Iron oxide nanoparticles alleviate cold stress in rice by reducing oxidative damage and enhancing antioxidant defense systems, and transcriptional networks

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Given the need for novel strategies to improve crop cold tolerance, we evaluated the efficacy of iron oxide nanoparticles (FeO) in enhancing rice cold stress resilience. The plant nano-bionics strategy employs sub-12.5 nm iron oxide nanoparticles with a negative ζ-potential (− 37.6 mV), which achieve high colocalization within chloroplasts to confer cold tolerance in rice by enhancing photosynthetic efficiency and ROS scavenging. The reported mechanisms involve promoting plant growth and development, alleviating oxidative stress and inducing defense responses. Using RNA-seq, we analyzed the physiological and transcriptomic responses of rice to cold stress and Fe₂O₃ treatment. Under cold stress, the NPs elicited a strong antioxidant response-elevating superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities-which led to a marked reduction in oxidative damage, as shown by decreased ROS and MDA levels. Further, the NPs concurrently restored photosynthetic function and ameliorated cold-induced phenotypic damage. RNA-sequencing revealed that NPs application significantly alters a comprehensive transcriptomic reprogramming, enriching pathways for carbohydrate metabolism, photosystem, plant hormone signaling, and glutathione biosynthesis. Collectively, our findings establish that Fe₂O₃ nanoparticles ameliorate cold stress by preserving chloroplast structure, stomatal architecture, reduce oxidative stress marker, enhancing antioxidant defense system and stabilize photosystem, and providing a promising nanozyme-based approach for rice protection against cold induce damage. cold stress iron oxide nano-biotechnology rice oxidative stress RNA-seq ultrastructure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Rice ( Oryza sativa L) one of the most staple food crop widely grown in the world. Over the past century, global rice consumption increases due to increase per capita consumption and population growth ( 1 ). However, in the mountainous regions of the tropics and the temperate rice-growing zones, cold stress remains a significant limitation to enhancing rice production ( 2 , 3 ). Approximately 15 million hectares of rice-growing land in 24 countries are vulnerable to cold-induced crop damage ( 4 ). Rice subjected to cold stress exhibits symptoms such as yellowing, slow seedling growth, stunting, withering, reduced tillering, and ultimately diminished productivity, particularly in cold-sensitive varieties. Plant nano-bionics, an interdisciplinary field integrating nanotechnology with plant biology, augments plant function by embedding nanomaterials directly into tissues and organelles. This approach offers an influential strategy for engineering abiotic stress tolerance ( 5 ). Although the physiological effects of nanoparticles on plants are increasingly documented, however the influence on molecular processes remains poorly understood. This knowledge gap is critical to address in rice, a chilling-sensitive staple crop that feeds half the world, to reveal nanotechnology’s potential for engineering climate-resilient varieties ( 6 ). “Late spring coldness,” a recurrent climatic threat in China’s, double-season rice regions, inflicts severe damage on early seedlings ( 7 ). This stress cascade stunted growth, reduces tillering, and causes considerable yield losses, establishing low-temperature resilience as a critical research significance ( 8 ). To meet this challenge, we demonstrate that precisely engineered iron oxide nanoparticles can directly intervene in this stress cascade, offering a nano-bionics strategy to enhance rice chilling tolerance at the molecular level. Nano-agrochemicals have emerged as a promising strategy for enhancing global food security, offering advantages over conventional methods through their improved efficacy, reduced application requirements, and lower environmental toxicity ( 9 , 10 ). Different nanoparticles work as strong elicitors in agriculture, activating plant innate immunity to combat abiotic stress. For instance, silver and silver-silica nanoparticles enhance resistance by stimulating phenolic synthesis, boosting antioxidative enzyme activity, and upregulating systemic acquired resistance (SAR) genes ( 11 – 13 ). Evidence confirms that nanomaterials like Fe₂O₃, TiO₂, and carbon-based NPs can directly suppress pathogens and enhance plant growth. Iron oxide Fe 2 O 3 show increase in antioxidant enzymatic activity superoxide dismutase mimetic, scavenge hydrogen peroxide to water and molecular oxygen ( 14 ). Significant progress has been made toward understanding plant-nanoparticle interactions on physiological level in other cereal crops, but progress on rice molecular behavior toward iron oxide nanoparticle (Fe 2 O 3 ) is poorly understood. Therefore, RNA-Seq technology and gene expression profiling are powerful tools for identifying cold-tolerant genes in rice under abiotic stress, enabling comprehensive analysis of expression profiles, single-nucleotide polymorphisms (SNPs), and alternative splicing events ( 15 , 16 ). Transcriptomic analysis of two Indica rice genotypes, cold-tolerant and cold-sensitive revealed that cold-tolerant seedlings exhibit enhanced biological processes, including membrane transport, sucrose synthesis, hormone and Ca²⁺ signaling. In contrast, cold-sensitive seedlings primarily upregulate heat shock proteins and dehydrins in response to low-temperature (LT) stress ( 17 ). Additionally, transcriptome profiling of chilling-tolerant and chilling-sensitive rice genotypes highlighted the coordinated involvement of multiple regulatory pathways under LT conditions ( 18 ). These findings underscore the effectiveness of RNA-Seq in elucidating the molecular basis of cold stress tolerance in rice, which is crucial for sustaining its productivity. Elucidating the mechanisms by which iron oxide nanomaterials (Fe₂O₃) increase cold stress tolerance could optimize sustainable agricultural practices. However, existing research on Fe₂O₃ NPs primarily examines physiological and morphological responses. Therefore, this study was conducted to compare cold stress exposures and nanoparticle application of rice by performing morpho-logical, physiological, and transcriptomic analyses in rice, focusing on the response mechanism of GSH to scavenge ROS, photosystem, carbohydrate metabolism and plant hormones signal transduction. The specific objectives of the present study were to ( 1 ) investigate the influence of cold stress on the growth and photosynthetic pigments and carbon fixation ( 2 ) investigate the influence of cold stress and nanoparticles application on the ROS accumulation, antioxidant enzymatic activity and GSH pathway to scavenge rice ( 3 ) perform transcriptomic analysis to investigate the response of nanoparticles and cold stress on carbohydrate metabolism and plant hormones related genes. Materials and methods Preliminary screening and dose optimization Nanoparticles (Zno, Fe 2 O 3 , Tio 2 , and CeO 2 ) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. ( https://www.aladdinsci.com/ ), Shanghai China. The effectiveness of nanoparticles was pre-optimized on seedling stage on rice. Seeds of four rice cultivars (LLY-7108, LLY-32, XZX-06 and ZJZ-17) was provided by crop physiology and production center (CPPC), Huazhong Agriculture University (114.37 °E, 30.48 °N), Wuhan, China. In the preliminary experiment, the seed of each cultivar were grown in pots under normal condition. Foliar application of nanoparticles (Zno, Fe2O 3 , Tio 2 , and CeO 2 ) with different concentrations were tested under cold stress to optimize best level of NPs ( 19 ) (Text S1, Table S1 ). The selection of cold-sensitive and tolerant cultivars, best nanoparticles and best stage was based on second experiment (Text S2, Table S2 ). The cultivars were chosen to assess the effectiveness of foliar application of best nanoparticles on morphological and physiological responses to mitigate cold stress in rice. Plant materials and growth conditions Pot experiments were conducted using two early indica rice cultivars with contrasting cold tolerance: the cold-sensitive Zhongjiazao-17 (ZJZ-17), bred by the China National Rice Research Institute, and the cold-tolerant Lingliangyou-7108 (LLY-7108), developed by the Hunan Institute of Yahua Seed Industry ( 20 ). The present study was carried out in Huazhong Agricultural University, Wuhan, China. The experiment used pots (23 cm diameter, 15 cm height) filled with 5 kg of soil per pot. Nutrients were supplied by mixing 0.96 g nitrogen (N), 0.92 g phosphorus (P), and 112 g potassium (K) into the soil two days before seedling establishment. Soil samples were collected from the topsoil layer (0–20 cm depth) of the rice paddy experimental field. The soil had a pH of 7.1, organic matter content of 6.7 g kg − 1 , Olsen phosphorus of 6.27 mg kg − 1 , exchangeable potassium of 129 mg kg − 1 , and total nitrogen of 0.63%. Experimental design and treatments Rice ( Oryza sativa L.) seeds were soaked into ultrapure water at 30°C for two days. Each pot was sown with five seeds and maintained under standard growth conditions. To ensure uniform competition, seedlings were thinned to three plants per pot following germination. A foliar spray of Fe₂O₃ nanoparticles (50 mg/L) was applied three days prior to the induction of cold stress. The experiment treatments include one control (CK), one cold stress (CS) treatments groups and one Fe 2 O 3 . For chilling stress treatment, 14-day-old rice seedlings were exposed to low-temperature conditions (12 h light at 14°C and 12 h dark at 10°C) for five days. Following stress exposure, seedlings were sampled for phenotypic, physiological, biochemical, and molecular analyses. Three plants per treatment (three biological replicates) were used to measure plant height and fresh and dry biomass, while 3–6 plants per replicate, depending on sample weight requirements, were used for other physiological, biochemical, and molecular assessments. Iron oxide (FeO) NPs characterization The physicochemical characteristics of Fe 2 O 3 nanoparticles were determined using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Powder XRD analysis was performed using an XPERT-3 diffractometer operated at 40 kV and 40 mA. Diffraction patterns were recorded over a 2θ range of 10–80° with a step size of 0.01° and a scanning rate of 0.02° s − 1 , employing a monochromatized Cu-Kα radiation source (λ = 1.54178 Å). The size, morphology, and mesoporous structure of Fe 2 O 3 nanoparticles were examined using a JEOL-2100 transmission electron microscope, while surface morphology was further analyzed with a Nano Magnetic (hpSPMv1.5) atomic force microscope. Hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZS90 (Malvern Instruments) based on dynamic light scattering, following sonication of the nanoparticle suspension for 15 min to ensure complete dispersion. XPS analysis was conducted using a Kratos AXIS ULTRADLD system equipped with a monochromatic Al Kα X-ray source and a 165 mm mean-radius hemispherical energy analyzer, under ultra-high vacuum conditions (< 3 × 10 − 9 Torr). Dried Fe 2 O 3 nanoparticle samples were mounted on carbon tape, and spectral deconvolution and data analysis were performed using CasaXPS software (version 2.3.18, Casa Software Ltd.). Microscopic localization analysis of FeO nanoparticles Fe 2 O 3 was labeled using a 1,1’-octacoalkyl-3,3,3’, 3-tetramethylindole carbocyanine perchloric acid drought (Dil) fluorescent dye. Dil- Fe 2 O 3 was synthesized as follows: 0.8 mL 50mg/L Fe 2 O 3 and 7.2 mL of deionized water were mixed in a 50 mL glass beaker and stirred on a magnetic stirrer (500 rpm/min .24 µL Dil dye solution (2.5 mg / mL, dissolved in dimethyl sulfoxide (DMSO) was added to 176 ul DMsO solution to obtain Dil dye solution (0.3 mg /mL). The Dil dye solution was added to the Fe 2 O 3 solution drop by drop and stirred for 1 min at room temperature at 1000 rpm/min on a magnetic mixer, Transfer the mixed solution (Dil- Fe 2 O 3 ) into a 15 mL 10 kD ultrafiltration tube, add deionized water to the final volume of 15 mL, Dialyze in a beaker of ultra-pure water at 200 rpm/min for 24 h. Finally, the Dil- Fe 2 O 3 solution is obtained. Store in a 4℃. To verify the absorption and transport of Fe 2 O 3 in rice leaves inside chloroplast was cultured with fluorescent PSNPs for 3 h in dark. Leaves of rice seedlings were collected and cut in approximately 0.05 mm thick sections and each placed in a drop of distilled water and covered with a coverslip. The location and distribution of Fe 2 O 3 in rice leaves were observed using the confocal system FV3000 (Olympus, Tokyo, Japan). Growth attribute and photosynthetic pigment Three seedlings per treatment were randomly sampled, with three biological replicates. Plant height was measured, and fresh weight was determined immediately after washing. Dry weight was recorded after oven-drying the samples to a constant mass. The dry weight of the above ground parts was determined after oven-drying at 80°C to achieve a constant weight. Chlorophyll content in rice leaves was determined following the method described by ( 21 ). Fresh leaf tissue (0.5 g) was homogenized in 10 mL of 80% (v/v) acetone. The homogenates were incubated in the dark for 2 h and subsequently centrifuged at 12,000 rpm for 10 min. The absorbance of the supernatant was recorded at 663 and 645 nm using a spectrophotometer, and chlorophyll concentrations were calculated as previously described. Electron microscopy: SEM and TEM approaches Fresh rice leaf samples were cut into small segments and immediately fixed in 2.5% (v/v) glutaraldehyde prepared in 0.05 M phosphate buffer (pH 7.0) at 4°C for 12–24 h. After fixation, samples were rinsed three times with phosphate-buffered saline (PBS; pH 7.4) for 15 min each. Tissues were then post-fixed in 1% osmium tetroxide (OsO 4 ) prepared in 0.1 M phosphate buffer (pH 7.4) at 4°C for 1–2 h, washed again with PBS, and sputter-coated with platinum for 20 min. Surface morphology was examined using a scanning electron microscope (Hitachi High-Tech, Tokyo, Japan) following the protocol of Zheng et al. ( 22 ). For ultrastructural analysis, transmission electron microscopy (TEM) was conducted on Fe 2 O 3 -treated and untreated rice seedlings subjected to cold stress. Samples were post-fixed in 1% OsO 4 for 1 h, washed 2–3 times with 0.1 M PBS (pH 7.4), and dehydrated through a graded ethanol series (50–100%). After critical point drying, tissues were embedded in Spurr’s resin according to the manufacturer’s instructions and polymerized at 70°C for 9 h. Ultrathin sections (~ 80 nm) were cut, mounted on copper grids, and examined using a JEOL 2100F transmission electron microscope (USA) at ×10,000 magnification. Staining visualization of photo‑oxidative stress Superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) localization in leaves was visualized using nitro-blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) ( 23 ). For NBT staining, leaves from both LLY-7108 and ZJZ-17 cultivars subjected to 5-day cold stress were immersed in staining solution containing 500 mg NBT dissolved in 500 mL PBS (0.01 M, pH 7.8) and incubated overnight at room temperature. The stained leaves were subsequently rinsed in 70% ethanol at 60°C with three washing cycles before imaging. For DAB staining, leaves were incubated in staining solution consisting of 500 mg DAB dissolved in 500 mL PBS supplemented with 500 µL H₂O₂ and 50 µL HCl, followed by overnight incubation at 25°C in darkness. After staining, leaves were cleared in 60% ethanol at 60°C with three washing cycles, and staining patterns were documented photographically. All experiments included three biological replicates. ROS, lipid peroxidation, and antioxidant enzyme determination Reactive oxygen species (ROS), lipid peroxidation (malondialdehyde, MDA), proline and antioxidant enzyme activities were quantified following established protocols ( 24 ) with minor modifications. Briefly, 100 mg (ROS and proline) and 500 mg (MDA, and antioxidant enzyme activity) of leaf tissue from each treatment group was homogenized in 1 and 5 mL of ice-cold saline extraction buffer (100 mM potassium phosphate, pH 7.8), respectively. The homogenates were vortexed for 2 min and centrifuged at 1,150 × g for 10 min at 4°C. The supernatants were aliquoted for subsequent assays of H₂O₂, MDA, SOD, POD, and CAT. ROS and MDA concentrations were determined using commercial assay kits (G0112W-H₂O₂ and G0110W-MDA, respectively; Geruisi Biotechnology, Suzhou, China), with absorbance measured at 450 nm (ROS) and 450, 532/600 nm (MDA). RNA-seq Analysis Leaf samples were collected in triplicate from LLY-7108 and ZJZ-17 cultivars after combined cold stress and nanoparticle treatment for de novo transcriptome assembly via RNA-seq and subsequent qRT-PCR validation. The manufacturer’s guideline was used to extract total RNA from tissues by performing extraction with TRIzol® Reagent from Invitrogen based in the USA. The 5300 Bioanalyzer (Agilent) and ND-2000 (NanoDrop Technologies) tools were used to determine RNA quality along with measuring RNA quantities. A total of 18 RNA sequencing libraries were built with 1 µL of RNA from LLY-7108 and ZJZ-17 samples using Illumina® Stranded mRNA Prep and ligation kit (Illumina, San Diego, CA). The library preparation service was conducted by Majorbio Biological Technology Shanghai China using their standardized protocols, as described previously ( 25 ). The purification of sequencing data involved raw data filtering followed by error rate correction together with GC content distribution analysis. Clean reads obtained data which underwent alignment against ( O. sativa L. IRGSP-10; Ensembl Plants) reference genome. The RSEM program version v1.3.1 calculated gene transcription levels by generating log 2 Fc value results. The DESeq R program version 1.24.0 found DEGs through analysis between normal LLY-7108 and ZJZ-17 leaf pairs (5 d), cold stress leaf pairs and Fe 2 O 3 application. The DEGs were identified through the pair-wise analysis by applying FDR < 0.05 threshold and fold change as the selection criteria. The Diamond software (v0.9.24) performed functional analysis of DEGs through database comparisons with NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG. Further details about DEGs could be uncovered through KEGG database enrichment tests. An adjusted P-value < 0.05 was used to determine significantly enriched GO terms and KEGG pathways, with the analysis performed via the Majorbio platform ( www.majorbio.com ). qPCR for transcriptomic data validation The RNA was extracted using RNAprep Pure Plant Kit (DP441, Tiangen, Beijing, China). 