Whole-Tree Water Use Efficiency Is Decreased by
Ambient Ozone and Not Affected by O3-Induced
Stomatal Sluggishness
Yasutomo Hoshika1, Kenji Omasa1, Elena Paoletti2*
1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 2 Institute of Plant Protection, National Research Council, Sesto Fiorentino,
Florence, Italy

Abstract
Steady-state and dynamic gas exchange responses to ozone visible injury were investigated in an ozone-sensitive poplar
clone under field conditions. The results were translated into whole tree water loss and carbon assimilation by comparing
trees exposed to ambient ozone and trees treated with the ozone-protectant ethylenediurea (EDU). Steady-state stomatal
conductance and photosynthesis linearly decreased with increasing ozone visible injury. Dynamic responses simulated bysevering of a leaf revealed that stomatal sluggishness increased until a threshold of 5% injury and was then fairly constant.
Sluggishness resulted from longer time to respond to the closing signal and slower rate of closing. Changes inphotosynthesis were driven by the dynamics of stomata. Whole-tree carbon assimilation and water loss were lower in trees
exposed to ambient O3 than in trees protected by EDU, both under steady-state and dynamic conditions. Althoughstomatal sluggishness is expected to increase water loss, lower stomatal conductance and premature leaf shedding of
injured leaves aggravated O3 effects on whole tree carbon gain, while compensating for water loss. On average, WUE oftrees exposed to ambient ozone was 2–4% lower than that of EDU-protected control trees in September and 6–8% lower in
October.
Citation: Hoshika Y, Omasa K, Paoletti E (2012) Whole-Tree Water Use Efficiency Is Decreased by Ambient Ozone and Not Affected by O3-Induced Stomatal
Sluggishness. PLoS ONE 7(6): e39270. doi:10.1371/journal.pone.0039270
Editor: Carl J. Bernacchi, University of Illinois, United States of America
Received February 24, 2012; Accepted May 22, 2012; Published June 18, 2012
Copyright: ß 2012 Hoshika et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: JSPS Fellowships for Young Scientists. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: e.paoletti@ipp.cnr.it

adaptation and competition [31]. Because of climate change,
forest ability of water control and carbon sequestration under O3
pollution is of rising importance [14].
Scalar and conceptual uncertainties still limit the current
understanding of the basic physiological mechanisms that underline responses of forests to O3 [32]. The scalar uncertainties are
due to transfer of results from seedlings in controlled environments
to mature trees in the field, while the conceptual uncertainties are
due to contrasting results about whole-tree water use responses toambient O3 [32–35]. In contrast, there is a general agreement
about O3 exposure as a factor of reduced tree carbon sequestration
and biomass [36], although the results usually come from steadystate measurements of photosynthesis.
Ozone visible injury of leaves may be used as a clear and easily
quantifiable proxy of O3 foliar damage and is the only method toassess O3 damage in the field [37]. Ozone visible injury has been
investigated in many European and North American tree andherbaceous species, and partly validated under controlled conditions [38,39]. There are few reports of relationship between
stomatal conductance and O3 visible injury. After onset of O3
visible injury, significant reductions in steady-state leaf gas
exchange were recorded for tree species in chamber experiments[40–42]. Omasa et al. (1981) did not report any correlation
between visible injury and stomatal O3 uptake in a leaf [43].
Dynamic stomatal response was slower in injured leaves (20%Introduction
Tropospheric ozone (O3) is an important phytotoxic air
pollutant and is also recognized as a significant greenhouse gas[1]. Tropospheric O3 level has been continuously increasing since
the first direct measurements in 1874 and its atmosphericconcentration is now twice or more than in the pre-industrial
age in the northern hemisphere [2–4]. Phytotoxic nature of O3 hasbeen well known for decades [5–12]. Ozone concentrations
recorded in rural areas are higher than those in the city [13] andthus O3 is now considered as the air pollutant with the highest
damage potential to forests [14].
As the penetration of O3 through the cuticle can be considered
as negligible [15], uptake through the stomata is a crucial factor
for assessing the adverse effect of O3 on plants [16–20]. However,effects of O3 on stomatal responses are not straightforward, as both
reductions and sluggish responses have been reported [21,22].
Reductions of stomatal conductance occur when measurements
are carried out under steady-state conditions [23]. Sluggishness
has been reported during dynamic stomatal responses tofluctuating photosynthetic photon flux density (PPFD) [22,24–27], vapor pressure deficit (VPD) [27], and severe water stress
imposed by severing a leaf [26,28–30]. Sluggish stomatal control
over transpiration may increase water loss. Plants live ina fluctuating environment. A fast gas exchange response to rapidchanges in the environmental stimuli is the key for successful plant
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Whole-Tree Ozone Effects and Stomata

