manganese nanoparticles: impact on non-nodulated plant...

Manganese Nanoparticles: Impact on Non-nodulated Plant as aPotent Enhancer in Nitrogen Metabolism and Toxicity Study both inVivo and in VitroSaheli Pradhan,*,† Prasun Patra,§ Shouvik Mitra,† Kushal Kumar Dey,# Sneha Jain,† Samapd Sarkar,†

Shuvrodeb Roy,† Pratip Palit,⊥ and Arunava Goswami†

†Biological Sciences Division, Indian Statistical Institute, 203 B.T. Road, Kolkata 700108, India§Centre for Research in Nanoscience and Nanotechnology, University of Calcutta, Kolkata 700098, India#Department of Statistics, University of Chicago, Chicago, Illinois 60637, United States⊥Plant Physiology Section, Central Research Institute for Jute and Allied Fibers, Indian Council of Agricultural Research, Barrackpore,Kolkata 700120, India

*S Supporting Information

ABSTRACT: Mung bean plants were grown under controlled conditions and supplemented with macro- and micronutrients.The objective of this study was to determine the response of manganese nanoparticles (MnNP) in nitrate uptake, assimilation,and metabolism compared with the commercially used manganese salt, manganese sulfate (MS). MnNP was modulated to affectthe assimilatory process by enhancing the net flux of nitrogen assimilation through NR-NiR and GS-GOGAT pathways. Thisstudy was associated with toxicological investigation on in vitro and in vivo systems to promote MnNP as nanofertilizer and canbe used as an alternative to MS. MnNP did not impart any toxicity to the mice brain mitochondria except in the partial inhibitionof complex II−III activity in ETC. Therefore, mitochondrial dysfunction and neurotoxicity, which were noted by excess usage ofelemental manganese, were prevented. This is the first attempt to highlight the nitrogen uptake, assimilation, and metabolism in aplant system using a nanoparticle to promote a biosafe nanomicronutrient-based crop management.

KEYWORDS: manganese nanoparticles, nitrogen metabolism, manganism, mitochondrial dysfunction

■ INTRODUCTION

Nitrogen assimilation is a vital process controlling plant growthand development. Plants biosynthesize both the suite ofnitrogenous compounds that are present in all living organismsandmany chemical species unique to plants. The most importantand rate-limiting step is the assimilation of inorganic nitrogeninto organic metabolites catalyzed by a series of enzymes througha high-flux process subjected to extensive regulation in all plants.1

Nitrogen moves along a complex branching and mergingpathway, which interacts at numerous sites with the carbonflow, pH regulation, and ions, assimilating flow at the cell withinwhole plant level. Superimposed on these metabolic fluxes,nitrate and nitrogen metabolism influences plant developmentand architecture including changes in root physiology, the timingof senescence, and flowering.2 The largest requirement ofnitrogen is the synthesis of amino acids, which function as thebuilding blocks of proteins as well as precursors to many othermetabolites. It is also prerequisite to nucleic acid, which is acofactor to many physiological processes and amajor componentof chlorophyll.3 In addition, several plant hormones containnitrogen and/or nitrogenous precursors.4 Plants can take upnitrogen from soil by roots in the form of nitrate ion (NO3

−) andutilize it in nitrogen metabolism. Assimilation of NO3

− requiressubsequent uptake, reduction, and conversion of NO3

− toammonium ion (NH4

+) and incorporation to organic com-pounds.5 However, the availability of nutrients includingnitrogen within the plant system is the limiting factor for its

productivity. Henceforth, greater attention is demanded for cropimprovement within the realm of nitrogen metabolism in bothnodulated and non-nodulated plants. Presently several nano-particles have been designed to work in agricultural industry bychanging their size, shape, and surface functionality. Most of theworks demonstrated the improvement of the biocidal activity of acompound so that it can break the resistance of pests andfungi,6−9 whereas others showed detrimental roles in plantphysiology and growth.10,11 Little attention has been paid in cropimprovement to modulating the structural or functionalproperties of existing plant nutrients for the atonement ofplant nutritional deficiency. It has already been established thatplant cells have the capacity to execute active transport of NH4

+

during nitrogen assimilation. Some literature has shown that lowavailability of NO3

− concentration results from the decrease inthe rate of nitrogen assimilation in acidic soil.12 Meanwhile,manganese salt (Mn), as a fertilizer for nitrogen metabolism, hasbeen used in acidic soil for many years, which also influences theassimilatory process as a cofactor.13

In this context, we introduce manganese nanoparticles(MnNP) within the framework that is already known tomodulate the photochemical pathways in photosynthesis evenbetter than its elemental/bulk counterpart.14 MnNP increase the

Received: February 3, 2014Accepted: August 15, 2014Published: August 15, 2014

Article

pubs.acs.org/JAFC

© 2014 American Chemical Society 8777 dx.doi.org/10.1021/jf502716c | J. Agric. Food Chem. 2014, 62, 8777−8785

pubs.acs.org/JAFC

activity of the electron transport chain by binding with the CP43protein chain of photosystem II.14 They also enhance the oxygenevolution process, being a part of the water splitting complex inlight reaction of photosynthesis; as a consequence, photo-phosphorylation capacity is improved, too.14 MnNP thusimprove the photosynthetic ability of the plants, therebyproducing more sugar, and can be useful in the agriculturalsector for the improvement of plant productivity. As anafterthought, we have tried to investigate the route of nitrateuptake, assimilation, ammonium assimilation, and furtherdownstream nitrogen metabolism in both source and sinktissues of legumes usingMnNP to abate the problems of nitrogenmetabolism. Thereafter, nitrogen reductase protein has beenisolated from leaf tissue of treated plants followed by proteinprofiling andWestern blot analysis to evaluate themode of actionof MnNP in accelerated nitrogen assimilation. Interestingly,MnNP are found to be more effective by both biochemical andmolecular techniques in nitrogen assimilation and metabolism incontrast to their bulk counterpart, manganese sulfate (MS). Tothe best of our knowledge this is the first effort to use MnNP inthe process of nitrogen metabolism for beneficial cropmanagement study.Mn is an essential trace element for human nutrition but also a

