Response to the Cold Stress Signaling of the Tea Plant (Camellia sinensis) Elicited by Chitosan Oligosaccharide

Round 1

Reviewer 1 Report

For context, it would be good to hear more about why it is thought that plants can sense chitosan and change their metabolism in response in the introduction.  It is a component of fungal cell walls and insect exoskeletons, so you might expect it to be inducing defense pathways evolved to respond to biotic stress. What does the literature say about why this helps with abiotic stress? I'd love to see a bit more about this in the introduction.

Explain why you chose Anji Bai Cha, a tea cultivar with a known mutation causing it to not produce chlorophyll in young leaves at cold temperatures and therefore alter its entire metabolic profile and stunt growth.  What makes this a good study system for studying cold stress?  It doesn't seem like a good choice to me since it is such an odd cultivar and your results are not likely to be generalizable to all tea plants. I think you'll need to spend quite a bit of time convincing the reader that it was a good choice, or make this manuscript more narrowly focused on albino cultivars (which are popular and interesting on their own).

Lines 139–148: This paragraph is difficult to read partly because of the organization. I'd recommend to start with general trends in the effect of cold stress (e.g. cold stress consistently increased SOD activity, but POD activity was highest at -4C) and the COS treatment (e.g. COS treatment consistently increased Chlorophyll content).  Then, describe the interactive effects of COS treatment and cold stress (e.g. For all parameters, the effects of COS were more pronounced under cold stress).

Explain why you chose the -4C treatment to do transcriptomics rather than the -8C treatment.

Lines 145–148: These two sentences would fit better in the discussion section than in the results.

Line 155: "The sequencing analyses were performed by Illumina." belongs in the methods.

Figure 3: What is "rich factor" and how should I interpret it?  Explain this briefly in methods or figure caption

Line 175–176: This is true, but if you look at individual GO terms aren't the differentially expressed genes mostly enriched in serine-type endoptidase activity (in the molecular function category)?  A quick search for "chitosan serine-type endopeptidase" makes me think that you might be missing something really important from the literature.

Line 188: What scatter plot?  Refer to plots with figure numbers.

For all the transcriptomics figures, the caption should remind the reader that the results are only from the -4C treatment.

"control" does not need to be abbreviated.  Replace "CK" with "control"

Similarly, "chlorophyll" does not need to be abbreviated as "Chl"

SOD, POD, and RPKM are never defined. Make sure acronyms and abbreviations are defined at least once (except maybe gene names)

Typo in figure 1 caption.  Should be "data" not "date"

Figure 4 should read "the color of the dots indicates the degree of rich factor enrichment"

 

Discussion:

You did this research in Anji Bai Cha, a cultivar with a known mutation that inhibits chlorophyll development at cold temperatures.  I don't see how your findings about chlorophyll enhancement by COS under cold temperatures are generalizable to other plants or even other tea cultivars that don't have such dramatic reductions in chlorophyll content under cold temperatures.  Regardless, you need to include more information about the Anji Bai Cha mutation for readers to correctly interpret your results.  Surely your results would be different with a non-albino cultivar, right?  You may want to read and cite the following:

Du Y, Chen H, Zhong W, Wu L, Ye J, Lin C, et al. Effect of temperature on accumulation of chlorophylls and leaf ultrastructure of low temperature induced albino tea plant. Afri J Biotechnol. 2010; 7:1881–5

Xiong L, Li J, Li Y, Yuan L, Liu S, Huang Ja, et al. Dynamic changes in catechin levels and catechin bio- synthesis-related gene expression in albino tea plants (Camellia sinensis L.). Plant Physiol Biochem. 2013; 71:132–43. doi: 10.1016/j.plaphy.2013.06.019 PMID: 23911731
5.

Li CF, Yao MZ, Ma CL, et al (2015) Differential metabolic profiles during the albescent stages of “Anji Baicha” (Camellia sinensis). PLoS One 10:e0139996. doi: 10.1371/journal.pone.0139996

I also think that the discussion could do a better job of connecting your results to the literature.  Sentences like "And this is consistent with previous research [15, 17, 27, 28]" are not very helpful, especially when those citations are not brought up again.  On the other hand, there are sections about the literature that do not connect well with your results (e.g. line 240–247).  Organize paragraphs and sentences to better connect your findings, your interpretations, and supporting (or contrasting) examples from the literature. The paragraph starting at line 248 does this really well.

