Plant Physiol. (1969) 44, 1368-1370
Short Comnmunication
Oxygen Requirement for Ethane Production In Vitro
by Phaseolus vu!garis' 2
Roy W. Curtis
Department of Botany and Plant Pathology, Purdue University, Lafayett:, Indiana 47907
Received December 20, 1968.
Ethane, which is normally produced by plant
tissues in minute quantities relative to ethylene, is a
normal product of apples and potatoes (9), and of
"apple" bananas (Musa sapientum L.) (2). "Ethylene" produced by cytoplasmic particles from apple
and tomato fruits (5) was shown to be a gas other
than ethylene (3), and subsequently identified as
ethane (6). Little is known concerning ethane
biosynthesis. Apple homogenates produce considerably more ethane than whole fruits (6), and ethane
production by particulate systems is stimulated by
thiomalic and thioglycolic acid and requires an
unsaturated fatty acid. In a model system producing
ethane or ethylene, activation and oxidation of
linolenic acid was a necessary requirement (7).
Although oxygen is required for ethylene production (1) a similar requirement for ethane production has not been demonstrated. I describe here
experiments which indicate that oxygen is both
required for and inhibitory to ethane production.
Materials and Methods
Seven to 10-day old seedlings of Phaseolus vulgaris L. cv Black Valentine were grown in vermiculite. Apical bud sections were cut 5 mm below the
bud and included 3 mm stumps of the primary leaf
petioles. Samples were 5 sections per treatment.
The sections were submerged in water, placed in
250 mm desiccators, and evacuated for 2 min by
water aspiration. The pressure was gradually released over a 2 min period. Controls were untreated
sections and "air-infiltrated" sections (i.e., vacuuminfiltrated in the a;bsence of solution). The sections
were incubated in 5 ml glass syringes containing
1) air, 2) nitrogen, and 3) nitrogen for 22 hr and
'Supported by grant GB.7158 from the National
Science Foundation.
2J ournal Paper No. 3665 of the Purdue Agrioultural
Experiment Station.
air between the twenty-second and twentv-ninth hr.
Ethane was determined after 4, 22, and 29 hr;
syringes were flushed after eaclh determination.
The production of ethane and propane by honiogenates was also studied. Five apical bud sections
were ground by hand in 1 ml of water or test soltution
for 30 sec in a mortar. The homogenate was poured
into a syringe, sealed, and incubated in the dark at
27°. Unground sections served as controls. To
determine the rate of ethane production, samples
were removed from the syringes after 30, 60, 120,
and 180 min for ethane analysis. The syringes were
flushed and sealed after each determination.
Ethane, ethylene, and propane were determined
by gas chromatography using an aluminum oxide
column (4) and were readily separated. The authenticity of the gases was determined by co-chromatography with authentic samples (Olin Matheson
Company), Neither authentic nor sample-produced
ethane and propane were removed by pre-treatment
with aqueous base and mercuric perchlorate, bromine
water, or mercuric nitrate. The amount of eachl gas
was proportional to peak height when 2 ml samples
of different concentration were analyzed.
Results and Discussion
The production of ethane by untreated and airinfiltrated apical bud sections maintained in air was
generally similar (table I). In both, production
was greater in nitrogen and declined when sections
were transferred from nitrogen to air after 22 hr,
as compared to sections maintained continually in
nitrogen. Although ethane production by waterinfiltrated tissues was greater than that of untreated
and air-infiltrated tissues in air or nitrogen for
22 hr, the rate of ethane production in nitrogen
declined beLween 22 and 29 hr. When water-infiltrited tissues were transferred to air after 22 hr,
ethane production increased markedly. Similar results were obtained when apical bud sections were
vacuum-infiltrated with 0.4 M sucrose. The slight
increase in ethane production by water-infiltrated
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1368
1369
C'URTIS-OXYGEN REQUIRED FOR ETHANE PRODUCTION
Table I. Effect of Atmosphere on Produiction of Ethane
by Apical Bud Sections of P. vulgaris
Treatment and
atmosphere
Ethane produced per hr
22-29 hr
4-22 hr
04 hr
ni/5 sectionts
Untreated
Air
No
N2
Air-Infiltrated
Air
N2
N2
0.08
0.16
0.15
0.02
0.22
0.151
0.02
1.09
0.19
0.08
0.17
0.15
0.02
0.28
0.111
0.02
0.40
0.27
Water-Infiltrated
0.13
0.03
0.10
0.08
0.34
0.20
3.60
0.151
0.19
N.,
' Atmosphere changed to air after 22 hr determination.
Average 6 determinations.
Air
N2
tissues in air was not observed when similar tissues
were maintained in oxygen. In all treatments ethylseverely inlhibited in nitrogen,
ene production
but recovered to varying degrees wlhen tissues were
tran ferred to air. In air, water-infiltrated tissues
produced approximately 50 % less etlylene -than tuntreated controls; in oxygen, ethylene production
recovered to approximately that of controls.
Honmogenates of apical bud sections produiced
80.4 times nmore ethane and 10 times more propane
than unground controls after 4 hr (average 14
determinations). To determine the rate of ethane
production. samples were removed from the syringes
after 30, 60, 120. and 180 min for ethane analvsis.
Approximately 98 % of the total ethane was produced wi.hin 2 hr. Incubation periods of 2 to 4 hr
were used subsequently. Homogenization in cold
was
or room
temperature wvater, and agitation of the
homogenates during incubation had little effect on
the yield of ethane. Tissue age influenced ethane
yield. Homogenates of 7 to 10 and 24 day old
primary leaf petiole sections produced 27.9 and 5.1
times as much ethane, respectively, as unground
controls (average 10 determinations).
