Reactive Oxygen Species (ROS) Metabolism and Nitric Oxide (NO) Content in Roots and Shoots of Rice (Oryza sativa L.) Plants under Arsenic-Induced Stress

Round 1

Reviewer 1 Report

I can not found all figures in downloaded your manuscript.

Therefore I can not review.

Author Response

No comments by this reviewer were made 

Reviewer 2 Report

This manuscript submitted to Agronomy describes the impact of arsenate stress on rice plants with focus on changes in the reactive oxygen species (ROS) metabolism and plant growth. The grew rice seedlings for 14 days and then supplemented for 12 days the medium with arsenate (AsV). They first analyzed in plant roots and shoots the content of different macro and micro nutrients during increasing AsV concentrations as well as the root growth and biomass production. Afterwards, they supplemented the seedlings with constant 50 µM AsV and monitored the altered activities of various antioxidant enzymes as well as the changing levels of antioxidant metabolites and the two signaling molecules H2O2 and NO. Overall, the experiments are comprehensively designed and performed, the manuscript is clearly written, the discussion is conclusive. Please find below my comments regarding the manuscript.

 

General comments:

The statistical significance is usually defined by 1-3 asterisks. As the data in several experiments appear to have high significance, it would be very helpful to introduce therefore more than just one P-threshold.

Figure 8: No statistical significance is given at all. As the significance is mentioned in the text, I assume that it was accidently not shown. Please insert. However, if the significance was not statistically relevant, one of the key messages in the abstract/discussion/conclusion would require rewriting.

 

Line by line comments:

Line 53: please insert: AsV is an analog TO phosphate…

Line 58: please correct: This reaction IS accompanied…

Line 62: please correct: …by virtue of the activity OF ABC…

Line 98: please insert: ….together WITH its derived….

Line 103: please insert: …together WITH that of MDA…

Line 149: please correct: ….either 5 mM KCN OR 5 mM H2O2…

Line 154: please insert a full stop at the end of the sentence.

Line 186: It seems that Fig. 1 is not cited in the text, but the first mentioned figure is Fig. 2A. Please insert the Figure 1 citation in the text.

Line 186 ff.: “Figure”, “Fig.” and “Fig” are randomly used (e.g. line 186, line 188, line 192). Please use consistently throughout the manuscript.

Line 210: please remove the first word in the line (the)

Line 227: please remove the first word in the line (only)

Line 228: please correct: … and NO changes…

Line 232: please correct: … Thus, IT was found…

Line 232: please correct: … their sensibility to THE specific inhibitors…

Line 242: please insert the correct POD isoform number (4)

Line 265/266: please correct: … root was the organ which underwent the most significant changes…

Line 281: please correct: … of rice plants HAS been reported…

Line 309: please correct: …This assertion is based ON results…

Line 412: please correct: … which triggers most…

Line 413: please correct the AsV concentration: according to the experimental procedures, this should be 50 µM instead of mM

Line 709 ff: the figure legend text seems to be duplicated. Please revise.

Line 733/Figure 4: please remove the PLOT AREA

Line 737/746/759/769 please correct: 50 µM instead of 50 mM.

 

Author Response

Dear Editor:

First of all, the authors want to thank to reviewers for their helpful and valuable comments and to improve this manuscript. We realice that it has gained clarity a better arrangement. Below you can find the specific reply one by one to all concerns made by the reviewers. Please be also aware that, following the reviewers’ suggestions, Figures 1, 3, 5, 6, 7 and 8 have been replaced by improved ones.

Best regards

José M. Palma

 

Reviewer 2

Comments and Suggestions for Authors

This manuscript submitted to Agronomy describes the impact of arsenate stress on rice plants with focus on changes in the reactive oxygen species (ROS) metabolism and plant growth. The grew rice seedlings for 14 days and then supplemented for 12 days the medium with arsenate (AsV). They first analyzed in plant roots and shoots the content of different macro and micro nutrients during increasing AsV concentrations as well as the root growth and biomass production. Afterwards, they supplemented the seedlings with constant 50 µM AsV and monitored the altered activities of various antioxidant enzymes as well as the changing levels of antioxidant metabolites and the two signaling molecules H2O2 and NO. Overall, the experiments are comprehensively designed and performed, the manuscript is clearly written, the discussion is conclusive. Please find below my comments regarding the manuscript.

