Biogenic Titanium Dioxide Nanoparticles Ameliorate the Effect of Salinity Stress in Wheat Crop
, Nilofar Mustafa 1, Riaz Khan 1, Zia-ur-Rehman Mashwani 1, Naveed Iqbal Raja 1,*, Mikhlid H. Almutairi 2, Lotfi Aleya 3, Amany A. Sayed 4, Shah Zaman 5, Laraib Sawati 6 and Sohail 7,*
Round 1
Reviewer 1 Report
This article studied the positive effects of Biogenic Titanium Dioxide Nanoparticles under salinity stress in wheat crop. They found that plants treated with this particle enhances physiological changes such as germination rate, germination indices, fresh and dry weight of plant, number of leaves, RWC and MSI, Chlorophyll, metabolic profiling with and without salt They concluded that outcomes of the research are notable, and Titanium Dioxide Nanoparticles can be a useful supply of fertilizer in the crop fields. While I agree with their findings, they need major improvement before it can be published.
1. Poor writing style. Many of their sentences were very hard to comprehend. They need to re-write the whole manuscript and review it with a native English speaker.
2. They should talk more about using Nanoparticles in agriculture, their positive and negative effects and cite more relevant articles.
3. Why they chose Duncan’s Multiple Range Test (DMRT) for the statistical analysis? I think Tukey HSD test would be more appropriate.
4. They mentioned they used P < 0.05, but they didn’t show which are significant in any of their figures and tables.
5. Table 2 and 3 are hard to comprehend. They should use figures to represent. They can still keep the tables as supplementary files and generate figures for the main doc. They don’t need to show all the data, rather focus on some of the important ones.
6. All the figure legends should be descriptive, so that reader could follow without going back and forth to the main text.
7. Figure 2a says TiO2 NPs+NaCl, but I see only TiO2 NPs in there. Check the other legends too. Also, check the spelling “concentration”
8. All the figures say both wheat varieties showed significant improvement correspondingly compared to control. I don’t see the corresponding controls in the figures. Also, how do the readers know which are statistically significant by looking at the figures?
9. Why they used bar diagram for figure 3 and 4, and line diagram for 5 and 6? Figure 7 is also different from the others. They should keep it consistent since they are basically showing the similar results.
10. They need to describe the results in a better way. Reading about percentage of improvement in every line makes it monotonous.
11. Overall, they need to re-write the whole manuscript and represent the data in a better way. There are typos, they should check.
Author Response
Response to Reviewer 1 Comments
Dear Reviewer 1,
Thank you for reviewing our manuscript entitled “Biogenic Titanium Dioxide Nanoparticles ameliorate the effect of salinity stress in wheat crop” we tried our best to incorporate the comment/suggestion as you recommend in the revision of manuscript.
Comment: 1. Poor writing style. Many of their sentences were very hard to comprehend. They need to re-write the whole manuscript and review it with a native English speaker.
Response: We are thankful to the reviewer for mentioning such errors, we improved the whole manuscripts in a better way and rewrite it in a simple words to be easier for the readers. The manuscript were intensively reviewed with a native English speaker.
Comment: 2. They should talk more about using Nanoparticles in agriculture, their positive and negative effects and cite more relevant articles.
Response: Thank you for highlighting such issues, we added more relevant description regarding role of different nanoparticles in agriculture, and provide a citations of relevant articles with an updated references. Also highlighted in track mode changes.
Comments: 3. Why they chose Duncan’s Multiple Range Test (DMRT) for the statistical analysis? I think Tukey HSD test would be more appropriate.
Response: we are thankful to the reviewer for this point, we have analyzed the data again using SPSS, and we performed ANOVA, and least significant difference were analyzed using LSD test. The data in the table are now presented with lettering indicating The data represented in the columns in the form of means having similar letters are identical and dissimilar letters are significantly different where p was kept p=<0.05.
Comments: 4. They mentioned they used P < 0.05, but they didn’t show which are significant in any of their figures and tables.
