Non-thermal plasma enhances rice seed germination, seedling development, and root growth under low-temperature stress

Recently, non‑thermal plasma (NTP) technologies have found widespread application across diverse fields, includ‑ ing plant growth, medical science, and biological and environmental research. Rice ( Oryza sativa L. ) is exceptionally sensitive to temperature changes. Notably, low‑temperature stress primarily affects the germination and reproduc‑ tive stages of rice, often leading to reduced crop yield. This study aimed to identify optimal conditions for enhancing rice seed germination and seedling growth under low temperatures using NTP technology. Our research indicated that NTP treatment at 15.0 kV for 30 s optimally promotes rice seed germination and growth under low‑temperature stress. Furthermore, NTP treatment increases the activity and expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), under low‑temperature conditions. Moreover, it downregu‑ lates the expression of β‑ketoacyl‑[acyl carrier protein] synthase I (KASI) and cis‑epoxy carotenoid dioxygenase 3 (NCED3) and upregulates the expression of alternative oxidase (AOX1B), BREVIS RADIX‑like homologous gene (BRXL2), WRKY transcription factor 29 (WRKY29), and EREBP transcription factor 2 (EREBP2) in roots after tandem 7 days low‑ temperature (16 ℃ ) and 7 days room‑temperature (28 ℃ ) treatments. Transcriptomic analysis revealed the involve‑ ment of various key genes in phosphotransferase activity, phosphate‑containing compound metabolic processes, and defense responses. These analyses provide comprehensive information on gene expression at the transcriptional level, offering new insights for a deeper understanding of candidate genes required for root growth in rice.


Introduction
Rice is a staple food for more than half of the global population [1].Rice growth and production are significantly influenced by various factors, including soil nutrients, water quality, and temperature variations [2].Given its predominant cultivation in tropical and temperate climate zones, rice is sensitive to temperature changes [3].It is more susceptible to low-temperature stress than other cereal crops due to its tropical origins.Cold stress can adversely affect rice crops in all three rice-growing areas-tropical, subtropical, and temperate-adversely affecting every growth stage, from germination to harvest [4].The consequences of cold stress manifest in yield losses resulting from poor germination and seed establishment, stunted growth patterns, plant failure, sterile spikelets, delayed flowering, and reduced seed filling [5].Root distribution and development play vital roles in facilitating water and nutrient access and absorption in rice [6].The rice root system synthesizes trans-zeatin, a phytohormone and potent cytokinin, which enhances leaf photosynthesis and seed-filling capacity [7].The root system serves as a hub for synthesizing numerous hormonal substances.Notably, the yield of a rice plant is closely related to the number and vitality of its roots [8].Optimal root growth occurs at temperatures equal to or exceeding room temperature (25-28 ℃); however, at lower temperatures, root growth ceases and the risk of root rot increases [9].Minimizing these yield losses, especially in cold-affected areas, necessitates the identification and development of high-yield rice varieties with tolerance to low temperatures.
Low-temperature (non-thermal) plasma is the fourth state of matter, following the gaseous, liquid, and solid states.Gas molecules typically disperse at ignition voltage, producing a mixture of states encompassing electrons, ions, atoms, and free radicals.Despite the high electron temperature during the discharge process, the temperature of the heavy particles remains low, creating a system characterized by a low-temperature state, called low-temperature (non-thermal) plasma [10].Nonthermal plasma (NTP) is a novel and promising agricultural technology that can improve plant performance by modulating gene expressions associated with seed germination, plant immune responses to abiotic stresses, pathogen resistance, and growth regulation [11].
Numerous strategies for improving plant growth processes and defense mechanisms have been explored [12].Among these methods, NTP stands out as a potential alternative to traditional seed treatments, such as physical scratching and thermal or chemical treatments [13].In 2017, Zhang et al. reported an increase in germination and growth rates of soybeans following NTP treatment [14].The application of NTP to improve seed germination and plant growth has been investigated in various species, including sunflower [15], corn, maize [16], rye [17], and pea [18].NTP treatment promotes seed germination by inducing the production of certain active substances, which, in turn, induce a range of biochemical changes.Furthermore, free radicals, such as reactive oxygen species (ROS) or reactive nitrogen species (RNS), produced through NTP treatments can interfere with the abscisic acid and gibberellin pathways, affecting redox homeostasis and breaking dormancy [19].Seed dormancy is a protective mechanism that facilitates seed survival in harsh environments.However, breaking seed dormancy is a prerequisite to seed germination.Following NTP treatments, UV radiation, chemical radical production, and chemical reactions alter the seed coat, promote root and stem development, stimulate root production, and accelerate the germination process [20].Under short-term NTP treatment, ROS and RNS act as signaling molecules regulating seed growth and development.
Some studies have demonstrated that NTP treatment alters the surface properties of rice seeds [21], accelerates germination speed, and increases water absorption.Moreover, it completely inactivates pathogenic fungi and other microorganisms, thereby improving germination rate and seedling quality [22].Low-pressure dielectric barrier discharge (LPDBD) plasma (produced using Ar + Air) increases ROS production in the leaves and roots of rice plants.The increase in ROS, which is meticulously regulated by the upregulation of CAT activity, ultimately promotes the germination and growth of rice seedlings [23].In the present study, we scrutinized the impact of argon dielectric barrier discharge (DBD) NTP treatment on seed germination and growth patterns in rice under cold stress.Specifically, we evaluated the effects of NTP generated at different voltages on rice seed germination and root growth and development under low temperatures.We also explored the role of NTP in regulating seed antioxidant enzyme activities and gene expressions related to seed growth and root development.Our findings offer supporting evidence for the effective application of NTP technology in rice cultivation.

