Ecophysiological and biochemical behavior in young plants of Parkia gigantocarpa Ducke subjected to waterlogging conditions

Ecophysiological and biochemical behavior in young plants of Parkia gigantocarpa Ducke subjected to waterlogging conditions Waldemar Viana de Andrade Júnior, Benedito Gomes dos Santos Filho, Cândido Ferreira de Oliveira Neto*, Angelo Cleiton da Costa Pereira, Raimundo Thiago Lima da Silva, Ismael de Jesus Matos Viégas, Kerolém Prícila Sousa Cardoso, Luma Castro de Souza, Tamires Borges de Oliveira and Ricardo Shigueru Okumura

and forest plantations.The low diversity of tree species that can be used for recovery of areas in waterlogging conditions is a major challenge for environmental managers, because of the difficulty of species with adaptations to tolerate conditions of absence of oxygen (Lopez and Kursar, 2003).Thus, the identification of species that tolerate the waterlogging, including tree species with economic and environmental potential, is a viable alternative for the recovery of degraded areas.
An alternative is the cultivation of Parkia gigantocarpa, characterized as a neotropical tree that naturally occurs in terra firme forest and waterlogging conditions.Due to present rapid growth, uniformity, low death rate, economic and ecological potential, the tree is indicated as promising species for recovery of altered or degraded areas, especially those of permanent preservation, too, can be used in forestry systems.The waterlogging condition promotes the absence of oxygen in the soil, reducing the root system and the vegetation growth of trees which negatively affects the physiological processes of plants, including stomatal conductance, photosynthesis, and the hydraulic conductivity of the root and the synthesis and translocation of assimilates (Parent et al., 2008).
The stomatal closure contributes to the preservation of leaf water potential, and decreasing the reduction of hydraulic conductivity of roots (Kreuzwieser et al., 2004).Thus, the stomatal closure, the reducing evaporation, and the maintenance of leaf water potential are strategies developed by plants in waterlogged conditions to minimize water loss (Davanso et al., 2002).The root system is the respiratory glucose dissimilation of glucose fermentation with low output power (Drew, 1997), resulting in decreased absorption of nutrients for the plants (Rocha et al., 2010), interfering with the absorption of nitrate, due to change in nitrate-reductase activity, thereby influencing the nitrogen metabolism, amino acids (Liao and Lin, 2001), proteins and enzymes (Sairam et al., 2008).Alam et al. (2011) observed that plants subjected to waterlogging conditions showed sugar decomposition processes, such as glycolysis and fermentation, associated with induction of synthesis of various stress proteins.The enzymes of fermentation pathways as lactate dehydrogenase, pyruvate decarboxylase, and alcohol-dehydrogenase are synthesized under conditions of absence of oxygen (Zabalza et al., 2009), thus, the activity of these enzymes are indicative of a possible adaptation of the plant response to waterlogging conditions, although it was not explained the extent of the contribution and how they relate to low-O 2 (Dennis et al., 2000).
Recommendation of P. gigantocarpa by farmers is necessary to obtain information about their ecophysiological and biochemical behavior in waterlogged conditions.The understanding about the behavior of tree species in the early stages of its development under waterlogging conditions can support reforestation of degraded areas.In this sense, the objective of the study was to evaluate the ecophysiological and biochemical behavior of young plants of P. gigantocarpa subjected to waterlogging conditions.

Plant materials
The P. gigantocarpa (Ducke) seeds were collected from location 03°41' 07" S and 48°38' 04" W. Seeds were scarified in the lateral region and adjacent to the hilum, and were immediately sown in plastic trays with capacity of 5 L, containing sterile sand.After emergence, the seedlings were transplanted to black opaque polythene bags with dimensions of 25 x 15 cm in height and diameter, respectively, with perforations in all sides, containing the substrate Plantmax ® (composed of composted pine bark, peat, vegetal charcoal, and vermiculite).The seedlings were grown for 60 days.After this period, the young plants were evaluated for height, stem diameter, number of leaves and number of leaflets, and transferred to plastic pots with a capacity of 14 kg, containing the same substrate used in transplanting.The pots with the young plants were taken to the greenhouse to acclimatize for a period of 45 days.

