A Phenomics and Metabolomics Investigation on the Modulation of Drought Stress by a Biostimulant Plant Extract in Tomato (Solanum lycopersicum)

Round 1

Reviewer 1 Report

The authors used a metabolomics approach to elucidate the mechanism of tomato drought stress alleviation by Biostimulant.

 

Metabolomics is a suitable method to reveal the effects of environmental stresses on crops, and it is an area of research that has received increasing attention in recent years due to the increasing number of reports.

 

The manuscript is well-written, and the discussion of the results is substantial and worthy of publication.

 

However, I believe that some minor improvements are needed.

 

 

1. I can't see the supplemental data. Please improve it.

 

2. The text in Figure is too small. The figure should be larger overall.

 

3. The vertical axis in Figure 2 is %?　The explanation is in %, but shouldn't the vertical axis be %?

 

4. Statistical analysis in 2.4.

It is stated that HCA and OPLS-DA were conducted, but the results are not shown in the figures and text. Please improve this.

What about Volcano Plot data?

Author Response

Please, find below author's comments:

I can't see the supplemental data. Please improve it.

Authors: supplementary material has been added, separately from the paper, during the submission process. From our account, we can regularly access the supplementary material (a zip folder including two excel files). Might this be related to the MDPI system?

The text in Figure is too small. The figure should be larger overall.

Authors: The figures have been enlarged.

The vertical axis in Figure 2 is %? The explanation is in %, but shouldn't the vertical axis be %?

Authors: Actually, the vertical axis is in µmol of photons m^2-1 s^-1 The light conditions of the growing chamber go from 0 to 60 % of its total intensity. The % of intensity per period (hours light-night) is shown below:

• 00.00-5.00: 0%
• 5.00-6.00: 10%
• 6.00-7.00: 20%
• 7.00-8.00: 30%
• 8.00-10.00: 40%
• 10.00-12.00: 50%
• 12.00-15.00: 60% (177,67 µmol of photons m^2-1 s^-1)
• 15.00-16.00: 50%
• 16.00-17.00: 40%
• 17.00-18.00: 30%
• 18.00-19.00: 20%
• 19.00-20.00: 10%
• 20.00-24.00: 0%

Statistical analysis in 2.4. It is stated that HCA and OPLS-DA were conducted, but the results are not shown in the figures and text. Please improve this.

Authors: Thank for the comment. Actually, the HCA and OPLS-DA were finally not included in the manuscript, since their output did not add so much to what already present. We apologize for the mistake; accordingly, we have deleted the sentence in the M&M section.

What about Volcano Plot data?

Authors: Thank the reviewer for the comment. The Volcano plot data is fully provided as supplementary table S2.

 

 

Reviewer 2 Report

The authors have presented a manuscript, which evaluated phenomics and metabolomics insight on the modulation of drought stress by a biostimulant plant extract in tomato. Cultivars were exposed to drought stress phase followed by a recovery phase. The manuscript presents interesting results concerning the selection of evaluated phenomics and metabolomics for monitoring response in the reponse of biostimulant plant extract in tomato, but they are some point which need to improve. Following, I have included some comments aimed to enhance the paper:

 ·    This work presents very interesting results and practice to increase the crop of tomato. I think that the authors can improve the format of results demonstration. The authors can highlight better the importance of the results obtained.

 

·    Conclusions Consider extending the conclusions and adding a Future works paragraph. The summary and Conclusions, it is better to combine them in only section of conclusions.  

 

Finally, the topic of this manuscript is interesting; since the selection of tomato species more tolerant to drought can contribute to the sustainability of water sources, but authors must improve the presentation of their results and discussion.

Author Response

This work presents very interesting results and practice to increase the crop of tomato. I think that the authors can improve the format of results demonstration. The authors can highlight better the importance of the results obtained.

Authors: Thanks for the comment. The results and discussion have been improved, especially making the tables and graph clearer.

Conclusions: Consider extending the conclusions and adding a Future works paragraph. The summary and Conclusions, it is better to combine them in only section of conclusions.

Authors: Thanks for the comment. The future works paragraph has been added to the manuscript.

Finally, the topic of this manuscript is interesting; since the selection of tomato species more tolerant to drought can contribute to the sustainability of water sources, but authors must improve the presentation of their results and discussion.

Authors: The results and discussion have been improved.

Reviewer 3 Report

Thanks to the authors for providing this useful study

This study provides valuable practical information on “phenomics and metabolomics insight on the modulation of drought stress by a biostimulant plant extract in tomato”. Therefore, the results of this study are important for researchers in this field. However, please pay attention to the following notes:

 

In the title: I see the replacement of the expression "insight" with an expression appropriate to the use of "biostimulant plant extract" in increasing the stress tolerance of tomato.

Also, in the title write the scientific name of the tomato in parentheses.

In abstract:

• As is well known, the abstract begins with an introduction on the subject of the study, with no more than two lines, then the study's objective, then the treatments that took place in the experiment in brief, then the most important results. That is, the abstract should give a complete and brief idea of the study.
• The results should be abbreviated in the abstract and an abbreviation of the treatments should be added.

In the introduction:

• Add a paragraph about any previous studies of the topic of this study on tomato.
• And another paragraph on the most important similar modern applications of tomato to increase its drought tolerance.

in Materials and Methods:

• Line 76: It is necessary to write the name of the tomato variety and the source.
• How was the amount of fertilizer converted from a hectare to a pot?
• Line 99: It should be added how the amount of irrigation water was estimated for each pot, based on the field capacity of the pot. That is, a paragraph must be added showing how the field capacity of the pot was calculated before planting in it.
• Line 131: How was the soil separated from the roots? There is a simple method (I do not know if it has been implemented) which is to put the pot in the water, so the whole roots are extracted after separating the soil from it by water without losing any part of it.
• Why was the content of proline not estimated? Which gives an indication of the extent of plant stress tolerance within the treatments that were applied in this study.

In Results:

• Line 222: If the content of chlorophyll is estimated the best, put its results here as well.
• All the figures "especially in the results" are not good clear (For example Figure 3, 4 and 5), so a different way must be found to place the figures in the text.
• Line 288: Table 6: The title of the table is long. It should be shortened, and it is enough to refer to what the table contains and not write a result within the table title.
• Also, in Table 6: Where is the statistical analysis and the significant differences?
• All Tables 7, 8 and 9: applies the same comments as above to Table 6.
• Line 266 and line 376: Title in Figure 5 & 7: The title is too long, this is not a title but a write of the result! It should be modified and shortened.
•  

In Discussion:

• Lines 402-409: There is no need to repeat the writing of results, but only discuss the topic of the result and compare it with the relevant references, and this does not exist.

In Conclusion: It is better to have a recommendation at the end of the conclusion.

Author Response

Please, find below the authors' comments:

In the title: I see the replacement of the expression "insight" with an expression appropriate to the use of "biostimulant plant extract" in increasing the stress tolerance of tomato. Also, in the title write the scientific name of the tomato in parentheses.

Authors: we amended the title as suggested.

In abstract:

• As is well known, the abstract begins with an introduction on the subject of the study, with no more than two lines, then the study's objective, then the treatments that took place in the experiment in brief, then the most important results. That is, the abstract should give a complete and brief idea of the study.

Authors: abstract has been revised as suggested by the reviewer

• The results should be abbreviated in the abstract and an abbreviation of the treatments should be added.

Authors: The results have been shortened in the abstract. Concerning the name of the treatment, we would prefer not to abbreviate it because “4-Vita” is the commercial name of the biostimulant.

In the introduction:

• Add a paragraph about any previous studies of the topic of this study on tomato.

Authors: Thank you for the suggestion. This part has been added.

• And another paragraph on the most important similar modern applications of tomato to increase its drought tolerance.

Authors: Thank you for the suggestion. This part has been added.

in Materials and Methods:

• Line 76: It is necessary to write the name of the tomato variety and the source.

Authors: The name of the tomato variety was already mentioned (Money maker) but wasn’t in italic. It has been changed. The source of the seed has also been added.

• How was the amount of fertilizer converted from a hectare to a pot?

Authors: Thank you for the comment. The data have been added to the manuscript.

• Line 99: It should be added how the amount of irrigation water was estimated for each pot, based on the field capacity of the pot. That is, a paragraph must be added showing how the field capacity of the pot was calculated before planting in it.

Authors: A paragraph with an explanation has been added.

• Line 131: How was the soil separated from the roots? There is a simple method (I do not know if it has been implemented) which is to put the pot in the water, so the whole roots are extracted after separating the soil from it by water without losing any part of it.

Authors: The roots were removed together with the soil. No separation was done between the soil and the roots., because the pictures were taken from each of the 4 faces. “After removing the shoot part (14 DAR), the root part was separated from the pot together with the soil. For each of the four faces of the pot, a picture was taken.”

• Why was the content of proline not estimated? Which gives an indication of the extent of plant stress tolerance within the treatments that were applied in this study.

Authors: In fact, amino acids (including proline) are amenable in UHPLC/QTOF metabolomics and is included in the database (PlantCyc) we used in compounds annotation. Therefore, the lack of information on proline can be related to a lack of significance in our experiments.

 

In Results:

• Line 222: If the content of chlorophyll is estimated the best, put its results here as well.

Authors: Thank reviewer for the suggestion. However, the chlorophyll content was not measured in our experiments, and we preferred to focus on photosynthetic performance instead. Nonetheless, this suggestion will be considered in future experiments.

• All the figures "especially in the results" are not good clear (For example Figure 3, 4 and 5), so a different way must be found to place the figures in the text.

Authors: The figures have been modified and are now clearer.

• Line 288: Table 6: The title of the table is long. It should be shortened, and it is enough to refer to what the table contains and not write a result within the table title.

Authors: Thanks for the comment. The title of the table is arranged based on the reviewer’s suggestion.

• Also, in Table 6: Where is the statistical analysis and the significant differences?

Authors: Table 6 shows the main metabolites involved in the biosynthesis processes of tomato treated with 4-Vita under drought stress. Since the Omics Dashboard analysis was carried out using only compounds that passed the volcano analysis, ANOVA P= 0.05 and fold change > 2, it means that all of these compounds and their fold change values are statistically significant.

• All Tables 7, 8 and 9: applies the same comments as above to Table 6.

Authors: Thanks for the comment. The title of tables is arranged based on the reviewer’s suggestion.

• Line 266 and line 376: Title in Figure 5 & 7: The title is too long, this is not a title but a write of the result! It should be modified and shortened.

Authors: The title of figures is modified based on the reviewer’s suggestion.

In Discussion:

• Lines 402-409: There is no need to repeat the writing of results, but only discuss the topic of the result and compare it with the relevant references, and this does not exist.

Authors: This part of the manuscript has been improved.

In Conclusion: It is better to have a recommendation at the end of the conclusion.

Authors: Thanks for the comment. The future prospective paragraph has been added to the conclusion part.

Round 2

Reviewer 3 Report

Thanks to the authors
All comments have been answered