Article pubs.acs.org/est
Bidirectional Flux of Methyl Vinyl Ketone and Methacrolein in Trees with Different Isoprenoid Emission under Realistic Ambient Concentrations Silvano Fares,*,† Elena Paoletti,‡ Francesco Loreto,§ and Federico Brilli∥ †
Council for Agricultural Research and EconomicsResearch Center for the Soil-Plant System, Rome 00184, Italy National Research Council, Institute for Sustainable Plant Protection (IPSP), Firenze 1-50019, Italy § National Research Council, Department of Biology, Agriculture and Food Science (DISBA), Rome 7-00185, Italy ∥ National Research Council, Institute of Agro-Environmental and Forest Biology (IBAF), Rome 7-00185, Italy ‡
ABSTRACT: Methyl vinyl ketone (MVK) and methacrolein (MAC) are key oxidation products (iox) of isoprene, the most abundant volatile organic compound (VOC) emitted by vascular plants in the atmosphere. Increasing attention has been dedicated to iox, as they are involved in the photochemical cycles ultimately leading to ozone (O3) and particle formation. However, the capacity of plants to exchange iox under low and realistic ambient concentrations of iox needs to be assessed. We hypothesized that a foliar uptake of iox exists even under realistic concentrations of iox. We tested the capacity of iox exchange in trees constitutively emitting isoprene (Populus nigra) or monoterpenes (Quercus ilex), or that do not emit isoprenoids (Paulownia imperialis). Laboratory experiments were carried out at the leaf level using enclosures under controlled environmental factors and manipulating isoprene and reactive oxygen species (ROS) production by using the isoprene specific inhibitor fosmidomycin, acute O3 exposure (300 ppbv for 4 h), and dark conditions. We also tested whether stress conditions inducing accumulation of ROS significantly enhance iox formation in the leaf, and their emission. Our results show a negligible level of constitutive iox emission in unstressed plants, and in plants treated with high O3. The uptake of iox increased linearly with exposure to increasing concentrations of ambient iox (from 0 to 6 ppbv of a 1:1 = MVK/MAC mixture) in all the investigated species, indicating iox fast removal and low compensation point in unstressed and stressed conditions. Plant capacity to take up iox should be included in global models that integrate estimates of iox formation, emission, and photochemical reactions in the atmosphere.
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isoprene with stress-induced ROS.7 MVK has been reported to accumulate in heat-stressed plants and to suitably indicate stress occurrence.8 MVK is by itself a potentially cytotoxic molecule9 and must be rapidly removed from the mesophyll.7 Within-leaf detoxification might be possible,10 as ecosystem-scale measurements and laboratory observations demonstrate enzymatic catabolism of iox.11 The exchange of gases (including VOCs) between leaves and the atmosphere can occur bidirectionally and is mainly regulated by the gradient of the gas between the internal concentration in leaves and the atmosphere, by boundary-layer resistance, and by stomatal resistance, stomata being the main entry/exit port for all gases.6 The uptake of VOCs by leaves occurs only when the VOC concentration is higher outside than inside the leaves. Uptake of aldehydes and ketones by leaves of several tree species was reported by Kondo et al.12 and Tani et al.13,14 in laboratory experiments. Seco et al.15 showed that plants take up a range of oxygenated VOCs, including
INTRODUCTION Plants produce Volatile Organic Compounds (VOCs), whose emissions to the atmosphere account for ∼1 Pg per year.1 Among plant VOCs, isoprenoids are the most abundant class, with isoprene representing about half of the total biogenic VOCs emitted globally.1 Isoprene oxidation generates peroxy radicals (RO2) which can react with HO2 and NO. The predominant reaction products are methacrolein (MAC) and methyl vinyl ketone (MVK) (referred to together as iox) when the atmosphere is affected by anthropogenic emissions (NOdriven reaction pathway), whereas organic hydroperoxides can be formed in a more pristine environment (HO2-driven reaction pathway).2−4 A series of complex photochemical reactions driven by iox ultimately leads to formation of tropospheric ozone (O3) and secondary organic aerosols, depending on concentrations of OH radicals, O3, and nitrogen oxides (NOx).5 The capacity of plants to produce iox has therefore received increasing attention. There is now concrete evidence that oxidation products may be formed within the leaf, when isoprenoids react with Reactive Oxygen Species (ROS) prior to their emission into the atmosphere (recently reviewed in ref 6). In particular, stress conditions were shown to increase the emission rate of iox, possibly because of the interaction of © XXXX American Chemical Society
