Biogenic synthesis and spatial distribution of silver nanoparticles in the legume mungbean plant (Vigna radiata L.)

Accepted Manuscript Biogenic synthesis and spatial distribution of silver nanoparticles in the legume mung bean plant (Vigna radiata L.) Rima Kumari, Jay Shankar Singh, Devendra Pratap Singh PII:

S0981-9428(16)30222-4

DOI:

10.1016/j.plaphy.2016.06.001

Reference:

PLAPHY 4572

To appear in:

Plant Physiology and Biochemistry

Received Date: 9 March 2016 Revised Date:

30 May 2016

Accepted Date: 1 June 2016

Please cite this article as: R. Kumari, J.S. Singh, D.P. Singh, Biogenic synthesis and spatial distribution of silver nanoparticles in the legume mung bean plant (Vigna radiata L.), Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Biogenic synthesis and spatial distribution of silver nanoparticles in the legume mungbean

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plant (Vigna radiata L.)

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Rima Kumari1, Jay Shankar Singh2, Devendra Pratap Singh1*

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226025, India

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Department of Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow-

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Lucknow-226025, India

Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar University,

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*Corresponding author. E-mail address: [email protected] (D.P. Singh)

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Abstract

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The present investigation aimed to study the in vivo synthesis of silver nanoparticles (AgNPs) in

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the legume Vigna radiata. The level of plant metabolites such as total phenolics, lipid, terpenoids,

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alkaloids and amino acid increased by 65%, 133%, 19%, 67% and 35%, respectively, in AgNO3

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(100 mg L-1) treated plants compared to control. Whereas protein and sugar contents in the treated 1

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plants were reduced by 38% and 27%, respectively. FTIR analysis of AgNO3 ( 20-100 mg L-1)

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treated plants exhibited changes in the IR regions between 3297-3363 cm-1, 1635-1619 cm-1,

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1249-1266 cm-1 and that corresponded to alterations in O-H groups of carbohydrates, O-H and N-

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H groups of amide I and II regions of protein, when compared with the control. Transmission

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electron micrographs showed the spatial distribution of AgNPs in the chloroplast, cytoplasmic

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spaces, vacuolar and nucleolar plant regions. Metal quantification in different tissues of plants

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exposed to 20-100 mg L-1 AgNO3 showed about a 22 fold accumulation of Ag in roots as

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compared to shoots. The phytotoxic parameters such as percent seed germination and shoot

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elongation remained almost unaltered at low AgNO3 doses (20-50 mgL-1). However, at higher

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levels of exposure (100 mg L-1), the percent seed germination as well as root and shoot elongation

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exhibited concentration dependent decline. In conclusion, synthesis of AgNPs in V. radiata

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particularly at lower doses of AgNO3, could be used as a sustainable and environmentally safe

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technology for large scale production of metal nanoparticles.

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Keywords: Silver nanoparticles, Vigna radiata, Plant metabolites, Spatial metal distribution,

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Phytotoxicity

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1. Introduction

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Nanotechnology is an important field of modern research dealing with the synthesis,

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applications and manipulations of nanoparticles. Metallic nanoparticles are known to have

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multiple applications such as targeted drug delivery and safety control, cancer therapy, bio-

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sensing and antimicrobial applications (Salata, 2004). Silver nanoparticles are of great scientific

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interest because of their unique optical, electromagnetic and physicochemical properties

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(Firdhouse and Lalitha, 2015). The wide range of AgNPs applications include the production of

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antimicrobial products, biosensors, composite fibers, cryogenic superconductors, cosmetics and

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many electronic gadgets (Korbekandi and Iravani, 2012). Due to their anti-microbial

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characteristics, AgNPs have been widely used in the field of agriculture and production of

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miscellaneous industrial products (Chen and Schluesener, 2008). An increasing demand for

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AgNPs due to their vast applications, there is need for large scale production of AgNPs by using

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low cost and environmentally safe technology. Recently, efforts are being made to develop a

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synthesis that is economical and environmentally friendly for the production of AgNPs through

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biogenic methods. This bio-mimetic approach utilizes microorganisms like bacteria, yeast and

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fungi (Salunke et al., 2011; Kowshik et al., 2003; Li et al., 2012) as well as plants (Makarov et al.,

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2014). Among all the bioresources, the plant-mediated synthesis of silver nanoparticles is being

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preferred over other methodologies due to its low cost and surplus availability of resources which

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can successfully meet the current market demand. It is also a green technology for the rapid

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production of silver nanoparticles that does not need any special, complex and multi-step

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procedure such as isolation, purification and culture preparation or culture maintenance (Iravani,

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2011).