injury) compared to control leaves (0% injury) for manna ash(Fraxinus ornus L.) [28].
Our main objectives were to improve our knowledge of steadystate and dynamic stomatal response to O3 visible injury in adulttrees in the field, and to assess whole-tree water loss and carbon
assimilation under ambient O3 impacts. Measurements werecarried out in an O3-sensitive poplar clone (Oxford, Populus
maximoviczii Henry 6 berolinensis Dippel) [44]. The amount of leaf
injury per tree was experimentally manipulated by applying theO3-protectant ethylenediurea (EDU, N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N’-phenylurea). EDU per se does not affect gas
exchange [45] and has been widely used to prevent O3 visible
injury and determine O3 effects in many plant species [39,45–
47].

Measurement of Steady-state and Dynamic Gas
Exchange
Fully expanded sun leaves (medium size) with visible injury from
0% to 50% at set positions from the terminal shoot (5th to 16th) of
WAT trees were measured in clear sky days of September andOctober 2010 between 10:00 and 15:00 CET. Preliminary
measurements did not show significant differences in gas exchangeof healthy leaves, i.e. without visible ozone injury, at those set
positions. Gas exchange was measured with a portable infra-red
gas analyzer (CIRAS-2 PPSystems, Herts, UK), equipped witha 2.5 cm2 leaf cuvette which controlled leaf temperature (24uC),
leaf-to-air vapour pressure deficit (1.0 kPa), saturating light(1800 mmol m22 s21) and CO2 concentration (375 ppm).
Steady-state light-saturated photosynthesis (Amax), stomatal conductance to water vapor (gs) and transpiration were measured in
41 leaves from WAT trees.
Dynamic measurements were carried out for 21 leaves from
WAT trees. When both gs and Amax reached equilibrium underconstant light at 1800 mmol m22 s21, the leaf petiole was severed
with a sharp scalpel, similar to the methodology in Paoletti (2005)[26]. The data were logged at 1 min intervals for 30 min after
severing. As the absolute value of gs and Amax varied amongindividual leaves, relative gs and Amax were expressed asa percentage of the average of the last 5 points at equilibrium,i.e., just before leaf severing. The following parameters were
estimated based on fittings of two linear lines to minimize the root
mean square error between measured and predicted values for gs
or A (Figure 1A): range of relative gs decrease at 30 min after
severing, Dgs; time to start gs decrease, Tresp (gs); rate of gs decreaseat 30 min from severing, Slope(gs) = Dgs/(30– Tresp (gs)); range ofrelative Amax decrease at 30 min after severing, DA; time to start
Amax decrease, Tresp (A); rate of Amax decrease at 30 min fromsevering, Slope(A) = DA/(30– Tresp (A)).
After measurements, the leaf area was measured by means ofa leaf area meter (AM300, ADC, Herts, UK) for assessing
a relationship between leaf size and the variation of gs in single
leaves. We hypothesized that the water content of a leaf may
depend on leaf size and affect gs response.

