RESEARCH ARTICLE

Establishing the redox potential of Tibouchina pulchra (Cham.)Cogn., a native tree species from the Atlantic Rain Forest,in the vicinity of an oil refinery in SE Brazil

Marisia Pannia Esposito & Marisa Domingos

Received: 24 July 2013 /Accepted: 10 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract The present study aimed to establish the seasonalvariations in the redox potential ranges of young Tibouchinapulchra plants growing in the Cubatão region (SE Brazil)under varying levels of oxidative stress caused by air pollut-ants. The plants were exposed to filtered air (FA) and non-filtered air (NFA) in open-top chambers installed next to an oilrefinery in Cubatão during six exposure periods of 90 dayseach, which included the winter and summer seasons. Afterexposure, several analyses were performed, including thefoliar concentrations of ascorbic acid and glutathione in itsreduced (AsA and GSH), total (totAA and totG) and oxidizedforms (DHA and GSSG); their ratios (AsA/totAA and GSH/totG); the enzymatic activities of superoxide dismutase(SOD), ascorbate peroxidase (APX), catalase (CAT) and glu-tathione reductase (GR); and the content of malondialdehyde(MDA). The range of antioxidant responses in T. pulchraplants varied seasonally and was stimulated by high or lowair pollutant concentrations and/or air temperatures. Glutathi-one and APX were primarily responsible for increasing planttolerance to oxidative stress originating from air pollution inthe region. The high or low air temperatures mainly affectedenzymatic activity. The content ofMDA increased in responseto increasing ozone concentration, thus indicating that the pro-oxidant/antioxidant balance may not have been reached.

Keywords Antioxidants . Air pollution . Air temperature .

Oil refinery . Redox potential . Open-top chambers

Introduction

Air pollution has become a very important risk factor to theenvironment, especially in urban and industrialized areas.Increasing emissions of primary pollutants in such areas haveinduced the accumulation of secondary pollutants in the tro-posphere, such as ozone (O3). In fact, new hot spots of O3 arearising in Asia, Central Africa and South America, includingBrazil (Emberson 2003; Cape 2008; Noyes et al. 2009;Orlando et al. 2010). Even in concentrations below the safetylimits established by the legislation in many countries,primary and secondary air pollutants can cause harmful effectsto human health and vegetation (Moura et al. 2008; Jasinskiet al. 2011).

Climatic factors, besides affecting directly the formation,concentration and dispersion of air pollutants, may interfereon plant responses to pollution stress. Many studies havehighlighted that intensity of leaf injury caused by O3 on plantsof Nicotiana tabacum "Bel-W3", for instance, may be modu-lated by the action of multiple climatic factors, which regulatethe plant's physiology and metabolism. Such effects on tobac-co plants may occur both in the northern hemisphere, inEuropean countries (Koppel and Sild 1995; Finnan et al.1996; Antonielli et al. 1997; Peñuelas et al. 1999; Yuskaet al. 2003; Klumpp et al. 2006) as in the southern hemisphere,in São Paulo, SE Brazil (Sant’Anna et al. 2008; Esposito et al.2009; Souza and Pagliuso 2009; Dias et al. 2011). Similarresponses were observed in plants of Ipomoea nil "ScarletO'Hara" exposed to chronic levels of O3 in São Paulo city(Dafré-Martinelli et al. 2011; Ferreira et al. 2012).

Gaseous pollutants, after entering plants through the sto-mata, quickly react with water and intensify the formation ofreactive oxygen species (ROS) at the cell wall interface, aneffect that may be enhanced by other environmental stressfactors, such as extremes of air temperature and relative hu-midity (RH) (Bray et al. 2000; Mittler 2002). Oxidative

Responsible editor: Philippe Garrigues

M. P. Esposito (*) :M. DomingosInstituto de Botânica, Núcleo de Pesquisa em Ecologia,PO Box 68041, 04045-972 São Paulo, SP, Brazile-mail: biompe@yahoo.com.br

Environ Sci Pollut ResDOI 10.1007/s11356-013-2453-8

disturbance caused by ROS on the lipids and proteins of theplasma membranes produces a burst of other free radicals andreactive intermediates, a process called lipid peroxidation,which is one of the earliest effects observed in plant cells(Kanofsky and Sima 2005; Pucckette et al. 2007; Jaleel et al.2009).

However, plants have a number of enzymatic and non-enzymatic antioxidants that may protect them against oxida-tive damage and control the level of ROS effects. Superoxidedismutase (SOD), catalase (CAT), ascorbate peroxidase(APX) and glutathione reductase (GR) are enzymatic antiox-idants, and glutathione, carotenoids and ascorbic acid (AsA)are non-enzymatic components (Caregnato et al. 2008). Mostnotably, the interaction between ROS production and itsresulting scavenging mechanisms determines the plant's redoxpotential and depends on the plant's physiological status anddevelopment and biochemical stimuli (Mittler 2002). Howev-er, only the total concentrations of antioxidants such as ascor-bic acid (totAA) and glutathione (totG) do not indicate theredox potential of plants or even their performance asbioindicators of air pollutants because the antioxidant charac-teristic of these substances is given especially by their reducedforms (AsA and GSH, respectively). The availability of thesesubstances in the cells depends essentially on the efficiency ofenzymes that regenerate their oxidized forms (Burkey et al.2006). Establishing the redox potential, by measuring lipidperoxidation and activity of enzymatic antioxidants and byestimating concentration ratios (AsA/totAA and GSH/totG),is therefore a measurement of the appropriate induction of theacclimation processes in plants to tolerate oxidative stress inpolluted areas (Apel and Hirt 2004; Shao et al. 2008; Potterset al. 2010; Zachgo et al. 2013).

