Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi

Round 1

Reviewer 1 Report

The authors present in this manuscript entitled Comprehensive analysis of GRAS gene family and their expression under GA3, drought stress and ABA treatment in Larix kaempferi the identification and characterization of 11 GRAS genes in Larix kaempferi. The authors were the gene expression of these genes in response to three different abiotic stresses. 

After a thorough analysis of the present manuscript, I believe that the results of this study are not innovative and consistent enough. 

In addition, one of my major concerns is related to the approach used to perform the quantitative PCRs. For species for which little information is available (partially known genome, little information on gene expression under different conditions, etc) it is especially necessary to test several potential reference genes (for an example, please see: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297944/). This is particularly true for your study since you had imposed different types of stress on the trees. 

With respect to the structure of your manuscript, I suggest reducing the number of paragraphs in the material and methods section and presenting Figures 4 and 5 as supplementary data. It would be appropriate to succinctly explain the purpose of each analysis performed in your study, to help the reader better understand how each analysis helps to answer the initial scientific hypotheses that were posed. I found the introduction and discussion sections to be well structured and well written. 

Author Response

We would also like to express our thanks to the reviewers for their time and constructive comments on our manuscript. We have implemented their comments and suggestions and wish to submit a revised version of the manuscript for further consideration in the journal. Changes were tracked for the modified parts and a modification was made in the manuscript. Below, we also provide a point-by-point response explaining how we have addressed each of the reviewers’ comments.

 

Comment 1

“After a thorough analysis of the present manuscript, I believe that the results of this study are not innovative and consistent enough.”

Our response: We thank the reviewer for this comment. After received this comment, we have also thoroughly analyzed our manuscript and found that the manuscript does not look innovative due to the inaccurate explanation in several parts. We have revised them as follows:

 - Page 1 Line 11-15

“The GRAS family transcription factors play important roles in regulating plant growth and responses to abiotic stress, which can be utilized to breed novel plants with improved abiotic stress resistance. But the GRAS gene family has been largely unexplored for tree species, particularly for Larix kaempferi that has high economic and ecological values, challenging practices for breeding abiotic stress-resistant L. kaempferi.”

    - Page 2 Line 79-91

“L. kaempferi is now recognized as important tree species for various economical uses such as timber and pulp production and papermaking, as well as afforestation and ornamental purposes. The problem is that recent climate change-derived abiotic stresses such as drought are severely challenging afforestation practices of L. kaempferi, which calls for breeding novel L. kaempferi varieties with improved abiotic stress resistance. The GRAS gene family is a candidate gene family that can be utilized to breed novel L. kaempferi varieties with improved abiotic stress resistance. However, GRAS gene family has not yet been largely explored in L. kaempferi due probably to unavailability of L. kaempferi genome information. Now, the whole genome of L. kaempferi has recently been sequenced [26] and it is, therefore, possible to perform genome-wide identification analysis for important TFs such as the GRAS TFs.

In this study, we for the first time identified the GRAS gene family in L. kaempferi whole genome and then performed comprehensive analyses”

    - Page 13 Line 359-360

“Notably, the GRAS gene family has been largely unexplored; only reported in cassava [42] and poplar [45].”

In addition, the GRAS gene family has previously not been identified in L. kaempferi and we for the first time identified and comprehensively analyzed the L. kaempferi GRAS gene family in this work. The experimental methods adopted in this work have been logically designed and the results were aligned accordingly with relevant discussion. Thus, we believe that the experimental methods, results and discussion of our manuscript are consistent enough.

Comment 2

“In addition, one of my major concerns is related to the approach used to perform the quantitative PCRs. For species for which little information is available (partially known genome, little information on gene expression under different conditions, etc) it is especially necessary to test several potential reference genes (for an example, please see: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297944/). This is particularly true for your study since you had imposed different types of stress on the trees.”

