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Carbon-bound iron oxide nanoparticles prevent calcium induced iron deficiency in Oryza sativa L. Abin Sebastian, Ashwini Nangia, and M. N. V. Prasad J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04634 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 6, 2017

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Journal of Agricultural and Food Chemistry

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Carbon-bound iron oxide nanoparticles prevent calcium induced iron deficiency in Oryza

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sativa L.

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Abin Sebastian †*, Ashwini Nangia †, M.N.V. Prasad ‡

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†

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Hyderabad, 500046, India.

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‡

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PO, Hyderabad, 500046, India

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*

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Abstract

School of Chemistry, University of Hyderabad, Prof.CR Rao Road, Central University PO,

Department of Plant sciences, University of Hyderabad, Prof. CR Rao Road, Central University

Correspondence e-mail: [email protected]

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Iron based nano-composites can be practical solution to combat iron deficiency in calcareous

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agricultural soil. In the present study, carbon bound iron oxide nanoparticle is synthesized by

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mixing ferric chloride and caffeic acid, and tested to correct Ca inducible Fe deficiency in rice.

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Physicochemical characterization point that nanoparticle is carbon coated semicrystalline Fe3O4.

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It is found that nanoparticle amendment enhances bioproductivity, photosynthetic electron

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transport, antioxidant enzyme activity, and Fe accumulation under Ca stress. Reduction in Ca

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accumulation via physical adsorption, Fe release from the particles, and maintenance of

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molecular responses related to Fe acquisition were the reason for above progressive growth

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effects. Thus it is concluded that nanoparticles synthesized in the study act as a potential

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ameliorant to correct Ca induced Fe deficiency in rice plants.

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ACS Paragon Plus Environment


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Keywords: iron nanoparticles, calcium stress, adsorption isotherms, type II iron uptake,

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photosynthesis

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Introduction

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Iron deficiency is common in calcareous soils. 1- 3 Excess of calcium carbonate (1-15 %) create

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Fe deficiency in these soils via precipitation of Fe in soil solution. Naturally occurring chelating

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compounds also decrease plant available Fe in calcareous soil through formation of insoluble

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complexes of Fe.4, 5 Iron deficiency disorder among plants grown in calcareous soils is known as

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lime-induced chlorosis. It is noteworthy that Fe nutrition is critical for plant growth because Fe is

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a cofactor of several metalloproteins involved in the synthesis of chlorophyll and photosynthetic

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electron transport. Iron deficiency thus causes alterations in photosynthetic functions which

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drastically decrease crop productivity6.

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Iron deficiency in calcareous soil is often ameliorated to some extent with foliar spray of Fe, and

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synthetic fertilizers.7-8 But the low pace of Fe transport in leaves, as well as, leaf burn act as a

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barrier to the foliar application of Fe salts.9 Inorganic sources of Fe such as ferrous sulfate

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(FeSO4) or ferric sulfate [Fe2(SO4)3] were proven to have the very limited effect to correct Fe

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deficiency unless applied very frequently at extremely high rates.5,

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fertilization rapidly increases Fe concentration in the rhizosphere for a short period, and the

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temporary rise of Fe would cease molecular responses related with high-affinity Fe uptake.11

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Hence it is clear that Fe supplement must be carried out through composites which have the

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ability to function as a slow releasing agent of Fe. This is because the slow release of Fe would

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not cease high-affinity Fe uptake mediated by IRT genes.

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Nanocomposites of Fe oxides can be promising soil remediation agent to combat lime-induced

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chlorosis because of long term effect ie these oxides release Fe slowly compare with Fe salts,

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and hence allow efficient uptake of Fe through high-affinity Fe uptake system. Functionalized 3


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This is because Fe


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nanoparticles also have the advantage of more surface area which can be exploited for Ca

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adsorption too. Nanoparticles treatments also found to boost plant metabolism.12, 13 Nano - Fe

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oxide is smaller than the common Fe oxides, and undergo faster mineralization compare with

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natural oxides.14 These properties of nano Fe oxides help to release ambient Fe for plant growth

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at a slower rate than common Fe salts. But the synthesis of nano-Fe oxide is laborious, and

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economically not feasible. Removal of impurities from synthetic Fe oxides is another drawback

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that limited application of nano-Fe oxides in the agricultural field. In the present study, Fe oxide

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nanoparticle synthesized from ferric chloride and caffeic acid - a naturally occurring phenolic

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acid, in water. So the synthetic approach followed in the present study is eco-friendly.