Tibouchina pulchra (Cham.) Cogn., a native tree speciesfrom the Atlantic rain forest in southeastern Brazil, maytheoretically tolerate the oxidative stress imposed by air pol-lutants as the levels observed in the industrial pole regionsettled in the city of Cubatão (SE Brazil). This tree speciespredominates in regions of the Atlantic forest in the vicinity ofthe industrial pole (Leitão-Filho et al. 1993), which remaincurrently affected by significant amounts of carbon monoxide(CO), hydrocarbons (CH), nitrogen oxides (NOx), sulfur ox-ides (SOx) and particulate matter (PM) (CETESB 2011). Thetolerance of T. pulchra to pollutants may result from a numberof physiological, structural and metabolic defense character-istics, as shown in biomonitoring studies performed in theregion during the 1990s. The species accumulates high levelsof toxic elements, such as heavy metals, sulfur and fluoride,and rarely shows visible symptoms (Klumpp et al. 1998,2000, 2002; Furlan et al. 1999, 2004, 2007; Moraes et al.2003; Domingos et al. 2003; Szabo et al. 2003). Visible injuryon T. pulchra leaves was only detected after an acute exposureto a 6,038 ppb/h cumulative dose of ozone (AOT40) 25 daysfrom the beginning of the experiment using open-top

chambers (OTP) (Furlan et al. 2008). However, parallel expo-sure experiments have been performed around the industrialpole of Cubatão employing T. pulchra as a biomonitor.Moraes et al. (2000) observed a reduction of the total AsAconcentration in the leaves, and Klumpp et al. (2000) ob-served an increase in peroxidase activity in plants grown nearfertilizer, cement and steel industries. Based on these results, itis possible to assume that T. pulchra has an efficient redoxpotential, which would enable it to tolerate any change in airquality in the region, as occurred around an important oilrefinery at the industrial pole because of the exchange be-tween its energy and steam production system (from boilerspowered by oil to a thermal power plant powered by naturalgas). Therefore, the present study, which was proposed toelucidate this hypothesis, aimed to establish the redox poten-tial range of T. pulchra, which grew in the region undervarying levels of oxidative stress caused by seasonal varia-tions in air pollutant concentrations and/or climatic factors, bymeasuring multiple indicators of the antioxidant defense sys-tem and oxidative injury. For this study, saplings of T. pulchrawere grown in OTP installed next to an oil refinery withfiltered (FA) and non-filtered air (NFA) and varying meteoro-logical conditions. Such a semi-controlled experimental de-sign permitted the authors to determine the interacting effectsof both sets of environmental variables that might exposeplants to oxidative stress in the region.

Materials and methods

Description of the study area and plant exposure

The field experiments in the OTP were developed in themunicipality of Cubatão, which is located on the Atlanticcoast in São Paulo state, southeastern Brazil (23°45′–23°55′S, 46°15′–46°30′W) at the base of the Serra do Mar mountainrange (Fig. 1). Cubatão is approximately 16 km from the cityof Santos, which has an important port that facilitates the tradeof industrial products. Development of the industrial pole inCubatão occurred from 1950 to 1970 and is currently com-posed of more than 20 industrial sources, including petro-chemical, steel and chemical companies.

The OTC were installed at the Environmental Center ofTraining and Research of Polytechnic School from the Uni-versity of São Paulo (CEPEMA) next to petrochemical indus-tries and railways with intense diesel truck traffic and lightvehicles powered by gasoline or ethanol (Fig. 1). The wear oftires and brakes and the formation of secondary aerosols fromgases emitted by vehicles are additional sources of air con-tamination in the region. Therefore, this area was highlyaffected by primary air pollutants, such as nitrogen and sulfuroxides (NOx and SOx), particulate matter (PM10) with variedcomposition and ozone (O3) (CETESB 2011).

Environ Sci Pollut Res

Four OTCs (3.0 m in diameter and 2.2 m high) were used.The homemade OTCs consisted of stainless steel frameswrapped in Teflon film, which transmits nearly 100 % ofvisible radiation wavelengths, in addition to allowing heatexchange. The entry and distribution of air within the cham-bers occurred from the base to the top of the cylinder and wasforced by fans installed in hermetically sealed stainless steelhousing. Two OTCs received NFA (NFA treatment), and theremaining two OTCs received FA (FA treatment) from filtersthat removed thick and fine particles and gases from the airand a series of chemical beds prepared with activated carbonand aluminum oxide pellets impregnated with potassium per-manganate to filter inorganic and organic gases. The entirefiltering and air flowing system was supplied by Purafil Inc.The airflowwas adjusted in the chambers bymanometers. Thefiltering efficiency was checked by measuring the O3 and NOx

concentrations in the chambers with HORIBA analyzers(models APOA-360CE and APNA-360CE, respectively),both manufactured in Japan.

The T. pulchra saplings for all experiments were acquiredfrom the same nursery. On average, the saplings were 20 cmhigh and had at least six leaves on the main stem. Before eachexperiment, the saplings were transplanted into 10-l potscontaining a mixture of standardized substrate that consisted

predominantly of pine bark and fine vermiculite in a ratio of3:1. The saplings were fertilized weekly with 100 ml ofHoagland solution and maintained for 1 month inside a green-house under FA and ideal meteorological conditions of tem-perature, RH and photosynthetic active radiation (PAR) (T=20 °C, RH=80% and PAR=443μmolm−2 s−1) at the Institutode Botânica in São Paulo.

Exposure of T. pulchra to the NFA and FA treatmentsinside the OTCs followed the model proposed by Arndt andSchweizer (1991). In each chamber, the potted plants werekept in boxes containing water and covered by rigid plasticplates. This apparatus was covered by a shadow screen (50 %brightness reduction). Strings inserted into the base of the potsremained immersed in the water stored in the boxes to ensure asuitable water supply to the plants.

Six exposure experiments of 90 days each were performed.Three of them occurred during less rainy and colder periods,covering autumn and winter seasons (April–June 2011, July–September 2011 and April–June 2012) and three during rainyand hotter periods, covering spring and summer seasons (No-vember 2010–January 2011, October–December 2011 andJanuary–March 2012). With the purpose of presenting theresults, two main treatments were proposed, generally referredto as winter and summer experiments, respectively. Resultsobtained during less rainy and colder periods were joined inthe first treatment, and those obtained during rainy and hotterperiods were grouped in the second treatment.

Figure 2 shows the monthly averages, maximum and min-imum of temperature and humidity and monthly amounts ofrainfall in the region of Cubatão during all the experimentalperiod. As expected, the average, maximum and minimumvalues of temperature and accumulated rainfall were higherduring spring and summer months (October to March) andlower during autumn and winter months (April to September).Higher amounts of rainfall were observed during the rainyperiod of 2010–2011. Values of RH (average, maximum andminimum) ranged from 60% to 90%, 70% to 99% and 50%to 76 %, respectively, during all the experimental period.

Each OTC experiment began with eight saplings ofT. pulchra per OTC. A subgroup of four plants were collectedfrom each OTC after 45 days of exposure, and the remainingfour plants were collected at the end of the exposure period(90 days) for analyses of indicators of the redox state andoxidative injury.

Analyses of the redox state and oxidative injury indicators

The redox state of the plants from all six OTC experimentswas determined by analyzing the CAT activity, SOD, APXand GR and measuring the concentrations of reduced AsA,dehydroascorbic acid (DHA), total ascorbic acid (totAA=AsA+DHA), reduced glutathione (GSH), oxidized glutathi-one (GSSG) and total glutathione (totG=GSH+GSSG). Lipid

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Fig. 1 Map of the area of study in Cubatão, SE Brazil (23°45′–23°55′S,46°21′–46°30′W). 1 CEPEMA, exposure site of T. pulchra in open-topchambers, 2 downtown area, 3 chemical industries, 4 petrochemicalindustries, 5 Atlantic Rain Forest, 6 major highways (source: ArcgizOnline, adapted)

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peroxidation was also analyzed and was measured by theaccumulation of malondialdehyde acid (MDA), which is anoxidative injury indicator.

These indicators were analyzed in a mixture of leaves fromeach plant, mainly by those inserted into the second and thirdnodes. A total of 192 leaf samples were analyzed for eachindicator. Analytical replicates were performed for CAT,APX, GR, MDA and glutathione.

Portions of fresh leaves (0.35 g) were homogenized withpotassium phosphate buffer (50 mM pH 7.0), triton (0.05 %),polyvinyl polypyrrolidone (PVPP; 10 %) and AsA (1 mM).The mixture was centrifuged at 10,000×g at 2 °C for 10 min,and the supernatant was stored at −80 °C to determine theSOD, APX and CAT activities.

The SOD activity was determined according to a slightlymodified method described by Reddy et al. (2004) by mea-suring the ability of the enzyme to inhibit the photochemicalreduction of nitroblue tetrazolium (NBT). A reaction mixturewas prepared by adding potassium phosphate buffer (100 mMpH 7.0), ethylenediamine tetraacetic acid (EDTA; 0.4 μM),methionine (1 mM), leaf extract, NBT (5 mM) and riboflavin(1 mM). The reaction was initiated by placing one fraction ofthis mixture under a fluorescence lamp (80 W) and the otherfraction in the darkness for 30 min. A second similar mixturewithout the leaf extract was prepared and placed under illu-minated and non-illuminated conditions to serve as a blank.The absorbance in the mixtures was measured to determinethe amount of enzyme required to inhibit 50 % of the reduc-tion of NBT.

