Volatile Profiling and Transcriptome Sequencing Provide Insights into the Biosynthesis of α-Pinene and β-Pinene in Liquidambar formosana Hance Leaves

Round 1

Reviewer 1 Report

The manuscript entitled “Volatile profiling and transcriptome sequencing provides insights into the biosynthesis of α-pinene and β-pinene in Liquidambar formosana Hance leaves” focuses on transcriptomic analysis and volatile oil components analysis of Liquidambar formosana leaves in order to understand the molecular regulation and biosynthesis of components in leaf fragrance. From a scientific point of view, an interesting study with relevant data and results with good analyses has been done however for more clarification I just leave the following comments:

1- There are some very related works that have not been cited in the present study:

Li, Y., Zhou, Y., Chen, H., Chen, C., Liu, Z., Han, C., Wu, Q. and Yu, F., 2022. Transcriptomic Analyses Reveal Key Genes Involved in Pigment Biosynthesis Related to Leaf Color Change of Liquidambar formosana Hance. Molecules, 27(17), p.5433.

Chen, S., Zhang, Y., Zhang, T., Zhan, D., Pang, Z., Zhao, J. and Zhang, J., 2022. Comparative Transcriptomic, Anatomical and Phytohormone Analyses Provide New Insights Into Hormone-Mediated Tetraploid Dwarfing in Hybrid Sweetgum (Liquidambar styraciflua× L. formosana). Frontiers in plant science, 13.

Mancarz, G.F.F., Laba, L.C., da Silva, E.C.P., Prado, M.R.M., de Souza, L.M., de Souza, D., Nakashima, T. and Mello, R.G., 2019. Liquidambar styraciflua L.: a new potential source for therapeutic uses. Journal of pharmaceutical and biomedical analysis, 174, pp.422-431.

2- However the manuscript is well-written it needs moderate English polishing for potential grammatical and structural errors throughout all parts of the manuscript. As in some sections, the sentences lack logical structure or lack relevant verbs. Please make a final check for possible grammatical or structural errors.

3- Please provide all exploited information in material and methods even default parameters of used software. This could include the Gene ID of the used sequence, gap penalty and gap extension used, the exact version of the software (in case of online tools mention access date), and other cases. On the other hand, use the past tense instead of the other in the material and method part.

4- Please specify in material and method the exact origin of plant material. Was it from a single tree? Does the tree belong to a commercial cultivar or it has previously been collected from nature?

5- The information provided in the introduction is not adequate and it could be furnished with more explanations of the genes and pathways responsible for terpenoids biosynthesis.

6- Can you please explain what were your criteria to select the candidate genes for expression analysis?

7- It was better to use corresponding the functional name of the candidate gene instead of accessions ID to have a better understanding of their functional roles.

8- The conclusion should be rewritten and revised as it doesn’t conclude the results as it solely describes the methods and results.

 

Providing addressing the above-mentioned questions.

Author Response

Response to Reviewer 1

 

1- There are some very related works that have not been cited in the present study:

Li, Y., Zhou, Y., Chen, H., Chen, C., Liu, Z., Han, C., Wu, Q. and Yu, F., 2022. Transcriptomic Analyses Reveal Key Genes Involved in Pigment Biosynthesis Related to Leaf Color Change of Liquidambar formosana Hance. Molecules, 27(17), p.5433.

Chen, S., Zhang, Y., Zhang, T., Zhan, D., Pang, Z., Zhao, J. and Zhang, J., 2022. Comparative Transcriptomic, Anatomical and Phytohormone Analyses Provide New Insights Into Hormone-Mediated Tetraploid Dwarfing in Hybrid Sweetgum (Liquidambar styraciflua× L. formosana). Frontiers in plant science, 13.

Mancarz, G.F.F., Laba, L.C., da Silva, E.C.P., Prado, M.R.M., de Souza, L.M., de Souza, D., Nakashima, T. and Mello, R.G., 2019. Liquidambar styraciflua L.: a new potential source for therapeutic uses. Journal of pharmaceutical and biomedical analysis, 174, pp.422-431.