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Rice plants adapt to Fe deficiency by Type II Fe uptake strategy mediated via phytosiderophores

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together with Type I Fe uptake.15-17 Excess Ca accumulation in plants is well known to cause

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chlorosis, malfunctioning of photosynthesis, and oxidative stress. 18-22 Activities of antioxidant

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enzymes such as superoxide dismutase and catalase are critical to cope up with oxidative stress

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and these enzymes are considered as biomarker of plant stress.23 Hence above mentioned

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components are monitored in the present study to explore the effect of nanoparticle treatment in

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the prevention of Fe deficiency and Ca stress in rice plants.

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Materials and methods

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Synthesis of iron oxide nanoparticle

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Iron oxide nanoparticle was synthesized by mixing caffeic acid and anhydrous ferric chloride

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through bottom scale up approach. Deionized water was used as a medium for synthesis. Due to

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sparingly soluble nature of caffeic acid in water, the temperature of water raised with help of a

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hot plate to 85 0C, and thereafter caffeic acid was dissolved. The mixture allowed to cool down

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to 30 0C, and an equal volume of 10 mM ferric chloride dissolved in water was added and kept in

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a rotary shaker at 160 rpm for 12 hrs. Black coloration of the solution indicated the formation of

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Fe oxide particle. The particles were separated from the mixture by centrifugation at 15000 rpm

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(REMI C-24 plus). The nanoparticles obtained were washed 3 times using deionized water and

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dried in a hot air oven at 65 0C.

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Physico-chemical characterization of nanoparticles

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Nanoparticles were subjected to surface plasmon resonance analysis to reveal chemical nature of

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the particles. This method monitors resonant oscillation of conduction electrons at the interface

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between a negative and positive permittivity material stimulated by incident light. The analysis

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was carried out on nanoparticles dispersed in water in the Uv-Visible range of 190-700 nm using

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a Uv-Visible spectrophotometer (Thermo scientific, Evolution 201). Electron paramagnetic

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resonance (EPR) studies on nanoparticles were carried out in the solid state. The spectra were

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measured at 9.64 GHz microwave frequency, 0.249 mW microwave power, 100.0

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modulation frequency, 0.4 mT modulation amplitude, and 200-6200 T scanning field in an

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EPR spectrometer (Bruker-EMX, Germany).

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Powder X-ray diffraction (XRD) studies on nanoparticle were performed for the phase

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identification. Particle was exposed to monochromatized Cu Kα radiation (λ = 1.54 Å) at a

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temperature of 25 0C, and the scanning was performed in the 2ɵ angle range from 5 to 90 in an

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XRD recorder (Bruker AXS D8, Germany). Morphology of nanoparticle was visualized with

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help of atomic force microscopy (NT-MDT, USA). Particles dispersed in water (20 µl) were

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loaded into a piece of the glass slide, and made into a layer with help of cover slip. The slide was 5


KHz


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dried in a vacuum oven and thereafter analyzed in atomic force microscope using Nova PX

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software.

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Elemental analysis of the particle was done using field-emission scanning electron microscope

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(Zeiss Merlin Compact) coupled to energy dispersive spectroscopy (X-max, Oxford). The

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dehydrated powder was mounted on aluminum stubs, and the elemental analyses were carried

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out at an operating voltage of 15 keV at a working distance of 10 mm with counts per

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sec >1000. Quantitative analysis of carbon content in the particles was measured with CHNS

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analyzer (EA Flash 1112, Thermo Finnigan, USA), and the carbon content was expressed in

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percentage. Thermal stability of the particle was analyzed with differential thermal analysis

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coupled with thermo-gravimetric analysis (DTA-TGA). This analysis reveals melting point,

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phase transition, and break down of the compound in response to temperature. DTA-TGA

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analysis was carried out in the temperature range 25 to 900 0C (Mettler Toledo, USA). To reveal

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nature of functional groups bound, the particle was subjected to infra-red spectroscopy. The

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particles were made into a pellet with KBr, and subjected to the instrument (Thermo-Nicolet

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6700, USA). Transmittance was monitored in the wave number range of 500 to 3500 cm-1.

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Plant culture

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The sand culture was conducted to study the influence of nanoparticle on Ca tolerance in rice.

