Assessing the Systematic Effects of the Concentration of Nitrogen Supplied to Dual-Root Systems of Soybean Plants on Nodulation and Nitrogen Fixation
Round 1
Reviewer 1 Report
The topic of this work is of significant interest to readers, but the objectives of this research are not stated, and the hypothesis is not clear. Moreover, the terms “systematically”, “short-term”, and “long-term” should be defined in the context of the research in the introduction. Without the above and the further address of my comments below, I can't recommend this article for publication.
Here are my comments and suggestions:
#5: space missing in “andChunmei Ma”
Abstract:
#9-10: Avoid the use of double negative “…soybean plants are not well unclear”
#11-12: Objective missing. Which is the objective of the work? It should be stated here.
#15: Is the “NH4NO3“ marked as in experiment I(15NH415NO3)?
#19-20: “specific nitrogenase activity (SNA)” and “acetylene reduction activity (ARA)” The definitions of variables should be in Material and Methods sentences rather than in result sentences.
#30-32: This is a methodology fact rather than a conclusive sentence. It should be improved.
Introduction:
#36-42: This article is also relevant to N limitation in high-yielding soybean. Suggested literature:
Salvagiotti, F., Cassman, K. G., Specht, J. E., Walters, D. T., Weiss, A., & Dobermann, A. (2008). Nitrogen uptake, fixation and response to fertilizer N in soybeans: A review. Field Crops Research, 108(1), 1-13.
Cafaro La Menza, N., Monzon, J. P., Specht, J. E., & Grassini, P. (2017). Is soybean yield limited by nitrogen supply?. Field Crops Research, 213, 204-212.
#43-46: Very short paragraph that could be merged with the 1st paragraph or with the 4th (71-80)
#47-70: Ok, but which is the research niche that has not been explored yet that justifies the publication of this article?
#81-83: “Split-root and dual-root systems… …. are not ideal for studying the systematic regulation of nitrogen” Then, why the authors used dual-root systems? It is either contradictory or not clear.
#83-87: These two sentences are in the wrong section. They seem to be discussion/conclusion.
#89: ”to generate to construct” chose between generate and construct.
#94-98: Hypotheses issues:
- The idea of “long-term” and “short-term” nitrogen effects are not previously defined nor explained in the introduction.
- The direction of the change should be stated. “…would systematically affect...” Positive? Negative?
- “different mechanisms” give examples of at least some of the mechanisms that the authors compare in this article.
- The second hypothesis is not clear. ARA, Ndfa%, and Naccumulation were not previously defined.
Materials and Methods
This section is not clear. The most important thing missing here is the experimental design (no mentioned at all). There is also no mention of seed inoculation. Were both sides of the root inoculated? Which was the commercial product or strain?
Explanation of the short and long-term experiments should be in relation to the hypothesis. Also, an explanation of the choice of crop stages V3, V5, and R3 for the treatment is missing.
#103-106: are the nodulated and non-nodulated variety genetically related? If they are not genetically related, could it influence the results?
#110: how many pots per treatment?
#135-139: Figure 1 should include a layout of the experimental design including Experiments I and II, treatments, and replicates.
#142: Were the 25 mg/L of N marked with 15N?
#154-155: “The nitrogen-containing nutrient solution was prepared by adding 15NH415NO3 to the nitrogen-free nutrient solution.” In which proportion?
#181-190: Here is some suggested literature about some issues related to the ARA assessments:
Peoples, M. B., Unkovich, M. J., Herridge, D. F., & Emerich, D. W. (2009). Measuring symbiotic nitrogen fixation by legumes. Nitrogen fixation in crop production’.(Eds D Emerich, H Krishnan) pp, 125-170.
Minchin, F. R., Witty, J. F., & Mytton, L. R. (1994). Reply to ‘Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay’by JK Vessey. Plant and Soil, 158(2), 163-167.
#200: Is it whole plant of aboveground?
#206: It is not clear how the authors calculated the Ndfa% for the nodulated and non-nodulated root side? Is it possible to do it with this experimental setting? An additional description is needed here.
#215-218: The correlation analysis of Figure 2 should be explained here.
