Plant surface reactions: an ozone defence mechanism impacting atmospheric chemistry

Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. Plant injuries have been linked to the uptake of ozone through stomatal pores and oxidative damage of the internal leaf tissue. But a striking question remains: can surface reactions limit the stomatal uptake of ozone and therefore reduce its detrimental effects to plants? 5 In this laboratory study we could show that semi-volatile organic compounds exuded by the glandular trichomes of different Nicotiana tabacum varieties are an efficient ozone sink at the plant surface. In our experiments, different diterpenoid compounds were responsible for a strongly variety dependent ozone uptake of plants under dark conditions, when stomatal pores are almost closed. Surface reactions of ozone were accompanied by a prompt release of oxygenated volatile organic com10 pounds, which could be linked to the corresponding precursor compounds: ozonolysis of cis-abienol (C20H34O) – a diterpenoid with two exocyclic double bonds – caused emissions of formaldehyde (HCHO) and methyl vinyl ketone (C4H6O). The ring-structured cembratrien-diols (C20H34O2) with three endocyclic double bonds need at least two ozonolysis steps to form volatile carbonyls such as 4-oxopentanal (C5H8O2), which we could observe in the gas phase, too. 15 Fluid dynamic calculations were used to model ozone distribution in the diffusion limited leaf boundary layer under daylight conditions. In the case of an ozone-reactive leaf surface, ozone gradients in the vicinity of stomatal pores are changed in such a way, that the ozone flux through the open stomata is strongly reduced. Our results show that unsaturated semi-volatile compounds at the plant surface should be consid20 ered as a source of oxygenated volatile organic compounds, impacting gas phase chemistry, as well as efficient ozone sink improving the ozone tolerance of plants.


Introduction
Tropospheric ozone (O 3 ) is formed as a product of photochemical reactions involving nitrogen oxides (NO x ) and volatile organic compounds (VOC) as precursors (Jenkin and Clemitshaw, 2000). 25 Increasing anthropogenic precursor emissions from fossil fuel and biomass burning have led to elevated ambient ozone concentrations over large portions of the earth's surface. Today, many regions experience near-ground ozone background levels greater than 40 parts per billion volume (ppbv) (Vingarzan, 2004), levels which may be responsible for cellular damage inside leaves (Hewitt et al., 1990;Wohlgemuth et al., 2002) adversely affecting photosynthesis and plant growth (Ashmore, 30 2005). Toxic ozone concentrations cause visible leaf injury, plant damage and reduction in crop yields with associated economic costs of several billion dollars per annum worldwide (Wang and Mauzerall, 2004;Van Dingenen et al., 2009). Future trends of tropospheric ozone strongly depend on the emission factors of the corresponding precursor compounds (i.e. VOC and NO x ) and indirectly also on land cover and characteristics of the vegetation (Dentener et al., 2006;IPCC, 2013;Fu 35 and Tai, 2015). Some recent studies revealed a stabilization or even a lowering of the tropospheric background ozone concentrations in parts of the industrialized western countries since the turn of the millennium (Logan et al., 2012;Parrish et al., 2012;Oltmans et al., 2013;IPCC, 2013). This is likely a result of preventive measures reducing ozone precursor emissions (Granier et al., 2011).
In contrast, ozone background concentrations are still rising in parts of Asia experiencing high eco-40 nomic growth and a concomitant increase in NO x emissions (Granier et al., 2011;Fu and Tai, 2015).
Land cover and land use changes, often determined by changing climatic conditions, could impact tropospheric ozone in different ways: A higher leaf area index of the vegetation would enhance dry deposition of ozone (Fu and Tai, 2015). In low NO x regions enhanced emissions of isoprene emitting species could decrease ozone concentrations, while they would lead to an ozone increase in high surfaces this approach is not fully correct and may lead to an overestimation of stomatal ozone uptake in the case of very reactive surfaces.
We present results from ozone fumigation experiments, in which intact leaves of different varieties of tobacco (Nicotiana tabacum) were exposed to elevated ozone levels (20-150 ppbv) under light and dark conditions in an exceptionally clean plant enclosure system (see Materials and methods section for experimental details). The Nicotiana tabacum species is famous for large differences in the ozone tolerance of the different varieties. For example, the Bel W3 is known to be very ozone sensitive (Heggestad, 1991;Loreto et al., 2001) and has therefore been used as an ozone indicator plant in earlier times (see Heggestad, 1991, and references therein). Conversely, the Bel B variety is known to be non-sensitive (Heggestad, 1991). The high ozone tolerance of this variety has been attributed to wider epidermal cells and more spongy mesophyll cell layers (Borowiak et al., 2010) 70 and to differences in the plant's ability to cope with oxidative stress once ozone has entered the stomata (Schraudner et al., 1998;Eltayeb et al., 2007).
Several studies were investigating the possibility to increase the ozone tolerance of plants by external application of ozone-scavenging compounds (Gilbert et al., 1977;Loreto et al., 2001;Vickers et al., 2009a;Singh and Agrawal, 2010;Agathokleous et al., 2014) or by enabling the emission of volatile 75 terpenoids in transgenic plants (Vickers et al., 2009b;Palmer-Young et al., 2015). We show here that some of the tobacco varieties investigated in our experiments are intrinsically equipped with ozone scavenging compounds located on their leaf cuticula. As is the case for many other plant species (Fahn, 1988), tobacco leaves possess glandular trichomes. In tobacco, various diterpenoids are the major compounds exuded by these secretory structures at the leaf surface (Sallaud et al.,80 2012). The exudates cover the plant leaves as a defence barrier, for example against arthropod pests (Wagner, 1991;Lin and Wagner, 1994); they were shown to have an anti-fungal (Kennedy et al., 1992) and insecticidal action (Kennedy et al., 1995). We show that in a tobacco variety secreting the diterpenoid cis-abienol, the exudates have a beneficial side-effect: they act as a powerful chemical protection shield against stomatal ozone uptake by depleting ozone at the leaf surface.

