Research
Linking photorespiration, monoterpenes and
thermotolerance in Quercus
Blackwell Science, Ltd
Josep Peñuelas and Joan Llusià
Unitat d’Ecofisiologia CSIC-CEAB-CREAF, CREAF (Center for Ecological Research and Forestry Applications), Edifici C, Universitat Autònoma de
Barcelona, E−08193 Bellaterra, Barcelona, Catalonia, Spain
Summary
Author for correspondence:
Josep Peñuelas
Tel: +34 3 5812199
Fax: +34 3 5811312
Email: [email protected]
Received: 1 January 2002
Accepted: 18 April 2002
• The functions of two important plant processes, photorespiration and monoterpene production remain controversial. Here, we investigated one possible function,
that of protection of plants from photodamage at high temperatures.
• Fluorescence, reflectance, monoterpene concentrations and visual leaf damage
were measured in Quercus ilex seedlings exposed to temperature increases from 25
to 50°C (in 5°C steps) under photorespiratory (21% O2) or nonphotorespiratory
(2% O2) atmospheres, and under control or terpene fumigation conditions.
• Lower variable to maximum fluorescence ratio (Fv : Fm: potential photochemical
efficiency of photosystem II, PSII) and electron transport rate (ETR) were found in
nonphotorespiratory conditions at temperatures greater than 35°C. Monoterpene
concentrations were also lower, and leaf damage greater, in the low O2 atmospheres. Monoterpene fumigation, which increased the foliar terpene concentrations
by two- to four-fold, increased the photochemical efficiency between 35°C and
50°C, and decreased leaf damage, only under the nonphotorespiratory conditions.
• These results provide evidence that: photorespiration decreases photodamage,
especially at high temperatures; photorespiration increases monoterpene production; plants are able to acquire exogenous monoterpenes and the acquisition
response to temperature follows the stomatal conductance response; and monoterpenes can replace photorespiration in protection from photodamage at high temperatures, possibly by scavenging oxygen-reactive species, but they do not provide
additional thermotolerance.
Key words: Fv : Fm, electron transport rate (ETR), photochemical reflectance
index (PRI), α-pinene, limonene, fumigation, photorespiration, monoterpenes,
thermotolerance.
© New Phytologist (2002) 155: 227–237
Introduction
High temperature may represent an important constraint
for plants, restricting growth and productivity (Boyer, 1982)
and influencing the distribution of species (Grace, 1987).
Models of global climate predict a further 1.5 – 5.5°C warming
for this century as a result of the increased atmospheric
concentrations of CO2 and other trace gases (IPCC, 2001).
Therefore, plants are likely to experience increasing hightemperature stress.
Plants thus need to protect themselves against the damaging effects of high temperature. Photorespiration, which lowers
© New Phytologist (2002) 155: 227– 237 www.newphytologist.com
the energy efficiency of photosynthesis, particularly in C3
plants (Ogren, 1984), seems to protect these plants from
photoinhibition by dissipation of excess light energy (Kozaki &
Takeba, 1996). While chloroplasts have superoxide dismutase
to detoxify oxygen radicals, it is apparently insufficient to
protect the leaf in high light intensity when CO2 fixation is
limited by heat (or drought) stress, stomata close and CO2 cannot
enter the leaf. Photorespiration can continue in leaves with
external and internally produced oxygen. Photorespiration thus
provides internal CO2 for re-fixation, and the Calvin cycle
keeps running. This photoprotective role for photorespiration
is, however, somewhat controversial since sometimes has been
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not demonstrated (Powles et al., 1979; Ogren, 1984; Wu et al.,
1991; Demmig-Adams & Adams, 1992; Aro et al., 1993;
Long & Humphries, 1994; Brestic et al., 1995; Heber et al.,
1996; Migge et al., 1999; Niyogi, 1999).
Temperature increases the production and emission rates
of most terpenes exponentially up to maximum by enhancing
the synthesis enzymatic activities, raising the terpene vapor
pressure and decreasing the resistance of emission pathway
(Tingey et al., 1991; Loreto et al., 1996; Peñuelas & Llusià,
2001). Even for nonstored volatile organic compounds
(VOCs) such as α-pinene in Quercus ilex, emission has been
found to increase threefold when temperature raises from 20
to 30°C (Loreto et al., 1996). Terpenes serve several functions, including deterring herbivores and attracting pollinators, but the function of terpene production and emission by
plants that do not store them remains elusive. One potential
function of isoprene and terpenes is the stabilization and
protection of plant membranes against high temperatures
(Sharkey & Singsaas, 1995; Loreto et al., 1998; Singsaas, 2000)
even described in neighbor nonemitting species, although at
unlikely high air concentrations (Delfine et al., 2000). However, such thermotolerance enhancement has not always
been found (Logan & Monson, 1999). A second hypothesis
of a protective role of isoprene and monoterpenes is that they
serve as antioxidants in leaves. However, this hypothesis has
little evidence to support or reject it (Sharkey & Yeh, 2001).