Study location
The experiment was conducted in a greenhouse belonging to Instituto de Ciências Agrárias (ICA) of the Universidade Federal Rural da Amazônia (UFRA) in Belém city, State of Pará, Brazil (01°28' 03"S, 48°29'18"W) during July until November of 2012.The biochemical analyzes were performed at the Plant Physiology Laboratory at the Universidade Federal Rural da Amazônia (UFRA) in Capitão Poço city, State of Pará, Brazil.

Experimental design
The experimental design was completely randomized with two water conditions (control and waterlogging) combined with five evaluation times (0, 4, 8, 12, and 16-days waterlogging conditions), with five replicates, and 50 experimental units in total.Each experimental unit consisted of one plant per pot.

Waterlogging application and plant treatments
After the acclimation period, all the four-month-old P. gigantocarpa were subjected to two water conditions and the control plants were irrigated daily with 2.5 L of water to replace the water lost by evaporation, made individually for each pot, and considering the daily weighting set (pot+plant+soil).In the treatment under waterlogging conditions, the plants were placed in pots without holes to avoid water drainage, with the water level maintained at 5 cm above the soil surface.Control and waterlogging plants remained for a period of 16 days under these conditions.
Leaf specific hydraulic conductance: The leaf specific hydraulic conductance was calculated in agreement with equation: , where KL = leaf specific hydraulic conductance, gsmd = stomatal conductance in midday, Δwmd = variation in water saturation during midday, pΨ = leaf water potential in predawn and mdΨ = leaf water potential in midday.The measurements were carried out during 0, 4, 8, 12, and 16-days of waterlogging conditions.
Nitrate concentration: Nitrate was determined with 100 mg of leaf dry matter powder incubated with 5 mL of sterile distilled water at 100°C for 30 min; after the homogenized mixture was centrifuged at 10,000 g for 15 min at 25°C and the supernatant was removed.The quantification of the nitrate was carried out at 410 nm in agreement with Cataldo et al. (1975), KNO3 (Sigma Chemical) was used as standard.

Nitrate-reductase activity:
The extraction of the nitrate-reductase enzyme was carried out with leaf disks until the weight of 200 mg was reached, the samples were incubated in 5 mL of extraction mix [0.1M KH2PO4, 50mM KNO3, isopropanol at 1% (v/v) and pH 7.5] by 30 min at 30°C, and all the procedures were carried out in dark.The quantification of the enzyme activity was made by the method of Hageman and Hucklesby (1971) with absorbance at 540 nm using spectrophotometer (Quimis, model Q798DP), nitrite (Sigma Chemicals) was used as standard.

Glutamine-synthetase activity:
The extraction of glutaminesynthetase enzyme was carried out with 200 mg leaf tissue ground in liquid nitrogen, the samples were incubated in 5 ml of extraction mix [Tris-HCl buffer pH 7.6 containing 10 mM MgCl2. 10 mM βmercaptoethanol, 5% (w/v) PVP and 5 mM EDTA]; after the homogenized mixture was centrifuged at 30,000 g for 10 min and the supernatant was removed.All the procedures were carried out in the interval of 0 to 4°C.The quantification of the enzyme activity was made by the method of Kamachi et al. (1991) with absorbance at 540 nm; γ-glutamylhydroxamate (Sigma Chemicals) was used as standard.
Total soluble amino acids: Determination of amino acids was performed using 50 mg of leaf dry matter powder, and was incubated with 5 ml of sterile distilled water at 100°C by 30 min, the homogenized was centrifuged to 2,000 g by 5 min at 20°C, and supernatant was removed.Quantification of the total soluble amino acids was carried out at 570 nm according to Peoples et al. (1989), L-asparagine and L-glutamine (Sigma Chemicals) were used as standard.
Proline: Proline level was determined with 50 mg of leaf dry matter poder, which was incubated with 5 ml of sterile distilled water at 100°C by 30 min after the homogenized was centrifuged to 2,000 g by 5 min at 20ºC.Quantification of proline was carried out at 520 nm according to Bates et al. (1973), in which L-proline (Sigma Chemicals) was utilized as standard.

Glycine-betaine:
The determination of glycine-betaine was carried out with 25 mg of powder incubated with 2 ml of distilled water.The homogenized mixture was kept in agitation for 4 h at 25°C, after this period t was centrifuged at 10,000 g for 10 min at 25°C, and subsequently the supernatant was removed.The quantification of glycine-betaine was carried out at 365 nm in agreement with Grieve and Grattan (1983), glycine-betaine (Sigma Chemicals) was used as standard.

Alcohol-dehydrogenase and lactate-dehydrogenase activities:
Enzymes alcohol-dehydrogenase and lactate-dehydrogenase were extracted from 200 mg of leaf and root tissues.Samples incubated in 2 ml of extraction mix (Tris-HCl buffer at 50mM, tiamina pirofosfato at 0.5mM, dithiothreitol at 2mM, EDTA at 1mM, NaCl at 110mM, MgCl2 at 2.5 mM, with pH adjusted to 6.8).After homogenization, samples were centrifuged at 10,000 g for 10 min, and the supernatant was removed.All the procedures were carried out in the interval of 0 to 4°C.Quantification for alcoholdehydrogenase was based on the method of Bertani et al. (1980), and lactate-dehydrogenase was based on methodology described by Hoffman and Hanson (1986), with both determinations under absorbance at 340 nm, with NADH (Sigma Chemicals) as a standard.

Data analysis
The data were subjected to variance analysis and significant differences between means were determined by F test at 5% level of error probability (Steel et al., 2006).The standard deviations were calculated to each treatment in all evaluation points.The correlation analysis was performed by the Pearson parametric method, and the statistical procedures were carried out with the SAS software (SAS Institute Inc, 2008).

Water potential (Ψ) in plants subjected to waterlogging
In assessing the p Ψ and x Ψ it was observed significant difference between treatments (P≤0.001) and between the evaluated periods (P≤0.001)further showing interaction between the factors (P≤0.001)(Figure 1).The p Ψ (4:30-5:30 h) in the control plants subjected to waterlogging conditions was -0.28 and -0.40 MPa, respectively, giving a decrease of 42.85% in plants in waterlogged conditions compared to the control (Figure 1A).In times of 10:00 to 12:00 h the x Ψ was -0.84 and -1.69 MPa, respectively, resulting in decrease of 101.19% in plants subjected to waterlogging compared to the control (Figure 1B).

Influence of waterlogging conditions on hydraulic conductivity of P. gigantocarpa plants
Figure 2 showed a significant interaction between treatments (P≤0.001) and periods of exposure to stress (P≤0.05), as well as the interaction between the factors (P≤0.001).In control and flooded plants, the values obtained were 0.29 and 0.02 mol m -2 s -1 Mpa -1 , respectively, thus, the plants grown in the absence of oxygen conditions showed a decrease of 93.1% compared to control plants.

Interference induced by waterlogging conditions on nitrate concentration in young plants
The analysis of the nitrate concentration in young plants  of P. gigantocarpa showed significant differences between treatments (P≤0.001) and periods of exposure to stress (P≤0.001) as well as the interaction between the factors (P≤0.001)(Figure 3).In plant leaves from control and waterlogging conditions the values obtained were 0.48 and 0.13 µmoles g -1 of N0 3 -, respectively (Figure 3A).While, in the root system the nitrate concentrations were 0.63 and 0.14 µmoles g -1 of N0 3 -the control and flooded plants, respectively (Figure 3B).Thus, though the results showed a decrease of 72.92 and 77.80% in plants subjected to waterlogging conditions compared to control plants.

Effect of waterlogging conditions on nitratereductase activity (NRA) in leaves and roots of P. gigantocarpa
The nitrate-reductase activity was significantly decreased in the flooded plants, regardless of the sampled vegetative parts, thus, the absence of oxygen promoted reduction of the nitrate-reductase activity in both leaves and roots of young plants of P. gigantocarpa (Figure 4).The values obtained for the NRA variable showed significant differences between treatments (P≤0.001) and periods of exposure to stress (P≤0.001), as well as the interaction between the factors (P≤0.001).In plant leaves from control and waterlogging conditions, the values obtained were 0.83 and 0.58 µmoles g -1 of NO 2 -, respectively, thus, showing reduction of 30.12% in plants subjected to waterlogging conditions compared to control plants (Figure 4A).In the roots, the values were 0.85 and 0.63 µmoles g -1 of NO 2 -, respectively, resulting in reduction of 25.88% in the waterlogging conditions compared to the control plants (Figure 4B).