Received: February 6, 2015 Revised: May 27, 2015 Accepted: June 1, 2015
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DOI: 10.1021/acs.est.5b00673 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
for 10−15 min until observing stable rates of gs. Leaves were maintained at a temperature of 30 °C, and the RH was adjusted to be stable at ∼50% at the cuvette outlet, using the control system detailed by Loreto et al.17 Precise concentrations of iox (five concentration steps of 0, 0.75, 1.5, 3, and 6 ppbv of a 50% MVK and 50% MAC gas mixture) were added to the airflow entering the cuvette using a certified gas cylinder (SIAD s.r.l., Bergamo, Italy) connected to mass-flow controllers (Brooks Instr., The Netherlands). For each concentration level, fumigations lasted 5 min or more, depending on the time needed to reach a steady state iox uptake inside the cuvette. Between fumigations, pure air was flushed in the cuvette with the intent to test whether exposure to increasing concentrations of iox led to foliar emissions of these reaction products. The concentration of CO2 and H2O in the cuvette incoming and outgoing air was monitored continuously using an LI-7000 infrared gas analyzer (Li-Cor, Lincoln, NE, U.S.A.). The concentration of VOCs was also monitored online by diverting part of both the inlet and the outlet air into a proton transfer reaction mass-spectrometer (PTR-MS; Ionicon, Innsbruck, Austria). The PTR-MS can detect VOC-protonated ions or fragments at the ppt level.18 For our experiments, we used an E/N (where E is the electric field in the drift tube, and N is the gas number density) ≈ 130 Td, by setting a drift-pressure of 2.2 mbar drift temperature of 40 °C, and a drift voltage of 600 V. In addition, the PTR-MS was set in a single-ion detection mode (MID) to improve the recording of traces of protonated formaldehyde (m/z 31), methanol (m/z 33), acetaldehyde (m/ z 45), acetone (m/z 59), isoprene (m/z 69), MVK+MAC (m/z 71), monoterpenes (m/z 137), and their main fragment (m/z 81), and green leaf volatile fragments (m/z 81 and 83). For each VOC, the PTR-MS was calibrated daily with gaseous standards (Apel-Riemer, CO, U.S.A.). In order to account for possible effects of (slightly) different levels of RH on the PTRMS sensitivity for the measured VOC-protonated ions, we normalized the PTR-MS raw signals (cps) to ncps, as previously reported.19,20 Fluxes of VOCs were calculated by the difference between the inlet and the outlet normalized concentration as described by Fares et al. 21 Possible contaminations due to release or absorption of VOCs by cuvette walls were assessed daily by measuring the VOC concentration exiting the empty cuvette, in the presence or absence of iox fumigation. These measurements never revealed significant differences between inlet and outlet concentrations of iox for the VOCs analyzed. The detection limit of PTR-MS for m/z 71 was 0.26 ± 0.05 ppbv (obtained by setting a dwell time of 1 s). In an additional experiment, the isoprene inhibitor fosmidomycin (Sigma-Aldrich, MO, U.S.A.) was fed to P. nigra detached leaves as an aqueous solution (5 μmol) which was taken up through the transpiration stream following the procedure reported by Velikova et al.22 Gas exchanges were analyzed using the system shown above. In a third experiment, entire branches of P. nigra were fumigated with O3 in a custom-made dynamic enclosure (100 L). The branches were exposed to synthetic air made as described above, and enriched with O3 produced by a UV light source (Heliozon, Milan, Italy) at a rate of 7 L min−1, mixed with a mass flow controller (Model GFC171, Aalborg, New York, U.S.A.) and monitored by an O3 sensor (Model 205, 2B Technologies, Boulder CO, U.S.A.). O3 concentration in the enclosure was maintained at 300 ppbv for 4 h. Measurements of the gases exchanged were carried out immediately after
