Introduction
Tropospheric ozone (O3) is formed as a product of photochemical
reactions involving nitrogen oxides (NOx) and volatile organic compounds
(VOC) as precursors . 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) , levels which may be
responsible for cellular damage inside leaves
, adversely affecting photosynthesis and
plant growth . 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
. Future trends of tropospheric ozone
strongly depend on the emission factors of the corresponding precursor
compounds (i.e. VOC and NOx) and indirectly also on land cover and
characteristics of the vegetation . 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 . This is likely a result of preventive
measures reducing ozone precursor emissions . In contrast,
ozone background concentrations are still rising in parts of Asia
experiencing high economic growth and a concomitant increase in NOx
emissions .
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
. In low NOx regions enhanced emissions of isoprene-emitting
species could decrease ozone concentrations, while they would lead to an
ozone increase in high NOx regions .
Traditionally, the risk of ozone damage to plants is estimated on the basis
of the accumulated ozone exposure above 40 ppbv (AOT 40) .
However, the negative effects of ozone on vegetation have been observed to be
more closely related to the effective dose, i.e. the stomatal
flux × time minus the portion of ozone which can be detoxicated by
the plant defence system . In the expected CO2
richer and warmer future atmosphere , plants may reduce
stomatal conductance and thus indirectly alleviate ozone damage
.
However, accurate experimental quantification of the stomatal uptake of ozone
is complicated by the presence of other ozone sinks, either in the gas phase
or on the plant surface . In previous studies
the ozone flux through the stomata was calculated by multiplying the stomatal
ozone conductance with the ambient ozone concentration see,
e.g., assuming similar
gradient profiles of ozone and H2O close to the stomata. As we will
show, for ozone-reactive leaf 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 Sect. 2 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
and has therefore been used as an ozone
indicator plant in earlier times seeand references
therein. Conversely, the Bel B variety is known to
be non-sensitive . The high ozone tolerance of this
variety has been attributed to wider epidermal cells and more spongy
mesophyll cell layers and to differences in the plant's
ability to cope with oxidative stress once ozone has entered the stomata
.
Several studies were investigating the possibility to increase the ozone
tolerance of plants by external application of ozone-scavenging compounds
or by
enabling the emission of volatile terpenoids in transgenic plants
. 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 , tobacco leaves possess glandular
trichomes. In tobacco, various diterpenoids are the major compounds exuded by
these secretory structures at the leaf surface . The
exudates cover the plant leaves as a defence barrier, for example against
arthropod pests ; they were shown to have an
anti-fungal and insecticidal action .
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.
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.
Materials and methods
Plant material
We used the following four tobacco cultivars: Ambalema, secreting
only the diterpenoid cis-abienol (C20H34O, see
Fig. ), BYBA secreting α- and
β-cembratrien-diols (CBTdiols, C20H34O2, see
Fig. ), and Basma Drama, secreting all these
compounds . The new 3H02 line does not exude
diterpenoids at all (see Appendix ).
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.
Ozonolysis of diterpenoids exuded by the trichomes of the
investigated tobacco plants. The BYBA variety releases
α- and β-cembratrien-diols (C20H34O2), the
Ambalema variety cis-abienol
(C20H34O); the Basma Drama variety exudes all
these compounds. Ozonolysis of the cembratriendiols requires at
least two ozonolysis steps to form short-chained, volatile
carbonyls, e.g. 4-oxopentanal (C5H8O2). Ozonolysis of
cis-abienol leads to the formation of volatile formaldehyde
(HCHO) and methyl vinyl ketone (C4H6O).
The background image shows glandular trichomes on a tobacco leaf.
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 Sect. 2.2).
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
significant stress signals in form of green leaf volatiles were detected.
In total, combined dark and light ozone fumigation experiments were conducted
with five Ambalema, two Basma Drama, one BYBA and
three 3H02 samples. Moreover, experiments under solely light conditions
were conducted with eight Ambalema, four Basma Drama, four
BYBA, and two 3H02 plants. Each sample plant was tested only once.
