The Effect of Water Stress on the Glucosinolate Content and Profile: A Comparative Study on Roots and Leaves of Brassica oleracea L. Crops

Round 1

Reviewer 1 Report

Dear editor,

The study evaluates the effects of water deficit on a set of accessions belonging to the Brassica core and also analyzed the variation in the amount and profile of the glucosinolates (GLSs) compounds on the root and the leaf tissues. The study was well designed and suitable for publishing in the Agronomy journal but the authors should modify some corrections. Also, for improving manuscript quality for readers and better identification of results, I recommend to adding more physiological data which are written in the comments. Therefore, my final recommendation is "Major revision".

Abstract

- L26-27: This sentence should be deleted from this section and should be added in materials and methods section.

- L28-32: These sentences are not clear. The authors noted that ‘The GLSs profile varied significantly among both genotypes and portions of the plant analyzed ….’ Which was the highest?

- L28-32: The authors noted that ‘Plants grown under drought stress, for the last two weeks of the growing cycle under consideration, showed increased GLSs levels compared to well-watered plants.’ How many percentage?

- L34-35: What is the best recommendation for organic breeders?

Introduction

- This section is good. However, the linkage between paragraphs is missed. The authors should be added the negative impacts of drought stress to plants.

- L96 and L97: The citation format is not true. Please check all text.

- Highlight the novelty of this study.

- The hypothesis should be added at the end of this section.

- The experimental design, factors and treatments should be added in this section clearly.

- I recommend to adding more physiological data such as proline content, ROS compounds concentration, antioxidant enzymes activity, etc.

- How many repetitions were used for measuring studied traits.

Results

- Please add the means comparison results for determining the significance or non-significance among means.

- I recommend to delete the %var from tables.

- Please add the increasing or decreasing percentage between treatments.

- The tables and figures should be cited in text.

- I recommend to adding more physiological data such as proline content, ROS compounds concentration, antioxidant enzymes activity, etc.

Discussion

- This section should be deeply revised. The authors should be discussed why the studied traits, especially agronomic traits, affected negatively by drought stress conditions.

- The authors should be discussed how plant adapt to drought stress through increasing or decreasing GLs.

- The citation format is not true in this section.

L 456-470: I can see too many paragraphs in this section that are reporting the previous studies reports. This is not a really discussion. Please try your best to illustrate the possible reasons after this.

Conclusion

- This section is repetitive and should be rewritten.

- Please make sure your conclusions' section underscores the scientific value-added of your paper, and/or the applicability of your findings/results. Highlight the novelty of your study.

- What suggestions do you have for future research in this field?

Author Response

List of responses to the Editorial board member’s comments

All changes proposed by the reviewer 1 were tacked

List of responses to the Reviewer 1’s comments

Comments to the manuscript entitled ‘The Effect of Water Stress on the Glucosinolate Content and Profile: a comparative study on roots and leaves of Brassica oleracea L. crops’ written by Hajer Ben Ammar, Donata Arena, Simone Treccarichi, Maria Concetta Di Bella, Nadia Ficcadenti, Roberto Lo Scalzo, Sonia Marghali and Ferdinando Branca (Manuscript Number: agronomy-2166897).

The study evaluates the effects of water deficit on a set of accessions belonging to the Brassica core and also analysed the variation in the amount and profile of the glucosinolates (GLSs) compounds on the root and the leaf tissues. The study was well designed and suitable for publishing in the Agronomy journal, but the authors should modify some corrections.

General comments

1. The study evaluates the effects of water deficit on a set of accessions belonging to the Brassica core and also analysed the variation in the amount and profile of the glucosinolates (GLSs) compounds on the root and the leaf tissues.

Response: In the BRESOV project, we are interested to glucosinolate, and our aim was focused on this molecule to select the varieties of Brassica oleracea that showed a high amount of this bioactive compound with an interesting profile. As water stress increases the level of several bioactive component, we were interested to evaluate the effect of water deficit on the amount and profile of glucosinolate, and we will use those data in the future work of breeding.