Line 240–247: Were these genes upregulated or downregulated by COS?  How does this information relate to your study?

Line 268–270: I was expecting a paragraph about which genes in the phenylpropanoid pathway were up/downregulated.  What do cysteine and methionine have to do with the phenylpropanoid pathway? Isn't phenylalanine the start of the phenylpropanoid pathway?

Author Response

Response to Reviewer 1

Dear esteemed reviewer,

Thank you so much for the helpful comments and suggestions. We make meticulous investigation and response to your comments as listed below.

1)For context, it would be good to hear more about why it is thought that plants can sense chitosan and change their metabolism in response in the introduction.  It is a component of fungal cell walls and insect exoskeletons, so you might expect it to be inducing defense pathways evolved to respond to biotic stress. What does the literature say about why this helps with abiotic stress? I'd love to see a bit more about this in the introduction.

Response: The manuscript has been revised according to your suggestion. This part was revised as below:

Line 40-57: “Chitosan oligosaccharide (COS) prepared from chitosan, is an environmentally friendly plant growth regulator and stress tolerance inducer [14-18]. Chitosan is a linear polysaccharide composed of β-1,4-glucosamines. Hydrolysis of the glycosidic chitosan chains yields oligosaccharides, including the water-soluble oligochitosan [15, 16]. Chitosan and COS has a rich history of being researched for applications in agriculture, primarily for plant defense and yield increase [17,18]. As a natural biocontroler and elicitor of defense responses, COS can boost the innate ability of plants to defend themselves by stimulating secondary metabolite synthesis, and increasing the chlorophyll content and photosynthetic ability [14,15], enrich the soluble sugar in plant [19], and enhancing the activities of antioxidant enzymes [19,21]. COS stimulated that signaling pathways disease resistance in rice [22], and its role in TMV resistance in Arabidopsis has been investigated [23]. And studies have shown that COS enhances carbon metabolism, nitrogen metabolism, photosynthesis, and defense response against abiotic stress in plants [24].

COS is able to mitigate the effects of abiotic stresses in plant, including salt, cold and drought [20-26]. The mechanism of COS in increasing abiotic stress tolerances was summarized as enhancing the activities of antioxidant enzymes [19], photosynthesis, and stimulate secondary metabolite synthesis [25]. For example, COS has been applied to wheat seedlings for improved chilling tolerance by enhancing antioxidant activities of SOD and POD, and increasing content of chlorophyll.”

2)Explain why you chose Anji Bai Cha, a tea cultivar with a known mutation causing it to not produce chlorophyll in young leaves at cold temperatures and therefore alter its entire metabolic profile and stunt growth. What makes this a good study system for studying cold stress?  It doesn't seem like a good choice to me since it is such an odd cultivar and your results are not likely to be generalizable to all tea plants. I think you'll need to spend quite a bit of time convincing the reader that it was a good choice, or make this manuscript more narrowly focused on albino cultivars (which are popular and interesting on their own).

You did this research in Anji Bai Cha, a cultivar with a known mutation that inhibits chlorophyll development at cold temperatures.  I don't see how your findings about chlorophyll enhancement by COS under cold temperatures are generalizable to other plants or even other tea cultivars that don't have such dramatic reductions in chlorophyll content under cold temperatures.  Regardless, you need to include more information about the Anji Bai Cha mutation for readers to correctly interpret your results.  Surely your results would be different with a non-albino cultivar, right?

Response: We had thought of this issue before the design of the research. We had found that the Anji baicha is less cold-resistant than other tea varieties and its response pattern were similar(except the albino at early spring around 15-20℃ ) while Anji baicha is more sensitive to temperature, which might make it be more suitable to be the research model of cold stress. So considering your wisdom suggestion, we deleted the particular description of  ‘Anji Baicha’ with a brief mention at the material and method part.  

