Free-Air Controlled Exposure Research Articles

Allocation of Nutrients and Leaf Turnover Rate in Poplar under Ambient and Enriched Ozone Exposure and Soil Nutrient Manipulation.

An excess of ozone (O3) is currently stressing plant ecosystems and may negatively affect the nutrient use of plants. Plants may modify leaf turnover rates and nutrient allocation at the organ level to counteract O3 damage. We investigated leaf turnover rate and allocation of primary (C, N, P, K) and secondary macronutrients (Ca, S, Mg) under various O3 treatments (ambient concentration, AA, with a daily hourly average of 35 ppb; 1.5 × AA; 2.0 × AA) and fertilization levels (N: 0 and 80 kg N ha-1 y-1; P: 0 and 80 kg N ha-1 y-1) in an O3-sensitive poplar clone (Oxford: Populus maximowiczii Henry × P. berolinensis Dippel) in a Free-Air Controlled Exposure (FACE) experiment. The results indicated that both fertilization and O3 had a significant impact on the nutrient content. Specifically, fertilization and O3 increased foliar C and N contents (+5.8% and +34.2%, respectively) and root Ca and Mg contents (+46.3% and +70.2%, respectively). Plants are known to increase the content of certain elements to mitigate the damage caused by high levels of O3. The leaf turnover rate was accelerated as a result of increased O3 exposure, indicating that O3 plays a main role in influencing this physiological parameter. A PCA result showed that O3 fumigation affected the overall allocation of primary and secondary elements depending on the organ (leaves, stems, roots). As a conclusion, such different patterns of element allocation in plant leaves in response to elevated O3 levels can have significant ecological implications.

Comet assay as an early predictor tool to detect ozone enhanced sensitivity of vegetation in a free-air controlled long-term exposure

Although ozone (O3) is a strong oxidant with serious impacts on plants, few studies have investigated the effects on DNA damage. Here we tested the Comet assay combined with the enzymatic isolation of leaf protoplasts. Two species differing in O3 sensitivity were used, i.e. the moderately tolerant Arbutus unedo and the very sensitive Populus maximowiczii Henry × berolinensis (or Oxford clone), exposed to three O3 levels (ambient [40.3 ppb], 1.5 times ambient O3 concentration, twice ambient O3 concentration) for one (A. unedo) or two (Oxford clone) growing seasons in an O3 Free Air Controlled Exposure (FACE) facility. We found that O3 has strong plant genotoxicity capacity and the negative effects increased with increasing exposure or stomatal uptake in both species. Interestingly, both ecophysiological and DNA damage were detected in the sensitive species, while no ecophysiological injury was observed in A. unedo although DNA was damaged before other symptoms occurred. Overall, DNA damage increased when the ecophysiological parameters decreased, confirming that DNA damage is a good proxy of the functional status of a plant. We conclude that the enzymatic isolation and Comet assay could be used as a predictor tool to reveal plant O3 DNA damage even in moderately O3 tolerant species, able to recover the abiotic stress.

Functional responses of two Mediterranean pine species in an ozone Free-Air Controlled Exposure (FACE) experiment.

Effects of the phytotoxic and widespread ozone (O3) pollution may be species specific, but knowledge on Mediterranean conifer responses to long-term realistic exposure is still limited. We examined responses regarding to photosynthesis, needle biochemical stress markers and carbon and nitrogen (N) isotopes of two Mediterranean pine species (Pinus halepensis Mill. and Pinus pinea L.). Seedlings were grown in a Free-Air Controlled Exposure experiment with three levels of O3 (ambient air, AA [38.7p.p.b. as daily average]; 1.5×AA and 2.0×AA) during the growing season (May-October 2019). In P. halepensis, O3 caused a significant decrease in the photosynthetic rate, which was mainly due to a reduction of both stomatal and mesophyll diffusion conductance to CO2. Isotopic analyses indicated a cumulative or memory effect of O3 exposure on this species, as the negative effects were highlighted only in the late growing season in association with a reduced biochemical defense capacity. On the other hand, there was no clear effect of O3 on photosynthesis in P. pinea. However, this species showed enhanced N allocation to leaves to compensate for reduced photosynthetic N- use efficiency. We conclude that functional responses to O3 are different between the two species determining that P. halepensis with thin needles was relatively sensitive to O3, while P. pinea with thicker needles was more resistant due to a potentially low O3 load per unit mass of mesophyll cells, which may affect species-specific resilience in O3-polluted Mediterranean pine forests.

