Emission of Isoprene from Salt-Stressed Eucalyptus globulus Leaves

Abstract

Eucalyptus spp. are among the highest isoprene emitting plants. In the Mediterranean area these plants are often cultivated along the seashore and cope with recurrent salt stress. Transient salinity may severely but reversibly reduce photosynthesis and stomatal conductance of Eucalyptus globulus leaves but the effect on isoprene emission is not significant. When the stress is relieved, a burst of isoprene emission occurs, simultaneously with the recovery of photosynthetic performance. Later on, photosynthesis, stomatal conductance, and isoprene emission decay, probably because of the onset of leaf senescence. Isoprene emission is not remarkably affected by the stress at different light intensities, CO₂ concentrations, and leaf temperatures. When CO₂ was removed and O₂ was lowered to inhibit both photosynthesis and photorespiration, we found that the residual emission is actually higher in salt-stressed leaves than in controls. This stimulation is particularly evident at high-light intensities and high temperatures. The maximum emission occurs at 40°C in both salt-stressed and control leaves sampled in ambient air and in control leaves sampled in CO₂-free and low-O₂ air. However, the maximum emission occurs at 45°C in salt-stressed leaves sampled in CO₂-free and low-O₂ air. Our results suggest the activation of alternative non-photosynthetic pathways of isoprene synthesis in salt-stressed leaves and perhaps in general in leaves exposed to stress conditions. The temperature dependence indicates that this alternative synthesis is also under enzymatic control. If this alternative synthesis still occurs in the chloroplasts, it may involve a thylakoid-bound isoprene synthase.

The ecophysiology of isoprene emission from plants has been studied for years (Jones and Rasmussen, 1975). These studies unraveled the environmental control over the emission (Monson and Fall, 1989; Loreto and Sharkey, 1990; Fall and Monson, 1992; Sharkey and Loreto, 1993; Monson et al., 1994) and helped in elucidating the biochemical pathway of isoprene formation (Sharkey et al., 1991), which more recently has been conclusively identified (Lichtenthaler et al., 1997). This information is in turn useful for predicting the emission of this highly reactive biogenic compound and the consequent potential formation of tropospheric ozone (Guenther et al., 1991, 1995).

The genus Eucalyptus is one of the highest emitters of isoprene (Benjamin et al., 1996). Unlike many other isoprenoid-emitting plants, Eucalyptus spp. emit both isoprene and monoterpenes, but the emission of isoprene is by far more prevalent (Guenther et al., 1991; Pio et al., 1996; Street et al., 1997; He et al., 2000). Because of these characteristics, Eucalyptus has been chosen as the plant species more suitable for modeling purposes (Guenther et al., 1991).

Eucalyptus spp. are native to Australia (Zacharin 1978) but are nowadays among the more widely distributed tree genera around the world. In Mediterranean environments, in particular, they rapidly colonized large areas, particularly in the Iberian peninsula (Pio et al., 1996; Street et al., 1997). Because of their fast growth, Eucalyptus spp. trees were planted for commercial purposes, or for limiting land erosion of deforested lands. They became one of the main plant species in urban forests, city parks, and green belts around conurbation. Along the coastal shores of the Mediterranean, Eucalyptus spp. were planted because of two other peculiar functions: to break saline wind, and to adsorb saline water and potentially drain soils otherwise unexploitable for agricultural or tourism purposes. Consequently, Eucalyptus spp. groves often cope with saline stresses.

Environmental stresses have a strong effect on isoprene emission by plants. Isoprenoid emission is stimulated at elevated temperatures (Loreto and Sharkey, 1990), probably because of the concurrent activation of isoprene synthase (Monson et al., 1992). Water stress seems to have limited effect on isoprene emission even if photosynthesis is severely inhibited. When water stress is relieved, however, isoprene emission can transiently increase 3- to 4-fold over the prestress emission rate (Sharkey and Loreto, 1993).

