Germinating Peanut (Arachis hypogaea L.) Seedlings Attenuated

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Germinating Peanut (Arachis hypogaea L.) Seedlings Attenuated Selenite-induced Toxicity by Activating the Antioxidant Enzymes and Mediating the Ascorbate-glutathione Cycle Guang Wang, Hong Zhang, Furao Lai, and Hui Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05945 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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Journal of Agricultural and Food Chemistry

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Germinating Peanut (Arachis hypogaea L.) Seedlings Attenuated Selenite-induced

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Toxicity by Activating the Antioxidant Enzymes and Mediating the

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Ascorbate-glutathione Cycle

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Guang Wang, Hong Zhang, Furao Lai, Hui Wu *

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College of Light Industry and Food Sciences, South China University of Technology,

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Guangzhou 510640, China

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Corresponding author:

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*Hui Wu, College of Light Industry and Food Sciences, South China University of

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Technology, Wushan Road 381, Guangzhou, Guangdong, China.

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E-mail: [email protected], Phone: (+86)20-87112853.

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ACS Paragon Plus Environment


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Abstract: Selenite can enhance selenium nutrition level of crops, but excessive selenite

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may be toxic to plant growth. To elucidate the mechanisms underlying the role of

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selenite in production and detoxification of oxidative toxicity, peanut seedlings were

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developed with sodium selenite (0, 3, and 6 mg/L). The effects of selenite on

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antioxidant capacity, transcript levels of antioxidant enzyme genes, and enzyme

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activities in hypocotyl were investigated. The CuZn-SOD, GSH-Px, GST, and APX

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gene expression levels and their enzyme activities in selenite treatments were 1.0 to 3.6

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folds of the control. Selenite also significantly increased the glutathione and ascorbate

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concentrations by mediating the ascorbate-glutathione cycle, and the selenite-induced

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hydrogen peroxide may act as a second messenger in the signaling pathways. Our work

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has revealed a complex antioxidative response to selenite in peanut seedling.

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Understanding these mechanisms may help future research in increasing selenite

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tolerance and selenium accumulation in peanut and other crops.

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Keywords: peanut; selenium; selenite; ascorbate-glutathione cycle; antioxidant enzyme;

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gene expression.

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Introduction

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Selenium (Se) is a naturally occurring metalloid element, and its concentration in

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most soils of the world ranges from 0.01 to 2 mg/kg (average 0.4 mg/kg), but higher

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concentrations (>2 mg/kg) can occur in so called seleniferous soils which is widely

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distributed throughout the world 1, 2. Selenate and selenite are the two main inorganic Se

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forms in soil. Studies indicate that organic Se compounds are the better supplements for

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animals and humans than inorganic Se compounds because organic Se compounds are

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less toxic

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inorganic Se to organic Se, and selenite has been used as one of the Se fertilizers for

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enhancing Se nutrition level of crops 5. Additionally, plants that accumulate Se can be

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used as a Se-delivery system to provide dietary Se in individuals with Se deficiency 6.

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Se-accumulator crops may be utilized to improve nutrition and prevent disease in

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animals and humans, but plants themselves are not thought to have a requirement for Se

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7

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are Se accumulators that can accumulate very high levels of Se 8, most plants are Se

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non-accumulators and are even Se-sensitive when grown in seleniferous soils. Se

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sensitive plants may exhibit symptoms of injury, including stunting growth, and

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withering and drying of leaves when cultivated in soils with high concentrations of Se 6.

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Se toxicity in plants has been attributed to the excessive formation of non-specific

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selenoproteins within their tissue. However, there are increasing evidences suggesting

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that Se-induced oxidative stress also contributes to toxicity in plants 9. The mechanisms

3, 4

. Plants play a pivotal role in organic Se production since they can convert

. However, plants differ in their abilities to accumulate Se. While some plant species

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behind this stress involves the prooxidant ability of selenium to induce the oxidation of

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thiols (such as glutathione (GSH)), and simultaneously generate superoxide free radicals

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(O2.-) and hydrogen peroxide (H2O2)

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toxicity may also involve in the antioxidant capacity of plants. However, previous

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studies have focused on the mechanisms of stress generation induced by Se rather than

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exploring the cellular homeostasis of plants when they were developed in Se-rich

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environments.

10

. Therefore, the detoxification of Se-induced

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Plants have a complex system to protect themselves from the environment stress,

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which including antioxidant enzymes and a number of metabolic pathways. The

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ascorbate-glutathione cycle is a major H2O2 scavenging pathway and an effective

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detoxifying mechanism in plant cells 11. Ascorbate (AsA), glutathione (GSH), and their

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associated enzymes play an important role in enhancing the oxidative stress tolerance 12.

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Research have shown that the addition of Se enhanced the levels of GSH and related

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enzymes in rapeseed, and AsA content also showed a significant increase by

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Se-pretreated 13. Se may inhibit lipid peroxidation in rape and soybean through GSH-Px

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and nonenzymatic reactions

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essential part of the gluthatione peroxidise (GSH-Px) molecule in higher plants, Se may

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have particular biological functions in plants. Se regulated GSH, AsA, and their

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associated enzymes raised the question that what’s the physiological role of Se involved

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in the toxicity and detoxification in plant growth. With regard to reports indicating the

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importance of the ascorbate-glutathione cycle for plant stress tolerance

14

. Although there is no evidence to show that Se is an

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, we

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hypothesize that at low concentration Se may play a regulatory role in Se detoxification

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by influencing the antioxidant defense system and the ascorbate-glutathione cycle.

