Journal of Experimental Botany, Vol. 49, No. 318, pp. 115–123, January 1998
The impact of ozone, isoprene and propene on
antioxidant levels in two leaf classes of
velvet bean (Mucuna pruriens L.)
N.J. Stokes1, G.M. Terry and C.N. Hewitt
Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK
Received 6 February 1997; Accepted 22 August 1997
Abstract
Introduction
Four-week-old velvet beans (Mucuna pruriens L.) were
fumigated with ozone, isoprene and propene both
singly and in combination (hydrocarbon+oxidant) in
controlled-environment chambers for a 4 week period.
Measurements were made of total soluble protein,
ascorbate, dehydroascorbate, lipid peroxidation, and
glutathione reductase activity in mature and young
velvet bean leaves.
Significant increases in soluble protein concentration with respect to the controls were found in plants
fumigated with propene and isoprene+ozone for
young leaves, and ozone only for mature leaves. The
analysis of ascorbate concentrations in young leaves
showed a significant increase in total ascorbate for
propene-fumigated plants, a significant decrease in
the level of the oxidized form (dehydroascorbate) for
ozone and isoprene+ozone-treated plants, and a significant increase in the reduced form for plants fumigated with propene, propene+ozone and isoprene+
ozone. The analysis of ascorbate levels in mature
leaves showed no significant effect from any fumigation regime.
When compared to control material the specific
activity of the enzyme glutathione reductase was found
to be significantly reduced in young leaves treated
with propene, isoprene+ozone and ozone alone.
However, in mature leaves this effect is lost and there
are no significant differences with respect to the
control.
The impact of various atmospheric pollutants on plant
physiology and biochemistry has been under investigation
for many years. Until very recently much of this work
was based on defining the impact of individual pollutants
in the absence of other compounds. Gas phase reactive
hydrocarbons, of biogenic and anthropogenic origins,
have been shown to cause deleterious effects in some
plants at concentrations as low as 10 ppbv (109 by vol.)
(Abeles and Heggestad, 1973). These compounds are also
important reactants in tropospheric chemistry. The oxidation of non-methane hydrocarbons (NMHC ) can result
in increased ozone levels, when emitted into a polluted
environment (Fehsenfeld et al., 1992). In addition to the
phytotoxicity of ozone, its oxidation of hydrocarbons can
result in the production of organic peroxides (ROO) and
hydroperoxides (ROOH ). These highly reactive species
have been implicated in plant damage, especially in those
plants which are emitters of biogenic alkenes (Hewitt and
Kok, 1991; Hewitt et al. 1990a, b).
The chemistry of the interactions between ozone and
NMHCs are not well understood, but some suggested
mechanisms are discussed by Salter and Hewitt (1992).
Experiments with the biogenic NMHCs ethylene and
isoprene show that ROOH can be formed in both the gas
and aqueous phases by reaction with ozone. Hewitt and
Kok (1991) have shown that the reaction of isoprene and
propene with ozone generates a hydroperoxide yield of
approximately 35% of the initial alkene. They identified
four reaction products, hydroxymethyl hydroperoxide
(dominant), methyl hydroperoxide, ethyl hydroperoxide,
and 2-hydroxypropyl hydroperoxide. An unknown product, believed to be a substituted hydroxybutyl hydroperoxide, was also detected. Further experiments with plants
Key words: Ozone, hydrocarbons, velvet bean, Mucuna
pruriens, antioxidants.
1 To whom correspondence should be addressed. Fax: +44 1524 382212. E-mail: [email protected]
© Oxford University Press 1998
116
Stokes et al.
that emit NMHCs have shown that ozone damage can
be reduced or prevented by treatments which inhibit the
production of endogenous hydrocarbons (Mehlhorn
et al., 1991; Mehlhorn and Wellburn, 1987). These observations support the hypothesis that hydroperoxides
and/or other reactive hydrocarbon oxidation products
produced by the reaction of ozone and biogenic hydrocarbons inside the plant cause damage. Therefore, it follows,
that the more stable hydroperoxides formed in the troposphere may be available for uptake by plants and that
these may then initiate damage.
