Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript with the title of “Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi 3 Shuanghui’” is a research article. The ‘Yinbi Shuanghui’ is a colored leaf variety of Osmanthus fragrans, with yellow and green parts on one leaf. The authors investigated the expression of PAO and leaf color-related genes in different parts of the leaves, and found that the PAO was significantly up-regulated in the yellow part of the colored leaves. The PAO gene from ‘Yinbi 3 Shuanghui’” was transiently and stably transformed into tobacco. In the tobacco transiently transformed, the genes involved in chlorophyll degradation were up-regulated and the genes related to chlorophyll synthesis were down-regulated. For stable transgenic lines, overexpression of PAO gene promotes the degradation of chlorophyll in tobacco. The promoter sequence of the PAO gene was analyzed and the methylation level of CHH in the promoter was found lower in the yellow part of the colored leaves. PAO appeared to be involved in chlorophyl degradation. The findings are interesting. However, the English of the manuscript is really bad. There are many problems in the manuscript writing. Long sentences are often used. The meanings of many sentences are not clear and with grammatical errors. The data are not fully explained. Title of some figure is missing. Figure qualities need to be improved. Based on current quality, the manuscript cannot be accepted before it is thoroughly revised.

1. For the Abstract, the last 2nd sentence (line 25-35) is too long. 
2. In the 2nd paragraph of the Introduction section, the sentence “Darkness-induced senescence of isolated leaves of Arabidopsis thaliana revealed up-regulation of PAO expression, protein level expression, and it is believed that PAO regulates chlorophyll degradation mainly through transcriptional level [6].” (line 67-69) is not accurate. PAO is an enzyme catalyzing chlorophyll degradation, how can it regulate chlorophyll degradation through transcriptional level? 
3. In the Introduction section, “eas1” in line 70 and 71 should be written in italic type. 
4. In the first paragraph of Materials and Methods section, is the phrase “in the feld at Liyangg” (line 93) correct? 
5. In the Materials and Methods section 2.5, the 2nd sentence (line 148-152) is too long. It needs to be revised to make it easier to be understood. 
6. In the Materials and Methods section 2.8, the last sentence (line 174-177) needs to be revised to make its meaning clearer. 
7. In the Results section 3.1, the sentence “Results suggested that(Fig.1B), the OfCRD1 ……, which might be related to a decrease in chlorophyll synthesis in the leaves.” (line 183-189) is too long and with grammatic error. It needs to be revised and make the meaning clearer. 
8. For the Results section 3.1, in the first paragraph, the author analyzed the expression levels of 24 leaf color related genes, while the data was not fully explained, only mentioning the expression of 2 genes except OfPAO.  
9. For Figure 1, “A” and “B” in the figure should be cited in the figure legend at appropriate position.  
10. In the Results section 3.2, the sentence “The cloned OfPAO sequence was translated into an amino acid sequence by DNAMAN, ……, with a CDS sequence similarity of 80.33% between them.” is too long. It needs to be revised. 
11. For Figure 2, the title of the figure legend is missing. Meanwhile, Fig. 2B is small with low definition. 
12.  For the 2nd paragraph of the Results section 3.2, the meaning of the first sentence “Compared the homology between the amino acid sequence encoded by the OfPAO 213 and the amino acid sequence encoded by PAO in other plants (Fig.3).” (line 213-214) is not clear.
    
