Comparative Study of Green-Synthesized and Market-Procured CuO and FeO Nanoparticles on the Growth and Stress Tolerance in Rice Seedlings

Comparative Study of Green-Synthesized and Market-Procured CuO and FeO Nanoparticles on the Growth and Stress Tolerance in Rice Seedlings | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Study of Green-Synthesized and Market-Procured CuO and FeO Nanoparticles on the Growth and Stress Tolerance in Rice Seedlings Padmaja Rai, Kanchan Vishwakarma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7025406/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The widespread use of nanoparticles (NPs) resulting from industrial activities has led to significant environmental challenges, including biodiversity loss, climate change, soil pollution (both agricultural and saline), and contamination of water bodies. Iron oxide nanoparticles (FeO NPs) and copper oxide nanoparticles (CuO NPs) are extensively used across various industries such as biomedical devices, glass manufacturing, paint production, and as doping materials in semiconductors due to their unique properties. This widespread application necessitates an assessment of their impact on plant growth and agricultural systems. In this study, we investigated the comparative effects of green-synthesized versus market-procured CuO and FeO NPs on rice seedlings. The green synthesis of CuO and FeO NPs was achieved using a plant extract from Amragandhi Haridra (Curcuma amada). Several analytical techniques, including ultraviolet spectrometry (UV-vis), particle size analysis (PSA), X-ray diffraction (XRD), field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FE-SEM EDX), and Fourier transform infrared spectroscopy (FTIR), were employed to characterize the size, shape, functional groups, and crystalline structure of the green-synthesized nanoparticles. Our findings revealed that market-procured CuO NPs (50 µM) and FeO NPs (200 µM) significantly impaired rice seedling growth, reducing shoot length, root length, and fresh weight. These nanoparticles also decreased the levels of chlorophyll a, chlorophyll b, and carotenoids compared to green-synthesized CuO (50 µM) and FeO NPs (200 µM). Moreover, market-procured nanoparticles at the same concentrations induced the generation of reactive oxygen species (ROS), disrupting plant metabolism by interfering with photosynthesis, respiration, and electron transport activities. In contrast, green-synthesized CuO and FeO NPs at lower concentrations (50 µM) enhanced plant growth and offered protection against oxidative stress. In conclusion, these findings highlight the superior potential of green-synthesized NPs in protecting rice crops from oxidative stress, in contrast to market-procured NPs, representing a significant advancement toward sustainable agricultural practices. CuO NPs FeO NPs ecofriendly green synthesized market based rice seedlings Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Stress in plants refers to any adverse condition or factor that disrupts a plant's normal growth, development, metabolism or physiological functions caused by unfavorable environmental conditions (Zhang et al., 2020 ). Just like humans and animals, plants can experience stress, and it can be caused by various environmental or biological factors. Plants can experience various types of stress, which can be broadly categorized into abiotic and biotic stress based on their origin. Abiotic stress lowers the growth, yield and developmental pattern of crop plant. It is caused by non-living factors in the plant's environment, while biotic stress is caused by living organisms (Gull et al., 2019 ). Apart from various abiotic and biotic stresses, nanopollution is one of the major areas that raise concern on the detrimental consequences of nanoparticles pollution in crop plants. Nanopollution refers to the potential hazards linked to the introduction of NPs into the agricultural soil (Jan et al., 2022 ). NPs are extremely small particles with at least one dimension less than 100 nanometers (nm), often manufactured for various industrial, commercial and consumer applications due to their unique properties at the nanoscale (Jeevanandam et al., 2018 , Jan et al., 2022 ). The concern with nanopollution arises from the fact that NPs can behave differently compared to their bulk counterparts (Wang et al., 2009 ). The compact dimensions and the elevated ratio of surface area to volume they possess might result in augmented reactivity, mobility and potential toxicity within the plant system. Some sources of nanopollution include industrial processes, consumer products and environmental releases during the manufacturing, use and disposal (Buzea et al., 2007 ). In addition, the risk of NPs being discharged into the environment during the manufacture of products, including its distribution and disposal is also higher than that of other bulk materials due to its smaller size (Wang, 2012 , Wang et al., 2013a , Reinhart et al., 2010 , Yang et al., 2013 ). Among the various types of metal oxide NPs, Copper oxide (CuO) and Iron oxide (FeO) based NPs have been widely used in industrial sectors. These NPs are unique among the most widely used NPs as they exhibit many interesting and useful physicochemical properties. The yearly global production of CuO NPs ranged from 200 to 830 tons in 2010, and it is anticipated to rise in a range of 330 to 1600 tons by the year 2025 (Liu et al., 2018 ). Compared to other metal oxide NPs, the consumption and production of CuO and FeO NPs are higher in catalysts and biomedical devices (Janković & Plata 2019 ). In present years, large amount of money has been spent in nano-industry. Therefore, in coming years this has led to increase in more production of NPs which further released into our environment and goes into the agriculture fields. Unintentional way of NPs to reach environment and soil system is through volcanic emissions, agriculture, wastewater, and accidental release during manufacturing industries and vehicles (Klaine et al. 2008 ). This results in the movement of NPs into the soil and water bodies thereby affecting entire food chain. Nowadays, investigations have been focused on understanding the mechanism and phytotoxic effect of CuO NPs and FeONPs on different crops. Saif et al. ( 2016 ) stated that CuO NPs are used by many of the industries for various purposes like antimicrobials, biosensors, surfactants, catalysis etc. (Ahamed et al., 2014 , Nasrollahzadeh et al., 2016 ) and different types of FeO NPs like Fe 3 O 4 as magnetite, γ-Fe 2 O 3 (maghemite) and α-Fe 2 O 3 (hematite) act as a good promising agent for biomedical applications because of high magnetization and superparamagnetic properties (Karimzadeh et al., 2016). The nanoparticles size < 100 nm has shown detrimental effects on plants (Tripathi et al., 2017). Disposal of these nanoparticles from industries and laboratories is causing major harm to the plant system and disturbing water ecosystem. In the present time, main aim is to protect the plant system and environment from detrimental effects of nanoparticles and other substances formed through them. It is also important to find better approach for safely utilization of nanoparticles. It is reported that 90% biomass reduction was observed by exposure to copper nanoparticles (50 nm) at concentration of 1,000 mg L − 1 in Cucurbita pepo (Zucchini) (Stampoulis et al., 2009 ). Similarly, it was reported by Musante and White ( 2012 ) on treatment with CuO NPs the growth and transpiration of C. pepo was significantly inhibited by 60–70% as compared to control. The insufficient work on impact of metal oxide NPs on plant is a major limitation which requires carrying out extensive research in this area. High concentration of FeNPs causes cytotoxic effect to many plants due to formation of reactive oxygen species (ROS), lipid peroxidation on pumpkin ( Cucurbita mixta ) and perennial ryegrass ( Lolium perenne L.) (Wang et al., 2011 ). These NPs are absorbed in the roots of plants which are further transported and used by different parts of the plant. On the other hand, high amount of FeO NPs is found to be harmful for the plants. These NPs in roots are transported by the apoplastic pathway and collected into the cell walls. But excess of these particles prevents the availability of mineral nutrients in plants as they get blocked in the cell wall (Yuan et al., 2018). Souza et al. ( 2019 ) have stated that chlorophyll content were reduced by impact of FeO NP at high concentrations, which stopped the light absorption process on the aquatic plant Lemna minor and ultimately harm the nutrient uptake in L. minor . Therefore, market procured FeO NPs and CuO NPs have detrimental effects on the growth of crops and more attention is needed to find better approaches to reduce NPs toxicity in plants. NPs exert toxic effects on plant growth and seed germination depending on their properties and concentration (García-Gómez and Fernández 2019 ). The effect of these NPs depends on type, amount and specimen plants used for experiment. When plant is under any type of stress, the balance between activation of antioxidant enzymes in plants and free radical formation is disturbed and it leads to oxidative stress and cell death in plants, this further result in lipid peroxidation and protein degradation in plants (McCord JM, 2000 , Kapoor et al., 2019 ). The communication between different NPs and plant crops are still in its infancy stage (Pallavi et al., 2016 ). Different studies are seen in crops with respect to translocation, toxicity, biotransformation, and absorption of NPs (Husen and Siddiqi, 2014 ). Still the research studies are carried out to understand impact of NPs on different crops (Rajput et al., 2018 , Souza et al., 2019 ). There are various methods to synthesize NPs via chemical, physical and biogenic. Among these biogenic synthesis approaches is an ecofriendly way to synthesize NPs which can be further used to enhance the plant growth (Begum and Jayawardana, 2023 ). The choice of synthesis method depends on the material to be synthesized, desired NPs characteristics (size, shape and composition), scalability, cost and intended application. Hence, it's important to consider the properties and potential applications of the NPs during the selection of the appropriate synthesis method. Safety precautions and proper waste management are crucial considerations during NPs synthesis to ensure the safety of the environment (Kamali et al., 2019 ). Integrating the principles of green chemistry into nanoscience has helped to identify eco-friendly substances which act as a reducing agent as well as a capping agent to synthesize stabilised NPs (Iravani, 2011 , Soltys et al., 2021 ). Different chemical and physical methods are applied for synthesis of different NPs e.g., thermal reduction, chemical and physical synthesis (Dhas et al., 1998 , Feldmann and Jungk 2001 ). At the same time, green synthesis method uses substances from plants which are not only eco friendly, but also cost effective (Xiong et al., 2011 ). Copper oxide nanoparticles have been produced by utilizing extracts derived from Cassia auriculata leaves (Valli and Geetha, 2016 ), Aloe vera leaf Extract (Kumar et al., 2015 ), Daphnia magna (Saif et al., 2016 ). Similarly, FeO NPs are synthesised from Lawsonia inermis and Gardenia jasminoides leaves extract (Naseem and Farrukh, 2015 ). The green synthesis approach is a better way to synthesize NPs due to their eco-friendly and non-toxic behaviour. Also, its application on the plant system is safe to promote growth and maintain agricultural sustainability (Parveen et al., 2016, Vishwakarma et al., 2017 ). Green synthesized NPs boost the antioxidant enzyme which helps to remove the ROS generated in cells of plants under stressed seedlings and reduce its toxic effect (Ramzan et al., 2023 ). Green-synthesized NPs made by using natural extracts, plants, microbes or other biological agents to reduce and stabilize metal ions into NPs, results in less toxicity and enhanced biocompatibility as compared to engineered and chemically synthesize NPs (Parveen, et al., 2016, Ijaz et al., 2020 ). The plant extracts or biological agents used in green synthesis often contain bioactive compounds that can contribute to the synthesis process and potentially enhance the properties of the resulting NPs with respect to physical or chemical synthesis (Parveen, et al., 2016). These NPs may be more readily absorbed by plants due to their biocompatible nature, leading to improved uptake and potential benefits for plant growth and health. Rice ( Oryza sativa L.) is one of the staple food crops, which nourish more than half of the world’s population (Kennedy, 2002 ). As it has significant role in the food security of human kind therefore the measurement of the toxicity due to metal oxide-based NPs on this vital crop holds great importance in relation to human health. Plant material taken is Amragandhi haridra ( Curcuma amada ) for the green synthesis of CuO NPs and FeO NPs. Phytochemical constituents present in this plant such as Carbohydrates, Alkaloids, Amino acids, Saponin, Glycosides, Flavonoids, Steroids and Tannins which act as a capping and reducing agent for the synthesis of CuO NPs and FeO NPs. Therefore, the aim of this study is to determine the effect of green synthesized and market based CuO NPs and FeO NPs on rice seedlings. Nanoparticle toxicity examinations have been showed that green-synthesized CuO NPs and FeO NPs are less toxic than market one, providing insight into a new mode to synthesizing more environmentally friendly NPs for numerous uses. Although, it still unclear whether market procured CuO NPs and FeO NPs toxicity is due to ion dissolution or deposition of oxide form of CuO NPs and FeO NPs themselves. Therefore, study needs more investigation to find the proper deposition of CuO NPs and FeO NPs in plant cells. Hence, the work aims on measuring growth attributes, biochemical changes, ROS metabolism (qualitative and quantitative production of oxidative stress markers) and gene expression changes caused by both green- synthesized and market based CuO NPs and FeONPs on rice plant. Therefore, this is the first study to assess comparative effect of green synthesized and Market based CuO NPs and FeO NPs on rice seedlings. 2. Material and Methods 2.1 Green synthesis of CuO and FeO NPs The plant selected for the green synthesis (GS) of CuO and FeO NPs was Amragandhi haridra ( Curcuma amada ), a prominent herb in Ayurveda. This plant's vibrant yellow spice is commonly referred to as "haldi" in Hindi. The rhizomes of Curcuma amada were obtained from a local Ayurveda store in Prayagraj, India and were carefully stored in sterile plastic bags. The rhizomes were thoroughly cleaned with deionized water, dried and finally coarsely ground. Subsequently, an extraction process was carried out using 80% methanol as the solvent, employing the Maceration method. After 24 hours of incubation, the extract was filtered through Whatman No.1 filter paper. The plant extract was then dried and stored at 4°C for future use (Saif et al., 2016 ). In order to finetune the process for synthesizing CuO and FeO NPs, various concentrations within a range of 0 to 1000mM in their respective precursors i.e., CuSO 4 and FeCl 3 were dissolved in 50 ml of deionized water (Saif et al., 2016 ). In the case of CuO NPs, 4 ml of a freshly prepared plant extract was added into 2 ml of the 5mM CuSO 4 solution. Subsequently, the total volume was brought to 50 ml by adding MilliQ water. Similarly, for synthesizing FeO NPs, plant extract was mixed with 100 mM FeCl 3 (15 ml) and the final volume was adjusted to 300 ml through the addition of MilliQ water. Further, the process involved repeated shaking for 1 hour, followed by incubation for varying durations (10 minutes, 20 minutes, 30 minutes and 60 minutes) at a temperature range of 50–80°C on a hot plate, with sample analysis conducted at each 10 minute intervals. During this time, the color transformation was observed, with a shift to a dark brownish indicating the formation of CuO NPs and a dark blackish shade suggesting the presence of FeO NPs. Subsequently, the solution was subjected to centrifugation at speeds exceeding 12,000 rpm for 15 minutes, resulting in a pellet that was reconstituted in deionized water. Thereafter, to remove any residual impurities from both types of NPs, acetone was added on the pellet and subjected to two rounds of centrifugation at 12000 rpm for 2 minutes. Following this, the CuO and FeO NPs were dried in an oven for 24–78 hours and they were further processed in a muffle furnace for 10–15 minutes to obtain the final dried form of both NPs (Devi et al., 2019 , Amin et al., 2021 , Alhalili, 2022 ). 2.2.2 Characterization of CuO NPs and FeO NPs The green synthesized CuO and FeO NPs were subsequently characterized. They were subjected to ultrasonication in a bath sonicator for 30 minutes. Following this, scanning was performed within the wavelength range of 300–700 nm using an Ultraviolet-visible spectrophotometer (Eppendorf Bio Spectrometer). De-ionized water was employed as a reference (blank) during these measurements. The dimensions of the CuO and FeO NPs were determined using a Particle Size Analyzer (PSA). Both commercially procured and green synthesized NPs were accurately dissolved in distilled water, with the addition of 1 ml of diluted 0.1% DMSO followed by ultrasonication for 30 minutes (Devi et al., 2019 , Amin et al., 2021 , Alhalili, 2022 ). In addition, a powdered form of green synthesized CuO and FeO NPs was utilized to categorize the diverse biomolecules contained within them. This enabled the identification of the capping agents responsible for effectively stabilizing both types of NPs. For this, the analysis was conducted using Fourier Transform Infrared Spectroscopy (FTIR) with PerkinElmer Spectrum Version 10.4.00, following the method outlined by Rajendran and Sengodan (2017). Further the NPS were characterized by X-Ray diffraction analysis (XRD) to know the crystalline structure of NPs. Additionally, FE-SEM-EDS were conducted to determine the surface structure and elemental phase composition. Furthermore, sizes of both types of NPs were detected by TEM (Devi et al., 2019 , Amin et al., 2021 , Alhalili, 2022 ). 