Materials and Methods
Experimental Site and Plant Material
The study was carried out in an experimental field site locatedin central Italy (Antella: 43u44’ N, 11u16’ E, 50 m a.s.l., 14.7uC asmean annual temperature and 1233 mm as total annual precipitation in 2010). Forty root cuttings of the O3-sensitive Oxford
clone were planted in two lines in 2007. Every week over thegrowing seasons 2008–2010, each tree was irrigated with 1 to 2 L
of water (WAT, control line) or 450 ppm EDU solution (EDU,
treated line), according to the successful application of EDU as soil
drench to adult trees [48]. In 2010, the mean tree height was
2.9 m, and the mean stem diameter at breast height was 19 mm.
Soil moisture was measured in the root layer (30 cm depth) by
EC5 sensors equipped with an EM5b data logger (Decagon
Devices, Pullman WA, USA). On average, soil moisture was
21.160.2% during the gas exchange measurements (SeptemberOctober) and 24.560.1% during the growing season (April to
October). The values were between field capacity (25.5%) andwilting point (17.5%) for this type of soil, i.e. sandy clay loam. Air
temperature, relative humidity and precipitation were recorded bya 110-WS-16 modular weather station (NovaLynx corp., AuburnCA, USA). Average vapor pressure deficit during daylight hours
and total precipitation were 1.02 kPa and 197 mm in September
to October and 1.42 kPa and 625 mm from April to October,
respectively. Ozone concentrations were continuously recorded at
canopy height (2.0 m) by an O3 monitor (Mod. 202, 2B
Technologies, Boulder CO, USA). The AOT40 value (accumulated exposure above a threshold concentration of 40 ppb during
daylight hours) during the growing season (April to October) was
25.8 ppm?h and the maximum hourly O3 concentration reached
118 ppb.

Tree Level ModelingTo assess effects of O3 visible injury on leaf gas exchange at treelevel, we constructed a simple model to scale up from single–leaf
steady-state and dynamic gas exchange. The model was applied to
the five trees per treatment (WAT and EDU) whose leaves werecounted and assigned to a 5%-step visible injury class. Steady-state
leaf water loss and photosynthesis at tree level, i.e. Wloss: mol H2O
tree21 s21, and Atree: mmol CO2 tree21 s21, were estimated asfollows:

Assessment of Ozone Visible Injury
Ozone visible injury occurred as dark stippling on the upper
leaf surface since early September 2010. The injury was
identified as O3-like because it was missing in shaded leaves
and more severe in older than in younger leaves [38]. The
symptoms were similar to those caused by ambient O3 in Populus
nigra [42]. In September (22th to 28th) and October (23th to 28th),
all 9502 leaves from five trees per treatment (WAT and EDU)
were counted and assigned to 5%-step injury classes by the sametwo observers. Photoguides quantifying visible injury (0,100%)by image analysis processing were used [38,39]. Pest, pathogen
and mechanical injury occurred in both EDU and WAT trees
and was assessed to be ,5% of total leaves. Leaves formeasurements of gas exchange showed O3 visible injury only
and were evaluated on a 1%-step basis.

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Wloss ~

Atree ~X
Tr inj :LA:Ninj

X

Amax inj :LA:Ninj

ð1Þ



ð2Þ

where Tr_inj and Amax_inj are transpiration rate (mmol m22 s21)
and photosynthesis (mmol m22 s21), respectively, at
1800 mmol m22 s21 constant light for leaves showing O3 visible
injury. Ninj is the number of leaves in each 5%-step injury class.
LA is the average leaf area per leaf (0.003 m2 leaf21), calculated
from subsamples of 30 randomly collected leaves per tree.

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Whole-Tree Ozone Effects and Stomata

Statistical Analysis
Effects of O3 visible injury on steady-state leaf gas exchange anddynamic responses after severing a leaf were tested with a regression analysis. Correlation between variables of dynamic
stomatal response was tested. Two-way analysis of variance(ANOVA) was used to assess the effects of measuring month and
EDU treatments on number of leaves, ozone visible injury and gas
exchange parameters at whole tree level. Differences amongmeans were tested by Tukey HSD test. Percents were arcsine
square root transformed prior to analysis. Data were checked fornormal distribution (Kolmogorov-Smirnov D test) and homogeneity of variance (Levene’s test). Results were consideredsignificant at p,0.05. All statistical analyses were performed withSTATISTICA software (6.0, StatSoft Inc., Tulsa, OK, USA).

