Germination, Growth, and Secondary Metabolite Contents of Radish Sprouts (Raphanus sativus L.) as Affected by Plasma Activated Water Process Time

doi:10.21203/rs.3.rs-1993356/v1

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Germination, Growth, and Secondary Metabolite Contents of Radish Sprouts (Raphanus sativus L.) as Affected by Plasma Activated Water Process Time

https://doi.org/10.21203/rs.3.rs-1993356/v1

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This study was performed to investigate the effects of plasma-activated water on germination, growth, and functional properties of radish sprouts. The experimental design involved radish seeds cultivated in different concentrations of plasma-activated water for seven days, with plasma water generated by 10, 20, or 30 minutes of plasma discharge in water. The underwater plasma discharge produced nitrate ions and hydrogen peroxide(H₂O₂) and changed the pH and electrical conductivity. The plasma-activated water improved germination, and the longer the underwater plasma generation time, the better the effect. In terms of growth, plasma-activated water treatment oxidized the roots and negatively affected plants. Functional substance analysis showed that the levels of indolic and aliphatic glucosinolates increased, with 30 min of plasma treatment leading to the highest results. Similarly, the phenolic content increased with increasing plasma discharge time, with the 30 min treatment increasing phenolic content by 288.8% compared to the control. The results of this study suggest that plasma-activated water can increase the functional components of plants during hydroponic cultivation. However, more research is required to solve these growth disorders observed with plasma-activated water use.

Plasma refers to a highly reactive gaseous state in which ions, electrons, and neutral particles are generated under high- or low-pressure conditions^1,2. Plasma refers to both high-temperature plasmas used in ultra-high-temperature heat sources or fusion power generation and low-temperature plasma (non-thermal plasma) generated at a low temperature of less than 100°C³. It is widely used in sterilization, virus, and mold removal, and in the medical field, namely low-temperature plasma^4,5. Recently, research using plasma has been conducted in the agricultural field. Liu et al. (2020) reported that plasma can reduce aerobic bacteria, molds, yeasts, and coliforms in fresh-cut apples, whereas plasma-activated water (PAW) treatment was able to limit fruit spoilage and maintain the quality of Chinese bayberries during post-harvest storage^6,7. Zhou et al. (2016) reported that plasma treatment resulted in high mung bean seed germination and seed growth⁸. In agriculture, plasma activated water is one of form using plasma and is divided into dielectric barrier discharge (DBD) and pulsed corona discharge (PCD), depending on the discharge method used.

When plasma is processed in water, active species generated by the reaction between the liquid material and gaseous plasma are dissolved, and this is defined as plasma activated water^9,10. Reactive nitrogen species (RNS) such as nitrate (NO₃^-), and reactive oxygen species (ROS) such as OH radicals, ozone (O₃) and hydrogen peroxide (H₂O₂) are generated as by-products^11–13. Among the active nitrogen species generated through PAW, nitrate ions are one of the nitrogen sources used by plants, and they affect the biosynthesis of hormones by participating in cytokinin and auxin production in the plant root zone¹⁴. The nitrate absorbed by the plant induces the accumulation of trans-zeatin in the root and acts as part of the signal to regulate the rhizosphere¹⁵, promoting the transport of auxin in the body, which is involved in the growth of the entourage and main roots¹⁶. The expression of genes can be regulated by nitrate ions in plants, where the expression of specific proteins can be controlled through mRNA and transcription pathways and can act as a signal transduction factor in plant metabolism^17–19. It plays an important role in the biosynthesis of secondary metabolites in plants, and it has been reported that the growth period from flowers to flowering is shortened when nitrogen content increases²⁰. Another PAW by-product, H₂O, is produced by the reactive oxygen species. H₂O₂ is the most stable ROS and acts as a signalling molecule in plants²¹. It is produced in plants grown under physical, chemical, biological, and abiotic stress conditions, and is involved in cellular redox signaling through peroxiredoxins, glutaredoxins, thioredoxins, or NADPH²². It has been reported that low concentrations of H₂O₂ can regulate photosynthesis, stomatal conductance, respiration, growth, and development of plants and upregulate antioxidant production pathways under stressful conditions^21,23. However, because high concentrations of H₂O₂ can induce severe oxidative stress and even lead to cell death, the PAW process time that can generate the appropriate concentration is important.

