Protection by isoprene against singlet oxygen in leaves.

Abstract

Isoprene (2-methyl-1,3-butadiene) protection against effects of singlet oxygen was investigated in Myrtus communis and Rhamnus alaternus. In M. communis, singlet oxygen produced in the leaves by Rose Bengal (RB) led to a 65% decrease in net assimilation rates within 3 h, whereas isoprene emission rates showed either a 30% decrease at ambient CO2 concentrations or a 70% increase under high CO2. In both cases, these changes led to an increase in calculated internal isoprene concentrations. The isoprene protection effect was directly demonstrated by fumigation of young (non-emitting) leaves, treated with RB or bromoxynil (simulating photoinhibition). There was 42% and 29% reduction in the damage to net assimilation compared with non-fumigated leaves for RB or bromoxynil, respectively. In R. alaternus, similar effects of RB on net assimilation were observed, and additional fluorescence measurements showed a significantly smaller decrease in Fv/Fm in isoprene-fumigated young leaves treated with RB (from 0.78 to 0.52), compared with non-fumigated leaves (from 0.77 to 0.27). The internal isoprene concentrations used in this study and possible rate of 1O2 production in leaves indicate that the protective effects observed should be beneficial also under natural conditions.

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Protection by Isoprene against Singlet Oxygen in Leaves
Hagit P. Affek and Dan Yakir*
Department of Environmental Sciences and Energy Research, Weizmann Institute of Science,
Rehovot 76100, Israel
Isoprene (2-methyl-1,3-butadiene) protection against effects of singlet oxygen was investigated in Myrtus communis and
Rhamnus alaternus. In M. communis, singlet oxygen produced in the leaves by Rose Bengal (RB) led to a 65% decrease in net
assimilation rates within 3 h, whereas isoprene emission rates showed either a 30% decrease at ambient CO2concentrations
or a 70% increase under high CO2. In both cases, these changes led to an increase in calculated internal isoprene
concentrations. The isoprene protection effect was directly demonstrated by fumigation of young (non-emitting) leaves,
treated with RB or bromoxynil (simulating photoinhibition). There was 42% and 29% reduction in the damage to net
assimilation compared with non-fumigated leaves for RB or bromoxynil, respectively. In R. alaternus, similar effects of RB
on net assimilation were observed, and additional fluorescence measurements showed a significantly smaller decrease in
Fv/Fmin isoprene-fumigated young leaves treated with RB (from 0.78 to 0.52), compared with non-fumigated leaves (from
0.77 to 0.27). The internal isoprene concentrations used in this study and possible rate of1O2production in leaves indicate
that the protective effects observed should be beneficial also under natural conditions.
Isoprene (2-methyl-1,3-butadiene) is emitted by a
variety of plant species (Harley et al., 1999; Kes-
selmeier and Staudt, 1999). It influences the trace-gas
composition of the troposphere by reacting with OH
radicals and NOXto generate tropospheric ozone
(Trainer et al., 1987; Chameides et al., 1988; Ryerson
et al., 2001). Isoprene also affects the oxidizing capac-
ity of the atmosphere by scavenging OH radicals
(Jacob and Wofsy, 1988) and may indirectly influence
atmospheric methane accumulation. Models predict-
ing ozone concentrations such as that of Trainer et al.
(1987) use emission rates and inventories of isoprene,
as well as other volatile organic compounds, and
must consider physiological and environmental in-
fluences on emission rates.
Isoprene production can consume a few percent of
the carbon fixed in photosynthesis but, despite much
research, the role of isoprene emission is not fully
understood. It was suggested to be protection against
sharp temperature increases by dissolving in the thy-
lakoid membranes and stabilizing hydrophobic inter-
actions (Sharkey and Singsaas, 1995; Singsaas et al.,
1997; Sharkey et al., 2001). However, this effect was
not observed in all studies (Logan and Monson,
1999). Isoprene was also suggested to potentially pro-
vide a more general protection against stress condi-
tions (Sharkey and Loreto, 1993) and in particular
against photooxidative stress (Zeidler et al., 1997)
and was recently shown to protect leaves against
ozone (Loreto et al., 2001; Loreto and Velikova, 2001).
Other alkenes were also found to increase thermotol-
erance in leaves (Sharkey et al., 2001). The well-
known ability of alkenes, such as isoprene, to react
with singlet oxygen (1O2), ozone and OH radicals led
us to test the hypothesis that isoprene may help to
protect leaves against oxidative stress by reacting
with1O2and other radicals.
Singlet oxygen is produced in leaves by interaction
of molecular oxygen with triplet state chlorophyll,
which is formed under conditions of excessive exci-
tation. This may occur under high-light intensities or
because of environmental stress that limits the use of
the absorbed sunlight (Demmig-Adams, 1990). Here,
we used Rose Bengal (RB) as a photosensitizer that
produces
herbicide bromoxynil (BX) that increases the sensi-
tivity of PSII to light, thus, producing1O2through
photoinhibition (Krieger-Liszkay and Rutherford,
1998).
In this study, we used the Mediterranean shrubs
Myrtus communis and Rhamnus alaternus. Both are
evergreen shrubs that grow in northern Israel and
throughout the Mediterranean region (Heler and
Livneh, 1982). M. communis is a high isoprene emitter
(Hansen et al., 1997) and emits only small amounts of
monoterpenes (Owen et al., 1997). To our knowledge,
there is no information of emission from R. alaternus,
however, two other Rhamnus spp. are known to emit
isoprene (Kesselmeier and Staudt, 1999).
1O2upon absorbing green light and the
RESULTS
Isoprene Emission from M. communis
To characterize isoprene emission from M. commu-
nis, we examined rates of emission, seasonality, phe-
nology, and CO2response. Large seasonal variations
were observed in isoprene emission rates (Fig. 1a)
with maximal rates during the summer and autumn
* Corresponding author; e-mail dan.yakir@weizmann.ac.il; fax
972–8–9344124.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010909.
Plant Physiology, May 2002, Vol. 129, pp. 269–277, www.plantphysiol.org © 2002 American Society of Plant Biologists269

