Alleviation Mechanism of Melatonin in Chickpea (Cicer arietinum L.) under the Salt Stress Conditions

Round 1

Reviewer 1 Report

Horticulturae-1998099：

This manuscript investigates different does of MT on the growth, biochemical and physiological properties of chickpea under salt stress. The manuscript is worthy of publication in HORTICULTURAE journal, but the major revision with structural change listed below needs to be addressed before publication. Nonetheless, my concrete comments are given below manuscript section-wise, which are needed to solve and improve the manuscript quality.

Overall comments:

a.    Authors should read the whole manuscript several times after correcting the comments below section-wise with full concentration. And then, the whole manuscript needs to refine/rewrite/reorganize sentences to maintain sentence consistency and subsequently easy understanding for scientific readership from the preceding sentence to the running sentence.

b.    References should be checked for improvement with the currently published article. And the order and number of the references in the manuscript were wrong and chaos. For example, Line 18, “[18]”? Where is the [1]???

Special comments:

Abstract:

1.    The abstract section needs final scrutiny for better readership as if it looks like a mirror of the whole manuscript with the appropriate sentence structure as it is the heart of the manuscript. The results in the abstract were too tedious, and need refine.

2.    In the end of abstract, the authors should emphasize the conclusion and significance of the current study.

Introduction:

3.  The active literature review, the current research status regarding the manuscript title and the research gap are totally absent in the introduction section; furthermore. Authors should carefully meet the aforementioned requirement for publication in an international journal.

4.    Line 49, should be Melatonin (N-acetyl-5-methoxytryptamine, MT).

5.  The third and fourth paragraphs can be combined. All of them were about the MT.

6.  Line 74-76, are the authors sure this study was the first study about the MT application in chickpeas under saline conditions. Please confirm carefully after refer to the newest literatures.

Materials and Methods:

7. Why only choose the 50 and 100 uM MT as target concentration?

8. Line 104, add “.” In the end of 150 mM does

9. Line 113, should be “48 h”.

10. Line 144, change H2O2 to H₂O₂.

11. Why choose 45 days as experimental period?

Results and Discussion:

12. Table 1, what the meaning of “3.74 a-c”? Is it should be “3.74ac”?

13. Table 1 and 2 needs to be modified, just as neat as Table 3.

14. Add significant difference in the figure 3.

15. The response of oxidative stress of plant under different MT treatment is very interesting, and maybe the main mechanism in the benefit effect of MT. Therefore, please add more description and explanation about the oxidative stress and antioxidant enzymes. These two articles are for your reference: https://doi.org/10.1016/j.chemosphere.2021.130130; https://doi.org/10.1016/j.jhazmat.2022.129640

Conclusions:

16. In my opinion, Line 385-387 seems unnecessary.

17. Merge the conclusion into one paragraph.

18. Line 390-391 make reader a litter confuse. Please rewrite.

Author Response

Dear Editor,

On behalf of all of the co-authors, I would like to thank the editor and the reviewers for careful reading, and constructive suggestions for our manuscript. According to comments from editor and reviewers, we comprehensively revised our manuscript. We are looking forward to your feedback and further comments/suggestions are welcome.

The reviewers' comments are reproduced in black; our responses are detailed below in red:

RESPONSE TO REVIEWERS 

First of all, I would like to extend our gratitude to the anonymous reviewers for forwarding very constructive criticism of the MS and giving us an opportunity to improve the standard of the MS. We are glad to state that all suggestions forwarded by the reviewers have been incorporated in their entirety and none has been left unaddressed.

Horticulturae-1998099：

This manuscript investigates different does of MT on the growth, biochemical and physiological properties of chickpea under salt stress. The manuscript is worthy of publication in HORTICULTURAE journal, but the major revision with structural change listed below needs to be addressed before publication. Nonetheless, my concrete comments are given below manuscript section-wise, which are needed to solve and improve the manuscript quality.