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Surface-assisted ozonolysis not only protects plants from uptake of phytotoxic ozone through stomata, but also acts as a source of volatile carbonyls into the atmosphere, impacting atmospheric chemistry. To our knowledge, our study reports for the first time on detailed measurements of plant surface-assisted ozonolysis of semi-volatile diterpenoids forming volatile carbonyl products.   (Sallaud et al., 2012). The new 3H02 line does not exude diterpenoids at all (see Appendix A).

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Seeds of the tobacco cultivars were obtained from the Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Halle. The plants were grown in the green houses of the Institute of Ecology of the University of Innsbruck for 8-10 weeks in standard soil.
Before being used in the experiments the sample plants were allowed to adapt 1-4 weeks in the laboratory, obtaining light from the same true light lamp type as used during the measurements (see 100 Setup section).
Plants were installed into the plant enclosure used for ozone fumigation the evening before the actual experiment, so they could adapt to the system and recover from possible stress during installation. The sample plants were well watered and in a good physiological condition and showed no visible signs of damage. At the beginning of the experiments, when no ozone was added, no 105 significant stress signals in form of green leaf volatiles were detected.
In total, combined dark and light ozone fumigation experiments were conducted with five Am-

Setup
In the present ozone experiments we used only inert materials such as Teflon ® , PEEK ® or Duran ® glass in order to minimise artificial side-reactions of ozone with unsaturated compounds, present in e.g. sealing materials like rubber. Moreover, special care was taken to avoid fingerprints, which could result in side reactions of ozone with skin oils (Wisthaler and Weschler, 2010). Ozone loss, 115 estimated from measured ozone concentrations at the inlet and outlet of the empty plant enclosure, was typically less than 5 %.
For plant fumigation, synthetic air 5.0 grade was mixed with CO 2 4.8 grade (both Messer Austria GmbH, Gumpoldskirchen, Austria). By bubbling the air in distilled water and passing it by a subsequent thermoelectric cooler (TEC) the relative humidity was set. Before entering the plant 120 enclosure, the air was flushed through an ozone generator (UVP, Upland (CA), USA). The enclosure system consisted of a desiccator (Schott Duran ® ) of 17.3 L volume, turned upside-down, and two end-matched PTFE ® ground plates. A central hole served as feed-through for the plant stem, possible gaps were sealed with Teflon ® tape. The (single-sided) leaf area enclosed was typically in the range of 250-850 cm 2 .

