Transcriptome Analysis Provides Insights into Lignin Biosynthesis in Styrax tonkinensis Branches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Lignin is one of the most essential polymer (with cellulose) contributing to the structure and shape of most perennial plants on the Earth. Moreover, the synthesis and activity of many enzymes involved in the lignification pathway, e.g. phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), or peroxidase (POD) are also induced to significant levels by the biotic and abiotic stresses, implying that lignin synthesis plays a crucial role in plant defense mechanisms. The KEGG pathway analysis consubstantially helps in understanding the conserved subpathways (pathway motifs) which are often encoded by positionally coupled orthologue genes on the chromosome, and which are especially useful in predicting gene functions.

The Author confirmed the well-known observation about the seasonal increase of lignin content (at the beginning of vegetation season) and its decrease (in autumn), as well as the key-enzymes involved in lignin synthesis, i.e. CCoAOMT (caffeoyl-CoA O-methyltransferase), COMT (caffeic acid O-methyltransferase), POD (peroxidase), and F5H (ferulate 5-hydroxylase). Surprisingly, no significant participation in the lignin biosynthesis pathway was observed for: PAL (phenylalanine ammonia-lyase), cinnamyl alcohol dehydrogenase (CAD), nor 4CL (4-coumarate: CoA ligase), which are typically associated with cell wall reinforcement through lignification.

The novelty of the paper lies in a simultaneous exploration of various regulatory mechanisms using Gene Ontology and the KEEG pathway analysis in Styrex plant from Jiangsu Province.

The manuscript was prepared correctly. The title, abstract, and keywords clearly reflect the paper's content. The introduction explicitly presents the problem. The methodology and analysis of results rather don't raise any objections except for the limited repetition in years of the experiment (performed in only one year) and limited space (only one experimental plot a Xiashu Forestry Farm. Therefore, how one can be sure that the following years (with different environmental conditions) will generate similar results? Broader studies are necessary to formulate definite conclusions. But the Authors are conscious about this mentioning at the end of the introduction that “our study could give a theoretical framework for a deeper comprehension of the S. tonkinensis lignin biosynthesis mechanism and the improvement of its wood properties”. Nevertheless, the results obtained in the carried study depicted some patterns of the key enzymes involved in the biosynthesis of lignin during 3 months of the most intensive growing season.

The manuscript was prepared correctly, but some points can be improved:

Line 108 and others: adjust the format of all subtitles from 2.1. to 3.6. according to the Journal rules or unify their typing (i.e. all words in capitals)

Line 136: give a reference for the acetylation method, because not all readers are familiar with it

Lines 143-151: the same for the RNA processing, I understand that the methodology was performed according to the manufacturer's instruction

Line 226: put “Styrax tonkinensis” in Italic font

Line 228: ”in figures 5 & 6”

Line 278 – develop the abbreviation of POD when first time cited (apart in the abstract), not all readers see this as peroxidase which is essential in the last stage of lignification

Line 325 – put “Syringyl” in minuscule letter

Line 340 - "Populus tremula ×Poppulus  alba" in Italic font

Line 376 – give full reference for “Gui et al.” mentioning about transcription factor LTF1 associated with lignin synthesis because the reference numbered as 11 refers to Zhang et al.

Author Response

Dear Editor and Reviewers,

Thank you very much for your valuable comments and suggestions on our manuscript. Following the reviewers’ comments, we have modified and improved our manuscript according to your kind advices and referee’s detailed suggestions. In order to distinguish and find easily, we use four colors to highlight the modified part. Responses to the first reviewer correspond to “yellow”. See details in word. 

Thank you for all your help and we sincerely hope this manuscript will be acceptable to be published on forests.

Best regards,

Chao Han

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments:

The manuscript "Transcriptome analysis provides insights into lignin biosynthesis in Styrax tonkinensis branches” by Han et al. is extremely important and current. The study sheds light on the genetic mechanisms underlying lignin biosynthesis in Styrax tonkinensis, a valuable plant species with potential industrial applications. The findings could have significant implications for biofuel production and lignin engineering research. Despite some important data, the manuscript lacks fundamental information on the experimental methods used in the study, which could limit reproducibility and further research in this area. Additionally, further studies could explore the potential applications of lignin biosynthesis in other plant species to expand our understanding of this important process. Furthermore, investigating the regulation of lignin biosynthesis in different environmental conditions could provide valuable insights into its potential role in plant adaptation strategies. 

Specific comments:

 

1. Abbreviations must be given with the full name for the first time.
2. In abstract, numerical or quantitative data is required.
3. Referencing is improper throughout the paper. Please carefully provide references for specific statements, for example, statements in lines 32–34, 34–37, 64, 65–66, etc. Please see the entire manuscript for similar errors.
4. Line 44: "prior," replace it with "previous/earliar.".
5. Texts from lines 96–100 are repeated in lines 129–131. Please delete either of them.
6. Which standard method was used for determination of lignin content, please provide reference.
7. In the statistical analysis section, PCA analysis is not mentioned. How PCA analysis was performed and which software was used were clearly mentioned.
8. The results are too lengthy and descriptive. It would be more effective to provide a concise summary of the findings. This will make it easier for readers to grasp the main points.
9. 289-290 is not clear; please rewrite.
10. Line 336, "Paeonia ostii," should be in italic font.
11. Line 343: After Zhao et al. reference is missing, please correct it as Zhao et al. [29].
12. A schematic representation of the pathway involved could provide a more clear and better understanding to readers. Please provide a pathway diagram. 
13. The discussion should be expanded and research compared in depth with clear language and a logical arrangement of information to improve the interpretation of results. Contextual explanations and highlighting of the findings' significance are essential to capture readers' attention and deliver significant impact.
14. Strictly format references according to journal guidelines.
15. Moderate English language revision is required. This includes correcting grammar, punctuation, and sentence structure to improve clarity and coherence. Additional attention may be needed for word choice and phrasing to enhance overall readability and effectiveness. 

