Plant Physiol. (1979) 63, 142-145
0032-0089/79/63/0142/04/$00.50/0
Ethylene and Ethane Production from Sulfur Dioxide-injured
Plants'
Received for publication May 2, 1978 and in revised form July 14, 1978
GALEN D. PEISER AND SHANG FA YANG
Department of Vegetable Crops, University of California, Davis, California 95616
ABSTRACT
After alfalfa (Medicago sativa) seedlings were exposed to approximately
0.7 microliter per liter SO2 for 8 hours, elevated ethylene and ethane
production was observed. Ethylene production peaked about 6 hours and
returned to control levels by about 24 hours following the fumigation, while
ethane production peaked about 36 hours and was still above control levels
48 hours after the fumigation. Light had an opposite effect upon the
production of the two gases: ethane production rates were higher from
plants held in light, whereas ethylene production rates were higher from
those held in the dark. Peak ethylene and ethane production rates from
SOs-treated plants were about 10 and 4 to 5 times greater, respectively,
than those of the control plants. Ethylene appeared to be formed primarily
from stressed yet viable leaves and ethane from visibly damaged leaves.
The different time courses and light requirements for ethylene and ethane
production suggest that these two gases were formed via different mechanisms. Light appears to have a dual role. It enhances S02-induced cellular
damage and plays a role for repairs.
Ethylene is produced from plants under normal conditions in
relatively low amounts, but when plants are perturbed or injured,
elevated ethylene production rates usually occur (1, 23). Stressinduced ethylene production has been reported to occur in plants
exposed to air pollutants, such as ozone (5, 21) and SO2 (3, 4).
Stress ethylene from ozone-injured plants reached a maximum
within 2 to 4 hr, and returned to control levels by 24 hr after
exposure to ozone (5, 21). Bressan et al. (3, 4) exposed leaf tissues
of cucurbits to bisulfite solution or SO2 gas and found that
ethylene production greatly increased in slightly injured leaves,
but declined in severely injured leaves. Both ethylene and ethane
can be produced from freeze-injured tissue (8). We have recently
reported that both of these gases were produced in a chemical
system consisting of linolenic acid hydroperoxide and bisulfite
(15). In the present study we examined ethylene and ethane
production from intact alfalfa plants exposed to SO2.
of 160 1/hr. S02-air mixture was prepared and the flow rate was
maintained by a capillary flowmeter (14). At this flow rate a
considerable amount of S02 was absorbed by the plants as revealed by the drop in SO2 concentration after the plants were
placed in the chambers. Typical S02 concentrations, determined
at the exit chamber port immediately before and at 1 and 5 hr
after the plants were placed in the chambers, were 1.5, 0.5 and 0.6
,ul/l, respectively. Gas samples were collected in 2-liter Erlenmeyer
flasks and S02 concentration was measured by the West-Gaeke
method as modified by Scaringelli et al (18). The temperature
and light intensity during the fumigation were the same as that at
which the plants were grown.
To determine the ethylene and ethane production rates, each
pot of plants was enclosed periodically in a 1-liter glass chamber
for 4 hr over a period both before and after the S02 fumigation.
A 3-ml gas sample was withdrawn from each chamber at the
beginning and end of each 4-hr period, and the gas concentrations
were measured by gas chromatography employing an alumina
column and a flame ionization detector. Between each 4-hr period
the chambers were left open for 30 min and then reclosed for the
next determination.
The thiobarbituric acid assay for malonaldehyde was conducted
according to Dillard and Tappel (6) with the exception that leaf
samples were homogenized in 20%Yo (w/v) trichloroacetic acid, and
aliquots of this crude homogenate were used in the assay. The
content of thiobarbituric acid reactants (malonaldehyde) was calculated assuming that the molar absorptivity at 532 nm is 150,000
(16).
