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.
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 188.8.131.52), α-(EC 184.108.40.206) and β-galactosidases (EC 220.127.116.11), and endo-β-1,4-glucanase (EC 18.104.22.168). The appearance of polygalacturonase (EC 22.214.171.124) 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.
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.
Wenjing Guan, Xin Zhao and Donald J. Huber
Interspecific hybrid squash (Cucurbita maxima × Cucurbita moschata) is a well-known cucurbit rootstock for controlling soilborne diseases and improving abiotic stress tolerance. However, reduced fruit quality has been reported on certain melon (Cucumis melo) cultivars when grafted with squash rootstocks. In this study, a field experiment was designed to explore fruit development and quality attributes of galia melon ‘Arava’ by grafting with hybrid squash rootstock ‘Strong Tosa’. Grafted plants with ‘Strong Tosa’ showed delayed anthesis of female flowers by ≈8–9 days, but harvest dates were unaffected compared with non- and self-grafted ‘Arava’ plants. Early and total yields were not significantly different between grafted and nongrafted plants. Grafted plants with ‘Strong Tosa’ rootstock exhibited accelerated fruit development and greater vegetative growth. During the harvest period, ≈27% of grafted plants with ‘Strong Tosa’ wilted, which was determined as nonpathogenic. Grafting with ‘Strong Tosa’ rootstock resulted in reduced fruit total soluble solids (TSS) and consumer rated sensory properties.
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.
James W. Rushing and Donald J. Huber
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 126.96.36.199). 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.
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.
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.
Donald J. Huber and Ken C. Gross
The absence of endo-α-1,4-d-galacturonanase (PG, EC 188.8.131.52) 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.