Development of flavor-related metabolites in cherimoya (Annona cherimola Mill.) fruit and their relationship with ripening physiology

Postharvest Biology and Technology 94 (2014) 58–65

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Development of flavor-related metabolites in cherimoya (Annona cherimola Mill.) fruit and their relationship with ripening physiology b ˜ Daniel A. Manríquez a,∗ , Pablo Munoz-Robredo , Orianne Gudenschwager b , b b Paula Robledo , Bruno G. Defilippi a b

AgroFresh, South Cone, Avenida Américo Vespucio 100, piso 6, Santiago, Chile Unidad de Postcosecha, Instituto de Investigaciones Agropecuarias (INIA-La Platina), Casilla 439/3, Santiago, Chile

a r t i c l e

i n f o

Article history: Received 3 December 2013 Accepted 6 March 2014 Keywords: AAT Esters Flavor Fruit quality

a b s t r a c t Flavor is one of the most important attributes of fresh fruit for the consumer, and is affected by several factors, including genotype, maturity stage, and environmental conditions. Flavor-related metabolites were characterized in two important cherimoya varieties, cv. Concha Lisa and cv. Bronceada, during fruit ripening. The most important sugars present were glucose, fructose and sucrose, and only fructose and glucose increased during ripening. The most important acids were tartaric, malic and citric acids, and all increased as ripening progressed. Overall aroma profile was mainly determined by esters and terpenes in both varieties. Ester compounds such as ethyl hexanoate, butyl butyrate and hexyl propanoate increased during ripening. The activity of alcohol acyl transferase also increased during fruit ripening concomitant with ester accumulation. Terpenes, such as ␣ and ␤-pinene, showed a reduction during ripening, whereas others, such as myrcene and limonene, increased. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The cherimoya (Annona cherimola Mill.) is a subtropical fruit of the genus Annona. The most important cherimoya varieties grown in Chile are ‘Concha Lisa’ and ‘Bronceada’. In the cherimoya, as in other Annona species, flavor, defined primarily by aroma, sweetness and sourness, is the most important attribute at the consumer level (Díaz, 1991; Palma et al., 1993; Gardiazabal and Cano, 1999). As in other climacteric fruit, many of the changes related to quality occur during fruit ripening. Ethylene plays an important role in the coordination of many of these changes. In the cherimoya, respiration exhibits a particular pattern during ripening, described by a double-sigmoid curve. The first increase occurs soon after harvest and is followed by a plateau, which in turn is followed by a second peak. Both the respiration rate and ethylene production are very high during ripening, with ethylene production increasing at the beginning of the plateau observed for respiration (Paull et al., 1983; Wills et al., 1984; Martínez et al., 1993; Palma et al., 1993; Alique and Oliveira, 1994; Gutiérrez et al., 1994). Many of the changes related to organoleptic quality, such as softening, sugar and acid increases and aroma biosynthesis, appear to begin after the first

∗ Corresponding author. Tel.: +56 2 4404831; fax: +56 2 4404831. E-mail address: [email protected] (D.A. Manríquez). http://dx.doi.org/10.1016/j.postharvbio.2014.03.004 0925-5214/© 2014 Elsevier B.V. All rights reserved.

respiratory peak, earlier than the increase in ethylene production. Sugars and acids influence flavor properties of cherimoya, imparting sweetness and sourness, respectively. Malic acid has been found to be the predominant acid, with tartaric and citric acids present at lower concentrations. The development of sweetness is associated with an increase in the content of soluble solids. This increase is related primarily to an increase in sugars, which results from the conversion of starch into simple sugars such as fructose, glucose and sucrose. Aroma is one of the most important attributes at the consumer level in many fruit including cherimoya. Esters, alcohols and terpenes are the most important compounds in the aroma profile for Annona species (Idstein et al., 1984; Wyllie et al., 1987; Iwaoka and Zhang, 1993; Ferreira et al., 2009; Pino et al., 2003). The role of ethylene in aroma development has previously been described in many fruit (Defilippi et al., 2004; El-Sharkawy et al., 2005; Manríquez et al., 2006). Regarding the synthesis of esters, ethylene can regulate the activity of two key enzymes involved in volatile production, alcohol dehydrogenase (ADH) and alcohol acyltransferase (AAT). AAT regulates the last step in ester biosynthesis (Defilippi et al., 2004; El-Sharkawy et al., 2005; Manríquez et al., 2006). This work contributes to a better understanding of the relationships among ethylene, the respiratory pattern and the changes in flavor-related metabolites during ripening in two varieties of cherimoya grown in Chile. Within volatile production, new insights

