Planta
Planta 144, 5 0 3 - 5 0 7 (1979)
9 by Springer-Verlag 1979
The Compartmentation of Ethylene in Developing Cotyledons of Phaseolus vulgaris L. P.H. Jerie*, A.R. Shaari**, and M.A. Hall Department of Botany and Microbiology, University College of Wales, Aberystwyth, Dyfed SY23 3EB, U.K.
Abstract. Isolated cotyledons of Phaseolus vulgar& L. cv. Canadian Wonder accumulated t4C2H 4 (0.7-1 ~tl 1-1) from air to give partition coefficients of 1 to 4, which greatly exceeded the value obtained with steam killed cotyledons (0.05) and with water (0.11). After 1 4 C 2 H 4 treatment, 98% of the 14C in the tissue remained a s 1 4 C 2 H 4. The labelled ethylene accumulated by cotyledons was released only slowly (1-10% h-~) either in an air stream or into toluene. Heating to 60 ~ C for 2 h, but not freezing and thawing, caused the immediate release of 14C2H 4 from the tissue. Propylene and vinyl chloride competitively inhibited the accumulation of 1 4 C 2 H 4. Cotyledons emanated endogenous ethylene at a very low rate but after heating (although not freezing and thawing) 13 nl of ethylene per g fresh mass were released within minutes. It was concluded that french bean cotyledons hold ethylene in a compartmented form in sufficient amount to account for at least 200 h of emanation. Key
words:
Compartmentation
-
Ethylene
-
Phaseolus.
Introduction The ethylene status of a tissue is commonly determined by measuring the rate of emanation of the gas, which it is assumed equals the rate of ethylene synthesis. Implicit in this approach are the assumptions that the concentration of ethylene at its site of action bears a constant relation to its concentration *
Present address: Irrigation Research Institute Tatura, Victoria,
Australia Present address : Malaysian Agricultural Research and Development Institute, Serdang, Selangor, Malaysia
**
Abbreviation: PPO = diphenyloxazole
in the rest of the cell and that in turn there is a constant resistance to diffusion of ethylene between the cell and the internal air spaces of the tissue. Recently, we reported evidence that, in Vicia faba, the assumptions referred to above appear to be invalid (Zeroni etal., 1977; Jerie et al., 1978a, b). It is, however, uncertain to what extent these results were influenced by the metabolism of ethylene to ethylene oxide which was subsequently demonstrated in this species (Jerie and Hall, 1978). In this report we describe the compartmentation of ethylene in cotyledons of Phaseolus vulgaris L. a species which does not metabolise ethylene in detectable quantities.
Materials and Methods Plant Material
Plants of Phaseolus vulgar& L. cv. Canadian Wonder, were grown in a heated glasshouse in 10 cm pots of John Innes No 2 compost. When flowering commenced the plants were repotted in 15 cm pots and were transferred to a growth cabinet under an 18 h photoperiod and a 20/16~ C day/night temperature regime. Pods were harvested as required and their relative maturity judged by eye. In the most mature pods the seed coat showed visible traces of red pigment. The seed coats were carefully peeled, the two cotyledons separated and the embryonic axis removed. On some occasions each cotyledon was cut lengthways with no noticeable effect on the accumulation or release of 14C2H 4. Treatment with 14C2H4
Labelled ethylene (14C2H4, A m e r s h a m Radiochemicai Centre 119.8 mCi mmol 1.11,870 disintegrations per minute (dmin 1 n l - 1) was used without further purification. The cotyledon segments were weighed and enclosed in a glass tube (18mi) with a ' s u b a s e a l ' cap. In some cases a 20 ml glass syringe was used as a variable sized chamber. This was fitted with a silicone rubber septum on the needle taper and the syringe plunger was made gas tight by wetting with distilled water. The required a m o u n t of 14C2H r was injected and the tubes held for 2.5 h in the light at 20 ~ C unless otherwise stated. When the effect of ethylene ana-
0032-0935/79/0144/0503/$01.00
504
P.H. Jerie et al. : Ethylene in Developing Cotyledons
logues was studied, these materials were injected just prior to the injection of 14C2H 4.
Measurement of 14C2H 4 With gaseous samples, 1 ml was injected directiy into a gas-tight vial of toluene containing 5 g/1 2,5 diphenyloxazole (PPO). To determine toluene-solubie radio-activity in the tissue, chambers containing the latter were opened under a saturated solution of ammonium sulphate, air bubbles were removed and the tissue was quickly transferred to gas tight vials as above. The vials were heated to 60 ~ C for 2 h before determination of radioactivity in a Beckman scintillation spectrometer using the sample channel ratio method of quench correction. Toluene-insoluble radioactivity was determined by taking the tissue through several changes of toluene followed by grinding in 5 ml of water and counting the entire homogenate suspended in the gel formed by adding to it 10 ml of Insta-Gel (Packard Instrument Company) (Jerie et al., 1978a). To identify the chemical form of the radioactivity, ~4C2H 4treated cotyledons were removed from the feeding chamber, sealed into a fresh glass tube and a sample of air above the tissue was analysed by radio-gaschromatography (radio gc) (Jerie and Hall, 1978) both before and after heating the tube to 60 ~ C. Alternatively, to determine whether the ~4C released from the cotyledons could be trapped in mercuric perchlorate, released with NaC1 and retrapped in mercuric acetate, as would be expected for ~4C2H4, the cotyledons were transferred to a tube containing an inner vial and, after heating, 4 ml of 0.2 M mercuric perchlorate (Young et al., 1952) was injected into the inner via1. After 16 h at room temperature a 2 ml aliquot of the perchlorate was transferred to another gas tight vial with an inner tube containing mercuric acetate (Gibson, 1964). Two ml o f 4 M NaC1 was added to the mercuric perchlorate and the vial was allowed to stand overnight. For scintillation counting, 0.5 ml aliquots were added to 2 ml of water and 6 ml of Insta Gel. The internal standard method was used for quench correction. Emanation of endogenous ethylene was measured by enclosing tissue in suitable containers as described above. To determine the effect of heat killing on ethylene emanation, vials containing the tissue were stood in a boiling water bath for 5 min. Ethylene in the air above the tissue was anaiysed by gas chromatography as previously described (Zeroni et al., 1977).
