Review Reports - Deciphering the Regulatory Mechanism of PmMYB21 in Early Flowering of Prunus mume through Dap-Seq and WGCNA Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

8. The manuscript titled “Deciphering the Regulatory Mechanism of PmMYB21 in Early Flowering of Prunus mume Through Dap-seq and WGCNA Analysis”. The authors investigated the binding site and genes associated with the transcription factor PmMYB21 and also screened key genes that closely related to flowering. Overall, this research is potentially full of interest, as it addresses the relevant topic but this study is not drafted in intelligible fashion. Throughout the manuscript, there are several grammatical errors that need to be corrected. Below are some suggestions that I believe would benefit the readers with greater precision.

1. Rewrite the sentence with better clarity “Our analysis identified 8533 potentially bound by PmMYB21 with motif1 sequence type in the promoter region.”

2. Line 60-77, this paragraph suits better in the discussion section.

3. Add growth conditions in materials and methods

4. A separate paragraph in the introduction section should integrate the relationship of the genome and transcriptome to molecular regulation mechanism of flowering trait in horticulture plants.

5. Check fonts theme between line 263-267

You may cite or mention some relevant published work that may be discussed and also can provide contradictions and similarities of these below published work with you results.

10.3389/fpls.2022.1036221

10.1016/j.xplc.2021.100232

https://doi.org/10.3390/plants11243473

6. References style is not uniform

7. Tables provided in the supplementary file did not open. Provide word file in revision

8. Conclusions should be added

Wish you luck

should be added

Wish you luck

Comments on the Quality of English Language

The english language required a crosscheck throughout the manuscript.

Author Response

Comments1: Rewrite the sentence with better clarity “Our analysis identified 8533 potentially bound by PmMYB21 with motif1 sequence type in the promoter region.”

Response: Thank you very much for your reminder. I have modified the sentence and highlighted them in yellow. See line 21-22.

Comments2: Line 60-77, this paragraph suits better in the discussion section.

Response: Agree with this comment. I have moved the paragraph to the Discussion section and made appropriate revisions. This paragraph is highlighted in yellow. See line 261-279.

Comments3: Add growth conditions in materials and methods

Response: Agree. Section 2.2 growth conditions has been added. Furthermore, if you require additional information about the ambient temperature during sample collection, it can be found in the Table S1 Sampling Data."

Comments4: A separate paragraph in the introduction section should integrate the relationship of the genome and transcriptome to molecular regulation mechanism of flowering trait in horticulture plants.

Response: Agree with this comment. I have added a separate paragraph between lines 70-81 in the introduction section. The revised parts have been highlighted in yellow.

Comments5: Check fonts theme between line 263-267

You may cite or mention some relevant published work that may be discussed and also can provide contradictions and similarities of these below published work with you results.

10.3389/fpls.2022.1036221

10.1016/j.xplc.2021.100232

https://doi.org/10.3390/plants11243473

Response: Thank you for your reminder. The font of the original lines 263-267 has been modified and highlighted in yellow. See line 289-292.

Thank you very much for your careful recommendation. The recommended literature 10.1016/j.xplc.2021.100232 and 10.3390/plants11243473 has been cited, in lines 70-81, lines 282-285 and highlighted in green.

The paper “10.3389/fpls.2022.1036221” shares some similarities with my work, yet the results are markedly different. I have endeavored to analyze the reasons for these discrepancies. However, due to the absence of detailed descriptions of the cultivation sites, sampling method, and the specific phenotypic characterization of the flower buds in the aforementioned article, it is challenging to ascertain the causes of these differences. Also, I am unable to determine what specific issues their results actually reflect (environmental conditions or the dormancy stages). I am encountering difficulties in citing this paper and hope for your understanding.

Comments6: References style is not uniform

Response: Thank you for for pointing this out, the citation format throughout the text has been corrected.

Comments7: Tables provided in the supplementary file did not open. Provide word file in revision

Response: Agree with this comment. I have submitted the Word document for the supplementary file.

Comments8: Conclusions should be added

Response: The conclusion section has been added, and highlighted in yellow. See line 335-344.

Thank you for your attentive reminder. I will request the polish from MDPI before the publication.

Thanks again for your kindly help! ❤

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Deciphering the Regulatory Mechanism of PmMYB21 in Early Flowering of Prunus mume Through Dap-seq and WGCNA Analysis" is well written and reports interesting information about the regulatory mechanism of PmMYB21 adequately supported by experimental data.

- Lines 243-244 Please, rephrase the sentences, in my opinion they are unclear.

- Line 257 Change “gens” to “genes”

- Verify that all gene names in the text are written in italics

- Line 319 Please, italicize "arf6"... "arf12"... "arf6"... "arf12"

- Please, indicate in which database the raw data from the sequencing are stored

Author Response

Reply to Authors 2

Comments1: Lines 243-244 Please, rephrase the sentences, in my opinion they are unclear.

Response: Thank you for pointing this out. I have rewritten the sentence in line 227-228 and highlighted them in yellow. I will request the polish from MDPI before the publication.

Comments2: Line 257 Change “gens” to “genes”

Response: Thank you for your attentive reminder. The content has been modified and highlighted in yellow at Line 242.

Comments3: Verify that all gene names in the text are written in italics

Response: Agree. I have modified the gene names in the text and highlighted in yellow. Additionally, I have also made same modification in Figure 3b to italics, Figure S1 and Table S2. Thanks again.

Comments4: Line 319 Please, italicize "arf6"... "arf12"... "arf6"... "arf12"

Response: Thank you very much for your kindly reminder. I have modified the gene names to italics and highlighted in yellow. See line 328-329.

