Postharvest Biology and Technology 78 (2013) 4047

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Postharvest Biology and Technology

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Diffusivity of 1-methylcyclopropene in spinach and bok choi leaf tissue, disks of tomato and avocado fruit tissue, and whole tomato fruit
Xiaoqing Dong a,b , Maricruz Ramrez-Snchez a , Donald J. Huber a, , Jingping Rao b , Zhengke Zhang a,c , Sun Tay Choi a,d , James H. Lee a
a

Horticultural Sciences Department, IFAS, University of Florida, Gainesville, FL 32611-0690, USA College of Horticulture, Northwest A&F University, Yangling, Shaanxi Province 712100, China Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, China d National Horticultural Research Institute, Rural Development Administration, 475 Imok-Dong, Jangan-Gu, Suwon 440-706, Republic of Korea
b c

a r t i c l e

i n f o

a b s t r a c t
Gaseous 1-methylcyclopropene (1-MCP) has been widely employed for delaying ripening and senescence of harvested fruit and vegetables; however, details on ingress of gaseous1-MCP in plant tissues, which might contribute to differences in responsiveness of different horticultural commodities to 1-MCP, have not been reported. In this study, we used spinach and bok choi leaves, disks from tomato epidermis, stemscar and avocado-exocarp tissues, and whole tomato fruit to examine ingress of gaseous 1-MCP. Using a dual-ask system, equilibration of 20 L L1 (831 mol m3 ) 1-MCP through leaf tissue was reached within 12 h, and paralleled 1-MCP transfer through glass-ber lter paper. For disks derived from fruit tissues, changes in 1-MCP concentrations in the dual-ask system showed anomalous patterns, declining as much as 70% in source asks with negligible accumulation in sink asks. The pattern of 1-MCP distribution was markedly different from that of ethylene, which approached equal distribution with tomato stem-scar and avocado exocarp but not tomato epidermis tissues. 1-MCP ingress was further addressed by exposing whole tomato fruit to 20 L L1 1-MCP followed by sampling of internal fruit atmosphere. Tomato fruit accumulated internal gaseous 1-MCP rapidly, reaching approximately 89 L L1 within 36 h at 20 C. Internal 1-MCP concentration ([1-MCP]) declined around 74 and 94% at 1 and 3 h after exposure, respectively. Ingress was similar at all ripening stages and reduced by 45% in fruit coated with commercial wax. Blocking 1-MCP ingress through stem- and blossom-scar tissues reduced accumulation by around 60%, indicating that ingress also occurs through epidermal tissue. Fruit preloaded with 1-MCP and immersed in water for 2 h retained about 45% of post-exposure gaseous [1-MCP], indicating that 1-MCP is not rapidly sorbed or metabolized by whole tomato fruit. Rapid ingress of gaseous 1-MCP was also observed in tomato fruit exposed to aqueous 1-MCP. Both accumulation and post-exposure decline in internal gaseous [1-MCP] are likely to vary among different fruit and vegetables in accordance with inherent sorption-capacity, surface properties (e.g., waxes, stoma), volume and continuity of gas-lled intercellular spaces, and tissue hydration. 2012 Elsevier B.V. All rights reserved.

Article history: Received 17 October 2012 Accepted 19 December 2012 Keywords: Avocado Diffusion Metabolism 1-Methylcyclopropene Ripening Tomato

1. Introduction 1-Methylcyclopropene (1-MCP), a powerful ethylene antagonist (Sisler, 2006), is typically applied as a gas and tacitly assumed to rapidly ingress and diffuse through plant tissues. Indeed, the efcacy of 1-MCP at delaying ripening of Galia (Ergun et al., 2005) and Athena melon (Cucumis melo) fruit (Jeong et al., 2007) and preventing ethylene-induced placental-tissue watersoaking in

Corresponding author at: Horticultural Sciences Department, PO Box 110690, University of Florida, IFAS, Gainesville, FL 32611-0690, USA. Tel.: +1 352 273 4779; fax: +1 352 392 6479. E-mail address: djhuber@u.edu (D.J. Huber). 0925-5214/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.12.008

watermelon (Citrullus lanatus) fruit (Mao et al., 2004), attests to ingress of 1-MCP even in commodities of small surface/volume ratio. The capacity of woods and cardboards and intact and fresh-cut fruit and vegetables to deplete gaseous 1-MCP in closed systems provides evidence for non-target binding and/or metabolism (Dauny et al., 2003; Vallejo and Beaudry, 2006; Nanthachai et al., 2007; Choi and Huber, 2009; Rodrguez-Prez et al., 2009; Huber et al., 2010; Ambaw et al., 2011; Lee et al., 2012) and raises uncertainties regarding the extent to which gaseous 1-MCP accumulates in the internal atmosphere of targeted horticultural commodities. The objective of this study was to examine 1-MCP diffusivity in spinach and bok choi leaves, disks of avocado exocarp, tomato epidermis and stem-scar tissues, and whole tomato fruit. For whole

