Nano‐pyrite as a plant hormone regulator: Emulating seed hormopriming

‘Pre‐sowing seed priming’ and ‘pre‐transplanting root priming’ are promising strategies to improve agriculture productivity. Farmers, seed production enterprises, and seedling producers constantly search for economic priming agents that help improve yield and crop health. Recently, several nanomaterials have emerged as economical seed and root priming agents, with nanoiron pyrite standing out as a particularly promising molecule. The observed enhancement in germination of nano‐pyrite treated seeds across various plant species indicate a shared underlying mechanism. We conducted evaluations of gibberellic acid (GA) and abscisic acid (ABA) content in red radish and soybean seeds subjected to nano‐pyrite priming revealing a notable increase in the GA: ABA ratio compared to the control group. In addition, mature red radishes cultivated from nano‐pyrite primed seeds exhibited elevated anthocyanin content and a remarkable 25.46% increase in yield. The aqueous pyrite suspension utilised in the process generates trace peroxide, and we propose that this trace peroxide plays a crucial role in orchestrating the increased GA: ABA ratio, anthocyanin content, and crop yield. These results position nano‐pyrite as a plant hormone regulator, effectively mimicking the seed hormo‐priming strategy. Considering the widespread presence of pyrite in the earth's crust, using pyrite as a commercial seed and root priming agent emerges as a potentially sustainable approach to enhance food production.

Among these nanomaterials, nano pyrite (FeS 2 ) stands out as the first nanomaterial evidence showcasing its efficacy as a seed-priming agent, improving germination and crop yield. 8[10][11][12][13][14][15][16] Notably, nano pyrite also positively impact root nodule formation in chickpeas, thereby enhancing the plant's inherent nitrogen-fixing capability by emulating nitrogenase enzyme. 13][27] Previous observations of nano pyrite's substantial impact on seed vigour across multiple crops led us to explore its potential influence on gibberellic acid (GA) and abscisic acid (ABA), key regulators of seed germination and dormancy.9][30][31][32][33][34][35][36][37] Building on this, our hypothesis suggests that nano pyrite may shift the GA: ABA balance in favour of GA, thus resulting in improved germination vigour.To investigate this hypothesis, we quantified the concentrations of GA and ABA in red radish and soybean seeds at the onset of germination.Additionally, we tracked anthocyanin content and crop yield in mature red radishes to gain a comprehensive understanding of nano pyrite's impact.

Nano-pyrite and nano cerium oxide synthesis
6][27] Briefly, 100 mL of 0.8 M NaOH solution was divided into two equal parts.In an inert chamber, hydrogen sulfide gas was bubbled into one part until the solution turned pale yellow, generating an equivalent amount of NaSH.The other half of the NaOH solution was added to generate an equal amount of Na 2 S. Into this, 1.28 g sulphur powder (0.04 mol) was added and stirred until dissolved to produce a yellowish-orange polysulfide solution at room temperature.The polysulfide solution was stored in an airtight amber-coloured glass vessel at 4°C to prevent oxidation.
Further, in a 100 mL (0.04 M) iron(III) chloride solution, the pH was set to 5.6 by adding 100 mL of CH 3 COONa/CH 3 COOH buffer (0.2 M, 5.6 pH).This buffered iron solution in a flask was placed into an oil bath at room temperature with constant stirring in an argon environment for 1 h.15 mL polysulfide solution (as described above) was slowly added while raising the oil bath temperature to 120°C, and the reaction mixture was stirred continuously.Further, the oil bath temperature was raised to 180°C for another 2 h.Then, the nanoparticle suspension was allowed to settle down and was sequentially washed with hydrochloric acid, toluene, and acetone.1][12][13][14][15][16][25][26][27] As noted, Cerium (III) nitrate and hexamethylenetetramine (HMTA) were used for nanoceria synthesis. 3800 mL aqueous solutions of 0.025 M of (Ce(NO 3 ) 3.6H 2 O) and 0.025 M HMTA each were mixed in a glass container and heated to 75°C in a water bath for 3 h to yield nanoceria.The solution was concentrated by centrifugation and washed with water and acetone. 38