Results
#240-242: This is a great example of a sentence with a clear message and were the direction of the changes is stated.
#262-263: Therefore… Is it a direct or indirect effect of the N concentration? Also, this statement does not apply to Table 3 or there may be a different explanation for that.
#299 Table 5: Was 100 % on N0 assumed or measured? Also, how was the Ndfa% determined for roots and nodules? It should be explained in the Material and Method section.
#331-334: Authors may want to be more specific about the “far less”. How much 10%, 50%, 90%?
#350: typo “Thethe”
#364: Table 7: It looks like columns are in the wrong place. The N0 has 100% on N derived from fertilizer and it should be the opposite.
#410-412: Delete these lines that are not from the article.
Discussion
It looks OK, but I am unable to provide much feedback on this section due to the problems that this article has in the previous sections. Authors may consider adding a scheme with the different mechanisms that “systematically” regulate this trade-off between N supply and N concentration.
Conclusions
This section is long, and authors should consider shortening it. Some sentences belong to the results and discussion sections.
#528-529: Delete these lines that are not from the article.
#536-538: This is a good conclusion, but the article does not provide all the information, or it is not clear. The authors may consider rephrasing this sentence.
Author Response
Dear Editors and Reviewer 1:
Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Assessing the systematic effects of the concentration of nitrogen supplied to dual-root systems of soybean plants on nodulation and nitrogen fixation” (ID agronomy-790344). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked by using "Track Changes" function in Microsoft Word. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
We have revised the text, figures, tables, references in the manuscript as required, and added the Supplementary Materials to make the manuscript more completed. We have revised section two of my manuscript to avoid too high level of coincidence with other published materials. After careful revision of the article, we chose American Journal Experts for professional English editing, to make the article more in line with the native English expression.
Response to Reviewer 1 Comment
Point 1: #5: space missing in “andChunmei Ma”
Response 1: We are very sorry for our negligence, we added space in“and Chunmei Ma”.
Point 2：#9-10: Avoid the use of double negative “…soybean plants are not well unclear”
Response 2: As Reviewer suggested that we have changed “…soybean plants are not well unclear”to “…soybean plants are unclear”.
Point 3：#11-12: Objective missing. Which is the objective of the work? It should be stated here.
Response 3: Considering the Reviewer’s suggestion, we added the objective of the work here.
Point 4：#15: Is the “NH4NO3“ marked as in experiment I(15NH415NO3)?
Response 4:Thanks to the reviewer for the comments, the “NH4NO3” is not marked in experiment II.
Point 5：#19-20: “specific nitrogenase activity (SNA)” and “acetylene reduction activity (ARA)” The definitions of variables should be in Material and Methods sentences rather than in result sentences.
Response 5:The comments given by the reviewer are very correct, but the Agronomy requested the abbreviations have been defined in parentheses the first time they appear in the abstract, main text, and in figure or table captions, I hope the you can agree to Keep original.
Point 6：#30-32: This is a methodology fact rather than a conclusive sentence. It should be improved.
Response 6: We modified this sentence based on the comments of the reviewers, make it be a conclusive sentence
Point 7：#36-42: This article is also relevant to N limitation in high-yielding soybean. Suggested literature:
Salvagiotti, F., Cassman, K. G., Specht, J. E., Walters, D. T., Weiss, A., & Dobermann, A. (2008). Nitrogen uptake, fixation and response to fertilizer N in soybeans: A review. Field Crops Research, 108(1), 1-13.
Cafaro La Menza, N., Monzon, J. P., Specht, J. E., & Grassini, P. (2017). Is soybean yield limited by nitrogen supply?. Field Crops Research, 213, 204-212.
Response 7: Considering the Reviewer’s suggestion, we added these two literatures.
Point 8：#43-46: Very short paragraph that could be merged with the 1st paragraph or with the 4th (71-80)
Response 8: As Reviewer suggested that we merged with the 1st paragraph.
Point 9：#47-70: Ok, but which is the research niche that has not been explored yet that justifies the publication of this article?
Response 9: Considering the Reviewer’s suggestion, we have revised the innovation of Introduction in the manuscripts
Point 10：#81-83:“Split-root and dual-root systems… …. are not ideal for studying the systematic regulation of nitrogen” Then, why the authors used dual-root systems? It is either contradictory or not clear.
