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

Round 1

Reviewer 1 Report

The submitted manuscript presents results from experiments investigating the effects of PAW treatment of thale cress seedlings growth. The study includes physiological analysis of seedling root development (root length, root hair number and length) and development of cotyledons (size, number and size of palisade cells). Gene expression analysis of some selected genes relevant for root hair development underpins the physiological observations on root development. In the presented study, a surface DBD device operating in ambient air was used to treat one liter of deionized water for different treatment times ranging from five to 40 minutes. By analysis of plasma with OES and by chemical analysis of PAW, the authors detected essentially nitrogen species and especially nitrate within the PAW. The authors conclude that these altered characteristics of plasma treated water explains the positive effects on the observed seedling development for water treatment times shorter 12 minutes. Longer treatment times had negative effects on seedling development.

Major points: In general, the authors’ research topic is of high relevance for better understanding of how PAW can influence specifically root development under growth conditions on agar with deionized water. For potential future applications, it is important to learn more about the effects and the effective dosage of PAW on plant development. In this respect, Arabidopsis thaliana L. is a suitable model for such in-depth studies while such studies are rare and worthwhile to be published. The experimental methods applied to elucidate the effects on Arabidopsis seedlings root and cotyledon development are of a high and professional standard. However, the manuscript needs to be improved in many sections ranging from linguistic to content related issues.

First, information should be added to section 2 and 3 concerning description of the plasma source (e.g. reactor configuration, electrical parameters applied) and water treatment (e.g. distance of treatment zone to plasma/electrode). A scheme of the SDBD device would help to understand the experimental set-up. If already published, the respective reference should be cited. It is hard to imagine where the OES spectra exactly was taken (e.g. which region of the plasma, water contact zone?); again, a scheme would help for better understanding. Moreover, if the SDBD device can treat one liter of water, what is the surface area of the treated water exposed to plasma and does the water needs to be stirred during treatment for homogeneous treatment? Such information could be added as well.

Second, the authors should address the general nutrient status of Arabidopsis plants grown under chosen experimental cultivation conditions. Control plants are grown in deionized water. It seems to be that no further nutrients are applied to growth media and substrate. Thus, it is reasonable to ask whether plants are grown under nutrient starvation conditions and whether osmotic stress occurred. Starvation and ionic imbalance in growth substrates can lead to altered root development e.g. effecting lateral root growth. Usually, agar media (is supplemented with micro- and macronutrients e.g. SM media) to avoid starvation. If in the presented study, minimal growth conditions as part of experimental-setup are intended, a clear statement and description in the manuscript should be done, if true. In this respect, the authors should rethink about the usage of the term “ideal conditions” when compared to control groups under starvation conditions. Moreover, observed effects in PAW19 and PAW40 could be a result of metabolic deregulation due to multi-nutrient starvation, which would be probably not detectable under nutrient supply containing essential micro- and macroelements. Moreover, it should be shortly mentioned in the manuscript if an addition of further ions, such as potassium, is able to affect plant growth as well, e.g. by changing osmolarity in the media.

Third, figures and tables needs improvement. In all figures with photos presented (including supplementary figures), scale bars are too flat with little contrast and therefore hardly visible.

Please specify the values for “root hair number” in Table 2, 4 and 5. Usually this information is given as root hair density with the information about size and location of measured region along the root axis. Please specify “n=6”, does it mean number of roots from six individual plants. Please add this information to the table captions or to section 2.

Similar question to “root hair lengths” (Table 2, 4, 5): How many individual plants were chosen to analyze root hair lengths for in total n=100?

Table 3: What was the size in mm2 of tissue cross sections used for the measurement of palisade cell number? Please add this information. Of how many individual cotyledons the palisade cell size was measured. Please add this information to the manuscript.

Fourth, language is in some parts misleading and confusing. Some sentences should be proofed in respect to semantics and structure. Usage of tenses and usage of plural vs. singular of nouns and auxiliary verbs needs to be checked as well. There is an extensive usage of sentences beginning with an infinitive “To…”

 Abstract:

Lines 25-26: Please avoid descriptions of nomenclatures in the Abstract. Instead, give precise information of different plasma treatment times in minutes.

Lines 31-33: The expression of AUX1 and LAX3 is not mentioned in the abstract, but was also significantly influenced by the PAW treatment.

1. Introduction:

The introduction section should be restructured and more focused on the definition of low-temperature atmospheric plasmas. Information about the plasma source applied in this study should be placed to section 2 and 3.

Line 43: “and plasma is closely related to us” is unintelligible

Lines 44 ff.: it should be known that electrodes are negatively charged; please revise the definition of plasma. There are more than the listed components, e.g. ions, neutral atoms and molecules, radicals and in general reactive oxygen and nitrogen species. Moreover, plasma can consist of different kinds of radiation (heat, UV, Vis) and/or electromagnetic fields etc.

Lines 49 ff.: repetition, compare to lines 39 ff.

Lines 53-54: Plasma is classified according to temperature and density of electrons and particles

Lines 59-66: This paragraph is part of section 2 and 3.

Line 67: Please specify if plasma is already applied in agricultural practice. Are there companies to be named?

Lines 68-69: Language revision needed.

Lines 69-70: Delate superfluous “indirect”, PAW treatment is an indirect treatment mode.

Lines 72-73: Nitrogen species should be mentioned as well.

Line 75: Delate “activated” and write only “…water treated with…”

Lines 77-79: H, O and C are elements and components of nutrients; those are the main components for all biomolecules and organisms on earth and not exclusively for plants. Moreover, water is taken up by the roots and not to be considered as a nutrient. It should be specified which nitrogen containing components are taken up by plants, which are usually dissolved in water. Thus, the sentence needs revision.

Lines 87-88: specify to which plants Arabidopsis is smaller, otherwise eliminate vague statements.

Line 92: vague statement “began with future insights”.

Lines 92-94: sentence revision needed.

Lines 98-03: Further results are mentioned that are not presented in the abstract. Does abstract needs completion?

2. MM:

Add information to section 2.1 about plasma source and treatment condition as suggested.

Lines 114-119: The method for hydrogen peroxide measurement is missing. Please add the description of techniques and express the values in m/v or molar concentration. Dionex standards for IC measurement of nitrate and nitrite allow estimation of mass concentrations in m/v. Please indicate the concentration of nitrate in “mg/l” or “g/l”. Otherwise state, why “ppm” is used instead.

Lines 133-140: Please indicate here from which seed stock center the plant material was received. Please indicate the accession background of the double mutant. In addition, literature should be cited for construction of WER::GFP plants and of WER::GFP/cobl9‐1 double mutant. Please give a reference to supplementary material for primers needed for genotyping.

Line 152-153: “double mutants of Columbia 0 (Col‐0) and WEREWOLF (WER)::GFP/cobl9‐1” is confusing, are there two different double mutants?

3. Results:

Lines 185-186: add “time in minutes”

Table 1: In all other tables standard deviation, statistics and number of replicates are given. Explain, why only single values are presented in Table 1. Please present data in mass or molar concentrations if possible.

Line 198: Please define chemical components more precisely, nitrate and nitrite ions are not being considered as “RNS”, unlike “NO” or “peroxynitrite”.

Line 203-205: revise sentence, e.g. “greatest phenotypic difference” is unintelligible.

Lines 211-213: repetition of lines 199-203 and ff.

Figure 3: There is no reference in the text for this figure.