Results
Number of Leaves and Ozone Visible Injury
In September, EDU trees had 83% more leaves per tree than
WAT trees (Figure 2A). In October, leaf abscission had progressed
faster in WAT trees (236% of leaves relative to September) thanin EDU trees (215%), resulting in EDU trees showing significantly
more leaves (+144%) than WAT trees. The percentage of injured
leaves (.5% of visible injury) was significantly higher in WAT
trees than in EDU trees in both September and October(Figure 2B). In October, the percentage of injured leaves was
3.13 and 7 times higher than in September in WAT and EDU
trees, respectively.

Figure 1. Examples of dynamic response of gs and Amax after
detachment of the leaf (A: calculation of the dynamic
parameters in a leaf with 0% visible injury, B: time courses ofabsolute values in gs, C: time courses of absolute values in A).
Dgs and DA show the range of gs and Amax variation, respectively, over
30 min from the leaf severing. Tresp (gs) and Tresp(A) show the time tostart decrease of gs and Amax, respectively. Slope(gs) and Slope(A) show
the rate of decrease for gs and Amax, respectively, over 30 min.
doi:10.1371/journal.pone.0039270.g001

Whole-tree leaf water loss and carbon assimilation under thesevere water stress simulated by severing a leaf (Wloss_st: mol H2O
tree21 s21, and Atree_st: mmol CO2 tree21 s21) were estimated by
the following equations:X
Tr inj ::LA:Ninj

ð3Þ

X

ð4Þ

Wloss st ~Atree st ~Amax inj :LA:Ninjwhere Tr inj is the average transpiration rate (mmol m22 s21) andAmax inj is the average photosynthesis (mmol m22 s21) at
1800 mmol m22 s21 constant light during the 30 min after
severing a leaf with O3 visible injury.

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Figure 2. Total number of leaves (A) and percentage of ozone
injured leaves (more than 5% of injured surface) (B) per tree(+SE) (WAT: water treated plants; EDU: EDU treated plants).
* and *** denote significance at the 5% and 0.1% level, respectively; n.s.
indicates no significance. Different letters above the bars indicate
significant differences among bars (Tukey HSD test, P,0.05, n = 5 trees).
doi:10.1371/journal.pone.0039270.g002

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Whole-Tree Ozone Effects and Stomata

healthy leaves to 25 min in a leaf with 50% injury. Table 1 shows
correlation between the Amax and gs variables obtained fromdynamic response after severing of a leaf. The magnitude ofchange in Amax (DA) increased with increasing Dgs. The rate of
reduction in Amax, i.e. Slope(A), was positively correlated with
Slope(gs). The response times to start decrease of Amax and gs, i.e.,
Tresp, were not significantly correlated, although they showeda statistical tendency to a positive correlation (p,0.1).

Steady-state gs and Amax
Steady-state leaf gas exchange for both gs and Amax decreased
with increasing O3 visible injury (Figure 1, 3). In healthy leaves(0% injury), gs was 400 to 800 mmol m22 s21 whereas it was lessthan 100 mmol m22 s21 in leaves with 50% injury (Figure 3A).
Leaves with higher injury (.50% injury) were tested but did notshow a measurable gs. In control leaves, Amax was 5 to
15 mmol m22 s21, and dropped to around 0 mmol m22 s21 inleaves with more than 35% injury (Figure 3B).

Carbon Assimilation and Water Loss at Tree Level
In September, Atree and Wloss were significantly lower in WAT
trees, being half of the values in EDU trees (Figure 5A–B). In
October, the difference between WAT and EDU trees became
even larger. Whole-tree water use efficiency (Atree/Wloss) at steadystate was significantly higher in EDU trees than in WAT trees bothin September and October (Figure 5C). WUE decreased overtime, but the decrease was larger in WAT (26%) than in EDU
trees (22%), resulting in a significant Time x EDU interaction.
Both in September and October, both Atree_st and Wloss_st, i.e.
whole-tree carbon assimilation and water loss under the simulated
severe water stress, were significantly lower in WAT trees(Figure 6A–B), similarly to the results from steady-state measurements (Figure 5A–B). Whole-tree instantaneous water use
efficiency, expressed as Atree_st/Wloss_st, was significantly higher
in EDU trees than in WAT trees both in September and October(Figure 6C). Again, the decrease of WUE_st over time was larger
in WAT (28%) than in EDU trees (23%).

