The Regulatory Effect of Se-Cd Interaction on Tea Plants (Camellia sinensis (L.) O. Kuntze) Under Cadmium Stress
, Yueling Zhao 1,2, Hongyu Zhou 1,2, Faxing Li 1,2, Yuanyuan Wang 1,2 and Xiao Du 1,2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors wrote an important piece of manuscript, “The regulatory effect of Se-Cd interaction on tea plants under cadmium stress”. The parameters studied in the manuscript are impressive but lacks depth in terms of analysis. Further, it lacks the following points:
1. Page 2 Line 69-71 Remove repetitive sentences for e.g.
Selenium compounds have antagonistic effects against the toxicity of elements such as
mercury (Hg), arsenic (As), cadmium (Cd), and lead (Pb) in humans, but there is little
research on their antagonistic effects against heavy metal toxicity in plants. Se and heavy metals like Cd often exhibit antagonistic relationships.
2. Avoid long sentences. For example,
Page 2 Line 78-80
Therefore, studying the ions that exhibit antagonistic or inhibitory effects during the absorption and accumulation of heavy ions such as lead (Pb), copper (Cu), chromium (Cr), and Cd by plants, and exploring the strength of their inhibitory effects, is of both theoretical and practical significance.
Page 13: Line 339-341
The accumulation of heavy metals in tea trees mainly comes from the absorption by the root system, while the cell walls of the root system can fix most of the heavy metals, thereby reducing their transport to the above-ground parts.
3. In Material and Methods
1. What is the basis of selection of Cd: Cultured with 20 mg/L CdSO4 nutrient solution?
2. What is the basis of selection of Cd + Se1.5: Cultured with 20 mg/L CdSO4 + 1.5 mg/L Na2SeO3 nutrient solution
3. The discussion is shallow, lacks depth and cited less references.
4. Make the conclusion a little short.
5. Add more references in the introduction and discussion section.
Therefore, manuscript needs thorough revision and reconsider after major revision.
Comments on the Quality of English Language
The authors wrote an important piece of manuscript, “The regulatory effect of Se-Cd interaction on tea plants under cadmium stress”. The parameters studied in the manuscript are impressive but lacks depth in terms of analysis. Further, it lacks the following points:
1. Page 2 Line 69-71 Remove repetitive sentences for e.g.
Selenium compounds have antagonistic effects against the toxicity of elements such as
mercury (Hg), arsenic (As), cadmium (Cd), and lead (Pb) in humans, but there is little
research on their antagonistic effects against heavy metal toxicity in plants. Se and heavy metals like Cd often exhibit antagonistic relationships.
2. Avoid long sentences. For example,
Page 2 Line 78-80
Therefore, studying the ions that exhibit antagonistic or inhibitory effects during the absorption and accumulation of heavy ions such as lead (Pb), copper (Cu), chromium (Cr), and Cd by plants, and exploring the strength of their inhibitory effects, is of both theoretical and practical significance.
Page 13: Line 339-341
The accumulation of heavy metals in tea trees mainly comes from the absorption by the root system, while the cell walls of the root system can fix most of the heavy metals, thereby reducing their transport to the above-ground parts.
3. In Material and Methods
1. What is the basis of selection of Cd: Cultured with 20 mg/L CdSO4 nutrient solution?
2. What is the basis of selection of Cd + Se1.5: Cultured with 20 mg/L CdSO4 + 1.5 mg/L Na2SeO3 nutrient solution
3. The discussion is shallow, lacks depth and cited less references.
4. Make the conclusion a little short.
5. Add more references in the introduction and discussion section.
Therefore, manuscript needs thorough revision and reconsider after major revision.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsReview
The regulatory effect of Se-Cd interaction on tea plants under cadmium stress
Yanyun Sun, Yueling Zhao, Hongyu Zhou, Faxing Li, YuanYuan Wang and Xiao du
The relevance of the study of the problem of protecting tea plants from cadmium stress is beyond doubt. Heavy metals disrupt the vital activity of plant cells, reduce their growth and productivity, and worsen the quality of agricultural crops. This study is interesting because, along with studies of the causes of oxidative stress (increased levels of hydrogen peroxide and superoxide, lipid peroxidation), the authors study the protective mechanisms of cells of various organs in response to the action of heavy metals (cadmium) with the participation of selenium. The protection includes activation of the osmotic system (proline) and the antioxidant protection system of enzymatic nature (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase). The state of photosynthetic parameters (pigment content, characteristics of the functioning of photosystem 2) was assessed. Of interest are the data on the localization of cadmium and selenium in the cells of the root, stem and leaves of tea. We agree with the authors that selenium reduces the stress effect of cadmium, but it is not clear how this affects the quality of tea if the cadmium content in the leaf does not change.