Response: we are thankful to the reviewer for identifying this issue, we described the significant values and identified in all figures and tables of the manuscript. The data represented in the columns in the form of means having similar letters are identical and dissimilar letters are significantly different where p was kept p=<0.05. Also highlighted in track mode changes.
Comments: 5. Table 2 and 3 are hard to comprehend. They should use figures to represent. They can still keep the tables as supplementary files and generate figures for the main doc. They don’t need to show all the data, rather focus on some of the important ones.
Response: We again thankful to the reviewer for raising such comments, we used the tables for germination and agronomical parameters to make it easier for the readers. Instead of figures, we added tables for these two attributes to reduce complexion, and make the paper more beautiful and interesting. We discussed about the important data by skipping irrelevant descriptions highlighted in track mode changes.
Comments: 6. All the figure legends should be descriptive, so that reader could follow without going back and forth to the main text.
Response: We are again thankful to the reviewer for highlighting such issues, all figures legend were changed and discussed in a comprehensive form to become more appropriate for the readers to understand. And highlighted in a track mode changes.
Comments: 7. Figure 2a says TiO2 NPs+NaCl, but I see only TiO2 NPs in there. Check the other legends too. Also, check the spelling “concentration”
Response: we are pleased to the reviewer for identifying this error, this was done mistakenly during applying a software; we made changes in Figure 2a legends and correction in the spelling “concentration” has done also highlighted in track mode changes.
Comments: 8. All the figures say both wheat varieties showed significant improvement correspondingly compared to control. I don’t see the corresponding controls in the figures. Also, how do the readers know which are statistically significant by looking at the figures?
Response: We are grateful to the reviewer for mentioning such issues, we have described significant values in a simple mode in all parameters. 0 µg/ml is indication control; you can see the 0 µg/ml in every graph which is basically control condition. We divided the figures into three different categories in order to make it easier for the readers. Also highlighted in track mode changes.
Comments: 9. Why they used bar diagram for figure 3 and 4, and line diagram for 5 and 6? Figure 7 is also different from the others. They should keep it consistent since they are basically showing the similar results.
Response: we are thankful to reviewer for this comment, the reason for making different graphs was to highlight and make it easier for the reader, so we used different presentation styles for every group parameter. This increase the readability in order to avoid confusion while using same format for the graphs. I hope the reviewer ail understand here that why we use graphs with bars and lines.
Comments: 10. They need to describe the results in a better way. Reading about percentage of improvement in every line makes it monotonous.
Response: We are thankful to the reviewer for identifying such issues, we checked and rewrite the result where necessary, and tried to remove confusing lines throughout the descriptions of results about the percentages of improvement as highlighted in track mode changes.
Comments: 11. Overall, they need to re-write the whole manuscript and represent the data in a better way. There are typos, they should check.
Response: We are thankful to reviewer for highlighting such an important error, the manuscript has been checked and re-write the whole manuscript by discussing data in a better way. We tried our best to clean out the irrelevant words, improved the manuscript from typos and grammatical errors as we can. Any changes in the spelling, line, or in a characters done were highlighted in a track mode changes.
Author Response File: 
Author Response.docx
Reviewer 2 Report
A suitable application rate of TiO2 NPs can alleviate the effect of salinity stress in wheat crop, which is very conducive to improve crop productivity under salt stress conditions. However, the authors need to carefully revise the manuscript based on the below advice before publication.
1. L61: “cause leads to…”, please check this statement.
2. L151: “Wheat”, not need to capitalize the first letter.
3. Please replace “titanium dioxide nanoparticles” with “TiO2 NPs” in the next text after first abbreviation.
4. Please accurately describe the effect of application of TiO2 NPs on all related parameters in this manuscript (L329-331, L344-345, L381-383, L407-408, …). From the data presented in the manuscript, only 50 µ g/ml of TiO2 NPs has positive effects germination and morphological parameters of wheat varieties under without salt stress; 25 and 50 µ g/ml of TiO2 NPs have positive effects under 100 mM salt solution; 25, 50 and 75 µ g/ml of TiO2 NPs have positive effects under 100 mM salt solution; all application rates of TiO2 NPs have positive effects under 150 mM salt solution. I am very confused which one is the corresponding control, and I can’t obtain the same results the authors presented in the text (L346-358, L409-425, …). Such problems also appeared in ABSTRACT and the section of physiological parameters.
