Postharvest Biology and Technology 94 (2014) 104–111
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Postharvest Biology and Technology
journa l h om epa ge : www.elsev ier .com/ locate /postharvbio
ostharvest ethylene conditioning as a tool to reduce quality loss oftored mature sweet oranges
aría T. Lafuente ∗, Fernando Alférez, Paco Romeronstituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Consejo Superior de Investigaciones Científicas, Av. Agustín Escardino, 7, 46980aterna-Valencia, Spain
r t i c l e i n f o
rticle history:eceived 11 February 2014ccepted 23 March 2014
Ethylene is related to senescence but also induces protective mechanisms against stress in plants. Thecitrus industry only applies the hormone to induce fruit degreening. The aim of this work was to deter-mine the effect of ethylene on the quality of colored citrus fruit stored under commercial conditions toextend postharvest life, since it protects them from stress causing postharvest disorders such as chillinginjury (CI) and non-chilling peel pitting (NCPP). The effect of conditioning mature Navelate and LaneLate sweet oranges (Citrus sinensis L. Osbeck) for 4 days with 2 �L L−1 ethylene at 12 ◦C, rather than athigher temperatures used for degreening, on the quality of fruit stored at 2 or 12 ◦C, was examined. Theethylene conditioning (EC) treatment did not increase color but reduced calyx abscission and NCPP infruit of both cultivars stored at 12 ◦C, and also CI in Navelate fruit at 2 ◦C. Lane Late fruit did not develop CIbut showed a new disorder in EC fruit held at 2 ◦C. This disorder began as scalded areas around the fruitstem end and extended over the fruit surface during storage. EC had no deleterious effect on the qualityof Navelate oranges stored at either 2 or 12 ◦C. Similar results were found in Lane Late fruit althoughEC slightly increased off-flavor perception at 2 ◦C and the maturity index at 2 and 12 ◦C. Moreover, ECslightly increased the content of bioactive flavonoids in the pulp of Navelate fruit but significant differ-
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ences between control and EC fruit were only found after prolonged storage at 2 C. In Lane Late fruit,EC avoided the initial decrease in flavonoid content found in control samples. Results show, therefore,that EC at 12 ◦C may be a tool to extend postharvest life of NCPP and CI-sensitive oranges, and that thetolerance of citrus cultivars to the combined effect of EC and non-freezing low temperature (2 ◦C) shouldbe tested to select the proper storage temperature.
Several types of postharvest physiological disorders affectingeel quality have been described in citrus fruit but factors causinghem and how they are related to each other is not understoodGrierson, 1986; Lafuente and Zacarías, 2006; Magwaza et al.,013). Efforts have been made to reveal factors responsible forifferent postharvest citrus fruit disorders and to develop newtrategies to control them. However, currently no proven method-logies exist to reduce their incidence. Most studies on reduction
f physiological peel disorders have been related to methodologiesiming at reducing chilling injury (CI), and the effect on citrus fruit
quality has been well characterized (Schirra et al., 2005; Mulas andSchirra, 2007).
CI and non-chilling peel pitting (NCPP), also known as rind-staining, are two main postharvest physiological disorders in citrusfruit (Lafuente and Zacarías, 2006). NCPP develops at temperatureshigher than those causing CI, which in citrus fruit may occur at tem-peratures ranging between 1 and 12 ◦C. Sweet oranges from theNavel group may develop these physiological disorders (Lafuenteand Sala, 2002; Alférez et al., 2003; Holland et al., 2005). Navelateorange (Citrus sinensis (L.) Osbeck) is prone to develop NCPP as wellas CI after prolonged cold storage. In this cultivar, NCPP is character-ized by depressed areas in the peel affecting both the albedo (innerpart of the peel) and the flavedo (outer part of the peel), whicheventually turn bronze in color; while CI is manifested as superfi-
cial scalding or flavedo bronze non-depressed areas (Alférez et al.,2005). The postharvest performance of other late maturing sweetNavel orange cultivars, which have expanded very quickly in manycountries to extend the citrus season, is less known. This is the case
f the late-maturing Lane Late orange (C. sinensis (L.) Osbeck) thatas good fruit quality characteristics.
