NaOAc and 30 m m NaCl (pH 4.5) for 30 min at 34 °C. Reducing groups generated by PG were assayed using the method of Milner and Avigad (1967) , and PG activity was expressed as moles of D-galacturonic acid equivalents produced per milligram protein per
Eunkyung Lee, Steven A. Sargent and Donald J. Huber
Ming-Wei S. Kao, Jeffrey K. Brecht, Jeffrey G. Williamson and Donald J. Huber
-PG (EC 22.214.171.124) removes monomer units from the non-reducing end of the pectin chain and its activity increases only after extensive fruit softening for MF peaches ( Downs and Brady, 1990 ; Orr and Brady, 1993 ). Exo-PG activity can be similar or higher
Lucia E. Villavicencio, Sylvia M. Blankenship, G. Craig Yencho, Judith F. Thomas and C. David Raper
PG was not responsible for fruit softening. However, in these studies, suppression of PG activity was not complete ( Brummell and Harpster, 2001 ). Postharvest factors in transgenic tomato fruit with reduced PG activity were improved ( Schuch et al
Shulin Li and Preston K. Andrews
The activities of the fruit ripening enzymes cellulase, polygalacturonase (PG) and pectin methylesterase (PME) were detected during the development of sweet cherry (Prunus avium L.) fruit. Cellulase and PG activities of pericarp tissue increased 4-10 times between hypanthium abscission and harvest. PME activity remained high throughout this period of fruit development. There was a positive correlation between the anthocyanin content of the pericarp and both cellulase and PG activities. Concomitant with the increases in the activities of these ripening enzymes was a decrease in fruit firmness. The increases in cellulase and PG activities were checked following two-weeks storage at 10 C after harvest. The purification and characterization of the putative cellulase and PG enzymes will be discussed, together with attempts to chemically inhibit their activities and modify fruit softening.
Navjot K. Mangat and Jiwan P. Palta
The pericarp tissue of red mature tomato (Lycopersicon esculentum cv. Gagliano) was used to exctract polygalacturonase (PG) enzyme. The technique for assaying PG activity involves measurement of released reducing groups that were linked together in pectin. Since the crude extract of PG from pericarp will contain considerable reducing groups, we found that repeated washings of the cell wall pulp removed much of the sugars and thus minimized the background absorbance without loss of PG activity. There is an inherent perplexity concerning the selection of blank for PG assay. This is because (i) the enzyme extract contains both the substrate (pectin) and product (free reducing groups) involved in the reaction; (ii) the color development with cyanoacetamide requires heating for 10 min. Thus, even though the reaction is terminated with borate buffer (pH 9.0) the breakdown of pectin continues chemically by heat; (iii) the absorbance from both pectin and enzyme together at zero time termination was always lower than the sum of absorbances from pectin alone and enzyme alone. This suggests that when together in the same tube, the enzyme appears to protect the pectin from physical breakdown during the period of 10 min. boil needed to develop color using the cyanoacetamide. Thus, the most appropriate blank is processing separately the solutions of enzyme alone and substrate pectin alone for color development and then adding the two absorbances. Using this improved assay we found that lysophosphatidylethanolamine (LPE) inhibited tomato PG activity. This inhibition appears to depend on the ripening stage of the fruit. Our results suggest that LPE is able to impart firmness to tomato fruit by reducing the PG activity, which in turn could protect the pectin/middle lamellae from enzymic breakdown. The effects of LPE on PG activity are distinct from those of Triton X-100 and lysophosphatidylcholine.
Eunkyung Lee, Steven A. Sargent and Donald J. Huber
Roma tomatoes (`Sunoma') were hand-harvested at the mature-green color stage and treated with 100 μL·L-1 ethylene for 60 h at 20 °C and 90% RH. Tomatoes at breaker ripeness stage (<10% red coloration) were sorted by weight (about 100 g) and half of the fruits were treated with 1-methylcyclopropene (1-MCP; 1 μL·L-1 for 24 h at 22 °C). After 1-MCP treatment, individual fruits were subjected to double impacts over the marked locular surface with force equivalent to a 40-cm height drop using a pendulum impactor. In non-1-MCP treated fruit, impacts increased the maximum respiration rate by 27% (to 39.1 mL·kg-1 per h) and ethylene production by 24% (to 5.5 μL·kg-1 per h). Treatment with 1-MCP decreased relative production of both CO2 (56%) and ethylene (54%) over non-1-MCP treated fruit, while the ripening period (as measured by softening and color development) was extended 2.5 times, to about 8 d. Fruits treated with 1-MCP had increased TTA (about 40%; 0.58% citric acid equivalent), decreased pH (5%), and no difference in soluble solids content (3.7 °Brix); double impacts did not affect these values. Double impacts accelerated the onset of polygalacturonase (PG) activity by about 100% (to 99.8 mol·kg-1 per min*10-5 D-galacturonic acid) at day 6 over non-impacted control fruit. 1-MCP treatment delayed the onset of increased PG activity by 10 d over non-1-MCP treated fruit. Although 1-MCP alleviated the impact-induced increase in PG activity, PG activity recovered to rates similar to those of non-1-MCP treated fruit during the final 4 d of ripening.
