Foliar Application of Melatonin Improves Photosynthesis and Secondary Metabolism in Chenopodium quinoa Willd. Seedlings Under High-Temperature Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I suggest to include at line 124 the mainly steps used for chlorophyll and carotenoids measurement

Line 140 “stored quinoa leaves” how the laves were stored?

Line 149 and line 155 wat was the concentration range used for calibration?

Line 157 Fresh leaves?

Line 156- The method used was the authors own method? The same for line 168

Line 357- Correlation analysis of the photosynthetic parameters- very useful

 

Author Response

Responses to reviewer can be seen in the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction: This section is well-written and provides a comprehensive summary of the relevant literature related to the topic.

Material and Methods: This section is informative and detailed. At the same time, I have a few questions and remarks:

Lines 97–99: My question related to this sentence is the following: How did the authors ensure that melatonin solutions of different concentrations were applied in equal amounts in every treatment and to each test plant?

Lines 123–125: The methodology used for measuring or determining all examined parameters (such as anthocyanin, polyphenols, flavonoid content, PPO activity, saponin content) is described in great detail. However, in the case of photosynthetic pigment content, only a literature reference is provided. While this is not necessarily a major issue, in my opinion, it would be more consistent and effective if this method were also described in detail.

Results: The Results section is also well-written and well-structured. I have a few comments here as well:

• Since the evaluations are based on significant differences, it would be advisable to indicate below each table and figure that different letters denote statistically significant differences. Or something similar: “The letters (a, b, c...) indicate statistically different groups for each treatment.”
• Some figures have frames or partial frames, which should be removed for consistency.
• Line 297: The authors state that "...but a significant decrease was observed in RQ-MT100." However, based on the figure, it seems to me that an increase was observed. This should be double-checked and revised if necessary.
• Lines 339–355: In my opinion, the interpretation of the correlation analysis is, in several respects, not accurate. The relationship between Pn and WUE is very weak, and therefore, it should not be highlighted. Similarly, only weak or very weak correlations exist between Pn and Chl a/b, as well as PPO activity. The correlations between anthocyanin content and Chl a, Chl b, carotenoids, and Chl(a+b) also do not appear to be significant. Furthermore, WUE does not seem to show a positive correlation with carotenoids. In my assessment, the relationship between saponin content and Pn, WUE, PPO activity, and flavonoid content is negative, not positive. This part of the manuscript, therefore, needs to be reconsidered thoroughly. It is worth noting that a strong correlation is generally considered to fall within the range of ±0.8 to ±1.0. In the current analysis, values in this range are only found among Chl a, Chl b, carotenoids, and Chl(a+b). Relatively strong correlations can also be observed between: Pn and Gs (0.74), Pn and Tr (0.66), Gs and Tr (0.97), Pn and flavonoids (0.66). However, in most cases, the correlation coefficients are very low – for instance, Pn and Chl a/b: 0.35, Pn and PPO: 0.54 – which indicates only a negligible or virtually no meaningful relationship. This is true for most of the examined parameters. For the reasons above, I strongly recommend reconsidering and revising the interpretation of the correlation analysis.

Discussion: The section is well-written, and the references used are relevant, closely related to the research topic, and up-to-date.

Author Response

Responses to the reviewer can be seen in the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Title:

• My opinion is that the title must declare results. ‘Effects’ and ‘Impacts’ are words that do not define your work, only methods. Please reformulate your title including what was the most important result. Melatonin helps what????
• The scientific authority (Willd.) has to be written in regular letters.
• Why seedlings in uppercase letters?

Abstract:

• You can delete the first phrase of your abstract because you did not investigate the diet.
• Use the verbs in the past for all that considered your work. The work aimed, not aims… Please, apply in the whole manuscript.
• Where the experiment was executed, where the temperatures were set and why?
• Never use dots to separate parts of units here and in the whole text, for example

 μmol·L^-1 must be μmol L^-1

• First define Pn (never write like that, but A or P_n), Gs (never write like this, please write g_s) and Tr (idem E or T_r).
• In the case of gas exchanges are not levels but rates.
• Lines 27-28: Does it your main discovery? If yes, use it in the title

Keywords:

• Please, use the parameters from the experiment and results and do not repeat the terms from the title.

Introduction:

• Lines 37-39: Improve the phrase, not finished.
• Line 53: Here you can introduce the abbreviated terms for leaf gas exchanges.
• Line 58: flavonoids and anthocyanins are also pigments.
• Line 61: What is Jingle?
• Lines 66-67: Not clear, with melatonin, or simply increasing yield?
• Line 68: However – please delete and never start the phrases with words that link phrases. Paragraph has autonomy bringing a new idea.
• Lines 68-70: What are general responses to melatonin in other species than quinoa? Which concentration/doses were applied? Please, introduce known about the substance effects on other species and based on that give hypotheses that are missing in your manuscript.
• Lines 70-76: Completely falling from the sky. Nothing was introduced. Please, improve.

