Functional Interpretation of Cross-Talking Pathways with Emphasis on Amino Acid Metabolism in Rhizosphere Microbiome of the Wild Plant Moringa oleifera

Round 1

Reviewer 1 Report

I find the work very interesting but the way it is presented in some parts the reader loses interest because there are several parts that are not connected and/or sections that are too descriptive.

 

The objectives of the work and the gaps that it intends to fill are not objectively presented. However, I must recognize that the authors have carried out an extensive and thorough investigation of the results obtained using a technique that has been little studied so far.

Check spelling and concordance of genera and number, throughout the manuscript there are a lot of these incoherences. For instance, rhizobacteria is plural, thus in line 32 instead of “this rhizobacteria” should be “these rhizobacteria”. Line 33, line 41 (these wild plants, as it is should be this wild plant, without S). There are just some examples, check in the MS. 

 

The introduction should be focused on agriculture, and how this specific plant may be useful in this field of researcher, based on what has been previously discover in other field studies (as described in the first paragraph of the introduction). Instead, the authors have included general information about its role in human medicine for instance. They should have focused on agricultural deficits, and how the study of the rhizobiome of this plant may be used on behalf of the difficulties that we are facing in the agro field.  Moreover, the different paragraphs of the introduction should be connected, rather they are presented watertight, each presenting its own subject. The introduction is lacking clear statements of gaps knowledge in the field and how this work may contribute.

The technique is of course very interesting, but without clearly stating what are the advantages and how does it fits on the work carried out in this field of research so far, the introduction is merely a brief and shallow highlight of the work.

Line 90-104: were the three samples then pooled or treated independently? The bulk soil was collected how far from the plant root? 10 meters? This was based on previous literature? How do you define that distance?

 

Regarding the presentation of the results, would it be possible to do a multivariate analysis so that an overall visualization of the data could be possible?

 

Line 203-204: this description on colours should be presented in the graph but maybe as a legend on the side of the figure? Also check Figure 4 legend: shouldn’t it be violet instead of violate (break rule, disregard)?

 

Discussion is very long and very detailed. Although after careful reading I noticed that authors had the care to “educate” the reader in very small aspects that are actually fundamental to understand these pathways. But the major results and/or findings of the work are lost within. Accordingly, I would suggest the authors to revise very carefully each section of the discussion and start each section by clearly stating the major finding.

 

Line 301-303: Which? Is there any literature supporting this hypothesizes?

Line 308-311: The authors state this here but it is supported by your data? Much information is presented like this throughout the results, but the reader must always go back to check if this is related to the data presented in this study or not.  

 

Line 338-340: information like this should be opening the section, clearly presenting a brief description of the results obtained, and only then explaining and relation how they contributed.

A conclusion section should be provided. It should include major results and achievements for crop improvement as well as limitations of this study. For instance, this type of analysis may not be available to every scientist. Alternatives?

Author Response

Comments and Suggestions for Authors

Comment 1: I find the work very interesting but the way it is presented in some parts the reader loses interest because there are several parts that are not connected and/or sections that are too descriptive.

Response: We would appreciate it very much if the reviewer referred to these specific parts in order to fix them accordingly.

Comment 2: The objectives of the work and the gaps that it intends to fill are not objectively presented. However, I must recognize that the authors have carried out an extensive and thorough investigation of the results obtained using a technique that has been little studied so far.

Response: Thanks for the valuable comment. Objectives were revised and changed based on the reviewer’s suggestion.

Comment 3: Check spelling and concordance of genera and number, throughout the manuscript there are a lot of these incoherences. For instance, rhizobacteria is plural, thus in line 32 instead of “this rhizobacteria” should be “these rhizobacteria”. Line 33, line 41 (these wild plants, as it is should be this wild plant, without S). There are just some examples, check in the MS.  

Response: Concordance of genera and number was revised and spelling fixed accordingly. Thanks to the reviewer.

Comment 4: The introduction should be focused on agriculture, and how this specific plant may be useful in this field of researcher, based on what has been previously discover in other field studies (as described in the first paragraph of the introduction). Instead, the authors have included general information about its role in human medicine for instance. They should have focused on agricultural deficits, and how the study of the rhizobiome of this plant may be used on behalf of the difficulties that we are facing in the agro field.  Moreover, the different paragraphs of the introduction should be connected, rather they are presented watertight, each presenting its own subject. The introduction is lacking clear statements of gaps knowledge in the field and how this work may contribute.