Results
The partition coefficient for ethylene between tissue and air was calculated as described by Jerie et al., (1978).
Preliminary experiments had shown that the level of radioactivity in the cotyledons reached equilibrium before 2.5 h after injection of t~C2H 4. Bean cotyledons treated with 1 4 C 2 H 4 a t 0.7 to 1.0 pl/1 for 2.5 h gave partition coefficients ranging from 1 for young cotyledons up to 4 for older cotyledons (Table 1). When cotyledons were steam killed before 14C2H ~ treatment, the partition coefficient fell to 0.05. The partition coefficients for living tomato pericarp, blood and frog muscle were similar to that for water. The 1r 4 absorbed by living cotyledons was only slowly and incompletely released into toluene at 20~ but could all be released if the tissue was heated to 55 60 ~ C (Fig. 1). Similarly, when the cotyledons were transferred to a rapid, humidified air stream the rate of emanation of 14C2H4, estimated by measuring the residual radioactivity at intervals over 100 min, was low and variable with typical results of between 1% and 10% perh. This was in marked contrast to segments of tomato pericarp or sycamore petiole which under the same conditions emanated essentially all the 1~C in 20 min and 50 min respectively (Fig. 2). When treated bean cotyledons were transferred to a sealed 20 ml glass syringe the concentration of 14C2H 4 in the air increased for about 60 min but subsequently stabilized. After venting the syringe this pattern was repeated (Fig. 3). The radioactivity released from treated bean cotyledons appeared to be ~4C2H4 in that it could be trapped in mercuric perchlorate reagent. Of the a~C thus trapped, 99% was released after adding an equal volume of 4M NaC1 and of this, 93% was re-trapped in mercuric acetate. Further, when the 14C2H4treated cotyledons were transferred to a new gas-tight container and a sample of the air was analysed by radio gc, a single small peak of activity was observed at the retention time of ethylene. After the tissue was heated and a sample of air again injected, a single but much larger peak was obtained, again at the retention time of ethylene.
Table 1. Partition coefficients for 14CzH ~ between bean cotyledon tissue and air. 14C2H 4 was supplied at 0.7 to 1.0 ~1 1 1 for 2 h. Figures are based on radioactivity soluble in toluene after heating tissue to 55 ~ C for 2 h in toluene. Tissue age is relative and was judged by eye. The figures in brackets are standard deviations Tissue
Fresh mass (g)
14C in tissue d min- 1
Partition coefficient
1~C insoluble in toluene (d m i n - 1 in tissue)
French bean cotyledons Y o u n g French bean cotyledons Older French bean cotyledons killed ~ Tomato pericarp Blood (human) Frog muscle
0.113 0.175 0.131 0.362 -
1,540 4,700 130 622 -
1.38 3.46 0.052 0.114 0.176 0.085
44 34 40 -
a
(0.015) (0.006) (0.014) (0.034)
(486) (374) (13) (61)
(0.32) (0.22) (0.003) (0.027) (0.034) (0.008)
Tissue was killed prior to treatment with 14C2H 4 by suspending it for 20 min in a flask containing boiling water
P.H. Jerie et al. : Ethylene in Developing Cotyledons
505
30
2.5
A
T o
r~ g ..~ 2.01-
/
'o x
20
/0/.01.0 0 ~ 0 ~ 0 ~ 0 ~ 0
Tc~
/
~0
y %
~0-0-0~0~0~
'~ 1.5 "o
/o
1,0
o
i0.5 t
/o/ )
0
2F5
510
75
1E)O
0
1.25
0
Time (h)
1.0 10 z,
0/5
1 .P5
i
2.0
14C2 Hz,(dpm m[-I)
Fig. 1. The elution of radioactivity from single 14C2H 4 pretreated bean cotyledons into toluene at room temperature. Treatments involved heating the tissue in toluene-PPO to 60 ~ C for 2 h (arrows) at zero time (~) and after l l 0 m i n (9 The cotyledons for both treatments were taken at random from a single pod. Each point shows the mean of 4 replicates, recounted at the times indicated. Vertical bars are standard deviations
2.5
G
B
9
E
=~ 2.0 'iN
E
100
"S ~2
,~ 50 u)
9 ~=
m'--'-~mL..~an
\
1.0
E
2.___D_______~
o
~
o
c c)
0 0
20
40
60
80
0!5
100
Time (rain)
Fig. 2. The loss of 14C2H 4 by emanation after transferring 1~C2H 4 pre-treated tissue directly to a moist air stream; bean cotyledons from two pods (,,, e), tomato pericarp segments (A), sycamore petiole segments (A). Zero time levels of 14C2H ~ in the tissue (100%) were determined in separate samples transferred to toluene-PPO under ammonium sulphate. The points show the residual 1~C2H4 g-1 fresh mass as a percentage of the level at zero time. Before scintillation counting samples were heated to 60~ for 2 h in toluene-PPO
1.10 104 14C2 H4 (dpm m[ -I)
1/5
210
Fig. 4A and B. The effect of ethylene analogues on the incorporation of 1~C2H ~ in bean cotyledons. Double reciprocal plots for (A) propylene 50 pl 1 1 and (B) vinyl chloride 100 gl 1 1. Control (o), plus inhibitor (o)
200C
T
150(
ojO j ~ 1 7 6 /
/~ jO~
o
o~O
~176
E
o_ 100(
0/ 0~
0
o/
~
50s
'~
120 I;0 Time (rain)
2;o
2~o 2;o
Fig. 3. The emanation of 14C2H 4 from t~C2H a pre-treated cotyledons (3.5 g) after transfer to a gas-tight glass syringe (air space 15.5 ml). 1 ml samples of air were withdrawn at intervals to determine the concentration of 1~C2H ~. The arrows show the times at which the syringe was vented. At transfer the tissue contained 320,000 dmin-1. The experiment was repeated four times. The points shown are from a single typical experiment
506