Exploring a New O3 Index as a Proxy for the Avoidance/Tolerance Capacity of Forest Species to Tolerate O3 Injury

Tropospheric ozone (O3) is a detrimental air pollutant causing phytotoxic effects. Several O3 indices are used to assess the risk for vegetation, e.g., the exposure-based AOT40 (accumulated ozone exposure over a threshold of 40 ppb) and the stomatal-flux based POD1 (Phytotoxic Ozone Dose above a threshold of 1 nmol m−2 s−1). Leaf Mass per Area (LMA) is recommended as a simple index to explain the plant tolerance capacity to O3. We therefore tested a new species-specific O3 index (Leaf Index Flux—LIF: calculated as stomatal O3 flux/LMA) as a proxy of the avoidance/tolerance capacity against O3 stress according to datasets of visible foliar injury (VFI) in forest monitoring and a manipulative Free-Air Controlled Exposure (FACE) experiment. For the forest monitoring, AOT40, POD1, and LIF were calculated from hourly O3, soil moisture, and meteorological measurements at nine Italian forest sites over the period 2018–2022. The results were tested for correlation with the O3 VFI annually surveyed at the same sites along the forest edge (LESS) or inside the forest (ITP) and expressed as relative frequency of symptomatic species in the LESS (SS_LESS) and Plant Injury Index per tree in the plot (PII_ITP). Based on VFI occurrence at ITP and LESS, Fagus sylvatica was considered the most O3-sensitive species, whereas conifers (Pinus pinea and Picea abies) and other deciduous/evergreen broadleaf (Quercus petraea, Q. cerris, Q. ilex, and Phyllirea latifolia) showed rare and no O3 VFI. Shrub species such as Rubus spp. and Vaccinium myrtillus were O3-sensitive, as they showed VFI along the LESS. AOT40 did not show significant correlations with the VFI parameters, POD1 increased with increasing SS_LESS (p = 0.005, r = 0.37) and PII_ITP (p < 0.001, r = 0.53), and LIF showed an even higher correlation with SS%_LESS (p < 0.001, r = 0.63) and PII_ITP (p < 0.001, r = 0.87). In the FACE experiment, PII was investigated for five deciduous and three evergreen tree species following one growing season of exposure to ambient and above-ambient O3 levels (PII_FACE). Moreover, PII_FACE resulted better correlated with LIF (r = 0.67, p < 0.001) than with POD1 (r = 0.58, p = 0.003) and AOT40 (r = 0.35, p = 0.09). Therefore, LIF is recommended as a promising index for evaluating O3 VFI on forest woody species and stresses high O3 risk potential for forest species with high stomatal conductance and thin leaves.

Chronic ozone exposure impairs the mineral nutrition of date palm (Phoenix dactylifera) seedlings

Chronic ozone (O3) exposure in the atmosphere preferentially disturbs metabolic processes in the roots rather than the shoot as a consequence of reduced photosynthesis and carbohydrate allocation from the leaves to the roots. The aim of the present study was to elucidate if mineral nutrition is also impaired by chronic O3 exposure. For this purpose, date palm (Phoenix dactylifera) plants were fumigated with ambient, 1.5 × ambient and 2 × ambient O3 in a free air controlled exposure (FACE) system for one growing season and concentrations of major nutrients were analyzed in leaves and roots. In addition, concentrations of C and N and their partitioning between different metabolic C and N pools were determined in both organs. The results showed that calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), sodium (Na) and potassium (K) acquisition by roots was diminished by O3 exposure of the shoot. For Ca, Mg, Fe and Zn reduced uptake by the roots was combined with reduced allocation to the shoot, resulting in a decline of foliar concentrations; for Na and K, allocation to the shoot was maintained at the expense of the roots. Thus, elevated O3 impaired both mineral uptake by the roots and partitioning of minerals between roots and shoots, but in an element specific way. Thereby, elevated O3 affected roots and shoots differently already after one growing season. However, considerable changes in total C and N concentrations and their partitioning between different metabolic pools upon chronic O3 exposure were not observed in either leaves or roots, except for reduced foliar lignin concentrations at 2 × ambient O3. Significant differences in these parameters were shown between leaves and roots independent of O3 application. The physiological consequences of the effects of chronic O3 exposure on mineral acquisition and partitioning between leaves and roots are discussed.