3)Lines 139–148: This paragraph is difficult to read partly because of the organization. I'd recommend to start with general trends in the effect of cold stress (e.g. cold stress consistently increased SOD activity, but POD activity was highest at -4C) and the COS treatment (e.g. COS treatment consistently increased Chlorophyll content).  Then, describe the interactive effects of COS treatment and cold stress (e.g. For all parameters, the effects of COS were more pronounced under cold stress)

Response: With your suggestions, in order to precisely analyze the physiological parameter change relationship in our manuscript, we re-write section 3.1 as “To analyze the effects of COS on tea plant growth, we measured the change in activity of SOD, and POD enzymes and content of chlorophyll, soluble sugar in COS-treated tea plant and their respond to low temperature stress, with sterile distilled water served as control. As shown in Figure 1, under low temperature, tea plant response to cold stress with all the physiological parameters changed and COS enhanced freeze protection. As in control group, low temperature caused increasing of those physiological parameters. As shown in Figure 1A, enzyme activity of SOD was significantly increased by 24.04% at -4℃ and 32.68% at -8℃. Similarly, enzyme activity of POD was significantly increased by 38.05% at -4℃ and 8.81% at -8℃. Cold stress significantly reduced the chlorophyll content by 20.18% and 21.96% at -4℃ and -8℃, respectively (Figure 1C). Moreover, soluble sugar content was significant increased by 29.87% at -4℃ and 28.16% at -8℃, respectively (Figure 1D). The results showed that cold stress consistently increased SOD and POD activity, and soluble sugar content, when the temperature was switched from 25℃ to -4℃ or -8℃, but POD activity was highest at -4℃.

When exogenous COS was used, it consistently enhanced SOD and POD activities, and soluble sugar of content, chlorophyll content in tea plant. For example, COS improved SOD activity by 11.75% at 25℃, 25.93% at -4℃ and 9.21% at -8℃, respectively as compared with control. Similarly, POD activity was enhanced by 19.91%, 19.23% and 30.09% on 25℃, -4℃ and -8℃, respectively.

For all the tested parameters, the effects of COS were more pronounced under cold stress. When tea plants were treated with COS combined with cold stress, SOD enhanced by 56.21% and 44.91% at -4℃ and -8℃, respectively. Similarly, POD increased 37.26% and 18.04%. Content of soluble sugar also increased 45.22% and 40.25% at -4℃ and -8℃, respectively. Chlorophyll content was decreased by 13.47% and 14.99%, respectively. The results showed that COS treatment consistently increased chlorophyll content, but three parameters of SOD, POD and soluble sugar were highest at -4℃ of cold stress combined with COS.”

4)Explain why you chose the -4C treatment to do transcriptomics rather than the -8C treatment.

Response: Considering the physiological changes of tea plant both at -4℃ and -8 ℃ were similar， and the limited weather conditions in Guizhou, we chose the -4℃ treatment to do transcriptomics rather than the -8℃ treatment.

5）Lines 145–148: These two sentences would fit better in the discussion section than in the results.

Response: The manuscript has been revised according to your suggestion. Line 145-148 “These findings indicated utilization of COS can positively affect these physiological parameters in tea plant. Hence COS can beneficially regulate the natural defense system and improve growth and developmental processes of tea plant under cold stress." was moved to “Discussion” and revised as: “Those results indicated that utilization of COS can positively affect these physiological parameters in tea plant, and beneficially regulate the natural defense system and improve growth and developmental processes of tea plant under cold stress”

6）Line 155: "The sequencing analyses were performed by Illumina." belongs in the methods.

Response: Sure，we did accordingly.

7）Figure 3: What is "rich factor" and how should I interpret it?  Explain this briefly in methods or figure caption.

Response: We have made an annotation of “rich factor” in Figure 3.

Line 203-205: “rich factor” represents the ratio between the number of different genes enriched in the term and the background genes in GO term.

8）Line 175–176: This is true, but if you look at individual GO terms aren't the differentially expressed genes mostly enriched in serine-type endoptidase activity (in the molecular function category)?  A quick search for "chitosan serine-type endopeptidase" makes me think that you might be missing something really important from the literature.

Response: Thank you for your kindly suggestion, we added “Besides, “serine-type endoptidase activity” (GO: 0004252) was mostly enriched in the molecular function category.” to Line 200-201. We know too little about what it does in plants, and we can't explain it in detail. We are sorry for it.

9）Line 188: What scatter plot?  Refer to plots with figure numbers.

Response: We had carefully checked our manuscript, and the unclear expression was changed to Line 207-208.

Line 207: “The scatter plot” was changed as “The KEGG enrichment scatter plot”.

Line 208: have added the figure numbers “Figure 4”.

10）For all the transcriptomics figures, the caption should remind the reader that the results are only from the -4C treatment.

