Environmental and Experimental Botany 88 (2013) 19–23

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Environmental and Experimental Botanyjournal homepage: www.elsevier.com/locate/envexpbot

Both ozone exposure and soil water stress are able to induce stomatalsluggishness
Yasutomo Hoshika a , Kenji Omasa a , Elena Paoletti b,∗ab

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Istituto Protezione Piante, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019 Sesto Fiorentino, Italya r t i c l e

i n f oArticle history:
Received 29 August 2011
Received in revised form
20 November 2011
Accepted 1 December 2011
Keywords:
Tropospheric ozone
Stomatal conductance
Stomatal sluggishness
Snapbeana b s t r a c t
We tested whether short-term exposure to realistic ozone pollution (≤150 ppb, 1 h) and soil water stress(soil water content ≤15%) slow stomatal dynamics in an ozone-sensitive cultivar of snapbean. Both ozone
exposure and water stress caused stomata to be sluggish in the degree of closure after leaf severing, whileozone also delayed the time the closing signal was perceived. Ozone-induced aberrations lasted up to
the night and caused incomplete closure of stomata. No synergic effect was observed in the dynamic
measurements. In contrast, at steady-state, water stress protected the plants from the negative ozone
effects on stomatal conductance. Ambient ozone peaks may thus cause sluggish stomatal response andincrease leaf water loss both under well watered and drought conditions.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Tropospheric ozone (O3 ) is an important secondary pollutant
resulting from emission of volatile organic compounds and nitrogen oxides, and is also recognized as a signiﬁcant greenhouse gas(Bytnerowicz et al., 2007; Serengil et al., 2011). Ozone is seriously
phytotoxic and causes negative effect on plants (e.g., NIES, 1980,
1984; Omasa et al., 2002; Paoletti, 2007). Stomatal O3 uptake iscrucial for assessing the adverse effect of O3 on plants (Omasa
et al., 1979; UNECE, 2004; Paoletti and Manning, 2007; Grulke et al.,2007a; Cieslik et al., 2009). However, our understanding about stomatal responses to O3 is still imperfect (Paoletti and Grulke, 2005).
Previous studies reported that O3 generally induces both stomatal closure (Wittig et al., 2007, 2009) and sluggishness of stomatal
response to change of environmental factors (Paoletti and Grulke,
2005, 2010). Because plants live in a ﬂuctuating environment, both
steady-state stomatal conductance and stomatal dynamics play
an important role in regulating leaf gas exchange. Ozone-induced
sluggishness of stomata has been reported in response to change
in photosynthetic photon ﬂux density (PPFD) (Reich and Lassoie,
1984; Reiling and Davison, 1995; Paoletti, 2005; Grulke et al.,2007a; Paoletti and Grulke, 2010), vapor pressure deﬁcit (VPD)(Grulke et al., 2007b) and severe water stress imposed by severing
a leaf (Paoletti, 2005; Paoletti et al., 2009; Mills et al., 2009). Suchaberrations may increase nighttime transpiration, as reported for∗ Corresponding author. Tel.: +39 55 5225 591; fax: +39 55 5225 666.
E-mail address: e.paoletti@ipp.cnr.it (E. Paoletti).
0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2011.12.004

several tree species exposed to daytime O3 exposure in controlled(Skärby et al., 1987; Matyssek et al., 1995; Wieser and Havranek,
1995; Günthardt-Goerg et al., 1997; Grulke et al., 2007c) and ambient conditions (Grulke et al., 2004). Ozone is known to induce
up-regulation of ethylene emission, which has been suggested to be
responsible for a reduction in stomatal sensitivity to abscisic acid
(ABA) and thus to closure (Wilkinson and Davies, 2010). Several
stressors, including water deﬁcit, are known to stimulate ethylene
production by plants (Morgan and Drew, 1997).
Climate change is expected to increase O3 levels and alter
precipitation regimes, regionally increasing the probability ofdrought (Ciais et al., 2005). Drought has the capacity to limit O3
injury through stomatal closure (Tingey and Hogsett, 1985), while
O3 -induced impairment of stomatal response may increase susceptibility to drought (Nali et al., 2004).
Our main objective was to test whether short-term exposure to
realistic O3 pollution and soil water stress, singly and in combination, may slow stomatal dynamics in an ozone-sensitive cultivar of
snapbean (Phaseolus vulgaris, S156).
2. Materials and methods
Seeds of the ozone sensitive cultivar S156 of snapbean developed at the Raleigh USDA-ARS (Burkey and Eason, 2002; Flowers
et al., 2007; Booker et al., 2009), were planted in 17-cm (1.7–1)
pots, ﬁlled with sand:peat:soil = 1:1:1 (v:v:v). Seed were planted
over several days so that same-age (4-week old) plants were usedin the experiment. All plants were grown in a room with controlled environmental conditions (air temperature of 20 ◦ C, PPFD

