Resistances and Physiological Responses of Impatiens uliginosa to Copper Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article sheds light on a new plant species that can be used to remediate Cu contamination in water. However, the reviewer has several concerns about the research design and materials & methods. Please see below.

1. By keeping the seedlings in soil for 50 days and then transferring them into hydroponic solutions may cause many problems. First, washing and cleaning the roots causes significant mechanical stress for the plants. Also, samples from the soil could carry other contaminants that cause microbial growth in the hydroponic solutions, which can induce biotic stress. Both stresses could result in an alteration of the following Cu stress response the authors want to observe. The Cu treatments should either be performed in the soil environment (for soil remediation) or in the hydroponic system with the plants grown in the same systems from the beginning (for water remediation). If there are any specific purposes for the experimental design (e.g., this is the standard water remediation procedure), the authors need to justify it.

2. The concentration of Cu in the solution must be in uM. Grams per liter does not represent the real concentration of Cu in the solution. Especially in the introduction, the authors discussed Cu stress for plants in "uM," not mg/L. In this regard, the authors might want to convert their Cu treatments from 5-25 mg/L to the concentrations of Cu in uM and explain if the plants were grown in the range of Cu toxicity. 

3. The authors must provide more detailed information regarding section 2.4 with at least a brief description of their methods.

4. The authors must provide the number of replicates for statistical analysis for all figures.

5. Since MDA represents oxidative stress response, the authors may consider justifying why they measured MDA in the tissues in section 3, not only in the discussion.

Other comments:

1. Table 3 shows the concentration of all elements the authors analyzed. However, only Cu data need to be presented, considering the authors want to discuss the feasibility of I. uliginosa in Cu remediation.

 

2. The authors may also consider changing the way they present data. The tables are impressive but hard to read or analyze.

Comments on the Quality of English Language

The quality of English is good. This article might need minor edits, though.

Author Response

Comments 1:

Dear reviewer, we appreciate your insightful feedback and meticulous revisions of our manuscript. The remarkable ability of Impatiens uliginosa to thrive in environments with high copper concentrations has piqued the interest of our research team. Consequently, we intend to subject I. uliginosa to copper stress in order to investigate its resistance and physiological responses, with the ultimate goal of offering a novel approach to tackling heavy metal pollution in Dianchi Lake. I. uliginosa, an annual herbaceous plant, thrives in humid environments but is not suited for aquatic growth, flourishing only along streams and riverbanks. In contrast to soil-based cultivation, hydroponic systems offer a more visually intuitive and efficient means of capturing data on the plant's underground components. Hence, we opted to initially conduct experiments in soil culture followed by hydroponic setups to better replicate its natural habitat and observe morphological indicators under stress.

Based on the initial findings of our research group, the development of I. uliginosa typically progresses through three distinct stages: seed germination, seedling growth, and maturation. The seed germination phase generally lasts between 2 to 12 days, while the seedling growth phase is characterized by rapid development but also vulnerability. Toxicological reactions in the seedlings of I. uliginosa start to manifest when the concentration exceeds 5 mg·L⁻¹. To ensure data stability, we opted to extend the cultivation period to 50 days and initiate stress experiments once growth had stabilized. Prior to conducting stress experiments, we meticulously prepared a substantial number of samples to minimize damage during transplantation and promptly replaced any plants that were harmed during the process to reduce interference caused by mechanical damage. Recognizing that plant growth is influenced by various factors, and that external factors may be introduced during transplantation, we chose to employ pure water for cleaning in the experiment. However, mitigating the impact of external factors on test results remains a persistent challenge for our research team. We are committed to further exploration and investigation of this issue.

We hope that the above explanation addresses the teacher's inquiries on this matter. We eagerly anticipate your guidance and response. Thank you.

 

Comments 2:

The concentration of Cu in the solution must be in uM. Grams per liter does not represent the real concentration of Cu in the solution. Especially in the introduction, the authors discussed Cu stress for plants in "uM," not mg/L. In this regard, the authors might want to convert their Cu treatments from 5-25 mg/L to the concentrations of Cu in uM and explain if the plants were grown in the range of Cu toxicity.

