Exogenous Activation of the Ethylene Signaling Pathway Enhances the Freezing Tolerance of Young Tea Shoots by Regulating the Plant’s Antioxidant System
, Yajun Yang 1, Xinchao Wang 1 and Xinyuan Hao 1,*Round 1
Reviewer 1 Report
In this interesting study, the authors investigated the involvement of ethylene in cold responses of tea plants. This is a reasonably sound work, mostly (but not always) well written. The conclusions are in part consistent with the results, but unfortunately a key result (Figure 5, effects of ethephon on the expression of ethylene-related genes) is not included in the manuscript (insetad, Fig. 6 is reported twice).
Maybe the weakest point is the attempt to explain the positive effects of ethephon treatments in terms of ROS production and scavenging. Many recent studies confirm that ROS have an important role as signalling molecules (see e.g. Ron MIttler's "ROS are good", doi: 10.1016/j.tplants.2016.08.002). This part of the paper should be reconsidered and improved. Further detailed comments are given below.
Line 14 (Abstract) Cammellia
Line 152 (Methods) HPLP? Supposedly, HPLC
Fig. 1 and figure legend Lines 214-221). Several typos here. In the figure, green house and out door should be written greenhouse and outdoor, respectively. “Normal condation” should be corrected, but maybe “control conditions” or just “control” would be even better. In the text, please specify the control temperature. Line 215: “low” does not fit to control, you could just write “under different temperature…”. Line 217: The word “promoter” might be misleading, why not using “enhancer” or “inducer”? Lines 217-219, the treatment details are a bit confusing, please rephrase.
Line 228. Exogenous neither… sounds odd, please rephrase.
Line 232. What does “plants in one and a bud stage” mean? Maybe something is missing here.
Figure 2. Some abbreviations are not consistent with those used in Fig. 1. CK is used to indicate control conditions. Relative electrical conductivity (as in Fig. 1) for the y axis (A). Indicate the unit (%) in the y axis of B. Here it is cold-spell stress, in figure 1 it is different temperature treatment, please unify. Biological replicates here, independent experiments in figure 1. Please make the figures consistent to avoid confusion.
Line 255. The expression of antioxidant genes is shown in Fig.6, please mention it here.
Fig. 5 which is supposed to show the expression of ethylene related genes, is missing. The expression of ROS-related genes is shown twice, as Fig. 5 and 6.
Data shown in Fig.6 are not consistent with the statement that “antioxidant enzyme activities (SOD, POD, and CAT) of ethephon-treated plants were significantly higher than those of the control plants, consistently with the level of expression of antioxidant enzyme-related genes induced by ethephon” (Lines 253-254). Only CsAPX-6 apparently responds significantly. However, it is not clear the reason why this gene, among different ascorbate peroxidase-encoding genes, was chosen for this study. Not long ago, it was reported that APX-1, a cytosolic APX found in tea plants, had apparently an important role in regulating ascorbate metabolism (https://doi.org/10.1016/j.plantsci.2020.110500). Picking within a gene family is not a good strategy, as different APX (but this also applies to SOD other genes/enzymes represented in multiple copies/isoforms) may behave differently under different stress conditions.
Of course, nothing can be said about the effects of treatments on ethylene-related genes (the data are not shown anywhere).
In general, the language is clear and understandable, but especially some parts of the results, but also the legends to figures, are less good and require revision.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
This paper explores shows that ethylene treatment (ethephon) can induce processes linked to cold acclimation in tea.
The problem that with the term “cold” one can refer to two different phenomena: chilling or freezing. “Chilling” is for low temperatures but positive ones (above 0°C) while “freezing temperatures” refer to temperature below 0°C. For clarity, I highly recommend authors not to use “cold” but either chilling or freezing. Except for the accepted expression “cold-acclimation” that means freezing resistance attained by chilling exposure.
The authors indicate that -5°C is chilling… well, for me it is freezing.
The experiments are very badly explained. We do not understand if the freezing exposure is maintained through what they call “days after treatments”. They confuse “days of treatments” (ie the treatments continue) with “days after treatments (the treatment is stopped).
Figures 5 and 5 are identical so one is missing
The way data are expressed in figures 2, 3 and 4 is not clear. What do they mean by “change”. Are data relative to day0. If it is so, why do not we have “1” at day 0?
In the current stage the papers can not be accepted.