Mathematical models of ozone diffusion into the leaf
and of rates of depletion by reaction with endogenous
hydrocarbons in the cell wall have been used to suggest
that only a small fraction of the total ozone flux reacts
with alkenes before reaching the plasmalemma
(Chameides, 1989). The validity of these calculations and
conclusions have been questioned by Salter and Hewitt
(1992).
These problems can be addressed by analysing experimentally the response of isoprene-emitting and nonemitting species to hydrocarbon and oxidant gas mixtures.
The velvet bean (Mucuna pruriens L.) is relatively unusual
because its leaves emit large quantities of isoprene between
approximately 5 and 21 days of age (Grinspoon et al.,
1991). Outside this age range the emission rate for
isoprene is zero for younger leaves and negligible for
older leaves. Propene is an anthropogenic alkene with a
significant UK mass emission and photochemical ozone
creation potential (Derwent and Jenkin, 1991).
Determining the biochemical response of these two leaf
classes (young: non-emitting and mature: emitting) to
fumigation with isoprene and propene, both singly and
in combination with ozone, allows the impact of endogenous hydrocarbons on oxidizing stress to be assessed.
Measurement of glutathione reductase activity, total
soluble protein levels, ascorbate concentrations, and lipid
peroxidation should enable the way in which hydrocarbons interact with the antioxidant mechanisms of plants
to be better understood.
Fumigation and growth conditions
A specialized fumigation facility which allows the growth of
plant material under controlled conditions (Stokes et al., 1993)
was used for the experiments. In brief, eight 1 m3 Teflon-lined
controlled-environment chambers are supplied with air via a
temperature/humidity control unit, which is then filtered
through a mixed (50/50) purafil/charcoal bed. All plants in all
treatments were maintained at constant growth conditions
(humidity, 50%; light intensity, 200 mmol m−2 s−1; daylength,
16 h; temperature, 23±0.5 °C day/17±0.5 °C night). Accurately
metered quantities of the gas phase pollutants are passed to the
individual chambers via Teflon lines and mass flow controllers
(MKS Instruments, UK ). The hydrocarbons, propene and
isoprene, were supplied from 1% hydrocarbon in nitrogen stock
cylinders (Linde Gases, UK ). The bottled gases have a shelf
life in excess of 12 months and are produced gravimetrically
using weights traceable to NPL standards. There are no
contaminants present that exceed the detection limits of the
anaytical instrumentation employed. Ozone was generated by
the high voltage dissociation of oxygen. The ozone/air stream
produced in this way was bubbled through a water trap to
remove nitrogen oxides. All air pollutant gases were added to
the purified air supply at a constant level of 100 ppbv for 6
h d−1. Stomatal conductances of 65 mmol m−2 s−1 and
110 mmol m−2 s−1 were commonly obtained for the young and
mature leaves, respectively (see also Grinspoon et al., 1991).
These values give approximate pollutant doses of
0.03 mg m−2 s−1 and 0.05 mg m−2 s−1 for the two age classes of
leaf (young and mature, respectively).
Fumigation chamber concentrations of ozone were monitored
by UV absorption using a Dasibi analyser (Environmental
Corp., Glendale, CA). Hydrocarbon levels were determined by
gas chromatography with flame ionization detection (GC-FID)
with thermal desorption (Perkin Elmer, UK ). Chamber air
samples were collected over 10 min periods at 100 ml min−1 on
tubes packed with Tenax TA (0.2 g) and Carbotrap (0.1 g).
Table 1 gives full details of the desorption and GC conditions
employed during this series of experiments.