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The biogenic synthesis of nanoparticles in living plants is an emerging field of

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nanotechnology, where the natural ability of plants to accumulate metals and transform them into

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desired nanocrystals can be easily exploited (Iravani, 2011). Gardea-Torresdey et al. (2002), for

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the first time, reported the in vivo synthesis of nanoparticles in Medicago sativa (alfalfa) and

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suggested that the plants have the ability to synthesize metal nanoparticles of different shapes and

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sizes. Shah et al. (2015) suggested that an interaction between the plant metabolites and metal

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ions was responsible for the synthesis of stable metal nanoparticles. However, any in vivo

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synthesis of metal nanoparticles also necessitates a complete knowledge about the phytotoxic

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response of the selected plants against metal toxicity.

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Plants can be used as a “bio-factory” for in vivo synthesis of metallic nanoparticles with no

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additional maintenance cost (Rupiasih et al., 2013). However, the in vivo synthesis of

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nanoparticles by plants has added an advantage over the in vitro processes as it can also prevent

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the spillover of toxic metals in the environment. There are limited references available on the in 3

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vivo synthesis and fate of silver nanoparticles by living plants (Marchiol et al., 2014), but there are

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no reports related to the in vivo synthesis of AgNPs by the leguminous crop plant V. radiata. The

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present investigation aimed to study the in vivo synthesis of AgNPs by the legume crop V. radiata

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and the role of plant metabolites. In addition, the phytotoxic effect of AgNPs on legume plant V.

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radiata was also evaluated including the spatial distribution of silver nanoparticles in different

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plant tissues.

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Materials and Methods

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2.1 Experimental plant

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Vigna radiata L. is commonly known as green gram or mung bean and it is a major edible

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legume crop in Asia and Southern Europe. It is a rich source of minerals, vitamins and proteins

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(Deshpande, 1992). Seeds of the mung bean (V. radiata L.) [Wilczek] was purchased from Kisan

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Seeds Limi ted, Lucknow.

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2.2 Chemicals

Silver as AgNO3 was obtained from Labline traders, Lucknow, India. Different dilutions

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of AgNO3 suspensions (0-100 mg L-1) were prepared in deionized water.

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2.3 AgNO3 treatment to plant seedlings

Seeds of mung bean (V. radiata) were pretreated with 10% (v/v) sodium hypochlorite

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solution for 10 min. and then rinsed thoroughly with distill water to remove dust particles. Seeds

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were used directly afterwards. Different concentrations of AgNO3 (0, 20, 50, 100 mg L-1) were

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prepared in distilled water. The viable seeds were first pretreated with AgNO3 solution for at least

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4h. Ten seeds were kept for germination in Petri dishes containing water soaked filter paper on

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bottom) and then 1.0 mL of different concentrations of AgNO3 suspensions were added for

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treatments. Distilled water was used in control. Moisture in the petri dishes was maintained by

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adding distilled water as required. After germination in Petri-plates, seedlings were transferred in

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soil pot culture.

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2.4 Plant metabolites assay

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Plant metabolites i.e., carbohydrate, protein and amino acid, alkaloids, saponins, steroids,

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tannins, total phenolics, flavonoids, glycosides and tri-terpenoids were analyzed in the control as

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well as AgNO3 treated plants analyzed by following standard biochemical methodologies

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(Horbone, 1984; Kokate, 1995).

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2.4.1 Phytochemical screening

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below:

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Test for carbohydrates

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Phytochemical screening of active metabolites in V. radiata was carried out as given

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In 2.0 mL of aqueous plant extracts, few drops of Molisch’s reagent and 1.0 mL conc.

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H2SO4 were added slowly along the side of the test tube. The appearance of red ring at the

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junction of two layers indicated the presence of carbohydrate.

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Test for protein and amino acids

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A freshly prepared solution of 0.2% (w/v) ninhydrin reagent was added to 3.0 mL of

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aqueous plant extract and mixture was heated to boiling temperature. The appearance of pink or

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purple colour indicated the presence of proteins, peptides or amino acids.

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Test for alkaloids (Mayer’s test)

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About 0.2 g fresh leaf sample was boiled with 5 mL of 2% hydrochloric acid on a water

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bath for 5 min. In 3.0 mL of filtrate, 2-3 drops of Mayer’s (potassium mercuric iodide solution)

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reagent was added. A creamy white colored precipitate after adding Mayer’s reagent indicated

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the presence of alkaloid.

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Test for saponin (Foam test)

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0.5 mL of plant extract was diluted with 100 ml of distilled water and shaken well for 15

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min. Formation of foam or no foam was considered indicator for the presence and absence of

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saponins, respectively.