Many thanks for this stimulating appraisal. Please find below the reply to all queries and comments.

 

General comments:

The statistical significance is usually defined by 1-3 asterisks. As the data in several experiments appear to have high significance, it would be very helpful to introduce therefore more than just one P-threshold.

Thanks for this valuable comment. This has been addressed and the statistical analysis improved as it can be followed in the new manuscript. See new paragraph “2.7. Statistical analysis” and corresponding figures.

 

Figure 8: No statistical significance is given at all. As the significance is mentioned in the text, I assume that it was accidently not shown. Please insert. However, if the significance was not statistically relevant, one of the key messages in the abstract/discussion/conclusion would require rewriting.

The statistical analysis has now been included in this figure. We realize that it was an unwanted mistake on our behalf. We apologize for that. Accordingly, and due to the significance meaning of these results, the text in the abstract and discussion has been maintained.

 

Line by line comments:

Line 53: please insert: AsV is an analog TO phosphate…

This has been corrected.

 

Line 58: please correct: This reaction IS accompanied…

This has been corrected.

 

Line 62: please correct: …by virtue of the activity OF ABC…

This has been corrected.

 

Line 98: please insert: ….together WITH its derived….

This has been corrected.

 

Line 103: please insert: …together WITH that of MDA…

This has been corrected.

 

Line 149: please correct: ….either 5 mM KCN OR 5 mM H2O2…

This has been corrected.

 

Line 154: please insert a full stop at the end of the sentence.

This has been corrected.

 

Line 186: It seems that Fig. 1 is not cited in the text, but the first mentioned figure is Fig. 2A. Please insert the Figure 1 citation in the text.

Figure 1 was already cited in the Materials and methods section, line 111

 

Line 186 ff.: “Figure”, “Fig.” and “Fig” are randomly used (e.g. line 186, line 188, line 192). Please use consistently throughout the manuscript.

Thanks for this comment. The term Fig. has been now homogenized throughout the text.

 

Line 210: please remove the first word in the line (the)

This has been corrected.

 

Line 227: please remove the first word in the line (only)

This has been corrected.

 

Line 228: please correct: … and NO changes…

This has been corrected.

 

Line 232: please correct: … Thus, IT was found…

This has been corrected.

 

Line 232: please correct: … their sensibility to THE specific inhibitors…

This has been corrected.

 

Line 242: please insert the correct POD isoform number (4)

This has been corrected.

 

Line 265/266: please correct: … root was the organ which underwent the most significant changes…

This has been corrected.

 

Line 281: please correct: … of rice plants HAS been reported…

This has been corrected.

 

Line 309: please correct: …This assertion is based ON results…

This has been corrected.

 

Line 412: please correct: … which triggers most…

This has been corrected.

 

Line 413: please correct the AsV concentration: according to the experimental procedures, this should be 50 µM instead of mM

This has been corrected.

 

Line 709 ff: the figure legend text seems to be duplicated. Please revise.

This has been corrected. Thanks for this assessment

 

Line 733/Figure 4: please remove the PLOT AREA

This has been corrected. Thanks for this assessment. The former figure has been replaced by a new one where “PLOT AREA” image was removed.

 

Line 737/746/759/769 please correct: 50 µM instead of 50 mM.

This has been corrected. Also the amounts of proteins loaded on gels (80 mg).

Reviewer 3 Report

The main idea of the work has been to assess the enzymatic activity and lipid peroxidation level of rice under As treatment in order to follow ROS metabolism and NO content. Some parts of the manuscript need improvement.

 

My detailed comments are stated below.

 

Title: I propose to add ‘’content” after ‘(NO’’).

 

The manuscript lacks the goal of the study. Please, write it clearly.

Why AsV was chosen?

 

Abstract

Line 13 – I propose to delete the sentence.

Line 18 – ‘’some” should be deleted.

In the abstract, results should be written more pricesly.

 

Please, improve the sentence order, e.g.:

- line 40 – I propose ‘’distributed worldwide”;

- line 149 – I propose to put ‘’was carried out’’ at the end of the sentence;

- line 227 – Please improve ‘’only were only’’;

- line 264 – The first word of the sentence should be ‘’analyzed’, but later, in the same line, the same word appears. Please, omit repetitions.

