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Metabolism of ethylene to ethylene oxide by cell-free preparations from vicia faba L
Plant Science Letters, 17 (1979) 109--114 © Elsevier/North-Holland Scientific Publishers Ltd.
109
METABOLISM OF ETHYLENE TO ETHYLENE OXIDE BY C E L L - F R E E P R E P A R A T I O N S F R O M VICIA F A B A L.
J O H N H. D O D D S , S U L A F A
K. M U S A , P E T E R H. JERIE* and M I C H A E L
A. H A L L
Department of Botany and Microbiology, University College of Wales. Penglais, Aberystwyth, Dyfed SY23 3 DA (United Kingdom) (Received July 17th, 1979 ) (Revision received and accepted September 17th, 1979)
SUMMARY
Ethylene oxide is produced as a metabolite of ethylene by cell free preparations from cotyledons of Vicia faba L. cv Aquadulce. The system has a requirement for molecular oxygen and shows a high affinity for ethylene. The reaction is competitively inhibited by propylene.
INTRODUCTION
It has recently been demonstrated [1] that ethylene is rapidly metabolised to ethylene oxide by mature cotyledons of Vicia faba. The earlier w o r k of Jerie and Hall [ 1 ] was carried out on whole cotyledons of Vicia faba; in this paper we wish to present data on some of the properties of a cell-free system derived from c o t y l e d o n tissue which catalyses the conversion of ethylene to ethylene oxide. MATERIALS AND METHODS
Plants of Vicia faba cv. Aquadulce were grown under a 16 h p h o t o p e r i o d in a greenhouse at 20°C. Seeds were sown in 8-cm pots containing John Innes No. 2 c o m p o s t and the plants were grown on until flowering began, at which time they were transferred to 12-cm pots. Seeds from well developed pods approaching maturity or dried seeds soaked in running tap water for 30--40 h until fully imbibed, were used. C o t y l e d o n segments were obtained by removing the testa and cutting out the r o o t and s h o o t axes. The isolated cotyledons were weighed and homogenised in buffered sucrose (1 g fresh w t tissue/1 cm 3 buffered sucrose). * Present address : Irrigation Research Institute, Tatura, Victoria, Australia.
110 The buffered sucrose solution consisted of 10% (w/v) sucrose + 50 mM N-2-Hydroxyethylpiperazine propane sulphonic acid (EPPS) + 1 mM MgCl: (pH 8.5). The tissue was homogenised with reciprocating razor blades at 4°C until a slurry was obtained which was then squeezed through a single layer of miracloth into 16.5 cm 3 centrifuge tubes. The homogenate was centriguged in a 16.5 X 6 SW 16 s w i n g ~ u t rotor at 3500 g for 45 min at 4°C. The pellet was discarded and the supernatant re-centrifuged at 10 000 g for 45 min at 4°C. The pellet from this step constitued the active enzyme fraction used for further analysis. The pellet from each tube was resuspended in 2 cm 3 of 10% (w/v) sucrose + 50 mM EPPS + 1 mM MgCl2 (pH 8.5). Aliquots (1 cm 3) of sample were placed in standard glass scintillation vials, fitted with a metal screwcap incorporating a r u b b e r septum and injection hole. Where appropriate, the pH o f extracts was adjusted with HC1 or NaOH. Radioactive ethylene (Amersham J4C2H4 119.9 Ci/mol, 11870 dpm/nl) was used w i t h o u t purification and added to the vials at varying concentrations within the range 0.1--2.0 ~1/1. Vials were incubated in the light for 1 h at 20°C with shaking. After incubation, the radioactivity in 1 cm 3 of air above the sample was estimated by the mini vial m e t h o d of Jerie et al. [2]. The vials were then vented with an air stream for 10--15 s before 10 cm 3 o f seintiUant cocktail was added (scintillant = toluene : triton 3 : 1 + 5 g/1 2-5
111 RESULTS
