Identification of AP2/ERF Transcription Factor Family Genes and Expression Patterns in Response to Drought Stress in Pinusmassoniana

Round 1

Reviewer 1 Report

In paper, the authors have successfully identified PmAP2/ERFs genes from Pinus massoniana and done bioinformatic analysis. The gene expression pattern analysis with the help of qRT PCR has shown its role in drought stress and in response to hormones. The study can be the basis for further investigation into this plant for abiotic stress tolerance, and I recommend for acceptance of the manuscript

Author Response

Reply：Thanks very much for the comments from reviewer. You gave good advice to edit this paper better. We will be happy to edit the text further, based on helpful comments from the reviewers. We also thank you for your recognition and decision on the manuscript, which is a confirmation of our work and scientific research. We will continue to carry out our subsequent scientific research with scientific rigor. Thank you again for reviewing our paper and for your recognition of us.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

The manuscript contains valuable information; the method is well described, and the results are formulated. Even so, several aspects need to be improved, as is the case of the introduction, where the main objective and working hypothesis are missing. The figure needs to be further adjusted since several are unclearly presented, and the explanation is missing. Point by point, I detailed the observation and suggestions in the following lines.

Introduction

L33 – "other abiotic stressors" please be more specific; the expression is ambiguous.

L61-63 there are discussed two annual plants, A. thaliana and Oryza sativa L. and in the literature exist several articles discussing the AP2/ERF genes implication in various functions in trees, e.g. for oaks (Susana T Lopes, Daniel Sobral, Bruno Costa, Pedro Perdiguero, Inês Chaves, Augusta Costa, Célia M Miguel, Phellem versus xylem: genome-wide transcriptomic analysis reveals novel regulators of cork formation in cork oak, Tree Physiology, Volume 40, Issue 2, February 2020, Pages 129–141, https://doi.org/10.1093/treephys/tpz118), for Liriodendron chinense  (Zong, Y., Hao, Z., Tu, Z., Shen, Y., Zhang, C., Wen, S., ... & Li, H. (2021). Genome-wide survey and identification of AP2/ERF genes involved in shoot and leaf development in Liriodendron chinense. BMC genomics, 22(1), 1-17.), for Taxus× media (Zhang, K., Jiang, L., Wang, X., Han, H., Chen, D., Qiu, D., & Yang, Y. (2021). Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating taxol biosynthesis in Taxus× media. Industrial Crops and Products, 171, 113972.), for poplar (Chen, N., Qin, J., Tong, S., Wang, W., & Jiang, Y. (2022). One AP2/ERF Transcription Factor Positively Regulates Pi Uptake and Drought Tolerance in Poplar. International journal of molecular sciences, 23(9), 5241.). Thus, please change this association between studies on annual shrubs and trees.

L82 – please cite at least one study to demonstrate this sentence "… P. massoniana related transcription factors involved in drought resistance from molecular level study was rarely reported".

L82-87 I suggest deleting this sentence which appears as a conclusion. Please insert a sentence with the study's primary objective and the hypothesis of work, which are missing in the present stage.

Material and method

L94 – "greenhouse" this word is repeated; please double check

All figures are missing the explanation.

The quality of figure 2 is deficient. Can it be changed?

The information from figure7 and 8 is very compressed and difficult to read. The font needs to be increased.

Conclusions need to be rephrased since the main results which describe the "Expression Patterns in Response to Drought Stress" are missing.

Please double-check the references. For example in the manuscript the refernce 43 is "Kaikai, Z., Luyuan, J., Xu, W., Hua, H., Duan, C., De, Q., Yan, F. Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating Taxol biosynthesis in Taxus×media. Industrial Crops & Products 2021, 171, 113972."

and in reality is “Zhang, K., Jiang, L., Wang, X., Han, H., Chen, D., Qiu, D., & Yang, Y. (2021). Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating taxol biosynthesis in Taxus× media. Industrial Crops and Products, 171, 113972."

Author Response

Thanks very much for the comments from reviewer. You gave good advice to edit this paper better. we will be happy to edit the text further, based on helpful comments from the reviewers. Following the instruction of the reviewer, we revised the contents of the article. Then we listed specific changes in this paper below following every questions and advice from reviewer.

Question 1: L33 – "other abiotic stressors" please be more specific; the expression is ambiguous.

Reply 1: Thanks to the reviewers, We agree with the reviewer’ suggestion. Thank you for your comment of supplementing abiotic stress, which we did not expect again before. Therefore, we have added low temperature and salt stress to the text and cited relevant literature to support our presentation. Your comments make the preface more comprehensive and complete, and we thank you again for your comments.