As they are in the spotlight as a functional food, sprout vegetables are becoming more popular with people pursuing a healthy lifestyle change, and interest in them is increasing^24,25. Sprout vegetables are sprouts and root plants grown from seeds that have germinated for a minimum of two days and a maximum of seven days, and harvested while the cotyledons are still underdeveloped²⁶. It has also been reported to contain more secondary metabolites, such as antioxidants, phenols, vitamins, and minerals than seeds or mature vegetables²⁷. Cruciferous plants contain high levels of glucosinolates and phenolic compounds. Radish sprouts are foods with very high nutritional value as they contain indole compounds known as anticancer substances, vitamins, and alkaline elements, such as Ca, P, Fe, and Mg^28–30. In addition, it has been reported that glucosinolate in radish sprouts has a high antioxidant capacity and is effective in anticancer and antiviral activities, thereby enhancing human health and immunity^31,32. Although the demand for and interest in sprout vegetables is rapidly increasing, research on sprout vegetable cultivation technology that contains various large amounts of bioactive substances is insufficient. This study was conducted to investigate the physicochemical properties of PAW and the changes in the germination rate, growth, and functional substance content of radish sprouts grown using PAW.

Measurement of change in properties of PAW

Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure ions immediately after treatment according to the plasma process time. Comparative analysis of tap water before and after plasma treatment shows that EC increased to 0.28 after 30 min of treatment, but there was no difference in EC as the time increased (Table 1). The pH decreased with process time, from 7.62 in tap water to 7.24 in PAW. NO^2- ion concentration measured as 0.03 at 20 and 30 min. NO^3- ion concentration increased with time to 45.01 at 30 min, which was 4.97 times more when compared to tap water. The H₂O₂ ion concentration was 0.006 in tap water but showed a 10-fold increase to 0.06 after 30 min of PAW treatment. It was possible to obtain increased reactive oxygen and nitrogen species according to the PAW process time.

Table 1

Physicochemical properties of TW and PAW for 10, 20, 30 min process time.
Water	EC	pH	NO₂^- (mg·L^-1)	NO₃^- (mg·L^-1)	H₂O₂ (mg·L^-1)
TW^z	0.16	7.62	0.0080	9.04	0.0060
PAW 10 min	0.25	7.45	0.0280	41.10	0.0480
PAW 20 min	0.26	7.31	0.0300	43.40	0.0520
PAW 30 min	0.28	7.24	0.0300	45.01	0.0600
^zTW: Tap water, PAW: Plasma activated water

Germination of radish sprouts according to PAW process time

Germination rate increased with increasing PAW generation time (Fig. 1). The germination rate (GR) on the first day after sowing the control was 25.7%, but the 30 min PAW treatment showed significantly higher GR (55%). On the harvest day (7th day), there was no significant difference between the PAW 20 min and 30 min treatments at 91.7% and 93%, but the results were 18% and 19.7% higher than the control group. Germination energy (GE) was calculated as the total number of germinated seeds up to the 4th day (Table 2). Similar to GR, the longer the underwater plasma discharge time, the higher the GE, and the 30 min PAW treatment resulted in significantly higher results at 91.33. The mean germination time (MGT) was the shortest in the 30 min PAW treatment, and there was no significant difference between the 10 min and 20 min treatments. The germination speed (GS) of 20 min PAW treatment was significantly faster than the 10 min treatment (14.5%), and the 30 min treatment, which showed the fastest GS, was 52.8% higher than that of the control group. Conversely, the mean daily germination speed (MDG) did not show a significant difference between the PAW treatments, and the control showed a significantly lower value at 11.1.

Table 2

Effect of plasma activated water process time on seeds.
Plasma treatment	Germination
Plasma treatment	GR^w (%)	GE	MGT	GS	MDG
Control^x	77.7 ± 1.8 c^y	72.67 ± 1.5 c	1.88 ± 0.03 c	47.17 ± 0.92 d	11.1 ± 0.25 b
10min	87.0 ± 1.2 b	83 ± 0.3 b	1.69 ± 0.06 b	56 ± 1.28 c	12.71 ± 0.41 a
`20min	91.7 ± 0.3 ab	88.67 ± 0.7 a	1.65 ± 0.01 b	64.1 ± 0.\9 b	13.1 ± 0.05 a
30min	93.0 ± 0.6 a	91.33 ± 0.3 a	1.44 ± 0.01 a	72.08 ± 0.61 a	13.29 ± 0.08 a
Significant^z	*	*	*	*	*
^wGR: Germination rate, GE: Germination energy, MGT: Mean germination time, GS: Germination speed, MDG: Mean daily germination
^xControl: Tap water, 10min: Plasma activated water 10 minutes, 20min: Plasma activated water 20 minutes, 30min: Plasma activated water 30 minutes.
^yMean separation within column's Tukey's multiple range test.
^zSignificant at * p ≤ 0.05 (n = 3)

