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CuNP nanochain arrays with a reduced toxicity response: a biophysical, biochemical outlook on Vigna radiata. Saheli Pradhan, Prasun Patra, Shouvik Mitra, Kushal Kumar Dey, Satakshi Basu, Sourov Chandra, Pratip Palit, and Arunava Goswami J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504614w • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 20, 2015

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Journal of Agricultural and Food Chemistry

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CuNP nanochain arrays with a reduced toxicity response: a biophysical, biochemical outlook on

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Vigna radiata.

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Saheli Pradhan*a, Prasun Patrab, Shouvik Mitraa, Kushal Kumar Deyc, Satakshi Basua, Sourov

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Chandraa, Pratip Palitd, Arunava Goswamia

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a

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b

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Kolkata,700098, India.

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c

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d

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Biological Sciences Division, Indian Statistical Institute, 203 B.T. Road, Kolkata 700108, India. Centre for Research in Nanoscience and Nanotechnology, University of Calcutta,

Department of Statistics, University of Chicago Plant Physiology Section, Central Research Institute for Jute and Allied Fibres, Indian Council

of Agricultural Research, Barrackpore, Kolkata 700 120, India

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ACS Paragon Plus Environment


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ABSTRACT

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Copper deficiency or toxicity in agricultural soil circumscribes plant’s growth and physiology

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hampering photochemical and biochemical network within the system. So far copper sulfate

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(CS) has been used widely despite its toxic effect. To get around this long-standing problem

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copper nanoparticles (CuNP) have been synthesized, characterized and tested on mung bean

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plants along with commercially available salt CS, to observe morphological abnormalities

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enforced if any. CuNP enhanced photosynthetic activity by modulating fluorescence emission,

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photophosphorylation, electron transport chain (ETC) and carbon assimilatory pathway under

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controlled laboratory conditions, as revealed from biochemical and biophysical studies on treated

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isolated mung bean chloroplast. CuNP at recommended dose worked better than CS treated

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plants in terms of basic morphology, pigment contents, antioxidative activities. CuNP showed

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elevated nitrogen assimilation compared to CS. Even at higher doses CS was found to be toxic to

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the plant system, while CuNP did not impart any toxicity to the system including morphological

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and/or physiological alterations. This newly synthesized polymer encapsulated CuNP can be

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utilized as nutritional amendment to balance the nutritional disparity enforced by copper

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imbalance.

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Key words: Copper nanoparticles, encapsulated, photosynthesis, light reaction, dark reaction

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INTRODUCTION

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Copper (Cu) is an essential plant micronutrient in both algae and higher plants, and plays

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important role in numerous metabolic processes including photosynthesis, respiration, plant

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growth and development;1 although at higher concentration, Cu may act as “food poison”.2

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Excess Cu inhibits a large number of enzymes and interferes with several aspects of plant

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biochemistry, including photosynthesis, pigment synthesis, membrane integrity. Perhaps its most

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important set back is to block photosynthetic electron transport chain (ETC) leading to

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production of radicals which start peroxidative chain reactions involving membrane integrity.3

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However, not a single effective strategy has been developed to combat this severe nutritional

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imbalance.

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With recent developments in new technology, several smart nanoparticles have been introduced

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in order to improvise the nutritional imbalance within the plant system. Manganese nanoparticle

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has been used to enhance the photosynthetic efficacy in mung bean system;4 nano-titanium

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dioxide (TiO2) enhanced the photosynthetic activity as well as nitrogen metabolism in spinach.5

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Although typical nanoparticles are used for crop management but their phytotoxic effects cannot

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be ruled out. Zinc oxide nanoparticles and nanoscale zinc have been found to inhibit seed

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germination as well as root growth of six higher plant species Raphanus sativus, Brassica napus,

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Lolium multiflorum, Lactuca sativa, Zea mays and Cucumis sativus.6 Zinc oxide nanoparticles

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are readily internalized within the apoplast and tonoplast of the root endodermis and stele

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resulting shrinkage of root tip of ryegrass. Use of nanoparticles may give rise to environmental

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toxic response; in this context to Doshi et al. have injected two types of nanoalumina into the

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sand column to evaluate ecotoxicological assessment.7 Several works have also been performed



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on various plant model systems like wheat, rice, spinach, onion to evaluate the potential efficacy

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of copper and copper oxide nanoparticles (CuO NPs).8 But CuO NPs induce oxidative stress

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mediated damage within plant system.9 Therefore, significant efforts are being needed to design

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the smart copper based nanoparticles for crop management with a reduced toxic response in

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order to eliminate the aforementioned setbacks that copper oxide nanoparticles reinforced to the

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system.

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In this study, we demonstrate an alternative strategy to combat the Cu-induced nutritional

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disorder, with enhanced plant growth, photochemical, carbon and nitrogen assimilatory activities

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along with a reduced toxic response. In order to address the critical issues, copper nanoparticles

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(CuNP) have been synthesized by a facile method with modifications. Synthesized CuNPs have

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been tested against mung bean plants and as a comparison commercially available bulk

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counterpart, copper sulfate (CS) salt is used under controlled laboratory condition. At 0.05 mg/L

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concentration, CS promotes the plant growth and development as per earlier literature. It also

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elevates the function of photosynthetic machineries, rate of carbon and nitrogen assimilation as

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Cu is known cofactor in these metabolic processes.10 But at or above 0.5 mg/L of CS treatment,

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it impaired severe tissue injuries causing reduction in growth, even death of the plants.11 Cu

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toxicity is a threat worldwide because of its tendency to produce harmful Cu-chelates during

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interaction with soil colloids and nutrients, which are detrimental to the surrounding

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microflora.12 In this study, we demonstrate a single step designed encapsulation of CuNP within

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a biocompatible polymer to reduce its toxic response. Sustained release of nutrient interacts with

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biological sites which controls the toxic response. Biochemical experiments have been carried

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out on isolated chloroplast (Chl) of mung bean plant treated with CuNPs which demonstrates that

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CuNPs are more effective than CS to some extent. In a tandem process the effect of both nano-


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and bulk counterpart of Cu in carbon and nitrogen assimilation has been checked. Interestingly

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CuNP shows its greater efficacy in contrast to CS. A series of biophysical techniques are used to

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confirm the bioavailability and uptake within the system. Oxidative stress markers, both

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antioxidative and non-antioxidative enzymes level, show that CuNP does not inhibit any toxic

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response to the treated plant system. CuNP, both in physiological and biochemical aspects,

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worked significantly better than CS, which caused cellular toxicity because of dysfunctional

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metabolic pathways. Whole study is assisted by a probable mechanistic interpretation to outline

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its beneficial response. In light of these aforementioned analyses we envision CuNPs future

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prospect in crop management with reduced toxic response in contrast to its bulk counterpart. To

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best our knowledge this is the first effort to use biocompatible polymer encapsulated CuNP for

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augmentation of photosynthesis; elevated carbon, nitrogen assimilation; improved morphological

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and physiological outputs within plant system.