Modifications produced by waterlogging conditions on glutamine-synthetase (GS) of P. gigantocarpa plants
The waterlogging conditions significantly reduced the activity of glutamine-synthetase in leaves and roots of flooded plants (Figure 5).The values obtained for GS variable showed significant difference between treatments (P≤0.001), and the periods of exposure to stress (P≤0.001), as well as the interaction between factors (P≤0.001).In leaves of control and flooded plants, the concentration of glutamine-synthetase was 27.8 and 10.8 mmoles kg -1 of GGH, respectively, corresponding to a 61.15% reduction in waterlogging conditions compared to control plants (Figure 5A).In the roots, the concentration of glutamine-synthetase was 25.1 and 6.7 mmoles kg -1 de GGH in control and flooded plants, respectively, with a decrease of 73.3% in the waterlogging conditions (Figure 5B).

Impact of waterlogging conditions on total soluble amino acids in plants
The total soluble amino acid concentrations in leaves and roots of young plants of P. gigantocarpa varied significantly between the treatments (Figure 6).The evaluations showed significant differences between the treatments (P≤0.001), and periods of exposure to stress (P≤0.001), as well as the interaction between the factors (P≤0.001).The analysis of the total soluble amino acid in the leaves of the control plants showed a concentration of 36.8 µmol g -1 of amino acid, while the plants subjected to waterlogging conditions provided value of 23.4 µmol g -1 of amino acid (Figure 6A).By the information presented in Figure 6A, a decrease of 93.47% in plants subjected to waterlogging conditions compared to the control plants was observed.In the roots of young plants of P. gigantocarpa were observed values 52.23 and 17.45 µmol g -1 of amino acid in control and flooded plants, respectively, representing a decrease of 66.59% in plants subjected to waterlogging conditions compared with the control plants (Figure 6B).

Effects on proline in plants subjected to waterlogging conditions
By the information presented in Figure 7, increase in proline concentrations in leaves and roots of plants subjected to waterlogging conditions was observed.The results showed significant differences between the treatments (P≤0.001), and periods of exposure to stress (P≤0.001), as well as the interaction between the factors (P≤0.001).In leaves of control and flooded plants the concentrations were 1.37 and 5.16 µmol g -1 of proline, respectively, an increase of 276.64% of proline in plants subjected to waterlogging conditions compared the control plants (Figure 7A ).While, in the roots of young plants of P. gigantocarpa were obtained values of 1.52 µmol g -1 of proline in control plants, and 4.19 µmol g -1 of proline in plants subjected to waterlogging conditions, an increase of 175.6% in plants subjected to waterlogging conditions compared to control plants (Figure 7B).

Effects promoted by waterlogging conditions on glycine-betaine of P. gigantocarpa plants
For the variable glycine-betaine results showed significant differences between the treatments (P≤0.001), and periods of exposure to stress (P≤0.001), as well as the interaction between the factors (P≤0.001)(Figure 8).The evaluation of the concentration of glycine-betaine in the leaves of control and flooded plants showed values of 4.78 e 15.19 µg g -1 of glycine-betaine, respectively, thus, an increase of 217.78% in plants subjected to waterlogging conditions were compared to control plants (Figure 8A).For the roots, the values obtained were 6.57 and 15.29 µg g -1 of glycine-betaine in the control and flooded plants, respectively, thus, an increase of 132.72% in plants subjected to waterlogging conditions compared to control plants (Figure 8B).

Interference induced by waterlogging conditions on alcohol-dehydrogenase (ADH) and lactatedehydrogenase activity (LDH) in young plants of P. gigantocarpa
The absence of oxygen significantly affected the activity of ADH (Figure 9) and LDH (Figure 10) of young plants of P. gigantocarpa, both the aerial part and the root system.The analysis of the activity of enzymes dehydrogenase alcohol-dehydrogenase and lactate-dehydrogenase activity showed significant differences between treatments (P≤0.001), and periods of exposure to stress (P≤0.001), as well as the interaction between the factors (P≤0.001).In the leaves of ADH, activity was 0.06 and 2.88 moles of NADH H + kg -1 of protein min -1 in control and flooded plants, respectively, with an increase of 97.92% compared to the control plant (Figure 9A).In the roots of the enzyme activity in the control and flooded plants were 2.6 and 12.0 moles of NADH H + kg -1 of protein min plants subjected to waterlogging conditions compared to control plants (Figure 9B).The LDH activity in leaves were 0.08 and 2.21 moles of NADH H + kg -1 of protein min -1 in control and flooded plants, respectively, with a 96.38% increase compared to the control plant (Figure 10A).The roots values obtained were 2.16 and 7.6 moles of NADH H + kg -1 of protein min -1 in control and flooded plants, respectively, thus, an increase of 71.58 % was observed in plants subjected to waterlogging conditions compared to control plants (Figure 10B).

