Transcriptomic Analysis of Yunwu Tribute Tea Leaves under Cold Stress

Round 1

Reviewer 1 Report

The manuscript number cimb-2083673 entitled "Transcriptomic Analysis of Yunwu Tribute Tea Leaves Under Cold Stress" explained the experimental analyze the transcriptome data of Yunwu Tribute Tea, an endemic tea species in Guizhou Province, China, under low temperature stress comprehensively analyzed. The results will help to further understand the cold-responsive molecules of Yunwu Tribute Tea mechanism, and provide a new theoretical basis for cold-resistant germplasm screening and cold-resistant breeding of tea trees. In general, the article is worthy of acceptance, and has a certain significance for the study of cold resistance of tea trees, and can also attract the attention of the audience.However, in my opinion there are a few minor errors that need to be improved: 1.Camellia sinensis (L.)Kuntze var.niaowangensis Q.H.Chen is the Latin for Yunwu Tribute Tea, I suggested to use the Latin in the title. 2.How many plants are kept for temperate treatment?Authors mentioned upper leaves. Is it a leaf or leaf bud? Why did authors select upper leaves? 3.Please include more papers for Tea plant. For example, research on cold resistance of tea trees, the tea plant germplasm . 1）Samarina LS, Malyukova LS, Efremov AM, Simonyan TA, Matskiv AO, Koninskaya NG, Rakhmangulov RS, Gvasaliya MV, Malyarovskaya VI, Ryndin AV, Orlov YL, Tong W, Hanke MV. Physiological, biochemical and genetic responses of Caucasian tea (Camellia sinensis (L.) Kuntze) genotypes under cold and frost stress. PeerJ. 2020 Aug 28;8:e9787. 2）Zhao Y, Wang R , Liu Q , et al. Genetic Diversity of Ancient Camellia sinensis (L.) O.Kuntze in Sandu County of Guizhou Province in China[J]. Diversity, 2021, 13(6):276. 3）Zhao M, Jin J, Wang J, Gao T, Luo Y, Jing T, Hu Y, Pan Y, Lu M, Schwab W, Song C. Eugenol functions as a signal mediating cold and drought tolerance via UGT71A59-mediated glucosylation in tea plants. Plant J. 2022 Mar;109(6):1489- 1506.

Author Response

1.Camellia sinensis (L.)Kuntze var.niaowangensis Q.H.Chen is the Latin for Yunwu Tribute Tea, I suggested to use the Latin in the title.

Reply：Yunwu Tribute Tea is literally translated from English, the Latin name [Camellia sinensis (L.)Kuntze var.niaowangensis Q.H.Chen]  has been indicated in line 56 of the article. I think the literal English name should still be used in the title of the paper.

2.How many plants are kept for temperate treatment?Authors mentioned upper leaves. Is it a leaf or leaf bud? Why did authors select upper leaves?

Reply：In the previous experiments of different low temperature stress treatments, we used 20 plant samples for each temperature treatment, which was explained in the reference at line 106 of the paper. For transcriptome sequencing of plant leaves, samples from fresh, tender and vigorous growth parts should be selected for rapid collection. Therefore, in this paper, young leaves with vigorous growth at the upper part were collected, and three biological replicates were carried out, as explained in line 114 of the paper.

3.Please include more papers for Tea plant. For example, research on cold resistance of tea trees, the tea plant germplasm . 

Reply：Although the three literatures proposed by the reviewer are relatively new and belong to Guizhou tea tree, they are not very relevant to the content of my paper. I will add some new references and list them in the revised draft submitted.

Reviewer 2 Report

Wang and collaborators investigate the process of cold-stress response in Tea plants (Yunwu Tribute variety) by transcriptomic approach. Differential gene expression analysis and gene ontology analysis revealed several interesting molecular pathways that might be involved in response to cold stress.

The research question is relevant and meaningful, and it would provide new dataset for research group that work on cold stress adaptation also in different plant species. The data presented seems robust and promising, and the experimental design and tools used for the analysis are clearly explained. Anyway, my big concern regards the way results are displayed and discussed in the manuscripts.

Major comments:

The results section severely suffers for ‘shopping-list’ effects, since the gene and category found by the RNA-seq are only listed but not explained in a way that the reader could understand the most relevant pathway and genes founded among the DEGs.

Then in my opinion the discussion section seems a separate review about cold stress response in plants, being completely unlinked from the results obtained in this work.

Genes that are already known to be involved in cold stress response in other plants (such as the ICE1-COR-DREB) are not even mentioned in the introduction or in the results sections but only proposed at the end in Figure 14 but again unlinked form results obtained. Authors should analysis the expression of the homologous of those genes in their dataset to investigate whether those genes are also involved in cold stress response in Tea plants. 

Then among the genes confirmed by RT-PCR authors should focus on few of them and explain why they are interesting and in which way they could represent a target for future application in identification of cold stress Tea tolerant variety.

Category presented in GO analysis (Figure 3) are too general. I would suggest to remove general category such as ‘biological regulation/cellular process’ in order to highlight other less general category that might be more interesting.

 

Minor comments:

Line 62: please explain more in detail the effect of low temperature stress on cell membrane system.

Results: I strongly suggest explaining in the results section the characteristic of the samples collected for the RNA-seq analysis (developmental stage of the leaves, number of biological replicates,…). The part that is now mentioned in material and methods line 99-101 should be explain in the details in the Result section. Names given to samples DW: cold-treated group and CK: control group are misleading.

Line 252- 266: I suggest talking before about Protein coding sequence prediction (Paragraph 3.4.) and then going into the details of transcription factors (Paragraph 3.3.). So invert order of figure 4 and 5. 

Line 221: space missing

Line 225: Explain ‘having general function’

Line 272: space missing

Line 291: the enzyme system involved in eliminating reactive oxygen species should be explain in the detail in the first part of paragraph 3.5.1 to help the non-expert readers in understanding the genes and category mentioned then below (such as FAR, CAT, SOD).

Line 413: gene

Line: 416 space

Line 417- 420 I suggest discussing more in the details the gene mentioned, in which pathway are they involved? Does it fix with the enriched categories found in the GO analysis?

Figure 6. What does it mean the category ‘global and overview maps’?

Explain what green and red colors mean in figure 8-9-10-11.

Author Response

1.The Discussion and References sections have been revised, please see the attachment.

2. DW: cold-treated group and CK: control group only stand for abbreviation. It is noted in line 37 of the article, which will not cause readers to misunderstand.

3.Red colors mean up-regulated and green colors mean down-regulated in figure 8-9-10-11.These are illustrated in the Figure legends.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thanks to the more detailed explanation added to the manuscript I think the manuscript has improved and it is now easier to follow. Anyway in my opinion the discussion remains a bit disconnected since it does not present any clear reference to the results obtained. This aspect does not compromise the validity of the work and the quality of the data but only lowers the presentation quality.   minor comments: minor spell check required (like space error in line 416 and 583).