Nitrogen Modulates the Effects of Short-Term Heat, Drought and Combined Stresses after Anthesis on Photosynthesis, Nitrogen Metabolism, Yield, and Water and Nitrogen Use Efficiency of Wheat

Round 1

Reviewer 1 Report

The overall objective of this study was to test the effects of heat, drought and nitrogen supply in wheat. The paper is interesting, and well written. The authors perform a large number of experiments aimed at proving their hypothesis. In general the paper meets the requirements and quality to be accepted. The material and methods are complete and exhaustive; the results are extensive and the discussion is very broad and perfectly explains the results obtained.

Below are some comments to be taken into account:

As there are a large number of treatments, it is initially difficult for the reader to understand the treatments, as the legend in figure 1 does not correspond to the legend in the other figures. I don't know if there is a way to make it a little more comprehensible; if so, it would be better. 

Although many bibliographical references are provided, I think it would be interesting to describe a few more reasons why the N2 treatment is usually better than N1 and N3 in the discussion. Although some reasons are mentioned, I think that the paper would improve the quality by providing more information instead of simply providing the bibliographic reference.

Regarding figure 8 and table 3, perhaps it would be better to include it as supplementary material, as it is not mentioned as such in the material and methods, and it is unusual to find a figure and a table in the discussion.

Review the text, as scientific names should be written in italics; for example on page 2 in Plant material and growth conditions, Triticum aestivum should be written in italics.

In general, the bibliography should be revised, as there are some mistakes:

References 11, 15, 24, 29, 32, 34 and 57: remove full stops in abbreviated journal names.

References 12: journal name in italics.

References 22: scientific name in italics and year of publication in bold.

References 26, 37, 50, 53, 61 and 66: scientific name in italics.

Author Response

Dear Editors and Reviewers:

On behalf of my co-authors, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Nitrogen modulates the effects of short-term heat, drought and combined stresses after anthesis on photosynhesis, nitrogen metabolism, yield, and water and nitrogen use efficiency of wheat”. (Water-1678043). We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments which we hope meet with approval. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to express our great appreciation to you and reviewers for comments on our paper.The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Responds to the reviewer’s comments:

• As there are a large number of treatments, it is initially difficult for the reader to understand the treatments, as the legend in figure 1 does not correspond to the legend in the other figures. I don't know if there is a way to make it a little more comprehensible; if so, it would be better.

Response: Thank you for your comment. We have modified Figure 1 in order to better understand the contents in the figure according to the comments of reviewer. In addition, a detailed explanation of the 12 treatments was presented in the 2.2 "Experimental design" in Materials and methods.

• Although many bibliographical references are provided, I think it would be interesting to describe a few more reasons why the N2 treatment is usually better than N1 and N3 in the discussion. Although some reasons are mentioned, I think that the paper would improve the quality by providing more information instead of simply providing the bibliographic reference.

Response: Thanks a lot for the reviewer’s comments, your comment is very helpful to improve the quality of our papers. We have added more reasons and interpretations on the the regulatory effect of nitrogen on wheat in the “discussion” section according to the comments of reviewer.

• Regarding figure 8 and table 3, perhaps it would be better to include it as supplementary material, as it is not mentioned as such in the material and methods, and it is unusual to find a figure and a table in the discussion.

Response: We are grateful for your comment. According to the comments of reviewer, we have removed figure 3 and table 3 from the discussion section. Figure 8 and table 3 are one of the main contents of this study, moreover, this part is mentioned in 2.4.2 plant N and grain protein and 2.7 data analysis in materials and methods. Therefore, we think it may be more appropriate to put figure 8 and table 3 in 3.6 “The Relationship between photosynthetic rate and biological factors” of the results. We have analyzed this section in detail in section 3.6. We hope our decision can be approved by reviewer.

• Review the text, as scientific names should be written in italics; for example on page 2 in Plant material and growth conditions, Triticum aestivum should be written in italics.

Response: Thanks a lot for the reviewer’s comments. According to the comments of reviewer, Triticum aestivum has been written in italics.

5)In general, the bibliography should be revised, as there are some mistakes:

References 11, 15, 24, 29, 32, 34 and 57: remove full stops in abbreviated journal names.

References 12: journal name in italics.

References 22: scientific name in italics and year of publication in bold.

References 26, 37, 50, 53, 61 and 66: scientific name in italics.

Response: Thanks a lot for the reviewer’s comments. We have corrected all mistakes in the bibliography according to the comments of reviewer.

We have tried our best to revise and improve the manuscript according to the Reviwers′ good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval.

Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

Best regards.

