Growth and physiological responses... GROWTH AND PHYSIOLOGICAL RESPONSES OF TREE SPECIES (Hymenaea courbaril L., Peltophorum dubium (Spreng.) Taub. and Myroxylon peruiferum L. F.) EXPOSED TO DIFFERENT COPPER CONCENTRATIONS IN THE SOIL

1 Received on 13.06.2017 accepted for publication on 01.03.2018. 2 Universidade Federal de Lavras, Programa de Pós-Graduação em Botânica Aplicada, Lavras, MG-Brasil. E-mail: <danimarques.bio@gmail.com>. 3 Universidade José do Rosário Vellano, Instituto de Ciências Agrárias, Alfenas, MG-Brasil. E-mail: <adriano.silva@unifenas.br>, <jose.mantovani@unifenas.br> and <DalvanaSousa@hotmail.com>. 4 Universidade Federal de Alfenas, Instituto de Ciências da Natureza, Alfenas, MG-Brasil. E-mail: <thiagonepre@hotmail.com>. *Corresponding author.


INTRODUCTION
Copper (Cu) is an essential micronutrient for plant development, participating in metabolism and being a structural part of some enzymes (Gautam et al., 2016). The concentration of Cu in plant tissues may vary depending on plant species, development stage and environmental factors (Yruela, 2009). Marschner (1995) reported that, in general, Cu concentration in plant tissues is between 1 and 5 µg g -1 dry weight.
Cu contamination in soils due to anthropic activities such as the arbitrary use of pesticides, fungicides, wastewater-based irrigation, industrialization, urbanization, mineralization and the use of urban waste has caused an increase in the levels of this element (Mateos-Naranjo et al., 2013). High copper concentrations in the biosphere cause a reduction in the environmental quality and vitality of living beings, in addition to high persistence in the environment (Nagajyoti et al., 2010;Ali et al., 2013;Stankovic et al., 2014).
In plants, excess Cu can lead to physiological, biochemical and morpho-anatomical disturbances in plants, causing inhibition of their development (Sánchez-Pardo et al., 2014;Gautam et al., 2016), besides oxidative stress in plants, due to the increase in the production of highly toxic oxygen free radicals that damage lipids and cell membrane proteins, amino acids and nucleic acids (Thounaojam et al., 2012;Moraes et al., 2015), decreasing agricultural productivity (Gao et al., 2010).
Plants have different mechanisms to grow in soils contaminated with Cu. The accumulation of this heavy metal in the roots (Malik and Biswas, 2012) is one of the main tolerance strategies to prevent its translocation to the photosynthetic tissues , which can alter chlorophyll levels in the leaves. The vacuole and the ion flow to the plasma membrane are channels that may decrease the presence of heavy metals in the cytoplasm (Sheoran et al., 2010). Other mechanisms promote the binding of carboxylic acids, amino acids, peptides, metallothioneins and phytochelatins with heavy metals (Sheoran et al., 2010), which act in the sequestration and inactivation of metallic ions before their entry into the cytoplasm (Hossain et al., 2012).
Cu toxicity can cause oxidative stress in plants and increase the production of oxygen free radicals, which are highly toxic (Yruela, 2009). Plants designed to prevent the production of these free radicals produce antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and guaicol peroxidase (Adrees et al., 2015). The knowledge of the behavior of plant antioxidant enzymes exposed to heavy metals is important for the selection and use of tree species in the recovery of degraded areas.
In this context, the objective of this study was to evaluate the growth and physiological responses of the tree species Hymenaea courbaril ("Jatobá"), Peltophorum dubium ("Canafístula") and Myroxylon peruiferum ("Óleo Bálsamo"), subjected to different copper concentrations in the soil.

Growth conditions and experimental design
The experiments were conducted in a greenhouse with a plastic cover and 50% shading (Sombrite ® ), at Universidade José do Rosário Vellano (UNIFENAS), Alfenas, Minas Gerais, with geographic coordinates: 21º 25' 45" S, 45º 56' 50" W, and average altitude of 881m. In the growing environment, the average temperature was 28.9ºC and relative humidity (RH) around 68%. The substrate consisted of a medium-acidity Eutrophic Haplic Cambisol with a 0 -20 cm deep layer, air dried, crushed, sieved (4-mm mesh sieve), homogenized and packed in 8-dm 3 pots. The results of soil chemical analysis are shown in Table 1.
Sowing was carried out in 180-cm 3 tubes, filled with commercial substrate Plantmax ® and base fertilization consisting of 3 kg m -3 Yorim Master ® (16% P 2 O 5 , 18% Ca, 7% Mg). Three cover fertilization procedures were carried out at 45, 60 and 90 days after sowing with: 20 g of N and 15 g of K 2 O, with urea and potassium chloride, respectively, as the source, which were dissolved in 10 L water, and 10 mL solution per plant were applied.
Plants with approximately 120 days of age from the three species under study were transferred to the pots. The different copper concentrations (CuSO 4 .5H 2 O) were applied to the soil at 30 days of transplanting. During the conduction of the experiment, soil moisture was controlled every two days, by weighing the pots and replacing the water, to maintain soil moisture at 70% of the retention capacity.

