Seasonal Eco-Physiology Characteristics of Four Evergreen Rhododendron Species to the Subalpine Habitats

Round 1

Reviewer 1 Report

General comments

I have read the manuscript (forest -1655041). Entitle: Eco-physiology regulation strategies of four evergreen Rhododendron species to the subalpine habitats written by Huie Li et. al., for publication of forest MDPI. In this study, the author investigates the eco-physiological regulation characteristics by determining their seasonal variation of antioxidant enzyme activity, osmotic adjustment substrates content, pigment content, and photosynthetic efficiency. In this manuscript author mainly found the regulation trends were slightly different among the four Rhododendrons, but the general trends were similar. Overall, the maximum quantum yield of PSII, the actual quantum yield of PSII, the non-photochemical chlorophyll fluorescence quenching, and chlorophyll content increased in warm summer. Meanwhile, the protective enzyme activity, and total soluble sugar content, proline content, and carotenoid content increased in cold spring, autumn, and winter. The overall conclusion stated that the Rhododendrons can adapt to subalpine habitats.

The overall research is well conducted, and research is obvious application potential for the readers because that helps to the baseline for the conservation and application of these valuable ornamental evergreen broadleaf Rhododendrons, in the plant Eco physiological aspect in the high altitudes. In this sense, the manuscript is much valuable. However, I found some points, especially the flow of the text is not smooth and sometimes I found the shallow writing and lack of potential references, and lack of connection of story in different paragraphs, especially in the introduction and discussion sections. In discussion, the author should be deal with the physiological perspectives that properly cover the plant biological parts. I also found the lack of potential and appropriate references to support the findings. The author should provide enough examples and their interpretation of different traits of physiological and biochemical responses. I mention some suggestions and recommend some literature. I hope these suggestions will help to improve this manuscript quality better than before. Overall after I evaluate this manuscript, I request the author for the “MAJOR REVISION” and, I request to authors for revision according to the rules of the journal and correct the bibliography.

 Major suggestions

1)Abstract Issue: The author wrote the important finding in their abstract, but the text seems more confusing. Author was unable to figure out the regulation trend and general trend figure out between the four Rhododendron species.  The author mentions that the regulation trend was different but what are those and trends in the species is unclear. Please revise this part in the abstract. Please see the Ln. 20 and 21 in the abstract. The author should focus on the novelty of the finding in the abstract while comparing the Rhododendron species. Moreover, the presentation of the result parts is somewhat shallow and less conciseness. Please remember that the abstract should more logical, short, concise, and informative. Your abstract should reflect your study and major findings while shortly observed by readers. Please make the necessary corrections. Please address all those comments in the abstract for further clearance of your research to the readers.

2) Introduction: Starting of the introduction of Rhododendron and its geographical coverage very well in the first paragraph. However, author should focus general text which is related to the physiological and biochemical perspectives in the second paragraph. Some part is included but that is still not enough to the background for Eco physiological and biochemical parts. Refer this article https://doi.org/10.1016/j.scienta.2017.12.006 and improved the text in like “physiological performance specially Pn and Gs increased due to the leaf thickness and increase the Chlorophyll content because those help to capture the better light and higher amount of light due to Chl. then higher possibility of Pn because of conversation of light energy change into the chemical energy”. The author should present these things and related literature.

3) Hypothesis and objectives of the study: Author should rephrase the text in the last paragraph of the introduction. Author should more focus on the hypothesis of the study then at the same time author should connect the objectives of the study based on the hypothesis. Please revise the text from lines no 83-87. I saw the author mention the hypothesis and objective, but this expression is quite not enough and not the sequential flow. The hypothesis of the study is an important thing, and it gives another strength for the introduction. The hypothesis should be very clear in the introduction sections because, without appropriate literature, questions, or hypotheses in the introduction section the entire text will be unclear. The author should give special attention and the sequential presentation of the content in the introduction with presenting the hypothesis of the study.

4) Concise the text in the whole manuscript: The author should be more careful about the conciseness of the text in throughout the manuscript. Author should conciseness the text in the introduction and discussion section.

Some others comment

5) Line 136-137:  Author should include the author’s name at least even unable to describe the full methodological parts to determine the biochemical traits such as SOD, POD, and CAT activities.

6) Line 161:  Author should revise the subtitle of 3.2, actually the subtitle is not clear, please rephrase this.