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An ozone detector (Model 49i, Thermo Fisher Scientific Inc. Franklin (MA), USA) and an infrared gas analyser (LI-840A CO 2 /H 2 O Analyzer, LI-COR ® inc., Lincoln (NE), USA) were sampling at 2 min intervals from either the inlet or outlet of the enclosure. Plant enclosure inlet ozone concentrations were typically kept constant throughout each experiment and were adjusted to obtain real-istic ambient ozone concentrations at the enclosure outlet during light conditions (e.g. ∼ 60 ppbv in  Examples of differentiable isomers are aldehydes and ketones. In the H 3 O + reagent ion mode, aldehydes and ketones both exhibit proton transfer and thus e.g. methyl vinyl ketone (MVK) and 150 methacrolein (MACR) are both detected as C 4 H 7 O + (m/z 71.050). In NO + reagent ion mode, most aldehydes exhibit hydride ion transfer and ketones clustering reactions, comparable to the ionization mechanisms in a SIFT instrument (Španěl et al., 1997). Thus MVK is detected as C 4 H 6 O q NO + (m/z 100.040), whereas MACR is detected as C 4 H 5 O + (m/z 69.034).
In addition to isomeric separation, the high flow through the drift tube (here: ∼ 500 mL min −1 155 compared to 10-20 mL min −1 in a standard instrument) allows for the first time the detection of semi-volatile compounds such as the diterpenoid cis-abienol (C 20 H 34 O).
The SRI-ToF-MS was operated under standard conditions, 60 • C drift tube temperature, 540 or 350 V drift voltage and 2.3 mbar drift pressure, corresponding to an E/N of 120 or 78 Td (E being the electric field strength and N the gas number density; 1 Td = 10 −17 V cm 2 ) in H 3 O + or 160 NO + reagent ion mode, respectively. The instrument was calibrated approximately once a week by dynamic dilution of VOC using 2 different gas standards (Apel Riemer Environmental Inc., Broomfield (CO), USA), containing ca. 30 different VOC of different functionality distributed over the mass range of 30-204 amu. Full SRI-ToF-MS mass spectra were recorded up to m/z 315 with a 1 s time resolution. Raw data analysis was performed using the PTR-ToF Data Analyzer v3.36 and v4.17 165 (Müller et al., 2013).

cis-abienol identification
For the identification of cis-abienol a pure standard was acquired (Toronto Research Chemicals, Toronto, Canada). The powder was dissolved in n-hexane and applied on the surface of a glass container, which was put into the enclosure system and treated like the plant samples. In H  , respectively. Ozonolysis of the pure cis-abienol standard yielded the same primary ozonolysis products (see below) as in the case of Ambalema plants.

Leaf stripping 180
In order to relate the observed ozonolysis carbonyls to plant surface reactions, leaf exudates of untreated tobacco plants were stripped off by dipping leaves (of similar area) of untreated Ambalema, Basma Drama and 3H02 plants into n-hexane (∼ 100 mL for 1000 cm 2 leaf area) for ∼ 1 min. The nhexane -leaf exudate solution was then distributed as evenly as possible onto the inner surface of the empty desiccator serving as plant enclosure. n-hexane evaporated quickly and was further reduced 185 by flushing the glass cuvette with pure synthetic air. Afterwards, ozone fumigation experiments were performed similar to the experiments with intact plants.

GC-MS analysis
Non-volatile ozonolysis products and unreacted surface compounds were analysed by GC-MS (see also Supplement). Directly after the ozone fumigation experiments we extracted leaf exudates and 190 low volatility ozonolysis products from the fresh tobacco leaves (see Leaf Stripping section). 1 µL portions of the samples were then injected directly into a GC-MS for analysis on a 6890 N gas chromatograph coupled to a 5973 N mass spectrometer (Agilent Technologies) according to the procedures described elsewhere (Sallaud et al., 2012).
Tobacco diterpenoids were identified on the basis of their mass spectra, as described in the litera-195 ture (Enzell et al., 1984).

