Green-Synthesized Zinc Oxide Nanoparticles Mitigate Salt Stress in Sorghum bicolor

Round 1

Reviewer 1 Report

Dear Authors, in your interesting manuscript, the following points should be added/changed to further improve it:

1. Introduction: I have a comment on the sentence “Metal oxide nanoparticles, specifically Zinc oxide nanoparticles (ZnO NPs) has re-49 cently gained lot of attention in nanoscience due to their unique physiochemical properties and their various application in biology, chemistry, medicine and physics [11].” What does an article with the title " Green synthesis of nickel oxide, palladium and palladium oxide synthesized via Aspalathus linearis natural extracts: physical properties & mechanism of formation" quoted in this sentence have to do with ZnO. I suggest you cite relevant review articles about ZnO that extensively describe the properties and applications of nano ZnO (DOI: 10.1515/ntrev-2018-0067, DOI:10.3390/nano10061086).
2. Introduction: I have a comment on the sentence “This study is the first to investigate the application of green synthesized ZnO NPs in plant growth under salt stress.” How does the indicated novelty of the authors' work relate to the article “The potential mitigation effect of ZnO nanoparticles on [Abelmoschus esculentus L. Moench] metabolism under salt stress conditions, DOI: 10.1016/j.sjbs.2020.08.005.”?
3. Biosynthesis of Zinc Oxide Nanoparticles: Please explain exactly why the authors believe they have carried out "Biosynthesis of Zinc Oxide Nanoparticles"?. Please refer to the reliable definition of biosynthesis.
4. Biosynthesis of Zinc Oxide Nanoparticles: I have a comment on the sentence “Zinc nitrate hexahydrate [N2O6Zn] purchased from Sigma-Aldirich (Cat# 96482-88 500G, Lot # BCBJ7666V) was used as a precursor for the synthesis of ZnO nanoparticles 89 (NPs), while the Buchu extract served as the capping and reducing agent.” Please explain to me why the authors believe that buchu Extract served as a reducing agent? Did a redox reaction take place during the synthesis of ZnO?
5. Biosynthesis of Zinc Oxide Nanoparticles: Whether the described ZnO synthesis is an original method or is a modified method based on the work of other authors (34. Thema, F., et al., Green synthesis of ZnO nanoparticles via Agathosma betulina natural extract. Materials Letters, 2015. 161: p. 124-586 127)?
6. Characterization of Zinc Oxide Nanoparticles: Please add information about the zeta potential measurement method and the analyser (model, manufacturer). Please add information about the sample preparation for zeta potential measurement (concentration, conductivity, information about water composition).
7. Characterization of Zinc Oxide Nanoparticles: Please add information about the EDX analyser (model, manufacturer).
8. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The Debye Scherrer's 197 formula (shown below) was used to determine the crystalline size of the synthesized ZnO 198 NPs.” Please correct the name of the equation (DOI:10.1038/nnano.2011.145). The description of the equation should be moved to the section "Characterization of Zinc Oxide Nanoparticles"
9. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The XDR spectrum of the synthe-195 sized ZnO NPs confirms that all the diffraction peaks are indexed to the hexagonal wurtz-196 ite structure of ZnO (JCPDS Card No 36-1451) as shown in Figure 1c.” I disagree with the authors, the XRD plot (figure 1(c)) shows that the sample has additional peaks from an unidentified phase(s).
10. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The average particle size of 201 the prepared ZnO NPs is 26.03 nm and matched with the TEM micrograph, which re-202 vealed the size of ZnO NPs to be between 20 nm and 30 nm (Figure S1c).” Is the result of 26.03 nm the average size of the crystallites or the average size of the nanoparticles? Please round the result to whole numbers.
11. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The surface charge of the synthesized ZnO NPs was determined using 208 Zeta Potential Analyzer, which showed a mean Zeta Potential of −1.71 mV indicating that 209 the ZnO NPs are moderately stable (Figure 1e).” I disagree with the authors, a zeta potential value of - 1.7 mV indicates the electrostatic instability of ZnO NPs in the sample. Taking into account the measurement error, a value equal to 0 mV may be assumed here. It cannot be said here that the sample "are moderately stable". Please add the standard deviation for the zeta potential result.