7) Line no. 231(Discussion): Author should Improve subtitle 4.1 further. Author should specially focus on the reason for ROS formation and its possible reasons behind it because the main theme of antioxidant and secondary metabolites formation especially SOD, POD CAT, and other secondary metabolites under the environmental stress condition not only for the cold acclimation.  The author should to refer and cite these two articles for better clarify accurate examples for the stress condition (1) https://doi.org/10.1016/j.scitotenv.2021.146466 (2) https://doi.org/10.1038/s41598-019-55889 Author should mention that “abiotic stress especially environmental stress (I.e. drought) plant produces the ROS when these plant exposed to the stress condition and plant produce antioxidant, flavonoids, and secondary metabolites play to the role for protecting the plant for detoxifying ROS and protect the plant to protect the abnormal condition (i.e. stress) and protein and amino acid stabilization”.

 8) Line no. 272 (Discussion)

The author should Improve this subtitle further. Especially author should by deal with the plant-water relation as well with the dealing the Fv/Fm, PS11, Pn, gs because to control the water use efficiency in the different canopy, stomatal conductance (gs) plant the very important role by controlling the mechanism of stomatal closing. I request to author to refer to these articles 1) https://doi.org/10.1016/j.envexpbot.2020.104111 (2) doi: 10.3390/plants8070232. describe the controlled by the stomata opening and closing because stromal full opening and partial closing cause the turgid the plant part”. Moreover, it may be varying the transpiration rate from the leaf canopy level, this story you mention clearly by referencing the above references somewhere in 4.2 sections.

9) Line 260:  Why author did not include the statistical analysis in the figure 4 E, F, G, H, with in the same figure please try to make consistent of the statistical analysis.

10) Line 266-268:  Author repeated the “13 eco-physiological indicators in the same figure footnote, on this behalf author just mentions one with the include the broad categories of those traits that might be better. Or for more clarification, the PCA analysis author may also include all those traits in the figure footnote.

11) Conclusion section (Line no. 339)

The conclusion for me comes off as repetitive of the abstract or a summary of the results section. I would love to read striking points and take-home messages that will linger in the readers’ minds. What is the novelty, how does the study elucidate some questions along this field, and the contributions the paper may offer to the scientific community?

 12) Line no. 367: Reference: please double-check the citations, their style, and spell check, and other grammatical errors. moreover, I request to authors for revision throughout the manuscript according to the journal rules.

Good Luck!

Author Response

Response: We thank three reviewers for the valuable comments and suggestions for improving. All suggestions have been carefully revised in our revised manuscript.

Reviewer1

Major suggestions

1)Abstract Issue: The author wrote the important finding in their abstract, but the text seems more confusing. Author was unable to figure out the regulation trend and general trend figure out between the four Rhododendron species. The author mentions that the regulation trend was different but what are those and trends in the species is unclear. Please revise this part in the abstract. Please see the Ln. 20 and 21 in the abstract. The author should focus on the novelty of the finding in the abstract while comparing the Rhododendron species. Moreover, the presentation of the result parts is somewhat shallow and less conciseness. Please remember that the abstract should more logical, short, concise, and informative. Your abstract should reflect your study and major findings while shortly observed by readers. Please make the necessary corrections. Please address all those comments in the abstract for further clearance of your research to the readers.

Response: We thank the reviewer for this suggestion. We carefully revised the abstract from Ln 20 to 26. In this part, we focused on every Rhododendron species to clarify its physiological characteristics in order to present the novelty of the finding. Moreover, we further improved the language to shape the concise abstract. All the revised places were addressed in the MS.

2) Introduction: Starting of the introduction of Rhododendron and its geographical coverage very well in the first paragraph. However, author should focus general text which is related to the physiological and biochemical perspectives in the second paragraph. Some part is included but that is still not enough to the background for Eco physiological and biochemical parts. Refer this article https://doi.org/10.1016/j.scienta.2017.12.006 and improved the text in like “physiological performance specially Pn and Gs increased due to the leaf thickness and increase the Chlorophyll content because those help to capture the better light and higher amount of light due to Chl. then higher possibility of Pn because of conversation of light energy change into the chemical energy”. The author should present these things and related literature.

Response: We thank the reviewer for this directive suggestion. In the second paragraph, we added the content related to physiological and biochemical parts in Ln 62 to 73. Meanwhile, we referred these valuable articles (https://doi.org/10.1016/j.scienta.2017. 12.006). According the related literatures (Reference 8,9, and 10), we improved the text about physiological performance in Ln 86 to 90, especially the regulation of net photosynthetic rate, stomatal conductance in Ln 87. 