Biomass and biometric measurements
At 90 days after copper application, height (A), stem diameter (SD), shoot dry matter (SDM) and root dry matter (RDM) were evaluated. Measurements were established using a caliper and tape measure. To obtain leaf dry matter, stems and roots were washed to remove any particles, and brought to a forced circulation oven at 60°C until reaching constant weight.

Chlorophyll content, antioxidant enzymes and copper content
For the analysis of chlorophyll content, two fully expanded leaves were collected, by replication; 0.1 g leaf was ground in 5 mL of 80% acetone. The extract was filtered on glass wool and the volume was made up to 10 mL with 80% acetone.
The enzymatic analysis on the leaves was according to Souza et al. (2012) and the supernatants collected were used in the analysis of all enzymes. The activities of the enzymes were evaluated spectrophotometrically and expressed in milligrams (mg) of proteins, which were determined by the method of Bradford (1976), using a standard curve of bovine serum albumin (BSA).
The activity of superoxide dismutase (SOD, EC 1.15.1.1) was evaluated by the ability to inhibit the photoreduction of nitro blue tetrazolium (NBT), proposed by Giannopolitis and Ries (1977). Catalase (CAT, EC 1.11.1.6) activity was determined by the consumption of H 2 O 2 at 240 nm for 3 minutes (Havir and Mchale, 1987). The activity of ascorbate peroxidase (APX, EC 1.11.1.11) was determined by monitoring the oxidation of ascorbate at 290 nm for 3 minutes (Nakano and Asada, 1981). The activity of guaiacol peroxidase (GPX, EC 1.11.1.7) was determined by the oxidation of guaiacol at 470 nm, according to Lin and Wang (2002), with modifications. All analyses were performed at the end of the ninety days of culture. Cu contents in plant tissues and soil were quantified by atomic absorption spectrophotometry, according to Carmo et al. (2000).

Data analysis
For all analyzed variables, the means and ± standard error (SE) were calculated. For statistical analysis of the results, when significant, linear and non-linear regression analysis were used, as well as ANAVA, using the Sisvar software (version 4.3) (Universidade Federal de Lavras, Lavras, Brazil).

Biomass and biometric measurements
The use of copper (Cu) in the different species did not present a significant effect (p>0.05) on plant growth (Table 2). H. courbaril had a mean height of (H=74.00 cm),  Table 2).

Chlorophyll content and antioxidant enzymes
The content of chlorophyll "a" showed a peak at a Cu concentration of 100 mg kg -1 in all the tree species studied and, at concentrations of 200 and 400 mg kg -1 , a reduction in the content of this pigment was observed in the plants ( Figure 1A).   Growth and physiological responses...
Regarding chlorophyll "b", no common behavior was observed among the species studied. In H. courbaril, the content of chlorophyll "b" was reduced from the concentration of 50 mg Kg -1 . In M. peruiferum, the reduction in chlorophyll "b" was observed from 100 mg Kg -1 . In P. dubium plants, the contents of chlorophyll "b" and total chlorophyll increased at higher concentrations (200 and 400 mg Kg -1 ). The curves for the content of total chlorophyll had a behavior similar to that observed for chlorophyll "b" (Figure 1B, 1C). In general, the leaves of the species under study showed a higher content of chlorophyll "b" in relation to chlorophyll "a" (Figure 1A, 1B).
With the increase in Cu in the soil, H. courbaril plants showed an increase in the activity of catalase (CAT), when compared to the control plants ( Figure  2C). SOD showed peak activity in the plants maintained in soil with 200 mg Kg -1 Cu, being inhibited at the highest Cu concentration in the soil (400 mg Kg -1 ). Even with Cu increase in the soil, APX showed similar activity to that observed in the control plants ( Figure 2B). It is noteworthy that guaiacol peroxidase (GPX) did not show activity, and that CAT was probably the main enzyme responsible for preventing oxidative stress in this species ( Figure 2C, 2D).