Nano-pyrite characterisation
Scanning electron microscopy (SEM, Carl Zeiss, Model Number-EVO18) was utilised to analyse the surface morphology and size of the nano-pyrite crystals.X-ray diffraction (XRD, X'pert powder PANalytical) spectrum was recorded to study the characteristic crystal planes of the nanomaterial.X-ray Photoelectron Spectroscopy (XPS, PHI 5000 Versa Prob II, FEI) was used to observe the surface-chemical properties of the nanomaterial.

Germination test and seedling length analysis of red radish and soybean
Forty-four red radishes (Imperial Hybrid Seeds™ variety, Imperial Agro Genetics, Delhi, India; www.imperialagroindia.com)and 40 soybean seeds (JS 2024 Variety) were used for the germination test.The seeds were divided into two equal parts (test and control groups), resulting in the treatment of 22 red radishes and 20 soybean seeds with 0.1 mg ml −1 FeS 2 suspension for the test group and the treatment of the remaining seeds with distilled water for the control group.The seed germination rate was measured after 96 h of treatment.To measure seedling lengths, soybean, and red radish seeds were further grown for 8 days and 12 days, respectively.

Analysis of gibberellic acid and abscisic acid
For both GA and ABA analysis, 1000 mg red radish and 40 soybean seeds were separately taken and divided into two parts (test and control groups).Gibberellic acid and ABA levels were measured using the highperformance liquid chromatography (HPLC) analysis on FeS 2 -treated red radish and soybean seeds against control seeds.The seed samples were analysed 120 h after germination for GA and 24 h after germination for ABA.The germinated seeds were crushed in 50 mL, 80% methanol, and allowed to stand overnight for GA expression quantification.The sample was filtered, and the solid was again resuspended in 50 mL, 80% methanol for 1 h, and filtered.Both the filtrates were pooled and vacuum dried at 50°C with constant stirring to obtain a residual aqueous solution, which was mixed with 1 mL 1M HCl and 15 mL ethyl acetate.The organic layer was obtained three times by repeating this extraction process, and the organic fractions were pooled together.This organic layer was dried using anhydrous sodium sulphate and later vacuum-dried.The remaining residue was dissolved in 10 mL methanol, filtered using a polyvinylidene fluoride (PVDF) filter (pore size 0.22 μm), and stored for HPLC analysis.For ABA extraction, the 24-h-old germinated seeds were crushed in 100 mL of 1% acetic acid in acetone and left in solution overnight.The solution was filtered and dried under a vacuum in a water bath (40°C), and the remnant was dissolved in 10 mL 1% acetic acid and filtered using a PVDF syringe filter (pore size 0.22 μm).It was then stored in vials for HPLC injection. 39,40Waters 515 HPLC Pump and 2998 Photodiode Detector were employed for the HPLC analysis.The isocratic mobile phase with 0.74 mL min −1 water and 0.26 mL min −1 acetonitrile was utilised for GA.The isocratic mobile phase with 0.5 mL min −1 water and 0.5 mL min −1 methanol was used for ABA.Three recordings were taken per experiment, and each experiment was repeated three times.The area under the curve between the test and the control was compared for analysis and shown as a fold change upon normalisation to controls.Fold change values are represented as Average � standard error (p-value).Student's t-test was performed for statistical analysis. 39,40