2 µg of total RNA was reversely transcribed into cDNA using the TRUEscript first Strand cDNA Synthesis Kit (PC5402, Aidlab, Beijing, China). The amplification of qRT-PCR products was performed in a reaction mixture of 12.5 µL SYBR Green qPCR Mix (PC3302, Aidlab, Beijing, China) according to the manufacturer’s instructions. The qRT-PCR analysis was performed on the Bio-Rad CFX Connect Real-Time PCR System (Bio-Rad, California, USA). Three technical replicates were used for each investigated gene. The relative gene expression was calculated using the 2 −ΔΔCt method. The primers used for qRT-PCR are shown in Additional file Table S3 . Identification of co-expression network and hub genes Weighted gene co-expression network analysis (WGCNA) was performed to investigate the relationships between gene expression patterns and phenotypic traits. In the scale-free weighted gene co-expression network, nodes represent differentially expressed genes (DEGs), while edges denote pairwise connections defined by Pearson correlation coefficients between gene expression profiles. A soft-thresholding power (β = 9) was selected according to the scale-free topology criterion and used to construct the adjacency matrix. Network construction and module detection were carried out using a signed network with the following parameters: minimum module size of 30, minimum module eigengene-based connectivity (kME) of 0.3, and a merge cut height of 0.25. Hub genes within each module were identified based on intramodular connectivity using visual network analysis with default settings (connectivity threshold = 30; weight value = 0.02). In the resulting networks, each node represents a gene, with node size proportional to the number of connections, such that highly connected genes were considered hub genes due to their central regulatory roles. Associations between gene modules and phenotypic traits were further assessed by integrating module eigengenes with phenotypic data to calculate gene and module significance. Statistical analysis The experiment was conducted using randomized complete block design with three biological replicates per treatment. Quantitative data for chlorophyll content, reactive oxygen species (ROS), antioxidant enzyme activities, and gene expression were analyzed for statistical significance using two-way analysis of variance (ANOVA) at P ≤ 0.05. Post hoc comparisons were performed using Tukey’s multiple range test to identify significant differences among treatments. Principal component analysis (PCA) was employed to examine the relationships between treatments and the control. Results Nanoparticles Fe 2 O 3 characterization, localization and mechanisms of transport to chloroplasts The X-ray diffraction (XRD) analysis reveals single-phase, crystalline structure of iron oxide nanoparticles (Fig. 1a). In the XRD diffractogram, the characteristic peaks of the samples are at 2θ = 29.7◦, 35.50◦, 43.25◦, 57.56◦, 57.14◦, and 64.73◦. The DLS size of Fe 2 O 3 nanoparticles is 10 nm with the zeta potential of -24.3 mV (Fig. 1b). The delivery of Fe 2 O 3 nanoparticles to leaf mesophyll chloroplasts was performed by a simple method of infiltration through the stomata pores into the leaf lamina (Fig. 1c). An iron oxide nanoparticle concentration of 50 mg L − 1 was selected for leaf infiltration. For confocal imaging of nanoparticle localization in leaf mesophyll cells, Fe₂O₃ nanoparticles were labeled with the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), while chloroplast autofluorescence was used to visualize photosynthetic pigments (Fig. 1d?). Confocal z-stack images were acquired from leaf mesophyll cells at a 2 µm optical step size following 3 h of dark incubation after leaf infiltration. The fluorescence signals revealed clear colocalization of iron oxide nanoparticles with chloroplasts (Fig. 1e). Iron oxide appears to move rapidly from leaf cell extracellular spaces, through mesophyll cell walls and plasma membranes into chloroplasts. The two varieties show different localization of iron oxide in chloroplast (Fig. 1f). We propose that the observed differences in nanoparticle colocalization are partly attributable to electrostatic interactions between the nanoparticle ζ-potential and the plasma membrane potential. The outer surface of the plasma membrane carries a net positive charge, which favors the association and uptake of anionic nanoparticles. Figure 1. Nanoparticle characterization i.e. ( a ) XRD-pattern, ( b ) DLS size and zeta potential, ( c ) Transmission electron microscopy (TEM), ( d ) Schematic representation of nanoparticle movement in plants. Fe 2 O 3 nanoparticles are delivered into the leaf via stomatal infiltration (step 1) and subsequently migrate through the mesophyll cell wall matrix (step 2). The nanoparticles then associate with the outer surface of the mesophyll cell plasma membrane, where electrostatic interactions between the negatively charged Fe 2 O 3 nanoparticles and the positively charged membrane surface facilitate binding (step 3). Internalization and subsequent transport of Fe 2 O 3 nanoparticles into the cytosol and chloroplasts occur via a non-endocytotic pathway and are influenced by the plasma membrane potential (step 4), ( e ) localization of Fe 2 O 3 in chloroplast and schematic representation of Fe 2 O 3 transport inside the chloroplast ( f ) Translocation intensity in two cultivars. Growth parameters and photosynthetic pigments of LLY-7108 and ZJZ-17 under cold stress and nanoparticles application Cold stress induced significant changes in leaf phenotypes and growth, particularly in the ZJZ-17 cultivar compared to LLY-7108. Iron oxide nanoparticles (Fe₂O₃ NPs) significantly increased plant growth and photosynthetic pigments under cold stress. Cold stress reduced plant height by 39.2% in LLY-7108 and 41.0% in ZJZ-17 relative to their respective controls (CK). Foliar application of Fe₂O₃ nanoparticles significantly mitigated this reduction, most notably in LLY-7108, where the decrease was lowered to 20.7% (Fig. 2 a). Foliar application of iron oxide nanoparticles significantly mitigated cold-induced fresh weight loss in both rice varieties. While cold stress alone reduced fresh weight by 51.5% in LLY-7108 and 64.9% in ZJZ-17, nanoparticle treatment dramatically lessened this deficit to 20.7% in ZJZ-17 (Fig. 2 b). In LLY-7108, dry weight of rice seedling due to cold stress is reduce (41.2%) as compared to CK (normal temperature) (Fig. 2 c), while this reduction in susceptible variety (ZJZ-17) is 50.5%. Application of iron oxide nanoparticles to rice leaves reduce this decrease in dry weight due to cold stress in both cultivars. The photosynthetic pigments (chlorophyll a, chlorophyll b, and total chlorophyll) were significantly influenced by cold stress and nanoparticle application. After 5 days of cold stress, leaf yellowing was most severe in ZJZ-17 and mildest in LLY-7108, correlating with significant differences in chlorophyll loss between the two cultivars. Specifically, chlorophyll a content in LLY-7108 and ZJZ-17 leaves under cold stress decreased by 43.1%, and 51.8%, respectively, compared to CK (normal temperature) leaves (Fig. 2 d). Likewise, chlorophyll b levels were approximately 38.97% and 48.1% lower as compare to control temperature, while the nanoparticles application reduce the damage to photosynthetic pigment due to cold stress (Fig. 2 d-f). The reduction in total chlorophyll content is almost 41.2% and 50.1% in LLY-7108 and ZJZ-17 respectively (Fig. 2 f). In term of cultivars the LLY-7108 perform best as compare to ZJZ-17 under cold stress and cold stress + nanoparticles application. Effect of Fe 2 O 3 NPs on stomatal traits and chloroplast ultrastructure Fe₂O₃ nanoparticle treatment counteracted cold stress-induced stomatal damage, preserving stomatal structure (index, length, width) and mitigating functional impairment to gas exchange (Fig. 3 a-f). These findings reveal that Fe₂O₃ NPs mitigate cold stress damage to stomata by modulating guard cell ROS and Ca²⁺ signaling, thereby supporting water relations, protecting chloroplast integrity, and preserving photosynthetic function. Cold stress (CS) significantly reduced stomatal length and width in both rice cultivars (LLY-7108 and ZJZ-17) (Fig. 3 g-h). The smallest stomata were observed under CS, indicating impaired stomatal development. Application of Fe₂O₃ nanoparticles under CS significantly increased both stomatal length and width compared with CS alone, partially restoring values toward the control (CK). Overall, LLY-7108 exhibited longer and wider stomata than LLY-7108 across treatments, suggesting greater tolerance to cold stress. Transmission electron microscopy (TEM) analysis revealed pronounced ultrastructural alterations in plant cells under cold stress, indicating its deleterious effects on cellular integrity, including structural deformation and a reduction in mesophyll cell size. Cold stress induced pronounced chloroplast damage, characterized by autophagic-like degradation, shrinkage, and severe structural deformation (Fig. 4 ). Affected chloroplasts exhibited disrupted grana stacks, disorganized stroma lamellae, and enhanced plastoglobule accumulation In contrast, Fe₂O₃ nanoparticle treatment under cold stress markedly alleviated these ultrastructural impairments, preserving chloroplast size and overall structural integrity. Fe₂O₃ NP-treated leaves displayed well-organized grana and stroma lamellae with clearly defined dense layers, along with increased plastoglobule formation. Collectively, these results indicate that Fe₂O₃ nanoparticles promote the maintenance and regeneration of mesophyll cell and chloroplast architecture, thereby enhancing rice tolerance to cold stress (Fig. 4 ). Transcriptomic profiling of LLY-7108 and ZJZ-17 under cold stress A comprehensive analysis was performed on 18 samples to investigate the response of LLY-7108 and ZJZ-17 under cold stress. Following read refinement and adapter trimming, 206.04 Gb of clean data was generated (Q20 > 98.83%; Q30 > 96.17%) (Table S5). A PCA based on log 2 |(foldchange)|≥ 1 from each library revealed distinct variations between the treatments under cold stress. Principal components PC1 and PC2 explained approximately 71.85% of the total variation (Fig. 5 a). A total of 28504 genes were identified, with transcription levels log10 (FPKM + 1) in both cultivars ranging from − 2 to 5. Comparisons of gene expression revealed 13134 (treatment vs control), 2,336 (Foliar vs control), and 13,009 (Foliar vs treatment) genes (Fig. 5 b). In total, 428 and 470 DEGs were detected in LLY-7108 and ZJZ-17 under cold stress, respectively, while under cold stress + nanoparticle application in total, 357 and 202 DEGs were detected in LLY-7108 and ZJZ-17, with 14191 DEGs common to both cultivars across all the treatments (Fig. 5 c). Volcano plot analysis (Fig. 5 d–f) showed extensive transcriptional changes under cold stress, with a large number of significantly up- and downregulated genes in the CK vs CS comparison. In contrast, Fe₂O₃ nanoparticle treatment reduced the number of cold stress–induced DEGs, indicating mitigation of stress-related transcriptional disruption. However, the Fe₂O₃ + CS comparison still displayed pronounced differential expression, suggesting active nanoparticle-mediated regulation of cold-responsive genes. Functional annotation and enrichment analysis of differentially expressed genes (DEGs) The transcriptional profiles of differentially expressed genes (DEGs) in the rice cultivars LLY-7108 and ZJZ-17 were functionally annotated using six major databases, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), EggNOG, NR, Swiss-Prot, and Pfam, to elucidate their biological roles. Sequence alignment against the GO (26,853), KEGG (12,014), EggNOG (28,670), NR (31,961), Swiss-Prot (23,714), and Pfam (20,905) databases yielded robust functional annotations for the majority of DEGs, confirming their involvement in well-characterized biological processes. GO and KEGG enrichment analyses revealed significant enrichment of DEGs in both cultivars across all treatments. GO analysis showed predominant enrichment in biological processes such as “cellular process” (GO:0009987) and “metabolic process” (GO:0008152) (Table S6, Figure S1 ). KEGG pathway analysis further indicated significant enrichment in pathways related to carbohydrate metabolism, glutathione biosynthesis, plant hormone signal transduction, and photosynthesis antenna proteins (Figure S2 ). Moreover, GO enrichment analysis highlighted glutathione metabolism as a significantly enriched biological process under both cold stress and nanoparticle treatments. KEGG pathway enrichment of cultivar-specific DEGs revealed predominant enrichment in photosynthesis antenna proteins (map00196), starch and sucrose metabolism (map00500), plant hormone signal transduction (map04075), and glutathione metabolism (map00480) in both LLY-7108 and ZJZ-17 (Fig. 6). Figure 6. Overview of transcriptomic analysis in LLY-7108 and ZJZ-17 leaves under cold stress. GO slim–based functional categorization and pathway enrichment analysis under different treatments. (a) CK, (b) Fe₂O₃ nanoparticle treatment, and (c) cold stress. Bubble size indicates the number of genes enriched in each KEGG pathway, while color intensity represents the level of statistical significance according to the scale bar. Corrected P values < 0.05 were considered statistically significant for both GO and KEGG enrichment analyses. KEGG pathways of rice leaves of different treatments i.e. CK (d), Fe 2 O 3 (e) and CS (f). The size of the bubble represents the number of genes in the KEGG pathway, and color intensity corresponds to different P-value ranges, as indicated by the scale bar. Lipid peroxidation, ROS accumulation, and antioxidant responses under cold stress and nanoparticles application The accumulation of ROS, MDA, and antioxidants was significantly influenced by the cold stress and nanoparticles application. Thus, the transcription patterns of antioxidant-related genes in LLY-7108 and ZJZ-17 under cold stress and nanoparticles application were examined. In the cold stress and nanoparticles application, rice leaves were further assessed using NBT and DAB histochemical staining (Fig. 7 a). Notable accumulation of superoxide radicals was observed in ZJZ-17 as compared to LLY-7108. Likewise, DAB staining revealed a substantial increase in H 2 O 2 in ZJZ-17 as compared to LLY-7108. Overall, the enhanced activities of enzymatic antioxidants in LLY-7108 led to a reduction in ROS and MDA, suggesting that LLY-7108 exhibited greater cold stress tolerance than the ZJZ-17 cultivar. In physiological examination of lipid peroxidation, ROS, and proline revealed higher concentration in cold stress treated plants, in contrast the ROS, MDA, and proline content reduce with nanoparticles application under cold stress. The increase in ROS in LLY-7108 due to cold stress is 3.43-fold as compared to CK (normal temperature), while this increase in ROS under nanoparticles application is 1.96-fold (Fig. 7 b). In ZJZ-17 the increase in ROS content is 4.88-fold due to cold stress as compared to CK (normal temperature), while the nanoparticles application reduces the ROS content by 40.4% as compared to cold stress. Similarly, MDA and proline levels showed significant increases over the course of cold stress, with MDA and proline content significantly higher in ZJZ-17 than in LLY-7108 of cold stress (Fig. 7 c-d). The increase in MDA and proline content in LLY-7108 due to cold stress is 2.27 and 2.17-fold, respectively as compared to CK (normal temperature), while this increase in MDA and proline under nanoparticles application is 1.47 and 1.28-fold (Fig. 7 c-d). To understand the changes in ROS levels and homeostasis, the activities of SOD, POD, and CAT were investigated, consistent with the GSH pathway. The antioxidant enzymatic activity i.e. SOD, POD and CAT were significantly influence by cold stress and nanoparticles application. The activity of these enzymatic antioxidant was at lowest in the crop under cold stress, while nanoparticles application increases these anti-oxidants enzymatic activity (Fig. 7 e-f). The decrease in SOD, POD and CAT activity in LLY-7108 due to cold stress is 45.8%, 43.2% and 30.7%, respectively as compared to CK (normal temperature), while Fe 2 O 3 nanoparticle application increase the antioxidant enzyme inside the plants by 52.1%, 38.1% and 26.9%, respectively as compared to CS (cold stress) (Fig. 7 e-f). In ZJZ-17 this decrease in SOD, POD and CAT activity due to cold stress is 56.8%, 54.2% and 44.3%, respectively as compared to CK (normal temperature) (Fig. 7 e-g). In GSH pathway under cold stress the expression of gene Os07g0462000 , Os12g0263000 and Os06g0232650 is lower, while under CK (normal temperature) and Fe 2 O 3 nanoparticles application the expression of these genes is high, which indicate to lower the accumulation of ROS and reduce membrane damage due to cold stress (Fig. 7 h). GSH participates in ROS detoxification through glutathione peroxidase, generating GSSG, which is then reduced back to GSH by OsGR1 . GSH catabolism through GGT1 and OsARP contributes to the γ-glutamyl cycle. OsDHAR1/2 contribute to the ascorbate–glutathione cycle. Heatmap beside each gene encode differential expression across treatments and cultivars, showing stronger induction of antioxidant-related genes in the tolerant cultivar, particularly with Fe₂O₃-NP treatment. The comparison of both cultivars revealed the best performance of LLY-7108 across all the treatments as compared to ZJZ-17 (Fig. 7 h). Effect of Fe 2 O 3 NPs foliar application on Photosynthesis, photosystem stabilization and genes expression Excessive ROS accumulation under cold stress damage chloroplast and photosynthetic machinery such as the D1 protein, oxygen evolving complex in PSII, thylakoid membrane lipids, and chloroplast DNA. Therefore, we assessed the effect of cold stress on photosynthetic parameters in leaves treated with Fe 2 O 3 NPs application. Cold stress significantly reduces the photosynthesis rate, stomatal conductance and internal CO 2 concentration, while Fe 2 O 3 NPs foliar application increase these parameters (Fig. 8 c-e). To clarify the mechanisms of cold stress tolerance, photosynthesis pathway was found significantly enriched (p ≤ 0.05) under cold stress and nanoparticles application. To elucidate the underlying mechanisms of photosynthesis alteration under cold stress, 39 DEGs related photosystem II (PSII), photosystem I (PSI), electron transport chain and F-type ATPase were identified in both LLY-7108 and ZJZ-17 leaves (Fig. 8 a). In the photosystem II (18 DEGs), photosystem I (9 DEGs), electron transport chain (9 DEGs), Cytochrome b6f complex and F-type ATPase (2 DEGs) were found upregulated. The heatmap analysis of photosynthesis-related genes revealed distinct transcriptional responses across treatments and between the tolerant (V₁) and susceptible (V₂) rice cultivars. Most Photosystem II (PSII) genes, including PsbA , PsbB , PsbC , PsbD , and corresponding nuclear-encoded homologs, were markedly downregulated under cold stress in both cultivars, with a stronger suppression observed in the susceptible genotype. However, Fe₂O₃ nanoparticle treatment partially restored the expression of several PSII components in V₁, indicating enhanced stability of the light-harvesting and reaction-center complexes under stress. A similar trend was observed for Photosystem I (PSI) genes (e.g., PsaA , PsaB , PsaD , and PsaF ), where cold stress sharply reduced expression, especially in ZJZ-17, whereas Fe₂O₃ treatment led to moderate upregulation or recovery in LLY-7108 but not in ZJZ-17, suggesting better preservation of PSI-mediated electron flow in the tolerant cultivar. Genes associated with the cytochrome b6f complex ( PetB , PetC , PetD , and PetF ) and photosynthetic electron transport (PC, Fd family) also showed significant downregulation under cold stress, with the decline being more severe in the susceptible cultivar. Fe₂O₃ nanoparticles again mitigated this reduction in LLY-7108, maintaining higher transcription levels of key electron carriers and supporting more efficient plastoquinone-cytochrome electron transfer. Similarly, genes encoding the F-type ATP synthase subunits ( AtpA , AtpB , AtpF , and AtpH ) exhibited strong repression under cold stress, particularly in ZJZ-17. Notably, Fe₂O₃-treated LLY-7108 plants showed enhanced or recovered expression of several ATP synthase subunits, indicating improved capacity for ATP generation under low-temperature conditions. Overall, the results demonstrate that cold stress impairs the transcription of core photosynthetic machinery in rice, with the susceptible genotype exhibiting the strongest decline. In contrast, Fe 2 O 3 nanoparticles significantly alleviate the cold-induced suppression of photosynthetic genes in the tolerant cultivar, maintaining the integrity of PSII, PSI, cytochrome b6f, electron transport components, and ATP synthase complexes, thereby supporting more efficient photosynthetic performance under stress. After analyzing the transcriptome photosynthetic carbon fixation pathway in photosynthetic organisms, significantly influenced by cold stress and nanoparticles application (Fig. 8 b). Studies on the differentially expressed genes related to the photosynthetic carbon sequestration pathway under cold stress showed that the expression of Os03g0432100 , Os01g0723400 , Os01g0110700 and Os01g0723400 were upregulated in nanoparticle-treated plant and CK, while cold-stressed plants have lower expression of these genes. In sum, it showed us that the carbon fixation in plants under normal temperature and nanoparticles application is more as compare to cold stress . Carbohydrate metabolism KEGG pathway analysis revealed that the most significantly enriched processes were starch/sucrose metabolism, glycolysis, and the TCA cycle, highlighting a concerted rerouting of carbon for energy production under combined cold and nanoparticle treatment (Fig. 9 ). Assessment of carbohydrate metabolism demonstrated clear transcriptional reprogramming across starch and sucrose metabolism, glycolysis, and the TCA cycle in both ‘LLY-7108’ and ‘ZJZ-17’ under cold stress and Fe₂O₃ nanoparticle treatments compared with the CK (normal temperature) (Fig. 9 a-d). In the starch and sucrose metabolism pathway (Fig. 9 b), cold stress substantially increased several key genes, such as INV , HK , and SPP -as reflected by the dominant blue color patterns in both cultivars to maintain the cell energy level. In contrast, Fe₂O₃ nanoparticle application (Fe₂O₃) partially restored the expression of sucrose-cleaving and starch-biosynthetic genes, including SUS , GBE , WAXY , and glgA . This enhancement was notably stronger in the tolerant cultivar ‘LLY-7108’, suggesting a more efficient carbon assimilation response under stress when supplemented with nanoparticles. Additionally, genes involved in sucrose phosphate cycling ( SPS and SPP ) and UDP-glucose interconversion exhibited moderate transcriptional activation