Genome-Wide Identification and Transcriptional Expression Analysis of Annexin Genes in Capsicum annuum and Characterization of CaAnn9 in Salt Tolerance

Round 1

Reviewer 1 Report

The authors propose a manuscript titled “Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance”. The authors have evaluated the role of Annexin (Ann), which plays key roles in plant growth, development and stress response, but we have a limited a comprehensive understanding of CaAnn genes of pepper (Capsicum annuum) at the genome-wide level. The authors based on the available pepper genomic information, have identified some members of CaAnn protein family and in particular that: 1) the phylogenetic analysis showed that CaAnn proteins could be categorized into four different orthologous groups; 2) the quantitative RT-PCR analysis showed that the CaAnn genes were tissue-specific and were widely expressed in pepper leaves after treatments with cold, salt, and drought as well as exogenously applied MeJA and ABA; 3) the function of CaAnn9 was further explored using the virus-induced gene silencing (VIGS) technique. The manuscript is original in the data compared to other similar articles and is able to be published on international audience. However I believe it is necessary to implement the manuscript with few formal concepts that the authors will have no problem to accepting as they are designed to improve the work.

Title

Please reports the scientific name in the complete way with the author: Capsicum annuum L.

1. Introduction

Well done, it’s referenced in the correct way. Few observations

• Lines 35-36. Please add a references for this statement: “…numerous plant Anns have been discovered (CHOOSE A REFERENCES)
• Line 46. “…In genus Arabidopsis” instead “Arabidopsis”. Please pay attention for the correct nomenclature. Remember that the scientific name must be reported in italic except the author. Check whole document in this way.
• Line 56. See my previous comment. Brassica juncea (L.) Czern.
• Line 78. Pleaee check and complete in the suggeste way: rapa, B. oleracea, and B. napus
• Capsicum annuum L. Now is ok, but please report the complete scientific name only the firt time when cite the taxon.
• Lines 80-81. Please complete the period in the suggested way: “Ann genes, however, are yet to be characterized in pepper, differently than already been done in other plant groups crucial to humans, as genus Aegilops, Triticum L. [Perrino et al 2014, Hsiao et al. 1995],….. add other plant group if you have a knowledge.

References to be added:

• Perrino, E.V., Wagensommer, R.P.; Medagli, P. The genus Aegilops (Poaceae) in Italy: taxonomy, geographical distribution, ecology, vulnerability and conservation. Systematics and Biodiversity, 2014, 12, 331-349. https://doi.org/10.1080/14772000.2014.909543
• Hsiao, C.; Chatterton, N.J.; Asay, K.H.; Jensen, K.B. Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae), inferred from nuclear rDNA (internal transcribed spacer) sequences. Genome, 1995, 38, 211-223.

2.2. Materials and methods

Well done, no obseravtions

• Lines 93, 110. Arabidopsis thaliana, Solanum lycopersicum, Arabidopsis thaliana, Oryza sativa. Please see my previous comment.

3. Results

No observations. The figures and tables are clears.

4. Discussion

• Lines 396-397. Gossypium raimondii and Vitis vinifera. Please correct
• Line 422. Arabidopsis thaliana. Also here
• Line 455. napus. Add author
• Lines 477-478. Please complete the period in this way: “Under different stresses and intensities, there are differences in the antioxidant enzyme activities, as also been observed in other botanical families such as lamiaceae (Valerio et al. 2021), Apiaceae (CHOOSE A REFERENCE), …”. Check and add other family with relative reference.

References to be added:

• Valerio, F.; Mezzapesa, G.N.; Ghannouchi, A.; Mondelli, D.; Logrieco, A.F.; Perrino, E.V. Characterization and Antimicrobial Properties of Essential Oils from Four Wild Taxa of Lamiaceae Family Growing in Apulia. Agronomy 2021, 11, 1431. https://doi.org/10.3390/agronomy11071431

Conclusion

Some more information on prospective studies

Reference

Please see the guidelines of the journal

Author Response

Dear referee1,

Thanks very much for your comments concerning our manuscript entitled " Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance ". (ijms-1326986). We gratefully thank the editor and all reviewers for their time making their constructive comments, which has significantly raised the quality of the manuscript and enable us to improve the manuscript. Each suggested revision and comment, brought forward by the reviewers, was accurately considered and revised. Below the comments of the reviews are responsive point by point and the revisions are indicated.