Variation of gs and Amax after Detachment of a Leaf
After detachment of a leaf, two phases of gas exchange response
were observed (Figure 1A): no response until Tresp and then a linear
decrease. The magnitude of change in gs at 30 min after severing
a leaf (Dgs) did not depend on leaf size (data not shown: R2 = 0.02,
p = 0.537) and thus on the total water content of a leaf.
Figure 4 shows the relationships between O3 visible injury anddynamic response of gs and Amax. Dgs showed a non-linear
response to O3 visible injury (Figure 4A). It sharply decreased from45–60% in healthy leaves (0% injury) to 15–30% in leaves with
.5% visible injury. Slope(gs) sharply decreased from 2.5–3.2%
min–1 in healthy leaves (0% injury) to 0.8–1.8% min–1 in leaves
with .5% visible injury, and did not vary in leaves with 5–50% of
injury (Figure 4B). The response time to start stomatal closing(Tresp (gs)) was linearly correlated to O3 visible injury (Figure 4C).
Tresp (gs) increased from about 10 min in healthy leaves to.13 min in leaves with .20% injury. The magnitude of decreasein photosynthetic rate (DA) sharply decreased from about 55% in
healthy leaves to about 25% in leaves with .5% visible injury(Figure 4D). Slope(A) sharply decreased from about 3.3% min–1 inhealthy leaves to about 1.6% min–1 in leaves with .5% visible
injury (Figure 4E). There was a linear relationship between the
response time to start decrease of photosynthesis (Tresp (A)) and O3
visible injury (Figure 4F). Tresp (A) increased from 5–13 min in

Discussion
According to previous reports in different species [28,40–42],
the steady-state measurements indicated that gs and Amax linearlydecreased with increasing leaf visible injury in the O3-sensitive
Oxford clone (Figure 3). Amax dropped to around 0 mmol m22 s21
in leaves with more than 35% injury and gs was not measurable in
leaves with more than 50% injury. In a previous field study, leaves
of manna ash with 20% visible injury showed a 33% reduction in
gs and Amax relative to healthy leaves (measurement in September)[28]. The result of the present study showed a larger reduction in
gs (about 39%) and Amax (about 54%) of 20% injured leaves,suggesting effects of O3 visible injury on gas exchange are speciesspecific. Paoletti et al. (2009a) suggested that the modifications ofstomatal conductance in O3 injured leaves were driven by the
structural alterations found in the mesophyll rather than bystructural changes in stomata or other epidermal cells [28]. Omasa
et al. (1981) suggested that stomatal opening in leaves with O3
visible injury varied with changes in the pressure balance between
guard cells and epidermal cells caused by the water-soaking of
epidermal cells [43]. The most likely changes, however, are due tophotosynthetic impairment [21,49].
When analyzing dynamic gs response to severing of a leaf,
stomata of injured leaves were shown to be slower than those ofhealthy leaves in responding to the closing signal (Tresp (gs)) and in
the rate of closing (Slope(gs)) (Figure 4B–C). These combined
effects translated in a lower ability of injured leaves to close
stomata, i.e. in a lower Dgs than healthy leaves, resulting ina sluggish stomatal control over water loss. In a previous study,
Paoletti et al. (2009a) also reported a slower response of stomata to
severing in leaves of manna ash with O3 visible injury [28], eventhough only leaves with 0% and 20% injury were compared.
Here, we compared leaves with a range of O3 visible injury, i.e.
from no injury (control) until a measurable gs was recorded (,50%
injury) and showed that Dgs decreased sharply above 5% injury
and did not change any more (Figure 4A).