One of the primary effects of high-temperature stress is
the damage to photosynthetic electron transport. A review by
Berry & Björkman (1980) found the electron transport
through photosystem II (PSII) to be the component of photosynthesis most susceptible to irreversible high-temperature
damage. Chlorophyll fluorescence and reflectance methods
allow the quantification of such stress effects on plant photosynthesis, with the advantage of being nondestructive and
rapid, thereby only minimally affecting the object of study
(Bolhar-Nordenkampf et al., 1989; Bilger et al., 1995;
Peñuelas & Filella, 1998). Changes in chlorophyll fluorescence induced by high temperature have been widely used as
an indicator of thermal damage (Bilger et al., 1984). These
changes seem to be produced by dislocation between the
light-harvesting complexes and PSII reaction centers because
of excessive membrane fluidity (Armond et al., 1980) or/and
denaturation of PSII reaction center proteins (Yamane et al.,
1997). The xanthophyll cycle is another photoprotective
mechanism for excess of energy that lowers the photochemical
efficiency of PSII (Demmig-Adams & Adams, 1992; Long
& Humphries, 1994; Peñuelas et al., 1995). PSII efficiency can
be monitored by changes in reflectance at 531 nm (Gamon
et al., 1992; Peñuelas et al., 1995; Peñuelas & Filella, 1998).
High-temperature stress is especially evident in Mediterranean area where high temperatures coincide with drought
conditions in summer (Peñuelas & Llusià, 1999; Larcher,
2000). Quercus ilex is one of the most typical dominant
Mediterranean forest species. Its leaves may suffer from thermal
stress above 35°C (Larcher, 2000). Usually, CO2 uptake suddenly decreases at 40–45°C, but Q. ilex still grows on sites
where the maximum air temperatures reach 49°C (Seville,
Spain). This species seems to present higher photorespiration
rates than other co-occurring species under the high temperatures
and dry conditions of the Mediterranean summer (Filella et al.,
1998; Peñuelas et al., 1998), and its emissions of biogenic
terpenes seem to have an enhancement effect on the thermotolerance of leaves (Loreto et al., 1998; Peñuelas & Llusià, 1999).
We measured fluorescence, reflectance, monoterpene concentrations and visual leaf damage of Q. ilex whole seedlings
exposed to temperature increases from 25 to 50°C in 5°C
steps in atmospheres under photorespiratory (21% O2) or
nonphotorespiratory conditions (2% O2), and under control
or terpene fumigation conditions. Our general aim was to
study the effects of (1) photorespiration, (2) monoterpenes,
and (3) their interactions on thermotolerance. Our detailed
aims were to determine (1) whether photorespiration protects
from photodamage at high temperatures, (2) whether there is
a link with monoterpene foliar concentrations, (3) whether
plants are able to take up monoterpenes and increase the foliar
concentrations, and (4) whether monoterpenes can replace or
add to photorespiration in protection from photodamage at
high temperature.
Materials and Methods
Experimental system
To avoid dysfunction and mismatching between the functioning
of single leaf and the whole plant, we conducted the
experiments with whole-seedling plants inside the fumigation
chambers. Two-yr-old plants of Q. ilex L., previously grown in
a nursery (Forestal Catalana, Breda, Spain) in Mediterraneanlike environmental conditions, were transplanted to 5-l pots
with a substrate composed of peat, Perlite and vermiculite
(2 : 1 : 1), and were well watered, well fertilized with
osmocote (Scott O.M., Tarragona, Spain) and maintained in
Mediterranean-like environmental conditions in a greenhouse
until experimentation. Then, they were wholly placed inside
a specifically designed chamber-system sealed with Terostat
gum (Casadesús, 1995; Nogués et al., 2001). They were then
exposed to a temperature ramp of 50 °C steps from 25 to
50 °C. The plants were maintained at each temperature for
30 min before measuring leaf chlorophyll fluorescence, leaf
reflectance, and air terpene concentration and sampling leaves
for VOC concentration analyses.
