Variations in Plant Growth Characteristics Due to Oxygen Plasma Irradiation on Leaf and Seed

Round 1

Reviewer 1 Report

The manuscript submitted by Hayashi and Yamamoto deals with the study of the effect of chemically active species produced by low-temperature on plant grow characteristics. The topic addressed in the work is relevant for the Agronomy journal. The article has a strongly interdisciplinary character and contains a significant part dealing with the formation of chemically reactive particles by means of electric discharges. I can only comment on this part. In the current state, the part of the text that focuses on electric discharge requires several corrections and amendments.

Comments:

1. Page 2, lines 46-61: Please add few more details concerning low-pressure setup, such as discharge power and O2 flow. Add also some info on your spectrometric setup.

2. Page 2, lines 62-75: As described, it seems that authors use surface DBD in coaxial geometry with forced axial flow of working gas. In my opinion, the authors should avoid using ‘torch-type’ simply because at the exit of the DBD there is no plasma by only discharge products such as O3 or NxOy . Please also add information on the determination of the discharge power

3. I would suggest that the authors, after describing the two discharge setups, make explicit and clear statements on nature of the treatment. In the case of the low-pressure setup, the seeds interact directly with oxygen plasma and most important interaction mechanisms include surface etching and irradiation by VUV-UV photons On the other hand, in the case of atmospheric-pressure setup, there in absolutely no direct contact with DBDS plasma or with VUV-UV photons, and plants/leafs interact only with effluents containing RONS! See the classification of the treatment approaches in 10.1140/epjd/s10053-021-00206-4 ... Clear classification of the treatment type might be important for your followers!

4. Page 3, l. 87-88: please add few more details. Specify what means ‘active oxygen’!

5. Figure 3 caption and related text: please improve description of this figure (integration time, slit width, how and from where did you collect the spectra?).

6. Page 4, lines 130-132: While I agree with the assignment of the 309, 762 and 777 peaks, I have some doubts on your assignment of the 527, 559 and 636 peaks. In my opinion, these and other peaks should be assigned to bands the O2+ ions (see figure 5 in 10.1007/s11090-010-9245-4 ). Please clarify/correct!

7. Page 5, line 137: The excitation energy of the O(3p 5P) state which is responsible for the 777nm emission is NOT 5.1 eV. It is more than 10 eV (see figure 1 in 10.1088/1361-6463/aadcd1 )! Please clarify/correct!

8. Page 4, l. 159: What is transport time of species to reach leave? What do you mean by ‘OH* and O(5P) deactivate?

9. Page 4, lines 163-165: I cannot agree with this statement. Under low pressure conditions, the etching (see 10.1088/1361-6463/aa5c21) and irradiation by VUV and UV cannot be neglected. Please explain better why the O(1D) should dominate …

10. Page 5, l. 197: Leafs are not exposed to plasma using atmospheric-pressure setup … only to O3. Please rephrase.

11. Page 9, l. 332: Leafs are not exposed to plasma using atmospheric-pressure setup … only to O3. Please rephrase.

12. Page 11, l. 387: Leafs are not exposed to plasma using atmospheric-pressure setup … only to O3. Please rephrase.

13. Page 13, l. 437-438: Leafs are not exposed to plasma using atmospheric-pressure setup … only to O3. Please rephrase.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

A brief summary
The manuscript “Variations in Plant Growth Characteristics due to Oxygen 2 Plasma Irradiation on Leaf and Seed” describes the plasma treatments applied on seeds and leaves. The study follows the effects of gases generated in plasma on plant growth. The topic is important to understand the functions and enzymes sensible of type of treatment.
The title fitted the aspects presented and the topic is almost covered in the sections.
Ultimately, this work could be suitable for Agronomy journal, following major revisions relating to the robustness of gene expression evaluation and the statistical analysis.

Specific comments
I have few suggestions regarding the manuscript. some of them are regarding the presentation of the methods,

Comments regarding the aspects that needs major revisions:

1) Which were the methods used for gene expression analysis. The results were well detailed but in order to understand the results at least few words about the methods/equipment used is necessary.

The details from tables are not clary explained in text. For example:

2) Table 1 shows the gene annotations those have changed statistically significantly among expression-variable genes.’ (line 229) but in table 1 are presented only some conclusion. How were determined/observed the variations of expression genes after irradiating? The same question I have for table 4.

3) How was calculate the Enrichment Score and what significance has it in this context? What does mean the other parameters from tables (RT, count, p_value, Bejamini)? (Tables 2, 3, 5, 6). The terms should be explained if even these parameters are common in this type of analysis.

4) Also, you must present the obtained results in text. Which are the limits for that the gene activities are increased, respectively inhibited?

5) How many seeds/plants were used for this research? Are the number of it enough for statistical analysis? Which method was used for statistical analysis? In this type of experiments is mandatory to have a section for statistical analysis in order to be sure that stochastic oscillation was avoid.

Comments regarding the aspects that needs minor revisions:

6) I recommend to use the complete name of plant, ”Arabidopsis thaliana”, in text.

7) The leaf area is only one bioindicator used for evaluation the growth process (line 100). In agriculture/horticulture, the most use bioindicator is the plant height. The growth parameters depend by environmental condition, but I suppose that all plants were maintained in the same conditions.

8) The gas used as dielectric discharge medium is ‘pure oxygen’ (line 55,73), but in figure 2 the input gas in chamber is ‘Ozone’. The term of pure oxygen it is used for molecular oxygen (O2), not for describe the ozone molecules (O3). Please clarify this aspect.

9) The plants were irradiated with the fluorescent light of 3000 Lux. The Arabidopsis thaliana is long day plant, is it enough 3000 lux for optimal growth condition? (line 93)

10) The blue bar columns from images of tables 1 and 4 don’t offer any information. The reader can guess that these bars show the magnitude of one parameter. Moreover, the middle parts of image from those tables are almost empty (tables 1 and 4). You should transcript the values into editable table in order to be able to adjust the columns size (tables 2, 3, 5, 6).

11) In generally, the electron orbital energy levels of atoms (O(s), O(p) and O(d)) are notated with lowercase, not with uppercase as were notated in manuscript. (page 4)

Figures:

13) What represent the dash line and the two arcs of a circle from figure 5?

14) Why you presented in figure 5 only the ozone concentrations into 0-2 min range, if the applied time in experiment were 3 and 30 minutes? The concentration increasing linear till the 30 minutes? I suppose that not.

15) I didn’t find so relevant the sequences from plasma irradiation (lines 315-326 and 384-394)

16) The figure 3 and 4 should be move on page 4.

English or Typos:

• double verbs ”is placed is installed” (line 50)
• repeated text ‘After the irradiation for 30 min, After the irradiation for 30 60 min,’ (line 60)
• ”Discharge voltage and current are measured using the high voltage probe and the Rogowski coil with a oscilloscope, respectively” is unclear sentence (line 70-71)
• There are some sentences where was used coma before and (”, and” 144, 145, 148, 228).
• The sentence from line 144-145 should be rephrased.
• The dot used as symbol for multiply is too big. In text should be used the symbol (line 157)
• The term cultivation period is not properly to describe the vegetative stage.
• The subtitle 3.2 and 3.3 are the same. (page 6)
• (un irradiated) is (unirradiated) (line 210).

Author Response

Please see the attachment.

Author Response File: Author Response.docx