Responses of selected birch (Betula pendula Roth) clones to ozone change over time

A long-term free air ozone fumigation experiment was conducted to study changes in physiological ozone responses during tree ontogeny and exposure time in ozone sensitive and tolerant clones of European white birch (Betula pendula Roth), originated from south and central Finland. The trees were grown in soil in natural microclimatic conditions under ambient ozone (control) and 1.4-1.7 x ambient (elevated) ozone from May 1996 to October 2001, and were measured for stem and foliage growth, net photosynthesis, stomatal conductance, stomatal density, visible injuries, foliar starch content and bud formation. After 6 years of exposure, the magnitude of ozone-induced growth reductions in the sensitive clone was 12-48% (significant difference), levels similar or greater than those reported earlier for 2- and 3-year-old saplings undergoing shorter exposures. In the tolerant clone, growth of these larger trees was reduced by 1-38% (significant difference in stem volume), although the saplings had previously been unaffected. In both clones, ozone stress led to significantly reduced leaf-level net photosynthesis but significantly increased stomatal conductance rates during the late summer, resulting in a lower carbon gain for bud formation and the onset of visible foliar injuries. Increasing ozone sensitivity with duration of exposure was explained by a change in growth form (relatively reduced foliage mass), a lower photosynthesis to stomatal conductance ratio during the late summer, and deleterious carry-over effects arising from the reduced number of over-wintering buds.     

Responses of Leaf Processes in a Sensitive Birch (Betula pendulaRoth) Clone to Ozone Combined with Drought

Saplings of an ozone sensitive clone of birch (Betula pendulaRoth,KL-5-M) were well-watered or exposed to mild drought-stresscombined with ambient or elevated (1.5xthe ambient) ozone for11 weeks in open-field conditions in central Finland. Stomatalresponse, visible injury, chlorophyll and nutrient content,and changes in cellular anatomy and plant growth were studied.Drought stress alone, in ambient ozone, reduced stomatal densityand stomatal conductance. Drought stress and ozone effects wereadditive, reducing total leaf number, foliage area and starchformation in mesophyll cells. Drought stress and ozone effectswere additive, increasing the N concentration in the leaves,the thickness of the upper epidermal cell wall, the number ofpectinaceous projections of mesophyll cell walls, and the vacuolartannin-like depositions and phenolic droplets, regarded as signsof activated stress defence mechanisms. The increase in specificfoliage mass, cytoplasmic lipids (younger leaves), and a condensedappearance of the upper epidermal mucilaginous layer were causedby both drought and ozone, but were not additive. The resultsshow that combined drought stress contributed to birch responsesto 1.5xcurrent ambient ozone concentrations, corresponding tocritical-level ozone exposure. The only beneficial effect ofdrought stress was the slight reduction of visible leaf symptomsinduced by ozone in autumnal leaves.Copyright 1998 Annals ofBotany Company Birch,Betula pendula, sensitive clone, ozone, drought, microscopy.     

Ozone exposure over two growing seasons alters root-to-shoot ratio and chemical composition of birch (Betula pendula Roth)

Physiological and chemical responses of 17 birch (Betula pendula Roth) clones to 1.5–1.7 × ambient ozone were studied in an open‐field experiment over two growing seasons. The saplings were studied for growth, foliar visible injuries, net photosynthesis, stomatal conductance, and chlorophyll, carotenoid, Rubisco, total soluble protein, macronutrient and phenolic concentrations in leaves. Elevated ozone resulted in growth enhancement, changes in shoot‐to‐root (s/r) ratio, visible foliar injuries, reduced stomatal conductance, lower late‐season net photosynthesis, foliar nutrient imbalance, changes in phenolic composition, and reductions in pigment, Rubisco and soluble protein contents indicating accelerated leaf senescence. Majority of clones responded to ozone by changing C allocation towards roots, by stomatal closure (reduced ozone uptake), and by investment in low‐cost foliar antioxidants to avoid and tolerate ozone stress. A third of clones, showing increased s/r ratio, relied on inducible efficient high‐cost antioxidants, and enhanced leaf production to compensate ozone‐caused decline in leaf‐level net photosynthesis. However, the best ozone tolerance was found in two s/r ratio‐unaffected clones showing a high constitutive amount of total phenolics, investment in low‐cost antioxidants and N distribution to leaves, and lower stomatal conductance under ozone stress. The results highlight the importance of phenolic compounds in ozone defence mechanisms in the birch population. Depending on the genotype, ozone detoxification was improved by an increase in either efficient high‐cost or less efficient low‐cost antioxidative phenolics, with close connections to whole‐plant physiology.     

