2017 IEEE International Conference on Technological Advancements in Power and Energy ( TAP Energy)
Green Synthesized Iron Nanoparticles and its Uptake in Pennisetum glaucum - A Nanonutriomics Approach Siji S, Njana J, Amrita PJ, Athira Raj, Dalia Vishnudasan School of Biotechnology Amrita Vishwa Vidyapeetham, Clappana P O, Kollam, Kerala.
[email protected] Abstract - Green nanotechnology, deals with the use of biological systems such as plants, bacteria, fungi, yeast, algae etc. for the synthesis of Nanoparticles (Nps). The aim of our study was to undertake Green synthesis of Iron nanoparticles/magnetite (Fe3O4) from a marine algae namely Chaetomorpha antennina. Two types of Iron Nps were synthesized and characterized– bare FeNp and citrate coated FeNp. Further the uptake mechanism of green synthesised Iron Nps were evaluated on Pennisetum glaucum (Pearl millet). An overall increase in the plant growth was observed. It was also observed that with increasing concentrations of FeNps (Fe3O4), the seedlings showed a concomitant increase in chlorophyll as well as soluble sugar content, suggesting that the iron uptaken by the plants is used up for producing photoassimilates. The iron content in the plants shoots and roots were estimated, which further confirmed that the plants readily uptook FeNp. Histochemical assays (undertaken in the roots as well as the shoots sections) involved looking at localization study of Iron, starch, lignin and suberization. Throughout the experiment no toxic effect was observed in the plants. This confirms the potential of FeNp to be used as an eco-friendly fertilizer. Hence we would like to conclude with the present study that FeNp could be potentially used as a nano-fertilizer hence paving the way towards “plant nanonutriomics”. Solutions such as this could effectively solve the present agricultural issues, especially pertaining to iron deficiency in the human population and its amelioration. It would definitely prove to be a worthy challenge to Genetically Modified food. Index Terms—marine algae, Pennisetum glaucum, Chaetomorpha antennina, Iron nanoparticles (Fe3O4), plant nanonutriomics
I. INTRODUCTION Agriculture is the backbone of India and with the current population growth it becomes pertinent to use the modern technologies that can boost the agricultural yield. In this context fertilizers are vital for plant growth and development and yet most of the applied fertilizers are unavailable to plants due to leaching, degradation, insolubility and decomposition. Hence in the given context Nanofertilizers or nanoencapsulated nutrients have the ability to effectively to release
978-1-5386-4021-0/17/$31.00 ©2017 IEEE
Manoj P K Department of Physics TKM College of Arts and Sciences, Kollam, Kerala.
[email protected] nutrients and chemical fertilizers on demand to up-regulate plant growth [1]. On a global scale, over three billion people suffer from micronutrient deficiencies (MNDs) of essential minerals and vitamins [2]. Deficiencies in iron (Fe) and zinc (Zn) are two of the most common and widespread MNDs [3]. Recent studies have proved a direct link between celiac disease and malabsorption of Fe and Zn via the small intestine. The prevalence of celiac disease is estimated to be between 0.5 and 1% in various parts of the world [4], but is especially prominent in grain dependent communities in India and Africa [5]. Pennisetum glaucum (Common name: Pearl millet, Bajra; family: Poaceae, subfamily: Panicoideae) is a multi-purpose cereal crop grown in the arid and semi-arid regions [5]. It has a 2530 Mb genome size and a diploid chromosome number of 7 x 2n = 14 [6] [7]. Pearl millet is recommended in the treatment of celiac diseases; furthermore, it may be employed for improving Fe and Zn content in staple foods through biofortification so as to meet the target levels of Fe and Zn in human populations [8]. The aim of biofortification is to make crops more nutritious as they grow, rather than adding nutrients when processing them into foods. In the present study we intend to systematically address topics such as - Green synthesized FeNp and its feasibility of use as a fertilizer (fertigation). We chose Pearl millet as it naturally has more iron content. We also wanted to evaluate the barriers, pathways and transport processes that FeNps face in pearl millet plants? How (fast) do – particularly non-dissolving – FeNps translocate within plants? Where in plants will FeNps accumulate or would it assimilate all of it? And do plants, at all, excrete insoluble FeNps again, through hydathodes? Furthermore, we want to establish the feasibility of using FeNp as nanofertilizer, both in aquaponics as well as a soil amendment application. Perhaps the bare FeNp could be best used along with a slow release fertilizer, while the citrate capped FeNp could be used in hydroponics.
