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Pre-harvest drought stress treatment improves antioxidant activity and sugar accumulation of sugar apple at harvest and during storage
Accepted Manuscript Pre-harvest drought stress treatment improves antioxidant activity and sugar accumulation of sugar apple at harvest and during storage Laddawan Kowitcharoen, Chalermchai Wongs-Aree, Sutthiwal Setha, Ruangsak Komkhuntod, Satoru Kondo, Varit Srilaong PII:
S2452-316X(17)30315-0
DOI:
10.1016/j.anres.2018.06.003
Reference:
ANRES 175
To appear in:
Agriculture and Natural Resources
Received Date: 10 July 2017 Revised Date:
18 October 2017
Accepted Date: 17 November 2017
Please cite this article as: Kowitcharoen L, Wongs-Aree C, Setha S, Komkhuntod R, Kondo S, Srilaong V, Pre-harvest drought stress treatment improves antioxidant activity and sugar accumulation of sugar apple at harvest and during storage, Agriculture and Natural Resources (2018), doi: 10.1016/ j.anres.2018.06.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pre-harvest drought stress treatment improves antioxidant activity and sugar
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accumulation of sugar apple at harvest and during storage
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Laddawan Kowitcharoena,e,†, Chalermchai Wongs-Areea,b, Sutthiwal Sethac, Ruangsak
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Komkhuntodd, Satoru Kondoe,†, Varit Srilaonga,b,*
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Division of Postharvest Technology, School of Bioresources and Technology, King
Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand b
Postharvest Technology Innovation Center, Commission of Higher Education, Bangkok
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a
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10400, Thailand
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Chiang Rai 57100, Thailand
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d
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Postharvest Technology Program, School of Agro-Industry, Mae Fah Luang University,
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Research Center (Pakchong), Kasetsart University, Nakhon Ratchasima 30320, Thailand
Graduate School of Horticulture, Chiba University, Chiba 271-8510, Japan
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Received 10 July 2017
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Accepted 17 November 2017
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Available online
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Keywords:
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Abscisic acid (ABA);
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Ascorbic acid;
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Ethylene;
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Storage;
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Tropical fruit
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*Corresponding author.
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E-mail address: [email protected] (V. Srilaong)
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Abstract
33 Physico-chemical and quality changes in 72 sugar apple (Annona squamosa Linn.)
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fruits subjected to pre-harvest drought stress were analyzed at harvest and during storage at
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10ºC or 15ºC, with 90–95% relative humidity. At harvest, the ascorbic acid, sugar and
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endogenous abscisic acid concentrations increased while the concentration of the substrate
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indicating a 50% loss in 2,2-diphenyl-2-picrylhydrazyl scavenging activity (DPPH EC50)
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decreased in fruit from drought-treated trees compared with fruit from well-watered trees
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(control). The fresh weight loss of fruit stored at 15ºC was higher than at 10ºC, with no
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significant effect of drought treatment. In contrast, fruit firmness was reduced by drought
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treatment compared with the control during storage at both temperatures. Respiration,
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ethylene production and the endogenous abscisic acid and total sugar concentrations were
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higher in fruit from the drought-treated trees kept at 15ºC. The total ascorbic acid
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concentration was higher in fruit from drought-stressed trees kept at 10ºC compared with
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other treatments. This was concomitant with the DPPH EC50 value, which was lowest in fruit
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from drought-stressed trees stored at 10ºC. These results implied that pre-harvest drought
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stress treatment activated antioxidant activity and increased sugar concentration in sugar
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apple fruit. In addition, pre-harvest drought stress hastened fruit ripening. Thus, based on the
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results, storage of sugar apple fruit at 10ºC is recommended as this induces antioxidant
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activity which delays chilling injury for 8 d.
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Introduction
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The incidence of drought stress during fruit and vegetable production is occurring
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more frequently with climate change patterns from global warming, which in turn are leading
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to limited water resources (Whitmore, 2000). Water stress during the production of some
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agricultural products can influence fruit physiology and morphology, which may affect fruit
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quality (Toivonen and Hodges, 2011). Abscisic acid (ABA) synthesis is one of the
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mechanisms in response to water stress in plants; ABA is synthesized and then triggers
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stomatal closure, thereby reducing water loss via transpiration (Wilkinson and Davies, 2010).
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Furthermore, the onset of fruit ripening is also accelerated under water deficit conditions,
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such as in peach (Prunus persica L.; Mercier et al., 2009) and apple (Malus domestica Borkh.;
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El-Soda et al., 2014). These effects contribute to the production of ethylene, which is
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coordinated with fruit-ripening processes in many fruits, such as banana (Musa × paradisiaca
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L.) and strawberry (Fragaria × ananassa D.) according to Barry and Giovannoni (2007).
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There are many reports of the effect of water stress on fruit quality. Miller et al. (1998)
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observed that water stress during fruit set decreased the fruit weight but increased the total
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soluble solids concentration in kiwifruit (Actinidia deliciosa cv. Hayward). Terry et al. (2007)
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also found higher fructose and glucose concentrations in strawberry subjected to water deficit
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conditions. Moreover, Pérez-Pastor et al. (2007) showed the benefits of deficit irrigation
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treatments on apricot (Prunus armeniaca L.), when they observed a slight increase in total
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soluble solids and firmness at harvest and during cold storage. Under stress conditions,
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ascorbic acid causes plants to resist stress by reducing the reactive oxygen species constituted
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by the stress (Ahmed et al., 2014). Thus, the antioxidative system in plants plays an important
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role in eliminating free radicals from plants under stress conditions. However, the effect of
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water stress on fruit quality and postharvest change is still complex and variable as water
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stress affects so many biological processes in plants. Apart from pre-harvest environmental
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factors, the postharvest control conditions, specifically temperature, greatly affect the visual
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quality, chemical composition and eating quality of fresh produce. Good management of
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temperature is the most important and simplest procedure for delaying the deterioration of
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fresh fruits and vegetables. In addition, the optimum storage temperature can retard softening
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and color changes of fruits and vegetables, as well as slow down metabolic changes and
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moisture loss (Nunes, 2008).
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Sugar apple (Annona squamosa Linn.) is a drought tolerant plant cultivated in
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subtropical and tropical areas (Egydio-Brandão and Santos, 2016). It is an important
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commercial crop in Thailand, where it is mainly cultivated in the northeast, which is an arid
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area (Pratcharoenwanich et al., 2014). Drought stress decreased the stomatal conductance and
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CO2 assimilation rate and increased the soluble sugar and free amino acid concentrations in
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young sugar apple plants (Rodrigues et al., 2010). A more recent previous study found that
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endogenous ABA and ascorbic acid concentrations in the leaves and fruit of the sugar apple
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tree increased under drought conditions (Kowitcharoen et al., 2015). In addition, changes in
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the respiration rate and the sugar and chlorophyll concentrations in sugar apple fruit during
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development have also been reported (Pal and Kumar, 1995). With regard to postharvest
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research, the ripening rate of ‘Balanagar’ sugar apple fruit was delayed when the storage
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temperature decreased (Vishnu Prasanna et al., 2000). Sugar apple is perishable; therefore an
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optimum postharvest storage temperature is critical to ensure improved storage life and
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prevent chilling injury (CI). However, the optimum storage temperature varies in the range
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7–20°C (Vishnu Prasanna et al., 2000). Broughton and Guat (1979) suggested that storage
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temperatures below 15°C cause CI in sugar apple. In addition, Chunprasert et al. (2006)
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reported that sugar apple is susceptible to CI at temperatures lower than 13°C. Although several studies have described the physiological and biochemical changes
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that occur in sugar apple during growth, drought stress and storage, there is little information
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on the influence of pre-harvest drought treatment on the postharvest quality changes in sugar
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apple. Therefore, the current study aimed to investigate the effect of pre-harvest drought
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stress treatment on sugar apple performance at harvest and during storage at low temperatures.
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Materials and Methods
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109 Plant material and treatments
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The plant material consisted of 6-year-old, fruit-bearing sugar apple trees (Annona
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squamosa L., cv. ‘Fai’) trained using a modified central leader system, grown in an orchard
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with sandy loam soil at the Pakchong Research Center, Faculty of Agriculture, Kasetsart
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University (Nakhon Ratchasima, Thailand) located at 14 ᵒN, 101 ᵒE at an altitude of 317 m
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above mean sea level. The experiment was carried out in a randomized 2 × 2 factorial design.