P.H. Jerie et al. : Ethylene in Developing Cotyledons
Table 2. Ethylene emanation from living tissue and release after heat killing of tissue. Figures show rates of ethylene emanation calculated for the time interval preceding the respective time of measurement. The tissue was killed by standing the tubes containing it in a boiling water bath for 5 min Tissue
Time of measurement (min) 30 70 190 Rates of C2H4 emanation ( n l g 1 f . m . h 1)
C2H 4 released after boiling (nl g 1 f.m.)
Whole bean pod
a
0.032
0.047
-
Bean pericarp segments
0.101
0.608
1.06
1.20
Entire bean seed
a
0.750
2.44
9.47
Bean seed coat
0.216
2.81
-
1.62
Bean cotyledons
0.0
a
0.055
Tomato pericarp
-
-
-
1.45
Meat (beef)
-
-
-
2.06
11.3
a Ethylene detected but amounts inadequate for reliable measurement
The presence of the ethylene analogues propylene and vinyl chloride inhibited the incorporation of 1 4 C 2 H 4 into bean cotyledons, giving 50% inhibition at 12 and 100 gl1-1 respectively. A more detailed investigation with propylene and vinyl chloride indicated that the inhibition was competitive (Fig. 4). In these experiments the tissue was treated with 1~C2H 4inhibitor combinations for only 40 rain. Preliminary experiments had shown the rate of incorporation was approximately constant over this time interval. Whole pods emanated endogenous ethylene at a low rate (Table 2). The rate of ethylene emanation from entire seeds, seed coats or injured pericarp increased with time, making it impossible to estimate ethylene production before dissection. Isolated cotyledons emanated little or no ethylene. Killing individual tissues in gas-tight glass tubes by standing these in a boiling water bath for 5 min caused an immediate release of ethylene in all samples but cotyledons released at least 8 times more ethylene than other tissues. Neither accumulated 1 4 C 2 H 4 n o r endogenous ethylene were released in response to freezing in liquid nitrogen or at - 20 ~ C followed by thawing. However, subsequent heating of the thawed tissue released ethylene from both sources, as in other experiments.
Discussion
Isolated bean cotyledons actively accumulate 14C2H 4 and after release from the tissue into toluene or air, the radioactivity still appears to be in the form of 14C2I-I4. No metabolism to ethylene oxide could be detected, which is in marked contrast to the situation in Viciafaba (Jerie and Hall, 1978). Due to the presence of radioactive impurities we cannot determine the significance of a minor component such as the toluene-insoluble 14C (Table 1) and whether or not this indicates a metabolism of ethylene such as that described by Beyer (1975). Endogenous ethylene appears to be held in cotyledons in a similar manner to that of exogenous 1 4 C 2 H 4 as indicated by the similarity in rates of emanation and in the effect of heat and cold. It has been suggested that heating will cause many biological materials to release ethylene (Abeles, 1973, Table 2), however, because in the cotyledons, heating has a parallel effect on endogenous ethylene and previously accumulated 1~C2H4, it would seem unlikely that ethylene arising as an artefact in this way materially affected the result. The ethylene must be bound or otherwise sequestered in the living cotyledons because the amounts involved greatly exceed the levels normally soluble in biological material. In living tissues of several other species, we have also observed significantly high partition coefficients (Jerie et al., 1978). When 'compartmented', the 14CzH4 is tightly held in the cotyledons so that the loss by emanation is far slower than the rate of gain during 1 4 C 2 H 4 treatment and is also strikingly slower than the rate of emanation from tomato pericarp or sycamore petiole segments although the intermediate position of sycamore petioles may be significant because this tissue initially has a partition coefficient of about 0.3 (Jerie et al., 1978a) as opposed to 0.13 for tomato pericarp. The ethylene analogues propylene and vinyl chloride inhibited the compartmentation of 14CZH4 (0.7 to 1.0 lal 1-1) by 50% when present at 12 and 100 lal 1-1 respectively. This compares well with the relative potency of these materials as mimickers of ethylene effects on the growth of peas (Burg and Burg, 1967) and on abscission (Abeles and Gahagan, 1968). The analogues were about 10 times more effective in inhibiting incorporation of 14C2H4 than in substituting for ethylene in the abscission or pea growth systems, but this may simply reflect the fact that the latter processes involve a far more complex interaction of events than occurs in compartmentation. A more detailed study showed that the inhibition by propylene and vinyl chloride was competitive and also that the process leading to compartmentation has a
P.H. Jerie et al. : Ethylene in Developing Cotyledons