ABSTRACT
Lines 19/20: this sentence is repetitive with what comes next
Line 30: “…did not seem relevant to the response to cold”: replace by “…did not seem relevant to cold acclimation in response to ethephon”
INTRODUCTION
Line 51. Why would a “sudden temperature decrease prevents full cold acclimation”. What is the ref for that? Besides, why in early spring would you want your plants to attain full cold acclimation, that is the capacity to resist to freezing? In cold stress, you have two things: resistance to freezing (attained by cold acclimation) and resistance to chilling. Understanding resistance to chilling is also important.
Line 59 : this “cold” is meant for chilling or freezing?
Line 61 : this “cold” is meant for chilling or freezing?
Line 64: what do you mean by cold hardiness? Chilling resistance? Cold acclimation (ie freezing resistance attained by chilling exposure)?
Lines 69, 71, 73, 75, 77, 84, 88, 91, 96, 97, 100, 102, 103, 207, 208, 218, 222, 235, 243 and so on: these “cold” are meant for chilling or freezing? Change “cold” by the appropriate term.
Line 11à: indicate light/dark
Line 117: define REC
Line 122: how do you control the spray process? Do you spray once, twice, same times for each treatment?
Line 146. What is EP?
Line 166. What is agent I? agent II?
RESULTS
Line 206: add “(REC)” after the full name. then only use the abbreviated form.
Line 211: what is “normal condition”?
Line 210: be more specific: what are the two possible “germinating conditions”? what is the link with “greenhouse” and “outdoor” which in the caption seem not to refer to germination condition but to where the cold treatments took place. Please clarify.
What is the conclusion of Fig1A. you say you need to optimize the conductivity measurements? What parameters have you tested? If I understand well, you tested to way to apply freezing, either outdoor (but how do you control temperature outdoor) or greenhouse. Is that correct (very stange). Anyway, what are the conditions that are OK and that you used for 1B and after?
Line 223; you should explain what ethephon is. How does it act? Does it spontaneously become ethylene? Or ACC?
In fig1B, where is the control????
Line 240. Why do not ACC have the same effect as ethephon?
Line 232/ what do you mean “plants in one and a bud stage”? one what??
Line 233: what is “experimental treatment”? freezing treatment? The experimental procedure is not well explained.
Figure 2. Is CK for cytokinins??? If you used it for “control” write “control” and not “CK” because it is confusing.
Line 244, 258 271 and after: -5°C is not chilling!!!!
Line 243; what do you mean by “application of ethephon for two days”? the ethephon is applied for two days? Or it is applied once, and two days later you do freezing treatment??
“Days after treatments” why “s” at “treatments”? when I read the caption, I think you mean “duration of -5°C exposure (days)”. Is that correct?
In figure 2B, what do you mean by “content changes under different treatments”??? you make the ration between what and what? Freezing treated versus not freezing treated??
Fig3 and 4 I have the same issue. How is “0” obtained? Is all expressed relative to 0D. why then we have not “1” but “0” in 0d??
Figures 5 and 6 are very small.
Line 280: figures 5 and 6 are the same!!
Is the day 7 pertinent? For GST3, having day 7 prevent seeing the other data.
What is “treat time “?
see my general comments
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
This work presents an interesting study on the regulation of ethylene pathway and the increase of cold tolerance in Camelia sinensis plants. This is useful research for agricultural applications.
There are some topics to be included to improve the overall quality of the manuscript, also some big mistakes in the presentation of results.
Introduction
- Include the minimum temperatures of the latest events of chilling that have injured the plants, and how frequently these events have occurred in the past five years.
- How significative have been the loses of tea leaves due chilling events, in terms of areas of damaged plants and its economic value. This is to highlight the relevance of this study for the development of new methods for plant tolerance to cold stress.
- Are there studies about the selection and cultivation of cold-tolerant varieties of C. sinensis?
- Please include more information about the possible transduction signals or metabolic pathways activated by the application of exogenous calcium and secondary metabolites.
- Regarding the studies about the application of ethylene to induce cold-tolerance, is it related to the increase of mitochondrial respiration or the activation of membrane-bound NADPH oxidases to produce heat?
Materials and methods
- In section 2.1 please include the light source, such as fluorescent, led, halogen.
- In section 2.2 please indicate the volume of each solution for the treatment of the plant tissue.
- In section 2.3 please include the details for ACC extraction from plant tissue. Also include the detailed information of the HPLC program of elution and detection of the molecule.
- Please include in Table 1 the primers for the housekeeping gene CsPTB.
Results
- The description of the methods should be improved. Including the details for reproducibility.