Related reference:

5. Bhusal, S.J.; Yoon, T.M. Comparisons of physiological and anatomical characteristics between two cultivars in bi-leader apple trees (Malus×domestica Borkh.). Sci. Hortic. 2018, 231, 73–81.
6. Rathore, N.; Thakur, D.; Chawla, A. Seasonal variations coupled with elevation gradient drives significant changes in eco-physiological and biogeochemical traits of a high altitude evergreen broadleaf shrub, Rhododendron anthopogon. Plant Physiol. Biochem. 2018, 132, 708–719.
7. Ran, F.; Zhang, X.; Zhang, Y.; Korpelainen, H.; Li, C. Altitudinal variation in growth, photosynthetic capacity and water use efficiency of Abies faxoniana Rehd. et Wils. seedlings as revealed by reciprocal transplantations. Trees 2013, 27, 1405–1416.
8. Guo, Q.Q.; Li, H.E.; Zhang, W.H. Variations in leaf functional traits and physiological characteristics of Abies georgei smithii along the altitude gradient in the Southeastern Tibetan Plateau. J Mt Sci. 2016, 13, 1818–1828.

3) Hypothesis and objectives of the study: Author should rephrase the text in the last paragraph of the introduction. Author should more focus on the hypothesis of the study then at the same time author should connect the objectives of the study based on the hypothesis. Please revise the text from lines no 83-87. I saw the author mention the hypothesis and objective, but this expression is quite not enough and not the sequential flow. The hypothesis of the study is an important thing, and it gives another strength for the introduction. The hypothesis should be very clear in the introduction sections because, without appropriate literature, questions, or hypotheses in the introduction section the entire text will be unclear. The author should give special attention and the sequential presentation of the content in the introduction with presenting the hypothesis of the study.

Response: We thank the reviewer for this valuable suggestion and particular reminder. We had carefully renewed the hypotheses in the introduction section. Based on our focus on our research object and giving the special attention, we presented two main questions, which were (1) how do Rhododendrons plants cope with seasonal climate change by regulating physiological parameters? (2) whether there are significant differences in their physiological regulation characteristics at different altitudes？ (Ln 98 to 104)

4) Concise the text in the whole manuscript: The author should be more careful about the conciseness of the text in throughout the manuscript. Author should conciseness the text in the introduction and discussion section.

Response: Thanks to the reviewer for your suggestions. We carefully read the MS again and revised the text in the revised MS. Special in the introduction and discussion section, the repetitive and wordy statements were simplified furtherly, such as, Ln 65-68, Ln 103-104, Ln 175-176 in 3.1 section, Ln 219-220 in 3.2 section, Ln 350-335 and Ln 368-369 in 4.2 section, Ln 394-401 and Ln 413-414 in discussion section.

5) Line 136-137:  Author should include the author’s name at least even unable to describe the full methodological parts to determine the biochemical traits such as SOD, POD, and CAT activities.

Response: Ok, thanks a lot. We added the detail text about the extract of the supernatant and the related method in Ln 160-165 in 2.4 section.

6) Line 161:  Author should revise the subtitle of 3.2, actually the subtitle is not clear, please rephrase this.

Response: We thank the reviewer for this suggestion. We revised the subsite of 3.2 in order to make it clear furtherly in Ln 195.

7) Line no. 231(Discussion): Author should Improve subtitle 4.1 further. Author should specially focus on the reason for ROS formation and its possible reasons behind it because the main theme of antioxidant and secondary metabolites formation especially SOD, POD CAT, and other secondary metabolites under the environmental stress condition not only for the cold acclimation.  The author should to refer and cite these two articles for better clarify accurate examples for the stress condition (1) https://doi.org/10.1016/j.scitotenv.2021.146466 (2) https://doi.org/10.1038/s41598-019-55889 Author should mention that “abiotic stress especially environmental stress (I.e. drought) plant produces the ROS when these plant exposed to the stress condition and plant produce antioxidant, flavonoids, and secondary metabolites play to the role for protecting the plant for detoxifying ROS and protect the plant to protect the abnormal condition (i.e. stress) and protein and amino acid stabilization”.

