Review Reports - Phenotypic and RNA-seq Profiles Identified Key Pathways and Genes Involved in Gleditsioside Biosynthesis in <i>Gleditsia sinensis</i> Lam.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study focuses on the changes in gleditsioside content, pod length and transcriptome analyses of Gleditsia sinensis, a widely used traditional herbal 42 medicine in China for treating various diseases, on six stages of pods from June to November. The researchers found the essential genes for the formation of gleditsioside in the pods of G. sinensis that can be useful for the breeding of some varieties with high-yield gleditsioside.

Overall, the research and experiments are clearly described and the analyses are complete and interesting. However, there are important flaws in the manuscript.

- The manuscript has major grammar and punctuation problems that need to be checked. In line 45, Velutina should be replaced with velutina. In line 170, “Bioinformatics Analysis of Key Genes Involved in Gleditsioside Biosynthesis in G. sinensis” should be replaced with “Bioinformatics analysis of Kkey genes involved in gleditsioside biosynthesis in G. sinensis”. In addition, in line 183, “Functional Validation of Three Genes via Transient Transformation in G. sinensis Pods” replaced with “Functional validation of three genes via transient transformation in G. sinensis pods”. The same in line 414.

-In line 161, four genes were mentioned to have been randomly selected for RT-qPCR, however, only three genes were named.

-It is unclear how many replications are taken for phenotypic and transcriptomic analyses.

- The scientific names of species written in complete form for the first time and for the rest, write the abbreviation of the genus. Therefore, in line 354, Panax ginseng should be replaced with P. ginseng, in line 356, Panax notoginseng with P. notoginseng. In line 420, Arabidopsis thaliana with A. thaliana. In line Populus trichocarpa with P. trichocarpa.

-In conclusion more explanation about the usefulness of these results for the breeding of G. sinensis high-yield gleditsioside is required.

Comments on the Quality of English Language

The manuscript has major grammar and punctuation problems that need to be checked.

Author Response

This study focuses on the changes in gleditsioside content, pod length and transcriptome analyses of Gleditsia sinensis, a widely used traditional herbal medicine in China for treating various diseases, on six stages of pods from June to November. The researchers found the essential genes for the formation of gleditsioside in the pods of G. sinensis that can be useful for the breeding of some varieties with high-yield gleditsioside.

Overall, the research and experiments are clearly described and the analyses are complete and interesting. However, there are important flaws in the manuscript.

Response: Thank you very much for your high evaluation of our work. According to your constructive suggestions below, we have modified the relevant parts in the revised version.

- The manuscript has major grammar and punctuation problems that need to be checked. In line 45, Velutina should be replaced with velutina. In line 170, “Bioinformatics Analysis of Key Genes Involved in Gleditsioside Biosynthesis in G. sinensis” should be replaced with “Bioinformatics analysis of key genes involved in gleditsioside biosynthesis in G. sinensis”. In addition, in line 183, “Functional Validation of Three Genes via Transient Transformation in G. sinensis Pods” replaced with “Functional validation of three genes via transient transformation in G. sinensis pods”. The same in line 414.

Response 1: Thanks for your kind comments. We have corrected these problems in the manuscript. Additionally, we have conducted a comprehensive check throughout the manuscript to ensure the accuracy of grammar.

-In line 161, four genes were mentioned to have been randomly selected for RT-qPCR, however, only three genes were named.

Response 2: According to your careful review, we have corrected it in line 161. The fourth gene is CL5936.Contig3 (MYB), which has been inserted into the corresponding sentence.

-It is unclear how many replications are taken for phenotypic and transcriptomic analyses.

Response 3: Thanks for your constructive comments. We have revised the manuscript to show the repetitions in line 111-114 in the methods section.

- The scientific names of species written in complete form for the first time and for the rest, write the abbreviation of the genus. Therefore, in line 354, Panax ginseng should be replaced with P. ginseng, in line 356, Panax notoginseng with P. notoginseng. In line 420, Arabidopsis thaliana with A. thaliana. In line Populus trichocarpa with P. trichocarpa.