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Two irrigation treatments were applied: well-watered (untreated control), where six sugar
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apple trees were irrigated during the experiment (30 L/tree/day), and a drought treatment,
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where six sugar apple trees were not watered for 30 d before harvest. The average amount of
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rainfall during the experiment period was 1.45 mm/day. Guard trees were grown between the
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untreated control and drought areas. In total, 72 sugar apple fruits, with uniform color and
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free from defects, were harvested from the six trees in each treatment (12 fruit/tree), 110 d
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after full bloom, and were composited in each treatment. Harvested fruits were transported to
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the Postharvest quality assurance laboratory, King Mongkut’s University of Technology
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Thonburi (Bangkok, Thailand) within 2 hr. The fruits were washed with tap water and dried
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at room temperature. After drying, the fruits in each group were unpacked and randomly re-
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divided into two test groups. In the first group, fruits were kept at 15ºC and 90–95% relative
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humidity, whereas the second group was kept at 10ºC and 90–95% relative humidity. Fruits
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from each treatment combination were randomly sampled at 2-day intervals to evaluate the
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fruit quality and biochemical changes, using three replicates. The collected samples were
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immediately frozen using liquid nitrogen and kept at -80ºC until analysis, and they were
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lyophilized before analysis.
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Measurement of soil water potential
135 The soil water potential was measured using a tensiometer (Eastern Agritec; Rayong,
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Thailand). Three tensiometers were randomly installed at 30 cm soil depth under the trees,
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and 60 cm distance from the trees in each treatment area. Three trees were used for the soil
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water potential investigation. The soil water potential (measured in bars) was measured at
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weekly intervals during the drought stress period.
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Analysis of total ascorbic acid concentration
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The total ascorbic acid concentration was measured following the method of Roe et
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al. (1948). A 5 g sample of pulp was homogenized in 20 mL of 5% (weight per volume, w/v)
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metaphosphoric acid. The homogenate was filtered through filter paper. A 0.4 mL sample of
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the filtrate was added to 0.2 mL 0.02% (w/v) indophenol solution, and then 2% (w/v)
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thiourea and 2% (w/v) 2, 4-dinitrophenylhydrazine solution were added, respectively. The
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mixed solution was incubated for 3 hr at 37ºC and then 1 mL 85% (volume per volume, v/v)
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sulfuric acid was added, and left for 30 min at room temperature. Absorbance was measured
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at 525 nm using a spectrophotometer (model: UV-1501; Shimadzu; Kyoto, Japan). The same
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procedure was repeated for a range of ascorbic acid solutions to obtain the standard curve.
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Analysis of 2,2-diphenyl-2-picrylhydrazyl-radical scavenging activity
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Peel and pulp samples (0.1 g dry weight, DW) were homogenized in 20 mL of 80%
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ethanol and filtered. Analysis of 2,2-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging
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activity was carried out according to the method of Kondo et al. (2004). A test sample of 20
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µL was added to 980 µL of 0.1 M DPPH in ethanol, and the combination was mixed and kept
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for 20 min at room temperature in the dark. The concentration of the antioxidant sample was
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made from zero to full inhibition at the point where 50% inhibition of reaction in the solution
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of the sample and DPPH, and the decrease in absorbance at 516 nm was monitored. The data
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were shown as EC50 [half maximum (50%) effective concentration] values.
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Analysis of sugar concentration
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The sugar concentration was analyzed as reported previously (Kondo et al., 2014). A
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1 g dried pulp sample in 10 mL 80% (v/v) ethanol was boiled for 15 min, cooled and then
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homogenized. The homogenate was filtered and evaporated. The residue was re-dissolved
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with 3 mL distilled water and analyzed using high performance liquid chromatography
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(model L-6200; Hitachi, Tokyo, Japan) with a Shodex ODP2 HP–4E column (Showa Denko;
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Tokyo, Japan; 4.6 mm internal dimater × 25 cm). The column temperature was set at 30ºC
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and the mobile phase flow rate was 1 mL/min (75% (v/v) acetonitrile). A refractive index
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detector was used to identify sugar components.
175 Measurement of ethylene production and respiration rate
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The sugar apple fruit was placed in an air-tight plastic box (700 mL volume) for 2 hr
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at 10ºC or 15ºC. A 1 mL sample of headspace gas was collected and analyzed. The
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respiration rate (production of carbon dioxide from the fruit) was determined using a gas
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chromatograph (GC–8A; Shimadzu, Kyoto, Japan) equipped with a 1.8 m packed column of
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WG–100 at 50ºC and a thermal conductivity detector. The injector temperature was 50ºC, the
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detector temperature was 80ºC and He gas was used as carrier gas. Ethylene production was
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analyzed using a gas chromatograph (GC–14B, Shimadzu, Kyoto, Japan) equipped with a 2
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m packed column of 80/100-mesh Porapak Q at 80ºC, and a flame ionization detector. The
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injector temperature was 120ºC, the detector temperature was 120ºC and N2 gas was used as
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carrier gas.
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Analysis of endogenous abscisic acid concentration
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The analysis of the endogenous ABA concentration was performed according to
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Kondo et al. (2014) with some modifications. A 0.3 g dried sample was homogenized in 20
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mL of cold 80% (v/v) methanol with 20 µg ABA–d6 as an internal standard. The homogenate
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was centrifuged and then filtered through filter paper, and the residue was washed with 20
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mL cold 80% (v/v) methanol, centrifuged, and filtered again. The filtrate was evaporated,
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then the aqueous residue was adjusted to pH 2.5 with 0.1 M hydrochloric acid and extracted
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three times with 20 mL 100% (v/v) ethyl acetate. The ethyl acetate phase was evaporated to
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dryness, re-dissolved three times in 1 mL ethyl acetate and dried. The residue was re-
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dissolved in 1 mL 4.8 M acetonitrile containing 20 mM acetic acid, filtered through a
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nitrocellulose filter, purified using high performance liquid chromatography using an ODS
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Mightysil RP-18 column (250 mm × 4.6 mm internal diameter) with a gradient of 4.8–9.6 M
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acetonitrile containing 20 mM acetic acid over a period of 30 min, and then held in 9.6 M
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acetonitrile for 5 min. The fraction containing ABA was collected, evaporated to dryness, re-
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dissolved three times in 0.5 mL methanol and dried in vacuo. The residue was re-dissolved
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using 1 mL 10% (v/v) methanol in diethyl ester and then methylated with diazomethane for
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10 min. The methyl ester of ABA was quantified and identified using gas chromatography-
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mass spectrometry-selected ion monitoring (model QP5000; Shimadzu; Kyoto, Japan) with
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an InertCap 1 MS column (GL Sciences; Tokyo, Japan; 0.25 mm internal diameter× 30 m,
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0.25 µm film thickness) and a linear helium flow of 50.2 cm/s. The column temperature was
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set as follows: 60°C for 2 min, then increasing from 60°C to 270°C at 10 ᵒC /min and finally
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270°C for 35 min. The ions were measured as ABA–d0 methyl ester/ABA- d6 methyl ester at
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m/z 190, 260 and 194. The ABA concentration was calculated from the ratio of the peak areas
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for m/z 190 (d0)/194 (d6). To identify ABA methyl ester in the samples, fragmentation ion
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patterns were compared with those of the chemical standard in total monitoring mode.
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Measurement of fresh weight loss
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Three replications of fruit in each treatment were separated for weight loss
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investigation. The initial weight of each fruit was measured and recorded before storage in
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the cold room. Fruit were weighed every 2 d. The fruit weight loss (%) was determined as the
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difference between the initial and final weights and compared with the initial weight.
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Measurement of fruit firmness
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Three replications of fruit in each treatment were measured for fruit firmness through
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the peel. Two measurements were taken on the two opposite sides of the fruit using a Texture
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Analyzer (model: TA-XTPlus, Stable Micro Systems Ltd.; Surrey, England) equipped with a
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5 mm diameter puncture probe. The penetration speed of the probe was fixed at 5 mm/s and
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the probe penetrated 10 mm into the fruit. The fruit firmness value was expressed in
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Newtons.