Ozone‐induced stomatal sluggishness changes stomatal parameters of Jarvis‐type model in white birch and deciduous oak

• Stomatal ozone flux is closely related to ozone injury to plants. Jarvis‐type multiplicative model has been recommended for estimating stomatal ozone flux in forest trees. Ozone can change stomatal conductance by both stomatal closure and less efficient stomatal control (stomatal sluggishness). However, current Jarvis‐type models do not account for these ozone effects on stomatal conductance in forest trees.
• We examined seasonal course of stomatal conductance in two common deciduous tree species native to northern Japan (white birch: Betula platyphylla var. japonica ; deciduous oak: Quercus mongolica var. crispula ) grown under free‐air ozone exposure. We innovatively considered stomatal sluggishness in the Jarvis‐type model using a simple parameter, s , relating to cumulative ozone uptake (defined as POD : phytotoxic ozone dose).
• We found that ozone decreased stomatal conductance of white birch leaves after full expansion (?28%). However, such a reduction of stomatal conductance by ozone fell in late summer (?10%). At the same time, ozone reduced stomatal sensitivity of white birch to VPD and increased stomatal conductance under low light conditions. In contrast, in deciduous oak, ozone did not clearly change the model parameters.
• The consideration of both ozone‐induced stomatal closure and stomatal sluggishness improved the model performance to estimate stomatal conductance and to explain the dose–response relationship on ozone‐induced decline of photosynthesis of white birch. Our results indicate that ozone effects on stomatal conductance (i.e . stomatal closure and stomatal sluggishness) are crucial for modelling studies to determine stomatal response in deciduous trees, especially in species sensitive to ozone.

     

Ageing-related Anatomical and Ultrastructural Changes in Leaves of Birch (Betula pendula Roth.) Clones as Affected by Low Ozone Exposure

Effects of ozone on the leaf anatomy and ultrastructure of fivebirch (Betula pendula Roth.) clones were studied during onegrowing season in open-field conditions. Cumulative ozone exposurewas 1·5 times higher than ambient. Ozone exposure decreasedtotal leaf thickness in one, ozone sensitive, clone. The effecton palisade spongy mesophyll thickness was clone-specific, whilethe amount of palisade intercellular space was reduced in allclones. A second effect was a change in the relative amountsof adaxial and abaxial epidermis. In palisade and spongy parenchymacells of all clones, ozone increased the number of irregularand spherical shaped chloroplasts, the electron density of chloroplaststroma, swelling and curling of thylakoids, translucency ofthe mitochondrial matrix and also the amount of cytoplasmiclipids. In the sensitive clone shorter chloroplasts and reducedamount of starch were observed in ozone-exposed plants, whilst,in the tolerant clone, the size of chloroplasts and the amountof starch were unaffected. Ozone effects on number, size andelectron density of plastoglobuli and vacuolar tannin were clone-dependent.At the ultrastructural level, the normal leaf ageing processprogressed at different rates in the birch clones. Ozone acceleratedsenescence-related structural changes, in accordance with earlierobservations of deciduous species.Copyright 1995, 1999 AcademicPress Betula pendula Roth., birch, clones, ageing, ozone, leaf anatomy, ultrastructure     

Northern environment predisposes birches to ozone damage

Ozone sensitivity of silver birch ( BETULA PENDULA Roth) has been thoroughly investigated since early 1990's in Finland. In our long-term open-field experiments the annual percentage reduction in basal diameter and stem volume increment were the best non-destructive growth indicators for ozone impact when plotted against AOTX. Remarkable differences in defence strategies, stomatal conductance, and defence compounds (phenolics), clearly indicate that external exposure indices are ineffective for accurate risk assessment for birch. For flux-based approaches, site-specific values for G(max) and G(dark) are necessary, and determinants for detoxification capacity, ageing of leaves, and cumulative ozone impact would be needed for further model development. Increasing CO(2) seems to counteract negative ozone responses in birch, whereas exposure to spring time frost may seriously exacerbate ozone damage in northern conditions. Therefore, we need to proceed towards incorporating the most important climate change factors in any attempts for ozone risk assessment.     