2.3.3 Screening experiment to optimize the NPs concentration for Hydroponics Studies The comparative assessment was carried out to evaluate the effect of both green synthesized and market procured CuO and FeO NPs on rice seedlings. Rice seeds were sown hydroponically in Hoagland solution within a plant growth chamber (M/s Ocean Life Science Corporation, India). The growth conditions in the chamber were maintained at a relative humidity of 65% and a temperature of 28 ± 2°C. The seeds of rice were surface sterilized for 20 minutes using a 2% (v/v) sodium hypochlorite solution. Afterward, the seeds were thoroughly rinsed with distilled water and soaked in a dark environment for 12 hours to break dormancy. Following this period, muslin cloth was used to wrap the seeds, which were then kept in the dark to facilitate proper germination (Hoagland and Arnon, 1950 ). Once the seeds had sprouted well after two days, healthy and uniformly sized seedlings were selected and placed in Petri plates containing Whatman No. 1 filter paper (150 mm, Riviera), which had been moistened with half-strength Hoagland solution. These seedlings were grown in the plant growth chamber (PGC) with a photon flux density (PFD) of 350 µmol photons m − 2 s − 1 , under a 12/12-hour day/night cycle and a relative humidity of 60%, all maintained at a temperature of 25 ± 2°C. This growth period lasted for duration of 8 days (Tripathi et al., 2021 ). Following this, both NPs were optimized for the experiment. Subsequently green synthesized and market procured NPs were dissolved in distilled water using bath sonication for 30 minutes. The experimental setup were included different concentrations (0, 20, 50, 100, 200, 500 and 1000 µM) of green synthesized and market procured CuO and FeO NPs in 40 ml Hoagland’s solution. The macro- and micronutrients used to make the Hoagland solution were represented in Appendix 1. Subsequently, seedlings were allowed to grow for 7 more days in a Plant growth chamber. The nutrient solution was changed periodically and was aerated daily to avoid hypoxia. Following the ending of the experiment, seedlings were collected and subjected to various analysis to assess different factors. The experiments were conducted in triplicates (Tripathi et al., 2021 ). 2.3.3.1 Morphological parameters The morphological parameters comprised of root length, shoot length as well as fresh weight of shoot and root were assessed. The seedlings were cut into separate shoots and roots for the determination of growth parameters under varying concentrations (0, 20, 50, 100, 200, 500 and 1000 µM) for green synthesized and market procured CuO and FeO NPs. The length of the rice seedlings was measured using a centimeter scale. Furthermore, the fresh weight was determined by individually collecting the roots and shoots and weighing them using a digital weighing balance (Tripathi et al., 2021 , Rai et al., 2021 ). Subsequently, green synthesized and market procured CuO NPs at a concentration of 50 µM and 200 µM for FeO NPs respectively were selected for further parameter testing. 2.3.3.2 Physiological parameters The physiological parameters encompassed the analysis of photosynthetic pigments and the assessment of Chlorophyll a fluorescence. Photosynthetic pigment estimation The photosynthetic pigments were assessed for the specified concentrations of green synthesized and market procured CuO and FeO NPs. The determination of total chlorophyll and carotenoid levels were obtained by using 20 mg of leaves from various treatments that were ground and placed in a 5 ml solution containing 80% (v/v) acetone. Following this step, the mixture was placed for centrifugation at 10,000 rpm for approximately 15 minutes at a temperature of 4°C. The quantification of chlorophyll a and b as well as carotenoids was done as per the protocol mentioned in Lichtenthaler ( 1987 ). Thereafter, the optical absorbance values of the solution were taken at wavelengths of 663.2, 646.5 and 470 nm using a UV–visible spectrophotometer Measurement of protein content Lowry et al. ( 1951 ) method is employed for determining the total soluble protein content. In this technique, both untreated and additional samples were ground in 50 mM potassium phosphate buffer (pH 6.8). Subsequently the mixture was centrifuged at 8,000 g for 15 minutes. The protein content in the various treatments and control samples, Bovine Serum Albumin (BSA) was used as a reference standard. 2.3.3.3 Chlorophyll a fluorescence analysis Additionally, the photosynthetic efficiency of rice seedlings was evaluated utilising a handheld leaf fluorometer (Fluor Pen FP 100, Photon System Instrument, Czech Republic). After subjecting the leaves to 30 minutes of darkness, fluorescence parameters from the JIP test, such as the maximum photochemical efficiency of PSII (Fv/Fm), photochemical quenching (qP) and non-photochemical quenching (NPQ) were analyzed by the procedure outlined in Strasser, ( 1995 ) and Strasser et al. ( 2000 ). 2.3.4 Biochemical parameters The biochemical parameters included the evaluation of oxidative stress markers namely SOR, H 2 O 2 and MDA. The assessment of anatomical characteristics involved the application of stress marker dyes for the purpose of evaluating cell viability through histochemical staining. Additionally, in vivo localization of reactive oxygen species and superoxide ions was conducted using fluorescence microscopy. Additionally, the antioxidative defense responses were assessed by measuring the activities of antioxidant enzymes. 2.3.4.1 Estimation of super oxide radical (SOR) The shoot samples were crushed with 3 ml of a 65 mM potassium phosphate buffer at pH 7.8. Following this, they were subjected to centrifugation at 10,000 g for 10 minutes, following the procedure outlined by Elstner and Heupel ( 1976 ). Subsequently the obtained liquid (1 ml) was combined with 0.9 ml of a 65 mM phosphate buffer and 0.1 ml of 10 milimolar hydroxylamine hydrochloride. After twenty minutes of incubation at 27 o C, 7 mM NEDD and 17 mM sulphanilamide were mixed into the incubation mixture. To eliminate interference from pigments, the components were meticulously blended and subsequently divided into two distinct layers by adding an equivalent volume of diethyl ether. The pink water phase in the lower layer was assessed for absorption at 530 nm. A standard curve, established with sodium nitrite to determine the SOR content. The quantity of SOR in the sample is expressed in nmol per gram of fresh weight (nmol g − 1 FW). 2.3.4.2 Hydrogen peroxide (H 2 O 2 ) Following Velikova et al. ( 2000 ) method, the assessment of H 2 O 2 was conducted. Fresh shoot samples were blended in 0.1% (v/v) Trichloroacetic acid (TCA) and then centrifuged at 15,000 g for 15 minutes. The obtained liquid (supernatant) was used for the H 2 O 2 analysis. The reaction mixture comprises of 10 milimolar potassium phosphate buffer (0.5 ml- pH 7.0), 1 M potassium iodide (KI- 1.0 ml) and extract (0.5 ml). Afterward, the absorbance at 390 nm was measured against a blank in each sample and determined using a standard curve made with H 2 O 2 . The amount of hydrogen peroxide in each sample is shown as nmol g − 1 FW. 2.3.4.3 Lipid peroxidation The assessment of lipid peroxidation involved estimating the concentration of MDA (malondialdehyde) equivalents, which serve as markers for the peroxidation products of unsaturated fatty acids. This was achieved by measuring thiobarbituric acid reactive metabolites Heath and Packer, ( 1968 ). 2.3.5 Anatomical parameters Further, the quantitative results of oxidative stress markers were further validated qualitatively by evaluating the cell viability by histochemical staining to visualize the production of radicals. Also, in vivo localization of ROS (reactive oxygen species) and O 2 •− by Olympus BX51 Fluorescence Microscopy were evaluated. 2.3.5.1 Histochemical staining The histochemical staining was performed as per the protocol of Thordal-Christensen et al., ( 1997 ). The application of 3, 30-diaminobenzidine (DAB ) was utilised to detect the presence of hydrogen peroxide (H 2 O 2 ) in rice root and leaf tips. The tips of root and leaf were washed with MQ water, subsequently put in 1% DAB (pH 3.8; Sigma, United States) for 8 hr at 25 0 C in the light. These samples were stained with the dye and then washed with MQ water. These were subsequently dipped fully in 95% ethanol and kept for boiling for 10 times. This step was repeated for two times. The slides were prepared, and photography was done by using Olympus compound dark-field microscope Furthermore, to assess membrane damage in plant cells, tips from the leaves and roots of rice seedlings were excised. Subsequently, these tips were washed with a sodium phosphate buffer at a pH of 7.4. The solution of Evan’s blue was prepared at a concentration of 10 mg/ml. The excised tips were then immersed in the Evan’s blue dye for duration of 30 minutes. After staining, the tips were rinsed with ethanol and MQ water and then mounted on a slide followed by imaging an Olympus compound dark-field microscope (Schützendübel et al., 2001 ). Schiff's staining was utilized to detect the lipid peroxidation in plants subjected to stress conditions. Fresh tips from the roots and leaves of rice seedlings were excised. Subsequently, these tips were washed with MQ water and then immersed in a 0.5% solution of Schiff's stain for a period of 3 hours. After staining, they were rinsed with a 0.5% solution of potassium silicate (K 2 SiO 3 ). Following this, the tips underwent washing with ethanol and MQ water. Images of the root tips and leaves were captured using an Olympus compound dark-field microscope by the method mentioned in Awasthi et al. ( 2018 ). 2.3.5.2 In vivo localization of ROS and O 2 •− by fluroscence microscopy - The visualization of the presence of O 2 − (superoxide) in rice roots was carried out using 10µM DHE stain, which was prepared in a 10 mM tris HCL (pH- 7.4) for 15 minutes (Sandalio et al., 2008 ). Subsequently, to remove any excess stain, the samples were washed three times with MQ water; each wash lasting for 10 minutes and were then observed using an Olympus BX51 fluorescent microscope. The observation was performed with excitation at 488nm and emission at 520nm. Additionally, the staining protocol mentioned by Morina et al., ( 2010 ) was employed to identify the occurrence of H 2 O 2 in various treatments. This was accomplished by dipping the root tips in 10µM DCF2DA stain for 15 minutes. The roots of the rice seedlings were rinsed twice in the identical buffer solution, each time for 15 minutes, and subsequently positioned on a microscope slide for observation using an Olympus BX51 Fluorescence Microscope. The microscope was configured to excite the sample at a wavelength of 485nm and capture the emitted light at 535nm. 2.3.6 Antioxidative defense responses This section encompasses assays for estimating the antioxidant enzymes activities. 2.3.6.1 Superoxide dismutase (SOD; EC 1.15.1.1) The assessment of SOD was done by the protocol of Giannopolitis and Ries, ( 1977 ). Enzyme activity was obtained by crushing the 100 mg of tissue from each sample in 100 mM phosphate buffer (pH 7.8) along with EDTA under refrigerated conditions. The mixture was gently inverted in tubes and thoroughly mixed. Following this, the mixture was centrifuged at 10,000g for 20 minutes. The resulting liquid was transfer to sterile test tubes and used for the enzyme assay. The reaction mixtures were prepared by combining 0.1 ml of the supernatant with riboflavin, L-methionine, Na 2 CO 3 (pH 10.2) and NBT with a concentration of 1.3 µM,, 13 mM, 0.05 M and 63 µM respectively to achieve a total volume of 3 ml. All the test tubes were then exposed to light at an intensity of 100 µmol photon m − 2 s − 1 . Afterwards, the absorbance of the final product was assessed at a wavelength of 560 nm in relation to the control. The enzyme concentration exhibited a direct correlation with the disparity in absorbance at 560 nm when comparing the presence and absence of the mixture. Furthermore, the unit of SOD activity was defined as the amount of enzyme that hindered the 50% decrease of NBT under the specified conditions. 2.3.6.2Catalase (CAT; EC 1.11.1.6) The measurement of CAT activity followed the methodology outlined by Aebi ( 1984 ). In this process, 50 milligrams of tissue from each sample were homogenized using 1 ml of a 50 millimolar PPB solution containing 1 mM EDTA at a pH of 7.0. The mixture underwent centrifugation at 10,000 g for 15 minutes. Subsequently, the liquid above the debris was utilized for enzyme activity analysis. The quantification of H 2 O 2 was carried out using the extinction coefficient (ε = 39.4 mM − 1 cm − 1 ). A unit is defined as the quantity of enzyme that can break down 1 nanomole of H 2 O 2 in one minute. 2.3.6.3 Dehydroascorbate reductase (DHAR; EC 1.8.5.1) The content of DHAR in each sample was estimated spectrophotometrically by adopting the procedure of Nakano and Asada ( 1981 ). The enzyme activity of MDHAR was measured by quantifying it using an extinction coefficient of (ε = 7.0 mM-1cm-1). 2.3.6.4 Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) The content of MDHAR was assessed by the protocol of Hossain et al. ( 1984 ). Activity of MDHAR was assessed utilizing an extinction coefficient of 6.2 mM − 1 cm − 1 . A single unit of enzyme activity is defined as the oxidation of 1 nmol of NADH per minute. 2.3.6.5 Glutathione reductase (GR; EC 1.6.4.2 ) The activity of enzyme was determined by following the process outlined in Schaedle and Bassham ( 1977 ). One unit of GR activity represents the quantity of enzyme that facilitates the oxidation of 1 nanomole of NADPH in a minute. 2.3.7 Gene expression analysis The gene expression analyses of antioxidant associated genes, copper and iron transporters were performed using quantitative real time PCR in different treated samples. Total RNA was obtained from the sample using the Spectrum Total Plant RNA isolation kit from Sigma. The RNA was reverse transcribed into complementary DNA (cDNA) following the method by Wormuth et al. ( 2006 ). Subsequently, quantitative real time PCR (qRT-PCR) was performed using the Step One Plus Real Time PCR System from Applied Biosystems. The pure RNA gets collected in the flow through and it was stored at -80 0 C for further use. The quality and quantity of RNA was checked through NanoDrop instrument. Finally, the 260/280 ratio was checked for RNA purity and integrity. Furthermore, the cDNA was prepared from the RNA using verso cDNA synthesis kit. Once the reaction mixture was prepared, it was subjected to incubation at 42°C for 30 minutes (1 cycle) and subsequently at 95°C for 2 minutes. The cDNA that was prepared was then diluted in DEPC water in a 1:4 ratio and employed for the analysis of gene expression via qRT-PCR. Following this, 5 µl of SYBR Green Supermix was added to the samples to make the reaction mixture a total of 10 µl. The actin gene was taken as the internal control. Further, after preparation of reaction mix the contents were transferred to qRT-PCR plate and the reaction was set in the machine. The expression studies in qRT-PCR were carried out by using Step One plus Real-Time PCR System. The entire volume of reaction mixture was 10µl and actin was taken as a housekeeping gene for normalization. The general conditions were set using following steps: 95°C for 1 min; 40× (95°C for 30s, 58°C for 40s, 72°C for 45s), 72°C for 10 min followed by a melting curve program (55–95°C in increasing steps of 0.5°C). Further, the relative quantification for gene expression levels after normalization with actin was calculated by comparative CT (Delta–Delta CT) method, as demonstrated by Livak and Schmittgen ( 2001 ). Primers were designed with primer 3 tool. The list of primers specific to particular genes designed is presented in Supplementary table 1 . 2.4 Statistical analyses The data presented in the thesis have been statistically analysed using one-way analysis of variance (ANOVA) by SPSS 16.0 software, following verification of the data's normal distribution. Variances among treatments were assessed through Duncan’s multiple range tests at a significance level of p < 0.05. The values depicted represent the means derived from three independent biological replicates (n = 3). The Graphs represented in the study were made by using sigma plot 14. 3. Results 3.1 Green synthesis and Characterization of green synthesized CuO NPs and FeO NPs Green synthesis of CuO and FeO NPs were successfully synthesized using plant i.e., Curcuma amada . The precursors, CuSO 4 and FeCl 3 were used at final concentrations of 5 mM and 100 mM respectively. Thereafter, the synthesis was carried out at temperatures of 55°C for CuO NPs and 60°C for FeONPs, each for duration of 20 minutes. The resulting CuO NPs displayed a brownish color, while the FeO NPs exhibited a slightly blackish color. The absorbance peaks were detected within the wavelength range of 250 to 500 nm using a UV-Visible spectrophotometer. Specifically, the absorbance maxima for green synthesized CuO NPs and FeO NPs were observed at 480 nm and 300 nm respectively (Fig. 1 A and 1 D). In addition, the mean particle size was observed at 33.7 nm for CuO NPs (Fig. 1 B) and 26.45 nm for FeO NPs (Fig. 1 E) respectively. The data of X-ray diffraction (XRD) revealed that all the diffraction peaks of NPs were consistent with the standard structure. The planes (021), (110), (111), (131) and (151) for green synthesized CuO NPs represent the monoclinic structure of CuO NPs. The planes (111), (400), (422), (511), (440), (533) for FeO NPs indicated the formation of a monoclinic crystalline structure (Fig. 1 C and Fig. 1 F). In addition, FTIR results demonstrated the presence of biomolecules in the plant extract, which acted as capping agents for the efficient stabilization of both NPs. The observed peaks at 602.92 cm − 1 and 509.19 cm − 1 corresponded to the characteristic stretching vibration of the Cu-O bond in Copper oxide (Fig. 2 A). Subsequently, the observed peaks at 666.01 cm − 1 and 477 cm − 1 corresponded to the characteristic stretching vibration of the Fe-O bond (Fig. 4.2D). The sharp peaks observed at 602.92 cm − 1 and 666.01 cm − 1 in the FTIR spectrum confirmed the formation of CuO NPs and FeO NPs (Fig. 2 A and 2 D). Surface morphology of the CuO NPs and FeO NPs was observed by FE-SEM (Fig. 2 B, 2 E). The EDS spectrum revealed the presence of copper (Cu) and oxygen (O 2 ) in CuO NPs, as well as iron (Fe) and oxygen (O 2 ) in FeO NPs. The detection of sulfur (S) and chlorine (Cl) atoms in the spectrum can be ascribed to the plant extract. The EDS spectrum also confirmed that the CuO NPs and FeO NPs are in oxide form and free from any other impurities. These results were further validated by TEM as depicted in (Fig. 2 C and Fig. 2 D) showing that the size of both NPs was below 50 nm and the shape was round. 