Identification and Tissue-Specific Expression Analysis of CYP720B Subfamily Genes in Slash Pine and Loblolly Pine
Round 1
Reviewer 1 Report
Thank you for the update. Yes, I checked the manuscript and the responses of the authors. The authors addressed the comments. I recommend accepting the manuscript.Author Response
Thank you for your valuable comments and best wishes for you.
Reviewer 2 Report
Comments to authors
Paper describes identification of CYP720B subfamily genes in two Pinus species.
Major comments to authors
1) M&M section 2.3 Plant materials and treatments? My question is what treatments? I don’t see in the description of this subsection that you performed any treatments. Description of the growth conditions in the greenhouse (light, photoperiod, temperature, PAR…) is missing. Please describe and define.
2) Add the pictures of two Pine species along with the pictures of young and mature needles , stems and roots of each of the analyzed species in one Figure.
3) Figure 5 and qPCR analyses. Please add statistical analyses for each of the gene analyzed by qPCR.
4) The part of Conclusion where you write about expression levels of CYP720B genes and DRAs should be more specific (lines 414-417). Mostly, your Conclusion is to general.
Minor comments to authors
Introduction
Lines 56-58 Rephrase the sentence. It is not clear.
Line 64 reference is missing regrading this statement
Lines 61-62 Write the species in which expression levels of these genes were measured.
Line 65 Correct the Latin name of the species (remove dot after species name)
Line 74 PtAO (define)
Line 121 Define what was biological replicate?
Table 1 Add Latin names of the species in Table header
Lines 209-210 This sentence makes no sense, rephrase
Figure 3. and 4. In header define in which species
Lines 269-270 In both species? Define
Table A2 Define species name in table
Author Response
Comment 1: M&M section 2.3 Plant materials and treatments? My question is what treatments? I don’t see in the description of this subsection that you performed any treatments. Description of the growth conditions in the greenhouse (light, photoperiod, temperature, PAR…) is missing. Please describe and define.
Response 1: Thanks for reminding, we have corrected the manuscript according to your comments.(line 114-123)
Comment 2: Add the pictures of two Pine species along with the pictures of young and mature needles , stems and roots of each of the analyzed species in one Figure.
Response 2: Thanks for reminding, we have corrected the manuscript according to your comments.(Appendix A)
Comment 3: Figure 5 and qPCR analyses. Please add statistical analyses for each of the gene analyzed by qPCR.
Response 3: Thank you for your reminding, and the corresponding modifications have been made.(Figure 5, line 144-145)
Comment 4: The part of Conclusion where you write about expression levels of CYP720B genes and DRAs should be more specific (lines 414-417). Mostly, your Conclusion is to general.
Response 4: Thanks for reminding, we have corrected the manuscript according to your comments.(line 422-438)
Comment 5: Lines 56-58 Rephrase the sentence. It is not clear.
Response 5: Thanks for reminding, we have corrected the manuscript according to your comments.(line 57-59)
Comment 6: Line 64 reference is missing regrading this statement
Response 6: Thanks for your valuable comments, the corresponding modifications have been made accordingly.(line 65)
Comment 7: Lines 61-62 Write the species in which expression levels of these genes were measured.
Response 7: Thanks for reminding, we have corrected the manuscript according to your comments.(line 62-63)
Comment 8: Line 65 Correct the Latin name of the species (remove dot after species name)
Response 8: Thank you for your reminding, and the corresponding modifications have been made.(line 66)
Comment 9: Line 74 PtAO (define)
Response 9: Thanks for reminding, we have corrected the manuscript according to your comments.(line 74)
Comment 10: Line 121 Define what was biological replicate?
Response 10: Thanks for reminding, we have rewritten it to make it a liitle bit clear. We carried out the GC-MS analysis for three individuals for each species. We regarded each individual as one biological replicate and three individuals as three biological replicates.
Comment 11: Table 1 Add Latin names of the species in Table header
Response 11: Thanks for your valuable comments, the corresponding modifications have been made accordingly.(line 190)
Comment 12: Lines 209-210 This sentence makes no sense, rephrase
Response 12: Thank you for your reminding, and the corresponding modifications have been made.(line 213)
Comment 13: Figure 3. and 4. In header define in which species
Response 13: Thanks for reminding, we have corrected the manuscript according to your comments.(line 231-234, 266)
Comment 14: Lines 269-270 In both species? Define
Response 14: Thanks for reminding, a total of 9 CYP720B genes in slash pine and 12 in loblolly pine, we have rewritten it to make it a little bit clearer.(line 277)
Round 2
Reviewer 2 Report
Authors accepted all sugestions and corrected manuscript text. Only minor lenguage editing is required. Therefore, I recomend acceptance of this manuscript.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Dear Editor:
Please receive my review for the manuscript entitled “Identification and Tissue-Specific Expression Analysis of 2 CYP720B Subfamily Genes in Slash Pine and Loblolly Pine” by Zhang et al.
