Analysis of Transcriptome and Expression of C4H and FLS Genes on Four Flower Colors of Impatiens uliginosa

Round 1

Reviewer 1 Report

There are many typing and grammar errors in the manuscript.

Material and Method section: lack of important information (see the attached file). 

Consider adding a brief discussion after presenting the results for each parameter to provide some context and interpretation of the data.

Should mention the limitations of the study and suggest directions for future research in the discussion section.

The authors used the wrong citation style in the text.

Please check the attached file carefully to see the comments.

Comments for author File: Comments.pdf

Moderate editing of English language required

Author Response

We feel great thanks for your professional review workon our article. As you are concerned, there are severalproblems that need to be addressed. According to yournice suggestions, we have made extensive corrections toour previous draft, the detailed corrections are listed below.

• There are many typing and grammar errors in the manuscript.

----Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made thecorrections to make the word harmonized within the whole manuscript.

• Material and Method section: lack of important information (see the attached file).

----Your suggestion is very important to us and we have added important information in the "Materials and Methods" section.

如："good growing condition" is "robust and free of pests and diseases".

Scale has been added for Figure 1.

Three biological replicates were set up for each sample in qRT-PCR.

The full name of ddH2O is "doble distilled water".

• Consider adding a brief discussion after presenting the results for each parameter to provide some context and interpretation of the data.

----Based on this suggestion of yours, we have refined the descriptions in the manuscript to achieve a connection between the context and the data.

• Should mention the limitations of the study and suggest directions for future research in the discussion section.

----The limitation of this study is that only the transcriptome and genes were used to investigate
the coloration mechanism of the four flower colors of I. uliginosa. The group has already
investigated the coloration mechanism of the four flower colors from the physiological and
biochemical perspectives, but this study is the first to investigate the four flower colors of I.
uliginosa from the transcriptome, which can lay the foundation for more in-depth molecular
studies in the later stage of the study.
(5) The authors used the wrong citation style in the text.
----We have revised the citation and reference formatting in the manuscript in accordance with
journal formatting specifications.

Author Response File: Author Response.pdf

Reviewer 2 Report

horticulturae-2950663-review

Analysis of Transcriptome and Expression of C4H and FLS  genes on Four Flower Colors of Impatiens uliginosa  

Xiaoli Zhang, Yi Tan,  Xinyi Li,  Zengdong Liu,  Fan Li,  Haiquan Huang,  Meijuan Huang

The authors of this manuscript studied plants from natural populations. They collected Impatiens uliginosa flowers with different colors (white, pink, red, and dark red). The authors analyzed the transcriptomes of the four colors in I. uliginosa flowers using RNA-Seq technology.

The authors conducted a large and painstaking effort to study the transcriptomes in four colored flowers by analyzing 100,705 unigenes based on data from five publicly available databases NCBI-Nr, Swiss-Prot, KEGG, InterPro, and KOG.

The Gene Ontology gene pool contained three sections of gene attributes: molecular function, biological process, and cellular component, which amounted to 58 different functional domains of the annotated genes. According to the Clusters of Orthologous Protein Groups database, the annotated genes were categorized into 25 classes according to their functions. The authors predicted Unigenes encoding transcription factors, among them transcription factors essential for the plant flavonoid metabolic pathway. Flavonol synthase (FLS) is known to affect the accumulation of anthocyanin glycosides and the synthesis of flavonols. In addition, C4H is a member of the cytochrome P450 (Cytochrome P450, CYP450) family of cytochrome oxidases and belongs to the CYP73 subfamily. C4H directly affects the synthesis of flavonoid compounds, lignin, and aromatic compounds.

At the molecular level, the authors identified the mechanisms of coloration of four colors in flowers. They showed that the C4H genes positively regulate red coloration of I. uliginosa flowers, whereas the FLS gene regulates white coloration. The expression of the FLS gene was shown to be gradually attenuated from white to dark red in flowers, which allowed it to be classified as a negative regulator of color.

 This study has fundamental theoretical significance, since the results obtained by the authors expand the understanding of the mechanisms of regulation of I. uliginosa flower color.

The manuscript is well illustrated.

Remarks:

Page 6, line 202. The designation NR in Figure 4 should be in accordance with the designation in the caption to the figure Nr.

Page 9, line 262. Please clarify the caption to Figure 7 in accordance with the designations in the figure. The caption to the Figure 7 shows 4 letters (A, B, C, D), while in the Figure 7 itself there are only 3 (A, B, C).

Page 12, line 336. Please correct the text. This phrase is incorrect.

Page 13, line 390. The link does not contain the year of publication.

References. References should be updated. The cited literature references from 2019-2024 are 15% (6 out of 40).