Response 10: The comments given by the reviewer are very correct, we changed“Split-root and dual-root systems”to“Split-root and dual-root (both sides were nodulated) systems”. We consider dual-root systems with nodules on both sides are not ideal for studying the systematic regulation of nitrogen , so we construct a dual-root system with a single nodulated side.
Point 11：#83-87: These two sentences are in the wrong section. They seem to be discussion/conclusion.
Response 11: As Reviewer suggested that we have moved these two sentences in correct section.
Point 12：#89: ”to generate to construct” chose between generate and construct.
Response 12: According to the Reviewer’s comments, we have changed “to generate to construct” to “to construct”
Point 13：#94-98: Hypotheses issues:
The idea of “long-term” and “short-term” nitrogen effects are not previously defined nor explained in the introduction.
The direction of the change should be stated. “…would systematically affect...” Positive? Negative?
“different mechanisms” give examples of at least some of the mechanisms that the authors compare in this article.
The second hypothesis is not clear. ARA, Ndfa%, and Naccumulation were not previously defined.
Response 13: Considering the Reviewer’s suggestion, we have added the definitions of long-term and short-term in introduction. And we have stated “would systematically inhibit” in here. We have also added the two examples of mechanisms. The second hypothesis has been revised again to make it clearer. We have ensured the ARA, Ndfa%, and Naccumulation were defined in abstract.
Point 14：This section is not clear. The most important thing missing here is the experimental design (no mentioned at all). There is also no mention of seed inoculation. Were both sides of the root inoculated? Which was the commercial product or strain?
Response 14: We have made correction according to the Reviewer’s comments. We have revised the content of this section, added the content of the single-factor experimental design, and the method of inoculation of soybeans has also been revised for clearer reading, all the roots both two sides were inoculated.
Point 15：Explanation of the short and long-term experiments should be in relation to the hypothesis. Also, an explanation of the choice of crop stages V3, V5, and R3 for the treatment is missing.
Response 15: We have rewritten the methods to reinterpret the short and long-term experiments and added explanations for the choice of each stages according to the Reviewer’s suggestion
Point 16：#103-106: are the nodulated and non-nodulated variety genetically related? If they are not genetically related, could it influence the results?
Response 16: The comments given by the reviewer are very correct, but the nodulated and non-nodulated variety genetically were not related, we are studying the effect of nitrogen supply concentration on soybean nodulation and nitrogen fixation, all the experimental treatments use the same two varieties for testing, so the two varieties are not genetically related should not affect the results.I hope the you can agree to my request.
Point 17：#110: how many pots per treatment?
Response 17: Thanks to the reviewers for the comments, we added the nunber of pots per treatment in Materials and Methods.
Point 18：#135-139: Figure 1 should include a layout of the experimental design including Experiments I and II, treatments, and replicates.
Response 18: Thanks to the reviewers for the comments, we have reworked Figure 1 in Supplementary Materials 1, and added key information about experiments.
Point 19：#142: Were the 25 mg/L of N marked with 15N?
Response 19:Thanks to the reviewers for the comments, Here 25mg / L is not marked by 15N.
Point 20：#154-155:“The nitrogen-containing nutrient solution was prepared by adding 15NH415NO3 to the nitrogen-free nutrient solution.” In which proportion?
Response 20:Thanks to the reviewers for the comments, we have added 15NH415NO3 to the nitrogen-free nutrient solution to make concentrations of nitrogen reach 25mg / L, 50mg / L, 75mg / L and 100mg / L.
Point 21：#181-190: Here is some suggested literature about some issues related to the ARA assessments:
Peoples, M. B., Unkovich, M. J., Herridge, D. F., & Emerich, D. W. (2009). Measuring symbiotic nitrogen fixation by legumes. Nitrogen fixation in crop production’.(Eds D Emerich, H Krishnan) pp, 125-170.
Minchin, F. R., Witty, J. F., & Mytton, L. R. (1994). Reply to ‘Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay’by JK Vessey. Plant and Soil, 158(2), 163-167.
