Skip to main content
NCBI home page
Physiology and Molecular Biology of Plants logoLink to Physiology and Molecular Biology of Plants
. 2014 Aug 15;20(4):435–447. doi: 10.1007/s12298-014-0255-1

H2O2 pretreated rice seedlings specifically reduces arsenate not arsenite: difference in nutrient uptake and antioxidant defense response in a contrasting pair of rice cultivars

Shekhar Mallick 1,✉, Navin Kumar 1, Sarita Sinha 1, Arvind Kumar Dubey 1, Rudra Deo Tripathi 1,✉, Vivek Srivastav 1,2
PMCID: PMC4185047  PMID: 25320467

Abstract

The study investigated the reduction in metalloid uptake at equimolar concentrations (~53.3 μM) of As(III) and As(V) in contrasting pair of rice seedlings by pretreating with H2O2 (1.0 μM) and SA (1.0 mM). Results obtained from the contrasting pair (arsenic tolerant vs. sensitive) of rice seedlings (cv. Pant Dhan 11 and MTU 7029, respectively) shows that pretreatment of H2O2 and H2O2 + SA reduces As(V) uptake significantly in both the cultivars, while no reduction in the As(III) uptake. The higher growth inhibition, higher H2O2 and TBARS content in sensitive cultivar against As(III) and As(V) treatments along with higher As accumulation (~1.2 mg g−1 dw) than in cv. P11, unravels the fundamental difference in the response between the sensitive and tolerant cultivar. In the H2O2 pretreated plants, the translocation of As increased in tolerant cultivar against AsIII, whereas, it decreased in sensitive cultivar both against AsIII and AsV. In both the cultivars translocation of Mn increased in the H2O2 pretreated plants against As(III), whereas, the translocation of Cu increased against As(V). In tolerant cultivar the translocation of Fe increased against As(V) with H2O2 pretreatment whereas, it decreased in the sensitive cultivar. In both the cultivars, Zn translocation increased against As(III) and decreased against As(V). The higher level of H2O2 and SOD (EC 1.15.1.1) activity in sensitive cultivar whereas, higher, APX (EC 1.11.1.11), GR (EC 1.6.4.2) and GST (EC 1.6.4.2) activity in tolerant cultivar, also demonstrated the differential anti-oxidative defence responses between the contrasting rice cultivars.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0255-1) contains supplementary material, which is available to authorized users.

Keywords: Hydrogen peroxide, Salicylic acid, Arsenite, Arsenate, Oryza sativa

Introduction

Arsenic is a non-threshold carcinogen, estimated to be transferred into the rice paddy fields to the tune of 1,000 tonnes annually in the rice growing areas of Indian subcontinent (soil As level ranging between 10.4 and 15.5 μg g−1) during dry season (Dwivedi et al. 2012) as a result of irrigation with As laden ground-water. Hydrogen peroxide (H2O2) is present endogenously as an important intermediate of the metabolic pathways in plants. It has an important role in plant’s response towards biotic and abiotic stresses. Primarily, H2O2 functions as a messenger in ethylene and salicylic acid (SA) signal transduction pathway during plant stress response (Foyer et al. 1997; Chamnogpol et al. 1998). Several studies have highlighted the role of H2O2 in enhancing plant’s tolerance towards abiotic stresses (Murphy et al. 2002; Uchida et al. 2002; Azevedo Neto et al. 2005; Wahid et al. 2007; Liu et al. 2010; Chou et al. 2012). At the molecular level H2O2 regulates expression of several genes encoding antioxidants, defense proteins, kinase signaling proteins, phosphatases and transcription factors that are individually as well as contentedly involved in plant defense and hypersensitive reactions (Kovtun et al. 2000; Hung et al. 2005). Salicylic acid (a hormone like endogenous regulator) acts with the involvement of H2O2 in one of several ways including gene expression during plant defense against biotic and abiotic stresses (Szalai et al. 2000). It induces the synthesis of antioxidants ascorbate (AsA) and glutathione (GSH), antioxidant enzymes glutathione-S-transferase (GST), glutathione peroxidase (GPX), ascorbate peroxidase (APX). Endogenous SA protects rice plants from oxidative damage caused by aging, biotic and abiotic stress (Yang et al. 2004). The mode of SA action is largely through generation of reactive oxygen species (ROS) and by inhibiting the activity of catalase (CAT), which is a H2O2 scavenging enzyme (Janda et al. 2012). Alternatively, H2O2-triggered programmed cell death (PCD) has been recognized as an essential process to maintain tissue or organ homeostasis in concert with cell proliferation, growth and differentiation (Mittler et al. 2004).

The role of H2O2 in stress response has been directly assessed through its exogenous application. This is reported to increase chilling tolerance (Murphy et al. 2002), salt tolerance (Wahid et al. 2007), tolerance to osmotic stress (Liu et al. 2010), and low light (Zhang et al. 2011). Alleviation of heavy metals induced oxidative stress have also been demonstrated during Cd treatment in rice, barley, maize, pea seedlings, Arabidopsis thaliana and Glycine max (Pal et al. 2002; Metwally et al. 2003; Choudhury and Panda 2004; Drazic and Mihailovic 2005; Noriega et al. 2012; Guo et al. 2007; Zawoznik et al. 2007; Popova et al. 2009; Xiao-Juan et al. 2010). Reduction of Pb and Hg induced membrane damage in Oryza sativa have been studied by pretreatment with SA (Mishra and Choudhuri 1999), pre-treatement of a rice cultivar with H2O2 has been reported to increase its tolerance towards Cd toxicity by increasing GSH, phytochelatins and non-protein thiols (Chou et al. 2012). However, the role of H2O2 or both H2O2 and SA, towards alleviation of heavy metal(loid) induced toxicity in plants has not been extensively studied. Therefore, investigating the role of H2O2 and SA in reducing heavy metal(loid) related toxicity in plants is of great importance, particularly with arsenic (As) stress in rice plants.

Among various strategies, pre-soaking and priming of seeds with stress alleviating compounds has been proposed as an easy, low cost, low risk and effective approach to overcome the abiotic stresses (Wahid and Shabbir 2005). It is well established that most of the biotic and abiotic stress response results into generation of H2O2, hence, priming rice seedlings with H2O2 would induce a well developed antioxidative defence mechanism prior to As exposure, and treatment with H2O2 + SA could also have additive effect, as the mode of SA in plants is through generation of H2O2. As exposure to rice interferes with uptake of mineral elements such as Cu, Zn, Fe, Ni and Se (Duan et al., 2013; Dwivedi et al. 2012). However, there are no reports about the influence of the plant hormomes such as jasmonic acid, salicylic acid or molecules like H2O2 on the uptake or translocation of these minerals in As challenged rice plants. In this backdrop, the current study attempts to explore the role of H2O2 and SA in abatement of As toxicity in contrasting cultivars of rice plants that differ in tolerance towards arsenic. Additionally, the study also investigates the translocation of As and essential metal nutrients, antioxidant pool and anti-oxidative enzyme activities in the H2O2 or H2O2 + SA pretreated plants.

Materials and methods

Growth conditions and experimental design

Popular rice cultivars, MTU 7029 (Nati mansuri) and cv. Pant Dhan 11 (P11) were obtained from Narendera Dev Agricultural University, Faizabad, Uttar Pradesh, and from Govind Ballav Pant Agricultural University, Panthnagar, Uttrakhand, respectively. The seeds were germinated and cultured as explained in Kumar et al. (Kumar et al. 2013). 50 uniform (10 cm) seedlings were placed in 150 ml beaker, containing 100 ml of 100 % Hewitt nutrient medium (HNM), 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), Mo (0.048). After 7d of growth in the treatments were provided as As(III), As(V) (both at the rate of 4 μg ml−1 which is equivalent to ~53.3 μM) in HNM using salts of NaHAsO4.7H2O (Merck) and NaAsO2 (Sigma, USA), respectively for another 14 days and harvested for morphological and biochemical analysis. Morphological parameters were also recorded after 7 days in a similar set of experiment separately.