A. Role of Iron in Plants Iron is one of the most essential micronutrient for plant growth and is the fourth most abundant element in the Earth’s crust. Despite the abundance, its availability to plant roots is very low. Because 30% of the world's cropland is too alkaline for optimal plant growth, and some staple crops, like rice, are especially susceptible to Fe deficiency, much research has focused on how plants cope with Fe limitation [9]. Studies show that the strategy of iron uptake differs on the type of plant; non graminaceous and graminaceous. Non graminaceous plants reduce Fe3+ via a membrane-bound reductase to make it accessible for uptake by a Fe2+ transporter, while graminaceous secrete phytosiderophores (PS) that readily bind Fe3+, and the Fe-PS complexes are then transported back into the roots [9] and references therein. Grasses like corn, rice, millet, bajra use the chelation based Strategy 2. This chelation strategy is considered to be more efficient than reduction based Strategy 1, thus helping grasses to survive better under iron deficiency. Grasses release compounds known as mugenic acid (MA) family of phytosiderophores in response to iron deficiency. The MA family includes MA, DMA and epi-HMA. The secretion of MAs depends on the plant species and is directly correlated to the ability of the plant to resist iron deficiency [10]. B. Iron Translocation FeNp translocation in plants involves various steps, including radial transport across the root tissues, including the symplastic transport through the Casparian strip; xylem loading, transport, and unloading; xylem-to phloem transfer; phloem loading, transport, and unloading. Physiological and molecular studies by other researchers suggest the involvement of principal chelators, such as citrate, nicotianamine (NA), and MAs. C. Strategies to Increase Uptake Different transgenic approaches are being analysed to biofortify food crops. Various transgenic as well as nontransgenic approaches have been employed for enhanced iron uptake in plants. Such as, expression of - storage protein ferritin gene under the control of endosperm specific promoter; over expression of NAS gene [11]; introduction of MAs from barley in to rice; employment of phytosiderophore synthase gene IDS3 and also overexpression of iron transporter gene IRT [12] . Secondary metabolites such as coumarins also play an important role in assisting plants to tolerate iron deficiency [13]. Flavins [i.e. riboflavin (Rbfl)] may also be secreted in the rhizosphere to further facilitate ferric ions uptake [14]. Together, these studies demonstrate that phytosiderophores exuded by roots are able to support nutrient acquisition directly or indirectly, in the vicinity of rhizosphere. D. Nanoparticle Uptake in Plants There are many reports concerning Nps uptake, translocation and toxicity in plants, but the published results are somewhat contradictory, showing variations depending on the Nps used, their size and the plant species [15]; [16]; [17]; [18]; [19]; [20]. The first FeNps uptake study was
demonstrated in pumpkin plants and their subsequent translocation and accumulation has also been noted [21]. In soybean plants, iron oxide Nps notably influenced photosynthesis reactions [22]. Uptake, transport and toxicity of nanomaterials into plant cells are complex processes that are currently still not well understood. A vast number of studies showed uptake, clogging, or translocation in the apoplast of plants, most notably of nanoparticles with diameters much larger than the commonly assumed size exclusion limit of the cell walls of approximately 5-20 nm. Important factors strongly affecting nanomaterials internalization are the cell wall composition, mucilage, symbiotic microorganisms (mycorrhiza), the absence of a cuticle (submerged plants) and stomatal aperture [23]. In the present study our endeavor is to assess nanoparticles uptake rates in pearl millet (Pennisetum glaucum) and to unravel plant physiological features favoring uptake. II. MATERIALS AND METHODS I. Green Synthesis of Nanoparticles a) Preparation of Algal Extract The seaweed was washed with water, dried and stored. To prepare the extract, 0.1 g of algal powder was added to 100 ml of Ultrapure water and was heated to 70-80°C. The crude algal extract was stirred continuously, then filtered and the supernatant thus obtained was used as the seaweed bioextract. b) Synthesis of Magnetite (Fe3O4) 0.1M FeCl2.4H2O and 0.1% algal extract were added in the ratio 2:3, in four different sets, each with a different pH. The pH was adjusted to 6, 8, 10 and 12 using NaOH. These reactions were maintained at temperature ranging from 6070°C. The synthesized Nps were washed thrice using 70% ethanol, dried in hot air oven for 24 hrs. To evaluate the effect of temperature on the nanoparticle synthesis, two different sets of reactions were performed at temperature ranging from 60°C and 27°C. The green synthesized bare FeNps were stored till further use. The bare FeNps were coated with Tri sodium citrate. Sodium citrate was prepared in 5 mg/10ml