Silicon improves growth and alleviates oxidative stress in rice seedlings (Oryza sativa L.) by strengthening antioxidant defense and enhancing protein metabolism under arsanilic acid exposure

Ecotoxicology and Environmental Safety 158 (2018) 266–273

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Silicon improves growth and alleviates oxidative stress in rice seedlings (Oryza sativa L.) by strengthening antioxidant defense and enhancing protein metabolism under arsanilic acid exposure

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Anjing Genga,b,1, Xu Wanga,b,1, Lishu Wuc, Fuhua Wanga,b, , Zhichao Wub,d, Hui Yanga,d, Yan Chena,d, Dian Wenb, Xiangxiang Liub a

Research Center of Trace Elements (Guangzhou), Huazhong Agricultural University, Guangzhou 510640, Guangdong, PR China Public Monitoring Center for Agro-product of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, Guangdong, PR China College of resource and environment, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China d Key Laboratory of Testing and Evaluation for Agro-Product Safety and Quality, Ministry of Agriculture, PR China, Guangzhou 510640, Guangdong, PR China b c

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A B S T R A C T

Keywords: Rice Arsanilic acid Silicon Growth Oxidative stress

Organoarsenic arsanilic acid (ASA) contamination of paddy soil is a serious but less concerned hazard to agriculture and health of people consuming rice as staple food, for rice is one major pathway of arsenic (As) exposure to human food. To date little research has studied the effect of ASA on biochemical process of rice. Silicon (Si) application is able to reduce the toxicities of heavy metals in numerous plants, but little information about ASA. This work investigated whether and how Si influenced alleviation of ASA toxicity in rice at biochemical level to have a better understanding of defense mechanism by Si against ASA stress. Results showed that ASA reduced rice growth, disturbed protein metabolism, increased lipid peroxidation but decreased the efficiencies of antioxidant activities compared to control plants, more severe in roots than in shoots. The addition of Si in ASAstressed rice plants noticeably increased growth and development as well as soluble protein contents, but decreased malondialdehyde (MDA) contents in ASA-stressed rice plants, suggesting that Si did have critical roles in ASA detoxification in rice. Furthermore, increased superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities along with elevated glutathione (GSH) and ascorbic acid (AsA) contents implied the active involvement of ROS scavenging and played, at least in part, to Si-mediated alleviation of ASA toxicity in rice, and these changes were related to rice genotypes and tissues. The study provided physio-chemical mechanistic evidence on the beneficial effect of Si on organoarsenic ASA toxicity in rice seedlings.

1. Introduction Organoarsenics are commonly thought less toxic than inorganic arsenic such as arsenate (As(V)) and arsenite (As(III)), but they can be converted to inorganic arsenic by biotic and abiotic processes. P-arsanilic acid (ASA) (4-aminophenylarsonic acid) is being an emerging but less concerned contaminant used widely in animal feeding industry. It can be transformed to more toxic metabolites after being excreted by animals, and contaminate soil and water when animal manure enters the environment, or when used as organic fertilizer in agricultural sites. Reports showed that organoarsenics accumulated in plants, and their degradation products As(V) and As(III) were stored up in vegetable (Yao et al., 2008). Arsenic (As) concentrations of animal manure were from the level below the detection limit to 50 mg kg−1 (Arai et al.,

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2003). So rice with high As accumulation ability grown in these areas was influenced by As toxicity, mainly because of As-contaminated soil and/or irrigating water. As accumulation by rice is of great attention, because the dietary intake of rice is contributing to the main As exposure pathway to people in countries where rice is a staple diet. It was reported that rice presented the highest inorganic As value in 215 samples, corresponding to 67% of dietetic inorganic As intake (Fontcuberta et al., 2011). Moreover, As-contaminated rice grains may cause their further processing products containing As. However, to date, the chain of feed additives (ASA) - excrement - soil/water - rice ( rice products) - human received little attention and little research was in this field. The entry of As into food chain poses severe threats to human health and has come into a major public concern. Therefore, there is an imperative need of seeking effective methods for minimizing

Correspondence to: No. 20 Jinying Road, Tianhe District, Guangzhou 510640, Guangdong, PR China E-mail address: [email protected] (F. Wang). Xu Wang is co-first author

https://doi.org/10.1016/j.ecoenv.2018.03.050 Received 14 November 2017; Received in revised form 11 March 2018; Accepted 23 March 2018 0147-6513/ © 2018 Published by Elsevier Inc.

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As contamination in rice. Plenty of ways have been practiced to remediate As polluted soil and the use of fertilizer is one multifunctional and easy way. Silicon (Si) fertilizer is widely used to improve soil conditions for plant growth. Potassium silicate has been reported as one citrate soluble silicate with a high capacity of cutting down As contents in rice and is suitable for the base fertilizer. It was reported that As could activate the generation of reactive oxygen species (ROS) and free radicals which brought about oxidative stress. However, the effects of Si on mediating ASA induced oxidative stress as a strategy for As tolerance remains at its infancy stage. In present study, we investigated the function of Si-based supplementation to growth and oxidative stress behaviors of three different kinds of rice seedlings under ASA exposure. By evaluating the biomass and lipid peroxidation (LPO) of shoots and roots, we revealed whether organoarsenic does harm to rice seedlings, and if so, we shed light to the physiological function of Si in protective mechanisms by assay of the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), the contents of ascorbic acid (AsA), glutathione (GSH) and soluble protein in rice seedlings under different treatments. 2. Materials and methods 2.1. Materials and treatments The spiked concentration of ASA was 100 mg L−1 selected based on the previous permissible additive amount in fodder and environmental realistic concentration (Arai et al., 2003; Mangalgiri et al., 2015) and was prepared freshly as C6H8AsNO3 (A.R.). Three types of rice (Oryza sativa L.), T (hybrid rice cultivar), Y (indica rice) and B (glutinous rice), were chosen for the test. Seeds of the three rice cultivars were obtained from Rice Research Institute, Guangdong Academy of Agricultural Science. Prior to germination, rice seeds were sterilized with 15% (v: v) sodium hypochlorite for 20 min, rinsed off with deionized water, then sprouted in Petri dishes containing moist filter paper at 25 ℃ darkly for 4 days. Subsequently, just healthy and consistent rice seedlings were transferred to cases (57 cm × 39 cm × 8 cm) with 14 L 1/2 Hoagland solution under natural condition (Hoagland and Arnon, 1950). The pH was adjusted to 5.5 by HCl. All solutions were replaced every 7 days. For treatment, 100 mg L−1 ASA (ASA), 168 mg L−1 sodium silicate (Si), 100 mg L−1 ASA and 168 mg L−1 Si (ASA + Si) were added into the culture solutions for 20 days. Left untreated rice (CK) was grown concurrently. Each treatment was replicated three times.