Analysis of yield, plant length, anthocyanin, and flavonol in mature red radish
Red radish seeds were primed with 0.1 mg ml −1 nano pyrite suspension for 12 h for the test group and distilled water for the control group.We used 20 seeds for the test and 20 seeds for the control.The seeds were sown in a plot (dimensions 1.82 m � 0.6 m) at the Institute Nursery at the Indian Institute of Technology Kanpur, Kanpur, India.The patch of land where the control and test trials were conducted has similar soil characteristics and elemental content.Mature plants were harvested after 40 days and were used for analysis.
Anthocyanin levels in red radish roots were measured using the pH differential method. 41Briefly, for extraction, 7 mL of 80% ethanol (in 0.1% HCl) was added to 2 g of crushed red radish.The extract was filtered, and the solution was red radish ethanol extract. 1 mL of red radish extract was dissolved separately each in 0.2 M KCl/HCl buffer (pH 1) and 0.2 M CH 3 COONa/CH 3 COOH buffer (pH 4.5) to make solutions of 25 mL volumes.The anthocyanin content (expressed in mg L −1 extract) was calculated using Equation ( 1) and the spectral reading of the above solutions at 525 and 700 nm. 41thocyanin Content where I = (OD525 nm -OD700 nm) pH = 1 -(OD525 nm -OD700 nm) pH = 4.5.
The flavanol content was deduced by comparing the absorbance of the ethanol extract prepared by crushing 2 g red radish root in 7 mL, 80% ethanol.The filtered extract was used for UV-vis spectrophotometry at 510 nm for flavanol detection. 41roxide detection in aqueous suspension of nano pyrite and peroxide quenching using nanoceria Nano pyrite was dissolved in varying amounts (50, 100, and 150 mg) in 10 mL distilled water and allowed to stand for 6 h to detect the release of trace amounts of hydrogen peroxide.The samples were centrifuged for 10 min at 7000 RPM to collect the supernatants.A colour-developing agent containing 0.5 g V 2 O 5 in 100 mL H 2 SO 4 was prepared and mixed with an equal volume of each of the supernatant solutions, and the corresponding absorbance was measured at 454 nm.The above procedure was repeated by adding 25 mg nano-CeO 2 to all three pyrite suspensions. 11

Statistical analysis
The data is represented as average � standard error (p-value), where n is the number of repetitions.Student's t-test was performed for statistical analysis, and the corresponding p-values are shown.

Synthesis of nano-pyrite and nano cerium oxide
Naturally occurring pyrite is common in several parts of the world.It is rare to find it as an accessory primary mineral in igneous rocks.Instead, it appears as a secondary mineral.The fact that it is found as a secondary mineral indicates that it formed at a later geological time through weathering by hydrothermal alterations.Interestingly, the slow formation of pyrite in nature results in the stable incorporation of other elements in pyrite, namely copper, arsenic, and cobalt.These elements mentioned above need to be removed to use natural pyrite for experimental use.So to simplify the experimental protocol, we developed a low-energy chemical process to synthesise pure iron pyrite so it is ready for translation to the field.Nano FeS 2 was synthesised using a low-energy two-step process.Freshly prepared nanoparticles were used for all the studies.Cerium oxide nanoparticles were synthesised for the peroxide quenching experiment.

Seed germination and seedling lengths
In all our earlier experiments, we have observed that nano pyrite seed treatment prior to sowing resulted in improved germination and faster emergence.We selected soybeans, keeping in mind that soybean has inherent problems with germination.Thus, any improvement in germination will have tremendous economic potential for soybean growers.The core idea of selecting red radish is to test whether improved germination could influence the anthocyanin content of the mature radish.An improved anthocyanin yield could improve the nutritional status of red radish.For red radish, we observed an average germination of 22 � 1.73% in the control seeds after 96 h, while pyritetreated scored 57 � 2.08% germination (p = 0.0002, 2; unpaired two-tailed T-test; n = 3).In soybean seeds, we observed an average germination of 35.67 � 2.08% in the control seeds after 96 h, while pyrite-treated scored 65 � 1.15% germination (p = 0.0002; n = 3).Upon nano pyrite seed treatment, we observed a significant improvement in red radish and soybean germination.The treated plants emerge faster than their untreated counterparts and offer scope for uniform crop standing.The representative images of red radish and soybean seeds in test and control groups are shown in Figure 2a,b.
Once we observed improved germination by nano pyrite treatment, we were keen to compare the growth dynamics during the early development of the plants.These early development phases are exceptionally critical for the plant's long-term growth.So, we assayed for the plant lengths after 12 days.A representative image showing red radish seeds 12 days after germination with their lengths labelled is shown in Figure 3a.(Figure 3c).For soybean seedlings grown for 8 days after germination, an average length of 11.56 � 0.95 cm (n = 12) for the test and 9.91 � 0.78 cm (n = 12) for the control group (p = 0.19) was observed (Figure 3d).Thus, quite early in the development, we