Author Response

Dear Reviewer

We thank you for taking your time to review our manuscript, your comments are valuable and have greatly improved the manuscript. 

Thank you

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

The manuscript "Green Synthesized Zinc Oxide Nanoparticles mitigate Salt Stress in Sorghum bicolor" dealt with an interesting aspect for plant physiology and biochemistry  under salt conditions. The presented evidences about the effects of Zinc oxide NP for improvement of plant performance are clear and demosntrate the positive impact that use of nanotechnology to crop protection on such stress can bring. However, some important points must be discussed:

1. Is the research novelty as indicated in the manuscript? (line 442-443, references 6, 7).
2. I did not found the description of methods for element distribution. How the results of figure 3C were obtained. 
3. Lane 114, disinfected.
4. Please, verify the comments on Lane 168-169.
5.  Lane 216-217: ...in relation to the salt-treated plants.
6. I suggest that the results on dry mass are showed, it can be discused in relation to soluble sugar content.
7. Lane 229-255: the informations should be revised.
8. Lane 301, 10 mg.L
9. Lane 330-332. This comment should be included in Discussion section. However, measurements of osmotic potential were not carried out.  I consider that interpretation must be written in another way.
10. If the incubation of the sorghum seeds with Zinc oxide NP induced the tolerance responses to salt stress, it is likely that are absorved and accumulated in plant tissues. This accumulation was verifed by the authors through mineral distribution? Zn or ZnO in plant tissues was  analyzed? 
11. The discussion must be based on the zeta potential of zn, whose value was indicated on lane 365. Zn acts as reductor agent that could have minimized tha oxidative effects induced by salinity and thus a reduced need for proline accumulation was observed. This point couls be correlated with a high accumulation of Zn or ZnO NP in plants, as indicated previously.

With kind regards

Author Response

Dear Reviewer

We thank you for taking your time to review our manuscript, your comments are valuable and have greatly improved the manuscript. 

Thank you

Author Response File: Author Response.docx

Reviewer 3 Report

The article by the authors Tessia Rakgotho, Nzumbululo Ndou, Takalani Mulaudzi, Emmanuel Iwuoha, Noluthando Mayedwa and  Rachel Fanelwa Ajayi "Title Green Synthesized Zinc Oxide Nanoparticles mitigate 2 Salt Stress in Sorghum bicolor" contains a large amount of experimental material and is broadly in line with Journal Agriculture Special Issue:Abiotic Stresses, Biostimulant and Plant Activity I have a few suggestions.

1. Please insert in the text the size (height, diameter and other) of the pots used for the experiment.
2. Line 123 concentration of salt was 400mM, but what was the volume applied to experiment each second day of salt solution
3. Don't use dots when writing a formula.
4. Line 201 the sentence "The average particle size of 201 the prepared ZnO NPs is 26.03 nm...." start from the new line.
5. Figure 1 , I suggest authors to redraw the figure 1e and to make it the same appearance as 1a and 1c.
6. Figure2a there are some mismatches in the text under the photo and on the photo. On the photo is given NPs concentration +salt concentration and under the photo oppositely.
7. Figure2. Figures 2b and 2c / 2d and 2e should be merged because value given for 400mM is the same in the 2b and 2c / 2d and 2e.
8. Figure 4 c and d should be merged as given above.
9. Figures 6 and 7 I suggest to authors of the manuscript to merge figures because some values in different graphs are the same.

Author Response

Dear Reviewer

We thank you for taking your time to review our manuscript, your comments are valuable and have greatly improved the manuscript. 

Thank you

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

1. Introduction: I have a comment on the sentence “Metal oxide nanoparticles, specifically Zinc oxide nanoparticles (ZnO NPs) has re-49 cently gained lot of attention in nanoscience due to their unique physiochemical properties and their various application in biology, chemistry, medicine and physics [11].” What does an article with the title " Green synthesis of nickel oxide, palladium and palladium oxide synthesized via Aspalathus linearis natural extracts: physical properties & mechanism of formation" quoted in this sentence have to do with ZnO. I suggest you cite relevant review articles about ZnO that extensively describe the properties and applications of nano ZnO (DOI: 10.1515/ntrev-2018-0067, DOI:10.3390/nano10061086).

