Inhibition of ethylene action by 1-methylcyclopropene extends postharvest life of “Bartlett” pears

Postharvest Biology and Technology 32 (2004) 193–204

Inhibition of ethylene action by 1-methylcyclopropene extends postharvest life of “Bartlett” pears G.D. Trinchero a,1 , G.O. Sozzi b,∗,1 , F. Covatta b , A.A. Fraschina a a

Cátedra de Bioqu´ımica, Departamento de Biolog´ıa Aplicada y Alimentos, Facultad de Agronom´ıa, Universidad de Buenos Aires, Avda. San Mart´ın 4453, Buenos Aires C1417DSE, Argentina b Cátedra de Fruticultura, Departamento de Producción Vegetal, Facultad de Agronom´ıa, Universidad de Buenos Aires, Avda. San Mart´ın 4453, Buenos Aires C1417DSE, Argentina Received 4 February 2003; accepted 15 November 2003

Abstract Preclimacteric European pears (Pyrus communis L. cv. “Bartlett”) were untreated or treated with 0.4 ␮l l−1 1-methylcyclopropene (1-MCP) for 10 h at 20 ◦ C and then kept at 20 ◦ C, or stored at 1 ◦ C for 30 or 60 days before transfer to 20 ◦ C. Other lots were treated with 0.4, 0.8, 1.2, or 1.6 ␮l l−1 1-MCP after 30 or 60 days of storage at 1 ◦ C. 1-MCP-treated pears kept at 20 ◦ C had lower ethylene production and slower softening than untreated fruit. Treated fruit were more than 75 N firmer than control fruit after a 6 day storage period. Fruit color changes were also delayed by 1-MCP treatment. However, additional color sorting may be necessary to reduce variability in response of fruit to 1-MCP in commercial situations. 1-MCP-treated fruit had lower total glycosidase (␣- and ␤-d-galactosidase, ␣-l-arabinofuranosidase, ␤-d-xylosidase, and ␤-d-glucosidase) activities. When 1-MCP was applied to fruit prior to cold storage (CS) at 1 ◦ C, the synergistic interaction of cold and 1-MCP resulted in an extended postharvest life after transfer to room temperature, with concomitant delayed ethylene and respiratory level increases, retarded color development and retention of firmness. In contrast, application of 0.4–1.6 ␮l l−1 1-MCP after 30 or 60 days of cold storage did not affect most ripening indices. These findings point to the experimental and commercial utility of 1-MCP in “Bartlett” pear postharvest management. © 2003 Elsevier B.V. All rights reserved. Keywords: Ethylene; Glycosidases; 1-Methylcyclopropene; Pear; Ripening; Softening

1. Introduction “Bartlett” pears (Pyrus communis L.) require cold storage (CS) (approximately −1 to 0 ◦ C) and controlled atmospheres (1–2% O2 and 0–0.5% CO2 ) to diminish losses during long-term storage and transport ∗ Corresponding author. Tel.: +54-11-4524-8055; fax: +54-11-4514-8739. E-mail address: [email protected] (G.O. Sozzi). 1 These two authors contributed equally to this work.

(Richardson and Kupferman, 1997; Thompson et al., 2000). In many regions worldwide, the availability of controlled atmospheres or refrigeration equipment is limited. Utilization of these technologies may not be possible under every marketing condition, and their improper use can result in fruit losses. In addition, most pear cultivars including “Bartlett” are sensitive even to slightly elevated storage temperatures. 1-Methylcyclopropene (1-MCP) is a synthetic cyclic olefin capable of inhibiting ethylene action. This simple organic compound probably acts as a

0925-5214/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2003.11.009

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competitor of ethylene, blocking its access to the ethylene-binding receptor (Sisler and Serek, 1997). 1-MCP, a gaseous non-toxic product, is being studied as a tool to extend postharvest life, delay softening and improve poststorage quality of different climacteric fruits (Mitcham, 2001; Watkins and Miller, 2003). Few reports on the effects of 1-MCP on pear fruit are available. Baritelle et al. (2001) found that “Bartlett” pears exposed to 2 ␮l l−1 1-MCP for 16 h ripened and softened over a 10-day period. Furthermore, Lelièvre et al. (1997) reported that 1-MCP blocked ethylene action during chilling and reduced the chilling-induced accumulation of 1-aminocyclopropane-1-carboxylic synthase and oxidase transcripts in “Passe–Crassane” pears. Attention has also been paid to the understanding of the regulatory mechanisms of ethylene biosynthesis and respiration by 1-MCP in pears (de Wild et al., 1999). Texture is a major attribute that has a strong effect on consumer perception of pear fruit quality (Kappel et al., 1995). Different factors affect pear textural properties, among them cell wall polysaccharide composition. A substantial decrease in cell wall-bound glycosyl residues is one of the most evident cell wall compositional changes during fruit ripening. In pear fruit, 71, 36, and 60% of the cell wall arabinose, galactose, and glucose, respectively, may be released in the period between the pre-climacteric and the post-climacteric stage (Gross and Sams, 1984). Some fruit glycosidases, mainly ␤-galactosidases and ␣arabinofuranosidases, have been studied with regard to their potential for removing glycosyl residues from cell wall polymers (e.g. Pressey, 1983; Kitagawa et al., 1995; Tateishi et al., 2001; Sozzi et al., 2002), and their impact in fruit metabolism and softening is now being demonstrated using transgenic plants (Smith et al., 2002). While some members of a given glycosidase family may target cell wall components, it is possible that not all of them are involved in cell wall metabolism during ripening. The search is now underway to identify these ripening-specific glycosidases and their functions in vivo. 1-MCP can provide a convenient means for testing the ethylene responsiveness of many physiological processes (Golding et al., 1998), including whether this plant growth regulator modulates the expression of genes encoding ripeningrelated cell wall-degrading enzymes in climacteric fruits (Dong et al., 2001a,b; Jeong et al., 2002).

