Influence of 1-methylcyclopropene on the bIochemIcal response and rIpenIng of ‘ solo ’ papayas 1

The market demand for tropical fruits has been growing steadily over the past two decades and global papaya production has grown significantly over the last few years. This sector, however, suffers greatly from postharvest losses due to reduced quantity and quality of fruits between harvest and consumption. The use of ethylene inhibitors after harvest could improve the final quality of the fruit to satisfy the consumer and also minimize waste. The physiological and biochemical responses of ‘Solo’ papayas treated with the ethylene inhibitor 1-methylcyclopropene (1-MCP) to extend storage shelf life and maintain quality during long-term storage are deeply discussed in this study. Papaya fruits arrived at Cranfield University (CU) and received a 24 h 1-MCP, being stored at 20 oC for 10 days. The ethylene inhibitor 1-MCP application significantly delayed ‘Solo’ papaya ripeness on fruit storage by reducing respiration rate and ethylene production. There was a delay from 7 days in fruit firmness loss and the retention of green peel colour was increased. Inhibition of ethylene perception by 1-MCP did not prevent the accumulation of sugars and the mean values were similar and higher than those found for control fruits, which are possibly due to the lower reaction speed, leading to a higher accumulation.


IntroductIon
The quality of fruits and vegetables is closely associated with their physical features, such as surface colour, shape and firmness.These characteristics are associated with metabolism and respiration rate during ripening, in which living cells from harvested fruits use oxygen from its own reserves and from the environment, releasing ethylene and carbon dioxide (ROUPhAEL et al., 2010;NAYIK AND MUZAFFAR, 2014).Although the changes that take place during ripening are important, uncontrolled ripening process can lead to rapid degradation in quality, which that contributes to postharvest losses.Thus, it is important to control the ripening process during storage and distribution in order to maintain fruit quality at the best possible level until reaching the consumer (hAMzAh et al., 2013).In recent years, several techniques have been developed to regulate the effect of ethylene action (MEyER & TERRy, 2010;GARDIn et al., 2012;WATKINS & NoCK, 2012;ZHANG et al., 2013;ChIARA et al., 2014;RAzzAQ et al., 2014;CAO et al., 2015).
1-methylcyclopropene (1-MCP) is a compound employed as inhibitor of ethylene and plant growth regulator for many fruits, showing to be highly effective for fruit ripening and senescence control (ChIABRAnDO & GIACALOnE, 2011;ZHANG et al., 2012).1-MCP is a gas that blocks the ethylene action by binding to its receptor on the cell membrane (In et al., 2013), severely reducing the changes associated with ripening and extending the postharvest life of fruits and vegetables (PAUL et al., 2010;vIEIRA et al., 2012;WAGhMARE & AnnAPURE, 2013).This product is considered one of the most important tools in postharvest technology, both in storage and in the transportation of ethylenesensitive fruits, maintaining quality as if they were freshly harvested (EGEA et al., 2010;TIWARI & PALIyATh, 2011;TREvISAn et al., 2013).The compound concentration required to promote inhibition of the ethylene action varies according to the species, cultivar, maturation stage, temperature and exposure time, and the production of new ethylene receptors on the cell membranes (WATKINS AND NoCK, 2012;PEREIRA et al., 2013).
Thus, this research aimed at studying the effects of 1-MCP as inhibitor in the ripening and postharvest quality of 'Solo' papayas.Comparisons of physiological (respiration rate, ethylene production, firmness, colour measurement) and biochemical responses (sugar analysis) of papaya edible (flesh) and non-edible parts (peel and seed separately) were carried out with 1-methylcyclopropene (1-MCP), aiming at extending storage shelf life and maintain fruit quality.

materIals and methods
Plant material 'Solo' papayas (Carica papaya L.) from South Africa were obtained from UNIVEG Katope Ltd. (UK) and transported properly packed in cardboard boxes to Cranfield University, UK within 1 h.Fruits were selected according to size, colour and external maturation stage 2 (corresponding to ¼ ripening fruit, with 15 to 25% of the yellow peel surface) discarding those with any physical and/or biological damage.The fruits were not pretreated with 1-MCP.