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Oryza sativa L. cv MTU-7029 seeds were obtained from seed research center, Professor

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Jayashankar Telangana State Agricultural University, Hyderabad, India. MTU-7029 is largely

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cultivated in calcareous soils of the semi arid Deccan plateau of India. This variety had

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synchronous tillering and resistance to bacterial leaf blight. The seeds were sterilized with 5 %

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hydrogen peroxide for 15 min and thereafter washed with double distilled water. Germination 6


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was carried out in a germination box contain wet sand. Five-day old seedlings were planted in

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acid washed sand. Hoagland nutrient media (10 ml at an interval of 3 days) was used to provide

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nutrients for plant growth throughout 21 days of growth.

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Nanoparticle and calcium treatments

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Seedlings grown in sand mixed with 10 % Hoagland solution (pH 6.5) contain 53.7 µM Fe stand

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as control for all treatments (+Fe). It must be noted that usage of diluted media at regular interval

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is preferred in sand culture to minimize fluctuations of nutrient concentration in the medium and

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in plant tissue. Seedlings grow in sand treated with 10 % Hoagland solution having 5µM Fe

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correspond plants exposed to Fe deficiency (-Fe). Ability of nanoparticle to prevent Fe

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deficiency monitored with addition of 20 mg nanoparticle to plants reared under Fe deficiency (-

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Fe+NP). Calcareous nature of sand (1%) was established by addition of calcium carbonate. This

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approach makes the growth substrate similar to calcareous soils where powdery calcium

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carbonate is mixed in the soil. Seedlings grow calcareous sand was nurtured with 10 % Hoagland

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media contain 53.7 µM Fe (+Fe+Ca) to monitor Ca inducible Fe deficiency.

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nanoparticles to prevent Ca inducible Fe deficiency was checked by amending 20 mg

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nanoparticles to calcareous sand (+Fe+Ca+NP). The pH of water extract of the sand (1:2 w/v)

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was recorded using a pH meter (Table.1). Light intensity (500 µmol photons m-2 s-1),

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photoperiod (18 hrs light / 6 hrs dark), temperature (27 0C), and relative humidity (50 + 10

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%) were maintained throughout the growth period.

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Ability of


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Biomass and metal content analysis

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The fresh weight or dry weight biomass of the sample was weighed using a weigh balance

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(Sarotius, Germany). For metal content analysis, plants were cleaned with deionized water

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followed by wash in 0.5 M EDTA, and kept in an oven at 80 0C for 12 days. Sample was

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transferred into 150 mL conical flask for acid digestion using HNO3 - H2Cl4 (3:1).The mixture

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was digested at 95 0C using hotplate under a fume hood. The dry powder obtained was dissolved

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in 0.1N HCl, and used for quantitative estimation of elements by atomic absorption spectrometer

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(GBC 932, Australia) calibrated with Poplar leaf NCS DC 73550 reference. The concentration of

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metals was expressed with respect to dry weight of the plant material. DTPA extraction method

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was used for the analysis of Fe release from nanoparticles. Sand used for plant culture was air

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dried after the harvest of the plants. Fe in the sand (5.0 g) was extracted with 10 ml of 10.0 mM

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DTPA solution. The extract was subjected to Atomic absorption spectrophotometer for the

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quantitative estimation of Fe.

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Batch adsorption studies

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Adsorption experiment was performed by mixing nanoparticles and calcium chloride in water.

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For isotherm studies, mixtures containing varying concentration of Ca were incubated at 30 0C in

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a rotary shaker till equilibrium time. Experiments were also performed at different pH values

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range from 2.0 to 10.0, contact time range from 5 to 120 min, adsorbent dosage range from 10.0

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to 50.0 mg, temperature range from 10 to 50 0C, and initial metal concentrations range from 2 to

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10 mg L−1. To determine the amount of Ca adsorbed, particles were separated from the mixture

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by centrifugation at 18000 rpm. The amount of Ca ions adsorbed at the equilibrium, qe (mg g−1)

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is represented by the following equation, qe = ((Ci−Ce)V)/X , where Ci and Ce are the initial and 8


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equilibrium concentrations of the metal, V is the volume of the metal solution, and X is the

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weight of the adsorbent.24

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Reverse transcription-polymerase chain reaction (RT-PCR)

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For RT-PCR analysis, total RNA was isolated from the roots of seedlings using TRIzol reagent

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(Sigma), according to supplier`s recommendations. The reverse-transcription reaction carried

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out with 100 ng of total RNA in a PCR system (Eppendorf vapoprotect, Germany).The gene-

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specific primers were designed from the 3‘UTR of the rice genes. The sequences used are listed

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in supplementary data (Supporting information.1).