Comments for author File: Comments.pdf

Author Response

We feel great thanks for your professional review workon our article. As you are concerned, there are severalproblems that need to be addressed. According to yournice suggestions, we have made extensive corrections toour previous draft, the detailed corrections are listed below.

• Page 6, line 202. The designation NR in Figure 4 should be in accordance with the designation in the caption to the figure Nr.

----We apologize for our carelessness! In response to your reminder, we have standardized all "Nr" to "NR" in the text.

• Page 9, line 262. Please clarify the caption to Figure 7 in accordance with the designations in the figure. The caption to the Figure 7 shows 4 letters (A, B, C, D), while in the Figure 7 itself there are only 3 (A, B, C).

----Figure 7 does contain only "A, B, C", and we have checked and removed the redundancy.

• Page 12, line 336. Please correct the text. This phrase is incorrect.

----We have changed“Flavonols are important auxins that affect petal coloration”to“Flavonols are one of the major co-pigments that enhance the stability of anthocyanin glycosides and influence flower color formation. In the metabolic pathway of flavonoid compounds, flavonols share a pre-synthesis pathway with anthocyanin glycoside biosynthesis”.

• Page 13, line 390. The link does not contain the year of publication.

----We have added the missing "year" in the references.

• References should be updated. The cited literature references from 2019-2024 are 15% (6 out of 40).

----We have updated the references to cite the period 2019-2024 where possible.

Author Response File: Author Response.pdf

Reviewer 3 Report

The question investigated by the authors is really very important both theoretically and for practice. It would be very important to know the mechanism of formation of a particular flower color.

The authors chose four colors of Impatiens uliginosa (white, pink, red and dark red). To elucidate the mechanism of color formation, the authors created transcriptomes to identify differentially regulated genes (DEGs) that would probably be responsible to some extent for the formation of a particular color of Impatiens uliginosa.

They were able to identify DEGs probably involved in anthocyanin biosynthesis and a number of trans-factors that are also probably important.

The standard approach used for the current stage of science is the creation of transcriptomes. Nowadays, most often all the work of creating and analyzing transcriptomes is done by scientific firms. These methods are so well practiced now that there are practically no errors in this part of the work. Despite the complexity and costliness of this approach, the result is data only on the comparative content of transcripts of different genes. To confirm the correctness of the transcriptome data, the transcript levels of the studied genes are determined by RT-qPCR, which the authors have done. The approach taken is the initial stage of the work, followed by work with specific genes and encoded proteins. Although the authors write in the abstract that "the mechanisms of coloration of the four colors have been uncovered at the molecular level," this is not true. No mechanisms have been uncovered by the authors, nor could they. They only showed the relative level of transcripts of a number of DEGs in the variants studied. No other information is available in the paper. Usually this approach goes together with other methodological techniques and in the complex really important information is obtained.

Was new information obtained? Yes, it is. It is useful, but it is only at the level of transcripts. In fact, the time of such articles has passed.

Many journals write in their rules for authors that articles that are based only on the transcript level will not be accepted. However, this depends on the policy of the particular journal. If the editor of a given journal feels that the article should be accepted, then I would not object. This is the journal's decision, however, if he feels that the manuscript should not be accepted, then I will also agree with him.

If the manuscript is accepted, I request that in the text fragment (lines 172-179) the length of the transcripts be expressed in nucleotides, not in nucleotide pairs, because RNA is a single-stranded molecule.

Thus, the solution is completely passed to the editor, taking into account the above comments.

Author Response

We feel great thanks for your professional review workon our article. As you are concerned, there are severalproblems that need to be addressed. According to yournice suggestions, we have made extensive corrections toour previous draft, the detailed corrections are listed below.

• Although the authors write in the abstract that "the mechanisms of coloration of the four colors have been uncovered at the molecular level," this is not true. No mechanisms have been uncovered by the authors, nor could they.

----It is difficult to fully explain the coloration mechanism of the four flower colors in I. uliginosa by transcriptome data alone, but this study can lay the foundation for the subsequent molecular coloration mechanism. We will conduct more experiments to investigate the coloration mechanism of the four flower colors in I. uliginosa.

If the manuscript is accepted, I request that in the text fragment (lines 172-179) the length of the transcripts be expressed in nucleotides, not in nucleotide pairs, because RNA is a single-stranded molecule.

----Thank you for this valuable comment. The transcriptome lengths described in the text are already nucleotides, not nucleotide pairs.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Accept in the present form.

Reviewer 3 Report

Dear Editor,

My review had a serious doubt about the possibility of accepting this manuscript for publication in the journal and two minor comments. However, it is up to you to decide on my serious objection, as it depends on the policy of your journal. Judging by the fact that the manuscript was re-sent to me without major revision, it means that you have made a favourable decision. In that case, I have no reason to disagree with your decision. My minor remarks have been corrected.

I wish you all the best