The revisions made to the manuscript have marked up using the “Track Changes” function, which we hope meet with your approval. Thank you again!

We wish safety and health to you and the reviewers, given the current COVID-19 pandemic!

Yours sincerely,

H.D. Ding

Reviewer 1

Comments and Suggestions for Authors

The authors propose a manuscript titled “Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance”. The authors have evaluated the role of Annexin (Ann), which plays key roles in plant growth, development and stress response, but we have a limited a comprehensive understanding of CaAnn genes of pepper (Capsicum annuum) at the genome-wide level. The authors based on the available pepper genomic information, have identified some members of CaAnn protein family and in particular that: 1) the phylogenetic analysis showed that CaAnn proteins could be categorized into four different orthologous groups; 2) the quantitative RT-PCR analysis showed that the CaAnn genes were tissue-specific and were widely expressed in pepper leaves after treatments with cold, salt, and drought as well as exogenously applied MeJA and ABA; 3) the function of CaAnn9 was further explored using the virus-induced gene silencing (VIGS) technique. The manuscript is original in the data compared to other similar articles and is able to be published on international audience. However I believe it is necessary to implement the manuscript with few formal concepts that the authors will have no problem to accepting as they are designed to improve the work.

Title

Please reports the scientific name in the complete way with the author: Capsicum annuum L.

Reply: Thanks, the complete scientific name has added to title.

Introduction

Well done, it’s referenced in the correct way. Few observations

Lines 35-36. Please add a reference for this statement: “…numerous plant Anns have been discovered (CHOOSE A REFERENCES)

Reply: Thanks, the cited reference has been added.

Line 46. “…In genus Arabidopsis” instead “Arabidopsis”. Please pay attention for the correct nomenclature. Remember that the scientific name must be reported in italic except the author. Check whole document in this way.

Reply: Thanks. Generally, when Arabidopsis is represented in plants, the genus name is generally not added. The full name of Arabidopsis thaliana needs italics, while Arabidopsis generally does not use italics. At present, we still adopt a common approach.

Line 56. See my previous comment. Brassica juncea (L.) Czern.

Reply: We have modified the writing of scientific name.

Line 78. Pleaee check and complete in the suggest way: rapa, B. oleracea, and B. napus

Capsicum annuum L. Now is ok, but please report the complete scientific name only the firt time when cite the taxon.

Reply: Thanks. The gene names of the 3 species have first appeared in line 56, so he taxon abbreviation was used here. And “Capsicum annuum L.” in line 79 have changed to “C. annuum L.”

Lines 80-81. Please complete the period in the suggested way: “Ann genes, however, are yet to be characterized in pepper, differently than already been done in other plant groups crucial to humans, as genus Aegilops, Triticum L. [Perrino et al 2014, Hsiao et al. 1995],….. add other plant group if you have a knowledge.

References to be added:

Perrino, E.V., Wagensommer, R.P.; Medagli, P. The genus Aegilops (Poaceae) in Italy: taxonomy, geographical distribution, ecology, vulnerability and conservation. Systematics and Biodiversity, 2014, 12, 331-349. https://doi.org/10.1080/14772000.2014.909543

Hsiao, C.; Chatterton, N.J.; Asay, K.H.; Jensen, K.B. Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae), inferred from nuclear rDNA (internal transcribed spacer) sequences. Genome, 1995, 38, 211-223.

Reply: Thanks for you commend. It is crucial to emphasize the important of this research. Cited references have added and have insert the corresponding reference number order.

Materials and methods

Well done, no obseravtions

Lines 93, 110. Arabidopsis thaliana, Solanum lycopersicum, Arabidopsis thaliana, Oryza sativa. Please see my previous comment.

Reply: It has been modified as required.

Results

No observations. The figures and tables are clears.

Discussion

Lines 396-397. Gossypium raimondii and Vitis vinifera. Please correct

Line 422. Arabidopsis thaliana. Also here

Reply: It has been modified as required.

Line 455. napus. Add author

Reply: Thanks for remind. The author and time information are at the beginning of the sentence.