- In section 3.1 please include the percentage of increase of electrical conductivity of -5°C treatment compared to control and compared to -2°C treatment.
- Also include the percentage of change of electrical conductivity to compare the post- and pre-treatments shown in figure 1B.
- In section 3.2 include the percentage or fold of change between treatments and control conditions shown in figure 2A and 2B.
- In figure 2A indicate the meaning of “CK” in the figure legend.
- In section 3.3 when explaining the results shown in figure 3 please include the percentage of increase of the ethephon treatment compared to control at day 7. Also, CAT activity was higher than ethephon treatment, which contradicts what is exposed in the text (lines 253-254).
- Figure 6 has an error, the genes shown in the graphs are the same as figure 5.
- For all the gene expression experiments please indicate the fold of increase or fold of change compared to control condition at the time of maximum differences.
Discussion
- Please include information about the effect of the inhibitors of ethylene production and why some of them presented a mild inhibition compared to silver nitrate.
- Regarding the overexpressed genes of the ethylene signaling pathway, what are the tolerance mechanisms activated by those genes? What other stress-tolerance mechanisms are they involved in?
- As it was determined by your experimental data, the sugars are not involved in the tolerance against chilling. But what is known about other compatible metabolites related to chilling-tolerance, such as small peptides and amino acids? And what about cell wall modifications? Or the secretion of substances for the protection of leaf epidermis?
- I do believe the mechanisms is yet to be further analyzed, since your results partially explain the tolerance against chilling. You can propose new experiments and lines of research to complement this work or future publications.
The English writing has some grammar mistakes and some incorrect use of vocabulary.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors improved the manuscript. Further minor improvements are required, especially in the language. a few examples:
1. Figure 5, Panel F. MocK
2. Lines 325-329. Understandable, but awkward. Please rephrase.
3. Lines 414-415. "THe genes... will be further determined". Not clear who is going to further determine what. The reference here [38] tells something different.
Many more examples here and there, please have your manuscript revised by a native speaker/professional editing service
Many minor issues. Please have your manuscript revised by a native speaker/professional editing service
Author Response
Response to Reviewer Comments
Chen et al. “Exogenous activation of the ethylene signalling pathway enhances freezing tolerance of young tea shoots by regulating the plant antioxidant system”.
Manuscript ID: horticulturae-2444936
#Reviewer1
The authors improved the manuscript. Further minor improvements are required, especially in the language. a few examples:
- Figure 5, Panel F. MocK
Response: We thank the Reviewer for the careful reading of our text and apologise for the typographical error. ‘MocK’ in Figure 5 has been changed to ‘Mock’ as noted.
- Lines 325-329. Understandable, but awkward. Please rephrase.
Response: We appreciate this comment. This paragraph was rephrased as suggested in Line 336-346.
- Lines 414-415. "THe genes... will be further determined". Not clear who is going to further determine what. The reference here [38] tells something different.
Response: The sentence was modified as ‘ROS plays a dual role in plant biology and is closely related to plant stress response’ (lines 429).
- Many more examples here and there, please have your manuscript revised by a native speaker/professional editing service.
Response: We appreciate the Reviewer’s excellent advice and apologise for the errors in our original text. To ensure the language quality of the revised manuscript, it has been submitted to a professional editing service (Editage) and reviewed by a native English speaker.
Comments on the Quality of English Language
Many minor issues. Please have your manuscript revised by a native speaker/professional editing service
Author Response File: Author Response.docx
Reviewer 2 Report
This is a revised version.
Some of my previous comments have been taken into account. Captions are better. Yet all my remarks have not been considered. There are still a lot of remarks to be done, and to be honest it is the job of authors to make sure what they submit is good. I cannot spend my time writing down what they should do.
line 29: explain what is ethephon.
lines 47/48. “Generally, plants can acquire freezing or chilling resistance through cold acclimation”. This is not true. Cold acclimation is a specific term that defines the fact that plants can acquire resistance to freezing by a previous exposure to chilling. See the papers/reviews by Thomashow or Ruelland.
The definition given of cold acclimation lines 40/41 is not correct either? why “brief”? why “pulse”? Besides, what does “low temperature” stand for? it is chilling here: once again, Cold acclimation is a specific term that defines the fact that plants can acquire resistance to FREEZING by a previous exposure to CHILLING.
Lines 48/50 are not clear. what is the point, I think, is the fact that you can have mild winter, not cold enough to induce Cold acclimation (ie to set up the acquisition of freezing tolerance) followed by one freezing period.