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tomato fruit, properties examined included accumulation and post-exposure persistence of internal gaseous 1-MCP, ingress through stem scar and epidermis, and effects of commercial wax coating. 2. Material and methods Plant material for experiments analyzing transfer of gaseous 1MCP in leaf tissue and fruit tissue-disks was obtained from local retail sources. Material tested included tomato (Solanum lycopersicum L.) and avocado fruit (Persea americana Mill. cv. Hass), and baby spinach (Spinacia oleracea L.) and bok choi (Brassica rapa L.) leaves. Experiments with whole tomato fruit employed unwaxed Sanibel fruit obtained from a packinghouse in Homestead, FL. 2.1. 1-MCP transfer through glass-ber lter paper, and disks of baby spinach and bok choi leaves, tomato epidermis and stem-scar, and avocado exocarp Experiments with lter paper employed Whatman GF/C glassber lter paper trimmed to 36 mm diameter. Glass-ber paper shows no 1-MCP-binding capacity, consistent with lack of binding of 1-MCP to glass jars (Nanthachai et al., 2007; Ambaw et al., 2011). Glass-ber lters were tested dry or after hydration with several mL diH2 O. Disks (diameter 36 mm, thickness 1.5 0.3 mm) of tomato epidermis and avocado exocarp were prepared from the equatorial region of the fruit using a scalpel. Tomato stem-scar disks were excised with a sufcient quantity of surrounding epidermal tissue to permit afxing in the dual-ask system. Disks of baby spinach and bok choi were prepared from the centermost region of healthy, turgid leaves. Filter paper or freshly prepared tissue disks were placed over the neck of a 125 mL side-arm ask (Pyrex No. 5360) with the edge of the disk extending to the outer limit of the ange. The ask has a at, 4-mm diameter ange, aiding in support of the disk and ensuring a relatively tight seal. Disks were secured in place with several layers of paralm. A second ask was inverted over the bottom ask, and the junction sealed with several layers of paralm. The asks were further secured using ring stand-mounted clamps positioned to provide moderate opposing force between the two asks. Tissues were oriented with adaxial (leaves) or external surfaces (fruit) facing the upper (source) ask. 1-MCP was injected with a syringe through the side-arm septum of the source ask at 20 L L1 (831 mol m3 ). 1-MCP transfer was monitored by GC analysis of 1 mL samples removed at intervals through side-arm septa of both upper and lower asks. Preliminary experiments established that 1-MCP injected into the lower ask (adaxial/surface tissues oriented downward) did not alter gas transfer. In experiments with avocado and tomato tissue disks, ethylene was injected into the source ask at 20 L L1 and concentrations monitored along with 1-MCP. Disks were pre-incubated in 40 mL aqueous 10 mM aminoethoxyvinylglycine (AVG) for 20 min to suppress ethylene biosynthesis (Saltveit, 2005). All experiments with the dual-ask system employed three replicates and were repeated two times. Data are expressed as average standard error of the mean (SEM) of 1-MCP or ethylene concentration (% of initial). 1-MCP and ethylene concentrations were measured using gas chromatography as described in detail in Lee et al. (2012) and Choi et al. (2008), respectively. 2.2. 1-MCP ingress in intact tomato fruit 2.2.1. Internal [1-MCP] in response to exposure time Sixteen tomato fruit (pink stage) were placed individually in 1.76 L glass jars with lids tted with septa. 1-MCP was applied at 20 L L1 . After exposure for 1, 3, 6 and 24 h [n = 4 jars (fruit) per

