Zinc Oxide Nanoparticles Enhance Vigor of Aged Naked Oat Seeds: Transcriptomic Insights into Antioxidant and Metabolic Reprogramming

Abstract

Naked oat (Avena nuda L.) is an important dual-purpose crop for grain and forage in cold regions; however, its high fatty acid content renders seeds prone to deterioration during storage. This study aimed to investigate the protective effects of zinc oxide nanoparticles (ZnO NPs) on artificially aged naked oat seeds and elucidate the underlying molecular mechanisms. Non-aged seeds (Naged) were subjected to artificial aging at 45 °C and 100% relative humidity for 24 h (Aged), followed by priming with 30 mg L⁻¹ ZnO NPs for 6 h (Daged). Antioxidant enzyme activities were determined spectrophotometrically, and transcriptome sequencing was performed on an Illumina platform to identify differentially expressed genes (DEGs) and enriched pathways. We found that ZnO NPs increased catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities by 3–4-fold, restored germination rate from 75% to 98%, and enhanced seed vigor index. A total of 21,403 DEGs were detected, with 15,841 stably expressed in response to nano-priming. Reactive oxygen species (ROS) burst rapidly induced up-regulation of AP2/EREBP transcription factor family members, which subsequently activated antioxidant enzyme genes to maintain cellular redox homeostasis. Metabolic pathway analysis demonstrated that the phenylpropanoid pathway was reprogrammed, characterized by down-regulated lignin biosynthesis and up-regulated flavonoid production, thereby enhancing ROS scavenging capacity. Additionally, the pentose phosphate pathway was activated to provide additional NADPH for antioxidant defense, and up-regulated ADP-glucose pyrophosphorylase (AGPase) facilitated starch accumulation. Notably, the 40S ribosomal protein S13 exhibited the highest connectivity in protein–protein interaction networks, was up-regulated 2.1-fold, and was enriched in post-translational modification processes. These findings suggest that nano-priming with ZnO NPs represents a promising biotechnological strategy for enhancing seed vigor and storability in naked oat, with potential applications in sustainable agriculture and the seed industry.

1. Introduction

Naked oat (Avena nuda L.), an annual member of the Poaceae family, is cultivated worldwide as a dual-purpose crop for both human food and livestock feed [1]. It is predominantly grown in the saline–alkaline regions of Inner Mongolia, Qinghai, and Gansu, China, where its tolerance to infertile, drought-prone, and cold environments enables reliable production under marginal soil conditions. The species is characterized by a husk-free caryopsis that threshes free of lemma and palea, which provides superior processing quality for food applications [2]. The grain contains a well-balanced protein fraction, high levels of soluble dietary β-glucan, unsaturated fatty acids, vitamins, and minerals essential for human health [3]. When used as forage, its low acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents contribute to high digestibility and palatability for ruminants [4]. However, the seeds are rich in lipids and possess membranes that are highly susceptible to lipid peroxidation, resulting in poor storability [1]; viability declines sharply as storage duration increases. This deterioration has markedly raised seed costs for both the seed industry and the forage sector, posing a significant challenge to sustainable crop and livestock production.

Seed aging is a ubiquitous physiological process that occurs during the storage of oats [5]. It is a natural and irreversible progression that intensifies over time, leading to reduced germinability, impaired grain quality, and lower field yields, all of which seriously undermine oat-based production systems. Currently, seed enhancement technologies, most notably seed priming and low-temperature storage, are the primary strategies employed to counteract aging. The concept of seed priming was first introduced by Heydecker et al. [6] and is defined as a controlled pre-sowing treatment in which seeds are allowed to imbibe water slowly and then dried back under regulated conditions [7]. During this process, seeds are maintained at the lag phase of germination (stage II), enabling pre-germinative physiological and biochemical metabolism, membrane and organelle repair, DNA restoration, and enzyme activation, thereby positioning the seed in a metabolically ready-to-germinate state [8].

Nano-priming utilizes nanoparticles as priming agents and has emerged as a novel seed enhancement strategy, demonstrating benefits in improving seed vigor, stress tolerance, disease control, and nutrient use efficiency [9]. Due to their high surface-area-to-volume ratio and ability to modulate reactive oxygen species (ROS), nanoparticles can initiate ROS signaling cascades in seeds, promote enzymatic activity, and facilitate membrane repair, collectively enhancing germination performance and stress resilience [10]. For instance, treating aged onion (Allium cepa L.) seeds with gold nanoparticles (Au NPs) significantly improved germination compared to untreated controls after 21 days, indicating a pronounced rejuvenating effect [11]. Similarly, priming aged peanut (Arachis hypogaea L.) seeds with zinc oxide nanoparticles (ZnO NPs) increased germination from 60% to 77% and enhanced the vigor index from 692 to 3067 [12]. These nanoparticles activate antioxidant enzymes, including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), which scavenge free radicals and mitigate cellular damage. As a result, ZnO NPs are emerging as key agents for stimulating plant growth and conferring tolerance against a wide array of abiotic stresses.