toxicant at high concentration. Therefore, its biosafety impacthas to be evaluated in light of in vivo experimentation for futureresearch and development studies. As a consequence, MnNPhave been injected intravenously to Swiss albino mice, and after15 days of observation, the animals were sacrificed for biosafetystudies following standard guidelines. Mn is also known toinduce mitochondrial dysfunction and causes neurotoxicity,called manganism.15 Manganism is characterized by neuro-psychiatric symptoms and extrapyramidal dysfunction such ashypokinesia, rigidity, and tremor that often resemblesParkinson’s disease.16 Therefore, detailed toxicity profiling ofMnNP is required related to mitochondrial dysfunction andneurotoxicity; as a result, the effect of MnNP in energymetabolism and ETC activity in isolated brain mitochondriahas been assessed. Biosafety studies with newly synthesizedengineered nanoparticles are important because they may befound to be hazardous for biological systems; if it is related to theagricultural sector, then it is necessarily obligatory to profile theentire biosafety experiments, which ensure the completescreening of the particles in mammalian systems. In a recentwork MnNP have been found to decimate the level of dopamine,imparting mitochondrial dysfunctioning and generation of moreROS in vivo as a result of metal toxicity in PC-12 cells such as freeMn2+ ions.17 Besides, there are several studies on metal toxicityperformed in cell lines18−20 and mammalian systems21−24 toaccess the biological importance and environmental hazardmeasurements. In our previous paper we have reported that ourcustom-made MnNP are biosafe on the basis of acute oraltoxicity parameter, although detailed research is explicitlydemanded for its future endeavor in agricultural sectors. Herein,histopathological analysis of MnNP-treated mice is performedafter intravenous injection with emphasis on brain mitochondrialtoxicity studies. A series of detailed in vitro and in vivo studiesincluding studies on human lymphocytes, hemolysis, and murinemodel system with an emphasis on brain mitochondria has beencarried out. Our studies reveal that severe toxicity symptoms areavoided in the treated sets with minor alterations noted incomplex II−III activity in ETC. Excess oxidative stress, severemitochondrial dysfunction, and neurotoxicity are avoided,thereby paving the way toward the development of a biosafe

crop management study based on nitrogen assimilation andmetabolism with the aid of MnNP.

■ MATERIALS AND METHODSCharacterization of MnNP. Custom-made manganese nano-

particles were purchased from MK Implex, Canada. The surfacemorphology was determined by field emission scanning electronmicroscope (FESEM; FEI Quantum-200 MK-2), and particle size wasobserved through a transmission electron microscope (TEM) (JEOL2010). Surface topology of MnNP was obtained using atomic forcemicroscopy (AFM). Hydrodynamic size distribution was obtained bydynamic light scattering (DLS) spectroscopy.

Experiments with Plant Model System. Seeds of mung bean(Vigna radiata) were purchased from Berhampur Pulse and OilResearch Centre, West Bengal. These seeds were selected from Chaitimung. Seeds were soaked in 5% sodium hypochloride solution forsurface sterilization and imbibed with the treatment solutions (control;MnNP, 0.05, 0.1, 0.5, and 1 mg/L; MS, 0.05, 0.1, 0.5, and 1 mg/L) for aminimum of 4−6 h prior to germination for various analyses. After 24 hof germination, seeds were planted in pots filled with perlitesupplemented with Hoagland’s solution with or without manganesesolution (control plants were treated without manganese solution) for15 days in a growth cabinet (GC-300, Lab companion) with 14 hdaytime temperature of 25 °C and night temperature of 20 °C at arelative humidity of 40−60% and a light intensity of 440 μmol/m2/s.After 15 days of treatments, plants were uprooted and used to carry outfurther experiments.

Preparation of Leaf and Root Enzyme Extract for the Assaysof Nitrogen Metabolism. Plant samples were ground with a ratio of1:10 (w/v) in 50 mM phosphate buffer (pH 7.5) containing 2 mMEDTA, 1.5% (w/v) soluble casein, 2 mMDDT, and 1% (w/v) insolublePVPP at cold. The homogenate was filtered and centrifuged at 3000g for5 min. Supernatant was then centrifuged at 30000g for 20 min. Theextract was then used for enzymatic assays.

Determination of NO3−. Enzyme extract was added to 10% salicyclic

acid solution dissolved in 12 N sulfuric acid (w/v). NO3− content was

measured as described by Cataldo et al.25 The results were expressed asmicromoles per gram of fresh weight.

Determination of Nitrate Reductase (NR) Activity. The reactionmixture contained 100 mM phosphate buffer (pH 7.5), 100 mMKNO3,10 mM cysteine, 2 mM NADH, and enzyme extract. After 30 min ofincubation at 30 °C, reaction was stopped by 1 M zinc acetate. Thenitrite formed was determined at 540 nm after azocoupling withsulfanilamide and naphthylethylenediamine dihydrochloride. The NRactivity was expressed as micromoles of NO2

− formed per gram freshweight per hour.

Determination of Nitrite Reduction (NiR) Activity. The reactionmixture contained 50 mM phosphate buffer (pH 7.5), 20 mM KNO2, 5mM methyl viologen (MV), 300 mM NaHCO3, and enzyme extract.After 30 min of incubation at 30 °C, NiR activity was determined by thedisappearance of NO2

− from the reaction medium and expressed asmicromoles of NO2

− per gram of fresh weight per hour.Determination of Glutamine Synthetase (GS) Activity. Enzyme

extract was mixed with the reaction mixture containing 100 mMphosphate buffer (pH 7.5), 4 mM EDTA, 1 M sodium glutamate, 450mM MgSO4·7H2O, 300 mM hydroxylamine, and 100 mM ATP. Twocontrols were prepared, one without glutamine and another withouthydroxylamine, and all of the treatments were incubated for 30min at 28°C. The formation of glutamylhydroxamate was measured at 540 nmafter complexing with acidified ferric chloride. GS activity was expressedas formed glutamylhydroxamate in grams of fresh weight per hour.