Response 21: As Reviewer suggested that we added these two literatures.
Point 22：#200: Is it whole plant of aboveground?
Response 22:Thanks to the reviewers for the comments, it is whole plant of aboveground.
Point 23：#206: It is not clear how the authors calculated the Ndfa% for the nodulated and non-nodulated root side? Is it possible to do it with this experimental setting? An additional description is needed here.
Response 23: Considering the Reviewer’s suggestion, We have revised the expression of Ndfa% calculation to make it easier to understand to ensure that it can be carried out in this experimental setting.
Point 24：#215-218: The correlation analysis of Figure 2 should be explained here.
Response 24: The comments given by the reviewer are very correct, but We did not do Pearson’s correlations analysis in Figure 2, I hope the you can agree to Keep original
Results
Point 25：#240-242: This is a great example of a sentence with a clear message and were the direction of the changes is stated.
Response 25: Thanks to the reviewers for the comments, we modified other unclear expression sentences according to this sentence.
Point 26：#262-263: Therefore… Is it a direct or indirect effect of the N concentration? Also, this statement does not apply to Table 3 or there may be a different explanation for that.
Response 26: It is really true as reviewer suggested that we determined that it was a systemic effect, and we revised the expression.
Point 27：#299 Table 5: Was 100 % on N0 assumed or measured? Also, how was the Ndfa% determined for roots and nodules? It should be explained in the Material and Method section.
Response 27: Thanks to the reviewers for the comments, InTable 5, the 100 % on N0 was assumed, We have explained the Ndfa% determined for roots and nodules in the Material and Method section according to the reviewer’s suggestion .
Point 28：#331-334: Authors may want to be more specific about the “far less”. How much 10%, 50%, 90%?
Response 28:We have re-written this paragraph so that the degree of reduction is expressed as a percentage according to the reviewer’s suggestion.
Point 29：#350: typo “Thethe”
Response 29:We are very sorry for our incorrect writing and we have modified the typo.
Point 30：#364: Table 7: It looks like columns are in the wrong place. The N0 has 100% on N derived from fertilizer and it should be the opposite.
Response 30: The comments given by the reviewer are very correct, we have changed N0 to N100.
Point 31：#410-412: Delete these lines that are not from the article.
Response 31:Thanks to the reviewers for their comments,we have deleted these lines that are not from the article
Discussion
Point 32：It looks OK, but I am unable to provide much feedback on this section due to the problems that this article has in the previous sections. Authors may consider adding a scheme with the different mechanisms that “systematically” regulate this trade-off between N supply and N concentration.
Response 32:Thanks to the reviewers for the comments, we have modified the previous problem, and modified some sentences in the Discussion to make it easier to understand
Conclusions
Point 33：This section is long, and authors should consider shortening it. Some sentences belong to the results and discussion sections.
Response 33:We have re-written the Conclusions to make it brief according to the Reviewer’s suggestion
Point 34：#528-529: Delete these lines that are not from the article.
Response 34:Thanks to the reviewers for their comments,we have deleted these lines that are not from the article
Point 35：#536-538: This is a good conclusion, but the article does not provide all the information, or it is not clear. The authors may consider rephrasing this sentence.
Response 35: As Reviewer suggested that, we rephrased this sentence.
Special thanks to you for your good comments.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in revised paper.
We appreciate for Editors and Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The work by Xiaochen et al. showed the study of the systematic regulation of nitrogen using a modified version of the known soybean dual-root system, at which they added increasing concentrations of a nitrogen source to the non-nodulated root part during a short- (experiment II) or a relatively long-term (experiment I). Authors made a deep analysis by measuring the nodule number, nodule dry weight and nitrogenase activity of nodulated root side, as well as the quantification of the 15N abundance, nitrogen derived from atmosphere (Ndfa%), and nitrogen accumulation. Statistical analysis was made for each measure. Results obtained showed that by a short-term treatment with increasing concentrations of nitrogen, only specific nitrogenase activity (SNA) and the acetylene reduction activity (ARA) parameters were significantly reduced. On the other hand, a significant reduction on the number and dry weight of root nodules, which they correlated to a reduction on SNA and ARA was observed with a long-term treatment with increasing concentration of nitrogen. Besides, they found that fertilizer nitrogen was distributed from the non-nodulated roots, from which the nitrogen source was taken, to other part of the roots (nodulated roots and nodules), and the shoot. Finally, they suggested that ARA probably reflected nitrogen derived from atmosphere, but not the accumulation of nitrogen fixed by nodules.