 

Line 42 – The sentence should be started from the full name. Now, it is an abbreviation ‘’As’’. Please, improve it elsewhere in the text.

 

Pleae, be careful about spelling, e.g.:

- line 58 – ‘’in’’ or ‘’is’’?;

- line 235 – ‘’isoyzmes”?;

- line 276 – ‘’is’’ or ‘’in’’?;

- line 289 – do you mean ‘’previously’’?

 

Line 108 - How many plants per pot?

Line 120 - How long digested?

 

Please, do not repeat the explanations of the abbreviations. Improve all full names and abreviations usage elsewhere in the text, e.g.:

- line 144 – Full name and abbreviation of SOD appear but it also appeared earlier – see lines 72-73;

- line 157 – GSSG – see line 141;

- line 172 – MDA – see line 91.

 

Please, be careful about editorial mistakes, like unnecessary double space bar (e.g. line 150), lacking full stop (e.g. the end of line 154), unnecessary coma (e.g. line 330 – before ‘’[28]’’; line 401 – before ‘’other’’).

Lines 155-156 - – I propose to put ‘’(Bio-Rad)” after ‘’solution’’ and to write ‘’protein assay’’ (lowercased).

 

Lines 177-179 (TF) should be moved to 2.2.

 

I propose to add separate paragraph ‘’2.7 Statistical analysis’’ and explain the differences in presentation of the statistical results, e.g. Fig. 2 has letters, Fig. 3 - asterisks.

Fig. 8 – lack of symbols of statistical significance. According to the order in which it is mentioned in the text, I propose to put MDA at the end as (C).

 

Fig. 4 – In the middle of the figure ‘’Plot Area’’ appears. Please, remove it.

Fig. 6 – Fe-SOD and POD7 are invisible. Please, improve the quality of the figures.

It can be written the range of the analyzed data  - ‘’at least three’’ means from 3 to …?

 

Line 188 – Fig. 2 or Fig. 2A?

Line 188-189 – The statement is not obvious  from Fig. 2A and 2B (roots).

Lines 189-191 – Please, be more precise. The senence is too general.

Line 195 – Add ‘’In roots,” before ‘’K …’’.

Line 196 – Add ‘’(Fig. 3A)’’ at the end of the sentence.

Line 198 – Remove ‘’(0-200…)”.

Please, complete the information, e.g.:

- lines 199-201 and 201-203 – both statements are correct from 25 µM;

- lines 205-207 – from 10 µM in shoots, but only 200 – for roots.

 

Line 208 – ‘’visible symptoms’’ – which one? Please, explain.

Line 224 – Please, remove ‘’most’’.

 

Lines 237, 239 – It is impossible to evaluate Fe-SOD presence because of the low quality of the figure. Similarly, line 245 – POD 7.

Line 242 – POD 1, POD 2 and POD ???

Lines 253-254 – ‘’As … in roots” – Do you mean ‘’elevated’’? Please, specify the tendency clearly.

Lines 258-262 – It can be checked after completing the statistical data in the figure.

 

Lines 265-268 – These sentences are repetitions of the results. Please, delete them.

Lines 283-285 – It is not the right part of the discussion. Please, rewrite or delete.

Line 298 – ‘’higher ratio’’ than what? Please, explain.

Line 300 – Is it correct? It starts from 0.047. Please, improve the statement.

Line 317 – ‘’oppositely’’ is misleading. Please, rewrite the sentence.

Line 343 – See GR – roots. Please, rewrite.

Line 354 – ‘’the’’ instead of ‘’these’’.

Line 355 – Please, delete ‘’conditions’’.

Lines 379-381 – There are much more variables. Take under consideration others, like length of application, the plant age, etc.

 

Conclusions should be partially rewritten to be based on the Author’s research more than on other manuscripts.

Lines 414-417 – Please, write 2 sentences instead of 1.

Line 419 – ‘’This perspective’’ – which one?  Unclear.

Lines 419-422 – Please, delete. It is not the cocnlusions part.

 

Figure 1 – Please, unify the font size.

 

Lines 714-719 – Please, delete.

 

Bibliography also needs editorial improvement.

 

 

 

 

 

 

Author Response

Dear Editor:

First of all, the authors want to thank to reviewers for their helpful and valuable comments and to improve this manuscript. We realice that it has gained clarity a better arrangement. Below you can find the specific reply one by one to all concerns made by the reviewers. Please be also aware that, following the reviewers’ suggestions, Figures 1, 3, 5, 6, 7 and 8 have been replaced by improved ones.

