Regulatory Effects of Exogenous Trehalose on the Growth and Photosynthetic Characteristics of Celery (Apium graveolens L.) Under Salt Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please see the attached reoprt.

Comments for author File: Comments.pdf

Author Response

The study entitled “Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress” focusses on improvement in photosynthetic performance of celery plants treated with salinity due to trehalose pre-treatment. Trehalose, being a critical component of plant sugar sensing, as well as an osmolyte has been widely explored in literature. Recent studies have shown that exogenous application of trehalose can relieve osmotic stress (salinity, drought etc.) effects in different plant species. Considering the increasing soil salinity and current agricultural sustainability goals, studies focussed on identification of specific solutions and mechanisms of improved tolerance in different crops should be encouraged. In this context, the basic idea of the authors is appreciated. Also, the general presentation of given data is decent. However, major shortcomings are evident in the experimental design, presentation of study objectives, conclusions derived and language (in some parts) etc. Specific comments are given below:

 

Response : Thank you for your encouraging remarks and valuable comments on our manuscript entitled “Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress” (ID: plants-3995547). These suggestions are all of great value and will be of great help to the revision and improvement of our thesis. Based on your opinions and suggestions, we have made extensive revisions to the manuscript and hope to obtain your approval. We used the "Track Changes" function in Microsoft Word to modify our manuscript, and the modified parts were marked in red.  

 

Point 1: Please clarify if trehalose was sprayed every day for the mentioned 7 days! (the expression is not clear). In any case, in the interpretation of positive effects of trehalose, analysis of a “control” with only trehalose-application parallel to other plants with/without salt-trehalose combination is missing. It is evident in literature that trehalose excess, considering it is an important metabolite, can drastically shift the metabolic balance and hence influence growth rate or photosynthesis. Its interaction or lack of it with salinity cannot be justified without analysis of plants that received only trehalose! Therefore, it

would be ideal if authors, after a brief presentation of salinity and trehalose concentration dependence of growth (as in Fig. 1, 2), compare only CK (no treatment), N (100 mM NaCl), T3 (10 mM trehalose + 100 mM NaCl), and Trehalose-alone (10 mM) for all other.

 

Response 1: Thank you for your valuable suggestion. We sprayed trehalose solution once a day for 7 days. Modify the text as "After 7 days of transplanting, celery seedlings were foliar sprayed with different concentrations of trehalose (Tr; 0, 1, 5, 10, and 15 mM) once a day at 09:00 am, and continued for 7 consecutive days." (Lines 433-435). Thank you again for your suggestions on the experimental design of this paper. As you said, "After briefly introducing the dependence of salinity and trehalose concentration on growth (as shown in Figures 1 and 2), It would be ideal to compare only CK (untreated), N (100 mM NaCl), T3 (10 mM trehalose + 100 mM NaCl), and all other cases of trehalose alone (10 mM).  Our research approach is exactly the same as yours.  This study is divided into three steps.  The first two steps are pre-experiments, aiming to lay the foundation for in-depth analysis of the role of trehalose.  The first step is to screen out the moderate salt stress concentration (100 mM NaCl).  The second step is to screen out the appropriate trehalose concentration based on the moderate salt stress concentration.  Then, the third step is to conduct the formal experiment.  Make an in-depth and systematic comparison of CK (no treatment), N (100 mM NaCl), T3 (10 mM Trehalose + 100 mM NaCl), and Trehalose-alone (10 mM).  Comprehensively explain the effects of trehalose, as an important osmotic regulatory substance, on the growth, photosynthesis and other indicators of celery under moderate salt stress.

 

Point 2: Further, considering that relative metabolic significance of trehalose can vary from one plant species to another, as can the sucrose to trehalose ratio, it is required that:

1. a) a background trehalose content is determined and its significance to metabolism in celery is placed in context of the current findings.
2. b) it needs to be presented clearly that the trehalose and salinity application is not simultaneous, but that it’s a trehalose pre-treatment aimed at some sort of osmotic priming of plants (authors have written the experimental design in methods part, but it needs to be made clear in figures, figure legends too).
3. c) also, in context of above, after trehalose pre-treatment and before salinity exposure, a comparison of internal trehalose content would be helpful. It would help identify if celery plants accumulate trehalose as osmolyte or it is metabolized and the effects seen are of downstream reprograming of the growth rate!