Response: A statement was provided in the “Introduction” section to indicate that this plant represents a model as to how plant species can benefit from their rhizobiome at the agricultural level.

Additionally, we preferred to start “Introduction” section by indicating the importance of this wild plant species at the medicinal and pharmaceutical levels, on one hand, and the high throughput technology that allowed us to detect plant-microbe interaction versus non-interactive microbes in the bulk soil, on the other hand. However, we have rephrased parts of the “Introduction” section to make the connection requested by the reviewer feasible.

Comment 5: The technique is of course very interesting, but without clearly stating what are the advantages and how does it fits on the work carried out in this field of research so far, the introduction is merely a brief and shallow highlight of the work.

Response: We have indicated the advantage of the technology we have used in order to get accurate figure on the structure of soil rhizobiome in order to give suggestions as to how we can benefit from these microbes via non-traditional methods, like metabolic engineering.

Comment 6: Line 90-104: were the three samples then pooled or treated independently? The bulk soil was collected how far from the plant root? 10 meters? This was based on previous literature? How do you define that distance?

Response: Samples were treated individually as stated in “M & M” section and in several figures and tables. But, in some cases, we preferred to introduce mean values of microbiome samples of either type of soil. We figured the safe distance of spot to collect bulk soil to be 10 meters apart from the spot of rhizosphere collection as this wild plant can have its roots extended laterally for at least 3-5 meters. There is no prior information about the standard safe distance as it differs from one plant to the other based on their root architecture.

Comment 7: Regarding the presentation of the results, would it be possible to do a multivariate analysis so that an overall visualization of the data could be possible?

Response: In fact, the experiment was designed to do univariate analysis, which includes the factor of soil type, e.g., rhizosphere versus bulk. We will be glad to know what might be the other factors to study based on the data recovered from this work.

Comment 8: Line 203-204: this description on colors should be presented in the graph but maybe as a legend on the side of the figure? Also check Figure 4 legend: shouldn’t it be violet instead of violate (break rule, disregard)?

Response: In each of the Supplementary figures, a color scale (between red for high enrichment to blue for low enrichment) was inserted on the side of each figure to indicate the level of enzyme enrichment. Spelling of violet was corrected. Thanks to the reviewer.

Comment 9: Discussion is very long and very detailed. Although after careful reading I noticed that authors had the care to “educate” the reader in very small aspects that are actually fundamental to understand these pathways. But the major results and/or findings of the work are lost within. Accordingly, I would suggest the authors to revise very carefully each section of the discussion and start each section by clearly stating the major finding.

Response: Thanks for the nice suggestion. We completely appreciate the comment made by the reviewer. Therefore, we found it important to subdivide the discussion section into 10 sub-sections to avoid overlapping or confusion among sub-sections. However, we further revised and modified these sub-sections as requested by the reviewer.

Comment 10: Line 301-303: Which? Is there any literature supporting this hypothesizes?

Response: No, there is no information in the literature to support our claim. We added a statement on the necessity to support this claim by further experimentation.

Comment 11: Line 308-311: The authors state this here but it is supported by your data? Much information is presented like this throughout the results, but the reader must always go back to check if this is related to the data presented in this study or not.  

Response: If we correctly understood this comment, nitrogen seems to be strongly produced in the present study from nitric oxide in the rhizosphere microbiome of M. oleifera and efficiently converted to ammonia via action of nitrogenase enzyme (EC 1.18.6.1) as shown in pathway “Nitrogen metabolism” of Figure S23. We emphasized these actions to refer to the mutual benefits between plant and surrounding rhizosphere microbes.

Comment 12: Line 338-340: information like this should be opening the section, clearly presenting a brief description of the results obtained, and only then explaining and relation how they contributed.

Response: Rearrangement of the first paragraph in “Chemotaxis” section was done based on the valuable suggestion of the reviewer.

Comment 13: A conclusion section should be provided. It should include major results and achievements for crop improvement as well as limitations of this study. For instance, this type of analysis may not be available to every scientist. Alternatives?

Response: Thanks for the valuable comment. A new conclusion section was inserted to indicate the merits of the study as well as the limitations and subsequent recommendations.

Author Response File: Author Response.pdf

Reviewer 2 Report

Line 41-44: The key research object is rhizosphere microbiome, what is the intention of emphasizing“especially when they are not in the form available for plant root absorption”?

Line 47-85: In Introduction, maybe some previous research results related to this article should be mentioned.

