Genome-Wide Profiling of the Genes Related to Leaf Discoloration in Zelkova schneideriana

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The MS under consideration "Genome-wide profiling of the genes related to leaf discoloration in Zelkova schneideriana" makes an interesting story where the researchers  makes important investigations in a landscaping flowering plant. Through RNA analyses, they reported several DEGs  between red and green lines.  Furthermore, it was found that four transcription factors, positively regulated the anthocyanin synthesis genes. In the end they concluded that the information is helpful in breeding colorful plants. 

Overall, I have found the MS with appropriate merit as it concludes efficiently and an easy follow up of the article. The introduction, mat and methods and results including discussion are of appropriate length. I have made 3-4 comments in the attached file for your reference to correct before submitting the final version.

Best regards,

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Overall, I have found the MS with appropriate merit as it concludes efficiently and an easy follow up of the article. The introduction, mat and methods and results including discussion are of appropriate length. I have made 3-4 comments in the attached file for your reference to correct before submitting the final version.

Reply: Modifications were performed according to the comments

Reviewer 2 Report

Comments and Suggestions for Authors

This research discusses the molecular mechanisms underlying the discoloration of Zelkova schneideriana leaves in the fall season, focusing on the differential gene expression related to anthocyanin and chlorophyll biosynthesis. Results show that the transcriptome sequencing identified many differentially expressed genes involved in pathways.

The analysis of the published data was provided with a sufficient level of scientific novelty. The text of the paper is factual concrete, realistic, and understandable.

However, there are important flaws in the manuscript listed below:

 -In the materials and methods section, the sampling methods are unclear. What is the genetic difference between the red line (the leaves turned red) and the green line (the leaves remained green)? Are both of them similar? If not, how can we conclude that the gene expression differences are due to color or any other characteristics? How many technical and biological replicates were considered for transcriptome analysis? Regarding Table 1, three samples were considered for each genotype, but in line 133 of the materials and methods section, it is mentioned that twelve seedlings were considered and in lines 138-139, it is mentioned that five seedlings were considered for each genotype. More clarification about the sampling is needed.

 -According to the KEGG analysis, lines 250-251 show that DEGs are mainly enriched in the plant-pathogen interaction pathway. More explanation is needed to understand this result. What is the relationship between color and plant-pathogen interaction?

 -In Table 3, “Gene number” should be replaced with “Number of genes”.

 -In some references such as No. 29 and 30 the scientific names of the journal need to be italicized.

Author Response

Reviewer 2

 -In the materials and methods section, the sampling methods are unclear. What is the genetic difference between the red line (the leaves turned red) and the green line (the leaves remained green)? Are both of them similar? If not, how can we conclude that the gene expression differences are due to color or any other characteristics? How many technical and biological replicates were considered for transcriptome analysis? Regarding Table 1, three samples were considered for each genotype, but in line 133 of the materials and methods section, it is mentioned that twelve seedlings were considered and in lines 138-139, it is mentioned that five seedlings were considered for each genotype. More clarification about the sampling is needed.

Reply: The Zelkova seeds were collected from the same provenance, which are of similar genotype but have different leaf color exhibition. The both red and green lines have green leaves in warm season but the red lines turned to red and the green lines remained green in fall season. The leaf color changes were accordingly due to the gene expression difference that were confirmed in some other species (cited in the introduction section). We selected three plants (genotype) with similar performance as the replicates of the red or green line for transcriptome analysis and then screened the differential expressed genes (DEGs) between the red lines and green lines, which will possibly find out the real DEGs. They were also used for the physiological index tests and pigment contents measurement.

Since Zelkova genome were not sequenced, we then did the full-length transcriptome sequencing with the mixed leaf samples from twelve Zelkova seedlings with different leaf color, resistance to chilling stress, ground diameter, and more. We can then gain more gene sequence information and used for assembling the gene fragments by the second-generation transcriptome sequencing.

Some modifications were performed in the material section.

 

 -According to the KEGG analysis, lines 250-251 show that DEGs are mainly enriched in the plant-pathogen interaction pathway. More explanation is needed to understand this result. What is the relationship between color and plant-pathogen interaction?