The APX activity was assayed according to the method ofReddy et al. (2004) with a fewmodifications. The activity wasdetermined at 30 °C in a reaction mixture with potassiumphosphate buffer (100 mM pH 7.0), EDTA (1 mM), AsA(5 mM), hydrogen peroxide (2 mM) and the above-mentioned thawed supernatant. The oxidation of AsA wasmeasured by the decrease in absorbance (ΔA) at 290 nm for2 min using a UV–Vis spectrophotometer. APX activity wasdefined as the change in the absorbance at two different timesin the linear region of the curve.

CAT activity was determined using spectrophotometry asdescribed by Kraus et al. (1995) with some modificationsproposed by Azevedo et al. (1998). At 25 °C, the above-mentioned thawed supernatant was added to a reaction mix-ture of 1 ml potassium phosphate buffer (100 mM pH 7.5) andhydrogen peroxide (1 mM) that was prepared immediatelybefore use. The activity was determined by following thedecomposition of H2O2 per minute through changes in absor-bance at 240 nm.

The activity of GR was determined in fresh leaves (1.0 g)that had been homogenized with potassium phosphate buffer(50 mM pH 7.8), AsA (5 mM), EDTA (5 mM) and 1.4-dithiothreitol (DTT; 5 mM) according to the method of Reddyet al. (2004). The homogenate was centrifuged at 10,000×g at2 °C for 10 min, and the supernatant was frozen at −80 °C forsubsequent analyses. Activity was determined at 30 °C in areaction mixture with potassium phosphate buffer (100 mMpH 7.5), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB; 1 mM),nicotinamide adenine dinucleotide phosphate (NADPH;0.1 mM), GSSG (1 mM), GR and the thawed supernatant.In the presence of NADPH, GR reduced GSSG to GSH, andthis molecule was oxidized by DTNB. The product of thisreaction was measured by the increase in absorbance (ΔA) at412 nm for 20 s using a UV–Vis spectrophotometer. The GRactivity was defined as the change in the absorbance at twodifferent times in the linear region of the curve.

AsA and totAAwere analyzed using the chromatographicmethod described by Lopez et al. (2005) and an HPLC(Metrohm) connected to a UV–Vis detector. A 4×15 mm(5 μg) Prontosil column (C18) was used. The mobile phaseused was water brought to pH 2.3 with phosphoric acid; theflow rate used was 1.0 ml min−1; and the detection wavelengthused was 245 nm. Fresh portions of leaves (0.25 g) werehomogenized with metaphosphoric acid (6 %) and EDTA-Na2 (0.5 mM). The mixture was centrifuged at 10,000×g at2 °C for 10 min. The supernatant was filtered through a paperfilter (Whatman no. 41), and the filtrate was diluted withwater. To determine the AsA, a fraction of this extract wasfiltered through a 0.45-μm filter (Millipore), and this filtrate

Fig. 2 Monthly maximum,average and minimum values oftemperature (T) and relativehumidity (RH) and amounts ofrainfall in the region of Cubatão,during the experimental period

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was then injected into the chromatographic system. To deter-mine totAA, 1.4-DTT (0.2 %) in sodium phosphate buffer(0.2 M pH 7.0) and dipotassium hydrogen phosphate buffer(pH 7.0) were added to an additional portion of the extract.The mixture was incubated for 10 min, and the reaction wasstopped by the addition of phosphoric acid (2M). The mixturewas then filtered through a 0.45-μm filter, and the filtrate wasinjected into the chromatographic system. DHAwas deducedfrom the difference between totAA and AsA and the AsA/totAA ratios were calculated.

The GSH and GSSG content were determined according tothe method described by Israr et al. (2006). Samples of leaves(1.0 g), frozen at −80 °C, were homogenized withsulfosalicylic acid (0.1 %) and centrifuged at 12,000×g at2 °C for 20 min. A mixture of phosphate buffer (100 mMpH 7.0), EDTA (0.5 mM) and DTNB (3 mM) was added to analiquot of the supernatant. After 5 min, the absorbance wasmeasured at 412 nm using a UV–Vis spectrophotometer(Shimadzu) to determine GSH content. NADPH (0.4 mM)and GR were then added to the mixture, and the absorbancewas measured after 20 min to determine totG. The GSSGcontent was calculated by subtracting the GSH content fromthe totG concentrations. The GSH/totG ratios were alsocalculated.

The determination of lipid peroxidation followed the meth-od proposed by Heath and Packer (1968) and Buege and Aust(1978) with some modifications. The plant material was ho-mogenized in trichloroacetic acid (0.1 %) containing PVPPfollowed by centrifugation at 10,000×g for 5 min. Trichloro-acetic acid containing thiobarbituric acid was added to thesupernatant, which was maintained for 30 min at 95 °C in awater bath. The samples were then rapidly cooled on ice. Analiquot was centrifuged at 10,000×g for 10 min to separate theresidue formed during heating and to lighten the sample. Theabsorbances of the extracts were measured using a spectro-photometer at 535 and 600 nm to determine theMDA content.

