Effects of Seasonal Variation on Nitrogen Use in Brazilian Cerrado Grass Communities

Round 1

Reviewer 1 Report

The present research evaluates the impact of seasonal variation on nitrogen use of several grass species in Brazil

The introduction is complete, making a link between the knowledge on nitrogen cycle and on the particular topic of the soils in Brazil and the availability for grasses. It presents clearly the research question. Minor correction in the document attached.

The material and method section is complete, except the description about the sampling date to be associated to the associated seasonal climate-weather conditions. Also is missing how the climate was characterized.

The results are well presented, with good figures and tables with the associated statistical analysis. But what the analysis of the climate-weather conditions and the impact on the resultas has never been studied, only there is a line on the conclusion, saying that the climate has a big impact. So it's necessary to add some analysis, either comparing the two seasons, and the year with the climate, and then make a good analysis at least in the discussion.

The conclusions are in line with the results obtained

Comments for author File: Comments.pdf

Author Response

The present research evaluates the impact of seasonal variation on nitrogen use of several grass species in Brazil.

The introduction is complete, making a link between the knowledge on nitrogen cycle and on the particular topic of the soils in Brazil and the availability for grasses. It presents clearly the research question. Minor correction in the document attached.

Response: We appreciate your detailed feedback. We have been committed to addressing the issues raised and have implemented the suggestions provided to the best of our ability. Your contribution has been fundamental to the ongoing refinement of the manuscript.

The material and method section is complete, except the description about the sampling date to be associated to the associated seasonal climate-weather conditions. Also is missing how the climate was characterized.

Response: We appreciate your valuable feedback. Following your suggestions, we have made the following modifications to the manuscript: the sampling dates for both dry and wet periods have been added. Additionally, we have included a climatic diagram for better contextualization of the study, which also addressed the comment regarding accumulated precipitation during the period under analysis.

The results are well presented, with good figures and tables with the associated statistical analysis. But what the analysis of the climate-weather conditions and the impact on the results has never been studied, only there is a line on the conclusion, saying that the climate has a big impact. So it's necessary to add some analysis, either comparing the two seasons, and the year with the climate, and then make a good analysis at least in the discussion.

Response: We understand the concern raised. To address this issue, we have made additions to the study concerning a more detailed characterization of the seasonal climatic conditions of the Cerrado and have included arguments in the results section regarding the relationship between this climate seasonality and nitrogen use by plants. This has enabled us to provide a better discussion of the observed patterns and their implications, reinforcing the assertions made in the conclusions of the paper.

The conclusions are in line with the results obtained

Response: We appreciate the review conducted. Your observations and suggestions have been extremely valuable and assist us in improving the quality of the work.

Reviewer 2 Report

Comments and Suggestions for Authors

Title: Effects of seasonal variation on nitrogen use in Brazilian Cerrado
grass communities

Dear Authors and Editors

The scope of research presented in the manuscript is consistent with the Nitrogen publishing profile.

The aim of this study was to characterize nitrogen availability and its use by Cerrado’s native grasses, and to check for a climatic seasonality effect over nitrogen presence and use by native grasses. The Material and Methods section requires significant additions. The Discussion and Conclusion sections are well written.

Remarks

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

§  Methods and Materials section: Line 98 …(Keller 2019, data not published). Such a citation should not be included because it cannot be verified. Please consider removing the last column in Table 1. For each device described in the Material and Methods section, add its place of production (country and city). Why were soil samples only taken from a depth of 0-10 cm? The rules for determining soil chemical fertility should be described. The ANR unit must be verified and recorded correctly. There is a need to supplement the principles of methods for determining all parameters presented in section 3. For what purpose were samples prepared in MCW solution for the determination of N-NO₃^-?

§  Results: On what basis was the low or high content of OM, P, and Fe determined? Maybe tables of limit numbers should be quoted. Table 2 - explain pHps. Which pH values are shown in Figure 1A? Nitrate Reductase Activity (ANR or NRA) should be correct. Table 3 - explanations should be placed under the table. Figure 2- units should be written in the SI system. Lines 222-231 should be included in the appropriate place in the MS.