Line 86-88: What is the meaning of detecting metabolic processes and core metabolites? It is better to summarize it briefly.

Line 91-99: How many biological replicates were taken from two soil types?

Line 633-635: For a conclusion, this statement is a bit thin. It is necessary to develop the conclusion.

How were the figures and tables in the article made? What softwares were used? Maybe the "Data analysis" section should be added.

Author Response

Comments and Suggestions for Authors

Comment 1: Line 41-44: The key research object is rhizosphere microbiome, what is the intention of emphasizing“especially when they are not in the form available for plant root absorption”?

Response: The term “especially” was removed to avoid confusion.

Comment 2: Line 47-85: In Introduction, maybe some previous research results related to this article should be mentioned.

Response: We thought to introduce a brief information regarding different subjects of this work, while intensively discussed the previous research results in comparison to ours in the “Discussion” section to reach a more solid conclusions regarding the interaction between plant and its rhizobiome.

Comment 3: Line 86-88: What is the meaning of detecting metabolic processes and core metabolites? It is better to summarize it briefly.

Response: We have rephrased the sentence to indicate that these metabolic processes and core metabolites are major players in pathways that were discussed in this study and represent the link for occurrence of cross-talking among pathways.

Comment 4: Line 91-99: How many biological replicates were taken from two soil types?

Response: We used three biological replicates for each soil type, but in some cases we preferred to display means of these replicates as soil type representatives in comparing microbiomes of the two soil types. This approach was followed in editing Figures 2-5 and all supplementary figures.

Comment 5: Line 633-635: For a conclusion, this statement is a bit thin. It is necessary to develop the conclusion.

Response: Thanks for the valuable comment. A separate section was inserted to conclude our work and refer to current limitations and subsequent recommendations.

Comment 6: How were the figures and tables in the article made? What softwares were used? Maybe the "Data analysis" section should be added.

Response: All embedded figures in the text were edited by excel based on the data available in the different supplementary tables (excel files) of this study. Original files of supplementary figures are downloaded from the open source of Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Reviewer 3 Report

General comments:

In this work, the authors evaluated the wild plant Moringa oleifera and its rhizosphere microbiome were studied via metagenomic whole genome shotgun sequencing (mWGS) in comparison with bulk soil microbiome. The results indicated high gene abundance of the four categories “Cellular Processes”, “Environmental Information Processing”, “Genetic Information Processing” and “Metabolism” in rhizosphere microbiome. Amino acids in the rhizosphere might stand mainly as an intermediate switcher for the direction of soil nitrogen cycle. This studying signatured and metabolic capabilities of plant 633 rhizosphere microbiome of wild plants helps in deciphering new approaches for im-634 proving plant growth and defense mechanisms against pathogens. The research theme is significate and innovative, and the manuscript is generally well-written.

Furthermore comments:

1. The discussion sections are too long, the authors can reduce them.

2. Too many references, the authors can reduce the number of references.

Author Response

Comments and Suggestions for Authors

General comments:

In this work, the authors evaluated the wild plant Moringa oleifera and its rhizosphere microbiome were studied via metagenomic whole genome shotgun sequencing (mWGS) in comparison with bulk soil microbiome. The results indicated high gene abundance of the four categories “Cellular Processes”, “Environmental Information Processing”, “Genetic Information Processing” and “Metabolism” in rhizosphere microbiome. Amino acids in the rhizosphere might stand mainly as an intermediate switcher for the direction of soil nitrogen cycle. This studying signatured and metabolic capabilities of plant 633 rhizosphere microbiome of wild plants helps in deciphering new approaches for im-634 proving plant growth and defense mechanisms against pathogens. The research theme is significant and innovative, and the manuscript is generally well-written.

Response: Thanks to the reviewer for his kind and encouraging statement.

Furthermore comments:

1. The discussion sections are too long, the authors can reduce them.

Response: Analysis of high throughput datasets generates tremendous amount of information that are required to be introduced and discussed properly. To avoid confusion in the “Discussion” section due to our previous statement, we thought to sub-divide it to 10 sub-sections referring to the most important phenomena to be considered for discussion. If we reduced the capacity of any sub-section, we are afraid we might disregard an important issue that requires our attention. However, if the reviewer is not convinced with this response, we shall work on reducing capacity of this section and disregard some issues along with their citations.

2. Too many references, the authors can reduce the number of references.

The number of references is now 130 rather than 147. We wish the reviewer consider this as a sufficient response to his/her comment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have complied with the suggestions. I do not have any further comments.