Page 2

(up to 30 nmol m?2s?1) and close to detection limit
(approximately 0.2 nmol m?2s?1) in winter. Only
small changes in net assimilation rates were observed
over the same time (Fig. 1a).
Net assimilation and isoprene emission rates were
measured in branches that developed from leaves
marked at “age zero” (approximately 5 mm2). Both
net assimilation and isoprene emission rates in-
creased with leaf age up to 2 months (Fig. 1b). Iso-
prene emission was first detected, however, only
about 1 week later than net CO2uptake in both sun
and shade leaves. The lack of isoprene emission in
young leaves was useful in fumigation experiments
(see below). Throughout the experiment, both net
assimilation and isoprene emission rates were higher
in sun leaves than in shade leaves.
The short-term response of net CO2assimilation
and isoprene emission rates to changes in the inter-
cellular CO2concentration (ci) was measured at two
leaf temperatures (26 ? 0.5°C and 34 ? 0.5°C). Emis-
sion rates were relatively constant at civalues be-
tween 20 and 300 ?L L?1, whereas net assimilation
rates increased with ci(Fig. 2). At civalues of approx-
imately 300 to 900 ?L L?1, isoprene emission rates
decreased by 55% to 75% (at 26°C), whereas net
assimilation appeared to be CO2saturated. The de-
crease of isoprene emission rates at high civalues
was much smaller at 34°C, and isoprene emission
rates decreased only by approximately 25% between
300 and 1,000 ?L L?1(Fig. 2b).
Effects of Singlet Oxygen in M. communis
RB was used as a sensitizer to produce1O2under
light, and its effect on net assimilation and isoprene
emission rates was clearly concentration dependent
(Fig. 3). Treatments with RB at ambient CO2concen-
tration resulted in a rather fast decrease in both net
assimilation and isoprene emission rates (Figs. 3 and
4a), whereas lesions on the leaves were observed
only after 2 d. In control plants (no RB), net assimi-
lation and isoprene emission rates remained rela-
tively constant throughout the measurement day
(Fig. 3).
An important check was made by repeating the
treatment and control measurements using light fil-
tered through purple zelofan but maintaining the
same total photosynthetically active radiation at the
leaf level (the zelofan filter had a broad absorption
peak around ?max ? 567 nm, as compared with
?max ? 547 nm of RB). The filter prevented most of
the photochemical formation of1O2by RB, and no
effect of RB on net assimilation and isoprene emis-
sion rates was observed in this case. Net assimilation
rates after 2.5 h of RB feeding were 36% of control
with no filter (Fig. 4) but 70% of control with the
filter. The decrease in net assimilation with filter was
similar to that observed in untreated leaves. The
Figure 1. Seasonal variations (a) and effects of branch age (b) on net assimilation (A) and isoprene emission rates (Is) from
a shrub of M. communis. Measurements were performed at leaf temperature of 26°C and light intensity of 1,000 ?mol m?2
s?1. The seasonal variations were measured at intercellular CO2concentration (ci) values between 450 and 500 ?L L?1, and
the effect of branch age was measured at ambient CO2concentrations (approximately 350 ?L L?1).
Figure 2. Effects of cion net assimilation (a) and isoprene emission
rates (b) from branches of M. communis measured at 26°C (circles;
two different branches) and 34°C (triangles) and light intensity of
1,000 ?mol m?2s?1.
Affek and Yakir
270 Plant Physiol. Vol. 129, 2002

