Leaves of the monoterpene emitter Quercus ilex were exposed to a temperature ramp with 5 °C steps from 30 to 55 °C while maintained under conditions in which endogenous emission of monoterpenes was allowed or suppressed, or under fumigation with selected exogenous monoterpenes. Fumigation with monoterpenes reduced the decline of photosynthesis, photorespiration and monoterpene emission found in non-fumigated leaves exposed to high temperatures. It also substantially increased respiration when photosynthesis and photorespiration were inhibited by low O2 and CO2-free air. These results indicate that, as previously reported for isoprene, monoterpenes may help plants cope with heat stress. Monoterpenes may enhance membrane stability, thus providing a rather non-specific protection of photosynthetic and respiratory processes. Monoterpene emission was maximal at a temperature of 35 °C and was inhibited at higher temperatures. This is likely to be the result of the temperature dependency of the enzymes involved in monoterpene synthesis. In contrast to other monoterpenes, cis- and trans-β-ocimene did not respond to exposure to high temperatures. Cis-β-ocimene also did not respond to low O2 or to fumigation. These results indicate that cis and trans-β-ocimene may have a different pathway of formation that probably does not involve enzymatic synthesis.
Many plants emit isoprene and monoterpenes. Monoterpenes may serve to attract pollinators, or as a deterrent, to protect plants against insects and animals (Harborne 1991). Isoprene emission from leaves may be related to a flowering stimulus (Terry et al. 1995). One of the most intriguing hypotheses is that isoprene is formed and emitted to protect leaves against high-temperature damage (Sharkey & Singsaas 1995). Isoprene could stabilize cell membranes by binding either the lipid bilayer or the protein membrane interface, or the photosystem II subunits (Sharkey 1996). This could offer a general protection against environmental constraints, including water stress (Sharkey & Loreto 1993).
Monoterpenes, as well as isoprene, are emitted at higher rates under high temperatures (Tingey, Turner & Weber 1991; Loreto et al. 1996a). Laboratory studies generally report the emission of monoterpenes from Quercus leaves at temperatures lower than 30 °C (Staudt & Seufert 1995; Loreto et al. 1996a). The total emission of monoterpenes has been measured at temperatures up to 45 °C in the field (Bertin et al. 1997). However, this field study was carried out with branch cuvettes and under conditions that did not allow a careful separation of the effects on monoterpene formation and emission of temperature, light, humidity and leaf age and health. Temperature dependence of isoprene emission is caused by the thermal activation of the isoprene synthase enzyme (Monson et al. 1992). Monoterpene synthesis, however, is likely to be controlled by many enzymes (monoterpene cyclases) whose thermal activation is unknown.
The present experiments were designed to determine the response of monoterpene emission to high temperatures and to understand whether monoterpene increases plant thermotolerance. For our experimental species we chose Quercus ilex, a tree that does not emit isoprene in detectable amount but emits monoterpenes formed, as in the case of isoprene, from freshly fixed carbon but not stored in a reservoir (Staudt & Seufert 1995; Loreto et al. 1996a,b). Q. ilex grows in the Mediterranean area where it has to cope with dry and hot summers and monoterpene synthesis may have evolved as a mechanism to tolerate these conditions. We measured gas exchange, fluorescence characteristics and monoterpene emissions of Q. ilex leaves exposed to temperatures increasing from 30 to 55 °C. We maintained the leaves under conditions that allow or inhibit monoterpene emission. As most monoterpenes emitted by Q. ilex leaves are formed by carbon just fixed by photosynthesis (Loreto et al. 1996b), we inhibited monoterpene emission by turning off carbon fixation (using low O2 and CO2-free air). Finally, we checked if monoterpene protection against high temperatures could be induced or restored by fumigation with exogenous monoterpenes. We fumigated leaves exposed to increasing temperatures with monoterpenes selected from those emitted by Q. ilex. These included the most abundant monoterpene emitted (α-pinene), a monoterpene that may be interconvertable to other monoterpenes (sabinene), and limonene and cis-β-ocimene, which may be formed from different carbon sources or whose synthesis is related to stress factors (Loreto et al. 1996c).
MATERIAL AND METHODS
Plant material and analysis of experiments
Plants of Q. ilex were grown for 3 years in 50 dm3 pots in commercial soil and were regularly fertilized and watered. Potted plants were maintained in a climatized greenhouse at the Environment Institute of the EC-Joint Research Centre (Ispra, Va, Italy). This setting was chosen to simulate growth in the Mediterranean environment but also to avoid environmental stresses. Temperatures never dropped below 5 °C during winter nor exceeded 35 °C during summer.
