Effect of Plasma Activated Water on Peanut Seed Germination, Seedling Biomass, Morphology, and Gene Expression

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Priatama, Taewon Yang, Seung-Won Lee, Kibum Kim, Hyunjoo Hwang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3820334/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plasma-activated water (PAW) has been shown to enhance seed germination and seedling growth across various plants. This study investigates the impact of PAW on peanut ( Arachis hypogaea L.) seed germination, viability, growth and gene expression in sprouts. Seeds were treated with PAW for 0, 40, and 80 minutes and sown in fermented oak sawdust. Germination rate, weight, length, and seedling vigor index were assessed. Among the different PAW treatments, the longest true leaf with epicotyl and the longest hypocotyl were observed in PAW80 (7.3 cm and 3.1 cm, respectively). Specifically, the root length was observed the longest in PAW80 (10.4 cm). The PAW80 had the highest vigor index (1385.1), followed by PAW40 (1048.7). Gene ontology analysis revealed that shoot growth related gene expression in PAW40 and PAW80 peanut groups relatively higher than PAW 0 group. Overall, PAW80 provided the most favorable conditions for peanut sprout growth, promoting shoot-growth related gene expression. Our findings suggest that PAW positively affects peanut and seedling growth by the regulation of shoot promoting genes. Arachis hypogaea Peanut sprout Plasma activated water Vigor index Transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Plasma-activated water (PAW) is generated by cold atmospheric plasma (CAP) treatment of water with a plasma plume above or below the water surface [1]. The plasma generated ionized gas interact with water molecules and trigger several chemical reactions to create a unique biochemical reactive species. During PAW generation, energetic particles in the plasma phase are trapped in the aqueous liquid and a series of reactions are initiated at the gas liquid interface, leading to a variety of primary and secondary reactive species that are soluble in the liquid [2,3]. These reactive species, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are responsible for the chemical and biological effects of PAW. In recent years, PAW has been found to possess outstanding biological and agricultural sectors [4,5]. Plasma direct or indirect treatment on seeds promotes seed germination and growth in plants due to the reactive species, electric field, charged particles, photons and changed physicochemical properties of PAW [6]. PAW with beneficial physico-chemical properties has effects on promoting seed germination and plant growth [7]. Seed germination is a process frequently applied to increase beneficial biochemical contents and nutrients [8]. Peanut sprouts are rich in amino acids, minerals, carbohydrates, fatty acids, and antioxidants, while containing less fat than seeds. Seed germination, crucial for plant sprouts, is influenced by environmental factors such as soil, light, temperature, water, and pH [9]. In our previous studies, it was optimized to increase the seed germination rate, sprout biomass, and sprout morphology quality following the seed sowing direction and fermentation period of oak sawdust [10,11]. However, PAW's impact on peanut seed germination and seedling growth remains unexplored. In plasma agriculture, several studies have performed gene expression analysis to explore the underlying mechanisms of plasma treatment in molecular levels. These analyses often involve the use of advanced techniques such as quantitative real-time PCR and transcriptome sequencing, which allow researchers to understand how plasma treatment affects various biological processes, from seed germination to plant growth and development. Recent studies using transcriptome analysis of plasma treated Arabidopsis seeds has revealed the regulation of germination trough stress regulation pathways [12,13]. Transcriptome analysis serves as a powerful tool for studying global gene expression patterns, providing insights into the regulatory networks and pathways activated by PAW treatment [14]. This approach facilitates the identification of differentially expressed genes (DEGs) and the functional annotation of these genes, shedding light on the molecular processes involved in PAW-induced seed germination and growth. This analysis not only enhances our understanding of the molecular mechanisms underlying the beneficial effects of PAW on peanut seed germination and growth, but also lays a foundation for future research on the application of PAW in agriculture. In this study, we investigated the effects of PAW on peanut seed germination and seedling growth followed by a transcriptome analysis to identify differentially expressed genes (DEGs) in peanut seedlings post PAW treatment. Our final objective was to elucidate the identified DEGs and explore into an analysis of the associated biological processes and pathways. This approach provided us with a deeper understanding of the molecular mechanisms at play, furthering our knowledge of the impact of PAW on plant growth and development. Materials and Methods PAW generation and physicochemical analysis PAW was generated using plasma reactor devices consist of two surface dielectric barrier discharge (SDBD) which an alternating current (AC) was used as a power source. The plasma generator has been described by [15,16]. The production of PAW 0, 40, and 80 was based on plasma treatment time (0, 40, and 80 minutes, respectively). Immediately after PAW generation, the anion content of PAW was measured using ion chromatography (ICS-2100, Thermo Dionex, Sunnybale, CA, USA). Hydrogen peroxide content was measured using LR method as described previouslyb [15]. Plasma activated water cultivation system for seed germination Peanut ( Arachis hypogaea L.) seeds were sown in trays (50×30×10 cm; Yesan, Korea). Seeds were planted side by side using 1,500 ml of 60 days fermented oak sawdust [17] mixed with 1,500 ml of PAW 0, PAW40, and PAW80. The seed row spacing, and sowing depth were set to 3 cm. All 18 seeds were sown in the same direction, 3 per row. The tray was moved to a dark room at 30℃, and 200 ml of each PAW were given twice per day for 10 days. Measuring seed germination, seedling weight, and tissue length Seedlings were removed from each tray after 10 days of culture to determine seedling weight and true leaf with epicotyl, hypocotyl, and root length. Whole seedlings were washed with tap water and blotted using paper towels (Yuhan Kimberly, Seoul, Korea). The seed germination rate, true leaf with epicotyl, hypocotyl, and root length were measured. Vigor index The vigor index of peanut seedling groups treated with three different PAWs (PAW 0, PAW40, and PAW80) were investigated to determine which PAW conditions positively give the best seedling biomass production. The vigor index was calculated using the formula (germination rate × length of each tissue part) [18]. Morphological Observation The peanut sprouts were uprooted on 10 days after seedling. The harvested peanut sprouts were placed on black superfine fibers, and photographed using an Apple iPhone camera (Apple, Cupertino, CA, USA) Total RNA extraction from peanut sprouts Total RNA was extracted from peanut seedlings grown under the PAW 0, PAW40, and PAW80 treatment for 10 days, respectively. Fresh seedlings were sampled and ground in liquid nitrogen. The RNA was extracted using the MiniBEST Plant RNA Extraction Kit, as previously described (Takara, Seoul, Korea) [19]. Extracted RNA was stored in a deep freezer (-70°C) for further study. RNA-sequencing and Gene Ontology Analysis RNA quantity and quality were analyzed using NanoDrop8000 system (NanoDrop Technologies, Wilmington, DE, USA) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively. One µg of total RNA was used in library construction using a Truseq Stranded mRNA Prep Kit (Illumina, Inc. San