2. For improving manuscript quality for readers and better identification of results, I recommend to adding more physiological data which are written in the comments.

Response: In this paper, we are interested to glucosinolate as a very important molecule of Brassica oleracea. We have analyzed some morphometric traits to detect the phenotypic resistance to water stress. Regarding the physiological data we will plan to analyze it.

Specific comments

Abstract

1. L26-27: This sentence should be deleted from this section and should be added in materials and methods section.

Response: As requested, this sentence was removed from the abstract and was inserted in the material and method section (2.3. High-Performance Liquid Chromatography (HPLC) Analysis).

2. L28-32: These sentences are not clear. The authors noted that ‘The GLSs profile varied significantly among both genotypes and portions of the plant analyzed ….’ Which was the highest?

Response: A clarification has been added in L31-L33: Plants grown under drought stress, for the last two weeks of the growing cycle under consideration, showed higher amount of GLS in leaves (190.1 ± 8.9 µmol. g-1 d.w) comparing to roots (17.3 ± 1.9 µmol. g-1 d.w).

3. L28-32: The authors noted that ‘Plants grown under drought stress, for the last two weeks of the growing cycle under consideration, showed increased GLSs levels compared to well-watered plants.’ How many percentages?

Response: A sentence has been added in L34-L36 to clarify how many percentages of GLSs levels increasing: Under water stress conditions, the highest increase of the glucosinolate amount was detected in roots of broccoli (accession BR1) with 85.4% and in leaves of cauliflower (accession CV1).

4. L34-35: What is the best recommendation for organic breeders?

Response: The recommendation for organic breeders have been improved as requested by the reviewer focusing on the aim of our work which is the screening and the selection of the best tolerant Brassica oleracea genotypes to water stress with high amount of Glucosinolates (especially glucoraphanin and sinigrin that had a high potential in health) and the use of those crops for crossing.  The following sentence was added in the text: The chemotypes selection of Brassica oleracea allow important time reduction during the organic breeding programs after crossing accessions with specific profile and high amount of glucosinolates.

Introduction

1. This section is good. However, the linkage between paragraphs is missed. The authors should be added the negative impacts of drought stress to plants.

Response: The linkage between paragraphs is added; in the end of each paragraph we add one sentence about the next section. Moroever, we have added in the introduction the negative impacts of drought stress in plant in lines L64-81.

2. L96 and L97: The citation format is not true. Please check all text.

Response: The citations have been checked and written in the propriate format in all text.

3. Highlight the novelty of this study.

Response: All the introduction has been improved to highlight the novelty of the study. Please see the text in the manuscript.

4. The hypothesis should be added at the end of this section.

Response: In the end of the introduction section, the hypothesis has been added in line L136. Please see the text in the manuscript.

Material and Methods

1. - The experimental design, factors and treatments should be added in this section clearly.

Response: In order to clarify the approach of this study we had explained the experimental design factors, which are: the genotypes (17 accessions) of Brassica oleracea studied and the Irrigation system (IRR), with the irrigated plants (IRR) and the not irrigated (NIR) which are considered respectively as control and stressed samples. Then we have evaluated the interaction between the factors in the statistical analysis.

2. - I recommend to adding more physiological data such as proline content, ROS compounds concentration, antioxidant enzymes activity, etc.

Response: As recommended by the reviewer, we have inserted in this section more physiological data that had improved the introduction.

3. - How many repetitions were used for measuring studied traits.

Response: As mentioned in the experimental design and the statistical analysis, we have used 3 replicates for measuring studied traits.

Results

1. Please add the means comparison results for determining the significance or non-significance among means.

Response: As requested by the reviewer, the means comparison results realized by ANOVA have been added, represented by a table for determining the significance or non-significance among means.

2. - I recommend to delete the %var from tables.