Determination of NADH-Glutamate Synthase (GOGAT) Activity.The reaction mixture contained 50 mM phosphate buffer (pH 7.5) with0.1% (v/v) mercaptoethanol, 1 mM EDTA, 18.75 mM 2-oxoglutarate,15 mM aminoxyacetate, 1.5 mM NADH, 75 mM L-glutamine, andenzyme extract. Two controls, without ketoglutarate and withoutglutamine, were used to correct for endogenous NADH oxidation. Thedecrease in absorbance was recorded for 5min at 28 °C. The activity wasexpressed as micromoles of NAD per gram fresh weight per hour.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf502716c | J. Agric. Food Chem. 2014, 62, 8777−87858778

SDS-PAGE andWestern Blot of Isolated NR Protein.NRprotein waspurified from mung bean leaves following a standard procedure.26 NRprotein was dissolved in a buffer containing 0.09% (w/v) n-decyl-β-D-maltopyranoside (β-DM), 20 mM NaCl, 70% (v/v) glycerol, and 20mM HEPES (pH 7.5). After isolation of the protein, 12% SDS-PAGEwas performed using a Bio-Rad protein gel apparatus, and subsequentWestern blot analysis was also carried out by using NR antibody(Agrisera).Experiments with Mouse Model System. Healthy young

nulliparous, Swiss albino mice (average body weight = 20 g) of 8weeks of age were kept in the animal house at 22 ± 2 °C), 60 ± 10%relative humidity, and 12 h light/dark cycle. The animals were kept inclean polypropylene cages and were provided with commercial rat pelletdiet and deionized water. After 7 days of acclimatization, the mice wererandomly assigned to control and treatment groups. There were fivemice of each sex in each group. Water as control andMnNP as treatmentwere injected intravenously, and animals were weighed before and afterthe completion of the experiment. The animals were kept under closeobservation for 14 days. Skin and fur changes, eye secretion, andbehavior patterns of the mice were observed. Special attention was paidto the clinical signs of toxicity including tremors, convulsions, salivation,nausea, vomiting, diarrhea, lethargy, etc. At the end of 14 days, they weresacrificed. Blood and serum from control and treated mice wereanalyzed for total count (TC), differential count (DC), platelet count(PLT), lactate dehydrogenase (LDH), creatinine, alkaline phosphatase(ALP), total protein (TP), cholesterol, triglyceride (TG), uric acid,blood urea nitrogen (BUN), serum glutamic oxaloacetic transaminase(SGOT), serum glutamic pyruvic transaminase (SGPT), andphosphorus. Brain, heart, lungs, liver, stomach, kidney, spleen, uterus,and testis were carefully removed and fixed in 10% formalin solutioncontaining neutral phosphate-buffered saline (PBS). Thereafter, theorgans were embedded in paraffin, stained with eosin−hematoxylin, andexamined under a light microscope.Isolation of Mice Brain Mitochondria. Mouse brain mitochondria

(after 24 h of treatment) were isolated by a method based on differentialcentrifugation with digitonin treatment. The final mitochondrial pelletwas suspended in isotonic buffer A (145 mM KCl, 50 mM sucrose, 1mM EGTA, 1 mM magnesium chloride, 10 mM phosphate buffer, pH7.4) and used immediately for the experiments related to measurementof membrane potential and phosphorylation capacity.27 For themeasurement of activity of respiratory complexes, the mitochondrialpellet was suspended in 50 mM phosphate buffer, pH 7.4, stored at 20°C, and used within 3 days.Measurement of Transmembrane Potential of Mouse Brain

Mitochondria. Mitochondrial transmembrane potential was measuredby using a mitochondrial membrane potential sensitive carbocyaninedye, 5,5′,6,6′-tetrachloro-1,10,3,30-tetraethylbenzamidazolocarbocya-nine iodide (JC-1). When excited at 490 nm, the monomeric form ofJC-1 had an emission maximum at 527 nm, but the aggregated form (J-aggregates) showed an emission maximum at 590 nm. Briefly, an aliquotof mitochondria (control or MnNP treated) was incubated at 37 °C for30 min in isotonic buffer A containing 10 mM pyruvate, 10 mMsuccinate, and 1 mM ADP in the presence of 5 μM JC-1. At the end ofthe incubation, the dye-loaded mitochondria were collected bycentrifugation and resuspended in the same buffer in appropriatedilution, followed by the measurement of fluorescence intensity (λex 490nm, λem 590 nm) in a spectrofluorometer.Measurement of Phosphate Utilization of Mitochondria. In a total

volume of 250 μL, an aliquot of 25 μL of mitochondrial suspension fromcontrol or MnNP-treated mitochondria was diluted into a mediumcontaining 125 mMKCl, 75 mM sucrose, 0.1 mM EGTA, 1 mMMgCl2,10 mMHEPES, 2 mM phosphate, 0.3% bovine serum albumin, 0.5 mMADP, 5 mM pyruvate, 10 mM succinate, and 10 mM glucose, followedby the immediate addition of 5 units of hexokinase, and the incubationwas continued at 37 °C for 30 min. The incubation was terminated bythe addition of 5% ice-cold trichloroacetic (TCA) acid, and the amountof inorganic phosphate was estimated spectrophotometrically. A 0 minsample was also assayed for inorganic phosphate content wherehexokinase addition was immediately followed by treatment with 5%ice-cold TCA. Glucose and hexokinase in the reaction mixture acted as a

trap for ATP to maintain the level of ADP in the system and also toprevent the release of free inorganic phosphate from ATP by the actionof various phosphatases.

Measurement of Respiratory Chain Activity of Isolated BrainMitochondria after MnNP Treatment. The activity of NADH−ferricyanide reductase (complex I) was assayed by using ferricyanide asthe electron acceptor in a system containing 0.17 mM NADH, 0.6 mMferricyanide, and Triton X-100 (0.1% v/v) in 50 mM phosphate buffer,pH 7.4, at 30 °C. The reaction was initiated by the addition ofmitochondrial suspension (10−30 μg of protein) to the sample cuvette,and the rate of oxidation of NADH was measured by the decrease inabsorbance at 340 nm.

The activity of succinate cytochrome c reductase (complex II−III)was assayed by following the succinate-supported reduction offerricytochrome c to ferrocytochrome c at 550 nm in an assay mixturecontaining 100 mM phosphate buffer, 2 mM succinate, 1 mM KCN, 0.3mM EDTA, and 1.2 mg/mL cytochrome c in a total volume of 1 mL.The reaction was initiated by adding mitochondrial suspension (10−30μg) into the sample cuvette.

The activity of cytochrome c oxidase (complex IV) was assayed bymeasuring the rate of decrease in absorbance at 550 nm at roomtemperature following the oxidation of reduced cytochrome c (50 μM)in 10 mM phosphate buffer, pH 7.4. Ferricyanide (1 mM) was added tooxidize ferrocytochrome c in the blank cuvette and the reaction initiatedin the sample cuvette by the addition of mitochondrial suspension (10−30 μg). The activity of the enzyme was calculated from the first-orderrate constant.

(a) Measurement of Mitochondrial Respiratory Control Ratio(RCR). Polarographic study of oxygen consumption using freshlyisolated mitochondria allows measurement of respiratory control ratios(RCR) calculated as the ratio of state III to state IV. The chamber of theoxygraph was filled with fresh respiratory buffer along with ADP,glutamate, pyruvate, maleate, succinate, and DNP. A low (state IV)oxygen consumption rate was achieved. After that, 100−200 nmol withor without treated mitochondria was added for inducing state IIIrespiration. After 2 min, the data were recorded, and RCRwas calculatedaccordingly.

