Influence of No-Till System with or without Cover Crops on Stomata Sensitivity of Glyphosate-Tolerant Soybeans to Vapor Pressure Deficit
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
Correction suggestions are inserted in the text.
Best regards,
Comments for author File: 
Comments.pdf
Dear Authors,
Correction suggestions are inserted in the text.
Best regards,
Author Response
RESPONSE TO REVIEWER 1
Abstract
It is important to highlight that the vulnerability of soybeans to drought will depend on the intensity and duration of water stress.
Q01: What are the main problems caused by drought in the soybean crop?
Answer01: Clarification has been made following reviewer’s recommendation (Lines 11-13)
Introduction
The introduction is well-founded on the theme proposed in the manuscript, however it has very old references, so it is extremely important to search the literature for current articles.
Q02: I suggest the author raise the hypothesis and justification of the work. It was not clear from the text.
Answer02: We have added hypotheses in the introduction in response to the comments (Lines 55-58). We also added more recent references.
Q03: The no-tillage system reduces mechanical tillage and soil erosion by how much %?
Answer03: Details were given following reviewer’s recommendation (Lines 32-34)
Q04: Author said: “Vapor pressure deficit has negative impact on crop production”
It is interesting to bring quantitative data to support this decline in vegetation.
Answer04: Clarification is now given following reviewer’s recommendation (lines 64-66)
Q05: Since what year has this problem been discussed?
Answer05: Clarification is now given following reviewer’s recommendation (lines 64-66)
Q06: Since this factor is directly linked to climate change, what should be done so that producers do not have a loss in production?
Answer06: Clarification is now given following reviewer’s recommendation (lines 76-81)
Materials and method
Q07: Why did you work with glyphosate-tolerant soybeans?
Answer07: Clarification is now given following reviewer’s recommendation (lines 98-102)
Q08: Format the caption of Figure 1
Answer08: We have formatted the caption of figure 1
2.2 Sampling and measurements
Q09: Stomatal conductance was measured at different time intervals (48h, 7, 14, 21 and 28 days), what was the parameter used in this determination?
Answer09: We aim to have an equivalent interval of days between the start of the safe exposure period after GBH application and before the majority of plants were in an advanced reproductive stage. Plants may change their gas exchange strategy between the vegetative and reproductive stages (i.e. filling period), which could have led to a bias between the values measured as a function of Vpd.
Also, 5 measurement periods represent an interesting compromise, in order to avoid trampling and compacting soil nearby the monitored plants, which could potentially have influenced their stomatal function and Gs values.
Q10: Why was anatomical characterization and histochemical tests not performed?
Answer10: We performed an anatomical characterization when we observed the various stomatal and foliar features under the scanning electron microscope. However, we did not carry out a histochemical test in this article, but the idea is worth being considered for a future work.
2.3. leaf trait
Q11: Why was 70% ethanol used instead of hydrated chloral?
Answer11: We used a protocol taken from another published article. We added the reference. We haven't considered hydrated chloral, but the idea is worth being considered in a future work.
Q12: How long were the samples left in the Safranin stain?
Answer12: For 30 minutes or until we obtained a sufficient staining of the foliar veins. We added the length of time the leaves were kept in the safranin stain (Line 159).
Q13: Why didn't you do physiological analysis?
Answer13: We plan to carry out further physiological analyses to quantify the impact of Vpd and glyphosate-based herbicide on cellular processes that may influence stomatal activity (e.i. ROS production, abscisic acid and other players involved in the redox stress cycle). However, this will presented in another manuscript in preparation.
Conclusion
Q14: In conclusion, you say that cover crops are promising to minimize the reduction of gaseous exchanges in soybeans triggered by spraying herbicides during the dry period.
How much % is this reduction? It's important information. I didn't find it in the results.
Answer14: No percentage is mentioned in the text regarding this matter. We did not specifically measure this in this study.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
The study conducted by Brillon et al. aims to investigate how stomata sensitivity changes according to seeding strategy (with soil cover crops x without). The study is interesting and the manuscript is well written. However, I have major suggestions regarding the methods description and other questions. The manuscript can be accepted after revisions. You can find my suggestions below:
Title
Productivity is not the focus of this paper. I recommend altering the title to something along the lines of Impact of No-Till Systems with or without Cover Crops on Stomata Sensitivity of Glyphosate-Tolerant Soybeans to Vapor Pressure Deficit. This title better reflects your primary and outcomes.
Abstract
Overall, the abstract is well-written. Nevertheless, I recommend refraining from utilizing abbreviations within this section. The abstract should be as reader-friendly as feasible, and an excessive reliance on abbreviations could challenge comprehension.
L. 14: what GBH means?
Introduction
Very well-written and clear introduction. However, a hypothesis is missing. What do authors expect from previous studies?
Methods
L115: Three different plants form each plot? Please, clarify.
L118: What was the duration of the gs measurements? As gs exhibits high sensitivity to hourly fluctuations, please, incorporate the time range from the beginning of the measurements to their conclusion.
