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Donald J. Huber

The discovery of 1-methylcyclopropene (1-MCP) as an inhibitor of ethylene action has provided yet another effective tool for understanding the role of this hormone in the development of higher plants. In the nearly 12 years since the description of the effects of this growth regulator on ethylene action, the subsequent introduction of stable formulations has resulted in an explosive increase in 1-MCP-targeted research, particularly, although not exclusively, in the discipline of postharvest science. The vicinal relationship between 1-MCP and postharvest biology is understandable in view of the established roles of ethylene, both beneficial and detrimental, in the handling and storage behaviors of a vast majority of harvested fruit and vegetative organs. The use of 1-MCP is proving to be supplemental to molecular approaches for identifying and understanding the spectrum of senescence and ripening processes under the direct control of ethylene perception. Climacteric fruits have served as the predominant target for investigations of 1-MCP, and the responses of these fruits have confirmed that the antagonist operates in opposition to ethylene. Studies of nonclimacteric fruits challenged with 1-MCP and other ethylene action inhibitors have identified both ethylene-dependent and ethylene-independent ripening processes and have posed interesting questions regarding the canonical distinctions between climacteric and nonclimacteric fruits.

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Linchun Mao* and Donald J. Huber

Having been hold in 10 μL·L-1 1-MCP or air for 18 h, seedless watermelon (Citrullus lanatus Matsum and Nakai, cv. Millionaire) fruits were cut to obtain tissue cylinders which were rinsed with 2% CaCl2 or deionized water. Respiration rate, ethylene production, firmness, electrolyte leakage, total soluble solids, titratable acidity, microbial growth rates (aerobic bacteria and yeast counts), and activities of ACS, ACO, PLC, PLD, LOX were determined during 7 days at 10°C to investigate the effects of 1-MCP and CaCl2. Ethylene was not detected in cylinders, while wound-induced respiration rates increased over time. Although 1-MCP stimulated ACS activity, it completely inhibited ACO activity and lowered respiration rate. CaCl2 had little effect on ACS activity, but stimulated ACO activity and maintained tissue firmness throughout storage. 1-MCP abolished the effect of CaCl2 in retaining or stimulating PLC, PLD and LOX activities, but inhibited aerobic bacteria synergically with CaCl2. Results suggest that 2% CaCl2 stimulated activities of PLC, PLD and LOX, which are key enzymes catalyzing phospholipid degradation. Results also provide evidence indicating that 1-MCP counteracts CaCl2 in aspect of lipolytic enzymes through unknown mechanisms. Furthermore, we provide results that 1-MCP and CaCl2 have a synergic effect in inhibiting the growth of aerobic bacteria. We suggest that CaCl2 may be applied together with 1-MCP as a complex treatment to extend shelf life of fresh-cut products.

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Jiwon Jeong and Donald J. Huber

