Fruit Volatile Profiles of Two Citrus Hybrids Are Dramatically Different

Article pubs.acs.org/JAFC

Fruit Volatile Profiles of Two Citrus Hybrids Are Dramatically Different from Those of Their Parents José Luis Rambla,†,∥ M. Carmen González-Mas,*,‡,∥ Clara Pons,† Guillermo P. Bernet,§,⊥ Maria José Asins,§ and Antonio Granell† †

Instituto de Biología Molecular y Celular de Plantas, CSICUniversidad Politécnica de Valencia, Ciudad Politécnica de la Innovación, Edificio 8 E, Ingeniero Fausto Elio, 46022 Valencia, Spain ‡ Fundación Agroalimed-Centro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Náquera-Moncada, Km. 4.5, 46113 Moncada, Valencia, Spain § Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Náquera-Moncada, Km. 4.5, 46113 Moncada, Spain S Supporting Information *

ABSTRACT: Volatile compounds released from the fruit of two hybrid Citrus genotypes (FxCh90 and FxCh77) were compared to those from their parental varieties, Fortune mandarin and Chandler pummelo. A series of 113 compounds were identified, including 31 esters, 23 aldehydes, 20 alcohols, 17 monoterpenoids, and other compounds. The differences in the volatile profile among these four genotypes were essentially quantitative. The most striking result was that the volatile profile of the hybrids was not intermediate between their parents and completely differed from that of Chandler, but came closer to Fortune. This was because 56 of the 113 volatile compounds in the hybrids showed significantly higher or lower levels than in any of the parents. Such transgressive behavior in these hybrids was not observed for other fruit quality traits, such as acidity or soluble solid content. The combination of volatile profiling and chemometrics can be used to select new Citrus genotypes with a distinct volatile profile. KEYWORDS: aroma, VOCs (volatile organic compounds), gas chromatography−mass spectrometry, Citrus, Fortune, Chandler pummelo, hybrid

■

INTRODUCTION One of the main characteristics of Citrus fruit quality is defined by the aroma of its juice. The aroma of fresh juice is due to the complex combination of a large number of compounds, which includes esters, aldehydes, alcohols, ketones, and monoterpene and sesquiterpene hydrocarbons, generically termed VOCs (volatile organic compounds).1−4 The volatile composition in Citrus fruit has been thoroughly studied,5−11 but not many studies address how the volatile pattern is modified in hybrids. Two hybrids, FxCh90 and FxCh77, resulting from the cross between Fortune mandarin and Chandler pummelo, are both important varieties for the world’s fresh Citrus market and were obtained as part of the IVIA Citrus breeding program. The characterization of the metabolites underlying the organoleptic characteristics of these new hybrids was studied as a first step to establish their potential commercial value. The volatile composition of both parents, Fortune mandarin,12,13 and Chandler pummelo juice,13 has been previously reported. The aroma composition of juices of other pummelo cultivars has been described, including Nakon pummelo,14 and one whitefleshed and one pink-fleshed pummelo cultivar (Citrus grandis (C. grandis) (L.) Osbeck).15 In the present work, we describe the characterization of the volatile profile for the juice of FxCh90 and FxCh77 hybrids and compare their profiles with those of their parents. From these results, we conclude that it is possible to obtain Citrus hybrids with a volatile profile that considerably differs from any of their parents and that the HSSPME-GC-MS metabolomics and chemometrics approach used © 2014 American Chemical Society

herein is a powerful auxiliary technology to identify and characterize new volatile profiles in Citrus breeding programs.

■

MATERIALS AND METHODS

Plant Material, Citrus Juice, and Agronomic Characterization. A segregating population of 201 full-sib hybrids was obtained by crossing each parental genotype in the spring of 2000 and 2001. Parents were graft-propagations of two commercial varieties: Fortune (F) and Chandler (Ch). The female parent, Fortune, is a hybrid mandarin derived from the cross between C. clementina Hort. ex Tan. and C. tangerina Hort. ex Tan. The male parent, Chandler, is a hybrid pummelo derived from a cross between two accessions of C. grandis Osbeck. The genetic makeup of each hybrid was determined for 171 molecular markers (three of which were from cytoplasmic organelles) and was used to obtain the genetic linkage maps of parental species and genetic distances within their progeny.16 All the hybrids were grafted on Carrizo rootstock and were grown in the same orchard under the same cultural conditions. The experimental orchard is located at the Experimental Station of the Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain, with a Mediterranean climate (full rainfall of 61.6 mm and average temperature of 10.6 °C for winter 2009). Mature fruits were collected after reaching maximum juice content (Regulation EC No. 1799/2001, Sep. 12, 2001) from the trees of Chandler (first week of January), Fortune (first week of March), and Received: Revised: Accepted: Published: 11312

April 25, 2014 October 13, 2014 October 22, 2014 October 22, 2014 dx.doi.org/10.1021/jf5043079 | J. Agric. Food Chem. 2014, 62, 11312−11322

Journal of Agricultural and Food Chemistry

Article

two hybrids, FxCh77 and FxCh90 (fourth week of February), in 2009. In all cases, three biological replicates per genotype were obtained, all representing at least four different fruits each. Juice was obtained using a hand extractor to avoid squeezing the flavedo and to prevent juice becoming contaminated with peel components. Juice samples of 10 mL aliquots each were placed in 22 mL crimp cap headspace vials and kept frozen at −20 °C until analyzed. Two 10 mL aliquots corresponding to technical replicates of each sample were analyzed. The total number of analysis was 24 (3 biological samples × 2 technical replicates for all 4 genotypes). Eleven fruit quality traits were also evaluated, as usual, in the Citrus breeding program: fruit weight (FW, g); fruit diameter (FD, mm), measured in the transversal section including flavedo and albedo; rind thickness (RT, mm), including albedo; juice volume (JV, mL), quantified as the volume of juice without pulp; number of seeds per fruit (SN); juice content (JC) as JV% of FW; total soluble solids (TSS, degrees Brix), measured with a digital refractometer (PR-101α; Palette, Atago; Tokyo, Japan); acidity (A), determined by titration with 0.4 N NaOH and a phenolphthalein indicator; maturation index as the ratio between TSS and A (Brix/A); and color of rind and juice (Rcol and Jcol, respectively), evaluated by visual comparisons with the chromatic circle (from red, 1, to yellow, 20). All of these traits were used for the principal component analysis (see Figure 2). HS-SPME Extraction Conditions. Immediately before the analysis, samples were thawed at 20 °C for 10 min and were then subjected to headspace solid phase microextraction (HS-SPME). Extraction was carried out using 10 mL of sample placed into a 22 mL crimp cap headspace vial. A 50/30 μm DVB/CAR/PDMS (Supelco, Bellefonte, PA, USA) fiber was used in all cases. Samples were preincubated for 10 min at 50 °C, and volatiles were extracted from the vial headspace for an additional 20 min period at the same temperature, as previously described.13 Desorption was performed for 1 min at 250 °C in the splitless mode. Gas Chromatography−Mass Spectrometry Conditions. VOCs were analyzed by GC-MS using a COMBI-PAL autosampler (CTC Analytics, Zwingen, Switzerland), and a 6890N gas chromatograph coupled to a 5975B mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), equipped with a J&W Scientific DB-5 ms fused silica capillary column (5−95 phenyl−dimethylpolysiloxane as the stationary phase; 60 m length; 0.25 mm i.d.; 1 μm film thickness) (Folsom, CA, USA). Oven temperature conditions were 40 °C for 2 min, with 5 °C/min ramp until 250 °C, and were then held isothermally at 250 °C for 5 min. Helium was used as the carrier gas at 1.2 mL/min constant flow. Detection was performed by an Agilent mass spectrometer in the EI mode (ionization energy, 70 eV; source temperature, 230 °C). Acquisition was performed in the scan mode (mass range, m/z 35−220; 7 scans/s). Considering that the molecular weight of the identified compound with the highest mass (nootkatone) was 218 Da, a wider m/z range was not necessary. Chromatograms and spectra were recorded and processed using the Enhanced ChemStation for GC-MS software (Agilent). For the unequivocal identification of volatile compounds, both standards and samples were also injected into a 6890N gas chromatograph (Agilent) coupled to a Pegasus 4D time-of-flight (TOF) mass spectrometer (LECO, St. Joseph, MI, USA) under the following chromatographic conditions: BPX35 column (30 m, 0.32 mm, 0.25 μm), helium (1.5 mL/min) constant flow, and the same oven temperature ramp as previously described. Ionization was performed by EI (ionization energy, 70 eV; source temperature, 200 °C), and acquisition was performed within the mass range m/z 35−300. Compound Identification. Compound identification in each sample was based on the comparison of both mass spectrum and retention time in two columns of different polarity with those of pure standards. The retention times of the identified compounds in each column are detailed in Supporting Information (SI) Table S1. From 113 compounds, 103 compounds were identified in the Citrus samples when analyzed with each of the two columns. The other 10 volatiles were overlapped by more abundant compounds in the BPX35 column chromatograms, and therefore their identity could not be confirmed. The identification of these 10 compounds was based on the similarity

