Characterization of the MADS-Box Gene CmFL3 in chrysanthemum

Round 1

Reviewer 1 Report

Summary:  

The authors provide the results of an applied genetics study wherein the apply knowledge of the flower induction effects of the Arabidopsis FRUITFULL (FUL) gene to a commercial Chrysanthemum moriflorium species.  Several previous studies had predicted a role for homologs of the AtFUL gene in promoting flowering in Chrysanthemum.  Here they report that overexpression of the CmFUL gene in C. morifolium itself caused earlier flowering and that downstream flowering genes were also upregulated. The findings are not surprising given the earlier work of overexpressing the CDM111 FUL gene in C. morifolium.  However manipulation of genes for agronomic or horticultural practice favors the manipulation of endogenous genes by methods such as gene editing - therefore this study identifies a suitable gene for such manipulation and is a useful step forward.

Major and Minor Comments

Introduction: OK - they acknowledge previous work in the filed including closely related work.

Results:

Fig 1 I agree from the MSA and phylogenetic tree that the CmAFL3 protein is very similar to the AtFUL protein and different from the CDM111 protein used in the earlier study.  They should be sure to provide a GenBank number for the newly isolated Chrysanthemum morifolium genes/proteins - I note that they are not provided in the supplementary file.

Fig 2. Here they provide qRT-PCR evidence that the expression of CmFL3 increases significantly in the receptacle region of the flower during short day flowering conditions.

The in situ image is incorrectly referred to as Fig 2D instead of Fig 2C and they omit to point out the FM in the image.

Fig 3. Here report that a CmFL:GFP fusion protein localizes to both the cell membrane and the nucleus during transient expression in onion epidermal cells.  Although that may be true this part of the study is weakened by the lack of controls for subcellular localization and the fact that it is transient expression only in a different species and non-flowering tissue.

They should discuss their findings in the light of the much more detailed study by Urbanus et al from 2009 using GFP fusion transgenics and confocal microscopy in Arabidopsis.

Urbanus, S.L., de Folter, S., Shchennikova, A.V. et al. In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana. BMC Plant Biol 9, 5 (2009). https://doi.org/10.1186/1471-2229-9-5

Fig 4: I agree that in Fig 4A the CmFL3 protein does not appear to provide transactivation activity in yeast although this is often the case in a heterologous system.  Fig 4B does indicate that CmFL3 may act as a repressor of gene activity - however I would have liked to have seen better quantification in this assay in terms of units of the reporter enzyme - also the color gradient is not labelled - what are the units of 4061 and 1398?

Fig 5: Here they show evidence that overexpression of CmFL3 causes earlier flowering.  In Fig 5A and 5B RT-PCR data are shown.  They should clarify if Fig5B is the result of three biological replicates or three technical replicates. Overall this is the most valuable figure in the paper as it indicates CmFL3 as a suitable target for future manipulation.

Fig 6:  Here they report on the effect of overexpressing CmFL3 in Chrysanthemum on suspected target genes.  This is not too surprising but they should be careful in stating their conclusions since the effect may be indirect - they have shown direct binding of CmFL3 to the promoters of these genes.

Fig 7: Here overexpressing the CmFL3 heterologously in Arabidopsis creates the expected phenotype of earlier flowering and altered downstream genes. This is supportive of the conservation of this function across species and again helps support the idea that targeting this gene is suitable for altering flowering time in Chrysanthemum. 

Discussion: OK although the last paragraph about Gerbera may be omitted. 

Materials and Methods:

Generally OK as most of the methods have bene previously described and use. 

References:  OK - but add in the one that I requested above. 

Supplementary:

Supplementary results are OK except the GenBank numbers need to be provided for newly isolated Cm genes.

Minor Spelling and grammar errors

Abstract          Fifth and sixth sentences should not start with "And "  - See also other places in the manuscript like the legend of Fig 5.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present study demonstrated the function of CmFL3 in regulating the flowering time and further revealed that it could affect the expression of flowering-related genes CmAFT and CmFTL3. Although this work's overall interest and visibility, some aspects should still be considered to improve the quality and objectiveness.

1.      The background of the study should be made very clear. Provide more details of the introduction and review of the work.

2.      Please speculate about the reasons for the obtained results. The discussion needs to improve.

3.      In Conclusion, the authors should add the potential practical application.

4.      The article should be reviewed for English language proficiency and grammar. There are a lot of sentences without sense, misspelled words, and punctuation errors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The study has great potential but it needs to be text edited before it can be accepted. Many sentences are incomplete, are missing determinants/articles or are being used incorrectly. Scientifically, I have the following major comments:

- It has not particularly easy to follow that SD refers to short-day conditions. I understand that this has been written in the introduction (once) but then there is also a SD/-His-Ade medium. So, I assumed that the "SD treatment" refers to short-day conditions though this is not specified in M&Ms. However, Arabidopsis was planted in "LD conditions"?? In sum: it is necessary to detail the treatments involved in this study. 

- It was very hard to understand how many plants or replicates were used in the experiments?

- Statistical results are also indicated but these need to be detailed in M&Ms. Sometimes the authors mention a Student test and sometimes a Duncan test (?) - not sure why this refers to posthoc tests after ANOVA (not mentioned). 

- Please explain the axis of Figure 6. What are the numbers in the x-axis referring to?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf