Application of glycine reduces arsenic accumulation and toxicity in Oryza sativa L. by reducing the expression of silicon transporter genes

Ecotoxicology and Environmental Safety 148 (2018) 410–417

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Application of glycine reduces arsenic accumulation and toxicity in Oryza sativa L. by reducing the expression of silicon transporter genes

MARK

Arvind Kumar Dubeya,b, Navin Kumara, Ruma Ranjana, Ambedkar Gautama, Veena Pandeb, ⁎ Indraneel Sanyala, Shekhar Mallicka, a b

CSIR-National Botanical Research Institute, Lucknow, India Department of Biotechnology, Kumaun University, Bhimtal Campus, Nainital 263136, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Arsenic Oryza sativa Glycine Accumulation Glutaredoxin Silicon transporters

The present study was intended to investigate the role of amino acid glycine in detoxification of As in Oryza sativa L. The growth parameters such as, shoot length and fresh weight were decreased during As(III) and As(V) toxicity. However, the application of glycine recovered the growth parameters against As stress. The application of glycine reduced the As accumulation in all the treatments, and it was more effective against As(III) treatment and reduced the accumulation by 68% in root and 71% in shoot. Similarly, the translocation of As from root to shoot, was higher against As(III) and As(V) treatments, whereas, reduced upon glycine application. The translocation of Fe and Na was also affected by As, which was lower under As(III) and As(V) treatments. However, the application of glycine significantly enhanced the translocation of Fe and Na in the shoot. Besides, the expression of lower silicon transporters i.e. Lsi-1 and Lsi-2 was observed to be significantly suppressed in the root with the application of glycine against As treatment. Similarly, the expression of three GRX and two GST gene isoforms were found to be significantly increased with glycine application. Simultaneously, the activities of antioxidant enzymes i.e. L-arginine dependent NOS, SOD, NTR and GRX were found to be significantly enhanced in the presence of glycine. Increased activities of antioxidant enzymes coincided with the decreased level of TBARS and H2O2 in rice seedlings. Overall, the results suggested that the application of glycine reduces As accumulation through suppressing the gene expression of lower silicon transporters and ameliorates As toxicity by enhancing antioxidants defense mechanism in rice seedlings.

1. Introduction Arsenic (As) is a metal(loid), poses a significant threat to plants, particularly in rice, causing health hazards to rice patrons (Williams et al., 2009; Zhao et al., 2010). Arsenic naturally exists in two abundant inorganic forms i.e., arsenite [As(III)] and arsenate [As(V)]. Arsenite is predominant under anoxic paddy fields, easily uptaken by plants, whereas, As(V) in aerobic conditions. As(III) is 2–10 times more toxic than As(V), causing morphological and physiological disorders in plants (Tripathi et al., 2007; Ahsan et al., 2008; Tchounwou et al., 2012). The As related problem is very acute in South East Asian countries and these areas produces 90% of world rice. Rice is a major staple food to half of the world population. It accumulates As more efficiently than other cereals crops (Williams et al., 2007) as it grows under flooded conditions that lead to As(III) uptake through nodulin-26 like intrinsic protein (NIP) aquaporin channel (Lsi-1 and Lsi-2) (Ma et al., 2008). However, As(V) enters into the cell through specific phosphate

⁎

transporters (Zhao et al., 2010). In most of the As-resistant organisms, As(V) is reduced to As(III) and expelled outside the cell or sequestered into the vacuoles facilitated by glutathione–S-transferase (GST) (Dubey et al., 2016). To detoxify the toxicity induced by the As, plants increases its antioxidant enzymes and non-enzymatic products like ascorbateglutathione pools (Alscher et al., 2002; Azevedo et al., 2002). Plants under As stress also synthesise amino acids, thiol-containing proteins and other metabolites to counter against the stress (Cobbett, 2001; Hassinen et al., 2011). Amino acids (AAs) such as glycine, proline, cysteine and histidine plays a crucial role in providing tolerance to the plants under stress conditions. The level of these amino acids increases under heavy metal exposure in plants (Davies et al., 1987; Rai, 2002). Some stress responsive amino acids act as an osmoregulator and phytochelator against various stresses (Tripathi et al., 2013). Dave et al. (2013) have demonstrated that glutamic acid (Glu), Gly and cysteine (Cys) are involved in As detoxification by the formation of glutathione and