Best regards

José M. Palma

Reviewer 3

Comments and Suggestions for Authors

The main idea of the work has been to assess the enzymatic activity and lipid peroxidation level of rice under As treatment in order to follow ROS metabolism and NO content. Some parts of the manuscript need improvement.

My detailed comments are stated below.

 

Title: I propose to add ‘’content” after ‘(NO’’).

The term “content” has been added to title. Thanks.

 

The manuscript lacks the goal of the study. Please, write it clearly.

Thanks for this valuable comment. This has been addressed and the aim of this work has been explained in the new text.

 

Why AsV was chosen?

As already reported by Duan et al. 2013 (Reference 32 in the manuscript), “As(III) is the predominant As form under anaerobic flooded conditions” like those governing rice culture. Under these conditions, “As(III) uptake by roots and translocation from roots to shoots is of great importance in rice As accumulation”. However, these were not our experimental conditions which used hydroponic well-aerated culture.

On the other hand, the same authors postulated that, “since P fertilization is the common agricultural strategy to improve crop yield, especially for rice…, it is important to understand As uptake and accumulation by rice plants when P fertilizer is supplied”, mainly because “phosphate transporters are involved in As(V) uptake and translocation”. And these are our conditions and one of the final aims of our study. These authors go further “The competition of uptake and translocation between P and As(V) proposes a potential strategy for reducing As uptake and accumulation in grains”.

Taking into consideration all these premises, we set As(V) as the ideal arsenic form to carry out our study. In a very short formula, this has been specified in the new manuscript.

 

Abstract

Line 13 – I propose to delete the sentence.

This sentence was removed.

Line 18 – ‘’some” should be deleted.

This has been corrected.

 

In the abstract, results should be written more pricesly.

The results in the Abstract have been written more concisely with a better precission.

 

Please, improve the sentence order, e.g.:

- line 40 – I propose ‘’distributed worldwide”;

This has been corrected.

 

- line 149 – I propose to put ‘’was carried out’’ at the end of the sentence;

This has been corrected.

 

- line 227 – Please improve ‘’only were only’’;

This has been corrected.

 

- line 264 – The first word of the sentence should be ‘’analyzed’, but later, in the same line, the same word appears. Please, omit repetitions.

This sentence has been rewritten.

 

Line 42 – The sentence should be started from the full name. Now, it is an abbreviation ‘’As’’. Please, improve it elsewhere in the text.

This has been reviewed throughout the text.

 

Please, be careful about spelling, e.g.:

- line 58 – ‘’in’’ or ‘’is’’?;

This has been corrected.

 

- line 235 – ‘’isoyzmes”?;

This has been corrected.

 

- line 276 – ‘’is’’ or ‘’in’’?;

This has been corrected.

 

- line 289 – do you mean ‘’previously’’?

This has been corrected.

 

Line 108 - How many plants per pot?

As indicated in the new text, 36 seedlings per pot were transplanted.

Line 120 - How long digested?

As indicated in the text, the digestion took 90 min as total time.

Please, do not repeat the explanations of the abbreviations. Improve all full names and abreviations usage elsewhere in the text, e.g.:

- line 144 – Full name and abbreviation of SOD appear but it also appeared earlier – see lines 72-73;

This has been properly corrected.

- line 157 – GSSG – see line 141;

This has been properly corrected.

 

- line 172 – MDA – see line 91.

This has been corrected.

 

Please, be careful about editorial mistakes, like unnecessary double space bar (e.g. line 150), lacking full stop (e.g. the end of line 154), unnecessary coma (e.g. line 330 – before ‘’[28]’’; line 401 – before ‘’other’’).

This has been properly corrected.

 

Lines 155-156 - – I propose to put ‘’(Bio-Rad)” after ‘’solution’’ and to write ‘’protein assay’’ (lowercased).

Thanks. The commercial name is “Bio-Rad protein assay”, so we have left this name but have used lower cases for protein assay.

 

 Lines 177-179 (TF) should be moved to 2.2.

This has been properly moved. Thanks for this re-arrangement of the text.