3.2 Morphological Parameters The impact of green synthesized and market procured CuO NPs and FeO NPs was studied on the morphological parameters of rice seedlings. The morphological attributes of rice seedlings were analyzed by measuring the length and fresh weight of shoots and roots to evaluate the effects of green synthesized and market procured CuO NPs and FeO NPs at different concentrations. Obtained results revealed that with increasing concentration, both shoot and root length, as well as shoot and root fresh mass, showed a decreasing trend with both CuO NPs and FeO NPs. However, the impact was more significant in both the market procured NPs as compared to green synthesized (P < 0.05). Based on the screening experiment, following doses of green synthesized (GS) and market procured (MP) CuO and FeO NPs were selected for further studies (Supplementary Fig. 1 and Fig. 2 ). Control GS CuO NPs- 50 µM MP CuO NPs − 50 µM GS FeONPs − 200 µM MP FeO NPs- 200 µM The concentration of 50 µM green synthesized CuO NPs showed a significant augment in shoot length and fresh weight, by 15.09% and 17.06%, respectively with respect to the control. Similarly, the root length and fresh weight also increased under green synthesized CuO NPs (50 µM) by 16.95% and 14.48%, respectively with respect to the control (P < 0.05) (Fig. 3 ). Similarly, a concentration of 200 µM green synthesized FeO NPs led to an increase in shoot length and root length by 10.00% and 4.80% respectively with respect to the control (Fig. 3 ). In contrast, at a concentration of 50 µM and 200 µM market procured CuO NPs and FeO NPs respectively, there was a significant decrease in shoot length by 18.37% and 18.65% and fresh mass by 31.53% and 34.32% respectively as compared to the control (P < 0.05) (Fig. 3 ). However, the shoot length and fresh mass at a concentration of 200 µM market procured FeO NPs were significantly decreased by 31.53% and 34.32% respectively as compared to the control. 3.3 Effect of treatments on photosynthetic properties and total protein content The physiological parameters were assessed through the measurement of photosynthetic pigment levels and Chlorophyll a fluorescence. Green synthesized CuO NPs resulted in a notable increase in chlorophyll and carotenoid levels by 2.90% and 1.55%, respectively with respect to control (Fig. 4 ). In contrast, market procured CuO and FeO NPs showed a significant decrease in chlorophyll by 30.4%, 26.80% and carotenoid by 33.9%, 37.00% with respect to control (Fig. 4 ). Furthermore, photosynthetic pigments wee further validated by Chlorophyll a fluorescence parameters. This was calculated by measuring the parameters such as Fv/Fm, qP and NPQ. The results were depicted in the spider plot shown in Fig. 5 . Significant differences were observed between green synthesized and market procured NPs for all the parameters. The green synthesized CuO NPs and FeO NPs were increased in Fv/Fm by 16.30% and 13.10% as compared to control. Similarly, green synthesized CuO NPs and FeO NPs were increased qP by 9.13% and 9.92% with respect to control (Fig. 5 ). However, NPQ showed a reverse effect under green synthesized both the NPs as compared to the control. Additionally, market procured NPs decreased the content of Fv/Fm and qP as compared to the control as represented in Fig. 5 . The total protein content in rice seedlings was shown to be significantly decreased (p < 0.05) in both the market procured NPs with respect to green synthesized counterparts. 3.4 Impact of treatments on reactive oxygen species and cellular damage The in vitro levels of ROS, such SOR, H 2 O 2 and MDA for lipid peroxidation have been depicted in (Fig. 6 A,B,C). Rice seedlings exposed with green synthesized CuO NPs and FeO NPs showed a significant decrease in SOR levels by 32.20% and 33.70%, respectively (Fig. 6 A) as compared to their market procured counterparts (P < 0.05). Additionally, green synthesized CuO NPs and FeO NPs showed decreased in the content of H 2 O 2 by 18.47% and 32.29% respectively as compared to market procured NPs. Lipid peroxidation, measured in terms of MDA, was also significantly decreased under green synthesized CuO NPs and FeO NPs treatments by 9.40% and 15.40% respectively as compared to market procured both NPs (P < 0.05) (Fig. 6 C). The staining with Schiff’s stain (Fig. 7 A,B), DAB (Fig. 7 C,D) and Evan’s blue (Fig. 7 E,F) for ROS mediated damage to the plant cells (leaf and root tips) depicted lesser deposition in selected concentrations of green synthesized CuONPs and FeONPs in comparison to market procured CuONPs and FeONPs (Fig. 7 ). The markers of oxidative stress were affirmed through the application of fluorescent probes during histochemical and fluorescence staining of the root tips. Lighter fluorescent intensity (red fluorescence) was noticed for SOR (Fig. 8 a) as well as total ROS (green fluorescence) as represented in (Fig. 8 b) under green synthesized CuO and FeO NPs as compared to market one. The present findings also highlighted that under market procured NPs exposure, a significant diminution in the quantity of plant cells was seen under fluorescence microscopy (Fig. 8 a, b). Maximum cell death in plant cells was recorded due to more absorption of DHE stain, which might be correlated with the reduced growth of rice plants under market procured NPs treatments as compared to green synthesized one. 3.5 Impact of treatments on antioxidant enzymes The results indicated that the activity of SOD in green synthesized CuO NPs and FeO NPs were increased by 20.65% and 18.78%, respectively as compared to market procured NPs (Fig. 9 A). Similarly, CAT activity was also enhanced under green synthesized CuO NPs and FeO NPs as compared to the market counterparts as showed in Fig. 9 B. Additionally, the enzymatic activities of MDHAR and DHAR were increased under green synthesized CuO NPs and FeO NPs as compared to market procured NPs, as represented in Fig. 9 C and 9 D. Furthermore, GR activity was increased under green synthesized CuO NPs and FeO NPs by 38.16% and 39.09% respectively as compared to market procured NPs (Fig. 9 E). Overall, rice seedlings under market procured NPs treatment showed a diminution in the activity of SOD, CAT, MDHAR, DHAR and GR due to an increase in cellular ROS production, as well as associated damage to seedlings, which is also indicated in the in vivo imaging of O 2 •− and H 2 O 2 radicals (Fig. 8 and Fig. 9 ). 3.6 Regulation of gene expression The analyzed genes are associated with stress and detoxification mechanisms and their expression was assessed through qRT-PCR. It was observed that genes related to antioxidant activity namely DHAR (dehydroascorbate reductase), MDHAR (monodehydroascorbate reductase), GR (glutathione reductase) and APX (ascorbate peroxidase) , showed up-regulation in response to green synthesized CuO NPs and FeO NPs as compared to market procured NPs (Fig. 4.11). In contrast, the regulation of COPT7 (Copper transporter) and YSL15 (Iron transporter) genes were significantly up regulated under market procured CuO NPs and FeO NPs as compared to green synthesized NPs (Fig. 10 ). These antioxidant genes could not maintain the integrity of genes inside the stressed rice seedlings (Fig. 10 ). This suggests that the expression pattern of antioxidant genes responded differentially to green synthesized and market procured NPs treatments in diverse ways. 4. Discussion Nowadays, there is a growing focus on synthesizing NPs through green synthesis approaches because they are eco-friendly, pollution-free methods that promote a sustainable environment. Currently, the interplay between nanoparticles and crops has emerged as a key subject of exploration among nanotechnologists and plant scientists owing to how nanoparticles behave within plant cells (Tripathi et al., 2017b ). The findings of the current research are corroborated by the findings of Souza et al. 2019 who employed a chemically synthesized approach to create FeO nanoparticles using FeCl 3 salt. Similarly, (Fazlzadeh et al., 2017 ) synthesized zero-valent Fe NPs via the green synthesis approach using extracts of Rosa damascene, Thymus vulgari and Urtica dioica . These NPs also used to mitigate abiotic stress in plants (Singh et al., 2021 ). The current findings state that the round shape of CuO NPs and FeO NPs have been confirmed using the FE SEM and TEM technique. FESEM (Field Emission Scanning Electron Microscopy) and EDX (Energy-Dispersive X-ray Spectroscopy) are two common techniques utilised to analyze the surface and elemental composition of NPs. EDX is often used in conjunction with FESEM to analyze the elemental composition of a NPs surface (Restivo et al., 2014 ). The characterization was further validated by EDX to confirm the presence of Cu and O 2 for CuO NPs and Fe and O 2 for FeO NPs. These results are also in conformity with the findings of Kumar et al., 2015 used Aloevera leaf extract to synthesize CuO NPs. Their study revealed an average particle size of 20 nm through TEM analysis. X-ray diffraction (XRD) is a method employed for examining the atomic and molecular arrangement within crystalline substances (Dorofeev et al., 2012 ). Also, powder X-ray diffraction indicated the monoclinic phase of the NPs. They observed that the SEM images displayed the presence of larger particles, potentially arising from the aggregation or overlapping of smaller nanoparticles with sizes within 100 nm. Further (Kumar et al., 2015 ) showed that the chemical composition of the CuO NPs having an atomic percent of 54% for Cu and 45% for O by EDS spectra. Another study by Tandon et al., 2013 demonstrated that zero valent iron (ZVI) nanoparticles, averaging a particle size of 59.08 ± 7.81 nm were produced through the reaction between ferric nitrate and Mentha spicata L. tea extract. These particles exhibited absorption peaks at 360 and 430 nm, a confirmation made via UV-Vis analysis. The findings of the current result draws support from the study of Prasad et al. 2014 who reported that the size range of FeNPs falls below 50 nm when using TEM. The results mentioned in Fig. 1 and Fig. 2 , as reported by numerous authors demonstrate that plants are capable of efficiently forming stable metal oxide nanoparticles, making them ideal candidates for rapid and large scale synthesis compared to alternative methods (Iravani 2011 ; Nair et al., 2022 ; Vijayaram et al., 2023 ). The possible reason for the greater toxicity of market procured NPs is that they were more soluble than green synthesized NPs, resulting in a higher rate of ions release as compared to the plant synthesized nanoparticles (Saif et al., 2016 ). Several other studies have also highlighted the severe detrimental impacts of nanoparticles, impeding not just growth but also detrimently influence the metabolic, physiological and molecular characteristics of various plant (Du et al., 2017 ; de la Rosa et al., 2017 ). The findings of the present result draw support from the findings of Shi et al., 2014 who demonstrated that the CuO NPs detrimented the growth of plant and are likely to accumulate in the root cells and leaf cells of E. splendens . However, in a different study Da Costa and Sharma 2016 , found that engineered CuO NPs at a dose of 100 mg/L severely inhibited plant growth in terms of root and shoot development at elevated doses of CuO NPs as compared to the control. The biomass of rice shoots is also declined by 31% on a fresh mass (FM) basis and 14% on a dry mass (DM) basis. Similarly, another study by Ren et al., 2011 also suggests that FeO NPs (20 mg/L) enhanced the speed of seed germination, shoot length and root length in Chinese mung beans. The outcomes align with Anwaar et al., 2016 who used the leaf solution of Azadirachta indica in the synthesis of CuO NPs, showing substantial positive role on plant growth parameters at a concentration of 10 mg/lit CuONPs. A similar study was highlighted by Saif et al., 2016 to investigate the biotoxic consequences of plant synthesized and engineered CuO NPs on the water flea Daphnia and revealed that plant synthesized NPs exhibited greater stability as compared to their engineered counterparts. Physiological parameters in plants refer to various factors and processes that are associated with the functioning and health of a plant. This includes quantification of photosynthetic pigments that capture light energy for photosynthesis (Fernandez-Marin et al., 2018 ). Another important parameter to consider is the evaluation of chlorophyll a fluorescence, a critical process in photosynthesis that is widely used in plant physiology to evaluate the health and photosynthetic activity of plants. This assessment provides valuable information about the efficiency of photosynthesis and the overall stress levels of plants. This provides insights into the efficiency of photosynthesis and the physiological state of plants by measuring the fluorescence emitted by chlorophyll molecules (Kalaji et al., 2017 ). This information is essential for managing the risk of NPs in plant crops. Chlorophyll a fluorescence is an essential parameter for evaluating the performance of the photosynthetic machinery in rice seedlings (Tsai et al., 2019 ). Similarly, previous findings (Ren et al., 2011; Vishwakarma et al., 2017 ) have also shown that application of Ag NPs and CuO NPs in mustard and mung beans at 100 mg/L significantly declined the total chlorophyll contents. The present results also confirmed that the values of Fv/Fm (a reliable indicator of photosynthetic efficiency) and qP noticeably decreased under the exposure to market procured NPs as compared to green synthesized NPs. This reduction might be related to the decrease in total chlorophyll (Fig. 4.5A). The present data showed that disturbed electron flow between the photosystems ultimately reduced the Fv/Fm of the seedlings (Fig. 4.6). Additionally, Genty et al., 1990 observed that the down regulation of photosystem II was the main factor leading to increased NPQ levels, which may be due to the reduced need for electrons through NADPH when subjected to stressful conditions. The data demonstrated that NPQ was significantly enhanced under both the market procured NPs treatments, while its enhancement was less pronounced under green synthesized NPs at the same concentration, suggesting that green synthesized NPs allow for the appropriate performance of the electron transport chain. In another study by Singh et al., 2017a , NPs were synthesized using a green approach, utilizing flower extracts from Morus alba . It was indicated that application of biosynthesized CuO NPs at a doses of 10 mg/L exhibited the highest seedling growth in terms of increased radicle and plumule length and photosynthetic parameters. CuO NPs inhibited the growth of H. sativum by affecting the maximal quantum yield of photosystem (Rajput et al., 2018 ). Similar study reported by Singh et al., 2017a that exposure of plants to 100 and 500 mg L − 1 of biosynthesized CuO NPs significantly diminished the total chlorophyll and sugar content. Yet, when present at a concentration of 10 mg L − 1 of CuO NPs led to a marginal rise in pigment and sugar content in tomato plants. Biochemical parameters were assessed by evaluating the content of oxidative stress markers viz. , SOR, H 2 O 2 and MDA. Oxidative stress markers in plants are indicators used to assess the level of oxidative stress within plant cells. Oxidative stress is a condition that occurs when there is a disparity between the formation of ROS and the plant's capacity to detoxify or repair the damage caused by these molecules (Sharma et al., 2019 ). Elevated levels of Reactive oxygen species at its elevated level have the potential to harm cellular elements such as proteins, lipids and DNA, which can ultimately harm the plant's overall growth (Garg and Manchanda, 2009 ). ROS are generated in plants under oxidative stress, making it an important parameter to assess the consequences of NPs in rice seedlings (Tripathi et al., 2017b ). Similar findings for ROS species which generate oxidative stress under nanoparticles were also revealed by other authors (Ren et al., 2011; Vishwakarma et al., 2017 ; Pandey et al., 2022 ; Azhar et al., 2023 ). Likewise, the presence of CuO NPs at a dose of 5 mg/L resulted in higher ROS production, possibly attributed not only to the ions from CuSO 4 but also the NPs present in the rice (Wang et al., 2015 ). Tang et al., 2016 demonstrated that exposure to CuO NPs at a dose of 10 mg/L for 2 hours resulted in much higher ROS generation in root tips compared to the corresponding treatment with Cu 2+ ions at a concentration of 0.8 mg/L. In this context, Afzal et al. 2021 indicated that green synthesized FeO NPs revealed the existence of a critical concentration of NPs. Under this threshold, rice crop growth receives a boost, yet no additional improvement occurs beyond this level. In this context (Laouini and Bouafia 2021; Murugesan et al., 2022 ) reported that green synthesized NPs based on their concentration are more biocompatible and eco-friendlier due to the occurrence of phytochemicals in the plant extract, making these NPs non-toxic for plants. Subsequently, the presence of oxidative stress markers were supported by evaluating anatomical characteristics, including the in vivo localization of ROS and superoxide ions using histochemical dyes and fluorescent probes, through the use of fluorescence microscopy. Histochemical dyes like DAB (3,3'-diaminobenzidine), NBT (nitroblue tetrazolium), Evans Blue, and Schiff's reagent are used in to visualize ROS production in the cellular structure of plant cells under stress (Yadav et al., 2021 ). These staining techniques can provide valuable understanding regarding the localization and intensity of stress-induced changes in plant tissues (Yadav et al., 2021 , Basu et al., 2021 ). The results indicated a notable decline in the growth pattern of rice seedlings when exposed to market procured NPs, which might be related to the increased formation of oxidative stress markers. Previous research has similarly demonstrated that stressors expedite the production of ROS, encompassing lipid peroxidation, membrane impairment, and the production of H 2 O 2 (Xie et al., 2019; Tripathi et al., 2021 ). Treatment of seedlings with green synthesized NPs represented an augmentation in the growth pattern and reduced damage to biomolecules through ROS formation as compared to market procured NPs. These biomolecules are important markers in the photosynthetic pathway in plants, as reported by Sharma et al. 2012 , Tripathi et al. 2017b . Fluorescent probes like DHE (dihydroethidium) and DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) are commonly used to assess oxidative stress production and ROS levels in the cells of plant roots (Kováčik and Babula 2017 ). Wang et