This experimental system (Fig. 1) is similar to the systems
described by McCree (1986) and Smart et al. (1994). It consists of two independent Plexiglas chambers (0.5 × 0.5 ×
0.5 m, 0.125 m3 volume) whose environmental conditions
(photosynthetic photon flux density (PPFD), relative humidity and temperature) can be programmed independently by
an automated control mechanism (Fig. 1). Light was supplied
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Fig. 1 Experimental system for submitting whole seedlings to temperature ramps under atmospheres with different O 2 and monoterpene
concentrations.
by three 500 W halogen lamps (Model 64702; Osram,
Madrid, Spain) per chamber, each supplying a PPFD of about
500–860 µmol m−2 s−1 on a plane at half the height of the
plant during a 10-h photoperiod. The lamps were placed
about 50 cm above the plants and 7 cm of deionized water
and 1 cm of Plexiglas in the container filtered the radiation.
Air temperature and relative humidity in the chambers were
measured with two HMP 112Y sensors (Vaisala Oy, Helsinki,
Finland). The relative humidity in each chamber was regulated
by controlling the water content of the air entering the chamber
with a bypass control valve (Fig. 1). Air entered the chambers
at about 355 µmol mol−1 CO2. This concentration was
controlled by using an infrared gas analyzer (IRGA) and a
manometer to regulate CO2 flux from a CO2 tank (Fig. 1).
Air entering the chambers was cooled at 4°C to reduce its
water content and a regulated proportion (independently
determined for each chamber) passed through a bubble tank
at 18°C before entering the chamber. For each chamber, the
amount of air passed through the bubbler was regulated by a
progressive valve (Model VXG41.15R; Landis & Gyr, Geneva,
Switzerland) controlled by computer. The air temperature in
each chamber was automatically regulated by adjusting the
© New Phytologist (2002) 155: 227– 237 www.newphytologist.com
temperature bath where the air incoming to the chambers
circulated. The airflow was measured with a mass flow meter
(Model 5811N; Brooks Instruments, Veenendaal, The
Netherlands) attached to each chamber. The temperature
difference between the air and a chosen leaf of each plant was
measured with a pair of thermocouples (Model LI-6000TC;
Li-Cor, Lincoln, NE, USA). Data acquisition and process
control of the entire system were carried out with the aid of a
computerized data logger (Model HP3412A; Hewlett Packard,
Palo Alto, CA, USA).
O2 atmospheres and terpene fumigation
Four different atmospheres were assayed as result of the
combination of 21% and 2% O2 and of terpene-fumigation
and non-fumigation. Monoterpene fumigation was applied
each time to one of the Plexiglas chambers with a peristaltic
pump that fluxed air through a flask containing 1 cm3 of
α-pinene and 1 cm3 of limonene (99% purity; Fluka, Buchs,
Switzerland). These monoterpenes are two of the most
abundant monoterpenes produced by Q. ilex (Peñuelas &
Llusià, 1999; Llusià & Peñuelas, 2000). This flask with
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α-pinene and limonene was placed in a thermostatic bath at
20°C (Fig. 1). The concentration in the fumigated chamber
was 2.6 ± 0.3 µg l−1 (n = 10) for α-pinene and 1.9 ± 0.19
µg l−1 (n = 10) for limonene, whereas the concentrations in
the control chamber were 0.019 ± 0.006 µg l−1 for α-pinene
and 0.013 ± 0.0048 µg l−1 for limonene. The system was
allowed to equilibrate for 1 h before starting the measurements.
For each one of these four different atmospheres, the
temperature ramp experiment was repeated five or six times
(five or six different plants).
Terpene analysis
Part of the air exiting the chamber flowed through a T-system
to a glass tube (11.5 cm long and 0.4 cm internal diameter)
manually filled with terpene adsorbents Carbotrap C (300
mg), Carbotrap B (200 mg), and Carbosieve S-III (125 mg)
(Supelco, Bellefonte, PA, USA) separated by plugs of quartz
wool. The hydrophobic properties of the tubes were supposed
to minimize sample displacement by water. In these tubes,
terpenes did not suffer chemical transformations, as checked
with standards (α-pinene, β-pinene, camphene, myrcene,
p-cymene, limonene, sabinene, camphor and dodecane).
Prior to use, these tubes were conditioned for 3 min at 350°C
with a stream of purified helium. The sampling time was
5 min, and the flow varied between 100 cm3 min−1 and
200 cm3 min−1, depending on the glass tube adsorbent and
quartz wool packing. The flow passing through the VOC
adsorbents were measured with a potentiometer and calibrated
with a bubbler flowmeter. The trapping and desorption
efficiency of liquid and volatilized standards such as α-pinene,
β-pinene or limonene was almost 100%.