concentration. Requisite amount of prepared citrate solution is added to the synthesized FeNp, sonicated and then dried. II. Characterization a) UV−Visible Absorption Spectra UV−visible absorption spectroscopy measurements were performed in aqueous buffer using a Shimadzu double beam monochromator spectrometer (UV-2540). UV−visible absorption spectra of the algal extracts were also undertaken. b) X-ray Diffraction The Fe3O4 nanoparticles were analysed for phase composition using X-ray powder diffraction (Rigaku miniflex 600) over the 2 θ range from 20–600 at rate of 50/min, using Cu-Ka radiation (l ¼ 1.54060 A˚). The average particle sizes of Fe3O4-Nps were estimated using the Debye-Scherrer equation: d = kλ/(β·cosθ) Where d is the particle size of the crystal, k is Sherrer constant (0.9), λ is the X-ray wavelength (0.15406 nm), β is the
width of the XRD peak at half-height, and θ is the Bragg diffraction angle. c) FTIR Fourier transform infrared spectrometer (Shimadzu Affinity 1S) was performed to analyze the characteristics of the FeNp. d) Scanning Electron Microscopy The FeNp synthesized at pH 8- both bare and citrate capped, were analyzed using SEM and structural data were studied. e) Charge Characterization of FeNps Agarose gel electrophoresis was performed to analyze the charge of the synthesized FeNp. 2.5% agarose gel was prepared in 1x TAE buffer. The samples were loaded and the gel was run at 100V. III. Evaluation of Nanoparticles uptake in plants a) Germination and Growth Conditions Seeds of Pennisetum glaucum (Pearl millet) were purchased from WellGate Organics. The seeds were sterilized using sodium hypochlorite for 10 min followed by thorough washing with autoclaved distilled water. The seeds were germinated on either on sterilized cotton or in sterilized cocopeat in plastic glasses, with requisite treatment conditions. Nanoparticle suspension solutions (pH 8) were prepared at concentrations 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L and 50 mg/L with distilled water using both citrate capped and bare FeNp. About 7- 8 ml of the requisite solution was provided to the seeds. Plants were harvested after 5 days and subsequent tests were done. Control plants treated with plain water was compared with other controls- Citrate treatment only (0.1 M and 0.01 M).The plants were allowed to grow at 16/8 h light/dark at 27°C. The glasses were tightly covered with cling film to maintain optimal humidity. Hydathode exudation was collected from day 2 onwards. All biological experiments were repeated thrice. b) Chlorophyll Assay The chlorophyll assay was done as per the protocol Hiscox and Israelstam, [24] and the subsequent measurements were done as per the calculation mentioned in Sumanta et al 2014. The shoots of three plants were extracted with 1 mL DMSO and incubated for 20 min at 63°C. The absorbance of the solutions was read at 665, 649 and 480 nm for chlorophyll a, chlorophyll b and carotenoids respectively in Biomek plate reader. c) Iron Assay The Iron assay was estimated as per the method of Zohlen [25]. 250 μL of 4% HCl was added to two harvested shoots and incubated overnight at room temperature. 200 μL of 2% hydroxyl ammonium chloride, 200 μL of acetate buffer (pH4.6) and 100 μl of 0.2 M, 1,10 phenanthroline hydrochloride monohydrate were added to the solution and absorbance was read at 510 nm in Biomek plate reader. d) Soluble Sugar Assay The soluble sugar was estimated by the method of Dey [26]. 0.5 ml of 90% alcohol was added to the harvested single leaves in an eppendorf and incubated for 1 hour at 65°C. 0.5 ml 5% phenol, and 2.5 ml concentrated H2SO4 were added to the
solution and mixed thoroughly. The absorbance at 490 nm was measured in Biomek plate reader. e) Perls’ Staining Staining solution was prepared by adding 4% potassium ferrocyanide (K4[Fe(CN)6].3H2O) and 4% HCl in 1:1 ratio. Thin hand-sections of plant root were mounted on glass slides with a drop of the staining solution. Perls’ method has been reported to stain Fe at concentrations of 35 μM from both Fe(II) and Fe(III) [27]. f) Iodine staining Thin hand-sections of 5 day old leaves and roots were mounted on glass slides with Lugol’s iodine and observed under microscope. g) Lignin Stain Thin hand-sections of 5 day old leaves and roots were stained with Toluidine blue solution, a metachromatic stain (1 mg/ml). A similar test was performed with phloroglucinol-HCl solution. III. RESULTS AND DISCUSSION In the field of agriculture, the use of nanomaterials is relatively new [28] and its potential applications include increased effectiveness of herbicides and pesticides administered at lower doses [29], as fertilizer to increase crop productivity [30], for crop protection and production, as well as detection of pathogens and pesticide/ herbicide residues [28] ; [31]. In the present study, our aim was to evaluate the mechanisms of uptake, transport and impact of FeNp in Pennisetum glaucum (bajra). TABLE I: THE TABLE SHOWS THE VARIATION IN GREEN SYNTHESIZED FENP PROPERTIES, OBTAINED FROM CHAETOMORPHA ANTENNINA WITH RESPECT TO VARYING PH.