al., 2015 using 7 DCFH–DA dye, observed that treatment with NPs resulted in green fluorescence primarily located in the meristem zone, indicating that the meristem zone is the part where ROS were primarily generated in rice roots. Similarly, Dang et al. 2018 have used NBT and DHE for visualizing superoxide (O 2 •– ). Further, DAB and DCF2DA were used to observe the production of H 2 O 2 and gain mechanistic insights into the regulation of ROS homeostasis in the development of microtubules in A. thaliana under oxidative stress. Additionally, H 2 O 2 is also involved in senescence and stress signaling (Tripathy and Oelmüller 2012 ; Jurdak et al., 2022 ). However, the proper translocation and dissolution of these NPs in the plant system are not yet well understood. DHE is permeable to cell membranes and interacts with reactive species, converting to ethidium, which then binds with nucleic acids, giving the nucleus a red color. This could result from the breakdown of the photosynthetic apparatus and the liberation of electrons to oxygen that augments the level of ROS (Hajiboland, 2014 ; Sgherri et al., 2017 ). The antioxidative defense responses were assessed by measuring the activities of antioxidant enzymes. The data for variations in enzymatic activities are represented in Fig. 4.10 (A-E). The regulation of defense mechanisms in plants is maintained through the enzymatic activity of the AsA-GSH cycle, which is intricately linked to the production of oxidative stress (Pandey et al., 2015 ; Hossain et al., 2022 ). The AsA-GSH cycle constitutes a crucial element within the plant's inherent antioxidant system, tasked with handling and alleviating the impacts of oxidative stress (Tripathi et al., 2021 ). In line with the current findings, Koca et al., 2018 found that the levels of CAT and APX activities were boosted by the accumulation of CuO NPs at (2 ppm) in E. canadensis . However, it's possible that doses above 2 ppm have a toxic impact on plants. Similarly, Wang et al., 2011 reported that Fe 3 O 4 NPs often induce additional oxidative stress as compared to Fe 3 O 4 bulk particles in pumpkin and ryegrass plants. SOD activity in ryegrass drastically declined under treatment with 100 mg/L Fe 3 O 4 NPs. Likewise, Iannone et al., 2016 revealed that the activities of antioxidant enzymes in NPs treated plants significantly increased in both the root and the aerial parts, further leading to the prevention of oxidative damage caused by Fe 3 O 4 nanoparticles at 20 mg/L on hydroponically grown wheat ( Triticum aestivum L.). However, it is possible that at higher concentrations of Fe 3 O 4 NPs, there's a likelihood of a detrimented impact on wheat growth. Similar study by Ren et al. (2011) revealed that FeO NPs at doses of 20 mg/L enhanced the activity of antioxidant enzymes, namely CAT, SOD and peroxidase. Likewise, study by Tripathi et al. ( 2017a ) revealed the application of ZnO NPs in wheat seedlings resulted in a drastic inhibition in enzyme activities of AsA–GSH enzymes due to the increase in the quantity of ROS and lipid peroxidation. Whereas, very few studies have been found that evaluates the comparative effects of green synthesized and market procured CuO and FeO NPs on rice with respect to antioxidant enzyme activities. Molecular studies involve the analysis of a plant's genetic and molecular makeup, which can yield a deeper understanding of its responses to various conditions and stresses (Praveen et al., 2018 ; Tripathi et al., 2021 ). Gene regulation helps the plant control different metabolic processes under stress conditions (Chaves et al., 2003 ). However, NPs at higher concentrations lead to stressful conditions in plants. They disturb the internal network of processes in different crops, which cannot be observed through morphological changes or biochemical assays (Al-Khayri et al., 2023 ). In this context, Gopalakrishnan Nair et al., 2014 found that under high dosees of CuO NPs, the levels of SOD and CAT genes in the roots was significantly decreased, whereas the expression of the APX gene was significantly up regulated in the roots at lower doses of CuO NPs as compared to the control in mung bean. Another study reported by Plaksenkova et al., 2019 on rocket Eruca sativa showed that increasing the concentration of Fe 3 O 4 NPs reduced the expression level of miR159c. In various plant species, treatment with Fe 3 O 4 NPs elicits different responses in miR159c expression. The activation of genes responding to oxidative stress following NPs exposure suggests either the direct generation of ROS or free radicals by NPs, or the indirect signaling of cellular stress (Dimkpa et al., 2013 ; Frazier et al., 2014 ; Pandey et al., 2022 ). Therefore, when the stress exceeds the threshold level, this antioxidant enzyme/gene machinery fails to provide tolerance capacity to the plants. Conclusion The findings of this study reveal that rice seedlings exhibit improved growth when treated with green-synthesized CuO and FeO NPs compared to their market-based counterparts. This highlights the potential benefits of employing green synthesis methods for various nanoparticle applications in the future. Conversely, the use of market-based CuO and FeO NPs significantly hindered growth patterns, impacted photosynthetic parameters, elevated reactive oxygen species (ROS) accumulation, reduced antioxidant enzyme levels, and downregulated genes linked to the AsA-GSH cycle in rice seedlings. The study strongly indicates that stress induced by market-sourced CuO and FeO NPs poses a notably higher level of toxicity compared to both green-synthesized nanoparticles. Consequently, our results advocate for green synthesis as a superior approach for producing stable nanoparticles. Additionally, the study emphasizes the necessity of developing strategies to mitigate nano pollution, aiming to provide stress-resistant crop plants and enhance overall production. Declarations Ethical approval Not applicable Consent to Publish Not applicable Consent to participate Not applicable Author contributions- PR and KV designed the manuscript. PR performed the experiment and wrote the manuscript. KV also help with experiments and editing in manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments Authors are thankful to the Director MNNIT Allahabad, Prayagraj for facilitating necessary resources for accomplishment of this work. Authors also acknowledge CSIR Project no- 38(1460)/18/EMR-II for providing financial assistance. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7025406","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482702833,"identity":"a9323b72-e9f8-4f7a-a563-d3c61da8f32c","order_by":0,"name":"Padmaja Rai","email":"data:image/png;base64,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","orcid":"","institution":"Motilal Nehru National Institute of Technology Allahabad","correspondingAuthor":true,"prefix":"","firstName":"Padmaja","middleName":"","lastName":"Rai","suffix":""},{"id":482702836,"identity":"9fc79e6f-c186-4e8b-b0ec-04169850a1a0","order_by":1,"name":"Kanchan Vishwakarma","email":"","orcid":"","institution":"Motilal Nehru National Institute of Technology Allahabad","correspondingAuthor":false,"prefix":"","firstName":"Kanchan","middleName":"","lastName":"Vishwakarma","suffix":""}],"badges":[],"createdAt":"2025-07-02 05:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7025406/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7025406/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86437493,"identity":"d62a9501-8558-4604-948e-7b07a8ea7d19","added_by":"auto","created_at":"2025-07-10 15:48:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":495258,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of green synthesized- \u0026nbsp;\u003cstrong\u003eCuO NPs\u003c/strong\u003e by (A) UV-Visible spectrophotometer, , (B) particle size analyzer, (C) XRD; \u0026nbsp;\u003cstrong\u003eFeO NPs\u003c/strong\u003e - (D) Uv-Vis, (E) PSA, and (F) XRD.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/52d8ee863631fd7bef342ffd.png"},{"id":86437497,"identity":"3adda1f0-afe6-4f9a-bf1a-9f196aa86e05","added_by":"auto","created_at":"2025-07-10 15:48:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":488705,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of green synthesized \u003cstrong\u003eCuO NPs\u003c/strong\u003e- A- FTIR, B- FESEM-EDAX, C- TEM; \u003cstrong\u003eFeONPs\u003c/strong\u003e- D-FTIR, E FESEM-EDAX, F- TEM.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/e5479b0e94302140c9995f71.png"},{"id":86437495,"identity":"49ed4d78-ff1f-4fc9-acdb-c10f5c1282c0","added_by":"auto","created_at":"2025-07-10 15:48:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157893,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of green synthesized CuO and FeO NPs (50 µM) and market purchased CuO NPs and FeO NPs (200 µM) \u0026nbsp;in terms of (A) Shoot length, Root Length and (B) Shoot and Root fresh mass. Prefix ‘GS’ stands for green synthesized NPs and ‘MP’ stands for market purchased NPs. Data are mean ± standard error (SE) of four biological replicates. Bars with different letters show significant differences at P \u0026lt; 0.05 between treatments according to DMRT.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/5884f55ad148a06cc796d639.png"},{"id":86438267,"identity":"654e2d52-1993-436f-95e8-229fbdab9ccd","added_by":"auto","created_at":"2025-07-10 15:56:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40168,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of selected concentration of green synthesized CuO and FeO NPs (50 µM) and market purchased CuO NPs and FeO NPs (200 µM) on (A) Total chlorophyll and carotenoid (B) Total protein. Data are mean ± standard error (SE) of four biological replicates. Bars with different letters show significant differences at P \u0026lt; 0.05 between treatments according to DMRT.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/5ceb3779b1ac3e4702b80bac.png"},{"id":86437500,"identity":"0c191d0e-025b-466e-9e9a-fd9c01d23089","added_by":"auto","created_at":"2025-07-10 15:48:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":200675,"visible":true,"origin":"","legend":"\u003cp\u003eSpider plot represents the Fv/Fm, qP and NpQ parameters under green synthesized CuO and FeO NPs (50 µM) and market purchased CuO NPs and FeO NPs (200 µM).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/dd0b2d6604f84bed44482483.png"},{"id":86438268,"identity":"7671f18b-e645-407b-9feb-aee9e97e72d4","added_by":"auto","created_at":"2025-07-10 15:56:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115795,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of selected concentration of green synthesized CuO and FeO NPs (50 µM) and market purchased CuO NPs and FeO NPs (200 µM) on reactive oxygen species (a) SOR, (b) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and (c) MDA.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/6fee7e2f913fefd339398ab2.png"},{"id":86437504,"identity":"3826daae-819d-4a54-8c6e-60b533b75fa4","added_by":"auto","created_at":"2025-07-10 15:48:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":266587,"visible":true,"origin":"","legend":"\u003cp\u003eImages of leaf and root tips under different treatments after staining with (a) Schiffs, (b) DAB and (c) Evans dye\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/ed32d9b87b640dcc454159d7.png"},{"id":86437502,"identity":"7aecf68d-6b24-4be2-92dc-b8fdbad9517f","added_by":"auto","created_at":"2025-07-10 15:48:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":102921,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopic images of root tips under different treatments after staining with (a) DCFDA and (b) DHE\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/86a82bcef39ece613a66ccc9.png"},{"id":86438273,"identity":"a24d0d81-694b-494a-a4b4-5348c89042aa","added_by":"auto","created_at":"2025-07-10 15:56:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":291938,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of selected concentration of 50 µM CuO NPs (GS) and 200 µM FeO NPs (GS); 50 µM CuO NPs (MP) and 200 µM FeO NPs (MP) on antioxidant enzymes ((A) Super oxide dismutase (SOD) (B) Catalase (CAT) (C) Monodehydro ascorbate reductase (MDHAR) (D) Dehydroascorbate reductase (DHAR) and (E) Glutathione reductase (GR). Data are mean ± standard error (SE) of four biological replicates. Bars with different letters show significant differences at P \u0026lt; 0.05 between treatments according to DMRT.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/fc0fcfa5c03f3d3ec6944f3c.png"},{"id":86438274,"identity":"ad30a0c0-d1c2-4490-ae7a-db3a3578dfbd","added_by":"auto","created_at":"2025-07-10 15:56:23","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":193970,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of selected concentration of 50 µM CuO NPs (GS) and 200 µM FeO NPs (GS); 50 µM CuO NPs (MP) and 200 µM FeO NPs (MP) on gene expression of (A) \u003cem\u003eAPX, DHAR\u003c/em\u003e(B \u003cem\u003eMDHAR, GR\u003c/em\u003e (C) \u003cem\u003eCOPT7\u003c/em\u003e and \u003cem\u003eYSL15\u003c/em\u003e. Data are mean ± standard error (SE) of four biological replicates. Bars with different letters show significant differences at P \u0026lt; 0.05 between treatments according to DMRT.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/fb9b4c810510932d91d47808.png"},{"id":108605696,"identity":"c26fa54b-4b8a-4cc1-8f25-a9c6491d0198","added_by":"auto","created_at":"2026-05-06 12:13:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2952023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/6f26156b-af51-486b-a032-2d99ca9a276c.pdf"},{"id":86438566,"identity":"efb0fa21-8c0f-4e7e-8599-2580eb7ca942","added_by":"auto","created_at":"2025-07-10 16:04:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":470815,"visible":true,"origin":"","legend":"","description":"","filename":"supplmentaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7025406/v1/3d61d716e9c1ca6f8a0076c0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Study of Green-Synthesized and Market-Procured CuO and FeO Nanoparticles on the Growth and Stress Tolerance in Rice Seedlings","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStress in plants refers to any adverse condition or factor that disrupts a plant's normal growth, development, metabolism or physiological functions caused by unfavorable environmental conditions (Zhang et al., \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Just like humans and animals, plants can experience stress, and it can be caused by various environmental or biological factors. Plants can experience various types of stress, which can be broadly categorized into abiotic and biotic stress based on their origin. Abiotic stress lowers the growth, yield and developmental pattern of crop plant. It is caused by non-living factors in the plant's environment, while biotic stress is caused by living organisms (Gull et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eApart from various abiotic and biotic stresses, nanopollution is one of the major areas that raise concern on the detrimental consequences of nanoparticles pollution in crop plants. Nanopollution refers to the potential hazards linked to the introduction of NPs into the agricultural soil (Jan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). NPs are extremely small particles with at least one dimension less than 100 nanometers (nm), often manufactured for various industrial, commercial and consumer applications due to their unique properties at the nanoscale (Jeevanandam et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Jan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The concern with nanopollution arises from the fact that NPs can behave differently compared to their bulk counterparts (Wang et al., \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The compact dimensions and the elevated ratio of surface area to volume they possess might result in augmented reactivity, mobility and potential toxicity within the plant system. Some sources of nanopollution include industrial processes, consumer products and environmental releases during the manufacturing, use and disposal (Buzea et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In addition, the risk of NPs being discharged into the environment during the manufacture of products, including its distribution and disposal is also higher than that of other bulk materials due to its smaller size (Wang, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e, Reinhart et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Yang et al., \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Among the various types of metal oxide NPs, Copper oxide (CuO) and Iron oxide (FeO) based NPs have been widely used in industrial sectors. These NPs are unique among the most widely used NPs as they exhibit many interesting and useful physicochemical properties. The yearly global production of CuO NPs ranged from 200 to 830 tons in 2010, and it is anticipated to rise in a range of 330 to 1600 tons by the year 2025 (Liu et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Compared to other metal oxide NPs, the consumption and production of CuO and FeO NPs are higher in catalysts and biomedical devices (Janković \u0026amp; Plata \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In present years, large amount of money has been spent in nano-industry. Therefore, in coming years this has led to increase in more production of NPs which further released into our environment and goes into the agriculture fields. Unintentional way of NPs to reach environment and soil system is through volcanic emissions, agriculture, wastewater, and accidental release during manufacturing industries and vehicles (Klaine et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This results in the movement of NPs into the soil and water bodies thereby affecting entire food chain. Nowadays, investigations have been focused on understanding the mechanism and phytotoxic effect of CuO NPs and FeONPs on different crops. Saif et al. (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) stated that CuO NPs are used by many of the industries for various purposes like antimicrobials, biosensors, surfactants, catalysis etc. (Ahamed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Nasrollahzadeh et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and different types of FeO NPs like Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e as magnetite, γ-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e (maghemite) and α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e (hematite) act as a good promising agent for biomedical applications because of high magnetization and superparamagnetic properties (Karimzadeh et al., 2016). The nanoparticles size\u0026thinsp;\u0026lt;\u0026thinsp;100 nm has shown detrimental effects on plants (Tripathi et al., 2017). Disposal of these nanoparticles from industries and laboratories is causing major harm to the plant system and disturbing water ecosystem.