Specific comments

All units should be corrected as required by the publisher. The record of citations should be improved according to publishing requirements.

Line 130 - Bieleski and Turner (1966) - not included in References.

Line 139 and 144 (Sokal and Rohlf 1995) - not included in References.

The list of references should be prepared in accordance with Nitrogen publishing requirements. The MS must be corrected according to the editorial requirements of the publisher.

Best regards

Author Response

Title: Effects of seasonal variation on nitrogen use in Brazilian Cerrado
grass communities

Dear Authors and Editors

The scope of research presented in the manuscript is consistent with the Nitrogen publishing profile.

The aim of this study was to characterize nitrogen availability and its use by Cerrado’s native grasses, and to check for a climatic seasonality effect over nitrogen presence and use by native grasses. The Material and Methods section requires significant additions. The Discussion and Conclusion sections are well written.

Remarks

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

• Methods and Materials section: Line 98 …(Keller 2019, data not published). Such a citation should not be included because it cannot be verified.
• Response: The reference Keller (2019) has been added and can be consulted at https://repositorio.unifesp.br/items/b08eb344-2633-435c-bbac-7793a6b37798.
•  
• Please consider removing the last column in Table 1.
• Response: We appreciate the suggestion. However, the other reviewer requested clarification regarding the Importance Value (IV). In response, we have added information to the text, explaining the IV and its relationship with variables such as relative frequency and relative coverage of the studied species. This has allowed us to provide a broader insight into the structure of a small portion of the campo sujo community.
•  
• For each device described in the Material and Methods section, add its place of production (country and city).
• Response: Understood, we have made the necessary additions in the Materials and Methods section, including the country and city, to the best of our knowledge, regarding the production of each described device.
•  
• Why were soil samples only taken from a depth of 0-10 cm?
• Response: The soil samples were collected at a depth of 0 to 10 cm because this superficial layer is where most of the soil nutrients are concentrated. This approach was adopted to characterize the chemical fertility of the soil at the study site. Collecting a single composite soil sample per plot in this superficial layer allowed for a representative assessment of soil fertility across the entire study area, while minimizing variability within plots. All of these aspects have been included in the text, and we appreciate the feedback.
•  
• The rules for determining soil chemical fertility should be described.
• Response: The official Brazilian reference on soil analysis standardization (H. G., dos Santos, P. K. T., Jacomine, L. H. C., Dos Anjos, V. A., De Oliveira, J. F., Lumbreras, M. R., Coelho,... & T. J.F., Cunha. Sistema brasileiro de classificação de solos. Brasília, DF: Embrapa, 2018) has been included.
•  
• The ANR unit must be verified and recorded correctly.
• Response: The unit of measurement for Nitrate Reductase Activity (NRA) was initially expressed in pkat g-1 FW, following the guidelines of previous works conducted by GR Stewart and various collaborators, as well as subsequent researchers, including studies carried out in Brazil in different vegetation formations such as the Forested Savanna, the Semideciduous Forest, and the Rainforest, as well as savanna formations in Australia. This will allow us to perform numerical comparisons in future works. However, if you are referring to the typographical error in "ANR," we would like to clarify that this has been corrected throughout the text, replacing it with "NRA." We acknowledge that it was a mistake on our part and assure you that it has been duly corrected.
•  
• There is a need to supplement the principles of methods for determining all parameters presented in section 3.
• Response: Reference bibliographies have been included to complement the methods, including the one that best explains the determination of NRA.
•  
• For what purpose were samples prepared in MCW solution for the determination of N-NO₃^-?

• RESPOSTA: Os teores de N-NO3- das plantas foram analisados utilizando material vegetal fresco extraído usando MCW (metanol:clorofórmio:água 12:5:3) e armazenado a -20°C por 24 horas ou mais antes de ser processado posteriormente. Esse mesmo MCW também foi destinado para os teores de aminoácidos livres, cujos dados ainda não forma publicados. Como trabalhamos com uma escassa quantidade de material vegetal fresco, seguimos a sugestão de Pereira-Silva et al. 2019a (Pereira-Silva, E. F. L., Casals, P., Sodek, L., Delitti, W. B. C., & Vallejo, V. R. (2019). Post-fire nitrogen uptake and allocation by two resprouting herbaceous species with contrasting belowground traits. Environmental and Experimental Botany, 159, 157-167) e Pereira-Silva 2019b (Pereira-silva, E. F. L., Hardt, E. Biral, M. B. Keller, V. C. e Delitti, W. “Effects of recent fire on soil conditions and nutrient use of a native and an invasive grass in the Brazilian savanna”, Écoscience).