Page 3

results confirmed that the effect of RB was via the
photochemical reaction that yields1O2, and not, for
example, by a chemical poisoning.
Comparing the time response of isoprene emission
and net assimilation rates showed that, at ambient
CO2concentrations, the decrease in isoprene emis-
sion rates was delayed by 1 to 2 h, and was smaller in
magnitude, relative to that in net assimilation (Fig.
4a). As a result, the fraction of fixed C allocated to
isoprene production increased from 0.5% to 1.1%
within 3 h, as did estimated isoprene concentration in
the leaf airspaces (Fig. 4c; calculated as by Singsaas et
al. [1997]; note that consideration of stomatal patch-
iness may have small effect on our results). At high
CO2concentrations, net CO2assimilation rates began
to decrease approximately 1.5 h after the beginning
of RB feeding, but, in contrast to ambient CO2, iso-
prene emission rates either did not change or in-
creased (Fig. 4b).
At high CO2concentrations, the response of iso-
prene emission to RB treatments was well correlated
with changes in ci. In untreated leaves, isoprene
emission normally decreased with increasing ci(at
26°C; Fig. 2); whereas in RB-treated leaves, a decrease
in ciwith time, because of RB effect, led to an increase
in isoprene emission rates (comparison of absolute
emission rates between Figs. 2 and 4 is difficult be-
cause of large differences among the different
branches used in the different ciexperiments). This
was most apparent over the seasonal cycle. During
autumn and summer, cidecreased and isoprene
emission rates increased in response to RB treat-
ments (Fig. 4, b and d). In contrast, during spring,
both ciand isoprene emission rates did not change
(data not shown). Furthermore, in an RB treatment
in autumn at 34°C and high CO2concentration, a
large decrease in net assimilation and in cibut no
change in isoprene emission rates were observed
(data not shown). This is consistent with the notion
of reduced effect of cion isoprene emission rate at
elevated temperatures (Fig. 2b).
Isoprene Fumigation in M. communis
The possibility that isoprene may provide protec-
tion against1O2damage was further tested by iso-
prene fumigation of young, non-emitting branches of
M. communis treated by RB or BX. In both isoprene-
fumigated and non-fumigated young leaves, RB
treatment (0.4 ?m) led to a decrease in net assimila-
tion rates. However, in isoprene-fumigated leaves
(1–2 ?L L?1), the decrease in net assimilation was
one-half that of non-fumigated leaves (Table I).
Higher isoprene concentration (4–5 ?L L?1) did not
increase the protection effects, and similar treatments
in mature (isoprene-emitting) leaves showed no clear
fumigation effects.
RB may produce
tions that do not occur naturally. A more natural
cause for1O2production in the leaves is photoinhi-
bition, during which the rate of absorption of pho-
tons is higher than the rate of utilization of the exci-
tation energy. This leads to formation of triplet state
chlorophyll that reacts readily with oxygen, yielding
1O2(Demmig-Adams, 1990). To better simulate pho-
toinhibition and
PSII, we repeated the isoprene fumigation experi-
ments in leaves treated with the herbicide BX (50
?m). BX increases the sensitivity of PSII to light,
leading to production of
fumigated leaves, the treatment resulted in approxi-
mately 50% decrease in net assimilation rates within
4 h (beginning 1.5–2 h after feeding). Isoprene-
fumigated leaves (1–15 ?L L?1) showed approxi-
mately 30% smaller decrease in net assimilation due
to BX (Table I).
1O2at concentrations and loca-
1O2production in the vicinity of
1O2near PSII. In non-
Isoprene Fumigation in R. alaternus
The isoprene
tested in a different isoprene-emitting species, R. alat-
ernus. Similar fumigation experiments were carried
out with young shade leaves treated with RB (0.1
?m). Young leaves of R. alaternus emitted small
amounts of isoprene, and isoprene concentrations
used in fumigation were up to 20 ?L L?1. In non-
1O2protection effect was further
Figure 3. Effects of different concentration of RB in the feeding
solution on net assimilation (a) and isoprene emission rates (b) of M.
communis at leaf temperature of 26°C, light intensity of 1,000 ?mol
m?2s?1, and civalues of 200 to 250 ?L L?1. The vertical line
indicates the beginning of RB feeding. Values were normalized to the
average before RB feeding.
Singlet Oxygen Protection by Isoprene
Plant Physiol. Vol. 129, 2002271