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Peanuts (Arachis hypogaea L.) are an important oil crop all over the world, it can

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also be used as a Se accumulator 17. Adding selenite to soil can increase Se in the peanut

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kernel, thereby producing an organic Se resource that is beneficial to human and animal

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health 18. However, it is unknown how selenite affects the formation and detoxification

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of oxidative stress in peanut seedlings. There are no reports associated with the

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mechanism of scavenging selenite-induced oxidant stress with the metabolism of the

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ascorbate-glutathione cycle in germinating peanuts. Therefore, further studies are

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needed to elucidate the molecular mechanism and signaling pathways underlying the

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role of selenite in the peanut seedlings. To better understand the effects of selenite on

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the antioxidant defense system and the ascorbate-glutathione cycle, we investigated the

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effects of selenite treatments on total antioxidant capacity, changes of AsA and GSH

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content, enzyme activities involved in the ascorbate-glutathione cycle, and the

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expression of related genes in peanut seedlings. The results of this study may clarify the

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effects of selenite toxicity in peanut seedlings and the metabolic adjustments needed to

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mediate a selenite-response.

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MATERIALS AND METHODS

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Chemicals. Sodium selenite (Na2SeO3) was purchased from Sigma Co. (St. Louis,

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MO, USA). 2, 2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,

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1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Aladdin biological

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technology Co. (Shanghai, China). Hydrogen peroxide (H2O2), malondialdehyde

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(MDA), CuZn Superoxide dismutase (CuZn-SOD) and peroxidase (POD) kits were

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purchased from the Nanjing Jiancheng Institute of Biotechnology (Jiangsu, China).

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Glutathione peroxidase (GSH-Px) and glutathione (GSH) kits were purchased from the

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Beyotime Institute of Biotechnology (Shanghai, China). A plant RNA isolation kit was

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purchased from Aidlab Company (Beijing, China). The RNase-free DNase I and

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First-strand cDNA synthesis kits were purchased from Takara (Dalian, China). A

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FastStart Essential DNA Green Master was purchased from Roche (Mannheim,

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Germany). Ultrapure water was obtained from a Milli-Q water purification system

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(Millipore, Bedford, MA, USA). Other chemicals were obtained from local sources and

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were of analytical grade.

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Plant materials and selenite treatments. Peanut seeds were purchased from local

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market in Guangzhou, China. Healthy seeds were selected and sterilized with 75%

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ethanol followed by washing several times with distilled water. Peanut seeds were then

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soaked in distilled water for 8 h at 25°C. Excess water was removed and seeds were

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pre-germinated at 25°C and 90% relative humidity (RH) in the dark for 24 h. The seeds

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were then sowed into a sprouter (370 × 270 × 60 mm) using quartz sand as the culture

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medium, which were irrigated with different concentrations of sodium selenite water

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solution (0, 3, 6, 12, and 24 mg/L), and peanut seeds were developed at 25°C and 80%

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RH in the dark for 10 days. Samples were obtained every 24 h from the second day to

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the tenth day after sowing. The hypocotyls were cut from peanut seedlings and then

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ground into a fine powder in the presence of liquid nitrogen. The powder samples were

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stored at -80°C for further use.

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Measurement of MDA and H2O2 levels. We used the kits from the Nanjing

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Jiancheng Institute of Biotechnology (Jiangsu, China) to assay the MDA and H2O2 levels.

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The MDA content was assayed applying the thiobarbituric acid (TBA) test based on the

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absorbance at the 532nm of a red-violet complex formed with MDA. The H2O2 content

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was measured based on the generation of the color complex when H2O2 was bound with

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molybdenic acid. The absorbance of the color solutions could be measured with a

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spectrophotometer (Shimadzu UV1800, Japan). And the content of MDA and H2O2 were

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calculated in accordance with the instructions of the test kits.

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Total antioxidant activity evaluation. For the measurements of total antioxidant

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activity of different treatments, the powder samples of our three different treatments

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were mixed with phosphate-buffered saline (PBS pH 7.0) and then centrifuged at 10000

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× g for 15 min at 4°C. The supernatants were collected and used to evaluate the DPPH

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and ABTS radical scavenging capacity.

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Method of DPPH radical scavenging capacity assay was closely followed those

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described by Miliauskas et al. 2004 19, where DPPH was dissolved in ethanol to a 0.1

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mM concentration, and 3 ml of this solution was mixed with 100 µl supernatant of each

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sample. The reaction mixtures were incubated for 20 min in the dark at 30°C, and the

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decrease in absorbance at 515 nm was measured (AA). The absorbance of a blank

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sample that contained 100 µl of PBS and 3ml DPPH solution was also measured (AB).

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Radical scavenging activity was calculated using the following formula: % (inhibition)

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= [(AB –AA)/ AB] × 100.