of mass spectrum and retention time in only one column, and should therefore be considered tentative, as indicated in Table 1. The compounds used as a reference were of analytical grade and purchased from Sigma-Aldrich Quı ́mica (Madrid, Spain), except for 2-carene, which was obtained from Extrasynthese (Genay, France). In addition to the commercial compounds, four esters (ethyl heptanoate, methyl octanoate, methyl nonanoate, and ethyl nonanoate) were synthesized by acid-catalyzed esterification, according to González-Mas et al.13 For relative quantification, one specific ion for each compound was selected, and the corresponding peak from the extracted ion chromatogram was integrated. The ion selected for the integration of each peak was that with the highest signal-to-noise ratio and one that was specific enough to provide good peak integration for that particular region of the chromatogram. An admixture reference sample was prepared by mixing equal amounts of each sample corresponding to the four analyzed genotypes. This admixture was aliquoted (10 mL in 22 mL headspace vials), stored, and further treated as any other sample. This admixture contained all of the compounds identified in any of the samples at an intermediate concentration between those in the individual samples. The admixture aliquots were injected regularly (one admixture for every five to six samples) as part of the injection series and were used as a reference to normalize for temporal variation and fiber aging. All of the samples were analyzed using the same SPME fiber. Finally, the normalized results (corrected for temporal variation and fiber aging) for each compound were expressed as the relative levels to the average levels present in the Chandler juice. If a compound was not detected in Chandler, the ratio was calculated to the levels in a genotype that contained it, as indicated (Table 1). Statistical Analysis. A principal component analysis (PCA) and a hierarchical cluster analysis (HCA) were done to analyze the volatile data set, which included all of the replicates and samples. In them both, the ratio of the levels of each volatile in a sample to the average of the four genotypes was log2-transformed. For the PCA, SIMCA-P version 11 (Umetrics, Umea, Sweden) was used with Pareto normalization. For the HCA, Acuity 4.0 (Axon Instruments, Union City, CA, USA) was used with the distance metrics based on the Pearson correlation. To represent the phenotypic traits (either volatile levels or quality traits) that were intermediate or significantly under or over the parental levels, the Z-scores of the average values for each genotype were calculated. Data were expressed as the values relative to the corresponding level in Chandler, log2-transformed (log RFortune or log Rhybrid), and then Z-score standardization was performed for each metabolite in all the samples. To calculate the fold change between hybrids and Fortune, the Z-score for the difference was calculated using ZD = log Rhybrid − log RFortune/(SE2hybrid + SE2Fortune)1/2, where SE2 is the squared standard error of the mean. The Z-score for each phenotypic trait (volatile level or other) in both of the two hybrids was plotted against the corresponding Z-scores in Fortune (Figure 1 and SI Figure S2). The between, under, or over parental sectors were defined by the X and Y axes, and the slope 1 diagonal, where the phenotypic traits with the Z-score hybrid > 0 (y > 0) and ZD > 0 were in the overparent sector and the phenotypic traits with the Z-score hybrid < 0 and ZD < 0 in the underparent sector. To determine the significance of the differences relating to both Chandler and Fortune, the p-value associated with each Z-score and ZD was calculated. The metabolites and physiological parameters in each sector were considered statistically significant with a p-value < 0.05 (SI Table S2).

■

RESULTS AND DISCUSSION Table 1 lists the VOCs detected by the HS-SPME-GC-MS technique and the relative volatile levels in hybrids FxCh90 and FxCh77 as compared to those in the parental genotypes: Fortune mandarin and Chandler pummelo. In all, 113 compounds were identified: 31 esters (22 aliphatic and 9 monoterpene acetates), 23 aldehydes (18 aliphatic, 4 monoterpene, and 1 norisoprenoid), 20 alcohols (11 aliphatic and 9 monoterpene), 14 monoterpene hydrocarbons, 13 11313

dx.doi.org/10.1021/jf5043079 | J. Agric. Food Chem. 2014, 62, 11312−11322


Article

Table 1. Relative Levels (Fold Changes) of the VOCs Detected in the Juices of Two Citrus Hybrids and Their Parental Varietiesa code

cluster

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

A1 A1 A1 A1 A1 A1 A1 A1 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 B B B B B B B B B B B B B B B B B B

volatile organic compound linaloolc β-citronellolc perillaldehydec α-terpineolc β-citronellalc,d hexyl acetatee geraniolc nerolc heptyl acetated 1-decanold 1-nonanol sabinene α-terpinyl acetatec,f nonyl acetate linalyl acetate nerale 1-octanol methyl decanoate octyl acetate decyl acetate terpinen-4-olc γ-terpinene (E)-carvyl acetatec dodecanal geranyl acetatec terpinolene α-terpinene citronellyl acetatec α-pinene limonene α-phellandrene myrcene (Z)-carvyl acetatec neryl acetatec camphene 3-carene eucalyptol (1,8-cineole)e (Z)-3-hexenal bornyl acetatec β-pinene α-copaene decanal undecanal (Z)-limonene oxideg 1-penten-3-one (E)-2-hexenal (E)-2-pentenal (E)-limonene oxideg (Z)-carveolc carvonec (E)-carveolc dihydrocarvonec ethyl decanoate acetaldehyde ethanol (E)-2-decenal ethyl butanoate ethyl octanoate ethyl propanoate propyl acetatee,f ethyl 2-methylbutanoatef

family codeb/no.

retention time (min)

specific ion (m/z)

Alc/1 Alc/2 Ald/1 Alc/3 Ald/2 Est/1 Alc/4 Alc/5 Est/2 Alc/6 Alc/7 Mt hd/1 Est/3 Est/4 Est/5 Ald/3 Alc/8 Est/6 Est/7 Est/8 Alc/9 Mt hd/2 Est/9 Ald/4 Est/10 Mt hd/3 Mt hd/4 Est/11 Mt hd/5 Mt hd/6 Mt hd/7 Mt hd/8 Est/12 Est/13 Mt hd/9 Mt hd/10 Mt cyc ether/1 Ald/5 Est/14 Mt hd/11 Sqt/1 Ald/6 Ald/7 Mt cyc ether/2 Ket/1 Ald/8 Ald/9 Mt cyc ether/3 Alc/10 Ket/2 Alc/11 Ket/3 Est/15 Ald/10 Alc/12 Ald/11 Est/16 Est/17 Est/18 Est/19 Est/20