NTP-generating device and treatment
A DBD-based NTP generator, operating at atmospheric pressure, was utilized as previously described.A schematic representation of the device is depicted in Fig. 1A.Pure argon gas was fed into the NTP generator at a flow rate of 5 L/min.The output voltages were set at 0, 13.9, 15.0, or 19.7 kV, with a fixed treatment time of 30 s.

Seed germination and growth analysis
Full, intact rice seeds (Long Jing, Daqing Branch of the Heilongjiang Academy of Agricultural Sciences, Daqing, China; n = 90) were treated with NTP at the indicated voltages (0, 13.9, 15.0, or 19.7 kV) for 30 s at 25 °C.Seeds were soaked in water for 12 h before NTP treatment.The germination rate was recorded daily during the tandem 7 days low-temperature (16 °C) and 7-d roomtemperature (28 °C) treatments, with seeds incubated at 100% relative humidity.The germination rate was calculated using the following formula: Germination Rate (%) = (Number of Germinated Rice Seeds/Total Number of Rice Seeds) × 100%.Additionally, another set of seeds, previously subjected to NTP treatment, was sown in the soil, one plant per pot, and grown in an artificial climate room with 25 °C temperature, 10/14 h photoperiod, and 75% relative humidity.Seedling emergence was recorded when the rice seedlings emerged from the soil.The length and fresh weight of both roots and stems were measured 14 days after sowing.The emergence rate was recorded and calculated using the same formula as that employed for the germination rate.The experimental design followed a completely randomized layout with three replications.

Superoxide dismutase (SOD) and catalase (CAT) activity assays
The control group consisted of rice seeds without NTP treatment, whereas the experimental group encompassed rice seeds treated with 13.9, 15.0, or 19.7 kV NTP for 30 s.Following 14 days of growth, the rice seeds were homogenized in cold phosphate-buffered saline and then centrifuged at 12,000 rpm at 4 °C.The resulting supernatant was collected for soluble protein concentration analysis.SOD and CAT activities were analyzed using the corresponding assay kits from Sigma-Aldrich, according to the manufacturer's instructions.