 Response:  Revised sentence: “Metal oxide nanoparticles, specifically Zinc oxide nanoparticles (ZnO NPs) has recently gained a lot of attention in nanoscience due to their unique physicochemical properties and their various application in biology, chemistry, medicine and physics ( Bandeira et al., 2021;  Agarwal et al., 2020; Jiang et al., 2018; Mirzaei and Darroudi, 2016)” We have added four references to support the effectiveness of green-synthesized ZnO NPs: •        Bandeira, M., Giovanela., M., Roesch-Ely, N., Devine., D., M and Crepo., J., S. (2020). Green synthesis of zinc oxide nanoparticles: A review of the synthesis methodology and mechanism of formation. Sustainable chemistry and pharmacy, 15, 100223. •        Agarwal., H., Kumar, S., V., and Rajeshkumar., S. (2017). A review on green synthesis of zinc oxide nanoparticles –An eco-friendly approach. Resource-efficient technologies, 2(4); 406-413.[ •        Jiang, J., Pi., J., and Cai, J. (2018). The advancing of Zinc oxide Nanoparticles for biomedical application. Hindawi, Bioinorganic chemistry and application.1; 18 https://doi.org/10.1155/2018/1062562 •       Mirzaei., H., and Darroudi, M. (2016). Zinc oxide nanoparticles; Biological synthesis and biomedical applications. Ceramics International, 43(1); 907-914.

Reviewer's reply: I note that the microwave method of obtaining ZnO is also included in green chemistry (green synthesis).

2. Introduction: I have a comment on the sentence “This study is the first to investigate the application of green synthesized ZnO NPs in plant growth under salt stress.” How does the indicated novelty of the authors' work relate to the article “The potential mitigation effect of ZnO nanoparticles on [Abelmoschus esculentus Moench] metabolism under salt stress conditions, DOI: 10.1016/j.sjbs.2020.08.005.”?

Response: The study is novel based on three main points, firstly green-synthesized ZnO NPs were applied for the first time to alleviate effects of salt stress in Sorghum bicolor L. Moench. NB: ZnO NPs previously applied to improve growth of other crops under salt stress, such as soybean (Gaafar et al., 2020) and Brassica napus (El-Badri et al., 2020) were synthesized using chemical methods.

Secondly, although both studies (our study and Alabdallah and Alzahrani, 2020) applied green synthesized ZnO NPs on plant growth under salt stress, nanoparticles were synthesized from different green materials i.e Alabdallah and Alzahrani, 2020, in their study used leaflet extracts of Phoenix dactylifera L. var. Khalas, while in our study, Agathosma betulina leaves were used to synthesize the nanoparticles.

Thirdly, the green synthesized nanoparticles were applied on two different plants, our study investigated the effects of green –synthesized ZnO NPs on Sorghum bicolor L. Moench, which is a cereal crop also known as sorghum whereas Alabdallah and Alzahrani, 2020 studied ZnO NPs effects on Abelmoschus esculentus L. Moench, which is a vegetable crop also known as Okra.

Finally, their study only assessed the effects of ZnO NPs by looking at photosynthetic, osmolyte and antioxidant enzyme activities, whereas our study additionally looked at different growth parameters (shoot length and Fresh weights and anatomical structure), oxidative stress markers (superoxide anion, hydrogen peroxide and lipid peroxidation) and biomolecules using FTIR.

Reviewer's reply: Please explain to me why the authors did not add the article I indicated (DOI:10.1016/j.sjbs.2020.08.005) and the article mentioned in the reply (Alzahrani, 2020) to the description of the state of the art review?

3. Biosynthesis of Zinc Oxide Nanoparticles: I have a comment on the sentence “Zinc nitrate hexahydrate [N2O6Zn] purchased from Sigma-Aldirich (Cat# 96482-88 500G, Lot # BCBJ7666V) was used as a precursor for the synthesis of ZnO nanoparticles 89 (NPs), while the Buchu extract served as the capping and reducing agent.” Please explain to me why the authors believe that the buchu Extract served as a reducing agent? Did a redox reaction take place during the synthesis of ZnO?

