Role of glutathione in tolerance to arsenite in Salvinia molesta , an aquatic fern

In many plant species, tolerance to toxic metals is highly dependent on glutathione, an essential metabolite for cellular detoxifi cation. We evaluated the responses of glutathione metabolism to arsenite (AsIII) in Salvinia molesta, an aquatic fern that has unexplored phytoremediation potential. Plants were exposed to diff erent AsIII concentrations in nutrient solution for 24 h. AsIII caused cell membrane damage to submerged leaves, indicating oxidative stress. Th ere was an increase in the glutathione content and Υ-glutamylcysteine synthetase enzyme activity in the submerged and fl oating leaves. Th e glutathione peroxidase and glutathione sulfotransferase enzymes also showed increased activity in both plant parts, whereas glutathione reductase only showed increased activity in the submerged leaves. Th ese fi ndings suggest an important role for glutathione in the protection of S. molesta against the toxic eff ects of AsIII, with more eff ective tolerance responses in the fl oating leaves.


Introduction
Human populations are mainly exposed to arsenic (As) through consumption of contaminated water.Estimates suggest that between 150 and 200 million people are exposed to As concentrations above the 10 µg/L, the maximum recommended by the World Health Organization (WHO) (Naujokas et al. 2013).India and Bangladesh are the most aff ected countries in the world, where prolonged ingestion of As-contaminated water has been the cause of many health problems, including skin diseases, respiratory diseases, and cancer (Chakraborti et al. 2016a;b).
In aquatic environments, the inorganic As forms predominate, occurring mainly as arsenate (As V ) or arsenite (As III ).Arsenite is the most toxic form and it is more commonly found in naturally contaminated groundwater sources (Zheng et al. 2017).The precipitation, ion exchange, adsorption, and membrane-based fi ltrations are physicochemical technologies that are available to remove As from water.However, they all have environmental and economic losses, as well as being more effi cient for As V than As III (Nicomel et al. 2016).
An alternative method of removing As from the ecosystems is via plant uptake and accumulation of the pollutant in their roots and/or leaves.This technique is known as phytoremediation and presents some advantages, such as lower implementation cost, low environmental impact and applicability in vast contaminated areas (Bernardino et al. 2016).Several macrophyte species have shown potential for As phytoremediation from contaminated water, including 1 Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Av.Peter Henry Rolfs, s/n, 36570-000, Viçosa, MG, Brazil 2 Departamento de Biologia Geral, Universidade Federal de Viçosa, Av.Peter Henry Rolfs, s/n, 36570-000, Viçosa, MG, Brazil 3 Laboratório de Ecofi siologia Vegetal, Instituto Federal de Educação, Ciência e Tecnologia Goiano-Campus Rio Verde, 75901-970, Rio Verde, GO, Brazil Adinan Alves da Silva, Juraci Alves de Oliveira, Fernanda Vidal de Campos, Cleberson Ribeiro and Fernanda dos Santos Farnese Eichhornia crassipes (Alvarado et al. 2008), Pistia stratiotes (Farnese et al. 2013), and Lemna gibba (Leão et al. 2014).The As III absorbed by the plant affects physiological processes such as photosynthesis and growth, besides promoting cell damage (Farooq et al. 2016).Despite being a non-redox metalloid, it is known that As III causes oxidative stress by increasing the generation of reactive oxygen species (ROS).This response is considered to be one of the most evident symptoms of As toxicity in plants (Zhao et al. 2010;Sharma et al. 2012).
Arsenite also features a high affinity for protein sulfhydryl groups and binds to transcription factors, signal transduction proteins, proteolytic and structural proteins, and metabolic and regulatory enzymes.This binding changes the structure and activity of these components in the plant cell (Finnegan & Chen 2012).For example, the enzymatic complex pyruvate dehydrogenase is an important cellular target that is inhibited by As III (Bergquist et al. 2009).
An essential mechanism for As tolerance in plants involves the complexes formed between the metalloid and rich SH-group compounds, especially the reduced glutathione molecule (GSH).The As-GSH complexes can be safely stored in vacuoles, to prevent cellular damage (Seth et al. 2012).Moreover, GSH is a key player in oxidative stress avoidance, by acting as a cofactor for antioxidant enzymes, directly scavenging ROS, or participating in the ascorbate-glutathione cycle, which is an essential pathway for maintenance of non-toxic levels of hydrogen peroxide in plant cells (Hossain et al. 2012;Zagorchev et al. 2013;Singh et al. 2015).
Some floating ferns of the Salvinia genus (Salvinia natans, Salvinia minima, and Salvinia molesta) have already shown the ability to absorb and tolerate toxic metals, including As V (Rahman et al. 2008;Estrella-Gómez et al. 2012;Hariyady et al. 2013).However, limited information is available about As III -tolerant macrophytes and the behavior of glutathione metabolism in these species.
To evaluate the capacity of S. molesta to As III phytoextraction and potential to tolerate high As III concentrations, we exposed the plants to the pollutant and studied the antioxidative functions of glutathione under these conditions.We tested the hypothesis that S. molesta is able to tolerate As III and this capacity is related to increases in GSH metabolism.