following Fe₂O₃ treatment, indicating nanoparticle-mediated stabilization of sucrose metabolism. In glycolysis (Fig. 9 c), cold stress caused significant expression of rate-limiting glycolytic enzymes including PFK , ALDO , GAPDH , and ENO particularly in the susceptible cultivar ‘ZJZ-17’. Conversely, Fe₂O₃ treatments restore the expression of multiple glycolytic genes in both cultivars, with a stronger recovery in ‘LLY-7108’. Increased expression of PGK , GAPDH , ENO , PK , and PDHB under Fe₂O₃ + cold conditions indicate enhanced carbon flux from hexose phosphates toward pyruvate formation. The upregulation of ACSS, DLAT , and ALDH7A1 under cold stress treatment further suggests improved acetyl-CoA formation, helping maintain energy metabolism under cold stress. The transcriptional profiles of TCA cycle related genes (Fig. 9 d) exhibited a comparable trend, wherein cold stress alone led to the up-regulation of key dehydrogenases and core cycle enzymes, including MDH, fumC, SDH4 , and DLST . In contrast, Fe₂O₃ nanoparticle treatment markedly restores the expression of ACO , IDH , OGDH , and CS/ACLY in both cultivars, with a more pronounced induction observed in the tolerant ‘LLY-7108’. This upregulation indicates that nanoparticle application may stimulate mitochondrial function and help sustain TCA cycle flux under low-temperature stress. Furthermore, elevated expression levels of LSC2 and OGDH support enhanced NADH production, which likely contributes to improved ATP synthesis and overall metabolic resilience. Overall, the integrated pathway analysis demonstrates that cold stress markedly impairs carbohydrate metabolism in both rice cultivars; however, Fe₂O₃ nanoparticle application effectively alleviates these disruptions by maintaining the expression of key genes associated with sucrose metabolism, glycolysis, and the TCA cycle. The tolerant cultivar ‘LLY-7108’ exhibits consistently stronger transcriptional recovery and metabolic stabilization compared with the susceptible ‘ZJZ-17’, highlighting a cultivar-specific improvement in metabolic homeostasis in response to nanoparticle supplementation under cold stress conditions. Plant hormones signal transduction pathways during cold stress and nanoparticles application Plant hormonal response is the first line of defense against abiotic stress; plant hormones control abiotic stress response by altering transcriptional programs. The plant hormone signaling transduction network depicted in the Fig. 10 provides a comprehensive representation of the molecular interactions and transcriptional regulation involved in coordinating plant development and stress responses. To explain the mechanisms of signal transduction via plant hormones in rice seedlings under cold stress and nanoparticles application, the KEGG pathway (plant hormones) was observed under different treatments. An analysis of the expression patterns of DEGs associated with the plant hormone signal transduction pathway of the rice revealed the identification of 14 DEGs in rice leaves under cold stress and nanoparticles application (Fig. 10 ). These genes were found to be enriched in five distinct hormonal signaling pathways, including IAA, gibberellin (GA), ABA, Cytokinin and JA. The highest count of DEGs were involved in the Indole-3-acetic acid (IAA), Abscisic acid (ABA) and Gibberellin (GA) signaling pathways. Auxin signaling, mediated through key components such as OsLAX3 , OsIAA16 , and OsGH3-7 , is primarily associated with cell enlargement and vegetative growth. The OsLAX3 , OsIAA16 , and OsGH3-7 genes were found in higher expression inside the IAA pathway across control and nanoparticle application as compare to cold stress (Fig. 10 a), in the tolerant cultivar, indicating a better maintenance of auxin-mediated cell elongation during stress. In parallel, cytokinin phosphotransferase protein AHP1 and response regulator ORR3 were upregulated, stimulating meristematic activity and shoot initiation (Fig. 10 b). The GA pathway shows changes primarily at DELLA -associated regulatory genes. Genes such as OsGID1 , OsPIFs ( OsPIF11 , OsPIF14 , and OsPIF12 ), and DELLA repressors exhibit mixed but treatment-specific responses. Fe₂O₃ nanoparticles enhance GA-related expression more strongly in tolerant cultivar, consistent with improved GA-mediated stem elongation and stress compensation (Fig. 10 c). ABA biosynthesis genes ( OsPYL and OsPYR ) and ABA-activated MAP kinases ( SAPK1 , SAPK2 , SAPK6 , SAPK9 , and SAPK10 ) show substantial induction under cold stress, with Fe₂O₃-NPs amplifying several signals. The tolerant cultivar shows a markedly stronger activation of ABA receptor genes and signaling nodes, indicating more robust stomatal control and stress adaptation capacity than the susceptible cultivar (Fig. 10 d). JA biosynthesis and signaling genes, including AOS , COI1b, JAZ repressors, and MYC2 , show strong modulation under cold stress and nanoparticle treatments. In particular, OsJAZ2 , OsJAZ8 , and OsbHLH009 show cultivar-dependent differences, with tolerant cultivar showing stronger upregulation. Activation of the JA pathway leads to induction of ORCA3 , a major regulator of stress-responsive genes. The expression patterns indicate that JA-mediated defense signaling is significantly more active in the tolerant cultivar during cold stress, and further enhanced by Fe₂O₃ nanoparticles application. Together, these transcriptional adjustments underscore a multifaceted hormonal crosstalk that balances enhanced growth with adaptive stress responses under cold stress condition due to Fe 2 O 3 nanoparticles application. Validation of RNA-Seq Analysis by qRT-PCR. Quantitative real-time PCR (qPCR) analysis was performed to validate the RNA-seq expression profiles of selected cold-responsive genes, including psbP , psbQ , PSBW , OsLAX3 , OsIAA16 , and OsCOI1b . The qPCR expression trends were highly consistent with the FPKM values obtained from RNA-seq data across treatments, confirming the reliability and accuracy of the transcriptomic analysis. Cold stress markedly downregulated photosynthesis-related genes, whereas Fe₂O₃ nanoparticle application significantly restored or enhanced their expression in both cultivars. Similarly, genes involved in hormone signaling and stress responses exhibited concordant expression patterns between qPCR and RNA-seq datasets, supporting the robustness of the sequencing results (Figure S4). Profiling of hub DEGs involved in cold tolerance under nanoparticles application To assess sample correlations and the relationships between gene modules and co-expressed genes, weighted gene co-expression network analysis (WGCNA) was performed on differentially expressed genes (DEGs identified under cold stress and nanoparticle foliar application. After removing unannotated DEGs, seven distinct modules comprising 16,250 highly co-expressed genes were identified (Table S7; Figure S4). Among these, the green, magenta, and turquoise modules exhibited negative correlations with reactive oxygen species (ROS), proline, and MDA contents, but showed significant positive correlations with antioxidant enzyme activities and photosynthetic parameters (Fig. 11 a-b). For network construction, 31 hub genes from the green module were selected; these hub genes displayed high intramodular connectivity (kME) and were extensively connected within the interaction network. Similarly, in the magenta module, correlation coefficients ranged from 0.0227 under control conditions to − 0.341 under cold stress, with 29 hub genes showing strong interactions within the network. In the turquoise module, correlation coefficients ranged from 0.681 under nanoparticle foliar application to − 0.818 under cold stress, and 29 hub genes exhibited extensive connectivity within the interaction network (Fig. 11 c-e). Discussion Cold stress is a primary limiter of rice productivity, a legacy of the crop’s tropical origins that renders seedlings especially vulnerable. Understanding the genetic and physiological mechanisms of cold adaptation is therefore crucial. Utilizing a plant nano-bionics approach, our study reveals that nanoparticle application enhances cold tolerance by activating key pathways. We pinpoint the reduction of oxidative damage alongside the modulation of antioxidants, phytohormones, and photosynthetic antenna proteins as central to this fortified response in LLY-7108 and ZJZ-17 rice varieties. Fe₂O₃ NPs enhance chilling resilience by bolstering antioxidant systems and preserving photosynthetic function Chloroplasts and mitochondria are the primary sites of hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 − ) production during abiotic stress conditions ( 26 ). Excessive accumulation of ROS induces lipid peroxidation, mainly through the oxidation of unsaturated fatty acids, leading to the formation of toxic aldehydes such as MDA. In the present study, cold stress caused severe damage to ZJZ-17 leaves, as evidenced by reduced cell viability and significantly elevated ROS and MDA levels (Fig. 7 a–b). These results indicate that cold stress enhanced ROS production, disrupted membrane integrity, and exacerbated leaf injury in this cold-sensitive cultivar. In contrast, LLY-7108 exhibited comparatively lower H 2 O 2 and MDA accumulation, suggesting the presence of a more efficient ROS scavenging system and enhanced membrane stability. Genotype-dependent variation in MDA and proline accumulation under low-temperature stress has been widely reported, reflecting differences in chilling tolerance among cultivars. Previous studies demonstrated that nanoparticle applications, particularly TiO₂ nanoparticles, improve electrolyte leakage, photosynthetic performance, and membrane integrity under cold stress through transcriptional regulation in chickpea ( 27 – 29 ). Hasanpour et al. ( 30 ) suggest that TiO 2 NPs application to plants increase the tolerance of plants to cold stress due to controlling the pressure of the temperature drop injury and altered metabolism for plant growth. The harmful effects of cold stress are reduced and glycyrrhizin content is enhanced when using TiO 2 NPs in licorice plants ( 31 ). The use of chitosan nanoparticles was found to be effective in reducing the ROS with the accumulation of osmoprotectants in plants under cold-stress conditions( 32 ). Furthermore, in rice plants, the foliar application of ZnO NPs may reduce cold stress through the antioxidative system and transcription factors involved in the chilling response( 33 ). Consistent with these reports, both rice cultivars in the present study exhibited significant increases in MDA and proline under cold stress; however, the cold-sensitive cultivar ZJZ-17 accumulated substantially higher levels of ROS, MDA, and proline than LLY-7108. Importantly, Fe₂O₃ nanoparticle application markedly reduced ROS and MDA accumulation under cold stress, highlighting their role in enhancing antioxidative defense capacity. This protective effect may be attributed to the provision of bioavailable iron, which is essential for the activity of antioxidative enzymes and associated metabolic processes ( 34 ). Collectively, ROS, MDA, and proline profiling supports the classification of LLY-7108 as a cold-tolerant cultivar and ZJZ-17 as cold-sensitive. Antioxidant enzymes are essential for scavenging ROS and mitigating lipid peroxidation ( 35 ). Increased activities of SOD, POD, and CAT following nanoparticle application under cold stress have been reported in rice ( 33 ). In the present study, LLY-7108 exhibited significantly enhanced SOD, POD, and CAT activities, along with upregulation of glutathione (GSH) pathway-related genes. These enzymatic antioxidants efficiently scavenge H₂O₂ and O₂⁻ across multiple cellular compartments, thereby maintaining redox homeostasis ( 36 ). Enhanced antioxidant capacity in LLY-7108 effectively mitigated oxidative damage and conferred improved cold tolerance (Fig. 7 e–h), in agreement with earlier findings in rice ( 33 ). Influence of Fe₂O₃ NPs on chloroplast ultrastructure and photosystem stabilization under cold stress Foliar application of Fe₂O₃ NPs preserved chloroplast ultrastructure under cold stress, as evidenced by the maintenance of well-organized grana stacks, intact stroma lamellae, and plastoglobule integrity, indicating effective mitigation of cold-induced photooxidative damage ( 37 ). Enhanced chloroplast structural stability facilitated efficient light harvesting and sustained photosynthetic performance, thereby counteracting cold-mediated impairment of leaf photosynthesis. These observations are consistent with reports in lettuce, where carbon-based nanoparticles safeguarded chloroplast membranes under stress by stabilizing thylakoid architecture, reinforcing lipid peroxidation defense systems, and maintaining photosystem II functionality, ultimately preserving chloroplast integrity and photosynthetic efficiency ( 38 ). Furthermore, increased chlorophyll contents (chlorophyll a, b, and carotenoids) reflect an improved light-harvesting capacity, in agreement with findings in rice exposed to nanoparticle treatments under cold stress conditions ( 39 ). Cold stress has been reported to decrease PSII abundance, electron flow, and Rubisco activity ( 40 ). Our results indicate that cold stress markedly reduce photosynthesis rate, internal CO 2 concentration and stomatal conductance while NPs application increase this parameter significantly (Fig. 8 c-e). Plant ROS are mainly produced by chloroplasts, mitochondria, peroxisomes, NADPH oxidases, and class III peroxidases ( 41 ). Among these, chloroplasts are a source of hydroxyl radical production in leaves under stress conditions ( 41 , 42 ). The iron oxide NPS with large surface to volume ratios catalytically scavenges ROS produced by the chloroplasts such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, the most destructive ROS in plant cells. Cold stress cause oxidative damage to chloroplast components inhibiting the repair of PSII, the most temperature-sensitive component of the photosynthetic apparatus ( 5 ). Iron oxide NPs protect both the light and carbon reactions of photosynthesis from ROS damage in plants under stress condition. Cold stress and nanoparticle treatments also significantly influenced photosynthesis antenna proteins and carbon fixation pathways in rice. Genes associated with photosynthesis antenna proteins and carbon fixation exhibited higher expression levels under normal temperature (CK) and nanoparticle treatments compared with cold stress. Cold-induced lipid modifications compromise membrane integrity, affecting essential physiological processes such as photosynthesis, gas exchange, and transpiration. Reduced CO₂ assimilation and chlorophyll content under cold stress are often linked to inhibition of the large subunit and degradation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) ( 8 ). This is also due to the ROS accumulation in chloroplasts, damage the photosynthetic apparatus, particularly PSII, leading to imbalances in photosynthetic redox processes during cold stress. In cold-tolerant genotypes, efficient ROS scavenging by SOD, POD, and CAT alleviates oxidative damage in chloroplasts and mitochondria, restoring electron transport between PSII and PSI and reducing lipid peroxidation ( 43 ). The higher expression of photosynthetic and energy metabolism proteins alleviates their reduced activity due to cold temperature stress. The energy metabolism is shifted from aerobic to anaerobic pathway and the oxaloacetate formed by the PEPC enzyme is reduced to malate (Fig. 8 c), which is then degraded by dismutation in the mitochondria. Such a metabolic model reduces the electrolyte leakage index ( 44 ). In line with these earlier studies, the findings here suggest that LLY-7108 enhances ROS scavenger activity to sustain PSII photosynthetic function and electron transport, thereby reducing photoinhibition. Despite extensive research, the precise mechanisms by which plants perceive low-temperature signals remain unclear. However, alterations in membrane fluidity are believed to play a critical role in cold perception. Signal transduction from the plasma membrane to the nucleus likely involves protein phosphorylation cascades mediated by calcium-dependent protein kinases (CPKs) and mitogen-activated protein kinases (MAPKs), which are activated by increases in cytosolic calcium levels under cold stress ( 45 ). Accumulating evidence further highlights the involvement of phytohormones as central regulators that integrate multiple signaling pathways, thereby coordinating plant growth, development, and adaptive responses to cold stress. Metabolic reconfiguration to sustain energy homeostasis and cellular protection To explain the mechanisms of carbohydrate metabolism and energy production in rice seedling under cold stress and nanoparticles application, three KEGG pathways (starch and sucrose metabolism, glycolysis and TCA cycle) were found highly enriched (P-value ≤ 0.5) across all the treatments. Cold stress triggers extensive metabolic reprogramming in rice leaves proved by the differential regulation of carbohydrate and nitrogen metabolism genes. Leaves are primary sites of photosynthesis and are highly sensitive to cold stress-induced damage, particularly in chilling-sensitive rice varieties. Our findings reveal distinct shifts in starch, sucrose, trehalose/raffinose metabolism, and nitrogen turnover, reflecting strategies to maintain energy homeostasis, protect cellular structures, and mitigate oxidative stress under cold conditions. The repression of hexokinases ( Os01g0190400 and Os07g0197100 ) and phosphofructokinase ( Os08g0345700 ) in CS suggests a slowdown in glycolytic flux, likely due to the inhibition of enzyme activity at low temperatures. In contrast, the upregulation of fructose-1,6-bisphosphatase ( Os11g0236100 ) in Fe₂O 3 foliar application implies enhanced gluconeogenesis, potentially to sustain sucrose synthesis for phloem transport and cryoprotection. This aligns with studies showing that cold-tolerant rice genotypes accumulate soluble sugars to stabilize chloroplast membranes( 46 ). The expression of trehalose-6-phosphate (T6P) synthases ( Os08g0414700 and Os09g0397300 ) in CK and Fe₂O₃ but not in CS suggests genotype-specific roles for T6P in cold adaptation. T6P is a key regulator of sucrose metabolism and stress responses in leaves, and its suppression in CS may reflect severe carbon limitation. Conversely, the upregulation of raffinose synthases ( Os12g0555400 and Os03g0808900 ) and α-galactosidases ( Os06g0229800 ) across treatments highlights the importance of raffinose family oligosaccharides (RFOs) as leaf cryoprotectants, consistent with their accumulation in cold-stressed Oryza sativa L . The expression of granule-bound starch synthases ( Os06g0133000 and Os06g0133100 ) suggests transient starch accumulation, possibly due to impaired phloem loading under cold stress. The upregulation of β-glucosidases ( Os06g0320200 ) and endoglucanase ( Os03g0329500 ) in CK and Fe 2 O 3 points to cell-wall loosening, a response to cold-induced membrane rigidification, while their repression in CS may indicate cell-wall damage. Phytohormonal reprogramming: A key mechanism in NPs-induced cold tolerance Plant hormones are pivotal in regulating growth and stress tolerance, particularly under abiotic stress conditions ( 47 , 48 ). IAA and JA play a key role in cold stress response and nanoparticles application. Auxin influx carrier OsLAX3 and repressor OsIAA16 were significantly induced, alongside OsGH3-7 , driving cell enlargement and biomass accumulation ( 49 ). Moreover, studies indicate the expression of the auxin-responsive marker IAA2-GUS and a direct auxin transport assay verified that CS initially targets intracellular-auxin transport ( 50 , 51 ). JA and SA are critical signaling molecules that modulate defense responses by regulating antioxidants and during abiotic stress ( 52 ). Exogenous JA has been shown to enhance abiotic stress tolerance in Arabidopsis. In our study the ABA related genes are significantly down regulated in cold stressed plants as compared to normal temperature (CK) and nanoparticles application (Fig. 10 ). For instance, exogenous ABA (1× 10 –5 mol L –1 ) increased CAT, SOD, POD, APX, and GR activities, reducing H 2 O 2 production in wheat plants under CS (0℃ to − 24℃) ( 53 ). Furthermore, ABA promoted cold tolerance in rice by increasing putrescine biosynthesis( 54 ), suggesting that ABA can regulate plant polyamine levels ( 55 ). Abscisic acid protects the photosynthetic apparatus in plants subjected to CS through ultrastructural alterations in chloroplasts ( 56 ). In our study genes related to SA ( OsNPR5 ) and cytokinin ( ORR3 and AHP1 ) was found in higher expression under normal temperature (CK) and NPs application (Fig. 10 ). Recent studies have revealed that exogenous SA can enhance CS tolerance mechanisms in different plant species ( 57 , 58 ). This alteration in genes expression due to NPs foliar application indicate the ability of Fe 2 O 3 to enhance CS tolerance in rice in seedling stage. Conclusions In conclusion, our study highlights the role of iron oxide nanoparticle (Fe 2 O 3 ) in cold stress resilience of rice seedling. We provided an overview of different molecular changes between different treatments in cold-tolerant and cold susceptible cultivars under cold stress (Fig. 12 ). RNA-seq data indicated that the transcription in response to cold relatively differed between different treatments. our study highlights the role of iron oxide nanoparticle (Fe 2 O 3 ) in regulating antioxidant defense, photosynthesis, hormonal regulation and carbohydrate metabolism under cold stress, these findings provide new insights into the complex mechanisms of rice seedlings under cold stress and nanoparticle application, thereby advancing our understanding of chilling adaptation in rice. Future research should focus on validating several candidate genes, hormones, and their associated crosstalk networks, alongside further investigation into specific adaptation strategies that contribute to increased cold resilience in rice due to Fe 2 O 3 nanoparticle application. Declarations Ethics statement: This study did not involve human participants or vertebrate animals. All experimental procedures were conducted in accordance with institutional, national, and international guidelines for research integrity and laboratory safety. Funding: This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-01) and the Fundamental Research Funds for the Central Universities (2662025YJ011). Author Contribution Author Contributions: Conceptualization, H.J.L. and D.L.X.; methodology, H.J.L and S.U.; software, S.U and A.K.; validation, E.H.K., A.K. and M.W.; formal analysis, S.L.W.; data curation, S.U and H.J.L. writing—original draft preparation, S.U., M.A. and M.W; writing—review and editing, S.U. N.Y. and M.A.; visualization, D.L.X.; supervision, H.J.L. project administration, H.J.L.; funding acquisition, H.J.L. All authors have read and agreed to the published version of the manuscript.” Data Availability The datasets collected and/or analyzed in the present study are available from the corresponding author upon reasonable request. 