Physiological, Transcriptomic Investigation on the Tea Plant Growth and Yield Motivation by Chitosan Oligosaccharides
Round 1
Reviewer 1 Report
Dear Authors,
Your manuscript needs some improvements based on some suggestions and comments presented on the file. The main ones to be regarding material and methods section.
Comments for author File: 
Comments.pdf
Author Response
Response to reviewer 1
Dear reviewer, we are so appreciated your scrupulous review and great advice. We have taken care of all the revisions accordingly.
- L3 “Promotion Motivated” was changed to “Motivtion”.
- L26 has added “natural biostimulants have practical importance in cultivation system and crops production”.
- L46 has been modified accordingly.
- L57 “are” was changed to “were”.
- L58 “. And” was changed to “, and”.
- L58 “is” was changed to “was”.
- L59 Thanks for your valuable comments and suggestions. We responded in L60-63. Thanks
- L61 In the trial tea garden, the tea plants will be pruned every year after the end of the autumn tea. After cutting, the spring tea must be kept for one season before picking. Before we apply COS, the tea plants have only a little tea and no tea buds. The spring tea has high nutrients and is economical. The benefit is better, and the yield of spring tea is particularly important. Therefore, COS should be applied before spring tea picking to promote the growth of spring tea and increase the yield.
- L62 Before spring tea budding, it was applied once every 5 days in 15 days and total for 3 times.
- L64. “All experiments” refer to the COS application experiments
Reviewer 2 Report
The presented manuscript deals with the use of biological substances and their application to plants. With regard to monitoring the quality and safety of food, biologically sustainable production is a topical issue. This solved problem and the obtained results have a considerable potential for further study of the mentioned problem. Teapots treated with chitosan oligosaccharides were used as a model plant. The manuscript is written relatively carefully. The introduction is relatively short. I think he should describe this issue more. The methodology completely lacks a description of the methodology for determining SOD, POD, soluble sugars and chlorophyll content. This needs to be added. The relation of the results describing the enzymatic activity, the content of soluble sugars and chlorophylls to the solved problem is not entirely clear from the text. This is also reflected in the description of the results. Graph 7 does not have a readable x-axis description. The authors cite literature from the first decade of this century. You may want to use more current data. Furthermore, journals - abbreviations and full names of journals - are not uniformly cited.
Author Response
Response to reviewer 2：
The presented manuscript deals with the use of biological substances and their application to plants. With regard to monitoring the quality and safety of food, biologically sustainable production is a topical issue. This solved problem and the obtained results have a considerable potential for further study of the mentioned problem. Teapots treated with chitosan oligosaccharides were used as a model plant. The manuscript is written relatively carefully. The introduction is relatively short. I think he should describe this issue more.
Responses: Thanks for your valuable comments and suggestions. We have added more descriptions in the introduction.
The methodology completely lacks a description of the methodology for determining SOD, POD, soluble sugars and chlorophyll content. This needs to be added.
Responses: Thanks. As suggested, we add detail description.
The tea leaves (0.2g) was grounded with 1mL stilled water for both the control and COS treatment group. And the homogenate was then transferred into centrifuge tube and centrifuged at 8000g for 10min at 4 °C. The supernatant was subjected to a spectrophotometer which analyzes the liquid by measuring the amount of light that is absorbed by it. Thus, the enzyme activity, soluble sugar and chlorophyll content were accordingly obtained by following provisions in bioassay kits as reported[24]. The involved bioassay kits were purchased from Solarbio, Beijing, China and the Cat. No. was BC0175, BC0095, BC0995, BC0035, respectively.
The relation of the results describing the enzymatic activity, the content of soluble sugars and chlorophylls to the solved problem is not entirely clear from the text. This is also reflected in the description of the results.
Responses: Done. Thanks for your valuable comments and suggestions. We have re-described this issue in the results section 3.2.
“The contents of SOD, POD enzyme activity, chlorophyll and soluble sugar in tea leaves were evaluated in response to COS treatment. A significant increase in COS group were shown in comparison to control group (Figure 3). In tea plants, the SOD content in control group was calculated to be 147.23 U/g FW, however in COS group its activity was 262.94 U/g and increased by 69.32% (Figure 4A). Similarly, the POD activity in control group was 91.80 U/g FW and the enzyme level in COS increased by 48.45% and reached 136.28(Figure 4B). Likewise, chlorophyll content in control was 0.35 mg/g FW and increased by 10.27% after application of COS (Figure 4C). Additionally, soluble sugar content was calculated to be 38.37 mg/g FW in control and was promoted to 56.95 mg/g FW in COS treatment and increased by 48.42% (Figure 4D). It is obvious that application of 5% COS systematically promoted the activity of SOD and POD and the content of soluble sugar and chlorophyll in the tea plant compared with the CK.”
Graph 7 does not have a readable x-axis description.
Responses: We replaced the new graph with high resolution.
The authors cite literature from the first decade of this century. You may want to use more current data.
