ree-air O3 eXposure

FO3X (Free-air O3 eXposure) is a multidisciplinary study to assess the effects of increasing tropospheric ozone and other stress factors on vegetation under ambient conditions. FO3X is an ecosystem-level manipulative research facility, that ensures long-term operability, fully replicated treatment plots connected with state-of-the-art ecophysiology and genomics labs, and is available for undertaking new collaborative projects.

Ozone is a photochemical, highly-reactive, phytotoxic gas pollutant. Its concentrations in the troposphere are more than doubled relative to the pre-industrial age. In Europe, and in particular in Mediterranean climate, ozone is considered as the pollutant of major concern to plants. Formation increases with increasing air temperature and solar radiation, i.e. two factors that stimulate plant metabolism and growth. When absorbed into the plant tissues, ozone immediately generates reactive oxygen species. In contrast with all the other pollutants, in fact, ozone does not accumulate into plant tissues and induces aspecific symptoms. These characteristics make challenging the study of ambient ozone effects on plants e.g. by epidemiological investigations. Simulation of realistic plant exposure to ozone is also challenging, because of the high ventilation needed for keeping a constant O3 concentration inside closed or open-top chambers. Low light, higher-than-ambient temperature, altered hydrology, high humidity, young plants, non-limiting conditions except for O3, exclusion of competition and parasites, and control plants in O3-free air are among the main weaknesses of controlled-condition experiments. In recent years, free-air systems for plant exposure to ozone have been developing, similar to the FACE (Free-Air Controlled Exposure) facilities used for simulating elevated atmospheric concentrations of CO2. A basic difference is the much higher reactivity of O3, which makes the control of O3 concentrations in FACE systems technically more complex than the control of CO2 concentrations. This is why at present there are only a few ozone FACE experiments in the world.

FO3X is a last-generation, 3D ozone FACE, with ozone diffusion through microholes, pre-mixing of air and ozone, an ozone generator system with integral oxygen generator. FO3X is one of the few ozone FACEs at present available in the world, the only one in Mediterranean climate, and one of the very few ozone FACEs investigating more than one stressor at a time.

FO3X is located nearby Florence, central Italy, within the CNR campus.
It is a joint facility developed by three CNR institutes of the Department of Bio-AgroFood: Institute of Sustainable Plant Protection (IPSP, Elena Paoletti), Institute of Biosciences and Bioresources (IBBR, Cristina Vettori), and Institute of Biometeorology (IBIMET, Alessandro Materassi, Gianni Fasano).

FO3X welcomes national and international scientists for treating plants, collecting samples, getting measures and testing specific experimental hypotheses. Laboratories of plant genomics and ecophysiology are available for supporting research at FO3X. Support for transnational access can be provided.

Key questions to be addressed by FO3X are:

Are interactions of ozone and other stressors (water deficit, nitrogen deposition, phosphorus imbalance) additive or synergic?
Which are the combined effects of ozone and water stress on carbon sequestration and allocation?
Which are ozone effects on the nutraceutical quality of food?
Which is the effect of ozone exposure on stomatal functioning, water control and the global water budget?
Why and how does ozone induce stomatal sluggishness?


FO3X consists of nine 5x5x2m blocks in which the concentrations of tropospheric ozone and the availability of water in the soil can be controlled. The design is a split-plot experiment, that provides the ability to assess the effects of ozone and other stressors, alone and in combination, on many plant attributes, including growth, root characteristics, gas exchanges, biogenic volatile organic compounds, nutrients, antioxidants. At present, plants are potted, but can be planted in the ground if needed. Each block consists of a network of vertical ventpipes which disperses ozone or normal air across the entire block, to simulate three ozone treatments: ambient air (AA); x1.2 ambient ozone (x1.2) and x1.4 ambient ozone (x1.4). Each ozone treatment is replicated three times. Plants in each block may be then divided into other stress treatments, e.g.: full irrigation (water stress 0), 1/3 irrigation (water stress 1), 2/3 irrigation (water stress 2). A computer-controlled system uses signal feedback technology to adjust gas release and water supply. All main ambient variables are continuously recorded (every 15 min to 1 hr), subject to quality assurance procedures and stored in a local system.

FO3X started its activity in June 2015.


