Comparative Primary Metabolite Profiling of Setaria viridis Reveals Potential Markers to Water Limitation
, Luana Beatriz dos Santos Nascimento 2,*,†, Mara Priscila Costa 2, Andrew Macrae 2,3, Marcio Alves-Ferreira 1,2, Camila Caldana 4 and Fernanda Reinert 2,5Round 1
Reviewer 1 Report
The information about C4 grasses Setaria viridis in response to water limitation is limited. The authors conducted the primary metabolic profile of roots, leaves, and panicles in response to three watering levels. Metabolites, such as proline, were selected out as drought bioindicator candidates. However, the manuscript is not easy to follow. The authors mixed results and discussion together. It was not easy to identify the results. The physiological and biochemical results are limited, but the authors conducted too much discussion of metabolic results. The modification was needed.
2. Materials and Methods
In the 2.1, methods of plant material and measurement of physiological parameter (dry weight and stomatal conductance) were suggested to separate. Not in one section.
Line 90, plants were watered with a commercial plant nutrient solution. What the components of the commercial plant nutrient solution?
3. Results and discussion
Overall, the authors mixed results and discussion together, it was not clear to follow. It was suggested to separate the results and discussion.
R Supplemental Figure S1 representative plants of each treatment was suggested to show in the manuscript. If so, please provide better photos of representative plants.
Did the authors have the dry weight for the shoots and roots. Not the whole plants.
Normally, the weight of roots probably increased under water limited condition.
Table 1
The authors stated the efficient photosynthetic activity and stem reserve accumulation during the vegetative phase is crucial for the formation of reproductive organs, water deficit can cause delay in or inhibition of floral development or early flowering, called drought scape in 162-174. But, the manuscript is lack of photosynthetic indexes of Setaria viridis under different water levels.
In line 186-187, Tricarboxylic Acid Cycle (TCA) intermediates? What are TCA intermediates? What these metabolites include?
Histograms in Fig. 4 and 5 is not easy to follow, heatmap is suggested.
Author Response
Responses to the reviewers (manuscript: agriculture-2212289)
Dear editor and reviewer,
Please find below a point-by-point reply to the reviewers’ comments. All changes in the manuscript we conducted using the track-changes tool.
We are grateful for the useful comments on our manuscript and we hope it will be now suitable for publication in Agriculture.
I look forward to hearing from you.
Yours sincerely,
Fernanda Reinert.
Referee 1
The information about C4 grasses Setaria viridis in response to water limitation is limited. The authors conducted the primary metabolic profile of roots, leaves, and panicles in response to three watering levels. Metabolites, such as proline, were selected out as drought bioindicator candidates.
a) However, the manuscript is not easy to follow. The authors mixed results and discussion together. It was not easy to identify the results.
- We thank the reviewer for the comment. However, we sincerely do not believe that the manuscript will benefit with the separation of the sections, because we built the rational of the study based on sequential blocks of complementary statistical approaches. Thus, the manuscript brings information regarding the primary metabolites profile of the different plant parts, followed by the comparison among the water treatments, and finally defining important bioindicators. Additionally, this aspect was taken care of by modifying some sentences, such as in lines 163, 191, 227, 239, 265, 305, 310 and others.
b) The physiological and biochemical results are limited, but the authors conducted too much discussion of metabolic results.
- Total yield (dry weight) and stomata conductance are two parameters included to show the degree of impact of each water regime. The study intended solely the characterization of primary metabolite profile of Setaria viridis in response to water limitation. It is a metabolomic study.
c) Materials and Methods. In the 2.1, methods of plant material and measurement of physiological parameter (dry weight and stomatal conductance) were suggested to separate. Not in one section.
- Thank you. Done (section 2.2).
d) Line 90, plants were watered with a commercial plant nutrient solution. What the components of the commercial plant nutrient solution?
- Thanks for the comment. We added the information of the nutrient solution used (Hoagland).
e) RSupplemental Figure S1 representative plants of each treatment was suggested to show in the manuscript. If so, please provide better photos of representative plants.
- These are the best photos available. That´s why we placed them in the supplemental section.
f) Did the authors have the dry weight for the shoots and roots. Not the whole plants.
- No. The intention of the total plant yield (dry weight) measurement was applied to ensure that the drought treatments were effective (i.e. that the plants suffered with the water limitation). The total plant dry weight does not add to our metabolomic analysis of the plant parts. This study did not aim for a physiological approach for the impact of drought.
g) Normally, the weight of roots probably increased under water limited condition.