The effect of various compounds on yield of
ethane from apical bud sections, homogenized in 1 ml
of test solution, was compared with water-homogenized controls. IAA (10-4, 10-5 M), 2,4-D (10-4,
10-5 M), and 0.4 M sucrose had no appreciable effect.
Methylene blue, 10-1 and 10-8 M, inhibited ethane
production 90 and 25 %, respectively; oxygen inhibited ethane production 50 %; nitrogen stimulated
ethane production 42 %. The effect of thioglycolic
acid, which stimulates ethane and propane production
by particles prepared from tomato fruit (8), on
apical bud sections treated by vacuum-infiltration.
soaking, and homogenization was examined (table
II). Because thioglycolic acid itself liberated significant quantities of these compounds, syringes containing only the solutions served as controls and
were used as correction factors. Both ethane and
propane were stimulated by thioglycolic acid; ethylene
production was unaffected. Sections steamed for
20 min prior to homogenization produced negligible
quantities of these gases. Although the amount of
ethane produced by apical bud sections homogenized
in 1 ml of thioglycolic acid was considerably greater
than that produced by vacuum-infiltrated tissues,
the amount of thioglycolic acid available to vacuuminfiltrated sections was only 0.054 ml (determined
by weighing apical bud sections before and after
infiltration). When ethane production by vacuuminfiltrated sections was calculated per ml of thioglycolic acid, the amount of ethane and propane
released was greater than that from homogenates.
Thus, the efficiency of ethane release from thioglycolic acid by uninjured tissues was greater than
that of homogenates. No estimation of thioglycolic
acid uptake by soaked tissues was attempted.
Generally, the production of ethane by apical bud
sections, and homogenates of these tissues, was
inhibited by conditions favoring oxidation (02,
methvlene blue) and stimulated by conditions favoring reduction (N., thioglycolic acid). However, the
decline in ethane production by water-infiltrated
tissues after 22 to 29 hr under nitrogen, and the
dramatic rise in ethane production when transferred
to air after 22 hr, suggeFts an oxygen requirement
for eihane production. The accumulation of an
ethane precursor, such as "non-activated" linolenic
acid, under anaerobic conditions could account for
the high ethane yield. Alternatively, reduction of
unsaturated fatty acids during prolonged anaerobiosis
may accotunt for the failure of the tissues to release
ethane in substantial quantities when kept in nitrogen.
Restoration of oxidative conditions presumably allows
the formation of unsaturated fatty acids required for
ethane synthesis. The decline in ethane production
by homogenates in the presence of oxygen and
methylene blue may reflect inhibition of a subsequent
reductive step leading to ethane synthesis.
Table II. Production of Ethane and Propane by Apical
Buqd Sections of P. vulgaris Homogenized, Soaked and
Vacuum-infiltrated With Thioglycolic Acid
Average of 8 determinations.
Treatment
Gas produced per interval
3-6 hr
0-3 hr
Ethane Propane Ethane Propane
n1/5 sections
0.23
Whole sections
Homogenized in H20 16.90
Homogenized in 10-2 M 243.4
thioglycolic acid
33.3
Soaked in 10-2 M
thioglycolic acid
Vacuum-infiltrated with 17.4
thioglycolic acid
10-2
M
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7.15
0.10
0.65
11.85
2.25
1.50
41.50
2.05
0.55
3.6
0.25
0.04
0.45
0.0
0.0
1370
PLAN'T
I'HYSIOLOGY
The failure of untreated sections and sections
vacuum-infiltrated with air to liberate quantities of
ethane after prolonged anaerobiosis and return to
air may be due to sufficient oxygen in the tissues to
prevent the accumulation of ethane precursors,
whereas sections vacuum-infiltrated with aqueous
solutions undoubtedly contain less oxygen. Vacuunminfiltration per se does not significantly injure the
tissues, since ethane production increases only moderately following vacuum-infiltration with water as
compared to the large increase induced by homogenization. Because oxygen reduced both the inhibitory effect of water-infiltration on ethylene production and the stimulatory effect on ethane production.
changes in the rate of production of these gases after
water-infiltration may represent oxygen deficiency.
The inhibitory action of water on oxygen uptake of
corn tissue has been attributed to a decreased rate
of oxygen diffusion into the tissue (10).
Acknowledgment
The technical assistance of Mrs. P'hyllis Phillips is
gratefully acknowledged.
Literature Cited
1. BURG, S. P. 1962. The physiology of ethylene
formation. Ann. Rev. Plant Physiol. 13: 265-302.
2. BURG, S. AND E. BURG. 1965. Relationship between
ethylene production and ripening in bananas.
Botan. Gaz. 126: 200-04.
3. BURG, S. AND E. BURG. 1961. Ethylene evolution
and sub-cellular particles. Nature 191: 967-69.
4. CURTIS, R. W. 1968. Mediation of a plant response to malformin by ethylene. Plant Physiol.
43: 76-80.
5. LIEBERMAN, M. AND C. CRAFTS. 1961. Ethylene
production by cytoplasmic particles from apple and
tomato fruits in the presence of thiomalic and thioglycolic acid. Nature 189: 243.
6. LIEBERMAN, M. AND L. MAPSON. 1962. Fatty
acid control of ethane prcduction by sub-cellular
particles from apples and its possible relationship
to ethylene biosynthesis. Nature 195: 1016-17.
7. LIEBERMAN, M. AND L. MAPSON. 1964. Genesis
and biogenesis of ethylene. Nature 204: 34345.
8. MEIGH, D. F. 1962. Problems of ethylene metabolism. Nature 196: 345-47.
9. MEIGH, D. F. 1959. Nature of olefines produced
by apples. Nature 184: 1072-73.
10. OHMURA, T. AND R. HOWELL. 1960. Inhibitory effect of water on oxygen consumption by plant
materials. Plant Physiol. 35: 184-88.
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