Growth and physiological responses to ozone and mild drought stress of tree species with different ecological requirements

An open-top chamber experiment was carried out in Curno (Northern Italy) in 2004 and 2005 on seedlings of Fagus sylvatica (FS), Quercus robur (QR), and an ozone-sensitive Populus (POP) clone, to investigate the role of two stress factors: tropospheric ozone and water shortage. Treatments were filtered air to achieve a 50% reduction in the environmental ozone concentrations (charcoal filtered, CF); and non-filtered air, with a 5% reduction in the environmental ozone concentrations (non-filtered, NF). Overall ozone exposure (AOT40) in open air (April–September) was 26,995 ppb h in 2004 and 25,166 ppb h in 2005. The plants were either watered (W) or not watered (dry, D). We investigated the above-ground biomass, tree-ring growth, stable carbon isotopes ratio, i.e. δ¹³C of tree rings, and the photosynthetic parameter Driving forces (DF_ABS), derived from chlorophyll a fluorescence analysis. Ozone-induced growth reduction (in terms of biomass) in POP, and that reduction was more pronounced in D plots. A synergistic effect of ozone and drought stress was evidenced by DF_ABS in POP and QR, but not in FS. The water availability was revealed as the main factor influencing the isotopic ratio δ¹³C. In drought-stressed seedlings, the increase in δ¹³C value was accompanied by the reduction in stomatal conductance and increased DF_ABS. Fast-growing plant species with high water requirements are more susceptible to ozone and drought stress.     

Effects of ozone and mild drought stress on gas exchange, antioxidants and chloroplast pigments in current-year needles of young Norway spruce [Picea abies (L.) Karst.]

 To investigate the effects of ozone exposure and soil drought, singly and in combination, on gas exchange, antioxidant contents and pigments in current-year needles of Norway spruce [Picea abies (L.) Karst.] 4-year-old seedlings were fumigated in growth chambers with either charcoal-filtered air or with 100 nl l^–1 ozone for 106 days. After 3 weeks a 20% reduction in gas exchange was observed in ozone-treated seedlings. However, no further decrease occurred in spite of continued ozone exposure. Whole needle ascorbate and apoplastic ascorbate increased until the end of the experiment and contents were 62% and 82%, respectively, higher than in ozone-free controls. This increase in ascorbate might have protected net photosynthesis from further decline. Ozone pre-treated plants and ozone-free controls were subjected to soil drought for 38 days which caused stomatal narrowing. Thereby ozone uptake was reduced when compared to well watered seedlings. At the end of the experiment drought alone, and even more in combination with ozone, had also caused an increase in ascorbate. Glutathione increased only in drought-stressed seedlings. The redox states of the ascorbate and the glutathione pools were not affected by any treatment. Superoxide dismutase activity declined under both stresses but was most reduced by ozone alone. While chlorophyll and neoxanthin contents remained unchanged, carotenes were significantly decreased upon drought. The combination of O₃ and drought induced increased lutein contents, an increased pool size of the xanthophyll cycle as well as an increased epoxidation status of the xanthophyll cycle. These results suggest that spruce needles seem to be able to acclimate to ozone stress but also to drought stress by increasing their ascorbate pools and protecting pigments. Received: 15 September 1997 / Accepted: 24 March 1998     

Distinct roles of electric and hydraulic signals on the reaction of leaf gas exchange upon re-irrigation in Zea mays L

The hypothesis that electric and hydraulic long-distance signals modify photosynthesis and stomatal aperture upon re-irrigation in intact drought-stressed plants was examined. Maize plants (Zea mays L.) were exposed to drought conditions by decreasing the soil water content to 40-50% of field capacity. The decrease in water content resulted in a decline in stomatal conductance to 50-60% of the level in well-watered plants. Re-irrigation of the plants initiated both hydraulic and electric signals, followed by a two-phase response of the net CO2 uptake rate and stomatal conductance of leaves. The transitional first phase (phase 1) is characterized by a rapid decrease in both levels. In the second phase (phase 2), both parameters gradually increase to levels above those of drought-stressed plants. Elimination of either the hydraulic signal by compensatory pressure application to the root system, or of the electric signal by cooling of the leaf blade gave evidence that the two signals (1) propagated independently from each other and (2) triggered the two-phase response in leaf gas exchange. The results provided evidence that the hydraulic signal initiated a hydropassive decrease in stomatal aperture and for the involvement of electric signals in the regulation of photosynthesis of drought-stressed plants.     