Diego, CA, USA), according to manufacturer's protocol. Libraries from 70–370 bp (mainly 150 bp) were constructed and sequenced using Illumina NovaSeq 6000 sequencing system (Illumina, Inc. San Diego, CA, USA) to generate 2x100 bp paired-end reads. FastQC v0.11.7 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) was used to check the quality of the raw reads, and Trimmomatic v0.36 was used to remove adaptor sequences and low-quality reads. An alignment process was performed using Tophat (v2.0.13) mapped to Arachis hypogaea v1.0 database. The abundance calculation and differentially expressed genes (DEG) was analyzed using Cuffdiff (v2.2.0). Further analysis was performed using DEG that satisfies 2-fold change and p value < 0.05 and FDR < 0.1. The Venn diagram analysis was carried out using the VENNY (v2.1) webtool, accessible at https://bioinfogp.cnb.csic.es/tools/venny/ . The Gene Ontology (GO) analysis was performed using the ShinyGO (version 0.77) software, as described by [20]. The heatmap analysis was performed utilizing the 'pheatmap’ v1.0.12 package in R using Rstudio 2023.03.0 Build 386. Results Physicochemical properties of PAW Analysis of physicochemical properties revealed an increase in several chemical components of PAW that was linearly related to the duration of plasma treatment (Table 1 ). The pH values for PAW40 and PAW80 were lower, at 2.52 and 2.28 respectively, than the control, which was 6.96. In terms of nitrate (NO3-, mg/L), PAW80 demonstrated a higher mean level (831) compared to PAW40 (383). This trend was also observed for nitrite (NO2-, mg/L), with PAW80 (5.12) displaying a higher mean level than PAW40 (3.017). For conductivity (µs/cm), PAW80 (3,252) again showed a higher mean level than PAW40 (1,558). No traces of nitrate or nitrite were detected in the control (PAW0). However, the concentration of hydrogen peroxide (H 2 O 2 ) did not appear to be noticeably affected by the plasma treatment. Table 1 Physicochemical properties of the PAW 0, PAW40, and PAW80 Treatment time pH Nitrate NO 3 - (mg/L) Nitrite NO 2 -(mg/L) H 2 O 2 (mg/L) Conductivity (µs /cm) 0 6.96 ± 0.09 nd nd nd 1.48 ± 0.18 40 2.52 ± 0.08 383 ± 5.88 3.07 ± 0.80 0.03 ± 0 1558 ± 53.90 80 2.28 ± 0.08 831 ± 10.41 5.12 ± 1.74 0.01 ± 0 3252 ± 89.02 The results of the Table 1 are the mean ± standard deviation (SD). nd, not detected. Effect of PAW on Peanut Seedling Peanut seedlings were uprooted, washed, and weighed 10 days after sowing. Phenotypically an obvious phenotypic variation was clearly shown in PAW treated seedlings (Fig. 2 a-c). To measure seedling length, three different seedling tissue were classified as true leaf with epicotyl, hypocotyl, and root. In seedling true leaf in PAW80 condition started to come out first at 6 days after sowing. In the mean length of true leaf with epicotyl, the seedlings in PAW80 (7.3 cm) showed the longest compared to PAW 0 (2 cm) and PAW40 (4.7 cm) (Fig. 2 d). In the mean of hypocotyl length, the seedlings in PAW40 (5.3 cm) and PAW80 (3.1 cm) had significantly longer lengths than PAW 0 (2.3 cm) (Fig. 2 e). In root length, the seedling in PAW80 (10.4 cm) showed the longest length than the others [PAW 0 (2 cm) and PAW40 (3.8 cm)] (Fig. 2 f). The seedling with PAW80 had the highest mean weight (5.1 g) than the others. The mean seedling weights of the PAW 0 and 40 were 3.3, and 4.5 g, respectively (Fig. 2 g). Effect of PAW on Vigor Index To identify the optimal plasma exposure time to water for efficient peanut sprout biomass production, vigor indexes of seedlings in PAW 0, 40, and 80 were computed and are presented in Table 2 and Fig. 3 . The highest total vigor index was observed in the PAW80 group, registering a value of 1385.1. Conversely, the PAW 0 group, which received no plasma treatment, had the lowest total vigor index, with a value of 213.0. The PAW40 group, with a plasma exposure time of 40 minutes, recorded a total vigor index of 1048.7, ranking second highest among the groups. It was noted that the total vigor index values for the groups treated with plasma-activated water (PAW 40 and PAW 80) were higher than the group without plasma treatment (Fig. 3 ). Table 2 Calculation of Vigor Index (VI) and total VI on seedling each water exposed to plasma time. Length Vigor Index (VI) PAW Z Germination rate (%) True Leaf with Epicotyl Hypocotyl Root True Leaf with Epicotyl Hypocotyl Root Total VI 0 33.3 ± 1.4 2.0 ± 0.4 2.3 ± 0.1 2.1 ± 0.3 33.3 ± 1.4 66.7 ± 3.5 51.6 ± 4.9 151.6 ± 9.8 40 77.7 ± 1.4 4.7 ± 0.2 5.1 ± 1.0 3.7 ± 0.2 358.4 ± 2.6 235.1 ± 2.6 292.4 ± 0.3 885.9 ± 5.4 80 66.6 ± 1.4 7.3 ± 0.3 3.1 ± 0.1 10.4 ± 0.4 514.1 ± 8 213 ± 5.1 680.4 ± 18 1407.4 ± 31.1 Z PAW; 0: water unexposed to plasma, 40: 40-hour water exposed to plasma, 80: 80-hour water exposed to plasma. y Vigor Index (VI) = Germination Rate (%) × Length (cm). x Total = VI (True leaf with epicotyl) + VI (Hypocotyl) + VI (Root) Gene regulations in PAW-induced peanut sprouts To investigate the regulation of gene expression of seedlings growth in PAW treatment, the RNA sequencing data were generated from peanut samples including DW, PAW40 and PAW80. From the average of 41 million reads, around 37 million clean reads (~ 91%) per library were retained, with approximately 91% mapping rate to the A. hypogea reference genome (Additional file 1: Table 1 ). As shown in Fig. 4 a-b, the identified gene expressions are plotted, and the volcano plot displays the fold-change significance for each gene between control and treatment. By filtering of 2fold & p -value < 0.05 & FDR < 0.1, we generated heatmap to demonstrate the samples from each replicate were clustered together despite of the variations among replicates (Fig. 4 c-d). In detail, the analysis revealed that in total 5428 and 7911 genes were differentially expressed in PAW40 and PAW80, respectively (Additional file 1: Fig. S1 ). Among them, 734 and 2313 genes were uniquely upregulated in PAW40 and PAW80 respectively, 1624 genes were upregulated in both PAW40 and PAW80 (Fig. 4 e). Additionally, 1453 genes were uniquely downregulated in PAW40, 2505 were downregulated in PAW80, and 2655 genes were downregulated in both treatments (Fig. 4 f). To obtain a general overview of the PAW regulation in peanut seedling growth, Gene ontology analysis (GO) was performed using the overlapped DEG of PAW40 and PAW80. We separately analyzed the upregulated and downregulated DEGs and showed the top 20 GO terms which are highly enriched (Fig. 5 a-c, and Additional file 1: Fig. S2 ). In GO: plasma treatment affects various biological processes categories including shoot system development, organelle organization, post-embryonic development, RNA processing, and more (Fig. 6 a). In cellular components, various cellular component functions are enriched (Fig. 6 b). In addition, the molecular function also revealed enrichment of enzyme activity and signaling pathway (Fig. 6 c). In contrast, the overlapped downregulated genes showed enrichment in function related to response to external stimulus such as biotic stimulus, hormone and lipid (Additional file 1: Fig. S2 b). Whereas the cellular component and molecular function show various enrichment in extracellular region and kinase activity, respectively (Additional file 1: Fig. S2 ). Given that PAW treatment dramatically affects GO term of shoot system development. We further analyze the GO-network of the upregulated genes in shoot system developments. The network showed the relation of each enriched pathway, which overall significant connection to the plant development (Fig. 6 d). Additionally, we isolated shoot system development genes which are highly enriched and confirm the expression by generating heatmap as shown in Fig. 6 e. The heatmap showed more genes were upregulated in the shoot system development both in PAW40 and PAW80 and the most upregulated genes were found in PAW80. To confirm more specific regulation of PAW in the affecting seedling development. We isolated several marker genes and GO term