Physiological and Biochemical Properties of Cotton Seedlings in Response to Cu2+ Stress
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe figures and tables are missing.
Also methods should be described in more details (missing instruments used…).
Please, resubmit the manuscript.
In the manuscript entitled " Physiological and biochemical properties of cotton seedlings in response to copper stress" the authors investigated the tolerance mechanism of cotton under copper stress between a hybrid cotton variety (Zhongmian 63) and two parent lines with different Cu2+ concentrations.
I recommend major revision of the manuscript. Major comments:
1. English should be corrected by native English speaker. The paper contains a typo and grammatical errors which should be fixed.
2. Latin names of the plants mentioned should be written in italic.
3. The phrase "copper" is incorrect, as actually a copper salt was dissolved. Hence, this is a solution containing copper ions. To be strict, an oxidation state should also be indicated, e.g. copper(II) or Cu2+.
4. Materials and methods should be described in more details, every technique and instrument used should be mentioned.
For example: 2.4 Determination of copper content “:…. followed by Cu estimation using an atomic absorption spectrophotometer.” Instrument is missing, describe used method.
5. The conclusion part should be separated from the discussion and re-written, Also, the authors should add the potential practical application
Author Response
- The figures and tables are missing
ANSWER: all figures and tables are presented in the manuscript.
- Also methods should be described in more details (missing instruments used…).
ANSWER: methods have described in more details in the revised manuscript.
- Please, resubmit the manuscript
ANSWER: the revised manuscript has been resubmitted.
Major comments:
- English should be corrected by native English speaker. The paper contains a typo and grammatical errors which should be fixed.
Answer：English language has been edited by MDPI. The text has been checked for correct use of grammar and common technical terms in the revised manuscript.
- Latin names of the plants mentioned should be written in italic.
Answer：All are modified in the revised manuscript.
- The phrase "copper" is incorrect, as actually a copper salt was dissolved. Hence, this is a solution containing copper ions. To be strict, an oxidation state should also be indicated, e.g. copper(II) or Cu2+.
Answer：Yes, all Cu are replaced for Cu2+ in the revised manuscript
- Materials and methods should be described in more details, every technique and instrument used should be mentioned.
For example: 2.4 Determination of copper content “:…. followed by Cu estimation using an atomic absorption spectrophotometer.” Instrument is missing, describe used method.
Answer：the section ‘Materials and Methods’ have been detailedly described by the authors, or are cited according to a reference and are properly modified by the authors.
- The conclusion part should be separated from the discussion and re-written, Also, the authors should add the potential practical application
Answer：We modified the format according to the guidance o
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper entiltled ''Physiological and biochemical properties of cotton seedlings in response to copper stress'' was a very interesting paper which dealing with a well known economic crop that is grown all over the world. The paper also investigated the tolerence mechanism of cotton under copper stress between a hybrid cotton variety. The methodology was repetable and the results was well written.
Author Response
- The paper entiltled ''Physiological and biochemical properties of cotton seedlings in response to copper stress'' was a very interesting paper which dealing with a well-known economic crop that is grown all over the world. The paper also investigated the tolerence mechanism of cotton under copper stress between a hybrid cotton variety. The methodology was repetable and the results was well written.
ANSWER: Thank you for your comments.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript is very important in field
But need improved language editing
Where the figures not found in manuscript
Author Response
- The manuscript is very important in field but need improved language editing where the figures not found in manuscript.
ANSWER: English language has been edited by MDPI. The text has been checked for correct use of grammar and common technical terms in the revised manuscript.
Reviewer 4 Report
Comments and Suggestions for AuthorsComments for author File: 
Comments.pdf
Author Response
- In the chapter 'Materials and Methods', protocols are almost always given without specifying who developed them (it is only mentioned in section 2.3). Indicate a reference, or whether it is a protocol developed (or modified) by the authors based on their own experience (R72-89, R90-94, R103-108).
ANSWER: in the section ‘Materials and Methods’, 2.1, 2.2, 2.4, 2.7, 2.8 methods have been detailedly described by the authors, and 2.3, 2.5, 2.6 methods are cited according to a reference and are properly modified by the authors.