•  
• Results: On what basis was the low or high content of OM, P, and Fe determined? Maybe tables of limit numbers should be quoted.
• Response: This interpretation should be considered locally, within the context of a tropical savanna environment, and was based on previous studies conducted in the same location. Additionally, other studies conducted by the group in other vegetation formations show savanna soils as being acidic and dystrophic (V% <30), with deficiencies in elements such as phosphorus (P < 2 and 3.3 mg/dm3). The high acidity is typical of tropical soils, possibly caused by higher aluminum availability, as observed in this study. Our other works show that the content of H+ + Al+3 and the low cation exchange capacity (CTC <190 cmolc L-1) suggest low chemical reactivity of organic matter (OM <60 g/dm3) and, therefore, relatively low soil fertility. In other areas, such as tropical forests, although dystrophic (m% <50, V% <30, OM <60 g/dm3), the soil has closer chemical thresholds but is more influenced by high rainfall, with lower content of H+ + Al+3 and lower CTC, exerting a subtle influence on concentrations of Ca+2, Mg+2, and K+ when compared to SF, thus being subtly more fertile soils. These references supported the interpretation of the soil in the studied area.
•  
• Table 2 - explain pHps.
• Response: The acronym pHps has been adjusted to pHss = soil solution pH.
•  
• Which pH values are shown in Figure 1A?

• Response: The value displayed in Figure 1A is the pH (H2O).

   

   
• Nitrate Reductase Activity (ANR or NRA) should be correct.
• Response: The text has been extensively revised, and the acronym for NRA has been adjusted.
•  
• Table 3 - explanations should be placed under the table.
• Response: The change has been made. Thank you for pointing that out.
•  
• Figure 2- units should be written in the SI system.
• Response: The unit of measurement used for Nitrate Reductase Activity (NRA), as well as for nitrate, was expressed in pkat g-1 FW and mg/g FW, following the protocols adopted by GR Stewart and other researchers in previous studies, and other studies that followed the same units. We emphasize that these standards have also been applied in subsequent research, including those conducted in Brazil in different types of vegetation, such as Forested Savanna, Semideciduous Forest, and Rainforest, as well as in savanna ecosystems in Australia and particularly for some botanical families. This approach will allow us to make numerical comparisons in future studies.
•  
• Lines 222-231 should be included in the appropriate place in the MS.
• Response: Change made, thank you for pointing that out.

Specific comments

All units should be corrected as required by the publisher. The record of citations should be improved according to publishing requirements. Line 130 - Bieleski and Turner (1966) - not included in References. Line 139 and 144 (Sokal and Rohlf 1995) - not included in References.

Response: A comprehensive review of the bibliography was conducted, and the missing references have been included.

The list of references should be prepared in accordance with Nitrogen publishing requirements. The MS must be corrected according to the editorial requirements of the publisher.

Response: I would like to thank you very much for your review and the care with your suggestions and points to improve this manuscript.

Best regards

Reviewer 3 Report

The manuscript entitled "Effects of Seasonal Variation on Nitrogen Use in Brazilian 2 Cerrado Grass Communities" is interesting and important, however, the manuscript needs revisions as follows.

1) Abstract: Line 16:  The abbreviation "ANR" for nitrate reductase activity should be "NRA".

2) Introduction: Line28-30; The authors referred amino acids as a representative of organic N forms, but the major form of organic N may be humic substances rather than amino acids.

3) Line 49: Nitrite Reductase (NiR) on chloroplasts may be Nitrite Reductase (NiR) in chloroplasts and plastids.

4) Materials and methods: Line 85: Please add the latitude and longitude of experimental field.