Responses: Done. Thanks for your valuable comments and suggestions. We have made changes and added more current references in the article.
Furthermore, journals - abbreviations and full names of journals - are not uniformly cited.
Responses: Thanks for your valuable comments and suggestions. We have united all related as abbreviations version made correction on some wrong description.
Author Response File: 
Author Response.docx
Reviewer 3 Report
In this manuscript, the authors evaluated the effects of chitosan oligosaccharides on tea plant. The positive results of the COS treatment on tea yield were further investigated by the transcriptomic analysis. The work provided a basic overview of the physiological changes of the COS-treated tea plant.
Overall comments:
The discussion is mainly a repetition of results and needs improvement. Are the results in accordance with other studies in different plant species? The explanation of the changes in phytohormone signaling is adequate; however, there should be more discussion on the photosynthesis and carbon metabolism since these are also the main differences caused by COS treatment on tea plant. The phenotype data are positive. The conclusion would be more persuasive if the authors could provide some discussion linking the physiology data and the transcriptomic data
Minor
- 58: Please check the grammar and the brackets of the sentence.
- 60: According to what reference the concentration of COS solution was decided? Have you ever tried other COS solution concentration?
- 77-85: Please provide more details about the experiments.
- 122-125: Which temperatures and how many cycles did you use?
- Figure 1: Is there a clear standard that you define new bud? Is there any possibility that the results were caused by the enhanced growth rates of buds in COS-treated tea rather than promoting the bud density?
- 138: the bud ‘density’
- Besides the effect on tea yields, is there any other changes on the phenotype of the COS-treated group compared with CK?
- In Figure 4, the chlorophyll contents were higher in the buds of COS-treated tea. Does the color of bud also vary between these experiment groups? The abbreviations of DEGs, SOD, POD, and FW should be defined for the first appearance in the manuscript.
- 181: please check for the number ‘6..89’.
- 191-192: Please check for the writing of this sentence.
- 193: Do you mean ‘COS-treated plant’?
- 230-237: How do you choose these genes and what are the GO function of these genes?
- Figure 9: Does the log 2 FC values of these genes in transcriptomic data also close to the qRT-PCR results?
- 259-260: Since the regulation of ROS by antioxidant enzymes were complicated, did you also examine the activity of other antioxidant enzymes such APX or CAT?
- 263-266: The description of the function of photosynthesis is more related to the result of chlorophyll content below. The authors are suggested to adjust the arrangement of this sentence.
- 268, 270: What do you mean compatible sugar? That should not be the correct name of soluble sugar.
- 276: It should be a period at the end of sentence rather than a comma.
- 282-310: Only gene can be induced and all the name of genes shown in the paragraph should be written in italic form. Please check.
- 306: ‘can catalyze the catalyze the oxygenation’? Please correct the sentence.
- The p value should be lower case and italic in all figure legends.
- What’s the importance of the cultivar you used in this study? Did you ever conduct similar experiments on other tea cultivars?
- Please check the color corresponding to the regulated gene in the figure legend of Figure 6. There were no green dots.
- Please add some descriptions on why did you choose these eight genes and their functions in the plant in “ 3.3.5. qRT-PCR verification.
- Please correct the word on line 308 “EDGs”.
Author Response
Response to reviewer 3：
L58: Please check the grammar and the brackets of the sentence.
Responses: Done. Thanks for your valuable comments and suggestions.
L60: According to what reference the concentration of COS solution was decided?
Responses: Thanks for your valuable comments and suggestions. The recommended dose was referred the provisions instructions which included application in various crops. For tea plant, the 5% COS agent was diluted 800 times by volume with water consumption 32kg in every 667m2 (mu). So, the applied concentration of 5% COS is 40mL / 667m2. We have supplemented it in section 2.2.
Have you ever tried other COS solution concentration?
We did try other diluted concentration in early work,like 500,800, 1000,1200 time in plot testing. Among them, dilution of 800 time performed best. It’s easy to understand that 800 worked well than both 1000 and1200. However, high concentration of 500 spoil the effect (rather than increasing the production yield) So we applied the most effective 800-time solution in this report.
77-85: Please provide more details about the experiments.
Responses: Done. Thanks for your valuable comments and suggestions. The revision is as following:
The tea leaves (0.2g) was grounded with 1mL stilled water for both the control and COS treatment group. And the homogenate was then transferred into centrifuge tube and centrifuged at 8000g for 10min at 4 °C. The supernatant was subjected to a spectrophotometer which analyzes the liquid by measuring the amount of light that is absorbed by it. Thus, the enzyme activity, soluble sugar and chlorophyll content were accordingly obtained by following provisions in bioassay kits as reported [24]. The involved bioassay kits were purchased from Solarbio, Beijing, China and the Cat. No. was BC0175, BC0095, BC0995, BC0035, respectively.
122-125: Which temperatures and how many cycles did you use?
Responses: 95 ◦C, 60 ◦C, 72 ◦C and 40 cycles. We have supplemented it with L122-125.