Y. Hoshika et al. / Environmental and Experimental Botany 88 (2013) 19–23

b
c

20

a

a
ab

-0.3

cd

de

Water stressed condition
ef

f

fg

abc abc

-0.4

g

abc

10

bc

-0.5

bc

bc

Soil water content
Leaf water potential

0

0

2

4

6

gh gh

8

c
10

(A)

150

Relative g s (%)

Soil water content (%)

30

-0.2

Well-watered conditionaLeaf water potential (MPa)20

M agnitude of WWR
Magnitude
100

Δgs
50

Duration of
WWR

-0.6

0

Time (days)of 500 ␮mol m−2 s−1 , and relative humidity of 55%). Well-wateredplants were supplied with water every day and in particular immediately before the ozone exposure, while water-stressed plants
received no irrigation for 10 days before O3 fumigation. Soil watercontent was more than 30% in well-watered plants and 6–15%
in water-stressed plants, corresponding to a predawn leaf water
potential of −0.32 MPa and −0.53 MPa, respectively (Fig. 1). Water
content was measured in 18 pots with an ECH2O EC-5 soil moisture sensor (Decagon Devices, Pullman WA, USA) at root level. Leaf
water potential was measured at dawn in a selection of 2–3 plants
per day by means of an SKPM 1400 chamber pressure (Skye, Powys,
UK).
The fully expanded central leaf of the second trifoliate leaf wasselected as a target leaf. After 1-h exposure to 1000 ␮mol m−2 s−1light, steady state leaf gas exchange was measured with a portableinfra-red gas-analyzer (CIRAS-2 PP Systems, Herts, UK), equippedwith a 2.5 cm2 leaf cuvette which controlled leaf temperature(20 ◦ C), leaf-to-air vapour pressure deﬁcit (0.9 kPa), saturating light(1800 ␮mol m−2 s−1 ) and CO2 concentration (365 ppm). Ozone
exposure to one of four levels (±SD), i.e. low (48 ± 6.7 ppb), middle (87 ± 9.4 ppb), high (150 ± 10.9 ppb), and control (0 ppb, no O3 ),was then carried out for 60 further min by a web fumigation system (Velikova et al., 2005; Pinelli and Tricoli, 2008). Ozone wasadded by an O3 generator (Model Heliozon, Milano, Italy) to thefumigating air for the target leaf through a teﬂon tube. The concentration around the leaf was recorded with an ozone monitor (Mod.
205, 2B Technologies, Boulder CO, USA), and adjusted through mass
ﬂow controllers (Mod. GFC171S A alborg). At 30 min after the endof O3 exposure, steady-state stomatal conductance (gs ) was measured again. When gs reached equilibrium under constant light at
1800 ␮mol m−2 s−1 , the methodology described by Paoletti (2005)
was applied to assess dynamic variations of gs after cutting the leaf
petiole (Fig. 2). Data were logged at 1 min intervals in the 45 minafter severing. In the experiment, two phases of gs response wereobserved (Fig. 2). At ﬁrst, gs increased for Iwanoff effect. This transient increase called as the transient ‘wrong-way response’ (WWR)
is due to a difference in turgor pressure between guard cell andepidermal cells (Omasa and Maruyama, 1990; Powles et al., 2006).
Subsequently, stomatal conductance decreased with increasing
leaf water stress. WWR duration and magnitude of WWR and gs
decrease at 45 min (gs ) were recorded.
After O3 exposure and steady-state measurements, 22 plants
were placed in the dark for 10 h. Nocturnal steady-state gs wasmeasured with leaf temperature of 20 ◦ C, leaf-to-air vapour pressure deﬁcit of 0.9 kPa, no exposure to light and CO2 concentrationof 365 ppm.
Data were checked for normal distribution and homogeneity of variance (Levene’s test). Percents were arcsine square root

(B)

150

Relative gs (%)

Fig. 1. Changes in soil water content (±S.D., n = 18) (%)and predawn leaf water
potential (±S.D., n = 2–3) after irrigation until ﬁeld capacity. Different letters indicate
signiﬁcant differences among mean values (Tukey HSD test, P < 0.05).