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Test for tannins

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3 mL of aqueous plant extract was mixed with 5-6 drops of 1% (w/v) solution of gelatin

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containing 10% of sodium chloride. Formation of white precipitates indicated the presence of

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tannins.

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Test for phenols

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In 3.0 mL of aqueous plant extract, few drops of neutral 5% ferric chloride solution were added. A dark green colour indicated the presence of phenolic compounds.

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Test for flavanoids

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In 5.0 mL of aqueous plant extract, 2.0 mL of the 10% sodium hydroxide solution was

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added to produce a yellow colouration. A change in colour from yellow to colourless after adding

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few drops of dilute hydrochloric acid was an indication for the presence of flavonoids (Trease and

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Evans, 2002).

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Test for anthocyanin

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3.0 mL of methanolic plant extract was mixed with 5.0 mL of 1 N sodium hydroxide. Solution color changed to blue that indicates the presence of anthocyanin.

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Test for glycosides

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Keller Killiani test for glycosides was carried out using 3.0 mL of plant extracts treated

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with 2.0 mL of glacial acetic acid containing one drop of ferric chloride solution. This was

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followed by addition of 1.0 mL of concentrated sulphuric acid. Formation of two distinctly

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coloured layers, comprised of lower reddish brown layer and upper bluish green layer, indicated a

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positive test for glycosides.

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Test for Phytosterols

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Salkowski’s test: Plant extract mixed with 10 mL of chloroform was filtered and was

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supplemented with 5-6 drops of concentrated H2SO4. The mixture was shaken gently until it

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turned golden red in colour, which was indicative of positive test for the presence of phytosterol.

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Test for terpenoids

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Liebermann – Burchard test: 3.0 mL of plant extract was mixed with 3.0 mL of chloroform, 1.0

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mL acetic anhydride and few drops of H2SO4. The mixture turned dark green in colour, which

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indicated the presence of terpenoids.

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2.4.2 Quantitative determination of plant metabolites

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Quantitative assay of plant metabolites in control and AgNO3 treated plants were carried out spectrophotometrically by following standard methodologies as given below-

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Determination of carbohydrate content

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The total sugar content was determined by the anthrone reagent method (Dreywood,

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1946). The anthrone reagent was freshly prepared by dissolving 0.2 g of anthrone (0.2%) in 100

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mL of ice cold concentrated sulfuric acid. In 1.0 mL of aqueous plant extract, 4.0 mL of anthrone

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reagent was added. The reaction mixture was kept in boiling water bath (100ºC) for 10 to 15 min.

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The colour intensity of reaction mixture was measured at wavelength 620 nm against appropriate

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blank. Glucose solution was used for preparation of standard curve. Total carbohydrate content of

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plant sample was expressed in term of µg carbohydrate mg-1 fresh weight.

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Determination of protein content

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For protein extraction, fresh leaves were homogenized in Tris buffer (0.1 M)and that was

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followed by addition of trichloro acetic acid (10%). The precipitate was collected by

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centrifugation and dissolved into 0.1 M sodium hydroxide. Protein content of the plant extract

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was determined by following the standard method of Lowry et al. (1951).

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Determination of amino acid content

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For quantitative determination of amino acids, 1.0 mL of the ninhydrin solution was added

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to 5.0 mL of plant extract. Test tubes were properly covered with a piece of paraffin film to avoid

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the loss of solvent due to evaporation. Reaction solution was heated gently at 80-100ºC for 4-7

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minutes. After cooling to room temperature, absorbance of reaction mixture was measured at 570

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nm (Yemm et al., 1955).

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Determination of total phenol and lipid content 7

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Methanolic extract of the plant (3.0 mL) was mixed with 2.5 mL of 1 N Folin- Ciocalteu

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reagent and 2.0 mL of 7.5% sodium carbonate. Phenol content in the reaction mixture was

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determined spectrophotometrically at 760 nm (Singleton et al., 1999). For quantifying the total

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phenol, known concentrations of gallic acid served as standard. Total phenolic content were

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expressed as mg g-1 fresh wt. (GAE).

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gravimetrically by following the method described by Bligh and Dyer (1959).

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Determination of alkaloids

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Total lipid content was extracted and measured

For determination of alkaloid content, 3.0 mL of aqueous plant extract was mixed with

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5.0 mL ethanol- H2SO4 (1:1) solution, followed by 5.0 mL of 60% tetraoxosulphate and 5.0 mL of

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formaldehyde. Absorbance of the reaction mixture was measured at 565 nm (Harborne, 1976).