RESULTS
In preliminary experiments we observed that following the SO2
fumigation the production rate of ethylene was greater when
plants were incubated in the dark, while the ethane production
rate was greater in light. We have therefore examined the effect of
light and dark on ethylene and ethane production by control and
by fumigated plants. Figure 1 illustrates that both ethylene and
ethane production rates increased following the SO2 fumigation.
Although comparable pots of plants were fumigated in the same
chamber, the plants subsequently incubated in the dark had about
twice the rate of ethylene production as those incubated in continuous light (Fig. IA). For these same plants, ethane production
(Fig. 1B) started earlier in light and more than twice the amount
of ethane was produced in light than in darkness. In control
plants, there was little difference between the rates of either
ethylene or ethane production in light and dark. The important
role of light in stimulating ethane production by the fumigated
plants is further illustrated by the change in the ethane production
rate when the plants were reversed with respect to the light-dark
environment (as indicated by arrows in Fig. 1B). The dark-incubated plants transferred to light exhibited a greatly increased
ethane production rate, while the light-incubated plants transferred to darkness showed a decreased production rate. Similar
MATERIALS AND METHODS
Alfalfa plants (Medicago sativa L.) were grown in Vermiculite
in plastic pots, which were placed in a controlled temperature
chamber maintained at 25 + 2 C. A 16-hr daylength, with an
illuminance of about 300 ft-c, was provided by cool-white fluorescent lamps. Air was continuously renewed.
When the plants were about 19 days old, two pots (15 plants
per pot with a total shoot fresh weight of about 1.8 g) were
fumigated with air (control) and two pots with air containing SO2
for about 8 hr, in a 9-liter cylindrical glass chamber, at a flow rate
' This work was supported by United States Public Health Service
Grant ES 01045 and National Science Foundation Grant PCM 75-14444.
142
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Plant Physiol. Vol. 63, 1979
C2H4 AND C2H6 FROM SO2-INJURED PLANTS
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FIG. 1. Increases in ethylene (A) and ethane (B) production by alfalfa
plants following SO2 fumigation. Two pots of plants (A, A) were exposed
to S02 for 7.5 hr and two pots ofcontrol plants (0, 0) were correspondingly
exposed to air only. The SO2 concentration was 1.6, 0.6, and 0.8 pd/l at 0,
1, and 5 hr, respectively, during the fumigation. Fumigation began 2 hr
after the beginning of light period and continued for 6 hr. At the end of
the fumigation period, one S02-treated pot (A) and one control pot (0)
were placed in continuous light, while the other SOrtreated pot (A) and
control pot (0) were placed in the dark. Forty-five hr after fumigation, as
indicated by arrow, the pot of SOrtreated plants kept in the dark (A) was
transferred to light, and that in light (A) transferred to the dark.
results were obtained when visibly damaged leaves excised from
fumigated plants were incubated in dark or light (Fig. 2C). Leaves
incubated in the light achieved ethane production rates three to
six times greater than leaves incubated in the dark, and this effect
was reversible by reversing the light-dark environment.
When fumigated plants were exposed to continuous light, the
ethylene production rate increased to a peak within 4 to 8 hr and
then declined to the basal level within 16 to 20 hr following the
fumigation period (Fig. IA). Similar results were observed in each
of the four other experiments conducted, although there were
slight variations in the time periods required for ethylene production to reach a maximum and subsequently to decline to the basal
level, due to variation in exposure time and/or SO2 concentration.
Following the fumigation period, plants incubated in the dark
reached a peak ethylene production rate at about the same time
(4-8 hr) as those in the light, but sustained this higher rate over a
longer period and required 36 hr to decline to the basal level (Fig.
IA). In comparison, the ethane production rate from plants in the
dark peaked much later (24-36 hr after fumigation) and was
sustained for a longer time than that for ethylene (Fig. 1B). When
fumigated plants were exposed to continuous light, the high ethane
production rate was maintained as long as the experiment was
continued, about 48 hr after fumigation.