Gibberellic acid and abscisic acid estimation
Based on the improved germination, growth, and vigour upon pyrite treatment, the most critical question is the cause of the above positive effects.One possible explanation we tested is whether the plant's GA and ABA hormonal profile alters at the very onset of germination.These two essential hormones that play a critical role in germination and vigour are GA and ABA.An increase in GA improves germination.Therefore, in our next set of experiments, we tested the level of these hormones in freshly germinated red radish and soybean seeds upon pyrite treatment and compared the results with untreated control seeds.For red radish, the ABA content (Figure 4a) was higher in the control group, whereas the GA (Figure 4b) was higher in the test group.It is represented as fold change and was measured using the average area under the curve in the chromatogram normalised to the control group.The fold change in the test for ABA was 0.53 � 0.067 (p = 0.00005), and for GA in the test group was 2.05 � 0.140 (p = 0).
For soybean seeds, the control group showed higher ABA content (Figure 4c), and the test group had higher GA content (Figure 4d).For ABA, the fold change in the test normalised to controls was 0.67 � 0.046 (p = 0.000037), and for GA, the fold change in the test group was 1.66 � 0.143 (p = 0).

Yield, plant length, anthocyanin, and flavonol in mature red radish
Upon observing an increased GA level in red radish during germination following nano pyrite treatment, we followed the crop till maturity to evaluate the anthocyanin content and the subsequent yield.The reason to follow up on the anthocyanin content is to test the possibility of whether a brassinosteroid-like pathway is getting activated following pyrite treatment.A representative image of the red radish crops in the field is shown in Figure 5a,  b. Figure 5c shows the representative pictures of mature red radishes from the test and control plots after harvest.After harvesting, the fresh weight of the control group (n = 19) was 1.563 kg, whereas the fresh weight of the test group was 2.097 kg (n = 17); this translates to a 25.46% increase from the control (where n = number of red radish's plants).We observed a 28.64% increase in the average length of the plant from 47.06 � 2.73 cm in F I G U R E 4 Pyrite treatment led to reduced ABA and increased GA contents in the seeds.This figure compares the GA: ABA ratio in red radish and soybean in freshly germinated seeds upon nano pyrite treatment with the control seeds.High-performance liquid chromatography chromatograms for ABA and GA estimation and their corresponding area under the curve for control and test groups in (a) Red radish ABA, (b) Red radish GA, (c) Soybean ABA, (d) Soybean GA.ABA, abscisic acid; GA, gibberellic acid.control to 60.55 � 2.24 cm in the test (p = 0.00055) (Figure 5d).The total anthocyanin content (Figure 5e) from three independent experiments (n = 3) for nano pyrite-treated red radish is 1.659 � 0.04 mg/L versus the control is 1.138 � 0.03 mg/L (p = 0.008; two-tailed unpaired T-test).Average flavanol absorption at 510 nm differed as 1.386 in the test and 1.025 in the control (n = 2), respectively.

Peroxide detection in nano pyrite suspension
The next critical question from these results is, 'What links pyrite treatment with GA and ABA hormones?'The argument we put forward is how a benign nanomaterial alters the hormonal profile of the germinating seeds.One cue we obtained from our earlier work was that an aqueous suspension of nano pyrite generates a trace amount of peroxide and multiple anionic and cationic species of iron and sulphur.Literature documents that peroxide influences the GA: ABA ratio-following up on this, we went ahead to detect the presence of trace amounts of peroxide in the aqueous suspension of pyrite.A trace amount of peroxide was detected by the standard measuring the absorption of a vanadium peroxide complex at 454 nm against control (Figure 6).