Plant material and treatment conditions
Salvinia molesta D. S. Mitchell plants were collected in an artificial lake with an area of approximately 30 m 2 and 60 cm in height, located in the Botanic Garden of the Federal University of Viçosa (20°45'24"S 42°52'23"W).In this lake several species of aquatic plants are cultivated, including S. molesta.The water supplying the lake comes from the municipal water supply network.
Salvinia molesta is a floating aquatic fern that is native to southeastern Brazil.The plants are born from a rhizome and grow attached to it.From each node in this rhizome, two floating leaves originate and a set of modified submerged leaves constitutes a plant.The two leaf groups differ in their morphology.Floating leaves are green, oblong-shaped, and covered by papillae and hairs that repel water, while the submerged leaves are brown, long, and filiform, and present as numerous segments densely covered with hairs.These hairs are multicellular and have the ability to absorb water and nutrients, acting as a root.However, true roots are absent (Miranda & Schwartsburd 2016).
The plants were acclimatized for 7 days in a growth chamber in diluted Clark nutrient solution (1/4 strength) (Clark 1975) at pH 6.5, with a controlled temperature (25 ± 2 °C) and irradiance (250 µmol m -2 s -1 ), and with a 16-h light photoperiod.Plants with similar sizes were then exposed to the following treatments: control (Clark nutrient solution 1/4 strength) and three As concentrations (5, 10 and 20 µM), which were provided in arsenite sodium form (NaAsO 2 ) in a nutrient solution for 24 hours, under the same conditions described above.
At the end of exposure, plants were harvested, and floating leaves and submerged leaves were separated, washed in deionized water and immediately analyzed or stored at −80°C.Experiments were set up with three replicates, each consisting of 4 g of fresh weight plants in glass pots with 0.5 L of solution.

Determination of arsenic concentration
After treatment, floating and submerged leaves were dried in a conventional oven at 80°C, until a constant dry mass was obtained.The oven-dried plant materials were finely ground in a steel electric grinder and 0.1 g was digested in a nitric-perchloric acid mixture (2:1).A blank sample was also digested (Marin et al. 1993).The As concentration was determined through inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Optima 3300 DV, Perkin-Elmer, Norwalk, CT, USA).The accuracy of the analytical technique was verified by analyzing the certified reference material (Tomato leaves -NIST, SRM 1573a, Gaithersburg, MD, USA).

Lipid peroxidation
Lipid peroxidation was determined by estimating the malondialdehyde (MDA) content, using the method described by Heath & Packer (1968), with modifications.An approximately 80 mg sample of floating and submerged leaves was homogenized in 2 mL 0.1 % trichloroacetic acid (TCA) and centrifuged at 10,000 g for 20 min.Then, 1 mL of 0.5 % thiobarbituric acid in 20 % TCA was added to a 0.5 mL supernatant aliquot.The reaction mixture was heated to 95 ºC for 30 min and then cooled quickly in an ice bath.