Response:  We believe that the Buchu extract indeed served as capping and reducing agent in this study. This is because several authors have indicated that the use of green materials (plant extracts, tea extracts etc.), provide essential biomolecules (e.g flavonols, polyphenols, phenolic compounds), which have capping and reducing capabilities. The following references, which have been updated in our corrected manuscript, support our above statement ( Gupta et al., 2018; Shamila et al., 2019; Khan et al., 2019; Ogunyemi et al., 2019) have been added on the manuscript. In addition, these biomolecules have played an important role in the reduction of Zn2+ to ZnO, as supported by the data illustrated in Figure 1a (FTIR analysis). The data illustrates the presence of these biomolecules in the absence of zinc nitrate salt and after the completion of the synthesis of the nanoparticles.

Reviewer's reply: A misunderstanding has crept in here. I ask the authors to read the definition of a reduction (redox) reaction. There is no evidence in the results discussed that metallic Zn was formed. The formation of ZnO is a simple chemical reaction. Please do not reproduce the error.

4. Characterization of Zinc Oxide Nanoparticles: Please add information about the zeta potential measurement method and the analyzer (model, manufacturer). Please add information about the sample preparation for zeta potential measurement (concentration, conductivity, information about water composition).

Response:  Description of Zeta potential methodology has been supplied” The size was of ZnO NPs also analyzed using the Zeta potential Zetasizer (Malvern Instruments Ltd., UK) measured at 25 °C. About 0.5 µg/ml synthesized ZnO NPs prepared by dissolving in 2 ml distilled water and sonicated for 30 minutes at 25°C. The data was analysed using Zetasizer software version 7.11“  The conductivity was not measured in this study.

Reviewer's reply: The authors are wrong because the conductivity was measured and was reported in the Zeta Potential Report.

5. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The Debye Scherrer's 197 formula (shown below) was used to determine the crystalline size of the synthesized ZnO 198 NPs.” Please correct the name of the equation (DOI:10.1038/nnano.2011.145). The description of the equation should be moved to the section "Characterization of Zinc Oxide Nanoparticles" Response: A description of the Debye-Scherre’s formula has been corrected and moved to the methods section.

Reviewer's reply: Please correct the name of the equation (DOI:10.1038/nnano.2011.145). Indeed, strictly speaking, there is no Debye-Scherrer equation. The first scientist to investigate the effect of limited particle size on X-ray diffraction patterns was Paul Scherrer, who published his results in a paper that included what became known as the Scherrer equation. Please do not reproduce the error.

6. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence ““The XDR spectrum of the synthe-195 sized ZnO NPs confirms that all the diffraction peaks are indexed to the hexagonal wurtz-196 ite structure of ZnO (JCPDS Card No 36-1451) as shown in Figure 1c.” I disagree with the authors, the XRD plot (figure 1(c)) shows that the sample has additional peaks from an unidentified phase(s). I disagree with the authors, the XRD plot (figure 1(c)) shows that the sample has additional peaks from an unidentified phase(s).

Response: The two additional peaks indicated as (004) and (202) in the XRD plot are characteristic of ZnO NPs. They are in good agreement with reported literature as illustrated below with the chosen references. The peaks have been re-labelled and the diffraction patterns included. As such, there are no unidentified phase(s).

Al‑Kordy, H. M. H., Sabry, S. A., Mabrouk, M. E. M. Statistical optimization of experimental parameters for extracellular synthesis of zinc oxide nanoparticles by a novel haloalaliphilic Alkalibacillus sp.W7. Scientific Reports, 11 (2021)10924.

Shashanka, R., Esgin, H., Yilmaz, V. M., Caglar, Y. Fabrication and characterization of green synthesized ZnO nanoparticle based dye-sensitized solar cells. Journal of Science: Advanced Materials and Devices, 5 (2020) 185-191.

Reviewer's reply: I do not agree with the authors' explanations. The two peaks from the XRD results do not match the ZnO reference data (JCPDS Card No 36-1451). Please check the appendix for review.

7. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The surface charge of the synthesized ZnO NPs was determined using Zeta Potential Analyzer, which showed a mean Zeta Potential of −1.71 mV indicating that the ZnO NPs are moderately stable (Figure 1e).” I disagree with the authors, a zeta potential value of - 1.7 mV indicates the electrostatic instability of ZnO NPs in the sample. Considering the measurement error, a value equal to 0 mV may be assumed here. It cannot be said here that the sample "is moderately stable". Please add the standard deviation for the zeta potential result.

Response: The zeta potential standard deviation of our ZnO NPs is 7.6 and according to literature the stability of nanoparticle is between -30 and +30. However, since the zeta potential of ZnO NPs is -1.7 mV, an indication of instability, however based on the standard deviation we can suggest that the ZnO NPs are moderately stable, and after consultation we have decided to remove any data relating to Zetapotential since the size was obtained from the TEM and XDR data, we believe it is sufficient. Since the results were misinterpreted, in that case the Zeta Potential figure was moved to supplementary data (Figure S1d).

Reviewer's reply: I ask that the authors comment honestly on the results obtained. Your current comment does not refer to the mean value but to the standard deviation of the zeta potential measurements. I note that it is not possible to ascertain instability or stability 100% on the basis of zeta potential. This is about determining the value of electrostatic interactions. Please insert your train of thought in the text of the manuscript and add the word "presumably or probably". For your information, a Turbiscan analyser is used to test the stability of suspensions.

Good luck!

Comments for author File: Comments.docx

Author Response

Dear Reviewer 1,

We would like to thank you for taking your precious and valuable time to thoroughly re-evaluate our Manuscript (agriculture-1659089), and for the insightful comments and suggestions that greatly improved it. Below we provide a point-to-point response of the reviewer’s comments. Changes in the MS are highlighted in red (tracked changes), another copy with the accepted changes (clean) in PDF format is also supplied.

REVIEWERS COMMENTS AND SUGGESTIONS FOR AUTHORS

REVIEWER 1

1. Introduction: I have a comment on the sentence “Metal oxide nanoparticles, specifically Zinc oxide nanoparticles (ZnO NPs) has re-49 cently gained lot of attention in nanoscience due to their unique physiochemical properties and their various application in biology, chemistry, medicine and physics [11].” What does an article with the title " Green synthesis of nickel oxide, palladium and palladium oxide synthesized via Aspalathus linearis natural extracts: physical properties & mechanism of formation" quoted in this sentence have to do with ZnO. I suggest you cite relevant review articles about ZnO that extensively describe the properties and applications of nano ZnO (DOI: 10.1515/ntrev-2018-0067, DOI:10.3390/nano10061086).

Response:

Revised sentence: “Metal oxide nanoparticles, specifically Zinc oxide nanoparticles (ZnO NPs) has recently gained a lot of attention in nanoscience due to their unique physicochemical properties and their various application in biology, chemistry, medicine and physics (Bandeira et al., 2021;  Agarwal et al., 2020; Jiang et al., 2018; Mirzaei and Darroudi, 2016)”

We have added four references to support the effectiveness of green-synthesized ZnO NPs:

• Bandeira, M., Giovanela., M., Roesch-Ely, N., Devine., D., M and Crepo., J., S. (2020). Green synthesis of zinc oxide nanoparticles: A review of the synthesis methodology and mechanism of formation. Sustainable chemistry and pharmacy, 15, 100223.
• , H., Kumar, S., V., and Rajeshkumar., S. (2017). A review on green synthesis of zinc oxide nanoparticles –An eco-friendly approach. Resource-efficient technologies, 2(4); 406-413.[
• Jiang, J., Pi., J., and Cai, J. (2018). The advancing of Zinc oxide Nanoparticles for biomedical application. Hindawi, Bioinorganic chemistry and application.1; 18 https://doi.org/10.1155/2018/1062562
• , H., and Darroudi, M. (2016). Zinc oxide nanoparticles; Biological synthesis and biomedical applications. Ceramics International, 43(1); 907-914.

Reviewer's reply: I note that the microwave method of obtaining ZnO is also included in green chemistry (green synthesis).