Response: Thanks for the relative study. As suggested, we cited these three excellent articles in line401, line381, and line432.

• However the manuscript is well-written it needs moderate English polishing for potential grammatical and structural errors throughout all parts of the manuscript. As in some sections, the sentences lack logical structure or lack relevant verbs. Please make a final check for possible grammatical or structural errors.

Response: Thanks for the comment. As suggested, we made a final check on possible grammatical or structural errors. The language presentation was improved with assistance from a native English speaker with appropriate research background.

• Please provide all exploited information in material and methods even default parameters of used so This could include the Gene ID of the used sequence, gap penalty and gap extension used, the exact version of the software (in case of online tools mention access date), and other cases. On the other hand, use the past tense instead of the other in the material and method part.

Response:Thanks for the comment. As suggested, We have revised.（line 200-209 and line 220-221）

• Please specify in material and method the exact origin of plant material. Was it from a single tree? Does the tree belong to a commercial cultivaror it has previously been collected from nature?

Response: Thanks for the comment. The tree belong to a commercial cultivar in  forestry station, and materials collected from a single tree. We have supplemented according to the suggestion.（line119-120）

• The information provided in the introduction is not adequate and it could be furnished with more explanations of the genes and pathways responsible for terpenoids biosynthesis.

Response: Thanks for the comment. As suggested.We have supplemented the introduction according to the suggestion.

• Can you please explain what were your criteria to select the candidate genes for expression analysis?

Response:

1) It belongs to significantly differentially expressed genes

2) KEGG enrichment results showed that terpenoid synthesis pathways were enriched

3）Contains 10 motifs that are identical to known TPSs

4）Pfam domain prediction containing typical TPS domain

5）Gene expression was significantly correlated with the content of major metabolites

• It was better to use corresponding the functional name of the candidate gene instead of accessions ID to have a better understanding of their functional roles.

Response: Thanks for the comment. As suggested, We have revised the entire text.

8- The conclusion should be rewritten and revised as it doesn’t conclude the results as it solely describes the methods and results.

Response: Thanks for the comment. As suggested ,We have revised.（line465-470）

Reviewer 2 Report

Results presented by Authors are important and new. However, there are a problems related to description of Material and Methods  or Results presentation. Also some obtained results could be corrected or removed because they are hard to evaluate. Therefore I suggest the following major revisions.

 

I have no access to the Supplement material- provide it.

Line 29 Correct the sentence from „Bioinformatic results….”  to  „Bioinformatic and transcriptomic results….” It will be more accurate.

Line 49, 81, 96, 121 etc, provide the proper spacing, check the same in the entire text.

At the end of introduction section provide in several sentences the description of major results that are achieved, for example number of DEG, identified genes of crucial importance for α and β-pinene biosynthesis, they correlation with α or β-pinene concentration.

In Section 2.1 Authors should clearly state that plants were divided into two groups;  low and high pinene content based on HS-SPME-GC-MS results. Provide number of plants in each L and H sample.

Line 140: provide meaning of the „>10g”.

In the material and methods Authors should shortly descibe, for example in a short separate subsection, the  statistics used to analyse  concentration of both pinenes and RT-PCR results.

Section 3.2; Provide volume and concentration of applied libraries. Provide total length of clean reads. Provide inclusion/exclusion criteria of raw reads. Provide approximate length of raw and clean reads. Provide % GC content of raw and clean reads.

Figure 1- provide in the description the meaning of asterisks presented in the Figure1.

Fig. 4a The standard deviation values in some samples are very high, resulting with the problems with results assessment. For example relative expression of Lq31161 in sample L3, expression of Lq19266 in  sample H2, also the expression of Lq19265 in sample L3 or Lq15225 in sample L1. These results should be removed from Figure 4a and the text should be corrected appropriately. Instead of relative express Authors could use shortage form relative expr.

Fig 4b- correct the meyalonate to mevalonate, remove space in acetoacetyl

Fig 4c- remove et al. from the bracket at the bottom of Figure 4c. In line 313 Authors write that Lq23365 and Lq15225 are members of pathway branches different from TPS. Why they are classified as TPS on Fig. 5c?