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MATERIALS AND METHODS

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CuNP synthesis

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0.5 g of copper sulfate was allowed to dissolve in 2 mL of water; to it 20 mL of PEG-200 was

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added. The resultant mixture was sonicated in a sonication bath (100 Hz) for about 5 min in

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order to obtain a homogeneous dispersion. To the resultant dispersion aqueous solution of

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sodium borohydride (0.1 M) was added drop wise and the reaction mixture was subjected to a

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microwave heating (450 watt) until a characteristic reddish color was developed. Then mixture

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was centrifuged and washed with ethanol thrice in order to remove impurities and placed in the

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hot-air oven at 80 ºC to obtain polymer encapsulated CuNP.



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Plant material and growth conditions

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Mung bean seeds (Vigna radiate var. Sonali) were purchased from Berhampur Pulse and Oil

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Research Centre. Seeds (20 seeds/replicate; 3 replicates in total) were washed in deionized

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double distilled water and surface sterilized with 5 % sodium hypochloride solution for about 20

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min. Then the seeds were washed thoroughly with deionized double distilled water and imbibed

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with the treatment solutions (control; CuNP:0.05 mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L; CS 0.05

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mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L) in dark for 4-6 h, prior to the germination. Detailed growth

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conditions of plants are described in SI.

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Morphological parameters

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Root and shoot length, fresh and dry weight of CuNP and CS treated plants were measured. Dry

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weight was recorded by drying the plants at 80 °C for 24 h.

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Estimation of Photosynthetic Pigment content

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Control and treated leaves were centrifuged twice with 80 % alkaline acetone at 6000 g for 20

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min and chlorophyll content were estimated with the supernatant spectrophotometrically13 in

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terms of µg chlorophyll/ gm fresh tissue. Carotenoid was estimated according to the method of

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Davies with little modification14 and the data were expressed in terms of OD /gm fresh wt.

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Isolation of chloroplast

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Mung bean leaves were homogenized in isolation buffer (330 mM mannitol, 30 mM HEPES, 2

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mM EDTA, 3 mM MgCl, and 0.1 % w/v BSA, pH 7.8.15 The homogenate was filtered and

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centrifuged. Supernatant was further centrifuged at 1000 g for 5 min at 4 ºC.


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Estimation of Fluorescence and PI of chloroplast by photoluminescence (PL) Spectra

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Chloroplast suspension contained 100 µM potassium phosphate buffer (pH 7.8), 2 µM NaCN,

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and 1.25 mM sucrose. The fluorescence spectra of the control and treated chloroplasts were

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recorded at 273 K under 440 nm excitation wavelength. Photosynthesis versus irradiance (PI) of

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treated and untreated chloroplasts was calculated accordingly.16

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Photo reduction Activities

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The electron transport through the whole chain of photosynthesis was measured

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polarographically with an Oxygraph oxygen electrode (Hansatech Instruments, UK).17 Oxygen

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evolution was assayed in a medium containing chloroplasts equivalent to 378 µg/mL of

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chlorophyll. Hill activity was measured according to the method of Vishniac.18 Ferricyanide and

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Nicotinamide adenine dinucleotide phosphate (NADP) reduction were determined by the

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spectrophotometric methods.19 Light-induced Adenosine triphosphate (ATP) content of

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chloroplasts was measured by comparing the ATP level in the dark and 1 min illumination.20

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Carbon assimilation assay

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Chloroplast NADP-glyceraldehydes-3-phosphate dehydrogenase (GPDHase) activity was

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determined following Taylor et al.21 The enzyme was activated by a solution containing 0.2 M

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Tricine (pH 8.4) and 20 mM ATP to 0.1 mL of the solution containing the extracted enzymes.

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After 5 min, reaction medium (62.5 mM Tricine [pH 8.4], 16 mM MgCl2, 7.8 mM ATP, and 0.2

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mM NADPH) was added to it. 50 mM glycerate 3-P was then added to intiate the reaction and

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the oxidation of NADPH monitored at 340 nm. Fructose-1, 6-bisphosphatase (FBPase) was

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activated by preincubation of enzyme extract with reaction medium (0.14 M Tricine (pH 7.9), 14



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mM MgCl2, and 7 mM DTT). 60 mM fructose-1, 6-bisphosphate was added after 10min and

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terminated after 15 min by the addition of 0.5 mL of 10 % (w/v) TCA. This solution was cleared

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by centrifugation for 10 min at 12,000 g in order to determine the enzymatic activity. Ribulose-

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5-phosphate kinase (RBPase) activity was analyzed by 0.1 M Tricine (pH 7.6) and 15 mM DTT

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for 1 h. After that reaction medium (66 mM Tricine (pH 7.6), 13 mM MgC12, 1.3 mM P-pyruvic

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acid, 1.3 mM ATP, 0.33 mM NADH, and 2 units each of phosphoriboisomerase, pyruvate

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kinase, and lactic dehydrogenase) was added. The reaction was initiated by 30 mM ribose-5-P

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and the oxidation of NADH monitored at 340 nm.

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Nitrogen metabolism

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Preparation of leaf and root enzyme extract

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Control and treated plants were homogenized with a ratio of 1:10 (w/v) in 50 mM phosphate

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buffer (pH 7.5) containing 2 mM EDTA, 1.5 % (w/v) soluble casein, 2 mM DDT and 1 % (w/v)

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insoluble PVPP at cold and centrifuged at 3000 g for 5 min. Supernatant was again centrifuged at

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30,000 g for 20 min. The extract was then used for enzymatic assays.

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Determination of NO3-

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Enzyme extract was added to 10 % salicyclic acid solution dissolved in 12 N sulphuric acid

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(w/v). NO3- content was measured as described by Cataldo et al.22 The results were expressed as

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µmol g-1 fresh wt.