Chronic ozone exposure preferentially modifies root rather than foliar metabolism of date palm (Phoenix dactylifera) saplings

In their natural environment, date palms are exposed to chronic atmospheric ozone (O3) concentrations from local and remote sources. In order to elucidate the consequences of this exposure, date palm saplings were treated with ambient, 1.5 and 2.0 times ambient O3 for three months in a free-air controlled exposure facility. Chronic O3 exposure reduced carbohydrate contents in leaves and roots, but this effect was much stronger in roots. Still, sucrose contents of both organs were maintained at elevated O3, though at different steady states. Reduced availability of carbohydrate for the Tricarboxylic acid cycle (TCA cycle) may be responsible for the observed reduced foliar contents of several amino acids, whereas malic acid accumulation in the roots indicates a reduced use of TCA cycle intermediates. Carbohydrate deficiency in roots, but not in leaves caused oxidative stress upon chronic O3 exposure, as indicated by enhanced malonedialdehyde, H2O2 and oxidized glutathione contents despite elevated glutathione reductase activity. Reduced levels of phenolics and flavonoids in the roots resulted from decreased production and, therefore, do not indicate oxidative stress compensation by secondary compounds. These results show that roots of date palms are highly susceptible to chronic O3 exposure as a consequence of carbohydrate deficiency.

Stress markers and physiochemical responses of the Mediterranean shrub Phillyrea angustifolia under current and future drought and ozone scenarios

Mediterranean plants are particularly threatened by the exacerbation of prolonged periods of summer drought and increasing concentrations of ground-level ozone (O3). The aims of the present study were to (i) test if selected markers (i.e., reactive oxygen species, ROS; malondialdehyde, MDA; photosynthetic pigments) are able to discriminate the oxidative pressure due to single and combined stress conditions, and (ii) elucidate the physiochemical adjustments adopted by Phillyrea angustifolia (evergreen woody species representative of the maquis, also known as narrow-leaved mock privet) to perceive and counter to drought and/or O3. Plants were grown from May to October under the combination of two levels of water irrigation [i.e., well-watered (WW) and water-stressed (WS)] and three levels of O3 [i.e., 1.0, 1.5 and 2.0 times the ambient air concentrations, i.e. AA (current O3 scenario), 1.5 × AA and 2.0 × AA (future O3 scenarios), respectively], using a new-generation O3 Free Air Controlled Exposure (FACE) system. Overall, this species appeared relatively sensitive to drought (e.g., net CO2 assimilation rate and stomatal conductance significantly decreased, as well as total chlorophyll and carotenoid contents), and tolerant to O3 (e.g., as confirmed by the absence of visible foliar injury, the unchanged values of total carotenoids, and the detrimental effects on stomatal conductance, total chlorophylls and terpene emission only under elevated O3 concentrations). The combination of both stressors led to harsher oxidative stress. Only when evaluated together (i.e., combining the information provided by the analysis of each stress marker), ROS, MDA and photosynthetic pigments, were suitable stress markers to discriminate the differential oxidative stress induced by drought and increasing O3 concentrations applied singly or in combination: (i) all these stress markers were affected under drought per se; (ii) hydrogen peroxide (H2O2) and MDA increased under O3per se, following the gradient of O3 concentrations (H2O2: about 2- and 4-fold higher; MDA: +22 and + 91%; in 1.5 × AA_WW and 2.0 × AA_WW, respectively); (iii) joining together the ROS it was possible to report harsher effects under 2.0 × AA_WS and 1.5 × AA_WS (both anion superoxide and H2O2 increased) than under 2.0 × AA_WW (only H2O2 increased); and (iv) MDA showed harsher effects under 2.0 × AA_WS than under 1.5 × AA_WS (increased by 49 and 18%, respectively). Plants activated physiological and biochemical adjustments in order to partially avoid (e.g., stomatal closure) and tolerate (e.g., increased terpene emission) the effects of drought when combined with increasing O3 concentrations, suggesting that the water use strategy (isohydric) and the sclerophyllous habit can further increase the plant tolerance to environmental constraints in the Mediterranean area.