 

Response 2: Thank you for your kind comment, in this study, the determination of trehalose background content was derived from a large number of similar studies on other crops in the previous period. Based on their research results, we designed different concentration gradients and screened out the appropriate trehalose concentration based on indicators such as growth and photosynthesis. In the subsequent experiments, we will measure the safety quality, flavor quality and other indicators of celery, and then explain its significance to human metabolism. We have supplemented in the caption of each figure that "the application of trehalose and salt is not simultaneous; salt stress is carried out after the application of trehalose." (Figure 2- Figure 10). It is helpful to compare the internal trehalose content before trehalose pretreatment and salinity exposure. This suggestion is of great value and we fully agree. It can not only clearly determine the basic content of trehalose in celery, but also clearly identify whether trehalose accumulates or is metabolized as an osmotic substance. In the subsequent experiments, we will measure the trehalose content inside celery based on your suggestion.

 

Point 3: Another question pertaining to experimental design concerns missing analysis of any of the cellular markers of salinity, e.g. Na/K ratio, cellular osmotic imbalance or ROS status. Plant growth in this case is a final output of a complex metabolic reprogramming over 23 days from start of trehalose exposure, while photosynthesis is more directly influenced by the trehalose itself with or without salinity. Therefore, considering that authors have chosen roots as primary salinity exposure tissue, while leaves were sprayed with trehalose, analysis of salinity markers could help identify any direct correlation between the two as well as differentiate the local and systemic effects of NaCl and trehalose, both!.

 

Response 3:Thank you very much for your valuable suggestions on the experimental design again. Trehalose, as a osmotic regulatory substance, it is very important to clarify the osmotic imbalance and ROS status of cells. After screening out the appropriate trehalose concentration in this study, We will conduct experiments of CK (no treatment), N (100 mM NaCl), T (10 mM Trehalose + 100 mM NaCl), and Trehalose-alone (10 mM).Based on growth and photosynthesis, we will focus on the Na/K ratio, cellular osmotic imbalance and ROS status of different tissues (roots and leaves), and then determine the direct connection between trehalose and salt as well as distinguish the local and systemic effects of NaCl and trehalose.

 

Point 4: All the figure legends are very brief and must be expanded to make them “stand alone”, which is a pre-requisite..

 

Response 4: We have supplemented and improved all the graphic legends to make them " stand alone ".(Figure 1- Figure 10)

 

Point 5: The primary objectives of the study should be better defined and should be focussed on celery and not in general on vegetables. Further, introduction section should first clearly define the metabolic significance of trehalose, the metabolite, before exploring its use as a growth boosting agent. How much trehalose to spray can be an objective, as has been identified by authors, but it needs to be placed in context of how much trehalose, in general, can be in celery plants normally and if external application increases its accumulation? If not, which trehalose metabolic pathway is responsible for priming plants?

 

Response 5: Thank you for your valuable suggestion. Our main research objective is to explore the alleviating effect of exogenous proline on celery salt stress.  As you mentioned, focusing on celery would be more meaningful.  However, there are few reports on celery at present, so we try to refer to vegetables of the same family as much as possible.  In the introduction, we have already supplemented the metabolic significance of trehalose as a metabolite.  That is, "Trehalose metabolism is of great significance for plants to respond to abiotic stress.  It is an important signaling molecule and cell protective agent.  Meanwhile,  an appropriate concentration of trehalose can reduce Na ion accumulation and maintain ROS balance”.  To determine the appropriate concentration of trehalose, we referred to a large number of references in the early stage and then designed this experiment in order to screen out the appropriate concentration for alleviating salt stress in celery to the greatest extent.  Generally, the content in celery plants is relatively low.  Mastering the trehalose content in celery can accurately determine whether the externally applied trehalose accumulates, which is of great significance for our subsequent research.

 

Point 6: Mlultiple spelling errors, extra or no spaces and correct use of tenses should be revised.

 

Response 6: We have carefully checked and corrected the "spelling mistakes" in the text, deleted the redundant words, filled the places without Spaces, and comprehensively checked and corrected the tense issues.

 

Point 7: 3.1 needs to be shortened, too much repetition of the results. Other discussion parts can be revised in the light of above comments, after addition of missing data.

