Environ Sci Pollut Res (2014) 21:54845495

DOI 10.1007/s11356-013-2453-8

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 / Published online: 10 January 2014
# Springer-Verlag Berlin Heidelberg 2014

Abstract The present study aimed to establish the seasonal

variations in the redox potential ranges of young Tibouchina
pulchra plants growing in the Cubato region (SE Brazil)
under varying levels of oxidative stress caused by air pollutants. The plants were exposed to filtered air (FA) and nonfiltered air (NFA) in open-top chambers installed next to an oil
refinery in Cubato during six exposure periods of 90 days
each, which included the winter and summer seasons. After
exposure, several analyses were performed, including the
foliar concentrations of ascorbic acid and glutathione in its
reduced (AsA and GSH), total (totAA and totG) and oxidized
forms (DHA and GSSG); their ratios (AsA/totAA and GSH/
totG); the enzymatic activities of superoxide dismutase
(SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR); and the content of malondialdehyde
(MDA). The range of antioxidant responses in T. pulchra
plants varied seasonally and was stimulated by high or low
air pollutant concentrations and/or air temperatures. Glutathione and APX were primarily responsible for increasing plant
tolerance to oxidative stress originating from air pollution in
the region. The high or low air temperatures mainly affected
enzymatic activity. The content of MDA increased in response
to increasing ozone concentration, thus indicating that the prooxidant/antioxidant balance may not have been reached.

Keywords Antioxidants . Air pollution . Air temperature .

Oil refinery . Redox potential . Open-top chambers

Responsible editor: Philippe Garrigues

M. P. Esposito (*) : M. Domingos
Instituto de Botnica, Ncleo de Pesquisa em Ecologia,
PO Box 68041, 04045-972 So Paulo, SP, Brazil
e-mail: biompe@yahoo.com.br

Introduction
Air pollution has become a very important risk factor to the
environment, especially in urban and industrialized areas.
Increasing emissions of primary pollutants in such areas have
induced the accumulation of secondary pollutants in the troposphere, such as ozone (O3). In fact, new hot spots of O3 are
arising in Asia, Central Africa and South America, including
Brazil (Emberson 2003; Cape 2008; Noyes et al. 2009;
Orlando et al. 2010). Even in concentrations below the safety
limits established by the legislation in many countries,
primary and secondary air pollutants can cause harmful effects
to human health and vegetation (Moura et al. 2008; Jasinski
et al. 2011).
Climatic factors, besides affecting directly the formation,
concentration and dispersion of air pollutants, may interfere
on plant responses to pollution stress. Many studies have
highlighted that intensity of leaf injury caused by O3 on plants
of Nicotiana tabacum "Bel-W3", for instance, may be modulated by the action of multiple climatic factors, which regulate
the plant's physiology and metabolism. Such effects on tobacco plants may occur both in the northern hemisphere, in
European countries (Koppel and Sild 1995; Finnan et al.
1996; Antonielli et al. 1997; Peuelas et al. 1999; Yuska
et al. 2003; Klumpp et al. 2006) as in the southern hemisphere,
in So Paulo, SE Brazil (SantAnna et al. 2008; Esposito et al.
2009; Souza and Pagliuso 2009; Dias et al. 2011). Similar
responses were observed in plants of Ipomoea nil "Scarlet
O'Hara" exposed to chronic levels of O3 in So Paulo city
(Dafr-Martinelli et al. 2011; Ferreira et al. 2012).
Gaseous pollutants, after entering plants through the stomata, quickly react with water and intensify the formation of
reactive oxygen species (ROS) at the cell wall interface, an
effect that may be enhanced by other environmental stress
factors, such as extremes of air temperature and relative humidity (RH) (Bray et al. 2000; Mittler 2002). Oxidative

Environ Sci Pollut Res (2014) 21:54845495

disturbance caused by ROS on the lipids and proteins of the

plasma membranes produces a burst of other free radicals and
reactive 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 nonenzymatic antioxidants that may protect them against oxidative damage and control the level of ROS effects. Superoxide
dismutase (SOD), catalase (CAT), ascorbate peroxidase
(APX) and glutathione reductase (GR) are enzymatic antioxidants, and glutathione, carotenoids and ascorbic acid (AsA)
are non-enzymatic components (Caregnato et al. 2008). Most
notably, the interaction between ROS production and its
resulting scavenging mechanisms determines the plant's redox
potential and depends on the plant's physiological status and
development and biochemical stimuli (Mittler 2002). However, only the total concentrations of antioxidants such as ascorbic acid (totAA) and glutathione (totG) do not indicate the
redox potential of plants or even their performance as
bioindicators of air pollutants because the antioxidant characteristic of these substances is given especially by their reduced
forms (AsA and GSH, respectively). The availability of these
substances in the cells depends essentially on the efficiency of
enzymes that regenerate their oxidized forms (Burkey et al.
2006). Establishing the redox potential, by measuring lipid
peroxidation and activity of enzymatic antioxidants and by
estimating concentration ratios (AsA/totAA and GSH/totG),
is therefore a measurement of the appropriate induction of the
acclimation processes in plants to tolerate oxidative stress in
polluted areas (Apel and Hirt 2004; Shao et al. 2008; Potters
et al. 2010; Zachgo et al. 2013).
Tibouchina pulchra (Cham.) Cogn., a native tree species
from the Atlantic rain forest in southeastern Brazil, may
theoretically tolerate the oxidative stress imposed by air pollutants as the levels observed in the industrial pole region
settled in the city of Cubato (SE Brazil). This tree species
predominates in regions of the Atlantic forest in the vicinity of
the industrial pole (Leito-Filho et al. 1993), which remain
currently affected by significant amounts of carbon monoxide
(CO), hydrocarbons (CH), nitrogen oxides (NOx), sulfur oxides (SOx) and particulate matter (PM) (CETESB 2011). The
tolerance of T. pulchra to pollutants may result from a number
of physiological, structural and metabolic defense characteristics, as shown in biomonitoring studies performed in the
region during the 1990s. The species accumulates high levels
of 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 injury
on T. pulchra leaves was only detected after an acute exposure
to a 6,038 ppb/h cumulative dose of ozone (AOT40) 25 days
from the beginning of the experiment using open-top