FIG. 2. Ethane production rates by leaves detached from control plants
(A), SOrtreated plants without visible damage (B), and SOrtreated plants
with visible damage (C). Intact alfalfa plants were fumigated as described
in Figure 1. Twenty-two hr after the S02 fumigation, at which time
ethylene production rates from treated plants had returned to the basal
level, leaves were detached and placed in vials with 1 ml of water and
incubated in either light (0, A) or dark (0, A) for four consecutive 2-hr
periods. At the end of the second 2-hr period, as indicated by arrow, leaves
incubated in the light were transferred to the dark and those in the dark
transferred to light.
Under the conditions used for fumigation, some of the leaves
exhibited visible damage (a water-soaked appearance) during the
fumigation period. To determine which leaves were responsible
for the ethane production, visibly and nonvisibly damaged leaves
were excised from fumigated plants and their ethane production
rates compared. This experiment was conducted when the ethane
production rate was high, 22 to 32 hr after fumigation. The results
demonstrate that visibly damaged leaves produced much more
ethane (10 times, or more) than the nonvisibly damaged leaves
from the same treated plants (Fig. 2). The nonvisibly damaged
leaves produced only slightly more ethane than did the controls.
By varying the length of the fumigation period in five experiments, with a total of 10 pots of treated plants, the percentage of
visibly damaged leaves ranged from about 10 to 60%. Over this
whole range, the ethane production rate increased as the number
of leaves showing visible damage increased. This observation,
together with the results obtained from excised leaves (Fig. 2),
suggests that ethane production was closely related to visible
damage. For the production of ethylene a similar relationship was
observed only when fewer than 40 to 50%1o of the leaves had
suffered visible damage; there was a decline in ethylene production
rate as the visible damage increased to include more than half the
number of leaves.
Ethane is known to be produced during the peroxidation of
linolenic acid (7, 17), and the level of thiobarbituric acid reactants
(malonaldehyde) is known to serve as an index of lipid peroxidation. We have therefore compared ethane production rates with
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144
Plant Physiol. Vol. 63, 1979
PEISER AND YANG
the level of thiobarbituric acid reactants in the control leaves and
in fumigated leaves which were visibly or not visibly damaged.
The results (Fig. 2 and Table I) indicate that both the ethane
production rate and the level of thiobarbituric acid reactants were
highest in leaves showing damage caused by SO2 fumigation.
Following the fumigation, the condition and number of leaves
showing visible damage were influenced both by the light-dark
environment during incubation and by the duration of incubation.
When scored visually for discoloration and desiccation 1 day after
fumigation, leaves from light-incubated plants were much further
deteriorated than those from the dark. Three days after the
fumigation, more leaves from dark-incubated plants than from
light-incubated plants showed visible damage.
DISCUSSION
The results indicate that the production rates of both ethylene
and ethane increased after exposure of alfalfa plants to SO2 (Fig.
1). Since both gases were produced, the possibility exists that they
could have been formed by the same mechanism. In chemical
systems both ethylene and ethane can be produced from linolenic
acid hydroperoxide (7, 17). However, the kinetics and the influence of light for ethylene and ethane production in the present
system were quite different (Fig. 1), suggesting that the two gases
were not produced by the same mechanism. It is generally thought
that methionine is the precursor of both stress- and normally
produced ethylene (1, 2, 10, 11, 23) while ethane is produced from
peroxidized linolenic acid (7, 13, 17). Wilson et aL (22) reported
that a rhizobitoxin analog, an inhibitor of ethylene synthesis from
methionine, partially inhibited the emission of ethylene but not
the emission of ethane from leaf tissues of cucurbits exposed to
bisulfite solution. These observations are consistent with the view
that the two gases are produced largely by the different pathways.
These results do not exclude the possibility that some ethylene
may arise from peroxidized linolenic acid especially in those
tissues which are badly injured in light and produce large amounts
of ethane.