Remarks
Abstract
At the beginning of the Abstract, you should state the relevance and purpose of your research, and only then provide the parameters studied and the results of the research.
Ð .1, L.13.
Please remove the coefficient in the abbreviation for selenium, as the study uses a single concentration of the element and the extra numbers clutter the text.
Ð .1, L.21.
Please enter enzyme names at first mention.
Keywords: Tea plant
The Latin name of the tea plant should be entered, omitting the word "plant"
P.2, L. 50
Genes should be specified.
P.2, L. 69-70
The Latin name of the tea plant must be provided.
2.2. 2.2.1, L. 111-138
The Methodology should expand the description of the methods, indicating their authors. Specify the name of the kits for determining substances, as well as the devices used to analyze the physiological indicators of tea plants.
The methodology should include a decoding of the photosynthetic parameters.
Ð .4, L.151-153.
Results: The text of the manuscript does not correspond to the data presented in Fig. 1a and 1c. Please clarify.
Ð .5, L.179
Please clarify, as the trend of active forms content is opposite.
Ð .6, L.185
Please clarify heading 3.3.1.
Ð .6, L.197
Replace with "experimental options"
Ð .11, L.264
Please clarify heading 3.4.2.
When comparing parameters, one should rely not only on %, but also on statistical data. For example, if the NPG and Gp parameters differ under cadmium stress and combined treatment with cadmium and selenium, then one should not write that they decrease by %. Fo and Fm were not the lowest, since Fo Cd + Se = CK, Fm Cd + Se = Cd.
Ð .12, L.271
In Fig. 8 the accumulation of cadmium and selenium should be indicated with arrows.
What tissue cells are shown in Fig. 8?
Ð .12, L.286-288, Ð .13, L.314-317.
In the Discussion, the authors should analyze their data and, if necessary, provide data for other plants that are consistent with the data obtained for tea plants. And not vice versa.
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn a publication entitled "The regulatory effect of Se-Cd interaction on tea plants under cadmium stress", the authors describe an important problem of heavy metal accumulation in plants. In their work, the authors chose tea as a model plant. This is particularly important in the context of the widespread use of tea by many consumers and thus the potential occurrence of heavy metals in food products.
Below is a review of the paper:
Introduction:
In the introduction, the authors should include information on what are the sources of heavy metals in tea cultures. Are they natural or artificial sources introduced during agrotechnical treatments?
It is also important in the context of this paper to outline how Cd ions can accumulate in the plant and whether the enzymes described by the authors are involved.
The authors also mention that selenium (Se) can be toxic to plants. What doses of Se are already toxic and what doses are not yet toxic?
Materials and methods
Line 101 - Citation. Even well-known methods need to be cited. Preferably the original paper where this substrate was first used.
Chapter 2.2.2
This chapter needs to be rewritten by the authors. A scientific paper is not a technical research report. The methodology requires citations to original or modified papers on which the authors have based their research, or a detailed description of the research carried out so that the methodology can be replicated by other researchers. As a reviewer, suggest that the authors provide a detailed description of the methodology used in the paper.
Why did the authors use selenium (Se) at 1.5 mg/L? Provide an explanation.
Results:
The main objection to this chapter is the way in which the graphs have been prepared. All bar graphs in the paper need to be changed. The use of bars that blur from bottom to top makes them unreadable. In addition, very similar colours have been used for each bar. This also needs to be changed to make the graphs more readable. The font used is also very unreadable. There are also green rectangles in the titles of the figures. In every graph. What does this mean?
The authors have used lines in each graph. The descriptions give the impression that the authors are comparing the trend of changes between the variants under investigation. This is a mistake. You cannot use correlations between the groups studied if there are not many variables in them. This is what the statistics used, in this case Duncan's test, are for. The authors should remove these lines and change the description of the results.