5485

chambers (OTP) (Furlan et al. 2008). However, parallel exposure experiments have been performed around the industrial
pole of Cubato employing T. pulchra as a biomonitor.
Moraes et al. (2000) observed a reduction of the total AsA
concentration in the leaves, and Klumpp et al. (2000) observed an increase in peroxidase activity in plants grown near
fertilizer, cement and steel industries. Based on these results, it
is possible to assume that T. pulchra has an efficient redox
potential, which would enable it to tolerate any change in air
quality in the region, as occurred around an important oil
refinery at the industrial pole because of the exchange between its energy and steam production system (from boilers
powered by oil to a thermal power plant powered by natural
gas). Therefore, the present study, which was proposed to
elucidate this hypothesis, aimed to establish the redox potential range of T. pulchra, which grew in the region under
varying levels of oxidative stress caused by seasonal variations in air pollutant concentrations and/or climatic factors, by
measuring multiple indicators of the antioxidant defense system and oxidative injury. For this study, saplings of T. pulchra
were grown in OTP installed next to an oil refinery with
filtered (FA) and non-filtered air (NFA) and varying meteorological conditions. Such a semi-controlled experimental design permitted the authors to determine the interacting effects
of both sets of environmental variables that might expose
plants 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 the
municipality of Cubato, which is located on the Atlantic
coast in So Paulo state, southeastern Brazil (23452355
S, 46154630W) at the base of the Serra do Mar mountain
range (Fig. 1). Cubato is approximately 16 km from the city
of Santos, which has an important port that facilitates the trade
of industrial products. Development of the industrial pole in
Cubato occurred from 1950 to 1970 and is currently composed of more than 20 industrial sources, including petrochemical, steel and chemical companies.
The OTC were installed at the Environmental Center of
Training and Research of Polytechnic School from the University of So Paulo (CEPEMA) next to petrochemical industries and railways with intense diesel truck traffic and light
vehicles powered by gasoline or ethanol (Fig. 1). The wear of
tires and brakes and the formation of secondary aerosols from
gases emitted by vehicles are additional sources of air contamination in the region. Therefore, this area was highly
affected by primary air pollutants, such as nitrogen and sulfur
oxides (NOx and SOx), particulate matter (PM10) with varied
composition and ozone (O3) (CETESB 2011).

Environ Sci Pollut Res (2014) 21:54845495

disturbance caused by ROS on the lipids and proteins of the

plasma membranes produces a burst of other free radicals and
reactive 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 nonenzymatic antioxidants that may protect them against oxidative damage and control the level of ROS effects. Superoxide
dismutase (SOD), catalase (CAT), ascorbate peroxidase
(APX) and glutathione reductase (GR) are enzymatic antioxidants, and glutathione, carotenoids and ascorbic acid (AsA)
are non-enzymatic components (Caregnato et al. 2008). Most
notably, the interaction between ROS production and its
resulting scavenging mechanisms determines the plant's redox
potential and depends on the plant's physiological status and
development and biochemical stimuli (Mittler 2002). However, only the total concentrations of antioxidants such as ascorbic acid (totAA) and glutathione (totG) do not indicate the
redox potential of plants or even their performance as
bioindicators of air pollutants because the antioxidant characteristic of these substances is given especially by their reduced
forms (AsA and GSH, respectively). The availability of these
substances in the cells depends essentially on the efficiency of
enzymes that regenerate their oxidized forms (Burkey et al.
2006). Establishing the redox potential, by measuring lipid
peroxidation and activity of enzymatic antioxidants and by
estimating concentration ratios (AsA/totAA and GSH/totG),
is therefore a measurement of the appropriate induction of the
acclimation processes in plants to tolerate oxidative stress in
polluted areas (Apel and Hirt 2004; Shao et al. 2008; Potters
et al. 2010; Zachgo et al. 2013).
Tibouchina pulchra (Cham.) Cogn., a native tree species
from the Atlantic rain forest in southeastern Brazil, may
theoretically tolerate the oxidative stress imposed by air pollutants as the levels observed in the industrial pole region
settled in the city of Cubato (SE Brazil). This tree species
predominates in regions of the Atlantic forest in the vicinity of
the industrial pole (Leito-Filho et al. 1993), which remain
currently affected by significant amounts of carbon monoxide
(CO), hydrocarbons (CH), nitrogen oxides (NOx), sulfur oxides (SOx) and particulate matter (PM) (CETESB 2011). The
tolerance of T. pulchra to pollutants may result from a number
of physiological, structural and metabolic defense characteristics, as shown in biomonitoring studies performed in the
region during the 1990s. The species accumulates high levels
of 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 injury
on T. pulchra leaves was only detected after an acute exposure
to a 6,038 ppb/h cumulative dose of ozone (AOT40) 25 days
from the beginning of the experiment using open-top