Response: We made a change in the information in the article by repeatedly checking the Figure 2, Figure 3 and the Figure 4 in the caption. The unclear expression was corrected.

Line 186, 202, 223 has added “under -4℃ treatment”.

11）"control" does not need to be abbreviated.  Replace "CK" with "control". Similarly, "chlorophyll" does not need to be abbreviated as "Chl".

Response: We are sorry for the error.

“CK” was replaced to “control”.

“Chl” was replaced to “chlorophyll”.

12）SOD, POD, and RPKM are never defined. Make sure acronyms and abbreviations are defined at least once (except maybe gene names).

Response: “SOD” and “POD” were defined in Abstract part when it appeared first time, “superoxide dismutase (SOD) activity, peroxidase (POD) activity.

Reads Per Kilobase of transcript per Million “RPKM” are defined Line 113.

13)Typo in figure 1 caption.  Should be "data" not "date"

Response: The “date” was corrected as “data”.

14)Figure 4 should read "the color of the dots indicates the degree of rich factor enrichment".

Response: We are sorry for the error. The “the depth of the dots indicates the degree of rich factor enrichment” was changed to “the color of the dots indicates the degree of rich factor enrichment”.

15)I also think that the discussion could do a better job of connecting your results to the literature. Sentences like "And this is consistent with previous research [15, 17, 27, 28]" are not very helpful, especially when those citations are not brought up again. 

Response: according to your suggestion, we additionally describe previous research to onnecting our results to the literature.  

And it was revised as in Line 237-253: “Cold stress can cause excessive production of reactive oxygen species (ROS), disrupt the normal physiological and metabolic balance of plants, lead to the increase of membrane lipid peroxidation and damage to vital biomolecules [36-37]. Plants have evolved complex mechanisms to combat against the damage induced by ROS, including improve the antioxidant enzymes [38-39]. In this study, under cold stress, the tea plant natively reacted to protect themselves by increasing the activity of SOD and POD enzyme, and the application of COS provided external assistance plant. Chlorophyll content in COS-treated tea plant was higher than in control, which indicated that COS application mitigated the cold-induced decline in chlorophyll content. Soluble sugar can maintain the osmotic balance, and the soluble sugar in COS treated tea plant was higher than that without COS treatment, suggesting that COS can stabilize cell membrane and enhance cold resistance of plant. Those results indicated that utilization of COS can positively affect these physiological parameters in tea plant, and beneficially regulate the natural defense system and improve growth and developmental processes of tea plant under cold stress. And this were also demonstrated in wheat seedlings where application of COS could enhance the activities of antioxidant enzymes and the content of chlorophyll and alleviate the damage of abiotic stress in wheat [19-21]. In wheat, COS could enhance the activities of antioxidant enzymes and the content of chlorophyll, alleviate plant the damage of abiotic stress [19-21]”

16)On the other hand, there are sections about the literature that do not connect well with your results (e.g. line 240–247).  Organize paragraphs and sentences to better connect your findings, your interpretations, and supporting (or contrasting) examples from the literature. The paragraph starting at line 248 does this really well. Line 240–247: Were these genes upregulated or downregulated by COS?  How does this information relate to your study?

Response: We made an appropriate addition to revise this part. It was changed as foolwing:

Line 266-271: “Besides, Chlorophyll a/b binding protein can participate in light uptake, transfer energy to the reaction centers of the photosystem I and photosystem II, and regulate the excitation energy distribution to maintain the structure of the thylakoid membrane [44]. And all of 23 chlorophyll a/b binding protein genes were also up-regulated, which can imply the recovery of photosynthesis activities by COS treatment under cold stress [45].”

17)Line 268–270: I was expecting a paragraph about which genes in the phenylpropanoid pathway were up/downregulated.  What do cysteine and methionine have to do with the phenylpropanoid pathway? Isn't phenylalanine the start of the phenylpropanoid pathway?