Setup
In the present ozone experiments we used only inert materials such as
Teflon®,
PEEK® or
Duran® glass in order to minimize
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
. Ozone loss, 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 CO2 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 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 cm2.
An ozone detector (Model 49i, Thermo Fisher Scientific Inc. Franklin (MA),
USA) and an infra-red gas analyser (LI-840A CO2/H2O 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 realistic
ambient ozone concentrations at the enclosure outlet during light conditions
(e.g. ∼60 ppbv in Fig. ). Relative
humidity in the plant enclosure ranged from typically ∼ 55 % in
dark experiments up to ∼95 % in light experiments.
VOC were quantitatively detected at the enclosure outlet by
a Selective Reagent Ionization Time-of-Flight Mass Spectrometer
(SRI-ToF-MS, see next section) which was switched every 6 min between
H3O+ and NO+ reagent ion mode.
Sample plants were illuminated by a true light lamp (Dakar, MT/HQI-T/D,
Lanzini Illuminazione, Brescia, Italy). Infra-red light was shielded off by
a continuously flushed water bath in order to prevent heating of the plant
enclosure. Photosynthetically active radiation (PAR) was measured with
a sunshine sensor (model BF3, Delta T Devices Ltd, Cambridge, UK) and
temperature on the outer plant enclosure surface with K-type thermocouples.
SRI-ToF-MS
The UIBK Advanced SRI-ToF-MS (University of Innsbruck Advanced Selective
Reagent Ionization Time-of-Flight Mass Spectrometer,
) combines the high mass resolution of PTR-ToF-MS
with the capability to separate isomeric compounds having
specific functional groups. For this purpose, the SRI-ToF-MS makes use of
different chemical ionization pathways of a set of fast switchable primary
ions (here: H3O+ and NO+). Moreover, the employment of
different primary ions could help to differentiate molecules suffering from
fragmentation onto the same mass to charge ratio in the standard
H3O+ mode .
Examples of differentiable isomers are aldehydes and ketones. In the
H3O+ reagent ion mode, aldehydes and ketones both exhibit proton
transfer and thus, e.g. methyl vinyl ketone (MVK) and methacrolein (MACR) are
both detected as C4H7O+ (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
. Thus MVK is detected as
C4H6O ⚫ NO+ (m/z 100.040), whereas MACR is
detected as C4H5O+ (m/z 69.034).
In addition to isomeric separation, the high flow through the drift
tube (here: ∼500 mLmin-1 compared
to 10–20 mLmin-1 in a standard instrument) allows for
the first time the detection of semi-volatile compounds such as the
diterpenoid cis-abienol (C20H34O).
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; 1Td=10-17 Vcm2) in H3O+ or NO+ reagent ion mode,
respectively. The instrument was calibrated approximately once a week by
dynamic dilution of VOC using two 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 .
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
H3O+ reagent ion mode, the major cis-abienol derived signal
was detected on m/z 273.258 (C20H33+); like many other
alcohols, cis-abienol is losing H2O after the protonation
reaction. Minor fragment signals in the range of a few percent were detected
at m/z 191.180 (C14H23+), m/z 163.149
(C12H19+) and m/z 217.196 (C16H25+),
respectively.
In NO+ reagent ion mode, the major cis-abienol derived
signals were detected at m/z 272.250 (C20H32+) and
m/z 178.172 (C13H22+). Minor signals were measured at
m/z 163.149 (C12H19+) and m/z 134.101
(C10H14+), 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
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 cm2 leaf area) for ∼1 min. The n-hexane – 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 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 low-volatility ozonolysis products
from the fresh tobacco leaves (see Sect. 2.5). 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
.
Tobacco diterpenoids were identified on the basis of their mass spectra, as
described in the literature .
Calculation of leaf gas exchange parameters
For the calculation of the gas exchange parameters we followed well-established procedures by and .