Experimental design
The experiment was a completely randomized design and was carried out in a temperature controlled room at 20 ºC.Samples were collected on arrival (baseline sampling; n = 5) before the treatments.Papaya fruits were then treated with 1-MCP (1 μL L -1 ) for 24 h.After the 24 h 1-MCP treatment, fruits were stored in 13 L boxes.Samples were taken at 3, 5, 7 and 10 days of storage for physiological assessments and biochemical analysis.The 24 h 1-MCP (1 μL L -1 ) treatment was performed in 264 L water-sealed air tight polypropylene chambers.Electric fans (nidec Beta SL, Nidec, Japan) were used to circulate the gas in the boxes.The 1-MCP was applied by adding 1.47 g SmartFresh (0.14%, Rohm and Haas, PA) to a 100 mL conical flask.To release 1 μL L -1 1-MCP gas, 16.30 mL warm (50 ºC) water was injected into the conical flask (CooLS et al., 2011).Gas samples were taken periodically by using 60 mL plastic syringes, and were analyzed (TERRY et al., 2007) by extraction and injection into a GC 8340 gas chromatograph (Carlo Erba Instruments, herts, UK).After 24 h of 1-MCP treatment, 5 fruits from each box were analyzed and the remaining fruits were distributed in 13 L sealed polypropylene boxes, being stored at 20 ºC for 10 days.The experiment was conducted using a randomized design with 2 factorial treatments, 6 sampling days, 5 fruits per experimental unit, 3 types of fruit tissue (flesh, peel and seed), 3 fruit sections (top, middle and bottom), and the duplicate samples storage at -40 ºC and -80 ºC.

Physiological parameters
Respiration rate.Respiration rate (mL kg -1 h -1 ) of papaya fruits was measured using a Sable Respirometry System (model 1.3.8Pro, Sable Influence of 1-methylcyclopropene on the bIochemIcal... Systems International, nv, USA).Five papayas were used per treatment and each fruit was placed in a 3 L air-tight glass jar.Three values were obtained per fruit and per treatment.Respiration rates were calculated using ExpeData (Release 1.3.8,version: PRO).
Ethylene production.Papaya fruits were placed in 3 L glass jars containing air-tight lids and septum for 1 h at room temperature.Gas samples (n = 2 per jar) were taken with repeated withdrawalinjection displacements using 60 mL plastic syringes (CooLS et al., 2011) and were injected into a gas chromatography (GC Model 8340, Carlo Erba Instruments, Herts, UK).
Firmness.Firmness measurement was performed using an Instron Uniaxial Testing Machine (model 5542, Instron, Norwood, USA), equipped with a calibrated 500 N load cell and fitted with an 8 mm diameter cylindrical flat probe.The machine was programmed (Bluehill 2, version 2.11, Instron) with a crosshead speed set at 50 mm min -1 (ERGUn et al., 2006) and the force (n) at bioyield produced was recorded.Three penetrations were performed on each fruit (top, middle and bottom) after removing a small piece of skin carefully.
Colour measurement.objective colour (lightness; L*), chroma (colour saturation; C*), and hue angle (Hº) of each fruit were determined as in Meyer and Terry (2010), using a Minolta CR-400 colorimeter and DP-400 data processor (Minolta Co. Ltd., osaka, Japan).The colour measurement was spatially processed, at the top, middle and bottom of each fruit, resulting in an average of the colour parameters.The ΔE, C* and h were calculated using the following equation (RUFIÁN-HENARES et al., 2006):

Biochemical parameters
Sample preparation, extraction and standard solutions preparation.Papayas were cut into 3 sectional slices divided as top, middle and bottom.The fruits were divided into 3 types of tissue: flesh, peel and seed, snap-frozen in liquid nitrogen and stored at two temperatures: -40 ºC and -80 ºC respectively.Frozen tissues (-40 ºC) were freezedried (Scav Vac, Västerås, Sweden) in the dark at -50 ºC for 10 days and they were used for the biochemical analysis.Fresh and dry weights were recorded before and after lyophilisation and were subsequently ground into fine powder.Sugars were extracted according to Downes et al. (2010).For each 150 mg of lyophilized sample, 3 mL of 62.5:37.5 methanol:water (v/v) solution (High-performance liquid chromatography, HPLC grade) were added and homogenized in plastic vials, which was then incubated in a low-speed shaking water bath at 55 ºC for 15 minutes, vortexing for 20 s at every 5 min.The samples were then allowed to cool at room temperature, filtered through 0.2 µm pore syringe filters, and stored at -20 ºC.Samples were diluted 1:10 before injection into HPLC.The calibration standard solution with a concentration of 2.5 mg mL -1 was prepared by weighing 250 mg of each sugar: D-(+) sucrose, glucose and D-(-) fructose (Sigma) into a 100 mL volumetric flask and filled up with HPLC grade water.1, 5, 10 and 25 mL of standard solution were pipetted into 50 mL volumetric flasks, which were filled up with HPLC grade water to give final standard concentrations of 0.05; 0.25; 0.50; 1.25 and 2.5 mg L -1 (sucrose, glucose and fructose, respectively).From each standard, 1.0 mL aliquots were dispensed into HPLC vials and stored at -40 ºC until required (TERRY et al., 2007).