Although biological nitrogen fixation by leguminous crop plants field has extensively explored, authors made an effort to obtain a system to improve data resolution, providing a solid and reliable reference for rational application of nitrogen fertilizer.
Specific comments:
- Since authors suggest that both sides of split-root or dual-root systems used in previous studies can form nodules and fix nitrogen, I would conduct simultaneous experiments using a regular soybean dual-root system and the system described on the present manuscript with a single nodulated side to compare both systems. These data could be a quantitatively way the show the advantages of using the dual-root system with a single nodulation side.
- Authors should make an effort to improve the style, the structure, and quality of writing of the manuscript, trying to expose better main ideas and results. Specially, abstract and results sections are very hard to read. Authors should delete the title enumeration and organization, since it does not correspond to the scientist style. Titles from discussion section should be deleted for the same reason.
- Authors developed two experiments: long- (experiment I).and short-term (experiment II) nitrogen treatments. Please, authors must be more precise and concise in describing each experiment. Instead experiment I and II, I would refer them as short- and long-term treatments. Short-term nitrogen supply experiment was conducted by 5 days, but what about long-.term nitrogen supply experiment? In addition, you could add a workflow in material and method section in order to facilitate the reader how each of them have been developed (i.e: Jiménez-Guerrero et al., 2015. MPMI. Figure S1).
- Tables and Figures: revise that all tables and figures are named in the text before they appear in the manuscript (i.e: Table 1 appears before it is named). Please, indicate in all tables to which experiment the represented results belong (experiment I and II, or long-term nitrogen treatment).
- Please, add a brief description of plant growth stages (V3, V4, V5, R3, etc) in material and methods section.
- Figure 1 should be improved. Authors should improve the quality of the photo showed in this figure and they could add an arrow indicating where the grafted point is located. They should include the varieties of soybean plants used in the figure legend.
- Last reference was from year 2017. Authors should make an effort to add new recently published information.
Author Response
Dear Editors and Reviewer:
Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Assessing the systematic effects of the concentration of nitrogen supplied to dual-root systems of soybean plants on nodulation and nitrogen fixation” (ID agronomy-790344). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked by using "Track Changes" function in Microsoft Word. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
We have revised the text, figures, tables, references in the manuscript as required, and added the Supplementary Materials to make the manuscript more completed. We have revised section two of my manuscript to avoid too high level of coincidence with other published materials. After careful revision of the article, we chose American Journal Experts for professional English editing, to make the article more in line with the native English expression.
Response to Reviewer 2 Comment
Point 1：Since authors suggest that both sides of split-root or dual-root systems used in previous studies can form nodules and fix nitrogen, I would conduct simultaneous experiments using a regular soybean dual-root system and the system described on the present manuscript with a single nodulated side to compare both systems. These data could be a quantitatively way the show the advantages of using the dual-root system with a single nodulation side.
Response 1: Considering the Reviewer’s suggestion, we have added the regular soybean dual-root system and the system described on the manuscript with a single nodulated side to compare both systems in Supplementary Materials 3.
Point 2：Authors should make an effort to improve the style, the structure, and quality of writing of the manuscript, trying to expose better main ideas and results. Specially, abstract and results sections are very hard to read. Authors should delete the title enumeration and organization, since it does not correspond to the scientist style. Titles from discussion section should be deleted for the same reason.
Response 2: Considering the Reviewer’s suggestion, we have revised the style, the structure, and quality of writing of the manuscript, we also have re-written Abstract and Results section according to the reviewer’s suggestion to make it more clear and concise. We have revised the title tomake it more standardized.
