An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry

Round 1

Reviewer 1 Report

In the manuscript “A R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry" Li et al. address the regulatory role of a R2R3-MYB TF RoMYB10 in black raspberry (Rubus occidentalis. L) in promoting the synthesis of anthocyanins. They first cloned the transcript RoMYB10 in black raspberry. Then tested the expression levels of the RoMYB10 genes in four different color raspberry varieties and different tissues of these raspberry. Ectopic expression of the RoMYB10 impairs plant growth in tobacco which accumulate much higher levels of anthocyanin. Genes regulate anthocyanin synthesis, NtCHS, NtCHI, NtF3H, NtANS and NtUFGT were up regulated in RoMYB10-OE plants, which suggested RoMYB10 involves in regulating these genes directly or indirectly. The genesis of this paper is uncovering the function of RoMYB10 involved in regulating anthocyanin synthesis. The title and abstract are appropriate for the content of the text.

However, the text is not always easy to follow as the manuscript is poorly structured with unclear logic and suffers from excessive language deficiencies. The experiments were not well conducted. The authors should revise the language and logic of manuscript to improve readability.

As explained below, the concerns are listed below.

1.     Line 12-14, “And the gene sequence, the physical and chemical properties, phylogenetic relationship and the subcellular localization of RoMYB10 protein were analyzed.” This sentence is confusing, please rewrite it.

2.     Line 23, “In addition, the expression of several key enzyme genes in tne anthocyanin synthesis were detected.” Is the highlighted “tne” a typo?

3.     Line 24, Is it confusing to readers “Among these genes, NtCHS, NtCHI, NtF3H, NtANS and NtUFGT were significantly higher in transgenic tobacco plants.” it is better to specify the transcripts or expression levels are higher in RoMYB10 overexpression plants.

4.     Line 31-33, It should be more reasonable and logical to swap the order the two sentences:  As a shrubby small berry fruit tree, raspberry is widely cultivated in northeast China and southwest China. It is also widely distributed in Europe, North America and parts of Asia.

5.     Line 38, please delete the “and” in anti-cancer and anti-mutation.

6.     Line 45-46, “MYB TFs commonly play a central role in distinguishing the target gene in the MBW complex [12,13].” The highlighted gene should be genes.

7.     Line 67, “could bind to the G-box motifs of PyHY5 and promoted the anthocyanin synthesis in pear.” The highlighted promoted should be “promote”.

8.     Line 70-71 “These studies confirmed the important role of MYB10 in regulating anthocyanin accumulation” This sentence is not grammatically correct, please rewrite it.

9.     Line 73-74, Ectopic expression of PoMYB10 from Populus promoted secondary cell wall thickening and inhibits anthocyanin accumulation. Please rewrite this sentence with the correct tense. 

10.  Line 79, “peach, strawberry and kiwi fruit and so on” the highlighted and should be replaced with a comma. Please also check the whole draft.

11.  Line 89, “желтый гигант” should be a yellow giant.

12.  Figure1A, please provide detailed information on the different color indicated in protein alignment.

13.  Line 181-182, please change the order of the raspberries to fit the order in Fig. 2A.

14.  Fig. 2A, the figure legend is not consistent with Fig. 2A.

15.  Fig. 2B, it is not easy to interpret, please use one reference to analyze the relative expression and normalize the reference/control, like root, as 1, the transcript levels in the rest of samples are relative to reference/control.

16.  Fig. 3A, the brightfield images do not seem clear enough, please change them.

17.  Line 215, please rewrite “This result was the same as Feng’s [6]”

18.  Fig. 5, please reformat NtDFR, NtLAR and NtANR as the numbers of the y-axis it way too high. 

 

 

 

 

 

 Extensive editing of English language required. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled “A R2R3-MYB Transcription Factor RoMYB10 Regulates Antho- 2 cyanin Biosynthesis in Black Raspberry” is informative regarding anthocyanin because of its diverse biological functions. Results are clear. This study is interesting for readers.

However, the following issues need to be addressed:

A)    I have founded many typing and grammar mistakes. Please check and revise the whole manuscript.

B)    The language of manuscript is poor. I have found many meaningless sentences. Please  check and revise throughout the manuscript  manuscript.

C)    Please replace the keywords with appropriate keywords. These keywords are looking attractive for readers.

D)    Please provide the methods in detail, especially “Subcellular localization of the RoMYB10 protein and Pigment analysis” sections.

1.     “'желтый гигант'” ?

2.     In line 101, please check the format “manufacturer’s instructions”.

3.     Please remove the space between “The” and “qRT-PCR”

4.     In lines 127-129. “The fusion construct was transformed into tobacco (Nicotiana benthamiana) leaves using a needless syringe and the 128 empty pCambia 2300 vector was as a control” . This information is not enough. Please provide the methods in detail. If there need, please also provide the reference.