Growth parameters

Shoot length, root length, shoot fresh weight, and shoot dry weight were measured seven days post sowing according to the PAW process time. Shoot length was the longest (4.72 cm) in the 10 min PAW group, and there was no significant difference from the control (Fig. 2A). As the PAW process time increased, shoot length decreased, with the 20 min and 30 min treatments being 12.4% and 25.2% shorter, respectively. These are significantly lower than that of the 10 min treatment. Root length showed a negative effect of PAW treatment compared to the control. The length of roots was the longest at 5.23 cm in the control group and was significantly higher (146.7%) when compared to the 30 min treatment, which was the shortest. During the growth period, root length was shortened as the underwater plasma discharge time increased, and in the case of the 20 min and 30 min treatments, the rhizosphere was oxidized to red and showed a tendency to shorten. There were no significant differences in fresh weights between the control and 10 min treatment group, and both 20 min and 30 min treatments showed a tendency to decrease significantly (21.1%) compared to the control (Fig. 2B). Similarly, dry weight was not significantly different between the control and treated, with the 30 min treatment group being the highest.

Total glucosinolate contents

The total and individual glucosinolate contents were determined in radish sprouts. Three types of indolic glucosinolates (4-methoxyglucobrassicin, glucobrassicin, and neoglucobrassicin) and three types of aliphatic glucosinolates (progoitrin, sinigrin, and gluconapin) were detected in the control and the three PAW treatments. Glucobrassicin content was not significantly different between the control and the PAW groups. The content of 4-methoxyglucobrassicin increased linearly with increasing process time, and the 30 min treatment increased to 40.2% compared to the control (Fig. 3A). Neoglucobrassicin content also showed the same trend as 4-methoxyglucobrassicin content, and the 30 min treatment showed 45.3% higher concentrations than that of the control (Fig. 3B). In aliphatic glucosinolate, there was no significant difference in progoitrin content between the control and PAW treatment, and the content of sinigrin increased by 19.5% after 30 min of treatment compared to the control (Fig. 4A). Similarly, the 30 min treatment also had the highest gluconapin content and significantly increased it by 45.3% compared to the control (Fig. 4B). There was no significant difference in the total glucosinolate content per unit dry weight for 10 and 20 min, but 30 min increased by 9.5% compared to that of the control. Total glucosinolate content per plant dry weight after 30 min of treatment significantly increased by 28.9% compared to the control (Fig. 5).

Total phenolic contents

Methanol extracts of radish sprouts cultivated in different PAW processing times (10, 20, and 30 min) were analyzed by HPLC. Chlorogenic acid, benzoic acid, epicatechin, trans-cinnamic acid, and rutin were detected in the control and PAW-treated samples. The chlorogenic acid and benzoic acid concentrations tended to increase as the processing time increased, and these were observed at their highest values at 30 min of treatment, and significantly increased by 21% and 94%, respectively, compared to the control (Fig. 6A). The epicatechin concentration was also the lowest in the control and was significantly higher (21.1%) after 30 min of treatment. Trans-cinnamic acid concentration increased linearly with process time in the same tendency as benzoic acid, and the 30 min treatment increased 69.5% compared to the control (Fig. 6B). Rutin concentration was increased by 29.9% with 20 min of treatment and decreased at 30 min as compared to the control (Fig. 7A). To analyze the total phenolic acid concentrations and contents, the contents per unit dry weight and per plant dry weight were measured. Total phenolic acid content per unit dry weight increased by 29.9% after 30 min of treatment compared to the control, and the content per plant dry weight was significantly higher (288.8%) than that of the control (Fig. 7B). As the PAW processing time increased, the lengths of the shoot and root parts decreased in radish sprouts, but the total glucosinolate and total phenolic acid content increased.

