Slightly elevated ozone exposure causes cell structural changes in needles and roots of Scots pine

Scots pine (Pinus sylvestris L.) seedlings were fumigated with 1.2–1.5 x ambient ozone over 2 seasons in an open-air experiment. Fumigation started in the early spring and continued into late autumn during both years. Needle and root cell structures were analyzed in the summer, autumn and early winter following the second fumigation period. Under the light microscope an increase in the intercellular space and disintegrating cells in the mesophyll tissue near the stomata and stomatal cavities were observed in the ozone-exposed needles. Darkening of chloroplast stroma, increased plastoglobulus size and decreased chloroplast size were characteristic ultrastructural changes associated with ozone exposure. In addition, less dense grouping of the chloroplasts in the needles of elevated ozone-exposed seedlings as compared to the controls (background ozone) was observed in the early winter. Fewer starch grains and an increased accumulation of tannin-like substances were detected in both mycorrhizal and uninfected roots of ozone-exposed seedlings as compared to the control seedlings. For the first time, we were able to show that the ozone-induced darkening of needle chloroplast stroma is a reversible symptom. An increased frequency of frost injury symptoms indicated that the winter hardening process was disturbed in the needles of ozone-treated seedlings.     

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     

Experiments on the water balance of individual attached twigs of Picea abies (L .) Karst. in pure and ozone-enriched air

 Rates of transpiration and xylem sap flow were continuously measured in individual twigs in the upper crown of an 18-year-old spruce. Two gas exchange chambers were run simultaneously under identical conditions. One of two equivalent twigs was exposed to pure air whereas the other received the ozone-enriched air of the site. A third gas exchange chamber in mid-crown ran independently with normal outside air and was used for basic experiments. At certain times needles were sampled for water potential measurements. Chamber humidity was reduced step by step and the transpiration and xylem sap flow rates were permanently compared. It turned out that sap flow keeps up with transpiration without lagging as long as chamber humidity is high and the twigs outside the chamber are not subject to substantial evaporative demand. However, in warm summer weather and with high flow rates sap flow is no longer sufficient. As the balance quotient (uptake/release) of 0.8 was reached the stomata began to close and water balance improved. The flux quotient increased far above 1.0 without water potential of the needles, which had decreased before, increasing significantly. The balance quotient of the twig in ozone-enriched air fell to relatively low values and only increased again correspondingly slowly due to lagging stomatal closure. Despite increased water uptake after the light phase, the ozone-treated twig ran into a water deficit in the daily balance during the course of an uninterrupted drought period. Water deficit increased from day to day and only disappeared with the next rainfall. For providing insight into the ozone effect shown in this study it was thought necessary to discuss comprehensively the controversial views of the stomatal control mechanism. The results show that the stomata react to small scale changes in water status as expected for an effective negative feedback system. The less sensitive reaction of the stomata under the influence of ozone confirms earlier results. Increased severity and duration of the daily drought stress loads were attributed to a lag in adjustment due to poor stomatal control. The demonstrated function mechanism shows that the detrimental effect of ozone is dependent on weather conditions. Received: 12 April 1996 / Accepted: 24 June 1996     

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 concentration in leaf intercellular air spaces is close to zero

Transpiration and ozone uptake rates were measured simultaneously in sunflower leaves at different stomatal openings and various ozone concentrations. Ozone uptake rates were proportional to the ozone concentration up to 1500 nanoliters per liter. The leaf gas phase diffusion resistance (stomatal plus boundary layer) to water vapor was calculated and converted to the resistance to ozone multiplying it by the theoretical ratio of diffusion coefficients for water vapor and ozone in air (1.67). The ozone concentration in intercellular air spaces calculated from the ozone uptake rate and diffusion resistance to ozone scattered around zero. The ozone concentration in intercellular air spaces was measured directly by supplying ozone to the leaf from one side and measuring the equilibrium concentration above the other side, and it was found to be zero. The total leaf resistance to ozone was proportional to the gas phase resistance to water vapor with a coefficient of 1.68. It is concluded that ozone enters the leaf by diffusion through the stomata, and is rapidly decomposed in cell walls and plasmalemma.     

Delignification of subsidiary and guard cell walls of Picea abies (L.) Karst. by fumigation with ozone

Summary After fumigation with ozone, the exterior periclinal walls of the stomatal apparatus of Picea abies appear to be partially delignified, as in earlier experiments with SO₂. This shows up cytophotometrically as reduced UV absorption. Random samples from the stand named Wank in the Bavarian Alps clearly showed a relation between the degree of lignification of the stomatal cells and the grading of the respective trees in their damage classes. The significance of delignification for the regulatory capacity of the stomata is discussed, and a hypothesis is proposed for a specific disturbance of hydroregulation by ozone.     