Precipitation Amount of Precipitation Ferromagnetism Particle size Yield Nano Size (XRD Data)
pH6 Incomplete +
pH8 complete ++
pH10 complete +++
pH12 complete ++
+ + 0.086g 8.89nm
+++ ++ 0.171g 13.62-15.86 nm
++ +++ 0.210g 16.15nm
++ +++ 0.143g 10nm
A. Iron Nanoparticle Synthesis: The Chaetomorpha antennina (green algae) bioextract, was used for synthesizing the green FeNps. Varying pH, temperature, precursor concentration (FeCl2.4H2O) as well as algal extract concentration resulted in green synthesized FeNp having differing features, as shown in Table 1. In the present study we focused on obtaining very small green synthesized FeNps that could be easily uptaken by plants. The Fe-Nps were prepared using ferrous chloride as iron precursor and Algal extract as reducing agent and stabilizer. The addition of ferrous chloride solution as an iron precursor to the Algal extract containing sulphated polysaccharides as a major component which has sulphate, hydroxyl and aldehyde group may cause the oxidation of Fe2+ and stabilization of the nanoparticles. A decrease in pH during the formation of Fe-Nps signifies the involvement of the OH group in the reduction process. Optimal
0
60 -200
50
400
200
40
[12], d=1.4742(5), 2-theta=63.00(2),
[12], d=1.4742(5), 2-theta=63.00(2),
70
Intensity (cps)
Intensity (cps)
80
600
200
[11], d=1.604(2), 2-theta=57.40(8), H=14
800
400
90
[11], d=1.604(2), 2-theta=57.40(8), H=14
100
Meas. data:Cheto 8 Calc. data:Cheto 8
[10], d=2.098(9), 2-theta=43.1(2), H=137
600
[10], d=2.098(9), 2-theta=43.1(2), H=137 [9], d=2.5046(9), 2-theta=
800
[9], d=2.5046(9), 2-theta=
C. Iron Nanoparticle Characterization a) UV−visible absorption spectra The characteristic surface plasmon band (SPR) of Fe3O4 is centered at 190-250 nm, and hence the iron oxide formed is Fe3O4 (Figure 1a). Since there is near infrared absorption, the complex is confirmed as Fe3O4 because Fe2O3 which also has two ionic species does not show absorption at near-infrared region. UV−visible absorption spectroscopy measurements were performed in aqueous buffer using a Shimadzu double beam monochromator spectrometer (UV-2540) equipped with an integrated sphere assembly ISR-240A in the range of 200800 nm (Figure 1a). b) FT-IR Fourier transform infrared spectrometer (Shimadzu Affinity 1S) was used to analyze the characteristics of the Fe3O4 nanoparticles (Figure 1b). Signals obtained by us are similar to the results obtained by other researchers. The band at 1,641 cm−1 was attributed to the binding of a C=O group with the nanoparticles [32]. The formation of Fe3O4 is characterized bands at 535 and 307 cm−1 which correspond to the Fe–O bond in magnetite [33].
[8], d=2.937(13), 2-theta=30.41(14), H=101
B. Factors Affecting Synthesis (TABLE I): Many factors were noted to effect synthesis of FeNps. a) Biological material: Different Bioextracts of Algal species can give variations in particle size, morphology, property etc. We observed that concentration of algal extract (0.1%) is very important for Nanoparticles synthesis. Higher concentration of algal extract (1%) leads to higher concentration of reducing and capping agent in water, hence more chances of bulky sized FeNp. b) Concentration of FeCl2.4H2O: Since FeCl2.4H2O is employed as the precursor in this synthesis, its higher concentration results in agglomeration; further leading to bulky sized FeNp. c) Effect of pH on Green synthesized FeNp: The data (Table 1) represents the variation in properties of Np observed with respect to changes in pH. Irrespective of the algae used, it is observed that the Nps synthesized at pH 8 shows a higher remanence. The magnetic properties of the Nps show a huge variation; Nps synthesized at pH 6 shows the least magnetism. Nps synthesized at pH 8 were used for further evaluations in pearl millet due to its higher remanence and small size. Moreover the size of particles at pH 8 is much smaller. d) Effect of temperature on Green synthesized FeNp
resulted in smaller FeNp. The properties of Np were observed to change with respect to temperature. For the synthesis of Np with higher remanence and better magnetism, a higher temperature is preferred. Thus, throughout the experiment, a temperature ranging from 60-70°C is maintained during the synthesis.
[8], d=2.937(13), 2-theta=30.41(14), H=101
pH conditions were maintained by using concentrated NaOH. The proposed green synthesis method for Fe3O4-Nps was found to be constructive and extremely reproducible.
Meas. data:Cheto 8 Calc. data:Cheto 8
0 20
30
40
30
50
60
70
2-theta (deg)
-200
20 10 0
a)
b)
20
30
40
50
60
70
2-theta (deg)
5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 1 2 3 4 5 6 8 9 0 1 2 1 2 2 3 3 4 4 6 5 7 6 8 7 9 8 0 0 1 1 2 3 4 5 6 7 9 0 1 1 1 1 1 1 1 1 1 2 2
c)
+ve electrode
d)
e)
f)
C
B
-ve electrode
Fig. 1: a) UV-Vis Spectra, b) FTIR spectra, c) XRD Data, SEM image of bare Fe3O4 Np (d) and citrate capped Fe3O4 Np (e), f) Charge mobility of citrate capped (C) and bare Fe3O4 Np (B).