Role of glutathione in tolerance to arsenite in Salvinia molesta, an aquatic fern
After an additional centrifugation at 10,000 g for 15 min, the absorbance of the supernatant was taken at 532 and 600 nm.The lipid peroxidation level was calculated using the molar extinction coefficient 155 mM −1 cm −1 (Hodges et al. 1999) and results were expressed in nmol MDA g -1 fresh weight.

Total glutathione content (GSH + GSSG)
The total glutathione content (reduced (GSH) + oxidized (GSSG)) was determined according to the method described by Griffith (1980).Approximately 250 mg of fresh mass samples were homogenized in 2 mL of extraction medium, consisting of 0.1 M HCl containing 1 mM ethylenediaminetetraacetic acid (EDTA).The extract was centrifuged for 15 minutes at 12,000 g and 4° C. Aliquots with 200 µL of the supernatant were added in a reaction mix, containing 200 µL of 125 µM sodium phosphate buffer with disodium salt of 6.3 mM EDTA at pH 7.5, 500 µL 0.3 mM nicotinamide adenine dinucleotide phosphate reduced (NADPH), and 100 µL 6.0 mM 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB).The absorbance was measured at 412 nm for 1 minute, after addition of 0.5 units of reductase glutathione enzyme.The total glutathione content was determined using a calibration curve and results were expressed in nmol glutathione g -1 fresh weight.

Υ-glutamylcysteine synthetase activity (Υ-ECS, EC 6.3.2.2)
Υ-ECS activity was evaluated according to Rüegsegger & Brunold (1992), using approximately 250 mg of plant material.Samples were homogenized in 2.0 mL of the extraction medium, consisting of 0.10 M Tris-HCl buffer and 5.0 mM EDTA at pH 8.0.The enzyme activity was determined by adding 100 µL of crude enzyme extract to 0.9 mL of a reaction medium containing 10 µM sodium glutamate, 10 µM L-aminobutyrate, 2.0 mM EDTA, 0.2 mg bovine serum albumin (BSA), 20 µM magnesium chloride (MgCl 2 ), 5.0 µM disodium salt ATP, and 100 mM Tris-HCl buffer at pH 8.2.The mixture was incubated at 37 °C for 30 minutes and the reaction was stopped by adding 100 µL 50 % TCA.Then, the mixture was centrifuged at 10.000 g for 10 min, and the inorganic phosphate content in the supernatant was determined at 720 nm using the phosphomolybdate method (Lindeman 1958).
The GR activity was determined by adding 100 µL of crude enzyme extract to 0.9 ml of a reaction medium consisting of 0.10 M potassium phosphate bufferpH 7.5, 1.0 mM EDTA, 1.0 mM oxidized glutathione (GSSG), and 0.10 mM NADPH in 0.5 mM Tris-HCl buffer at pH 7.5 (Carlberg & Mannervik 1985).The decrease in absorbance was measured during the first minute of the reaction and the enzyme activity was calculated using the molar extinction coefficient 6.22 mM -1 cm -1 (Foyer & Halliwell 1976).The results were expressed in nmol NADPH oxidized min -1 g -1 fresh weight.
Glutathione peroxidase activity (GPX,EC 1.11.1.9)To analyze the activity of GPX, approximately 250 mg of fresh mass samples were homogenized in an extraction medium, consisting of 0.1 M Tris-HCl buffer at pH 7.5, 1.0 mM EDTA, and 10 mM MgCl 2 .The enzyme activity was measured by adding 100 µL of crude enzyme extract to 0.9 mL of a reaction medium, consisting of 50 mM potassium phosphate buffer at pH 7.0, 1.0 mM EDTA sodium chloride, 1.0 mM reduced glutathione (GSH), 0.2 mM NADPH, 0.25 mM hydrogen peroxide (H 2 O 2 ), and 1 unit of glutathione reductase enzyme (Nagalakshmi & Prasad 2001).The absorbance was measured at 340 nm during the first minute of the reaction and the GPX activity was calculated using the molar extinction coefficient of 6.22 mM -1 cm -1 (Anderson & Davis 2004).The results were expressed in nmol NADPH oxidized min -1 g -1 fresh weight.
Glutathione-S-transferase activity (GST,EC 2.5.1.18)The GST activity was determined according to the methodology proposed by Habig et al. (1974) and Habig & Jakoby (1981), using approximately 250 mg of fresh mass.Samples were homogenized with 2.0 mL of an extraction medium, with 0.2 M Tris-HCl buffer at pH 7.8, 1.0 mM EDTA, 1.0 mM DTT, 0.10 mM PMSF, and 5 % PVPP.GST activity was determined following addition of 0.1 mL of the crude enzyme extract to 0.9 mL of a reaction medium consisting of 0.20 M potassium phosphate buffer at pH 6.5, 20 mM GSH, and 0.10 M 1-chloro-2,4-dinitrobenzene (CDNB).The absorbance was measured at 340 nm and the enzyme activity was calculated using the molar extinction coefficient 9.6 mM -1 cm -1 and expressed in nmol min -1 g -1 fresh weight.