\u003c/p\u003e\u003cp\u003eIn the present time, main aim is to protect the plant system and environment from detrimental effects of nanoparticles and other substances formed through them. It is also important to find better approach for safely utilization of nanoparticles. It is reported that 90% biomass reduction was observed by exposure to copper nanoparticles (50 nm) at concentration of 1,000 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in \u003cem\u003eCucurbita pepo\u003c/em\u003e (Zucchini) (Stampoulis et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Similarly, it was reported by Musante and White (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) on treatment with CuO NPs the growth and transpiration of \u003cem\u003eC. pepo\u003c/em\u003e was significantly inhibited by 60\u0026ndash;70% as compared to control. The insufficient work on impact of metal oxide NPs on plant is a major limitation which requires carrying out extensive research in this area. High concentration of FeNPs causes cytotoxic effect to many plants due to formation of reactive oxygen species (ROS), lipid peroxidation on pumpkin (\u003cem\u003eCucurbita mixta\u003c/em\u003e) and perennial ryegrass (\u003cem\u003eLolium perenne\u003c/em\u003e L.) (Wang et al., \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These NPs are absorbed in the roots of plants which are further transported and used by different parts of the plant. On the other hand, high amount of FeO NPs is found to be harmful for the plants. These NPs in roots are transported by the apoplastic pathway and collected into the cell walls. But excess of these particles prevents the availability of mineral nutrients in plants as they get blocked in the cell wall (Yuan et al., 2018). Souza et al. (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) have stated that chlorophyll content were reduced by impact of FeO NP at high concentrations, which stopped the light absorption process on the aquatic plant \u003cem\u003eLemna minor\u003c/em\u003e and ultimately harm the nutrient uptake in \u003cem\u003eL. minor\u003c/em\u003e. Therefore, market procured FeO NPs and CuO NPs have detrimental effects on the growth of crops and more attention is needed to find better approaches to reduce NPs toxicity in plants.\u003c/p\u003e\u003cp\u003eNPs exert toxic effects on plant growth and seed germination depending on their properties and concentration (Garc\u0026iacute;a-G\u0026oacute;mez and Fern\u0026aacute;ndez \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The effect of these NPs depends on type, amount and specimen plants used for experiment. When plant is under any type of stress, the balance between activation of antioxidant enzymes in plants and free radical formation is disturbed and it leads to oxidative stress and cell death in plants, this further result in lipid peroxidation and protein degradation in plants (McCord JM, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Kapoor et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The communication between different NPs and plant crops are still in its infancy stage (Pallavi et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Different studies are seen in crops with respect to translocation, toxicity, biotransformation, and absorption of NPs (Husen and Siddiqi, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Still the research studies are carried out to understand impact of NPs on different crops (Rajput et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Souza et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere are various methods to synthesize NPs via chemical, physical and biogenic. Among these biogenic synthesis approaches is an ecofriendly way to synthesize NPs which can be further used to enhance the plant growth (Begum and Jayawardana, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The choice of synthesis method depends on the material to be synthesized, desired NPs characteristics (size, shape and composition), scalability, cost and intended application. Hence, it's important to consider the properties and potential applications of the NPs during the selection of the appropriate synthesis method. Safety precautions and proper waste management are crucial considerations during NPs synthesis to ensure the safety of the environment (Kamali et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Integrating the principles of green chemistry into nanoscience has helped to identify eco-friendly substances which act as a reducing agent as well as a capping agent to synthesize stabilised NPs (Iravani, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Soltys et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Different chemical and physical methods are applied for synthesis of different NPs e.g., thermal reduction, chemical and physical synthesis (Dhas et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Feldmann and Jungk \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). At the same time, green synthesis method uses substances from plants which are not only eco friendly, but also cost effective (Xiong et al., \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Copper oxide nanoparticles have been produced by utilizing extracts derived from \u003cem\u003eCassia auriculata\u003c/em\u003e leaves (Valli and Geetha, \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), Aloe vera leaf Extract (Kumar et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eDaphnia magna\u003c/em\u003e (Saif et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, FeO NPs are synthesised from \u003cem\u003eLawsonia inermis\u003c/em\u003e and \u003cem\u003eGardenia jasminoides\u003c/em\u003e leaves extract (Naseem and Farrukh, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The green synthesis approach is a better way to synthesize NPs due to their eco-friendly and non-toxic behaviour. Also, its application on the plant system is safe to promote growth and maintain agricultural sustainability (Parveen et al., 2016, Vishwakarma et al., \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Green synthesized NPs boost the antioxidant enzyme which helps to remove the ROS generated in cells of plants under stressed seedlings and reduce its toxic effect (Ramzan et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Green-synthesized NPs made by using natural extracts, plants, microbes or other biological agents to reduce and stabilize metal ions into NPs, results in less toxicity and enhanced biocompatibility as compared to engineered and chemically synthesize NPs (Parveen, et al., 2016, Ijaz et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The plant extracts or biological agents used in green synthesis often contain bioactive compounds that can contribute to the synthesis process and potentially enhance the properties of the resulting NPs with respect to physical or chemical synthesis (Parveen, et al., 2016). These NPs may be more readily absorbed by plants due to their biocompatible nature, leading to improved uptake and potential benefits for plant growth and health.\u003c/p\u003e\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) is one of the staple food crops, which nourish more than half of the world\u0026rsquo;s population (Kennedy, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). As it has significant role in the food security of human kind therefore the measurement of the toxicity due to metal oxide-based NPs on this vital crop holds great importance in relation to human health. Plant material taken is Amragandhi haridra (\u003cem\u003eCurcuma amada\u003c/em\u003e) for the green synthesis of CuO NPs and FeO NPs. Phytochemical constituents present in this plant such as Carbohydrates, Alkaloids, Amino acids, Saponin, Glycosides, Flavonoids, Steroids and Tannins which act as a capping and reducing agent for the synthesis of CuO NPs and FeO NPs. Therefore, the aim of this study is to determine the effect of green synthesized and market based CuO NPs and FeO NPs on rice seedlings. Nanoparticle toxicity examinations have been showed that green-synthesized CuO NPs and FeO NPs are less toxic than market one, providing insight into a new mode to synthesizing more environmentally friendly NPs for numerous uses. Although, it still unclear whether market procured CuO NPs and FeO NPs toxicity is due to ion dissolution or deposition of oxide form of CuO NPs and FeO NPs themselves. Therefore, study needs more investigation to find the proper deposition of CuO NPs and FeO NPs in plant cells.\u003c/p\u003e\u003cp\u003eHence, the work aims on measuring growth attributes, biochemical changes, ROS metabolism (qualitative and quantitative production of oxidative stress markers) and gene expression changes caused by both green- synthesized and market based CuO NPs and FeONPs on rice plant. Therefore, this is the first study to assess comparative effect of green synthesized and Market based CuO NPs and FeO NPs on rice seedlings.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Green synthesis of CuO and FeO NPs\u003c/h2\u003e\u003cp\u003eThe plant selected for the green synthesis (GS) of CuO and FeO NPs was Amragandhi haridra (\u003cem\u003eCurcuma amada\u003c/em\u003e), a prominent herb in Ayurveda. This plant's vibrant yellow spice is commonly referred to as \"haldi\" in Hindi. The rhizomes of \u003cem\u003eCurcuma amada\u003c/em\u003e were obtained from a local Ayurveda store in Prayagraj, India and were carefully stored in sterile plastic bags. The rhizomes were thoroughly cleaned with deionized water, dried and finally coarsely ground. Subsequently, an extraction process was carried out using 80% methanol as the solvent, employing the Maceration method. After 24 hours of incubation, the extract was filtered through Whatman No.1 filter paper. The plant extract was then dried and stored at 4\u0026deg;C for future use (Saif et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn order to finetune the process for synthesizing CuO and FeO NPs, various concentrations within a range of 0 to 1000mM in their respective precursors i.e., CuSO\u003csub\u003e4\u003c/sub\u003e and FeCl\u003csub\u003e3\u003c/sub\u003e were dissolved in 50 ml of deionized water (Saif et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the case of CuO NPs, 4 ml of a freshly prepared plant extract was added into 2 ml of the 5mM CuSO\u003csub\u003e4\u003c/sub\u003e solution. Subsequently, the total volume was brought to 50 ml by adding MilliQ water. Similarly, for synthesizing FeO NPs, plant extract was mixed with 100 mM FeCl\u003csub\u003e3\u003c/sub\u003e (15 ml) and the final volume was adjusted to 300 ml through the addition of MilliQ water. Further, the process involved repeated shaking for 1 hour, followed by incubation for varying durations (10 minutes, 20 minutes, 30 minutes and 60 minutes) at a temperature range of 50\u0026ndash;80\u0026deg;C on a hot plate, with sample analysis conducted at each 10 minute intervals. During this time, the color transformation was observed, with a shift to a dark brownish indicating the formation of CuO NPs and a dark blackish shade suggesting the presence of FeO NPs. Subsequently, the solution was subjected to centrifugation at speeds exceeding 12,000 rpm for 15 minutes, resulting in a pellet that was reconstituted in deionized water. Thereafter, to remove any residual impurities from both types of NPs, acetone was added on the pellet and subjected to two rounds of centrifugation at 12000 rpm for 2 minutes. Following this, the CuO and FeO NPs were dried in an oven for 24\u0026ndash;78 hours and they were further processed in a muffle furnace for 10\u0026ndash;15 minutes to obtain the final dried form of both NPs (Devi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Amin et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Alhalili, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Characterization of CuO NPs and FeO NPs\u003c/h2\u003e\u003cp\u003eThe green synthesized CuO and FeO NPs were subsequently characterized. They were subjected to ultrasonication in a bath sonicator for 30 minutes. Following this, scanning was performed within the wavelength range of 300\u0026ndash;700 nm using an Ultraviolet-visible spectrophotometer (Eppendorf Bio Spectrometer). De-ionized water was employed as a reference (blank) during these measurements. The dimensions of the CuO and FeO NPs were determined using a Particle Size Analyzer (PSA). Both commercially procured and green synthesized NPs were accurately dissolved in distilled water, with the addition of 1 ml of diluted 0.1% DMSO followed by ultrasonication for 30 minutes (Devi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Amin et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Alhalili, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, a powdered form of green synthesized CuO and FeO NPs was utilized to categorize the diverse biomolecules contained within them. This enabled the identification of the capping agents responsible for effectively stabilizing both types of NPs. For this, the analysis was conducted using Fourier Transform Infrared Spectroscopy (FTIR) with PerkinElmer Spectrum Version 10.4.00, following the method outlined by Rajendran and Sengodan\u003c/p\u003e\u003cp\u003e(2017). Further the NPS were characterized by X-Ray diffraction analysis (XRD) to know the crystalline structure of NPs.\u003c/p\u003e\u003cp\u003eAdditionally, FE-SEM-EDS were conducted to determine the surface structure and elemental phase composition. Furthermore, sizes of both types of NPs were detected by TEM (Devi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Amin et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Alhalili, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Screening experiment to optimize the NPs concentration for Hydroponics Studies\u003c/h2\u003e\u003cp\u003eThe comparative assessment was carried out to evaluate the effect of both green synthesized and market procured CuO and FeO NPs on rice seedlings. Rice seeds were sown hydroponically in Hoagland solution within a plant growth chamber (M/s Ocean Life Science Corporation, India). The growth conditions in the chamber were maintained at a relative humidity of 65% and a temperature of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. The seeds of rice were surface sterilized for 20 minutes using a 2% (v/v) sodium hypochlorite solution. Afterward, the seeds were thoroughly rinsed with distilled water and soaked in a dark environment for 12 hours to break dormancy. Following this period, muslin cloth was used to wrap the seeds, which were then kept in the dark to facilitate proper germination (Hoagland and Arnon, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1950\u003c/span\u003e). Once the seeds had sprouted well after two days, healthy and uniformly sized seedlings were selected and placed in Petri plates containing Whatman No. 1 filter paper (150 mm, Riviera), which had been moistened with half-strength Hoagland solution. These seedlings were grown in the plant growth chamber (PGC) with a photon flux density (PFD) of 350 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, under a 12/12-hour day/night cycle and a relative humidity of 60%, all maintained at a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. This growth period lasted for duration of 8 days (Tripathi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Following this, both NPs were optimized for the experiment. Subsequently green synthesized and market procured NPs were dissolved in distilled water using bath sonication for 30 minutes. The experimental setup were included different concentrations (0, 20, 50, 100, 200, 500 and 1000 \u0026micro;M) of green synthesized and market procured CuO and FeO NPs in 40 ml Hoagland\u0026rsquo;s solution. The macro- and micronutrients used to make the Hoagland solution were represented in Appendix 1.\u003c/p\u003e\u003cp\u003eSubsequently, seedlings were allowed to grow for 7 more days in a Plant growth chamber. The nutrient solution was changed periodically and was aerated daily to avoid hypoxia. Following the ending of the experiment, seedlings were collected and subjected to various analysis to assess different factors. The experiments were conducted in triplicates (Tripathi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.1 Morphological parameters\u003c/h2\u003e\u003cp\u003eThe morphological parameters comprised of root length, shoot length as well as fresh weight of shoot and root were assessed. The seedlings were cut into separate shoots and roots for the determination of growth parameters under varying concentrations (0, 20, 50, 100, 200, 500 and 1000 \u0026micro;M) for green synthesized and market procured CuO and FeO NPs. The length of the rice seedlings was measured using a centimeter scale. Furthermore, the fresh weight was determined by individually collecting the roots and shoots and weighing them using a digital weighing balance (Tripathi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Rai et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Subsequently, green synthesized and market procured CuO NPs at a concentration of 50 \u0026micro;M and 200 \u0026micro;M for FeO NPs respectively were selected for further parameter testing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.2 Physiological parameters\u003c/h2\u003e\u003cp\u003eThe physiological parameters encompassed the analysis of photosynthetic pigments and the assessment of Chlorophyll a fluorescence.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhotosynthetic pigment estimation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe photosynthetic pigments were assessed for the specified concentrations of green synthesized and market procured CuO and FeO NPs. The determination of total chlorophyll and carotenoid levels were obtained by using 20 mg of leaves from various treatments that were ground and placed in a 5 ml solution containing 80% (v/v) acetone. Following this step, the mixture was placed for centrifugation at 10,000 rpm for approximately 15 minutes at a temperature of 4\u0026deg;C. The quantification of chlorophyll a and b as well as carotenoids was done as per the protocol mentioned in Lichtenthaler (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Thereafter, the optical absorbance values of the solution were taken at wavelengths of 663.2, 646.5 and 470 nm using a UV\u0026ndash;visible spectrophotometer \u003cb\u003eMeasurement of protein content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLowry et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1951\u003c/span\u003e) method is employed for determining the total soluble protein content. In this technique, both untreated and additional samples were ground in 50 mM potassium phosphate buffer (pH 6.8). Subsequently the mixture was centrifuged at 8,000 g for 15 minutes. The protein content in the various treatments and control samples, Bovine Serum Albumin (BSA) was used as a reference standard.