The effect of temperature on green synthesis was evaluated in all the three algal extracts and it was observed that high temperature 60°C (in comparison to low temperature 20°C)
c)
X-ray diffraction
The phase identification and crystalline structures of the nanoparticles was characterized by X-ray powder diffraction (Fig. 1c). It is found that there exist strong diffraction peaks with 2θ values of 30.4°, 35.8°, 43.1°, and 57.4° 63.0°, corresponding to the crystal planes of (200), (311), (400) and (511) of crystalline Fe3O4-Nps, respectively. The results show the spinel phase structure of magnetite and are in agreement with the XRD standard for the magnetite nanoparticles. (JCPDS file No. 19 -0629). Using the Scherrer equation the average crystallite sizes of the magnetic Fe3O4-Nps were found to be in the range of 8–16 nm (see Table 1) as has been observed to be similar to other reports [33]. d) Scanning Electron microscopy Morphology of nanoparticle was visualized with help of Scanning Electron microscopy undertaken at Amrita Centre for Nanosciences, AIMS, Kochi. Figure 1d shows bare FeNp and Figure 1e shows capped FeNp. e) Charge characterisation of Fe3O4-Nps In the present study it was observed that the FeNps were negatively charged when citrate capping was undertaken. However, uncoated FeNps were positively charged when observed on 2.5% Agarose gel electrophoresis. Hence the negatively charged citrate FeNps uptake in plant would differ with respect to uncoated FeNps (Fig. 1f).
D. Effect of Fe3O4-Nps in Plant Shoot The focus of our study involved the use of bare (uncapped) FeNp as well as the capped FeNp for evaluating the uptake in Pennisetum glaucum (Pearl millet).Various models have been put forth to show the conditions which affect the iron bioavailability [34] taking into account the dissociation, complexation and precipitation reactions occurring in solutions and the dynamic equilibrium [34]. With increasing concentration of FeNp the seedlings showed a concomitant in increase in plant height (Fig. 2a), shoot weight (Fig. 2b) as well as root length (Fig. 2c). It was also observed that, plants/seedlings provided with citrate capped FeNp showed similar performance in height, Chlorophyll a content (Fig. 3a), iron content (data not shown), and also soluble starch assay (Fig. 3b) when compared to the seedlings treated with bare Np. This further implies that the capped Nps are being absorbed and utilised better in the plants. Nanoparticle treatments were found to increase biomass of Pennisetum glaucum seedlings when treated with FeNps. Two factor ANOVA tests clearly show the influence of FeNp treatments, whether it is statistically relevant or not (Fig. 3b). 14 12 10 8
D 6 O 4 2 0 -2
a)
c h lo ro p h y ll a
c h lo r o p h y ll b
a)
b)
b)
c)
c) Fig. 2. Seedlings grown under different FeNp treatment conditions (bare as well as capped) were evaluated for a) Shoot Height (cms), b) Shoot weight (gm) and c) Root Length (cms).
d)
Fig. 3. Seedlings treated with varying concentrations of FeNp (bare as well as capped) were evaluated for a) Chlorophyll a and Chlorophyll b, b) Two Factor ANOVA analysis for Chlorophyll a and Soluble Sugar, c) Perls’ stained root section and d) Lugol’s Iodine stained root section.
E. Effect of Fe3O4-Nps in Plant Roots Root system architecture depends on nutrient availability that shapes primary and lateral root development in a nutrientspecific manner. To better understand how nutrient signals are integrated into root developmental programs, we investigated
the morphological response in Pennisetum glaucum roots to FeNps (Fig. 3c). Local symplastic Fe gradient in lateral roots is known to upregulate AUX1, so as to accumulate auxin in lateral root apices as a prerequisite for lateral root elongation in Arabidopsis [35] . In the present study, the seeds were planted on cotton as well as cocopeat with varying concentrations of FeNps. Plants grown on cotton could be analysed for root morphology, better than those grown in cocopeat; especially with respect to collecting root exudate. Formation of Lateral Roots could be observed in FeNp treated plants and these roots lacked endodermis. Such lateral roots would enable the plant to uptake more Fe. The roots also showed fluorescence at high concentration of FeNp, possibly due to the exudation of phytosiderophores. F. Chlorophyll assay and soluble sugar assay Chlorophyll is vital for capturing of light in plants. In the present study Chlorophyll a, Chlorophyll b, and also the Carotenoid levels were observed to increase during the course of FeNp treatment (Fig. 3a). Biomass productivity depends on photosynthesis. Therefore increase levels of Chlorophyll a also resulted in higher photosynthetic efficiency (as measured by soluble sugar assay). Plants treated with higher concentrations of FeNp (5-50 mg/L) showed much higher soluble sugar content compared to the control plants (Fig. 3b). It was also observed that the seedling roots have a higher concentration of starch compared to the shoots. This might be due to starch produced in the leaf being translocated and stored in the root which is why the roots show a higher starch content. G. Iron Assay Iron assay was performed on both shoots and roots of the seedlings (Data not shown). The results show a homeostasis in the plants irrespective of the concentration of FeNp treatment. We also observed the iron in the hydathode excretion. This further implies that the plant is maintaining homeostasis to overcome the deleterious effects of too much iron which may trigger Fenton Reaction, an unwanted phenomenon. H. Starch Lugol's iodine Soluble sugar assay highlights the roots to be the reservoirs of photoassimilates, hence Lugol’s iodine staining was done in both leaf and root cross sections. Intense stained starch reservoirs could be observed in Pennisetum glaucum roots (Fig. 3d), while in the leaves only the bundle sheath cells showed some staining (data no shown). The root cross sections stained with Lugol’s iodine also showed an autofluorescence (Fig. 3d), perhaps due to the secretion of mucilage and phytosiderophores. In plants grown with higher concentration of FeNp, the intensity of fluorescence was observed to be higher. This clearly indicates that when plants were given higher concentration of FeNp, more mucilage/phytosiderophore production occurs to facilitate the absorption.