L125: did the authors use any fixation method? Or authors use fresh leaves?
L126: If I’m not wrong, soybean has amphistomatic leaves (stomata on both leaf surfaces). Which leaf surface did the authors use? Please, clarify and make it more transparent throughout the entire manuscript. In addition, why not use both leaf surfaces for analysis? The ratio between adaxial stomatal density/ abaxial stomatal density is an important trait and may change due to your treatments.
L127: If electron microscopy was used, is presumably assumed that authors used fixation methods and samples were processed. Please, include the detailed techniques used.
L126: Those three locations were all located within the detached area in figure 2b?
Figure 2b: how this detached area used for measurement was standardized in all the leaves? Please, clarify.
L129: What’s the difference between stomata size, length, and width? Stomata size is total area of the stomata? Stomata pores? How was length measured? Maximum length of guard cells? Please, clarify how each measurement was conducted.
L148: how gsmax was determined?
L162: how dry mass production was determined. This method is missing in material and methods section. Moreover, I can’t find dry mass data within the paper.
Results
Figure 4: Can authors make each point with colors? Red for DS and blue for DSCC. I think it would improve the clarity of your data.
Discussion
Hypothesis testing is missing. Was your hypothesis corroborated or not?
The rest of the discussion is excellent.
Author Response
RESPONSE TO REVIEWER 2
Title
Q01: Productivity is not the focus of this paper. I recommend altering the title to something along the lines of Impact of No-Till Systems with or without Cover Crops on Stomata Sensitivity of Glyphosate-Tolerant Soybeans to Vapor Pressure Deficit. This title better reflects your primary and outcomes.
Answer01: The title has been changed following the suggestion of the reviewer 2
Abstract
Q02: Overall, the abstract is well-written. Nevertheless, I recommend refraining from utilizing abbreviations within this section. The abstract should be as reader-friendly as feasible, and an excessive reliance on abbreviations could challenge comprehension.
Answer02: We made small modifications that allow us to remove GBH, DS and DSCC as abbreviations in the abstract, which made it more reader-friendly.
Q03: L. 14: what GBH means?
Answer03: It means glyphosate-based herbicide. We removed this abbreviation in the abstract.
Introduction
Q04: Very well-written and clear introduction. However, a hypothesis is missing. What do authors expect from previous studies?
Answer04: We have added our hypotheses to the introduction (Lines 55-58)
Methods
Q05: Three different plants form each plot? Please, clarify.
Answer05: Yes. Clarification has been made (Line 124)
Q06: What was the duration of the gs measurements? As gs exhibits high sensitivity to hourly fluctuations, please, incorporate the time range from the beginning of the measurements to their conclusion.
Answer06: Clarifications (e.i. time range) have been made according to the recommendations (lines 131-133)
Q07: L125: did the authors use any fixation method? Or authors use fresh leaves?
Answer07: Clarifications have been made according to the recommendations (lines 141-144)
Q08: If I’m not wrong, soybean has amphistomatic leaves (stomata on both leaf surfaces). Which leaf surface did the authors use? Please, clarify and make it more transparent throughout the entire manuscript. In addition, why not use both leaf surfaces for analysis? The ratio between adaxial stomatal density/ abaxial stomatal density is an important trait and may change due to your treatments.
Answer08: Yes. Soybean has amphistomatic leaves but the number of stomata on the abaxial surface is significantly higher compared to adaxial surface (Bernier Brillon et al. 2023). Moreover, the majority of gas exchange occurs via stomata on the abaxial surface in the case of soybeans Stomata on abaxial surfaces are also the most sensitive to environmental variations. Also, in order to reduce the time required to perform stomatal conductivity measurements and to avoid potential bias, a compromise had to be made and only the abaxial surface was considered in this study.
Clarifications have been made according to the recommendations (Line 140) and we do specify the abaxial surface throughout the text.
Q09: If electron microscopy was used, is presumably assumed that authors used fixation methods and samples were processed. Please, include the detailed techniques used.
Answer09: The fresh leaf material was mounted directly on a slide using double-sided adhesive tape (Lines 141-144)
Q10: Those three locations were all located within the detached area in figure 2b?
Answer10: Yes. Clarifications have been made according to the recommendations (Lines 141-144)
Q11: Figure 2b: how this detached area used for measurement was standardized in all the leaves? Please, clarify.
Answer11: Clarifications have been made according to the recommendations (Lines 141-144). This detached area was measured approximatively considering that the surface area does not influence the number of stomata observed. We did not take into account stomata measuring less than 20 um found on this surface area otherwise this approach would be far too time-consuming. An average of the stomatal density observed following sampling at the three locations was calculated, providing the necessary information (Lines 149-151).
Q12: What’s the difference between stomata size, length, and width? Stomata size is total area of the stomata? Stomata pores? How was length measured? Maximum length of guard cells? Please, clarify how each measurement was conducted.
Answer12: Clarifications have been made according to the recommendations (lines 146-148)
Q13: How Gsmax was determined?