which potentially co-related with the improved growth in PAW treatment. In Fig. 6 a, plant development related marker genes and nitrate transport are shown the DE gene expression. LATERAL ORGAN BOUNDARIES (LOB), CUP SHAPED COTYLEDON (CUC), GIGANTEA, SHOOT-GRAVITROPISM 5 (SGR5), AUXIN RESPONSE FACTOR (ARFs), PHYTOCHROME-INTERACTING FACTOR (PIFs), NITRATE TRANSPORTER (NRTs) genes are generally up-regulated in PAW40 and PAW80. Interestingly, those genes are more strongly enhanced in PAW80 condition. Since PAW mainly contains nitrate, we show additionally the expression of genes that are related to nitrogen response of the cells, nitrogen compound catabolic process and cell response to nitrogen. Indeed, it showed a similar pattern of induction in both PAW treatments (Fig. 6 b). Furthermore, we identified downregulated genes which appear to function as negative regulators in nitrate response and shoot development by PAW treatment (Fig. 6 c-d). Such as family member of auxin response factor (ARFs) family, flowering locus K, and brassinosteroid insensitive 1 (BR1). This suggests that shoot system development downregulation of specific genes might contribute to the enhancement of seedling growth and affected by PAW treatment. Discussion In this study, we aimed to investigate the effects of PAW on peanut seed germination, sprout tissue biomass, morphology, and gene expression of PAW treatment according to the peanut phenotype. Determining optimal PAW conditions is crucial for efficient seedling growth, as various plant species exhibit unique responses to PAW at different exposure times. In addition, understanding the gene regulation through transcriptome analysis can provide valuable insights into the mechanism of PAW in the regulation of growth in peanut sprouts. In the seedling weight, the PAW80 showed the highest level, followed by the PAW40. In the seedling length, the PAW80 had the longest lengths of true leaf with epicotyl, hypocotyl, and root. The PAW80 yielded the highest vigor index, followed by the PAW40. It is interesting that in peanut seedlings treated with PAW80, the enhanced root formation was clearly observed. This morphological observation is not unexpected since it was reported in previous studies that plants treated with PAW had enhanced root growth [15,16]. The PAW contains chemical molecules such as nitrate, and nitrate with high electrical conductivity which can be used as inorganic chemical nutrients for plant growth developmental physiology. In this study, PAW80 with nitrate contents (835 ppm) showed the best peanut plant seeding growth physiology. This PAW condition and nitrate concentration in this study are differs to previous studies which reported the beneficial effects of PAW5, PAW12, and PAW19 in augmenting root growth in Arabidopsis, with PAW12 being the optimal condition for tobacco seedlings [15,16]. It indicates that proper plasma condition with nitration concentration differently appear depending on the crop. GO analysis of plasma treatment focusing on the shared differentially expressed genes (DEGs) between PAW40 and PAW80, provided a comprehensive overview of the possible regulatory impacts of PAW on peanut seedling growth. The results highlight that PAW treatment influences a multitude of biological processes, encompassing shoot system development, organelle organization, post-embryonic development, and RNA processing. These findings align with prior research illustrating the broad-spectrum impacts of PAW on plant biological processes. In addition to the fact that the most enriched go terms are shoot system development, in the gene expression analysis, nitrate transporter genes were also highly affected. This finding suggests that the positive effects of plasma treatment on plant growth may be attributed to the activation of specific genes that are involved in regulating growth and may occur through the nitrogen pathway. Nitrogen is a critical component of plant growth, as it is an essential element to produce amino acids, nucleic acids, and proteins [21,22]. As a powerful signal, nitrate can regulate plant growth and development, stimulating primary root growth through enhanced meristem activity and cytokinin signaling [23]. Nitrate availability serves as a primary environmental factor for plant growth, development, and stress responses, interacting with endogenous phytohormones to manage plant growth and development [24]. Therefore, nitrate in PAW holds a critical role in plant growth and development by inducing changes in root system architecture, fostering lateral root growth, and boosting primary root growth via increased meristem activity and cytokinin signaling. In contrast to the upregulated gene, the downregulated genes were primarily involved in responses to external stimuli, including biotic stimulus, hormones, and lipids. This implies that PAW treatment potentially modulates the expression of genes related to these processes, thereby influencing plant growth. It is essential to note that while our findings provide an intriguing insight into the potential role of these downregulated genes, their exact function remains to be explored. Further studies are needed to elucidate the precise molecular mechanisms and regulatory networks on biological roles as well as their potential implications for plant growth and development. Recent transcriptome analysis of plasma-treated Arabidopsis seeds showed an enrichment in genes related to secondary metabolites and cell wall lignin, both crucial for plant defense [12,13]. Interestingly, our study on peanut seeds revealed a substantially different gene ontology when compared to the Arabidopsis seed treatment. This underlines the distinct sensitivity of peanut seeds to PAW and distinct pathways of plasma treatment. To deepen our understanding, it's essential to carry out a thorough investigation of species-specific plasma treatments, while simultaneously exploring the underlying molecular mechanisms. This dual approach has the potential to decipher and customize plasma treatments for plants, shedding light on the molecular aspects of seedling growth. It could also lead to the identification of new targets that can enhance crop growth. Conclusion In conclusion, our study elucidates the beneficial impact of PAW on peanut sprout cultivation, which is evidenced by significant enhancements in weight, length, and seedling vigor index. Additionally, our transcriptome analysis has provided insights into the molecular regulation by PAW, highlighting the significance of gene expression related to shoot growth in improving peanut seed germination and seedling growth. More specifically, an 80-minute PAW treatment has demonstrated promising results in these areas. These findings underscore the potential of PAW as a potent tool in agricultural practices, particularly in the context of improving seed germination and seedling growth rates, and open path for future investigations into species-specific plasma treatment protocols. Declarations Acknowledgement This research was supported by R&D Program of "R&D Program of ‘Plasma Advanced Technology for Agriculture and Food (Plasma Farming) (EN2325)" through the Korea Institute of Fusion Energy (KFE) funded by the Government funds, Republic of Korea. his research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [2021R1F1A1063869]. This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) NRF-2019K1A3A1A18116087 Author contribution Conceptualization: Y.K.L., and Ks.K. Investigation and data curation: R.A.P., T.Y., S-W.L., Kb.K., H.H., Y.K. Writing-original draft: R.A.P., T.Y., and Ks.K. Writing-review and editing: R.A.P., Y.K.L., and Ks.K. Supervision: Y.O., M.-S.K., Ks.K. All authors have read and agreed to the published version of the manuscript. 