Response: We thank the reviewer for this suggestion. Firstly, we improved the subtitle 4.1 further in Ln 270. Secondly, we furtherly discussion the reason for ROS formation and its possible reasons in Ln 271-278, especially SOD, POD, CAT and their protective functions. Thirdly, the two articles (1. https://doi.org/10.1016/j.scitotenv.2021. 146466; 2. https://doi.org/10.1038/s41598-019-55889) gave us so many important information about plant producing antioxidant and secondary metabolites, which played the role for protecting the plant for detoxifying ROS and protect the plant to protect the abnormal condition. Therefore, we didn’t hesitate to cite these literatures. As a whole, our knowledge about abiotic stress of plant had also been further enriched by learning the previous research.

Related reference:

26. Ahmad, P.; Sarwat, M.; Sharma, S. Reactive oxygen species, antioxidants and signaling in plants. J Plant Biol. 2008, 51, 167–173.
27. Hashempour, A.; Ghasemnezhad, M.; Fotouhi Ghazvini, R.; Sohani, M.M. Olive (Olea europaea L.) freezing tolerance related to antioxidant enzymes activity during cold acclimation and non acclimation. Acta Physiol. Plant. 2014, 36, 3231–3241.

8) Line no. 272 (Discussion)

The author should Improve this subtitle further. Especially author should by deal with the plant-water relation as well with the dealing the Fv/Fm, PS11, Pn, gs because to control the water use efficiency in the different canopy, stomatal conductance (gs) plant the very important role by controlling the mechanism of stomatal closing. I request to author to refer to these articles 1) https://doi.org/10.1016/j.envexpbot.2020.104111 (2) doi: 10.3390/plants8070232. describe the controlled by the stomata opening and closing because stromal full opening and partial closing cause the turgid the plant part”. Moreover, it may be varying the transpiration rate from the leaf canopy level, this story you mention clearly by referencing the above references somewhere in 4.2 sections.

Response: We thank the reviewer for this suggestion. We improve the subtitle 4.2 in Ln 317. We improve the related exposition in detail about the regulation characteristics of photosynthesis and chlorophyll fluorescence meanwhile, we cited the helpful references (1.https://doi.org/10.1016/j.envexpbot.2020.104111; 2. https://doi:10.3390/ plants8070232.) to clarify the controlling the mechanism of stomatal closing. The revised place was located in Ln 320-324, page 9.

Related reference:

35．Bhusal, N.; Kim, H.S.; Han, S. G.; Yoon, T.M. (2020). Photosynthetic traits and plant–water relations of two apple cultivars grown as bi-leader trees under long-term waterlogging conditions. Environ. Exp. Bot. 2020, 176, 104111.

36. Bhusal, N.; Lee, M.; Lee, H.; Adhikari, A.; Han, A.R.; Han, A.; Kim, H.S. Evaluation of morphological, physiological, and biochemical traits for assessing drought resistance in eleven tree species. Sci. Total Environ. 2021, 779, 146466.

9) Line 260:  Why author did not include the statistical analysis in the figure 4 E, F, G, H, with in the same figure please try to make consistent of the statistical analysis.

Response: We thank the reviewer for pointing this out, and apologize for our carelessness. We improved the statistical analysis (Figure 4 E, F, G, H) in our revised manuscript (Figure 4).

10) Line 266-268:  Author repeated the “13 eco-physiological indicators in the same figure footnote, on this behalf author just mentions one with the include the broad categories of those traits that might be better. Or for more clarification, the PCA analysis author may also include all those traits in the figure footnote.

Response: We thank the reviewer for this suggestion. We improved the caption of Figure 5 in the PCA analysis, and the 12 physiological indicators have been added to the figure caption in Ln 311.

11) Conclusion section (Line no. 339)

The conclusion for me comes off as repetitive of the abstract or a summary of the results section. I would love to read striking points and take-home messages that will linger in the readers’ minds. What is the novelty, how does the study elucidate some questions along this field, and the contributions the paper may offer to the scientific community?

Response: We thank the reviewer for this constructive advice. We rewrite the conclusion section. The tedious text has been removed, and we focused on our study to elucidate the novelty find in the revised MS in Ln 403-408.

 12) Line no. 367: Reference: please double-check the citations, their style, and spell check, and other grammatical errors. moreover, I request to authors for revision throughout the manuscript according to the journal rules.

Response: We thank the reviewer for pointing this out. According to Forests’ format, we rechecked all the citations carefully and corrected the errors (Related reference 10,13, 18,15,43).