Preparation of leaf extracts
At the end of the 4 week fumigation period samples of the two
leaf classes were removed from each plant for analysis. As the
harvesting process took >2 h (around midday) one replicate
plant from each treatment was harvested in turn until all
Table 1. Hydrocarbon desorption and analysis conditions
Desorption conditions
Primary desorption
Secondary (cold trap) desorption
Materials and methods
Plant material
Velvet beans (M. pruriens L.) were grown from seed in 3 l pots
using a multipurpose compost (Levington) incorporating a slow
release fertiliser (Ozmocote) at 2 g l−1. Nine replicate plants
were grown in individual pots for each treatment for 4 weeks
in control standard air prior to fumigation. Ten days after the
onset of fumigation a juvenile leaf on each plant was tagged to
provide assurance that the mature leaf harvested after 4 weeks
treatment was sufficiently young to be emitting significant
quantities of isoprene. Young non-emitting leaves were selected
from those between 2 and 4 days of age.
280 °C for 5 min to the cold
trap—Tenax at 30 °C
25 °C min−1 to 280 °C held
12 min
GC-FID conditions
Carrier gas
Column
Temperature programme
Detector
Temperature
Flame gases
Make-up gas
Helium (1–2 ml min−1)
PLOT (50 m×0.32 mm I.D.)
120 °C for 1 min, heated
3 °C min−1 to 165 °C, heated
45 °C min−1 to 200 °C, held for
10 min
Flame ionization
225 °C
Hydrogen+air
Nitrogen
Antioxidant levels in velvet bean 117
treatments had been completed. Mature leaves were divided
lengthways and each half weighed immediately. One half was
dried at 60 °C for 48 h to provide a fresh weight/dry weight
ratio and the other half frozen in liquid nitrogen. As a single
young leaf was too small for the determination of the fresh
weight/dry weight ratio and the biochemical analyses, one
trifoliate was selected from each replicate, one-third being used
for fresh weight/dry weight and the remainder for biochemical
assay.
The frozen leaf tissue was prepared by grinding in a chilled
mortar and pestle with a small quantity of sharp sand in 5 ml
of ice-cold phosphate buffer (100 mM ), containing 1 mM
EDTA (pH 7.5). The macerated tissue was separated by
centrifugation at 13 000 rpm for 2 min. All assays were then
carried out on the supernatant.
Biochemical analyses
Reduced ascorbate levels were determined spectrophotometrically at 525 nm after acidification and reaction with bipyridyl
and Fe3+ ions (Law et al., 1983). Sample cuvettes contained
0.45 ml plant extract, 0.3 ml trichloroacetic acid (TCA) (10%,
v/v), 0.3 ml phosphoric acid (44%, v/v), 0.3 ml bipyridyl (4%,
w/v) in 70% ethanol and 0.15 ml ferric chloride (3%, w/v).
Total ascorbate was measured by reduction of the oxidized
form, the concentration of the oxidized ascorbate being
calculated by the difference between total and reduced ascorbate.
Standards were prepared from commercially available ascorbic
acid (r2=0.997, n=12).
Glutathione reductase (EC 1.6.4.3) was determined spectrophotometrically at 412 nm by measuring the rate of the reaction
between glutathione and 5, 5∞ dithiobis 2-nitrobenzoic acid
(DTNB) (Akerboom and Sies, 1981; Smith et al., 1988). Each
analysis contained 0.1 ml plant extract, 0.1 ml NADPH
(3.6 mM ), 0.1 ml DTNB (7.6 mM ), 0.1 ml oxidized glutathione
(GSSG, 15 mM ), and 0.6 ml phosphate buffer (described
above). Purified GR from spinach was used to generate a
standard calibration (r2=0.997, n=7).
Lipid peroxidation was estimated spectrophotometrically at
532 nm by measurement of malondialdehyde (MDA) equivalents following acidification and reaction with thiobarbituric
acid (TBA) (Gutteridge and Halliwell, 1990). The reaction
volume comprised 1.0 ml plant extract, 1.0 ml TCA (20%, v/v)
and 2.0 ml TBA (0.67%, w/v). Calculation of the TBA reactive
equivalents in leaf extract was made via the molar absorbance
of MDA.
Total soluble protein levels (non-structural ) were determined
spectrophotometrically at 595 nm using the Bradford assay
(Bradford, 1976), the only variation being the use of a
commercially available concentrated protein indicator (Biorad,
UK ). A standard calibration curve was constructed using
bovine serum albumen (r2=0.984, n=5). Unless otherwise
stated all chemicals used in these methods were obtained from
the Sigma Chemical Co ( UK ).