The authors sometimes use the abbreviation APx, sometimes Apx. It should be APX throughout the paper.
Section 3.3.1 and 3.3.3
The authors describe many parameters by their abbreviations. There is no explanation of what these parameters mean in the methodology. This should be added
Chapter 3.3.2
What do the blue, green, yellow and red zones mean? The fluorescence relationship described here should be explained in detail.
Figure 7 - The authors cannot use a line graph to compare test results from different study groups.
Figure 8 - The first part of Figure 8 (page 11, just after line 275) appears to be detached from the rest of the figure. There is no description or numbering. It looks like a detached part of the whole. The authors need to check this.
Discussion:
The authors have obtained many intermediate results in their work, but have not discussed them as much as is possible with the literature data. The effects of heavy metals have been studied for many years. On plants, microorganisms and humans. It is necessary to extract more data from the literature. One such example is: Why does the activity of SOD, APX, POD and CAT decrease after the application of Cd, but return to the initial level after the addition of Se?
The authors should clarify this.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe study of the problem of Se–Cd interaction in the cultivation of various plant species, including tea, has been the subject of many scientific publications, which indicates the relevance of this research topic. The authors presented their research material logically and obtained important results indicating the important role of selenium in reducing the negative effects of cadmium contained in soil contaminated with heavy metals.
There are requests, questions and comments on the manuscript.
Be sure to include the Latin name of the tea plant in the abstract, keywords, materials and methods.
The abstract should be edited, and information about the cadmium concentration, which cadmium salt was used, the method of treating plants with cadmium, which selenium salt was used, the conditions for growing plants (hydroponics), etc. should be included. How long after cadmium treatment were the analyses carried out?
L.19-21. The authors write "Cd stress treatment significantly increased the content of peroxides in tea tree leaves and significantly decreased the activities of four antioxidant enzymes, SOD, POD, CAT, and Apx; Cd+Se1.5 treatment significantly reduced the peroxide content in tea trees under cadmium stress and significantly increased the activities of SOD, POD, CAT, and Apx.;". At the same time, see L. 54-56, it is written that "Following Cd treatment, the activities of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in tea plants are significantly elevated". The authors do not explain this contradiction - what determines the increase or decrease in enzyme activity - the method of cadmium treatment (root, foliar), the concentration of cadmium or other factors.
L.31-35. The conclusion of the abstract should be changed based on the title of the manuscript.
In the Introduction, the authors did not show the role of selenium in the vital activity of higher plants - as an important chemical element included in many enzymes.
References to literary sources are needed (L.40-46).
The topic of Se-Cd interaction is poorly covered - references to literary sources are not provided (L.83-84.)
In Materials and Methods, the choice of this particular type of tea for the study should be substantiated (L.92-94).
L.101. A reference to the article by Shigeki Kobayashi (1986) is needed.
Section 2.2. Methods - should be expanded, the essence of each method should be indicated, a reference to a literary source or Kit should be given (indicate its number and manufacturer). This is a very important section, the presentation of the results and discussion depend on it, so it should be presented in detail.
L.114. On what basis was this concentration of 20 mg / L CdSO4 taken?
L.127. The authors write "superoxide anion content". It is impossible to measure superoxide anion content, since its lifetime is 10-11 seconds. This is a gross error. Usually the rate of superoxide anion generation is determined. Correct here and further in the text (e.g. 3.2. Effects of Se-Cd Interaction on the Regulation of Antioxidant System in Tea Plants under Cadmium Stress; Figure 2.)
L.133. Specify which Chlorophyll fluorescence parameters were determined. The following parameters should be specified in Materials and Methods - Fo, Fm, Fv/Fm, Fv´/Fm´, Y(â…¡), OE, Qp, NPQ, Y(NO), Y(NPQ).
For example, Fo is the initial fluorescence level, etc. In addition, explain the Light Energy Absorption indices.
At the end of the methodological section (or in the captions to the tables and figures), statistical information about the presented results must be provided - what do the numbers and points on the graphs represent - the average of how many replicates (biological or analytical) of a single experiment or a typical experiment from a series; the average of how many repeated experiments? The methodology must indicate what the plus/minus in the tables and the bars on the graphs mean: standard error or standard deviation.
