Biomass Production and Antioxidative Enzyme Activities of Sunflower Plants Growing in Substrates Containing Sediment from a Tropical Reservoir

Many Brazilian reservoirs are intensely submitted to the silting process, particularly the small and medium size ones. The study aimed to examine the feasibility of using silt sediment to grow sunflower plants under conditions of water stress, by evaluating its effects on the relative chlorophyll contents, dry matter and antioxidative enzyme system. The study was conducted under greenhouse conditions at the Instituto Federal do Ceará Campus Maracanaú, Brazil. The sunflower seeds were sown in buckets containing 1) sand; 2) sand + manure/mixed organic fertilizer; 3) sand + 91.8 g of sediment, and 4) sand + 183.6 g of sediment. The sediment was collected from the Tijuquinha reservoir, Northeast of Brazil. The plants were watered daily to 70% field capacity. At 16 days after sowing, irrigation to half of each group of seedlings was suspended. The experimental design was completely randomized in a 2 × 4 factorial with five replicates. The data of each harvest time were analysed by analysis of variance and the means were compared by Tukey’s test (P ≤ 0.05). The addition of silt sediment improved the variables (relative chlorophyll content, and shoot and total dry matters) compared to plants grown in substrate containing sand and sand + compost/mixed organic fertilizer, respectively. In general, a greater increase in the variables was observed with the 200% nitrogen recommendation treatment than the other treatments studied. It is possible that the silt sediment from reservoirs can be an alternative to chemical fertilizers for plant cultivation, reducing production costs, providing improvements in the quality of potable water and restoring the storage capacity of surface reservoirs lost by siltation.


Introduction
Dams are the main water storage forms in the Brazilian semiarid region.These reservoirs complement water abstraction for population supply in addition to meeting the needs of agricultural activities in an area dominated by intermittent rivers (Araújo & Medeiros, 2013).Over 90% of the water in the state of Ceará is provided by surface reservoirs (Araújo et al., 2006).
However, the water quality in these reservoirs is negatively affected by siltation, which compromises its use for human consumption and can lead to eutrophication.One of the main causes of siltation is the leaching of soil nutrients by rainfall that is then retained in the bottom of the reservoirs.These sediments are derived from erosion of exposed soil, due to the removal of vegetation or to inadequate soil management.Carvalho et al. (2000) found that the sediments serve as catalysts, carriers and pollutant fixing agents.According to Cabral et al. (2005), many Brazilian reservoirs are intensely submitted to the silting process, particularly the small and medium size ones. et al., 2006).In the state of Ceará, siltation generates an average 2% reduction in the capacity of the reservoir every 10 years (Araújo & Medeiros, 2013).Thus, preventive and corrective actions are essential to maintaining the quality and supply of water in these water bodies.Irregular rainfall and inadequate water quality are among the main factors affecting agricultural production in the Northeastern region of Brazilbecause, among the resources used by plants, water is considered the most critical.
The semiarid region of Ceará contains soils with low organic matter and nutrients, there by, reducingcrop yields (Feitosa et al., 2013).Conversely, dam sediment contains a high concentration of nutrients, such as nitrogen, phosphorus and organic matter, which allow the use of this type of waste in Brazilian agriculture (Feitosa et al., 2013).The application of this sediment may improve the chemical and physical characteristics of the soil, thus, improving the quality and crop yields and reducing production costs (Abreu-Júnior et al., 2005).
The sunflower (Helianthus annus L.) is a plant of the Asteraceae family, which originates from North America and has been cultivated in large areas throughout the world.According to Guerra and Picksius (2005), sunflower oil is among the potential sources of biofuel production in Brazil.
The currentstudy aimed to assessthe viability of usingsilt sediment from the Tijuquinha dam in Ceará, Brazil, to grow sunflower plants under conditions of water restriction, by evaluating its effects on the relative chlorophyll contents, dry mass production, antioxidative enzyme system and lipid peroxidation in the plants.

