4.3.1 Materials (See ' General Materials and Methods')
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The time � experiments carried out were written in the titles of tables or figures. Fruit of maturity at harvest time, colour grade 2-3 (Japanese pear colour chart), fruit weight 1 80±5g for Hosui, and 'a' value (-20±0.5), starch grade 2-3 and fresh weight (means:1 85g) for Granny Smith were used for experiments.
4.3.2 Preparation of fruit discs (See 'General Materials and Methods') 4.3.3 Ethylene and carbon dioxide measurement (See 'General Materials and Methods')
4.3.4 Methods
4.3.4. 1 Wound discs
Six discs were placed in each of 5 X 36 ml vials and incubated in a shaking waterbath ( 1 20 strokes/min) at 27°C. Ethylene concentrations in vials were measured after 0.5, 1 , 2, 3, 4, 6, 8 hours incubation. For each
measurement, vials were sealed for 30 minutes before withdrawing a 1 ml gas sample for ethylene measurement using G LC. Immediately after each sampling, vials were flushed with air for 20 seconds to prevent C02
accumulation and then returned to incubate at 27°C until the next ethylene determination was required.
4.3.4 .2 Isotonic solution assessment
The osmotic potential of the tissue was determined using a
gravimetric method (Harker, 1 986). Five discs were placed i n each of three petrif dishes and incubated in the buffers or mannitol solution for 2 hours at 27°C. Buffers used were K2HP04-KH2P04 buffer (pH 6.5); and Na2HP04- citric acid buffer (pH 6.5) (see Chapter 2). Mannitol (pH 6.5) concentrations used were 0.2 M, 0.3 M, 0.4 M and 0.8 M for Hosui and 0.4 M and 0.8 M for Granny Smith tissues. Fresh weight of each disc was
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easured accurately before and after incubation by electronic balance (Metter PE 360) to obtainan indication of the osmotic potential of the tissues.
4.3.4.3 EFE in 0.4 M or 0.8 M mannitol solution at 27°C
Six discs were immersed in each of 4 X 36 ml vials containing 3 ml of 0.4 M or 0.8 M mannitol solution which contained 3 mM ACC and 1 m M cyclohexmide (CHI); the vials were incubated for 2 hou rs at 27°C, before being sealed for 1 hour at 27°C when a 1 ml sample were withdrawn fro m each vial for ethylene analysis by GLC.
4.3.4.4 of methods for fruit tissue with ACC
To determine the concentration of ACC which saturated Hosui and Granny Smith tissues, six discs were placed in each of 3 X 36 ml vials containing 3 ml of medium and incubated at 27°C for 2 hours. The medium contained K+-phosphate buffer (pH 6.5) and a range of ACC concentrations (0, 0.005, 0.02, 0.05, 0. 1 , 0.2, 0.5, 1 .0, 2.0, 5.0 mM). Discs were then dried on tissue paper and each of six disc replicates were placed dry and sealed in each of 3 X 36 ml vials for a further 1 hou r incubation at 27°C. A 1 ml sample was then taken from each vial and ethylene concentration was measured by GLC.
To evaluate the most efficient method of saturating tissue with ACC, several methods were used to expose discs to a medium containi ng 5 mM ACC in K+-phosphate buffer (pH 6.5).
The methods used were:
(a) I m mersion method: as a control, discs were immersed in above medium for 2 hours at 27°C.
(b) Vacuum-infiltration method: Discs were infiltrated with the medium by vacuu m (90 Kpa) followed by incubation for 2 hours at 27°C. The vacuum infiltration time were :
- 3 minutes; - 2 minutes;
- 1 minute;
(c) Discs were incubated in the medium for 2 hours at 27°C, then vacuum i nfiltrated with the same medium. The vacuum-infiltration time were :
- 3 minutes; - 2 minutes; - 1 minute ;
- Intermittent-vacuum : 1 min vacuum, then release, repeated for 3 times. After treatment with ACC, discs were dried on tissue paper. Six discs were then sealed in each of 3 X 36 ml vials for 1 hour at 27°C before
ethylene concentrationf in a 1 ml sample withdrawn from each vial , was m easured by GLC.
4.3.4.5 of three methods for E FE
To obtain the highest EFE activity, three methods for measuring EFE activity were tested. After saturation with 5 mM ACC by immersion of discs i n the medium for 2 hours at 27°C, six discs were sealed in each of 4 X 36 ml vials with the following conditions:
(a) Discs were immersed in 3 ml medium.