All of the present experiments were conducted in triplicate by choosing one sample on three different plants. We present results as means ± SD. The significance of differences between means was tested using a t-test. In Fig. 1, single measurements are reported to make the figure less crowded and as clear as possible, emphasizing the temperature dependence of different monoterpenes rather than the mean differences among terpenes. The replications confirmed the temperature dependence shown in Fig. 1. ANOVA of the three replications indicated that the standard error of measurements reported in Fig. 1 was in all cases lower than 15%.
Protocol of the experiments, gas exchange and fluorescence measurements
A single plant was transferred to a walk-in growth chamber (Heraeus Voetsch, HB410 MOPS, Belingen, Germany) in which temperature, photon flux density, and relative humidity could be varied between 5 and 60 °C, 0 and 2000 μmol photons m–2 s–1 and 0 and 80% RH, respectively. A group of two to four leaves was enclosed in a gas-exchange cuvette (Walz, Effeltrich, Germany) that also allowed insertion of a polyfurcated optical fibre through a window in the top. One branch of the optical fibre was connected to a light source (KL1500, Schött) that generated a saturating (10 000 μmol photons m–2 s–1) pulse of light. Two other branches of the optical fibre were attached to a PAM 101 modulated fluorometer (Walz) to convey the measuring light emitted by the instrument to the leaf and the fluorescence emitted by the leaf to the instrument. The cuvette system is described in full detail elsewhere (Loreto et al. 1996a).
The cuvette temperature was raised in 5 °C steps from 30 to 55 °C by using Peltier thermoelectric modules (Walz). Each temperature could be reached within 3–5 min. The leaves were maintained at each temperature for 30 min before measuring gas-exchange and chlorophyll fluorescence. After the measurement at 55 °C, the cuvette temperature was returned to 30 °C and the recovery of the gas exchange and fluorescence parameters was measured after 2 h of adaptation. During measurements, the rest of the plant was maintained at a temperature 5 °C lower than the temperature of the cuvette to maintain a minimum gradient of temperature between the leaves where measurements were performed and the other leaves. Before starting the temperature ramp, the plant was maintained in the dark for 15 min and the ratio between variable and maximal fluorescence (Fv/Fm) was measured to ascertain that the photochemical efficiency of the chosen leaves was not impaired. Then the growth chamber lights were switched on and the whole plant was maintained under a photon flux density of 700 μmol m–2 s–1 throughout the temperature ramp.
In the cuvette, the humidity was maintained at about 40% throughout the measurements by bubbling the air through water and condensing the excess humidity by passing the gas stream through a coil in a water bath. The air composition was set by mixing N2, O2, and 100 mmol mol–1 CO2 in N2 with mass flow controllers (Brooks 5850 series E, Veenendaal, Holland). The temperature ramp was repeated on leaves exposed to four different air compositions, each of these corresponding to a treatement. Treatment (1) was ambient (200 mmol mol–1 O2, 350 μmol mol–1 CO2), and treatment (2) was low O2 (20 mmol mol–1) and CO2-free air. Leaves exposed to these two treatments will be referred to as controls. Controls at 30 °C were also exposed to non-photorespiratory conditions (low O2 and ambient CO2) to investigate if stimulation of photosynthesis caused an increase of monoterpene emission. The other two treatments were: (3) ambient + fumigation with selected monoterpenes, and (4) low O2 and CO2-free air + fumigation with selected monoterpenes. Leaves exposed to these two treatments will be referred to as fumigated leaves.
Net photosynthesis was measured according to von Caemmerer & Farquhar (1981) using a differential infrared gas analyser (Binos, Leybold-Heraeus, Germany) to measure the difference between the CO2 concentration in the air flowing in and out the cuvette. Another Binos instrument was used to control absolute concentration of CO2 entering the cuvette.
Each measurement of photosynthesis was coupled to a measurement of the ratio of (maximal – steady state) to maximal fluorescence (ΔF/Fm), which represents the quantum yield of photosystem II (PSII) and which multiplied by the absorbed light intensity (αPAR) and by a factor (0·45) that accounts for the excitation partitioning between photosystems, allows calculation of the electron transport rate (J):
J =ΔF/Fm×αPAR × 0·45.
The electron transport rate driving photosynthesis and photorespiration may be estimated by comparing J and photosynthesis under photorespiratory and non-photorespiratory conditions, provided that the electron transport driving the direct photoreduction of O2 is negligible (Di Marco, Iannelli & Loreto 1994).