Page 4

fumigated leaves, net assimilation decreased signifi-
cantly and with increasing rates of decrease over time
(i.e. increase in the slope of the time response curve)
to 0.53 ? 0.02 of control after 4 h (n ? 4). In isoprene-
fumigated leaves, net assimilation rates decreased to
0.66 ? 0.05 of control (n ? 4; Fig. 5; the difference
betweenisoprene-fumigated
leaves was significant at the P ? 0.04 level). Interest-
ingly, there was a consistent difference in response
between isoprene-fumigated
leaves even before RB feeding, in which non-
fumigated leaves of R. alaternus consistently showed
a decrease in net assimilation rates from the onset of
theexperiment,whereas
leaves, net assimilation rates began decreasing only
after RB feeding (Fig. 5).
Isoprene protection in R. alaternus was also clearly
observed in chlorophyll fluorescence measurements
(Fv/Fm, an indicator of PSII efficiency, compared be-
fore and after each RB treatment experiment). Fv/Fm
andnon-fumigated
and non-fumigated
inisoprene-fumigated
decreased
leaves than under isoprene fumigation (from 0.765 ?
0.010 to 0.274 ? 0.027, n ? 5; and from 0.778 ? 0.003
to 0.520 ? 0.069, n ? 4, in non-fumigated and
isoprene-fumigated leaves, respectively; P ? 0.006).
Slightly better recovery of Fv/Fmafter 24 h in low
light was observed in isoprene-fumigated leaves
(Fv/Fmafter 24 h was 0.700 ? 0.020 and 0.639 ? 0.016
in isoprene-fumigated and non-fumigated leaves, re-
spectively; P ? 0.03).
significantlymore innon-fumigated
DISCUSSION
Characteristics of isoprene emission in M. commu-
nis reported here are consistent with reports on other
plants (Sharkey et al., 1991; Sharkey and Loreto, 1993;
Harley et al., 1994; Monson et al., 1994; Schnitzler et
al., 1997). Such characteristics included pronounced
seasonality, absence of isoprene emission in young
leaves, delayed onset of emission with respect to
Figure 4. Effects of singlet oxygen in leaves of
M. communis under ambient CO2concentra-
tion (approximately 350 ?L L?1; a and b) and
high CO2concentration (approximately 800 ?L
L?1; c and d) on net assimilation and isoprene
emission rates (a and c) and on changes in the ci
and isoprene (Isopi; b and d) at leaf temperature
of 26°C and light intensity of 1,000 ?mol m?2
s?1. The vertical line indicates the beginning of
RB feeding.
Table I. Effect of isoprene fumigation on net assimilation rates (A) after 4 h of1O2treatment in young (non-emitting) leaves of M. communis
Data are presented as the percentage of A left after the treatment. Also presented is fumigation effect on percentage of A, as the ratio between
consecutive fumigation and non-fumigation experiments. Light intensity was 1,000 ?mol m?2s?1and leaf temperature was 26°C. Exogenous
isoprene was added to give isoprene concentrations of 1 to 5 and 1 to 15 ?L L?1in the leaf air spaces in the RB and BX experiments, respectively.
The fumigation effect was significant at P ? 0.005 for RB and P ? 0.025 for BX (one tailed paired Student’s t test). Data represent results of two
shrubs each in the RB and BX experiments.
Rose Bengal (0.4 ?M)Bromoxynil (50 ?M)
Fumigation
87
79
109
92 ? 9
No fumigation
64
49
84
66 ? 10
Ratio
1.36
1.61
1.30
Fumigation
66
62
57
62 ? 3
No fumigation
43
49
54
49 ? 3
Ratio
1.53
1.27
1.53
Average ? SE
1.42 ? 0.10 1.29 ? 0.14
Affek and Yakir
272Plant Physiol. Vol. 129, 2002

