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Plasma-Activated Water Modulates Root Hair Cell Density via Root Developmental Genes in Arabidopsis thaliana L.

Plasma-Activated Water Modulates Root Hair Cell Density via Root Developmental Genes in... Article Plasma-Activated Water Modulates Root Hair Cell Density via Root Developmental Genes in Arabidopsis thaliana L. 1,2,† 1,† 1,3 4 1 2 Dong Hyeun Ka , Ryza Aditya Priatama , Joo Young Park , Soon Ju Park , Seong Bong Kim , In Ah Lee and 1, Young Koung Lee * Institute of Plasma Technology, Korea Institute of Fusion Energy, 37 Dongjangsan-ro, Gunsan-si, Jeollabuk-do 54004, Korea; kdh9799@kfe.re.kr (D.H.K.); priatama@kfe.re.kr (R.A.P.); pjy901113@gmail.com (J.Y.P.); sbkim@kfe.re.kr (S.B.K.) Department of Chemistry, College of Natural Science, Kunsan National University, 558 Daehak-ro, Gunsan-si, Jeollabuk-do 54150, Korea; leeinah@kunsan.ac.kr Surface Materials Division, Korea Institute of Materials Science, 797, Changwon-daero, Seongsan-gu, Changwon-si, Gyeongsangnam-do 51508, Korea Division of Biological Sciences and Research Institute for Basic Science, Wonkwang University, Iksan, Jeollabuk-do 54538, Korea; sjpark75@wku.ac.kr * Correspondence: leeyk@kfe.re.kr † Dong Hyeun Ka and Ryza Aditya Priatama contributed equally to this work. Abstract: Low-temperature atmospheric pressure plasma technology has been used in agriculture and plant science by direct and indirect treatment of bio-samples. However, the cellular and mo- lecular mechanisms affected by plasma-activated water (PAW) are largely unexplored. In this study, PAW generated from a surface dielectric barrier discharge (SDBD) device was used for plant development. Physicochemical analysis was performed to confirm the PAW properties that corre- lated with the plasma treatment time. Arabidopsis thaliana L. was utilized to study the effect of the Citation: Ka, D.H.; Priatama, R.A.; PAW treatment in the early developmental stage. The plasma-activated water samples are denoted Park, J.Y.; Park, S.J.; Kim, S.B.; Lee, I.A.; Lee, Y.K. Plasma-Activated as PAW5 time in minutes (min), PAW7 min, PAW12 min, PAW19 min and PAW40 min with the Water Modulates Root Hair Cell plasma treatment time. Seedlings grown in the PAW5, PAW7 and PAW12 had increased root Density Via Root Developmental lengths while the root lengths were decreased in the PAW19 and PAW40. In the cellular level ob- Genes in Arabidopsis thaliana L. servation, the PAW treatment specifically increased the root hair numbers per unit of the root but Appl. Sci. 2021, 11, 2240. suppressed the root hair length in the PAW, indicating that PAW mainly modulates the root hair https://doi.org/10.3390/app11052240 cell density in the root. Furthermore, we found that the root hair density and length at PAW5 in maximal observed conditions were positively regulated by root developmental-related genes in- Academic Editor: Gyungsoon Park cluding COBRA-LIKE9 (COBL9), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (XTH9), XTH17, AUXIN1 (AUX1) and LIKE-AUXIN (LAX3). Received: 27 January 2021 Accepted: 26 February 2021 Keywords: plasma agriculture; plasma-activated water; Arabidopsis thaliana L.; nitrate; root cell Published: 3 March 2021 number Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and insti- tutional affiliations. 1. Introduction Low-temperature, atmospheric pressure, plasma technology has emerged as a new technology that potentially enhances plant growth and defense [1–3]. Bio-samples can be treated by a plasma device through gas or water [4]. Plasma-activated water (PAW) Copyright: © 2021 by the authors. treatment changes the chemical properties, and PAW containing an altered chemical Licensee MDPI, Basel, Switzerland. composition is utilized with plants [5,6]. In this indirect method, the development of This article is an open access article low-temperature plasma technology is used for plant cultivation in agronomy [7–9]. distributed under the terms and PAW has differences in electrical conductivity, pH, nitrite, nitrate and H2O2 compared conditions of the Creative Commons with deionized water (DW). As for the application of PAW in plants, ionized oxygen and Attribution (CC BY) license nitrogen species dissolve in the water to create an acidic environment due to a lower pH (http://creativecommons.org/licenses [10,11]. Moreover, water treated with low-temperature plasma contains a high concen- /by/4.0/). Appl. Sci. 2021, 11, 2240. https://doi.org/10.3390/app11052240 www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 2240 2 of 16 tration of nitrate. Therefore, it has the role of a nitrogen (N) source for plant growth and could be possibly used as a liquid fertilizer [12,13]. Nitrogen is an important macronu- trient for plant growth and an essential component in the basic metabolic process [14–16]. The use of nitrogen fertilizer has a critical role in the green revolution by improving plant yield. Several reports have been published on using plasma treatment as an eco-friendly technology that produces nitrogen and functions as a liquid fertilizer [12,13,17,18]. Direct plasma application changes the appearance of the seed coat surface, resulting in a reduced time for germination and rapid growth in the development stage [7,19]. In tomatoes, a horticultural crop, stem growth differs from the controls according to the helium plasma treatment time [20]. The difference between the germination rate and growth rate for PAW in Raphanus sativus, Solanum lycopersicum and Capsicum annum has been reported to be due to the effects of a long PAW treatment time [18]. Both direct and indirect processing of plasma is considered to have a positive effect on plant develop- ment [21,22]. We selected Arabidopsis thaliana L. (Arabidopsis) as a model plant to understand the effect of PAW on its molecular mechanism during the plant development process. The growth cycle of Arabidopsis thaliana L. is very short, 4–6 weeks. In addition, the number of seeds in the plant is about 1500, which is a good model for studying genetics. Ara- bidopsis has a relatively small 135 Mbp genome, 5 pairs of chromosomes and 33,602 genes, which make it easy to analyze the genome, and can be used for other crop appli- cations. The Arabidopsis genome sequence was first decoded in December 2000. Since then, the advancement of plant science has accelerated at a faster pace [23,24]. This ad- vancement was due to the influence of Arabidopsis as a good model plant to understand the fundamental phenomena of plants. In this study, we examined the phenotype, cytological observation, and gene ex- pression after PAW treatment to elucidate the molecular mechanism of Arabidopsis dur- ing the development process. As a plasma generating device, the plasma-activated water was generated using a surface dielectric barrier discharge (SDBD) to dissolve ionized gas in deionized water. In particular, hydrogen peroxide (H2O2), nitrate (NO3-), pH and electrical conductivity were altered during the plasma treatment time. The altered phys- icochemical properties affected the vegetative and root development. In the cotyledon, PAW treated palisade cells were regulated by cell expansion, not cell proliferation, and the root hair number was dramatically increased in the root cells. In the present study, we deduced that PAW modulates the root hair cell density induced by the expression of root developmental-related genes, COBL9, XTH9, XTH17, AUX1 and LAX3. 2. Materials and Methods 2.1. Plasma-Activated Water Preparation and Physicochemical Analysis The plasma source used in this study was previously described in Lee et al., 2020 [25]. A surface dielectric barrier discharge (SDBD) in an air-tight container was used as the plasma treatment device. The electrodes of the SDBD generator were made up of two parallel metals, one of which was covered with a dielectric layer, and the plasma was generated using an alternating current (AC) flowing through the electrodes. The electrode consisted of powered and grounded stainless steel, and an aluminum oxide plate (1 mm-thick) was placed between the electrodes. Each electrode was attached at the top of the reactor with a 10 W average consumed power, an 8 kVpp voltage ampli- tude and a 17 kHz frequency. A 12 cm fan below the electrodes was used (15-LED 120, Aone, China) to circulate the air inside the reactor. In the reactor, the distance between the two electrodes and the water surface was 12 cm. The optical emission spectrum (OES) was obtained by an ultraviolet–visible (UV-VIS) spectrometer (Oceanoptics, maya 2000 pro) within the range of 200–600 nm. An optical lens (Ocean optics, UV-74) was used to collimate the emission spectrum. The lens was placed in front of the SDBD at Appl. Sci. 2021, 11, 2240 3 of 16 1.5–2.0 cm. The OES was obtained with an integration time of 1 s which was averaged 50 times. PAW was produced by adding 1 L of deionized to the reactor. The PAW was pre- pared according to the plasma treatment time for 5, 7, 12, 19 and 40 min The plas- ma-activated water samples in this paper are denoted as PAW5, PAW7, PAW12, PAW19 and PAW40 with the number indicating the plasma treatment time. To confirm the chemical properties of the PAW, the anion content was measured one day after PAW generation using ion chromatography (ICS-2100, Thermo Dionex, Sunnybale, CA, USA). To quantify the anions, the standard sample [Dionex Seven Anion Standard II (in deionized water)] was measured with a three-point method (25, 50, and 100 mg/L) [24], and the pH and conductivity were measured using Orion™ Versa Star Pro™ (Thermo Scientific, Waltham, MA, USA). To meet the appropriate pH for plant growth, the pH was adjusted to pH 5.6~5.8 with 0.1 N and 1 N KOH solution. An AQ- UAfast AQ3700 Colorimeter (ORION AQ3700, Thermo Scientific Orion, USA) was used to measure the hydrogen peroxide (H2O2) using the hydrogen peroxide LR method (Tintometer Lovibond, UK). All chemical properties measurement was performed using at least 3 replicates. 2.2. Plant Material and Growth Conditions To prevent contamination, the Arabidopsis thaliana L. (Columbia 0, Col-0, ecotype background) seeds were surface sterilized with 20% commercial bleach followed by washing with sterilized deionized water and then stored at 4 °C for 3 days for the cold treatment before placing them onto the media. The sterilized seeds were placed onto agar medium and allowed to dry for 30 min before moving the plates into the growth chamber. The growth chamber setting was set at 22 °C, 60% relative humidity, and 16 h/8 h, light/dark condition, respectively. The seeds were in the growth chamber for 15 days. Solid medium was prepared using plant agar (Duchefa Biochemie) at a 0.8% con- centration with deionized water (DW) and PAW (5, 7, 12, 19, 40 min). Seedlings were grown on the minimal growth condition media at the vertical position for the root length examination and the horizontal position for the cotyledon size examination. All steriliza- tion and planting procedures were carried out on a clean bench. To observe the root morphology, the midpoint in the longitudinal direction of the root was observed for the optimal root hair initiation and length phenotype. WER::GFP/cobl9-1 (CS67758) and cobl9-1 (Salk_099933) were acquired from the Ara- bidopsis Biological Resource Center (ABRC), www.arabidopsis.org, to examine the root hair. Two T-DNA insertional mutants, WER::GFP and cobl9-1, were Col-0 background. The double mutant WER::GFP/cobl9-1 (CS67758) was obtained by crossing the single loss of mutant line (cobl9-1, Salk-099933) and the WER::GFP mutant. The COBRA-LIKE 9 (AT5G49270) gene is involved in root hair [26,27]. Genotyping was performed using polymerase chain reaction (PCR) to confirm the deletion of the cobl9-1 mutant and the insertion of the GFP gene. AccuPower Taq PCR PreMix (BIONEER) was used, and the temperature and time were as follows: 95 °C for 5 min for the initial denaturation, 35 cy- cles of 95 °C for 20 s, 55 °C for 20 s, and 72 °C for 1 min, and a final extension step of 72 °C for 5 min Gel loading was performed using 1xTAE, Red safe (1×), and 1.5% Agarose (Duchefa Biochemie, The Nederland). 2.3. Morphological Analysis To count the palisade cells and measure their size in the cotyledon, samples were prepared from 10-day-old Arabidopsis grown in the horizontal growth condition. The cotyledon leaves were preserved in FAA solution (5% acetic acid, 45% ethanol and 5% formaldehyde) and fixed at 4 °C overnight. An ethanol series (50%, 60%, 70%, 80%, 90%, 95%, 99% and 100%) was sequentially used for the sufficient dehydration. Then, a 50:50 ratio of ethanol and methyl salicylate (Sigma-Aldrich, St. Louis, MI, USA) was added to the leaves which then sat for an hour, and finally, 100% methyl salicylate was added and Appl. Sci. 2021, 11, 2240 4 of 16 substituted. The prepared cotyledon leaves were fixed using slide glass and cover glass, and the palisade cells of the cotyledon were photographed using a differential interfer- ence contrast (DIC) microscopy (ZEISS) system to count the number and measure the size. 2.4. Microscopy To confirm the appearance and shape of the root hair development of Arabidopsis thaliana L., Columbia 0 (Col-0) and WEREWOLF (WER)::GFP/cobl9-1 (CS67758) were grown in Petri dishes for 5 and 7 days. The root morphology was examined using a Ni- kon-Fi3 stereo microscope with a 5× zoom magnification, objective lens (10×), 40 ms ex- posure, and 2.0× analog gain. To check the GFP fluorescence of the WER::GFP/cobl9-1 lines, we selected the middle area of the roots with the optimal root hair initiation and elongation present. The GFP signals were detected under a Nikon-A1 confocal micro- scope at 488 nm with eyepiece lens (10×), objective lens (20×) and a FITC filter with the laser set to 40% and the analog gain to 80%. The quantification was performed using the photograph image of the microscopy. 2.5. RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Analysis For the gene expression analysis, the samples were grown for 15 days and then sampled and immediately frozen in liquid nitrogen and crushed using a bead beater followed by RNA extraction from the roots using the Trizol reagent (Thermo Fisher Sci- entific, USA) method. The purified RNA was then treated with the Turbo DNA-free kit (Invitrogen, Carlsbad, CA, USA). For the reverse transcription, 500 ng of RNA were used for the cDNA synthesis using the SuperScript III First-strand Synthesis System for RT-PCR (Invitrogen, USA) following the manufacturer’s instructions. Then, 1 μL of cDNA was used for real-time PCR, and the results were quantified with the CFX Man- TM ager software (Bio-Rad Laboratories, Inc., Hercules, CA, USA, http://www.bio-rad.com/ (accessed on 2 March 2021)), and Ubiquitin 10 was used for normalization. The primers used in this study are listed in Supplementary Table S1. 