Yours sincerely,

Author Response File: Author Response.pdf

Reviewer 2 Report

In the paper entitled "Nitrogen modulates the effects of short-term heat, drought and combined stresses after anthesis on photosynthesis, nitrogen metabolism, yield, and water and nitrogen use efficiency of wheat ", the authors presented an interesting study on plant responses to combined abiotic stresses in wheat. They investigated several growth traits and physiological parameters in drought and control conditions, in low and high temperatures, and under different nitrogen supplies.

Overall, the manuscript looks good, but I have several major comments to make.

Major comments:

1. The experiment design shows a big flaw. Indeed, the authors aimed to compare 4 conditions: control (SF), drought (SD), heat stress (HF), and combined heat and drought stress (HD). Plants under SF and SD were in a first greenhouse while plants under HF and HD were in a second greenhouse. With this experiment design, when the authors tested the effect of HF and HD compared to SF, they also tested a location effect. They could not distinguish the difference in condition from the difference in location. In that case, I expect to have a second independent experiment to confirm their observations.
2. In figures 3 to 6, please indicate more clearly the four conditions (SF, SD, HF, and HD) on the x-axis.
3. What are the data analyzed in the ANOVAs (data of 3, 6, 9, or 12 days of stress treatment) in figures 3 to 5?
4. The section 3.5 called “Growth analysis” is confusing and needs to be rewritten. For instance, data in Table 1 does not support the sentence “plant height under the HF and SD treatments increased with increasing N application”.
5. Section 3.6 called “Yield, water and N use efficiency”, second paragraph. The authors wish to compare the effect of treatments and their interactions. They cannot do this based on the significance of the ANOVA, since the degree of freedom varies between own effects and interactions. I suggest analyzing the proportion of variance explained by each factor and interaction. The authors can do it using the sums of squares in ANOVA.
6. Section 3.7 called “Principal Component Analysis”. Because the authors discussed the correlation between yield-related traits and PSII-related traits, I suggest including the latter group of traits in the PCA. The authors have mainly interpreted axis 1 of the PCA by analyzing the traits contributing to this axis. Can they also interpret axis 2?
7. Figure 8 and Table 3 are results that are currently presented in the discussion section and should be presented in the results section.
8. In the discussion, the authors developed a nice discussion about the relationship between reduced photosynthesis and plant response to drought and heat. They mentioned a good explanation of this relation by the damage of chloroplast by heat and drought. Other authors have referred to stomatal responses that prevent transpiratory water loss under heat and drought, and can be discussed by the authors.

Author Response

Dear Editors and Reviewers:

On behalf of my co-authors, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Nitrogen modulates the effects of short-term heat, drought and combined stresses after anthesis on photosynhesis, nitrogen metabolism, yield, and water and nitrogen use efficiency of wheat”. (Water-1678043). We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments which we hope meet with approval. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to express our great appreciation to you and reviewers for comments on our paper.The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Responds to the reviewer’s comments:

1)The experiment design shows a big flaw. Indeed, the authors aimed to compare 4 conditions: control (SF), drought (SD), heat stress (HF), and combined heat and drought stress (HD). Plants under SF and SD were in a first greenhouse while plants under HF and HD were in a second greenhouse. With this experiment design, when the authors tested the effect of HF and HD compared to SF, they also tested a location effect. They could not distinguish the difference in condition from the difference in location. In that case, I expect to have a second independent experiment to confirm their observations.

Response: Thank you for your comment. In this experiment, two identical intelligent growth chambers (RQS-15) were used, with temperature control range 0-50°C, temperature resolution 0.1°C, temperature uniformity ±1°C, error ≤0.5°C; humidity control range was 40~85%, error ±3%RH. During the stress period, temperature and relative humidity and other environmental factors were observed daily in the microcomputer in the growth chamber. The control of different environmental factors was stable and precise in both growth chambers. In addition, 24 time points in a 24-h day were set to better simulate the process of daily changes in temperature. the actual temperature in the two growth chambers was able to be consistent with the pre-set temperature. The daily variation of temperature in Fig. 2a shows that the actual temperature in both growth chambers corresponded to the pre-set ambient temperature, and the temperature fluctuations were within the error tolerance. The two growth chambers did not show any anomalies in the environmental parameters during the experiment. The control of environmental factors in both high temperature and control growth chambers was consistent and both had high accuracy. The differences caused by the location effects between them were minimal compared to the effects of the environment (heat, drought and combined stress) on wheat plants. In addition, when reading the literature on this field, we found that in previous studies on the effects of heat on plants, two growth chambers were usually used for controlling heat and control temperatures, respectively, where heat-stressed and control plants were always in the heat and control growth chambers during the stress period, respectively. For example, Wang (2011), doi.org/10.1016/j.plaphy.2013.11.014;

Ullah (2019), doi.org/10.1016/j.plaphy.2019.08.020;

Liu (2019), doi.org/10.3389/ fpls.2019.00357;

Abdelhakim (2021), doi.org/10.1016/j.plaphy.2021.02.015;

He (2017), doi.org/10.1016/j.sajb.2017.05.032;

Pérez-Jiménez (2019), doi.org/10.1016/j.scienta.2018.09.072;

Mendanha (2018), doi.org/10.1016/j.plaphy.2018.09.002;

Liu (2016), doi:10.1111/jac.12190.