Lines 477-478. Please complete the period in this way: “Under different stresses and intensities, there are differences in the antioxidant enzyme activities, as also been observed in other botanical families such as lamiaceae (Valerio et al. 2021), Apiaceae (CHOOSE A REFERENCE), …”. Check and add other family with relative reference.

References to be added:

Reply: Thanks. The cited references have added and have inserted the corresponding reference number order.

Reviewer 2 Report

The study was focused on genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance. Based on the available pepper genomic information, the Authors identified 15 members of CaAnn protein family. Phylogenetic analysis showed that CaAnn proteins could be categorized into four different orthologous groups. Real-time quantitative RT-PCR analyses evidenced that the CaAnn genes were tissue-specific and were widely expressed in pepper leaves after treatments with cold, salt, and drought as well as exogenously applied MeJA and ABA. In addition, the function of CaAnn9 was further explored using the virus-induced gene silencing (VIGS) technique. CaAnn9-silenced pepper seedlings were more sensitive to salt stress, reflected by the degradation of chlorophyll, the accumulation of reactive oxygen species (ROS), and the decrease of antioxidant defense capacity.

I recommend the following improvements of the manuscript in order to increase its scientific soundness and overall readability:

• The Introduction is too long, I suggest to present it in more concise form,
• I recommend including the electropherograms presenting the RNA bands in agarose gels in the manuscript or in the Supplementary file – it would provide information regarding quality of RNA samples,
• The Authors used SYBR Green fluorescent dye during gene expression studies, hence, it is obligatory to perform Melting Curve Analysis, and results of this examination should be added in the manuscript or Supplementary file (e.g., JPG or TIFF file),
• In the paragraph “2.5. Expression analysis of CaAnn genes” - description of real-time qRT-PCR analyses should be profoundly extended. In addition, there is the lack citation of 2-ΔΔCT method (Livak, Schmittgen 2001),
• in the paragraph “2.9. Statistical Analysis” – ANOVA testing and post-hoc test should be described in more details,
• discussion of the results is superficial and should be thoroughly revised,
• there is the lack of statistical test used under some figures,
• colour figures are suggested,
• moderate English changes by the native speaker are required.

Author Response

Dear referee2,

Thanks very much for your comments concerning our manuscript entitled " Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance ". (ijms-1326986). We gratefully thank the editor and all reviewers for their time making their constructive comments, which has significantly raised the quality of the manuscript and enable us to improve the manuscript. Each suggested revision and comment, brought forward by the reviewers, was accurately considered and revised. Our revised manuscript has been carefully reviewed by the native speaker and modified appropriately. Below the comments of the reviews are responsive point by point and the revisions are indicated.

The revisions made to the manuscript have marked up using the “Track Changes” function, which we hope meet with your approval. Thank you again!

We wish safety and health to you and the reviewers, given the current COVID-19 pandemic!

Yours sincerely,

H.D. Ding

Reviewer 2

The study was focused on genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance. Based on the available pepper genomic information, the Authors identified 15 members of CaAnn protein family. Phylogenetic analysis showed that CaAnn proteins could be categorized into four different orthologous groups. Real-time quantitative RT-PCR analyses evidenced that the CaAnn genes were tissue-specific and were widely expressed in pepper leaves after treatments with cold, salt, and drought as well as exogenously applied MeJA and ABA. In addition, the function of CaAnn9 was further explored using the virus-induced gene silencing (VIGS) technique. CaAnn9-silenced pepper seedlings were more sensitive to salt stress, reflected by the degradation of chlorophyll, the accumulation of reactive oxygen species (ROS), and the decrease of antioxidant defense capacity.

I recommend the following improvements of the manuscript in order to increase its scientific soundness and overall readability:

The Introduction is too long, I suggest to present it in more concise form,

Reply: Good suggestion. The introduction has been appropriately deleted

I recommend including the electropherograms presenting the RNA bands in agarose gels in the manuscript or in the Supplementary file – it would provide information regarding quality of RNA samples,

Reply: Thanks! We have added the data information for assess the quality of RNA in supplementary Table S1 and provided the electropherograms presenting the RNA bands in supplementary Figure S1.

The Authors used SYBR Green fluorescent dye during gene expression studies, hence, it is obligatory to perform Melting Curve Analysis, and results of this examination should be added in the manuscript or Supplementary file (e.g., JPG or TIFF file),

Reply: We added the figures of Melting Curve data in supplementary Table S2, and correspondingly modified the serial number of supplementary.