27.15 31.15 33.43 30.75 28.92 23.86 31.95 31.28 27.27 32.50 29.36 23.09 35.10 33.47 31.82 31.81 26.00 33.94 30.47 36.29 30.38 26.03 35.30 36.55 35.50 27.05 24.65 34.69 21.67 25.11 24.29 23.28 34.50 34.97 22.44 24.43 25.38 15.92 33.66 23.46 36.41 30.59 33.67 28.77 11.57 18.17 14.30 28.84 31.83 32.31 31.41 30.89 35.91 4.77 5.64 32.43 15.88 30.04 12.47 12.52 17.87

93 81 68 59 69 56 69 93 43 115 70 93 121 98 93 84 56 74 70 70 93 93 84 57 69 121 121 95 93 108 93 91 84 69 93 93 154 69 121 93 119 57 57 67 55 83 83 94 109 82 109 95 88 43 45 70 88 88 57 61 102 11314

Chandler 1 1 1 1

± ± ± ±

0.69 0.53 0.29 0.59

a a a a

1 ± 0.86 a 1 ± 0.63 a 1 ± 0.47 a

1 ± 0.37 a 1 ± 0.53 a 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.35 0.47 0.55 0.33 0.82 0.53 1.51 0.77 0.61 1.17 2.35 0.96 0.40 0.52 0.62 0.41 0.25 0.41 0.42 0.46 0.55 1.04 0.56 0.62 0.89 1.17 0.88 0.58 0.19 0.18 1.15 0.52 0.70 0.45 0.77 0.49 0.68 0.36 0.40 0.78 0.25 0.59 0.37 2.04 0.55 1.08

a a a a a a a a a a a a a a a a a a a a a a a a a a b b a b a a a b a a a a a a a a a a a a

FxCh90 37.57 6.32 5.58 4.07

± ± ± ±

2.12 0.27 1.18 0.81

FxCh77 c c b b

32.96 ± 11.01 c 4.02 ± 0.50 b 2.79 ± 0.20 b 8.67 ± 2.44 b 15.23 ± 3.47 b 8.23 ± 0.90 c 8.42 ± 4.11 c 38.06 ± 11.68 b 487.1 ± 147.7 c 217.92 ± 60.01 c 46.90 ± 10.02 c 37.82 ± 1.14 b 9.23 ± 1.03 d 574.5 ± 171.6 c 727.3 ± 219.4 c 34.86 ± 7.42 d 16.56 ± 3.96 d 43.19 ± 18.72 b 78.60 ± 24.86 c 41.21 ± 22.00 c 6.84 ± 1.30 c 12.26 ± 4.37 c 95.15 ± 29.37 c 11.09 ± 2.41 b 2.66 ± 0.21 bc 8.62 ± 1.63 c 4.42 ± 0.50 c 4.47 ± 1.37 b 4.80 ± 1.25 c 2.99 ± 0.62 bc 2.47 ± 0.19 b 2.19 ± 0.36 b 1.44 ± 0.47 a 5.59 ± 1.57 c 1.04 ± 0.35 b 1.21 ± 0.37 b 3.65 ± 2.44 b 1.62 ± 0.59 c 2.87 ± 0.64 b 0.60 ± 0.04 a 0.64 ± 0.06 a 0.35 ± 0.05 a 3.54 ± 0.70 b 6.32 ± 3.50 c 5.10 ± 1.01 b 2.00 ± 0.08 b 3.06 ± 0.50 b 3.09 ± 0.25 b 1.57 ± 0.25 b 2.39 ± 0.67 b 1.44 ± 0.34 b 3.58 ± 1.14 a 0.96 ± 0.06 a 1.52 ± 0.38 a

131.14 16.57 22.44 8.14 1 17.91 7.47 3.98 1 1 1.89 4.76 1.19 22.58 22.38 6.23 2.18 1.98 16.72 43.00 5.35 3.53 3.72 22.92 18.59 5.21 5.11 91.39 15.50 2.89 11.15 5.20 5.32 4.57 3.41 1.40 3.08 0.95 3.82 1.25 1.15 0.90 0.65 4.75 2.98 1.27 1.44 6.11 2.89 5.64 2.46 5.76 2.96 1.55 1.66 0.87 2.92 1.46 0.31

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

18.44 d 0.64 d 10.67 d 2.06 d 0.23 3.96 b 1.05 c 0.71 c 0.77 a 0.20 a 0.37 b 2.35 bc 0.57 a 8.53 b 1.43 b 0.98 b 0.95 a 0.85 b 0.93 ab 8.12 ab 1.63 b 1.09 b 1.20 a 9.32 b 8.14 b 1.33 b 1.65 b 37.02 c 5.37 c 0.28 c 3.15 d 0.96 d 1.90 b 2.04 c 1.00 c 0.54 a 0.07 b 0.26 a 1.63 b 0.34 b 0.43 b 0.98 a 0.12 a 0.53 c 1.94 b 0.58 a 0.83 b 1.11 c 0.34b 1.17 b 0.32 b 1.11 c 1.31 b 0.40 b 0.59 ab 0.29 a 2.79 a 1.20 a 0.48 a

Fortune 26.77 4.53 15.19 5.67

± ± ± ±

1.66 0.45 5.28 0.69

b b c c

0.98 ± 0.22 a 1.50 ± 0.47 a 1.86 ± 2.07 b traces 0.82 1.34 1 22.54 7.29 6.21 2.18 3.14 64.08 68.70 15.86 5.16 7.36 20.38 14.09 4.78 4.75 47.41 9.17 2.46 5.64 3.21 4.27 1.90 1.95 1.39 0.30 0.44

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.30 0.82 0.23 6.43 0.92 0.69 0.26 0.92 0.30 11.4 2.59 0.85 4.56 8.79 1.39 0.33 1.80 4.01 1.18 0.10 0.58 0.28 1.58 0.19 0.20 0.64 0.06 0.14

a ab a b ab b a c b b c c a b b b b b b b b b b b b a a a

0.17 0.35 0.78 0.76 3.47 2.45 1.02 1.45 6.81 6.91 20.89 5.35 10.25 16.43 2.75 6.05 2.15 27.11 4.05 22.36 1 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.02 0.22 0.09 0.14 0.59 0.16 0.25 4.59 0.85 1.02 0.71 0.38 3.04 0.22 1.64 0.38 5.07 0.81 7.40 0.53 0.25

a a a ab b b a b bc c c c d c c c c b b b



Article

Table 1. continued code

cluster

62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

B B B C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1

78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

C1 C1 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2

101 102 103 104 105 106 107 108 109 110 111 112 113

C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2

volatile organic compound ethyl acetate 3-methylfuran ethyl nonanoate geranylacetoneh (Z)-3-hexen-1-ol geranialc,e (E,E)-2,4-decadienal 1-penten-3-ol β-cyclocitrale,h β-iononeh 3-pentanone hexyl butanoate hexyl hexanoateI butyl hexanoateI nootkatonej α-humulene (α-caryophyllene) β-caryophyllene (Z)-linalool oxidec,k methyl nonanoate isobornyl acetatec,e valencene 2-methylfuran 2-ethylfuran (E)-2-octenal ethyl heptanoate pseudocumene 1-hexanol hexanal pentanal 2-pentylfuran 1-octen-3-ol 3-octanoneI nonanal (E)-2-nonenal 2-nonanoneI heptanal 2,3-pentanedione 1-heptanol 6-methyl-5-hepten-2onee,h 1-pentanol ethyl hexanoate methyl hexanoate methyl octanoate octanal 1-octen-3-one (E)-2-heptenale p-cymene (Z)-ocimene (E,E)-2,4-nonadienal (E)-linalool oxidec,k 2-carenee γ-dodecalactone

retention time (min)

specific ion (m/z)

Est/21 Fur/1 Est/22 Ket/4 Alc/13 Ald/12 Ald/13 Alc/14 Ald/14 Ket/5 Ket/6 Est/23 Est/24 Est/25 Ket/7 Sqt/2

9.18 9.16 33.05 37.62 18.16 32.60 34.17 11.46 31.63 38.90 11.97 29.94 35.72 29.92 47.87 38.82

