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Keywords:

  • carotenoids;
  • dimethylallyl diphosphate (DMADP);
  • elevated CO2;
  • free air CO2 enrichment (FACE);
  • isoprene;
  • ozone (O3) uptake;
  • Populus

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Isoprene is the most important nonmethane hydrocarbon emitted by plants. The role of isoprene in the plant is not entirely understood but there is evidence that it might have a protective role against different oxidative stresses originating from heat shock and/or exposure to ozone (O3). Thus, plants under stress conditions might benefit by constitutively high or by higher stress-induced isoprene emission rates.
  • • 
    In this study, measurements are presented of isoprene emission from aspen (Populus tremuloides) trees grown in the field for several years under elevated CO2 and O3. Two aspen clones were investigated: the O3-tolerant 271 and the O3-sensitive 42E.
  • • 
    Isoprene emission decreased significantly both under elevated CO2 and under elevated O3 in the O3-sensitive clone, but only slightly in the O3-tolerant clone.
  • • 
    This study demonstrates that long-term-adapted plants are not able to respond to O3 stress by increasing their isoprene emission rates. However, O3-tolerant clones have the capacity to maintain higher amounts of isoprene emission. It is suggested that tolerance to O3 is explained by a combination of different factors; while the reduction of O3 uptake is likely to be the most important, the capacity to maintain higher amounts of isoprene is an important factor in strengthening this character.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isoprene is the most abundant hydrocarbon emitted by vegetation, and this emission may have large consequences for the chemistry of the atmosphere (Guenther et al., 1995; Wang & Shallcross, 2000; Karl et al., 2004; Yokouchi & Ambe, 2007). The genus Populus includes some of the highest isoprene-emitting species (Lenz et al., 2001). Populus species are widely used in short-rotation plantations that are becoming common in agroforestry (FAO, 2005), thus contributing heavily to the global isoprene pool released into the atmosphere. Global change is expected to influence heavily isoprene emission from plants. Global warming is expected to stimulate isoprene emission, but the effects of rising atmospheric CO2 and tropospheric ozone (O3) episodes remain uncertain, in particular because of the indirect and feedback effects that might occur at the ecosystem level (Arneth et al., 2008). A series of experiments showed a decrease of basal isoprene emission under elevated CO2 (Rosenstiel et al., 2003; Possell et al., 2004; Scholefield et al., 2004). Using different models, it was estimated that the positive effect of warming on global isoprene emission might be balanced by the negative effect of rising CO2 concentration (Arneth et al., 2008), with crucial repercussions on tropospheric ozone and aerosols (Liao et al., 2006). While we are aware of the important role of isoprene on the formation of O3, very little is known about the effect of elevated tropospheric O3 on the emission of isoprene or volatile organic compounds. In a few laboratory studies it was observed that isoprene emission might increase following exposure to a high level of O3 (Velikova et al., 2005a), although the effect seems to be modulated by the position and the age of the leaf (Fares et al., 2006). Different responses to O3 in terms of monoterpene emission rates were observed in various Mediterranean species (Llusiàet al., 2002). The role of isoprene in protection against oxidative stress deriving from O3 exposure has been more thoroughly investigated (Loreto & Velikova, 2001; Velikova et al., 2005b). In particular it has been shown that when isoprene is provided to nonemitting plants, O3 damage is considerably reduced, whereas O3 damage is induced when isoprene synthesis is inhibited in isoprene emitters (Loreto & Fares, 2007). Lerdau (2007) suggested that increasing the concentration of atmospheric O3 will favour isoprenoid-emitting species over nonemitting species because of the protective role of isoprenoids on O3 stress.

