Circadian Clock Contributes to Modulate Salinity Stress-Responsive Antioxidative Mechanisms and Chloroplast Proteome in Spinacia oleracea

Round 1

Reviewer 1 Report (New Reviewer)

Journal: Agriculture (ISSN 2077-0472)

Manuscript ID: agriculture-2073525

Type: Article

Title: Circadian clock contributes to modulate salinity stress-responsive antioxidative mechanisms and chloroplast proteome in Spinacia oleracea

 

Circadian clocks are 24-h biological oscillators, which generally enable organisms to anticipate predictable, daily changes that are linked to the rotation of the earth. Circadian regulation may provide a sophisticated, tunable mechanism to ensure an optimum compromise between plant growth and salinity stress tolerance. Adjustments in clock function in response to changes in the environment may allow spinach plants to re-adjust this balance and to ensure a sustained increase in salinity stress tolerance when needed. If these hypotheses are correct, such mechanisms may in the future be exploited for improving salinity stress tolerance in spinach and other crops without reducing yield.

 

This topic is investigated in the literature, and there is a very few of reference published. However, this paper gives significant contribution to the current knowledge in related field. The data are sound and it deserves to be published, after minor revisions as:

 

??? Abstract is not written as per MDPI Journal Format. Plenty of errors observed. Use www.turnitin.com to find and eliminate unnecessary self-repetition and any copied text.

 

??? Keywords should not be the same as mentioned in the title or abstract.

 

??? Use the passive voice not possessive (we, our, etc.) such as in abstract Page-1, line-32 e.g., “our” can be replaced.  

 

??? The text has many typing and grammatical errors, capitalization issues.

 

??? No proper formatting, spacing issues and formalities etc. have not been seen. Please read the Instructions to Authors of MDPI Journal Agriculture as to settle arrangement problems.

 

??? English style and language requires a profound revision. However, the readability of the manuscript needs to be improved, preferably carefully reviewing by a native English speaker.

 

??? Abbreviations must be described completely at first mention alongwith brackets, afterwards use Abbreviations throughout the manuscript.

 

??? The materials and methods section is very brief. Please add details for analytical methodologies to make it reproducible.

 

 

??? Quality assurance of data is mandatory!!! How many batch, repeats, amendment/chemical grade and for used instruments manufacturers’ user manual and instructions were strictly followed or not!!!

 

Data is sound one. It deserves to be published.

 

??? Very Minute Scientific Discussion. It can be improved viz I would have expected slightly greater discussion of how exactly plants growth was affected, more detail on the mechanisms and logical reasoning is required. The current discussion says very little about “Circadian clock contributes to modulate salinity stress-responsive antioxidative mechanisms and chloroplast proteome in Spinacia oleracea”

 

??? For discussion section, not much detailed discussion is going on. This is just restating the observations and results. There is much more scope here for discussing the implications of what these results mean.

 

 

??? Please cite Figure No. or Table No. in brackets at suitable places for a better connectivity in results and discussion sections as to facilitate the reader.

 

??? Please improve the figures/graphs with legends. Please mention replications n = ???

 

??? The Tables must be as per MDPI Journal Agriculture format.

 

??? Novelty of this research work is again questionable with reference to practical significance and economic feasibility must be worked and mentioned.

 

??? A few very old references have been used. These must be updated with recent research findings or removed. Proper formatting is questionable. It must be according to MDPI Journal Agriculture  References formatting are inconsistent. A few DOI missing. Verify each reference from original source and cross check references in the text and reference section.

Author Response

Reviewer 1

Circadian clocks are 24-h biological oscillators, which generally enable organisms to anticipate predictable, daily changes that are linked to the rotation of the earth. Circadian regulation may provide a sophisticated, tunable mechanism to ensure an optimum compromise between plant growth and salinity stress tolerance. Adjustments in clock function in response to changes in the environment may allow spinach plants to re-adjust this balance and to ensure a sustained increase in salinity stress tolerance when needed. If these hypotheses are correct, such mechanisms may in the future be exploited for improving salinity stress tolerance in spinach and other crops without reducing yield.

 

This topic is investigated in the literature, and there is a very few of reference published. However, this paper gives significant contribution to the current knowledge in related field. The data are sound and it deserves to be published, after minor revisions as:

 

??? Abstract is not written as per MDPI Journal Format. Plenty of errors observed. Use www.turnitin.com to find and eliminate unnecessary self-repetition and any copied text.