Thank you very much

Overall comments:

1. Authors should read the whole manuscript several times after correcting the comments below section-wise with full concentration. And then, the whole manuscript needs to refine/rewrite/reorganize sentences to maintain sentence consistency and subsequently easy understanding for scientific readership from the preceding sentence to the running sentence.

Please see that the whole manuscript was refine/rewrite/reorganized.

1. References should be checked for improvement with the currently published article. And the order and number of the references in the manuscript were wrong and chaos. For example, Line 18, “[18]”? Where is the [1]???

 Please see that we have corrected the references in the text

Special comments:

Abstract:

1. The abstract section needs final scrutiny for better readership as if it looks like a mirror of the whole manuscript with the appropriate sentence structure as it is the heart of the manuscript. The results in the abstract were too tedious, and need refine.

Abstract was re-written.

2. In the end of abstract, the authors should emphasize the conclusion and significance of the current study.

 Done

Introduction:

3. The active literature review, the current research status regarding the manuscript title and the research gap are totally absent in the introduction section; furthermore. Authors should carefully meet the aforementioned requirement for publication in an international journal.

Please see that required corrections were made.

4. Line 49, should be Melatonin (N-acetyl-5-methoxytryptamine, MT).

Done

5. The third and fourth paragraphs can be combined. All of them were about the MT.

Done

6. Line 74-76, are the authors sure this study was the first study about the MT application in chickpeas under saline conditions. Please confirm carefully after refer to the newest literatures.

We have reviewed the literature.  To our best knowledge this is the first.

Materials and Methods:

7. Why only choose the 50 and 100 uM MT as target concentration?

We have chosen these concentrations according to our previous studies and literature.

8. Line 104, add “.” In the end of 150 mM does

Done

9. Line 113, should be “48 h”.

Done

10. Line 144, change H2O2 to H₂O₂.

Done

11. Why choose 45 days as experimental period?

 The symptoms were visible.

Results and Discussion:

12. Table 1, what the meaning of “3.74 a-c”? Is it should be “3.74ac”?

Done

13. Table 1 and 2 needs to be modified, just as neat as Table 3.

Done

14. Add significant difference in the figure 3.

Done

15. The response of oxidative stress of plant under different MT treatment is very interesting, and maybe the main mechanism in the benefit effect of MT. Therefore, please add more description and explanation about the oxidative stress and antioxidant enzymes. These two articles are for your reference: https://doi.org/10.1016/j.chemosphere.2021.130130; https://doi.org/10.1016/j.jhazmat.2022.129640

 We have benefited from the mentioned articles.

Conclusions:

16. In my opinion, Line 385-387 seems unnecessary.

We deleted line 385-387

17. Merge the conclusion into one paragraph.

Done

18. Line 390-391 make reader a litter confuse. Please rewrite.

Done

Author Response File: Author Response.docx

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Dear Editor,

On behalf of all of the co-authors, I would like to thank the editor and the reviewers for careful reading, and constructive suggestions for our manuscript. According to comments from editor and reviewers, we comprehensively revised our manuscript. We are looking forward to your feedback and further comments/suggestions are welcome.

The reviewers' comments are reproduced in black; our responses are detailed below in red:

RESPONSE TO REVIEWERS 

First of all, I would like to extend our gratitude to the anonymous reviewers for forwarding very constructive criticism of the MS and giving us an opportunity to improve the standard of the MS. We are glad to state that all suggestions forwarded by the reviewers have been incorporated in their entirety and none has been left unaddressed.

The topic of the article is relevant. The purpose of the work is set clearly; the chosen methods correspond to the goal. The manuscript contains new knowledge in the field of plants stress physiology. The results are of great practical importance.

Thank you very much

However, this manuscript has a number of comments and questions.

1. L. 156-157 “First, plant and root samples were subjected to combustion in nitric acid-hydrogen peroxide…”. It is necessary to clarify whether shoot or leaves were used for analysis?