We measured isoprene emission in an O3-tolerant clone (271) and an O3-sensitive clone (42E) of aspen grown for several years under elevated O3 and elevated CO2, alone or in combination, at the AspenFACE facility. In a previous manuscript we showed that isoprene synthase (ISPS), the enzyme responsible for isoprene synthesis, was inhibited at both transcriptional and translational levels by treatment with O3. In this study we present data deriving from a large campaign carried out in early summer and aimed to establish whether the different O3 sensitivity of clones is related to O3 uptake and isoprene emission.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Site description

The experiment was carried out at the AspenFACE facility located in Rhinelander (Northern Wisconsin, USA). The AspenFACE experiment started in 1998 when 12 experimental plots were planted that underwent four different treatments, as follows: ambient CO2 and O3 (control); elevated CO2 and ambient O3 (CO2); ambient CO2 and elevated O3 (O3); and elevated CO2 and elevated O3 (CO2+ O3). The target for elevated CO2 at the AspenFACE is 560 ppm and for elevated O3 is 1.5× ambient concentration. Details on the layout of the plots and of the fumigation performances are given in the AspenFACE website (http://aspenface.mtu.edu). In our study, samples were collected only within the sectors occupied by the trembling aspen (Populus tremuloides Michx.) clones. Two clones were investigated, namely 271 (O3 tolerant) and 42E (O3 sensitive) (Isebrands et al., 2001; Karnosky et al., 2003). These two clones exhibited the most contrasting responses to O3 in a previous campaign focusing on the isoprene synthase gene and isoprene synthase protein (Calfapietra et al., 2007).

Leaf gas-exchange measurements

An intensive campaign was carried out from 26 June to 12 July 2006 to measure leaf gas exchange in six sun-exposed leaves from the upper canopy of each clone in each plot. Measurements were carried out on sunny days and from 10:00 to 16:00 h every day in order to measure parameters under homogeneous environmental and physiological conditions.

A LI-COR 6400 (LI-COR Inc., Lincoln, NE, USA) infrared gas analyzer was used for measurements of photosynthesis, stomatal conductance to water vapor diffusion (gs) and emission of isoprene under environmental conditions that were previously standardized (basal emission, Guenther et al., 1991). Leaf temperature was set at 30°C and photosynthetic photon flux density (PPFD) at 1000 µmol m−2 s−1. Measurements were carried out at the O3 and CO2 concentrations at which plants were growing. Peak hourly O3 concentrations during the campaign were 46 ppb and 65 ppb in the ambient and elevated O3 plots, respectively. To avoid CO2 oscillations during measurements, a fixed CO2 concentration of 560 and 370 ppm, simulating growth conditions in the different plots, was set with the LI-COR 6400 equipment. The outlet of the LI-COR 6400 cuvette was connected to a Fast Isoprene Sensor (Hills Scientific, Boulder, CO, USA) for online measurements of isoprene emission, as described by Hanson & Sharkey (2001).

Ozone uptake was calculated by multiplying data of O3 concentration and stomatal conductance to O3 during leaf gas-exchange measurements (Emberson et al., 2000). Stomatal conductance to water vapor diffusion (gs) was converted to O3 conductance by dividing the gs value by the coefficient of molecular diffusivity of O3 in water vapor (1.68), and assuming that the concentration of O3 inside the intracellular spaces of the leaf is approximately zero (Laisk et al., 1989).

After measurements were taken, leaves were immediately frozen in dry ice and then stored at −80°C until used in biochemical assays.

Biochemical assays

The assay for dimethylallyl diphosphate (DMADP) was carried out on tissue samples previously ground and subjected to acid hydrolysis with 1 m H3PO4 at 60°C for 30 min. The powdered leaf samples were incubated with 500 µl of H2O and 500 µl of H3PO4 in 4-ml vials sealed with inert caps. Pure N2 was continuously flushed in the vials at a flux of 100 ml min−1. The air exiting the vial was injected directly into the Proton Transfer Reaction-Mass Spectrometer (PTR-MS; Ionicon, Innsbuck, Austria). The amount of DMADP was calculated after measuring the amount of isoprene evolved by the hydrolysis (protonated m/z 69), as indicated by Fisher et al. (2001) and Brüggeman & Schnitzler (2002).

Standard DMADP (1 mg ml−1; Sigma-Aldrich, St Louis, MO, USA) was used at amounts of 2.5, 5, 10 and 20 µl for calibrations of the PTR-MS.

Total carotenoids were extracted in 96% ethanol and detected spectrophotometrically, as described in Calfapietra et al. (2003).