Effect of drought stress on growth and water relations of the mycorrhizal association Helianthemum almeriense-Terfezia claveryi

 Plants of Helianthemum almeriense were micropropagated on MS medium and inoculated in vitro with Terfezia claveryi mycelium on MH medium and vermiculite. Mycorrhizal (M) and non-mycorrhizal (NM) plants were subjected to a drought stress period of 3 weeks in greenhouse conditions with the soil matric potential maintained at –0.5 MPa. Drought stress did not affect the amount of mycorrhizal colonization. The survival rate of M plants at the end of the drought stress period was higher than that of NM plants. The water potential was higher in M plants than in NM plants by 14% in well-watered and 26% in drought-stressed plants. Transpiration, stomatal conductance and net photosynthesis were higher in M plants than in NM plants. Transpiration was 92% higher in M plants than in NM plants under drought-stress conditions and 40% when irrigated. Stomatal conductance was 45% and 14% higher and net photosynthesis 88% and 54% higher, respectively, in M than in NM plants. Drought-stressed M plants accumulated more N, P and K than drought-stressed NM plants. Reduced negative effects of drought stress on H. almeriense by the desert truffle T. claveryi could be ascribed to specific physiological and nutritional mechanisms, suggesting that this mycorrhizal symbiosis aids adaptation to arid climates. Accepted: 7 July 2000     

Ozone and water deficit reduced growth of Aleppo pine seedlings

The effects of ambient and elevated ozone (O₃) levels on photosynthesis, growth, pigment, biomass and element contents of Aleppo pine (Pinus halepensis Mill.) were studied for two growing seasons (1997, 1998). Two-year-old seedlings were exposed to elevated O₃ in open-top chambers. The treatments were charcoal-filtered air and non-filtered air + 50 nl l^–1 O₃ (24 h per day, 7 days per week). In summer 1998, half of the seedlings were drought-stressed (leaf water potential down to approximately –2 MPa), while the other half were kept well-watered. At the beginning of the season (1998), current (c) and previous-year (c + 1) needles under O₃ stress showed an increase in stomatal conductance and net photosynthesis. During the drought period, only stomatal conductance increased in both needle age-classes, whereas the net photosynthesis decreased. At the end of the measuring period, both parameters were reduced in the O₃ treatment. Both O₃ and drought decreased chlorophyll a and b concentrations, growth and biomass. A carry-over effect of O₃ on pigments was also observed. Needle K content was increased in the O₃ treatment. Drought protected Aleppo pine against O₃ (less chlorotic mottle and less decrease of stem and branch biomass).     

Improvement of heat and drought photosynthetic tolerance in wheat by overaccumulation of glycinebetaine

Within their natural habitat, crops are often subjected to drought and heat stress, which suppress crop growth and decrease crop production. Causing overaccumulation of glycinebetaine (GB) has been used to enhance the crop yield under stress. Here, we investigated the response of wheat (Triticum aestivum L.) photosynthesis to drought, heat stress and their combination with a transgenic wheat line (T6) overaccumulating GB and its wild-type (WT) Shi4185. Drought stress (DS) was imposed by controlling irrigation until the relative water content (RWC) of the flag leaves decreased to between 78 and 82%. Heat stress (HS) was applied by exposing wheat plants to 40°C for 4 h. A combination of drought and heat stress was applied by subjecting the drought-stressed plants to a heat stress as above. The results indicated that all stresses decreased photosynthesis, but the combination of drought and heat stress exacerbated the negative effects on photosynthesis more than exposure to drought or heat stress alone. Drought stress decreased the transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO₂ concentration (Ci), while heat stress increased all of these; the deprivation of water was greater under drought stress than heat stress, but heat stress decreased the antioxidant enzyme activity to a greater extent. Overaccumulated GB could alleviate the decrease of photosynthesis caused by all stresses tested. These suggest that GB induces an increase of osmotic adjustments for drought tolerance, while its improvement of the antioxidative defense system including antioxidative enzymes and antioxidants may be more important for heat tolerance.     