Fig. 1. Effects of different treatments [non-stressed control (CK), 168 mg L−1 silicon (Si), 100 mg L−1 arsanilic acid (ASA), 100 mg L−1 arsanilic acid and 168 mg L−1 silicon (ASA + Si)] on fresh weights of shoots (A), roots (B) in three different kinds of rice seedlings. Values (+ SD) following different lowercase and capital letters indicated significant differences at p < 0.05 among different treatments and different cultivars, respectively.

reported in Rao et al. (1996). 2.5. Measurements of AsA, GSH and soluble protein contents Ascorbic acid contents were determined by spectrophotometer method obtaining the absorbance at 525 nm (Hodges et al., 1997) and GSH at 412 nm (Anderson, 1985). Soluble protein concentration was measured by using coomassie brilliant blue staining (Marion, 1976).

2.2. Measurements of growth parameters

2.6. Statistical analyses

After treatment, rice seedlings were harvested, then washed thoroughly with distilled water, and separated into shoots and roots, which were measured using electric balance.

All the determinations were analyzed statistically by one-way ANOVA tests using SPSS 18.0 software followed by Duncan test. The probability level of significance was set at p < 0.05.

2.3. Measurements of LPO

3. Results and discussion

Shoots and roots samples from 20-d-old rice seedlings were used to study LPO. LPO was evaluated as malondialdehyde (MDA) using thiobarbituric acid method (Heath and Packer, 1968). The concentration of MDA was gained from the formula: C (µmol g−1) = [6.452 * (A532 A600) − 0.56 * A450] * VT / (V0 * W), for A532, A600, A450, VT, V0 and W represented the absorbance at 532, 600 and 450 nm, and the total extract volume, the determined volume and the rice seedling weight, respectively.

3.1. Effects of Si addition on fresh weights of shoots and roots Based on variance analysis, there were significant differences of fresh weights in shoots (Fig. 1 A) and roots (Fig. 1 B) among different treatments and different rice cultivars (p < 0.05). Maximum fresh weights were observed in rice supplied with Si (p < 0.05). ASA has inhibited growth significantly of all rice cultivars compared to control (p < 0.05). And the inhibition effects of ASA were more severe in roots than in shoots of all rice plants and more serious in conventional rice plants (B cultivar and Y cultivar) than in hybrid rice (T cultivar). But Si addition significantly increased fresh weights of rice seedlings under ASA treatments in comparison to only subjected to ASA stress (p < 0.05).

2.4. Measurements of SOD, CAT and POD activities SOD activities were analyzed using nitroblue tetrazolium reduction method (Shi et al., 2005). 50% inhibition showed the expression of 1 unit enzyme. CAT activities were estimated by using spectrophotometer method (Chance and Maehly, 1954), POD activities were assayed as 267

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Many previous investigations showed that As decreased yield, photosynthesis and pigments in plants (Hu et al., 2013; Tripathi et al., 2013; Begum et al., 2016; Pandey et al., 2016; Raza et al., 2016). This research made clear that ASA exposure inhibited rice growth and development significantly. It was inferred that organic As could cause harm to rice as well. It was likely that ASA disturbed the biochemical and metabolic processes in rice (Hu et al., 2013), because As could inhibit the parameters of gas exchange (stomatal conductance, water use efficiency, net photosynthesis and transpiration rate), the contents of chlorophyll and carotenoids (Li et al., 2007; Pisani et al., 2011; Hu et al., 2013). Reduced growth under As exposure has been reported in other plants (Mangalgiri et al., 2015; Raza et al., 2016). It is well known that Si is valuable for growth and yield by enhancing resistance to biotic stress (biological pests and diseases), as well as abiotic stress (temperature, drought, salt and metal toxicity) (Ma et al., 2006; Liu et al., 2009; Shen et al., 2010; Shekari et al., 2015; Kang et al., 2016; Helaly et al., 2017; Li et al., 2017). Results from this investigation also have demonstrated that Si application significantly raised biomass under ASA stress (Fig. 1, p < 0.05), proving the conducive effects on rice growth and development. Such advantageous effects of Si could be indirect (e.g. strengthens disease resistance) or direct (e.g. improves nutrition). Reportedly, Si could induce growth promotion by strengthening extensibility of rice cell wall (Hossain et al., 2002). The conducive influences of Si on growth have been confirmed in numerous plants (Guo et al., 2005; Farooq et al., 2013; Vaculikova et al., 2014; Raza et al., 2016; Ju et al., 2017; Li et al., 2017). 3.2. Effects of Si on MDA contents of rice seedlings The LPO was measured in the light of MDA contents in shoots (Fig. 2 A) and roots (Fig. 2 B) of rice seedlings. Upon ASA exposure, MDA contents significantly rose in shoots and roots of three rice seedlings compared with CK (p < 0.05), and more in roots than in shoots. The most increase was detected in roots of Y cultivar. Si addition decreased MDA contents significantly in shoots of all rice plants (p < 0.05). For ASA + Si treatment, MDA contents decreased significantly in shoots and roots of all rice cultivars compared with ASA treatments (p < 0.05), and more reduction was found in roots of B cultivar and T cultivar. There were also significant differences among different rice cultivars (p < 0.05). Generally speaking, for most plants, active oxygen free radicals are produced unavoidably in non-polluted plants, and plants have evolved effective eliminating systems (antioxidant enzymes and non-enzymatic antioxidants) for controlling ROS (inducer of plant cell death) levels in order not to accumulate excessively. However, the balance could be broken by environmental stress. According to many studies, excessive heavy metal in plants caused active oxygen free radicals accumulation. As accumulation stimulates ROS increase, which influences enzyme activity, membrane permeability, photosynthetic reactions, chlorophyll synthesis and biomass. ROS induced by As can attack directly hydrogen atom on the methylene group next to an unsaturated carbon atom, then induce chain-like peroxidation of the polyunsaturated fatty acids (more susceptible on free radical damage) in the membrane, and degenerate fatty acids and produce lipids peroxides lastly. MDA, one major degradation product of polyunsaturated fatty acids hydroperoxides, is commonly considered as a dependable biomarker for LPO in plants and animals and an indirect indicator of oxidative stress. In this study, MDA contents increased in all cultivars under ASA treatments compared with CK. This indicated that ASA could induce generation of superoxide radicals and increase LPO products, showing higher speed of LPO and stronger susceptibility to oxidative stress compared with non-stress condition. The increased MDA contents provided the clear proof that rice functioned under oxidative stress condition. Moreover, MDA contents in Y cultivar increased more than in B and T cultivars, showing that Y cultivar possessed weaker resistance to ASA stress. It could be related to rice genotype, as well as rice tissues, growth stages and