5485

chambers (OTP) (Furlan et al. 2008). However, parallel exposure experiments have been performed around the industrial
pole of Cubato employing T. pulchra as a biomonitor.
Moraes et al. (2000) observed a reduction of the total AsA
concentration in the leaves, and Klumpp et al. (2000) observed an increase in peroxidase activity in plants grown near
fertilizer, cement and steel industries. Based on these results, it
is possible to assume that T. pulchra has an efficient redox
potential, which would enable it to tolerate any change in air
quality in the region, as occurred around an important oil
refinery at the industrial pole because of the exchange between its energy and steam production system (from boilers
powered by oil to a thermal power plant powered by natural
gas). Therefore, the present study, which was proposed to
elucidate this hypothesis, aimed to establish the redox potential range of T. pulchra, which grew in the region under
varying levels of oxidative stress caused by seasonal variations in air pollutant concentrations and/or climatic factors, by
measuring multiple indicators of the antioxidant defense system and oxidative injury. For this study, saplings of T. pulchra
were grown in OTP installed next to an oil refinery with
filtered (FA) and non-filtered air (NFA) and varying meteorological conditions. Such a semi-controlled experimental design permitted the authors to determine the interacting effects
of both sets of environmental variables that might expose
plants 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 the
municipality of Cubato, which is located on the Atlantic
coast in So Paulo state, southeastern Brazil (23452355
S, 46154630W) at the base of the Serra do Mar mountain
range (Fig. 1). Cubato is approximately 16 km from the city
of Santos, which has an important port that facilitates the trade
of industrial products. Development of the industrial pole in
Cubato occurred from 1950 to 1970 and is currently composed of more than 20 industrial sources, including petrochemical, steel and chemical companies.
The OTC were installed at the Environmental Center of
Training and Research of Polytechnic School from the University of So Paulo (CEPEMA) next to petrochemical industries and railways with intense diesel truck traffic and light
vehicles powered by gasoline or ethanol (Fig. 1). The wear of
tires and brakes and the formation of secondary aerosols from
gases emitted by vehicles are additional sources of air contamination in the region. Therefore, this area was highly
affected by primary air pollutants, such as nitrogen and sulfur
oxides (NOx and SOx), particulate matter (PM10) with varied
composition and ozone (O3) (CETESB 2011).

5486

Environ Sci Pollut Res (2014) 21:54845495

5
3
6

6
2

Fig. 1 Map of the area of study in Cubato, SE Brazil (23452355S,

46214630W). 1 CEPEMA, exposure site of T. pulchra in open-top
chambers, 2 downtown area, 3 chemical industries, 4 petrochemical
industries, 5 Atlantic Rain Forest, 6 major highways (source: Arcgiz
Online, adapted)

Four OTCs (3.0 m in diameter and 2.2 m high) were used.

The homemade OTCs consisted of stainless steel frames
wrapped in Teflon film, which transmits nearly 100 % of
visible radiation wavelengths, in addition to allowing heat
exchange. The entry and distribution of air within the chambers occurred from the base to the top of the cylinder and was
forced by fans installed in hermetically sealed stainless steel
housing. Two OTCs received NFA (NFA treatment), and the
remaining two OTCs received FA (FA treatment) from filters
that removed thick and fine particles and gases from the air
and a series of chemical beds prepared with activated carbon
and aluminum oxide pellets impregnated with potassium permanganate to filter inorganic and organic gases. The entire
filtering and air flowing system was supplied by Purafil Inc.
The airflow was adjusted in the chambers by manometers. The
filtering 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 acquired
from the same nursery. On average, the saplings were 20 cm
high and had at least six leaves on the main stem. Before each
experiment, the saplings were transplanted into 10-l pots
containing a mixture of standardized substrate that consisted

predominantly of pine bark and fine vermiculite in a ratio of

3:1. The saplings were fertilized weekly with 100 ml of
Hoagland solution and maintained for 1 month inside a greenhouse under FA and ideal meteorological conditions of temperature, RH and photosynthetic active radiation (PAR) (T=
20 C, RH=80 % and PAR=443 mol m2 s1) at the Instituto
de Botnica in So Paulo.
Exposure of T. pulchra to the NFA and FA treatments
inside the OTCs followed the model proposed by Arndt and
Schweizer (1991). In each chamber, the potted plants were
kept in boxes containing water and covered by rigid plastic
plates. This apparatus was covered by a shadow screen (50 %
brightness reduction). Strings inserted into the base of the pots
remained immersed in the water stored in the boxes to ensure a
suitable 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 (AprilJune 2011, July
September 2011 and AprilJune 2012) and three during rainy
and hotter periods, covering spring and summer seasons (November 2010January 2011, OctoberDecember 2011 and
JanuaryMarch 2012). With the purpose of presenting the
results, two main treatments were proposed, generally referred
to as winter and summer experiments, respectively. Results
obtained during less rainy and colder periods were joined in
the first treatment, and those obtained during rainy and hotter
periods were grouped in the second treatment.
Figure 2 shows the monthly averages, maximum and minimum of temperature and humidity and monthly amounts of
rainfall in the region of Cubato during all the experimental
period. As expected, the average, maximum and minimum
values of temperature and accumulated rainfall were higher
during spring and summer months (October to March) and
lower during autumn and winter months (April to September).
Higher amounts of rainfall were observed during the rainy
period of 20102011. Values of RH (average, maximum and
minimum) 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 of
T. pulchra per OTC. A subgroup of four plants were collected
from each OTC after 45 days of exposure, and the remaining
four plants were collected at the end of the exposure period
(90 days) for analyses of indicators of the redox state and
oxidative injury.
Analyses of the redox state and oxidative injury indicators
The redox state of the plants from all six OTC experiments
was determined by analyzing the CAT activity, SOD, APX
and GR and measuring the concentrations of reduced AsA,
dehydroascorbic acid (DHA), total ascorbic acid (totAA=
AsA+DHA), reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (totG=GSH+GSSG). Lipid

