The seed stimulant effect of nano iron pyrite is compromised by nano cerium oxide: regulation by the trace ionic species generated in the aqueous suspension of iron pyrite

Abstract

A brief seed pretreatment of 12 hours, in an aqueous suspension of synthesized nano iron pyrite (FeS₂), significantly increases the yield of spinach and other crops. The effector mechanism is not clear. An aqueous suspension of FeS₂, produces very trace amounts of H₂O₂, Fe₂O₃, FeS, FeSO₄, Fe(SO₄)₃, SO₂, S and H⁺ ionic species. Thus for 12 hours, seeds are exposed to this complex aqueous suspension. Among these trace species, H₂O₂ and Fe₂O₃ are known seed stimulants and plant growth promoters respectively. In this work, an attempt has been made to quench one of these trace compounds generated in the aqueous suspension of FeS₂, viz., H₂O₂; and the long-term effect on the mature plant was monitored. To test this, along with FeS₂, an agriculturally relevant inorganic peroxide scavenger viz., nano cerium oxide (CeO₂) was introduced into this system. Four seed pretreatment regimens were followed for the spinach viz., (i) control (water), (ii) FeS₂ + water, (iii) CeO₂ + water, (iv) FeS₂ + CeO₂ + water; and growth was monitored for the next 80 days. It was found that, at maturity, CeO₂ and FeS₂ + CeO₂, resulted in significantly smaller leaves, as compared to the control and FeS₂; furthermore, FeS₂ resulted in leaves with increased chlorophyll and carbohydrate. Thus, the data indicates that by quenching the H₂O₂, the seed-stimulant effect of FeS₂ is compromised. So, while the FeS₂ + water suspension functions as a seed vigor enhancer, CeO₂ + water on the contrary, functions as a ‘seed vigor reducer’. It is noteworthy, that CeO₂ is used by Chinese farmers, as a micro-nutrient to increase crop production. Current data indicates that, it delays germination of seeds, whereas FeS₂ hastens germination. Thus such an approach could be used for hastening or delaying germination, manipulating weed population, seed storage in critical conditions, timing the life-cycle of a plant and developing more energy-efficient plants, especially in regions, where there is limited sunlight during significant parts of the year.

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1. Introduction

Nano iron pyrite (FeS₂) is a seed biostimulant. Pyrite treated seeds raise the yields of spinach, sesame and fenugreek in field trials and chickpea in laboratory trials.^1–4 The mechanism of action of FeS₂ is not clear. In this work, the possible chemical mechanism of action of FeS₂ was explored.

The current practice of nano FeS₂ seed pre-treatment is the following: nano FeS₂ is prepared by a low temperature route.^1–5 Before sowing, the seeds are pretreated in an aqueous suspension of FeS₂ (80–100 μg mL⁻¹) for 12 hours. The key question is ‘how FeS₂ + H₂O is acting on the seeds, during this brief 12 hour exposure?’. There are two possibilities; the first possibility is, FeS₂ is acting directly on the surface of the seeds through certain un-explored receptors or pathways; the second possibility is, FeS₂ in the presence of water, is generating a certain effector molecule/molecules in very trace amounts viz., H₂O₂, Fe₂O₃, FeS, FeSO₄, Fe(SO₄)₃, SO₂, S and H⁺ ionic species;^2,4,6 which is/are influencing the seed metabolism positively. The second possibility seems more probable, since among these trace ionic species, H₂O₂ (ref. 7–16) and nano Fe₂O₃ (ref. 17) are known seed stimulant and plant growth promoter respectively. Based on this available knowledge about the aqueous chemistry of FeS₂, an attempt was made to dissect out the action of trace amounts of H₂O₂ generated in an aqueous suspension of FeS₂, by quenching it using another agriculturally relevant inorganic compound viz., CeO₂.^18–26

In the following paragraph, the existing literature on peroxide and seed germination have been discussed. Peroxide has two modes of action during seed germination; an endogenous and an exogenous mode. In the endogenous mode, seeds are equipped with an inbuilt molecular machinery to produce trace amounts of peroxide during the time of germination, which subsequently helps in the emergence process.^7–9 In addition, if the germinating seeds are incubated in an inhibitor of catalase, the antioxidant enzyme responsible for the breakdown of the peroxide, then germination rate can be accelerated.¹⁰ Thus the corollary is that ‘if the germinating seeds are incubated in the presence of exogenous catalase, the seed germinating vigor will be low and germination rate will fall’. Moreover, in the exogenous mode, if the seeds are pretreated in peroxide, it helps in breaking dormancy and promotes germination.^10–16 So based on the current understanding of seed physiology, peroxide is emerging as a critical switch during the time of germination and by tweaking this switch, it is possible to modulate the fate of the plant at the very onset of germination.