Statistical analysis
Statistical analysis was performed using Statistical Analysis System software (SAS InSTITUTE, 2010).Analysis of variance (AnOvA) was used to demonstrate the main effects to a probability of 5%.

Physiological parameters
Respiration rate and ethylene production.According to Table 1, 1-MCP significantly reduced papayas respiratory rate (p ≤ 0.05), immediately on the 1 st day of analysis (corresponding to Day 0, after 24 h 1-MCP).Respiration can also be affected by cultivar, maturity, tissue, and storage temperature (TERRy et al., 2007).For ethylene production, 1-MCP inhibitor treatment presented lower average values compared to control treatment, even in non-significant levels.1-MCP inhibited this hormone production, delaying production peak until the 5 th storage day (Table 2).Ethylene rates in ripening papayas are 6 to 10 μL kg -1 h -1 (PAULL, 1993;TREVISAN et al., 2013), showing an effective production peak delay in all treated fruits.Comparing to other studies, the respiratory and ethylene climacterics were delayed in avocados treated with 1-MCP at 1 µL L -1 for 24 h at 20 ºC (PATHIRANA et al., 2011), and it was also reduced in mangosteens exposed to 1 µL L -1 for 6 h at 15 or 25 ºC (PIRIyAvInIT et al., 2011) and tomatoes treated with 1 µL L -1 for 24 h at 20 ºC (WAnG et al., 2010).In addition, the maturation stage at which the fruits were treated influenced 1-MCP effective action.The maturation stage 2 set for papayas is early in the ripening, and presents higher chances of effective responses to the ethylene inhibitor 1-MCP.
Studies have shown less effective responses of this inhibitor in fruits at advanced maturation stages (LU et al., 2013;JUNG AND WATKINS, 2014).This was observed for pears as more advanced stage of the fruit development provided shorter inhibition time of ethylene production (GAMRASnI et al., 2010;CHIRIBoGA et al., 2013), which indicates that fruit maturation stage interferes with 1-MCP effective response on ethylene production.1-MCP is characterized as a competitor for the cell ethylene binding site.When applied at the correct time, 1-MCP occupies ethylene binding sites and prevents their effects, which include for example, synthesis of degradative enzymes, increase of respiratory rate and ethylene production (autocatalysis).horticulture product conservation capability is inversely related to respiratory rate and, in many cases, with ethylene production rate (KELLER et al., 2013).Reductions of respiratory rate and ethylene production explain the higher preservation of fruits treated with 1-MCP (zhAnG et al., 2010;yAn et al., 2011;TREvISAn et al., 2013;SCOLARO et al., 2015).
Firmness.Fruits treated with 1-MCP tend to keep firmness for longer periods, significantly differing from control, as displayed in Table 3. Fruits do not ripen uniformly, starting from its internal tissue, and then proceeding toward external tissue, progressing from the bottom to the calyx (KLEE AND CLARK, 2010).Fruit flesh firmness is determined by the cohesion strength between pectins.Pectinolytic enzymes are released during the ripening evolution, which transform insoluble pectin into soluble and promote fruit softening (YoSHIoKA et al., 2011).
Fruit softening is one of the ripening processes more sensitive to ethylene (PAyASI AnD SAnWAL, 2010).The greater firmness of fruits treated with 1-MCP is probably associated with a reduction of pectinolytic enzymes activity, caused by the lower ethylene action.Similar results were obtained with apples (hEnDGES et al., 2011al., ), bananas (yAn et al., 2011) ) and papayas (ThUMDEE et al., 2010) treated with 1-MCP inhibitor.Firmness is an important quality attribute for papayas and direct influence fruit storage shelf life and consumer acceptance.Low firmness fruits present less resistance to transport, storage and manipulation, which makes it more vulnerable to mechanical injuries susceptible to diseases (AhMADI-AFzADI et al., 2013).The firmness average of treated papayas (18 N) was higher than in control fruits, with about 10 N (Table 3).In 1-MCP treatment, there was a reduction in the fruit firmness loss, especially until the 7 th storage day, corresponding to the analysis 4. on the 7 th storage day, 1-MCP treated fruits showed firmness values of about 20 N, compared to 7 N of control fruits (Table 3).In this treatment, the typical softening of papaya fruit during ripening was impaired by the presence of the ethylene competitor, giving papaya flesh fruit an undesirable hardness.Although there were changes in peel and flesh colour and fruits started to deteriorate, no additional softening was observed.Apparently, papaya fruit could not recover from 1-MCP treatment and became sensitive to ethylene.Colour measurement.1-MCP treatment significantly reduced green colour of fruits (p ≤ 0.05), affecting the measured colour parameters.The evaluation of 'Solo' papayas peel colour using L parameter, which indicates lightness, 1-MCP treatment yielded difference of control, and both control and treated fruits had L values higher than 50 (Table 4).As the scale ranges from 0 to 100, these treated fruits presented darker peel colour than control fruits, which had higher values, being DOI 10.1590/0100-29452016791 March/April 2016 Jaboticabal -SP Influence of 1-methylcyclopropene on the bIochemIcal... lighter.Chroma defines colour intensity, in which the positive values correspond to red, 0 (zero) to gray and negative values to green colour (IBRAhEEM et al., 2012).Chroma remained lower in all fruits treated with 1-MCP, while the hue angle remained higher, demonstrating that there was a higher retention of green colour in these fruits.It was observed a trend towards reduction in the green colour of 1-MCP treated fruits between the 3 rd and 5 th storage day.From then until the 10 th day, chroma parameter presented values tending to reduce green colour, as well as hue angle values (Table 4), indicating intense process of peel yellowing.
Degreening is due to chlorophyll degradation, mainly caused by chlorophyllase activity (WAnG et al., 2015).The increase in this enzyme activity is generally associated with ethylene production during fruit ripening (CHARoENCHoNGSUK et al., 2015).1-MCP binds to the cell ethylene binding site, avoiding hormone action on the ripening physiological processes (SU; FInLAySOn, 2012).Green colour reduction, which results from the normal ripening process, was delayed by 1-MCP application.Peel colour is a factor that has an essential influence on papayas acceptance.The consumer has preference for fruits with smooth and yellow or orange bright colour peel in relation to light and green peel fruits.Consumers usually relate fruit colour with sweetness and other desirable attributes, which leads the purchase preference (PATHARE et al., 2013).Green colour retention in papaya fruits treated with 1-MCP was also verified by Krongyut et al. (2011) andTrevisan et al. (2013) as well as in other fruits, such as banana (yAn et al., 2011), avocado (MEyER;TERRy, 2010), plum (SInGh & SINGH, 2012), mango (SIVAKUMAR et al., 2012) and tomato (WAnG et al., 2010).The reduction of ethylene production observed in papayas treated with 1-MCP (Table 2) may have a direct influence on reducing the fruit green colour.