Statistics

Analysis of variance (ANOVA) was carried out to determine the effects of elevated CO2, elevated O3 and clone. The systat 11 software (Systat Software Inc., Richmond, CA, USA) general linear models procedure was used. Thereafter, the significance within each combination of clone, CO2 and O3 treatments was calculated using Fischer's multiple comparison procedure. Differences between means were considered significant at a P value of < 0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plants grown at elevated CO2 had higher photosynthesis but lower isoprene emission than controls grown at ambient CO2. The CO2 effect on photosynthesis was significant for both clones, whereas the effect on isoprene was only significant for the O3-sensitive clone 42E (Fig. 1, Table 1). Under conditions of elevated CO2, DMADP concentrations dropped considerably in both clones compared with the control, whereas the amount of carotenoids was only slightly affected.

image

Figure 1. Treatment effect calculated as treated (T) over control (C) for O3 uptake (squares), assimilation rates (triangles) and isoprene emission (circles) by aspen (Populus tremuloides) trees. Closed symbols, O3-tolerant clone 271; open symbols, O3-sensitive clone 42E.

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Table 1.  Values of leaf gas exchange, isoprene emission, and dimethylallyl diphosphate (DMADP) and carotenoid contents of the leaves in the aspen (Populus tremuloides) clones 271 (O3-tolerant) and 42E (O3-sensitive) in the four treatments
 ControlCO2O3CO2 + O3P values
27142E27142E27142E27142ECO2O3Clone
  1. The effect of CO2, O3 and clone is indicated by the P values in the last three columns at the right of the table (a significant effect is shown in bold). The interactions between these factors are never significant and therefore are not reported. Letters in superscript indicate differences identified from the post hoc multiple comparison using the Fischer's test. For each parameter, values with the same letter are not significantly different. Values are means (SE) (n = three plots). FW, fresh weight.

Assimilation (µmol m−2 s−1)12.9ab (0.3)13.7ab (0.3)17.4cd (0.7)20.2d (0.5)11.7b (1.1)12.7ab (1.6)15.4ac (1.4)15.1ac (1.8)0.0000.0100.184
gs (mol m−2 s−1) 0.20ab (0.01) 0.24ab (0.02) 0.15b (0.01) 0.26ab (0.03) 0.19ab (0.04) 0.28a (0.08) 0.16b (0.03) 0.20ab (0.05)0.2310.8780.027
Ci (ppm)207a (5)214a (8)292b (12)354b (15)203a (23)221a (32)299b (27)335b (28)0.0000.8720.054
Ozone uptake (nmol m−2 s−1) 5.2a (0.3) 6.3a (0.6) 3.5a (0.19) 6.1a (0.6) 7.9ab (2.5)11.7b (4.4) 6.3ab (1.7) 8.0ab (2.2)0.2330.0440.137
Isoprene emission (nmol m−2 s−1)47.1a (0.7)44.5a (2.6)41.6ab (3.6)34.4c (3.4)40.4abc (3.1)35.5bc (0.7)36.2bc (1.5)23.9d (1.3)0.0000.0000.001
DMADP (ng mg−1 FW) 2.6a (0.2) 2.5a (0.2) 1.8b (0.2) 1.8b (0.2) 1.9b (0.1) 2.0b (0.1) 1.8b (0.1) 2.1ab (0.1)0.0030.1730.561
Carotenoids (mg g−1 FW) 0.73a (0.03) 0.61a (0.08) 0.69a (0.06) 0.56a (0.03) 0.69a (0.10) 0.53a (0.03) 0.70a (0.01) 0.56a (0.04)0.8060.8050.043

Stomatal conductance to water vapor diffusion (gs) and O3 uptake were not significantly affected by elevated CO2, although a decrease was observed in clone 271 (Fig. 1, Table 1).

Elevated O3 induced a drop in both photosynthesis and isoprene emission, the latter being more affected. The DMADP concentration also decreased under conditions of elevated O3, but to a lesser extent than under elevated CO2. Ozone uptake was largely increased under elevated O3, especially in clone 42E which also showed a slight O3-induced increment of gs.