MARQUES DM et al.
In P. dubium, a high activity of SOD and CAT was observed in plants growing in soil at a concentration of 200 mg Kg -1 Cu, being inhibited at the concentration of 400 mg Kg -1 . APX activity was similar to the control plants (Figure 2A, 2B, 2C). GPX also showed no activity in the evaluated plants ( Figure 2D). Thus, it can be observed that SOD and CAT were the main enzymes responsible for the elimination of reactive oxygen species (ROS) for this species (Figure 2A, 2C).
M. peruiferum plants showed higher APX activity, especially at the highest Cu concentrations in the soil, and an increase in GPX up to the concentration of 50 mg Kg -1 was observed, followed by inhibition and decline with increasing Cu concentrations ( Figure 2B, 2D). SOD and CAT had constant activity, similar to the plants submitted to the control treatment (Figure 2A, 2C). For this species, the high APX activity showed that it was the main enzyme in preventing oxidative stress ( Figure 2B).

Cu content in the plants
In general, Cu bioaccumulation in the species occurred in the plant root system. Increased Cu accumulation was observed in plants growing in soils treated with 400 mg Kg -1 of the element; H. courbaril accumulated 4.39 mg plant -1 , P. dubium 5.56 mg plant -1 and M. peruiferum 1.56 mg plant -1 (Figure 3). H. courbaril had 56 to 92% of the copper retained in the roots, and the same behavior was observed for P. dubium (77-91%) and M. peruiferum (19-64%).
Regarding the total copper content, H. courbaril plants had a linear increase in this chemical element in the tissues with the increase in copper concentrations in the soil (Figure 3). P. dubium had a quadratic behavior, showing high Cu contents in the tissues of plants grown in soils with Cu (400 mg Kg -1 ), bioaccumulating Cu in all tissues, mainly in the roots, reaching 4.47 mg kg -1 (Figure 3). M. peruiferum showed a linear behavior, with an increase in Cu contents in the tissues with increasing Cu concentrations in the soil (Figure 3).