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ABTS radical scavenging capacity was determined using the methods described by

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Dudonne S et al.20, where ABTS was dissolved in deionized water to a concentration of

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7 mM. ABTS radical cation (ABTS•+) was produced by reaction of the ABTS solution

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and potassium persulfate (final concentration of 2.45 mM), allowing the mixture to be

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stored in the dark at room temperature for 12-16 h before use. The ABTS•+ solution was

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diluted in a phosphate buffer (pH 7.4) to an absorbance of 0.7 (±0.02) at 734 nm, and an

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appropriate solvent blank reading (AB) was taken. After supernatant of each treatment

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was added to the ABTS•+ solution, the absorbance reading (AA) was taken at 30°C, 10

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min after the initial mixing. The percentage of inhibition of ABTS•+ was calculated

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using the above formula.

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Measurement of ascorbate (ASA), dehydroascorbate (DHA), and glutathione

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(GSH) levels. Powder samples (1.0 g for each treatment) were homogenized in a 3 ml

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ice-cold acidic extraction buffer (6% metaphosphoric acid containing 1 mM

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ethylenediaminetetraacetic acid, EDTA). Homogenates were centrifuged at 10000 × g

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for 15 min at 4°C, and the supernatants were collected for the analysis of ascorbate

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(ASA), dehydroascorbate (DHA), and glutathione (GSH) levels.

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AsA and DHA were determined using the methods described by Kampfenkel et al. 1995

21

. The assay of AsA was based on the reduction of Fe3+ to Fe2+ by AsA and the

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spectrophotometric detection of Fe2+ complexed with 2,2’ -dipyridyl. DHA was reduced

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to AsA by pre-incubating the sample supernatant with dithiothreitol (DTT). The excess

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DTT was removed with N-ethylmaleimide to determine the total ascorbate present.

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DHA levels were estimated on the basis of the differences between total ascorbate and

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AsA values.

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GSH content was measured with a GSH kit from Beyotime Institute of

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Biotechnology (Shanghai, China). The method is based on the generation of a yellow

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color when 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was reacted with GSH. The

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absorbance of the mixture was measured at 412 nm after 10 min of reaction, and the

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GSH content was calculated according to the instructions of the test kit.

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Enzymes extraction and determination. Powder samples (1.0 g for each treatment)

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were homogenized in 3 ml of 50 mM ice-cold K-phosphate buffer (pH 7.0) that

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contained 1 mM EDTA, 3 mM β-mercaptoethanol, and 2% (w/v) polyvinylpyrrolidone

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(PVP). The homogenates were centrifuged at 10000 × g for 15 min at 4°C, and the

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supernatants were collected and used to determine enzyme activities.

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APX (EC: 1.11.1.11) activity was determined according to the method of Nakano

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and Asada 1981 22, which was measured by observing the decrease in absorbance at 290

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nm with a spectrophotometer. The oxidation rate of ascorbate was estimated from 1 to

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60 seconds after starting the reaction with the addition of H2O2. The reaction mixture

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contained 50 mM K-phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.6 mM H2O2 and

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0.1 ml enzyme extract in a final volume of 3.0 ml.

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GR (EC: 1.6.4.2) activity was assayed spectrophotometrically as described by

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Knörzer et al. 1996 23. The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5),

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5 mM MgCl2, 0.2 mM NADPH, 1.0 mM oxidized glutathione (GSSG), and 0.1 ml

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enzyme extract for a final volume of 3 ml. The reaction was initiated with the addition

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of GSSG, and the decrease in absorbance at 340 nm (due to NADPH oxidation) was

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recorded for 60 seconds. 24

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GST (EC: 2.5.1.18) activity was measured by the method of Habig et al. 1974

.

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The reaction mixture contained 100 mM K-phosphate buffer (pH 6.5), 30 mM

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1-chloro-2,4,-dinitrobenzene (CDNB), 30 mM GSH, and 0.1 ml enzyme extract for a

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final volume of 3 ml. The enzyme reaction was initiated by the addition of GSH. The

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increase in absorbance was measured at 340 nm at 10 s intervals for 3 min.

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CuZn-SOD (E.C. 1.16.1.1) activity was assayed with a kit from Nanjing Jiancheng

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Institute of Biotechnology (Jiangsu, China). There are three main forms of SODs in

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plant, including CuZn-SOD, Mn-SOD, and Fe-SOD. The CuZn-SOD enzyme was

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extracted by a reagent of the test kit that inactivates the Mn-SOD and Fe-SOD but

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without influencing the CuZn-SOD activity. In the reactive system, O2·− was produced

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via the reaction of xanthine with xanthine oxidase, which can oxidize hydroxylamine to

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produce nitrite that develops the red-violet by combining with dye-forming reagent of

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the test kit. The CuZn-SOD activity was determined by measuring the inhibiting rate of

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the enzyme to the O2·−, which was evaluated by a decrease of absorbance of the

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red-violet solution at 550 nm with a spectrophotometer. The CuZn-SOD activity was

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calculated according to the manufacturer's instructions.