Jerie et al. [ 1 ] showed that whole c o t y l e d o n tissue metabolised ethylene to ethylene oxide at a very high rate, with 85% of the ethylene added being converted to ethylene oxide within 2 h. The metabolism was n o t a result of microbial contamination. Table I shows the relative ability of various fractions from cell free preparations of Vicia faba to oxidise ethylene. The 3000--10 000 g pellet contains nearly half of the total activity and was used as a suspension in the following work. When 1 cm 3 of cell free extract was incubated with 1.0 pl/1 of [ 14C] ethylene in the gaseous phase production of ethylene oxide was linear over the first hour. In all subsequent experiments samples were incubated for this period at 20°C in the light. On dilution of the e n z y m e preparation with 10% w/v sucrose + 50 mM EPPS + 1 mM MgCI2 (pH 9.0) formation of ethylene oxide was found to be proportional to enzyme concentration, thus in subsequent experiments either aliquots of 0.5 or 1.0 cm 3 of enzyme extract were placed in each vial for incubation in [ 14C]ethylene. Heating extracts in a water bath at 100°C for 5 min completely abolished enzyme activity. The pH of 1 cm 3 fractions of extract were adjusted by the addition of HC1 or NaOH, to give a range of pH values from 4.5 to 11.5. Following the standard incubation procedure it was f o u n d that the enzyme showed a sharp pH o p t i m u m of about 8.5. (Fig. 1). In all subsequent experiments incubation was carried out at pH 8.5. Lineweaver-Burk analysis of incubations of enzyme preparations with a range of ethylene concentrations yielded the results shown in Fig. 2. A Km value of 4.17 × 10 -~° M for ethylene was calculated by regression analysis. The enzyme showed a requirement for molecular oxygen and the analysis shown in Fig. 3 gave a Km of 3.63 × 10 -3 M. Several co-factors were tested for their effect on the rate of ethylene metabolism. Cofactors tested included NADH, NADPH and a wide range of cations i.e. magnesium,
TABLE I E T H Y L E N E OXIDISING A C T I V I T Y O F V A R I O U S F R A C T I O N S O B T A I N E D B Y CENTRIFUGATION FROM CELL-FREE EXTRACTS Samples were centrifuged for 45 rnin at 4°C. Pellets were resuspended in 10% (w/v) sucrose + 50 mM EPPS (pH 9.0) + 1 mM MgC12. Fraction
m g protein/ml of sample
ethylene oxidising activity (% of total)
3 0 0 0 - - 1 0 000 g 10 000--14 000 g 14 0 0 0 - - 2 3 000 g 23 000--32 O 0 0 g 32 000--60 000 g 60 0 0 0 g supernatant
2.1 1.5 0.5 0.5 0.5 4.2
48.5 16.4 14.3 10.3 10.5 0
112 15 C~ 1,3 c-
~13
$
--O E rl
l
c 9 7 0
5
S o
~
l/
0
3
1.1 0.9
r" 0 rtl
~07 O
./
gO.5
-g w
~ 03 O
°~o~a
O--O
eO
a:01 2
45- 615
pH
8.5
10.5
10
14
1
1 Ethy[ene c o n c e n t r a t i o n
0
2
26
(M_Ix 10-9 )
Fig. 1. E f f e c t o f pH u p o n the rate o f e t h y l e n e o x i d a t i o n . Fig. 2. Lineweaver-Burk p l o t o f rate o f e t h y l e n e o x i d a t i o n versus e t h y l e n e c o n c e n t r a tion in the liquid phase. C o n d i t i o n s o f i n c u b a t i o n as in t e x t .
3.5
0
E e~
~, c - 2 5
!o ~1.5
~ 0.5 I
-25
-20
-15
-10
-5
0
I
I
5
10
1
Oxygen concentration
( N
1 15
i 20
i 25
3
i0
315
-I x 10-3 )
Fig. 3. L i n e w e a v e r - B u r k p l o t o f rate o f e t h y l e n e o x i d a t i o n versus o x y g e n c o n c e n t r a t i o n in the liquid phase. C o n d i t i o n s o f i n c u b a t i o n as in t e x t .
113
2.~ 2.0 c~
1.6
1.2 aJ
~,~ 0'~
• o/~ o~/o
• '/O
o
1 ( ri_1× 10-9 ) efhylene concenfrafion Fig. 4. Lineweaver-Burk plot of effect of propylene ( 1 . 4 8 x 10 -s M liquid phase, 613/~1]1 gaseous phase) on rate of e t h y l e n e oxidation. Conditions o f incubation as in text: control ( ~-------o ); plus propytene (e ; ).