Corresponding author. E-mail address: [email protected] (S. Mallick).

http://dx.doi.org/10.1016/j.ecoenv.2017.10.047 Received 2 August 2017; Received in revised form 11 October 2017; Accepted 22 October 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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1.6.4.2) activity was assayed following Smith et al. (1988) and represented as U mg−1 protein, where 1U is the conversion of 1 mM of oxidized glutathione (GSSG) min−1 to reduced glutathione (GSH).

phytochelatins (PC). Dwivedi et al. (2010b) demonstrated that higher exposure of As to the rice plants decreases the levels of some AAs. However, the few AAs play a crucial role in amelioration of heavy metal toxicity. Glycine is a hydrophobic amino acid, which has no alkyl group. It is involved in the biosynthesis of glutathione whose, concentration increases during metal stress. To detoxify the toxicity generated by As, plants increases its antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutaredoxin (GRX) and non-enzymatic products like hydrogen peroxide (H2O2), thiobarbituric acid reactive substance (TBARS) and glutathione (GSH) pools (Alscher et al., 2002; Azevedo et al., 2002). Plants under As stress synthesise AA, thiolcontaining proteins and other metabolites for defending the stress (Cobbett, 2001; Hassinen et al., 2011). As an abundant source of nitrogen, glycine acts as a direct precursor of ammonium after urea, which regulates the nitrogen cycle of plants (Schiller et al., 1998). The accumulation of glycine also influences the level of other AAs e.g. serine and glutamate. Waditee et al. (2005) reported that the application of glycine in Arabidopsis also enhances the levels of betaine, which reduces the toxic effects of different stresses. In lights of these findings, the present study was aimed to investigate the effects of glycine on the As accumulation, modulation of As(III) specific transporters and responses of antioxidant defense mechanism against As stress.

2.3. Elemental analysis The metal(loid) contents (As) were determined, following the method of Kumar et al. (2013). Plant tissues (leaf ~500 mg and root ~300 mg) were digested on a hot plate using HNO3:HClO4 (3:1). Elements [Cu, Fe, Zn and Mn (µg ml−1)] were analyzed using AAS (GBC Avanta ∑), whereas, for As (µg l−1), AAS was fitted with a hydrate generator (MDS 2000) using NaH2BO4+NaOH (3 M) and HCl (3 M). The values are presented in µg g−1 dw (dry weight) and the translocation factor (TF) is the ratio of the elements in shoot divided by root. 2.4. Gene expression analysis using qRT-PCR Total RNA (ribonucleic acid) from the shoot after 7 days of treatment, was extracted using the Spectrum Plant Total RNA Kit (SigmaAldrich, USA), followed by treatment with RNAase-free DNase I (deoxiribonuclease I) (Sigma-Aldrich, USA). Quantitative real-time PCR (qRT-PCR) was carried out using 2 µl of the cDNA (complementary DNA) corresponding to the set of selected genes in a reaction containing 2×PCR Master Mix (Thermo Scientific, USA). Lower silicon transporters, namely Lsi-1 and Lsi-2, two CC (cystein-cystein) type GRX (Os01g27140 and Os01g13950), one CPYC (cystein, proline, tyrosine and cystein) type (Os02g40500) and one GRL (glutaredoxin like) type GRX (Os01g61350) genes were taken for the study. The expression of two glutathione-S-transferase (GST) genes, namely (Os09g20220 and Os02g38160) was also studied using specific set of primers. Three technical replicates of each biological replicate were taken for the qRTPCR analysis. The primers for rice ubiquitin gene were used as an internal control to ensure that equal amounts of cDNA were used in all the reactions. The PCR reaction was carried out using the following cycle conditions: an initial denaturation at 94 °C for 2 min, 40 PCR cycles were performed at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a final 5-min extension at 72 °C. After obtaining the “ct” value for each reaction, the relative expression was calculated by 2^delta Ct method. The list of selected genes and oligonucleotide primers (Eurofins, India) used for each gene are listed in the additional file (S. Table 1).