After VOC sampling, the adsorbent tubes were stored at
−30°C until analysis (within 24–48 h). There were no observable changes in terpene concentrations after storage of the
tubes, as checked by analysing replicate samples immediately
and after 48-h storage. Terpene analyses were conducted by gas
chromatography–mass spectrometry (GC-MS) (Hewlett Packard
HP59822B, California). Trapped monoterpenes were desorbed
(Thermal Desorption Unit, Model 890/ 891; Supelco) at
250°C over 2 min and injected into a 30-m 0.25 × 0.25 mm
film thickness capillary column (Supelco HP-5, Crosslinked
5% pH Me Silicone). After sample injection, the initial
temperature (46°C) was increased at 30°C min−1 up to 70°C,
and thereafter at 10°C min−1 up to 150°C, which was maintained for further 5 min. Helium flow was 1 cm3 min−1. The
identification of monoterpenes was conducted by GC-MS
and comparison with standards from Fluka (Chemie AG,
Buchs, Switzerland), literature spectra, and GCD Chemstation
G1074A HP. Internal standard dodecane, which did not mask
any terpene, together with frequent calibration with common
terpene standards (α-pinene, β-pinene, 3-carene, β-myrcene,
p-cymene, limonene and sabinene) once every three analyses
were used for quantification. Terpene calibration curves (n = 4
different terpene concentrations) were always highly significant
(r 2 > 0.97) in the relationship between signal and terpene
concentration. The most abundant terpenes had very similar
sensitivity (differences were 5%).
Fluorescence measurements
The variable to maximum fluorescence ratio, Fv : Fm in the
nonenergized state after darkness is a reliable measure of the
potential efficiency of PSII photochemistry. It is used as an
estimate of the functional state of the photosynthetic
apparatus at a given environmental situation. A decrease
in Fv : Fm indicates photoregulation of PSII (Oliveira &
Peñuelas, 2001). An indicator of the actual photochemical
activity is the electron transport rate (ETR). Genty et al.
(1989) have shown that PSII radiation-use efficiency may be
determined with the expression:
∆F/F′m= (F′m − F)/F′m
Eqn 1
derived from measurements of leaf fluorescence under
ambient light (F), and under saturating light pulses (F′m).
This ∆F/F′m can be used to calculate relative ETR, which
represents the apparent photosynthetic electron transport
rate in µmol electrons m−2 s−1 (Schreiber et al., 1995), by
multiplying the quantum yield of PSII (∆F/F′m) by leaf
absorbance (A) and by half PPFD, since it is assumed that
radiant energy is equally absorbed by the two photosystems:
ETR = ∆F/F′m × PPFD × 0.5 × A
Eqn 2
At each temperature of the experimental ramp, chlorophyll
fluorescence of apical mature leaves was determined using a
portable modulated fluorometer PAM-2000, including the
leaf clip holder part 2030-B (Heinz Walz, Effeltrich, Germany).
Minimal fluorescence of a dark adapted leaf with all PSII
reaction centers fully open (Fo), maximal fluorescence yield of
a dark-adapted leaf with all PSII reaction centers fully closed
(Fm), maximal (potential) photochemical efficiency of PSII
(given by the Fv : Fm ratio) after dark adaptation for 20 min,
and actual photochemical efficiency of PSII (F′m − Fs)/F′m
or ∆F/F′m were measured, as well as leaf temperature and
incident PPFD.
Reflectance measurements
Another indirect optical measure of photosynthetic performance
with similar nondestructive and speed properties can be
derived from the reflectance changes near 531 nm (Gamon
et al., 1992; Peñuelas et al., 1995; Peñuelas & Filella, 1998).
These changes are associated with xanthophyll pigment
interconversion and chloroplast conformational changes, and
correlate with both epoxidation state of the xanthophyll
cycle pigments and photosynthetic radiation-use efficiency
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(Gamon et al., 1992; Peñuelas et al., 1995; Peñuelas & Filella,
1998). The de-epoxidation state of the xanthophyll cycle,
DPS = (A + Z)/(V + A + Z), is an additional indicator of the
functional state of chloroplasts. When light becomes
excessive, violaxanthin (V) is de-epoxidized to zeaxanthin (Z)
through antheraxanthin (A).
Leaf spectral radiance was measured by directing a fiber
optic probe to the leaf surface at the same angle of the leaf clip
holder of the PAM-2000 fluorometer. The other end of the
fiber optic was attached to a spectroradiometer (Spectral analysis VIS/NIR portable equipment UniSpec; PP-Systems,
Haverhill, MA, USA). Reflectance was calculated by normalizing leaf radiance by the radiance of a 99% reflective standard
(Spectralon; Labsphere, North Sutton, NH, USA).