When elevated O3 was combined with elevated CO2, the stimulating effect of CO2 on photosynthesis was almost completely suppressed, especially in the case of clone 42E. The combination of elevated CO2 and O3 induced a large decrease in the isoprene emission rate, especially in clone 42E. The concentrations of DMADP were also reduced in comparison with the control, whereas the amount of carotenoids did not change significantly with treatments, but were lower in clone 42E than in clone 271.

Isoprene emission rates decreased linearly with the increase of intercellular CO2 concentration (Ci) for both clones and in both O3 treatments. At a given value of Ci, isoprene emission was lower in elevated O3 than in ambient O3, and was also lower in clone 42E than in clone 271 (Fig. 2).

image

Figure 2. Relationship between intercellular CO2 concentration (Ci) and isoprene emission rates in aspen (Populus tremuloides) O3-tolerant clone 271 (circles) and in the O3-sensitive clone 42E (triangles) under ambient (closed symbols) or elevated (open symbols) O3. Each symbol represents the value of one plot. R2 values range from 0.60 and 0.85 and P values from 0.04 to 0.008.

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Differences between clones became more evident when different isoprene emission rates and O3 uptake were compared. Ozone uptake was higher in clone 42E than in clone 271, whereas isoprene emission was higher in clone 271 than in clone 42E at all isoprene emission levels (Fig. 3). Interestingly, differences in O3 uptake between clones increased with the increase of O3 uptake (slope significantly different from 1, intercept not significantly different from 0). Differences in isoprene emission rates between clones were larger at low rates (corresponding to values under elevated O3) than at higher rates, as shown by the intercept being significantly different from 0.

image

Figure 3. Comparison between the aspen (Populus tremuloides) O3-tolerant clone 271 and the O3-sensitive clone 42E in O3 uptake (squares) and isoprene emission (circles). Each point represents the values of the two clones in each plot. The dotted line represents the 1:1 line. Fit line for O3 uptake has R2 = 0.91 and P < 0.0001, whereas for isoprene emission R2 = 0.72 and P < 0.001.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Both elevated CO2 and O3 induced a decrease in isoprene emission rates in closed canopy free air CO2 enrichment (FACE)-grown trees. However, the effect of elevated CO2 was significant for the O3-sensitive clone but not for the O3-tolerant clone. A reduction of isoprene emission under elevated CO2 was previously found in experiments in closed environments (Rosenstiel et al., 2003; Possell et al., 2004), in natural springs (Scholefield et al., 2004) and at different FACE sites (Monson et al., 2007), but no significant effect of elevated CO2 on isoprene emission was observed for Populus alba in the POP-EUROFACE experiment (Loreto et al., 2007). A decrease of isoprene emission with increasing Ci has been measured previously (Loreto et al., 2007; Monson et al., 2007). Here we show that this relationship exists in different clones and also under conditions of elevated O3. This confirms the observed CO2 sensitivity of isoprene synthesis, probably because the cytosolic source of carbon for isoprene is more efficiently competed for by respiratory processes under high CO2 (Rosenstiel et al., 2003; Loreto et al., 2007). For each value of Ci, isoprene emission was higher in the O3-tolerant clone than in the O3-sensitive clone, and higher under ambient O3 than under elevated O3.

The high Ci value measured in clone 42E was associated with a high gs value, which is typical of this clone (Darbah, 2007). The effect of clone on Ci was not fully significant (P = 0.054) because of the high variability of measurements. However, O3 uptake was higher in clone 42E than in clone 271 in all plots. In addition, the gs value of clone 42E plants was not reduced under elevated O3, whereas this is usually the case in other aspen clones (Karnosky et al., 2003). High Ci might be also the consequence of inefficient photosynthesis (Evans & Loreto, 2000). However, photosynthesis in the O3-sensitive and O3-tolerant clones was similar within each treatment and does not explain the differences observed of Ci. Variability in Ci was quite high and differences were strong both among clones and CO2 treatments, where values of the Ci/Ca ratio (where Ca is the ambient CO2 concentration) were found to be lower under ambient CO2 than under elevated CO2. This might be attributed to the onset of a hot and dry period that reached a peak later in the season and that affected trees under ambient CO2 to a major extent, mainly as a result of generally higher stomatal conductance.