manganese, copper, cobalt, ferrous, ferric and calcium (all at 1 mM). None of these co-factors had any significant effect on the rate of ethylene oxidation. The presence of propylene inhibited the rate of ethylene metabolism under the usual conditions used by 50% at 500 ~1/1. A more detailed investigation (Fig. 4) showed that the inhibition was competitive. The Ki for propylene was found to be 1.8 × 10 -6 M. Preparations which had been incubated with propylene alone (500 ~1/1 gaseous phase) for 5 h were heated and a sample of the gaseous phase analysed by GSC on Porapak Q. In addition to propylene two other smaller peaks eluted, one of which cochromatographed with authentic propylene oxide. DISCUSSION The results clearly demonstrate the presence of enzyme activity in preparations from Vicia faba cotyledons capable of oxidising ethylene to ethylene oxide. The enzyme appears to be an oxygenase. This is the first demonstration of such an enzyme in higher plants although similar enzymes are known in microorganisms [ 5]. The Km of the system for ethylene corresponds to a concentration of the gas of 0.096 ~1/1 in the gaseous phase, which corresponds well with values for endogenous ethylene concentrations in the air spaces of plant tissues (e.g. ref. 6). The high affinity of the system for ethylene makes it likely
114 t h a t the latter is its true substrate. This is also borne o u t by the Ki for the structural analogue pr opyl ene, which has been shown to be oxidised by higher plants [7]. Pr opyl e ne is approx. 50 times less effective in inhibiting e t h y len e o x id ation than it is in mimicking the effects of the growth regulator in various developmental systems. It is o f interest to n o t e t h a t t h e system derived from Echinocystis lobata which metabolises abscisic acid is also an oxygenase [4] ; this e n z y m e differs fr o m th at oxidising e t h y l e n e in having a r e q u i r e m e n t for NADPH. The f u n c t i o n o f t he e n z y m e in the plant is unclear as is its relationship to the e t h y l e n e metabolising systems d e m o n s t r a t e d by Beyer [3], but as shown elsewhere [1] there is no d o u b t t ha t the system has a p r o n o u n c e d e f f ect u p o n the e t h y l e n e status of the developing broad bean cot yl edon. ACKNOWLEDGEMENT We wish to t h a n k the Science Research Council for financial Assistance. REFERENCES 1 2 3 4 5 6 7
P.H. Jerie and M.A. Hall, Proc. R. Soc., B 200 (1978) 87. P.H. Jerie, A.R. Shaari, M. Zeroni and M.A. Hall, New Phytol, 81 (1978) 499. E. Beyer Jr., Plant Physiol., 56 (1975) 273. D.F. Gillard and D.C. Walton, Plant Physiol., 58 (1976) 790. J.A.M. DeBont and W. Harder, FEMS Lett., 3 (1978) 89. M. Zeroni, P.H. Jerie and M.A. Hall. Planta, 134 (1977) 119. E. Beyer Jr., Plant Physiol., 61 (1978) 893.
109
METABOLISM OF ETHYLENE TO ETHYLENE OXIDE BY C E L L - F R E E P R E P A R A T I O N S F R O M VICIA F A B A L.
J O H N H. D O D D S , S U L A F A
K. M U S A , P E T E R H. JERIE* and M I C H A E L
A. H A L L
Department of Botany and Microbiology, University College of Wales. Penglais, Aberystwyth, Dyfed SY23 3 DA (United Kingdom) (Received July 17th, 1979 ) (Revision received and accepted September 17th, 1979)
SUMMARY
Ethylene oxide is produced as a metabolite of ethylene by cell free preparations from cotyledons of Vicia faba L. cv Aquadulce. The system has a requirement for molecular oxygen and shows a high affinity for ethylene. The reaction is competitively inhibited by propylene.
INTRODUCTION
It has recently been demonstrated [1] that ethylene is rapidly metabolised to ethylene oxide by mature cotyledons of Vicia faba. The earlier w o r k of Jerie and Hall [ 1 ] was carried out on whole cotyledons of Vicia faba; in this paper we wish to present data on some of the properties of a cell-free system derived from c o t y l e d o n tissue which catalyses the conversion of ethylene to ethylene oxide. MATERIALS AND METHODS
Plants of Vicia faba cv. Aquadulce were grown under a 16 h p h o t o p e r i o d in a greenhouse at 20°C. Seeds were sown in 8-cm pots containing John Innes No. 2 c o m p o s t and the plants were grown on until flowering began, at which time they were transferred to 12-cm pots. Seeds from well developed pods approaching maturity or dried seeds soaked in running tap water for 30--40 h until fully imbibed, were used. C o t y l e d o n segments were obtained by removing the testa and cutting out the r o o t and s h o o t axes. The isolated cotyledons were weighed and homogenised in buffered sucrose (1 g fresh w t tissue/1 cm 3 buffered sucrose). * Present address : Irrigation Research Institute, Tatura, Victoria, Australia.