F I G U R E 6
Trace amounts of peroxide are present in the aqueous suspension of pyrite and nano-cerium oxide can be used to quench the peroxide.In this figure, we showed the presence of trace amounts of peroxide in the aqueous suspension of pyrite and a method to quench the peroxide using nano-cerium oxide.So we have two nanoswitches in the form of pyrite and ceria; while one produces a trace amount of peroxide, the other quenches the peroxide.Peroxide Detection Assay: Absorption of 'FeS 2 released H 2 O 2 ' at 454 nm for different concentrations and with the addition of fixed nano CeO 2 (n = 3).
MODERN AGRICULTURE absorption of the vanadium peroxide complex was reduced to 0.14 � 0.002, 0.18 � 0.004, and 0.19 � 0.004, respectively.Based on our results, we conclude that the trace amounts of peroxide generated in the aqueous suspension of pyrite play a key role in altering the GA: ABA ratio.Nanoceria quenching experiments offer a molecular switch to dampen the beneficial effects of pyrite.Furthermore, since pyrite is used for seed treatment, we are not influencing the soil directly.Instead, we alter the metabolic profile of the seed.In the truest sense, we use a molecular switch in the form of pyrite that modifies the seed hormonal profile (GA: ABA ratio) and brings long-term beneficial effects in plant production.

CONCLUSION AND DISCUSSIONS
In this study, we have uncovered a 'peroxide link' between pyrite seed priming and the increased GA: ABA ratio, leading to elevated anthocyanin levels in red radish, and a substantial increase in yield.This highlights the novel role of nano pyrite as a regulator of plant hormones.
Our observations revealed an increase in the GA: ABA ratio in nano-pyrite-primed seeds of red radish and soybean coupled with higher anthocyanin content and a remarkable 25.46% increase in yield.The central question around the results is, 'How does pyrite seed priming influence the GA: ABA ratio, anthocyanin pigments, and yield'.
Based on the present findings, we postulate that nano-pyrite activates at least two pathways in the germinating seeds.The first pathway leads to an increased GA: ABA ratio, while the second leads to increased anthocyanin by activating the brassinosteroid and jasmonic acid synthesis pathway.Notably, peroxide, identified as a key molecule in this dual activation, 30-32 is released in trace amounts during pyrite suspension.We have also shown that peroxide quenching abolishes the growth-promoting effects of pyrite. 11The trace peroxide potentially augments the GA: ABA ratio in red radish and soybean crops, leading to an improved ratio and subsequently increased crop yield.
Furthermore, our study delves into the intricate chemistry of pyrite-induced peroxide generation, emphasising two distinct reactions involving iron in different oxidation states.FeS 2 generates hydrogen peroxide in water without external oxygen (Reactions i and ii).
Such FeS 2 -catalysed traces of the generated H 2 O 2 could be sufficient in promoting the germination process of seed in water.Such H 2 O 2 generation from water catalysed by FeS 2 should not be confused with the other prevalent reaction of FeS 2 with aerial oxygen (Reactions iii and iv).
Schematics showing nano-pyrite as plant growth hormone regulator.Schematics showing nano-pyrite as plant growth hormone regulator.The observed enhancement in germination of nano-pyrite-treated seeds across various plant species indicates a shared underlying mechanism.Nano-pyrite priming results in a notable increase in the GA: ABA ratio compared to the control group in soybean and red radish crops.In addition, mature red radishes cultivated from nano-pyrite primed seeds exhibited elevated anthocyanin content and a remarkable 25.46% increase in yield.The aqueous pyrite suspension utilised in the process generates trace peroxide, and we propose that this trace peroxide plays a crucial role in orchestrating the increased GA: ABA ratio, anthocyanin content, and crop yield.ABA, abscisic acid; GA, gibberellic acid.
The interplay between these reactions, utilising Fe (II) and Fe(III) states of pyrite, suggests their mutual dependence and underscores the environmental relevance of these processes.The involvement of oxygen in one reaction adds an intriguing dimension, especially considering natural selection scenarios where buried seeds can undergo air diffusion, triggering the oscillation between Fe(II)S 2 and Fe(III)S 2 .This sustained generation of H 2 O 2 aids plant growth, with nature's build-in defence mechanisms ensuring controlled H 2 O 2 levels to prevent harm to seeds.The kinetics and overall optimisation of these reactions warrant further exploration.
The second noteworthy aspect of the study lies in the elevated concentrations of anthocyanins and other flavonols in red radish.The increase in anthocyanin levels suggests that peroxide may activate the brassinosteroid and jasmonic acid pathways, 30,31,33,34,36 demonstrating the dual role of trace peroxide generated from pyrite.The schematic in Figure 7 illustrates the potential mode of action of nano pyrite.
'Pre-sowing seed priming' with hormones (hormonepriming) like auxins, cytokinins, gibberellins, ABA, salicylic acid, and ethylene are currently used to enhance germination, viability, vigour, abiotic stress tolerance, and improved yield. 42In parallel to the traditionally hormone-priming strategy, our study introduces nanopyrite as an agent that alters the hormonal profile, specifically affecting GA and ABA.In essence, nanopyrite emulates seed hormone-priming, shedding light on its potential application in enhancing germination, viability, vigour, abiotic stress tolerance, and overall improved yield.These findings collectively unveil the multifaceted roles of nanopyrite, positioning it as a pivotal player in seed priming and a potential sustainable strategy for agricultural enhancement.