Please see that we have corrected

2. In fig. 2 in the legend there is no designation of the 3rd column (150 mM NaCl).

Please see that we have corrected

3. L. 245-246. Based on the data in Fig. 2 cannot be said that LRWC decreased under 150 mM NaCl compared to control. There are no significant differences between the variants (0 and 150 mM NaCl).

Please see that we have corrected

4. L. 246-248. How can a decrease of the LRWC affect root biomass under saline conditions?

Please see that we have corrected

5. It is better to point out that under 75 mM NaCl, MT treatment of plants does not positively affect membrane permeability and LRWC in plants.

Done

6. L. 252-253. “LRWC of 100 μM MT treated chickpea seedlings increased by 2.7%, compared to non-MT treated plants under 150 mM salt-treated, respectively”. These differences are not significant.

Done

7. L. 255-257. It is not stated that all of these parameters also increased at 75 mM NaCl.

Done

8. L. 273-274 Clarification: Plants have one antioxidant system, which includes enzymatic and non-enzymatic components.

Please see that we have corrected as required.

9. L. 309-312 “Since MT is a strong water-soluble antioxidant that can mitigate stress-induced ROS damage through different mechanisms, a reduction in antioxidant enzyme activity in chickpea seedlings under salinity stress conditions was determined when MT was applied.” If MT itself is a strong antioxidant, how can one explain its stimulatory effect on the antioxidant enzymes activity?

Hamid, A. A., Aiyelaagbe, O. O., Usman, L. A., Ameen, O. M., & Lawal, A. (2010). Antioxidants: Its medicinal and pharmacological applications. African Journal of pure and applied chemistry, 4(8), 142-151.

Fernando Antunes, L.Ross C. Barclay, Keith U. Ingold, Mary King, James Q. Norris, J.C. Scaiano, Fengde Xi, On the antioxidant activity of melatonin, Free Radical Biology and Medicine, Volume 26, Issues 1– 2, 1999, Pages 117-128

Hacışevki, A., & Baba, B. (2018). An overview of melatonin as an antioxidant molecule: a biochemical approach. Melatonin molecular biology, clinical and pharmaceutical approaches, 5, 59-85.

The literature shown above suggests that MT is an antioxidant.

10. L. 343-344. The authors write that many studies reported that exogenous application of MT decreased Na+ accumulation and increased K+ content in the above-ground parts of various species grown under saline conditions. But the reference is given to only one literary source.

Please see that we have corrected as required.

11. L. 346-347. “K+ uptake increased due to the decrease in Na+ uptake as an effect of MT”. The possible mechanism for this effect is unclear. It is known that with an excess of sodium ions in the environment, there is competition with K ions for binding sites on the cell surface. How can treatment of plants with MT increase the intake of K+ under these conditions?

Melatonin plays a key role in maintaining ion homeostasis. The salt tolerance of M.26 (an important dwarf rootstock of apple (Malus domestica)) was enhanced by melatonin via up-regulation of MdNHX1 (Malus vacuolar Na+/H+ antiporter gene) [114]. The in-ward-rectifying channel AKT1 (Arabidopsis K+ transporter 1) mediates the relative uptake rates of Na+ and K+ under high salinity (Junpeng Li, Jing Liu, Tingting Zhu, Chen Zhao, Lingyu Li and Min Chen., The Role of Melatonin in Salt Stress Responses Int. J. Mol. Sci. 2019, 20, 1735

12. L. 376-377. “The protective roles of MT in salt stress were investigated by comparing the growth, yield, and physiological responses in chickpea plants”. But the authors did not study the yield of plants.

Please see that we have corrected as required.

13. L. 379-380. “Also, 100 μM MT application displayed more effective protective roles than 50 μM MT in reducing Na+ and Cl- contents…”. The Cl- content was not determined.

Please see that we have corrected as required.

14. L. 391-394 “In conclusion, under saline conditions, exogenous MT treatments effectively provide protection against cellular and macromolecular damage in chickpea seedlings and improve plant tolerance to salt stress.” Since the authors found the most effective concentration of MT for treating plants under saline conditions, it may be indicated. For example, “In conclusion, under saline conditions, exogenous MT treatments (100 мкМ) effectively …”

Please see that the suggestion was considered.