 

Question 2: L61-63 there are discussed two annual plants, A. thaliana and Oryza sativa L. and in the literature exist several articles discussing the AP2/ERF genes implication in various functions in trees, e.g. for oaks (Susana T Lopes, Daniel Sobral, Bruno Costa, Pedro Perdiguero, Inês Chaves, Augusta Costa, Célia M Miguel, Phellem versus xylem: genome-wide transcriptomic analysis reveals novel regulators of cork formation in cork oak, Tree Physiology, Volume 40, Issue 2, February 2020, Pages 129–141, https://doi.org/10.1093/treephys/tpz118), for Liriodendron chinense  (Zong, Y., Hao, Z., Tu, Z., Shen, Y., Zhang, C., Wen, S., ... & Li, H. (2021). Genome-wide survey and identification of AP2/ERF genes involved in shoot and leaf development in Liriodendron chinense. BMC genomics, 22(1), 1-17.), for Taxus× media (Zhang, K., Jiang, L., Wang, X., Han, H., Chen, D., Qiu, D., & Yang, Y. (2021). Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating taxol biosynthesis in Taxus× media. Industrial Crops and Products, 171, 113972.), for poplar (Chen, N., Qin, J., Tong, S., Wang, W., & Jiang, Y. (2022). One AP2/ERF Transcription Factor Positively Regulates Pi Uptake and Drought Tolerance in Poplar. International journal of molecular sciences, 23(9), 5241.). Thus, please change this association between studies on annual shrubs and trees.

Reply 1: Thanks to the reviewers, We agree with the reviewer’ suggestion. In the review section of the text, we add the progress of AP2/ERF Transcription Factor in woody plants as suggested by the reviewers, and better cite the findings of these literature in the discussion in order to improve the manuscript.

 

Question 3: L82 – please cite at least one study to demonstrate this sentence "… P. massoniana related transcription factors involved in drought resistance from molecular level study was rarely reported".

Reply 3: We thank you for your suggestion to add citations, and we will make changes. Most of the current studies on drought resistance in Pinus massoniana have been conducted from physiological and other perspectives-Citation 5, while transcription factor-related studies in Pinus massoniana have gradually increased in recent years, involving studies on low phosphorus stress and pine nematode disease, while relatively few studies have been conducted on the regulatory mechanisms of transcription factors involved in drought resistance in Pinus massoniana. The number of AP2/ERF genes is large and their functions are comprehensive, therefore, in this study, the AP2/ERF gene family of Pinus massoniana was investigated. The AP2/ERF gene family was identified in this study, and the drought response mechanism of AP2/ERF was subsequently investigated.

 

Question 4: L82-87 I suggest deleting this sentence which appears as a conclusion. Please insert a sentence with the study's primary objective and the hypothesis of work, which are missing in the present stage.

Reply 4: Thank you for your suggestion, the primary objective and the hypothesis of work expression in this paper are not comprehensive enough, and we have revised this section following your suggestion. AP2/ERF gene plays an important role in abiotic stresses such as drought, and since genome-wide data are not yet available for Pinus massoniana, the AP2 /ERF gene family identification is a feasible approach. In this study, we performed gene family identification by identifying PmAP2/ERFs genes in Pinus massoniana, looked for relevant drought resistance genes, and explored the possible involvement of genes in signaling pathways.

 

Question 5:L94 – "greenhouse" this word is repeated; please double check

Reply 5: Thanks to your suggestion, the duplicate "greenhouse" has been removed and we have proofread the whole paper. Thanks to your review and suggestions, the quality of our research paper has been ensured.

 

Question 6: All figures are missing the explanation.

Reply 6: Thank you for your suggestion. Due to our negligence, the images of the Word document were compressed, resulting in a blurry picture of the paper. We have replaced the images in the full text with high resolution images. We look forward to your feedback and thank you again for your suggestions.

 

Question 7: The quality of figure 2 is deficient. Can it be changed?

Reply 7: Thank you for your suggestion, we have replaced the fuzzy figure 2 with the new HD figure 2.

 

Question 8: The information from figure7 and 8 is very compressed and difficult to read. The font needs to be increased.

Reply8: Thank you for your suggestion, we have re-arranged the images so that each small image is clearer and can clearly see the differences in expression patterns and expression levels of each gene in different tissues and under different treatments. Thank you again for your suggestion, which makes the results of figure7 and 8 to be presented in a clearer way.