Response>

Authors have revised the abstract as per suggestions raised by respected reviewer  

??? Keywords should not be the same as mentioned in the title or abstract.

Response>

Author have revised the keywords which is now distinct from the title and abstract

 ??? Use the passive voice not possessive (we, our, etc.) such as in abstract Page-1, line-32 e.g., “our” can be replaced.  

Response>

Authors have revised whole manuscript by making using the passive voice not possessive nouns.

??? The text has many typing and grammatical errors, capitalization issues.

Response>

Authors have revised the whole manuscript by changing the grammatical errors and capitalization issues

??? No proper formatting, spacing issues and formalities etc. have not been seen. Please read the Instructions to Authors of MDPI Journal Agriculture as to settle arrangement problems.

Response>

Authors have revised whole manuscript by changing the formatting in MDPI Journal Agriculture format after checking the Instructions to Authors

??? English style and language requires a profound revision. However, the readability of the manuscript needs to be improved, preferably carefully reviewing by a native English speaker.

Response>

 Authors have tried to revised the English language by taking help from native speaker and using grammerly

??? Abbreviations must be described completely at first mention alongwith brackets, afterwards use Abbreviations throughout the manuscript.

Response>

Authors have revised the whole manuscript by mentioning the Abbreviations first completely along with brackets, afterwards the Abbreviations

 ??? The materials and methods section is very brief. Please add details for analytical methodologies to make it reproducible.

Response>

Authors have revised the manuscript by adding the details of analytical methodologies in the materials and methods part. Please see highlighted sections

 ??? Quality assurance of data is mandatory!!! How many batch, repeats, amendment/chemical grade and for used instruments manufacturers’ user manual and instructions were strictly followed or not!!!

Response>

Authors have revised the manuscript by adding the chemical grade and repeats in the materials and methods part in the revised manuscript

 Data is sound one. It deserves to be published.

 Response>

We thank respected reviewer for her/his appreciation towards our research work

??? Very Minute Scientific Discussion. It can be improved viz I would have expected slightly greater discussion of how exactly plants growth was affected, more detail on the mechanisms and logical reasoning is required. The current discussion says very little about “Circadian clock contributes to modulate salinity stress-responsive antioxidative mechanisms and chloroplast proteome in Spinacia oleracea”

Response>

Authors have revised the manuscript by adding the few recent data in the discussion part.  

??? For discussion section, not much detailed discussion is going on. This is just restating the observations and results. There is much more scope here for discussing the implications of what these results mean.

Response>

Authors have revised the discussion part as suggested by reviewer.

 ??? Please cite Figure No. or Table No. in brackets at suitable places for a better connectivity in results and discussion sections as to facilitate the reader.

Response>

Authors have revised the manuscript by adding the Figure no. in brackets at suitable places in both results and discussion parts

  ??? Please improve the figures/graphs with legends. Please mention replications n = ???

Response>

Authors have revised the manuscript by mentioning the replications using ‘n’ in figure legends

??? The Tables must be as per MDPI Journal Agriculture format.

Response>

Authors have not used any tables in the manuscript; figures and figure legends are in the MDPI Journal Agriculture format.

 ??? Novelty of this research work is again questionable with reference to practical significance and economic feasibility must be worked and mentioned.

Response>

 Authors have given the practical feasibility of this research work in conclusion section

??? A few very old references have been used. These must be updated with recent research findings or removed. Proper formatting is questionable. It must be according to MDPI Journal Agriculture  References formatting are inconsistent. A few DOI missing. Verify each reference from original source and cross check references in the text and reference section.

Response>

Authors have referred new references in the revised manuscript

Authors have revised the manuscript by changing the reference according to MDPI Journal Agriculture references; also, the references in the text and reference part were cross checked.

Reviewer 2 Report (Previous Reviewer 4)

 

The manuscript brings a comprehensive overview of the morphological, physiological and biochemical changes that occur in two spinach genotypes (during day time) when challenged against salt stress. I do understand that this is a discovery driven study but due to the extensive and diverse type of analysis I found hard to follow a line and make clear cut conclusions. Paper is dense and although it contains important information, I have some concerns that hamper me to endorse the publication.