Pre-ripe `Booth 7' avocado (Persea americana Mill.) fruit, a cross of West Indian and Guatemalan strains, were treated with 0.9 μL·L-1 1-methylcyclopropene (1-MCP) for 12 hours at 20 °C. After storage for 18 days in air at 13 °C, at which time whole fruit firmness values averaged about 83 N, half of the 1-MCP-treated fruit were treated with 100 μL·L-1 ethylene for 12 hours and then transferred to 20 °C. 1-MCP delayed softening, and fruit treated with 1-MCP retained more green color than air-treated fruit when full ripe (firmness 10 to 15 N). 1-MCP affected the activities of pectinmethylesterase (EC 3.2.1.11), α-(EC 3.2.1.22) and β-galactosidases (EC 3.2.1.23), and endo-β-1,4-glucanase (EC 3.2.1.4). The appearance of polygalacturonase (EC 3.2.1.15) activity was completely suppressed in 1-MCP-treated fruit for up to 24 days, at which time the firmness of 1-MCP-treated fruit had declined nearly 80% compared with initial values. The effect of exogenous ethylene applied to partially ripened 1-MCP-treated fruit differed for different ripening parameters. Ethylene applied to mid-ripe avocado exerted no effect on the on-going rate or final extent of softening of 1-MCP-treated fruit, even though polygalacturonase and endo-1,4-β-glucanase activities increased in response to ethylene. β-galactosidase decreased in 1-MCP-treated fruit in response to ethylene treatment. 1-MCP delayed the increase in solubility and depolymerization of water- and CDTA (1,2-cyclohexylenedinitrilotetraacetic acid)-soluble polyuronides, likely due to reduced polygalacturonase activity. At the full-ripe stage, the levels of arabinose, galactose, glucose, mannose, rhamnose, and xylose associated with the CDTA-soluble polyuronide fraction were similar among all treatments. In contrast, the galactose levels of water-soluble polyuronides declined 40% and 17% in control and 1-MCP treated fruit, respectively. Hemicellulose neutral sugar composition was unaffected by 1-MCP or ethylene treatment. The data indicate that the capacity of avocado fruit to recover from 1-MCP-mediated suppression of ripening can be only partially amended through short-term ethylene application and differs significantly for different ripening parameters.

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Daniel A. Stanley and Donald J. Huber

In previous studies, 1-methylcyclopropene (1-MCP) was shown to significantly suppress peel degreening and appearance of senescent spotting of banana fruit (Stanley and Huber, 2004). In the present study, the effect of the ethylene antagonist on banana pulp soluble sugar levels and on peel soluble and total phenolics was measured. One hundred and sixty hands (10 boxes) of banana fruit (Musaacuminata cv. Cavendish) were treated with ethylene (300 μL·L-1, 24 h, 15 °C, 90% RH) at a commercial ripening facility in Bradenton, Fla., and transported by truck (15 °C) to the University of Florida. Fruit were sorted and placed in 174-L ripening chambers, where 80 hands received 500 nL·L-1 1-MCP for two 12-h periods at 18 °C, while the other 80 hands (controls) were maintained in identical containers without 1-MCP for equal time periods at 18 °C. Mean whole fruit firmness in both treatment groups was 140 N and decreased to 15 N (controls) and 30 N (1-MCP) by day 12. Soluble sugars in the pulp of control fruit achieved levels between 160–180 mg·g-1 fresh weight by day 8, while 1-MCP treated fruit required about 12 days to achieve similar soluble sugar levels. Total phenolic compounds present in peel tissue of control and 1-MCP treated fruit required 10 and 14 days, respectively, to achieve levels of about 4000 μg·g-1 fresh weight. Chlorogenic acid levels, a subset of total peel phenolic compounds, peaked above 500 μg·g-1 by day 10 in control fruit and by day 12 in 1-MCP treated fruit. Maintenance of fruit firmness along with the achievement of acceptable sugar levels of 1-MCP treated fruit demonstrate possible benefits of suppression of ethylene action for retail and processing markets for banana fruit.

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Jong-Pil Chun and Donald J. Huber

The high catalytic potential of PG evident in reactions with soluble pectic polymers is typically not expressed in vivo. In this study, the binding and catalytic properties of PG isozyme 2, and the influence of the B-subunit protein, were investigated using cell walls prepared from tomato fruit expressing the B-subunit antisense gene. Cell walls were prepared from mature-green fruit and treated to remove/inactivate endogenous enzymes. Walls were then preloaded with rate-limiting quantities of purified PG 2, and incubated under catalysis-promoting conditions over the range of pH from 4.5 to 6.0. Cell walls of both B-subunit antisense and wild-type fruit retained comparable quantities of loaded PG 2. The enzymic release of pectin from PG-loaded walls was proportional to the quantity of wall-bound PG 2. In walls lacking the B-subunit protein, the quantity of pectin released by a given dose of wall-associated PG was as much as 2-fold higher than from wild-type walls. The B-subunit protein also influenced the extent of pectin depolymerization during ripening. The release of pectin from cell walls during periods of catalysis was not the sole indicator of the range of pectins hydrolyzed. Treating postcatalytic loaded cell walls to inactivate PG, and subsequent extraction of residual wall pectins using 50 mm CDTA solutions solubilized polymers of significantly lower mol mass compared with pectins solubilized directly from nonloaded cell walls.