Page 5

photosynthetic activity, and higher activity of sun
leaves. The effect of cion rates of net assimilation and
isoprene emission at both high and moderate tem-
peratures is similar to that observed in aspen and red
oak (Monson and Fall, 1989; Loreto and Sharkey,
1990). This was related to ATP limitation at high ci
and moderate temperatures, which is reduced at high
temperatures (Loreto and Sharkey, 1990). However,
no decrease in isoprene emission rates was observed
in M. communis at CO2free air (ciof approximately 20
?L L?1) for extended time periods, similar to obser-
vations in aspen but not in red oak.
The results presented here show for two plant spe-
cies and using two
isoprene can reduce oxidative damage to the photo-
synthetic apparatus. The beneficial effects are evident
in the reduced effect of1O2on net assimilation rates
and on PSII fluorescence yield (Fv/Fm) in the pres-
ence of isoprene.
In discussing the isoprene protection effects ob-
served in this study, it is important to consider the
relevance of the experimental results to natural con-
ditions. For this purpose, we consider the concentra-
tions used, the production rates of isoprene as com-
pared with possible production rates of1O2in leaves,
and other sources for oxidative stress such as O3and
water stress.
A first indirect indication for the usefulness of iso-
prene in protection against1O2is an increase in in-
ternal concentration during1O2stress. Using RB as a
photosensitizer, we observed a decrease in net assim-
ilation and in stomatal conductance. Markedly
smaller reduction in isoprene emission than in net
assimilation rates (at ambient CO2) or even enhance-
ment (at high CO2) was observed. As a consequence,
the plant apparently invested more of the fixed car-
bon in isoprene formation, and the reduction in sto-
1O2production methods that
matal conductance led to an increase in isoprene
concentrations in the leaf air spaces during stress.
Such increases in intercellular isoprene concentra-
tions may indicate enhancement of isoprene potential
to scavenge1O2.
More directly, isoprene protection against1O2was
observed in isoprene-fumigated young leaves (little
or no endogenous isoprene emission; Table I; Fig. 5).
Fumigation experiments are physiologically more
relevant if the concentration of isoprene used for
fumigation is consistent with concentrations that oc-
cur naturally within the leaf air spaces. The concen-
trations we used for fumigation (1–20 ?L L?1) were
higher than that occurring regularly in leaves of M.
communis but were not unusual in mature stressed
leaves (Fig. 4) and in leaves of other plants such as
white oak and kudzu (?10 ?L L?1; Singsaas et al.,
1997). We, therefore, concluded that isoprene concen-
trations that are expected in the leaf air spaces of
isoprene-emitting leaves, are effective in reacting
with and scavenging1O2, as observed under the ex-
perimental conditions.
In addition to physiologically relevant concentra-
tions, it also seems that potential rates of1O2scav-
enging by isoprene are physiologically relevant. Al-
though detailed evidence is lacking, results from
isolated PSII reaction centers show that approxi-
mately 30% of excitation yields3P680, and most of it
is reflected as1O2(Durrant et al., 1990; Telfer et al.,
1994). In intact leaves, however, about 85% of the
excitation of the reaction center leads to photochem-
istry (Papageorgiou, 1975), and in this case, one can
assume that only a few percent of the excitation
could yield
production of1O2in the chloroplasts should, at least
a priori, be comparable with the production of iso-
prene that can constitute up to a few percent of the
carbon assimilation.
The fumigation experiments reported here seemed
to be more efficient with RB than with BX. This, in
fact, is consistent with the expected isoprene concen-
tration gradients from a source in the atmosphere to
the chloroplasts. RB-produced
spread across the leaf, and an external source of
isoprene would be more efficient in reacting with it
than with1O2produced specifically near PSII (i.e. by
BX). Under natural conditions, however, a gradient
in the opposite direction is likely to exist. In this case,
isoprene production is in the chloroplasts, where
protection against photoinhibition (simulated here
by BX treatment) is expected to be more efficient.
Isoprene protection against1O2was not limited to
M. communis, and similar effects were observed in
young leaves of R. alaternus. Interestingly, non-
fumigated leaves of this plant consistently showed a
decrease in net assimilation rates even before RB
treatment. This was probably due to exposure of
shade-adapted leaves to high light intensity during
measurements. Our interpretation is supported by
1O2. Therefore, the naturally occurring
1O2is expected to
Figure 5. Effect of RB (0.1 ?M) on net assimilation rates (full symbols)
and chlorophyll fluorescence yield (Fv/Fm; white symbols) in young
shade leaves of R. alaternus at leaf temperature of 26°C, light inten-
sity of 1,000 ?mol m?2s?1. The vertical line indicates the beginning
of RB feeding. The fumigation effect was significant at P ? 0.04 and
P ? 0.006 in net assimilation and fluorescence, respectively. The
error bars of Fv/Fmbefore the RB treatment are smaller than the
symbols.
Singlet Oxygen Protection by Isoprene
Plant Physiol. Vol. 129, 2002273

