The Rice CHD3/Mi-2 Chromatin Remodeling Factor Rolled Fine Striped Promotes Flowering Independent of Photoperiod

Round 1

Reviewer 1 Report

In the submitted manuscript, the authors dissected the role of RSF gene in the flowering process of rice through the deep study of a rsf mutant. They showed the role of RSF in the upregulation of two florigen genes, Hd3a e RFT1. Moreover, the authors performed an expression analysis on different flowering genes identifying a possible role of RSF in the regulation of Ghd7 and Ehd1 expression. They investigated the role of RFS in the histone methylation pattern of Ehd1 and Ghd7 genes concluding that it controls the expression of Ghd7 and Ehd1 through a different mechanism. Finally, the authors evaluated some agronomic traits of rsf mutant showing a decreased grain yield and number of secondary panicle branches compared to wild type.

The study is clear and well-conducted. The findings are convincing and the proposed model of the flowering regulatory pathway controlled by RSF is interesting.

Author Response

Reviewer 1:

In the submitted manuscript, the authors dissected the role of RSF gene in the flowering process of rice through the deep study of a rfs mutant. They showed the role of RSF in the upregulation of two florigen genes, Hd3a e RFT1. Moreover, the authors performed an expression analysis on different flowering genes identifying a possible role of RSF in the regulation of Ghd7 and Ehd1 expression. They investigated the role of RFS in the histone methylation pattern of Ehd1 and Ghd7 genes concluding that it controls the expression of Ghd7 and Ehd1 through a different mechanism. Finally, the authors evaluated some agronomic traits of rfs mutant showing a decreased grain yield and number of secondary panicle branches compared to wild type.

The study is clear and well-conducted. The findings are convincing and the proposed model of the flowering regulatory pathway controlled by RSF is interesting.

Answer: Thank you for your careful reading and recommending our manuscript to be published in IJMS without further revision.

Reviewer 2 Report

The presented manuscript provides evidences of involvement of a rice CHD3/Mi-2 Chromatin Remodeling Factor in flowering by affecting the expression of the flower repressor Ghd7 (a CCT domain protein).
This finding is novel and original, as the respective rice gene rfs has been assigned to completely different functions so far.
The results are clearly presented and the experimental data support the hypotheses. Style and language of the paper are correct and the quality of figures also meet the expectations of a state-of-the-art scientific journal.

However, outline and articulation should be improved, as in its present form the manuscript is difficult to interpret and follow.

In the Introduction the Authors provide a compact, but informative overview on the scientific background of flowering regulation in rice and model plants.
However, in my opinion, they did not pay attention to properly draw up the experimental setup and conception of their own work.
For instance, at the end of the Introduction they just shortly mention that ".. two rfs mutants exhibited delayed flowering independent of the photoperiod" and push the more detailed description of the two mutants to the beginning of the Results. In my opinion, it would better to dedicate a paragraph (possibly the last paragraph of the Introduction) for describing the background, aims and scopes as well as initial hypothesis of the experiments.

Generally, the Authors should pay attention to more clearly separate Introduction-, Results- and Discussion-related contents. (Shortly: There is unnecessarily too much introductory and discussive content in the Results section. Sentences that not just describe, but interpret experimental results, should rather be moved to the Discussion).
On the other hand, the Authors should also attempt to re-formulate the Discussion chapter in a more structured way. (Perhaps, by adding sub-titles to the main paragraphs of the Discussion and finally adding a Conclusion section.)

Upon such revising and re-editing the manuscript is highly recommended to publish in MDPI IJMS.

Author Response

Reviewer 2:

The presented manuscript provides evidences of involvement of a rice CHD3/Mi-2 Chromatin Remodeling Factor in flowering by affecting the expression of the flower repressor Ghd7 (a CCT domain protein). This finding is novel and original, as the respective rice gene rfs has been assigned to completely different functions so far.

The results are clearly presented and the experimental data support the hypotheses. Style and language of the paper are correct and the quality of figures also meet the expectations of a state-of-the-art scientific journal. However, outline and articulation should be improved, as in its present form the manuscript is difficult to interpret and follow.

Comment 1: In the Introduction the Authors provide a compact, but informative overview on the scientific background of flowering regulation in rice and model plants. However, in my opinion, they did not pay attention to properly draw up the experimental setup and conception of their own work.

For instance, at the end of the Introduction they just shortly mention that ".. two rfs mutants exhibited delayed flowering independent of the photoperiod" and push the more detailed description of the two mutants to the beginning of the Results. In my opinion, it would better to dedicate a paragraph (possibly the last paragraph of the Introduction) for describing the background, aims and scopes as well as initial hypothesis of the experiments.

Answer 1: Thank you for your comments. As you suggested, we revised a last paragraph of the Introduction section as follow (P2 L85-L92) and a first sentence of the Results section (P2 L95-L96).

“Here, we found that two rfs mutants (rfs-1 and rfs-2) derived from different genetic backgrounds exhibited late flowering independent of the photoperiod, implying that RFS is involved in flowering-time regulation in rice. Although numerous genes that control rice flowering have been reported, few epigenetic regulator that modulate flowering has not been identified in rice. To reveal the regulatory function of RFS in rice flowering, we analyzed the expression and histone methylation levels of flowering-time genes in rfs-2 mutant. Reverse transcription-quantitative PCR (RT-qPCR) showed that RFS promotes flowering by downregulating Ghd7 in LDs and upregulating Ehd1 in SD and LD conditions. Our results suggest a new molecular function of RFS as a floral regulator in rice.”

Comment 2: Generally, the Authors should pay attention to more clearly separate Introduction-, Results- and Discussion-related contents. (Shortly: There is unnecessarily too much introductory and discussive content in the Results section. Sentences that not just describe, but interpret experimental results, should rather be moved to the Discussion).

Answer 2: Thank you for your comments. We deleted unnecessary introductory contents (P3 L110; P5 L140) and revised a sentence (P5 L143) in the Results section. We also moved discussive contents from the Results section (P7 L196) to the Discussion section to clarify possible functions of RFS on epigenetic regulations (P12 L309-312).

Comment 3: On the other hand, the Authors should also attempt to re-formulate the Discussion chapter in a more structured way. (Perhaps, by adding sub-titles to the main paragraphs of the Discussion and finally adding a Conclusion section.)

Answer 3: Thank you for your suggestion. In the Discussion section, we added subtitles in main paragraphs (P10 L257; P11 L288; P12 L321) and revised a fourth paragraph (P11 L305-319).