Our response: Thank you for this suggestion and for your reference articles. We thank the reviewer for pointing this out. We apologize that the reference gene information was missing in our manuscript. It was an error that we wrote as “The whole-genome sequencing of L. kaempferi gene (Whole Genome Shotgun (WGS):  INSDC: WOXR00000000.2) was used as a reference gene.” in the original manuscript. It is not a reference gene, but the target genome. L. kaempferi gene (INSDC: WOXR00000000.2) is the genome data that includes LKGRAS family genes. As a reference gene, GAPDH gene was originally used in this manuscript. Actually, our laboratory had selected and verified the reference genes for real-time quantitative PCR analysis of Larix olgensis under abiotic stress previously and obtained several optimal genes that can be used as reference genes. We have tested these genes and selected the most suitable gene as the reference gene for this study. The primers for GAPDH gene were listed in Table S1 and the relevant text in ‘Materials and Methods’ section reads as follows.

“The whole-genome sequencing of GRAS genes in L. kaempferi gene (Whole Genome Shotgun (WGS):  INSDC: WOXR00000000.2) was used as target genes. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene [30].”

Comment 3

“With respect to the structure of your manuscript, I suggest reducing the number of paragraphs in the material and methods section and presenting Figures 4 and 5 as supplementary data. It would be appropriate to succinctly explain the purpose of each analysis performed in your study, to help the reader better understand how each analysis helps to answer the initial scientific hypotheses that were posed. I found the introduction and discussion sections to be well structured and well written.”

Our response: We thank the reviewer for this suggestion. We have reduced the number of paragraphs in the material and methods section. In addition, we have made minor revisions on the “Results” section to facilitate better understanding of our manuscript. All the revisions in “Materials and methods” and “Results” sections were highlighted and tracked using MS Word track-change mode. And we have presented Figures 4 and 5 as supplementary data and revised figure numbers accordingly.

Reviewer 2 Report

Members of the GRAS family play important roles in plant growth, defence, abiotic stress, phytohormone signaling and also in symbiosis. In the manuscript, the authors examined the molecular evolution of the GRAS family of transcription factors in L. kaempferi, analyzed their phylogenetic relationship with other plant members, identified conserved motifs, and regulatory elements in the promoter. Additionally, they also examined the expression patterns of LkGRAS genes in different tissues and also under different stress treatments, including GA3, ABA and drought stress. The results reported here are very interesting. Study design and methodology are appropriate. English is fine. The manuscript requires few minor revisions prior to publication. I suggest the authors address the following points and improve the manuscript further.

 

1.   The authors must add economic and ecological importance of L. kaempferi in the introductory part. Also, highlight the importance of the conducting the present work with possible explanation.

2.   In the Materials and Methods section, the authors performed RNA extraction from plant samples by the CTAB (cetyltrimethylammonium bromide) method. Add appropriate reference for this protocol.

3.   The error bar represents deviations from biological replications missing in the Figure 7 and Figure 9.

 

4.    Many of the eukaryotic transcription factors (TFs) are intron-less, but few have. This is optional. Authors may add exon-intron structures and their relationships between the GRAS gene family members in L. kaempferi.

Author Response

We would also like to express our thanks to the reviewers for their time and constructive comments on our manuscript. We have implemented their comments and suggestions and wish to submit a revised version of the manuscript for further consideration in the journal. Changes were tracked for the modified parts and a modification was made in the manuscript. Below, we also provide a point-by-point response explaining how we have addressed each of the reviewers’ comments.

 

Comment 1

“The authors must add economic and ecological importance of L. kaempferi in the introductory part. Also, highlight the importance of the conducting the present work with possible explanation.”

Our response: We thank the reviewer for pointing this out. We supplemented following contents to the ‘Introduction’ section.

    - Page 2 Line 72-86

“L. kaempferi belongs to conifer species, generally called larch trees with great value for wood production and ecological afforestation. Larch trees constitute forests in large areas of China, Eastern Europe, and Western North America. Among larch trees, L. kaempferi has several superiorities over others; it grows faster at juvenile, has longer fibrous and denser wood and can adapt more easily to environment than other larch trees. Thus, L. kaempferi is now recognized as important tree species for various economical uses such as timber and pulp production and papermaking, as well as afforestation and ornamental purposes. The problem is that recent climate change-derived abiotic stresses such as drought are severely challenging afforestation practices of L. kaempferi, which calls for breeding novel L. kaempferi varieties with improved abiotic stress resistance. The GRAS gene family is a candidate gene family that can be utilized to breed novel L. kaempferi varieties with improved abiotic stress resistance. However, the GRAS gene family has not yet been largely unexplored in L. kaempferi due probably to the unavailability of L. kaempferi genome information. Now, the whole genome of L. kaempferi has recently been sequenced [26] and it is, therefore, possible to perform genome-wide identification analysis for important TFs such as the GRAS TFs.”