Line 261: DIC microscopy should be first mentioned in the MM section and should be spelled out for the first time.

Line 272-273: “…not following the origin of the cells..” is unintelligible.

Line 275-282: It should be made clearer for what reason such double mutant was chosen for the study e.g. GFP expressing line for better visualization of epidermal cells without affecting the root development. It is not clear, why plants with cobl9 mutations were chosen. Therefore, the phenotype of the individual lines and the double mutant should be mentioned and what changes are to be expected compared to the wild type, including those associated with PAW treatments compared to the DW control.

Lines 282-284 and 286-287: revise sentence structures.

Table 4 (5 day old seedlings) and Table 5 (7 day old seedlings): It is surprising that the values of “root hair number” and “root hair lengths” are much lower in the 7-day-old plants compared to the 5-day-old plants. This is not easy to understand! Please give a brief explanation.

Figure 5: Explain the meaning of arrows pointing to the hair cells in the pictures.

4. Discussion:

Lines 346-347: Revise sentence in respect to semantics.

Lines 372-374: This important information about analysis of PAW after one day needs to be presented in section 2 and 3. Is there any proof that nitrite was present in PAW directly after plasma treatment?

1. Line 378: According to element composition of chlorophylls, the major components are carbon and hydrogen. Thus, such statements should be handled with care. More important is the general function of nitrogen for plants metabolism as for e.g. nitrogen in biomolecules such as proteins as part of mandatory functional groups (amino groups in amino acids) or in purines and pyrimidines of energy metabolism (ATP, NAD) and RNA/ DNA etc.

Lines 383-384: Revise sentence in respect to semantics.

Lines 386-389: sentence too long, revise structure

Line 395: Revise the term “the roots become activated”.

Lines 395 ff.: In Table 1, it is interesting to see that values of e.g. nitrate (and the conductivity) for PAW19 and PAW40 are much higher compared to PAW5-12. The authors should address this. Is there any correlation (e.g. osmolarity, pH, excess of nitrogen species in an environment of other nutritional starvation) with the observed alterations in plant growth as PAW19 and PAW40 treated plants display reduced root development?

Lines 401-403 and 303-304: Sentences seem to be incomplete, please revise language.

Line 405-408: Here, the discussion could shortly review research findings on plant’s nitrate/nitrogen acquisition and findings about nitrate/nitrogen starvation and nutrient conditions in excess. How such conditions affects root growth?

Line 429: Avoid term “ideal conditions”. It is better to speak about the maximal observed effects on root development by PAW5 treatment compared to all PAW generation times. Plasma treatment times shorter five minutes were not tested; theoretically, it can be not excluded that shorter plasma treatment times could further improve growth.

Supplementary Materials: Indicate the age of roots from which photos were taken in Figures S1 and S2.

Author Response

Response to Reviewer 1 Comments

The submitted manuscript presents results from experiments investigating the effects of PAW treatment of thale cress seedlings growth. The study includes physiological analysis of seedling root development (root length, root hair number and length) and development of cotyledons (size, number and size of palisade cells). Gene expression analysis of some selected genes relevant for root hair development underpins the physiological observations on root development. In the presented study, a surface DBD device operating in ambient air was used to treat one liter of deionized water for different treatment times ranging from five to 40 minutes. By analysis of plasma with OES and by chemical analysis of PAW, the authors detected essentially nitrogen species and especially nitrate within the PAW. The authors conclude that these altered characteristics of plasma treated water explains the positive effects on the observed seedling development for water treatment times shorter 12 minutes. Longer treatment times had negative effects on seedling development.

Major points: In general, the authors’ research topic is of high relevance for better understanding of how PAW can influence specifically root development under growth conditions on agar with deionized water. For potential future applications, it is important to learn more about the effects and the effective dosage of PAW on plant development. In this respect, Arabidopsis thaliana L. is a suitable model for such in-depth studies while such studies are rare and worthwhile to be published. The experimental methods applied to elucidate the effects on Arabidopsis seedlings root and cotyledon development are of a high and professional standard. However, the manuscript needs to be improved in many sections ranging from linguistic to content related issues.

First, we would like to thank Reviewer #1 for the critical and detailed comments on our manuscript. We are glad that the reviewer's views on our research are relevant and important to the study of PAW effects on A. thaliana development when using different plasma exposure times to generate the various PAW conditions. Based on the suggestions and comments from Reviewer #1, we have revised our manuscript and addressed the comments and suggestions from the reviewers. Please find our detailed responses below.

First, information should be added to section 2 and 3 concerning description of the plasma source (e.g. reactor configuration, electrical parameters applied) and water treatment (e.g. distance of treatment zone to plasma/electrode). A scheme of the SDBD device would help to understand the experimental set-up. If already published, the respective reference should be cited. It is hard to imagine where the OES spectra exactly was taken (e.g. which region of the plasma, water contact zone?); again, a scheme would help for better understanding. Moreover, if the SDBD device can treat one liter of water, what is the surface area of the treated water exposed to plasma and does the water needs to be stirred during treatment for homogeneous treatment? Such information could be added as well.

> We really appreciate your critical and detailed comment. Following your comment, we have revised the manuscript to include more details on the plasma source which was already published by our group. We added a citation for the previous paper and added information where the OES spectra position is placed. We did not use stirrers during the treatment, but we used a fan below the electrodes inside the reactor. More detailed information has been added to the materials and methods section as shown below.

“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 are made up of two parallel metals, one of which is covered with a dielectric layer, and the plasma is generated using alternating current (AC) flowing through the electrodes. The electrode consisted of a 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 amplitude 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 a 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 1.5 - 2.0 cm. The OES was obtained with an integration time of 1 second which was averaged 50 times”.

 

 

Second, the authors should address the general nutrient status of Arabidopsis plants grown under chosen experimental cultivation conditions. Control plants are grown in deionized water. It seems to be that no further nutrients are applied to growth media and substrate. Thus, it is reasonable to ask whether plants are grown under nutrient starvation conditions and whether osmotic stress occurred. Starvation and ionic imbalance in growth substrates can lead to altered root development e.g. effecting lateral root growth. Usually, agar media (is supplemented with micro- and macronutrients e.g. SM media) to avoid starvation. If in the presented study, minimal growth conditions as part of experimental-setup are intended, a clear statement and description in the manuscript should be done, if true.

In this respect, the authors should rethink about the usage of the term “ideal conditions” when compared to control groups under starvation conditions. Moreover, observed effects in PAW19 and PAW40 could be a result of metabolic deregulation due to multi-nutrient starvation, which would be probably not detectable under nutrient supply containing essential micro- and macroelements. Moreover, it should be shortly mentioned in the manuscript if an addition of further ions, such as potassium, is able to affect plant growth as well, e.g. by changing osmolarity in the media.

> Thank you for this very good comment. The study of Arabidopsis seedlings grown in minimal growth condition has been done in many previous studies to observe the dynamics of more severe effects and the effects of various treatments. For example, Réthoré et al. 2019 showed that Arabidopsis can grow for weeks in water without an external supply of nutrients. Following your suggestion, we have revised to the materials and methods and discussion section to add the minimal growth conditions.

In addition, we did not observe any starvation during the early Arabidopsis developmental stage. We had data from an Arabidopsis experiment using ½ MS media and DW and PAW (not published data). There was a significant difference in the phenotype between the DW and PAW that showed a similar pattern with PAW19, 40 and DW. In longer treatment conditions, root growth was inhibited. We do not completely exclude the possibility of starvation, but it might be a minor issue of this paper.

