Biosynthesis of fatty acids-derived volatiles in ‘Hass’ avocado is modulated by ethylene and storage conditions during ripening

Scientia Horticulturae 202 (2016) 91–98

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Biosynthesis of fatty acids-derived volatiles in ‘Hass’ avocado is modulated by ethylene and storage conditions during ripening Miguel García-Rojas a , Alejandra Morgan b , Orianne Gudenschwager a , Sofía Zamudio a , Reinaldo Campos-Vargas b , Mauricio González-Agüero a , Bruno G. Defilippi a,∗ a b

Instituto de Investigaciones Agropecuarias, INIA-La Platina, Santa Rosa 11610, Santiago, Chile Universidad Andres Bello, Centro de Biotecnología Vegetal, Facultad de Ciencia Biológicas, República 217, Santiago, Chile

a r t i c l e

i n f o

Article history: Received 30 September 2015 Received in revised form 11 February 2016 Accepted 15 February 2016 Keywords: Persea americana Aroma Cold storage Volatile Fatty acids and ethylene

a b s t r a c t The aroma in avocados (Persea americana Mill. cv. ‘Hass’) is mainly defined by fatty acids-derived volatile compounds that change according to the fruit maturity stage which is modulated by ethylene. In order to understand the changes in fatty acid substrates and gene expression involved in the synthesis of the key aroma-volatile compounds in avocado after harvest, we performed two trials using avocados harvested with 11% oil content. In the first trial avocados were ripened immediately after harvest at 20 ◦ C until reaching the ready-to-eat stage, and in the second trial, fruit were stored at 5 ◦ C for 30 days and then ripened at 20 ◦ C. In addition, to assess the ethylene effect in the volatile compounds and transcript levels measured, a 100 ␮l L−1 ethylene application was carried out, at harvest or after storage for trial 1 and 2, respectively. The concentration of the key volatile compounds and fatty acids were performed by gas chromatography, and the changes in the expression of genes related to lipoxygenase derived compounds were measured by q-PCR. The results obtained indicated that ethylene modulated the production of linolenic acid and hexanal, whereas at the gene expression level, only PamLOX transcript changes responded to ethylene application, although its changes were maturity/ripening dependent. The cold storage did not generate significant changes in fatty acids and gene expression levels, but a decreased in the concentration of hexanal during the ripening was observed. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The ripening of climacteric fruit, such as avocado, involves a series of coordinated metabolic events that affect the morphology, biochemistry, physiology and gene expression (Giovannoni, 2001), and these alterations modify many quality attributes, such as the color, texture and flavor of the fruit (Cai et al., 2006). The hormone that modulates a wide range of responses during fruit ripening and senescence in climacteric fruit is ethylene (Khan, 2006). In avocado, the “peak” in the production of ethylene during ripening has been specifically associated with an increase in the respiratory rate, softening of the flesh, color change and development of aroma compounds (Kassim et al., 2013; Zamorano et al., 1994). In many fruits, the use of ethylene inhibitors has demonstrated that volatile compounds are affected differently. For example, in avocado, Pereira et al. (2013) demonstrated that the ethylene inhibitor 1-MCP delayed ripening and mainly affected the flavor of the fruit

∗ Corresponding author. Fax: +56 22 7575104. E-mail addresses: bdefi[email protected], bdefi[email protected] (B.G. Defilippi). http://dx.doi.org/10.1016/j.scienta.2016.02.024 0304-4238/© 2016 Elsevier B.V. All rights reserved.

in the middle stage of ripening. Cold storage is the most effective tool to reduce the rate of respiration and ethylene production in fruit, thus reducing the rates of metabolic processes and extending the postharvest life of the fruit (Kassim et al., 2013). Aroma-volatile compounds have a well-established role in determining the characteristic flavor of a wide variety of fruits, including apple (Malus domestica), melon (Cucumis melo), banana (Musa sp.), mango (Mangifera indica), papaya (Carica papaya L.) and tomato (Lycopersicon esculentum Mill) (Defilippi et al., 2009; Lewinsohn et al., 2001). Studies of strawberry (Fragaria x ananassa) fruit have demonstrated that the storage temperature significantly affects aroma compounds (Ayala-Zavala et al., 2004). There are also studies in climacteric fruits showing that during ripening the production of aroma volatiles are mainly derived from fatty acids modulated by ethylene (Defilippi et al., 2009; Yahia, 1994). These free fatty acids, or those liberated by lipase activity and further metabolized by ␤-oxidative enzymes and/or lipoxygenase (LOX), are generally regarded as being the main precursors of esters, alcohols and aldehydes (Fellman et al., 2000). These compounds change as the fruit develops; for example, short-chain aldehydes are predominant in immature avocados, particularly hexanal