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Statistical analysis
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The data were presented as mean values ± SE. The SPSS analysis of variance
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procedure (SPSS Inc.; Chicago, IL, USA) was used to determine the treatment effects, and
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mean separations were analyzed using Duncan’s multiple range test at p < 0.05. A t-test
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(independent) at the 5% level was used to determine treatment mean differences.
238 Results and Discussion
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Soil water potential
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The soil water potentials in the drought-treated area were significantly lower than
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those in the untreated control area, and gradually decreased over the time of treatment (Table
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1). In addition, it was found that the value of the soil water potential of the untreated control
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at 4 wk after treatment was lower than that for 1‒3 wk. This may have been due to the higher
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water demand during fruit development. Furthermore, the vapor pressure deficit (VPD) was
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higher in the fourth week (data not shown). VPD drives transpiration in the plant which is
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influenced by relative humidity and temperature (Gates et al., 1998).
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Total ascorbic acid concentration and 2,2-diphenyl-2-picrylhydrazyl radical scavenging
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activity
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Ascorbic acid is one of the most important nutritional factors in fruits and vegetables
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as it is known to have many biological functions in the human body, acting as an antioxidant
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which could reduce the risk of many diseases such as cardiovascular disease and cancer
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(Harris, 1996). Moreover, it is also involved in plants undergoing growth and development
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processes, including their responses to environmental stress (Lee and Kader, 2000). From the
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results, the highest total ascorbic acid concentrations were found in sugar apple fruit from
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drought-exposed trees at harvest and at 2 d after storage at 15ºC or 10ºC. On day 6 and day 8
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after storage, the total ascorbic acid concentration in fruit from drought-exposed trees kept at
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10ºC was highest, followed by fruit from untreated control trees kept at 10ºC and the fruit
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from drought-exposed trees kept at 15ºC (Fig. 1A). The increase in ascorbic acid in sugar
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apple fruit from the drought-treated trees and also in fruit at lower storage temperature may
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have been caused by abiotic stress conditions. Normally, the stress conditions can induce
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ABA biosynthesis which can promote H2O2 production during periods of stress; H2O2 is
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classified as a kind of stress signaling that may induce the antioxidant system in plant to
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maintain or increase the ascorbic acid content (Bayoumi, 2008). Gallie (2013) reported that
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maintaining a normal level of ascorbic acid in plant cells is a consequence to the tolerance of
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reactive oxygen species (ROS) from stress conditions without increasing sensitivity to
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drought conditions. Radical scavenging activity is an indicator of antioxidant functionality and activity
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and is related to the presence of bioactive compounds (Heim et al., 2002). EC50 refers to the
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concentration of substrate that indicates a 50% loss in DPPH scavenging activity. A high
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EC50 value indicates low antioxidant activity. Drought stress for 30 d before harvesting
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significantly decreased the DPPH EC50 values in both the peel and pulp of sugar apple at
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harvest (Fig. 1B and 1C). Subsequently, the DPPH EC50 value significantly decreased in the
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peel of fruit from drought-exposed trees kept at 10ºC, and this was lower than those in other
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treatments (Fig. 1B).
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The fruit from drought-exposed trees kept at 10ºC delayed an increase in the DPPH
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EC50 value in pulp and had a lower value than in other treatments. The DPPH EC50 value in
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pulp of fruit from drought-exposed trees kept at 15ºC gradually increased throughout the
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storage period. At the end of storage, the DPPH EC50 values in pulp of fruit kept at 10ºC were
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lower than at 15ºC, with no significant differences between treatments (control and drought
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treatment) at the same storage temperature (Fig. 1C). Alali et al. (1999) reported that sugar
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apple peel contains many bioactive compounds and that the peel is an important part of the
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fruit which is exposed to environment, and functions as protection against infection by
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pathogens and pests. Therefore, to understand sugar apple fruit physiology, the current study
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involved the measurement of the DPPH scavenging activity in the peel separately from the
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pulp.
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The generation of ROS has been shown to be induced by water deficit (Shao et al.,
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2008). The enhancement of production and the ability of antioxidants may play an important
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role in the detoxification of ROS. Samieiani and Ansari (2014) reported that water stress
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raised the DPPH radical scavenging activity in many groundcover plants. As noted, ascorbic
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acid has a function as an antioxidant (Noctor and Foyer, 1998). In the current study, the
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ascorbic acid concentration and DPPH radical scavenging activity were enhanced in fruit
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from drought-exposed trees at harvest, and 2 d after being held at 15ºC or 10ºC. This
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suggested that an increase in ascorbic acid concentrations may have a role in the scavenging
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of free radicals, which accumulate under drought conditions as mentioned earlier. In general,
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the degradation of ascorbic acid is rapid after harvesting and increases as the storage time and
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temperature increases (Nunes, 2008). Previous study reported that ascorbic acid concentration
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in broccoli stored at 1ºC decreased progressively during storage (Serrano et al., 2006). In
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contrast, the ascorbic acid concentration in citrus fruits (lemon, orange and lime) was higher
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at 20ºC than at 30ºC (Njoku et al., 2011). These results indicated that the change in the
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ascorbic acid concentration in a plant is temperature dependent. The current study found that
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there were higher ascorbic acid concentrations and lower DPPH EC50 values in sugar apple
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fruit stored at lower temperature after 4 d of storage, especially with fruit from drought-
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exposed trees. Moreover, the DPPH EC50 values were lower in the peel than in the pulp (Fig.
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1B and 1C), indicating that sugar apple peel had a higher antioxidant activity than sugar apple
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pulp. This finding may imply that sugar apple fruit accumulates various antioxidants in the
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peel to prevent infestation of pests and diseases (Manochai et al., 2014). Although the sugar
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apple peel is not usually a consumed part of the fruit, as it has high free radical scavenging
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effects, it is possible that it can be used as a source of antioxidant in the pharmaceutical and
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food industry. In addition, for greater understanding of the effect of drought stress treatment
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on the antioxidative system in sugar apple fruit, changes in the enzymatic antioxidant activity
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should be investigated in future work.
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Sugar concentration
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Soluble sugar is one of the major osmotic compounds that accumulate in the fruit of
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many kinds of fruit trees (Ripoll et al., 2014). The sugar concentration in sugar apple is an
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important parameter that relates to fruit quality, especially to the taste and has also been
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related to the response to drought stress (Kowitcharoen et al., 2017). The current study found
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that the fructose, glucose and total sugar concentrations significantly increased in the fruit
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from drought-stressed trees at harvest. (Fig. 2A ̶ 2C). Previous studies also reported that
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drought stress induced sugar accumulation in Satsuma mandarin (Citrus unshiu Marc.
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‘Okitsu-Wase’; Yakushiji et al., 1998) and peach (Kobashi et al., 2000). This suggested that
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the increase in sugar concentrations may be associated with plant defense mechanisms
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against drought stress. Sugar acts as a compatible solute that accumulates in cells, which has
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a role in osmotic adjustment, preventing turgor loss in tissue (Clifford et al., 1998). Moreover,
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sugar enhancement by drought may improve the quality of sugar apple fruit, as such fruit was
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sweeter. During storage, the fructose, glucose and total sugar concentrations in fruit from the
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drought-exposed trees stored at 15ºC significantly increased and were higher than those for
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the other treatments during the first 4 d of storage (Fig. 2A ̶ 2C). Thereafter, the fructose,
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glucose and total sugar concentrations increased at 15ºC, and corresponded with increasing
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ethylene production. At the end of storage, there was no difference in the levels of fructose
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and glucose in the fruit from control trees stored at 15ºC and the fruit from drought-exposed
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trees stored at 15ºC or 10ºC, but these levels were significantly higher than those in the fruit
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from control trees stored at 10ºC (Fig. 2A and 2B). However, the total sugar concentrations
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were significantly higher in fruit from both control and drought-exposed trees stored at 15ºC
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compared with fruit stored at 10ºC (Fig. 2C). Vishnu Prasanna et al. (2000) reported an
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elevated sugar concentration at high temperatures (25ºC and 20ºC) compared with low
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temperatures (15ºC or 10ºC). Ethylene has been implicated as having a role in the conversion
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of starch to sugar in fruit (Watkins, 2003). During the ripening process of climacteric fruit,
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the fruit emit ethylene and the ethylene signal causes the hydrolysis of starch into soluble
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sugars such as sucrose and glucose, associated with acceleration of amylase in order to
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increase sweetness (Koning, 1994). This could imply that induction of sugar accumulation in
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sugar apple fruit during storage was associated with ethylene production. Although high
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sugar and antioxidant activity were observed in sugar apple fruit from drought-stressed trees,
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reductions in fruit size and weight were found (data not presented). Cells were smaller in pear
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fruit (Pyrus communis L.) that had experienced water deficit (Lopez et al., 2011).