Western blotting
Proteins extracted from rice leaves and roots (15 μg) were separated on 12% SDS-PAGE and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).The membranes were incubated with primary antibodies overnight at 4 °C and then washed 5 times using Tris-buffered saline with 0.05% Tween 20 (TBST).The membranes were then incubated with skim milk for 10 min and with secondary antibodies for 1 h at room temperature (28 °C).Excess secondary antibodies were washed off using TBST, and specific binding was detected using a chemiluminescence detection system (Amersham, Berkshire, UK).

Quantitative real-time PCR (qPCR)
RNA from leaves of both the control and experimental groups was extracted using TRIzol (Gibco BRL, Life Technologies, Roskilde, Denmark).cDNA was synthesized using the cDNA Synthesis SuperMix (Human Innovagene Biotech Co. Ltd., Hunan, China).Primers were synthesized based on gene sequences (Table 1).qPCR was performed using SYBR Green QPCR Mix (Human Innovagene Biotech Co. Ltd.) on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

RNA sequencing
RNA was extracted using TRIzol reagent (Sigma, St. Louis, MO, USA) and analyzed via mRNA sequencing on a HiSeq system (personal, Nanjing, China).The differences in the gene expression levels between the two sample groups (0 and 15 s) were compared using DESeq2.Pathway analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Statistical analysis
Data are presented as the mean ± standard deviation from three independent experiments.Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS version 19.0; IBM, Armonk, NY, USA) and the two-way ANOVA (p < 0.05 was considered significant) analysis.

NTP treatment accelerates rice seed germination
We investigated the impact of NTP treatment on the germination rate of rice seeds under low-temperature conditions.The germination of rice seeds was recorded on days 6, 7, and 8, with photos taken for comprehensive documentation (Fig. 1B).The growth of rice seedlings was recorded on day 14, accompanied by photographic documentation (Fig. 1C).Analysis of the germination rate data revealed that NTP treatment at 15.0 kV for 30 s significantly increased rice seed germination in the early stages (6-7 days).Nevertheless, the total germination rates did not exhibit any significant differences (Fig. 1D).

NTP treatment promotes rice seedling growth
Low temperatures can prolong the germination time of rice, resulting in frail, decay-prone seedlings with a higher susceptibility to diseases.We observed accelerated rice seed germination following NTP treatment and further explored the impact of NTP treatment on rice traits.NTP-treated and control seeds were sown in Petri dishes and subjected to tandem 7 days low-temperature (16 °C) and 7 days room-temperature (28 °C) treatments in seed germination boxes with constant temperature and humidity.Compared to the other groups, the group treated with NTP at 15.0 kV for 30 s did not show a significant difference in plant fresh weight (Fig. 2A), leaf dry weight (Fig. 2C), or root fresh weight (Fig. 2E).However, the leaf fresh weight (Fig. 2B) and root dry weight (Fig. 2F) in the 15.0 kV for 30 s group were higher than those in the other groups.NTP treatment also increased the leaf length (Fig. 2D), taproot length (Fig. 2G), and lateral root length (Fig. 2H) of rice seedlings but did not affect the average number of lateral roots (Fig. 2I).These results suggest that NTP treatment at 15.0 kV for 30 s significantly enhances the growth of rice seedlings.

NTP treatment increases root soluble protein levels and antioxidant enzyme activities
Plant growth and development in low-temperature environments are intricately linked to the activity of antioxidant enzymes.Our experiments revealed that NTP treatment increased root SOD and CAT activities but did not affect leaf SOD and CAT activities (Fig. 3A,  B).Furthermore, we analyzed the concentrations of soluble protein in roots and leaves.Compared with the control group, NTP treatment at 15.0 kV for 30 s significantly increased the soluble protein concentration in roots but not in leaves (Fig. 3C).The protein expression levels of SOD and CAT, assessed through Western blotting, were significantly higher in the roots of the NTP treatment group than in those of the control group (Fig. 3D, E).These results demonstrate that NTP treatment at 15.0 kV for 30 s effectively reduces ROS levels in the roots of rice seedlings.