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denaturation for 5 min, then 30 cycles of 94 ℃ for 30 s, 54 or 58 0C for 30 s, 72 ℃ for 30 s, 72

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℃ extension for 5 min, and finally at 16 ℃. The PCRs were optimized for a number of

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cycles to ensure product intensity within the linear phase of amplification. PCR products

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were resolved by electrophoresis in 2 % agarose gel. The gel images were digitally captured with

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gel documentation system (UVitec Ltd, UK).

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Oxidative stress analysis

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Lipid peroxidation in leaf was determined in terms of malonyl dialdehyde (MDA) content.25

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Leaves or roots (0.5g) were extracted in 4 mL of 20 % trichloroacetic acid (TCA) containing

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0.5% 2-thio-barbituric acid. Mixture was heated at 95 0C for 30 min, and the homogenate was

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centrifuged at 10000 rpm for 10 min. Absorbance of the supernatant was taken at 532 and 600

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nm. MDA content was calculated by using extinction coefficient of 155 mM-1cm-1. For the

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analysis of antioxidant enzymes, leaves were homogenized in 100 mM potassium phosphate

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buffer (pH 7.5) contains 40 mM PMSF and 2 % PVPP. The extract was centrifuged at 13,000

The

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RT-PCR program was 94

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x g for 20 min, and the resultant supernatant was used for assays of superoxide dismutase

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and catalase.26

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Photosynthetic pigments

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Chlorophyll and carotenoids were estimated from extract of intact leaves kept in Acetone -

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DMSO mixture (50:50) in dark. Absorbance of extract was taken at 470, 646, and 663 nm. The

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amounts of pigments were calculated using following formulae.27 Total chlorophyll (µg/ml) =

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20.2 (A645) + 8.02 (A663); Chlorophyll a (µg/ml) = 12.21 (A663) - 2.81(A645); Chlorophyll b

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(µg/ml) = 20.13 (A645) - 5.03 (A663); Carotenoids (µg/ml) = (1000 A470 - 3.27 [chl a] -104

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[chl b]) / 227.

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Chlorophyll fluorescence

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Chlorophyll fluorescence measurements were carried out using PAM 2500 (Heinz Walz,

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Germany). Measurements for PSII mediated functions were carried out in light curve

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mode. After dark adaptation of the leaves for 30 min, the maximum fluorescence was monitored

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by application of a 0.8 seconds saturating light pulse (6,000 µmol photons m-2s-1). The steady

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state fluorescence yield was monitored through exposure of leaf to actinic light range starting

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from 8 to 1200 µmol photons m-2s-1. Electron transports rate (ETR), and non-photochemical

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quenching (NPQ) were tabulated, and plotted against photosynthetically active radiation (PAR).

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Statistical analysis

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Statistical significance of each parameter studied was analyzed by ANOVA with the Duncan’s

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multiple range test. Result is represented as in the form of alphabets where a, b, c, d, and e

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represent first, second, third, fourth, and fifth levels of statistical significance. All analysis is

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considered significant at P < 0.05.

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3. Results and Discussion

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3.1. Physico-chemical analysis confirm formation of carbon bound iron oxide nanoparticles

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Iron oxides are colored.28 Black coloration of the reaction mixture after mixing of 10 mM caffeic

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acid with 10 mM ferric chloride indicates formation of Fe oxide (Supporting information. 2). It

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could be the reduction of Fe3+ to Fe2+ by caffeic acid, and the spontaneous dehydration of ferric

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and ferrous hydroxides formed in the mixture that lead formation of iron oxide nano particles.29,

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the iron oxide formed is Fe3O4 (Figure 1a-b).31 Since there is near infrared absorption, the

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complex is confirmed as Fe3O4 because Fe2O3 which also have two ionic species does not show

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absorption at near-infrared region.30 Transient absorption bands were also noticed between the

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wavelengths 360 - 320, 320 - 290, 290 - 270, and 270 - 250 nm. These absorption bands indicate

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formation of carbon functionalized nanoparticle.32 Carboxylic carbonyl group of organic acids

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binds with Fe3O4.