For convenience, the treatments were abbreviated as (P11) for Pant Dhan 11, MTU for MTU 7029, “C” for control, “AsIII(4)” for arsenite 4 μg ml−1 (~53.3 μM), “AsIII(4)H2O2” for arsenite 4 μg ml−1 pretreated with H2O2, “AsIII(4)H2O2 + SA” for arsenite 4 μg ml−1 pretreated with SA and H2O2. Similarly, “AsV(4)” for arsenate 4 μg ml−1 (~53.3 μM), “AsV(4)H2O2” for arsenate 4 μg ml−1 pretreated with H2O2, “AsV(4)H2O2 + SA” for arsenate 4 μg ml−1 pretreated with both SA and H2O2. The pre-treatment of the seedlings with H2O2 was made with 1 mM of H2O2 prepared in HNM for 48 h and for H2O2 + SA, pretreatments were made by 1 mM of SA + 1 mM of H2O2 prepared in HNM for 48 h, prior to exposure to various As treatments as mentioned above.

Biochemical analysis

Chlorophyll and protein content was estimated by Arnon (1949) and Lowry et al. (1951) methods, respectively. TBARS content (mmol g−1 fw) was estimated according to Heath and Packer (1968) using ε = 0.155 M g−1 fw for MDA-TBA adduct. H2O2 content (nmol g−1 fw) by Velikova et al. (2000) and ascorbic acid (μM g−1 fw) by Shukla et al. (1979). Reduced and total glutathione (GSH and total GSH) were estimated by Anderson (1985) methods by estimating the stoichiometric formation of 5-thio-2-nitrobenzioc acid (TNB) at λ = 412 nm and comparing with a standard curve prepared with GSH (Sigma).

For estimation of antioxidant enzyme activities, fresh samples of leaves (~300 mg each) were used following Kumar et al. (2013). 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 1 U of SOD activity is the amount of protein required to inhibit 50 % of initial reduction of nitro-blue tetrazolium (NBT) under light. Ascorbate peroxidase (APX) (EC 1.11.1.11) activity was measured following Nakano and Asada (1981) using ε = 2.8 mM−1 cm−1 and the enzyme activity was expressed as μ moles of ascorbate oxidized min−1 mg−1 protein. Catalase (CAT) (EC 1.11.1.6) activity was measured by method of Scandalios et al. (1983) and was expressed as mM min−1 mg−1 protein. Guaiacol peroxidase (POD) (EC 1.11.1.7) activity was assayed at 470 nm following Kato and Shimizu (1987) using ε = 26.6 mM−1 cm−1 and expressed as μmoles of guaiacol oxidized min−1 mg−1 protein. Glutathione reductase (GR) (EC 1.6.4.2) activity was assayed following Smith et al. (1988) and represented as U mg−1 protein, where 1U is conversion of 1 mM of oxidized glutathione (GSSG) min−1 to reduced glutathione (GSH). Ascorbate oxidase (AO) (EC 1.10.3.3) activity was assayed following Oberbacher and Vines (1963) and represented as U mg−1 protein, where 1U is oxidation of 1 μmol of the ascorbic acid min−1. Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) activity was assayed following Vanacker et al. (1998) by monitoring the formation of monodehydroascorbate at λ = 340 nm, due to the action of ascorbate oxidase (0.4 unit; 1 unit; 1 mmol of ascorbate oxidized per min) and represented as mM mg−1 protein. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) activity by Doulis et al. (1997) at λ = 265 nm using ε = 7.0 mM−1 cm−1 and represented as μM mg−1 protein. Glutathione S-transferase (GST; EC 2.5.1.13) activity by Habig et al. (1974) using the ε = 9.6 mM−1 cm−1 of the product formed and was expressed as μM of CDNB conjugated mg−1 protein.

Elemental and radical analysis

The metal(loid) contents (As) were determined following Kumar et al. (2013). Plant tissues (leaf ~ 500 mg and root ~ 300 mg) were digested using HNO3: HClO4 (3:1) in borosilicate glass beakers (50 ml) kept on hot plate temperature not exceeding 120 °C . Cu, Fe, Zn and Mn (μg ml−1) were analysed 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 elements in leaves upon its roots.

Statistical analysis and analytical quality control

The whole experiment was set in randomized block design with four replicates for each treatment. The presented data is an average of two experiments conducted separately at different time intervals. The data were subjected to one way ANOVA through Duncan’s Multiple Range Test (DMRT) for the analysis of significant difference between the treatments. Analytical data quality of the elements, was ensured through repeated analysis (n = 6) of Standard Reference Material (CRM 028–050) Resource Technology Corporation, USA (Lot No. IH 028). The values obtained (10 times) varied between −3.97 to 22.86 % error. The blanks were used to eliminate background noise.

Morphological parameters

The fresh-weight (fw) was measured by blot-drying roots, on an electrical balance (Mettler-Toledo) in milligrams (mg), shoot length and roots in millimetres (mm). All the growth parameters were measured after both 7th and 14th day after treatments, whereas, the biochemical parameters were measures only after 14d.

Results

Growth parameters

Pretreatment of the cultivars P11(tolerant) and MTU(sensitive) with H2O2 and H2O2 + SA did not show any significant change in root and shoot lengths (Table 1; S Fig. 1). However, in case of fresh weight a marginal increase was observed at both 7d and 14d of treatment in cultivar P11. Relative to the As(III) treatment, in this cultivar, pretreatment with H2O2 and H2O2 + SA showed ca. 14 % and ca. 35 % increase respectively at 7d, while corresponding values at 14d were ca. 17 and ca. 18 %, respectively.

Table 1.

Effect of the pretreatment with H2O2 and H2O2 + SA in AsIII and AsV treated on the growth parameters of Oryza sativa seedlings, cv. P11 and cv. MTU. The values ate mean of four independent replicates + SD