To follow the reflectance changes at 531 nm associated
with the photosynthetic radiation-use efficiency and with the
dissipation of excess energy, we used the ‘photochemical
reflectance index’ PRI (Gamon et al., 1992; Peñuelas et al.,
1995; Peñuelas & Filella, 1998), calculated as follows:
experiment between 25°C and 50°C by measuring the
damage area of all the plant leaves. Leaf area (separating
damaged and healthy) was measured in the laboratory using
a Li-Cor LI-3100 area meter. The leaf dry mass was determined after drying at 60°C until constant mass.
PRI = (R531 − R570)/(R531 + R570)
Photorespiration and thermotolerance
Eqn 3
where R is reflectance and the numbers for the wavelength in
are in nm.
We also measured other reflectance indices such as normalized difference vegetation index (NDVI) (R900 – R680)/
(R900 + R680), indicative of greenness, water index (WI)
(R900/R970) indicative of water content or normalized total
pigment to chlorophyll ratio index (NPCI) (R485 – R680)/
(R485 + R680) indicative of the ratio between carotenoids
and chlorophyll (Peñuelas & Filella, 1998).
Leaf sampling and terpene concentration analysis
Individual leaves were sampled at each temperature after the
measurement of fluorescence and reflectance and were
immediately submerged in liquid nitrogen. For extraction of
leaf terpenes, these leaves were submerged in liquid nitrogen
in Teflon tubes. They were afterwards heated in a water bath
at 100°C while a flow of 166 cm3 min−1 of nitrogen drew
volatiles towards an adsorbent tube similar to those described
for analysis of terpenes in the atmospheres of the different
treatments. The absence of breakthrough was checked by
placing two traps in series and by verifying that no
monoterpenes were collected in the second one. Standards of
the different monoterpenes were also frozen and extracted
with the same method to check for absence of losses.
Monoterpenes trapped in the adsorbent tubes were desorbed
and measured by GC as described above.
Leaf measurements: damage, area and dry weight
The percentage of damaged (dicolored or brown) leaves was
measured immediately at the end of each temperature-ramp
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Statistical analyses
All the temperature-ramp experiments described were
repeated five or six times on different plants. Repeated
measures analyses of variance (ANOVAs), and regression
analyses were conducted using STATISTICA version 5.0 for
Windows (StatSoft, Tulsa, Ok, USA). To test for treatment
effects at each temperature (or at each O2 concentration and
at each fumigation treatment when analysing leaf visual
damage), one-way ANOVAs were also conducted.
Results and Discussion
Under photorespiratory conditions, the Fv : Fm index,
fluorescence measure of the potential photochemical efficiency
of PSII (Oliveira & Peñuelas, 2001) was maximum at 25°C,
decreased slowly up to 40°C, and thereafter it decreased
rapidly (Fig. 2). A similar pattern was found for ETR (Fig. 2)
although at 45°C it presented a stronger decrease. This
temperature of 45°C is close to the critical temperature of 47–
48°C derived from chlorophyll fluorescence in other studies
of Q. ilex (Méthy et al., 1997). These results confirm that one
of the primary effects of high-temperature stress is the damage
to photosynthetic electron transport through PSII (Berry
& Björkman, 1980). Heat damage arises from inactivation of
the highly sensitive water-splitting reaction, disconnection of
PSII centers from the bulk pigments, thermal uncoupling
of photophosphorylation, and biomembrane lesions (Berry
& Björkman, 1980). The greatest damage was found close to
50°C. Necrosis appeared at this temperature, as has also been
reported by Larcher (2000). This was also indicated by NDVI
and WI values in the visually undamaged leaves, which
strongly decreased from 0.83 ± 0.01 to 0.7 ± 0.06, and from
1.03 ± 0.003 to 1.01 ± 0.007 (data not shown), respectively,
indicating foliar loss of green pigments and water (Peñuelas &
Filella, 1998).
When photorespiration was inhibited (under 2% O2
atmospheres), Fv : Fm decreased (indicating a decrease of
potential photochemical efficiency of PSII) markedly already
at 35°C, and Fo was always greater (indicating reduction of
the energy trapping by PSII centers) and ETR was always
smaller than under 21% O2 atmospheres, as expected from
the inhibition of photorespiration (Fig. 2). The photochemical reflectance index, the reflectance index of photochemical
efficiency linked to zeaxanthin formation, did not change in the
whole range of temperatures tested in control atmospheres.