Statistical analysis
The experiment was performed using a randomized design.Data were analyzed using a two-way analysis of variance (ANOVA) followed by a means comparison using Tukey's test.P < 0.05 was considered to be significant.The statistical analyses were performed using the statistical software SAEG ® 9.1 (Universidade Federal de Viçosa, Viçosa, Brazil).

As accumulation
As accumulation in the plants (absorbed and adsorbed) increased both in the submerged leaves and floating leaves as the As III concentration increased in the nutrient solution.However, the As content was significantly higher in the submerged leaves compared with the floating leaves in all As III treatments (Tab.1).

Integrity of cell membranes
The concentration of malondialdehyde (MDA) in the floating leaves only differs from control in plants that were exposed to 10 µM As III , while in submerged leaves, there was an increase in MDA for all As III treatments (Fig. 1).

Total glutathione content
The total glutathione content increased on in the floating and submerged leaves in all As III treatments.In plants exposed to higher As III concentrations, the content of glutathione in the floating leaves was approximately two times higher than that observed in control, while in the submerged leaves, the GSH concentration was more than three times higher than control.Regardless of the treatment, the total glutathione content was always higher in floating leaves compared with submerged leaves (Fig. 2).

Glutathione metabolism enzymes
The activity of glutathione metabolism enzymes showed distinct responses, which were influenced by the plant part analyzed (Fig. 3).
The Υ-ECS activity in floating leaves only increased in plants exposed to higher As III concentration.In the submerged leaves, there was an increase in all As treatments, with the highest enzyme activity observed in 20 µM As III , but there was no remarkable difference between 5 and 10 µM As III .In the 10 µM As III treatment, there was no difference in enzyme activity between the floating leaves and submerged leaves (Fig. 3A).
The As III concentration did not affect GR activity in the floating leaves.However, the enzyme activity in submerged leaves increased in all As III treatments.GR activity did not differ between the evaluated plant parts exposed to 20 µM As III , but it was higher in floating leaves in all the other treatments (Fig. 3B).
Table 1.Arsenic concentration (µg g -1 DW) on S. molesta floating leaves and submerged leaves after 24 hours of As III exposure.Values are means of three replicates (n= 3) ± SD.Means followed by different capital letters for the same column and small letters for the same row, show significant difference at P< 0.05, according to Tukey test.nd*-non detected.

Role of glutathione in tolerance to arsenite in Salvinia molesta, an aquatic fern
The GPX activity in S. molesta floating leaves showed increases at 10 and 20 µM As III concentrations, while in the submerged leaves, there was a noticeable decrease in GPX activity with these same treatments.Comparing the enzyme activities between plant parts, there was increased GPX activity in the submerged leaves at lower As III concentrations and higher activity in floating leaves at 20 µM As III (Fig. 3C).
Exposure to As III induced increases in GST activity in floating leaves and submerged leaves.We observed that in submerged leaves, the enzyme activity decreased at the highest As III concentration compared to the other As III treatments (Fig. 3D).