Ethylene is usually related to senescence but it plays also a rolerotecting plants from stress causing damage (Yang and Hoffman,984). Although this hormone may enhance a senescence-relatedhysiological disorder, rind breakdown in Nules Clementine man-arin (Cronjé et al., 2011), or other peel defects such as zebra skinr horseshoe/green rings in other citrus cultivars (Krajewsky andittaway, 2002), it also protects citrus fruit from stresses caus-ng CI and NCPP (Lafuente et al., 2001; Lafuente and Sala, 2002).he industry only applies this hormone to early-maturing citrusruit for degreening purposes although it may reduce physiologi-al disorders in mature fruit harvested after color break (Lafuentend Sala, 2002; Lafuente et al., 2004; Cajuste and Lafuente, 2007).he effect on the quality of citrus fruit of postharvest degreen-ng at 90% RH and 20–22 ◦C using 2–4 �L L−1 ethylene, has beenocumented (Carvalho et al., 2008; Mayuoni et al., 2011a; Sdirit al., 2012a,b; Moscoso-Ramírez and Palou, 2014). Previous datalso show that conditioning mature citrus fruit with ethylene con-entrations ranging between 1 and 10 �L L−1 considerably reducesCPP in fruit stored afterwards at 20–22 ◦C and increases peel phe-olic content (Lafuente and Sala, 2002; Cajuste and Lafuente, 2007).oreover, it is known that physiological responses of citrus fruit to
thylene may vary with maturation stage (Lisker et al., 1983; John-aruppiah and Burns, 2010). However, there is no information on
he effect of ethylene on the quality of fully mature fruit stored forrolonged periods at lower temperatures under commercial condi-ions required to extend postharvest life. Experiments performedo evaluate the effect of degreening have been limited to exam-ning changes occurring after a single quarantine treatment (1 ◦Cor 16 days) in fruit of citrus cultivars that did not develop physi-logical disorders (Sdiri et al., 2012a), while storage duration andemperature affects citrus fruit quality (Mulas and Schirra, 2007;benland et al., 2008). Therefore, the aim of this work was to eval-ate the effect of treating full colored mature sweet Navelate andane Late orange cultivars, which are not degreened for commer-ialization, for 4 days with 2 �L L−1 ethylene at 90–95% relativeumidity (RH) on the quality of fruit stored for up to 60 days at
or 12 ◦C and 90–95% RH. This ethylene conditioning (EC) treat-ent was performed at 12 ◦C rather than at the temperature used
or commercial degreening (20–21 ◦C) (Mayuoni et al., 2011a; Sdirit al., 2012a,b). The degreening temperature is recommended tonable color development after ethylene treatment. Fruit in thistudy were already fully colored, and therefore a lower temper-ture (12 ◦C) was used to reduce fruit quality loss. The effects ofhe treatment on color, firmness, weight loss and internal qualityf the fruit, as well as the incidence of calyx abscission, decay andhysiological disorders were evaluated. Considering that ethylene
ncreases the activity of the enzyme phenylalanine ammonia-lyasePAL) in citrus fruit, especially in the more mature fruit (Liskert al., 1983), that this is a key enzyme at the entry point of phenyl-ropanoid metabolism, and the health benefits of phenolics (Tripolit al., 2007), we have also evaluated in the fruit edible portion theffect of the EC treatment on the accumulation of total phenos andavonoids, which are the most abundant phenolics in citrus fruitNogata et al., 2006).
. Materials and methods
.1. Plant material and postharvest treatments
Fully mature Navelate and Lane Late sweet oranges (C. sinen-is (L.) Osbeck) were harvested in March from the same orchardt Lliria (Valencia, Spain), and immediately delivered to the lab-ratory. Fruit were surface-sterilized with a commercial bleach
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solution (Ballester et al., 2013) and then randomly divided into 2groups that were treated with ethylene (group 1) or held in air(control fruit, group 2). These groups were further subdivided intotwo subgroups to evaluate the effect of the storage temperature (2and 12 ◦C). Fruit in each subgroup were divided into two lots. Threereplicates of 10 fruit were included in the first lot to estimate peri-odically the incidence and severity of physiological disorders andof calyx abscission. The second lot contained 3 replicates of 10 fruitper storage period since they were used to determine changes inpeel color but also in other quality attributes in the pulp requiringdestructive methods. Fruit within the first group were conditionedfor 4 days with 2 �L L−1 ethylene at 90–95% RH and 12 ◦C (EC fruit),and then stored at 2 (subgroup 1) or 12 ◦C (subgroup 2). Controlfruit were maintained for 4 days in air at 90–95% RH and 12 ◦C (ACfruit) before being stored at 2 or 12 ◦C. EC and AC fruit were keptfor 8 weeks at 2 ◦C or for 6 weeks at 12 ◦C and then transferred to20 ◦C for 4 days to simulate a shelf-life (SL) period.