Author Response File: Author Response.docx

Reviewer 2 Report

The presented manuscript is very topical from the point of view of understanding the ecological adaptation of plants to the negative effects of the environment.

This is a hitherto completely unknown and undescribed part of plant responses to environmental factors, especially with regard to plant metabolism, enzymatic activity. I think it will have very good citation potential after adjustments. I have the following comments and remarks on this manuscript.

The introductory part describes in detail the locality of interest, but unfortunately in a smaller part, it is devoted to the issues addressed. I recommend focusing on this part.

Workflows are described briefly, with links to resources. Is it sufficient? I somewhat lack information on which sheets the parameters were measured on. What is the age of the leaves? What was their position on the plant? The location was described in terms of climate, but not the weather. It would be appropriate to supplement the weather within the observed period. The text states that summer is referred to as the warm season and the other three as the cold season. On what basis was this defined?

The results are based on a description of their graphical representation. It would be appropriate to add values ​​or relative values ​​to the text.

Graphs are necessary, but graphs 2 and 3 are small, harder to read. Graph 5 is also harder to read. I would also recommend cross-correlations of the obtained results.

The discussion is rather general. It is appropriate to focus on the analysis of the obtained data.

It is necessary to check the citations, as not all journals are written in abbreviations and capital letters.        

Author Response

This is a hitherto completely unknown and undescribed part of plant responses to environmental factors, especially with regard to plant metabolism, enzymatic activity. I think it will have very good citation potential after adjustments. I have the following comments and remarks on this manuscript.

Response: We thank the reviewer for her/his affirmation and the valuable comments. However, there were still a lot of imperfections in the MS, and we tried our best to revise them in order to reach the publication level.

The introductory part describes in detail the locality of interest, but unfortunately in a smaller part, it is devoted to the issues addressed. I recommend focusing on this part.

Response: We thank the reviewer for this suggestion. We focused on the physiological and biochemical characteristics to clarify the scientific questions. Especially in the second paragraph, we cited the related reference to elaborate the plant responses of leaf phenotype, photosynthesis, and biochemistry under the abiotic stress. The revised parts were located in Ln 61-74, Ln 86-87, and Ln 99-104.

Workflows are described briefly, with links to resources. Is it sufficient? I somewhat lack information on which sheets the parameters were measured on. What is the age of the leaves? What was their position on the plant? The location was described in terms of climate, but not the weather. It would be appropriate to supplement the weather within the observed period. The text states that summer is referred to as the warm season and the other three as the cold season. On what basis was this defined?

Response: We thank the reviewer for this suggestion. We added the necessary text to explain the age and position of the leaves on the plant in 2.2 section (Ln 130-132). About the climate, we added the related text in Ln 115-120. About the defined season, we usually based on the growing season of native wood plant, which were divided into the warm season (the growing season) and the cold season (the nongrowing season). Meanwhile，we also supplemented the supporting reference (Reference 16) in Ln 136-139. 

The results are based on a description of their graphical representation. It would be appropriate to add values ​​or relative values ​​to the text.

Response: We thank the reviewer for this suggestion. We added the text to clarify the enzyme activities (Ln 179-182 in 3.1 section), proline content (Ln 199-204 in 3.2 section), chF parameters (Ln 225-226, Ln 233-234 in 3.3 section) in the revised manuscript. The specific values are easy to understand for reader.

Graphs are necessary, but graphs 2 and 3 are small, harder to read. Graph 5 is also harder to read. I would also recommend cross-correlations of the obtained results.

Response: We thank the reviewer for this suggestion. We replotted the color graphic2 and 3 so that the reader can observe the change trend clearly. Meanwhile, graph 5 include so much information that we have to enlarge it just to be clear exhibition.

The discussion is rather general. It is appropriate to focus on the analysis of the obtained data.

Response: We thank the reviewer for this suggestion. According to the obtained data, we reorganized the discussion section to make them more concise and focused. In the 4.1 section, we added the antioxidants enzymes generate and function (Ln 271-278). In the 4.2 section, we added the elaboration about photosynthesis and chF (Ln 320-323 and Ln 335-338). Meanwhile, the content about pigment content were cut.

It is necessary to check the citations, as not all journals are written in abbreviations and capital letters.        

Response: We thank the reviewer for this reminder. We checked all the citations carefully and corrected the errors (Related reference 10,13, 18,15,43).