Figure 1: Is there a clear standard that you define new bud?
Responses: The newly beared top 1-2 leave in the branch.
Is there any possibility that the results were caused by the enhanced growth rates of buds in COS-treated tea rather than promoting the bud density?
Responses: Our field investigation found that the number of buds in the area treated with COS was significantly higher than that of CK. The statistics of the data survey showed that the average number of bud teeth in three different areas was 1433.33, 1263.33 and 1841.33, which increased by 18.46%, 13.81% and 23.16% respectively compared with the control.
Besides the effect on tea yields, is there any other changes on the phenotype of the COS-treated group compared with CK?
Responses: Our field investigation found that compared with ck, the area treated with COS had thicker and shiny tea leaves. The quality was improved, and the tea flavor was highly promoted.
In Figure 4, the chlorophyll contents were higher in the buds of COS-treated tea. Does the color of bud also vary between these experiment groups?
Responses: Yes. The new tea leaves in the area treated with COS seemed greener and shinier.
The abbreviations of DEGs, SOD, POD, and FW should be defined for the first appearance in the manuscript.
Responses: Done.
181: please check for the number ‘6.89’.
Responses: Done.
191-192: Please check for the writing of this sentence.
Responses: Done. ‘DEGs between control and COS P-value < 0.05 and | log2 (fold change) | > 1 were defined as’ was changed to ‘Genes with p-value < 0.05 and |log2(Foldchange)| > 1 were defined as DEGs between control and COS’.
193: Do you mean ‘COS-treated plant’?
Responses: Yes.
230-237: How do you choose these genes and what are the GO function of these genes?
Responses: According to the experimental purpose, the genes related to plant growth were screened out, and then 8 genes were randomly selected from these genes for verification.
Figure 9: Does the log 2 FC values of these genes in transcriptomic data also close to the qRT-PCR results?
Responses: Some genes are not close, such as LOC114312375. Log2FC is positive in the transcriptome, which is an up-regulated gene, while log2FC is negative in qRT-PCR, which is a down-regulated gene. However, the change trend of the remaining seven DEGs in the transcriptome is consistent with that in qRT-PCR.
259-260: Since the regulation of ROS by antioxidant enzymes were complicated, did you also examine the activity of other antioxidant enzymes such APX or CAT?
Responses: We only measured the activities of SOD and pod.
263-266: The description of the function of photosynthesis is more related to the result of chlorophyll content below. The authors are suggested to adjust the arrangement of this sentence.
Responses: Thanks for your valuable comments and suggestions. We have adjusted this sentence and re described the whole paragraph.
268, 270: What do you mean compatible sugar? That should not be the correct name of soluble sugar.
Responses: Thanks.‘compatible’ was changed to ‘soluble’.
276: It should be a period at the end of sentence rather than a comma.
Responses: Done.
282-310: Only gene can be induced, and all the name of genes shown in the paragraph should be written in italic form. Please check.
Responses: Done.
306: ‘can catalyze the catalyze the oxygenation’? Please correct the sentence.
Responses: Done. ‘catalyze the catalyze the oxygenation’ was changed to ‘analytic oxidation’.
The p value should be lower case and italic in all figure legends.
Responses: Done.
What’s the importance of the cultivar you used in this study?
Responses: Fuding Dabai tea plant is a cross-pollinated economic crop with high genomic heterozygosity, excellent germination rate, cold resistance, drought resistance, heat resistance, and resistance to diseases and insect pests.
Did you ever conduct similar experiments on other tea cultivars?
Responses: No, because most tea gardens in Guizhou Province are planted with Fuding Dabai tea plants, which have high yields, good quality, and considerable economic benefits, so we mainly conduct research on them.
Please check the color corresponding to the regulated gene in the figure legend of Figure 6. There were no green dots.
Responses: Thanks for your valuable comments and suggestions. Figure 6. “green dots” was changed to “blue dots”. “blue dots” was changed to “gray dots”.
Please add some descriptions on why did you choose these eight genes and their functions in the plant in “ 3.3.5. qRT-PCR verification.
Responses: Thanks for your valuable comments and suggestions. We have made a supplementary description in 3.3.5. qRT-PCR verification.
Please correct the word on line 308 “EDGs”.
Responses: Done. “EDGs” was changed to “DEG
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors modified the text of the manuscript according to the opponents' comments. These comments have improved the quality of the manuscript, which now has a higher scientific standard. In its current form, the manuscript is already suitable for publication
Author Response
Dear reviewer,
Thank you very much for you great comments that highly promoted our paper.
Sincerely
Linhong Jin
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China.
E-mail: [email protected].
Tel: +186-8517-4719.
Reviewer 3 Report
The manuscript has been sufficiently improved, and thus acceptable for publication.
Author Response
Dear reviewer,
We appreiciated so much for your kind comments and early great suggestion on our MS revision.
Sincerely
Linhong Jin
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China.
E-mail: [email protected].
Tel: +186-8517-4719.