DISCUSSION
The reduction of the root system of young plants of P. gigantocarpa, contributed in reducing x Ψ, which continued until the 8th day of waterlogging conditions (Figure 1B).This period observed relative stabilization of x Ψ.This can be explained by the appearance of hypertrophic lenticels, which improves aeration of the roots and minimizes decrease in x Ψ. Folzer et al. (2006) studying in young plants of Quercus petraea in waterlogged conditions for 14 days, found out that the increased in x Ψ in flooded plants, coincided with the appearance of hypertrophic lenticels after 10 days of water saturation.Islam et al. (2010) studying the effect of waterlogged conditions in young plants of two genotypes of Vigna radiata (GK48 e BARImung5), observed a significant reduction of x Ψ in flooded plants compared to control plants.These authors attributed the smaller reduction of x Ψ in the GK48 genotype production of a bigger amount of adventitious roots.In this study, the relative stabilization of x Ψ was not influenced by the formation of adventitious roots, since the reduction in the decrease of x Ψ began on the 8th day of waterlogging conditions (Figure 1B), in which the adventitious roots were identified in the last evaluation (16th day of waterlogged conditions).
Through the information presented in Table 1, negative correlation was observed (P≤0.001) between x Ψ with proline and glycine-betaine.Thus, the increase of compatible osmolytes, possibly, contributed to reduce the water potential of the plant tissue, indicating a possible occurrence of osmotic adjustment in young plants of P. gigantocarpa.Gimeno et al. (2012) studying young plants of Jatropha curcas L. submitted the waterlogged conditions for 10 days, observed a significant reduction in x Ψ of flooded compared to the control plant, as a result of plant subject to waterlogged conditions did not increase the concentration of proline.Similar results of x Ψ were described by Alves et al. (2012) in Tabebuia serratifolia (Vahl) Nicholson submitted to waterlogged conditions for 9 days, in which they observed values of -2.3 MPa in plants subjected to waterlogged conditions.The hydraulic conductivity of the root was significantly correlated with the x Ψ, stomatal conductance and transpiration plant (Table 2).Thus, the decrease in hydraulic conductivity possibly influenced the reducing of x Ψ, and stomatal closure of P. gigantocarpa in order to avoid the internal water deficit.Then, the hydraulic conductivity worked as a corregulador while the x Ψ, stomatal conductance and plant transpiration in plants were subjected to waterlogged conditions.
The young plants of P. gigantocarpa showed significant reduction of nitrate-reductase activity, in the roots and the leaves, with increasing time of exposure to waterlogged conditions.Possibly, the water saturation in the soil caused an increase of inorganic phosphate, as a function of the reduction of ATP, promoting the phosphorylation of nitrate reductase, providing its connection with the 14-3-3 protein, resulting in decreased enzyme activity in the plant.The results obtained for the nitrate-reductase activity in young plants of P. gigantocarpa corroborate with the studies described by Alves et al. (2012), in which they observed a reduction of this enzyme in roots and leaves of Tabebuia serratifolia (Vahl) Nicholson subjected to waterlogged conditions for 9 days.
For the induction of nitrate-reductase activity, it required the presence of NO 3 -, thus, the nitrate-reductase activity is regulated by NO 3 -metabolism located in the cytoplasm (Allègre et al., 2004).The decrease in the concentration of NO 3 -with the period of exposure to waterlogged conditions of young plants of P. gigantocarpa limited the nitrate-reductase activity (Figures 3 and 4), affecting the assimilation of NO 3 -and influenced on nitrogen metabolism, following the reduction in glutamine-synthetase (Figure 5) amino acids at the root and the leaves of the plants.
The reduction of nitrate-reductase activity in leaves of young plants of P. gigantocarpa subjected to waterlogged conditions, is related to low translocation of NO 3 -from the root (Alaoui-Sosse et al., 2005), demonstrating the dependence of nitrate-reductase by NO 3 -carried in the transpiration stream, which in this study was affected, probably, by reducing the water potential of the leaf xylem (Figure 1B) of stomatal conductance and hydraulic conductivity (Figure 2) in young plants of P. gigantocarpa subjected to waterlogged conditions.However, the reduction of the nitrate-reductase activity in flooded plants is related to the increase in alcoholic fermentation, evidenced by the increase in the ADH activity (Figure 9), the resulting pH increase, thus, inhibiting the activity of nitrate-reductase.
The reduced activity of glutamine-synthetase in the flooded plants, is related to the reduced availability of ATP, since this enzyme is strongly dependent on energy derived from ATP phosphorylated produced in the glycolytic pathway, in mitochondrial oxidative phosphorylation and during the photosynthetic activity in leaves.In T. serratifolia, plants were subjected to waterlogged conditions, the activity of this enzyme in the roots and leaves, significantly reduced compared to the activity in plants in aerobic conditions (Alves et al., 2012).The authors attributed the decrease in activity of this enzyme, and reduced synthesis of ATP in the cell tissues.