I. Root Anatomy and FeNps Casparian strip, within the root endodermis is able to block the diffusion of solutes between the outer environment and the stele, thereby forcing uptake of nutrients through the endodermal plasma membrane [36]. In addition to possessing the Casparian strip, the cell wall of endodermal cells undergoes a secondary differentiation consisting of suberin deposition on the entire cell surface. The plasticity of suberization is therefore an adaptive new response to Fe deficiency, which enables the plant to modulate the radial movement of Fe through its control of both the apoplastic pathway and the transcellular pathway involving efflux and reimport of Fe between the cortex and endodermis [37]. In the present study we could observe direct relationship between iron uptake (from FeNp) and lignification of cell walls. With increasing concentration of FeNp treatment more lignification was observed (Fig. 3d). But despite the lignification the iron uptake and re-mobilization from FeNp was not hindered. In the present study we could observe localization of Perl stain in pericycle cells facing towards the stele region (Fig. 3c). The use of FeNps for medical [51], environmental applications [52], [53] would result in release of Nps to the environment which furthermore necessitates the need to look at their uptake in plants. IV. CONCLUSION In summary, we demonstrated that a significant amount of FeNps suspended in a liquid medium can be taken up by Bajra/Pearl millet plants. FeNps coated with tri-Sodium citrate showed better uptake and consequently better photosynthetic efficiency than bare FeNps. However, homeostasis is the norm in the shoots, as plants always maintain an optimal steady state. This requisite maximal amount of iron was sufficient for optimal photosynthesis, as observed in the soluble sugar assay. Interestingly, these Nps did not show any toxicity response towards the plants. ACKNOWLEDGMENT The seaweeds were identified by kind help from Dr P Sophiammal Nettar, Fatima Mata National College. UV−Visible spectroscopy was undertaken with the kind support of Dr Asoke Banerji, Distinguished Professor, Amrita School of Biotechnology and Mr Chinchu Bose. Scanning Electron Microscopy was undertaken at Amrita Centre for Nanosciences, AIMS, Kochi, with the kind assistance of Dr Raja Biswas and Mr Sajin. REFERENCES [1] N. S. Singh A, “Plant-nanoparticle interaction: an approach to improve agricultural practices and plant productivity.,” Int J Pharm Sci Invent, vol. 4 (8). pp. 25–40, 2015. [2] C. T. Chasapis, A. C. Loutsidou, C. A. Spiliopoulou, and M. E. Stefanidou, “Zinc and human health: an update.,” Archives of toxicology, vol. 86, no. 4, pp. 521–534, Apr. 2012. [3] R. L. Bailey, K. P. West, and R. E. Black, “The epidemiology of global micronutrient deficiencies.,” Annals of nutrition & metabolism, vol. 66 Suppl 2, pp. 22–33, Jun. 2015.
[4] N. Gujral, H. J. Freeman, and A. B. R. Thomson, “Celiac disease: prevalence, diagnosis, pathogenesis and treatment.,” World journal of gastroenterology, vol. 18, no. 42, pp. 6036–6059, Nov. 2012. [5] H. R. Manwaring, H. F. J. Bligh, and R. Yadav, “The Challenges and Opportunities Associated with Biofortification of Pearl Millet (Pennisetum glaucum) with Elevated Levels of Grain Iron and Zinc.” [6] M. Bennett, “Nuclear DNA Amounts in Angiosperms and their Modern Uses—807 New Estimates,” Annals of Botany, vol. 86, no. 4. [7] I. Oumar, C. Mariac, J.