In addition, we tested the configuration and duration of the treatment. The wilting of Arabidopsis grown on minimal agar medium occurred approximately 3 weeks after sowing. Furthermore, by using this approach, we could see the possibilities of using the plasma treatment as an alternative method when limited resources are only available.

Regarding the osmolarity, previously, we did not consider that different osmolalities would affect the root growth significantly in this study because the adjustment of pH using KOH or NaOH has been routinely performed in plant studies. Furthermore, the osmolarity of media has not been considered as a main factor unless a study’s focus was on osmolarity effects or certain genes in the osmolarity pathway. However, we do not exclude the possibility of osmolarity affecting plant growth; the study of osmolarity effects in plasma activated water treatments could be a very interesting topic to further explore in the future.

“Regarding the use of the term “ideal condition,” we agree that this term is not exactly accurate. Therefore, we revised this term to “maximal observed conditions” as the reviewer suggested.

Réthoré, E., d'Andrea, S., Benamar, A., Cukier, C., Tolleter, D., Limami, A.M., Avelange‐Macherel, M.‐H. and Macherel, D. (2019), Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self‐sufficient for weeks. Plant J, 99: 302-315. https://doi.org/10.1111/tpj.14325

 

Third, figures and tables need improvement. In all figures with photos presented (including supplementary figures), scale bars are too flat with little contrast and therefore hardly visible.

> We have revised and improved the graph and table including a clear bar chart and detailed captions in the table.

Please specify the values for “root hair number” in Table 2, 4 and 5. Usually this information is given as root hair density with the information about size and location of measured region along the root axis. Please specify “n=6”, does it mean number of roots from six individual plants. Please add this information to the table captions or to section 2.

> We briefly provided in the materials and methods section 2.1 the plant material and growth conditions for the root morphology observation, and we now added the measured region and microscopy magnification for the root hair number to the table captions. We have revised the table captions to clearly indicate the number of plants used in the quantifications.

Similar question to “root hair lengths” (Table 2, 4, 5): How many individual plants were chosen to analyze root hair lengths for in total n=100?

> Thank you for pointing out this important information. We chose 6 individual plants and counted 100 root hairs, and the root hair length was from the 6 plants. We have updated our table caption to clearly indicate the information about the number of plants.

Table 3: What was the size in mm2 of tissue cross sections used for the measurement of palisade cell number? Please add this information. Of how many individual cotyledons the palisade cell size was measured. Please add this information to the manuscript.

> Thank you for pointing out this important information. We have updated our table captions to indicate the information on the number of individual plants and other details in the table captions.

Fourth, language is in some parts misleading and confusing. Some sentences should be proofed in respect to semantics and structure. Usage of tenses and usage of plural vs. singular of nouns and auxiliary verbs needs to be checked as well. There is an extensive usage of sentences beginning with an infinitive “To…”

> We have edited the manuscript revising sentence structure and semantics. In addition, we have submitted the manuscript for English Proofreading to address this issue.

Abstract:

Lines 25-26: Please avoid descriptions of nomenclatures in the Abstract. Instead, give precise information of different plasma treatment times in minutes.

> As you suggested, we edited the manuscript in line 25-26.

Lines 31-33: The expression of AUX1 and LAX3 is not mentioned in the abstract, but was also significantly influenced by the PAW treatment.

> Thank you for this suggestion. We initially thought we did not need to present all the results in the abstract and wanted to emphasize the COBL9 and XTH genes in the abstract. However, we agree that the expression of these genes is significantly influenced by the PAW treatment. Therefore, we have added AUX1 and LAX3 to the abstract.

1. Introduction:

The introduction section should be restructured and more focused on the definition of low-temperature atmospheric plasmas. Information about the plasma source applied in this study should be placed to section 2 and 3.

Line 43: “and plasma is closely related to us” is unintelligible

> As suggested by the other reviewers to remove lines 38-66, we have deleted lines 38 to 66 in our revised manuscript. The plasma source information was added in the materials and methods section.

Lines 44 ff.: it should be known that electrodes are negatively charged; please revise the definition of plasma. There are more than the listed components, e.g. ions, neutral atoms and molecules, radicals and in general reactive oxygen and nitrogen species. Moreover, plasma can consist of different kinds of radiation (heat, UV, Vis) and/or electromagnetic fields etc.

Lines 49 ff.: repetition, compare to lines 39 ff.

> Following one of the reviewers' comments, we deleted this sentence.

Lines 53-54: Plasma is classified according to temperature and density of electrons and particles

> Following one of the reviewers' comments, we deleted this sentence.

Lines 59-66: This paragraph is part of section 2 and 3.

> Following one of the reviewers' comments, we deleted this sentence.

Line 67: Please specify if plasma is already applied in agricultural practice. Are there companies to be named?  

> At our institute, we call it a family company in which we communicate and develop plasma technology together from an industry aspect. Those company names are Smart Tech, Kyung-nong Incorporation, etc.

Lines 68-69: Language revision needed.

> We have revised the sentences in line 39 -40.

Lines 69-70: Delete superfluous “indirect”, PAW treatment is an indirect treatment mode.

> We deleted ‘ indirect’ in formerly line 40-41 and modified sentence.

Lines 72-73: Nitrogen species should be mentioned as well.

> We added ‘nitrite and nitrate’ in formerly line 44-45.

Line 75: Delete “activated” and write only “…water treated with…”

> We deleted ‘activated’ in formerly line 47.

Lines 77-79: H, O and C are elements and components of nutrients; those are the 6main components for all biomolecules and organisms on earth and not exclusively for plants. Moreover, water is taken up by the roots and not to be considered as a nutrient. It should be specified which nitrogen containing components are taken up by plants, which are usually dissolved in water. Thus, the sentence needs revision.

> We have revised the introduction by deleting sentences from lines 49-53. In the revised manuscript, we discussed the main role of nitrate as a nitrogen source in plants and that plasma treatment could potentially be used as a fertilizer application.

Lines 87-88: specify to which plants Arabidopsis is smaller, otherwise eliminate vague statements.

> We eliminated ‘Arabidopsis thaliana L. is smaller than other plants’ and modified the sentence in the manuscript.

Line 92: vague statement “began with future insights”.

> We have revised the sentences (line 66-69).

Lines 92-94: sentence revision needed.

> We have revised the sentences (line 66-69).

 

Lines 98-03: Further results are mentioned that are not presented in the abstract. Does abstract needs completion?

> We have revised both the abstract and further results in this section to include AUX1 and LAX3.

2. MM:

Add information to section 2.1 about plasma source and treatment condition as suggested.

> We have added section 2.1 which includes the plasma source and treatment conditions.

Lines 114-119: The method for hydrogen peroxide measurement is missing. Please add the description of techniques and express the values in m/v or molar concentration. Dionex standards for IC measurement of nitrate and nitrite allow estimation of mass concentrations in m/v. Please indicate the concentration of nitrate in “mg/l” or “g/l”. Otherwise state, why “ppm” is used instead.

> Thank you for this comment. In our revised manuscript, we have added the measurement method for hydrogen peroxide in section 2, and we converted all ppm to mg/L.