With this background information, the mechanism of action of synthetic FeS₂ on seed germination and subsequent growth was explored in the current study. To test the role of peroxide, an agriculturally relevant nano-peroxide scavenger cum anti-oxidant viz., cerium oxide (CeO₂) was introduced into the system. Cerium is used for increasing paddy production in China and other parts of the world.²⁰ It is noteworthy that recent findings have shown that CeO₂ helps the leaves to reduce oxidative damage in the chloroplast and further increasing the rate of photosynthesis. The central idea behind introducing another plant friendly nanomaterial is the following: could the fate of a germinating seed be modulated externally, with a nanomaterial having peroxide generating (FeS₂) and another with peroxide scavenging (CeO₂) properties. One has to also keep in mind that, both these nano-materials are agriculturally relevant and have showed growth stimulatory effects at different phases of plant development. But whether both of them have the same effect on seed germination and subsequent growth is yet to be addressed systematically.

2. Experimental

2.1. Synthesis and characterization of pyrite and ceria nano particles

FeS₂ nanoparticles was synthesized by following formerly reported method. It follows a nucleophilic reaction between the aqueous solution of 0.8 M sodium polysulfide and 0.4 M aqueous solution of FeCl₃·6H₂O in argon atmosphere, at a temperature around 100 °C for 8 hours. Tri-sodium-citrate was used as a capping agent to prevent agglomeration of the nano particles in an aqueous medium. Acetic acid–sodium acetate buffer solution was used to maintain a pH around 6. The obtained greyish black precipitate was centrifuged and thoroughly washed with 2 M of HCl to remove other iron sulfur impurity (as FeS₂ is not soluble in 2 M HCl). Following this, the particles are washed with water, toluene and acetone to remove non-reacted sulphur. The purified FeS₂ precipitate was dried under vacuum and used for further experiment.^1–5

Nano ceria was synthesized by reacting 0.025 M aqueous solution of Ce(NO₃)₃·6H₂O with 0.025 M aqueous solution hexamethylene-tetra-amine (HMTA) at a temperature of 75–80 °C for 4 hours, as reported in earlier work. The white turbid solution obtained, was centrifuged at 12 000 rpm for 2 minutes and washed with water and acetone for 3 times and stored in vacuum desiccator for drying and further use.¹⁸

The particles are characterized by HRTEM (JEOL Transmission JEM2000FX with STM equipped with a Quantum detector), XRD (PANanalytical XRD), Raman spectroscopy, atomic force microscopy (Agilent 5500), zeta potential analyzer (Microtrac) and X-ray photoelectron spectroscopy (XPS).

XPS characterization was done using PHI 5000 Versa Prob II, FEI Inc. equipped with AlK-alpha monochromatic source. All peaks were fitted using a Lorentzian: Gaussian line shape and with a Shirley background.

Atomic force microscopy (AFM) was performed using an Agilent Technologies Atomic Force Microscope (Model 5500) operating in ACAFM mode. The scanner model N9520A-USO8380185.xml was calibrated and used for the imaging. Micro fabricated silicon nitride cantilevers with resonant frequency [f] of 181 kHz and 349 kHz were used. The images were taken at room temperature with a scan speed of 2.0 lines per s. Data acquisition and analysis was done using Pico View® 1.8 and Pico Image® Basic software respectively. Sparingly soluble solution of FeS₂ (1 mg mL⁻¹) was sonicated for about 2 minutes and was heated at 65 °C in water-bath for 2 minutes. Further, it was centrifuged using Spin-wine-microcentrifuge at 13 500 rpm for 1 minute. 15 μL of clear supernatant was drop casted over freshly cleaved mica surface and it was dried at room temperature followed by AFM imaging. Sparingly soluble solution of CeO₂ HMTA (1 mg mL⁻¹) was sonicated about 2 minutes and it was heated at 65 °C in waterbath for 2 min. Further, it was centrifuged using Spinwine-microcentrifuge at 13 500 rpm for 1 minutes. 15 μL of clear supernatant was drop casted over freshly cleaved mica surface and it was dried at room temperature followed by AFM imaging.

