Identification and Functional Analysis of the Flower Development-Related TCP Genes in Erycina pusilla
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe work “Studies on the Flower Development Related TCP Transcription Factors of Erycina pusilla” described the characterization of TCP transcription factors in orchid, with emphasis in flower development/architecture, the work includes the use of the “model orchid” Erycina pusilla. Authors describe the identification of TCP candidate sequences using available transcriptome datasets for the model and TCP protein sequences of Arabidopsis; the work also covers differential expression of these candidates in E. pusilla, and heterologous expression of TCP relevant (differential expression, DE) candidate gene sequences using the A. thaliana Col-0 system.
These studies and the trait flower development is a very attractive field of research, making the paper interesting for the community due to the species under study.
Introduction. In general, the section is well organized, and includes relevant issues needed to proceed with the study. The first paragraphs in the section are adequate and achieve the explanation of the area of the work, which is flower development from genes´ perspective. However, a single sentence about Cyc-like genes as belonging to the TCP family should be convenient to be indicated/included, considering more general readers (Lines 56-58 do this job, however there is a jump from the previous paragraph). Also, authors need to remember that results will lead us to a broader spectrum than Cyc. For instance, lines 255-257 fit quite well at this rate.
Methods.
2.3. Actin gene sequence used as a reference for qPCR is not clear; please, confirm (ref 32) or include an accession number.
2.4. The indicated accession numbers consider mRNA sequences for these candidate TCP genes. In the text (lines 110-111), please, clarify how CDS were cloned into pCAMBIA1302 vector; primers and cloning details for full CDS generation have been indicated. Hygromycin concentration also needs to be established.
Results.
3.1. The section indicates that 15 sequences were found in the E. pusilla dataset, and that sequences are compared to 20 Arabidopsis AtTCPs; Supplementary Table 1S gives information for ten AtTACP protein sequences, why these discrepancies between At protein and nucleic acid data? This information should be included in captions for this figure.
3.2. “I” and “B” stages were previously defined (referenced), or authors organized these developmental stages according to their own experience? This is important because it seems that inflorescences and buds can be perfectly assigned in an opposite way.
Figure 2 should be better organized. A (flowers/buds images) B (qPCR).
3.3. As for the previous section, support of text (lines 166-168) on Figure 3 (it should be 3A) needs to be properly indicated. In the same line, including a flower scheme and organ correspondence (in 3A) will improve the relevance of the experiment and findings. This is important because in the following section authors are going for transgenics with 3 candidate genes.
Discussion. The section looks some disorganized. First, a paragraph establishing the novelty level of the main findings should improve the starting point of this section, and then covering (in a more organized way) the following points. For instance, a possibility can be covering these questions: 2. Is this the first report for these genes in the system? Then, 3. Did phylogenetic tree deductions agree to functional relevance found for some TCPs considering DE? And 4, indeed, the effect of selected TCP genes in Arabidopsis is well covered in the section, including potential interactions.
Comments on the Quality of English Language
Checking of English editing should be carried out. Some sections were not clear enough due to this reason.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study entitled:"Studies on the Flower Development Related TCP Transcription Factors of Erycina pusilla" by Tang et al., discusses the role of TCP transcription factors in the development of orchid flowers, particularly in the context of zygomorphy.
The study focuses on the expression patterns of TCP-domain genes (EpTCPs) in various tissues and floral organs of the orchid Erycina pusilla, and their potential involvement in floral morphogenesis. The results show that CYC-like genes (EpTCP25) and CIN-like genes (EpTCP11 and EpTCP26) are highly expressed in inflorescences but lowly expressed in leaves and roots. The novelty in this study lies in the identification and characterization of TCP-domain genes (EpTCPs) from the orchid Erycina pusilla, which are involved in the development of zygomorphic flowers.
Introduction :
- Can you provide more detail on the specific research question or hypothesis that this study aims to address, and how the results will contribute to our understanding of orchid floral development (in the last paragraph of the introduction)?
Materials and Methods :
1. Consider adding more detail on the specific primers used for qPCR, including their sequences and the annealing temperatures.
2. Consider adding more detail on the Agrobacterium-mediated plant transformation, including the specific strains used and the selection methods for transgenic plants.
3. The statistical part of this section is poor, please add more details on the statistical analysis used to calculate the relative expression levels, including the specific software used and the number of replicates.
4. Can the authors provide more information on the quality control measures used to ensure the integrity of the RNA samples and the accuracy of the qPCR assays, it is not necessary to include this information in the manuscript.