Lines 133-140: Please indicate here from which seed stock center the plant material was received. Please indicate the accession background of the double mutant. In addition, literature should be cited for construction of WER::GFP plants and of WER::GFP/cobl9‐1 double mutant. Please give a reference to supplementary material for primers needed for genotyping.

> Thank you for this suggestion. As suggested, we added more information about the seed stock center in the material section as below. Background of the double mutant was Col-0. We provided literature for the construction of WER::GFP, and the WER::GFP/cobl9-1 double mutant was cited in the manuscript. For the reference on the primers, we added a reference containing paper that we use for the primer.

“WER::GFP/cobl9‐1 (CS67758) and cobl9-1 (Salk_099933) were acquired from the Arabidopsis Biological Resource Center (ABRC), www.arabidopsis.org, to examine the root hair. And 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. And COBRA-LIKE 9 (AT5G49270) gene is involved in root hair [26,27].”.

Line 152-153: “double mutants of Columbia 0 (Col‐0) and WEREWOLF (WER)::GFP/cobl9‐1” is confusing, are there two different double mutants?

> Thank you for comments. You are correct. We have removed the “double mutants of” to clear any confusion (line 144-145).

3. Results:

Lines 185-186: add “time in minutes”

> We added time in minutes in line 177-178.

Table 1: In all other tables standard deviation, statistics and number of replicates are given. Explain, why only single values are presented in Table 1. Please present data in mass or molar concentrations if possible.

> Thank you for the suggestion. We measured the physicochemical characteristics of the PAW at least 3 times, so we have updated the table according to the measured data.

Line 198: Please define chemical components more precisely, nitrate and nitrite ions are not being considered as “RNS”, unlike “NO” or “peroxynitrite”.

> Thank you. We changed the term RNS to nitrogen ions.

Line 203-205: revise sentence, e.g. “greatest phenotypic difference” is unintelligible.

> We have removed “greatest phenotypic difference” and rewrote the sentences as shown below.

“At 7 days after planting, the root lengths in the PAW5 treated plants were increased to about 33% compared to DW (Figure 2)”.

 

Lines 211-213: repetition of lines 199-203 and ff.

> We have removed the repetitive sentences, lines 211-213, and modified the sentences.

Figure 3: There is no reference in the text for this figure.

> Thank you for your comments. We have revised the text of the results and discussion adding the text “Figure 3” to the results and discussion section.

Line 261: DIC microscopy should be first mentioned in the MM section and should be spelled out for the first time.

> Thank you for the suggestion. We have revised our materials and methods section to clarify this term.

Line 272-273: “…not following the origin of the cells..” is unintelligible.

> Thank you for the suggestion. We have revised the sentence at line 265-266.

Line 275-282: It should be made clearer for what reason such double mutant was chosen for the study e.g. GFP expressing line for better visualization of epidermal cells without affecting the root development. It is not clear, why plants with cobl9 mutations were chosen. Therefore, the phenotype of the individual lines and the double mutant should be mentioned and what changes are to be expected compared to the wild type, including those associated with PAW treatments compared to the DW control.

> Thank you for this comment. We have added more detailed information and an explanation for using the double mutant. We also added to the discussion how we interpreted the data using WER::GFP/cobl9-1 in the results section (line 268-277 and line 413-417).

Line 268-277

“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 formation during development [33]. By using the double mutant WER::GFP/cobl9-1, we expected to see a clear effect of the PAW treatment in epidermal cell patterning which can help 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”.

 

Line 413-417:

 

“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 regulation of root hairs under PAW treatment involves sophisticated genetic networks, not simply a single pathway.”

 

Lines 282-284 and 286-287: revise sentence structures.

> We have revised the sentence in line 278-283.

Table 4 (5 day old seedlings) and Table 5 (7 day old seedlings): It is surprising that the values of “root hair number” and “root hair lengths” are much lower in the 7-day-old plants compared to the 5-day-old plants. This is not easy to understand! Please give a brief explanation.

> Thank you for this comment. Data were created using the taken photos. “The root morphology was examined using a Nikon-Fi3 stereo microscope with a 5x zoom magnification, objective lens (10x), 40 ms exposure, and 2.0x analog gain (Table 4). The GFP signals were detected under a Nikon-A1 confocal microscope at 488 nm with eyepiece lens (10x), objective lens (20x) and a FITC filter with the laser set to 40% and the analog gain to 80% (Table 5)”. And so, the photo size of the Confocal is 690.56 ųm x 690.56 ųm, and the photo size of the stereo microscope is 2401.73 ųm x 1758.13 ųm. Consequently, we measured the root phenotype in a different area presented in Tables 4 and 5. We added this information in the materials and methods and table caption section.

 

Figure 5: Explain the meaning of arrows pointing to the hair cells in the pictures.

> We have added more information to the caption of Figure 5.

 

4. Discussion:

Lines 346-347: Revise sentence in respect to semantics.

> We revised the sentence.

Lines 372-374: This important information about analysis of PAW after one day needs to be presented in section 2 and 3. Is there any proof that nitrite was present in PAW directly after plasma treatment?

> Following your suggestion, we moved line 372 to the materials and methods section and revised the manuscript by adding more details to sections 2 and 3. 

After we received your comment about nitrite, we communicated with our group and performed the experiment again. We made a mistake in the nitrite measurement because of the delayed retention time in the Ion Chromatography. We changed the manuscript to reflect that a small amount of nitrite is present in the PAW. We have repeated and confirmed the measurement with 3 replicates. We modified the data in Table 1 and results section in line 180-181. 

 

Line 378: According to element composition of chlorophylls, the major components are carbon and hydrogen. Thus, such statements should be handled with care. More important is the general function of nitrogen for plants metabolism as for e.g. nitrogen in biomolecules such as proteins as part of mandatory functional groups (amino groups in amino acids) or in purines and pyrimidines of energy metabolism (ATP, NAD) and RNA/ DNA etc.

>We have revised the sentences to reflect the more general importance of nitrogen to plants.

Lines 383-384: Revise sentence in respect to semantics.

> We have revised the sentence as shown below.

Line 391-394 : “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)”.

Lines 386-389: sentence too long, revise structure

>We have revised the sentence as shown below:

Line 394-398: “In detail, the morphological differences 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 treatment condition by 63% compared to the DW control, indicating that root growth occurred independent of the plasma treatment time used to generate the PAW. Thus, Arabidopsis needs specific growth conditions (Figures 2 and Figure 3)”.

 

Line 395: Revise the term “the roots become activated”.

> We have revised the sentence. “the roots are elongated”

Lines 395 ff.: In Table 1, it is interesting to see that values of e.g. nitrate (and the conductivity) for PAW19 and PAW40 are much higher compared to PAW5-12. The authors should address this. Is there any correlation (e.g. osmolarity, pH, excess of nitrogen species in an environment of other nutritional starvation) with the observed alterations in plant growth as PAW19 and PAW40 treated plants display reduced root development?

> Good suggestion about the discussion.

We agree that the amount of the other components in the PAW may have a correlation with the phenotype. But, based on our previous study and in this study, we were focused on nitrate as a macronutrient. Previously, in tobacco seedlings grown with plasma activated water, they also showed a similar pattern of activation and suppression in the root. Therefore, we hypothesized that the main factor was due to the availability of nitrate as a macronutrient that affects the nitrate-responsive root developmental genes. In our revised manuscript, we added a discussion about this and emphasized further evaluation of the osmolarity effect needs to be explored.