Response: We are sorry for the error. We made repeatedly checking the phenylpropanoid-related genes. We found that in phenylalanine biosynthesis pathway, three enzymes (phenylalanine ammonia-lyase, PAL; cinnamate 4-hydroxylase, C4H; 4-coumaric acid coenzyme A ligase, 4CL) involved in the production of important secondary metabolites of plant development and defense responses were down-regulated. Thus, we deleted this part from the manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript examines the activity of several enzymes with known roles in ROS metabolism, and transcriptomics impact of COS treatment under control (in the ROS enzyme assays) and freezing stress conditions (for ROS and RNAseq). The manuscript provides some insights I to the mechanisms underlying the protection from freezing damage provided by COS on tea plants. There was adequate replication. The RNAseq data however needs to be submitted to a publicly accessible database such as the SRA database at NCBI before this publication is acceptable for publishing. The authors also attempted to confirm the RNAseq results with a small number of genes by quantitative RT-PCR. With one exception, the primers used failed to show significant differences in gene expression. I would suggest dropping this analysis from the manuscript. As I note in the marked-up manuscript (attached), confirmation of RNAseq by qRT-PCR is not necessary because RNAseq I more accurate and less fraught with confounding affects and uncontrollable factors than qRT-PCR. Some minor language suggested changes are noted in the manuscript. Additionally, some suggested additional analyses are suggested such as the separation of up and down-regulated genes for the gene set enrichment analyses, and some tighter levels of significance (FRD-p values vs uncorrected p values and some limits on poorly expressed genes) is needed.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2

Dear esteemed reviewer,

Thank you so much for the helpful comments and suggestions. We make meticulous investigation and response to your comments as listed below.

To make it clear, we refer the comments from attached pdf file as “From the PDF”. We have accepted all your suggestions listed in PDF file, and the manuscript has been revised according to your suggestion.

 

From the PDF

• OK, but not needed. RNAseq is much more reliable than RT-PCR. If there were any difference, one would assume that the RT-PCR was wrong since it is a hybridization-based procedure and thus more prone to improper binding of primers or the multitude of other factors that can alter reverse transcription fidelity/activity or amplification efficiency.

Ans.: Line 21-22 “And the RNA-Seq results were confirmed by quantitative real-time PCR (qRT-PCR).” was deleted.

• The "b" looks significant in figure 1D. Please check this figure for accuracy.

Ans.: We repeatedly checked and calculated the enzyme activity data, and found no problems. The data is based on the results of three replicate experiments. The vertical bars refer to the mean ± SD (n = 3). The letters above the bars represent significant differences at p < 0.05. Means followed by the same letter over the bars are not significantly different at the 0.5% level, according to Least Significant Difference (LSD) test.

• How many reads mapped as pairs? This is important to ensure that there were no issues with the paired-end sequencing.

Ans.: “Unique mapped reads were over 88.02% (Table 1).” was changed to

Line 173-176: “The ratio of total mapped reads between control and COS-treatment was 94.69% - 94.90% and 94.85% - 95.20% for Camellia sinensis Genome Database. Unique mapped reads were 91.48% –92.10% in control and 88.02% - 90.66% in COS-treatment group (Table 1).”

• This is too low a cut off. The standard is to use a FDR p value of less than 0.05. It is also wise to limit your significant genes to those that had reasonable levels of expression. I generally use a cut of of an RPKM >2 for all replicates of at least one treatment.

Ans.: We have defined “an FDR p value of less than 0.05” used in this analysis, and some up and down-regulated genes were enriched.

• Something that I also find helpful is to run the GSEA on the up and down-regulated genes separately. Some processes are enriched in both, but often, there will be useful insight gained from looking at the positively or negatively regulated processes.

Ans.: We have analyzed the up and down-regulated genes separately, but we found that the down-regulated genes did not contribute much to the paper, so they were not discussed in the paper.

• Should probably be "enrichment factor"?

Ans.: We repeatedly checked the Figure 3, and “rich factor” is no clear. To make it clear, we add “the rich factor represents the ratio between the number of different genes enriched in the term and the background genes in GO term” in the Figure 3 legend.

• format error?

Ans.: It was corrected.

• From the PDF: This should be dropped. First, it does not confirm that these genes (with the exception of GH3 was differentially expressed. Second, the reason is most likely because the RT-QPCR is inaccurate due to primers binding to non-regulated gene family members of these genes and/or other factors that impacted the cDNA production and/or amplification efficiency.
• The authors also attempted to confirm the RNAseq results with a small number of genes by quantitative RT-PCR. With one exception, the primers used failed to show significant differences in gene expression. I would suggest dropping this analysis from the manuscript. As I note in the marked-up manuscript (attached), confirmation of RNAseq by qRT-PCR is not necessary because RNAseq I more accurate and less fraught with confounding affects and uncontrollable factors than qRT-PCR.