high affinity for ethylene, in the range of 4 8.10 8 M, although it is difficult to estimate affinities accurately with data in this form (Fig. 4). Although the degree of ~4CzH 4 compartmentation has been measured initially as partition coefficients it is unlikely that the whole cell forms the compartment because the t 4 C 2 H 4 is not released after grinding the tissue. The nature of the ethylene compartmentation in bean cotyledons is uncertain at this stage. We cannot at present exclude the possibility that ethylene is sequestered in an organelle. However, there are similarities to some characteristics of the site of auxin binding. Heating (Ray et al., 1977) but not Triton X 100 (Batt et al., 1976) nor freezing (Ray et al., 1977) will disrupt auxin binding. The action of Triton X 100 on membrane structure may be similar to that of toluene which did not elute a 4 C z H 4 from cotyledons much faster than the observed rates of emanation in the air. Whatever the form of the compartmentation, we have not as yet investigated its relation to physiological events except to observe that the level of sequestered ethylene increases with tissue age (Table 1). It is conceivable that the compartmentation is involved in ethylene action or alternatively represents a mechanism for controlling ethylene levels inside the cell, thus allowing these levels to be independent of the current rate of ethylene synthesis. The literature is devoid of reference to the effects described here but 'autoinhibition' of ethylene synthesis in Sycomore Fig described by Zeroni et al., (1976) shows some similarities. These authors found that in non-ripening stages of fruit development, Sycomore Fig would (a) absorb ethylene when the gas was present in the atmosphere at 5 lal 1-~ (b) in a closed container the fruit would emanate ethylene only until an equilibrium was attained at 0.6 pl I- 1. This system appears similar to bean cotyledons where exogenous ethylene is absorbed and when re-emanated in a closed container an equilibrium is formed between 14"C2H 4 i n the tissue and that in the air. It is tempting to suggest that the mechanism o f ' autoinhibition' is not the control of ethylene synthesis, but the formation of an equilibrium between compartmented ethylene and air. There are a number of reports in the literature where the authors argue that ethylene does not control certain physiological events because emanation of the gas does not alter at the expected time. This report and our previous results on ethylene metabolism (Jerie and Hall, 1978) demonstrate mechanisms
507
whereby the cell may regulate its 'ethylene status' without the change being reflected in the rate of ethylene emanation. Until therefore, the occurrence and role of these phenomena are fully elucidated it is not permissible to assume a constant relationship between the effective concentration of ethylene in a particular tissue and the levels of ethylene determined by using conventional sampling techniques. We wish to thank the SRC and ARC for financial support towards some of this work.
References Abeles, F.B. : Ethylene in plant biology. London : Academic Press, 1973 Abeles, F.B., Gahagan, H.E.: Abscission: The role of ethylene, ethylene analogues, carbon dioxide, and oxygen. Plant Physiol. 43, 1255-1258, (1968) Batt, S., Wilkins, M.B., Venis, M.A.: Auxin binding to corn coleoptile membranes: kinetics and specificity. Planta 130, 7 13, (1976) Beyer, E.M.Jr. : ~4C2H4: its incorporation and metabolism by pea seedlings under aseptic conditions. Plant Physiol. 56, 273~78, (1975) Beyer, E.M.Jr., Morgan, P.W.: A method for determining the concentration of ethylene in gas phase of vegetative plant tissue. Plant Physiol. 46, 352-354, (1970) Burg, S.P., Burg, E.A. : Molecular requirements for the biological activity of ethylene. Plant Physiol. 42, 144 152, (1967) Gibson, M.S. : Incorporation of pyruvate-C 14 into ethylene by Penicillium digitatum Sacc. Arch. Biochem. Biophys. 106, 3t2-316, (1964) Jerie, P.H., Hall, M.A.: The identification of ethylene oxide as a major metabolite of ethylene in Vicia faba L. Proc. Roy. Soc. Lond. B 200, 87 94, (1978) Jerie, P.H., Shaari, A.R., Zeroni, M., Hall, M.A.: The partition coefficient of 14C2H 4 in plant tissue as a screening test for metabolism or compartmentation of ethylene. New Phytol. (1978), in press Jerie, P.H., Zeroni, M., Hall, M.A.: Movement and distribution of ethylene in plants in relation to the regulation of growth and development. Pest Sci. 9, 162 168, (1978a) Jerie, P.H., Zeroni, M., Hall, M.A. : Aspects of the role of ethylene in fruit ripening. Acta Hort. 80, (1978b), in press Ray, P.M., Dohrman, U., Hertel, R.: Characterisation of naphthaleneacetic acid binding to receptor sites on cellular membranes of maize coleoptile tissue. Plant Physiol. 59, 357-364, (1977) Young, R.E., Pratt, H.K., Biale, J.B. : Manometric determination of low concentrations of ethylene. Anal. Chem. 24, 551 555, (1952) Zeroni, M., Galil, T., Ben-Yehoshua, S.: Autoinhibition of ethylene formation in non-ripening stages of the fruit of Sycomore Fig (Ficus sycomorus L). Plant Physiol. 57, 647 650, (1976) Zeroni, M., Jerie, P.H., Hall, M.A. : Studies on the movement and distribution of ethylene in Vicia faba L. Planta 134, 119 125 (1977) Received 27 September; accepted 10 October 1978
Planta 144, 5 0 3 - 5 0 7 (1979)
9 by Springer-Verlag 1979
The Compartmentation of Ethylene in Developing Cotyledons of Phaseolus vulgaris L. P.H. Jerie*, A.R. Shaari**, and M.A. Hall Department of Botany and Microbiology, University College of Wales, Aberystwyth, Dyfed SY23 3EB, U.K.
