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- Author or Editor: Donald J. Huber 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.
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.
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.
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.
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.
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.
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.
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.
Enzymically active cell wall isolated from mature-green and ripening tomato (Lycopersicon esculentum Mill cv. `Rutgers') fruit was employed to investigate the mobility of the enzyme polygalacturonase (PG, EC 18.104.22.168). Cell walls from mature-green `Rutgers' fruit or from the ripening mutant rin, which alone exhibits little or no release of pectin, were unaffected by the addition of enzymically active cell wall from ripening `Rutgers' fruit, indicating that PG is either not transferred at all or is not transferred to sites of pectin hydrolysis. The quantity of pectin released by the addition of soluble PG to enzymically active wall depended on the quantity of enzyme added. Similar data were obtained using purified PG2. Pectin solubilization from all wall isolates exhibiting enzymically mediated pectin release diminished with time; however, transfer to fresh buffer initiated a resumption of autolytic activity, indicating that an inhibitor is released during the course of pectin hydrolysis.
A vacuum infiltration technique that allowed precise control of both infiltration rate and amount of solution administered to whole tomato (Lycopersicon esculentum) fruit was developed. Controlled volumes of 5 mm solutions of CuSO4, Cu(NO3)2, HgCl2, CaSO4, KNO3, (NH4)2C2O4, Na2HPO4, citrate and 1 mm EDTA or EGTA were infiltrated into intact, mature-green tomato fruit and evaluated with regard to their effect on the pattern of tomato ripening. Copper significantly accelerated lycopene accumulation and influenced both the timing and magnitude of climacteric ethylene production. Infiltration with HgCl2 elicited similar effects as copper, but severe phytotoxicity was observed. In contrast, CaSO4, KNO3, and chelators had no significant effect on the pattern of ripening. Copper initiated wound ethylene production in the ripening mutant rin that reached up to 50% of the wound levels observed in normal fruit, but rin was not induced to ripen.