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Supplementary Files 1.SupplementaryData.pdf 2.SupplementaryTableS2.DEGofControlvsPAW40.xlsx 3.SupplementaryTableS3.DEGofControlvsPAW80.xlsx SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Priatama","email":"","orcid":"","institution":"Korea Institute of Fusion Energy","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ryza","middleName":"A.","lastName":"Priatama","suffix":""},{"id":265027112,"identity":"f1f5e354-0022-45fe-91aa-628fda7fb89e","order_by":1,"name":"Taewon Yang","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Taewon","middleName":"","lastName":"Yang","suffix":""},{"id":265027113,"identity":"a7780083-7c82-467a-ab2e-4ee1e47e3e58","order_by":2,"name":"Seung-Won Lee","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seung-Won","middleName":"","lastName":"Lee","suffix":""},{"id":265027114,"identity":"044fc597-be80-450e-bb9c-2a8be363ab5f","order_by":3,"name":"Kibum Kim","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kibum","middleName":"","lastName":"Kim","suffix":""},{"id":265027115,"identity":"3a8c90cd-dd78-4b4f-8388-c3dec9fff305","order_by":4,"name":"Hyunjoo Hwang","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyunjoo","middleName":"","lastName":"Hwang","suffix":""},{"id":265027116,"identity":"4a7f8130-6b94-415b-8b78-8c470d26b10e","order_by":5,"name":"Yerin Kim","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yerin","middleName":"","lastName":"Kim","suffix":""},{"id":265027117,"identity":"426a5bb7-0135-4077-8343-ca761f364267","order_by":6,"name":"Yoojin Oh","email":"","orcid":"","institution":"Johannes Kepler University Linz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoojin","middleName":"","lastName":"Oh","suffix":""},{"id":265027119,"identity":"3c41cceb-fd71-4448-b26b-795ea9da9c0c","order_by":7,"name":"Myoung-Shin Kim","email":"","orcid":"","institution":"Myongji University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Myoung-Shin","middleName":"","lastName":"Kim","suffix":""},{"id":265027120,"identity":"69bdba27-9c25-4218-8137-4619b5ef32cb","order_by":8,"name":"Young Koung Lee","email":"","orcid":"","institution":"Korea Institute of Fusion Energy","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Young","middleName":"Koung","lastName":"Lee","suffix":""},{"id":265027121,"identity":"a06df193-cbe0-499a-a38f-000d8e35d910","order_by":9,"name":"Kisung Ko","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACCQY2BoYPUA5jA7FaGGeQrIWZhyQtkv3HEh/b1NglNrAffsA4cw8RWqQl0g4b5xxLTmzgSTNg3PCMCC1yEuxt0rkNzIkNDDkMjA8OEKOF/3ibtGVDfWID/xsitUgzpB2TZmw4nNggAbRlAzFaJGekJRv2HDtu3CbxzODgDGK0SJw/ZvjgR021bD9/8sOHPcRogQNgImAgScMoGAWjYBSMAjwAAEXtMyJA1j5OAAAAAElFTkSuQmCC","orcid":"","institution":"Chung-Ang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kisung","middleName":"","lastName":"Ko","suffix":""}],"badges":[],"createdAt":"2023-12-29 08:44:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3820334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3820334/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49207030,"identity":"49c83292-d061-48d3-b0b1-7feb018c8b40","added_by":"auto","created_at":"2024-01-05 07:31:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of SDBD Plasma Activated Water (PAW) reactor.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/3ab4b9a76db01818d6d9c49e.png"},{"id":49207176,"identity":"5976c455-3c7d-43e9-a559-1df6348590a5","added_by":"auto","created_at":"2024-01-05 07:39:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":513714,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of plasma activated water exposure time on the phenotype of the germinated seedling. \u003cstrong\u003e(a)\u003c/strong\u003e water unexposed to plasma for 0 minutes \u003cstrong\u003e(b)\u003c/strong\u003ewater exposed to plasma for 40 minutes \u003cstrong\u003e(c)\u003c/strong\u003e water exposed to plasma for 80 minutes. Scale bar = 2 cm. \u003cstrong\u003e(d-f) \u003c/strong\u003eQuantification of the phenotypic characters of PAW-treated seedlings; \u003cstrong\u003e(d)\u003c/strong\u003e true leaf with epicotyl length, \u003cstrong\u003e(e)\u003c/strong\u003e hypocotyl length; \u003cstrong\u003e(f)\u003c/strong\u003e primary root length.\u003cstrong\u003e(g)\u003c/strong\u003e Seedling weight. Asterisk indicates statistically significant by \u003cem\u003et\u003c/em\u003e-test (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, and ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/eb5eb30b9c321d55a587e699.png"},{"id":49207032,"identity":"96167c25-f9ad-4994-98ca-2e41172f1b6a","added_by":"auto","created_at":"2024-01-05 07:31:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54900,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of water exposed to plasma time on vigor index (VI) water unexposed to plasma (0), 40-hour water exposed to plasma (40), 80-hour water exposed to plasma (80). \u003cstrong\u003e(a)\u003c/strong\u003e True leaf with epicotyl; \u003cstrong\u003e(b)\u003c/strong\u003eHypocotyl; \u003cstrong\u003e(c)\u003c/strong\u003e Root. Each water exposed to plasma was statistically compared with 0 (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, and ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/9c4089b66b7ddade25a45b93.png"},{"id":49207178,"identity":"f9b3ddde-f846-4444-a2cd-60cbeb31c2e3","added_by":"auto","created_at":"2024-01-05 07:39:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215697,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed genes (DEG) isolated from PAW treatment compared to control. \u003cstrong\u003e(a-b)\u003c/strong\u003e Volcano plot displays fold-change versus significance for each gene between control and treatment. The y-axis represents the negative log of the \u003cem\u003ep\u003c/em\u003e-value (base 10), with highly significant points at the top. The x-axis represents the log of the fold change, allowing changes in both directions to appear equidistant from the center. (Red if padj\u0026lt;0.05, orange if log2FC\u0026gt;1, green if both). \u003cstrong\u003e(c-d)\u003c/strong\u003e Heatmap of the DEGs isolated from PAW40 vs control and PAW80 vs control, respectively. The heatmap illustrates the expression patterns (Z-scaled FPKM values) hierarchically clustered according to their relative expression profile. Each column represents an individual sample, while each row corresponds to a gene. The upper axis displays the sample clusters, and the clusters of genes are shown on the left vertical axis. The color scale signifies the relative gene expression: green denotes low relative expression levels; red signifies high relative expression levels; black indicates zero (no change). \u003cstrong\u003e(e)\u003c/strong\u003eVenn diagram of upregulated genes in DEG of PAW40 and PAW80. \u003cstrong\u003e(f)\u003c/strong\u003e Venn diagram of downregulated genes in DEG of PAW40 and PAW80.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/690e0a59e91b93e25a9094c9.png"},{"id":49207515,"identity":"6f2debe4-86de-4791-aad0-26e5362b187f","added_by":"auto","created_at":"2024-01-05 07:47:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":344292,"visible":true,"origin":"","legend":"\u003cp\u003eGene ontology (GO) analysis of PAW-treated peanut seedlings. \u003cstrong\u003e(a)\u003c/strong\u003e Biological process, \u003cstrong\u003e(b)\u003c/strong\u003e Cellular component, \u003cstrong\u003e(c)\u003c/strong\u003e Molecular function, \u003cstrong\u003e(d)\u003c/strong\u003e GO-network of biological process emphasized on shoot system development. \u003cstrong\u003e(e)\u003c/strong\u003eOverview of the heatmap isolated from gene with GO function in Shoot system development.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/1b7ae5ec7b7e15f9a511d614.png"},{"id":49207037,"identity":"f6d55c59-c01c-4a22-af49-43a824d7905f","added_by":"auto","created_at":"2024-01-05 07:31:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":962644,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of PAW treatment on gene expression. \u003cstrong\u003e(a)\u003c/strong\u003e Gene expression of marker genes in nitrate and shoot developments. \u003cstrong\u003e(b) \u003c/strong\u003eGene expression of isolated genes from gene ontology: cellular nitrogen compound catabolic process. \u003cstrong\u003e(c-d) \u003c/strong\u003eRepresentation of the expression from downregulated gene in response to PAW treatment. The heatmap illustrates the expression patterns (Z-scaled FPKM values) hierarchically clustered according to their relative expression profile. Each column represents an individual sample, while each row corresponds to a gene.