2.2. Peroxide detection in an aqueous suspension of synthesized pyrite

The spectroscopic method which has been followed in this study is based on the instant reaction between H₂O₂ and V₂O₅ (vanadium pentoxide) in the presence of sulfuric acid, thus resulting in the formation of a peroxovanadate complex, which has an absorption maximum at 454 nm.²⁷ Synthesized FeS₂ particles were used for this experiments. 100 mg FeS₂ was suspended in 10 mL distilled water, sonicated for 30 seconds and left undisturbed for 2 hours. In order to avoid any contamination and loss of generated H₂O₂, the tubes were sealed with parafilm. A coloring agent was prepared by dissolving 0.2 g V₂O₅ in 100 mL 0.5 M H₂SO₄. FeS₂ suspension was centrifuged for 10 minutes at 5000 rpm and supernatant was collected. Equal volumes (5 mL) of coloring agent and the supernatant were mixed in separate tubes. Absorbance was recorded at 454 nm. The control samples consisted of coloring agent and distilled water taken in equal volumes (5 mL each). Control was done at auto-zero mode and comparative test readings were taken. In another set of experiment, along with 100 mg FeS₂, an additional 100 mg CeO₂ was added, to verify whether CeO₂ quenches the peroxide generated by the pyrite.

2.3. Spectroscopic detection of the peroxide quenching ability of nano ceria

Peroxide quenching ability of ceria was spectroscopically verified by the above method, as described in the previous section. In the control experiment, pure H₂O₂ was reacted with 0.2 g V₂O₅ in 100 mL 0.5 M H₂SO₄, and the absorbance was recorded at 454 nm. In the test sample, 100 mg cerium was incubated along with H₂O₂ and to this mixture 0.2 g V₂O₅ in 100 mL 0.5 M H₂SO₄ was added.

2.4. Studying the quenching of peroxide by ceria using cyclic voltammetry (CV)

Peroxide quenching ability of ceria was further verified with CV. The cerium working electrode was prepared by spraying 30 mg of CeO₂ (prepared by mixing and sonicating in 3 mL isopropyl alcohol and 30 μL of 6× diluted Nafion solution for 3 hours, where Nafion functions as a binder) on one side of the ITO sheet. Phosphate buffer saline (adjusted at a pH of 7.4) was used as the electrolyte. The CV was carried out using Epsilon Basi C3 cell stand with conventional three electrode configuration, where Ag/AgCl was the reference electrode, and platinum as the counter electrode. CV was performed at different scan rate from 10 mV s⁻¹ to 300 mV s⁻¹ at −0.8 V to 0.8 V voltage window.

2.5. Spinach seed pretreatment, field trial, sample collection and statistical analysis

Four seed pretreatments (12 hours) protocols were followed, viz. control (water), FeS₂ (80 μg mL⁻¹), CeO₂ (80 μg mL⁻¹), FeS₂ + CeO₂ (equal amounts of both the nanoparticles: 80 μg mL⁻¹ + 80 μg mL⁻¹ respectively); to evaluate how CeO₂ alone, and CeO₂ in combination with FeS₂, influences the germination and long-term growth of the spinach plant. Germination analysis were performed on day 5, post seed pre-treatment. Long term analysis was performed on day 50, on the basis of average individual leaf area of spinach following different treatments. The total leaf area of spinach plants obtained from the experimentation was calculated. A random sample of 10 plants were chosen from each of the plots, viz., control, FeS₂, CeO₂, and FeS₂ + CeO₂ plots. While the control plot represents the plants germinated from untreated seeds, the rest of the plots represented the different particle that was used for seed pretreatment. Since the sample size was 10 for each case, paired t-test for unequal variance was used to compare the average individual leaf area from each treatment level. The null hypothesis was that there is no significant difference between the average leaf areas among the treatment levels; whereas, the alternate hypothesis claimed that FeS₂ pretreated seeds produced plants that have significantly larger leaf area. For chlorophyll and carbohydrate estimation, leaf samples were collected at two different time points, at day 50 and at day 80 following seed sowing.

2.6. Chlorophyll and carbohydrate estimation

2.6.1. Chlorophyll estimation. Total chlorophyll content was measured following the protocol described earlier.⁴ Briefly, 100 mg of fresh leaves were cut into small pieces and homogenized manually in a mortar in the presence of 10 mL of 80% acetone, till the color was released from the tissue. To prevent pheophytin formation, a pinch of CaCO₃ was added to the homogenate. A clear supernatant was collected after centrifugation (5000 rpm for 10 minutes at room temperature) and volume was made up to 10 mL. All the tubes were protected from light to prevent chlorophyll degradation. Total chlorophyll a (Chl a) and chlorophyll b (Chl b) was measured in a spectrophotometer at OD_{663 nm} and OD_{645 nm} respectively. The spectrophotometric readings were converted to chlorophyll concentration using Arnon's equation.