Response2:

Dear reviewer, thank you for your thoughtful revisions and insightful feedback on our manuscript. We have thoroughly reviewed and discussed your comments. The average concentration of Cu²⁺ in Dianchi Lake water is 105.32 µg·L^-1, significantly higher than that in non-polluted water bodies, which is 2 µg·L^-1. This high concentration of Cu²⁺ does not hinder the healthy growth of I. uliginosa, which is why we selected this plant as our research subject. Research indicates that copper toxicity levels for most plants fall within the range of 0.19 mg·L⁻¹ to 6.4 mg·L⁻¹. Therefore, we established a concentration range of 0-25 mg·L⁻¹ to investigate the tolerance and resistance of I. uliginosa to copper stress. While we carefully considered your suggestion regarding unit replacement, we found that the converted values were not suitable for the concentration gradient settings typically used in our research. As a result, we opted to maintain the unit as "mg·L⁻¹" in the text. For instance, we converted "between 3 µM and 100 μM" in line 62 to "between 0.19 mg·L⁻¹ and 6.4 mg·L⁻¹". We eagerly await your response and any further suggestions. Thank you.

 

Comments 3:

The authors must provide more detailed information regarding section 2.4 with at least a brief description of their methods.

Response 3:

Dear reviewer, thank you for your feedback. We have included additional details on the test method in Section 2.4 as per your suggestion. Please refer to lines 137-147 of the revised manuscript for review. Thank you.

 

Comments 4:

The authors must provide the number of replicates for statistical analysis for all figures.

Response 4:

Dear reviewer, thank you for your feedback. We have revised the contents of the figures and tables in the study as per your recommendations. The data related to statistical analysis has been properly annotated with the number of replicates.

 

Comments 5:

Since MDA represents oxidative stress response, the authors may consider justifying why they measured MDA in the tissues in section 3, not only in the discussion.

Response 5:

Dear reviewer, thank you for your suggestion. We have incorporated a description of the role of MDA in the first part of the middle section of Section 3.3, as recommended. This addition can be found in lines 238-239 of the revised manuscript. We kindly request your review. Thank you.

 

Comments 6:

Table 3 shows the concentration of all elements the authors analyzed. However, only Cu data need to be presented, considering the authors want to discuss the feasibility of I. uliginosa in Cu remediation.

Response 6:

Dear reviewer, thank you for your insightful feedback. Our design concept for this section aims to illustrate the impact of copper stress on plants by examining the changes in metal elements within I. uliginosa. Throughout the experiment, we were intrigued to observe that all elements, with the exception of copper, remained relatively stable within the body of I. uliginosa., while copper exhibited fluctuations following a discernible pattern. This observation further supports our hypothesis that one of the mechanisms through which I. uliginosa. responds to copper stress is by mitigating damage through the redistribution of copper elements within its body.

 

Comments 7:

The authors may also consider changing the way they present data. The tables are impressive but hard to read or analyze.

Response 7:

Dear reviewer, we have carefully considered your feedback and made revisions to the presentation of data in our manuscript. The original Table 1 has been transformed into a visual representation on pages 5-6, now referred to as Figure 1. As for Tables 2, 3, and 4, we believe it may be more appropriate to keep this data in tabular form. We eagerly await your guidance and response. Thank you for your valuable input.

 

Comments 8:

The quality of English is good. This article might need minor edits, though.

Response 8:

Dear reviewer, hello! We are very honored to receive your recognition. We will continue to revise and improve the article. Thank you.

 

Special thanks to you for your good comments.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, 

I have carefully read your manuscript. To my mind, this paper is interesting and rather well prepared, however, certain parts of it could be improved. 

Please, find my comments in the uploaded manuscript.

To summarize, several places in the abstract, discussion and especially conclusions should be revised. I found some inconsistencies or unclear meanings there. 

Apart from this, pay attention to my suggestion for the title and presentation of plants' growth data. 

There are also some minor shortcomings in the section of Materials and Methods (incomplete information), as well as some mistakes in the text throughout the paper.

 

Kind regards,

Comments for author File: Comments.pdf

Comments on the Quality of English Language

English language is almost fine, however, several mistakes and suggestions are indicated in the text.

Author Response

Comments 1:

this part of the title is confusing. I suggest to emphasize the resistance and phytoremediation potential of this plant in the title.

Response 1:

Dear reviewer, in response to your query about the title, our research group collectively agreed to modify it to 'Resistances and Physiological Responses of Impatiens uliginosa to Copper Stress' following thorough deliberation. Additionally, we have crafted two alternative titles for consideration. Would this be acceptable?