Comments on the Quality of English Language

Moderate English language revision is required. This includes correcting grammar, punctuation, and sentence structure to improve clarity and coherence. Additional attention may be needed for word choice and phrasing to enhance overall readability and effectiveness. 

Author Response

Dear Editor and Reviewers,

Thank you very much for your valuable comments and suggestions on our manuscript. Following the reviewers’ comments, we have modified and improved our manuscript according to your kind advices and referee’s detailed suggestions. In order to distinguish and find easily, we use four colors to highlight the modified part. Responses to the second reviewer correspond to “green”. See details in word.

Thank you for all your help and we sincerely hope this manuscript will be acceptable to be published on forests.

Best regards,

Chao Han

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article by Chao Han et al. presents data from transcriptomic analysis to elucidate the characteristics of lignin synthesis depending on the age of the branches and time of year. The work as a whole corresponds to the profile of the journal. The authors presented new results. However, the manuscript contains many typos and requires careful reading.

Line 64 for m3 “3” should be superscript

Line 176 please, check it is repetition: in the supplementary table 1(table S1).

Fig 3 is titled “PCA analysis of eight samples…” sorry, but only 6 samples (in triplicate) are presented.

Legend for Figure 4 “Styrax tonkinensis” should be in italics

Line 256-258. Please, check, what is the difference in the sentence: “with 3 encoding caffeoyl-CoA O-methyltransferase [EC:2.1.1.104], 2 encoding caffeoyl-CoA O-methyltransferase [EC:2.1.1.104],…”

Line 280, 320 “S. tonkinensis” – should be  in italics

Lines 299-302  The sentence “These enzymes are part of a complex network of genes and gene families, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate: CoA ligase (4CL),…” needs correction, as enzymes for sure are not the part of genes network.

Line 304 “p-coumaroyl…” “p” should be in italics. The same, line 340 - italics

Line 313, 325, 327, 332, 346 it should be the gap between the last word in the sentence and citation “peroxide[18]”, “lignin[19,20]” etc.

Line 336 please, check punctuation (flax、jute、tea). Paeonia ostii should be in italics

Line 347-348.  Please, check the sentence “POD is crucial in the last stage of lignin synthesis, and oxidative polymerization of monolignols[30].” Is the oxidative polymerization of monolignols  last stage? Or POD is crucial in both, last stage and polymerization?  If so, please, clarify, what is the last stage. Also, it should be space in front of [30]

Line 377-378. Please, check the sentence “associated with lignin, known as LTF1, Modification of LTF1 for…” It is a capital letter in the middle.

Supplementary table 3. The column called "KEGG Gene Name" mainly represents enzymes. The column name should be changed. Genes listed in this column should be italicized.

Comments on the Quality of English Language

I'm not a native English speaker, so I can't make a clear assessment. But in general, the article is written in clear language, it is not difficult to understand the content. However, in my opinion, there are some errors that authors can eliminate on their own.

Author Response

Dear Editor and Reviewers,

Thank you very much for your valuable comments and suggestions on our manuscript. Following the reviewers’ comments, we have modified and improved our manuscript according to your kind advices and referee’s detailed suggestions. In order to distinguish and find easily, we use four colors to highlight the modified part. Responses to the third reviewer correspond to “light blue”. See details in word.

Thank you for all your help and we sincerely hope this manuscript will be acceptable to be published on forests.

Best regards,

Chao Han

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

   The manuscript by Han et al. is devoted to transcriptome analysis of lignin biosynthesis in Styrax tonkinensis branches. Results of the work is potentially interesting because this analysis provides potential mechanisms of the lignin biosynthesis and its regulation; however, I have some minor comments.

   1.  Introduction: Description of background and novelty of tasks of of this work should be additionally stressed.

   2. P. 4. Lines 144-145: “Three biological replicates were sampled at each time for a total of 18 samples.” Were three biological repetitions enough? Usually, at least 5-6 repetitions are used in investigations. It should be clarified.

   3. 2.7 Statistics Analysis: Why was Tukey selected for this analysis?

Author Response

Dear Editor and Reviewers,

Thank you very much for your valuable comments and suggestions on our manuscript. Following the reviewers’ comments, we have modified and improved our manuscript according to your kind advices and referee’s detailed suggestions. In order to distinguish and find easily, we use four colors to highlight the modified part. Responses to the forth reviewer correspond to “grey”. See details in word.

Thank you for all your help and we sincerely hope this manuscript will be acceptable to be published on forests.

Best regards,

Chao Han

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have incorporated all of the suggestions into the revised manuscript. I have no additional comments. The manuscript can be accepted in its present form.