(4) FACTORS INFLUENCING SENSITIVITY OF CLIMACTERIC FRUITS TO 1-METHYLCYCLOPROPENE

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1 (4) FACTORS INFLUENCING SENSITIVITY OF CLIMACTERIC FRUITS TO 1-METHYLCYCLOPROPENE Donald Huber, Sun Tay Choi, Zhengke Zhang, Brandon Hurr ABSTRACT The response intensity of different climacteric fruits to 1-MCP applied after ripening initiation varies markedly. We have been investigating factors that contribute to response variation, including non-specific sorption and possible metabolism, and competition with internal ethylene levels. 1-MCP sorption varies markedly among different fruits, with high sorption exhibited by tissues of avocado, plantain and apple fruits, and low sorption by tomato and cantaloupe fruits. Lignins, oils and highly methylated pectin serve as primary targets for sorption of 1-MCP in vitro. Transient increases in internal ethylene levels in tomato fruit result in strongly dampened responses to 1-MCP, suggesting that naturally occurring levels of internal ethylene can significantly alter 1-MCP efficacy INTRODUCTION 1-Methylcyclopropene (1-MCP), an antagonist of ethylene action (Sisler, 2006), has been investigated for many horticultural crops but has been most effectively employed for climacteric fruits (Blankenship and Dole 2003; Watkins 2006; Huber, 2008). Nevertheless, the use of 1- MCP to regulate ripening of climacteric fruits is not without problems. As reported for several climacteric fruits, application of 1-MCP at the preclimacteric stage can result in incomplete or otherwise compromised ripening (Bassetto et al., 2005; Hurr et al., 2005; Bagnato et al., 2003; Jacomin et al., 2007). For this reason, studies with climacteric fruits have focused on application of 1-MCP after ripening initiation. Some fruits including tomato, papaya and cantaloupe retain sensitivity to 1-MCP when applied throughout the course of ripening (Wills et al., 2002; Ergun and Huber, 2004; Jeong et al., 2007). Other fruits including banana (Jiang et al., 1999) and more so avocado (Adkins et al., 2005) become increasingly unresponsive to 1-MCP applied after ripening initiation. The objective of this paper is to review some of our recent findings regarding factors other than ethylene receptors that might modulate fruit sensitivity to 1-MCP. MATERIAL AND METHODS 1-MCP sorption analysis. Avocado (cv. Hass), cantaloupe, tomato, plantain, orange, nectarine, and coconut fruits, and asparagus were obtained from local retail sources. Late Booth avocado fruit were obtained from a commercial source near Homestead, FL. 1-MCP sorption to mesocarp tissue of Late Booth avocado was tested at pre-ripe (firmness > 120 N) and full-ripe stages (firmness 10 to 20 N). Fruits and asparagus were processed into pieces of approximately 0.5 cm 3 (mesocarp, asparagus stems) or 1 cm diameter (exocarp of avocado, cantaloupe and plantain, pericarp of orange), and 20 g fresh weight used for 1-MCP sorption analysis. Horticultural Sciences Dept., IFAS, University of Florida, Gainesville, FL USA 27

2 Polysaccharides used for 1-MCP sorption analysis included Avicel cellulose powder, cotton, apple pectin (70 to 75% degree of esterification), citrus polygalacturonic acid (10 to 15% d.e.), xyloglucan, and corn starch. Sorption of 1-MCP was measured using 3 g of dry or hydrated (6 ml dih 2 O) polysaccharides. Spruce (commercial) and plantain peel lignin (prepared as described in Luo et al., 2007) were tested for 1-MCP sorption. Dry (3 g spruce and plantain lignin, 10 g coconut and nectarine endocarp in approximately 1 g pieces) and hydrated (6 ml dih 2 O) samples were tested for 1- MCP sorption. Detailed methods for measuring sorption of 1-MCP generated from SmartFresh SM Quality System (0.14% a.i., AgroFresh, Inc., Philadelphia, PA) can be found in Choi and Huber (2009). Briefly, tissue or other materials were placed in 130 ml Wheaton glass jars with lids fitted with septa. 