（1）Tolerance and Physiological Responses of Impatiens uliginosa to Copper stress

（2）Diferential physiological responses and tolerance of Impatiens uliginosa to Copper stress

 

Comments 2:

unclear meaning, revise is necessary

Response 2:

Dear reviewer, we appreciate your insightful feedback and have made the necessary revisions as per your suggestions. The updated content can be found in lines 29 to 31 of the revised manuscript. We kindly request your review. Thank you.

 

Comments 3:

growth conditions before/during the treatment

Response 3:

Dear reviewer, greetings! At present, the project is in the seedling stage prior to exposure to copper stress. Owing to oversight in the experimental design, the data pertaining to this aspect has not been incorporated into the statistical analysis yet, resulting in a lack of data support in this particular area. We sincerely apologize for any inco

 

Comments 4:

Was this instrument used for measuring of Cu in tissues/growth medium? Please, indicate the method of determination.

Response 4:

Dear reviewer, greetings!The magnetic floating agitator is not utilized for measuring metal elements. Its primary purpose is to heat and stir low-viscosity solutions, speeding up the acid volatilization process and facilitating the determination of metal element content. As a result, the usage method has not been included in the article.

 

Comments 5:

I suggest to draw the linear diagrams of these data, or percentage data, that are presented in the text. I think that this table is rather big, not sufficiently informative, most of the averages vary statistically insignificantly... However, the percentage data in the text seems to be interesting but underrepresented

Response 5:

Dear reviewer, we have carefully considered your feedback and made revisions to the presentation of data in our manuscript. The original Table 1 has been transformed into a visual representation on pages 5-6, now referred to as Figure 1. As for Tables 2, 3, and 4, we believe it may be more appropriate to keep this data in tabular form. We eagerly await your guidance and response. Thank you for your valuable input.

 

Comments 6:

Please, add the units here and elsewhere

Response 6:

Dear reviewer, we have diligently incorporated the recommended additions to the article's sections. The latest content can be found at lines 237, 241, 248, 249, 270, 271, and so forth in the revised manuscript. We kindly request your review. Thank you.

 

Comments 7:

revision needed (partitioning of Cu to the leaf?)

Response 7:

Dear reviewer, I appreciate your insightful feedback. We have addressed the ambiguity in the mentioned section by reorganizing the content. The revised content can now be found in lines 393-396 of the manuscript. Your review of this section would be greatly appreciated. Thank you.

 

Comments 8:

revision needed (the highest rate of Cu²⁺ partitioning to the leaves was...?.)

Response 8:

Dear reviewer, I appreciate your insightful feedback. We have addressed the ambiguity in the mentioned section by reorganizing the content. The revised content can now be found in lines 393-396 of the manuscript. Your review of this section would be greatly appreciated. Thank you.

 

Comments 9:

my suggestion: the treatment with low concentration (5 mg L)

Response 9:

Dear esteemed reviewer, we sincerely appreciate your insightful feedback and have diligently incorporated the suggested modifications. The revised content can now be found on line 402 of the manuscript. We eagerly await your review. Thank you.

 

Comments 10:

the end of the sentence contradicts its beginning. Does Cu have no effect on absorption of these minerals, or still prevents their absorption?

Response 10:

Dear esteemed reviewer, we sincerely appreciate your insightful feedback and have diligently incorporated the suggested modifications. The section emphasizes that copper stress treatment does not affect the K and Mg contents in different parts of I. uliginosa., as well as the absorption of Ca, Na, and Zn in stems, leaves, and roots. There was ambiguity in our initial presentation, which has now been clarified. The revised content can be found in lines 506-509 of the manuscript. We kindly request your review. Thank you.

 

Comments 11:

unclear meaning. What do you mean by starting "Alternatively..." . Apart from this, affinity of the plant for specific elements was not mentioned in the text above, in contrast, Cu²⁺ effects should be summarized here...

Response 11:

Dear reviewer, hello! Thank you for your valuable comments. We have revised the ambiguous content based on your suggestions. The updated content can be found in lines 560-565 of the revised manuscript. We look forward to your review. Thank you.

 

Comments 12:

unclear meaning, the revision is needed. please, clearly summarize the effects of Cu for mineral balance of the plant, as well as Cu accumulation and transfer in different parts of the plant..

Response 12:

Dear reviewer, hello! Thank you for your valuable comments. We have revised the ambiguous content based on your suggestions. The updated content can be found in lines 560-565 of the revised manuscript. We look forward to your review. Thank you.