Point 3：Authors developed two experiments: long- (experiment I).and short-term (experiment II) nitrogen treatments. Please, authors must be more precise and concise in describing each experiment. Instead experiment I and II, I would refer them as short- and long-term treatments. Short-term nitrogen supply experiment was conducted by 5 days, but what about long-.term nitrogen supply experiment? In addition, you could add a workflow in material and method section in order to facilitate the reader how each of them have been developed (i.e: Jiménez-Guerrero et al., 2015. MPMI. Figure S1).
Response 3: Considering the Reviewer’s suggestion, we have revised the description of each experiment,We added the number of days of long-term nitrogen supply experiment, We have revised the material and method so that two experiments can be read more easily. And we added workflow of material and method section in Supplementary Materials 1 to make more clearly.
Point 4：Tables and Figures: revise that all tables and figures are named in the text before they appear in the manuscript (i.e: Table 1 appears before it is named). Please, indicate in all tables to which experiment the represented results belong (experiment I and II, or long-term nitrogen treatment).
Response 4: Thanks to the reviewer for the comments, we have revised all sequence of tables and figures, and we also have indicated in all tables to which experiment the represented results belong.
Point 5：Please, add a brief description of plant growth stages (V3, V4, V5, R3, etc) in material and methods section.
Response 5: As Reviewer suggested that we have added a brief description of plant growth stages (V3, V4, V5, R3) in material and methods section.
Point 6： Figure 1 should be improved. Authors should improve the quality of the photo showed in this figure and they could add an arrow indicating where the grafted point is located. They should include the varieties of soybean plants used in the figure legend.
Response 6: We have made correction according to the reviewer’s comments. we have added an arrow indicating where the grafted point is located, we also added the the varieties of soybean plants used in the figure legend in Supplementary Materials 1.
Point 7：Last reference was from year 2017. Authors should make an effort to add new recently published information.
Response 7:As Reviewer suggested that we added literature as follow:
Lyu X , Xia X , Wang C , et al. Effects of changes in applied nitrogen concentrations on nodulation, nitrogen fixation and nitrogen accumulation during the soybean growth period[J]. Soil Science and Plant Nutrition, 2019:1-11.
Shinji I , Yuki O , Norikuni O , et al. Transcriptome and Metabolome Analyses Reveal That Nitrate Strongly Promotes Nitrogen and Carbon Metabolism in Soybean Roots, but Tends to Repress It in Nodules[J]. Plants, 2018, 7(2):32.
Yamashita N , Tanabata S , Ohtake N , et al. Effects of Different Chemical Forms of Nitrogen on the Quick and Reversible Inhibition of Soybean Nodule Growth and Nitrogen Fixation Activity[J]. Frontiers in Plant Science, 2019, 10.
Special thanks to you for your good comments.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in revised paper.
We appreciate for Editors and Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions
Round 2
Reviewer 1 Report
Abstract:
#26-29: This sentence is now conclusive. However, it is not being conclusive about the objective of the study somehow stated in lines 10 to 12. How the results of this experiment could provide a ‘solid and reliable reference for the rational application of N fertilizer’?
Introduction:
This section has been improved. However, there is no justification for how the authors came up with the idea of study the short- and long-term effects. This deserves a whole new paragraph between lines #119 and 120. Otherwise, the hypotheses make no sense or are not well supported for the information provided in the introduction.
#120-177: Here there is a problem with the line number, but I am referring to the last paragraph of the introduction on the following comment. The objective of the study should be explicitly stated here like “The objective of this study was…”. It looks like there is a general objective of ‘investigating the relationship between nitrogen, nodulation and nitrogen fixation to provide a solid and reliable reference for the rational application of nitrogen fertilizer’ (somehow stated in the abstract), and a more specific objective of studying the mechanisms underlying the short and long term systematic regulation of nitrogen uptake from fixation and fertilizer in soybean. Is that correct? The authors should clarify the objective/s of the study in the introduction and abstract.
Material and Methods:
#192-200 It is not clear on whether the authors performed the experiment I and II in the same pot or there were a different set of pots for the experiment I and for experiment II. Please, clarify that.
#201-206 Is there a specific way or protocol that the authors followed for watering the pots and adding the N solution? This is a very important explanation that is needed because an overflow of one side of the pot to the other could have increased the N concentration of the N- side. If the authors have soil analysis of both sides of the pot that can help to clarify this issue, I encourage them to briefly incorporate that data in a sentence.