Mei (Prunus mume `Daqinghe') fruit were immersed in 20 °C (control), 47 °C (HWT47), 50 °C (HWT50), or 53°C (HWT53) water for 3 min after harvest, then stored at 20 °C. Firmness, peel color, chlorophyll, chlorophyllase activity, soluble solids content (SSC), titratable acidity (TA), respiration, ethylene production, and pectinmethylesterase (PME) and polygalacturonase (PG) activity were monitored to determine the effects of hot water treatment in delaying fruit ripening. Control fruit displayed a typical climacteric pattern of respiration and ethylene production. Peak CO2 production and ethylene production were observed 6 days after harvest. Fruit softening was accompanied by decreases in hue angle, chlorophyll content, SSC, and TA and increases in chlorophyllase and PME and PG activity. Hot water treatment delayed the onset of the climacteric peaks of CO2 and ethylene production. The delays were associated with delays in fruit softening, consistent with lags in the rise of PME and PG activity; delays in yellowing and chlorophyll breakdown, consistent with lags in the rise of chlorophyllase activity; and delays in loss of SSC and TA. The shelf life of fruit increased by 6 days, or 60%, with HWT47, and by 8 days, or 80%, with HWT50 or HWT53.
Andrew Proctor and Terrence J. Miesle
Polygalacturonase (PG, E.C. 126.96.36.199) and pectinmethylesterase (PME, E.C. 188.8.131.52) activities, berry size, and texture were measured in fruit of developing high-bush blueberries (Vaccinium corymbosum L.). Peak PME activity occurred in red berries and preceded peak PG activity, which was observed in blue-red fruit. Extensive softening occurred with the transition in berry color from red to blue-red. Both peak enzyme activities and maximum softening occurred by the time the fruit were =70$%0 of their maximum fresh weight.
H. Yoshioka, K. Aoba and Y. Kashimura
The concentrations of water-soluble polyuronides in apples [Malus domestica Borkh.) and pears (Pyrus communis L.) increased, but those of EDTA- and HCl-soluble polyuronides decreased during softening. Total polyuronide content decreased slightly during softening in both fruits. Depolymerization of polyuronides was observed only in the water-soluble fraction in pear fruit during softening, concomitant with an increase in polygalacturonase (PG) activity. No detectable depolymerization was observed in any of the polyuronide fractions during softening of apple fruit nor was any PG activity detected. The polyuronide fractions extracted from pear and apple cell walls contained various amounts of methoxyl groups. Polyuronides with a high degree of methoxylation were preferentially lost from EDTA- and HCl-soluble polyuronides during softening of both fruit. The water-soluble polyuronide had a lower degree of methoxylation than those lost in the EDTA- and HCl-soluble fractions. These results suggest de-esterification of polyuronides with a high degree of methoxylation rather than the depolymerization of polyuronides in the solubilization of polyuronides during ripening of apples and pears.
Ramon A. Arancibia and Carl E. Motsenbocker
`McIlhenny Select' (easy detachment) and `Hard Pick' are two lines of tabasco pepper (Capsicum frutescens L.) that differ in the fruit detachment characteristics. Cellulase (Cx) and polygalacturonase (PG) activity, extracted from the fruit abscission zone, correlated inversely with the force needed to separate the fruit from the pedicel. A trend of higher Cx and PG is associated with the lower detachment force in the McIlhenny Select line. Differences in the fruit cell wall protein profile between both lines occurred during ripening. Two bands of 23 kDa and 40 kDa were higher in `McIlhenny Select'. A band of approximately 30 kDa was higher in `Hard Pick', while a band of ≈70 kDa increased in both lines. Isolation and characterization of these bands as well as Cx and PG is needed to understand the factors affecting fruit detachment in tabasco pepper.