Effects of Exogenous Trehalose on Plant Growth, Physiological and Biochemical Responses in Gardenia Jasminoides Seedlings During Cold Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigated how exogenous trehalose improved cold resistance in Gardenia jasminoides seedlings exposed to low-temperature stress. The findings provide a theoretical basis for using trehalose to improve cold tolerance in ornamental plants. Thus, I believe the present manuscript is suitable for Horticulturae after revision.

• The abstract is too extended; I invite the authors to drastically reduce the abstract to 250-300 words (or check the requirements of the journal)
• Without lines number its very tough to review this paper
• They should reorganize their abstract
• The numeric results are redundant and overly detailed; try to focus on the most important results concisely
• The final sentence of your abstract should provide a general conclusion and/or perspectives
• The same pattern was observed in the introduction section, which is too long
• Reduced drastically the background about oxidative stress, osmoregulation and trehalose, also don’t repeat these concepts in different paragraphs
• What were the hypotheses tested in this study?
• In the first sentence of the Introduction the authors claimed that “There are about 250 species of Gardenia jasminoides in the world”, the authors might confuse genus and species ?
• Merge paragraph 2 and 4 and please make it concise
• In the paragraph about photosynthesis, please avoid providing examples that are unrelated to your study (brassinosteroids, etc). It can help you to reduce the length and focus more on your topic
• In the last paragraph, the authors should show the main objectives and hypotheses of their study
• In the Materials and Methods (MM) why used POTS with uppercase letters?
• In MM described the light incubators (Conviron? Or etc. need more information)
• Where did you get the seedlings, and describe the substrate in which they grew
• “Multifunctional leaf measuring instrument” described how you measured the pigments and apparatus used.
• You said that you have two factors, so which type of ANOVA you used?
• The results are very well organized; please revise the grammatical errors and make sure that the tense is always in the past
• At the bottom of each table please make sure that the standard deviation or error are mentioned, as the number of replicates and the statistical test, and do the same for the figure’s legends
• The discussion needs to be edited. Some words are not appropriate. Here are a few, examples: cultivation measures, effective means, exogenous application of trehalose solution, the participation of many pigments, transformed in the body, When ROS is generated too much etc.
• This paragraph about photosynthesis is too long and unnecessary: “Photosynthesis of plants requires the participation of many pigments, and photo-synthetic pigments are a crucial component. Photosynthetic pigments mainly include chlorophyll and carotenoids, and light energy is transferred and transformed in the body after being absorbed by them during photosynthesis [33]. Chlorophyll is divided into chlorophyll a and chlorophyll b, which are responsible for the capture and transfer of light energy. When plants encounter low temperature stress, it will have adverse effects on their own photosynthesis process. It is generally believed that the original chlorophyll will be destroyed by low temperature, and the chlorophyll content will be forced to de-crease, which will eventually weaken the photosynthetic capacity of plants and inhibit the carbon assimilation pathway, resulting in slow plant growth [34].”
• Please re-edit your discussion section and make it more concise

Good Luck!!!!!

Author Response

Dear Editor and reviewers,

Thank you very much for consideration of this manuscript and peer review with helpful suggestions. We have revised the manuscript according to your suggestions with red background that you can immediately recognize where the changes have been made.

Now, I will answer reviewers’ comments one by one.

Response to the comments of Reviewer #1:

The authors investigated how exogenous trehalose improved cold resistance in Gardenia jasminoides seedlings exposed to low-temperature stress. The findings provide a theoretical basis for using trehalose to improve cold tolerance in ornamental plants. Thus, I believe the present manuscript is suitable for Horticulturae after revision.

1. The abstract is too extended; I invite the authors to drastically reduce the abstract to 250-300 words (or check the requirements of the journal). Without lines number its very tough to review this paper. They should reorganize their abstract.

 

Response: Thank you for pointing out this problem in our manuscript. We have already condensed the Abstract. And we have added lines number. Thanks very much.

 

 

2. The numeric results are redundant and overly detailed; try to focus on the most important results concisely. The final sentence of your abstract should provide a general conclusion and/or perspectives.

 

Response: Thank you for your suggestion. We have deleted some unimportant numeric results in Abstract. At the same time, we have already condensed the final sentence in Abstract.

 

3. The same pattern was observed in the introduction section, which is too long. Reduced drastically the background about oxidative stress, osmoregulation and trehalose, also don’t repeat these concepts in different paragraphs.