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Determination of nitrate reduction (NR) activity

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Enzyme extract was mixed in the reaction mixture containing 100 mM phosphate buffer (pH

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7.5), 100 mM KNO3, 10 mM cysteine, 2 mM NADH. After 30 min of incubation at 30 °C, 1 M


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zinc acetate was added. The nitrite formed was determined at 540 nm and NR activity was

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expressed as µmol NO3- formed g-1 fr wt. h-1

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Determination of nitrite reduction (NiR) activity

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To the reaction mixture (50 mM phosphate buffer (pH 7.5), 20 mM KNO2, 5 mM methyl

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viologen (MV), 300 mM NaHCO3) enzyme extract was added at 30 °C. After 30 min NiR

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activity was determined by disappearance of NO2- from the reaction medium and expressed as

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µmol NO2- formed g-1 fr wt. h-1.

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Determination of glutamine synthetase (GS) activity

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Enzyme extract was added in the reaction mixture containing 100 mM phosphate buffer (pH

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7.5), 4 mM EDTA, 1 M sodium glutamate, 450 mM MgSO4, 7H2O, 300 mM hydroxylamine and

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100 mM ATP. The formation of glutamylhydroxamate was measured at 540 nm after

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complexing with acidified ferric chloride. GS activity was expressed as formed

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glutamylhydroxamate in g-1 fr wt. h-1.

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Determination of NADH-glutamate synthase (GOGAT) activity

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The reaction mixture contained 50 mM phosphate buffer (pH 7.5) with 0.1 % (v/v)

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mercaptoethanol, 1 mM EDTA, 18.75 mM 2-oxoglutarate, 15 mM aminoxyacetate, 1.5 mM

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NADH, 75 mM L-glutamine and enzyme extract. The decrease in absorbance was recorded for 5

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min at 28 °C. The activity was expressed as µmol NAD g- I fr wt. h-1

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Uptake of CuNP by Fluorescence microscopy study

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CuNP were amine functionalized and conjugated Fluorescein-B-isothiocyanate (FITC) for uptake

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study. Ultrathin cross section of treated plant samples were fixed in 2 % glutaraldehyde followed

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by washing in graded ethanol and observed under confocal microscope.5



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Electron Micrographic study

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Transmission electron microscopy (TEM) and Field emission scanning electron microscope

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(FESEM) of treated and untreated chloroplast were performed for analyzing the interaction

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between chloroplast and CuNP in photosynthesis. Chloroplasts were fixed with glutaraldehyde,

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stained with osmium tetraoxide and washed in graded ethanol to observe under electron

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microscope.

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Cu release study by ICPOES

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Cu release from CuNP was monitored by using inductively coupled plasma optical emission

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spectroscopy (ICP-OES) at pH 7 i.e. the pH of plant growth medium. 100 mL of CuNP

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dispersion of aforementioned concentrations in milli-Q water was allowed to stir for 24 h. The

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resultant dispersion was centrifuged, subjected to digestion in presence of ultrapure nitric acid

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followed by estimation against a standard.5

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Enzyme assays related to oxidative stress

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Control and treated plants were ground in Tris-HCl buffer (pH 7.5) containing EDTA and

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polyvinylpyrrolidone followed by centrifugation at 0 ºC. The supernatant was used for enzymatic

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assays. Superoxide dismutase (SOD) activity was estimated following standard procedure.23

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Peroxidase (POD) activity was assayed according to Chance et al..24 The total peroxide content

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of plant tissue was estimated according to the method of Thurman et al.25 Glutathione reductase

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(GR) was assayed according to Wang.26 Catalase (CAT) activity was assayed according to Beers

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et al.27 Activity of Polyphenol oxidase (PPO) was assayed following standard methods.28

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Estimation of proline was done according to the method of Bates et al.29 Phenol content was

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estimated according to the method of Malik et al.30


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Estimation of total amino acid (AA), lipid, protein and carbohydrate content

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AA content in plant sample was estimated according to the ninhydrin method. Protein contents

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were estimated according to Lowry et al. Lipid content was estimated according to Bligh

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method. Total carbohydrate content was estimated following Loewus.

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RESULTS AND DISCUSSIONS

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Physico-chemical characterization of CuNP

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Here in we demonstrated a designed synthesis of CuNP encapsulated within a polymeric matrix

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followed by its evaluation in photosynthetic efficacy, carbon and nitrogen assimilation and

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biosafety study in plant system. In correlation bulk counterpart CS was also considered for a

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brief comparison. FESEM was used to evaluate the morphology of CuNP which justified

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spherical particles were sheathed within a polymeric matrix (Figure 1a). TEM image showed that

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the small spherical particles of size almost 20 nm were formed during the synthesis (Figure 1b).

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In situ encapsulation of CuNP within PEG-200 resulted in a chain like unique morphology. This

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unique Cu nanochain-array was comprised of spherical CuNP encapsulated within PEG-200 to

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yield uniform dispersion which was visible in the high resolution image (Figure 1b inset). It was

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well documented that CuNP nanochain-array was originally reddish colored; during synthesis a

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drastic blue to reddish coloration signified the formation of CuNP. A subsequent UV-Vis

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absorbance at around 450-480 nm was noted which also justified the formation of CuNP (Figure

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1c) and corroborated with previous experimental findings. Polymeric encapsulation within PEG-

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200 resulted suppression in crystallinity within CuNP in X-ray diffraction (XRD) pattern (SI

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Figure S1). This amorphous nature of encapsulating agent was also consistent with literature.



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Energy dispersive X-ray (EDX) analysis also justified that Cu is present as main constituent.

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Additional peaks of impurities were avoided which confirmed that CuNPs were obtained in its

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pure form (SI Figure S2). The surface topology and height profile was obtained by atomic force

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microscopy (AFM). AFM image (SI Figure S3) justified that the average height of the PEG-200

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encapsulated CuNPs were 6-8 nm. Surface functionality of CuNP was confirmed from fourier

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transformed infrared (FTIR) spectra (Figure 1d). CuNPs were hydrophilic with existence of

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surface hydroxyl, carbonyl/carboxyl groups. Spectral pattern confirmed C-H stretching, O-H

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bending, C=O stretching, C-O stretching which aroused due to surface PEG-200 during

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encapsulation of CuNPs. A sustained release of 0.0136 ppm from predesigned 0.05 mg/L CuNP

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nanochain-arrays after 24 h was noted by ICP-OES under mimicking environment sealed inside

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a dialysis bag (SI Figure S4). Sustain release of Cu from polymer encapsulated nanochain-array

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resulted lower availability of Cu to in vitro system in contrast to its bulk counterpart CS. As a

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result toxicity symptom was reduced to some extent in this smart designed nanochain-array.