Effect of ozone exposure on polyamines in Scots pine trees

Effects of ozone exposure on polyamines in Pinus sylvestris L. were studied in a long-term experiment. Ten- to 15-year-old Scots pines were exposed to target ozone levels which began at ambient + 40 ppb in May, decreasing to ambient air only by September for 3 growing seasons. The amount of ozone applied followed the natural pattern of variation in ozone concentrations in Northern Finland. The free, soluble conjugated and insoluble conjugated polyamines were analyzed during the experiment and shortly after termination of exposure as well as at the beginning of the following growing season. A carry-over effect was observed as ozone-induced reduction of free spermidine in the oldest needle year class, which developed during the first exposure season of the experiment. This reduction was observed both after the second and the third ozone exposure season. Conversely, after termination of the experiment, levels of free polyamines increased in the following growing season, and soluble conjugated polyamines decreased in the developing needles. The post-treatment changes in polyamine concentrations are hypothesized to be caused by stress-induced injuries or delayed recovery of metabolic processes rather than protective responses. It is noteworthy that some responses in polyamines were found in the developing needles nine months after terminating the ozone exposure. This suggests that stress-induced injuries to older needles affected metabolism of new developing needles.     

Structural changes in the vascular bundles of light-exposed and shaded spruce needles suffering from Mg deficiency and ozone pollution

The correlation between structural changes of the vascular bundles and needle yellowing was examined for needles of damaged spruce (Picea abies (L.) Karst.) growing at a Mg-deficient and ozone polluted mountain site in the Central Black Forest (840m a.s.l.). In the previous year's sun-exposed needles, the following sequence of events was observed: (1) rapid needle yellowing, (2) hypertrophy and anomalous divisions of cambium cells, (3) phloem collapse, and, (4) production of atypical xylem tracheids. Under defined shade (reduction of the photosynthetically active photon flux density of the ambient light by 85-90%), the needles remained green, while the phloem collapsed completely within the first 6 weeks of shading; subsequently, a reversal of the collapse was observed. Under both light conditions, the content of Mg not bound to chlorophyll (Mg(free)) was in the range of 0.1 mg g(-1) needle dry matter, and hardly changed throughout the investigation period. After Mg fertilization, the Mg(free) level of the previous year's needles increased to 0.2 mg g(-1) dry matter, the light-exposed needles remained green, and the vascular bundles developed no anomalies. The data show that the rapid needle yellowing of ozone-exposed Mg-deficient needles did not depend on the collapse of the phloem. Mg deficiency played a key role in the development of anomalous vascular bundles under light, and also appears to explain the transient changes in sieve cell structure under shade. The role of Mg deficiency, rather than ozone pollution, in the damage of the sieve cells was confirmed in a long-term ozone exposure experiment with young clonal spruce growing under defined conditions.     

The role of the mesophyll cell wall in leaf transpiration

Summary Evidence is presented which suggests that the mesophyll cell walls of cotton leaves may influence observed rates of transpiration.The net diffusive flux of water vapour, from the upper and lower surfaces of a leaf, was compared with the flux of nitrous oxide through a leaf and evidence obtained of an extra resistance in the water-vapour pathway associated with water transport in the mesophyll cell walls.This extra resistance appeared to be insignificant at low transpiration rates and in turgid leaves, but increased with transpiration rate and dehydration. The most likely explanation for its origin appeared to be a reduction in hydraulic conductivity across the internal cuticle which lines the outer surfaces of the mesophyll cell walls. In turn this served to reduce the relative vapour pressure at the sites of evaporation.The experiments were conducted under conditions where stomatal opening was induced by CO₂-free air. Under normal conditions stomatal closure would tend to reduce the development of this extra resistance. Even so, the results throw doubt on the validity of the long-standing assumption that the water-vapour pressure at the evaporation sites is equal to the saturation vapour pressure under all conditions.     

