Nitrogen Metabolism and Antioxidant System in Young Plants of Parkia pendula Submitted to Drought

The (Parkia pendula (Willd.) Benth. ex Walp.) species most know as visgueiro is a native specie from the Amazon region, belongs to the family Fabaceae-mimosoideae and has been scientifically studied because it is used in landscape projects and the restoration of degraded areas. Intrinsic responses of this species due to water stress is still very superficial, lacking a larger scientific approach in nitrogen and oxidative parameters. For this, a greenhouse experiment was set up in the Federal Rural University of Amazon with seedlings, where they were submitted to water restriction. The experimental design was completely randomized in a 2 × 3 factorial design (control and water deficit, and three evaluation times), with 5 replications, totaling 30 experimental units. The results showed that the RWC reduced during the experiment from 73.5% to 52.99%, evidencing a lower amount of water in these plants. This condition caused the plants to respond positively to the increase of antioxidant enzymes (catalase, APX and SOD), making the plant defense system efficient. As well as for some variables of nitrogen metabolism. The results showed that the RWC reduced during the experiment from 73.5% to 52.99%, evidencing a lower amount of water in these plants. This condition caused the plants to respond positively to the increase of antioxidant enzymes (catalase, APX and SOD), making the plant defense system efficient. As well as for some variables of nitrogen metabolism. The species in this experimental condition was considered as sensitive to the water stress condition.


Introduction
The Amazon is considered a region with high rates of deforestation and high consumption of wood.This has led to a marked decrease in forest species.Parkia hangs up (Willd.)Benth.ex Walp.It is among these species (Silva et al., 2014).
It is a tree of significant size, 20 to 30 m high, unmistakable by the tabular aspect of its canopy.The shaft is cylindrical, rectilinear, occasionally with small sapopemas; the leaves are composed; the inflorescence is of the capitular type, with dark red flowers; the fruits are hung by long peduncles and are of the vegetable type, exuding, when ripe, a viscous resin; the seeds are small, rounded and long (Loureiro et al., 2000).It is widely used in construction and shipbuilding (Souza et al., 1997), taboos, boxwood, slabs for plywood and canoes (Loureiro et al., 2000).It occurs naturally in the states of Pará, Amazonas, Acre, Mato Grosso, Rondônia and Maranhão (Souza et al., 1997).Besides these functions, they have been indicated for the recovery of degraded areas in the Amazon because they present, among other characteristics, rapid growth over open areas and economic potential (Hopinks, 1986;Oliveira et al, 2006).
Water is one of the most fundamental natural resources for the development and growth of plants, since it contributes to the maintenance of the activities of biological molecules, cellular, tissues and organisms (Marenco & Lopes, 2005).Once its absorption in plants is limited, it triggers a series of implications that can lead to tolerance or sensitivity.Knowing the biochemical characteristics of this species under conditions of water stress, broadens the horizons to base it with a forest species of great value in economic, social and environmental aspects.The water stress condition decreases the availability of nitrogen in plant tissues, considering that this element is constituent of structures such as proteins, coenzymes, nucleic acids, chlorophyll, pigments and by-products (Ferreira et al., 2007), essential structures for the growth and development of plants (Pavinato et al., 2008).
Water stress can also generate oxidative stress, due to the stomatal closure, which results in the limitation of photosynthesis.However, this closure does not prevent the photons from being absorbed by the pigments located in the antenna complex.Thus, the energy from the excitation of chlorophyll molecules that has not been used in photosynthesis or eliminated in the form of heat causes a production of reactive oxygen species (ROS), such as superoxide (O 2 -), hydrogen peroxyde (H 2 O 2 ) and singlet oxygen ( 1 O) (Assada, 1999).These ROS are highly harmful to the plant as they can react with any molecule in the cell, such as proteins, deoxyribonucleic acids and lipid peroxidation causing a cellular disorder (Soares & Machado, 2007).
In order to clarify the responses of this plant to water stress, the objective was to evaluate the nitrogen and antioxidative behavior of the species Parkia pendula.

Location and Experimental Conduction
Developed in a greenhouse, from March to August 2015, using visgueiro seedlings (Parkia pendula (Willd.)Benth.Ex Walp) at approximately seven months of age.They were irrigated daily to maintain them in the field capacity for a period of one month (Fernandes & Sykes, 1968) and 5 mL of macro and micronutrients were applied in the form of Arnon's nutrient solution before the treatments.
The plants were submitted to two water regimes: irrigated (control) and water deficiency (suspension of irrigation in 10 days), considering time 0 (zero days of water deficiency), time 1 (5 days of water deficiency) and time 2 (10 days of water deficiency), with 5 replicates, totaling 30 experimental units, each unit being composed of one plant/vessel.

Relative Water Content
The leaf relative water content was evaluated using leaf disks with 10 mm of diameter and it was carried out in each plant, in which 40 disks were removed and the calculation was done in agreement with the formula proposed by Slavick (1979): Where, FM1 is fresh matter, FM2 is turgid matter evaluated after 24 h and saturation in deionized water at 4 °C in dark, and DM is the dry matter determined after 48 h in oven with forced air circulation at 80 °C.