(b) Measurement of Reactive Oxygen Species (ROS). ROSgeneration in treated and untreated brain was determined using thenitrotetrazolium blue (NBT) reduction assay.28 The assay is based onthe reduction of yellow water-soluble NBT powder to a blue insolublesubstance upon reduction. The treated and control brains wereincubated in NBT solution for 1 h. The reaction was stopped by theaddition of acetic acid solution. After centrifugation (1 min, 12000g),intracellular reduced content was solubilized by adding 50% (v/v) aceticacid to the cell pellet followed by vortexing for 5 min. Cell debris wasthen pelleted, and the absorbance of the supernatant was determined at595 nm using a UV−vis spectrophotometer.

(c) Protein Estimation. The protein was estimated after themembranes had been solubilized in 1% SDS according to Lowry’smethod.

Experiments with Lymphocytes. Isolation of Lymphocyte fromBlood. Human peripheral blood was obtained by venipuncture fromhealthy volunteers into heparinized vacutainers. Lymphocytes wereisolated from fresh blood according to themethod of Boyum et al.29 withsome modifications using Histopaque.30 The cells were washed withPBS and resuspended in RPMI-1640 medium at a concentration of 106

cells mol−1 for further use.Treatment of Lymphocytes. Freshly isolated human lymphocytes

were treated with different concentrations ofMnNP and incubated for atleast 3 h at 37 °C in RPMI-1640 medium. After incubation, cytotoxicitystudies were carried out with treated lymphocytes.

(a) Cell Proliferation Assay (WST).The number of mononuclear cellsremaining alive after reacting with the cellular mitochondrialdehydrogenase could be quantified by using a Biovision cell proliferationassay kit bymeasuring the absorbance of the dye solution at 440 nm. Theformazan dye is produced by the breakdown of tetrazolium salt, and theamount of dye generated by the activity of dehydrogenase was directlyproportional to the number of living cells.



(b) NO Content.Measurement of NO involved a two-step process; inthe first step, nitrate was converted to nitrite by nitrate reductase, and inthe second step a deep purple azo compound was developed by Griessreagents, supplied externally to the system. The intensity of theazochromophore developed by this process could be measuredspectroscopically to determine the amount of NO present in thesamples.31

(c) LDH Cytotoxicity Assay. LDH cytotoxicity assay was performedusing Cayman’s LDH Cytotoxicity Assay Kit by reducing a tetrazoliumsalt to highly colored formazan, which absorbed strongly at 490−520nm.(d) ROS/RNS Assay. Total ROS/RNS generated within the

treatments could be measured from fluorescence intensity. DCF couldbe used as standard, and the free radical content generated could becompared with the predetermined standard intensity using a Cell BiolabAssay Kit.Hemolysis. To perform in vitro hemolysis experiments, human blood

was centrifuged twice at 3000 rpm for 15 min to remove residual plasmafrom the erythrocyte-enriched fraction of blood. RBCs were washedthree times with PBS, pH 7.4, and resuspended in the same buffer. Sixhundred and ninety microliters of the above RBC stock was suspendedwith 10 μL of MnNP treatments (1, 10, and 25 mg/L) along withcontrol, and then the volume was adjusted to 1.5 mL with PBS for eachof the treatments. The cells were then centrifuged again at 3000 rpm for15 min at 4 °C and washed three times with PBS before measurement at540 nm in a UV−vis spectrophotometer.32Statistical Analysis. We tested for the effect of MnNP and MS for

different biochemical and biosafety features. For each of the biosafetyexperiments, we considered three levels of concentration of thenanoparticles, 1, 10, and 25 mg/L, and compared the effects of thevarious concentrations with respect to the control and also relative toeach other. For each concentration in the design, we had three replicatesand so, the total numbers of experiments conducted for a specificnanoparticle were n = 12 (3 replicates for the 4 levels: control and 1, 10,and 25 mg/L). Although the data size was small, from a biological pointof view, normality of the replicate data for each experiment could beassumed. Because there were three replicates for each concentration,two errors were attached to each concentration with a total error degrees

of freedom of 8. The degrees of freedom for the prediction space was(number of levels− 1) 3. Therefore, in an ANOVA table, we performedthe F3,8 distribution and calculated the P value corresponding to thisunderlying distribution. In the case of biochemical assays of plants, fourof each MnNP and MS concentrations were taken into account alongwith the control, so we had nine replicates altogether and for this case n= 27 (control, four concentrations of each MnNP and MS).

If the ANOVA F test was found to be significant, then the next stepwould be to check which level significantly affected the response,thereby influencing the F test to reject the null. This was achieved byusing Tukey’s post hoc called HSD (honestly significant difference) test.We used alphabetic labels to check if one level was significantly differentfrom the previous one as concentrations of the nanoparticles wereconsidered as ordered factors. We also observed the box plot responsefor various levels to get a better indicative picture.

■ RESULTSCharacterization ofMnNP. The morphology of MnNP was

investigated by using FESEM and TEM images. Cubic-shapedMnNP of size around 20.88 ± 0.44 nm were more or lesshomogeneously distributed as revealed from FESEM (Figure 1a)and TEM (Figure 1b) images, respectively. It was previouslymentioned that the surface of MnNP contained hydroxyl groups,which contributed to their hydrophilic character. Hydrophiliccharacter was one of the key features of MnNP to study theirinteraction with plant systems.14Although due to the higherreactivity and oxophilicity of Mn,33 synthesis of MnNP was quitedifficult; however, these custom-made MnNP were stable for along time. Hydrodynamic radius measurement revealed ahydrodynamic radius of around 101.5 ± 13.395 nm, whichchanged insignificantly over time (Supporting Information (SI)Figure S1). Surface topology of MnNP was obtained from anAFM micrograph (Figure 1c), which justified the average 10point height of around 6 nm. Chemical purity of thenanoparticles was determined by EDX analysis (SI Figure S2),where Mn was the major component in the sample powder.

Figure 1. Characterization of MnNP: (a) FESEM image; (b) TEM image; (c) AFM micrograph of MnNP.