Terpene fumigation and analysis
The air reaching the cuvette was enriched with terpenes using the open tube diffusion system described by Staudt et al. (1995). Briefly, silanized glass tubes were filled with a single terpene. Ten cm3 of α-pinene, sabinene, cis-β-ocimene, trans-β-ocimene and limonene (Fluka, Buchs, Switzerland, 99% purity) were used. The tubes were of different size to account for the different diffusion rates of the monoterpenes used (Staudt et al. 1995). They were maintained in a water bath. Water temperature regulated the terpene concentration in the flowing air. We chose combinations of tube size and water temperature (10 °C) at which the terpene concentration in the air leaving the cuvette was two to four times higher than that of the endogenous emission (i.e. about 10 nmol m–2 s–1) The relative amount of the terpenes fumigated was maintained similar to that of emitted terpenes. A flux of pure N2 (50 cm3 min–1) continuously flushed the diffusion system. At the beginning of the experiment, this flux was added to the synthetic air reaching the cuvette. The total flux entering the cuvette was adjusted to 1 dm3 min–1 and the system was left to equilibrate for at least 1 h before starting the measurements. We substituted the fumigation system with monoterpene-free air to check if exogenous monoterpenes were adsorbed by the Teflon-coated cuvette or by other parts of the gas-exchange instrumentation. We found no monoterpene in the air exiting the empty cuvette 30 min after removing the fumigation system.
Part of the air exiting the cuvette was pumped at a rate of 100 cm3 min–1 through a water trap placed in ice and a Tenax TA trap placed above the ice bath to avoid water interference and breakthrough of volatile compounds (Staudt et al. 1995). After collecting 1 dm3 of air the trap was removed, thermally desorbed through a cold trap injector (TCT/PTI CP 4001, Chrompack, Middelburg, The Netherlands) and terpene content was analysed by gas chromatography using a fused silica capillary column (CP 9001 and CP-Sil 8 CB, Chrompack) and a flame ionization detector. Other details of the method are reported in Staudt et al. (1995).
The summed emission of the seven most abundant monoterpenes (α-pinene, β−pinene, sabinene, myrcene, cis-β-ocimene, trans-β-ocimene, limonene) was maximum at 35 °C and rapidly decreased at higher temperature (Fig. 1a). The relative contribution of the single monoterpenes was similar to that found in laboratory and field experiments (Loreto et al. 1996b; Bertin et al. 1997) with α-pinene the most abundant monoterpene emitted (not shown). The emission of single monoterpenes responded differently to increasing temperatures (Fig. 1b). α-Pinene, β-pinene, sabinene, myrcene and limonene emission was stimulated by temperatures up to 35 °C but reduced at higher temperatures. At 55 °C the emission of myrcene and limonene was higher than that measured at 30 °C. The emission of α-pinene, β-pinene and sabinene at 50 °C, on the other hand, was less than that measured at 30 °C and this reduction was more evident at 55 °C. Cis-β-ocimene and trans-β-ocimene emission were not significantly affected by the increasing temperatures. All monoterpenes showed an emission lower than the initial one when the temperature of 30 °C was restored.
Photosynthesis rapidly decreased when the leaves were exposed to temperatures higher than 30 °C (Fig. 2a). At 55 °C a loss of CO2 rather than an uptake was noticed. Recovery on return to 30 °C after the exposure to high temperatures was less than 50% in the short term (2 h). Fumigation with monoterpenes caused a small but non-significant increase in photosynthesis at 30 °C (12·1 ± 1·3 and 12·8 ± 1·1 μmol m–2 s–1 in control and fumigated leaves, respectively). However, fumigation improved the photosynthetic performances at temperatures higher than 30°C and the fumigated leaves recovered the original photosynthetic rates slightly but not significantly more than controls when temperature was again decreased to 30 °C. The estimated quantum yield of PSII (ΔF/Fm) of fumigated leaves was higher than in control leaves at all temperature (Fig. 2b). The difference was statistically significant at temperatures lower than 45 °C.
When the leaves were exposed to CO2-free and low O2 air, photosynthesis and photorespiration were inhibited. As a result, only a small CO2 evolution consequent to respiratory activity was observed (Fig. 3a). CO2 evolution increased with leaf temperature and was significantly higher in fumigated leaves than in control leaves. Recovery of initial photosynthesis at 30 °C was lower than in leaves maintained under ambient CO2 and O2, and again the recovery of fumigated leaves and control leaves was not different (compare upper panels of Figs 2 & 3). Chlorophyll fluorescence indicated again a slightly higher quantum yield of PSII in fumigated leaves than in control leaves but differences between the two treatments were small (Fig. 3b).