Treatments Root length (cm) Shoot length (cm) Fresh weight (gm) Total Chlorophyll (mg g−1 Fw) Carotenoid (mg g−1 Fw) Protein (mg g−1 Fw)
7d 14d 7d 14d 7d 14d 7d 14d 7d 14d 7d 14d
C(P11) 3.8 ± 0.5ab 3.4 ± 0.8abcde 24.5 ± 1.7a 32.5 ± 0.6e 10.9 ± 2.1abcd 18.1 ± 1.2c 3.8 ± 0.2bcd 3.4 ± 0.2a 1.2 ± 0.11abc 1.1 ± 0.0b 3.3 ± 0.1bc 5.5 ± 0.0e
AsIII(4)(P11) 3.0 ± 0a 4.1 ± 0.3def 22.0 ± 2.9a 30.1 ± 2.1de 10.3 ± 2.2abcd 17.1 ± 1.4c 3.4 ± 0.1ab 4.2 ± 0.2ab 1.1 ± 0.12a 1.4 ± 0.1c 2.8 ± 0.1a 4.4 ± 0.7abc
AsIII(4)H2O2(P11) 3.8 ± 0.9ab 3.9 ± 0.5cdef 22.5 ± 3.3a 30.7 ± 0.5de 12.1 ± 2.5bcd 18.7 ± 1.5c 3.3 ± 0.3a 4.1 ± 0.1a 1.2 ± 0.16ab 1.3 ± 0.1c 3.4 ± 0.1c 3.9 ± 0.1ab
AsIII(4)H2O2 + SA (P11) 4.0 ± 0.8ab 4.3 ± 0.5ef 23.5 ± 0.6a 32.0 ± 1.6e 13.9 ± 1.4d 20.1 ± 1.4c 3.6 ± 0.2abc 3.4 ± 0.2a 1.2 ± 0.08ab 1.2 ± 0.1b 3.2 ± 0.2bc 3.8 ± 0.1a
AsV(4)(P11) 3.58 ± 0.6ab 3.75 ± 0.3bcdef 24.0 ± 1.1a 32.0 ± 0.82e 13.8 ± 1.5d 19.1 ± 0.9c 3.6 ± 0.0abc 3.4 ± 0.2a 1.2 ± 0.03ab 1.1 ± 0.1b 3.4 ± 0.3c 4.3 ± 0.0abc
AsV(4)H2O2(P11) 3.8 ± 0.5ab 4.8 ± 1.0f 23.5 ± 1.0a 30.0 ± 0.82de 13.0 ± 1.4cd 17.7 ± 0.8c 3.7 ± 0.3abc 3.9 ± 0.3a 1.2 ± 0.06abc 1.2 ± 0.1b 3.0 ± 0.0ab 5.4 ± 0.0e
AsV(4)H2O2 + SA(P11) 3.8 ± 0.5ab 3.7 ± 0.7bcdef 24.5 ± 1.2a 31.5 ± 1.2e 13.1 ± 0.9cd 20.2 ± 1.8c 3.5 ± 0.1abc 3.4 ± 0.1a 1.1 ± 0.02a 1.1 ± 0.0b 3.3 ± 0.0bc 4.5 ± 0.1bc
C(MTU) 3.8 ± 0.9ab 4.8 ± 0.5f 22.5 ± 1.0a 27.2 ± 1.2cd 8.6 ± 0.4ab 12.1 ± 2.1b 4.1 ± 0.1d 3.8 ± 0.1a 1.4 ± 0.06c 1.2 ± 0.1b 3.4 ± 0.2c 5.3 ± 0.0de
AsIII(4)(MTU) 5.0 ± 0.0b 4.9 ± 0.3f 20.2 ± 2.2a 21.8 ± 3.2abc 8.0 ± 1.7a 9.7 ± 2.8ab 3.9 ± 0.2cd 3.8 ± 0.1a 1.3 ± 0.08abc 1.2 ± 0.1b 3.8 ± 0.2d 4.3 ± 0.0abc
AsIII(4)H2O2(MTU) 4.7 ± 0.5b 2.5 ± 0.4ab 23.2 ± 1.2a 24.0 ± 1.4abc 9.4 ± 1.08abc 7.9 ± 1.2a 3.7 ± 0.1bcd 3.8 ± 0.0a 1.2 ± 0.04ab 1.1 ± 0.1b 3.2 ± 0.1bc 4.7 ± 0.0cd
AsIII(4)H2O2 + SA(MTU) 4.5 ± 0.6ab 2.8 ± 0.3abc 21.7 ± 3.1a 21.6 ± 1.2ab 8.5 ± 1.9ab 9.7 ± 1.5ab 3.7 ± 0.1abc 3.5 ± 0.7a 1.1 ± 0.05a 1.0 ± 0.0a 3.2 ± 0.1bc 4.3 ± 0.0abc
AsV(4)(MTU) 4.3 ± 0.5ab 2.9 ± 0.3abcd 24.0 ± 1.8a 24.5 ± 1.7bc 8.6 ± 1.1ab 9.9 ± 1.0ab 3.8 ± 0.1cd 3.6 ± 0.2a 1.2 ± 0.02ab 1.1 ± 0.1b 3.4 ± 0.3c 4.4 ± 0.2abc
AsV(4)SA(MTU) 4.8 ± 0.5b 2.1 ± 0.5a 22.0 ± 1.4a 20.6 ± 1.2a 8.5 ± 1.8ab 7.4 ± 1.5a 3.9 ± 0.1cd 3.7 ± 0.3a 1.3 ± 0.01bc 1.1 ± 0.0b 4.1 ± 0.2d 4.6 ± 0.4c
AsV(4)H2O2 + SA (MTU) 4.3 ± 0.9ab 2.1 ± 0.3a 22.0 ± 2.9a 23.7 ± 1.7abc 7.4 ± 1.2a 7.5 ± 0.5a 3.6 ± 0.1abc 3.5 ± 0.0a 1.2 ± 0.09ab 1.2 ± 0.0b 3.4 ± 0.2d 4.5 ± 0.4bc
Open in a new tab

Values marked with same letter letters are not significantly different (DMRT, p < 0.05)

Arsenic accumulation and variation on essential elements

In the tolerant cultivar (P11), in comparison to the As accumulation (μg g−1 dw) in the leaves of AsV(4), the accumulation in the H2O2 pretreated plants receiving AsV(4), was significantly lower (ca. 42 %) (Fig. 1a). Similarly, significant reduction (ca. 30 %) in As accumulation was also observed in sensitive cultivar (MTU) with H2O2 pretreatment and also with H2O2 + SA pretreatment (ca. 16 %), in comparison to its AsV(MTU). On the contrary, the uptake and accumulation of As in As(III) treated plants was not reduced by pretreatment of H2O2 and SA in any of the cultivars. Arsenic uptake in the sensitive cultivar pretreated with H2O2+ SA receiving AsIII [AsIII(4)H2O2 + SA(MTU)], increased (ca. 63 %), as compared to AsIII(4)MTU. Comparison of the levels of As accumulation in the leaves of AsIII(4) and AsV(4) between the two cultivars, show that accumulation in the sensitive cultivar was ca. 16 % and ca. 64 % higher than their respective As(III) and As(V) treatments in tolerant cultivar. On the contrary, As level in the roots of tolerant cultivar, of AsIII(4) and AsV(4) treatments were ca. 54 % and ca. 56 % higher than those in sensitive cultivar, respectively. Hence, the TF of As in the tolerant cultivar against As(III) (0.31) and As(V) (0.49), were lower than those of sensitive cultivar, which was 0.79 and 0.88, respectively (Table 2). However, the reduction of TF of As with H2O2 pre-treatments in AsV(4)(P11) was ca. 12 % and for AsIII(4)(MTU) and AsV(4)(MTU) was ca. 38 % and ca. 33 %, respectively.

Fig. 1.

Fig. 1

Open in a new tab

Levels of As, (μg g−1 dw) in the leaves and roots of H2O2 and SA pretreated seedlings of rice cv. P11 and cv. MTU (A); PO4 − level (mg g−1 dw) of cv. P11 and cv. MTU (B), Cu level (C), Fe level (D), Mn level (E), Zn level (F). All bars are mean of four replicates + SD. Bars marked with same letters are not significantly different (Duncan’s test, p < 0.05).

Table 2.

Effect of the pretreatment with H2O2 and H2O2 + SA in AsIII and AsV treated on the TF of various nutrient, in Oryza sativa seedlings, cv. P11 and cv. MTU

Treatments As Leaves/Root Cu Leaves/Root PO4 3− Leaves/Root Fe Leaves/Root Mn Leaves/Root Zn Leaves/Root
C(P11) – 0.29 ± 0.01b 1.31 ± 0.02d 0.34 ± 0.01a 2.09 ± 0.02g 0.19 ± 0.04a
AsIII (4) (P11) 0.35 ± 0.01a 0.35 ± 0.01de 1.74 ± 0.11ef 0.56 ± 0.02bc 3.17 ± 0.02i 0.37 ± 0.03b
AsIII (4) H2O2 (P11) 0.54 ± 0.01d 0.41 ± 0.02g 1.76 ± 0.07f 0.52 ± 0.03b 3.59 ± 0.07j 0.66 ± 0.02ef
As III (4) H2O2 + SA (P11) 0.47 ± 0.02c 0.37 ± 0.01e 1.57 ± 0.02e 0.57 ± 0.02bcd 4.28 ± 0.04k 0.72 ± 0.06fg
AsV (4) (P11) 0.37 ± 0.01a 0.37 ± 0.02e 1.17 ± 0.01c 0.51 ± 0.02b 3.25 ± 0.03i 0.73 ± 0.05fg
AsV (4) H2O2 (P11) 0.36 ± 0.03a 0.42 ± 0.02g 0.95 ± 0.02a 1.08 ± 0.01h 1.67 ± 0.07e 0.59 ± 0.02de
AsV (4) H2O2 + SA (P11) 0.40 ± 0.01b 0.40 ± 0.03g 1.03 ± 0.01b 0.61 ± 0.02de 1.30 ± 0.04d 0.56 ± 0.02d
C (MTU) – 0.19 ± 0.01a 1.40 ± 0.05d 1.04 ± 0.04h 1.17 ± 0.06c 0.48 ± 0.03c
AsIII (4) (MTU) 0.69 ± 0.01f 0.35 ± 0.02de 1.40 ± 0.03d 0.62 ± 0.05e 0.54 ± 0.05a 0.77 ± 0.07g
AsIII (4) H2O2 (MTU) 0.51 ± 0.02d 0.34 ± 0.01d 1.39 ± 0.02d 0.79 ± 0.05g 1.18 ± 0.07c 1.00 ± 0.05h
As III (4) H2O2 + SA (MTU) 0.40 ± 0.03b 0.31 ± 0.01d 1.62 ± 0.01ef 0.70 ± 0.06f 0.93 ± 0.02b 0.63 ± 0.03ef
AsV (4) (MTU) 0.79 ± 0.01g 0.34 ± 0.02c 1.08 ± 0.03b 0.69 ± 0.04f 1.78 ± 0.03f 1.13 ± 0.04i
AsV (4) H2O2 (MTU) 0.53 ± 0.05d 0.39 ± 0.02f 1.22 ± 0.02c 0.67 ± 0.05cde 2.12 ± .06g 0.72 ± 0.04fg
AsV (4) H2O2 + SA (MTU) 0.64 ± 0.02e 0.31 ± 0.01c 1.59 ± 0.01e 0.71 ± 0.06f 2.47 ± 0.08h 1.28 ± 0.04j
Open in a new tab