Fig. 2. Effects of different treatments [non-stressed control (CK), 168 mg L−1 silicon (Si), 100 mg L−1 arsanilic acid (ASA), 100 mg L−1 arsanilic acid and 168 mg L−1 silicon (ASA + Si)] on the contents of MDA of shoots (A), roots (B) in three different kinds of rice seedlings. Values (+ SD) following different lowercase and capital letters indicated significant differences at p < 0.05 among different treatments and different cultivars, respectively.

conditions, concentrations and duration of exposure to As, As species, etc (Begum et al., 2016). Similarly, higher MDA contents were noticed in rice under other stress (Song et al., 2011), and in other plants under As and various stresses (Li et al., 2007; Liu et al., 2009; Vaculikova et al., 2014; Tripathi et al., 2015; Wu et al., 2017). Si has a prominent role in alleviating varieties of abiotic stresses, including As toxicity, via physiological mechanisms. Such protection system against oxidative stress may be able to prevent plants from injury. In present study, MDA contents in shoots and roots of three rice cultivars decreased under ASA + Si treatment compared with ASA exposure, suggesting that exogenous Si was involved in ROS metabolism of rice exposed to stressful environment and alleviated ASA toxicity. The reason might that 1) Si decreased the accumulation of superoxide radical (O2·) and H2O2 and thereby reduced LPO in ASAstressed rice, and improved antioxidant defence system. This result was supported by higher activities of SOD, POD and CAT (Fig. 3) and larger amounts of GSH and AsA (Fig. 4) in ASA + Si treated rice plants; 2) exogenous Si minished the permeation abilities of the cell plasma membranes by enhancing lipids stabilities and preventing the deteriorations of structures and functions of rice cell membranes when rice was under stress. This result was in line with the previous reports (Shi et al., 2005, 2014; Gunes et al., 2007; Liu et al., 2009; Song et al., 2009, 2011; Li et al., 2012; Bharwana et al., 2013; Farooq et al., 2013; Tripathi et al., 2013, 2015; Vaculikova et al., 2014; Cao et al., 2015; Wu et al., 2017).

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Fig. 3. Effects of different treatments [non-stressed control (CK), 168 mg L−1 silicon (Si), 100 mg L−1 arsanilic acid (ASA), 100 mg L−1 arsanilic acid and 168 mg L−1 silicon (ASA + Si)] on activities of superoxide dismutase (SOD) in shoots (A), roots (B), catalase (CAT) in shoots (C), roots (D), peroxidase (POD) in shoots (E), roots (F) of three different kinds of rice seedlings. Values (+ SD) following different lowercase and capital letters indicated significant differences at p < 0.05 among different treatments and different cultivars, respectively.

rice plants compared with CK (p ＞ 0.05), but increased SOD activities in all rice plants significantly under ASA treatments compared with single treatments with ASA (p < 0.05). The increase was T＞B＞Y. Abiotic stresses, such as heavy metals and metalloids are well known to induce ROS and oxidative stress. The induction of oxidative stress by As is the chief toxicity process in plants. ROS include O2·, H2O2 and OH·, that attack important metabolic functions by degrading vital

3.3. Effects of Si on SOD activities of rice seedlings The effects of Si on SOD activities were presented in Fig. 3 (A – B). It was observed that SOD activities significantly decreased in shoots and roots of all rice cultivars during ASA exposure compared with CK (p < 0.05), and most decrease was found in root of Y cultivar. Si addition did not show significant differences of SOD activities in shoots of 269

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Fig. 4. Effects of different treatments [non-stressed control (CK), 168 mg L−1 silicon (Si), 100 mg L−1 arsanilic acid (ASA), 100 mg L−1 arsanilic acid and 168 mg L−1 silicon (ASA + Si)] on the contents of ascorbate (AsA) of shoots (A), roots (B), glutathione (GSH) of shoots (C), roots (D) in three different kinds of rice seedlings. Values (+ SD) following different lowercase and capital letters indicated significant differences at p < 0.05 among different treatments and different cultivars, respectively.