2^nd rebuttal Author’s Response: In this study, the microwave method was not used for synthesis nor for any other application. Instead, the ZnO nanoparticles (NPs) were calcinated using a furnace at 600 Degree Celsius with the purpose to increase the crystallization of the NPs. Since this value is less than 1000 Degrees Celsius, it not is considered higher energy. The reference by Jiang and Cai (2018), was used to support the statement that ZnO NPs can be used in the medical field. The authors further mentioned different methods that can be used to synthesize ZnO NPs using green chemistry, where the microwave is one of them.

1. Introduction: I have a comment on the sentence “This study is the first to investigate the application of green synthesized ZnO NPs in plant growth under salt stress.” How does the indicated novelty of the authors' work relate to the article “The potential mitigation effect of ZnO nanoparticles on [Abelmoschus esculentus L. Moench] metabolism under salt stress conditions, DOI: 10.1016/j.sjbs.2020.08.005.”?

Response: The study is novel based on three main points, firstly green-synthesized ZnO NPs were applied for the first time to alleviate effects of salt stress in Sorghum bicolor L. Moench. NB: ZnO NPs previously applied to improve growth of other crops under salt stress, such as soybean (Gaafar et al., 2020) and Brassica napus (El-Badri et al., 2020) were synthesized using chemical methods.

Secondly, although both studies (our study and Alabdallah and Alzahrani, 2020) applied green synthesized ZnO NPs on plant growth under salt stress, nanoparticles were synthesized from different green materials i.e Alabdallah and Alzahrani, 2020, in their study used leaflet extracts of Phoenix dactylifera L. var. Khalas, while in our study, Agathosma betulina leaves were used to synthesize the nanoparticles.

Thirdly, the green synthesized nanoparticles were applied on two different plants, our study investigated the effects of green –synthesized ZnO NPs on Sorghum bicolor L. Moench, which is a cereal crop also known as sorghum whereas Alabdallah and Alzahrani, 2020 studied ZnO NPs effects on Abelmoschus esculentus L. Moench, which is a vegetable crop also known as Okra.

Finally, their study only assessed the effects of ZnO NPs by looking at photosynthetic, osmolyte and antioxidant enzyme activities, whereas our study additionally looked at different growth parameters (shoot length and Fresh weights and anatomical structure), oxidative stress markers (superoxide anion, hydrogen peroxide and lipid peroxidation) and biomolecules using FTIR. 

Reviewer's reply: Please explain to me why the authors did not add the article I indicated (DOI:10.1016/j.sjbs.2020.08.005) and the article mentioned in the reply (Alzahrani, 2020) to the description of the state of the art review?

2^nd rebuttal Author’s Response: Authors apologize for forgetting to include this reference, it is significant and very relevant to the literature and discussion of this study. We have added it in the introduction and discussion sections as reference no. [8].

1. Biosynthesis of Zinc Oxide Nanoparticles: I have a comment on the sentence “Zinc nitrate hexahydrate [N2O6Zn] purchased from Sigma-Aldirich (Cat# 96482-88 500G, Lot # BCBJ7666V) was used as a precursor for the synthesis of ZnO nanoparticles 89 (NPs), while the Buchu extract served as the capping and reducing agent.” Please explain to me why the authors believe that the buchu Extract served as a reducing agent? Did a redox reaction take place during the synthesis of ZnO?

Response:

We believe that the Buchu extract indeed served as capping and reducing agent in this study. This is because several authors have indicated that the use of green materials (plant extracts, tea extracts etc.), provide essential biomolecules (e.g flavonols, polyphenols, phenolic compounds), which have capping and reducing capabilities. The following references, which have been updated in our corrected manuscript, support our above statement (Gupta et al., 2018; Shamila et al., 2019; Khan et al., 2019; Ogunyemi et al., 2019) have been added on the manuscript. In addition, these biomolecules have played an important role in the reduction of Zn²⁺ to ZnO, as supported by the data illustrated in Figure 1a (FTIR analysis). The data illustrates the presence of these biomolecules in the absence of zinc nitrate salt and after the completion of the synthesis of the nanoparticles.

Added references:   

                Gupta, M., et al., Effective antimicrobial activity of green ZnO nano particles of Catharanthus roseus. Frontiers in microbiology, 2018: p. 2030.