Calculation of leaf gas exchange parameters
For the calculation of the gas exchange parameters we followed well established procedures by Caemmerer and Farquhar (1981) and Ball (1987). Transpiration rate E, assimilation rate A, total ozone flux F tot,O3 and total water vapour conductance g l,H2O were calculated from (1) and is typically assumed to be the saturation mole fraction at leaf temperature (Ball, 1987).
For the calculation of the total ozone conductance we applied a ternary diffusion model as has been 210 proposed by Caemmerer and Farquhar (1981). Thereby, pairwise interactions between ozone, water vapour and air are considered (for the sake of simplicity we neglected interactions with CO 2 ). Interactions of ozone molecules with water vapour are important only for that portion of ozone, which is entering the stomatal pores and not for that lost in reactions at the leaf surface. However, in the latter case the consideration of binary diffusion between ozone and water leads to an overestimation of the 215 total ozone conductance in the range of < 1 %.
Total ozone conductance g l,O3 is then defined by  from leaf models made of chromatography paper (see Ball 1987). However, in our experiments this was not really practical for all sample plants which were all complexly and differently shaped. Consequently, also the stomatal water vapour and ozone conductances could not be inferred from the calculated total water vapour conductance (Eq. 4).
As we show in the Supplement, even if stomatal and boundary layer ozone conductances are known, 240 for semi-reactive leaf surfaces the calculation of stomatal and non-stomatal parts of the total ozone flux is not feasible.
For these reasons we report here only total ozone conductance values (Eq. 6), normalized to the single-sided leaf area or to the area of the enclosure covered with leaf exudates in experiments with pure leaf surface compounds (see Sec. 2.5).

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In the microscopic model the air flow was neglected and a pure diffusion process was simulated.
Stomata were modelled as 100 µm long and 40 µm wide eye-shaped openings recessed 20 µm deep into the leaf surface. The simulation domain with 500 000 cells covered an area of 300 µm square around the stoma and extended 2 mm from the leaf surface into the surrounding gas. A single stoma with cyclic boundaries was used to represent a whole leaf with stomata spread repeatedly over its 255 surface. The ozone-reactive bottom of the stomata was modelled as 100 % efficient sink (Laisk et al., 1989) with a constant ozone concentration of zero, while the side walls of the stomata were assumed not to absorb ozone and set to zero gradient. The top of the measurement domain acting as ozone inlet from the surrounding was set to one. The leaf surface around the stomata was set to zero gradient or to a fixed concentration of zero, representing two idealised plant types with either non-reactive or 260 reactive leaf surface. "scalarTransportFoam" was run on this grid with a uniform zero velocity field until a steady state was reached.
For the macroscopic model (see Supplement) a laminar flow around the plant was simulated using the steady state Reynolds averaged Navier-Stokes solver "simpleFoam", the transport of ozone in the resulting flow velocity field was studied using the "scalarTransportFoam" solver. The simulated 265 gas volume consisted of a cube with 20 cm edge length with the shape of an exemplary tobacco plant cut out of its volume (see Fig. S3). The resulting simulation domain was divided into a hexahedrondominant grid of 3.7 million cells with the finest granularity around the stomata and the leaf surfaces with the OpenFOAM tool "snappyHexMesh". The domain was divided into eight subdomains for parallel computation. Stomata were represented by small patches spread equally over the leaf sur- In the case of the ring structured CBTdiols with three endocyclic double bonds, produced by the Basma Drama and BYBA plants, at least two ozonolysis steps are needed to form volatile carbonyls.
The three smallest carbonyl products are shown in Fig. 1, whereby 4-oxopentanal (C 5 H 8 O 2 ) is ex-295 pected to be the most volatile one (Goldstein and Galbally, 2007).

Ozone fumigation experiments with pure leaf surface compounds
In order to relate a release of carbonyls to surface chemistry only and to exclude stimulated emissions caused, e.g. by the plant ozone defence system, we investigated ozone reactions with pure leaf surface extracts. Leaf surface compounds were extracted with n-hexane and subsequently applied 300 onto the inner surface of an empty plant enclosure and fumigated with ozone (see Materials and methods section).
Ambalema leaf extracts showed a weak signal of cisabienol (we refer to the Materials and methods section for the identification of this compound), which disappeared during ozone fumigation while MVK and formaldehyde were prominently observed. These carbonyls are produced by surface-305 assisted ozonolysis of cis-abienol (see Fig. 1 into account the humidity dependent sensitivity (Hansel et al., 1997). In the NO + reagent ion mode formaldehyde cannot be ionized (Španěl et al., 1997), consequently we detected no signal.