2. Materials and methods 2.1. Growth conditions and experimental design The experimental design consists of different treatments of As(III) and As(V) along with glycine lower (L) and glycine higher (H) and their respective controls. The experimental control is wholly untreated plants and designated as “C” while the treatments of glycine are designated as Gly(L) and Gly(H), reported as glycine controls. Rice cultivar, Usar-3, was obtained from Narendra Dev Agriculture University, Faizabad, Uttar Pradesh. The seeds were germinated and grown as explained by Dubey et al. (2016). Twenty five uniform (10 cm) seedlings were placed in 150 ml beaker, containing 100 ml of 100% Hewitt nutrient solution (HNS), prepared with Milli-Q water (pH 6.8–7.0) (Hewitt, 1966). The composition (µg ml−1) of Hewitt nutrient solution included N (168), P (41), K (156), Mg (36), Ca (160), S (48), Fe (2.8), Mn (0.55), B (0.54), Cu (0.064), Zn (0.065) and Mo (0.048). All the treatments contained four biological replicates. After 7d of growth in the nutrient solution, different treatments were provided as As(III) (4 µg ml−1) and As(V) (4 µg ml−1), using salts of NaAsO2, Na2HAsO4·7H2O (Sigma-Aldrich, USA) respectively. For convenience, the treatments were abbreviated as “C” for wholly untreated plants, As(III) (4) for arsenite 4 µg ml−1 (~53.3 µM), As(III) (4)+Gly(L)” for arsenite 4 µg ml−1 and 3 mM Gly, As(III)(4)+Gly(H) for arsenite 4 µg ml−1 and 4 mM Gly, As(V)(4) for arsenate 4 µg ml−1 (~53.3 µM), As(V)(4)+Gly(L) for arsenate 4 µg ml−1 and Gly 3 mM, As (V)(4)+Gly(H) for arsenate 4 µg ml−1 and Gly 4 mM.

2.5. Statistical analysis All the values are average of four replicates. The data were subjected to Duncan's Multiple Range Test (DMRT) for the analysis of significant difference between the means (p < 0.05). All the values are represented as percentage increase or percentage decrease with respect to the respective values in wholly untreated control seedlings, or otherwise mentioned. 3. Results

2.2. Determination of antioxidants and stress markers 3.1. Glycine affected the morphology and accumulation of elements TBARS content (mmol g−1 fw) was measured as mentioned in Heath and Packer (1968) using ε=0.155 M g−1 fw given for MDA-TBA adduct. The H2O2 content (nmol g−1 fw) was estimated according to the method described by Velikova et al. (2000). For estimation of antioxidant enzyme activities, fresh samples of leaves (~300 mg each) were used, following the procedure described by Dubey et al. (2016). Superoxide dismutase (SOD) (EC 1.15.1.1) activity was measured spectrophotometrically at 560 nm following Beauchamp and Fridovich (1971) and presented as U mg−1 protein, where 1U of SOD activity is the amount of protein required to inhibit 50% of initial reduction of nitro-blue tetrazolium (NBT). NOS activity was performed according to the method of Corpas et al. (2006). Glutathione reductase (GR) (EC

Both the treatments of As i.e. As(III) and As(V) reduced the growth parameters of rice seedlings, where As(III) was found to be more toxic. However, with the application of glycine, shoot length and fresh weight were recovered (Table 1). The maximum recovery in shoot length (11%) and fresh weight (23%) was observed with the application of Gly (H) to the As(V) treated rice seedlings, as compared to their respective control. However, Gly(L) applied to As(III) recovered the root length (21%), shoot length (5%) and fresh weight (17%), as compared to the As(III) treatment alone. Accumulation of the As in root was higher than shoot against both the As species (Table 2). The application of glycine reduced the As 411

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Table 1 Effect of different treatments of As(III) and As(V) under glycine (L and H) conditions on, (A) Root length (cm); (B) Shoot length (cm); (C) fresh weight (gm); ((D) leaf TBARS level (μmol g−1 fw) and (E) H2O2 (nM g−1FW). Significance of the mean values have been compared for each parameter separately (Duncan's test, p < 0.05) where bars marked with same letters are not significantly different. Treatments

Root length

Shoot length

Fresh weight

TBARS

H2O2

C As(III) As(V) Gly (L) Gly (H) As(III)+Gly (L) As(III)+Gly(H) As(V)+Gly(L) As(V)+Gly(H)

4.38 ± .48b 3.63 ± .25a 4.10 ± .36ab 4.00 ± .41ab 4.00 ± .50ab 4.38 ± .25b 4.25 ± .29b 4.67 ± .29b 4.13 ± .25ab

