Alternative First Exons Drive Enzymatic Activity Variation in Chalcone Synthase 3 of Dendrobium sinense

Round 1

Reviewer 1 Report

This manuscript describes the functional characterization of chalcone synthase gene in Dendrobium sinense. There are several problems should be addressed.

- The authors stated that pET28a vector was used to express DsCHS3 proteins containing a C-terminal HisTag in E. coli. But pET28a vector is not proper to express the C-terminal HisTag fusion protein. The authors also said that DsCHS3 genes were inserted between XbaI and ScI sites of pET28a vector. However, the XbaI site is before "rbs" in pET28a vector, which seems that the inserted genes are not properly expressed. So, the authors should carefully check the vector used and cloning strategy for E. coli expression of DsCHSs in this experiments.

- The resolution of all figures are poor.

- In page 3, line 135-142, there are several "HisTig"s. They should be "HisTag".

Author Response

Dear reviewers:

Thank you for the comments concerning our manuscript. The main corrections in the paper and the responds to the reviewer’s comments are as following:

- The authors stated that pET28a vector was used to express DsCHS3 proteins containing a C-terminal HisTag in E. coli. But pET28a vector is not proper to express the C-terminal HisTag fusion protein. The authors also said that DsCHS3 genes were inserted between XbaI and ScI sites of pET28a vector. However, the XbaI site is before "rbs" in pET28a vector, which seems that the inserted genes are not properly expressed. So, the authors should carefully check the vector used and cloning strategy for E. coli expression of DsCHSs in this experiments.

Response: Thank you for your detailed feedback and concerns regarding the expression of DsCHS3 proteins. The choice of adding a C-terminal HisTag was based on the previous study of BBS gene from the same type III PKS family, where C-terminal HisTag fusion proteins were successfully expressed.  (Chen et al., 2022). This approach was adopted in this study, and indeed, we were able to achieve successful expression of the fusion protein.

When we got these two CHS3 genes, we found that there were similar RBS-like sites in front of the start codon, as illustrated in following figure. Thus, we conducted a preliminary experiment and the protein was expressed normally, indicating that the similar RBS-like sites in 5’ UTR of CHS3 genes have similar functions with "rbs" in pET28a vector. Compared with CHS3-2 gene, the CHS3-1 gene has a more similar RBS-like site with pET28a vector. This may be the reason why CHS3-1 protein was more expressed compared to CHS3-2 (Figure 3). To ensure the integrity of the desired protein and avoid potential uncertainties from additional amino acid residues, we made the decision to use the XbaI and SacI sites for insertion.

Chen, Y.; Wang, Y.; Liang, C.; Liu, L.; Song, X.; Zhao, Y.; Wang, J.; Niu, J. Characterization of the Key Bibenzyl Synthase in Dendrobium sinense. Int. J. Mol. Sci. 2022, 23, 6780, doi:https://doi.org/10.3390/ijms23126780

 

- The resolution of all figures are poor.

Response: Thank you for your feedback. We have addressed the issue by enhancing the resolution of all figures in the paper, as per your recommendation.

 

- In page 3, line 135-142, there are several "HisTig"s. They should be "HisTag".

Response: Thank you for pointing that out. We have revised the text as per your suggestion. The occurrences of "HisTig" on page 3, lines 135-142 have been corrected to "HisTag". We appreciate your review and guidance.

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.

We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval.

Yours sincerely,

Niu Jun

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Alternative first exons in chalcone synthase gene affects its activity for flavonoid biosynthesis in Dendrobium sinense” focused on the identification of type III polyketide synthase (PKS) that catalyzes the formation of key intermediates in secondary metabolites. In my opinion interesting research has been undertaken with novel findings on type III polyketide synthase (PKS). which adds to the existing body of knowledge in the field. This originality is particularly evident in [specific section or experiment], where the authors introduce a new methodology or propose a novel interpretation of the data.  Therefore, there are some important questions which need to be addressed:

1- The title could be revised as it comprising all performed analyses.