Chl a (mg g⁻¹) = [(12.7 × A₆₆₃) − (2.6 × A₆₄₅)] × mL acetone per mg leaf tissue

Chl b (mg g⁻¹) = [(22.9 × A₆₄₅) − (4.68 × A₆₆₃)] × mL acetone per mg leaf tissue

Total Chl = Chl a + Chl b.

2.6.2. Carbohydrate estimation. The total soluble carbohydrates were measured according to the method described earlier.⁴ Briefly, to 1 g of macerated sample, 10 mL of water and 15 mL of 52% perchloric acid were added and the solution was stirred vigorously for 30 minutes at room temperature. The solution was filtered though Whatman filter paper and subsequently, freshly prepared anthrone solution was added to the filtrate. The absorbance was measured at OD_{620 nm} and carbohydrate concentrations were estimated using a standard curve of glucose.

3. Results and discussion

3.1. Characterization of the synthesized FeS₂ and CeO₂ particles

3.1.1. TEM and XRD. The representative TEM image of FeS₂ particles are shown in Fig. 1A. The powder XRD pattern (Fig. 1B) of the FeS₂ particles exhibited peaks at 28.5, 32.9, 36.9, 40.7, 47.4 and 56.2; which were indexed to 111, 200, 210, 211, 220 and 311 planes respectively and matches with previously published results (JCPDS no. 42-1340).^1–5 The representative TEM image of CeO₂ particles are shown in Fig. 1C. The powder XRD pattern (Fig. 1D) of the CeO₂ particles exhibited peaks at 28.4, 33.1, 47.6 and 56.8; which were indexed to 111, 200, 220, and 311 planes respectively, and matches with previously published results (JCPDS no. 81-0792).^4,18,28

Fig. 1 TEM and XRD of FeS₂ and CeO₂ nanoparticles. (A) Representative TEM image of FeS₂ nanoparticles. The average size of the particles varies from 250–500 nm. The scale bar shown in the micrograph is 250 nM. (B) The representative XRD spectra of FeS₂. (C) Representative TEM picture of CeO₂ nanoparticles. The average particle size varies from 20–40 nM. The scale bar shown in the micrograph is 10 nM. (D) The representative XRD spectra of CeO₂.

3.1.2. XPS of the synthesized nanoparticles. The representative XPS spectra of FeS₂ and CeO₂ are shown in Fig. 2A–D. Fig. 2A showed the Fe2p XPS spectrum having a sharp narrow peak at 707.3 eV for Fe2p_3/2 which is similar to marcasite Fe2p_3/2 peak. The peak at 707.3 and 720.1 eV attributed to Fe2p_3/2 and Fe2p_1/2 respectively for Fe²⁺ state. A small peak at 708.7 eV might be possibly due to electron-deficient Fe²⁺ sites created by the breaking of Fe–S bonds. The broad structure to higher binding energy is due to presence of some Fe³⁺ states from oxidation of the surface. Fig. 2B, showed the S2p XPS spectrum, having two maxima originated from spin–orbit signal of S atoms. The binding energy of S2p_3/2 and S2p_1/2 are 161.7 and 162.8 eV respectively similar to the metal sulfides. Fig. 2C and D showed the XPS spectra of CeO₂. The broad XPS peaks could be de-convoluted to the peaks related to the spin–orbit coupling.^4,18,28 The observed peaks denoted as v₀, v, v′, v′′ and v′′′ are signatures of the Ce3d_5/2, while the u, u′, u′′, and u′′′ were signatures of Ce3d_3/2 ionization. Ce⁴⁺ oxidation states were denoted by v, v′′, v′′′, u, u′′ and u′′′; while v₀, v′ and u′ are assigned to Ce³⁺ species. Thus XPS spectra showed the dual oxidation state of CeO₂ (Ce⁴⁺ and Ce³⁺), thus further supporting the proposed auto-catalytic activity of CeO₂, by continuously changing its oxidation state at nano-dimension, and thereby scavenging H₂O₂ and other free radicals.