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POD (E.C. 1.11.1.7) activity was measured based on the change of absorbance at

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420 nm by catalyzing H2O2. POD activity was calculated used the formula according to

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the test kit from Nanjing Jiancheng Institute of Biotechnology (Jiangsu, China).

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GSH-Px (E.C. 1.11.1.9) activity was measured using H2O2 as a substrate. The

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enzyme activity were determined by measuring the oxidation of glutathione in a coupled

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assay using glutathione reductase and saturating concentrations of NADPH. The

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experiments were performed at 25°C and the disappearance of NADPH was monitored

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at 340nm with a spectrophotometer. GSH-Px activity was expressed as nmoles of

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NADPH min-1 mg-1 protein. All the measurement steps were according to the test kit

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instructions from Beyotime Institute of Biotechnology (Shanghai, China).

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Determination of protein concentration. The protein concentration in the

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hypocotyl of peanut seedlings was assayed according to the methods described by

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Bradford 1976 25, using bovine serum albumin (BSA) as a protein standard.

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mRNA isolation and cDNA preparation for quantitative real-time PCR

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(qPCR). Total RNA was isolated from the hypocotyl using a plant RNA isolation kit

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(Aidlab Company, Beijing, China) and then treated with a RNase-free DNase I (Takara,

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Dalian, China) to eliminate the residual genomic DNA. We quantified total RNA using a

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spectrophotometer and its quality was checked by agarose-formaldehyde gel

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electrophoresis (see Fig. 1A). Total RNA was converted into cDNA using the

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First-strand cDNA Synthesis Kit (Takara, Dalian, China). The cDNA solution was taken

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as the template for qPCR analysis.

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Primer design and PCR product identity. Primer pairs for qPCR were designed

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using primer premier 5 software. The gene sequences are from peanuts (Arachis

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hypogaea L.) that are available in GenBank (see Table 1 for details). Gene-specific

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primers were chosen so that the resulting PCR products all had approximately the same

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size, ranging between 100–200 bp. The quality of PCR products were visually inspected

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by 2% agarose gel electrophoresis (see Fig. 1B), and the generation of only one single

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band of the expected size was taken as a criterion for specificity.

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Quantitative real-time PCR and quantification of mRNA levels. The qPCR was

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performed following the instructions of FastStart Essential DNA Green Master (Roche).

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The 20 µl of reaction mixture contained 10 µl of Master Mix, 2 µl of qPCR primers, 5 µl

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of cDNA template, and 3 µl of RNase-free water. Amplification of qPCR products were

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monitored using an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems,

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Foster City, CA, USA). The following program was applied: the initial polymerase

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activation was at 95°C for 10 min, followed by 45 cycles at 95°C for 10 s, 60°C for 10 s,

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and 72°C for 10 s. PCR conditions were optimized for high amplification efficiency

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(95%) for all primer pairs used. The cycle threshold (Ct) values were analyzed using the

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SDS 1.4 software (ABI Prism 7500, SDS User Bulletin; Applied Biosystems). The

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expressions of the target genes were normalized using actin as an endogenous control.

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The relative quantification of gene expressions were analyzed by the 2–∆∆CT method and

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the results were expressed as extent of change with respect to control values

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sample was tested in triplicate to obtain an average Ct value.

26

. Each

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Statistical Analysis. All the experiments were carried out in triplicate, and all data

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were expressed as means ±standard deviation (SD). Statistical analysis was performed

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by SPSS 19 (SPSS Inc., Chicago, IL, USA). The probabilities of less than 5% (P < 0.05)

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were considered significant.

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RESULTS AND DISCUSSION

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The selenite-induced toxicity during peanut seedlings development. Selenite can

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be used as a Se fertilizer to enhance Se nutrition level of crops; research show that 5-10

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mg/kg selenite treatments in the soil can increase by an average of 16.1 mg Se/kg in the

260

peanut kernel

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seedlings. As shown in Fig. 2A-B, peanut seedlings growing on high concentrations of

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selenite (12 and 24 mg/L) showed some visible toxic symptoms such as stunted growth.

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As can be seen in the photographs, high concentrations of selenite not only inhibited the

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hypocotyl and root elongation, but also stunted the lateral root growth. The effect of

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selenite on inhibition of peanut seedlings growth showed a dose-dependent manner.

266

These results were in agreement with the previous report showing that selenite

267

decreased the root length and lateral root numbers in Arabidopsis plants

18

. Nevertheless, excessive selenite would cause toxicity in peanut

13


27

. However,


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there were no significant differences in peanut seedling morphology between the low

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concentrations of selenite treatment (3 and 6 mg/L) and the untreated control during

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seedlings development (Fig. 2C-D). Therefore, we applied the concentrations of 0, 3,

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and 6 mg/L of selenite treatment for the further research in our present work.