The objectives of this work were to evaluate (1) the effect of 1-MCP at potential commercial concentrations on the postharvest life of “Bartlett” pears, (2) the incidence of 1-MCP treatment on different ripening and quality indices (ethylene production, respiration rate, firmness, color, soluble solids, and titratable acidity), (3) the effectiveness of 1-MCP when applied before and/or after cold storage, and (4) the effect of 1-MCP on the activity of various cell wall glycosidases that may have critical roles in fruit ripening: ␣and ␤-d-galactosidase (␣- and ␤-Gal; EC 3.2.1.22 and EC 3.2.1.23, respectively), ␣-l-arabinofuranosidase (␣-Af; EC 3.2.1.55), ␣- and ␤-d-glucosidase (␣- and ␤-Glc; EC 3.2.1.20 and EC 3.2.1.21, respectively), and ␣- and ␤-d-xylosidase (␣- and ␤-Xyl; EC 3.2.1.and EC 3.2.1.37, respectively). 2. Materials and methods 2.1. Plant material Preclimacteric “Bartlett” pears (100 count per 18 kg box; mean fruit weight = 180 g) were harvested in the Alto Valle del R´ıo Negro, Argentina, on 20 January 2002 at optimum maturity for long-term storage and immediately forwarded to our laboratory in Buenos Aires where the experiments were performed. Pears were allowed to equilibrate at 20 ◦ C overnight. Fruit, packed in vented commercial boxes, were divided into three different groups and exposed to various conditions as described below. 2.2. 1-MCP and storage treatments 1-MCP was supplied as SmartFreshTM powder (Rohm and Hass S.A., Argentina). 1-MCP was released from vials containing weighed amounts of SmartFreshTM powder (0.14% active ingredient; Rohm and Hass) by adding a buffering agent provided by the manufacturer. The solution was injected through a port inserted in the wall of the chambers that was connected to the vials. This system allowed the addition of the solution without opening the hermetic chamber. Small fans inside the chambers ensured a rapid diffusion of the gas. The 1-MCP concentration in each chamber was quantified on a gas chromatograph (Hewlett Packard 5890 Series II) as previously described (Jiang et al., 2001), using iso-butylene as

G.D. Trinchero et al. / Postharvest Biology and Technology 32 (2004) 193–204

standard. The atmosphere in the chambers was circulated through a CO2 trapping system containing pellets of NaOH. CO2 accumulation was checked by gas chromatography as described below, and did not exceed 0.2%. 2.2.1. Experiment 1: application of 1-MCP and subsequent storage at 20 ◦ C Pears were randomly divided into a 250-fruit control lot that was kept in air at 20 ◦ C, and another 750-fruit lot that was placed in a chamber at 20 ◦ C. These fruit were treated overnight (10 h) with 0.4 ␮l l−1 1-MCP (called 0.4) and, after ventilation, were kept in the same room as the control fruit. After 13 days, the partially used 1-MCP-treated lot (630 pears) was further divided into three 210-pear sets. One set was left in air at 20 ◦ C. The second set was placed in a chamber and submitted to a second application of 0.4 ␮l l−1 1-MCP overnight (called 0.4 + 0.4). The third set was placed in a sealed plastic cabinet and treated with 400 ␮l l−1 ethylene (called 0.4+ethylene). The concentration of ethylene required was obtained by injecting appropriate amounts of pure ethylene (AGA, Argentina) into the experimental 60 l container. Fruit were kept in that gaseous atmosphere for 6 h at 20 ◦ C. After each treatment, fruit were ventilated and held in vented boxes in the dark, at 20 ◦ C and ∼90% RH. Afterwards, an additional 400-fruit consignment was treated with 0.4 ␮l l−1 1-MCP and its response to the ethylene-blocking agent was commercially evaluated. After ventilation, fruit were kept at 20 ± 1 ◦ C in a commercial chamber without the addition of ethylene. The storage atmosphere was checked before sampling the fruit and trace amounts of ethylene from fruit ripening were detected (0.1 ± 0.02 ␮l l−1 ). These levels were similar to those found in retail outlets (Wills et al., 2000). 2.2.2. Experiment 2: application of 1-MCP before and after 30 days storage at 1 ◦ C Pears were divided into five 120-fruit lots. Treatments were as follows: (1) 0 + CS + 0: untreated fruit were placed in a chamber, kept in air at 1 ± 0.5 ◦ C (CS) for 30 days and transferred to a chamber at 20 ◦ C until evaluated;