 

Response 7: Thank you for your valuable suggestion. We have shortened 3.1, deleted the repetitive parts, and carefully revised other discussion sections.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors Dear Author, The manuscript titled "Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress" was evaluated for scientific merit and typing formatting. The manuscript studied the ameliorative effects of different trehalose doses on celery under salt stress. Significant differences were observed in plant development and biochemical processes depending on the doses used. The most promising results were obtained with the application of 10 mM trehalose. This study will contribute to the existing literature on this subject. However, some spelling and wording errors were determined in the manuscript. Corrections and recommendations were shown to the main text. The manuscript may be suitable for publication after revision in accordance with the recommendations.      

Comments for author File: Comments.pdf

Author Response

Dear Author, The manuscript titled "Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress" was evaluated for scientific merit and typing formatting. The manuscript studied the ameliorative effects of different trehalose doses on celery under salt stress. Significant differences were observed in plant development and biochemical processes depending on the doses used. The most promising results were obtained with the application of 10 mM trehalose. This study will contribute to the existing literature on this subject. However, some spelling and wording errors were determined in the manuscript. Corrections and recommendations were shown to the main text. The manuscript may be suitable for publication after revision in accordance with the recommendations.

 

Response 1:Thank you for your and valuable comments on our manuscript entitled “Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress” (ID: plants-3995547). These suggestions were valuable and very helpful in revising and improving our paper. Based on the corrections and suggestions you made in the main text, we have carefully revised the spelling and wording errors in the manuscript and hope to obtain your approval. We used the "Track Changes" function in Microsoft Word to modify our manuscript, and the modified parts were marked in red.

 

Response 1: Thank you for your valuable suggestion, we have added " osmotic stress ". (Line 73)

 

Response 2: We have added "of ". (Lines 635-638)

 

Response 3: We have changed the word "stem" to "stalk" in the full paper.

 

Response 4: We have changed "stem" to "stalk" in the full paper.

 

Response 5: We have uniformly changed all the "Fig." and "Figure" in the entire text to "Figure".

 

Response 6: We have changed "(F)" to "(I)".

 

Response 7: We have added " concentrations ".

 

Response 8: We have changed "forks" to "branching" in the full paper.

 

Response 9: We have removed " analysis".

 

Response 10: We have changed "value" to "Value".

 

Response 11: We have added "leaf ".

 

Response 12: We have removed "growth".

 

Response 13: We have changed "chlroposhyll" to " chlorophyll ".

 

Response 14: We have changed "organs" to "system".

 

Response 15: We have changed "20 cm heigh × 20 cm diameter " to "volume, 6.28 L".

 

Response 16: We have added "(Tr) ".

 

Response 17: We have changed "Celery" to "celery" in the full paper.

 

Response 18: In this study, three replicates were set for each treatment, with 15 seedlings in each replicate. In the text, it has been changed to" The treatments were organized in a completely randomized design, each including five three replications and 15 seedlings in each replicate".

 

Response 19: We have changed "carbon" to "internal".

 

Response 20: We have changed "Illustrations" to "Description".

 

Response 21: We have carefully checked the spelling of " graveliens " and it is appropriate.

 

Response 22: We have changed " Ginkgo biloba " to " Ginkgo biloba ".

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article investigates aspects of the alleviation of negative effects of high salinity by priming of celery plantlets with the disaccharide trehalose, applied in different concentrations. The results are aimed to improved growth and photosynthetic performance of celery when cultivation occurs on saline soils.

Here are some specific comments aimed to improve the scientific quality of the article:

1. Row 14 of the Introduction: Fv/Fo does not represent the effective photochemical efficiency of PS II (which is Φ = ΔF / Fm’ from the modulated chlorophyll fluorescence parameters); Fv/Fo is just an alternative expression form of the potential or maximal quantum yield of PS II in dark-adapted leaves, less used than the Fv/Fm form; please correct the name of Fv/Fo throughout the text of the manuscript
2. In the section of Introduction, a short paragraph should be dedicated to the most characteristic manifestations of salt stress in plants, in order to facilitate interpretation of the results concerning salinity-induced physiological modifications.
3. At the end of the Introduction, a working hypothesis should be formulated and one or two questions should be asked, for which the research seeks pertinent answers.
4. In the title of figures, please mention what the vertical bars and the different letters represent (considering that every figure has to be self-explanatory).
5. In the title of 2.2, please change the term “different trehalose” to “different trehalose concentrations”.
6. In the second row above Figure 2, it is unclear what the term “forks” is intended to mean, please replace it with the appropriate scientific term.
7. In the legend of Figure 2.C, please replace the term “overground” with “aboveground”.
8. In subsection 3.2, please specify what was “the optimal concentration” of trehalose identified in the experiments.
9. In the first row of 3.3, please rephrase the affirmation that “chloroplasts facilitate photosynthesis”, because these organelles perform all the processes of photosynthesis, not only facilitate them.
10. In row 7 of 3.3, please consider that transpiration rate is not related to photosynthesis, but to water economy of plants.
11. In subsection 3.3, if mineral nutrients that are “crucial for chlorophyll biosynthesis”, please include in the list the iron, which is particularly needed for the biosynthetic pathway of chlorophylls.
12. In subsection 3.3, please correct the construction “the light energy absorbed by chloroplasts” to the more correct form: “the light energy absorbed by chlorophylls”.
13. At the beginning of the second paragraph of 3.3, please replace “chlorophyll fluorescence” with “induced chlorophyll fluorescence”, because this is the correct name of the parameter registered with the described method.
14. In the first row of page 13, please replace the term “oxidizing side of PSII” with the more correct term “donor side pf PSII”.
15. In the first paragraph of 3.4, please reconsider and replace the formulation “notable increase in overall fluorescence intensity”, because, as it is shown in figure 7A, there are very small differences between the different OJIP curves.
16. In the title of paragraph 4.2 please replace the term “morphological indices” with “growth” indices, because the mentioned parameters are related to plant growth and not to changes of the shape of different organs (morphology being the study of the shape of different body parts)
17. In section 4.2 please indicate the temperature and the time period of drying the plant material.
18. In section 4.3 please mention that Ci represents carbon dioxide concentration in the intercellular spaces of the leaf blade.
19. In section 4.4 the light intensities used for chlorophyll fluorescence measurements should be expressed as µmol m^-2 s^-1, not as µmol/ms (…ms stands for milliseconds, which is not the case here)
20. Because the data in Table 5 do not originate from the authors of the manuscript, at the end of the table title please give the reference from which the parameters are taken.

   21. Concerning the statistical analysis of experimental data sets, before choosing the ANOVA and Duncan’s test, evaluation of homogeneity of variances and analysis of normality of data sets distribution should be performed. After these, ANOVA and Duncan’s test can be used only if the distribution of data sets was proved to be normal. Were these tests performed before ANOVA, and if not, please explain why not.

Author Response

The article investigates aspects of the alleviation of negative effects of high salinity by priming of celery plantlets with the disaccharide trehalose, applied in different concentrations. The results are aimed to improved growth and photosynthetic performance of celery when cultivation occurs on saline soils.

Here are some specific comments aimed to improve the scientific quality of the article:

 

Response : Thank you for your valuable comments on our manuscript entitled “Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress.” (ID: plants-3995547). These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have revised the manuscript substantially and hope it will be approved. We revised our manuscript using the “Track Changes” function in Microsoft Word and the revised portions were marked in red color.

 

 

Point 1: Row 14 of the Introduction: Fv/Fo does not represent the effective photochemical efficiency of PS II (which is Φ = ΔF / Fm’ from the modulated chlorophyll fluorescence parameters); Fv/Fo is just an alternative expression form of the potential or maximal quantum yield of PS II in dark-adapted leaves, less used than the Fv/Fm form; please correct the name of Fv/Fo throughout the text of the manuscript

 

Response 1: Thank you for your valuable suggestion. We have changed the name of Fv/Fo to "maximal quantum yield of PSII in dark-adapted".

 

Point 2: In the section of Introduction, a short paragraph should be dedicated to the most characteristic manifestations of salt stress in plants, in order to facilitate interpretation of the results concerning salinity-induced physiological modifications.

 

Response 2: Thank you very much! We have briefly introduced the typical manifestations of plant salt stress and deleted the redundant parts.

 

Point 3: At the end of the Introduction, a working hypothesis should be formulated and one or two questions should be asked, for which the research seeks pertinent answers.

 

 

Response 3: Thank you very much. We have added the assumption "What are the effects of salt stress on the growth and photosynthesis of celery? Can trehalose positively alleviate the salt stress of celery? " after the introduction.

 

Point 4: In the title of figures, please mention what the vertical bars and the different letters represent (considering that every figure has to be self-explanatory).

 

Response 4: Thank you very much for your suggestion. We have marked the meanings of different letters in the captions of each chart to ensure that every chart is self-explanatory.

 

Point 5: In the title of 2.2, please change the term “different trehalose” to “different trehalose concentrations”.

 

Response 5: We have changed the term “different trehalose” to “different trehalose concentrations”.

 

Point 6: In the second row above Figure 2, it is unclear what the term “forks” is intended to mean, please replace it with the appropriate scientific term.

 

Response 6: We have changed “forks”to“branching”in the full paper.

 

Point 7: In the legend of Figure 2.C, please replace the term “overground” with “aboveground.

 

Response 7: We have replaced the term “overground” with “aboveground.

 

Point 8: In subsection 3.2, please specify what was “the optimal concentration” of trehalose identified in the experiments.

 

Response 8: We have supplemented in section 3.2 that the "optimal concentration" of trehalose is "10 mM trehalose concentration".

 

Point 9:    In the first row of 3.3, please rephrase the affirmation that “chloroplasts facilitate photosynthesis”, because these organelles perform all the processes of photosynthesis, not only facilitate them.

 

Response 9: Thank you for your valuable suggestion. We have changed them to " Chloroplasts are the main sites for photosynthesis in plants and are also one of the most salt-sensitive organelles in plant cells ".

 

Point 10: In row 7 of 3.3, please consider that transpiration rate is not related to photosynthesis, but to water economy of plants.

 

Response 10: We have removed " transpiration rate ".

 

Point 11: In subsection 3.3, if mineral nutrients that are “crucial for chlorophyll biosynthesis”, please include in the list the iron, which is particularly needed for the biosynthetic pathway of chlorophylls.

 

Response 11: We have added iron, it is particularly needed for the biosynthetic pathway of chlorophylls.

 

Point 12: In subsection 3.3, please correct the construction “the light energy absorbed by chloroplasts” to the more correct form: “the light energy absorbed by chlorophylls”.

 

Response 12: We have changed “the light energy absorbed by chloroplasts” to “the light energy absorbed by chlorophylls”.

 

Point 13: At the beginning of the second paragraph of 3.3, please replace “chlorophyll fluorescence” with “induced chlorophyll fluorescence”, because this is the correct name of the parameter registered with the described method.

 

Response 13: Thank you very much, we have replaced “chlorophyll fluorescence” with “induced chlorophyll fluorescence”.

 

Point 14: In the first row of page 13, please replace the term “oxidizing side of PSII” with the more correct term “donor side pf PSII”.

 

Response 14: We have replaced the term “oxidizing side of PSII” with the more correct term “donor side pf PSII”.

 

Point 15: In the first paragraph of 3.4, please reconsider and replace the formulation “notable increase in overall fluorescence intensity”, because, as it is shown in figure 7A, there are very small differences between the different OJIP curves.

 

Response 15: We have changed “notable” to “slight”.

 

Point 16: In the title of paragraph 4.2 please replace the term “morphological indices” with “growth” indices, because the mentioned parameters are related to plant growth and not to changes of the shape of different organs (morphology being the study of the shape of different body parts).

 

Response 16: We changed “morphological indices” to “growth indices”.

 

Point 17: In section 4.2 please indicate the temperature and the time period of drying the plant material.

 

Response 17: We have added the temperature and the time period of drying the plant material, it is “Then, the aboveground and underground parts of the plants were placed in a 105℃ oven for blanching for 30 minutes, followed by drying at 85℃ for one week. Finally, the dry weight was measured”.

 

Point 18: In section 4.3 please mention that Ci represents carbon dioxide concentration in the intercellular spaces of the leaf blade.

 

Response 18: We have added that Ci represents carbon dioxide concentration in the intercellular spaces of the leaf blade.

 

Point 19: In section 4.4 the light intensities used for chlorophyll fluorescence measurements should be expressed as µmol m-2 s-1, not as µmol/ms (…ms stands for milliseconds, which is not the case here).

 

Response 19: We have changed “µmol/ms” to “µmol m^-2 s^-1” in section 4.4.

 

Point 20: Because the data in Table 5 do not originate from the authors of the manuscript, at the end of the table title please give the reference from which the parameters are taken.

 

Response 20: We have given the references to these parameters at the end of the headings of Table 5.

 

Point 21: Concerning the statistical analysis of experimental data sets, before choosing the ANOVA and Duncan’s test, evaluation of homogeneity of variances and analysis of normality of data sets distribution should be performed. After these, ANOVA and Duncan’s test can be used only if the distribution of data sets was proved to be normal. Were these tests performed before ANOVA, and if not, please explain why not.

 

Response 21: Thank you for your valuable suggestion, first, we evaluated the uniformity of data variance and the normality of dataset distribution. The distribution of all datasets was normal. Then, analysis of variance (ANOVA) was conducted using SPSS 20.0 software (SPSS Institute Inc., USA). Duncan's multiple range test was used to compare the significant differences of the treatment methods at a probability level of 0.05. All data were collated using Excel 2020 (Microsoft Corporation). The experiment was conducted three times, and the results were expressed as mean ± standard error. All figures were constructed using Origin Pro 2022 software (Origin Lab Corporation, USA).

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. Authors have improved figure legends, but also some other parts of the text, as was suggested in last review. Minor language issues persist.
2. The bigger issue is the experimental design. The author responses to original queries/suggestions 1, 2, 3, and 5 are not adequate. Authors do not show any changes or effects of Trehalose application alone on the plant growth rate, especially in the context of information they have provided in the responses that 'trehalose is generally low in celery'. A seven day external trehalose application surely would change the growth rate, with or without salinity. Therefore, the conclusions on Trehalose-dependent alleviation of salinity-tolerance are not completely supported. Further, measurements of constitutive and modified trehalose levels on celery would be necessary.
3. Further, as the trehalose application and salinity exposure are in two different plant tissues, it is crucial to have internal markers of salinity (Na, Cl, ROS, Osmotic potential etc), as has been originally requested, quantified to substantiate the claims.

Author Response

Thank you again for your valuable comments on our manuscript titled " Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress” (ID: plants-3995547). Based on the opinions and suggestions you raised in the main text, we have carefully revised the manuscript and hope to receive your recognition. We used the "Track Changes" function in Microsoft Word to modify our manuscript. The modified parts are marked in red.

 

 

Point 1: Authors have improved figure legends, but also some other parts of the text, as was suggested in last review. Minor language issues persist.

 

Response 1: Thank you for your valuable suggestion, we inspected all the graphics and supplemented the figure legends and graphic text to ensure that all the graphics are independent and complete. All the authors have carefully checked, revised and read  the language in the text again to ensure that all the language is professional.

 

Point 2: The bigger issue is the experimental design. The author responses to original queries/suggestions 1, 2, 3, and 5 are not adequate. Authors do not show any changes or effects of Trehalose application alone on the plant growth rate, especially in the context of information they have provided in the responses that 'trehalose is generally low in celery'. A seven day external trehalose application surely would change the growth rate, with or without salinity. Therefore, the conclusions on Trehalose-dependent alleviation of salinity-tolerance are not completely supported. Further, measurements of constitutive and modified trehalose levels on celery would be necessary.

 

Response 2: Thank you for your kind comment, Based on the previous salt concentration screening tests, this study used N (0 mM Tr + 100 mM NaCl) as the control and designed different concentrations of Tr. The main purpose was to screen out the appropriate Tr concentration that could alleviate 100 mM NaCl stress.  If trehalose is sprayed under salt stress compared with that sprayed under normal conditions, In this way, there are two variables.  Additionally, in this study, CK (0 mM Tr + 0 mM NaCl) was used as the control without salt stress and without spraying Tr, precisely to further reveal the mitigating effect of trehalose when celery was exposed to an environment of 100 mM NaCl.  The main focus is to explore whether trehalose can be alleviated to the normal level or close to the normal level.  Our research group has completed similar studies on tomatoes.  Under normal conditions, spraying Tr has no significant effect on the growth of tomatoes. Its main effect is manifested under salt stress.  Related achievements: "Trehalose alleviates salt tolerance by improving photosynthetic performance and maintaining mineral ion homeostasis "in tomato plants" has been published in the journal Frontiers in Plant Science.  Meanwhile, we reviewed a large number of relevant studies [1,2,3,4,5].  Trehalose, as an osmotic regulator, mainly functions under salt stress.  Spraying trehalose alone has no significant effect on the growth of plants.  As you mentioned, the content of trehalose is very important. On this basis, we will subsequently conduct experiments with treatments of CK (untreated), N (100 mM NaCl), T3 (10 mM trehalose + 100 mM NaCl), and trehalose alone (10 mM).. In the experiment, we listed the level of trehalose as an important indicator to further explore the mechanism of trehalose salt tolerance. Thank you again sincerely for your valuable opinions and suggestions, which will play an important guiding role in our subsequent research.