110 The buffered sucrose solution consisted of 10% (w/v) sucrose + 50 mM N-2-Hydroxyethylpiperazine propane sulphonic acid (EPPS) + 1 mM MgCl: (pH 8.5). The tissue was homogenised with reciprocating razor blades at 4°C until a slurry was obtained which was then squeezed through a single layer of miracloth into 16.5 cm 3 centrifuge tubes. The homogenate was centriguged in a 16.5 X 6 SW 16 s w i n g ~ u t rotor at 3500 g for 45 min at 4°C. The pellet was discarded and the supernatant re-centrifuged at 10 000 g for 45 min at 4°C. The pellet from this step constitued the active enzyme fraction used for further analysis. The pellet from each tube was resuspended in 2 cm 3 of 10% (w/v) sucrose + 50 mM EPPS + 1 mM MgCl2 (pH 8.5). Aliquots (1 cm 3) of sample were placed in standard glass scintillation vials, fitted with a metal screwcap incorporating a r u b b e r septum and injection hole. Where appropriate, the pH o f extracts was adjusted with HC1 or NaOH. Radioactive ethylene (Amersham J4C2H4 119.9 Ci/mol, 11870 dpm/nl) was used w i t h o u t purification and added to the vials at varying concentrations within the range 0.1--2.0 ~1/1. Vials were incubated in the light for 1 h at 20°C with shaking. After incubation, the radioactivity in 1 cm 3 of air above the sample was estimated by the mini vial m e t h o d of Jerie et al. [2]. The vials were then vented with an air stream for 10--15 s before 10 cm 3 o f seintiUant cocktail was added (scintillant = toluene : triton 3 : 1 + 5 g/1 2-5
111 RESULTS
Jerie et al. [ 1 ] showed that whole c o t y l e d o n tissue metabolised ethylene to ethylene oxide at a very high rate, with 85% of the ethylene added being converted to ethylene oxide within 2 h. The metabolism was n o t a result of microbial contamination. Table I shows the relative ability of various fractions from cell free preparations of Vicia faba to oxidise ethylene. The 3000--10 000 g pellet contains nearly half of the total activity and was used as a suspension in the following work. When 1 cm 3 of cell free extract was incubated with 1.0 pl/1 of [ 14C] ethylene in the gaseous phase production of ethylene oxide was linear over the first hour. In all subsequent experiments samples were incubated for this period at 20°C in the light. On dilution of the e n z y m e preparation with 10% w/v sucrose + 50 mM EPPS + 1 mM MgCI2 (pH 9.0) formation of ethylene oxide was found to be proportional to enzyme concentration, thus in subsequent experiments either aliquots of 0.5 or 1.0 cm 3 of enzyme extract were placed in each vial for incubation in [ 14C]ethylene. Heating extracts in a water bath at 100°C for 5 min completely abolished enzyme activity. The pH of 1 cm 3 fractions of extract were adjusted by the addition of HC1 or NaOH, to give a range of pH values from 4.5 to 11.5. Following the standard incubation procedure it was f o u n d that the enzyme showed a sharp pH o p t i m u m of about 8.5. (Fig. 1). In all subsequent experiments incubation was carried out at pH 8.5. Lineweaver-Burk analysis of incubations of enzyme preparations with a range of ethylene concentrations yielded the results shown in Fig. 2. A Km value of 4.17 × 10 -~° M for ethylene was calculated by regression analysis. The enzyme showed a requirement for molecular oxygen and the analysis shown in Fig. 3 gave a Km of 3.63 × 10 -3 M. Several co-factors were tested for their effect on the rate of ethylene metabolism. Cofactors tested included NADH, NADPH and a wide range of cations i.e. magnesium,
TABLE I E T H Y L E N E OXIDISING A C T I V I T Y O F V A R I O U S F R A C T I O N S O B T A I N E D B Y CENTRIFUGATION FROM CELL-FREE EXTRACTS Samples were centrifuged for 45 rnin at 4°C. Pellets were resuspended in 10% (w/v) sucrose + 50 mM EPPS (pH 9.0) + 1 mM MgC12. Fraction
m g protein/ml of sample
ethylene oxidising activity (% of total)
3 0 0 0 - - 1 0 000 g 10 000--14 000 g 14 0 0 0 - - 2 3 000 g 23 000--32 O 0 0 g 32 000--60 000 g 60 0 0 0 g supernatant
2.1 1.5 0.5 0.5 0.5 4.2
48.5 16.4 14.3 10.3 10.5 0
112 15 C~ 1,3 c-
~13
$
--O E rl
l
c 9 7 0
5
S o
~
l/
0
3
1.1 0.9
r" 0 rtl
~07 O
./
gO.5
-g w
~ 03 O
°~o~a
O--O
eO
a:01 2
45- 615
pH
8.5
10.5
10
14
1
1 Ethy[ene c o n c e n t r a t i o n
0
2
26
(M_Ix 10-9 )
Fig. 1. E f f e c t o f pH u p o n the rate o f e t h y l e n e o x i d a t i o n . Fig. 2. Lineweaver-Burk p l o t o f rate o f e t h y l e n e o x i d a t i o n versus e t h y l e n e c o n c e n t r a tion in the liquid phase. C o n d i t i o n s o f i n c u b a t i o n as in t e x t .