The extent of the stress or damage that tissues experience
appears to determine whether they will produce ethylene or
ethane. Elstner and Konze (8), working with point frozen leaf
discs, observed that ethane production increased linearly as the
frozen area increased from 0 to 100%1o; in contrast, ethylene production increased as the amount of frozen tissue increased from 0
to 40%, but declined as the area frozen increased beyond 50%o.
They concluded that ethylene was produced from stressed yet
viable tissue, while ethane was produced from decompartmented
(killed) cells. Our results are consistent with this conclusion.
Ethane was produced primarily from visibly damaged leaves (Fig.
2) after they were in a deteriorated condition, as indicated by their
low respiration rate compared to the control rate (Table I).
In the present system light was found to inhibit stress ethylene
Table I.
Respiration rates and malonaldehyde levels in excised
leaves in relation to different degrees of SO2 injury.
The excised leaves were prepared as described in Figure 2.
Respiration rates were determined as CO2 production during each
2-hr period from detached leaves incubated in the dark. The
values are averages from 4 incubation periods. Malonaldehyde
was assayed witn thiobarbituric acid reagent after homogenization of the leaf samples at the end of the incubation period.
C02 production
wl/g fresh wt-hr
Malonaldehyde
tmol/g
fresh wt
Control leaves
242 ± 38
145 ± 31
Treated leaves with
no visible injury
352 ± 51
188 ± 33
Treated leaves with
visible injury
103 t 15
292
38
production but to stimulate ethane production from injured tissue.
Tingey et al. (21) found that plants exposed to ozone produced
more ethylene when they were incubated in the dark than in the
light. In the excised leaf tissues of cucurbits exposed to bisulfite
solution, Wilson et al. (22) reported that light enhanced both
ethane and ethylene production. The causes of such variations are
not clear.
Ethane is thought to be produced from peroxidized linolenic
acid via a free radical mechanism (7, 13, 17). Both higher amounts
of thiobarbituric acid reactants and a greater production of ethane
occurred in those leaves which were visibly damaged than in
either nonstressed controls or in leaves which were treated but not
visibly damaged (Table I). These results are in parallel with the
notion that ethane could have resulted from the peroxidation of
lipids. It is to be noted that during the aerobic oxidation of
bisulfite in vitro, linolenic acid is cooxidized to linolenic acid
hydroperoxide via a free radical mechanism (M. C. Lizada and S.
F. Yang, unpublished results). Such a free radical-mediated oxidation of bisulfite can be initiated in vitro photochemically by Chl
(9, 15) and result in Chl destruction (15). These observations lead
to the speculation that SO2 absorbed by leaves may undergo
oxidation and generate radicals which in turn may cause tissue
damage, lipid peroxidation, ethane production and Chl destruction. In addition to the regulation of stomata opening by light,
and the consequent SO2 absorption during exposure to SO2, light
may also accelerate the radical-mediated oxidation of dissolved
S02/bisulflte, and accentuate injury and ethane formation. We
observed, 1 day after fumigation, that visibly damaged leaves
deteriorated more rapidly when incubated in the light than in the
dark. Three days after fumigation, there were more visibly damaged leaves on plants incubated in the dark than on plants
incubated in light. This leads us to speculate that light must also
play a role in a repair mechanism. Recovery from free radical
damage and prevention of damage by free radicals are known to
involve thiol compounds (20), such as GSH. Light-dependent
reduction of oxidized glutathione in chloroplast is well known (12,
19). The concentration of GSH in chloroplast would increase
during illumination. Light appears to have a dual though counteractive role: it enhances cellular damage by accelerating the
radical-mediated oxidation of S02/bisulfite, but it also plays an
important role in providing necessary metabolites for repair or
recovery. The relative contribution of these two counteractive
mechanisms will thus be influenced by the level of S02/bisulfite
in the tissues. Further investigation is needed to test this hypothesis.
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Plant Physiol. Vol. 63, 1979
C2H4 AND C2H6 FROM S02-INJURED PLANTS
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