Abstract. Isolated cotyledons of Phaseolus vulgar& L. cv. Canadian Wonder accumulated t4C2H 4 (0.7-1 ~tl 1-1) from air to give partition coefficients of 1 to 4, which greatly exceeded the value obtained with steam killed cotyledons (0.05) and with water (0.11). After 1 4 C 2 H 4 treatment, 98% of the 14C in the tissue remained a s 1 4 C 2 H 4. The labelled ethylene accumulated by cotyledons was released only slowly (1-10% h-~) either in an air stream or into toluene. Heating to 60 ~ C for 2 h, but not freezing and thawing, caused the immediate release of 14C2H 4 from the tissue. Propylene and vinyl chloride competitively inhibited the accumulation of 1 4 C 2 H 4. Cotyledons emanated endogenous ethylene at a very low rate but after heating (although not freezing and thawing) 13 nl of ethylene per g fresh mass were released within minutes. It was concluded that french bean cotyledons hold ethylene in a compartmented form in sufficient amount to account for at least 200 h of emanation. Key
words:
Compartmentation
-
Ethylene
-
Phaseolus.
Introduction The ethylene status of a tissue is commonly determined by measuring the rate of emanation of the gas, which it is assumed equals the rate of ethylene synthesis. Implicit in this approach are the assumptions that the concentration of ethylene at its site of action bears a constant relation to its concentration *
Present address: Irrigation Research Institute Tatura, Victoria,
Australia Present address : Malaysian Agricultural Research and Development Institute, Serdang, Selangor, Malaysia
**
Abbreviation: PPO = diphenyloxazole
in the rest of the cell and that in turn there is a constant resistance to diffusion of ethylene between the cell and the internal air spaces of the tissue. Recently, we reported evidence that, in Vicia faba, the assumptions referred to above appear to be invalid (Zeroni etal., 1977; Jerie et al., 1978a, b). It is, however, uncertain to what extent these results were influenced by the metabolism of ethylene to ethylene oxide which was subsequently demonstrated in this species (Jerie and Hall, 1978). In this report we describe the compartmentation of ethylene in cotyledons of Phaseolus vulgaris L. a species which does not metabolise ethylene in detectable quantities.
Materials and Methods Plant Material
Plants of Phaseolus vulgar& L. cv. Canadian Wonder, were grown in a heated glasshouse in 10 cm pots of John Innes No 2 compost. When flowering commenced the plants were repotted in 15 cm pots and were transferred to a growth cabinet under an 18 h photoperiod and a 20/16~ C day/night temperature regime. Pods were harvested as required and their relative maturity judged by eye. In the most mature pods the seed coat showed visible traces of red pigment. The seed coats were carefully peeled, the two cotyledons separated and the embryonic axis removed. On some occasions each cotyledon was cut lengthways with no noticeable effect on the accumulation or release of 14C2H 4. Treatment with 14C2H4
Labelled ethylene (14C2H4, A m e r s h a m Radiochemicai Centre 119.8 mCi mmol 1.11,870 disintegrations per minute (dmin 1 n l - 1) was used without further purification. The cotyledon segments were weighed and enclosed in a glass tube (18mi) with a ' s u b a s e a l ' cap. In some cases a 20 ml glass syringe was used as a variable sized chamber. This was fitted with a silicone rubber septum on the needle taper and the syringe plunger was made gas tight by wetting with distilled water. The required a m o u n t of 14C2H r was injected and the tubes held for 2.5 h in the light at 20 ~ C unless otherwise stated. When the effect of ethylene ana-
0032-0935/79/0144/0503/$01.00
504
P.H. Jerie et al. : Ethylene in Developing Cotyledons
logues was studied, these materials were injected just prior to the injection of 14C2H 4.
Measurement of 14C2H 4 With gaseous samples, 1 ml was injected directiy into a gas-tight vial of toluene containing 5 g/1 2,5 diphenyloxazole (PPO). To determine toluene-solubie radio-activity in the tissue, chambers containing the latter were opened under a saturated solution of ammonium sulphate, air bubbles were removed and the tissue was quickly transferred to gas tight vials as above. The vials were heated to 60 ~ C for 2 h before determination of radioactivity in a Beckman scintillation spectrometer using the sample channel ratio method of quench correction. Toluene-insoluble radioactivity was determined by taking the tissue through several changes of toluene followed by grinding in 5 ml of water and counting the entire homogenate suspended in the gel formed by adding to it 10 ml of Insta-Gel (Packard Instrument Company) (Jerie et al., 1978a). To identify the chemical form of the radioactivity, ~4C2H 4treated cotyledons were removed from the feeding chamber, sealed into a fresh glass tube and a sample of air above the tissue was analysed by radio-gaschromatography (radio gc) (Jerie and Hall, 1978) both before and after heating the tube to 60 ~ C. Alternatively, to determine whether the ~4C released from the cotyledons could be trapped in mercuric perchlorate, released with NaC1 and retrapped in mercuric acetate, as would be expected for ~4C2H4, the cotyledons were transferred to a tube containing an inner vial and, after heating, 4 ml of 0.2 M mercuric perchlorate (Young et al., 1952) was injected into the inner via1. After 16 h at room temperature a 2 ml aliquot of the perchlorate was transferred to another gas tight vial with an inner tube containing mercuric acetate (Gibson, 1964). Two ml o f 4 M NaC1 was added to the mercuric perchlorate and the vial was allowed to stand overnight. For scintillation counting, 0.5 ml aliquots were added to 2 ml of water and 6 ml of Insta Gel. The internal standard method was used for quench correction. Emanation of endogenous ethylene was measured by enclosing tissue in suitable containers as described above. To determine the effect of heat killing on ethylene emanation, vials containing the tissue were stood in a boiling water bath for 5 min. Ethylene in the air above the tissue was anaiysed by gas chromatography as previously described (Zeroni et al., 1977).
