METABOLISM OF ETHEPHON

(2-CHLOROETHYLPHOSPHONIC ACID) AND

RELATED COMPOUNDS IN HEVEA BRASILIENSIS
B. G. AUDLEY, B. L. ARCHER AND I. B. CARRUTHERS

R. R. I. M. Biochemistry Unit,
Malaysian Rubber Producers' Research Association,
Brickendonbury, HerOCord SGI3 8NP, HerOCordshire, England.

Ethephon (I) is used commercially to prolong the flow of latex from the rubber tree
after tapping (Yield stimulation). The compound is applied to the bark in the region of
the tapping cut and the effect on latex flow is due to the ethylene released by chemical
decomposition, since gaseous ethylene itself is also a very effective stimulant. When
NC-I is applied to the bark of a young Hevea seedling, it is absorbed into the plant by
processes which appear to be largely non-metabolic. Ethylene formation commences
immediately at the site of application, and the gas is quickly translocated throughout
the plant. Translocation of I to all parts of the plant also occurs and the accumulation
of 14C in the bark above the zone of application is greater than that below. Chromatog-
raphic analysis has shown that compounds other than I remain in the plant tissue.

Experiments using 14C-I have shown that detached leaves are able to convert a
considerable proportion of the compound to at least twelve non-volatile acid products.
One of these is a conjugate of I with an unidentified material. A major component of
the products is 2-hydroxyethylphosphonic acid (II), which is itself converted to a
number of compounds in leaves. The application of I to bark from mature Hevea,
results in the formation of a single substance which is also a conjugate of I. Neither I
nor II is effective in inducing the formation of ethylene from endogenous precursors in
vegetative Hevea tissue.

Ethylene is poorly metabolized by Hevea leaves and the evidence available indicates
that it is unlikely that any of the compounds produced from I are metabolites of
ethylene.

Until about four years ago, practically the only c o m p o u n d s used c o m m e r c i a l l y to

stimulate the yield o f latex from rubber trees were herbicides such as 2,4-dichloro-
and 2 , 4 , 5 , - t r i c h l o r o p h e n o x y a c e t i c acid. It is now k n o w n that these p r o b a b l y act by
inducing the f o r m a t i o n o f ethylene within the tissues o f the trunk to which they are
applied, and that the gas itself is the actual ,yield stimulatory agent. In 1968 a
c o m p l e t e l y new type o f stimulant was d i s c o v e r e d which was capable o f direct
c h e m i c a l d e c o m p o s i t i o n into ethylene under p h y s i o l o g i c a l conditions. ( A b r a h a m et
al. 1968, C o o k e and Randall 1968). This c o m p o u n d is 2 - c h l o r o e t h y l p h o s p h o n i c acid

Presented at the Third International Congress of Pesticide Chemistry (IUPAC), Helsinki, Finland,
1974.

Archives of Environmental Contamination 183

and Toxicology Vol. 4, 183-200 (1976)
9 1976 by Springer-Verlag New York Inc.
184 B.G. Audley et al

(ethephon), which is marketed by Amchem Inc., USA, as a formulation under the

trade name Ethrel for use as a general plant growth regulator (Amchem Products
Inc. 1969) An even later development has been a method of applying ethylene itself
to the tree by pre-adsorption of the gas on a suitable carrier (Dickenson 1973).

When a rubber tree is tapped, the initial rapid flow of latex from the cut ends of
the latex vessels fails to zero in about an hour. If the vessels are reopened before the
flow has ceased, there is an immediate increase in rate which again decreases. This
pattern of flow has been explained by postulating that restriction of flow (plugging)
occurs at the open ends of the latex vessels (Boatman 1966) The application of a
yield stimulant increases the duration of the flow and decreases the plugging, since
reopening the latex vessels of a stimulated tree has little effect. It is at present
believed that certain organelles (lutoids) in latex, which contain a system capable of
coagulating rubber are involved, and that ethylene stabilizes these particles, thus
reducing the plugging (Southern 1969, Ribaillier 1970). There is no evidence that
yield stimulants affect the rate of biosynthesis of rubber.