2.2. Estimation of postharvest physiological disorder severity andincidence
Three different physiological disorders were identified underthe above mentioned storage conditions. Since their symptomswere different, severity was evaluated independently during hold-ing of AC and EC fruit at 2 and 12 ◦C, and after the SL period.Depending on the cultivar, postharvest conditioning treatment orstorage condition, fruit showed: (1) NCPP, manifested as collapsedsurface areas that became dark brown with time (Supplemen-tal Fig. S1A); (2) CI, manifested as bronzed non-depressed areas(scalding) of the fruit surface (Supplemental Fig. S1B); and/or (3) aphysiological disorder, which as far as we know has not been previ-ously described in citrus fruit, that begins as scalded areas aroundthe stem end of the fruit (Supplemental Fig. S1C) and extendsthrough the fruit surface during storage (Supplemental Fig. S1D).This disorder, named SECI (from stem end chilling injury), showsnon-depressed areas in the fruit stem end, and therefore it is dif-ferent to stem-end-rind-breakdown (SERB). A rating scale from 0(no damage) to 4 (severe damage) (Vicente et al., 2013) was used todetermine the average severity damage index of each physiologi-cal disorder. In this scale, only fruit showing a damage score higherthan 1 would be rejected by consumers. The same fruit were used atthe various evaluation dates. The severity indexes were calculatedby adding up the products of the number of fruit in each categorymultiplied by its score, and then dividing the total obtained by thenumber of fruit, evaluated as described by Lafuente et al. (1997).Moreover, the commercial incidence of each physiological disor-der was estimated by calculating the percentage of fruit showinga damage score higher than 1. The results were the means of 3replicate samples containing 10 fruit each ± S.E.M.
Supplemental Fig. S1 related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.postharvbio.2014.03.011.
2.3. Analysis of quality attributes
Changes in fruit quality were assessed by measuring the acid-ity and SSC in the pulp, peel fruit color, and fruit firmness andweight loss as described by Holland et al. (1999) and Lafuenteet al. (2011). Soluble solids (◦Brix) were determined from fruit juicewith an Atago/X-1000 digital refractometer (Atago Co. Ltd., Tokyo,Japan) and acid content was titrated with 0.1 N NaOH using phenol-phthalein as indicator and expressed as g of anhydrous citric acid
in 100 mL of juice. The maturity index was calculated by dividingthe ◦Brix of the extracted juice by its acid content. Peel color wasanalyzed by using a Minolta CR-300 Chromameter (Konica MinoltaInc, USA) with a measuring area of 8 mm at four locations around
Fig. 1. Non-chilling peel pitting (NCPP) index of Navelate and Lane Late orangesstored at 2 or 12 ◦C after being conditioned at 12 ◦C for 4 days with 2 �L L−1 ethylene(EC, white) and of their respective controls maintained 4 days in air at 12 ◦C priorstorage (AC, black). Dashed line indicates transfer of fruit at 20 ◦C to simulate shelf-
06 M.T. Lafuente et al. / Postharvest Bio
he equatorial plane of the fruit. The color index was expressed ashe a/b Hunter ratio that is classically used for color measurementn citrus fruit (Stewart and Wheaton, 1972). This ratio is negativeor green fruit and positive for orange fruit, while zero value cor-esponds to yellow fruit at the midpoint of color break period.
eight loss was expressed as the cumulative percentage of fruiteight loss during fruit storage and fruit firmness determined using
TA-XT2 Texture Analyser (Stable Micro Systems, Godalming, UK)nd expressed as the percentage of fruit deformation resulting from
pressure of 10 N on the fruit longitudinal axis at a constant speedSdiri et al., 2013). Along with these quality attributes, sensory eval-ation was performed by eight semi-trained panelists as describedy Sdiri et al. (2012a) using a rating scale from 1 to 10, where0 = excellent, 5 = fair (commercially acceptable), and 1 = extremelynpleasant. The average flavor acceptance was calculated follow-
ng the same procedure described above to calculate the averagendex severity of peel disorders.
.4. Evaluation of calyx abscission and decay incidence
The incidence of calyx abscission and decay was evaluated afteronditioning the fruit with ethylene at 12 ◦C, during fruit storage at
and 12 ◦C and after the SL period. Calyx abscission incidence wasxpressed as the percentage of fruit without calyx compared to theotal number of fruit in each replicate sample and decay incidencestimated by quantifying the percentage of decayed fruit.
.5. Analysis of total phenolics and flavonoids
Total phenolic content was determined following a methodased on that described by Swain and Hillis (1959) as reported byajuste and Lafuente (2007). Briefly, 500 �L juice were extractedith 1500 �L of ethanol using a Mini Beadbeater 8 Cell Disruptor
Biospec Products, Inc.). The extract was centrifuged at 13,000 × gor 5 min at 4 ◦C and the supernatant used to estimate phenolicontent. Two sample aliquots of 250 �L of 5-fold diluted super-atant were incubated at room temperature with 250 �L of 1 Nolin-Ciocalteau and 2500 �L of 2% Na2CO3. After centrifugation at3,000 × g for 5 min at 4 ◦C, absorbance was recorded at 724 nm andhe total phenolic content determined by using a standard curveeveloped with chlorogenic acid.