Building on these findings, the present study aimed to investigate the protective effects of ZnO NPs on artificially aged naked oat seeds and elucidate the underlying molecular mechanisms. Specifically, we hypothesized that (1) ZnO-NP priming would restore seed vigor through enhanced antioxidant enzyme activities, and (2) transcriptome analysis would reveal differentially expressed genes and enriched pathways associated with ROS scavenging, metabolic reprogramming, and stress tolerance. To test these hypotheses, we performed physiological assays combined with transcriptome sequencing on an Illumina NovaSeq platform to systematically identify differentially expressed genes and their enriched pathways [13]. This integrated approach provides robust data for elucidating the molecular networks underlying nano-priming-enhanced seed vigor in naked oat.

2. Materials and Methods

2.1. Plant Material

Seeds of the naked oat cultivar “Baiyan 2” were obtained from the College of Pratacultural Science at Gansu Agricultural University. The seeds were harvested in 2024 and stored under ambient warehouse conditions, characterized by continuous ventilation, a temperature range of 10–12 °C, and relative humidity levels of 45–56%.

2.2. Experimental Design

2.2.1. Synthesis of ZnO Nanoparticles

ZnO nanoparticles were commercially purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Catalog No. S52531, Lot No. C2518577, Shanghai, China). According to the manufacturer’s Certificate of Analysis, the nanoparticles had an average particle size of 41.73 nm (specification: 50 ± 10 nm), a purity of 99.8% based on trace metals analysis, a Zn content of 79.52% (complexometric titration), a total metallic impurity of 31 ppm, a specific surface area of 18.19 m²/g, and a crystal structure conforming to the standard ZnO structure as verified by X-ray diffraction (XRD). The nanoparticles appeared as white powder and were stored at room temperature. For seed priming, 30 mg of the ZnO nanoparticles were dispersed in 1 L of ultrapure water via ultrasonication to obtain a homogeneous suspension at a concentration of 30 mg L⁻¹.

2.2.2. Seed Priming

Uniform, plump seeds with an initial moisture content of 7.65% (as determined by ISTA rules [14]; designated as “Naged”) were placed in nylon mesh bags and artificially aged in a 45 °C water bath for 24 h. Aged but unprimed seeds served as the control group (denoted as “Aged”). For priming, the remaining aged seeds were immersed in an optimal 30 mg L⁻¹ ZnO-NP suspension (previously determined in pilot tests) and incubated at 25 °C for 6 h with gentle shaking. After priming, the seeds were rinsed three times with distilled water, blotted dry with filter paper, and equilibrated in darkness at 20 °C until their moisture content returned to the initial level [15] (designated as “Daged”). The primed seeds were then used for subsequent germination assays and physiological analyses.

2.2.3. Germination Assay

Standard germination tests were conducted on the primed naked oat seeds. Forty plump seeds were placed in Petri dishes lined with double-layer filter paper and incubated in a light chamber at 20 °C, with three replicates per treatment. Morphological, physiological, and biochemical indices, including germination percentage, germination index, and vigor index, were determined or calculated accordingly. Starting from the day of seed germination, the number of germinated seeds was recorded daily at regular intervals.

2.3. Determination of Physiological Indices

2.3.1. Germination Indices

Germination percentage (%) = (number of germinated seeds at final count/total seeds) × 100% [16].

Germination index (GI) = $\sum Gt/Dt$, where Gt is the number of seeds germinated on day t and Dt is the corresponding germination day [16].

Vigor index (VI) = GI × SS, with SS being the mean shoot length (cm) of 10 selected seedlings [16].

2.3.2. Biochemical Assays

Sample Preparation. For biochemical analyses, 0.3 g of whole naked oat seeds from each of the three treatments (Naged, Aged, and Daged) was ground in a pre-chilled mortar with 3 mL ice-cold extraction buffer (50 mM PBS, pH 7.8, 1 mM EDTA-Na₂, 1% PVP-40, 0.5 mM DTT). The homogenate was transferred to a 2 mL centrifuge tube and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant (crude enzyme extract) was either used immediately or snap-frozen in liquid nitrogen and stored at −80 °C to avoid repeated freeze–thaw cycles [17].

Soluble Protein Content. Soluble protein (SP) content was quantified using the Coomassie Brilliant Blue G-250 method [17].

Soluble Sugar Content. Soluble sugar (SS) content was determined by the anthrone colorimetric method [17].

Antioxidant Enzyme Activities. Superoxide dismutase (SOD) activity was determined by the nitro-blue tetrazolium (NBT) photochemical reduction method [17]. Peroxidase (POD) activity was measured using the guaiacol oxidation assay [17]. Catalase (CAT) activity was assayed by monitoring the rate of H₂O₂ decomposition at 240 nm with a UV spectrophotometer [17].