Ethephon has been applied to many species other than Hevea brasiliensis to
control growth, flowering, fruit abscission, etc. (Amchem. Products Inc. 1969, Fritz
and Evans 1970). Yamaguchi et al. (1971) applied ethephon-14C to summer squash,
cucumber and tomato plants and observed translocation. With tomato, most of the
residual 14C was found within the treated leaves and fruits on the same branch; no
metabolites were detected. In squash, a single unidentified metabolite appeared in
the leaves two days after ethephon-14C application, and after a further four days, all
the translocated radioactivity was present in this metabolite. After fourteen days, the
14C present in the tissue was only 3% of that applied to the plant and the overall
yield of t4CO~ was 0.2%. In cucumber, the residual radioactivity in the fruits
totalled only 0.3%, and no further analysis of the material was possible. Weaver et
al. (1972) detected no metabolites seven days after ethephon was injected into the
berries of a grape vine. In young plants, phloem translocation of radioactivity was
observed. In the case of walnut, translocation from a leaflet to the kernel occurred,
but no metabolites were found (Martin et al. 1972). Ethephon-14C was applied to the
leaf and fruit surfaces of apples and cherries by Edgerton and Hatch (1972), who
found that most of the ethephon in the fruits had moved there from adjacent leaves
and only small amounts penetrated the surface of the fruits. Again no metabolites
were present. In peach fruits, Lavee and Martin (1974) concluded that compounds
originally thought to be ethephon metabolites, were impurities in the tracer used or
were formed during the isolation procedure. Our investigation into ethephon
metabolism in H. brasiliensis yielded results quite different from those described
above, for it was shown that 5 to 20% of the ethephon applied to vegetative tissue
was converted, probably by enzymic processes, into compounds other than ethylene.
(Archer et al. 1973).

Work carried out over a number of years on the metabolism of ethylene in plants
has been recently reviewed by Abeles (1972, 1973) and the balance of the evidence
indicates that ethylene is metabolized poorly, or not at all. In this paper we present
 Metabolism of Ethephon and Related Compounds t85

further results of experiments on ethephon metabolism and translocation, and de-

scribe some preliminary work on the nature of the compounds formed.

Material and methods

Two-year-old seedlings of Hevea brasiliensis grown in a greenhouse in this

country were used, together with leaves and segments of stem cut therefrom. Pieces
of mature bark were kindly supplied by our colleagues in Malaysia, from trees in
normal tapping.

Ethephon-:4C was the same as that described in our previous publication (Archer
et al. 1973). 2-Hydroxyethylphosphonic acid-~4C(2-HEPA-t4C) was prepared from
ethephon-14C by the method of Vogt (1970), and was purified by TLC using solvent
(e) below. The radiochemical purity was >98% and the specific activity 2.2 mCi/
mmol.

Radioactivity was measured by scintillation counting as described previously

(Archer et al. 1973). Highly colored or insoluble samples were burned in oxygen
and the resulting x4CO~ counted by the method of Jeffay and Alvarez (1961).
Ethylene-X4C which had been trapped in mercuric perchlorate, was released by
adding lithium chloride and then estimated as before (Ygung et al. 1952, Archer et
al. 1973).

Treatment of tissue with x4C-compounds. (1) Stem segments. A solution of the

compound in 5% aqueous glycerol was soaked into a piece of filter paper which was
fitted round the bark and covered with polythene (Field and Peel 1971).

(2) Mature bark. For metabolism experiments the surface of the bark was
scraped to below the green layer and ethephon-X4C applied as an aqueous solution.
For studies on radial translocation, agar discs containing ethephon-14C were cast on
the surface of the bark.

(3) Isolated leaves. These were treated with ethephon-X4C and 2-HEPA-t4C as
described previously (Archer et al. 1973) Ethylene-14C was fed directly using the
chambers described below, into which the gas was administered from a break-seal
tube attached to the chamber. The concentration of ethylene-:4C (50/xCi) in the air
surrounding the leaf was 43 ppm. All incubations were carried out at 25~ under
continuous illumination (4500 lx).

(4) Whole seedlings. An aqueous solution of ethephon-~4C was applied to a

20-mm length of the stem which had been scraped lightly with a scalpel to remove
the outer suberised layers.

Extraction of 14C-compounds from Hevea tissue. Whole plants were frozen in

solid carbon dioxide to arrest translocation of 14C, before being separated into
leaves, petioles, and roots. The stem was cut into 40-ram sections while still frozen,
186 B.G. Audley et al

and the bark separated from the wood. All samples were then disintegrated in 0.01M
H2SO4 containing 0.3% ethephon as carrier, using a Willems Polytron, type PT20
and then centrifuged.

Isolated leaves were disintegrated and extracted with water, or methanol-

chloroform-water (MCW) or methanol-chloroform-formic acid (MCF) and the ex-
tracts dried (Bielski and Young 1963).

Mature bark was distintegrated and extracted in chloroform-water, as water alone

caused aggregation of the rubber and the formation of intractable lumps.

Chromatography of plant extracts. In the early stages of this work it was

shown that anion-exchange chromatography of plant extracts was absolutely essen-
tial if satisfactory TLC's were to be obtained. Later it was shown that QAE-
Sephadex A-25 gave better separations than the Bio-Rad AG 1-X 10 used previously.

Two methods of column chromatography were employed.

(1) Columns of QAE-Sephadex A-25 were equilibrated with imidazole hyd-

rochloride buffer at pH 6.6 [C1-] = 0.01 M. These are referred to as chloride
columns. Solutions of tissue extracts were sorbed under the above conditions and the
compounds eluted with the same buffer at a chloride concentration o f 0.05 M.
Fractions from the columns required for TLC were passed down columns o f Bio-Rad
AG50W X-12 H + and the effluents freeze-dried.