D.A. Manríquez et al. / Postharvest Biology and Technology 94 (2014) 58–65

about the relationship between ester biosynthesis and AAT enzyme activity in both varieties are provided. 2. Materials and methods 2.1. Plant material and ripening conditions The cherimoya (Annona cherimola Mill.) varieties ‘Concha Lisa’ and ‘Bronceada’ were harvested at maturity from a commercial orchard located in the Valparaiso region, Chile. Immediately after harvest, the fruit were transported to the Postharvest Laboratory at the Institute for Agricultural Research (INIA-La Platina) in Santiago, Chile. The fruit were placed at 20 ◦ C and 50% R.H. until they reached the ripe (ready-to-eat) stage and underwent senescence. Each day, a sample of fruit was collected to measure the respiration rate, ethylene production, and quality parameters, as described below. For the assays of volatiles, sugars, organic acids and enzyme activity, pulp tissue was frozen with liquid nitrogen and stored at −80 ◦ C prior to analysis. 2.2. Maturity parameters The flesh firmness of peeled whole fruit, expressed in terms of newtons (N), was measured on opposite sides of the fruit with a penetrometer (Effegi, Milan, Italy). At harvest, a 4 mm plunger was used, and an 8 mm probe was used when the fruit approached the ripe stage. The total soluble solids (TSS) were measured with a manual temperature-compensated refractometer (ATC-1E, Atago, Tokyo, Japan) in a sample of the juice; the results were expressed as a percentage (%). The titratable acidity (TA) was obtained through the titration of 10 mL of juice with 0.1 N NaOH until the organic acids were neutralized to pH 8.2–8.3. In this case, the results were expressed as a percentage of malic acid equivalents. 2.3. Ethylene production and respiration rate Ethylene production and the respiration rate were measured for intact fruit with a static system. On each sampling date, seven fruit were weighed and placed in 2.6 L airtight jars. The jars were sealed and kept at 20 ◦ C for 30–60 min prior to measurement. The concentrations of carbon dioxide (mg CO2 kg−1 h−1 ) and ethylene (␮L C2 H4 kg−1 h−1 ) in the jar headspace were then determined using a gas analyzer (PBI-Dansensor Checkmate 9900, Ringsted, Denmark) and a gas chromatograph (Shimadzu 8A, Tokyo, Japan) equipped with a flame ionization detector (FID), respectively. The GC-FID was equipped with a Supelco 80/100 Porapak costum column (75 cm × 5 mm × 3 mm). The injector and detector temperatures were 150 ◦ C and 40 ◦ C, respectively. The oven temperature kept at 40 ◦ C, and the nitrogen was used as the carrier gas at 98.08 kPa. 2.4. Sugar and acid extraction and measurement The samples to be analyzed for sugars and organic acids were prepared from a homogeneous sample of 60 g of tissue per fruit, and six replicates per variety at each sampling time were considered for both metabolites. Sugars and organic acids were analyzed according to the method of Pérez et al. (1997). Briefly, 10 g of tissue was homogenized in a fruit crusher (Polytron) with 25 mL of cold 95% ethanol for 3–5 min. The sample was centrifuged at 12,000 rpm for 20 min and vacuum-filtered through two layers of Whatman N◦ 1 paper. The solution was brought up to a volume of 50 mL with 80% ethanol. An aliquot of 10 mL was then dried under a nitrogen stream at 50 ◦ C. The residue was dissolved in 2 mL of 0.2 N H2 SO4 with 0.05% EDTA. The sample was loaded onto an activated Sep-Pak C-18 cartridge, and the eluate was collected. The sample