5. Please check all the results when describing the effects of application of TiO2 NPs in combination with three salt solutions.
6. Please double check and keep the same decimal point for the same parameters in all tables.
Author Response
Response to Reviewer 2 Comments
Dear Reviewer 2,
Thank you for reviewing our manuscript entitled “Biogenic Titanium Dioxide Nanoparticles ameliorate the effect of salinity stress in wheat crop” we tried our best to incorporate the comment/suggestion as you recommend in the revision of manuscript.
Comment: 1. L61: “cause leads to…”, please check this statement.
Response: We are thankful to the reviewer for mentioning such errors, we have checked the statement and made changes in it by highlighted in track mode changes.
Comment: 2. L151: “Wheat”, not need to capitalize the first letter.
Response: Thank you for highlighting such issues, we have changed the first letter of the word “Wheat” into small letter, highlighted in track mode changes.
Comments: 3. Please replace “titanium dioxide nanoparticles” with “TiO2 NPs” in the next text after first abbreviation.
Response: We are thankful to the reviewer for raising such errors, and made changes in “titanium dioxide nanoparticles” with “TiO2 NPs” in the next text throughout the manuscript. Also highlighted in track mode changes.
Comments: 4. Please accurately describe the effect of application of TiO2 NPs on all related parameters in this manuscript (L329-331, L344-345, L381-383, L407-408,). From the data presented in the manuscript, only 50 µ g/ml of TiO2 NPs has positive effects germination and morphological parameters of wheat varieties under without salt stress; 25 and 50 µ g/ml of TiO2 NPs have positive effects under 100 mM salt solution; 25, 50 and 75 µ g/ml of TiO2 NPs have positive effects under 100 mM salt solution; all application rates of TiO2 NPs have positive effects under 150 mM salt solution. I am very confused which one is the corresponding control, and I can’t obtain the same results the authors presented in the text (L346-358, L409-425,). Such problems also appeared in ABSTRACT and the section of physiological parameters.
Response: we are thankful to the reviewer for identifying this issue, we have accurately described the effect of application of TiO2 NPs with and without salt stress on all related parameters in the whole manuscript highlighted by reviewer. Treatment 50 µg/ml of TiO2 NPs have positive effects in alone treatment; and in combination with salt stress, 50 µg/ml of TiO2 NPs under 50 mM salt solution also provide a positive effect in all parameters. We did changes in abstract and in the section of physiological parameters. Also highlighted in track mode changes.
Comments: 5. Please check all the results when describing the effects of application of TiO2 NPs in combination with three salt solutions.
Response: We again thankful to the reviewer for raising such comments, the manuscript has been checked and investigated for describing the effects of application of TiO2 NPs in combination with three salt solutions. A short correction to the paragraph has been incorporated where needed according to the need and beauty of the manuscript. As highlighted in the track mode changes.
Comments: 6. Please double check and keep the same decimal point for the same parameters in all tables.
Response: we are thankful to the Reviewer for raising this comment, here in table 2 and 3, we have checked and made the decimal point same for the same parameters in all tables. We tried our best to sort out this issue. As highlighted in the track mode changes.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors, investigated the potential significance of Titanium dioxide nanoparticles prepared by green synthesis, in the alleviation of salt stress in two wheat cultivars. The research topic is of increasing interest, for the potential benefits on crop performance, but also for the not completely known consequence of nanoparticles release on the environmental safety and human health.
The work describe the effects of many (19!!) salts and titanium treatments on an extended list of physiological parameters stating that many of them undergo positive variations following the application of titanium.
RESULTS
Reading and understanding the results is very difficult.
There is not a clear description of which treatments are compared all over the manuscript.
As an example see rows 346-349 that describe results of table 2 : “Biogenic titanium dioxide nanoparticles at 50 µg/ml in combination with 50 mM salt solution showed pronounced increase in germination percentage (72% and 68%) germination index (74% and 72%), seedling vigor index (58% and 51%) seedling length (34% and 27%) and seedling fresh weight (30% and 25%) of both wheat varieties respectively”.