RNA sequencing analysis of the rice transcriptome
To investigate the effects of NTP treatment on seed growth, root development, and cold resistance genes, we analyzed the whole transcriptome of the control and NTP treatment (15.0 kV for 30 s) groups using RNA-Seq technology.The RNA-Seq data, analyzed using Pearson's correlation coefficient, revealed strong correlations in both the control and NTP treatment groups (Fig. 4A), validating their suitability for further experimentation.We utilized DESeq for differential gene expression analysis, identifying differentially expressed genes (DEGs) with the criteria: |log2FoldChange|> 1 and P value < 0.05.Compared to the control group, 3498 DEGs (1660 upregulated and 1838 down-regulated) were identified in the NTP treatment (15.0 kV for 30 s) group (Fig. 4B).The Euclidean method was used to calculate distances and hierarchical clustering was performed using the longest distance method (complete linkage) (Fig. 4C).The DEGs were visualized on a heatmap (Fig. 4D).Two-way cluster analysis of cascaded DEG sets and samples further delineated gene expression levels in different samples and the expression patterns of different genes in the same sample.Furthermore, we analyzed the KEGG pathways of all DEGs (Fig. 4E) and screened for significant signalling pathways.The top 20 gene ontology (GO) term entries, selected based on the lowest false discovery rate (FDR) values, indicated significant enrichment in transferase activity, defense response, kinase activity, and phosphatecontaining compound metabolic processes, which are integral to rice seed development, root growth, and cold tolerance (Fig. 4F).

Effect of NTP treatment on germination-associated gene expressions against cold stress in rice seeds
We further analyzed RNA-Seq data from the control and NTP treatment (15.0 kV for 30 s) groups, focusing on DEGs related to seed germination, root development, and cold tolerance and validated through qPCR.
The expression levels of the plant antioxidant system enzymes CAT, SOD, and POD in the treatment group were higher than those in the control.In contrast, the expression levels of the fatty acid synthesis inhibitor gene β-ketoacyl-[acyl carrier protein] synthase I (KASI) (Fig. 5D) and a key enzyme in abscisic acid (ABA) anabolism, 9-cis-epoxycarotenoid dioxygenase (NCED3) (Fig. 5F), in the treatment group were lower than those in the control.Meanwhile, the expression levels of the alternative oxidase gene (AOX1B) (Fig. 5E), BREVIS RADIX-like homologous gene (BEXL2) (Fig. 5G), WRKY transcription factor 29 (WRKY29) (Fig. 5H), and EREBP transcription factor 2 (EREBP2) (Fig. 5I) in the treatment group were higher than those in the control.These data indicate that NTP treatment promotes seed germination and root development and increases cold tolerance

NTP treatment promotes rice seedling growth in the soil
Previous experiments in this study confirmed the positive effect of NTP on rice seed germination.To further simulate rice cultivation conditions and assess the influence of soil composition on the effects of NTP, rice seeds from both the control (NC) group and those treated with NTP (0, 13.9, 15.0, or 19.7 kV for 30 s) were grown in the soil.The growth period included 7 d at low temperature (16 °C) followed by 7 days at room temperature (28 °C).
Seedlings were assessed and photographed on day 14 (Fig. 6A).Seedlings with lengths greater than 0.1 cm or 0.5 cm were considered for quantifying germination rates.The group treated with 15.0 kV for 30 s exhibited higher germination rates compared to the other groups (Fig. 6B, C).Additionally, NTP treatment significantly increased plant (Fig. 6E) and root fresh weights (Fig. 6I) and root length (Fig. 6H).However, no significant differences were observed among the groups in terms of plant height (Fig. 6A), leaf length (Fig. 6F), and leaf fresh weight (Fig. 6G).