Page 6

the results that fumigation always prevented this
effect, apparently protecting the leaves against pho-
toinhibition. We, therefore, speculate that the results
show, in fact, two levels of protection before and
after RB feeding.
Increased thermotolerance and ozone protection by
isoprene were suggested to be achieved through
strengthening of the thylakoid membranes (Loreto et
al., 2001; Sharkey et al., 2001) and preventing peroxi-
dation of membrane lipids (Loreto and Velikova,
2001). In R. alaternus, the fluorescence yield (Fv/Fm)
indicated significantly lower damage to the photosyn-
thetic apparatus in isoprene-fumigated as compared
with non-fumigated leaves. This also suggest that iso-
prene may protect the photosynthetic apparatus
embedded in the thylakoid membranes, but, whereas
the actual cause for the thermal or ozone damage is
not clear, here, it was directly related to1O2.
Although not the only protection mechanism avail-
able, isoprene may have some specific advantages.
For example, chloroplast membranes are highly sen-
sitive to photooxidative damage that occurs because
of excessive light intensity leading to1O2production.
The extent of this damage/protection is related to the
amount of ?-carotene bound to the PSII reaction cen-
ters (Telfer et al., 1994). In that sense, isoprene may
provide a more dynamic protection mechanism, con-
sidering the increase in isoprene emission rates with
light intensities (Guenther et al., 1993). This enables
the plant to rapidly raise the protection level when
needed and before changes in carotenoid synthesis
are effective.
The products of reactions between isoprene and
1O2are likely to be toxic hydroperoxides similar to
those produced in reactions with O3(Mehlhorn and
Wellburn, 1987; Hewitt et al., 1990; Salter and Hewitt,
1992). However, we show here that isoprene can
protect leaves against1O2, and isoprene was shown
lately to protect leaves against ozone by preventing
the collapse of mesophyll cells and chloroplasts
membranes (Loreto et al., 2001) and decreasing the
amounts of H2O2and lipid peroxidation (Loreto and
Velikova, 2001). The reaction products are apparently
less toxic than1O2itself or are efficiently scavenged
by other agents. Also, the possibility that isoprene
could protect against1O2by strengthening the mem-
branes, whereas the direct reaction with1O2is less
significant, cannot be ruled out (isoprene protection
against O3was suggested to be achieved by both
mechanisms; Loreto et al., 2001).
Photoinhibition and1O2stress are often associated
with high light intensities together with low temper-
atures, as observed in clear winter days. It was con-
sequently shown that in some plants, levels of the
carotenoids of the xanthophyll
epoxidation state are higher in winter than in sum-
mer (e.g. Adams and Demmig-Adams, 1994). How-
ever,isoprene emission,
protection mechanism, was minimal in winter in M.
cycleandde-
proposed hereasa
communis. But note that in Mediterranean plant spe-
cies, the xanthophyll cycle de-epoxidation state was
shown to be higher in summer, well correlated with
low water potential (Kyparissis et al., 2000). In the
eastern Mediterranean region, photoinhibition and
oxidative stress are likely to be more pronounced in
summer, when plants are exposed to high light and
water stress, whereas winter conditions are optimal
for growth. Low stomatal conductance during water
stress periods would also enhance intercellular iso-
prene concentrations (Sharkey and Loreto, 1993;
Fang et al., 1996) and the potential protective
capacity.
We note in closing that the protective aspects of
isoprene discussed above may also be consistent
from an evolutionary perspective. Carotenoids of the
xanthophyll cycle are known to scavenge both triplet
chlorophyll and singlet oxygen (Demmig-Adams,
1990; Havaux and Niyogi, 1999). Zeaxanthin was
suggested to decrease membrane fluidity and to in-
crease thermotolerance (Tardy and Havaux, 1997;
Havaux, 1998) as was suggested for isoprene (Shar-
key et al., 2001; Sharkey and Singsaas, 1995; Singsaas
et al., 1997). Isoprene shares a common biochemical
production pathway with carotenoids, and it can be
speculated that isoprene was a primitive protection
mode against1O2that evolved into these more ded-
icated radical scavengers.
MATERIALS AND METHODS
Plant Material
Net assimilation and isoprene emission rates were mea-
sured for cut branches of Myrtus communis from plants
growing on the campus of the Weizmann Institute of Sci-
ence (Rehovot, Israel) and for attached branches from
plants grown in pots under similar light and temperature
conditions. Net CO2assimilation (6.5 ? 0.5 and 7.5 ? 0.5
?mol m?2s?1) and isoprene emission rates (6.0 ? 0.5 and
6.5 ? 2.0 nmol m?2s?1) were similar in attached and cut
branches, respectively. Hence, branches were cut under
water and were kept with the stem immersed in deionized
water for the experiments reported here. Cut branches
containing young leaves from a shade growing shrub of
Rhamnus alaternus in the Weizmann campus were used in
isoprene fumigation experiments.
Gas-Exchange Measurements
The sampling system was centered on a flow-through
cuvette (volume of approximately 60 mL) in which the
branch was sealed. The cuvette was equipped with a
magnet-operated fan for mixing the air. Light (1,000 ?mol
m?2s?1photosynthetically active radiation) was supplied
by a halogen lamp (250 W, Quartzline lamp, General Elec-
tric Co., Cleveland, OH); the infrared radiation was filtered
out using a water bath (3 cm thick). The temperature in the
cuvette was controlled by water circulation through a cool-
ing bath (Haake D8-V, Karlsruhe, Germany) and through
Affek and Yakir
274Plant Physiol. Vol. 129, 2002