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Fig. 2 Effect of increasing temperature on Quercus ilex Fo
(minimal fluorescence of dark adapted leaves), Fv : Fm
(potential photochemical efficiency of photosystem II) and electron
transport rate (ETR, in µmol electrons m−2 s−1) under photorespiratory
(circles, 21% O2) and nonphotorespiratory (squares, 2% O2)
conditions. Error bars indicate SEM; n = 5–6 temperature ramp
treatments (i.e. five or six different plants). In each of these treatment
replications a different plant was measured and three different
leaves were measured for each of the temperatures tested. Statistical
significance of the difference between O2 treatments: *, P < 0.05,
**, P < 0.01.
However, PRI abruptly decreased at 50°C under nonphotorespiratory conditions (Fig. 3), indicating enhancement of
the dissipation of excess of energy through the xanthophyll
de-epoxidation cycle (Gamon et al., 1992; Peñuelas et al.,
1995; Peñuelas & Filella, 1998). Finally, the inhibition of
photorespiration increased leaf visual damage threefold after
the temperature treatment (Fig. 4).
These results confirm the decreased ETR and increased
dissipation as heat found in other studies under nonphotorespiratory conditions. Moreover, these other studies reported
accumulation of photorespiratory metabolites, decreased
Leaf visual damage (%)
Fig. 3 Effect of increasing temperature on Quercus ilex
photochemical reflectance index (PRI; indicative of xanthophyll
cycle dissipation energy and radiation-use efficiency) under
photorespiratory (circles, 21% O2) and nonphotorespiratory
(squares, 2% O2) conditions. Error bars indicate SEM; n = 5 – 6
temperature ramp treatments (i.e. five or six different plants).
In each of these treatment replications a different plant was measured
and three different leaves were measured for each of the
temperatures tested. Statistical significance of the difference
between O2 treatments: *, P < 0.05, **, P < 0.01.
100
80
**
60
40
20
0
2
21
Atmospheric O2 concentrations (%)
Fig. 4 Leaf visual damage after the temperature treatment
(ramp increase up to 50°C) under photorespiratory (21% O2) and
nonphotorespiratory (2% O2) conditions. Error bars indicate SEM;
n = 5 –6 temperature ramp treatments (i.e. five or six different
plants). Statistical significance of the difference between O2
treatments: **, P < 0.01. DM, dry matter.
number of starch grains in chloroplasts (among other changes
in plant morphology) and lower growth under nonphotorespiratory conditions (Kozaki & Takeba, 1996; Migge et al.,
1999; Niyogi, 1999). It is likely that when the supply of CO2
to leaves is cut off by stomatal closure at high temperatures,
and photorespiration does not work, the Calvin–Benson C3
cycle will not operate. Under those conditions, light energy
may be diverted to produce active oxygen, which damage the
photosynthetic apparatus (Aro et al., 1993; Kozaki & Takeba,
1996).
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Fig. 5 Effect of increasing temperature on α-pinene and limonene
leaf concentrations under photorespiratory (circles, 21% O2) and
nonphotorespiratory (squares, 2% O2) conditions. Error bars indicate
SEM; n = 5– 6 temperature ramp treatments (i.e. five or six different
plants). In each of these treatment replications a different plant was
measured and three different leaves were measured for each of the
temperatures tested. Statistical significance of the difference between
O2 treatments: *, P < 0.05, **, P < 0.01.
Monoterpenes and thermotolerance
Loreto et al. (1998) found monoterpene emissions to be
stimulated by low atmospheric O2 concentrations. Here, we
found decreased leaf concentrations (a higher rate of emissions
could lead to these lower concentrations, but this hypothesis
should be tested in further studies with simultaneous
measurements of concentrations and emissions). The leaf
concentrations of α-pinene and limonene were smaller (less
than half ) in the absence of photorespiration, especially at the
highest temperatures, when leaf concentrations were also the
highest (Fig. 5). As the protective effect of photorespiration
was accompanied by these two to three times greater foliar
terpene concentrations at the highest temperatures, we
experimentally increased the concentrations of α-pinene and
limonene, two of the most abundant monoterpenes produced
by Q. ilex (Peñuelas & Llusià, 1999). Under fumigation, the
concentrations of α-pinene in the chamber air increased from
0.019 ± 0.006 to 2.6 ± 0.3 µg l−1, and the concentration of
limonene increased from 0.013 ± 0.0048 to 1.9 ± 0.19 µg l−1.
As a result, α-pinene and limonene concentrations in the
Q. ilex leaves increased between two- and four-fold relative to the
endogenous concentrations ( Fig. 6). The maximum increase
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occurred at 40°C and then decreased again in parallel with the
stomatal conductance response of the seedlings of this
species to temperature (Larcher, 1995; Ogaya & Peñuelas,
unpublished; J. Llusià & J. Peñuelas, unpublished). These
increases in leaf concentration showed that there was terpene
acquisition by Q. ilex plants and that it probably occurred
through stomata. Many factors affect stomatal conductance.