Experimental assessment of ozone risk on ecotypes of the tropical tree Moringa oleifera

Ozone (O3) is an oxidative air pollutant that affects plant growth. Moringa oleifera is a tree species distributed in the tropical and subtropical regions. This species presents high morphological plasticity, which increases its ability to tolerate stressful conditions, but with no O3 risk assessment calculated so far. The present study assessed the O3 risk to different M. oleifera ecotypes using exposure-based index (AOT40) or flux-based index (PODy - where y is a threshold of O3 uptake). PODy considers the O3 uptake through the stomata and the consequence of environmental climate conditions on stomatal conductance (gsto); thus, it is efficient in assessing O3 risk. Five M. oleifera ecotypes were subjected to ambient (Amb.); middle (Mid. X1.5), and High (x2.0) O3 concentrations for 77 days in a free-air controlled exposure facility (FACE). Leaf biomass (LB) was evaluated, and the biomass loss was projected assuming a clean atmosphere (10 ppb as 24 h O3 average). The gsto parameterization was calculated using the Jarvis-type multiplicative algorithm considering several climate factors, i.e., light intensity, air temperature, air vapor pressure deficit, and AOT40. Ozone exposure harmed the LB of all ecotypes. The high gsto (~559 mmol H2O m−2 s−1) can be considered the reason for the species' O3 sensitivity. M. oleifera is adapted to hot climate conditions, and gsto was restricted with air temperature (Tmin) below ~ 9 °C. As expected, the PODy index performed better than the AOT40 for estimating the O3 effect on biomass losses. We recommend a y threshold of 4 nmol m−2 s−1 to incorporate O3 effects on M. oleifera LB. To not exceed a 4% reduction of LB for any M. oleifera genotype, we recommend the critical levels of 1.1 mmol m−2 POD4.

Date palm responses to a chronic, realistic ozone exposure in a FACE experiment

Date palms are highly economically important species in hot arid regions, which may suffer ozone (O3) pollution equivalently to heat and water stress. However, little is known about date palm sensitivity to O3. Therefore, to identify their resistance mechanisms against elevated O3, physiological parameters (leaf gas exchange, chlorophyll fluorescence and leaf pigments) and biomass growth responses to realistic O3 exposure were tested in an isoprene-emitting date palm (Phoenix dactylifera L. cv. Nabut Saif) by a Free-Air Controlled Exposure (FACE) facility with three levels of O3 (ambient [AA, 45 ppb as 24-h average], 1.5 x AA and 2 x AA). We found a reduction of photosynthesis only at 2 x AA although some foliar traits known as early indicators of O3 stress responded already at 1.5 x AA, such as increased dark respiration, reduced leaf pigment content, reduced maximum quantum yield of PSII, inactivation of the oxygen evolving complex of PSII and reduced performance index PITOT. As a result, O3 did not affect most of the growth parameters although significant declines of root biomass occurred only at 2 x AA. The major mechanism in date palm for reducing the severity of O3 impacts was a restriction of stomatal O3 uptake due to low stomatal conductance and O3-induced stomatal closure. In addition, an increased respiration in elevated O3 may indicate an enhanced capacity of catabolizing metabolites for detoxification and repair. Interestingly, date palm produced low amounts of monoterpenes, whose emission was stimulated in 2 x AA, although isoprene emission declined at both 1.5 and 2 x AA. Our results warrant more research on a biological significance of terpenoids in plant resistance against O3 stress.

Ozone impairs the response of isoprene emission to foliar nitrogen and phosphorus in poplar

Tropospheric ozone (O3) impairs physiological processes of plants while nitrogen (N) deposition may cause imbalances in soil N and other nutrients such as phosphorus (P) suggesting an increase of P demand for plants. However, the combined effect of O3, soil N and P on isoprene emission from leaves has never been tested. We therefore examined isoprene emission in leaves of Oxford poplar clone exposed to O3 (ambient, AA [35.0 nmol mol−1 as daily mean]; 1.5 × AA; 2.0 × AA), soil N (0 and 80 kg N ha−1) and soil P (0, 40 and 80 kg P ha−1) in July and September in a Free-Air Controlled Exposure (FACE) facility. We also investigated the response of isoprene emission to foliar N, P and abscisic acid (ABA) contents in September because the 2-C-methylerythritol-5-phosphate (MEP) pathway of isoprenoid biosynthesis produces ABA. We found that O3 increased isoprene emission in July, which was associated to increased dark respiration, suggesting an activation of metabolism against O3 stress as an initial response. However, O3 decreased isoprene emission in September which was associated to reduced net photosynthesis. In September, isoprene emission was positively correlated with leaf N content and negatively correlated with leaf P content in AA. However, no response of isoprene emission to foliar N and P was found in elevated O3, suggesting that the isoprene responses to foliar N and P depended on the O3 exposure levels. Isoprene emission rate in 1.5 × AA and 2.0 × AA increased with increasing leaf ABA content, indicating accelerated senescence of injured leaves to favor new leaf growth when high O3 and nutritional availability in the soil were combined. Even though foliar N and P usually act as a proxy for isoprene emission rate, the impact of recent abiotic factors such as O3 should be always considered for modeling isoprene emission under climate change.