Histological examination of spruce needles from a long-term gas exchange experiment in pure and polluted air in the field

Summary At the end of a 4-year period of gas exchange measurements in a natural stand in the Lower Bavarian Forest, needles of an adult spruce [Picea abies (L.) Karst.] were harvested from two chambers, one with pure air and the other with ambient air. The needles were examined as to their histological properties in the stomatal apparatus and in the bundle sheath. In needles from the polluted air UV absorbance at 280 nm was decreased in the walls of the stomatal apparatus. Simultaneously, the deposition of compounds with an absorption maximum at 310 nm increased within the encrusted plate-like thickenings of the subsidiary cells. The contents of the lumina of hypodermal cells and of the bundle sheath exhibited a greater degree of autofluorescence in ambient-air material than in pure-air leaf organs. Differences between needles exposed to pure and polluted air are gradual. The damaged condition is rare in pure air, common in polluted air. The needles from outside the chambers occupied an intermediate position between pure-air and ambient-air needles. This fact is traced to an unnaturally high pollutant load in the liquid phase of the needle surfaces within the ambient-air chamber because in order to compensate pollutant losses within the system, SO₂ and O₃ were added even during periods of irrigation. The reduction of absorption capacity at 280 nm in the walls of the stomatal apparatus is attributed to destruction of lignin due to the high reactivity of the pollutants in the liquid phase on the damp needle surface. The importance of delignification with regard to hydroregulation is discussed.     

Poor stomatal control of water balance and the abscission of green needles from a declining stand of spruce trees [Picea abies (L.) Karst.] from the northern Alps

On a site on the west slope of the Wank in the northern Alps changes in water potential, osmotic potential and transpiration rate were measured in spruce trees during the dry summer months of 1991. The pattern of decrease in water potential and osmotic potential on days of varying evaporative demand from trees of widely different decline conditions was used to describe the relative ability of the trees to withstand drought stress. Stress diagrams served as a tool for interpreting the state of health of each tree. The criterion is independent of the water situation of the tree and the other external conditions of the respective experiment. These diagrams clearly show that the foliage of spruces with high needle losses reaches the limit of endurance relatively early. For equal evaporative demand much lower turgor levels were observed in spruces with high needle loss compared to undamaged trees. Associated with the occurrence of low turgor values was the shedding of green needles. The abscission zone was shown in sections. The accumulation of highly fluorescent substances in the bundle sheath cells of the same material was described. Incomplete to non-existent stomatal control over water loss was attributed to modifications in the cell walls of the stomata which appear to alter the ability of the guard cells to sense changes in either atmospheric or cellular hydration. Our studies point to the following situation: air pollution directly affects stomatal control, the loss of stomatal control changes the drought avoidance abilities of the foliage and, as a consequence, low turgor levels occur and premature needle abscission is induced. As site water balance decreases, either due to a dry year or to poor moisture holding abilities of the soil, these conditions become apparent.     

Cytological and structural changes in Pinus pines L. needles following the application of an anionic surfactant

In many coastal areas, synthetic surfactants are present as pollutants in the chemical composition of sea spray. Their phytotoxic action in synergism with sea salt is well known. This paper examines the early effects of an anionic surfactant (sodium alkylbenzene sulphonate = ABS) on the anatomy and ultrastructure of Pinus pinea L. needles. Three successive treatments in the open field on Pinus pinea trees about 2 m in height were carried out in the month of August. The crowns of the trees were sprayed with an aqueous solution containing 1000 g m^?3, and subsequently the current-year needles were observed by light microscopy, as well as by scanning and transimission electron microscopy. A few days after the treatment the needles had turned yellow. The results of the microscopical analysis show that the surfactant is absorbed prevalently through the stomata and that it causes deterioration of the epistomatal wax structures and induces alterations in the stomatal guard cell walls. Lastly, it causes degeneration of the protoplasm in cells of the mesophyll, beginning with the cells closest to the stomatal openings. The efficiency of both photosynthesis and gaseous exchange processes may be affected.     

Direct Measurements of Turgor Pressure Potentials of Guard Cells: II. THE MECHANICAL ADVANTAGE OF SUBSIDIARY CELLS, THE SPANNUNQSPHASE, AND THE OPTIMUM LEAF WATER DEFICIT

Evidence of the mechanical advantage of subsidiary cells wasobtained by simultaneous measurements of turgor pressure potentialsin adjacent subsidiary and guard cells using injection circuitswith two separate needles. In Tradescantia virginiana the mechanicaladvantage approaches two. Using the same technique evidencewas obtained that the Spannungsphase is, in the first place,a turgor relations phenomenon due to the mechanical advantageof epidermal or subsidiary cells. In addition, the evidenceindicated that the elastic properties of guard cell walls mayundergo changes during the Spannungsphase when potassium iontransport commences. During these measurements it was confirmedthat the optimum leaf water deficit for maximum stomatal openingoccurs when the epidermal turgor is near zero. Under these conditionsthe width of the stomatal pore is a function of the turgor pressureof the guard cells, since at zero turgor of the subsidiary cellstheir mechanical advantage has disappeared.     