195

(2) 0 + CS + 0.4: fruit were kept as described in (1). After transfer to 20 ◦ C in air, pears were allowed to rewarm (20 ◦ C) and then treated with 0.4 ␮l l−1 1-MCP for 10 h. The chamber was then ventilated and fruit were kept at 20 ◦ C until used; (3) 0.4 + CS + 0: fruit were treated with 0.4 ␮l l−1 1-MCP at 20 ◦ C for 10 h prior to a 30-day CS and then transferred to 20 ◦ C until utilized; (4) 0.4 + CS + 0.4: fruit were treated with 0.4 ␮l l−1 1-MCP at 20 ◦ C for 10 h before and after exposure to a 30-day CS. Fruit were allowed to rewarm to 20 ◦ C before the second exposure to 1-MCP. Fruit were then kept at 20 ◦ C until used; (5) 0.4 + CS + 0.8: fruit were treated as described in (4), except that they were submitted to 0.8 ␮l l−1 1-MCP after exposure to cooling conditions. 2.2.3. Experiment 3: application of 1-MCP before and after 60 days storage at 1 ◦ C A 600-pear group was pretreated with 0.4 ␮l l−1 1-MCP at 20 ◦ C for 10 h and then kept in air at 1 ± 0.5 ◦ C for 60 days. After transfer to 20 ◦ C, pears were allowed to warm and were randomly divided into three 200-pear lots. Two lots were treated with 1.2 and 1.6 ␮l l−1 1-MCP (called 0.4+CS+1.2 and 0.4+CS+ 1.6, respectively). The third lot (called 0.4 + CS + 0) was placed in a control chamber containing no 1-MCP. 2.3. Ethylene and CO2 determination Ethylene and CO2 production of individual fruit were measured by the headspace technique using 1.5 l glass containers. One milliliter of the headspace gas was extracted after 1 h and ethylene was quantified on a gas chromatograph (Hewlett Packard 5890 Series II) fitted with a FID and a stainless steel Porapak N column (3.2 mm × 2 m; 80/100 mesh). The injector, column and detector temperatures were 110, 90, and 250 ◦ C, respectively. N2 was used as the carrier gas. Linear gas velocity was 4.5 cm s−1 . Six independent replicates per treatment and sampling time were evaluated. Results were expressed as nanogram of ethylene produced per kilogram of fruit in 1 s. The respiration rate was measured using a Hewlett Packard 4890 gas chromatograph fitted with a TCD and equipped with a CarboplotTM (Chrompack) column (0.53 mm × 25 m; 25 ␮m thick). Analysis was performed isothermally at 100 ◦ C, with the injector

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and the detector temperatures held at 80 and 200 ◦ C, respectively. He was used as the carrier gas. Linear gas velocity was 68 cm s−1 . Six independent replicates per treatment and sampling time were evaluated. Results were expressed as microgram of CO2 produced per kilogram of fruit in 1 s. 2.4. Firmness, color, and other quality evaluations Fruit firmness was determined by measuring the force required to penetrate each pear, with the skin removed, to a depth of 1 cm, using an Instron Universal Testing Machine (Model 4442, Canton, MA). Each fruit was placed on a stationary steel plate. Two spots located on opposite sides of a fruit sector perpendicular to the most sun-exposed area were punctured to a depth of 10 mm. The average of those two measurements was considered as one replicate. We avoided measuring firmness on light-exposed sites to standardize the sampling procedure. Puncture tests involved use of a 7.9-mm probe on a drill base with a crosshead setting of 50 mm min−1 . Twelve pears (two measurements per fruit) per treatment and sampling time were evaluated. Surface color measurements were obtained from the equatorial region of intact fruit with a Minolta chroma meter (model CR-300; Osaka, Japan) using CIE illuminant C lighting conditions and an 8 mm-diameter measuring area. The chroma meter was calibrated to a white calibration plate (CR-A43). L∗ a∗ b∗ were recorded and a∗ and b∗ were converted to hue angle (h◦ ) (McGuire, 1992). Sixteen to twenty pears per treatment and sampling time were evaluated. A longitudinal wedge was removed from each fruit and pressed through cheesecloth. The juice from wedges of three pears was pooled, analyzed for soluble solids concentration (SSC) using a hand-held temperature-compensated refractometer (Atago Co., Tokyo). Titratable acidity (TA) was determined by titrating a 10-ml juice sample with 0.05N NaOH utilizing a titrator to an endpoint of pH 8.1 as indicated by phenolphthalein, and was calculated as mmol H+ per liter juice. Three replications per treatment and sampling time were evaluated. 2.5. Glycosidase activity Three composite (three-fruit) mesocarp samples (150 g) per treatment and date were homogenized in