5. Provide more information on the specific software used for data analysis, including the version numbers and the specific parameters used to ensure the replicability of this significant study.
Results and Discussion :
I have some concerns regarding the results reported in the study, this would not undermine their quality, rather my questions seek to enrich the discussion :
- Can you provide more details on the characterization of the Arabidopsis transgenic lines, such as the confirmation of transgene integration and expression levels?
- Did you include any wild-type Arabidopsis plants or empty vector controls in your ectopic expression experiments, and how did these serve as controls for your observations?
- What were the criteria used to select the three EpTCP genes (EpTCP11, EpTCP25, and EpTCP26) for the ectopic expression experiments, and how do you plan to further investigate the roles of these genes in orchid floral development?
- Can you comment on the potential limitations of the Arabidopsis system for studying the functions of orchid TCP-domain genes, and how you plan to validate your findings in the native orchid system?
Conclusion and perspectives :
How do the findings of this study contribute to our understanding of the evolution of floral asymmetry in orchids, and what are the implications for our understanding of the 'Orchid Code' and 'Perianth Code' models? consider adding a conclusion to this study.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript titled “Studies on the Flower Development Related TCP Transcription Factors of Erycina pusilla” successfully identifies gene members from the TCP family of transcription factors in Erycina pusilla. The authors have provided a detailed description of the expression pattern in several tissues for each one of the genes identified. The functional validation of three genes was carried out by overexpressing them in Arabidopsis thaliana, showing interesting phenotypes. However, with the information provided in the methods and the results sections, I have a major concern about the functional validation and the phenotypes observed in the overexpressing lines from Arabidopsis thaliana.
The authors present results for three different lines per gene, but it was not reported the expression level of the transgene on any of those lines. The authors have provided clear pictures of several phenotypes, but without evidence showing that the transgene is being expressed on those lines, it is not possible to draw conclusions about the possible function of the gene. Following up on this idea, it was not mentioned the criteria to select those three lines per gene, was it due to phenotypic differences between those lines and the others?
Minor observations:
Could the authors provide which arabidopsis accession they used?
Could the authors add to the methods section which database was used to store the sequences they identified?
Are all the sequences identified in this work publicly available or just the three sequences with accession numbers?
Comments on the Quality of English Language
There are a few minor grammatical issues that need to be addressed, I will just mention a few:
Line 72: remove “on”
Line 73: a missing comma: in behavior “,” and
Line 73: a missing its: of “its” short growth
Line 76: development-related genes
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsI have thoroughly reviewed the manuscript titled "Studies on the Flower Development Related TCP Transcription Factors of Erycina pusilla" and find it to be scientifically valuable with important experiments and analyses. The manuscript studies the expression patterns of EpTCPs isolated from a reported Erycina pusilla transcriptome data. Moreover, three flower development related genes (EpTCP11, EpTCP25, and EpTCP26) were ectopically expressed in Arabidopsis to analyze their functions in plant growth and flower development. Altogether, interesting research with relevant data has been performed. However, I recommend minor revisions before publication. Firstly, I suggest improving the quality of the provided figures (in particular Figure 3) as they currently appear to be of low resolution. Clear, high-quality figures are essential for conveying the results effectively. Additionally, I encourage the authors to enhance the Materials and Methods section by providing more details on the performed experiments. This would improve the reproducibility and clarity of the study. Overall, with these minor revisions, I believe the manuscript will be well-suited for publication in its current form.
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the current version of the manuscript, authors have incorporated most of the suggestions and comments raised during my previous review. I thank authors this consideration.
From my point of view, the work, and its graphical display are valuable for publication in the Journal now. Please, take a look on Figure 1´s captions, I recommend, at this rate (paper finished!), to include some self-explanatory text about sequences´ sources (briefly, as indicated in the response letter should be OK).
Nice work, congrats!
Author Response
I would like to thank you for taking the time out of your busy schedule to review this manuscript again. Please find the detailed responses below and the corresponding revisions highlighted changes in red bold in the re-submitted manuscript.
Comment 1: From my point of view, the work, and its graphical display are valuable for publication in the Journal now. Please, take a look on Figure 1´s captions, I recommend, at this rate (paper finished!), to include some self-explanatory text about sequences´ sources (briefly, as indicated in the response letter should be OK).
Response 1: Agree. We added some text about the sources of sequences in Figure 1’s caption of the revised manuscript (line 150-157, marked in red bold).
