar trends were observed in “Somerset” fruit for the evolution of the main types of cutin monomers, although some differences were found for particular compounds. For example, the content of oleic acid was higher after cold storage, in contrast with findings for “Celeste” fruit, and the increase in alkane content was also more perceptible (Table 5). Although the content of α,ω-dicarboxylic and ω-hydroxy acids recovered after 3 days at 20 °C, the declining trend for the percentage of these families of cutin monomers along cold storage was accentuated in fruit kept at 0 °C for a longer period (data not shown), after which the contents of these compounds remained low. A little percentage of alkanes was noticed among the cutin constituents detected after cold storage, particularly for “Somerset” samples (data not shown). This may be an artifact representing wax constituents not completely released during the dewaxing procedure, which may have remained enclosed within the cutin matrix even though the chloroform extraction was repeated three times. In summary, the most abundant components identified in cuticular waxes of “Celeste” and “Somerset” fruit agree with previous reports for other cherry cultivars,6,16 but remarkable differences were found as to the main types of cutin monomers. These data suggest cultivar-related variability in the specific composition and in the abundance of particular compounds in cuticles of sweet cherry fruit. Such dissimilarities in the chemical composition of fruit cuticles and in their evolution during postharvest preservation of produce might account, at least partially, for some of the quality or storability characteristics of each cultivar, including weight loss, firmness, or susceptibility to infections or physiological disorders.2 To the best of our knowledge, this is the first study reporting changes in cuticle composition of sweet cherry fruit during cold storage and one of the few works addressing postharvest modifications in the components of this outer layer of fruit surface. Further work on modifications in the relative amounts of the different cuticle components in response to internal and external factors will be required to shed light on the physiological consequences of these compositional changes, thus helping dissect the contribution of each particular compound family to the biological role of fruit cuticles.

Many metabolic processes in fruit are enhanced or redirected in response to low temperatures. Data shown herein put forward the possibility that cuticle formation is one of such processes. A few reports have described the changes in cuticular waxes after cold storage of apple8,12−14 and shown considerable cultivarassociated variability for this fruit species. For instance, total wax contents after long-term cold storage were found to remain unchanged in “Sturmer” but to increase in “Granny Smith” and “Dougherty” apples,12 while in contrast a sharp decline was observed for “Red Fuji” samples.8 Changes in the composition of cuticular waxes during cold storage of apples were modulated in response to usual postharvest procedures such as controlled atmosphere storage13 or 1-methylcyclopropene treatment.8,14 However, we are not aware of any similar works for other fruit species. To the best of our knowledge, cutin changes after cold storage have not been previously reported for any fruit commodities either. We therefore analyzed the fate of wax and cutin components in cuticles isolated from “Celeste” and “Somerset” fruit kept at 0 °C. The modifications described below are indicative of intense alterations taking place in cuticle composition, and hence probably also in cuticle structure and properties, during cold storage. The content of triterpenes (μg cm−2) in cuticular waxes of “Celeste” fruit was unchanged after cold storage (Figure 1A). Still, owing to lower wax content in cuticles of cold-stored fruit as compared with values at harvest (roughly 25 vs 33 μg cm−2, respectively), the percentage of triterpenes increased, ursolic and oleanoic acids representing approximately 58 and 10% of total waxes, in that order (Table 4). Although to a lower extent, some increase was also found in alkane percentage, particularly in that of n-nonacosane, the main alkane in quantitative terms, even though the absolute content was likewise unaffected (Figure 1A). Similar results were obtained for phytosterols, while in contrast the content of fatty acids decreased sharply, the main reduction being found for linoleic acid (Table 4). The total content of triterpenes after cold storage was also lower in cuticles of “Somerset” cherries, both in absolute (Figure 1B) and relative (Table 4) terms, while the percentage of alkanes and sterols increased. This is interesting in the light of previous suggestions that the waterproofing properties of fruit cuticles are defined primarily by the n-alkane components.34 Reduced wax alkanes and enhanced triterpenoids would lead to increased content of amorphous waxes, thus impairing the transpiration barrier function of cuticles,29 and in fact the ratio of alkanes to triterpenoids plus sterols has been found to correlate inversely with dehydration rates in pepper fruit.26 Indeed, this ratio was 0.18 in “Celeste” and 0.33 in “Somerset”, which would be consistent with the observation of higher weight loss rates for “Celeste” (15.8%) than for “Somerset” (7.2%) after cold storage (Table 3). In relation to cutin monomers, some differences in the evolution of the content of particular compounds were also found between both cultivars considered. In “Celeste” samples, the percentage of monocarboxylic fatty acids was higher in fruit kept at 0 °C for 2 weeks, resulting from parallel increases in palmitic and linoleic acids (Table 5). However, the percentage of total fatty acids decreased again after 3 days of shelf life. In spite of a significant augment in the amount of α,ω-dicarboxylic and ωhydroxy acids per unit surface area (Figure 2), the percentage of these compounds (particularly those of the C18-type) over total cutin mass decreased after cold storage. This is indicating that actual production of these cutin monomers must have taken place during the experimental period but that biosynthesis rates

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 973 702526. E-mail: [email protected]. Funding

B. Belge is the recipient of a FI-DGR grant from AGAUR (Generalitat de Catalunya). This work was supported through the AGL2010−14801/ALI project, funded by the Ministerio de Ciencia e Innovación (MICINN) of Spain. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Fruit samples were provided by J. M. Camats (Cireres de Corbins Camats-Carpi). G



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Article

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