a Waring blender (45 s) and then an Omnimixer (45 s) with 1 vol. of cold 200 mM sodium acetate buffer, pH 4.5, containing 1.4 M NaCl, 0.1% (w/v) ascorbic acid and 1.5% (w/v) PVPP. The subsequent steps were performed at 4 ◦ C. The suspension was filtered through several layers of Miracloth (Calbiochem Corp., La Jolla, CA) and centrifuged at 12,000 × g for 20 min. Aliquots of centrifuged extract were assayed for total glycosidase activity using the corresponding p-nitrophenyl glycoside (Sigma Chemical Co., St. Louis, MO) as substrate. Reaction mixtures contained 250 ␮l of 0.1 M citrate buffer, pH 4.5, 200 ␮l of 0.1% bovine serum albumin, 50 ␮l of enzyme solution (or an appropriate dilution, in the case of ␣- and ␤-Gal) and 200 ␮l of 13 mM substrate solution, with incubation at 37 ◦ C (Sozzi et al., 2002). The generation of free p-nitrophenol was linear for at least 2 h. Activities reported are based on rates determined after 1 h for ␣-Af, ␣- and ␤-Glc, and ␣- and ␤-Xyl, and 15 min for ␣- and ␤-Gal, with the reaction stopped by addition of 1 ml of 0.2 M sodium carbonate. Absorbance was measured at 400 nm. Free p-nitrophenol was used as standard. One unit of each glycosidase was defined as the amount of enzyme hydrolyzing 1 nmol s−1 of p-nitrophenyl glycoside. Data of enzyme activities were expressed on a per kilogram fresh weight basis. Three replications per treatment and sampling time were evaluated.

3. Results and discussion 3.1. Experiment 1: application of 1-MCP and subsequent storage at 20 ◦ C The onset of ethylene climacteric of fruit treated with 0.4 ␮l l−1 1-MCP and kept at 20 ◦ C was delayed by 15 days compared with control fruit (Fig. 1A). Exposure of 1-MCP-treated fruit to 400 ␮l l−1 ethylene did not consistently enhance ethylene production compared with non-exposed fruit. In contrast, a second application of 0.4 ␮l l−1 1-MCP after 13 days prevented ethylene biosynthesis for additional 6 days. Major deterioration was found at the end of the experimental period in all fruit but 90% control pears were found to be deteriorated after 12 days. Fruit softening proceeded in parallel with ethylene production. Pear softening was greatly inhibited by

100 0 (Control) 0.4 0.4 + 0.4 0.4 + Ethylene

80 60

74

L*

-1

(A)

70

40

66

20

62

0

197

78

-1

Ethylene Production (ng kg s )

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0

3

6

9

12 15 18 21 24 27

58 (C)

0

3

6

9

12 15 18 21 24

0

3

6

9

12 15 18 21 24

112 108

60

Hue Angle

Firmness (N)

80

40 20 0

(B)

104 100 96 92 88

0

3

6

9 12 15 18 21 24 27 Time (days)

(D)

Time (days)

Fig. 1. Ethylene production (A), firmness (B), and color (C and D) of pears whether exposed or not to 1-MCP and stored at 20 ◦ C. Numbers in the legend indicate the concentration (␮l l−1 ) of 1-MCP used in each treatment. A set of 1-MCP-treated fruit was exposed to a second application of 0.4 ␮l l−1 1-MCP (day 13, arrow) and allowed to ripen. Simultaneously, another set was submitted to 400 ␮l l−1 ethylene (day 13, arrow) and allowed to ripen. Values represent the means ± S.D., where bars are not shown, the S.D. does not exceed the size of the symbol.