3. Results 3.1. Plasma Diagnostics and Plasma-Activated Water (PAW) Characteristics The emission spectrum of the plasma from SDBD was measured using optical emission spectroscopy (OES). The result shows the intensive molecular spectra of a ni- trogen second positive system (N2 SPS) and nitrogen first negative (N2 FNS) in the 300– 400 nm region during the air discharge. An OH radical peak (309 nm) was not observed; thus, the air discharge may not produce OH radicals (Figure 1). Because N2 SPS and N2 FNS were detected by OES, it is estimated that a large amount of nitrogen ions will be generated by this plasma system. When these NOx react with H2O, they become ionized - - into NO2 and NO3 , which were detected in the PAW. The physical and chemical prop- erties of DW and the PAW were analyzed (see the Materials and Methods section), and the results are presented in Table 1. To produce the proper PAW for Arabidopsis, plas- ma-activated water samples were generated using five different PAW treatment times and used in the experiment. The plasma-activated water samples in this paper are de- noted as PAW5 time in minutes (min), PAW7, PAW12, PAW19 and PAW40 with the number indicating the plasma treatment time. The result showed that the pH decreased (increased acidity) as the PAW processing time was extended due to the change in the ion content of the water. Furthermore, we confirmed that a small amount of nitrite was detected in the PAW, whereas the concen- tration of nitrate ions and their conductivity dramatically increased in proportion to the plasma treatment time in the PAW. In the case of hydrogen peroxide (H2O2), it was not detected in the DW, but it increased slightly depending on the plasma treatment time (Table 1). Appl. Sci. 2021, 11, 2240 5 of 16 Figure 1. Optical emission spectrum of the surface dielectric barrier discharge (SDBD) with air from 200 to 600 nm. Table 1. Physicochemical properties of the plasma-activated water (PAW). − − Nitrite NO2 H2O2 Nitrate NO3 Conductivity pH (mg/L) (mg/L) (mg/L) (µs/cm) Deionized Water(DW) 5.74 ± 0.06 0.00 0.00 0.00 1.53 ± 0.13 PAW5 min(PAW5) 3.62 ± 0.02 1.09 ± 0.11 0.09 ± 0.01 25.29 ± 2.88 118.10 ± 2.26 PAW7 min(PAW7) 3.34 ± 0.03 1.24 ± 0.12 0.14 ± 0.01 49.05 ± 2.61 218.50 ± 9.64 PAW12 min(PAW12) 2.94 ± 0.08 1.85 ± 0.07 0.27 ± 0.02 102.67 ± 6.30 460.33 ± 15.25 PAW19 min(PAW19) 2.62 ± 0.07 3.68 ± 0.12 0.88 ± 0.04 204.87 ± 8.74 972.93 ± 32.41 PAW40 min(PAW40) 2.37 ± 0.04 5.17 ± 0.16 1.31 ± 0.04 389.08 ± 12.24 1847.00 ± 70.19 The results of the Table 1 are the mean ± standard deviation (SD). 3.2. PAW Increases the Total Root Length and Root Hair Number in the Arabidopsis Root To understand how the PAW treatment affects the kinetics on root elongation in Arabidopsis during development, we monitored the changes in root length using solid media to see a more severe and significant effect of PAW component affecting the growth. The changes in the compositions of the nitrogen ions in the PAW were predict- ed to affect plant growth and development. As the result shows, the root length became significantly longer with the PAW5, 7, and 12 treatments from day 7 to day 15 after planting compared to the DW treatment (Figure 2). In contrast to the root length for the PAW5, 7, and 12 treatments, the root length was significantly shorter for the PAW 19 and 40 treatments compared to the DW treatment. At 7 days after planting, the root lengths in the PAW5 treated plants were increased to about 33% compared to DW (Fig- ure 2). Furthermore, at 15 days after planting, the root lengths for the DW-treated seeds were 22.14 ± 2.62 mm, and for PAW5, they were 41.38 ± 3.44 mm showing an 87% dif- ference in length (Figure 3). In contrast, the root lengths under the PAW19 and 40 condi- tions were suppressed compared to the DW treatment (Figure 2). The root length at 7 days after planting was 9.82 ± 1.07 mm, which was about 28% shorter compared to the DW treatment, while at 15 days after planting, it was 39% shorter, indicating that the PAW40 condition was the most suppressed condition. It is indicated that the appropri- Appl. Sci. 2021, 11, 2240 6 of 16 ate plasma treatment time is essential for plant growth and development, and if exces- sive, it inhibits growth (Figures 2 and 3). Figure 2. Plasma-activated water promotes root elongation during early root development in Ara- bidopsis thaliana L. Figure 3. PAW effects on root length during the developmental stage of Arabidopsis thaliana L. Seedlings were vertically grown for 15 days on agar medium. (a) DW, (b) PAW5, (c) PAW7, (d) PAW12. (e) PAW19, (f) PAW40. One square size indicates 2 × 2 cm. Because the difference in the root length was only clearly visible 7 days after plant- ing, we further checked more detailed morphological characteristics in the earlier stage of plant growth. At 5 days after planting, Col-0 grown on the plant medium did not show any differences in the root length. However, under closer examination using a ste- reo microscope, the number of root hairs increased by 84.83 ± 9.21 for the DW treatment and by 112.00 ± 5.25, 107.83 ± 5.74, 100.50 ± 8.11 and 92.66 ± 6.18 for the PAW5, PAW7, PAW12, and PAW19 treatments, respectively (Table 2). Furthermore, the length of the root hairs was 550.01 ± 138.61 μm for the DW treatment and 284.06 ± 63.12, 188.12 ± Appl. Sci. 2021, 11, 2240 7 of 16 46.67, and 161.50 ± 38.05 μm for the PAW5, PAW12, and PAW19 treatments, which are shorter than that of the DW treatment by about 48% and 71% for the PAW5 and PAW19 treatment, respectively, (Table 2, Supplementary Figure S1,). Therefore, the results show that the PAW treatment increases the number of root hairs while it shortens the length of the root hairs at 5 days after planting. Table 2. Total root length, root hair number and root hair length A. thaliana L. (Col.0) 5 days after planting. DW PAW5 PAW7 PAW12 PAW19 9.91 ± 0.85 8.19 ± 0.68 ns 8.04 ± 1.40 ns 8.92 ± 0.82 ns 7.95 ± 0.77 ns Root length(mm) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) 84.83 ± 9.21 112.00 ± 5.25 *** 107.83 ± 5.74 *** 100.50 ± 8.11 ** 92.66 ± 6.18 ns Root hair number (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) 550.01 ± 138.61 284.06 ± 63.12 *** 193.20 ± 33.45 *** 188.12 ± 46.67 *** 161.50 ± 38.05 *** Root hair length (μm) (N = 100) (N = 100) (N = 100) (N = 100) (N = 100) The midpoint in the longitudinal direction of the root was observed for the root hair number and root hair length by examining the most optimum root hair initiation and length phenotype. For the root length and root hair number, “n” indicates the number of individual plants. For root hair length, “N” indicates the individual root hair measured from total 6 plants. The results of the Table 2 are the mean ± SD (Student’s t-test compared with D.W. p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). 3.3. PAW Treatment Increases the Cotyledon Size Dependent on Palisade Cell Enlargement To further confirm the PAW effect on early vegetative development in Arabidopsis, the cotyledon was selected for cellular level examination. The size of the cotyledon was significantly increased in the PAW treated samples as examined by stereo microscope (Figure 4a–d). Quantification of the cotyledon size confirmed the enlargement of the cotyledon in the leaf length and leaf width directions for all PAW treatment conditions (Table 3). It was found that the cotyledon size for the PAW40 treatment condition was increased by about 60% in the leaf length direction (longitudinal direction) and by about 65% in the leaf width direction compared to the DW treatment condition. Figure 4. PAW treatment increases the Arabidopsis cotyledon dependent on the palisade cell size. (a–d) cotyledon size examined by a stereo microscope. (e,f) Palisade cells were examined from a cleared cotyledon sample under a differential interference contrast (DIC) microscope. (a,e) Deionized water (DW), (b,f) PAW5, (c,g) PAW19, (d,h) PAW40. (a–d) Bar = 1000 μm. (e–h) Bar = 50 μm). Table 3. Cotyledon size and palisade cell size and number of Arabidopsis grown in the DW and PAW treatment condition at 10 days after planting. DW PAW5 PAW7 PAW12 PAW19 PAW40 Cotyledon size in the 0.60 ± 0.03 0.77 ± 0.09 *** 0.81 ± 0.09 *** 0.84 ± 0.09 *** 0.88 ± 0.10 *** 0.96 ± 0.14 *** leaf length direction (n = 30) (n = 21) (n = 25) (n = 30) (n = 15) (n = 23) Appl. Sci. 2021, 11, 2240 8 of 16 (mm) Cotyledon size in the 0.46 ± 0.03 0.62 ± 0.07 *** 0.64 ± 0.06 ** 0.67 ± 0.07 *** 0.63 ± 0.05 *** 0.76 ± 0.08 *** leaf width direction (n = 30) (n = 21) (n = 25) (n = 30) (n = 25) (n = 23) (mm) ns ns ns ns 48.20 ± 6.83 43.20 ± 8.19 45.40 ± 5.59 ns 47.40 ± 5.85 48.40 ± 5.59 40.40 ± 5.31 Palisade cell number (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) 15.47 ± 1.50 24.63 ± 2.34 *** 25.07 ± 2.39 ** 25.59 ± 2.00 *** 31.35 ± 3.93 *** 42.88 ± 3.36 *** Palisade cell size (μm) (N = 300) (N = 300) (N = 300) (N = 300) (N = 300) (N = 300) For the cotyledon size and palisade cell number, “n” indicates the number of individual plants, and for the palisade cell size, “N” indicates the individual palisade cells measured from a total of 5 individual samples. Single cotyledons were measured from each biological sample. The palisade cell number was counted from the cotyledon length direction. Re- sults are the mean ± SD (Student’s t-test compared with DW; p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). There are two important factors that generally determine plant morphology. One is the number of cells involved in cell proliferation, and the other is the size of the cells in- volved in elongation [28]. To determine the main factor that affects the size of the coty- ledon, we observed the cell size and number of the palisade cells of the cotyledon using the clearing method and examined it under a DIC microscope (Figure 4e,f). As shown in Table 3, the difference in the number of palisade cells in the leaf length direction was not significant between the DW and PAW treatment conditions. However, the size of the palisade cells in the leaf length direction growth was 15.47 ± 1.50 μm in the DW treat- ment condition and 42.88 ± 3.36 μm in the PAW40 treatment condition, showing a 277% difference between DW and PAW40. The size of the palisade cells increased in propor- tion to the treatment time of the PAW. Therefore, it was found that plasma-activated water modulates the morphology of the cotyledon, which is dependent on cell size reg- ulation, not cell proliferation. 3.4. PAW Modulates the Root Hair Density through Root Developmental Genes Root hair is one of the important organs in plants. It controls osmotic pressure to absorb moisture and increases the area where the root comes into contact with the soil [29]. In Arabidopsis, the development of root hairs is determined by the location of the epidermal cells [30]. The epidermis cells adjacent to two cortical cells develop into root hairs, while epidermis adjacent to one cortical cell does not develop into root hairs [31,32]. GFP fusions with target genes such as GLABRA2 (GL2), WEREWOLF (WER), and CAPRICE (CPC) expressed in the root epidermal have been used to study root epidermal patterns in a previous study [26]. The visualization of the GFP helps to differentiate the cell structure and development process and clearly shows any alteration in the root. In Arabidopsis, cobl9-1 was shown to have root hair defects. The COBL9 gene is one of the important biomarkers as a root hair-specific gene and has a crucial role in root hair for- mation during development [33]. By using the double mutant WER::GFP/cobl9-1, we ex- pected to see a clear effect of the PAW treatment in epidermal cell patterning which can help us to understand the morphological structure at the single cell level, especially the root hairs. Furthermore, PAW treatment via cobl9 can help to confirm if the root hair phenotype is dependent on a single pathway or broader gene regulatory networks. The difference in the morphology of the root hairs between the DW and PAW treatments at days 5 and 7 of the developmental stage was confirmed using the WER::GFP/cobl9-1 mutant line. The results showed at 5 days after planting that there were no differences in the root length, but the root hair number and root hair length were changed between the DW and PAW treatment. In detail, the number of root hairs was increased by 14.5 ± 1.37 in the DW treatment. This was increased by 40.66 ± 5.71 and 27.50 ± 5.54 for the PAW5 and PAW19 treatments, respectively. The length of the root hair was decreased by 138.35 ± 11.11 and 85.05 ± 11.65 ųm for the PAW5 and PAW19 treatments compared with 261.21 ± 100.37 ųm for the DW treatment. These results show that the PAW treatment did not affect the total root length, but the root hair phenotypes Appl. Sci. 2021, 11, 2240 9 of 16 were altered for the plant harboring the mutation in the COBRA-LIKE9 genes, especially during the early developmental stage. In addition, the length of the root hairs was shortened, but the number of root hairs was increased for all PAW treatment conditions (Table 4, Supplementary Figure S2,). Table 4. Root hair number and length of Arabidopsis (WER::GFP/cobl9-1) at 5 days after planting. DW PAW5 PAW7 PAW12 PAW19 ns ns ns ns 9.75 ± 0.96 10.07 ± 0.75 10.10 ± 0.63 9.78 ± 0.95 10.08 ± 0.66 Root length (mm) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) ns 14.50 ± 1.37 40.66 ± 5.71 *** 33.16 ± 3.6 *** 31.00 ± 3.84 ** 27.50 ± 5.54 Root hair number (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) 261.21 ± 100.37 138.35 ± 11.11 *** 108.25 ± 11.54 *** 99.50 ± 13.65 *** 85.05 ± 11.65 *** Root hair length (μm) (N = 60) (N = 60) (N = 60) (N = 60) (N = 60) For root length and root hair number, “n” indicates the number of individual plants as biological replicates measured; for root hair length, “N” indicates the individual root hair length measured from a total of 6 samples. The results are the mean ± SD (Student’s t-test compared with D.W. p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). Because the differences in the root hair phenotype between the DW and PAW treatment conditions were significant at 5 days after planting (Table 4), a confocal mi- croscope was used for imaging the root epidermal cell shape at 7 days after