Effects of Soil Application of Chitosan and Foliar Melatonin on Growth, Photosynthesis, and Heavy Metals Accumulation in Wheat Growing on Wastewater Polluted Soil

Round 1

Reviewer 1 Report

 

The authors studied effects of soil amedments, such as chitosan and melatonin on wheat heavy metal concentrations in roots and shoots, growth parameters, protein, fat, carbohydrates, fiber, phytate, polyphenols content in grain as well as their effects on photosinthesis and chlorophyll content. The writing, clarity and english language in the manuscript are correct and satisfactory. Generally, MS is well-written. There are only minor corrections. I gave some suggestions in the Annotated Manuscript (pdf version).

 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We would like to thank the Reviewer 1 for the evaluation of this manuscript. We are grateful for the detailed comments, which helped us improving the manuscript. All minor corrections were provided to the text of the manuscript eg. we corrected the subtitles, we changed the order of the subtitles sections, we added more detailed methodology of the grain biochemistry as well as we added the statistical analysis section to the text of this manuscript. All the changes were implemented using Track Changes” function. We hope that updated version of our manuscript will be accepted by the Reviewer.

With best regards,

Authors

Reviewer 2 Report

Dear authors,

I received the revised form of the manuscript and analyzed this version, some observations were argued in the letter to the review without making any changes to the text. I understood that these cannot be performed at this stage in the research and that they will be parts of analysis in your future research.

The current form of the manuscript is consistent and presents information of interest to the scientific community in the field of environmental sciences.

Also, I recommend to analyze some relevant studies for future research:

·         Melatonin Stimulates Activities and Expression Level of Antioxidant Enzymes and Preserves Functionality of Photosynthetic Apparatus in Hickory Plants (Carya cathayensis Sarg.) under PEG-Promoted Drought, October 2019, Agronomy 9(11):702, DOI: 10.3390/agronomy9110702.

·         Effect of Wastewater Irrigation on Photosynthesis, Growth, and Anatomical Features of Two Wheat Cultivars (Triticum aestivum L.), February 2020Water 12(2):607, DOI: 10.3390/w12020607.

·         Melatonin Modulates Plant Tolerance to Heavy Metal Stress: Morphological Responses to Molecular Mechanisms, Int J Mol Sci. 2021 Nov; 22(21): 11445, doi: 10.3390/ijms222111445

Best regards!

Author Response

Dear Reviewer,

We would like to thank the Reviewer 2 for understanding and acceptation our answers, which we send in previous resposnes. We also appreciate for the articles recommendation, which we already added to the text. Your advice in each step of the manuscript revision not only helped to improve the text but also will help us in our future research. Thank you very much.

With best regards,

Authors

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors studied effects of soil amedments, such as chitosan and melatonin on wheat heavy metal concentrations in roots and shoots, growth parameters, protein, fat, carbohydrates, fiber, phytate, polyphenols content in grain as well as their effects on photosinthesis and chlorophyll content. The writing, clarity and english language in the manuscript are correct and satisfactory. Generally, MS is well-written. There is only minor corrections. I gave some suggestions in the Annotated Manuscript (pdf version).

Comments for author File: Comments.pdf

Author Response

We would like to thank the Reviewer 1 for the evaluation of this manuscript and we are grateful for the detailed comments, which helped us improving the manuscript. All minor corrections were provided to the text of the manuscript eg. additional keywords, rephrase subtitles and some sentences, insertion of the chlorophyll abbreviation and more. Additionally, we added the methodology of chlorophyll determination. We hope that updated version of our manuscript will be accepted by the Reviewer.

Reviewer 2 Report

Comments and Suggestions for authors

The present study Effects of soil application of chitosan and foliar melatonin on growth, photosynthesis, and heavy metals accumulation in wheat growing on wastewater impacted soil, deals with evaluation the efficiency of exogenous application of MT and chitosan and other soil amendments at wheat under heavy metal stress conditions, and to further explore the heavy metals (HMs) accumulation, growth, nutritional quality and photosynthesis in wheat.

The manuscript addresses a very current and interesting topic of research relating to the presence of metal ions in soil and bioconcentration in plant tissues.

I may recommend to the authors to determine certain physiological parameters of the plant such as carotenoid pigments, proline, enzymatic activity etc. which could provide data about the HM stress mechanism.

However, I have some comments, which are mainly associated with the methodological part and the structure of the manuscript. In my opinion the methods should be explained more in detail.

My comments and suggestions are listed below:

• I recommend to change impacted soil with “polluted soil” (title section)
• Line 93-95, must be removed;
• I suggest firstly to describe the Material and methods and after that the Results, line 311-377 should be introduced after Introduction.
• Line 328 – provide tradename;
• There are some errors in the numbering of the subsections – line 175, 180, 219, 310-311...
• Section 2.1. The determination chemical methods must be described in details not only mentioned.
• I suggest to use carotenoids and proline as indicator of plant stress induced by HM or enzymatic activity.
• Line 187-218 are general considerations not results and should be introduced in the Introduction.
• Section 5 – Conclusions:  I suggest to emphasize better the practical importance of the study and the results obtained in your analysis.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

This is an interesting and complex study under real soil conditions with several additional treatments. Mainly the use of melatonin and combined treatments with it are fine. I have, however, several essential comments for revision:

- where the cultivation was carried out? It is unclear from methods (lab or greenhouse? PAR and other parameters?)

- some treatments considerably affected growth based on the provided data in table 1, I would ask to add a photo of the experimental plants to show the impact on visible phenotype. THIS IS A MANDATORY REQUEST and I hope that photo of plants is routine evidence for each study, mainly if growth is enhanced   

- you used 5 wheat seeds per pot, then growth data in table 1 would be better per plant, not per pot

- plant-available metals in the fig. 1 show columns with very narrow SE, were results from individual pots really such homogenous? I do not have similar experiences

- fig. 4 is the most important outcome of the study but individual graphs are rather chaotic. PLEASE USE SIMPLE COLUMNS for shoot/root/grains with various colors. ALTERNATIVELY, USE A TABLE TO PRESENT THESE DATA, WHICH WILL BE EASILY AVAILABLE FOR READERS AND CITABLE

- chlorophyll content in leaves seems to be low and a/b ratio is low too, verify measurement and calculation. Which leaves were used for analyses of photosynthetic parameters? Data in fig. 3 are again very homogenous  

- comparison of heavy metal uptake by other crops and species under soil cultivation could increase the outcome, I recommend e.g.:

https://pubs.acs.org/doi/10.1021/jf5010176

https://www.sciencedirect.com/science/article/abs/pii/S0981942818304777

https://www.sciencedirect.com/science/article/abs/pii/S0269749116307631

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Authors provided replies but no significant changes. Based on the photo showing plants of rather low vitality, I have doubts about strong homogeneity of all results as previously mentioned and I tested many crops where such homogeneity was not observed. Growth changes are not clearly visible from the photo and changes of metal contents are all in the same trend and thus expectable. Several independent repetitions of the whole study must be done. At the same time, addition of 3% of exogenous material has no practical usability. Actually, much lower doses are tested (for any compound) to mimic real agriculture. Authors report both 0.005 M and 0.5 mM DTPA, this is not the same unit. Application of ML and duration of the experiment is unclear. If you state that “Our experiment is not about boosting the agronomic parameters of wheat under the influence of treatments”, it is then question what the aim of study is.