Fig. 2 XPS of FeS₂ and CeO₂ nanoparticles. (A) Fe2p XPS spectrum of FeS₂. (B) S2p XPS spectrum of FeS₂. (C) Ce³⁺ and Ce⁴⁺ states of CeO₂. (D) Oxygen of CeO₂.

3.1.3. XPS spectra validating the presence of Fe₂O₃, FeS, FeSO₄, Fe(SO₄)₃, SO₂, S on FeS₂ particles after recovery from an aqueous suspension. A comparative XPS spectra has been presented in Fig. 3A–D, highlighting the generation of Fe₂O₃, FeS, FeSO₄, Fe(SO₄)₃, SO₂, S following 12 hours of FeS₂ + H₂O treatment, in the absence of seed, as reported in earlier studies.^2,4

Fig. 3 XPS spectra of FeS₂ particles before and after water treatment. (A) Fe2p XPS spectrum of FeS₂ before water treatment. (B) Fe2p XPS spectrum of FeS₂ after water treatment, showing the peaks of Fe₂O₃. (C) S2p XPS spectrum of FeS₂ before water treatment. (D) S2p XPS spectrum of FeS₂ after water treatment showing the FeSO₄, SO₂ and Fe₂(SO₄)₃ peaks.

3.1.4. Raman spectroscopy. The Raman spectra of FeS₂ showed three distinct peaks at 335, 380 and 433 cm⁻¹, which corresponds to symmetric mode (A_g), a doubly degenerate (E_g), and three triply degenerate modes (T_g) respectively (Fig. 4A). The A_g mode represents the in-phase stretching vibration of the S–S dimer. The E_g mode represents the perpendicular displacement of the sulfur atoms along the dimer axes. The T_g mode corresponds to the various librational and stretching vibrations or their combinations. Similar Raman spectra for FeS₂ was reported earlier.⁵ The Raman spectra of CeO₂ (Fig. 4B) was recorded between 200 and 1400 cm⁻¹ shift. A sharp peak was observed at 458 cm⁻¹, representing the F_2g mode. The F_2g mode shows the symmetric breathing mode of the oxygen atoms around each cation; and indicates the formation of cubic ceria nano-crystals. Further a broad band around 600 cm⁻¹ shows the oxygen vacancies and defects caused at the nano-dimensions.²⁸

Fig. 4 Raman spectra of (A) FeS₂ (B) CeO₂ nanoparticles.

Fig. 5 AFM, zeta potential and particle size analysis: (A) representative AFM image of FeS₂. (B) Representative AFM image of FeS₂. (C) Representative AFM image of FeS₂ showing two different size of population of particles, which could be distinctly observed in the depth profiles of the micrograph. (D) Smaller size CeO₂ particles with fewer bigger particles. (E) Quantitative analysis of the particle sizes showed the presence of two populations of FeS₂; one population was found to be less than 100 nm, the other between 300 and 400 nm. (F) CeO₂ also had two distinct sizes of particles; one population was around 50 nm, the other population was around 300 nm. (G) The hydro-dynamic radius as determined by particle size analyzer for FeS₂, indicated two peaks; one lies around 900 nm whereas the other lies around 1100 nm. (H) The hydro-dynamic radius as determined by particle size analyzer for CeO₂, indicated two peaks; one lies around 100 nm, whereas the other lies around 900 nm.

3.1.5. AFM, zeta potential and particle size analyzer (Fig. 5). The AFM data indicated that the FeS₂ particles are larger than the CeO₂ particles. Both the nanoparticles existed in two different sized populations. Further the particle size analyzer data corroborated the AFM findings, by showing the two population of hydrodynamic radius of the particles. The zeta potential lies between 18 and 93 mV with an average value of 66.5 mV for CeO₂ and it was observed that the values remain positive for all the trials (n = 10). The average zeta potential for FeS₂ is 1.39 mV with a range varying from 1.16 to 1.82 mV, as observed in 10 different trials and the observed values were always positive.

3.2. Detection of peroxide in an aqueous suspension of synthesized iron pyrite

Formation of significant amount of peroxovanadate complex was detected,²⁷ thus indicating the formation of H₂O₂ in an aqueous solution of FeS₂ (Fig. 6A). In Fig. 6B, the concentration of the peroxo-vanadate complex reduces significantly, in the presence of CeO₂ along with FeS₂. Thus indicating that CeO₂ is possibly quenching H₂O₂ in this system.