                Sharmila, G., M. Thirumarimurugan, and C. Muthukumaran, Green synthesis of ZnO nanoparticles using Tecoma castanifolia leaf extract: characterization and evaluation of its antioxidant, bactericidal and anticancer activities. Microchemical Journal, 2019. 145: p. 578-587.

                Khan, Z.U.H., et al., Greener synthesis of zinc oxide nanoparticles using Trianthema portulacastrum extract and evaluation of its photocatalytic and biological applications. Journal of Photochemistry and Photobiology B: Biology, 2019. 192: p. 147-157.

Ogunyemi, S.O., et al., Green synthesis of zinc oxide nanoparticles using different plant extracts and their antibacterial activity against Xanthomonas oryzae pv. oryzae. Artificial cells, nanomedicine, and biotechnology, 2019. 47(1): p. 341-352.

Reviewer's reply: A misunderstanding has crept in here. I ask the authors to read the definition of a reduction (redox) reaction. There is no evidence in the results discussed that metallic Zn was formed. The formation of ZnO is a simple chemical reaction. Please do not reproduce the error

2^ND rebuttal Author’s Response: The following articles (https://doi.org/10.1515/gps-2016-0190; https://doi.org/10.1155/2014/523869 ) have details of possible mechanisms of the formation of ZnO NPs from zinc nitrate using plant extracts. Based on the request by the reviewer we have refrained from using the word ‘reduction’ and instead used ‘synthesized’ or ‘formation’.

1. Characterization of Zinc Oxide Nanoparticles: Please add information about the zeta potential measurement method and the analyzer (model, manufacturer). Please add information about the sample preparation for zeta potential measurement (concentration, conductivity, information about water composition).

Response:

Description of Zeta potential methodology has been supplied” The size was of ZnO NPs also analyzed using the Zeta potential Zetasizer (Malvern Instruments Ltd., UK) measured at 25 °C. About 0.5 µg/ml synthesized ZnO NPs prepared by dissolving in 2 ml distilled water and sonicated for 30 minutes at 25°C. The data was analysed using Zetasizer software version 7.11“

The conductivity was not measured in this study.

Reviewer's reply: The authors are wrong because the conductivity was measured and was reported in the Zeta Potential Report.

2^ND rebuttal Author’s response: Authors have decided to remove any information pertaining to Zeta Potential analysis until they obtain comprehensive interpretation of the results in a more scientific manner. In our future manuscripts, we will greatly appreciate any help from the reviewer regarding this kind of data analysis. We believe that the different techniques used including, FTIR, XRD, EDX, HRSEM, HRTEM, and UV are enough to support the synthesis of ZnO NPs and interrogated both their structure and size.

5. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The Debye Scherrer's 197 formula (shown below) was used to determine the crystalline size of the synthesized ZnO 198 NPs.” Please correct the name of the equation (DOI:10.1038/nnano.2011.145). The description of the equation should be moved to the section "Characterization of Zinc Oxide Nanoparticles"

Response: A description of the Debye-Scherre’s formula has been corrected and moved to the methods section. 

Reviewer's reply: Please correct the name of the equation (DOI:10.1038/nnano.2011.145). Indeed, strictly speaking, there is no Debye-Scherrer equation. The first scientist to investigate the effect of limited particle size on X-ray diffraction patterns was Paul Scherrer, who published his results in a paper that included what became known as the Scherrer equation. Please do not reproduce the error.

2^nd rebuttal Author’s Response: We have corrected the equation, instead of “Debye-Scherrer’s” equation, we wrote “Scherrer’s” equation, based on the understanding that the Ph.D. student “Paul Scherrer” discovered the equation. In addition, this reference (DOI:10.1038/nnano.2011.145) was added to support this change.

1. Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence ““The XDR spectrum of the synthe-195 sized ZnO NPs confirms that all the diffraction peaks are indexed to the hexagonal wurtz-196 ite structure of ZnO (JCPDS Card No 36-1451) as shown in Figure 1c.” I disagree with the authors, the XRD plot (figure 1(c)) shows that the sample has additional peaks from an unidentified phase(s). I disagree with the authors, the XRD plot (figure 1(c)) shows that the sample has additional peaks from an unidentified phase(s).