Fig 5b Lq23365 and Lq15225 have only very short – marked in pink motifs (pink bar) found in TPSs, they lack many other motifs found in other TPSs. Why they are presented on Fig. 5c as TPS? Provide meaning of the pink color code. Authors write in Fig. 5 description that TPSs (Fig. 5a) are marked with red dots- these dots are rather brown, not red. Correct it in the figure description or make clearly red dots.

Fig. 6 Instead of using gray lines of different thickness, Authors could use lines of different thickness and different color, or just different color, results will be more clearly presented.

Author Response

Dear Reviewer, 

       Please browse the attachment of review report.

      Thank you very much!

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Authors strongly improved the manuscript. However, some issues should be sitill corrected. Following improvements are recommended:

1. 89, 92, 96, 149, 419; correct the spacing

2. Line 62 use shortened plant name L. formosana

3. Line 65 L. formosana  write in italics

4. Line 168- before word „Total” put dot, not comma.

5. Line 224- instead of 3 Authors should write „Three”

6. Lines 229-230; Authors could build a subsection nr 2.8 entitled for example  „Graphical analysis of obtained results”. Then change sentences in lines 229-230, for example like this: „ Microsoft Excel (2013) was used to draw pictures. Volcano map, heat map, histogram and bubble map were prepared using R (2009-2020), provided by RStudio (PBC, v 230 4.0.3 )”.

7. Lines 240/241; Following fragment : (LF6, H1; LF12, H2; LF11, H3) and L groups (LF2, L1; LF7, L2; LF5, L3), exchange as follows: (LF6 → H1; LF12 → H2; LF11 → H3) and L groups (LF2 → L1; LF7 → L2; LF5 → L3). Now Readers will quickly and intuitively understand the origin of H1, H2, H3, L1, L2, L3.

8. Lines 314, 316, 320- instead of numbers as 9 or 5 put words, Nine/nine or five.

9. Figure 4a; Authors provided below the gene name values of R, for example below LfTPS1 is R= 0.270. Which results are correlated? Describe it in Figure 4 description.

Standard errors are too high in some samples of RT-PCR analysis; for example H3 and L1 for LFTPS1, H3 and L1 for LFTPS3, H3 and L1 for LFTPS2,L3 for LFTPS5. In my opinion Authors should try to repeat or re-calculate this fragment of RT-PCR analysis. Calculate/re-calculate once more mean and standard errors, they should be roughly/generally  within the range of up to 5-15 % of mean.

Then draw a Figure 4a containing these repeated/re-calculated RT-PCR results, with smaller standard errors,  where the mean will be in the middle of error bar, and standard errors will be shown as higher and lower to mean. Now you show only the upper part of the standard error.

10. Line 389-390 and Fig 5a. Authors wrote that LFTPS4 and 5 have motifs (pink color) in C-terminal fragment. However, the Picture 5 a shows that it is in the N-terminal part. Correct the text and Fig 5a.

11. Line 393; do not capitalize Formosana.

 

Supplementary material:

12. Suppl fig 1; put space after Pct (axis 0Y and 0X)

13. Suppl table 1; mark names of genes using LfTPS1, LfTPS2 etc.

14. Suppl table 5

Authors wrote in material and methods, that RT-PCR exp were performed in triplicate from three biological replicates, which means 3x3=9 total repetitions for H1,H2,H3, L1, L2 and L3 for each analysed LfTPS1-5 genes. However, in Suppl table 5 is shown only 3  replicates (biological or technical). Authors should name this table (Suppl 5) as providing sample, representative RT-PCR data, remove it or add lacking replicates to complete it. Try to use such completed/re-calculated RT-PCR data to provide mean and much smaller errors as stated in comments to Fig 4a (point nr 9). Remove signs after delta CT in column E of Suppl table 5.

15. Suppl table S6; In column B put not A or B pinene but α- or β-pinene.

Author Response

Response to reviewer 2

1. 89, 92, 96, 149, 419; correct the spacing

Response: Thanks for the comment. As suggested, we have corrected the spacing.

2. Line 62 use shortened plant name  formosana

Response: Thanks for the comment. As suggested, We have revised.