Assessment of the environmental variables

During the experimental period, the temperature and humiditywithin the chambers were monitored once a week using athermo-hygrometer, following identical procedures to stan-dardize the measurements. Ozone (O3), nitrogen oxide (NO)and dioxide nitrogen (NO2) were also monitored continuouslyand sequentially within all four chambers by HORIBA ana-lyzers, which permitted filtering efficiency to be checked.Moreover, air quality data were obtained from a monitoringstation that belonged to the Company of Environmental San-itation Technology (CETESB) that was situated in the centerof Cubatão and near CEPEMA. The acquired data helpedcharacterize the level of air pollution in the study area duringthe experimental period.

Statistical analyses

Significant differences in each indicator of the redox statebetween OTC treatments (NFA and FA; factor 1), seasons(winter and summer; factor 2) and the interacting effects ofboth factors were identified by a two-way analysis of variancefollowed by a post-hoc multiple comparison test (Dunn's test).The data obtained during the three winter experiments werecombined and the data for the summer experiments weresimilarly combined to statistically evaluate the effect of factor2. If necessary, an appropriate transformation of the data wasperformed to generate a normal distribution and/or equalvariances.

A principal component analysis (PCA) was applied to theresults of all indicators of the redox potential measured in theplants of T. pulchra exposed to FA and NFA treatments,during all seasonal experiments, after log10 transformation.This analysis permitted to determine the total variability of theredox potential of the plants and to know which indicatorspreponderantly explained such variability.

Multiple linear regression analyses were performed to as-certain whether the redox state in T. pulchra plants exposed toNFA in the OTCs might be predicted by oscillations in mete-orological variables (air temperature and RH) and/or NO2 andO3 concentrations. The data from all six experiments werejointly analyzed with a backward stepwise method using theindicators of the redox state as the dependent variables (fol-lowing an appropriate transformation when necessary) and theenvironmental conditions as the independent variables. Ateach step, the adjustment and the significance of the variableswere evaluated and only those that significantly contributed toexplaining the remaining variation were retained. Only themost explicative model for each indicator (the one with thehighest determination coefficient and R2) was selected andpresented in Table 1.

Results

Environmental conditions during the experimental period

The highest temperature monitored inside the OTP (FA andNFA) occurred during the summer experiments (24 °C onaverage) and the highest RH during the winter experiments(91 % on average), as was expected. The lowest temperaturevalues occurred during the winter experiments (21 °C onaverage) and the lowest RH occurred during the summerexperiments (87 % on average). Measuring campaigns indi-cated that air temperature was, on average, 1.5 °C and 3 °Chigher within the chambers than in the external environmentduring the winter and summer experiments, respectively. TheRH was generally reduced in 5 % within the chambers than in

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the outside environment, throughout the entire experimentalperiod.

The highest average concentrations of pollutants weremeasured inside the OTCs used for the NFA treatment. Aver-age, minimum and maximum NO2 concentrations were 38.9,13.7 and 56.2 μg m3, respectively, during winter experimentsand 33.9, 12.5 and 51.4 μg m3, respectively, during thesummer experiments. Average, minimum and maximum O3

concentrations varied from 12.8, 7.9 and 32.3, μg m3, respec-tively, in the winter to 39.0, 21.8 and 72.4 μg m3, respectively,in the summer. Therefore, NO2 and O3 in the NFA treatmentwere more concentrated during the winter and summer, re-spectively, as was expected. The reduction percentage ofpollutant concentrations in the FA OTCs, compared to theconcentrations in the NFA OTCs, varied from 62 % to 45 %for NO2 and from 50 % to 26 % for O3.

Indicators of redox potential

The results obtained in the winter and summer experimentsperformed in the OTP are presented as box plots. Each boxshows the 25th to 75th percentiles of the dataset of eachtreatment, the medians, the error bars and the outlier values

(●). All of the analyzed indicators varied between both OTCtreatments (FA and NFA) and/or seasonal treatments (winterand summer) (Figs. 3–6).

Leaf MDA was measured in lower amounts in saplingsmaintained inside the NFA chambers during winter experi-ments and in higher levels in the same treatment duringsummer. A significantly lower content of MDAwas observedin samplings from the NFA treatment during winter thanduring summer experiments. No seasonal differences weredetected in plants exposed to FA (Fig. 3).

The activities of CAT and APX (Fig. 4a and b) increasedand the activities SOD and GR decreased (Fig. 4c and d) inplants exposed to the NFA treatment during the winter exper-iments compared to the results obtained for plants in the FAtreatment. Enzymatic activities were similar in the plantsgrown in both NFA and FA chambers in the summer experi-ments (Fig. 4a–d). CAT and APX were less expressive inwinter saplings exposed to both FA and NFA (Fig. 4a andb). Similar results were observed for SOD and GR, but only inplants from the NFA chambers (Fig. 4c and d).