Materials and Methods
The experiment was conducted from August to December 2015 under greenhouse conditions at the Instituto Federal de Educação, Ciência e Tecnologia do Ceará (IFCE) in Maracanaú,Ceará,Brazil (03º52′S;38º37′W).The mean values of temperature and relative air humidity in the greenhouse were 33.3 ºC and 54%, respectively.
The sediment was collected in October 2015 from the Tijuquinha dam, in the city of Baturité, Ceará.After collection, the sediment was placed in an oven at 60 °C to complete drying.The sediment was then macerated to obtenir homogeneous mixture and finally added to the treatments in the proportions described above.
The amount of mixed organic fertilizer obtained commercially, and sediment (91.8 g) were calculated according to the recommendation for planting, which is 80 kg nitrogen per hectare, in addition to the compounds nitrogen concentrations.However, to verify a possible increase in plant growth, this concentration was doubled in the plants treated with 183.6 g of dam sediment.Table 1 presents the results of the chemical analysis of the sediments.
Table 1.Chemical analysis of the Tijuquinha dam sediment used in the composition of the substrates for growing Helianthus annuus L. plants The plants were irrigated daily to 70% of field capacity.At 16 days after sowing (DAS), the irrigation to half of each group of seedlings described above, was suspended.
Two evaluations were performed, at 21 (5 days under water restriction) and 23 DAS (7 days under water restriction), respectively.The chlorophyll relative contents were measured in the first fully expanded leaf using an SPAD-502 chlorophyll meter.Later, a group of plants was harvested and the material was dried in a forced-air oven at 60 ºC to complete drying for the determination of shoot (leaves + petiole + stems) (SDM), root (RDM) and total (shoot + roots) (TDM) dry matter, respectively.
For biochemical analyses, fresh leaves and roots were frozen in liquid nitrogen and stored frozen (-25 °C).Later, extracts of plant materials were prepared for the determination of antioxidative enzyme activities.Leaf tissue (1 g) was homogenized in a mortar and pestle with 4 ml of ice-cold extraction buffer (100 mM potassium phosphate buffer, pH 7.0, 0.1 mM EDTA).For ascorbate peroxidase (APX) estimation, 2 mM ascorbic acid was added to the extraction buffer.
The activities of catalase (CAT), guaiacol peroxidase (GPX), APX and superoxide dismutase (SOD) were determined.CAT activity was determined according to Haver and McHale (1987), by the decrease in absorbance at 240 nm due to the consumption of hydrogen peroxide (H 2 O 2 ).The method described by Kar and Mishra method (1976) was used to measure the GPX activity.This assay is based on an increase in absorbance at 470 nm due to the formation of tetraguaiacol.The APX activity was evaluated by the method of Nakano and Asada (1981), which measures the oxidation of ascorbate by the decrease in absorbance at 290 nm.The SOD activity was determined by Beauchamp and Fridovich's method (1971), and the reaction measured by the increase in absorbance at 560 nm due to the photoreduction of p-nitrobluetetrazolium (NBT) toblue formazan.
The activities of CAT, APX and GPX, were expressed as mol H 2 O 2 /min/g fresh mass (FM), and SOD in activity units (U)/g FM, where U is defined as the amount of enzyme required to cause 50% inhibition of NBT photoreduction.Each extract was measured in duplicate.
Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) produced by the thiobarbituric acid (TBA) reaction, according to Buege and Aust (1978).The results were expressed as nmol MDA/FM.
The experimental design was completely randomized, arranged in a 2 (irrigated or non-irrigated) × 4 (sand, sand + fertilizer, sand + 91.8 g of dam sediment, sand + 183.6 g of dam sediment) factorial, with five replicates.Each replicate consisted of a bucket containing two plants.The data for each harvest time were analysed separately by analysis of variance (ANOVA) and the means were compared by Tukey's test (P ≤ 0.05) using Sigma Plot 11.0 software.

Results
Figure 1 shows the leaf chlorophyll relative contents of the sunflower plants.It was found that water restriction did not reduce this variable in relation to their respective controls.However, at 23 DAS, higher means were observed for plants with sediment in their substrate.This increase was most pronounced for the treatment with 200% nitrogen recommendation (NR) in sediment, which was 15 and 23% superior to sand treatment under control conditions and water restriction, respectively.4A), it was found that at the first harvest, the 100% NR treatment was superior to the other treatment sunderboth irrigation conditions.At the second harvest, the 200% NR treatment showed excellent performance, with averages 38 and 58% higher than the sand treatment under control conditions and water restriction, respectively.
It was observed that at 21 DAS, the water restriction did not cause an increase in the APX activity in the roots.However, at 21 DAS the APX activity was higher in the 200% NR treatment.At 23 DAS, the 100% NR treatment differed significantly from the sand treatment under both conditions of irrigation, respectively, and the sand treatment average was 35 and 15% lower than the 100% NR treatment in plants irrigated and non-irrigated, respectively (Figure 4B).
In general, GPX activity in the roots was higher in plants grown under water restriction conditions.It was observed that the 200% NR treatment was superior to the other treatments at the two harvest times under both control and water restriction conditions, respectively.The 200% NR treatment was on average, 174 and 40% higher compared to the sand treatment at the first harvest and 312 and 311% higher than the sand treatment at the second harvest, under control and water restriction conditions, respectively (Figure 4C).