(2) Alternatively, the anion-exchanger in the formate form was used and the
compounds eluted with ammonium formate buffer, pH 6.6, containing a total formic
acid plus formate ion concentration of 0.05M. These columns are referred to as
formate columns. In this case the volatile buffer was removed by vacuum sublima-
tion only.

TLC was carried out on cellulose MN300 using the following solvents: (a) 72%
w/w phenol in water, (b) n-butanol-propionic acid - water (26:12:17), (c) ethanol-
0.880 ammonia-water (38:5:8), (d) eucalyptol-n-propanol-formicacid-water (10:10:
4:1) (e) n-butanol-aceticacid-water (12:3:5).

Phosphorus compounds were detected by the method of Burrows e t a l . (1952).

Measurement of transpired ethylene-14C in translocation experiments. (1)

Leaves. In determining the evolution of ethylene-14C from the various parts of the
plant, the main problem was the counting of small amounts of the gas in a relatively
large volume of air surrounding the plar~t. This was overcome by using 'Perspex'
leaf chambers as shown in Figure 1. The internal thickness of the chambers was 12
mm and the laminae of the leaves were kept from touching the walls by small plastic
balls cemented to the surface. The two halves of the chambers were sealed together
with liquid silicone rubber and held in position by bull-dog clips. A small fan
 Metabolism of Ethephon and Related Compounds 187

prevented the build-up of high localized concentrations of ethylene-X4C in the

chamber. Moist air was drawn through each chamber, carrier ethylene added, and
the gas stream passed through a sintered b u b b l e r c o n t a i n i n g mercuric perchlorate
(Young et al. 1952). In all, ten leaves were each fitted with a leaf-chamber and
bubbler, any other leaves being removed. The bubblers were replaced every 24 hr.

(2) Stem. Two-piec.e stem chambers were m a c h i n e d from 'Perspex' blocks as

shown in Figure 1. The soft butyl rubber gaskets, cut from a three-mm sheet were
sealed onto the stem with silicone rubber. The standard counting vials contained 0.1
ml of mercuric perchlorate to absorb ethylene-14C evolved from the stem and every
24 hr they were replaced and the solution counted as above. The stem chamber over

28
~ 20~

(i) 45 25 a I b

Fig. 1. (i) Ethylene absorption chamber for fitting to stem of plant. Machined from two
blocks of 'Perspex' 14 x 45 x 45 mm held together by four 2BA bolts and wing nuts. a =
stem; b = scintillation counting vial containing 0.! ml of 0.25M Hg(C104, c = soft rubber
gaskets; d = counting vial cap.
(ii) Ethylene absorption chamber for covering leaf of H. brasiliensis plant. Vacuum
formed from two pieces of 3mm 'Perspex'. Internal thickness of chamber 12 mm. e = leaf; f
= air inlet; g = air outlet; h = 12 mm rod for mounting on retort stand, j = fan, driven by an
extemaUy mounted electric motor (Escap type 15 and F15 reduction gear) k = petiole fitted
through a preformed orifice in the chamber rim. m = 3 mm polystyrene beads cemented on
the inner face of the chamber to separate the leaf from the wall; n = bulldog clips; p = liquid
rubber seal. All dimensions in mm.
 188 B.G. Audley et al

the site of application of the ethephon-~4C was fitted with an inlet and outlet to
enable the transpired ethylene-~4C to be measured by the flow method used for the
leaves. This procedure was adopted because of the higher rate of ethylene produc-
tion from this site. The production of ethylene-t4C from the whole stem was
estimated by graphical integration of the individual stem chamber results. Prelimi-
nary experiments had shown that the amount of ethylene evolved from the petioles
was negligible.

(3) R o o t s . Any volatile radioactivity emanating from the roots was collected in a
slow air-stream drawn upwards through the peat in which the plant was grown. A
'Perspex' plate was sealed over the top of the pot, from which a tube led to an
absorber as used for the leaves.

Results

Absorption of ethephon-X4C by H. brasiliensis stems. Since it is standard

commercial practice to apply yield stimulants to the trunks of rubber trees, much of
the work described below has been carded out on stems of young seedlings or on
excised bark from mature trees.

Preliminary experiments indicated that the absorption of ethephon through bark

tissue is a reversible process which is markedly assisted by removing the largely
hydrophobic outer layers. In one experiment on young stem segments the rate of
uptake of ethephon-~4C was accelerated tenfold by lightly scraping the cuticle.
Changing the pH of the applied solution from two to five had little effect on the rate
of uptake, as did altering the polarity of the solvent used. Analyses of samples taken
at various depths through mature bark suggested that the radial movement of
ethephon into the tissue is a physical phenomenon and this was substantiated by the
results of an experiment in which the absorption of ethephon by stem segments
incubated under a variety of conditions was measured (Table I).

