PsFT, PsTFL1, and PsFD Are Involved in Regulating the Continuous Flowering of Tree Peony (Paeonia × lemoinei ‘High Noon’)

Round 1

Reviewer 1 Report

This study analyzed the genetic mechanism of the continuous flowering behavior of tree peonies. A detailed analysis of PsFT, PsFD, and PsTFL1 (previously identified genes related to flower induction and differentiation in other species) revealed the role of PsTFL1 as a flowering inhibitor since it was overexpressed in the cultivar that flowers once a year. The authors also established an interaction between the protein and its cellular localization.

 

I find this work interesting since through light on the regulation of different flowering behaviors. All the work is well presented and discuss. I suggest some changes in order to improve the manuscript (see the comments on the manuscript).

Comments for author File: Comments.pdf

Author Response

Thanks for the advice. We have studied comments carefully and have made correction. Please see the attachment. Revised portions are marked in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Zhang et al. presents an analysis of the continuous flowering mechanisms in tree peony. The authors consider three main regulators of flowering in plants, namely, the homologues of the Arabidopsis FT, FD and TFL1 genes. The earlier studies have shown that an absence of the TFL1 homologue causes continuous flowering in rose. The authors propose the similar role of this gene in tree peony, however, provide no data on TFL1 mutants in peony. The manuscript considers the mRNA expression of FT, FD and TFL1, as well as the protein-protein interactions of FT and TFL1 proteins with the FD protein and the subcellular localization of these proteins. The RT-PCR results suggest that the FT, FD and TFL1 genes may be involved in the continuous flowering, however, this part of the paper does not have a clear connection with the analysis of the protein products of these genes.

Overall, the authors should rewrite the Discussion and Conclusion to get rid of conclusions, which are not supported by the experimental results.

Comments:

1)     The authors did not use the template with the line numbering and I hope that the phrases from the text that I cited in my comments will be easy to find.

 

Introduction:

2)     “The transition from vegetative to reproductive development (flowering) requires environmental signals, such as photoperiod and vernalization, which triggers the expression and regulation of endogenous genes..”  What do the authors mean by “endogenous genes”?

3)     “Structural studies and in vivo analyses of these genes have revealed that they form an external loop along with an adjacent peptide.” Which in vivo studies do the authors mention? What is meant by the adjacent peptide?

4)     “FT with FD binds to the promoter of APETALA1 (AP1)”. “APETALA1 (AP1)” should be in italics.

5)     “In rice, 14-3-3” (the FT-like gene in rice)”.  14-3-3 should be in italics.

 

Materials and Methods:

6)     “The apical buds used for expression analysis were collected using paraffin sectioning to detect the developmental stage.” Please provide the details of paraffin sectioning. Was the total RNA extracted from paraffin sections?

 

Results:

 

7)     What is shown by green color in Figure 1?

8)     Figure 1b: “After the first flower appears at the early of May, a portion of the buds continue germinating, and floral induction occurs in early July. CF bud differentiation is completed shortly after, and flowering occurs between late August and early October. “ To my mind, these two periods (early May-early July and late August - early October) are not shown precisely. In my opinion, the continuous flowering proceeds to mid-October.

 

Section 3.3.

9)     “In the three cultivars (species), PsFT or PsFD shared overall similar expression pattern during the flower development, albeit the difference in levels.” It is evident that HN has different expression patterns of FT and FD (Figure 3).

10)  “The results indicate that the expression levels of the other two genes were higher during the S3 stage in CF ‘HN’, with the exception of PsTFL1, which had a relatively low expression level.” Please correct this phrase, it is not evident what is meant by «the other two genes with the exception of TFL1»

11)  “These observations implied that, unlike PsFT-PsFD, PsTFL1-PsFD might repress flowering.” ??? Where does such a conclusion come from?

12)  Figure 4:  What does the chlorophyll autofluorescence show? Why is it absent from the two upper panels (PsFT-GFP and PsTFL1-GFP)?