Physiological, stomatal and ultrastructural ozone responses in birch (Betula pendula Roth.) are modified by water stress

The physiological, stomatal and ultrastructural responses to ozone and drought of ozone-sensitive and more ozone-tolerant birch ( Betula pendula Roth.) clones were studied singly and in combination, in a high-stress chamber experiment and in a low-stress open-field experiment. In the chamber experiment, well watered (WW), moderately watered (MW) or drought-stressed (DS) saplings were exposed for 36 d to 0 or 130 nmol mol^∠1 ozone. In the open-field experiment, well watered or drought-stressed saplings were grown for one growing season in ambient air or exposed to 1·8 × ambient ozone. Drought stress reduced growth rate, stomatal conductance, stomatal density and the proportion of starch and thylakoids in chloroplasts, but stimulated net photosynthesis, Rubisco and chlorophyll quantity at the end of the growing season, and increased the size and density of plastoglobuli. Ozone fumigations caused more variable, clone- and exposure-dependent responses in growth, decreased stomatal conductance and net photosynthesis, an increased number of stomata, visible and ultrastructural chloroplast injuries, and enhanced autumn yellowing of the leaves. Ozone-induced changes in plastoglobuli, starch and thylakoids resembled drought responses. The two experiments revealed that, depending on the experimental conditions and the variable, the response to drought and ozone stress can be independent, additive or interactive. Drought protected the plants from ozone injuries under high-stress conditions in the chamber experiment. In the low-stress, open-field experiment, however, enhanced ozone damage was observed in birch saplings grown under restricted water supply.     

Changes in stomatal frequency and size during elongation of Tsuga heterophylla needles

BACKGROUND AND AIMS: The inverse relationship between the number of stomata and atmospheric CO2 levels observed in different plant species is increasingly used for reconstructions of past CO2 concentrations. To validate this relationship, the potential influence of other environmental conditions and ontogenetical development stage on stomatal densities must be investigated as well. Quantitative data on the changes in stomatal density of conifers in relation to leaf development is reported. METHODS: Stomatal frequency and epidermal cells of Tsuga heterophylla needles during different stages of budburst were measured using computerized image analysis systems on light microscope slides. KEY RESULTS: Stomata first appear in the apical region and subsequently spread basipetally towards the needle base during development. The number of stomatal rows on a needle does not change during ontogeny, but stomatal density decreases nonlinearly with increasing needle area, until about 50 % of the final needle area. The total number of stomata on the needle increases during the entire developmental period, indicating that stomatal and epidermal cell formation continues until the needle has matured completely. CONCLUSIONS: Epidermal characteristics in developing conifer needles appear to be fundamentally different from angiosperm dicot leaves, where in general leaf expansion in the final stages is due to cell expansion rather than cell formation. The lack of further change in either stomatal density or stomatal density per millimetre needle length (the stomatal characteristic most sensitive to CO2 in conifers) in the final stages of leaf growth indicates that in conifers the stage of leaf maturation would not influence CO2 reconstructions based on stomatal density.     

On the response of pigments and antioxidants of Pinus halepensis seedlings to Mediterranean climatic factors and long-term ozone exposure

An experiment was carried out in open-top chambers located in eastern Spain. One-yr-old Pinus halepensis Mill. seedlings were exposed during three consecutive summers to the following ozone (O₃) treatments: charcoal-filtered air (CFA), non-filtered air (NFA) or non-filtered air plus 40 nl l⁻¹ O₃, 9 h d⁻¹, 5 d wk⁻¹ (NFA+40). Seasonal variations in Aleppo pine performance were observed since reductions in chlorophyll and cellular peroxidase levels associated with increases in superoxide dismutase activity, were recorded during the summer. Similarly, a reduction in epoxidation state was found at midday during the summer, derived from an activation of the xanthophyll cycle associated to an increment in radiation and temperature levels.
The first O₃-induced effects were recorded in previous-year needles (1991) during the first summer exposure as an increase in extracellular and total peroxidase activities and in zeaxanthin levels in the NFA+40 treatment along with a trend to a higher SOD activity in this treatment. A carry-over effect was detected since a lower winter recovery of chlorophyll levels was found in the NFA+40 seedlings along with a reduction of xanthophyll levels. A reduction in chlorophyll levels was observed in the previous-year needles (1992) from the NFA+40 treatment at the end of the second fumigation period. Realistic ozone exposures induced alterations in plant antioxidative systems and plant pigments as shown in this paper. These observations together with the reductions in stomatal conductance and net photosynthesis recorded in the same experiment, indicate that Aleppo pine is a species sensitive to ozone.     