exposure time] at 20 C, a single jar from the rst treatment (1h exposure) was opened and the fruit immediately immersed in plastic bins containing 8.5 L diH2 O. Replicate jars of each treatment (exposure time) remained sealed and were opened in sequence (about 1.5 min intervals) following internal sampling of the prior replicate. Data for 1-MCP concentrations in response to exposure duration (see Fig. 4) indicate that the time required for processing fruit from all replicates in a single experiment would have had negligible inuence on internal 1-MCP concentration (designated here as [1-MCP]). Three samples (1 mL each) of internal atmosphere were removed from the submerged fruit at three equidistant points approximately 1 cm from the outer edge of the stem scar using a 1 mL syringe with a 23-gauge, 2.5 cm needle inserted to a depth of 1 cm. The 1 mL samples were immediately injected into 5 mL React-vials tted with Teon-lined septa (Thermo Scientic, Catalog No. TS-12718). One-milliliter samples were removed from the vials and analyzed for 1-MCP. Data are reported as average (four fruit three samplings per fruit for each exposure time) internal [1-MCP] ( L L1 ) SEM. 2.2.2. Internal [1-MCP] at different sampling depths Twelve tomato fruit (breaker-turning stage) were placed individually in 1.76 L glass jars with lids tted with septa. 1-MCP was applied at 20 L L1 . Each treatment had 4 jars (replications), each with a single fruit. Fruit were exposed to gaseous 1-MCP for 3 h at 20 C. The jars were opened in sequence as described in Section 2.2.1. One-milliliter samples of internal atmosphere were removed from three equidistant points approximately 1 cm from the outer edge of the stem scar of each fruit to a depth of 1, 2 or 3 cm (targeting the columella) using 1 mL syringes with 23 gauge needles of 2.5 cm (for 1 and 2 cm sampling depth) or 3.75 cm length (3 cm depth). GC septa were placed onto the needles to control sampling depth. Three samples of single measuring depth were removed from each replicate fruit (n = 4). The samples were immediately injected into 5 mL vials and analyzed for 1-MCP. Data are reported as average (four fruit three samplings of equal depth per fruit) internal [1MCP] ( L L1 ) SEM. 2.2.3. Internal [1-MCP] in different regions of tomato fruit Eight tomato fruit (pink stage) were individually placed in 1.76 L jars and 1-MCP applied at 20 L L1 . After 3 h, jars were opened and fruit processed for analysis of internal [1-MCP]. In four of the fruit, three 1 mL samples of internal atmosphere were removed at depth of 1 cm from three points at the blossom end. For the other four fruit, samples were removed through the stem end as described above. Data are reported as average (four fruit three samplings per fruit) internal [1-MCP] ( L L1 ) SEM. Six tomato fruit (breaker stage) were individually placed in 1.76 L jars and 1-MCP applied at 20 L L1 . After 3 h, jars were opened and fruit processed for analysis of internal 1-MCP. For each of the six fruit, six 1 mL samples were taken by insertion of a needle to a depth of 1 cm in six equidistant equatorial regions in radial pericarp or locule gel. Locule samplings sometimes yielded less than 1 mL of atmosphere, presumably due to the higher hydration of this tissue. After sampling, fruit were sliced in half to conrm needle placement (radial pericarp versus locule tissue). Data are reported as average (six fruit three samplings/each for radial pericarp and locule gel) internal [1-MCP] ( L L1 ) SEM. 2.2.4. Internal [1-MCP] and post-exposure changes in internal [1-MCP] in tomato at different ripening stages Nine tomato fruit each at green, pink, or red stage were placed in 3.24 L glass jars (3 fruit per jar) oriented horizontally to avoid fruitfruit contact. The jars were sealed with lids containing septa and injected with 1-MCP at 20 L L1 . Samples were treated 3 h at 20 C. Afterward, replicate jars of each ripening stage were