planting. To visualize the cell type in the root, we used three different treatment conditions, DW, PAW5, and PAW19. The roots of the Arabidopsis WER::GFP/cobl9-1 line were grown for 7 days. Figure 5 shows the tagged WER promoter with GFP, which resulted in the ap- pearance of fluorescence in all the epidermal layers of the root cells. Quantification of the root hair numbers showed an increment in all the PAW treatment conditions com- pared to the DW treatment condition. In contrast, the root hair length was decreased in the PAW treatment condition compared to the DW treatment condition (Table 5, Sup- plementary Figure S2). Interestingly, the length of the root epidermal cells was elongat- ed with a similar pattern to the root length phenotype after the PAW treatment, which is consistent with the root enhancement in the PAW5 and PAW7 and the reduction in the PAW19 treatment condition compared to the DW treatment condition. It is believed that there will be a specific mechanism regulating the number of root hairs and length of the root hair in the PAW condition during the plant development process. Moreover, the results indicate that PAW5 is the optimized condition for plant growth and develop- ment. Table 5. Root hair number and length of A. thaliana L. (WER::GFP/cobl9-1) grown for 7 days after planting. DW PAW5 PAW19 15.14 ± 2.47 24.57 ± 3.10 *** 26.85 ± 5.33 *** Root hair number (n = 7) (n = 7) (n = 7) 162.92 ± 29.10 77.40 ± 7.21 *** 54.33 ± 7.83 *** Root hair length (μm) (N = 50) (N = 50) (N = 50) 133.01 ± 12.67 141.33 ± 11.45 *** 110.46 ± 9.38 *** Root epidermal cell (μm) (N = 50) (N = 50) (N = 50) For the root hair number, “n” indicates the number of individual plants; for the root hair length from the root epidermal cells, “N” indicates the individual root hairs measured from a total of 7 samples. The GFP signals for root hair umber, root hair length and root epidermal cell were meas- ured under a Nikon-A1 confocal microscope at 488 nm with a eyepiece lens (10×), objective lens (20×). The results in the Table are the mean ± SD (Student’s t-test compared with DW p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). Appl. Sci. 2021, 11, 2240 10 of 16 Figure 5. PAW regulates the number of root hairs and epidermal cell size in the Arabidopsis root (WER::GFP/cobl9-1). Ar- abidopsis seedlings were grown in solid media for 7 days. (a) DW, (b) PAW5, (c) PAW19. The white arrows indicate the individual root hairs that were used in the quantification. Bar = 100 μm. To further understand the mechanism of PAW affecting the root development in Arabidopsis, we confirmed the expression of several marker genes related to root hair ini- tiation and elongation. Previously, it was shown that the nitrate concentration could in- duce the expression of COBL9 which activates root hair initiation and elongation [34]. NT_COB9 was upregulated by PAW treatment in tobacco [25]. As expected, the expres- sion of COBL9 was significantly increased in the PAW5 treatment condition compared to the DW treatment condition. In contrast, the expression was suppressed in the PAW19 treatment condition. AUXIN1 (AUX1) and LIKE-AUXIN (LAX3) are the key auxin influx carriers and are multi-membrane transmembrane proteins that transfer the auxin in- volved in cell growth [35]. AUX1 and LAX3 both had up-regulated expression patterns in PAW5 while AUX1 was down-regulated in the PAW19 treatment condition. For OBF BINDING PROTEIN 4 (OBP4), its expression was down-regulated in the PAW5 and PAW19 treatment conditions. OBP4 is known as a negative regulator of root growth and differentiation including the root hairs [33,34], and it also functions as a negative regu- lator in PAW. Furthermore, XYLOGLUCAN ENDOTRANSGLUCOSYL- ASE/HYDROLASE (XTH) gene family members are factors involved in cell wall modifi- cation [36–38]. Interestingly, two genes, XTH9 and XTH17, showed high expression for PAW5 but low expression for the PAW19 treatment condition (Figure 6). We concluded that the root architecture under PAW is modulated by root developmental genes in- cluding COBL9, XTH9 and XTH17. Appl. Sci. 2021, 11, 2240 11 of 16 Figure 6. PAW treatment induces gene expression of root hair initiation and elongation genes in Arabidopsis. Student’s t-test compared with D.W. p < 0.05 *, p < 0.01 **, p < 0.001 ***. 4. Discussion We investigated the effect of plasma-activated water (PAW) on the early growth and development of Arabidopsis in this study. The physicochemical properties of the plasma-activated water were detected according to the plasma exposure time, and the effect of the altered physicochemical properties of the PAW on plant growth was exam- ined in Arabidopsis. The morphological differences in the roots and vegetative tissues, differences at the cellular level, and molecular biological differences were investigated. In addition, possible molecular mechanisms underlying the effect of the PAW treatment on plant growth were explored in Arabidopsis based on the phenotype. We found that PAW facilitated the growth of the Arabidopsis by modulating developmental genes at an early developmental stage. In recent years, plasma devices have emerged as an eco-friendly alternative method to improve plant growth and development. The application of plasma-related treat- ments in plants has been studied for improving seed germination, plant growth, plant height and yield in crops. The differences in plant phenotypes are caused by the differ- ence in physical and chemical properties with increased biochemical components, al- tered seed coat morphology, biochemical activities, and altered gene expression [5–8,18]. The degree of difference in a plasma-induced plant phenotype is due to a variety and range of RONS (reactive oxygen and nitrogen species) generated by plasma with differ- ent feeder gases or just air [39]. Low pressure dielectric barrier discharge generates Ar/O2, and plants treated with Ar/O2 produce increased H2O2 in the roots and shoots of wheat [40]. Cylinder double dielectric barrier discharge (DBD) reactors generate nitrate (NO3 ) and hydrogen peroxide (H2O2) that positively regulate growth and germination in seeds [18,19]. Even in Arabidopsis as a model plant, PAW generated by air dielectric barrier discharge activates growth and development such as the leaf area, the number of flowering plants and the leaf number over a long-term observation period, and this phenomenon is also partly explained by the elevated hydrogen peroxide and nitrate [7]. In this study, the plasma generated by the SDBD device was confirmed to have a nitrogen molecule spectrum in the region of 290~400 nm (Figure 1). Nitrogen ions are mainly produced due to the plasma effect and react with H2O. When they ionize into - − NO2 and NO3 , the pH decreases, and the conductivity increases. Compared to other studies on both direct and indirect plasma applications, ROS (reactive oxygen species) Appl. Sci. 2021, 11, 2240 12 of 16 are the main factor affecting germination and plant growth and defense [7,40,41]. The SDBD generator device used in this study mainly produced a higher NO3 concentration when a longer plasma treatment time was used. In addition, the change of pH and con- ductivity was also significant at longer plasma treatment. This change was predicted to affect the plant growth and development. In a previous study, tobacco grown on me- dium containing plasma treated water had increased cotyledon growth but suppressed primary root hair growth at PAW40 [25]. Arabidopsis seedlings showed self-sufficient under minimal conditions for weeks without external supply of nutrients [42]. There- fore, we used the minimal media of only DW and PAW to study the more dramatic ef- fect of the PAW treatment. By using this method, we expected to see more specific pathways that are affected by PAW. Even though it is important to study the effect of all component changes in PAW such as conductivity and osmolality, in this study we fo- cused on the changes in the nitrate concentration which is a main nitrogen source that affects plant growth. Nitrogen is an essential macronutrient for plants and an important constituent of key molecules such as amino acids, nucleic acid and chlorophyll [43]. Ni- trogen availability in the soil is a major factor in plant growth, and its availability is a key determinant of plant yield [43–46]. From this perspective, the increment of the ni- trate concentration from plasma treatment potentially can serve as a very important ni- trogen source for plant growth and development. Concerning root development, the PAW treatment condition (PAW5, 7 and 12) promoted an increased total root length compared with the DW treatment condition in Arabidopsis; however, root development was inhibited by the PAW that was generated with a longer plasma treatment time (PAW19 and 40). In detail, the morphological dif- ferences at 15 days after planting in the root showed that the root length for the PAW5 treatment condition increased by 87%; however, it was suppressed by the PAW40 treat- ment condition by 63% compared to the DW control, indicating that root growth oc- curred independent of the plasma treatment time used to generate the PAW. Thus, Ara- bidopsis needs specific growth conditions (Figures 2 and 3). In contrast to the specific pattern of activation and inhibition of root growth, the effect of the PAW treatment in the cotyledon tissue did not show any inhibition. The cotyledon size increased according to the plasma treatment time of the PAW due to an enlarged palisade cell size, not by cell number. In tobacco, the cotyledon size was also regulated by cell size not cell prolif- eration at 10 days after planting [25]. This result implies that they share the same mech- anism in cotyledon development in dicotyledonous plants. In the seedlings, the roots become activated in the PAW5, 7 and 12 treatment conditions and inhibited in the PAW19 and 40 treatment conditions, and the cotyledon expands according to the plasma treatment time of the PAW as a tissue-specific plasma effect. Therefore, we need to un- derstand it as an overall balance of the plant developmental growth and investigating the optimal treatment conditions for Arabidopsis is important to achieve the desired ef- fect. Interestingly, the PAW treatments showed an increased number of root hairs in all the PAW conditions; however, they showed a decreased root hair cell length shown by cell length measurements, in other words, a decreased trichoblast cell length (Table 2). In addition, root hair number in the WER::GFP/cobl9-1 line increased induced by the PAW treatment while root cell elongation shortened. In the Col-0 background, the root hair number increased by 18~32% dependent on the PAW treatment time (Table 2). Root hair number in the cobl9-1 mutant background was dramatically increased by at least two times by the PAW treatment at 5 days after planting (Table 4). It implies that the regula- tion of root hairs under PAW treatment involves sophisticated genetic networks, not simply a single pathway. Moreover, this result indicates the increased root hair number induced by PAW, which results in securing more space for root epidermal cells to come into contact with nutrients per unit of root cell. These differences may lead to improved growth because the root hairs are in response to the availability of NO3 which has an important role in nitrogen (N) source nutrient acquisition when plants are exposed to Appl. Sci. 2021, 11, 2240 13 of 16 nitrate-rich soils. It is known that nitrate also can act as signaling molecules that can control downstream gene expression in plants. Previously, the change in the root archi- tecture in plants by the nitrate concentration was presented as an example of plasticity in the developmental process in response to the environment [36,47]. For example, the in- crement of nitrate concentration of 0.1 to 1 mM KNO3 was known to induce root hair and lateral root formation [48–50]. In contrast, high nitrate concentration suppressed the root formations [47,48,51]. At the molecular level, we confirmed the effect of the PAW treatment by checking the expression of marker genes related to root development. One of the key genes of root hair initiation and root hair elongations, COBL9, was induced in the PAW5 and sup- pressed in the PAW19 treatment condition. This expression result confirmed our finding of the different phenotypes in root hair number and root length between the Col-0 and cobl9-1 mutants which were affected by the COBL9 expression (Tables 2 and 4, Figure 6). and our PAW generated from SDBD mainly affects the nitrate concentration. In plants, auxin has a major role in root hair development, and the auxin trans- porter proteins, AUX1 and LAX3, regulate lateral root formation [35,50]. Short root hair is due to mutations of AUX1 genes indicating AUX1 controls elongation of the root hair [52]. We checked the expression of two auxin transport genes, AUX1 and LAX3, which were induced in the PAW5 treatment condition; however, the expression was sup- pressed in the PAW19 treatment condition only in AUX1. This expression pattern of the AUX1 gene is in line with a previous report that AUX1 predominantly regulates root hair elongation whereas LAX3 mostly regulates the lateral root formation [35]. Further- more, we confirmed the expression of the OBP4, XTH9 and XTH17 genes that are known to regulate root hair initiation and elongation and are sensitive to the nitrate concentra- tion in the media [50,53]. In detail, OBP4 acts as a negative regulator of root growth, while XTH9 and XTH17 act as a positive regulator of root growth through cell expan- sion. The expression showed that these genes were affected by the PAW treatments. All the PAW treatments suppressed the expression of OBP4. Although the expressions of XTH9 and XTH17 were suppressed in the PAW19 treatment condition, the maximal ob- served effect was observed on the root development by the PAW5 treatment compared to all the other PAW generation times (PAW7, 12, 19 and 40). Therefore, the PAW5 condi- tion affected the root growth by positively regulating the expression of XTH9 and XTH17 which may mainly be caused by the root cell expansion. Moreover, as showed in the Figure 5 and Table 5, the cell expansion of PAW19 was severely reduced, thus corre- lating with lower expression of XTH genes. It is suggested that PAW-mediated root ar- chitecture regulates the root developmental genes in Arabidopsis, and it is at least in- volved in the root auxin transporter signaling system. Taken together, the molecular mechanism of the PAW treatment increases the ni- trate concentration in PAW which induces the activation of the nitrate-responsive and cell expansion genes leading to the induction of the root hair density and root length. However, the mechanism we explored may still be affected by other characters such as a change in the osmolarity of the medium. Therefore, further detailed analyses still need to be done on each component of the PAW and their effects on plant growth. In addi- tion, to further understand PAW regulation at the molecular level, a detailed study us- ing current technology such as RNA-sequencing and small-RNA profiling is necessary for future applications. By revealing these mechanisms, further fine-tuned applications of plasma treatment for specific bio-samples will be elucidated from the specific target pathways in a precise manner. 