61 82 88 43 82 69 81 57 137 177 57 89 117 117 121 80

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.42 0.32 0.91 0.26 0.92 0.41 0.68 0.64 0.42 0.44 0.63 0.71 0.45 1.10 0.33 0.64

a a a b b b b b c b b b b b b b

0.44 0.78 0.36 0.60 0.10 0.44 0.32 0.28 0.27 0.20 0.11 0.57 0.33 0.54 0.14 0.14

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.12 0.17 0.13 0.11 0.06 0.23 0.30 0.01 0.05 0.03 0.04 0.15 0.08 0.16 0.01 0.03

a a a a a a a a a a a ab a ab ab a

0.19 1.10 1.13 1.33 1.20 1.10 0.79 0.54 0.58 0.92 0.55 1.05 0.59 0.73 0.43 0.10

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.28 0.53 0.57 1.09 0.63 0.54 0.11 0.17 0.50 0.21 0.23 0.15 0.24 0.15 0.05

a a a c b b ab ab ab b b b a ab ab a

Sqt/3 Alc/15 Est/26 Est/27 Sqt/4 Fur/2 Fur/3 Ald/15 Est/28 Ar/1 Alc/16 Ald/16 Ald/17 Fur/4 Alc/17 Ket/8 Ald/18 Ald/19 Ket/9 Ald/20 Ket/10 Alc/18 Ket/11

37.87 26.47 30.91 33.48 39.69 8.84 12.17 25.79 26.82 23.85 18.59 16.01 12.04 23.36 22.85 23.10 27.30 29.21 26.79 20.00 11.91 22.44 23.03

133 111 74 121 133 82 81 70 88 105 56 56 58 138 57 99 57 70 58 70 100 70 108

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.57 0.24 0.51 0.87 0.56 0.25 0.21 0.24 0.67 0.18 0.70 0.22 0.24 0.19 0.32 0.41 0.15 0.28 0.38 0.26 0.28 0.55 0.40

b b a a b b b c b b b b b c b b c b c c c b b

0.04 0.03 1.09 0.91 0.39 0.25 0.14 0.11

± ± ± ± ± ± ± ±

0.01 0.01 0.14 0.07 0.04 0.06 0.04 0.02

a a a a a a a a

0.18 0.06 0.09 0.09 0.08 0.06 0.03 0.25 0.15 0.09 0.06 0.03 0.24 0.03

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.01 0.02 0.01 0.02 0.05 0.01 0.02 0.01 0.01 0.01 0.02 0.04 0.01

a a a a a a a a a a a a a a

0.04 0.03 0.64 0.56 0.29 0.51 0.30 0.24 0.13 0.12 0.17 0.12 0.13 0.16 0.04 0.04 0.17 0.14 0.09 0.07 0.03 0.17

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.18 0.21 0.07 0.18 0.09 0.11 0.20 0.05 0.13 0.02 0.04 0.07 0.03 0.02 0.05 0.04 0.02 0.03 0.02 0.05

a a a a a a a a a a a a a ab a a a a a a a a

Alc/19 Est/29 Est/30 Est/31 Ald/21 Ket/12 Ald/22 Mt hd/12 Mt hd/13 Ald/23 Alc/20 Mt hd/14 Ket/13

14.60 23.37 20.68 27.74 23.77 22.82 22.14 24.91 24.87 31.10 27.00 24.05 43.71

41 88 74 74 57 70 83 119 93 81 111 93 85

1 ± 0.48 1 ± 0.66 1 ± 0.46 1 ± 0.49 1 ± 0.22 1 ± 0.46 1 ± 0.26 1 ± 0.85 1 ± 0.61 1 ± 0.31 1 ± 0.22 1 ± 0.25 traces

b b b b b b b b

0.03 0.08 0.02 0.64 0.42 0.22 0.15

± ± ± ± ± ± ±

0.05 0.01 0.02 0.15 0.20 0.01 0.01

a a a ab a a a

0.28 0.06 0.51 0.19 0.12 0.19 0.02

± ± ± ± ± ± ±

0.33 0.05 0.23 0.13 0.01 0.09 0.01

a a a a a a a

family codeb/no.

Chandler

FxCh90

FxCh77

Fortune 40.71 2.91 5.59 0.58 0.33 0.39 0.24 0.54 0.78 0.88 0.59 0.31 0.22 0.45 0.02 0.04

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.12 0.94 1.51 0.14 0.09 0.11 0.08 0.14 0.13 0.18 0.19 0.07 0.06 0.06 0.01 0.01

b b b a ab a a ab bc b ab a a a a a

traces 1.02 0.73 1.16 1.13 1.22 0.51 1.76 0.24 0.22 0.23 0.23 0.29 0.14 0.10 0.60 0.98 0.49 0.42 0.53 0.58 0.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.11 0.13 0.32 0.29 0.24 0.15 0.31 0.01 0.18 0.02 0.04 0.05 0.03 0.05 0.06 0.20 0.08 0.01 0.16 0.32 0.02

a a b b b b b a a a a b a a b b b b b a a

0.11 0.62 0.07 0.44 0.39 0.19 0.23

± ± ± ± ± ± ±

0.02 0.11 0.02 0.08 0.05 0.07 0.05

a ab a a a a a

traces

a Data are normalized to the mean response values in the Chandler variety, unless otherwise indicated. Mean corresponding to n = 6 values. Means followed by different letters in the same row are significantly different (p < 0.05) in the Duncan’s text. bCode family: Ald, aldehyde; Ket, ketone; Alc, alcohol; Est, ester; Fur, furane; Mt hd, monoterpene hydrocarbon; Sqt, sesquiterpene; Ar, aromatic hydrocarbon; Mt cyc ether, monoterpene cyclic ether. cMonoterpene-derived compounds. dData are normalized to the mean response calculated for FxCh77 since this compound was not present in the Chandler variety. eTentative identification based on coincidence of mass spectrum and retention time in only one capillary column. fData are normalized to the mean response calculated for the Fortune variety. gIn addition to the alcohol group, it has a tetrahydrofuran group. h Norisoprenoid compounds. INew compounds reported for the first time in Citrus juices. jSesquiterpene compounds. kIts cyclic ether group is an epoxy group.

11315



Article

Figure 1. Values for the different metabolites and phenotype traits in hybrid FxCh90 in relation to the values in Fortune. Values are expressed as standardized Z-score values of log2 relative to Chandler (value = 0). The blue sector defines those traits whose value in the hybrid fell between the values of the parents. The pale orange and pale yellow sectors correspond to the underparent sector and the overparent sector, respectively. Volatile compounds are represented in black; phenotypic traits are represented in red.