 

I propose to add separate paragraph ‘’2.7 Statistical analysis’’ and explain the differences in presentation of the statistical results, e.g. Fig. 2 has letters, Fig. 3 - asterisks.

This has properly been addressed and corresponding figures appropriately modified. So, paragraph “2.7. Statistical analysis” has been added to the new manuscript. Thanks for this valuable comment.

 

Fig. 8 – lack of symbols of statistical significance. According to the order in which it is mentioned in the text, I propose to put MDA at the end as (C).

According to the above comment, asterisks have now been used and the arrangement of graphs changed as suggested.

 

Fig. 4 – In the middle of the figure ‘’Plot Area’’ appears. Please, remove it.

This has been corrected. Thanks for this assessment. The former figure has been replaced by a new one where “PLOT AREA” image was removed.

 

Fig. 6 – Fe-SOD and POD7 are invisible. Please, improve the quality of the figures.

Figure 6 has been improved and has replaced the former one. Fe-SOD and POD7 are now visible.

 

It can be written the range of the analyzed data  - ‘’at least three’’ means from 3 to …?

This has been properly changed.

 

Line 188 – Fig. 2 or Fig. 2A?

Fig. 2A. It shows the picture of rice plants grown at the As concentrations used in this work.

 

Line 188-189 – The statement is not obvious  from Fig. 2A and 2B (roots).

The text has been slightly modified to make the statement clearer. Thanks for the comment. The figure legend has also been modified accordingly.

 

Lines 189-191 – Please, be more precise. The senence is too general.

This has been more precisely written.

 

Line 195 – Add ‘’In roots,” before ‘’K …’’.

This was corrected. Thanks.

 

Line 196 – Add ‘’(Fig. 3A)’’ at the end of the sentence.

We have written Fig. 3A only once at the end of the text concerning K to avoid repetitions.

 

Line 198 – Remove ‘’(0-200…)”.

It has been removed.

 

Please, complete the information, e.g.:

- lines 199-201 and 201-203 – both statements are correct from 25 µM;

Both sentences have been properly corrected as suggested.

 

- lines 205-207 – from 10 µM in shoots, but only 200 – for roots.

This statement has also been modified according to the suggestion.

 

Line 208 – ‘’visible symptoms’’ – which one? Please, explain.

This sentence has been rewritten.

 

Line 224 – Please, remove ‘’most’’.

It has been removed.

Lines 237, 239 – It is impossible to evaluate Fe-SOD presence because of the low quality of the figure. Similarly, line 245 – POD 7.

See reply above: Figure 6 has been improved and has replaced the former one. Fe-SOD and POD7 are now visible.

 

Line 242 – POD 1, POD 2 and POD ???

This has been corrected. POD 4 has now been written.

 

Lines 253-254 – ‘’As … in roots” – Do you mean ‘’elevated’’? Please, specify the tendency clearly.

This has been properly corrected.

 

Lines 258-262 – It can be checked after completing the statistical data in the figure.

Figure 8 has been properly corrected. Thanks.

 

Lines 265-268 – These sentences are repetitions of the results. Please, delete them.

This sentence has been rewritten as indicated above.

 

Lines 283-285 – It is not the right part of the discussion. Please, rewrite or delete.

This sentence has been deleted.

 

Line 298 – ‘’higher ratio’’ than what? Please, explain.

This has been changed according to the comment.

 

Line 300 – Is it correct? It starts from 0.047. Please, improve the statement.

This has been appropriately changed. Thanks.

 

Line 317 – ‘’oppositely’’ is misleading. Please, rewrite the sentence.

This has been changed and we think that now is clearer.

 

Line 343 – See GR – roots. Please, rewrite.

We have re-read this statement several times and, looking at the corresponding figure, we do not understand what the reviewer means. Indeed, the only significant change in MDAR and GR was found in shoots. MDAR slightly increased in roots, but it was not significant. Thus, we prefer to maintain this statement, unless we are specifically said what is the proposed change.

 

Line 354 – ‘’the’’ instead of ‘’these’’.

This has been corrected.

 

Line 355 – Please, delete ‘’conditions’’.

It has been deleted.

 

Lines 379-381 – There are much more variables. Take under consideration others, like length of application, the plant age, etc.

Thanks. These other issues have also been cited.

 

Conclusions should be partially rewritten to be based on the Author’s research more than on other manuscripts.