How ozone affects stomatal functioning
Understandings about stomatal responses to O3 is crucial because stomata play an important role in regulating O3 uptake into plants causing harmful effects on growth. We assessed effects of O3 on stomatal response (i.e., leaf gas exchange parameters of CO2 and water vapor as photosynthetic rate, A, and stomatal conductance, gs) at both day- and night-time in an O3-sensitive poplar clone (Oxford: Populus maximoviczii Henry × berolinensis Dippel). All plants were supplied with water at 1-3 day intervals to avoid water stress. Preliminary results show that O3 induced a decrease of gs and A in June. In July and August, no significant difference of gs was recorded, although A was significantly decreased by O3. Ozone increased night-time gs especially in August (+95% and +119% in 1.2x and 1.4x, compared to ambient, respectively). Furthermore, continuous recording of gs during night-time (every 5 min) suggested the loss of closing response of stomata at night rather than slower stomatal closure under dark conditions. Our results indicate that incomplete stomatal closure at night may finally enhance stomatal O3 uptake.

Responsible: Dr. Yasutomo Hoshika, IPSP-CNR

Collaborations: Dr. Yves Jolivet, University Lorraine, Nancy, France

Grant: Life+ project FO3REST (LIFE10 ENV/FR/000208)

Ozone visible foliar injury in poplar


Does drought protect oaks from ozone damage?
Aim of the study was assessing mechanisms and effects of the interaction between ozone and water stress. Oak species representative of the Mediterranean region were selected for their different strategy in water use and leaf traits: the deciduous Quercus robur is water spending; the deciduous Quercus pubescens is water saving; and the evergreen Quercus ilex is water saving. Measurements of leaf gas exchange, Volatile Organic Compound emission, biomass, growth, ozone visible foliar injury, wood properties, mychorrizae, nutrients, and decomposition were carried out from June to November 2015.

Responsible: Dr. Elena Paoletti, IPSP-CNR

Collaborations: Dr. Marcello Vitale, La Sapienza University (Rome, Italy), Dr. Silvano Fares, RPS-CREA (Rome, Italy), Dr. Alessio Giovannelli, IVALSA-CNR (Florence, Italy), Dr. Ovidiu Badea, ICAS (Bucarest, Romania), Dr. Hojka Krajgher, SFI (Ljubljana, Slovenia), Prof. Cristina Nali, University Pisa (Pisa, Italy), Dr. Yves Jolivet, University Lorraine (Nancy, France)

Grant: Life+ project FO3REST (LIFE10 ENV/FR/000208)


Effects of ozone on sugarcane
The effect of O3 on sugarcane (Saccharum spp.), a leader crop on production worldwide, was unknown until now. We assessed growth, leaf anatomy, gas exchange and antioxidants in two varieties, differing in water requirements, during three months of exposure to three levels of O3 (AA; x1.2 and x1.4). Preliminary results point out a reduction of biomass and suggest that sugarcane is ozone-sensitive, even though it is a C4 species. Our final aim is to assess ozone risk to sugarcane production.

Responsible: Dr. Bárbara B. Moura, FAPESP, Brazil

Collaborations: Dr. Rafael V. Ribeiro (Institute of Biology, University of Campinas (UNICAMP), Brazil), Dr. Pierre Vollenweider (Swiss Federal Institute for Forest, Snow and Landscape Research, Birmensdorf, Switzerland)

Grant: São Paulo Research Foundation (FAPESP 2014/23839-9 and 2014/13524-0)



Effects of ozone and nutrients on poplar
This is the first experiment investigating the combined effects of ozone (ambient, ambient x 1.5, and ambient x 2.0), phosphorus (0, 40 and 80 kg P ha-1 year-1) and nitrogen (0 and 80 kg ha-1 year-1) on plants. As a target species, we use an ozone sensitive poplar clone, as poplar is a model tree species, and an ecologically and economically important deciduous broadleaf species. Our main hypotheses are: 1. optimal nutritional condition allows plants to mitigate O3-induced decline of CO2 uptake by accelerating leaf turn over. On the other hand, trees under nutritional imbalance condition cannot replace O3-injured leaves due to limited available nutrients. 2. O3-induced increase of night-time water loss may help the acquisition of nutrients (especially P) for new leaf development. Therefore, this night-time water loss will change the available P conditions.