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opened and three fruit of each ripening stage sampled for internal 1-MCP. Additional groups of three fruit of each ripening stage were measured after holding in air for 1 or 3 h. One-milliliter samples of internal atmosphere were removed from 1-cm depth at two points near the stem scar and injected into 5 mL vials. One-milliliter samples were removed from the vials and analyzed for 1-MCP. Data are reported as average (three fruit two samplings per fruit) internal [1-MCP] ( L L1 ) SEM. 2.2.5. 1-MCP ingress through epidermis and stem/blossom scar tissues Six tomato fruit (pink stage) were employed in this experiment. At the bottom of each of three 1.76 L jars was placed a 9-cm diameter petri plate containing a sufcient volume of diH2 O water (10 mL) to ensure coverage of the blossom-scar region. After placing individual fruit into the petri plates, about 1 mL of diH2 O was carefully layered onto the stem end to fully cover the stem-scar. Three additional fruit were individually placed into jars containing 11 mL diH2 O water not in contact with the fruit. The jars were sealed and 1-MCP applied at 20 L L1 for 3 h at 20 C. Afterward, the jars were opened and the fruit immersed in diH2 O. One-mL of internal atmosphere was removed from two points in the stem scar and analyzed for 1-MCP. Data are reported as average (three fruit two samplings/fruit) internal [1-MCP] ( L L1 ) SEM. 2.2.6. Post-exposure retention of gaseous 1-MCP in internal atmosphere Twelve tomato fruit (light-red stage) were placed in four 3.24 L glass jars (3 fruit per jar) oriented horizontally to avoid fruitfruit contact. The jars were sealed and 1-MCP applied at 20 L L1 for 3 h at 20 C. Afterward, jars were opened and two fruit from each jar (replicate) immediately immersed in 8.5 L diH2 O (separate solutions for each jar/replicate). The third fruit from each jar was held in air and, after 2 h, immersed in water for sampling of internal gaseous 1-MCP as described above. Of the two immersed fruit from each treatment, one was sampled immediately for internal 1-MCP. The remaining fruit were maintained under water with the aid of a weighted perforated plastic plate and, after 2 h, sampled for internal gaseous 1-MCP. 1-MCP was sampled at three points (1-cm depth) for each fruit. Data are reported as average (four fruit three samplings) internal [1-MCP] ( L L1 ) SEM. 2.2.7. Ingress and post-exposure loss of gaseous 1-MCP in waxed and unwaxed tomato fruit Two groups of nine tomato fruit (green stage) were separately immersed for 23 s in diH2 O or a wax suspension [50:50 wax/water (v/v)] prepared using Sta-Fresh 711 (FMC FoodTech, FMC Technologies, Inc. Fresh Produce Technologies). Fruit were placed over PTFE Laboratory Matting (Saint Gobain Chemware) in trays and held for 24 h at 20 C for drying. Afterward, waxed and unwaxed fruit were placed in six 3.24 L jars (three fruit per jar, three jars per treatment). 1-MCP was applied at 20 L L1 . After 3 h at 20 C, the jars were opened and one fruit from each jar immersed in diH2 O and sampled for internal gaseous 1-MCP. Additional groups of three fruit per treatment were held in air for 1 and 3 h after 1MCP exposure and similarly processed for internal [1-MCP]. Data are reported as average (three fruit two samplings/fruit) internal [1-MCP] ( L L1 ) SEM. 2.2.8. Internal gaseous 1-MCP in tomato fruit treated with aqueous 1-MCP Three groups of four tomato fruit (turning stage) were separately placed in potato bags and immersed in 6 L of 1 mg L1 (18.5 mmol m3 ) or 2 mg L1 (37.0 mmol m3 ) aqueous 1-MCP prepared from AFXRD-038 powder (3.8% active ingredient, AgroFresh, Inc., Rohm and Haas, Philadelphia, PA) (Choi et al., 2008). The

aqueous 1-MCP solution was used no earlier than 15 min after preparation. Fruit were dipped for 1 min (1 and 2 mg L1 ) or 2 min (1 mg L1 ). The fruit were rinsed for several seconds in diH2 O and then transferred to fresh diH2 O for sampling internal atmosphere. Data are reported as average (four fruit three samplings/fruit) internal [1-MCP] ( L L1 ) SEM. 2.2.9. Preparation of gaseous 1-MCP 1-MCP gas was prepared using solutions of AFXRD-038 powder (3.8% active ingredient,) as described in Choi and Huber (2009). The solutions were prepared in side-arm asks with septa, immediately sealed, and gently stirred for 2 h prior to use. After measuring headspace 1-MCP concentrations, volumes of stock gaseous 1-MCP were injected into the treatment asks or jars at a concentration of around 20 L L1 . 2.3. Statistical analysis All data represent means of replicates (replicate number specied above for each experiment) SEM. Each experiment was repeated a minimum of two times. Representative results are presented. 3. Results 3.1. Transfer of gaseous 1-MCP through glass-ber lter paper or excised leaf and fruit tissues Transfer of gaseous 1-MCP occurred rapidly through glass-ber paper, reaching a distribution of about 48 and 48% of initial in the source and sink asks, respectively, within 1 h and 45 and 45% after 24 h (Fig. 1). Transfer was strongly suppressed through hydrated lter paper, with distribution reaching 98 and 1.6% of initial in source and sink asks, respectively, after 1 h and 70 and 23% after 24 h. Approximately 710% of system 1-MCP was lost during experiments with dry or hydrated lter paper, possibly due to leakage or binding of 1-MCP to septa (Choi et al., 2008). Paralm shows slight sorption of 1-MCP, but exposed paralm within the sealed system was negligible. As with dry lter paper, 1-MCP transfer through baby spinach and bok choi leaves was quite rapid, reaching equilibrium within 2 and 1 h, respectively (Fig. 2). 1-MCP transfer through disks of tomato epidermis and stemscar tissues and avocado exocarp tissue (Fig. 3AC) exhibited patterns markedly different from those observed for leaf tissues. Transfer of 1-MCP and ethylene through tomato epidermis occurred slowly, with about 74 and 89%, respectively, of initial concentrations remaining in the source asks after 24 h. Concentrations of both gases reached about 7% of initial in sink asks. For stem-scar tissue, 1-MCP and ethylene in the source ask declined to about to 73 and 80% of initial over the rst 6 h and 28 and 49% of initial after 24 h. Ethylene concentrations in the sink ask reached 10 and 35% after 6 and 24 h, respectively, whereas 1-MCP accumulation in the sink ask reached only 3 and 5% of initial concentrations after 6 and 24 h. With disks of avocado exocarp, 1-MCP and ethylene in the source ask declined to about 54 and 62% of initial concentrations, respectively, after 6 h and 18 and 41% after 24 h. Concentrations of ethylene in the sink ask increased to 33 and 34%, respectively, after 6 and 24 h. 1-MCP in the sink ask accumulated to only about 3% of initial levels. By contrast with 1-MCP transfer in leaf tissue and dry lter paper, the data for tomato and avocado tissue disks indicated that 1MCP in the dual-ask system was being signicantly depleted. For this reason, experiments were designed to measure 1-MCP ingress using whole tomato fruit exposed to gaseous 1-MCP and subsequently sampled for internal gaseous [1-MCP]. These experiments employed sampling procedures previously employed to measure