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Fig. 1. Ethylene production rate of the ‘Hass’ avocado. The fruits were evaluated after reaching each range of firmness at 20 ◦ C, in (A) harvest trial and (B) cold storage trial. Each concentration value is the mean of four fruits that were individually measured ± standard errors. The fruits were treated with 100 ␮l L−1 of ethylene for 24 h at 20 ◦ C. Each firmness ranges corresponds at: F1 (150–250N); F2 (50–150N) and F3 (1–50N). Values followed by different letters within the same firmness group were determined to be significantly different at P ≤ 0.05 by the LSD test.

and E-2-hexenal compounds; then their concentrations decrease during maturation, contributing to the decline of the herbaceous aroma (El-Mageed, 2007; Obenland et al., 2012). Reduction in the amount of hexanal and E-2-hexenal has been associated with the low expression of LOX, hydroperoxide lyase (HPL) and alcohol dehydrogenases (ADH) (Zhang et al., 2010). LOX belongs to a multigene family that catalyzes the incorporation of molecular oxygen into polyunsaturated fatty acids, such as linoleic and linolenic acids (Chen et al., 2004), and ADH participates in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters (Speirs et al., 1998). The tomato ADH2 enzyme is one of two ADH enzymes described, having a higher activity in later stages of ripening, combined with the increase in flavor volatiles in the fruit. This suggests that ADH may play an important role in flavor development (Longhurst et al., 1990). During the development of ‘Hass’ avocado, the concentrations of some volatile compounds, such as acetaldehyde, nonanal methyl acetate, b-myrcene and 2, 4-heptadienal increase (Obenland et al., 2012). In late harvest fruits and during ripening, the volatiles 1-penten-3-ol (alcohol) and 1-penten-3-one (ketone) exhibit significant increases in production (Obenland et al., 2012). In contrast, ripe or overripe cv. ‘Fuerte’ avocados had higher levels of terpenes and esters than less mature fruit (El-Mageed, 2007). This work focuses on the synthesis of fatty acid volatile derivatives contributing to the aroma in avocado after storage. Being cold storage a common commercial practice for this fruit affecting the organoleptic quality at the consumer level, the role played by cold storage and ethylene on evolution of quality parameters of fatty acid volatile was studied. Additionally, because there are few studies regarding the behavior of volatile compounds in avocado, and even less at the level of transcripts, we study the 6-carbon aldehydes, mainly hexanal and E-2-hexenal, which are produced from linolenic and linoleic acids in the synthetic pathway of lipoxygenase. 2. Materials and methods 2.1. Plant material Avocado fruit (Persea americana Mill. cv. Hass) were harvested from a commercial orchard located in Melipilla, Metropolitan Region, Chile. The fruit size and oil content were used as the harvesting indexes. Medium-sized fruit were selected and transported to the Postharvest Laboratory facility at the Institute of Agricultural Research (INIA). The average dry matter and oil contents of twenty

avocado fruit were 26.5% and 11%, respectively. For the postharvest storage experiments, two trials were performed: I. the fruit was ripened immediately after harvest at 20 ◦ C and 45% relative humidity (RH) (harvest trial) and II. the fruit was stored at 5 ◦ C and 92% RH for 30 days with subsequent exposure at 20 ◦ C until the fruit was ripe (cold storage trial). For ethylene application, the fruit was stored in a container and 100 ␮l L−1 of ethylene was applied for 24 h at 20 ◦ C. After treatment the container was opened and ventilated for 1 h before storage in both trials. 2.1.1. Sampling procedure Due to the important effect of exogenous ethylene application on the ripening process; sampling was based on segregation of fruit according to three flesh firmness ranges. Flesh firmness was selected since it is a suitable parameter indicating the progress of ripening in avocado (Bower and Cutting, 1988). Despite skin color was modulated by ripening, only showed significant changes in later ripening stages, therefore it was not used for assessing ripening evolution in earlier stages with firm fruit. Thus, during ripening at 20 ◦ C, fruit were sorted according to the degree of softening into three firmness ranges: F1 (150–250N), F2 (50–150N) and F3 (1–50N). After reaching each firmness range, each fruit was evaluated for the different quality parameters, and the flesh of six fruits from each condition was frozen in liquid nitrogen, homogenized and stored at −80 ◦ C until use for volatile, fatty acid and molecular analyses. 2.1.2. Fruit quality parameters Avocados were evaluated for dry matter content; physiological disorders, including external damage and internal browning; color changes; and flesh firmness. Dry matter content was determined by drying the samples in an oven at 103 ◦ C for 24 h until a constant weight was reached (Gudenschwager et al., 2013). External damage was assessed after cold storage at 5 ◦ C, and internal browning and flesh firmness were assessed after cold storage and again after ripening at 20 ◦ C. For external and internal damage a hedonic scale from 1 to 5 (1: no occurrence, 2: slight, 3: moderate, 4: moderately severe and 5: severe) was used (García-Rojas et al. (2012)). Skin color was assessed visually using a hedonic scale with scores from 1 (green) to 5 (black). Flesh firmness was measured at the equatorial levels at opposite sites using a penetrometer (Effegi, Milan, Italy) equipped witha 4- or 8-mm plunger tip. 2.1.3. Respiration and ethylene production rate For measurement of ethylene, avocados were placed in a 1.6 L plastic container and sealed for approximately 3 h at 20 ◦ C, thereby