1-MCP was injected at 18.6 µl L MCP sorption was determined by removing 1 ml headspace with a syringe and analyzed using a FID gas chromatograph equipped with a stainless steel column packed with Chromosorb 103 (60/80 mesh). Injector, oven, and detector were operated at 125, 125, and 150 C, respectively. The carrier gas (N 2 ), H 2, and air were 0.4, 0.6 and 3.8 ml s -1, respectively. Isobutylene was used as a standard (Jiang et al., 1999). Influence of ethylene on 1-MCP sensitivity of tomato fruit. Combination 1-MCP/ethylene treatment: Sebring tomato fruit were obtained from local packinghouses. Breaker-turning fruit were placed in 174-L chambers and provided with ethylene at 10 or 100 µl L -1. Half of the chambers were injected with 1-MCP at 500 nl L -1. After 6 h, the fruit were transferred to 20 C and monitored for ripening. Ethylene treatment followed by aqueous 1-MCP (AFxRD-038): Breaker-turning Florida 47 tomato fruit were treated with 100 µl L -1 ethylene for 6 h. Afterward, some of the ethylenetreated fruit were immersed for 1 min in aqueous 200 µg L -1 1-MCP (Choi et al., 2008) immediately or 1 or 6 h after ethylene treatment. Ripening was monitored as changes in firmness, surface color, and ethylene production at 20 C as described in Zhang et al. (2009). Internal ethylene measurement: Breaker-turning fruit were immersed in dih 2 O and 1 ml of internal atmosphere collected via insertion of a 2.0 cm long, 23-gauge hypodermic syringe into the blossom end of the fruit. Insertion was at a point within 5 mm of the blossom-end scar. The 1 ml samples were injected into 5 ml vials and 1 ml aliquots measured for ethylene concentration. RESULTS 1-MCP sorption to fruit and vegetable tissues, and cell wall polymers. All tissues sorbed 1- MCP, with avocado tissues, plantain peel and asparagus spears showing significantly higher sorption than cantaloupe, tomato and orange fruits (Figure 1). Even for the latter three fruits, however, outer tissues sorbed to a much greater extent than inner tissues. Sorption rates on a fresh weight basis ranged from highs of 34.2 ± 0.0, 24.1 ± 0.7 and 34.2 ± 0.0 ng kg -1 s -1 in avocado exocarp, mesocarp and seed tissues, respectively, 34.1 ± 0.2 ng kg -1 s -1 in plantain peel, 20.9 ± 0.7 and 19.4 ± 1.4 ng kg -1 s -1 in asparagus tips and basal sections, to lows of 6.8 ± 0.2 and 5.2 ± 0.7 ng kg -1 s -1 in cantaloupe exocarp and mesocarp and 4.2 ± 0.3 and 5.1 ± 0.4 ng kg -1 s -1 in tomato outer and inner pericarp, respectively. Sorption rates and capacities were significantly inhibited (50 to 80%) in all tissues in response to tissue drying, but recovered to various extents 28

3 upon rehydration. Sorption of 1-MCP to avocado fruit has been attributed to the high levels of oil (Dauny et al., 2003); however, avocado exocarp and mesocarp subjected to lipid removal by solvent extraction (chloroform/methanol) retained 99% and 84%, respectively, of their 1-MCP sorption capacity. Sorption to Late Booth avocado mesocarp was developmentally dependent, with sorption rate declining during ripening from 28.7 ± 0.7 to 15.8 ± 0.8 ng kg -1 s -1. Oil content of avocado fruit changes negligibly during ripening (El-Zeftawi, 1978). The data collectively indicate that multiple factors/sites contribute to tissue 1-MCP sorption. Cell wall polymers exhibited low (<20%) and comparable 1-MCP sorption capacities with the notable exception of high methoxy pectin (HMP) that sorbed nearly 90% of gaseous 1-MCP over 24 h (Figure 2). Sorption to HMP also paralled the sorption trends of fruit and vegetable tissues in being sensitive to hydration. Sorption to lignin and lignin-rich tissues (coconut and nectarine endocarp) was among the highest of the materials examined (Figure 3). Influence of ethylene on 1-MCP sensitivity of tomato fruit. Figure 4 illustrates that ethylene at 10 or 100 µl L -1 was without effect on the progression of tomato ripening, as expected for ripening-initiated climacteric fruit (Rhodes, 1980). In contrast, ethylene at 100 µl L -1 nearly completely negated the efficacy of 500 nl L -1 1-MCP at delaying softening and hue angle decline. Figure 5 illustrates the influence on ripening (external color) of exposure to aqueous 1- MCP (200 µg L -1 ) at intervals following removal of fruit from ethylene. 