orange and tangerine hybrids juices and mandarin peel.9,11,27 The other four compounds are described here for the first time in the juice of a Citrus species (Table 1). Hexyl butanoate and butyl hexanoate are typical apple fruit volatile compounds that can be used as a maturity index in this fruit; butyl hexanoate is also an insect attractant.28,29 Regarding 3-octanone, it has been described as having a mushroom odor, similar to 1-octen-3-one, this being another aliphatic ketone identified frequently in Citrus juice.3,9,30 The other new ketone, 2-nonanone, has been identified in several matrixes such as kiwifruit juices, in the essential oil of several Rutaceae species, in yellowfin tuna, or even as a component of cheese aroma.31−35 The compound 2nonanone has been described to have nematicidal and antifungal properties.36,37 In fact an active packaging system based on the release of 2-nonanone is used to increase the postharvest shelf life of fresh wild strawberries in the marketing stage; excessive levels of this volatile can, however, affect the taste of berries and lead consumers to reject the product.38 As seen in Table 1, almost all of the identified compounds were detected in the four genotypes. Only four compounds were identified exclusively in the Chandler parental genotype ((Z)-ocimene, (E,E)-2,4-nonadienal, (E)-linalool oxide, and γdodecalactone; m 109, m 110, m 111, and m 113, respectively). Similarly, propyl acetate (m 60) and ethyl 2-methylbutanoate (m 61) were compounds identified exclusively in the parental Fortune genotype. Regarding hybrids, β-citronellal (m 5) was the only exclusive compound identified in the FxCh77 hybrid,

ketones (7 aliphatic, 2 norisoprenoid, 3 monoterpene, and 1 sesquiterpene), 4 sesquiterpene hydrocarbons, 3 monoterpene cyclic ethers, 4 furans, and 1 aromatic hydrocarbon. Although more than 300 VOCs have been reported in other Citrus juice,9,15,17−19 some were identified only tentatively.9,11,20−22 To assign chemical names to our data set, in this work we used the actual compounds to verify the identification of the VOCs analyzed by confirming and expanding the power of our volatile profiling platform.13 All of these compounds were unequivocally identified based on the coincidence of both mass spectrum and retention time with those of pure standards in two capillary columns of different polarity, with the only exception of 10 compounds not identified in the second column (Table 1). A representative chromatogram is shown in SI Figure S1. The majority of the identified compounds were described in our previous Citrus juice studies, which included Chandler pummelo, Fortune and Clemenules mandarins, and Powell orange.13 In this work, eight new compounds were identified in hybrids FxCh90 and FxCh77 and their parental genotypes: (E)2-decenal (m 56), isobornyl acetate (m 81), dihydrocarvone (m 52), hexyl butanoate (m 73), butyl hexanoate (m 75), hexyl hexanoate (m 74), 3-octanone (m 93), and 2-nonanone (m 96). The first four compounds have been previously identified in other Citrus genotypes: (E)-2-decenal in tangerine hybrids juices and in pummelo, lime, orange, and tangerine peel oils,9,23,24 isobornyl acetate in pummelo peel,25 dihydrocarvone in orange and mandarin juices,9,20,26 and hexyl butanoate in 11316



Article

while no exclusive compound was detected in the FxCh90 hybrid. The relative levels of each compound for all of the genotypes are shown in relation to the level of each compound in the Chandler genotype, because this is the genotype where the largest number of compounds was identified (107 compounds, with γ-dodecalactone, m 113, only detected in trace amounts). The relative levels of those compounds not detected in Chandler are expressed in relation to a genotype containing it (Table 1). Although very few qualitative differences were observed in any of the four genotypes, differences in the levels of accumulation were observed for almost all of the compounds, indicating that the characteristic volatile profile of each genotype relies on quantitative rather than qualitative differences. Although it seemed reasonable to expect that hybrids would show that the levels of most volatiles are intermediate between those of the two parents, the results revealed that 56 of the 113 compounds were present at transgressive levels in at least one of the hybrids. Indeed, 40 compounds in the hybrids presented significantly higher levels than in any of their parents, and 16 compounds were present at statistically significantly lower levels. The remaining 57 volatiles were present at levels that were either intermediate or not significantly different from one of the parentals. The most extreme behavior was shown by ester compounds hexyl acetate (m 6), α-terpinyl acetate (m 13), or linalyl acetate (m 15), present in hybrid FxCh90 at levels of 30-fold or higher than in any of their parentals. Compounds such as ethyl heptanoate (m 86), 6-methyl-5-hepten-2-one (m 100), and 1-pentanol (m 101), which were present at different levels well above the threshold levels in both parentals, were not be detected in at least one of the hybrids, while 2,3pentanedione (m 98) was reduced 17-fold in the hybrids (Table 1). This transgressive behavior can be observed graphically for FxCh90 and FxCh77 in Figure 1 and SI Figure S2, respectively, where the volatiles that over- or underaccumulated in the hybrids if compared to their parents are shown in the three defined areas (overaccumulating zone, underaccumulating zone, and interparents zone). The statistical significance of these differences is shown in Table 1 and SI Figure S3. However, the transgressive behavior described for the levels of volatile compounds in these hybrids was not observed for other fruit quality traits (see those traits in the interparental zone in the plots, in Figure 2 and in SI Figure S4). A similar transgressive behavior in the volatile profile has been observed in a number of peach siblings derived from a cross also involving one hybrid as a parent39 and also in apple,40 which probably indicates that volatile traits are especially prone to this type of transgressive behavior. To globally verify whether the volatile profiles can be used to differentiate both hybrids from the parental genotypes and to identify the volatiles that contributed the most to the separation between the new materials generated by breeding and the parental genotypes, a PCA was performed using the complete data set. Based on the volatile profile, Figure 3 shows that all four genotypes are completely separated from each other. Principal component 1 accounts for the majority of total variance (53.6% of variance), and mainly separates the parent Chandler pummelo from the other three genotypes, and also the parent Fortune from their derived hybrids to a lesser extent. The relative position of the samples in the PCA space is not consistent with any of the hybrids, with a volatile phenotype intermediate shown between the two parentals. In fact,

Figure 2. Principal component analysis score plot based on a data set of 11 fruit quality traits. Principal components 1 and 2 are represented.

principal component 1 confirmed that both hybrids showed rather transgressive behavior, with a volatile profile opposed to that of the male parental Chandler pummelo, which was even more distant to Chandler than the other parental, Fortune mandarin. Of the two hybrids, FxCh90 was seen to be slightly more transgressive than FxCh77. An analysis of the loading plot revealed the compounds to be responsible for the separation between samples (Figure 4). The volatile compounds that most contributed to the first component were a group of monoterpenoid compounds (p-cymene, (Z)-ocimene, (E)linalool oxide, 2-carene; m 108, m 109, m 111, m 112, respectively) and (E,E)-2,4-nonadienal (m 110), which are almost exclusive of Chandler pummelo. This group of compounds also differentiated Chandler pummelo from Clemenules mandarin and Powell orange, as previously reported,13 and some have also been identified in the other few pummelo juice and flavedo studies.14,15,25,41 According to the literature, 2-carene has never been detected in orange and mandarin aroma juices, and (Z)-ocimene, linalool oxides, and (E,E)-2,4-nonadienal have been detected only very occasionally.1,11,14,21,22,42 In contrast, p-cymene has been frequently isolated in these juices.6,9,11,12,18,19,43 Fortune mandarin, and the two hybrids in particular, show higher levels of acetic esters octyl acetate (m 19), nonyl acetate (m 14), decyl acetate (m 20), α-terpinyl acetate (m 13), linalyl acetate (m 15), geranyl acetate (m 25), and citronellyl acetate (m 28), the monoterpene alcohol linalool (m 1), and long-chain lipid derivatives 1decanol (m 10) and dodecanal (m 24). Principal component 2 explained about 22.4% of variance and clearly separated Fortune from the other three genotypes (SI Figure S5). What differentiated Fortune from the rest was mainly the higher levels of propyl acetate (m 60) and a series of ethyl esters: ethyl 2-methylbutanoate (m 61), ethyl acetate (m 62), ethyl propanoate (m 59), ethyl butanoate (m 57), and ethyl nonanoate (m 64) (SI Figure S6). Principal component 3, accounting for only 10.2% of variance, revealed that it is possible to distinguish the two hybrids from each another in accordance with specific volatiles (Figure 3). According to the loading plot corresponding to PC3, hybrid FxCh90 was characterized by higher levels of acetate compounds heptyl acetate (m 9), octyl acetate (m 19), nonyl acetate (m 14), decyl acetate (m 20), methyl decanoate (m 18), α-terpinyl acetate (m 13) and (E)-carvyl acetate (m 23), and also long-chain lipid 11317



Article

Figure 3. Principal component analysis score plot based on 113 volatile compounds. Principal components 1 and 3 are represented.

Figure 4. Principal component analysis loading plot based on volatile composition for principal components 1 and 3. Each number corresponds to a particular volatile compound, as indicated in Table 1.