24.50 ± 2.38d 20.00 ± 1.41b 21.75 ± .35bc 23.67 ± .58c 23.33 ± .58c 21.00 ± 1.41bc 17.17 ± 2.08a 23.50 ± 1.80c 24.13 ± 1.65cd

0.16 ± .02cd 0.12 ± .01a 0.13 ± .01ab 0.16 ± .02cd 0.16 ± .02ab 0.14 ± .01b 0.13 ± .01ab 0.15 ± .01c 0.16 ± .01cd

10.51 ± .27a 12.48 ± 1.58ab 20.15 ± 1.22d 11.28 ± .81ab 10.39 ± .79a 15.45 ± 1.85c 12.07 ± .57ab 13.20 ± .06b 16.36 ± .53c

1037.02 ± 111.96a 1170.77 ± 97.59ab 1248.41 ± 155.73ab 1557.05 ± 113.92c 1324.80 ± 39.98b 1177.00 ± 92.38ab 1023.99 ± 47.00a 1198.15 ± 113.09ab 1131.15 ± 86.96ab

other hand, the expression of these transporters was suppressed with the application of glycine i.e. As(III)+Gly(L) (1.4 fold), As(III)+Gly(H) (1.60 fold), As(V)+Gly(L) (1.75 fold) and As(V)+Gly(H) (1.10 fold). Whereas, the expression of Lsi-2 was found not to be significantly altered with the treatment of glycine. The expression of all GRX genes was higher in As(V)+Gly(H) treatment, except GRX Os01g13950, which was suppressed against all treatments (Fig. 1C, D, E, F). However, the expression of GRX gene Os01g27140 was upregulated by 2.58 fold in As(V)+Gly(H) treatment. Similarly, the expression of Os02g40500 gene was upregulated by 4.48 fold, under As(V)+Gly(H) treatment. However, there were no significant changes in the expression of GRX Os01g13950 and nitrate reductase genes. The GST gene expression were significantly higher and upregulated by glycine application. GST Os02g20220 was found to be 7.64 fold higher in As(V)+Gly(H), while 5.18 fold higher in As(III)+Gly(H) (Fig. 1G, H), as compared to the control. Similarly, the expression of GST Os02g38160 was found to be 10.30 fold higher in As(III)+Gly(H), while 3.11 fold higher under As(V)+Gly(H) treatment.

accumulation in all the As treatments. Glycine was more effective against As(III), where it significantly reduced the As accumulation in shoot (71%) and root (68%), as compared to their respective control. Further, the ratio of As accumulation of root to shoot, is mentioned as translocation, was higher in shoot with the As(III) (0.17) than the As(V) (0.15) treatment. The application of Gly(L) to the As(III) significantly decreased the translocation of As (0.13) while Gly(H) and (L) with As (V) increases the translocation of As in the shoot (0.22 and 0.27), respectively. Among the essential elements, the accumulation of Fe in shoot and root was decreased against all the treatments involving glycine and As, except it increased (15%) in the root with the As(V)+Gly(L) treatment (Table 2). The translocation of Fe was affected by As stress, which was found to be lower under As(V) and As(III) stress (0.26 and 0.23), respectively (Table 2). The application of Gly(L) and Gly(H) increased the translocation of Fe (0.37 and 0.28), respectively, in the shoot. The translocation of Fe was significantly increased in shoot of the rice seedlings receiving As(III)+Gly(L) and As(III)+Gly(H) (0.32 and 0.47), respectively, as compared to the As(III) alone (Table 2). The level of the element Na, was also affected by the As stress and glycine application. The rice seedlings treated with As(III)+Gly(L) showed a higher level of Na (1.50 and 1.60) in shoot and root, respectively, as compared to wholly untreated control (1.09 and 1.58).

3.3. Application of glycine reduced the As toxicity The level of H2O2 in the As-treated seedlings was significantly increased, (19%) and (24%) with As(III) and As(V), respectively, as compared to the wholly untreated control (Table 1). However, the application of Gly(H) alongwith As(III) and As(V) reduced the H2O2 level significantly in rice seedlings (Table 1). The maximum reduction in H2O2 (15%) was observed with the As(III)+Gly(H), as compared to the As(III) alone. Similar to the H2O2, the TBARS level was higher in plants receiving As(III) and As(V), as compared to the wholly untreated control, but it was significantly higher in the case of As(V) (48%). However, the application of Gly(L) with As(V), significantly reduced the TBARS level (34%) in comparison to the As(V) alone.