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Fig. 2. Diffusion of 1-MCP through baby spinach leaf (A) and baby bok choi leaf (B). Values represent the average of three replications SEM. Fig. 1. Diffusion of 1-MCP through dry and hydrated lter paper. Values represent the average of three replications standard error of the mean (SEM). (A) Transfer over 24 h. (B) Transfer over initial 3 h.

internal ethylene and CO2 concentrations in tomato fruit (Burg and Burg, 1962; Bergevin et al., 1993; Kapotis et al., 2004; Zhang et al., 2009, 2011). 3.2. Ingress of gaseous 1-MCP in tomato fruit Experiments with whole tomato fruit involved treatment with gaseous 1-MCP at 20 L L1 . Gaseous [1-MCP] in treatment jars declined no more than 25% in exposure periods of 24 h. Most of the experiments involved exposure periods of 3 h. 3.2.1. Internal [1-MCP] in response to 1-MCP exposure time, sampling depth and fruit region Maximum values for internal [1-MCP] were consistently attained at 3 (7.9 L L1 ) to 6 h (8.9 L L1 ), followed by declines following longer exposure (Fig. 4A). Experiments were routinely performed using 3 h exposure. [1-MCP] in samples from the stemregion were highest at 1 cm depth (7.8 L L1 ), followed by 26% and 32% lower values at 2 (5.7 L L1 ) and 3 cm (5.2 L L1 ), the latter representing the central-most region of the fruit (Fig. 4B). Following 12 h exposure, concentrations at 3-cm depth remained 32% lower than values at 1 cm (not shown), indicating that spatial differences in internal 1-MCP distribution are persistent. Following 3 h exposure, internal [1-MCP] was 56% higher at the stem (8.6 L L1 ) compared with blossom end (5.5 L L1 ) (Fig. 4C), and 73% higher in equatorial radial pericarp (6.3 L L1 ) compared with locule tissue (3.6 L L1 ) (Fig. 4D).

3.2.2. Internal [1-MCP] in fruit at different ripening stages Internal [1-MCP] was not affected by stage of ripening, with green, pink and red fruit showing similar ingress and post-exposure depletion in internal 1-MCP (Fig. 5). Internal [1-MCP] averaged about 8.3 L L1 immediately after exposure, declining to averages of 2.2 (74% decline) and 0.47 L L1 (94%) at 1 and 3 h following exposure. 3.2.3. 1-MCP ingress through stem- and blossom-scar tissues compared with epidermal tissue 1-MCP transfer through hydrated glass-ber lter paper (Fig. 1) was strongly suppressed, demonstrating that water constitutes an effective barrier to 1-MCP diffusion. Hydrated lter paper reduced by nearly 93% the quantity of 1-MCP transferred from source to sink asks in the initial 3 h period. As shown in Fig. 6A, covering the stem- and blossom-scar regions of tomato fruit with water prior to exposure to gaseous 1-MCP for 3 h reduced 1-MCP ingress by 60% compared with control fruit, indicating that 1-MCP ingress occurs through epidermal tissue. Subsequent experiments examined post-exposure changes in internal gaseous [1-MCP] in fruit exposed to gaseous 1-MCP for 3 h and then immersed in water to minimize off-gassing. As shown in Fig. 6B, fruit fully immersed in water for 2 h after exposure to 1MCP retained 45% of post-exposure gaseous 1-MCP concentrations compared with 9% for fruit held in air. 3.2.4. Ingress of gaseous 1-MCP in fruit treated with commercial wax Control fruit (unwaxed) showed typical ingress and postexposure loss of internal gaseous 1-MCP, declining from about