It is impossible to understand which the compared treatments are, to calculate the percentage variations. Are they T5 and T9? How are the percentages calculated? Moreover, admitting that the T5 treatment can give a significant contribution to the reduction of salt damage on germination it does not seem the same on seedling vigor indices, seedling length, and seedling fresh weight.
I suggest to only describe the comparisons that show significant differences, bearing in mind that to analyze several variables that act simultaneously it is correct the use of the ANOVA factorial analysis. Instead of the cumbersome calculation of percentages, simply the Authors must show the results of the Duncan test (by inserting letters next to the averages) so allowing the reader to quickly identify the effective or deleterious treatments.
It also would be appropriate to avoid results interpretation (delete rows: 403-406; 454-460) and citations of obvious knowledge (delete the citation Gul and Weber,1999). Correct is the reference to similar results obtained by other authors however the descriptive sentences of other author results in rows: 426-435; 465-468; 485-488; 499-500; 507-513; 522-524; 532-538; 596-598; 608-613; 620-624; 626-627, etc., should be moved in the introduction.
I also have some doubts about the correctness of the method used for the calculation of the RWC. Being an estimate of the relative turgor (i.e. compared to the maximum achievable by the leaf tissues) RWC can never be higher than 100%, while in Fig.3 it reaches peaks of 150.
Fig.7 is without legend
DISCUSSION
Is lacking
CONCLUSIONS
The work would seem to prove the potential efficacy of treatment with TiO2 NPs, in alleviating salt with moderate intensity effects, on some but not all the analyzed traits. Hence, the conclusions should be rewritten after verification if the morphological and physiological parameters have been correctly estimated (see RWC) and significatively affected, by the different treatments. There is also the necessity to underly that the genotype effect exists, and to verify, by future works on natural saline soils, if the same physiological benefits obtained by TiO2 NPs in controlled conditions, can be achieved in field.
In my opinion, the paper re-writing according to suggestions I pointed out, could overcome its present deficiencies.
Author Response
Response to Reviewer 3 Comments
Dear Reviewer 3,
Thank you for reviewing our manuscript entitled “Biogenic Titanium Dioxide Nanoparticles ameliorate the effect of salinity stress in wheat crop” we tried our best to incorporate the comment/suggestion as you recommend in the revision of manuscript.
Comment 1: Reading and understanding the results is very difficult.
Response: We are thankful to the reviewer for mentioning such errors, we improved the whole manuscripts in a better way and rewrite it in a simple word to be easier for the readers. The manuscript was intensively reviewed with a native English speaker.
Comment 2: There is not a clear description of which treatments are compared all over the manuscript.
Response: We are thankful to the reviewer for identifying such issues, we checked and rewrite the result where necessary, and tried to remove confusing lines throughout the descriptions of results about the percentages of improvement as highlighted in track mode changes.
Comment 3: As an example see rows 346-349 that describe results of table 2: “Biogenic titanium dioxide nanoparticles at 50 µg/ml in combination with 50 mM salt solution showed pronounced increase in germination percentage (72% and 68%) germination index (74% and 72%), seedling vigor index (58% and 51%) seedling length (34% and 27%) and seedling fresh weight (30% and 25%) of both wheat varieties respectively”.
Response: The error in these lines has been fixed, and the entire manuscript has been checked over as well as rewritten in order to eliminate any reading difficulties that may have been caused. Highlighted in track mode changes.
Comment 4: It is impossible to understand which the compared treatments are, to calculate the percentage variations. Are they T5 and T9? How are the percentages calculated? Moreover, admitting that the T5 treatment can give a significant contribution to the reduction of salt damage on germination it does not seem the same on seedling vigor indices, seedling length, and seedling fresh weight.