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/effb3f4cc654b72789f6e9d2.png"},{"id":62771437,"identity":"9b21dc01-7e31-4346-9838-e143a9fa2dd5","added_by":"auto","created_at":"2024-08-19 09:29:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2602751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/13c4a10b-9f01-4506-a998-1cadf934b7d7.pdf"},{"id":49207179,"identity":"2f71d2bd-e296-417e-9fce-1a508aa273a4","added_by":"auto","created_at":"2024-01-05 07:39:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":394860,"visible":true,"origin":"","legend":"","description":"","filename":"1.SupplementaryData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/854122ef37d9ddade04c5b62.pdf"},{"id":49207038,"identity":"ea6a394d-0e31-4afe-b61b-49e8cb5753d8","added_by":"auto","created_at":"2024-01-05 07:31:23","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":867129,"visible":true,"origin":"","legend":"","description":"","filename":"2.SupplementaryTableS2.DEGofControlvsPAW40.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/fa16629272c3bc4e4b6a0723.xlsx"},{"id":49207040,"identity":"e9d63b59-ec05-4146-97f8-57bade87075f","added_by":"auto","created_at":"2024-01-05 07:31:23","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1257824,"visible":true,"origin":"","legend":"","description":"","filename":"3.SupplementaryTableS3.DEGofControlvsPAW80.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/aa99c6414740cec771a6eece.xlsx"},{"id":49207031,"identity":"885c9f4f-f6c4-47b0-aefd-23681d0f1258","added_by":"auto","created_at":"2024-01-05 07:31:23","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12564,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3820334/v1/ea4de39469cecec45d09a3b2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Plasma Activated Water on Peanut Seed Germination, Seedling Biomass, Morphology, and Gene Expression","fulltext":[{"header":"Background","content":"\u003cp\u003ePlasma-activated water (PAW) is generated by cold atmospheric plasma (CAP) treatment of water with a plasma plume above or below the water surface [1]. The plasma generated ionized gas interact with water molecules and trigger several chemical reactions to create a unique biochemical reactive species. During PAW generation, energetic particles in the plasma phase are trapped in the aqueous liquid and a series of reactions are initiated at the gas liquid interface, leading to a variety of primary and secondary reactive species that are soluble in the liquid [2,3]. These reactive species, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are responsible for the chemical and biological effects of PAW. In recent years, PAW has been found to possess outstanding biological and agricultural sectors [4,5]. Plasma direct or indirect treatment on seeds promotes seed germination and growth in plants due to the reactive species, electric field, charged particles, photons and changed physicochemical properties of PAW [6]. PAW with beneficial physico-chemical properties has effects on promoting seed germination and plant growth [7].\u003c/p\u003e \u003cp\u003eSeed germination is a process frequently applied to increase beneficial biochemical contents and nutrients [8]. Peanut sprouts are rich in amino acids, minerals, carbohydrates, fatty acids, and antioxidants, while containing less fat than seeds. Seed germination, crucial for plant sprouts, is influenced by environmental factors such as soil, light, temperature, water, and pH [9]. In our previous studies, it was optimized to increase the seed germination rate, sprout biomass, and sprout morphology quality following the seed sowing direction and fermentation period of oak sawdust [10,11]. However, PAW's impact on peanut seed germination and seedling growth remains unexplored.\u003c/p\u003e \u003cp\u003eIn plasma agriculture, several studies have performed gene expression analysis to explore the underlying mechanisms of plasma treatment in molecular levels. These analyses often involve the use of advanced techniques such as quantitative real-time PCR and transcriptome sequencing, which allow researchers to understand how plasma treatment affects various biological processes, from seed germination to plant growth and development. Recent studies using transcriptome analysis of plasma treated Arabidopsis seeds has revealed the regulation of germination trough stress regulation pathways [12,13]. Transcriptome analysis serves as a powerful tool for studying global gene expression patterns, providing insights into the regulatory networks and pathways activated by PAW treatment [14]. This approach facilitates the identification of differentially expressed genes (DEGs) and the functional annotation of these genes, shedding light on the molecular processes involved in PAW-induced seed germination and growth. This analysis not only enhances our understanding of the molecular mechanisms underlying the beneficial effects of PAW on peanut seed germination and growth, but also lays a foundation for future research on the application of PAW in agriculture.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effects of PAW on peanut seed germination and seedling growth followed by a transcriptome analysis to identify differentially expressed genes (DEGs) in peanut seedlings post PAW treatment. Our final objective was to elucidate the identified DEGs and explore into an analysis of the associated biological processes and pathways. This approach provided us with a deeper understanding of the molecular mechanisms at play, furthering our knowledge of the impact of PAW on plant growth and development.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePAW generation and physicochemical analysis\u003c/h2\u003e \u003cp\u003ePAW was generated using plasma reactor devices consist of two surface dielectric barrier discharge (SDBD) which an alternating current (AC) was used as a power source. The plasma generator has been described by [15,16]. The production of PAW 0, 40, and 80 was based on plasma treatment time (0, 40, and 80 minutes, respectively). Immediately after PAW generation, the anion content of PAW was measured using ion chromatography (ICS-2100, Thermo Dionex, Sunnybale, CA, USA). Hydrogen peroxide content was measured using LR method as described previouslyb [15].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasma activated water cultivation system for seed germination\u003c/h2\u003e \u003cp\u003ePeanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.) seeds were sown in trays (50\u0026times;30\u0026times;10 cm; Yesan, Korea). Seeds were planted side by side using 1,500 ml of 60 days fermented oak sawdust [17] mixed with 1,500 ml of PAW 0, PAW40, and PAW80. The seed row spacing, and sowing depth were set to 3 cm. All 18 seeds were sown in the same direction, 3 per row. The tray was moved to a dark room at 30℃, and 200 ml of each PAW were given twice per day for 10 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMeasuring seed germination, seedling weight, and tissue length\u003c/h2\u003e \u003cp\u003eSeedlings were removed from each tray after 10 days of culture to determine seedling weight and true leaf with epicotyl, hypocotyl, and root length. Whole seedlings were washed with tap water and blotted using paper towels (Yuhan Kimberly, Seoul, Korea). The seed germination rate, true leaf with epicotyl, hypocotyl, and root length were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVigor index\u003c/h2\u003e \u003cp\u003eThe vigor index of peanut seedling groups treated with three different PAWs (PAW 0, PAW40, and PAW80) were investigated to determine which PAW conditions positively give the best seedling biomass production. The vigor index was calculated using the formula (germination rate \u0026times; length of each tissue part) [18].