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.
Donald J. Huber
Senescence has long been a concern of postharvest scientists. The essential interests are two-fold. On the one hand, the premature senescence of many commodities, often accelerated due to adverse handling and storage conditions, renders them useless to the consumer. On the other hand, for many other commodities, notably fruits and cut flowers, the early manifestations of senescence form the basis of desirable quality attributes. The objectives of postharvest handling practices in the latter case are to: a) arrest continued developm ent during storage and b) ensure that normal development occurs following storage. For these reasons, an appreciation of the underlying causes of senescence becomes crucial to our understanding of how best to control and regulate the process. The symposium speakers will focus on the mechanisms and control of senescence in three horticultural commodity groups: leafy vegetables, fruits, and cut flowers.
Donald J. Huber
Cellulose contents in acetone powders derived from tomato (Lycopersicon esculentum Mill. ‘Sunny’) fruit pericarp and locular gel were measured. In the pericarp, cellulose increased throughout development, whereas Cx-cellulase activity increased during ripening. Locular gel cellulose content was lower than that in pericarp at all developmental stages, increasing through the breaker stage. Beyond the breaker stage, during the terminal period of gel formation, locular gel cellulose decreased. Levels of Cx-cellulase were high in gel tissue, and maximum activity was attained prior to the decrease in gel cellulose indicating that Cx-cellulase and cellulose may be important features of gel formation.
Donald J. Huber
Hemicelluloses and polyuronides from the cell wall of ripening tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit were examined using gel-filtration chromatography. Gel filtration of polyuronides revealed that these polymers were extensively degraded during ripening, as evidenced by the increase in the quantity of polymers that fractionated on the gel. Low molecular weight polyuronides were first evident in fruit harvested at the turning stage and they constituted the major portion of the polyuronides obtained from fruit at more advanced stages of ripening. The appearance of degraded polyuronides corresponded well with the activity of endo-D-galacturonanase, which appeared to be solely responsible for the degradation of these wall polymers. The cell wall hemicelluloses were also affected during ripening; gel-filtration analyses revealed marked changes in the molecular-weight distribution of these polymers. Hemicelluloses from immature green and mature green fruit were similar chromatographically, whereas those from fruit harvested during ripening showed progressively lower quantities of high molecular weight polymers and higher quantities of low molecular weight polymers (<40,000). These changes coincided with the degradation of the pectic polysaccharides; however, in vitro studies using isolated cell wall showed that pectin degradation occurred independently of the changes in hemicelluloses.
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.
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 22.214.171.124), α-(EC 126.96.36.199) and β-galactosidases (EC 188.8.131.52), and endo-β-1,4-glucanase (EC 184.108.40.206). The appearance of polygalacturonase (EC 220.127.116.11) 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.
Domingos P. F. Almeida and Donald J. Huber
Pectin solubility in ripening tomato fruit is typically studied in vitro, employing isolated cell walls; however, it is unknown whether in vitro studies address the actual changes in the status of pectins in the fruit in situ. In vivo pectin solubilization was examined in a pressure-extracted apoplastic fluid obtained from ripening and chill-injured tomato fruit with down-regulated polygalacturonase (PG) activity and untransformed wild-type. Pectin levels in apoplastic fluid increased 3-fold during ripening and were not affected by PG levels. In contrast, PG strongly affected pectin levels in bulk, enzymically active pericarp fluid. There was a 14-fold increase in bulk pectin levels during ripening of PG-antisense fruit and a 36-fold increase in wild-type fruit. Pectin levels in the apoplastic fluid of fruit stored at 5 °C for 14 days were 40% lower than that of freshly harvested mature-green fruit, but increased significantly upon transfer of fruit to 15 °C. Monomeric galactose in the apoplastic fluid increased from 41 mg·mL–1 at the mature-green stage to 67 mg·mL–1 in ripe fruit. Bulk levels of galactose were 3- to 4-fold higher than apoplastic levels. After low-temperature storage galactose levels were 50% and 20% lower than in freshly harvested fruit for the bulk and apoplastic fluids, respectively. These results indicate that in vivo pectin solubilization is restricted and largely independent of PG. Low-temperature storage reduces in vivo pectin solubilization, an effect that is reversed upon transfer of fruit to higher temperature following cold storage.
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.
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.
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.