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Although at low concentrations, selenite treatments would cause ROS generation in

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peanut seedlings. As shown in Fig. 2E, H2O2 was significantly up-regulated from

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selenite treatments (3 and 6 mg/L) in the early stage of seedlings growth. The H2O2

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content in the 6 mg/L selenite treatment was much greater than the 3 mg/L selenite

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treatment and the control (from day 2 to day 4). However, the H2O2 contents of all

277

treatments returned to nearly normal levels by day 5. MDA is a decomposition product

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of the peroxidized polyunsaturated fatty acid component of the membrane lipid. MDA

279

formation corresponded to the H2O2 generation in peanut seedlings, but did not form

280

until a day later (see Fig. 2F). From day 3 to 5, selenite treatments (3 and 6 mg/L)

281

slightly increased MDA formation compared to the untreated control; however, there

282

were no significant differences between selenite treatments and the control from day 6

283

to 10. These results demonstrated that the selenite treatments significantly induced H2O2

284

generation, thereby promoting the MDA formation in peanut seedlings at an early

285

growth stage. However, the level of H2O2 was mediated by physiological regulatory

286

actions, resulting in a decrease of MDA formation to nearly normal levels by day 6.

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Plants have a complex response to protect themselves against selenite-induced

288

toxicity. Studies have demonstrated that a potential strategy for enhancing plant Se

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tolerance rather than accumulation, is the up-regulation of Se via volatilization 28. Due

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to the chemical similarity of sulfur (S) and Se, plants are able to take up inorganic and

291

organic forms of Se and metabolize them to volatile forms via the S assimilation

292

pathway

293

non-enzymatically with GSH to form GS-Se-SG and subsequently to GS-Se−, which can

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then be used as a physiological substrate to form SeCys by the action of Cys synthase 6.

295

An alternative fate of SeCys is to be converted to selenomethionine (SeMet).

296

Furthermore, biological volatilization has the advantage of converting Se into relatively

297

non-toxic forms, such as dimethylselenide (DMSe), which is produced by the

298

methylation of SeMet 29. This may be one of the detoxification mechanisms of selenite

299

in plants. However, a consequence of selenite reacting with glutathione is the

300

production of reactive oxygen species (ROS) such as O2.- and

301

capacity to suppress ROS accumulation is overwhelmed or impaired, oxidative stress

302

can result in the oxidation of cellular macromolecules such as lipids, nucleic acids, and

303

proteins. In the present work, selenite treatments (3 and 6 mg/L) may induce a small

304

oxidant stress in germinating peanut seedlings in the early stage of seedling growth.

305

Nevertheless, the morphology of peanut seedlings and the results of H2O2 and MDA

306

indicated that low concentrations of selenite treatment did not cause metabolic disorder

307

in peanut seedlings.

29

. As depicted in Fig. 2G, when selenite is taken up by plants, it reacts

H2O2

10

. If a plant’s

308

Effect of selenite on the mRNA expression profiles of antioxidant genes. The

309

expression levels of the antioxidant enzyme genes (CuZn-SOD, APX, and GSH-Px,

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GST) were investigated in selenite treatments and control peanut seedlings by qPCR. As

311

shown in Fig. 3A-D, we found that the expression levels of these genes were

312

significantly over-expressed in selenite treatments (3 and 6 mg/L) from day 2 to 10

313

during seedlings growth. In particular, expression levels of CuZn-SOD and APX genes

314

in selenite treatments were 1.1-3.1 folds and 1.2-3.4 folds of the untreated control,

315

respectively. Also, GSH-Px and GST gene expression levels were 1.3-3.6 folds and

316

1.0-2.5 folds higher than those observed in untreated samples, respectively. The

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maximal accumulation of mRNA in these genes appeared on day 10 of peanut seedling

318

growth. Comprehensive analysis of antioxidant gene expression provided insight into

319

the transcriptional changes triggered by a specific stimulus or perturbation

320

proposed that the effects on these antioxidant enzyme genes were due to a higher

321

efficiency of mRNA transcription which triggered by the selenite treatments.

30

. We

322

In plants, SODs (including CuZn-SOD, Mn-SOD and Fe-SOD) are the first line of

323

defense against superoxide radicals (O2.–), which is extremely important in maintaining

324

cellular homeostasis

325

chloroplasts, Mn-SOD and Fe-SOD are present in the mitochondria and chloroplast,

326

respectively

327

light-dependent. The previous research showed that Se alters the transcript

328

accumulation of CuZn-SOD (but not Mn-SOD and Fe-SOD) in potato plant

329

Therefore, the CuZn-SOD in the cytosol was extremely important since peanut

330

seedlings were developed with sodium selenite in the dark in our experiments. As

31

. In most plants, CuZn-SOD is present in the cytosol and

32

. Chloroplast can’t develop in the dark because its formation is

16


33

.

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described above, the non-enzymatic reduction of selenite was likely mediated by

332

glutathione and consequently generated ROS (such as O2.– and H2O2). The up-regulation

333

of the SOD gene can be induced by its substrate, the superoxide anion itself 34, or by Se

334

directly

335

Therefore, we hypothesized that CuZn-SOD gene expression was up-regulated by Se

336

directly or by selenite-induced superoxide anion when peanut developed with sodium

337

selenite.

33

. Studies have shown that Se induces SOD activity in many plants

33, 35

.