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was washed thoroughly with an additional 4 mL of the solution. The eluate was filtered through a 0.45 ␮m filter and analyzed by HPLC. For quantification, calibration curves were designed based on standards for each compound. Calibration curves for d-(−)-fructose (Sigma–Aldrich, USA), d-(+)-glucose and sucrose (Supelco Analytical, USA) were used to quantify the sugars. Calibration curves for citric acid, d-malic acid (Supelco Analytical, USA) and d-(−)-tartaric acid (Fluka Analytical, USA) were used to quantify the acids. The sugars were analyzed using a chromatography system composed of an ELSD (Evaporative Light Scattering Detector) detector, a Sedex 60 lt ELSD (Sedere) and an LC-NET II/ADC interface (JASCO, Japan). The separation of the sugars was performed with a Kromasil 100 5NH2 amino column (250 mm × 4.6 mm) (AkzoNobel, Bohus, Sweden) with a mobile phase of 77% acetonitrile and 23% HPLC-grade water, which had been degassed and ultrasonicated. The conditions during the analysis were held constant, with a flow rate of 1.8 mL min−1 for 12 min at 20 ◦ C under a pressure of 13.2 kPa. The injection volume was 20 ␮L. Organic acids were analyzed in a chromatography system with an L-4250A UV-VIS ultraviolet detector (Merck-Hitachi, USA) to measure the absorbance at 195 nm with a D-6000 interface (Merck-Hitachi, Tokyo, Japan). The separation of the acids was performed using a Symmetry C-18 column (4.6 mm × 250 mm, 5.0 ␮m) (Waters, Ireland). The data were analyzed with D-7000 HSM software. The mobile phase used was 0.0085 N H2 SO4 that had been degassed and ultrasonicated. The conditions during the analysis were held constant, with a flow rate of 0.4 mL min−1 for 24 min at 20 ◦ C at an average pressure of 8.4 kPa. The injection volume was 20 ␮L. 2.5. Volatile extraction and quantification Different volatile compounds present in the fruit pulp were analyzed based on a sample of 8 g of pulp frozen in liquid nitrogen and homogenized in 16 mL of 2 mM sodium fluoride with a homogenizer (Ultra-Turrax, Staufen, Germany). The homogenized tissue was filtered through four layers of cheesecloth and centrifuged at 20,000 × g for 20 min at 4 ◦ C. A total of 10 mL of supernatant was filtered (Whatman paper N◦ 2); 9.8 mL of filtrate was collected, and 0.2 mL of 1-octanol was used as an internal standard (1 ␮L mL−1 ). The volatile compounds were extracted with 10 mL of pentane and vortexed for 1.5 min, and the pentanolic phase was concentrated. The quantification of the compounds was performed with gas chromatography (GC); 1 ␮L of the concentrated pentanolic phase was injected into the GC equipped with a Clarus 500 flame ionization detector (FID) (Perkin Elmer, Shelton, USA). The GC-FID was equipped with a Supelco SPB-5 cross-linked polyethylene glycol column (30 m × 0.25 mm × 0.25 ␮m). The injector and detector temperatures were 250 ◦ C. The oven temperature was programmed to increase from 40 ◦ C for 1 min to 60 ◦ C for 1 min at a rate of 2 ◦ C min−1 and finally to 190 ◦ C for 5 min at a rate of 10 ◦ C min−1 . Nitrogen was used as the carrier gas at 100 kPa. The compounds were identified by comparing the retention times with those of authentic standards. Quantification was performed using calibration curves that were made by adding standards from each of the quantified volatiles to water, and using the ratios of standard and aroma volatiles to the internal standard (1octanol) for the calculations. 2.6. Alcohol acyltransferase (AAT) activity of cherimoya fruit crude protein extract The total protein was extracted from cherimoya pulp cv. Bronceada using the method described by El-Sharkawy et al. (2005) with modifications. Five grams of mesocarp tissue with 1 mL of extraction buffer (250 mM Tris/HCl, pH 7.5, 1 mM DTT) was ground

