Ethylene Regulates Combined Drought and Low Nitrogen Tolerance in Wheat: Proteomic Analysis

Round 1

Reviewer 1 Report

Ethylene regulates combined drought and low nitrogen tolerance in wheat: proteomic analysis

iji Yan , Daoping Wang , Shuyu Kang , Zhang He , Xin Li , Wensi Tang , Kai Chen , Yinghong Pan , Yongbin Zhou , Zhaoshi Xu , Jun Chen , Youzhi Ma , Ming Chen * , Chunhua Pang

Wheat yield, as well as yield quality, is severely affected by drought in combination with low nitrogen supply worldwide. Elucidation of molecular mechanisms of plant response to these stresses could provide the basis for elaborating the measures to alleviate the effect of these stresses. Because of the research of Yan et al. is of importance.

 The authors used the proteomics methods, supplemented them with analysis of expression of certain genes involved in the response and obtained evidence for involvement of ethylene in drought and low nitrogen tolerance.

Some corrections are recommended for the manuscript.

1.There is no supplementary table S3  in the file with the supplementary information.

2. In the Fig. 1 description, some letters (E-G) do not correspond to the diagrams:

lane 182 (E) is to be replaced by (F) and (F), by (G);

lane 183 (G) is to be replaced by (E)

3. In Fig.2 . lane 206, ‘seeding’ should be replaced by ‘seedling’

4. Do dots in Fig.2A correspond to replicates within a group? There are four dots for each groups, but only three replicates were mentioned in Materials and Methods. How this could be explained?

5. In Fig,2G, replicate DSL-2 strongly differs from replicates DSL-1 and DSL-3. Did this affect the conclusions on DAPs? And how is this relevant to the conclusion on Lane 199 “Strong correlations were obtained among all three replicates per sample (R >0.96)”?

6. It would be very interesting to know whether AAC could increase stress tolerance in the sensitive cultivar Shi4185. If there are such data, they could be added to this manuscript.

7. Is there any literature (or your previous) data about drought and low nitrogen tolerance of the cultivars Chang6878 and Shi4185 in the field? If there are, please, mention them in the Discussion.

Similarly, are there literature data on correlation of drought and low nitrogen tolerance of other wheat cultivars at the 2-leaf stage and the adult plant stage?

 

Author Response

1. The Supplementary Table S3 has been included with the files.
2. I have corrected this issue.
3. I have corrected this issue.
4. In Fig. 2A, points with the same color represent replicates within each group. Initially, each sample had four independent biological replicates, and we described this in the Materials and Methods. Based on the PCA (Fig. 2A), we selected three similar replicates from each sample for further analysis.
5. There is inherent variation between biological replicates, and replicate DSL-2 strongly differed from replicates DSL-1 and DSL-3. However, this did not affect comparisons between DSL and other groups, and thus did not impact the conclusions regarding DAPs. Fig. 2G is a heatmap of the DAPs. Lane 199 describes the results of Fig. 2B, which is a correlation analysis of the overall proteomic data. These two figures cannot be compared as they represent different analyses.
6. When verifying ACC for ethylene synthesis, the ethylene inhibitor silver nitrate and the ethylene signal transduction inhibitor 1-MCP were tested for their effects on drought and low nitrogen dual stress in wheat. Both varieties were treated, and it was found that the sensitive Shi4185 did not show significant differences after treatment. Therefore, this part of the results was not presented in the manuscript.
7. There is currently no literature or previous data on the drought tolerance and low nitrogen traits of the long 6878 and stone 4185 varieties under field conditions. This highlights the necessity for future research in this area. Subsequent field experiments and comparative studies can provide valuable insights, helping us understand the performance of these varieties under challenging environmental conditions.
8. Similarly, are there literature data on correlation of drought and low nitrogen tolerance of other wheat cultivars at the 2-leaf stage and the adult plant stage?

        We added a section to the Discussion.

