Ozone Impact on Emission of Biogenic Volatile Organic Compounds in Three Tropical Tree Species From the Atlantic Forest Remnants in Southeast Brazil.

Plants emit a broad number of Biogenic Volatile Organic Compounds (BVOCs) that can impact urban ozone (O3) production. Conversely, the O3 is a phytotoxic pollutant that causes unknown alterations in BVOC emissions from native plants. In this sense, here, we characterized the constitutive and O3-induced BVOCs for two (2dO3) and four (4dO3) days of exposure (O3 dose 80 ppb) and evaluated the O3 response by histochemical techniques to detect programmed cell death (PCD) and hydrogen peroxide (H2O2) in three Brazilian native species. Croton floribundus Spreng, Astronium graveolens Jacq, and Piptadenia gonoacantha (Mart.) JF Macbr, from different groups of ecological succession (acquisitive and conservative), different carbon-saving defense strategies, and specific BVOC emissions. The three species emitted a very diverse BVOC composition: monoterpenes (MON), sesquiterpenes (SEQ), green leaf volatiles (GLV), and other compounds (OTC). C. floribundus is more acquisitive than A. graveolens. Their most representative BVOCs were methyl salicylate-MeSA (OTC), (Z) 3-hexenal, and (E)-2-hexenal (GLV), γ-elemene and (-)-ß-bourbonene (SEQ) ß-phellandrene and D-limonene (MON), while in A. graveolens were nonanal and decanal (OTC), and α-pinene (MON). Piptadenia gonoachanta is more conservative, and the BVOC blend was limited to MeSA (OTC), (E)-2-hexenal (GLV), and ß-Phellandrene (MON). The O3 affected BVOCs and histochemical traits of the three species in different ways. Croton floribundus was the most O3 tolerant species and considered as an SEQ emitter. It efficiently reacted to O3 stress after 2dO3, verified by a high alteration of BVOC emission, the emergence of the compounds such as α-Ionone and trans-ß-Ionone, and the absence of H2O2 detection. On the contrary, A. graveolens, a MON-emitter, was affected by 2dO3 and 4dO3, showing increasing emissions of α-pinene and ß-myrcene, (MON), γ-muurolene and ß-cadinene (SEQ) and H2O2 accumulation. Piptadenia gonoachanta was the most sensitive and did not respond to BVOCs emission, but PCD and H2O2 were highly evidenced. Our results indicate that the BVOC blend emission, combined with histochemical observations, is a powerful tool to confirm the species' tolerance to O3. Furthermore, our findings suggest that BVOC emission is a trade-off associated with different resource strategies of species indicated by the changes in the quality and quantity of BVOC emission for each species.

Biogenic Volatile Organic Compounds Emission of Brazilian Atlantic Tree Grown Under Elevated Ozone in Ambient Controlled and Field Conditions.

Croton floribundus (L.) Spreng trees were exposed to accumulated ozone (O3) levels under laboratory and field conditions and monitored the foliar visible symptoms and BVOC emissions. Plants exposed to O3 in the laboratory presented more substantial damage and significant increase in the BVOC emissions than plants in the field. Caryophyllene and 3-hexen-1-ol emissions were significantly increased in plants exposed to O3 in the laboratory. Under field conditions, methyl salicylate (MeSA) was the majority compound emitted. A positive correlation among the meteorological conditions, O3 and MeSA emission was observed in the field conditions, which may represent a mechanism of tolerance by C. floribundus to deal with long-term exposure to O3.

Ozone affects leaf physiology and causes injury to foliage of native tree species from the tropical Atlantic Forest of southern Brazil.

In southern Brazil, the recent increase in tropospheric ozone (O3) concentrations poses an additional threat to the biodiverse but endangered and fragmented remnants of the Atlantic Forest. Given the mostly unknown sensitivity of tropical species to oxidative stress, the principal objective of this study was to determine whether the current O3 levels in the Metropolitan Region of Campinas (MRC), downwind of São Paulo, affect the native vegetation of forest remnants. Foliar responses to O3 of three tree species typical of the MRC forests were investigated using indoor chamber exposure experiments under controlled conditions and a field survey. Exposure to 70ppb O3 reduced assimilation and leaf conductance but increased respiration in Astronium graveolens while gas exchange in Croton floribundus was little affected. Both A. graveolens and Piptadenia gonoacantha developed characteristic O3-induced injury in the foliage, similar to visible symptoms observed in >30% of trees assessed in the MRC, while C. floribundus remained asymptomatic. The underlying structural symptoms in both O3-exposed and field samples were indicative of oxidative burst, hypersensitive responses, accelerated cell senescence and, primarily in field samples, interaction with photo-oxidative stress. The markers of O3 stress were thus mostly similar to those observed in other regions of the world. Further research is needed, to estimate the proportion of sensitive forest species, the O3 impact on tree growth and stand stability and to detect O3 hot spots where woody species in the Atlantic Forest are mostly affected.