5. Conclusions PAW generated from a SDBD reactor can be used to enhance Arabidopsis growth in the early seedling stage. The PAW treatments were shown to affect the root hair density mediated by the COBL9, XTH9 and XTH17 genes. The maximal observed condition for Arabidopsis root development was identified as PAW5 in which potential target genes Appl. Sci. 2021, 11, 2240 14 of 16 were up-regulated while root development was suppressed by the PAW19 treatment condition. The findings of this study will provide new insights at the molecular level in- to developmental stage plants and accelerate the study of applying plasma technology to crop plants. Supplementary Materials: The following are available online at www.mdpi.com/2076-3417/11/5/2240/s1, Supplementary Figure S1: PAW regulates number of root hair in (Col-0) Arabidopsis (a) DW, (b) PAW5, (c) PAW7, (d) PAW12, (e) PAW19. Seedlings were grown on solid media for 5 days. Bar = 100 μm. Figure S2: PAW regulates number of root hair in (WER::GFP/cobl9-1) Arabidopsis (a) DW, (b) PAW5, (c) PAW7, (d) PAW12, (e) PAW19. Seedlings were grown on solid media for 5 days. Bar = 100 μm. Table S1: List of primers used in this study. Author Contributions: Conceptualization, Y.K.L., I.A.L., S.B.K., and R.A.P.; experiment and data curation, D.H.K., R.A.P., J.Y.P., and S.J.P.; writing—original draft preparation, D.H.K., R.A.P., and Y.K.L.; writing—review and editing, R.A.P. and Y.K.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by R&D Program of “Plasma Advanced Technology for Agriculture and Food (Plasma Farming) (EN2025)-1711124797)” through the Korea Institute of Fu- sion Energy(KFE) funded by the Government funds, Republic of Korea, and a grant from the ‘Na- tional Research Foundation of Korea (grant no. NRF-2016R1C1B2015877) funded by the Ministry of Science, ICT & Future Planning’ to S.J.P. Conflicts of Interest: The authors declare no conflict of interest. References 1. Adhikari, B.; Adhikari, M.; Park, G. The Effects of Plasma on Plant Growth, Development, and Sustainability. Appl. Sci. 2020, 10, 6045, doi:10.3390/app10176045. 2. Ito, M.; Ohta, T.; Hori, M. Plasma Agriculture. J. Korean Phys. Soc. 2012, 60, 937–943, doi:10.3938/jkps.60.937. 3. Ohta, T. Plasma in Agriculture. In Cold Plasma in Food and Agriculture; Elsevier: Amsterdam, The Netherlands, 2016; pp. 205– 221, ISBN 978-0-12-801365-6. 4. Starič, P.; Vogel-Mikuš, K.; Mozetič, M.; Junkar, I. Effects of Nonthermal Plasma on Morphology, Genetics and Physiology of Seeds: A Review. Plants 2020, 9, 1736, doi:10.3390/plants9121736. 5. Adhikari, B.; Adhikari, M.; Ghimire, B.; Park, G.; Choi, E.H. Cold Atmospheric Plasma-Activated Water Irrigation Induces Defense Hormone and Gene Expression in Tomato Seedlings. Sci. Rep. 2019, 9, 16080, doi:10.1038/s41598-019-52646-z. 6. Fan, L.; Liu, X.; Ma, Y.; Xiang, Q. Effects of Plasma-Activated Water Treatment on Seed Germination and Growth of Mung Bean Sprouts. J. Taibah Univ. Sci. 2020, 14, 823–830, doi:10.1080/16583655.2020.1778326. 7. Bafoil, M.; Jemmat, A.; Martinez, Y.; Merbahi, N.; Eichwald, O.; Dunand, C.; Yousfi, M. Effects of Low Temperature Plasmas and Plasma Activated Waters on Arabidopsis Thaliana Germination and Growth. PLoS ONE 2018, 13, e0195512, doi:10.1371/journal.pone.0195512. 8. Sergeichev, K.F.; Lukina, N.A.; Sarimov, R.M.; Smirnov, I.G.; Simakin, A.V.; Dorokhov, A.S.; Gudkov, S.V. Physicochemical Properties of Pure Water Treated by Pure Argon Plasma Jet Generated by Microwave Discharge in Opened Atmosphere. Front. Phys. 2021, 8, 614684, doi:10.3389/fphy.2020.614684. 9. Belov, S.V.; Danyleiko, Y.K.; Glinushkin, A.P.; Kalinitchenko, V.P.; Egorov, A.V.; Sidorov, V.A.; Konchekov, E.M.; Gudkov, S.V.; Dorokhov, A.S.; Lobachevsky, Y.P.; et al. An Activated Potassium Phosphate Fertilizer Solution for Stimulating the Growth of Agricultural Plants. Front. Phys. 2021, 8, 618320, doi:10.3389/fphy.2020.618320. 10. Traylor, M.J.; Pavlovich, M.J.; Karim, S.; Hait, P.; Sakiyama, Y.; Clark, D.S.; Graves, D.B. Long-Term Antibacterial Efficacy of Air Plasma-Activated Water. J. Phys. D Appl. Phys. 2011, 44, 472001, doi:10.1088/0022-3727/44/47/472001. 11. Zhou, R.; Zhou, R.; Wang, P.; Xian, Y.; Mai-prochnow, A.; Lu, X.P.; Cullen, P.; Ostrikov, K. (Ken); Bazaka, K. Plasma Activated Water (PAW): Generation, Origin of Reactive Species and Biological Applications. J. Phys. D Appl. Phys. 2020, 53, 303001, doi:10.1088/1361-6463/ab81cf. 12. Zhang, S.; Rousseau, A.; Dufour, T. Promoting Lentil Germination and Stem Growth by Plasma Activated Tap Water, De- mineralized Water and Liquid Fertilizer. RSC Adv. 2017, 7, 31244–31251, doi:10.1039/C7RA04663D. 13. Graves, D.B.; Bakken, L.B.; Jensen, M.B.; Ingels, R. Plasma Activated Organic Fertilizer. Plasma Chem. Plasma Process. 2019, 39, 1–19, doi:10.1007/s11090-018-9944-9. 14. Gaudinier, A.; Rodriguez-Medina, J.; Zhang, L.; Olson, A.; Liseron-Monfils, C.; Bagman, A.M.; Foret, J.; Abbitt, S.; Tang, M.; Li, B.; et al. Transcriptional Regulation of Nitrogen-Associated Metabolism and Growth. Nature 2018, 563, 259–264, doi:10.1038/s41586-018-0656-3. 15. Ueda, Y.; Yanagisawa, S. Engineering Nitrogen Utilization in Crop. Plants; Springer International Publishing: Basingstoke, UK, 2018; ISBN 978-3-319-92957-6. Appl. Sci. 2021, 11, 2240 15 of 16 16. Chandran, A.K.N.; Priatama, R.A.; Kumar, V.; Xuan, Y.; Je, B.I.; Kim, C.M.; Jung, K.H.; Han, C.D. Genome-Wide Transcriptome Analysis of Expression in Rice Seedling Roots in Response to Supplemental Nitrogen. J. Plant Physiol. 2016, 200, 62–75, doi:10.1016/j.jplph.2016.06.005. 17. Sarinont, T.; Katayama, R.; Wada, Y.; Koga, K.; Shiratani, M. Plant Growth Enhancement of Seeds Immersed in Plasma Acti- vated Water. MRS Adv. 2017, 2, 995–1000, doi:10.1557/adv.2017.178. 18. Sivachandiran, L.; Khacef, A. Enhanced Seed Germination and Plant Growth by Atmospheric Pressure Cold Air Plasma: Combined Effect of Seed and Water Treatment. RSC Adv. 2017, 7, 1822–1832, doi:10.1039/C6RA24762H. 19. Song, J.-S.; Lee, M.J.; Ra, J.E.; Lee, K.S.; Eom, S.; Ham, H.M.; Kim, H.Y.; Kim, S.B.; Lim, J. Growth and Bioactive Phytochemicals in Barley (Hordeum Vulgare L.) Sprouts Affected by Atmospheric Pressure Plasma during Seed Germination. J. Phys. Appl. Phys. 2020, 53, 314002, doi:10.1088/1361-6463/ab810d. 20. Adhikari, B.; Adhikari, M.; Ghimire, B.; Adhikari, B.C.; Park, G.; Choi, E.H. Cold Plasma Seed Priming Modulates Growth, Redox Homeostasis and Stress Response by Inducing Reactive Species in Tomato (Solanum Lycopersicum). Free Radic. Biol. Med. 2020, 156, 57–69, doi:10.1016/j.freeradbiomed.2020.06.003. 21. Thirumdas, R.; Kothakota, A.; Annapure, U.; Siliveru, K.; Blundell, R.; Gatt, R.; Valdramidis, V.P. Plasma Activated Water (PAW): Chemistry, Physico-Chemical Properties, Applications in Food and Agriculture. Trends Food Sci. Technol. 2018, 77, 21– 31, doi:10.1016/j.tifs.2018.05.007. 22. Thirumdas, R.; Sarangapani, C. Cold Plasma : A Novel Non-Thermal Technology for Food Processing. Food Biophys. 2015, 10, 1–11, doi:10.1007/s11483-014-9382-z. 23. Meinke, D.W.; Cherry, J.M.; Dean, C.; Rounsley, S.D.; Koornneef, M. Arabidopsis Thaliana: A Model Plant for Genome Anal- ysis. Science 1998, 282, 662–682, doi:10.1126/science.282.5389.662. 24. The Arabidopsis Genome Initiative Analysis of the Genome Sequence of the Flowering Plant Arabidopsis Thaliana. Nature 2000, 408, 796–815, doi:10.1038/35048692. 25. Lee, Y.K.; Lim, J.; Hong, E.J.; Kim, S.B. Plasma-Activated Water Regulates Root Hairs and Cotyledon Size Dependent on Cell Elongation in Nicotiana Tabacum L. Plant Biotechnol. Rep. 2020, 14, 663–672, doi:10.1007/s11816-020-00641-6. 26. Lee, M.M.; Schiefelbein, J. Cell Pattern in the Arabidopsis Root Epidermis Determined by Lateral Inhibition with Feedback. Plant Cell 2002, 14, 611–618, doi:10.1105/tpc.010434. 27. Bruex, A.; Kainkaryam, R.M.; Wieckowski, Y.; Kang, Y.H.; Bernhardt, C.; Xia, Y.; Zheng, X.; Wang, J.Y.; Lee, M.M.; Benfey, P.; et al. A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis. PLoS Genet. 2012, 8, e1002446, doi:10.1371/journal.pgen.1002446. 28. Horiguchi, G.; Ferjani, A.; Fujikura, U.; Tsukaya, H. Coordination of Cell Proliferation and Cell Expansion in the Control of Leaf Size in Arabidopsis Thaliana. J. Plant Res. 2006, 119, 37–42, doi:10.1007/s10265-005-0232-4. 29. Aiken, R.M.; Smucker, A.J.M. Root System Regulation of Whole Plant Growth. Annu. Rev. Phytopathol. 1996, 34, 325–346, doi:10.1146/annurev.phyto.34.1.325. 30. Grierson, C.; Nielsen, E.; Ketelaarc, T.; Schiefelbein, J. Root Hairs. Arab. Book 2014, 12, e0172, doi:10.1199/tab.0172. 31. Datta, S.; Kim, C.M.; Pernas, M.; Pires, N.D.; Proust, H.; Tam, T.; Vijayakumar, P.; Dolan, L. Root Hairs: Development, Growth and Evolution at the Plant-Soil Interface. Plant Soil 2011, 346, 1–14, doi:10.1007/s11104-011-0845-4. 32. Julkowska, M. Releasing the Cytokinin Brakes on Root Growth. Plant Physiol. 2018, 177, 865–866, doi:10.1104/pp.18.00660. 33. Parry, G.; Marchant, A.; May, S.; Swarup, R.; Swarup, K.; James, N.; Graham, N.; Allen, T.; Martucci, T.; Yemm, A.; et al. Quick on the Uptake: Characterization of a Family of Plant Auxin Influx Carriers. J. Plant Growth Regul. 2001, 20, 217–225, doi:10.1007/s003440010030. 34. Schindelman, G. COBRA Encodes a Putative GPI-Anchored Protein, Which Is Polarly Localized and Necessary for Oriented Cell Expansion in Arabidopsis. Genes Dev. 2001, 15, 1115–1127, doi:10.1101/gad.879101. 35. Swarup, R.; Bhosale, R. Developmental Roles of AUX1/LAX Auxin Influx Carriers in Plants. Front. Plant Sci. 2019, 10, 1306, doi:10.3389/fpls.2019.01306. 36. Xu, P.; Cai, W. Nitrate-Responsive OBP4-XTH9 Regulatory Module Controls Lateral Root Development in Arabidopsis Tha- liana. PLoS Genet. 2019, 15, e1008465, doi:10.1371/journal.pgen.1008465. 37. Lee, Y.K.; Rhee, J.Y.; Lee, S.H.; Chung, G.C.; Park, S.J.; Segami, S.; Maeshima, M.; Choi, G. Functionally Redundant LNG3 and LNG4 Genes Regulate Turgor-Driven Polar Cell Elongation through Activation of XTH17 and XTH24. Plant Mol. Biol. 2018, 97, 23–36, doi:10.1007/s11103-018-0722-0. 38. Maris, A.; Suslov, D.; Fry, S.C.; Verbelen, J.-P.; Vissenberg, K. Enzymic Characterization of Two Recombinant Xyloglucan En- dotransglucosylase/Hydrolase (XTH) Proteins of Arabidopsis and Their Effect on Root Growth and Cell Wall Extension. J. Exp. Bot. 2009, 60, 3959–3972, doi:10.1093/jxb/erp229. 39. Ahn, C.; Gill, J.; Ruzic, D.N. Growth of Plasma-Treated Corn Seeds under Realistic Conditions. Sci. Rep. 2019, 9, 4355, doi:10.1038/s41598-019-40700-9. 40. Rahman, M.M.; Sajib, S.A.; Rahi, M.S.; Tahura, S.; Roy, N.C.; Parvez, S.; Reza, M.A.; Talukder, M.R.; Kabir, A.H. Mechanisms and Signaling Associated with LPDBD Plasma Mediated Growth Improvement in Wheat. Sci. Rep. 2018, 8, 10498, doi:10.1038/s41598-018-28960-3. 41. del Río, L.A. ROS and RNS in Plant Physiology: An Overview. J. Exp. Bot. 2015, 66, 2827–2837, doi:10.1093/jxb/erv099. Appl. Sci. 2021, 11, 2240 16 of 16 42. Réthoré, E.; d’Andrea, S.; Benamar, A.; Cukier, C.; Tolleter, D.; Limami, A.M.; Avelange-Macherel, M.-H.; Macherel, D. Ara- bidopsis Seedlings Display a Remarkable Resilience under Severe Mineral Starvation Using Their Metabolic Plasticity to Re- main Self-Sufficient for Weeks. Plant J. 2019, 99, 302–315, doi:10.1111/tpj.14325. 43. Tegeder, M.; Masclaux-Daubresse, C. Source and Sink Mechanisms of Nitrogen Transport and Use. New Phytol. 2018, 217, 35– 53, doi:10.1111/nph.14876. 44. Zhao, M.; Zhao, M.; Gu, S.; Sun, J.; Ma, Z.; Wang, L.; Zheng, W.; Xu, Z. DEP1 Is Involved in Regulating the Carbon–Nitrogen Metabolic Balance to Affect Grain Yield and Quality in Rice (Oriza Sativa L.). PLoS ONE 2019, 14, e0213504, doi:10.1371/journal.pone.0213504. 45. Kumar, V.; Kim, S.H.; Priatama, R.A.; Jeong, J.H.; Adnan, M.R.; Saputra, B.A.; Kim, C.M.; Je, B.I.; Park, S.J.; Jung, K.H.; et al. NH4+ Suppresses NO3–-Dependent Lateral Root Growth and Alters Gene Expression and Gravity Response in OsAMT1 RNAi Mutants of Rice (Oryza Sativa). J. Plant Biol. 2020, 63, 391–407, doi:10.1007/s12374-020-09263-5. 46. Xuan, Y.H.; Priatama, R.A.; Huang, J.; Je, B.I.; Liu, J.M.; Park, S.J.; Piao, H.L.; Son, D.Y.; Lee, J.J.; Park, S.H.; et al. Indeterminate Domain 10 Regulates Ammonium-Mediated Gene Expression in Rice Roots. New Phytol. 2013, 197, 791–804, doi:10.1111/nph.12075. 47. Boer, M.D.; Santos Teixeira, J.; Ten Tusscher, K.H. Modeling of Root Nitrate Responses Suggests Preferential Foraging Arises From the Integration of Demand, Supply and Local Presence Signals. Front. Plant Sci. 2020, 11, 708, doi:10.3389/fpls.2020.00708. 48. Zhang, H. Regulation of Arabidopsis Root Development by Nitrate Availability. J. Exp. Bot. 2000, 51, 51–59, doi:10.1093/jexbot/51.342.51. 49. Canales, J.; Contreras-López, O.; Álvarez, J.M.; Gutiérrez, R.A. Nitrate Induction of Root Hair Density Is Mediated by TGA1/TGA4 and CPC Transcription Factors in Arabidopsis Thaliana. Plant J. 2017, 92, 305–316, doi:10.1111/tpj.13656. 50. Quint, M.; Gray, W.M. Auxin Signaling. Curr. Opin. Plant Biol. 2006, 9, 448–453, doi:10.1016/j.pbi.2006.07.006. 51. Asim, M.; Ullah, Z.; Xu, F.; An, L.; Aluko, O.O.; Wang, Q.; Liu, H. Nitrate Signaling, Functions, and Regulation of Root System Architecture: Insights from Arabidopsis Thaliana. Genes 2020, 11, 633, doi:10.3390/genes11060633. 52. Salazar-Henao, J.E.; Vélez-Bermúdez, I.C.; Schmidt, W. The Regulation and Plasticity of Root Hair Patterning and Morpho- genesis. Development 2016, 143, 1848–1858, doi:10.1242/dev.132845. 53. Itoh, J.-I.; Hibara, K.-I.; Sato, Y.; Nagato, Y. Developmental Role and Auxin Responsiveness of Class III Homeodomain Leucine Zipper Gene Family Members in Rice. 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Plasma-Activated Water Modulates Root Hair Cell Density via Root Developmental Genes in Arabidopsis thaliana L.

