Chlorophyll , nitrogen and antioxidant activities in Cumaru ( Dipteryx odorata ( Aubl . ) Willd ) ( Fabaceae ) in two water regimes

Chlorophyll, nitrogen and antioxidant activities in Cumaru (Dipteryx odorata (Aubl.) Willd) (Fabaceae) in two water regimes Bruno Moitinho Maltarolo*, Ellen Gleyce da Silva Lima, Vitor Resende do Nascimento, Kerolém Prícila Sousa Cardoso, Ana Ecídia de Araújo Brito, Tamires Borges de Oliveira, Glauco André dos Santos Nogueira, Karollyne Renata Souza Silva, Wander Luiz da Silva Ataíde, Cândido Ferreira de Oliveira Neto, Waldemar Viana De Andrade Junior and Benedito Gomes dos Santos Filho

reforestation of degraded areas, which in addition to the ecological benefits, increase the supply of wood from reforestation in the region, increasing the income on the farm and reducing the pressure on the remaining natural forest dependent of water resources (Shimizu, 1998).
As water resources become scarce, the commercial exploration of plants tolerant to drought becomes a priority for obtaining high yields (Matos et al., 2012).The impact of drought in the forestry and agricultural activities is an important socioeconomic consequence that affects millions of people around the world (Elliott et al., 2013).Among the various factors affecting the production plant, the water deficit occupies a prominent position, as well as affect the water balance in plants by altering their metabolism, is a phenomenon that occurs in large extensions of arable areas (Nogueira et al., 2001).
Among the many implications of drought on plant development, the restriction on the acquisition of nutrients and water is commonly recognized (Manivannan et al., 2008).Evidence suggests that drought causes oxidative stress in various plants, in which reactive oxygen species (ROS) such as superoxide radical (O 2 -), hydroxyl radical (OH -), hydrogen peroxide (H 2 O 2 ) and singlet oxigen ( 1 O 2 ), are produced (Jaleel et al., 2007).
To minimize the cytotoxic effects of ROS, the plants causes a complex antioxidant system where specific enzymes act by neutralizing the action of these radicals, starting with the superoxide dismutase (SOD), which inmute radical O 2 to H 2 O 2 .This, in turn, undergoes action of various enzymes such as catalase (CAT), responsible for the conversion of H 2 O 2 to H 2 O and O 2 , and peroxidase, ascorbate peroxidase (APX) reducing the H 2 O 2 to H 2 O (Apel and Hirt, 2004).Besides, in water restriction, the plants should be able to handle ROSs particularly to prevent oxidative damage to lipids, proteins and nucleic acids; if there is an inability to adequately handle ROSs, oxidative damage may result in cell death (Demidchik, 2015).
Several methods are adopted by researchers to identify species tolerant to water stress, being more common selection through ecological descriptors associated with physiological and biochemical descriptors.According Pincelli (2010) water deficiency is one of the environmental stresses responsible for the reduction of pigments in the leaves, making the plant life cycle changes.Among these, related to the antioxidant system and osmotic adjustment are supported substantially in identifying promising species, and consequently, the progress of culture of works for improving drought resistance (Azevedo Neto et al., 2009).
The antioxidant enzyme activity is usually enhanced to promote better elimination of ROSs and promote increased cellular protection against oxidative damage (Jaleel et al., 2009).Considering then that collaboration between antioxidant enzymes should provide better protection against the deleterious effects of ROS, minimizing oxidative damage (Blokhina et al., 2003).
Given the above, the study aimed to analyze the content of chlorophyll A and B, ammonium, nitrate and proline as well as the activity of oxidative enzymes in evaluation tolerance of cumaru plants subjected to drought.

Location and experimental conduction
The experiment was conducted in a greenhouse at the Federal Rural University of Amazonia (UFRA) belonging to the Institute of Agricultural Sciences (IAS), located in Belém, Pará, in the period from March to July 2015.The seedlings of cumaru (Dipteryx odorata (Aubl.)Willd.), from seeds were provided by AIMEX (Association of Industries Exporters of Wood in Pará) with four months old, they were placed in plastic pots with a capacity of 3.6 L. The substrate consisted of yellow dystrophic Latosol (EMBRAPA, 2013).Before the start of treatment, all plants were irrigated daily for three months, corresponding to the acclimation time.5 ml of solution cocktail containing macro and micronutrients (Table 1) was added to all the samples at the start of acclimation, in the form of nutrient solution (Hoagland and Arnon, 1950), modified in Biodiversity Studies Laboratory in Higher Plants (EBPS), UFRA.
The plants were subjected to two water regimes: Irrigated (control) and water deficit, in which the imposition of water deficit was obtained by suspension of irrigation in 21 days, and the time 0 (zero days of drought), time 1 (7 days of drought), time 2 (14 days of drought) and 3 time (21 days of drought).During the period of analysis, control plants were irrigated daily to replace the water lost by evapotranspiration.There was also the weed control manually.It was not detected occurring nutritional deficiency symptoms, as well as the attack of pests and pathogens.