Material and Methods:

• Line 98: When did you start with spaying? You sprayed for 22 days started from?????
• Table 1 and all tables and figures must have the captions complete and well descriptive, what is the content. For example, in the Table 1 is missing what is RK, BK, or WQ, what are such temperatures (day/night) and what is MT. Take that guide for the whole manuscript.
• Lines 107-110: They have to be in the next subchapter, and the next. Please, organize…
• Line 114: Why did you use 800 µmol of CO2???? Today is 420???? Are you sure that you know what you are talking about?
• How did you use 4°C of the chamber to measure the photosynthesis at 35°C??? Those are ´perturbing situations.
• Lines 117-120: Please, read new papers and improve the abbreviations.
• Line 120: The PPFD of 180 µmol m^-2 s^-1 is extremely low!
• Line 126: Give a description of the spectrometer, not at line 131.
• Line 148: What was determined?
• Line 155: What is the meaning of rutin content? Pattern?
• Line 169: Please, give a reference.
• Previously explain well when you collected leaves and which leaves were collected for the biochemical analyses.
• Statistical analysis must start with the exact number of repetitions for each kind of is. You had 3, 5 and 6 repetitions!
• Didi you analyzed data using ANOVA before the Duncan test? What did you apply, which exact package?
• Give the description of SPSS 20.0.
• Please explain well statistics, because your ‘ms’ is pretty messy and disorganized. Interactions using ANOVA two way??? Explain the levels of each factor. Start with the general and develop to more specific.
• What is type III of SS? SS troupes in the II World War????????? Do you know that you MUST be scientific and search for types, explain them and please, do not offer such bad, bad statistics as here. Please, ask for help from a statistician.
• In results you had the statistical impact of replacates. That’s inadmissible.

Results:

• Repetition of the Table 1???
• Always, in every paragraph, indicate where data are. Terrible structure.
• In Table 2 give all necessary details. -1 is written ^-1.
• Statistics in Table 2 is wrong. YOU CANNOT compare all treatments as one-way ANOVA (letters up to infinity), because you had two factors and their interactions. So, compare all levels of MT in each cultivar, and all levels of cultivars in each MT level.
• Lines 189-336: Wrong statistics. You cannot use two-way ANOVA and compare it with the Duncan test as it was one way ANOVA. Serious error.
• At all paragraphs the reference of Table/Figure is missing.
• Lines 337-358: Correlations and their constructions (all values, means) had not been explained in the M&M!

Discussion:

• Not useful to read. Leaf gas exchanges were analyzed in the very wrong way (800 ppm of CO₂ and 4°C when you worked heat effect!!!!), and all statistical analyses are also wrong. So, what discussion can you give???
• Start with the novelty of your work, not some photosynthesis under what?
• Use the verbs in the past for your data, please… All published data (Line 378) MUST be present.
• Line 367 and all others: Use unpersonal reference citation. Only statements, not history about authors.
• Line 389: What is Peak?????
• Lines 402-404: You had to hypothesize in Introduction. Here you are explaining…
• The discussion must be written by basic form where each paragraph supports one idea that is indicated in the beginning of the paragraph, developed and compared to your data and finished with some conclusion about the subject of the paragraph.

Conclusions:

Here you must respond to your hypotheses, not repeat the results. Please reformulate and try to write in one concise paragraph. You can recommend at the end, but only based on your results, not something that you imagine that can be in the future.

Details are in the pdf file

Comments for author File: Comments.pdf

Author Response

Response to the reviewer can be seen in the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did not improve the statistics. They are committing a crime in science, because they declared that they made one-way ANOVA and they had two factors and interactions between them (shown in Tables!!!!). Telling ‘not true’ only to calm a reviewer... Inadmissible.

In One-way ANOVA does not exist two factors, only one!!!!!!!!!!!!!!!!

The statistics had not improved.  Authors declare one-way ANOVA in M&M, because I commented that their comparisons are one-way ANOVA (all data was compared with all). In tables of Results they are showing two-way ANOVA and interactions, but the comparisons continue as one-way. In Results and in Discussion they are talking about each factor separately, which is the issue of two-way ANOVA, and they are showing two-way ANOVA in Tables 3-7 with  comparison everywhere as one way ANOVA (Table 2 and figures). The essential was to work on their results and discussion. They improved hypotheses and Introduction, as I asked, but the essential is still not OK. I am working a lot with statisticians and they are teaching me about such rigor. Also, I asked that authors describe how they make correlations. 

The authors continue to declare that CO2 concentration was elevated (500, that's how the concentration will be in 80 years, they made very complex confusion as they never do not have the basic knowledge about gas-exchanges), and the temperature was 4 ºC in one reference about climate changes and low temperatures. They are additionally set at 21.5 °C, that's inadmissible for leaf gas-exchanges.at elevated temperatures, as declared here! 35 °C is 35 °C, not 4 °C or 21.5 °C.

In Conclusions they MUST respond to their hypotheses, not to make a list, in lines, as PowerPoint presentation, of results.

Comments for author File: Comments.pdf

Author Response

Response to reviewer can be seen in the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

You did not improve leaf gas exchange methodology. Your manuscript worked with climate changes and 500 μmol of CO₂. That's not OK.

Your temperature was 21.5° C working with 25° C and 35° C as your levels (lines 154-156). So, error. What was your PPFD?  500 maybe was the internal flux during measurements? Please, look at your data, please take care, you are wrong in many points.

Your statistics did not make the differences of MT level in each variety (for example lowercase letters) and among varieties (uppercase letters for each mean), or between temperatures (third sign).

In figure captions describe the whole content, without any note. Read good papers, please.

Comments for author File: Comments.pdf

Author Response

Reply to the review report can be seen in the attachment.

Author Response File: Author Response.pdf

Round 4

Reviewer 3 Report

Comments and Suggestions for Authors

I am pleased with the great improvements of the manuscript. I would recommend that authors delete : "... and the means values did not shower in Figure" They showed mean values! And as the means differ among factors, normally are used the estimated means of all factors used in the study (for example mean (MT|temperature).

The statistics and methods are still problematic, because authors included MT treatment in heat treatment, without control of the heat without MT. That's highly wrong and any good journal would accept such wrong methodology. But, the work was done, and I would not stop it if editors would permit.

Comments for author File: Comments.pdf

Author Response

Response of point by point reply can be seen in the attachment.  

Author Response File: Author Response.pdf