Another justification for the reduction of the glutaminesynthetase activity in plants subjected to waterlogged conditions is the decrease in glutamate synthase and, of glutamate, in the chloroplast or the cell plastids (Horchani and Aschi-Smiti, 2010), or the reduction of the nitratereductase activity, caused by the ion NH 4 + limitation (Alves et al., 2012).The main pathway of uptake of NH 4 + in plants occurs by means of glutamine-synthetase activity, which is dependent of ATP, acting as a catalyst of binding of NH 4 + with glutamic acid to form glutamine (Masclaux-Daubresse et al., 2010), this amino acid is the main source of organic nitrogen transported from roots to leaves through the xylem to the biosynthesis of all nitrogenous compounds (Okumoto and Guillaume, 2011), justifying the results obtained in this study, in which there was an observed reduction in glutamine-synthetase activity in plants P. gigantocarpa subjected to waterlogged conditions (Figure 5), simultaneously with NH 4 + accumulation and reducing nitrogen compounds, such as amino acids.In plants of S. lycopersicum subjected to waterlogged conditions, the activity of glutamine-synthetase was significantly inhibited in the leaves, together with the increase of concentration of NH 4 + in the plant tissue compared with plants grown under aerobic conditions (Horchani and Aschi-Smiti, 2010).According to these authors, the reduction in glutamine-synthetase activity in the leaves of S. lycopersicum plants is related to a reduction of glutamate, which was used in the synthesis of proline and glycinebetaine, as an alternative for maintaining the metabolism and osmotic adjustment.
The glutamine-synthetase is an enzyme precursor in the formation of all amino acids at the root and leaves of higher plants (Taiz and Zeiger, 2013), therefore, in this study the young plant of P. gigantocarpa decreased the glutamine-synthetase activity (Figure 5), justifying the reduction of total soluble amino acids in the leaves and roots of P. gigantocarpa plant (Figure 6).Alves et al. (2012) observed that decreased glutamine-synthetase activity promoted reduction of 87.6% of amino acids in roots and 76.2% in leaves of young plants T. serratifolia subjected to waterlogged conditions for 9 days.Another possible explanation for the reduction of amino acids is due to waterlogged conditions which has promoted the reduction in ATP synthesis, resulting in a decrease in the absorption of nitrate, or, according to Kreuzwieser et al. (2009) the reduction of amino acids in plants subjected to waterlogged conditions is due to dynamic changes of the transcriptional levels that encode enzymes involved in its metabolism.In Table 1, a significant positive correlation coefficient (P<0.001) between the proline and the concentration of ammonium was observed.In plants subjected to waterlogged conditions, the increased ammonium concentration, or protein breakdown by proteolytic enzymes, possibly, was responsible for the high concentration of free proline, in which it is important in the protection of cellular structures against oxidative damage caused by free radicals (Kavi-Kishor et al., 2005).Furthermore, the increase in proline may have acted as carbon and nitrogen source for plant growth (Silva-Ortega et al., 2008) or, in which it is an osmoprotectors amino acid, assisted in reducing the water potential of tissues, reducing dehydration.The results obtained in P. gigantocarpa corroborate with the studies by Horchani et al. (2010) and Parvin and Karmoker (2013) observed an increase of proline in roots and leaves of plants of S. lycopersicume and C. capsularis L. subjected to waterlogged conditions.
In the present study, a significant negative correlation (P≤0.001) between the glycine-betaine and the x Ψ in plants subjected to waterlogged conditions (Table 1) was observed.The increase of glycine-betaine in leaves and roots is probably related to osmotic adjustment hyaloplasm plants subjected to stress (Jaleel et al., 2007), thus, the accumulation of glycine-betaine enable the reduction of cellular water potential during periods of osmotic stress (Taiz and Zeiger, 2013), favoring the absorption and soil water transport to aerial part of the plant, thereby, protecting the plant tissues and physiological processes, improving plant tolerance to abiotic stress.Another factor that contributes to the increase in glycine-betaine concentrations was the synthesis of amino acids due to the breakdown of proteins and increased ammonia concentrations promoted by photorespiration (Colmer et al., 2009).
The high activity of ADH with the exposure period to waterlogged conditions (Figure 9), demonstrates that P. gigantocarpa, requires, among other mechanisms, rapid and permanent use of fermentative pathway metabolism as a way of maintaining, regenerating the reducing power and ATP production, having an efficient anaerobic respiration when subjected to lack of oxygen in roots.The high activity of LDH (Figure 10), and consequently, the signaling mechanism is required to initiate or promote the activity of ADH in the stimulation of ethanolic fermentation, by consuming more protons than lactic, increasing the cytosolic pH survivability of plants subjected to waterlogged conditions (Dolferus et al., 2008), which explains the results obtained in this study.
The results obtained in this study, the increase of ADH and LDH activity in leaves and roots is probably a strategy for continued growth and survival of young plants of P. gigantocarpa under waterlogging conditions.Furthermore, the synthesis of enzymes ADH and LDH act as an alternative to compensate the depletion of proteins (Zabalza et al., 2009) in which it occurs during oxygen deficiency.