1-MCP, the treated fruit being 75 N firmer than untreated fruit after a 6-day ripening period (Fig. 1B). Firmness in 1-MCP-treated fruit was not significantly affected by exogenous ethylene but differences between ethylene-treated pears and fruit treated twice with 1-MCP were significant. Successive applications of 1-MCP have been found to slow down softening in apples (Mir et al., 2001) and could be an interesting alternative to be tested in “Bartlett” pears if cost effective. In control fruit, L∗ increased and h◦ decreased rapidly (Fig. 1C and D). Application of 1-MCP delayed h◦ decline and the upsurge in L∗ . In control fruit, the maximum L∗ value was reached after a 5-day storage while 1-MCP-treated fruit showed a similar value after 24 days (Fig. 1C). A second

1-MCP application failed to show any statistical effect on lightness. Exposure to ethylene did not lead to significant differences in L∗ values (Fig. 1C). In contrast, a significant difference for h◦ was measured in days 14–21, between the set exposed to a second application of 1-MCP and the ethylene-treated set (Fig. 1D). In control fruit, SSC increased from 12.2±0.14% at the beginning of the experiment to 13.77±0.84% after a 7-day storage. In 1-MCP treated fruit, no significant changes were observed after 7 days (12.15 ± 0.32%). TA decreased from 65.97 ± 1.04 mmol l−1 to 48.06 ± 1.94 mmol l−1 in control fruit after a 7-day storage, but did not change significantly in 1-MCP-treated fruit after 7 days (64.03 ± 4.93 mmol l−1 ). A second 1-MCP application or exposure to ethylene of 1-MCP-treated

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Table 1 Effects of treatment with 1-MCP on postharvest life of a “Bartlett” pear commercial consignment Treatment

Stage

Firmness (N)

Percentage

Zone

L∗

Apex and equatorial region

75.35 ± 2.25

88.68 ± 0.78

1.9 ± 0.5

10.00 (n = 40)

1

Apex Equatorial region

69.57 ± 3.05 69.84 ± 1.28

105.28 ± 1.74 104.17 ± 1.21

53.3 ± 18.5

84.50 (n = 338)

2

Apex Equatorial region

73.33 ± 2.28 69.59 ± 2.67

92.76 ± 2.03 101.59 ± 1.65

32.4 ± 11.4

10.25 (n = 41)

3

Apex Equatorial region

74.85 ± 2.63 74.32 ± 2.79

92.06 ± 1.39 93.72 ± 2.05

17.6 ± 9.7

5.25 (n = 21)

Control 1-MCP

Color h◦

A lot of 400 mature-green pears showing no apparent variations was treated with 0.4 ␮l l−1 1-MCP and kept in air at 20 ◦ C. Ethylene in air was checked by gas chromatography, and was ∼0.1 ␮l l−1 . Pears were visually sorted by color after 12 days, applying the following selection criteria: stage 1, the surface was completely green; stage 2, there was a definite change in color from green to yellow at the stem-end; stage 3, the entire fruit surface became yellow. After visual classification, color and firmness were objectively measured. Each value represents the mean ± S.D. of 10 replicate samples. After 12 days at 20 ◦ C, 90% untreated pears were severely deteriorated, with external signs of fungal attack or physiological decay. Firmness and color are reported using control fruit with no visible deterioration.

fruit did not have any additional effect on SSC and TA. These indices may be accompanying other physiological flavor changes. 1-MCP-treated pears did not develop flavor to the same extent as control pears according to preference test results based on hedonic scaling (data not shown). Exposure of “Bartlett” pears to 1-MCP may alter the emanation pattern of volatile fractions, as is the case for bananas (Golding et al., 1998), apples (Fan and Mattheis, 1999; Rupasinghe et al., 2000), apricots (Fan et al., 2000), and Charentais cantaloupe melons (Flores et al., 2002). The effectiveness of 1-MCP varied within a lot at the same maturity stage, sampled from the same orchard and kept at 20 ◦ C for 12 days (Table 1). While 1-MCP shows commercial potential, this different response to 1-MCP could be a limiting factor in any commercial consignment and additional electronic color sorting (or separation by hand) may be necessary for 1-MCP-treated fruit. These results may reflect physiological differences not easily detectable under present harvest methods. In the context of the potential use of 1-MCP as an alternative for pear short-term storage protocols under non-cooling conditions, 1-MCP application time, concentration, and frequency deserve further examination. For medium- or long-term storage, quick cooling and good temperature management practices are essential to slow physiological deterioration of “Bartlett” pears though 1-MCP may be a useful supplement, as stated below.

To verify our results, one lot of 100 mature-green pears was treated with 0.4 ␮l l−1 1-MCP for 10 h and held in air at 20 ◦ C. After a 12-day storage, 90 pears were sorted for green color and allowed to ripen while the others which displayed a yellow stem-end were discarded. After 28 days, the lot was evaluated for color (90 independent replicates) and firmness (20 independent replicates). Our results (L∗ = 76.05±1.28; h◦ = 90.31 ± 1.24; firmness = 6.30 ± 1.94 N mean ± S.D.) confirmed those obtained in the first experiment. 3.2. Experiment 2: application of 1-MCP before and/or after 30 days storage at 1 ◦ C After exposure to a 30-day CS, control “Bartlett” pears displayed a sharp ethylene peak on day 4 (Fig. 2A). At that time, ethylene production was inhibited over 33-fold in fruit treated with 1-MCP prior to storage. In contrast, no significant differences between control fruit and fruit exposed to 1-MCP after CS were found (Fig. 2A). The pattern of ethylene biosynthesis was similar in fruit submitted to 0.4 + CS + 0, 0.4 + CS + 0.4, and 0.4 + CS + 0.8. Fruit treated with 1-MCP before a 30-day CS did not show a peak in ethylene production but only a gradual rise towards the end of the experimental period (Fig. 2A), as observed in 1-MCP-treated fruit stored at 20 ◦ C (Fig. 1A). In all cases, major deterioration of the fruit was found at the end of the experimental period.