Fig. 6 FeS₂, CeO₂ and H₂O₂ chemistry. (A) Generation of peroxide in an aqueous suspension of pyrite. The formation of peroxovanadate complex indicating peroxide formation in the system. (B) Cerium oxide is quenching the peroxide, and reducing the concentration of peroxovanadate complex, when ceria is introduced along with pyrite. (C) Pure H₂O₂ is quenched by CeO₂, as seen by comparing the control (only H₂O₂) and test (H₂O₂ + ceria). (D) Cyclic voltammetry of CeO₂ in phosphate buffer saline at voltage range of −0.9 V to 0.6 V and at a scan rate of 10 mV s⁻¹ to 300 mV s⁻¹. (E) Comparison of the current densities at the CeO₂ electrode before and after addition of H₂O₂ (100 μM). Upon addition of peroxide the peak current increased significantly.

3.3. Peroxide quenching by ceria

Next spectroscopically peroxide quenching ability of ceria was detected (Fig. 6C). In control experiments, pure H₂O₂ was allowed to react to form peroxovanadate complex. In the test, 100 mg of CeO₂ was added along with same amount of H₂O₂, and the concentration of peroxovanadate complex was detected. There was a significant reduction in the concentration of the peroxovanadate complex, indicating that CeO₂ is possibly quenching H₂O₂.

3.4. Electro-catalytic activity of ceria in the presence of ceria

The electro-catalytic potential of the ceria was further studied for H₂O₂ redox reactions (Fig. 6D and E). Fig. 6D showed the CV of CeO₂ electrode under optimal conditions. In Fig. 6E, CV was shown before and after injection of an aliquot of H₂O₂. As it could be seen in Fig. 6E, the peak current value for the redox couple at the CeO₂ electrode increased significantly, following addition of H₂O₂. This clearly suggests that H₂O₂ oxidation is significantly facilitated by CeO₂ nanoparticles.

3.5. Effect of nano pyrite and ceria following seed pretreatment

We used four seed pretreatments viz. control, FeS₂, CeO₂, FeS₂ + CeO₂. First, the germination test was performed. The data showed that maximum germination at day 5 was observed in following FeS₂ seed pre-treatment and least in the case of CeO₂ seed pre-treatment (Fig. 7).

Fig. 7 Germination test following pre-treatment of the spinach seeds in H₂O (control), FeS₂ + H₂O, CeO₂ + H₂O, FeS₂ + CeO₂ + H₂O. Maximum germination at day 5 was observed in FeS₂ seed pre-treatment and the least in CeO₂.

It is interesting to note that upon maturity, CeO₂ and FeS₂ + CeO₂ seed pretreatment resulted in significantly smaller leaf as compared to the control and FeS₂ (Fig. 8, Table 1). Why there is such a marked antagonistic effect of CeO₂? One of the possible reasons could be that, CeO₂ is scavenging the endogenous H₂O₂ produced by the seeds during the time of germination, as well as exogenous H₂O₂ (produced by FeS₂) in FeS₂ + CeO₂ treatment. Thus CeO₂ by its autocatalytic activity, is mimicking the activity of catalase and nullifying the effect of FeS₂-induced peroxide formation.

Fig. 8 Spinach plant growth following nano pyrite and ceria following seed pretreatment. Quantifying the leaf area in spinach plant following seed pre-treatments with control (water), FeS₂, CeO₂ and FeS₂ + CeO₂. There is a significant reduction in the average individual leaf area upon CeO₂ and FeS₂ + CeO₂ seed pretreatments. Further the representative plant picture in the field (below the graph) indicate the same.

Table 1 It is evident that all p-values are much less than the assumed level of significance (alpha) of 0.01; allowing us to reject the null hypothesis and accept the alternate hypothesis. The near zero p-values suggest strong statistical evidence that FeS₂ pretreated spinach seeds have larger leaf area than all other treatments compared in this study

Sl. no	Pair-wise comparison	Average individual leaf area (mm^2)	p-Value	t-Statistic
1	FeS2 vs. control	4897.3 vs. 2143.3	0.000000720	7.96
3	FeS2 vs. CeO2	4897.3 vs. 635	0.000000036	13.90
4	FeS2 vs. (FeS2 + CeO2)	4837.3 vs. 646.5	0.000000039	13.76

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3.6. Chlorophyll and carbohydrate content in the mature spinach leaves

Since FeS₂ seed pretreatment resulted in higher biomass, so we estimated the chlorophyll content of the spinach leaves. The data were collected from 6 different plants at two different time points (day 50 and 80) and the average value of total chlorophyll content (mg per g of tissue) is plotted (Fig. 9). We found that FeS₂ pretreated seeds, resulted in plants having higher chlorophyll concentration in the leaves. Next we estimated the total carbohydrate in the spinach leaves at day 50. The control sample has an average total carbohydrate content of 1.5 g/100 g of plant tissue (n = 3), whereas the FeS₂ pretreated samples has 1.8 g/100 g of plant tissue (n = 3). Thus an increase in chlorophyll and total carbohydrate upon seed pretreatment with FeS₂ was observed. In Fig. 10, a proposed pictorial depiction of the interaction between pyrite and ceria in an aqueous medium is being shown.