2.4. Transcriptome Sequencing and Bioinformatics Analysis

2.4.1. RNA Extraction and Library Construction

Total RNA was extracted from each of the three treatments (Naged, Aged, and Daged) using the Tiangen RNA prep kit (TIANGEN Biotech (Beijing) Co., Benjing, China). RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technonlogies, Santa Clara, CA, USA). Poly-adenylated mRNA was enriched with oligo(dT) magnetic beads and reverse-transcribed into double-stranded cDNA using random hexamers. The cDNA was subjected to end-repair, A-tailing, adapter ligation, size selection and PCR amplification to generate sequencing libraries. Library concentration was initially quantified with a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), insert-size distribution was verified on an Agilent 2100 Bioanalyzer, and absolute quantification was performed by qRT-PCR. Qualified libraries were sequenced on an Illumina platform. Clean reads were assembled using Trinity; assembly completeness was evaluated with BUSCO. Functional annotation was performed against the NCBI-NR database. Raw sequence data have been deposited in NCBI under BioProject ID PRJNA1404124.

2.4.2. Expression Quantification and Differential Analysis

High-quality clean reads were mapped to the assembled transcriptome. Differentially expressed genes (DEGs) between pairwise comparisons were identified using DESeq2 [18] with thresholds of |log2fold-change| ≥ 1 and adjusted p-value (padj) < 0.05. DEGs were annotated and enriched against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (padj < 0.05). MapMan v3.5.1R2 was employed to classify and visualize DEGs involved in transcriptional regulation, phytohormone pathways, kinases, protein modification, and protein degradation [19].

2.4.3. qRT-PCR Validation

Twenty DEGs were randomly selected for validation. Total RNA was re-extracted using the RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China) and reverse-transcribed with PrimeScript RT Reagent Kit (TaKaRa, v3.0). Primers were designed with NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 5 February 2026) (sequences listed in Table S1). SYBR Green qRT-PCR was performed with Actin as the internal reference, following the protocol described by Chen et al. [20]. Relative expression levels were calculated using the 2^−∆∆Ct method [21]. qPCR data were analyzed using Bio-Rad CFX Maestro v2.3 software.

3. Results

3.1. Antioxidant-Enzyme Activities

Artificially aged naked oat seeds exposed to 30 mg L⁻¹ ZnO NPs exhibited significant differences (p < 0.05) in both germination parameters and antioxidant enzyme activities (Figure 1). Compared to the non-aged control group (Naged), the aged group (Aged) demonstrated a reduction in germination percentage, germination index, vigor index, and enzyme activities, whereas the nanoprimed aged group (Daged) displayed pronounced improvements. Among the antioxidant enzymes (Figure 1a–c), the most substantial increase was noted in catalase (CAT) activity, which rose by 433% in Daged compared to Aged, while the corresponding increase in superoxide dismutase (SOD) activity was slightly lower at 342%.

With respect to seed germination (Figure 1d–g), ZnO-NP priming markedly elevated the germination percentage of artificially aged naked oat seeds to 96%. Additionally, seed vigor improved notably (p < 0.05), rising from 347.9 to 392.1, which is statistically comparable to the non-aged control value of 388.5. In line with these enhancements, the contents of soluble proteins and soluble sugars also exhibited significant increases relative to the aged seeds: soluble protein levels rose from 1.1 to 1.3 mg/g dry weight (DW), while soluble sugars increased from 0.32% to 0.67%.

3.2. Sequencing Data Quality Control and Identification of Differentially Expressed Genes (DEGs)

Nine libraries representing the Naged, Aged, and Daged seed lots were subjected to RNA sequencing. Following stringent quality filtering, a total of 64.26 Gb of clean data were retained (

Table 1. Quality control statistics of sequencing data for each sample.

Sample	Raw Reads	Raw Bases (Gb)	Clean Reads	Clean Bases (Gb)	Q20 (%)	Q30 (%)	GC Pct (%)
Naged 1	50290752	7.54	48949068	7.34	99.16	96.46	53.48
Naged 2	47343232	7.1	46151416	6.92	99.17	96.46	53.68
Naged 3	50894812	7.63	49698154	7.45	99.12	96.26	54.83
Aged 1	47722446	7.16	45420376	6.81	99.07	96.25	55.08
Aged 2	47452018	7.12	45780270	6.87	99.17	96.43	54.47
Aged 3	49612996	7.44	47913218	7.19	99.14	96.38	54.52
Daged 1	49988470	7.50	49149444	7.37	99.12	96.44	56.65
Daged 2	40942224	6.14	38932050	5.84	99	96.13	55.37
Daged 3	44199824	6.63	42310034	6.35	99.04	96.27	54.96

). The average clean read yield per sample was 6.90 Gb, with 48,266,213, 46,371,288 and 43,463,843 high-quality reads obtained for the Naged, Aged, and Daged seed lots, respectively. The Q20 and Q30 values exceeded 99% and 96.13%, respectively, while the GC content ranged from 53.48% to 56.65%. These metrics confirm the reliability of the transcriptomic data and its suitability for downstream analyses.