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Determination of total terpenoids

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The total terpenoid content was also determined by spectrophotometric method by

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following standard procedure (He et al., 2007). The plant extract (3.0 mL) was mixed with 0.3 mL

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of 5% glacial acetic acid added to 1.0 mL perchloric acid solution and mixture was heated for 15

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min at 60 ºC. After cooling, the mixture was again supplemented with 5.0 mL glacial acetic acid

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and absorbance of the solution was measured at 542 nm.

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2.5 Fourier transform infrared (FTIR) analysis

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Fourier transform infrared (FTIR) analysis of control and AgNO3 treated plant sample was

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performed by KBr pellets methods operated on FTIR spectrophotometer (FTIR; Nicole 6700,

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Thermo Scientific, USA) to investigate the treatment induced changes in functional groups of

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plant biomolecules (lipids, proteins, carbohydrates, lignin etc.,) in V. radiata and overview into

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interaction of possible binding sites with silver nanoparticles. For FTIR analysis, plant samples

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were washed and rinsed with Millipore water, and oven dried at 70°C for at least 72 h. Plant

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samples were grounded with potassium bromide (KBr) in the ratio of 100:1 (100 mg KBr + 1 mg

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dried plant sample) in an agate motor to make the pellets by using a hydraulic press (CAP-15T) at

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a pressure of 10 tons. Powdered samples were placed on the spectrophotometer and IR spectra

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were recorded in a frequency range from 4000 to 400 cm-1 and at a resolution of 1.0 cm-1. Three

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scans were recorded for each sample against KBr background, using the OMNIC software. All

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spectra were smoothened using the standard ‘‘automatic smooth’’ function of the above software,

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and then the baseline correction was done using the ‘‘automatic baseline correction’’ function.

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2.6 Transmission electron microscopy (TEM) analysis

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Transmission electron microscopy (TEM) analysis was used for in vivo localization of

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AgNP in plant parts. Spatial localization of AgNPs in plant and induced structural modifications/

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damage in the ultra-structure of plant was also examined by TEM following a well-established

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method (Bozzola and Russell, 1999). Fresh samples of the plant were collected from the tip region

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of roots and fully expanded leaves after 4 days exposure of 100 mg L-1 AgNO3 treatment. Plant

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samples were excised, sectioned (2 × 3 mm) and fixed for 2 h at 4°C in 0.1% (w/v) sodium

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phosphate buffer. Thereafter, the samples were prefixed in, 2% glutaraldehyde at pH 7.2 and left

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overnight at room temperature. Then the samples were post- fixed with 1% osmium tetroxide in

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50 mM phosphate buffer for 40 min and were washed twice with 50 mM phosphate buffer. The

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plant samples were dehydrated with ethanol series and embedded in epoxy resin. Ultrathin

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sections from each sample were mounted on Cu grids after staining with uranyl acetate and lead

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citrate and the samples were then observed under Tecnai G2 sprit Twin transmission electron

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microscope equipped with CCD Camera, (The Netherlands) operating at 80 kV. More than 10

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sections cut from different roots and leaf samples were examined.

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2.7 Analysis of silver (Ag) concentration in plant

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Atomic absorption spectroscopic (AAS) analysis was conducted to measure the silver

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content accumulated by plants treated with different concentrations of AgNO3 (0, 20, 50, 100 mg

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L-1) for 1-2 weeks. Silver content analysis of the acid digested samples was carried by using

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Flame- Atomic Absorption Spectrometer (Zeenit 700, Analytik Jena, Germany). For Ag analysis,

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plant samples were dried overnight at 110ºC, ground, sieved and digested in HNO3 in

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a microwave digestion system (Buchmann et al., 2000). The digested samples were filtered 9

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through Whatman® paper before analysis. Blank sample was processed in the similar manner

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and analyzed. NIST traceable 'certipur' certified reference Material from Merck, KGaA, Germany

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was used for calibration of the instrument and standards were run after every five samples for

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stability of the measurements.

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2.8 AgNO3 toxicity on seed germination and plant growth traits

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After 4 days of AgNO3 treatment, the percentage seed germination was recorded in

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untreated (control) as well as AgNO3 treated plants. After germination, seedlings were planted in

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pots having soil-sand mixture. Different concentrations of AgNO3 (20, 50, 100 mg L-1) was

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prepared in double distilled water for treatments. The soil (1.0 kg) was treated with 10 mL of

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varying concentrations of AgNO3 solution by irrigation on every alternate day. Moisture in the pot

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culture was maintained by water as and when required. All the experiments were carried out in

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triplicate under field conditions. Root and shoot lengths of control as well treated plants were

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measured for two weeks.

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2.9 Statistical analyses

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The statistical evaluation was performed using SPSS 13.0 (SPSS Inc., USA). All the data were

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presented as mean ± SD (standard deviation) based on at least three replicates per treatment in

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multiple experiments. Significance of difference was calculated on one-way ANOVA.