Transpiration rate E, assimilation rate A, total ozone flux
Ftot,O3 and total water vapour conductance
gl,H2O were calculated from
E=ues⋅wo-we1-wo×10-3,[mmolm-2s-1]A=ues⋅ce-1-we×10-31-wo×10-3⋅co,[µmolm-2s-1]Ftot,O3=ues⋅oe-1-we×10-31-wo×10-3⋅oo,[nmolm-2s-1]gl,H2O=103⋅E1-wo+wi2×103wi-wo,mmolm-2s-1,
with ue the molar flow of air entering the enclosure in
[mols-1], s the leaf area in [m2],
we/ce/oe and
wo/co/oo the mole fraction of water
vapour/CO2/ozone entering respectively leaving the plant enclosure in
[mmolmol-1], [µmolmol-1] and
[nmolmol-1], respectively. wi is the mole fraction of
water vapour inside the leaf in [mmolmol-1] and is typically
assumed to be the saturation mole fraction at leaf temperature
.
For the calculation of the total ozone conductance we applied a ternary
diffusion model as has been proposed by . Thereby,
pairwise interactions between ozone, water vapour and air are considered (for
the sake of simplicity we neglected interactions with CO2).
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 total ozone conductance in the range of <1%.
Total ozone conductance gl,O3 is then defined by
gl,O3=-103⋅Ftot,O3+oa+oi2⋅Eoa-oi,[mmolm-2s-1],
with oi and oa the mole fractions of ozone
inside the leaf (at the leaf surface for reactive leaf surfaces) and
in the surrounding air, respectively. oa equals the
ozone mole fraction oo measured at the outlet of the plant
enclosure. Typically, we consider
oi≈0 and therefore
Eq. () simplifies further to
gl,O3=-103⋅Ftot,O3+oa2⋅Eoa.
Quantification of the ozone depletion capability of individual plants
In our fumigation experiments the ozone concentrations in the plant enclosure
varied between the different experiments and within experiments switching
from light to dark conditions. In order to compare the ozone depletion
capability (i.e. surface plus stomatal sinks) of different plants or of the
same plant under dark and light conditions, it is therefore important to use
a concentration independent measure. As for a given ozone conductance the
ozone flux increases with the ambient ozone concentration (cf.
Eqs. and ), we follow others see
e.g. and use the ozone conductance values instead. In
experiments with plants having an ozone reactive surface, the total ozone
conductance gl,O3 (Eq. ) comprises
boundary layer conductance, stomatal conductance, and cuticular conductance.
Stomatal and boundary layer ozone conductances can be calculated from those
of water vapour by correcting for the different diffusivities of the two
gases. The boundary layer water vapour conductance could be determined by
measuring temperature and evaporation rate from leaf models made of
chromatography paper (see ). 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. ).
As we show in the Supplement, even if stomatal and boundary layer ozone
conductances are known, 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. ), 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 Sect. ).
Statistical analysis
Data (gl,O3, A, gl,H2O)
were tested for statistically significant differences between dark and light
experiments (using the same variety) and between different tobacco varieties
(in either dark or light experiments), respectively, using the
Wilcoxon-Mann-Whitney test in Matlab®. Due
to the partially small sample size, probabilities p < 0.1 are reported
as marginally significant. Lacking replicates of dark experiments with
BYBA plants, in the statistical analysis this type of experiment
was omitted.
Fluid dynamic calculations
In order to visualize the ozone concentration gradients caused by plant ozone
uptake, two idealized setups were simulated: a macroscopic plant model in an
ambient air flow and a microscopic model for the stomatal gas exchange. The
simulations were done using the open-source CFD code OpenFOAM
(www.openfoam.com).