Responsible: Dr. Yasutomo Hoshika (IPSP-CNR)

Collaborations: Prof. Lu Zhang (Northeast Agricultural University, Harbin, China); Dr. Silvano Fares, RPS-CREA (Rome, Italy), Dr. Federico Brilli, Dr. Alessandra Podda (IPSP-CNR), Dr. Ovidiu Badea, ICAS (Bucarest, Romania), Dr. Hojka Krajgher, SFI (Ljubljana, Slovenia), Prof. Cristina Nali, University Pisa (Italy), Dr. Rita Baraldi (IBIMET-CNR, Bologna, Italy), Dr. Anna De Carlo (IVALSA-CNR)

Grant: LIFE15 IT/ENV/000183 project MOTTES


Testing antiozonant chemicals
To protect sensitive plants from ozone damage, several agrochemicals have been assessed. We compared here ethylenediurea (EDU) and kinetin (KIN) in a short term experiment on an O3 sensitive line (S156) of snap bean (Phaseolus vulgaris), with focus on photosynthetic performances.

Responsible: Prof. Lu Zhang (Northeast Agricultural University, Harbin, China)

Collaborations: Dr. Yasutomo Hoshika, Dr. Elisa Carrari (IPSP-CNR)

Grant: State Scholarship Fund of China No. 201606615002



Ecophysiological and molecular responses to salinity and ozone in pomegranate (Punica granatum)
Salinity and ozone are major stressors in the Mediterranean area. Pomegranate fruits are of great nutraceutical interest. Pomegranate is tolerant to salinity and may be considered for cultivation in marginal areas subject to salinization. Its tolerance to ozone, in contrast, in unknown. Ecophysiological (photosynthesis, stomatal conductance, transpiration, photorespiration) and molecular responses (gene expression, proteomics) will be investigated.

Responsible: Dr. Bianca Elena Maserti (IPSP)

Collaborations: Dr. Yasutomo Hoshika, Dr. Elisa Carrari, Dr. Alessandra Podda (IPSP-CNR)

Grant: LIFE15 IT/ENV/000183 project MOTTES


Responses of a vine species (Passiflora edulis) to ozone stress
Knowledge on vine species responses to ozone is very limited. Passiflora edulis seedlings will be exposed to ozone, for evaluating enzymatic and non-enzymatic compounds, biomarkers of oxidative stress, phenolic compounds, visible injuries, structural markers of oxidative stress, leaf gas exchange, photochemistry, stomatal flux, and reproduction biology. In case of fruiting, the nutritional characteristics of the maracuja fruits will be also investigated.

Responsible: Dr. Marisa Domingos, Instituto de Botânica de São Paulo, Brazil

Collaborations: Dr. Yasutomo Hoshika, Dr. Elisa Carrari (IPSP-CNR), Miss. Marisia Esposito, Francine Fernandes, Marcela Engela (Instituto de Botânica de São Paulo, Brazil)

Grant: Fundação de Desenvolvimento da Pesquisa do Agronegócio – FUNDEPAG


DNA integrity under ozone stress
A novel methodology to reveal DNA integrity (Kuzminsky et al., 2016 Frontiers in Plant Science) will be tested for detecting ozone effects on Arbutus unedo, a Mediterranean evergreen tree.

Responsible: Prof. Elena Kuzminsky (Univ. Tuscia, Viterbo, Italy)

Collaborations: Dr. Yasutomo Hoshika, Dr. Elisa Carrari (IPSP-CNR)




Simulating ozone visible foliar injury and parameterizing stomatal ozone flux in two broadleaf tree species
Within the dominant or ozone-injured tree species investigated in the LIFE project MOTTLES, details on ozone visible foliar injury and stomatal ozone flux are not available for the deciduous broadleaf species Sorbus aucuparia and Alnus glutinosa. Potted plants are thus characterized in terms of leaf injury and stomatal conductance.

Responsible: Dr. Elisa Carrari (IPSP-CNR)

Collaborations: Dr. Yasutomo Hoshika (IPSP-CNR)

Grant: LIFE15 IT/ENV/000183 project MOTTES


Ozone impacts on European blueberry fruits
Our understanding of ozone effects on the quality of yield is still imperfect. We are exposing Vaccinium myrtillus plants to three levels of ozone and evaluating the effects on berry nutraceutical and antioxidant properties. In addition visible foliar injury and stomatal ozone uptake will be characterized.