All measurements were made using a Pharmacia Ultrospec
III UV/VIS spectrometer with a six position temperature
controlled cell unit. For all analyses this was maintained at
25 °C and kinetic analyses were performed using the manufacturer’s computer software package.
Statistical analysis
The significance of differences between points was determined
by analysis of variance and, if appropriate, a calculation of
least significant differences (LSD).
Results
The results of the total soluble protein determinations on
the two leaf classes are shown in Fig. 1. In young (nonemitting) leaves, treatment with propene alone or
isoprene+ozone produced a significant increase in levels
of total soluble proteins, with respect to the control and
isoprene alone. In the mature leaves, treatment with
ozone produced a significant increase in soluble protein
level over all treatments except isoprene+ozone. The
combination of isoprene+ozone also produced a significantly greater protein level than found in leaves treated
with just propene.
Glutathione reductase (GR) activity in the two leaf
classes from fumigated and control plants are shown in
Fig. 2. When these data are expressed in terms of the
specific activity, i.e. GR on a ‘per mg protein’ basis, the
activity of the enzyme in young leaves treated with
isoprene+ozone, ozone alone and propene alone is significantly lower than the enzyme activity in control material.
Fumigation of young leaves with isoprene also produced
a significantly greater specific activity than material
treated with isoprene+ozone and ozone alone. In mature
leaves, fumigation with ozone produced a significant
increase in the specific activity of GR compared to plants
fumigated with isoprene or propene alone.
The quantification of total ascorbate, reduced ascorbate
and dehydroascorbate in mature leaves proved inconclusive: no significant treatment effects were detected.
However, in young leaves a number of significant effects
were found and these are shown in Fig. 3. Fumigation
with propene alone produced a significant increase in
total ascorbate concentration compared to the control
and plants fumigated with isoprene alone. The quantification of reduced ascorbate levels shows that fumigation
with propene alone and isoprene+propene produced a
significant increase, with respect to the control material.
The concentration of reduced ascorbate in young leaves
treated with isoprene+ozone is also significantly higher
than in plants treated with isoprene alone. The measurement of dehydroascorbate shows that treatment with
ozone alone significantly reduces levels compared to all
other treatments, with the exception of isoprene+ozone.
The latter fumigation regime significantly reduces the
dehydroascorbate concentration when compared to treatment with both propene and isoprene alone.
Fumigation with isoprene produced a significant reduction (Fig. 4) in the fresh weight/dry weight ratio of young
leaves when compared to all other treatments, excepting
ozone alone. However these two fumigation regimes are
significantly different at P<0.10.
The analysis of lipid peroxidation via the TBA test was
inconclusive (data not shown): no significant effects were
detected in either age class for any treatment.
Examination of all plants in all treatments indicated
118
Stokes et al.
Fig. 1. Total soluble protein concentrations in extracts from young (non-emitting) and mature (isoprene emitting) velvet bean leaves fumigated
over a 4 week period with propene, isoprene and ozone, singly and in combination. Data represent mean value±standard error (n=9).
that fumigation had ceased prior to to the onset of
visible damage.
Discussion
These experiments show that fumigation with relatively
low concentrations of pollutant gases can produce quantifiable changes to biochemical processes. The levels of
hydrocarbons selected are within approximately one order
of magnitude of ambient urban levels for propene (DoE,
1993) and rural levels for isoprene (Fehsenfeld et al.,
1992). It has been shown that the emission of isoprene
from broadleaved species occurs via the stomata, but that
stomatal conductance has a limited impact on the rate of
emission (Fall and Monson, 1992). This results from an
increased intercellular isoprene concentration when
stomata close that negates the impact of decreased
stomatal conductance. In this instance the internal isoprene concentration may increase from approximately
70 ng g−1 dry weight to in excess of 800 ng g−1 dry weight
( Fall and Monson, 1992). This indicates that the concentration of externally applied isoprene in these experiments
is likely to be below the levels naturally found within
mature leaves. The isoprene emission rate from the mature
leaves of the velvet bean has been measured and found
to be approximately 1.5 nmol m−2 s−1. The concentration
Antioxidant levels in velvet bean 119
Fig. 2. Glutathione reductase specific activity (units per mg protein) in extracts from young (non-emitting) and mature (isoprene emitting) velvet
bean leaves fumigated over a 4 week period with propene, isoprene and ozone, singly and in combination. Data represent mean value±standard
error (n=9).
of ozone employed (100 ppbv) represents a high, but not
excessive, summertime level often found in the UK
(PORG, 1993).