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Article Plasma-Activated Water Modulates Root Hair Cell Density via Root Developmental Genes in Arabidopsis thaliana L. 1,2,† 1,† 1,3 4 1 2 Dong Hyeun Ka , Ryza Aditya Priatama , Joo Young Park , Soon Ju Park , Seong Bong Kim , In Ah Lee and 1, Young Koung Lee * Institute of Plasma Technology, Korea Institute of Fusion Energy, 37 Dongjangsan-ro, Gunsan-si, Jeollabuk-do 54004, Korea; kdh9799@kfe.re.kr (D.H.K.); priatama@kfe.re.kr (R.A.P.); pjy901113@gmail.com (J.Y.P.); sbkim@kfe.re.kr (S.B.K.) Department of Chemistry, College of Natural Science, Kunsan National University, 558 Daehak-ro, Gunsan-si, Jeollabuk-do 54150, Korea; leeinah@kunsan.ac.kr Surface Materials Division, Korea Institute of Materials Science, 797, Changwon-daero, Seongsan-gu, Changwon-si, Gyeongsangnam-do 51508, Korea Division of Biological Sciences and Research Institute for Basic Science, Wonkwang University, Iksan, Jeollabuk-do 54538, Korea; sjpark75@wku.ac.kr * Correspondence: leeyk@kfe.re.kr † Dong Hyeun Ka and Ryza Aditya Priatama contributed equally to this work. Abstract: Low-temperature atmospheric pressure plasma technology has been used in agriculture and plant science by direct and indirect treatment of bio-samples. However, the cellular and mo- lecular mechanisms affected by plasma-activated water (PAW) are largely unexplored. In this study, PAW generated from a surface dielectric barrier discharge (SDBD) device was used for plant development. Physicochemical analysis was performed to confirm the PAW properties that corre- lated with the plasma treatment time. Arabidopsis thaliana L. was utilized to study the effect of the Citation: Ka, D.H.; Priatama, R.A.; PAW treatment in the early developmental stage. The plasma-activated water samples are denoted Park, J.Y.; Park, S.J.; Kim, S.B.; Lee, I.A.; Lee, Y.K. Plasma-Activated as PAW5 time in minutes (min), PAW7 min, PAW12 min, PAW19 min and PAW40 min with the Water Modulates Root Hair Cell plasma treatment time. Seedlings grown in the PAW5, PAW7 and PAW12 had increased root Density Via Root Developmental lengths while the root lengths were decreased in the PAW19 and PAW40. In the cellular level ob- Genes in Arabidopsis thaliana L. servation, the PAW treatment specifically increased the root hair numbers per unit of the root but Appl. Sci. 2021, 11, 2240. suppressed the root hair length in the PAW, indicating that PAW mainly modulates the root hair https://doi.org/10.3390/app11052240 cell density in the root. Furthermore, we found that the root hair density and length at PAW5 in maximal observed conditions were positively regulated by root developmental-related genes in- Academic Editor: Gyungsoon Park cluding COBRA-LIKE9 (COBL9), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (XTH9), XTH17, AUXIN1 (AUX1) and LIKE-AUXIN (LAX3). Received: 27 January 2021 Accepted: 26 February 2021 Keywords: plasma agriculture; plasma-activated water; Arabidopsis thaliana L.; nitrate; root cell Published: 3 March 2021 number Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and insti- tutional affiliations. 1. Introduction Low-temperature, atmospheric pressure, plasma technology has emerged as a new technology that potentially enhances plant growth and defense [1–3]. Bio-samples can be treated by a plasma device through gas or water [4]. Plasma-activated water (PAW) Copyright: © 2021 by the authors. treatment changes the chemical properties, and PAW containing an altered chemical Licensee MDPI, Basel, Switzerland. composition is utilized with plants [5,6]. In this indirect method, the development of This article is an open access article low-temperature plasma technology is used for plant cultivation in agronomy [7–9]. distributed under the terms and PAW has differences in electrical conductivity, pH, nitrite, nitrate and H2O2 compared conditions of the Creative Commons with deionized water (DW). As for the application of PAW in plants, ionized oxygen and Attribution (CC BY) license nitrogen species dissolve in the water to create an acidic environment due to a lower pH (http://creativecommons.org/licenses [10,11]. Moreover, water treated with low-temperature plasma contains a high concen- /by/4.0/). Appl. Sci. 2021, 11, 2240. https://doi.org/10.3390/app11052240 www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 2240 2 of 16 tration of nitrate. Therefore, it has the role of a nitrogen (N) source for plant growth and could be possibly used as a liquid fertilizer [12,13]. Nitrogen is an important macronu- trient for plant growth and an essential component in the basic metabolic process [14–16]. The use of nitrogen fertilizer has a critical role in the green revolution by improving plant yield. Several reports have been published on using plasma treatment as an eco-friendly technology that produces nitrogen and functions as a liquid fertilizer [12,13,17,18]. Direct plasma application changes the appearance of the seed coat surface, resulting in a reduced time for germination and rapid growth in the development stage [7,19]. In tomatoes, a horticultural crop, stem growth differs from the controls according to the helium plasma treatment time [20]. The difference between the germination rate and growth rate for PAW in Raphanus sativus, Solanum lycopersicum and Capsicum annum has been reported to be due to the effects of a long PAW treatment time [18]. Both direct and indirect processing of plasma is considered to have a positive effect on plant develop- ment [21,22]. We selected Arabidopsis thaliana L. (Arabidopsis) as a model plant to understand the effect of PAW on its molecular mechanism during the plant development process. The growth cycle of Arabidopsis thaliana L. is very short, 4–6 weeks. In addition, the number of seeds in the plant is about 1500, which is a good model for studying genetics. Ara- bidopsis has a relatively small 135 Mbp genome, 5 pairs of chromosomes and 33,602 genes, which make it easy to analyze the genome, and can be used for other crop appli- cations. The Arabidopsis genome sequence was first decoded in December 2000. Since then, the advancement of plant science has accelerated at a faster pace [23,24]. This ad- vancement was due to the influence of Arabidopsis as a good model plant to understand the fundamental phenomena of plants. In this study, we examined the phenotype, cytological observation, and gene ex- pression after PAW treatment to elucidate the molecular mechanism of Arabidopsis dur- ing the development process. As a plasma generating device, the plasma-activated water was generated using a surface dielectric barrier discharge (SDBD) to dissolve ionized gas in deionized water. In particular, hydrogen peroxide (H2O2), nitrate (NO3-), pH and electrical conductivity were altered during the plasma treatment time. The altered phys- icochemical properties affected the vegetative and root development. In the cotyledon, PAW treated palisade cells were regulated by cell expansion, not cell proliferation, and the root hair number was dramatically increased in the root cells. In the present study, we deduced that PAW modulates the root hair cell density induced by the expression of root developmental-related genes, COBL9, XTH9, XTH17, AUX1 and LAX3. 2. Materials and Methods 2.1. Plasma-Activated Water Preparation and Physicochemical Analysis The plasma source used in this study was previously described in Lee et al., 2020 [25]. A surface dielectric barrier discharge (SDBD) in an air-tight container was used as the plasma treatment device. The electrodes of the SDBD generator were made up of two parallel metals, one of which was covered with a dielectric layer, and the plasma was generated using an alternating current (AC) flowing through the electrodes. The electrode consisted of powered and grounded stainless steel, and an aluminum oxide plate (1 mm-thick) was placed between the electrodes. Each electrode was attached at the top of the reactor with a 10 W average consumed power, an 8 kVpp voltage ampli- tude and a 17 kHz frequency. A 12 cm fan below the electrodes was used (15-LED 120, Aone, China) to circulate the air inside the reactor. In the reactor, the distance between the two electrodes and the water surface was 12 cm. The optical emission spectrum (OES) was obtained by an ultraviolet–visible (UV-VIS) spectrometer (Oceanoptics, maya 2000 pro) within the range of 200–600 nm. An optical lens (Ocean optics, UV-74) was used to collimate the emission spectrum. The lens was placed in front of the SDBD at Appl. Sci. 2021, 11, 2240 3 of 16 1.5–2.0 cm. The OES was obtained with an integration time of 1 s which was averaged 50 times. PAW was produced by adding 1 L of deionized to the reactor. The PAW was pre- pared according to the plasma treatment time for 5, 7, 12, 19 and 40 min The plas- ma-activated water samples in this paper are denoted as PAW5, PAW7, PAW12, PAW19 and PAW40 with the number indicating the plasma treatment time. To confirm the chemical properties of the PAW, the anion content was measured one day after PAW generation using ion chromatography (ICS-2100, Thermo Dionex, Sunnybale, CA, USA). To quantify the anions, the standard sample [Dionex Seven Anion Standard II (in deionized water)] was measured with a three-point method (25, 50, and 100 mg/L) [24], and the pH and conductivity were measured using Orion™ Versa Star Pro™ (Thermo Scientific, Waltham, MA, USA). To meet the appropriate pH for plant growth, the pH was adjusted to pH 5.6~5.8 with 0.1 N and 1 N KOH solution. An AQ- UAfast AQ3700 Colorimeter (ORION AQ3700, Thermo Scientific Orion, USA) was used to measure the hydrogen peroxide (H2O2) using the hydrogen peroxide LR method (Tintometer Lovibond, UK). All chemical properties measurement was performed using at least 3 replicates. 2.2. Plant Material and Growth Conditions To prevent contamination, the Arabidopsis thaliana L. (Columbia 0, Col-0, ecotype background) seeds were surface sterilized with 20% commercial bleach followed by washing with sterilized deionized water and then stored at 4 °C for 3 days for the cold treatment before placing them onto the media. The sterilized seeds were placed onto agar medium and allowed to dry for 30 min before moving the plates into the growth chamber. The growth chamber setting was set at 22 °C, 60% relative humidity, and 16 h/8 h, light/dark condition, respectively. The seeds were in the growth chamber for 15 days. Solid medium was prepared using plant agar (Duchefa Biochemie) at a 0.8% con- centration with deionized water (DW) and PAW (5, 7, 12, 19, 40 min). Seedlings were grown on the minimal growth condition media at the vertical position for the root length examination and the horizontal position for the cotyledon size examination. All steriliza- tion and planting procedures were carried out on a clean bench. To observe the root morphology, the midpoint in the longitudinal direction of the root was observed for the optimal root hair initiation and length phenotype. WER::GFP/cobl9-1 (CS67758) and cobl9-1 (Salk_099933) were acquired from the Ara- bidopsis Biological Resource Center (ABRC), www.arabidopsis.org, to examine the root hair. Two T-DNA insertional mutants, WER::GFP and cobl9-1, were Col-0 background. The double mutant WER::GFP/cobl9-1 (CS67758) was obtained by crossing the single loss of mutant line (cobl9-1, Salk-099933) and the WER::GFP mutant. The COBRA-LIKE 9 (AT5G49270) gene is involved in root hair [26,27]. Genotyping was performed using polymerase chain reaction (PCR) to confirm the deletion of the cobl9-1 mutant and the insertion of the GFP gene. AccuPower Taq PCR PreMix (BIONEER) was used, and the temperature and time were as follows: 95 °C for 5 min for the initial denaturation, 35 cy- cles of 95 °C for 20 s, 55 °C for 20 s, and 72 °C for 1 min, and a final extension step of 72 °C for 5 min Gel loading was performed using 1xTAE, Red safe (1×), and 1.5% Agarose (Duchefa Biochemie, The Nederland). 2.3. Morphological Analysis To count the palisade cells and measure their size in the cotyledon, samples were prepared from 10-day-old Arabidopsis grown in the horizontal growth condition. The cotyledon leaves were preserved in FAA solution (5% acetic acid, 45% ethanol and 5% formaldehyde) and fixed at 4 °C overnight. An ethanol series (50%, 60%, 70%, 80%, 90%, 95%, 99% and 100%) was sequentially used for the sufficient dehydration. Then, a 50:50 ratio of ethanol and methyl salicylate (Sigma-Aldrich, St. Louis, MI, USA) was added to the leaves which then sat for an hour, and finally, 100% methyl salicylate was added and Appl. Sci. 2021, 11, 2240 4 of 16 substituted. The prepared cotyledon leaves were fixed using slide glass and cover glass, and the palisade cells of the cotyledon were photographed using a differential interfer- ence contrast (DIC) microscopy (ZEISS) system to count the number and measure the size. 2.4. Microscopy To confirm the appearance and shape of the root hair development of Arabidopsis thaliana L., Columbia 0 (Col-0) and WEREWOLF (WER)::GFP/cobl9-1 (CS67758) were grown in Petri dishes for 5 and 7 days. The root morphology was examined using a Ni- kon-Fi3 stereo microscope with a 5× zoom magnification, objective lens (10×), 40 ms ex- posure, and 2.0× analog gain. To check the GFP fluorescence of the WER::GFP/cobl9-1 lines, we selected the middle area of the roots with the optimal root hair initiation and elongation present. The GFP signals were detected under a Nikon-A1 confocal micro- scope at 488 nm with eyepiece lens (10×), objective lens (20×) and a FITC filter with the laser set to 40% and the analog gain to 80%. The quantification was performed using the photograph image of the microscopy. 2.5. RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Analysis For the gene expression analysis, the samples were grown for 15 days and then sampled and immediately frozen in liquid nitrogen and crushed using a bead beater followed by RNA extraction from the roots using the Trizol reagent (Thermo Fisher Sci- entific, USA) method. The purified RNA was then treated with the Turbo DNA-free kit (Invitrogen, Carlsbad, CA, USA). For the reverse transcription, 500 ng of RNA were used for the cDNA synthesis using the SuperScript III First-strand Synthesis System for RT-PCR (Invitrogen, USA) following the manufacturer’s instructions. Then, 1 μL of cDNA was used for real-time PCR, and the results were quantified with the CFX Man- TM ager software (Bio-Rad Laboratories, Inc., Hercules, CA, USA, http://www.bio-rad.com/ (accessed on 2 March 2021)), and Ubiquitin 10 was used for normalization. The primers used in this study are listed in Supplementary Table S1. 