- The English language leaves something to be desired, there are many errors, and this makes the reading unclear in places. There is hardly any number matching (verbs in the third person singular without the's' or plural subjects with the verb in the singular). Furthermore, 50% of the bibliography is older than 10 years.
ANSWER: The English language errors have been corrected and edited by MDPI. In addition, and current bibliography has been added in the revised manuscript.
- When inserting a reference as subject or complement in the discourse, indicate at least the first name et al., the year, and then the reference in square brackets: R96 (n.21), R111 (n.22), R114 (n.2), R122 (n.23). For example, in R96: "Chlorophyll and carotenoid concentrations were determined according to Lichtenthhaler et al., 1983 [21] with a few modifications". Otherwise, use as in R125 (No 24).
ANSWER: Improper citations formats are all corrected in the revised manuscript.
- At the plagiarism check, the sentence in R87-88 "the pH value was adjusted to 5.6-5.7 with 0.1 M HCl or 0.1 M NaOH" is plagiarised. In my opinion, it can also remain so, as it is a sentence in the chapter "Materials and Methods" that does not detract from the merit of others, but I have a duty to point this out in the general report and the editor must decide. Also plagiarised is the sentence at R347 "(...) levels of Cu-induced oxidative stress increase H2O2 production in cotton seedlings. On the other hand, when compared with the parent line, the hybrid lines effectively increased (...)' and this must be changed.
ANSWER: Mentioned plagiarism sentences have been modified in the revised manuscript.
- Move the tables to the chapter 'Results' when they are first mentioned: it is better to look for them when reading the discussion than when reading the results
ANSWER: The tables have been moved to the chapter 'Results' and been put in a place where they are first mentioned.
Reviewer 5 Report
Comments and Suggestions for AuthorsThe study investigated how cotton plants cope with high concentrations of copper, which can be toxic to them. The authors looked at a hybrid cotton variety and two parent lines with different levels of copper. They found that increasing copper concentrations reduced growth rates and caused copper accumulation in the roots, stems, and leaves of all three cotton genotypes. However, the hybrid cotton variety, Zhongmian 63, was found to be more tolerant to copper stress compared to the parent lines. Excess copper also affected the cellular redox homeostasis, but the hybrid variety showed an increase in antioxidant enzyme activity. These findings suggest that Zhongmian 63 is a good candidate for planting in copper-contaminated soils.
Overall, the study provides valuable information about how cotton plants cope with copper stress. The study highlights the importance of understanding the molecular mechanisms of plant tolerance to stressors.
1. The study only looked at one hybrid cotton variety and two parent lines. Is there any specific reason the author use this verieties for the study? a few description would give readers better understanding the variety effects.
2. While the findings in the young plants shows significant effect, how is the long-term long-term effects of copper stress on cotton plants? A few introductions or discussion would be good. Is there any plan from the authors to examine the long term effect?
3. The paper could benefit from a more thorough discussion of the implications of the study's findings. For example, how might the findings inform agricultural practices or policy decisions? Because the study limited to the young plants.
4. The authors used hydroponic culture to grow the cotton plants, which is a controlled environment. However, in the field, plants are exposed to many other factors that can influence their growth and physiological responses. A discussion on the limitations of using hydroponic culture and the implications of the study's findings in a field setting could be interesting.
5. Addition to the experimental findings presented in this study, a molecular analysis of gene expression could provide further insight into the mechanisms by which copper stress affects cotton growth and development. The authors may consider exploring the gene expression changes in response to copper stress, which could reveal important regulatory pathways and molecular mechanisms involved in copper stress response in cotton plants. This could further enhance the understanding of the plant's response to copper stress and provide valuable information for future breeding and genetic engineering efforts aimed at developing copper-resistant cotton varieties.
Author Response
- The study only looked at one hybrid cotton variety and two parent lines. Is there any specific reason the author use this verieties for the study? a few description would give readers better understanding the variety effects.
