Natural Variation of OsHd8 Regulates Heading Date in Rice
, Xiao Wei 1, Wei Wang 1, Fengfeng Fan 1, Licheng Zhang 1, Zhikai Wang 2, Zhihong Tian 2 and Shaoqing Li 1,*
Round 1
Reviewer 1 Report
The manuscript by Yuan et al. reports the identification and functional analysis of OsHd8, regulates heading date in rice. The authors performed fine mapping of the OsHd8 locus and identified a transcription factor from the HAP family as a candidate gene. The authors also provided evidence that OsHd8 encodes a transcriptional repressor. Overexpression of this gene could prolong flowering time. The authors also report natural variations in the promoter of the gene. Although the results of this study are interesting and of good quality, I have some comments, which are listed below.
Comments
- In many places there is a lack of information on plant material and methodology.
- P4:L100, how many F2 lines were used?
- Please provide more details on growth condition, growth duration, and day length.
- P4:L108, does “hybrid progeny population” refer to the F2 population.
- Please provide more details on method for map-based cloning and genetic analysis.
- P4:L111-112, please provide the number of F2 plants with each extreme phenotype and define the criteria for determining the two extreme phenotypes.
- P6:L171, please provide details on genetic linkage in the Materials and Methods section.
- Please also provide the methods and tools for sequencing and variant detection.
- In the results in Section 3.2, Figure 3, please explain the different levels of gene expression in the different complementation and overexpressed lines.
- P10:L229-230, please provide more details/methodology for sequence analysis.
- Figure 8: Please provide the details on the genomic region of OsHd8 to support Figure 8A.
Author Response
Reviewer 1
Comments and Suggestions for Authors
The manuscript by Yuan et al. reports the identification and functional analysis of OsHd8, regulates heading date in rice. The authors performed fine mapping of the OsHd8 locus and identified a transcription factor from the HAP family as a candidate gene. The authors also provided evidence that OsHd8 encodes a transcriptional repressor. Overexpression of this gene could prolong flowering time. The authors also report natural variations in the promoter of the gene. Although the results of this study are interesting and of good quality, I have some comments, which are listed below.
Comments
- In many places there is a lack of information on plant material and methodology.
Response: Thank you for your kind suggestion, the manuscript have checked again and added it in this revised version.
- P4:L100, how many F2 lines were used?
Response: 4500 F2 individuals were used to clone OsHd8, the information has been added in Page4, Line104.
- Please provide more details on growth condition, growth duration, and day length.
Response: The details of growth condition, growth duration, and day length were shown in line 100-107 (“All the rice plants were grown in the EZhou experimental field (30°40’N, 114°88’E) of Wuhan University.
- P4:L108, does “hybrid progeny population” refer to the F2 population.
Response: The “hybrid progeny population” refers to the F2 population, the content has been corrected in Page4, Line112.
- Please provide more details on method for map-based cloning and genetic analysis.
Response: The related content has been added in Page4, Line117-120.
- P4:L111-112, please provide the number of F2 plants with each extreme phenotype and define the criteria for determining the two extreme phenotypes.
Response: Thanks for your kind suggestion, all the 606 F2 plants are short heading date plants and, the extreme phenotype of short growth period was defined as the first 15% of heading in the whole F2 population. The related content has been corrected in Page4, Line115.
- P6:L171, please provide details on genetic linkage in the Materials and Methods section.
Response: The related content has been added to the Supplementary Table S2 in the revised version.
- Please also provide the methods and tools for sequencing and variant detection.
Response: The methods and tools for sequencing and variant detection have been added in Page4, Line122-141.
- In the results in Section 3.2, Figure 3, please explain the different levels of gene expression in the different complementation and overexpressed lines.
Response: Thank you for your kind comment. The previous results demonstrated that the difference in the expression of OsHd8 led to the difference in the heading date, which was delayed with the increase in the expression of OsHd8. In Figure 3, compared with JH2B, the expression levels of different complementation and overexpressed lines were increased to different degrees, and the growth period was also significantly delayed.
- P10:L229-230, please provide more details/methodology for sequence analysis.
Response: The related content has been added in Page112, Line263-264.
- Figure 8: Please provide the details on the genomic region of OsHd8 to support Figure 8A.