Author Response File: Author Response.docx

Reviewer 2 Report

The MS entitled “Ethylene regulates combined drought and low nitrogen tolerance in wheat: proteomic analysis” with authors Jiji Yan, Daoping Wang, Shuyu Kang, Zhang He, Xin Li, Wensi Tang, Kai Chen, Yinghong Pan, Yongbin Zhou, Zhaoshi Xu, Jun Chen, Youzhi Ma, Ming Chen, and Chunhua Pang presents original new data and is interesting but needs minor improvement to be published.

Wheat (Triticum aestivum L.) is the world’s second most cultivated cereal crop, after rice, and is consumed as a staple food in many countries. Wheat is one of the major grains in the diet, and plays a significant role in the nutritional quality of the diet and in human health. Among different abiotic stresses, drought stress has harmful effects on growth and development of wheat crop. The authors' focus is to evaluate the mechanism underlying drought and LN resistance in wheat (using two wheat varieties), and the results may serve to facilitate the breeding of drought and LN resistant wheat. It is important to search and study existing and induced biodiversity with a view to finding sources of agronomically valuable traits. In this regard, the research done in the presented manuscript merits attention.

The article follows the order required by the journal: Introduction, Materials and Methods, Results and Discussion, Conclusions. Abstract is informative; the Introduction is good; the purpose of the study is clearly formulated. Materials and methods are well described. Over 84 % of the cited literature is from the last 10 years, оf which over 50% are from the last 5 years. It is essential to improve data presentation. I recommend that authors take in mind the notes I have made below.

The manuscript is not formatted according to the requirements of the Journal. References must be numbered in order of appearance in the text (including table captions and figure legends) and listed individually at the end of the manuscript. Please, check carefully “Instructions for Authors”  https://www.mdpi.com/journal/agronomy/instructions .

L15 – Please, remove the name of corresponding author, to be written just the e-mail and Author's abbreviation. The section “Author Contributions” - similarly.

I have some minor comments on the text:

L88 – Please, give more information about both Chang6878 and Shi4185 wheat varieties. Please, indicate if the seeds used for analysis are from one plant or from several.

L90 – Please, fix the formula

L130 - ”(FC) > 1.2 or < 0.83” Why did you choose these thresholds for up and down-regulation of proteins?

Author Response

The manuscript is not formatted according to the requirements of the Journal. References must be numbered in order of appearance in the text (including table captions and figure legends) and listed individually at the end of the manuscript. Please, check carefully “Instructions for Authors” https://www.mdpi.com/journal/agronomy/instructions .

Response: I have modified the format of the references.

 

L15 – Please, remove the name of corresponding author, to be written just the e-mailand Author's abbreviation. The section “Author Contributions” - similarly.

Response: We have corrected those parts.

 

I have some minor comments on the text:

L88 – Please, give more information about both Chang6878 and Shi4185 wheat varieties. Please, indicate if the seeds used for analysis are from one plant or from several.

Response: The origin and characteristics of Chang6878 and Shi4185 have been described in more detail in the Materials and methods.

L90 – Please, fix the formula

Response: I have revised this formula.

L130“(FC) > 1.2 or < 0.83” Why did you choose these thresholds for up and down-regulation of proteins?

Response: Many proteins with significant differences in abundance could serve as DAPs, but to minimize the number of DAPs, we selected FC > 1.2 or < 0.83 as additional up- and downregulation thresholds for screening.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript is well written only a minor modifications are required as explained below

1- add prospects of study and some relevance to food security in abstract

- add some more reviews especially food security and climate change in introduction

-captions of the figures and tables must be self explanatory 

 

It is fine, only minor changes are required 

Author Response

1. add prospects of study and some relevance to food security in abstract

Response: I have explained the prospects of study and mentioned the relevance of our work to food security in the Abstract.

2. add some more reviews especially food security and climate change in introduction

Response: I have referred to some more reviews regarding food security and climate change in the Introduction.

3. captions of the figures and tables must be self explanatory

Response: I have checked the captions of the figures and tables to make sure that they are self explanatory.

Author Response File: Author Response.docx