Total flavonoids were determined in the same supernatant usedo analyze total phenolics by following the method described byonzález-Aguilar et al. (2007) with some modifications. Two super-atant aliquots of 100 �L were diluted with 400 �L of nanopureater and 30 �L of 5% NaNO2 were added to each aliquot. After
min incubation at room temperature, 30 �L of 10% AlCl3 weredded and at 6 min the reaction was stopped by adding 200 �Lf 1 N NaOH. Flavonoid contents were determined by comparinghe absorbance at 350 nm with that of prepared blanks, calculatedsing a standard curve prepared with hesperidine, which is theajor flavonoid in the pulp, and results expressed as mg 100 g−1.
he total phenolic and flavonoid contents were determined in theame fruit used to analyze quality attributes. Results are the meansf three biological replicate samples ± S.E.M.
.6. Statistical analysis
A mean comparison using the Tukey’s test was performed toetermine if means values were significantly different (P ≤ 0.05)etween EC and AC control fruit for each cultivar and at each exper-
mental storage temperature and sampling period.
life (SL). Results represent the means of three replicates samples ± S.E.M. Asterisksindicate significant differences (P < 0.05) between EC and their respective controlsamples (AC) for the same storage period.
3. Results and discussion
3.1. Effect of ethylene conditioning on postharvest physiologicaldisorders of citrus fruit stored at chilling and non-chillingtemperatures
The effects of ethylene degreening on quality attributes of dif-ferent still-green citrus fruit cultivars have been reported (Carvalhoet al., 2008; Mayuoni et al., 2011a; Sdiri et al., 2012a,b). However,these effects were not evaluated after prolonged storage, whichfavors the development of physiological disorders. Moreover, still-green citrus fruit are less sensitive to develop CI (Lafuente et al.,1997) and NCPP (Alférez and Zacarías, 2013) than mature coloredfruit. The efficacy of EC treatments at 20 ◦C reducing NCPP in matureNavelina and Navelate sweet oranges stored under non-stressfulenvironmental conditions (20 ◦C and 90–95%RH) has been shownduring different citrus seasons (Lafuente and Sala, 2002; Cajusteet al., 2010; Vicente et al., 2013). Nevertheless, the effect of EC at12 ◦C on the incidence of physiological peel disorders and otherquality attributes of fruit that do not need to be degreened, storedat lower temperatures, remains unknown. Two storage tempera-tures were selected in the present study. Fruit were stored at 2 ◦C,to maximize the storage period and to test the combined effectof ethylene and low temperature stress, and at 12 ◦C which mightavoid the appearance of CI and delay fruit aging as compared toholding the fruit at 20 ◦C.
NCPP symptoms were evident by 20 days at 12 ◦C in the ACNavelate and Lane Late oranges and increased thereafter (Fig. 1).While the susceptibility of Navelate fruit to postharvest NCPP iswell known (Alférez et al., 2003; Sala et al., 2005; Romero et al.,
2012), as far as we know, this is the first study showing that LaneLate orange is also susceptible to the disorder although its suscep-tibility is lower (Fig. 1). By day 20, the NCPP index of AC Navelateoranges held at 12 ◦C was higher than 1, indicating the existence
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Table 1Effect of ethylene-conditioning (EC) as compared to air-conditioned (AC) controlfruit on the percentage (%) of fruit showing non-commercial external quality causedby NCPP during fruit storage at 2 and 12 ◦C and a subsequent shelf-life (SL) periodof 4 days at 20 ◦C. AC: air-conditioned control fruit. EC: ethylene-conditioned fruit.
Fig. 2. Chilling injury (CI) index of Navelate oranges and stem-end chilling injury(SECI) index of Lane late oranges stored at 2 ◦C after being conditioned at 12 ◦C for 4days with 2 �L L−1 ethylene (EC, white) and of their respective controls maintained4 days in air at 12 ◦C prior storage (AC, black). Dashed line indicates transfer of fruit
◦
esults represent means of three replicate samples. Values labeled with asteriskithin the same row and storage temperature are significantly different at the 5%
evel according to Tukey’s test.
f fruit lacking the quality required for commercialization. Underhese conditions, 39% of AC Navelate fruit could be rejected becauseow external quality (Table 1). The number of unacceptable Laneate oranges was much lower by this period (11.5%, Table 1). Thisifference between cultivars decreased by 40 days but a lower valueas still found after the SL period in Lane Late fruit. As expected
Lafuente and Sala, 2002; Alférez et al., 2005), keeping AC fruit fromoth cultivars at 2 ◦C delayed the appearance of non-commercialCPP (Table 1) and reduced NCPP severity (Fig. 1) and incidence
Table 1). At this temperature, NCPP index was lower than 0.8 by0 days storage in Navelate oranges and than 0.6 by 60 days in Laneate fruit.