Results:
#631 ‘various parts’. The authors should be more specific.
#650 ‘other four treatments’ which ones? Specify.
#867 Table 7: It looks like columns are in the wrong place. The N0 has 100% on N derived from fertilizer and it should be the opposite. I already made this comment. The Authors said they changed it, but the tables remain the same. Please, check that the data and labels are correct. An N0 treatment means no fertilizer. Therefore, it is not possible that 100% of the N was derived from fertilizer.
Discussion:
#1006-1017 This should be the last paragraph. The last sentence (#1015-1017) is a good closing sentence for the discussion that recall the objectives of the paper.
#1018-1078 It is not a good paragraph to end the discussion. My suggestion is to move it up.
Conclusions:
# 1089 ‘providing a theoretical basis and guidance’ This is not supported by results and discussion. I suggest modifying this sentence by something like ‘providing new insights for the rational and proper use of nitrogen fertilizer in soybean’ or something similar.
Author Response
Response to Reviewer 1 Comment
Abstract:
Point 1：#26-29: This sentence is now conclusive. However, it is not being conclusive about the objective of the study somehow stated in lines 10 to 12. How the results of this experiment could provide a ‘solid and reliable reference for the rational application of N fertilizer’?
Response 1: Thanks to the reviewer for the comments, We think the conclusions given by the reviewer are excellent ‘providing new insights for the rational and proper use of nitrogen fertilizer in soybean’, so we modified the purpose of the study in the abstract. Long-term and short-term nitrogen experiments effects on nodulation, can provid the mechanisms underlying the systematic regulation of nitrogen uptake from fixation and fertilizer in soybean. #26-29 The evaluation of the three indicators can clarify the limitations of the acetylene reduction method for the determination of ARA, and the nitrogen fixation research after this study can choose the measurement indicators suitable for their research purposes. So the results of this experiment could provid a new insights for the rational and proper use of nitrogen fertilizer in soybean. I hope the reviewer can agree to our revises.
Introduction:
Point 2：This section has been improved. However, there is no justification for how the authors came up with the idea of study the short- and long-term effects. This deserves a whole new paragraph between lines #119 and 120. Otherwise, the hypotheses make no sense or are not well supported for the information provided in the introduction.
Response 2: Thanks to the reviewer for the comments, we added the idea of study the short- and long-term effects in new paragraph, and this paragraph to express the importance of studying different nitrogen supply time and constructed soybean dual-root system (single nodulated side). This paragraph can better support the information provided in the introduction. I hope the reviewer can agree to our revises.
Point 3：#120-177: Here there is a problem with the line number, but I am referring to the last paragraph of the introduction on the following comment. The objective of the study should be explicitly stated here like “The objective of this study was…”. It looks like there is a general objective of ‘investigating the relationship between nitrogen, nodulation and nitrogen fixation to provide a solid and reliable reference for the rational application of nitrogen fertilizer’ (somehow stated in the abstract), and a more specific objective of studying the mechanisms underlying the short and long term systematic regulation of nitrogen uptake from fixation and fertilizer in soybean. Is that correct? The authors should clarify the objective/s of the study in the introduction and abstract.
Response 3: Thanks to the reviewer for the comments, we added the objective of the study in this paragraph. With the reviewer ‘s suggestion, we believe ‘study the mechanisms underlying the short and long term systematic regulation of nitrogen uptake from fixation and fertilizer in soybean’ is a specific objective, ‘provid new insights for the rational and proper use of nitrogen fertilizer in soybean’ is a further objective, these two sentences were added to the article to make the purpose of the experiment more clear. I hope the reviewer can agree to our revises.
Material and Methods:
Point 4：#192-200 It is not clear on whether the authors performed the experiment I and II in the same pot or there were a different set of pots for the experiment I and for experiment II. Please, clarify that.
Response 4: Thanks to the reviewer for the comments, Five pots were used for each treatment，the treatments in the experiment I and experiment II have a different set of pots
Point 5：#201-206 Is there a specific way or protocol that the authors followed for watering the pots and adding the N solution? This is a very important explanation that is needed because an overflow of one side of the pot to the other could have increased the N concentration of the N- side. If the authors have soil analysis of both sides of the pot that can help to clarify this issue, I encourage them to briefly incorporate that data in a sentence.