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.3 Chlorophyll a fluorescence analysis\u003c/h2\u003e\u003cp\u003eAdditionally, the photosynthetic efficiency of rice seedlings was evaluated utilising a handheld leaf fluorometer (Fluor Pen FP 100, Photon System Instrument, Czech Republic). After subjecting the leaves to 30 minutes of darkness, fluorescence parameters from the JIP test, such as the maximum photochemical efficiency of PSII (Fv/Fm), photochemical quenching (qP) and non-photochemical quenching (NPQ) were analyzed by the procedure outlined in Strasser, (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and Strasser et al. (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 Biochemical parameters\u003c/h2\u003e\u003cp\u003eThe biochemical parameters included the evaluation of oxidative stress markers namely SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA. The assessment of anatomical characteristics involved the application of stress marker dyes for the purpose of evaluating cell viability through histochemical staining. Additionally, \u003cem\u003ein vivo\u003c/em\u003e localization of reactive oxygen species and superoxide ions was conducted using fluorescence microscopy. Additionally, the antioxidative defense responses were assessed by measuring the activities of antioxidant enzymes.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section4\"\u003e\u003ch2\u003e2.3.4.1 Estimation of super oxide radical (SOR)\u003c/h2\u003e\u003cp\u003eThe shoot samples were crushed with 3 ml of a 65 mM potassium phosphate buffer at pH 7.8. Following this, they were subjected to centrifugation at 10,000 g for 10 minutes, following the procedure outlined by Elstner and Heupel (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Subsequently the obtained liquid (1 ml) was combined with 0.9 ml of a 65 mM phosphate buffer and 0.1 ml of 10 milimolar hydroxylamine hydrochloride. After twenty minutes of incubation at 27\u003csup\u003eo\u003c/sup\u003eC, 7 mM NEDD and 17 mM sulphanilamide were mixed into the incubation mixture. To eliminate interference from pigments, the components were meticulously blended and subsequently divided into two distinct layers by adding an equivalent volume of diethyl ether. The pink water phase in the lower layer was assessed for absorption at 530 nm. A standard curve, established with sodium nitrite to determine the SOR content. The quantity of SOR in the sample is expressed in nmol per gram of fresh weight (nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section4\"\u003e\u003ch2\u003e2.3.4.2 Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/h2\u003e\u003cp\u003eFollowing Velikova et al. (\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) method, the assessment of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was conducted. Fresh shoot samples were blended in 0.1% (v/v) Trichloroacetic acid (TCA) and then centrifuged at 15,000 g for 15 minutes. The obtained liquid (supernatant) was used for the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e analysis. The reaction mixture comprises of 10 milimolar potassium phosphate buffer (0.5 ml- pH 7.0), 1 M potassium iodide (KI- 1.0 ml) and extract (0.5 ml). Afterward, the absorbance at 390 nm was measured against a blank in each sample and determined using a standard curve made with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The amount of hydrogen peroxide in each sample is shown as nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section4\"\u003e\u003ch2\u003e2.3.4.3 Lipid peroxidation\u003c/h2\u003e\u003cp\u003eThe assessment of lipid peroxidation involved estimating the concentration of MDA (malondialdehyde) equivalents, which serve as markers for the peroxidation products of unsaturated fatty acids. This was achieved by measuring thiobarbituric acid reactive metabolites Heath and Packer, (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1968\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.3.5 Anatomical parameters\u003c/h2\u003e\u003cp\u003eFurther, the quantitative results of oxidative stress markers were further validated qualitatively by evaluating the cell viability by histochemical staining to visualize the production of radicals. Also, \u003cem\u003ein vivo\u003c/em\u003e localization of ROS (reactive oxygen species) and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e by Olympus BX51 Fluorescence Microscopy were evaluated.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section4\"\u003e\u003ch2\u003e2.3.5.1 Histochemical staining\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe histochemical staining was performed as per the protocol of Thordal-Christensen et al., (\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The application of 3, 30-diaminobenzidine (DAB\u003cb\u003e)\u003c/b\u003e was utilised to detect the presence of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in rice root and leaf tips. The tips of root and leaf were washed with MQ water, subsequently put in 1% DAB (pH 3.8; Sigma, United States) for 8 hr at 25\u003csup\u003e0\u003c/sup\u003eC in the light. These samples were stained with the dye and then washed with MQ water. These were subsequently dipped fully in 95% ethanol and kept for boiling for 10 times. This step was repeated for two times. The slides were prepared, and photography was done by using Olympus compound dark-field microscope\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFurthermore, to assess membrane damage in plant cells, tips from the leaves and roots of rice seedlings were excised. Subsequently, these tips were washed with a sodium phosphate buffer at a pH of 7.4. The solution of Evan\u0026rsquo;s blue was prepared at a concentration of 10 mg/ml. The excised tips were then immersed in the Evan\u0026rsquo;s blue dye for duration of 30 minutes. After staining, the tips were rinsed with ethanol and MQ water and then mounted on a slide followed by imaging an Olympus compound dark-field microscope (Sch\u0026uuml;tzend\u0026uuml;bel et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSchiff's staining was utilized to detect the lipid peroxidation in plants subjected to stress conditions. Fresh tips from the roots and leaves of rice seedlings were excised. Subsequently, these tips were washed with MQ water and then immersed in a 0.5% solution of Schiff's stain for a period of 3 hours. After staining, they were rinsed with a 0.5% solution of potassium silicate (K\u003csub\u003e2\u003c/sub\u003eSiO\u003csub\u003e3\u003c/sub\u003e). Following this, the tips underwent washing with ethanol and MQ water. Images of the root tips and leaves were captured using an Olympus compound dark-field microscope by the method mentioned in Awasthi et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section4\"\u003e\u003ch2\u003e\u003cb\u003e2.3.5.2\u003c/b\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003elocalization of ROS and O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003csup\u003e\u003cb\u003e\u0026bull;\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eby fluroscence microscopy\u003c/b\u003e-\u003c/h2\u003e\u003cp\u003eThe visualization of the presence of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (superoxide) in rice roots was carried out using 10\u0026micro;M DHE stain, which was prepared in a 10 mM tris HCL (pH- 7.4) for 15 minutes (Sandalio et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Subsequently, to remove any excess stain, the samples were washed three times with MQ water; each wash lasting for 10 minutes and were then observed using an Olympus BX51 fluorescent microscope. The observation was performed with excitation at 488nm and emission at 520nm.\u003c/p\u003e\u003cp\u003eAdditionally, the staining protocol mentioned by Morina et al., (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) was employed to identify the occurrence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in various treatments. This was accomplished by dipping the root tips in 10\u0026micro;M DCF2DA stain for 15 minutes. The roots of the rice seedlings were rinsed twice in the identical buffer solution, each time for 15 minutes, and subsequently positioned on a microscope slide for observation using an Olympus BX51 Fluorescence Microscope. The microscope was configured to excite the sample at a wavelength of 485nm and capture the emitted light at 535nm.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.3.6 Antioxidative defense responses\u003c/h2\u003e\u003cp\u003eThis section encompasses assays for estimating the antioxidant enzymes activities.\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section4\"\u003e\u003ch2\u003e2.3.6.1 Superoxide dismutase (SOD; EC 1.15.1.1)\u003c/h2\u003e\u003cp\u003eThe assessment of SOD was done by the protocol of Giannopolitis and Ries, (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Enzyme activity was obtained by crushing the 100 mg of tissue from each sample in 100 mM phosphate buffer (pH 7.8) along with EDTA under refrigerated conditions. The mixture was gently inverted in tubes and thoroughly mixed. Following this, the mixture was centrifuged at 10,000g for 20 minutes. The resulting liquid was transfer to sterile test tubes and used for the enzyme assay.\u003c/p\u003e\u003cp\u003eThe reaction mixtures were prepared by combining 0.1 ml of the supernatant with riboflavin, L-methionine, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (pH 10.2) and NBT with a concentration of 1.3 \u0026micro;M,, 13 mM, 0.05 M and 63 \u0026micro;M respectively to achieve a total volume of 3 ml. All the test tubes were then exposed to light at an intensity of 100 \u0026micro;mol photon m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Afterwards, the absorbance of the final product was assessed at a wavelength of 560 nm in relation to the control. The enzyme concentration exhibited a direct correlation with the disparity in absorbance at 560 nm when comparing the presence and absence of the mixture. Furthermore, the unit of SOD activity was defined as the amount of enzyme that hindered the 50% decrease of NBT under the specified conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section4\"\u003e\u003ch2\u003e2.3.6.2Catalase (CAT; EC 1.11.1.6)\u003c/h2\u003e\u003cp\u003eThe measurement of CAT activity followed the methodology outlined by Aebi (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). In this process, 50 milligrams of tissue from each sample were homogenized using 1 ml of a 50 millimolar PPB solution containing 1 mM EDTA at a pH of 7.0. The mixture underwent centrifugation at 10,000 g for 15 minutes. Subsequently, the liquid above the debris was utilized for enzyme activity analysis. The quantification of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was carried out using the extinction coefficient (ε\u0026thinsp;=\u0026thinsp;39.4 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). A unit is defined as the quantity of enzyme that can break down 1 nanomole of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in one minute.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section4\"\u003e\u003ch2\u003e2.3.6.3 Dehydroascorbate reductase (DHAR; EC 1.8.5.1)\u003c/h2\u003e\u003cp\u003eThe content of DHAR in each sample was estimated spectrophotometrically by adopting the procedure of Nakano and Asada (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The enzyme activity of MDHAR was measured by quantifying it using an extinction coefficient of (ε\u0026thinsp;=\u0026thinsp;7.0 mM-1cm-1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section4\"\u003e\u003ch2\u003e2.3.6.4 Monodehydroascorbate reductase (MDAR; EC 1.6.5.4)\u003c/h2\u003e\u003cp\u003eThe content of MDHAR was assessed by the protocol of Hossain et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Activity of MDHAR was assessed utilizing an extinction coefficient of 6.2 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A single unit of enzyme activity is defined as the oxidation of 1 nmol of NADH per minute.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section4\"\u003e\u003ch2\u003e\u003cb\u003e2.3.6.5 Glutathione reductase (GR; EC 1.6.4.2\u003c/b\u003e)\u003c/h2\u003e\u003cp\u003eThe activity of enzyme was determined by following the process outlined in Schaedle and Bassham (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). One unit of GR activity represents the quantity of enzyme that facilitates the oxidation of 1 nanomole of NADPH in a minute.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e2.3.7 Gene expression analysis\u003c/h2\u003e\u003cp\u003eThe gene expression analyses of antioxidant associated genes, copper and iron transporters were performed using quantitative real time PCR in different treated samples. Total RNA was obtained from the sample using the Spectrum Total Plant RNA isolation kit from Sigma. The RNA was reverse transcribed into complementary DNA (cDNA) following the method by Wormuth et al. (\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Subsequently, quantitative real time PCR (qRT-PCR) was performed using the Step One Plus Real Time PCR System from Applied Biosystems.\u003c/p\u003e\u003cp\u003eThe pure RNA gets collected in the flow through and it was stored at -80\u003csup\u003e0\u003c/sup\u003eC for further use. The quality and quantity of RNA was checked through NanoDrop instrument. Finally, the 260/280 ratio was checked for RNA purity and integrity. Furthermore, the cDNA was prepared from the RNA using verso cDNA synthesis kit.\u003c/p\u003e\u003cp\u003eOnce the reaction mixture was prepared, it was subjected to incubation at 42\u0026deg;C for 30 minutes (1 cycle) and subsequently at 95\u0026deg;C for 2 minutes. The cDNA that was prepared was then diluted in DEPC water in a 1:4 ratio and employed for the analysis of gene expression via qRT-PCR. Following this, 5 \u0026micro;l of SYBR Green Supermix was added to the samples to make the reaction mixture a total of 10 \u0026micro;l. The actin gene was taken as the internal control.\u003c/p\u003e\u003cp\u003eFurther, after preparation of reaction mix the contents were transferred to qRT-PCR plate and the reaction was set in the machine. The expression studies in qRT-PCR were carried out by using Step One plus Real-Time PCR System. The entire volume of reaction mixture was 10\u0026micro;l and actin was taken as a housekeeping gene for normalization. The general conditions were set using following steps: 95\u0026deg;C for 1 min; 40\u0026times; (95\u0026deg;C for 30s, 58\u0026deg;C for 40s, 72\u0026deg;C for 45s), 72\u0026deg;C for 10 min followed by a melting curve program (55\u0026ndash;95\u0026deg;C in increasing steps of 0.5\u0026deg;C). Further, the relative quantification for gene expression levels after normalization with actin was calculated by comparative CT (Delta\u0026ndash;Delta CT) method, as demonstrated by Livak and Schmittgen (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Primers were designed with primer 3 tool. The list of primers specific to particular genes designed is presented in Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Statistical analyses\u003c/h2\u003e\u003cp\u003eThe data presented in the thesis have been statistically analysed using one-way analysis of variance (ANOVA) by SPSS 16.0 software, following verification of the data's normal distribution. Variances among treatments were assessed through Duncan\u0026rsquo;s multiple range tests at a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The values depicted represent the means derived from three independent biological replicates (n\u0026thinsp;=\u0026thinsp;3). The Graphs represented in the study were made by using sigma plot 14.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Green synthesis and Characterization of green synthesized CuO NPs and FeO NPs\u003c/h2\u003e\n \u003cp\u003eGreen synthesis of CuO and FeO NPs were successfully synthesized using plant i.e., \u003cem\u003eCurcuma amada\u003c/em\u003e. The precursors, CuSO\u003csub\u003e4\u003c/sub\u003e and FeCl\u003csub\u003e3\u003c/sub\u003e were used at final concentrations of 5 mM and 100 mM respectively. Thereafter, the synthesis was carried out at temperatures of 55\u0026deg;C for CuO NPs and 60\u0026deg;C for FeONPs, each for duration of 20 minutes. The resulting CuO NPs displayed a brownish color, while the FeO NPs exhibited a slightly blackish color. The absorbance peaks were detected within the wavelength range of 250 to 500 nm using a UV-Visible spectrophotometer. Specifically, the absorbance maxima for green synthesized CuO NPs and FeO NPs were observed at 480 nm and 300 nm respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, the mean particle size was observed at 33.7 nm for CuO NPs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) and 26.45 nm for FeO NPs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE) respectively.\u003c/p\u003e\n \u003cp\u003eThe data of X-ray diffraction (XRD) revealed that all the diffraction peaks of NPs were consistent with the standard structure. The planes (021), (110), (111), (131) and (151) for green synthesized CuO NPs represent the monoclinic structure of CuO NPs. The planes (111), (400), (422), (511), (440), (533) for FeO NPs indicated the formation of a monoclinic crystalline structure (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n \u003cp\u003eIn addition, FTIR results demonstrated the presence of biomolecules in the plant extract, which acted as capping agents for the efficient stabilization of both NPs. The observed peaks at 602.92 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 509.19 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to the characteristic stretching vibration of the Cu-O bond in Copper oxide (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, the observed peaks at 666.01 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 477 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to the characteristic stretching vibration of the Fe-O bond (Fig. 4.2D). The sharp peaks observed at 602.92 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 666.01 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the FTIR spectrum confirmed the formation of CuO NPs and FeO NPs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eSurface morphology of the CuO NPs and FeO NPs was observed by FE-SEM (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). The EDS spectrum revealed the presence of copper (Cu) and oxygen (O\u003csub\u003e2\u003c/sub\u003e) in CuO NPs, as well as iron (Fe) and oxygen (O\u003csub\u003e2\u003c/sub\u003e) in FeO NPs. The detection of sulfur (S) and chlorine (Cl) atoms in the spectrum can be ascribed to the plant extract. The EDS spectrum also confirmed that the CuO NPs and FeO NPs are in oxide form and free from any other impurities. These results were further validated by TEM as depicted in (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD) showing that the size of both NPs was below 50 nm and the shape was round.