Comment 2

“In the Materials and Methods section, the authors performed RNA extraction from plant samples by the CTAB (cetyltrimethylammonium bromide) method. Add appropriate reference for this protocol.”

Our response: We thank the reviewer for pointing this out. We have added appropriate reference to CTAB method in the “Materials and Methods” section in Page 3 Line 145. Many thanks for your comments.

Comment 3

“The error bar represents deviations from biological replications missing in the Figure 7 and Figure 9.”

Our response: We thank the reviewer for pointing this out. I am really sorry for the missing in the manuscript. We have supplemented “Error bars represent the deviations from three biological replicates”. Many thanks for your comments.

Comment 4

“Many of the eukaryotic transcription factors (TFs) are intron-less, but few have. This is optional. Authors may add exon-intron structures and their relationships between the GRAS gene family members in L. kaempferi.”

Our response: We thank the reviewer for pointing this out. In fact, the sequences of LkGRAS genes and the corresponding promoter sequence were downloaded from NCBI (Whole Genome Shotgun (WGS):  INSDC: WOXR00000000.2). We used the online tool ‘Gene Structure Display Server’ predicting the gene structure and found that they were all CDS sequences. At present, we tentatively believe that the gene sequence we downloaded may be only containing the CDS sequences. Therefore, we did not add this part about exon-intron structures in LkGRAS genes. We also tried to contact the uploader to obtain a complete genome annotation file, but we have not yet received the reply.

We also learned the gene structure of GRAS family in other species. In 117 soybean (Glycine max) GRAS genes, almost all GRAS genes contained very few or no introns; 80.34% of the GmGRAS genes was free of introns, which is similar to the lack of introns in members of this family in other species. For example, 88, 83.3, and 80.23% of GRAS genes in grapevine, Chinese cabbage, and maize, respectively, have no introns. In Barley (Hordeum vulgare L.), most HvGRAS genes had no intron 74.2% (46 out of 62). In orchardgrass Twenty-six (56.52%) DgGRAS genes did not have any introns, 15 (32.6%) had one intron, four (8.70%) had two introns, and only one (2.17%) had three introns. In cucumber (Cucumis sativus L.), the proportion of GRAS genes without introns was 54.05%, while the other genes contained only 1–2 introns. Therefore, we also preliminarily judged that the most of genes structures of GRAS family are intron-less. Many thanks for your comments.

Reviewer 3 Report

In the present article entitled “Comprehensive Analysis of GRAS Gene Family and Their Expression Under GA3, Drought Stress and ABA Treatment in Larix kaempferi” Ma et al has presented the expression profiling of GRAS Gene Family in Larix kaempferi. Overall the manuscript is fine, and the results are presented well. I have few concerns with the manuscript which need to be addressed before its further consideration

-Authors should clearly present the aim and objective of the study

-I found an inconsistency in the italicised format for gene name. Also, the binominal nomenclature should be italicised throughout manuscript.

-Information regarding the endogenous reference gene used is missing, author should elaborate the information about the stable expression of reference gene used. Also, add the primer sequence of reference gene in the table S1 where all the primer sequences are enlisted.

- Check entire manuscript for grammatical errors.

Author Response

We would also like to express our thanks to the reviewers for their time and constructive comments on our manuscript. We have implemented their comments and suggestions and wish to submit a revised version of the manuscript for further consideration in the journal. Changes were tracked for the modified parts and a modification was made in the manuscript. Below, we also provide a point-by-point response explaining how we have addressed each of the reviewers’ comments.

 

Comment 1

“Authors should clearly present the aim and objective of the study.” 