Radish sprouts of the cruciferous family are richer in bioactive compounds than adult plants and may increase phytochemicals by at least 2 to 10-fold depending on environmental conditions^33,34. Germination was promoted when PAW was applied to radish sprout cultivation. It is known that H₂O₂, a major constituent of plasma water, can be delivered to plant cell membranes by diffusion dependent on membrane lipid composition, and can also be delivered by aquaporin, a membrane protein that promotes water transport³⁵. H₂O₂ delivered to plants is involved in seed metabolism, antioxidant enzyme activity, or cell signaling. H₂O₂ can break dormancy by downregulating abscisic acid (ABA) and upregulating gibberellic acid (GA) biosynthesis by participating in seed germination³⁶. When mung bean seeds were treated with PAW for 15 min, the germination rate increased significantly by 5.6% compared to distilled water and decreased when the treatment time was longer³⁷. It has been reported that the germination rate of Arabidopsis thaliana seeds significantly increased when treated with oxygen-mediated PAW³⁸. In this experiment, the germination of seeds increased when the plasma processing time was increased, however, the effects on the seeds of the active oxygen species and the content of active nitrogen species generated when the processing time is long, should be considered.

It is known that a low concentration of H₂O₂ has a positive effect on root development, seed germination and plant growth³⁹. In contrast, high concentrations of H₂O₂ have been reported to inhibit root-hair extension and induce oxidative stress^40,41. A high concentration of H₂O₂ oxidizes ascorbic acid and glutathione and induces apoptosis, which negatively affects growth and damages proteins, lipids, and nucleic acids essential for cell development^42,43. Therefore, the reason why the roots turned red and the length shortened in the 20 min and 30 min process time treatments in PAW is due to the increased H₂O₂ concentration according to the treatment time, and it is thought that the growth of the shoot was lowered because the development of the rhizosphere was insufficient.

H₂O₂ generated during underwater plasma discharge accounts for the largest portion of various reactive oxygen species and has been reported to be a stable molecule⁴⁴. The stability, reaction, and diffusivity of H₂O₂ are signaling molecules that oxidize the methionine and thiol groups of cysteine to affect the redox state of the protein and act as mediators to change its function to activation or inactivation^22,45,46. H₂O₂ modulates carbonylation, which alters protein activity and increases the susceptibility to protein degradation^47–49. Sewelam et al. (2014) found that indolic glucosinolate biosynthesis genes were upregulated and aliphatic glucosinolate was downregulated when plants produced H₂O₂, since it induced an initial signal transduction response, including transcription factors and biosynthetic genes involved in the production of secondary signal transmitters⁵⁰. Balazadeh et al. (2012) reported that indolic glucosinolate and camalexin biosynthesis genes could be expressed when H₂O₂ content was increased in plants⁵¹. In addition, glucosinolates contained in Cruciferae consist of sulfur and nitrogen components^52,53, and it is known that glucosinolate biosynthesis can be regulated according to the concentration of sulfur and nitrogen in the plant⁵⁴. NO₃⁻ absorbed by plants as a nitrogen source is involved in indolic glucosinolate and aliphatic glucosinolate biosynthesis to increase its content⁵⁵.

Seed priming is a mechanism that can enhance plant tolerance to biological stress. It has been reported that chemical immersion treatment at the early stage of growth can enhance stress tolerance by participating in various stress response pathways, such as the physiological regulation and detoxification of ROS and methylglyoxal^56–59. In particular, exogenous H₂O₂ and ROS stimulation activate the mitogen-activated protein kinase (MAPKs) pathway and generate defense mechanism metabolites under oxidative stress conditions^60,61. The increase in glucosinolate and phenolic acid content is considered to be a product of a defense mechanism against radish sprouts because it was grown under oxidative stress by plasma-activated water.

Design of non-thermal plasma model and water quality characterization

PAW was generated using corona discharge, as shown in Fig. 8A. The radius of the electrode was 2 mm, and the quartz tube surrounding the electrode had a radius of 4 mm (Fig. 8B). It was designed such that the voltage is regulated through the power supply and amplified through a transformer to discharge. The frequency of the transformer used for plasma generation was 25 kHz, and the maximum voltage was 4.2 kV with a sine wave (Fig. 8C). Oxygen gas was used as the gas injected during the plasma water generation time at a rate of 2 L ·min^-1. The amount of water used was 500 mL and the processing time was 1 h. PAW water was measured using ICP-MS and ion chromatography to measure water quality characteristics and ions when plasma discharge occurred in tap water.

Seed treatments and assessment of germination and growth

The study complied with local and national regulation.

