Transcriptomics Reveal an Integrated Gene Regulation Network of Early Flowering Development in an Oil Sunflower Mutant Induced by Heavy Ion Beam

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study offers valuable insights into the regulatory mechanisms governing early flowering in oil sunflower mutants. By elucidating the roles of plant hormones, sucrose signaling, and flowering genes, the findings contribute to our understanding of the molecular pathways underlying flowering time control. The manuscript has the potential to make a significant contribution to the field of plant developmental biology.

Suggestions for Improvement:

1. The Statistical Analysis section i M& M is missing.
2. The study mentions the potential for genetic improvement of sunflower, it could elaborate further on how the insights gained from this study could be practically applied in breeding programs to develop sunflower genotypes with desirable flowering traits.

Author Response

1. The Statistical Analysis section i M& M is missing.

Answer: Thanks for your reminder. We added 2.6. statistical analysis to illustrate the data analysis and drawing method.

2.6. Statistical analysis

Correlation analysis between RNA-seq and qRT-PCR and bar chart drawing were conducted using Excel (Office 2021). The Venn diagram used the website (http://bioinformatics.psb.ugent.be/webtools/Venn/) to draw. The heatmap was completed using the conditional format color scale function of Excel or the BMKCloud analysis plat-form (https://www.biocloud.net/). Enrichment analysis was accomplished using BMK-Cloud platform. Finally, for image combination and adding notes, use Adobe Illustrator 2020.

2. The study mentions the potential for genetic improvement of sunflower, it could elaborate further on how the insights gained from this study could be practically applied in breeding programs to develop sunflower genotypes with desirable flowering traits.

Answer: Thanks for your advice. We add the potential contribution of this study to the genetic improvement of sunflower in the discussion and conclusion part.

“It can be seen from our research results that the comprehensive external environment of plant growth, plant age, exogenous application of hormones, sugar sources, signal inhibitors, and genetic engineering methods to promote or inhibit flowering genes can all play a role in regulating plant flowering time. It is possible to develop new composite chemical agents for sunflower flowering and apply them to production practice.”

“The results provide useful information for understanding the functional genes of flowering, and the early flowering mutant obtained in this study can be further cultivated as a new oil sunflower variety. Hybridization of the ef mutant with other excellent cultivars is expected to obtain new sunflower varieties with short growth period or early maturity. Further mapping of the major gene linked to early flowering will enrich the functional gene research in sunflower and contribute to molecular assisted breeding.”

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer comments:

The transcriptomics study to identify an integrated gene regulation network of early blooming development in an oil sunflower mutant generated by a heavy ion beam is described in the current article by Liu et al.  Oil sunflower cultivar ‘133211311’ originated from Sunflower Research Institute of Jilin Province, China was used as experimental material. Overall, the work is original, and it looks that the bioinformatics work, statistical measurements, and experimental setup were well-planned and executed.

The following questions need to be clarified-

 1.      What is the duration of the oil sunflower cultivar ‘133211311’?

2.      Authors have screened visible early flowering mutant candidates in the M3 population throughout comparing the whole growth period and flowering time. Quantification can be done by counting the days needed to add the full plant's growth period, including any mutations that resulted in the first flower or 50% of the flower.

3.      Line 120-2-121:  The candidate mutants were self-pollinated to produce homozygous mutants in the subsequent M3 to M6 generation. Finally, one stable early flowering mutant was obtained. How many mutants in total were identified? how many were self-pollinated?

4.      Line 188-189: The early flowering is a stable inherited mutant line selected after heavy ion beam irradiation. It blooms 15 days earlier than the control WT in the field (Figure 1A) needs to be expressed in days.   

5.      Authors identified 63 key genes involved in flowering induction, including flowering integrator genes, photoperiod pathway, hormone pathway, aging pathway, autonomous pathway and temperature pathway and flower development genes may be enlisted in the article may be as supplementary file.

6.      A small number of additional genes can be added, as the expression level of the 15 chosen genes that were confirmed by qRT-PCR is lower.

 I suggest that the article be published in the Agriculture journal with minor revision.

 Author Response

1. What is the duration of the oil sunflower cultivar ‘133211311’?

Answer: The whole growth period of the oil sunflower variety ‘133211311’ is 108 days. We add the relevant information in Sections 2.1 and 3.1. “In accordance with the planting practices of timely sowing, the whole growth period of this variety is about 108 days.”. “60 days after sowing, the ligulate flowers of the ef mutant were fully expanded and tubular florets were already visible, while the WT only appeared as a flower bud with a diameter of about 5.0 cm. The whole growth period of the ef mutant is 91 days, shorter than that of WT (108 days)”

2. Authors have screened visible early flowering mutant candidates in the M3 population throughout comparing the whole growth period and flowering time. Quantification can be done by counting the days needed to add the full plant's growth period, including any mutations that resulted in the first flower or 50% of the flower.