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMorphological Observation\u003c/h2\u003e \u003cp\u003eThe peanut sprouts were uprooted on 10 days after seedling. The harvested peanut sprouts were placed on black superfine fibers, and photographed using an Apple iPhone camera (Apple, Cupertino, CA, USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction from peanut sprouts\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from peanut seedlings grown under the PAW 0, PAW40, and PAW80 treatment for 10 days, respectively. Fresh seedlings were sampled and ground in liquid nitrogen. The RNA was extracted using the MiniBEST Plant RNA Extraction Kit, as previously described (Takara, Seoul, Korea) [19]. Extracted RNA was stored in a deep freezer (-70\u0026deg;C) for further study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing and Gene Ontology Analysis\u003c/h2\u003e \u003cp\u003eRNA quantity and quality were analyzed using NanoDrop8000 system (NanoDrop Technologies, Wilmington, DE, USA) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively. One \u0026micro;g of total RNA was used in library construction using a Truseq Stranded mRNA Prep Kit (Illumina, Inc. San Diego, CA, USA), according to manufacturer's protocol. Libraries from 70\u0026ndash;370 bp (mainly 150 bp) were constructed and sequenced using Illumina NovaSeq 6000 sequencing system (Illumina, Inc. San Diego, CA, USA) to generate 2x100 bp paired-end reads.\u003c/p\u003e \u003cp\u003eFastQC v0.11.7 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to check the quality of the raw reads, and Trimmomatic v0.36 was used to remove adaptor sequences and low-quality reads. An alignment process was performed using Tophat (v2.0.13) mapped to \u003cem\u003eArachis hypogaea\u003c/em\u003e v1.0 database. The abundance calculation and differentially expressed genes (DEG) was analyzed using Cuffdiff (v2.2.0). Further analysis was performed using DEG that satisfies 2-fold change and \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.1. The Venn diagram analysis was carried out using the VENNY (v2.1) webtool, accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The Gene Ontology (GO) analysis was performed using the ShinyGO (version 0.77) software, as described by [20]. The heatmap analysis was performed utilizing the 'pheatmap\u0026rsquo; v1.0.12 package in R using Rstudio 2023.03.0 Build 386.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical properties of PAW\u003c/h2\u003e \u003cp\u003eAnalysis of physicochemical properties revealed an increase in several chemical components of PAW that was linearly related to the duration of plasma treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The pH values for PAW40 and PAW80 were lower, at 2.52 and 2.28 respectively, than the control, which was 6.96. In terms of nitrate (NO3-, mg/L), PAW80 demonstrated a higher mean level (831) compared to PAW40 (383). This trend was also observed for nitrite (NO2-, mg/L), with PAW80 (5.12) displaying a higher mean level than PAW40 (3.017). For conductivity (\u0026micro;s/cm), PAW80 (3,252) again showed a higher mean level than PAW40 (1,558). No traces of nitrate or nitrite were detected in the control (PAW0). However, the concentration of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) did not appear to be noticeably affected by the plasma treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of the PAW 0, PAW40, and PAW80\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNitrate NO\u003csub\u003e3\u003c/sub\u003e- (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNitrite NO\u003csub\u003e2\u003c/sub\u003e-(mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConductivity\u003c/p\u003e \u003cp\u003e(\u0026micro;s /cm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e383\u0026thinsp;\u0026plusmn;\u0026thinsp;5.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1558\u0026thinsp;\u0026plusmn;\u0026thinsp;53.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e831\u0026thinsp;\u0026plusmn;\u0026thinsp;10.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3252\u0026thinsp;\u0026plusmn;\u0026thinsp;89.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eThe results of the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). nd, not detected.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of PAW on Peanut Seedling\u003c/h2\u003e \u003cp\u003ePeanut seedlings were uprooted, washed, and weighed 10 days after sowing. Phenotypically an obvious phenotypic variation was clearly shown in PAW treated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). To measure seedling length, three different seedling tissue were classified as true leaf with epicotyl, hypocotyl, and root. In seedling true leaf in PAW80 condition started to come out first at 6 days after sowing. In the mean length of true leaf with epicotyl, the seedlings in PAW80 (7.3 cm) showed the longest compared to PAW 0 (2 cm) and PAW40 (4.7 cm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In the mean of hypocotyl length, the seedlings in PAW40 (5.3 cm) and PAW80 (3.1 cm) had significantly longer lengths than PAW 0 (2.3 cm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). In root length, the seedling in PAW80 (10.4 cm) showed the longest length than the others [PAW 0 (2 cm) and PAW40 (3.8 cm)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The seedling with PAW80 had the highest mean weight (5.1 g) than the others. The mean seedling weights of the PAW 0 and 40 were 3.3, and 4.5 g, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of PAW on Vigor Index\u003c/h2\u003e \u003cp\u003eTo identify the optimal plasma exposure time to water for efficient peanut sprout biomass production, vigor indexes of seedlings in PAW 0, 40, and 80 were computed and are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The highest total vigor index was observed in the PAW80 group, registering a value of 1385.1. Conversely, the PAW 0 group, which received no plasma treatment, had the lowest total vigor index, with a value of 213.0. The PAW40 group, with a plasma exposure time of 40 minutes, recorded a total vigor index of 1048.7, ranking second highest among the groups. It was noted that the total vigor index values for the groups treated with plasma-activated water (PAW 40 and PAW 80) were higher than the group without plasma treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalculation of Vigor Index (VI) and total VI on seedling each water exposed to plasma time.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eVigor Index (VI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAW\u003csup\u003eZ\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGermination rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrue Leaf with Epicotyl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypocotyl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTrue Leaf with Epicotyl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypocotyl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTotal VI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e66.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e51.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e151.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e77.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e358.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e235.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e292.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e885.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e66.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e514.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e213\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e680.