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- Author or Editor: Donald J. Huber x
- HortScience x
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.
Abstract
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.
Abstract
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.
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.
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.
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.
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.
Hydrolytic activities in liquefying locule tissue of mature-green tomato (Lycopersicon esculentum Mill. `Solar Set') fruit were studied in pursuing the understanding of mechanisms involved in the rheological changes occurring in this tissue. Ethanol-insoluble solids (EIS) were prepared with and without enzyme-inactivating treatment. The release of uronic acids from enzymically active EIS incubated under autolysic conditions was 5-fold higher than recoveries from inactive EIS. Uronic acid release was partially inhibited by 1 mm Hg2+. Cell-free proteins extracts from active EIS exhibited hydrolytic activity against inactive EIS. Pectins released from active EIS showed no evidence of main-chain hydrolysis. Neutral sugars recovered as 80% ethanol-soluble products of autolytic reactions included glc, gal, rha, ara, xyl, and man. Gal was recovered at significant higher levels in autolysates of active EIS. Glycosidases present at high activities in locule tissue included α- and β-galactosidases, β-mannosidase, β-arabinosidase, and β-glucosidase. The results confirm our earlier findings that the metabolism of water, chelator, and alkali-soluble pectins in tomato locule tissue involves deglycosylation with no apparent depolymeriation. These changes alone appear to be inadequate in explaining the unique rheological characteristics in locule gel tissue.
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.
The absence of endo-α-1,4-d-galacturonanase (PG, EC 3.2.1.15) in some fruits and the molecular suppression of PG in tomato fruit have collectively provided evidence that this protein is neither required nor sufficient to achieve normal softening in fleshy fruits. On the other hand, initial claims that down-regulation of PG was without effect on tomato softening were overstated. The influence of PG on softening does appear to be minimal during the initial stages of ripening, during which time changes in the locule tissues can significantly alter texture as monitored in whole fruit. Enzymes, including pectinmethylesterases, cellulases, rhamnogalacturonanase, and glycosidases may also play pivotal roles in softening. β-galactosidases have attracted much attention as potential determinants of fruit texture; however, conclusive evidence for this role is lacking, and increased levels of β-galatosidase (and net cell wall galactosyl residue loss) have been noted in senescing vegetative and floral organs as well as in fruit. Apoplastic pH, ionic activity, and composition are likely to contribute to tissue and wholeorgan texture through weakening of polymer aggregates and/or through modulation of cell wall enzyme activity. During the latter stages of ripening and overripening, the role of PG is apparent from the persistent structural integrity of fruit transformed with PG antisense constructs. Patterns of softening and deterioration in normal tomato fruit suggest that the catalytic activity of PG in vivo is initially queued and does not parallel the accumulation of PG protein. Developmental changes in membrane permeability, physical injury, and other stress conditions can alter the apoplastic environment, releasing constraints on PG action.