Concentrations of Nitrate
The Cataldo et al. (1975) method was used for the determination of nitrate.The absorbance was determined at 410 nm and the concentration of nitrate was obtained from a standard curve with increasing concentrations of NO 3 -(0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 μmol mL -1 ).The results were expressed in μmoles of NO 3 -g -1 DM of tissue.

Nitrate Reductase Activity
The collection of plants was scheduled at 05:30 AM for the determination of nitrate reductase activity (RN), which was performed in vivo by selecting in the greenhouse.The fully expanded primary leaves were selected from each of the repetitions, according to the method described by Hageman and Hucklesby (1971).The reading was performed at 540 nm and the result of the activity estimated by producing NO 2 -in 851. the reaction middle, expressed in μmoles NO 2 -g -1 MF h -1 from a standard curve obtained by KNO 2 p.a (Sigma).

Concentrations of Free Ammonium
50 mg of lyophilised leaves weighted and placed in test tubes containing the total extract, solution A and solution B after shaking.The free ammonium concentrations were estimated from the standard curve constructed with (NH4) 2 SO 4 p.a. (Sigma) according to method described by (Weatherburn, 1967).
2.2.4 Concentrations of Total Soluble Amino Acids 50 mg of previously lyophilised leaves weighted then buffered solution and reagent ninhydrin were added.The total free amino acid levels were determined based on a standard curve adjusted from increasing concentrations of a standard mixture of L-glutamine according to the method described by Peoples et al. (1989).

Concentrations of Total Soluble Proteins
Determination of the total soluble proteins was carried out with 100 mg of powder, incubated with 5 mL of extraction buffer.This was homogenized and kept in agitation for 2 h, and centrifuged to 2.000 g for 10 minutes at 20 ºC.Quantification of the total soluble proteins was carried out at 595 nm in accordance with Bradford (1976) with albumin bovine (Sigma Chemicals) as standard.
2.2.6 Concentrations of Glycine Betaine 25 mg of lyophilised leaves weighted and H 2 SO 4 2N added into the test tubes and KI-I2 iced.A standard curve was used of Glycine-Betaine according to the method of Grieve and Grattan (1983).
2.2.7 Concentrations of Proline 50 mg of lyophilised leaves weighted and placed in the test tubes containing total extract, ninhydrin acid and glacial acetic acid.It was determined through a calibration curve proline and proline result expressed in mmol g -1 dry matter (DM) according to Bates et al. (1973).

Antioxidant System
(1) Extraction The extract for the determination of the activity of the SOD, APX and CAT enzymes were obtained from the homogenization in mortar at 4 °C of 0.1 g of lyophilized leaf powder and root with 5 mL of potassium phosphate buffer solution (at 4 °C) at 0.1 mM, pH 7.0, containing 0.1 mM EDTA, followed by homogenization for 4 min.
The additions of the phosphate buffer were made in a fragmented form, 50% of the total volume of this solution (2.5 ml) being used in the homogenization for 2 min, after which the other 50% were immediately added, the mixture being homogenized in time equivalent to the previous one.The homogenate was filtered on nylon tissue and transferred to test tubes, and kept at 4 °C for two hours, with occasional shaking.The filtered homogenate was centrifuged at 12,000 × g for 15 min at 4 °C.The supernatant, the crude extract, was stored in a freezer at -80 °C until used in enzyme activity assays.
(2) Superoxide Dismutase The SOD activity was determined by inhibition of photoreduction of nitroblue tetrazolium chloride (NTC) according to Giannopolitis and Ries (1977).
(3) Ascorbate Peroxidase The APX activity was determined by the method of Nakano and Asada (1981).
(4) Catalase CAT activity was determined by the method of Beers Jr. and Sizer (1952) with modifications.

Experimental Design
The experimental design was completely randomized in a 2 × 3 factorial scheme (two water conditions: control and water deficit, and three evaluation times), with 5 replications, totaling 30 experimental units.

Statistical analysis of the data
The experimental results were submitted to analysis of variance (ANOVA), and when significant differences were verified the averages were compared by the Tukey test at the 5% level of significance.Regression analysis was performed on the variables, whose significance was verified by the F-test (P < 0.05).Statistical analyzes were performed using the Assistat 7.7 program.

Relative Water Content
The plants submitted to water deficiency reduced their relative water content in the mesophyll when compared to the control plants (Figure 1), the values presented in the experimental periods 0, 5 and 10 days were 73.5%, 62.47% and 52, 99% respectively.It was below the percentage of control plants that had an average of 76%.
By obeying the physiological principles, water movement occurs by the potential gradient, and for water to flow, the water potential of the soil must be higher than that of the plant.However, under water deficit, the water potential of the soil is reduced and for roots to absorb water, plants reduce their water potential to continue absorption, this mechanism was observed in Jatropha curcas plants (Moura et al., 2016).This reduction can be attributed to the osmotic adjustment due to the accumulation of low molecular weight organic solutes, as observed in Hymenaea courbaril (Nascimento et al., 2011).