100

50

0

0

5

10

15

20

25

30

35

40

45

Time after detachment (min)Well wat, control (0 pp b)

Well wat, O3 (150 ppb)

Wat str, control (0 ppb)

Wat str, O 3 (150 ppb)

Fig. 2. Time courses of stomatal conductance (gs ) after severing a leaf at time 0.
Graph A shows an example of calculation of WWR magnitude and duration, and of
the degree of gs decrease (gs ). At ﬁrst, gs showed a transient increase called ‘wrongway response’ (WWR) and a subsequent decrease with increasing leaf water stress.
Graph B shows the time course of average gs (±SE) under well-watered condition(soil water content > 30%) and water-stressed condition (soil water content = 6–15%)
in control (0 ppb O3 ) and ozone-exposed (150 ppb O3 ) leaves.

transformed prior to analysis. Effects of soil water status and O3
exposure were tested using two-way analysis of variance (ANOVA).
Results were considered signiﬁcant at p < 0.05. Differences amongmeans were tested by Tukey’s HSD test. Statistical analysis was
performed with STATISTICA software (6.0, StatSoft Inc., Tulsa, OK,
USA), according to Statsoft (2001).

3. Results
3.1. Steady-state and dynamic stomatal responses
Ozone exposure induced a decline of steady-state gs under wellwatered conditions (Fig. 3). gs was 73% in the low O3 treatment
and 25% in the high O3 treatment compared to the control plants.
A reduced soil water availability signiﬁcantly reduced gs relative towell-watered plants and resulted in no effect of O3 on gs .
After severing a leaf, two phases of gs response were observed(Fig. 2): a transient increase as WWR and then a linear decrease.
Ozone exposure increased WWR duration from 11.9 ± 2.7 min in
the control plants to 17.1 ± 3.2 min in the high O3 treatment, while
the effect of water stress was not signiﬁcant (Fig. 4A). Magnitudeof WWR was not affected by both O3 and soil water availability (Fig. 4B). Ozone reduced the degree of stomatal closure overtime (gs ) in both soil water conditions (Fig. 4C). Also soil waterdeﬁcit reduced gs relative to the optimal soil water availability. In well-watered plants, the high O3 treatment resulted insmaller stomatal closure than in control leaves (48% vs. 75%). In

Y. Hoshika et al. / Environmental and Experimental Botany 88 (2013) 19–23

Fig. 3. Effect of 1-h exposure to four levels of O3 (control: 0 ppb; low: 45 ppb; middle: 90 ppb; high: 150 ppb) and soil water availability (well-watered 30% watercontent; water-stressed: 6–15% water content) on steady-state stomatal conductance (gs ). Data are means (N = 5–8) ± SE. * denotes signiﬁcance at 5% levels; **denotes signiﬁcance at the 1% levels; n.s. indicates no signiﬁcance. Different letters
indicate signiﬁcant differences among bars (Tukey HSD test, P < 0.05).

21

Fig. 5. Effects of 1-h O3 exposure (Control: 0 ppb; low: 45 ppb; middle: 90 ppb;
high: 150 ppb) and soil water availability (well-watered 30% water content; waterstressed: 6–15% water content) on nocturnal steady-state stomatal conductance (gs )relative to the pre-exposure daytime value. Data are means (N = 2–4) ± SE. * denotes
signiﬁcance at the 5% levels; n.s. indicates no signiﬁcance.

water-stressed plants, high O3 and control treatment resulted in
41% and 61% stomatal closure, respectively.
3.2. Nocturnal stomatal conductanceFig. 5 shows nocturnal steady-state gs relative to daytime preexposure gs . Ozone exposure caused an increase of nocturnal gs . A
reduced soil water availability did not affect nocturnal gs . Nocturnal
gs increased in well-watered plants from 7% in the control plants
to 23% in the high O3 treatment.
4. Discussion

Fig. 4. Effects of 1-h O3 exposure (Control: 0 ppb; low: 45 ppb; middle: 90 ppb;
high: 150 ppb) and soil water availability (well-watered 30% water content; waterstressed: 6–15% water content) on dynamic gs response after severing a leaf (A:duration of transient increase in gs after severing; B: magnitude of transient increasein gs after severing; C: degree of gs decrease at 45 min after severing). Data are means(N = 3–4) ± SE. * denotes signiﬁcance at the 5% levels; n.s. indicates no signiﬁcance.