Figure 3. Relationships between steady-state leaf gas exchange (A: stomatal conductance (gs), B: light-saturated
photosynthesis (Amax)) and visible ozone foliar injury.
doi:10.1371/journal.pone.0039270.g003

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Whole-Tree Ozone Effects and Stomata

Figure 4. Relationships between visible ozone foliar injury anddynamic response of stomatal conductance (gs) and photosynthesis (Amax) over 30 min after leaf severing (A: Dgs at
30 min; B: Slope(gs); C: Tresp (gs); D: DA; E: Slope(A); F: Tresp (A)).
doi:10.1371/journal.pone.0039270.g004

Literature results highlight several mechanisms by which O3
may induce sluggishness. Omasa (1990) reported a slight increasein permeability of epidermal cell membranes and alteration of theosmotic pressure after O3 exposure, that may modulate a balancein turgor between guard and subsidiary cells [50]. Vahisalu et al.
(2010) found that Ca2+-dependent signaling and O3-induced
stomatal movements were independent, and highlighted a temporary desensitization of the guard cells due to blocking of the K+
channels [51]. Another cause of sluggishness may be O3-induced
lower rates of transpiration in which leaves take longer to perceive
the same change in water status following petiole excision [26,28–
30] or light variation [22,26]. All the above mechanisms, however,cannot explain the non-linear response of Dgs to visible injury
observed in the present study. Ozone may also delay stomatal
responses by stimulating ethylene production and reducing
stomatal sensitivity to ABA [52]. Ethylene production is known
to increase with increasing O3 visible injury [53,54]. In tomato
plants, concentration of ACC (1-aminocyclopropane-1-carboxylicacid), a precursor of ethylene, increased when visible injury
reached 5% and remained constant until the maximum injury
recorded in the experiment, i.e. 35% [55]. A sharp rise of ethylene
emission as soon as visible injury reaches 5% and a constantemission over this threshold would explain why Dgs decreased
sharply above 5% injury and did not change any more wheninjury was .5% (Figure 4A). Tuomainen et al. (1997) also showedthat ethylene emission from detached leaves was enhanced
fourfold in ozone-treated plants, while no changes were observedin control leaves that were similarly cut at the petiole [55].
Sluggish Amax responses with increasing O3 visible injury werealso found in the measurements of dynamic leaf gas exchange(Figure 4D–F). The response of Amax was similar to that of gs after
severing a leaf (Figure 1), i.e. no response until Tresp and thena linear decrease during stomatal closure. Although the responsetime to start reduction of Amax was not significantly correlated withthe response time to closing stomata, the magnitude and rate of
reduction in Amax were linearly correlated to those of stomatalclosure (Table 1). Heber et al. (1986) showed that photosynthetic
rate decreased following stomatal closure after severing of a leaf
[56]. Slightly shorter Tresp(gs) than Tresp(A) confirmed that the
reduction of Amax was mediated by the response of gs. The slower
reduction of Amax in injured leaves than in healthy leaves would
increase carbon assimilation under water stress conditions andmay be interpreted as a feedback mechanism to maximize
Table 1. Correlation between Amax vs. gs variables obtained
during the dynamic response to severing of a leaf (D:magnitude of change in Amax and gs over 30 min from the leaf
severing; Tresp: time to start decrease in Amax and gs after
severing a leaf; Slope: rate of Amax and gs decrease).

Parameter

Pearson coefficientLevel of significance

D

0.626

0.002**

Slope

0.622

0.003**

Tresp

0.371

0.098 n.s.