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In the ozone fumigation experiments using Basma Drama leaf extracts, besides MVK and formaldehyde as ozonolysis products of cis-abienol, also the most volatile CBTdiol ozonolysis product -4-oxopentanal -was detected in the gas phase by SRI-ToF-MS. 4-oxopentanal was detected at m/z 101.060 (C 5 H 9 O + 2 ) in H 3 O + and m/z 99.045 (C 5 H 7 O + 2 ) in NO + reagent ion mode, respectively.

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No significant amount of volatile carbonyls was observed from ozonolysis of 3H02 leaf extracts.
Consistently, the total ozone conductance was far less than in experiments with extracts from diterpenoid-exuding tobacco varieties (see Fig. 2). This is in line with the results from the corresponding experiments with intact plants (see below). The ozone depletion efficiency of the 3H02 exudates was decreasing fast, while the presence of cis-abienol in Ambalema leaf exudates kept the 320 ozone conductance at elevated levels for many hours (cf. Fig. 2).

Ozone fumigation experiments with diterpenoid exuding tobacco varieties
Also in experiments with intact plants we observed a prompt release of volatile carbonyls as soon as the tobacco leaves were fumigated with ozone. The Ambalema and Basma Drama varieties released MVK and formaldehyde. In addition, we detected sclaral, a non-volatile compound, in surface ex-325 tracts obtained from ozone fumigated plants of the same varieties (see Materials and methods and Supplement). Sclaral is an isomerisation product of the C 16 carbonyl formed in cis-abienol ozonolysis (cf. Fig. 1). All these compounds can therefore be attributed again to surface-assisted ozonolysis of cis-abienol (see Fig. 1).
In experiments using Basma Drama and BYBA plants we detected the CBTdiol ozonolysis product  In plant experiments using diterpenoid exuding tobacco varieties, the carbonyl emission and consequently the total ozone conductance and flux (under constant light) eventually reached a steady state, 340 when the diterpenoid production by the trichomes (leading to a permanent deposition of those onto the plant surface) and plant surface reactions were in equilibrium (cf. Fig. 3). This is in contrast to experiments with pure leaf surface compounds, in which the diterpenoids were slowly consumed as ozone fumigation progressed (see Sec. 3.2).
Simulating diurnal ozone variations over two days in experiments with Ambalema and Basma Drama 345 plants, we could show that the reactive layer at the plant surface is a large pool and not quickly consumed (see Supplement and Fig. S2). We therefore assume that the diterpenoids released are likely to represent a long term ozone protection for these varieties.

Variety specific ozone depletion during dark and light phases
In further experiments we investigated the ozone depletion by different tobacco varieties under dark 350 and light conditions.
In dark experiments, when stomatal pores are almost closed, the Ambalema variety showed the highest total ozone conductance under steady-state conditions (cf. Fig. 4a). This is a direct indication for the high ozone depletion capacity of the surface of this variety.
Due to the lack of reactive diterpenoids on the leaf surface of 3H02 plants, the surface ozone sink 355 plays a minor role for this tobacco line. However, we cannot totally exclude the presence of other unsaturated compounds at the surface of this variety.
The low surface reactivity of the Basma Drama and BYBA varieties correlates with the lower amount of detected ozonolysis carbonyls compared to that of the Ambalema variety in dark conditions. This might be related to a lower diterpenoid surface coverage of these two varieties and the expected 360 lower reactivity of the CBTdiols having endocyclic double bonds (Atkinson and Arey, 2003).
The Ambalema variety also shows a higher g l,H2O and dark respiration than the other varieties (cf. Fig. 4b+c). g l,H2O linearly correlates with the stomatal water vapour conductance and therefore also with the stomatal ozone conductance. However, higher stomatal conductance during dark conditions cannot explain the large differences in g l,O3 between the plant types. While g l,H2O of the Ambalema 365 variety in dark conditions is about twice as high as that of the 3H02 variety, the corresponding g l,O3 is four times as high.
When switching from dark to light conditions we assume cuticular conductance not to change significantly and thus an increase in the calculated g l,O3 is attributable mainly to an increasing stomatal ozone conductance. In the case of Ambalema, switching the light on increased the total conductance 370 by ∼ 55 % (see Fig. 4). In contrast, in the 3H02 case, switching on the light triggered an substantial increase in the total ozone conductance by ∼ 340 % (cf. Fig. 4).
During light conditions the total ozone conductances of the different tobacco varieties were in a comparable range; slightly higher values were observed for the diterpenoid exuding lines Ambalema, Basma Drama and BYBA.