Conditioning the fruit with 2 �L L−1 ethylene at 12 ◦C consid-rably increased the tolerance of oranges of both cultivars to NCPPFig. 1). The treatment was very effective reducing the developmentf NCPP at both storage temperatures since the NCPP index of ECruit was lower than 1 during fruit storage at 2 or 12 ◦C and afterimulation of SL (Fig. 1). This is in concordance with previous find-ngs showing the effect of ethylene reducing NCPP in citrus fruit at0 ◦C (Lafuente and Sala, 2002; Vicente et al., 2013). Furthermore,
t shows that despite ethylene-induced responses being tempera-ure dependent, it is possible to decrease the temperature of theC treatment to reduce NCPP incidence and other negative effectsn quality attributes occurring at higher temperatures. As shown inable 1, the percentage of fruit showing commercially unacceptableCPP at 12 ◦C was lower than 8% even after the SL period in bothultivars while the percentage was higher than 50% in the AC fruit.
Fruit storage at 2 ◦C caused CI in Navelate orange by 40 days,lthough CI severity was very low (Fig. 2, upper panel). The effectf conditioning citrus fruit with ethylene on reducing CI in very chil-ing sensitive citrus cultivars is low (Lafuente et al., 2001). However,I was reduced in the less chilling sensitive citrus cultivar Navelatey the EC treatment (Fig. 2, upper panel). Thus, EC Navelate orangesid not show commercially relevant CI for up to 60 days and itseverity and incidence were low (data not shown).
Results also showed that AC Lane Late oranges did not developI (data not shown) and that fruit from this cultivar conditionedith ethylene developed SECI when stored at 2 ◦C (Fig. 2, loweranel). To the best of our knowledge, SECI has not been previouslyescribed as a postharvest disorder in citrus fruit. It appears in theruit stem-end areas as scalded patches and extends superficiallylong the fruit. Therefore, SECI differs from SERB, a non-chilling
elated peel disorder that manifests as depressed areas in the stemnd is associated with senescence and enhanced by dehydrationGrierson, 1986; Romero et al., 2013). Since SECI was not observedt 12 ◦C, results indicate that it may be caused by the combined
at 20 C to simulate shelf-life (SL). Results represent the means of three replicatessamples ± S.E.M. Asterisks indicate significant differences (P < 0.05) between EC andtheir respective control samples (AC) for the same storage period.
effect of ethylene and chilling in Lane Late oranges, while Navelateoranges are more tolerant of such a combination in spite of develop-ing some CI. Fruit of both cultivars were harvested the same dateand from the same orchard. Therefore, the different responses ofEC Navelate and Lane Late oranges to cold should be more likelyrelated to genetic factors than to different preharvest conditions.
Overall results indicate that applying ethylene immediatelyafter fruit harvest at 12 ◦C could be a tool to reduce the incidence ofphysiological disorders in mature citrus fruit. However, the effectof EC followed by extreme low non-freezing storage temperaturesshould be tested in each citrus cultivar. Thus, result from this workshow that conditioning the NCPP prone Navelate orange with eth-ylene and storing it at 12 ◦C could be a feasible method to reducethis peel disorder. Similar results might be obtained in EC Navelatefruit stored at 2 ◦C since CI was very low. Therefore, storage tem-perature of Navelate oranges conditioned with ethylene should beselected also considering changes in other fruit quality attributes.In contrast, conditioning Lane Late fruit with ethylene could causeSECI if fruit are stored at very low temperatures (2 ◦C), althoughEC fruit kept at 12 ◦C in this cultivar showed very low peel dam-age. Alternatively, storing AC Lane Late fruit at 2 ◦C would also beeffective in reducing NCPP without causing other peel disorders.
3.2. Effect of ethylene conditioning on calyx abscission and
disease incidence
Calyx abscission is an important external fruit quality factorsince it affects freshness and general appearance of fruit. The
108 M.T. Lafuente et al. / Postharvest Biology a
Fig. 3. Calyx abscission incidence (%) in Navelate and Lane Late oranges stored at2 or 12 ◦C after being conditioned at 12 ◦C for 4 days with 2 �L L−1 ethylene (EC,white) and of their respective controls maintained 4 days in air at 12 ◦C prior storage(AC, black). Dashed line indicates transfer of fruit at 20 ◦C to simulate shelf-life (SL).Results represent the means of three replicates samples ± S.E.M. Asterisks indicates(
scidmaiIeftocnfbccie1totfeyrLaat(f
the EC treatment did not consistently modify this quality parameter
ignificant differences (P < 0.05) between EC and their respective control samplesAC) for the same storage period.