3.2. Effect on gene expression The expression of lower silicon transporters i.e., Lsi-1 (Os02g0745100) and Lsi-2 (Os03g0107300), GRX genes (Os01g27140, Os01g13950, Os02g40500 and Os01g61350), GST (Os02g38160, Os02g20220) and nitrate reductase (Os02g53130) were analyzed using qRT-PCR under different treatments of As and glycine. The expression of lower silicon transporters (Lsi-1 and Lsi-2) were higher (3.0 and 1.47 fold change) with As(III) treatment, as compared to wholly untreated control C (Fig. 1A, B). However, only the Lsi-1 transporter gene expression was increased (1.48 fold change) with As(V) treatment. On the

Table 2 Arsenic (µg g−1 dw) accumulation in shoot and root of As(III) and As(V) treated rice seedlings supplemented with glycine(L & H). Significance of the mean values have been compared for each parameter separately (Duncan's test, p < 0.05) where bars marked with same letters are not significantly different. Accumulation (µg g−1 dw) Treatments

As Root

As Shoot

As TF

C As(III) As(V) Gly (L) Gly (H) As(III)+Gly (L) As(III)+Gly(H) As(V)+Gly(L) As(V)+Gly(H)

– 733.85 ± 22.68d 944.57 ± 92.59e – – 314.10 ± 35.81ab 231.46 ± 12.14a 523.58 ± 19.73c 405.84 ± 51.89b

– 126.91 ± 13.52c 150.89 ± 21.19c – – 41.13 ± 3.73a 37.06 ± 2.43a 143.61 ± 13.65c 93.18 ± 11.28b

– 0.17 0.16 – – 0.13 0.16 0.27 0.23

Fe Root

Fe Shoot f

1.14 ± .10 0.88 ± .15de 0.98 ± .07e 0.70 ± .02abc 0.76 ± .05bcd 0.65 ± .09ab 0.55 ± .05a 1.31 ± .09g 0.84 ± .06cde

412

Fe TF c

0.29 ± .03 0.23 ± .03b 0.23 ± .03ab 0.26 ± .03bc 0.22 ± .01ab 0.21 ± .02ab 0.26 ± .03bc 0.18 ± .03a 0.17 ± .02a

0.25 0.26 0.23 0.38 0.29 0.33 0.46 0.13 0.21

Na root

Na shoot a

1.58 ± .14 1.61 ± .13a 2.50 ± .10c 1.66 ± .04a 1.74 ± .17ab 1.67 ± .12a 1.97 ± .15b 1.67 ± .25a 1.62 ± .19a

Na TF a

1.09 ± .12 1.41 ± .16c 1.46 ± .17c 1.02 ± .14a 1.15 ± .11ab 1.50 ± .20c 0.97 ± .07a 0.97 ± .08a 1.37 ± .15bc

0.69 0.88 0.58 0.62 0.66 0.90 0.49 0.58 0.84

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Fig. 1. Gene expression of Low silicon transporter (Lsi-1 and Lsi-2) (A, B), GRX (C, D, E, F), and GST (G, H) in rice seedlings through qRT-PCR against As(III) and As(V) under low and high glycine conditions. Significance of the mean values have been compared for each parameter separately (Duncan's test, p < 0.05) where bars marked with same letters are not significantly different.

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Fig. 2. : Effect of As(III), As(V) treatments and glycine (L and H) on the specific activities of different antioxidant enzymes in shoots of rice (A) GRX (μmol g−1 Fw); (B) GR(μmol g−1 Fw); (C) NR (μmol g−1 Fw); (D) SOD (μmol g−1 Fw), (E) NOS (μmol g−1 Fw) and (F) NTR (μmol g−1 Fw). Significance of the mean values have been compared for each parameter separately (Duncan's test, p < 0.05) where bars marked with same letters are not significantly different.

to their respective glycine control. On the contrary, there was no significant difference observed in the activity of NTR, except against As(V) +Gly(H). Similarly, the activity of GR showed no significant change against any treatments. However, the specific activity of SOD was increased significantly with a combination of As(V)+Gly(H) (97%), as compared to the control (Fig. 2D).