In general, the article can be published in the journal after revision.

Thank you very much for The Reviewers suggestions

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript submitted by Dadasoglu et al. discusses the effect of melatonin foliar spray on the response of chickpea to salinity stress in greenhouse conditions. The manuscript shows the effect of melatonin on plant growth, relative water content, chlorophyll and carotenoid content, electrolytic leakage, antioxidant activity and K/Na and Ca/Na ratios. The experiment is reasonably well design, and the data is in line other publications on the topic. Unfortunately, this topic has been extensively researched and many publications can be easily found in the literature. The only factor of novelty is the use of chickpea, however, considering that the effect of melatonin in salt-treated plants seems to be extremely consistent among the many species in which it has been tested, I fail to see the necessity of repeating the same analysis in other species. Unless the authors can highlight the novelty behind this manuscript, I do not think that is worth being published in Horticulturae.

These are some other points that require the author’s attention:

-If numbers are used for the in-text citations, they should be ordered according to their position in the manuscript, not alphabetically.

-L83. Does the 20-35 °C temperature refer to the night/day temperature?

-L102. Authors should specify how old the plants were when the Melatonin and Salinity treatments were applied.

-Authors should specify in which tissue each of the measurement/quantifications were done. The table and figure captions only mention “seedlings”. Does this mean that they were measured on the whole plant? Authors should clearly explain this and implement it in the discussion accordingly.

-L151. What extraction method was used for the MDA quantification?

-L174-180. I would suggest moving this section to the part in which LRWC is discussed.

-L215-216. These two sentences should be rephrased. Is the decrease in chlorophyll caused by Fe and Mg deficiency or by accumulation of ROS and Na? Or are these two causes related to each other? If so, how?

-L218. By “chlorophyll membranes” do authors mean “chloroplast membranes”?

-Figure 1. The scale does not work particularly well since the values below 0 are considerably more condensed that those above. I would suggest using a logarithmic scale to counteract this. In addition, the colors at the very top and bottom of the scale are very similar and easy to mix. Lastly, I believe that the bottom two labels should be “150S-50MT” and “150S-100MT”.

-Figure 2. 150 mM NaCl treatment is missing in the legend. Error bars should be included.

-L247. According to figure 2, the LRWC did not significantly decrease in salt-stressed plant without Melatonin treatment.

-L248. It is not correct to claim that plant fresh and dry weight decreased because of the decrease in RWC. In any case it would be the other way around, since RWC is a function of dry and fresh biomass.

-L313. Since the effect of melatonin has already been shown to activate enzymatic and non-enzymatic antioxidants, it could be said that this results “support” it, rather than “suggest” it. Proper citations to the already published works should be provided.

-L340. This sentence should be deleted, as it just repeats what was stated in the previous two sentences.

-L346-347. The authors only show K/NA and Ca/Na ratios, not the individual concentrations, so it is not possible for the reader to confirm whether K uptake increased. Also, citations regarding the role of K in water balance should be provided.

-Figure 3. Error bars and statistical analysis should be included.

-L360. The Na content is not available in the data, only ratios. Also, it is not clear whether figure 3 shows the ratios found in the roots, shoots, or whole plant.

-L362-364. The figure 3 does not show any differentiation between roots and shoots, therefore it cannot be claimed that the effect of melatonin happened first on the roots.

-L384. Delete “combined”

-L388-389. Delete this sentence, as it is already shown in the previous paragraph.

Author Response

Dear Editor,

On behalf of all of the co-authors, I would like to thank the editor and the reviewers for careful reading, and constructive suggestions for our manuscript. According to comments from editor and reviewers, we comprehensively revised our manuscript. We are looking forward to your feedback and further comments/suggestions are welcome.