Separation of ozone surface and gas phase reactions
In order to qualify the measured total ozone fluxes for the calculation of g l,O3 values, we had to take into account the possibility of homogeneous gas phase ozonolysis of the semi-volatile diterpenoids exuded by the tobacco varieties.
To assess the significance of gas phase ozonolysis to our results, we connected the plant enclosure 380 containing a diterpenoid emitting tobacco plant with a second empty enclosure downstream and added ozone only to the second enclosure. Only negligible carbonyl signals were observed once the initial burst from deposited diterpenoids faded away (see Supplement and Fig. S1). This result indicates that with our setup gas-phase reactions of the diterpenoids were not significant.
This observation can be explained theoretically, too. The air in our enclosure system was exchanged 385 every ∼ 5 min. Therefore, only extremely fast gas phase ozone -alkene reactions have to be considered. For an ozone concentration of 100 ppbv, a reaction rate of 1.35 × 10 −15 cm 3 s −1 results in an alkene ozonolysis lifetime of 5 min. Such fast ozonolysis rates have only been measured for a few very reactive sesquiterpenes (Atkinson and Arey, 2003). We found no reaction rates of cis-abienol and CBTdiols with ozone in the literature to exclude the possibility of a gas phase contribution to 390 total ozone loss in our experiments a priori. Nonetheless, taking into account the estimated vapour pressures of cis-abienol (∼ 10 −9 bar) and CBTdiol (∼ 10 −12 bar) (Goldstein and Galbally, 2007) we can state that the bulk of the exuded diterpenoids stayed at the leaf surface and that other surfaces (e.g. the inner surface of the plant enclosure and the tubing system) were very slowly covered by condensed diterpenoids. This is also the explanation for the bursts of volatile ozonolysis products 395 at the beginning of every ozone fumigation (see e.g. Fig. 3). We therefore assume that gas phase reactions are unlikely to have played a major role in our experiments.  Figure 5a and b show the resistance schemes used to describe the ozone flux to the leaves in the two scenarios, which were the basis for our simulations. Ambient ozone has to overcome the boundary 410 layer resistance R b and the stomatal resistance R s before being destroyed in the stomatal cavity (for the sake of simplicity we neglected here the mesophyl resistance, which comprises diffusion through inner air spaces and dissolution of the gas in the cell wall water, followed by losses in the aqueous phase, penetration of plasmalemma or chemical reactions in the cell, cf. Neubert et al., 1993). In the case of a non-reactive leaf surface, ozone depletion within the stomata is the sole ozone sink (see 415 Fig. 5a).

Fluid dynamic model calculations
In the case of an ozone-reactive leaf surface, an additional surface chemical resistance R sc has to be introduced, which is parallel to the stomatal resistance (see Fig. 5b). R sc inversely correlates with the reactive uptake coefficient of ozone at the leaf surface. In the case of the model plant having a non-reactive surface, R sc is very large (R sc → ∞) and ozone flux to the leaf surface can be omitted.

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Conversely, R sc is small for reactive surfaces.
The porous leaf surface architecture has special relevance for the gas uptake of plants. For gases having a negligible leaf surface sink (or source) -like e.g. CO 2 -steep concentration gradients parallel and perpendicular to the surface develop in close proximity to the stomata. These gradients enhance the gas transport in the diffusive leaf boundary layer towards the pores. This effect is exten-425 sively described in the literature as the "paradox of pores" (see e.g. Monson and Baldocchi, 2014). It enables plants to effectively harvest CO 2 for photosynthesis, but in the same manner also "funnels" phytotoxic ozone through the stomata into the plant leaves (see Fig. 5c).
In the case of an ozone-reactive leaf surface, R sc is small compared to R s and only surface-parallel ozone concentration isosurfaces develop (black lines in Fig. 5d). Concentration gradients (white 430 lines) close to the stomata are exclusively perpendicular to the surface. Consequently, the ozone transport in the diffusive leaf boundary layer is equally distributed over the whole leaf surface and the ozone concentration in this layer is strongly reduced (see Fig. 5d). Similarly, also macroscopic model calculations show that this effect broadens the space of reduced ozone concentrations surrounding a plant with opened stomata (see Supplement and Fig. S3).