Response 5: The comments given by the reviewer are very correct, the gap between the plate and the pot was sealed with glue. Two drainage holes 1 cm in diameter were drilled at the bottom of the pot, one for each side of the partition plate. The nitrogen-containing nutrient solution was prepared by adding NH4NO3 to the nitrogen-free nutrient solution. Every time irrigated the nutrient solution slowly, we make sure that the nutrient solution does not flow into the opposite side. Since we are cultivating in the sand, we did not conduct chemical analysis to the sand. We believe that the way of pouring the nutrient solution will not affect the test results.
Results:
Point 6：#631 ‘various parts’. The authors should be more specific.
Response 6: Thanks to the reviewer for the comments, we have added specific organs.
Point 7：#650 ‘other four treatments’ which ones? Specify.
Response 7: Thanks to the reviewer for the comments, we have added specific treatments.
Point 8：#867 Table 7: It looks like columns are in the wrong place. The N0 has 100% on N derived from fertilizer and it should be the opposite. I already made this comment. The Authors said they changed it, but the tables remain the same. Please, check that the data and labels are correct. An N0 treatment means no fertilizer. Therefore, it is not possible that 100% of the N was derived from fertilizer.
Response 8: We are very sorry for our negligence, we have revised the Table 7, the ‘Nitrogen derived from atmosphere’ and ‘Nitrogen derived from the fertilizer’ already in the proper place.
Discussion:
Point 9：#1006-1017 This should be the last paragraph. The last sentence (#1015-1017) is a good closing sentence for the discussion that recall the objectives of the paper.
Response 9: Thanks to the reviewer for the comments, We have adjusted the order of the discussion.
Point 10：#1018-1078 It is not a good paragraph to end the discussion. My suggestion is to move it up.
Response 10: Thanks to the reviewer for the comments, We have adjusted the order of the discussion.
Conclusions:
Point 11：# 1089 ‘providing a theoretical basis and guidance’ This is not supported by results and discussion. I suggest modifying this sentence by something like ‘providing new insights for the rational and proper use of nitrogen fertilizer in soybean’ or something similar.
Response 11: Thanks to the reviewer for the comments, we have modified this sentence. I hope the reviewer can agree to our revises.
Special thanks to you for your good comments.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in revised paper.
We appreciate for Editors and Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.
Reviewer 2 Report
All suggestions have been included. Thus, I think that the manuscript would be published in the present form after correction of some minor points:
- I would include the advantages of using a soybean dual-root system with a single side nodulated at the beginning of the discussion section instead the results section (S3).
- Authors should revise the new material and check the small spelling errors that may be (capital letters, numbers as subscript in S2…).
- Please, name the Table 2 in the text.
- Line 392: …accumulation of nitrogen…
- Line 631: By using the 15N abundance and Naccumulation of various parts of the plant at the… (it´s redundant)
Author Response
Response to Reviewer 2 Comment
Point 1：I would include the advantages of using a soybean dual-root system with a single side nodulated at the beginning of the discussion section instead the results section (S3).
Response 1: Thanks to the reviewer for the comments, we have added the advantages of using a soybean dual-root system with a single side nodulated (S3) in the discussion section.
Point 2：Authors should revise the new material and check the small spelling errors that may be (capital letters, numbers as subscript in S2…).
Response 2: Thanks to the reviewer for the comments, we have revised the new material and checked the small spelling errors.
Point 3：Please, name the Table 2 in the text.
Response 3: Thanks to the reviewer for the comments, we have named the Table 2 in the text.
Point 4：Line 392: …accumulation of nitrogen…
Response 4: As Reviewer suggested that we have changed ‘Naccumulation’ to ‘accumulation of nitrogen’.
Point 5：Line 631: By using the 15N abundance and Naccumulation of various parts of the plant at the… (it´s redundant)
Response 5: As Reviewer suggested that we have deleted redundant sentences.
Special thanks to you for your good comments.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in revised paper.
We appreciate for Editors and Reviewers’ warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.