Volatile organic compounds and nitric oxide as responses of a Brazilian tropical species to ozone: the emission profile of young and mature leaves.

The emission profile of volatile organic compounds (VOCs) and nitric oxide (NO) in young and mature leaves of Croton floribundus was assessed in plants exposed to filtered air (FA) and ozone-enriched filtered air (FA+O3). After the period of exposure, leaves were enclosed in polyethylene terephthalate bags and VOCs were collected in young and mature leaves. Both young and mature leaves constitutively emitted the same VOC, but the concentrations were higher in young leaves. O3 exposure induced the emission of sesquiterpenes (mainly ß-caryophyllene) known as antioxidant compounds that may scavenge O3. Young leaves were the highest emitters of sesquiterpenes. O3 induced a rapid accumulation of NO in different tissues and leaf developmental stages; this accumulation was marked in palisade and spongy parenchyma cells in young and mature leaves, respectively. O3 altered the levels of the signaling compound methyl salicylate (MeSA). Moreover, our data showed that NO together with VOC emissions, such as geranyl acetate, α-cadiene, trans-farnesol, cis-ß-farnesene, and MeSA, participate of plant defense mechanisms against the oxidative damage caused by O3.

Protective effect of Mn(III)-desferrioxamine B upon oxidative stress caused by ozone and acid rain in the Brazilian soybean cultivar Glycine max "Sambaiba".

This study aimed to investigate the effects of the Mn complex (Mn(III)-desferrioxamine B (MnDFB)) on oxidative stress in the Brazilian soybean cultivar Glycine max "Sambaiba" following exposure to ozone and acid rain. We determined the suitable dose of MnDFB to apply to G. max seedlings using a dose-response curve. The highest superoxide dismutase (SOD) activity and Mn content in leaves were found upon the application of 8 µM MnDFB. Thus, G. max seedlings pretreated with 8 µM MnDFB were individually exposed to ozone and acid rain simulated. Pretreatment with MnDFB reduced lipid peroxidation upon ozone exposure and increased SOD activity in leaves; it did not alter the metal content in any part of the plant. Conversely, following acid rain exposure, neither the metal content in leaves nor SOD enzyme activity were directly affected by MnDFB, unlike pH. Our findings demonstrated that exogenous MnDFB application before ozone exposure may modulate the MnSOD, Cu/ZnSOD, and FeSOD activities to combat the ROS excess in the cell. Here, we demonstrated that the applied dose of MnDFB enhances antioxidative defenses in soybean following exposure to acid rain and especially to ozone.

Ozone-induced responses in Croton floribundus Spreng. (Euphorbiaceae): metabolic cross-talk between volatile organic compounds and calcium oxalate crystal formation.

Here, we proposed that volatile organic compounds (VOC), specifically methyl salicylate (MeSA), mediate the formation of calcium oxalate crystals (COC) in the defence against ozone (O3) oxidative damage. We performed experiments using Croton floribundus, a pioneer tree species that is tolerant to O3 and widely distributed in the Brazilian forest. This species constitutively produces COC. We exposed plants to a controlled fumigation experiment and assessed biochemical, physiological, and morphological parameters. O3 induced a significant increase in the concentrations of constitutive oxygenated compounds, MeSA and terpenoids as well as in COC number. Our analysis supported the hypothesis that ozone-induced VOC (mainly MeSA) regulate ROS formation in a way that promotes the opening of calcium channels and the subsequent formation of COC in a fast and stable manner to stop the consequences of the reactive oxygen species in the tissue, indeed immobilising the excess calcium (caused by acute exposition to O3) that can be dangerous to the plant. To test this hypothesis, we performed an independent experiment spraying MeSA over C. floribundus plants and observed an increase in the number of COC, indicating that this compound has a potential to directly induce their formation. Thus, the tolerance of C. floribundus to O3 oxidative stress could be a consequence of a higher capacity for the production of VOC and COC rather than the modulation of antioxidant balance. We also present some insights into constitutive morphological features that may be related to the tolerance that this species exhibits to O3.

Response of Brazilian native trees to acute ozone dose.

Ozone (O3) is a toxic secondary pollutant able to cause an intense oxidative stress that induces visual symptoms on sensitive plant species. Controlled fumigation experiment was conducted with the aim to verify the O3 sensibility of three tropical species: Piptadenia gonoachanta (Mart.) Macbr. (Fabaceae), Astronium graveolens Jacq. (Anacardiaceae), and Croton floribundus Spreng. (Euphorbiaceae). The microscopical features involved in the oxidative stress were recognized based on specific histochemical analysis. The three species showed visual symptoms, characterized as necrosis and stippling between the veins, mostly visible on the adaxial leaf surface. All the studied species presented hypersensitive-like response (HR-like), and peroxide hydrogen accumulation (H2O2) followed by cell death and proanthocyanidin oxidation in P. gonoachanta and A. graveolens. In P. gonoachanta, a decrease in chlorophyll autofluorescence occurred on symptomatic tissues, and in A. graveolens and C. floribundus, a polyphenol compound accumulation occurred. The responses of Brazilian native species were similar to those described for sensitive species from temperate climate, and microscopical markers may be useful for the detection of ozone symptoms in future studies in the field.