We have changed the heading of this chapter. Thus, considering future research in this subject, the Conclusion chapter has been also opened to “future prospects”. The first part of the conclusions is focused in a series of considerations that the authors think should be taken into account to address the research in this relevant pollution threat. Then, we set how our work contributes to this field and, finally, the authors propose that future strategies to alleviate arsenic effects need perspectives. These include signaling events within plants, alternative practices, etc. The future prospect that the authors compile are based not only in our own data but also on reports from other researchers.

 

Lines 414-417 – Please, write 2 sentences instead of 1.

This has been changed as suggested.

Line 419 – ‘’This perspective’’ – which one?  Unclear.

The reviewer is right. This paragraph has been reformulated, so the present arrangement is more logical.

 

Lines 419-422 – Please, delete. It is not the conclusions part.

See above comment on the new paragraph “Conclusions and future prospects”.

 

Figure 1 – Please, unify the font size.

This has been done

 

Lines 714-719 – Please, delete.

These lines have been deleted.

 

Bibliography also needs editorial improvement.

Thorough revision of bibliography has been done.

Reviewer 4 Report

General Comments: This manuscript deals with "ROS and NO reaction in rice due to the As stress in rice". It is an interesting topic but needs some revisions before considering for publication.

Heavy metal contamination is not only a threat to living organisms but also a global environmental concern. Arsenic (As) is amongst the most hazardous metals that could be poisonous to humans. As enters agricultural lands via natural sources, and anthropogenic sources. Rice is more seriously affected by As pollution than other crop plants. 

 

1. Abstract:

1.1. Page 1, Line 14; "...although it is well known that there are high As concentrations in rice plants and grains." It is better to edit it as "although it is well known that rice can uptake high amount of As in comparing with other crops. 

1.2. Page 1, Line 15; "Commonly, agronomic species not tolerant to arsenic...". It is better to edit it as "Commonly agronomic species with low tolerance against arsenic show...."

1.3. Page 1, Line 21; "A decrease in root length as well as plant biomass was observed with AsV treatment..." Mention to decreasing value.

 

2. Introduction:

2.1. Page 1, Line 40; "Arsenic (As) is a major environmental pollutant worldwide distributed..." It is better to edit it as "Arsenic (As) is one of the major environmental pollutant worldwide distributed..."

2.2. Page 2, Line 78; "Commonly, agronomic species not tolerant to arsenic show..." It was copied from the Abstract! Please re-write it

 

3. Materials and Methods

3.1. Page 3, Line 108; "...Healthy and vigorous seedlings were selected and grown in 4 L plastic.." They were planted in plastic pots OR transplanted? In my opinion they were transplanted since in the previous sentence was mentioned that "Rice (Oryza sativa L) seeds were planted and grown in vermiculite for 14 d"

 

Author Response

Dear Editor:

First of all, the authors want to thank to reviewers for their helpful and valuable comments and to improve this manuscript. We realice that it has gained clarity a better arrangement. Below you can find the specific reply one by one to all concerns made by the reviewers. Please be also aware that, following the reviewers’ suggestions, Figures 1, 3, 5, 6, 7 and 8 have been replaced by improved ones.

Best regards

José M. Palma

 

Reviewer 4

Comments and Suggestions for Authors

General Comments: This manuscript deals with "ROS and NO reaction in rice due to the As stress in rice". It is an interesting topic but needs some revisions before considering for publication.

Heavy metal contamination is not only a threat to living organisms but also a global environmental concern. Arsenic (As) is amongst the most hazardous metals that could be poisonous to humans. As enters agricultural lands via natural sources, and anthropogenic sources. Rice is more seriously affected by As pollution than other crop plants.

We appreciate the comment made by the reviewer to highlight the importance of investigating this subject. We totally agree.

 

1. Abstract:

1.1. Page 1, Line 14; "...although it is well known that there are high As concentrations in rice plants and grains." It is better to edit it as "although it is well known that rice can uptake high amount of As in comparing with other crops.

This has been modified following the reviewer’s suggestion.

 

1.2. Page 1, Line 15; "Commonly, agronomic species not tolerant to arsenic...". It is better to edit it as "Commonly agronomic species with low tolerance against arsenic show...."

This has been changed as suggested.