Discussion
Salvinia molesta was able to absorb and/or adsorb substantial amounts of As in short period of exposure to the treatments.Aquatic plants can help to reduce heavy metals in the environment in an active way through As absorption and passively by As adsorption to the surface of their roots.The adsorption promotes pollutant phytostabilization by reducing its mobility and migration (Rai & Tripathi 2011;Dixit et al. 2015).
Once the whole plant is removed from the water, the absorption and adsorption process can contribute to environmental pollutant removal.Thus, S. molesta is an interesting plant in the phytoremediation processes, and a higher As content was observed in its submerged leaves compared with macrophytes commonly used in As phytoremediation such as Hydrilla verticillata (Srivastava et al. 2007) and Ceratophyllum demersum (Khang et al. 2012), under similar experimental conditions.Moreover, the As content in S. molesta increased in all treatments, indicating no saturation of Bars represent standard deviation (n= 3).Means followed by different capital letters for the same plant part and small letters between plant parts, show significant difference at P< 0.05, according to Tukey test.

Cleberson Ribeiro and Fernanda dos Santos Farnese
As removal capacity by the plant under the evaluated conditions (Zhao et al. 2009).The higher accumulation of As in S. molesta submerged leaves, which plays the role of roots, follows a trend usually observed in macrophytes (Zhao et al. 2009;Farnese et al. 2013) and acts as a defense strategy (Rahman & Hasegawa 2011).A key mechanism involved in the As III retention in root cells is the complexation with phytochelatins (PCs), or GSH.These complexes are sequestered in vacuoles, which reduces As transportation to the shoots and protects the photosynthetic tissues (Liu et al. 2010).
Several studies have shown that As accumulation promotes an increase in ROS generation in plants (Talukdar 2013;Upadhyaya et al. 2014;Tripathi et al. 2015).ROS are reactive molecules that are capable of damaging cell biomolecules, and whose preferred targets are biological membranes.Lipid peroxidation is a common injury resulting from As stress and MDA is an oxidative stress biochemical marker in plants (Sharma 2012;Sytar et al. 2013).Therefore, we can say that As III that accumulated in the S. molesta floating and submerged leaves promoted oxidative stress and cell damage in plants.Although cellular damage occurred only 24 h after exposure to treatments, the As III concentrations used in this test are much higher than those naturally found in regions where there are water sources with high levels of As contamination (Chakraborti et al. 2016c).
As exposure can induce glutathione synthesis in plants, which was verified in S. molesta.Increases in glutathione levels were also related to the As response in other macrophytes, such as C. demersum (Mishra et al. 2008) and Wolffia globosa (Zhang et al. 2012).Glutathione is directly linked to the plants' ability to tolerate stress caused by toxic metals and it may be used primarily as a substrate for PC synthesis and to promote acclimatization and survival (Hernández et al. 2015).
GSH synthesis occurs mainly in the cytosol, chloroplasts, and mitochondria, and Υ-ECS is the first enzyme involved in this process (Noctor et al. 2012).Increases in Υ-ECS activity in submerged leaves sustained the increase in total glutathione content in S. molesta.Enhanced GSH synthesis enzyme activity is an important tolerance response to As stress to meet the higher demand for this metabolite in cellular defense antioxidant mechanisms (Hernández et al. 2015).
The increase in the GSH content of floating leaves in all As treatments, despite an increase in Υ-ECS activity only at the highest concentration of the pollutant may have resulted from transport of glutathione synthesized in the submerged leaves to the floating leaves.Indeed, GSH can be transported over long distances through the phloem, for example, between root and shoot, within a few hours (Tausz et al. 2004;Li et al. 2006;Gigolashvili & Kopriva 2014).