Isotopic and Water Relation Responses to Ozone and Water Stress in Seedlings of Three Oak Species with Different Adaptation Strategies

The impact of global changes on forest ecosystem processes is based on the species-specific responses of trees to the combined effect of multiple stressors and the capacity of each species to acclimate and cope with the environment modification. Combined environmental constraints can severely affect plant and ecological processes involved in plant functionality. This study provides novel insights into the impact of a simultaneous pairing of abiotic stresses (i.e., water and ozone (O3) stress) on the responses of oak species. Water stress (using 40 and 100% of soil water content at field capacity—WS and WW treatments, respectively) and O3 exposure (1.0, 1.2, and 1.4 times the ambient concentration—AA, 1.2AA, and 1.4AA, respectively) were carried out on Quercus robur L., Quercus ilex L., and Quercus pubescens Willd. seedlings, to study physiological traits (1. isotope signature [δ13C, δ18O and δ15N], 2. water relation [leaf water potential, leaf water content], 3. leaf gas exchange [light-saturated net photosynthesis, Asat, and stomatal conductance, gs]) for adaptation strategies in a Free-Air Controlled Exposure (FACE) experiment. Ozone decreased Asat in Q. robur and Q. pubescens while water stress decreased it in all three oak species. Ozone did not affect δ13C, whereas δ18O was influenced by O3 especially in Q. robur. This may reflect a reduction of gs with the concomitant reduction in photosynthetic capacity. However, the effect of elevated O3 on leaf gas exchange as indicated by the combined analysis of stable isotopes was much lower than that of water stress. Water stress was detectable by δ13C and by δ18O in all three oak species, while δ15N did not define plant response to stress conditions in any species. The δ13C signal was correlated to leaf water content (LWC) in Q. robur and Q. ilex, showing isohydric and anisohydric strategy, respectively, at increasing stress intensity (low value of LWC). No interactive effect of water stress and O3 exposure on the isotopic responses was found, suggesting no cross-protection on seasonal carbon assimilation independently on the species adaptation strategy.

Flux-Based Ozone Risk Assessment for a Plant Injury Index (PII) in Three European Cool-Temperate Deciduous Tree Species

This study investigated visible foliar ozone (O3) injury in three deciduous tree species with different growth patterns (indeterminate, Alnus glutinosa (L.) Gaertn.; intermediate, Sorbus aucuparia L.; and determinate, Vaccinium myrtillus L.) from May to August 2018. Ozone effects on the timing of injury onset and a plant injury index (PII) were investigated using two O3 indices, i.e., AOT40 (accumulative O3 exposure over 40 ppb during daylight hours) and PODY (phytotoxic O3 dose above a flux threshold of Y nmol m−2 s−1). A new parameterization for PODY estimation was developed for each species. Measurements were carried out in an O3 free-air controlled exposure (FACE) experiment with three levels of O3 treatment (ambient, AA; 1.5 × AA; and 2.0 × AA). Injury onset was found in May at 2.0 × AA in all three species and the timing of the onset was determined by the amount of stomatal O3 uptake. It required 4.0 mmol m−2 POD0 and 5.5 to 9.0 ppm·h AOT40. As a result, A. glutinosa with high stomatal conductance (gs) showed the earliest emergence of O3 visible injury among the three species. After the onset, O3 visible injury expanded to the plant level as confirmed by increased PII values. In A. glutinosa with indeterminate growth pattern, a new leaf formation alleviated the expansion of O3 visible injury at the plant level. V. myrtillus showed a dramatic increase of PII from June to July due to higher sensitivity to O3 in its flowering and fruiting stage. Ozone impacts on PII were better explained by the flux-based index, PODY, as compared with the exposure-based index, AOT40. The critical levels (CLs) corresponding to PII = 5 were 8.1 mmol m−2 POD7 in A. glutinosa, 22 mmol m−2 POD0 in S. aucuparia, and 5.8 mmol m−2 POD1 in V. myrtillus. The results highlight that the CLs for PII are species-specific. Establishing species-specific O3 flux-effect relationships should be key for a quantitative O3 risk assessment.