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Meanwhile hydrophilic character produced aqueous stability for in vitro experiment. Altogether

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this report claims well characterized, stable, polymer encapsulated designed CuNPs to be used

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for in vitro and in vivo plant experiments.

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CuNP at a recommended dose promoted the growth of 15days treated mung bean plants

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Cu is a known essential micronutrient in both algae and higher plants.31 The bulk counterpart of

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CuNP, i.e., CS has now been extensively applied in the field to abate Cu-induced nutritional

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disorder. But at or above certain concentration limit, it impairs toxicity to the plants resulting in

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decrease in plant yield; external toxic symptom is the consequence of periodically or

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permanently changed cellular metabolism. Both CuNP and CS at different physiological doses


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were tested on mung bean plants and several growth parameters were checked thereafter. In all

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cases, CuNP worked better than CS in terms of root and shoot length, fresh and dry weight as

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noted in Table 1. Figure 2a shows the digital images of 15 days treated plants in contrast to the

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control ones. Both CuNP and CS had positive effect on plant at a dose of 0.5 mg/L and 0.1 mg/L

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with respect to control; but beyond that dose, plant’s growth was affected by CS treatment.

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Plants are very sensitive to Cu toxicity displaying metabolic disturbances and growth inhibition

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with Cu contents slightly higher than the normal levels in tissues. CuNP-treated plants were

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found to be healthy in all doses without any chlorotic or necrotic symptoms. Therefore no

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significant abnormalities or stress response were noted in CuNP treated sets, in fact CuNP

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exhibited growth promotion of the plants.

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Increased in Chlorophyll and Carotenoid contents in CuNP treated plants even at a dose of

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1 mg/L

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It is well established that in both Cu deficient and excess plants; change in chlorophyll (Chl)

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content alters chloroplast structure and thylakoid membrane composition in leaves of spinach,

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rice, wheat, and bean in experimental growth conditions.1 Cu interferes with the biosynthesis of

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the photosynthetic machinery modifying the pigment and protein composition of photosynthetic

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membranes. This is attributed to the reduction of Chl content and subsequent substitution of the

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central Mg ion of Chl by Cu in vivo, leading to the inhibition of photosynthesis.32 In our

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experiment both in Cu-deficient and CS treated plants (at higher doses), similar results were

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observed. In case of CuNP treated plants, Chl-a content was increased specifically in all

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treatments, indicating CuNPs might have a momentous effect in photosynthesis, as chl-a is key-

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player in the light dependant photosynthetic pathway (Figure 2b). Carotenoid the other important



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factor which protects the photosynthetic machinery from any abiotic stress was simultaneously

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checked. In CuNP treated plants, both xanthophyll and carotene were found to be increased

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significantly with respect to its bulk counterpart and the control one (Figure 2c). Therefore

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photosynthetic machinery was prevented from unnecessary external stress during CuNP

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treatment.

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CuNP @0.05 mg/L enhanced the activity of light reaction in photosynthesis

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In natural photosynthesis, incident light electronically excites a membrane bound protein-

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pigment complex, called photosystem (PS). The photogenerated electrons are rapidly delivered

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to reaction centers (both PSI and PSII) with the help of antenna pigments along the electron

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transport chain (ETC) through a series of oxidizing agents for regenerating NADPH cofactors.

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These cofactors drive redox enzymatic reactions to synthesize organic compounds in Calvin

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cycle.

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At the initial stage of light dependent reaction, some associated pigments, called antenna

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pigments along with some light harvesting proteins capture solar energy and transfer them to the

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reaction center. When the energy stored in chlorophylls in the excited state is rapidly dissipated

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by excitation transfer, the excited state is said to be quenched.

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originate directly from light harvesting pigment bed and is influenced more by CuNP treatment

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than its bulk counterpart. Therefore more intense energy transfer to the reaction center in the

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form of non-photosynthetic fluorescence was noted in CuNP treatment in contrast to CS

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treatment (SI Figure 5). If the excited state of chlorophyll is not rapidly quenched by excitation

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transfer, it can react with molecular oxygen to form singlet oxygen. The extremely reactive

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singlet oxygen can damage many cellular components and hamper the photo-efficiency of PSII.


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This quenching is believed to

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Photosynthesis irradiance (PI) response was evaluated (SI Figure S6) to understand the

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relationship between CuNP and pigment-efficiency in terms of energy capture and subsequent

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transfer of exciton. Enhancement in response curve after CuNP treatment with respect to control

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supported that CuNP did not encourage photochemical damage on PSII; the subsequent

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quenching (SI figure S6) was due to non-photosynthetic change during energy transfer. This

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initial stimulation after CuNP treatment provided a strong basis to trace out next sets of

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photochemical assays to address photosynthetic efficiency.

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From reaction center, the excited PSII transfers an electron to Pheophytin. On the oxidizing site

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of PSII, it is re-reduced by oxidation of water, generating molecular oxygen which is a by-

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product of photosynthesis. Pheophytin transfers electrons to a series of electron acceptors to

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Cytochromeb6f complex. A Cu-protein component, called Plastocyanin then transfer electrons

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from Cytochrome-complex to PSI. Thereafter, series of electron acceptors transfer electron to

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produce NADPH which is essential component to be used in later stage, i.e.; dark reaction of

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photosynthesis. Therefore photosynthetic efficacy was measured in lights of a series of

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biochemical reaction in presence of different electron acceptors.

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Cu, being a metalloprotein component of Plastocyanin, induces structural changes in

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photosynthetic membrane leading to the inhibition of electron flux. As a result, at any alteration

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of doses, it inhibits sites of photosynthesis starting from Hill reaction, i.e.; process of oxygen

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evolution, photosynthetic ET and photophosphorylation to dark reaction. Excess Cu indirectly

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disturbs iron incorporation into QA-Fe sites in Chl causing inhibition of PSII acceptor sites.34 In

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our experiments, at higher doses of CS treated Chl showed impaired photosynthesis in all aspects

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of light reactions starting from Hill reaction, whole chain ET, PS I and II activity to



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photophosphorylation. CuNP at all doses was found to enhance light reaction, outclassing the

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harmful effect of Cu deficiency and/or toxicity. Polarographic study for efficiency of whole

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chain electron transport (Figure 3a) and subsequent oxygen evolution (Figure 3b) in light

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reaction corroborated with the previous results. Effect was maximum at the dose of 0.05 mg/L

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compared to CS counterpart in Hill reaction (Figure 3c), Fericyanide reduction assay (PSII

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activity) (Figure 3d), Fericyanide-NADP reduction (PSI activity) (Figure 3e).