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Fig. 4. Internal [1-MCP] in tomato fruit treated with 20 L L1 1-MCP. (A) Internal [1-MCP] in tomato (pink stage) in response to exposure time. (B) Internal [1-MCP] in tomato (breaker-turning) at different sampling depth. (C) Internal [1-MCP] in tomato (pink) sampled at stem and blossom end. (D) Internal [1-MCP] in tomato fruit (breaker) sampled at radial wall and locule gel. Values represent the average of 12 (four fruit three samplings per fruit, A and B), eight (four fruit two samplings per fruit, C) or 18 (six fruit three radial and three locule samplings per fruit, D) samplings SEM.

Fig. 3. Diffusion of 1-MCP and ethylene through disks of tomato epidermis (A), tomato stem-scar (B) and avocado exocarp (C) tissues. Values represent the average of three replications SEM.

8.1 L L1 immediately following exposure to 1.5 (81% decline) and 0.26 L L1 (97% decline) after 1 and 3 h, respectively (Fig. 7). Postexposure internal [1-MCP] in fruit subjected to commercial wax application averaged 4.7 L L1 , 42% lower than control fruit. Postexposure loss of internal [1-MCP] was markedly delayed in waxed fruit, declining 52% after 1 h compared with 81% in unwaxed fruit. 3.2.5. Internal gaseous [1-MCP] following exposure to aqueous 1-MCP Internal gaseous [1-MCP] increased in a time/concentrationdependent manner during exposure to aqueous 1-MCP (Fig. 8). Immersion for 1 or 2 min in 1 mg L1 aqueous 1-MCP or 1 min in 2 mg L1 averaged 3.8, 5.8 and 8.8 L L1 , respectively. 4. Discussion The high efcacy of relatively low concentrations of 1-MCP at inhibiting ripening and other ethylene-dependent phenomena has provided clear evidence that 1-MCP ingress in fruit and vegetables is not restricted (Blankenship and Dole, 2003; Watkins, 2006;

Huber, 2008). The consumption of gaseous 1-MCP in static systems with intact and processed (fresh-cut) fruit and vegetables (Dauny et al., 2003; Nanthachai et al., 2007; Choi and Huber, 2009), while not a direct indicator of ingress per se, provides evidence for strong interactions between 1-MCP and biological targets. These interactions might involve free diffusion coupled with irreversible binding (Vallejo and Beaudry, 2006; Ambaw et al., 2011) and/or enzymic or nonenzymic oxidation of 1-MCP (Huber et al., 2010; Lee et al., 2012). Long-term off-gassing of 1-MCP from avocado fruit pretreated with gaseous 1-MCP (Dauny et al., 2003) demonstrated that sorption also involves reversible interactions. Quantities of gaseous

Fig. 5. Internal [1-MCP] in green, pink and red tomato fruit over a 3-h period following exposure to 20 L L1 1-MCP for 3 h. Values represent the average of six (three fruit two samplings per fruit) samplings SEM.

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Fig. 8. Internal [1-MCP] in tomato fruit (turning stage) following exposure to aqueous 1-MCP. Fruit were exposed to aqueous 1-MCP at 1 mg L1 for 1 or 2 min and 2 mg L1 for 1 min. Values represent the average of twelve (four fruit three samplings/fruit) samplings SEM.

Fig. 6. Internal [1-MCP] in tomato fruit exposed to 20 L L1 1-MCP for 3 h. (A) Prior to 1-MCP exposure, half of the fruit (all fruit at pink stage) were provided with water barriers over stem and blossom-scar regions. (B) Following 3 h exposure to 20 L L1 1-MCP, some of the fruit (all fruit at light-red stage) were measured immediately while others were held for 2 h in air or fully immersed in water. Values represent the average of six (three fruit two samplings/fruit, A) or twelve (four fruit three samplings/fruit, B) samplings SEM.