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3.

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3.1 Root uptake of AgNO3 and its translocation in different plant parts

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Results

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Plants take up the silver metal (Ag+) from the soil (1.0 Kg) spiked with 10 mL AgNO3

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solution of varying concentrations (20-100 mg L-1). Results (Figure 1) showed that Ag content in

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plants increased significantly with increasing concentration of AgNO3 and exposure time. After

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14 days exposure, total Ag content in root and shoot tissues registered three and two fold increase,

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respectively, at 100 mg L-1 concentration of AgNO3 when compared with the results obtained at

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20 mgL-1 concentration of AgNO3.

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The translocation ratio of Ag i.e., Ag content in shoot/ Ag content in root was very low

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(0.022-0.35), suggesting the maximum accumulation of Ag (more than 90%) in roots, whereas

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translocation of Ag from root to shoot part was very low i.e., even lesser then 10% of the total Ag

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taken up by the plant.

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3.2 TEM analysis of Vigna radiata

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TEM analysis was used to visualize the presence of silver nanoparticles inside the V.

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radiata plant. These nanoparticles were predominantly found in the sub-cellular compartments of

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the plant as electron dense rounded structures with approximate radius of 10-35 nm in size (Figure

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2). These nanospots were subsequently identified as silver using EDX (EDX data is not shown).

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AgNPs in the plant cells were found to have different shapes and size. They were observed either

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as discrete small particles or aggregated large particles.

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TEM analysis of different plant parts was carried out to study the bioaccumulation and

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spatial localization of AgNPs within the cells. The results indicated that Ag nanoparticles inside

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the plants were accumulated in certain specific zones and were found to be mostly associated with

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the cell walls, chloroplasts, plasmalemma, cytoplasmic vacuoles and nuclear region (Figure 4,

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arrows), indicating specific sites of bioaccumulation and translocation route. Interestingly, most

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of the AgNPs were accumulated in alignment across the plasma membrane. Thus, the present

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results indicated preferential association of the AgNPs at the membrane level.

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Ultra-structural studies carried out by TEM also showed modifications/ damage to the

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structural integrity of root and leaf i.e., compressed cellular integrity and shrinkage, structural

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distortion and disorganization in chloroplast structure (Figure 3).

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3.4 FTIR analysis

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Fourier Transform Infrared Spectroscopy (FTIR) measurement was performed to identify

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the possible interaction of biomolecules with silver metal within the plant tissues. The results

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showed shift in certain characteristic IR absorbance peaks as well as emergence of few new IR

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absorbance peaks due to interaction of biomolecules with silver metal (Figure 5). As shown in 11

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Table 1, a major IR peak (3297 cm-1), mainly associated with conformational changes in O-H, C-

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H, and –NH- (amide group) of protein, shifted towards higher frequency (3363 cm-1) by 58-66

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cm-1. There was no significant change in IR peaks associated with lipids (at 2929 cm-1) due to

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interaction with silver ions. Due to interaction with Ag metal, there was emergence of new IR

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peaks at 1544 and 1547 cm-1, attributed to O-H and N-H groups of Amide II region of the

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proteins. The results demonstrated a change in the conformational structure of the protein due to

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its interaction with silver metal. The change in the IR peak at 1635 cm-1 was also indicative of

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changes in Amide I region of the proteins. A vibrational shift in the IR absorbance peak at 1249

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cm-1 (from 1249 to 1266 cm-1) after silver treatment of the tissues indicated alterations in C=O

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functional group of the oligo and polysaccharides, carboxylic groups, O-H stretching, NH bending

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(amide III). The changes in the IR peaks (1068 and 1154 cm-1), associated with carbohydrates

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were indicative of C-O stretching, symmetric stretching of phosphodiester group in the nucleic

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acid and glycogen. Presence of new IR peaks in silver treated plants observed at 826 -832 cm-1,

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assigned to phosphate and hydrogen bonds of nucleic acid regions, suggested changes in the

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characteristic functional groups. These findings suggested that many biomolecules, particularly

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proteins and carbohydrates, and their function groups might be associated with preferential

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binding and reduction of silver metal in plants.

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3.5 Plant metabolites

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The qualitative screening of plant compounds showed that the plant V. radiata was rich in

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carbohydrates, phenols, flavonoids, alkaloids, terpenoids, lignins, anthocyanins, glycosides,

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alkaloids (Table 2). The AgNO3 treated plant showed variation in the metabolites when compared

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with untreated control plant. The results showed that silver nitrate treatment of plant exhibited

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increase in the phenols (65%), lipid (133%), terpenoids (19%), alkaloids (67%) and amino acids

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(35%) over that of the control plant. Whereas protein and sugar contents in the treated plant were

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decreased (27 and 28%, respectively) when compared with the control. One way analysis results

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(ANOVA) also indicated the significant variations among treatments.