The reviewers' comments are reproduced in black; our responses are detailed below in red:

RESPONSE TO REVIEWERS 

First of all, I would like to extend our gratitude to the anonymous reviewers for forwarding very constructive criticism of the MS and giving us an opportunity to improve the standard of the MS. We are glad to state that all suggestions forwarded by the reviewers have been incorporated in their entirety and none has been left unaddressed.

The manuscript submitted by Dadasoglu et al. discusses the effect of melatonin foliar spray on the response of chickpea to salinity stress in greenhouse conditions. The manuscript shows the effect of melatonin on plant growth, relative water content, chlorophyll and carotenoid content, electrolytic leakage, antioxidant activity and K/Na and Ca/Na ratios. The experiment is reasonably well design, and the data is in line other publications on the topic. Unfortunately, this topic has been extensively researched and many publications can be easily found in the literature. The only factor of novelty is the use of chickpea, however, considering that the effect of melatonin in salt-treated plants seems to be extremely consistent among the many species in which it has been tested, I fail to see the necessity of repeating the same analysis in other species. Unless the authors can highlight the novelty behind this manuscript, I do not think that is worth being published in Horticulturae.

We agree with the Reviewer tiara are studies salinity stress and melatonin applications. However, to our best knowledge there is no study on this subject. The present study firstly was carried out to focus on the effects of spraying seedlings with MT on enhancing salt stress tolerance of chickpea plants, secondly, to reveal the mechanism of MT effect on the growth of chickpea seedling under salt stress.

These are some other points that require the author’s attention:

-If numbers are used for the in-text citations, they should be ordered according to their position in the manuscript, not alphabetically.

Please see that we have corrected

-L83. Does the 20-35 °C temperature refer to the night/day temperature?

Yes, we have added

-L102. Authors should specify how old the plants were when the Melatonin and Salinity treatments were applied.

Please see that we have corrected

-Authors should specify in which tissue each of the measurement/quantifications were done. The table and figure captions only mention “seedlings”. Does this mean that they were measured on the whole plant? Authors should clearly explain this and implement it in the discussion accordingly.

Please see that we have corrected as leaves

-L151. What extraction method was used for the MDA quantification?

Extraction of plant tissues was carried out essentially as described previously for the analysis of plant l-ascorbic acid (L-AA) using a metaphosphoric acid (MPA) extraction buffer (M.W. Davey, C. Franck, J. Keulemans Distribution, developmental, and stress responses of antioxidant metabolism in Malus Plant Cell Environ., 27 (2004), pp. 1309-1320)

-L174-180. I would suggest moving this section to the part in which LRWC is discussed.

These sentences explain decrease of growth under salt stress.

-L215-216. These two sentences should be rephrased. Is the decrease in chlorophyll caused by Fe and Mg deficiency or by accumulation of ROS and Na? Or are these two causes related to each other? If so, how?

Please see that we have corrected

-L218. By “chlorophyll membranes” do authors mean “chloroplast membranes”?

Please see that we have corrected

-Figure 1. The scale does not work particularly well since the values below 0 are considerably more condensed that those above. I would suggest using a logarithmic scale to counteract this. In addition, the colors at the very top and bottom of the scale are very similar and easy to mix. Lastly, I believe that the bottom two labels should be “150S-50MT” and “150S-100MT”.

Done

-Figure 2. 150 mM NaCl treatment is missing in the legend. Error bars should be included.

Please see that we have corrected

-L247. According to figure 2, the LRWC did not significantly decrease in salt-stressed plant without Melatonin treatment.

Please see that we have corrected

-L248. It is not correct to claim that plant fresh and dry weight decreased because of the decrease in RWC. In any case it would be the other way around, since RWC is a function of dry and fresh biomass.

Please see that we have corrected

-L313. Since the effect of melatonin has already been shown to activate enzymatic and non-enzymatic antioxidants, it could be said that this results “support” it, rather than “suggest” it. Proper citations to the already published works should be provided.

Please see that we have corrected

-L340. This sentence should be deleted, as it just repeats what was stated in the previous two sentences.