 

1. Data presentation is confusing and hard to follow from a biological perspective. A suggestion would be using a multivariate approach in order to find specific patterns with true biological meaning;

 

 Response>

Authors have revised whole manuscript and added how statistical analysis (Multivariate analysis) was done. We have also revised results and figures, figure legends to make it more undertakable among all biological replicates. Please see the revised manuscript (all changes are highlighted in red)

 

Reviewer>

Data presentation is still confusing and hard to understand from a global perspective. I suggested the use of a Multivariate Analysis (e.g. PCA, clustering….not only ANOVA/MANOVA) in order to find patterns and facilitate visual interpretation of data.

 

2. Authors did not look into real “circadian clock” changes as only data from day light period was sampled. It would have been nice to read about changes during the night time. As it is, I can affirm that changes were due to light intensity changes throughout the day;

 

Response>

Circadian loop have morning and evening loops and we have checked only those two loops, and changes are definitely because of circadian rhythmic periods because all works based on photoperiodism as reviewer suggested it could be based on light intensity (photoperiodism is based on loops). We have not checked the night loop due to some ethical restrictions.

 

Reviewer>

I am aware that authors collected data from the morning and evening loops only (central loop?). However, when I asked about sampling during the night time, I was looking for consistency throughout the experiment that could support the oscillatory phenomenon expected for a circadian study. If night sampling was/is not possible more sampling points distributed throughout a larger period of time (not only at day 5 and day 10) would suffice (2-3 days).

 

3. Is seems that circadian events can only be determined when you leave plants in the dark and oscillation still occur for a while (system left w/o entrainment);

 

Response>

Circadian loop does not work on dark conditions but there is morning, evening and night loop and all works based on photoperiodism.

 

Reviewer>

Circadian usually refers to biological variations that occur throughout a 24 hours cycle apart from the presence of light. It seems to me that if one wants to show circadian oscillations, a night time period has to be added to the assay. Otherwise only morning/central/evening loops are revealed (which is valid if, again, the experiment is carried out for a longer period of time).

 

4. I found hard to make conclusions out of the morphological data as plants were grown from seeds (plants have different genome backgrounds);

 

Response>

Plants are grown from seeds only unless you are following any other vegetative method of propagation like grafting, budding etc. We do not understand reviewers comments why she/he asked this question. Definitely all plants will have different genome backgrounds with some similarities.

 

Reviewer>

Sorry if I did not make that clear. What I meant is that is morphological data was very variable and this was possible due to the use of seeds as the starting material for the experiments.

 

5. Data sometimes does not reflect author´s statements and conclusions;

 

Response>

We have revised whole manuscript to make consistent statements reflecting the data present in this manuscript.

 

Reviewer>

It seems to me that authors only added a few sentences within each section.

 

6. Protein analysis: Authors cannot infer quantitative differences based on the staining intensity. Also, protein identification should not be based solely on the MW of the bands. Finally, differences seem to be too slight and not significant. A proper image or mass spectrometry analysis should have been carried out.

 

Response>

We have revised the gel figures by indicating differences by arrows among all treatments. We have not identified proteins which were up-regulated or down-regulated and will be taken into consideration for next set of experiments which is currently going on in our lab.

 

Reviewer>

Authors added red arrows in the gel figure/s. However, protein identification is still only based on the MW. Quantitative differences are still only based on staining intensities.

 

 

Author Response

Reviewer 2

The manuscript brings a comprehensive overview of the morphological, physiological and biochemical changes that occur in two spinach genotypes (during day time) when challenged against salt stress. I do understand that this is a discovery driven study but due to the extensive and diverse type of analysis I found hard to follow a line and make clear cut conclusions. Paper is dense and although it contains important information, I have some concerns that hamper me to endorse the publication.

 

1. Data presentation is confusing and hard to follow from a biological perspective. A suggestion would be using a multivariate approach in order to find specific patterns with true biological meaning;

 

Response>

Authors have revised whole manuscript and added how statistical analysis (Multivariate analysis) was done. We have also revised results and figures, figure legends to make it more undertakable among all biological replicates. Please see the revised manuscript (all changes are highlighted in red)

 

Reviewer>

Data presentation is still confusing and hard to understand from a global perspective. I suggested the use of a Multivariate Analysis (e.g. PCA, clustering….not only ANOVA/MANOVA) in order to find patterns and facilitate visual interpretation of data.