3.4. Glycine modulates the defense mechanisms of rice seedlings against As stress Most of the antioxidant enzymes's activity was found to be significantly modulated during all the treatments as compared to the wholly untreated control. The activities of GRX and NR were enhanced against As(III) and As(V) treatments. However, the activities of GR, NOS and NTR were decreased under As(III) and As(V) treatments, as compared to wholly untreated control (Fig. 2B, E & F). The application of Gly(L) and (H) alongwith As(III) and As(V) significantly increased the activity of GRX, respectively, as compared to As(III) and As(V) alone (Fig. 2 A). The highest activity was observed against As(V)+Gly(H) (42%), as compared to their respective control. Similarly, the activity of NOS was found to be increased when plants were treated with Gly(H) alone (55%). The application of Gly(L) and (H) along with As(III) increased the activity of NOS by 75% and 12%, respectively, as compared

4. Discussion Amino acids are the primary constituent of biomolecules, which triggers different signaling responses in the cell and maintain cellular homeostasis. Some stress responsive amino acids such as glycine, proline, glutamic acid, and cysteine, play a crucial role in the plants stress management (Kumar et al., 2014). In present study, the application of glycine enhanced the tolerance response against As stress. The AA glycine is known to elevate the level of betaine, which protect the 414

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against As(III)+Gly(L) and As(V), which demonstrates the detoxification role of glycine via stimulation of GRX gene. Although, there was no difference observed in the expression of Os01g13950 but the expression of other GRX i.e. Os01g61350 and Os01g27140 was very high against As (V)+Gly(H) that exhibits the differential expression of GRX under different conditions. Glutathione-S-transferase is a cytoplasmic enzyme that thiolates As (III) and pumps it into vacuole in an ATP-dependent manner (Tripathi et al., 2012). In plants, GST is known to be induced by heavy metal exposure (Chakrabarty et al., 2009; Moons, 2003). Out of 79 GST genes, as reported by Jain et al. (2010), expression of two GST genes i.e. Os09g20220 and Os02g38160 (TAU class) were studied to find the modulation of GST genes in the presence of exogenous glycine for detoxification of arsenic. The higher expression of GST Os09g20220 in the presence of Gly (L and H) supports that the presence of glycine enhances the expression of GST via synthesis of GSH. The higher expression of Os09g20220 against As(V) and Os02g38160 against As(III) show the specificities of these two GST towards As(III) and As(V) stress which is in agreement with our previous study (Dubey et al., 2016). Similar to Os09g20220, the expression of Os02g38160 was also higher in the presence of Gly(H), however, there is no significant expression in seedlings receiving Gly(L) only. The higher expression of GST Os09g20220 against As(V)+Gly (L and H) and Os02g38160 against As (III)+Gly(L and H) shows that the thiolation and sequestration of As into the vacuoles are very specific to the GST and are regulated by different species of As in the cell. This is contrary to the report of upregulation of GST against As(V) exposure (Chakrabarty et al., 2009).