Response: The related content has added Supplementary Table S3 in the revised version.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript is of a high degree of interest. The work as a whole is presented very well. The problems solved in this work are relevant and modern. The methods used correspond to the tasks set. The results are clear and well presented. This manuscript is recommended for publication with the correction of minor comments:
1. Line 122 needs a link to the calculation method 2–ΔСt, it is.
2. Figure 8 is very beautiful, but not readable at all.
3. There is not enough chapter Conclusions with brief conclusions on the work.
Author Response
Reviewer 2
Comments and Suggestions for Authors
This manuscript is of a high degree of interest. The work as a whole is presented very well. The problems solved in this work are relevant and modern. The methods used correspond to the tasks set. The results are clear and well presented. This manuscript is recommended for publication with the correction of minor comments:
- Line 122 needs a link to the calculation method 2–ΔСt, it is.
Response: Thank you for your kind suggestion, the related content has made revised in Page6, Line152.
- Figure 8 is very beautiful, but not readable at all.
Response: Thank you for your kind comment, the previous experimental results show that OsHd8 plays an important role in the regulation of heading date, so we will explore the role of OsHd8 in genetic evolution. The results showed that OsHd8 might be subjected to natural selection and suggested strong genetic differentiation between indica and japonica subspecies at the OsHd8 locus, which laid a theoretical foundation for us to continue to explore the function and utilization of this gene.
- There is not enough chapter Conclusions with brief conclusions on the work.
Response: A brief conclusions have been added in Page22, Line440-448.
Author Response File: Author Response.docx
Reviewer 3 Report
The heading date is critical in the yield determination and significant factor for the adaptability of plants to specific natural environments. Molecular characterization of the rice genes that regulate heading is important to better understand rice transition into generative phase and sexual reproduction but also benefits breeding programs. Therefore I've found this work important for the community. Hovewer I would like to give some comments to this work. Title: “Natural variation of OsHd8 regulates heading date in rice.” Since you claim that mutations in the promoter are the source of variation, please reconsider the changes to make the title more specific.
Line 43 - “key component functioning factor” - key component or key factor
Line 44 - FT (FLOWERING LOCUS T), a major floral activator, encodes RAF kinase inhibitor, which is activated by CO to promote flowering
The regulatory pathway that controls flowering in response to light is well characterized in Arabidopsis thaliana. FT (FLOWERING LOCUS T) is a member of PEBP gene family that shares homology with RAF kinase inhibitor proteins (RKIPs). I think that more accurate way for summarizing flowering promotion can be find in the recent papers by Jin et al. (2021) https://doi.org/10.1016/j.semcdb.2020.05.007 or Nakamura et al. (2019) https://doi.org/10.1016/j.isci.2019.10.045
Line 46: Ortholog replace by ortholog
Line 47: short-Day replace by short day (SD)
Line 49: What you mean by phytochromes B? Are there more than one?
· Talking about Ehd1 please include the newest data from Shrestha et al., 2014 ( DOI: 10.1093/aob/mcu032), Nemoto et al., 2016, and Chai et al. 2021 ( doi: 10.1111/pbi.13462.)
· Line 89 – “MERISTEM 20), encodes” – delete the comma
· Line 114 to 122 – It is indicated that the relative expression levels for candidate genes were studied. What kind of the reference gene was used for quantification? Was the DNAse treatment included? How many technical replicates per sample were used?
· Line 137 and Line 160:
Following sentences needs re-writing:
“Subsequently, the vectors co-transformed with GV3101 (pSoup-p19) chemically competent cells, Overnight A. tumefaciens were cultured at 28 o 138 C and collected by centrifugation and re-suspended in MS medium with OD600 =1.5, and incubated at 28 o 139 C and 200 rpm for 3 h.”
“All the assays were performed three biological replicates.”
Line 244: “C)The main structure” Please include a space.
Line 264: “Figure 5. Sequence variations in the OsHd8 promote.” Change to “promoter”
Figure 6: What is the reason behind indicating twice the on the A-Panel the function (Molecular Function, Biological Process, Cellular Component) If you decide to leave as it is, please change the color appropriately. There are different fonts used to indicate OsHd8 related genes in heading date between JH2B and OE-OsHd8.
Author Response
Reviewer 3
Comments and Suggestions for Authors
The heading date is critical in the yield determination and significant factor for the adaptability of plants to specific natural environments. Molecular characterization of the rice genes that regulate heading is important to better understand rice transition into generative phase and sexual reproduction but also benefits breeding programs. Therefore, I've found this work important for the community. However, I would like to give some comments to this work.