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mechanically in liquid nitrogen. The protein extract was thawed in ice and centrifuged at 45,000 × g for 20 min at 4 ◦ C. The supernatant phase was desalted with SephadexTM G-25 columns (Amersham Biosciences, Sweden) and eluted with buffer (Tris–HCl 50 mM, pH 7.5, 10% (v/v) glycerol, 1 mM DTT). Total proteins were quantified according to the method of Bradford (Bradford, 1976). AAT activity was assayed in a 500 ␮L total volume containing 300 ␮L of the soluble fraction of the protein extract, 2 mM alcohol (ethanol or 1-butanol) and 250 ␮M acyl-CoA. The sample was brought up to the final volume with elution buffer (Tris–HCl 50 mM, pH 7.5, 10% (v/v) glycerol, 1 mM DTT). The mixture was incubated at 30 ◦ C for 30 min. Immediately after the reaction, 20 ␮L of the internal standard (1-octanol 0.5 ␮L L−1 ) was added to the mixture. The volatile compounds produced by the reaction were extracted with 500 ␮L of pentane and vortexed for 1.5 min. The pentanolic phase was concentrated, and the volatile quantification was performed as described above. 2.7. Data analysis Each fruit was considered an experimental unit. For maturity and physiological parameters seven fruit were analyzed per day during ripening at 20 ◦ C. For sugars, organic acids, and enzyme activity 6 replicates were considered. Differences between analyzed parameters were statistically evaluated using an analysis of

variance (ANOVA) and the mean comparisons between varieties were determined by Student’s t-test at p < 0.05 using JMP 10 (SAS Institute Inc., Duxbury, USA) statistical program (Maalekuu et al., 2006). 3. Results 3.1. Ethylene production and respiration rate The ‘Concha Lisa’ and ‘Bronceada’ fruit showed an increase in ethylene production during ripening and higher rates were observed for both varieties when the fruit reaching a ready-to-eat stage. The respiration rate in both varieties followed a doublesigmoid curve. The first peak occurred immediately after exposure to 20 ◦ C in ‘Concha Lisa’ and after 3 days in ‘Bronceada’. A plateau in the respiration rate was observed after this first increase. The plateau was longer in ‘Concha Lisa’ than in ‘Bronceada’. The last phase of the curve was characterized by a second increase, which coincided with the maximum level of ethylene production in ‘Concha Lisa’ (Fig. 1A). 3.2. Ripening and quality parameters Pulp firmness was very high at harvest, reaching values of 131 N and 125 N in ‘Concha Lisa’ and ‘Bronceada’ fruit, respectively. Later,

Fig. 1. Respiration rate, ethylene production and firmness during fruit ripening at 20 ◦ C for cherimoya cv. Concha Lisa (A) and cv. Bronceada (B). Bars at each sampling point show standard deviations of the mean. Arrow indicates beginning of the ready to eat stage.

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Fig. 2. Soluble solids and sugar changes during ripening of cv. Concha Lisa (A) and cv. Bronceada cherimoyas (B). Bars at each sampling point show standard deviations of the mean.

a rapid softening was observed in both varieties after 1 day at 20 ◦ C. This change coincided with an increase in ethylene production in both varieties (Fig. 1A and B). As expected, increases in the TSS and TA were observed during ripening (Figs. 2 and 3). The ready-toeat stage was mainly determined when the fruit reached a pulb firmness value below 10 N, i.e. after 3 and 4 days for ‘Concha Lisa’ and ‘Bronceada’, respectively. Similarly, the TA at the ready-to-eat stage was three times greater than at harvest. These increases in TSS and TA levels were observed during the entire process of fruit ripening. The maximum values were reached after 5 and 4 days in Concha Lisa’ and ‘Bronceada’, respectively (Figs. 2 and 3). Fructose and glucose were the main sugars identified (Fig. 2). These sugars showed the highest concentrations during ripening and followed a pattern similar to that observed for the TSS. The concentration of sucrose was higher during the first days of ripening and then decreased after 2 and 3 days at 20 ◦ C for ‘Concha Lisa’ and ‘Bronceada’, respectively. This decrease was greater in the ‘Concha Lisa’ fruit (Fig. 2). The analysis of the acids showed that malic and citric acids were predominant, and their concentrations increased during ripening. In ‘Concha Lisa’, malic acid was the predominant acid during the first 3 days at 20 ◦ C, whereas citric acid was predominant during the final stage of ripening. In contrast, citric acid