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Fig. 2. Gene expression analysis of PamLOX transcripts in avocado for the harvest trial (A) and cold-storage trial (B). The fruits were treated with 100 ␮l L−1 of ethylene for 24 h at 20 ◦ C. Each firmness ranges corresponds at: F1 (150–250N); F2 (50–150N) and F3 (1–50N). The transcript accumulation of PamLOX was determined by qPCR in fruit at different firmness ranges. The relative abundance of each mRNA was normalized to the PamTCPB gene. Bars in the graphs correspond to the standard error (SE) from six biological samples, assayed in duplicated. Different letters within each firmness group represent significant differences at P ≤ 0.05 as determined by the LSD test.

avoiding excessive accumulation of carbon dioxide. Then, 1 mL of gas was taken from the headspace and injected into a gas chromatograph (Shimadzu GC 8A, Tokyo, Japan) equipped with an alumina column (Supelco 80/100 Porapak custom column of dimensions 75 cm × 5 mm × 3 mm) and a flame ionization detector (FID). The oven and injector temperatures were 40 ◦ C and 150 ◦ C, respectively. The results were expressed in ␮L C2 H4 kg −1 h −1 . The concentrations of carbon dioxide in the same plastic container was measured by taking 1 mL of gas from the head space and injecting it in a gas analyzer (PBI-Dansensor Checkmate 9900, Ringsted, Denmark). Results were expressed in ml CO2 kg −1 h −1 . 2.1.4. Oil extraction and quantification of fatty acids Using the methodology described by Meyer and Terry (2008), 1 g of each sample was lyophilized and homogenized, and 30 mL of hexane was subsequently added before incubating for 1 min at room temperature. Then, the sample was vacuum filtered with a Buchner flask and a funnel with 0.1-mm filter paper (FisherQL100). The powdered residue was recovered from the filter paper and washed with 20 mL of hexane. The sample was then evaporated in a rotary evaporator at 49 ◦ C under pressure. The recovered (oil) sample was weighed and stored at −80 ◦ C until it was analyzed. Transesterification was done by adding 3 mL of methanolic 0.3 N sodium metoxide to the avocado oil extract diluted in hexane. The composition of fatty acids was obtained from the oil by gas chromatography using the official A.O.C.S. Ce 1-62 (1993) method. Gas chromatography was performed in a Clarus 500 gas chromatograph (using H2 as the carrier gas). The capillary column used was of SPTM 2560 fused silica (length 75 m and internal width 0.18 mm with a film thickness of 0.14 ␮m). The oven temperature was programmed from 160 to 220 ◦ C with a heating rate of 2 ◦ C min. The temperature of the injector and detector was fixed at 250 ◦ C. Calibration curves and response factors were calculated for the different fatty acids already reported in avocado (oleic 18:1, palmitic 16:0, palmitoleic 16:1, stearic 18:0, linoleic 18:2, linolenic 18:3). All standards were obtained from Sigma–Aldrich. 2.1.5. Isolation of volatile compounds using solid-phase microextraction (SPME) Ten grams of the avocado mesocarp were homogeneized with an Ultra-Turrax T-25 homogenizer (IKA, Germany) with 20 mL of a saturated sodium chloride solution. Subsequently, 10 mL of the homogenate was transferred to 20 mL vials, 0.2 mL of the internal standard 1-octanol at a concentration of 1 ␮L mL−1 was added, and the vials were sealed. Then, a 65 ␮-diameter polydimethylsilox-