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Jong-Pil Chun and Donald J. Huber

The catalytic capacity of tomato polygalacturonase (PG) toward soluble pectic polymers is in excess of activity expressed in vivo; however, in vitro assays of PG have traditionally been performed under conditions (pH 4.0 to 4.5, 150 mM NaCl) that likely do not reflect the apoplastic environment of ripening tomato fruit. In this study, hydrolysis of pectin by purified tomato PG (isozyme 2) was examined in response to K+ (the predominate apoplastic cation) and over the pH range from 3.0 to 6.0. In the presence of K+, PG activity toward polygalacturonic acid measured reductometrically increased nearly 3.5-fold from pH 4.0 to pH 5.5. In the presence of Na+, activity decreased 90% over the same pH range.

PG-mediated degradation of cell wall from mature-green fruit showed divergent hydrolytic patterns in response to pH and K+. At pH 4.5 in the presence of K+ (as KCl), catalysis resulted in both solubilization and extensive depolymerization of cell wall pectin, with oligomers accounting for a significant portion of the hydrolysis products. At pH 5.5, the total quantity of wall pectin released in response to PG2 was similar to that at pH 4.5; however, oligomer production was strongly suppressed at the higher pH. At pH values favoring extensive depolymerization, low mol mass products were produced at 5 mM K+ and increased to a maximum at 100 mM K+. At higher pH, hydrolysis patterns were not affected by [K+]. pH and ionic effects may contribute to the distinctive patterns of pectin hydrolysis observed for different fruits.

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Jiwon Jeong, James Lee, and Donald J. Huber

This study was performed to characterize the physiological responses of tomato (Lycopersicon esculentum L.) fruit harvested at either 10% to 30% or 30% to 60% color change and treated with two forms of 1-methylcyclopropene (1-MCP). Tomato fruit were treated either by submersion for 1 min in 1-MCP aqueous solution at the ambient temperature or by exposure for 12 h at 20 °C in air with 1-MCP gas, then stored at 20 °C. The concentrations (1.0, 5.0, or 10.0 μL·L-1) in 1-MCP aqueous solution were achieved through addition of 0.5, 2.5, or 5.0 g of AFxRD-300 powder (2.0% formulation, Agro-Fresh, Inc.) to 10 L of the de-ionized water, following manufacturer's instructions. 1-MCP (0.5 μL·L-1) gas in a 174-L container was achieved through addition of 0.22 g of SmartFresh® powder (0.14% formulation, Agro-Fresh, Inc.) to 100 mL of tap water. Both forms of 1-MCP significantly delayed ripening of fruit at the two initial ripeness stages, as noted by a significant delay in fruit softening and peel color change. The firmness of 30% to 60% color change tomatoes was significantly retained in response to gaseous or aqueous 1-MCP. Control fruit softened rapidly and reached the minimum marketable firmness value (about 5 N) within 8 days of storage at 20 °C, whereas fruit treated with gaseous 1-MCP (0.5 μL·L-1) or aqueous 1-MCP (1.0 or 5.0 μL·L-1) reached the same stage after 16, 20, or 24 days, respectively. Firmness retention was also highly significant for 10% to 30% color change tomatoes treated with both forms of 1-MCP. The highest concentration of aqueous 1-MCP (10.0 μL·L-1) did not result in a further delay in ripening compared with treatment at 5.0 or 1.0 μL·L-1 1-MCP.