When the complete data set of fruit quality traits was used (Figure 2), the PCA showed that both hybrids displayed mostly an intermediate behavior between their parental genotypes for these traits, as revealed by principal component 1, which accounted for most of the total variability (75.8%). The HCA done over the same volatile data set confirmed that FxCh90 and FxCh77 shared the most similar volatile profile (Figure 5). Consistently with the PCA results, the volatile profile of the Fortune genotype in HCA clustering also fell between Chandler and the hybrids. HCA also allowed us to analyze the compounds to check if clustering revealed any

derivatives 1-octanol (m 17), decanal (m 42), and undecanal (m 43) (Figure 4). Hybrid FxCh77, however, was characterized by lower levels of these compounds, together with higher levels of monoterpenoids linalool (m 1), β-citronellol (m 2), perillaldehyde (m 3), α-terpineol (m 4), β-citronellal (m 5), geraniol (m 7), (Z)- and (E)-limonene oxides (m 44 and m 48, respectively), and geranial (m 67), norisoprenoids geranylacetone (m 65), and β-ionone (m 71), C5 and C6 lipid derivatives 1-penten-3-one (m 45), (E)-2-pentenal (m 47), 3-pentanone (m 72), and (Z)-3-hexen-1-ol (m 66) (Figure 4). 11318



Article

Figure 5. Hierarchical cluster analysis of both samples and volatile compounds. Samples grouped according to varieties: Ch, Chandler; F, Fortune; FxCh90 and FxCh77 hybrids. Volatiles grouped into clusters A, B, and C, and subclusters A1, A2, C1, and C2. Colors in the heatmap are related to fold change, in accordance with the scale shown at the bottom: red for higher levels; green for lower levels.

literature,13 (E)-2-decenal (m 56), 3-methylfuran (m 63), and ketones carvone (m 50) and dihydrocarvone (m 52). Seven ethyl esters, e.g., ethyl octanoate (m 58), ethyl 2-methylbutanoate (m 61), or ethyl acetate (m 62), were more abundant in the Fortune genotype, which is consistent with the results of the loading plots for PC2. According to the literature, these ethyl esters have been frequently identified in many Citrus juices.3,11,17,18,20,24,45 It should be noted that the Fortune samples analyzed herein were relatively rich in ethanol as compared to the other genotypes. This observation might explain the high levels of ethyl esters as they can be produced by esterification of ethanol with the acyl groups derived from fatty acid and amino acid metabolism.46 Finally, cluster C included the compounds that were more abundant in Chandler than in the other three genotypes. Subcluster C1 included the compounds found more abundantly in Chandler but also in hybrid FxCh77 (the most similar hybrid to Chandler), such as geranylacetone (m 65) or geranial (m 67). Subcluster C2 included the compounds that were more abundant in the Chandler genotype than in the other three genotypes. A number of aliphatic aldehydes (pentanal to nonanal, m 90, m 89, m 97, m 105, and m 94, respectively) and olefins (E)-2-heptenal, (E)-2-octenal, and (E,E)-2,4-nonadienal; m 107, m 85, and m 110, respectively), several aliphatic alcohols (1-pentanol, 1-hexanol, 1-heptanol, and 1-octen-3-ol; m 101, m 88, m 99, and m 92, respectively), and some aliphatic ketones (3-octanone, 2-nonanone, 2,3-pentanedione, 6-methyl5-hepten-2-one, and 1-octen-3-one; m 93, m 96, m 98, m 100, and m 106, respectively) abounded in this genotype if compared to the other three genotypes. The higher levels of aliphatic aldehydes (octanal, nonanal) can, in part, be responsible for peely and fatty notes of pummelo juices.15

underlying relationship between them. Compounds were organized by HCA into three clusters, namely, A, B, and C, with some subclusters (named A1, A2, C1, and C2), as shown in Table 1. The compounds in cluster A were generally present with higher values in the hybrids than in their parentals. All of these compounds have been generally described in Citrus juices.13,18 Subcluster A1 included the compounds generally present at the highest levels in hybrid FxCh77. These compounds are principally several monoterpene alcohols such as linalool (m 1), β-citronellol (m 2), or geraniol (m 7), and monoterpene aldehydes such as β-citronellal (m 5). Actually this latter compound is exclusive for the FxCh77 genotype and has been identified only in very few mandarins, orange, or hybrids juices.13,19,43,44 Subcluster A2 mainly included the compounds that accumulated preferentially in the FxCh90 genotype and included α-terpinyl acetate (m 13), nonyl acetate (m 14), neral (m 16), α-terpinene (m 27), 3-carene (m 36), or decanal (m 42). Interestingly, most of these compounds are aliphatic and monoterpene acetates and monoterpene hydrocarbons. The results from the compounds in cluster A indicated that the two hybrids showed higher levels than their parental genotypes of many monoterpenoid compounds, such as linalool (m 1), β-citronellol (m 2), geraniol (m 7), citronelly acetate (m 28), or α-phellandrene (m 31). The odor of these last compounds has been described as floral, fruity, or citrus,15 which are positive aromatic notes that can make these two hybrids more appealing to consumers. Nevertheless, a taste panel will be necessary to confirm this term. Cluster B was defined principally by the compounds found more abundantly in Fortune than in the other three genotypes. These include compounds such as propyl acetate (m 60), which has never been described in other Citrus genotypes in the 11319



Article

istics of their parents, which the genetic analysis neither predicted nor anticipated. The HS-SPME-GC-MS analysis of Citrus juice has proven to be a tool that allows genotype differentiation that can be used in the assessment of Citrus breeding programs.

The higher levels of both 3-octanone and 1-octen-3-one can confer a hint of mushroom to the characteristic pummelo juice aroma.30 These compounds were detected in Fortune and in hybrids FxCh90 and FxCh77 at almost trace levels. Several esters, such as some alkyl hexanoates (m 74, m 103), and compound 2-pentylfuran (m 91), were also present in larger quantities in Chandler than in Fortune or the hybrids. Moreover, as we noted in the analysis of the loading plots, some monoterpene compounds, such as p-cymene (m 108), (Z)-ocimene (m 109), 2-carene (m 112), or (Z)- and (E)linalool oxide (m 79 and m 111, respectively), were also more abundant in Chandler juices, and some were not even detected in the hybrids. Nevertheless, Chandler juice generally showed lower levels of monoterpene compounds than the other three genotypes. Regarding sesquiterpenes, α-humulene (m 77) and β-caryophyllene (m 78) (subcluster C1) were much more profuse in the Chandler genotype than in any of the other three genotypes here studied or in Powell orange and Clemenules mandarin, according to a previous study. 13 However, sesquiterpene compounds α-copaene (m 41) and nootkatone (m 76), which served to differentiate Chandler from Powell orange, Clemenules mandarin, or Fortune juices in a previous study,13 were herein found at similar levels in their hybrids FxCh90 and FxCh77. Therefore, these compounds are unsuitable to differentiate Chandler from their hybrids. The differential terpene profile can explain, at least partially, why pummelo juices exhibit the most differential volatile profile as compared to orange and mandarine juices, and a more similar profile to that of grapefruit.13,25,41 It is interesting to note that some volatile compounds described in the only other previous study into pummelo juice15 were not detected in our study (2ethylhexanol, dimethylsulfone, and (E)-2-dodecenal). This might be due to biological variability, differences in growth conditions, or the chemical nature of the fiber used for volatile extraction, among other causes. This study basically indicates that the differences in the volatile profile between the juice of the two Citrus parental genotypes (Chandler pummelo and Fortune mandarin) and their two hybrids are mainly quantitative. Each genotype exhibited a specific volatile profile that can be used for classification or identification purposes. As also noted in our previous study into Citrus aroma, there seems to be only a small number of Citrus genotype-specific compounds.13 Most remarkably, the two hybrids characterized herein showed a transgressive (i.e., not intermediate) volatile profile when compared to those of the parents, which completely differed from that of Chandler pummelo and was more similar to that of Fortune mandarin. FxCh90 is especially rich in aliphatic and monoterpene acetates, while FxCh77 contains higher levels of monoterpene hydrocarbons and monoterpene alcohols than any of their parents. In our case the most characteristic VOCs of the Fortune genotype are 3-methylfuran (m 63), (E)-2decenal (m 56), carvone (m 50), and dihydrocarvone (m 52), and some ethyl esters (m 53, m 57, m 58, m 59, m 61, m 62, and m 64). Finally, the juice of the Chandler genotype is more abundant in aliphatic compounds (aldehydes, alcohols, and ketones), some monoterpene hydrocarbons (p-cymene, (Z)ocimene, and 2-carene; m 108, m 109, and m 112, respectively), and sesquiterpenes, such as α-humulene (m 77) and βcaryophyllene (m 78). In conclusion, the volatile analysis platform used has allowed us to identify genotypes with differences in the aromatic profile that either enhance or inhibit some of the volatile character-