Aqueous dispersion of MnNPs was fairly stable. No significantchange in the hydrodynamic radius was noted up to 24 h underexperimental condition. Even its XRD pattern was unchanged inaqueous dispersion after 24 h, which corroborated our previousreport (SI Figure S3).14 To produce some response in nitrogenassimilation and metabolism, MnNP should be bioavailable tothe root system. In our previous study we have alreadydemonstrated the bioavailability of MnNP in roots throughconfocal microscopy;14 therefore, bioavailable MnNP on theroot system could execute some response, which also triggered usto focus on a series of biochemical andmolecular assays related tonitrogen assimilation and metabolism discussed below. Small-sized MnNP resulted in a better bioavailability with theirnanosized effect, which was also consistent with the literature.Effect on Nitrogen Metabolism in Treated Plants. The

maximum NO3− concentration was observed at the highest dose

of MS in both root and leaf tissues, whereas a significant decreasein NO3

− concentration was noted in all of theMnNP-treated setsas shown in Figure S4 (SI). The amount of NO3

− present in theroot was more than in the foliar tissue in all treatments. However,there were increases in both NR and NiR activities in root andleaf tissue ofMnNP-treated plants in all treated sets, although the0.05 mg/L MnNP dose was the most effective (Figure 2a,b). Innon-nodulated plants activity of NR-NiR is the initial step fornitrogen metabolism, which is also a determining factor for theassimilation of inorganic nitrogen to organic metabolites in living

tissues. Interestingly, NO3− concentration in 0.05 mg/L MnNP

treatment was lowest but NR and NiR activities were bothmaximum in the aforementioned dose. MnNP possiblyinfluenced the process of enzymatic reduction during the courseof assimilation; hence, NR and NiR activities were enhanced.The responses of GS andGOGAT, in the synthetic process of AAfrom the assimilated product of NR−NiR activities (Figure 2c,d),were similar. Maximum activity was found in 0.05 mg/L MnNPtreatment in both root and leaf tissues, although functions of allof these enzymes were much greater in root tissue compared tofoliar tissue. In all of the experiments, the highest doses of MSwere found to be toxic to the plant tissues as expected.Elevated NR activity was further confirmed by protein

profiling and Western blot techniques shown in Figure S5 (SI).NR, a homodimer or homotetramer of 110 kDa protein, ispresent in shoot mesophyll cells and is tightly regulated to reducenitrate to nitrite.34 It plays a pivotal role in the nitrogenassimilatory pathway in plants. It was interesting to note that NRactivity was increased biochemically; the result was furtherconfirmed by SDS-PAGE and subsequent Western blot analysisof the protein. In both cases MnNP-treated plants showedenhanced activity with respect to control. This justified ourhypothesis that MnNPmodulated the activity of NR protein, thefirst assimilatory enzyme and also presumptive candidateresponsible for nitrate inhibition step of nitrogen fixation.They bind to and inhibit nitrogenase and leghemoglobin,35

Figure 2. Effect of MnNP and MS on nitrogen metabolism of 15-day-treated mung bean plants. (a) NR activity. MnNP leaves: F = 20.42, P < 0.0001.MnNP roots: F = 7.59, P < 0.01. MS leaves: F = 2.64, P < 0.1. MS roots: F = 8.18, P < 0.01. (b) NiR activity. MnNP leaves: F = 14.16, P < 0.01. MnNProots: F = 8.83, P < 0.01. MS leaves: F = 19.95, P < 0.0001. MS roots: F = 8.09. P < 0.01. (c) GS activity. MnNP leaves: F = 39.21, P < 0.00001. MnNProots: F = 23.66, P < 0.0001. MS leaves: F = 31.68, P < 0.0001. MS roots: F = 23.44. P < 0.0001. (d) GOGAT activity. MnNP leaves: F = 20.02, P <0.0001. MnNP roots: F = 122.23, P < 0.0000001. MS leaves: F = 26.03, P < 0.0001. MS roots: F = 223.94 P < 0.000000001. Within each type oftreatment, mean data (±SE, n = 3) followed by the same upper case letter are not significantly different for a particular dose; within each dose, meansfollowed by the same lower case letter are not significantly different (Tukey−Kramer HSD test).



simultaneously increasing the activities of all interlinked proteinsin the course of nitrogen metabolism.Toxicity Study of MnNP with Mouse Model System.

MnNP was injected intravenously at different doses, and themice were sacrificed after 15 days of observation. No significantchanges were noted in general appearance or weight gainbetween control and treated mice during the observationalperiod (SI Table 1). Treated animals did not show histologicalabnormalities except in brain tissue at very high doses (SI FigureS6), and even pathological changes (SI Table 2) were negligible.Thus, MnNP were considered to be safe at the recommendeddosage after 15 days of treatment, which justified itsbiocompatibility for future perspectives. Anomalies in braintissues at higher MnNP concentration forced us to conductdetailed brainmitochondrial assays as it might trigger manganismto the mammalian system. In all toxicity studies we have onlyaccounted for MnNP, as MS is a known toxicant to mammaliansystems at experimental doses according to the literature.15,16

Experiments with Mouse Brain Mitochondria. As shownin Figure 3a, there was a dose-dependent loss of mitochondrialmembrane potential after MnNP treatment, although significantinhibition was observed at the highest dose of 25mg/L. A gradualincrease in inorganic phosphate utilization was observed in dose-dependent manner with MnNP treatment shown in Figure 3b.Complex I activity of treated brain mitochondria (Figure 3c) wasnot significantly altered, but the activities of complex II−III andcomplex IV were significantly increased at the highest dose ofMnNP treatment (Figure 3d,e). MnNP at much higher dosesmight impair the activity of complex II−III and/or complex IV,resulting in a reduction in energy conversion through electrontransport chain (ETC). RCR was left unaltered even after MnNP

treatment, showing no negative influence in the respiratoryprocess as shown in Figure 3f. However, more ROS were foundto be generated at 25 mg/L MnNP treatment (Figure 4),whereas the lowest dose (1 mg/L) showed no significant changein ROS level with respect to control.

Experiments with Lymphocyte and Hemolysis. Figure5a represents the cytotoxicity of mononuclear cells by MnNPtreatment; minor alteration was noted in treated sets with respect

Figure 3. Effect of MnNP on mice brain mitochondria. (a) Mitochondrial membrane potential. (b) Inorganic phosphate utilization. F = 76.18, P <0.00001. (c) Complex I activity. F = 33.41, P < 0.0001. (d) Complex II−III activity. F = 746.35, P < 0.000000001. (e) Complex IV activity. F = 197.69, P< 0.0000001. (f) RCR. F = 43.07, P < 0.0001. Within each type of treatment, mean data (±SE, n = 3) followed by the same upper case letter are notsignificantly different for a particular dose; within each dose, means followed by the same lower case letter are not significantly different (Tukey−KramerHSD test).