Response: This recommendation has been considered and we removed the % variation from tables.

3. - Please add the increasing or decreasing percentage between treatments.

Response: The decreasing and increasing percentage between treatments had been calculated following the formula cited in the material and method Increasing percentage = -(100-(Control/stress *100)).

4. The tables and figures should be cited in text.

Response: all the tables and figures are cited in the text

5. I recommend to adding more physiological data such as proline content, ROS compounds concentration, antioxidant enzymes activity, etc.

Response: As explained in general comments, our study is focused on the Glucosinolates profile and content to select the best tolerant Brassica oleracea genotypes to water stress characterized by specific chemotype and high amount of glucosinolates. We will plan to study the physiological mechanism related to water stress in the future research.

Discussion

1. This section should be deeply revised. The authors should be discussed why the studied traits, especially agronomic traits, affected negatively by drought stress conditions.

Response: All the discussion has been rewritten and deeply revised as requested by the reviewer. In addition, in the paragraph L605-614, the impact of water stress on the agronomic traits has been discussed.

2. The authors should be discussed how plant adapt to drought stress through increasing or decreasing GLS.

Response: As reported in the discussion, there are distinct differences in the total amount and profile of glucosinolates among accessions in relation to water stress. In fact, the GLSs content was twice higher in water-stressed plants compared to the control. We can say that drought stress increases glucosinolates accumulation and this mechanism could be a part of plant response through the process of osmotic adjustment. Besides, we had mentioned others studies that highlighted the increase of Glucosinolate as a response to water stress.

3. The citation format is not true in this section.

Response: the citation has been changed following the request format

4. L 456-470: I can see too many paragraphs in this section that are reporting the previous studies reports. This is not a really discussion. Please try your best to illustrate the possible reasons after this.

Response: Many thanks for your suggestion, this part of the discussion was improved focusing on the comparison of our results to the other studies not just reporting.

Conclusion

1. This section is repetitive and should be rewritten.

Response: All the section of the conclusion has been rewritten as requested.

2. Please make sure your conclusions' section underscores the scientific value-added of your paper, and/or the applicability of your findings/results. Highlight the novelty of your study.

Response: The conclusions have been improved as requested to highlight the novelty of this study. In fact, we have added the scientific value of this study. The obtained results indicate that the accumulation of Glucosinolate is higher in leaves than in roots and it is increased under water stress conditions. Besides according to our funding, each variety is characterized by a specific profile that varies in relation to water deficit. This approach could be helpful for breeding programs. Indeed, the experimental factors analyzed offer the opportunity to improve the knowledge related to Glucosinolate.

3. What suggestions do you have for future research in this field?

Response: The conclusions have been improved and rewritten as requested by the reviewer. In fact, as mentioned at the end of the conclusion, further research using a genomic and transcriptomic approaches targeting some candidate genes is needed to confirm this finding.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors show the glucosinolate content of roots and leaves of B. oleracea.

 First of all, I think that the number of materials analyzed in this experiment is small.

 It's such a great idea to analyze the roots and leaves. Nevertheless, is the root of B. oleracea being used for food?

 The authors mentioned SPAD in the results related to Table 2, but they are missing in materials and methods.

 Line 215: Please check if the description of CV3 is correct.

 s there any way to express Figure 1 with high readability? How about representing it as a circle?

Please explain a bit more about PCA. You might want to try clustering.

Table 5 shows the correlation results. Why don't you explain the results of this and the metabolic pathway of glucosinolate synthesis in a discussion together?

Overall, the description of the results is poor.

Author Response

List of responses to the Editorial board member’s comments

List of responses to the Reviewer 2’s comments

Comments to the manuscript entitled ‘The Effect of Water Stress on the Glucosinolate Content and Profile: a comparative study on roots and leaves of Brassica oleracea L. crops’ written by Hajer Ben Ammar, Donata Arena, Simone Treccarichi, Maria Concetta Di Bella, Nadia Ficcadenti, Roberto Lo Scalzo, Sonia Marghali and Ferdinando Branca (Manuscript Number: agronomy-2166897).