Page 7

the bottom of the cuvette and was set to give the desired
leaf temperature (normally 26 ? 0.5°C). Air temperature
was measured by a shaded thermocouple in the cuvette,
and the leaf temperature was measured by a thermocouple
touching the abaxial side of the leaf (type T thermocouple
digital thermometer, HH82, Omega, Stamford, CT; preci-
sion of 0.1°C). Dry air with various CO2concentrations was
supplied by mixing cylinder air with no CO2and cylinder
air having 1% or 2.5% (v/v) CO2using two mass flow
controllers (MKS1179A, Andover, MA; 1,000 mL min?1
and 100 mL min?1full scale, respectively, used in a relative
mode). Total flow rate through the cuvette was 200 mL
min?1. Both air flows passed through activated charcoal
traps to remove hydrocarbons. The tubing (0.25- and 0.125-
inch stainless steel) carrying the air exiting the cuvette was
heated to 80°C to avoid condensation of water vapor.
CO2and H2O concentrations in the air entering and
leaving the leaf cuvette were measured by an infrared gas
analyzer (Li-6262, LI-COR, Lincoln, NE), at a precision of
?1 ?mol mol?1for CO2and ?0.1 mmol mol?1for water
vapor. Net assimilation, stomatal conductance, and ciwere
calculated according to von Caemmerer and Farquhar,
(1981) assuming that stomatal patchiness has only small
effects (van Kraalingen, 1990). Projected leaf area was es-
timated at the end of each experiment.
Isoprene Measurements
For hydrocarbons measurements (Greenberg et al., 1994;
R.K. Monson, personal communication) an aliquot of the
air stream exiting the cuvette was pumped (bypassing the
infrared gas analyzer) into a pre-evacuated glass bulb (2 L,
60 mtorr), through a trapping loop (0.125-inch stainless
steel, 27 cm long in which the central approximately 9 cm
was packed with 212- to 300-?m glass beads [Sigma, St.
Louis], with a 2-?m filter [Valco, Houston, TX] placed at
the outlet) connected to a six-port valve (Valco). The loop
was cooled with either liquid nitrogen or a mixture of
ethanol and dry ice (?75°C) for trapping the hydrocarbons
in the air. The pressure (pressure transducer, Omega) at the
entrance to the glass bulb was used to estimate the amount
of air sampled (typical pressure used was 100 torr, namely
the sample size was approximately 250 mL). After trap-
ping, the valve was switched to a flow of helium (1.5 mL
min?1), the loop was rapidly heated (hot sand, 200°C), and
the trapped hydrocarbons passed directly to a gas chroma-
tography (GC) column (HP 5890, Wilmington, DE).
The hydrocarbons were separated using a polar GC col-
umn (30 m long, 0.25 mm i.d., 0.25 ?m film; Supelcowax 10,
Supelco, Bellefonte, PA; temperature program: 35°C for 1
min, temperature increase at 10°C min?1to 170°C for 2
min). Isoprene was detected using a flame ionization de-
tector kept at 250°C. The area of the peaks obtained was
recorded and analyzed by a chromatography software
(Borwin, version 1.21.60, JMBS developments, La Fontenil,
