Fe₃O₄ and ZnO nanoparticles on cotton-stalk-derived cellulose microfibers enable a nanopriming strategy to enhance drought resilience in cotton

Abstract

Drought is one of the major environmental stressors that significantly limits the germination, seedling growth, and productivity of cotton, particularly in arid and semi-arid regions. Recently, nanopriming has emerged as a novel and environmentally friendly seed treatment strategy to enhance plant resilience to drought stress. This study investigated the efficacy of nanopriming with Fe₃O₄ and ZnO nanoparticles (NPs) incorporated into cellulose microfibres (CMF) in improving drought tolerance of cotton (Gossypium hirsutum L.) under osmotic stress induced by polyethylene glycol (PEG). CMF extracted from cotton stalk residues was used as a sustainable carrier for Fe₃O₄ and ZnO NPs, yielding Fe/CMF and Zn/CMF composites. Cotton seeds were primed with Fe/CMF (50 and 100 ppm), Zn/CMF (150 and 200 ppm), and a non-primed control, and exposed to three water regimes (0%, 10%, and 15% PEG) to simulate increasing drought severity during germination and early seedling establishment. A comprehensive set of germination, growth, physiological, and biochemical traits was evaluated, including germination percentage, mean germination time, seedling vigour index, root and shoot growth, relative water content (RWC), proline accumulation, oxidative stress markers (malondialdehyde and hydrogen peroxide), and antioxidant enzyme activities (SOD, POD, and CAT). Nanopriming, particularly with 50 ppm Fe/CMF and 200 ppm Zn/CMF, significantly enhanced germination performance, seedling vigour, and biomass accumulation under moderate and severe PEG-induced drought stress. Moreover, nanoprimed seedlings maintained higher RWC, exhibited strongly upregulated antioxidant enzyme activities, accumulated greater proline levels, and showed marked reductions in lipid peroxidation and hydrogen peroxide content, indicating improved osmotic adjustment, redox homeostasis, and membrane stability under drought conditions. These results demonstrate that CMF-mediated delivery of Fe₃O₄ and ZnO NPs effectively mitigates drought-induced physiological and oxidative constraints during early cotton establishment, highlighting Fe/CMF and Zn/CMF nanopriming as a promising, sustainable strategy for enhancing early-stage drought tolerance in cotton-based production systems.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-47735-9.

Introduction

Drought is one of the most serious abiotic constraints limiting agricultural productivity worldwide, especially in arid and semi-arid regions. It impairs water uptake, delays seedling emergence, disrupts enzymatic and hormonal activities and reduces photosynthetic efficiency, resulting in overall lower biomass formation and yield potential^{1, 2}. In the face of increasing climate variability and water scarcity, the development of innovative, sustainable agronomic strategies is crucial to ensure global food and fiber security^{3, 4}.

Seed priming has proven to be a simple, cost-effective and physiologically robust technique to increase seed vigour, synchronise germination and improve stress tolerance. By activating the metabolism early without distending the radicle, priming prepares the seeds for rapid germination and better seedling establishment^{5, 6}. Various methods such as hydro-priming, halo-priming, osmo-priming, hormone priming and nanoparticle (NP)-based priming have been shown to improve the growth and resistance of seedlings to abiotic stress⁷. These strategies improve physiological traits such as relative water content (RWC), leaf area index, photosynthetic activity and nutrient uptake, thus improving overall plant performance⁸.

Among the emerging seed enhancement technologies, NP-based priming has attracted great interest due to its particular physicochemical properties, such as surface-to-volume ratio, reactivity and controlled release of nutrients or active molecules⁹. Nanoscale interventions facilitate water uptake and starch mobilisation via the formation of nanopores, enhance reactive oxygen species (ROS)-mediated signalling and increase the activity of enzymes involved in early seed metabolism¹⁰. They also regulate aquaporin gene expression and maintain ROS homeostasis in membranes, which contributes to improved water uptake and cell function under stress conditions¹¹. Of particular importance are metal-based NPs, such as zinc (Zn) and iron (Fe), which actively bind to the surface of plant cells and improve the redox balance and nutrient supply during the critical early stages of development¹².

Iron and zinc are essential micronutrients and the two most abundant transition metals in biological systems¹³. Their functional roles range from enzymatic catalysis to the attenuation of oxidative stress. In particular, zinc NPs priming ensures efficient Zn supply to embryonic tissues and improves membrane integrity, water uptake and nutrient mobilization during early development¹⁴. It also supports the biosynthesis of auxin by acting as a precursor for tryptophan, thus promoting root and shoot elongation¹⁵. In addition, Zn acts as a cofactor for antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), which play a central role in the elimination of excess ROS during seed germination¹⁶. Zinc also contributes to auxin biosynthesis by functioning as an enzymatic cofactor in key metabolic pathways, thereby promoting root and shoot elongation during early seedling development. In addition, Zn increases the activity of hydrolytic enzymes such as proteases and amylases, facilitating the mobilisation of storage proteins and carbohydrates that are important for seedling establishment^{17, 18}.

Fe-based NPs have also shown promise in alleviating stress during germination¹⁹. Fe₃O₄ NPs have been reported to improve seedling emergence and drought tolerance in cereals such as wheat and maize by increasing RWC, chlorophyll content and antioxidant enzyme activities²⁰. ZnO NPs also improve seedling vigour and oxidative defense in maize under water stress, while similar benefits have been observed in soybean and rice, where NP priming improved photosynthetic efficiency and osmotic balance under stress²¹. Although there is growing evidence for NP priming in cereals and legumes, its use in cotton has not yet been sufficiently explored, despite cotton being an economically important crop and being particularly sensitive to drought stress in the early stages. One challenge in the application of nanoparticles, especially magnetic NPs, is the difficulty of synthesising relatively monodisperse and disaggregated NPs with uniform morphology and size distribution. Therefore, protective strategies are usually utilized in the synthesis of NPs, such as encapsulation by a carbon material, coating with surfactants, or incorporation on carriers to avoid the aggregation of the synthesised NPs^{22, 23}.

On the other hand, cellulose microfibres (CMF), derived from lignocellulosic biomass, have recently gained attention as a sustainable primer due to their large surface area, biocompatibility and exceptional water retention capacity²⁴. Its application has been shown to improve seed hydration and uniformity of germination while minimizing ecological risk²⁵. Therefore, anchoring Fe and Zn NPs in CMF could inhibit their aggregation and lead to the innovative CMF-supported Fe/Zn-NP hybrids for seed priming applications. In addition, the potential synergy between CMF and Fe/Zn NPs represents a novel, environmentally friendly strategy to enhance the benefits of seed priming, especially under conditions of water scarcity.

Nevertheless, there are still concerns about the widespread use of NPs in agriculture. These include issues of uneven distribution, potential phytotoxicity and uncertain long-term effects on soil health and crop safety^{26, 27}. Therefore, the determination of optimal NP concentrations is crucial to maximise physiological benefits while minimising environmental and agronomic risks²⁸. With this in mind, the present study investigates the efficacy of seed priming with Fe and Zn NPs incorporated in nanocellulose to improve the drought resistance of cotton (G. hirsutum L.). Two concentrations of Fe/CMF (50 and 100 ppm) and Zn/CMF (150 and 200 ppm) as well as a non-primed control were used for the experiment. Seeds were primed and then germinated under two levels of PEG 6000-induced drought stress (0% and 15%) under laboratory conditions. A comprehensive set of morphological, physiological and biochemical traits was evaluated, including percentage of germination, mean germination time (MGT), root and shoot length, fresh and dry biomass and vigour index. In addition, stress-related biomarkers such as proline, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and RWC were measured together with the activities of the main antioxidant enzymes (SOD, CAT and POD). Chlorophyll content and chlorophyll fluorescence (Fv/Fm) were also analyzed to assess photochemical efficiency and overall stress adaptation.

Materials and methods

Synthesis and characterization of incorporated zinc oxide (ZnO) and magnetite (Fe₃O₄) NP_S

CMF were synthesised from cotton stalks by alkaline treatment and chemical oxidation. First, raw cotton stalks were crushed to a fine powder using a blender (Panasonic, Osaka, Japan). Four grams of the powder were added to 160 mL of sodium hydroxide solution (4% w/v; Merck, Darmstadt, Germany) and stirred overnight at room temperature using a magnetic stirrer (IKA, Staufen, Germany). The precipitates were collected by centrifugation (Eppendorf 5810R, Hamburg, Germany), washed several times with distilled water, and dried in a forced-air oven (Memmert, Schwabach, Germany) at 70 °C. The dried, alkali-treated powder (1 g) was dispersed in dilute aqueous sulfuric acid solution (50 mL, 1%; Merck, Darmstadt, Germany), followed by the addition of potassium permanganate (2 g; Sigma–Aldrich, St. Louis, MO, USA) and oxalic acid (1 g; Merck, Darmstadt, Germany). The mixture was stirred at 50 °C for 4 h using a temperature-controlled hotplate stirrer (IKA, Germany). The resulting precipitates were centrifuged and washed repeatedly with distilled water until the pH of the supernatant reached approximately 5, and finally dried at 70 °C, yielding CMF. This oxidative treatment increased the surface density of hydroxyl (–OH) and carboxyl (–COOH) functional groups on the CMF backbone, providing abundant active sites for metal ion coordination and nanoparticle anchoring.

All NP precursor solutions were prepared using distilled water as the solvent, in accordance with widely adopted aqueous-phase synthesis protocols for metal oxide NPs to ensure chemical stability, environmental compatibility, and reproducibility^{29, 30}. To prepare Fe/CMF, as-prepared CMF powder (500 mg) was suspended in distilled water (50 mL) using an ultrasonic bath (Elmasonic S 30 H, Elma Schmidbauer GmbH, Germany), for 5 min. A separate solution was prepared by dissolving FeSO₄·7 H₂O (210 mg; Sigma-Aldrich, USA), and FeCl₃·6 H₂O (400 mg; Merck, Germany) in distilled water (20 mL). The iron solution was added dropwise to the CMF suspension, and the reaction temperature was raised to 85 °C. Ammonium hydroxide (NH₄OH) (30%; Merck, Germany) was then added dropwise until the pH reached 10, and the mixture was stirred continuously for 1 h. During this in situ co-precipitation process, Fe²⁺ and Fe³⁺ ions were electrostatically attracted to the negatively charged hydroxyl and carboxyl groups of CMF, followed by nucleation and growth of Fe₃O₄ NPs directly on the CMF surface. The resulting Fe/CMF nanohybrid was stabilised primarily through coordination bonding, hydrogen bonding, and electrostatic interactions, which effectively limited NP agglomeration and ensured uniform surface anchoring. The resulting black precipitates were magnetically separated using a neodymium magnet, washed three times with absolute ethanol (Merck, Germany), and dried overnight at 70 °C in a vacuum oven (Binder, Tuttlingen, Germany).

To prepare Zn/CMF, zinc acetate dihydrate (Zn(CH₃COO)₂·2 H₂O, 2 g; Sigma-Aldrich, USA) was added to the CMF suspension (1 wt%) prepared in distilled water, following established aqueous synthesis routes for ZnO NPs–cellulose hybrid systems³¹, and stirred for 30 min. The mixture was subsequently sonicated for 2 h at 35 °C using an ultrasonic bath (Elmasonic S 30 H, Germany). Zn²⁺ ions interacted strongly with CMF surface hydroxyl and carboxyl groups through coordination and hydrogen bonding, promoting the homogeneous nucleation of ZnO NPs along the CMF matrix. This interfacial interaction facilitated effective anchoring of ZnO NPs within the CMF network, enhancing colloidal stability and preventing NPs aggregation. The resulting precipitates were collected by centrifugation (Eppendorf 5810R, Germany), washed three times with distilled water, and dried at 70 °C in an oven (Memmert, Germany). Morphological and structural characterisation of CMF, Fe/CMF, and Zn/CMF was performed using scanning electron microscopy (SEM; ZEISS EVO MA15, Carl Zeiss, Germany; accelerating voltage 20 kV), Fourier-transform infrared spectroscopy (FTIR; Bruker Tensor 27, Bruker Optics, Germany), X-ray diffraction (XRD; Philips PW1730, PANalytical, Netherlands; Cu Kα radiation, λ = 1.5406 Å), dynamic light scattering analysis (DLS; Particle Metrix, Stabisizer 200, Germany), and Brunauer–Emmett–Teller surface area analysis (BET; BELsorp-mini II, Microtrac BEL, Japan).

Cotton materials and treatments

Cotton seeds (Gossypium hirsutum L., cv. Paroto) were used in this study. Seeds were obtained from the Cotton Research Institute of Iran (CRII, Gorgan, Iran). The cultivar Paroto is a commercially available variety with no intellectual property restrictions or licensing requirements; therefore, no specific permissions or material transfer agreements (MTAs) were required for its use in research. Seeds were selected based on uniformity of size, colour, and plumpness prior to treatment. Before priming, seeds were surface sterilised with 1% (v/v) sodium hypochlorite solution (Merck, Darmstadt, Germany) for 5 min, followed by thorough rinsing with distilled water. Seeds were then primed in NP suspensions containing Fe/CMF (50 and 100 ppm) and Zn/CMF (150 and 200 ppm), respectively, while control seeds were primed with distilled water only. To ensure homogeneous dispersion and minimise NP agglomeration, all suspensions were sonicated for 30 min using an ultrasonic bath (Elmasonic S 30 H, Elma Schmidbauer GmbH, Germany) prior to priming. Seeds were soaked in the respective solutions for 12 h at 25 °C, a priming duration commonly employed in cotton and other oilseed crops³², after which they were air-dried at room temperature in the shade to reach their original moisture content.

For germination assessment, twenty seeds per treatment were placed in each Petri dish, and each treatment was performed in three independent biological replicates (i.e., three separate Petri dishes prepared and treated independently). Within each biological replicate, all measurements were conducted in three technical replicates (e.g., three measurements per seedling or three aliquots for biochemical assays), ensuring analytical precision and reproducibility. Petri dishes were 9 cm sterile (SPL Life Sciences, Pocheon, South Korea), lined with double layers of Whatman No. 1 filter paper (GE Healthcare, Little Chalfont, UK), and moistened with 5 mL of polyethylene glycol solution (PEG-6000; Sigma-Aldrich, St. Louis, MO, USA) at three osmotic stress levels: 0% (control), 10%, and 15%, corresponding to non-stress, moderate, and severe drought-mimicking conditions, respectively². Petri dishes were incubated for seven days in a controlled-environment growth chamber (Binder KBWF series, Tuttlingen, Germany) maintained at 25 ± 1 °C, 70% relative humidity, and a 12 h light/12 h dark photoperiod.

Overall experimental design and workflow

The study followed a stepwise experimental framework to investigate whether CMF-mediated delivery of Fe₃O₄ and ZnO NPs could enhance drought tolerance during cotton germination and early seedling establishment. Nanocellulose derived from cotton stalk residues was employed as a sustainable carrier matrix for the stabilisation and delivery of metal oxide NPs, enabling controlled seed exposure during the priming phase.

Following nanopriming, cotton seeds were subjected to a gradient of PEG-induced osmotic stress to mimic increasing drought severity under controlled conditions. This design enabled simultaneous evaluation of nanopriming effects across multiple stress intensities, allowing treatment × stress interactions to be assessed. A comprehensive set of response variables was analysed, including germination behaviour, early seedling growth performance, plant water status, photosynthetic pigment composition, and biochemical markers of oxidative stress and osmotic adjustment. The experiment was conducted using a randomised complete block design with three independent biological replicates per treatment, and the resulting dataset was subjected to statistical analysis to determine the significance of nanopriming-mediated stress mitigation effects (Fig. 1).

Fig. 1.

Schematic overview of the experimental workflow illustrating CMF-mediated Fe₃O₄ and ZnO NPs seed priming, followed by PEG-induced osmotic stress during cotton germination and early seedling establishment, and subsequent physiological and biochemical assessments.

Evaluation of germination and seedling growth traits

After seven days of incubation under PEG-induced osmotic stress conditions, germination and early seedling growth traits were evaluated. Germination percentage was calculated as the proportion of seeds exhibiting visible radicle emergence (≥ 2 mm) relative to the total number of seeds sown. Three independent Petri dishes per treatment were considered as biological replicates, and within each biological replicate, all measurements were performed in three technical replicates to ensure precision and reproducibility. Mean germination time (MGT) was calculated using the formula of Ellis and Roberts (1981)³³, providing an index of germination speed. The seedling vigour index (SVI) was computed as the product of the germination percentage and the sum of the mean root and shoot lengths. Seedling growth traits, including root length and shoot length, were measured using a digital Vernier calliper (Mitutoyo Corp., Kawasaki, Japan) from ten randomly selected seedlings per treatment within each biological replicate. Fresh weight was recorded immediately after harvesting using an analytical balance (Sartorius Entris II, Göttingen, Germany), while dry weight was determined after oven-drying the samples at 70 °C to constant mass in a forced-air drying oven (Memmert UF110, Schwabach, Germany):

Speed of Germination was calculated using the equation:

where X1, X2, X3,… are the number of seedlings germinated on days n1, n2, n3,…, respectively, and n is the total number of days.

Seedling vigour index was calculated according to Abdul-Baki and Anderson (1973) and expressed as a whole number. The formulas are provided below:

Determination of chlorophyll and carotenoids

The Arnon method was used to determine chlorophyll and carotenoid contents in fresh leaf samples³⁴. Fresh leaf tissue (0.5 g) was homogenised using a chilled mortar and pestle with 10 mL of 80% (v/v) acetone (Merck, Darmstadt, Germany). The homogenate was transferred to centrifuge tubes and incubated at 4 °C for 12 h in darkness to ensure complete pigment extraction. Each measurement was performed in triplicate (technical replicates) for each biological replicate. The extract was then centrifuged at 4000 rpm for 5 min using a refrigerated centrifuge (Eppendorf 5424R, Hamburg, Germany). Absorbance of the clear supernatant was recorded at 663, 645, and 470 nm using a UV–visible spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents were calculated according to Lichtenthaler and Wellburn³⁵ using the following equations:

Measurement of antioxidant enzyme activities and MDA, H₂O₂, O₂−, and proline contents

Leaf samples were collected after six days of PEG-induced osmotic stress to determine the activities of SOD, peroxidase (POD), and CAT, as well as the contents of MDA, superoxide anion (O₂⁻), H₂O₂, and proline (Pro). All biochemical analyses were performed using commercially available, plant-specific assay kits obtained from Solarbio Life Sciences (Beijing, China), including the Superoxide Dismutase (SOD) Activity Assay Kit (Catalogue No. BC0175), Catalase (CAT) Activity Assay Kit (Catalogue No. BC0205), POD Activity Assay Kit (Catalogue No. BC0095), MDA Content Assay Kit (Catalogue No. BC0025), H₂O₂ Content Assay Kit (Catalogue No. BC3595), O₂⁻ Content Assay Kit (Catalogue No. BC1295), and Pro Content Assay Kit (Catalogue No. BC0295). All assays were conducted strictly in accordance with the manufacturer’s instructions. For enzyme extraction, 0.1 g of fresh leaf tissue was homogenised in 1 mL of the proprietary ice-cold extraction buffer supplied with each respective assay kit using a chilled mortar and pestle. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C using a refrigerated centrifuge (Eppendorf 5424R, Germany), and the resulting supernatant was immediately used for biochemical analyses. Antioxidant enzyme activities (SOD, CAT, and POD) were expressed as activity units per gram fresh weight (U g⁻¹ FW), as defined by the respective assay kits, whereas MDA, H₂O₂, O₂⁻, and proline contents were expressed on a fresh weight basis (µmol g⁻¹ FW or nmol g⁻¹ FW, as appropriate). Reaction mixtures were incubated in 96-well microplates (Corning Inc., Corning, NY, USA), and absorbance was measured at kit-specified wavelengths using a microplate reader (BioTek Synergy HTX, Winooski, VT, USA). Each treatment comprised three independent biological replicates, with each biochemical measurement performed in triplicate as technical replicates, and mean values were used for statistical analyses to ensure analytical robustness and reproducibility.

Quantification of MDA and H₂O₂ contents

For MDA quantification, 1 g of frozen leaf tissue was homogenised in 2 mL of 0.1 M potassium phosphate buffer (pH 7.0) prepared using analytical-grade reagents (Sigma-Aldrich, St. Louis, MO, USA). The homogenate was centrifuged at 9,000 rpm for 10 min at 4 °C using a refrigerated centrifuge (Eppendorf 5810 R, Eppendorf AG, Hamburg, Germany). The reaction mixture contained 200 µL of 20% (w/v) trichloroacetic acid (TCA), 200 µL of 0.5% (w/v) thiobarbituric acid (TBA), and 200 µL of enzyme extract. TCA and TBA were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA).

The mixture was incubated in a boiling water bath at 95 °C for 20 min using a thermostatically controlled water bath (Memmert WNB series, Memmert GmbH + Co. KG, Schwabach, Germany), rapidly cooled on ice for 5 min, and centrifuged at 4,000 rpm for 10 min. Absorbance was measured at 532 and 600 nm using a UV–visible spectrophotometer (Shimadzu UV-1800, Shimadzu Corporation, Kyoto, Japan) with 1 cm quartz cuvettes. MDA content was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹³⁶.

For hydrogen peroxide (H₂O₂) determination, 1 g of frozen leaf tissue was homogenised in 10 mL of 1% (w/v) TCA and centrifuged at 4,000 rpm for 10 min using the same refrigerated centrifuge. Five millilitres of the supernatant were mixed with 5 mL of 50 mM potassium phosphate buffer (pH 7.0) and 10 mL of 1 M potassium iodide (KI). Potassium iodide and buffer components were analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). The reaction mixture was incubated in the dark for 1 h at room temperature (25 ± 2 °C), and absorbance was recorded at 390 nm using the same UV–visible spectrophotometer. A standard curve prepared with H₂O₂ (Merck, Darmstadt, Germany) was used for quantification³⁷.

Total proline content assay

Proline content was estimated following the method of Y Sun, MFB Mfarrej, X Song, J Ma, B Min and F Chen³⁸, with minor modifications. Fresh leaf tissue (0.3 g) was homogenised in 5 mL of 3% (w/v) sulfosalicylic acid (Sigma-Aldrich, St. Louis, MO, USA) using a pre-chilled mortar and pestle. After centrifugation at 4,000 rpm for 10 min using the refrigerated centrifuge described above, 2 mL of the supernatant were mixed with 2 mL of acid ninhydrin reagent (prepared from 0.31 g ninhydrin, 7.5 mL glacial acetic acid, and 5 mL of 6 M phosphoric acid), 2 mL of 96% (v/v) acetic acid, and 1 mL of 3% sulfosalicylic acid. Ninhydrin, glacial acetic acid, phosphoric acid, and toluene were of analytical grade and purchased from Merck (Darmstadt, Germany). The reaction mixtures were incubated in a boiling water bath for 1 h using the thermostatically controlled water bath described above, cooled in an ice bath, and 4 mL of toluene was added. The samples were vortexed using a bench-top vortex mixer (Vortex-Genie^® 2, Scientific Industries, Bohemia, NY, USA), and the chromophore-containing upper phase was quantified at 520 nm using the Shimadzu UV-1800 UV–visible spectrophotometer. Proline concentration was calculated from a standard curve and expressed as µmol g⁻¹ fresh weight (FW).

Measurement of leaf RWC

Leaf relative water content (RWC) was measured following the protocol described by I Torres, M-T Sanchez, M Benlloch-Gonzalez and D Perez-Marin³⁹. Freshly harvested leaves were placed in sealed polyethylene sample bags and immediately transferred to ice to minimise transpirational water loss. In the laboratory, leaves were weighed to obtain the fresh weight (FW) using an analytical balance (± 0.0001 g accuracy; Sartorius Cubis^® MSA224S, Sartorius AG, Göttingen, Germany). The samples were then immersed in 20 mL of distilled water produced using a Milli-Q^® water purification system (Milli-Q Advantage A10, Merck Millipore, Darmstadt, Germany) in sterile conical flasks and maintained at 4 °C in a refrigerated incubator (Memmert ICP series, Memmert GmbH + Co. KG, Schwabach, Germany) for 24 h to obtain the turgid weight (TW). Subsequently, leaves were oven-dried at 70–80 °C in a forced-air drying oven (Memmert UF series, Memmert GmbH + Co. KG, Schwabach, Germany) until a constant weight was achieved to determine the dry weight (DW). RWC was calculated using the following equation:

Statistical analyses

Preliminary calculations were performed using Microsoft Excel (Microsoft Corp., Redmond, WA, USA) to organise the experimental data. All experiments were conducted using three independent biological replicates per treatment, where each biological replicate corresponded to an independently primed and stress-exposed experimental unit. For each biological replicate, all quantitative measurements were performed in triplicate as technical replicates, and the mean of the technical replicates was used for subsequent statistical analyses. Data were analysed using a two-way analysis of variance (ANOVA) to evaluate the main effects of nanopriming treatments and PEG-induced osmotic stress levels, as well as their interaction effects. Statistical analyses were performed using IBM SPSS Statistics v29.0 (IBM Corp., Armonk, NY, USA). When significant main or interaction effects were detected, mean comparisons were conducted using Duncan’s multiple range test at p < 0.05. Graphical representation of the data was generated using GraphPad Prism v10 (GraphPad Software, San Diego, CA, USA).

Results

Characterization of the as-prepared NCs, Fe/CMF and Zn/CMF

Cotton stalk (CS) is a globally abundant agricultural residue (~ 50 million tonnes/year production) with low economic importance. Recently, the high cellulose, hemicellulose and lignin content of this agricultural waste, together with its environmental friendliness and biocompatibility, has led to considerable interest in its utilisation as a promising resource for the synthesis of green nanomaterials, bioenergy/biogas, etc^{40, 41}. Therefore, in this study, cotton stalks were used for cellulose extraction and synthesis of NP/cellulose hybrids for cotton seed priming. Figure 2 shows the SEM images and BET analyses of the cotton stalks and extracted cellulose, which confirm the morphological and surface changes of CS after alkali treatment and chemical oxidation. The raw cotton stalk showed a distinct tubular hollow structure composed of cell walls and numerous pores, with a linear multi-cavity structure that can form a bridge between the pores, creating complex porous network connections (Fig. 2a). After alkali treatment and oxidation, a significant portion of the lignin and hemicelluloses were degraded, revealing the significant transformation into cellulose microfibre bundles with shorter and thinner fibril diameters in the range of 2–10 μm (Fig. 2b). These structural changes can be explained by the fact that during the oxidation reaction, the combination of KMnO₄, dilute sulphuric acid and oxalic acid produces stabilised, strong oxidative species that remove the binders and amorphous regions of the cellulose⁴². This process simultaneously enriches the CMF surface with hydroxyl and carboxyl functional groups, which are known to act as effective coordination and anchoring sites for metal ions and NPs.

Fig. 2.

SEM images of (a) raw CS, and (b) CMF samples; BET isotherms and the pore size distributions (inset) of (c) raw CS, and (d) CMF samples.

BET analyses were carried out to evaluate the porosity and specific surface area of the samples. Hysteresis loops of type IV and III isotherms were observed, indicating that adsorption of N₂ occurs in a single layer and multiple layers on the mesoporous structure (Fig. 2c,d)⁴². In addition, BET analyses showed that CS had a low specific surface area (6.46 m² g⁻¹) and pore volume (0.0091 m³ g⁻¹), while the synthesised CMF had more than 15 times the surface area (105.75 m² g⁻¹) and more than 15 times the pore volume (0.1485 m³ g⁻¹). This highly mesoporous architecture with increased surface area provides abundant active sites for metal ion adsorption, thereby facilitating homogeneous nucleation and stabilisation of Fe₃O₄ and ZnO NPs while suppressing their aggregation.

In the next step, SEM analysis was performed to confirm the incorporation of NPs on the CMF surface and to investigate their size distribution and morphology (Fig. 3). At lower magnification, the SEM images of both samples showed cellulose fibres covered by spherical NPs. In particular, the NPs were densely and uniformly attached to each cellulose fibril as bright spots (Fig. 3a,c). The intimate and continuous coverage of NPs along the CMF backbone suggests strong interfacial interactions rather than physical mixing. At higher magnification, the spherical and well-dispersed NPs with diameters of 300 nm–3 μm and 600 nm–4 μm for Fe/CMF and Zn/CMF, respectively, were observed (Fig. 3b,d). Shah et al. reported a similar co-precipitation-driven arrangement of microcrystalline cellulose with magnetite NPs⁴³, while Jayachandiran et al. demonstrated ultrasound-assisted anchoring of ZnO NPs onto CMF⁴⁴. In addition, DLS analyses was employed to confirm particle size and dispersion uniformity of samples. Due to the fact that DLS undertakes spherical particle geometry and analyses hydrodynamic diameter through Brownian motion analysis, its results may be differ from SEM analysis. However, it is considered a useful tool for comparative size measurement of cellulosic dispersions. The average diameter of the particles was measured to be 112 nm, 65.9 nm, and 99.10 nm for pristine CMF, Fe/CMF, and Zn/CMF, respectively. These results confirmed the SEM results as Fe/CMF suspension had a smaller particle size distribution than the Zn/CMF sample. The larger average particle sizes of pristine CMF before its incorporation with Fe/Zn particles, may be because of several factors, such as reduction of length of CMF fibres during the chemical reactions of loading NPs, or its rapid accumulation of the particles. Moreover, this should be noticed that to distinguish between the sizes of CMF and ZnO or Fe₃O₄ NPs in Zn/CMF and Fe/CMF, SEM analysis is more reliable tool.

Fig. 3.

SEM images of (a, b) Fe/CMF, and (c, d) Zn/CMF samples, at different magnifications. (e) Size distribution from DLS study of CMF, Fe/CMF, and Zn/CMF samples.

To compare chemical composition and functional groups, FTIR spectra of CMF, Fe/CMF and Zn/CMF were analysed (Fig. 4a). In all spectra, bands around 3410 and 2920 cm⁻¹ corresponded to stretching vibrations of –OH and –CH groups, respectively. The peak at 1620 cm⁻¹ was attributed to in-plane –OH deformation of CMF⁴⁴. Peaks at 1450, 1218 and 1108 cm⁻¹ reflected cellulose crystallinity⁴⁵. Following NP incorporation, distinct spectral changes were observed, indicating the formation of CMF–NP interfacial interactions. New bands at 580 and 490 cm⁻¹ were assigned to Fe–O and Zn–O vibrations in Fe/CMF and Zn/CMF, respectively⁴⁶. Moreover, the downshift of the broad –OH stretching band from 3427 cm⁻¹ (CMF) to 3414 cm⁻¹ (Fe/CMF) and 3407 cm⁻¹ (Zn/CMF) confirms coordination and hydrogen bonding interactions between CMF functional groups and metal oxide NPs, supporting their stable anchoring onto the CMF matrix.

Fig. 4.

(a, left panel) FTIR spectra, (b, right panel) XRD patterns of CMF, Fe/CMF and Zn/CMF samples.

XRD analysis further confirmed NP loading and crystalline structure (Fig. 4b). CMF exhibited characteristic cellulose peaks at 2θ = 17.6° (110) and 22.3° (200). After Fe NPs incorporation, additional peaks corresponding to Fe₃O₄ crystal planes appeared at 2θ = 30.45º (2 2 0), 36.03º (3 1 1), 44.71º (4 0 0), 54.23º (4 2 2), 57.36º (5 1 1), 63.29º (4 0 0) which were in conformity with the (JCPDS) card No. 19–0629, confirming successful formation of Fe₃O₄–CMF hybrids⁴³. Similarly, Zn/CMF sample showed characteristic ZnO diffraction peaks at 2θ = 33.67º (1 0 0), 35.41º (0 0 2), 37.85º (1 0 1), 56.91º (0 0 2), 61.37º (1 1 0), 70.47º (1 0 3) consisting with the (JCPDS) card No. 36–1451, and alongside cellulose signals⁴⁴. The coexistence of cellulose and metal oxide diffraction peaks without peak broadening or phase distortion suggests that NP anchoring occurred on the CMF surface without disrupting the cellulose crystalline framework.

Effects of ZnO and Fe₃O₄ NPs on phenotypic characteristics of cotton seedlings under drought stress

Under PEG-induced drought stress, the root fresh weight of Gossypium hirsutum seedlings declined substantially compared with the non-stressed control. Root fresh weight decreased by approximately 40–45% under 10% PEG (0.21 g vs. 0.37 g) and by approximately 50% under 15% PEG (0.19 g vs. 0.37 g) (P < 0.05) (Fig. 5a). Under non-stressed conditions (0% PEG), seed nanopriming with Zn/CMF, Fe/CMF, or their combinations resulted in root fresh weight values comparable to the non-primed control, indicating that priming alone did not affect basal root biomass.

Fig. 5.

Effects of Zn/CMF and Fe/CMF on the morphological parameters of cotton seedlings under drought stress. Quantitative representation of (a) root fresh weight, (b) root dry weight, (c) root length, (d) shoot fresh weight, (e) shoot dry weight, and (f) shoot length. Vertical bars indicate standard error (SE). Different letters denote statistically significant differences at P ≤ 0.05 according to Duncan’s multiple range test. Abbreviations (Zn for Zn/CMF, and Fe for Fe/CMF) were used to avoid cluttering the diagrams.

Under drought stress, seed nanopriming mitigated the reduction in root fresh weight in a treatment- and dose-dependent manner. At 10% PEG, Zn200 and Zn150 + Fe50 treatments increased root fresh weight to 0.29–0.31 g, corresponding to increases of approximately 35–50% relative to the drought-stressed control (P < 0.05). Zn150, Zn200 + Fe50, and Zn150 + Fe100 treatments also produced significant improvements, whereas Fe50 and Fe100 alone resulted in comparatively smaller increases (P < 0.05). At 15% PEG, Zn150 + Fe50 and Zn200 treatments exhibited the highest root fresh weight values (0.28–0.31 g), representing increases of approximately 45–65% compared with stressed controls (P < 0.05).

Drought stress also caused a marked decline in root dry weight. Relative to the non-stressed control (0.168 g), root dry weight decreased by approximately 50% under 10% PEG (0.080 g) and by approximately 55–60% under 15% PEG (0.072 g) (P < 0.05) (Fig. 5b). Priming-alone treatments (0% PEG) again showed root dry weight values similar to the control, confirming that nanopriming did not alter root dry matter accumulation in the absence of stress. Under 10% PEG, Zn150 + Fe50, Zn200, and Zn200 + Fe100 treatments increased root dry weight by approximately 55–80% relative to the drought-stressed control (P < 0.05), while under 15% PEG, corresponding increases ranged from approximately 60% to 85% (P < 0.05).

Root length exhibited a comparable response to drought stress and nanopriming. PEG-induced stress reduced root length by approximately 30% under 10% PEG (11.0 cm vs. 15.5 cm) and by approximately 35% under 15% PEG (10.0 cm vs. 15.5 cm) (P < 0.05). Seed nanopriming significantly improved root elongation under both stress levels, with Zn200 + Fe50 and Zn200 treatments increasing root length by approximately 25–45% under 10% PEG and by approximately 40–50% under 15% PEG relative to stressed controls (P < 0.05). Other Zn-containing treatments also resulted in moderate but significant increases.

Shoot biomass parameters followed a similar trend. Shoot fresh weight declined by approximately 45–50% under 10% PEG (1.10 g vs. 2.10 g) and by approximately 60% under 15% PEG (0.85 g vs. 2.10 g) (P < 0.05). Nanopriming with Zn200, Zn150 + Fe50, Zn200 + Fe50, and Zn150 treatments significantly increased shoot fresh weight under drought stress, with values ranging from 1.85 to 2.10 g under 10% PEG and from 1.75 to 1.90 g under 15% PEG (P < 0.05).

Shoot dry weight responded similarly. Relative to the non-stressed control (0.903 g), shoot dry weight decreased by approximately 50% under 10% PEG (0.473 g) and by approximately 60–65% under 15% PEG (0.323 g) (P < 0.05). Seed nanopriming significantly enhanced shoot dry matter accumulation under drought, with Zn200, Zn200 + Fe50, and Zn150 + Fe50 treatments producing the highest values under both stress levels, corresponding to approximately two-fold increases relative to drought-stressed controls at 15% PEG (P < 0.05).

Shoot length was also significantly reduced by PEG-induced drought stress. Compared with the non-stressed control (24.0 cm), shoot length declined by approximately 30% under 10% PEG (17.0 cm) and by approximately 40–45% under 15% PEG (14.0 cm) (P < 0.05). Nanopriming significantly promoted shoot elongation under drought conditions, with Zn200 + Fe50 and Zn200 treatments increasing shoot length to 25–26 cm under 10% PEG and to 24–25 cm under 15% PEG (P < 0.05), while other Zn-containing treatments produced moderate but significant improvements. These results demonstrate that Zn/CMF- and Fe/CMF-based seed nanopriming does not affect seedling growth under non-stressed conditions but confers pronounced, dose-dependent protection against PEG-induced drought stress, with combined Zn–Fe formulations generally outperforming single-metal treatments.

Chlorophyll, carotenoid, and anthocyanin content

Under non-stress conditions (0% PEG), seed nanopriming with Fe/CMF, Zn/CMF, or their combinations resulted in only slight numerical increases in chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, and anthocyanin content compared with the non-primed control (Table S1; Fig. 6). These differences were not statistically significant (P > 0.05), indicating that nanopriming alone did not alter basal pigment composition under optimal water availability.

Fig. 6.

Influence of Zn/CMF and Fe/CMF on physiological attributes of cotton seedlings. Quantification of (a) chlorophyll content, (b) carotenoid content, (c) anthocyanin content, and (d) relative water content (RWC). Vertical bars indicate SE. Different letters indicate statistically significant differences at P ≤ 0.05 (Duncan’s test). Abbreviations (Zn for Zn/CMF, and Fe for Fe/CMF) were used to avoid cluttering the diagrams.

PEG-induced osmotic stress caused a significant decline (P < 0.05) in photosynthetic pigment contents in non-primed plants (Table S1; Fig. 6). Chlorophyll a decreased by approximately 40% under 10% PEG and by about 49% under 15% PEG relative to non-stressed controls, while chlorophyll b declined by approximately 43% and 66%, respectively. Total chlorophyll content followed a similar trend, confirming pronounced drought-induced impairment of the photosynthetic pigment system.

Seed nanopriming mitigated drought-induced reductions in chlorophyll content, with the magnitude of recovery dependent on treatment and stress intensity (Table S1; Fig. 6). Under 10% PEG, chlorophyll a increased from 0.222 mg g ⁻¹ FW in drought-stressed controls to 0.281–0.336 mg g⁻¹ FW in nanoprimed plants, corresponding to increases of approximately 27–51%. Under 15% PEG, chlorophyll a increased from 0.187 mg g⁻¹ FW to 0.227–0.322 mg g⁻¹ FW, representing improvements of approximately 21–72% relative to drought-stressed controls. The highest chlorophyll a values under both stress levels were consistently observed in 200 ppm Zn/CMF and combined Zn/CMF + Fe/CMF treatments.

Chlorophyll b exhibited greater drought sensitivity but a strong recovery response to nanopriming (Table S1; Fig. 6). Under 10% PEG, chlorophyll b increased from 0.108 mg g⁻¹ FW in drought-stressed controls to 0.139–0.179 mg⁻¹ FW in nanoprimed plants. Under 15% PEG, chlorophyll b increased from 0.064 mg⁻¹ FW to 0.114–0.167 mg g⁻¹ FW, with Zn200-based and combined treatments showing increases exceeding 150% relative to drought-stressed controls. Total chlorophyll content mirrored these trends, with Zn200-based and combined treatments maintaining the highest total chlorophyll levels under both drought intensities.

Carotenoid content declined significantly (P < 0.05) under PEG-induced drought stress, decreasing from 0.31 mg⁻¹ FW in non-stressed plants to 0.12 mg g⁻¹ FW at 10% PEG and to 0.085 mg g⁻¹ FW at 15% PEG (Table S1; Fig. 6). Seed nanopriming enhanced carotenoid accumulation under stress, increasing values to 0.159–0.244 mg g⁻¹ FW at 10% PEG and to 0.136–0.238 mg g⁻¹ FW at 15% PEG. The largest increases were observed in 200 ppm Zn/CMF and combined treatments.

Anthocyanin content increased in response to PEG-induced stress and was further enhanced by seed nanopriming (Table S1; Fig. 6). Compared with drought-stressed controls, anthocyanin levels increased from 20.2 to 24.4–27.8 nmol g⁻¹ FW under 10% PEG and from 25.8 to 32.7–36.9 nmol g⁻¹ FW under 15% PEG, with the highest accumulation consistently recorded in Zn200-based and combined Zn/CMF + Fe/CMF treatments.

Leaf relative water content (LRWC)

PEG-induced drought stress significantly reduced (P < 0.05) leaf relative water content (LRWC) relative to non-stressed plants. Specifically, LRWC declined by approximately 30% under 10% PEG and by about 40% under 15% PEG compared with the non-stressed control (Table S1, Fig. 6). Seed nanopriming improved LRWC under drought conditions, with the magnitude of improvement varying among treatments and stress intensity. Under 10% PEG, LRWC increased from 68.2% in drought-stressed control plants to 78.4–81.5% in nanoprimed plants. The highest LRWC values were recorded in 200 ppm Zn/CMF and combined Zn/CMF + Fe/CMF treatments, corresponding to improvements of approximately 18–20% relative to the drought-stressed control.

Under severe drought stress (15% PEG), nanopriming similarly enhanced LRWC, increasing values from 58.5% in drought-stressed controls to 71.0–76.5% across treatments. The largest improvements (approximately 29–31%) were observed in 200 ppm Zn/CMF and combined Zn/CMF + Fe/CMF treatments, while lower-dose single-element treatments also showed moderate but consistent increases in LRWC. Overall, Zn200-based and combined treatments were most effective in maintaining leaf water status under osmotic stress (Fig. 6).

Modulation of antioxidant enzymes under drought stress using Zn/CMF and Fe/CMF

Under non-stress conditions (0% PEG), seed nanopriming with Zn/CMF, Fe/CMF, or their combinations caused only slight numerical changes in catalase (CAT), POD, and SOD activities, as well as in H₂O₂, MDA, and proline content, compared with non-primed controls (Table S1; Fig. 7). These differences were not statistically significant (P > 0.05), confirming that priming alone does not perturb basal antioxidant enzyme activity or oxidative stress markers, thereby addressing the requirement for priming-only controls.

Fig. 7.

Modulation of antioxidant enzyme activities and stress indicators in cotton seedlings under drought stress following treatment with Zn/CMF and Fe/CMF. Quantification of (a) CAT, (b) SOD, (c) POD, (d) H₂O₂, (e) MDA, and (f) proline. Vertical bars represent SE, and different letters indicate statistically significant differences at P ≤ 0.05 (Duncan’s test). Abbreviations (Zn for Zn/CMF, and Fe for Fe/CMF) were used to avoid cluttering the diagrams.

PEG-induced drought stress caused significant alterations in antioxidant enzyme activities and stress markers (P < 0.05). CAT activity increased from 2.1 U g⁻¹ FW in non-stressed controls to 2.4 U g⁻¹ FW under 10% PEG and remained similar (2.4 U g⁻¹ FW) at 15% PEG, indicating a stress-induced enhancement of approximately 14% at both drought levels. POD and SOD activities also increased under drought stress, from 2.8 to 3.1–3.4 U g⁻¹ FW (POD) and from 4.1 to 4.7–4.7 U g⁻¹ FW (SOD) at 10–15% PEG, respectively, confirming that drought triggers antioxidant defense responses (Table S1; Fig. 7).

Seed nanopriming significantly enhanced antioxidant enzyme activity (P < 0.05), with effects dependent on treatment and stress intensity. Under 10% PEG, CAT activity increased to 2.5–3.7 U g⁻¹ FW, POD to 3.0–4.2 U g⁻¹ FW, and SOD to 4.2–6.0 U g⁻¹ FW across different nanopriming treatments, corresponding to relative increases of 4–54% compared with drought-stressed controls. At 15% PEG, the highest activities were observed in 200 ppm Zn/CMF and combined 200 ppm Zn/CMF + 50 ppm Fe/CMF treatments, with CAT reaching 3.8–4.2 U g⁻¹ FW, POD 4.4–4.9 U g⁻¹ FW, and SOD 6.3–7.1 U g⁻¹ FW, representing 58–75% increases relative to stressed controls.

H₂O₂ content rose significantly under drought from 0.65 nmol g⁻¹ FW (control) to 1.45 nmol g⁻¹ FW at 10% PEG and 1.75 nmol g⁻¹ FW at 15% PEG. Nanopriming with 200 ppm Zn/CMF and combined Zn/CMF + Fe/CMF significantly reduced H₂O₂ levels to 1.00–1.05 nmol g⁻¹ FW (10% PEG) and 0.85–0.98 nmol g⁻¹ FW (15% PEG), representing 28–51% decreases relative to drought-stressed controls (P < 0.05).

MDA content, a marker of lipid peroxidation, increased from 1.24 nmol g⁻¹ FW (control) to 1.98 nmol g⁻¹ FW (10% PEG) and 2.45 nmol g⁻¹ FW (15% PEG). Nanopriming with 200 ppm Zn/CMF and combined treatments reduced MDA to 1.38–1.40 nmol g⁻¹ FW at 10% PEG and 1.42–1.45 nmol g⁻¹ FW at 15% PEG, corresponding to 29–42% reductions relative to drought-stressed controls (P < 0.05).

Proline content increased under drought from 0.11 µmol g⁻¹ FW (control) to 0.16 µmol g⁻¹ FW at both 10% and 15% PEG. Nanopriming further enhanced proline accumulation in a stress- and treatment-dependent manner, reaching 0.21–0.28 µmol g⁻¹ FW (10% PEG) and 0.21–0.34 µmol g⁻¹ FW (15% PEG), representing 31–112% increases relative to drought-stressed controls. The highest proline levels were consistently observed in 200 ppm Zn/CMF and combined 200 ppm Zn/CMF + 50 ppm Fe/CMF treatments (Table S1; Fig. 7). Overall, 200 ppm Zn/CMF and combined Zn/CMF + Fe/CMF treatments were consistently most effective in enhancing antioxidant defenses, reducing oxidative stress markers, and improving osmoprotectant accumulation under PEG-induced drought (Fig. 7).

Correlation matrix and principal component analysis

A correlation matrix and principal component analysis (PCA) were conducted to elucidate the interrelationships among the measured physiological and biochemical traits across treatment groups. In the correlation matrix (Fig. 8a), the colour intensity reflects the strength and direction of Pearson correlation coefficients, with statistically significant correlations (P < 0.05) indicated by deeper colour intensities, whereas lighter or pale shades represent weak or statistically non-significant associations (P > 0.05). Notably, H₂O₂ and MDA exhibited strong and statistically significant negative correlations (P < 0.05) with key morphophysiological parameters, including germination percentage, MGT, shoot and root length, fresh and dry biomass, RWC, and antioxidant enzyme activities such asSOD, POD, and CAT. These significant inverse relationships indicate that elevated oxidative stress markers are associated with reduced seedling vigour and physiological performance.

Fig. 8.

Correlation and variability among different treatments (A) Correlation matrix, (B) Principal component analysis.

PCA was subsequently performed to reduce data dimensionality and identify dominant patterns of trait variation among treatments. The first two principal components (PCs), both with eigenvalues greater than 1, were retained for interpretation (Fig. 8b). PC1 explained 60.4% of the total variance, while PC2 accounted for 29.2%, together capturing 89.6% of the overall variability. The PC1 loading plot showed that morphophysiological traits, including CAT, POD, SOD, proline, RWC, and shoot and root biomass parameters (SFW, RFW, SDW, RDW), were strongly and positively loaded, whereas MDA, and H₂O₂, were negatively loaded, indicating a clear separation between stress-mitigating and stress-inducing traits.

In contrast, PC2 displayed a differential loading pattern, with antioxidant enzymes (CAT, POD, SOD), RWC, and root dry weight loading positively, while EL, MDA, and H₂O₂ were negatively associated, suggesting that PC2 primarily discriminates treatments based on oxidative damage versus physiological resilience.

Each point in the PCA biplot represents a unique treatment combination of Fe/CMF and Zn/CMF NPs priming under varying PEG-induced drought conditions. Along PC1, non-stressed and NP-primed treatments were positioned toward the positive axis, reflecting statistically supported associations with improved physiological performance, whereas PEG-stressed treatments clustered toward the negative axis, corresponding to significantly elevated oxidative stress indicators. Along PC2, most NP-primed treatments clustered closely regardless of PEG level, indicating shared antioxidant and osmoprotective response patterns induced by seed nanopriming. This multivariate clustering further supports the role of Fe/CMF- and Zn/CMF-based nanopriming in coordinating physiological responses that mitigate drought-induced oxidative stress.

Discussion

Nanotechnology-based seed priming is increasingly viewed as a functional intervention for enhancing early-stage stress adaptation rather than a general growth-promoting strategy in crop plants⁴⁶. In the context of drought stress, the present findings demonstrate that priming cotton seeds with Fe/CMF and Zn/CMF nanohybrids markedly alleviates PEG-induced osmotic constraints by modulating key morphophysiological and biochemical processes associated with early stress adaptation. In cotton, where seed germination and early seedling establishment represent highly drought-sensitive developmental phases, such improvements are agronomically significant, as stress-induced impairment at these stages often leads to poor stand establishment and irreversible yield penalties under field conditions^{47, 48}. The enhanced seedling performance observed in this study likely reflects the coordinated regulation of water status, antioxidant defence systems, and metabolic resilience, processes previously implicated in NP-mediated stress tolerance^{6, 49}. Importantly, the efficacy of the nanopriming response appears closely linked to NP physicochemical attributes, including composition, concentration, surface characteristics, and exposure duration^{50, 51}. Consistent with earlier reports on ZnO and Fe₃O₄ NPs³⁰, their nanoscale dimensions facilitate improved bioavailability and cellular interaction, enabling more effective modulation of redox homeostasis, micronutrient dynamics, and enzyme activation under osmotic stress⁵². Beyond their role as micronutrient sources, the present results support the emerging view that metal-based nanomaterials function as regulators of stress-responsive signalling pathways, thereby contributing to improved physiological stability during drought exposure. From an applied perspective, enhancing tolerance during the germination-to-establishment transition is particularly valuable, as this developmental window represents a critical physiological bottleneck determining crop uniformity and productivity in water-limited agroecosystems. These findings position nanocellulose-integrated metal NPs as a promising and scalable platform for nano-enabled seed technologies aimed at advancing climate-smart cotton production systems.

Effect of nano priming on germination and seedling growth under drought stress

Drought stress imposes a major constraint on seed germination and early seedling establishment in cotton (G. hirsutum L.), a developmental phase highly sensitive to cellular dehydration and metabolic disruption⁵³. PEG-induced osmotic stress markedly suppressed seedling growth, reflecting the well-documented inhibition of cell expansion, reduced turgor pressure, and impaired reserve mobilization that typically accompany water limitation. However, priming with Zn/CMF and Fe/CMF nanohybrids substantially mitigated these inhibitory effects, indicating a pronounced protective influence during the critical transition from germination to autotrophic growth. The superior performance of the combined 200 ppm Zn/CMF + 50 ppm Fe/CMF treatment suggests a coordinated physiological response that promotes recovery of seedling architecture and biomass accumulation under osmotic stress. Such improvements are most plausibly attributed to enhanced osmotic regulation, improved cellular hydration, and greater micronutrient bioavailability facilitated by nano-enabled delivery systems⁵⁴. Metal oxide NPs have been shown to regulate transmembrane water movement through aquaporin modulation, stabilize membrane structures, and activate stress-responsive signalling cascades, collectively supporting early seedling establishment under drought conditions^{55, 56, 57}. The enhanced elongation of root and shoot tissues observed here therefore likely reflects improved metabolic activity and sustained cell expansion, both of which are essential for drought adaptation during early ontogeny⁵⁸. Notably, individual applications of Zn/CMF and Fe/CMF also produced substantial growth stimulation, reinforcing the independent physiological roles of zinc and iron in stress mitigation. Zinc is widely associated with enzyme activation, membrane stabilization, and auxin metabolism, whereas iron plays a central role in electron transport, chloroplast development, and redox regulation. Their combined application may therefore generate complementary biochemical effects that optimize ROS detoxification and metabolic continuity under water-deficit conditions. Similar NP-mediated enhancements in enzymatic activity and micronutrient signalling have been reported in Basella alba and soybean, supporting the conserved role of metal-based nanomaterials in promoting stress resilience across species^{59, 60}. The differential response observed across NP concentrations further indicates the existence of a dose-dependent physiological threshold, beyond which stress-mitigating benefits are maximized. Suboptimal responses at intermediate concentrations may reflect restricted NP uptake, altered surface reactivity, or insufficient activation of drought-responsive metabolic pathways, underscoring the importance of precise dose calibration in nano-priming strategies⁶¹. Such concentration-dependent behaviour is increasingly recognised as a defining characteristic of NP–plant interactions and warrants careful optimisation for translational deployment.

Restoration of shoot growth under NP treatments further highlights the role of nano-priming in maintaining turgor-driven expansion and whole-plant water balance during osmotic stress. Improvements in shoot elongation are consistent with reports that ZnO and Fe₃O₄ NPs promote cell division, cellular elongation, and hormone-mediated growth regulation under adverse environmental conditions⁶². Overall, the enhanced biomass accumulation observed in primed seedlings suggests that nano-enabled micronutrient delivery helps sustain cellular hydration, preserve membrane functionality, and maintain nutrient assimilation, thereby attenuating drought-induced growth arrest^{63, 64}. Importantly, the recovery of early seedling growth observed in this study is unlikely to be driven solely by improved water relations; rather, it likely reflects upstream stabilization of cellular redox homeostasis. Effective control of reactive oxygen species is fundamental for maintaining membrane integrity, enzyme functionality, and metabolic flux under drought stress. Consequently, the growth-promoting effects of Fe/CMF and Zn/CMF nanohybrids may be mechanistically linked to the enhanced antioxidant capacity discussed in subsequent sections, suggesting that nano-priming supports drought tolerance through coordinated regulation of both structural development and oxidative stress defence. Taken together, these findings indicate that priming with Zn/CMF and Fe/CMF nanohybrids reinforces structural and metabolic resilience in cotton seedlings exposed to water deficit. Mechanistically, this protection likely arises from integrated regulation of stress signalling networks, antioxidant capacity, and nutrient-dependent metabolic processes⁶⁵, Strengthening seedling vigour at this early developmental stage is particularly consequential for cotton, as robust establishment directly influences stand uniformity, resource capture, and ultimately yield stability in drought-prone production systems.

Drought-induced physio-biochemical changes and their mitigation by NPs

The relative water content (RWC) of cotton seedlings decreased under PEG-induced drought stress, probably due to a disruption of cell membrane integrity, a known consequence of drought-induced damage. This disruption results from an imbalance in the homeostasis of ROS, with excessive ROS formation leading to lipid peroxidation and impaired membrane function⁶⁶. Exogenous administration of iron and zinc NPs improved membrane stability and significantly reduced water loss, especially at higher concentrations of NPs. The extent of cellular damage and excessive ROS accumulation was determined by quantifying the concentrations of MDA and H₂O₂ produced after lipid peroxidation of the cell membrane. As drought stress causes the production and accumulation of ROS, it leads to the peroxidation of lipids in cellular and organellar membranes, which ultimately leads to cell death⁶⁷. ROS remove electrons from the hydrogen atoms of polyunsaturated fatty acids and increase the loss of electrolytes from the cells, leading to a loss of membrane integrity⁶⁸. MDA is an important biochemical marker that indicates the extent of cell membrane damage⁶⁹. PEG-induced desiccation stress significantly increased H₂O₂ and MDA content, while the addition of Fe/CMF and Zn/CMF decreased MDA content. This pattern of an initial increase followed by a decrease after NP treatment is consistent with the existing literature that abiotic stress increases oxidative markers and NP supplementation attenuates them⁷⁰. Similar observations were made in Brassica napus, where the combined application of selenium and melatonin effectively reduced MDA and H₂O₂ concentrations under abiotic stress⁷¹.

Several antioxidant enzymes such as SOD, POD and CAT were activated due to excessive ROS production in response to drought stress and stimulated natural defense mechanisms. Superoxide dismutase is a metalloenzyme that provides primary defense by detoxifying the free superoxide radical (O₂-) into peroxide (H₂O₂), which is then degraded to H₂O and O₂ by peroxidase and catalase⁷². According to our findings, although the activities of antioxidant enzymes increased slightly in response to PEG-induced stress due to the innate stress response, these levels were not sufficient to fully counteract oxidative toxicity. In addition, drought stress can impair enzyme function by oxidative modification of thiol groups, leading to inhibition of ROS scavenging activity⁷³. The application of Fe/CMF and Zn/CMF NPs significantly enhanced the activities of antioxidant enzymes (Fig. 7), probably by structurally stabilizing the enzymes and providing essential metallic cofactors. These enhancements mirror previous reports in which exogenous application of magnesium, selenium and melatonin nanoparticles significantly increased antioxidant responses in Oryza sativa and B. napus under abiotic stress conditions⁷⁴.

In addition to enzymatic antioxidants (SOD, POD, CAT), the content of non-enzymatic antioxidants such as proline increased under drought stress, contributing to osmotic adjustment and cellular protection against dehydration-induced damage. Proline functions as a compatible solute that stabilizes membranes, preserves protein structure, and supports redox balance under water-deficit conditions. Treatment with Fe/CMF and Zn/CMF further enhanced proline accumulation in cotton seedlings, indicating improved osmoprotective capacity. Comparable results were reported in tomato plants, where selenium application mitigated cadmium stress through proline-mediated alleviation of oxidative damage⁷⁵. Anthocyanins, a major class of flavonoids, possess strong antioxidant properties due to their ability to scavenge ROS and chelate metal ions, thereby supporting cellular redox homeostasis during abiotic stress. The observed increase in anthocyanin content under drought stress, particularly following NP application, suggests an enhanced secondary antioxidant defence contributing to improved stress tolerance⁷⁶. Similar stress-associated accumulation of anthocyanins has been widely reported in plants exposed to adverse environmental conditions⁷⁷. In addition to anthocyanins, drought stress drastically inhibited photosynthesis due to alterations in the light-harvesting complex⁴⁷. Stress can also replace Mg ions in chlorophyll, thereby disrupting photosynthetic function⁷⁸. Drought stress affected the content of chlorophyll a, b, total chlorophyll and carotenoids in cotton seedlings. Administration of Fe/CMF and Zn/CMF mitigated stress-induced declines in photosynthetic pigments, likely by supporting nutrient availability and maintaining pigment biosynthesis. This restoration of pigment levels suggests that NP priming helps preserve the structural and functional integrity of the photosynthetic apparatus under abiotic stress⁸. These results are consistent with previous reports in which drought stress reduced chlorophyll content, photosynthetic rate, stomatal conductance and transpiration in Coriandrum sativum, whereas exogenous selenium improved physiological performance. Similarly, protein content decreased in response to drought stress⁷⁹.

The results of our study indicate that Fe/CMF and Zn/CMF NPs have significant potential to alleviate drought stress in cotton seedlings. Metallic NPs can interact with antioxidant systems and transporters and promote detoxification and compartmentalization of ROS or toxic intermediates. They can also form complexes with ROS or stress-induced toxins and facilitate their compartmentalization. The correlation matrix and PCA plot also show that, except MDA and H₂O₂, all morphophysiological parameters were positively correlated and showed a strong positive correlation between the control and the Zn/CMF 200 ppm + Fe/CMF 50 ppm treatments. Taken together, these results support the hypothesis that priming with ZnO and Fe₃O₄ NPs modulates physiological and biochemical metabolic pathways to improve the drought tolerance of cotton. The proposed mechanism of drought-induced toxicity and attenuation by NPs in cotton is summarised in Fig. 9.

Fig. 9.

Proposed general mechanism of drought-induced stress and its alleviation by Fe/CMF and Zn/CMF nanohybrids-based seed priming in cotton (Gossypium hirsutum) seedlings. The schematic illustrates the key physiological and biochemical metabolic pathways modulated by nanopriming, including enhanced antioxidant defence, improved osmotic adjustment, membrane stabilisation and maintenance of photosynthetic efficiency under drought stress.

Conclusions

This study provides convincing evidence that nanopriming with iron (Fe/CMF) and zinc (Zn/CMF) nanohybrids significantly increases the drought resistance of cotton by modulating key physiological and biochemical attributes. Priming seeds with Fe/CMF (50 and 100 ppm) and Zn/CMF (150 and 200 ppm), especially under osmotic stress induced by PEG (15%), resulted in marked improvements in germination percentage, mean germination time, seedling growth parameters, and biomass accumulation. In addition, nanoprimed seeds exhibited enhanced activities of key antioxidant enzymes (SOD, POD, and CAT), reduced lipid peroxidation as indicated by lower MDA levels, and improved osmotic adjustment through increased proline accumulation. Nanopriming further contributed to higher relative water content and reduced hydrogen peroxide accumulation, reflecting improved water status and attenuation of oxidative stress under drought conditions. The most pronounced effects were observed with Fe/CMF at 50 ppm and Zn/CMF at 200 ppm, suggesting a synergistic role of these micronutrients in strengthening antioxidant defence systems and maintaining cellular integrity under water-deficit stress. Overall, these findings highlight the potential of NP-mediated seed priming as a promising, cost-effective, and scalable strategy for improving early-stage drought tolerance in cotton and support its prospective application in nano-enabled, climate-resilient agricultural systems.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

**RH** and **EM** Conceptualization, Data curation, Formal analysis, Writing original draft. **ZGH** : Investigation, Validation. **OA, EM** review & editing. The author(s) read and approved the final manuscript.

Funding

This study was funded by the Agricultural Biotechnology Research Institute of Iran (ABRII) and the Cotton Research Institute of Iran (CRII).

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All experimental studies on plants were conducted in compliance with relevant institutional, national, and international guidelines and legislation.