Ozone-induced impairment of night-time stomatal closure in O3-sensitive poplar clone is affected by nitrogen but not by phosphorus enrichment.

Nocturnal transpiration may be a key factor influencing water use in plants. Tropospheric ozone (O3) and availability of nutrients such as nitrogen (N) and phosphorus (P) in the soil can affect daytime water use through stomata, but the combined effects of O3, N and P on night-time stomatal conductance (gs) are not known. We investigated the effects of O3 and soil availability of N and P on nocturnal gs and the dynamics of stomatal response after leaf severing in an O3-sensitive poplar clone (Oxford) subjected to combined treatments over a growing season in an O3 free air controlled exposure (FACE) facility. The treatments were two soil N levels (0 and 80 kg N ha-1; N0 and N80), three soil P levels (0, 40 and 80 kg P ha-1; P0, P40 and P80) and three O3 levels (ambient concentration, AA [35.0 ppb as hourly mean]; 1.5 × AA; 2.0 × AA). The analysis of stomatal dynamics after leaf severing suggested that O3 impaired stomatal closure execution. As a result, nocturnal gs was increased by 2.0 × AA O3 in August (+39%) and September (+108%). Night-time gs was correlated with POD0 (phytotoxic O3 dose) and increased exponentially after 40 mmol m-2 POD0. Such increase of nocturnal gs was attributed to the emission of ethylene due to 2.0 × AA O3 exposure, while foliar abscisic acid (ABA) or indole-3-acetic acid (IAA) did not affect gs at night. Interestingly, the O3-induced stomatal opening at night was limited by N treatments in August, but not limited in September. Phosphorus decreased nocturnal gs, although P did not modify the O3-induced stomatal dysfunction. The results suggest that the increased nocturnal gs may be associated with a need to improve N acquisition to cope with O3 stress.

The passion fruit liana (Passiflora edulis Sims, Passifloraceae) is tolerant to ozone

Passiflora edulis Sims is a liana species of high economic interest and is an interesting model plant for understanding ozone action on disturbed vegetation. In this work we hypothesized that P. edulis has adaptive responses to oxidative stress that enable it to tolerate ozone damage based on its capacity to grow under a diversity of environmental conditions and to dominate disturbed areas. We exposed seedlings to three levels of ozone in a Free-Air Controlled Exposure (FACE) system (22, 41 and 58 ppb h AOT40 and 13.52, 17.24 and 20.62 mmol m−2 POD0, over 97 days) for identifying its tolerance mechanisms. Anatomical (leaf blade structure and fluorescence emission of chloroplast metabolites), physiological (leaf gas exchange, growth rate and biomass production) and biochemical (pigments, total sugars, starch, enzymatic and non-enzymatic antioxidant metabolites, reactive oxygen species and lipid peroxidation derivatives) responses were assessed. Ozone caused decreased total number of leaves, hyperplasia and hypertrophy of the mesophyll cells, and accelerated leaf senescence. However, O3 did not affect carbohydrates content, net photosynthetic rate, or total biomass production, indicating that the carboxylation efficiency and associated physiological processes were not affected. In addition, P. edulis showed higher leaf contents of ascorbic acid, glutathione (as well high ratio between their reduced and total forms), carotenoids, and flavonoids located in the chloroplast outer envelope membrane. Our results indicate that P. edulis is an O3-tolerant species due to morphological acclimation responses and an effective antioxidant defense system represented by non-enzymatic antioxidants, which maintained the cellular redox balance under ozone.

Effects of nitrogen and phosphorus imbalance on photosynthetic traits of poplar Oxford clone under ozone pollution.