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products exists in the mixture after reaction of ferric chloride with caffeic acid.29 Thus it is clear

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that carboxyl carbonyl group containing breakdown products of caffeic acid bind with Fe3O4, and

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result in the formation of functionalized Fe oxide.

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Solid state EPR spectrum of the particles confirmed formation of Fe3O4. There was two spin

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transitions at G value near 1500 and 3500 respectively (Figure1c). This indicates presence of

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both Fe3+ and Fe2+ ionic species in the particle as well as superparamagnetic nature. Hysteresis

The characteristic surface plasmon band (SPR) of Fe3O4 is centered at 190-250 nm, and hence

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It is also reported that carboxylic carbonyl group contain breakdown

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closure between 2800-2900 G in the EPR spectra is a characteristic feature of Fe3O4.34 The

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absence of sharp lines in the powder XRD line pattern point semi-crystalline nature of the

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particles. But a broad peak was visible in the XRD diagram at 2ɵ angle 300 with count 405 which

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is characteristic of Fe3O4 (Figure.1d, JCPDS file No. 19 -0629).

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FTIR transmittance peak of Fe oxides fall in the wave number range of 600-500 cm-1, and it was

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noticed in the present study too (Figure 1e). Transmittance peaks at wave number 3480, 2910,

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1628, 1260, 1095, 773, and 600 cm-1 also support functionalized nature of iron oxide.35 Melting

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peak of the particles observed at 390 0C, and the particle degradation started at 504 0C (Figure 1f,

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Supporting information 3a). Hence it is concluded that the particle pose considerable thermal

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stability. The DTA-TGA pattern is also matching to a carbon conjugated Fe3O4 nanoparticle.36

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Atomic force microscopic image confirmed that size of the particles is below 10 nm (Figure 2a).

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Elemental analysis indicated presence of carbon, oxygen, and Fe in the particles (Figure 2b).

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Quantitative elemental analysis through CHNS analyzer revealed that 41.13 % weight of the

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particles is carbon (Supporting information. 4).Thus the physicochemical analyses confirmed

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that the nanoparticle formed is carbon bound Fe3O4.This kind of particle is in demand for soil

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remediation due to high ion adsorption capacity, and low rate of mineralization.21

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3.2 Amendment of nanoparticles enhance bioproductivity of rice plants

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Nanoparticle treatment found to increase biomass of rice seedlings under Fe deficiency and Ca

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stress (Figure 2c, Table.1). Biomass productivity of plants depends on photosynthesis, and it is

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found that progressive change in functional components of photosynthesis during nanoparticle

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treatment38. Chlorophyll is vital for capturing of light in plants. Chlorophyll a, Chlorophyll b,

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and total chlorophyll increased during nanoparticles treatment (Figure 3a-c). Nanoparticle 12


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treatment also increased carotenoids (Figure 3d).These progressive changes are attributed as the

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result of more Fe accumulation and higher photosynthetic efficiency.37

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Chlorophyll fluorescence studies figure out that linear electron transport rate as well as non-

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photochemical quenching capacity increased during nanoparticle treatment under Ca stress

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(Figure 4a-b). Linear electron transport is critical for maintenance of both photophosphorylation

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and generation of reducing power essential for carbon fixation.38 On the other hand, non-

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photochemical quenching (NPQ) allow plants to avoid photo-oxidative stress with help of

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carotenoids.39 Nanoparticle exposed plants had higher rate of linear electron transport, and low

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NPQ compare with control plants under Ca stress. Above results indicate normal functioning of

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photosynthesis during nanoparticle treatment. Low NPQ observed among Ca stressed plants was

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the result of decrease in carotenoids content. Excess cytosolic Ca causes oxidative stress in

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plants.11, 40 Treatment of the nanoparticles decreased malonyl dialdehyde (MDA), an indicator of

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oxidative stress, in roots and leaves (Table 1). The reason for above effect was enhancement in

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the activity of antioxidant enzymes (Figure 4c-d). Enhancement in antioxidant activity was due

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to entry of more Fe in the cells which evokes Fenton-type reactions.

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3.3 Nanoparticles treatment increase iron accumulation in plants

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The foremost side effect of Ca stress is the blockage of Fe uptake.