formic and acetic acids, acetone, formaldehyde, acetaldehyde, methanol, and ethanol. It was demonstrated that VOCs with low gas−liquid partition coefficients are prevalently removed by plants via stomata, while cuticle deposition is minimal.15 In all cases, the uptake of VOCs stops when a compensation point (CP, the point at which the internal and external concentration of a VOC are equivalent) is reached. A CP characterizes VOCs constitutively produced in leaves or formed from within-leaf oxidation of primary VOCs, or gases that are taken up through stomata but then accumulate in the mesophyll, without being metabolized. Iox also follow this general rule, and changes in the iox CP may reveal the presence of within-leaf oxidation of constitutively produced isoprenoids, or the accumulation of iox taken up by leaves and insufficiently scavenged within the leaves. Tiny changes of iox CP could be relevant for the iox budget in the plant−atmosphere continuum, since the atmospheric concentration of iox is very small, ranging from 0 to 3 ppb.16 So far, very few studies have focused on the uptake of iox under realistic iox concentrations typically recorded in air.14 In order to test our hypothesis that plants may take up iox even under realistic iox concentrations, we designed a laboratory experiment using dynamic enclosures that allowed iox exchanges to be measured in leaves exposed to accurately set concentrations of iox under controlled environmental conditions. We used tree species that were selected either for their capacity to produce isoprenoids (Populus nigra and Quercus ilex as isoprene and monoterpenes emitters, respectively), or that do not produce isoprenoids (Paulownia imperialis). The specific goals of our experiments were to test whether (a) the capacity to exchange iox depends on isoprenoid emission and (b) the exchange of iox by isoprene emitters is affected by a short but acute exposure to high O3 concentration that may enhance isoprene oxidation and formation of iox within leaves.
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MATERIALS AND METHODS Plant Material. One-year-old seedlings of P. nigra and three-year-old seedlings of Q. ilex and P. imperialis were purchased from local nurseries, and potted in buckets of 15 L volume. Seedlings were grown in a greenhouse for one year, before they were used for experiments. All plants were potted in the same commercial soil and irrigated as well as fertilized regularly to prevent drought stress and nutrient shortage. Experimental Setup and PTR-MS Measurements. We used a 0.5 L gas exchange cuvette (Waltz, Germany) that guarantees control of the environmental parameters (light intensity, temperature, relative humidity-RH, and CO 2 concentration). Due to limited boundary layer development, this cuvette allows faster and more precise calculations of stomatal conductance (gs) and photosynthesis (A)17 than the large enclosures flushed with low airflow employed to determine iox exchange. One or more leaves covering a surface ranging from 35 to 50 cm2 were enclosed in the cuvette, and flushed with a 1.5 L min−1 flux of synthetic air. Synthetic air was prepared by precisely mixing gases with mass flow controllers (Model GFC171, Aalborg, New York, U.S.A.) to reconstitute ambient air composition (80% N2, 20% O2 and 390 ppmv CO2). The air was cleaned by contaminants (e.g., VOC) using a zero-air generator (Model 737−12A, Aadco, Cleves OH, U.S.A.). During the experiments, leaves were either exposed to a full-spectrum light (Osram-Power Star 1000 HQT source, Osram, Munich, Germany) providing a photosynthetic active radiation (PAR) of 700 μmol photons m−2 s−1), or darkened B
DOI: 10.1021/acs.est.5b00673 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology exposure to O3 in the 0.5 L gas exchange cuvette, using the experimental setup described above. Data Analysis. All experiments were replicated 7 times on leaves belonging to different individual plants for each treatment. Data were tested using a one-way analysis of variance (ANOVA). Means were compared among plant species and treatments according to the Holm−Sidak method with a significance level = 0.05 using Sigma Plot version 11.0 (Systat Software Inc., San Jose, CA, U.S.A.). After testing for normality (Kolmogorov−Smirnov test) and homoscedasticity (Levene’s test), slopes of the significant regression lines were compared by analysis of covariance (ANCOVA). When the slopes were significantly different, it was not possible to test whether the intercepts (i.e., the compensation points) were significantly different.
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RESULTS VOC Emission Rates and Physiological Parameters. The main VOCs that were constitutively emitted from the tree species selected for our study, under physiological (unstressed) conditions (leaf temperature = 30 °C and PAR = 700 μmol m−2 s−1), are shown in Figure 1a. Leaves of P. imperialis showed negligible constitutive emissions of VOCs (