The concentration of reduced (AsA) and oxidized (DHA)forms of AsA were significantly higher in plants exposed toNFA than in plants from the FA treatments in the winter, analteration that was not observed in the summer experiments.Significantly higher AsA and DHA levels were observed inplants from both FA and NFA chambers during the summerexperiments than in plants from the winter experiment (Fig. 5aand b). The total AA leaf concentration (Fig. 5c) was similarin both treatments (FA and NFA) and seasons (winter andsummer). Increased AsA/totAA ratios were observed in sap-lings exposed to NFA as compared to saplings exposed to FA

Table 1 The predictive environmental variables for the fluctuations in theredox potential indicators in Tibouchina pulchra plants exposed to non-filtered air in open-top chambers (data of all six experiments included)

Indicators T RH NO2 O3 R2

(P<0.001)

AsA ni ni − − 0.26

totAA ni ni − − 0.27

Rank(DHA) ni ni − − 0.29

Sqrt(AsA/totAA) ni ni − - 0.22

Ln(GSH) ni ni + + 0.41

Ln(totG) ni ni + + 0.40

Rank(GSSG) − ni + + 0.32

Sqrt(GSH/totG) − ni + + 0.24

Ln(APX) + ni ni + 0.44

Ln(GR) − ni ni − 0.31

Exp(SOD) + ni ni ni 0.26

Rank(CAT) − ni ni ni 0.49

Rank(MDA) ni ni ni + 0.25

AsA reduced ascorbic acid, totAA total ascorbic acid, DHA oxidizedascorbic acid, AsA/totAA ascorbic acid ratio, GSH reduced glutathione,totG total glutathione, GSSG oxidized glutathione, GSH/totG glutathioneratio, APX ascorbate peroxidase, GR glutathione reductase, SOD super-oxide dismutase,CATcatalase,MDAmalondialdehyde, T temperature,RHrelative humidity, NO2 nitrogen dioxide, O3 ozone, R2 determinationcoefficient, ni variable not included in the linear model; (−) significantnegative relationship and (+) significant positive relationship. Whennecessary, the data were linearized by rank, square root (Sqrt), linear(Ln) or exponential (Exp) transformations

FAw NFAw FAs NFAs

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Fig. 3 A box plot of the malondialdehyde (MDA) content in Tibouchinapulchra leaves exposed to filtered air (FA) and non-filtered air (NFA) inopen-top chambers during the winter (w) and summer (s) experiments.Distinct uppercase letters indicate significant differences between the FAand NFA treatments during the identical season.Distinct lowercase lettersindicate significant differences between the seasons in the same treatment(FA or NFA) (P<0.001)

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during the winter experiments. Opposite results were obtainedin experiments from the summer (Fig. 5d).

The concentrations of reduced (GSH), oxidized (GSSG)and total (totG) glutathione were significantly higher in plantsfrom the NFA treatment than plants from the FA treatment inthe winter. The levels of all forms of glutathione were similarin the saplings exposed to both FA and NFA during thesummer experiments (Fig. 6a, b and c). Similar GSH/totGratios were estimated in plants grown under FA and NFA air inboth seasons. However, the ratio estimated for saplings ex-posed to the FA treatment was higher in the summer experi-ments compared to the values calculated in the winter exper-iments (Fig. 6d).

The PCA summarized 40 % of the total variability of thedata on the first two axes (Fig. 7). Glutathione in its total (r=−0.79), reduced (r=−0.72) and oxidized (r=−0.62) forms,AsA in its oxidized (r=0.73), reduced (r=0.60) and total (r=0.51) forms, as well as AsA/totAA ratio (r=−0.64) showed thehighest correlation with axis 1. Oxidized AsA (r=−0.59) andglutathione (−0.62) and AsA/totAA ratio (r=0.70) alsoshowed high correlation with axis 2. The majority of sampling

unities of FA treatment from the winter experiments (▽),located in the positive side of axis 1, were characterized bylower AsA/totAA ratio, lower levels of AsA and GSH andhigh concentrations of DHA. In opposition, the sampling unitsof both chamber treatments (NFA: ▲ and FA: ■) from sum-mer experiments and of NFA treatment from winter experi-ments (○), located in the negative side of axis 1 and positiveside of axis 2, were generally characterized by higher redoxstate of AsA (indicated by AsA/totAA ratio), high levels ofreduced AsA and glutathione (GSH) and low concentrationsof oxidized ascorbic acid (DHA).

The relationship between redox potential and environmentalconditions

The multivariate regression analyses indicated that variationsin the indicators of the redox potential in T. pulchra plantsgrown in the NFA chambers were mainly explained by sig-nificant linear combinations of the oscillations of NO2 and O3

levels and of the meteorological variables included in theanalyses with the exception of the relative air humidity. The

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Fig. 4 The activities of catalase (a), ascorbate peroxidase (b), superoxidedismutase (c) and glutathione reductase (d) in Tibouchina pulchra leavesexposed to filtered air (FA) and non-filtered air (NFA) in open-top cham-bers during the winter (w) and summer (s) experiments. Distinct

uppercase letters indicate significant differences between the FA andNFA treatments during the identical season. Distinct lowercase lettersindicate significant differences between the seasons in the same treatment(FA or NFA) (P<0.001)

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most explicative models were proposed for reduced (r2=0.41)and total (r2=0.40) forms of glutathione, APX (r2=0.44) andCAT (r2=0.49). The least explicative models were proposedfor AsA and SOD (r2=0.26) andMDA (r2=0.25), as shown inTable 1.