Ans.: We appreciate that you pointed out the weakness of this part and according to your suggestion we delete the figure and much part of the description. And it was limited to Line 229-231: “Five randomly selected transcripts were selected for RT-PCR analysis, which used to confirm validity and accuracy the RNA-Seq data. The results showed that the trend of qRT-PCR were consistent with results of RNA-Seq in Figure S1.”

• The proof that COS improves stress tolerance is the impact on phenology (growth yield etc.). The authors should provide the references that support this statement. This manuscript is focused on mechanisms. The authors should not confuse these points.

Ans.:  We provided the references that support this statement COS improves stress tolerance.

“The present study indicated that, at low temperature, COS could enhance improved antioxidant activity, increase accumulation of sugar content and chlorophyll content of tea plant, which confirmed that COS could play an important role in promoting plant growth and improving stress tolerance of plant. And this is consistent with previous research [15,17,27,28].” was changed to Line 237-253: “Cold stress can cause excessive production of reactive oxygen species (ROS), disrupt the normal physiological and metabolic balance of plants, lead to the increase of membrane lipid peroxidation and damage to vital biomolecules [36-37]. Plants have evolved complex mechanisms to combat against the damage induced by ROS, including improve the antioxidant enzymes [38-39]. In this study, under cold stress, the tea plant natively reacted to protect themselves by increasing the activity of SOD and POD enzyme, and the application of COS provided external assistance plant. Chlorophyll content in COS-treated tea plant was higher than in control, which indicated that COS application mitigated the cold-induced decline in chlorophyll content. Soluble sugar can maintain the osmotic balance, and the soluble sugar in COS treated tea plant was higher than that without COS treatment, suggesting that COS can stabilize cell membrane and enhance cold resistance of plant. Those results indicated that utilization of COS can positively affect these physiological parameters in tea plant, and beneficially regulate the natural defense system and improve growth and developmental processes of tea plant under cold stress. And this were also demonstrated in wheat seedlings where application of COS could enhance the activities of antioxidant enzymes and the content of chlorophyll and alleviate the damage of abiotic stress in wheat [19-21]. In wheat, COS could enhance the activities of antioxidant enzymes and the content of chlorophyll, alleviate plant the damage of abiotic stress [19-21]”

• This manuscript examines the activity of several enzymes with known roles in ROS metabolism, and transcriptomics impact of COS treatment under control (in the ROS enzyme assays) and freezing stress conditions (for ROS and RNAseq). The manuscript provides some insights I to the mechanisms underlying the protection from freezing damage provided by COS on tea plants. There was adequate replication.

Ans.: Thanks for your good comments.

• The RNAseq data however needs to be submitted to a publicly accessible database such as the SRA database at NCBI before this publication is acceptable for publishing.

Ans.: We will submit the RNAseq data to NCBI before the article is published.

• Some minor language suggested changes are noted in the manuscript.

Ans.: We have checked and polished the manuscript by a foreigner expect and all the changes are marked in the revised edition.

• Additionally, some suggested additional analyses are suggested such as the separation of up and down-regulated genes for the gene set enrichment analyses, and some tighter levels of significance (FRD-p values vs uncorrected p values and some limits on poorly expressed genes) is needed.

Ans.: Thank you for your kind suggestions, we have defined “a FDR p value of less than 0.05” used in this analysis, and some up and down-regulated genes were enriched.

Author Response File: Author Response.docx

Reviewer 3 Report

The scientific name may be placed first and the English name in parenthesis like Camellia sinensis (L.) O. Kuntze (Tea plant). In same line, crops should be crop. A brief introduction regarding the cultivation of tea plant should be added (Global cultivation and production and in the understory country). What type of agroclimatic is required for the better development and quality? Any range of pH, optimum temperature etc? In Introduction section, nothing is mentioned about the role of transcriptome assembly in such studies?? 38-39: From those studies add a little about the resistant varieties. Is there any literature about colt tolerant varieties? If yes, should be added. Line 70: “The plants were grown under normal environmental in a growth chamber”…. Should be rephrased like “ Plants were grown under the normal environment in a growth chamber”. Also verify the that the plants were grown in normal conditions of the study area? Or the optimum conditions required for the better growth?  Last line of section “2.1. Plant Materials and Cold Treatments” should be placed as 2^ndlast. Section “2.2. Physiological response assay” Line 01 should be rephrased. Line 85: should be merged in the previous section (section 2.1). Line 87: “We select tea leaves” should be “We selected tea leaves….). And what was the criteria for selection of leaves? Section “3.1. Physiological Parameter Change in Tea Plant at Low temperature” should be rephrased “3.1. Physiological Parameters Response to Low temperature”. Line 136-137: For example, these four checked physiological parameters were all changed??? Re-write it. The changes were significantly different??? The transcriptomic section is well written, some English and typographic edits are required, it should be better if proof read by a native speaker.