Differentially expressed genes (DEGs) were identified through two pairwise comparisons: Naged versus Aged, and Daged versus Aged. The first comparison yielded 5562 DEGs, while the second produced 18,385 DEGs. Venn analysis of these gene sets (Figure 2) revealed that 15,841 DEGs were consistently regulated in aged seeds following ZnO NPs priming. These stably expressed DEGs represent strong candidate genes potentially underlying the molecular response of artificially aged naked oat seeds to ZnO NPs treatment.

3.3. Comparative Transcriptional Regulation

The MapMan annotation of the 18,385 differentially expressed genes (DEGs) identified in the comparison between Daged and Aged revealed a comprehensive reprogramming of transcriptional networks (Figure 3). A total of 1071 differentially expressed transcription factors (DETs) were implicated in the priming response, with 589 being up-regulated and 482 down-regulated. Additionally, protein-modification processes involved 505 DEGs (171 up-regulated and 334 down-regulated), while 698 DEGs were associated with protein degradation.

Hormone-biosynthesis pathways were also extensively remodeled: 421 DEGs were mapped to hormone-related processes, including 93 and 73 DEGs that were significantly enriched in abscisic-acid (ABA) and ethylene (ETH) metabolism, respectively. In addition, large sets of receptor-like kinase genes, Ca²⁺-signaling components, and G-protein signaling genes were markedly regulated, indicating that ZnO NPs priming coordinates a multi-layered transcriptional cascade to counteract aging-induced deterioration in naked oat seeds.

3.4. Transcription Factor Profiling

To identify the differentially expressed transcription factors (DETs) driving the priming response, we assigned 1071 DETs to 66 plant-specific gene families (Figure 4). The largest group identified was the RTPR family, which stands for RNA regulation of transcription—putative transcription regulators, comprising 98 DETs (51 up-regulated and 47 down-regulated). The AP2/EREBP superfamily ranked second, with 77 DETs (54 up-regulated and 23 down-regulated), followed by the MYB family, which included 53 DETs known for their role in regulating flavonoid and phenylpropanoid biosynthesis, as well as reactive oxygen species (ROS) scavenging. In contrast, only one DET from the RTSG family, which pertains to RNA regulation of transcription silencing group, was detected, and it was down-regulated.

3.5. KEGG Pathway Enrichment Analysis

KEGG enrichment analysis was performed on the 18,385 DEGs identified in the Daged vs. Aged comparison (Figure 5). Pathways were ranked by ascending p-value (cut-off < 0.05), and the top 20 are reported. Metabolism-related routes dominated the list, accounting for 17 of the 20 pathways. These included phenylpropanoid biosynthesis, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, galactose metabolism, α-linolenic acid metabolism, fatty acid degradation, pyruvate metabolism, fatty acid metabolism, the pentose phosphate pathway, taurine and hypotaurine metabolism, cyanoamino acid metabolism, phenylalanine metabolism, nucleotide sugar biosynthesis, phenylalanine/tyrosine/tryptophan biosynthesis, biosynthesis of various plant secondary metabolites, and diterpenoid biosynthesis. A single pathway corresponded to photosynthesis (carbon fixation in photosynthetic organisms), whereas two pathways were associated with genetic information processing, namely ribosome biogenesis in eukaryotes and DNA replication.

Among the enriched routes, the pentose phosphate pathway (PPP) operates as a central metabolic switch during seed priming. It is rapidly activated at the onset of imbibition, generating large amounts of NADPH and ribulose-5-phosphate (Ru5P). The former supplies the reducing power required for reactive-oxygen-species (ROS) scavenging, whereas the latter provides the nucleotide precursors essential for early DNA/RNA synthesis. The PPP is tightly coupled to amino sugar and nucleotide sugar metabolism, which channels UDP-glucose (UDP-Glc) and related nucleotide sugars into cell wall biogenesis, thereby furnishing the structural basis for embryonic cell elongation.

Phenylalanine metabolism occupies a pivotal junction between primary and secondary metabolism. On the one hand, it supplies phenylalanine as a building block for the burst of protein synthesis that accompanies priming; on the other, it funnels carbon into the phenylpropanoid pathway via phenylalanine ammonia-lyase (PAL). The resulting flavonoids and anthocyanins accumulate as protective antioxidants, enhancing seed tolerance to priming-induced stress. Given these strategic roles, we conducted hierarchical clustering and normalization analyses focused on the four aforementioned pathways.