2- However the manuscript is well-written in some parts but it could benefit from some moderate grammatical and linguistic revisions.

3- Introduction is somewhat short and it can be furnished with more information on the studied species and even the flavonoid biosynthesis pathway. You can show the pathways as figures in manuscript.

4- Please provide all exploited information in material and methods even default parameters of used software. This could include Gene ID of used sequence, gap penalty and gap extension used, exact version of software (in case of on-line tools mention access date) and other cases. Moreover please determine all the sequence used for phylogeny analysis, belong to which taxa?

5- Why you didn’t analyze flavonoid compounds via HPLC technique to trace change in the content of different flavonoid compounds instead of total flavonoid content?

6- The quality of the provided dendrogram and figures is very poor. Pleas use high-quality figures.

7- What is the role and place of drought stress in the present manuscript? There is no information regarding drought stress treatment in the Material and Methods.

 

 

Moderate ploishing is required.

Author Response

Dear reviewers:

Thank you for the comments concerning our manuscript. The main corrections in the paper and the responds to the reviewer’s comments are as following:

1- The title could be revised as it comprising all performed analyses.

Response：Thank you for your suggestion regarding the title. We have revised it to encompass all the analyses conducted in the paper. Title is as follows “Alternative First Exons Drive Enzymatic Activity Variation in Chalcone Synthase 3 of Dendrobium sinense”.

2- However the manuscript is well-written in some parts but it could benefit from some moderate grammatical and linguistic revisions.

Response：Thank you for your feedback. We have made the necessary grammatical and linguistic revisions.

3- Introduction is somewhat short and it can be furnished with more information on the studied species and even the flavonoid biosynthesis pathway. You can show the pathways as figures in manuscript.

Response：Thank you for your valuable feedback. We have extended the Introduction section to include more details about the Dendrobium sinense (line 43-49) and the flavonoid biosynthesis pathway (line 86-93). Additionally, we have incorporated visual representations of the flavonoid biosyntheisis pathwayas within the manuscript (Figure 1).

4- Please provide all exploited information in material and methods even default parameters of used software. This could include Gene ID of used sequence, gap penalty and gap extension used, exact version of software (in case of on-line tools mention access date) and other cases. Moreover please determine all the sequence used for phylogeny analysis, belong to which taxa?

Response：Thank you for your insightful input. We have revised the Material and Methods section to include comprehensive details, encompassing all utilized information, default parameters of employed software, and specific data, such as Gene IDs and gap penalties. The exact versions of software tools have been specified, along with the respective access dates for online tools. Furthermore, we have explicitly identified all sequences employed in the phylogeny analysis.

 5- Why you didn’t analyze flavonoid compounds via HPLC technique to trace change in the content of different flavonoid compounds instead of total flavonoid content?

Response：We appreciate your question. In our study, we focused on the analysis of total flavonoid content primarily due to the catalytic nature of CHS, which rapidly converts its product, chalcone, into various other flavonoid compounds. Consequently, CHS activity directly influences the total flavonoid content. While analyzing specific flavonoid components would indeed provide a more comprehensive understanding of changes pre- and post-transgenically, we believe that the analysis of total flavonoid content effectively reflects the enzymatic differences between the two splice variants under investigation. We acknowledge the importance of examining individual flavonoid compounds and plan to conduct such analyses in subsequent experiments.

6- The quality of the provided dendrogram and figures is very poor. Pleas use high-quality figures.

Response：Thank you for your feedback. We have replaced the existing figures with high-quality versions, addressing the issue of poor image quality.

7- What is the role and place of drought stress in the present manuscript? There is no information regarding drought stress treatment in the Material and Methods.

Response：Thank you for your question. The main focus of this party is to determine the predominant forms of the two splice variants, rather than investigating the response to drought stress. The inclusion of drought stress data is primarily due to the availability of third-generation sequencing results only under drought conditions. However, we understand the need for clarity and have added details about the drought stress treatment in the Experimental Methods section to address this concern (line 119-121).

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.

We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval.

Yours sincerely,

Niu Jun