 

Lines 401-403 and 303-304: Sentences seem to be incomplete, please revise language.

> Thank you, we have revised the sentence in revised line 409-411 and 298-302.

 

Line 405-408: Here, the discussion could shortly review research findings on plant’s nitrate/nitrogen acquisition and findings about nitrate/nitrogen starvation and nutrient conditions in excess. How such conditions affects root growth?

> We have revised our manuscript by adding a research review of nitrate's role and how its concentration affects root growth in the discussion as shown below (line 421-426).

“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 architecture 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 increment 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]”.

 

Line 429: Avoid term “ideal conditions”. It is better to speak about the maximal observed effects on root development by PAW5 treatment compared to all PAW generation times. Plasma treatment times shorter five minutes were not tested; theoretically, it can be not excluded that shorter plasma treatment times could further improve growth.

> Following your suggestion, we edited the sentence as shown below.

Line 31-34

“Furthermore, we found that the root hair density and length at PAW5 in maximal observed conditions were positively regulated by root developmental related genes including COBRA-LIKE9 (COBL9), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (XTH9), XTH17, AUXIN1 (AUX1) and LIKE-AUXIN (LAX3).”

 

Line 446-448

“Although the expressions of XTH9 and XTH17 were suppressed in the PAW19 treatment condition, the maximal observed effect was observed on the root development by the PAW5 treatment compared to all the other PAW generation times (PAW7, 12, 19 and 40).”

Line 467-469

“The maximal observed condition for Arabidopsis root development was identified as PAW5 in which potential target genes were up-regulated while root development was suppressed by the PAW19 treatment condition”.

 

In addition, we performed an experiment in which plasma treatment times of 1 and 3 minutes were used to generate PAW1 and 3. Their effect on root length was investigated and compared with PAW5. The root length for PAW5 was not significantly different from those of PAW1 and 3. We do not think shorter plasma treatment times improve the root growth more than PAW5 during early developmental stages.

 

Supplementary Materials: Indicate the age of roots from which photos were taken in Figures S1 and S2.

> Thank you for the suggestion. We have revised the sentence.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The Authors present a very interesting study on the modulation of root and cotyledon growth by means of plasma activated water. The topic is timely and the presented results are of relevance. The set of diagnostic techniques adopted in the work is impressive. Nevertheless, too many details are missing from the material and methods section. Furthermore, the Authors hypothesis on the key role of nitrate is lacking the support of significant controls or comparisons with appropriate literature works. The manuscript would deserve publication after some major issues are addressed.

1. Section 1: In the Introduction there are too many parts that describe the plasma source and the selected plant model. These should be moved to the Material and Method section.
2. Section 2.1: The plasma reactor description is missing any information concerning the high voltage excitation system. What is the voltage amplitude and frequency? What is the average consumed power?
3. Section 2.1: The plasma reactor description is also missing any information concerning its configuration, dimension and materials. Also the relative position of the water and the electrodes is absent. All these information must be added in the materials and methods section.
4. Section 2.1: In which part of the plasma reactor was the OES measurement effectuated? With which exposure time?
5. Figure 1: Presents an unnecessary large amount of blank space. I suggest the Authors to vary the vertical scale to enlarge the significant section of the spectra.
6. Figure 1: Are peaks characteristic of NO detected? The dimension of the Figure doesn’t allow to discern it.
7. Table 1: It is very strange that there is absolutely no detection of nitrite even for the longest treatment time. How long after the treatment were the measurement effectuated? The Authors should try to compare their results with the literature and try at least to formulate some hypothesis explaining this peculiar trend.
8. Section 2.1: The 0.1 N and 1 N KOH solutions were added only to the PAW cases? Do the Author evaluated the possible influence of these solutions with an appropriate control? Was the osmolality of control and PAW solution comparable?
9. Figure 2: The Authors should add to Figure 2 the data at day 5 that they show in Table 5. This would allow to better understand the growth trend.
10. Section 4: The time lapse between PAW production and nitrate measurement should be in the M&M section, not here.
11. Control: The Author hypothesis is that nitrate plays a key role in mediating the effect of PAW on seeds. To prove this it would be enough to compare PAW results with that obtained by a nitrate enriched solution produced by chemical means (no plasma). Knowing the nitrate concentration in PAW that should be relatively easy. In the absence of this control, the authors should try to compare their results with literature works using solutions of similar nitrate concentration. In this sense reference 32 is certainly very interesting but is only very briefly cited in the text. Quickly comparing the concentration reported in the reference and those used in the work the trends appears to generally agree. An extended comparison would greatly benefit the discussion section.

Author Response

Response to Reviewer 2 Comments

Comments and Suggestions for Authors

The Authors present a very interesting study on the modulation of root and cotyledon growth by means of plasma activated water. The topic is timely and the presented results are of relevance. The set of diagnostic techniques adopted in the work is impressive. Nevertheless, too many details are missing from the material and methods section. Furthermore, the Authors hypothesis on the key role of nitrate is lacking the support of significant controls or comparisons with appropriate literature works. The manuscript would deserve publication after some major issues are addressed.

First, we would like to thank Reviewer #2 for the kind words and interest in this study. Based on the comments of the reviewers, we agree that there were missing details in the materials and method sections in our previous manuscript. In addition, we did not discuss the key role of nitrate shown in previous studies. Your comments really helped us to improve and present our research in a better way. We have revised our manuscript and responded to the reviewer’s comments shown below.

1. Section 1: In the Introduction there are too many parts that describe the plasma source and the selected plant model. These should be moved to the Material and Method section.

> We eliminated lines 38 to 59 from the introduction in the original manuscript as recommended by reviewer 3’s comment. We added the plasma source to the materials and methods section.

2. Section 2.1: The plasma reactor description is missing any information concerning the high voltage excitation system. What is the voltage amplitude and frequency? What is the average consumed power?

> We added a description to the materials and methods section as shown below

“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 are made up of two parallel metals, one of which is covered with a dielectric layer, and the plasma is generated using alternating current (AC) flowing through the electrodes. The electrode consisted of a 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 amplitude 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 a 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 1.5 - 2.0 cm. The OES was obtained with an integration time of 1 second which was averaged 50 times”.

”.

 

3. Section 2.1: The plasma reactor description is also missing any information concerning its configuration, dimension and materials. Also the relative position of the water and the electrodes is absent. All this information must be added in the materials and methods section.

> The plasma reactor information was published in a previous paper, and we cited this paper in the materials and methods section. We added the plasma reactor information including the position of the water and the electrodes to the materials and methods section.

4. Section 2.1: In which part of the plasma reactor was the OES measurement effectuated? With which exposure time?

> We have revised our manuscript in section 2.1 to clearly describe the measurement method.

5. Figure 1: Presents an unnecessary large amount of blank space. I suggest the Authors to vary the vertical scale to enlarge the significant section of the spectra.

> Thank you for the suggestion. We have modified the Y axis of this graph to enlarge the significance of the spectra (Figure 1).

6. Figure 1: Are peaks characteristic of NO detected? The dimension of the Figure doesn’t allow us to discern it.

> Thank you. No, we did not detect NO peaks in our spectrum. As mentioned in the response for comment No. 3 above, we modified the Y axis of the graph.

7. Table 1: It is very strange that there is absolutely no detection of nitrite even for the longest treatment time. How long after the treatment were the measurement effectuated? The Authors should try to compare their results with the literature and try at least to formulate some hypothesis explaining this peculiar trend.