Please see that we have deleted.

-L346-347. The authors only show K/NA and Ca/Na ratios, not the individual concentrations, so it is not possible for the reader to confirm whether K uptake increased. Also, citations regarding the role of K in water balance should be provided.

PLaese see that We have made corrections as required.

-Figure 3. Error bars and statistical analysis should be included.

Done

-L360. The Na content is not available in the data, only ratios. Also, it is not clear whether figure 3 shows the ratios found in the roots, shoots, or whole plant.

Done

-L362-364. The figure 3 does not show any differentiation between roots and shoots, therefore it cannot be claimed that the effect of melatonin happened first on the roots.

Please see that we have corrected

-L384. Delete “combined”

Done

-L388-389. Delete this sentence, as it is already shown in the previous paragraph.

Done

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Horticulturae-1998099：

This manuscript have been revised according to reviewers’ comments. However, there still reminds some problems.

Special comments:

Materials and Methods:

1. Why only choose the 50 and 100 uM MT as target concentration? Please give more detail introduction.

Results and Discussion:

2.  Please add standard deviation in the Table 1-3 and Figure 2-3.

Conclusions:

3. The conclusions was too short and simple. Please add the implications of the results and prospects for future applications

Comments for author File: Comments.pdf

Author Response

Dear Editor,

On behalf of all of the co-authors, I would like to thank the editor and the reviewers for careful reading, and constructive suggestions for our manuscript. According to comments from editor and reviewers, we comprehensively revised our manuscript. We are looking forward to your feedback and further comments/suggestions are welcome.

The reviewers' comments are reproduced in black; our responses are detailed below in red:

RESPONSE TO REVIEWERS 

First of all, I would like to extend our gratitude to the anonymous reviewers for forwarding very constructive criticism of the MS and giving us an opportunity to improve the standard of the MS. We are glad to state that all suggestions forwarded by the reviewers have been incorporated in their entirety and none has been left unaddressed.

This manuscript has been revised according to reviewers’ comments. However, there still reminds some problems.

 Special comments:

Materials and Methods:

1. Why only choose the 50 and 100 uM MT as target concentration? Please give more detail introduction.

 Done

Results and Discussion:

2. Please add standard deviation in the Table 1-3 and Figure 2-3.

 Please see that we have added standard deviations

Conclusions:

3. The conclusions was too short and simple. Please add the implications of the results and prospects for future applications

 Please see that we have added the implications of the results and prospects for future applications

Author Response File: Author Response.docx

Reviewer 3 Report

The quality of the manuscript has improved after the changes implemented by the authors, even if the novelty remains fairly low. If the editors of Horticulturae can look past the low novelty score, I believe that the manuscript could be publishable after making few more corrections.

I would suggest having the manuscript checked by a native English speaker or an English editing service, as it would improve the clarity of the text.

L26. “suggested for decreasing”

L47-49. Consider rephrasing the sentence.

L82. Please, specify how often and how much water was added to the plants, if any.

L91. Please specify the volume of solution sprayed.

L98, Please specify the volume of saline irrigation applied and how often they were applied.

Figure 1. This figure has not been modified, except for the correction in the axis label, so my previous comments still stand:  The scale does not work particularly well since the values below 0 are considerably more condensed that those above. I would suggest using a logarithmic scale to counteract this. In addition, the colors at the very top and bottom of the scale are very similar and easy to mix. Also, please indicate the value of the bottom part of the scale.

L189-191. I would suggest writing “The accumulation of Na+ and ROS in plants under salt stress may lead to negative effects and contribute to the reduction of photosynthetic pigments”

L208. Authors do not show any photosynthetic measurement in this manuscript, and chlorophyll content alone is not enough to claim changes in photosynthesis.

L209. What does Pn stand for? If authors mean photosynthetic rates, I would suggest rephrasing this sentence, since they would be claiming that photosynthesis is modulated by photosynthesis.