Among the essential elements, the level of Cu (μg g−1 dw) in the roots of H2O2 pretreated plants of tolerant cultivar receiving AsIII(4), decreased by ca. 26 %, against that in AsIII(4) (Fig. 1c). However, when compared with the control, the TF of Cu increased in H2O2 pretreated plants receiving AsIII(4) and AsV(4), were 0.41 and 0.42, respectively (Table 2). The level of Fe increased significantly in the leaves of the sensitive cultivar (ca. 57 %) treated with AsV(4), as compared to AsIII(4) (Fig. 1d). The TF of Fe in all the plants of tolerant cultivar receiving AsIII(4) and AsV(4), were higher than its control, whereas, in sensitive cultivar the TF declined. The level of Mn (μg g−1 dw) in H2O2 pretreated plants of the tolerant cultivar receiving AsIII(4), was significantly higher (ca. 25 %) than its control [P11(C)], whereas, in the sensitive cultivar, it was significantly lower (ca. 42 %) in AsIII(4) and As(III) + H2O2 + SA treated plants (Fig. 1e). Compared between the Zn level (μg g−1 dw) in the leaves of control plants of the two cultivars, the level in sensitive cultivar was significantly higher (ca. 122 %) than that in the tolerant cultivar (Fig. 1f). With respect to the level of Zn in control of tolerant cultivar, except for AsIII(4) the levels in the leaves of all the treated plants was significantly higher and that in the roots were significantly lower, however, same pattern was not evident in sensitive cultivar. Comparing the Zn levels between AsIII(4) and AsV(4) treated seedlings of the two cultivars, in sensitive cultivar the level increased significantly (ca. 45 %) in plants receiving AsV(4) than in AsIII(4). In tolerant cultivar, the TF of Zn (0.37) in AsIII(4) receiving plants was higher than C(P11) which increased further consistently with H2O2 (0.65) and H2O2 + SA treatment (0.72), whereas, in AsV(4) treated plants, the TF although higher than C(P11), but the values in H2O2 (0.59) and H2O2 + SA(0.55) treated plants were less than AsV(4) (0.73). The TF of Zn in the sensitive cultivar (0.48) was relatively higher than that in tolerant cultivar (0.19) as compared between the control plants, and also the TF in AsIII(4) (0.77) and AsV(4) (1.13) were higher than the respective treatments in the tolerant cultivar. Similarly, the TF of Zn in AsIII(4) + H2O2 (1.0) and AsV(4) + H2O2 (0.71) in sensitive cultivar, were higher than the respective values in tolerant cultivars. The levels of PO4 in the leaves and roots of the two cultivars did not vary significantly, as compared to their respective controls or between the various As treatments (Fig. 1b).

Effect on H2O2 and TBARS

The level TBARS (mmol g−1 fw) in the sensitive cultivar were relatively higher than in the tolerant cultivar (Fig. 2a). No significant difference was observed between the treatments, except in H2O2 and H2O2 + SA pretreated plants of sensitive cultivar receiving AsIII(4), where, it was higher by ca. 80 % and ca. 72 % respectively, as compared to AsIII(4) (Fig. 2a). Unlike in sensitive cultivar, the level of lipid peroxidation in tolerant cultivar receiving AsIII(4), decreased with H2O2 and H2O2 + SA pretreatment, as compared to plants receiving only AsIII(4). Alternatively, while comparing with its control [C(P11)], the TBARS levels of the tolerant cultivar increased by ca. 43 % and ca. 36 % in AsIII(4) of AsV(4), respectvely.

Fig. 2.

Fig. 2

Open in a new tab

Effect of pretreatment of H2O2 and H2O2 + SA on the activities of different antioxidant enzymes activity in leaves of AsIII and AsV treated rice cv. P11 and MTU after 14d. A, leaf TBARS level (mmol g−1 fw); B, leaf H2O2 level (nmol g−1 fw); C: APX: (μmol min−1 mg−1 protein); D, POD (μmol min−1 mg−1 protein); E: CAT (M min−1 mg−1 protein); F: SOD (U mg−1 protein). All the values are means of four replicates + SD. Bars indicated by same letters are not significantly different (DMRT, p < 0.05)

The level of H2O2 (nmol g−1 fw) in the control plants of the two cultivars did not vary significantly, however, its level in As(III) and As(V) treated seedlings of the sensitive cultivar, were relatively higher than the corresponding values in tolerant cultivar (Fig. 2b). In the tolerant cultivar, the H2O2 content in both the As treatments, i.e. AsIII(4)(P11) and AsV(4)(P11) were significantly lower than its control, i.e. ca. 42 % and ca. 54 %, respectively. Whereas, in the sensitive cultivar, significant increase was observed only in plants treated with AsIII(4) (ca. 36 %), whereas, with AsV(4) treatment, it increased by ca. 21 %, as compared to C(P11). H2O2 levels in the H2O2 preteated plants receiving AsV(4), were higher over their respective AsV(4) values in both the cultivars i.e. ca. 42 % in cv. P11 and ca. 10 % in cv. MTU.

Antioxidant activity

Specific activity of SOD (U mg−1 protein) of the tolerant cultivar was lower than that in the sensitive cultivar, as evident from the difference in the values between control plants (Fig. 2f). When compared between plants treated with AsIII(4) and AsV(4) without any pretreatments, the enzyme activity in As(V)(4) treated plants of tolerant cultivar was significantly lower (ca. 62 %) than in AsIII(4). Whereas, in the sensitive cultivar, it was ca. 9 % lower in AsV(4) than AsIII(4). The specific activity of SOD in AsIII(4) treated plants of tolerant cultivar was ca. 42 % higher, while, it was ca. 24 % lower in sensitive cultivar, as compared to their respective controls. The activity of SOD in H2O2 pretreated plants receiving AsV(4), of both tolerant and sensitive cultivars was ca. 80 % and ca. 20 %, higher than their respective plants receiving only AsV(4) without any pretreatment.

The specific activity of APX (μmol min−1 mg−1 protein) in the H2O2 pretreated plants of tolerant cultivar receiving AsV(4) was ca.10 % and ca.137 % higher than its AsV(4) and C(P11), respectively (Fig. 2c). On the contrary, sensitive cultivar, the values in the corresponding treatments were ca. 6 % and ca. 42 % lower than its AsV(4)(MTU) and C(MTU), respectively. Similarly, when compared with their respective AsIII(4) treatments, the APX activites in AsV(4)P11 and AsV(4)MTU were ca. 39 % and ca.15 % lower, respectively. In the sensitive cultivar, the activity was significantly higher (ca. 68 %) in plants treated with AsV(4), as compared to AsIII(4), whereas, it was lower (ca. 35 %) in plants pretreated with H2O2 + SA receiving AsV(4), as compared to AsV(4) without pretreatment (Fig. 2e). The specific activity of POD (μmol min−1 mg−1 protein) in tolerant cultivar pretreated with H2O2 and H2O2 + SA receiving AsIII(4), were ca. 76 % and ca.106 % respectively, higher than in AsIII(4) without pretreatment. On the contrary in sensitive cultivar, the activity decreased by ca. 23 % and ca. 56 %, in the corresponding treatments, as compared to its AsIII(4) values (Fig. 2d).