Most of them (irradiance, air humidity and CO2 partial
pressure) were the same for the different treatments, but low
O2 concentrations and terpene fumigation may affect stomatal
conductance. However, for the same O2 and terpene fumigation
conditions, the changes in terpene acquisition and previous
measurements of stomatal conductance response to temperature
(Larcher, 1995; Ogaya & Peñuelas, unpublished; J. Llusià &
J. Peñuelas, unpublished) indicated that stomatal conductance
changes were mostly driven by temperature changes.
Monoterpene fumigation, and consequent two- to fourfold increased monoterpene leaf concentrations, did not
enhance thermotolerance of Q. ilex plants, as measured by Fo,
Fv : Fm and ETR under normal atmospheres (Fig. 7). Delfine
et al. (2000) did not find improved thermotolerance when
internal content was similar to the endogenously formed.
They only found thermotolerance when high fumigation
doses yielded about fivefold the endogenous pool of Q. ilex.
High concentrations might be needed to achieve thermotolerance under standard photorespiratory conditions. These
high monoterpene concentrations could be reached in field
conditions under typical summer stress in the Mediterranean
region, where drought closes stomata and favors monoterpene
accumulation because they are less volatile than other VOCs
such as isoprene.
However, monoterpene fumigation did increase thermotolerance (Fo was 25–35% lower, Fv : Fm was 0.1–0.2 units
higher and ETR was 20–40 µmol electrons m−2 s−1 higher)
under nonphotorespiratory conditions at temperatures higher
than 35°C (or than 40°C for Fo) (Fig. 7). Under these nonphotorespiratory conditions, and with monoterpene fumigation, PRI recovered the standard value at 50°C (Fig. 8)
indicating that zeaxanthin formation was not enhanced.
Terpenes would have membrane-stabilizing properties
similar to this other isoprenoid, zeaxanthin, at high temperatures (Singsaas, 2000) and could substitute xanthophyll
de-epoxidation protection under nonphotorespiratory conditions and very high temperatures (50°C). Under the nonphotorespiratory conditions, monoterpene fumigation decreased
the leaf visual damage after the temperature treatment from
77% to 50% (Fig. 9). This was also indicated by NDVI and
WI reflectance indices, which did not decrease at 50°C. Values
of 0.85 and 1.03 were maintained for NDVI and WI (data not
shown), respectively, when under the nonphotorespiratory
conditions plants were monoterpene-fumigated.
Thus, fumigation with monoterpenes at physiologically
realistic concentrations was able to improve tolerance to temperatures higher than 35°C only when photorespiration was
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234 Research
Fig. 6 Increase in α-pinene and limonene
leaf concentrations in Quercus ilex
seedlings at fumigated atmospheres under
photorespiratory (21% O2) and
nonphotorespiratory (2% O2) conditions,
and at different temperatures. Open
symbols, control; closed symbols,
monoterpene-fumigated atmospheres.
statistical significance of the difference
between terpene fumigated and
non-fumigated treatments: *, P < 0.05,
**, P < 0.01.
absent. Sharkey & Singsaas (1995) also found that the protective effect of exogenous isoprene from thermal stress was
enhanced under conditions that inhibited endogenous formation. We speculate that monoterpenes may have, at least in
part, a photorespiration-like protection role that is enhanced
when photorespiration is absent.
There is not enough evidence of the mechanism or mechanisms and the interactions involved in this phenomenon of
enhanced PSII thermotolerance by photorespiration and
monoterpenes. Some xanthophylls, which are also isoprenoids, are known to increase thermotolerance. Zeaxanthin,
when present in the membranes, reduces photon leakage at
high temperatures (Tardy & Havaux, 1997). The hypothesis
is that zeaxanthin may act as a solute in the membrane, which
stabilizes its structure at high temperatures. Although terpenes
are extremely hydrophobic they could have similar membranestabilizing properties (Singsaas, 2000). It is likely that the
thermotolerance achieved results from both the terpeneinduced thermostable conformations of the membraneconnected thylakoid protein subunits and the terpene-induced
adjustment of membrane lipid fluidity. However, our data for
monoterpene enhancement of thermotolerance only under
nonphotorespiratory conditions fits better with a mechanism
linked to the monoterpene capacity to scavenge photosynthesisderived reactive oxygen species. These reactive oxygen species raise their concentration inside the leaf during periods of
high temperature and light, especially when photorespiration
is not active in inhibiting their formation (Aro et al., 1993;
Kozaki & Takeba, 1996). The monoterpenes would use their
capacity to scavenging the radical oxygen species produced
under high temperatures.