had the highest concentration in ‘Bronceada’ fruit throughout the ripening period. Tartaric acid was present in both varieties, but its concentration was very low from harvest to the last stages of ripening (Fig. 3). 3.3. Changes in volatiles The volatile profile in both varieties was dominated by esters, terpenes and alcohols. These groups of compounds represented between 78% and 83% of the volatiles occurring at the ready-to-eat stage in ‘Concha Lisa’ and ‘Bronceada’ fruit, respectively (data not shown). Based on odor activity values (OAV) reported in the literature, i.e. the ratio of the concentration and odor threshold of each individual compound, the compounds with the highest OAV occurring in ripe fruit were the esters ethyl octanoate, ethyl hexanoate, ethyl butyrte, butyl butyrate, hexyl butyrate, hexyl propanoate, methyl decanoate, ethyl heptanoate and isobutyl isobutyrate, and the terpenes ␣-pinene, linalool, myrcene, ␤-pinene, limonene and caryophyllene. In Table 1 we provide the odor thresholds for the volatiles considered in this study (Guth, 1997; Ferreira et al., 2000; Culleré et al., 2004; Pet‘ka et al., 2006; Gomez-Miguez et al., 2007; Loscos et al., 2007). The concentrations of these compounds and

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Fig. 3. Titratable acidity and acid changes during ripening in cv. Concha Lisa (A) and cv. Bronceada cherimoyas (B). Bars at each sampling point show standard deviations of the mean.

Table 1 Changes in volatiles (␮mol kg−1 ) during the ripening of cv. Concha Lisa cherimoyas.

Ethyl butyrate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Butyl butyrate Hexyl butyrate Hexyl propanoate Methyl decanoate Linalool ␣-Pinene ␤-Pinene Myrcene Limonene Caryophyllene a b

Odor threshold (mg L−1 )

Harvest

20a 14 2.2 2.0 100 250 8 2 6 6 140 14 10 64

0.066b b 0.016a 0.099b 1.819a 0.326b 0.304b 0.196b 3.010b 9.016a 0.236a 0.134a 0.074ab 0.296ab 0.367ab

Days at 20 ◦ C 1

2

3

4

5

0.070ab 0.045a 0.161b 1.648ab 0.269b 0.334b 0.281b 3.259b 6.703a 0.185ab 0.040b 0.063b 0.232ab 0.670a

0.131ab 0.075a 0.094b 0.727c 0.249b 0.394b 0.425b 9.493a 2.424a 0.041c 0.007b 0.049b 0.137b 0.264b

0.106ab 0.055a 0.091b 1.884a 0.278b 0.311b 0.815a 2.525b 1.800b 0.072bc 0.012b 0.084b 0.701ab 0.385ab

0.217ab 0.167a 0.124b 1.373abc 0.408b 0.451b 0.222b 2.188b 2.187b 0.025c 0.011b 0.110b 0.471ab 0.328ab

0.318a 0.396a 6.236a 0.712bc 2.231a 2.915a 0.129b 2.686b 1.552b N.D. 0.014b 0.151a 2.049a

Odor thresholds from literature (Guth, 1997; Ferreira et al., 2000; Culleré et al., 2004; Pet‘ka et al., 2006; Gomez-Miguez et al., 2007; Loscos et al., 2007). Values are means of six replicates. Means within the same compound followed by different letters are significantly different (ANOVA, Student’ t-test, p < 0.05).

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Table 2 Changes in volatiles (␮mol kg−1 ) during the ripening of cv. Bronceada cherimoyas.

