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.

Tillandsia usneoides: A successful alternative for biomonitoring changes in air quality due to a new highway in São Paulo, Brazil.

Tillandsia usneoides is an aerial epiphytic bromeliad that absorbs water and nutrients directly from the atmosphere by scales covering its surface. We expanded the use of this species as a broader biomonitor based on chemical and structural markers to detect changes in air quality. The usefulness of such comprehensive approach was tested during the construction and opening of a highway (SP-21) in São Paulo State, Brazil. The biomonitoring study was performed from 2009 to 2012, thus comprising the period during construction and after the highway inauguration. Metal accumulation and structural alterations were assessed, in addition to microscopy analyses to understand the metal chelation in plant tissues and to assess the causes of alterations in the number and shape of scale cells. Altogether, our analyses support the use of this species as a wide biomonitor of air quality in urbanized areas.

Synergism between ozone and light stress: structural responses of polyphenols in a woody Brazilian species.

Microscopic studies on isolated ozone (O3) effects or on those in synergy with light stress commonly report the induction of polyphenols that exhibit different aspects within the vacuole of photosynthesizing cells. It has been assumed that these different aspects are randomly spread in the symptomatic (injured) regions of the leaf blade. Interestingly, secretory ducts that constitutively produce polyphenols also exhibit these same variations in their vacuolar aspect, in a spatial sequence related to the destiny of these cells (e.g., programmed cell death (PCD) in lytic secretion processes). Here, we demonstrate that the deposition pattern of polyphenols prior to the establishment of the hypersensitive-like response, a type of PCD caused by O3, follows the same one observed in the epithelial cells of the constitutive lysigenous secretory ducts. Astronium graveolens, an early secondary Brazilian woody species, was selected based on its susceptibility to high light and presence of secretory ducts. The synergism effects were assessed by exposing plants to the high O3 concentrations at an urban site in São Paulo City. Confocal, widefield and light microscopies were used to examine polyphenols' occurrence and aspects. The spatial pattern of polyphenols distribution along the leaflets of plants submitted to the synergism condition, in which a dense vacuolar aspect is the target of a cell destined to death, was also observed in the constitutive secretory cells prior to lysis. This similar structural pattern may be a case of homology of process involving both the constitutive (secretory ducts) and the induced (photosynthesizing cells) defenses.

Tillandsia usneoides: a successful alternative for biomonitoring changes in air quality due to a new highway in São Paulo, Brazil.

Tillandsia usneoides is an aerial epiphytic bromeliad that absorbs water and nutrients directly from the atmosphere by scales covering its surface. We expanded the use of this species as a broader biomonitor based on chemical and structural markers to detect changes in air quality. The usefulness of such comprehensive approach was tested during the construction and opening of a highway (SP-21) in São Paulo State, Brazil. The biomonitoring study was performed from 2009 to 2012, thus comprising the period during construction and after the highway inauguration. Metal accumulation and structural alterations were assessed, in addition to microscopy analyses to understand the metal chelation in plant tissues and to assess the causes of alterations in the number and shape of scale cells. Altogether, our analyses support the use of this species as a wide biomonitor of air quality in urbanized areas.

Temporal dynamics of the cellular events in tobacco leaves exposed in São Paulo, Brazil, indicate oxidative stress by ozone.

Nicotiana tabacum 'Bel-W3' is widely used as an ozone bioindicator species, showing typical necrosis preceded by microscopic markers of oxidative stress. This study aimed to follow the development of symptoms in tobacco exposed in São Paulo highlighting the temporal dynamics of the cellular events. Leaves with and without necrosis were processed according to standard techniques for anatomical analyses. Leaves from the site with higher SUM00 presented thinner palisade parenchyma, fewer layers of spongy parenchyma, higher stomatal density, clusters of vessel elements in the midrib, erosion of cuticular waxes and stomatal damage. The sequence of microscopic events from the third day of exposure were condensation of the cytoplasm in parenchyma tissue, sinuosity of anticlinal walls, pectinaceous cell wall protrusions, chromatin condensation and changes in chlorophyll autofluorescence. On the 14th day of exposure, these events finally led to cell death in the palisade parenchyma and necrosis on the leaf. The markers observed indicated oxidative stress caused by 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.

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.