Glutathione acts as a cellular antioxidant when in its reduced state.Therefore, GR enzyme activity, which converts oxidized glutathione (GSSG) to reduced glutathione (GSH), plays a key role in the defense system against oxidative stress (Sytar et al. 2013).The stimulus for GR activity in the presence of As is a defense mechanism reported for different plant species (Srivastava et al. 2005;Singh et al. 2007;Shri et al. 2009), according to observations in the S. molesta submerged leaves.In the floating leaves, other antioxidant mechanisms may have been activated beyond the GSH.Scavenging ROS enzymes, such as superoxide dismutase (SOD) and catalase may be upregulated by As III (Mishra et al. 2011).In addition, there is an alternative possible way to convert GSSG back to GSH through Class III peroxidases, a group of enzymes that are unique to plants (Liu et al. 2015).
The GSH is the substrate used by the enzymes GPX and GST to the removal of organic radicals and ROS (Meyer & Hell 2005;Chi et al. 2011).GPX is considered to be a biomarker for heavy metal toxicity and an intrinsic defense mechanism against As in hyperaccumulating plants (Radotic et al. 2000;Srivastava et al. 2005).The GPX showed participation in antioxidant defenses in the S. molesta floating leaves.Similar results have been observed in Lemna gibba subjected to As V (Leão et al. 2014) and rice seedlings treated with As III (Mishra et al. 2011).Conversely, decreases in submerged leaves and may have contributed to further cellular damage in these organs.As III has the ability to inhibit the activity of various enzymes (Shen et al. 2013) and GPX is considered to be As-sensitive (Chouchane & Snow 2001).
The increase in GST activity indicates that the complex formation between GSH and As is an important defense mechanism in S. molesta, especially in floating leaves.GSTs are enzymes that are capable of catalyzing xenobiotic complexation with GSH, making the pollutant less reactive in the intracellular environment.High GST activity can be considered to be a biomarker of As tolerance in plants (Bianucci et al. 2017).Although submerged leaves have experienced increases in GST activity, the arsenic amount in their tissues may have generated a stress that exceeded their tolerance ability.
The present results suggest that GSH confers tolerance to As III in S. molesta plants.The floating leaves showed a more efficient defense against oxidative damage.When in direct contact with the plants, toxic metals can be immediately retained and detoxified in the roots, mainly using phytochelatins at the expense of glutathione.This may lead to a delay in the response to the metal by the aerial part of the plant.In addition, the change in the ratio between reduced and oxidized glutathione (GSH:GSSG) can generate a redox signal, and once it is perceived by the aerial part, it allows more time for the activation of defense mechanisms against stress (Jozefczak et al. 2014).The floating leaves also had lower As accumulation, as well as a high glutathione content and action of the detoxification enzymes GPX and GST.
Despite the clear role of glutathione metabolism in submerged leaves, such as increasing the GSH concentrations Role of glutathione in tolerance to arsenite in Salvinia molesta, an aquatic fern and GR and GST activities, the large As III accumulation and its high toxicity promoted greater cellular damage in this part of the plant.Submerged leaves accumulated between 5 and 7 times more As than floating leaves.
To draw conclusions that are more reliable about the As III tolerance level in S. molesta and validate their use for As phytoremediation, a long-term study with other As III concentrations are required.Finally, this study reinforces the relevant role of glutathione in plant tolerance to As III and provides a basis for genetic breeding for toxic metal phytoremediation.

Figure 1 .
Figure 1.Malondialdehyde concentration on floating leaves (black bar) and submerged leaves (grey bar).Bars represent standard deviation (n= 3).Means followed by different capital letters for the same plant part and small letters between plant parts, show significant difference at P< 0.05, according to Tukey test.

Figure 2 .
Figure 2. Content of total glutathione on floating leaves (black bar) and submerged leaves (grey bar).Bars represent standard deviation (n= 3).Means followed by different capital letters for the same plant part and small letters between plant parts, show significant difference at P< 0.05, according to Tukey test.

Figure 3 .
Figure 3. Activity of enzymes: Υ-ECS (a), GR (b), GPX (c) and GST (d) on floating leaves (black bar) and submerged leaves (grey bar).Bars represent standard deviation (n= 3).Means followed by different capital letters for the same plant part and small letters between plant parts, show significant difference at P< 0.05, according to Tukey test.