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3.6 Phytotoxicity response of Vigna radiata against silver nitrate (AgNO3) Effect of different concentrations of AgNO3 (0-100 mg L-1) was tested on the seed

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germination, root/ shoot elongations in case of V. radiata with an aim to establish the phytotoxic

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concentration of silver. It was observed that seed germination remained unaffected at lower

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concentrations (20-50 mg L-1) of AgNO3, but the seed germination at higher concentration (100

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mg L-1) of AgNO3 decreased by 20±3% (Figure 6). Similar trend was observed for shoot length,

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exhibiting about 40% decrease in the shoot length at higher dose (100 mg L-1) of AgNO3 after 14

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days of germination (DAG). On the contrary, root length elongation showed 42% increase in root

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length at 50 mg L-1 concentration of AgNO3 (Figure 7). The stimulatory response of AgNO3 on

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root elongation at particular dose may be attributed to hormesis (stimulatory effect at lower doses

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of toxicants). But at 100 mg L-1 of AgNO3, the root length also showed declining pattern similar

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to shoot length. The overall results indicated that 100 mg L-1 concentration of AgNO3 is toxic to

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V. radiata plant.

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4.

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Discussion

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Preliminary observations show a quick absorption and/or adsorption of AgNO3 by root tissues

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that result into fast progressive browning of roots. Despite the short term exposure, the silver

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(Ag+) is taken up quickly by the roots and is subsequently transported to other parts of plant in a

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time dependent manner (Haverkamp and Marshall, 2009). The present results exhibiting a low

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translocation factor (TF) for (0.022-0.35) also suggests that major fraction of Ag ions is retained

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by the roots and a limited apoplastic translocation, depending upon the time factor, is considered a

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possible route for upward translocation of silver to shoot region. Yin et al. (2011) have also

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suggested that major portion of Ag is retained by the roots and a small fraction of total Ag is

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translocated from root to shoot. Further, ionic form of silver reaching to different plant parts is

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converted to silver nanoparticles through bioreduction of silver ions by plant metabolites

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(Makarov et al., 2014; Aromal and Philip, 2012). The present results on quantitative screening of

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metabolites in AgNO3 treated plants show substantial increase in the amino acid, lipids, phenols,

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terpenoids and alkaloids except sugar and protein contents. The changes in the level of plant

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metabolites due to interaction with silver metal suggest their possible involvement in the process

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of in vivo synthesis of nanoparticles as suggested by Prasad (2004) and Marchiol (2012). The

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reduced level of protein and sugar content after silver treatment may be due to their role in

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reduction of silver metal and formation of silver nanoparticles. Earlier Saxena et al. (2010) have

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also suggested that amine groups of proteins and reducing sugars play important role in the

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reduction of silver ions into silver nanoparticles. The results on FTIR spectra of AgNO3 treated

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tissues also corroborate the observation on the involvement of proteins and sugars in interaction

343

with silver metals as evident from very strong absorption band around 3297-3363cm–1,

344

corresponding to N–H, C–H, O–H stretching of amines and amides of proteins (Mc Cann et al.,

345

1997; Stuart, 2004; Movasaghi et al., 2008). The dominant bands at 1635 cm–1 and 1547 cm–1,

346

attributed to amide І and ІІ groups of proteins, are also altered due to interaction with silver metal

347

(Ducor, 1992). The IR peaks at 1062 and 1077 cm–1, assigned to the vibration modes of CH2OH

348

and stretching/ bending of the C-O functional groups associated with carbohydrates (Yang et al.,

349

1995). The IR absorption peaks positioned at 777 and 826-832 cm-1 in silver treated plants are

350

reported to be specifically assigned to polysaccharides, particularly the peak at 832 cm–1, belongs

351

to the characteristic peak of α-glucose (Chen et al., 2001).