13)  Section 3.5: “In contrast, the number of bolting leaves in wild-type (WT)..” Rosette leaves?

14)  Figure 5 legend: “Ectopic expression of PsTFL1 in tree peonies induced late flowering in A. thaliana”. Should be: “PsTFL1 from tree peonies induced late flowering in A. thaliana”.

 

Discussion:

15)  Section 4.1. should be divided into paragraphs for readability.

16)  “This structure can act as a switch between two downstream possibilities in Arabidopsis”. What did the authors mean by “two downstream possibilities”?

17)  “In line with the findings in Arabidopsis[14], we concluded that PsFT-PsFD or PsTFL1-PsFD may promote or delay flowering in tree peony.” The authors may suggest it, but this conclusion does not follow from their results.

18)  “Unlike other species with more than one copy[39,40], this single copy of PsFT in tree peony functions in common bud differentiation, CF flowering. These findings indicate that PsFT acts as a florigen that is essential for flowering and floral induction in tree peony.” This statement has not been confirmed either.

19)  I do not think that the authors should compare the number of FT and TFL genes in peony with those in Medicago or pea, since the increased number of copies of these flowering regulators is a property of legumes.

20)  “This indicates that the low expression of PsTFL1 in CF tree peonies could allow them to flower continuously under suitable conditions.” This might suggest….

21)  “According to the overlap in the expression curves of PsFD and PsFT during the bud differentiation of CF ‘HN’, the expression of PsTFL1 was reduced gradually when the expression of PsFT increased, which coincides with the expression of PsFD.” The meaning of this phrase is unclear.

22)  “The results of our study showed that the regulatory mechanism of PsFT, PsTFL1, and PsFD may be related to the CF trait in the tree peony ‘HN’.” This article did not show the regulatory mechanism.

23)  “The florigen and repressor protein could form a complex with PsFD simultaneously, and the expression of PsFD was detected in both the common and CF flowering of ‘HN’.” This phrase mentions the expression of FD mRNA and the binding properties of FD protein, which are not connected with each other.

24)   Conclusion: “Our findings suggest that PsFT and PsTFL1 can interact with PsFD to form a complex, and the gene regulation patterns of PsFD, PsFT, and PsTFL1 may be involved in the CF flowering of ‘HN’, as this mechanism was only detected in the CF tree peony cultivar ‘HN’.” The manuscript does not contain any evidence of a regulatory mechanism.

Author Response

Thanks for the advice. We have studied comments carefully and have made correction. Please see the attachment. Revised portions are marked in the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript by Zhang et al. has been revised, and the authors satisfactory addressed almost all comments.

The only remaining issue is the text inserted on page 9 at the end of Section 3.3: “It has been reported that TFL1 lacks recognizable nuclear localization signals, which significantly reinforces the notion that TFL1, along with FT, functions as a transcription mediator molecule[14,27]. Furthermore, FD, a member of the bZIP transcription factor family, is the one of candidate transcription factors that may interact with TFL1. Therefore, we conducted subsequent studies related to subcellular localization, ectopic expression and protein interactions”.

The authors tried to link the section on FT, TFL1, and FD mRNA expression with the analysis of subcellular localization and interactions of the protein products of these genes, but I do not think that such a link is necessary. All this information is presented in the “Introduction”, in exactly the same phrases (“Additionally, TFL1 lacks recognizable nuclear localization signals, DNA-binding domains, as well as activation or repression domains. These findings significantly reinforce the notion that TFL1, along with FT, functions as a transcriptional mediator molecule[14,27]” (see page 2).

So I suggest to remove this text from Section 3.3.

Regarding the content of the above paragraph from the “Introduction”, I cannot agree that “TFL1 lacks recognizable nuclear localization signals”. Please check the paper by Zhu et al. (https://www.nature.com/articles/s41467-020-18782-1) which writes:  “Our data point to a prominent nuclear role for TFL1 and show that FD recruits TFL1 to the chromatin of target loci.”