The authors studied the CYP720B and mechanism of resin synthesis pathway. The authors identified 17 genes in slash pine and 19 in loblolly pine were identified and assumed to be CYP720B candidate genes. The results are valuable for the scientific community and provided further explanation related to the role of CYP720B and CYP720B genes and resin mechanism pathways. The manuscript needs revision regarding statistics and software used. I suggest the authors creates a separate section and name it “Experimental Design and Statistical Analysis” and write in details how they design the experiment (design, replicates, software used, and so on). Also, they need to revise the entire text, beginning from the abstract for errors such as (….the rosin synthesis pathway…” for “resin” and “rosin”, and so on. All abbreviation in tables or figures should be defined; tables and figures are self-explanatory.
References are mixed for journal names (journal source); sometimes abbreviated, sometimes not, and sometimes mixed up (see below . So, please revise the entire reference list and follow the journal instructions for writing names and formatting (see below for examples how the journal names are mixed up. Therefore, I am recommend accepting the manuscript with minor revision.
Pham, T.; Chen, H.; Dai, L.; Vu, T.Q.T. Isolation a P450 Gene in Pinus armandi and Its Expression after Inoculation ofLeptographium qinlingensis and Treatment with Methyl Jasmonate. Russian Journal of Plant Physiology 2016, 63, 118-125, doi: 10.7868/S0015330316010139
De, L.; Fernanda, D. C.; TN Füller; Silva, R.; Kerber, M. R.; Lima, M. S.; Fett, J.P.; Fett-Neto, A.G. Reference Genes for qPCR 474 Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis. Front. Plant Sci. 2016, 7, doi: 10.3389/fpls.2016.00849 476 25.
Yang, S.; Wang, H.; Sathyan, P.; Stasolla, C.; Loopstra, C.A. Real-time RT-PCR Analysis of Loblolly pine (Pinus taeda) Arabino-galactan-protein and Arabinogalactan-protein-like Genes. Physiol. Plantarum 2005, 124, 91-106, doi: 10.1111/j.1399-3054.2005.00479.x 479 26.
Sheng, Z.; D, F.R. Comprehensive Algorithm for Quantitative Real-time Polymerase Chain Reaction. Journal of computational biology : a journal of computational molecular cell biology 2005, 12,1047-64 .doi: 10.1089/cmb.2005.12.104727. Ivo, R.; J., P.S. Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics. The Plant Cell 2009, 21, 1031-1033. doi:10.1105/tpc.109.066001
Author Response
We thank the reviewer for your constructive suggestions and detailed corrections. We have made corresponding corrections based on the revised opinions in our manuscript. All the modified places have been marked as red in the manuscript. Please have a check.
Detailed reply to reviewers' comments:
Reviewer 1:
Comment 1: The manuscript needs revision regarding statistics and software used. I suggest the authors create a separate section and name it “Experimental Design and Statistical Analysis” and write in detail how they design the experiment (design, replicates, software used, and so on).
Response 1: Thank you for your valuable suggestion, additional information has been added in the Materials and Methods part (line 87-104).
Comment 2: They need to revise the entire text, beginning from the abstract for errors such as (….the rosin synthesis pathway…” for “resin” and “rosin”, and so on.
Response 2: Thanks for your valuable comments, the corresponding modifications have been made accordingly. (line 15, 78, 318, 320)
Comment 3: All abbreviations in tables or figures should be defined; tables and figures are self-explanatory.
Response 3: Thanks for your kind suggestion, corresponding modifications have been made in tables and figures. (line 238-241, 273-278, 316-317)
Comment 3: References are mixed for journal names (journal source); sometimes abbreviated, sometimes not, and sometimes mixed up. So, please revise the entire reference list and follow the journal instructions for writing names and formatting
Response 3: Thanks for reminding, we have corrected the references according to the journal instructions for writing names and formatting. ( line 440, 445, 453, 454, 459, 473, 485, 497, 505, 512, 532, 539)
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript# forests-1442809 discusses important objectives “Identification and Tissue-Specific Expression Analysis of CYP720B Subfamily Genes in Slash Pine and Loblolly Pine”. In this study the authors demonstrated a comprehensive analysis of CYP720B candidate genes in splash pine and their structural and phylogenetic features candidate genes were further classified into four main clades. The findings described in the paper are sound and will be considered for publication in this journal. There are some queries/corrections which need to be addressed/corrected in the revised version.