Monoterpene emission at 30 °C after the temperature ramp was generally lower than that measured before the heat stress (Fig. 4). Only in the case of cis- and trans-β-ocimene was no significant difference found. Fumigation noticeably increased the emission of monoterpenes after the temperature ramp with respect to the emission from control leaves. This effect was noticeable also for those monoterpenes that were not included in mixture used for fumigation (e.g. β-pinene). Only myrcene emission was not affected by fumigation. The emission of cis-β-ocimene, limonene and sabinene in fumigated leaves was comparable to that found before the heat stress in control leaves. In the case of α-pinene and β-pinene the emission from fumigated leaves was still lower than that found before the heat stress in control leaves.
Monoterpene emission was generally stimulated by low O2 but ambient CO2 concentration in the air (Fig. 5). The stimulation ranged from about 40–50% for α-pinene and β-pinene to about 20% in the case of limonene. This stimulation was similar to that of photosynthesis (12·1 ± 1·2 and 17·1 ± 1·0 μmol m–2 s–1 in ambient and low O2, respectively). Cis-β-ocimene was a notable exception as its emission was similar whether leaves were exposed to ambient or low O2 concentration.
Monoterpene emission under high temperatures
Exposure of Q. ilex to high temperature revealed that cis and trans-β-ocimene emissions are not sensitive to temperature. On the contrary, the other monoterpenes showed a strong temperature dependence with an optimum of 35°C. As α-pinene, β-pinene, sabinene and myrcene constitute the bulk of the emitted monoterpenes, the total emission also peaked at 35°C. This is consistent with the reported maximal emission of Q. ilex branches in the field (Bertin et al. 1997). However, the best fit of the experimental data collected in that field study show no emission at 45°C. In contrast, at 45°C we measured an emission reduced by about 30% compared with that measured at 35°C. Perhaps other environmental factors, such as water stress or photoinhibition, contribute to inhibit emission in the field at high temperatures. Even at high temperatures our measurements fit reasonably to the algorithm developed for predicting the temperature dependence of isoprene emission (Guenther et al. 1993; Bertin et al. 1997).
We interpret these data to indicate that monoterpene synthesis is generally under enzymatic control, with the exception of ocimenes. Cis and trans-β-ocimene are acyclic terpenes that can be formed from their intermediates (geranyl pyrophosphate and linalyl or neryl pyrophosphate, respectively) by simple and reversible protonation and deprotonation (Gleizes et al. 1982). Other terpenes taken into consideration in this study (α-pinene, β-pinene, sabinene) are apparently synthesized by means of cyclases (McGarvey & Croteau 1995). Perhaps cyclases act at a later stage using the acyclic compounds as substrata and irreversibly forming cyclic monoterpenes (Gleizes et al. 1982). Our results indicate that only this step is controlled by temperature.
Loreto et al. (1996a) showed that α-pinene emission increased with temperatures between 20 and 30 °C and attributed the temperature dependence to cyclization of geranyl pyrophosphate (Croteau 1987). Our results confirm and expand this indication. If monoterpene emission is not regulated, for instance by the substrate availability, the temperature dependence of isoprenoid cyclization may be that of the overall process. In this case, presumably, the cyclases’ optimum temperature is 35 °C. This temperature is, however, lower than the optimum temperature for isoprene synthase, which catalyses the formation of isoprene (Silver & Fall 1991).
Monoterpene fumigation and leaf thermotolerance
Fumigation with monoterpenes apparently is able to improve tolerance to high temperature because it increases the ability to photosynthesize at temperature higher than 30 °C. It may also facilitate recovery after exposure to extreme temperatures, although our measurements show that this was not statistically significant. The comparison between the effect of fumigation on photosynthesis and on the electron transport rate (as calculated from ΔF/Fm) suggests that both photosynthesis and photorespiration are stimulated. Sharkey & Singsaas (1995) first reported that isoprene formation may induce tolerance to heat stress by stabilizing membranes. Monoterpenes may have the same function. Monoterpene emitter plants like Q. ilex grow in the Mediterranean region and have to cope with frequent water (Bertin & Staudt 1996) and heat stress. Monoterpenes are less volatile than isoprene and may be found in abundance in the leaves of emitting species (Loreto et al. unpublished results). Therefore the synthesis of monoterpenes may be an evolutionary adjustment to enhance membrane protection with respect to that offered by the more volatile isoprene.