significantly in this study (Fig. 3 A - B, p < 0.05), indicating that rice seedlings have suffered serious oxidative stress induced by ASA toxicity. It might be due to 1) stress causing severe damage to rice cells, leading to inhibition of engaging larger amounts of SOD; 2) the serious oxidative stress damaging SOD, hence SOD activities were diminished; 3) metal bindings with rice either on the surface or in the compartmentalization of the cells; 4) enhancing bio-geochemical and nutrient cycle within the rhizosphere of rice and 5) providing a minimum area for ASA accumulation. This tendency was in keeping with previous reports (Shen et al., 2010; Farooq et al., 2013; Wu et al., 2017). Moreover, SOD activities reductions were detected more in roots than in shoots under ASA stress, showing SOD activities reductions were related to rice tissues. Similar result was reported that noticeable SOD activities reduced in roots, while little effects were found in leaves (Zeng et al., 2011). Si addition could ameliorate oxidative stress induced by As in rice seedlings partly by improving thiolic and antioxidant systems (Tripathi et al., 2013). In this study, Si application significantly increased SOD activities, indicating that Si treatment could activate SOD to remove the excess free radicals (Hu et al., 2013), and effectively enhance the defence capacities against oxidative stresses induced by ASA toxicity in rice seedlings. Supporting evidence also concluded that exogenous Si enhanced SOD activities in rice under other stresses (Song et al., 2009;

biomacromolecules like proteins, nucleic acids and lipids. These oxidative damages have been reported in rice and other plants. Luckily, plants actually possess many defence mechanisms to counter ROS. The antioxidative defence in plants falls into two classes: 1) various enzymes such as SOD, POD and CAT, and 2) low molecular weight compounds that function as antioxidants such as AsA and GSH. These antioxidants not only detoxificate ROS, but also remove free radicals, which are in charge of cellular signalings. The effects of SOD, POD and CAT are highly specialized, because usually the product of one of these enzymes instantly becomes the substrate of another enzyme, which leads to obtaining nontoxic substances. The view dominated that the first defending line of organisms against toxic ROS included metalloenzymes generally referred to SOD. SOD catalyzes the disproportionate reaction of O2˙ˉ into hydrogen peroxide and oxygen, and acts as one most important defence mechanism against ROS and xenobiotics. Therefore, SOD can be a good indicator to assess As toxicity in the environment. SOD is used as the scavenger of superoxide free radical. Under the moderate adverse circumstance, SOD activity is induced to increase the ability of stress resistance and adaptation of the plants. As ROS increase, plants improve SOD activities and/or contents to clear ROS, showing their adaptive responses. But SOD activities decreased 270

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also detoxify H2O2 using various substrates (such as phenol) as electron donors. POD could participate in lignin biosynthesis to establish physiological barriers against toxic heavy metals stresses. Changes of POD activities are considered as the most reliable indicator of the oxidative stress emergence. The decline of POD activity might be as a result of the severe oxidative stress damage to POD. Suppression of POD activity has also been recorded in cotton upon cadmium exposure (Farooq et al., 2013). But POD activities increased under ASA + Si treated rice, suggesting that addition of Si was great beneficial on activities of POD and effectively strengthened the defence capacity against oxidative stress caused by ASA exposure in rice seedlings. Similar conclusions were found in rice under chromium (Zeng et al., 2011), simulated acid rain (Ju et al., 2017) exposures, as well as in several plants such as Brassica chinensis L. (Song et al., 2009), peanut (Shi et al., 2010), barley (Gunes et al., 2007), peas (Rahman et al., 2017), cotton (Bharwana et al., 2013; Farooq et al., 2013), pakchoi (Zhang et al., 2013), banana (Li et al., 2012), dill (Shekari et al., 2015), pistachio (Habibi and Hajiboland, 2013), xerophyte Zygophyllum xanthoxylum (Kang et al., 2016) under heavy metals (Song et al., 2009; Shi et al., 2010; Li et al., 2012; Bharwana et al., 2013; Farooq et al., 2013; Zhang et al., 2013; Rahman et al., 2017) and other stresses (Gunes et al., 2007; Habibi and Hajiboland, 2013; Shekari et al., 2015; Kang et al., 2016). Besides, the effects of Si addition were dependent on cultivars (Shi et al., 2010; Zeng et al., 2011), tissues (Shi et al., 2010; Zeng et al., 2011) and stress strength (Zeng et al., 2011; Kang et al., 2016).

