Round 1

Reviewer 1 Report

Dear Author,

I really enjoyed reviewing the manuscript entitled "The effects of exogenously applied copper in tomato plants' oxidative and nitrogen metabolisms under organic farming conditions" by Alexandre Alves, Rafael Ribeiro, Mario Cunha and Jorge Teixeira.

The purpose of the research presented in this manuscript was to determine the effects of exogenously applied copper in tomato plant grown in organic farming systems.

It's been a long time since I reviewed such a well-written manuscript. The subject of research is important from the point of view of the development of sustainable agriculture.

The theoretical introduction to the research is satisfactory. In my opinion, the research methods are appropriately selected and well presented in the Materials and Methods chapter. The results are clearly presented and graphically presented in a very readable way. I like the discussion very much. I have only a few minor remarks that Authors should take into account:

1) In the Abstract chapter, please add a short research hypothesis.

2) In the Introduction chapter in the last paragraph, please also formulate the research hypothesis.

3) Table 1 - please explain the abbreviations GR and EF1.

4) Figures 1-10 - the explanation of the abbreviation "ns" is missing, please complete. And also in all the figures above the first bars "C" there is no "*" or "ns"... is it supposed to be like that???

6) The Conclusions chapter - it's interestingly written, but in my opinion it needs to be shortened a bit!

7) The manuscript is fairly well adapted to the template used in the journal Horticulturae, but please pay more attention to the References section.

In conclusion, I believe that the editorial board of Horticulturae should strongly consider publishing this manuscript.

Author Response

Reviewer #1 (R1)

Major comments

R1.M1

« In the Abstract chapter, please add a short research hypothesis.»

Response:

• The authors would like to thank the Reviewer’s interest in the work presented in our paper.
• Regarding the concern raised by the Reviewer, the research hypothesis was added in lines (23-24).
•  

R1.M2

« In the Introduction chapter in the last paragraph, please also formulate the research hypothesis. »

Response:

• In the introduction section, research hypothesis was added as suggested in line (131-135).

R1.M3

• « Table 1 - please explain the abbreviations GR and EF1. »

Response:

• Altered Table 1 legend: “Gene-specific primers for GR (Glutathione Reductase: accenssion number NM 001231394 1 and EF1 (Elongation Factor 1 [45]) and expected amplicon sizes produced by each primer pair for the performed semi-quantitative polymerase chain reaction”, lines (303-305).

R1.M4

«I felt Figures 1-10 - the explanation of the abbreviation "ns" is missing, please complete. And also, in all the figures above the first bars "C" there is no "*" or "ns"... is it supposed to be like that???»

Response:

• Altered figures legend to contain “ns” (example: line 334). All the copper treatments were compared with the control (C), which doesn't receive supplementary cooper. Multiple comparisons were not performed. Therefore, the significance symbol was not placed at the top of the column C.

R1.M5

«I felt The Conclusions chapter - it's interestingly written, but in my opinion, it needs to be shortened a bit! »

Response:

• The authors would like to thank the Reviewer’s suggestions regarding potential improvements to the applicability and operationalization of the approach proposed in our paper. As such, the altered conclusion is presented in lines (630-647). Lines (648-651) were removed.
•  

R1.M6

«The manuscript is fairly well adapted to the template used in the journal Horticulturae, but please pay more attention to the References section. »

Response:

• All invalid references were corrected in lines: Reference 1, line 671; references (12-14), lines (695-702); reference 19, line 713; new references were added considering reviewed manuscript (38,39 and 45).
•  

Reviewer 2 Report

In this paper the authors are providing new information about how copper treatments and copper toxicity can affect nitrogen metabolism in tomato plants. The authors address this research from the farming practices side. Personally, I found this paper very interesting, but the authors should provide more information to clarify some aspects and improve the manuscript. 

Here are my requests (major comments):

1.- The authors should quantify the copper content in the leaves and roots of the plants in all the treatments. This will help the authors to improve their discussion, especially the section with the proline quantification (lines 520-529).