Due to the brief presentation of the methods and the incomplete presentation of the statistical analysis, additional questions arise.
For example,
Figure 1a - what does "Electrolyte permeability, %" mean. If this is % of the total yield of electrolytes from leaf cuttings, then values ​​from 2 to 3.5 are very low - this cannot be! Moreover, an increase in Electrolyte permeability by 20.87% is apparently within the error limits, statistically unreliable.
Figure 3. The word "enzymatic" can be removed from the ordinate axis and from the figure caption.
L.210. Replace "Figrues 5" with "Figures 5"
L.211. Apparently not "in 2.3.1", but "in 3.3.1"
L.215-216. The authors write "The decrease in Fv/Fm indicates that the PSII reaction centers are inhibited or the photosynthetic apparatus is damaged to some extent." However, Figure 4c shows an increase in Fv/Fm, which is known to depend on NPQ, which decreases - Figures 4e. At the same time, the proportion of open reaction centers of photosystem 2 also decreases. This should be discussed in the Discussion section.
Figure 7. The ordinate axis should start at 0, not “-10”. It is better to swap the leaf and root graphs - this will be logically correct.
L.241. "Accumulation Distribution"- apparently error.
L.244-247. The authors write "Compared with the CK control treatment, the Cd content in the tea plant root system under Cd stress significantly increased by 78.86%; the Cd content in the stem significantly increased by 56.36%; and the Cd content in the leaves significantly increased by 48.17%.". It is not clear how this was calculated, given that there was no cadmium in the control (CK). In this case, it is correct to use absolute values ​​of cadmium content in the experimental variants.
Figure 8b - nothing is visible!
Section 4. Discussion contains few references to literary sources. The discussion of the results should be strengthened.
L.351. What do the authors mean by "osmiophilic granules" - plastoglobuli? If yes, then plastoglobuli should be written.
It should be noted that the list of references consists of only 23 articles - this is very little.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsReview 2
The regulatory effect of Se-Cd interaction on tea plants (Camellia sinensis) under cadmium stress
Yanyun Sun, Yueling Zhao, Hongyu Zhou, Faxing Li, YuanYuan Wang and Xiao Du
The authors have clarified many of the comments on the text.
They described the methods and inserted references to the authors.
Table 1 and Table 2 have deciphered the parameters of chlorophyll fluorescence and the distribution of energy score points.
Comments
Methodology
In the Methodology there is no reference to the authors of the method for determination of electrolyte leakage rate. What is meant by this rate if the leaf is shredded?
It is desirable to decipher the abbreviations of enzymes in the Methodology on page 4, line 168, and to remove the abbreviation from page 23, lines 390-391. The same applies to malonic dialdehyde and proline.
Results
The authors did not change title 3.4.2.
The text talks about the distribution of metals in the cell, so we should insert the word “cell” of different organs in the heading. In addition, the cells of leaf and root are organized into tissues that perform different functions. These functions are especially pronounced in the root. I would like to know which tissue cells we are talking about in the pictures. The authors did not give an explanation.
In Figure 8, it is not clear what color cadmium and selenium inclusions are represented. The reviewer suggested the authors to insert arrows, but it is possible to describe the color of the phenocrysts.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors of the paper entitled "The regulatory effect of Se-Cd interaction on tea plants (Camellia sinensis) under cadmium stress" have addressed almost all of the reviewers' comments, thereby improving the quality of the paper. However, the authors still need to make some minor corrections before publication.
Section 2.2.2 - Auotrzy described the methodology used, but the description itself is not done correctly for a scientific paper. In scientific papers, all activities should be described impersonally in the past tense. The style of the paper should be improved here.
Results - in many places they use terms like: showed a pattern of low-high-low. The results presented do not lend themselves to such interpretations. Reversing the test samples will change the order but will not affect the results. Please check this carefully.
Figure 7 - auotrs did not address the reviewer's comments about changing the appearance. However, it cannot be a line graph. Any sub-chart must be converted to a bar chart. Or a cumulative bar chart.
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe authors answered all the reviewer's questions, made corrections to the manuscript, made an additional table, and expanded the list of references.
However, there is 1 question -
L. 179. The authors write "Superoxide anion generation rate", while in Figure 2b it says "Content of O2-", please correct the figure. In addition, make a change to L.232.
Author Response
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