Identification and Expression Analysis of the Ethylene Response Factor Gene Family in Tea Plant (Camellia sinensis)
Round 1
Reviewer 1 Report
The research presented by Zhang et al focus on genome-wide analysis and expression analysis of ERF gene family in tea plant. Through transcriptome analysis, the authors propose that seventeen up and five down regulated ERF genes are responsive to cold stress. My main criticism to this manuscript is the significant redundancy between the submission and Liu et al. 2023 (doi: 10.1186/s12870-023-04221-y). Liu et al. 2023 is not cited in the current submission either. While I appreciate the aims of the study, there are several issues that should be considered including improvements in experimental design, data presentation, and language revision.
I have listed some comments and suggestions which help authors to improve their manuscript.
1) Abstract section: L14” were screened by bioinformatics” should be “ bioinformatics tools”. L17 “ elements were found” should be elements were predicted
2) L19-L20: “Subcellular localization predicts that 76 members are in the nucleus, 8 in chloroplasts, 5 in mitochondria and 1 in plasmid” what means CsERF gene located in plasmid?.
3) L29-L32 : should be rephrased
4) The quality of Fig.4 should improved and duplicated genes must be clearly highlighted
5) scientific names should be written in italics
6) I am concerned about the quality of the phylogenetic inference. The adjacency method is not the most robust phylogenetic method, so I suggest re-running phylogenetic analyses with a different, more robust method (like maximum likelihood, Bayesian, or maximum parsimony) and using CsERF genes only.
7) Fig. 7, Fig 8 and elsewhere “ERF gene family players” should be “ERF gene family members”
8) Fig. 7 and Fig.8 : what about the expression scale? Log2FC?
9) Fig 8 should be replaced with differential expression analysis using DESeq2. The differential ERF genes involved in cold responses will be clearly distinguished
10) Validation experiment of the result obtained though transcriptome analysis should be conducted using qPCR.
11) All the results should be compared and discussed with those reported vy Liu et al. 2023 (doi: 10.1186/s12870-023-04221-y).
12) Ectopic-/ heterologous expression of some candidate ERF genes can highlight their function in response to cold stress
Language revision
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
The experiment was well organized and performed and the theme has some novelty and is very interesting, but some improvements are needed. Overall, the manuscript will meet the publishing standard of the journal after revisions as follows:
Title:
- It is preferable to be satisfied with the plant's scientific name only in the title.
Abstract:
- Lines 11-12: Please correct to …….. widely present in plants, and have crucial regulatory importance …………………
- Line 19: ….., and 85 members …….
- Line 24,25, and 30: tea tree or tea plants? Please unify throughout the manuscript.
Keywords:
- Please do not repeat the title words in the keywords.
Introduction:
- Line 37: Please end the sentence at the reference [1] and then start a new sentence (It is ………..).
- Lines 53-54: Preferably, all plants should be written in "scientific names" or in "English".
- In introduction and throughout the manuscript: please consider the space between the last word of the sentence and the [reference].
- Line 78: …….. elements are ………
Materials and methods:
- Lines 99 and 101 (introduction), 104 and 107: tea trees or tea plants? Unify
Results:
- Fig. 4 needs to be high resolution.
Discussion:
- Lines 254-257: Long sentence, please divide it into at least two sentences.
- Line 262: Chen and others[45] should be Chen et al. [45].
- Lines 261-263: please rewrite the sentence to be correct.
- Throughout the discussion section: please unify the plant names; in scientific or in English (also throughout the manuscript).
- Throughout the discussion section: please write the gene names in italics (also throughout the manuscript).
Moderate editing of the English language is required.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The study of Zhang et al, discusses 90 members of the CsERF gene family that were identified through bioinformatics and named CsERF 1 to CsERF 90, in tea plant. These genes' molecular characteristics and evolutionary relationships were investigated, along with their tissue expression patterns and promoter cis-acting elements. The results revealed that most CsERF proteins had conserved motifs, with some members also possessing additional motifs. The proteins exhibited a range of theoretical isoelectric points (pI) and stability, with a majority predicted to be located in the nucleus. The promoter sequences of CsERFs contained cis-acting elements related to hormone regulation, low-temperature response, and light response, indicating their involvement in plant growth and abiotic stress. Phylogenetic analysis showed the division of tea tree ERF gene families into six groups, with varying member counts. Chromosomal location analysis indicated that most CsERF genes were distributed across 15 chromosomes. Collinearity analysis revealed homologous gene pairs among CsERFs, suggesting potential functional similarities. Furthermore, the expression of CsERFs was found to vary in response to cold stress and across different tissues, implying their roles in tea plant growth and adaptation to cold stress.
This is a very interesting paper that may serve as a foundation for further exploration of CsERF functions. Yet, the authors are invited to include the following suggestions, in their paper, for the sake of enhancing clarity for all the readers, before considering it for publication in Agronomy.
Introduction
· The description of the ERF gene family and its involvement in cold stress regulation is somewhat vague. More specific details about the functions and mechanisms of ERF genes in response to cold stress should be included to enhance the understanding of the topic.
· Line 48: There is no Ethiopian Response Factor. Do you mean ETHYLENE RESPONSE FACTOR, which belongs to the transcription factor family APETALA2/ERF?
· Line 49: Write AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR)
· Consider writing genes’ full names (in uppercase for the full form, and Italicized for the abbreviations)
Materials and methods
· Ambiguous description of screening and selection processes: Clarify the criteria used for screening and selecting target sequences. Explain the specific parameters or thresholds applied during the screening steps.
· The authors did not mention any method validation of the retrieved data, the authors are invited to include a section on how the methods were validated or their limitations. Discuss potential biases, error rates, or caveats associated with each analysis.