Zeng et al., 2011; Tripathi et al., 2013; Ju et al., 2017), and in other plants upon many stresses (Shi et al., 2005, 2010, 2014; Gunes et al., 2007; Liu et al., 2009; Li et al., 2012; Bharwana et al., 2013; Bokor et al., 2013; Habibi and Hajiboland, 2013; Zhang et al., 2013; Shekari et al., 2015; Tripathi et al., 2015; Kang et al., 2016; Helaly et al., 2017; Wu et al., 2017). In this study, exogenous Si enhanced SOD activities more in T and B cultivars than Y variety, showing that the function of Si was related to rice genotype. 3.4. Effects of Si on CAT activities of rice seedlings In Fig. 3 (C – D), CAT activities fell off in shoots and roots of all rice seedlings significantly, but went up significantly under Si treatments compared with control (p < 0.05), and the decrease was T < B < Y, and the increase was Y < B < T. When treated with Si and ASA, CAT activities increased both in shoots and in roots of three rice cultivars compared with ASA exposure (p < 0.05), and the most increase was found in shoots of T cultivar. CAT can converse H2O2 into H2O and O2 not requiring a reluctant in peroxisomes, so CAT mostly plays a primary role in cutting down excessive H2O2 levels in plant cells to prevent cells from being damaged. For this reason, CAT is considered as a basic enzyme responsible for removing H2O2 from cells. CAT may be a high expressing enzyme in facilitating ROS scavenging in rice, which made it an indispensable part of ROS detoxification system and linked in the main defence mechanism against ROS toxicity. So CAT activity is always subjected to a considerable increase under oxidative stress (Wang et al., 2015). However, opposite results were reached in this study upon ASA exposure (Fig. 3(C - D)), showing that rice seedlings were under severe stress and damaged. The lower activity of CAT manifested that 1) ROS level was higher than its scavenging by CAT and 2) CAT might play a less important part in detoxification of ASA in rice. This might be attributed to 1) ASA inhibiting the rice growth when single treatment; 2) severer oxidative damage to CAT. Similar consequences were not only reported in rice (Zeng et al., 2011), but also in other plants under heavy metal stress (Shi et al., 2005; Song et al., 2009; Zeng et al., 2011; Farooq et al., 2013; Tripathi et al., 2015; Rahman et al., 2017). In this study, Si increased CAT activities when compared with ASA treatment alone. It was supposed that the function of Si was attributed to enforcing synthesis of CAT. This induction of CAT was compatible with decreasing MDA contents and increasing SOD activities, suggesting that higher CAT activities induced by Si – based addition might help scavenge the excessive ROS as well as H2O2 and protect rice tissues from membrane oxidative damage upon ASA exposure. Similar outcomes were reported in rice and other plants under various stresses (Shi et al., 2005, 2014; Gunes et al., 2007; Song et al., 2009, 2011; Zeng et al., 2011; Li et al., 2012; Bharwana et al., 2013; Farooq et al., 2013; Habibi and Hajiboland, 2013; Zhang et al., 2013; Vaculikova et al., 2014; Shekari et al., 2015; Tripathi et al., 2015; Kang et al., 2016; Ju et al., 2017; Rahman et al., 2017; Wu et al., 2017), and this tendency was related to cultivars (Song et al., 2009; Zeng et al., 2011; Li et al., 2012), tissues (Shi et al., 2010; Song et al., 2011), grow stages (Shi et al., 2014), duration time (Shi et al., 2005), concentrations, stress intensities and so on (Shi et al., 2005; Kang et al., 2016; Wu et al., 2017).

3.6. Effects of Si on AsA contents of rice seedlings As seen in Fig. 4 (A - B), during ASA treatment, AsA contents decreased significantly in shoots and roots of three rice cultivar seedlings (p < 0.05) compared with control. The most decrease was detected in roots of Y cultivar, and least reduction was found in shoots of T cultivar. But the addition of Si significantly increased AsA contents in shoots and roots of three rice cultivar seedlings under ASA treatment (p < 0.05), as well as under control condition. Ascorbate is one of the major non-enzymic antioxidants, which plays an important protective part to plants in clearing up ROS and inhibiting membrane LPO under stress conditions. AsA serves as one substrate for ascorbate peroxidase in cytosol, mitochondria, cytoplasm, peroxisomes and chloroplasts through AsA – GSH cycle to detoxify H2O2. In this study, AsA contents reduced under ASA stress, suggested that acute or chronic exposure to abiotic stress commonly led to AsA depletion. It might because that 1) ASA increased oxidation; 2) AsA formation was prevented during ASA stress; 3) when oxygen free radical increased by ASA stress, AsA that eliminated the oxygen free radical needed more, thus AsA content decreased. The reduction reflected the anti-stress ability. The less reduction, the stronger anti-stress ability. In this study, T and B cultivars processed a higher ability of anti-stress than Y cultivar, showing that Y suffered more severely from damage by ASA and more susceptible to ASA stress. This was reported genotypeand concentration dependent (Shi et al., 2005; Song et al., 2009; Wu et al., 2017). In this study, AsA contents went up in ASA + treatments when compared with ASA treatments alone, showing that Si was conducive to cope with oxidative damage induced by ASA. It may because Si 1) increased DHAR activity; 2) lowered oxidative stress; 3) enhanced the possible function of AsA in H2O2 detoxification for AsA was involved phytochelatin synthase; 4) improved the activities of ROS sweepers and raised ASA tolerance. This phenomenon has been backed by higher activities of SOD, POD and CAT (Fig. 3) and lower MDA contents (Fig. 2) in ASA + Si treated rice plants. Similar results were also reported in vegetables and fruits under metals and other stresses (Shi et al., 2005; Liu et al., 2009; Li et al., 2012; Wang et al., 2015; Ashfaque et al., 2017), and this enhancing effect was related to genotype (Song et al., 2009).

3.5. Effects of Si on POD activities of rice seedlings From Fig. 3 (E – F), it can be seen that there was a downside of activities of POD in shoots and roots of all rice cultivar seedlings (p < 0.05) during ASA exposure. POD activities decreased most roots of Y cultivar, and least in shoots of T cultivar. Si addition climbed POD activities under ASA treatment compared with ASA treatment (p < 0.05), but decreased POD activities under control condition. When CAT activity does not protect properly against high H2O2 amount in some situation, plants engage next enzyme – POD, which can 271