Ozone effects on the ultrastructure of peatland plants: Sphagnum mosses, Vaccinium oxycoccus, Andromeda polifolia and Eriophorum vaginatum

BACKGROUND AND AIMS: Ozone effects on peatland vegetation are poorly understood. Since stress responses are often first visible in cell ultrastructure, electron microscopy was used to assess the sensitivity of common peatland plants to elevated ozone concentrations. METHODS: Three moss species (Sphagnum angustifolium, S. magellanicum and S. papillosum), a graminoid (Eriophorum vaginatum) and two dwarf shrubs (Vaccinium oxycoccus and Andromeda polifolia), all growing within an intact canopy on peat monoliths, were exposed to a concentration of 0, 50, 100 or 150 ppb ozone in two separate growth chamber experiments simulating either summer or autumn conditions in central Finland. After a 4- or 5-week-long exposure, samples were photographed in a transmission electron microscope and analysed quantitatively using image processing software. KEY RESULTS: In the chlorophyllose cells of the Sphagnum moss leaves from the capitulum, ozone exposure led to a decrease in chloroplast area and in granum stack thickness and various changes in plastoglobuli and cell wall thickness, depending on the species and the experiment. In E. vaginatum, ozone exposure significantly reduced chloroplast cross-sectional areas and the amount of starch, whereas there were no clear changes in the plastoglobuli. In the dwarf shrubs, ozone induced thickening of the cell wall and an increase in the size of plastoglobuli under summer conditions. In contrast, under autumn conditions the cell wall thickness remained unchanged but ozone exposure led to a transient increase in the chloroplast and starch areas, and in the number and size of plastoglobuli. CONCLUSIONS: Ozone responses in the Sphagnum mosses were comparable to typical ozone stress symptoms of higher plants, and indicated sensitivity especially in S. angustifolium. The responses in the dwarf shrubs suggest stimulation of photosynthesis by low ozone concentrations and ozone sensitivity only under cool autumn conditions.     

Phosphorus application and elevated CO2 enhance drought tolerance in field pea grown in a phosphorus-deficient vertisol

Background and Aims Benefits to crop productivity arising from increasing CO₂ fertilization may be offset by detrimental effects of global climate change, such as an increasing frequency of drought. Phosphorus (P) nutrition plays an important role in crop responses to water stress, but how elevated CO₂ (eCO₂) and P nutrition interact, especially in legumes, is unclear. This study aimed to elucidate whether P supply improves plant drought tolerance under eCO₂.Methods A soil-column experiment was conducted in a free air CO₂ enrichment (SoilFACE) system. Field pea (Pisum sativum) was grown in a P-deficient vertisol, supplied with 15 mg P kg⁻¹ (deficient) or 60 mg P kg⁻¹ (adequate for crop growth) and exposed to ambient CO₂ (aCO₂; 380–400 ppm) or eCO₂ (550–580 ppm). Drought treatments commenced at flowering. Measurements were taken of soil and leaf water content, photosynthesis, stomatal conductance, total soluble sugars and inorganic P content (Pi).Key Results Water-use efficiency was greatest under eCO₂ when the plants were supplied with adequate P compared with other treatments irrespective of drought treatment. Elevated CO₂ decreased stomatal conductance and transpiration rate, and increased the concentration of soluble sugars and relative water contents in leaves. Adequate P supply increased concentrations of soluble sugars and Pi in drought-stressed plants. Adequate P supply but not eCO₂ increased root length distribution in deeper soil layers.Conclusions Phosphorus application and eCO₂ interactively enhanced periodic drought tolerance in field pea as a result of decreased stomatal conductance, deeper rooting and high Pi availability for carbon assimilation in leaves.     

Responses to ozone on Populus “Oxford” clone in an open top chamber experiment assessed before sunrise and in full sunlight

The effects of ambient levels of ozone and summer drought were assessed on a poplar clone (Populus maximowiczii Henry X P. × berolinensis Dippel — Oxford clone) in an open top chamber experiment carried out at the Curno facilities (Northern Italy). Chlorophyll (Chl) a fluorescence parameters (from both modulated and direct fluorescence) were assessed at different hours of the day (predawn, morning, midday, afternoon, and evening), from June to August 2008. This paper compares the results from predawn (PD, before sunrise) and afternoon (AN, in full sunlight) measurements, in order to evaluate the role of high sunlight as a factor influencing responses to ozone stress. Sunlight affected the maximum quantum yield of primary photochemistry (decrease of F_v/F_m) thus indicating photoinhibition. The effective quantum yield (Φ_PSII) and the photochemical quenching (q_P) were enhanced in the afternoon with respect to the predawn, whereas the nonphotochemical quenching (NPQ) was reduced. The effect of ozone was detected with fluorescence on well watered plants in the first week of July, before the onset of visible symptoms. As far as F_v/F_m are concerned, the differences between ozone-treated and control plants were statistically significant in the predawn, but not in the afternoon. Ozone exerted only minor effects on drought exposed plants because of the reduced stomatal ozone uptake, but effects on the IP phase of the fluorescence transient were observed also in drought-stressed plants.     