3.6. Phenylpropanoid Biosynthesis

The phenylpropanoid pathway serves as a metabolic hub that provides precursors for lignin, flavonoids, anthocyanins, and benzoate derivatives, thereby influencing seed coat hardness, dormancy, pigment deposition, and tolerance to biotic stress. KEGG annotation identified 180 DEGs significantly enriched in this pathway (p < 0.05; Figure 6).

Topological analysis of the pathway revealed that the DEGs were predominantly clustered in the branch directing phenylalanine towards flavonoid biosynthesis. Among the 66 enzyme-encoding DEGs, 24 were up-regulated, while 42 were down-regulated. Seven DEGs encoded phenylalanine ammonia-lyase (PAL), all of which were suppressed; the most repressed isoform, novel.6840, exhibited a 2.3-fold decrease in transcript abundance. Cinnamate-4-hydroxylase (C4H; novel.8327) and 4-coumarate-CoA ligase (4CL; AVESA_00001b_r3_6Cg0001902) were down-regulated by 7.2- and 1.3-fold, respectively. Forty-three DEGs encoded peroxidases (PERs), with AVESA_00001b_r3_4Dg0000971 showing the strongest repression (5.1-fold), whereas AVESA_00001b_r3_4Ag0003736 exhibited the most induction (3.1-fold). Thirteen DEGs corresponded to cinnamoyl-CoA reductase (CCR), with the largest change observed for novel.8489, whose expression declined by 10.4-fold.

3.7. Amino Sugar and Nucleotide Sugar Metabolism

The amino sugar and nucleotide sugar metabolic network serves as the primary donor pool for cell wall polysaccharides, glycoproteins, and glycosylated secondary metabolites. By supplying UDP-glucose, UDP-xylose, UDP-arabinose, GDP-fucose, and related substrates, this pathway regulates endosperm cell wall remodeling, mucilage deposition, and osmotic-stress tolerance in seeds. KEGG annotation identified 170 DEGs that were significantly enriched in this pathway (p < 0.05; Figure 7).

Topological mapping revealed that the DEGs were predominantly localized in the branch diverting fructose-6-phosphate towards starch granules. Transcripts encoding HXK and AGPase were significantly up-regulated, suggesting that fructose-6-phosphate is directed into starch biosynthesis. Specifically, HXK (AVESA_00001b_r3_1Cg0000549) and AGPase (AVESA_00001b_r3_5Dg0001733) increased by 1.5- and 1.3-fold, respectively. In contrast, genes encoding phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPA) were down-regulated, indicating a reduced carbon flux through the main nucleotide sugar axis; PGM (AVESA_00001b_r3_7Cg0000984) and UGPA (AVESA_00001b_r3_5Dg0002678) decreased by 3.1- and 1.1-fold, respectively. These expression patterns suggest that, following priming, fructose-6-phosphate is preferentially diverted into starch granule synthesis to repair aging-induced damage.

3.8. Pentose Phosphate Pathway (PPP)

The PPP is the primary source of NADPH and pentose phosphates (R5P, Xu5P) in plants. By furnishing reducing equivalents for fatty-acid, lignin, and flavonoid biosynthesis and by supplying carbon skeletons for nucleotide synthesis, DNA repair, and Calvin-cycle regeneration, the pathway controls early-germination energy reserves, redox buffering, and the rate of endosperm cell division. KEGG annotation identified 64 DEGs that were significantly enriched in the PPP (p < 0.05; Figure 8).

Topological analysis revealed that the DEGs were concentrated in the segment channeling fructose-6-phosphate toward 3-phosphoglycerate. Transcript AVESA_00001b_r3_7Ag0002160, encoding pyrophosphate-dependent phosphofructokinase (PFP), was up-regulated 1.1-fold, thereby accelerating the conversion of Fru-1,6-BP to F6P and preventing germination arrest. Concurrently, most genes encoding fructose-1,6-bisphosphatase (FBPase) were down-regulated, restricting gluconeogenic back-flow and ensuring that a larger proportion of carbon skeletons is directed toward nucleotide and histone synthesis.

3.9. qRT-PCR Validation

To corroborate the RNA-seq data, nine genes involved in phenylpropanoid biosynthesis were randomly selected for qRT-PCR analysis (

Table 2. The relative expression of genes verified by qRT-PCR.