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3.7. Effects of Si on GSH contents of rice seedlings As represented in Fig. 4 (C - D), GSH contents decreased in all shoots and roots of three rice seedlings during ASA exposure compared with control (p < 0.05), and the most and least changes of GSH contents were found in roots of Y cultivar and in shoots of T cultivar, respectively. But Si addition significantly raised GSH contents in shoots and roots of all rice cultivars under ASA treatment (p < 0.05), especially in shoots of B cultivar and T cultivar. GSH is one foremost source of nonprotein thiols in plant cells, required as the substrate for synthesizing metalloid chelating ligands - the phytochelatins. It also acts as the substrate of glutathione peroxidase involving ROS detoxification. GSH plays a crucial role in scavenging H2O2 in AsA – GSH cycle and modulating cellular redox- and thioldisulfide status of proteins. It is known to take a key part in invalidating metal stress as one major non-enzymic antioxidant involving cellular defence against toxicants in plants. GSH protects the plants against toxicants by conjugating them or their metabolites through glutathioneS-transferases. Meanwhile, GSH plays a role in As biotransformation, for example, it is used as reductant for enzymatic or nonenzymatic reduction of As(V) into As(III), that is known to increase under metal stresses. So GSH can be a good indicator to assess As toxicity in the environment. GSH can chelate As directly for the sake of avoiding As toxicity to plants. The binding of inorganic GSH to As is the mechanism that GSH can impair As toxicity. In present study, the presence of ASA distinctly declined GSH contents in three rice cultivars, indicating that the ability of GSH to remove ROS was reduced so much so that rice was injured. The decreased levels of GSH might due to 1) its depletion or conversion to phytochelatins by either chronic or acute exposure to abiotic stress; 2) short of NAD(P)H as a substrate in the reaction probably (Shi et al., 2005), because GSH could be oxidized to GSSG when DHA converts to AsA, which relies on GR to transform GSH through oxidizing NAD(P)H into NAD(P)+; 3) involvement in AsA regeneration from its oxidized form during the AsA – GSH cycle, for GSH is an electron donor. Components in AsA – GSH circle have been reported in mitochondria, cytoplasm and peroxisomes, and represented an important antioxidant protection system of fighting oxidative damage generated in those organelles under stresses. Reportedly, Si took a key part in restraining the activities declines of ROS scavenging enzymes in chloroplasts in AsA – GSH pathway (Cao et al., 2015). In this study, Si addition raised GSH contents in rice, showing activity enhancements of GSH by Si application. This indicated that 1) Si heightened the ability of GSH to eliminate ROS in ASAstressed rice; 2) Si was involved in ROS metabolism when rice was under stressful environment. It was also backed by higher activities of SOD, POD, and CAT (Fig. 3), higher AsA concents (Fig. 4 (A – B)) and lower MDA concents (Fig. 2) in ASA + Si treated rice plants. It was similar for rice under cadmium exposure (Wang et al., 2015), as well as for plants under other stresses (Shi et al., 2005; Liu et al., 2009; Li et al., 2012; Rahman et al., 2017). The more increases of GSH contents were found in shoots of B and T cultivars, showing that the effects of Si on GSH contents were depended on cultivars and tissues (Song et al., 2009), as well as doses reported (Shi et al., 2005).

Fig. 5. Effects of different treatments [non-stressed control (CK), 168 mg L−1 silicon (Si), 100 mg L−1 arsanilic acid (ASA), 100 mg L−1 arsanilic acid and 168 mg L−1 silicon (ASA + Si)] on contents of soluble protein of shoots (A), roots (B) in three different kinds of rice seedlings. Values (+ SD) following different lowercase and capital letters indicated significant differences at p < 0.05 among different treatments and different cultivars, respectively.

protein contents decreased in three rice seedlings, showing that rice seedlings were harmed and the protein metabolisms were disturbed by ASA stress. It was indicated that the oxidative damages to proteins were caused by ASA-induced ROS such as •OH or O2•−. The reduction of soluble protein contents may because of 1) more oxidative harm that restrained the protein synthesis; 2) ASA reaction with the sulphydryl groups of proteins which resulted in abscission in rice tissues; 3) severe inhibition of rice growth and photosynthetic activity; 4) accelerated degradation by disturbing the membrane system; 5) interference with enzymes and tissue proteins to malfunction, because heavy metal ions could combine with proteins, nucleic acids and enzymes, which cause inhibition of metabolism, respiration, photosynthetic activity and cell division; 6) reduction of As(V) into As(Ⅲ) in cells responsible for protein damage by thiol groups oxidation. The decreased protein contents were also found in rice under chromium stress (Zeng et al., 2011) and in other plants under various stresses (Pisani et al., 2011; Bharwana et al., 2013; Farooq et al., 2013; Ashfaque et al., 2017; Rahman et al., 2017). In this study, Si addition into ASA solution increased significantly soluble protein contents of three rice cultivars compared with ASA treatment alone (p < 0.05), suggesting that Si took important roles in counteracting the toxicities of overproduced ROS and alleviating ASA stress, thus lowered oxidative damage, decreased the proteins decomposition, improved the protein synthesis and enhanced the protein metabolism under stress. These observations were in accordance with the results of researchers who have found the convinced impacts of Si on stabilizing protein contents in rice and other plants under multifarious stresses (Zeng et al., 2011; Bharwana et al., 2013; Farooq et al., 2013; Ashfaque et al., 2017), and this increase was also concentration dependent (Ashfaque et al., 2017).

3.8. Effects of Si on soluble protein contents of rice seedlings As illustrated in Fig. 5, soluble protein contents in both shoots and roots of the rice plants were significantly reduced by the addition of ASA compared to control (p < 0.05). The most decrease was found in roots of Y cultivar. However, the reduction was significantly alleviated by the exogenous application of Si compared with ASA treatment (p < 0.05). Moreover, in absence of ASA, exogenous Si application also increased the soluble protein contents in both shoots and roots as compared with the control. The correlation between As stress and protein change was less studied compared with antioxidant enzymes. In this study, the soluble 272