Response: Respected, reviewer Thank you for bringing this up; the treatment details are provided in Table No. 1. T0 represents the control, and each treatment% percentage was calculated using control values. The treatment T5 is simply the application of salt, which reduces and negatively affects the growth/germination parameters, which is why no positive response is observed. Similarly, the T9 is the effect of TiO2 NPs on plant growth under salt stress, and the TiO2 NPs showed better results or a significant value because the NPs relieved the plant from abiotic stress in the current study, and a number of studies have reported this effect of NPs under abiotic stress (i.e. Neelofar etal., 2020). I hope this explanation will clear your objection.
effect of nanoparticles is varied in different condition,
Comment 5: I suggest to only describe the comparisons that show significant differences, bearing in mind that to analyze several variables that act simultaneously it is correct the use of the ANOVA factorial analysis. Instead of the cumbersome calculation of percentages, simply the Authors must show the results of the Duncan test (by inserting letters next to the averages) so allowing the reader to quickly identify the effective or deleterious treatments.
Response: we are thankful to the reviewer for this point, we have analyzed the data again using SPSS, and we performed ANOVA, and least significant difference were analyzed using LSD test. The data in the table are now presented with lettering indicating The data represented in the columns in the form of means having similar letters are identical and dissimilar letters are significantly different where p was kept p=<0.05.
Comment 6: It also would be appropriate to avoid results interpretation (delete rows: 403-406; 454-460) and citations of obvious knowledge (delete the citation Gul and Weber,1999). Correct is the reference to similar results obtained by other authors however the descriptive sentences of other author results in rows: 426-435; 465-468; 485-488; 499-500; 507-513; 522-524; 532-538; 596-598; 608-613; 620-624; 626-627, etc., should be moved in the introduction.
Response: we are thankful to the reviewer for this point, we have deleted the extra interpretation of the results, and fixed it with proper citation by deleting the suggested citations. We have change the whole paragraphs and these line with new and appropriate statements. As highlights in track mode changes.
Comment 7: I also have some doubts about the correctness of the method used for the calculation of the RWC. Being an estimate of the relative turgor (i.e. compared to the maximum achievable by the leaf tissues) RWC can never be higher than 100%, while in Fig.3 it reaches peaks of 150.
Response: Dear reviewer, thank you for this comment, but here I would like to notice you that the relative water content is not above 100, the values of control and all treatments are below 100 can be seen in the graphs, actually we have applied combine graphs in which single bars of the graph showed two varieties with different color, that is why you seen the values goes up to 150, but if look the same bare there are two color, one represents the 1st variety and 2nd do the another variety. I hope this explanation is enough to satisfy these graphs.
Comment 8: Fig.7 is without legend
Response: we have added the legends to the figure 7. Highlighted in track mode changes
Comment 9: DISCUSSION Is lacking
Response: a detailed and logical discussion according to the need of the study has been added in the manuscript under heading Discussion. As highlighted in track mode changes.
Author Response File: Author Response.docx
Reviewer 4 Report
The references before 2018 are not acceptable.
Author Response
Response to Reviewer 4 Comments
Dear Reviewer 4,
Thank you for reviewing our manuscript entitled “Biogenic Titanium Dioxide Nanoparticles ameliorate the effect of salinity stress in wheat crop” we tried our best to incorporate the comment/suggestion as you recommend in the revision of manuscript.
Dear Reviewer We are thankful to the reviewer for mentioning such errors, we improved the whole manuscripts in a better way and rewrite it in a simple word to be easier for the readers. The manuscript was intensively reviewed with a native English speaker.
Comment 1: The references before 2018 are not acceptable
Response: we are thankful to the reviewer for raising thus issue. All the references have been updated starting from 2018 to 2022.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
No comment.
Author Response
Dear Reviewer 2,
we are thankful for your valuable suggestions, we have checked the manuscript for linguistic errors, and all the spelling mistakes have been corrected and doubled checked for grammatical errors.
thank you
Reviewer 4 Report
improved the English
Author Response
Dear Reviewer 4,
we are thankful for your valuable suggestions, we have checked the manuscript for linguistic errors, and all the spelling mistakes have been corrected and doubled checked for grammatical errors.
we also improve all the section where need in order to maintain the beauty of the manuscript.
thank you