Gene ID	Log2FC in RNA-Seq	Log2FC in RT-qPCR
AVESA_00001b_r3_6Cg0001341	1.571929518	3.274619737
AVESA_00001b_r3_3Ag0000799	1.318599259	2.274936218
AVESA_00001b_r3_4Dg0000922	1.716227173	1.438628361
AVESA_00001b_r3_3Dg0002255	1.954554907	1.286481264
AVESA_00001b_r3_7Dg0000654	3.579764764	2.397831628
AVESA_00001b_r3_7Ag0001222	−1.766967635	−3.219361892
AVESA_00001b_r3_5Cg0000118	1.22709215	2.291739279
AVESA_00001b_r3_5Ag0001692	−1.205459998	−1.218374923
AVESA_00001b_r3_6Ag0000958	1.156041808	2.219739364
AVESA_00001b_r3_7Ag0002437	2.114624007	1.291837494
AVESA_00001b_r3_1Cg0001784	−2.901883267	−4.129749127
AVESA_00001b_r3_7Cg0002534	1.40169125	1.394792434
AVESA_00001b_r3_4Ag0002352	1.686660369	1.932056924
AVESA_00001b_r3_5Ag0001296	2.032417925	2.497924153
AVESA_00001b_r3_6Cg0001962	2.188758089	3.312781464
AVESA_00001b_r3_7Dg0000793	−1.727274037	−0.134681274
AVESA_00001b_r3_6Ag0002240	1.204548979	2.149797234
AVESA_00001b_r3_7Cg0003030	1.396781501	1.492648273
AVESA_00001b_r3_7Cg0003031	1.885230252	1.497259438
AVESA_00001b_r3_6Ag0002948	−5.428265308	−3.216498324

; Figure 9). The expression trends obtained by qRT-PCR were consistent with those derived from the transcriptome dataset, confirming the accuracy, reliability, and biological validity of the sequencing results.

3.10. Protein–Protein Interaction Network Analysis

Potential interactions among the differentially expressed genes (DEGs) were analyzed using the STRING database. The resulting network identified 40S ribosomal protein S13 (RPS13) as the most highly connected node, followed by several members of the 60S large-subunit family, including RPL10A, RPL9, RPL11, and RPL18A, as well as cinnamoyl-CoA reductase (CCR) and enolase-1 (ENO1), along with additional 40S ribosomal proteins (Figure 9). These hub genes are believed to play crucial roles in mediating the seed’s response to aging stress.

4. Discussion

Priming artificially aged naked oat seeds with 30 mg L⁻¹ ZnO NPs significantly restored seed vigor and simultaneously activated the antioxidant defense network. In comparison to the aged control, primed seeds demonstrated a 23% increase in germination percentage, an elevation in seed vigor from 347.9 to 392.1, and increments of 2.1-fold and 1.1-fold in soluble sugar and soluble protein contents, respectively. The activities of the three principal antioxidant enzymes—catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)—increased three- to four-fold, corroborating previous reports that nanoparticles can enhance antioxidant capacity in stressed seeds [22].

Excessive ROS accumulation during aging triggers lipid peroxidation, protein oxidation, and DNA lesions that ultimately erode seed viability. The pronounced stimulation of CAT activity observed here underscores the importance of rapid H₂O₂ detoxification for the resurrection of aged seeds. Transcriptomic evidence further supports this notion: three DEGs encoding the core antioxidant enzymes SOD (AVESA_00001b_r3_5Ag0002328), POD (AVESA_00001b_r3_4Ag0003736) and CAT (AVESA_00001b_r3_7Cg0000067) were up-regulated 1.6- to 3.1-fold. These data confirm that ZnO NPs activate the antioxidant system at the transcriptional level and lend support to the nano-priming immunity hypothesis [12]. Acting as an abiotic nano-elicitor, ZnO NPs penetrate the seed coat, redistribute within the embryo, and re-establish the ROS scavenging/producing balance that is otherwise disrupted in aged seeds [23].

In this study, ZnO-NP priming significantly reprogrammed 1071 differentially expressed transcription factors (DETs) across 66 gene families. Within the AP2/EREBP superfamily, represented by 77 differentially expressed genes (DEGs), 54 were found to be up-regulated. Notably, the gene Avesa_00001b_r3_5cg0002586 exhibited the strongest induction, with a 6.6-fold increase, indicating a robust AP2/EREBP-mediated response to ZnO-NP treatment in aged naked oat seeds.

Members of the DREB and ERF sub-clades within the AP2/EREBP superfamily serve as central hubs that integrate signals related to seed aging and priming [24]. Under aging stress, the accumulation of reactive oxygen species (ROS) rapidly induces the expression of AP2/EREBP; the encoded transcription factors (TFs) directly bind to the promoters of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), enhancing intracellular ROS scavenging and re-establishing redox homeostasis. Concurrently, these TFs promote the extensive synthesis of late embryogenesis abundant (LEA) proteins, which stabilize membranes and protein structures, thereby mitigating dehydration injury. Consequently, ZnO-NP priming significantly reduces the oxidative damage index during germination and ultimately improves both the emergence percentage and uniformity, highlighting the AP2/EREBP family as a critical molecular target for nano-priming technology.