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Ethylene production and respiration rate
At harvest, the drought stress treatment had no significant effect on ethylene
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production and the respiration rate in sugar apple fruit (Fig. 3A and 3B). The ethylene
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production rate in fruit from the drought treatment that was kept at 15ºC sharply increased to
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a peak on day 4. The changes in ethylene production in fruit from the untreated control kept
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at 15ºC or 10ºC showed a similar trend, which increased and reached a peak on day 6 and
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decreased thereafter (Fig. 3A). This finding agreed with a previous report that water deficit
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causes an increase of endogenous ethylene, which leads to accelerated ripening of fruit such
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as bananas (Burdon et al., 1994). Storage temperature affected the rate of ethylene production,
363
where a higher rate was observed in fruit held at 15ºC compared with fruit held at 10ºC,
364
indicating that higher temperatures accelerated physiological changes. These results
365
suggested that drought stress may induce fruit ripening by enhancing ethylene production,
366
especially at higher storage temperatures and as a consequence of the accumulation of a
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higher sugar concentration.
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Drought stress had no effect on the respiration rate during storage. In fact, increasing
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the respiration rate in sugar apple fruit positively correlated with increasing the storage time
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(Fig. 3B). The rate of increase was significantly higher at 15ºC than at 10ºC. These results
371
suggested that low temperatures may slow down the metabolic activities and consequently
372
delay fruit senescence as reported in many studies (Mworia et al., 2012; Freitas and Mitcham,
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2013; Li et al., 2017). Storage at 10ºC effectively reduced the respiration and ethylene
374
production rate resulting in delayed fruit ripening and retardation of sugar accumulation.
375 376
Endogenous abscisic acid concentration
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ABA is an important plant hormone associated with fruit development, physiology
379
and drought stress tolerance (Ripoll et al., 2014). ABA synthesis is a rapid response to
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drought stress in plants and is also a health-promoting phytochemical that can be found in
381
fruits and vegetables and is effective against diseases such as type II diabetes, obesity-related
382
inflammation and atherosclerosis-induced hypertension (Guri et al., 2007; 2010). To
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understand sugar apple fruit physiology, changes in the ABA concentration in fruits subjected
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to drought stress during storage were analyzed in the peel separately from the pulp. This
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study found that the endogenous ABA concentrations in the peel and pulp of sugar apple fruit
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from drought-exposed trees at harvest were significantly higher than those in the control,
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well-watered trees (Fig. 4A and 4B). The increasing ABA concentration may be associated
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with drought stress tolerance systems (Kowitcharoen et al., 2015). This result was consistent
389
with a previous report that endogenous ABA concentrations in peach fruit increased
390
significantly under drought stress (Kobashi et al., 2000). The synthesis of ABA appeared to
391
change over time during the first 4 d of storage; the ABA concentrations in the peel and pulp
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of fruit from drought-exposed trees stored at either 15ºC or 10ºC remained higher than those
393
of fruit from untreated trees stored at these temperatures (Fig. 4A and 4B), indicating that
394
drought stress affected the ABA accumulation in the peel and pulp of sugar apple. However,
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over the following days of storage, temperature also seemed to affect ABA accumulation,
396
where fruit from drought-exposed and untreated trees held at 15ºC had increased ABA
397
concentrations. This result may have been due to drought stress and the higher temperatures
398
inducing fruit senescence, which led to an increase in ABA synthesis. Kondo et al. (2002)
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reported that trans-ABA may increase with mangosteen fruit senescence. The change in the
400
ABA concentrations in the current experiment implied that a pre-harvest drought stress
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treatment may provide a way to stimulate the synthesis and accumulation of this beneficial
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phytochemical in sugar apple.
403 404
Fresh weight loss and fruit firmness
405 The percentage of fresh weight loss in sugar apple fruits from the untreated control
407
and drought-exposed trees kept at 15ºC substantially increased with storage time. While a
408
gradual increase in the weight loss was recorded in sugar apple fruit from untreated control
409
and drought-treated trees kept at 10ºC, there was a significant difference in the fresh weight
410
loss between storage temperatures (Fig. 5). A higher weight loss at higher temperature could
411
be related to the acceleration of transpiration, respiration and ripening as previously reported
412
by Lebibet et al. (1995) and Vishnu Prasanna et al. (2000). In the current study, the rate of
413
respiration increased rapidly at 15ºC (Fig. 3B) and may have been the main factor influencing
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the weight loss that was observed.
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Drought stress treatment for 30 d before harvesting the sugar apple fruit had no
416
significant effect on fruit firmness at harvest (Fig. 6). However, the fruit firmness in all
417
treatments decreased as storage progressed and as storage temperature increased. Fruit from
418
drought-exposed tree stored at 15ºC showed rapid loss of firmness compared with that of
419
other treatments. This could be ascribed to drought stress accelerating fruit softening as
420
mentioned above, and this response may be associated with the induction of ethylene
421
production in response to drought stress. An ethylene-mediated response may up-regulate cell
422
wall modification enzymes and subsequently accelerate softening (Toivonen and Hodges,
423
2011). In addition, the current study found that the decrease in fruit firmness in all treatments
424
had a very high correlation with fresh weight loss (R2 = 0.93, data not shown); this change
425
was due to the fresh weight loss causing a reduction in turgor pressure (Harker and Hallett,
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1994). These results were in agreement with Shackel et al. (1991) who found a decrease in
427
turgor pressure of tomato (Lycopersicon esculentum Mill.) coincided with losses in fruit
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firmness.
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In summary, ascorbic acid, sugar and ABA concentrations and antioxidant activity
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increased in fruit from drought-exposed trees at harvest. In addition, drought stress also
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activated antioxidant activity, enhanced sugar accumulation and induced fruit ripening in
433
sugar apple fruit during low temperature storage. The changes in fruit qualities were
434
temperature dependent; at 15ºC the sugar apple ripened faster than at 10ºC. These results
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suggested that pre-harvest drought stress treatments may enhance the eating quality of sugar
436
apples, especially the increase of antioxidant activity and sugar concentration. However, the
437
induction of fruit ripening by drought stress treatment must be considered for application in
438
proper postharvest technology.
439 Conflict of interest
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There is no conflict of interest.
443 Acknowledgments
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The authors thank the Thailand Research Fund through the Royal Golden Jubilee PhD
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program under grant No. PHD/0039/2554 and the Japan Student Service Organization
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(JASSO) for their financial support.
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Table 1 Values (mean ± SE) of soil water potential in untreated control and drought-treated
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areas within the sugar apple orchard.
602 603 Soil water potential (bar) Untreated control Drought 604 605 -0.09 ± 0.006 ns -0.09 ± 0.006 606 -0.10 ± 0.023 * -0.37 ± 0.029 607 -0.10 ± 0.023 * -0.47 ± 0.040 608 609 -0.06 ± 0.023 * -0.57 ± 0.052 610 -0.31 ± 0.020 * -0.62 ± 0.046 611 * Significant at the 5% level using t-test † ns: non-significant
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Fig. 1 Total ascorbic acid (A) concentration in sugar apple fruit, and DPPH radical
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scavenging activity (EC50 value) in peels (B) and pulps (C) of sugar apple fruit subjected to
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drought stress at harvest and during storage at 10ºC or 15ºC. Data are means ± SE of three
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test at 5% test level.