In the paragraph “2.5. Expression analysis of CaAnn genes” - description of real-time qRT-PCR analyses should be profoundly extended. In addition, there is the lack citation of 2-ΔΔCT method (Livak, Schmittgen 2001),

Reply: We have added the detail experimental process of qRT-PCR and add the correspond reference (Reference 29).

in the paragraph “2.9. Statistical Analysis” – ANOVA testing and post-hoc test should be described in more details,

Reply: The details have been added.

discussion of the results is superficial and should be thoroughly revised,

Reply: Thank you very much. The functional mechanism of ANN protein has been deeply studied in recent years. In the discussion section, we have a detailed discussion and cite the latest references. We have modified and deleted some contents in the discussion section. We look forward to your support.

there is the lack of statistical test used under some figures

Reply: The statistical test has been added.

colour figures are suggested,

Reply: Good suggestion. Some color figures have been added.

moderate English changes by the native speaker are required.

Reply: Good suggestion. Our revised manuscript has been carefully reviewed by the native speaker and modified appropriately

Reviewer 3 Report

I have reviewed the manuscript and please find suggestion on the manuscript of Wu et al. entitled " Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance”

This study reports the identification and characterisation of CaAnn genes in the Pepper plants grown under different stress. The authors showed the expression of candidate Ann genes under different stress conditions and show that CaAnn genes regulates stress tolerance in plants by modulation physiological and biochemical processes. The research conducted in this study was well supported by the experimental and statistical findings. The manuscript has some inconsistencies in terms of formatting and English. The scientific content of the manuscript is good but needs to be presented in a better way and should be addressed before publication in journal IJMS. It needs careful reading before submission. My minor concerns are listed in the manuscript itself. Please look at the comments throughout the manuscript and needs to be addressed before acceptance in the manuscript. Manuscript can be accepted after addressing the comments mentioned in the manuscript.

Comments for author File: Comments.pdf

Author Response

Dear referee3,

Thanks very much for your comments concerning our manuscript entitled " Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance ". (ijms-1326986). We gratefully thank the editor and all reviewers for their time making their constructive comments, which has significantly raised the quality of the manuscript and enable us to improve the manuscript. Each suggested revision and comment, brought forward by the reviewers, was accurately considered and revised. Below the comments of the reviews are responsive point by point and the revisions are indicated.

The revisions made to the manuscript have marked up using the “Track Changes” function, which we hope meet with your approval. Thank you again!

We wish safety and health to you and the reviewers, given the current COVID-19 pandemic!

Yours sincerely,

H.D. Ding

Reviewer 3

I have reviewed the manuscript and please find suggestion on the manuscript of Wu et al. entitled " Genome-wide identification and transcriptional expression analysis of annexin genes in Capsicum annuum and characterization of CaAnn9 in salt tolerance”

This study reports the identification and characterisation of CaAnn genes in the Pepper plants grown under different stress. The authors showed the expression of candidate Ann genes under different stress conditions and show that CaAnn genes regulates stress tolerance in plants by modulation physiological and biochemical processes. The research conducted in this study was well supported by the experimental and statistical findings. The manuscript has some inconsistencies in terms of formatting and English. The scientific content of the manuscript is good but needs to be presented in a better way and should be addressed before publication in journal IJMS. It needs careful reading before submission. My minor concerns are listed in the manuscript itself. Please look at the comments throughout the manuscript and needs to be addressed before acceptance in the manuscript. Manuscript can be accepted after addressing the comments mentioned in the manuscript.

Thank you very much. We have made changes according to your suggestions.

Lines 140 (137)

Reply: Thanks! Give the complete name of the SuperScript double stranded-cDNA synthesis kit.

Lines 213, 227

Reply: Thanks! Change MEGA 7.0 to MEGA 6.0.

Lines 334 (329)

Reply: The bracket in this sentence were not need and have been deleted.

Lines 413 (409):

Reply: Correct and give the right spell of “introns”.

Lines 154 (151)

Reply: Change word Agro to GV3101

Lines 164 (161)

Reply: Add the method for estimate the gene silence level

Lines 196 (199)

Reply: This is the real gene number in the database. It has not been modified.