Response 4: According to your careful comments, we have corrected the form of the scientific names of species. In line 354, Panax ginseng has been replaced with P. ginseng, in line 356, Panax notoginseng has been with P. notoginseng. In line 420, Arabidopsis thaliana has been with A. thaliana, and Populus trichocarpa has been with P. trichocarpa.

-In conclusion more explanation about the usefulness of these results for the breeding of G. sinensis high-yield gleditsioside is required.

Response 5: Thanks for your constructive comments. We have added the explanation in “in conclusion” section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The present work is devoted to the study of key pathways and genes involved in gleditsioside biosynthesis in Gleditsia sinensis plants. The topic is interesting and its investigation is complex where many aspects are considered. However, in my opinion, the study is too detailed and there is too much information. I recommend consider this MS for publication after some minor revisions.

1. There are incorrect references to the Figures and Tables in the text. I encourage the authors to check this.

For example,

L. 207-208. Incorrect reference to Table 1.

L. 382. Table S9?

2. L. 192-199. It looks like it's a manual. Why is it here?

3. Maybe it would be better if the abbreviations (GS1. June, GS2. July, GS3. August, GS4. September, GS5. October, GS6. November) will be given in the text.

4. It seems that there are more references to the Supplementary material in the text than to the main illustrations.

5. Materials and methods.

No information on statistical analysis is provided.

Comments on the Quality of English Language

The English should be checked by expert proofreader.

Author Response

Response: We are very grateful for your expert comments on our manuscript. According to your suggestions, we have modified the relevant parts in the revised version.

There are incorrect references to the Figures and Tables in the text. I encourage the authors to check this.

For example,

207-208. Incorrect reference to Table 1.
382. Table S9?

Response 1: Thank you for your careful suggestions, and we have corrected the incorrect parts. In line 211, we have replaced Table 1 with Figure 1B. In line 385 and in the supplementary materials, we have deleted Table S9.

L. 192-199. It looks like it's a manual. Why is it here?

Response 2: Thanks for your careful comments, and we have corrected the paragraph in lines 195-196.

Maybe it would be better if the abbreviations (GS1. June, GS2. July, GS3. August, GS4. September, GS5. October, GS6. November) will be given in the text.

Response 3: We have added the abbreviations (GS1. June, GS2. July, GS3. August, GS4. September, GS5. October, GS6. November) in the text.

It seems that there are more references to the Supplementary material in the text than to the main illustrations.

Response 4: Thank you for pointing out this problem. Since there are redundancies of Table S8 with Figure 5, Table S9 with Table 1, we have removed Table S8 and Table S9 along with the references to them in the text.

Materials and methods.

No information on statistical analysis is provided.

Response 5: According to your expert comments, we have added“2.10 Statistical analysis”in Materials and methods.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

• L51. There is not enough information about gleditsioside. Why were they measured in pods? In what parts is it synthesized and accumulated?
• L112. A completely incomprehensible method. It is necessary to describe in detail and provide a reference. It is also necessary to justify why HPLC was not used. What was used as a standard and why. Manufacturer of the standard. In this form, data on gleditsioside content are not of interest and raise more questions.
What was the instrument for qPCR?
L404. The chapter doesn't give any information. Have the sequences been verified and deposited in the genebank? Why is there no statistical analysis? Why doesn't the chapter describe what genes are being studied and why? How many biological replicates were used? It's better to delete the chapter
L431. This chapter is not credible. Chemical analysis does not confirm gleditsioside, transient expression is not suitable for these purposes, expression does not confirm gene transfer. It's better to delete the chapter

Best regards

Comments on the Quality of English Language

language and professional editing required

Author Response

L51. There is not enough information about gleditsioside. Why were they measured in pods? In what parts is it synthesized and accumulated?