Clone 42E was more sensitive to O3 than clone 271 in several experiments, showing decreased photosynthesis, decreased growth rates and increased visible symptoms (Isebrands et al., 2001; Wustman et al., 2001; Karnosky et al., 2003). In this study, however, the expected decline of photosynthesis under O3 was not observed. This may be attributed to the fact that the campaign was carried out quite early in the season. Noormets et al. (2001) showed that the negative effect of O3 becomes evident only in leaves with a high leaf plastochrone index (i.e. on older leaves undergoing a long exposure to O3). This finding is particularly important because it shows that the effect of O3 on isoprene synthesis is greater, or occurs earlier, than that on assimilation. Unfortunately, data on specific leaf area are not available for all clones and for all treatments and therefore it is not possible to assess whether variations observed on a leaf area basis for these clones would have been different on a leaf mass basis.

High O3 uptake is probably the main reason why clone 42E is very sensitive to O3 (Karnosky et al., 2003). However, isoprene emission may also play a role in O3 sensitivity. It has been demonstrated that plants emitting higher amounts of isoprenoids are more protected against oxidative stress, probably because isoprenoids can increase the cohesion between cellular structures and can scavenge O3 inside leaves (Loreto & Velikova, 2001; Loreto & Fares, 2007). This second hypothesis might lead to the idea that decreased isoprene emission rates under elevated O3 are a result of the fact that isoprene is reacting with O3 before exiting the leaf. However, it has been explained that the lifetime of isoprene in our measurement conditions is c. 8 h (Calfapietra et al., 2007) and therefore the contribution to O3 uptake as a result of scavenging by isoprenoids is important for monoterpenes but negligible for isoprene (Fares et al., 2008).

Isoprene emission was considerably lower in the O3-sensitive clone, particularly when the emission was reduced by elevated O3. Therefore, we suggest that the capacity to maintain higher levels of isoprene emission contributes to the O3 tolerance of clone 271.

Moreover, carotenoid content was higher in clone 271 leaves than in clone 42E leaves. Carotenoids are formed through the same biochemical pathway as isoprene (Laule et al., 2003) and are able to quench singlet oxygen and other reactive oxygen species (Asada, 2006). The finding that both isoprene and carotenoids are higher in clone 271 suggests that resistance to O3 is related to the activation of the entire metabolic pathway of isoprene formation, including both volatile and nonvolatile compounds with antioxidant action. However, our experiments do not rule out that isoprene emission and the amount of carotenoids remain higher in O3-tolerant lines because of independent O3 tolerance mechanisms that preserve isoprene emission capacity and carotenoids amount independently.

In a previous paper (Calfapietra et al., 2007) we showed that the level of isoprene synthase gene expression and amount of isoprene synthase protein were reduced in aspen clones grown under elevated O3, especially in sensitive clones. This report, on the other hand, shows that DMADP, the substrate for isoprene synthesis, decreased significantly only under elevated CO2. Therefore, the decline of isoprene emission observed when elevated CO2 and elevated O3 co-occur may be exacerbated by a combination of substrate limitation and enzyme limitation. Moreover the amount of DMADP was very similar between clones, suggesting that differences in isoprene emission between clones might be caused more by differences in the enzyme amount or activity rather than by the substrate concentration.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by a Marie Curie International Fellowship within the 6th European Community Framework Programme (‘GLOBALVOC’, contract MOIF-CT-2005-007692) and by the Italy-USA Bilateral Programme on Climate Change of the Italian Ministry of Environment. The AspenFACE experiment is funded by the Office of Science (BER), US Department of Energy, the National Science Foundation, the USFS Global Change Program, Michigan Technological University, and the USFS North Central Research Station. The authors wish to thank Tanja Falbel, Amy Wiberley, Angela Elwell and Isabel Nogues for help during the field and laboratory activities and Prof. Rich Lindroth for providing useful equipment during the field campaign.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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