- Line 20: ……closely related to adversity resistance.
- Line 119: This study was carried out in greenhouse? Please correct the sentence…. ‘In this study, two-year-old slash pine and loblolly pine seedlings of approximately 50 cm in height growing in a greenhouse at the Research Institute of Subtropical Forestry, Chinese Academy of Forestry, located in Hangzhou, Zhejiang Province, China (30°3'N, 119°6'E) were used as tissue-specific expression analysis materials’.
- Line 129: What about the RNA extraction from other materials (root and stem). Should be corrected as ‘The plant materials (50-100 mg) were ground……’
- Line 134: Please provide the name of ‘city and country, of manufacturer
- Line 245: ‘plant hormone’
- Line 274-282: No need to repeat the abbreviations many times. Just spell out the full term with the abbreviation in the first appearance and then use the abbreviation only throughout the text.
- Line 291-292: Did the author examined the content of diterpenes in the same seedlings used for expression analysis or not? Not clear in either materials and methods or results section. If not, no conclusion or correlation between changes in CYP720Bs expression and diterpene contents in the studied tissues.
- Line 293-295: This should be a part of discussion. Several parts like this sentence are found in the results section. These sentences are obviously discussion and should be moved to the discussion section (Line 25, Line 205-207 etc. please check the whole results.
- Section 4.3: More comprehensive discussion about the potential roles of identified genes based on their differential expression in studied tissues and the content of diterpenes is needed.
- Line 394-399: Repetition from abstract. Please re-write.
Author Response
We thank the reviewers for your constructive suggestions and detailed corrections. We have made corresponding corrections based on the revised opinions in our manuscript. All the modified places have been marked as red in the manuscript. Please have a check.
Detailed reply to reviewers' comments:
Reviewer 2:
Comment 1: Line 20: ……closely related to adversity resistance.
Response 1: Thanks for reminding, we have corrected the manuscript according to your comments.(line 20-21)
Comment 2: Line 119: This study was carried out in greenhouse? Please correct the sentence…. ‘In this study, two-year-old slash pine and loblolly pine seedlings of approximately 50 cm in height growing in a greenhouse at the Research Institute of Subtropical Forestry, Chinese Academy of Forestry, located in Hangzhou, Zhejiang Province, China (30°3'N, 119°6'E) were used as tissue-specific expression analysis materials’.
Response 2: Thanks for reminding, we have corrected the manuscript according to your comments. (line 92-95)
Comment 3: Line 129: What about the RNA extraction from other materials (root and stem). Should be corrected as ‘The plant materials (50-100 mg) were ground……’
Response 3: Thanks for your valuable comments, the corresponding modifications have been made accordingly.(line137)
Comment 4: Line 134: Please provide the name of ‘city and country, of manufacturer
Response 4: Thanks for your reminding, the corresponding modifications have been made accordingly.(line 137, 143, 150)
Comment 5: Line 245: ‘plant hormone’
Response 5: Thanks for your comment. The word has been revised as ‘plant hormone’ accordingly. (line 256)
Comment 6: Line 274-282: No need to repeat the abbreviations many times. Just spell out the full term with the abbreviation in the first appearance and then use the abbreviation only throughout the text.
Response 6: Thanks for your valuable comments, the corresponding modifications have been made accordingly.(line 283-295)
Comment 7: Line 291-292: Did the author examined the content of diterpenes in the same seedlings used for expression analysis or not? Not clear in either materials and methods or results section. If not, no conclusion or correlation between changes in CYP720Bs expression and diterpene contents in the studied tissues.
Response 7: Thank you for your question, the material used for expression quantitative analysis and diterpene content determination was the same test material, We have made the corresponding changes in the manuscript. (line 95-97)
Comment 8: Line 293-295: This should be a part of the discussion. Several parts like this sentence are found in the results section. These sentences are obviously discussion and should be moved to the discussion section (Line 25, Line 205-207，etc. please check the whole results.
Response 8: Thank you for your suggestion, and the corresponding modifications have been made.(line 341-342, 364-366, 407-411)
Comment 9: Section 4.3: More comprehensive discussion about the potential roles of identified genes based on their differential expression in studied tissues and the content of diterpenes is needed.
Response 9: Thank you for your reminding, and the corresponding modifications have been made. (line 395-414)
Comment 10: Line 394-399: Repetition from the abstract. Please re-write.
Response 10: Thanks, we have taken your suggestions into consideration, now the corresponding modifications have been made. (line 417-421)
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The manuscript has been considerably improved. I would like to recommend for publication in its current form.