usceptibility to postharvest calyx abscission varies among citrusultivars (Carvalho et al., 2008). Results from degreening exper-ments indicate that the effect of ethylene on calyx abscissionepends on the ethylene doses, the citrus cultivar and the fruitaturity stage (Sdiri et al., 2012a). Our results showed that calyx
bscission was very low at 2 ◦C and that the EC treatment reducedt in fully mature fruit from both cultivars stored at 12 ◦C (Fig. 3).n contrast, treating Clemenules mandarins with 2 �L L−1 ethyl-ne at 21 ◦C for degreening purposes favored calyx abscission inruit stored for 16 days at 1 ◦C (Sdiri et al., 2012a). Results fromhe same authors showed that the treatment had a different effectn the early-ripening Navelina orange. In this cultivar, it inducedalyx abscission in deep green fruit but its effect was variable andegligible in more mature fruit, which is in concordance with the
act that the less mature fruit (deep green), the more suscepti-le to abscission (Sdiri et al., 2012a). Further research should beonducted to elucidate whether the effect of ethylene on reducingalyx abscission in mature Navelate and Lane Late (Fig. 3) orangess related to fruit maturity stage and/or the temperature at whichthylene was applied. The ethylene treatment was performed at2 ◦C rather than at 21 ◦C since the aim of the EC treatment waso reduce physiological disorders and minimize fruit aging insteadf favoring degreening at 21 ◦C. In this context, it should be notedhat PAL activity increases in the calyx abscission zone of citrus fruitavoring wound healing and thus reducing abscission (Kostenyukt al., 2002), and that the rise in PAL activity in response to eth-lene or other stress factors inducing the hormone is much lesselevant in green than in mature citrus fruit (Lisker et al., 1983;afuente et al., 2003). It is also known that hormonal balances play
role in controlling abscission (Meir et al., 2010), that indole-3-cetic acid (IAA) may reduce abscission (Yuan et al., 2003) and
hat ethylene stimulates the degradation of IAA in citrus tissuesWiner et al., 2000). Since the capacity of flavedo of citrus fruitor IAA catabolism decreases at the beginning of fruit senescence
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(Chamarro et al., 2001), fully mature fruit might be less suscepti-ble to ethylene-induced degradation of IAA. The complexity of theabscission process is highlighted by results of Sdiri et al. (2012a).These authors found that abscission was higher in less mature cit-rus fruit despite young citrus fruit having higher IAA levels in theabscission zone than mature fruit (Yuan et al., 2003). Therefore, fur-ther research should be conducted to understand factors balancingcitrus fruit responses to ethylene at 12 ◦C and its consequences incalyx abscission.
Exogenous application of ethylene may also affect disease inci-dence. Degreening with high hormone levels may exacerbateDiplodia stem-end rot incidence (Smilanick et al., 2006). Con-versely, ethylene does not stimulate Penicillium digitatum growthand can even reduce green mold incidence in mature citrus fruit(Marcos et al., 2005). Fruit used in the present work were imme-diately disinfected after harvest and held in aseptic cold rooms.Therefore, the incidence of rots, which was mainly caused by P.digitatum, was negligible both in AC and EC fruit (data not-shown).Diplodia stem-end rot is not common in citrus fruit growing inMediterranean climate so it was not surprising the absence of rotscaused by this pathogen. Nevertheless, the incidence of the EC treat-ment on decay should be further tested in other citrus producingareas with wet climate and high incidence of Diplodia stem-endrot.
3.3. Effect of ethylene conditioning on fruit quality attributes
Weight loss was very low, and similar in AC and EC fruit, dur-ing the whole storage period in both Navelate (Table 2) and LaneLate oranges (Table 3) kept either at 2 or 12 ◦C. The increase inweight loss occurring after SL simulation was higher in Lane Lateoranges, which were less susceptible than Navelate oranges toNCPP. Although weight loss was very low, this result reinforces theidea that dehydration is not the only factor responsible for NCPPdevelopment (Cajuste and Lafuente, 2007), and points out the rel-evance of genetic factors in the susceptibility of citrus fruit to thisphysiological disorder.
Fruit firmness slightly decreased after 20 days in fruit held at12 ◦C but barely changed at 2 ◦C in Navelate fruit. Moreover, con-ditioning fruit of this cultivar with ethylene had little effect on thisquality attribute (Table 2). A similar effect was observed in Lane Lateoranges (Table 3), which showed higher fruit firmness. Whetherpeel ultrastructure and strength are related to the lower suscep-tibility of this citrus cultivar to NCPP is unknown but this resultagrees with previous findings encouraging research aimed at iden-tifying cell wall influence on citrus fruit susceptibility to developNCPP (Vicente et al., 2013).
The EC treatment slightly reduced pulp acidity in Navelate fruit(4 days, Table 2) but such difference was lost during fruit storage.The EC Lane Late oranges had slightly lower acidity values duringstorage (Table 3). This effect was lost after SL in fruit stored at 12 ◦C.On the other hand, results show that keeping Navelate and LaneLate oranges at 2 ◦C delayed the decline in pulp acidity with respectto fruit held at 12 ◦C (Tables 2 and 3).