The specific activity of GR (U mg−1 protein) was relatively higher in tolerant cultivar than that in sensitive cultivar, both in the control and treated plans (Fig. 3a). In both, H2O2 and H2O2 + SA pretreated plants of tolerant cultivar receiving AsIII(4), the GR activity were significantly higher by ca. 27 % and ca. 50 %, respectively, as compared to AsIII(4), while plants receiving AsIII(4) without any pretreatment, the increase was ca. 38 % over its control [C(P11)]. Similarly, with AsV(4) treatments, the activity increased significantly (ca. 72 %) in H2O2 pretreatment, as compared to AsV(4). On the contrary, in the sensitive cultivar, the activity decreased significantly (ca. 53 %) in plants pretreated with H2O2 + SA and receiving AsIII(4). Quite similar to the pattern of GR activity, the specific activity of GST (μM mg−1 protein) was higher in the tolerant cultivar than the sensitive cultivar, as compared between their treatments and their control plants (Fig. 3b).

Fig. 3.

Fig. 3

Open in a new tab

Effect of the pretreatment of H2O2 and H2O2 + SA on the activities of different biochemical parameter antioxidant enzymes activity in leaves of AsIII and AsV treated rice cv. P11 and MTU after 14d. A: GR activity (U mg−1 protein); B, GST activity (μM mg−1 protein); C, DHAR activity (μM mg−1 protein); D, MDHAR activity (mM mg−1 protein); E: AO (U mg−1 protein); F, AsA level (μM g−1 fw); G, GSH level (μg g−1 fw); H, total GSH (μg g−1 fw). All values are mean of four replicates + SD. Bars marked with same letters are not significantly different (Duncan’s test, p < 0.05)

The specific activity of DHAR (μM mg−1 protein) was observed to significantly decline in H2O2 and H2O2 + SA pretreated plants of the sensitive cultivar receiving AsIII(4), viz ca. 54 % and ca. 67 %, respectively (Fig. 3c). On the contrary, the specific activity of MDHAR (mM mg−1 protein), decreased significantly in H2O2 pretreated plants receiving AsV(4), in either of the cultivars viz. ca. 44 % in both tolerant and ca. 44 % in sensitive, as compared to their respective AsV(4) treated plants which was without any pretreatment (Fig. 3d). The specific activity of AO (U mg−1 protein) was significantly higher in AsV(4)H2O2(P11), in comparison to AsV(4)(P11), while no such change was observed in cv. MTU. The activity in AsIII(4)H2O2 + SA was significantly higher in either of the cultivars with respect to their respective control plants (Fig. 3e).

Ascorbate, GSH and GSSG

In the tolerant cultivar, the level of AsA (μM g−1 fw) in AsIII(4)H2O2 and AsIII(4)H2O2 + SA were significantly higher i.e. ca. 85 % and ca. 88 %, respectively than C(P11). However, the level in AsV(4)(P11) were significantly lower than AsIII(4)(P11) (Fig. 3f). On the contrary, in sensitive cultivar, the level was significantly higher (ca. 26 %) in AsV(4) plants than C(MTU), contrarily, the level in AsV(4)H2O2(MTU) and AsV(4)SA + H2O2(MTU) was significantly lower than AsV(4)(MTU). The level of total GSH (μg g−1 fw) in either of the cultivars were significantly higher than their respective controls (Fig. 3g, h). However, the level did not differ significantly between the AsIII(4) and AsV(4) treated seedlings.

Discussion

Comparison of the growth parameters between the tolerant (P11) and sensitive (MTU) cultivars, reveals that the tolerant cultivar was phenotypically more profound in growth than the sensitive cultivar, as the root length, shoot length and fresh weight were higher in the tolerant than sensitive cultivar. The growth inhibition in sensitive cultivar was higher after 14d, against different treatments of both As(III) and As(V), compared to the same parameters in the respective treatments of tolerant cultivar. Similarly, higher translocation of As(III) and As(V) in the leaves of tolerant cultivar than in the sensitive cultivar, along with higher levels of TBARS and H2O2, shows cv. MTU is sensitive than cv. P11. Besides, the accumulation of As being relatively higher in cv. P11 along with its lower level of H2O2 and lipid peroxidation in comparison to cv. MTU, exhibits its tolerance towards As. Doncheva et al. (2009) considered Mn tolerant and Mn sensitive cultivars of maize, based upon its tolerance towards high concentrations Mn in the tissue and not by virtue of avoidance but by exclusion. On the contrary in rice, Dave et al. (2013) have reported that, As sensitive rice cultivar accumulates higher As but has low Thiol-As molar ratio as compared to as tolerant cultivar having low As accumulation but higher thiol-As molar ratio. Hence, the higher As accumulation cv. P11 and less toxic manifestation qualifies it as tolerant than the other cultivar towards As stress.

Decline in fresh weight and shoot length of plants receiving AsIII(4) and AsV(4), both after 7d and 14d, in either of the cultivars, is attributed to the As induced toxicity. However, exception being increase in the root length in AsIII(4) receiving plants. Also, the root length in the H2O2 pre-treated plants of tolerant cultivar receiving AsV(4), significantly increased after 14d, in comparison to the plants treated only with AsV(4) without any pretreatment, which exhibits the amelioration of the toxicity due to H2O2 pretreatment. Whereas, in absence of any such phenomenon in the plants pretreated with both H2O2 + SA and receiving AsV(4), demonstrates that there is no added advantage with SA pretreatment. Hence, the pretreatment of H2O2 could induce tolerance and enhance growth of the plants. On the contrary, the significant decline in root lengths in all the treatments in sensitive cultivar after 14d, demonstrates the higher sensitivity of the cultivar towards As toxicity. Alternatively, increase in fresh weight of tolerant cultivar, exposed to both AsIII(4) and AsV(4) with H2O2 and SA + H2O2 pretreatment, also confirms its enhanced tolerance. Increase of shoot lengths in plants of tolerant cultivar pretreated with H2O2 and H2O2 + SA receiving AsIII(4), on the other hand, absence of similar effect in sensitive cultivar supports the conclusion. Several studies indicate that either exogenous application or pretreatment of H2O2 or SA can alleviate abiotic stress in plants. Murphy et al. (2002) observed that exogenous application of H2O2 increases chilling tolerance by enhancing the GSH level in mung bean seedlings. Azevedo Neto et al. (2005) reported that addition of H2O2 to the nutrient solution induces salt tolerance by enhancement of antioxidants activities and reduced peroxidation of membrane lipids in leaves and roots of maize. Guo et al. (2007) have also observed that pretreatment with SA significantly alleviated the growth inhibition and transpiration suppression in rice plants caused by Cd stress, although the underlying mechanism was not fully explained. Increase in the length of shoot and root of rice seedlings was observed by Jing et al. (2007) by pre-treating seeds with SA (0.1 mmol L−1 SA for 48 h before Pb stress) against Pb, however under long-term exposure the effect was not maintained. Although, there are reports of increase in photosynthetic rate in mustard plant (Fariduddin et al. 2003), wheat (Arfan et al. 2007), and in tomato plants (Poor et al. 2011), however, non significant increase in the total chlorophyll content in H2O2 pre-treated seedlings of the present study, thus remains inconclusive.

With H2O2 and H2O2 + SA pretreatment, As translocation to the leaves of tolerant cultivar increased in plants receiving As(III), this shows that the P11 being tolerant could overcome the toxicity due to the increased As(III) uptake translocation in the leaves. An important observation result of in the present study was that in both the cultivars, which showed significant decrease in As translocation in plants receiving As(V) by pretreatment with H2O2 for 48 h. This is in agreement with the earlier studies where plants with exogenous application of H2O2 or SA have ameliorated Cd toxicity (Guo et al. 2007, 2009), where they have attributed this to SA-induced H2O2 signaling in mediating Cd tolerance. Several studies have supported a major role of SA in modulating plant responses to abiotic stresses including heavy metal toxicity (Guo et al. 2007; Janda et al. 2012; Shi and Zhu 2008). The higher As level in the leaves of sensitive cultivar and the reverse in tolerant cultivar can be explained in the light of leaf GSH level. The leaf GSH level in tolerant cultivar was less than sensitive cultivar, hence the higher GSH level in sensitive cultivar could have chelated the As in roots and rendered it inactive, on the contrary the reverse happened in tolerant cultivar. The transport of As(III) takes place across aquaporin and As(V) across PO4 transporters. It is quite likely that the PO4− transporters are down regulated with H2O2 and H2O2 + SA pretreatments, across which transportation of both As(V) and PO4 takes place. Consecutively, the TF of PO4 in As(V) followed by H2O2 pretreated plants were lower than that of AsIII receiving plants. Hence, it could also be inferred that H2O2 pretreatment in rice plants may reduce As(V) translocation albeit however, compromising the uptake of PO4−.