L224. "Toxicity"

L225. "Increased" (or increase in)

Figure 2. How come the statistical analysis is different from the previous version of the manuscript? Authors should indicate in the figure caption the type of statistical test used (also in Figure 3).

L275. Write only “MT content of plants increases under salt stress.”

L281. Please revise and rewrite this sentence.

L330. The authors only show K/Na and Ca/Na ratios, not the individual concentrations, so it is not possible for the reader to confirm whether K uptake increased. Technically, K/Na ratio could decrease even if K concentration itself decreases, provided Na concentration decreases in a bigger extent. I would suggest that authors include K, Na and Ca concentrations as supplementary material in order to support their claims. 

Author Response

Dear Editor,

On behalf of all of the co-authors, I would like to thank the editor and the reviewers for careful reading, and constructive suggestions for our manuscript. According to comments from editor and reviewers, we comprehensively revised our manuscript. We are looking forward to your feedback and further comments/suggestions are welcome.

The reviewers' comments are reproduced in black; our responses are detailed below in red:

RESPONSE TO REVIEWERS 

First of all, I would like to extend our gratitude to the anonymous reviewers for forwarding very constructive criticism of the MS and giving us an opportunity to improve the standard of the MS. We are glad to state that all suggestions forwarded by the reviewers have been incorporated in their entirety and none has been left unaddressed.

The quality of the manuscript has improved after the changes implemented by the authors, even if the novelty remains fairly low. If the editors of Horticulturae can look past the low novelty score, I believe that the manuscript could be publishable after making few more corrections.

Thank you very much

I would suggest having the manuscript checked by a native English speaker or an English editing service, as it would improve the clarity of the text.

Please see that I have the manuscript checked by a native English speaker

L26. “suggested for decreasing”

Done

L47-49. Consider rephrasing the sentence.

Done

L82. Please, specify how often and how much water was added to the plants, if any.

Soil moisture meter (WET Sensor) was used to calculate irrigation water amounts. Pots were irrigated to reach field capacity at each irrigation.

L91. Please specify the volume of solution sprayed.

Done

L98, Please specify the volume of saline irrigation applied and how often they were applied.

Soil moisture meter (WET Sensor) was used to calculate irrigation water amounts. Pots were irrigated to reach field capacity at each irrigation.

Figure 1. This figure has not been modified, except for the correction in the axis label, so my previous comments still stand:  The scale does not work particularly well since the values below 0 are considerably more condensed that those above. I would suggest using a logarithmic scale to counteract this. In addition, the colors at the very top and bottom of the scale are very similar and easy to mix. Also, please indicate the value of the bottom part of the scale.

Please see that we have corrected

L189-191. I would suggest writing “The accumulation of Na+ and ROS in plants under salt stress may lead to negative effects and contribute to the reduction of photosynthetic pigments”

Done

L208. Authors do not show any photosynthetic measurement in this manuscript, and chlorophyll content alone is not enough to claim changes in photosynthesis.

Yes The reviewer wright, We cited earlier study here.

L209. What does Pn stand for? If authors mean photosynthetic rates, I would suggest rephrasing this sentence, since they would be claiming that photosynthesis is modulated by photosynthesis.

Please see that we have corrected

L224. "Toxicity"

Done

L225. "Increased" (or increase in)

Done

Figure 2. How come the statistical analysis is different from the previous version of the manuscript? Authors should indicate in the figure caption the type of statistical test used (also in Figure 3).

Sorry about that inconvenience, we have reviewed the data and graphs, we added standard deviations also

L275. Write only “MT content of plants increases under salt stress.”

Please see that we have corrected

L281. Please revise and rewrite this sentence.

Please see that we have revised

L330. The authors only show K/Na and Ca/Na ratios, not the individual concentrations, so it is not possible for the reader to confirm whether K uptake increased. Technically, K/Na ratio could decrease even if K concentration itself decreases, provided Na concentration decreases in a bigger extent. I would suggest that authors include K, Na and Ca concentrations as supplementary material in order to support their claims.

Done

Author Response File: Author Response.docx