-L. Pham, and Y. Vigouroux, “Phylogeny and origin of pearl millet (Pennisetum glaucum [L.] R. Br) as revealed by microsatellite loci.,” TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, vol. 117, no. 4, pp. 489–497, Aug. 2008. [8] G. Velu, K. N. Rai, V. Muralidharan, V. N. Kulkarni, T. Longvah, and T. S. Raveendran, “Prospects of breeding biofortified pearl millet with high grain iron and zinc content,” Plant Breeding, vol. 126, no. 2. [9] J. Morrissey and M. L. Guerinot, “Iron uptake and transport in plants: the good, the bad, and the ionome.” [10] V. Römheld, “The role of phytosiderophores in acquisition of iron and other micronutrients in graminaceous species: an ecological approach.” [11] H. Masuda and N. N. MS Aung, “Iron biofortification of rice using different transgenic approaches.” [12] S. A. Kim and M. L. Guerinot, “Mining iron: iron uptake and transport in plants.” [13] S. Clemens and M. Weber, “The essential role of coumarin secretion for Fe acquisition from alkaline soil,” Plant Signaling & Behavior, vol. 11, no. 2, Feb. 2016. [14] P. Sisó-Terraza, J. J. Rios, J. Abadía, A. Abadía, and A. Álvarez-Fernández, “Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.,” New Phytol., vol. 209, no. 2, pp. 733–745, Jan. 2016. [15] S. C. C. Arruda, A. L. D. Silva, R. M. Galazzi, R. A. Azevedo, and M. A. Z. Arruda, “Nanoparticles applied to plant science: a review.,” Talanta, vol. 131, pp. 693–705, Jan. 2015. [16] G. Chichiriccò and A. Poma, “Penetration and Toxicity of Nanomaterials in Higher Plants.,” Nanomater., vol. 5, no. 2, pp. 851– 873, May 2015. [17] D. Lin and B. Xing, “Phytotoxicity of nanoparticles: inhibition of seed germination and root growth.,” Environ. Pollut., vol. 150, no. 2, pp. 243–250, Nov. 2007. [18] C. Ma, J. C. White, O. P. Dhankher, and B. Xing, “Metalbased nanotoxicity and detoxification pathways in higher plants.,” Environ. Sci. & Technol., vol. 49, no. 12, pp. 7109–7122, Jun. 2015. [19] P. Miralles, T. L. Church, and A. T. Harris, “Toxicity, Uptake, and Translocation of Engineered Nanomaterials in Vascular plants.,” Environ. Sci. & Technol., vol. 46, no. 17, pp. 9224–9239, Sep. 2012. [20] H.-X. Ren et al., “Physiological investigation of magnetic iron oxide nanoparticles towards Chinese mung bean.,” J. Biomed. Nanotechnol., vol. 7, no. 5, pp. 677–684. [21] H. Zhu, J. Han, J. Q. Xiao, and Y. Jin, “Uptake, translocation, and accumulation of manufactured iron oxide nanoparticles by pumpkin plants.,” J. Environ. Monit. JEM, vol. 10, no. 6, pp. 713–717, Jun. 2008. [22] M. H. Ghafariyan, M. J. Malakouti, M. R. Dadpour, P. Stroeve, and M. Mahmoudi, “Effects of magnetite nanoparticles on soybean chlorophyll.,” Environ. Sci. & Technol., vol. 47, no. 18, pp. 10645–10652, Sep. 2013.
[23] F. Schwab, G. Zhai, M. Kern, A. Turner, J. L. Schnoor, and M. R. Wiesner, “Barriers, pathways and processes for uptake, translocation and accumulation of nanomaterials in plants--Critical review.,” Nanotoxicology, vol. 10, no. 3, pp. 257–278, Jun. 2016. [24] J. D. Hiscox and G. F. Israelstam, “A method for the extraction of chlorophyll from leaf tissue without maceration,” Can. J. Bot., vol. 57, no. 12, Jun. 1979. [25] A. Zohlen, “Use of 1,10-phenanthroline in estimating metabolically active iron in plants,” Commun. Soil Sci. Plant Anal., vol. 31, no. 3–4, pp. 481–500, Feb. 2000. [26] P. . DEY, “Oligosaccharides,” in Carbohydrates, Elsevier, 1990, pp. 189–218. [27] H. Roschzttardtz, G. Conéjéro, C. Curie, and S. Mari, “Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo.,” Plant Physiol., vol. 151, no. 3, pp. 1329–1338, Nov. 2009. [28] L. R. Khot, S. Sankaran, J. M. Maja, R. Ehsani, and E. W. Schuster, “Applications of nanomaterials in agricultural production and crop protection: A review,” Crop. Prot., vol. 35. [29] C. M. Rico, S. Majumdar, M. Duarte-Gardea, J. R. PeraltaVidea, and J. L. Gardea-Torresdey, “Interaction of nanoparticles with edible plants and their possible implications in the food chain.,” J. Agric. Food Chem., vol. 59, no. 8, pp. 3485–3498, Apr. 2011. [30] C. O. Dimkpa and P. S. Bindraban, “Fortification of micronutrients for efficient agronomic production: a review,” Agron. Sustain. Dev., vol. 36, no. 1, Jan. 2016. [31] R. Mohammadinejad, S. Karimi, S. Iravani, and R. S. Varma, “Plant-derived nanostructures: types and applications,” Green Chem., vol. 18, no. 1, 2016. [32] R. R. . Kannan, W. . Stirk, and J. Van Staden, “Synthesis of silver nanoparticles using the seaweed Codium capitatum P.C. Silva (Chlorophyceae),” South Afr. J. Bot., vol. 86. [33] M. Mahdavi and M. A. F