ane/divinylbenzene (PDMS/DVB) fiber was exposed to the open space of the vial for 30 min at 50 ◦ C with agitation. Finally, the fiber was injected into a Clarus 500 gas chromatograph (PerkinElmer, USA) equipped with an SPB-5 capillary column (Supelco, 30 m, 0.25 mm diameter, 0.25 ␮m thick film), a flame ionization detector at 250 ◦ C, an injector at 250 ◦ C and an oven with a temperature gradient of 40–190 ◦ C for sample analysis. The compounds were identified by comparing the retention times with those of authentic standards. Quantification was performed using external calibration curves from authentic standards for each compound identified. 2.1.6. Ribonucleic acid (RNA) isolation and complementary deoxyribonucleic acid (cDNA) synthesis Total RNA was isolated from 3–4 g of frozen tissue using the modified hot borate method (Gudenschwager et al., 2013). The quantity and quality of the RNA were assessed with a Qubit® 2.0 fluorometer (InvitrogenTM by Life Technologies) by measuring the A260 /280 ratio and by electrophoresis on a 1.2% formaldehydeagarose gel. The first strands of cDNA were obtained by reverse transcription reactions with 2 ␮g of total RNA as the template using MMLV-RT reverse transcriptase (Promega, Madison, WI) and oligo dT primers according to standard procedures. The concentration of cDNA was assessed by measuring the absorbance at 260 nm. Each cDNA sample was diluted to 50 ng ␮L−1 prior to use in the real-time quantitative PCR (qPCR) assays. 2.1.7. Real-time qPCR assays The genes analyzed in this study were the following: PamLOX (lipoxygenase, GenBank AD001673); PamADH1 (alcohol dehydrogenase 1, GenBank KT246104); PamADH2 (alcohol dehydrogenase 2, GenBank KT246105); PamADH3 (alcohol dehydrogenase 3, GenBank KT246106); and PamTCPB (T-complex protein 1 subunit beta, GenBank KT246107). The transcript abundance was analyzed via real-time PCR with the LightCycler® 96 system from Roche (Roche Diagnostics, Mannheim, Germany) using SYBR Green to measure the amplified RNA-derived DNA products as described in García-Rojas et al. (2012). Gene-specific primers were designed using the Primer Premier 5.0 software package (Premier Biosoft International, Palo Alto, CA) and were synthesized by IDT (Integrated DNA Technologies). The primer sequences used in this work were the following: PamLOX(F) 5 -CGACTCCATGCAGCAGTCAAC3 , PamLOX(R) 5 -GATCTCCACCAGCTTCTTTCCA-3 , PamADH-1(F) 5 -ATCCAAAGGATCACAATCGACCA-3 , 5 -ATTGGCTTTGTCATGAAAACAGC-3 , PamADH-1(R) PamADH-2(F) 5 -TGTTGAGATGACTGGTGGAGGA-3 ,

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PamADH-2(R) 5 -CAGATCAGAGCGTGGCTTGTAG-3 , PamADH3(F) 5 -TTCAAGAGCCGTTCACAAGTCC-3 , PamADH-3(R) 5 -AAATAAAGCACGCCTAATACTCAA-3 , PamTCPB(F) PamTCPB(R) 5 5 -CGACAGTAGGGTTTTGCTTGGT-3 ,  ATGATTGTTGGTATCGCCTGGAG-3 . 2.1.8. Experimental design and statistical analysis A fully randomized experimental designed was used considering four replicates of 30 avocados per treatment. For quality attributes, 10 fruits per replicate were used. qPCR was performed on each of the six biological samples in duplicate. The gene expression values were normalized to the PamTCPB expression, similar to Vitis vinifera (González-Aguero et al., 2013). Finally, the data were subjected to statistical analyses of variance, and the means were separated by a least significant difference (LSD) test at 5% significance using the Statgraphics Centurion Plus 5 software package (Manugistics Inc., Rockville, USA). 3. Results and discussion 3.1. Selection of firmness Avocado flesh firmness is one of the most important parameters that reflects the progression of ripening. Thus, it was used in this study to compare the different treatments at the same stage of ripeness during storage at 20 ◦ C. Three firmness ranges which were representative of the evolution of fruit softening from a high degree of firmness (F1) to ready-to-eat fruit (F3), were determined. Table 1 lists the number of days to reach the firmness range for each treatment at 20 ◦ C within each trial. Fruit ripened at 20 ◦ C immediately after harvest took a longer time to reach each firmness range than fruit ripened after storage. This behavior is mainly caused by the advanced ripening stage of the fruit after 40 days at 5 ◦ C, increasing softening rate and color change development (data not shown). In contrast, in the ethylene treatments, the firmness range was reached in a shorter period of time than the control fruit, especially in the harvest trial at 20 ◦ C, in which the time to reach F3 was shortened by 10 days for the ethylene-treated fruit. These results are consistent with studies in which avocado stimulated with ethylene exhibited increased flesh softening (Adkins et al., 2005; Feng et al., 2000; Jeong et al., 2003; Pereira et al., 2013). In addition to the flesh firmness, changes in other quality attributes, such as peel color, were also observed in the ethylene treated avocados. No incidence of physiological disorders was observed in this study (data not shown), similar to the results of Zauberman et al. (1988). 3.2. Respiration and ethylene production rate For the harvest trial, the rate of ethylene production immediately after harvest increased concomitantly with a decrease in flesh firmness, with a peak in the F2 range (Fig. 1A) and no differences among treatments within the same group of firmness. In the cold-storage trial, a similar behavior for ethylene production was observed, with a more rapid progression of ripening compared with the control fruit, with the exception of the ethylene-treated fruit (Fig. 1B). Based on the major impact of this treatment in stimulating the ripening of avocado and the relatively lower values of ethylene production compared with the control fruit for F2 and F3, it can be hypothesized that the ethylene peak occurred in the early stages of ripening with firmer fruit, and the peak could not be identified using the static sampling procedure used in this study. Respiration increased for all treatments with the progression of ripening in both trials, without significant changes among treatments (data not shown). Previous studies showed that ethylene ripened avocado had a characteristic pattern of respiration for climacteric fruits, coinciding also with an increase in ethylene production rate and