Among the essential elements, lower level of Cu in the roots of the tolerant cultivar receiving both AsIII(4) and AsV(4), as compared to C(P11), and further reduction with H2O2 and H2O2 + SA pretreatment along with their reduced translocation to leaves, all signifies that Cu translocation in rice is reduced both by As(III) and As(V) and pretreating the plants with H2O2 compliments in reducing the translocation further. Similar, trend in the sensitive cultiver, (cv. MTU) also supports the fact. However, in sensitive cultivar the increase in Cu level against AsIII(4) treatment is in congruence with the corresponding increase in SOD levels, where Cu acts as a co-factor (CuSOD isoform). Apart from Cu, Fe and Zn are also cofactors of a SOD, hence, the concomitant increase in translocation of these elements in the tolerant cultivar against AsV(4) treatments supports the rise in the specific activity of SOD. Inspite of the relatively higher TF of Fe in the leaves of sensitive cultivar, however, its translocation in the tolerant cultivar did not decline upon treating with As or pre-treating with H2O2. This could be due to inter-varietal difference of Fe accumulation with response to As(III) or As(V) and not due to the pretreatments. Similar inter-varietal difference in Fe content has been earlier reported in different rice cultivars (Dwivedi et al. 2010). The translocation of both Mn and Zn increased with As(V) treatments in both the cultivars, however, in the tolerant cultivar pretreating plants with H2O2 and H2O2 + SA, the translocation of both the elements decreased in plants with AsV(4) exposure, on the contrary it increased with As(III) exposure. In agreement to this, Williams et al. (2009) surveyed the concentrations of As and trace mineral elements in rice grains collected from Bangladesh and found that Zn content in rice grain significantly declined with increasing As concentration. Hence, it can be concluded that there exists a certain degree of specificity imparted by As(III) and As(V) towards influencing the translocation of elements in rice plant. Hayat and Ahamed (Hayat and Ahmad 2007) have reported that decrease in PO4 uptake in plants upon exogenous application of SA. Although, in the present study, PO4 content in roots of both the cultivars treated with As(III) declined. Similarly, PO4 content was also declined by SA, which signifies that SA and As(III) could inhibit PO4 transport. However, in sensitive cultivar, the higher accumulation of As could be due to higher expression of PO4 transporters and thus contributing towards higher As(V) accumulation. Thus, it is apparent that H2O2 has a role in down regulation of PO4 transporters which manifesting in reduced uptake of both As(V) and PO4−.

Decreased level of H2O2 in the leaves of the tolerant cultivar when treated with both AsIII(4) and AsV(4), could be correlated with increased activity of APX. Whereas, the higher level of H2O2 in sensitive cultivar treated with AsV(4), correlates well with its lower APX activities. Guo et al. (2007) have attributed the SA signaling pathway in plants being associated with increased H2O2 levels. In both the cultivars, the higher level of H2O2 in plants pretreated with H2O2 + SA receiving AsIII(4), over their respective plants pretreated only with H2O2 receiving AsIII(4), and no such increase in their corresponding As(V) treatments [AsV(4)H2O2 + SA and AsV(4)H2O2, respectively], show that with AsV there is little role of exogenous H2O2 pretreatment on the plant’s endogenous H2O2 level. Alternatively, meager increase of H2O2 levels in both As + H2O2 treated seedlings along with their non significant increase in APX and SOD activities, over their AsIII(4) or AsV(4) values, supports that exogenous H2O2 pretreatment does not elevate the endogenous H2O2 levels significantly. Similar observation was also reported by Popova et al. (2009) where they observed no major changes in H2O2 level in Cd- treated plants or pretreated with SA before exposure to Cd.

The observed variation in SOD activity between the two contrasting cultivars shows that the tolerant cultivar, was responsive towards As(III) stress than the sensitive cultivar, however, the progressive increase in SOD activity from AsIII(4) to AsIII(4)H2O2 and further to AsIII(4)H2O2 + SA in the sensitive cultivar can be attributed to its higher sensitivity towards As(III). Similarly, higher APX activity in tolerant cultivar than in sensitive cultivar conjunction with lower H2O2 levels, also demonstrates the existence of differential response between the two cultivars. Higher level of POD and GR activities in As(III) treated plants of tolerant cultivar than in sensitive cultivar, also supports existence of differential detoxification mechanism between the two cultivars. Alternatively, significantly higher POD and GR activities in AsIII(4) + H2O2vis-a-vis AsV(4) + H2O2 seedlings demonstrates a differential response towards the two species of As, as well. Increase in DHAR activity in AsIII(4)H2O2 and AsIII(4)H2O2 + SA treated seedlings of tolerant cultivar in contrast to decrease in its activity in sensitive cultivar, could be due to similar reason. Similarly, the significant decline in MDHAR activity in AsV(4)H2O2 as compared to AsV(4) in both the cultivars and not with AsIII(4)H2O2 is yet another evidence supporting the differential response towards two species of As.

Among the antioxidant levels, the significantly higher levels of AsA in AsIII(4) and AsIII(4)H2O2 treated seedlings of the tolerant cultivar than AsV(4) and AsV(4) + H2O2 shows that tolerant cultivar is more responsive towards As(III) than As(V), whereas the sensitive cultivar is more responsive towards As(V). The higher level of total GSH in the treated seedlings of sensitive cultivar than in tolerant cultivar shoes than GSH plays a greater role in the sensitive cultivar.

Analysis of the results thus demonstrates that the pre-treatment of the rice seedlings with H2O2 increases fresh-weight against As(III) treatment whereas, it significantly reduces the As(V) translocation to the leaves. The study also differentiates sensitive verses tolerant cultivar on the basis of the biochemical and growth parameters, where growth inhibition, lipid peroxidation and H2O2 content in the sensitive cultivar (cv. MTU) was higher than the tolerant (cv. P11) observed against As(III) and As(V) stress. The As accumulation against AsIII and AsV treatments, were lower in sensitive cultivar, however, the As translocation to its shoots were higher than that in tolerant cultivar. In both the cultivars, pretreatment of the seedlings with H2O2 decreased the As(V) uptake and translocation significantly. Cu uptake and translocation decreased considerably in tolerant cultivar with both As(III) and As(V) which increased with H2O2 and H2O2 + SA pre-treatments. The higher uptake of Cu in the sensitive cultivar could be is attributed to higher activity of CuSOD. In absence of any additive effect with H2O2 + SA pretreatment, it is concluded that both H2O2 or H2O2 + SA yields similar response in terms of reduction of As(V) uptake. CAT had little role in either of the cultivars, however, APX played a significant role towards H2O2 detoxification in tolerant cultivar. On the contrary, higher H2O2 levels in different treatments of sensitive cultivar were attributed to higher activity of SOD. Also the higher APX, GR and GST activity in tolerant cultivar contributed towards higher tolerance towards As. Redundancy of antioxidant enzymes existed between tolerant cultivar and sensitive cultivar, where, APX, GR and GST dominated in the former and SOD in the later.