Author Response File: Author Response.docx

Reviewer 3 Report

A descriptive work presenting seasonal changes in some stress parameters in 4 species of Rhododendron in subalpine habitats. Plant material and its habitats are very nice but descriptions of all other components of the ms are very far from scientific value.

Introduction is schematic. A great majority (if not all) literature data are described as “adaptations” (p.2. 2^nd paragraph). For example: "Rhododendron decorum in Yunnan-Guizhou Plateau of Southwest China adapts to seasonal change by adjusting its PSII activity." (l.61) or "Three plant species, namely, Populus cathayana, Salix longistamina, and Ulmus pumila, adapt to the QTP by regulating the characteristics of respiration and, photosynthesis."(l.68). This is very superficial way and not proper. These are list of parameters/cues which have been measured. The Authors need to undertake an effort to understand deeper the deliveries of these papers.

English needs a strong improvement. The words “eco-physiological” and “meanwhile” are highly abused throughout the ms. For example, in the goal of the paper (l.87-90) the word “eco-physiological” is used twice, as “eco-physiological regulation” and  “eco-physiological responses”. The Authors use many scientific words but the meaning of the sentences has not much sense. An example of that is the above-mentioned formulation of the goal of the paper – which is finally superficial and not convincing (only pretending to sound scientific).

Similarly to the goal, the title is unacceptable “Eco-physiology regulation strategies..”. There are no strategies described or their regulations, but just the seasonal changes in some well-known stress parameters.

There are many nonsensical expressions and sentences in the text. For example: “leaves with healthy function..” (p.3.l.115) or "The differences in SOD activities of R. wardii or R. triflorum were not significant was the not significant during autumn and winter." (p.4.l.151-152) or "Different photosynthetic pigment performs different functions and resistances to adversity." (p.5.l.183).

Also, as a rule, the formulations used in figure legends are not good. For example: “Figure 2. Seasonal protective enzyme SOD (a), POD (B), and CAT (C) activity..” (p.4.l.157).

The subtitles in M&M are not precise enough or misleading, for example: ”Lab detection of leaf substances” (p.4.l.131).

Several descriptions in M & M are not sufficient. Methodology for chlorophyll fluorescence parameters ignores the intensity of actinic light, longevity of the induction curve etc. These characteristics and their proper adjustment are really crucial to obtain a valuable information. This in particular relates to plants in subalpine locations (above 3000 m). It also raises some doubts whether the temperature used for chlF measurements, namely 25 deg C, is proper in view of an average of 8.15 deg C in summer for this habitat.

Description of methodology for antioxidant enzymes activities is unacceptable: “The activity of protective enzymes SOD, POD, and CAT activities were detected following the methods in references.." (136-137). The reviewer cannot judge whether these methods are proper or not and how the Units of enzymatic activities are calculated.

Changes in CAT activities are not described in Results.

Discussion is not very informative and demands a deeper literature mining. There are many papers describing stress parameters and protective strategies in alpine plants – but they are largely ignored. In part describing chlF parameters there is no mention about PSII photoinhibition. Authors should also take into consideration how changes in the poll of open and closed PSII reaction centers affect qP. Discussion on changes in chlorophyll pigments and their ratio is not scientific at all.

The theory about influence of shorter and longer light wavelengths affect the composition of chl a and chl b in totally not convincing. Firstly, the Authors should explain the difference between adaptation to high and low light in regard to chlorophylls content and their ratio. The striking conclusion given on page 9 (l.311-312), namely, “R. aganniphum distributed at relatively higher altitudes can grow in a shorter time than R. triflorum; thus, it needs additional chl a” need a better explanation to be convincing. This is also of importance to verify whether such increase in chlorophyll content/FW does not result from the increased content of dry mass in leaves. The mechanism regulating the size of photosynthetic antenna in response to light intensity does also to be explored.

In regard to carotenoids, it is of importance to understand that their protective role is not due their higher stability than chlorophylls but due to their ability to dissipate excess excitation energy. Such information is missing.

A concise conclusion about differences in relation to different locations above the sea level is missing.

The chapter “5.Conclusions” (p.10) is written like the abstract and does not deliver any concise conclusion from this paper. This chapter is also rich in expressions such as “eco-physiological regulation characteristics..”, “eco-physiological regulation trends..” but these mean nothing. The claim written at the end of Conclusion (p.10.l.354-357) that "These finding .. enrich theory of plant eco-physiology in the high altitudes of QTP." - is totally not supported by the data.

