Antioxidant Enzymes Activity in the Elaeis guineensis Jacq. Submitted to Drought

Oil palm is a very responsive culture in relation to climate change that intensifies or lowers its productivity. Thus, the objective of this study is to evaluate the activity of antioxidant enzymes in two genotypes of E. guineensis, both under water deficiency. The experiment conducted in a greenhouse at UFRA used genotypes 2528 and 2501 of E. guineensis submitted to water deficiency from the 10 day. The biochemical analysis was evaluated at the 5% level of significance by the Tukey test. The antioxidant variables analyzed were superoxide dismutase, catalase activity, ascorbate peroxidase activity, Malondialdeído (MDA), Glutathione and ascorbic acid content. In view of the obtained results, it was observed increases of the antioxidant enzymes when the genotypes were submitted to the water deficiency, presented significance for the results. Therefore, the study suggests that oil palm had a good use and adaptation when submitted to water deficit and that genotype 2528 was more responsive to maintain its vital biochemical activities. Keyword: biochemistry, hydric stress, oxidative stress


Introduction
Drought is one of the environmental factors of greater agricultural importance, as it causes many physiological, metabolic and morphological alterations causing numerous deleterious effects on plant growth, water relations and photosynthesis (Hasanuzzaman et al., 2014).Concomitant to this, the oil palm responds negatively when subjected to drought affecting the productive processes of the species (Al-Amin et al., 2011).
The oil palm (Elaeis guineensis Jacq.) is an oleaginous plant belonging to the Arecaceae family and cultivated mainly in tropical regions such as Latin America, Southeast Asia and Africa (Luis et al., 2010).Because it is a cultivation of manual and long cycle cultivation, it has great productive potential, standing out for the high productivity of oil, used in food, industrial and agro-energy processes (Zimmer, 2010).
Several studies indicate that under conditions of water deficiency the activity of photosynthesis is reduced, regardless of the type of metabolism (Cha-um et al., 2010;Cao et al., 2011;Son et al., 2011;Zlatev & Lindon, 2012;Ashraf & Harris, 2013).Thus, the reduction of palm oil production can be directly related to the inhibition of the photosynthetic rate, which is caused, among other factors, by the low relative water content (Fahramand et al., 2014;Zain et al., 2014).
As a consequence, decreases in CO 2 fixation under stress conditions can cause photochemical and biochemical imbalances of photosynthesis (Asada & Badger, 1984).Resulting in excessive production of reactive oxygen species (ROS) which are unstable molecules capable of causing enzymatic damage affecting proteins, carbohydrates, lipids and nucleic acids (Silva & Gonçalves, 2010).
These molecules can be formed as a result of excitation, producing singlet oxygen ( 1 O 2 ) or reducing O 2 to the anionic superoxide radical (O 2 •-), hidroperoxylic radical (HO 2

•
), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH • ) (Bhattacharjee, 2010).Despite the importance of O 2 for the performance of cellular functions, under stress conditions there is ROS formation in metabolic events (Karuppanapandian et al., 2011).However, plants have a complex antioxidant system to contain the deleterious effects of reactive oxygen species, in which specific enzymes act to neutralize the action of these radicals (Miller et al., 2010), among the antioxidant enzymes superoxide dismutase (SOD), ascorbato peroxidase (APX), catalase (CAT) among others, and among the main antioxidant metabolites are ascorbic acid (AsA) and the glutathione (GSH) (Kim & Kwak, 2010).
Therefore, due to the great importance of the oil palm it is necessary to understand its physiological or biochemical functions when submitted to regions with water restriction.Thus, the objective of this study is to evaluate the activity of antioxidant enzymes in two genotypes of E. guineensis, both under water deficiency.

Plant Material and Treatment
Young plants of E. guineensis with 10 months from Embrapa Amazônia Oriental with similar aspects and sizes were selected and placed in 20 L pots filled with soil and bovine manure (3:1 v/v) substrate.The experiment was carried out in a greenhouse at the Federal Rural University of Amazônia, Capitão Poço campus (Latitude 1°44′47″S and Longitude 47°3′34″W), Brazil, and biochemical analyzes in laboratory biodiversity studies of higher plants.
The plants were submitted to two water regimes: irrigated (control) and water deficit (total irrigation suspension at the beginning of the experiment), in a period of 30 days.During the experimental period the plants called control were irrigated daily to replace the lost water, in which the volume of water applied was due to the size of the vessel used.