Evolution of ethylene-14C from treated plants. Preliminary experiments had

shown that ethylene-14C was released from the application zone very soon after
applying ethephon-14C and within four hr the gas was detected in quantity coming
from the upper leaves, the stem near the application zone and the apical region.
Ethylene-:4C evolution was barely detectable in the central part of the stem and
from the lower leaves nearby, but within 24 hr all parts of the stem and leaves had
started to evolve ethylene. As might be expected, translocation of ethylene itself
was very rapid, the gas being detectable around a leaf within 30 min of an injection
of ethylene into a stem chamber fitted 26 cm away. Half of the gas had disappeared
from the chamber within 3.5 hr and only 0.1% remained after 48 hr. These
observations are consistent with an initial movement of ethephon in the transpira-
tion stream, and with decomposition of the compound in all parts of the plant to
which it had penetrated.
 Metabolism of Ethephon and Related Compounds 189

Table I. Uptake of ethephon -14C into bark and wood of seedling stem segmentsa

Incubation conditions 14 C absorbed as % applied

Wood Bark

1. 16 b r a t 25~ under
continuous illumination 6.4 17.2
2. As I after heating stem
at 50~ for 30 min. before
application of ethephon 8.1 15.9
3. As 1 but tissue treated with
ethephon solution containing
0.1M NaF 3.9 16.0
4. 16 br at 250C in dark 5.9 14.8
5. As 4 but under N 2 7.0 12.6

Mean + S.D 6.3 -- 1 . 6 15.6 + 2.6

aEthephon-14C (13200 dps) in glycerol-water was applied via a filter paper pad to a
one-cm lengths of seven-mm diameter scraped stem (see Methods). Each result is the
mean of five experiments.

14C2H4
Yield
%

I x e

10

I I I
5 10 15
Days
Fig. 2. Evolution of ethylene-t4C from various sites on a seedling treated on the stem with an
aqueous solution of ethephon-14C, pH3 (1 /zmol, 2.5 /zCi).
 190 B . G . Audley et al

Figure 2 shows the dependence on time, o f the evolution o f ethylene-X4C from the
leaves, the application site, and the remainder o f the stem in a typical experiment
with ethephon-X4C. A total o f 12% o f the applied 14C was recovered as ethylene-14C
from the application site, 23% from the remainder of the stem and 16% from the
leaves. One percent-was recovered from t h e p e a t surrounding the r o o t s . I n all, 51%
o f the radioactivity initially applied to the stem was recovered as ethylene-14C (See
Table II). Fig. 3 emphasizes that the stem near to the application site evolves most
o f the ethylene and that the lower part of the stem also produces considerable
amounts.

Table II. Distribution o f Radioactivity Recovered From a Hevea Brasiliensis Seedling

Treated with Ethephon "14C (94,300 dps) for 15 daysa

dps As % of
Ethylene- 14 C ethephon
applied

Stem, application zone 11 000 I 1.7

Stem, remainder 21 300 22.6
Stem total 34.3
Leaves 14 900 15.8
Roots 1 190 1.3
Total ethylene -14C 51.4

Residual radioactivity
Specific activity
of tissue dps/g
Stem fresh weight

Bark, application zone 4 010 4.3 11 100

" above . . . . 1 850 2.0 166
" below . . . . 70 <0.01 34
Wood application zone 4 920 5.2 4 060
" above . . . . 2 740 3.1 115
" below . . . . 160 <0.02 28
Stem total 14.6

Leaves 2 680 2.8 106

Petioles 620 0.7 83
Roots 360 0.4 15
Peat 320 0.3 - ~

4.2

a Total radioactivity recovered 66,120 dps, 70.2%

 Metabolism of Ethephon and Related Compounds 191

13o0

1200

I
7O
T
=..
3OO
"1"
r

200

100

.7. : C

2'5 7'5
A A
Ground Application site Height (em) Tip
Fig. 3. Daily yield of ethylene-14C from the stem (a) I day, (b) 2 days, (c) 4 days, (d) 11
days after treatment of the same seedling as in Fig. 2.

Residual radioactivity in a seedling treated with ethephon-14C. Fifteen days

after treatment with ethephon-14C, 19% of the applied 14C was still present in the
plant as nonvolatile water-soluble material and chromatographic analyses of extracts
of individual tissues showed the presence of labelled substances in addition to
ethephonJ~C. The highest proportion of radioactivity in the tissue was present in
the stem especially at the application site (Table II). Figure 4 shows the distribution
of residual radioactivity along the stem. It appears that translocation of non-volatile
radioactivity is mainly in an upward direction. Further experiments are needed to
confirm this observation.