13. For figure 4, the phylogenetic analysis is performed using PAO from different species, it is not correct to use the title "Phylogenetic tree of OfPAO". "OfPAO" is suggested to be changed to "PAO".
14. In the 1st paragraph of the Results section 3.3, the meaning of the sentence “The expression level of transient expression tobacoo was detected (Fig. 5C), and the results showed that the expression level of OT was significantly higher than that in WT.” (line 239-241) is not clear. It needs to be revised. What is the meaning of “OT” in this sentence? The results shown in figure 5 should be explained, in the text, in the order of Fig.5A to 5D. 
15. For figure 5A, what are the samples in the gel other than P1 to P6? If the samples are not related to the manuscript, they should not be included in the figure 5A.  
16. For the figure 5, the figure legend (D) “Regulate the relative expression of chlorophyll pathway genes in transgenic tobacco.” (line 255-256) should be revised to make its meaning clearer. 
17. For Figure 7, the Fig. 7B is small with low definition. It needs to be enlarged or re-organized to make it clearer. 
18. In the 2nd paragraph of the Result section 3.7, for the last 2nd sentence “Combining the CHH methylation level …… showed that the promoter of OfPAO in Osmanthus fragrans ‘Yinbi Shuanghui’ was significantly lower than that in green leaves.” (line 334-337), is the “promoter” herein referred to the methylation of the promoter? It is shown in Figure 9B that the methylation levels of four CHH sites (but one at 145 bp position) were higher in the yellow parts of the colored leaves than those in green leaves. How did the authors conclude that “the promoter of OfPAO in Osmanthus fragrans ‘Yinbi Shuanghui’ was significantly lower than that in green leaves.” (i.e., the last 2nd sentence)? 
19. For the Figure 9, the phrase “CG: Green parts of colored leaves; CY: Yellow parts of colored leaves; G: Green leaves” (line 342) in the figure legend should be deleted because of duplication.  
20. For the legend of Fig.9C, Is it the relative expression of OfPAO gene or promoter? 
21. In the discussion section 4.1, the meaning of the first sentence (line 350-352) is not clear. It needs to be revised to make its meaning clearer.
    

 

Comments on the Quality of English Language

 

The English of the manuscript is really bad and needs to be thoroughly improved.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1.      The aim of the work is to study the genetic mechanism of leaf color formation in O.fragrans  'Yinbi Shuanghui, focusing on the OfPAO gene involvement. The potential regulatory role of OfPAO in Tobacco was also examined.

2.      Currently, studies on the relationship between the expression of PAO, a key gene for chlorophyll degradation, and DNA methylation have not been sufficiently elucidated. The study is addressed to cover this gap. However, a critical discussion of the work and comparisons to existing research works in the field are absent. Authors are invited to underline what improvements their work provide.

3.      The work is described in detail and the adopted methodological approach is appropriate.

4.      The conclusions are consistent with the results and the topic treated.

5.      The references are appropriate

6.      Tables and figures are sufficiently clear.

However, some major revisions are necessary.

In fact:

·        the abstract is very dispersive and unorganized. Please focus the abstract on the main objectives, methods and results of the work

·        in the introduction it not clearly specified what previously works f the author research group have developed and what intend to use the achieved results. Overall, the introduction contents are not well organized.

·        Furthermore, it is absolutely necessary to improve the form of many sentences in order to make them clearer.

Finally, the manuscript format is not coherent with the one required by the journal. The abstract is more than 200 words and the reference format is wrong. Please edit them. Also, please, refer to the author’s instructions available at

https://www.mdpi.com/journal/horticulturae/instructions

Comments on the Quality of English Language

 Extensive editing of English language required

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

Authors analysed the functions of OfPAO gene of Osmanthus fragrans ‘Yinbi 3 Shuanghui’ in chlorophyll catabolism and leaf coloration changes. Differences in gene methylation status are inversely correlated with the  OfPAO promoter activity and gene expression.

Study is interesting to persons worki in the particular field. Results are important and significant. Obtained results support conclusions. Study is well planned and performed. Figures are of good quality. Following comments sholud be addressed before publication:

1. Correct typographical errors (lack of space, double space etc) in lines: 30,37, 49, 63, 105, 120, 184, 216-220, 244, 245, 314, 315, 351, 352. Check the entire text.

2. Rewrite sentences in lines:  93-97, 99-103, 244-245, 259-260, 363-365, title for table S2. Check the entire text for English language mistakes.