advanced fruit ripening (Feng et al., 2000; Bill et al., 2014), similar to that observed in both trials for control fruit (Fig. 1). 3.3. Fatty acid content The concentration profiles of five major unsaturated fatty acids detected in samples were measured from the two trials. Oleic (18:1) acid was the major fatty acid in the ‘Hass’avocado flesh, independent of the trial or treatments tested (Table 2), followed in decreasing order of concentration by palmitic (16:0), linoleic (18:2), palmitoleic (16:1) and linolenic (18:3) acids, similar to the results of previous studies (Eaks, 1990; Meyer and Terry, 2008; Ozdemir and Topuz, 2004). Among the unsaturated fatty acids, linolenic and linoleic acids are involved in the production of volatile compounds. A small reduction during softening was observed in the concentration of linolenic for ethylene-treated fruit from the harvest trial (Table 2). The significant reduction from 109 to 6 g/mL between firmness groups F2 and F3 indicates that linolenic acid (18:3) is used either as a substrate in a metabolic pathway (i.e., aroma volatile compounds) or as a precursor of an important regulator (e.g., jasmonates and phosphoinositides), as suggested by Pedreschi et al. (2014) and Song and Bangerth (2003). In the case of linoleic acid (18:2), we did not observe significant differences among treatments or trials (Table 2). Linolenic and linoleic fatty acids, as free fatty acids or liberated by lipase activity and further metabolized by ␤-oxidative enzymes and/or lipoxygenase, are generally regarded as the main precursors of volatile esters, alcohols and aldehydes produced by apple fruit during development and maturation (Fellman et al., 2000). Further, in transgenic apples suppressed in ethylene synthesis, the exogenous application of ethylene during ripening caused an increased in fatty acids concentrations and the fatty acids-derived volatile compounds (Defilippi et al., 2005). Villa-Rodriguez et al. (2011) studied the effect of the maturity stage on the content of fatty acids in ‘Hass’ avocado, observing variation in the fatty acids content during ripening due to oxidative degradation (Richard et al., 2008). However, Meyer and Terry (2008) suggested that fatty acids would not be related to the ripening event in avocado because no considerable changes in response to ethylene scavenger treatment were observed, and several factors, such as cultural practices, growing location, transportation and storage conditions can influence ripening (Landahl et al., 2009). 3.4. Changes in aroma-volatile compounds during maturity The profile of the volatile compounds was determined in mature and ripe ‘Hass’ avocado. Of the 15 identified aroma volatiles, only hexanal and E-2-hexenal exhibited significant changes in concentration during ripening (Supplementary Table 1). These two volatile compounds have been described as providing a particular ‘grassy aroma’ with a low aroma threshold (Obenland et al., 2012). Table 3 lists the changes in the concentrations for both compounds during fruit growth, with a peak for hexanal in the early stages of development and with a significant reduction until the fruit reached maturity at harvest. E-2-hexenal did not exhibit major changes during fruit development, and it maintained lower concentrations than hexanal. For both trials, there was a significant reduction in the concentrations of both compounds as ripening progressed, especially for hexanal (Table 4). Fruit from the cold-storage trial had a smaller concentration and a greater reduction in the hexanal concentration than the fruit from the harvest trial. In terms of treatments, exogenous application of ethylene caused a major reduction in the hexanal levels between the F1 and F2 firmness ranges in fruit from the harvest trial, which could be related to the differences in the advancement of the ripening stage (Pech et al., 2012).

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Fig. 3. Gene expression analysis of three isoforms of gene PamADH in avocado for the harvest trial (A–C) and cold storage trial (D–F). The fruits were treated with 100 ␮l L−1 of ethylene for 24 h at 20 ◦ C. Each firmness ranges corresponds at: F1 (150–250N); F2 (50–150N) and F3 (1–50N). The accumulation of PamADH1 (A–D), PamADH2 (B–E) and PamADH3(C–F) was measured by qPCR in fruit at different firmness stages. The relative abundance of each mRNA was normalized to that of the PamTCPB gene. Bars in the graphs correspond to the standard error (SE) from six biological samples, assayed in duplicated. Different letters within each firmness group represent significant differences at P ≤ 0.05 as determined by the LSD test. Table 1 Time (days) required to reach the flesh firmness ranges during ripening at 20 ◦ C for both trials. Harvest triala

Firmness ranges (N)

F1 F2 F3

150–250 50–150 1–50

Cold storage triala

Control

Ethyleneb

Control

Ethyleneb

9 12 19

7 8 9

6 7 8

2 3 4

a Time (days) the fruits took to reach each range of firmness at 20 ◦ C for the harvest trial (fruit was ripened at 20 ◦ C) and cold storage trial (fruit was stored at 5 ◦ C for 30 days with subsequent storage at 20 ◦ C until the fruit was ripe). The value represents the means of four replicates of six fruits for each treatment. N: Newton. b 100 ␮l L−1 of ethylene were applied for 24 h at 20 ◦ C.