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Photophosphorylation is the process of converting energy from a light-excited electron into the

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pyrophosphate bond of an adenosine diphosphate (ADP) molecule. During water splitting

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liberated proton is bypassed to the ATPase complex by a proton gradient to form ATP from ADP

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which is an important material for the dark reactions. In CuNP treated Chl, ATP content was

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found to be greater than its bulk counterpart CS. No apparent toxicity was observed even at

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highest dose of CuNP as noted in CS treated Chl (Figure 3f).

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In brief, when light falls on the green cells CuNP encouraged the energy-packets present in

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pigment bed to transfer the electron readily to the reaction center without hampering the pigment

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machinery, i.e., PS II and/ or PSI. CuNP positively enhanced the efficiency of chloroplast in light

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mediated entire electron transfer process (30.29% more than control Chl when measured

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polarographically). It stimulated the PSII activity (20.5% more activity was observed w.r.t

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control) resulting greater oxygen evolution. CuNP also promoted the PSI activity probably

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because of its interaction between Cu-protein components, Plastocyanin. As a result

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photophosphorylation activity was also improved after CuNP treatment. CuNP thus modulated

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the entire light reaction positively with effective influence on electron transport chain along with

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phosphorylation activity as seen in Schematic representation 1.


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343

CuNP enhanced the activity of light regulating enzymes in dark reaction of photosynthesis

344

In light independent reaction, CO2 and water are enzymatically combined with a 5-C molecule to

345

generate 2 molecules of 3-C intermediate which is reduced to carbohydrate by enzymatic

346

reactions driven by ATP and NADPH generated photochemically. CuNP was found to facilitate

347

the light-dark modulation providing an on-off switch for three key enzymes in Calvin cycle, viz.,

348

Fructose-1,6-bisphosphate phosphatase (FBPase), Ribulose-5-phosphate kinase (RBPase) and

349

NADP-glyceraldehyde-3-phosphate dehydrogenase (GPDHase).When CuNP treated Chl was

350

subjected to evaluation of GPDHase (Figure 4a), RBPase (Figure 4b) and FBPase activity

351

(Figure 4c); it was noted that CuNP exhibited greater efficacy in contrast to its bulk counterpart

352

CS. Interestingly 0.05 mg/L was found to be most effective amongst all the dosages although

353

CuNP had strong influence on FBPase activity among these three enzymes. This result indicated

354

that CuNP might play a pivotal role in dark reaction of photosynthesis.

355

CuNP in nitrogen metabolism

356

Plant growth and photosynthetic ability can be improved by enhancing the nitrogen supply

357

within the system; and therefore it is essential to evaluate the interactive effect of CuNP on

358

nitrogen metabolism in the plant. The first step of this process is the reduction of nitrate in

359

cytosol and the reaction is catalyzed by nitrate reductase (NR) in which nitrate ion is converted

360

to nitrite ion. Since nitrite is a highly reactive, potentially toxic ion so plant cells immediately

361

transport the nitrite generated by NR from cytosol into chloroplastic leaves and plastids in

362

roots.35 Here with the help of nitrite reductase (NiR), nitrite is transferred to ammonium ion. To

363

avoid ammonium toxicity, plants rapidly convert ammonium ion into amino acids (AAs) with the

364

help of glutamine synthase (GS) and glutamate synthase (GOGAT). GS combines ammonium



Page 18 of 39

365

with glutamate to form glutamine whereas GOGAT assimilates glutamine translocated from

366

roots to leaves. Once assimilated into glutamate and glutamine, nitrogen is incorporated to AA

367

via transamination reactions. Therefore the activities of all four enzymes are very imperative in

368

nitrogen assimilation and can be stimulated by essential heavy metals.36 In this report, CuNP at

369

modulated dose was found to be pertinent for the usage as an integral component in nitrogen

370

fertilizer as it enhanced the activity of NR (Figure 5a), NiR (Figure 5b), GS (Figure 5c) and

371

GOGAT (Figure 5d). As free nitrate ion concentration was much less in the system compared to

372

the control (SI Figure S7), it ruled out the possibility of nitrate toxicity leading to cellular injuries

373

within the system. CuNP treatment at all doses had outclassed the bulk counterpart at their

374

respective concentrations. Therefore, it justified that CuNP might have significant and implicit

375

role in both photosynthesis and nitrogen metabolism as it offset all ominous effect of Cu toxicity

376

caused in agricultural sector.

377

Bioavailability and uptake of CuNP

378

Due to strong binding of Cu by organic matter and other soil colloids, its mobility is severely

379

restricted and the fraction of total Cu available for plant uptake is usually very low. Therefore

380

uptake and distribution of the CuNP within plant system was important to execute the response.

381

Compared to the bulk counterpart, availability of CuNP into the rhizosphere is expected to be

382

very high because of its size, high surface by volume ratio and unique physico-chemical

383

properties.37 Bioavailability of CuNP within the plant sample was justified by confocal

384

microscopy. In order to demonstrate its bioavailability CuNP was conjugated with FITC

385

following our previous report.5 FITC conjugated CuNP treated mung bean plants were then

386

observed under confocal microscope. Leaf and root section (SI Figure S8a and S8b)

387

demonstrated bright green color under confocal microscope at 488 nm excitation wavelength;


Page 19 of 39


388

although leaf section appeared to be brighter under confocal microscope. A direct biomass

389

distribution of CuNP treated mung bean sample was observed by ICPOES shown in SI Table S1.

390

Distribution of Cu was more in leaf than root which corroborated with the confocal results as

391

well.

392

We assessed the morphology of treated and control Chl by electron microscopic experiments.

393

Treated samples retained similar spherical morphology under scanning electron microscope

394

(SEM). No significant alterations in morphology were observed in the post treated samples (SI

395

Figure S9a). EDX analysis associated with SEM justified the presence of CuNP within the Chl

396

(SI Figure S9b). Finally TEM image demonstrated the presence small sized CuNPs over the

397

surface of Chl without altering its uniform spherical morphology (Figure 6).