· Lack of references for online tools: Include proper references or citations for the online tools used in the study. This helps readers access and understand the tools used.
Results and discussion
· From all the data in the paper, summarize the functioning of ERFs in a model of the ethylene (ET) signaling pathway to ERFs, in the presence of ethylene in Camellia sinensis.
· Lines 254 – 275: The section compares the number of CsERFs with other plant species (rice, Arabidopsis, ginger, strawberry, and durian). It would be helpful to briefly discuss the significance or implications of the CsERFs count being less than some species and greater than others. It is not clear why this comparative analysis is conducted without drawing any conclusions of it!.
· Lines 287 – 295: The section mentions the phylogenetic analysis of CsERF members and their categorization into six groups. It would be beneficial to explain the significance of this categorization and how it relates to the overall understanding of CsERFs. Similarly, when discussing gene expression patterns under cold stress, provide more details on the specific genes that were upregulated or downregulated and their potential roles in cold stress tolerance. It is not clear within the text.
Conclusion
· consider summarizing only the main findings and their implications for the understanding of CsERFs in Camellia sinensis
Comments for author File: 
Comments.docx
Author Response
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Reviewer 4 Report
The manuscript titled with "Identification and Expression Analysis of ERF Gene Family in Tea Plant (Camellia sinensis)" is considered as a one of the bioinformatic evaluation for the tea plant transcriptome. The idea and the work which approached in this manuscript are very good. But I have some comments which could help in the manuscript improvement such as;
1-The Title should be changed into; Identification and Expression Analysis of ERF Gene Family in Tea Plant (Camellia sinensis) virtually" because most of the bioinformatics results is kind of expectation not in real.
2-At the end of the abstract, lines 32-33; This study provides a basis to go a step further seeking the functions of CsERFs, yes this is the conclusion for your work but where is convey for the readers, the stone from which the others will start work under this subject. The missing part are presented at the end of the introduction " to provide a theoretical foundation for the study of tea tree growth and development and low temperature resistance " you can add this sentence at the end of the abstract please.
3- Introduction lines 78-82 needs references.
Lines from 86-88 needs refrences.
Results; The authors studied 90 ERF genes and study the cis-acting elements among the examined genes. Is there any of Trans-acting elements are existed among the studied genes or not. Especially the genes are founded in 15 different chromosomes and in different organelles inside the tea cells.
Discussion: written in very good manner but needs more citation especially in 2023.
Conclusion: is too long and should be summarized and contains the convey and the sold results in final.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
In their revised version of the manuscript, the authors have addressed some of the minor concerns; however, the most important issues are still open. In particular, the responses to the major concerns related to the transcriptomic analysis and validation of in silico obtained findings, as they raise doubts about the reliability and objectivity of this scientific research and the accuracy of the results and conclusions drawn.
Critical points must be revised and clarified and the authors should consider the following aspects.
1- In their rebuttal letter, the authors reported that “the Subcellular localization is the result of website prediction, which is not completely true”. So, the authors should use more than one subcellular component annotator to augment the power of the plant protein subcellular localization prediction
2- Fig.4: the duplicated genes must be clearly highlighted. The scale for the lower part of the fig.4 should be checked.
3- The adjacency method is not the most robust phylogenetic method, so the authors should re-running phylogenetic analyses with a different, more robust method (like maximum likelihood, neighbor-joining, Bayesian, or maximum parsimony) and using CsERF genes only. Several published papers (listed below) on Tea genes support the use of a robust method to build phylogenetic tree.
doi: 10.1515/biol-2022-0466
https://doi.org/10.1186/s12870-023-04221-y
https://doi.org/10.1371/journal.pone.0166727
https://doi.org/10.3389/fpls.2022.1008408
4- In their rebuttal letter, the authors reported that “the expression level was estimated by FPKM”; however this information was not mentioned either in the scale presented in fig.7 and 8 nor in the M&M section
5- The transcriptomic data was expressed on FPKM in the public database.
The scientific research was never a simple narrative presentation of already public data but rather a presentation of new findings derived from analysis using public data. The annotated genome of Tea plant genome (http://eplant.njau.edu.cn/ ) and the transcriptomic data (on FPKM) are publicly available, so the transcript count can be simply generated and differential expression analysis using DESeq2 can be conducted.
6- qPCR validation experiment of in silico transcriptomic analysis is strongly recommended and is not acceptable anymore to publish an in silico analysis without validation. Several published paper support this direction:
Liu, Y., Chen, S., Chen, J. et al. Comprehensive analysis and expression profiles of the AP2/ERF gene family during spring bud break in tea plant (Camellia sinensis). BMC Plant Biol 23, 206 (2023). https://doi.org/10.1186/s12870-023-04221-y
An Y, Xia X, Jing T, Zhang F (2022) Identification of gene family members and a key structural variation reveal important roles of OVATE genes in regulating tea (Camellia sinensis) leaf development. Front Plant Sci 13:1008408. doi:10.3389/fpls.2022.1008408.
Chen, Y., Niu, S., Deng, X. et al. Genome-wide association study of leaf-related traits in tea plant in Guizhou based on genotyping-by-sequencing. BMC Plant Biol 23, 196 (2023). https://doi.org/10.1186/s12870-023-04192-0
7- The reported results should be discussed with those reported by Liu et al. 2023 (Liu, Y., Chen, S., Chen, J. et al. Comprehensive analysis and expression profiles of the AP2/ERF gene family during spring bud break in tea plant (Camellia sinensis). BMC Plant Biol 23, 206 (2023). https://doi.org/10.1186/s12870-023-04221-y )
Minor
Author Response
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