Figure 4. Effect of MnNP on ROS content of mice brain mitochondria:F = 27.79, P = 0.0001. Within each type of treatment, mean data (±SE, n= 3) followed by the same upper case letter are not significantly differentfor a particular dose; within each dose, means followed by the samelower case letter are not significantly different (Tukey−Kramer HSDtest).



to control. Meanwhile, in the case of NO content (Figure 5b),LDH cell viability assay (Figure 5c), and ROS generation (Figure5d), no significant alterations were noted between control andtreated sets. Altogether MnNP did not impair dose-dependentcytotoxicity in any of the above-mentioned enzymatic assaysexpect in ROS content, although cytoxicity was seen at higherdoses in treated lymphocytes. Similar results were noted inhemolysis experiments. No significant hemolysis was noted inany of the treated sets (SI Figure S7); the highest dose of MnNPtreatment (25 mg/mL) exhibited 5.31% hemolysis, which wascomparable with the control one (4.96%).

■ DISCUSSIONThis study revealed that MnNP might modulate the nitrogenmetabolism process and thereby could be used as a suitablealternative for MS. MnNP, because of its size and properties, wasabsorbed readily by the root from soil and transported to the leaf,where it acted as a cofactor in the series of enzymatic reactionsduring the assimilation of nitrate salt into organic nitrogencompounds. That was probably the reason it did not participatein the process of NO3

− uptake and the level of NO3− in the plant

tissue was not influenced by uptake of MnNP. Nitrogenmetabolism in plants is a linear pathway that involves the uptakeand subsequent transport of nitrate within plant tissues, followedby nitrogen and ammonium assimilation and ultimately aminoacid and protein synthesis. However, there had been complexitiesrelated to storage and remobilization of nitrate in different partsof the plants: de novo ammonium assimilation and its recycling

in the course of photorespiration;36 unpredictable interactionswith carbon assimilatory pathways, which allowed malate toinhibit alkalization37 and also 2-oxoglutarate to act as the primaryacceptor of ammonium in GOGAT pathways.38 These complex-ities made it difficult to develop new methodologies andinnovations to enrich the status of nitrate assimilation withinthis complex biological network.The first step in nitrogen metabolism was the reduction of

NO3− to NO2

− in the cytosol by NR. MnNP at all doses directlyor indirectly increased NR activity in foliar tissue with respect tocontrol, whereas no significant alterations were noted in roottissue even at the highest dose. Therefore, MnNP could influencethe entry of NO3

− to the cells, resulting in the hyperpolarizationof the plasma membrane. The influence of MnNP over NRactivity was supported by biochemical and molecular techniques.Enhanced expression of NR protein after MnNP treatment withrespect to the control validated our speculation thatMnNP couldenhance protein activity. This in turn improved nitrogen status inthe mung bean crop. It might stimulate NR activity, which wasfurther validated by the enhanced activity of NiR, an enzymerequired for the conversion of NO2

− to NH4+ in the cytosol. Both

NR and NiR were induced by the same factors, explaining theirsimilar responses to the micronutrient treatments.Plants assimilate inorganic nitrogen into nitrogen-transport

amino acids (AAs), glutamate and glutamine, which are destinedto transfer nitrogen from source to sink tissues and to build upreserves during periods of nitrogen availability for subsequentuse in growth, defense, and reproductive processes. It also

Figure 5. Effect of MnNP on human lymphocyte cells. (a) Cytotoxicity assay by WST method. F = 10.57, P < 0.01. (b) NO content. F = 197.69, P <0.000001. (c) Cell viability assay by LDH method. F = 10.57, P < 0.01. (d) ROS generation. F = 21.26, P < 0.001. Within each type of treatment, meandata (±SE, n = 3) followed by the same upper case letter are not significantly different for a particular dose; within each dose means followed by the samelower case letter are not significantly different (Tukey−Kramer HSD test).



decimated the level of ammonium toxicity. The immediate stepthereafter was to convert NH4

+ to amino acid with the help ofglutamine synthetase (GS) and glutamate synthase (2-oxoglutarate aminotransferase, or GOGAT). These two veryimportant enzymes are involved in the primary assimilation ofinorganic nitrogen from soil along with reassimilation of freeammonium into the plants. GS catalyzed the ATP-dependentassimilation of NH4

+ into glutamine using glutamate as asubstrate. Mn is a known cofactor in this reaction, and therebythe presence of MnNP triggered more GS activity and also theactivity of GOGAT. Because GOGAT was known to bestimulated by elevated plastid levels of glutamine duringammonium assimilation, GOGAT was required to produce 2-oxoglutamate, which ultimately produced dicarboxylic acids ascarbon skeletons for glutamate, aspartate synthesis, and theircorresponding amides. A couple of studies demonstrated theinfluence of high GS activity on vegetative growth andphotosynthesis upliftment;39 even earlier flower and seeddevelopment were also observed under accelerated GS activity.40

MnNP triggered enhanced GS activity; thereby, it may functionas an enzymatic cofactor in nitrogen assimilation in non-nodulated plants. It might work as a boon for agriculture as it wasable to overcome the drawbacks of the commercially availableMS salt at higher doses.MnNP was already established as a micronutrient fertilizer

over commercially available MS, but it was well recognized thatexcess use of Mn salt could induce manganism; therefore, thetoxicity issues associated withMnNP could not be ruled out. As aconsequence, we carried out a series of detailed toxicologicalevaluations on mononuclear cells and murine model system atrelatively high dosages (1, 10, and 25 mg/mL) in contrast to theone used in nitrogen assimilation and metabolism. In murinemodel system MnNP was injected intravenously, and after 15days of observation, mice were sacrificed for histopathologicalevaluation. From the results we confirmed that MnNP wasbiosafe for further implementation, although minor anomalieswere noted in brain tissues. Therefore, we deliberately carried outa brain mitochondrial assay in the murine model system in lightof manganism. The assays were carried out after 24 h of injectionfollowed by isolation of brain tissues. In all of the experiments itwas observed that 25 mg/L MnNP partially impaired the activityof complex II−III, but it had no effect in mitochondrial complexI, complex IV protein domains, and RCR activity. This justifiedthat MnNP limited the efficiency of brain mitochondrialfunctions to some extent but did not hamper the entirephysiological process. There was no loss of mitochondrialmembrane potential at any of the dosages; increasedphosphorylation capacity ensured no loss of phosphateutilization and hence ATP synthesis. The key function of themitochondrial ETC was to supply reducing equivalents becausethe majority of electrons were provided by NADH. In ourexperiments, MnNP did not show any alteration in complex Iactivity of brain mitochondrial ETC like it did with its elementalcounterpart. Therefore, MnNP could easily overcome theproblem of inactivation of energy metabolism leading tomitochondrial dysfunction influenced by Mn salts. Partiallyinhibited complex II−III activity decreased electron transferactivity leading to ROS by leaking electrons from ETCcomplexes during respiration, particularly in complex III.In addition, a detailed in vitro cytotoxicity experiment on

human lymphocytes was carried out using MnNP ofaforementioned dosages. No significant cytotoxic response wasfound in any of the treated sets with respect to control as noted

from our detailed systematic evaluation. These results wereconsistent with the in vivo experiments mentioned earlier, whichsignified the biocompatibility of MnNP for biological perspec-tives. Interestingly, hemolysis experiments justified its bio-compatibility as well, which also corroborated the results notedfor mononuclear cells.To the best of our knowledge, this is the first study in which