Biochemical Analyses of the Samples
The relative water content (RWC) was determined according to Slavick (1974).The activity of superoxide dismutase (SOD) was determined according to Giannopolitis and Ries (1977).Aliquots of 0.1 mL of the protein extract were transferred to test tubes containing reaction medium composed of potassium phosphate buffer 100 mM (TFK; pH 7.8); 0.1 mM of EDTA; 0.1% (v/v) of 2-Mercaptoetanol; 0.1% (v/v) of Triton X-100; 30 mg of Polivinilpirrolidona (PVP); and, 20 mM from ascorbate.The reaction was initiated by addition of 13 mM of methionine (pH 7.8); 2 μM of riboflavin; 0.075 mM of nitrotetrazolium blue (NBT).After 5 min, the readings were performed at 560 nm and the activity of SOD expressed in (enzymatic unit) U mg -1 protein.

Extraction: For Leaves and Roots
The extract for the determination of the activity of the SOD, APX and CAT enzymes was 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 held 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.

Determination of the Concentration of Malonic Aldehyde (MDA)
Determination of the concentration of malonic aldehyde (TCA) (0.1% p/v).The homogenate was centrifuged at 10,000 ×g for 15 min at 4 °C and the supernatant was collected and used in the determination of the concentrations of MDA.

Extraction of GSH From Crude Yeast Extracts by ATPS
A total volume of 1 mL crude yeast extracts containing 0.22 g/L glutathione was added into aqueous two-phase systems, and the final concentration of GSH in ATPS was 20 mg/L.The systems containing different salts were prepared by directly dissolving the salt powder into the system.The systems were mixed thoroughly and centrifuged at 3,000 rpm for 30 min to assist phase separation.The centrifuged systems were then allowed to settle for 5 min to separate into two clear phases.Samples from top and bottom phases were then carefully removed and assayed for glutathione concentration (Wu et al., 2004).
For the determination of catalase activity (CAT) the methodology was used according to Havir and Mchale (1987), which the consumption of H 2 O 2 was based on the decrease of the absorbance at 240 nm.20 μL aliquots of the extract were added to 3 mL of reaction medium consisting of 50 mM potassium phosphate buffer (pH7.0) at 30 °C, plus 12.5 mM H 2 O 2 .The CAT activity was expressed in nmol H 2 O 2 g -1 DM min -1 .jas.ccsenet.

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The activi restriction showed in significant org ure the activit g the absorban sferred to test te.The reactio rbate g -1 DM m A was determin μL of the extr mples were rea nmol g -1 FM. genotypes as a response to metabolize the radicals produced superoxides (Jaleel et al., 2007).SOD, which may be mitochondrial, cytosolic or chloroplastidic, is responsible for the dismutation of O 2 converting it to hydrogen peroxide and oxygen (Grob et al., 2013).
Ascorbate peroxidase and catalase (CAT), which are enzymes that will detoxify the H 2 O 2 compounds generated by oxidative stress in order to generate other products that are not toxic to the plant cell (Bhattacharjee, 2010) are required.That is, the combined actions of SOD and CAT are crucial to mitigate the effects of oxidative stress, since the first involves the dismutation of O 2 in H 2 O 2 and the latter decomposes H 2 O 2 in water and O 2 , and may indicate an important role in the control of accumulation of H 2 O 2 from oil palm subjected to water deficiency.
The APX enzyme may also have been directly involved in the detoxification components of H 2 O 2 , due to the stressful condition (Bhatt & Tripathi, 2011).Thus, it is possible to observe that the increase of the enzymatic activities, during the studied period, shows the defense of the plant in the destruction of free radicals as a form of prevention to more severe damages.
With the permanence of the water suspension, there was possibly loss of cell compartmentalization in the two oil palm genotypes, which is accompanied by the increase in the lipid peroxidation caused by ROS being this peroxidation estimated by the concentration of MDA, that is, the accumulation of MDA in a tissue is widely used to estimate cell damage (Sung & Jeng, 1994).
Thus, it is possible to observe that the concentrations of MDA increased significantly when the genotypes were under treatment with water suspension, inferring the increase of the lipid peroxidation.However, with the action of the antioxidant enzymes it was observed a reduction of the oxidative damages in the tissues of the genotypes under water deficiency.

Content of Glutathione and Ascorbic Acid
The importance of glutathione (GSH) has been studied because it is a tripeptide that is central to the cellular redox state in plants (Noctor et al., 2011).Due to the fundamental role in plant cells including redox signaling and homeostasis, we observed high concentrations of this component in leaves and roots of oil palm when submitted to water deficiency (Koprivova et al., 2010).Therefore, it is possible that glutathione has acted in favor of the reduction of ROS acting as another plant defense system and being proportional to ascorbic acid (AsA) levels.From this, it is noted that some studies indicate that these two components act together in the ascorbate-glutathione cycle (ASC-GSH), doing the recycling of oxygenated compounds, showing increasing values for both roots and leaves in the two genotypes (Noctor & Foyer, 1998;Serkedjieva, 2011).

Conclusion
The study suggests that oil palm had a good use and adaptation when submitted to water deficit and that genotype 2528 was more responsive to maintain its vital biochemical activities.