Luehea divaricata Martius et Zuccarini Is a Sensitive Species to Aluminum , Not Presenting Phytoremediation Potential

The aim of this work was to evaluate the effect of different concentrations of aluminum (Al) on physiological and biochemical parameters of Luehea divaricata seedlings grown in a hydroponic system under greenhouse conditions to verify the possible tolerance to Al or phytoremediation potential of this species. Seeds of Luehea divaricata were placed to germinate in commercial substrate and after 30 days the seedlings were transferred to a hydroponic system with a complete nutrient solution, pH at 4.5±0.1, with daily adjustment. After 20 days of acclimatization, homogenous plants were selected and transferred to a new nutrient solution (without phosphorus (P) and pH at 4.5±0.1) with different concentrations of Al: 0, 25, 50, 75 and 100 mg L, each treatment being composed of 10 replicates of one plant each. The experiment was conducted in a completely randomized design. After seven days of exposure to the treatments, plants were collected for physiological and biochemical analyzes. Aluminum promoted a significant reduction in fresh and dry weight of roots, stems and leaves; in plant height; leaf number; leaf area; and pigment content. On the other hand, Al promoted an increase in lipid peroxidation and guaiacol peroxidase enzyme activity. Therefore, the presence of Al in the growth medium, for the studied conditions, altered significantly both physiological and biochemical parameters in Luehea divaricata seedlings, presenting a sensitive behavior to this element. Due to these characteristics, the studied species does not show phytoremediation potential.


Introduction
Acidic soils, which occupy about 30% of ice-free areas in the world (Brunner & Sperisen, 2013), directly influence on plant development.A large proportion of these soils is covered by forests (Fao & Iiasa, 2007).These soils are present in several parts of the world, including in Brazil, the state of Rio Grande do Sul being one where they appear more significantly (Abreu et al., 2003).This acidity promotes a decrease in plant growth due to aluminum (Al) toxicity, as well as low base saturation, and deficiency of phosphorus, calcium, magnesium and molybdenum (Poschenrieder et al., 2008).
Aluminum is the third most abundant element in Earth's crust, after oxygen and silicon.In acidic soils, where pH is less than or equal to 5.5, Al is able to solubilize itself and become possibly toxic to some plants (Singh et al., 2017).In addition, it is noteworthy that the effect of this toxicity on the root system is that it inhibits its growth, fixes phosphorus in forms which are less available in the soil and/or roots, and reduces root respiration (Gupta et al., 2013;Schmitt et al., 2016).Furthermore, it has the potential to interpose in enzymatic reactions, to promote oxidative stress, as well as to interfere in the absorption, transportation and utilization of other nutrients, inducing nutritional deficiency in the plant (Tabaldi et al., 2009;Dorneles et al., 2016;Singh et al., 2017).molecular and biochemical mechanisms of Al tolerance in plants have been proposed to try to explain the tolerance presented by some plants to the excess of this metal (Foy et al., 1978;Sousa et al., 2016).
Unlike annual crops (Tabaldi et al., 2009;Carlin et al., 2012;Bojórquez-Quintal et al., 2014), little attention has been given to forest species, especially the native ones, in relation to their behavior in relation to Al (Alves et al., 2001;Brunner & Sperisen, 2013).In synthesis, the effect of this element varies according to the studied species or even to the genotype within the same species.Forest species that are able to grow and complete their life cycle on acid soils rich in Al can be grown in these soils, which are often unsuitable for growing food or medicinal plants.Therefore, the selection of species of trees tolerant to Al toxicity may be an option for plant restoration in acidic soils and metal contaminated areas, besides being used as phytoremediation species.
The Luehea divaricata (Martius et Zuccarini) species, popularly known as açoita-cavalo in Brazil, is native to the state of Rio Grande do Sul (RS), belonging to the Malvaceae family (Carvalho, 2008).It is of great importance, especially in the Mixed Ombrophilous Forest of this state.Due to its easy adaptability, it is a target species for the recovery of degraded areas and reforestation.Its wood is widely used for construction, furniture and energy production, as well as medicinal uses (IBGE, 2012).Its vast use, along with its natural distribution in the soils of RS, suggests that the species could present a certain tolerance to Al and, therefore, have phytoremediation potential.
The previous selection of seedlings exposed to toxic concentrations of Al in a hydroponic system may provide adequate information about the survival capacity of the seedlings after their transplantation to the field.Thus, the objective of this study was to evaluate the effect of different concentrations of Al on physiological and biochemical parameters of Luehea divaricata seedlings grown in a hydroponic system to verify the possible tolerance or phytoremediation potential of this species.