Table 2 Changes in concentration of the main fatty acids identified in “Hass” avocado mesocarp at each flesh firmness range during ripening. Harvest trial1 Fatty acids (g/ml)4

Cold storage trial2 Ethylene3

Control

89.0 ± 15.1 24.8 ± 5.6b 29.2 ± 1.4b

44.0 ± 4.5 56.5 ± 10.2a 42.5 ± 8.8a

43.2 ± 8.3 42.2 ± 11.1a 64.0 ± 9.3a

58.7 ± 8.7a 45.4 ± 8.1a 40.9 ± 7.5a

F1 F2 F3

10.5 ± 3.9a 4.1 ± 1.1a 4.1 ± 0.6a

5.3 ± 1.1a 8.0 ± 0.1a 4.6 ± 0.9a

6.0 ± 0.7a 6.6 ± 1.6a 8.1 ± 1.9a

8.1 ± 1.1a 5.9 ± 1.0a 5.1 ± 1.2a

Oleic (18:1)

F1 F2 F3

172.6 ± 21.1a 101.5 ± 24.3a 115.9 ± 19.8a

115.6 ± 12.7a 177.0 ± 6.6a 166.4 ± 9.5a

155.4 ± 8.6a 155.7 ± 9.8a 166.0 ± 13.7a

160.7 ± 7.8a 114.4 ± 11.3a 131.8 ± 6.0a

Linoleic (18:2)

F1 F2 F3

60.8 ± 4.1a 26.5 ± 3.5b 28.5 ± 2.5b

32.6 ± 8.4a 54.2 ± 2.1a 42.9 ± 4.2a

41.9 ± 4.4a 43.6 ± 8.0a 44.1 ± 6.4a

47.5 ± 4.2a 35.0 ± 4.9a 32.1 ± 4.5a

Linolenic (18:3)

F1 F2 F3

8.4 ± 1.9a 4.0 ± 1.3a 5.2 ± 1.6a

65.4 ± 16.9a 108.7 ± 4.2a 6.1 ± 1.8b

5.5 ± 0.7a 5.6 ± 1.3a 5.7 ± 1.5a

6.6 ± 0.5a 5.1 ± 0.2ab 4.4 ± 0.6b

Firmness ranges (N)

Control

Palmitic (16:0)

F1 F2 F3

Palmitoleic (16:1)

a

a

Ethylene3 a

Harvest trial: fruit was ripened at 20 ◦ C. Cold storage trial: fruit was stored at 5 ◦ C for 30 days with subsequent storage at 20 ◦ C until the fruit was ripe. Each concentration value is the mean of three replicates ± standard errors. The means with different superscript letters within the same column between lines were determined to be statistically significant at P ≤ 0.05 by the LSD test. N: Newton. Each firmness ranges corresponds at: F1 (150–250N); F2 (50–150N) and F3 (1-50N). 3 100 ␮l L−1 of ethylene were applied for 24 h at 20 ◦ C. 4 Concentration in 100 g of lyophilized sample. 1 2

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Table 3 Aroma-volatile concentration in ‘Hass’ avocado mesocarp evaluated during fruit growth before harvest. Aroma volatile (pmol Kg−1 )

Sampling date

Hexanal E-2-Hexenal

January

April

July

October

March

6600 ± 50b 1900 ± 300a

12,700 ± 1,400a 1100 ± 300b

12,300 ± 700a 1500 ± 100ab

6400 ± 200b 1000 ± 100b

1100 ± 200c 900 ± 100b

Values represent the means of three replicates ± standard errors. Each value followed by different letter for a given compound in the same row was determined to be significantly different at P ≤ 0.05 by the LSD test. Tabla 4 Changes in the concentration of key aroma-volatile during ripening of ‘Hass’ avocado without (harvest trial) or with storage (cold storage trial) at 5 ◦ C. Harvest trial1 −1

Cold storage trial2

Firmness ranges(N)