Comments5: Please, indicate in which database the raw data from the sequencing are stored

Response: Agreed with your suggestions. The database information has been included in the Data Availability Statement section and highlighted in yellow. See line 354-356.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript, titled “Deciphering the Regulatory Mechanism of PmMYB21 in Early Flowering of Prunus mume Through Dap-seq and WGCNA Analysis,” presents a study to identify genes involved in regulating the flowering process of Prunus mume. This study provides valuable insight into the molecular basis of the role of PmMYB21 in regulating Mei flowering at low temperatures.

Comments and questions:

Based on research results, the role and response to temperature in Mei PmSEP3 is not entirely clear. The authors noted that PmSEP3 was mainly expressed in flowers and fruits and was significantly higher in petals and stamens at low temperature (≤16℃). Does this mean that PmSEP3 is tied to growing conditions and its expression will change depending on the growing region and the time of bud release from dormancy?

Was the influence of the rootstock and the amount of precipitation during critical periods of the growing season on the results obtained?

Have mutant forms of Prunus mume been studied and have decreased fertility and changes in stamen development been noted?

Can the results obtained from studying Prunus mume varieties be transferred to Prunus armeniaca?

Author Response

Comments1: Based on research results, the role and response to temperature in Mei PmSEP3 is not entirely clear. The authors noted that PmSEP3 was mainly expressed in flowers and fruits and was significantly higher in petals and stamens at low temperature (≤16℃). Does this mean that PmSEP3 is tied to growing conditions and its expression will change depending on the growing region and the time of bud release from dormancy?

Response: Uh, to be precise, the AtSEP3-overexpression Arabidopsis can ignore low temperature (16℃) and maintain the same flowering phenotype as Arabidopsis at 23°C. This suggests that SEP3 can promote the development of petals or stamens under low temperatures. In original sentence “SEP3 overexpression showed temperature-insensitive flowering at 23°C and 16°C. This suggests that Altered SEP3 activity affects ambient temperature-responsive flowering”.

In our results, PmSEP3 also has a relatively high expression level even under low temperatures (0°C/0°C/7°C/13°C/23°C during sampling). We speculate that this is related to the characteristic of mei flower bud development under low temperatures.

I think that the expression level of PmSEP3 is influenced by the environment, as the flowering process of mei buds is such a process. Also, PmMYB21, which we predict to regulate the expression of PmSEP3, has its expression level regulated by temperature and hormones. And hormones are also related to the endo-dormancy state. However, the role of PmSEP3 and its response to temperature still require further experimental verification.

Comments2: Was the influence of the rootstock and the amount of precipitation during critical periods of the growing season on the results obtained?

Response: Thank you, this is an interesting question. The growth season for Prunus mume is after flowering, that is, after March. From June to August, flower buds are formed and grow to a visible size, enter endo-dormancy in September, break dormancy in December, and entering the flowering phase from January to March. Theoretically, the total number of flower buds formed may be influenced by the rootstock and the amount of rainfall. After the full break of the endo-dormancy, the flowering process of the flower buds is limitedly related to the influence of the growth season.

Our results come from the flowering phase of Prunus mume in January to March. The rootstock and rainfall during the flowering phase may affect this process. However, our test plants have the same species of rootstock (mountain peach), the same age, and are planted within a distance of no more than 15 meters on the same hillside, almost having completely identical natural conditions. Therefore, the influence of rootstock and precipitation during growing season is considered to be minimal on our results.

Comments3: Have mutant forms of Prunus mume been studied and have decreased fertility and changes in stamen development been noted?

Response: Within the scope of my knowledge, no such mutant varieties of Prunus mume... Perhaps my knowledge is not yet extensive enough. However, I have observed some filial generation with exceptionally long stamen filaments, a characteristic that has not yet been documented.

Comments4: Can the results obtained from studying Prunus mume varieties be transferred to Prunus armeniaca?

Response: Sure. Very happy to answer this question.

Within the Rosaceae family, which includes Malus, Rosa, and Prunus, etc., the CDs sequences of genes related to flowering exhibit a high degree of identity. Indicate that their gene functions are likely to be very similar. Therefore, the key differences in their flowering trait may be concentrated in the expression regulation of these genes.

As members of the Prunus that flower in the spring of the following year with separate flower buds, Prunus armeniaca and Prunus mume are likely to have very similar flowering regulatory mechanisms. The paper recommended by Reviewer 1, titled 'DNA features beyond the transcription factor binding site specify target recognition by plant MYC2-related bHLH proteins' shows that transcription factors with similar sequences have similar recognition sites. This indicates the reference value of our results for apricot trees.

If flowering regulatory mechanisms of Prunus mume can be deciphered, it may be possible to make Prunus armeniaca flowering earlier, or conversely, to delay the flowering of some apricots cultivar, to avoid the negative impact of cold spell and persistent rainy days in later spring.

It should be noted that under the same natural conditions described in this paper, the apricot's flowering time is significantly later than that of the Prunus mume. This is mainly due to a significantly longer endo-dormancy period and a slower flowering process (the duration from bud break to full bloom). The latter is more closely related to the content of this paper. I have seen some reports mentioning almond that bloom very early, perhaps they have a shorter endodormancy period? In this case, our results would be more valuable for reference.

I have included the aforementioned content in lines 282-286 (highlight in green) and in the Conclusion section.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is suitable for publication

References style is still not uniform

Check the journal name of 5,6,7 references with that of 8