Experimental design and statistical analysis
The experimental design was completely randomized in split plot in time (four times evaluation and two water conditions: Control and drought), with 5 repetitions, totaling 40 experimental units, each experimental unit was composed of a plant/pot.Analysis of variance of the results was applied and when there was a significant difference, the means were compared by Tukey test at 5% significance level.Moreover, the standard deviations were calculated for each treatment, and statistical analyzes performed by Assistant Version 7.7 Beta program.

Relative water content (RWC)
The RWC was determined at 06:00h a.m in each collect.The method used was that described by Slavick (1979).Results were expressed as a percentage, according to the formula: Where, FM1 = Fresh mass 1; FM2 = Fresh mass 2 with saturation; DM = Dry mass.

Determining the ammonium content
50 mg of previously lyophilised leaves and roots were weighed and put in a test tube containing 400 ml of total extract + 2.5 ml of solution A (5 g phenol + 0.025 g of sodium nitroprusside / 500 ml  distilled water) and homogenized by vortexing, adding 2.5 ml of Solution B (2.5 g NaOH + 12.6 ml of sodium hypochlorite / 500 ml distilled water), respectively.The free ammonium concentrations of the total extract were estimated from the standard curve constructed with (NH4)2SO4 p.a. (Sigma) according to the method described by Weatherburn (1967).

Determination of nitrate
50 mg each of previously lyophilized leaves and roots was weighed and mixed with extract containing 100 mL + 200 of salicylic acid 5% solution (w / v) in concentrated sulfuric acid.After stirring vigorously in a vortex stirrer was slowly added 4700 uL of 2N NaOH.The concentration of nitrate was obtained from a standard curve with increasing concentrations of NO3 (0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 μmol ml -1 ) according the method described by Cataldo et al. (1975).
Determining the proline content 50 mg were weighed of previously lyophilised leaves and roots by adding in the test tubes the total extract, 1 ml of ninhydrin acid and 1 ml of glacial acetic acid 99.5%.It was determined through a standard calibration curve using proline and proline contents in samples were extrapolated from the curve and expressed in mmol g -1 .The dry matter (DM) was determined according to Bates et al. (1973).

Determination of photosynthetic pigments
The determination of photosynthetic pigments was realized according to Lichtenthaler (1987).The concentrations of chlorophyll A, B and total (mg.L -1 ) were calculated using the formulas:

Membrane integrity (leak electrolytes)
The degree of membrane integrity was estimated by electrolyte leak according Blum and Ebercon (1981).The electrolyte leak was estimated by the following equation:

Superoxide dismutase (SOD)
The SOD activity was determined by inhibition of photoreduction of nitroblue tetrazolium chloride (NTC) according to Giannopolitis and Ries (1977).

Catalase (CAT)
CAT activity was determined by the method of Beers Jr. and Sizer (1952) with modifications.

Ascorbate peroxidase (APX)
The APX activity was determined by the method of Nakano and Asada (1981).

Relative water content (RWC)
The relative water contents present in the leaves of cumaru under water stress decreased as the weeks went by, on average control plants showed water percentage between 87.7 and 85.5%, and plants under drought between 88.3 and 37.5%, representing a decrease of 50.8%.The relative water content present in the leaves represent the water availability in the soil as well as the efficiency of the plant in pick up water in adverse conditions and maintaining water in the system reducing losses.Cumaru seedlings have the high water content in the leaf under normal conditions but had a sharp decrease due to lack of water.The decrease was significant from the 7 th day of water suspension and decreasing over the 21 days of stress, as shown in Figure 1.

Photosynthetic pigments
The chlorophyll A contents do not vary significantly throughout the experiment (Tukey test at 5% significance level), while the chlorophyll B and total had a significant reduction in plants under drought compared to control , while total chlorophyll was 6.37and 5.08 mmol.m -2 .s -1 in control plants and plants under drought, respectively (Figure 2).Representing a decrease of 34% to chlorophyll A, 55% for chlorophyll B and 45% for total chlorophyll compared the two water conditions on the 21st day of the experiment.According to Morais et al. (2007), chlorophylls A and B are interconverted in the chlorophyll cycle and form complexes of chlorophyll-protein, that are important in the regulation and organization of the photosystem.Chlorophylls play an important role in photosynthesis, are responsible for capturing light energy, especially chlorophyll A as the main pigment of complex light collectors (LHC) for the photochemical reactions (Taiz and Zeiger, 2013).
Under reduced stomata conductance and consequently lower influx of CO 2 proceeds in reduction of net assimilation rate, which directly affects the biochemistry of photosynthesis and reduces the photochemical energy consumption (Carmo et al., 2014).In these situations there is constant production of reactive oxygen species and other chlorophyll degradation agents (Matos et al., 2012).Chlorophyll degradation occurs according to the level of stress in the plants are submitted and the implication is leaf senescence, occurrence found in this study (Carmo et al., 2014).
In this work the chlorophyll A showed no significant difference, a fact that may be in accordance with the statement of Dinakar et al. (2012), in which the chloroplasts are particularly susceptible to oxidative damage and when it comes to tolerance to drought periods as well as the production of antioxidants, chlorophyll content is maintained after the drying, to prevent the formation of reactive oxygen species (ROS's).

Ammonium, nitrate and proline content
The ammonium and nitrate concentrations had no significant change throughout the experiment in plants under drought and the control plants.Proline already had a significant increase from the 14th day in the leaves and 21th day in roots.The values for ammonium in the last day of collection were 11.2 and 11.5 mmol of NH 4 + .Kg -1 DM in roots and 7.2 and 6.4 mmol of NH 4 + .Kg -1 DM in leaves, control and drought, respectively (Figure 3A).Nitrate was of 0.07 and 0.08 mmol from NO 3 -.Kg -1 DM in roots and 0.06 and 0.06 mmol of NO 3 -.Kg -1 DM in leaves, control and drought, respectively (Figure 3B).Proline was of 3.8 and 20.8 mmol of Pro.g -1 DM in roots and 2.3 and 29.8 mmol of Pro.g -1 DM in leaves, control and drought, respectively (Figure 3C).
Most plants have a preference for nitrate ion as a nitrogen source, so it is common their levels were lower than those found ammonium levels (Martinelli, 2003;Araújo et al., 2004), corroborating with these results.
The ammonium and the nitrate are the main forms of nitrogen available to plants, reduction processes and nitrogen assimilation can be absorbed both in the leaves and in the roots simultaneously or between these bodies becoming an essential process for the plant, since it is through it that It is controlled growth and development of the plant (Shan et al., 2012).
As a result, various forms of N available in the substrate can affect the morphological, physiological and biochemical plant, possibly in root growth, photosynthetic rates and catalytic activity of several enzymes (Li et al., 2013).In studies comparing the nutrition with nitrate (NO 3-) or ammonium (NH 4 + ) show that these nitrogen sources can induce different metabolic responses (Patterson et al., 2010).
The accumulation of soluble solutes in plant cells provides a type of response to water deficit, called osmotic adjustment, which allows more negative water Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.potential in leaves, thus helping to keep the movement of water to the leaves (Silva et al., 2014).Proline has been highlighted as a compatible solute occurs in plants in response to environmental stresses that solute accumulates variety of plant species in response to stresses such as drought, heavy metals, extreme temperatures, salinity and ultraviolet radiation (Siripornadulsil et al., 2002).It is possible to note a proline increased much more significant in the leaves than the roots that this fact can be given by the need of the plant to have a more negative potential in the leaves so that the water can reach the highest parts of the plant.Proline contents only had increased from 14 days even if there is already a significant reduction in the RWC on the 7th day, this may be because the proline can be a compatible solute (osmoprotectors organic compound and osmoregulator) more secondary role in Cumaru species as it was the case in the study of Pinhão-manso (Sousa et al., 2012), which highlighted the glycine main osmoregulator and osmoprotectors.