Reply: A paragraph was added in the discussion section

 

 -In Table 3, “Gene number” should be replaced with “Number of genes”.

Reply: It was corrected

 -In some references such as No. 29 and 30 the scientific names of the journal need to be italicized.

Reply: All were corrected

Reviewer 3 Report

Comments and Suggestions for Authors

The authors' article Yuhan Sun, Longfeng Gong, Xiao Liu, Jiayu Hu, Xiaoxiong Lu, Jichen Xu «Genome-wide profiling of the genes related to leaf discoloration in Zelkova schneideriana “ It is devoted to changes in the content of pigments (chlorophylls, anthocyanins, carotenoids) and their ratio with seasonal changes in light and temperature.

The topic is interesting, reflecting the individual reaction of individual species, cultures and samples. However, there are disadvantages.

Introduction

 

- It is necessary to clearly identify the purpose of the study, its relevance and novelty. Next, it is necessary to indicate the possibility of further use of the obtained results and their practical significance.

 

Materials and methods

- It is unclear how many plants were taken in the study as a material.

-How many plants with initially green, red and red-green leaves were taken into the study, as noted in Table 1.

-Describe in more detail the conditions of sample’s reproduction (location of reproduction, soil and climatic conditions), which is very important for the pigment’s formation.

- Were the pigments detected on a spectrophotometer? It is necessary to indicate the brand of the device, the country of its manufacture.

- when determining brightness, the authors write about red/green phase a*, and yellow/blue phase 93 b*. Describe in more detail what this means and the characteristics of the device (Brand, country of manufacture).

- Which sugar was used to build the calibration model for determining soluble sugars.

- please note whether Zelkova schneideriana belongs to deciduous plant forms.

- Lines 352-354 The sentence is unclear. «Obviously, the chlorophyll content 352 and the chlorophyll/anthocyanin ratio influenced the appearance of plant leaves in the red 353 period». How does chlorophyll/anthocyanin ratio affect leaf appearance? Apparently the sentence is inaccurately formulated, it needs to be rephrased.

- The introduction and conclusion do not reflect the relevance and perspective of the practical use of the results

Author Response

Reviewer 3

- It is necessary to clearly identify the purpose of the study, its relevance and novelty. Next, it is necessary to indicate the possibility of further use of the obtained results and their practical significance.

Reply: Some modifications were performed in abstract, introduction and conclusion

 

- It is unclear how many plants were taken in the study as a material.

Reply: Some modifications were made in the material and method section

 

-How many plants with initially green, red and red-green leaves were taken into the study, as noted in Table 1.

Reply: It was noted in table 1.

 

-Describe in more detail the conditions of sample’s reproduction (location of reproduction, soil and climatic conditions), which is very important for the pigment’s formation.

Reply: They were modified in the material and method section

 

- Were the pigments detected on a spectrophotometer? It is necessary to indicate the brand of the device, the country of its manufacture.

Reply: The pigments were detected on a spectrophotometer. More details about the device were added in Material and methods section.

 

- when determining brightness, the authors write about red/green phase a*, and yellow/blue phase 93 b*. Describe in more detail what this means and the characteristics of the device (Brand, country of manufacture).

Reply: More details about the device were added in Material and methods section.

 

- Which sugar was used to build the calibration model for determining soluble sugars.

Reply: Glucose was used to build the calibration model for determining soluble sugars. Modifications were performed in Material and methods section.

 

- please note whether Zelkova schneideriana belongs to deciduous plant forms.

Reply: It was described in abstract and introduction section

 

- Lines 352-354 The sentence is unclear. «Obviously, the chlorophyll content 352 and the chlorophyll/anthocyanin ratio influenced the appearance of plant leaves in the red 353 period». How does chlorophyll/anthocyanin ratio affect leaf appearance? Apparently the sentence is inaccurately formulated, it needs to be rephrased.

Reply: The sentence was modified and the reason of “chlorophyll/anthocyanin ratio affect leaf appearance” was explained in the discussion section

- The introduction and conclusion do not reflect the relevance and perspective of the practical use of the results

Reply: Some modifications were performed

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the issues I raised and the manuscript has been modified accordingly.