2.- Since the authors are studying the effect of copper on nitrogen metabolism, they should provide pictures of the treated plants to observe their fitness. Is the biomass of the plants affected by the treatments? Did the authors check whether the yield is affected by the treatments? These results will help to improve and strengthen their results.

3.- How do the authors explain the higher amount of NO3- in the SP condition than the S in roots (303-306)? The authors do not discuss these results properly. 

(minor comments)

4.- The authors should provide the data from the reference genes used in the qPCRs.

5.- Which is the concentration of copper used in the contaminated media (S) (lines 129-131)?

 

 

Author Response

Reviewer #2 (R2)

Major comments

R2.M1

« The authors should quantify the copper content in the leaves and roots of the plants in all the treatments. This will help the authors to improve their discussion, especially the section with the proline quantification (lines 520-529)..»

Response:

• Copper quantification protocol was added in lines (151-160), and its results in lines (320-340).

R2.M2

« Since the authors are studying the effect of copper on nitrogen metabolism, they should provide pictures of the treated plants to observe their fitness. Is the biomass of the plants affected by the treatments? Did the authors check whether the yield is affected by the treatments? These results will help to improve and strengthen their results. »

Response:

• New information was added in lines (315-317). Unfortunately, we don’t have figures to insert in the manuscript.

R2.M3

« How do the authors explain the higher amount of NO3- in the SP condition than the S in roots (303-306)? The authors do not discuss these results properly. »

Response:

• In roots, the increase in nitrate levels is proportional to the copper levels observed. New data was added (as suggested by the reviewer), explaining the nitrate levels observed in the two situations. Information was added in lines (553-554).

Minor comments

R2.01

« The authors should provide the data from the reference genes used in the qPCRs..»

Response:

• The requested information was added to Table 1 legend, lines (303-305). For clarification, in our paper, the PCR made were semi-quantitative (sq-PCRs).

R2.02

« Which is the concentration of copper used in the contaminated media (S) (lines 129-131)? »

Response:

• Copper concentration in the contaminated media (S) was 10 mg of Cu ions for Kg soil. Information added at lines (181-182).

Reviewer 3 Report

Abstract with minor revision, in stating the main data values that support the main objectives. 

Overall results and discussion are in good order and commendable work. A baseline value of Cu in tomato can be included, if possible; to justify the extent/level of Cu exogenous exposure towards plant mechanism. This can be done by cross-referencing other studies with similar approaches. 

AOX mechanism relationship with CU application. 

If possible, conduct Pearson correlation or any other statistical analysis to further observe the extent (strength) of the interactions. 

Minor revision in the syntax and grammar. The tables and figures are well presented. However if possible, please include the threshold values as well. 

 

 

Author Response

Reviewer #3 (R3)

Minor comments

R3.01

« Abstract with minor revision, in stating the main data values that support the main objectives.»

Response:

• Formatted abstract with requested values at lines (32-35).

R3.02

« Overall results and discussion are in good order and commendable work. A baseline value of Cu in tomato can be included, if possible; to justify the extent/level of Cu exogenous exposure towards plant mechanism. This can be done by cross-referencing other studies with similar approaches. »

Response:

• Insufficient data pretending to copper exposure in tomato plants can be found, with the existing one being far higher than the concentrations used in this work. Furthermore, this work is intended to be the springboard for future research in this area.
•  

R3.03

« AOX mechanism relationship with CU application. »

Response:

• Information requested as added to the introduction in lines (124-126).
•  
•  
•  

R3.04

« If possible, conduct Pearson correlation or any other statistical analysis to further observe the extent (strength) of the interactions. »

Response:

• The ANOVA analysis was performed for the distinctive non-continuous few classes of copper. Therefore, the Pearson correlation does not provide relevant information for interpreting results.
•  
•  
•  
•  

R3.05

« Minor revision in the syntax and grammar.

Response:

Minor revision in the syntax and grammar was included.

 

R3.06

 

 

The tables and figures are well presented. However, if possible, please include the threshold values as well. »

Response:

• This paper did not intend to determine the threshold values for copper’s impact on Nitrogen use efficiency and AOX mechanisms.
•  
•  

Round 2

Reviewer 2 Report

The authors did a great job, so I will accept the manuscript as it is.