Response: The two additional peaks indicated as (004) and (202) in the XRD plot are characteristic of ZnO NPs. They are in good agreement with reported literature as illustrated below with the chosen references. The peaks have been re-labelled and the diffraction patterns included. As such, there are no unidentified phase(s).

Al‑Kordy, H. M. H., Sabry, S. A., Mabrouk, M. E. M. Statistical optimization of experimental parameters for extracellular synthesis of zinc oxide nanoparticles by a novel haloalaliphilic Alkalibacillus sp.W7. Scientific Reports, 11 (2021)10924.

Shashanka, R., Esgin, H., Yilmaz, V. M., Caglar, Y. Fabrication and characterization of green synthesized ZnO nanoparticle based dye-sensitized solar cells. Journal of Science: Advanced Materials and Devices, 5 (2020) 185-191.

Reviewer's reply: I do not agree with the authors' explanations. The two peaks from the XRD results do not match the ZnO reference data (JCPDS Card No 36-1 451). Please check the appendix for review.

[41] Shashanka, R., Esgin, H., Yilmaz, V. M., Caglar, Y. Fabrication and characterization of green synthesized ZnO nanoparticle based dye-sensitized solar cells. Journal of Science: Advanced Materials and Devices, 5 (2020) 185-191.

[42] Kordy, H. M. H., Sabry, S. A., Mabrouk, M. E. M. Statistical optimization of experimental parameters for extracellular synthesis of zinc oxide nanoparticles by a novel haloalaliphilic Alkalibacillus sp.W7. Scientific Reports, 11 (2021)10924.

Results - Characterization of Zinc oxide Nanoparticles: I have a comment on the sentence “The surface charge of the synthesized ZnO NPs was determined using Zeta Potential Analyzer, which showed a mean Zeta Potential of −1.71 mV indicating that the ZnO NPs are moderately stable (Figure 1e).” I disagree with the authors, a zeta potential value of - 1.7 mV indicates the electrostatic instability of ZnO

1. NPs in the sample. Considering the measurement error, a value equal to 0 mV may be assumed here. It cannot be said here that the sample "is moderately stable". Please add the standard deviation for the zeta potential result.

Response: The zeta potential standard deviation of our ZnO NPs is 7.6 and according to literature the stability of nanoparticle is between -30 and +30. However, since the zeta potential of ZnO NPs is -1.7 mV, an indication of instability, however based on the standard deviation we can suggest that the ZnO NPs are moderately stable, and after consultation we have decided to remove any data relating to Zetapotential since the size was obtained from the TEM and XDR data, we believe it is sufficient. Since the results were misinterpreted, in that case the Zeta Potential figure was moved to supplementary data (Figure S1d).

Reviewer's reply: I ask that the authors comment honestly on the results obtained. Your current comment does not refer to the mean value but to the standard deviation of the zeta potential measurements. I note that it is not possible to ascertain instability or stability 100% on the basis of zeta potential. This is about determining the value of electrostatic interactions. Please insert your train of thought in the text of the manuscript and add the word "presumably or probably". For your information, a Turbiscan analyser is used to test the stability of suspensions.

2^ND rebuttal Author’s response: Authors have decided to remove any information pertaining to Zeta Potential analysis until they obtain comprehensive interpretation of the results in a more scientific manner. In our future manuscripts, we will greatly appreciate any help from the reviewer regarding this kind of data analysis. We believe that the different techniques used including, FTIR, XRD, EDX, HRSEM, HRTEM, and UV are enough to support the synthesis of ZnO NPs and interrogated both their structure and size.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Authors, 

The manuscript was improved according to my comments.

Author Response

Dear Reviewer 3,

We would like to thank all the reviewers for taking their precious and valuable time to thoroughly re-evaluate our Manuscript (agriculture-1659089), and for the insightful comments and suggestions that greatly improved it. 

REVIEWER 3

Dear Authors, 

The manuscript was improved according to my comments.

2^ND Author’s response: Thank you to the reviewer for taking their time to work thoroughly on our manuscript.