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4. Conclusions

Hossain, M.T., Soga, R.M.K., Wakabayashi, K., Kamisaka, S., Fujii, S., Yamamoto, R., Hoson, T., 2002. Growth promotion and an increase in cell wall extensibility by silicon in rice and some other Poaceae seedlings. J. Plant Res. 115, 23–27. Hu, H.C., Zhang, J.T., Wang, H., Li, R.C., Pan, F.S., Wu, J., Feng, Y., Ying, Y.Q., Liu, Q.P., 2013. Effect of silicate supplementation on the alleviation of arsenite toxicity in 9311 (Oryza sativa L. indica). Environ. Sci. Pollut. Res. Int. 20, 8579–8589. Ju, S., Yin, N., Wang, L., Zhang, U., Wang, Y., 2017. Effects of silicon on Oryza sativa L. seedling roots under simulated acid rain stress. PLoS One 12, e0173378. Kang, J.J., Zhao, W.Z., Zhu, X., 2016. Silicon improves photosynthesis and strengthens enzyme activities in the C3 succulent xerophyte Zygophyllum xanthoxylum under drought stress. J. Plant Physiol. 199, 76–86. Li, C.X., Feng, S.L., Shao, Y., Jiang, L.N., Lu, X.Y., Hou, X.L., 2007. Effects of arsenic on seed germination and physiological activities of wheat seedlings. J. Environ. Sci. 19, 725–732. Li, G., Maozhong, Z., Jianfeng, T., SHIM, H., Chao, C., 2017. Effects of Silicon on Arsenic Concentration and Speciation in Different Rice Tissues. Pedosphere. Li, L.B., Zheng, C., Fu, Y.Q., Wu, D.M., Yang, X.J., Shen, H., 2012. Silicate-mediated alleviation of Pb toxicity in banana grown in Pb-contaminated soil. Biol. Trace Elem. Res. 145, 101–108. Liu, J.J., Lin, S.H., Xu, P.L., Wang, X.J., Bai, J.G., 2009. Effects of exogenous silicon on the activities of antioxidant enzymes and lipid peroxidation in chilling-stressed cucumber leaves. Agric. Sci. China 8, 1075–1086. Ma, J.F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara, M., Ishiguro, M., Murata, Y., Yano, M., 2006. A silicon transporter in rice. Nature 440, 688–691. Mangalgiri, K.P., Adak, A., Blaney, L., 2015. Organoarsenicals in poultry litter: detection, fate, and toxicity. Environ. Int. 75, 68–80. Marion M, B., 1976. A-rapid-and-sensitive-method-for-the-quantitation-of-microgramquantities-of-protein. Anal. Biochem. 72, 248–254. Pandey, C., Khan, E., Panthri, M., Tripathi, R.D., Gupta, M., 2016. Impact of silicon on Indian mustard (Brassica juncea L.) root traits by regulating growth parameters, cellular antioxidants and stress modulators under arsenic stress. Plant Physiol. Biochem. 104, 216–225. Pisani, T., Munzi, S., Paoli, L., Backor, M., Loppi, S., 2011. Physiological effects of arsenic in the lichen Xanthoria parietina (L.) Th. Fr. Chemosphere 82, 963–969. Rao, M.V., Paliyath, G., Ormrod, D.P., 1996. Ultraviolet-Band ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol 110, 125–136. Rahman, M.F., Ghosal, A., Alam, M.F., Kabir, A.H., 2017. Remediation of cadmium toxicity in field peas (Pisum sativum L.) through exogenous silicon. Ecotoxicol. Environ. Saf. 135, 165–172. Raza, M.M., Ullah, S., Ahmad, Z., Saqib, S., Ahmad, S., Bilal, H.M., Wali, F., 2016. Silicon mediated arsenic reduction in rice by limiting its uptake. Agric. Sci. 7, 1–10. Shekari, F., Abbasi, A., Mustafavi, S.H., 2015. Effect of silicon and selenium on enzymatic changes and productivity of dill in saline condition. J. Saudi. Soc. Agric. Sci. Shen, X.F., Zhou, Y.Y., Duan, L.S., Li, Z.H., Eneji, A.E., Li, J.M., 2010. Silicon effects on photosynthesis and antioxidant parameters of soybean seedlings under drought and ultraviolet-B radiation. J. Plant Physiol. 167, 1248–1252. Shi, G.R., Cai, Q.S., Liu, C.F., Wu, L., 2010. Silicon alleviates cadmium toxicity in peanut plants in relation to cadmium distribution and stimulation of antioxidative enzymes. Plant Growth Regul. 61, 45–52. Shi, Q.H., Bao, Z.Y., Zhu, Z.J., He, Y., Qian, Q.Q., Yu, J.Q., 2005. Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase. Phytochemistry 66, 1551–1559. Shi, Y., Zhang, Y., Yao, H.J., Wu, J.W., Sun, H., Gong, H.J., 2014. Silicon improves seed germination and alleviates oxidative stress of bud seedlings in tomato under water deficit stress. Plant Physiol. Biochem. 78, 27–36. Song, A.L., Li, P., Li, Z.J., Fan, F.L., Nikolic, M., Liang, Y.C., 2011. The alleviation of zinc toxicity by silicon is related to zinc transport and antioxidative reactions in rice. Plant Soil 344, 319–333. Song, A.L., Li, Z.J., Zhang, J., Xue, G.F., Fan, F.L., Liang, Y.C., 2009. Silicon-enhanced resistance to cadmium toxicity in Brassica chinensis L. is attributed to Si-suppressed cadmium uptake and transport and Si-enhanced antioxidant defense capacity. J. Hazard. Mater. 172, 74–83. Tripathi, D.K., Singh, V.P., Prasad, S.M., Chauhan, D.K., Dubey, N.K., 2015. Silicon nanoparticles (SiNp) alleviate chromium (VI) phytotoxicity in Pisum sativum (L.) seedlings. Plant Physiol. Biochem. 96, 189–198. Tripathi, P., Tripathi, R.D., Singh, R.P., Dwivedi, S., Goutam, D., Shri, M., Trivedi, P.K., Chakrabarty, D., 2013. Silicon mediates arsenic tolerance in rice (Oryza sativa L.) through lowering of arsenic uptake and improved antioxidant defence system. Ecol. Eng. 52, 96–103. Vaculikova, M., Vaculik, M., Simkova, L., Fialova, I., Kochanova, Z., Sedlakova, B., Luxova, M., 2014. Influence of silicon on maize roots exposed to antimony - Growth and antioxidative response. Plant Physiol. Biochem. 83, 279–284. Wang, S.H., Wang, F.Y., Gao, S.C., 2015. Foliar application with nano-silicon alleviates Cd toxicity in rice seedlings. Environ. Sci. Pollut. Res. Int. 22, 2837–2845. Wu, Z.C., Liu, S., Zhao, J., Wang, F.H., Du, Y.Q., Zou, S.M., Li, H.M., Wen, D., Huang, Y.D., 2017. Comparative responses to silicon and selenium in relation to antioxidant enzyme system and the glutathione-ascorbate cycle in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis) under cadmium stress. Environ. Exp. Bot. 133, 1–11. Yao, L.X., Li, G.L., Dang, Z., He, Z.H., Zhou, C.M., Yang, B.M., 2008. Arsenic speciation in turnip as affected by application of chicken manure bearing roxarsone and its metabolites. Plant Soil 316, 117–124. Zeng, F.R., Zhao, F.S., Qiu, B.Y., Ouyang, Y.N., Wu, F.B., Zhang, G.P., 2011. Alleviation of chromium toxicity by silicon addition in rice plants. Agric. Sci. China 10, 1188–1196. Zhang, S.R., Li, S., Ding, X.D., Li, F.B., Liu, C.P., Liao, X.R., Wang, R.P., 2013. Silicon mediated the detoxification of Cr on pakchoi (Brassica chinensis L.) in Cr-contaminated soil. J. Food Agric. Environ. 11, 814–819.