 

Response: Thank you for your suggestion. We have reduced drastically the background about oxidative stress, osmoregulation and trehalose in Introduction section.

 

4. What were the hypotheses tested in this study?

 

Response: The hypotheses tested in this study is Tre can enhance the cold resistance of Gardenia through various pathways such as photosynthesis, antioxidant system, endogenous hormone metabolism and respiration.

 

5. In the first sentence of the Introduction the authors claimed that “There are about 250 species of Gardenia jasminoides in the world”, the authors might confuse genus and species ?"

Response: Thank you for pointing out this problem in our manuscript. We have deleted this sentence.

 

6. Merge paragraph 2 and 4 and please make it concise.

Response: Thank you for your suggestion. We have merged and made it concise.

 

7. In the paragraph about photosynthesis, please avoid providing examples that are unrelated to your study (brassinosteroids, etc). It can help you to reduce the length and focus more on your topic.

Response: Thank you for your suggestion. We have deleted the sentence about brassinosteroids.

 

8. In the last paragraph, the authors should show the main objectives and hypotheses of their study.

Response: Thank you for your suggestion. We have added the hypotheses and objective in the last paragraph.

 

9. In the Materials and Methods (MM) why used POTS with uppercase letters?

Response: Thank you for pointing out this error. We have changed it to pots.

 

10. In MM described the light incubators (Conviron? Or etc. need more information).

Response: Thank you for pointing out this error. We have changed it to ‘illumination incubator (18/6 h day/night)’.

 

11. Where did you get the seedlings, and describe the substrate in which they grew

Response: The seedlings were given by Jiangxi Academy of Forestry and we have added it in MM. The substrate was sterilized river sand (have added in MM), and Hoaglang nutrient solution was irrigated from the day of planting.

 

12. “Multifunctional leaf measuring instrument” described how you measured the pigments and apparatus used..

Response: Thanks. The chlorophyll fluorescence parameters were determined by IMAGING-PAM (M-series modulated chlorophyll fluorescence meter, Heinz Walz GmbH, Nuremberg, German). We have added it in MM.

 

13. You said that you have two factors, so which type of ANOVA you used?

Response: Thank you. Data were subjected to a 2-way ANOVA using the GLM procedure of SAS software. We have added it in MM.

 

14. The results are very well organized; please revise the grammatical errors and make sure that the tense is always in the past.

Response: Thank you for your suggestion. We have carefully revised the grammatical errors.

 

15. At the bottom of each table please make sure that the standard deviation or error are mentioned, as the number of replicates and the statistical test, and do the same for the figure’s legends.

Response: Thank you for your suggestion. We have added complete information in the bottom of Table 1 and noted that ‘The same below’. However, there is no data analysis in the figures, so this information was not added below the figures.

 

16. The discussion needs to be edited. Some words are not appropriate. Here are a few, examples: cultivation measures, effective means, exogenous application of trehalose solution, the participation of many pigments, transformed in the body, When ROS is generated too much etc.

Response: Thank you for your suggestion. We have carefully revised them and other non-appropriate words.

 

17. This paragraph about photosynthesis is too long and unnecessary: “Photosynthesis of plants requires the participation of many pigments, and photo-synthetic pigments are a crucial component. Photosynthetic pigments mainly include chlorophyll and carotenoids, and light energy is transferred and transformed in the body after being absorbed by them during photosynthesis [33]. Chlorophyll is divided into chlorophyll a and chlorophyll b, which are responsible for the capture and transfer of light energy. When plants encounter low temperature stress, it will have adverse effects on their own photosynthesis process. It is generally believed that the original chlorophyll will be destroyed by low temperature, and the chlorophyll content will be forced to de-crease, which will eventually weaken the photosynthetic capacity of plants and inhibit the carbon assimilation pathway, resulting in slow plant growth [34].”

Please re-edit your discussion section and make it more concise.

Response: Thank you very much. We have carefully re-edit the Discussion section.

Author Response File: Author Response.doc

Reviewer 2 Report

Comments and Suggestions for Authors

This study aimed to evaluate the mitigating effects of exogenous trehalose on Gardenia jasminoides seedlings subjected to cold stress. The paper’s strength is the evidence that trehalose can significantly alleviate cold-induced damage, particularly at –3°C, by enhancing photosynthesis, reducing oxidative stress, and modulating hormone and metabolic responses. The manuscript is well-structured, even if using only a single trehalose concentration limits understanding of a dose-response relationship. The experimental approach is comprehensive, covering multiple physiological layers (growth, ROS, photosynthesis, hormone balance, and metabolism), and the results support the conclusions drawn. The writing is sometimes repetitive, but the topic is intriguing and relevant to fundamental plant physiology and practical horticulture under cold stress conditions.  

