CfCHLM, from Cryptomeria fortunei, Promotes Chlorophyll Synthesis and Improves Tolerance to Abiotic Stresses in Transgenic Arabidopsis thaliana

Round 1

Reviewer 1 Report

The manuscript entitled “CfCHLM, from Cryptomeria fortunei, promotes chlorophyll synthesis and improves tolerance to abiotic stresses in transgenic  Arabidopsis thaliana” required revision before its acceptance in forests.

All the stresses should be elaborated in the abstract

Homogenize the use of terminology throughout the manuscript

CHLM gene should be italized throughout the manuscript

Agrobacterium tumefaciens should be italized

2.6 heading add heat stress in the section

2.8 heading heat stress or salt stress treatment are different than 2.6 sections why authors’ uses different treatments it should be uniform. How authors selected these doses add references

CAT, SOD, and POD in results, their units should be uniform throughout the results

chl fluorescence parameters decreased under stress the reason for decrease should be discussed in discussion section

Conclusion should be improved

References style is not uniform

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The article entitled “CfCHLM, from Cryptomeria fortunei, promotes chlorophyll synthesis and improves tolerance to abiotic stresses in transgenic Arabidopsis thaliana” is devoted to the study of the CfCHLM gene from C. fortune. Authors firstly obtained and characterized the CfCHLM gene from C. fortune, verified its response to such abiotic stresses as cold, heat, salt and drought using genetic engineering approach. Authors showed that CfCHLM gene is essential for chlorophyll synthesis and photosynthesis in plants and enhances the tolerance of Arabidopsis to abiotic stresses.

There are several critical points, which should be explained in details.

Research papers dealing with transgenic plants should present a clear proof of transgenesis. Authors present only one figure with PCR results for transgene detection. For each transgenic line authors give two lanes on the electrophoresis. It is nit clear why? Did you set up the same PCR reaction twice? What for? Obtained PCR product should be gel purified and sequenced to prove the primer specific binding to the gDNA ant the real transgene presence in the genome. Also, both negative (without DNA) and positive (with subcloned target gene) PCRs should be done and presented. Otherwise, it is not possible to prove transgenesis. Why do you have bands in the WT plant at the same position as in transgenes? Did you sequence obtained bands from WT plants to find out that this is Arabidopsis’ gene? How did you control DNA cross contamination during gDNA extraction?

The qPCR should be described more detailed. What type of RNA did you use (total RNA or mRNA). How RNA was reverse transcribed (using oligo dT or random hexamer primers)? What was the efficiency of qPCR primers? Which qPCR did you use – regular qPCR with cDNA or qPCR with reverse transcription? What KIT was used for qPCR? How did you control the absence of gDNA in RNA fraction? Did you make DNAse treatment of RNA fraction? Did you set up a control PCR with RNA as a template to check gDNA absence?

Also MS contains several mistakes which should be corrected.

The word “Arabidopsis” is written in different styles through the text, both with capital and lowercase letters and Italic and not Italic style. Please unify.

L117: What is “bioinformatics function of CfCHLM sequence”?

L132: “Agrobacterium tumefaciens” – change type to Italic.

L138: “in a 29 °C incubator “ – change to “in an incubator at 29 °C”

L140: “PBI121” change to “pBI121”

L144: “50 mg ml-1 kanamycin” – please check for the mistake, sure that ml but not L? Change “kanamycin” to “of  kanamycin”.

L145: “planted into soil after 10 d of culture in an incubator”.  What temperature was used?

Fig 1(A): Increase a quality. Hard to read.

Fig 1(D): Please give an explanations to the colors used at the figure (blue, pink, orange, green).

L303: “gene to to drought” change to “gene to drought”

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript regards a study aimed at investigating the role of the CfCHLM gene in promoting chlorophyll synthesis so from improving tolerance to abiotic stresses.

Overall, I found the paper to be well-written and informative, also the experimental activity was well carried out. However, I have a few comments and suggestions for publication on Forest.

The information on the length of the full-length cDNA and the ORF is already contained in the text, as well as the length of the protein, the molecular weight, and the isoelectric point for this reason I would suggest eliminating Table 1.

Indicate on which model the was built the tertiary structure of the CfCHLM protein.

Which tissue was considered for expression analysis in response to different abiotic stresses? Is not specified.

I suggest replacing "Chl content" with "Chl a+b".

The caption is joined to Figure 2, please insert a space.

Please review the manuscript carefully for grammatical errors, punctuation, and especially for italics. Some examples: lines 26, 27, 29, 132, 148, 153, 215, 224, 263, 267, 313....

The references are quite old, only four references in the period 2020 – 2024. I suggest including more recent references.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The Comments are adequately addressed and the manuscript is suitable for publication

Reviewer 2 Report

I would like to thank authors for the deep analyses of the comments and questions and full answers. Authors have carefully answered all the questions addressed to the manuscript. The transgenesis was proved by presented materials. All mistakes were corrected, manuscript reviewed and improved.