Ethylene Production (ng kg-1 s-1)

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50

0 + CS + 0 0 + CS + 0.4 0.4 + CS + 0 0.4 + CS + 0.4 0.4 + CS + 0.8

40 30 20 10 0

(A)

0

3

6

9

12 15 18 21 24 27

0

3

6

9

12 15 18 21 24 27

0

3

6

-1

-1

Respiration Rate (µg kg s )

50 40 30 20 10 0

(B)

Firmness (N)

80 60 40 20 0 (C)

9 12 15 18 21 24 27 Time (days)

Fig. 2. Ethylene production (A), respiration rate (B), and firmness (C) of pears at 20 ◦ C after storage at 1 ◦ C for 30 days. Before storage, fruit were either not treated or treated with 0.4 ␮l l−1 1-MCP. After rewarming, fruit were treated with 0, 0.4, or 0.8 ␮l l−1 1-MCP. Numbers in the legend indicate the concentration (␮l l−1 ) of 1-MCP utilized in each treatment before and after cold storage (CS). Values represent the means ± S.D., where bars are not shown, the S.D. does not exceed the size of the symbol.

199

1-MCP application prior to CS affected both timing and magnitude of the respiratory climacteric (Fig. 2B). High respiration rates were detected in 0 + CS + 0 and 0 + CS + 0.4-treated fruit 4 days after rewarming. A suppressed respiration peak (days 1 and 2 after rewarming) was detected in fruit exposed to 0.4 ␮l l−1 1-MCP prior to a 30-day CS, but that peak declined abruptly below initial values by day 4. Then, the respiration rate for 0.4 + CS + 0, 0.4 + CS + 0.4, and 0.4 + CS + 0.8-treated fruit remained stable during at least 10 days (Fig. 2B). Treatment with 1-MCP slowed softening, both during and after CS (Fig. 2C). Pear firmness seemed to be influenced by the synergistic interaction of cold × 1-MCP. 1-MCP extended the postharvest life of pears 3 weeks when applied prior to CS. On the other hand, the effectiveness of 1-MCP greatly decreased when applied after medium-term CS (Fig. 2C). The epicarp of “Bartlett” pears became lighter during a 30-day CS (Figs. 1C and 3A), but this change was at least partially counteracted when 1-MCP was applied prior to CS. After transference to 20 ◦ C, untreated pears rapidly shifted from green to yellow and became lighter in color (Fig. 3). 1-MCP did not slow these changes when applied after CS. On the contrary, pears exposed to 1-MCP prior to CS remained greener throughout the experimental period (Fig. 3B). A second application of 1-MCP (0.4 + CS + 0.4 and 0.4 + CS + 0.8) showed delayed h◦ value changes for 15 days (Fig. 3B). In general, storing treated fruit under a 30-day CS before 1-MCP treatment reduced the pear response to 1-MCP. Pears from all treatments ripened normal with almost no physiological internal disorders. Neither the 1-MCP prestorage treatment nor a poststorage exposure to 1-MCP affected SSC and TA significantly after ripening (data not shown). 3.3. Experiment 3: application of 1-MCP before and after 60 day storage at 1 ◦ C Application of up to 1.6 ␮l l−1 1-MCP after a 60-day CS only showed a slight delay on ethylene increase (Fig. 4A). Fruit treated with 1-MCP before a 60-day CS did not show a peak in ethylene production but only a gradual rise towards the end of the experimental period, as observed in the experiments 1 (Fig. 1A) and 2 (Fig. 2A). In all cases, major

G.D. Trinchero et al. / Postharvest Biology and Technology 32 (2004) 193–204

-1

Ethylene Production (ng kg s )

200

-1

77 75

L*

73 71 69

0 + CS + 0 0 + CS + 0.4 0.4 + CS + 0 0.4 + CS + 0.4 0.4 + CS + 0.8

67 65 63 (A)

0

7

14

21

(A)

108

0.4 + CS + 1.2

12

0.4 + CS + 1.6

9 6 3 0

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10 15 Time (days)

20

25

-1

Respiration Rate (µg kg s )

-1

96

7

14

21

Time (days)