Namvar R.Mohamad, “Green biosynthesis and characterization of magnetic iron oxide (Fe3O4) nanoparticles using seaweed (Sargassum muticum) aqueous extract.” [34] C. A. Krohling et al., “Ecophysiology of iron homeostasis in plants,” Soil Sci. Plant Nutr., vol. 62, no. 1, pp. 39–47, Jan. 2016. [35] R. F. H. Giehl, J. E. Lima, and N. von Wirén, “Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution.,” Plant cell, vol. 24, no. 1, pp. 33–49, Jan. 2012. [36] N. Geldner, “Casparian strips.,” Curr. Biol. CB, vol. 23, no. 23, pp. R1025–R1026, Dec. 2013. [37] C. Curie and S. Mari, “New routes for plant iron mining.,” New Phytol., vol. 214, no. 2, pp. 521–525, Apr. 2017. [38] Z. Cifuentes et al., “Absorption and translocation to the aerial part of magnetic carbon-coated nanoparticles through the root of different crop plants.,” J. nanobiotechnology, vol. 8, p. 26, Nov. 2010. [39] T. S. Walker, H. P. Bais, E. Grotewold, and J. M. Vivanco, “Update on Root Exudation and Rhizosphere Biology Root Exudation and Rhizosphere Biology 1,” Society, vol. 132, no. May, pp. 44–51, 2003. [40] A. Driouich, M.-L. Follet-Gueye, M. Vicré-Gibouin, and M. Hawes, “Root border cells and secretions as critical elements in plant host defense.,” Curr. Opin. Plant Biol., vol. 16, no. 4, pp. 489– 495, Aug. 2013. [41] F. Battke, K. Leopold, M. Maier, U. Schmidhalter, and M. Schuster, “Palladium exposure of barley: uptake and effects.,” Plant Biol., vol. 10, no. 2, pp. 272–276. [42] F. Schwabe et al., “Dissolved cerium contributes to uptake of Ce in the presence of differently sized CeO2-nanoparticles by three crop plants.,” Met. Integr. biometal Sci., vol. 7, no. 3, pp. 466–477.
[43] P. Zhang et al., “Comparative toxicity of nanoparticulate/bulk Yb2O3and YbCl3 to cucumber (Cucumis sativus).,” Environ. Sci. & Technol., vol. 46, no. 3, pp. 1834–1841, Feb. 2012. [44] F. Schwab et al., “Are carbon nanotube effects on green algae caused by shading and agglomeration?,” Environ. Sci. & Technol., vol. 45, no. 14, pp. 6136–6144, Jul. 2011. [45] Z. Zhang et al., “Uptake and distribution of ceria nanoparticles in cucumber plants.,” Met. Integr. biometal Sci., vol. 3, no. 8, pp. 816–822, Aug. 2011. [46] Z.-J. Zhu et al., “Effect of surface charge on the uptake and distribution of gold nanoparticles in four plant species.,” Environ. Sci. & Technol., vol. 46, no. 22, pp. 12391–12398, Nov. 2012. [47] L. Zhao et al., “Effect of surface coating and organic matter on the uptake of CeO2 NPs by corn plants grown in soil: Insight into the uptake mechanism.,” J. Hazard. Mater., vol. 225–226, pp. 131– 138, Jul. 2012. [48] B. Xia, C. Dong, W. Zhang, Y. Lu, J. Chen, and J. Shi, “Highly efficient uptake of ultrafine mesoporous silica nanoparticles with excellent biocompatibility by Liriodendron hybrid suspension cells.,” Sci. China. Life Sci., vol. 56, no. 1, pp. 82–89, Jan. 2013.
[49] J. Hong et al., “Evidence of translocation and physiological impacts of foliar applied CeO2 nanoparticles on cucumber (Cucumis sativus) plants.,” Environ. Sci. & Technol., vol. 48, no. 8, pp. 4376– 4385, Apr. 2014. [50] M. F. Iannone, M. D. Groppa, M. E. de Sousa, M. B. Fernández van Raap, and M. P. Benavides, “Impact of magnetite iron oxide nanoparticles on wheat (Triticum aestivum L.) development: Evaluation of oxidative damage,” Environ. Exp. Bot., vol. 131. [51] S. Fazal, B. Paul-Prasanth, S. V. Nair, and D. Menon, “Theranostic Iron Oxide/Gold Ion Nanoprobes for MR Imaging and Noninvasive RF Hyperthermia,” ACS Appl. Mater. & Interfaces, vol. 9, no. 34, pp. 28260–28272, Aug. 2017. [52] N. K., D. A. Sathish, P. S. Kumar, and D. R. T., “Fast kinetics and high adsorption capacity of green extract capped superparamagnetic iron oxide nanoparticles for the adsorption of Ni(II) ions,” Journal of Industrial and Engineering Chemistry (IF – 4.4), 2017. [53] D. S. K. M. and D. D. Sahu, “Effect of iron oxide nanoparticle in bio digestion of a portable food-waste digester,” Journal of Chemical and Pharmaceutical Research, vol. 7, 2015.