Method
The experiments were carried out in the Plant Biotechnology Laboratory and in greenhouses belonging to the Biology Department of the Federal University of Santa Maria, RS.Seeds of Luehea divaricata were germinated in plastic trays with commercial substrate Plantmax and irrigated daily with a complete nutrient solution, keeping pH at 4.5±0.1, with daily adjustment.The nutrient solution had the following composition (in μM): 6090.5 of N; 974.3 of Mg; 4986.76 of Cl; 2679.2 of K; 2436.2 of Ca; 359.9 of S; 243,592 of P; 0.47 of Cu; 2.00 of Mn; 1.99 of Zn; 0.17of Ni; 24.97 of B; 0.52 of Mo; 47.99 of Fe (FeSO 4 /Na-EDTA).
After 30 days, the initial growth period of the seedlings, 75 homogenous plants with the height of about 10cm were transferred to trays in a hydroponic system, where they were fixed by means of sponges in polystyrene plates, for acclimatization.The same nutrient solution was used, with pH at 4.5±0.1, with daily adjustment and constant aeration system.The volume of the trays was replaced daily and the solution changed every 3 days.
At 20 days of acclimatization, new homogeneous plants were selected, which were transferred to the final hydroponic system, in 1 L pots, with one plant each.In a new nutrient solution (without P and pH at 4.5±0.1),five treatments were applied: 0, 25, 50, 75 and 100 mg L -1 of Al, each consisting of 10 replicates.The experiment was conducted in a completely randomized design, the nutrient solution was replaced every two days, with daily replenishment and pH level adjusted at each replacement.After seven days of exposure to the treatments, the plants were collected for physiological and biochemical analyzes.

Physiological Parameters
For the physiological evaluations, four plants per treatment were collected, having their roots washed in distilled water, and then divided into leaves, stem and root system.It was determined the number of leaves, aerial part length (using a ruler graduated in millimeters), leaf area (with leaf area integrator), and fresh and dry weight of leaves, stems and roots.The parts were collected separately and immediately weighed in a digital scale to determine fresh weight.Then they were dried in an oven at 65 °C with forced ventilation until constant weight, and the appropriate dry weights were measured.

Biochemical Parameters
For the biochemical analyzes, six plants per treatment were collected and separated into leaves and roots.The material was washed with distilled water, immediately frozen in liquid nitrogen and then stored in a freezer at -80 °C in order to maintain its characteristics.Chlorophylls a and b and carotenoids were extracted according to the method of Hiscox and Israelstan (1979) and estimated using the equation of Lichtenthaler (1987).
The lipid peroxidation was determined in the plants root system, according to the method of El-Moshaty et al. (1993).For the preparation of extracts for the determination of antioxidant enzymes activity, it was initially used the protocol of Zhu et al. (2004) to determine the enzyme activity, and later the protocol of Bradford (1976) to determine the concentration of proteins.The guaiacol peroxidase (POD) activity was determined according to Zeraik et al. (2008) and the superoxide dismutase (SOD) activity was determined according to the spectrophotometric method, described by Giannopolitis and Ries (1977).

Tolerance Index
The Al tolerance index was calculated according to Trannin et al. (2001), through Equation 1: where, TI = tolerance index; TDW Al = total dry weight in each concentration of aluminum; TDW C = total dry weight in control.