Results
The partition coefficient for ethylene between tissue and air was calculated as described by Jerie et al., (1978).
Preliminary experiments had shown that the level of radioactivity in the cotyledons reached equilibrium before 2.5 h after injection of t~C2H 4. Bean cotyledons treated with 1 4 C 2 H 4 a t 0.7 to 1.0 pl/1 for 2.5 h gave partition coefficients ranging from 1 for young cotyledons up to 4 for older cotyledons (Table 1). When cotyledons were steam killed before 14C2H ~ treatment, the partition coefficient fell to 0.05. The partition coefficients for living tomato pericarp, blood and frog muscle were similar to that for water. The 1r 4 absorbed by living cotyledons was only slowly and incompletely released into toluene at 20~ but could all be released if the tissue was heated to 55 60 ~ C (Fig. 1). Similarly, when the cotyledons were transferred to a rapid, humidified air stream the rate of emanation of 14C2H4, estimated by measuring the residual radioactivity at intervals over 100 min, was low and variable with typical results of between 1% and 10% perh. This was in marked contrast to segments of tomato pericarp or sycamore petiole which under the same conditions emanated essentially all the 1~C in 20 min and 50 min respectively (Fig. 2). When treated bean cotyledons were transferred to a sealed 20 ml glass syringe the concentration of 14C2H 4 in the air increased for about 60 min but subsequently stabilized. After venting the syringe this pattern was repeated (Fig. 3). The radioactivity released from treated bean cotyledons appeared to be ~4C2H4 in that it could be trapped in mercuric perchlorate reagent. Of the a~C thus trapped, 99% was released after adding an equal volume of 4M NaC1 and of this, 93% was re-trapped in mercuric acetate. Further, when the 14C2H4treated cotyledons were transferred to a new gas-tight container and a sample of the air was analysed by radio gc, a single small peak of activity was observed at the retention time of ethylene. After the tissue was heated and a sample of air again injected, a single but much larger peak was obtained, again at the retention time of ethylene.
Table 1. Partition coefficients for 14CzH ~ between bean cotyledon tissue and air. 14C2H 4 was supplied at 0.7 to 1.0 ~1 1 1 for 2 h. Figures are based on radioactivity soluble in toluene after heating tissue to 55 ~ C for 2 h in toluene. Tissue age is relative and was judged by eye. The figures in brackets are standard deviations Tissue
Fresh mass (g)
14C in tissue d min- 1
Partition coefficient
1~C insoluble in toluene (d m i n - 1 in tissue)
French bean cotyledons Y o u n g French bean cotyledons Older French bean cotyledons killed ~ Tomato pericarp Blood (human) Frog muscle
0.113 0.175 0.131 0.362 -
1,540 4,700 130 622 -
1.38 3.46 0.052 0.114 0.176 0.085
44 34 40 -
a
(0.015) (0.006) (0.014) (0.034)
(486) (374) (13) (61)
(0.32) (0.22) (0.003) (0.027) (0.034) (0.008)
Tissue was killed prior to treatment with 14C2H 4 by suspending it for 20 min in a flask containing boiling water
P.H. Jerie et al. : Ethylene in Developing Cotyledons
505
30
2.5
A
T o
r~ g ..~ 2.01-
/
'o x
20
/0/.01.0 0 ~ 0 ~ 0 ~ 0 ~ 0
Tc~
/
~0
y %
~0-0-0~0~0~
'~ 1.5 "o
/o
1,0
o
i0.5 t
/o/ )
0
2F5
510
75
1E)O
0
1.25
0
Time (h)
1.0 10 z,
0/5
1 .P5
i
2.0
14C2 Hz,(dpm m[-I)
Fig. 1. The elution of radioactivity from single 14C2H 4 pretreated bean cotyledons into toluene at room temperature. Treatments involved heating the tissue in toluene-PPO to 60 ~ C for 2 h (arrows) at zero time (~) and after l l 0 m i n (9 The cotyledons for both treatments were taken at random from a single pod. Each point shows the mean of 4 replicates, recounted at the times indicated. Vertical bars are standard deviations
2.5
G
B
9
E
=~ 2.0 'iN
E
100
"S ~2
,~ 50 u)
9 ~=
m'--'-~mL..~an
\
1.0
E
2.___D_______~
o
~
o
c c)
0 0
20
40
60
80
0!5
100
Time (rain)
Fig. 2. The loss of 14C2H 4 by emanation after transferring 1~C2H 4 pre-treated tissue directly to a moist air stream; bean cotyledons from two pods (,,, e), tomato pericarp segments (A), sycamore petiole segments (A). Zero time levels of 14C2H ~ in the tissue (100%) were determined in separate samples transferred to toluene-PPO under ammonium sulphate. The points show the residual 1~C2H4 g-1 fresh mass as a percentage of the level at zero time. Before scintillation counting samples were heated to 60~ for 2 h in toluene-PPO
1.10 104 14C2 H4 (dpm m[ -I)
1/5
210
Fig. 4A and B. The effect of ethylene analogues on the incorporation of 1~C2H ~ in bean cotyledons. Double reciprocal plots for (A) propylene 50 pl 1 1 and (B) vinyl chloride 100 gl 1 1. Control (o), plus inhibitor (o)
200C
T
150(
ojO j ~ 1 7 6 /
/~ jO~
o
o~O
~176
E
o_ 100(
0/ 0~
0
o/
~
50s
'~
120 I;0 Time (rain)
2;o
2~o 2;o
Fig. 3. The emanation of 14C2H 4 from t~C2H a pre-treated cotyledons (3.5 g) after transfer to a gas-tight glass syringe (air space 15.5 ml). 1 ml samples of air were withdrawn at intervals to determine the concentration of 1~C2H ~. The arrows show the times at which the syringe was vented. At transfer the tissue contained 320,000 dmin-1. The experiment was repeated four times. The points shown are from a single typical experiment