ANWSER: they have been carefully descripted in the section ‘materials and methods’ in the revised manuscript.
- While the findings in the young plants show significant effect, how is the long-term effect of copper stress on cotton plants? A few introductions or discussion would be good. Is there any plan from the authors to examine the long term effect?
ANSWER: the long-term effect of Cu on cotton genotypes is currently under investigation in the laboratory as literatures on this area of studies is limited.
- The paper could benefit from a more thorough discussion of the implications of the study's findings. For example, how might the findings inform agricultural practices or policy decisions? Because the study limited to the young plants.
ANSWER: Thanks for the suggestion. I think this suggestion will be more suitable for a review discussion article, which is currently in progress. However, a few paragraphs on the implications of heavy metals and recommended agricultural policies on the effect of heavy metals on plant seedlings have been added to the discussion section.
- The authors used hydroponic culture to grow the cotton plants, which is a controlled environment. However, in the field, plants are exposed to many other factors that can influence their growth and physiological responses. A discussion on the limitations of using hydroponic culture and the implications of the study's findings in a field setting could be interesting.
ANSWER: Although hydroponic culture was used in this study, it wasn’t the primary aim of this study. Therefore, writing a discussion section on hydroponic culture didn't come to mind. The objective of this study was to observe the responses of cotton seedlings to Cu toxicity, and it required us to have a controlled environment to monitor and regulate the desired Cu amount, unlike an external environment, where it will be difficult to know the exact amount of Cu in the soil. Nonetheless, a few paragraphs on our opinion of hydroponically cultivating cotton seedlings have been added to the discussion section. In addition, this suggestion has been taken into consideration and will be added to our subsequent research.
- Addition to the experimental findings presented in this study, a molecular analysis of gene expression could provide further insight into the mechanisms by which copper stress affects cotton growth and development. The authors may consider exploring the gene expression changes in response to copper stress, which could reveal important regulatory pathways and molecular mechanisms involved in copper stress response in cotton plants. This could further enhance the understanding of the plant's response to copper stress and provide valuable information for future breeding and genetic engineering efforts aimed at developing copper-resistant cotton varieties.
ANSWER: Thank you for this suggestion, and we are considering exploring the transcriptome and metabolome analysis of cotton in response to copper stress, which is currently in progress.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have corrected according to comments but the instruments used for the analysis are still missing. Please add for each measurement.
Author Response
Missing instruments used for the analysis have been added in the revision.
Reviewer 3 Report
Comments and Suggestions for Authorsthe author modified the papper
so i agree
Author Response
Thank you for your comments.
Reviewer 4 Report
Comments and Suggestions for AuthorsComments for author File: Comments.pdf
Author Response
1) In the chapter 'Materials and Methods', protocols are almost always given without specifying who developed them (it is only mentioned in section 2.3). Indicate a reference, or whether it is a protocol developed (or modified) by the authors based on their own experience (R72-89, R90-94, R103-108).
Answer: in the section ‘Materials and Methods’, 2.1, 2.2, do not require any source because these were personally done by the author and it’s well detailed. For instance plant roots and shoots were measured using a ruler. I think this is self-explanatory and a normal measurement that can been easily done, which don’t require any referencing. And in sections 2.4, 2.7 methods are cited according to a reference added in the revised manuscript and are properly modified by the authors.
3) Reviewer's reply: The way of entering references in reported cases is as used in line 107. It should also be corrected in lines 122 (change to "Described by Liu et al. 2018 [28]") and 133 (change to "Method in Zhang et al. [29]").
Answer: These mistakes have been corrected in the revised manuscript.
Specific notes:
Answer: All specific notes mentioned have been carefully corrected in the revised manuscript.
R95-96 Why are the tested concentrations so much higher than the control? I had already suggested in the first comment to you to justify this choice.
Answer: Cu2+ is an essential micronutrient which can meet the needs of plant growth with a concentration of 0.2 μM, but when the content of Cu2+ reach 100 μM, excess Cu2+concentrations shall cause the toxicity.