Response 1: Thanks for your expert comments. In introduction section, we have explained the reason why we measured saponins in the pods and added information about the synthesis and accumulation parts of gleditsioside mainly in the pods of G. sinensis.

L112. A completely incomprehensible method. It is necessary to describe in detail and provide a reference. It is also necessary to justify why HPLC was not used. What was used as a standard and why. Manufacturer of the standard. In this form, data on gleditsioside content are not of interest and raise more questions.

What was the instrument for qPCR?

Response 2: Thank you very much for your expert suggestions. We have detailed the methods, information about the standards, and their manufacturers for measuring gleditsioside content in Section “2.2. Gleditsioside extraction and quantification” of the Materials and Methods. In this investigation, we mainly focused on the change trends of total gleditsioside and the total diffenrentially expressed gene profiles. According to your construcive suggestions, we will further study the pattern of specific gleditsioside combined with HPLC and the corresponding gene regulation in our further research. Additionally, we have added details about the instrument used for qPCR.

L404. The chapter doesn't give any information. Have the sequences been verified and deposited in the genebank? Why is there no statistical analysis? Why doesn't the chapter describe what genes are being studied and why? How many biological replicates were used? It's better to delete the chapter

Response 3: A lot of thanks for your comments. We have verified and deposited the sequences in the National Genomics Data Center.

, and we have added the statistical analysis. These differentially expressed genes were randomly selected to verify the accuracy of the RNA-seq results. The experiment was conducted with three biological replicates, and we have added it to the figure legends of Figure 7.

L431. This chapter is not credible. Chemical analysis does not confirm gleditsioside, transient expression is not suitable for these purposes, expression does not confirm gene transfer. It's better to delete the chapter

Best regards

Response 4: According to your expert comments, We have addressed your concerns by including three additional methodological references in section 2.9 of the Methods. These references provide further evidence and support for the experimental approaches described in our study.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This is my first review of the manuscript entitled "Phenotypic and RNA-seq profiles identified key pathways and genes involved in gleditsioside biosynthesis in Gleditsia sinensis Lam." by Wang et al.

The manuscript is focused on studying the gleditsioside byosynthesis pathways in Gleditsia on the transcriptome level. The topic is applied and have future importance for designing optimised biotechnology and/or agriculture protocols to increase gleditsioside yield. The methodology of the study is mostly clearly described, as well as the results and the short discussion. The language of the manuscript allows for smooth reading and does not limit the understanding of the contents. Some points, however, should be addressed by the authors to facilitate the readability of the manuscript and correct some inconsistencies in terminology. These points for improvement are provided below.

In Materials and Methods section 2.2, authors refer to a chromogenic reaction (L116) used to determine gleditsioside level, but do not provide any reference or protocol details. The manuscript must be supplemented with this information.

Below the formula, the context suggests that variable 'M' corresponds to the sample mass of Gleditsia sinensis, but not the 'powder' substance as the authors specify.

In addition, the contents of the paragraph on lines 192–199 looks like copy-pasted snippet from the protocol or instruction, and most probably corresponds to the previous question. If that is right, this fragment should be rewritten and incorporated into section 2.2 of M&Ms.

Figures

Figures provided in the manuscript and supplementary materials are relevant, however, many of them should be subjected to editing such as increase of text size, readability enhancement. For instance:

Fig. 1, L219: the 'A' and 'B' parts of the image panel have radically different text size. Text labels of the left part (A) must be enlarged 1.5–2 times, whereas text size on the right part could be even slightly smaller. Also authors are welcome to increase the thickness of scale bars and to remove the text labels above them, given that the size of the scale bar is provided in the figure caption.

Fig. 2, L242: Labels above the bars in 'B' subfigure are barely visible. Please mind to increase them two times. The same applies to fig. 2C, and to the key legend of fig. 2D.

Figure caption would benefit if included the list of databases.

Fig. 3, L312: Authors are welcome to increase the readability of text within the image, especially the lines to the right of heatmaps, and the labels next to color scale legend.