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presence of nanoparticle reduces Ca accumulation, and similar results also noticed among plants

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grow without Ca treatment (Figure 5b). These data point that nanoparticle had Ca binding

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property which reduced Ca accumulation in plants.

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nanoparticle treatment increase iron accumulation in root and leaf (Figure 5a). These effects

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It is noteworthy that

Metal content analysis also indicated that

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were due to increase in plant available Fe in the growth media upon nanoparticle exposure, and

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maintenance of molecular process associated with Fe uptake (Table1, Figure. 6).

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3.4 Adsorption to nanoparticles reduce calcium accumulation in plants

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Nanoparticles are reported to pose high surface area and ion adsorption capacity.43 It is also

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reported that functionalized nanoparticle efficiently remove metal ion from solution by

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adsorption.44 The equilibrium relationship between nanoparticles and Ca ions in the solution was

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explained using Freundlich and Langmuir isotherms. The equilibrium studies were carried with

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Ca concentrations ranging from 2 to 10 ppm (pH 6.5) with fixed adsorbent dosage (10.0 mg).

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Freundlich isotherm assumes a monolayer sorption with a heterogeneous energetic distribution

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of active sites accompanied by interaction between adsorbed molecules. The graph plotted with

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log qe versus log Ce gave straight line with correlation coefficients (R2) of 0.996 for Ca

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adsorption (Figure 5c). KF (mg g−1) and n are constants representing the adsorption capacity and

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intensity of adsorption in the linear form of Freundlich adsorption equation, log qe = log KF +

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(1/n) log Ce. The KF and n value were 3.47 and 2.21 respectively in the present study. The

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value of n >1 obtained from Freundlich model suggest

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surface, and indicate a favorable adsorption. But difference between KF value (3.47) and

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observed maximum absorption value qmax (6.15) indicated that the Freundlich model is

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inadequate to describe the adsorption event.45

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Langmuir isotherm assumes the uptake of metal ions on a homogenous surface by monolayer

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adsorption without any interaction between adsorbed ions. The plots of Ce/qe versus Ce gave

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straight lines with correlation coefficients (R2) of 0.986 for adsorption of Ca (Figure 5d). qmax

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and b are the maximum adsorption capacity and the equilibrium Langmuir constant in the linear

heterogeneity of the adsorbent

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form of Langmuir equation, Ce/qe = 1/bqmax + Ce/qmax. The qmax and b value were 7.57 and 0.02

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respectively. Langmuir isotherm model well fitted to adsorption where experimental qmax (6.15)

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doesn`t vary far from the empirical qmax (7.57). The

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model implies that monolayer adsorption took place without any interaction between the

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adsorbed Ca ions. The value of equilibrium Langmuir constant (b) less than 1 indicates non-

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spontaneous nature of adsorption too.46 Adsorption on nanoparticles enhanced during an

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increment of Ca ions (Supporting information 3b). An increase in conc. of the nanoparticles in

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the solution also increased the adsorption (Supporting information 3c).The equilibrium time of

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the absorption process was 90 min (Supporting information 3d). It is also noticed that particles

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pose significant adsorption at environmentally relevant pH range and temperature (Figure 5e-f).

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These results point out that the synthesized particle is an efficient Ca binding agent, and the

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property limited plant available Ca during the experiment.

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3.5 Nanoparticles treatment helped to maintain expression of Fe uptake genes

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Rice plants respond to Fe deficiency via type II Fe uptake.

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muigenic acid complex through YSL family of Fe transporters. Methionine is essential for the

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synthesis of muigenic acids.8 Nanoparticle exposed plants had low expression of genes involved

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in recycling of methionine such as OsMTK and OsIDI.

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expression of OsFDH during nanoparticle treatment indicates respiratory cycle dependent

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progressive synthesis of methionine.50 Nicotianamine synthase (NAS), and mugineic acid

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synthase (MAS) are involved in the conversion of methionine to muigenic acids.

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expression is crucial for conversion of S-adenosyl-Met molecules to form nicotianamine.