Increasing O3 exposure under optimal water availability progressively decreased steady-state gs . Although the mechanismsthat regulate stomatal responses to acute and chronic O3 exposure may differ, the unifying result is a reduction of steady-state gs ,in both crops and trees, under different experimental conditions(e.g., Grulke et al., 2007a; Wittig et al., 2007). Dynamic measurements of gs in Arabidopsis, in contrast, showed a rapid decrease
triggered by acute exposure (Vahisalu et al., 2010) and followed byreopening to overshooting values (Moldau et al., 2011). Soil water
stress is usually considered to reduce O3 injury because it reduces
gs and thus O3 entering into a leaf (Tingey and Hogsett, 1985). The
result of our steady-state measurements supports this conclusion.
Simulated water deﬁcit resulted in no effect of O3 on gs (Fig. 3).
During the dry summer of 2003 in Central Europe, no difference
in steady-state gs was reported for beech trees exposed to ambient and twice-ambient O3 in free air (Löw et al., 2006). Figuresobtained from steady-state observations have been used to model
plant responses to O3 in a changing climate (e.g., Ollinger et al.,2002).
Measurements of dynamic stomatal responses revealed that
exposure to increasing O3 concentrations made stomata sluggish
i.e. progressively increased the duration of WWR and reduced thedegree of stomatal closure over time (Fig. 4A and C). After leaf
severing, duration of WWR and following linear reduction in gs
are related to induction and execution of guard cell osmoregulation, respectively (Powles et al., 2006). Omasa (1990) reported thatstomatal response was affected by the O3 -induced effects such asslight increase in permeability of epidermal cell membranes andalteration of the osmotic pressure modulating a balance in turgor
between the guard and subsidiary cells. Ozone may also delay stomatal responses by stimulating ethylene production and reducing

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Y. Hoshika et al. / Environmental and Experimental Botany 88 (2013) 19–23

stomatal sensitivity to ABA (Wilkinson and Davies, 2010), or temporarily desensitizing the guard cells by blocking the K+ channels(Vahisalu et al., 2010).
We also demonstrated, for the ﬁrst time that also a mild soil
water stress is able to cause stomatal sluggishness by reducing thedegree of stomatal closure, while no effect was recorded on the time
the closing signal is perceived. Following Powles et al. (2006), this
response suggests that water stress affects execution rather than
induction of guard cell osmoregulation. In contrast with steadystate gs , water deﬁcit did not provide any protection from O3 effects
on stomatal dynamics.
The effect of water stress on stomatal sluggishness, however,lasted shorter than the effect of ozone exposure. The imperfect stomatal closure at night, in fact, with higher nocturnal steady-state
gs after short-term O3 exposure in well-watered plants, may beconsidered as a long-lasting effect of the aberrations induced byozone. Enhanced nocturnal gs induced by O3 has been reported forseveral plant species (Skärby et al., 1987; Matyssek et al., 1995;
Wieser and Havranek, 1995; Grulke et al., 2004, 2007c). Ozoneinduced incomplete closure of stomata at night may translate into
losing control of water efﬂux at dark (Skärby et al., 1987; Grulke
et al., 2007c). Increase in nocturnal gs may also enhance O3 uptake
at night. Günthardt-Goerg et al. (1997) reported that considerable
O3 uptake at night induced leaf injury in Betula pendula, Populus × euramericana, and Alnus glutinosa. Matyssek et al. (1995)
reported that birch species exposed to O3 at night showed great
reductions in growth.
Ozone peaks may exceed 0.1 ppm h in suburban and rural areasof California in the United States (Heath et al., 2009), southern
Europe (Paoletti, 2006) and East Asia such as Japan (Takigawa et al.,2007). The present study revealed that realistic short-term O3 exposure (≤150 ppb) induced stomatal sluggishness with or without
drought stress. Although the effect is lighter than for ozone, alsosoil water deﬁcit can induce stomatal sluggishness. Climate change
brings about the risk of drought and ﬂooding (Bytnerowicz et al.,2007). Ozone- and drought-induced loss of stomatal function may
enhance both leaf water loss and O3 uptake. Current modeling
efforts of O3 effects on plants have been developed using steadystate parameters (Emberson et al., 2000; Grünhage et al., 2001) andthe O3 -induced losing control of dynamic stomatal response wasignored. The results presented here suggest to reconsider the roleof O3 pollution on leaf gas exchange and highlight complex interactions between ozone and drought. Further improvement about our
understanding of stomatal response to O3 and drought will contribute to assess climate change impacts on plant water balanceand susceptibility to stress.
Acknowledgements
This work was supported by JSPS Fellowships for Young Scientists. We thank Bill Manning for providing the seeds of the sensitive
cultivar of snapbean.
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