The AsA in its reduced, oxidized and total forms and theAsA/totAA ratio were not explained by meteorological vari-ations but by both pollutants (negative relationship). Changesin the levels of reduced and total glutathione were also notexplained by meteorological variations but by both pollutants(positive relationship). The oxidized glutathione and GSH/totG ratio were explained by the temperature reduction andby the increased concentration of NO2 and O3.

The SOD and APX activities increased following increasesin average temperature and ozone levels. Changes in CATactivity in the plants were negatively explained by variationsin temperature only. The GR activity was negatively explainedby variations in temperature and O3 concentrations, and theMDA content was explained by increases in O3

concentrations.

Discussion

The obtained results indicated that the antioxidant responsesin T. pulchra plants varied seasonally and appeared to bestimulated by seasonal variations in air pollutant concentra-tions and/or climatic factors.

The variance analyses, PCA and multivariate analyses alsoindicated distinct redox potentials during the winter and sum-mer experiments. During the winter experiments, when theconcentrations of primary air pollution tended to be higher andmilder temperatures and higher humidity were registered in-side the NFA chambers, the redox potential of T. pulchrasaplings appeared to increase, by investing in the reducedforms of AsA and glutathione, relative to levels of oxidizedforms and/or total levels. In parallel, reducing activities ofSOD, CAT and GR and lower levels of lipid peroxidationwere observed in those plants. However, a similar stimulationof antioxidant defenses was not clearly observed in the plantssubjected to polluted air in the NFA chambers during thesummer experiments, when increases in ozone concentrations

FAw NFAw FAs NFAs

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/totA

A

0,0

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1,0 Bb Aa Aa Bb

a b

c d

Fig. 5 A box plot of the concentrations of reduced (AsA; a), oxidized(DHA; b) and total (totAA; c) ascorbic acid and the AsA/totAA ratios (d)in Tibouchina pulchra leaves exposed to filtered air (FA) and non-filteredair (NFA) in open-top chambers during the winter (w) and summer (s)

experiments. Distinct uppercase letters indicate significant differencesbetween the FA and NFA treatments during the identical season. Distinctlowercase letters indicate significant differences between the seasons inthe same treatment (FA or NFA) (P<0.001)

Environ Sci Pollut Res

and temperature and decreases in RH were registered. Thisresult indicated that more extreme weather conditions ap-peared to mostly affect the redox potential of plants in theNFA treatments more than the air pollutants at the levelsobserved in the region during the summer. In fact, the majorityof the antioxidant responses were significantly more intense inthe plants during the warmer and rainy periods (referred to assummer treatment), even in those exposed to FA, probably inresponse to higher production of ROS caused by morestressing climatic conditions. As pointed out by Pieper et al.(2011), the climatic stress during these periods might haveenhanced due to the confinement of plants inside the OTP.

These seasonally marked stimulations of antioxidant re-sponses in plants exposed to air pollutants, which are typicalof plant species that are tolerant to oxidative stress, were alsoobserved by other authors, including Burkey et al. (2006),Hofer et al. (2008) and Heath et al. (2009). This result mightbe an attempt to achieve a new state of cellular homeostasisfollowing exposure to varying levels of oxidative stress, im-posed by both pollution and/or climate variables, as empha-sized by Apel and Hirt (2004), Shao et al. (2008), De Garaet al. (2010), Miller et al. (2010) and Zachgo et al. (2013).

Multivariate and PCA analyses applied to the data obtainedin the six experiments performed in the NFA chambers, re-gardless of whether they were performed during winter orsummer, emphasized the overall dynamics of the redox po-tential of T. pulchra and the multiple linear interference ofenvironmental factors on it.

Increases in NO2 and O3 caused increases in the redox stateof glutathione, as indicated by the positive relationships(shown in Table 1). However, these oxi-reduction reactionsinvolving glutathione were not significantly influenced bymeteorological oscillations. Therefore, the oxi-reduction reac-tions involving this antioxidant compound appear to be a keymechanism in T. pulchra, which would guarantee their sur-vival in the polluted areas of Cubatão, independent of meteo-rological conditions.

