Different Heat Tolerance of Two Creeping Bentgrass Cultivars Related to Altered Accumulation of Organic Metabolites
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript titled 'Different heat tolerance of two creeping bent grass cultivars related to altered accumulation of organic metabolites' is fairly well written. It seeks to compare the physiological changes of two creeping bent grass cultivars in response to persistent heat stress and also identify differential organic metabolites associated with thermotolerance in leaf tissue.
May the authors consider the following comments;
Line 86 -please provide the complete composition of the half strength nutrient solution
Line 94-what was used to irrigate the plants under heat stress. It's unclear from the description provided
Line 110- may you indicate the name of the equipment used to take the absorbance readings
Line 118-may you also include information on the equipment used for GC-MS
What are the main uses of the results obtained. The authors show both common and unique features of the grass responses to heat stress. May they suggest possible uses of the data. For example, can the data be utilised in breeding programs to improve other grass species responses to heat stress? Is it possible to utilise molecular techniques in future studies to better understand and validate the observed changes? If so, what do the authors recommend to further advance the study beyond the current scope?
Author Response
The manuscript titled 'Different heat tolerance of two creeping bent grass cultivars related to altered accumulation of organic metabolites' is fairly well written. It seeks to compare the physiological changes of two creeping bent grass cultivars in response to persistent heat stress and also identify differential organic metabolites associated with thermotolerance in leaf tissue.
Response: Thank you very much for professional review and giving us some good suggestions to improve our study. We have revised the manuscript carefully according to all suggestions.
May the authors consider the following comments:
(1) Line 86 - please provide the complete composition of the half strength nutrient solution
Response: Thanks. The complete composition of the half strength nutrient solution has been provided in revised manuscript (line 97-99).
(2) Line 94 - what was used to irrigate the plants under heat stress. It's unclear from the description provided
Response: Thanks. Tap water was used to irrigate the plants during heat stress. We have added the information in revised manuscript according your suggestion (line 107).
(3) Line 110 - may you indicate the name of the equipment used to take the absorbance readings
Response: Thanks. The name of the equipment used to take the absorbance readings has been added in revised manuscript (line 117).
(4) Line 118 - may you also include information on the equipment used for GC-MS
Response: Thanks. The information on the equipment used for GC-MS has been added in revised manuscript (line 133).
(5) What are the main uses of the results obtained. The authors show both common and unique features of the grass responses to heat stress. May they suggest possible uses of the data. For example, can the data be utilised in breeding programs to improve other grass species responses to heat stress? Is it possible to utilise molecular techniques in future studies to better understand and validate the observed changes? If so, what do the authors recommend to further advance the study beyond the current scope?
Response: Thank you very much for good suggestion. Yes, the data can be utilised in breeding programs to improve other grass species responses to heat stress. We also totally agree with your the point of view: It’s better to utilise molecular techniques in future studies to better understand and validate the observed changes. We have added the further advance beyond the current scope in the section of abstract and conclusions according to your suggestions (line 27-31; 408-412).
Reviewer 2 Report
Comments and Suggestions for Authors
The manuscript has a scientific contribution but there are some queries that need to be addressed:
- Line 27: Please add summary sentence highlighting potential future directions or practical implications from the research finding.
- Line 32: long sentence.
- Line 54: long sentence.
- In the introduction section, the authors do not clearly outline the hypotheses or research questions of this study. What makes this study innovative or unique compared to previous research
- Line 81 “Sods” should be seeds.
- Line 88: The experimental design is missing.
- Line 109: What the basis of selecting these temperatures treatment (38°C/33°C, day/night) for 15 days in both cultivars.
- Line 97: Only a few physiological parameters were measured in this study. What was the reason for not including antioxidant activity in the analysis?
- The quality of Figure 3 need improvement.
- The discussion thoroughly analyzes the results but could be more concise. A clearer comparison with recent studies is needed to highlight key similarities and differences. Additionally, many references are outdated and should be updated to reflect current literature.
- The conclusion is long, it should be more concise, briefly summarizes the main findings.
Author Response
The manuscript has a scientific contribution but there are some queries that need to be addressed:
Response: Thank you very much for professional review and giving us some good suggestions to improve our study. We have revised the manuscript carefully according to all suggestions.
- Line 27: Please add summary sentence highlighting potential future directions or practical implications from the research finding.
Response: Thank you very much for good suggestion. Summary sentence and practical implications have been added in the section of conclusions according to your suggestion (line 408-412).
- Line 32: long sentence.
Response: Thanks. This sentence has been shortened (line 37-43).
- Line 54: long sentence.
Response: Thanks. This sentence has been modified and shortened (line 59-63).
- In the introduction section, the authors do not clearly outline the hypotheses or research questions of this study. What makes this study innovative or unique compared to previous research
Response: Thank you very much for good suggestion. The research question has been mentioned in the last paragraph of introduction (line 77-79). We also added the hypotheses according to your suggestion (line 83-85).
- Line 81 “Sods” should be seeds.
Response: Thanks. The “Sods” is correct. Creeping bentgrass was planted by using sods instead of seeds in our study.
- Line 88: The experimental design is missing.
Response: Thanks. The experimental design has been clearly presented in the manuscript (line 100-106).
- Line 109: What the basis of selecting these temperatures treatment (38°C/33°C, day/night) for 15 days in both cultivars.
Response: Thank you very much for good question. We selected the temperatures treatment (38°C/33°C, day/night) for 15 days, because two creeping bentgrass genotypes showed differential physiological and metabolic changes after 15 days of heat stress (38°C/33°C, day/night).
- Line 97: Only a few physiological parameters were measured in this study. What was the reason for not including antioxidant activity in the analysis?
Response: Thank you very much for good question. We did not detect antioxidant activity because many previous studies have well revealed differential antioxidant response to heat stress in creeping bentgrass.
- The quality of Figure 3 need improvement..
Response: Thanks. The quality of Figure 3 has been improved in revised manuscript.
- The discussion thoroughly analyzes the results but could be more concise. A clearer comparison with recent studies is needed to highlight key similarities and differences. Additionally, many references are outdated and should be updated to reflect current literature.
Response: Thanks. Yes, the discussion is concise in the manuscript. We also comparison current findings with recent studies and deleted some unnecessary references in revised manuscript.
(11) The conclusion is long, it should be more concise, briefly summarizes the main findings.
Response: Thank you very much for good suggestion. The conclusion has been simplified according to your suggestion, briefly summarizing the main findings (line 392-412).
Reviewer 3 Report
Comments and Suggestions for Authors
In this manuscript, the authors investigated several physiological parameters in heat-tolerant and heat-sensitive cultivars of Agrostis stolonifera, comparing the different response patterns observed in the two cultivars. The results aid in understanding the effect of heat stress on physiological processes in creeping bentgrass. However, the findings are insufficient to construct a novel and compelling narrative. Most parameters have been identified in many other plant species, and the response patterns are pretty similar to those published previously. The unique or specific heat-tolerant mechanisms of Agrostis stolonifera were not elucidated. Although the authors compared the differences between the two cultivars, the reasons for their varied responses to heat stress, such as different genetic backgrounds, were not verified.
Comments on the Quality of English Language
Fine.
Author Response
In this manuscript, the authors investigated several physiological parameters in heat-tolerant and heat-sensitive cultivars of Agrostis stolonifera, comparing the different response patterns observed in the two cultivars. The results aid in understanding the effect of heat stress on physiological processes in creeping bentgrass. However, the findings are insufficient to construct a novel and compelling narrative. Most parameters have been identified in many other plant species, and the response patterns are pretty similar to those published previously. The unique or specific heat-tolerant mechanisms of Agrostis stolonifera were not elucidated. Although the authors compared the differences between the two cultivars, the reasons for their varied responses to heat stress, such as different genetic backgrounds, were not verified.
Response: Thank you very much for professional review. Yes, out study investigated several physiological parameters and metabolite profile in heat-tolerant and heat-sensitive cultivars of Agrostis stolonifera, comparing the different response patterns observed in the two cultivars. The results aid in understanding the effect of heat stress on physiological processes in creeping bentgrass.
- As reviewer mentioned,most physiological parameters have been identified in many other plant species and the response patterns are pretty similar to those published previously, so these parameters were used to prove that the heat tolerance of these two creeping bentgrass genotypes are different. This is why we detect these physiological parameters in this study. If not, how could you prove that heat tolerance of these two genotypes are significantly different?
- Yes, the unique or specific heat-tolerant mechanisms of Agrostis stoloniferawere elucidated in this study. In the abstract of this study, we have summarized the unique or specific heat-tolerant mechanisms: heat-tolerant PROVIDENCE accumulated more sugars (fructose, tagatose, lyxose, ribose, and 6-deoxy-D-glucose), amino acids (norleucine, allothreonine, and glycine), and other metabolites (lactic acid, ribitol, arabitol, and arbutin) than heat-sensitive PENNEAGLE. These metabolites play positive roles in energy supply, osmotic adjustment, antioxidant, and membrane stability. Heat stress significantly decreased accumulations of tricarboxylic acid cycle-related organic acids in two cultivars resulting in metabolic deficit for energy production. However, both PROVIDENCE and PENNEAGLE significantly up-regulated the accumulation of stigmasterol related to the stability of cell membrane systems under heat stress.
- Yes, we compared the differences between the two cultivars (heat-tolerant PROVIDENCE and heat-sensitive PENNEAGLE). The reasons for their varied responses to heat stress, such as different genetic backgrounds, have been explained in the introduction: Our prior study systematically evaluated heat tolerance of 41 creeping bentgrass genotypes. Among them, LOFTSL-93, PROVIDENCE, and 13M were identified as the top three heat-tolerant genotypes, whereas PENNEAGLE, W66569, and W66570 were ranked as heat-sensitive genotypes. Further findings showed that better tolerance of PROVIDENCE was related to improved Chl biosynthesis and reduced Chl degradation compared with other genotypes under high-temperature condition. However, it is still unclear whether or not superior heat tolerance of PROVIDENCE is associated with alterations in the accumulation and conversion of global organic metabolites.
Reviewer 4 Report
Comments and Suggestions for Authors
I have had the pleasure of reviewing your manuscript titled "
Different heat tolerance of two creeping bentgrass cultivars related to altered accumulation of organic metabolites." I must commend your comprehensive and forceful approach to this pertinent issue in the realm of agricultural science. The issue of how do different creeping bentgrass cultivars handle heat stress at a metabolic level is indeed a pressing one, and your research not only highlights the problem but provides a tangible and innovative solution. While the study presents interesting results, some revisions are required to enhance clarity, strengthen the analysis, and address specific points:
Point 1: My first comment pertains to the figure numbering/caption consistency. There appears to be a mismatch in figure numbering towards the end. The caption on line 259 refers to Figure 7. “Changes in contents of other metabolites….” However, the caption on line 267 also refers to Figure 7. A diagram of metabolic pathways..." This pathway diagram should logically be Figure 8. Please check and correct the numbering and any in-text citations accordingly.
Point 2: My second comment pertains to the discussion on TCA Cycle. The discussion rightly points out the decrease in TCA cycle intermediates (lines 323-328) as indicative of a metabolic deficit for energy production. While true, it might be strengthened by briefly speculating if the heat-tolerant cultivar (PROVIDENCE) shows any signs of compensating for this energy deficit more effectively than PENNEAGLE, perhaps through the alternative pathways or metabolites it accumulates more of. It's just a thought !!
Author Response
I have had the pleasure of reviewing your manuscript titled "
Different heat tolerance of two creeping bentgrass cultivars related to altered accumulation of organic metabolites." I must commend your comprehensive and forceful approach to this pertinent issue in the realm of agricultural science. The issue of how do different creeping bentgrass cultivars handle heat stress at a metabolic level is indeed a pressing one, and your research not only highlights the problem but provides a tangible and innovative solution. While the study presents interesting results, some revisions are required to enhance clarity, strengthen the analysis, and address specific points:
Response: Thank you very much for professional review and giving us some good suggestions to improve our study. We have revised the manuscript carefully according to all suggestions.
(1) Point 1: My first comment pertains to the figure numbering/caption consistency. There appears to be a mismatch in figure numbering towards the end. The caption on line 259 refers to Figure 7. “Changes in contents of other metabolites….” However, the caption on line 267 also refers to Figure 7. A diagram of metabolic pathways..." This pathway diagram should logically be Figure 8. Please check and correct the numbering and any in-text citations accordingly.
Response: Thank you very much for careful review. We have revised the typing error and further checked and corrected the numbering and any in-text citations accordingly throughout the entire manuscript.
(2) Point 2: My second comment pertains to the discussion on TCA Cycle. The discussion rightly points out the decrease in TCA cycle intermediates (lines 323-328) as indicative of a metabolic deficit for energy production. While true, it might be strengthened by briefly speculating if the heat-tolerant cultivar (PROVIDENCE) shows any signs of compensating for this energy deficit more effectively than PENNEAGLE, perhaps through the alternative pathways or metabolites it accumulates more of. It's just a thought !!
Response: Thank you very much for good suggestion. We have added relevant discussions about the possible compensation effect for energy deficit in the heat-tolerant PROVIDENCE compared with the heat-sensitive PENNEAGLE (line 346-358).
Reviewer 5 Report
Comments and Suggestions for Authors
I have read the manuscript titled “Different heat tolerance of two creeping bentgrass cultivars related to altered accumulation of organic metabolites” and evaluate it. The study presented some promising results. There are questions and comments on the manuscript to better understand the essence of the work.
- In the abstract, using more precise language to describe the physiological results section, adding data support is necessary.
- In the full text, the Latin name was changed to the correct format.
- In the Materials and Methods, add the screening criteria for differential metabolites.
- In the data analysis, please clarify whether the data was analyzed using two-way ANOVA together with the Tukey Test at P ≤05 or P < 0.05.
- Please add full metabolite reproducibility analysis (e.g. PCA plot).
- How were the 38 differential metabolites screened in the metabolome results?
- There are two Figure7 in the text.
- Some Figures use the Full name, some use abbreviations, please standardize.
- In both the Results and Analysis section and the Discussion section the Figure 7 of the metabolic pathway description are too brief.
- There are formatting problems in line 247.
- Add references related to metabolomics in the Discussion section.
- Add references related to “defective energy metabolism” caused by decreased intermediates of the TCA cycle in the Discussion.
13.There are many errors in the format of the references, please check the full text carefully and correct them.
Author Response
I have read the manuscript titled “Different heat tolerance of two creeping bentgrass cultivars related to altered accumulation of organic metabolites”and evaluate it. The study presented some promising results. There are questions and comments on the manuscript to better understand the essence of the work.
Response: Thank you very much for professional review and giving us some good suggestions to improve our study. We have revised the manuscript carefully according to all suggestions.
(1) In the abstract, using more precise language to describe the physiological results section, adding data support is necessary.
Response: Thanks. The introduction has been improved by adding data support according to your suggestion (line 16-18).
(2) In the full text, the Latin name was changed to the correct format.
Response: Thanks. We have checked the Latin name throughout the manuscript and they are correct.
(3) In the Materials and Methods, add the screening criteria for differential metabolites.
Response: Thank you very much for good suggestion. The screening criteria for differential metabolites have been added in revised manuscript (line 142-143).
(4) In the data analysis, please clarify whether the data was analyzed using two-way ANOVA together with the Tukey Test at P ≤05 or P < 0.05.
Response: Yes, the data was analyzed using two-way ANOVA together with the Tukey Test at P≤05. We have clearly clarified in the section of statistical analysis.
(5) Please add full metabolite reproducibility analysis (e.g. PCA plot).
Response: Thank you very much for good suggestion. The PCA plot has been added as a supplementary figure S1 in revised manuscript.
(6) How were the 38 differential metabolites screened in the metabolome results?
Response: Thanks. The screeing criteria for differential metabolites have been added in revised manuscript (line 142-143).
(7) There are two Figure 7 in the text.
Response: Thank you very much for careful review. This is a typing error. The second Figure 7 has been revised as Figure 8 in revised manuscript.
(8) Some Figures use the Full name, some use abbreviations, please standardize.
Response: Thanks. We have standardized all figures with abbreviations if the full name has a short name as mentioned in the manuscript.
(9) In both the Results and Analysis section and the Discussion section the Figure 8 of the metabolic pathway description are too brief.
Response: Thank you very much for good suggestion. We have added more descriptions about the Figure 8 of the metabolic pathways in Results section (line 276-278) and Discussion section (line 319; 334-335; 346-358).
(10) There are formatting problems in line 247.
Response: The formatting problems have been fixed. The text is formatted with full justification, so the spacing between two words may be slightly wider than normal spacing in this sentence.
(11) Add references related to metabolomics in the Discussion section.
Response: Thanks for this suggestion. References related to metabolomics have been added in the Discussion section according to your suggestion (line 334-336; 346-348; 353-356).
(12) Add references related to “defective energy metabolism” caused by decreased intermediates of the TCA cycle in the Discussion.
Response: Thank you very much for good suggestion. The references related to “defective energy metabolism” caused by decreased intermediates of the TCA cycle have been added in the Discussion (line 351-358).
(13) There are many errors in the format of the references, please check the full text carefully and correct them.
Response: Thank you very much for careful review. The format of all references have been corrected according to the journal Agronomy.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
I think the revised version of the manuscript is acceptable
Reviewer 3 Report
Comments and Suggestions for Authors
Sorry for not being able to accept the revision, although the revised manuscript appeared improved compared to the original version. I don’t agree to conduct a repeated investigation just by using different material. Although the authors argue for the novelty of the work, I believe that such novelty is not sufficient to make significant sense.