I suggest acceptance after minor revisions.

In particular:

Pag 1

The abstract is too extended and reports too many results

Line 13: after Juss. "trealose" in capital letters

Line 20 Check: To provide a theoretical basis for trehalose to improve the cold resistance of plants.

Pag 2 Introduction

Line 61 and line 74: "disaster" and "body" do not seem appropriate terms

Line 77 Check: Or a series of physiological and biochemical reactions in the body can cause serious effects

Line 115: plants are stressed by stress… I would eliminate "by stress" or specify what kind of stress is (by biotic factors or by drought, salinity, extreme temperatures, nutrient deficiencies.... ), but “plants stressed” is enough

Line 151: MDA: I assume it means malondialdehyde, the first time you should explain

Line 166: It also……lowercase letter

Lines 174-176: "The main significance of this study was to provide  new technology and theoretical support for cold resistance cultivation of gardenia from the perspective of exogenous trehalose"…… may be that "for cultivation of gardenia under low-temperature conditions" sounds better

Pag 4 Materials and methods

A curiosity that has nothing to do with the review: why exactly 15mmol/l? Thanks

Line 182: ‘POTS’ is in capital letters

Line 122: OD (Optical Density)

Line 241: The phrase is not grammatically correct; something is missing - Endogenous hormones were determined using...etc.

Pag 6 Results

Line 261: the effect….’was shown’…you use the past tense even in other lines, but for example in lines 336 and 570, you use the present ‘are shown’ - make uniform. I prefer the present, but it is up to you.

Lines 271, 282 and often in the results, you begin with: It can also be seen….It is worth noting that. It's correct and fine, but it makes reading a bit monotonous.

Lines 287-289: check the percentages and calculations, the reduction is excessive; you probably forgot a step (if so, recheck all the percentages - if sometimes you mention data instead of percentages, it becomes easier for the reader to follow, but it's your choice)

Line 380: improves or improved

Line 500: Pro - in line 226 proline (Pro)

Line 504: omega?

Discussion

Line 662:  between the abiotic stresses, include heavy metal toxicity, to connect Razzaq in line 681

Line 726: LOX activity ( lipoxygenase enzymes)

Conclusion

These are too long, and "trehalose treatment under low temperature stress" is repeated too many times.

Author Response

Dear Editor and reviewers,

Thank you very much for consideration of this manuscript and peer review with helpful suggestions. We have revised the manuscript according to your suggestions with red background that you can immediately recognize where the changes have been made.

Now, I will answer reviewers’ comments one by one.

Response to the comments of Reviewer #2:

This study aimed to evaluate the mitigating effects of exogenous trehalose on Gardenia jasminoides seedlings subjected to cold stress. The paper’s strength is the evidence that trehalose can significantly alleviate cold-induced damage, particularly at –3°C, by enhancing photosynthesis, reducing oxidative stress, and modulating hormone and metabolic responses. The manuscript is well-structured, even if using only a single trehalose concentration limits understanding of a dose-response relationship. The experimental approach is comprehensive, covering multiple physiological layers (growth, ROS, photosynthesis, hormone balance, and metabolism), and the results support the conclusions drawn. The writing is sometimes repetitive, but the topic is intriguing and relevant to fundamental plant physiology and practical horticulture under cold stress conditions. I suggest acceptance after minor revisions.

 

1. The abstract is too extended and reports too many results.

Response: Thank you for pointing out this problem in our manuscript. We have already condensed the Abstract. Thanks very much.

 

2. Line 13: after Juss. "trealose" in capital letters.

Response: Thanks. This problem has been resolved.

 

3. Line 20 Check: To provide a theoretical basis for trehalose to improve the cold resistance of plants.

Response: Thanks. We have deleted this sentence.

 

4. Line 61 and line 74: "disaster" and "body" do not seem appropriate terms.

Response: Thank you for pointing out these problem in our manuscript. We have revised them.

 

5. Line 77 Check: Or a series of physiological and biochemical reactions in the body can cause serious effects.

Response: Thank you for pointing out this error. We have deleted this sentence.

 

6. Line 115: plants are stressed by stress… I would eliminate "by stress" or specify what kind of stress is (by biotic factors or by drought, salinity, extreme temperatures, nutrient deficiencies.... ), but “plants stressed” is enough.