Biochemical parameters 24 h control and 1-MCP treatments.
The results for biochemical parameters, as sugar levels quantification (sucrose, glucose and fructose) of 'Solo' papayas treated with ethylene inhibitor 1-MCP for 24 hours are shown in Table 5 and Figs. 1 to 3.Besides flesh softening, sugar content is another important quality attribute of papaya fruit.
In contrast to the effect on the firmness, inhibition of ethylene perception by 1-MCP did not preclude the accumulation of sugars.However, the differences observed can provide some clues regarding the metabolism of soluble sugars during ripening.
Apparently, sucrose synthesis is operative during ripening, although rates of synthesis were lower than those described for fruit development (DE OLIvEIRA AnD vITÓRIA, 2011).Moreover, when the results were compared to those of papaya fruit exposed to radiation (GOMEz et al., 2002), the same conclusion of an operative sucrose synthesis during ripening can be achieved.Because the trace amounts of starch in the flesh of papayas could not account for the accumulation of soluble sugars, it is possible to speculate that some mechanisms of cell wall disassembly (NoGUEIRA et al., 2012;RAZALI et al., 2013) could provide a source of carbon for sugar synthesis during ripening, including sucrose.The similar profiles for glucose and fructose could be an indication that those sugars come from the accumulated sucrose, especially by the action of invertases in fully ripe fruits (PAULL et al., 2011).The intermittent sucrose level increase in 1-MCP treated fruit would also be in agreement with this idea.Additionally, the observed proportions between sucrose, glucose, and fructose content increase in 1-MCP treated fruit could be an indication of an additional source besides sucrose (FABI et al., 2007).