Discussion
The application of NTP has shown promising results in plasma medicine, particularly in enhancing wound healing and blood coagulation and eliminating microorganisms [24].These findings have spurred widespread interest in the potential benefits of NTP in agricultural production.In addition to purifying seed surfaces, NTP treatment has been shown to promote seed germination and plant growth [25].Research on the role of NTP in plant growth has demonstrated its effectiveness in boosting the seed germination and seedling growth of various plants.The impacts of NTP have been examined in several plant species, such as wheat [26], pepper [27], maize [16], peas, and oilseed rape [28], with studies consistently showing an increase in both germination rate and speed.Starič et al. studied the effect of NTP on the germination and early growth of two winter wheat cultivars (Triticum aestivum and Bezostaya) [29].However, the influence of NTP on the germination and growth of rice seeds under low-temperature conditions has not been studied previously.This study addresses this gap by investigating the changes in the germination rate and growth vigor of rice seeds treated with NTP (produced at different voltages) Plants exposed to various stressors typically experience ROS formation and accumulation, leading to oxidative stress.POD, SOD, and CAT are key enzymes in the plant's antioxidant system, and their activity levels are indicative of plant stress levels [30].SOD catalyzes the conversion of the superoxide anion into hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ), playing a pivotal role in free radical scavenging within living organisms.Subsequently, both CAT and POD function to scavenge H 2 O 2 .Thus, these three enzymes act synergistically to maintain the free radical content within plant tissues at a homeostatic level, thereby preventing physiological and biochemical changes induced by free radicals [31].Plants typically mitigate the adverse effects of stress by increasing the expression or activity of both enzymatic and nonenzymatic antioxidants.Notably, the treatment of soybean seeds with argon NTP has been demonstrated to increase the activities of SOD, POD, and CAT.A similar increase in antioxidant enzyme activity was also noted post-NTP treatment in wheat [32].
The results of our study also indicate that the antioxidant capacity of rice roots increased following NTP treatment, whereas the leaves showed no change, at low temperatures.This enhancement in root antioxidant capacity may contribute to the improved germination and growth of rice seeds, facilitating water and nutrient uptake and bolstering resistance to low temperatures.Plants respond to external stresses, such as drought, pests, diseases, and low temperatures, through multiple mechanisms, including alterations in gene expression.Zhang et al. studied the effects of NTP treatment on the expression of specific genes and found increased expression in genes related to ATP synthesis, GRF1-6 (growthregulating factor), and TOR (target of rapamycin); this increased expression was correlated with decreased methylation at these loci [14].These genes play crucial roles in metabolism and growth regulation; therefore, these results indicate that NTP treatment may have a regulatory impact on these processes at the molecular level.NTP treatment also influences the expression of drought-response genes, such as LEA1, SnRK2, and P5CS [33], and genes encoding antioxidant enzymes [34].These findings point to a complex mechanism by which NTP achieves enhanced germination, seedling development, and root growth in rice under low-temperature conditions.RNA sequencing analysis revealed that the expression of β-ketoacyl-[acyl carrier protein] synthase I (KASI) and cis-epoxy carotenoid dioxygenase 3 (NCED3) decreased, whereas that of AOX1B, BEXL2, WRKY29, and EREBP2 increased post-NTP treatment.OsKASI [35], which is involved in fatty acid synthesis, impacts root and seed development, with KASI mutants showing altered root lengths, tiller numbers, spike lengths, and fatty acid content [35].Abscisic acid (ABA) plays a crucial role in seed germination, dormancy, and stress response.Disruptions in ABA synthesis, which mainly occur in chloroplasts, via direct and indirect pathways significantly affect physiological activities [36,37].Knockdown of OsNCED3, an enzyme in ABA biosynthesis, alters ABA and gibberellin levels, affecting seed dormancy and germination [38,39].The cyanide-insensitive alternate oxidase (AOX) pathway for the mitochondrial electron transport chain involves AOX1B, which enhances cold tolerance in rice [40].The BREVIS RADIX gene family, including OsBRXL2, is involved in root growth and stress responses [41,42].WRKY29, a transcription factor, regulates ethylene production pathway genes, influencing stress response and seed dormancy [43,44].The EREBP subfamily genes are induced under low temperatures and drought and enhance plant resistance and tolerance to these conditions [45].These results suggest that NTP treatment increases cold resistance in rice by promoting seed germination and enhancing root growth and development.While we identified the genes influenced by NTP treatment under low-temperature conditions, the underlying mechanisms remain to be elucidated and will be the focus of future research.NTP has the potential to improve crop seed vitality, germination, and crop yield, reducing reliance on pesticides, hormones, and chemical fertilizers and minimizing environmental pollutants.As NTP technology advances, it can offer diverse applications in agricultural production, reducing cultivation costs and expanding arable land for rice cultivation, both of which are crucial for addressing food security challenges.