Line 61. what “cold “ is it? freezing? chilling ? one or the other?
lines 70, 73 : what “cold “ is it? freezing? chilling ? one or the other?
delete “EP” abbreviation. write Eppendorf each time.
sentences lines 236 to 241 are not necessary
Is it necessary to have two digits????
What you measure is “ion leakage”, and you measure it through REC.
CK is not a term for “control”. I do not see any “k” in “control”. so just write “control” or nothing.
What about the fact that AAC and ethephon have not the same effects.
Lines 252-255 are confusing. “The results showed that compared with pre-treatment, the electrical conductivity of post-ethephon treatment was decreased 2.97 times”: so here it is compared to control, right? Then next sentence “. After application with ACC, AVG and 253
CoCl2, the decrease fold is 1.97, 1.72 and 1.00 times” it is not compared to control??? why? Choose to be consistent. either you only give the numbers of the ratio or compared to control. Anyway, what do you mean by “Ratio of electrical conductivity in comparison of pots – vs pre- treatment”??? treatment meaning what : cold and molecule. the best would be to have the ratio REC -5°C in presence of molecules vs REC -5°C with no molecules
You do not even tell if panel B is -5°C or -2°C! The same for figure 2!
caption figure 1. what is the stat test done?
So now, for figure 2, we have another way to present the data. why in the mock there is such a decrease in ion leakage? is mock for “0.005% Tween control”??
line 267: is “ethylene” meant for “ethephon”?
line 270. “the ACC content of ethylene synthesis precursor…” not good. “the content of ACC, ethylene synthesis precursor, …”
Fig 2. what is the test? T-test?
Activity and metabolites shoud be expressed as ratio to the control (day 0). meaning that it should be “1” for day 0 (like they did for gene expression).
not qualified
Author Response
Response to Reviewer Comments
Chen et al. “Exogenous activation of the ethylene signalling pathway enhances freezing tolerance of young tea shoots by regulating the plant antioxidant system”.
Manuscript ID: horticulturae-2444936
#Reviewer2
This is a revised version.
Some of my previous comments have been taken into account. Captions are better. Yet all my remarks have not been considered. There are still a lot of remarks to be done, and to be honest it is the job of authors to make sure what they submit is good. I cannot spend my time writing down what they should do.
We greatly appreciate the Reviewer’s professional assistance for improving our manuscript, and for taking the time to evaluate our data and presentation. We are making sincere efforts to incorporate the excellent advice and complete the revision to the Reviewers’ and Editor’s satisfaction.
- line 29: explain what is ethephon.
Response: We appreciate this comment, and have added the relevant information (‘2-chloroethylphosphonic acid, an ethylene release reagent’) in line 23, the site of first mention in the Abstract, and in line 101, at first mention in the main text for clarity.
- lines 47/48. “Generally, plants can acquire freezing or chilling resistance through cold acclimation”. This is not true. Cold acclimation is a specific term that defines the fact that plants can acquire resistance to freezing by a previous exposure to chilling. See the papers/reviews by Thomashow or Ruelland.
Response: We thank the Reviewer for this clarification, and apologise for our misstatement. The sentence was rewritten in the revised manuscript as follows (lines 50–54):
‘Generally, plants can acquire freezing resistance through previous exposure to chilling. However, as extreme weather events are occurring with increasing frequency, de-acclimated plants or newly generated tissues without the prior chilling exposure necessary to obtain freezing resistance capacity are particularly susceptible to a sudden temperature decrease in early spring (such as a freezing cold spell).’
- The definition given of cold acclimation lines 40/41 is not correct either? why “brief”? why “pulse”? Besides, what does “low temperature” stand for? it is chilling here: once again, Cold acclimation is a specific term that defines the fact that plants can acquire resistance to FREEZING by a previous exposure to CHILLING.
Response: We appreciate the Reviewer’s clear explanation and have rephrased the sentence accordingly in the revised manuscript (lines 43–44).
- Lines 48/50 are not clear. what is the point, I think, is the fact that you can have mild winter, not cold enough to induce Cold acclimation (ie to set up the acquisition of freezing tolerance) followed by one freezing period.
Response: We apologise that our previous description was unclear. As noted by the Reviewer, a particularly mild winter may be insufficient to allow plants to acquire freezing tolerance. Usually, however, plants can survive freezing during winter owing to the establishment of cold acclimation in the autumn or early winter. However, in spring, plants become de-acclimated; moreover, the new shoots of tea plants have never experienced chilling during the period of sprouting and therefore have not acquired freezing tolerance. Therefore, de-acclimated plants or newly generated tissue (specifically tea shoots germinated in spring) are particularly susceptible to a sudden temperature decrease in early spring. We have modified the text in the revised manuscript to clarify this issue (lines 50–54).