338

ROS (such as hydrogen peroxide) plays a dual role in plants, where it causes

339

oxidative damage at high concentrations, but at low concentrations, hydrogen peroxide

340

acts as a second messenger in the signaling induced by abiotic and biotic stressors

341

Studies have shown that the expression of GSH-Px and GST genes increased after

342

treatment with hydrogen peroxide in soybeans and Arabidopsis

343

expressions of the APX gene by hydrogen peroxide was also reported

344

researches have demonstrated that hydrogen peroxide is an important messenger

345

involved in the expression of antioxidant enzymes. Taking into consideration the

346

increasing production of hydrogen peroxide in response to selenite treatments in the

347

early stage of peanut seedling growth (Fig. 2E), it is conceivable that the increased

348

expression and the activity of GSH-Px, GST, and APX are functionally linked to the

349

increase of hydrogen peroxide during peanut seedling development. These results

350

suggest that the increase in mRNA levels of antioxidant enzymes (GSH-Px, GST, and

351

APX) induced by selenite treatments may be involved in signal transduction that is

17


34

.

36, 37

. The increased 38

. These


352

mediated by the selenite-induced hydrogen peroxide.

353 354

Selenite activated the antioxidant enzymes and enhanced total antioxidant

355

capacity. When seedlings were developed with selenite, an equilibrium between the

356

ROS generation and scavenging activities is necessary for the peanut cells to combat

357

selenite-induced oxidative stress. The ROS scavenging activity was achieved by the

358

plant’s antioxidant defense system that comprises enzymatic and non-enzymatic

359

antioxidants 39. As mentioned above, SOD is the first line of defense against superoxide

360

radicals, the selenite-induced O2.– may be dismutated to molecular oxygen (O2) and

361

hydrogen peroxide (H2O2) by SOD in peanut seedlings. H2O2 can then be degraded by

362

catalase (CAT), peroxidase (POD), or by glutathione peroxidase (GSH-Px) and

363

Glutathione S-transferase (GST)

364

activity in potato plant, even though this enzyme is not the selenoenzyme in plants 33.

365

These enzymes play an important role in cellular protection against selenite-induced

366

oxidative stress.

40

. Research has shown that Se enhanced GSH-Px

367

In our present study, we saw a linear increase in CuZn-SOD activity in all

368

treatments and the control as development progressed. CuZn-SOD activity in selenite

369

treatments (3 and 6 mg/L) was significantly greater than the control (P < 0.01) during

370

peanut seedlings growth (Fig. 4A), which was corresponding with CuZn-SOD gene

371

expression described above (Fig. 3A). These results were in agreement with the

372

previous report showing that selenite enhanced the CuZn-SOD gene expression and its

18


Page 18 of 43

Page 19 of 43


373

activity in potato plant 33. As described above, CuZn-SOD is the main form of SODs in

374

higher plant cells, we suggest that the selenite-induced O2.– may be converted to H2O2

375

mainly catalyzed by CuZn-SOD in peanut seedling cells. Studies have reported that

376

H2O2 treatments at non-toxic concentrations effectively induced the antioxidant

377

enzymes that maintain extreme antioxidant activities that are used to quench the toxic

378

ROS and in turn increase the plant’s environmental stress tolerance

379

the activities of POD, GSH-Px, and GST in selenite treatments were higher than the

380

control from day 6 to 10, which were not significant different from day 2 to 5 (Fig.

381

4B-D). The activities of these enzymes were corresponding with their gene’s expression

382

levels (Fig. 3C-D). In accordance with our observed changes of H2O2 and MDA levels

383

(Fig. 2E-F), the enhanced antioxidant enzyme activities and their relative genes explain

384

why the H2O2 and MDA were accumulated in the early growth stage but were

385

eliminated after day 5 of peanut seedling development. We suggest here that the

386

increasing peroxide scavenging capacity of peanut seedlings in our experiment is

387

attributed to the up-regulation of antioxidant enzyme activities from low concentrations

388

of H2O2, which were induced by our selenite treatments.

41, 42

. Interestingly,

389

To better understand the effect of selenite on the total antioxidant capacity of peanut

390

seedlings, DPPH and ABTS assays were used, which are common methods to evaluate

391

the antioxidant activities of the plant extracts in vitro. These methods are the easiest to

392

implement and yield the most reproducible results. We found that the DPPH radical

393

scavenging activities were not significantly different between selenite treatments (3 and

19



394

6 mg/L) and the control from day 2 to 5 (Fig. 4E-F). The ABTS radical scavenging

395

activities of selenite treatments (3 and 6 mg/L) were lower than the control from day 2

396

to 3, and was not significantly different from day 4 to 5. However, DPPH and ABTS

397

radical scavenging activities were all higher in selenite treatments than the control from

398

day 6 to 10. The change trend of DPPH and ABTS radical scavenging activities was

399

negative corresponding to the change of H2O2 and MDA levels (Fig. 2E-F), which could

400

also be explained by the change of antioxidant enzymes activities described above (Fig.

401

4B-D). These results demonstrated that the selenite treatments promoted generation of

402

H2O2 in the early stage of peanut seedling growth, but subsequently enhanced the total

403

antioxidants available for scavenging of free radicals and sustained the normal situation

404

of development. Although selenite is toxic at high concentrations, we found that low

405

concentrations of selenite have a stabilizing and protective effect on germinating peanut

406

seedlings against peroxidative damage. Therefore, selenium is considered an important

407

compound that plays a dual role in germinating peanut seedlings.