Fig. 4, L342: Labels on subfigure B should be increased. Figure caption for 4B must include information on what metric is depicted by circle size and what metric is visualized by color scale.

Fig. 6, L400: This figure seems to be the most problematic one. On the one hand, the information on this figure is the central to the results and the discussion in this manuscript, and only a small fraction of its information is enlisted in the text. On the other hand, the information here is the least readable among all the figures in the mansucript. Figure 6B, as well as 6C, contains text in such a small size and low raster quality, so that it is almost impossible to read. Authors must increase the size of the interaction network graph, and its labels.

Figure caption should include the information on what frequencies are depicted by circle size and color scale in Fig. 6B.

Fig. 8, L426: The reviewer suggests to be more precise in figure caption and indicate that at least fig. 8B shows the comparion of conserved motifs in protein products of key gene families.

Tables

Table 1 caption refers to the 'Absorbance' of extract, however, the neither row of the table provides absorbance. The one shows 'value' expressed in mL, to reviewer's surprise, and the second one shows the percentage of gleditsioside.

Table 2 caption consists of strange asterisk indication. Authors specify that double asterisk corresponds to a significant difference (q<0.05), while single asterisk 'represents extremely significant difference, q<0.1'. Please check it again and provide a commentary, as usually lower value of the statistical metri, such as p-value or q-value, corresponds to a higher significance of the test result.

L66: Authors' phrase 'algae plants mainly contain...' seems to be incorrect if we consider the cited references in that context (ref. No. 15). Referenced paper dealed with various microalgae, and the majority of them belong to different clades, other than Chlorophyta.

L72: chlorophyll, tocopherols and plastoquinones are mistakenly listed as phytohormones. The only phytohormones in the list are cytokinins and gibberellins.

L261: authors' statement that 'Most DEGs had enzymatic catalytic activity' is incorrect. Differentially expressed genes in this study encode proteins, which, in turn, could exhibit various enzymatic activities. Authors should correct this and look through the text for similar incosistencies.

L462: Authors should check the terminology and comment whether they discuss gene expression or protein level / enzyme activity.

L507: Authors should check the spelling of 'UDP glossyltransfer' (see L28 of Abstract).

Comments on the Quality of English Language

The language is generally fine, however, some sentences require authors' attention to reconsider punctuation, spell check or other minor issues. These are enumerated in the following:

L24: 'positive increase' looks strange: there could be either 'positive change' or 'increase'.

L27–28: the sentence lacks verb.

L30: It is not clear what do authors mean when say '...pathway was extended to ten pathways...'.

L42: Please use systematically accepted name for Fabaceae family. Refer to the L236 of the manuscript.

L46–47: what does 'economic tree species' mean?

L53: 'Triterpenoid saponin' should be put in plural form in the beginning of the sentence.

L72: The sentence lacks the closing bracket, therefore chlorophyll, tocopherols and plastoquinones are mistakenly listed as phytohormones. The only phytohormones in the list are cytokinins and gibberellins.

L135–136: the sentence is written in the inappropriate tense, should be in Past Tense.

L241: this sentence seem to lack verb.

L372: First word should be spelled as 'Differentially'.

L462–464: Authors should check punctuation and the structure of the sentence.

L507: Authors should check the spelling of 'UDP glossyltransfer' (see L28 of Abstract).

Author Response

Response: Thank you very much for your high evaluation of our work. According to your kind suggestions, we have modified the relevant parts in the revised version.

Response 1: According to your careful comments, we have added the method for determining gleditsioside level in Section 2.2 of the Materials and Methods section.

Below the formula, the context suggests that variable 'M' corresponds to the sample mass of Gleditsia sinensis, but not the 'powder' substance as the authors specify.

Response 2: We have made modifications according to your constructive suggestions. Additionally, the fragment has been revised accordingly in lines 192-199 and has been added into section 2.2 of M&Ms.