 

Question 9: Conclusions need to be rephrased since the main results which describe the "Expression Patterns in Response to Drought Stress" are missing.

Reply 9: Thanks to your suggestion, we have rewritten the conclusion and added the description "Expression Patterns in Response to Drought Stress" to the conclusion and changed to ‘In this study, we successfully identified 124 PmAP2/ERFs genes and analyzed the physicochemical properties, phylogeny, and conserved motifs of the gene family members’. We also analyzed the expression patterns of nine PmAP2/ERFs genes under drought treatment and found that all nine genes changed expression in response to drought stress, but the expression patterns were different. The expression patterns showed that PmAP2/ERF11, PmAP2/ERF14, PmAP2/ERF44, PmAP2/ERF77, PmAP2/ERF80 and PmAP2/ERF109 genes were up-regulated in response to drought stress and were positively regulated; PmAP2/ERF96 gene was negatively regulated. The expression pattern of PmAP2/ERFs genes induced by hormones differed in different tissues of different families, and the PmAP2/ERFs genes responded to at least one hormone signal, suggesting that the PmAP2/ERFs genes may positively or negatively regulate the response to hormone to improve drought tolerance in P. massoniana. ". Your suggestion completes the conclusions of this paper, and again, thank you for your suggestion.

 

Question 10: Please double-check the references. For example in the manuscript the refernce 43 is "Kaikai, Z., Luyuan, J., Xu, W., Hua, H., Duan, C., De, Q., Yan, F. Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating Taxol biosynthesis in Taxus×media. Industrial Crops & Products 2021, 171, 113972."and in reality is “Zhang, K., Jiang, L., Wang, X., Han, H., Chen, D., Qiu, D., & Yang, Y. (2021). Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating taxol biosynthesis in Taxus× media. Industrial Crops and Products, 171, 113972."

Reply 10: Thank you for your suggestion, and for your suggestion that there was a problem with the references, this was the result of an oversight on our part. We have revised it and checked all the literature done cited in this article to ensure the accuracy of the citations in this paper.

 

 

Reviewer 3 Report

Dear Authors,

The exploration of the regulatory background of drought tolerance has great importance. In this regard your paper present a good example with a well designed experiment.

Generally, I have no problems with the content of the paper. In its current form however, I am almost sure that only the experts of forest genetics will resonate with it. Some more explanations may help the understanding of a wider range of forestry experts. Consequently, a greater impact can be achieved in this way. If it is possible, major conclusions should be presented, which species may have similar regulatory pathways, and how your results can promote research of other species drought response.

Some more minor improvements are needed before the publication. You should carefully check for spelling and typing errors e.g. in line 94 your wrote greenhouse twice.

There is a more remarkable issue with the figures. The figures have no captions at all and most of them is not readable in the real size of the document. This should be corrected.

Author Response

Thanks very much for the comments from reviewer. You gave good advice to edit this paper better. we will be happy to edit the text further, based on helpful comments from the reviewers. Following the instruction of the reviewer, we revised the contents of the article. Then we listed specific changes in this paper below following every questions and advice from reviewer.

 

Question 1: If it is possible, major conclusions should be presented, which species may have similar regulatory pathways, and how your results can promote research of other species drought response.

Reply 1: Thank you for your suggestion. Currently, the identification of related transcription factor families based on transcriptome and whole genome has become a hot research topic, and our research species is Pinus massoniana, which belongs to gymnosperms. ERF genes and the molecular mechanisms involved in the drought regulation of Pinus massoniana. Because of the close kinship between gymnosperms and the similarity in phylogeny and gene functions, this study also provides a reference for investigating the molecular mechanisms of AP2/ERFs genes in response to drought in other gymnosperms. 

 

Question 2: Some more minor improvements are needed before the publication. You should carefully check for spelling and typing errors e.g. in line 94 your wrote greenhouse twice.

Reply 2: Thank you for your suggestion, we have checked the writing etc. of the full text, your suggestion is essential to improve the quality of this paper, thank you again for your suggestion.

 

Question 3: There is a more remarkable issue with the figures. The figures have no captions at all and most of them is not readable in the real size of the document. This should be corrected.

Reply 3: Thank you for your suggestion, due to our negligence, we did not find that the uploaded document did not contain the figure caption, we have added the figure caption figure notes for the full text images, thank you again for your suggestion.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear authors,

Thank you very much for considering my remarks and suggestion. I noted a significant value added in the final form of the manuscript.

Sincerely,