Answer: Thanks for your suggestion. This study was based on the stable early flowering mutant ef. According to our observation, the first flower of the mutant bloomed about 60 days after sowing, while the first flower of the control bloomed about 75 days after sowing. It is described in Section 3.1. “It (ef mutant) blooms 15 days earlier than the control WT in the field (Figure 1A). 60 days after sowing, the ligulate flowers of the ef mutant were fully expanded and tubular florets were already visible, while the WT only appeared as a flower bud with a diameter of about 5.0 cm.”

3. Line 120-2-121: The candidate mutants were self-pollinated to produce homozygous mutants in the subsequent M3 to M6 generation. Finally, one stable early flowering mutant was obtained. How many mutants in total were identified? how many were self-pollinated?

Answer: In the M₃ generation, we not only screened early-flowering mutants (only one early flowering mutant), but also focused on other visible mutant traits, such as plant height variation, yield variation, plant morphology variation, etc. In the M₃-M₆ generation, we used single flower bagging to self-pollinate sunflowers to achieve genotype purification. Bagging before flowering ensures that the seeds on a plant are originated from self-pollinated.

4. Line 188-189: The early flowering is a stable inherited mutant line selected after heavy ion beam irradiation. It blooms 15 days earlier than the control WT in the field (Figure 1A) needs to be expressed in days.

Answer: It has been modified according to your suggestion. “It (ef mutant) blooms 15 days earlier than the control WT in the field (Figure 1A). 60 days after sowing, the ligulate flowers of the ef mutant were fully expanded and tubular florets were already visible, while the WT only appeared as a flower bud with a diameter of about 5.0 cm. The whole growth period of the ef mutant is 91 days, shorter than that of WT (108 days)”

5. Authors identified 63 key genes involved in flowering induction, including flowering integrator genes, photoperiod pathway, hormone pathway, aging pathway, autonomous pathway and temperature pathway and flower development genes may be enlisted in the article may be as supplementary file.

Answer: Thanks for your suggestion. We have given these genes as Table S3, please see Revised Supplementary Materials.

6. A small number of additional genes can be added, as the expression level of the 15 chosen genes that were confirmed by qRT-PCR is lower.

Answer: Many experimental results have shown that there is a high correlation between qRT-PCR and RNA-seq results, but the two are not exactly the same. The reason may be that the two methods are different in gene expression quantification. The qRT-PCR experiment is based on a primer to amplify a gene region of about 200 bp to detect the expression level of a specific gene local region, and RNA-seq can theoretically achieve full-length gene expression quantification. In other words, the quantification of qRT-PCR is measured in the local region of the gene, and the quantification of RNA-seq is measured in the full-length region of the gene. On the one hand, our results show that there is a high correlation between qRT-PCR and RNA-seq results, and the up or down-regulation trend of gene expression is almost the same in results of qRT-PCR and RNA-seq. On the other hand, qRT-PCR verification experiments have consumed the same batch of tissue materials as RNA-seq, and re-sampling verification may cause large experimental errors.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Liu et al. presents a succinct investigation into the gene regulation of early-flowering mutants in oil sunflower. The study effectively highlights the identification of key genes and pathways involved in flowering induction in this crop species. The findings are interesting and merits publication. Only minor corrections are needed to enhance clarity and accuracy.

Abstract section: Some sentences could be rephrased for improved clarity and flow, particularly in the discussion of specific pathways and gene expression patterns.

Lines 162-165: “Genes were annotated by blast tool using databases NCBI non-redundant protein sequence (NR), Swiss-Prot protein (Swiss-Prot), Gene Ontology (GO), Clusters of Orthologous Groups (COG), Clusters of orthologous groups for eukaryotic complete genome (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG)” : You should add the link website you used for each one of these.

Figure 3: you showed the results of correlation analysis, the heatmap and Venn diagram. However in the methodology section, you did not provide detail about them. You should add the detail in the methodology section where you talk about the statistical analysis or the statistical tools that will be used and you enumerate them giving the objectives of each analysis done.  

Line 245: Avoid abbreviation from the subtitles (for all the manuscript)

Figure 5: is not clear, you have to use a picture with a higher resolution

Line 311: why “CORONATINE INSENSITIVE” is in capital letter !!!

Line 356 and 357: you have a lot of words in capital letters such “SUPPRESSOR OF OVEREXPRESSION OF”, “APETALA1” etc… And more in Lines 362 to 374 and so on…

So please verify all of them in the hole manuscript !!!

Conclusion section: Lines 530-532: you mentioned the useful information provided by your study. However, you did not mention if your findings hold practical implications for crop improvement strategies (such offering potential targets for genetic manipulation aimed at optimizing flowering time and enhancing yield in oil sunflower cultivars) which could adress agricultural challenges and meet the demands of a changing climate.

Line 532: Including a brief sentences on future research directions would enhance the conclusion.

 Author Response

1. Abstract section: Some sentences could be rephrased for improved clarity and flow, particularly in the discussion of specific pathways and gene expression patterns.