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Results on TEM analysis clearly demonstrate the presence of AgNPs of different sizes and

353

shapes, which ultimately result into formation of particle aggregates in different cellular

354

compartment. The findings of Corredor et al. (2009) have indicated that endocytosis is apparently

355

a reasonable way for internalization of the nanoparticles and their accumulation in clusters inside

356

the cells. Plants response to the presence of high density silver nanoparticles can be seen in terms

357

of distortion in structural integrity of sub-cellular organelles such as thylakoids, membranous

358

structures and chloroplast (Marchiol et al., 2014). The main evidence of AgNP induced structural

359

damage is observed in terms of detached plasma membrane from cell wall, collapse of vacuoles

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360

and changes in choloroplast structure accompanied by rupture of membrane, disorganization of

361

the lamellae and granal stacks. Application of plants as bio-factory for in vivo synthesis of AgNPs also necessitates the

363

knowledge about the plant response to silver toxicity. Thus, the present investigation also includes

364

the study on phyto-toxicity response of plants against AgNO3, which is measured in terms of seed

365

germination potency and root/shoot elongation. The results have shown that lower doses of

366

AgNO3 (20-50 mg L-1) were not inhibitory to root/shoot elongation or seed germination.

367

However, an increase in the root elongation at lower doses of AgNO3 may be attributed to

368

hormesis, a dose dependent response, wherein low doses of toxicants activate the metabolic

369

system as a part of defense strategies to compensate the adverse effect of toxicant and act

370

positively (Mattson, 2008). However, 100 mg L-1 concentration of AgNO3 is able to elicit a

371

phytotoxic response in V. radiata. It is therefore suggested that use of lower doses of AgNO3 is

372

safer for biogenic synthesis of AgNPs in V. radiata plant.

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5.

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Conclusion

Results suggest that V. radiata have ability to take up the silver ions from soil spiked with

375

AgNO3 solution through root absorption and transport them to above ground plant parts.

376

However, more than 90% silver metal remains associated with the roots. It was also observed

377

that V. radiata is sensitive to silver metal stress only at higher concentrations of AgNO3. We also

378

propose that silver is accumulated in plant parts mostly as metal nanoparticles. Study clearly

379

suggest that AgNO3 treated plants use their intracellular biochemical constituents to transform the

380

silver ions into silver nanoparticles (AgNPs). These results provide a clue for future application of

381

plants, particularly crop plants like V. radiata (mung bean) with short span of life cycle, for

382

phytomining of silver contaminated sites as well as in vivo synthesis of metal nanoparticles. The

383

in vivo synthesis of AgNPs by crop plants may be used as cost-effective and eco-friendly

384

technology.

385

Acknowledgements

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The author Rima Kumari acknowledges thanks to Department of Science and Technology

387

(DST), New Delhi for providing DST- Start Up Young Scientist research grant on the project

388

entitled “Assessing the impact of silver nanoparticles on crop plants V. radiata and Fagopyrum

389

esculentum: Morphological, Biochemical, Genotoxic and Proteomics aspects, (Project F.No.

390

SB/YS/LS-231/2013). We also acknowledge the Head, Department of Environmental Science and

391

Director, USIC, BBAU, Lucknow, for providing SEM, FTIR and IITR for providing TEM and

392

AAS facilities. Thanks due to Prof. S.P. Singh, Dept. of Botany, Banaras Hindu University for

393

improving the language quality of the article.

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Legend to figures

512

Figure 1 Dose (0, 20, 50 and 100 mg L-1 AgNO3) and time dependent (7 and 14 days) response of

513

AgNO3 treatment on Total Ag concentration in plant root and shoot parts.

514

Figure 2: Transmission electron microscopic (TEM) image of morphology of silver nanoparticles

515

(AgNP).

516

Figure 3 Ultra-structural changes in plant cells under treatment of AgNO3 in respective to control

517

plants. Accumulation of silver nanoparticles (AgNP) as well as distortion in cellular integrity and

518

chloroplast structure can be seen in the treated root and shoot cells.

519

Figure 4 Bioaccumulation and spatial localization of silver nanoparticles (AgNPs) under

520

treatments of AgNO3. AgNPs can be seen in the root (A-D) and shoot cells (E- I). PC- Plant cell;

521

N-Nuclear space; Chl.- Chloroplast; Cyt.- cytoplasm; V- vacuolar region.

522

Figure 5 FTIR spectra of untreated (control) and AgNO3 treated plants of Vigna radiata

523

(Control= 0; T1 =.20; T2 = 50; T3 =100 mg L-1 AgNO3).

524

Figure 6 Effect of different concentrations of AgNO3 (0, 20, 50, 100 mg L-1) on seed germination

525

% of Vigna radiata.

526

Figure 7 Response of different concentrations of AgNO3 (0, 20, 50, 100 mg L-1) treatments on

527

root and shoot length in Vigna radiata at 7 and 14 DAG (days after germination).