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Expression of this gene was not affected in the present study. But Ca stress blocked expression

fitness of

8, 47

48, 49

the

data with Langmuir

In this strategy, roots uptake Fe-

(Figure 6) But the relatively higher

15


50, 51

OsNAS


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of OsDMAS1 which is essential for the formation of deoxymugineic acid from nicotianamine

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(Figure 6). Thus the blockage of OsDMAS1 gene expression can be considered as the main

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reason that leads suppression of type II Fe uptake in Ca stressed plants. It is noteworthy that Ca

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stress disrupts OsTUB expression too.

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Overexpression of OsIRO2 is reported to confer tolerance to low Fe availability in calcareous

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soil.47, 52 This gene communicates Fe deficiency signal for the induction of genes involved in

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Type II Fe acquisition such as OsYSL15 which take part in Fe (III)-deoxymugineic acid uptake,

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and long distance transport of Fe.53, 54 Expression of IRO2, as well as OsYSL15, retarded in Ca

324

stressed plants and this account for very first step in blockage of type II Fe acquisition during Ca

325

stress. Expression of genes involved in high-affinity Fe acquisition such as OsIRT1 and

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OsNRAMP1 were also blocked by Ca stress. Hence it is concluded that plants grow in calcareous

327

environment failed to pose molecular response to Fe deficiency. But the absence of immense

328

alterations in expression of genes such as OsIRT1, OsNramp1, OsYSL15, and OsDMAS1 helped

329

to maintain Fe uptake during nanoparticle treatment.

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Acknowledgement

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Abin Sebastian gratefully acknowledges Dr. DS Kothari Postdoctoral fellowship (No. BL/14-

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15/0162), UGC, India for financial support. Thanks are due to Crystalin research Pvt. Ltd,

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Technology business incubator, the University of Hyderabad for facilities.

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Notes

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The authors declare no competing financial interest

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References

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465 466 467 468 469 470 471 22


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472

Table.1 Biomass, MDA content, sand pH, and Fe in the sand at harvest.

Parameter

+Fe

-Fe

-Fe+NP

+Fe+Ca

+Fe+Ca+NP

12.7 + 1.4a

9.3 + 0.9b

12.1 + 0.7a

5.6 + 0.9c

9.5 + 1.21b

4.6 + 0.9b

5.2 + 0.4b

4.9 + 0.2b

10.5 + 0.8a

5.7 + 0.1b

3.5 + 0.8c

6.8 + 0.4a

5.8 + 0.3b

7.0 + 0.7a

5.5 + 0.7b

6.5

6.5

6.5

11.5

11.5

1.90

0.25

3.15

2.2

4.1

Biomass (mg f.wt) MDA in root (nmoles / g f.wt) MDA in leaf (nmoles/ g f.wt)

pH

Fe-DTPA (mg/kg) 473

23



Graphic for manuscript 311x164mm (120 x 120 DPI)


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Figure.1 Physco-chemical characterization of nanoparticles. Absorption spectra of reaction mixtures of 10 mM caffeic acid with 0.0, 0.1, 1.0, and 10.0 mM FeCl3. Disappearance of characteristic absorption spectra of caffeic acid in the mixture which indicate formation of nanoparticle is clearly visible at 10 mM conc. of caffeic acid (a). Absorption spectra of isolated nanoparticles suspended in MQ water (b) EPR spectra (c) PXRD pattern (d) FTIR spectra (e), and Melting curve (f). 180x182mm (150 x 150 DPI)



Figure. 2 Properties of the nanoparticles. Atomic force microscope image of surface of nanoparticle aggregate. The scale indicates the size of individual particle is less than 20 nm (a). EDS spectra of the nanoparticles (b), and plant growth response (c). 241x165mm (150 x 150 DPI)


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Figure. 3 Changes in photosynthetic pigments. Chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), and carotenoid content (d). 218x176mm (150 x 150 DPI)



Figure.4 Chlorophyll fluorescence analysis. Linear electron transport rate (a), non-photochemical quenching (b), and antioxidant enzyme activity - SOD activity (c), and catalase activity (d). 238x175mm (150 x 150 DPI)


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Figure. 5 Metal accumulation in plants - Fe content (a), Ca content (b), and calcium adsorption characteristics -Freundlich isotherm (c), Langmuir isotherm (d), adsorption under varying pH (e), and adsorption under varying temperature (f). 182x184mm (150 x 150 DPI)



Figure. 6 Gene expression profile in the roots 209x140mm (150 x 150 DPI)


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Carbon-Bound Iron Oxide Nanoparticles Prevent ... - ACS Publications

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