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Plant Cell 21(12):3823–3838 Pandey V, Bhatt ID, Nandi SK (2019) Role and regulation of auxin signaling in abiotic stress tolerance. Elsevier, Plant signaling molecules, pp 319–331 Sultana S, Rahman MM, Das AK, Haque MA, Rahman MA, Islam SMN et al (2024) Role of salicylic acid in improving the yield of two mung bean genotypes under waterlogging stress through the modulation of antioxidant defense and osmoprotectant levels. Plant Physiol Biochem 206:108230 Yu C, Zhou F, Wang R, Ran Z, Tan W, Jiang L et al (2022) B2, an abscisic acid mimic, improves salinity tolerance in winter wheat seedlings via improving activity of antioxidant enzymes. Front Plant Sci 13:916287 Lee T-M (1997) Polyamine regulation of growth and chilling tolerance of rice (Oryza sativa L.) roots cultured in vitro. Plant Sci 122(2):111–117 Li Z, Qiu Z, Ge H, Du C (2022) Long-term dynamic of cold stress during heading and flowering stage and its effects on rice growth in China. Atmosphere 13(1):103 Venzhik Y, Talanova V, Titov A (2016) The effect of abscisic acid on cold tolerance and chloroplasts ultrastructure in wheat under optimal and cold stress conditions. Acta Physiol Plant 38(3):63 Kazemi-Shahandashti S-S, Maali-Amiri R, Zeinali H, Khazaei M, Talei A, Ramezanpour S-S (2014) Effect of short-term cold stress on oxidative damage and transcript accumulation of defense-related genes in chickpea seedlings. J Plant Physiol 171(13):1106–1116 Duan X, Zhu Z, Yang Y, Duan J, Jia Z, Chen F et al (2022) Salicylic acid regulates sugar metabolism that confers freezing tolerance in Magnolia wufengensis during natural cold acclimation. J Plant Growth Regul 41(1):227–235 Additional Declarations No competing interests reported. Supplementary Files TableS5.csv Secondaryfile.docx TableS6.csv Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 Apr, 2026 Reviews received at journal 13 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers invited by journal 23 Feb, 2026 Editor assigned by journal 12 Feb, 2026 Submission checks completed at journal 12 Feb, 2026 First submitted to journal 07 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-8812976","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595927034,"identity":"d9284b6e-fd1b-426d-8864-f9abafb4012f","order_by":0,"name":"Shafi Ullah","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shafi","middleName":"","lastName":"Ullah","suffix":""},{"id":595927036,"identity":"4f146465-0ce8-4f39-8b0a-79bb32467dfa","order_by":1,"name":"Muhammad Ateeq","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Ateeq","suffix":""},{"id":595927038,"identity":"5b21335c-809c-44dc-bc6b-75b15a8272a6","order_by":2,"name":"Dongliang Xiong","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dongliang","middleName":"","lastName":"Xiong","suffix":""},{"id":595927042,"identity":"70495e3a-5297-472e-b52d-c9548266680e","order_by":3,"name":"Atika khan","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Atika","middleName":"","lastName":"khan","suffix":""},{"id":595927044,"identity":"42e7f0e9-5c2f-4b2d-b3a7-3b8b0999e047","order_by":4,"name":"Sui Liwu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sui","middleName":"","lastName":"Liwu","suffix":""},{"id":595927046,"identity":"21adaaeb-ddff-4250-850f-8f24c9d3eb9b","order_by":5,"name":"Ehtisham Hassan Khan","email":"","orcid":"","institution":"The University of Agriculture Peshawar","correspondingAuthor":false,"prefix":"","firstName":"Ehtisham","middleName":"Hassan","lastName":"Khan","suffix":""},{"id":595927048,"identity":"4a8f466e-cc2a-4295-91f4-0e3b1ac8e718","order_by":6,"name":"Muhammad Waqas","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Waqas","suffix":""},{"id":595927052,"identity":"d22dabf6-f4fe-4d65-9961-1c602438fbe7","order_by":7,"name":"Na Yang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Yang","suffix":""},{"id":595927054,"identity":"4e551e9c-0491-420f-b5a2-7bf1031f8d7c","order_by":8,"name":"Huang Jianliang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACxhlgyoaBgZm5gRkqaECMljSgFkYitTBIgMnDIN1EamGe3WP4ueDX+Wj+dqCWgj92iQ3szdskGGru4HbYnDPG0jP7bufOOAzUMoMnObGB51iZBMOxZ3j8kmMgzdtzO7cBpIVHgjmxQSLHTIKx4TA+Lca/eXvO5c4HazGoT2yQf0NQi5k0z48DuRvAWhIOA23hIaQlrcyatyE5dyNQy2GeA8eN23jSii0SjuHWYjgjefNtnj92ufPOHz74mOdPtWw/++GNNz7U4NHSALKqDcI5ACLYQEQCTg0MDPJg8g8eFaNgFIyCUTAKALF8VAbCPI+9AAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Huang","middleName":"","lastName":"Jianliang","suffix":""}],"badges":[],"createdAt":"2026-02-07 06:53:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8812976/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8812976/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103538174,"identity":"3fe182cc-f1a0-4e08-bc5c-8e5d59364f55","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":458948,"visible":true,"origin":"","legend":"\u003cp\u003eNanoparticle characterization i.e. (\u003cstrong\u003ea\u003c/strong\u003e) XRD-pattern, (\u003cstrong\u003eb\u003c/strong\u003e) DLS size and zeta potential, (\u003cstrong\u003ec\u003c/strong\u003e) Transmission electron microscopy (TEM), (\u003cstrong\u003ed\u003c/strong\u003e)\u003csub\u003e \u003c/sub\u003eSchematic representation of nanoparticle movement in plants. Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles are delivered into the leaf via stomatal infiltration (step 1) and subsequently migrate through the mesophyll cell wall matrix (step 2). The nanoparticles then associate with the outer surface of the mesophyll cell plasma membrane, where electrostatic interactions between the negatively charged Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003enanoparticles and the positively charged membrane surface facilitate binding (step 3). Internalization and subsequent transport of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003enanoparticles into the cytosol and chloroplasts occur via a non-endocytotic pathway and are influenced by the plasma membrane potential (step 4), (\u003cstrong\u003ee\u003c/strong\u003e) localization of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003ein chloroplast and schematic representation of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003etransport inside the chloroplast (\u003cstrong\u003ef\u003c/strong\u003e) Translocation intensity in two cultivars.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/8dedce08bf27d905822e5e61.png"},{"id":103538176,"identity":"e00bbe18-05e6-42b8-82be-0f24f1d3431e","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":322586,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of nanoparticles foliar application and cold stress treatment on plant height (a), fresh weight (b), dry weight (c), chlorophyll a (d), chlorophyll b (b) and total chlorophyll content (c) of rice seedling under cold stress. Different lowercase letters above the bars indicate significant differences among treatments within each cultivar, as determined by LSD test at P \u0026lt; 0.05. Bars sharing the same letter are not significantly different.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/0a6b8bdc1772fb027d2efd5c.png"},{"id":104397722,"identity":"6151de15-c193-44a7-b342-6864667d61db","added_by":"auto","created_at":"2026-03-11 11:55:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":889829,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Fe₂O₃ nanoparticles on stomatal architecture of LLY-7108 and ZJZ-17 under cold stress. (a-f) Scanning electron microscopy (SEM) images showing stomatal structural variations under different treatments, captured at 3000× magnification with a 5 μm scale bar. (g) Quantitative analysis of stomatal length. (h?) Quantitative analysis of stomatal width.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/5126e35885b1bc5fc9f82164.png"},{"id":104397767,"identity":"d97ecb75-cf84-4a49-8e80-a97709329e60","added_by":"auto","created_at":"2026-03-11 11:56:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":825628,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs on the ultrastructure of rice leaves under cold stress. The images were captured via TEM at 3000x (a-f) and 10000× magnification (g-l).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/7c387b6c32d4520bd633c22c.png"},{"id":104397609,"identity":"928bc2ac-59d0-4a78-bc21-9fad8b00b24d","added_by":"auto","created_at":"2026-03-11 11:52:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":363656,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of transcriptomic analysis in LLY-7108 and ZJZ-17 leaves under cold stress. PCA analysis based on gene transcription (FPKM values) from RNA-seq in LLY-7108 and ZJZ-17 leaves (a). The spatial distribution of samples reflects their degree of similarity, with greater distances between sample points indicating increased dissimilarity. The number of differentially expressed genes (DEGs), including both upregulated and downregulated genes, was compared between LLY-7108 and ZJZ-17 (b). Venn diagram displaying the overlap of DEGs across all stress treatments (c). Volcano scatter plot for different treatments under cold stress and nanoparticles application (d-f).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/4f3b5be17378a2c70ac94dd4.png"},{"id":103538178,"identity":"e20ed01f-672b-4306-87cc-a915282b585d","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":693427,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of transcriptomic analysis in LLY-7108 and ZJZ-17 leaves under cold stress. GO slim–based functional categorization and pathway enrichment analysis under different treatments. (a) CK, (b) Fe₂O₃ nanoparticle treatment, and (c) cold stress. Bubble size indicates the number of genes enriched in each KEGG pathway, while color intensity represents the level of statistical significance according to the scale bar. Corrected P values \u0026lt; 0.05 were considered statistically significant for both GO and KEGG enrichment analyses. KEGG pathways of rice leaves of different treatments i.e. CK (d), Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e(e) and CS (f). The size of the bubble represents the number of genes in the KEGG pathway, and color intensity corresponds to different P-value ranges, as indicated by the scale bar.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/c2956aafcb6d8d7a41d822c3.png"},{"id":103538179,"identity":"80632366-2860-48b5-8040-20aafe2f4562","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":464205,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of superoxide radicals via NBT and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e imaging using DAB histochemical staining in LLY-7108 and ZJZ-17 leaves under cold stress (a). Quantification of ROS (b), MDA (malondialdehyde) (c), Proline (d), SOD (superoxide dismutase) activity (e), POD (peroxidase) activity (f) and CAT (catalase) activity (g) content in rice leaves under cold stress. GSH pathway to scavenge ROS and their genes expression under cold stress and nanoparticle application (h).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/bd63c3ce2b0288ee98438400.png"},{"id":104397784,"identity":"21a6df7f-bed7-4a3e-93e5-43bc0bba170b","added_by":"auto","created_at":"2026-03-11 11:56:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":555879,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosystem of rice seedlings showing up-regulated (Red) and down regulated (blue) genes (a). (b) Heatmap showing relative expression profiles of genes associated with PSII, PSI, cytochrome b6f complex, photosynthetic electron transport, and F-type ATP synthase under control (CK), cold stress (CS), and iron oxide nanoparticle (Fe₂O₃ NPs) treatments. Color scale represents normalized expression levels, with red indicating upregulation and blue indicating downregulation. The heatmap was generated using TBtools v0.6696 software. The effect of iron oxide NPs on net photosynthetic rate (c), internal CO\u003csub\u003e2\u003c/sub\u003e concentration (b) and stomatal conductance (e) of rice seedling under cold stress and NPs application. Different lowercase letters above the bars indicate significant differences among treatments within each cultivar, as determined by LSD test at P \u0026lt; 0.05. Bars sharing the same letter are not significantly different.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/1de069f9fd0eaed650ffae2b.png"},{"id":104398350,"identity":"fbfe77d8-8b3e-4cc7-a988-ab8f5e210fe8","added_by":"auto","created_at":"2026-03-11 12:01:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":834375,"visible":true,"origin":"","legend":"\u003cp\u003eCarbohydrate metabolic pathways in ‘LLY-7108’ and ‘ZJZ-17’ leaves under cold stress and nanoparticles application as compared to CK (normal temperature) (a). Heat map represents the DEGs in different pathways i.e. (b) starch and sucrose metabolism, (c) Glycolysis metabolism, (d) citrate or TCA cycle. The color alteration reflects the fold change: blue denotes decreased, and yellow denotes increased levels of gene transcription (log\u003csub\u003e2\u003c/sub\u003e fold change). Each heatmap row corresponds to a gene, and each column represents the treatment sequences as specified. Gene IDs and annotations related to carbohydrate metabolism are provided on the right. The heatmap was generated using TBtools v0.6696 software.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/18f92b0f2069ba7b0e437e8e.png"},{"id":103538187,"identity":"6e8e3991-ec27-4af2-b31f-e4b766a5c094","added_by":"auto","created_at":"2026-02-26 19:02:09","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":344906,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional and metabolic responses of hormones in ‘LLY-7108’ and ‘ZJZ-17’ leaves under cold stress and nanoparticles application as compared to CK (normal temperature). Heatmaps displaying the transcriptional profiles (FPKM-1) of genes involved in Auxin (a), cytokinin (b), Gibberellin (c), ABA (d) and JA signaling pathways. The color alteration reflects the fold change: blue denotes decreased, and red denotes increased levels of gene transcription (log\u003csub\u003e2\u003c/sub\u003e fold change). The heatmap was generated using TBtools v0.6696 software.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/f205e0b975c9664419c3f832.png"},{"id":104397880,"identity":"7190960c-1c55-49c8-9c58-4b516fca4003","added_by":"auto","created_at":"2026-03-11 11:58:33","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1057871,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of hub genes associated with ROS scavenging, phytohormone signaling, photosynthesis, and carbohydrate metabolism using weighted gene co-expression network analysis (WGCNA). (a) Dendrogram showing clustering of genes included in the co-expression module analysis. (b) Heatmap of Pearson’s correlation coefficients illustrating the relationships between gene modules and phenotypic traits. (c-e) Gene interaction networks of the gray module, which exhibited strong associations with all evaluated traits; candidate genes are labeled according to functional annotations.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/2fb779485effe7892b1379bb.jpeg"},{"id":104398283,"identity":"af926b97-7b18-4311-b04e-fc424c6db02e","added_by":"auto","created_at":"2026-03-11 12:01:17","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":570237,"visible":true,"origin":"","legend":"\u003cp\u003eProposed schematic model illustrating rice responses to cold stress. The cold-tolerant cultivar LLY-7108 exhibits enhanced resilience through multiple coordinated mechanisms, including increased activities of antioxidant enzymes (POD, SOD, and CAT) to maintain reactive oxygen species (ROS) homeostasis; upregulation of differentially expressed genes (DEGs) associated with carbohydrate metabolism and glutathione pathways, leading to enhanced metabolic activity and reactivation of cell wall metabolism; and activation of transcriptional networks involved in abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) signaling, thereby strengthening adaptive responses to cold stress. Abbreviations: H₂O₂, hydrogen peroxide; MDA, malondialdehyde; POD, peroxidase; SOD, superoxide dismutase; CAT, catalase; ROS, reactive oxygen species.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/221ba7b6d67f78d085b26b01.png"},{"id":103538185,"identity":"090e7cfc-855d-43b6-b62b-0c60e5d56fda","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":58608,"visible":true,"origin":"","legend":"Cold stress significantly impairs the rice ( L.) growth and yield, particularly in temperate regions where abrupt temperature fluctuations often occur during the early growth stages. Given the need for novel strategies to improve crop cold tolerance, we evaluated the efficacy of iron oxide nanoparticles (FeO) in enhancing rice cold stress resilience. The plant nano-bionics strategy employs sub-12.5 nm iron oxide nanoparticles with a negative ζ-potential (\u0026minus;\u0026thinsp;37.6 mV), which achieve high colocalization within chloroplasts to confer cold tolerance in rice by enhancing photosynthetic efficiency and ROS scavenging. The reported mechanisms involve promoting plant growth and development, alleviating oxidative stress and inducing defense responses. Using RNA-seq, we analyzed the physiological and transcriptomic responses of rice to cold stress and Fe₂O₃ treatment. Under cold stress, the NPs elicited a strong antioxidant response-elevating superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities-which led to a marked reduction in oxidative damage, as shown by decreased ROS and MDA levels. Further, the NPs concurrently restored photosynthetic function and ameliorated cold-induced phenotypic damage. RNA-sequencing revealed that NPs application significantly alters a comprehensive transcriptomic reprogramming, enriching pathways for carbohydrate metabolism, photosystem, plant hormone signaling, and glutathione biosynthesis. Collectively, our findings establish that Fe₂O₃ nanoparticles ameliorate cold stress by preserving chloroplast structure, stomatal architecture, reduce oxidative stress marker, enhancing antioxidant defense system and stabilize photosystem, and providing a promising nanozyme-based approach for rice protection against cold induce damage.","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/20fce8227d93e456fbe2132a.png"},{"id":104407512,"identity":"f412dd8e-064b-4414-a014-237835660b23","added_by":"auto","created_at":"2026-03-11 12:38:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9052558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/028407fb-26bd-4f74-9265-c90848b18bcf.pdf"},{"id":103538175,"identity":"4d5610c8-0a25-420d-a8d4-319c95f08038","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2618,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.csv","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/aba0f306d33741a8fc7f3eea.csv"},{"id":104398837,"identity":"af25cfd4-440b-456a-9cc3-ed457ab9bcc4","added_by":"auto","created_at":"2026-03-11 12:03:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":402813,"visible":true,"origin":"","legend":"","description":"","filename":"Secondaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/cbdf125382d026c344c0ee6d.docx"},{"id":103538183,"identity":"e33cb795-4b7e-4049-8a54-1024fd464777","added_by":"auto","created_at":"2026-02-26 19:02:08","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":118639,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.csv","url":"https://assets-eu.researchsquare.com/files/rs-8812976/v1/ff8a65275f4e251d5380eac4.