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Fig. 2 Fructose (A), glucose (B), and total sugar (C) concentrations in sugar apple fruit
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subjected to drought stress at harvest and during storage at 10ºC or 15ºC. Data are means ±
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SE of three replicates. Mean separation in each storage period determined using Duncan’s
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Fig. 3 Ethylene production (A) and respiration rate (B) of sugar apple fruit subjected to
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Fig. 5 Fresh weight loss of sugar apple fruit subjected to drought stress at harvest and during
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storage at 10 or 15ºC. Data are means ± SE of three replicates. Mean separation at each
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S2452-316X(17)30315-0
DOI:
10.1016/j.anres.2018.06.003
Reference:
ANRES 175
To appear in:
Agriculture and Natural Resources
Received Date: 10 July 2017 Revised Date:
18 October 2017
Accepted Date: 17 November 2017
Please cite this article as: Kowitcharoen L, Wongs-Aree C, Setha S, Komkhuntod R, Kondo S, Srilaong V, Pre-harvest drought stress treatment improves antioxidant activity and sugar accumulation of sugar apple at harvest and during storage, Agriculture and Natural Resources (2018), doi: 10.1016/ j.anres.2018.06.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT 1
Pre-harvest drought stress treatment improves antioxidant activity and sugar
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accumulation of sugar apple at harvest and during storage
3 4
Laddawan Kowitcharoena,e,†, Chalermchai Wongs-Areea,b, Sutthiwal Sethac, Ruangsak
5
Komkhuntodd, Satoru Kondoe,†, Varit Srilaonga,b,*
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Division of Postharvest Technology, School of Bioresources and Technology, King
Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand b
Postharvest Technology Innovation Center, Commission of Higher Education, Bangkok
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8
a
10
10400, Thailand
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c
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Chiang Rai 57100, Thailand
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d
14
e
Postharvest Technology Program, School of Agro-Industry, Mae Fah Luang University,
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RI PT
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Research Center (Pakchong), Kasetsart University, Nakhon Ratchasima 30320, Thailand
Graduate School of Horticulture, Chiba University, Chiba 271-8510, Japan
15 Article history:
17
Received 10 July 2017
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Accepted 17 November 2017
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Available online
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Keywords:
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Abscisic acid (ABA);
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Ascorbic acid;
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Ethylene;
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Storage;
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Tropical fruit
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*Corresponding author.
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E-mail address: [email protected] (V. Srilaong)
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ACCEPTED MANUSCRIPT 32
Abstract
33 Physico-chemical and quality changes in 72 sugar apple (Annona squamosa Linn.)
35
fruits subjected to pre-harvest drought stress were analyzed at harvest and during storage at
36
10ºC or 15ºC, with 90–95% relative humidity. At harvest, the ascorbic acid, sugar and
37
endogenous abscisic acid concentrations increased while the concentration of the substrate
38
indicating a 50% loss in 2,2-diphenyl-2-picrylhydrazyl scavenging activity (DPPH EC50)
39
decreased in fruit from drought-treated trees compared with fruit from well-watered trees
40
(control). The fresh weight loss of fruit stored at 15ºC was higher than at 10ºC, with no
41
significant effect of drought treatment. In contrast, fruit firmness was reduced by drought
42
treatment compared with the control during storage at both temperatures. Respiration,
43
ethylene production and the endogenous abscisic acid and total sugar concentrations were
44
higher in fruit from the drought-treated trees kept at 15ºC. The total ascorbic acid
45
concentration was higher in fruit from drought-stressed trees kept at 10ºC compared with
46
other treatments. This was concomitant with the DPPH EC50 value, which was lowest in fruit
47
from drought-stressed trees stored at 10ºC. These results implied that pre-harvest drought
48
stress treatment activated antioxidant activity and increased sugar concentration in sugar
49
apple fruit. In addition, pre-harvest drought stress hastened fruit ripening. Thus, based on the
50
results, storage of sugar apple fruit at 10ºC is recommended as this induces antioxidant
51
activity which delays chilling injury for 8 d.
52
54
Introduction
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The incidence of drought stress during fruit and vegetable production is occurring
56
more frequently with climate change patterns from global warming, which in turn are leading
57
to limited water resources (Whitmore, 2000). Water stress during the production of some
58
agricultural products can influence fruit physiology and morphology, which may affect fruit
59
quality (Toivonen and Hodges, 2011). Abscisic acid (ABA) synthesis is one of the
60
mechanisms in response to water stress in plants; ABA is synthesized and then triggers
61
stomatal closure, thereby reducing water loss via transpiration (Wilkinson and Davies, 2010).
62
Furthermore, the onset of fruit ripening is also accelerated under water deficit conditions,
63
such as in peach (Prunus persica L.; Mercier et al., 2009) and apple (Malus domestica Borkh.;
64
El-Soda et al., 2014). These effects contribute to the production of ethylene, which is
65
coordinated with fruit-ripening processes in many fruits, such as banana (Musa × paradisiaca
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L.) and strawberry (Fragaria × ananassa D.) according to Barry and Giovannoni (2007).
67
There are many reports of the effect of water stress on fruit quality. Miller et al. (1998)
68
observed that water stress during fruit set decreased the fruit weight but increased the total
69
soluble solids concentration in kiwifruit (Actinidia deliciosa cv. Hayward). Terry et al. (2007)
70
also found higher fructose and glucose concentrations in strawberry subjected to water deficit
71
conditions. Moreover, Pérez-Pastor et al. (2007) showed the benefits of deficit irrigation
72
treatments on apricot (Prunus armeniaca L.), when they observed a slight increase in total
73
soluble solids and firmness at harvest and during cold storage. Under stress conditions,
74
ascorbic acid causes plants to resist stress by reducing the reactive oxygen species constituted
75
by the stress (Ahmed et al., 2014). Thus, the antioxidative system in plants plays an important
76
role in eliminating free radicals from plants under stress conditions. However, the effect of
77
water stress on fruit quality and postharvest change is still complex and variable as water
78
stress affects so many biological processes in plants. Apart from pre-harvest environmental
79
factors, the postharvest control conditions, specifically temperature, greatly affect the visual
80
quality, chemical composition and eating quality of fresh produce. Good management of
81
temperature is the most important and simplest procedure for delaying the deterioration of
82
fresh fruits and vegetables. In addition, the optimum storage temperature can retard softening
83
and color changes of fruits and vegetables, as well as slow down metabolic changes and
84
moisture loss (Nunes, 2008).
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Sugar apple (Annona squamosa Linn.) is a drought tolerant plant cultivated in
86
subtropical and tropical areas (Egydio-Brandão and Santos, 2016). It is an important
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commercial crop in Thailand, where it is mainly cultivated in the northeast, which is an arid
88
area (Pratcharoenwanich et al., 2014). Drought stress decreased the stomatal conductance and
89
CO2 assimilation rate and increased the soluble sugar and free amino acid concentrations in
90
young sugar apple plants (Rodrigues et al., 2010). A more recent previous study found that
91
endogenous ABA and ascorbic acid concentrations in the leaves and fruit of the sugar apple
92
tree increased under drought conditions (Kowitcharoen et al., 2015). In addition, changes in
93
the respiration rate and the sugar and chlorophyll concentrations in sugar apple fruit during
94
development have also been reported (Pal and Kumar, 1995). With regard to postharvest
95
research, the ripening rate of ‘Balanagar’ sugar apple fruit was delayed when the storage
96
temperature decreased (Vishnu Prasanna et al., 2000). Sugar apple is perishable; therefore an
97
optimum postharvest storage temperature is critical to ensure improved storage life and
98
prevent chilling injury (CI). However, the optimum storage temperature varies in the range
99
7–20°C (Vishnu Prasanna et al., 2000). Broughton and Guat (1979) suggested that storage
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temperatures below 15°C cause CI in sugar apple. In addition, Chunprasert et al. (2006)
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reported that sugar apple is susceptible to CI at temperatures lower than 13°C. Although several studies have described the physiological and biochemical changes
103
that occur in sugar apple during growth, drought stress and storage, there is little information
104
on the influence of pre-harvest drought treatment on the postharvest quality changes in sugar
105
apple. Therefore, the current study aimed to investigate the effect of pre-harvest drought
106
stress treatment on sugar apple performance at harvest and during storage at low temperatures.
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Materials and Methods
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109 Plant material and treatments
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The plant material consisted of 6-year-old, fruit-bearing sugar apple trees (Annona
113
squamosa L., cv. ‘Fai’) trained using a modified central leader system, grown in an orchard
114
with sandy loam soil at the Pakchong Research Center, Faculty of Agriculture, Kasetsart
115
University (Nakhon Ratchasima, Thailand) located at 14 ᵒN, 101 ᵒE at an altitude of 317 m
116
above mean sea level. The experiment was carried out in a randomized 2 × 2 factorial design.