Environ Sci Pollut Res (2014) 21:54845495

5487

Fig. 2 Monthly maximum,

average and minimum values of
temperature (T) and relative
humidity (RH) and amounts of
rainfall in the region of Cubato,
during the experimental period

peroxidation was also analyzed and was measured by the

accumulation of malondialdehyde acid (MDA), which is an
oxidative injury indicator.
These indicators were analyzed in a mixture of leaves from
each plant, mainly by those inserted into the second and third
nodes. A total of 192 leaf samples were analyzed for each
indicator. Analytical replicates were performed for CAT,
APX, GR, MDA and glutathione.
Portions of fresh leaves (0.35 g) were homogenized with
potassium phosphate buffer (50 mM pH 7.0), triton (0.05 %),
polyvinyl polypyrrolidone (PVPP; 10 %) and AsA (1 mM).
The mixture was centrifuged at 10,000g at 2 C for 10 min,
and the supernatant was stored at 80 C to determine the
SOD, APX and CAT activities.
The SOD activity was determined according to a slightly
modified method described by Reddy et al. (2004) by measuring the ability of the enzyme to inhibit the photochemical
reduction of nitroblue tetrazolium (NBT). A reaction mixture
was prepared by adding potassium phosphate buffer (100 mM
pH 7.0), ethylenediamine tetraacetic acid (EDTA; 0.4 M),
methionine (1 mM), leaf extract, NBT (5 mM) and riboflavin
(1 mM). The reaction was initiated by placing one fraction of
this mixture under a fluorescence lamp (80 W) and the other
fraction in the darkness for 30 min. A second similar mixture
without the leaf extract was prepared and placed under illuminated and non-illuminated conditions to serve as a blank.
The absorbance in the mixtures was measured to determine
the amount of enzyme required to inhibit 50 % of the reduction of NBT.
The APX activity was assayed according to the method of
Reddy et al. (2004) with a few modifications. The activity was
determined at 30 C in a reaction mixture with potassium
phosphate buffer (100 mM pH 7.0), EDTA (1 mM), AsA
(5 mM), hydrogen peroxide (2 mM) and the abovementioned thawed supernatant. The oxidation of AsA was
measured by the decrease in absorbance (A) at 290 nm for
2 min using a UVVis spectrophotometer. APX activity was
defined as the change in the absorbance at two different times
in the linear region of the curve.

CAT activity was determined using spectrophotometry as

described by Kraus et al. (1995) with some modifications
proposed by Azevedo et al. (1998). At 25 C, the abovementioned thawed supernatant was added to a reaction mixture of 1 ml potassium phosphate buffer (100 mM pH 7.5) and
hydrogen peroxide (1 mM) that was prepared immediately
before use. The activity was determined by following the
decomposition of H2O2 per minute through changes in absorbance 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 1.4dithiothreitol (DTT; 5 mM) according to the method of Reddy
et al. (2004). The homogenate was centrifuged at 10,000g at
2 C for 10 min, and the supernatant was frozen at 80 C for
subsequent analyses. Activity was determined at 30 C in a
reaction mixture with potassium phosphate buffer (100 mM
pH 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, and
this molecule was oxidized by DTNB. The product of this
reaction was measured by the increase in absorbance (A) at
412 nm for 20 s using a UVVis spectrophotometer. The GR
activity was defined as the change in the absorbance at two
different times in the linear region of the curve.
AsA and totAA were analyzed using the chromatographic
method described by Lopez et al. (2005) and an HPLC
(Metrohm) connected to a UVVis detector. A 415 mm
(5 g) Prontosil column (C18) was used. The mobile phase
used was water brought to pH 2.3 with phosphoric acid; the
flow rate used was 1.0 ml min1; and the detection wavelength
used was 245 nm. Fresh portions of leaves (0.25 g) were
homogenized with metaphosphoric acid (6 %) and EDTANa2 (0.5 mM). The mixture was centrifuged at 10,000g at
2 C for 10 min. The supernatant was filtered through a paper
filter (Whatman no. 41), and the filtrate was diluted with
water. To determine the AsA, a fraction of this extract was
filtered through a 0.45-m filter (Millipore), and this filtrate