Salt stress often mimics water stress in two ways. First, when moderate, both stresses limit CO₂ entry by reducing stomatal and mesophyll conductance; and second, when severe, they impair carbon metabolism (Delfine et al., 1998, 1999). We investigated the unknown effect of salt stress on isoprene emission of Eucalyptus globulus. Because of the wide distribution of E. globulus groves in saline soil, if salt stress affects isoprene emission, this environmental stress may have a relevant impact on the isoprene presence in the atmosphere over the Mediterranean countries. In particular, we wanted to test if, as in the case of water stress, recovery from moderate exposure to salt stress could lead to bursts of isoprene emission that are not taken into account by models predicting isoprene emission. We report results indicating that the processes leading to isoprene emission are by far more resistant than photosynthesis to salt stress, and that a secondary source of isoprene, likely to be independent of photosynthesis, is stimulated by salt-stress conditions.

RESULTS

Na accumulated rapidly in the leaves of E. globulus and was more than 8% of the leaf dry weight after 3 weeks of NaCl feeding (Fig. 1a). Irrigation with salt-free water decreased the internal concentration of Na but the content was still about 4% of the leaf dry weight after a 1-month recovery.

Figure 1.

Accumulation of Na (a) and effects of salinity on stomatal conductance (b), photosynthesis, and isoprene emission (c) of E. globulus leaves. Means ± se (n = 5 in a; n = 4 in b and c) are presented. Irrigation with water supplemented with 1% (w/v) NaCl was initiated at d 0. The dashed vertical line indicates the day at which the NaCl treatment was ended and plants were irrigated with salt-free water (recovery). b and c only refer to salt-stressed leaves.

Leaf stomatal conductance (Fig. 1b) and photosynthesis (Fig. 1c) were inhibited by Na accumulation. After 3 weeks both parameters were very low. Following a 2-week irrigation with salt-free water, leaf stomatal conductance and photosynthesis substantially recovered but these high values were not maintained and slowly dropped until the end of the experiment.

Isoprene emission was not severely affected by Na accumulation (Fig. 1c). We observed a slight reduction of the emission during the stress, which did not promptly recover when the stress was relieved. However, coincident with the maximum recovery of photosynthesis and stomatal conductance, isoprene emission showed a clear peak. The emission then dropped consistent with the reduction of the other physiological parameters.

Photosynthesis of salt-stressed leaves was inhibited at all light intensities with respect to controls (Fig. 2a). Isoprene emission, in contrast, was not inhibited at low-light intensity. At high light the emission of stressed leaves was marginally reduced with respect to the emission of controls. After inhibiting photosynthesis and photorespiration by CO₂ and O₂ removal (Fig. 2b), isoprene emission dropped with respect to that measured in ambient conditions, but it was still measurable. In particular, isoprene emission of salt-stressed leaves was significantly higher than that of controls at light intensities >200 μmol m⁻² s⁻¹. At the highest light intensity the emission of salt-stressed leaves was twice that of controls.

Figure 2.

Response of photosynthesis (black symbols) and isoprene emission (white symbols) to light intensity in control (circles) and salt-stressed leaves (triangles) under ambient air conditions (a) and when photosynthesis and photorespiration were inhibited by removing CO₂ and lowering O₂ concentration in the air (b). Means ± se (n = 4) are shown.

Photosynthesis was artificially reduced by lowering CO₂ concentration only. At the same intercellular CO₂ concentrations, photosynthesis was by far lower in salt-stressed leaves than in controls, but the compensation point between photosynthesis and photorespiration did not change (Fig. 3). Isoprene emission was not inhibited until intercellular CO₂ concentration was lower than 140 μmol mol⁻¹ in controls. In salt-stressed leaves, isoprene emission started to drop significantly only at intercellular CO₂ concentrations lower than 80 μmol mol⁻¹. Both in controls and salt-stressed leaves a significant amount of isoprene was still emitted in CO₂-free air.

Figure 3.