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The surface-parallel concentration isosurfaces are the reason why we can use the same reference concentration c b, r for both the stomatal and the surface chemical resistance, (cf. Fig. 5b). How-ever, this approach does only hold if the leaf surface is a complete ozone sink (see Supplement and The different ozone concentration patterns in the two modelled scenarios have important implica-440 tions for the stomatal ozone uptake. Typically, the stomatal conductance of ozone g s,O3 is estimated from that of water g s,H2O , by correcting for the different diffusivity of the two gases (see e.g. Ball, 1987;Neubert et al., 1993). The stomatal ozone flux F s,O3 can then be calculated with the following formula: 445 with c i,O3 being the ozone concentration in the leaf intercellular space and c b,O3 the ozone concentration in the leaf boundary layer. For high ambient ozone concentrations c i,O3 was found to be positive (Moldau and Bichele, 2002;Loreto and Fares, 2007), but typically it is assumed to be close to zero (Laisk et al., 1989). Therefore, Eq. (7) simplifies to 450 If now surface reactions drastically reduce c b,O3 (cf. Fig. 5b+d), the effective stomatal ozone flux and with that the effective ozone dose are also reduced, which eventually determine the phytotoxic effects of ozone to plants (Massman, 2004). At this point, it is important to note that the uptake of non surface-reactive gases such as CO 2 is not affected by the altered ozone gradients.
Thus, whenever surface loss plays a role, both surface and stomatal ozone uptake by plants have to 455 be considered together. Previous studies might therefore have overestimated stomatal ozone uptake (e.g. Kurpius and Goldstein, 2003;Cieslik, 2004;Goldstein et al., 2004;Fares et al., 2012). Hence, their reported stomatal ozone flux values should be considered as upper limits.
In future studies investigating the ozone depositions to vegetation, it might be worth to analyse also the surface composition of the plants. If the surfaces are covered with substantial amounts 460 of unsaturated organic compounds, surface loss has to be considered right from the beginning in order not to overestimate stomatal ozone uptake. Due to the fact that surface reactions reduce ozone concentrations in the leaf boundary layer, it is not correct to calculate stomatal ozone loss applying the resistance scheme shown in Fig. 5a and to eventually define the surface loss of ozone as that portion of the total loss which is not explainable by gas phase reactions and stomatal uptake.

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For real plants the altered ozone gradient profile shown in Fig. 5d is less pronounced depending on stomata depth, which reduces the total stomatal uptake, and reactive surface compounds, which show smaller surface reaction rates than assumed for the idealised 100 % efficient ozone depleting surface (see Supplement). In the case of such a semi-reactive leaf surfaces a more sophisticated resistance scheme has to be used, which strongly complicates the calculation of stomatal and non-stomatal