Testing for stress-dependent inbreeding depression in Impatiens capensis (Balsaminaceae)

The relevance of inbreeding depression to the persistence of plant populations can depend upon whether stress magnifies inbreeding depression for fitness-related traits. To examine whether drought stress exacerbates inbreeding depression in gas exchange traits and biomass, we grew selfed and outcrossed progeny of inbred lines from two populations of Impatiens capensis in a greenhouse experiment under water-limited and moist soil conditions. Drought stress did not magnify the degree of inbreeding depression for any of the traits measured. In fact, in one population there was a trend for stronger inbreeding depression under well-watered, benign conditions. Furthermore, significant inbreeding depression for carbon assimilation rate and stomatal conductance was only detected in the lines from one population. In contrast, inbreeding depression for biomass was detected within both populations and differed among lines. Drought stress exerted significant selection on physiological traits, favoring increased carbon assimilation rates and decreased stomatal conductance in drought-stressed plants. Patterns of selection did not differ between inbred and outcrossed plants but did differ marginally between populations. Thus, estimates of selection were not biased by the mixed mating system per se, but may be biased by combining individuals from populations with different histories of selection and inbreeding.     

Effects of nitrogen deficiency on leaf photosynthetic response of tall fescue to water deficit

Abstract. The objective of the present work was to study the effect of nitrogen deficiency on drought sensitivity of tall fescue plants. The authors compared photosynthetic and stomatal behaviour of plants grown at either high (8 mol m⁻³) or low (0.5 mol m⁻³) nitrogen levels during a drought cycle followed by rehydration. Other processes investigated were stomatal and non-stomatal inhibition of leaf photosynthesis, water use efficiency and leaf rolling. Plants were grown in pots in controlled conditions on expanded clay. A Wescor in situ hygrometer placed on the leaf base outside the assimilation chamber permitted, simultaneously to leaf gas exchange measurements, monitoring of leaf water potential. Drought was imposed by withholding water from the pot. CO₂ uptake and stomatal conductance decreased and leaves started to roll at a lower leaf water potential in the high-N than in the low-N grown plants. Stomatal inhibition of leaf photosynthesis seemed larger in the low-N than in the high-N plants. Water-use efficiency increased more in the high-N than in the low-N grown plants during the drought. The decrease of photosynthesis was largely reversible after rehydration in low-N but not in high-N leaves. The authors suggest that low-N plants avoid water deficit rather than tolerate it.     

Distinct responses to ozone of abaxial and adaxial stomata in three Euramerican poplar genotypes

Ozone induces stomatal sluggishness, which impacts photosynthesis and transpiration. Stomatal responses to variation of environmental parameters are slowed and reduced by ozone and may be linked to difference of ozone sensitivity. Here we determine the ozone effects on stomatal conductance of each leaf surface. Potential causes of this sluggish movement, such as ultrastructural or ionic fluxes modification, were studied independently on both leaf surfaces of three Euramerican poplar genotypes differing in ozone sensitivity and in stomatal behaviour. The element contents in guard cells were linked to the gene expression of ion channels and transporters involved in stomatal movements, directly in microdissected stomata. In response to ozone, we found a decrease in the stomatal conductance of the leaf adaxial surface correlated with high calcium content in guard cells compared with a slight decrease on the abaxial surface. No ultrastructural modifications of stomata were shown except an increase in the number of mitochondria. The expression of vacuolar H⁺/Ca²⁺‐antiports (CAX1 and CAX3 homologs), β‐carbonic anhydrases (βCA1 and βCA4) and proton H⁺‐ATPase (AHA11) genes was strongly decreased under ozone treatment. The sensitive genotype characterized by constitutive slow stomatal response was also characterized by constitutive low expression of genes encoding vacuolar H⁺/Ca²⁺‐antiports.