Fig. 3. Color of pears at 20 ◦ C after storage at 1 ◦ C for 30 days. Color is presented as L∗ (A) and h◦ (B) values. Before storage, fruit were either not treated or treated with 0.4 ␮l l−1 1-MCP. After rewarming, fruit were treated with 0, 0.4, or 0.8 ␮l l−1 1-MCP. Numbers in the legend indicate the concentration (␮l l−1 ) of 1-MCP utilized in each treatment before and after cold storage (CS). Values represent the means±S.D., where bars are not shown, the S.D. does not exceed the size of the symbol.

deterioration of the fruit was found at the end of the experimental period. Low respiration rates were found in 1-MCP-treated fruit exposed to a 60-day CS, with a gradual increase towards the end of the experimental period (Fig. 4B). The effect of 1-MCP on firmness was similar in pears exposed to 1 ◦ C for 30 (Fig. 2C) or 60 days (Fig. 4C). Increasing the concentration of 1-MCP from 0 to 1.6 ␮l l−1 after cold storage did not increase the time taken before the fruit would soften (Fig. 4C). The response to a specific concentration and exposure to 1-MCP may be influenced by the duration of the previous storage at cooling conditions and/or by the age of the fruit. A 2–3 days exposure to cooling conditions

(B)

20

10

0

80 70 Firmness (N)

Hue Angle

100

92

(B)

0.4 + CS + 0

15

30

104

88 0

18

60 50 40 30 20 10 0

(C)

Fig. 4. Ethylene production (A), respiration rate (B) and firmness (C) of pears at 20 ◦ C after storage at 1 ◦ C for 60 days. Before storage, fruit were treated with 0.4 ␮l l−1 1-MCP. After rewarming, fruit were treated with 0, 1.2, or 1.6 ␮l l−1 1-MCP. Numbers in the legend indicate the concentration (␮l l−1 ) of 1-MCP utilized in each treatment before and after cold storage (CS). Values represent the means ± S.D., where bars are not shown, the S.D. does not exceed the size of the symbol.

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201

Table 2 Percentage of consumable pears as affected by increasing concentrations of 1-MCP applied after 2-month cold storage 1-MCP concentration (␮l l−1 )

Time of storage (days) at 20 ◦ C after cold storage (CS) (%) 24

28

32

37

0 1.2 1.6

96.0 (n = 72) 98.7 (n = 74) 98.7 (n = 74)

46.7 (n = 35) 65.3 (n = 49) 78.7 (n = 59)

24.0 (n = 18) 32.0 (n = 24) 61.3 (n = 46)

0.0 (n = 0) 0.0 (n = 0) 18.7 (n = 14)

Three lots of 100 mature-green pears each were treated with 0.4 ␮l l−1 1-MCP and held in air at 1 ◦ C for 2 months. Then, pears were allowed to rewarm at 20 ◦ C and were subsequently submitted to 0, 1.2, or 1.6 ␮l l−1 1-MCP. After a 12-day storage at 20 ◦ C, only 75 pears per lot were selected for green color and allowed to ripen. This selection was performed to avoid the different initial response to 1-MCP previously described (Table 1). The percentage of pears free from fungal and physiological decay is reported. An additional control lot was never treated with 1-MCP and showed external signs of fungal attack or physiological decay after a 12-day storage at 20 ◦ C.

3.4. 1-MCP and glycosidase activity ␣-Xyl and ␣-Glc activities were not detected throughout the experimental period. ␣-Gal and ␤-Gal (Fig. 6A) as well as ␣-Af, ␤-Xyl, and ␤-Glc (Fig. 6B) activities tended to an increase as fruit ripened, in close agreement to results previously reported for “Bartlett” pear (Ahmed and Labavitch, 1980) and Japanese pear (Yamaki and Kakiuchi, 1979; Tateishi et al., 1996). All these glycosidase activities are present before the onset of ripening (Fig. 6, day 1), as happens in other fruit (Sozzi et al., 2002; Sozzi, 2004). Total ␤-Gal involves five different isoforms in Japanese pear (Kitagawa et al., 1995). All of them are active against native cell wall polysaccharides, but only

78 76

L*

74 72 0.4 + CS + 0

70

0.4 + CS + 1.2

68 (A)

66

0.4 + CS + 1.6

0

7

14

21

0

7

14 Time (days)

21

104 Hue Angle

after harvest did not affect the effectiveness of a subsequent 1-MCP treatment (data not shown). In contrast, 1.6 ␮l l−1 1-MCP after CS was required to obtain some delay in fruit decay (Table 2). Results suggest that the main consequence of 1-MCP application after CS may be less spoilage during the over-ripening stage. Higher (4 ␮l l−1 ) concentrations of 1-MCP almost completely inhibited ethylene in “Passe–Crassane” pears, even after a 27-day storage at 0 ◦ C (Lelièvre et al., 1997). Application of increasing 1-MCP concentrations after a 60-day CS delayed changes in h◦ values during the first 14 days at 20 ◦ C, but only during 7 days in L∗ values (Fig. 5). In all cases, pear fruit were likely to be less responsive even to high concentrations of 1-MCP after CS. Pretreatment with 1-MCP delayed pear degreening during CS, particularly when exposure to cooling conditions exceeded a 60-day period (data not shown).