408

Selenite mediated the metabolism of ascorbate-glutathione cycle. Besides the

409

antioxidant enzymes described above, free radical scavenging and cellular homeostasis

410

are also regulated by a complex antioxidant system, namely the ascorbate-glutathione

411

cycle

412

both in chloroplasts and the cytosol

413

cycle is shown in Fig. 6. H2O2 is reduced to water by APX using AsA as a reductant,

414

thereby producing an ascorbyl radical (monodehydroascorbate, MDHA) and an

43

. The ascorbate-glutathione cycle is an effective detoxifying pathway operates 44

. The mechanism of the ascorbate-glutathione

20


Page 20 of 43

Page 21 of 43


415

oxidized form of ascorbate (dehydroascorbate, DHA) 45. The regeneration of AsA from

416

MDHA and DHA are catalyzed by the NADH-dependent monodehydroascorbate

417

reductase (MDHAR) and the GSH-dependent dehydroascorbate reductase (DHAR),

418

respectively.

419

NADPH-dependent glutathione reductase (GR), so as to maintain a normal GSH level 47.

420

The central role of GSH is to regenerate AsA through reduction of DHA, hence GR can

421

maintain a high level of AsA via the ascorbate-glutathione cycle 48. GSH and AsA are

422

considered the most powerful ROS scavengers since their ability to donate electrons in a

423

great number of enzymatic and non-enzymatic reactions

424

regeneration is extremely important for the balance of the ascorbate-glutathione cycle.

425

In our work, we observed that GR activity significantly increased in selenite treatments

426

(3 and 6 mg/L) during peanut seedling growth (Fig. 5B). This is important because GR

427

plays an extremely important role in the maintenance of GSH levels. As shown in Fig.

428

5A, the GSH concentration in selenite treatments (3 and 6 mg/L) was consistently

429

higher than the control (except for day 2). We propose that the GSH then ensured

430

enough AsA regeneration via the ascorbate-glutathione cycle. This would also explain

431

why selenite treatments significantly enhanced AsA levels and the AsA/DHA rate

432

during seedling growth, as the AsA concentrations in selenite treatments were far higher

433

than that in control (Fig. 5C-D). As mentioned above, H2O2 was triggered by selenite

434

treatment in the early stage of seedling growth, which can be reduced to water by APX

435

using AsA as a reductant and then generated DHA. As a result, DHA content in the

43, 46

. The GSSG generated in this procedure is then reduced by the

13

. Therefore, GSH and AsA

21



Page 22 of 43

436

selenite treatments was increased from day 2 to 6 (Fig. 5E). However, the DHA

437

contents of selenite treatments were decreased by day 7, which may be because the

438

excessive DHA was reduced back to AsA via the ascorbate-glutathione cycle. These

439

results also explained the increasing AsA/DHA rate from day 7 to 10 (Fig. 5D), and the

440

enhancing total antioxidant capacity in peanut seedlings (Fig. 4E-F). In the present work,

441

we also found that APX activity significantly increased during peanut seedlings

442

developed with selenite treatments (Fig. 5F). These results suggested that the

443

ascorbate-glutathione cycle was extremely important in the detoxification of selenite in

444

peanut seedlings.

445

In the previous studies, GSH-Px was initially defined as GSH dependent; studies

446

have shown that GSH is necessary for the effective scavenging of toxic H2O2 by

447

GSH-Px

448

sometimes exclusively use the thioredoxin (Trx) system for reduction rather than GSH,

449

which could also contribute to scavenging the H2O2 51. The regeneration of the reduced

450

Trx in plant varies in tissues. In chloroplast, the reducing power provided by light is

451

mediated by the ferredoxin/thioredoxin reductase. However in mitochondria and

452

cytoplasm, thioredoxins could be reduced by NADP-thioredoxin reductase (NTR).

453

Particular thioredoxins could also be reduced by the thioredoxin and glutaredoxin (Grx)

454

systems which are interlinked with the ascorbate-gluathione cycle via NADPH

455

this system Trx is directly reduced by Grx, which are then reduced by GSH (Fig. 6).

456

Because we administered selenite treatments in the dark in our experiments, chloroplast

49, 50

. But recently there was evidence showed that GSH-Px preferentially and

22


52

. In

Page 23 of 43


457

of peanut seedlings can’t develop because its formation is light-dependent. Therefore

458

the thioredoxin and glutaredoxin systems in mitochondria and cytoplasm are more

459

important for the regeneration of the reduced Trx when peanut seedling was growth on

460

Se in the dark. As shown in Fig. 5A, GSH content was enhanced by selenite treatments

461

during peanut seedling growth, the increase of GSH content may contribute to the Trx

462

regeneration via the thioredoxin system. Associated with the results described above:

463

selenite enhanced the activity of GSH-Px (Fig. 4C) and reduced the H2O2 content from

464

day 6 to 10 of the seedling growth (Fig. 2E), we hypothesis that GSH-Px may also

465

contributed to scavenging of free radicals use Trx as a substrate. These results suggested

466

that selenite played an important role in modulating free radicals and its related

467

processes such as the thioredoxin system by involving the ascorbate-glutathione cycle.