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 surface. The ozone-reactive bottom of the stomata was modelled as
100 % efficient sink 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 idealized plant types with either non-reactive or
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 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 hexahedron-dominant 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 surfaces, covering 10 % of the total
leaf area. The boundary conditions for the gas flow simulation consisted of
an inlet with 2 mms-1 velocity entering on one face of the cube
and a constant pressure boundary condition outlet on the opposite face. The
gas velocity on the plant surface was set to zero. Initial conditions for the
flow simulation were calculated with “potentialFoam” to speed up
convergence of the “simpleFoam” solver. The simulation was run until the
flow velocity field reached a steady state. For the diffusion calculations
a relative initial concentration of ozone was set to one at the inlet and to
zero on the stomata patches. Like in the microscopic model calculations, the
leaf surface was either a zero concentration gradient boundary (for an
idealized 3H02 plant type) or a fixed concentration value of zero (for an
idealized Ambalema plant type). In the previously calculated
velocity field the ozone transport was simulated until a steady state was
reached, too.
Results and discussion
Expected ozonolysis products of cis-abienol and cembratrien-diols
Apart from the 3H02 variety, the investigated tobacco varieties secrete
different unsaturated diterpenoids (see Sect. ).
According to the Criegee mechanism , ozone attacks the
carbon double bonds of alkenes forming primary carboyls and so-called Criegee
Intermediates (see Supplement). Criegee Intermediates are, however, expected
to be too short-lived to be detected directly by the instruments used in our
experiments (see Supplement). We were therefore interested primarily in the
stable, volatile ozonolysis carbonyls, which could be detected in real-time
by our SRI-ToF-MS.
For the semi-volatile diterpenoid cis-abienol with two exocyclic
double bonds, exuded by the Ambalema and Basma Drama
varieties, we expected the formation of formaldehyde (HCHO) and methyl
vinyl ketone (MVK, C4H6O, see Fig. ).
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. , whereby
4-oxopentanal (C5H8O2) is expected to be the most volatile one
.
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 onto the
inner surface of an empty plant enclosure and fumigated with ozone (see
Sect. 2.5).
Ambalema leaf extracts showed a weak signal of cis- abienol
(we refer to Sect. 2.4 for the identification of this compound), which
disappeared during ozone fumigation while MVK and formaldehyde were
prominently observed. These carbonyls were produced by surface-assisted
ozonolysis of cis-abienol (see Fig. ). MVK was
detected at m/z 71.050 (C4H7O+) and m/z 100.040
(C4H6O ⚫ NO+) in the H3O+ and
NO+ reagent ion mode of the SRI-ToF-MS, respectively. Formaldehyde was detected
only using H3O+ as reagent ion at m/z 31.018 (CH3O+),
taking into account the humidity dependent sensitivity . In
the NO+ reagent ion mode formaldehyde cannot be ionized
, consequently we detected no signal.
Ozonolysis experiments with pure leaf exudates extracted from
non-ozone fumigated, unimpaired plants. The leaf extracts containing
the surface compounds were applied to the inner surface of the empty
plant enclosure system (see Sect. 2). During
ozone fumigation (grey shaded area), the total ozone conductance
gl,O3 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.
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 (C5H9O2+) in H3O+ and m/z 99.045
(C5H7O2+) in NO+ reagent ion mode, respectively.
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. ). 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
ozone conductance at elevated levels for many hours (cf.
Fig. ).
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 extracts obtained from ozone fumigated plants of the same varieties
(see Sect. 2 and Supplement). Sclaral is an isomerization product of the
C16 carbonyl formed in cis-abienol ozonolysis (cf.
Fig. ). All these compounds can therefore be attributed
again to surface-assisted ozonolysis of cis-abienol (see
Fig. ).
In experiments using Basma Drama and BYBA plants we
detected the CBTdiol ozonolysis product 4-oxopentanal, similar to the ozone
fumigation experiments with leaf surface extracts (see previous section).
Figure shows a typical result of an ozonolysis
experiment using Ambalema plants. Immediately after starting the
ozone fumigation, the cis-abienol signal decreased, while initial
bursts of MVK and formaldehyde were detected. These initial bursts can be
attributed to surface ozonolysis of cis-abienol deposited on
all surfaces (i.e. surfaces of the whole plant, the enclosure and
the enclosure outlet tubing) during plant acclimatization under ozone free
conditions lasting > 12 h (see Sect. and
Supplement).