Metabolism of ethephon-14C in excised leaves. The indications that ethephon is

metabolized by Hevea tissue were substantiated in a series of experiments with
isolated stem segments and leaves (Archer et al. 1973). Some results of further
investigations are given below.
 192 B. G. Audley et al

IPII

1000

100
E
T'I
- i-1
E I I.., ..-'= P'I r"=
t~. = n m m I I I
Q.
"0 - ~ "-~ r:~ I1"11 I
10 '-I "-"'"="'4 t-" I I "1 I
I,

I
25
i
50
9 II 9
75
=aa
}
100
/x
A a
Ground Application site Height (crn) Tip
Fig. 4. Kesidual z4C in the stem, 15 days after treatment of the same seedling as in Fig. 2:
wood; - - - bark; 9 attachment points of petioles.

200

x~
100

i
20 40
i
&
Fraction number
Fig. 5. Ion-exchange fractionation of leaf metabolites of ethephon-~4C. Six leaves were
incubated with 8.7 /zmol, 19/zCi of ethephon-taC (pH6) for 72 hr, extracted with water and
15% of the extract chromatographed on a column of QAE-Sephadex A-25 chloride (20 • 1.2
cm). The eluting buffer was imidazole hydrochloride, pH6.6 [CI-J = O.05M. Six-ml
fractions were collected.
 Metabolism of Ethephon and Related Compounds 193

The elution diagram (chloride column) of aqueous extracts made from leaves
which had been incubated with ethephon-Z4C for 72 hr showed four radioactive
peaks (Fig. 5). Peak 4 was identified as ethephon. Peaks 1, 2 and 3 (7.5% of the
ethephon-14C fed to the leaves) were analyzed by 2D-TLC in solvents (a) and (b).
At least 11 compounds were detected by autoradiography in peaks 1 and 2 and one
in peak 3 (Figure 6). The elution position of peak 3 suggested it contained 2-HEPA
and this was supported by 2D-cochromatography of peak 3 material and unlabelled
2-HEPA in solvents (a) and (b). Final proof was obtained by the addition of carder
2-HEPA and recrystallisation of the barium salt to constant specific activity.

Fractionation of the leaf extract on a formate column gave the elution diagram
shown in Figure 7; ethephon does not elute. 2D-TLC in this case gave poorer
chromatograms, but the resolution was sufficient to show that spot 8 (Figure 6) was
absent; 11 compounds in all were detected.

":~..:1 2

F
9 11
10
6
co
5 ol
8
O
r.~

~4
1 2

+ S o l v e n t (b) ~ B

Fig. 6. Autoradiograph of leaf metabolites (200 dps) of ethephon-t4C eluting in peaks 1 and 2
from a chloride column (see Fig. 5). Spot 8 = ethephon. 2-HEPA runs in position 4; 0 shows the
position of the compound isolated in the control experiment (see text); + is the origin, A and B
solvent fronts. The chromatogram was placed in contact with Kodirex X-ray film for 14 days.
 194 B.G. Audley et al

200

u!.
n

-6 100

| w

20 40 dO
Fraction number
Fig. 7. Ion-exchange fractionation of leaf metabolites of 14C-ethephon. Six leaves were
incubated with 8.7/zmol, 19/zCi of ethephon-14C (pH6) for 72 hr, extracted with water and
10% of the extract chromatographed on a column of QAE-Sephadex A-25 formate (20• 1.2
cm). The eluting buffer was 0.05M ammonium formate, pH6.6. Six-ml fractions were
collected.

Another experiment was carried out to obtain more comprehensive data on the
incorporation of 14C into various leaf constituents (see Figure 8). The leaves were
extracted by the MCF procedure of Bieleski and Young (1963) and the methanol-
water-solubles fractionated on a chloride column. The characteristic four-peak elu-
tion pattern was obtained and the 2D-TLC map of the labelled components of peaks
1 and 2 was qualitatively the same as that obtained previously. Peak 3 contained
2-HEPA and traces of other 14C-compounds, and peak 4 was again ethephon. The

Ethephon-14C fed to leaves

636000 dps = 100%

i I I
Methanol-Water Chloroform Insoluble
Soluble Soluble
29.6 0.02 0.8
! ! I I
Products Ethephon Soluble in Insoluble in Starch
I 20.3 hot alkali hot alkali
! I 0.6 0.14 <0.1
Basic Acidic Neutral
0.06 9.12 0.08
I
I I
Peaks 1 and 2 Peak 3
6.2 2.9

Fig. 8. Incorporation of 14C into various fractions isolated from six leaves incubated with
ethephon-14C (7.8/xmol, 17.3/xCi, pH6) for 72 hr. The leaves were extracted using the MCF
procedure and the extract fractionated as described in the text.
 Metabolism of Ethephon and Related Compounds 195