In addition to isoprene, monoterpenes and xanthophylls,
several heat-shock proteins accumulate in vegetative tissues in
response to heat stress (Vierling, 1991) and one of them, the
small chloroplasts’ heat-shock protein, has been demonstrated
to protect PSII from high-temperature damage (Heckathorn
et al., 1998). However, unlike the relatively rapid reversibility
of isoprene and terpene emission and xanthophyll epoxidation that should enhance thermotolerance during short periods
of high temperature, protection from heat-shock proteins
generally requires several hours for induction and the proteins
are around for a long time (Vierling, 1991, Hanson & Sharkey,
2001). Thus, it is unlikely that these heat-shock proteins
could mask some of the beneficial effects of photorespiration
or terpenes in the short-period high temperatures studied.
Moreover, under photorespiratory conditions and terpene
fumigation plants suffered significant damage (Fig. 9), with
no indication of possible protective role of heat-shock
proteins.
Finally, and to summarize these results, several important
conclusions can be drawn from this study. First, photorespiration seems to be necessary to avoid photochemical damage,
www.newphytologist.com © New Phytologist (2002) 155: 227– 237
Research
Fig. 8 Effect of increasing temperature on Quercus ilex
photochemical reflectance index (PRI, indicative of xanthophyll cycle
dissipation energy and radiation-use efficiency) under
photorespiratory (21% O2) and non-photorespiratory (2% O2)
conditions, and under control (open symbols) and monoterpenefumigated (closed symbols) atmospheres. Error bars indicate SEM;
n = 5 –6 temperature ramp treatment (i.e. 5 – 6 different plants). In
each of these treatment replications a different plant was measured
and three different leaves were measured for each of the
temperatures tested. Statistical significance of the difference between
terpene fumigated and nonfumigated treatments: *, P < 0.05,
**P < 0.01.
Fig. 7 Effect of increasing temperature on Quercus ilex Fo (minimal
fluorescence of dark adapted leaves), Fv : Fm (potential
photochemical efficiency of photosystem II) and electron transport
rate (ETR, in µmol electrons m−2 s−1) under photorespiratory (21%
O2) and nonphotorespiratory (2% O2) conditions, and under control
(open symbols) and terpene-fumigated (closed symbols)
atmospheres. Error bars indicate SEM; n = 5–6 temperature ramp
treatments (i.e. five or six different plants). In each of these treatment
replications a different plant was measured and three different leaves
were measured for each of the temperatures tested. Statistical
significance of the difference between terpene fumigated and
nonfumigated treatments: *, P < 0.05; **, P < 0.01.
especially at high temperatures. Second, terpene concentrations are higher under photorespiratory than under nonphotorespiratory conditions. Third, plants are able to acquire
exogenous monoterpenes and the acquisition response to
temperature follows stomatal conductance response. Fourth,
since we did not find thermotolerance enhancement under
standard photorespiratory conditions and instead found
thermotolerance enhancement under nonphotorespiratory
conditions, monoterpenes seem to replace photorespiration in
the plant’s protection against high temperatures. Fifth, the
© New Phytologist (2002) 155: 227– 237 www.newphytologist.com
Fig. 9 (a) Leaf visual damage (percentage of discolored or brown
total leaf area) after the temperature treatment (ramp increase up to
50°C) under photorespiratory (21% O2) and nonphotorespiratory
(2% O2) conditions, and under control (open columns) and
terpene-fumigated (filled columns) atmospheres. Error bars indicate
SEM; n = 5–6 temperature ramp treatments (i.e. five or six different
plants). Statistical significance of the difference between terpene
fumigated and nonfumigated treatments: **, P < 0.01. (b)
Photograph showing the lower leaf damage under terpene
fumigation in non-photorespiratory conditions.
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236 Research
monoterpene capacity to scavenge reactive oxygen species
seems a better explanation for these results than membranestabilization capacity. Sixth, these results warrant further studies to deal with the biochemical processes that are involved in
these terpene and photorespiration effects on thermotolerance (the competition for electrons between terpene synthesis
pathways and photorespiration is a possible one) and the
physiological and ecological role of differential terpene synthesis among species and conditions.
Acknowledgements
We thank F. Rodà and J. Terradas for insightful comments
on the manuscript, and J. Casadesús and Servei Camps
Experimentals Fac. Biologia University Barcelona for technical
assistance. This research was supported by CICYT grants
REN2000-0278/CLI and REN2001-0003/GLO, and Generalitat
grant DURSI-DMA IMMPACTE 1999.
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