The storage temperature had little impact on soluble sugars. The4 day ethylene treatment slightly decreased SSC (∼4%) in the pulpof Navelate fruit but not in Lane Late fruit. Slight differences inSSC were found between AC and EC stored fruit and they were lostafter the SL period. The EC treatment slightly increased the maturityindex in Navelate (Table 2) but not in Lane Late oranges (Table 3).After storage, the maturity index of EC Lane Late oranges was, ingeneral, slightly higher than that of the AC control fruit. However,
in Navelate fruit. After SL, the maturity index of ethylene-treatedfruit of these cultivars was higher than that of their respective ACfruit when they were stored at lowest temperature (2 ◦C).
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Table 2Effect of conditioning Navelate oranges for 4 days with 2 �L L−1 ethylene at 12 ◦C on changes in fruit quality attributes during fruit storage at 2 or 12 ◦C and a subsequentshelf-life (SL) period of 4 days at 20 ◦C. AC: air-conditioned control fruit. EC: ethylene-conditioned fruit.
T (◦C) Days Weight loss (%) Firmness Color (a/b) Acidity (%) % SSC Maturity index Flavor
Results represent means of three replicate samples. Values labeled with asterisk within the same row for each quality attribute are significantly different at the 5% levelaccording to Tukey’s test.
Table 3Effect of conditioning Lane Late oranges for 4 days with 2 �L L−1 ethylene at 12 ◦C on changes in fruit quality attributes during fruit storage at 2 or 12 ◦C and a subsequentshelf-life (SL) period of 4 days at 20 ◦C. AC: air-conditioned control fruit. EC: ethylene-conditioned fruit.
T (◦C) Days Weight loss (%) Firmness Color (a/b) Acidity (%) % SSC Maturity index Flavor
esults represent means of three replicate samples. Values labeled with asterisk wccording to Tukey’s test.
For both cultivars, flavor score decreased during fruit storage.torage at 12 ◦C was finished by 40 days because of deleteriousffects on fruit freshness appearance after this period, while stor-ge at 2 ◦C could be extended to 60 days. By day 40, flavor scoresf Navelate oranges stored at 2 and 12 ◦C were about 6.3 and 7.6,espectively (Table 2). Values found in Lane Late fruit were alsolose to 7 (Table 3). Negligible differences in fruit flavor were foundetween AC and EC fruit and any significant difference was lostfter simulation of SL, except in Lane Late fruit kept at 2 ◦C. In thisarticular sample, EC favored a significant decrease in flavor score,hich is coincident with major fruit SECI damage in this sample.
herefore, the combination of low temperature stress and ethyleneretreatment appears to affect not only the peel but also the pulp inruit of this cultivar. Previous reports indicate that degreening cit-us fruit at 21 ◦C followed by 16 days cold storage have little effectn internal fruit quality in early-ripening cultivars, which show stillreen peel but have reached commercial pulp maturity index (Sdirit al., 2012a,b). Results from the present work confirm this resultn more mature sweet oranges and further show that ethylene pre-reatment does not have a negative effect on internal quality eitherfter prolonged storage at 2 ◦C or at a higher temperature (12 ◦C)hich might enhance fruit aging and consequent quality loss with
espect to fruit held at 2 ◦C.It is well known that the effect of ethylene on citrus fruit color
hanges is highly dependent on the peel maturity stage and thattill green fruit must reach a threshold color to show proper com-ercial color after degreening (Jiménez-Cuesta et al., 1981; Sdiri
t al., 2012a). As expected, the EC treatment barely increasedruit color in fully colored mature Navelate and Lane Late orangesTables 2 and 3). The effect of the hormone was less relevant thanhat of storage temperature. Fruit color increased at both 2 ◦C and
the same row for each quality attribute are significantly different at the 5% level
12 ◦C and the rate of increase was faster at 12 ◦C, which is in con-cordance with results reported in Navelina oranges by Carmonaet al. (2012). Moreover, it has to be mentioned that the effect of theethylene treatment on the general fruit freshness appearance wasalso less relevant than that of the storage temperature.
The health benefits of phenolics are well recognized and it isknown that ethylene increases PAL activity and total phenolics inthe peel of mature Navelate oranges (Cajuste and Lafuente, 2007).Therefore, we examined in the pulp whether the EC treatmentwas able to increase total phenolic content (Fig. 4) and also totalflavonoids (Fig. 5), which are major phenolics in blond orange cul-tivars. At harvest, phenolic content in the edible portion of Navelatefruit was higher than that of Lane Late (Fig. 4). A low but significantincrease in total phenolics was found in response to ethylene (day4) in Lane Late but not in Navelate fruit. Phenolic changes at 2 or12 ◦C were largely irrelevant in AC fruit of both cultivars (Fig. 4). Themore marked effect of the ethylene pretreatment was observed inNavelate fruit kept at 2 ◦C. In this sample, ethylene pretreatmentdecreased phenolic content with respect to AC fruit, but signifi-cant differences were only found by 60 days. Results of this workshowing little effect of ethylene increasing phenolics in the pulpof mature fruit agree with those found in other citrus cultivarsharvested before color break (Mayuoni et al., 2011a; Sdiri et al.,2012b). The activity of PAL, at the entry point of phenylpropanoidbiosynthesis, increases in the peel of mature citrus fruit after ethyl-ene treatment, but not in fruit harvested before color break (Liskeret al., 1983). Therefore, changes found in phenolics in the pulp of
citrus fruit in response to exogenous ethylene might be more likelyrelated to the fact that the peel is an important physical barrierfor gas diffusion into the fruit than to its maturity stage. Neverthe-less, it has to be mentioned that the expression of genes involved
110 M.T. Lafuente et al. / Postharvest Biology a
Fig. 4. Total phenolic content in the pulp of Navelate and Lane Late oranges storedat 2 or 12 ◦C after being conditioned at 12 ◦C for 4 days with 2 �L L−1 ethylene (EC,white) and of their respective controls maintained 4 days in air at 12 ◦C prior storage(AC, black). Dashed line indicates transfer of fruit at 20 ◦C to simulate shelf-life (SL).Rs(
iepo
Faw(Rs(
esults represent the means of three replicates samples ± S.E.M. Asterisks indicateignificant differences (P < 0.05) between EC and their respective control samplesAC) for the same storage period.
n the synthesis of phenolics may increase in the fruit pulp during
thylene-induced degreening (Mayuoni et al., 2011b) despite totalhenolics not increasing (Mayuoni et al., 2011a). Similarly, a previ-us report showed that the hormone had a low and non-consistent
ig. 5. Total flavonoid content in the pulp of Navelate and Lane Late oranges storedt 2 or 12 ◦C after being conditioned at 12 ◦C for 4 days with 2 �L L−1 ethylene (EC,hite) and of their respective controls maintained 4 days in air at 12 ◦C prior storage
AC, black). Dashed line indicates transfer of fruit at 20 ◦C to simulate shelf-life (SL).esults represent the means of three replicates samples ± S.E.M. Asterisks indicateignificant differences (P < 0.05) between EC and their respective control samplesAC) for the same storage period.
nd Technology 94 (2014) 104–111
effect on the flavonoid content during degreening of different cit-rus cultivars (Sdiri et al., 2012b). The ethylene treatment slightlyincreased flavonoid content in the pulp of mature Navelate fruit butsignificant differences were only found by 40 days at 2 ◦C (Fig. 5).In Lane Late oranges, ethylene (4 days in Fig. 5) did not increaseflavonoids, but flavonoid content in EC fruit was higher than inAC fruit for up to 20 days at 2 ◦C. In contrast, no difference wasfound at 12 ◦C after transferring fruit of this cultivar from ethyleneto air (Fig. 5). Total flavonoids remained nearly constant for up to40 days at both temperatures in AC Navelate fruit. A substantialincrease in total flavonoids was only found after this period at 2 ◦C,which may be a consequence of prolonged temperature stress inthe pulp of this chilling susceptible citrus cultivar (Lafuente et al.,2011). In fact, flavonoids did not increase after prolonged storageat 2 ◦C in AC fruit of the chilling tolerant Lane Late fruit (Fig. 5). Inthis cultivar, flavonoid levels were significantly lower in AC thanin EC fruit at the beginning of cold storage (2 ◦C). Considering thestress imposed by the combination of ethylene plus cold in this cit-rus cultivar, and that ethylene by itself did not increase flavonoidlevels, further attention should be paid to understanding whetherthe higher flavonoid levels in EC fruit might be a defense responseto protect the fruit against such stress.
In conclusion, results indicate that EC at 12 ◦C had neither adeleterious effect on internal and external fruit quality nor on theconcentration of phenolics or flavonoids in Navelate orange storedat either 2 or 12 ◦C. Therefore, this treatment might be a useful andfeasible tool to extend postharvest life of NCPP and CI-sensitivecitrus cultivars. On the other hand, results from Lane Late orangesindicate that the tolerance of citrus cultivars to the combined effectof EC and extreme non-freezing low temperature (2 ◦C) should befurther tested to select the proper storage temperature. In these cul-tivars, storage temperatures ranging between 2 and 12 ◦C should betested to be able to reduce NCPP and loss of fruit freshness whileavoiding SECI.
Acknowledgements
This work was supported by research grants AGL2009-11969and CONSOLIDER 2007-00063 from the Comisión Interminis-terial de Ciencia y Tecnología (CICYT), Spain, and by PROME-TEO/2010/010 from the Generalitat Valenciana, Spain. Dr. F. Alférezacknowledges a Ramón y Cajal Contract (Spanish Governement andFondo Social Europeo). Dr. P. Romero was the recipient of a contractin the framework of the project AGL2009-11969.
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