Temporal evolution of selected VOC in an ozonolysis
experiment with an Ambalema plant and corresponding total ozone
deposition flux Ftot,O3. The yellow shaded area
denotes time ranges, in which the sample plant was illuminated.
Starting the fumigation
with ∼60 ppbv ozone (indicated by the black arrow) the
cis-abienol signal decreased quickly. At the same time, the
carbonyl products of cis-abienol ozonolysis, formaldehyde
and MVK (measured in H3O+ respectively NO+ reagent
ion mode of the SRI-ToF-MS), started to rise. The large scattering of
the formaldehyde signal derives from the strongly reduced
sensitivity of the SRI-ToF-MS under high humidity conditions towards
this compound. Two hours after the start of the ozone fumigation an
equilibrium between actual diterpenoid production and loss due to
surface reactions was established, resulting in stable signals of the
oxygenated VOC.
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, 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. ). This is in contrast to experiments
with pure leaf surface compounds, in which the diterpenoids were slowly
consumed as ozone fumigation progressed (see
Sect. ).
Simulating diurnal ozone variations over 2 days in experiments with
Ambalema and Basma Drama 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 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. , top
panel). This is a direct indication for the high ozone depletion capacity of
the surface of this variety.
Total ozone conductance gl,O3 (top),
total water vapour conductance gl,H2O (middle)
and assimilation rates A (bottom) of different tobacco varieties
during dark and light conditions. Error bars denote the standard error
of 5 (13), 2 (6), 1 (5) and 3 (5) replicates of Ambalema,
Basma Drama, BYBA respectively 3H02 in dark
(light) experiments. Different capital letters denote significant different means
in light and dark experiments of the same plant type and lower case letters
significant different means of different plant types in either dark or light
experiments, respectively (Wilcoxon-Mann-Whitney test, p < 0.1, see Sect. 2.9. Lacking replicates in the statistical analysis dark experiments
using BYBA plants were omitted).
Under dark conditions stomatal ozone conductance
is generally low and consequently surface reactions are the major
ozone sink. The surface sink is high for the Ambalema
tobacco line, which exudes cis-abienol and lower for
the other lines, exuding less reactive or no diterpenoids.
Due to the lack of reactive diterpenoids on the leaf surface of 3H02
plants, the surface ozone sink 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 lower reactivity of the CBTdiols having endocyclic
double bonds .
The Ambalema variety also shows a higher
gl,H2O and dark respiration than the other varieties
(cf. Fig. , middle and bottom panels).
gl,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 gl,O3 between the plant
types. While gl,H2O of the Ambalema variety in dark
conditions is about twice as high as that of the 3H02 variety, the
corresponding gl,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
gl,O3 is attributable mainly to an increasing
stomatal ozone conductance. In the case of Ambalema, switching the
light on increased the total conductance by ∼55 % (see
Fig. , top panel). In contrast, in the
3H02 case, switching on the light triggered a substantial increase in the
total ozone conductance by ∼340 % (cf.
Fig. , top panel).
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.
Statistical analysis confirmed the observed tendencies of the total ozone
conductance: only for the Ambalema variety was
gl,O3 under light conditions not significantly
different from the values measured under dark conditions (p > 0.1).
Conversely, gl,O3 calculated for the
Ambalema variety was significantly higher than that of the other
tobacco lines under dark conditions (p < 0.1, see
Fig. , top panel).
Volatile carbonyl yields from surface ozonolysis
In the ozone fumigation experiments the yield of volatile ozonolysis products
was generally in the low percentage range, e.g. for Ambalema plants
∼7 % under dark and ∼5 % under light conditions considering the
major volatile ozonolysis products MVK and formaldehyde quantified by
SRI-TOF-MS. The slight change from ∼7 to ∼5 % when switching from
dark to light conditions can be explained by the effect of the open stomata.