> Thank you for pointing out this issue. After we received your comment about nitrite, we communicated with our group and performed the experiment again. We made a mistake in the nitrite measurement because of the delayed retention time in the Ion Chromatography. We have repeated the measurement with more replicates and confirmed that nitrite was present in the PAW treatments. We changed the manuscript to reflect that a relatively small amount of nitrite is present in the PAW. We have repeated and confirmed the measurement with 3 replicates. We modified the data in Table 1. 

8. Section 2.1: The 0.1 N and 1 N KOH solutions were added only to the PAW cases? Do the Author evaluated the possible influence of these solutions with an appropriate control? Was the osmolality of control and PAW solution comparable?

> Thank you for this comment. The pH adjustment was performed in both the DW and PAW. We did not evaluate the effects of the addition of KOH into the medium because this practice is routinely performed in basic plant growth media preparation, especially for MS (Murashige and Skoog) media (Murashige and Skoog, 1962).The addition of KOH in the solution was considered a small amount that would not significantly affect plant growth. We agree that further study of the osmolality effect on plasma-activated water will show its effect on plants in detail.

9. Figure 2: The Authors should add to Figure 2 the data at day 5 that they show in Table 5. This would allow to better understand the growth trend.

> Thank you for the suggestion. We have updated the graph in Figure 2 with data at day 5 after planting.

10. Section 4: The time lapse between PAW production and nitrate measurement should be in the M&M section, not here.

> Thank you for the suggestion. We have moved and modified these details to the materials and methods section as recommended.

11. Control: The Author hypothesis is that nitrate plays a key role in mediating the effect of PAW on seeds. To prove this it would be enough to compare PAW results with that obtained by a nitrate enriched solution produced by chemical means (no plasma). Knowing the nitrate concentration in PAW that should be relatively easy. In the absence of this control, the authors should try to compare their results with literature works using solutions of similar nitrate concentration. In this sense reference 32 is certainly very interesting but is only very briefly cited in the text. Quickly comparing the concentration reported in the reference and those used in the work the trends appears to generally agree. An extended comparison would greatly benefit the discussion section.

> This is a very interesting point! Indeed, our hypothesis is mainly that nitrate is responsible for the effect of the PAW treatment on root growth in Arabidopsis. There are many well established studies related to the effect of nitrogen sources on Arabidopsis growth. For example, in Boer et al. 2020, they describe the root plasticity in response to the available nitrogen source in the agar media, in which the different concentrations of nitrate affect its root development. As suggested, we now added more discussion about how the nitrate concentration is related to our present work as shown below (Page 12, Line 421-427). In addition, in this paper, we focused on the plasma treatment time of the PAW and its effect on the regulation of root growth. Currently, we are preparing a more detailed experiment using a nitrate enriched solution, a chemical base, and the PAW for comparison. We would like to understand the molecular regulation of the PAW-mediated root growth in DW, PAW and chemical based treatment (KNO₃) using RNA sequencing and now performing this experiment. The hope is that the results will be in our next publication.

 

“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 architecture 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 increment 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]”.

 

Reference for Reviewer #2:

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.

Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. - Physiol. Plant.15: 473–497, 1962.

 

 

 

 

 

Response to Reviewer 2 Comments

Comments and Suggestions for Authors

The Authors present a very interesting study on the modulation of root and cotyledon growth by means of plasma activated water. The topic is timely and the presented results are of relevance. The set of diagnostic techniques adopted in the work is impressive. Nevertheless, too many details are missing from the material and methods section. Furthermore, the Authors hypothesis on the key role of nitrate is lacking the support of significant controls or comparisons with appropriate literature works. The manuscript would deserve publication after some major issues are addressed.

First, we would like to thank Reviewer #2 for the kind words and interest in this study. Based on the comments of the reviewers, we agree that there were missing details in the materials and method sections in our previous manuscript. In addition, we did not discuss the key role of nitrate shown in previous studies. Your comments really helped us to improve and present our research in a better way. We have revised our manuscript and responded to the reviewer’s comments shown below.

1. Section 1: In the Introduction there are too many parts that describe the plasma source and the selected plant model. These should be moved to the Material and Method section.

> We eliminated lines 38 to 59 from the introduction in the original manuscript as recommended by reviewer 3’s comment.  We added the plasma source to the materials and methods section.

2. Section 2.1: The plasma reactor description is missing any information concerning the high voltage excitation system. What is the voltage amplitude and frequency? What is the average consumed power?

> We added a description to the materials and methods section as shown below

“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 are made up of two parallel metals, one of which is covered with a dielectric layer, and the plasma is generated using alternating current (AC) flowing through the electrodes. The electrode consisted of a 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 amplitude 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 a 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 1.5 - 2.0 cm. The OES was obtained with an integration time of 1 second which was averaged 50 times”.

”.

 

3. Section 2.1: The plasma reactor description is also missing any information concerning its configuration, dimension and materials. Also the relative position of the water and the electrodes is absent. All this information must be added in the materials and methods section.

> The plasma reactor information was published in a previous paper, and we cited this paper in the materials and methods section. We added the plasma reactor information including the position of the water and the electrodes to the materials and methods section.

4. Section 2.1: In which part of the plasma reactor was the OES measurement effectuated? With which exposure time? 

> We have revised our manuscript in section 2.1 to clearly describe the measurement method.

5. Figure 1: Presents an unnecessary large amount of blank space. I suggest the Authors to vary the vertical scale to enlarge the significant section of the spectra.

> Thank you for the suggestion. We have modified the Y axis of this graph to enlarge the significance of the spectra (Figure 1).

6. Figure 1: Are peaks characteristic of NO detected? The dimension of the Figure doesn’t allow us to discern it.

> Thank you. No, we did not detect NO peaks in our spectrum. As mentioned in the response for comment No. 3 above, we modified the Y axis of the graph.

7. Table 1: It is very strange that there is absolutely no detection of nitrite even for the longest treatment time. How long after the treatment were the measurement effectuated? The Authors should try to compare their results with the literature and try at least to formulate some hypothesis explaining this peculiar trend.

> Thank you for pointing out this issue. After we received your comment about nitrite, we communicated with our group and performed the experiment again. We made a mistake in the nitrite measurement because of the delayed retention time in the Ion Chromatography. We have repeated the measurement with more replicates and confirmed that nitrite was present in the PAW treatments. We changed the manuscript to reflect that a relatively small amount of nitrite is present in the PAW. We have repeated and confirmed the measurement with 3 replicates. We modified the data in Table 1. 

8. Section 2.1: The 0.1 N and 1 N KOH solutions were added only to the PAW cases? Do the Author evaluated the possible influence of these solutions with an appropriate control? Was the osmolality of control and PAW solution comparable?

> Thank you for this comment. The pH adjustment was performed in both the DW and PAW. We did not evaluate the effects of the addition of KOH into the medium because this practice is routinely performed in basic plant growth media preparation, especially for MS (Murashige and Skoog) media (Murashige and Skoog, 1962).The addition of KOH in the solution was considered a small amount that would not significantly affect plant growth. We agree that further study of the osmolality effect on plasma-activated water will show its effect on plants in detail.

9. Figure 2: The Authors should add to Figure 2 the data at day 5 that they show in Table 5. This would allow to better understand the growth trend.

> Thank you for the suggestion. We have updated the graph in Figure 2 with data at day 5 after planting.