1-MCP applied immediately following ethylene treatment was significantly less effective compared with 1-MCP applied 1 and 6 h after ethylene. After 6 h, full resensitization to 1-MCP was observed, with efficacy being comparable to that for 1-MCP applied to fruit that had not received an ethylene pretreatment. Similar temporal shifts in 1-MCP sensitivity were also evident in climacteric ethylene production patterns (Figure 6). Noteworthy is that a 1 h delay in 1-MCP treatment (compare 0 h and 1 h after ethylene) resulted in an additional 2 d delay in the time to reach peak ethylene production. Internal ethylene concentrations prior to ethylene treatment averaged 7.05 ± 0.69 µl L -1. IEC averaged 32.4 ± 0.7 µl L -1 following removal from ethylene, declining to 18.3 ± 0.7 and 7.4 ± 0.4 µl L -1 at 1 and 6 h after ethylene treatment, respectively. DISCUSSION 1-MCP sorption to fruit and vegetable tissues, and cell wall polymers. The results of these studies demonstrate that 1-MCP sorption is a common feature of fruits and vegetables. Furthermore, 1-MCP sorption properties differ significantly among different tissues, with external tissues showing significantly higher sorption rate and capacity compared with internal tissues. Nanthachai et al. (2007) reported that sorption to a number of intact fruits and vegetables was correlated to dry weight, and suggested that cellulose might serve as a primary sorption target. We observed that dry weight/sorption relationships were not consistent for all tissues and that cellulose and most cell wall polymers showed low sorption. In contrast, sorption to HMP and more so lignins was quite high. The mechanism of 1-MCP sorption to lignins and HMP is unknown, but both polymers show high capacities for physical sorption of pesticides, phenols, fragrances, cholesterol and other hydrophobic compounds (Ludvík and Zuman, 2000; Van Beinum et al., 2006; Lewinska, 1994; Liu et al., 2007). In contrast to these interactions, wherein binding is readily reversible, attempts to release 1-MCP from lignins and HMP were unsuccessful. The apparent irreversibility of 1- MCP sorption to lignins and HMP also contrasts with the binding characteristics of 1-MCP in 29

4 commercial 1-MCP α-cyclodextrin inclusion formulations, in which hydration results in rapid release of free 1-MCP. The irreversible sorption to lignin, HMP as well as the plant tissues raises the possibility that 1-MCP is targeted by chemical or enzymic modification. The reactive groups in lignins (Carrott and Carrott, 2007) provide a possible means of destruction of 1-MCP through oxidation reactions. Similarly, the abiotic destruction of 1-MCP occurs through free radicalmediated oxidation reactions (Pest Management Regulatory Agency, 2004; Choi et al., 2009). Whether the sorption to lignins and HMP in vitro represents the basis for tissue sorption is unknown. The high sorption to asparagus spears and plantain peel, both rich sources of lignins (Anderson and Bridges, 1988; Emaga et al., 2008), is consistent with the idea that lignin is a major target for 1-MCP sorption to plant tissues. A lignin-based sorption model is also consistent with sorption of 1-MCP to woods and cardboard (Vallejo and Beaudry, 2006). Furthermore, sorption to HMP and lignins was enhanced in response to polymer hydration, a pattern also exhibited by plant