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e1407.4\u0026thinsp;\u0026plusmn;\u0026thinsp;31.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003eZ\u003c/sup\u003ePAW; 0: water unexposed to plasma, 40: 40-hour water exposed to plasma, 80: 80-hour water exposed to plasma. \u003csup\u003ey\u003c/sup\u003eVigor Index (VI)\u0026thinsp;=\u0026thinsp;Germination Rate (%) \u0026times; Length (cm). \u003csup\u003ex\u003c/sup\u003eTotal = VI (True leaf with epicotyl)\u0026thinsp;+\u0026thinsp;VI (Hypocotyl)\u0026thinsp;+\u0026thinsp;VI (Root)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGene regulations in PAW-induced peanut sprouts\u003c/h2\u003e \u003cp\u003eTo investigate the regulation of gene expression of seedlings growth in PAW treatment, the RNA sequencing data were generated from peanut samples including DW, PAW40 and PAW80. From the average of 41\u0026nbsp;million reads, around 37\u0026nbsp;million clean reads (~\u0026thinsp;91%) per library were retained, with approximately 91% mapping rate to the \u003cem\u003eA. hypogea\u003c/em\u003e reference genome (Additional file 1: Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b, the identified gene expressions are plotted, and the volcano plot displays the fold-change significance for each gene between control and treatment. By filtering of 2fold \u0026amp; \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026amp; FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.1, we generated heatmap to demonstrate the samples from each replicate were clustered together despite of the variations among replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-d). In detail, the analysis revealed that in total 5428 and 7911 genes were differentially expressed in PAW40 and PAW80, respectively (Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among them, 734 and 2313 genes were uniquely upregulated in PAW40 and PAW80 respectively, 1624 genes were upregulated in both PAW40 and PAW80 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Additionally, 1453 genes were uniquely downregulated in PAW40, 2505 were downregulated in PAW80, and 2655 genes were downregulated in both treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTo obtain a general overview of the PAW regulation in peanut seedling growth, Gene ontology analysis (GO) was performed using the overlapped DEG of PAW40 and PAW80. We separately analyzed the upregulated and downregulated DEGs and showed the top 20 GO terms which are highly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c, and Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In GO: plasma treatment affects various biological processes categories including shoot system development, organelle organization, post-embryonic development, RNA processing, and more (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In cellular components, various cellular component functions are enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In addition, the molecular function also revealed enrichment of enzyme activity and signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In contrast, the overlapped downregulated genes showed enrichment in function related to response to external stimulus such as biotic stimulus, hormone and lipid (Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). Whereas the cellular component and molecular function show various enrichment in extracellular region and kinase activity, respectively (Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven that PAW treatment dramatically affects GO term of shoot system development. We further analyze the GO-network of the upregulated genes in shoot system developments. The network showed the relation of each enriched pathway, which overall significant connection to the plant development (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Additionally, we isolated shoot system development genes which are highly enriched and confirm the expression by generating heatmap as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ee. The heatmap showed more genes were upregulated in the shoot system development both in PAW40 and PAW80 and the most upregulated genes were found in PAW80.\u003c/p\u003e \u003cp\u003eTo confirm more specific regulation of PAW in the affecting seedling development. We isolated several marker genes and GO term which potentially co-related with the improved growth in PAW treatment. In Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, plant development related marker genes and nitrate transport are shown the DE gene expression. LATERAL ORGAN BOUNDARIES (LOB), CUP SHAPED COTYLEDON (CUC), GIGANTEA, SHOOT-GRAVITROPISM 5 (SGR5), AUXIN RESPONSE FACTOR (ARFs), PHYTOCHROME-INTERACTING FACTOR (PIFs), NITRATE TRANSPORTER (NRTs) genes are generally up-regulated in PAW40 and PAW80. Interestingly, those genes are more strongly enhanced in PAW80 condition. Since PAW mainly contains nitrate, we show additionally the expression of genes that are related to nitrogen response of the cells, nitrogen compound catabolic process and cell response to nitrogen. Indeed, it showed a similar pattern of induction in both PAW treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Furthermore, we identified downregulated genes which appear to function as negative regulators in nitrate response and shoot development by PAW treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d). Such as family member of auxin response factor (ARFs) family, flowering locus K, and brassinosteroid insensitive 1 (BR1). This suggests that shoot system development downregulation of specific genes might contribute to the enhancement of seedling growth and affected by PAW treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e In this study, we aimed to investigate the effects of PAW on peanut seed germination, sprout tissue biomass, morphology, and gene expression of PAW treatment according to the peanut phenotype. Determining optimal PAW conditions is crucial for efficient seedling growth, as various plant species exhibit unique responses to PAW at different exposure times. In addition, understanding the gene regulation through transcriptome analysis can provide valuable insights into the mechanism of PAW in the regulation of growth in peanut sprouts.\u003c/p\u003e \u003cp\u003eIn the seedling weight, the PAW80 showed the highest level, followed by the PAW40. In the seedling length, the PAW80 had the longest lengths of true leaf with epicotyl, hypocotyl, and root. The PAW80 yielded the highest vigor index, followed by the PAW40. It is interesting that in peanut seedlings treated with PAW80, the enhanced root formation was clearly observed. This morphological observation is not unexpected since it was reported in previous studies that plants treated with PAW had enhanced root growth [15,16].\u003c/p\u003e \u003cp\u003eThe PAW contains chemical molecules such as nitrate, and nitrate with high electrical conductivity which can be used as inorganic chemical nutrients for plant growth developmental physiology. In this study, PAW80 with nitrate contents (835 ppm) showed the best peanut plant seeding growth physiology. This PAW condition and nitrate concentration in this study are differs to previous studies which reported the beneficial effects of PAW5, PAW12, and PAW19 in augmenting root growth in Arabidopsis, with PAW12 being the optimal condition for tobacco seedlings [15,16]. It indicates that proper plasma condition with nitration concentration differently appear depending on the crop.