Foliage visible injury in the tropical tree species, Astronium graveolens is strictly related to phytotoxic ozone dose (PODy).

The present study evaluates the development of visible injury related to phytotoxic ozone dose (PODy) in native tropical species Astronium graveolens Jacq. (Anacardiaceae) and validates the symptoms using structural markers attributed to oxidative burst and hypersensitive responses. Increasing POD0 was associated with increasing O3 visible injury using different metrics as the incidence (INC = number of injured plants/total number of plants × 100), severity (SF = number of injured leaves/total number of leaves on injured plant × 100), and severity leaflet (SFL = number of injured leaflets/total number leaflets injured plant × 100). The effective dose (ED), which represents the POD0 dose responsible for inducing 20 (ED20), 50 (ED50), or 80% (ED80) of visible injury, were used to demonstrate that for this species, the response is similar even when the plants are exposed to diverse climate environments. Further investigation of the INC and SF index may help in long-term forest monitoring sites dedicated to O3 assessment in forests, while the SFL index seems to be an excellent indicator to be used in the short term to investigate the effects of O3.

Metabolic and physiological alterations indicate that the tropical broadleaf tree Eugenia uniflora L. is sensitive to ozone.

Eugenia uniflora L. is an important fruit tree native to tropical South America that adapts to different habitats, thanks to its metabolic diversity and ability to adjust the leaf antioxidant metabolism. We hypothesized that this metabolic diversity would also enable E. uniflora to avoid oxidative damage and tolerate the enhanced ozone (O3) concentrations that have been registered in the (sub)tropics. We investigated whether carbohydrates, polyphenols and antioxidants are altered and markers of oxidative damage (ROS accumulation, alterations in leaf gas exchange, growth and biomass production) are detected in plants exposed to two levels of O3 (ambient air and twice elevated ozone level in a O3-FACE system for 75 days). Phytotoxic O3 dose above a threshold of 0 nmol m-2 s-1 (POD0) and accumulated exposure above 40 ppb (AOT40) were 3.6 mmol m-2 and 14.898 ppb h at ambient, and 4.7 mmol m-2 and 43.881 ppb h at elevated O3. Twenty-seven primary metabolites and 16 phenolic compounds were detected in the leaves. Contrary to the proposed hypothesis that tropical broadleaf trees are relatively O3 tolerant, we concluded that E. uniflora plants are sensitive to elevated O3 concentrations. Experimental POD0 values were lower than the critical levels for visible foliar O3, because of low stomatal conductance. In spite of this low stomatal O3 uptake, we found classic O3 injury, e.g. reduction in carbohydrates and fatty acids concentrations; non-significant changes in the polyphenol profile; inefficient antioxidant responses; increased contents of ROS and indicators of lipid peroxidation; reductions in stomatal conductance, net photosynthesis, root/shoot ratio and height growth. However, we also found some compensation mechanisms, e.g. increased leaf concentration of polyols for protecting the membranes, and increased leaf number for compensating the decline of photosynthetic rate. These results help filling the knowledge gap about tropical tree responses to O3.

Fluorescence emission spectra of target chloroplast metabolites (flavonoids, carotenoids, lipofuscins, pheophytins) as biomarkers of air pollutants and seasonal tropical climate.

Chloroplasts have luminescent metabolites-chlorophyll being the most known one-whose fluorescence emission may be a useful tool to assess the physiological status of the plant. Some antioxidants (flavonoids and carotenoids), and byproducts of membrane rupture (lipofuscins) and chlorophyll degradation (pheophytins), are chloroplasts' fluorescent metabolites directly involved in plant response to environmental stressors and pollutants and may act as a biomarker of stress. Here we hypothesized that climatic variations and air pollutants induce alterations in the emission profile of chloroplasts' fluorescent metabolites in Tillandsia usneoides (Bromeliaceae). To test this hypothesis, an active biomonitoring study was performed during 2 years in five polluted sites located at the Metropolitan Region of Campinas (São Paulo State, Brazil), aiming to identify target chloroplasts' fluorescent metabolites acting as biomarkers of environmental stress. In situ identification and quantification of the intensity of the fluorescence emission from target metabolites (flavonoids, carotenoids, lipofuscins, and pheophytins) were performed by the observation of fresh leaf sections under confocal laser scanning microscopy. Changes in the profile of fluorescence emission were correlated with local climate and air pollution data. The fluorescence emissions of flavonoids and carotenoids varied seasonally, with significant influence of rainfall and NO2. Our results expand the use of T. usneoides as a bioindicator by using alterations in the fluorescence emission profile of chloroplast metabolites. This application may be especially interesting for NO2 biomonitoring.

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.

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.