Control

Ethylene

Control

Ethylene3

Hexanal

F1 F2 F3

2200 ± 300a 1500 ± 300ab 1100 ± 100b

2200 ± 100a 320 ± 50b 120 ± 10b

1300 ± 100a 160 ± 10b 20 ± 2b

400 ± 10a 200 ± 15b 260 ± 25b

E2Hexenal

F1 F2 F3

1000 ± 100a 600 ± 80b 130 ± 10c

900 ± 150a 700 ± 150a 1000 ± 100a

610 ± 100a 990 ± 100a 30 ± 5b

600 ± 100a 200 ± 10b 500 ± 20a

Aroma volatiles (pmol Kg

)

3

Harvest trial: fruit was ripened at 20 ◦ C. Cold storage trial: fruit was stored at 5 ◦ C for 30 days with subsequent storage at 20 ◦ C until the fruit was ripe. Values represent the means of three replicates ± standard errors. Each value followed by different letter for a given compound within the same column between lines is significantly different at P ≤ 0.05 by the LSD test. N: Newton. Each firmness ranges corresponds at: F1 (150–250N); F2 (50–150N) and F3 (1–50N). 3 100 ␮l L−1 of ethylene were applied for 24 h at 20 ◦ C. 1 2

We studied two of the most abundant fatty acid-derived shortchain volatiles present in avocado (Obenland et al., 2012), which are associated with fruit development (Schwab et al., 2008; Chen et al., 2004). Pereira et al. (2013) observed that in cv. ‘Simmonds’, a West Indian avocado, the high emissions of volatiles resulted from the action of lipoxygenase (Chen et al., 2004) on linolenic acid (hexanal) and linoleic acid (cis-3-hexenal and cis-3-hexen-1ol). In general, C6 aldehydes are found in higher concentrations in the earlier stages of fruit development because of their role in plant defense (Bate and Rothstein, 1998), but both compounds are predominant in ‘Hass’ avocado, either during fruit development or in the ripening period. The concentrations decrease as the firmness of the fruit decreases (Table 4). In other fruits, this reduction in aldehydes as ripening progresses allows for contribution to the overall aroma from other volatile compounds, such as alcohols and esters that have constant concentrations during ripening (ElMageed 2007; Obenland et al., 2012; Pereira et al., 2013). When fruits were exposed to a cooling period (cold-storage trial), a reduction in hexanal and E-2-hexenal was observed with the progression of ripening in the control fruit (Table 4). This would suggest that these compounds could play a role in the synthesis of other aroma volatiles in avocado (Obenland et al., 2012; Schwab et al., 2008), which needs to be further demonstrated. Under the exogenous application of ethylene in the cold-storage trial, no major changes were observed, and steady state levels were maintained among the firmness ranges (Table 4). 3.5. Gene expression analyses The lipoxygenase pathway is important in the production of volatile aromas in fruits (Zhang et al., 2009, 2010). LOXs exhibit changes in enzyme activity and gene expression levels during maturity relative to ethylene response, depending on the species and variety studied (Leisso et al., 2013; Schaffer et al., 2007; Zhang et al., 2011; Schiller et al., 2015). In this work, the PamLOX expression increased as the fruit ripening progressed (Fig. 2A and B) as observed in other species (Liu et al., 2011). In fruit from the harvest trial, there were no significant differences among treatments within the same firmness range, except for the F3 group (ripe), in which a 3-fold decrease in the expression of this transcript was

observed in ethylene-treated fruit compared with control avocado (Fig. 2A). This change was concomitant with the pattern of hexanal production (Table 4) and linolenic acid concentration (Table 2) at the same firmness, which corresponded to the ready-to-eat stage. However, after a cold storage period of 30 days at 5 ◦ C, there was an increase in the transcript expression for all treatments until the F3 firmness range was reached (Fig. 2B), suggesting that ethylene modulates the transcription of this gene in the final stages of ripening (Chen et al., 2004). Zhang et al. (2010) observed this pattern in peaches, where one the LOX isoforms (PpLOX1) increase its transcription during ripening, whereas the concentration of hexanal decreased. However, when applying ethylene after a cold storage period (cold-storage trial), the transcription of PamLOX was enhanced in comparison to the non-treated fruit. Therefore, gene regulation of PamLOX may be induced by cold treatment, thus regulating the production of hexanal in ‘Hass’ avocados. This result suggests differential modulation by low temperatures in the regulation of LOX, but it is not clear whether the induction of LOX is due specifically to temperature or an indirect response to the accelerated ripening (Schaffer et al., 2007). Synthesis of volatile compounds belonging to the LOX pathway is activated during ripening, and the regulation is considered to be dependent on a lipase, which is still unidentified, and whose activity is promoted by regulatory signals, maturation and tissue degradation and damage (Schaffer et al., 2007). This would support an ethylene regulation in the production of these compounds in the final stages of ripening in ‘Hass’ avocado, specifically at the ready-to-eat stage. In this work, three isoforms of the gene alcohol dehydrogenase (ADH) were studied, and they did not exhibit a consistent pattern of transcript accumulation among treatments in both trials. Avocados exposed to ethylene had higher PamADH1 and PamADH2 transcript expression compared with other treatments at firmness F2 (Fig. 3A–B) in fruit from the harvest trial. In fruit from the coldstorage trial, this difference in the expression was observed among treatments in the F1 firmness range for PamADH1 and PamADH2 (Fig. 3D–E) and F2 firmness range for PamADH3 (3F) due to the enhancement in the ripening process after the fruit were exposed to cold (Leisso et al., 2013). PamADH3 did not exhibit significant changes in the harvest trial (Fig. 3C). For the three variants of PamADH, no relationship between the expression of these genes