Answer: Thanks for your suggestion. The abstract and other sections of the manuscript have been revised as suggested. The abstract section was modified as: “These DEGs mainly enriched in biological pathways, including plant hormone signal transduction, carbon metabolism, protein processing in endoplasmic reticulum, secondary metabolism, and photosynthesis. We also found significant differences in the expression levels of starch and sucrose metabolism-related genes in the ef mutant and WT, indicating that sugar signaling plays an important role in the early flowering of oil sunflower, especially SUC9 and sugar synthesis and degradation enzyme genes. In addition to hormone and sugar signals, flowering integration genes SOC1, AP1, FUL and LFY were up-regulated in the ef mutant, and genes in photoperiod, aging, autonomous and temperature pathways are also involved in the regulation of floral transition.” Other modifications are shown in the revised manuscript.

2. Lines 162-165: “Genes were annotated by blast tool using databases NCBI non-redundant protein sequence (NR), Swiss-Prot protein (Swiss-Prot), Gene Ontology (GO), Clusters of Orthologous Groups (COG), Clusters of orthologous groups for eukaryotic complete genome (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG)”: You should add the link website you used for each one of these.

Answer: Thanks for your suggestion. The link website of databases has been added. Genes were annotated by blast tool using databases NCBI non-redundant protein sequence (NR) (https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/), Swiss-Prot protein (Swiss-Prot) (https://www.expasy.org/resources/uniprotkb-swiss-prot), Gene Ontology (GO) (https://www.geneontology.org/), Clusters of Orthologous Groups (COG) (https://www.ncbi.nlm.nih.gov/research/cog/), Clusters of orthologous groups for eukaryotic complete genomes (KOG) (https://mycocosm.jgi.doe.gov/help/kogbrowser.jsf) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/).

3. Figure 3: you showed the results of correlation analysis, the heatmap and Venn diagram. However in the methodology section, you did not provide detail about them. You should add the detail in the methodology section where you talk about the statistical analysis or the statistical tools that will be used and you enumerate them giving the objectives of each analysis done.

Answer: Thanks for your reminder. We added 2.6. statistical analysis to illustrate the data analysis and drawing method.

2.6. Statistical analysis

Correlation analysis between RNA-seq and qRT-PCR and bar chart drawing were conducted using Excel (Office 2021). The Venn diagram used the website (http://bioinformatics.psb.ugent.be/webtools/Venn/) to draw. The heatmap was completed using the conditional format color scale function of Excel or the BMKCloud analysis plat-form (https://www.biocloud.net/). Enrichment analysis was accomplished using BMK-Cloud platform. Finally, for image combination and adding notes, use Adobe Illustrator 2020.

4. Line 245: Avoid abbreviation from the subtitles (for all the manuscript)

Answer: Thanks for your suggestion. We have changed the abbreviations of SAM and DEGs in 3.3, 3.4, subtitles to full names (the shoot apical meristem，differentially expressed genes).

5. Figure 5: is not clear, you have to use a picture with a higher resolution

Answer: Thanks for your suggestion. Figure 5 in the manuscript has been replaced. If it can be uploaded, we will provide a 300 dpi figure with .tif file.

6. Line 311: why “CORONATINE INSENSITIVE” is in capital letter !!!

Answer: Thanks for your suggestion. “CORONATINE INSENSITIVE” is the full name of the gene, and has been changed to lowercase.

7. Line 356 and 357: you have a lot of words in capital letters such “SUPPRESSOR OF OVEREXPRESSION OF”, “APETALA1” etc… And more in Lines 362 to 374 and so on. So please verify all of them in the hole manuscript !!!

Answer: According to your suggestion, the full name of the gene in the manuscript has been changed to lowercase. Please see the revised manuscript

8. Conclusion section: Lines 530-532: you mentioned the useful information provided by your study. However, you did not mention if your findings hold practical implications for crop improvement strategies (such offering potential targets for genetic manipulation aimed at optimizing flowering time and enhancing yield in oil sunflower cultivars) which could adress agricultural challenges and meet the demands of a changing climate.

Answer: Thanks for your suggestion. We add the potential contribution of this study to the genetic improvement of sunflower in the discussion and conclusion part.

“It can be seen from our research results that the comprehensive external environment of plant growth, plant age, exogenous application of hormones, sugar sources, signal inhibitors, and genetic engineering methods to promote or inhibit flowering genes can all play a role in regulating plant flowering time. It is possible to develop new composite chemical agents for sunflower flowering and apply them to production practice.”

“The results provide useful information for understanding the functional genes of flowering, and the early flowering mutant obtained in this study can be further cultivated as a new oil sunflower variety. Hybridization of the ef mutant with other excellent cultivars is expected to obtain new sunflower varieties with short growth period or early maturity. Further mapping of the major gene linked to early flowering will enrich the functional gene research in sunflower and contribute to molecular assisted breeding.”

9. Line 532: Including a brief sentences on future research directions would enhance the conclusion.

Answer: Thanks for your suggestion. We add future research directions in the conclusion part. “Further mapping of the major gene linked to early flowering will enrich the functional gene research in sunflower and contribute to molecular assisted breeding.”