3. Line 65  formosana write in italics

Response: Thanks for the comment.  As suggested, we have corrected the font.

4. Line 168- before word “Total” put dot, not comma.

Response: Thanks for the comment. As suggested, we have corrected the punctuation mark.

5. Line 224- instead of 3 Authors should write “Three”

Response: Thanks for the comment. As suggested, we have revised the figure.

6. Lines 229-230; Authors could build a subsection nr 2.8 entitled for example  “Graphical analysis of obtained results”. Then change sentences in lines 229-230, for example like this: “Microsoft Excel (2013) was used to draw pictures. Volcano map, heat map, histogram and bubble map were prepared using R (2009-2020), provided by RStudio (PBC, v 230 4.0.3 )”.

Response: Thanks for the comment. As suggested, We have revised.

7. Lines 240/241; Following fragment : (LF6, H1; LF12, H2; LF11, H3) and L groups (LF2, L1; LF7, L2; LF5, L3), exchange as follows: (LF6 →H1; LF12 → H2; LF11 → H3) and L groups (LF2 → L1; LF7 → L2; LF5 → L3). Now Readers will quickly and intuitively understand the origin of H1, H2, H3, L1, L2, L3.

Response: Thanks for the comment. As suggested, we have corrected these punctuation marks.

8. Lines 314, 316, 320- instead of numbers as 9 or 5 put words, Nine/nine or five.

Response: Thanks for the comment. As suggested, we have corrected these figures.

9. Figure 4a; Authors provided below the gene name values of R, for example below LfTPS1is R= 0.270. Which results are correlated? Describe it in Figure 4 description.

Standard errors are too high in some samples of RT-PCR analysis; for example H3 and L1 for LFTPS1, H3 and L1 for LFTPS3, H3 and L1 for LFTPS2,L3 for LFTPS5. In my opinion Authors should try to repeat or re-calculate this fragment of RT-PCR analysis. Calculate/re-calculate once more mean and standard errors, they should be roughly/generally  within the range of up to 5-15 % of mean.

Then draw a Figure 4a containing these repeated/re-calculated RT-PCR results, with smaller standard errors,  where the mean will be in the middle of error bar, and standard errors will be shown as higher and lower to mean. Now you show only the upper part of the standard error.

Response: Thanks for the comment. As suggested, we have re-calculated RT-PCR results and corrected Figure 4a.

9. Line 389-390 and Fig 5a. Authors wrote that LFTPS4 and 5 have motifs (pink color) in C-terminal fragment. However, the Picture 5 a shows that it is in the N-terminal part. Correct the text and Fig 5a.

Response: Thanks for the comment. As suggested, we have revised the text.

11. Line 393; do not capitalize Formosana.

Response: Thanks for the comment. As suggested, we have revised.

Supplementary material:

Suppl fig 1; put space after Pct (axis 0Y and 0X)

Response: Thanks for the relative study. As suggested, we have revised.(Suppl fig 1)

12. Suppl table 1; mark names of genes using LfTPS1, LfTPS2

Response: Thanks for the comment. As suggested, we have marked names of genes using LfTPS1, LfTPS2 etc, and revised the form.(Suppl table 1)

14. Suppl table 5

Authors wrote in material and methods, that RT-PCR exp were performed in triplicate from three biological replicates, which means 3x3=9 total repetitions for H1,H2,H3, L1, L2 and L3 for each analysed LfTPS1-5 genes. However, in Suppl table 5 is shown only 3  replicates (biological or technical). Authors should name this table (Suppl 5) as providing sample, representative RT-PCR data, remove it or add lacking replicates to complete it. Try to use such completed/re-calculated RT-PCR data to provide mean and much smaller errors as stated in comments to Fig 4a (point nr 9). Remove signs after delta CT in column E of Suppl table 5.

Response: Thanks for the comment. As suggested, we have revised the content and the name of the form.(Suppl table 5)

13. Suppl table S6; In column B put not A or B pinene but α- or β-pinene.

Response: Thanks for the comment. As suggested, we have revised.(Suppl table S6)

 

 

Author Response File: Author Response.pdf