Fig S-6 Double reciprocal plot (a) and data (b) of EFE against ACC concentrations in G ranny Smith fruit discs.
6.3 M ATERIALS AND METHODS
6.3.1 Materials (See Chapter 2)
6.3.2. Methods
6.3.2. 1 of fruit discs
6.3.2.2 Measurement of EFE and of
fruit discs
E FE activity, ethylene production and respiration of discs were m easured by sealing six discs in each of 3 or 4 X 36 ml vials for a certain tim e after vacuu m-infiltration with 0.4 M mannitol solution containing 5 mM AOA to inhibit ACC synthase, 1 mM CHI to inhibit EFE synthesis and with (for E FE), or without (for ethylene production) 5 mM ACC (as the substrate for EFE). After incubation at 27°C, 2 X 1 ml gas samples were removed from each vial. One sample was used for ethylene analysis, the other for C02 analysis using G LC (See Chapter 2).
6.3.2.3 Carbon dioxide treatment
To test the influence of C02 treatment on EFE activity, ethylene production and respiration of fruit discs, 3 or 4 X 36 ml vials and containing six discs were flushed for 20 seconds with a gas mixture which contained 23% C02, 21 % 02 and 66% N2. In control (C02-free) vials, small plastic tubes which contained a suspended piece of filter paper, wetted with 0 . 1 ml 1 0% KOH, was hung in each vial to prevent accumulation of C02. After i ncubation for a certain time, 1 ml gas was sampled for C02 measurement by G LC.
6 .3.2.4 Cobalt ion treatment
In order to reduce possible variation of internal cobalt which might have been caused by different tissue permeability to eo++, fruit discs were vacuum-infiltrated with a medium containing 5 mM ACC, 5 m M AOA and various eo++ concentrations.
For ethylene production measurements, six fruit discs for each treatment were vacuum-infiltrated (a vacuum of 90 Kpa was applied for 1
min then released; this procedure was carried out 3 times to ensure full penetration of the solution) in 3 X 36 ml vials containing 3 ml 0.4 M mannitol and the following concentrations of CoCI2: 0, 0.01 , 0.05, 0.1 , 0.5, 1 and 5 m M for Hosui and 0, 0.01 , 0. 1 , 1 , 3, 5 and 1 0 mM for Granny Smith.
To measure EFE activity, 5 mM ACC, 5 mM AOA and 1 mM CHI were
added to the above 0.4 M mannitol solutions.
After vacuum-infiltration, the fruit discs were dried on filter paper then six discs were sealed in each of 3 X 36 ml vials for 2 hours at 27°C before removal of 1 ml gas for c2H4 measurement.
To test the effect of C02 on EFE activity of discs in the presence of co++-treated discs, the same method of eo++ application was used as above with o nly one concentration, 1 m M CoCI2. After saturation with the medium, discs were treated with or without C02 for 1 and 4.5 hours for Hosui fruit, and sealed for 2 hours for Granny Smith fruit at 2JCC before removal of 1 ml gas for c2H4 measurement (method described above).
6.3.2.5 Silver ion treatment
Silver nitrate solutions were applied to fruit discs using the same m ethod as for the cobalt treatment. Concentrations of AgN03 in 3 ml of 0.4 M mannitol medium were 0, 0.02, 0.05, 0. 1 , 0.25, 0.5, 1 .0, 3.0 and 5.0 mM without (for ethylene production measurement) or with (for EFE activity measure ment) 5 m M ACC, 5 mM AOA and 1 m M CHI. After treatment six discs were dried and sealed in each of 4 X 36 ml vials for 2 hours at 27°C before removal of gas samples for c2H4 and C02 measurement.
To test the effect of C02 on EFE activity in Ag+-treated discs, the same method was applied except that only one concentration of Ag+No3 ( 1 m M) was used, and that the time o f C02 treatment was 2 hours.
6.3.2.6 treatment
In Hosui, discs were sampled from fruit that had been stored for 30 days at 1 °± 1 °C. In Granny Smith apple, discs were sampled from fruit that had been stored for 60 and 1 02 days at 1 °± 1 °C.
Six discs were placed in 4 X 36 ml vials containing 0.05 M K+ phosphate-sucrose buffer (pH 6.5), 5 mM ACC, 5 mM AOA and 1 mM CHI and vacuum-infiltrated for 3 minutes before removal and drying on filter paper. Six discs were sealed in each of 4 X 36 ml vials. Carbon dioxide was applied using the method described above. After sealing, NDE was injected into each vial with a syringe to give a concentration of 0.5% (v/v). After 2 hours incubation at room temperature (about 20°C) in the fume cabinet, 1 ml g as was sampled for ethylene analysis by GLC.
6.3.3 Statistical analysis of results
Each treatment had three or four replicates. Each experiment was repeated at least twice. A computing programme (SAS/STAT User's Guide) was used to analyse data for means and standard errors, and Duncan's m u ltiple comparisons (Steel and Torrie, 1 981 ).
6.4 RESULTS
6.4.1 Effects of CoCI2 on EFE activity
E FE activity in both fruits was stimulated, inhibited, or u naffected depending on the concentrations of cobalt applied (Fig.6-1 and Fig.6-2).
In Hosui fruit, EFE activity in discs increased from 1 6.1 4 (control) to 1 8.57 niC2H4/g/h at 0.01 mM CoCI2 (Fig.6-1 ), before declining to the control value at 0.05 mM. At higher concentrations, EFE activity decreased as eo++ increased. There was a rapid 58% decrease in E FE activity as eo++
increased from 0.05 to 1 mM, followed by a slower 31 % decrease between 1
to 5 mM CoCI2 (Fig.6-1 ).
I n contrast no significant increase in EFE occurred in G ranny Smith apple discs between control and 0.01 mM eo++ treatment (Fig.6-2). Over the range of 0. 1 -3 mM, EFE activity decreased rapidly by 64.9% from 32. 32 to 1 1 .33 niC2H4/g/h followed by a slower fall off (30.2%) in E FE activity between 3 and 1 0 mM CoCI2.
6.4.2 Interaction between CoCI2 and Carbon dioxide on EFE activity
Because 1 mM CoCI2 significantly reduced EFE activity, this concentration was selected for the experiment to test the interaction between eo++ and C02 on EFE activity.
In Hosui, ethylene production from discs was not detectable after 1
hour incubation at 27°C and only trace amounts of ethylene were measured during 4.5 hour incubation, thus the effect of eo++ on ethylene production could not be measured. After a 1 hour incubation C02 increased EFE activity by 42.76% (Table 6-1 ), but eo++ did not significantly reduce E FE activity. The combination of eo++ and C02 did not increase or decrease E FE activity compared with control but the addition of C02 did overcome the i nhibitory effect of eo++ alone.
EFE activity in control discs did not change betwee n 1 and 4.5 hours i ncubation. C02 also increased EFE activity after 4.5 hours. While eo++ again reduced EFE activity at this time, by 62%. However after 4.5 hours the combination of C02 and CoCI2 did not reverse the inhibitory effect of CoCI2.
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