DISCUSSION
Copper is an essential micronutrient to plants but, at high concentrations, above 200 mg Kg -1 in the soil, it causes pollution and can be toxic to plants, with potential direct or indirect risk to human health, when exposed to this scenario (CETESB, 2005). High levels of this metal in the soil are potentially toxic to most plants, with leaf chlorosis and growth inhibition as visible symptoms of Cu toxicity. However, in this study, these symptoms were not observed, even when the plants were exposed to high concentrations of this  (Table 2). This result is similar to that found by Cambrollé et al. (2013), working with wild vine growing on copper-contaminated soils.
Cu toxicity decreases biosynthesis, besides reducing chlorophyll content, due to damage to thylakoid membranes (Adrees et al., 2015). Several authors (Mateos-Naranjo et al., 2008;Gomes et al., 2011;Yruela, 2013;Nair et al., 2014;Adrees et al., 2015) reported the effect of excess copper on chlorophyll, resulting in the degradation of this pigment, which is in agreement with the results found in this research. However, in tolerant plants, chlorophyll content increases or does not undergo significant changes in response to the treatment with heavy metals (Borghi et al., 2008).
In the three species studied, there was a reduction trend in the levels of "a", "b" and total chlorophyll in the plants for the highest Cu concentration in the soil (400 mg kg -1 ), when compared to the plants of the control treatment. However, no visible chlorosis symptoms were observed in the leaves and plant growth was not adversely affected. It is possible that this result is related to the mechanisms that these plants used to prevent Cu translocation to the shoot, accumulating heavy metal in the root system, indicating a higher tolerance of these species to Cu.
The data in this study showed a higher content of chlorophyll "b" in relation to chlorophyll "a" in the leaves of the studied species, which is not common, since chlorophyll "b" is an accessory pigment. This response is possibly related to the fact that these plants are in a greenhouse, growing in a shaded environment, and may have some kind of compensatory mechanism, since the use of meshes reduces the luminous intensity in relation to full sun by 50%. The studied species probably invested in the production of pigments in an attempt to increase diffuse light capture and maintain photosynthetic rates. Leaves growing in shaded environments have a greater amount of chlorophyll "b", which is important to play a role in the absorption of diffuse radiation (Huang et al., 2016). Studies by Chang et al. (2016) with dragon fruit (Hylocereus polyrhizus (Weber) Britt. & Rose) plants report an increase in chlorophyll content, mainly chlorophyll "b" in relation to chlorophyll "a", with plants kept in different degrees of shading.
Another possible justification for the content of chlorophyll "b" being greater than chlorophyll "a" may be due to foliar senescence or some abiotic stress, such as the presence of heavy metals in the soil (Hörtensteiner, 2013). Mulisch and Krupinska (2013) reported that, in most cases of leaf senescence due to environmental stress, chlorophyll "a" is the first to be degraded. One of the mechanisms that performs this degradation is pheophorbide á oxygenase -PAO (Hörtensteiner and Kräutler 2011). In order for this metabolic pathway PAO to degrade chlorophyll "b", its conversion to chlorophyll "a" is necessary (Hörtensteiner, 2013). In this study, the species were kept under nursery conditions for a few months, and it may have occurred that, at the moment the leaves were sampled for pigment evaluation, they were in the initial senescence stage, resulting in a higher content of chlorophyll "b" in relation to chlorophyll "a".
Excess Cu can cause oxidative stress in plants due to increased production of highly toxic oxygen free radicals (Yruela, 2009). Reactive oxygen species (ROS), such as superoxide radicals (O 2 •-), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical (OH • ), cause damage to proteins and lipids of plant cell membranes (Thounaojam et al., 2012;Sánchez-Pardo et al., 2014). To prevent the production of ROS, plants have antioxidant mechanisms that include antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and guaiacol peroxidase (GPX), therefore preventing oxidative damage (Adrees et al., 2015). It is important to note that APX activity was higher to that of CAT for all species studied. This is due to the fact that, although CAT has high specificity for H 2 O 2 , its affinity is lower than that of APX (Meng et al., 2007;Sharma et al., 2012), being less efficient in the removal of H 2 O 2 , which possibly explains its lower activity in relation to APX.
H. courbaril plants showed increased CAT activity, from the lowest copper concentration (50 mg Kg -1 ). CAT is sensitive to copper stress which, at low concentrations, may stimulate the activity of this enzyme (Srivastava et al., 2006). It is interesting to observe that H. courbaril had compensatory defense mechanisms against oxidative stress (Cargnelutti et al., 2006), where CAT was the main enzyme.
The increase in the activity of SOD in the studied species, at the concentration of 200 mg Kg -1 , can be attributed to the accumulation of superoxide radicals (O 2 -) induced by copper. To catalyze the dismutation of the superoxide anion, which is the first ROS formed, SOD participates in the first line of defense against MARQUES DM et al.
oxidative stress (Gill and Tuteja, 2010). In addition, the removal of the superoxide radical by SOD is important to prevent the formation of hydroxyl radicals before their interaction with target molecules occurs (Xu et al., 2013).
For M. peruiferum plants, among the enzymes under study, APX played the main role in the removal of ROS. Lyubenova et al. (2015) reported increased APX in increasing Cu concentrations for Typha latifolia. However, the activity of antioxidant enzymes can be increased to a certain level and then decreased due to the increase in oxidative stress (Adrees et al., 2015), and this behavior was observed in P. dubium plants at the highest Cu concentration in the soil, for the enzymes CAT, SOD and APX.
Metal absorption by the plant depends on the availability of the soil metals and the physiological requirement of the plant (Dalcorso et al., 2014). Cu is absorbed by active transport and Cu ions tend to accumulate in the root system, mainly in protoplasms (Malavolta, 2006;Palmer and Gerinot, 2009). There are several proteins that are responsible for transporting these metallic ions to the cell membrane, such as Ptype ATPase, COPT and chaperones (Yruela, 2005). The three species studied were able to extract Cu from the soil and concentrate it in all studied organs: leaves, stems and roots. In the three species, copper bioaccumulation occurred mainly in the root system, and an increase in Cu contents in the tissue was observed with an increase in Cu concentrations in the soil. A study conducted with the species Peltophorum dubium, Parapiptadenia rígida, Enterolobium contortisiliquum (Silva et al., 2011), Bauhinia forficata Link, Pterogyne nitens Tul (Silva et al., 2015) and two apple cultivars (Malus hupehensis Rehd.) (Wang et al., 2016) showed that Cu accumulates preferentially in the root system, a result similar to that found in this study.
Copper accumulation in the root system may represent a tolerance strategy of the plant to the excess of heavy metal in the soil (Silva et al., 2011;Cambrollé et al., 2013), aiming at preserving the most metabolically active tissues (Branzini et al., 2012) and the photosynthetic machinery present in the leaves. Another possible explanation would be the apoplastic barriers of the exoderm and endoderm, since they play an important role in plant protection, minimizing metal translocation from the roots to the shoot (Gomes et al., 2011;Freitas et al., 2015). Cell wall thickening in the root system provides a greater retention area of heavy metals, reducing their translocation to the shoot (Adrees et al., 2015). Wang et al. (2106) worked with the cultivar Malus hupehensis Rehd. Ralls, and reported higher amounts of Cu sequestered mainly in the cell wall and vacuole of fibrous roots.

CONCLUSION
The growth of species H. courbaril, P. dubium and M. pereuiferum is not affected by the high Cu concentrations applied to the soil. In the studied species, the damage from excess Cu in the soil is prevented due to the increase in the activity of some enzymes of the antioxidant system and the accumulation of this heavy metal in the plant root system.