1-MCP sorbed to plant materials are estimated to be far in excess (factor of 106 ) (Nanthachai et al., 2007) of the anticipated number of ethylene binding sites (Sisler, 1979), suggesting that quantitative aspects of 1-MCP consumption do not reect binding to ethylene receptors.

Fig. 7. Internal [1-MCP] in tomato fruit (green stage) treated with 20 L L1 1-MCP following application of commercial wax. 1-MCP was measured following 3 h exposure to 20 L L1 1-MCP and after 1 and 3 h post-exposure in air. Values represent the average of six (three fruit two samplings per fruit) samplings SEM.

The present study rst addressed whether disks from leaf, tomato epidermis/stem scar and avocado exocarp tissues would serve as models for estimating 1-MCP ingress/diffusion. 1-MCP transfer occurred rapidly through baby bok choi and spinach leaves, equilibrating within one to two hours and paralleling diffusion through dry glass-ber paper. The strong suppression of transfer through hydrated glass paper indicates that water is an effective barrier to 1-MCP diffusion, consistent with the low Henrys Law constant for the gas (Pest Management Regulatory Agency, 2004). Accordingly, the rapid diffusion through spinach and bok choi leaf tissue likely occurs through intercellular spaces accessed directly via stoma or following partitioning of 1-MCP to cuticular waxes. Intercellular diffusion would be accompanied by partitioning into membranes, facilitating binding to ethylene receptors localized to endoplasmic reticulum (Chen et al., 2002). Changes in 1-MCP and ethylene concentrations showed highly variable patterns with disks of tomato epidermis/stem scar and avocado exocarp tissues. Tomato epidermal tissue showed minimum transfer of ethylene and only slight depletion of source-ask 1-MCP. By sharp contrast, ethylene approached equal distribution with stem-scar disks whereas 1-MCP was signicantly depleted in source ask with negligible accumulation in sink ask. Ethylene transfer through disks of stem-scar versus epidermis is consistent with models for ethylene diffusion in tomato fruit. Based on inux/efux of ethane gas in whole tomato fruit, Cameron and Yang (1982) determined that the stem scar accounted for nearly 97% of total gas exchange. Transfer of ethylene through avocado exocarp paralleled that noted for tomato stem-scar, indicating that avocado exocarp, unlike tomato epidermis, is permeable to ethylene. The marked depletion of 1-MCP in the avocado exocarp sourceask with negligible accumulation in sink ask was similar to that observed for tomato stem-scar tissue. We speculate that 1-MCP depletion in these systems involves irreversible sorption, possibly due to 1-MCP metabolism at the wounded surface of the disks. The failure of 1-MCP to accumulate in the sink asks indicates that reversible sorption processes as described for whole avocado fruit (Dauny et al., 2003) were not operative in the disk tissues. Further support for wound-related metabolism were the observations that 1-MCP sorption to apple slices was largely a cut-surface phenomenon and strongly suppressed by heating, tissue aging, anoxia, and the antioxidants ascorbate and hypotaurine (Lee et al., 2012). These factors would not be expected to uniformly negate sorption processes dependent on hydrophobic forces or physical entrapment.