■

ASSOCIATED CONTENT

S Supporting Information *

Figures showing the total ion count chromatogram of hybrid FxCh90(Figure S1), values for the different metabolites and phenotype traits in hybrid FxCh77 in relation to the values in Fortune (Figure S2), Z-score plots showing the traits with transgressive behavior above and below the Fortune range (Figure S3), fruit quality traits for the four genotypes (Figure S4), principal component analysis score plot based on volatile composition for principal components 1 and 2 (Figure S5), and principal component analysis loading plot based on volatile composition for principal components 1 and 2 (Figure S6) and tables listing retention times of the identified compounds under the two different chromatographic conditions used (Table S1) and statistical significance and values of the Z-scores shown in Figure 1 and Figure S2 (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: + 34 96 3424000. Fax: + 34 96 3424001. E-mail: [email protected]. Present Address ⊥

Instituto de Biologı ́a Molecular y Celular de Plantas, CSIC Universidad Politécnica de Valencia, Ciudad Politécnica de la Innovación, Edificio 8 E, Ingeniero Fausto Elio, 46022 Valencia, Spain. Author Contributions ∥

J.L.R. and M.C.G.-M. contributed equally to this work.

Funding

This work has been supported by Projects GVPRE/2008/164 of Conselleria d’Educació of the Valencian Community and RTA2011-00132-C02 of INIA. M.C.G.-M. is contracted by the Fundación Agroalimed (Conselleria d’Agricultura of VC). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank I. Herrero and T. Caballero for laboratory assistance. We also thank A. Gutiérrez for networking activities. Volatile profiling was performed at the Metabolomic facilities of the Instituto de Biologı ́a Molecular y Celular de Plantas, CSIC (Spain).

■

ABBREVIATIONS USED VOCs, volatile organic compounds; HS-SPME, headspace solid phase microextraction; DVB/CAR/PDMS, divinylbenzene/ carboxen/poly(dimethylsiloxane); m, metabolite; PCA, principal component analysis (PC2, principal component 2; PC3, principal component 3); HCA, hierarchical cluster analysis

■

REFERENCES

(1) Moshonas, G. P.; Shaw, P. E. Quantitation of volatile constituents in mandarin juices and its use for comparison with orange juices by multivariate analysis. J. Agric. Food Chem. 1997, 45, 3968−3972.

11320



Article

(2) Buettner, A.; Schieberle, P. Evaluation of key aroma compounds in hand-squeezed grapefruit juice (Citrus paradisi Macfayden) by quantitation and flavor reconstitution experiments. J. Agric. Food Chem. 2001, 49, 1358−1363. (3) Buettner, A.; Schieberle, P. Evaluation of aroma differences between hand-squeezed juices from Valencia Late and Navel oranges by quantitation of key odorants and flavor reconstitution experiments. J. Agric. Food Chem. 2001, 49, 2387−2394. (4) Rouseff, R. L.; Perez-Cacho, P. R.; Jabalpurwala, F. Historical review of Citrus flavor research during the past 100 years. J. Agric. Food Chem. 2009, 57, 8115−8124. (5) Arena, E.; Guarrera, N.; Campisi, S.; Asmundo, C. N. Comparison of odour active compounds detected by gas-chromatography-olfactometry between hand-squeezed juices from different orange varieties. Food Chem. 2006, 98, 59−63. (6) Barboni, T.; Luro, F.; Chiaramonti, N.; Desjobert, J.-M.; Muselli, A.; Costa, J. Volatile composition of hybrids Citrus juices by headspace solid-phase micro extraction/gas chromatography/mass spectrometry. Food Chem. 2009, 116, 382−390. (7) Barboni, T.; Muselli, A.; Luro, F.; Desjobert, J.-M.; Costa, J. Influence of processing steps and fruit maturity on volatile concentrations in juices from clementine, mandarin and their hybrids. Eur. Food Res. Technol. 2010, 231, 379−386. (8) Tietel, Z.; Porat, R.; Weiss, K.; Ulrich, D. Identification of aromaactive compounds in fresh and stored ‘Mor’ mandarins. Int. J. Food Sci. Technol. 2011, 46, 2225−2231. (9) Miyazaki, T.; Plotto, A.; Goodner, K.; Gmitter, F. G., Jr. Distribution of aroma volatile compounds in tangerine hybrids and proposed inheritance. J. Sci. Food Agric. 2011, 91, 449−460. (10) Jordan, M. J.; Tillman, T. N.; Mucci, B.; Laencina, J. Using HSSPME to determine the effects of reducing insoluble solids on aromatic composition of orange juice. LWT−Food Sci. Technol. 2001, 34, 244−250. (11) Brat, P.; Rega, B.; Alter, P.; Reynes, M.; Brillouet, J.-M. Distribution of volatile compounds in the pulp, cloud, and serum of freshly squeezed orange juice. J. Agric. Food Chem. 2003, 51, 3442− 3447. (12) Pérez-López, A. J.; Carbonell-Barrachina, A. A. Volatile odour components and sensory quality of fresh and processed mandarin juices. J. Sci. Food Agric. 2006, 86, 2404−2411. (13) González-Mas, M. C.; Rambla, J. L.; Alamar, M. C.; Gutiérrez, A.; Granell, A. Comparative analysis of the volatile fraction of fruit juice from different Citrus species. PLoS One 2011, 6, No. e22016. (14) Shaw, P. E.; Goodner, K. L.; Moshonas, M. G.; Hearn, C. J. Comparison of grapefruit hybrid fruit with parent fruit based on composition of volatile components. Sci. Hortic. (Amsterdam, Neth.) 2001, 91, 71−80. (15) Cheong, M. W.; Liu, S. Q.; Zhou, W.; Curran, P.; Yu, B. Chemical composition and sensory profile of pomelo (Citrus grandis (L.) Osbeck) juice. Food Chem. 2012, 135, 2505−2513. (16) Bernet, G. P.; Fernandez-Ribacoba, J.; Carbonell, E. A.; Asins, M. J. Comparative genome-wide segregation analysis and map construction using a reciprocal cross design to facilitate citrus germplasm utilization. Mol. Breed. 2010, 25, 659−673. (17) Bylaite, E.; Meyer, A. S. Characterisation of volatile aroma compounds of orange juices by three dynamic and static headspace gas chromatography techniques. Eur. Food Res. Technol. 2006, 222, 176− 184. (18) Ruiz Perez-Cacho, P.; Rouseff, R. L. Fresh squeezed orange juice odor: A review. Crit. Rev. Food Sci. 2008, 48, 681−695. (19) Tomiyama, K.; Aoki, H.; Oikawa, T.; Sakurai, K.; Kasahara, Y.; Kawakami, Y. Characteristic volatile components of Japanese sour citrus fruits: Yuzu, Sudachi and Kabosu. Flavour Fragrance J. 2012, 27, 341−355. (20) Selli, S.; Cabaroglu, T.; Canbas, A. Volatile flavour components of orange juice obtained from the cv. Kozan of Turkey. J. Food Compos. Anal. 2004, 17, 789−796.