Response: Thank you for pointing out this error. We have deleted this sentence.

 

7. Line 151: MDA: I assume it means malondialdehyde, the first time you should explain.

Response: Thank you for your suggestion. We have added the full name.

 

8. Line 166: It also……lowercase letter.

Response: Thank you for pointing out this error. We have re-write this sentence.

 

9. Lines 174-176: "The main significance of this study was to provide  new technology and theoretical support for cold resistance cultivation of gardenia from the perspective of exogenous trehalose"…… may be that "for cultivation of gardenia under low-temperature conditions" sounds better.

Response: Thank you for your advice. We have re-write this sentence according to your suggestion.

 

10. A curiosity that has nothing to do with the review: why exactly 15mmol/l? Thanks.

Response: Thank you for your question. The reason why we chose this concentration is based on the previous references which indicate that this concentration can enhance the stress resistance of ornamental plants.

 

11. Line 182: ‘POTS’ is in capital letters.

Response: Thank you for pointing out this problem in our manuscript. We have revised it.

 

12. Line 122: OD (Optical Density)

Response: Thank you for pointing out this problem in our manuscript. We have revised it.

 

13. Line 241: The phrase is not grammatically correct; something is missing - Endogenous hormones were determined using...etc.

Response: Thank you for pointing out this problem in our manuscript. We have re-write it.

 

14. Line 261: the effect….’was shown’…you use the past tense even in other lines, but for example in lines 336 and 570, you use the present ‘are shown’ - make uniform. I prefer the present, but it is up to you.

Response: Thank you for your advice. We have made it uniform in the past tense.

 

15. Lines 271, 282 and often in the results, you begin with: It can also be seen….It is worth noting that. It's correct and fine, but it makes reading a bit monotonous.

Response: Thank you for your advice. We have made careful revisions to this issue.

 

16. Lines 287-289: check the percentages and calculations, the reduction is excessive; you probably forgot a step (if so, recheck all the percentages - if sometimes you mention data instead of percentages, it becomes easier for the reader to follow, but it's your choice).

Response: Thank you for your advice. We have made revisions to this issue.

 

17. Line 380: improves or improved

Response: Thank you for your advice. We have changed it to ‘improved’.

 

18. Line 500: Pro - in line 226 proline (Pro)

Response: Thank you for your advice. We have added ‘(Pro)’ after proline at the first occurrence place.

 

19. Line 504: omega?

Response: Thank you for pointing out this error. We have deleted it.

 

20. Line 662:  between the abiotic stresses, include heavy metal toxicity, to connect Razzaq in line 68

Response: Thank you for your advice. We have added heavy metal toxicity in suitable position.

 

21. Line 726: LOX activity ( lipoxygenase enzymes)

Response:Thank you for your advice. We have added lipoxygenase enzymes in suitable position.

 

22. Conclusion. These are too long, and "trehalose treatment under low temperature stress" is repeated too many times.

Response: Thank you for your advice. We have already condensed the Conclusion. Thanks again.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article “Effects of exogenous trehalose on plant growth, physiological and biochemical responses in Gardenia jasminoides seedlings during cold stress” by Zhang et al. addresses how exogenous trehalose affect the growth, physiological, and biochemical responses of Gardenia jasminoides seedlings under cold stress conditions. The authors investigate whether trehalose can enhance cold tolerance in G. jasminoides by mitigating the negative effects of low temperature on growth, photosynthesis, oxidative stress, and root metabolism.

This study is relevant to the fields of plant physiology, stress biology, and horticultural science, particularly in the context of improving abiotic stress tolerance in ornamental and woody plants. The specific gap addressed is the lack of physiological and biochemical data on the use of trehalose to improve cold tolerance in Gardenia jasminoides, a species that has ornamental and medicinal value but is sensitive to cold.

Although trehalose has been studied in several herbaceous species under various abiotic stresses, its effect on woody or semi-woody evergreen plants, particularly in the context of cold stress, is underexplored. This study helps fill that gap.

The study adds a multifaceted understanding of how trehalose mitigates cold stress in G. jasminoides. Key novel contributions include:

- Comprehensive data showing that 15 mmol/L trehalose significantly improves both shoot and root biomass under -3°C stress.

- Insights into physiological mechanisms such as enhanced photosynthetic efficiency, antioxidant enzyme activities (SOD, POD, CAT), and hormone modulation (increased auxin, decreased zeatin).