5488

was then injected into the chromatographic system. To determine 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 was
stopped by the addition of phosphoric acid (2 M). The mixture
was then filtered through a 0.45-m filter, and the filtrate was
injected into the chromatographic system. DHA was deduced
from the difference between totAA and AsA and the AsA/
totAA ratios were calculated.
The GSH and GSSG content were determined according to
the method described by Israr et al. (2006). Samples of leaves
(1.0 g), frozen at 80 C, were homogenized with
sulfosalicylic acid (0.1 %) and centrifuged at 12,000g at
2 C for 20 min. A mixture of phosphate buffer (100 mM
pH 7.0), EDTA (0.5 mM) and DTNB (3 mM) was added to an
aliquot of the supernatant. After 5 min, the absorbance was
measured at 412 nm using a UVVis spectrophotometer
(Shimadzu) to determine GSH content. NADPH (0.4 mM)
and GR were then added to the mixture, and the absorbance
was measured after 20 min to determine totG. The GSSG
content was calculated by subtracting the GSH content from
the totG concentrations. The GSH/totG ratios were also
calculated.
The determination of lipid peroxidation followed the method proposed by Heath and Packer (1968) and Buege and Aust
(1978) with some modifications. The plant material was homogenized in trichloroacetic acid (0.1 %) containing PVPP
followed by centrifugation at 10,000g for 5 min. Trichloroacetic acid containing thiobarbituric acid was added to the
supernatant, which was maintained for 30 min at 95 C in a
water bath. The samples were then rapidly cooled on ice. An
aliquot was centrifuged at 10,000g for 10 min to separate the
residue formed during heating and to lighten the sample. The
absorbances of the extracts were measured using a spectrophotometer at 535 and 600 nm to determine the MDA content.

Environ Sci Pollut Res (2014) 21:54845495

Statistical analyses
Significant differences in each indicator of the redox state
between OTC treatments (NFA and FA; factor 1), seasons
(winter and summer; factor 2) and the interacting effects of
both factors were identified by a two-way analysis of variance
followed by a post-hoc multiple comparison test (Dunn's test).
The data obtained during the three winter experiments were
combined and the data for the summer experiments were
similarly combined to statistically evaluate the effect of factor
2. If necessary, an appropriate transformation of the data was
performed to generate a normal distribution and/or equal
variances.
A principal component analysis (PCA) was applied to the
results of all indicators of the redox potential measured in the
plants 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 the
redox potential of the plants and to know which indicators
preponderantly explained such variability.
Multiple linear regression analyses were performed to ascertain whether the redox state in T. pulchra plants exposed to
NFA in the OTCs might be predicted by oscillations in meteorological variables (air temperature and RH) and/or NO2 and
O3 concentrations. The data from all six experiments were
jointly analyzed with a backward stepwise method using the
indicators of the redox state as the dependent variables (following an appropriate transformation when necessary) and the
environmental conditions as the independent variables. At
each step, the adjustment and the significance of the variables
were evaluated and only those that significantly contributed to
explaining the remaining variation were retained. Only the
most explicative model for each indicator (the one with the
highest determination coefficient and R2) was selected and
presented in Table 1.

Assessment of the environmental variables

Results

During the experimental period, the temperature and humidity

within the chambers were monitored once a week using a
thermo-hygrometer, following identical procedures to standardize the measurements. Ozone (O3), nitrogen oxide (NO)
and dioxide nitrogen (NO2) were also monitored continuously
and sequentially within all four chambers by HORIBA analyzers, which permitted filtering efficiency to be checked.
Moreover, air quality data were obtained from a monitoring
station that belonged to the Company of Environmental Sanitation Technology (CETESB) that was situated in the center
of Cubato and near CEPEMA. The acquired data helped
characterize the level of air pollution in the study area during
the experimental period.

Environmental conditions during the experimental period

The highest temperature monitored inside the OTP (FA and
NFA) occurred during the summer experiments (24 C on
average) and the highest RH during the winter experiments
(91 % on average), as was expected. The lowest temperature
values occurred during the winter experiments (21 C on
average) and the lowest RH occurred during the summer
experiments (87 % on average). Measuring campaigns indicated that air temperature was, on average, 1.5 C and 3 C
higher within the chambers than in the external environment
during the winter and summer experiments, respectively. The
RH was generally reduced in 5 % within the chambers than in

Environ Sci Pollut Res (2014) 21:54845495

5489

Indicators

RH

NO2

O3

R2
(P<0.001)

AsA
totAA
Rank(DHA)
Sqrt(AsA/totAA)
Ln(GSH)
Ln(totG)
Rank(GSSG)
Sqrt(GSH/totG)
Ln(APX)
Ln(GR)
Exp(SOD)
Rank(CAT)
Rank(MDA)

ni
ni
ni
ni
ni
ni

ni

ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni

+
+
+
+
ni
ni
ni
ni
ni

+
+
+
+
+

ni
ni
+

0.26
0.27
0.29
0.22
0.41
0.40
0.32
0.24
0.44
0.31
0.26
0.49
0.25

AsA reduced ascorbic acid, totAA total ascorbic acid, DHA oxidized
ascorbic acid, AsA/totAA ascorbic acid ratio, GSH reduced glutathione,
totG total glutathione, GSSG oxidized glutathione, GSH/totG glutathione
ratio, APX ascorbate peroxidase, GR glutathione reductase, SOD superoxide dismutase, CATcatalase, MDA malondialdehyde, T temperature, RH
relative humidity, NO2 nitrogen dioxide, O3 ozone, R2 determination
coefficient, ni variable not included in the linear model; () significant
negative relationship and (+) significant positive relationship. When
necessary, the data were linearized by rank, square root (Sqrt), linear
(Ln) or exponential (Exp) transformations

the outside environment, throughout the entire experimental

period.
The highest average concentrations of pollutants were
measured inside the OTCs used for the NFA treatment. Average, minimum and maximum NO2 concentrations were 38.9,
13.7 and 56.2 g m3, respectively, during winter experiments
and 33.9, 12.5 and 51.4 g m3, respectively, during the
summer experiments. Average, minimum and maximum O3
concentrations varied from 12.8, 7.9 and 32.3, g m3, respectively, in the winter to 39.0, 21.8 and 72.4 g m3, respectively,
in the summer. Therefore, NO2 and O3 in the NFA treatment
were more concentrated during the winter and summer, respectively, as was expected. The reduction percentage of
pollutant concentrations in the FA OTCs, compared to the
concentrations in the NFA OTCs, varied from 62 % to 45 %
for NO2 and from 50 % to 26 % for O3.

(). All of the analyzed indicators varied between both OTC

treatments (FA and NFA) and/or seasonal treatments (winter
and summer) (Figs. 36).
Leaf MDA was measured in lower amounts in saplings
maintained inside the NFA chambers during winter experiments and in higher levels in the same treatment during
summer. A significantly lower content of MDA was observed
in samplings from the NFA treatment during winter than
during summer experiments. No seasonal differences were
detected in plants exposed to FA (Fig. 3).
The activities of CAT and APX (Fig. 4a and b) increased
and the activities SOD and GR decreased (Fig. 4c and d) in
plants exposed to the NFA treatment during the winter experiments compared to the results obtained for plants in the FA
treatment. Enzymatic activities were similar in the plants
grown in both NFA and FA chambers in the summer experiments (Fig. 4ad). CAT and APX were less expressive in
winter saplings exposed to both FA and NFA (Fig. 4a and
b). Similar results were observed for SOD and GR, but only in
plants 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 to
NFA than in plants from the FA treatments in the winter, an
alteration that was not observed in the summer experiments.
Significantly higher AsA and DHA levels were observed in
plants from both FA and NFA chambers during the summer
experiments than in plants from the winter experiment (Fig. 5a
and b). The total AA leaf concentration (Fig. 5c) was similar
in both treatments (FA and NFA) and seasons (winter and
summer). Increased AsA/totAA ratios were observed in saplings exposed to NFA as compared to saplings exposed to FA

50

Aa

The results obtained in the winter and summer experiments

performed in the OTP are presented as box plots. Each box
shows the 25th to 75th percentiles of the dataset of each
treatment, the medians, the error bars and the outlier values

Aa

FAs

NFAs

Bb

30

20

10

0
FAw

Indicators of redox potential

Ba

40
MDA (mg-1 fw)

Table 1 The predictive environmental variables for the fluctuations in the

redox potential indicators in Tibouchina pulchra plants exposed to nonfiltered air in open-top chambers (data of all six experiments included)

NFAw

Fig. 3 A box plot of the malondialdehyde (MDA) content in Tibouchina

pulchra leaves exposed to filtered air (FA) and non-filtered air (NFA) in
open-top chambers during the winter (w) and summer (s) experiments.
Distinct uppercase letters indicate significant differences between the FA
and NFA treatments during the identical season. Distinct lowercase letters
indicate significant differences between the seasons in the same treatment
(FA or NFA) (P<0.001)

5490

Environ Sci Pollut Res (2014) 21:54845495

b
50

Aa

180

Ba

40

30

APX (A min1 g-1 dw)

CAT (A min-1 g-1 dw)

160

Bb
Ab

20

10

Bb

Aa

Aa

FAs

NFAs

Ab

140
120
100
80
60
40
20

FAw
1,4

NFAs

FAw

Aa
Aa

1,2
SOD (unit g-1 dw)

FAs

Bb

1,0

NFAw

50
40

Aa

0,8
0,6
0,4

GR (A min-1 g-1 dw)

NFAw

30

Aa

Aa

Aa

FAs

NFAs

20

Bb
10
0

0,2
0,0

FAw

NFAw

FAs

NFAs

FAw

NFAw

Fig. 4 The activities of catalase (a), ascorbate peroxidase (b), superoxide

dismutase (c) and glutathione reductase (d) in Tibouchina pulchra leaves
exposed to filtered air (FA) and non-filtered air (NFA) in open-top chambers during the winter (w) and summer (s) experiments. Distinct

uppercase letters indicate significant differences between the FA and

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

during the winter experiments. Opposite results were obtained

in experiments from the summer (Fig. 5d).
The concentrations of reduced (GSH), oxidized (GSSG)
and total (totG) glutathione were significantly higher in plants
from the NFA treatment than plants from the FA treatment in
the winter. The levels of all forms of glutathione were similar
in the saplings exposed to both FA and NFA during the
summer experiments (Fig. 6a, b and c). Similar GSH/totG
ratios were estimated in plants grown under FA and NFA air in
both seasons. However, the ratio estimated for saplings exposed to the FA treatment was higher in the summer experiments compared to the values calculated in the winter experiments (Fig. 6d).
The PCA summarized 40 % of the total variability of the
data 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 the
highest correlation with axis 1. Oxidized AsA (r=0.59) and
glutathione (0.62) and AsA/totAA ratio (r= 0.70) also
showed 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 by
lower AsA/totAA ratio, lower levels of AsA and GSH and
high concentrations of DHA. In opposition, the sampling units
of both chamber treatments (NFA: and FA: ) from summer experiments and of NFA treatment from winter experiments (), located in the negative side of axis 1 and positive
side of axis 2, were generally characterized by higher redox
state of AsA (indicated by AsA/totAA ratio), high levels of
reduced AsA and glutathione (GSH) and low concentrations
of oxidized ascorbic acid (DHA).
The relationship between redox potential and environmental
conditions
The multivariate regression analyses indicated that variations
in the indicators of the redox potential in T. pulchra plants
grown in the NFA chambers were mainly explained by significant linear combinations of the oscillations of NO2 and O3
levels and of the meteorological variables included in the
analyses with the exception of the relative air humidity. The