- Yes, thank you. However, as mentioned above we did not collect these data.
h) Table 1
The authors stated the efficient photosynthetic activity and stem reserve accumulation during the vegetative phase is crucial for the formation of reproductive organs, water deficit can cause delay in or inhibition of floral development or early flowering, called drought scape in 162-174. But, the manuscript is lack of photosynthetic indexes of Setaria viridis under different water levels.
- Thank you for pointing that out. The sentence was removed.
i) In line 186-187, Tricarboxylic Acid Cycle (TCA) intermediates? What are TCA intermediates? What these metabolites include?
- Thanks for pointing out this issue. We included the information (line 190).
j) Histograms in Fig. 4 and 5 is not easy to follow, heatmap is suggested
- We thank your suggestion. However, all compounds present in the Figs. 4 and 5 are already present in the heatmap of the Fig. 2. In addition, the intention of figures 4 and 5 was to further analyze, in a more detailed way, the content of some compounds (bioindicators and common compounds in Venn diagram), which is better visualized by using a histogram. We improved the clarity of the Figs. 4 and 5, by removing the initials for the plant organs and replacing them by the full organ names.
Author Response File: 
Author Response.docx
Reviewer 2 Report
It is very interesting to detect metabolites under drought stress conditions, which will be useful for genetic investigation. However, there are some questions needed to further resolve. The main question is why only 65 metabolites were detected? Please clarify the metarials and methods. The another problem is large amount format errors in manuscript, such as line82, line129, and Table 1.
Author Response
Responses to the reviewers (manuscript: agriculture-2212289)
Dear editor and reviewer,
Please find below a point-by-point reply to the reviewers’ comments. All changes in the manuscript we conducted using the track-changes tool.
We are grateful for the useful comments on our manuscript and we hope it will be now suitable for publication in Agriculture.
I look forward to hearing from you.
Yours sincerely,
Fernanda Reinert.
Referee 2
It is very interesting to detect metabolites under drought stress conditions, which will be useful for genetic investigation. However, there are some questions needed to further resolve.
- We thank to the reviewer for the compliments and for the comments and suggestions.
a) The main question is why only 65 metabolites were detected?
We focused on the primary metabolites. The number of metabolites found depends on the plant species and on the methods used for extraction and analysis (including the limit of quantification and detection). We used methods extensively used for grasses (Ferreira et al., 2018), such as sugar cane, since S. viridis is a model for grasses. We focused on the quantification of 61 metabolites to compare their content among treatments. Other similar papers report comparable number of metabolites. For example, Song et al., 2022 (45 primary metabolites) and Yang et al., 2022 (73 primary metabolites).
Ferreira et al. 2018 - doi: 10.3389/fpls.2018.00857
Song et al. 2022 - https://doi.org/10.1111/tpj.15995
Yang et al. 2022 - DOI: 10.7717/peerj.13336
b) Please clarify the metarials and methods. The another problem is large amount format errors in manuscript, such as line82, line129, and Table 1.
We clarified the “Materials and methods” section (lines 96-100). We also checked the manuscript to correct any format mistakes, to assure the manuscript is within the journal’s template.
Author Response File: Author Response.docx
Reviewer 3 Report
Authors studied the change in metabolite profile in three different organ of S. viridis (as a C4 model plant) under drought stress to find a marker. The work is a novel study in metabolomics analysis of vegetative and reproductive organ of a C4 model plant. They measured relative concentration of 61 metabolites and analyzed using sPLS-DA, which only four metabolites were present in three organs. They highlighted twelve metabolites as biomarker candidates to response to water stress.
Author Response
Responses to the reviewers (manuscript: agriculture-2212289)
Dear editor and reviewer,
Please find below a point-by-point reply to the reviewers’ comments. All changes in the manuscript we conducted using the track-changes tool.
We are grateful for the useful comments on our manuscript and we hope it will be now suitable for publication in Agriculture.
I look forward to hearing from you.
Referee 3
Authors studied the change in metabolite profile in three different organ of S. viridis (as a C4 model plant) under drought stress to find a marker. The work is a novel study in metabolomics analysis of vegetative and reproductive organ of a C4 model plant. They measured relative concentration of 61 metabolites and analyzed using sPLS-DA, which only four metabolites were present in three organs. They highlighted twelve metabolites as biomarker candidates to response to water stress.
- We thank the reviewer for the comment and compliments.
Round 2
Reviewer 1 Report
The manuscript can be accepted after with language improvement.
Reviewer 2 Report
I have no additional comments.