 

Comments 13:

English language is almost fine, however, several mistakes and suggestions are indicated in the text.

Response 13:

Dear esteemed reviewer, We appreciate your insightful feedback on our manuscript. We have diligently revised the content to address any ambiguities as per your recommendations. Thank you.

 

Comments 14:

English language is almost fine, however, several mistakes and suggestions are indicated in the text.

Response 14:

Dear esteemed reviewer, we have diligently revised our manuscript in line with your insightful feedback. We acknowledge that certain sentences in our article may have been ambiguous due to writing constraints. We appreciate your valuable suggestions and are committed to further refining and enhancing the clarity of our work. Thank you.

 

Special thanks to you for your good comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “ Physiological responses of Impatiens uliginosa to Copper 2 stress and its characteristics of enrichment “ described the effect of copper stress in  physiology (growth, development, mineral status) and biochemistry (oxidative stress response) of Impatiens uliginosa. The experiment design and the results obtained are valuable. However, certain findings and the discussion and conjectures makes me question the conclusions. The authors have taken into consideration that the results showed in Tables 1 and 2 may be associated with the applied stress and the metabolism of Ethylene? Ethylene is a phytohormone that regulates various plant developmental processes as growth, and displays various signaling processes under environmental stress in plants. Such environmental stresses trigger ethylene biosynthesis and/or action, which influences the growth and development of plants as the results described in Tables 1 and 2.

The discussion is very long and with highly speculations. Perhaps, it would be more convenient to focus on the results and try to explain these.

Further information that must be taken into account. The grammar and English must be reviewed. For ex., line 40-42, this sentence is not clear, lacks clarity. According to the Kew Botanical Garden, I. uliginosa is identified as an annual herb, suggesting a potential omission of the term "perennial" (line 76). Moreover, it is desirable to maintain the coherence in the text and figures (for ex., results begin with the height and stem descriptions and, in Table 1, the height and stem should be positioned at the begin too).

Line 118, “The data are the average of three experiments.”, this sentence could be move to statistical determinations in M&M.

 

Comments on the Quality of English Language

The grammar and English must be reviewed.

Author Response

Comments 1:

The manuscript “ Physiological responses of Impatiens uliginosa to Copper 2 stress and its characteristics of enrichment “ described the effect of copper stress in physiology (growth, development, mineral status) and biochemistry (oxidative stress response) of Impatiens uliginosa.The experiment design and the results obtained are valuable. However, certain findings and the discussion and conjectures makes me question the conclusions. The authors have taken into consideration that the results showed in Tables 1 and 2 may be associated with the applied stress and the metabolism of Ethylene? Ethylene is a phytohormone that regulates various plant developmental processes as growth, and displays various signaling processes under environmental stress in plants. Such environmental stresses trigger ethylene biosynthesis and/or action, which influences the growth and development of plants as the results described in Tables 1 and 2.

Response 1:

Dear reviewer, I appreciate your insightful feedback regarding the complexity of plant growth and development. Indeed, various factors such as plant hormones, metal elements, root exudates, and external conditions play crucial roles in this intricate process. Considering the elevated copper levels in the unique regional setting of Dianchi Lake, we focused our research primarily on copper, overlooking the potential impact of ethylene and other variables. This oversight was a limitation in our experimental design, and your suggestions will undoubtedly guide our future investigations. Your valuable insights have sparked new research ideas, and I am sincerely grateful for your constructive critique. Thank you!

 

Comments 2:

The discussion is very long and with highly speculations. Perhaps, it would be more convenient to focus on the results and try to explain these.

Response 2:

Dear reviewer, thank you for your insightful feedback on our manuscript. We have carefully revised the discussion section based on your recommendations to improve clarity and conciseness. Specifically, we have streamlined and refined the content by removing speculative or unsupported statements, thereby strengthening the overall rigor of the paper. For instance, we have made revisions to lines 416-427 in section 4.1, lines 450-455 in section 4.2, and lines 506-509 and 560-565 in section 4.3 as per your suggestions.

 

Comments 3:

Further information that must be taken into account.The grammar and English must be reviewed. For ex., line 40-42, this sentence is not clear,lacks clarity.

Response 3:

Dear reviewer, we would like to express our sincere apologies for the previous typographical error. The revised section can now be found in lines 40-46 of the updated manuscript. We kindly request your review and feedback. Thank you.