Open stomata offer an alternative sink for ozone and for volatile carbonyls
produced in surface assisted reactions . The
reason why only a small percentage of the consumed ozone is detected as
volatile products indicates that most of the ozonolysis products are not
volatile enough to leave the plant surface (cf. Fig.
and Supplement). The fate of the Criegee Intermediates in surface ozonolysis
is discussed in detail in the Supplement.
Separation of ozone surface and gas phase reactions
In order to qualify the measured total ozone fluxes for the calculation of
gl,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 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 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 cm3s-1 results in an alkene ozonolysis lifetime of
5 min. Such fast ozonolysis rates have only been measured for a few very
reactive sesquiterpenes . 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 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) 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 at the beginning of every ozone
fumigation (see e.g. Fig. ). We therefore assume
that gas phase reactions are unlikely to have played a major role in our
experiments.
Fluid dynamic calculations of ozone uptake by stomata and
leave surface. (a and b) show the resistance
schemes for ozone uptake of leaves with non-reactive (nr) and
reactive (r) surfaces. ci, cc,
cb, and ca denote ozone concentrations in the
stomatal cavity, at the leaf surface, in the boundary layer and in
ambient air, respectively. Rs and Rb denote
the stomatal and boundary layer resistances. The surface chemical
resistance Rsc is infinite (Rsc=∞) on
a non-reactive surface. Fluid dynamic calculations reveal ozone
concentration gradients (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).
Fluid dynamic model calculations
Microscopic fluid dynamic model calculations (see Materials and methods)
revealed the principles responsible for the strong variety-dependent
partitioning between stomatal and non-stomatal ozone loss (see
Sect. ). The mixed convective and diffusive
ozone transport from the surrounding atmosphere to the plant surface and into
the stomata was simulated for two idealized plant types under light
conditions when the leaf stomata are open. The stomatal pores were
exemplarily modelled as small patches uniformly spread over the entire leaf
surface. For one model plant we assumed stomatal ozone uptake only,
corresponding to an idealized 3H02 variety plant lacking any reactive
surface compounds. The second model plant was representing an idealized
Ambalema variety. The surface acted as a perfect ozone sink with
every ozone molecule reaching it being lost, either on the leaf surface or
through the stomata.
Figure a 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 layer
resistance Rb and the stomatal resistance Rs before
being destroyed in the stomatal cavity (for the sake of simplicity we
neglected here the mesophyll 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. ). In the case of
a non-reactive leaf surface, ozone depletion within the stomata is the sole
ozone sink (see Fig. a).
In the case of an ozone-reactive leaf surface, an additional surface chemical
resistance Rsc has to be introduced, which is parallel to the
stomatal resistance (see Fig. b). Rsc
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,
Rsc is very large (Rsc→∞) and ozone
flux to the leaf surface can be omitted. Conversely, Rsc 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) – e.g. CO2 – 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 extensively described in the literature as
the “paradox of pores” (see, e.g. ). It enables plants
to effectively harvest CO2 for photosynthesis, but in the same manner
also “funnels” phytotoxic ozone through the stomata into the plant leaves
(see Fig. c).
In the case of an ozone-reactive leaf surface, Rsc is small
compared to Rs and only surface-parallel ozone concentration
isosurfaces develop (black lines in Fig. d).
Concentration gradients (white 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. d). 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).
The surface-parallel concentration isosurfaces are the reason why we can use
the same reference concentration cb, r for both the stomatal and
the surface chemical resistance, (cf. Fig. b). However,
this approach does only hold if the leaf surface is a complete ozone sink
(see Supplement and Fig. S5).
The different ozone concentration patterns in the two modelled scenarios have
important implications for the stomatal ozone uptake. Typically, the stomatal
conductance of ozone gs,O3 is estimated from that of
water gs,H2O, by correcting for the different
diffusivity of the two gases (see e.g. ). The
stomatal ozone flux Fs,O3 can then be calculated with
the following formula:
Fs,O3=gs,O3⋅(ci,O3-cb,O3),
with ci,O3 being the ozone concentration in the leaf
intercellular space and cb,O3 the ozone concentration
in the leaf boundary layer. For high ambient ozone concentrations
ci,O3 was found to be positive
, but typically it is assumed to be close to
zero . Therefore, Eq. () simplifies to
Fs,O3=-gs,O3⋅cb,O3.