days control and 1-MCP treatments.
Inhibition of ethylene perception by 1-MCP did not preclude the accumulation of sugars.Sugars analysis of 'Solo' papayas treated with 1-MCP after 3 storage days demonstrated that higher levels of sucrose, glucose and fructose were found in the flesh, followed by non-edible part, such as peel and seed, respectively.Even in non-significant levels, the mean values obtained for papayas treated with 1-MCP were similar and higher than those found for control fruits (Table 6).The lowest sugar values found in control treatment are possibly due to the faster reaction speed, leading to a lower accumulation.Soluble solid concentrations in treated products might be expected to be higher than in untreated products because of lower respiration rates, but they are also dependent on the product and the storage conditions (MAhAJAn et al., 2010;WAnG et al., 2010;zhAnG et al., 2012).Although the storage conditions of papayas were equal, 1-MCP treatment reduced speed sugars consumption for metabolism maintenance (sucrose hydrolysis and monosaccharides consumption), making higher levels of sucrose, glucose and fructose in the most of tissues (Figs. 4 to 6).Thumdee et al. ( 2010) also observed higher concentrations of sugar in 1-MCP treated papayas.As discussed previously, an additional supply of carbon could come from the cell wall disassembly or even organic acids.From sugar results, problems associated with fruit ripening interruption when using 1-MCP were not verified, contrarily to the negative and non-reversing effects on fruit softening.The pattern of sugar accumulation during 'Solo' papayas development (Table 6,Figs. 4 to 6) was similar to that observed by others (DE OLIvEIRA;vITÓRIA, 2011;nWOFIA et al., 2012;yAO et al., 2014).

conclusIon
The use of 1-MCP for fruits significantly increased 'Solo' papayas shelf life, as a consequence of the ability to inhibit ethylene action in tissues, delaying fruit ripening.Storage shelf life increase under environmental conditions is important, considering the high perishability of papaya after postharvest.The gain of seven days of shelf life allows fruit transportation over long distances and an extension of the marketing period.The use of 1-MCP provides strategies to increase fruit shelf life and improve efficiency/cost relation for fruits in general.Despite the positive results obtained for 1-MCP treated fruits, common ripening problems were still observed, leading for instance, to immature fruits even with the peel completely yellow, and fruits presenting green spots, even after 10 days of storage, demonstrating an irregular ripening.In contrast to the effect on the firmness, inhibition of ethylene perception by 1-MCP did not preclude the accumulation of sugars and the mean values obtained for papayas treated with 1-MCP were similar and higher than those found for control fruits, which are possibly due to the ripening delay.higher levels of sucrose, glucose and fructose were found in the flesh, followed by non-edible part, such as peel and seed, respectively.references AhMADI-AFzADI, M.; TAhIR, I.; nyBOM, h.Impact of harvesting time and fruit firmness on the tolerance to fungal storage diseases in an apple germplasm collection.postharvest biology and technology, Amsterdam, v.82, p.51-58, 2013.CAo, J.; LI, X.; WU, K.; JIANG, W.; QU, G. Preparation of a novel PdCl 2 -CuSO 4 -based ethylene scavenger supported by acidified activated carbon powder and its effects on quality and ethylene metabolism of broccoli during shelf-life.postharvest biology and technology, Amsterdam, v.99, p.50-57, 2015. CHARoENCHoNGSUK, N.;IKEDA, K.;ITAI, A.;oIKAWA, A.;MURAYAMA, H. Comparison of the expression of chlorophyll-degradation-related genes during ripening between stay-green and yellow-pear cultivars.scientia horticulturae, New York, v.181, p.89-94, 2015.

fIgure 6 -
fIgure 6 -Fructose concentrations of flesh, peel and seed in 'Solo' papayas treated with 1-MCP for 3 days.

acknowledgments
The authors are grateful to CnPq, FAPESP, CAPES and Embrapa from Brazil for all support.

table 1 -
Respiration rate in 'Solo' papayas treated with ethylene inhibitor 1-MCP, stored at 20 ºC for 10 days.Means followed by horizontal and vertical different letters differ significantly by the LSD test (p ≤ 0.05)

table 2 -
Ethylene production in 'Solo' papayas treated with ethylene inhibitor 1-MCP, stored at 20 ºC for 10 days.Means followed by the same letter do not differ significantly by the LSD test (p ≤ 0.05)
Means followed by horizontal and vertical different letters differ significantly by the LSD test (p ≤ 0.05) DOI 10.1590/0100-29452016791 March/April 2016 Jaboticabal -SP Influence of 1-methylcyclopropene on the bIochemIcal...

table 5 -
Sucrose, glucose and fructose levels of flesh, peel and seed in 'Solo' papayas treated with ethylene inhibitor 1-MCP, stored at 20 ºC for 24 h.

table 6 -
Sucrose, glucose and fructose levels of flesh, peel and seed in 'Solo' papayas treated with ethylene inhibitor 1-MCP, stored at 20 ºC for 3 days.
Means followed by horizontal different letters differ significantly by the LSD test (p ≤ 0.05) fIgure 1 -Sucrose concentrations of flesh, peel and seed in 'Solo' papayas treated with 1-MCP for 24 h.