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Iron oxide nanoparticles alleviate cold stress in rice by reducing oxidative damage and enhancing antioxidant defense systems, and transcriptional networks","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L) one of the most staple food crop widely grown in the world. Over the past century, global rice consumption increases due to increase per capita consumption and population growth (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). However, in the mountainous regions of the tropics and the temperate rice-growing zones, cold stress remains a significant limitation to enhancing rice production (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Approximately 15\u0026nbsp;million hectares of rice-growing land in 24 countries are vulnerable to cold-induced crop damage (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Rice subjected to cold stress exhibits symptoms such as yellowing, slow seedling growth, stunting, withering, reduced tillering, and ultimately diminished productivity, particularly in cold-sensitive varieties. Plant nano-bionics, an interdisciplinary field integrating nanotechnology with plant biology, augments plant function by embedding nanomaterials directly into tissues and organelles. This approach offers an influential strategy for engineering abiotic stress tolerance (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Although the physiological effects of nanoparticles on plants are increasingly documented, however the influence on molecular processes remains poorly understood. This knowledge gap is critical to address in rice, a chilling-sensitive staple crop that feeds half the world, to reveal nanotechnology\u0026rsquo;s potential for engineering climate-resilient varieties (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). \u0026ldquo;Late spring coldness,\u0026rdquo; a recurrent climatic threat in China\u0026rsquo;s, double-season rice regions, inflicts severe damage on early seedlings (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This stress cascade stunted growth, reduces tillering, and causes considerable yield losses, establishing low-temperature resilience as a critical research significance (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). To meet this challenge, we demonstrate that precisely engineered iron oxide nanoparticles can directly intervene in this stress cascade, offering a nano-bionics strategy to enhance rice chilling tolerance at the molecular level.\u003c/p\u003e \u003cp\u003eNano-agrochemicals have emerged as a promising strategy for enhancing global food security, offering advantages over conventional methods through their improved efficacy, reduced application requirements, and lower environmental toxicity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Different nanoparticles work as strong elicitors in agriculture, activating plant innate immunity to combat abiotic stress. For instance, silver and silver-silica nanoparticles enhance resistance by stimulating phenolic synthesis, boosting antioxidative enzyme activity, and upregulating systemic acquired resistance (SAR) genes (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Evidence confirms that nanomaterials like Fe₂O₃, TiO₂, and carbon-based NPs can directly suppress pathogens and enhance plant growth. Iron oxide Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e show increase in antioxidant enzymatic activity superoxide dismutase mimetic, scavenge hydrogen peroxide to water and molecular oxygen (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Significant progress has been made toward understanding plant-nanoparticle interactions on physiological level in other cereal crops, but progress on rice molecular behavior toward iron oxide nanoparticle (Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) is poorly understood. Therefore, RNA-Seq technology and gene expression profiling are powerful tools for identifying cold-tolerant genes in rice under abiotic stress, enabling comprehensive analysis of expression profiles, single-nucleotide polymorphisms (SNPs), and alternative splicing events (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Transcriptomic analysis of two Indica rice genotypes, cold-tolerant and cold-sensitive revealed that cold-tolerant seedlings exhibit enhanced biological processes, including membrane transport, sucrose synthesis, hormone and Ca\u0026sup2;⁺ signaling. In contrast, cold-sensitive seedlings primarily upregulate heat shock proteins and dehydrins in response to low-temperature (LT) stress (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Additionally, transcriptome profiling of chilling-tolerant and chilling-sensitive rice genotypes highlighted the coordinated involvement of multiple regulatory pathways under LT conditions (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). These findings underscore the effectiveness of RNA-Seq in elucidating the molecular basis of cold stress tolerance in rice, which is crucial for sustaining its productivity.\u003c/p\u003e \u003cp\u003eElucidating the mechanisms by which iron oxide nanomaterials (Fe₂O₃) increase cold stress tolerance could optimize sustainable agricultural practices. However, existing research on Fe₂O₃ NPs primarily examines physiological and morphological responses. Therefore, this study was conducted to compare cold stress exposures and nanoparticle application of rice by performing morpho-logical, physiological, and transcriptomic analyses in rice, focusing on the response mechanism of GSH to scavenge ROS, photosystem, carbohydrate metabolism and plant hormones signal transduction. The specific objectives of the present study were to (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) investigate the influence of cold stress on the growth and photosynthetic pigments and carbon fixation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) investigate the influence of cold stress and nanoparticles application on the ROS accumulation, antioxidant enzymatic activity and GSH pathway to scavenge rice (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) perform transcriptomic analysis to investigate the response of nanoparticles and cold stress on carbohydrate metabolism and plant hormones related genes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreliminary screening and dose optimization\u003c/h2\u003e \u003cp\u003eNanoparticles (Zno, Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, Tio\u003csub\u003e2\u003c/sub\u003e, and CeO\u003csub\u003e2\u003c/sub\u003e) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.aladdinsci.com/\u003c/span\u003e\u003cspan address=\"https://www.aladdinsci.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Shanghai China. The effectiveness of nanoparticles was pre-optimized on seedling stage on rice. Seeds of four rice cultivars (LLY-7108, LLY-32, XZX-06 and ZJZ-17) was provided by crop physiology and production center (CPPC), Huazhong Agriculture University (114.37 \u0026deg;E, 30.48 \u0026deg;N), Wuhan, China. In the preliminary experiment, the seed of each cultivar were grown in pots under normal condition. Foliar application of nanoparticles (Zno, Fe2O\u003csub\u003e3\u003c/sub\u003e, Tio\u003csub\u003e2\u003c/sub\u003e, and CeO\u003csub\u003e2\u003c/sub\u003e) with different concentrations were tested under cold stress to optimize best level of NPs (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) (Text S1, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The selection of cold-sensitive and tolerant cultivars, best nanoparticles and best stage was based on second experiment (Text S2, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The cultivars were chosen to assess the effectiveness of foliar application of best nanoparticles on morphological and physiological responses to mitigate cold stress in rice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant materials and growth conditions\u003c/h3\u003e\n\u003cp\u003ePot experiments were conducted using two early indica rice cultivars with contrasting cold tolerance: the cold-sensitive Zhongjiazao-17 (ZJZ-17), bred by the China National Rice Research Institute, and the cold-tolerant Lingliangyou-7108 (LLY-7108), developed by the Hunan Institute of Yahua Seed Industry (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The present study was carried out in Huazhong Agricultural University, Wuhan, China. The experiment used pots (23 cm diameter, 15 cm height) filled with 5 kg of soil per pot. Nutrients were supplied by mixing 0.96 g nitrogen (N), 0.92 g phosphorus (P), and 112 g potassium (K) into the soil two days before seedling establishment. Soil samples were collected from the topsoil layer (0\u0026ndash;20 cm depth) of the rice paddy experimental field. The soil had a pH of 7.1, organic matter content of 6.7 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Olsen phosphorus of 6.27 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, exchangeable potassium of 129 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and total nitrogen of 0.63%.\u003c/p\u003e\n\u003ch3\u003eExperimental design and treatments\u003c/h3\u003e\n\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) seeds were soaked into ultrapure water at 30\u0026deg;C for two days. Each pot was sown with five seeds and maintained under standard growth conditions. To ensure uniform competition, seedlings were thinned to three plants per pot following germination. A foliar spray of Fe₂O₃ nanoparticles (50 mg/L) was applied three days prior to the induction of cold stress. The experiment treatments include one control (CK), one cold stress (CS) treatments groups and one Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. For chilling stress treatment, 14-day-old rice seedlings were exposed to low-temperature conditions (12 h light at 14\u0026deg;C and 12 h dark at 10\u0026deg;C) for five days. Following stress exposure, seedlings were sampled for phenotypic, physiological, biochemical, and molecular analyses. Three plants per treatment (three biological replicates) were used to measure plant height and fresh and dry biomass, while 3\u0026ndash;6 plants per replicate, depending on sample weight requirements, were used for other physiological, biochemical, and molecular assessments.\u003c/p\u003e\n\u003ch3\u003eIron oxide (FeO) NPs characterization\u003c/h3\u003e\n\u003cp\u003eThe physicochemical characteristics of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles were determined using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Powder XRD analysis was performed using an XPERT-3 diffractometer operated at 40 kV and 40 mA. Diffraction patterns were recorded over a 2θ range of 10\u0026ndash;80\u0026deg; with a step size of 0.01\u0026deg; and a scanning rate of 0.02\u0026deg; s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, employing a monochromatized Cu-Kα radiation source (λ\u0026thinsp;=\u0026thinsp;1.54178 \u0026Aring;). The size, morphology, and mesoporous structure of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles were examined using a JEOL-2100 transmission electron microscope, while surface morphology was further analyzed with a Nano Magnetic (hpSPMv1.5) atomic force microscope. Hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZS90 (Malvern Instruments) based on dynamic light scattering, following sonication of the nanoparticle suspension for 15 min to ensure complete dispersion. XPS analysis was conducted using a Kratos AXIS ULTRADLD system equipped with a monochromatic Al Kα X-ray source and a 165 mm mean-radius hemispherical energy analyzer, under ultra-high vacuum conditions (\u0026lt;\u0026thinsp;3 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e Torr). Dried Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticle samples were mounted on carbon tape, and spectral deconvolution and data analysis were performed using CasaXPS software (version 2.3.18, Casa Software Ltd.).\u003c/p\u003e\n\u003ch3\u003eMicroscopic localization analysis of FeO nanoparticles\u003c/h3\u003e\n\u003cp\u003eFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was labeled using a 1,1\u0026rsquo;-octacoalkyl-3,3,3\u0026rsquo;, 3-tetramethylindole carbocyanine perchloric acid drought (Dil) fluorescent dye. Dil- Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was synthesized as follows: 0.8 mL 50mg/L Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and 7.2 mL of deionized water were mixed in a 50 mL glass beaker and stirred on a magnetic stirrer (500 rpm/min .24 \u0026micro;L Dil dye solution (2.5 mg / mL, dissolved in dimethyl sulfoxide (DMSO) was added to 176 ul DMsO solution to obtain Dil dye solution (0.3 mg /mL). The Dil dye solution was added to the Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e solution drop by drop and stirred for 1 min at room temperature at 1000 rpm/min on a magnetic mixer, Transfer the mixed solution (Dil- Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) into a 15 mL 10 kD ultrafiltration tube, add deionized water to the final volume of 15 mL, Dialyze in a beaker of ultra-pure water at 200 rpm/min for 24 h. Finally, the Dil- Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e solution is obtained. Store in a 4℃. To verify the absorption and transport of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e in rice leaves inside chloroplast was cultured with fluorescent PSNPs for 3 h in dark. Leaves of rice seedlings were collected and cut in approximately 0.05 mm thick sections and each placed in a drop of distilled water and covered with a coverslip. The location and distribution of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e in rice leaves were observed using the confocal system FV3000 (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGrowth attribute and photosynthetic pigment\u003c/h2\u003e \u003cp\u003eThree seedlings per treatment were randomly sampled, with three biological replicates. Plant height was measured, and fresh weight was determined immediately after washing. Dry weight was recorded after oven-drying the samples to a constant mass. The dry weight of the above ground parts was determined after oven-drying at 80\u0026deg;C to achieve a constant weight. Chlorophyll content in rice leaves was determined following the method described by (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Fresh leaf tissue (0.5 g) was homogenized in 10 mL of 80% (v/v) acetone. The homogenates were incubated in the dark for 2 h and subsequently centrifuged at 12,000 rpm for 10 min. The absorbance of the supernatant was recorded at 663 and 645 nm using a spectrophotometer, and chlorophyll concentrations were calculated as previously described.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElectron microscopy: SEM and TEM approaches\u003c/h3\u003e\n\u003cp\u003eFresh rice leaf samples were cut into small segments and immediately fixed in 2.5% (v/v) glutaraldehyde prepared in 0.05 M phosphate buffer (pH 7.0) at 4\u0026deg;C for 12\u0026ndash;24 h. After fixation, samples were rinsed three times with phosphate-buffered saline (PBS; pH 7.4) for 15 min each. Tissues were then post-fixed in 1% osmium tetroxide (OsO\u003csub\u003e4\u003c/sub\u003e) prepared in 0.1 M phosphate buffer (pH 7.4) at 4\u0026deg;C for 1\u0026ndash;2 h, washed again with PBS, and sputter-coated with platinum for 20 min. Surface morphology was examined using a scanning electron microscope (Hitachi High-Tech, Tokyo, Japan) following the protocol of Zheng et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). For ultrastructural analysis, transmission electron microscopy (TEM) was conducted on Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e-treated and untreated rice seedlings subjected to cold stress. Samples were post-fixed in 1% OsO\u003csub\u003e4\u003c/sub\u003e for 1 h, washed 2\u0026ndash;3 times with 0.1 M PBS (pH 7.4), and dehydrated through a graded ethanol series (50\u0026ndash;100%). After critical point drying, tissues were embedded in Spurr\u0026rsquo;s resin according to the manufacturer\u0026rsquo;s instructions and polymerized at 70\u0026deg;C for 9 h. Ultrathin sections (~\u0026thinsp;80 nm) were cut, mounted on copper grids, and examined using a JEOL 2100F transmission electron microscope (USA) at \u0026times;10,000 magnification.\u003c/p\u003e\n\u003ch3\u003eStaining visualization of photo‑oxidative stress\u003c/h3\u003e\n\u003cp\u003eSuperoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) localization in leaves was visualized using nitro-blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). For NBT staining, leaves from both LLY-7108 and ZJZ-17 cultivars subjected to 5-day cold stress were immersed in staining solution containing 500 mg NBT dissolved in 500 mL PBS (0.01 M, pH 7.8) and incubated overnight at room temperature. The stained leaves were subsequently rinsed in 70% ethanol at 60\u0026deg;C with three washing cycles before imaging. For DAB staining, leaves were incubated in staining solution consisting of 500 mg DAB dissolved in 500 mL PBS supplemented with 500 \u0026micro;L H₂O₂ and 50 \u0026micro;L HCl, followed by overnight incubation at 25\u0026deg;C in darkness. After staining, leaves were cleared in 60% ethanol at 60\u0026deg;C with three washing cycles, and staining patterns were documented photographically. All experiments included three biological replicates.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eROS, lipid peroxidation, and antioxidant enzyme determination\u003c/h2\u003e \u003cp\u003eReactive oxygen species (ROS), lipid peroxidation (malondialdehyde, MDA), proline and antioxidant enzyme activities were quantified following established protocols (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) with minor modifications. Briefly, 100 mg (ROS and proline) and 500 mg (MDA, and antioxidant enzyme activity) of leaf tissue from each treatment group was homogenized in 1 and 5 mL of ice-cold saline extraction buffer (100 mM potassium phosphate, pH 7.8), respectively. The homogenates were vortexed for 2 min and centrifuged at 1,150 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. The supernatants were aliquoted for subsequent assays of H₂O₂, MDA, SOD, POD, and CAT. ROS and MDA concentrations were determined using commercial assay kits (G0112W-H₂O₂ and G0110W-MDA, respectively; Geruisi Biotechnology, Suzhou, China), with absorbance measured at 450 nm (ROS) and 450, 532/600 nm (MDA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq Analysis\u003c/h2\u003e \u003cp\u003eLeaf samples were collected in triplicate from LLY-7108 and ZJZ-17 cultivars after combined cold stress and nanoparticle treatment for de novo transcriptome assembly via RNA-seq and subsequent qRT-PCR validation. The manufacturer\u0026rsquo;s guideline was used to extract total RNA from tissues by performing extraction with TRIzol\u0026reg; Reagent from Invitrogen based in the USA. The 5300 Bioanalyzer (Agilent) and ND-2000 (NanoDrop Technologies) tools were used to determine RNA quality along with measuring RNA quantities. A total of 18 RNA sequencing libraries were built with 1 \u0026micro;L of RNA from LLY-7108 and ZJZ-17 samples using Illumina\u0026reg; Stranded mRNA Prep and ligation kit (Illumina, San Diego, CA). The library preparation service was conducted by Majorbio Biological Technology Shanghai China using their standardized protocols, as described previously (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The purification of sequencing data involved raw data filtering followed by error rate correction together with GC content distribution analysis. Clean reads obtained data which underwent alignment against (\u003cem\u003eO. sativa\u003c/em\u003e L. IRGSP-10; Ensembl Plants) reference genome. The RSEM program version v1.3.1 calculated gene transcription levels by generating log\u003csub\u003e2\u003c/sub\u003eFc value results. The DESeq R program version 1.24.0 found DEGs through analysis between normal LLY-7108 and ZJZ-17 leaf pairs (5 d), cold stress leaf pairs and Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e application. The DEGs were identified through the pair-wise analysis by applying FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 threshold and fold change as the selection criteria. The Diamond software (v0.9.24) performed functional analysis of DEGs through database comparisons with NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG. Further details about DEGs could be uncovered through KEGG database enrichment tests. An adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used to determine significantly enriched GO terms and KEGG pathways, with the analysis performed via the Majorbio platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.aladdinsci.com/\" target=\"_blank\"\u003ewww.majorbio.