3.5
0
E e~
~, c - 2 5
!o ~1.5
~ 0.5 I
-25
-20
-15
-10
-5
0
I
I
5
10
1
Oxygen concentration
( N
1 15
i 20
i 25
3
i0
315
-I x 10-3 )
Fig. 3. L i n e w e a v e r - B u r k p l o t o f rate o f e t h y l e n e o x i d a t i o n versus o x y g e n c o n c e n t r a t i o n in the liquid phase. C o n d i t i o n s o f i n c u b a t i o n as in t e x t .
113
2.~ 2.0 c~
1.6
1.2 aJ
~,~ 0'~
• o/~ o~/o
• '/O
o
1 ( ri_1× 10-9 ) efhylene concenfrafion Fig. 4. Lineweaver-Burk plot of effect of propylene ( 1 . 4 8 x 10 -s M liquid phase, 613/~1]1 gaseous phase) on rate of e t h y l e n e oxidation. Conditions o f incubation as in text: control ( ~-------o ); plus propytene (e ; ).
manganese, copper, cobalt, ferrous, ferric and calcium (all at 1 mM). None of these co-factors had any significant effect on the rate of ethylene oxidation. The presence of propylene inhibited the rate of ethylene metabolism under the usual conditions used by 50% at 500 ~1/1. A more detailed investigation (Fig. 4) showed that the inhibition was competitive. The Ki for propylene was found to be 1.8 × 10 -6 M. Preparations which had been incubated with propylene alone (500 ~1/1 gaseous phase) for 5 h were heated and a sample of the gaseous phase analysed by GSC on Porapak Q. In addition to propylene two other smaller peaks eluted, one of which cochromatographed with authentic propylene oxide. DISCUSSION The results clearly demonstrate the presence of enzyme activity in preparations from Vicia faba cotyledons capable of oxidising ethylene to ethylene oxide. The enzyme appears to be an oxygenase. This is the first demonstration of such an enzyme in higher plants although similar enzymes are known in microorganisms [ 5]. The Km of the system for ethylene corresponds to a concentration of the gas of 0.096 ~1/1 in the gaseous phase, which corresponds well with values for endogenous ethylene concentrations in the air spaces of plant tissues (e.g. ref. 6). The high affinity of the system for ethylene makes it likely
114 t h a t the latter is its true substrate. This is also borne o u t by the Ki for the structural analogue pr opyl ene, which has been shown to be oxidised by higher plants [7]. Pr opyl e ne is approx. 50 times less effective in inhibiting e t h y len e o x id ation than it is in mimicking the effects of the growth regulator in various developmental systems. It is o f interest to n o t e t h a t t h e system derived from Echinocystis lobata which metabolises abscisic acid is also an oxygenase [4] ; this e n z y m e differs fr o m th at oxidising e t h y l e n e in having a r e q u i r e m e n t for NADPH. The f u n c t i o n o f t he e n z y m e in the plant is unclear as is its relationship to the e t h y l e n e metabolising systems d e m o n s t r a t e d by Beyer [3], but as shown elsewhere [1] there is no d o u b t t ha t the system has a p r o n o u n c e d e f f ect u p o n the e t h y l e n e status of the developing broad bean cot yl edon. ACKNOWLEDGEMENT We wish to t h a n k the Science Research Council for financial Assistance. REFERENCES 1 2 3 4 5 6 7
P.H. Jerie and M.A. Hall, Proc. R. Soc., B 200 (1978) 87. P.H. Jerie, A.R. Shaari, M. Zeroni and M.A. Hall, New Phytol, 81 (1978) 499. E. Beyer Jr., Plant Physiol., 56 (1975) 273. D.F. Gillard and D.C. Walton, Plant Physiol., 58 (1976) 790. J.A.M. DeBont and W. Harder, FEMS Lett., 3 (1978) 89. M. Zeroni, P.H. Jerie and M.A. Hall. Planta, 134 (1977) 119. E. Beyer Jr., Plant Physiol., 61 (1978) 893.