Conclusions
The waterlogged conditions adversely affect the water potential, hydraulic conductivity, concentration of nitrate, nitrate-reductase activity, glutamine-synthetase and concentration of total soluble amino acids in young plants of P. gigantocarpa.In contrast, the concentrations of proline, glycine-betaine and the activities of alcoholdehydrogenase and lactate-dehydrogenase showed an increase in plants grown under conditions of absence of oxygen.Thus, the young plants of P. gigantocarpa cannot be recommended for cultivation in waterlogged conditions.

Figure 1 .
Figure 1.Predawn water potential (A) and xylem water potential of the leaf (B) in young plants of P. gigantocarpa subjected to waterlogging conditions.Squares represent the mean values of five replicates, and error bars represent the mean standard errors.

Figure 2 .
Figure 2. Hydraulic conductivity in young plants of P. gigantocarpa subjected to waterlogging conditions.Squares represent the mean values of five replicates, and error bars represent the mean standard errors.

Figure 3 .
Figure 3. Nitrate concentration in the leaf (A) and the root (B) in young plants of P. gigantocarpa subjected to waterlogging conditions.Squares represent the mean values of five replicates, and error bars represent the mean standard errors.

Figure 4 .Figure 5 .Figure 6 .
Figure 4. Nitrate-reductase activity in leaf (A) and the root (B) in young plants of P. gigantocarpa subjected to waterlogging conditions.Squares represent the mean values of five replicates, and error bars represent the mean standard errors.

Figure 7 .
Figure 7. Proline in leaf (A) and the root (B) in young plants of P. gigantocarpa subjected to waterlogging conditions.Squares represent the mean values of five replicates, and error bars represent the mean standard errors.

Figure 8 .Figure 9 .Figure 10 .
Figure 8. Glycine-betaine in leaf (A) and the root (B) in young plants of P. gigantocarpa subjected to waterlogging conditions.Squares represent the mean values of five replicates, and error bars represent the mean standard errors.