Ozone (O3) pollution and the availability of nitrogen (N) and phosphorus (P) in the soil both affect plant photosynthesis and chlorophyll (Chl) content, but the interaction of O3 and nutrition is unclear. We postulated that the nutritional condition changes plant photosynthetic responses to O3. An O3-sensitive poplar clone (Oxford) was subject to two N levels (N0, 0kgNha- 1; N80, 80kgNha- 1), two P levels (P0, 0kgPha- 1; P80, 80kgPha- 1) and three levels of O3 exposure (ambient concentration, AA; 1.5 × AA; 2.0 × AA) over a growing season in an O3 free air controlled exposure (FACE) facility. The daily change of leaf gas exchange and dark respiration (Rd) were investigated at mid-summer (August). Chl a fluorescence was measured three times in July, August and September. At the end of the growing season, Chl content was measured. It was found that Chl content, the maximum quantum yield (Fv/Fm), Chl a fluorescence performance index (PI) and gas exchange were negatively affected by elevated O3. Phosphorus may mitigate the O3-induced reduction of the ratio of photosynthesis to stomatal conductance, while it exacerbated the O3-induced loss of Fv/Fm. Nitrogen alleviated negative effects of O3 on Fv/Fm and PI in July. Ozone-induced loss of net photosynthetic rate was mitigated by N in medium O3 exposure (1.5 × AA). However, such a mitigation effect was not observed in the higher O3 level (2.0 × AA). Nitrogen addition exacerbated O3-induced increase of Rd suggesting an increased respiratory carbon loss in the presence of O3 and N. This may result in a further reduction of the net carbon gain for poplars exposed to O3.

Ozone risk assessment is affected by nutrient availability: Evidence from a simulation experiment under free air controlled exposure (FACE)

Assessing ozone (O3) risk to vegetation is crucial for informing policy making. Soil nitrogen (N) and phosphorus (P) availability could change stomatal conductance which is the main driver of O3 uptake into a leaf. In addition, the availability of N and P could influence photosynthesis and growth. We thus postulated that the sensitivity of plants to O3 may be changed by the levels of N and P in the soil. In this study, a sensitive poplar clone (Oxford) was subject to two N levels (N0, 0 kg N ha−1; N80, 80 kg N ha−1), three P levels (P0, 0 kg P ha−1; P40, 40 kg P ha−1; P80, 80 kg P ha−1) and three levels of O3 exposure (ambient concentration, AA; 1.5 × AA; 2.0 × AA) for a whole growing season in an O3 free air controlled exposure (FACE) facility. Flux-based (POD0 to 6) and exposure-based (W126 and AOT40) dose-response relationships were fitted and critical levels (CLs) were estimated for a 5% decrease of total annual biomass. It was found that N and P availability modified the dose-response relationships of biomass responses to O3. Overall, the N supply decreased the O3 CLs i.e. increased the sensitivity of poplar to O3. Phosphorus alleviated the O3-caused biomass loss and increased the CL. However, such mitigation effects of P were found only in low N and not in high N conditions. In each nutritional treatment, similar performance was found between flux-based and exposure-based indices. However, the flux-based approach was superior, as compared to exposure indices, to explain the biomass reduction when all nutritional treatments were pooled together. The best O3 metric for risk assessments was POD4, with 4.6 mmol m−2 POD4 as a suitable CL for Oxford poplars grown under various soil N and P conditions.

Ozone risk assessment in three oak species as affected by soil water availability.

To derive ozone (O3) dose-response relationships for three European oak species (Quercus ilex, Quercus pubescens, and Quercus robur) under a range of soil water availability, an experiment was carried out with 2-year-old potted seedlings exposed to three levels of water availability in the soil and three levels of O3 pollution for one growing season in an ozone free-air controlled exposure (FACE) facility. Total biomass losses were estimated relative to a hypothetical clean air at the pre-industrial age, i.e., at 10ppb as daily average (M24). A stomatal conductance model was parameterized with inputs from the three species for calculating the stomatal O3 flux. Exposure-based (M24, W126, and AOT40) and flux-based (phytotoxic O3 dose (POD)0-3) dose-response relationships were estimated and critical levels (CL) were calculated for a 5% decline of total biomass. Results show that water availability can significantly affect O3 risk assessment. In fact, dose-response relationships calculated per individual species at each water availability level resulted in very different CLs and best metrics. In a simplified approach where species were aggregated on the basis of their O3 sensitivity, the best metric was POD0.5, with a CL of 6.8mmolm-2 for the less O3-sensitive species Q. ilex and Q. pubescens and of 3.5mmolm-2 for the more O3-sensitive species Q. robur. The performance of POD0, however, was very similar to that of POD0.5, and thus a CL of 6.9mmolm-2 POD0 and 3.6mmolm-2 POD0 for the less and more O3-sensitive oak species may be also recommended. These CLs can be applied to oak ecosystems at variable water availability in the soil. We conclude that PODy is able to reconcile the effects of O3 and soil water availability on species-specific oak productivity.