 

[1 ] Algopishi, U. B. "Mitigation of the Oxidative Effects of Salt Stress on Growth and Photosynthesis by the Exogenous Treatment of

Trehalose in Trigonella Foenum-gracum." Applied Ecology & Environmental Research 23.2 (2025).

[2 ] Shahbaz, M., Abid, A., Masood, A., & Waraich, E. A. (2017). Foliar-applied trehalose modulates growth, mineral nutrition, photosynthetic ability,

and oxidative defense system of rice (Oryza sativa L.) under saline stress. Journal of Plant Nutrition, 40(4), 584-599.

[3 ] Samadi, S., Habibi, G., & Vaziri, A. (2019). Exogenous trehalose alleviates the inhibitory effects of salt stress in strawberry plants. Acta Physiologiae

Plantarum, 41(7), 112.

[4 ] Islam, S., Mohammad, F., Siddiqui, M. H., & Kalaji, H. M. (2023). Salicylic acid and trehalose attenuate salt toxicity in Brassica juncea L. by activating the stress

defense mechanism. Environmental Pollution, 326, 121467.

[5] Karamzehi, R., & Einali, A. (2024). Trehalose-induced metabolic responses in basil (Ocimum basilicum) seedlings under salt treatment. Acta Botanica Croatica, 83(2), 0-0.

 

Point 3: Further, as the trehalose application and salinity exposure are in two different plant tissues, it is crucial to have internal markers of salinity (Na, Cl, ROS, Osmotic potential etc), as has been originally requested, quantified to substantiate the claims.

 

Response 3: Thank you very much for your valuable suggestions. This study mainly explores the effects of exogenous trehalose on the growth and photosynthesis of celery. The application of trehalose and salinity exposure are in two different plant tissues, and the main purpose is to directly reflect the differences through the morphological indicators of the plants and root systems in the following experiments, We will focus on quantifying the internal markers of salinity (Na, Cl, ROS, Osmotic potential, etc.). Based on growth and photosynthesis, we will further confirm the role of trehalose in alleviating salt stress through ROS metabolism, Na and Cl contents, osmotic regulation and trehalose metabolism. Your opinions have once again pointed out the direction for our research. Thank you very much.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Given in the attached report.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer

Thank you again for your valuable opinions and suggestions on our manuscript " Regulatory effects of exogenous trehalose on the growth and photosynthetic characteristics of celery (Apium graveolens L.) under salt stress” (ID: plants-3995547). Based on the opinions and suggestions you raised in the main text, we have had a serious discussion and made careful revisions to the manuscript. We hope to receive your recognition.

 

Point 1: Dear Authors,

Thanks for the responses, unfortunately the arguments presented are not sound.

I could access four out of five publications referenced by the authors in the response. Not surprisingly, all four have presented the control i.e., Trehalose-alone, which makes the experimental design complete (see attached images). It is not very unreasonable for the same to be requested for making the current manuscript suitable for publication. Among the four publications, three show an increased growth by trehalose-alone application, while the 4th one shows decrease. A plant species specific variation is evident. It becomes especially important when internal trehalose composition from the studied plant in not known/quantified/presented!  Also, quantification of at least Na/K contents and ratio is not unreasonable for a manuscript dealing with salinity stress and its recovery potentially through osmolytes.

 

Response 1: Thank you very much for your comments and suggestions on this paper again. Based on your opinions and suggestions, we have had a careful discussion and fully recognize your viewpoint. The sole application of trehalose is of great significance to this research. On the basis of this research, we are about to conduct subsequent experiments and will strictly follow your opinions and suggestions to add the treatment of trehalose's sole application. It is particularly important to further improve our research work and clarify the content of trehalose components inside plants. In addition, in the subsequent experiments, we will focus on determining the Na/K content and ratio, which is of guiding significance for this research.  All of our authors unanimously agree with your suggestions and have listed them as our key research targets. Thank you very much.

Author Response File: Author Response.docx