3. Results 3.1. Plasma Diagnostics and Plasma-Activated Water (PAW) Characteristics The emission spectrum of the plasma from SDBD was measured using optical emission spectroscopy (OES). The result shows the intensive molecular spectra of a ni- trogen second positive system (N2 SPS) and nitrogen first negative (N2 FNS) in the 300– 400 nm region during the air discharge. An OH radical peak (309 nm) was not observed; thus, the air discharge may not produce OH radicals (Figure 1). Because N2 SPS and N2 FNS were detected by OES, it is estimated that a large amount of nitrogen ions will be generated by this plasma system. When these NOx react with H2O, they become ionized - - into NO2 and NO3 , which were detected in the PAW. The physical and chemical prop- erties of DW and the PAW were analyzed (see the Materials and Methods section), and the results are presented in Table 1. To produce the proper PAW for Arabidopsis, plas- ma-activated water samples were generated using five different PAW treatment times and used in the experiment. The plasma-activated water samples in this paper are de- noted as PAW5 time in minutes (min), PAW7, PAW12, PAW19 and PAW40 with the number indicating the plasma treatment time. The result showed that the pH decreased (increased acidity) as the PAW processing time was extended due to the change in the ion content of the water. Furthermore, we confirmed that a small amount of nitrite was detected in the PAW, whereas the concen- tration of nitrate ions and their conductivity dramatically increased in proportion to the plasma treatment time in the PAW. In the case of hydrogen peroxide (H2O2), it was not detected in the DW, but it increased slightly depending on the plasma treatment time (Table 1). Appl. Sci. 2021, 11, 2240 5 of 16 Figure 1. Optical emission spectrum of the surface dielectric barrier discharge (SDBD) with air from 200 to 600 nm. Table 1. Physicochemical properties of the plasma-activated water (PAW). − − Nitrite NO2 H2O2 Nitrate NO3 Conductivity pH (mg/L) (mg/L) (mg/L) (µs/cm) Deionized Water(DW) 5.74 ± 0.06 0.00 0.00 0.00 1.53 ± 0.13 PAW5 min(PAW5) 3.62 ± 0.02 1.09 ± 0.11 0.09 ± 0.01 25.29 ± 2.88 118.10 ± 2.26 PAW7 min(PAW7) 3.34 ± 0.03 1.24 ± 0.12 0.14 ± 0.01 49.05 ± 2.61 218.50 ± 9.64 PAW12 min(PAW12) 2.94 ± 0.08 1.85 ± 0.07 0.27 ± 0.02 102.67 ± 6.30 460.33 ± 15.25 PAW19 min(PAW19) 2.62 ± 0.07 3.68 ± 0.12 0.88 ± 0.04 204.87 ± 8.74 972.93 ± 32.41 PAW40 min(PAW40) 2.37 ± 0.04 5.17 ± 0.16 1.31 ± 0.04 389.08 ± 12.24 1847.00 ± 70.19 The results of the Table 1 are the mean ± standard deviation (SD). 3.2. PAW Increases the Total Root Length and Root Hair Number in the Arabidopsis Root To understand how the PAW treatment affects the kinetics on root elongation in Arabidopsis during development, we monitored the changes in root length using solid media to see a more severe and significant effect of PAW component affecting the growth. The changes in the compositions of the nitrogen ions in the PAW were predict- ed to affect plant growth and development. As the result shows, the root length became significantly longer with the PAW5, 7, and 12 treatments from day 7 to day 15 after planting compared to the DW treatment (Figure 2). In contrast to the root length for the PAW5, 7, and 12 treatments, the root length was significantly shorter for the PAW 19 and 40 treatments compared to the DW treatment. At 7 days after planting, the root lengths in the PAW5 treated plants were increased to about 33% compared to DW (Fig- ure 2). Furthermore, at 15 days after planting, the root lengths for the DW-treated seeds were 22.14 ± 2.62 mm, and for PAW5, they were 41.38 ± 3.44 mm showing an 87% dif- ference in length (Figure 3). In contrast, the root lengths under the PAW19 and 40 condi- tions were suppressed compared to the DW treatment (Figure 2). The root length at 7 days after planting was 9.82 ± 1.07 mm, which was about 28% shorter compared to the DW treatment, while at 15 days after planting, it was 39% shorter, indicating that the PAW40 condition was the most suppressed condition. It is indicated that the appropri- Appl. Sci. 2021, 11, 2240 6 of 16 ate plasma treatment time is essential for plant growth and development, and if exces- sive, it inhibits growth (Figures 2 and 3). Figure 2. Plasma-activated water promotes root elongation during early root development in Ara- bidopsis thaliana L. Figure 3. PAW effects on root length during the developmental stage of Arabidopsis thaliana L. Seedlings were vertically grown for 15 days on agar medium. (a) DW, (b) PAW5, (c) PAW7, (d) PAW12. (e) PAW19, (f) PAW40. One square size indicates 2 × 2 cm. Because the difference in the root length was only clearly visible 7 days after plant- ing, we further checked more detailed morphological characteristics in the earlier stage of plant growth. At 5 days after planting, Col-0 grown on the plant medium did not show any differences in the root length. However, under closer examination using a ste- reo microscope, the number of root hairs increased by 84.83 ± 9.21 for the DW treatment and by 112.00 ± 5.25, 107.83 ± 5.74, 100.50 ± 8.11 and 92.66 ± 6.18 for the PAW5, PAW7, PAW12, and PAW19 treatments, respectively (Table 2). Furthermore, the length of the root hairs was 550.01 ± 138.61 μm for the DW treatment and 284.06 ± 63.12, 188.12 ± Appl. Sci. 2021, 11, 2240 7 of 16 46.67, and 161.50 ± 38.05 μm for the PAW5, PAW12, and PAW19 treatments, which are shorter than that of the DW treatment by about 48% and 71% for the PAW5 and PAW19 treatment, respectively, (Table 2, Supplementary Figure S1,). Therefore, the results show that the PAW treatment increases the number of root hairs while it shortens the length of the root hairs at 5 days after planting. Table 2. Total root length, root hair number and root hair length A. thaliana L. (Col.0) 5 days after planting. DW PAW5 PAW7 PAW12 PAW19 9.91 ± 0.85 8.19 ± 0.68 ns 8.04 ± 1.40 ns 8.92 ± 0.82 ns 7.95 ± 0.77 ns Root length(mm) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) 84.83 ± 9.21 112.00 ± 5.25 *** 107.83 ± 5.74 *** 100.50 ± 8.11 ** 92.66 ± 6.18 ns Root hair number (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) 550.01 ± 138.61 284.06 ± 63.12 *** 193.20 ± 33.45 *** 188.12 ± 46.67 *** 161.50 ± 38.05 *** Root hair length (μm) (N = 100) (N = 100) (N = 100) (N = 100) (N = 100) The midpoint in the longitudinal direction of the root was observed for the root hair number and root hair length by examining the most optimum root hair initiation and length phenotype. For the root length and root hair number, “n” indicates the number of individual plants. For root hair length, “N” indicates the individual root hair measured from total 6 plants. The results of the Table 2 are the mean ± SD (Student’s t-test compared with D.W. p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). 3.3. PAW Treatment Increases the Cotyledon Size Dependent on Palisade Cell Enlargement To further confirm the PAW effect on early vegetative development in Arabidopsis, the cotyledon was selected for cellular level examination. The size of the cotyledon was significantly increased in the PAW treated samples as examined by stereo microscope (Figure 4a–d). Quantification of the cotyledon size confirmed the enlargement of the cotyledon in the leaf length and leaf width directions for all PAW treatment conditions (Table 3). It was found that the cotyledon size for the PAW40 treatment condition was increased by about 60% in the leaf length direction (longitudinal direction) and by about 65% in the leaf width direction compared to the DW treatment condition. Figure 4. PAW treatment increases the Arabidopsis cotyledon dependent on the palisade cell size. (a–d) cotyledon size examined by a stereo microscope. (e,f) Palisade cells were examined from a cleared cotyledon sample under a differential interference contrast (DIC) microscope. (a,e) Deionized water (DW), (b,f) PAW5, (c,g) PAW19, (d,h) PAW40. (a–d) Bar = 1000 μm. (e–h) Bar = 50 μm). Table 3. Cotyledon size and palisade cell size and number of Arabidopsis grown in the DW and PAW treatment condition at 10 days after planting. DW PAW5 PAW7 PAW12 PAW19 PAW40 Cotyledon size in the 0.60 ± 0.03 0.77 ± 0.09 *** 0.81 ± 0.09 *** 0.84 ± 0.09 *** 0.88 ± 0.10 *** 0.96 ± 0.14 *** leaf length direction (n = 30) (n = 21) (n = 25) (n = 30) (n = 15) (n = 23) Appl. Sci. 2021, 11, 2240 8 of 16 (mm) Cotyledon size in the 0.46 ± 0.03 0.62 ± 0.07 *** 0.64 ± 0.06 ** 0.67 ± 0.07 *** 0.63 ± 0.05 *** 0.76 ± 0.08 *** leaf width direction (n = 30) (n = 21) (n = 25) (n = 30) (n = 25) (n = 23) (mm) ns ns ns ns 48.20 ± 6.83 43.20 ± 8.19 45.40 ± 5.59 ns 47.40 ± 5.85 48.40 ± 5.59 40.40 ± 5.31 Palisade cell number (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) 15.47 ± 1.50 24.63 ± 2.34 *** 25.07 ± 2.39 ** 25.59 ± 2.00 *** 31.35 ± 3.93 *** 42.88 ± 3.36 *** Palisade cell size (μm) (N = 300) (N = 300) (N = 300) (N = 300) (N = 300) (N = 300) For the cotyledon size and palisade cell number, “n” indicates the number of individual plants, and for the palisade cell size, “N” indicates the individual palisade cells measured from a total of 5 individual samples. Single cotyledons were measured from each biological sample. The palisade cell number was counted from the cotyledon length direction. Re- sults are the mean ± SD (Student’s t-test compared with DW; p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). There are two important factors that generally determine plant morphology. One is the number of cells involved in cell proliferation, and the other is the size of the cells in- volved in elongation [28]. To determine the main factor that affects the size of the coty- ledon, we observed the cell size and number of the palisade cells of the cotyledon using the clearing method and examined it under a DIC microscope (Figure 4e,f). As shown in Table 3, the difference in the number of palisade cells in the leaf length direction was not significant between the DW and PAW treatment conditions. However, the size of the palisade cells in the leaf length direction growth was 15.47 ± 1.50 μm in the DW treat- ment condition and 42.88 ± 3.36 μm in the PAW40 treatment condition, showing a 277% difference between DW and PAW40. The size of the palisade cells increased in propor- tion to the treatment time of the PAW. Therefore, it was found that plasma-activated water modulates the morphology of the cotyledon, which is dependent on cell size reg- ulation, not cell proliferation. 3.4. PAW Modulates the Root Hair Density through Root Developmental Genes Root hair is one of the important organs in plants. It controls osmotic pressure to absorb moisture and increases the area where the root comes into contact with the soil [29]. In Arabidopsis, the development of root hairs is determined by the location of the epidermal cells [30]. The epidermis cells adjacent to two cortical cells develop into root hairs, while epidermis adjacent to one cortical cell does not develop into root hairs [31,32]. GFP fusions with target genes such as GLABRA2 (GL2), WEREWOLF (WER), and CAPRICE (CPC) expressed in the root epidermal have been used to study root epidermal patterns in a previous study [26]. The visualization of the GFP helps to differentiate the cell structure and development process and clearly shows any alteration in the root. In Arabidopsis, cobl9-1 was shown to have root hair defects. The COBL9 gene is one of the important biomarkers as a root hair-specific gene and has a crucial role in root hair for- mation during development [33]. By using the double mutant WER::GFP/cobl9-1, we ex- pected to see a clear effect of the PAW treatment in epidermal cell patterning which can help us to understand the morphological structure at the single cell level, especially the root hairs. Furthermore, PAW treatment via cobl9 can help to confirm if the root hair phenotype is dependent on a single pathway or broader gene regulatory networks. The difference in the morphology of the root hairs between the DW and PAW treatments at days 5 and 7 of the developmental stage was confirmed using the WER::GFP/cobl9-1 mutant line. The results showed at 5 days after planting that there were no differences in the root length, but the root hair number and root hair length were changed between the DW and PAW treatment. In detail, the number of root hairs was increased by 14.5 ± 1.37 in the DW treatment. This was increased by 40.66 ± 5.71 and 27.50 ± 5.54 for the PAW5 and PAW19 treatments, respectively. The length of the root hair was decreased by 138.35 ± 11.11 and 85.05 ± 11.65 ųm for the PAW5 and PAW19 treatments compared with 261.21 ± 100.37 ųm for the DW treatment. These results show that the PAW treatment did not affect the total root length, but the root hair phenotypes Appl. Sci. 2021, 11, 2240 9 of 16 were altered for the plant harboring the mutation in the COBRA-LIKE9 genes, especially during the early developmental stage. In addition, the length of the root hairs was shortened, but the number of root hairs was increased for all PAW treatment conditions (Table 4, Supplementary Figure S2,). Table 4. Root hair number and length of Arabidopsis (WER::GFP/cobl9-1) at 5 days after planting. DW PAW5 PAW7 PAW12 PAW19 ns ns ns ns 9.75 ± 0.96 10.07 ± 0.75 10.10 ± 0.63 9.78 ± 0.95 10.08 ± 0.66 Root length (mm) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) ns 14.50 ± 1.37 40.66 ± 5.71 *** 33.16 ± 3.6 *** 31.00 ± 3.84 ** 27.50 ± 5.54 Root hair number (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) 261.21 ± 100.37 138.35 ± 11.11 *** 108.25 ± 11.54 *** 99.50 ± 13.65 *** 85.05 ± 11.65 *** Root hair length (μm) (N = 60) (N = 60) (N = 60) (N = 60) (N = 60) For root length and root hair number, “n” indicates the number of individual plants as biological replicates measured; for root hair length, “N” indicates the individual root hair length measured from a total of 6 samples. The results are the mean ± SD (Student’s t-test compared with D.W. p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). Because the differences in the root hair phenotype between the DW and PAW treatment conditions were significant at 5 days after planting (Table 4), a confocal mi- croscope was used for imaging the root epidermal cell shape at 7 days after planting. To visualize the cell type in the root, we used three different