- Line 61. what “cold “ is it? freezing? chilling ? one or the other?
Response: We appreciate the Reviewer bringing this issue to our attention. Generally, we intended ‘cold’ to indicate ‘freezing cold’. We have modified the text here and throughout the revised manuscript to specify the latter for clarity.
- lines 70, 73 : what “cold “ is it? freezing? chilling ? one or the other?
Response: As noted in our previous response, we have modified the text here and throughout the revised manuscript to specify the intended meaning of ‘cold’ as ‘freezing’ for clarity, as recommended.
- delete “EP” abbreviation. write Eppendorf each time.
Response: We appreciate this comment. As recommended, the abbreviation ‘EP’ was replaced by ‘Eppendorf’ throughout the revised manuscript.
- sentences lines 236 to 241 are not necessary
Response: We have deleted the specified sentences in the revised manuscript, as requested.
- Is it necessary to have two digits????
Response: We thank the Reviewer for bringing this to our attention. One digit was reserved.
- What you measure is “ion leakage”, and you measure it through REC.
Response: In the present study, electrical conductivity was detected and relative electrolyte leakage was calculated as described in section 2.3 of Materials and methods.
- CK is not a term for “control”. I do not see any “k” in “control”. so just write “control” or nothing.
Response: We appreciate this helpful advice. We have corrected Figure 1 in the revised manuscript as suggested.
- What about the fact that AAC and ethephon have not the same effects.
Response: We addressed this query in our response during the previous round of review, and have also provided additional discussion in the revised manuscript (lines 389–394). We agree that previous studies have generally found similar effects using ACC or ethephon; nevertheless, the two agents did not yield similar results in our experiment.
- Lines 252-255 are confusing. “The results showed that compared with pre-treatment, the electrical conductivity of post-ethephon treatment was decreased 2.97 times”: so here it is compared to control, right? Then next sentence “. After application with ACC, AVG and 253 CoCl2, the decrease fold is 1.97, 1.72 and 1.00 times” it is not compared to control??? why? Choose to be consistent. either you only give the numbers of the ratio or compared to control. Anyway, what do you mean by “Ratio of electrical conductivity in comparison of pots – vs pre- treatment”??? treatment meaning what : cold and molecule. the best would be to have the ratio REC -5°C in presence of molecules vs REC -5°C with no molecules
Response: We apologise for the lack of clarity in our original manuscript and have modified the text in accordance with the Reviewer’s suggestion (lines 258–261).
- You do not even tell if panel B is -5°C or -2°C! The same for figure 2!
Response: The relevant temperature (−5 °C) has been indicated in the captions for Figures 1 and 2 as instructed.
- 1 caption figure 1. what is the stat test done?
Response: We have added the relevant information (‘Different letters indicate significant differences as assessed by one-way ANOVA followed by Tukey’s HSD comparisons (p < 0.05)’) to the Figure 1 legend as recommended.
- 16. So now, for figure 2, we have another way to present the data. why in the mock there is such a decrease in ion leakage? is mock for “0.005% Tween control”??
Response: We thank the Reviewer for the careful assessment of our data presentation. The Reviewer is correct; ‘mock’ represents the ‘0.005% Tween control’. The tea shoots were in a growth status; therefore, the ion leakage varied at different days following treatment.
- line 267: is “ethylene” meant for “ethephon”?
Response: We have corrected this misspelling in the revised manuscript (line 274).
- line 270. “the ACC content of ethylene synthesis precursor…” not good. “the content of ACC, ethylene synthesis precursor, …”
Response: We appreciate this correction and have modified the revised manuscript accordingly (lines 277–279).
- 19. Fig 2. what is the test? T-test?
Response: The Reviewer is correct; the TTest was applied. The information has been added in all the figure captions.
- Activity and metabolites shoud be expressed as ratio to the control (day 0). meaning that it should be “1” for day 0 (like they did for gene expression).
Response: The data were expressed using ratio to the control (day 0).
- Comments on the Quality of English Language
not qualified
Response: To ensure the language quality of the revised manuscript, it has been submitted to a professional editing service (Editage) and reviewed by a native English speaker.
Author Response File: Author Response.docx