material not retained by the column (neutral fraction) contained little radioactivity.
A further portion of the methanol-water solubles was passed down a column of
Bio-Rad AG50W X-12 in the hydrogen form and the column then eluted with 2M
ammonia to obtain the basic fraction. Samples of the chloroform-solubles and the
insoluble residue were analyzed for a4C after combustion to 14CO2. Starch was
isolated by the method of Pucher et al. (1948). In a control experiment, ethephon-
14C was added to a leaf previously homogenized in MCF and the methanol-water
solubles fractionated on a chloride column. The 14C-material eluting in the peak 1
position (only 0.35% of the original ethephon) was analyzed by 2D-TLC in solvents
(a) and (b) after removal of buffer by the cation-column procedure. The position of
the single spot obtained as indicated on Figure 6.
Spot 8 (Figure 6) was identified from its position on 2D-TLC as ethephon and this
was confirmed by cochromatography in solvent (e) and by 2D-TLC with solvents (c)
and (d). The fact that this spot was only present when a cation-exchanger in the
hydrogen form was used to remove the buffer prior to TLC, suggested that the
ethephon was formed by the decomposition of one or more acid-labile derivatives of
ethephon synthesized in the leaves. Evidence for the presence of such compounds
was obtained by extracting a portion of the incubated leaves with MCW (see
" M e t h o d s " ) and fractionating the methanol-water solubles on a formate column.
After removal of buffer by vacuum sublimation, a sample from peak 1 was hyd-
rolyzed with N HC1 for one hr at 60"C and the hydrolysate chromatographed on a
chloride column. The elution diagram showed two peaks, one in the position of peak
1 and the other (50% of the 14C in the hydrolysate) in the ethephon position. The
unhydrolyzed control gave no ethephon peak.
Other candidates as labelled components of peaks 1 and 2 were carboxylic acids,
possibly produced as a result of ethylene metabolism. By 2D-TLC (solvents (c) and
(d) in the presence of marker compounds, of materials from peaks I and 2, it was
established that none of the following acids were labelled: glycollic, oxalic, suc-
cinic, malic, fumaric, a-ketoglutaric, citric, Glyoxylic acid, which was extracted
and chromatographed as its 2:4 DNP derivative, after addition of carrier, was also
inactive (El Hawary and Thompson 1953, Berlet 1968).
Incubation of ethephon-~4C with m a t u r e bark. A single peak eluting in the
same position as peak 1 of Figure 7 was found by fractionating the water-solubles of
incubated bark on a formate column, and 1D-TLC in solvents (c) and (e) showed the
presence of only one substance. The compound, which was converted back to
ethephon by mild acid hydrolysis, was not produced by bark which had been
steamed before incubation. The accumulation of the product in bark samples incu-
bated for different times is shown in Figure 9.
Incubation of ethylene-~4C with a leaf. After 72 hr incubation, the leaf was
extracted by the MCF procedure. The total incorporation of I4C into the fractions
examined was 0.16% of the initial ethylene. Of this 78% of the radioactivity was in
the methanol-water-solubles, 5% in the chloroform-solubles and 17% in the residual
solid. Ninety two percent of the ~4C in the methanol-water-solubles was in the
neutral fraction.
 196 B.G. Audley et al

Incubation of 2-HEPA-14C with leaves. The elution diagram (chloride column)

of an aqueous extract of leaves which had been incubated for 72 hr showed three
overlapping peaks. 2D-TLC in solvents (a) and (b) of these fractions indicated that
at least ten compounds were present in addition to 2-HEPA, which eluted last. The
total 14C in the products accounted for 20% of the 2-HEPA-~4C applied to the
leaves.

Non-induction of ethylene formation in H . brasiliensis by ethephon. Since

many growth regulators induce the formation of ethylene in plants from endogenous
precursors, it was of interest to see if ethephon behaved in this way. Lougheed and
Franklin (1970) attempted to measure the ethylene evolved from treated and un-
treated tomato fruits using unlabelled ethephon and obtained no evidence for the
induction of ethylene from tissue substrates as a result of ethephon treatment.
However a more satisfactory method of approaching this problem, is to treat the
tissue with ethephon-14C and to measure the specific activity of the ethylene-14C
evolved. Such measurements, when carried out on vegetative tissue (Table III)
provided no evidence of ethylene induction, in agreement with the results of the
previous authors.

25

0.15
20
e,i

e-
== 15 t:
0.1o w

c
O
r-
10 o
.c
c-
O i-
O
0.05 =
0
E

25 50 75 1O0
Hours

Fig. 9. Time-course of formation of an acid-labile derivative of ethephon-14C in mature bark.

Pieces of bark were treated with an aqueous solution of ethephon-14C (0.725 /zmol, 0.16
/zCi, per g of bark, pH3) and after incubation, the compound isolated on a column of
QAE-Sephadex A-25 formate.
 Metabolism of Ethephon and Related Compounds i97

Table III. Specific Activity of E thylene14 C Produced from H. Brasiliensis after

treatment with Ethephon'14 C for Various Periodsa

Specific activity of ethylene

pHof (dps//~moi) after days
applied
solution 1 2 3

SD SD SD

Stem segments 7 84.1 2.7 87.1 1.7 81.6 4.6

3 82.9 2.6 82.0 2.0 89.4 4.0
Leaves 7b 80.5 2.5 85.0 3.3 82.5 3.4

aMean specific activity of ethylene-t4C formed from ethephon -14 in the absence of plant
material = 83.1 dps//zmol (SD = 5.2, 46 determinations).
blnsuffieient ethylene-14C was obtained at pH3 to enable reliable measurements to be
made.
Stem segments (30 x 7ram) or excised leaves were surface treated with 0.4 and 9.0/amols
of ethephon-14C respectively and incubated as detailed under methods. Ethylene was
determined by GLC and counted as described previously (Archer et al. 1973). Each
value in the table is the mean obtained from five experiments.

