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Robert Bevacqua, Eugene Mielke, Timothy Facteau, Ruth Lavon and Paul Chen

An assay for pyruvate kinase (PK) was tested as a diagnostic tool for cork spot, a major physiological disorder in pear Pyrus communis L. cv. d'Anjou) fruit. PK activity and Ca and protein concentrations were measured in peel of normal and affected fruit during selected months in 2 years. Protein concentration was more closely associated with cork spot than PK activity or Ca concentration. These preliminary results suggested the PK assay was a poor diagnostic tool for cork spot.

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Juan M. Ruiz, Joaquín Hernández, Nicolas Castilla and Luis Romero

Potato (Solanum tuberosum L.) `Spunta' plants were grown with the root zone covered by different types of polyethylene plastic mulches. The plastic mulches used were transparent, white, co-extruded black and white, and black. As a control, plants were grown without plastic mulch. The parameters analyzed were soil temperature, root concentration of K and Ca, and enzymatic activities of ATPase and pyruvate kinase (PK), measured as basal and in the presence of K+ and Ca2+. The physical characteristics of the plastic mulches directly influenced soil and root temperatures in potato plants. In addition, the concentration of cations in the roots (particularly Ca2+) and basal ATPase activity were affected by soil temperature, whereas basal PK was not affected by soil temperature. The use of co-extruded black and white plastic mulch improved the nutritional status of Ca in the roots of potato plants. Finally, the basal ATPase and PK activities in the presence of K+ and Ca2+ were related with the root levels of these cations.

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Shiow Y. Wang, Hong J. Jiao and Miklos Faust

The activity of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphate gluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (ICDH), pyruvate kinase (PK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were studied in apple (Malus domestics Borkh.) buds during dormancy and thidiazuron-induced budbreak. When buds were dormant, the activity of the glycolytic enzymes GAPDH and PK and the tricarboxylic acid (TCA) cycle enzyme ICDH was low compared to that in nondormant buds. The activity of these enzymes increased during budbreak, peaked when buds were in the green tip stage just before the start of rapid expansion (at 8 days after thidiazuron treatment), and declined thereafter. The activity of pentose-phosphate cycle enzymes G6PDH and 6PGDH was higher in dormant buds than in nondormant buds. 6PGDH was about twice as high as G6PDH. During budbreak and resumption of growth, G6PDH and 6PGDH activity decreased.

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Brandon R. Smith and Lailiang Cheng

initiated with 5 m m isocitrate. NADP-IDH was assayed in a 1-mL mixture containing 100 m m Bicine-KOH pH 8.0, 5 m m MgSO 4 , 5 m m KCl, 0.5 m m NADP, and 50 μL of enzyme extract. The reaction was initiated with 5 m m isocitrate. Pyruvate kinase (PK

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George D. Nanos, Roger J. Romani and Adel A. Kader

The response of pear fruits and suspension-cultured pear fruit cells to 0% or 0.25% O2 is being examined to evaluate the feasibility of using such atmospheres for postharvest insect control. These treatments inhibited ethylene production, had no effect on acetaldehyde content, and increased ethanol production in pears kept at 20C for 10 days. The blossom end area of pear fruits was more prone to anaerobiosis, as indicated by increased alcohol dehydrogenase activity and ethanol content. Pear fruit plugs showed increased respiration and ethylene production rates when skin was present compared to plugs without skin. Methods for measuring activity of alcohol dehydrogenase, pyruvate decarboxylase, and pyruvate kinase have been modified and optimized and will be used to determine changes in pear fruit tissue during low O2 treatment and subsequent recovery in air.

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Y.H. Huang, David H. Picha and Charles E. Johnson

The glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose oxidase methods are commonly adapted for plant invertase assay. A disadvantage of the G-6-PDH assay is the relatively high cost of the coupling enzymes and cofactors. A disadvantage of the glucose oxidase method, which uses a glucose kit (Sigma, 510-A), is the presence of high activities of acid invertase and alkaline invertase in the PGO enzyme formula (peroxidase and glucose oxidase), which gives a falsely high invertase activity value. An alternative and inexpensive coupled assay was developed for enzymatic assay of plant invertases. In this assay, ADP produced from phosphorylation of glucose and fructose (hydrolysis products of invertases) is coupled to oxidation of NADH by the enzymes pyruvate kinase and lactate dehydrogenase in presence of phosphoenolpyruvate and NADH. This method was compared with the glucose-6-phosphate dehydrogenase method by using protein preparations derived from plant materials of three different species. Statistical analysis indicated that the alternative assay was similar in accuracy to the glucose-6-phosphate dehydrogenase method, with an advantage of reducing the cost from $0.85 to $0.35 per assay.