10. Section 4: The time lapse between PAW production and nitrate measurement should be in the M&M section, not here.

> Thank you for the suggestion. We have moved and modified these details to the materials and methods section as recommended.

11. Control: The Author hypothesis is that nitrate plays a key role in mediating the effect of PAW on seeds. To prove this it would be enough to compare PAW results with that obtained by a nitrate enriched solution produced by chemical means (no plasma). Knowing the nitrate concentration in PAW that should be relatively easy. In the absence of this control, the authors should try to compare their results with literature works using solutions of similar nitrate concentration. In this sense reference 32 is certainly very interesting but is only very briefly cited in the text. Quickly comparing the concentration reported in the reference and those used in the work the trends appears to generally agree. An extended comparison would greatly benefit the discussion section.

> This is a very interesting point! Indeed, our hypothesis is mainly that nitrate is responsible for the effect of the PAW treatment on root growth in Arabidopsis. There are many well established studies related to the effect of nitrogen sources on Arabidopsis growth. For example, in Boer et al. 2020, they describe the root plasticity in response to the available nitrogen source in the agar media, in which the different concentrations of nitrate affect its root development. As suggested, we now added more discussion about how the nitrate concentration is related to our present work as shown below (Page 12, Line 421-427). In addition, in this paper, we focused on the plasma treatment time of the PAW and its effect on the regulation of root growth. Currently, we are preparing a more detailed experiment using a nitrate enriched solution, a chemical base, and the PAW for comparison. We would like to understand the molecular regulation of the PAW-mediated root growth in DW, PAW and chemical based treatment (KNO₃) using RNA sequencing and now performing this experiment. The hope is that the results will be in our next publication.

 

“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 architecture 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 increment 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]”.

 

Reference for Reviewer #2:

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.

Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. - Physiol. Plant.15: 473–497, 1962.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Review of the article
"Plasma-activated water modulates root hair cell density via root developmental genes in Arabidopsis thaliana L"

The article investigates the effect of plasma-activated water (PAW) on the development of the root system of Arabidopsis thaliana L. The authors use several PAW samples prepared with a cold atmospheric plasma (CAP) generator based on dielectric barrier discharge (DBD). For each of the five samples of plasma-activated water and for the control sample (deionized water), pH, electrical conductivity, concentration of nitrite NO2-, nitrate NO3- and hydrogen peroxide H2O2 were measured. The effect of the concentration of NO3- in PAW on the length of the Arabidopsis thaliana L. root system, the length and density of the root hairs was studied. An analysis of gene expression was also carried out 15 days after the treatment of the planting material and it was found that PAW modulates the density and length of root hairs through the expression of genes COB9, XTH9 and XTH17, and PAW, activated by cold plasma for 5 minutes (PAW5), demonstrates the best among other samples have the ability to express genes COB9, XTH9 and XTH17. PAW prepared for 19 and 40 minutes (PAW19 and PAW40), in contrast, promotes the repression of these genes.
The methods used in the work are tested and well described in the literature. The correctness of their application and the correctness of the results are beyond doubt. The results obtained in the course of the experiments are of interest from the point of view of the introduction of the use of PAW in agriculture. The novelty of this study lies in the establishment of a relationship between the duration of PAW treatment and the expression of genes responsible for the development of the root system of Arabidopsis thaliana L., however, these results were not fully explained in the article. Two key points remain open:
1. Why PAW19 and PAW40 cause repression of the COB9, XTH9 and XTH17 genes? And also, where is the border between expression and repression of these genes in terms of the concentration of NO3 and other RONS?
2. What is the “ideal” concentration of NO3- (and other RONS) to ensure the most favorable conditions for the development of the root system of Arabidopsis thaliana L. PAW5 turned out to be the most effective of the given set of samples, but this does not mean that it is “ideal”, as stated by the authors ("The ideal condition for Arabidopsis root development was identified as PAW5 <...>").

The lack of answers to these questions reduces the significance of this article. However, the authors plan to provide answers in future publications. I believe that the presented results will be of interest to specialists involved in the study of the effects of CAP and PAW on biological objects, and the manuscript can be published after minor corrections.

1. I believe that the first two paragraphs of the introduction should be revised or deleted: lines 38-66. The authors make an attempt to describe what plasma is and in what ways it can be created, but they do this at an insufficiently high scientific level and admit a number of inaccuracies.
a) So, for example, any ionized state of matter is called plasma, although it is important to observe the condition of quasi-neutrality.
b) "There are radio frequency (RF) plasma torches with high energy discharge and an atmospheric pressure plasma jet (APPJ) with low energy discharge, and low temperature plasma torches." (lines 57-59)
High or low discharge energy is primarily determined by the amount of current flowing, and it can vary (if necessary) in any of the listed types of plasma sources.
с) "Because SDBD uses atmospheric pressure, it has the advantage of reducing maintenance costs by not using helium or argon [5]". (lines 63-64)
In this sentence, apparently, there was a typo, which led to an incorrect statement of the causal relationship. All CAP sources reviewed can operate at atmospheric pressure.
d). I believe that in the introduction you should cite articles 10.3389 / fphy.2020.614684; 10.3389 / fphy.2020.618320; 10.3390 / app10248971

2. A more detailed description of the CAP source used for sample preparation should be described. The article does not provide a diagram of the installation and its energy characteristics, as a result of which such a key parameter as the duration of PAW processing loses its physical meaning. Without a description of the source of the CAP or reference to an earlier description, sample preparation becomes non-reproducible. Also, the setup diagram would help clarify how the discharge glow spectra shown in Figure 1 were obtained.

Author Response

 

Response to Reviewer 3 Comments

"Plasma-activated water modulates root hair cell density via root developmental genes in Arabidopsis thaliana L"

The article investigates the effect of plasma-activated water (PAW) on the development of the root system of Arabidopsis thaliana L. The authors use several PAW samples prepared with a cold atmospheric plasma (CAP) generator based on dielectric barrier discharge (DBD). For each of the five samples of plasma-activated water and for the control sample (deionized water), pH, electrical conductivity, concentration of nitrite NO2-, nitrate NO3- and hydrogen peroxide H2O2 were measured. The effect of the concentration of NO3- in PAW on the length of the Arabidopsis thaliana L. root system, the length and density of the root hairs was studied. An analysis of gene expression was also carried out 15 days after the treatment of the planting material and it was found that PAW modulates the density and length of root hairs through the expression of genes COB9, XTH9 and XTH17, and PAW, activated by cold plasma for 5 minutes (PAW5), demonstrates the best among other samples have the ability to express genes COB9, XTH9 and XTH17. PAW prepared for 19 and 40 minutes (PAW19 and PAW40), in contrast, promotes the repression of these genes.

The methods used in the work are tested and well described in the literature. The correctness of their application and the correctness of the results are beyond doubt. The results obtained in the course of the experiments are of interest from the point of view of the introduction of the use of PAW in agriculture. The novelty of this study lies in the establishment of a relationship between the duration of PAW treatment and the expression of genes responsible for the development of the root system of Arabidopsis thaliana L., however, these results were not fully explained in the article. Two key points remain open:

First, we would like to thank Reviewer #3 for the kind words about the importance of this study which could establish the relationship between PAW treatment and gene expression in Arabidopsis roots. Your comments really helped us to improve and present our research in a better way. We have revised our manuscript and answer of the reviewer’s comments as follows.