The authors show the glucosinolate content of roots and leaves of B. oleracea.

1. I think that the number of materials analyzed in this experiment is small.

Response: Actually, the management of the experimental design is delicate as you have observed in this paper we have worked in control and water stress conditions. In addition, we analyzed two organs of each plant (roots and leaves) and the detection of 10 glucosinolates needs a lot of analysis and in any case, we have tried with different accessions of different species well used nowadays. It is important to note that it was a qualitative and quantitative study to screen and select the one with the highest amount of glucosinolate and a specific profile.

2. It's such a great idea to analyze the roots and leaves. Nevertheless, is the root of oleracea being used for food?

Response: Thank you for your question to clarify this point, actually its s not common to use the roots of Brasscia oleracea as a food, in our study we wanted to evaluate which organ accumulate more Glucosinolate and how it changes in relation to water stress, as the roots are the first detector of the deficient of the water. Regarding the nutritional potential of this part why not to use it (the powder of freeze-dried material) in cooking    

3. The authors mentioned SPAD in the results related to Table 2, but they are missing in materials and methods.

Response: A-the method of SPAD is inserted in the section material and method

4. Line 215: Please check if the description of CV3 is correct.

Response: The description is checked, it was a mistake BH2 showed the lowest variation of root weight not CV3

5. Is there any way to express Figure 1 with high readability? How about representing it as a circle?

Response: A-Many Thanks for your suggestion, the figure is replaced by circles to better represent the data

6. Please explain a bit more about PCA. You might want to try clustering.

Response: An explanation has been added to this section to clarify the result, this suggestion has improved our work and highlighted the result of PCA.

7. Table 5 shows the correlation results. Why don't you explain the results of this and the metabolic pathway of glucosinolate synthesis in a discussion together?

Response: Thank you for the suggestion that have improved our result, we will join the correlation with the metabolic pathway.

8. Overall, the description of the results is poor.

Response: A-The section of the results has been improved.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear editor,

In the revised version, the authors have appropriately edited and revised this earlier version according to the comments and suggestions from the reviewers and have reasonably addressed most of the concerns and issues in the review reports. The current version could be accepted for publication in this journal.

Best regards

Author Response

 Many thanks dear reviewer for your comments and your suggestions that improved our manuscript. Following your instructions gave us the opportunity to clarify our approach and highlight the results

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors responded appropriately to my comments and reflected them in the paper. Also, many corrections were made. Thank you very much for this part.

I think the content of the thesis is sufficient for publication.

However, it is recommended that some tables be moved to supplementary to improve readability for the reader. In addition, the PCA figure has two overlapping contents, so it is recommended to combine them into one.

In my opinion, it is important to show the results of the research well, but it is also important that the figures and tables harmonize like the contents.

Author Response

English language and style are fine/minor spell check required
Answer: The English had been checked by a native speaker
The conclusion should be improved 
Answer: As requested, this section is improved and you will find in track mode the modification 
*The authors responded appropriately to my comments and reflected them in the paper. Also, many corrections were made. Thank you very much for this part. I think the content of the thesis is sufficient for publication.
Answer: Many thanks dear reviewer for your comments. Your revision had improved the manuscript.
*However, it is recommended that some tables be moved to supplementary to improve readability for the reader. 
Answer: The tables are removed to supplementary data as requested; this detail improved the readability of the manuscript.
*In addition, the PCA figure has two overlapping contents, so it is recommended to combine them into one.
Answer: As requested, in the PCA figure the overlapping content has been combined and explained in the text.
*In my opinion, it is important to show the results of the research well, but it is also important that the figures and tables harmonize like the contents
Answer: the results of the research are shown in a clear way, as requested the tables are removed to supplementary data, and in this way the contents

Author Response File: Author Response.pdf