 

1.3. Page 1, Line 21; "A decrease in root length as well as plant biomass was observed with AsV treatment..." Mention to decreasing value.

Percentage decreasing values of both length and plant biomass have been given as requested.

2. Introduction:

2.1. Page 1, Line 40; "Arsenic (As) is a major environmental pollutant worldwide distributed..." It is better to edit it as "Arsenic (As) is one of the major environmental pollutant worldwide distributed..."

This has been changed as suggested.

 

2.2. Page 2, Line 78; "Commonly, agronomic species not tolerant to arsenic show..." It was copied from the Abstract! Please re-write it.

This has been changed according to the new text given in the abstract.

 

3. Materials and Methods

3.1. Page 3, Line 108; "...Healthy and vigorous seedlings were selected and grown in 4 L plastic.." They were planted in plastic pots OR transplanted? In my opinion they were transplanted since in the previous sentence was mentioned that "Rice (Oryza sativa L) seeds were planted and grown in vermiculite for 14 d"

The reviewer is right. It has been properly changed. Thanks.

Round 2

Reviewer 3 Report

The Authors modified the manuscript according to the suggestions, but some minor changes should be done. 

 

Abstract

Now abstract is too long, thus I propose some changes.

 

Line 13 – Please, delete “is well known that rice”

 

Lines 14-15 – Please, delete “. Commonly agronomic species with low tolerance against arsenic show” and match both sentences using for example “showing”.

 

Lines 17-20 – Please, delete the sentences “However, there is still scarce information to depict a wider view on the effect of As at shoot and root levels, and how the ROS-related parameters behave in the presence of such metalloid in those organs. Likewise, the possible role of nitric oxide (NO) in the As toxicity is also little known”.

 

Line 20 – Please, delete “Accordingly, the” and put “The”.

 

Line 44 – “pollutant” or “pollutants”?

 

Line 81 – “Commonly agronomic species” or “Common agronomic species”?

 

In the revised version of the manuscript, the goal of the study has been written in the abstract, but it is lacking in the body of the manuscript. Please, add it to the last paragraph of Introduction.

 

Line 276 – Delete “)” after “respectively”.

 

Line 361 – Please, check again if “0.06” or “0.05” is correct.

 

Line 404 – Please, delete “especially” – as you have mentioned it is true only for shoots.

Author Response

Dear Editor:

Once again, we would like to acknowledge to reviewers for their kind help to improve this manuscript. We realize that it has gained quality and scope, and hope it can help readers and researchers in the future.

Please find below all replies to comments made by the reviewers.

Best regards

Pepe

 

Reviewer 3

The Authors modified the manuscript according to the suggestions, but some minor changes should be done.

Abstract

Now abstract is too long, thus I propose some changes.

Line 13 – Please, delete “is well known that rice”

It has been deleted as suggested. Thanks.

 

Lines 14-15 – Please, delete “. Commonly agronomic species with low tolerance against arsenic show” and match both sentences using for example “showing”.

This has been corrected as suggested.

 

Lines 17-20 – Please, delete the sentences “However, there is still scarce information to depict a wider view on the effect of As at shoot and root levels, and how the ROS-related parameters behave in the presence of such metalloid in those organs. Likewise, the possible role of nitric oxide (NO) in the As toxicity is also little known”.

These sentences have been deleted.

 

Line 20 – Please, delete “Accordingly, the” and put “The”.

This has been corrected as suggested.

 

Line 44 – “pollutant” or “pollutants”?

Corrected to pollutants. Thanks

 

Line 81 – “Commonly agronomic species” or “Common agronomic species”?

It has been changed to “Commonly, agronomic species…”

 

In the revised version of the manuscript, the goal of the study has been written in the abstract, but it is lacking in the body of the manuscript. Please, add it to the last paragraph of Introduction.

This has been followed as suggested, so the last paragraph of Introduction makes a overview of the whole work. We, indeed acknowledge this appraisal.

 

Line 276 – Delete “)” after “respectively”.

It has been deleted.

Line 361 – Please, check again if “0.06” or “0.05” is correct.

Corrected to 0.05. Thanks again.

 

Line 404 – Please, delete “especially” – as you have mentioned it is true only for shoots.

It has been deleted.

 

Reviewer 4 Report

Reviewers's comments have been addressed.

Author Response

The authors deeply acknowledge the helpful comments made by the reviewer