506
P.H. Jerie et al. : Ethylene in Developing Cotyledons
Table 2. Ethylene emanation from living tissue and release after heat killing of tissue. Figures show rates of ethylene emanation calculated for the time interval preceding the respective time of measurement. The tissue was killed by standing the tubes containing it in a boiling water bath for 5 min Tissue
Time of measurement (min) 30 70 190 Rates of C2H4 emanation ( n l g 1 f . m . h 1)
C2H 4 released after boiling (nl g 1 f.m.)
Whole bean pod
a
0.032
0.047
-
Bean pericarp segments
0.101
0.608
1.06
1.20
Entire bean seed
a
0.750
2.44
9.47
Bean seed coat
0.216
2.81
-
1.62
Bean cotyledons
0.0
a
0.055
Tomato pericarp
-
-
-
1.45
Meat (beef)
-
-
-
2.06
11.3
a Ethylene detected but amounts inadequate for reliable measurement
The presence of the ethylene analogues propylene and vinyl chloride inhibited the incorporation of 1 4 C 2 H 4 into bean cotyledons, giving 50% inhibition at 12 and 100 gl1-1 respectively. A more detailed investigation with propylene and vinyl chloride indicated that the inhibition was competitive (Fig. 4). In these experiments the tissue was treated with 1~C2H 4inhibitor combinations for only 40 rain. Preliminary experiments had shown the rate of incorporation was approximately constant over this time interval. Whole pods emanated endogenous ethylene at a low rate (Table 2). The rate of ethylene emanation from entire seeds, seed coats or injured pericarp increased with time, making it impossible to estimate ethylene production before dissection. Isolated cotyledons emanated little or no ethylene. Killing individual tissues in gas-tight glass tubes by standing these in a boiling water bath for 5 min caused an immediate release of ethylene in all samples but cotyledons released at least 8 times more ethylene than other tissues. Neither accumulated 1 4 C 2 H 4 n o r endogenous ethylene were released in response to freezing in liquid nitrogen or at - 20 ~ C followed by thawing. However, subsequent heating of the thawed tissue released ethylene from both sources, as in other experiments.
Discussion
Isolated bean cotyledons actively accumulate 14C2H 4 and after release from the tissue into toluene or air, the radioactivity still appears to be in the form of 14C2I-I4. No metabolism to ethylene oxide could be detected, which is in marked contrast to the situation in Viciafaba (Jerie and Hall, 1978). Due to the presence of radioactive impurities we cannot determine the significance of a minor component such as the toluene-insoluble 14C (Table 1) and whether or not this indicates a metabolism of ethylene such as that described by Beyer (1975). Endogenous ethylene appears to be held in cotyledons in a similar manner to that of exogenous 1 4 C 2 H 4 as indicated by the similarity in rates of emanation and in the effect of heat and cold. It has been suggested that heating will cause many biological materials to release ethylene (Abeles, 1973, Table 2), however, because in the cotyledons, heating has a parallel effect on endogenous ethylene and previously accumulated 1~C2H4, it would seem unlikely that ethylene arising as an artefact in this way materially affected the result. The ethylene must be bound or otherwise sequestered in the living cotyledons because the amounts involved greatly exceed the levels normally soluble in biological material. In living tissues of several other species, we have also observed significantly high partition coefficients (Jerie et al., 1978). When 'compartmented', the 14CzH4 is tightly held in the cotyledons so that the loss by emanation is far slower than the rate of gain during 1 4 C 2 H 4 treatment and is also strikingly slower than the rate of emanation from tomato pericarp or sycamore petiole segments although the intermediate position of sycamore petioles may be significant because this tissue initially has a partition coefficient of about 0.3 (Jerie et al., 1978a) as opposed to 0.13 for tomato pericarp. The ethylene analogues propylene and vinyl chloride inhibited the compartmentation of 14CZH4 (0.7 to 1.0 lal 1-1) by 50% when present at 12 and 100 lal 1-1 respectively. This compares well with the relative potency of these materials as mimickers of ethylene effects on the growth of peas (Burg and Burg, 1967) and on abscission (Abeles and Gahagan, 1968). The analogues were about 10 times more effective in inhibiting incorporation of 14C2H4 than in substituting for ethylene in the abscission or pea growth systems, but this may simply reflect the fact that the latter processes involve a far more complex interaction of events than occurs in compartmentation. A more detailed study showed that the inhibition by propylene and vinyl chloride was competitive and also that the process leading to compartmentation has a
P.H. Jerie et al. : Ethylene in Developing Cotyledons