Environ Sci Pollut Res (2014) 21:54845495

5491

b
25

25
20

Aa
15

Aa

Aa

Bb
10

DHA (mg g-1 dw)

AsA (mg g-1 dw)

20

15
10

Aa
Aa

Bb

Aa

5
0

0
FAw

NFAw

FAs

NFAs

FAw

d
Aa

25

1,0

Aa
20

15

Bb

Aa

FAw

NFAw

FAs

NFAs

Aa

Bb

FAs

NFAs

0,8

Aa
Aa
AsA/totAA

totAA (mg g-1 dw)

NFAw

10

0,6

0,4

0,2

0,0

FAw

NFAw

FAs

NFAs

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-filtered
air (NFA) in open-top chambers during the winter (w) and summer (s)

experiments. Distinct uppercase letters indicate significant differences

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

most explicative models were proposed for reduced (r2 =0.41)

and total (r2 =0.40) forms of glutathione, APX (r2 =0.44) and
CAT (r2 =0.49). The least explicative models were proposed
for AsA and SOD (r2 =0.26) and MDA (r2 =0.25), as shown in
Table 1.
The AsA in its reduced, oxidized and total forms and the
AsA/totAA ratio were not explained by meteorological variations but by both pollutants (negative relationship). Changes
in the levels of reduced and total glutathione were also not
explained by meteorological variations but by both pollutants
(positive relationship). The oxidized glutathione and GSH/
totG ratio were explained by the temperature reduction and
by the increased concentration of NO2 and O3.
The SOD and APX activities increased following increases
in average temperature and ozone levels. Changes in CAT
activity in the plants were negatively explained by variations
in temperature only. The GR activity was negatively explained
by variations in temperature and O3 concentrations, and the
MDA content was explained by increases in O 3
concentrations.

Discussion
The obtained results indicated that the antioxidant responses
in T. pulchra plants varied seasonally and appeared to be
stimulated by seasonal variations in air pollutant concentrations and/or climatic factors.
The variance analyses, PCA and multivariate analyses also
indicated distinct redox potentials during the winter and summer experiments. During the winter experiments, when the
concentrations of primary air pollution tended to be higher and
milder temperatures and higher humidity were registered inside the NFA chambers, the redox potential of T. pulchra
saplings appeared to increase, by investing in the reduced
forms of AsA and glutathione, relative to levels of oxidized
forms and/or total levels. In parallel, reducing activities of
SOD, CAT and GR and lower levels of lipid peroxidation
were observed in those plants. However, a similar stimulation
of antioxidant defenses was not clearly observed in the plants
subjected to polluted air in the NFA chambers during the
summer experiments, when increases in ozone concentrations

5492

Environ Sci Pollut Res (2014) 21:54845495

b
25

25

20

20

15

Aa
10

Aa
Aa

Bb

GSSG (mol g-1 dw)

GSH (mol g-1 dw)

Aa

10

Aa

Aa

Bb

0
FAw

NFAw

FAs

NFAs

FAw

NFAw

FAs

NFAs

Ab

Aa

Aa

Aa

FAw

NFAw

FAs

NFAs

d
25

1,0

Aa

0,9

20

Aa

15
10

0,8

Aa
GSH/totG

totG (mol g-1 dw)

15

Bb

0,7
0,6
0,5

5
0,4
0

0,3
0,2
FAw

NFAw

FAs

NFAs

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) in
Tibouchina 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 differences

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

and temperature and decreases in RH were registered. This

result indicated that more extreme weather conditions appeared to mostly affect the redox potential of plants in the
NFA treatments more than the air pollutants at the levels
observed in the region during the summer. In fact, the majority
of the antioxidant responses were significantly more intense in
the plants during the warmer and rainy periods (referred to as
summer treatment), even in those exposed to FA, probably in
response to higher production of ROS caused by more
stressing climatic conditions. As pointed out by Pieper et al.
(2011), the climatic stress during these periods might have
enhanced due to the confinement of plants inside the OTP.
These seasonally marked stimulations of antioxidant responses in plants exposed to air pollutants, which are typical
of plant species that are tolerant to oxidative stress, were also
observed by other authors, including Burkey et al. (2006),
Hofer et al. (2008) and Heath et al. (2009). This result might
be an attempt to achieve a new state of cellular homeostasis
following exposure to varying levels of oxidative stress, imposed by both pollution and/or climate variables, as emphasized by Apel and Hirt (2004), Shao et al. (2008), De Gara
et al. (2010), Miller et al. (2010) and Zachgo et al. (2013).