Title: “Natural variation of OsHd8 regulates heading date in rice.” Since you claim that mutations in the promoter are the source of variation, please reconsider the changes to make the title more specific.
Response: Thank you for your kind suggestion, this study revealed that there were four SNPs and one InDel in the promoter region of OsHd8, which was involved in the regulation of heading date. Further evolutionary analysis showed that OsHd8 presents divergence between indica and japonica, showing artificial selection during the domestication of cultivated rice. These results indicate that Natural variation of OsHd8 plays an important role in the regulation of heading date, and the topic “Natural variation of OsHd8 regulates heading date in rice.” may be more appropriate.
Line 43 - “key component functioning factor” - key component or key factor
Response: Thank you for your kind comment, this is more appropriate with key factor. The key facto have corrected it in Page2, Line43.
Line 44 - FT (FLOWERING LOCUS T), a major floral activator, encodes RAF kinase inhibitor, which is activated by CO to promote flowering
The regulatory pathway that controls flowering in response to light is well characterized in Arabidopsis thaliana. FT (FLOWERING LOCUS T) is a member of PEBP gene family that shares homology with RAF kinase inhibitor proteins (RKIPs). I think that more accurate way for summarizing flowering promotion can be find in the recent papers by Jin et al. (2021) https://doi.org/10.1016/j.semcdb.2020.05.007 or Nakamura et al. (2019) https://doi.org/10.1016/j.isci.2019.10.045
Response: Thank you for your kind comment. The related content has been corrected in Page 2, Line 44-46.
Line 46: Ortholog replace by ortholog
Response: Thank you for your kind comment. Ortholog have corrected it in Page2:Line48.
Line 47: short-Day replace by short day (SD)
Response: Short day (SD) has corrected it in P2:L49.
Line 49: What you mean by phytochromes B? Are there more than one?
Response: Makoto et., al (2005) isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa), PhyB regulates Hd3a expression and is the main reason for long day inhibition of flowering in rice. PhyB is LOC_Os03g19590.
Talking about Ehd1 please include the newest data from Shrestha et al., 2014 (DOI: 10.1093/aob/mcu032), Nemoto et al., 2016, and Chai et al. 2021 (doi: 10.1111/pbi.13462.)
Response: These new data have been added in the revised version in P3:L59-60.
Line 89 – “MERISTEM 20), encodes” – delete the comma
Response: we are sorry for our mistake. The manuscript has been checked again and corrected it in this revised version.
- Line 114 to 122 – It is indicated that the relative expression levels for candidate genes
were studied. What kind of the reference gene was used for quantification? Was the DNAse treatment included? How many technical replicates per sample were used?
Response: Thank you for your kind suggestion. The actin gene was used as the internal control. All samples were treated with DNAase to remove genomic DNA and all assays were performed with three biological replicates. The related content has made revised in the revised version.
Line 137 and Line 160:
Following sentences needs re-writing:
“Subsequently, the vectors co-transformed with GV3101 (pSoup-p19) chemically competent cells, Overnight A. tumefaciens were cultured at 28 o 138 C and collected by centrifugation and re-suspended in MS medium with OD600 =1.5, and incubated at 28 o 139 C and 200 rpm for 3 h.”
“All the assays were performed three biological replicates.”
Response: Thank you for your kind comment. The related content hss corrected in Page6, Line 166-172.
Line 244: “C) The main structure” Please include a space.
Response: We are sorry for our mistake. The manuscript has been checked again and corrected it in this revised version.
Line 264: “Figure 5. Sequence variations in the OsHd8 promote.” Change to “promoter”
Response: We are sorry for our mistake. The manuscript has been checked again and corrected in this revised version.
Figure 6: What is the reason behind indicating twice the on the A-Panel the function (Molecular Function, Biological Process, Cellular Component) If you decide to leave as it is, please change the color appropriately. There are different fonts used to indicate OsHd8 related genes in heading date between JH2B and OE-OsHd8.
Response: We are sorry for the mistake. We have checked again and corrected the color appropriately and same font to indicate OsHd8 related genes in heading date between JH2B and OE-OsHd8 in this revised version.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors have addressed all my comments and accordingly revised the manuscript. I have no further comment on the current version of manuscript.