The concurrent enrichment of NAC and MYB families observed here further suggests that these transcription factors (TFs) also contribute to the response of aged naked oat seeds to ZnO-NP priming [25]. Beyond their canonical roles in plant development, both families play a significant role in modulating stress adaptation. For instance, ZmNAC2 enhances osmotic stress tolerance in transgenic Arabidopsis [26], while an R2R3-MYB factor from rice accelerates seed germination under varying sowing depths [27].

KEGG pathway enrichment assigns differentially expressed genes to well-characterized metabolic and signaling networks, thereby identifying the biological processes most significantly affected by a given treatment. In this study, a substantial proportion of differentially expressed genes (DEGs) were found to be involved in phenylpropanoid biosynthesis, the pentose phosphate pathway, and amino sugar/nucleotide sugar metabolism; each of these pathways is analyzed in detail below.

Phenylpropanoid biosynthesis is one of the most important secondary metabolic routes in plants [28]. One of its major branches channels phenylalanine into flavonoid biosynthesis. Flavonoids such as quercetin and kaempferol possess phenolic hydroxyl groups that donate hydrogen atoms to scavenge free radicals, thereby interrupting oxidative chain reactions. In addition, they chelate metal ions, suppress Fenton chemistry [29] and diminish de novo ROS formation. During seed aging, excessive ROS accumulation imposes oxidative stress that damages lipids, proteins, and nucleic acids and ultimately erodes seed vigor. Flavonoids therefore provide direct protection against oxidative injury, preserve membrane integrity, and stabilize enzyme conformation, making the flavonoid branch of phenylpropanoid metabolism a key determinant of naked oat seed viability.

Cinnamoyl-CoA reductase (CCR) is the rate-limiting enzyme of the flavonoid branch, whereas cinnamate-4-hydroxylase (C4H) controls the lignin branch. In the present study, three DEGs encoded C4H: two were down-regulated and one was up-regulated, indicating that C4H activity restricts the flux of phenylalanine into lignin biosynthesis. By contrast, nine DEGs encoded CCR; six were up-regulated and only three were repressed, demonstrating an accelerated flavonoid branch that diverts more phenylalanine toward flavonoid production. The resulting increase in flavonoid content lowers ROS levels, reduces membrane permeability, and elevates germination percentage. Zhang et al. [30] reported that abiotic stress down-regulated the entire phenylpropanoid pathway in potato (Solanum tuberosum), decreased lignin deposition, and spared phenylalanine for the synthesis of germination-essential proteins. The down-regulation of PAL observed here suggests that priming-induced repression is an energy-saving strategy that allows aged seeds to reallocate limited resources to repair and growth. Likewise, Xiao et al. [31] found that reduced C4H activity in salt-treated liquorice (Glycyrrhiza uralensis) inhibited lignin biosynthesis, but at the substrate level, promoted flavonoid accumulation.

The pentose phosphate pathway (PPP) is a cytosolic bypass of glycolysis that generates NADPH and ribose-5-phosphate (R5P) [32]. During germination, the massive demand for nucleotides required for DNA replication and RNA transcription makes R5P a critical metabolite; NADPH simultaneously fuels the antioxidant machinery. Gao et al. [33] reported that release of dormancy in orchid tree (Bauhinia variegata) seeds is accompanied by down-regulation of PFK1, which redirects carbon flux into the PPP to satisfy the need for nucleotide precursors and antioxidants. In the present study, the majority of DEGs encoding PFK were repressed. Reduced PFK activity lowers the rate of F6P → fructose-1,6-bisphosphate, leading to intracellular accumulation of F6P. The consequent increase in oxidative-phase flux accelerates the G6PDH and 6PGDH reactions, elevates NADPH output, and provides additional reducing power for the glutathione–ascorbate cycle, thereby enhancing ROS scavenging capacity [34].

Amino sugar and nucleotide sugar metabolism underpins the biosynthesis of numerous macromolecules essential for seed vigor [35]. Within this network, ADP-glucose pyrophosphorylase (AGPase) catalyzes the committed step that diverts glucose-1-phosphate into ADP-glucose for starch synthesis. Of the six DEGs encoding AGPase identified here, four were significantly up-regulated; AVESA_00001b_r3_5Ag0002180 showed a 1.4-fold increase, indicating elevated flux through this rate-limiting reaction. Consequently, a greater proportion of glucose-1-phosphate is channeled into starch biosynthesis, resulting in rapid deposition of amylose and amylopectin and replenishment of the seed’s reserve starch pool. The attendant rise in starch content strengthens carbon supply and osmotic adjustment, furnishing the energy and substrates required for repair metabolism and subsequent rapid germination of aged seeds. Wan et al. [36] similarly observed that up-regulation of AGPase activity elevates starch levels in Solomon’s seal (Polygonatum sibiricum) and enhances polysaccharide accumulation. Thus, the expanded starch reserves not only satisfy the ATP and reduce the power demands of naked oat seeds during the early phase of germination, but also mitigate aging-related damage through osmotic regulation, thereby providing an ample carbon and energy safeguard for aged oat seeds.