Measurement of germination and growth parameters

The experiment was carried out using radish seeds (Raphanus sativus L., Asia seed Co., Ltd., Korea) at plant factory in Chungnam national university (Daejeon, Korea). Radish seeds were immersed in running tap water for 2 h with 100 seeds for each treatment group, then placed in a growing tray and placed in a chamber. The environmental conditions in the chamber were set to 24 ± 0.5°C, a relative humidity of 75 ± 5%, and light density of 80 ± 5 µmol·m^-2·s^-1. To determine the effect of plasma water on seeds during the experimental period, GR, GE, MGT, GU, GS and MDG were investigated. After harvest, plant height and root length were measured using callipers (SD500-300PRO, Shin Con Co, Ltd., Korea). The live weight was investigated using an electronic scale (MW-2N, CAS Co., Ltd., Korea) on the above-ground part, and then the sample was placed in a bag and placed in a hot air dryer (HB-501M, Hanbaek Scientific Technology Co., Ltd., Korea) set to 70°C for 4 days and measured with the same electronic scale.

Analysis of glucosinolate contents

The glucosinolate content of the radish sprouts was analyzed using HPLC (1260 Infinity II, Agilent Technologies Co., Ltd., United States). The aboveground parts of radish sprouts grown for 7 d were dried for 4 d using a freeze dryer (TFD550, Ilshin BioBase Co., Ltd., Korea). The dried sample was pulverized using a blender (SMX 800SP, Shinil Co., Ltd., Korea) and then further powdered using a mortar and pestle. 100mg of powder was placed in a 2.0 mL tube, 70% (v/v) MeOH (1.5 ml) was added, and the mixture was vortexed. Glucosinolate was extracted by placing the extract in a water bath at 70°C for 5 min. The supernatant was extracted by centrifugation at 12,000 rpm for 10 min, and the same process was repeated 3 times. The DEAE-Sephadex A-25 (30g) was dissolved in ultrapure water and placed in a funnel. After adding 1.5 times (v/v) ultrapure water and filtering, 0.5M sodium acetate (68 g ·L^-1) was added. The mini-column was made by closing a 1 mL pipette tip with a degreased surface and inserting the DEAE-Sephadex A-25. The GSL extract was loaded into the mini column with a pasteur pipette, and 2 ml of ultrapure water was administered when all extracts were drained. When all the administered ultrapure water was removed, the lower part of the mini column was covered with a paraffin film, and 75 µL of arylsulfatase solution was loaded. The light was blocked for 16 h considering the enzyme reaction time, and the mixture was allowed to stand at room temperature. After 16 h, desulfo-GSLs were eluted by flowing ultrapure water (0.5 mL) three times into a 2.0 mL tube. The eluted sample was filtered through a 0.45 µm syringe filter and placed in an HPLC vial for analysis.

Analysis of phenolic acid contents

For the analysis of the phenolic acid content, it was dried and pulverized in a similar way as for the analysis of glucosinolate content. The control and treated radish sprout powder (100 mg) were placed in a 2.0 ml tube and placed in MeOH (80% of 2 ml), followed by sonication for 1 h and centrifugation at 13000 rpm for 10 min. The extracted supernatant was filtered using a syringe filter and placed in a vial for HPLC analysis. Using an HPLC column (Optimapak C18 column (250×4.6 mm 5 µm)), the column temperature was set to 30°C, flow rate was 1.0 mL/min, and detection wavelength was set to 280 nm. Solvent A contained 0.15% acetic acid and 80% MeOH as the mobile phase.

Statistical analysis

For the statistics of growth and functional substance content analysis, ANOVA was performed using the SPSS (Version 22.0.0.1, SPSS Inc., Illinois, USA) program, and the significance between the means was evaluated using Tukey’s multiple test method (p ≤ 0.05). A graph was prepared using Sigma Plot (10.0; Systat Software Inc., Chicago. IL, USA) program.

Funding

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) and Korea Smart Farm R&D Foundation(KosFarm) through Smart Farm Innovation Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) and Ministry of Science and ICT(MSIT), Rural Development Administration(RDA)(421034-04)

Data Availability

All relevant data are within the paper and posted at

https://doi.org/10.6084/m9.figshare.20723410.v2

Author Contributions Statement

S.J.K. and S.W.N. formulated the idea, set up the experiments, conducted data collection and analysis, wrote the original manuscript, and conducted the review and editing. D.W.K. prepared the manuscript, figure 8 and table 1. J.S.P. conducted project administration, supervised the construction of the idea, completed the experimental design, conducted data analysis, wrote the original manuscript, and conducted the review and editing. All authors have read and agreed to the published version of the manuscript.

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Germination, Growth, and Secondary Metabolite Contents of Radish Sprouts (Raphanus sativus L.) as Affected by Plasma Activated Water Process Time

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