468

The interconnections between these major redox regulating systems are probably

469

physiologically important.

470

Collectively, our study revealed the existence of a complex antioxidative response

471

to selenite toxicity at both the gene expressions and enzyme levels, showing how peanut

472

seedlings survive when growth on low concentrations of sodium selenite (3 and 6 mg/L).

473

Several lines of evidence indicate that selenite caused small oxidant stress and

474

metabolic adjustments in the peanut seedling hypocotyl. As summarized in Fig. 6,

475

selenite treatments up-regulated the CuZn-SOD gene expression and increased its

476

activity. Concurrently, selenite treatments resulted in a rapid accumulation of hydrogen

477

peroxide in the early stage of seedling growth, which may be a signal molecule involved

23



478

in the regulation of antioxidant enzyme gene expressions. Selenite treatments increased

479

the GSH-Px, GST, and APX enzymes activities and enhanced the concentration of

480

non-enzymatic antioxidants such as GSH and AsA probably by mediating the

481

ascorbate-glutathione cycle, thus subsequently increasing the free radical scavenging

482

activity. The increase of antioxidant capacity suppressed superoxide accumulation and

483

preserved metabolic homeostasis in peanut seedlings when developed with selenite. The

484

alleviation of the selenite-induced toxicity could be explained by the increase of DPPH

485

and ABTS scavenging activity, and the decrease in H2O2 accumulation and MDA

486

formation in peanut seedlings at the later period of development. Hence we

487

hypothesized that peanut seedling can attenuate the selenite-induced toxicity by the

488

cellular homeostasis at the low concentrations of selenite treatment, which may involve

489

in the regulation of the antioxidant enzyme gene expressions and their enzyme activities

490

and the mediation of ascorbate-glutathione cycle. Efforts to mitigate the detrimental

491

effects of selenite-induced oxidative stress in seedlings may ultimately improve

492

selenium tolerance and accumulation in peanut plant.

493

24


Page 24 of 43

Page 25 of 43


494

FUNDING SOURCES

495

This work was financially supported by the Open Project Program of Provincial Key

496

Laboratory of Green Processing Technology and Product Safety of Natural Products

497

(201304), National Natural Science Foundation of China (Grant No. 31201330), and

498

Key Science & Technology Brainstorm Project of Guangzhou (20130000202).

499 500

Notes

501

The authors declare no competing financial interest.

502

25



Page 26 of 43

503

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504

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33



658

Figure legends

659 660

Fig. 1. A photograph representing the results of electrophoresis. (A) Total mRNA

661

isolated from hypocotyl of germinating peanut seedling from day 2 through 10 of our

662

experiment. The quality was checked by agarose-formaldehyde gel electrophoresis. (B)

663

The PCR products from gene-specific primers that were confirmed by 2% agarose gel

664

electrophoresis.

665 666

Fig. 2. Peanut-seedling morphology; H2O2 and MDA content in peanut seedlings

667

over time (days) of growth on selenite; and a detoxification pathway for selenite in

668

plants. (A-B) Examples of the morphology of peanut seedlings are shown on day 3 and

669

day 7 during growth on high concentrations of selenite (0, 12, and 24 mg/L). (C-D)

670

Examples of the morphology of peanut seedlings are shown on day 2 and day 6 during

671

growth on low concentrations of selenite (0, 3, and 6 mg/L). (E) Selenite-induced H2O2

672

generation, and (F) MDA formation induced by selenite in peanut seedlings from day 2

673

to 10. * indicates P < 0.05, ** indicates P < 0.01 compared with control at a given time.

674

(G) One of the detoxification mechanisms of selenite in plants.

675 676

Fig. 3. Effect of selenite on the mRNA expression profiles of antioxidant genes in

677

germinating peanut seedlings over time (days). Selenite treatments (3 and 6 mg/L)

678

influenced (A) CuZn-SOD, (B) APX, (C) GSH-Px, and (D) GST mRNA expression in

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679

germinating peanut seedlings over a 10-day period. * indicates P < 0.05, ** indicates P

680

< 0.01 compared with control at a given time.

681 682

Fig. 4. Effects of selenite on antioxidant enzyme activities and free radical

683

scavenging rates in germinating peanut seedlings over time (days). We measured

684

how selenite (3 and 6 mg/L) influenced (A) CuZn-SOD, (B) POD, (C) GSH-Px, and (D)

685

GST activity in germinating peanut seedlings over 10 days. The total antioxidant

686

capacity of germinating peanut seedlings was quantified through the (E) DPPH and (F)

687

ABTS free radical scavenging rates. * indicates P < 0.05, ** indicates P < 0.01

688

compared with control at a given time.

689 690

Fig. 5. Effects of selenite on the ascorbate-glutathione cycle in germinating peanut

691

seedlings over time (days). Effects of selenite (3 and 6 mg/L) on (A) GSH content, (B)

692

GR activity, (C) AsA content, (D) AsA/DHA rate, (E) DHA content, and (F) APX

693

activity of peanut seedlings during development. * indicates P < 0.05, ** indicates P

Germinating Peanut (Arachis hypogaea L.) Seedlings Attenuated

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