 

Response>

 Authors are unable to provide PCA clustering.

2. Authors did not look into real “circadian clock” changes as only data from day light period was sampled. It would have been nice to read about changes during the night time. As it is, I can affirm that changes were due to light intensity changes throughout the day;

 

Response>

Circadian loop have morning and evening loops and we have checked only those two loops, and changes are definitely because of circadian rhythmic periods because all works based on photoperiodism as reviewer suggested it could be based on light intensity (photoperiodism is based on loops). We have not checked the night loop due to some ethical restrictions.

 

Reviewer>

I am aware that authors collected data from the morning and evening loops only (central loop?). However, when I asked about sampling during the night time, I was looking for consistency throughout the experiment that could support the oscillatory phenomenon expected for a circadian study. If night sampling was/is not possible more sampling points distributed throughout a larger period of time (not only at day 5 and day 10) would suffice (2-3 days).

 

Response>

The samplings were from morning, central and evening loops which is 6 and 10 am (morning loop); also, 2 pm (central loop) and 6 pm (evening loop). The sampling during the night time was not possible due to some ethical restrictions, as mentioned in our previous response. The sampling points were not distributed throughout a larger period of time, as there was a change in colour and nature of leave observed during day 10 of salt treatment.

3. Is seems that circadian events can only be determined when you leave plants in the dark and oscillation still occur for a while (system left w/o entrainment);

 Response>

Circadian loop does not work on dark conditions but there is morning, evening and night loop and all works based on photoperiodism.

 Reviewer>

Circadian usually refers to biological variations that occur throughout a 24 hours cycle apart from the presence of light. It seems to me that if one wants to show circadian oscillations, a night time period has to be added to the assay. Otherwise only morning/central/evening loops are revealed (which is valid if, again, the experiment is carried out for a longer period of time).

Response>

Authors do agree that circadian clock works throughout the 24 hours as mentioned above due to some ethical restrictions authors were unable to retrieve the experimental analysis for night loop. A specific permission is needed for night oscillation loops.

4. I found hard to make conclusions out of the morphological data as plants were grown from seeds (plants have different genome backgrounds);

 

Response>

Plants are grown from seeds only unless you are following any other vegetative method of propagation like grafting, budding etc. We do not understand reviewers’ comments why she/he asked this question. Definitely all plants will have different genome backgrounds with some similarities.

 

Reviewer>

Sorry if I did not make that clear. What I meant is that is morphological data was very variable and this was possible due to the use of seeds as the starting material for the experiments.

Response>

For any experiments we have to use seeds only at the starting till the treatments are given at some stage of the plants like we did at vegetative stage. Sorry if we are not able to understand your comment. We believe all morphological changes are not due to use of seeds

5. Data sometimes does not reflect author´s statements and conclusions;

 

Response>

We have revised whole manuscript to make consistent statements reflecting the data present in this manuscript.

 

Reviewer>

It seems to me that authors only added a few sentences within each section.

Response>

Authors have still tried to add more sentences, with recent references to make all data consistent with all statements. 

6. Protein analysis: Authors cannot infer quantitative differences based on the staining intensity. Also, protein identification should not be based solely on the MW of the bands. Finally, differences seem to be too slight and not significant. A proper image or mass spectrometry analysis should have been carried out.

 

Response>

We have revised the gel figures by indicating differences by arrows among all treatments. We have not identified proteins which were up-regulated or down-regulated and will be taken into consideration for next set of experiments which is currently going on in our lab.

 

Reviewer>

Authors added red arrows in the gel figure/s. However, protein identification is still only based on the MW. Quantitative differences are still only based on staining intensities.

Response>

Authors have revised the manuscript by changing the bar graph for more clarity and to quantify protein in micrograms. We are working on another set of experiments to identify the differentially expressed proteins

Reviewer 3 Report (Previous Reviewer 5)

The authors have addressed my queries satisfactorily. 

Author Response

Reviewer 3

The authors have addressed my queries satisfactorily. 

Response>

Authors thank you for the positive comments

Round 2

Reviewer 2 Report (Previous Reviewer 4)

Although data seem sound, conclusions still apear to be over extrapolated due to technical limitations (not fault) of the proposed experiments.