The experiments described here show that fumigation
of velvet beans with isoprene, propene and ozone, both
singly and in combination, produces a significant increase
in the concentration of total soluble protein. It is not
possible to determine from the data obtained whether the
increased levels represent the production of ‘stress’
proteins or compounds that act in a protective role.
Given the quantities of protein concerned it is most
likely that ribulose bisphosphate-carboxylase oxygenase
( RUBISCO) is implicated. As these elevated levels are
found in young, photosynthetically competent velvet bean
leaves (Grinspoon et al., 1991), this may represent a shift
in emphasis from the mature leaves, brought about by
the onset of premature senescence.
What is unclear is how the apparent stress in leaves
fumigated with just propene develops. A similar response
to this gas has been detected in our laboratory using wild
cherry (Prunus avium L.) seedlings ( Terry et al., 1995a).
Interestingly, whilst the response of young leaves to
propene and ozone are approximately equivalent the
effect is reversed in mature leaves. The biogenic reactive
hydrocarbon, isoprene, behaves differently to both ozone
and propene. This may be related to this compound’s
link with other important physiological processes, for
example, leaf protection under changing temperatures
(Sharkey, 1996). It has been found previously that isoprene promotes flowering in a number of different plant
species at concentrations equivalent to those used here,
which will undoubtedly impact on the biochemical processes occurring ( Terry et al., 1995b).
The developing picture of the way velvet beans respond
to the pollutant regimes employed is further confused by
the measurements of GR activity. If the increased protein
levels are a consequence of the production of protective
compounds one would expect to see a rise in GR activity
120
Stokes et al.
Fig. 3. Total, reduced and dehydroascorbate concentrations in young (non-isoprene emitting) velvet bean leaves fumigated over a 4 week period
with propene, isoprene and ozone, singly and in combination. Data represent mean value±standard error (n=9).
(Schmieden et al., 1993; Creissen et al., 1994). However,
this is not the case in young leaves fumigated with
isoprene alone and in combination with ozone. In mature
leaves, exposure to ozone produces the expected rise in
GR activity. Rao et al. (1995) have shown that prolonged
ozone exposure (5 weeks) produces an initial increase in
antioxidant activity, which lasts for 2 weeks in wheat, but
that this is followed by inactivation. This may indicate
the onset of senescence following the point at which the
antioxidant systems have been overrun by the flux of
ozone and its breakdown products. A similar situation
may have occurred in the velvet bean and analysis of GR
activity at an earlier time might have produced quite
different results.
Antioxidant levels in velvet bean 121
Fig. 4. Fresh weight/dry weight ratios from young (non-isoprene emitting) velvet bean leaves fumigated over a 4 week period with propene, isoprene
and ozone, singly and in combination. Data represent mean value±standard error (n=9).
It would be expected that any fumigation with oxidizing
species which produces a response from GR would also
produce some variation in ascorbate levels. However, this
was not observed in the mature velvet bean leaves and
cannot easily be explained. Alteration to the apoplastic
and whole leaf/needle ascorbate levels in response to
ozone has been reported in a number of differing species
(Luwe and Heber, 1995; Polle et al., 1995).