F I G U R E 1
The synthesised pyrites were characterised using SEM, XRD, and XPS.In this figure, we have presented the fundamental characterisation of the synthesised nanoparticles using SEM, XRD, and XPS.(a) SEM image of nano FeS 2 .(b) XRD Pattern of FeS 2 with 2θ angle 20°-60°.(c) High-resolution XPS spectra for the Fe2p region showing Fe2p1/2 (red) and Fe2p3/2 (green) peaks.(d) XPS highresolution spectra for the S2p region show S2p1/2 (red) and S2p3/2 (green) peaks.SEM, Scanning electron microscopy; XPS, X-ray Photoelectron Spectroscopy; XRD, X-ray diffraction.A representative image of soybean seeds 8 days after germination is shown in Figure 3b.The average length for red radish seeds grown for 12 days was noted as 17.90 � 1.04 cm (n = 19) in the test and 15.53 � 1.38 cm (n = 19) in the control group (p = 0.18)

F I G U R E 2
Nano Pyrite seed treatment prior to sowing resulted in improved germination and faster emergence.This figure shows a representative comparative picture of the germination test conducted on control and pyrite-treated red radish and soybean seeds.Representative image comparing germination in red radish seeds.Representative image showing germination in soybean seeds in control and test conditions.

F I G U R E 3
Pyrite-treated seedlings grew faster and taller than the control.In this figure, we have compared the growth of seedlings during the first 12 days of plants' life in control and pyrite-treated seeds.The core idea is to picture the effects of pyrite treatment on metabolic turnover, which in turn is quantified in the form of plant growth.(a) Red radish seedlings 12 days after germination and their lengths, (b) Representative image of soybean seeds 8 days after germination.Box and standard distribution plot comparing plant lengths between test and control groups for (c) Red radish and (d) Soybean seeds.MODERN AGRICULTURE observed that pyrite-treated plants grow faster and are taller than the control plants.

F I G U R E 5
Field trials of pyrite-treated red radish showed increased yield, plant length, and anthocyanin contents.In this figure, we present the image of mature red radish in the field, harvested red radish, and the total anthocyanin content in test and control crops.(a) Side view of red radish crop.(b) Front view of red radish crop where 'T' stands for test and 'C' stands for control rows.(c) Representative images of matured red radish from Test (T; n = 8) and Control (C; n = 9) groups.(d) Average plant length of mature red radish in test and control.(e) Total anthocyanin content in nanopyrite treated and control red radish.