treatment conditions, DW, PAW5, and PAW19. The roots of the Arabidopsis WER::GFP/cobl9-1 line were grown for 7 days. Figure 5 shows the tagged WER promoter with GFP, which resulted in the ap- pearance of fluorescence in all the epidermal layers of the root cells. Quantification of the root hair numbers showed an increment in all the PAW treatment conditions com- pared to the DW treatment condition. In contrast, the root hair length was decreased in the PAW treatment condition compared to the DW treatment condition (Table 5, Sup- plementary Figure S2). Interestingly, the length of the root epidermal cells was elongat- ed with a similar pattern to the root length phenotype after the PAW treatment, which is consistent with the root enhancement in the PAW5 and PAW7 and the reduction in the PAW19 treatment condition compared to the DW treatment condition. It is believed that there will be a specific mechanism regulating the number of root hairs and length of the root hair in the PAW condition during the plant development process. Moreover, the results indicate that PAW5 is the optimized condition for plant growth and develop- ment. Table 5. Root hair number and length of A. thaliana L. (WER::GFP/cobl9-1) grown for 7 days after planting. DW PAW5 PAW19 15.14 ± 2.47 24.57 ± 3.10 *** 26.85 ± 5.33 *** Root hair number (n = 7) (n = 7) (n = 7) 162.92 ± 29.10 77.40 ± 7.21 *** 54.33 ± 7.83 *** Root hair length (μm) (N = 50) (N = 50) (N = 50) 133.01 ± 12.67 141.33 ± 11.45 *** 110.46 ± 9.38 *** Root epidermal cell (μm) (N = 50) (N = 50) (N = 50) For the root hair number, “n” indicates the number of individual plants; for the root hair length from the root epidermal cells, “N” indicates the individual root hairs measured from a total of 7 samples. The GFP signals for root hair umber, root hair length and root epidermal cell were meas- ured under a Nikon-A1 confocal microscope at 488 nm with a eyepiece lens (10×), objective lens (20×). The results in the Table are the mean ± SD (Student’s t-test compared with DW p > 0.05 ns, p < 0.05 *, p < 0.01 **, p < 0.001 ***). Appl. Sci. 2021, 11, 2240 10 of 16 Figure 5. PAW regulates the number of root hairs and epidermal cell size in the Arabidopsis root (WER::GFP/cobl9-1). Ar- abidopsis seedlings were grown in solid media for 7 days. (a) DW, (b) PAW5, (c) PAW19. The white arrows indicate the individual root hairs that were used in the quantification. Bar = 100 μm. To further understand the mechanism of PAW affecting the root development in Arabidopsis, we confirmed the expression of several marker genes related to root hair ini- tiation and elongation. Previously, it was shown that the nitrate concentration could in- duce the expression of COBL9 which activates root hair initiation and elongation [34]. NT_COB9 was upregulated by PAW treatment in tobacco [25]. As expected, the expres- sion of COBL9 was significantly increased in the PAW5 treatment condition compared to the DW treatment condition. In contrast, the expression was suppressed in the PAW19 treatment condition. AUXIN1 (AUX1) and LIKE-AUXIN (LAX3) are the key auxin influx carriers and are multi-membrane transmembrane proteins that transfer the auxin in- volved in cell growth [35]. AUX1 and LAX3 both had up-regulated expression patterns in PAW5 while AUX1 was down-regulated in the PAW19 treatment condition. For OBF BINDING PROTEIN 4 (OBP4), its expression was down-regulated in the PAW5 and PAW19 treatment conditions. OBP4 is known as a negative regulator of root growth and differentiation including the root hairs [33,34], and it also functions as a negative regu- lator in PAW. Furthermore, XYLOGLUCAN ENDOTRANSGLUCOSYL- ASE/HYDROLASE (XTH) gene family members are factors involved in cell wall modifi- cation [36–38]. Interestingly, two genes, XTH9 and XTH17, showed high expression for PAW5 but low expression for the PAW19 treatment condition (Figure 6). We concluded that the root architecture under PAW is modulated by root developmental genes in- cluding COBL9, XTH9 and XTH17. Appl. Sci. 2021, 11, 2240 11 of 16 Figure 6. PAW treatment induces gene expression of root hair initiation and elongation genes in Arabidopsis. Student’s t-test compared with D.W. p < 0.05 *, p < 0.01 **, p < 0.001 ***. 4. Discussion We investigated the effect of plasma-activated water (PAW) on the early growth and development of Arabidopsis in this study. The physicochemical properties of the plasma-activated water were detected according to the plasma exposure time, and the effect of the altered physicochemical properties of the PAW on plant growth was exam- ined in Arabidopsis. The morphological differences in the roots and vegetative tissues, differences at the cellular level, and molecular biological differences were investigated. In addition, possible molecular mechanisms underlying the effect of the PAW treatment on plant growth were explored in Arabidopsis based on the phenotype. We found that PAW facilitated the growth of the Arabidopsis by modulating developmental genes at an early developmental stage. In recent years, plasma devices have emerged as an eco-friendly alternative method to improve plant growth and development. The application of plasma-related treat- ments in plants has been studied for improving seed germination, plant growth, plant height and yield in crops. The differences in plant phenotypes are caused by the differ- ence in physical and chemical properties with increased biochemical components, al- tered seed coat morphology, biochemical activities, and altered gene expression [5–8,18]. The degree of difference in a plasma-induced plant phenotype is due to a variety and range of RONS (reactive oxygen and nitrogen species) generated by plasma with differ- ent feeder gases or just air [39]. Low pressure dielectric barrier discharge generates Ar/O2, and plants treated with Ar/O2 produce increased H2O2 in the roots and shoots of wheat [40]. Cylinder double dielectric barrier discharge (DBD) reactors generate nitrate (NO3 ) and hydrogen peroxide (H2O2) that positively regulate growth and germination in seeds [18,19]. Even in Arabidopsis as a model plant, PAW generated by air dielectric barrier discharge activates growth and development such as the leaf area, the number of flowering plants and the leaf number over a long-term observation period, and this phenomenon is also partly explained by the elevated hydrogen peroxide and nitrate [7]. In this study, the plasma generated by the SDBD device was confirmed to have a nitrogen molecule spectrum in the region of 290~400 nm (Figure 1). Nitrogen ions are mainly produced due to the plasma effect and react with H2O. When they ionize into - − NO2 and NO3 , the pH decreases, and the conductivity increases. Compared to other studies on both direct and indirect plasma applications, ROS (reactive oxygen species) Appl. Sci. 2021, 11, 2240 12 of 16 are the main factor affecting germination and plant growth and defense [7,40,41]. The SDBD generator device used in this study mainly produced a higher NO3 concentration when a longer plasma treatment time was used. In addition, the change of pH and con- ductivity was also significant at longer plasma treatment. This change was predicted to affect the plant growth and development. In a previous study, tobacco grown on me- dium containing plasma treated water had increased cotyledon growth but suppressed primary root hair growth at PAW40 [25]. Arabidopsis seedlings showed self-sufficient under minimal conditions for weeks without external supply of nutrients [42]. There- fore, we used the minimal media of only DW and PAW to study the more dramatic ef- fect of the PAW treatment. By using this method, we expected to see more specific pathways that are affected by PAW. Even though it is important to study the effect of all component changes in PAW such as conductivity and osmolality, in this study we fo- cused on the changes in the nitrate concentration which is a main nitrogen source that affects plant growth. Nitrogen is an essential macronutrient for plants and an important constituent of key molecules such as amino acids, nucleic acid and chlorophyll [43]. Ni- trogen availability in the soil is a major factor in plant growth, and its availability is a key determinant of plant yield [43–46]. From this perspective, the increment of the ni- trate concentration from plasma treatment potentially can serve as a very important ni- trogen source for plant growth and development. Concerning root development, the PAW treatment condition (PAW5, 7 and 12) promoted an increased total root length compared with the DW treatment condition in Arabidopsis; however, root development was inhibited by the PAW that was generated with a longer plasma treatment time (PAW19 and 40). In detail, the morphological dif- ferences at 15 days after planting in the root showed that the root length for the PAW5 treatment condition increased by 87%; however, it was suppressed by the PAW40 treat- ment condition by 63% compared to the DW control, indicating that root growth oc- curred independent of the plasma treatment time used to generate the PAW. Thus, Ara- bidopsis needs specific growth conditions (Figures 2 and 3). In contrast to the specific pattern of activation and inhibition of root growth, the effect of the PAW treatment in the cotyledon tissue did not show any inhibition. The cotyledon size increased according to the plasma treatment time of the PAW due to an enlarged palisade cell size, not by cell number. In tobacco, the cotyledon size was also regulated by cell size not cell prolif- eration at 10 days after planting [25]. This result implies that they share the same mech- anism in cotyledon development in dicotyledonous plants. In the seedlings, the roots become activated in the PAW5, 7 and 12 treatment conditions and inhibited in the PAW19 and 40 treatment conditions, and the cotyledon expands according to the plasma treatment time of the PAW as a tissue-specific plasma effect. Therefore, we need to un- derstand it as an overall balance of the plant developmental growth and investigating the optimal treatment conditions for Arabidopsis is important to achieve the desired ef- fect. Interestingly, the PAW treatments showed an increased number of root hairs in all the PAW conditions; however, they showed a decreased root hair cell length shown by cell length measurements, in other words, a decreased trichoblast cell length (Table 2). In addition, root hair number in the WER::GFP/cobl9-1 line increased induced by the PAW treatment while root cell elongation shortened. In the Col-0 background, the root hair number increased by 18~32% dependent on the PAW treatment time (Table 2). Root hair number in the cobl9-1 mutant background was dramatically increased by at least two times by the PAW treatment at 5 days after planting (Table 4). It implies that the regula- tion of root hairs under PAW treatment involves sophisticated genetic networks, not simply a single pathway. Moreover, this result indicates the increased root hair number induced by PAW, which results in securing more space for root epidermal cells to come into contact with nutrients per unit of root cell. These differences may lead to improved growth because the root hairs are in response to the availability of NO3 which has an important role in nitrogen (N) source nutrient acquisition when plants are exposed to Appl. Sci. 2021, 11, 2240 13 of 16 nitrate-rich soils. It is known that nitrate also can act as signaling molecules that can control downstream gene expression in plants. Previously, the change in the root archi- tecture in plants by the nitrate concentration was presented as an example of plasticity in the developmental process in response to the environment [36,47]. For example, the in- crement of nitrate concentration of 0.1 to 1 mM KNO3 was known to induce root hair and lateral root formation [48–50]. In contrast, high nitrate concentration suppressed the root formations [47,48,51]. At the molecular level, we confirmed the effect of the PAW treatment by checking the expression of marker genes related to root development. One of the key genes of root hair initiation and root hair elongations, COBL9, was induced in the PAW5 and sup- pressed in the PAW19 treatment condition. This expression result confirmed our finding of the different phenotypes in root hair number and root length between the Col-0 and cobl9-1 mutants which were affected by the COBL9 expression (Tables 2 and 4, Figure 6). and our PAW generated from SDBD mainly affects the nitrate concentration. In plants, auxin has a major role in root hair development, and the auxin trans- porter proteins, AUX1 and LAX3, regulate lateral root formation [35,50]. Short root hair is due to mutations of AUX1 genes indicating AUX1 controls elongation of the root hair [52]. We checked the expression of two auxin transport genes, AUX1 and LAX3, which were induced in the PAW5 treatment condition; however, the expression was sup- pressed in the PAW19 treatment condition only in AUX1. This expression pattern of the AUX1 gene is in line with a previous report that AUX1 predominantly regulates root hair elongation whereas LAX3 mostly regulates the lateral root formation [35]. Further- more, we confirmed the expression of the OBP4, XTH9 and XTH17 genes that are known to regulate root hair initiation and elongation and are sensitive to the nitrate concentra- tion in the media [50,53]. In detail, OBP4 acts as a negative regulator of root growth, while XTH9 and XTH17 act as a positive regulator of root growth through cell expan- sion. The expression showed that these genes were affected by the PAW treatments. All the PAW treatments suppressed the expression of OBP4. Although the expressions of XTH9 and XTH17 were suppressed in the PAW19 treatment condition, the maximal ob- served effect was observed on the root development by the PAW5 treatment compared to all the other PAW generation times (PAW7, 12, 19 and 40). Therefore, the PAW5 condi- tion affected the root growth by positively regulating the expression of XTH9 and XTH17 which may mainly be caused by the root cell expansion. Moreover, as showed in the Figure 5 and Table 5, the cell expansion of PAW19 was severely reduced, thus corre- lating with lower expression of XTH genes. It is suggested that PAW-mediated root ar- chitecture regulates the root developmental genes in Arabidopsis, and it is at least in- volved in the root auxin transporter signaling system. Taken together, the molecular mechanism of the PAW treatment increases the ni- trate concentration in PAW which induces the activation of the nitrate-responsive and cell expansion genes leading to the induction of the root hair density and root length. However, the mechanism we explored may still be affected by other characters such as a change in the osmolarity of the medium. Therefore, further detailed analyses still need to be done on each component of the PAW and their effects on plant growth. In addi- tion, to further understand PAW regulation at the molecular level, a detailed study us- ing current technology such as RNA-sequencing and small-RNA profiling is necessary for future applications. By revealing these mechanisms, further fine-tuned applications of plasma treatment for specific bio-samples will be elucidated from the specific target pathways in a precise manner. 