Critical analysis of the potential of Ipomoea nil'Scarlet O'Hara' for ozone biomonitoring in the sub-tropics.

This study aimed to analyze critically the potential of Ipomoea nil'Scarlet O'Hara' for O(3) biomonitoring in the sub-tropics. Four field experiments (one in each season of 2006) were carried out in a location of the city of São Paulo mainly polluted by O(3). Each experiment started with 50 plants, and lasted 28 days. Sub-lots of five plants were taken at intervals between three or four days long. Groups of four plants were also exposed in closed chambers to filtered air or to 40, 50 or 80 ppb of O(3) for three consecutive hours a day for six days. The percentage of leaf injury (interveinal chloroses and necroses), the concentrations of ascorbic acid (AA) and the activity of superoxide dismutase (SOD) and peroxidases (POD) were determined in the 5th, 6th and 7th oldest leaves on the main stem of the plants taken in all experiments. Visible injury occurred in the plants from all experiments. Seasonality in the antioxidant responses observed in plants grown under field conditions was associated with meteorological variables and ozone concentrations five days before leaf analyses. The highest levels of antioxidants occurred during the spring. The percentage of leaf injury was explained (R(2) = 0.97, p < 0.01) by the reduction in the levels of AA and activity of POD five days before the leaf analyses and by the reduction in the levels of particulate matter, and enhancement of temperature and global radiation 10 days before this same day. Although I. nil may be employed for qualitative O(3) biomonitoring, its efficiency for quantitative biomonitoring in the sub-tropics may be compromised, depending on how intense the oxidative power of the environment is.

Sensitivity of Tradescantia pallida (Rose) Hunt. 'Purpurea' Boom to genotoxicity induced by ozone.

The aims of this study were to investigate the sensitivity of Tradescantia pallida 'Purpurea' to genotoxicity induced by ozone, by means of the micronucleus (MCN) bioassay, to verify whether the intensity of genotoxic responses in inflorescences is modulated by concentrations of ascorbic acid (AA) in their bracts, and/or by air temperature variations during the progress of the bioassay, and to define the time lag necessary after ozone exposure to observe maximal genotoxic effects. Flowering branches were exposed to filtered air (control) and to 60 ppb of ozone (ozone) for 3h in fumigation chambers during spring, autumn, winter and summer. After exposure, they were maintained for 24-120 h under filtered air for recovery. A sub-group of each treatment was taken every 24h, when MCN was scored in inflorescences and the levels of AA were determined in bracts. Ozone caused a significant increase in the frequency of MCN after 24-120 of recovery, compared to measurements in inflorescences from the control treatment, but maximal MCN rate was reached between 72 and 120 h of recovery. The highest percentages of MCN in both fumigation treatments were found during the winter experiment. Ozone exposure did not induce significant changes in the content of AA. However, it was positively influenced by daily amplitude of air temperature during the period of the bioassay. The intensity of genotoxic damage and the time lag necessary to visualize an enhanced number of MCN depended on the levels of ascorbic acid in bracts 24h before MCN scoring and on the daily amplitude of air temperature during the development of the bioassay. Narrower ranges between daily maximum and minimum temperatures (around 4 degrees C) during the days of experiment seemed to promote a more efficient diagnostic of genotoxiciy induced by ozone.

Potential of the Trad-MCN assay applied with inflorescences of Tradescantia pallida 'Purpurea' for evaluating air contamination by naphthalene.

The aims of this study were to determine clastogenic responses of Tradescantia pallida cv. Purpurea to naphthalene (NAPH) by means of the bioassay Trad-MCN with inflorescences of T. pallida cv. Purpurea and to verify if this assay might be an indicator of the potential risk imposed in a workplace, where solid insecticide containing NAPH is usually applied. The clastogenic potential of NAPH was assessed by using static and dynamic experimental systems. In both systems, increased micronucleus frequencies were observed in inflorescences submitted to increasing concentrations of solid or gaseous NAPH. The evident clastogenicity verified in inflorescences exposed experimentally to 25-50 mg m(-3) of NAPH during 6h points to a narrow threshold of plant sensitivity, indicating risks under lower NAPH levels than the standards established by OSHA and therefore revealing its suitability for biomonitoring purposes. However, the clastogenic risk should be carefully investigated by other monitoring methods if human health is taken into consideration.