(b) Discs were put on filter paper wetted by the medium.
(c) Discs were dried on filter paper, then placed into vials containing no medium.
After incubation for 1 hour at 27°C, 1 ml gas was taken from each vial for ethylene analysis. Fresh weight of the six discs from each vial was
determined and EFE activity was expressed as niC2H4/g/h.
4.3.4.6 media for determination of EFE in and tissues
To determine the optimum pH for EFE activity in Hosui and Granny S mith fruit discs, Na+ -citric acid buffer was used to obtain different pH values for the incubation media. The pH values used for Hosui discs were 3.0, 4.0, 5.0, 5.7, 6.2, 6.5, 7.2, and 8.2, while those for Granny S mith discs were 2.5, 3.0, 3.5, 4.0, 4.3, 4.8, 5.2, 5.7, 6.2, 7.2 and 8.2.
Six discs were saturated by incubation in each of 4 X 36 ml vials containing 3 ml of the buffer containing 5 mM ACC at each pH level for 2 hours at 27°C, then vacuu m-infiltrated at 90 Kpa for 1 min to reduce the possible difference of ACC uptake caused by different pHs. Discs were then sealed in the same vials and incubated for 1 hour before 1 ml gas was removed from each vial for ethylene analysis.
The pH values in the media were measured before and after incubation.
4.3.4.7 Time cou rse of effect of levels on EFE
Six discs were immersed and i ncubated at 27°C in each of 4 X 36 ml vials containing 3 ml Na+-citric acid buffer with 5 mM ACC. The pH values used for Hosui discs were 6.2, 7.2 and 8.2, those for Granny Smith discs were 4.0, 6.2 and 8.2. Ethylene concentrations were measured after 0.5, 1 , 2 and 3 hours incubation. For each measurement, vials were sealed for 30 minutes before withdrawing a 1 ml gas sample for ethylene measure ment using GLC. Immediately after each sampling, vials were flushed with air for 20 seconds to prevent C02 accumulation and then returned to incubate at 27°C until the next ethylene determination was required when the procedure was repeated.
4.3.4.8 Effect of on and EFE
The i nfluence of 0.4 M sucrose, fructose, glucose, mannitol and sorbitol on EFE activity and ethylene production was tested by addi ng each sugar separately to the incubation medium with (for EFE activity
measurement) or without (for ethylene production measurement) 1 m M CH I and 5 m M ACC, respectively. For EFE activity measurement a K+
phosphate buffer containing 1 m M CH I and 5 mM ACC was used as a control. For ethylene production, the same buffer was used but without ACC and CHI. The pH value of all solutions was 6.5. Six discs were placed in each of 3 X 36 ml vials containing 3 ml of the appropriate sugar solution for each treatment. After saturation of the discs with the appropriate media using the immersion method for 2 hours, discs were dried on tissue paper. Six discs were sealed and incubated in each of 3 X 36 ml vials for each treatment for 1 hour at 27°C before a 1 ml sample was removed for ethylene analysis.
4.3.4.9 Statistical
Data were analysed by Duncan's multiple comparison, paired comparison and linear regression (Steel and Torrie, 1 986) using the SAS statistical programs (SAS/STAT User's Guide, Release 6.03 Edition). 4.4 R ESULTS
4.4.1 Wound ethylene prod uction
Hosui fruit discs did not produce detectable ethylene in the first 3 hours after cutting (Table 4-2). After 4 hours incubation , trace amounts of ethylene were produced which increased to 5.3 and 7.24 nl/g/h after 6 and 8 hours at 27°C, respectively. In Granny Smith, fruit discs produced ethylene i mmediately after cutting; production increased to a maximum of 55.78 nl/g/h after 5 hours before decreasing to the initial level after 1 0 hours.
4.4.2 Isotonic solution fo r fruit discs
Fresh weights of Hosui fruit discs increased significantly in 0.2 M and 0.3 M mannitol (Table 4-3), but decreased in both Hosui and Granny Smith discs exposed to 0.8 M mannitol after 2 hour incubation (Table 4-3). No significant changes occurred in the fresh weights of either Hosui or Granny Smith fruit discs during the two-hour incubation in different buffers or 0.4 M mannitol solutions at 27°C. This indicated that the K+-phosphate buffer, the Na+ -citric acid buffer tested and the 0.4 M mannitol solution at pH 6.5 were isotonic for fruit discs of both Hosui and G ranny Smith fruits (Table 4-3).