Response of photosynthesis and isoprene emission to intercellular CO₂ concentrations at external CO₂ concentration decreasing from ambient (350 μmol mol⁻¹). Symbol legend as in Figure 2. Means ± se (n = 4) are shown.

Photosynthesis was maximal at a leaf temperature of 25°C in control leaves (Fig. 4a). Temperatures higher than 35°C inhibited photosynthesis. In salt-stressed leaves the rate of photosynthesis was very low at all temperatures. Isoprene emission was stimulated by high temperatures and was maximum at about 40°C but decreased at higher temperatures under non-photosynthetic and non-photorespiratory conditions. As observed in Figure 2b, when photosynthesis and photorespiration were inhibited by CO₂ and O₂ removal, isoprene emission was reduced but still measurable (Fig. 4b). As for the light dependence of this emission, salt-stressed leaves emitted more isoprene than controls at all temperatures. Remarkably, the maximum emission of salt-stressed leaves maintained under CO₂-free and low-O₂ air was not observed at 40°C, as in the respective controls and in all leaves maintained in ambient air, but at 45°C, a temperature at which the emission in ambient conditions was already significantly reduced.

Figure 4.

Response of photosynthesis and isoprene emission to leaf temperatures from 15°C to 50°C in control and salt-stressed leaves under ambient air conditions (a) and when photosynthesis and photorespiration were inhibited by removing CO₂ and lowering O₂ concentration in the air (b). Means ± se (n = 4) are shown. Symbol legend as in Figure 2.

DISCUSSION

Salt stress significantly affected carbon assimilation but did not reduce to a large extent the emission of isoprene by E. globulus leaves. The reduction of carbon assimilation is partly caused by the increased resistances to CO₂ diffusion because of stomatal closure (Fig. 1b). However, the slope of the response of photosynthesis to intercellular CO₂ concentration (Fig. 3) is lower in salt-stressed than in control plants, which indicates that carbon metabolism is also directly affected by the stress as often reported (Seemann and Sharkey, 1986; Brugnoli and Björkman, 1992). Delfine et al. (1998) demonstrated that the slope of the photosynthesis to intercellular CO₂ concentration ratio does not correctly indicate Rubisco activity if mesophyll resistance is high. They also showed that mesophyll resistance increased during salt-stress episodes and was partially eased during recovery from the stress, and they suggested that diffusion resistance is solely responsible for photosynthesis limitation under moderate salt stress (Delfine et al., 1999). As reported by Delfine et al. (1999), we also found that photosynthesis and stomatal conductance almost completely, though transiently, recovered when salt accumulation was reversed (Fig. 1). Thus the inhibition of photosynthesis in salt-stressed E. globulus leaves might only be attributable to diffusion (stomatal and mesophyll) resistances.

Photosynthesis inhibition may inhibit all processes metabolizing photosynthetic carbon. Isoprene is formed from photosynthetic carbon as indicated by the light and CO₂ dependence (Loreto and Sharkey, 1990), and by the fast labeling by ¹³C (Delwiche and Sharkey, 1993). Isoprenoid synthesis in the chloroplasts was recently found to share a unique pathway involving glyceraldehyde-3-P and pyruvate (Lichtenthaler et al., 1997). Despite isoprene dependence on photosynthetic carbon, isoprene inhibition was very limited in salt-stressed leaves. A low and delayed inhibition of isoprene emission was found in severely water-stressed kudzu leaves (Sharkey and Loreto, 1993). Water-stress recovery resulted in an isoprene emission up to 5 times higher than in controls (Sharkey and Loreto, 1993). We also found a peak of isoprene emission in E. globulus leaves 2 weeks after rewatering with salt-free water. Our results therefore confirm that environmental stresses do not significantly affect, and that recovery from stresses can temporarily stimulate, the emission of isoprene. After the peak, isoprene emission dropped in leaves recovering from salt stress. This drop occurred in concert with the drop of photosynthesis and stomatal conductance; we attribute these simultaneous effects to leaf aging. It is known that salt-stress accelerates aging of leaves (Delfine et al., 1999) and that isoprene emission is strongly dependent on leaf age (Sharkey and Loreto, 1993).

Both water and salt stress primarily affect stomatal conductance. The isoprene burst in recovering leaves could therefore reflect the release of isoprene synthesized but not emitted because of stomatal closure. Isoprene is emitted through stomata (Fall and Monson, 1992). However, there are numerous reports showing that stomata are not able to regulate isoprene emission even if they are tightly closed (Loreto and Sharkey, 1990; Sharkey, 1991; Fall and Monson, 1992). Isoprene builds-up in the leaves compensating for the increased stomatal resistances to its diffusion in air (Fall and Monson, 1992). We found that isoprene emission does not drop until a low-intercellular CO₂ concentration is reached (Fig. 3). This effect was even exacerbated in salt-stressed leaves that maintained a similar emission rate at intercellular CO₂ concentration higher than 80 μmol mol⁻¹.

Photosynthesis inhibition by salt stress was clear at different light intensities and leaf temperatures. However, we found no significant differences between isoprene emission in controls and salt-stressed leaves at reduced light intensities (Fig. 2a), at very low-intercellular CO₂ concentration (Fig. 3), and at increasing temperatures (Fig. 4a). On the contrary, we found that isoprene emission was actually increased in salt-stressed leaves with respect to controls when the light dependence and the temperature dependence were tested under non-photosynthetic and non-photorespiratory conditions (Figs. 2b and 4b). It is known that isoprene emission is severely reduced but not totally inhibited by simultaneous removal of CO₂ and O₂ (Loreto and Sharkey, 1990). This indicates that additional, non-photosynthetic sources of carbon can be used to form isoprene. The isoprene-like synthesis of monoterpenes in Quercus ilex leaves was not totally inhibited in the dark and exposure to darkness of leaves after a complete labeling of the emission resulted in a increasing amount of unlabeled monoterpenes emitted by and contained in the leaves (Loreto et al., 2000). These findings also indicate the presence of alternative routes of isoprenoid synthesis. Our results indicate that these routes are activated by salt stress and that perhaps a similar effect may be exerted by a range of environmental and biotic stresses. In fact, this would help explain why isoprene emission is sustained while photosynthesis is inhibited both in water- and salt-stressed leaves. The activation of photosynthesis-independent routes of isoprene synthesis makes it unclear if isoprene emission can be modeled on the basis of the leaf photosynthetic properties (Niinemets et al., 1999) under stress conditions.

Perhaps the most remarkable difference between the emission of control and salt-stressed leaves maintained under non-photosynthetic and non-photorespiratory conditions was observed at very high temperatures. Both controls and salt-stressed leaves showed the typical temperature dependence of isoprene emission (Monson et al., 1992; Sharkey and Loreto, 1993; Harley et al., 1996), which mirrors the activation of isoprene synthase (Monson et al., 1992). However, isoprene emission was maximal at 40°C in control leaves, but at 45°C in salt-stressed leaves. These different maxima correspond to the top activities of the stromal and thylakoid-bound isoprene synthases (Fall and Wildermuth, 1998). The observed temperature dependency might therefore reflect the increasing contribution of the thylakoid-bound isoprene synthase to isoprene emission in salt-stressed leaves. However, the high variability of temperature optima for isoprene synthases (Fall and Wildermuth, 1998) does not justify more than speculations. Certainly, the enzyme catalyzing isoprene synthesis in salt-stressed leaves resists temperature denaturation or down-regulation more effectively than in controls. This may have an ecological explanation if isoprene becomes more important in protecting membranes against thermal denaturation (Sharkey and Singsaas, 1995) in salt-stressed leaves.

In conclusion, our experiments show that isoprene synthesis is resistant to salt stress and, confirming previous experiments on water-stressed leaves, suggest that isoprene emission is rather unaffected by the stress. Isoprene emission can even be transiently stimulated by salt (and water) stress, perhaps because of the activation of alternative routes of isoprene synthesis under stress conditions. The regular and even enhanced investment of carbon into isoprene formation under stress conditions encourages the idea that isoprene is involved in resistance mechanisms and should be considered when modeling the emission from Mediterranean vegetation recurrently exposed to salt stress.

MATERIALS AND METHODS

Three-year-old plants of Eucalyptus globulus were grown in open air during summer 1999 at the Consiglio Nazionale delle Ricerche, Istituto di Biochimica ed Ecofisiologia Vegetali experimental field, near Rome (42° N), under the environmental conditions typical of summer in the Mediterranean area (maximal temperature >35°, absence of rain). Twenty plants were grown in 50-L pots filled with sandy and fertilized soil and were irrigated daily to restore evapotranspiration losses and avoid water stress. At the beginning of August one-half of the plants were irrigated with water supplemented with 1% (w/v) NaCl. After 3 weeks of salt accumulation, corresponding to a very low photosynthesis of salt-stressed plants, the NaCl treatment was suspended and the effect of salt dilution by irrigation with salt-free water was followed for 1 month.

Na accumulated in the leaves was extracted from 150 mg of dry leaf mass with the procedure described by Delfine et al. (1999) and was measured by atomic emission spectrometry (ICP Plasma 40, Perkin-Elmer, Foster City, CA).

Photosynthesis and stomatal conductance were measured using the gas-exchange system described by Delfine et al. (1998). The leaf portion (5 cm²) clamped in the gas-exchange cuvette was maintained at temperatures between 15°C and 50°C and at light intensities between 0 and 1,100 μmol photons m⁻² s⁻¹. The cuvette was flushed with a 1 L of synthetic air made by mixing N₂, O₂, and CO₂, and did not contain ozone, isoprenoids, and other contaminants. The CO₂ dependence of the emission was checked at CO₂ concentrations between zero and ambient (350 μmol mol⁻¹). A set of measurements was done in CO₂-free air and low O₂ (20 mmol mol⁻¹) to inhibit both photosynthesis and photorespiration. Measurements of gas-exchange and isoprene emission during the salt-stress treatment and the following recovery were done at a leaf temperature of 30°C and under a light intensity of 1,000 μmol m⁻² s⁻¹. The relative humidity of the air was maintained at 40% to 50% during all measurements. The leaf was maintained at least 30 min in each experimental condition to reach steady photosynthesis and isoprene emission before measurements.

Isoprene emission was measured by diverting a part of the air exiting the cuvette (40 mL) into a gas-chromatograph (Syntech GC855 series 600, Syntech, Groningen, The Netherlands). The air was pumped through a Graphite-Tenax (60–80 mesh, 8-cm) trap, desorbed at 240°C, transferred to a 13-m capillary column (i.d. 0.53 mm, packed with 95% [w/v] dimethylpolysiloxane, 5% [w/v] diphenylpolysiloxane) under a flow of pure N₂, and the isoprene present was detected after 198 s by photoionization at 10.6 eV. The gas chromatograph was calibrated with several concentrations of gaseous isoprene and monoterpenes and showed a linear response even at the highest monoterpene concentrations. Additional comparisons were done with simultaneous measurements of isoprene and monoterpenes trapped in carbon cartridges and analyzed by gas chromatography-mass spectrometry (Loreto et al., 1996).

All measurements were made on the last fully expanded leaf of tree branches. Na accumulation was measured in five leaf discs of different plants for each data point. Gas-exchange and isoprene emission measurements were repeated on four different leaves of different plants for each data point. Means and se of the measurements are shown. Measurements of photosynthesis, isoprene emission, and Na accumulation were repeated approximately every week during the stress and the following recovery as shown in Figure 1. Measurements of the light, CO₂, and temperature dependency (Figs. 2–4) were made in controls and salt-stressed leaves when Na accumulation was maximum (18–22 d after starting the treatment).