398

Consequently, excess Cu causes reduction of Chl content, connected with partial destruction of

399

grana and considerable modification of lipid-protein composition in thylakoid membrane

400

depending upon the duration of Cu action. As a consequence we checked the morphology of root

401

and leaf section under light microscope; no abnormalities in tissue anatomy were found which

402

corroborated with the previous results. In root tissues, epi- and endodermis pericycle and

403

vascular bundles were also normal in position and shape (SI Figure S10). In leaves, spongy and

404

palisade parenchyma were normal in position and shape with respect to control, even no sunken

405

stomata were observed (SI Figure S11). Therefore we conclude readily uptake of CuNP by the

406

root from rhizosphere and its subsequent transportation to leaves via shoot. CuNP did not impart

407

any structural changes in any of the root, leaf tissue, not even to the Chl signifying its

408

compatibility to the plant system.

409

Mechanistic interpretation or mode of action of CuNP



Page 20 of 39

410

Under physiological processes in plants, Cu exists as Cu2+/Cu1+ system.38 Cu acts as a structural

411

element in regulatory proteins and participates in photosynthetic ETC, mitochondrial respiration,

412

oxidative stress response, cell wall metabolism and hormone signaling. In absence, plants

413

develop specific deficiency symptoms which affect mostly in the early developmental stages of

414

plant and reproductive organs. Besides, the redox properties those make Cu an essential element

415

also contribute to inherent toxicity. Redox cycling between Cu2+ and Cu1+ can triggers the

416

production of highly toxic hydroxyl radical with subsequent damage in nucleic acid, protein and

417

other biomolecules. On the other hand Cu has strong affinity with thiols and can displace metals

418

in proteins.39 At the same time Cu is also prone to surface oxidation upon exposure to ambient

419

condition. Therefore we strategically encapsulated CuNP within a biocompatible polymer.

420

Biocompatible polymer PEG-200 was already approved by US FDA for biomedical applications.

421

This encapsulation prevented unnecessary leaching out of Cu from the constituents, at the same

422

time prevented oxidation of Cu under the conditions. Therefore the recycling of Cu2+/Cu1+ was

423

controlled and moderate amount of Cu would be released from the polymer matrix ensuring no

424

or little cellular damage within system. Cu from CuNP was already noted by ICPOES as shown

425

in Table 1 (SI). Meanwhile Cu being an inherent functional component of enzymatic reactions

426

including ETC pathway, nitrogen assimilation, CuNP was found to be enhanced and/or modulate

427

the significant biochemical pathways in the photosynthesis and nitrogen metabolism; a small but

428

significant amount of released Cu from nanochain-arrays was sufficient to execute the response.

429

Biosafety of CuNP in the plant system

430

As CuNP enhanced the nitrogen assimilation and protected the cellular machinery from

431

photochemical damages, it was obvious that the basic components of biomolecules like protein,


Page 21 of 39


432

AA, reducing sugar and total lipid content would be increased. The amount of reducing sugar

433

was increased in all the treated sets though maximum efficacy was observed @0.05 mg/L.

434

Increase in sugar content, an evidence of enhancement in photosynthesis is shown in SI Figure

435

S12a. Free AA content was elevated in CuNP treated plants with respect to control and their

436

respective bulk counterparts (SI Figure S12b). This was possibly because of the modulatory

437

effect of CuNP on nitrogen assimilation. CuNP directly or indirectly influenced many

438

physiological processes which were interconnected to photochemical reactions in nitrogen

439

metabolism. Thereafter protein (SI Figure S12c) and total lipid content (SI Figure S12d) were

440

also increased in CuNP treated plants compared to its control and bulk counterpart.

441

CuNP in oxidative stress

442

In plants, reactive oxygen species (ROS) are continuously produced as byproducts in various

443

metabolic pathways those are localized in different cellular compartments. Under physiological

444

steady-state conditions, these molecules are scavenged by different antioxidative defense

445

components those are often confined to particular compartments.40 Catalase (CAT) is involved in

446

scavenging hydrogen peroxide during photorespiration, β-oxidation of fatty acids. In both leaves

447

and root CAT activity was unaltered after CuNP treatment with respect to control; however in

448

higher doses of CS, it was decreased in alarming rate (Figure 7a). This result justified that even

449

after CuNP treatment stress response was minimum. Peroxidase (POD) participates in

450

developmental processes, ethylene biosynthesis and defense. POD levels were almost unchanged

451

between control and CuNP treated sets; while CS showed drastic changes with respect to control

452

(Figure 7b). Therefore in CuNP treated plants tissues (both root and leaf) were not significantly

453

altered maintaining normal homeostasis into the metabolic system; ROS mediated damage was



Page 22 of 39

454

insignificant. Superoxide dismutase (SOD) represents a group of multimeric metalloenzyme

455

catalyzing the disproportion of superoxide free radical, generated by univalent reduction of

456

molecular oxygen to hydrogen peroxide in different cellular compartments. No significant

457

deviations of enzymatic activities with respect to control were observed at CuNP treated plants at

458

recommended doses as observed in Figure 7c. GR is a member of flavoenzymes family which

459

catalyzes the NADPH dependent reduction of glutathione disulphide to glutathione. In CuNP

460

treated sets GR levels showed minor alterations with respect to control; while CS showed drastic

461

changes (Figure 7d). In both root and leaves after CuNP treatment GR activities were increased

462

with respect to control but not in a dose dependant manner, because of the high affinity of Cu to

463

bind with –SH group of the protein moiety. Non-antioxidative protein components like phenol

464

(SI Figure S13a), total peroxide (SI Figure S13b), proline (SI Figure S13c) and polyphenol

465

oxidase (SI Figure S13d) (PPO) were also determined as plants have a general strategies to

466

induce the activities of these non-antioxidative proteins to overcome the oxidative stress due to

467

imposition of abiotic stress. Except proline, no abnormal activities were observed in CuNP

468

treated root and leaf tissues. Proline is a known stress marker which counterbalances the

469

detrimental effect of stress to maintain the equilibrium of the cellular machineries. So the

470

elevated activities of proline in CuNP treated sets helped to surpass the possible harmful effect of

471

plants as enforced by the nanoparticle action. It ensured the protective baseline for the plant from

472

any abiotic stress that had been impaired at high concentration of CS to the system. Direct

473

exposure or release of Cu2+ from CS could result oxidative stress and damage within cellular

474

network, but deliberate embedding within polymeric matrix resulted controlled release of Cu

475

from CuNP to yield the beneficial response as a micronutrient for crop management.