If now surface reactions drastically reduce cb,O3
(cf. Fig. b and d), the effective stomatal ozone flux
(see Supplement) and with that the effective ozone dose are also reduced,
which eventually determine the phytotoxic effects of ozone to plants
. At this point, it is important to note that the uptake
of non surface-reactive gases such as CO2 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 be considered together. Previous studies might
therefore have overestimated stomatal ozone uptake (e.g.
). 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 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. a 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.
For real plants the altered ozone gradient profile shown in
Fig. d 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 idealized
100 % efficient ozone depleting surface (see Supplement). In the case of
such semi-reactive leaf surfaces a more sophisticated resistance scheme has
to be used, which strongly complicates the calculation of stomatal and
non-stomatal ozone fluxes (see Supplement and Fig. S5). Nonetheless, the
simulations explain the experimentally observed behaviour of different
tobacco plants very well.
Atmospheric implications
Over the last decade, several studies have shown discrepancies between
measured and expected ozone deposition fluxes. Large downward ozone fluxes
and high levels of oxidized VOC
have been taken as evidence for “unconventional
in-canopy chemistry” in a Ponderosa pine plantation, the Blodgett forest
site. Measured ozone deposition fluxes could not be explained by modelled
stomatal and known non-stomatal sinks, such as reactions with measured VOC in
the gas phase . The same observation was made by
in a Scots pine dominated field site in Hyytiälä. All
these studies assume the presence of yet unmeasured highly reactive semi- or
low-volatile compounds, which have a similar temperature-dependent emission
pattern as mono- and sesquiterpenes.
assumed that the unmeasured reactive compounds might be
unsaturated, cyclic terpenoids. Due to their low vapour pressure, the
measurement of semi- or low-volatile compounds represents a challenge, since
these substances strongly partition into the condensed phase and are
therefore easily lost in the inlet systems of most current VOC
instrumentation. However, were
able to identify several different sesquiterpenes in ambient air and in
branch enclosure experiments at the Blodgett forest site.
A large number of compounds with diterpenoid backbones were recently observed
for the first time in a different Ponderosa pine forest site during the
BEACHON-RoMBAS campaign 2011 . These unsaturated diterpenoids
contain the same backbone as abietic acid, a primary component of resin
acids. The observed temporal variations in concentrations were similar to
those of sesquiterpernoids, suggesting they are directly emitted from the
local vegetation.
Most recently, have shown that semi-and intermediate
volatility organic compounds measured for the first time at the same site
with a novel thermal desorption electron impact mass spectrometer (TD-EIMS)
could likely close the gap between observed and expected secondary aerosol
growth, estimated from gas-phase concentrations of the most abundant measured
VOC (mono- and sesquiterpenes, toluene/p-cymene, isoprene). We therefore
speculate that the high ozone deposition fluxes in such forest sites could be
a result of not only gas-phase reactions, but to a certain extent also of
ozone reactions with semi-volatiles emitted or redeposited onto the
vegetation surfaces.
Possible sources of the measured and unmeasured higher terpenoids are –
among others – constitutive plant emissions or resins, which are known to
contain high amounts of sesqui-, di- and triterpenoids
. Resins can be released during mechanical
stress, e.g. in the event of hail storms and could
eventually evaporate depending on their vapour pressure (and therefore
ambient temperature).
Di- and triterpenoids are also known constituents of surface waxes
. Moreover, it is
estimated that about 30 % of vascular plants have glandular trichomes,
which often exude higher terpenoid compounds, too .
Clearly, additional experiments are needed to better quantify the amount
of semi-volatiles deposited onto vegetation surfaces and their impact on
atmospheric chemistry.