Figures

Figures provided in the manuscript and supplementary materials are relevant, however, many of them should be subjected to editing such as increase of text size, readability enhancement. For instance:

Response 3: Thank you very much for your detailed comments. We have corrected the labels, fonts, and scale bars in Figure 1 as kindly suggested.

Fig. 2, L242: Labels above the bars in 'B' subfigure are barely visible. Please mind to increase them two times. The same applies to fig. 2C, and to the key legend of fig. 2D.

Figure caption would benefit if included the list of databases.
Response 4: According to your kind suggestion, we have corrected the labels and legends in Figure 2.

Fig. 3, L312: Authors are welcome to increase the readability of text within the image, especially the lines to the right of heatmaps, and the labels next to color scale legend.

Response 5: According to your comments, we have enhanced the clarity of the figure, particularly the lines on the right side of heatmaps and the labels adjacent to the color scale legend.

Fig. 4, L342: Labels on subfigure B should be increased. Figure caption for 4B must include information on what metric is depicted by circle size and what metric is visualized by color scale.

Response 6: We have increased the labels on subfigure B. Additionally, we have corrected the figure legend to include explanations of the circle sizes and color scales.

Fig. 6, L400: This figure seems to be the most problematic one. On the one hand, the information on this figure is the central to the results and the discussion in this manuscript, and only a small fraction of its information is enlisted in the text. On the other hand, the information here is the least readable among all the figures in the mansucript. Figure 6B, as well as 6C, contains text in such a small size and low raster quality, so that it is almost impossible to read. Authors must increase the size of the interaction network graph, and its labels.

Figure caption should include the information on what frequencies are depicted by circle size and color scale in Fig. 6B.

Response 7: Thanks for your kind comments. In accordance with your suggestions, we have expanded the results and discussion sections of this study. Moreover, we have enhanced the resolution and readability of the figure as best as we could. Additionally, we have added information on what frequencies are depicted by circle size and color scale in Fig. 6B.

Fig. 8, L426: The reviewer suggests to be more precise in figure caption and indicate that at least fig. 8B shows the comparion of conserved motifs in protein products of key gene families.

Response 8: Based on your suggestions, we have corrected the figure caption in Figure 8 and supplemented the comparison of conserved motifs in key gene family protein products.

Tables

Response 9: Thank you for your careful comments and we have corrected the caption of Table 1.

Response 10: We have corrected the significance represented by the asterisks in Table 2 accordingly.

Response 11: Thank you for your expert comments We replaced the phrase "while algae plants mainly contain the MEP/DOXP pathway" with "In contrast, certain algae may possess a novel plastidic MEP/DOXP pathway, deviating from the conventional routes" and updated the reference 15.

L72: chlorophyll, tocopherols and plastoquinones are mistakenly listed as phytohormones. The only phytohormones in the list are cytokinins and gibberellins.

Response 12: According to your comments, we have corrected the manuscript ensuring that only cytokinins and gibberellins are listed as plant hormones.

Response 13: Thank you for your constructive comments. We have corrected the statement and thoroughly checked the manuscript.

L462: Authors should check the terminology and comment whether they discuss gene expression or protein level / enzyme activity.

Response 14: This is an expert comment. We have checked the terminology and comment throughout the manuscript.

L507: Authors should check the spelling of 'UDP glossyltransfer' (see L28 of Abstract).

Response 15: We have corrected “UDP glossyltransfer” to “UDP-glucosyltransferase.”.

Comments on the Quality of English Language

The language is generally fine, however, some sentences require authors' attention to reconsider punctuation, spell check or other minor issues. These are enumerated in the following:

L24: 'positive increase' looks strange: there could be either 'positive change' or 'increase'.

Response 16: Based on your suggestion, we have corrected 'positive increase' to 'increase'.

L27–28: the sentence lacks verb.

Response 17: We have corrected the sentence accordingly.

L30: It is not clear what do authors mean when say '...pathway was extended to ten pathways...'.

Response 18: Thank you for your valuable suggestions. We have revised the sentence.