Previous studies have shown that under the precise control of the growth chamber, the differences caused by location on the plants are considered negligible. Moreover, both growth chambers in our experiment were able to provide precise environmental control. Therefore, the differences between SF and HF, HD treatments were mainly due to heat and combined heat and drought stress.

2)In figures 3 to 6, please indicate more clearly the four conditions (SF, SD, HF, and HD) on the x-axis.

Response: Thank you for your careful review. We have revised it according to the reviewers' comments. Figures 3-6 have been redrawn to more clearly show the four conditions on the x-axis.

3)What are the data analyzed in the ANOVAs (data of 3, 6, 9, or 12 days of stress treatment) in figures 3 to 5?

Response: Thank you for your careful review. In Figures 3-5, data from different measurement dates were pooled to analyze the effects of single factors and interaction on variables. For example, ANOVAs in figures 3 and 4 are from all the data of four measurement dates. ANOVAs in Figure 5 are from all data from 2 measurement dates. In addition, we analyzed the proportion of variance explained by each factor and interaction according to comment #5 from the reviewer.

4)The section 3.5 called “Growth analysis” is confusing and needs to be rewritten. For instance, data in Table 1 does not support the sentence “plant height under the HF and SD treatments increased with increasing N application”.

Response: Thanks a lot for the reviewer’s comments, we apologize for any confusion caused by our carelessness. We have rewritten 3.5 "Growth analysis" in accordance with the reviewer's comments.

5)Section 3.6 called “Yield, water and N use efficiency”, second paragraph. The authors wish to compare the effect of treatments and their interactions. They cannot do this based on the significance of the ANOVA, since the degree of freedom varies between own effects and interactions. I suggest analyzing the proportion of variance explained by each factor and interaction. The authors can do it using the sums of squares in ANOVA.

Response: Thanks a lot for the reviewer’s comments. This comment is very helpful to improve the quality of our papers. According to the comment of the reviewers, we analyzed the proportion of variance explained by each factor and interaction using the sums of squares in ANOVA. Specific modifications can be observed in figures 3-6 and Tables 1 and 2.

6)Section 3.7 called “Principal Component Analysis”. Because the authors discussed the correlation between yield-related traits and PSII-related traits, I suggest including the latter group of traits in the PCA. The authors have mainly interpreted axis 1 of the PCA by analyzing the traits contributing to this axis. Can they also interpret axis 2?

Response: We are grateful for your comment. According to the comments of reviewer, we have included the PSII-related traits (An, Fv/Fm, PSII and NPQ) in the PCA. Specific modifications can be seen in 3.5 "Principal component analysis". In the present study, principal component (PC1) corresponded to ADM, GNA, TKW, yield, SGN, An, GPY and Fv/Fm. These parameters contributed most of the variation observed in the dataset. The above variables mainly interpret axis 1 of the PCA, while they have relatively less interpretation for axis 2 of the PCA. WUEb, WUEg and NPQ corresponded to principal component 2, indicating that WUEb, WUEg and NPQ primarily interpreted axis 2 of the PCA.

7)Figure 8 and Table 3 are results that are currently presented in the discussion section and should be presented in the results section.

Response: Thanks a lot for the reviewer’s comments. According to the comments of reviewer, figure 8 and table 3 have been presented in the results section (3.6 The Relationship between photosynthetic rate and biological factors).

8)In the discussion, the authors developed a nice discussion about the relationship between reduced photosynthesis and plant response to drought and heat. They mentioned a good explanation of this relation by the damage of chloroplast by heat and drought. Other authors have referred to stomatal responses that prevent transpiratory water loss under heat and drought, and can be discussed by the authors.

Response: We are grateful for your comment. We added stomatal conductance (gs) data to Figure 3, and discussed the transpiratory water loss caused by stomatal response according to the comments of reviewer. Detailed supplementary contents can be seen in Section 4.1 of the discussion.

 

We have tried our best to revise and improve the manuscript according to the Reviwers′ good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval.

Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

Best regards.

Yours sincerely,

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have responded well to my comments.

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.