Multivariate and PCA analyses applied to the data obtained

in the six experiments performed in the NFA chambers, regardless of whether they were performed during winter or
summer, emphasized the overall dynamics of the redox potential of T. pulchra and the multiple linear interference of
environmental factors on it.
Increases in NO2 and O3 caused increases in the redox state
of glutathione, as indicated by the positive relationships
(shown in Table 1). However, these oxi-reduction reactions
involving glutathione were not significantly influenced by
meteorological oscillations. Therefore, the oxi-reduction reactions involving this antioxidant compound appear to be a key
mechanism in T. pulchra, which would guarantee their survival in the polluted areas of Cubato, independent of meteorological conditions.
The phytotoxic levels of O3 would be expected during
sunny and warm days in the experimental period based on
aspects emphasized by Collet (2012) because of the emissions
of its precursors from anthropogenic sources. O3 is a pollutant
that may cause oxidative injury in native tree species in this
region. The positive relationship between the accumulation of
MDA and O3 showed that this pollutant might have caused

Fig. 7 Principal components

analysis (PCA) of the indicators of
the redox potential (MDA
malondialdehyde, APX ascorbate
peroxidase, GR glutathione
reductase, CAT catalase, SOD
superoxide dismutase, AsA
reduced ascorbic acid, TotAA total
ascorbic acid, DHA oxidized
ascorbic acid, AsA/totAA ascorbic
acid ratio, GSH reduced
glutathione, totG total glutathione,
GSSG oxidized glutathione, GSH/
totG glutathione ratio) in leaves of
T. pulchra exposed to filtered (FA)
and non-filtered (NFA) air in
open-top chambers during the
summer and winter experiments.
Filled triangle NFA/summer,
filled square FA/summer, empty
circle NFA/winter, empty inverted
triangle FA/winter. The table
shows the correlation coefficients
of each indicator to axes 1 and 2

5493
Axis 2 = 17%

Environ Sci Pollut Res (2014) 21:54845495

AsA/totA

AsA

GSH/totG
1

-6

MDA
SOD
APX

-2
CAT
-1

Axis 1 = 23%

2 totAA

GR

GSH
totG
GSSG

DHA
-3

-5

Axis 1
Axis 2

MDA APX GR CAT SOD AsA TotAA DHA AsA/totAA GSH totG GSSG GSH/to tG
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
-0.03
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

lipid peroxidation in young T. pulchra grown inside NFA

chambers despite increased antioxidant defenses, such as the
higher GSH/totG ratios and in the activity of APX that followed increases in O3 levels. However, the enhanced MDA
accumulation in the leaf occurred in parallel with the decreased redox potential of AsA in response to enhanced O3
concentrations. This overall negative association between lipid peroxidation, AsA and O3 could indicate the low capacity
of AsA to scavenge ROS formed in the apoplast after O3
uptake, as is generally observed in tolerant plant species that
have 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, stomatal conductance and transpiration in young T. pulchra fumigated with high concentrations of O3 in OTP, which reinforced
the above-mentioned hypothesis. However, this hypothesis
must be tested more properly.
Reductions in AsA content are common responses in plants
fumigated with oxidative air pollutants, as observed by
Ferreira et al. (2012) in I. nil "Scarlet O'Hara" exposed to
O3. Increments in lipid peroxidation following O3 exposure
have 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 and
Agrawal 2011).
Finally, the multivariate analyses also indicated that oscillations in the air temperature inside the NFA chambers mainly
affected the enzymatic activity of T. pulchra, an effect also
observed in plants of N. tabacum "Bel W3" (Esposito et al.

2009; Dias et al. 2011), Caesalpinia echinata Lam. (Bulbovas

et al. 2010) and Ipomea nil "Scarlet O'Hara" (Dafr-Martinelli
et al. 2011; Ferreira et al. 2012) grown in sub-tropical sites
affected predominantly by O3. This meteorological factor
might affect the production of ROS during photosynthesis or
impose oxidative stress on the plants directly (Bulbovas et al.
2010; De Gara et al. 2010). In brief, based on the review by
Miller et al. (2010), the varying production of ROS and its
consequent influence on the redox state in sub-tropical species
of plants, such as T. pulchra, might be an overall acclimation
response to the natural variations of environmental conditions,
as characteristically observed during the summer experiments
performed in the present study.

Conclusion
The antioxidant responses of T. pulchra varied seasonally and
appeared to be stimulated by variations in air pollutant concentrations and/or air temperature. Glutathione and APX were
primarily responsible for increasing the tolerance of T. pulchra
to oxidative stress originating from air pollution in the region.
Variations in air temperature mainly affected the enzymatic
activity of T. pulchra. However, the content of MDA, which
was an indicator of oxidative damage to the plasma membranes in the plants, also increased in response to increasing
ozone concentration. This result appeared to indicate that the
pro-oxidant/antioxidant balance might not have been reached.

5494
Acknowledgements The authors gratefully acknowledge Petrobrs
(Petrleo Brasileiro) for financial support, FAPESP (So Paulo Research
Foundation: grant 2008/58682-1) for offering a Ph.D. scholarship to the
first author, CEPEMA (Environmental Center of Training and Research
of Polytechnic School from University of So Paulo) for permitting the
experimental installation on its property, CESP (Energy Company of So
Paulo) for donating the T. pulchra saplings, CETESB (Company of
Environmental Sanitation Technology) and EMAE (Metropolitan Enterprise of Water and Energy) for furnishing meteorological data.

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