Testing a ratio of photosynthesis to O3 uptake as an index for assessing O3-induced foliar visible injury in poplar trees.

Visible foliar injury by ozone (ozone visible injury) is known as a biomarker to assess potential phytotoxicity of ozone. We investigated ozone visible injury in an ozone-sensitive poplar (Oxford clone) under a 2-year free-air controlled exposure (FACE) experiment and calculated three ozone indices (i.e., accumulative ozone exposure over 40ppb during daylight hours (AOT40), phytotoxic ozone dose above a flux threshold of 0nmolm-2s-1 (POD0), and the cumulative value of the ratio of hourly ozone uptake to net photosynthesis (ΣU/P n ) to assess the critical level (CL) at the time of the first symptom onset of ozone visible injury. We tested the hypothesis that ozone injury depends both on the amount of ozone entering a leaf and on the capacity for biochemical detoxification or repair with photosynthesis as a proxy. The CLs at the time of the first symptom onset of ozone visible injury were 19ppmh for AOT40, 26mmolm-2 for POD0, and 1.2molmol-1 for ΣU/P n in Oxford clone at the ozone FACE experiment. Our findings were then verified by 4-year observation-based data in central Italy on Oxford clone and white poplar (Populus alba L.). These observation-based data indicated that we found ozone visible injury in Oxford clone even though AOT40 was relatively low (11.7ppmh). On the other hand, when values of POD0 and ΣU/P n exceeded over the CLs, the occurrence of initial symptoms in Oxford clone was shown. White poplar did not show ozone visible injury. ΣU/P n of white poplar at the field sites reached ~1.0molmol-1 (less than the CL=1.2molmol-1, which was obtained from O3 FACE) during May-September, although the values of POD0 were relatively high in white poplar (44-47mmolm-2 during May-September). The result implies that ozone injury may have occurred in poplars when stomatal ozone flux exceeded the critical range of tolerance due to the assimilate shortage for repair and defense against ozone stress.

A new-generation 3D ozone FACE (Free Air Controlled Exposure)

To artificially simulate the impacts of ground-level ozone (O3) on vegetation, ozone FACE (Free Air Controlled Exposure) systems are increasingly recommended. We describe here a new-generation, three-dimensional ozone FACE, with O3 diffusion through laser-generated micro-holes, pre-mixing of air and O3, O3 generator with integral oxygen generator, continuous (day/night) exposure and full replication. Based on three O3 levels and assumptions on the pre-industrial O3 levels, we describe principles to calculate relative yield/biomass and estimate impacts even at lower-than-ambient O3 levels. The case study is called FO3X, and is at present the only ozone FACE in Mediterranean climate and one of the very few ozone FACEs investigating more than one stressor at a time. The results presented here will give further impulse to the research on O3 impacts on vegetation all over the world.

Ozone-induced stomatal sluggishness changes carbon and water balance of temperate deciduous forests.

Tropospheric ozone concentrations have increased by 60–100% in the Northern Hemisphere since the 19th century. The phytotoxic nature of ozone can impair forest productivity. In addition, ozone affects stomatal functions, by both favoring stomatal closure and impairing stomatal control. Ozone-induced stomatal sluggishness, i.e., a delay in stomatal responses to fluctuating stimuli, has the potential to change the carbon and water balance of forests. This effect has to be included in models for ozone risk assessment. Here we examine the effects of ozone-induced stomatal sluggishness on carbon assimilation and transpiration of temperate deciduous forests in the Northern Hemisphere in 2006-2009 by combining a detailed multi-layer land surface model and a global atmospheric chemistry model. An analysis of results by ozone FACE (Free-Air Controlled Exposure) experiments suggested that ozone-induced stomatal sluggishness can be incorporated into modelling based on a simple parameter (gmin, minimum stomatal conductance) which is used in the coupled photosynthesis-stomatal model. Our simulation showed that ozone can decrease water use efficiency, i.e., the ratio of net CO2 assimilation to transpiration, of temperate deciduous forests up to 20% when ozone-induced stomatal sluggishness is considered, and up to only 5% when the stomatal sluggishness is neglected.