L42: Please use systematically accepted name for Fabaceae family. Refer to the L236 of the manuscript.

Response 19: Thank you very much for your professional suggestions. We have carefully reviewed the classification terminology used in our manuscript. The current International Code of Nomenclature for algae, fungi, and plants (ICN) which recommends “Fabaceae.” We have corrected the term to “Fabaceae” in the manuscript.

L46–47: what does 'economic tree species' mean?

Response 20: We have corrected “economic tree species,” to “a tree species with economic value”.

L53: 'Triterpenoid saponin' should be put in plural form in the beginning of the sentence.

Response 21: We have revised “triterpenoid saponins” to 'Triterpenoid saponins' in the manuscript.

Response 22: Thank you for your expert comments. We have corrected the manuscript ensuring that only cytokinins and gibberellins are listed as plant hormones.

L135–136: the sentence is written in the inappropriate tense, should be in Past Tense.

L241: this sentence seem to lack verb.

L372: First word should be spelled as 'Differentially'.

Response 23: Thank you for your careful comments. We have corrected the errors in tense, words, and sentence components in the manuscript.

L462–464: Authors should check punctuation and the structure of the sentence.

Response 24: We have revised the sentence to improve clarity and readability.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

The authors have done a great job; the article has become much better. However, before publication it is necessary to pay attention to some shortcomings.

Table 1. What was the age of the leaves?
Fig 9. A, statistical analysis? B, t-test? Please denote in the capture.
Line 576: was? Check English through text,

Best regards

Comments on the Quality of English Language

See Comments and Suggestions for Authors

Author Response

The authors have done a great job; the article has become much better. However, before publication it is necessary to pay attention to some shortcomings.

Response: Thank you very much for your high evaluation on our work and the revision. According to your kind suggestions below, we have modified the relevant parts in the revised version.

Table 1. What was the age of the leaves?

Response 1: The age of leaves has added in title of Table 1 and in “2.1. Plant materials”

Fig 9. A, statistical analysis? B, t-test? Please denote in the capture.

Response 2: Thanks for your careful comments. We have carried out a one - way analysis of variance (ANOVA) on Fig 9.A and a t-test on Fig 9.B. Moreover, we have added the descriptions of the statistical analyses in the legend section of Figure 9.

Line 576: was? Check English through text.

Response 3: Thank you for your suggestions. We have corrected “was” to “were” in line 504, and have thoroughly checked and corrected the errors in tense, words and sentence components in the revised manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

This is my second review of the manuscript entitled "Phenotypic and RNA-seq profiles identified key pathways and genes involved in gleditsioside biosynthesis in Gleditsia sinensis Lam." by Wang et al.

Authors have taken a good effort to address all the reviwers' comments to improve the manuscript. It is worth noting that the text reads smoother since authors have addressed the suggestions on the quality of the language. Happy to see the upgraded graphics in figures as well.
One point nevertheless requires the authors' attention. They renamed June...November timing names to GSx and that made the terminology of the manuscript more consistent.
Despite that figure 1 caption was edited as well, labels on the figure 1B itself still contain month names instead of pod development stage labels (June...November are still exist in place of GS1...GS6).

Author Response

This is my second review of the manuscript entitled "Phenotypic and RNA-seq profiles identified key pathways and genes involved in gleditsioside biosynthesis in Gleditsia sinensis Lam." by Wang et al.

Response: Thank you very much for your high evaluation on our work and the revision. According to your constructive advice, we have modified the relevant parts in the revised version.

One point nevertheless requires the authors' attention. They renamed June...November timing names to GSx and that made the terminology of the manuscript more consistent.

Despite that figure 1 caption was edited as well, labels on the figure 1B itself still contain month names instead of pod development stage labels (June...November are still exist in place of GS1...GS6).

Response 1: According to your kind suggestion, we have replaced “June...November” with the abbreviations (GS1, GS2, GS3, GS4, GS5, GS6) in Figure 1.