In young non-emitting leaves, pollutant fumigation
produced quite a marked response. Fumigation with
isoprene+ozone and ozone alone produced an ~20%
reduction in the level of dehydroascorbate, with respect
to the control. Polle and Junkermann (1994) suggest that
the apoplastic space contains between 4% and 20% of the
total ascorbate within a leaf. This would suggest that the
pollutant mixtures employed have impinged on biochemical processes within the symplastic space. The reduction
in the concentration of oxidized ascorbate in conjunction
with elevated levels of reduced ascorbate and a reduction
in the activity of GR lends some weight to this
proposition. The catalytic behaviour of GR is considered
to be the rate-limiting step within the Foyer and Halliwell
antioxidant cycle ( Foyer and Halliwell, 1976; Jablonski
and Anderson, 1981), which may account for the imbal-
122
Stokes et al.
ance in the ascorbate redox states. The addition of
propene to ozone appears to limit the impact of the latter,
both on ascorbate redox state and GR activity.
The data obtained from the thiobarbituric acid analyses
indicate that the pollutant regimes employed do not
impact on the plasmalemma in either leaf age class. This
would appear to suggest that the effects detected are
occurring within the apoplastic space. This does not
include consideration of the variable rate at which the
various antioxidant systems respond to pollutant-induced
stress. As far as we are aware, there are no data available
concerning the rate of response of antioxidant systems in
velvet beans. It would not be appropriate to consider
data obtained from other plant species as their response
to stress can vary markedly. For example, Wellburn and
Wellburn (1996) have shown that short-term ozone
fumigations of a range of plant species produces quite
different responses to the level of total ascorbate.
Similarly, it is inappropriate to consider comparison of
these data with those obtained from the treatment of
other plants with similar hydrocarbons, such as ethene.
In conclusion, the data obtained here suggest that the
impact of oxidizing air pollutants on plants can be
significantly altered by the type of plant species (i.e.
isoprene emitter or non-emitter) and the presence and
type of hydrocarbon. Much more data will be required
before it is possible to predict the likely effects of pollutant
mixtures on plant species.
Acknowledgements
The authors gratefully acknowledge financial support from the
Natural Environment Research Council (Grant: GR3/09739)
for N.J.S. We thank T.A. Mansfield for his comments and
encouragement.
References
Abeles FB, Heggestad HE. 1973. Ethylene—urban air pollutant.
Air Pollution Control Association Journal 51, 1082.
Akerboom TPM, Sies H. 1981. Assay of glutathione disulphide
and glutathione mixed disulphides in biological samples.
Methods in Enzymology 77, 373–82.
Bradford MM. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein–dye binding. Analytical Biochemistry
72, 248–54.
Chameides WL. 1989. The chemistry of ozone deposition to
plant leaves: role of ascorbic acid. Environmental Science and
Technology 23, 595–600.
Creissen GP, Broadbent P, Kular B, Reynolds H, Wellburn AR,
Mullineaux PM. 1994. Manipulation of glutathione-reductase
in transgenic plants: implications for plants responses to
environmental-stress. Proceedings of the Royal Society of
Edinburgh (section b-biological sciences) 102, 167–75.
Derwent RG, Jenkin ME. 1991. Hydrocarbons and the longrange transport of ozone and PAN across Europe.
Atmospheric Environment 25A, 1661–78.
DoE. 1993. Urban air quality in the United Kingdom. First
report of the Quality of Urban Air Group. Prepared at the
request of the UK Department of the Environment, Marsham
St, London, UK.
Fehsenfeld F, Calvert J, Fall R, Goldan AB, Hewitt CN, Lamb
B, Shaw L, Trainer M, Westberg H, Zimmerman P. 1992.
Emissions of volatile organic compounds from vegetation
and the implications for atmospheric chemistry. Global
Biogeochemical Cycles 6, 389–430.
Fall R, Monson RK. 1992. Isoprene emission rate and intercellular isoprene concentration as influenced by stomatal distribution and conductance. Plant Physiology 100, 987–92.
Foyer HC, Halliwell B. 1976. The presence of glutathione and
glutathione reductase in chloroplasts. A proposed role in
ascorbic acid metabolism. Planta 133, 21–5.
Grinspoon J, Bowman WD, Fall R. 1991. Delayed onset of
isoprene emission in developing velvet bean (Mucuna sp.)
leaves. Plant Physiology 97, 170–4.