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Table 1: FTIR peaks and corresponding functional groups in control and AgNO3 treated plants of Vigna radiata. Serial

FTIR peaks in different treatments (cm-1)

Number

C

T1

T2

T3

groups

3297

3355

3363

3297

O-H, N-H, C-H, Amid A (N-H stretching)

2

2929

2926

2927

2926

1635

1622

1619

1628

4

-

1544

-

1547

-CH2

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(methylene

asymmatric

1428

-

-

6

1385

1384

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-

1383

1383

Paluszkiewicz,

and

Kwiatek,

2001; Dovbeshko et al., 2002; Movasaghi et al., 2008 strech), Dovbeshko et al., 1997; HerediaGuerrero et al., 2014

NH2 bending (Amide I), C=O (ester)

Dukor, 2002; Stuart, 2004;

N–H bending & deformation (Amide II), Stuart, 2004; Garidel and Schott, C=N stretching

5

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aliphatic C–H stretch

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Assigned functional

2006; Mazurek et al., 2013

Deformation and bending of CH2 and CH3 Rehman et al., 2012 group COO- anti-symmetric stretching, deformation of C-H, N-H

Movasaghi et al., 2008

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1249

1260

1266

-

P=O streching of phopholipid, Carbxylic O-

Garidel

H streching, NH bending (amide III), CH2

Rehman et al., 2012

bending 8

1154

11450

1150

1152

C-O stretching

9

1068

1075

1077

1062

CHO

832

-

-

830

-

617

606

603

13

-

536

-

467

470

14

-

777

and

-

CH bending (out of plane), NH2 wagging

-

C represents control, T1= 20 mg L-1 AgNO3, T2 = 50 mg L-1 AgNO3, T3=100 mg L-1 AgNO3

2006;

symmetric Yang et al., 1995

CH bending (out of plane), NH2 wagging

P=S stretching, P–Cl stretching

Schott,

Movasaghi et al., 2008

Stuart, 2004; Movasaghi et al., 2008 Stuart, 2004; Movasaghi et al., 2008

623

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ACCEPTED MANUSCRIPT Table 2: Preliminary phytochemical screening of the plant extract of Vigna radiata. Test

Results

Molisch's reagent

++

2

Phenol

FeCl3 test

+++

3

Protein and amino acid

Ninhydrin test

++

5

Flavonoids

NaOH test

6

Glycosides

Keller Killiani Test, Bromine water test

++

7

Anthocyanin

NaOH test

+

8

Alkaloids

Mayer’s test & Dradenorff’s test

++

9

Sterols and terpenoids

Test with Chloroform + H2SO4

+

10

Tannin

Gelatin test

+

11

Saponin

Foam test

-

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+++

The notations (+++) refer the prominent presence (positive test within 5 min); (++) moderate amounts (positive at 5- 10 min); (+) trace amounts (late positive reaction after 10 min), (-) refer

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Parameter

Control

Total sugar

T1

T2

T3

58.94±2.71a

48.21± 1.8b

46.34± 2.6b

43.12± 1.2c

55.36± 1.7a

42.17± 1.2ab

38.23± 1.1b

38.21± 2.3b

30.25± 1.7a

29.87± 1.5a

37.32± 2.3b

40.74± 1.8c

1.08± 0.1c

1.67± 0.1b

1.42± 0.09c

2.52± 0.07a

98.73± 3.7c

123.4± 7.5b

117.8± 4.2bc

163.5± 12.7a

27.23± 1.3a

31.34± 0.9a

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control at 10 days of germination (DAG)

32.43± 1.2a

30.26± 0.8a

21.71± 1.3ab

24.76± 1.0a

Protein (mg/100 mg D.W) Total essential amino acid (mg/100 mg D.W) Total lipid content (mg/100 mg D.W) Total phenolic content

Terpenoids (mg/100 mg D.W)

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14.83 ± 1.21c

Alkaloids (mg/100 mg D.W)

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(mg/100 mg D.W)

16.29± 0.84b

Abbreviations: C represent control; T1- 20 mg L-1, T2 50 mg L-1, T3 100 mg L-1 AgNO3 suspension. Mean values followed by the different letter within same group indicate statistically

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ACCEPTED MANUSCRIPT Highlights

Phytoremediation of silver in Vigna radiata has been described.

•

Plant resources may be used for phytomining of toxic metals.

•

Plant metabolites contribute in synthesis of silver nanoparticles.

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V. radiata metabolic response indicates silver metal sensitivity.

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ACCEPTED MANUSCRIPT Author’s contribution: All Authors have been actively participated in terms of planning, experimental designing, interpretation and compilations of experimental data. i)

RK: contributed in designing and planning experiments, analysis and interpretation of

ii) iii)

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results. JSS: Describes the role of nano-materials in influencing the physiology and biochemistry of plants. DPS: Contributed significantly in designing and planning of experiments, conceptual

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suggestions and providing his opinion at different level of writing the article.

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