Our response: We apologize if our manuscript did not clearly present the aim and object of the study. We presented the aim and object of this work in the ‘Introduction’ and ‘Discussion’ sections of the manuscript as follows and believed that aim and object of this work were clearly presented:

- In the ‘Abstract’ section (Page 1 Line 12-13)

“In order to improve the stress resistance by regulating the transcription factors in Larix kaempferi”

- In the ‘Introduction’ section (Page 2 Line 73 Page 3 Line 101)

“L. kaempferi is an important fast-growing native tree species in northern China that has high economic and ecological values. L. kaempferi belongs to conifer species, generally called larch trees with great value for wood production and ecological afforestation. Larch trees constitute forests in large areas of China, Eastern Europe, and Western North America. Among larch trees, L. kaempferi has several superiorities over others; it grows faster at juvenile, has longer fibrous and denser wood and can adapt more easily to environment than other larch trees. Thus, L. kaempferi is now recognized as important tree species for various economical uses such as timber and pulp production and papermaking, as well as afforestation and ornamental purposes. The problem is that recent climate change-derived abiotic stresses such as drought are severely challenging afforestation practices of L. kaempferi, which calls for breeding novel L. kaempferi varieties with improved abiotic stress resistance. The GRAS gene family is a candidate gene family that can be utilized to breed novel L. kaempferi varieties with improved abiotic stress resistance. However, the GRAS gene family has not yet been largely unexplored in L. kaempferi due probably to the unavailability of L. kaempferi genome information. Now, the whole genome of L. kaempferi has recently been sequenced [26] and it is, therefore, possible to perform genome-wide identification analysis for important TFs such as the GRAS TFs.  ………………..  This study provides a comprehensive overview of the L. kaempferi GRAS gene family as well as a preliminary basis for further in-depth research on the roles of LkGRAS factors in regulating L. kaempferi responses to phytohormone and abiotic stresses. More importantly, this study provided valuable information for further studies of L. kaempferi to improve the stress resistance by regulating the transcription factors.”

- In the ‘Discussion’ section (Page 13 Line 359-Line 366)

“………  Notably, the GRAS gene family has been largely unexplored in tree species; only reported in cassava [42] and poplar [45]. In our work, we identified the GRAS gene family in L. kaempferi, which is an economically and ecologically important tree species in north-eastern China, for the first time. Then, we performed comprehensive analyses including phylogenetic analysis, conserved motif, and promoter cis-element analyses, tissue-specific and phytohormone and abiotic stress-triggered expression profile analysis, as well as protein interaction network prediction analysis for L. kaempferi GRAS gene family.”

 

In brief, our work aimed at identification and comprehensive (but preliminary) analysis of L. kaempferi GRAS gene family that has never been explored. The significance of this work is related to the high economic and ecological value of L. kaempferi and application potential of LkGRAS transcription factors for stress-resistant L. kaempferi breeding. And all of these provide preliminary method for further research of genome-wide identification, classification and expression in L. kaempferi.

Comment 2

“I found an inconsistency in the italicised format for gene name. Also, the binominal nomenclature should be italicised throughout manuscript.”

Our response: We thank the reviewer for this suggestion. We have scanned for the format errors carefully and then have revised. Several names that are identical to gene names but not italicized represent protein names. Many thanks for your comments.

Comment 3

“Information regarding the endogenous reference gene used is missing, author should elaborate the information about the stable expression of reference gene used. Also, add the primer sequence of reference gene in the table S1 where all the primer sequences are enlisted.”

Our response: We thank the reviewer for pointing this out. We have supplemented the contents related to reference genes and their primers to ‘Materials and methods’ section of the manuscript and supplemental dataset. Many thanks for your comments.

Comment 4

“Check entire manuscript for grammatical errors.”

Our response: We thank the reviewer for this suggestion. We have checked and fixed grammatical errors. Revisions have been made by MS Word ‘track-change’ mode. Many thanks for your comments.

Round 2

Reviewer 1 Report

I commend the authors for the changes made to the manuscript. They confirmed that the adopted method for quantitative PCR was robust, and  improved the materials and methods section. In addition, the changes to the introduction are relevant and better emphasize the novelty of the results. I believe that the improved version of this manuscript is suitable for publication in Forests.

It was a pleasure to help the authors to improve the quality of their paper. With kind regards, Claire Depardieu.