plants from wide range of abiotic stresses (Waditee et al., 2005). Glycinebetaine (GB) is a quaternary ammonium compound, accumulate against several environmental stresses such as drought, salinity, heat, UV radiation and heavy metals (Ashraf and Foolad, 2007; Chen and Murata, 2011). Some plants such as rice, mustard, Arabidopsis and tobacco have not able to synthesise GB (Rhodes and Hanson, 1993). However, genetic manipulation of gene related to GB synthesis and exposure of glycine in these types of plants could enhance the level of GB and protects plants from several stresses (Waditee et al., 2005). The exposure of glycine was also shown to enhance the development of root hairs that allows access to greater surface area and volume of soil, favoring both the acquisition of water and nutrients in Habanero pepper (Domínguez-May et al., 2013). However, according to Tian et al. (2009), the application of glycine enhances the transcript level of ACC oxidase, which regulates plant hormones thereby protecting plants from different stresses. In the present study, the application of glycine was found to be very helpful in reduction of As uptake and toxicity. This was in consonance with higher expression of GRX and GST genes which decreased the translocation of As from root to shoot through modulating Lsi-1 and Lsi-2 transporters gene expression. 4.1. Expression of lower silicon transporters (Lsi-1 and Lsi-2), glutaredoxin and glutathione-S-transferase genes against As stress In our study, the application of glycine suppressed the expression of lower silicon transporter genes (Lsi-1 and Lsi-2) against As, which was higher in As treatment alone. The suppression of these genes resulted in lowered accumulation of As in the root and shoot part. The application of glycine with As species reduced the expression of Lsi-1 to a greater extent than Lsi-2 which confers the inhibition of As transport from the medium to the cells. The Lsi-1 is an influx transporter abundantly expressed in the mature regions of root cells, modulation of this transporter gene expression, affects the accumulation of As(III) and silicon. On the other hand, the Lsi-2 is an efflux transporter, regulates transportation of As(III) and Si from the root to the apoplast of the plants. The variation in Lsi-2 transporter gene expression may influence the translocation of As(III) and Si to the shoot/grain of rice plants (Ma et al., 2008). The transporter proteins of the plants regulates the metal uptake in root cells and metal transfer between cells to the different organs depending on the availability of the elements (Thomine et al., 2000). Plants contain various specific and non-specific transporters for mineral transportation to from root to the shoot parts. In a similar study by Connolly et al. (2002), the higher expression of IRT1 transporter increased the accumulation of Cd and Fe in Oryza sativa. This is congruent with the our study, where we observed the higher accumulation of As with enhanced expression of Lsi-1 and Lsi-2 transporters. In our study, the gene expression of GRX and GST was found to be enhanced against As(III) and As(V) under glycine application, which is well known to ameliorate As stress in plants (Dubey et al., 2016). GRXs are ubiquitous small heat-stable disulfide oxidoreductase which acts as thioltransferases or transhydrogenase with a molecular mass of 10–15 kDa. (Fomenko and Gladyshev, 2002). Glutaredoxin/glutathione/glutathione reductase is one of the major systems involved in maintaining the cellular redox state. GRX can regulate a protein or enzyme activity by reversibly glutathionylating or reducing the disulfide bonds to prevent their critical cysteine residues from irreversible oxidation under oxidative stress (Meyer et al., 2009; Rouhier et al., 2008). The expression of four GRX genes, as represented by Garg et al. (2010) i.e. OsGRX4, OsGRX3, OsGRX9 and OsGRL2 was higher for OsGRX3 and OsGRX4 in all the seed developmental stages; OsGRX4, OsGRL2 and OsGRX9 in young leaves. The higher expression of Os02g40500 against As(V) and As(III) shows its greater specificity towards As(V) is in agreement with Chakrabarty et al. (2009). In our study, the presence of Gly (L and H) enhances the expression of Os02g40500, respectively, which demonstrates the dependency of GRX on glycine. Similarly, the expression of GRX gene was also higher

4.2. Accumulation and translocation of metals Accumulation of As depends on genetic variability of rice i.e. tolerant cultivar of rice accumulates more As than the sensitive cultivar (Norton et al., 2009). In our study, the accumulation of As was significantly affected by the application of glycine which was significantly lower in shoot while highest in root. The exogenous glycine relatively reduced the As(III) accumulation more than As(V). This may be due to lower expression of Lsi-1 and Lsi-2 transporter genes, which is the specific transporter for As(III) and silicon uptake (Ma et al., 2007, 2008). The translocation of As in shoots was higher in As-treated plants but reduced the translocation of As in shoots due to exogenous application of glycine with As(III) and As(V). This may be possibly due to the synthesis of GSH followed by thiolation and sequestration of As into the vacuoles. It is reported that the GSH chelates the As(III) followed by its sequestration into the vacuole using GST enzyme (Jain et al., 2010). The translocation of As alters the translocation of other essential elements (Dwivedi et al., 2010a). In our study, the Fe level was found to be decreased due to the translocation of As, as compared to control. The application of Gly(H) with As(III) enhanced the level of Fe in shoots which signifies the role of glycine in maintaining the level of essential elements for the survival of plants under As stress. Dwivedi et al. (2010b) have reported lower levels Fe in the shoot of two rice cultivars viz. Triguna and IET-4786, which accumulated higher As [As(V); 12 mg l−1], in contrast to the higher Fe level in the other two cultivars viz. IR-36 and PNR519, where As accumulation was found to be low. This strongly supports our data where the inverse relationship was observed between As and Fe translocation. The translocation of Na was also studied which was affected by As translocation. The translocation of Na was increased in shoots when plants were treated with As(III)+Gly(L) and As(V)+Gly(L), as compared to the control. Glycine is the main residue of Na+/K+ channel which is modulated during glycine rich medium (Mäser et al., 2002). 4.3. Toxicity and antioxidant response TBARS is a stress marker which is formed during lipid peroxidation due to a high level of reactive oxygen species (ROS) in the cell. The 415