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Morphological Parameters\u003c/h2\u003e\n \u003cp\u003eThe impact of green synthesized and market procured CuO NPs and FeO NPs was studied on the morphological parameters of rice seedlings. The morphological attributes of rice seedlings were analyzed by measuring the length and fresh weight of shoots and roots to evaluate the effects of green synthesized and market procured CuO NPs and FeO NPs at different concentrations. Obtained results revealed that with increasing concentration, both shoot and root length, as well as shoot and root fresh mass, showed a decreasing trend with both CuO NPs and FeO NPs. However, the impact was more significant in both the market procured NPs as compared to green synthesized (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eBased on the screening experiment, following doses of green synthesized (GS) and market procured (MP) CuO and FeO NPs were selected for further studies (Supplementary Fig. 1 and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eGS CuO NPs- 50 \u0026micro;M\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMP CuO NPs \u0026minus;\u0026thinsp;50 \u0026micro;M\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eGS FeONPs \u0026minus;\u0026thinsp;200 \u0026micro;M\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMP FeO NPs- 200 \u0026micro;M\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eThe concentration of 50 \u0026micro;M green synthesized CuO NPs showed a significant augment in shoot length and fresh weight, by 15.09% and 17.06%, respectively with respect to the control. Similarly, the root length and fresh weight also increased under green synthesized CuO NPs (50 \u0026micro;M) by 16.95% and 14.48%, respectively with respect to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, a concentration of 200 \u0026micro;M green synthesized FeO NPs led to an increase in shoot length and root length by 10.00% and 4.80% respectively with respect to the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, at a concentration of 50 \u0026micro;M and 200 \u0026micro;M market procured CuO NPs and FeO NPs respectively, there was a significant decrease in shoot length by 18.37% and 18.65% and fresh mass by 31.53% and 34.32% respectively as compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the shoot length and fresh mass at a concentration of 200 \u0026micro;M market procured FeO NPs were significantly decreased by 31.53% and 34.32% respectively as compared to the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Effect of treatments on photosynthetic properties and total protein content\u003c/h2\u003e\n \u003cp\u003eThe physiological parameters were assessed through the measurement of photosynthetic pigment levels and Chlorophyll a fluorescence. Green synthesized CuO NPs resulted in a notable increase in chlorophyll and carotenoid levels by 2.90% and 1.55%, respectively with respect to control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, market procured CuO and FeO NPs showed a significant decrease in chlorophyll by 30.4%, 26.80% and carotenoid by 33.9%, 37.00% with respect to control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, photosynthetic pigments wee further validated by Chlorophyll a fluorescence parameters. This was calculated by measuring the parameters such as Fv/Fm, qP and NPQ. The results were depicted in the spider plot shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Significant differences were observed between green synthesized and market procured NPs for all the parameters. The green synthesized CuO NPs and FeO NPs were increased in Fv/Fm by 16.30% and 13.10% as compared to control. Similarly, green synthesized CuO NPs and FeO NPs were increased qP by 9.13% and 9.92% with respect to control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). However, NPQ showed a reverse effect under green synthesized both the NPs as compared to the control. Additionally, market procured NPs decreased the content of Fv/Fm and qP as compared to the control as represented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe total protein content in rice seedlings was shown to be significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in both the market procured NPs with respect to green synthesized counterparts.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Impact of treatments on reactive oxygen species and cellular damage\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e levels of ROS, such SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA for lipid peroxidation have been depicted in (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA,B,C). Rice seedlings exposed with green synthesized CuO NPs and FeO NPs showed a significant decrease in SOR levels by 32.20% and 33.70%, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA) as compared to their market procured counterparts (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, green synthesized CuO NPs and FeO NPs showed decreased in the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by 18.47% and 32.29% respectively as compared to market procured NPs.\u003c/p\u003e\n \u003cp\u003eLipid peroxidation, measured in terms of MDA, was also significantly decreased under green synthesized CuO NPs and FeO NPs treatments by 9.40% and 15.40% respectively as compared to market procured both NPs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eThe staining with Schiff\u0026rsquo;s stain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA,B), DAB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC,D) and Evan\u0026rsquo;s blue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE,F) for ROS mediated damage to the plant cells (leaf and root tips) depicted lesser deposition in selected concentrations of green synthesized CuONPs and FeONPs in comparison to market procured CuONPs and FeONPs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The markers of oxidative stress were affirmed through the application of fluorescent probes during histochemical and fluorescence staining of the root tips. Lighter fluorescent intensity (red fluorescence) was noticed for SOR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea) as well as total ROS (green fluorescence) as represented in (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb) under green synthesized CuO and FeO NPs as compared to market one. The present findings also highlighted that under market procured NPs exposure, a significant diminution in the quantity of plant cells was seen under fluorescence microscopy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea, b). Maximum cell death in plant cells was recorded due to more absorption of DHE stain, which might be correlated with the reduced growth of rice plants under market procured NPs treatments as compared to green synthesized one.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Impact of treatments on antioxidant enzymes\u003c/h2\u003e\n \u003cp\u003eThe results indicated that the activity of SOD in green synthesized CuO NPs and FeO NPs were increased by 20.65% and 18.78%, respectively as compared to market procured NPs (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). Similarly, CAT activity was also enhanced under green synthesized CuO NPs and FeO NPs as compared to the market counterparts as showed in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB. Additionally, the enzymatic activities of MDHAR and DHAR were increased under green synthesized CuO NPs and FeO NPs as compared to market procured NPs, as represented in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD. Furthermore, GR activity was increased under green synthesized CuO NPs and FeO NPs by 38.16% and 39.09% respectively as compared to market procured NPs (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eE). Overall, rice seedlings under market procured NPs treatment showed a diminution in the activity of SOD, CAT, MDHAR, DHAR and GR due to an increase in cellular ROS production, as well as associated damage to seedlings, which is also indicated in the \u003cem\u003ein vivo\u003c/em\u003e imaging of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e radicals (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ch2\u003e3.6 Regulation of gene expression\u003c/h2\u003e\n \u003cp\u003eThe analyzed genes are associated with stress and detoxification mechanisms and their expression was assessed through qRT-PCR. It was observed that genes related to antioxidant activity namely \u003cem\u003eDHAR (dehydroascorbate reductase), MDHAR (monodehydroascorbate reductase), GR (glutathione reductase)\u003c/em\u003e and \u003cem\u003eAPX (ascorbate peroxidase)\u003c/em\u003e, showed up-regulation in response to green synthesized CuO NPs and FeO NPs as compared to market procured NPs (Fig. 4.11). In contrast, the regulation of \u003cem\u003eCOPT7\u003c/em\u003e (Copper transporter) and \u003cem\u003eYSL15\u003c/em\u003e (Iron transporter) genes were significantly up regulated under market procured CuO NPs and FeO NPs as compared to green synthesized NPs (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). These antioxidant genes could not maintain the integrity of genes inside the stressed rice seedlings (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). This suggests that the expression pattern of antioxidant genes responded differentially to green synthesized and market procured NPs treatments in diverse ways.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNowadays, there is a growing focus on synthesizing NPs through green synthesis approaches because they are eco-friendly, pollution-free methods that promote a sustainable environment. Currently, the interplay between nanoparticles and crops has emerged as a key subject of exploration among nanotechnologists and plant scientists owing to how nanoparticles behave within plant cells (Tripathi et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe findings of the current research are corroborated by the findings of Souza et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e who employed a chemically synthesized approach to create FeO nanoparticles using FeCl\u003csub\u003e3\u003c/sub\u003e salt. Similarly, (Fazlzadeh et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) synthesized zero-valent Fe NPs via the green synthesis approach using extracts of \u003cem\u003eRosa damascene, Thymus vulgari\u003c/em\u003e and \u003cem\u003eUrtica dioica\u003c/em\u003e. These NPs also used to mitigate abiotic stress in plants (Singh et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe current findings state that the round shape of CuO NPs and FeO NPs have been confirmed using the FE SEM and TEM technique. FESEM (Field Emission Scanning Electron Microscopy) and EDX (Energy-Dispersive X-ray Spectroscopy) are two common techniques utilised to analyze the surface and elemental composition of NPs. EDX is often used in conjunction with FESEM to analyze the elemental composition of a NPs surface (Restivo et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The characterization was further validated by EDX to confirm the presence of Cu and O\u003csub\u003e2\u003c/sub\u003e for CuO NPs and Fe and O\u003csub\u003e2\u003c/sub\u003e for FeO NPs. These results are also in conformity with the findings of Kumar et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e used Aloevera leaf extract to synthesize CuO NPs. Their study revealed an average particle size of 20 nm through TEM analysis. X-ray diffraction (XRD) is a method employed for examining the atomic and molecular arrangement within crystalline substances (Dorofeev et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Also, powder X-ray diffraction indicated the monoclinic phase of the NPs. They observed that the SEM images displayed the presence of larger particles, potentially arising from the aggregation or overlapping of smaller nanoparticles with sizes within 100 nm. Further (Kumar et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) showed that the chemical composition of the CuO NPs having an atomic percent of 54% for Cu and 45% for O by EDS spectra. Another study by Tandon et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2013\u003c/span\u003e demonstrated that zero valent iron (ZVI) nanoparticles, averaging a particle size of 59.08\u0026thinsp;\u0026plusmn;\u0026thinsp;7.81 nm were produced through the reaction between ferric nitrate and \u003cem\u003eMentha spicata\u003c/em\u003e L. tea extract. These particles exhibited absorption peaks at 360 and 430 nm, a confirmation made via UV-Vis analysis. The findings of the current result draws support from the study of Prasad et al. 2014 who reported that the size range of FeNPs falls below 50 nm when using TEM. The results mentioned in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, as reported by numerous authors demonstrate that plants are capable of efficiently forming stable metal oxide nanoparticles, making them ideal candidates for rapid and large scale synthesis compared to alternative methods (Iravani \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nair et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vijayaram et al., \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe possible reason for the greater toxicity of market procured NPs is that they were more soluble than green synthesized NPs, resulting in a higher rate of ions release as compared to the plant synthesized nanoparticles (Saif et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Several other studies have also highlighted the severe detrimental impacts of nanoparticles, impeding not just growth but also detrimently influence the metabolic, physiological and molecular characteristics of various plant (Du et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; de la Rosa et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe findings of the present result draw support from the findings of Shi et al., 2014 who demonstrated that the CuO NPs detrimented the growth of plant and are likely to accumulate in the root cells and leaf cells of \u003cem\u003eE. splendens\u003c/em\u003e. However, in a different study Da Costa and Sharma \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, found that engineered CuO NPs at a dose of 100 mg/L severely inhibited plant growth in terms of root and shoot development at elevated doses of CuO NPs as compared to the control. The biomass of rice shoots is also declined by 31% on a fresh mass (FM) basis and 14% on a dry mass (DM) basis. Similarly, another study by Ren et al., 2011 also suggests that FeO NPs (20 mg/L) enhanced the speed of seed germination, shoot length and root length in Chinese mung beans. The outcomes align with Anwaar et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e who used the leaf solution of \u003cem\u003eAzadirachta indica\u003c/em\u003e in the synthesis of CuO NPs, showing substantial positive role on plant growth parameters at a concentration of 10 mg/lit CuONPs. A similar study was highlighted by Saif et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e to investigate the biotoxic consequences of plant synthesized and engineered CuO NPs on the water flea Daphnia and revealed that plant synthesized NPs exhibited greater stability as compared to their engineered counterparts.\u003c/p\u003e\u003cp\u003ePhysiological parameters in plants refer to various factors and processes that are associated with the functioning and health of a plant. This includes quantification of photosynthetic pigments that capture light energy for photosynthesis (Fernandez-Marin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Another important parameter to consider is the evaluation of chlorophyll a fluorescence, a critical process in photosynthesis that is widely used in plant physiology to evaluate the health and photosynthetic activity of plants. This assessment provides valuable information about the efficiency of photosynthesis and the overall stress levels of plants. This provides insights into the efficiency of photosynthesis and the physiological state of plants by measuring the fluorescence emitted by chlorophyll molecules (Kalaji et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This information is essential for managing the risk of NPs in plant crops.\u003c/p\u003e\u003cp\u003eChlorophyll a fluorescence is an essential parameter for evaluating the performance of the photosynthetic machinery in rice seedlings (Tsai et al., \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, previous findings (Ren et al., 2011; Vishwakarma et al., \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) have also shown that application of Ag NPs and CuO NPs in mustard and mung beans at 100 mg/L significantly declined the total chlorophyll contents. The present results also confirmed that the values of Fv/Fm (a reliable indicator of photosynthetic efficiency) and qP noticeably decreased under the exposure to market procured NPs as compared to green synthesized NPs. This reduction might be related to the decrease in total chlorophyll (Fig.\u0026nbsp;4.5A). The present data showed that disturbed electron flow between the photosystems ultimately reduced the Fv/Fm of the seedlings (Fig.