tissues. Nevertheless, contribution of enzymic reactions to tissue sorption cannot be ruled out. Influence of ethylene on 1-MCP sensitivity of tomato fruit. Climacteric fruits treated with 1- MCP exhibit high and persistent insensitivity to applied ethylene (Sisler and Serek, 1997; Jeong and Huber, 2004; Adkins et al., 2005), consistent with the 30 d half-life estimates for 1-MCP receptor binding (Sisler and Serek, 1999). In contrast, 1-MCP efficacy at delaying ripening of breaker-turning tomato fruit was strongly influenced by internal ethylene levels at the time of 1- MCP exposure. Choi et al., (20080) reported that maximum delay of ripening of breaker-turning tomato fruit was achieved with gaseous 1-MCP at 500 nl L -1. As shown in the present study, effectiveness of saturating levels of 1-MCP was nearly completely negated when applied in the presence of 100 µl L -1 ethylene. These data indicate that ethylene concentrations in the range of those reported for internal atmospheres of a number of climacteric fruits (Burg and Burg, 1962) can completely suppress the effects of saturating doses of gaseous 1-MCP. It is evident that ethylene directly influences the capacity of 1-MCP to reach and associate with ethylene receptors. Breaker-turning tomato fruit pre-loaded with 100 µl L -1 ethylene prior to treatment with 1-MCP provided additional evidence for the influence in IEC on 1-MCP efficacy. Following ethylene treatment, IEC were elevated nearly 4.5-fold and sensitivity to 1-MCP was significantly reduced. Resensitization to 1-MCP occurred over 6 h, along with return of IEC to pretreatment values. The period required for return of IEC to pretreatment values is consistent with estimates for the dissociation half-life (minutes to hours) for ethylene binding (Schaller and Bleecker, 1995; Sisler and Serek, 1999). The data indicate that transiently increasing IEC followed by ethylene offgassing closely paralleled the pattern of desensitization and resensitization to 1-MCP. 30

5 ACKNOWLEDGEMENTS Figures 1, 2 and 3 reprinted from: Postharvest Biology and Technology, 52(1), Choi and Huber, Differential sorption of 1-methylcyclopropene to fruit and vegetable tissues, storage and cell wall polysaccharides, oils, and lignins. Pp , 2009, with permission from Elsevier. Figures 4, 5, 6 reprinted from Postharvest Biology and Technology, 54(1), Zhang, Huber, Hurr, and Rao, Delay of tomato fruit ripening in response to 1-methylcyclopropene is influenced by internal ethylene levels. Pp. 1-8, 2009, with permission from Elsevier. Supported in part by a grant from USDA-Tropical Subtropical Agricultural Research. The authors acknowledge AgroFresh, Inc. for providing the 1-MCP formulations used in these studies. LITERATURE CITED Adkins et al., 2005, Postharvest Biol. Technol., 35: Anderson, & Bridges, 1998, Am. J. Clin. Nutr., 47: Blankenship & Dole, 2003, Postharvest Biol. Technol., 28:1-25 Burg & Burg, 1962, Plant Physiol., 37: Carrott & Carrott, 2007, Bioresour. Technol., 98: Choi & Huber, 2009, Postharvest Biol. Technol., 52: Choi et al., 2008, Postharvest Biol. Technol., 48: Choi et al., 2009, Postharvest Biol. Technol., 53: Dauny et al., 2003, Postharvest Biol. Technol., 29: El-Zeftawi, 1978, Aust. J. Agric. Res., 29: Emaga et al., 2008, Bioresour. Technol., 99: Ergun & Huber, 2004, Eur. J. Hortic. Sci., 69: Huber, 2008, HortScience, 43: Jeong & Huber, 2004, J. Am. Soc. Hort. Sci., 129: Jeong et al., 2007, HortScience, 42: Jiang et al., 1999, Plant Growth Reg., 28: Lewinska et al., 1994, Artif. Organs, 18: Liu et al., 2007, Cellulose, 14: Ludvík & Zuman, 2000, Microchem. J., 64: Luo et al., 2007, Food Chem., 105: Nanthachai et al., 2007, Postharvest Biol. Technol., 43: Pest Management Regulatory Agency, Methylcyclopropene. Regulatory note, PMRA, Health Canada, Ottawa, Ont., 50 pp., 31

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