When photosynthesis and photorespiration were suppressed, the endogenous synthesis of monoterpenes was also inhibited (Loreto et al. 1996a). The protective effect of exogenous isoprene from thermal stress was enhanced under conditions that inhibited endogenous formation (Sharkey & Singsaas 1995). In the present study, however, the rate of recovery of photosynthesis at 30 °C after temperature stress was lower in leaves exposed to conditions of low O2 and CO2-free air than in leaves maintained under ambient conditions (compare upper panels of Figs 2 & 3). Fumigation did not increase recovery in the absence of photosynthesis and photorespiration. A small but insignificant increase of the photorespiratory component of the electron transport in fumigated leaves was evidenced by the higher ΔF/Fm when photosynthesis was either suppressed (during the temperature ramp) or recovered to a lower extent than in controls (Fig. 3). We speculate that when photosynthesis and photorespiration are inhibited the cumulative stress caused by membrane denaturation and inefficient use of light and carbon is too strong for monoterpene to protect against it.
Monoterpene fumigation of leaves under low O2 and CO2-free conditions stimulated their release of CO2 when compared with non-fumigated leaves. We interpret this as a stimulation of mitochondrial respiration, through an unknown mechanism. Indirectly, this result provides evidence that terpene protection involves membrane stability. If membranes are not damaged every physiological process is stimulated. When photosynthesis and photorespiration cannot be carried out, then the stimulating effect of terpenes on mitochondrial respiration is discerned by the gas exchange technique.
As we found that fumigation with monoterpenes stimulates all processes involved with carbon fixation and respiration, we also advance the hypothesis that monoterpenes may have a hormone-like action, with a rather non-specific series of effects. This could also account for the reported effect of isoprene on flowering (Terry et al. 1995).
We fumigated with terpenes belonging to different classes (Loreto et al. 1996c) and could not discriminate the effect of a single compound. It may be possible that thermal protection is offered by a single terpene or that complete protection is achieved only when the full spectrum of terpenes emitted by Q. ilex (Loreto et al. 1996b) is given. More studies are also needed to examine the dose effect or if the thermal protection is enhanced by fumigation with increasing monoterpene concentration beyond that given in the present study (about two to four times the endogenous production).
Terpene emission in heat-stressed control leaves was significantly lower than before the stress (Fig. 4). Fumigated leaves, on the other hand, almost recovered the initial emission rate when returned to 30 °C. This result indicates that, in the absence of monoterpene fumigation, the effect of heat stress on terpene emission was as negative as that on photosynthesis and that the emission did not rapidly recover. As monoterpene emission under ambient air is associated with photosynthetic activity, this result also supports the indication that monoterpene fumigation contributes to protect leaves against thermal damages. It may be worthy of note that the protective effect of monoterpene fumigation was almost or totally absent in the case of cis-β-ocimene, one of the acyclic terpenes. This may be explained by the absence of temperature dependence observed for the emission of this class of terpenes (Fig. 1).
Monoterpene emission under non-photorespiratory conditions
When the leaf was maintained under non-photorespiratory conditions, the emission of monoterpenes was stimulated. This stimulation was generally similar to photosynthetic stimulation. We interpret this result as a further indication that monoterpenes are rapidly formed from carbon fixed in photosynthesis (Loreto et al. 1996a, 1996b). Therefore monoterpene emission may also increase when photosynthesis is stimulated by high CO2. The stimulation of monoterpene emission under non-photorespiratory conditions may be extremely important to predict the emission by Quercus forests in an atmosphere with rising CO2 concentrations. However, exposure to high CO2 often triggers the onset of feedback conditions that inhibit both photosynthesis and isoprenoid emission. For instance, Loreto et al. (1996a) did not observe any increase of α-pinene emission in Q. ilex leaves exposed to CO2 concentrations higher than ambient. Under high CO2 the emission of isoprene was even depressed in Q. rubra (Loreto & Sharkey 1990).
Interestingly, cis-β-ocimene emission was not influenced by O2 removal. Unfortunately, trans-β-ocimene data for low O2 are not available. However, this peculiar oxygen insensitivity of cis-β-ocimene, together with the temperature insensitivity of the two ocimenes, indicates that these acyclic monoterpenes may have a different biosynthetic pathway, possibly involving non-enzymatic formation.
We thank Dieter Droste and Uwe Schwarz for valuable technical support and Dr Paolo Ciccioli for helpful discussions. F.L. was supported by a EC-Joint Research Centre contract no. 12251-96-10 F1EI ISP I and by a CNR project (Foreste e Produzioni Forestali nel Territorio Montano).