Ethyl butyrate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Butyl butyrate Hexyl butyrate Hexyl propanoate Methyl decanoate Linalool ␣-Pinene ␤-Pinene Myrcene Limonene Caryophyllene a b c

Odor threshold (␮g L−1 )

Harvest

20a 14 2.2 2.0 100 250 8 2 6 6 140 14 10 64

N.D.b 0.143c b 0.107ab 1.377a 0.297b 0.350b 0.169c 4.232a 2.537b 0.091b 0.028b 0.123a 0.285b 0.457a

Days at 20 ◦ C 1

2

3

4

5

6

0.055 0.032b 0.049b 0.908a 0.329b 0.379b 0.594bc 3.640a 4.804a 0.095a 0.078a 0.054a 0.742b 0.386a

N.D. N.D. 0.026b 1.411a 0.218b 0.382b 0.706abc 4.433a 1.924bc N.D. N.D. N.D. 2.013b 0.476a

N.D. 0.050b 0.132ab 1.070a 0.250b 0.383b 1.141ab 3.860a 1.057cd N.D. N.D. 0.557a 0.576b 0.542a

N.D. 0.130b 0.177ab 1.035a 0.246b 0.369b 1.564a 3.402a 1.067cd N.D. N.D. 0.042a 0.419b 0.361a

N.D. 0.740b 0.124ab 1.181a 4.153b 0.779b 1.323ab 3.455a 0.590d N.D. N.D. 0.129a 0.448b 0.408a

N.D. 2.426a 0.327a 2.220a 15.771a 2.004a 0.852abc 5.501a 0.887cd N.D. N.D. 0.074a 0.588b 0.759a

Odor thresholds from literature (Guth, 1997; Ferreira et al., 2000; Culleré et al., 2004; Pet‘ka et al., 2006; Gomez-Miguez et al., 2007; Loscos et al., 2007). Non-detected. Values are means of six replicates. Means within the same compound followed by different letters are significantly different (ANOVA, Student’s t-test, p < 0.05).

their change during ripening were different for each compound and variety. However, a similar pattern was observed for certain compounds in both varieties, although the actual concentrations differed (Tables 1 and 2). Esters such as ethyl hexanoate and ethyl butyrate showed an increase in their concentrations during ripening (Tables 1 and 2). This increase coincided with the increase in ethylene production that occurred after the plateau in respiration in both varieties. Other compounds, such as butyl butyrate, showed an increase in concentration at the end of ripening when the fruit were soft and reached the ready-to-eat stage and the respiration rates were the highest. Another pattern observed was in hexyl propanoate, whose concentration increased after 3 and 4 days at 20 ◦ C in ‘Concha Lisa’ and ‘Bronceada’, respectively. Some other esters, such as ethyl octanoate and methyl decanoate, were present during the entire ripening process. Their concentrations did not show a clear pattern of change (Tables 1 and 2). Among terpenes, linalool and ␣- and ␤-pinene concentrations decreased during ripening (Tables 1 and 2). In the ‘Concha Lisa’ fruit, the concentrations of myrcene and limonene increased after 5 days at 20 ◦ C, when the fruit were at the ready-to-eat stage. However, these compounds showed a peak in their concentrations after 2–3 days at 20 ◦ C in the’ Bronceada’ fruit (Tables 1 and 2). The increase in the concentration of caryophyllene during ripening also differed between the varieties. The maximum concentration in ‘Concha Lisa’ occurred after 1 day at 20 ◦ C, whereas in ‘Bronceada’ caryophyllene concentration reached a maximum at the ready-to-eat stage (Tables 1 and 2).

other species of the genus Annona, such as Annona muricata and Annona squamosa. This particular pattern could be related to the separation of the fruit from the plant at harvest and/or to differences in ovarian development and maturity, as described for A. muricata (Biale et al., 1965; Broughton and Tan, 1979; Reginato, 1980; Paull, 1982; Bruinsma and Paull, 1984; Brown et al., 1988;

3.4. Alcohol acyltransferase activity To evaluate AAT activity, a combination of two pairs of substrates was assayed in the crude protein extract from pulp extracted from cherimoyas cv. Bronceada in different ripening stages. In both pairs of substrates, an increase in AAT activity was observed during ripening (Fig. 4). This activity was highest at the later stages of ripening. The increase in AAT activity began after 2 or 3 days at 20 ◦ C and coincided with the increase observed in ethylene production during the plateau in respiration rate and when the rate of softening was higher (Figs. 1 and 4). 4. Discussion The high rate of respiration and the double-sigmoid pattern observed during ripening in both varieties has been described for other cherimoya varieties. These characteristics are also known in

Fig. 4. Alcohol acyl-transferase activity of crude protein extracts of cherimoya cv. Bronceada during ripening. (A) Measured acyl-transferase activity for ethanol and hexanoyl-CoA and (B) measured acyl-transferase activity for 1-butanol and butanoyl-CoA.