(21) Mahattanatawee, K.; Rouseff, R.; Valim, M. F.; Naim, M. Identification and aroma impact of norisoprenoids in orange juice. J. Agric. Food Chem. 2005, 53, 393−397. (22) Qiao, Y.; Xie, B. J.; Zhang, Y.; Fan, G.; Yao, X. L.; Pan, S. Y. Characterization of aroma active compounds in fruit juice and peel oil of Jinchen sweet orange fruit (Citrus sinensis (L.) Osbeck) by GC-MS and GC-O. Molecules 2008, 13, 1333−1344. (23) Minh Tu, N. T.; Thanh, L. X.; Une, A.; Ukeda, H.; Sawamura, M. Volatile constituents of Vietnamese pummelo, orange, tangerine and lime peel oils. Flavour Fragrance J. 2002, 17, 169−174. (24) Miyazaki, T.; Plotto, A.; Baldwin, E. A.; Reyes-De-Corcuera, J. I.; Gmitter, F. G., Jr. Aroma characterization of tangerine hybrids by gas-chromatography-olfactometry and sensory evaluation. J. Sci. Food Agric. 2012, 92, 727−735. (25) Cheong, M.-W.; Liu, S.-Q.; Yeo, J.; Chionh, H.-K.; Pramudya, K.; Curran, P.; Yu, B. Identification of aroma-active compounds in Malaysian pomelo (Citrus grandis (L.) Osbeck) peel by gas chromatography-olfactometry. J. Essent. Oil Res. 2011, 23, 34−42. (26) Elmaci, Y.; Altug, T. Flavor characterization of three mandarin cultivars (Satsuma, Bodrum, Clemantine) by using GC/MS and flavour profile analysis techniques. J. Food Qual. 2005, 28, 163−170. (27) Liu, C.; Cheng, Y.; Zhang, H.; Deng, X.; Chen, F.; Xu, J. Volatile constituents of wild Citrus Mangshanyegan (Citrus nobilis Lauriro) Peel oil. J. Agric. Food Chem. 2012, 60, 2617−2628. (28) Natale, D.; Mattiacci, L.; Pasqualini, E.; Dorn, S. Apple and peach fruit volatiles and the apple constituent butyl hexanoate attract female oriental fruit moth, Cydia molesta, in the laboratory. J. Appl. Entomol. 2004, 128, 22−27. (29) Villatoro, C.; Altisent, R.; Echeverría, G.; Graell, J.; López, M. L.; Lara, I. Changes in biosynthesis of aroma volatile compounds during on-tree maturation of ‘Pink Lady®’ apples. Postharvest Biol. Technol. 2008, 47, 286−297. (30) Cho, I. H.; Lee, S. M.; Kim, S. Y.; Choi, H.-K.; Kim, K.-O.; Kim, Y.-S. Differentiation of aroma characteristics of pine-mushrooms (Tricholoma matsutake Sing.) of different grades using Gas Chromatography-Olfactometry and sensory analysis. J. Agric. Food Chem. 2007, 55, 2323−2328. (31) Wan, X. M.; Stevenson, R. J.; Chen, X. D.; Melton, L. D. Application of headspace solid-phase microextraction to volatile flavour profile development during storage and ripening of kiwifruit. Food Res. Int. 1999, 32, 175−183. (32) Biniyaz, T.; Habibi, Z.; Masoudi, S.; Rustaiyan, A. Composition of the essential oils of Haplophyllum f urf uraceum Bge. ex Boiss. and Haplophyllum virgatum Spach. from Iran. J. Essent. Oil Res. 2007, 19, 49−51. (33) Edirisinghe, R. K. B.; Graham, A. J.; Taylor, S. J. Characterisation of the volatiles of yellowfin tuna (Thummus albacares) during storage by solid phase microextraction and GC-MS and their relationship to fish quality parameters. Int. J. Food Sci. Technol. 2007, 42, 1139−1147. (34) Dob, T.; Dahmane, D.; Gauriat-Desrdy, B.; Daligault, V. Volatile constituents of the essential oil of Ruta chalepensis L. subsp. Angustifolia (Pers.) P. Cout. J. Essent. Oil Res. 2008, 20, 306−309. (35) Poveda, J. M.; Sánchez-Palomo, E.; Pérez-Coello, M. S.; Cabezas, L. Volatile composition, olfactometry profile and sensory evaluation of semi-hard Spanish goat cheeses. Dairy Sci. Technol. 2008, 88, 355−367. (36) Gu, Y. Q.; Mo, M. H.; Zhou, J. P.; Zou, C. S.; Zhang, K. Q. Evaluation and identification of potential organic nematicidal volatiles from soil bacteria. Soil Biol. Biochem. 2007, 39, 2567−2575. (37) Neri, F.; Mari, M.; Brigati, S.; Bertolini, P. Fungicidal activity of plant volatile compounds for controlling Monilinia laxa in some fruit. Plant Dis. 2007, 91, 30−35. (38) Almenar, E.; Catalá, R.; Hernández-Muñoz, P.; Gavara, R. Optimization of an active package for wild strawberries bases on the release of 2-nonanone. LWT−Food Sci. Technol. 2009, 42, 587−593. (39) Sánchez, G.; Martínez, J.; Romeu, J.; García, J.; Monforte, A. J.; Badenes, M. L.; Granell, A. The peach volatilome modularity is 11321



Article

reflected at the genetic and environmental response level in a QTL mapping population. BMC Plant Biol. 2014, 14, 137. (40) Dunemann, F.; Ulrich, D.; Boudichevskaia, A.; Grafe, C.; Weber, W. E. QTL mapping of aroma compounds analysed by headspace solid-phase microextraction gas chromatography in the apple progeny ‘Discovery’ x ‘Prima’. Mol. Breed. 2009, 23, 501−521. (41) Sawamura, M.; Kuriyama, T. Quantitative determination of volatile constituents in the pummelo (Citrus grandis Osbeck forma Tosa-buntan). J. Agric. Food Chem. 1988, 36, 567−569. (42) Rinaldi, M.; Gindro, R.; Barbeni, M.; Allegrone, G. Pattern recognition and genetic algorithms for discrimination of orange juices and reduction of significant components from headspace solid-phase microextraction. Phytochem. Anal. 2009, 20, 402−407. (43) Tounsi, M. S.; Wannes, W. A.; Ouerghemmi, I.; Jegham, S.; Njima, Y. B.; Hamdaoui, G.; Zemni, H.; Marzouk, B. Juice components and antioxidant capacity of four Tunisian Citrus varieties. J. Sci. Food Agric. 2011, 91, 142−151. (44) Jordán, M. J.; Goodner, K. L.; Castillo, M.; Laencina, J. Comparison of two headspace solid phase microextraction fibres for the detection of volatile chemical concentration changes due to industrial processing of orange juice. J. Sci. Food Agric. 2005, 85, 1065−1071. (45) Plotto, A.; Margaría, C. A.; Goodner, K. L.; Baldwin, E. A. Odour and flavour thresholds for key aroma components in an orange juice matrix: Esters and miscellaneous compounds. Flavour Fragrance J. 2008, 23, 398−406. (46) Tietel, Z.; Lewinsohn, E.; Fallik, E.; Porat, R. Elucidating the roles of ethanol fermentation metabolism in causing off-flavors in mandarins. J. Agric. Food Chem. 2011, 59, 11779−11785.

■

NOTE ADDED AFTER ASAP PUBLICATION An additional Supporting Information file has posted for this paper on November 10, 2014.

11322


Fruit Volatile Profiles of Two Citrus Hybrids Are Dramatically Different

Recommend Documents