Author Response

Authors have tried to make conclusions confined to make the whole study understandable and also clear ideas what can be future prospects of this study. Please see the blue highlighted text of the revised manuscript 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors provide only a cursory description of the results; Moreover, there are many mistakes in the writing of the manuscript. I do not think the manuscript can be published in its current form. I suggest that the author write again, and then submit the manuscript for review.

The main comments:

1. Figure 2 Plant height determination results from how many plants? How does the author explain the variation in plant height? Why do root lengths change repeatedly?

2. The expressions of the full name and abbreviations should be the same, for example, “lipid peroxidation (LPO)”and“LPO (lipid peroxidation)”; Since the author has defined abbreviations, they should be used in the manuscript.

3. Line 68, ” Reactive oxygen species” changes into ROS;

4. Row 95, adding Latin name for species after " Spinach ";

5. Line 299, 303, "MgCl2" number 2 should be subscripted;

Reviewer 2 Report

Your paper has less innovations and more experiments need making for.The applied method need more citations.

Reviewer 3 Report

The authors have done good writing and papers is up to the mark. I don't have any comments.

Reviewer 4 Report

The manuscript brings a comprehensive overview of the morphological, physiological and biochemical changes that occur in two spinach genotypes (during day time) when challenged against salt stress. I do understand that this is a discovery driven study but due to the extensive and diverse type of analysis I found hard to follow a line and make clear cut conclusions. Paper is dense and although it contains important information, I have some concerns that hamper me to endorse the publication.

 

1. Data presentation is confusing and hard to follow from a biological perspective. A suggestion would be using a multivariate approach in order to find specific patterns with true biological meaning;

2. Authors did not look into real “circadian clock” changes as only data from day light period was sampled. It would have been nice to read about changes during the night time. As it is, I can affirm that changes were due to light intensity changes throughout the day;

3. Is seems that circadian events can only be determined when you leave plants in the dark and oscillation still occur for a while (system left w/o entrainment);

4. I found hard to make conclusions out of the morphological data as plants were grown from seeds (plants have different genome backgrounds);

5. Data sometimes does not reflect author´s statements and conclusions;

6. Protein analysis: Authors cannot infer quantitative differences based on the staining intensity. Also, protein identification should not be based solely on the MW of the bands. Finally, differences seem to be too slight and not significant. A proper image or mass spectrometry analysis should have been carried out.

 

 

 

 

Reviewer 5 Report

The study reporting the role of circadian clock in salinity response in spinach is interesting. The experiments conducted in this study generates sufficient data to conclude the roles and put forward a conceptual hypothesis. However, the manuscript has some major flaws in it. I have listed my comments in the followings to be considered by the authors to improve the manuscript.

Major

1. This is a data driven study as lots of experimental data has been generated. Thus, to find out the true effects, each data has to be statistically validated. The authors have not mentioned which statistical program they have used to validate these data (t-test, one way/two way ANOVA, GLM, etc.). As the study involves two salinity levels (20 & 50 mM) and two spinach varieties, the authors should employ a Two-way ANOVA analysis for deducing the statistical significance of the data. Please indicate the F value in the significant data when representing in the results.

2. Figure 2, 3, 7, 6, and 8 needs to be reconstructed. Please represent the data for day 5 and day 10 separately. Most importantly, use statistics to validate the data day wise, not for all. So, for each day the a, b, c, d, etc. should be separate. Use color gradients carefully so that easily it can be corelated to the graphs. Currently, the legend colors are too confusing.   

3. Figure 11 B ( and for all other picture where it is applicable), the PAGE gels look almost similar. Indicate the main or desired difference by using colored arrows. Improve the readability of all the picture. 

Minor

1. Sentence structuring and English of the manuscript is below average. There are numerous places ( lines 11-12, 13-14, 17, 135-140, 141-142, virtually almost everywhere) where the sentences needs to be revised or English needs to be improved. I strongly suggest the authors to form small sentences instead of long and complex ones. The revised version must be proof read by an English expert or a native speaker before submission. 

2. At many places, the short form is first written the followed by the full word. At some places the opposite style can also be found. And at some other places, no full form at all is there. I suggest the authors to carefully check the manuscript for all such errors and fix them. Use the full word first, followed by the abbreviation in bracket. 

3. Check the writing tense for the entire manuscript. At many places the tenses are not proper.