Page 23 of 39


476

In a nutshell, biochemical and physiological implication as well as efficacy of newly synthesized

477

polymer encapsulated CuNP on plant system had been established in comparison to its bulk

478

counterpart CS. It not only promised to improve the crop productivity by modulating

479

photochemical pathway, but also decimated the nutritional disorder by catalyzing the nitrogen

480

assimilatory pathway and carbon assimilation. It outclassed the toxicity issues developed by the

481

commercially available salt CS, as it was biosafe at the physiological dosages used in these

482

experiments. Ready uptake within plant system justified its bioavailability; at the same time

483

strategic encapsulation within biocompatible polymer matrix reduced the toxic response and

484

engrafted stability to some extent. At recommended dosages its efficacy in photosynthesis and

485

biocompatibility was ensured in contrast to its bulk counterpart CS; cellular machineries were

486

protected as well. Moreover polymer encapsulated CuNP might serve as a boon in agricultural

487

arena being a suitable alternative of copper micronutrient and hence it could be included in

488

fertilizers amalgamated with other necessary macro- and micronutrients. We believe systematic

489

modification in nanoparticle synthesis can lead to more of such beneficial response in near future

490

for crop management. Detailed toxicological profiling and thorough testing are also required

491

prior to its commercialization but it is an initial footstep which can provide a baseline for future

492

research for sustainable development in agri-economics.

493

AUTHOR INFORMATION

494

Corresponding Author

495

*Saheli Pradhan.

496

E-mail:[email protected], Tel/Fax: +91-332577-3227/+91-332577-3049.

497

ACKNOWLEDGEMENT



Page 24 of 39

498

This work was generously supported by major grants from the Department of Biotechnology,

499

Government

500

BT/BIPP0439/11/10). We are grateful to the National Agricultural Innovation Project (ICAR)

501

(grant nos. NAIP/Comp-4/C3004/2008-2014), the National Fund (Indian Council of Agricultural

502

Research) (grant no. NFBSFARA/GB-2019/2011−2015; NFBSFARA/CA-4-15/2013-146) and

503

ISI plan project funds (2008−2013) for providing financial support. We acknowledge Mr. Ashim

504

Dhar for his assistance during plant experiments. SM is thankful to CSIR, New Delhi for SRF.

505

PP acknowledges UGC-DSK postdoctoral fellowship.

506

ASSOCIATED CONTENT

507

Supporting Information. This information is available free of charge via the Internet at

508

http://pubs.acs.org.

509

REFERENCES

510

(1) Fernandes, J. C. and Henriques, F. S. Biochemical, Physiological, and Structural Effects of

511

Excess Copper in Plants. Bot Rev. 1991, 57, 246-273.

512

(2) Macsymiec, W. Effect of copper on cellular processes in higher plants. Photosynthetica 1997,

513

34, 321-342.

514

(3) Babu, T. S., Marder, J. B., Tripuranthakam, S., Dixon, D. G. and Greenberg, B. M.

515

Synergistic effects of a photooxidized polycyclic aromatic hydrocarbon and copper on

516

photosynthesis and plant growth: evidence that in vivo formation of reactive oxygen species is a

517

mechanism of copper toxicity. Environ Toxicol Chem. 2001, 20, 1351–1358.

of

India

(DBT)

(Grant

No:

BT/

PR15217/NNT/28/506/2011-2015;


Page 25 of 39


518

(4) Pradhan, S., Patra, P., Das, S., Chandra, S., Mitra, S., Dey, K. K., Akbar, S., Palit, P. and

519

Goswami, A. Photochemical Modulation of Biosafe Manganese Nanoparticles on Vigna radiata:

520

A Detailed Molecular, Biochemical, and Biophysical Study. Environ Sci Technol. 2013, 47,

521

13122–13131.

522

(5) Yang, F., Liu, C., Gao, F., Su, M., Wu, X., Zheng, L., Hong, F. and Yang, P. The

523

improvement of spinach growth by nano-anatase TiO2 treatment is related to nitrogen

524

photoreduction. Biol. Trace Elem. Res. 2007, 119, 77–88.

525

(6) Lin, D. and Xing, B. Phytotoxicity of nanoparticles: inhibition of seed germination and root

526

growth. Environ. Pollut. 2007, 150, 243–250.

527

(7) Doshi, R., Braida, W., Christodoulatos, C., Wazne, M. and O’Connor, G. Nano-aluminum:

528

transport through sand columns and environmental effects on plants and soil communities.

529

Environ. Res. 2008, 106, 296–303.

530

(8) Xiong, Z. T., Liu, C. and Geng, B. Phytotoxic effects of copper on nitrogen metabolism and

531

plant growth in Brassica pekinensis Rupr. Ecotox Environ Safe. 2006, 64, 273–280.

532

(9) Dimkpa, C. O., Mclean, J. E., Latta, D. E., Manangon, E., Britt, D. W., Johnson, W. P.,

533

Boyanov, M. I. and Anderson, A. J. CuO and ZnO nanoparticles: phytotoxicity, metal speciation,

534

and induction of oxidative stress in sand grown wheat. J. Nanopart. Res. 2012, 14, 1125/1-15.

535

(10) Llorens, N., Arola, L., Blade´, C. and Mas. A. Effects of copper exposure upon nitrogen

536

metabolism in tissue cultured Vitis vinifera. Plant Science 2000, 160, 159–163.

537

(11) Wong, M. H. and Bradshaw, A. D. A comparison of the toxicity of heavy metals, using root

538

elongation of rye grass, Lolium perenne. New phytol. 1982, 91, 255-261



539

(12) Johnson, A. C. and Naresh. Influence of Chelation on Cu Distribution and Barriers to

540

Translocation in Lolium perenne. Environ. Sci. Technol. 2013, 47, 7688−7695.

541

(13) Arnon, D. I. Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris.

542

Plant Physiol. 1949, 24(1), 1-15.

543

(14) Davies, B. H. Analysis of carotenoid pigments. In Chemistry and Biochemistry of Plant

544

Pigments (Goodwin, T.W. ed) Academic Press: New York, 1965.

545

(15) Heinemeyer, J., Eubel, H., Wehmhöner, D., Jänsch, L. and Braun, H. P. Proteomic

546

approach to characterize the supramolecular organization of photosystems in higher plants.