1. Why PAW19 and PAW40 cause repression of the COB9, XTH9 and XTH17 genes? And also, where is the border between expression and repression of these genes in terms of the concentration of NO3 and other RONS?

> Based on the root phenotype, the root length was dramatically decreased with the PAW19 and 40 treatments during the developmental stages. And we thought that target genes might be inactivated or downregulated in the PAW19 and PAW40 treatment conditions. The suppression of these genes with PAW19 and 40 was due to excessive amounts of nitrate in the plants. An excessive amount of nitrate is known to have negative effects on root formation, including the main/seminal root, lateral root, and root hairs. In addition, nitrate also functions as a signaling molecule to regulate downstream gene expression. In our revised manuscript, we added this part to the discussion with a relevant reference paper (Page 12, 421-426). In the case of COBL9, XTH9 and XTH17, they have an important role in cell initiation and cell expansion. In our phenotypic data (Figure 5 and Table 5), the cell expansion in PAW19 was severely reduced compared to PAW5 and DW. Thus, this reduced expansion is correlated with a lower expression of these XTH genes (Line 441-443).

The line between expression and repression is a very interesting point. Based on our recent study, we would say that the line is somewhere between the PAW12 and PAW19 condition. With the PAW12 treatment, the root phenotype is elongated, and it is inhibited with the PAW19 treatment. And so, the expression and repression of genes might be consistent with the elongated and inhibited phenotypes. Further study will be needed to find the exact point between expression and repression of these genes by nitrate concentration, and the whole gene expression profile will need to be examined by transcriptome analysis. We are now performing RNA sequencing. The hope is that the results will be in our next publication.

 

2. What is the “ideal” concentration of NO3- (and other RONS) to ensure the most favorable conditions for the development of the root system of Arabidopsis thaliana L. PAW5 turned out to be the most effective of the given set of samples, but this does not mean that it is “ideal”, as stated by the authors ("The ideal condition for Arabidopsis root development was identified as PAW5 <...>").

The lack of answers to these questions reduces the significance of this article. However, the authors plan to provide answers in future publications. I believe that the presented results will be of interest to specialists involved in the study of the effects of CAP and PAW on biological objects, and the manuscript can be published after minor corrections.

>Thank you for this good comment. After considering the comments, we agree to change the term “ideal” condition in the manuscript. We changed the term ideal condition to maximal observed condition. This term was originally intended to mean that among the treatments that we performed, the PAW5 treatment is the most suitable condition to increase Arabidopsis growth in this experiment. As for the “maximal observed” concentration of NO₃-, 25 mg/L is the best condition for root growth at the early developmental stage. It is consistent with previous studies on Arabidopsis showing that a nitrate concentration from 0.1 to 1 mM could induce root development in Arabidopsis (Zhang et al., 2000). We have added these discussions to our revised manuscript as shown below (Page 12, line 421-426).

“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 architecture 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 increment 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]”

 

Line 31-34

“Furthermore, we found that the root hair density and length at PAW5 in maximal observed conditions were positively regulated by root developmental related genes including COBRA-LIKE9 (COBL9), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (XTH9), XTH17, AUXIN1 (AUX1) and LIKE-AUXIN (LAX3).”

Line 447-452

“….the maximal observed 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 condition affected the root growth by positively regulating the expression of XTH9 and XTH17 which its expression may mainly cause by the root cell expansion. Moreover, as showed in the Figure 5 and Table 5, the cell expansion of PAW19 was severely reduced, thus corelate with lower expression of XTH genes.”

Line 467-469

“The maximal observed condition for Arabidopsis root development was identified as PAW5 in which potential target genes were up-regulated while root development was suppressed by the PAW19 treatment condition”.

 

 

1. I believe that the first two paragraphs of the introduction should be revised or deleted: lines 38-66. The authors make an attempt to describe what plasma is and in what ways it can be created, but they do this at an insufficiently high scientific level and admit a number of inaccuracies.
2. a) So, for example, any ionized state of matter is called plasma, although it is important to observe the condition of quasi-neutrality.
3. b) "There are radio frequency (RF) plasma torches with high energy discharge and an atmospheric pressure plasma jet (APPJ) with low energy discharge, and low temperature plasma torches." (lines 57-59)

High or low discharge energy is primarily determined by the amount of current flowing, and it can vary (if necessary) in any of the listed types of plasma sources.

с) "Because SDBD uses atmospheric pressure, it has the advantage of reducing maintenance costs by not using helium or argon [5]". (lines 63-64)

In this sentence, apparently, there was a typo, which led to an incorrect statement of the causal relationship. All CAP sources reviewed can operate at atmospheric pressure.

> We agree that the first two paragraphs of the introduction have several inaccuracies; therefore, we decided to remove this part as suggested by the other reviewers. The revised manuscript now contains a focused introduction about plasma technology applications and background from other studies.

d). I believe that in the introduction you should cite articles 10.3389 / fphy.2020.614684; 10.3389 / fphy.2020.618320; 10.3390 / app10248971

> Thank you for the suggestion. Indeed, your suggested papers are relevant to our topics. We have added the references in our revised manuscript.

 

2. A more detailed description of the CAP source used for sample preparation should be described. The article does not provide a diagram of the installation and its energy characteristics, as a result of which such a key parameter as the duration of PAW processing loses its physical meaning. Without a description of the source of the CAP or reference to an earlier description, sample preparation becomes non-reproducible. Also, the setup diagram would help clarify how the discharge glow spectra shown in Figure 1 were obtained.

> Thank you for the comment. Indeed, we agree that a diagram of the installation and energy characteristics is important to describe the sample preparation to make the experiment reproducible. In the previous version of manuscript, especially in the materials and methods section, we did not explain these in detail. This current setup for the generation of the PAW was published in our previous publication, in which a detailed diagram and the power settings are provided. Thus, in our revised manuscript, we provide more details and a reference to our previous paper so that the readers can further check and confirm the diagram, configuration and settings as shown below (Page 2, line 82-94).

“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 are made up of two parallel metals, one of which is covered with a dielectric layer, and the plasma is generated using alternating current (AC) flowing through the electrodes. The electrode consisted of a 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 amplitude 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 a 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 1.5 - 2.0 cm. The OES was obtained with an integration time of 1 second which was averaged 50 times”.

 

 

Reference for reviewer #3:

Hanma Zhang, Brian G. Forde, Regulation of Arabidopsis root development by nitrate availability, Journal of Experimental Botany, Volume 51, Issue 342, January 2000, Pages 51–59, https://doi.org/10.1093/jexbot/51.342.51

Zhang, H. Regulation of Arabidopsis Root Development by Nitrate Availability. J. Exp. Bot. 2000, 51, 51–59, doi:10.1093/jexbot/51.342.51.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript greatly improved and it is worth noting to be mentioned that the authors did additional experiments for filling up missing data! All sections are now presented, written and discussed in a proper way. The addition of further information to manuscript sections 1-3, to the tables and figures as well as the extended discussion (section 4) significantly improves the quality and the soundness of the study.

One comment is on the usage of the abbreviation for “minutes”. Instead of “min.”, the SI nomenclature “min” should be used. This should be discussed with the journal.

Reviewer 2 Report

The Authors succesfully addressed all comments. The manuscript deserve publication in the present form.

Reviewer 3 Report

The authors have significantly improved the text of the manuscript.