5) Line 97-98: Please explain importance value more acculately how to calculate from the data of frequency and coverage. What the IV means?

6) Table 1: Pease show the frequency and coverage data as well as importance value for each plant species harvested in the dry and wet season.

7) Line120-122: Please revise the analytical method to determine the in vivo nitrate reductase activity. The reference 49 by Stewart does not show the precise procedure for in vivo nitrate reductase activity. The reference of New Phytol. describe more precisely (1973) 72, 539-546- NITROGEN METABOLISM OF HALOPHYTES II. NITRATE AVAILABILITY AND UTILIZATION BY G. R. STEWART, J. A. LEE AND T . O. OREBAMJ O.

Please descrive the mathods here more precisely. 

8) Line 124: Please add the colorimetiric analysis of nitrite.

9) Discussion Line 293: Please explain what this sentence means "Competition and coexistence models indicate that the preference of a species for N-NH4 + or N-NO3 - can lead to its exclusion, coexistence, or dominance in a community".

Author Response

The manuscript entitled "Effects of Seasonal Variation on Nitrogen Use in Brazilian 2 Cerrado Grass Communities" is interesting and important, however, the manuscript needs revisions as follows.

1) Abstract: Line 16:  The abbreviation "ANR" for nitrate reductase activity should be "NRA".

RESPONSE: Regarding the typographical error in 'ANR,' we would like to clarify that this has been rectified throughout the text, replacing it with 'NRA.' We acknowledge that it was a mistake on our part and assure you that it has been duly corrected.

2) Introduction: Line28-30; The authors referred amino acids as a representative of organic N forms, but the major form of organic N may be humic substances rather than amino acids.

RESPONSE: The adjustment has been made, and the mention of humic acids has been included. Thank you for bringing it to our attention.

3) Line 49: Nitrite Reductase (NiR) on chloroplasts may be Nitrite Reductase (NiR) in chloroplasts and plastids.

RESPONSE: Thank you for the additional information. We are aware that Nitrite Reductase (NiR) can be present in both chloroplasts and plastids. The presence of NiR in chloroplasts and plastids suggests that this enzyme plays an important role in the assimilation and metabolism of nitrite in plants, contributing to fundamental processes such as nitrogen fixation and amino acid production.

4) Materials and methods: Line 85: Please add the latitude and longitude of experimental field.

Response: The coordinates have been added.

5) Line 97-98: Please explain importance value more acculately how to calculate from the data of frequency and coverage. What the IV means?

Response: The explanation has been included in the text. For clarification, the importance value, widely used in phytosociology as a structural index of plant communities, was determined using two main criteria: frequency and coverage. Frequency refers to the proportion of sample units in which the species was found, while coverage represents the sum of the coverage rates of individuals of each species relative to the sample unit. These two values were combined to calculate the Importance Value (IV), which is a measure that expresses the relevance of a species in a given environmental context.

6) Table 1: Pease show the frequency and coverage data as well as importance value for each plant species harvested in the dry and wet season.

Response: We have addressed the suggestion and included this information in table 1

7) Line120-122: Please revise the analytical method to determine the in vivo nitrate reductase activity. The reference 49 by Stewart does not show the precise procedure for in vivo nitrate reductase activity. The reference of New Phytol. describe more precisely (1973) 72, 539-546- NITROGEN METABOLISM OF HALOPHYTES II. NITRATE AVAILABILITY AND UTILIZATION BY G. R. STEWART, J. A. LEE AND T . O. OREBAMJ O.

Please descrive the mathods here more precisely. 

Response: We appreciate your feedback and the detailed observation regarding the analytical method used to determine in vivo NRA. We have revised and, as much as possible, supplemented the procedure, considering the suggested reference from New Phytol. from 1973 to obtain a more accurate description of the method.

8) Line 124: Please add the colorimetiric analysis of nitrite.

Response: The method for determining in vivo NRA has been detailed, and the colorimetric reading of nitrite has been included.

9) Discussion Line 293: Please explain what this sentence means "Competition and coexistence models indicate that the preference of a species for N-NH4 + or N-NO3 - can lead to its exclusion, coexistence, or dominance in a community".