 

Comments 4:

According to the Kew Botanical Garden,I. uliginosa is identified as an annual herb, suggesting a potential omission of the term "perennial" (line 76).

Response 4:

Dear esteemed reviewer, I appreciate your insightful comments. Following thorough research and extensive deliberation, we have collectively decided to classify I. uliginosa as a annual herbaceous plant in the article. The ambiguous section has been rectified and can now be found in line 78 of the revised manuscript. We kindly request your review and feedback. Thank you.

 

Comments 5:

Moreover, it is desirable to maintain the coherence in the text and figures (for ex., results begin with the height and stem descriptions and, in Table 1, the height and stem should be positioned at the begin too).

Response 5:

Dear esteemed reviewer, I appreciate your insightful comments.we have made the necessary adjustments to ensure coherence between the text and accompanying graphics, as per your recommendations. Kindly refer to Figure 2 for further details. Thank you for your attention.

 

Comments 6:

Line 118, “The data are the average of three experiments.”, this sentence could be move to statistical determinations in M&M.

Response 6:

Dear esteemed reviewer, we appreciate your insightful feedback. We have carefully considered your suggestion regarding the statement 'Data is the average of three experiments' on line 118. As this information was previously included in the Methods and Materials section, we have removed it from this section. The deletion has been clearly marked on line 122 of the revised manuscript. Thank you once again for your valuable guidance.

 

Response 7:

The grammar and English must be reviewed.

Response 7:

Dear esteemed reviewer, we have diligently revised our manuscript in line with your insightful feedback. We acknowledge that certain sentences in our article may have been ambiguous due to writing constraints. We appreciate your valuable suggestions and are committed to further refining and enhancing the clarity of our work. Thank you.

 

Special thanks to you for your good comments.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Starting plants in soil and doing hydroponics in the later growing stages raise many concerns and questions. Thus, incorporating the authors' feedback for comment 1 (the rationale of experimental design) into the manuscript is necessary.  

 

Comments on the Quality of English Language

The English read well. It might need some minor language edits.

Author Response

Comments 1:

Starting plants in soil and doing hydroponics in the later growing stages raise many concerns and questions. Thus, incorporating the authors' feedback for comment 1 (the rationale of experimental design) into the manuscript is necessary.

Response 1:

Dear esteemed reviewer, I would like to express my gratitude for your insightful feedback and constructive criticism. As per your recommendations, we have elaborated on the rationale behind the selection of the two culture methods in the experiment within the revised manuscript. Further information can be located in lines 98-100 of the revised text.

 

Comments 2:

The English read well. It might need some minor language edits.

Response 2:

Dear esteemed reviewer, I appreciate your diligent guidance and insightful feedback. Following your recommendations, we have thoroughly reviewed and refined the language editing issues in the text, specifically addressing lines 416-417 in the revised manuscript.

 

Special thanks to you for your good comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “ Physiological responses of Impatiens uliginosa to Copper 2 stress and its characteristics of enrichment “ was reviewed carefully by the authors, implementing the suggested corrections and incorporating the recommendations provided.

Two details to take into account:

Figure 1 and 2 shows the legend position on top, it could be desirable to move the same legend position in fig. 3, to prevent the fragmentation of the word “stem”.

 Line 446-447, it is desirable to review the sentence: “The results showed that with the increase in Cu2+ stress, the fresh weight and dry weight of the root system decreased with the increase in Cu2+ stress.”

Author Response

Comments 1:

Figure 1 and 2 shows the legend position on top, it could be desirable to move the same legend position in fig. 3, to prevent the fragmentation of the word “stem”.

Response 1:

Dear reviewer, thank you for your patient guidance and constructive suggestions. As per your recommendation, we have revised the location of the legend for Figure 3, now located on page 13 of the manuscript. Furthermore, we have provided additional details for the legends in Figures 1 and 2, which can be found on pages 5-6 and 8-10 respectively.

 

Comments 2:

Line 446-447, it is desirable to review the sentence: “The results showed that with the increase in Cu²⁺ stress, the fresh weight and dry weight of the root system decreased with the increase in Cu²⁺ stress.”

Response 2:

Dear esteemed reviewer, we sincerely appreciate your thoughtful guidance and insightful feedback. We have addressed the ambiguity mentioned by you in lines 416-417 of the revised manuscript.

 

Special thanks to you for your good comments.