Fig. 9 Chlorophyll content of the leaves in control and FeS₂ pretreated spinach plants. Total chlorophyll content was 0.8 mg g⁻¹ and 1.0 mg g⁻¹ in control plants at day 50 and 80 respectively, whereas in FeS₂ pretreated plants it is 1.5 mg g⁻¹ and 2.1 mg g⁻¹ at day 50 and 80 respectively (inset showing a control and FeS₂ pretreated plot at day 50).

Fig. 10 Proposed interaction between pyrite and ceria in an aqueous environment, which is eventually dictating the fate of the seed.

3.7. Proposed mechanism of action of FeS₂ and CeO₂ (Fig. 10)

Based on the experimental findings, the following mechanism has been proposed: in an aqueous suspension of FeS₂, the trace amounts of H₂O₂, Fe₂O₃ and S along with low pH due to H⁺ ions, is positively influencing the fate of the germinating seed. On the other hand, in the presence of CeO₂, the trace H₂O₂ is scavenged. Thus not only the positive influence of H₂O₂ derived from FeS₂ + H₂O is compromised, but the ability of H₂O₂ to disturb the lattice geometry of FeS₂ is ceased, and thereby generation of trace amounts of Fe₂O₃, S is also curtailed. Hence in the presence of CeO₂, the positive impact of FeS₂ is negated completely.

3.8. Significance of higher chlorophyll content upon FeS₂ seed pre-treatment

The exact pathway by which the chlorophyll is increasing needs further investigation. But more chlorophyll indicates the increased capability of plants to harvest more solar energy and subsequently more sugar turnover. Essentially this approach could have several economic ramifications viz., regions of the world, where sunlight is limited during significant part of the year, such a treatment could be a potential tool to obtain maximum growth and yield within a limited time window.

3.9. Economic significance of the work from the perspective of agro-sustainability and translational potential

This is a sustainable approach to reduce the fertilizer consumption, thereby reducing the cost input for the marginal farmers in the under-developed and developing world. Further reduction of fertilizer would help in maintaining a healthy, symbiotic soil micro-flora and fauna. It is worth discussing that, though it is known that H₂O₂ is a seed stimulant, but it can not be economically exploited till this date. Since H₂O₂ stand alone is highly corrosive difficult to carry over large distances. Thus because of these drawbacks, translating such a technology for the farmers is a challenge. On the contrary, approach of using FeS₂ + H₂O, is a eco-friendly approach and easily translatable to the farming community. Recently aqueous suspension of nano FeS₂ has also been used as a source of H₂O₂ to destroy organic waste and aqueous suspension of FeS₂ is nicknamed as ‘Fenton like reagent’.^2,4 Further FeS₂ is a multifunctional material: a seed stimulant, a bi-functional electrode for charge storage devices and a organic waste disposal material.^2,4,5

4. Conclusion

This study opens an unique peroxide-driven nano-window, to regulate the metabolic fate of a germinating seed, without manipulating the genes. It has been shown that by pre-treating the seeds with peroxide producing (iron pyrite) and peroxide scavenging (cerium oxide) nano particles, one could tune the future growth profile of a plant. Such an approach could be used for hastening or delaying germination, manipulating weed population, storing seeds and seedlings in critical conditions, timing the life-cycle of a plant and developing more energy-efficient plants, specially in the regions, where there is limited sunlight during significant parts of the year.

Acknowledgements

This work is part of GS's and AD's doctoral thesis. MR is supported by SB/FT/CS-199/2013 (DST-SERB, GOI).