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Robert F. Bevacqua

Cork spot is a serious physiological disorder in pear (Pyrus communis L. cv. d'Anjou) in the United States, but a reliable technique for diagnosing it has not been developed. A review of the scientific literature indicated the disorder was generally linked with low calcium concentration in the fruit. In the present study, mineral analyses were conducted in 1987 and 1988 on soil, leaves, and fruit peel from normal trees and trees prone to cork spot. Soil tests and leaf analysis did not provide measureable differences between the two groups of trees. Fruit analysis provided variable differences between normal and cork spotted fruit, but no single nutrient or ration of nutrients could be consistently associated with the disorder. An assay for pyruvate kinase was evaluated as a diagnostic tool for cork spot. The assay did not provide measureable differences between normal and cork spotted fruit. An important finding of this study was to learn cork spotted fruit had higher soluble protein concentration than normal fruit.

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F. Medina, E.M. Yahia, L. Vazquez and A.M. Calderón

The tolerance of “Keitt” mango to a modified atmosphere (MA) of < 0.5% O2 + 70-80% CO2 for 0 to 4 days and a controlled atmosphere (CA) of 2% O2 + 50% CO2 for 0 to 5 days was evaluated. MA and CA delayed the respiratory climacteric of the fruit. There was no significant fruit injury due to the low O2 or high CO2 atmosphere, Sensory evaluation tests did not indicate the presence of any off-flavors. Both CA and MA increased the activity of the enzyme ATP: Phosphofructokinase, did not effect the activity of pyruvate kinase, and MA reduced the activity of PPi: phosphofructokinase. MA reduced the levels of frutose 6-phosphate, while phosphoenolpyruvate and pyruvic acid were not effected by both atmosphere treatments Although insecticidal O2 and CO2 atmosphere resulted in changes in the glycolitic activity, there was no indication of any injury and fruits were ripened normally, This work indicates the potential of the application of M/CA for postharvest insect control in mango.

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F. Medina, E.M. Yahia, L. Vazquez and A.M. Calderón

The tolerance of “Keitt” mango to a modified atmosphere (MA) of < 0.5% O2 + 70-80% CO2 for 0 to 4 days and a controlled atmosphere (CA) of 2% O2 + 50% CO2 for 0 to 5 days was evaluated. MA and CA delayed the respiratory climacteric of the fruit. There was no significant fruit injury due to the low O2 or high CO2 atmosphere, Sensory evaluation tests did not indicate the presence of any off-flavors. Both CA and MA increased the activity of the enzyme ATP: Phosphofructokinase, did not effect the activity of pyruvate kinase, and MA reduced the activity of PPi: phosphofructokinase. MA reduced the levels of frutose 6-phosphate, while phosphoenolpyruvate and pyruvic acid were not effected by both atmosphere treatments Although insecticidal O2 and CO2 atmosphere resulted in changes in the glycolitic activity, there was no indication of any injury and fruits were ripened normally, This work indicates the potential of the application of M/CA for postharvest insect control in mango.

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George D. Nanos, Roger J. Romani and Adel A. Kader

`Bartlett' pears (Pyrus communis L.) at two physiological stages, climacteric minimum or approaching the climacteric peak as achieved via storage for 2 or 8 weeks in air at 0C, respectively, were either ripened at 20C in air immediately or after exposure to 0.25% 02 for 4 days at 20C. Fruit stored for 2 weeks had relatively stable phosphofructokinase (PFK), pyrophosphate: fru-6-P phosphotransferase (PFP), and pyruvate kinase (PK) activities but decreasing succinate dehydrogenase (SDH) activities during ripening in air. Similar fruit treated with 0.25% O2 had slightly increased PFK, PFP, and SDH activities and decreased PK activity. Fruit stored for 8 weeks exhibited higher levels of PFK and PFP activity upon transfer to 20C, in accordance with their more advanced physiological state. In general, the enzymic changes in these fruit upon exposure to 0.25% O2 and subsequent ripening in air were similar to those observed in the less-mature counterparts, most notable being an increase in mitochondrial SDH. Exposure of suspension-cultured pear fruit cells to hypoxia resulted in an accentuated rise in phosphoenolpyruvate carboxykinase activity and a dramatic rise in SDH activity upon transfer to air. Taken in concert, the enzymic analysis supports the hypothesis that the rise in succinate levels observed in hypoxic fruit tissues is the result of a partial reductive tricarboxylic acid cycle. Cytochrome oxidase activity did not change during hypoxia whereas soluble peroxidase decreased somewhat, perhaps a reflection of their Michaelis constants for O2.