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increased level of TBARS in As(III) and As(V) show the toxicity due to As in the cell. This corroborates with finding by Kumar et al. (2013, 2016), where enhanced level of TBARS was observed in rice seedlings under As toxicity. The significant decrease in the level of TBARS with the application of glycine showed the amelioration of As toxicity and this is also coincident with an enhanced level of antioxidant enzyme activity and decreased accumulation of As in the rice plants. The heavy metal detoxification by exogenous application of AA has also been reported in cotton and rice plants (Aslam et al., 2014; Wang et al., 2016). There could be enhanced level of glycine that increased the antioxidants e.g. GRX and other detoxifiers which eventually reduced the toxicity within the cell. H2O2 is a form of ROS which gets enhanced during the toxicity under different stresses. Enhanced level of H2O2 in the As-treated seedlings shows the enhanced oxidative stress against As. However, decreased level of H2O2 with the application of glycine shows the reduced toxicity of As in the cell. This is also congruent with the study of Wang et al. (2010), where they have observed lower level of H2O2 in the presence of glycine-betaine in wheat plants under drought and salinity stresses. In our study, the glycine treated plants also showed higher H2O2 which may be due to the enhanced signaling cascade as glycine and H2O2 both can act as signal molecule. Amino acids have been proposed as the protector of enzymes against salinity, drought, heat and metal poisoning under in vitro conditions (Sharma and Dietz, 2006; Chaffei-Haouari et al., 2009). This is because AA interacts with the three-dimensional structure of proteins and make it stable for catalysis. Thus AA could bind with these sidechain interactions and interfere with the activity of enzymes (Rai, 2002). Amino acids immensely modulate the detoxification of xenobiotic compounds and scavenging of free radicals and hence oxidative stress (Knights et al., 2007). The increased activities of antioxidant enzymes, i.e. NOS, SOD and GRX show the modulation of the antioxidant system using glycine. The NOS enzyme which is responsible for nitric oxide (NO) synthesis and nitrogen content regulation in the cells, are enhanced many folds, showing that the glycine also acts as a patron of nitrogen during stress conditions. The absence of any significant changes in the activity of GR and NTR shows that exogenous glycine does not alter the reducing environment of the cell during As stress, which is very essential for the survival of the cell (Noctor et al., 2012). The enhanced activity of glutaredoxin by the application of glycine with As(V) i.e. As(V)+Gly(H) signifies the detoxification role of GRX in the presence of glycine. A similar result was also observed in our previous study, where exogenous GSH enhanced the activity of GRX for reducing the As toxicity (Dubey et al., 2016). The activity of SOD dismutates the O2- to H2O2 which subsequently by the action of APX, POD or CAT is converted into water. The higher activity of SOD against As (V)+ Gly(H) indicates the scavenging of free radicals in the presence of glycine.

The authors are thankful to Director, CSIR-NBRI, Lucknow for the infrastructural support to carry out the research. The work is supported by research grant provided by SERB-DST, GOI, New Delhi (SR/SO/PS/ 81/2010). AKD acknowledges SERB-DST, GOI and CSIR, New Delhi for research fellowships. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.10.047. References Ahsan, N., Lee, D.G., Alam, I., Kim, P.J., Lee, J.J., Ahn, Y.O., Kwak, S.S., Lee, I.J., Bahk, J.D., Kang, K.Y., Renaut, J., 2008. Comparative proteomic study of arsenic‐induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress. Proteomics 8 (17), 3561–3576. Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 53, 1331–1341. Aslam, S. 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5. Conclusion The results obtained during this study, shows the ameliorating effect of glycine application on rice seedlings against As stress. Glycine application played a crucial role in the reduction of As accumulation via suppessing the expression of Lsi-1 and Lsi-2 transporter genes. The results also demonstrate that the glycine is more efficient to reduce As(III) accumulation than As(V). The stress responsive genes such as GRXs and GSTs showed higher expression during glycine application which signifies the glycine mediated regulation of tolerance machinary in rice plants against As toxicity. Simultaneously, enhanced activities of antioxidant enzymes (GRX, NOS, SOD) and lower level of TBARS and H2O2, show the overall modulation of defence system by the application of glycine.

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