Response: According to Boudsocq (2012), plants display diverse preferences for nitrogen sources, including nitrate, ammonium, or a combination of both. Within a community, the coexistence of species with different nitrogen utilization preferences can impact community dynamics, depending on their abundance and nitrogen resource preferences. Essentially, species with similar nitrogen utilization preferences may interact to either facilitate or compete with each other. Conversely, species with differing preferences for nitrogen utilization can result in varied outcomes such as exclusion, coexistence, or dominance within the community. These intricate interactions among species and their nitrogen utilization preferences play a pivotal role in shaping the structure and dynamics of plant communities.

Round 2

Reviewer 1 Report

The manuscrip has been corrected including weather conditions during the experiment in the material and method section and adding more discussion on this topic in the results/discussion section, which makes these section complete and more coherent the conclusion.

Two minor format comments to be checked/

Line 31: there is an extra "nitrogen" to be supressed

And line 128 pHH₂O: check the uniformity of the format along the document, maybe could be pH_H2O as is written in the same line pH_soil, later it appears as pH (H2O)

Author Response

The manuscript has been corrected including weather conditions during the experiment in the material and method section and adding more discussion on this topic in the results/discussion section, which makes these section complete and more coherent the conclusion.

Two minor format comments to be checked/

Line 31: there is an extra "nitrogen" to be suppressed.

Response: The repeated word was removed from the text.

And line 128 pHH2O: check the uniformity of the format along the document, maybe could be pHH2O as is written in the same line pHsoil, later it appears as pH (H2O)

Response: The text was checked, and the acronym was uniformly corrected to pH_H2O.

Reviewer 3 Report

The authors well-revised the manuscript followed by reviewer's comments.

The manuscript will be acceptable after some minor revisions.

1) Table 1 shows the result, so please move from the Materials and Method part to the Result part.

2) Line 145:  "After incubation, the assay mixture was analyzed for nitrite formation by spectrophotometry (λ = 540 nm), and the absorbance readings were converted to pkat g-1FW (Fresh Weight), considering the incubation time, the concentration of the reagents, and the amount of plant material used." may be changed to " After incubation, the assay mixture was analyzed for nitrite formation by After incubation, the assay mix- 145 ture was analyzed for nitrite formation by Saltman method [ref]. 1ml of mM sulfanilamide and 1 mL of mM N-(1-naphthyl)ethylenediamine solution were added to 1 ml of the assay mixture, and the induced azo dye was determined by spectrophotometry (λ = 540 nm) (Model, Company), and the absorbance readings were converted to pkat g-1FW (Fresh Weight), considering the incubation time, the concentration of the reagents, and the amount of plant material used."

Good

Author Response

The authors well-revised the manuscript followed by reviewer's comments.

The manuscript will be acceptable after some minor revisions.

1) Table 1 shows the result, so please move from the Materials and Method part to the Result part.

Response:  Thank you for the suggestion, and we have moved Table 1 to the results section and included a paragraph to contextualize this table (lines 182-190). Additionally, we have included in the Discussion a contextualization of the RC and RF from the table (lines 299-303).

2) Line 145:  "After incubation, the assay mixture was analyzed for nitrite formation by spectrophotometry (λ = 540 nm), and the absorbance readings were converted to pkat g-1FW (Fresh Weight), considering the incubation time, the concentration of the reagents, and the amount of plant material used." may be changed to " After incubation, the assay mixture was analyzed for nitrite formation by After incubation, the assay mix- 145 ture was analyzed for nitrite formation by Saltman method [ref]. 1ml of mM sulfanilamide and 1 mL of mM N-(1-naphthyl)ethylenediamine solution were added to 1 ml of the assay mixture, and the induced azo dye was determined by spectrophotometry (λ = 540 nm) (Model, Company), and the absorbance readings were converted to pkat g-1FW (Fresh Weight), considering the incubation time, the concentration of the reagents, and the amount of plant material used."

Response: Thank you for the writing suggestion, and we have seen that the nitrite analysis section has become clearer. We have made the requested changes.