100 96 92 88

(B)

Fig. 5. Color of pears at 20 ◦ C after storage at 1 ◦ C for 60 days. Color is presented as L∗ (A) and h◦ (B) values. Before storage, fruit were either not treated or treated with 0.4 ␮l l−1 1-MCP. After rewarming, fruit were treated with 0, 1.2, or 1.6 ␮l l−1 1-MCP. Numbers in the legend indicate the concentration (␮l l−1 ) of 1-MCP utilized in each treatment before and after cold storage (CS). Values represent the means ± S.D.

202

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-1

Activity (units kg )

3550 3100 2650 2200 1750

Alfa-Gal in Control Fruit Alfa-Gal in 1-MCP-Treated Fruit Beta-Gal in Control Fruit Beta-Gal in 1-MCP-Treated Fruit

1300 (A)

850 0

-1

4

6

8

10

Alfa-Af in Control Fruit Alfa-Af in 1-MCP-Treated Fruit Beta-Glc in Control Fruit Beta-Glc in 1-MCP-Treated Fruit Beta-Xyl in Control Fruit Beta-Xyl in 1-MCP-Treated Fruit

170 Activity (units kg )

2

150

4. Conclusion

130 110 90 70 50

(B)

0

2

4 6 Time (days)

1-MCP has been found to inhibit the increase in the activity of the cell wall-degrading enzyme polygalacturonase for up to 12 days in avocado (Jeong et al., 2002). In nectarines, polygalacturonase, and pectinmethylesterase mRNA abundance was partially inhibited 5 days after a 1-MCP treatment (Dong et al., 2001b). Endo-1,4-␤-d-glucanase, exo- and endo-polygalacturonase activities were higher in control than in 1-MCP-exposed plums (Dong et al., 2001a). Thus, 1-MCP application provides a suitable way to test ethylene responsiveness of cell wall enzymes, particularly in those species for which an ethylene synthesis-suppressed line is still not available.

8

10

Fig. 6. ␣- and ␤-d-galactosidase activity (A) and ␣-l-arabinofuranosidase, ␤-d-xylosidase and ␤-d-glucosidase activity (B) in pears whether untreated or treated with 0.4 ␮l l−1 1-MCP and kept at 20 ◦ C. Values represent the means ± S.D.

␤-Gal III activity increases during ripening (Kitagawa et al., 1995), in close correlation with the increase in JP-GAL transcript levels (Tateishi et al., 2001). 1-MCP was found to partially counteract the increase in total ␣-Gal, ␤-Gal (Fig. 6A), ␣-Af, ␤-Xyl, and ␤-Glc activity (Fig. 6B) during pear ripening at 20 ◦ C. The reduced increase in the total activity of these enzymes in 1-MCP-treated pears after reaching the mature-green stage reflects the reduction of ethylene synthesis and ethylene action. Activity of three glycosidases (␤-Gal, ␣-Af, and ␤-Xyl) increased in untreated kiwifruit during ripening but only to a limited extent, or not at all, in kiwifruit treated with 1 ␮l l−1 1-MCP (Boquete et al., 2004).

Our results allow the following conclusions: (1) a 0.4 ␮l l−1 1-MCP treatment results in the temporary inhibition of ethylene production, delayed climacteric, and a concomitant postponement of fruit softening and degreening; (2) reversal of these parameters with exogenous ethylene is not statistically significant; (3) a second treatment with 1-MCP further delays the onset of the climacteric, but its effectiveness is noticeably lower; (4) the effect of 1-MCP on pear fruit ripening appears not to be totally uniform, since a percentage of the 1-MCP-treated pears reach their climacteric peak and lose the green color prematurely, beginning at the stem-end; (5) exposure to 1-MCP prior to—though not after—a medium-term (30–60 days) CS (1 ◦ C) displays additive effects in the preservation of pears thus extending their postharvest life significantly when transferred to room temperature; and (6) the ripening-related increase in the activity of several glycosidases is inhibited when 1-MCP is applied. Summing up, these findings point to the experimental and commercial usefulness of 1-MCP in “Bartlett” pear postharvest conservation.

Acknowledgements The authors are grateful to Enrique, Bettina, and Cecilia Scholtz for kindly providing the “Bartlett” pears for the experiments, and to Walter S.P. Pereira

G.D. Trinchero et al. / Postharvest Biology and Technology 32 (2004) 193–204

of Rohm and Hass for the donation of SmartFreshTM . This work was supported by grants from the Universidad de Buenos Aires (UBACyT Program) and the Agencia Nacional de Promoción Cient´ıfica y Tecnológica, Argentina.

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