4.4.3 EFE activity in 0.4 M and 0.8 M man nitol so l ution
W�.5 EFE activity in both &{ Hosui fruit and Granny Smith fruits �
Table 4 -2 . Wound ethylene production by f ruit discs o f Hosui and Granny Smith at 2 7°C ( 1 9 8 9 ) . Time ( hour) 0 . 5 1 . 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 1 0 . 0 Ethylene production (nl /g/h)
Hosui Granny Smith
0 0 0 0 t race b 5 . 3 0 a 7 . 2 4 cd 2 3 . 93 cd 2 3 . 17 c 31 . 9 4 b 4 1 . 4 6 a 55 . 7 8 b 4 4 . 95 cd 2 3 . 31
*All values a re me ans (n=5 ) . Different characters within the s ame column represent s ignificant di fferences a t the 5 % level (Duncan ' s mult iple comparison ) .
( 1 9 0 0 ) . ( a ) . llosui .
ll'.rooh woight ( g )
K+ Na& + Mannitol s olution
Timo
-phollphn t o -oi trio o . a M 0 . 4 M 0 . 3 M 0 . 2 M - - - - a a initial 0 . 2 7 0 1 0 . 2 7 0 0 a a final 0 . 2 0 0 0 0 . 2 0 3 0 (b) . Granny Smit h . K+ Time -phoBphate a a 0 . 2 0 0 5 0 . 2 9 0 0 0 . 2 G 9 6 b b a a 0 . 2 4 1 0 0 . 3 0 4 4 0 . 2 97 9 Fresh weight ( g ) b 0 . 2 9 1 5 a 0 . 3 3 5 6 Na+ Mannitol s o lution -citric 0 . 0 M 0 . 4 M - - - � - - - - init ial final a 0 . 2 5 9 6 a 0 . 2 5 4 5 a 0 . 2 2 5 9 a 0 . 2 2 9 7 a 0 . 2 0 8 0 b 0 . 1 7 7 0 a 0 . 2 3 0 7 a 0 . 2 3 2 0
*All values a re means ( n=3 ) . Dif ferent characters within the s ame column for each fruit repre sent s ignificant diffe rences at the 5 % level between init ial and
f inal valueB (paired compa rison) . -...!
Table 4 - 4 . EFE a ctivity in fruit discs of Hosui and Granny Smith in 0 . 4 M and 0 . 8 M mannitol s olution at 27°C ( 1 9 9 0 ) .
Species Ho sui Granny Smith EFE activity ( n1C2H4 /g/ h ) in mannitol solution 0 . 4 M 0 . 8 M b 5 3 . 34 82 . 61 b 9 0 . 12 1 2 5 . 1 8 a a
A11 values are means (n=4 ) . Different characters in the s ame line represent significant differences at the 5% level (paired
comparison) .
4.4.4 Comparison of different methods to saturate ACC
In Hosui fruit, EFE activity increased from 1 2 nl/g/h at 0.02 mM ACC to 1 1 2 nl/g/h (Vmax) at 1 mM ACC concentrations (Fig.4-1 ) . 1n Granny Smith apple, the activity increased from 20 nl/g/h at 0.04 mM ACC to 1 03 nl/g/h (V max) at 1 mM (Fig.4-2). When ACC concentrations were higher than 1 mM, EFE activity in discs of both fruits remained unchanged. Thus the saturated concentration of ACC for EFE was 1 mM for both fruits. Results of a double reciprocal plot of EFE activity vs. ACC concentrations indicated that the apparent Km values of EFE for ACC were 0.1 66 m M for Hosui and 0.1 93
mM for Granny Smith apple (Fig.4-1 and Fig.4-2).
I n some experiments, it is necessary to use a vacuum-infiltration method to e nsure that (a) the discs were saturated with ACC, quickly to shorten the experimentaJ period to minimize disc ageing ; (b) ions as Ag+ and ea++ ions penetrated i nto the fruit tissue; (c) C02 or c2H4 were removed from fruit tissue after treatment. Discs were vacum-treated before or after a 2 hour incubation with the medium at 27°C to determine the effect of vacuu m infiltration on EFE activity.
No significant change of EFE activity occurred when discs of Hosui fruit were vacuum-infiltrated with 5 mM ACC before or after incubation compared with control discs which were incubated for 2 hours in the same medium (Table 4-SA).
0.08 0.07 0.06 0.05