MnNP has been used to enhance nitrogen metabolism in a plantmodel system. MnNP probably stimulated the activity of NRdirectly or indirectly as noted from biochemical and moleculartechniques. They also enhanced the function of both GS andGOGAT enzyme activities, being an active cofactor in thebiochemical reactions. They not only surpassed the difficultiesraised due to low availability of inorganic ions during nitrogenassimilation but also alleviated enzymatic activities of the samephysiological process. Hence, MnNP can be used as fertilizer forboth carbon and nitrogen assimilatory processes. Biosafetyexperiments were carried out in both mouse and humanmononuclear cells. Brain mitochondria of treated mice wereisolated to check functional activities in respiration and energyproduction. Results demonstrated thatMnNPwere fairly biosafe,although minor toxic responses were noted at a very high dosage.We have good reason to believe that MnNP could be used as afertilizer with great efficiency together with their biocompatibilityrelative to the commonly used salt counterpart, MS.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional figures and tables as noted in the text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*(S.P.) E-mail: [email protected].

FundingThis work was generously supported by major grants from theDepartment of Biotechnology, government of India (DBT)(Grants BT/PR15217/NNT/28/506/2011-2015; BT/BIPP0439/11/10). We are grateful to the National AgriculturalInnovation Project (ICAR) (Grant NAIP/Comp-4/C3004/2008-2014), the National Fund (Indian Council of AgriculturalResearch) (Grants NFBSFARA/GB-2019/2011−2015;NFBSFARA/CA-4-15/2013-14,,) and ISI plan project funds(2008−2013) for providing financial support. S.M. is thankful toCSIR, New Delhi, for SRF and P.P. is thankful to UGC-DSKPDF.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We acknowledge Ashim Dhar for his assistance during plantexperiments.

■ REFERENCES(1) Arnone, J. A.; Gordon, J. C. Effect of nodulation, nitrogen fixationand CO2 enrichment on the physiology, growth and dry mass allocationof seedlings of Alnus rubra Bong. New Phytol. 1990, 116, 55−66.(2) Hortensteiner, S.; Feller, U. Nitrogen metabolism and remobiliza-tion during senescence. J. Exp. Bot. 2002, 53 (370), 927−937.(3) Feller, U.; Fische, A. Nitrogen metabolism in senescing leaves. Crit.Rev. Plant Sci. 1994, 13 (3), 241−273.



http://pubs.acs.org

mailto:[email protected]

(4) Koch, K. Sucrose metabolism: regulatory mechanisms and pivotalroles in sugar sensing and plant development. Curr. Opin. Plant Biol.2004, 7, 235−246.(5) Robinson, S. A.; Slade, A. P.; Fox, G. G.; Phillips, R.; Ratcliffe, R. G.;Stewart, G. R. The role of glutamate dehydrogenase in plant nitrogenmetabolism. Plant Physiol. 1991, 95, 509−516.(6) Hansch, R.; Mendel, R. R. Physiological functions of mineralmicronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Curr. Opin. Plant Biol.2009, 12 (3), 259−266.(7) Patra, P.; Mitra, S.; Debnath, N.; Goswami, A. Biochemical-,biophysical-, and microarray-based antifungal evaluation of the buffer-mediated synthesized nano zinc oxide: an in vivo and in vitro toxicitystudy. Langmuir 2012, 28 (49), 16966−16978.(8) Pradhan, S.; Roy, I.; Lodh, G.; Patra, P.; Choudhury, S. R.; Samanta,A.; Goswami, A. Entomotoxicity and biosafety assessment of PEGylatedacephate nanoparticles: a biologically safe alternative to neurotoxicpesticides. J. Environ. Sci. Health., Part B 2013, 48, 559−569.(9) Debnath, N.; Das, S.; Seth, D.; Chandra, R.; Bhattacharya, S. C.;Goswami, A. Entomotoxic effect of silica nanoparticles against Sitophilusoryzae (L.). J. Pest Sci. 2011, 84, 99−105.(10) Clement, L.; Hurel, C.; Marmier, N. Toxicity of TiO2