Author Response

Response to Reviewer 3

Dear esteemed reviewer,

Thank you so much for the helpful comments and suggestions. We make meticulous investigation and response to your comments as listed below.

 

 

• The scientific name may be placed first and the English name in parenthesis like Camellia sinensis (L.) O. Kuntze (Tea plant). In same line, crops should be crop.

Ans.: The manuscript has been revised according to your suggestion.

“crops” was changed to “crop”

“Tea plant (Camellia sinensis (L.) O. Kuntze)” was commonly reported in the literature. We would like to keep it consistent.

Reference:

Integrative transcriptome, proteome, and microRNA analysis reveals the effects of nitrogen sufficiency and deficiency conditions on theanine metabolism in the tea plant (Camellia sinensis). Hortic. Res, 2020, 7, 65. doi: 10.1038/s41438-020-0290-8.

Transcriptomic analysis of the biosynthesis, recycling, and distribution of ascorbic acid during leaf development in tea plant (Camellia sinensis (L.) O. Kuntze). Sci. Rep. 2017, 7, 46212. doi: 10.1038/srep46212.

• A brief introduction regarding the cultivation of tea plant should be added (Global cultivation and production and in the understory country). What type of agroclimatic is required for the better development and quality? Any range of pH, optimum temperature etc?

Ans.: According to your comments, the cultivation of tea plant was added for providing sufficient background information on our manuscript：

Line 28-32: “The tea production in over 50 countries has reached over 5.95 million tons on 4.1 million hectares around the world [2]. Tea plant can grow in different agroclimates and adapted to optimal temperature of 18 to 30℃ and pH ranging from 4.5 to 5.5, but the thermophilic nature of tea plants confines their growth to temperate area [3-5].”

• In Introduction section, nothing is mentioned about the role of transcriptome assembly in such studies?

Ans.: Thanks for your suggestions. We added the sentences to part Introduction:

Line 58-64: “In addition, transcriptome sequencing has been widely applied to tea plant, which is has the advantage of highly accurate, highly efficient and sensitive profiling in recent years [27]. RNA sequencing (RNA-Seq) technology for measuring transcriptomes of organisms can analyze genes related to abiotic and biotic stress responses, growth, development and metabolites [28-31], to improve our understanding of the molecular mechanism of tea plant [6-9,20] [7-10,32], and RNA-Seq will also be a valuable tool to reveal the role of exogenous substances in tea plant cold resistance.”

• 38-39: From those studies add a little about the resistant varieties. Is there any literature about colt tolerant varieties? If yes, should be added.

Ans.: Line 38“cultivating cold-resistant tea plant varieties” was changed as “cultivating cold-resistant tea plant varieties (e.g. Fudingdabai, Shuchazao)”.

• Line 70: “The plants were grown under normal environmental in a growth chamber”…. Should be rephrased like “Plants were grown under the normal environment in a growth chamber”. Also verify the that the plants were grown in normal conditions of the study area? Or the optimum conditions required for the better growth?

Ans.: To precise the description, “The plants were grown under normal environmental in a growth chamber at the experimental of Guizhou University, Guizhou Province, China (16h day/8h night at 25℃/20℃ and relative humidity of 70%).” was changed to in Line 77-79: “Plants were grown in a growth chamber at the experimental of Guizhou University, Guizhou Province, China (16h day/8h night at 25℃/20℃ and relative humidity of 70%).”

• Last line of section “2.1. Plant Materials and Cold Treatments” should be placed as 2^nd Section “2.2. Physiological response assay” Line 01 should be rephrased. Line 85: should be merged in the previous section (section 2.1).

Ans.: We agree with your suggestion.  

Last line of section “2.1. Plant Materials and Cold Treatments” was changed to 2^nd last.

“2.2. Physiological response assay” of Line 01 “The tea leaves were physiological index, including the activities of SOD and POD, and content of Chl and soluble sugar were determined.” was changed to Line 87 “Physiological indexes of tea leaves (containing 1st, 2nd, 3rd leaf and old leaves), involving the activities of SOD and POD, and content of chlorophyll and soluble sugar, were determined.”.

Line 85 “Experiments were conducted with three biological replicates.” was merged in the previous section (section 2.1) Line 84 “Three independent biological repeats were collected for each treatment.”

• Line 87: “We select tea leaves” should be “We selected tea leaves….). And what was the criteria for selection of leaves?

Ans.: Line 102: “We select tea leaves” was changed to “We selected tea leaves”.

According to the literature, we added the criteria for selection of leaves to Line 87 “Physiological indexes of tea leaves (containing 1st, 2nd, 3rd leaf and old leaves)”

Reference: Integrative transcriptome, proteome, and microRNA analysis reveals the effects of nitrogen sufficiency and deficiency conditions on theanine metabolism in the tea plant (Camellia sinensis). Hortic. Res. 2020, 7, 65. doi: 10.1038/s41438-020-0290-8.

• Section “3.1. Physiological Parameter Change in Tea Plant at Low temperature” should be rephrased “3.1. Physiological Parameters Response to Low temperature”.

Ans.: Section “3.1. Physiological Parameter Change in Tea Plant at Low temperature” was changed to

Line 138 “3.1. Physiological Parameters Response to Low temperature”.

• Line 136-137: For example, these four checked physiological parameters were all changed??? Re-write it. The changes were significantly different?

Ans.: Thanks for your suggestions. It was rewriten as following:

“To analyze the effects of COS on tea plant growth, we measured the change in activity of SOD, and POD enzymes and content of chlorophyll, soluble sugar in COS-treated tea plant and their respond to low temperature stress, with sterile distilled water served as control. As shown in Figure 1, under low temperature, tea plant response to cold stress with all the physiological parameters changed and COS enhanced freeze protection. As in control group, low temperature caused increasing of those physiological parameters. As shown in Figure 1A, enzyme activity of SOD was significantly increased by 24.04% at -4℃ and 32.68% at -8℃. Similarly, enzyme activity of POD was significantly increased by 38.05% at -4℃ and 8.81% at -8℃. Cold stress significantly reduced the chlorophyll content by 20.18% and 21.96% at -4℃ and -8℃, respectively (Figure 1C). Moreover, soluble sugar content was significant increased by 29.87% at -4℃ and 28.16% at -8℃, respectively (Figure 1D). The results showed that cold stress consistently increased SOD and POD activity, and soluble sugar content, when the temperature was switched from 25℃ to -4℃ or -8℃, but POD activity was highest at -4℃.

When exogenous COS was used, it consistently enhanced SOD and POD activities, and soluble sugar of content, chlorophyll content in tea plant. For example, COS improved SOD activity by 11.75% at 25℃, 25.93% at -4℃ and 9.21% at -8℃, respectively as compared with control. Similarly, POD activity was enhanced by 19.91%, 19.23% and 30.09% on 25℃, -4℃ and -8℃, respectively.

For all the tested parameters, the effects of COS were more pronounced under cold stress. When tea plants were treated with COS combined with cold stress, SOD enhanced by 56.21% and 44.91% at -4℃ and -8℃, respectively. Similarly, POD increased 37.26% and 18.04%. Content of soluble sugar also increased 45.22% and 40.25% at -4℃ and -8℃, respectively. Chlorophyll content was decreased by 13.47% and 14.99%, respectively. The results showed that COS treatment consistently increased chlorophyll content, but three parameters of SOD, POD and soluble sugar were highest at -4℃ of cold stress combined with COS.”

• The transcriptomic section is well written, some English and typographic edits are required, it should be better if proof read by a native speaker.

Ans.: We appreciate that you pointed out the weakness of our manuscript, which can further enhance the quality of our manuscript. We have checked and polished the manuscript by a foreigner expect and all the changes are marked in the revised edition.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The manuscript has been revised significantly as the raised concerns have been covered in it. The manuscript is acceptable in present form.

Author Response

Response to Reviewer

Thank you for your confirmation of our manuscript. The revised manuscript is hereby submitted to be considered for publication. Thank you for your helpful coordination and understanding throughout the evaluation process of our manuscript.