Author Response

Introduction is schematic. A great majority (if not all) literature data are described as “adaptations” (p.2. 2^nd paragraph). For example: "Rhododendron decorum in Yunnan-Guizhou Plateau of Southwest China adapts to seasonal change by adjusting its PSII activity." (l.61) or "Three plant species, namely, Populus cathayana, Salix longistamina, and Ulmus pumila, adapt to the QTP by regulating the characteristics of respiration and, photosynthesis."(l.68). This is very superficial way and not proper. These are list of parameters/cues which have been measured. The Authors need to undertake an effort to understand deeper the deliveries of these papers.

Response: We thank the reviewer for this suggestion. We have recognized the text about “Rhododendron decorum” in Ln 72-73 of the revised MS, and more focused on the internal physiological mechanisms. Meanwhile，the part of "Three plant species, namely, Populus cathayana, Salix longistamina, and Ulmus pumila, adapt to the QTP by regulating the characteristics of respiration and, photosynthesis." has also been revised in Ln 77-83. We undertake an effort to present the more deeper information to readers.

English needs a strong improvement. The words “eco-physiological” and “meanwhile” are highly abused throughout the ms. For example, in the goal of the paper (l.87-90) the word “eco-physiological” is used twice, as “eco-physiological regulation” and “eco-physiological responses”. The Authors use many scientific words but the meaning of the sentences has not much sense. An example of that is the above-mentioned formulation of the goal of the paper – which is finally superficial and not convincing (only pretending to sound scientific).

Response: We thank the reviewer for this valuable suggestion. We furtherly improved the writing, and deleted the repetitive word in the introduction part (Ln 90-95 and Ln 106-109). Meanwhile, we also checked the whole MS and revised the similar statements.

Similarly to the goal, the title is unacceptable “Eco-physiology regulation strategies..”. There are no strategies described or their regulations, but just the seasonal changes in some well-known stress parameters.

Response: We thank the reviewer for this suggestion. We have changed the inaccurate statement and turned the title into“Seasonal eco-physiology characteristics of four ever-green Rhododendron species to the subalpine habitat” in the revised MS. Based on our research, we focused on the physiological responses of four Rhododendrons growing in the subalpine region of QTP. Therefore, the renewed text has been done in the whole MS.

There are many nonsensical expressions and sentences in the text. For example: “leaves with healthy function..” (p.3.l.115) or "The differences in SOD activities of R. wardii or R. triflorum were not significant was the not significant during autumn and winter." (p.4.l.151-152) or "Different photosynthetic pigment performs different functions and resistances to adversity." (p.5.l.183).

Response: We thank the reviewer for this suggestion. We revised the unsuitable expressions and sentences in Ln 135-136, Ln 183-192, and Ln 223-228.

Also, as a rule, the formulations used in figure legends are not good. For example: “Figure 2. Seasonal protective enzyme SOD (a), POD (B), and CAT (C) activity..” (p.4.l.157).

Response: We thank the reviewer for this suggestion. We have revised all the related figure legends, such as figure 2,3, and 4.

The subtitles in M&M are not precise enough or misleading, for example: ”Lab detection of leaf substances” (p.4.l.131).

Response: We thank the reviewer for this detailed suggestion. We revised the subtitle 2.4 in Ln 159.

Several descriptions in M & M are not sufficient. Methodology for chlorophyll fluorescence parameters ignores the intensity of actinic light, longevity of the induction curve etc. These characteristics and their proper adjustment are really crucial to obtain a valuable information. This in particular relates to plants in subalpine locations (above 3000 m). It also raises some doubts whether the temperature used for chlF measurements, namely 25 deg C, is proper in view of an average of 8.15 deg C in summer for this habitat.

Response: We thank the reviewer for this suggestion. We supplemented the statement about the methodology for chlorophyll fluorescence parameters in Ln 150-155. Meanwhile, we checked the temperature control of Li-Cor 6400 from the original record, the temperature used for chlF measurements was set at 15℃ at that time. We correct it in Ln 149 and apologized for our negligence.

Description of methodology for antioxidant enzymes activities is unacceptable: “The activity of protective enzymes SOD, POD, and CAT activities were detected following the methods in references.." (136-137). The reviewer cannot judge whether these methods are proper or not and how the Units of enzymatic activities are calculated.