high affinity for ethylene, in the range of 4 8.10 8 M, although it is difficult to estimate affinities accurately with data in this form (Fig. 4). Although the degree of ~4CzH 4 compartmentation has been measured initially as partition coefficients it is unlikely that the whole cell forms the compartment because the t 4 C 2 H 4 is not released after grinding the tissue. The nature of the ethylene compartmentation in bean cotyledons is uncertain at this stage. We cannot at present exclude the possibility that ethylene is sequestered in an organelle. However, there are similarities to some characteristics of the site of auxin binding. Heating (Ray et al., 1977) but not Triton X 100 (Batt et al., 1976) nor freezing (Ray et al., 1977) will disrupt auxin binding. The action of Triton X 100 on membrane structure may be similar to that of toluene which did not elute a 4 C z H 4 from cotyledons much faster than the observed rates of emanation in the air. Whatever the form of the compartmentation, we have not as yet investigated its relation to physiological events except to observe that the level of sequestered ethylene increases with tissue age (Table 1). It is conceivable that the compartmentation is involved in ethylene action or alternatively represents a mechanism for controlling ethylene levels inside the cell, thus allowing these levels to be independent of the current rate of ethylene synthesis. The literature is devoid of reference to the effects described here but 'autoinhibition' of ethylene synthesis in Sycomore Fig described by Zeroni et al., (1976) shows some similarities. These authors found that in non-ripening stages of fruit development, Sycomore Fig would (a) absorb ethylene when the gas was present in the atmosphere at 5 lal 1-~ (b) in a closed container the fruit would emanate ethylene only until an equilibrium was attained at 0.6 pl I- 1. This system appears similar to bean cotyledons where exogenous ethylene is absorbed and when re-emanated in a closed container an equilibrium is formed between 14"C2H 4 i n the tissue and that in the air. It is tempting to suggest that the mechanism o f ' autoinhibition' is not the control of ethylene synthesis, but the formation of an equilibrium between compartmented ethylene and air. There are a number of reports in the literature where the authors argue that ethylene does not control certain physiological events because emanation of the gas does not alter at the expected time. This report and our previous results on ethylene metabolism (Jerie and Hall, 1978) demonstrate mechanisms
507
whereby the cell may regulate its 'ethylene status' without the change being reflected in the rate of ethylene emanation. Until therefore, the occurrence and role of these phenomena are fully elucidated it is not permissible to assume a constant relationship between the effective concentration of ethylene in a particular tissue and the levels of ethylene determined by using conventional sampling techniques. We wish to thank the SRC and ARC for financial support towards some of this work.
References Abeles, F.B. : Ethylene in plant biology. London : Academic Press, 1973 Abeles, F.B., Gahagan, H.E.: Abscission: The role of ethylene, ethylene analogues, carbon dioxide, and oxygen. Plant Physiol. 43, 1255-1258, (1968) Batt, S., Wilkins, M.B., Venis, M.A.: Auxin binding to corn coleoptile membranes: kinetics and specificity. Planta 130, 7 13, (1976) Beyer, E.M.Jr. : ~4C2H4: its incorporation and metabolism by pea seedlings under aseptic conditions. Plant Physiol. 56, 273~78, (1975) Beyer, E.M.Jr., Morgan, P.W.: A method for determining the concentration of ethylene in gas phase of vegetative plant tissue. Plant Physiol. 46, 352-354, (1970) Burg, S.P., Burg, E.A. : Molecular requirements for the biological activity of ethylene. Plant Physiol. 42, 144 152, (1967) Gibson, M.S. : Incorporation of pyruvate-C 14 into ethylene by Penicillium digitatum Sacc. Arch. Biochem. Biophys. 106, 3t2-316, (1964) Jerie, P.H., Hall, M.A.: The identification of ethylene oxide as a major metabolite of ethylene in Vicia faba L. Proc. Roy. Soc. Lond. B 200, 87 94, (1978) Jerie, P.H., Shaari, A.R., Zeroni, M., Hall, M.A.: The partition coefficient of 14C2H 4 in plant tissue as a screening test for metabolism or compartmentation of ethylene. New Phytol. (1978), in press Jerie, P.H., Zeroni, M., Hall, M.A.: Movement and distribution of ethylene in plants in relation to the regulation of growth and development. Pest Sci. 9, 162 168, (1978a) Jerie, P.H., Zeroni, M., Hall, M.A. : Aspects of the role of ethylene in fruit ripening. Acta Hort. 80, (1978b), in press Ray, P.M., Dohrman, U., Hertel, R.: Characterisation of naphthaleneacetic acid binding to receptor sites on cellular membranes of maize coleoptile tissue. Plant Physiol. 59, 357-364, (1977) Young, R.E., Pratt, H.K., Biale, J.B. : Manometric determination of low concentrations of ethylene. Anal. Chem. 24, 551 555, (1952) Zeroni, M., Galil, T., Ben-Yehoshua, S.: Autoinhibition of ethylene formation in non-ripening stages of the fruit of Sycomore Fig (Ficus sycomorus L). Plant Physiol. 57, 647 650, (1976) Zeroni, M., Jerie, P.H., Hall, M.A. : Studies on the movement and distribution of ethylene in Vicia faba L. Planta 134, 119 125 (1977) Received 27 September; accepted 10 October 1978