Responsible: Dr. Elena Paoletti (IPSP-CNR)

Collaborations: Dr. Yasutomo Hoshika and Dr. Elisa Carrari (IPSP-CNR), Dr. Elisa Pellegrini and Prof. Cristina Nali (University of Pisa, Italy)

Grant: LIFE15 IT/ENV/000183 project MOTTES


Ozone and water stress effects on the Mediterranean maquis species Phyllirea angustifolia
Phyllirea is an evergreen shrub or small tree, used to face oxidative stressors. We study the morphological, physiological and biochemical responses to a combination of long-term exposure to ozone and water stress.

Responsible: Dr. Yasutomo Hoshika (IPSP-CNR)

Collaborations: Dr. Elisa Carrari (IPSP-CNR)

Grant: LIFE15 IT/ENV/000183 project MOTTES



Zhang, L., Hoshika, Y.*, Carrari, E., Badea, O., Paoletti, E. 2018 Ozone risk assessment is affected by nutrient availability: Evidence from a simulation experiment under free air controlled exposure (FACE). Environmental Pollution 23: 812-822

Moura, B.B.*, Hoshika, Y., Silveira, N.M., Marcos, F.C.C., Machado, E.C., Paoletti, E., Ribeiro, R.V. 2018 Physiological and biochemical responses of two sugarcane genotypes growing under free-air ozone exposure. Environmental and Experimental Botany 153: 72-79

Zhang L.*, Hoshika Y., Carrari E., Burkey K., Paoletti E.: 2018, Protecting the photosynthetic performance of snap bean under free-air ozone exposure. Journal of Environmental Sciences 66: 31-40

Moura B.B.*, Hoshika Y., Ribeiro R.V., Paoletti E.: 2018, Exposure- and flux-based assessment of ozone risk in two sugarcane genotypes. Atmospheric Environment 176: 252–260

Hoshika Y.*, Carrari E., Zhang L., Carriero G., Pignatelli S., Fasano G., Materassi A., Paoletti E.: 2018, Testing a ratio of photosynthesis to O3 uptake as an index for assessing O3-induced foliar visible injury of poplar trees. Environmental Science and Pollution Research, 25: 8113-8124

Hoshika Y., Moura B.B., Paoletti E.*: 2018, Ozone risk assessment in three oak species as affected by soil water availability. Environmental Science and Pollution Research, 25: 8125-8136

Hoshika Y.*, Watanabe M., Carrari E., Paoletti E., Koike T.: 2018, Ozone-induced stomatal sluggishness changes stomatal parameters of Jarvis-type model in white birch and deciduous oak. Plant Biology 20: 20-28

Paoletti E.*, Materassi A., Fasano G., Hoshika Y., Carriero G., Badea O., Silaghi D.: 2017, A new-generation 3D ozone FACE (Free Air Controlled Experiment). Science of the Total Environment 575: 1407–1414

Ochoa-Hueso R.*, Munzi S., Alonso R., Arróniz-Crespo M., Avila A., Bermejo V., Bobbink R., Branquinho C., Concostrina-Zubiri L., Cruz C., Cruz de Carvalho R., De Marco A., Dias T., Elustondo D., Elvira S., Estébanez B., Fusaro L., Gerosa G., Izquieta-Rojano S., Lo Cascio M., Marzuoli R., Matos P., Mereu S., Merino J., Morillas L., Nunes A., Paoletti E., Paoli L., Pinho P., Rogers I.B., Santos A., Sicard P., Stevens C.J., Theobald M.: 2017, Ecological impacts of atmospheric pollution and interactions with climate change in terrestrial ecosystems of the Mediterranean basin: current research and future directions. Environmental Pollution, 227: 194-206

Split-plot design of FO3X, with three squares per ozone level and randomized treatments with other stressors within each square





Project sponsored by

For more information on this project, contact the ozone FACE coordinators Dr. Yasutomo Hoshika (IPSP) at +39 055 5225 565 or mail to and Dr. Elena Paoletti (IPSP) at +39 055 5225 591 or mail to