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Fahad Al-Said and Donald J. Huber

A general feature of tomato fruit containing genetically reduced levels of polygalacmronase activity is decreased deterioration and cracking, particularly when handled at the ripe and over ripe stages. As fully ripe fruit are metabolically compromised and very prone to mechanical injuries, we investigated the influence of impact bruising on electrolyte leakage, pectin solubility, and depolymerization in ripening tomato fruit.

`Sunny' tomato fruit harvested at the mature-green, turning, and ripe stages of development and subjected to controlled impact injury exhibited elevated ethylene production at all developmental stages. Subsequent analyses were performed on discs prepared from bruised and uninjured pericarp tissue. Discs from bruised tissues exhibited enhanced electrolyte leakage and, in bruised tissues from ripe fruit, enhanced pectin efflux. Levels of soluble pectins derived from ethanol-insoluble powders were unaffected by bruising; however, pectins from bruised ripe fruit exhibited mol wt downshifts relative to those from nonbruised tissues.

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Guiwen W. Cheng and Donald J. Huber

Changes in the gel filtration behavior (apparent mol mass) of cell wall pectic polymers have been observed in a number of ripening fruits, including some that express little or no detectable polygalacturonase (PG). Pectins from tomato (Lycopersicon esculentum, Mill. v. Solar Set) fruit locule tissue show limited depolymerization during ripening, although alkali-soluble polymers are of reduced mol mass relative to water- and chelator-soluble polymers (Plant Physiol. 111:447). This study addressed whether the lower mol mass of alkali-soluble polymers was a consequence of extraction or specific metabolism of these wall polymers. Pectins from sequential water and chelator extractions of ethanol-insoluble solids from mature green tomato locule tissue were subjected to alkaline conditions. The size distribution of both water- and CDTA-soluble pectins treated with weak alkali were downshifted and similar to those extracted directly by weak alkali, indicating structural similarities of the three pectin fractions. Spectrophotometric analysis showed no involvement of β-elimination hydrolysis in the apparent mol mass reduction. The alkali-treated polymers were of greatly enhanced susceptibility to PG-mediated degradation. The alkali-associated changes also occurred in response to pectinmethylesterase hydrolysis. The results indicate that deesterification can strongly influence gel filtration behavior of pectins and may explain the apparent mol mass decreases of pectins in fruits not containing PG.

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Domingos P. F. Almeida and Donald J. Huber

Chilling injury limits the postharvest handling of many fruit and vegetables. In low-temperature storage trials, control treatments typically consist of fruit stored above the injury threshold. Since chilling exposures for tomato fruit often exceed 2 weeks, controls stored above the threshold continue to ripen, confounding comparisons with fruit maintained at low temperatures. In this study, the ethylene action inhibitor 1-MCP was used to arrest ripening to permit more valid comparisons between fruit stored under the two temperature regimes. Mature-green tomatoes were treated with EthylBloc and then stored at 5 or 15 °C for 2 or 3 weeks after which time the fruit stored at 5 °C were transferred to 15 °C to allow the expression of injury symptoms. 1-MCP inhibited ripening of fruit stored at 15 °C for 2 to 3 weeks. Color, pericarp firmness, and pectin solubilization of MCP-treated fruit stored at 15 °C remained at the values of mature-green fruit, validating their use as controls for these physiological characteristics. After 2 to 3 weeks at 15 °C, MCP-treated fruit resumed normal ripening. Comparing the fruit removed from low-temperature storage with nonripening controls at 15 °C revealed that storage at 5 °C for 2 to 3 weeks decreased the hue (yellowing) but did not affect chroma or lightness, maintained firmness, and did not affect pectin metabolism. Electrolyte leakage increased or remained unaffected by cold storage. MCP-treated fruit had slightly higher electrolyte leakage than non-MCP-treated fruit after storage at either 5 or 15 °C. We conclude that MCP-treated fruit provide adequate controls in experiments designed to study many aspects of low-temperature storage.