In summary, the results revealed that though organoarsenic had a relatively low toxicity, it also could do damage to rice at some concentration. ASA stress decreased the rice biomass and protein contents, due to increasing LPO, forming ROS and lowering the antioxidant capacities. While Si-based application could alleviate ASA stress by improving growth and decreasing LPO (by cutting down MDA contents) of rice seedlings. The biochemical mechanistic findings were partly that Si was able to enhance markedly the capacities of antioxidant defense and improve protein metabolism to moderate oxidative stress induced by ASA toxicity in rice seedlings, therefore contributing positively to strengthening organoarsenic stress tolerance. These suggested a potential use of Si fertilizer for rice contaminated by ASA. Acknowledgement This research was supported by the National Science Foundation for Young Scientists of China (grant number 41401367) and the Presidential Foundation of the Guangdong Academy of Agricultural Sciences (grant number 201617). We thank Dr. Bruce Chen (Symbio Alliance Laboratories of Australia), Dr. Mu Zhang (Institute of Agricultural Resource and Environment of Guangdong Academy of Agricultural Science, Guangzhou, PR China) and Dr. Ruixue Guo (School of Food Science and Engineering, South China University of Technology) for their thoughtful comments and constructive suggestions to improve the quality of this paper. References Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113, 548–555. Arai, Y., Lanzirotti, A., Sutton, S., Davis, J.A., Sparks, D.L., 2003. Arsenic speciation and reactivity in poultry litter. Environ. Sci. Technol. 37, 4083–4090. Ashfaque, F., Inam, A., Inam, A., Iqbal, S., Sahay, S., 2017. Response of silicon on metal accumulation, photosynthetic inhibition and oxidative stress in chromium-induced mustard ( Brassica juncea L.). S. Afr. J. Bot. 111, 153–160. Begum, M.C., Islam, M.S., Islam, M., Amin, R., Parvez, M.S., Kabir, A.H., 2016. Biochemical and molecular responses underlying differential arsenic tolerance in rice (Oryza sativa L.). Plant Physiol. Biochem. 104, 266–277. Bharwana, S., S, A., MA, F., N, I., F, A., MSA, A., 2013. Alleviation of lead toxicity by silicon is related to elevated photosynthesis, antioxidant enzymes suppressed lead uptake and oxidative stress in cotton. J. Bioremediat. Biodegrad. 4, 1000187–1000198. Bokor, B., Vaculík, M., Slováková, Ľ., Masarovič, D., Lux, A., 2013. Silicon does not always mitigate zinc toxicity in maize. Acta Physiol. Plant. 36, 733–743. Cao, B.L., Ma, Q., Zhao, Q., Wang, L., Xu, K., 2015. Effects of silicon on absorbed light allocation, antioxidant enzymes and ultrastructure of chloroplasts in tomato leaves under simulated drought stress. Sci. Hortic. 194, 53–62. Chance, B., Maehly, A.C., 1954. Assay of catalase and peroxidases. Methods Enzymol. 1, 358–424. Farooq, M.A., Ali, S., Hameed, A., Ishaque, W., Mahmood, K., Iqbal, Z., 2013. Alleviation of cadmium toxicity by silicon is related to elevated photosynthesis, antioxidant enzymes; suppressed cadmium uptake and oxidative stress in cotton. Ecotoxicol. Environ. Saf. 96, 242–249. Fontcuberta, M., Calderon, J., Villalbi, J.R., Centrich, F., Portana, S., Espelt, A., Duran, J., Nebot, M., 2011. Total and inorganic arsenic in marketed food and associated health risks for the Catalan (Spain) population. J. Agric. Food Chem. 59, 10013–10022. Gunes, A., Inal, A., Bagci, E.G., Coban, S., 2007. Silicon-mediated changes on some physiological and enzymatic parameters symptomatic of oxidative stress in barley grown in sodic-B toxic soil. J. Plant Physiol. 164, 807–811. Guo, W., Hou, Y.L., Wang, S.G., Zhu, Y.G., 2005. Effect of silicate on the growth and arsenate uptake by rice (Oryza sativa L.) seedlings in solution culture. Plant Soil 272, 173–181. Habibi, G., Hajiboland, R., 2013. Alleviation of drought stress by silicon supplementation in pistachio (Pistacia vera L.) plants. Folia Hortic. 25, 21–29. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Helaly, M.N., El-Hoseiny, H., El-Sheery, N.I., Rastogi, A., Kalaji, H.M., 2017. Regulation and physiological role of silicon in alleviating drought stress of mango. Plant Physiol. Biochem. 118, 31–44. Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants without soil. Cali. Agric. Exp. Stat. Cir. 347, 1–32. Hodges, D.M., Andrews, C.J., Johnson, D.A., Hamilton, R.I., 1997. Antioxidant enzyme responses to chilling stress in differentially sensitive inbred maize lines. J. Exp. Bot. 48, 1105–1113.

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