117
Two irrigation treatments were applied: well-watered (untreated control), where six sugar
118
apple trees were irrigated during the experiment (30 L/tree/day), and a drought treatment,
119
where six sugar apple trees were not watered for 30 d before harvest. The average amount of
120
rainfall during the experiment period was 1.45 mm/day. Guard trees were grown between the
121
untreated control and drought areas. In total, 72 sugar apple fruits, with uniform color and
122
free from defects, were harvested from the six trees in each treatment (12 fruit/tree), 110 d
123
after full bloom, and were composited in each treatment. Harvested fruits were transported to
124
the Postharvest quality assurance laboratory, King Mongkut’s University of Technology
125
Thonburi (Bangkok, Thailand) within 2 hr. The fruits were washed with tap water and dried
126
at room temperature. After drying, the fruits in each group were unpacked and randomly re-
127
divided into two test groups. In the first group, fruits were kept at 15ºC and 90–95% relative
128
humidity, whereas the second group was kept at 10ºC and 90–95% relative humidity. Fruits
129
from each treatment combination were randomly sampled at 2-day intervals to evaluate the
130
fruit quality and biochemical changes, using three replicates. The collected samples were
131
immediately frozen using liquid nitrogen and kept at -80ºC until analysis, and they were
132
lyophilized before analysis.
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Measurement of soil water potential
135 The soil water potential was measured using a tensiometer (Eastern Agritec; Rayong,
137
Thailand). Three tensiometers were randomly installed at 30 cm soil depth under the trees,
138
and 60 cm distance from the trees in each treatment area. Three trees were used for the soil
139
water potential investigation. The soil water potential (measured in bars) was measured at
140
weekly intervals during the drought stress period.
141 142
Analysis of total ascorbic acid concentration
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The total ascorbic acid concentration was measured following the method of Roe et
145
al. (1948). A 5 g sample of pulp was homogenized in 20 mL of 5% (weight per volume, w/v)
146
metaphosphoric acid. The homogenate was filtered through filter paper. A 0.4 mL sample of
147
the filtrate was added to 0.2 mL 0.02% (w/v) indophenol solution, and then 2% (w/v)
148
thiourea and 2% (w/v) 2, 4-dinitrophenylhydrazine solution were added, respectively. The
149
mixed solution was incubated for 3 hr at 37ºC and then 1 mL 85% (volume per volume, v/v)
150
sulfuric acid was added, and left for 30 min at room temperature. Absorbance was measured
151
at 525 nm using a spectrophotometer (model: UV-1501; Shimadzu; Kyoto, Japan). The same
152
procedure was repeated for a range of ascorbic acid solutions to obtain the standard curve.
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Analysis of 2,2-diphenyl-2-picrylhydrazyl-radical scavenging activity
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Peel and pulp samples (0.1 g dry weight, DW) were homogenized in 20 mL of 80%
157
ethanol and filtered. Analysis of 2,2-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging
158
activity was carried out according to the method of Kondo et al. (2004). A test sample of 20
159
µL was added to 980 µL of 0.1 M DPPH in ethanol, and the combination was mixed and kept
160
for 20 min at room temperature in the dark. The concentration of the antioxidant sample was
161
made from zero to full inhibition at the point where 50% inhibition of reaction in the solution
162
of the sample and DPPH, and the decrease in absorbance at 516 nm was monitored. The data
163
were shown as EC50 [half maximum (50%) effective concentration] values.
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Analysis of sugar concentration
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The sugar concentration was analyzed as reported previously (Kondo et al., 2014). A
168
1 g dried pulp sample in 10 mL 80% (v/v) ethanol was boiled for 15 min, cooled and then
169
homogenized. The homogenate was filtered and evaporated. The residue was re-dissolved
170
with 3 mL distilled water and analyzed using high performance liquid chromatography
171
(model L-6200; Hitachi, Tokyo, Japan) with a Shodex ODP2 HP–4E column (Showa Denko;
172
Tokyo, Japan; 4.6 mm internal dimater × 25 cm). The column temperature was set at 30ºC
173
and the mobile phase flow rate was 1 mL/min (75% (v/v) acetonitrile). A refractive index
174
detector was used to identify sugar components.
175 Measurement of ethylene production and respiration rate
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The sugar apple fruit was placed in an air-tight plastic box (700 mL volume) for 2 hr
179
at 10ºC or 15ºC. A 1 mL sample of headspace gas was collected and analyzed. The
180
respiration rate (production of carbon dioxide from the fruit) was determined using a gas
181
chromatograph (GC–8A; Shimadzu, Kyoto, Japan) equipped with a 1.8 m packed column of
182
WG–100 at 50ºC and a thermal conductivity detector. The injector temperature was 50ºC, the
183
detector temperature was 80ºC and He gas was used as carrier gas. Ethylene production was
184
analyzed using a gas chromatograph (GC–14B, Shimadzu, Kyoto, Japan) equipped with a 2
185
m packed column of 80/100-mesh Porapak Q at 80ºC, and a flame ionization detector. The
186
injector temperature was 120ºC, the detector temperature was 120ºC and N2 gas was used as
187
carrier gas.
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Analysis of endogenous abscisic acid concentration
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The analysis of the endogenous ABA concentration was performed according to
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Kondo et al. (2014) with some modifications. A 0.3 g dried sample was homogenized in 20
193
mL of cold 80% (v/v) methanol with 20 µg ABA–d6 as an internal standard. The homogenate
194
was centrifuged and then filtered through filter paper, and the residue was washed with 20
195
mL cold 80% (v/v) methanol, centrifuged, and filtered again. The filtrate was evaporated,
196
then the aqueous residue was adjusted to pH 2.5 with 0.1 M hydrochloric acid and extracted
197
three times with 20 mL 100% (v/v) ethyl acetate. The ethyl acetate phase was evaporated to
198
dryness, re-dissolved three times in 1 mL ethyl acetate and dried. The residue was re-
199
dissolved in 1 mL 4.8 M acetonitrile containing 20 mM acetic acid, filtered through a
200
nitrocellulose filter, purified using high performance liquid chromatography using an ODS
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Mightysil RP-18 column (250 mm × 4.6 mm internal diameter) with a gradient of 4.8–9.6 M
202
acetonitrile containing 20 mM acetic acid over a period of 30 min, and then held in 9.6 M
203
acetonitrile for 5 min. The fraction containing ABA was collected, evaporated to dryness, re-
204
dissolved three times in 0.5 mL methanol and dried in vacuo. The residue was re-dissolved
205
using 1 mL 10% (v/v) methanol in diethyl ester and then methylated with diazomethane for
206
10 min. The methyl ester of ABA was quantified and identified using gas chromatography-
207
mass spectrometry-selected ion monitoring (model QP5000; Shimadzu; Kyoto, Japan) with
208
an InertCap 1 MS column (GL Sciences; Tokyo, Japan; 0.25 mm internal diameter× 30 m,
209
0.25 µm film thickness) and a linear helium flow of 50.2 cm/s. The column temperature was
210
set as follows: 60°C for 2 min, then increasing from 60°C to 270°C at 10 ᵒC /min and finally
211
270°C for 35 min. The ions were measured as ABA–d0 methyl ester/ABA- d6 methyl ester at
212
m/z 190, 260 and 194. The ABA concentration was calculated from the ratio of the peak areas
213
for m/z 190 (d0)/194 (d6). To identify ABA methyl ester in the samples, fragmentation ion
214
patterns were compared with those of the chemical standard in total monitoring mode.
215 216
Measurement of fresh weight loss
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Three replications of fruit in each treatment were separated for weight loss
219
investigation. The initial weight of each fruit was measured and recorded before storage in
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the cold room. Fruit were weighed every 2 d. The fruit weight loss (%) was determined as the
221
difference between the initial and final weights and compared with the initial weight.
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Measurement of fruit firmness
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Three replications of fruit in each treatment were measured for fruit firmness through
226
the peel. Two measurements were taken on the two opposite sides of the fruit using a Texture
227
Analyzer (model: TA-XTPlus, Stable Micro Systems Ltd.; Surrey, England) equipped with a
228
5 mm diameter puncture probe. The penetration speed of the probe was fixed at 5 mm/s and
229
the probe penetrated 10 mm into the fruit. The fruit firmness value was expressed in
230
Newtons.
231 232 233
Statistical analysis
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The data were presented as mean values ± SE. The SPSS analysis of variance
235
procedure (SPSS Inc.; Chicago, IL, USA) was used to determine the treatment effects, and
236
mean separations were analyzed using Duncan’s multiple range test at p < 0.05. A t-test
237
(independent) at the 5% level was used to determine treatment mean differences.