3. MgCl2, line 119- should be MgCl₂

4. Agrobacterium rhizogenes -line 120-write in italice. Check the entire text.

5. Line 103- instead of extracted use obtained or prepared

6. Explain meaning  „XL10-Gold sensory state” in line 166

7. Section 2.7 Provide details of PCR reaction, name of applied DNA polymerase

8. Section 2.6 provide the amount of DNA as a template, details of PCR reaction

9. Sections 2.2 and 2.5

Provide following information:

I. the metod used to assure the quality of RNA,

II. details of RT reaction; duration, physical parameters, reaction mixture composition

III. details of PCR reaction; duration, physical parameters, reaction mixture composition, amount of RNA/cDNA per sample, name and manufacturer of PCR instrument

10. Numbers of Supplement tables are not consistent with their description, for example file described as Table S6 contains table S2  and file described as Table S7 contains table S3. Check all these suplement tables/files and correct it.  

 

 

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript needs a minor revision. I suggest some minor revisions to enhance its quality and align it more closely with the standards of our journal. Please consider the following recommendations:

Novelty and Future Implications:

While your manuscript provides valuable insights, it would benefit from a clearer articulation of its novelty. Explicitly state the unique contributions of your work and discuss how the findings could be applied in future research. This addition will help readers understand the significance of your study within the broader scientific context.

 

Figure Quality Improvement:

Figures 1, 5, 6, and 9 require improvements in quality to enhance their clarity and effectiveness. Pay particular attention to resolution, labeling, and overall presentation. High-quality Figures are essential for conveying your findings accurately. Please ensure that the revised Figures meet the standards expected for publication.

 

References Format and DOI Inclusion:

Update the format of your references to comply with the journal's requirements. Include DOI where applicable. This adjustment is crucial for readers who wish to access your cited works directly. Review the journal's guidelines for reference formatting to ensure compliance. 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript according to the reviewer's comments. The authors are suggested to read the manuscript carefully again to make it better.

Comments on the Quality of English Language

The English of the manuscript has been improved.

Author Response

I have carefully read the manuscript and made appropriate revisions to make it more perfect.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been sufficiently improved and all comments addressed.

About English, I appreciated the check by the MDPI English language editing support.

Author Response

I have carefully read the manuscript and made appropriate revisions to make it more perfect.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors significantly improved the manuscript. However, still some English language improvements should be done as follows:

  

1. Correct the desriptions to tables:

 

Table S1.  "Primer of qRT-PCR of chlorophyll metabolism related genes", correct to:

Sequences for primers used in qRT-PCR to analyse chlorophyll metabolism-related genes

Table S2. "Primer of target gene subcellular localization specific amplification", correct to:

Sequences for primers used to study target gene subcellular localization

 

Table S3. "Primer of gene promoter", correct to:

Sequences for primers used to amplify the PAO gene promoter

 

Table S4. "Primer of BSP methylation", correct to:

Sequences for primers used to analyse the BSP methylation

 

Table S5: Provide explanation not only for CG but also CY and G.

Table S6: I can not understand the meaning of the description to Table S6- correct it.

 

Table S7. "Relative expression level analysis of OfPAO gene in empty vector and transgenic Tobacco" correct to:

"Relative expression of OfPAO in transgenic tobacco transformed with the empty vector and overexpressing the OfPAO"

 

Table S8."Chlorophyll and Carotenoid content in transgenic tobacco leaves of OfPAO" correct to:

"Chlorophyll and carotenoid content in transgenic tobacco leaves overexpressing the OfPAO"

 

Table S9 should have description and separated A, B C parts, for example:

Table S9. DNA sequences of OfPAO cDNA (A) and OfPAO promoter (B).  Amino acid sequence of OfPAO (C).

PAO sequence of Osmanthus fragrans ‘Yinbi Shuanghui’ correct to: cDNA sequence of Osmanthus fragrans ‘Yinbi Shuanghui’ PAO

PAO amino acid sequence Osmanthus fragrans ‘Yinbi Shuanghui’, correct to: Amino acid sequence of Osmanthus fragrans ‘Yinbi Shuanghui’ PAO

PAO promoter sequence of Osmanthus fragrans ‘Yinbi Shuanghui’, correct to: Promoter DNA sequence of Osmanthus fragrans ‘Yinbi Shuanghui’ PAO

 

2. In line 107: Osmanthus fragrans write in italics

3. Comments nr 8 and 9 from the first revision should be introduced into the manuscript text.

 

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Please see the attachment

Author Response File: Author Response.pdf