Statistical Analysis
Data normality and homogeneity of variances were tested through the Shapiro-Wilk and Bartlett tests, respectively, both using the Action-Excel Software.The data were submitted to variance analysis and tested by the 5% probability of error regression models, through the SISVAR Software (Ferreira, 2011).The graphics program used was SigmaPlot 12.5.

Analysis of Fresh and Dry Weight
Based on tests of normality and homogeneity, the data showed to be normal and the variances are homogeneous.Tables 1 and 2 show the results of the analysis of variance for the physiological and biochemical parameters, demonstrating a significant effect at 5% of error probability in all study analyzes.the element, which led to a decrease no tolarance index.However, from the concentration of 50 mg / L, with the increase in stress caused by Al, it may also have increased the capacity of the plant to investigate defense mechanisms, a factor that resulted in the stability of the tolerance index in the highest concentrations studied.
Although the growth of the species occurs naturally in acid soils, this behavior suggests that it presents sensitivity to the element, being recommended its planting in places with low Al concentrations.Due to its low tolerance, the species has potential to be used as a signal for contaminated areas.However, Al concentrations employed in this study may have been higher than that found in acid soils.In addition, the use of the hydroponic system and daily adjustment of pH level, maintaining the constant availability of all ions to the plant, is different from what occurs in soils, factors that may have been crucial for such results.

Analysis of Leaves Number, Aerial Part Length and Leaf Area
The regression equation among the observed means for leaf number, aerial part length and leaf area are presented in Figure 2, where the quadratic function was the most adjusted to the model, as for plants height the linear function was the most adjusted to the model.The Al presence in growth medium reduced all parameters evaluated in L. divaricata seedlings.In an analogous way, in an experiment carried out by Alves et al. (2001), the presence of Al drastically reduced the variables of the shoot of Senna multijuga and Handroanthus stans plants.
The Al, damaging the root system of the plants, indirectly affects their absorption, translocation and transportation of nutrients to the aerial part, which can result in nutritional deficiency symptoms (phosphorus, potassium, calcium, magnesium and molybdenum) and consequent reduction of leaf area and leaf number (Poschenrieder et al., 2008).
The absorption of solar radiation in these plants may be impaired, causing lower photosynthetic rates and, with this, lower accumulation of biomass, as may be observed in Figure 1.The reduction of plant growth may be due to a decrease in photosynthetic activity, which in turn may be related to both stomatal and non-stomatal factors (Konrad et al., 2005). jas.ccsenet.

Metals wh aminolevu plant phot induces ca
The decrea forms (Oli impairs the and especi elements w Stojanovic In some s reactions.functional consequen photosynth

Analys
In relation as the best lipid perox a catalyst 2013).Th hydrogen p various bio Figure 4 The oxida obtaining t to Al conc of the elem observed b (2011) in significant previously

Table 1 .
Results of the analysis of variance for the physiological parameters in Luehea divaricata Martius et Zuccarini seedlings submitted to different aluminum concentrations Note.VF: Variation factor; CV: Coefficient of variation; DF: Degrees of freedom; RFW: Fresh weight of roots; LFW: Fresh weight of leaves; SFW: Fresh weight of stem; RDW: Dry weight of roots; LDW: Dry weight of leaves; SDW: Dry weight of stem; LN: Leaves number; APL: aerial part length; LA: leaf area.* Significant at 5% probability of error (Pr < 0.05).

Table 2 .
Results of the analysis of variance for the biochemical parameters in Luehea divaricata Martius et Zuccarini seedlings submitted to different aluminum concentrations Note.VF: Variation factor; CV: Coefficient of variation; DF: Degrees of freedom; MDA: malondialdehyde; POD: guaiacol peroxidase.* Significant at 5% probability of error (Pr < 0.05).

Table 3 .
Tolerance index for total dry weight of Luehea divaricata Martius and Zuccarini seedlings submitted to different aluminum concentrations