238 Results and Discussion
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Soil water potential
242
The soil water potentials in the drought-treated area were significantly lower than
244
those in the untreated control area, and gradually decreased over the time of treatment (Table
245
1). In addition, it was found that the value of the soil water potential of the untreated control
246
at 4 wk after treatment was lower than that for 1‒3 wk. This may have been due to the higher
247
water demand during fruit development. Furthermore, the vapor pressure deficit (VPD) was
248
higher in the fourth week (data not shown). VPD drives transpiration in the plant which is
249
influenced by relative humidity and temperature (Gates et al., 1998).
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Total ascorbic acid concentration and 2,2-diphenyl-2-picrylhydrazyl radical scavenging
252
activity
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Ascorbic acid is one of the most important nutritional factors in fruits and vegetables
255
as it is known to have many biological functions in the human body, acting as an antioxidant
256
which could reduce the risk of many diseases such as cardiovascular disease and cancer
257
(Harris, 1996). Moreover, it is also involved in plants undergoing growth and development
258
processes, including their responses to environmental stress (Lee and Kader, 2000). From the
259
results, the highest total ascorbic acid concentrations were found in sugar apple fruit from
260
drought-exposed trees at harvest and at 2 d after storage at 15ºC or 10ºC. On day 6 and day 8
261
after storage, the total ascorbic acid concentration in fruit from drought-exposed trees kept at
262
10ºC was highest, followed by fruit from untreated control trees kept at 10ºC and the fruit
263
from drought-exposed trees kept at 15ºC (Fig. 1A). The increase in ascorbic acid in sugar
264
apple fruit from the drought-treated trees and also in fruit at lower storage temperature may
265
have been caused by abiotic stress conditions. Normally, the stress conditions can induce
266
ABA biosynthesis which can promote H2O2 production during periods of stress; H2O2 is
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classified as a kind of stress signaling that may induce the antioxidant system in plant to
268
maintain or increase the ascorbic acid content (Bayoumi, 2008). Gallie (2013) reported that
269
maintaining a normal level of ascorbic acid in plant cells is a consequence to the tolerance of
270
reactive oxygen species (ROS) from stress conditions without increasing sensitivity to
271
drought conditions. Radical scavenging activity is an indicator of antioxidant functionality and activity
273
and is related to the presence of bioactive compounds (Heim et al., 2002). EC50 refers to the
274
concentration of substrate that indicates a 50% loss in DPPH scavenging activity. A high
275
EC50 value indicates low antioxidant activity. Drought stress for 30 d before harvesting
276
significantly decreased the DPPH EC50 values in both the peel and pulp of sugar apple at
277
harvest (Fig. 1B and 1C). Subsequently, the DPPH EC50 value significantly decreased in the
278
peel of fruit from drought-exposed trees kept at 10ºC, and this was lower than those in other
279
treatments (Fig. 1B).
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The fruit from drought-exposed trees kept at 10ºC delayed an increase in the DPPH
281
EC50 value in pulp and had a lower value than in other treatments. The DPPH EC50 value in
282
pulp of fruit from drought-exposed trees kept at 15ºC gradually increased throughout the
283
storage period. At the end of storage, the DPPH EC50 values in pulp of fruit kept at 10ºC were
284
lower than at 15ºC, with no significant differences between treatments (control and drought
285
treatment) at the same storage temperature (Fig. 1C). Alali et al. (1999) reported that sugar
286
apple peel contains many bioactive compounds and that the peel is an important part of the
287
fruit which is exposed to environment, and functions as protection against infection by
288
pathogens and pests. Therefore, to understand sugar apple fruit physiology, the current study
289
involved the measurement of the DPPH scavenging activity in the peel separately from the
290
pulp.
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The generation of ROS has been shown to be induced by water deficit (Shao et al.,
292
2008). The enhancement of production and the ability of antioxidants may play an important
293
role in the detoxification of ROS. Samieiani and Ansari (2014) reported that water stress
294
raised the DPPH radical scavenging activity in many groundcover plants. As noted, ascorbic
295
acid has a function as an antioxidant (Noctor and Foyer, 1998). In the current study, the
296
ascorbic acid concentration and DPPH radical scavenging activity were enhanced in fruit
297
from drought-exposed trees at harvest, and 2 d after being held at 15ºC or 10ºC. This
298
suggested that an increase in ascorbic acid concentrations may have a role in the scavenging
299
of free radicals, which accumulate under drought conditions as mentioned earlier. In general,
300
the degradation of ascorbic acid is rapid after harvesting and increases as the storage time and
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temperature increases (Nunes, 2008). Previous study reported that ascorbic acid concentration
302
in broccoli stored at 1ºC decreased progressively during storage (Serrano et al., 2006). In
303
contrast, the ascorbic acid concentration in citrus fruits (lemon, orange and lime) was higher
304
at 20ºC than at 30ºC (Njoku et al., 2011). These results indicated that the change in the
305
ascorbic acid concentration in a plant is temperature dependent. The current study found that
306
there were higher ascorbic acid concentrations and lower DPPH EC50 values in sugar apple
307
fruit stored at lower temperature after 4 d of storage, especially with fruit from drought-
308
exposed trees. Moreover, the DPPH EC50 values were lower in the peel than in the pulp (Fig.
309
1B and 1C), indicating that sugar apple peel had a higher antioxidant activity than sugar apple
310
pulp. This finding may imply that sugar apple fruit accumulates various antioxidants in the
311
peel to prevent infestation of pests and diseases (Manochai et al., 2014). Although the sugar
312
apple peel is not usually a consumed part of the fruit, as it has high free radical scavenging
313
effects, it is possible that it can be used as a source of antioxidant in the pharmaceutical and
314
food industry. In addition, for greater understanding of the effect of drought stress treatment
315
on the antioxidative system in sugar apple fruit, changes in the enzymatic antioxidant activity
316
should be investigated in future work.
317
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Sugar concentration
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Soluble sugar is one of the major osmotic compounds that accumulate in the fruit of
321
many kinds of fruit trees (Ripoll et al., 2014). The sugar concentration in sugar apple is an
322
important parameter that relates to fruit quality, especially to the taste and has also been
323
related to the response to drought stress (Kowitcharoen et al., 2017). The current study found
324
that the fructose, glucose and total sugar concentrations significantly increased in the fruit
325
from drought-stressed trees at harvest. (Fig. 2A ̶ 2C). Previous studies also reported that
326
drought stress induced sugar accumulation in Satsuma mandarin (Citrus unshiu Marc.
327
‘Okitsu-Wase’; Yakushiji et al., 1998) and peach (Kobashi et al., 2000). This suggested that
328
the increase in sugar concentrations may be associated with plant defense mechanisms
329
against drought stress. Sugar acts as a compatible solute that accumulates in cells, which has
330
a role in osmotic adjustment, preventing turgor loss in tissue (Clifford et al., 1998). Moreover,
331
sugar enhancement by drought may improve the quality of sugar apple fruit, as such fruit was
332
sweeter. During storage, the fructose, glucose and total sugar concentrations in fruit from the
333
drought-exposed trees stored at 15ºC significantly increased and were higher than those for
334
the other treatments during the first 4 d of storage (Fig. 2A ̶ 2C). Thereafter, the fructose,
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glucose and total sugar concentrations increased at 15ºC, and corresponded with increasing
336
ethylene production. At the end of storage, there was no difference in the levels of fructose
337
and glucose in the fruit from control trees stored at 15ºC and the fruit from drought-exposed
338
trees stored at 15ºC or 10ºC, but these levels were significantly higher than those in the fruit
339
from control trees stored at 10ºC (Fig. 2A and 2B). However, the total sugar concentrations
340
were significantly higher in fruit from both control and drought-exposed trees stored at 15ºC
341
compared with fruit stored at 10ºC (Fig. 2C). Vishnu Prasanna et al. (2000) reported an
342
elevated sugar concentration at high temperatures (25ºC and 20ºC) compared with low
343
temperatures (15ºC or 10ºC). Ethylene has been implicated as having a role in the conversion
344
of starch to sugar in fruit (Watkins, 2003). During the ripening process of climacteric fruit,
345
the fruit emit ethylene and the ethylene signal causes the hydrolysis of starch into soluble
346
sugars such as sucrose and glucose, associated with acceleration of amylase in order to
347
increase sweetness (Koning, 1994). This could imply that induction of sugar accumulation in
348
sugar apple fruit during storage was associated with ethylene production. Although high
349
sugar and antioxidant activity were observed in sugar apple fruit from drought-stressed trees,
350
reductions in fruit size and weight were found (data not presented). Cells were smaller in pear
351
fruit (Pyrus communis L.) that had experienced water deficit (Lopez et al., 2011).