Response: We thank the reviewer for this suggestion. We supplemented the statement about the determination of leaf inclusions according to the cited references, the revised text located in Ln 164-169.

Changes in CAT activities are not described in Results.

 Response: We thank the reviewer for this suggestion. We have improved the text and described about changes in CAT activities in the revised MS (Ln 189-192).

Discussion is not very informative and demands a deeper literature mining. There are many papers describing stress parameters and protective strategies in alpine plants – but they are largely ignored. In part describing chlF parameters there is no mention about PSII photoinhibition. Authors should also take into consideration how changes in the poll of open and closed PSII reaction centers affect qP. Discussion on changes in chlorophyll pigments and their ratio is not scientific at all.

Response: We thank the reviewer for this helpful suggestion. We collected the related references again, revised the content about PSII photoinhibition, and take into consideration how changes in the poll of open and closed PSII. The detail text was located in Ln 323-328 and Ln 339-342. Also, we cited the valuable reference [35] and [36].

The theory about influence of shorter and longer light wavelengths affect the composition of chl a and chl b in totally not convincing. Firstly, the Authors should explain the difference between adaptation to high and low light in regard to chlorophylls content and their ratio. The striking conclusion given on page 9 (l.311-312), namely, “R. aganniphum distributed at relatively higher altitudes can grow in a shorter time than R. triflorum; thus, it needs additional chl a” need a better explanation to be convincing. This is also of importance to verify whether such increase in chlorophyll content/FW does not result from the increased content of dry mass in leaves. The mechanism regulating the size of photosynthetic antenna in response to light intensity does also to be explored.

Response: We thank the reviewer for this suggestion, which encouraged us to lean the latest references in the field. Meanwhile, we deleted the irrelevant statement and revised the text in the revised MS (Ln 357-373).

In regard to carotenoids, it is of importance to understand that their protective role is not due their higher stability than chlorophylls but due to their ability to dissipate excess excitation energy. Such information is missing.

Response: We thank the reviewer for this suggestion. We collected the inform abut carotenoids which was helpful to improve the MS, and revised the text in Ln 371-373.

A concise conclusion about differences in relation to different locations above the sea level is missing.

Response: We thank the reviewer for this suggestion. We rewrite the conclusion and summarize the concise conclusion in Ln 414-415

The chapter “5.Conclusions” (p.10) is written like the abstract and does not deliver any concise conclusion from this paper. This chapter is also rich in expressions such as “eco-physiological regulation characteristics..”, “eco-physiological regulation trends..” but these mean nothing. The claim written at the end of Conclusion (p.10.l.354-357) that "These finding .. enrich theory of plant eco-physiology in the high altitudes of QTP." - is totally not supported by the data.

Response: We thank the reviewer for this valuable suggestion. We recognized the text and purified the conclusion in the revised MS. Certainly, we deleted the nonsense words. Meanwhile, the text was improved furtherly in Ln 418-422. Thanks for the reviewer again.  

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Author

I have read the revised manuscript (Forests -165541). Entitle: Eco-Physiology Regulation Strategies of Four Evergreen Rhododendron Species to the Subalpine Habitats publication in forest MDPI. This is the second submission made by the author. The author addressed all the questions and suggestions that I raised the issue in the review of the original manuscript. I satisfy the author’s revisions throughout the paper. Especially author improved the introduction and discussion section very well inflow. The abstract issue is also solved by the author. Now, this manuscript improved the flow of writing, which was comparatively shallow in the original version but in this revised copy author addressed all the quarries and suggestions very well. Before accepting this manuscript if there is anything needed to be revised by the author, especially English grammar or spell check, I request this manuscript is currently in “Minor Revision” and any grammatical error author may improve in this stage. Thank you.

Author Response

Response: We thank the reviewer for these suggestions. We carefully checked English grammar or spell check and revised them throughout the paper. All the revised parts in the manuscript adopt the revised mode. 

Reviewer 2 Report

The authors submitted a modified manuscript. In the attached letter, they comment on the individual changes and adjustments they have made to the text. All comments and comments of the reviewer were accepted and duly commented. The text is now modified as above. The text is now clearer and points to the results obtained. It is already possible to publish it in this form.

Author Response

Response: We thank the reviewer for this affirmation, which is which is a positive encouragement to us. Meanwhile, we also carefully checked the whole text, and corrected the spelling and formatting errors.