Fig. 1
Fig. 1 NTP treatment accelerates rice seed germination.A Schematic diagram of the NTP generator.B-D Germination of seeds treated with NTP at 0, 13.9, 15.0, and 19.7 kV for 30 s. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control ATA ACT CGA CGG ATC GC R: CTT GGA TGT GGT AGC CGT TT AOX1B F: AGG AGG ACG GCA CGG AAT GG R: CTT CGG CTC GTG GTG CTT CG EREBP2 F: CCT CCA GTC AAA CCT CTT GCT TCC R: GTG TCA TCC TCC CAA CCA AAG TCT G BRXL2 F: GCA CCA CTT CAA CCT CCT CAA CTC R: CGT CCT GCT CCA CCC ACT CC WRKY29 F: ACA CCT ACA GTC GCC AGT TTC ATT G R: CGC ATA ATC GTC CGC CTC CATC KASI F: CGC TTC GAC GCC TCC AAC TTC R: GCC TTC TTG CCG CTG ACG ATG Cu/Zn-SOD F: TGG CAG AGC CGT CGT TGT R: CGA TGA TCC CGC AAG CAA POD F: AGG CCC AGT GCT HCAMCTTC R: TTG GTG TAG TAG GCG TTG TC Fe-SOD F: CCA GAA GGT GGT GGG TCA R: CTG AGC GTA TAA ACT CTT CCCTA Mn-SOD F: TTT GGT TCA TTT GAG GCA CT R: GTC CTG GTT AGC AGT TGT TTCC CAT 1 F: ATA AGG CCA GAC AAT GTC AGATG R: CAG TGG CAT TAA TAC GCC AGTA CAT 2 F: GCA CAG TTT GAC AGG GAG R: GGT CTG AAC ACC AGG AGC CAT 3 F: CGG TGG TGA TTG CCA AGG AG R: CCT GTC AGC CTG AGA CCA GTA

Fig. 2
Fig. 2 NTP treatment enhances rice seedling growth.A Seedling height 14 days post-NTP treatment.B-D Changes in leaf fresh weight, leaf dry weight, and leaf length.E-I Changes in root fresh weight, root dry weight, root length, lateral root length, and lateral root number.Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control

Fig. 3
Fig. 3 NTP treatment increases root soluble protein levels and antioxidant enzyme activities.A Catalase (CAT) and (B) superoxide dismutase (SOD) activities in leaves and roots after 7 days at low temperature (16 °C) and 7 days at room temperature (28 °C) post-NTP treatment.C Protein concentrations in leaves and roots 14 days after NTP treatment at 15.0 kV for 30 s. D, E Catalase and SOD protein expressions in leaves 14 days post-NTP treatment.Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control

Fig. 4
Fig. 4 RNA sequencing analysis of the rice transcriptome.A Sample correlation analysis.B The number of differentially expressed genes (DEGs).C Differential gene clustering analysis.D Clusters of genes with representative expression patterns.E KEGG enrichment analysis based on gene count.F Gene expression profile grouped by characteristics.Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control

Fig. 6
Fig. 6 NTP treatment promotes rice seedling growth in the soil.A Seedling growth on day 14 post-NTP treatment at 15.0 kV for 30 s in the soil.B, C Germination rates categorized by leaf length.D-I Measurements of plant height, plant weight, leaf length, leaf fresh weight, root length, and root fresh weight after 7 days at low temperature (16 °C) and 7 days at room temperature (28 °C) post-NTP treatment.Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control