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Lahoz et al., 1993; Martínez et al., 1993; Palma et al., 1993). The rate of ethylene production was high, and a single peak was observed during ripening. In both varieties, an increase in ethylene production coincided with the plateau observed in respiration (Reginato, 1980; Paull, 1982; Bruinsma and Paull, 1984; Brown et al., 1988; Lahoz et al., 1993; Martínez et al., 1993; Palma et al., 1993). As in other climacteric fruit, such as tomato (Lycopersicon esculentum) and melon (Cucumis melo), ethylene appears to be involved in the coordination of particular processes during fruit ripening (Giovannoni, 2001; Alexander and Grierson, 2002; Pech et al., 2004, 2008). In both varieties, a high rate of softening was observed during the initial days at 20 ◦ C. This high softening rate coincided with the increase in ethylene production. The high rate of softening could be related to the regulation by ethylene of the genes involved in cell wall disassembly (Alique and Oliveira, 1994; Lahoz et al., 1993; Alique and Zamorano, 2000; Li et al., 2009). Espinoza (2005) showed that the ethylene antagonist 1-methylcyclopropene (1-MCP) reduced endogenous ethylene production and delayed softening in ‘Concha Lisa’ cherimoyas both during ripening in cold storage and during the shelf-life of the fruit. The increase in TSS observed in both cultivars could be a result of the high level of starch present in the fruit at harvest. The hydrolysis of the starch during cold storage is the principal source of sugars. This process increases the TSS concentration in the fruit, a phenomenon also observed in other climacteric fruit, such as apples (Malus × domestica) and kiwifruit (Actinia deliciosa) (Reginato, 1980; Knee, 1993; Lahoz et al., 1993; Martínez et al., ˜ et al., 2008). In apples and kiwifruit, 1993; Mitchell, 1994; Goni starch hydrolysis has been shown to be ethylene independent. The increase in TSS from the beginning of the ripening process indicates that the process of starch hydrolysis in cherimoya is also ethylene independent (Lahoz et al., 1993; Martínez et al., 1993; Manríquez et al., 2006; Watkins, 2006). Glucose and fructose, the most important sugars in terms of concentration, showed an increase during ripening. This pattern was similar to that found for the concentration of TSS. A decrease in sucrose was observed after 3 days at room temperature in both varieties, as described for other Annona species (Alique and Oliveira, 1994; Alique and Zamorano, 2000). In contrast to the pattern found in other climacteric fruit, an increase in TA during ripening was observed in both cherimoya varieties during fruit ripening. This increase reached a peak at the ready-to-eat stage (Reginato, 1980; Wills et al., 1984; Martínez et al., 1993; Alique and Oliveira, 1994; Alique and Zamorano, 2000). Malic and citric acids were the most abundant organic acids in the fruit during ripening. Citric acid was the most important in terms of concentration in both varieties. The changes in its concentration during ripening could be regulated by signals other than ethylene, as described for apples (Lau et al., 1986; Blankenship and Sisler, 1989; Fan et al., 1999). Esters and terpenes were two important groups that contributed to the aroma profile in both varieties. Esters are an important group of compounds in the aroma profile of other cherimoya varieties (Idstein et al., 1984; Iwaoka and Zhang, 1993; Ferreira et al., 2009). The increase in concentration observed for many of the esters present in both varieties began during the plateau in the respiration rate, at the same time as ethylene production, and also occurred when the fruit began to soften. The highest concentration occurred at the ready-to-eat stage. The activity of AAT, a key enzyme in ester biosynthesis, showed an increase during fruit ripening similar to that of many of the esters. Similar studies in other cherimoya varieties and in other Annona species found that the development of aroma in the fruit begins when the plateau in respiration occurs and when softening of the fruit is evident (Wills et al., 1984; Palma et al., 1993). Ethylene production increased at this point in the ‘Concha Lisa’ and ‘Bronceada’ fruit. The biosynthesis of esters and the role of ethylene as a coordinator of this process have been studied