Electronic supplementary material

Supplimentary Figure 1 (791.2KB, docx)

Effect of pretreatment of H2O2 and H2O2 + SA on AsIII and AsV exposed rice cv. P11 and MTU after 14d. A, Control cv. P11; B, AsIII(4)(P11); C, AsIII(4)H2O2(P11); D, AsIII(4)H2O2 + SA(P11); E, AsV(4)(P11); F, AsV(4)H2O2(P11); G, AsV(4)H2O2 + SA(P11); H, Control(MTU); I, AsIII(4)(MTU); J, AsIII(4)H2O2(MTU); K, AsIII(4)H2O2 + SA(MTU); L, AsV(4)(MTU); M, AsV(4)H2O2(MTU); N, AsV(4)H2O2 + SA. (DOCX 791 kb)

Acknowledgments

The authors are thankful to Director, CSIR-NBRI for necessary infrastructural support for carrying out the research work. CSIR, New-Delhi for the financial support. AKD is thankful to DST-GOI (SR/SO/PS-68/2009), New Delhi for providing Junior Research Fellowship.

Contributor Information

Shekhar Mallick, Phone: +915222297847, Email: shekharm@nbri.res.in.

Rudra Deo Tripathi, Email: tripathi_rd@rediffmail.com.

References

  1. Anderson ME. Determination of glutathione and glutathione disulfide in biological samples. Method Enzymol. 1985;113:548–555. doi: 10.1016/S0076-6879(85)13073-9. [DOI] [PubMed] [Google Scholar]
  2. Arfan M, Athar HR, Ashraf M. Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress. J Plant Physiol. 2007;164:685–694. doi: 10.1016/j.jplph.2006.05.010. [DOI] [PubMed] [Google Scholar]
  3. Arnon DI. Copper enzymes in isolated chloroplast, polyphenol oxidase in Beta vulgaris. Plant Physiol. 1949;24:1–15. doi: 10.1104/pp.24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Azevedo Neto AD, Prisco JT, Eneas-Filho J, Medeiros J-VR, Gomes-Filho E. Hydrogen peroxide pre-treatment induces stress acclimation in maize plants. J Plant Physiol. 2005;162:1114–1122. doi: 10.1016/j.jplph.2005.01.007. [DOI] [PubMed] [Google Scholar]
  5. Beauchamp C, Fridovich Y. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971;44:276–287. doi: 10.1016/0003-2697(71)90370-8. [DOI] [PubMed] [Google Scholar]
  6. Chamnogpol S, Willeekens H, Moeder W, Langebartels C, Sandermann H, Jr, Montage MV. Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Plant Biol. 1998;95:818–823. doi: 10.1073/pnas.95.10.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chou TS, Chao YY, Kao CH. Involvement of hydrogen peroxide in heat shock and cadmium induced expression of ascorbate peroxidase and glutathione reductase in leaves of rice seedlings. J Plant Physiol. 2012;169:478–486. doi: 10.1016/j.jplph.2011.11.012. [DOI] [PubMed] [Google Scholar]
  8. Choudhury S, Panda SK. Role of salicylic acid in regulating cadmium induced oxidative stress in Oryza sativa L. roots. Bulg J Plant Physiol. 2004;30:95–110. [Google Scholar]
  9. Dave R, Singh PK, Tripathi P, Shri M, Dixit G, Dwivedi S, Chakrabarty D, Trivedi PK, Sharma YK, Dhankher OP, Corpas FJ, Barroso JB, Tripathi RD. Arsenite tolerance is related to proportional thiolic metabolite synthesis in rice (Oryza sativa L.) Arch Environ Contam Toxicol. 2013;64:235–242. doi: 10.1007/s00244-012-9818-8. [DOI] [PubMed] [Google Scholar]
  10. Doulis AG, Debian N, Kingston-Smith AH, Foyer CH. Differential localization of antioxidants in maize leaves. Plant Physiol. 1997;114:1031–1037. doi: 10.1104/pp.114.3.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drazic G, Mihailovic N. Modification of cadmium toxicity in soybean seedlings by salicylic acid. Plant Sci. 2005;168:511–517. doi: 10.1016/j.plantsci.2004.09.019. [DOI] [Google Scholar]
  12. Duan G, Liu W, Chen, X, Hu Y, Zhu Y (2013) Association of arsenic with nutrient elements in rice plants. Metallomics 5:784–792. [DOI] [PubMed]
  13. Dwivedi S, Tripathi RD, Tripathi P, Kumar A, Dave R, Mishra S, Singh R, Sharma D, Rai UN, Chakrabarty D, Trivedi PK, Adhikari B, Bag MK, Dhankher OP, Tuli R. Arsenate exposure affects amino acids, mineral nutrient status and antioxidants in rice (Oryza sativa L.) genotypes. Environ Sci Technol. 2010;44:9542–9549. doi: 10.1021/es101716h. [DOI] [PubMed] [Google Scholar]
  14. Dwivedi S, Mishra A, Tripathi P, Dave R, Kumar A, Srivastava S, Chakrabarty D, Trivedi PK, Adhikari B, Norton GJ, Tripathi RD, Nautiyal CS. Arsenic affects essential and non-essential amino acids differentially in rice grains: Inadequacy of amino acids in rice based diet. Environ Int. 2012;46:16–22. doi: 10.1016/j.envint.2012.04.012. [DOI] [PubMed] [Google Scholar]
  15. Fariduddin Q, Hayat S, Ahmad A. Salicylic acid influences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity and seed yield in Brassica juncea. Photosynthetica. 2003;41:281–284. doi: 10.1023/B:PHOT.0000011962.05991.6c. [DOI] [Google Scholar]
  16. Foyer CH, Lopez-Delgado H, Dat JF, Scottm IM. Hydrogen peroxide and glutathione associated mechanism of acclamatory stress tolerance and signaling. Plant Physiol. 1997;100:241–254. doi: 10.1111/j.1399-3054.1997.tb04780.x. [DOI] [Google Scholar]
  17. Guo B, Liang YC, Zhu YG, Zhao FJ. Role of salicylic acid in alleviating oxidative damage in rice roots (Oryza sativa) subjected to cadmium stress. Environ Pollut. 2007;147:743–749. doi: 10.1016/j.envpol.2006.09.007. [DOI] [PubMed] [Google Scholar]
  18. Guo B, Liang Y, Zhu Y. Does salicylic acid regulate antioxidantdefense system, cell death, cadmium uptake and partitioning to acquire cadmium tolerance in rice? J Plant Physiol. 2009;166:20–31. doi: 10.1016/j.jplph.2008.01.002. [DOI] [PubMed] [Google Scholar]
  19. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130–7139. [PubMed] [Google Scholar]
  20. Hayat S, Ahmad . A salicylic acid a plant hormone. Dordrecht: Springer Publishers; 2007. [Google Scholar]
  21. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplast I. Kinetic and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198 [DOI] [PubMed]
  22. Hewitt EJ (1966) Sand and Water Culture Methods Used in the Study of Plant Nutrition. Technical Commun. 2nd edn. Commonveld Agr. Bureaux Far Royal, pp 547
  23. Hung SH, Yu CW, Lin CH. Hydrogen peroxide functions as a stress signal in plants. Bot Bull Acad Sin. 2005;46:1–10. [Google Scholar]
  24. Janda K, Hideg E, Szalai G, Kovacs L, Janda T. Salicylic acid may indirectly influence the photosynthetic electron transport. J Plant Physiol. 2012;169:971–978. doi: 10.1016/j.jplph.2012.02.020. [DOI] [PubMed] [Google Scholar]
  25. Jing C, Cheng Z, Li-ping L, Zhong-yang S, Xue-bo P. Effects of exogenous salicylic acid on growth and H2O2-metabolizing enzymes in rice seedlings under lead stress. J Environ Sci. 2007;19:44–49. doi: 10.1016/S1001-0742(07)60007-2. [DOI] [PubMed] [Google Scholar]
  26. Kato M, Shimizu S. Chlorophyll metabolism in higher plants. VII. Chlorophyll degradation in senescing tobacco leaves; phenolic dependent peroxidative degradation. Can J Bot. 1987;65:729–735. doi: 10.1139/b87-097. [DOI] [Google Scholar]