5. Conclusions PAW generated from a SDBD reactor can be used to enhance Arabidopsis growth in the early seedling stage. The PAW treatments were shown to affect the root hair density mediated by the COBL9, XTH9 and XTH17 genes. The maximal observed condition for Arabidopsis root development was identified as PAW5 in which potential target genes Appl. Sci. 2021, 11, 2240 14 of 16 were up-regulated while root development was suppressed by the PAW19 treatment condition. The findings of this study will provide new insights at the molecular level in- to developmental stage plants and accelerate the study of applying plasma technology to crop plants. Supplementary Materials: The following are available online at www.mdpi.com/2076-3417/11/5/2240/s1, Supplementary Figure S1: PAW regulates number of root hair in (Col-0) Arabidopsis (a) DW, (b) PAW5, (c) PAW7, (d) PAW12, (e) PAW19. Seedlings were grown on solid media for 5 days. Bar = 100 μm. Figure S2: PAW regulates number of root hair in (WER::GFP/cobl9-1) Arabidopsis (a) DW, (b) PAW5, (c) PAW7, (d) PAW12, (e) PAW19. Seedlings were grown on solid media for 5 days. Bar = 100 μm. Table S1: List of primers used in this study. Author Contributions: Conceptualization, Y.K.L., I.A.L., S.B.K., and R.A.P.; experiment and data curation, D.H.K., R.A.P., J.Y.P., and S.J.P.; writing—original draft preparation, D.H.K., R.A.P., and Y.K.L.; writing—review and editing, R.A.P. and Y.K.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by R&D Program of “Plasma Advanced Technology for Agriculture and Food (Plasma Farming) (EN2025)-1711124797)” through the Korea Institute of Fu- sion Energy(KFE) funded by the Government funds, Republic of Korea, and a grant from the ‘Na- tional Research Foundation of Korea (grant no. NRF-2016R1C1B2015877) funded by the Ministry of Science, ICT & Future Planning’ to S.J.P. Conflicts of Interest: The authors declare no conflict of interest. References 1. Adhikari, B.; Adhikari, M.; Park, G. The Effects of Plasma on Plant Growth, Development, and Sustainability. Appl. Sci. 2020, 10, 6045, doi:10.3390/app10176045. 2. Ito, M.; Ohta, T.; Hori, M. Plasma Agriculture. J. Korean Phys. Soc. 2012, 60, 937–943, doi:10.3938/jkps.60.937. 3. Ohta, T. Plasma in Agriculture. In Cold Plasma in Food and Agriculture; Elsevier: Amsterdam, The Netherlands, 2016; pp. 205– 221, ISBN 978-0-12-801365-6. 4. Starič, P.; Vogel-Mikuš, K.; Mozetič, M.; Junkar, I. Effects of Nonthermal Plasma on Morphology, Genetics and Physiology of Seeds: A Review. Plants 2020, 9, 1736, doi:10.3390/plants9121736. 5. Adhikari, B.; Adhikari, M.; Ghimire, B.; Park, G.; Choi, E.H. Cold Atmospheric Plasma-Activated Water Irrigation Induces Defense Hormone and Gene Expression in Tomato Seedlings. Sci. Rep. 2019, 9, 16080, doi:10.1038/s41598-019-52646-z. 6. Fan, L.; Liu, X.; Ma, Y.; Xiang, Q. Effects of Plasma-Activated Water Treatment on Seed Germination and Growth of Mung Bean Sprouts. J. Taibah Univ. Sci. 2020, 14, 823–830, doi:10.1080/16583655.2020.1778326. 7. Bafoil, M.; Jemmat, A.; Martinez, Y.; Merbahi, N.; Eichwald, O.; Dunand, C.; Yousfi, M. Effects of Low Temperature Plasmas and Plasma Activated Waters on Arabidopsis Thaliana Germination and Growth. PLoS ONE 2018, 13, e0195512, doi:10.1371/journal.pone.0195512. 8. Sergeichev, K.F.; Lukina, N.A.; Sarimov, R.M.; Smirnov, I.G.; Simakin, A.V.; Dorokhov, A.S.; Gudkov, S.V. Physicochemical Properties of Pure Water Treated by Pure Argon Plasma Jet Generated by Microwave Discharge in Opened Atmosphere. Front. Phys. 2021, 8, 614684, doi:10.3389/fphy.2020.614684. 9. Belov, S.V.; Danyleiko, Y.K.; Glinushkin, A.P.; Kalinitchenko, V.P.; Egorov, A.V.; Sidorov, V.A.; Konchekov, E.M.; Gudkov, S.V.; Dorokhov, A.S.; Lobachevsky, Y.P.; et al. An Activated Potassium Phosphate Fertilizer Solution for Stimulating the Growth of Agricultural Plants. Front. Phys. 2021, 8, 618320, doi:10.3389/fphy.2020.618320. 10. Traylor, M.J.; Pavlovich, M.J.; Karim, S.; Hait, P.; Sakiyama, Y.; Clark, D.S.; Graves, D.B. Long-Term Antibacterial Efficacy of Air Plasma-Activated Water. J. Phys. D Appl. Phys. 2011, 44, 472001, doi:10.1088/0022-3727/44/47/472001. 11. Zhou, R.; Zhou, R.; Wang, P.; Xian, Y.; Mai-prochnow, A.; Lu, X.P.; Cullen, P.; Ostrikov, K. (Ken); Bazaka, K. Plasma Activated Water (PAW): Generation, Origin of Reactive Species and Biological Applications. J. Phys. D Appl. Phys. 2020, 53, 303001, doi:10.1088/1361-6463/ab81cf. 12. Zhang, S.; Rousseau, A.; Dufour, T. Promoting Lentil Germination and Stem Growth by Plasma Activated Tap Water, De- mineralized Water and Liquid Fertilizer. RSC Adv. 2017, 7, 31244–31251, doi:10.1039/C7RA04663D. 13. Graves, D.B.; Bakken, L.B.; Jensen, M.B.; Ingels, R. Plasma Activated Organic Fertilizer. Plasma Chem. Plasma Process. 2019, 39, 1–19, doi:10.1007/s11090-018-9944-9. 14. Gaudinier, A.; Rodriguez-Medina, J.; Zhang, L.; Olson, A.; Liseron-Monfils, C.; Bagman, A.M.; Foret, J.; Abbitt, S.; Tang, M.; Li, B.; et al. Transcriptional Regulation of Nitrogen-Associated Metabolism and Growth. Nature 2018, 563, 259–264, doi:10.1038/s41586-018-0656-3. 15. Ueda, Y.; Yanagisawa, S. Engineering Nitrogen Utilization in Crop. Plants; Springer International Publishing: Basingstoke, UK, 2018; ISBN 978-3-319-92957-6. Appl. Sci. 2021, 11, 2240 15 of 16 16. Chandran, A.K.N.; Priatama, R.A.; Kumar, V.; Xuan, Y.; Je, B.I.; Kim, C.M.; Jung, K.H.; Han, C.D. Genome-Wide Transcriptome Analysis of Expression in Rice Seedling Roots in Response to Supplemental Nitrogen. J. Plant Physiol. 2016, 200, 62–75, doi:10.1016/j.jplph.2016.06.005. 17. Sarinont, T.; Katayama, R.; Wada, Y.; Koga, K.; Shiratani, M. Plant Growth Enhancement of Seeds Immersed in Plasma Acti- vated Water. MRS Adv. 2017, 2, 995–1000, doi:10.1557/adv.2017.178. 18. Sivachandiran, L.; Khacef, A. Enhanced Seed Germination and Plant Growth by Atmospheric Pressure Cold Air Plasma: Combined Effect of Seed and Water Treatment. RSC Adv. 2017, 7, 1822–1832, doi:10.1039/C6RA24762H. 19. Song, J.-S.; Lee, M.J.; Ra, J.E.; Lee, K.S.; Eom, S.; Ham, H.M.; Kim, H.Y.; Kim, S.B.; Lim, J. Growth and Bioactive Phytochemicals in Barley (Hordeum Vulgare L.) Sprouts Affected by Atmospheric Pressure Plasma during Seed Germination. J. Phys. Appl. Phys. 2020, 53, 314002, doi:10.1088/1361-6463/ab810d. 20. Adhikari, B.; Adhikari, M.; Ghimire, B.; Adhikari, B.C.; Park, G.; Choi, E.H. Cold Plasma Seed Priming Modulates Growth, Redox Homeostasis and Stress Response by Inducing Reactive Species in Tomato (Solanum Lycopersicum). Free Radic. Biol. Med. 2020, 156, 57–69, doi:10.1016/j.freeradbiomed.2020.06.003. 21. Thirumdas, R.; Kothakota, A.; Annapure, U.; Siliveru, K.; Blundell, R.; Gatt, R.; Valdramidis, V.P. Plasma Activated Water (PAW): Chemistry, Physico-Chemical Properties, Applications in Food and Agriculture. Trends Food Sci. Technol. 2018, 77, 21– 31, doi:10.1016/j.tifs.2018.05.007. 22. Thirumdas, R.; Sarangapani, C. Cold Plasma : A Novel Non-Thermal Technology for Food Processing. Food Biophys. 2015, 10, 1–11, doi:10.1007/s11483-014-9382-z. 23. Meinke, D.W.; Cherry, J.M.; Dean, C.; Rounsley, S.D.; Koornneef, M. Arabidopsis Thaliana: A Model Plant for Genome Anal- ysis. Science 1998, 282, 662–682, doi:10.1126/science.282.5389.662. 24. The Arabidopsis Genome Initiative Analysis of the Genome Sequence of the Flowering Plant Arabidopsis Thaliana. Nature 2000, 408, 796–815, doi:10.1038/35048692. 25. Lee, Y.K.; Lim, J.; Hong, E.J.; Kim, S.B. Plasma-Activated Water Regulates Root Hairs and Cotyledon Size Dependent on Cell Elongation in Nicotiana Tabacum L. Plant Biotechnol. Rep. 2020, 14, 663–672, doi:10.1007/s11816-020-00641-6. 26. Lee, M.M.; Schiefelbein, J. Cell Pattern in the Arabidopsis Root Epidermis Determined by Lateral Inhibition with Feedback. Plant Cell 2002, 14, 611–618, doi:10.1105/tpc.010434. 27. Bruex, A.; Kainkaryam, R.M.; Wieckowski, Y.; Kang, Y.H.; Bernhardt, C.; Xia, Y.; Zheng, X.; Wang, J.Y.; Lee, M.M.; Benfey, P.; et al. A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis. PLoS Genet. 2012, 8, e1002446, doi:10.1371/journal.pgen.1002446. 28. Horiguchi, G.; Ferjani, A.; Fujikura, U.; Tsukaya, H. Coordination of Cell Proliferation and Cell Expansion in the Control of Leaf Size in Arabidopsis Thaliana. J. Plant Res. 2006, 119, 37–42, doi:10.1007/s10265-005-0232-4. 29. Aiken, R.M.; Smucker, A.J.M. Root System Regulation of Whole Plant Growth. Annu. Rev. Phytopathol. 1996, 34, 325–346, doi:10.1146/annurev.phyto.34.1.325. 30. Grierson, C.; Nielsen, E.; Ketelaarc, T.; Schiefelbein, J. Root Hairs. Arab. Book 2014, 12, e0172, doi:10.1199/tab.0172. 31. Datta, S.; Kim, C.M.; Pernas, M.; Pires, N.D.; Proust, H.; Tam, T.; Vijayakumar, P.; Dolan, L. Root Hairs: Development, Growth and Evolution at the Plant-Soil Interface. Plant Soil 2011, 346, 1–14, doi:10.1007/s11104-011-0845-4. 32. Julkowska, M. Releasing the Cytokinin Brakes on Root Growth. Plant Physiol. 2018, 177, 865–866, doi:10.1104/pp.18.00660. 33. Parry, G.; Marchant, A.; May, S.; Swarup, R.; Swarup, K.; James, N.; Graham, N.; Allen, T.; Martucci, T.; Yemm, A.; et al. Quick on the Uptake: Characterization of a Family of Plant Auxin Influx Carriers. J. Plant Growth Regul. 2001, 20, 217–225, doi:10.1007/s003440010030. 34. Schindelman, G. COBRA Encodes a Putative GPI-Anchored Protein, Which Is Polarly Localized and Necessary for Oriented Cell Expansion in Arabidopsis. Genes Dev. 2001, 15, 1115–1127, doi:10.1101/gad.879101. 35. Swarup, R.; Bhosale, R. Developmental Roles of AUX1/LAX Auxin Influx Carriers in Plants. Front. Plant Sci. 2019, 10, 1306, doi:10.3389/fpls.2019.01306. 36. Xu, P.; Cai, W. Nitrate-Responsive OBP4-XTH9 Regulatory Module Controls Lateral Root Development in Arabidopsis Tha- liana. PLoS Genet. 2019, 15, e1008465, doi:10.1371/journal.pgen.1008465. 37. Lee, Y.K.; Rhee, J.Y.; Lee, S.H.; Chung, G.C.; Park, S.J.; Segami, S.; Maeshima, M.; Choi, G. Functionally Redundant LNG3 and LNG4 Genes Regulate Turgor-Driven Polar Cell Elongation through Activation of XTH17 and XTH24. Plant Mol. Biol. 2018, 97, 23–36, doi:10.1007/s11103-018-0722-0. 38. Maris, A.; Suslov, D.; Fry, S.C.; Verbelen, J.-P.; Vissenberg, K. Enzymic Characterization of Two Recombinant Xyloglucan En- dotransglucosylase/Hydrolase (XTH) Proteins of Arabidopsis and Their Effect on Root Growth and Cell Wall Extension. J. Exp. Bot. 2009, 60, 3959–3972, doi:10.1093/jxb/erp229. 39. Ahn, C.; Gill, J.; Ruzic, D.N. Growth of Plasma-Treated Corn Seeds under Realistic Conditions. Sci. Rep. 2019, 9, 4355, doi:10.1038/s41598-019-40700-9. 40. Rahman, M.M.; Sajib, S.A.; Rahi, M.S.; Tahura, S.; Roy, N.C.; Parvez, S.; Reza, M.A.; Talukder, M.R.; Kabir, A.H. Mechanisms and Signaling Associated with LPDBD Plasma Mediated Growth Improvement in Wheat. Sci. Rep. 2018, 8, 10498, doi:10.1038/s41598-018-28960-3. 41. del Río, L.A. ROS and RNS in Plant Physiology: An Overview. J. Exp. Bot. 2015, 66, 2827–2837, doi:10.1093/jxb/erv099. Appl. Sci. 2021, 11, 2240 16 of 16 42. Réthoré, E.; d’Andrea, S.; Benamar, A.; Cukier, C.; Tolleter, D.; Limami, A.M.; Avelange-Macherel, M.-H.; Macherel, D. Ara- bidopsis Seedlings Display a Remarkable Resilience under Severe Mineral Starvation Using Their Metabolic Plasticity to Re- main Self-Sufficient for Weeks. Plant J. 2019, 99, 302–315, doi:10.1111/tpj.14325. 43. Tegeder, M.; Masclaux-Daubresse, C. Source and Sink Mechanisms of Nitrogen Transport and Use. New Phytol. 2018, 217, 35– 53, doi:10.1111/nph.14876. 44. Zhao, M.; Zhao, M.; Gu, S.; Sun, J.; Ma, Z.; Wang, L.; Zheng, W.; Xu, Z. DEP1 Is Involved in Regulating the Carbon–Nitrogen Metabolic Balance to Affect Grain Yield and Quality in Rice (Oriza Sativa L.). PLoS ONE 2019, 14, e0213504, doi:10.1371/journal.pone.0213504. 45. Kumar, V.; Kim, S.H.; Priatama, R.A.; Jeong, J.H.; Adnan, M.R.; Saputra, B.A.; Kim, C.M.; Je, B.I.; Park, S.J.; Jung, K.H.; et al. NH4+ Suppresses NO3–-Dependent Lateral Root Growth and Alters Gene Expression and Gravity Response in OsAMT1 RNAi Mutants of Rice (Oryza Sativa). J. Plant Biol. 2020, 63, 391–407, doi:10.1007/s12374-020-09263-5. 46. Xuan, Y.H.; Priatama, R.A.; Huang, J.; Je, B.I.; Liu, J.M.; Park, S.J.; Piao, H.L.; Son, D.Y.; Lee, J.J.; Park, S.H.; et al. Indeterminate Domain 10 Regulates Ammonium-Mediated Gene Expression in Rice Roots. New Phytol. 2013, 197, 791–804, doi:10.1111/nph.12075. 47. Boer, M.D.; Santos Teixeira, J.; Ten Tusscher, K.H. Modeling of Root Nitrate Responses Suggests Preferential Foraging Arises From the Integration of Demand, Supply and Local Presence Signals. Front. Plant Sci. 2020, 11, 708, doi:10.3389/fpls.2020.00708. 48. Zhang, H. Regulation of Arabidopsis Root Development by Nitrate Availability. J. Exp. Bot. 2000, 51, 51–59, doi:10.1093/jexbot/51.342.51. 49. Canales, J.; Contreras-López, O.; Álvarez, J.M.; Gutiérrez, R.A. Nitrate Induction of Root Hair Density Is Mediated by TGA1/TGA4 and CPC Transcription Factors in Arabidopsis Thaliana. Plant J. 2017, 92, 305–316, doi:10.1111/tpj.13656. 50. Quint, M.; Gray, W.M. Auxin Signaling. Curr. Opin. Plant Biol. 2006, 9, 448–453, doi:10.1016/j.pbi.2006.07.006. 51. Asim, M.; Ullah, Z.; Xu, F.; An, L.; Aluko, O.O.; Wang, Q.; Liu, H. Nitrate Signaling, Functions, and Regulation of Root System Architecture: Insights from Arabidopsis Thaliana. Genes 2020, 11, 633, doi:10.3390/genes11060633. 52. Salazar-Henao, J.E.; Vélez-Bermúdez, I.C.; Schmidt, W. The Regulation and Plasticity of Root Hair Patterning and Morpho- genesis. Development 2016, 143, 1848–1858, doi:10.1242/dev.132845. 53. Itoh, J.-I.; Hibara, K.-I.; Sato, Y.; Nagato, Y. Developmental Role and Auxin Responsiveness of Class III Homeodomain Leucine Zipper Gene Family Members in Rice. Plant Physiol. 2008, 147, 1960–1975, doi:10.1104/pp.108.118679.

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