5.     In line 134, please ensure pCambia1300 or pCAMBIA1300?

6.     In lines 141-142 “The anthocyanin extraction was as the method of Bariola” this sentence is meaningless, please revise this sentence.

7.     In line 142-143“The measurement of 141 anthocyanin content was as Pirie and Mullins’s methods”.  Please revise it.

8.     In Figure A, why anthocyanin contents are lower in red fruits?  I think the anthocyanin contents should be higher in red cultivars. Please make it sure.

9.     This study more focused on anthocyanin. Have authors measured the contents of flavonol in this study?

10.  The Expression levels of NtLAR and NtANR in Figure 5 showed no significant results. is there any difference in proanthocyanin contents?

11.  In line 286-287 “The ectopic pigmentation was detected in leaves of these trans- 286 genic plants ”. Please make the clear meaning of this sentence.

12.  Why the authors do not provide the photo of flower pigmentation of transgenic tobacco?

13.  Have authors found any difference in flowers color in transgenic tobacco?

14.  “The anthocyanin extraction was as the method of Bariola [35]. The measurement of 141 anthocyanin content was as Pirie and Mullins’s methods [36]. Anthocyanin was moni- 142 tored at A553 and A600. Three biological replicates were conducted of each sample”. Autor’s need to improve this paragraph.

15.  The quality of figure 1 A is very poor. I am unable to read the amino acids. Please provide the quality figure.

16.  Please read the following the research articles, may be these research article will help to improve your manuscript.

17.  https://doi.org/10.1016/j.scienta.2023.112101

https://doi.org/10.3390/ijms20215456

 

https://doi.org/10.3390/molecules23040781

 

English need to imrove

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

In the manuscript "A R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry", the authors describe the role of the RoMYB10 gene in the biosynthesis of anthocyanins.  Next, I give my questions and comments.

 

•      Line 42-43. Is it correct to attribute anthocyanin reductase (ANR) to the enzyme of anthocyanin biosynthesis? In this case, I suggest replacing the word "biosynthesis and synthesis" with "metabolism".

 

·       In the introduction, the authors consider the role of transcription factor MYB10 in the biosynthesis of anthocyanins in various plant species. Further, the authors point out that the role of the transcription factor MYB10 has not yet been fully studied. What does it mean? I don't understand the novelty of this work.

Materials and methods

 

·       Line 98. Specify the light level at which the plants were grown.

·       Line 109-123. How was RNA isolated? How was cDNA obtained? Describe the qRT-PCR technique. By what method was sequencing performed in this work? Describe the techniques.

·       Chapter 2.2 is devoted to the isolation of the RoMYB10 gene isolation. I suggest calling the chapter "Isolation of RoMYB10 gene".

·       Usually, two housekeeping genes are used to normalize qRT-PCR data. Why do you use one gene? This can have a significant impact on the data.

·       Why was the HPLC-MS method not used to analyze anthocyanins? This approach would help to understand due to which substances there is an increase in the content of anthocyanins. It would also provide new data for discussion.

·       It is necessary to at least briefly describe the method of isolation and analysis of anthocyanins, and not only refer to other works.

 

Results

 

·       Line 148. Give a link to the primer sequences in the supplemental.

·       Figure 1. In the figures, the letters (A) and (B) should be in brackets. This remark is also for subsequent figures.

·       Perhaps the authors should present Figure 1 A in a more accessible form. Sequences and signatures are poorly distinguishable in this version.

·       Line 182. 'желтый гигант' the name of the variety should be written in Latin letters. It was 'желтый гигант', replaced by 'jeltii gigant'. Correct further in the text.

·       Figure 2. I propose to designate different varieties of raspberries with specific names, not colors.

·       Figure 2B, 4C, 5. In what units is 'Relative expression' given?

·        Figure 4B. In the photos of electrophoresis, it is necessary to use a ladder.

 

Discussion

·       Line 239. The expression of NtDFR was lower than that of WT. How can you explain this phenomenon? This needs to be discussed in more detail.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Thanks to the authors for the work done. Basically, the authors' answer suits me, but there is a question.

Response 7: Thank you for your valuable advice. We did analyze anthocyanins by HPLC-MS method, but due to an oversight, we made a mistake in the manuscripts. We have corrected it. Please forgive us. Line 182-196

If you analyzed the content using the HPLC-MS method, then why don't you describe the data obtained? This approach makes it possible to perform a qualitative and quantitative assessment of anthocyanins. The authors describe the data on the total content of anthocyanins, which can be obtained by simpler methods. If you have the results of mass spectrometry, I recommend that you provide detailed data on the content of anthocyanins.

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Thanks to the authors for the work done. This version of the manuscript is worthy of publication.