  27. Kovtun Y, Chiu WL, Tena G, Sheen J. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci U S A. 2000;97:2940–2945. doi: 10.1073/pnas.97.6.2940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kumar N, Mallick S, Yadava RN, Singh AP, Sinha S. Co-application of selenite and phosphate reduces arsenite uptake in hydroponically grown rice seedlings: Toxicity and defence mechanism. Ecotox Env Safety. 2013;91:171–179. doi: 10.1016/j.ecoenv.2013.01.027. [DOI] [PubMed] [Google Scholar]
  29. Liu ZJ, Guo YK, Bai JG. Exogenous hydrogen peroxide changes antioxidant enzyme activities and protects ultrastructure in leaves of two cucumber ecotypes under osmotic stress. J Plant Growth Regul. 2010;29:171–183. doi: 10.1007/s00344-009-9121-8. [DOI] [Google Scholar]
  30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–725. [PubMed] [Google Scholar]
  31. Metwally A, Finkemeier I, Georgi M, Dietz KJ. Salicylic acid alleviates the cadmium toxicity in barley. Plant Physiol. 2003;132:272–281. doi: 10.1104/pp.102.018457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mishra A, Choudhuri MA. Effect of salicylic acid on heavy metal-induced membrane deterioration mediated by lipoxygenase in rice. Biol Plant. 1999;42:409–415. doi: 10.1023/A:1002469303670. [DOI] [Google Scholar]
  33. Mittler R, Vanderauwera S, Gollery M, VanBreusegem F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004;9:490–496. doi: 10.1016/j.tplants.2004.08.009. [DOI] [PubMed] [Google Scholar]
  34. Murphy TM, Sung WW, Lin CH. H2O2 treatment induces glutathione accumulation and chilling tolerance in mung bean. Funct Plant Biol. 2002;29:1081–1087. doi: 10.1071/PP01264. [DOI] [PubMed] [Google Scholar]
  35. Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 1981;22:867–880. [Google Scholar]
  36. Noriega G, Caggiano E, Lecube ML, Cruz DS, Batlle A, Tomaro M, Balestrasse KB. The role of salicylic acid in the prevention of oxidative stress elicited by cadmium in soybean plants. Biometals. 2012;25:1155–1165. doi: 10.1007/s10534-012-9577-z. [DOI] [PubMed] [Google Scholar]
  37. Oberbacher MF, Vines HM. Spectrophotometric assay of ascorbic acid oxidase. Nature. 1963;197:1203. doi: 10.1038/1971203a0. [DOI] [PubMed] [Google Scholar]
  38. Pal M, Szalai G, Horvath E, Janda T, Paldi E. Effect of salicylic acid during heavy metal stress. Acta Biol Szegediensis. 2002;46:119–120. [Google Scholar]
  39. Poor P, Gemes K, Horvath F, Szepesi A, Simon ML, Tari I. Salicylic acid treatment via the rooting medium interferes with stomatal response, CO2 fixation rate and carbohydrate metabolism in tomato and decreases harmful effects of subsequent salt stress. Plant Biol. 2011;13:105–114. doi: 10.1111/j.1438-8677.2010.00344.x. [DOI] [PubMed] [Google Scholar]
  40. Popova LP, Maslenkova LT, Yordanova RY, Ivanova AP, Krantev AP, Szalai G, Janda T. Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings. Plant Physiol Biochem. 2009;47:224–231. doi: 10.1016/j.plaphy.2008.11.007. [DOI] [PubMed] [Google Scholar]
  41. Scandalios JG, Tsaftaris AS, Chandlee JM, Skadsen RW. Expression of the developmentally regulated catalase (Cat) genes in maize. Dev Genet. 1983;4:281–293. doi: 10.1002/dvg.1020040406. [DOI] [Google Scholar]
  42. Shi Q, Zhu Z. Effects of exogenous salicylic acid on manganese toxicity, element contents and antioxidative system in cucumber. Env Exp Bot. 2008;63:317–326. doi: 10.1016/j.envexpbot.2007.11.003. [DOI] [Google Scholar]
  43. Shukla VKS, Kokate CK, Srivastava KC. Spectrophotometric determination of ascorbic acid. Microchem J. 1979;24:124–126. doi: 10.1016/0026-265X(79)90048-1. [DOI] [Google Scholar]
  44. Smith IK, Vierheller TL, Thorne CA. Assay of glutathione reductase in crude tissue homogenates using 5, 5’-dithiobis (2-nitrobenzoic acid) Anal Biochem. 1988;175:408–413. doi: 10.1016/0003-2697(88)90564-7. [DOI] [PubMed] [Google Scholar]
  45. Sn D, Poschenrieder C, Zl S, Georgieva K, Velichkova M, Barceló J. Silicon amelioration of manganese toxicity in Mn-sensitive and Mn-tolerant maize varieties. Environ Exp Botany. 2009;65:189–197. doi: 10.1016/j.envexpbot.2008.11.006. [DOI] [Google Scholar]
  46. Szalai G, Tari I, Janda T, Pestenácz A, Páldi E. Effects of cold acclimation and salicylic acid on changes in ACC and MACC contents in maize during chilling. Biol Plant. 2000;43:637–640. doi: 10.1023/A:1002824721597. [DOI] [Google Scholar]
  47. Uchida A, Jagendrof AT, Hibino T, Takabe T. Effect of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice. Plant Sci. 2002;163:515–523. doi: 10.1016/S0168-9452(02)00159-0. [DOI] [Google Scholar]
  48. Vanacker H, Carver TLW, Foyer CH. Pathogen-induced changes in the antioxidant status of the apoplast in barley leaves. Plant Physiol. 1998;117:1103–1114. doi: 10.1104/pp.117.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Protective role of exogenous polyamines. Plant Sci. 2000;151:59–66. doi: 10.1016/S0168-9452(99)00197-1. [DOI] [Google Scholar]
  50. Wahid A, Shabbir A. Induction of heat stress tolerance in barley seedlings by pre-sowing seed treatment with glycinebetaine. Plant Growth Regul. 2005;46:133–141. doi: 10.1007/s10725-005-8379-5. [DOI] [Google Scholar]
  51. Wahid A, Perveen M, Gelani S, Basra SMA. Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins. J Plant Physiol. 2007;164:283–294. doi: 10.1016/j.jplph.2006.01.005. [DOI] [PubMed] [Google Scholar]
  52. Williams PN, Islam S, Islam R, Jahiruddin M, Adomako E, Soliaman AR, Rahman G K, Lu Y, Deacon C, Zhu Y G, Meharg AA (2009) 43: 8430–8436. [DOI] [PubMed]
  53. Xiao-Juan B, Li-Juan L, Chun-hua Z, Ying G, Wang-da C. Effect of H2O2 Pretreatment on Cd Tolerance of different rice cultivars. Rice Sci. 2010;17:29–35. [Google Scholar]
  54. Yang YN, Qi M, Mei CS. Endogenous salicylic acid protects rice plants from oxidative damage caused by aging as well as biotic and abiotic stress. Plant J. 2004;40:909–919. doi: 10.1111/j.1365-313X.2004.02267.x. [DOI] [PubMed] [Google Scholar]
  55. Zawoznik MS, Groppa MD, Tomaro ML, Benavides MP. Endogenous salicylic acid potentiates cadmium-induced oxidative stress in Arabidopsis thaliana. Plant Sci. 2007;173:190–197. doi: 10.1016/j.plantsci.2007.05.004. [DOI] [Google Scholar]
  56. Zhang X-L, Jia X-F, Yu B, Gao Y, Bai J-G. Exogenous hydrogen peroxide influences antioxidant enzyme activity and lipid peroxidation in cucumber leaves at low light. Sci Hort. 2011;129:656–662. doi: 10.1016/j.scienta.2011.05.009. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplimentary Figure 1 (791.2KB, docx)

Effect of pretreatment of H2O2 and H2O2 + SA on AsIII and AsV exposed rice cv. P11 and MTU after 14d. A, Control cv. P11; B, AsIII(4)(P11); C, AsIII(4)H2O2(P11); D, AsIII(4)H2O2 + SA(P11); E, AsV(4)(P11); F, AsV(4)H2O2(P11); G, AsV(4)H2O2 + SA(P11); H, Control(MTU); I, AsIII(4)(MTU); J, AsIII(4)H2O2(MTU); K, AsIII(4)H2O2 + SA(MTU); L, AsV(4)(MTU); M, AsV(4)H2O2(MTU); N, AsV(4)H2O2 + SA. (DOCX 791 kb)

Articles from Physiology and Molecular Biology of Plants are provided here courtesy of Springer

RESOURCES