Discussion

The decomposition of ethephon in vitro at physiological pH is generally accepted

as yielding only ethylene, phosphate and chloride ions, but Audley and Archer
(1973) showed that a small amount of 2-HEPA was also formed. Kinetic experi-
ments by Gregory and Higgins (1974) have shown that the half-lives of ethephon at
pH 7.0 and 6.0 are approximately one and six days respectively. Although the pH
values within the tissues of H. brasiliensis are not known, it is noteworthy that in
the experiments with whole plants described here, only half of the radioactivity
applied was recovered as ethylene-X4C in 15 days.

The present results support our previous conclusion that metabolism of ethephon
occurs to a significant extent in H. brasiliensis to produce a number of acidic
compounds. The high radiochemical purity of the ethephon used, the high conver-
sions obtained and the result of the control experiment, eliminate the possibility that
the compounds observed are radiochemical impurities in the starting material. Some
of the compounds may be formed from true metabolites as a result of the analyti-
cal procedure; for example the ethephon spot in Figure 6 is clearly formed by the decom-
position of an as yet unidentified material.

2-HEPA has been identified as a major component of the acidic products. Since
similar amounts of 2-HEPA are produced when ethephon is incubated in neutral
buffer, it would appear that 2-HEPA is formed in vivo primarily by a non-enzymic
process. However this may not be true, since this compound is itself extensively
198 B.G. Audley et al

metabolized and therefore its rate of formation in leaves may be considerably greater
than would appear from our present measurements.

The finding, in the leaf experiment, that 50% of the radioactivity of peak 1 from
the formate column (Figure 7), could be recovered as ethephon-~4C after mild acid
hydrolysis, indicates that at least one metabolite is a compound in which ethephon is
conjugated (probably via one of its -OH groups) to a material already present in the
leaf. The observation that this product is anionic suggests that only one of the
hydroxyl groups is utilized. However it is possible that both hydroxyls are involved,
and that the reactive plant compound is itself negatively charged. No 2-HEPA was
detected on hydrolysis of peak I material, so conjugates of 2-HEPA were apparently
absent unless such compounds were stable under the mild conditions of hydrolysis
employed. It is clearly possible that all the components of peak 1 are conjugates of
ethephon or 2-HEPA which have varying degrees of lability.

The possibility that some of the compounds of peak 1 are carboxylic acids,
arising from the oxidation of ethylene produced from ethephon in the leaf tissue, is
unlikely, since none of the common plant acids or amino acids formed therefrom
were labelled. The two-carbon acids, glycollic, glyoxylic or oxalic which might be
envisaged as oxidation products of ethylene were unlabelled, as was acetate, since
virtually no labelled lipid was formed. Formation of labelled carboxylic acids would
be expected to lead to the production of 1'CO2. and hence labelled sugar and starch.
The neutral fraction carried a small amount of label, but no 14C-starch was detected.
Furthermore when ethylene-~4C itself was incubated with a Hevea leaf, only 0.16%
of the 14C fed was utilized and only 8% of this, at most, could have been in the acid
fraction. Clearly the proportion of ethylene fixed in this experiment was so small
that it becomes necessary to consider whether the result is due to the presence of an
impurity in the labelled ethylene. If this were x4CO2, the labelling of the neutral
fraction would be explained.

Although immature green stem forms a range of compounds from ethephon

(Archer et.al. 1973), as do leaves, bark from mature rubber trees forms a single
compound, which like some of the products in leaves decomposes to ethephon on
acid hydrolysis. It is possible that the greater number of compounds formed in green
tissue is related to its photosynthetic ability, but tissue age may also be a contribut-
ory factor. Our finding, that Hevea tissues can substantially metabolise ethephon, is
contrary to the results of other workers who have used a wide variety of plants. It
seems unlikely that H. brasiliensis is unique in its ability to metabolize ethephon
and the disparity between our results and those of others is probably a consequence
of the different experimental techniques employed.

Acknowledgments
The Board of the Rubber Research Institute of Malaysia is thanked for permission
to publish this work. Thanks are also due to Mrs. P. M. Powter, Miss M. Wood-
 Metabolism of Ethephon and Related Compounds 199

cock, Mr. A. F. Carne, Mr. K. Subramaniam and Mr. S. Sivanayagam for technical
assistance.