References

1. G. Srivastava, A. Das, T. S. Kusurkar, M. Roy, S. Airan, R. K. Sharma, S. K. Singh, S. Sarkar and M. Das, Mater. Express, 2014, 4, 23.
2. G. Srivastava, C. K. Das, A. Das, S. K. Singh, M. Roy, H. Kim, N. Sethy, A. Kumar, R. K. Sharma, S. K. Singh, D. Philip and M. Das, RSC Adv., 2014, 4, 58495.
3. M. Das, G. Srivastava, C. Das, A. Dubey, N. Sethy, K. Bhargava, S. K. Singh and D. Philip, Iron pyrite as seed treatment biostimulant: The new revolution?, New AG International India, 2015, pp. 41–42.
4. G. Srivastava, Iron pyrite: A seed biostimulant, Doctoral thesis, Indian Institute of Technology Kanpur, India, 2016, Electronic Thesis and Dissertations (ETD) link http://172.28.64.70:8080/jspui/handle/123456789/15534.
5. A. Dubey, S. K. Singh, B. Tulachan, M. Roy, G. Srivastava, D. Philip, S. Sarkar and M. Das, RSC Adv., 2016, 6, 16859.
6. M. J. Borda, A. R. Elsetinow, M. A. Schoonen and D. R. Strongin, Astrobiology, 2001, 1, 283.
7. S. Puntarulo, M. Galleano, R. Sanchez and A. Boveris, Biochim. Biophys. Acta, 1991, 1074, 277.
8. C. Bailly, Seed Sci. Res., 2004, 14, 93.
9. C. Bailly, H. El-Maarouf-Bouteau and F. Corbineau, C. R. Biol., 2008, 331, 806.
10. S. B. Hendricks and R. B. Taylorson, Proc. Natl. Acad. Sci. U. S. A., 1975, 72, 306.
11. D. R. Hite, C. Auh and J. G. Scandalios, Redox Rep., 1999, 4, 29.
12. H. El-Maarouf-Bouteau and C. Bailly, Plant Signaling Behav., 2008, 3, 175.
13. G. Barba-Espín, J. A. Hernández and P. Diaz-Vivancos, Plant Signaling Behav., 2012, 7, 193.
14. G. Barba-Espin, P. Diaz-Vivancos, M. J. Clemente-Moreno, A. Albacete, L. Faize, M. Faize, F. Pérez-Alfocea and J. A. Hernández, Plant, Cell Environ., 2010, 33, 981.
15. P. Schopfer, Plant J., 2001, 28, 679.
16. R. Shin and D. P. Schachtman, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 8827.
17. K. Jeyasubramanian, U. U. Gopalakrishnan Thoppey, G. S. Hikku, N. Selvakumar, A. Subramania and K. Krishnamoorthy, RSC Adv., 2016, 6, 15451.
18. S. K. Ujjain, A. Das, G. Srivastava, P. Ahuja, M. Roy, A. Arya, K. Bhargava, N. Sethy, S. K. Singh, R. K. Sharma and M. Das, Biointerphases, 2014, 9, 031011.
19. M. Das, S. Patil, N. Bhargava, J. F. Kang, L. M. Riedel, S. Seal and J. J. Hickman, Biomaterials, 2007, 28, 1918.
20. X. Pang, D. Li and A. Peng, J. Soils Sediments, 2001, 1, 124.
21. C. T. Campbell and C. H. F. Peden, Science, 2005, 309, 713.
22. F. Esch, S. Fabris, L. Zhou, T. Montini, C. Africh, P. Fornasiero, G. Comelli and R. Rosei, Science, 2005, 309, 752.
23. T. Pirmohamed, J. M. Dowding, S. Singh, B. Wasserman, E. Heckert, A. S. Karakoti, J. E. S. King, S. Seal and W. T. Self, Chem. Commun., 2010, 46, 2736.
24. J. M. Dowding, T. Dosani, A. Kumar, S. Seal and W. T. Self, Chem. Commun., 2012, 48, 4896.
25. A. A. Boghossian, F. Sen, B. M. Gibbons, S. Sen, S. M. Faltermeier, J. P. Giraldo, C. T. Zhang, J. Zhang, D. A. Heller and M. S. Strano, Adv. Energy Mater., 2013, 3, 881.
26. J. P. Giraldo, M. P. Landry, S. M. Faltermeier, T. P. McNicholas, N. M. Iverson, A. A. Boghossian, N. F. Reuel, A. J. Hilmer, F. Sen, J. A. Brew and M. S. Strano, Nat. Mater., 2014, 13, 400.
27. Q. Zhang, S. Fu, H. Li and Y. Liu, BioResources, 2013, 8, 3699.
28. N. Padmanathan and S. Selladurai, RSC Adv., 2014, 4, 6527.

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Footnote

† CD, GS and AD are equal contribution first authors.

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