Electrolyte Leak
Results show that there was a significant increase in both the leaves and the roots that were under water deficit with values of 12.2 to 29.58% and 12.2 to 60.73% for the leaves (plant control and drought, respectively).As well as 24.56 to 28.55% and 24.6 to 51.29% for the roots (plants control and drought), respectively (Figure 4) with a 39.55% increase in percentage for leaves and roots 22.74% to the 21 day of experiment.Lack of water causes a decrease in liquid photosynthesis and in this case the sharp reduction of water in the cumaru plants probably caused this decrease in liquid photosynthetic rate and to produce more O 2 -and H 2 O 2 in chloroplasts (Blokhina et al., 2003;Reddy et al., 2004).The increased cellular leak in plants under drought is strongly related to the damage caused by free radicals O 2 -that attacks different parts of the plant as lipids and membrane proteins, nucleic acids and others causing cell death.

Superoxide dismutase (SOD)
Plants subjected to drought showed a significant increase when compared to the control plants over the 21 days of experiment (Figure 5).The values for the roots were 49.86 to 50.85 mg .protein(control plants and under drought), respectively.Plants have enzymatic systems of defense against reactive oxygen species, including SOD, CAT, APX.Activation of genes encoding these enzymes in response to oxidative stress was observed, for examples, tobacco (Bowler et al., 1991), soybean (Lee et al., 1999), and peanut (Sankar et al., 2007).Thus, increased activity of these enzymes is directly related to differential expression of the genes belonging to the antioxidant system, having as one of its functions to prevent H 2 O 2 accumulation in cells (Eyidogan and Oz, 2007;Vaidyanathan et al., 2003).

Catalase (CAT)
The enzyme catalase showed significant difference from the 14th day of the experiment (Figure 6), with values for the roots of 0.042 mg   Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.Akcay et al. (2010), who studied the CAT activity in creeping and erect peanut, found that the enzyme activity increased significantly when subjected to higher stress levels, confirming the results of this work.According to these authors, the CAT is one of the most effective defense enzymes in oxidative processes, since, in the resistant plants enables the integrity of the cell even when the stress is in a more rigorous stage.These results are reported in previous studies of water stress, salinity and other stresses, which reported that there is a reduced production of ROS in tolerant genotypes than in susceptible genotypes (Karabal et al., 2003;Chaitanya et al., 2002;Bhoomika et al., 2013).According to Sankar et al. (2007), as can be seen in his work, where an average increase of up to 230% of activity was obtained at the earliest material, when subjected to 10 days of water suppression.

Ascorbate peroxidase (APX)
The values of APX enzyme showed significant difference after 7 days of the experiment in plants were subjected to drought, when compared with control plants.The increase for the roots was from 0.0298 to 0.032 mmol.min - and of 0.0293 to 0.0376 mmol.min - in control plants and under drought, respectively.The leaves show values of 0.0315 to 0.0322 mmol.min - and of 0.0309 to 0.0405 mmol.min - in control plants and under drought, respectively (Figure 7).These results highlight that

2013).
Thus, the potential oxidative damage due to drought was adequately mitigated by the constitutive activity APX (Cruz et al., 2013).Possibly, this gives rise to the triggering of multiple strategies antioxidants (Silva et al., 2015).
The species studied presents different mechanisms to overcome the drought periods, either by maintaining low RWC values and photosynthetic pigments, or by the increased activity of oxidative enzymes which are variables that can be used as water stress sensitivity indicator.
Young plants of cumaru are not tolerant to more than 21 days of water stress, and respond very negatively to the conditions of low water availability in the soil.

Figure 1 .
Figure 1.Relative water content in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.
plants.Mean values for chlorophyll A were 3.31 mmol.

Figure 2 .
Figure 2. Chlorophyll content A (A), chlorophyll B (B) and chlorophyll total (C) in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.

Figure 3 .
Figure 3. Ammonium levels (A), nitrate (B) and proline (C) in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.

Figure 4 .
Figure 4. Electrolyte leak in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.
control plants and under drought), respectively.For the leaves the results were similar with values of 41.06 to 40.35 mg -1 .proteinand of 40.91 to 49,2 mg -1

Figure 5 .
Figure 5. Superoxide dismutase enzyme activity in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.

Figure 6 .
Figure 6.Enzyme catalase activity in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.

Figure 7 .
Figure 7. Enzyme peroxidase activity in young plants Cumaru subjected to water deficit.Capital letters show statistical differences between water conditions and lower statistical differences between the collections time.Tukey test p < 0.05 probability was used for comparison.

Table 1 .
Solution cocktail containing macro and micronutrients. 08