The phytotoxic levels of O3 would be expected duringsunny and warm days in the experimental period based onaspects emphasized by Collet (2012) because of the emissionsof its precursors from anthropogenic sources. O3 is a pollutantthat may cause oxidative injury in native tree species in thisregion. The positive relationship between the accumulation ofMDA and O3 showed that this pollutant might have caused

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Fig. 6 A box plot of the concentrations of reduced (GSH; a), oxidized(GSSG; b) and total (totG; c) glutathione and the GSH/totG ratios (d) inTibouchina pulchra leaves exposed to filtered air (FA) and non-filtered air(NFA) in the open-top chambers during the winter (w) and summer (s)

experiments. Distinct uppercase letters indicate significant differencesbetween the FA and NFA treatments during the identical season. Distinctlowercase letters indicate significant differences between the seasons inthe same treatment (FA or NFA) (P<0.001)

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lipid peroxidation in young T. pulchra grown inside NFAchambers despite increased antioxidant defenses, such as thehigher GSH/totG ratios and in the activity of APX that follow-ed increases in O3 levels. However, the enhanced MDAaccumulation in the leaf occurred in parallel with the de-creased redox potential of AsA in response to enhanced O3

concentrations. This overall negative association between lip-id peroxidation, AsA and O3 could indicate the low capacityof AsA to scavenge ROS formed in the apoplast after O3

uptake, as is generally observed in tolerant plant species thathave efficient detoxification capacity at the apoplast level(Burkey et al. 2003; Feng et al. 2010). Moraes et al. (2004),observed a significant reduction of net photosynthesis, stoma-tal conductance and transpiration in young T. pulchra fumi-gated with high concentrations of O3 in OTP, which reinforcedthe above-mentioned hypothesis. However, this hypothesismust be tested more properly.

Reductions in AsA content are common responses in plantsfumigated with oxidative air pollutants, as observed byFerreira et al. (2012) in I. nil "Scarlet O'Hara" exposed toO3. Increments in lipid peroxidation following O3 exposurehave also been reported in pea, wheat, spinach (Carlsson et al.1996; Rai and Agrawal 2008), lettuce (Calatayud et al. 2002),clover (Francini et al. 2007), radish and brinjal (Tiwari andAgrawal 2011).

Finally, the multivariate analyses also indicated that oscil-lations in the air temperature inside the NFA chambers mainlyaffected the enzymatic activity of T. pulchra, an effect alsoobserved in plants of N. tabacum "Bel W3" (Esposito et al.

2009; Dias et al. 2011), Caesalpinia echinataLam. (Bulbovaset al. 2010) and Ipomea nil "Scarlet O'Hara" (Dafré-Martinelliet al. 2011; Ferreira et al. 2012) grown in sub-tropical sitesaffected predominantly by O3. This meteorological factormight affect the production of ROS during photosynthesis orimpose oxidative stress on the plants directly (Bulbovas et al.2010; De Gara et al. 2010). In brief, based on the review byMiller et al. (2010), the varying production of ROS and itsconsequent influence on the redox state in sub-tropical speciesof plants, such as T. pulchra, might be an overall acclimationresponse to the natural variations of environmental conditions,as characteristically observed during the summer experimentsperformed in the present study.

Conclusion

The antioxidant responses of T. pulchra varied seasonally andappeared to be stimulated by variations in air pollutant con-centrations and/or air temperature. Glutathione and APX wereprimarily responsible for increasing the tolerance of T. pulchrato oxidative stress originating from air pollution in the region.Variations in air temperature mainly affected the enzymaticactivity of T. pulchra. However, the content of MDA, whichwas an indicator of oxidative damage to the plasma mem-branes in the plants, also increased in response to increasingozone concentration. This result appeared to indicate that thepro-oxidant/antioxidant balance might not have been reached.

AsA

totAA

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MDA APX GR CAT SOD AsA TotAA DHA AsA/totAA GSH totG GSSG GSH/to tGAxis 1 0.01 0.27 0.01 0.17 0.13 0.60 0.51 0.73 -0.64 -0.72 -0.79 -0.6 2 -0.03Axis 2 0.19 0.02 -0.15 -0.11 0.08 -0.28 -0.08 -0.59 0.70 -0.38 -0.55 -0.62 0.32

Fig. 7 Principal componentsanalysis (PCA) of the indicators ofthe redox potential (MDAmalondialdehyde, APX ascorbateperoxidase, GR glutathionereductase, CATcatalase, SODsuperoxide dismutase, AsAreduced ascorbic acid, TotAA totalascorbic acid, DHA oxidizedascorbic acid, AsA/totAA ascorbicacid ratio, GSH reducedglutathione, totG total glutathione,GSSGoxidized glutathione,GSH/totGglutathione ratio) in leaves ofT. pulchra exposed to filtered (FA)and non-filtered (NFA) air inopen-top chambers during thesummer and winter experiments.Filled triangleNFA/summer,filled square FA/summer, emptycircleNFA/winter, empty invertedtriangle FA/winter. The tableshows the correlation coefficientsof each indicator to axes 1 and 2

Environ Sci Pollut Res

Acknowledgements The authors gratefully acknowledge Petrobrás(Petróleo Brasileiro) for financial support, FAPESP (São Paulo ResearchFoundation: grant 2008/58682-1) for offering a Ph.D. scholarship to thefirst author, CEPEMA (Environmental Center of Training and Researchof Polytechnic School from University of São Paulo) for permitting theexperimental installation on its property, CESP (Energy Company of SãoPaulo) for donating the T. pulchra saplings, CETESB (Company ofEnvironmental Sanitation Technology) and EMAE (Metropolitan Enter-prise of Water and Energy) for furnishing meteorological data.

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