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and the production of E-2-hexenal, which is used as precursor for the synthesis of alcohols (Schwab et al., 2008), was observed. Strommet (2011) determined that short-chain ADH is related to the production of volatile aromas; however, the results obtained in this work do not indicate a relationship between the production of C6 aldehydes and the expression of the PamADH2 gene. 4. Conclusions The present study demonstrated that changes in the linolenic acid concentration, a primary substrate for volatile aroma biosynthesis, and the relative expression of PamLOX, the key enzyme in the lipoxygenase pathway, responded to ethylene modulation during storage in ‘Hass’ avocado, which modified the final production of hexanal as ripening progressed to the ready-to-eat stage. Similar to other major fruits, such as apple and pear, the storage temperature and storage period play a role in metabolism by affecting either the rate of ripening/senescence progression or the ethylene synthesis and action processes. The elucidation of this regulatory mechanism of volatile aromas should help researchers understand how the different technological practices available during postharvest handling (e.g., ethylene inhibitors, controlled atmosphere and forced ripening) affect the production of volatile-aroma compounds and their impact on the overall flavor of ‘Hass’ avocado. Acknowledgement This work was fully funded by FONDECYT 1130107. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2016. 02.024. References Adkins, M.F., Hofman, P.J., Stubbings, B.A., Macnish, A.J., 2005. Manipulating avocado fruit ripening with 1-methylcyclopropene. Postharvest Biol. Technol 35, 33–42. Ayala-Zavala, J.F., Wang, S.Y., Wang, C.Y., González-Aguilar, G.A., 2004. Effect of storage temperatures on antioxidant capacity and aroma compounds in strawberry fruit. LWT—Food Sci. Technol. 37, 687–695. Bate, N., Rothstein, S., 1998. C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes. Plant J. 16 (5), 561–569. Bill, M., Sivakumar, D., Thompson, A.K., Korsten, L., 2014. Avocado fruit quality management during the postharvest supply chain. Food Rev. Int. 30, 169–202. Bower, J.P., Cutting, J.G., 1988. Avocado fruit development and ripening physiology. Hortic. Rev. 10, 229–271. Cai, C., Chen, K.S., Xu, X.P., Zhang, W.S., Li, X., Ferguson, I., 2006. Effect of 1-MCP on postharvest quality of loquat fruit. Postharvest Biol. Technol. 40, 155–162. Chen, G., Hackett, R., Walker, D., Taylor, A., Lin, Z., Grierson, D., 2004. Identification of a specific isoform of tomato lipoxygenase (TomLoxC) involved in the generation of fatty acid-derived flavor compounds. Plant Physiol. 136, 2641–2651. Defilippi, B.G., Manríquez, D., Luengwilai, K., González-Agüero, M., 2009. Aroma volatiles: biosynthesis and mechanisms of modulation during fruit ripening. Adv. Bot. Res. 50, 1–37. Defilippi, B.G., Dandekar, A.M., Kader, A.A., 2005. Relationship of ethylene biosynthesis to volatile production: related enzymes and precursor availability in apple peel and flesh tissues. J. Agric. Food Chem. 53, 3133–3141. Eaks, I.L., 1990. Change in the fatty acid composition of avocado fruit during ontogeny cold storage and ripening. Acta Hortic. 269, 141–152. El-Mageed, M.A.A., 2007. Development of volatile compounds of avocado and casimiroa during fruit maturation. Arab. Univ. J. Agric. Sci. 15, 89–100. Feng, X., Apelbaum, A., Sisler, E.C., Goren, R., 2000. Control of ethylene responses in avocado fruit with 1-methylcyclopropene. Postharvest Biol. Technol. 16, 143–150. Fellman, J.K., Miller, T.W., Mattinson, D.S., Mattheis, J.P., 2000. Factors that influence biosynthesis of volatile flavor compound in apple fruits. HortScience 35, 1026–1033. García-Rojas, M., Gudenschwager, O., Defilippi, B.G., González-Agüero, M., 2012. Identification of genes possibly related to loss of quality in late-season ‘Hass’ avocados in Chile. Postharvest Biol. Technol. 73, 1–7.

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