\u003c/p\u003e \u003cp\u003eGO analysis of plasma treatment focusing on the shared differentially expressed genes (DEGs) between PAW40 and PAW80, provided a comprehensive overview of the possible regulatory impacts of PAW on peanut seedling growth. The results highlight that PAW treatment influences a multitude of biological processes, encompassing shoot system development, organelle organization, post-embryonic development, and RNA processing. These findings align with prior research illustrating the broad-spectrum impacts of PAW on plant biological processes. In addition to the fact that the most enriched go terms are shoot system development, in the gene expression analysis, nitrate transporter genes were also highly affected. This finding suggests that the positive effects of plasma treatment on plant growth may be attributed to the activation of specific genes that are involved in regulating growth and may occur through the nitrogen pathway. Nitrogen is a critical component of plant growth, as it is an essential element to produce amino acids, nucleic acids, and proteins [21,22]. As a powerful signal, nitrate can regulate plant growth and development, stimulating primary root growth through enhanced meristem activity and cytokinin signaling [23]. Nitrate availability serves as a primary environmental factor for plant growth, development, and stress responses, interacting with endogenous phytohormones to manage plant growth and development [24]. Therefore, nitrate in PAW holds a critical role in plant growth and development by inducing changes in root system architecture, fostering lateral root growth, and boosting primary root growth via increased meristem activity and cytokinin signaling.\u003c/p\u003e \u003cp\u003eIn contrast to the upregulated gene, the downregulated genes were primarily involved in responses to external stimuli, including biotic stimulus, hormones, and lipids. This implies that PAW treatment potentially modulates the expression of genes related to these processes, thereby influencing plant growth. It is essential to note that while our findings provide an intriguing insight into the potential role of these downregulated genes, their exact function remains to be explored. Further studies are needed to elucidate the precise molecular mechanisms and regulatory networks on biological roles as well as their potential implications for plant growth and development.\u003c/p\u003e \u003cp\u003eRecent transcriptome analysis of plasma-treated Arabidopsis seeds showed an enrichment in genes related to secondary metabolites and cell wall lignin, both crucial for plant defense [12,13]. Interestingly, our study on peanut seeds revealed a substantially different gene ontology when compared to the \u003cem\u003eArabidopsis\u003c/em\u003e seed treatment. This underlines the distinct sensitivity of peanut seeds to PAW and distinct pathways of plasma treatment. To deepen our understanding, it's essential to carry out a thorough investigation of species-specific plasma treatments, while simultaneously exploring the underlying molecular mechanisms. This dual approach has the potential to decipher and customize plasma treatments for plants, shedding light on the molecular aspects of seedling growth. It could also lead to the identification of new targets that can enhance crop growth.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study elucidates the beneficial impact of PAW on peanut sprout cultivation, which is evidenced by significant enhancements in weight, length, and seedling vigor index. Additionally, our transcriptome analysis has provided insights into the molecular regulation by PAW, highlighting the significance of gene expression related to shoot growth in improving peanut seed germination and seedling growth. More specifically, an 80-minute PAW treatment has demonstrated promising results in these areas. These findings underscore the potential of PAW as a potent tool in agricultural practices, particularly in the context of improving seed germination and seedling growth rates, and open path for future investigations into species-specific plasma treatment protocols.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eThis research was supported by R\u0026amp;D Program of\u0026nbsp;\u0026quot;R\u0026amp;D Program of \u0026lsquo;Plasma Advanced Technology for Agriculture and Food (Plasma Farming) (EN2325)\u0026quot; through the Korea Institute of Fusion Energy (KFE) funded by the Government funds, Republic of Korea.\u0026nbsp;his research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [2021R1F1A1063869]. This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) NRF-2019K1A3A1A18116087\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eConceptualization: Y.K.L., and Ks.K. Investigation and data curation: R.A.P., T.Y., S-W.L., Kb.K., H.H., Y.K. Writing-original draft: R.A.P., T.Y., and Ks.K. Writing-review and editing: R.A.P., Y.K.L., and Ks.K. Supervision: Y.O., M.-S.K., Ks.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eDeclaration of interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoni, A.; Choi, J.; Brightwell, G. 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Proc Natl Acad Sci U S A. 2017; 114, 46: E9989-E9998. http://dx.doi.org/10.1073/pnas.1710964114.\u003c/li\u003e\n\u003cli\u003eGe, S. X.; Jung, D. M.; Yao, R. A. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020; 36, 8: 2628-2629. http://dx.doi.org/10.1093/bioinformatics/btz931.\u003c/li\u003e\n\u003cli\u003eFan, H. M.; Quan, S. X.; Qi, S. D.; et al. Novel Aspects of Nitrate Regulation in Arabidopsis. Frontiers in Plant Science. 2020; 11. http://dx.doi.org/10.3389/fpls.2020.574246.\u003c/li\u003e\n\u003cli\u003eGaudinier, A.; Rodriguez-Medina, J.; Zhang, L. F.; et al. Transcriptional regulation of nitrogen-associated metabolism and growth. Nature. 2018; 563, 7730: 259-264. http://dx.doi.org/10.1038/s41586-018-0656-3.\u003c/li\u003e\n\u003cli\u003eNaulin, P.; Armijo, G.; Vega, A.; et al. Nitrate Induction of Primary Root Growth Requires Cytokinin Signaling in Arabidopsis thaliana. Plant \u0026amp; cell physiology. 2019; 61. http://dx.doi.org/10.1093/pcp/pcz199.\u003c/li\u003e\n\u003cli\u003eGuan, P. Z. Dancing with Hormones: A Current Perspective of Nitrate Signaling and Regulation in Arabidopsis. Frontiers in Plant Science. 2017; 8. http://dx.doi.org/10.3389/fpls.2017.01697.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Arachis hypogaea, Peanut sprout, Plasma activated water, Vigor index, Transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-3820334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3820334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlasma-activated water (PAW) has been shown to enhance seed germination and seedling growth across various plants. This study investigates the impact of PAW on peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.) seed germination, viability, growth and gene expression in sprouts. Seeds were treated with PAW for 0, 40, and 80 minutes and sown in fermented oak sawdust. Germination rate, weight, length, and seedling vigor index were assessed. Among the different PAW treatments, the longest true leaf with epicotyl and the longest hypocotyl were observed in PAW80 (7.3 cm and 3.1 cm, respectively). Specifically, the root length was observed the longest in PAW80 (10.4 cm). The PAW80 had the highest vigor index (1385.1), followed by PAW40 (1048.7). Gene ontology analysis revealed that shoot growth related gene expression in PAW40 and PAW80 peanut groups relatively higher than PAW 0 group. Overall, PAW80 provided the most favorable conditions for peanut sprout growth, promoting shoot-growth related gene expression. Our findings suggest that PAW positively affects peanut and seedling growth by the regulation of shoot promoting genes.\u003c/p\u003e","manuscriptTitle":"Effect of Plasma Activated Water on Peanut Seed Germination, Seedling Biomass, Morphology, and Gene Expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-05 07:31:18","doi":"10.21203/rs.3.rs-3820334/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"086e2191-7304-4b68-88aa-a8aace05e2b1","owner":[],"postedDate":"January 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T02:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-05 07:31:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3820334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3820334","identity":"rs-3820334","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}