Gutteridge JMC, Halliwell B. 1990. The measurement of lipid
peroxidation in biological systems. Trends in Biological
Science 15, 129–35.
Hewitt CN, Kok GL. 1991. Formation and occurrence of
organic hydroperoxides in the troposphere: laboratory and
field observations. Journal of Atmospheric Chemistry 12,
181–94.
Hewitt CN, Kok GL, Fall R. 1990a. Hydroperoxides in plants
exposed to ozone mediate air pollution damage to alkene
emitters. Nature 344, 56–8.
Hewitt CN, Lucas P, Wellburn AR, Fall R. 1990b. Chemistry of
ozone damage to plants. Chemistry and Industry August
1990, 478–81.
Jablonski PP, Anderson JW. 1981. Light-dependent reduction
of dehydroascorbate by ruptured pea chloroplasts. Plant
Physiology 67, 1239–44.
Law MY, Charles SA, Halliwell B. 1983. Glutathione and
ascorbic acid in spinach (Spinacia oleracea) chloroplasts.
Biochemical Journal 210, 899–903.
Luwe M, Heber U. 1995. Ozone detoxification in the apoplasm
and symplasm of spinach, broad bean and beech leaves at
ambient and elevated concentrations of ozone in air. Planta
197, 448–55.
Mehlhorn H, O’Shea JM, Wellburn AR. 1991. Atmospheric
ozone interacts with stress ethylene formation by plants to
cause visible plant injury. Journal of Experimental Botany
42, 17–24.
Mehlhorn H, Wellburn AR. 1987. Stress ethylene formation
determines plant-sensitivity to ozone. Nature 327, 417–18.
Polle A, Junkermann W. 1994. Does hydrogen peroxide
contribute to damage to forest trees? Environmental Science
and Technology 28, 812–15.
Polle A, Wieser G, Haveranek WM. 1995. Quantification of
ozone influx and apoplastic ascorbate content in needles of
Norway spruce trees (Picea abies L., Karst) at high altitude.
Plant, Cell and Environment 18, 681–8.
PORG. 1993. Ozone in the United Kingdom. Third report of the
Photochemical Oxidants Review Group prepared at the
request of the Air Quality Division, Department of the
Environment, Marsham St., London, UK.
Rao MV, Hale BA, Ormrod DP. 1995. Amelioration of ozoneinduced oxidative damage in wheat plants grown under high
carbon dioxide. Plant Physiology 109, 421–32.
Salter L, Hewitt CN. 1992. Ozone–hydrocarbon interactions in
plants. Phytochemistry 31, 4045–50.
Schmieden U, Schneider S, Wild A. 1993. Glutathione status
and glutathione reductase activity in spruce needles of healthy
Antioxidant levels in velvet bean 123
and damaged trees at two mountain sites. Environmental
Pollution 82, 239–44.
Sharkey TD. 1996. Emission of low molecular mass hydrocarbons from plants. TIPS 1, 78–82.
Smith IK, Vierheller TL, Thorne CA. 1988. Assay of glutathione
reductase in crude tissue homogenates using 5,5∞dithiobis(2-nitrobenzoic acid ). Analytical Biochemistry 175,
408–13.
Stokes NJ, Lucas PW, Hewitt CN. 1993. A controlled
environment closed-chamber facility for studies into the
effects of reactive hydrocarbons on plant growth and
physiology. Atmospheric Environment 27A, 679–83.
Terry GM, Stokes NJ, Lucas PW, Hewitt CN. 1995a. Effects
of reactive hydrocarbons and hydrogen peroxide on antioxidant activity in cherry leaves. Environmental Pollution 88,
19–26.
Terry GM, Stokes NJ, Hewitt CN, Mansfield TA. 1995b.
Exposure to isoprene promotes flowering in plants. Journal
of Experimental Botany 46, 1629–31.
Wellburn FAM, Wellburn AR. 1996. Variable patterns of
antioxidant protection but similar ethene emission differences
in several ozone-sensitive and ozone-tolerant plant selections.
Plant, Cell and Environment 19, 754–60.