**denotes the significance at 1% level; n.s. indicates no significant correlation.
doi:10.1371/journal.pone.0039270.t001

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Whole-Tree Ozone Effects and Stomata

Figure 5. Estimated steady-state carbon assimilation (A: Atree),water loss (B: Wloss) and instantaneous water use efficiency
expressed as Atree/Wloss (C: WUE) at tree level (+SE) (WAT:
water treated plants; EDU: EDU treated plants). * and *** denotesignificance at the 5% and 0.1% level, respectively; n.s. indicates nosignificance. Different letters above the bars indicate significant
differences among bars (Tukey HSD test, P,0.05, n = 5 trees).
doi:10.1371/journal.pone.0039270.g005

Figure 6. Estimated carbon assimilation (A: Atree_st), water loss
(B: Wloss_st) and instantaneous water use efficiency expressed
as Atree_st/Wloss_st (C: WUE_st) at tree level under severe water
stress imposed by severing a leaf (+SE) (WAT: water treatedplants; EDU: EDU treated plants). * and *** denote significance atthe 5% and 0.1% level, respectively; n.s. indicates no significance.
Different letters above the bars indicate significant differences amongbars (Tukey HSD test, P,0.05, n = 5 trees).
doi:10.1371/journal.pone.0039270.g006

photosynthesis under stress. However, severe O3 visible injury(.35%) shifted carbon sink to source because Amax was,0 mmol m22 s21 (Figure 3B).
At whole-tree level, the total carbon assimilation (Atree) andwater loss (Wloss) assessed under steady-state conditions weresignificantly lower in WAT trees exposed to ambient ozone than in
EDU-protected trees in both September and October (Figure 5A–
B). Such O3-induced reduction of photosynthesis and water loss
was in agreement with meta-analysis results [36]. Dynamic andsteady-state whole-tree WUEs showed a similar seasonal trend.
WUE was significantly higher in EDU trees than in WAT trees,
both in September and October and both when assessed understeady-state and dynamic conditions (Figure 5C and 6C). On
average, WUE of trees exposed to ambient ozone was 2–4% lower
than that of EDU-protected control trees in September and 6–8%
lower in October. The decrease of tree-level WUE over time, infact, was larger in WAT than in EDU trees, confirming the

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frequently reported decrease in leaf-level WUE in O3-exposed
plants [33] and O3-injured leaves [41]. Also whole-tree dynamic
carbon assimilation (Atree_st) and water loss (Wloss_st) weresignificantly lower in WAT trees than in EDU-protected trees(Figure 6A–B). In contrast, ozone-induced stomatal sluggishness
would be expected to increase whole-tree water loss. This
response, however, was balanced by lower gas exchange(Figure 3) and premature shedding of injured leaves. After the
onset of O3 visible injury in early September, ozone visible injury
increased quickly (Figure 2B). In parallel, leaf abscission also
progressed (Figure 2A), so that both whole-tree water loss andcarbon assimilation were reduced. However, McLaughlin et al.
(2007) reported that ambient O3 spikes significantly increased
water loss of trees, as assessed from sap-flow measurements,suggesting that ozone-induced aberrations in the stomatal
dynamics may differ depending on the species and the environmental conditions [32].
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Whole-Tree Ozone Effects and Stomatachange brings about the risk of drought and flooding [1]. The
results of this study contribute new knowledge about water control
and carbon sequestration of trees under ambient O3 exposure andsuggest that the effects of O3–induced stomatal sluggishness on thewhole-tree carbon and water balance are negligible.

Conclusions
One of the topical subjects in the assessment of O3 risk to forests
is scaling up from leaf level to the stand and landscape level [4].
Further improvement of our understanding about stomatal
responses to ambient O3 can be regarded as an essential factor
in modelling and predicting forest responses to both O3 andclimate [21]. Occurrence of O3 visible injury resulted in loss of
stomatal control for water loss, but was compensated by lower
stomatal conductance and premature leaf shedding. The resulting
decline in whole tree ability of transpiring and sequestering
atmospheric carbon is a significant effect of ambient ozone
pollution.
Stomata play a crucial role in regulating plant gas exchangewith the atmosphere, including O3 uptake [16–20]. Surface O3
concentrations are continuously increasing [4]. The climate

Acknowledgments
Prof. William J. Manning and Dr. Marcus Schaub are greatly
acknowledged for providing EDU and poplar cuttings, respectively.

Author Contributions
Conceived and designed the experiments: EP KO. Performed theexperiments: YH EP. Analyzed the data: YH EP. Contributed reagents/
materials/analysis tools: EP. Wrote the paper: YH EP.

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