- Biochemical evidence suggesting trehalose modulates root respiratory metabolism, notably increasing malic acid and reducing succinic acid levels under stress.

Compared with other studies focused primarily on foliar responses, this work highlights root-specific responses to trehalose under cold conditions, which is relatively less documented.

Overall, the conclusions are well-supported by the experimental evidence:

- Phenotypic recovery of growth traits aligns with improved chlorophyll content and photosynthetic parameters.

- The enhancement in antioxidant enzyme activities and reduction in ROS support the claim that trehalose mitigates oxidative damage.

- Hormonal and respiratory metabolite data align with the improved stress adaptation narrative.

This is a valuable contribution to the literature on cold stress physiology in ornamental species. It demonstrates convincingly that trehalose enhances cold tolerance in G. jasminoides, providing useful physiological and biochemical data. However, I suggest inserting the following minor revisions into a new, improved version:

- Following the journal guidelines, the abstract should be a total of about 200 words maximum, while this version presents an abstract with 539 words, more than double the limit. It is necessary to prepare a new revised abstract;

- It is not clear in the objectives nor the conclusion if the goal is to propose trehalose as a product to be applied to the plants or the study relies more in a theoretical view to better understand the inner responses in plant metabolism, please make it clearer in both end of introduction and conclusion;

- The experiment adopted a two-factor design, but the statistical analysis was performed as if it were a single-factor design. The correct way to analyze these data is to consider the interaction between the factors and, if this is significant, proceed to unfold the levels of one factor within the other;

- Lines should be numbered following the journal model;

- Preferably use the mM form for concentration, instead mmol/L;

- - Also, the form ng g-1 is preferable to ng/g;

- Figure 1: the caption (with the small piece of paper below) is not visible, it would be better to provide a proper editing of the image with the indication of the treatments in visible and clear text. Also, a scale bar, instead of just the ruler next to the plants, would be helpful;

- Figure 1: the caption (with the small piece of paper below) is not visible, it would be better to provide a proper editing of the image with the indication of the treatments in visible and clear text. Also, a scale bar, instead of just the ruler next to the plants, would be helpful;

- The same about the scale bars applies to Figure 2;

- The conclusion is very long and has many repetitions of discussions, it should be more concise.

Author Response

Dear Editor and reviewers,

Thank you very much for consideration of this manuscript and peer review with helpful suggestions. We have revised the manuscript according to your suggestions with red background that you can immediately recognize where the changes have been made.

Now, I will answer reviewers’ comments one by one.

Response to the comments of Reviewer #3:

1. Following the journal guidelines, the abstract should be a total of about 200 words maximum, while this version presents an abstract with 539 words, more than double the limit. It is necessary to prepare a new revised abstract;.

Response: Thank you for pointing out this problem in our manuscript. We have already condensed the Abstract. Thanks very much.

 

2. It is not clear in the objectives nor the conclusion if the goal is to propose trehalose as a product to be applied to the plants or the study relies more in a theoretical view to better understand the inner responses in plant metabolism, please make it clearer in both end of introduction and conclusion.

Response: Thank you for your suggestion. We have added the hypotheses and objective in the last paragraph of Introduction. And we also have already re-write the Conclusion.

 

3. The experiment adopted a two-factor design, but the statistical analysis was performed as if it were a single-factor design. The correct way to analyze these data is to consider the interaction between the factors and, if this is significant, proceed to unfold the levels of one factor within the other.

Response: Thanks. Data were subjected to a 2-way ANOVA using the GLM procedure of SAS software. We have added it in MM.

 

4. Lines should be numbered following the journal mode.

Response: Thank you for your suggestion. We have added lines number.

 

5. Preferably use the mM form for concentration, instead mmol/L. Also, the form ng g-1 is preferable to ng/g;

Response: Thank you for your suggestion. Since mmol/L was used in the figures (can't be changed), to maintain consistency, we decide use mmol/L and ng/g. 

 

6. Figure 1: the caption (with the small piece of paper below) is not visible, it would be better to provide a proper editing of the image with the indication of the treatments in visible and clear text. Also, a scale bar, instead of just the ruler next to the plants, would be helpful.The same about the scale bars applies to Figure 2

Response: Thank you for your suggestion. We have made modifications to the Figures 1 and 2 according to your suggestions.

 

7. The conclusion is very long and has many repetitions of discussions, it should be more concise.

Response: Thank you for your advice. We have already condensed the Conclusion. Thanks again.

Author Response File: Author Response.pdf