Discuss
The  Silva et al. (2007) studied the effects of four irrigation water depths (117.20, 350.84, 428.70 and 522.14 mm) over108 d and found a higher production, oil content and plant height for the 522.14 mm depth.Nobre et al. (2010) observed a linear increase in the SDM of sunflower, with increasing water depth.Castro et al. (2006) found that water stress decreased the dry mass yield of plants.
The beneficial effects of adding organic waste to the soil have been previously demonstrated in the literature.For instance, Mizobata et al. (2016) observed an increase in the SDM in H. stilbocarpa plants when organic residue was added to the substrate.Amaral et al. (2016) demonstrated that a substrate containing 50% plant compound provided higher RDM, SDM and TDM in plants of Leucaena leucocephala (Lam.) de Wit.However, there are limited studies published that evaluate the use of eutrophic dam sediments, such as that from the Tijuquinha dam, in the composition of substrates for plants.Thus, the current study proves pivotal to demonstrate the benefits of adding sediment to the sunflower plants soil, both under control and water restriction conditions, respectively.It is believed that the increase in plant growth associated with the sediment treatments may be due to the presence and availability of nutrients in their composition, such as nitrogen, phosphorus, calcium and magnesium, which increases soil fertility and enhances its chemical properties.
Plants use several integrated events to adjust to water stress conditions, which involves morphological, anatomical, cellular and biochemical changes.Among these events is the reduction of the stomatal aperture, which aims to reduce the loss of water by evapotranspiration (Gill & Tuteja, 2010).However, this process can lead to increased production of reactive oxygen species (ROS) including the radical superoxide ( • O 2 -), hydroxyl radical ( • OH), H 2 O 2 and singlet oxygen ( 1 O 2 ) (Jaleel et al., 2007).ROS production is a normal event during plant growth and development.These ROS species act as signalling molecules in plants.However, under biotic and abiotic stresses, the ROS production can be increased and cause damage to biomolecules (Apel & Hirt, 2004).
In response to ROS production, plants have an antioxidative enzyme system that is an important defence against free radicals generated under stress conditions.Some of the enzymes that scavenge ROS are SOD, which catalyses the dismutation of superoxide in H 2 O 2 and O 2 , as well as CAT, APX and GPX, which can split the H 2 O 2 molecule.The balance of this enzymatic system depends on several factors, such as the type, duration and intensity of the stress that the plant experiences (Larcher, 2000).
In the present study, there were increases in the activities of antioxidative enzymes in plants grown in substrates containing sediment.In leaves, CAT was the main enzyme responsible for H 2 O 2 scavenging, and this enzyme was much higher than the other enzymes evaluated.CAT activity was not detected in the roots, where GPX was the main enzyme responsible for H 2 O 2 scavenging.It is possible that the higher antioxidative enzymes activities in treatments with dam sediments may contribute to an increase in sunflower growth (dry mass) compared to plants grown in substrates containing sand or fertilizer.
Under water restriction, the production of free radicals is significantly increased, which can lead to a sequence of events that starts with lipid peroxidation, followed by degradation of the cell membranes and apoptosis (Greggains et al., 2000).However, the ROS scavenging by the antioxidative enzymes acts to minimize lipid peroxidation that can be considered the main symptom of oxidative damage to cell membranes (Hernandez et al., 2000).An increase in CAT activity could decrease the intracellular H 2 O 2 concentration, reducing lipid peroxidation and the damage to plant membranes under water restriction conditions.Therefore, it is possible that the increased enzymes activities in plants grown in substrates containing dam sediment caused the decrease in MDA contents observed in the sunflower leaves (Figure 5).
In the experimental conditions used in the current study, the addition of silt sediment from the Tijuquinha dam provided improvements in the relative chlorophyll content, SDM and TDM compared to plants grown in substrate containing sand and sand + compost/mixed organic fertilizer, respectively.Also, the 7-d water restriction was not capable of causing a drastic reduction in plant growth, independent of the substrate used.The application of double NR in the sediment (200% RN treatment) caused a greater increase in the variables analysed than the other treatments.Therefore, dam sediments can be an alternative to chemical fertilizers for plant cultivation.Furthermore, withdrawal of such material from dams can provide improvements in the quality of water supply and can recover the superficial reservoir storage, partially lost by the sedimentation process.

Conclusions
In the experimental conditions used in this experiment, the addition of silted dam sediment of the Tijuquinha dam provided improvements in the variables: relative chlorophyll content, shoot and total dry masses when compared to plants growing in substrate containing sand or sand + compost/mixed organic fertilizer.
It was found that the water restriction of 7 days was not capable of causing drastic reduction in plant growth, independent of the substrate used.
The application of double nitrogen recommendation in sediment (200% RN treatment) caused greater increase in the variables analyzed in comparison to the other treatments.
The dam sediments could be an alternative to chemical fertilizers for plant cultivation.Furthermore, withdrawal of such dams background material can provide improvements in the quality of water supply and can recover the superficial reservoir storage, partially lost by sedimentation process.

Figure
Figure 1 after susp (white bar the type o the subst Figure 2 harvest -5 Figure 4 harvest -5