Answer13: Clarifications have been made according to the recommendations. (Lines 170-171)
Q14: How dry mass production was determined. This method is missing in material and methods section. Moreover, I can’t find dry mass data within the paper.
Answer14: We removed this part of the sentence. This is a draft mistake. Dry biomass was not considered in this article.
Results
Q15: Figure 4: Can authors make each point with colors? Red for DS and blue for DSCC. I think it would improve the clarity of your data.
Answer15: The points have been filled with colors to facilitate understanding and interpretation of the figure.
Discussion
Q16: Hypothesis testing is missing. Was your hypothesis corroborated or not?
Answer16: We have added the validation of our hypotheses in the discussion section (Lines 259-265).
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsClimate change means that crop plants are increasingly exposed to abiotic stresses related to heat or drought. Various methods of increasing plant resistance to these unfavorable changes are sought. It can be the breeding of new varieties. It can also be the introduction of cultivation methods that promote the activation of physiological processes in the plant and morphological changes that increase resistance to stress factors. Therefore, in my opinion, the subject of the research presented in the manuscript is up-to-date. The Manuscript is generally well written, the results are statistically processed and presented in charts and tables in a clear manner. Thematically, the manuscript falls within the thematic scope of the journal ‘Physiologia’. However, in my opinion, it needs changes and additions.
Detailed notes:
1. Please correct the way of citation in accordance with the requirements of the journal
2. line [36] - Please correct this sentence, it is not the seeds that are resistant to glyphosate, but the plants
3. line [51-53] - Please delete this sentence. Should be in the Materials and Methods section
4. line [71-72] - Please delete this sentence and put it at the end of the 'Introduction' section
5. Line [86] - It is enough to enter values accurate to two decimal places
6. Line [87-90] – I suggest that you compile the soil properties in a table, it significantly improves the readability of the information provided. Please specify what are the forms of the elements, total or available? Carbon should be in g kg-1 if it is organic carbon. When and how were soil samples taken for analysis?. Please describe briefly whether the content is low or high, in accordance with the standards adopted in your country/region.
7. Line [95] - I don't understand . Rye sown before corn harvest? How? When?. Please describe it in more detail. ‘Autum rye’ or ‘Winter rye’?
8. Line [99] - in the first term, the herbicide was applied before sowing, so there is probably a mistake here. In 2020, it was applied on June 2 and soybeans were sown on May 26?
9. Line [298-323] - this part needs to be removed. It has nothing to do with the results reported in the manuscript. The authors did not study the effect of application of the herbicide with glyphosate on the activity and development of stomata.
10. Literature should be listed in order of citation, not alphabetically.
Author Response
RESPONSE TO REVIEWER 3
Q01: Please correct the way of citation in accordance with the requirements of the journal
Answer01: We correct the citation in accordance with the requirements of the journal
Q02: Please correct this sentence, it is not the seeds that are resistant to glyphosate, but the plants
Answer02: Correction made following the reviewer’s recommendation (Line 37)
Q03: Please delete this sentence. Should be in the Materials and Methods section
Answer03: We moved this sentence to the materials and methods section (Lines 85-87)
Q04: Please delete this sentence and put it at the end of the 'Introduction' section
Answer04: We put this sentence at the end of the ‘’introduction’’ section as suggested by the reviewer (Lines 79-81)
Q05: It is enough to enter values accurate to two decimal places
Answer05: We used two decimals as suggested by the reviewer (Line 92-93)
Q06: I suggest that you compile the soil properties in a table, it significantly improves the readability of the information provided. Please specify what are the forms of the elements, total or available? Carbon should be in g kg-1 if it is organic carbon. When and how were soil samples taken for analysis?. Please describe briefly whether the content is low or high, in accordance with the standards adopted in your country/region.
Answer06: We removed the soil properties in the text and compiled them in a table (Table 1). We have also changed mg ha-1 to g ha-1 in the case of carbon, considering that the proportion of inorganic C in the samples is negligible.
Clarification have been proposed concerning the methodological approach to obtain the mineral content of the soil (Lines 89-91 and 94-95)
Q07: I don't understand. Rye sown before corn harvest? How? When?. Please describe it in more detail. ‘Autum rye’ or ‘Winter rye’?
Answer07: Clarification have been proposed following reviewer’s recommendation (Lines 104-105)
Q08: In the first term, the herbicide was applied before sowing, so there is probably a mistake here. In 2020, it was applied on June 2 and soybeans were sown on May 26?
Answer08: Clarification have been proposed following reviewer’s recommendation
Q09: This part needs to be removed. It has nothing to do with the results reported in the manuscript. The authors did not study the effect of application of the herbicide with glyphosate on the activity and development of stomata.
Answer09: We have removed this part following the reviewer’s recommendation
Q10: Literature should be listed in order of citation, not alphabetically.
Answer10: We have made the necessary changes so that the references are presented in the order in which they appear in the text.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAurhots addressed all my suggestions and corrections. The paper can be accepted in its present form.
Reviewer 3 Report
Comments and Suggestions for AuthorsManuscrypt has been corrected and supplemented.