\u0026nbsp;4.6). Additionally, Genty et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e observed that the down regulation of photosystem II was the main factor leading to increased NPQ levels, which may be due to the reduced need for electrons through NADPH when subjected to stressful conditions. The data demonstrated that NPQ was significantly enhanced under both the market procured NPs treatments, while its enhancement was less pronounced under green synthesized NPs at the same concentration, suggesting that green synthesized NPs allow for the appropriate performance of the electron transport chain. In another study by Singh et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e, NPs were synthesized using a green approach, utilizing flower extracts from \u003cem\u003eMorus alba\u003c/em\u003e. It was indicated that application of biosynthesized CuO NPs at a doses of 10 mg/L exhibited the highest seedling growth in terms of increased radicle and plumule length and photosynthetic parameters. CuO NPs inhibited the growth of \u003cem\u003eH. sativum\u003c/em\u003e by affecting the maximal quantum yield of photosystem (Rajput et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similar study reported by Singh et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e that exposure of plants to 100 and 500 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of biosynthesized CuO NPs significantly diminished the total chlorophyll and sugar content. Yet, when present at a concentration of 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of CuO NPs led to a marginal rise in pigment and sugar content in tomato plants.\u003c/p\u003e\u003cp\u003eBiochemical parameters were assessed by evaluating the content of oxidative stress markers \u003cem\u003eviz.\u003c/em\u003e, SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA. Oxidative stress markers in plants are indicators used to assess the level of oxidative stress within plant cells. Oxidative stress is a condition that occurs when there is a disparity between the formation of ROS and the plant's capacity to detoxify or repair the damage caused by these molecules (Sharma et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Elevated levels of Reactive oxygen species at its elevated level have the potential to harm cellular elements such as proteins, lipids and DNA, which can ultimately harm the plant's overall growth (Garg and Manchanda, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). ROS are generated in plants under oxidative stress, making it an important parameter to assess the consequences of NPs in rice seedlings (Tripathi et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). Similar findings for ROS species which generate oxidative stress under nanoparticles were also revealed by other authors (Ren et al., 2011; Vishwakarma et al., \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pandey et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Azhar et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Likewise, the presence of CuO NPs at a dose of 5 mg/L resulted in higher ROS production, possibly attributed not only to the ions from CuSO\u003csub\u003e4\u003c/sub\u003e but also the NPs present in the rice (Wang et al., \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Tang et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2016\u003c/span\u003e demonstrated that exposure to CuO NPs at a dose of 10 mg/L for 2 hours resulted in much higher ROS generation in root tips compared to the corresponding treatment with Cu\u003csup\u003e2+\u003c/sup\u003e ions at a concentration of 0.8 mg/L. In this context, Afzal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e indicated that green synthesized FeO NPs revealed the existence of a critical concentration of NPs. Under this threshold, rice crop growth receives a boost, yet no additional improvement occurs beyond this level. In this context (Laouini and Bouafia 2021; Murugesan et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that green synthesized NPs based on their concentration are more biocompatible and eco-friendlier due to the occurrence of phytochemicals in the plant extract, making these NPs non-toxic for plants.\u003c/p\u003e\u003cp\u003eSubsequently, the presence of oxidative stress markers were supported by evaluating anatomical characteristics, including the \u003cem\u003ein vivo\u003c/em\u003e localization of ROS and superoxide ions using histochemical dyes and fluorescent probes, through the use of fluorescence microscopy. Histochemical dyes like DAB (3,3'-diaminobenzidine), NBT (nitroblue tetrazolium), Evans Blue, and Schiff's reagent are used in to visualize ROS production in the cellular structure of plant cells under stress (Yadav et al., \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese staining techniques can provide valuable understanding regarding the localization and intensity of stress-induced changes in plant tissues (Yadav et al., \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Basu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The results indicated a notable decline in the growth pattern of rice seedlings when exposed to market procured NPs, which might be related to the increased formation of oxidative stress markers. Previous research has similarly demonstrated that stressors expedite the production of ROS, encompassing lipid peroxidation, membrane impairment, and the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Xie et al., 2019; Tripathi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Treatment of seedlings with green synthesized NPs represented an augmentation in the growth pattern and reduced damage to biomolecules through ROS formation as compared to market procured NPs. These biomolecules are important markers in the photosynthetic pathway in plants, as reported by Sharma et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Tripathi et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFluorescent probes like DHE (dihydroethidium) and DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) are commonly used to assess oxidative stress production and ROS levels in the cells of plant roots (Kov\u0026aacute;čik and Babula \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Wang et al., \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2015\u003c/span\u003e using 7 DCFH\u0026ndash;DA dye, observed that treatment with NPs resulted in green fluorescence primarily located in the meristem zone, indicating that the meristem zone is the part where ROS were primarily generated in rice roots. Similarly, Dang et al. 2018 have used NBT and DHE for visualizing superoxide (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026ndash;\u003c/sup\u003e). Further, DAB and DCF2DA were used to observe the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and gain mechanistic insights into the regulation of ROS homeostasis in the development of microtubules in \u003cem\u003eA. thaliana\u003c/em\u003e under oxidative stress. Additionally, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is also involved in senescence and stress signaling (Tripathy and Oelm\u0026uuml;ller \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jurdak et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the proper translocation and dissolution of these NPs in the plant system are not yet well understood. DHE is permeable to cell membranes and interacts with reactive species, converting to ethidium, which then binds with nucleic acids, giving the nucleus a red color. This could result from the breakdown of the photosynthetic apparatus and the liberation of electrons to oxygen that augments the level of ROS (Hajiboland, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sgherri et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe antioxidative defense responses were assessed by measuring the activities of antioxidant enzymes. The data for variations in enzymatic activities are represented in Fig.\u0026nbsp;4.10 (A-E). The regulation of defense mechanisms in plants is maintained through the enzymatic activity of the AsA-GSH cycle, which is intricately linked to the production of oxidative stress (Pandey et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hossain et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The AsA-GSH cycle constitutes a crucial element within the plant's inherent antioxidant system, tasked with handling and alleviating the impacts of oxidative stress (Tripathi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In line with the current findings, Koca et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e found that the levels of CAT and APX activities were boosted by the accumulation of CuO NPs at (2 ppm) in \u003cem\u003eE. canadensis\u003c/em\u003e. However, it's possible that doses above 2 ppm have a toxic impact on plants. Similarly, Wang et al., \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2011\u003c/span\u003e reported that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs often induce additional oxidative stress as compared to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e bulk particles in pumpkin and ryegrass plants. SOD activity in ryegrass drastically declined under treatment with 100 mg/L Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs. Likewise, Iannone et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e revealed that the activities of antioxidant enzymes in NPs treated plants significantly increased in both the root and the aerial parts, further leading to the prevention of oxidative damage caused by Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles at 20 mg/L on hydroponically grown wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.). However, it is possible that at higher concentrations of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs, there's a likelihood of a detrimented impact on wheat growth. Similar study by Ren et al. (2011) revealed that FeO NPs at doses of 20 mg/L enhanced the activity of antioxidant enzymes, namely CAT, SOD and peroxidase. Likewise, study by Tripathi et al. (\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e) revealed the application of ZnO NPs in wheat seedlings resulted in a drastic inhibition in enzyme activities of AsA\u0026ndash;GSH enzymes due to the increase in the quantity of ROS and lipid peroxidation. Whereas, very few studies have been found that evaluates the comparative effects of green synthesized and market procured CuO and FeO NPs on rice with respect to antioxidant enzyme activities.\u003c/p\u003e\u003cp\u003eMolecular studies involve the analysis of a plant's genetic and molecular makeup, which can yield a deeper understanding of its responses to various conditions and stresses (Praveen et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tripathi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Gene regulation helps the plant control different metabolic processes under stress conditions (Chaves et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, NPs at higher concentrations lead to stressful conditions in plants. They disturb the internal network of processes in different crops, which cannot be observed through morphological changes or biochemical assays (Al-Khayri et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this context, Gopalakrishnan Nair et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e found that under high dosees of CuO NPs, the levels of \u003cem\u003eSOD\u003c/em\u003e and \u003cem\u003eCAT\u003c/em\u003e genes in the roots was significantly decreased, whereas the expression of the \u003cem\u003eAPX\u003c/em\u003e gene was significantly up regulated in the roots at lower doses of CuO NPs as compared to the control in mung bean. Another study reported by Plaksenkova et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e on rocket \u003cem\u003eEruca sativa\u003c/em\u003e showed that increasing the concentration of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs reduced the expression level of miR159c. In various plant species, treatment with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs elicits different responses in miR159c expression.\u003c/p\u003e\u003cp\u003eThe activation of genes responding to oxidative stress following NPs exposure suggests either the direct generation of ROS or free radicals by NPs, or the indirect signaling of cellular stress (Dimkpa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Frazier et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pandey et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, when the stress exceeds the threshold level, this antioxidant enzyme/gene machinery fails to provide tolerance capacity to the plants.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings of this study reveal that rice seedlings exhibit improved growth when treated with green-synthesized CuO and FeO NPs compared to their market-based counterparts. This highlights the potential benefits of employing green synthesis methods for various nanoparticle applications in the future. Conversely, the use of market-based CuO and FeO NPs significantly hindered growth patterns, impacted photosynthetic parameters, elevated reactive oxygen species (ROS) accumulation, reduced antioxidant enzyme levels, and downregulated genes linked to the AsA-GSH cycle in rice seedlings.\u003c/p\u003e\u003cp\u003eThe study strongly indicates that stress induced by market-sourced CuO and FeO NPs poses a notably higher level of toxicity compared to both green-synthesized nanoparticles. Consequently, our results advocate for green synthesis as a superior approach for producing stable nanoparticles. Additionally, the study emphasizes the necessity of developing strategies to mitigate nano pollution, aiming to provide stress-resistant crop plants and enhance overall production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions-\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePR and KV designed the manuscript. PR performed the experiment and wrote the manuscript. KV also help with experiments and editing in manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are thankful to the Director MNNIT Allahabad, Prayagraj for facilitating necessary resources for accomplishment of this work. Authors also acknowledge CSIR Project no- 38(1460)/18/EMR-II for providing financial assistance. Authors are deeply thankful to IIT Kanpur, IIT Mandi and MNIT Jaipur for their kind help in characterization of nanoparticles.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAebi, H. (1984). [13] Catalase in vitro. In \u003cem\u003eMethods in Enzymology\u003c/em\u003e (Vol. 105, pp. 121-126). Academic press.\u003c/li\u003e\n \u003cli\u003eAfzal, S., Sharma, D., \u0026amp; Singh, N. K. (2021). Eco-friendly synthesis of phytochemical-capped iron oxide nanoparticles as nano-priming agent for boosting seed germination in rice (Oryza sativa L.). Environmental Science and Pollution Research, 28, 40275-40287.\u003c/li\u003e\n \u003cli\u003eAhamed, M., Alhadlaq, H. A., Khan, M. A., Karuppiah, P., \u0026amp; Al-Dhabi, N. A. (2014). Synthesis, characterization, and antimicrobial activity of copper oxide nanoparticles. \u003cem\u003eJournal of Nanomaterials\u003c/em\u003e, \u003cem\u003e2014\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eAlhalili, Z. (2022). 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Thriving under stress: how plants balance growth and the stress response. \u003cem\u003eDevelopmental Cell\u003c/em\u003e, \u003cem\u003e55\u003c/em\u003e(5), 529-543.\u003c/li\u003e\n \u003cli\u003eZhang, W. X., \u0026amp; Elliott, D. W. (2006). Applications of iron nanoparticles for groundwater remediation. \u003cem\u003eRemediation Journal: The Journal of Environmental Cleanup Costs, Technologies \u0026amp; Techniques\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(2), 7-21.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CuO NPs, FeO NPs, ecofriendly, green synthesized, market based, rice seedlings","lastPublishedDoi":"10.21203/rs.3.rs-7025406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7025406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe widespread use of nanoparticles (NPs) resulting from industrial activities has led to significant environmental challenges, including biodiversity loss, climate change, soil pollution (both agricultural and saline), and contamination of water bodies. Iron oxide nanoparticles (FeO NPs) and copper oxide nanoparticles (CuO NPs) are extensively used across various industries such as biomedical devices, glass manufacturing, paint production, and as doping materials in semiconductors due to their unique properties. This widespread application necessitates an assessment of their impact on plant growth and agricultural systems.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the comparative effects of green-synthesized versus market-procured CuO and FeO NPs on rice seedlings. The green synthesis of CuO and FeO NPs was achieved using a plant extract from \u003cem\u003eAmragandhi Haridra\u003c/em\u003e (Curcuma amada). Several analytical techniques, including ultraviolet spectrometry (UV-vis), particle size analysis (PSA), X-ray diffraction (XRD), field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FE-SEM EDX), and Fourier transform infrared spectroscopy (FTIR), were employed to characterize the size, shape, functional groups, and crystalline structure of the green-synthesized nanoparticles.\u003c/p\u003e\u003cp\u003eOur findings revealed that market-procured CuO NPs (50 \u0026micro;M) and FeO NPs (200 \u0026micro;M) significantly impaired rice seedling growth, reducing shoot length, root length, and fresh weight. These nanoparticles also decreased the levels of chlorophyll a, chlorophyll b, and carotenoids compared to green-synthesized CuO (50 \u0026micro;M) and FeO NPs (200 \u0026micro;M). Moreover, market-procured nanoparticles at the same concentrations induced the generation of reactive oxygen species (ROS), disrupting plant metabolism by interfering with photosynthesis, respiration, and electron transport activities. In contrast, green-synthesized CuO and FeO NPs at lower concentrations (50 \u0026micro;M) enhanced plant growth and offered protection against oxidative stress.\u003c/p\u003e\u003cp\u003eIn conclusion, these findings highlight the superior potential of green-synthesized NPs in protecting rice crops from oxidative stress, in contrast to market-procured NPs, representing a significant advancement toward sustainable agricultural practices.\u003c/p\u003e","manuscriptTitle":"Comparative Study of Green-Synthesized and Market-Procured CuO and FeO Nanoparticles on the Growth and Stress Tolerance in Rice Seedlings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 15:48:18","doi":"10.21203/rs.3.rs-7025406/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e30922d8-bf94-41d5-abef-fb7ad5f784b4","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T12:11:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 15:48:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7025406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7025406","identity":"rs-7025406","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}