France).
The GC peak area of isoprene was found to vary linearly
with concentration, and to be constant over time. Isoprene
gaseous standards were prepared and measured every few
months. The precision of isoprene concentration measure-
ments was 4%. Standards were prepared by evaporating
isoprene (99%; Aldrich, Milwaukee, WI) into a pre-
evacuated 12-L bulb to a pressure of approximately 0.1
torr, on a vacuum line. Nitrogen was added to atmospheric
pressure. The bulb was evacuated to approximately 1 torr
and refilled with nitrogen to atmospheric pressure, to give
0.1 to 0.2 ?L L?1isoprene.
Singlet Oxygen Treatments
Singlet oxygen was produced in the leaves by RB
(4,5,6,7-tetrachloro-2?,4?,5?,7?-tetraiodofluorescein) or BX
(3,5-dibromo-4-hydroxybenzonitrile; Agan chemical man-
ufacturers, Ashdod, Israel) in the light. RB acts as a pho-
tosensitizer to singlet oxygen production upon absorbing
green light, ?max ? 547 nm. BX is a phenolic herbicide that
increases the sensitivity of PSII to excess light through
formation of P680 triplet (Krieger-Liszkay and Rutherford,
1998). Both chemicals were fed as aqueous solutions
through the petiole, and their effect on net assimilation and
isoprene emission rates was examined at ambient and high
CO2concentrations, 26°C, and at light intensity of 1,000
?mol m?2s?1. To use physiologically relevant1O2concen-
trations, RB concentration used in the fumigation experi-
ments (0.4 ?m in M. communis and 0.1 ?m in R. alaternus)
was the lowest to induce decrease in net assimilation rates
(Fig. 3), and BX was used as a source for1O2near PSII,
simulating photoinhibition more specifically than RB.
Isoprene fumigation was done together with the
treatment in young leaves, by passing the air entering the
leaf cuvette through a piece of permeable tubing that was
enclosed in an Erlenmeyer containing isoprene and kept in
an ice bath. The fumigation dose (1–20 ?L L?1) was deter-
mined by the permeability of the tubing and the isoprene
concentration in the Erlenmeyer.
1O2
Chlorophyll Fluorescence
Chlorophyll fluorescence yield (Fv/Fm) was measured in
dark-adapted leaves of R. alaternus before and after singlet
oxygen experiments, using a portable fluorometer (PAM-
2000, Walz, Germany).
Statistical Analysis
The data analysis add-in from Microsoft Excel 1998 (Mi-
crosoft Corp., Redmond, WA) was used to calculate Stu-
dent’s t test for determining the significance of difference
between fumigation and non-fumigation experiments.
ACKNOWLEDGMENTS
We thank Y. Rudich, F. Loreto, R.K. Monson, and two
anonymous reviewers for helpful comments and E.
Negreanu for technical help. We thank Agan Chemical
manufacturers for supplying BX.
Singlet Oxygen Protection by Isoprene
Plant Physiol. Vol. 129, 2002 275

Page 8

Received October 5, 2001; returned for revision December
16, 2001; accepted February 4, 2002.
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