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The differential rate and capacity of tomato epidermal, stemscar and avocado exocarp disks to deplete source-ask 1-MCP might be explained by compositional or physiological differences between the tissues. For example, depletion of headspace 1-MCP was much higher in avocado exocarp compared with mesocarp and tomato pericarp tissues, and much higher for outer versus radial tomato-pericarp tissues (Choi and Huber, 2009). Whereas 1-MCP diffusion through spinach and bok choi leaves occurred rapidly, possibly a consequence of minimal surface trauma to these tissues, the data collectively indicate that excised (wounded) fruit tissues are likely not suitable systems for estimating 1-MCP diffusivity in whole fruit. By contrast with the lack of 1-MCP transfer through tomato epidermis and stem-scar tissues, 1-MCP ingress occurred rapidly in whole tomato fruit. Following 3 h exposures to 20 L L1 1MCP, internal gaseous 1-MCP was detected in all fruit regions and independent of ripening stage. Internal [1-MCP] never exceeded 810 L L1 and declined following prolonged exposure. Under conditions restricting post-exposure off-gassing (fruit completely immersed in water), internal 1-MCP concentrations declined 55% in 2 h. This is consistent with depletion through sorption and/or metabolic processes (Dauny et al., 2003; Nanthachai et al., 2007; Choi and Huber, 2009; Huber et al., 2010; Lee et al., 2012). Loss through diffusive off-gassing is also indicated by the 91% decline in internal [1-MCP] in fruit held in air. Off-gassing appears not to represent free diffusion, however, since the post-exposure decline in internal [1-MCP] required considerably longer times than were required for off-gassing of ethane (>90% in <15 min) from tomato fruit (Cameron and Yang, 1982). The diffusion of ethylene in tomato fruit occurs largely (>95%) through the stem-scar (Cameron and Yang, 1982); however, as much as 40% of 1-MCP ingress occurred through epidermal tissue. Facile epidermal ingress is also supported by the report that asynchronous ripening following partial immersion of tomato fruit in aqueous 1-MCP was observed independently of direct exposure of the stem-scar (Choi et al., 2008). Nevertheless, the spatial distribution of internal 1-MCP indicates that the stem-scar region is the proportionally dominant ingress point. The sharp contrast between ingress through stem-scar of whole fruit and stem-scar tissue disks offers further evidence that wound effects (Lee et al., 2012) contribute to impeded ingress through disk tissues. Differences in internal [1-MCP] between radial pericarp and locule gel were particularly noteworthy given the similar proximity to epidermal- and stem scar-ingress points and sampling depth. The higher hydration and restricted intercellular spaces of this tissue (Ratanachinakorn et al., 1999) might explain the lower accumulation of gaseous 1-MCP in locule gel. Additionally, locule versus pericarp tissue contains much greater quantities of high methoxy pectin (HMP) (Huber and Lee, 1986), a strong sorption sink for 1-MCP in vitro (Choi and Huber, 2009). Indeed, the high hydration and sorption capacity of HMP for 1-MCP could contribute to both reduced accumulation as well as enhanced depletion of gaseous 1-MCP in locule tissue. These factors could additionally result in delayed egress of loculelocalized gaseous 1-MCP. Consistent with this idea, Mir et al. (2004) speculated that impeded lycopene accumulation in locule compared with surface tissues of 1-MCP-treated tomato fruit might reect restricted 1-MCP diffusion out of locule tissue. Reduction in 1-MCP ingress in fruit coated with commercial wax was somewhat unexpected. We anticipated that supplementing native waxes with commercial wax would facilitate ingress through enhanced 1-MCP partitioning (Pest Management Regulatory Agency, 2004) at the fruit surface. The sharp delineation in red color development at the immersion boundary of tomato fruit partially exposed to aqueous 1-MCP (Choi and Huber, 2008) is consistent with restricted movement of partitioned 1-MCP. Consistent with reduced 1-MCP ingress in waxed fruit, post-exposure

depletion of internal 1-MCP was delayed. We suggest that commercial wax serves as a reversible sorption sink for 1-MCP, as previously reported for avocado and safower oils (Dauny et al., 2003; Choi and Huber, 2009). Wax-partitioned 1-MCP might serve as a post-exposure reservoir of the gas, resulting in maintenance of comparatively high internal [1-MCP]. Aqueous 1-MCP formulations are effective at suppressing ripening following brief exposures (several minutes) (Manganaris et al., 2007; Choi and Huber, 2008). The present study shows that tomato fruit immersed in solutions of aqueous 1-MCP for 1 or 2 min accumulated gaseous 1-MCP at concentrations obtained with exposure to gaseous 1-MCP. The concentrations of gaseous (20 L L1 , 831 mol m3 ) and aqueous 1-MCP (1 mg L1 , 18.5 mmol m3 ; 2 mg L1 , 37.0 mmol m3 ) employed in the present study are around 1020-fold higher than concentrations effective at delaying ripening. The generation of comparable internal 1-MCP concentrations is consistent with the similar efcacy of proportionally lower levels of the gaseous (0.21 L L1 ) and aqueous formulations (100200 g L1 ) at delaying ripening. The results indicate that ingress and accumulation of gaseous 1-MCP in whole tomato fruit occur rapidly during exposure to gaseous or aqueous formulations. The post-exposure fate is likely due to multiple factors including off-gassing, metabolism, and reversible/irreversible physical sorption. Accordingly, we anticipate that quantitative aspects of gaseous 1-MCP accumulation and post-exposure depletion will likely differ markedly for different fruits, reecting inherent differences in physical sorption-capacity, surface properties (e.g., stoma, native and commercial waxes), volume and continuity of gas-lled intercellular spaces, and tissue hydration. Scrutiny of gaseous 1-MCP accumulation and depletion in the internal atmosphere of additional fruits may provide further insight into factors that control and modulate the responses to as well as recovery from the effects of 1-MCP. Acknowledgments Supported by grants from AgroFresh, Inc., USDA NIFA Award (SCRI) #2009-51181-05783, and by Federal formula funds NE1036. References
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