References

Abeles, F. B.: Biosynthesis and mechanism of action of ethylene. Ann. Rev. Plant
Physiol. 23, 259 (1972).
Abeles, F. B.: Ethylene in plant biology. New York: Academic Press (1973).
Abraham, P. D., P. R. Wycherley, and S. W. Pakianathan: Stimulation of latex
flow in Hevea brasiliensis by 4-amino-3,5,6-trichloropicolinic acid and
2-chloroethanephosphonic acid. J. Rubber Res. Inst. Malaya 20, 291 (1968).
Amchem Products Inc. : Technical service data sheet H.96 Ethrel. (1969).
Archer, B. L., B. G. Audley, and N. P. Mann: Decomposition of 2-chloroethyl-
phosphonic acid in stems and leaves o f H e v e a brasiliensis. Phytochemistry 12,
1535 (1973).
Audley, B. G., and B. L. Archer: Decomposition of 2-chloroethylphosphonic acid
in aqueous solution: formation of 2-hydroxyethylphosphonic acid. Chem. &
Ind. (London) p. 634 (1973).
Berlet, H. H.: Thin-layer chromatography of keto-acid dinitrophenylhydrazones of
biological interest. Anal. Biochem. 22, 525 (1968).
Bieleski, R. L., and R. E. Young: Extraction and separation of phosphate esters
from plant tissues. Anal. Biochem. 6, 54 (1963).
Boatman, S.G.: Preliminary physiological studies on the promotion of latex flow by
plant growth regulators. J. Rubber Res. Inst. Malaya 19, 243 (1966).
Burrows, S., F. S. M. Grylls, and J. S. Harrison: Paper chromatography of phos-
phoric esters. Nature 170, 800 (1952).
Cooke, A. R., and D. I. Randall: 2-Haloethanephosphonic acids as ethylene
releasing agents for the induction of flowering in pineapples. Nature 218, 974
(1968).
Dickenson, P. B.: Compositions containing plant growth regulants. British Patent
1315131 (1973).
Edgerton, L. J., and A. H. Hatch: Absorption and metabolism of 14C(2-chloroethyl)
phosphonic acid in apples and cherries. J. Am. Soc. Hort. Sci. 97, 112 (1972).
E1 Hawary, M. F. S., and R. H. S. Thompson: Separation and estimation of blood
keto-acids by paper chromatography. Biochem. J. 53, 340 (1953).
Field, R. J., and A. J. Peel: The metabolism and radial movement of growth
regulators and herbicides in willow stems. New Phytologist 70, 743 (1971).
Fritz, C. D., and W. F. Evans: Processes and compositions for the regulation of
plant growth. British Patent No. 1194433 (1970).
200 B.G. Audley et al

Gregory, M. J., and G. M. C. Higgins: The solvolytic fragmentation of 2-haloalkyl-

phosphonic acids. J. Chem. Soc. Perkin II, 711 (1974).
Jeffay, H., and J. Alvarez: Liquid scintillation counting of carbon-14. Anal. Chem.
33, 612 (1961).
Lavee, S., and Martin, G.C.: Ethephon (1,2-14C - (2-chlorethyl) phosphonic acid) in
peach fruits. 2. metabolism. J. Am. Soc. Hort. Sci. 99, 100 (1974).
Lougheed, E. C., and E. W. Franklin: Ethylene evolution from 2-chloroethyl-
phosphonic acid under nitrogen atmospheres. Can. J. Plant Sci. 50, 586
(1970).
Martin, G. C., H. A. Abdel-Gawad, and R. J. Weaver: The movement and fate of
(2-chlorethyl) phosphonic acid in walnut. J. Am. Soc. Hort. Sci. 97, 51
(1972).
Pucher, G. W., C. S. Leavenworth, and H. B. Vickery: Determination of starch in
plant tissues. Anal. Chem. 20, 850 (1948).
Ribaillier, D.: Importance of lutoids in flow of latex: Effect of stimulation. Rev.
Gen. Caoutchouc 47, 305 (1970).
Southorn, W. A.: Physiology of H e v e a (latex flow). J. Rubber Res. Inst. Malaya
21, 494 (1969).
Vogt, W.: Synthesis of fl-hydroxyethanephosphonic acid. Tetrahedron Letters No.
15., 1281 (1970).
Weaver, R. J., H. A. Abdel-Gawad, and G. C. Martin: Translocation and persis-
tence of 1,2-~4C-(2-chloroethyl) phosphonic acid (ethephon) in Thompson
seedless grapes. Physiol. Planatarum 26, 13 (1972).
Yamaguchi, M., C. W. Chu, and S. F. Young: Fate of 14C(2-Chloroethyl) phos-
phonic acid in summer squash, cucumber and tomato. J. Am. Soc. Hort. Sci.
96, 606 (1971).
Young, R. E., H. K. Pratt, and J. B. Biale: Manometric determination of low
concentrations of ethylene. Anal. Chem. 24, 551 (1952).

Manuscript received October I, 1974; November 23, 1974