547

Phytochem. 2004, 5, 1683-92.

548

(16) Lin, S. T., Guan, B. T. and Chang, T. Y. Fitting Photosynthesis Irradiance Response Curves

549

with Nonlinear Mixed-effects Models. Taiwan J. For Sci. 2008, 23, 55-69.

550

(17) Tripathy, B. C. and Mohanti, P. Zinc-inhibited Electron Transport of Photosynthesis in

551

Isolated Barley Chloroplasts. Plant Physiol. 1980, 66, 1174-1178.

552

(18) Vishniac, W. Methods for study of the Hill reaction. Methods Enzymol. (Colowick, S. P. and

553

Kaplan, N.O.eds.) Academic Press, New York, 1957, 4, 342-343.

554

(19) Terry, N. and Huston, R. P. Effects of Calcium on the Photosynthesis of Intact Leaves and

555

Isolated Chloroplasts of Sugar Beets. Plant Physiol. 1975, 55, 923-927.

556

(20) Wang, P., Duan, W., Takabayashi, A., Endo, T., Shikanai, T., Ji-Yu, Y. and Hualing, M.

557

Chloroplastic NAD(P)H Dehydrogenase in Tobacco Leaves Functions in Alleviation of

558

Oxidative Damage Caused by Temperature Stress. Plant Physiol. 2006, 141, 465–474.


Page 26 of 39

Page 27 of 39


559

(21) Taylor, S. T., Terry, N. and Huston. R. Limiting Factors in Photosynthesis, III. Effects of

560

iron nutrition on the activities of three regulatory enzymes of photosynthetic carbon metabolism.

561

Plant Physiol. 1982, 70, 1541-1543.

562

(22) Cataldo, D. A., Haroon, M., Schrader, L. E. and Young, V. L. Rapid colorimetric

563

determination of nitrate in plant tissue by nitration of salicylic acid. Comm Soil Sci Plant Anal.

564

1975, 6, 71-80.

565

(23) Giannopolitis, C. N. and Ries, S. K. Superoxide dismutase I. Occurrence in higher plants.

566

Plant Physiol. 1977, 59, 309-314.

567

(24) Chance, B. and Maehly, A. C. Assay of catalases and peroxidases. Methods in Enzymol.

568

(Colowick, S.P. and Kaplan, N.O. eds) 2, Academic Press, New York, 1955, 764–775.

569

(25) Thurman, R. G., Ley, H. G. and Scholz, R. Hepatic microsomal ethanol oxidation hydrogen

570

peroxide formation and the role of catalase. Eur. J. Biochem. 1972, 25, 420–430.

571

(26) Wang, C. Y. Temperature Preconditioning Affects Glutathione Content and Glutathione

572

Reductase Activity in Chilled Zucchini Squash. J. Plant Physiol. 1995, 145, 148-152.

573

(27) Beers, R. F. J. R. and Sizer, I. W. A spectrophotometric method for measuring the

574

breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952, 195, 133-140.

575

(28) Mayer, A. M. and Harel, E. Polyphenol oxidase in plants. Phytochem. 1979, 18, 193–215.

576

(29) Bates, L. S., Waldren R. P. and Teare, I. D. Rapid determination of free proline for water-

577

stress studies. Plant and Soil. 1973, 39, 205-207.

578

(30) Malik, C. P. and Singh, M. B. Plant enzymology and histo-enzymology. Kalyani Publishers:

579

New Delhi, 1980, 53.



Page 28 of 39

580

(31) Baszyński, T., Ruszkowska, M., Król, M., Tukendorf, A. and Wolińska, D. The effect of

581

copper deficiency on the photosynthetic apparatus of higher plants. Z Pflanzenphysiol. 1978, 89,

582

207–216.

583

(32) Stange, C. and Flores, C. Carotenoids and Photosynthesis - Regulation of Carotenoid

584

Biosyntesis by Photoreceptors, Advances in Photosynthesis - Fundamental Aspects (Najafpour,

585

M. ed). ISBN: 978-953-307-928-8, InTech Publisher, 2012.

586

(33) Maxwell, K. and Johnson, G. N. Chlorophyll fluorescence- a practical guide. J. Exp. Bot.

587

2000, 51, 659-668.

588

(34) Jegerschöld, C., MacMillan, F., Lubitz, W. and Rutherford, A. W. Effects of Copper and

589

Zinc Ions on Photosystem II Studied by EPR Spectroscopy. Biochemistry 1999, 38, 12439-

590

12445.

591

(35) Snowball, K. and Robson, A. D. The effect of copper on nitrogen fixation in subterranean

592

clover (Trifolium subterraneum) New Phytol. 1980, S5, 63-72.

593

(36) Abraham, Z. H. L., Lowe, D. J. and Smith, B. E. Purification and characterization of the

594

dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B.

595

11015): evidence for the presence of both type 1 and type 2 copper centres Cu in nitrogen

596

metabolism. Biochem. J. 1993, 295, 587-593.

597

(37) Lee, W. M., An, Y. J., Yoon, H. and Kweon, H. S. Toxicity and bioavailability of copper

598

nanoparticles to terrestrial plants Phaseolus radiatus (mungbean) and Triticum aestivum (wheat);

599

plant agar test for water-insoluble nanoparticles. Environ. Toxicol. Chem. 2008, 27, 1915–1921.


Page 29 of 39


600

(38) Manceau, A., Nagy, K., Marcu, M., Lanson, M., Geoffroy N., Jacquet, T. and

601

Kirpichtchikova, T. Formation of Metallic Copper Nanoparticles at the Soil-Root Interface.

602

Environ. Sci. Technol. 2008, 42, 1766–1772.

603

(39) Yruela, I. Copper in plants. Braz. J. Plant Physiol. 2005, 17, 145-156.

604

(40) Shigeoka, S., Ishikawa, T.,

605

Yoshimura, Y. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 2002,

606

53, 1305-1319.

Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y. and

607

608

609

Figure Captions

610

Figure 1. (a) FESEM image, (b) TEM image, (c) UV-Vis spectra and (d) FTIR spectra of CuNP.

611

Figure 2. (a) Morphology of control and treated plants after 15 days of treatment.(b) CuNP and

612

CS on chlorophyll content of 15days treated mung bean plants. Chlorophyll a: F= 76.5579, P

CuNP - dskpdf

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