nanoparticles to cladocerans, algae, rotifers and plants − effects of sizeand crystalline structure. Chemosphere 2013, 90, 1083−1090.(11) Josko, I.; Oleszczuk, P. Influence of soil type and environmentalconditions on ZnO, TiO2 and Ni nanoparticles phytotoxicity.Chemosphere 2013, 92, 91−99.(12) Boer, W. D.; Kowalchuk, G. A. Nitrification in acid soils: micro-organisms and mechanisms. Soil Biol. Biochem. 2001, 33 (7−8), 853−866.(13) Jiang, W. Z. Mn use efficiency in different wheat cultivars. Environ.Exp. Bot. 2006, 57 (1−2), 41−50.(14) Pradhan, S.; Patra, P.; Das, S.; Chandra, S.; Mitra, S.; Dey, K. K.;Akbar, S.; Palit, P.; Goswami, A. Photochemical modulation of biosafemanganese nanoparticles on Vigna radiata: a detailed molecular,biochemical, and biophysical study. Environ. Sci. Technol. 2013, 47(22), 13122−13131.(15) Wennberg, A.; Iregren, A.; Struwe, G.; Cizinsky, G.; Haqrnan, M.;Johansson, L. Manganese exposure in steel smelters a health hazard tothe nervous system. Scand. J. Work Environ. Health 1991, 17, 255−262.(16) Martin, C. J. Manganese neurotoxicity: connecting the dots alongthe continuum of dysfunction.NeuroToxicology 2006, 27 (3), 347−349.(17) Hussain, S. M.; Javorina, A. K.; Schrand, A. M.; Duhart, H. M.; Ali,S. F.; Schlager, J. J. The interaction of manganese nanoparticles with PC-12 cells induces dopamine depletion. Toxicol. Sci. 2006, 92 (2), 456−463.(18) Wang, J.; Rahman, M. F.; Duhart, H. M.; Newport, G. D.;Patterson, T. A.; Murdock, R. C.; Hussain, S. M.; Schlager, J. J.; Ali, S. F.Expression changes of dopaminergic system-related genes in PC12 cellsinduced by manganese, silver, or copper nanoparticles. NeuroToxicology2009, 30, 926−933.(19) Lanone, S.; Rogerieux, F.; Geys, J.; Dupont, A.; Maillot-Marechal,E.; Boczkowski, J.; Lacroix, G.; Hoet, P. Comparative toxicity of 24manufactured nanoparticles in human alveolar epithelial and macro-phage cell lines. Part. Fibre Toxicol. 2009, 6, 14.(20) Limbach, L. K.; Wick, P.; Manser, P.; Grass, R. N.; Bruinink, A.;Stark, W. J. Exposure of engineered nanoparticles to human lungepithelial cells: influence of chemical composition and catalytic activityon oxidative stress. Environ. Sci. Technol. 2007, 41 (11), 4158−4163.(21) Gilad, A. A.; Walczak, P.; McMahon, M. T.; Na, H. B.; Lee, J. H.;An, K.; Hyeon, T.; Zijl, P. C. M.; Bulte, J. W. M. MR tracking oftransplanted cells with “positive contrast” using manganese nano-particles. Magn. Reson. Med. 2008, 60 (1), 1−7.(22) Hussain, S. M.; Hess, K. L.; Gearhart, J. M.; Geiss, K. T.; Schlager,J. J. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol inVitro 2005, 19 (7), 975−983.(23) Yang, Z.; Liu, W.; Allaker, R. P.; Reip, P.; Oxford, J.; Ahmad, Z.;Ren, G. A review of nanoparticle functionality and toxicity on the centralnervous system. J. R. Soc. Interface 2010, 7, S411−S422.

(24) Lee, Y. C.; Chen, D. Y.; Dodd, S. J.; Bouraoud, N.; Koretsky, A. P.;Krishnan, K. M. The use of silica coated MnO nanoparticles to controlMRI relaxivity in response to specific physiological changes. Biomaterials2012, 3 (13), 3560−3567.(25) Cataldo, D. A.; Haroon, M.; Schrader, L. E.; Young, V. L. Rapidcolorimetric determination of nitrate in plant tissue by nitration ofsalicylic acid. Commun. Soil Sci. Plant Anal. 1975, 6, 71−80.(26) Crawford, N. M.; Smith, M.; Bellissimo, D.; Davis, R. W.Sequence and nitrate regulation of the Arabidopsis thaliana mRNAencoding nitrate reductase, a metalloflavoprotein with three functionaldomains. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5006−5010.(27) Banerjee, K.; Sinha, M.; Pham, C. L. L.; Jana, S.; Chanda, D.;Cappai, R.; Chakrabarti, S. α-Synuclein induced membrane depolariza-tion and loss of phosphorylation capacity of isolated rat brainmitochondria: implications in Parkinson’s disease. FEBS Lett. 2010,584, 1571−1576.(28) Tian, J.; Fu, F.; Geng, M.; Jiang, Y.; Yang, J.; Jiang, W.; Wang, C.;Liu, K. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebralischemia in rats. Neurosci. Lett. 2005, 374 (2), 92−97.(29) Boyum, A. Isolation of lymphocytes, granulocytes and macro-phages. Scand. J. Immunol. 1976, 5, 9−15.(30) Ghosh, M.; Bandyopadhyay, M.; Mukherjee, A. Genotoxicity oftitanium dioxide (TiO2) nanoparticles at two trophic levels: plant andhuman lymphocytes. Chemosphere 2010, 81, 1253−1262.(31) Chatter, R.; Othman, R. B.; Rabhi, S.; Kladi, M.; Tarhouni, S.;Vagias, C.; Roussis, V.; Guizani-Tabbane, L.; Kharrat, R. In vivo and invitro anti-inflammatory activity of neorogioltriol, a new diterpeneextracted from the red algae Laurencia glandulifera.Mar. Drugs 2011, 9,1293−1306.(32) Mitra, S.; Subia, B.; Patra, P.; Chandra, S.; Debnath, N.; Das, S.;Banerjee, R.; Kundu, S. C.; Pramanik, P.; Goswami, A. Porous ZnOnanorod for targeted delivery of doxorubicin: in vitro and in vivoresponse for therapeautic applications. J. Mater. Chem. 2012, 22, 24145−24154.(33) Bondi, J. F.; Oyler, K. D.; Ke, X.; Schiffer, P.; Schaak, R. E.Chemical synthesis of air-stable manganese nanoparticles. J. Am. Chem.Soc. 2009, 131, 9144−9145.(34) Crawford, N. M. Nitrate: nutrient and signal for plant growth.Plant Cell. 1995, 7, 859−868.(35) Carroll, B. J.; McNeil, D. L.; Gresshoff, P. M. Isolation andproperties of soybean [Glycine max (L.) Merr.] mutants that nodulate inthe presence of high nitrate concentrations. Proc. Natl. Acad. Sci. U.S.A.1985, 82, 4162−4166.(36) Stitt, M.; Muller, C.; Matt, P.; Gibon, Y.; Carillo, P.; Morcuende,R.; Scheible, W. R.; Krapp, A. Steps towards an integrated view ofnitrogen metabolism. J. Exp. Bot. 2002, 53 (370), 959−970.(37) Tcherkez, G.; Hodges, M. How stable isotopes may help toelucidate primary nitrogen metabolism and its interaction with (photo)respiration in C3 leaves. J. Exp. Bot. 2008, 59 (7), 1685−1693.(38)Muller, C.; Scheible,W. R.; Stitt, M.; Krapp, A. Influence of malateand 2-oxoglutarate on the NIA transcript level and nitrate reductaseactivity in tobacco leaves. Plant Cell Environ. 2001, 24, 191−203.(39) Soussi, M.; Ocana, A.; Lluch, C. Effects of salt stress on growth,photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.). J.Exp. Bot. 1998, 49 (325), 1329−1337.(40) Murray, D. R. Changes in activities of enzymes of nitrogenmetabolism in seedcoats and cotyledons during embryo development inpea seeds. Plant Physiol. 1980, 66, 782−786.



manganese nanoparticles: impact on non-nodulated plant...

Documents