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eqPCR for transcriptomic data validation\u003c/h2\u003e \u003cp\u003eThe RNA was extracted using RNAprep Pure Plant Kit (DP441, Tiangen, Beijing, China). 2 \u0026micro;g of total RNA was reversely transcribed into cDNA using the TRUEscript first Strand cDNA Synthesis Kit (PC5402, Aidlab, Beijing, China). The amplification of qRT-PCR products was performed in a reaction mixture of 12.5 \u0026micro;L SYBR Green qPCR Mix (PC3302, Aidlab, Beijing, China) according to the manufacturer\u0026rsquo;s instructions. The qRT-PCR analysis was performed on the Bio-Rad CFX Connect Real-Time PCR System (Bio-Rad, California, USA). Three technical replicates were used for each investigated gene. The relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The primers used for qRT-PCR are shown in Additional file Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of co-expression network and hub genes\u003c/h2\u003e \u003cp\u003eWeighted gene co-expression network analysis (WGCNA) was performed to investigate the relationships between gene expression patterns and phenotypic traits. In the scale-free weighted gene co-expression network, nodes represent differentially expressed genes (DEGs), while edges denote pairwise connections defined by Pearson correlation coefficients between gene expression profiles. A soft-thresholding power (β\u0026thinsp;=\u0026thinsp;9) was selected according to the scale-free topology criterion and used to construct the adjacency matrix. Network construction and module detection were carried out using a signed network with the following parameters: minimum module size of 30, minimum module eigengene-based connectivity (kME) of 0.3, and a merge cut height of 0.25. Hub genes within each module were identified based on intramodular connectivity using visual network analysis with default settings (connectivity threshold\u0026thinsp;=\u0026thinsp;30; weight value\u0026thinsp;=\u0026thinsp;0.02). In the resulting networks, each node represents a gene, with node size proportional to the number of connections, such that highly connected genes were considered hub genes due to their central regulatory roles. Associations between gene modules and phenotypic traits were further assessed by integrating module eigengenes with phenotypic data to calculate gene and module significance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experiment was conducted using randomized complete block design with three biological replicates per treatment. Quantitative data for chlorophyll content, reactive oxygen species (ROS), antioxidant enzyme activities, and gene expression were analyzed for statistical significance using two-way analysis of variance (ANOVA) at P\u0026thinsp;\u0026le;\u0026thinsp;0.05. Post hoc comparisons were performed using Tukey\u0026rsquo;s multiple range test to identify significant differences among treatments. Principal component analysis (PCA) was employed to examine the relationships between treatments and the control.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticles Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e characterization, localization and mechanisms of transport to chloroplasts\u003c/h2\u003e \u003cp\u003eThe X-ray diffraction (XRD) analysis reveals single-phase, crystalline structure of iron oxide nanoparticles (Fig.\u0026nbsp;1a). In the XRD diffractogram, the characteristic peaks of the samples are at 2θ\u0026thinsp;=\u0026thinsp;29.7◦, 35.50◦, 43.25◦, 57.56◦, 57.14◦, and 64.73◦. The DLS size of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles is 10 nm with the zeta potential of -24.3 mV (Fig.\u0026nbsp;1b). The delivery of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles to leaf mesophyll chloroplasts was performed by a simple method of infiltration through the stomata pores into the leaf lamina (Fig.\u0026nbsp;1c). An iron oxide nanoparticle concentration of 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was selected for leaf infiltration. For confocal imaging of nanoparticle localization in leaf mesophyll cells, Fe₂O₃ nanoparticles were labeled with the fluorescent dye 1,1\u0026prime;-dioctadecyl-3,3,3\u0026prime;,3\u0026prime;-tetramethylindocarbocyanine perchlorate (DiI), while chloroplast autofluorescence was used to visualize photosynthetic pigments (Fig.\u0026nbsp;1d?). Confocal z-stack images were acquired from leaf mesophyll cells at a 2 \u0026micro;m optical step size following 3 h of dark incubation after leaf infiltration. The fluorescence signals revealed clear colocalization of iron oxide nanoparticles with chloroplasts (Fig.\u0026nbsp;1e). Iron oxide appears to move rapidly from leaf cell extracellular spaces, through mesophyll cell walls and plasma membranes into chloroplasts. The two varieties show different localization of iron oxide in chloroplast (Fig.\u0026nbsp;1f). We propose that the observed differences in nanoparticle colocalization are partly attributable to electrostatic interactions between the nanoparticle ζ-potential and the plasma membrane potential. The outer surface of the plasma membrane carries a net positive charge, which favors the association and uptake of anionic nanoparticles.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1.\u003c/b\u003e Nanoparticle characterization i.e. (\u003cb\u003ea\u003c/b\u003e) XRD-pattern, (\u003cb\u003eb\u003c/b\u003e) DLS size and zeta potential, (\u003cb\u003ec\u003c/b\u003e) Transmission electron microscopy (TEM), (\u003cb\u003ed\u003c/b\u003e) Schematic representation of nanoparticle movement in plants. Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles are delivered into the leaf via stomatal infiltration (step 1) and subsequently migrate through the mesophyll cell wall matrix (step 2). The nanoparticles then associate with the outer surface of the mesophyll cell plasma membrane, where electrostatic interactions between the negatively charged Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles and the positively charged membrane surface facilitate binding (step 3). Internalization and subsequent transport of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles into the cytosol and chloroplasts occur via a non-endocytotic pathway and are influenced by the plasma membrane potential (step 4), (\u003cb\u003ee\u003c/b\u003e) localization of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e in chloroplast and schematic representation of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e transport inside the chloroplast (\u003cb\u003ef\u003c/b\u003e) Translocation intensity in two cultivars.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGrowth parameters and photosynthetic pigments of LLY-7108 and ZJZ-17 under cold stress and nanoparticles application\u003c/h2\u003e \u003cp\u003eCold stress induced significant changes in leaf phenotypes and growth, particularly in the ZJZ-17 cultivar compared to LLY-7108. Iron oxide nanoparticles (Fe₂O₃ NPs) significantly increased plant growth and photosynthetic pigments under cold stress. Cold stress reduced plant height by 39.2% in LLY-7108 and 41.0% in ZJZ-17 relative to their respective controls (CK). Foliar application of Fe₂O₃ nanoparticles significantly mitigated this reduction, most notably in LLY-7108, where the decrease was lowered to 20.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Foliar application of iron oxide nanoparticles significantly mitigated cold-induced fresh weight loss in both rice varieties. While cold stress alone reduced fresh weight by 51.5% in LLY-7108 and 64.9% in ZJZ-17, nanoparticle treatment dramatically lessened this deficit to 20.7% in ZJZ-17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In LLY-7108, dry weight of rice seedling due to cold stress is reduce (41.2%) as compared to CK (normal temperature) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), while this reduction in susceptible variety (ZJZ-17) is 50.5%. Application of iron oxide nanoparticles to rice leaves reduce this decrease in dry weight due to cold stress in both cultivars. The photosynthetic pigments (chlorophyll a, chlorophyll b, and total chlorophyll) were significantly influenced by cold stress and nanoparticle application. After 5 days of cold stress, leaf yellowing was most severe in ZJZ-17 and mildest in LLY-7108, correlating with significant differences in chlorophyll loss between the two cultivars. Specifically, chlorophyll a content in LLY-7108 and ZJZ-17 leaves under cold stress decreased by 43.1%, and 51.8%, respectively, compared to CK (normal temperature) leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Likewise, chlorophyll b levels were approximately 38.97% and 48.1% lower as compare to control temperature, while the nanoparticles application reduce the damage to photosynthetic pigment due to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f). The reduction in total chlorophyll content is almost 41.2% and 50.1% in LLY-7108 and ZJZ-17 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). In term of cultivars the LLY-7108 perform best as compare to ZJZ-17 under cold stress and cold stress\u0026thinsp;+\u0026thinsp;nanoparticles application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs on stomatal traits and chloroplast ultrastructure\u003c/h2\u003e \u003cp\u003eFe₂O₃ nanoparticle treatment counteracted cold stress-induced stomatal damage, preserving stomatal structure (index, length, width) and mitigating functional impairment to gas exchange (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f). These findings reveal that Fe₂O₃ NPs mitigate cold stress damage to stomata by modulating guard cell ROS and Ca\u0026sup2;⁺ signaling, thereby supporting water relations, protecting chloroplast integrity, and preserving photosynthetic function. Cold stress (CS) significantly reduced stomatal length and width in both rice cultivars (LLY-7108 and ZJZ-17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h). The smallest stomata were observed under CS, indicating impaired stomatal development. Application of Fe₂O₃ nanoparticles under CS significantly increased both stomatal length and width compared with CS alone, partially restoring values toward the control (CK). Overall, LLY-7108 exhibited longer and wider stomata than LLY-7108 across treatments, suggesting greater tolerance to cold stress. Transmission electron microscopy (TEM) analysis revealed pronounced ultrastructural alterations in plant cells under cold stress, indicating its deleterious effects on cellular integrity, including structural deformation and a reduction in mesophyll cell size. Cold stress induced pronounced chloroplast damage, characterized by autophagic-like degradation, shrinkage, and severe structural deformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Affected chloroplasts exhibited disrupted grana stacks, disorganized stroma lamellae, and enhanced plastoglobule accumulation In contrast, Fe₂O₃ nanoparticle treatment under cold stress markedly alleviated these ultrastructural impairments, preserving chloroplast size and overall structural integrity. Fe₂O₃ NP-treated leaves displayed well-organized grana and stroma lamellae with clearly defined dense layers, along with increased plastoglobule formation. Collectively, these results indicate that Fe₂O₃ nanoparticles promote the maintenance and regeneration of mesophyll cell and chloroplast architecture, thereby enhancing rice tolerance to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic profiling of LLY-7108 and ZJZ-17 under cold stress\u003c/h2\u003e \u003cp\u003eA comprehensive analysis was performed on 18 samples to investigate the response of LLY-7108 and ZJZ-17 under cold stress. Following read refinement and adapter trimming, 206.04 Gb of clean data was generated (Q20\u0026thinsp;\u0026gt;\u0026thinsp;98.83%; Q30\u0026thinsp;\u0026gt;\u0026thinsp;96.17%) (Table S5). A PCA based on log\u003csub\u003e2\u003c/sub\u003e |(foldchange)|\u0026ge; 1 from each library revealed distinct variations between the treatments under cold stress. Principal components PC1 and PC2 explained approximately 71.85% of the total variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). A total of 28504 genes were identified, with transcription levels log10 (FPKM\u0026thinsp;+\u0026thinsp;1) in both cultivars ranging from \u0026minus;\u0026thinsp;2 to 5. Comparisons of gene expression revealed 13134 (treatment vs control), 2,336 (Foliar vs control), and 13,009 (Foliar vs treatment) genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In total, 428 and 470 DEGs were detected in LLY-7108 and ZJZ-17 under cold stress, respectively, while under cold stress\u0026thinsp;+\u0026thinsp;nanoparticle application in total, 357 and 202 DEGs were detected in LLY-7108 and ZJZ-17, with 14191 DEGs common to both cultivars across all the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Volcano plot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u0026ndash;f) showed extensive transcriptional changes under cold stress, with a large number of significantly up- and downregulated genes in the CK vs CS comparison. In contrast, Fe₂O₃ nanoparticle treatment reduced the number of cold stress\u0026ndash;induced DEGs, indicating mitigation of stress-related transcriptional disruption. However, the Fe₂O₃ + CS comparison still displayed pronounced differential expression, suggesting active nanoparticle-mediated regulation of cold-responsive genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFunctional annotation and enrichment analysis of differentially expressed genes (DEGs)\u003c/h2\u003e \u003cp\u003eThe transcriptional profiles of differentially expressed genes (DEGs) in the rice cultivars LLY-7108 and ZJZ-17 were functionally annotated using six major databases, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), EggNOG, NR, Swiss-Prot, and Pfam, to elucidate their biological roles. Sequence alignment against the GO (26,853), KEGG (12,014), EggNOG (28,670), NR (31,961), Swiss-Prot (23,714), and Pfam (20,905) databases yielded robust functional annotations for the majority of DEGs, confirming their involvement in well-characterized biological processes. GO and KEGG enrichment analyses revealed significant enrichment of DEGs in both cultivars across all treatments. GO analysis showed predominant enrichment in biological processes such as \u0026ldquo;cellular process\u0026rdquo; (GO:0009987) and \u0026ldquo;metabolic process\u0026rdquo; (GO:0008152) (Table S6, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). KEGG pathway analysis further indicated significant enrichment in pathways related to carbohydrate metabolism, glutathione biosynthesis, plant hormone signal transduction, and photosynthesis antenna proteins (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Moreover, GO enrichment analysis highlighted glutathione metabolism as a significantly enriched biological process under both cold stress and nanoparticle treatments. KEGG pathway enrichment of cultivar-specific DEGs revealed predominant enrichment in photosynthesis antenna proteins (map00196), starch and sucrose metabolism (map00500), plant hormone signal transduction (map04075), and glutathione metabolism (map00480) in both LLY-7108 and ZJZ-17 (Fig.\u0026nbsp;6).\u003cb\u003eFigure 6.\u003c/b\u003e Overview of transcriptomic analysis in LLY-7108 and ZJZ-17 leaves under cold stress. GO slim\u0026ndash;based functional categorization and pathway enrichment analysis under different treatments. (a) CK, (b) Fe₂O₃ nanoparticle treatment, and (c) cold stress. Bubble size indicates the number of genes enriched in each KEGG pathway, while color intensity represents the level of statistical significance according to the scale bar. Corrected P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant for both GO and KEGG enrichment analyses. KEGG pathways of rice leaves of different treatments i.e. CK (d), Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e (e) and CS (f). The size of the bubble represents the number of genes in the KEGG pathway, and color intensity corresponds to different P-value ranges, as indicated by the scale bar.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLipid peroxidation, ROS accumulation, and antioxidant responses under cold stress and nanoparticles application\u003c/h2\u003e \u003cp\u003eThe accumulation of ROS, MDA, and antioxidants was significantly influenced by the cold stress and nanoparticles application. Thus, the transcription patterns of antioxidant-related genes in LLY-7108 and ZJZ-17 under cold stress and nanoparticles application were examined. In the cold stress and nanoparticles application, rice leaves were further assessed using NBT and DAB histochemical staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Notable accumulation of superoxide radicals was observed in ZJZ-17 as compared to LLY-7108. Likewise, DAB staining revealed a substantial increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in ZJZ-17 as compared to LLY-7108. Overall, the enhanced activities of enzymatic antioxidants in LLY-7108 led to a reduction in ROS and MDA, suggesting that LLY-7108 exhibited greater cold stress tolerance than the ZJZ-17 cultivar. In physiological examination of lipid peroxidation, ROS, and proline revealed higher concentration in cold stress treated plants, in contrast the ROS, MDA, and proline content reduce with nanoparticles application under cold stress. The increase in ROS in LLY-7108 due to cold stress is 3.43-fold as compared to CK (normal temperature), while this increase in ROS under nanoparticles application is 1.96-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). In ZJZ-17 the increase in ROS content is 4.88-fold due to cold stress as compared to CK (normal temperature), while the nanoparticles application reduces the ROS content by 40.4% as compared to cold stress. Similarly, MDA and proline levels showed significant increases over the course of cold stress, with MDA and proline content significantly higher in ZJZ-17 than in LLY-7108 of cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-d). The increase in MDA and proline content in LLY-7108 due to cold stress is 2.27 and 2.17-fold, respectively as compared to CK (normal temperature), while this increase in MDA and proline under nanoparticles application is 1.47 and 1.28-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-d). To understand the changes in ROS levels and homeostasis, the activities of SOD, POD, and CAT were investigated, consistent with the GSH pathway. The antioxidant enzymatic activity i.e. SOD, POD and CAT were significantly influence by cold stress and nanoparticles application. The activity of these enzymatic antioxidant was at lowest in the crop under cold stress, while nanoparticles application increases these anti-oxidants enzymatic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-f). The decrease in SOD, POD and CAT activity in LLY-7108 due to cold stress is 45.8%, 43.2% and 30.7%, respectively as compared to CK (normal temperature), while Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticle application increase the antioxidant enzyme inside the plants by 52.1%, 38.1% and 26.9%, respectively as compared to CS (cold stress) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-f). In ZJZ-17 this decrease in SOD, POD and CAT activity due to cold stress is 56.8%, 54.2% and 44.3%, respectively as compared to CK (normal temperature) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-g). In GSH pathway under cold stress the expression of gene \u003cem\u003eOs07g0462000\u003c/em\u003e, \u003cem\u003eOs12g0263000\u003c/em\u003e and \u003cem\u003eOs06g0232650\u003c/em\u003e is lower, while under CK (normal temperature) and Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles application the expression of these genes is high, which indicate to lower the accumulation of ROS and reduce membrane damage due to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). GSH participates in ROS detoxification through glutathione peroxidase, generating GSSG, which is then reduced back to GSH by \u003cem\u003eOsGR1\u003c/em\u003e. GSH catabolism through GGT1 and \u003cem\u003eOsARP\u003c/em\u003e contributes to the γ-glutamyl cycle. \u003cem\u003eOsDHAR1/2\u003c/em\u003e contribute to the ascorbate\u0026ndash;glutathione cycle. Heatmap beside each gene encode differential expression across treatments and cultivars, showing stronger induction of antioxidant-related