Atmospheric implications
Large downward ozone fluxes (Kurpius and Goldstein, 2003;Goldstein et al., 2004) and high levels of oxidized VOC (Holzinger et al., 2005) have been taken as evidence for "unconventional in-475 canopy chemistry" of unknown precursors in a Ponderosa pine forest site. We speculate that to a certain extent these unknown precursors could be reactive compounds emitted or deposited onto the vegetation surfaces. Most recent results support this speculation. A large number of compounds with diterpenoid backbones were recently observed for the first time also in a Ponderosa pine forest site during the BEACHON-RoMBAS campaign 2011 (Chan et al., 2015). These unsaturated diter- Resins contain high amounts of sesqui-, di-and triterpenoids (Dell and McComb, 1979;Langenheim, 2003); di-and triterpenoids are also known constituents of surface waxes (Estell et al., 1994a, 485 b; Altimir et al., 2008;Thimmappa et al., 2014). Moreover, it is estimated that about 30 % of vascular plants have glandular trichomes, which often exude higher terpenoid compounds, too (Wagner et al., 2004).
All these terpenoid classes contain carbon-carbon double bonds and are therefore reactive with O 3 , OH and NO 3 . Our results support the speculation that reaction rates of ozone with semi-volatiles 490 adsorbed at the surfaces are far higher than corresponding gas-phase ozonolysis rates. Thus, the fraction of volatile carbonyls produced in surface assisted ozonolysis of adsorbed semi-volatiles could compete with their respective gas-phase production rate from OH chemistry.
To some extend this source of carbonyls in form of exudates or resins at the surface of particular plants might be obscured by the immediate uptake of the volatile ozonolysis products by the plants 495 themselves (Karl et al., 2010;Niinemets et al., 2014).
Reactive surface compounds might also contribute to the varying ozone sensitivity of different conifer species (Schnitzler et al., 1999;Landolt et al., 2000) when exposed to the same cumulative ozone concentrations under light conditions. We anticipate therefore that surface ozonolysis plays an important role for the ozone tolerance of certain conifer species, too.

Conclusions
Our results reveal for the first time a powerful ozone protection mechanism of plants having an 510 ozone reactive leaf surface. This opportunistic defence mechanism, which is a beneficial side effect of semi-volatile terpenoids emitted onto the leaf surface, takes place before the phytotoxic gas enters the stomata. Plants emitting unsaturated semi-volatile compounds could have an advantageous effect for neighbouring plants as well: either directly by reducing overall ozone concentrations (see Supplement) or indirectly through the deposition of the semi-volatile compounds onto unprotected 515 neighbouring leaves (Schmid et al., 1992;Himanen et al., 2010;Chan et al., 2015).
Our findings have relevance not only for plants, but also for additional ozone-initiated processes that occur in the atmospheric boundary layer. The surface-assisted chemistry that we have elucidated for specific diterpenoids, linking for the first time volatile and non-volatile carbonyl products to semi-volatile precursors at the plant surface, is likely to occur also for other semi-volatile organic 520 compounds on different surfaces, e.g. soil with plant litter, aerosols, man-made structures and even human skin, as has been shown previously (Wisthaler and Weschler, 2010). We speculate that some of the ozonolysis-derived products may play important roles in atmospheric processes, influencing the budgets of OH radicals and ozone. Conversely, in our experiments we had no indication that surface ozonolysis itself releases detectable amounts of OH radicals into the gas phase (see Supple-525 ment). In order to assess the global impact of surface-assisted ozonolysis on atmospheric chemistry a more complete knowledge about the nature of reactive, semi-and low-volatile compounds at plant surfaces as well as the mechanisms triggering their release (e.g. constitutive vs. biotic and mechanical stress induced emission) is needed.
Appendix A: Generation of the 3H02 variety -a Nicotiana tabacum line without diterpenoids 530 The Ambalema variety which produces only cis-abienol and the Colorado variety which produces only CBTdiols (Sallaud et al., 2012) were crossed to produce hybrid F1 plants which produce both diterpenoids. Because the genetic loci responsible for the absence of CBTdiols and the absence of cis-abienol are distinct and unlinked, recombinant plants which produce neither diterpenoids could be recovered by analysing the leaf surface extracts by GC-MS in the selfed progeny of the F1 plants.

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One of these plants was selected, propagated over 2 generations by single seed descent and named line 3H02.
The Supplement related to this article is available online at doi:10.5194/acp-0-1-2015-supplement.  plants. The leaf extracts containing the surface compounds were applied to the inner surface of the empty plant enclosure system (see Materials and methods section). During ozone fumigation (grey shaded area), the total ozone conductance g l,O 3 to the enclosure surface was much higher for Ambalema leaf extracts (containing large amounts of the diterpenoid cis-abienol) than for 3H02 extracts. Moreover, it remained high for many hours. (white lines indicate their orientation) evolving parallel and perpendicular to the leaf surface around the stoma (located at the coordinate (0,0)) in this case (c). If the leaf surface is covered with ozone-reactive substances, the parallel fraction of the ozone gradients vanishes, resulting in isosurfaces of ozone concentration (black lines) parallel to the leaf surface and stronger ozone depletion in the leaf boundary layer (d).