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Ethylene production and respiration rate
At harvest, the drought stress treatment had no significant effect on ethylene
356
production and the respiration rate in sugar apple fruit (Fig. 3A and 3B). The ethylene
357
production rate in fruit from the drought treatment that was kept at 15ºC sharply increased to
358
a peak on day 4. The changes in ethylene production in fruit from the untreated control kept
359
at 15ºC or 10ºC showed a similar trend, which increased and reached a peak on day 6 and
360
decreased thereafter (Fig. 3A). This finding agreed with a previous report that water deficit
361
causes an increase of endogenous ethylene, which leads to accelerated ripening of fruit such
362
as bananas (Burdon et al., 1994). Storage temperature affected the rate of ethylene production,
363
where a higher rate was observed in fruit held at 15ºC compared with fruit held at 10ºC,
364
indicating that higher temperatures accelerated physiological changes. These results
365
suggested that drought stress may induce fruit ripening by enhancing ethylene production,
366
especially at higher storage temperatures and as a consequence of the accumulation of a
367
higher sugar concentration.
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Drought stress had no effect on the respiration rate during storage. In fact, increasing
369
the respiration rate in sugar apple fruit positively correlated with increasing the storage time
370
(Fig. 3B). The rate of increase was significantly higher at 15ºC than at 10ºC. These results
371
suggested that low temperatures may slow down the metabolic activities and consequently
372
delay fruit senescence as reported in many studies (Mworia et al., 2012; Freitas and Mitcham,
373
2013; Li et al., 2017). Storage at 10ºC effectively reduced the respiration and ethylene
374
production rate resulting in delayed fruit ripening and retardation of sugar accumulation.
375 376
Endogenous abscisic acid concentration
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ABA is an important plant hormone associated with fruit development, physiology
379
and drought stress tolerance (Ripoll et al., 2014). ABA synthesis is a rapid response to
380
drought stress in plants and is also a health-promoting phytochemical that can be found in
381
fruits and vegetables and is effective against diseases such as type II diabetes, obesity-related
382
inflammation and atherosclerosis-induced hypertension (Guri et al., 2007; 2010). To
383
understand sugar apple fruit physiology, changes in the ABA concentration in fruits subjected
384
to drought stress during storage were analyzed in the peel separately from the pulp. This
385
study found that the endogenous ABA concentrations in the peel and pulp of sugar apple fruit
386
from drought-exposed trees at harvest were significantly higher than those in the control,
387
well-watered trees (Fig. 4A and 4B). The increasing ABA concentration may be associated
388
with drought stress tolerance systems (Kowitcharoen et al., 2015). This result was consistent
389
with a previous report that endogenous ABA concentrations in peach fruit increased
390
significantly under drought stress (Kobashi et al., 2000). The synthesis of ABA appeared to
391
change over time during the first 4 d of storage; the ABA concentrations in the peel and pulp
392
of fruit from drought-exposed trees stored at either 15ºC or 10ºC remained higher than those
393
of fruit from untreated trees stored at these temperatures (Fig. 4A and 4B), indicating that
394
drought stress affected the ABA accumulation in the peel and pulp of sugar apple. However,
395
over the following days of storage, temperature also seemed to affect ABA accumulation,
396
where fruit from drought-exposed and untreated trees held at 15ºC had increased ABA
397
concentrations. This result may have been due to drought stress and the higher temperatures
398
inducing fruit senescence, which led to an increase in ABA synthesis. Kondo et al. (2002)
399
reported that trans-ABA may increase with mangosteen fruit senescence. The change in the
400
ABA concentrations in the current experiment implied that a pre-harvest drought stress
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treatment may provide a way to stimulate the synthesis and accumulation of this beneficial
402
phytochemical in sugar apple.
403 404
Fresh weight loss and fruit firmness
405 The percentage of fresh weight loss in sugar apple fruits from the untreated control
407
and drought-exposed trees kept at 15ºC substantially increased with storage time. While a
408
gradual increase in the weight loss was recorded in sugar apple fruit from untreated control
409
and drought-treated trees kept at 10ºC, there was a significant difference in the fresh weight
410
loss between storage temperatures (Fig. 5). A higher weight loss at higher temperature could
411
be related to the acceleration of transpiration, respiration and ripening as previously reported
412
by Lebibet et al. (1995) and Vishnu Prasanna et al. (2000). In the current study, the rate of
413
respiration increased rapidly at 15ºC (Fig. 3B) and may have been the main factor influencing
414
the weight loss that was observed.
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Drought stress treatment for 30 d before harvesting the sugar apple fruit had no
416
significant effect on fruit firmness at harvest (Fig. 6). However, the fruit firmness in all
417
treatments decreased as storage progressed and as storage temperature increased. Fruit from
418
drought-exposed tree stored at 15ºC showed rapid loss of firmness compared with that of
419
other treatments. This could be ascribed to drought stress accelerating fruit softening as
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mentioned above, and this response may be associated with the induction of ethylene
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production in response to drought stress. An ethylene-mediated response may up-regulate cell
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wall modification enzymes and subsequently accelerate softening (Toivonen and Hodges,
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2011). In addition, the current study found that the decrease in fruit firmness in all treatments
424
had a very high correlation with fresh weight loss (R2 = 0.93, data not shown); this change
425
was due to the fresh weight loss causing a reduction in turgor pressure (Harker and Hallett,
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1994). These results were in agreement with Shackel et al. (1991) who found a decrease in
427
turgor pressure of tomato (Lycopersicon esculentum Mill.) coincided with losses in fruit
428
firmness.
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In summary, ascorbic acid, sugar and ABA concentrations and antioxidant activity
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increased in fruit from drought-exposed trees at harvest. In addition, drought stress also
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activated antioxidant activity, enhanced sugar accumulation and induced fruit ripening in
433
sugar apple fruit during low temperature storage. The changes in fruit qualities were
434
temperature dependent; at 15ºC the sugar apple ripened faster than at 10ºC. These results
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suggested that pre-harvest drought stress treatments may enhance the eating quality of sugar
436
apples, especially the increase of antioxidant activity and sugar concentration. However, the
437
induction of fruit ripening by drought stress treatment must be considered for application in
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proper postharvest technology.
439 Conflict of interest
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There is no conflict of interest.
443 Acknowledgments
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The authors thank the Thailand Research Fund through the Royal Golden Jubilee PhD
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program under grant No. PHD/0039/2554 and the Japan Student Service Organization
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(JASSO) for their financial support.
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Table 1 Values (mean ± SE) of soil water potential in untreated control and drought-treated
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areas within the sugar apple orchard.
602 603 Soil water potential (bar) Untreated control Drought 604 605 -0.09 ± 0.006 ns -0.09 ± 0.006 606 -0.10 ± 0.023 * -0.37 ± 0.029 607 -0.10 ± 0.023 * -0.47 ± 0.040 608 609 -0.06 ± 0.023 * -0.57 ± 0.052 610 -0.31 ± 0.020 * -0.62 ± 0.046 611 * Significant at the 5% level using t-test † ns: non-significant
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Fig. 1 Total ascorbic acid (A) concentration in sugar apple fruit, and DPPH radical
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scavenging activity (EC50 value) in peels (B) and pulps (C) of sugar apple fruit subjected to
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drought stress at harvest and during storage at 10ºC or 15ºC. Data are means ± SE of three
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Fig. 2 Fructose (A), glucose (B), and total sugar (C) concentrations in sugar apple fruit
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Fig. 3 Ethylene production (A) and respiration rate (B) of sugar apple fruit subjected to
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Fig. 4 Endogenous ABA concentration in peel (A) and pulp (B) of sugar apple fruit subjected
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Fig. 5 Fresh weight loss of sugar apple fruit subjected to drought stress at harvest and during
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Fig. 6 Fruit firmness of sugar apple fruit subjected to drought stress at harvest and during
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