genes in the tolerant cultivar, particularly with Fe₂O₃-NP treatment. The comparison of both cultivars revealed the best performance of LLY-7108 across all the treatments as compared to ZJZ-17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEffect of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs foliar application on Photosynthesis, photosystem stabilization and genes expression\u003c/h2\u003e \u003cp\u003eExcessive ROS accumulation under cold stress damage chloroplast and photosynthetic machinery such as the D1 protein, oxygen evolving complex in PSII, thylakoid membrane lipids, and chloroplast DNA. Therefore, we assessed the effect of cold stress on photosynthetic parameters in leaves treated with Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs application. Cold stress significantly reduces the photosynthesis rate, stomatal conductance and internal CO\u003csub\u003e2\u003c/sub\u003e concentration, while Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs foliar application increase these parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003ec-e). To clarify the mechanisms of cold stress tolerance, photosynthesis pathway was found significantly enriched (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) under cold stress and nanoparticles application. To elucidate the underlying mechanisms of photosynthesis alteration under cold stress, 39 DEGs related photosystem II (PSII), photosystem I (PSI), electron transport chain and F-type ATPase were identified in both LLY-7108 and ZJZ-17 leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). In the photosystem II (18 DEGs), photosystem I (9 DEGs), electron transport chain (9 DEGs), Cytochrome b6f complex and F-type ATPase (2 DEGs) were found upregulated. The heatmap analysis of photosynthesis-related genes revealed distinct transcriptional responses across treatments and between the tolerant (V₁) and susceptible (V₂) rice cultivars. Most Photosystem II (PSII) genes, including \u003cem\u003ePsbA\u003c/em\u003e, \u003cem\u003ePsbB\u003c/em\u003e, \u003cem\u003ePsbC\u003c/em\u003e, \u003cem\u003ePsbD\u003c/em\u003e, and corresponding nuclear-encoded homologs, were markedly downregulated under cold stress in both cultivars, with a stronger suppression observed in the susceptible genotype. However, Fe₂O₃ nanoparticle treatment partially restored the expression of several PSII components in V₁, indicating enhanced stability of the light-harvesting and reaction-center complexes under stress. A similar trend was observed for Photosystem I (PSI) genes (e.g., \u003cem\u003ePsaA\u003c/em\u003e, \u003cem\u003ePsaB\u003c/em\u003e, \u003cem\u003ePsaD\u003c/em\u003e, and \u003cem\u003ePsaF\u003c/em\u003e), where cold stress sharply reduced expression, especially in ZJZ-17, whereas Fe₂O₃ treatment led to moderate upregulation or recovery in LLY-7108 but not in ZJZ-17, suggesting better preservation of PSI-mediated electron flow in the tolerant cultivar. Genes associated with the cytochrome b6f complex (\u003cem\u003ePetB\u003c/em\u003e, \u003cem\u003ePetC\u003c/em\u003e, \u003cem\u003ePetD\u003c/em\u003e, and \u003cem\u003ePetF\u003c/em\u003e) and photosynthetic electron transport (PC, Fd family) also showed significant downregulation under cold stress, with the decline being more severe in the susceptible cultivar. Fe₂O₃ nanoparticles again mitigated this reduction in LLY-7108, maintaining higher transcription levels of key electron carriers and supporting more efficient plastoquinone-cytochrome electron transfer. Similarly, genes encoding the F-type ATP synthase subunits (\u003cem\u003eAtpA\u003c/em\u003e, \u003cem\u003eAtpB\u003c/em\u003e, \u003cem\u003eAtpF\u003c/em\u003e, and \u003cem\u003eAtpH\u003c/em\u003e) exhibited strong repression under cold stress, particularly in ZJZ-17. Notably, Fe₂O₃-treated LLY-7108 plants showed enhanced or recovered expression of several ATP synthase subunits, indicating improved capacity for ATP generation under low-temperature conditions. Overall, the results demonstrate that cold stress impairs the transcription of core photosynthetic machinery in rice, with the susceptible genotype exhibiting the strongest decline. In contrast, Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles significantly alleviate the cold-induced suppression of photosynthetic genes in the tolerant cultivar, maintaining the integrity of PSII, PSI, cytochrome b6f, electron transport components, and ATP synthase complexes, thereby supporting more efficient photosynthetic performance under stress. After analyzing the transcriptome photosynthetic carbon fixation pathway in photosynthetic organisms, significantly influenced by cold stress and nanoparticles application (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Studies on the differentially expressed genes related to the photosynthetic carbon sequestration pathway under cold stress showed that the expression of \u003cem\u003eOs03g0432100\u003c/em\u003e, \u003cem\u003eOs01g0723400\u003c/em\u003e, \u003cem\u003eOs01g0110700\u003c/em\u003e and \u003cem\u003eOs01g0723400\u003c/em\u003e were upregulated in nanoparticle-treated plant and CK, while cold-stressed plants have lower expression of these genes. In sum, it showed us that the carbon fixation in plants under normal temperature and nanoparticles application is more as compare to cold stress .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eCarbohydrate metabolism\u003c/h2\u003e \u003cp\u003eKEGG pathway analysis revealed that the most significantly enriched processes were starch/sucrose metabolism, glycolysis, and the TCA cycle, highlighting a concerted rerouting of carbon for energy production under combined cold and nanoparticle treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Assessment of carbohydrate metabolism demonstrated clear transcriptional reprogramming across starch and sucrose metabolism, glycolysis, and the TCA cycle in both \u0026lsquo;LLY-7108\u0026rsquo; and \u0026lsquo;ZJZ-17\u0026rsquo; under cold stress and Fe₂O₃ nanoparticle treatments compared with the CK (normal temperature) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-d). In the starch and sucrose metabolism pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), cold stress substantially increased several key genes, such as \u003cem\u003eINV\u003c/em\u003e, \u003cem\u003eHK\u003c/em\u003e, and \u003cem\u003eSPP\u003c/em\u003e-as reflected by the dominant blue color patterns in both cultivars to maintain the cell energy level. In contrast, Fe₂O₃ nanoparticle application (Fe₂O₃) partially restored the expression of sucrose-cleaving and starch-biosynthetic genes, including \u003cem\u003eSUS\u003c/em\u003e, \u003cem\u003eGBE\u003c/em\u003e, \u003cem\u003eWAXY\u003c/em\u003e, and \u003cem\u003eglgA\u003c/em\u003e. This enhancement was notably stronger in the tolerant cultivar \u0026lsquo;LLY-7108\u0026rsquo;, suggesting a more efficient carbon assimilation response under stress when supplemented with nanoparticles. Additionally, genes involved in sucrose phosphate cycling (\u003cem\u003eSPS\u003c/em\u003e and \u003cem\u003eSPP\u003c/em\u003e) and UDP-glucose interconversion exhibited moderate transcriptional activation following Fe₂O₃ treatment, indicating nanoparticle-mediated stabilization of sucrose metabolism. In glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), cold stress caused significant expression of rate-limiting glycolytic enzymes including \u003cem\u003ePFK\u003c/em\u003e, \u003cem\u003eALDO\u003c/em\u003e, \u003cem\u003eGAPDH\u003c/em\u003e, and \u003cem\u003eENO\u003c/em\u003e particularly in the susceptible cultivar \u0026lsquo;ZJZ-17\u0026rsquo;. Conversely, Fe₂O₃ treatments restore the expression of multiple glycolytic genes in both cultivars, with a stronger recovery in \u0026lsquo;LLY-7108\u0026rsquo;. Increased expression of \u003cem\u003ePGK\u003c/em\u003e, \u003cem\u003eGAPDH\u003c/em\u003e, \u003cem\u003eENO\u003c/em\u003e, \u003cem\u003ePK\u003c/em\u003e, and \u003cem\u003ePDHB\u003c/em\u003e under Fe₂O₃ + cold conditions indicate enhanced carbon flux from hexose phosphates toward pyruvate formation. The upregulation of ACSS, \u003cem\u003eDLAT\u003c/em\u003e, and \u003cem\u003eALDH7A1\u003c/em\u003e under cold stress treatment further suggests improved acetyl-CoA formation, helping maintain energy metabolism under cold stress. The transcriptional profiles of TCA cycle related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) exhibited a comparable trend, wherein cold stress alone led to the up-regulation of key dehydrogenases and core cycle enzymes, including MDH, fumC, \u003cem\u003eSDH4\u003c/em\u003e, and \u003cem\u003eDLST\u003c/em\u003e. In contrast, Fe₂O₃ nanoparticle treatment markedly restores the expression of \u003cem\u003eACO\u003c/em\u003e, \u003cem\u003eIDH\u003c/em\u003e, \u003cem\u003eOGDH\u003c/em\u003e, and \u003cem\u003eCS/ACLY\u003c/em\u003e in both cultivars, with a more pronounced induction observed in the tolerant \u0026lsquo;LLY-7108\u0026rsquo;. This upregulation indicates that nanoparticle application may stimulate mitochondrial function and help sustain TCA cycle flux under low-temperature stress. Furthermore, elevated expression levels of LSC2 and \u003cem\u003eOGDH\u003c/em\u003e support enhanced NADH production, which likely contributes to improved ATP synthesis and overall metabolic resilience. Overall, the integrated pathway analysis demonstrates that cold stress markedly impairs carbohydrate metabolism in both rice cultivars; however, Fe₂O₃ nanoparticle application effectively alleviates these disruptions by maintaining the expression of key genes associated with sucrose metabolism, glycolysis, and the TCA cycle. The tolerant cultivar \u0026lsquo;LLY-7108\u0026rsquo; exhibits consistently stronger transcriptional recovery and metabolic stabilization compared with the susceptible \u0026lsquo;ZJZ-17\u0026rsquo;, highlighting a cultivar-specific improvement in metabolic homeostasis in response to nanoparticle supplementation under cold stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePlant hormones signal transduction pathways during cold stress and nanoparticles application\u003c/h2\u003e \u003cp\u003ePlant hormonal response is the first line of defense against abiotic stress; plant hormones control abiotic stress response by altering transcriptional programs. The plant hormone signaling transduction network depicted in the Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e provides a comprehensive representation of the molecular interactions and transcriptional regulation involved in coordinating plant development and stress responses. To explain the mechanisms of signal transduction via plant hormones in rice seedlings under cold stress and nanoparticles application, the KEGG pathway (plant hormones) was observed under different treatments. An analysis of the expression patterns of DEGs associated with the plant hormone signal transduction pathway of the rice revealed the identification of 14 DEGs in rice leaves under cold stress and nanoparticles application (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). These genes were found to be enriched in five distinct hormonal signaling pathways, including IAA, gibberellin (GA), ABA, Cytokinin and JA. The highest count of DEGs were involved in the Indole-3-acetic acid (IAA), Abscisic acid (ABA) and Gibberellin (GA) signaling pathways. Auxin signaling, mediated through key components such as \u003cem\u003eOsLAX3\u003c/em\u003e, \u003cem\u003eOsIAA16\u003c/em\u003e, and \u003cem\u003eOsGH3-7\u003c/em\u003e, is primarily associated with cell enlargement and vegetative growth. The \u003cem\u003eOsLAX3\u003c/em\u003e, \u003cem\u003eOsIAA16\u003c/em\u003e, and \u003cem\u003eOsGH3-7\u003c/em\u003e genes were found in higher expression inside the IAA pathway across control and nanoparticle application as compare to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003ea), in the tolerant cultivar, indicating a better maintenance of auxin-mediated cell elongation during stress. In parallel, cytokinin phosphotransferase protein \u003cem\u003eAHP1\u003c/em\u003e and response regulator ORR3 were upregulated, stimulating meristematic activity and shoot initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003eb). The GA pathway shows changes primarily at \u003cem\u003eDELLA\u003c/em\u003e-associated regulatory genes. Genes such as \u003cem\u003eOsGID1\u003c/em\u003e, \u003cem\u003eOsPIFs\u003c/em\u003e (\u003cem\u003eOsPIF11\u003c/em\u003e, \u003cem\u003eOsPIF14\u003c/em\u003e, and \u003cem\u003eOsPIF12\u003c/em\u003e), and \u003cem\u003eDELLA\u003c/em\u003e repressors exhibit mixed but treatment-specific responses. Fe₂O₃ nanoparticles enhance GA-related expression more strongly in tolerant cultivar, consistent with improved GA-mediated stem elongation and stress compensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003ec). ABA biosynthesis genes \u003cb\u003e(\u003c/b\u003e\u003cem\u003eOsPYL\u003c/em\u003e and \u003cem\u003eOsPYR\u003c/em\u003e) and ABA-activated MAP kinases \u003cb\u003e(\u003c/b\u003e\u003cem\u003eSAPK1\u003c/em\u003e, \u003cem\u003eSAPK2\u003c/em\u003e, \u003cem\u003eSAPK6\u003c/em\u003e, \u003cem\u003eSAPK9\u003c/em\u003e, and \u003cem\u003eSAPK10\u003c/em\u003e\u003cb\u003e)\u003c/b\u003e show substantial induction under cold stress, with Fe₂O₃-NPs amplifying several signals. The tolerant cultivar shows a markedly stronger activation of ABA receptor genes and signaling nodes, indicating more robust stomatal control and stress adaptation capacity than the susceptible cultivar (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003ed). JA biosynthesis and signaling genes, including \u003cem\u003eAOS\u003c/em\u003e, \u003cem\u003eCOI1b, JAZ\u003c/em\u003e repressors, and \u003cem\u003eMYC2\u003c/em\u003e, show strong modulation under cold stress and nanoparticle treatments. In particular, \u003cem\u003eOsJAZ2\u003c/em\u003e, \u003cem\u003eOsJAZ8\u003c/em\u003e, and \u003cem\u003eOsbHLH009\u003c/em\u003e show cultivar-dependent differences, with tolerant cultivar showing stronger upregulation. Activation of the JA pathway leads to induction of \u003cem\u003eORCA3\u003c/em\u003e, a major regulator of stress-responsive genes. The expression patterns indicate that JA-mediated defense signaling is significantly more active in the tolerant cultivar during cold stress, and further enhanced by Fe₂O₃ nanoparticles application. Together, these transcriptional adjustments underscore a multifaceted hormonal crosstalk that balances enhanced growth with adaptive stress responses under cold stress condition due to Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eValidation of RNA-Seq Analysis by qRT-PCR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eQuantitative real-time PCR (qPCR) analysis was performed to validate the RNA-seq expression profiles of selected cold-responsive genes, including \u003cem\u003epsbP\u003c/em\u003e, \u003cem\u003epsbQ\u003c/em\u003e, \u003cem\u003ePSBW\u003c/em\u003e, \u003cem\u003eOsLAX3\u003c/em\u003e, \u003cem\u003eOsIAA16\u003c/em\u003e, and \u003cem\u003eOsCOI1b\u003c/em\u003e. The qPCR expression trends were highly consistent with the FPKM values obtained from RNA-seq data across treatments, confirming the reliability and accuracy of the transcriptomic analysis. Cold stress markedly downregulated photosynthesis-related genes, whereas Fe₂O₃ nanoparticle application significantly restored or enhanced their expression in both cultivars. Similarly, genes involved in hormone signaling and stress responses exhibited concordant expression patterns between qPCR and RNA-seq datasets, supporting the robustness of the sequencing results (Figure S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eProfiling of hub DEGs involved in cold tolerance under nanoparticles application\u003c/h2\u003e \u003cp\u003eTo assess sample correlations and the relationships between gene modules and co-expressed genes, weighted gene co-expression network analysis (WGCNA) was performed on differentially expressed genes (DEGs identified under cold stress and nanoparticle foliar application. After removing unannotated DEGs, seven distinct modules comprising 16,250 highly co-expressed genes were identified (Table S7; Figure S4). Among these, the green, magenta, and turquoise modules exhibited negative correlations with reactive oxygen species (ROS), proline, and MDA contents, but showed significant positive correlations with antioxidant enzyme activities and photosynthetic parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003ea-b). For network construction, 31 hub genes from the green module were selected; these hub genes displayed high intramodular connectivity (kME) and were extensively connected within the interaction network. Similarly, in the magenta module, correlation coefficients ranged from 0.0227 under control conditions to \u0026minus;\u0026thinsp;0.341 under cold stress, with 29 hub genes showing strong interactions within the network. In the turquoise module, correlation coefficients ranged from 0.681 under nanoparticle foliar application to \u0026minus;\u0026thinsp;0.818 under cold stress, and 29 hub genes exhibited extensive connectivity within the interaction network (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003ec-e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCold stress is a primary limiter of rice productivity, a legacy of the crop\u0026rsquo;s tropical origins that renders seedlings especially vulnerable. Understanding the genetic and physiological mechanisms of cold adaptation is therefore crucial. Utilizing a plant nano-bionics approach, our study reveals that nanoparticle application enhances cold tolerance by activating key pathways. We pinpoint the reduction of oxidative damage alongside the modulation of antioxidants, phytohormones, and photosynthetic antenna proteins as central to this fortified response in LLY-7108 and ZJZ-17 rice varieties.\u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eFe₂O₃ NPs enhance chilling resilience by bolstering antioxidant systems and preserving photosynthetic function\u003c/h2\u003e \u003cp\u003eChloroplasts and mitochondria are the primary sites of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and superoxide (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) production during abiotic stress conditions (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Excessive accumulation of ROS induces lipid peroxidation, mainly through the oxidation of unsaturated fatty acids, leading to the formation of toxic aldehydes such as MDA. In the present study, cold stress caused severe damage to ZJZ-17 leaves, as evidenced by reduced cell viability and significantly elevated ROS and MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u0026ndash;b). These results indicate that cold stress enhanced ROS production, disrupted membrane integrity, and exacerbated leaf injury in this cold-sensitive cultivar. In contrast, LLY-7108 exhibited comparatively lower H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA accumulation, suggesting the presence of a more efficient ROS scavenging system and enhanced membrane stability. Genotype-dependent variation in MDA and proline accumulation under low-temperature stress has been widely reported, reflecting differences in chilling tolerance among cultivars. Previous studies demonstrated that nanoparticle applications, particularly TiO₂ nanoparticles, improve electrolyte leakage, photosynthetic performance, and membrane integrity under cold stress through transcriptional regulation in chickpea (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Hasanpour et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) suggest that TiO\u003csub\u003e2\u003c/sub\u003e NPs application to plants increase the tolerance of plants to cold stress due to controlling the pressure of the temperature drop injury and altered metabolism for plant growth. The harmful effects of cold stress are reduced and glycyrrhizin content is enhanced when using TiO\u003csub\u003e2\u003c/sub\u003e NPs in licorice plants (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The use of chitosan nanoparticles was found to be effective in reducing the ROS with the accumulation of osmoprotectants in plants under cold-stress conditions(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Furthermore, in rice plants, the foliar application of ZnO NPs may reduce cold stress through the antioxidative system and transcription factors involved in the chilling response(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Consistent with these reports, both rice cultivars in the present study exhibited significant increases in MDA and proline under cold stress; however, the cold-sensitive cultivar ZJZ-17 accumulated substantially higher levels of ROS, MDA, and proline than LLY-7108. Importantly, Fe₂O₃ nanoparticle application markedly reduced ROS and MDA accumulation under cold stress, highlighting their role in enhancing antioxidative defense capacity. This protective effect may be attributed to the provision of bioavailable iron, which is essential for the activity of antioxidative enzymes and associated metabolic processes (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Collectively, ROS, MDA, and proline profiling supports the classification of LLY-7108 as a cold-tolerant cultivar and ZJZ-17 as cold-sensitive. Antioxidant enzymes are essential for scavenging ROS and mitigating lipid peroxidation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Increased activities of SOD, POD, and CAT following nanoparticle application under cold stress have been reported in rice (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In the present study, LLY-7108 exhibited significantly enhanced SOD, POD, and CAT activities, along with upregulation of glutathione (GSH) pathway-related genes. These enzymatic antioxidants efficiently scavenge H₂O₂ and O₂⁻ across multiple cellular compartments, thereby maintaining redox homeostasis (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Enhanced antioxidant capacity in LLY-7108 effectively mitigated oxidative damage and conferred improved cold tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003ee\u0026ndash;h), in agreement with earlier findings in rice (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of Fe₂O₃ NPs on chloroplast ultrastructure and photosystem stabilization under cold stress\u003c/h2\u003e \u003cp\u003eFoliar application of Fe₂O₃ NPs preserved chloroplast ultrastructure under cold stress, as evidenced by the maintenance of well-organized grana stacks, intact stroma lamellae, and plastoglobule integrity, indicating effective mitigation of cold-induced photooxidative damage (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Enhanced chloroplast structural stability facilitated efficient light harvesting and sustained photosynthetic performance, thereby counteracting cold-mediated impairment of leaf photosynthesis. These observations are consistent with reports in lettuce, where carbon-based nanoparticles safeguarded chloroplast membranes under stress by stabilizing thylakoid architecture, reinforcing lipid peroxidation defense systems, and maintaining photosystem II functionality, ultimately preserving chloroplast integrity and photosynthetic efficiency (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Furthermore, increased chlorophyll contents (chlorophyll a, b, and carotenoids) reflect an improved light-harvesting capacity, in agreement with findings in rice exposed to nanoparticle treatments under cold stress conditions (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Cold stress has been reported to decrease PSII abundance, electron flow, and Rubisco activity (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Our results indicate that cold stress markedly reduce photosynthesis rate, internal CO\u003csub\u003e2\u003c/sub\u003e concentration and stomatal conductance while NPs application increase this parameter significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003ec-e). Plant ROS are mainly produced by chloroplasts, mitochondria, peroxisomes, NADPH oxidases, and class III peroxidases (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Among these, chloroplasts are a source of hydroxyl radical production in leaves under stress conditions (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The iron oxide NPS with large surface to volume ratios catalytically scavenges ROS produced by the chloroplasts such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, the most destructive ROS in plant cells. Cold stress cause oxidative damage to chloroplast components inhibiting the repair of PSII, the most temperature-sensitive component of the photosynthetic apparatus (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Iron oxide NPs protect both the light and carbon reactions of photosynthesis from ROS damage in plants under stress condition. Cold stress and nanoparticle treatments also significantly influenced photosynthesis antenna proteins and carbon fixation pathways in rice. Genes associated with photosynthesis antenna proteins and carbon fixation exhibited higher expression levels under normal temperature (CK) and nanoparticle treatments compared with cold stress. Cold-induced lipid modifications compromise membrane integrity, affecting essential physiological processes such as photosynthesis, gas exchange, and transpiration. Reduced CO₂ assimilation and chlorophyll content under cold stress are often linked to inhibition of the large subunit and degradation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This is also due to the ROS accumulation in chloroplasts, damage the photosynthetic apparatus, particularly PSII, leading to imbalances in photosynthetic redox processes during cold stress. In cold-tolerant genotypes, efficient ROS scavenging by SOD, POD, and CAT alleviates oxidative damage in chloroplasts and mitochondria, restoring electron transport between PSII and PSI and reducing lipid peroxidation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The higher expression of photosynthetic and energy metabolism proteins alleviates their reduced activity due to cold temperature stress. The energy metabolism is shifted from aerobic to anaerobic pathway and the oxaloacetate formed by the PEPC enzyme is reduced to malate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003ec), which is then degraded by dismutation in the mitochondria. Such a metabolic model reduces the electrolyte leakage index (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In line with these earlier studies, the findings here suggest that LLY-7108 enhances ROS scavenger activity to sustain PSII photosynthetic function and electron transport, thereby reducing photoinhibition.\u003c/p\u003e \u003cp\u003eDespite extensive research, the precise mechanisms by which plants perceive low-temperature signals remain unclear. However, alterations in membrane fluidity are believed to play a critical role in cold perception. Signal transduction from the plasma membrane to the nucleus likely involves protein phosphorylation cascades mediated by calcium-dependent protein kinases (CPKs) and mitogen-activated protein kinases (MAPKs), which are activated by increases in cytosolic calcium levels under cold stress (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Accumulating evidence further highlights the involvement of phytohormones as central regulators that integrate multiple signaling pathways, thereby coordinating plant growth, development, and adaptive responses to cold stress.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMetabolic reconfiguration to sustain energy homeostasis and cellular protection\u003c/h3\u003e\n\u003cp\u003eTo explain the mechanisms of carbohydrate metabolism and energy production in rice seedling under cold stress and nanoparticles application, three KEGG pathways (starch and sucrose metabolism, glycolysis and TCA cycle) were found highly enriched (P-value\u0026thinsp;\u0026le;\u0026thinsp;0.5) across all the treatments. Cold stress triggers extensive metabolic reprogramming in rice leaves proved by the differential regulation of carbohydrate and nitrogen metabolism genes. Leaves are primary sites of photosynthesis and are highly sensitive to cold stress-induced damage, particularly in chilling-sensitive rice varieties. Our findings reveal distinct shifts in starch, sucrose, trehalose/raffinose metabolism, and nitrogen turnover, reflecting strategies to maintain energy homeostasis, protect cellular structures, and mitigate oxidative stress under cold conditions. The repression of hexokinases (\u003cem\u003eOs01g0190400\u003c/em\u003e and \u003cem\u003eOs07g0197100\u003c/em\u003e) and phosphofructokinase (\u003cem\u003eOs08g0345700\u003c/em\u003e) in CS suggests a slowdown in glycolytic flux, likely due to the inhibition of enzyme activity at low temperatures. In contrast, the upregulation of fructose-1,6-bisphosphatase (\u003cem\u003eOs11g0236100\u003c/em\u003e) in Fe₂O\u003csub\u003e3\u003c/sub\u003e foliar application implies enhanced gluconeogenesis, potentially to sustain sucrose synthesis for phloem transport and cryoprotection. This aligns with studies showing that cold-tolerant rice genotypes accumulate soluble sugars to stabilize chloroplast membranes(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The expression of trehalose-6-phosphate (T6P) synthases (\u003cem\u003eOs08g0414700\u003c/em\u003e and \u003cem\u003eOs09g0397300\u003c/em\u003e) in CK and Fe₂O₃ but not in CS suggests genotype-specific roles for T6P in cold adaptation. T6P is a key regulator of sucrose metabolism and stress responses in leaves, and its suppression in CS may reflect severe carbon limitation. Conversely, the upregulation of raffinose synthases (\u003cem\u003eOs12g0555400\u003c/em\u003e and \u003cem\u003eOs03g0808900\u003c/em\u003e) and α-galactosidases (\u003cem\u003eOs06g0229800\u003c/em\u003e) across treatments highlights the importance of raffinose family oligosaccharides (RFOs) as leaf cryoprotectants, consistent with their accumulation in cold-stressed \u003cem\u003eOryza sativa L\u003c/em\u003e. The expression of granule-bound starch synthases (\u003cem\u003eOs06g0133000\u003c/em\u003e and \u003cem\u003eOs06g0133100\u003c/em\u003e) suggests transient starch accumulation, possibly due to impaired phloem loading under cold stress. The upregulation of β-glucosidases (\u003cem\u003eOs06g0320200\u003c/em\u003e) and endoglucanase (\u003cem\u003eOs03g0329500\u003c/em\u003e) in CK and Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e points to cell-wall loosening, a response to cold-induced membrane rigidification, while their repression in CS may indicate cell-wall damage.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003ePhytohormonal reprogramming: A key mechanism in NPs-induced cold tolerance\u003c/h2\u003e \u003cp\u003ePlant hormones are pivotal in regulating growth and stress tolerance, particularly under abiotic stress conditions (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). IAA and JA play a key role in cold stress response and nanoparticles application. Auxin influx carrier \u003cem\u003eOsLAX3\u003c/em\u003e and repressor \u003cem\u003eOsIAA16\u003c/em\u003e were significantly induced, alongside \u003cem\u003eOsGH3-7\u003c/em\u003e, driving cell enlargement and biomass accumulation (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Moreover, studies indicate the expression of the auxin-responsive marker \u003cem\u003eIAA2-GUS\u003c/em\u003e and a direct auxin transport assay verified that CS initially targets intracellular-auxin transport (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). JA and SA are critical signaling molecules that modulate defense responses by regulating antioxidants and during abiotic stress (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Exogenous JA has been shown to enhance abiotic stress tolerance in Arabidopsis. In our study the ABA related genes are significantly down regulated in cold stressed plants as compared to normal temperature (CK) and nanoparticles application (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). For instance, exogenous ABA (1\u0026times; 10\u003csup\u003e\u0026ndash;5\u003c/sup\u003e mol L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) increased CAT, SOD, POD, APX, and GR activities, reducing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production in wheat plants under CS (0℃ to \u0026minus;\u0026thinsp;24℃) (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Furthermore, ABA promoted cold tolerance in rice by increasing putrescine biosynthesis(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), suggesting that ABA can regulate plant polyamine levels (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Abscisic acid protects the photosynthetic apparatus in plants subjected to CS through ultrastructural alterations in chloroplasts (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). In our study genes related to SA (\u003cem\u003eOsNPR5\u003c/em\u003e) and cytokinin (\u003cem\u003eORR3\u003c/em\u003e and \u003cem\u003eAHP1\u003c/em\u003e) was found in higher expression under normal temperature (CK) and NPs application (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Recent studies have revealed that exogenous SA can enhance CS tolerance mechanisms in different plant species (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). This alteration in genes expression due to NPs foliar application indicate the ability of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e to enhance CS tolerance in rice in seedling stage.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our study highlights the role of iron oxide nanoparticle (Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) in cold stress resilience of rice seedling. We provided an overview of different molecular changes between different treatments in cold-tolerant and cold susceptible cultivars under cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e12\u003c/span\u003e). RNA-seq data indicated that the transcription in response to cold relatively differed between different treatments. our study highlights the role of iron oxide nanoparticle (Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) in regulating antioxidant defense, photosynthesis, hormonal regulation and carbohydrate metabolism under cold stress, these findings provide new insights into the complex mechanisms of rice seedlings under cold stress and nanoparticle application, thereby advancing our understanding of chilling adaptation in rice. Future research should focus on validating several candidate genes, hormones, and their associated crosstalk networks, alongside further investigation into specific adaptation strategies that contribute to increased cold resilience in rice due to Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticle application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics statement:\u003c/h2\u003e \u003cp\u003eThis study did not involve human participants or vertebrate animals. All experimental procedures were conducted in accordance with institutional, national, and international guidelines for research integrity and laboratory safety.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Earmarked Fund for China Agriculture Research System (CARS-01) and the Fundamental Research Funds for the Central Universities (2662025YJ011).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions: Conceptualization, H.J.L. and D.L.X.; methodology, H.J.L and S.U.; software, S.U and A.K.; validation, E.H.K., A.K. and M.W.; formal analysis, S.L.W.; data curation, S.U and H.J.L. writing\u0026mdash;original draft preparation, S.U., M.A. and M.W; writing\u0026mdash;review and editing, S.U. N.Y. and M.A.; visualization, D.L.X.; supervision, H.J.L. project administration, H.J.L.; funding acquisition, H.J.L. All authors have read and agreed to the published version of the manuscript.\u0026rdquo;\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets collected and/or analyzed in the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuo E, Wang L, Jiang S, Xiang H, Shi Y, Chen X et al (2022) Impacts of chilling at the tillering phases on rice growth and grain yield in Northeast China. J Agron Crop Sci 208(4):510\u0026ndash;522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao J (2025) Rice growth promotion and cold stress alleviation by an endophytic bacterium Microbacterium testaceum M15 isolated from rice seed\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNajeeb S, Mahender A, Anandan A, Hussain W, Li Z, Ali J (2021) Genetics and breeding of low- temperature stress tolerance in rice. 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Plant Physiol Biochem 206:108230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu C, Zhou F, Wang R, Ran Z, Tan W, Jiang L et al (2022) B2, an abscisic acid mimic, improves salinity tolerance in winter wheat seedlings via improving activity of antioxidant enzymes. Front Plant Sci 13:916287\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee T-M (1997) Polyamine regulation of growth and chilling tolerance of rice (Oryza sativa L.) roots cultured in vitro. Plant Sci 122(2):111\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Qiu Z, Ge H, Du C (2022) Long-term dynamic of cold stress during heading and flowering stage and its effects on rice growth in China. Atmosphere 13(1):103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenzhik Y, Talanova V, Titov A (2016) The effect of abscisic acid on cold tolerance and chloroplasts ultrastructure in wheat under optimal and cold stress conditions. Acta Physiol Plant 38(3):63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazemi-Shahandashti S-S, Maali-Amiri R, Zeinali H, Khazaei M, Talei A, Ramezanpour S-S (2014) Effect of short-term cold stress on oxidative damage and transcript accumulation of defense-related genes in chickpea seedlings. J Plant Physiol 171(13):1106\u0026ndash;1116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan X, Zhu Z, Yang Y, Duan J, Jia Z, Chen F et al (2022) Salicylic acid regulates sugar metabolism that confers freezing tolerance in Magnolia wufengensis during natural cold acclimation. J Plant Growth Regul 41(1):227\u0026ndash;235\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cold stress, iron oxide, nano-biotechnology, rice, oxidative stress, RNA-seq, ultrastructure","lastPublishedDoi":"10.21203/rs.3.rs-8812976/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8812976/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cold stress significantly impairs the rice ( L.) growth and yield, particularly in temperate regions where abrupt temperature fluctuations often occur during the early growth stages. Given the need for novel strategies to improve crop cold tolerance, we evaluated the efficacy of iron oxide nanoparticles (FeO) in enhancing rice cold stress resilience. The plant nano-bionics strategy employs sub-12.5 nm iron oxide nanoparticles with a negative ζ-potential (\u0026minus;\u0026thinsp;37.6 mV), which achieve high colocalization within chloroplasts to confer cold tolerance in rice by enhancing photosynthetic efficiency and ROS scavenging. The reported mechanisms involve promoting plant growth and development, alleviating oxidative stress and inducing defense responses. Using RNA-seq, we analyzed the physiological and transcriptomic responses of rice to cold stress and Fe₂O₃ treatment. Under cold stress, the NPs elicited a strong antioxidant response-elevating superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities-which led to a marked reduction in oxidative damage, as shown by decreased ROS and MDA levels. Further, the NPs concurrently restored photosynthetic function and ameliorated cold-induced phenotypic damage. RNA-sequencing revealed that NPs application significantly alters a comprehensive transcriptomic reprogramming, enriching pathways for carbohydrate metabolism, photosystem, plant hormone signaling, and glutathione biosynthesis. Collectively, our findings establish that Fe₂O₃ nanoparticles ameliorate cold stress by preserving chloroplast structure, stomatal architecture, reduce oxidative stress marker, enhancing antioxidant defense system and stabilize photosystem, and providing a promising nanozyme-based approach for rice protection against cold induce damage.","manuscriptTitle":"Iron oxide nanoparticles alleviate cold stress in rice by reducing oxidative damage and enhancing antioxidant defense systems, and transcriptional networks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-26 19:02:01","doi":"10.21203/rs.3.rs-8812976/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-14T09:42:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T05:01:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174310625071312289292729501328454576137","date":"2026-04-10T23:02:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107078870816333983020647248518021314073","date":"2026-03-31T09:37:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T09:25:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72744641404001390110645406105024948090","date":"2026-02-23T23:22:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-23T19:47:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T09:06:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T09:00:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2026-02-07T06:42:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74544ca0-6a57-4313-a2a9-7a67897c3857","owner":[],"postedDate":"February 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T13:26:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-26 19:02:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8812976","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8812976","identity":"rs-8812976","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}