and described for many climacteric fruit, including apples, melons and bananas (Musa spp.) (Golding et al., 1999; Fellman et al., 2000; Defilippi et al., 2005; El-Sharkawy et al., 2005). The biosynthesis of esters in cherimoya is also modulated by ethylene. Ethylene works at the molecular and enzymatic level to modulate the expression of particular genes coding for AAT and ADH (Flores et al., 2002; Defilippi et al., 2005, 2009; El-Sharkawy et al., 2005; Manríquez et al., 2006). However, the lack of an increase associated with ethylene production during ripening in certain compounds could result from the coordination of their synthesis by other factors. In the strawberry (Fragaria × ananassa), a non-climacteric fruit, ethylene does not play a key role in the coordination of ripening, and certain AATs have been described as ethylene independent (Pérez et al., 1996; Aharoni et al., 2000). In this study, changes in terpenes were observed to follow different patterns of increase during fruit ripening. However, the role of ethylene in modulating different pathways of terpene synthesis is still not clear in climacteric and non-climacteric fruit (Herianus et al., 2003; Ninio et al., 2003; Sharon-Asa et al., 2003; Sitrit et al., 2004; Harb et al., 2008). 5. Conclusions In the cherimoya, certain processes appear to be regulated by ethylene during the ripening of the fruit. These processes include softening and portions of the biosynthesis of the compounds responsible for the aroma of the fruit. However, other processes associated with ripening appear to be regulated by other signals. The plateau observed in the respiration rate during ripening appears to be an important period of fruit ripening because this plateau is associated with many of the changes associated with flavor, including softening, an increase in acidity, starch degradation and aroma biosynthesis. To understand the processes associated with flavor, it will be necessary to achieve a better understanding of the role of ethylene as a coordinator of aroma biosynthesis in cherimoya fruit. Acknowledgments This work was funded by Fondecyt Grant N◦ 11090098. We thank Pontificia Universidad Católica de Valparaiso, Research Station La Palma, for providing the fruit for the trials. We thank Prof. Dr. Jean Claude Pech and Dr. Alain Latché from INP-ENSAT France for the critical review of this paper. References Aharoni, A., Keizer, L.C.P., Bouwmeester, H.J., Sun, Z., Alvarez-Huerta, M., Verhoeven, H.A., Blass, J., Van Houwelingen, A.M.M.L., De Vos, R.C.H., Van der Voet, H., Jansen, R.C., Guis, M., Mol, J., Davis, R.W., Schena, M., Van Tunen, A.J., O’Connell, A.P., 2000. Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 12, 647–661. Alexander, L., Grierson, D., 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J. Exp. Bot. 53, 2039–2055. Alique, R., Oliveira, G., 1994. Changes in sugar and organic acids in cherimoya (Annona cherimola Mill.) fruit under controlled-atmosphere storage. J. Agric. Food Chem. 42, 799–803. Alique, R., Zamorano, J.P., 2000. Influence of harvest date within the season and cold storage on cherimoya fruit ripening. J. Agric. Food Chem. 48, 4209–4216. Biale, J.B., Young, R.E., Olmstead, A.J., 1965. Fruit respiration and ethylene production. Plant Physiol. 29, 168–174. Blankenship, S.M., Sisler, E.C., 1989. 2.5-Norbornadiene retards apple softening. HortScience 24, 313–314. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Broughton, W.J., Tan, G., 1979. Storage conditions and ripening of custard apple Annona squamosa L. Sci. Hort. 10, 73–82. Brown, B.I., Wong, L.S., George, A.P., Nissen, R.J., 1988. Comparative studies on the postharvest physiology of fruit from different species of Annona (custard apple). J. Hort. Sci. 63, 521–528.

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