The protein–protein interaction network generated in this study identified 40S ribosomal protein S13 (RPS13) as the node with the highest degree of connectivity, positioning it at the topological center of the ZnO-NP priming response. Salih and Johnson [37] demonstrated that, although moderate reactive oxygen species (ROS) stress reduces the abundance of 80S ribosomes in Arabidopsis, a functionally active subset is retained to sustain protein synthesis under stress. Furthermore, the degradation rate of most ribosomal protein subunits (RPS/RPL) is slowed, creating an ‘aging ribosome reservoir.’ The elevated centrality of RPS13 observed in the present study aligns with this scenario, suggesting that RPS13 is preferentially preserved during ROS bursts and participates in the assembly of a stress-tolerant ribosomal subpopulation. Such selective retention would enable translational reprogramming that accelerates the synthesis of antioxidant and repair proteins, thereby providing a critical molecular target through which ZnO-NP priming ultimately enhances seed vigor.

Future research should focus on several key areas to build upon these findings. First, the functional validation of RPS13 and other hub genes identified in the protein–protein interaction network through transgenic or gene-editing approaches will confirm their essential roles in ZnO NP-induced seed rejuvenation. Second, the optimization of ZnO NP concentration, particle size, and treatment duration for large-scale agricultural application requires systematic field trials across different oat cultivars and environmental conditions. Third, the long-term effects of ZnO NP nano-priming on seed storability, field emergence, and grain yield need to be evaluated to establish standardized protocols for commercial seed production. Additionally, comparative transcriptomic and metabolomic analyses between ZnO NPs and other priming agents (e.g., hormones, beneficial microbes) would elucidate the unique and shared mechanisms underlying different seed enhancement strategies. Finally, the biosafety and environmental fate of ZnO NPs in agricultural ecosystems warrant thorough investigation to ensure sustainable application of this technology.

Collectively, the aforementioned findings demonstrate that ZnO NP nano-priming effectively restores seed vigor through integrated physiological enhancement and extensive transcriptional reprogramming. While these molecular mechanisms underscore the promising application of nano-priming technology in the seed industry, the potential toxicity and environmental implications of nanoparticle application in agricultural systems warrant careful consideration. Previous studies have demonstrated that ZnO NPs can undergo dissolution and transformation in soil environments, releasing Zn²⁺ ions that may induce oxidative stress and affect soil microbial communities at high exposure levels [38]. Notably, phytotoxic effects, including root growth inhibition and reduced seed germination, have been observed at concentrations exceeding 100 mg L⁻¹ [39]. However, the concentration employed in this study (30 mg L⁻¹) is substantially lower than the reported toxic thresholds, suggesting a favorable safety margin for practical application. Moreover, the seed priming approach adopted herein minimizes direct soil exposure compared to conventional foliar spray or soil drench applications, thereby significantly reducing potential environmental risks. Nevertheless, the long-term fate of ZnO NPs in agricultural soils, their potential bioaccumulation in food chains, and their effects on soil ecosystem functions under field conditions remain to be systematically evaluated. Future research should focus on establishing standardized protocols for nano-priming, including optimized concentrations, particle sizes, and application methods, to ensure the sustainable and safe use of this technology in commercial seed production.

5. Conclusions

ZnO-NP priming effectively restores the vigor of artificially aged naked oat seeds by integrating physiological enhancement with extensive transcriptional reprogramming. Physiologically, priming increased CAT, POD, and SOD activities by 3–4-fold, elevated germination rate from 75% to 98%, and enhanced seed vigor index. Transcriptome analysis identified 21,403 differentially expressed genes, with 15,841 stably expressed as priming-specific responders. Key molecular mechanisms include: (1) ROS burst-induced up-regulation of AP2/EREBP transcription factors that activate antioxidant enzyme genes; (2) redirection of phenylpropanoid metabolism toward flavonoid biosynthesis via CCR up-regulation and C4H down-regulation; (3) enhanced pentose phosphate pathway flux providing NADPH for redox homeostasis; (4) AGPase-mediated starch synthesis replenishing energy reserves; and (5) RPS13-centered ribosomal remodeling facilitating repair protein synthesis. Nine functional anti-aging core genes were identified: SOD (AVESA_00001b_r3_5Ag0002328), POD (AVESA_00001b_r3_4Ag0003736), CAT (AVESA_00001b_r3_7Cg0000067), CCR (AVESA_00001b_r3_5Ag0001296), AGPase (AVESA_00001b_r3_5Dg0001733), and RPS13 (AVESA_00001b_r3_7Cg0003030/3031). These findings provide immediate targets for molecular breeding of anti-aging naked oats and theoretical support for standardizing nano-priming technology in seed storage.