The effect of vapor pressure on stomatal control of gas exchange in Douglas fir (Pseudotsuga menziesii) saplings

Summary Increasing leaf to air vapor pressure deficit (VPD) caused reductions in stomatal conductance of both current year and previous season needles of Pseudotsuga menziesii saplings. The stomata of current year needles were found to be more responsive to changes in VPD than those of previous season needles. The reductions in stomatal conductance of current year needles were not associated with decreases in xylem pressure potential. In fact, the reductions in stomatal conductance of current year needles were sometimes sufficient to reduce transpiration and thus raise xylem pressure potential even though VPD was increasing. There was a decline in stomatal responsiveness to VPD in current year needles between early and late summer. Pressure-volume curves determined for different age needles at different times of the year suggested that differences and changes in stomatal responsiveness to VPD may have been caused in part by differences and changes in needle water potential components. Hexane washes of current year needles during the late summer succeeded in partially restoring their VPD sensitivity, suggesting that changes in the water permeability of the external cuticle during needle maturation may also have played a role in causing the summer decline in VPD responsiveness.In both current and previous year needles VPD-induced changes in stomatal conductance had a greater relative effect on transpiration (q _w) than on net photosynthesis (Ph_N). In maturing needles the ratio of the sensitivities of transpiration and net photosynthesis to changes in stomatal conductance, (q _w/g _s)/Ph_N/g _s), remained nearly constant as VPD was varied. This provides experimental support for a recent hypothesis that stomata respond to environmental fluctuations in such a manner as to maintain the above ratio constant, which optimizes CO₂ uptake with respect to water loss.     

Stomatal density, anatomy and nutrient concentrations of Scots pine needles are affected by elevated CO2 and temperature

Stomatal density, anatomy and nutrient concentrations of Scots pine (Pinus sylvestris L.) needles were studied during 3 years of growth at elevated CO₂ (693 ± 30 µmol mol⁻¹), at elevated temperature (ambient +2·8–6·2 °C depending on the time of the year) and in a combination of elevated CO₂ and temperature in closed-top chambers. The treatments were started in August 1996. At elevated temperature, the needles that were grown in the first year (i.e. the 1997 cohort) were thinner, had thinner mesophyll in the abaxial side, thinner vascular cylinder and lower stomatal density than those grown at ambient temperature. The proportion of mesophyll area occupied by vascular cylinder or intercellular spaces were not changed. Lower stomatal density apparently did not lead to decreased use of water, as these needles had higher concentrations of less mobile nutrients (Ca, Mg, B, Zn and Mn), which could indicate increased total transpiration. In the 1997 and 1998 cohorts, elevation of temperature decreased concentrations of N, P, K, S and Cu. In the 1999 cohort, contradictory, higher concentrations of N and S at elevated temperature may be related to increased nutrient mineralization in the soil. Elevation of CO₂ did not affect stomatal density, needle thickness, thickness of epidermis or hypodermis, vascular cylinder or intercellular spaces. Concentrations of N, P, S and Cu decreased at elevated CO₂. Reductions were transient and most distinct in the 1997 cohort. The effects of CO₂ and temperature were in some cases interactive, which meant that in the combined treatment stomatal density decreased less than at elevated temperature, and concentrations of nutrients decreased less than expected on the basis of separate treatments, whereas the thickness of the epidermis and hypodermis decreased more than in the separate treatments. In conclusion, alterations in the anatomy and stomatal density of Scots pine needles were more distinct at elevated temperature than at elevated CO₂. Both elevated CO₂ and temperature-induced changes in nutrient concentrations that partly corresponded to the biochemical and photosynthetic alterations in the same cohorts ( Luomala et al. Plant, Cell and Environment 26, 645–660, 2003 ) Reductions in nutrient concentrations and alterations in the anatomy were transient and more evident in the needle cohort that was grown in the first treatment year.     

Water Stress Reduces Ozone Injury via a Stomatal Mechanism

Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at −35 or −80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes.