Exogenous Application of Melatonin and Strigolactone by Regulating Morphophysiological Responses and Gene Expression to Improve Drought Resistance in Fodder Soybean Seedlings
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors conducted a comprehensive study to analyze the effects of melatonin (MT) and strigolactone (SL) in mitigating drought stress in fodder soybean. The study is well written and useful for scientific community (agriculture sector). However, authors should address the following comments to improve the manuscript.
COMMENTS:
ABSTARCT
1. Authors are suggested to use the specific terms e.g. Authors mentioned "ecological function" and "multiple ecological function" in abstract section without indicating the type of ecological functions.
2. Author are suggested to replace the phrase “increased plant height, leaf area, and root system" with "enhanced plant growth parameters.
3. Authors should specify which metabolic processes are being studied.
4. Authors should summarize the key findings without extensive methodological details.
INTRODUCTION
5. Authors are suggested to avoid repetitive statements about the effects of drought stress and the role of MT and SL. Introduction should be concise and focused referring the research gaps and significance of the current study. Authors mentioned the research gaps but does not highlight the novelty of the research.
6. MT is a widely present endogenous hormone in plants, with many important biological functions, MT not only has extensive functions in plant germination, flowering, root growth but also plays a crucial role in plant resistance to various biotic and abiotic stresses." This sentence is lengthy and repetitive. Simplify and clarify the roles of MT.
METHODOLOGY
7. Authors should simplify the methodology section by focusing on key aspects and moving detailed protocols as a supplementary materials.
8. Authors must provide the statistical models used to represent their findings. Authors should ensure all the claims should be supported by appropriate statistical model.
DISCUSSION
9. The discussion should focus more on highlighting the similarities and differences with their findings and Provide a deeper analyses of the mechanisms by which MT and SL confer drought tolerance.
10. Authors should enlist the limitations of the study and these limitations should be addressed.
11. Authors may provide the practical implications of the study
12. Authors should ensure that all references updated and relevant.
13. Similarity index should be within acceptable limit (<20%)
Author Response
ABSTARCT
Comments 1: Authors are suggested to use the specific terms e.g. Authors mentioned "ecological function" and "multiple ecological function" in abstract section without indicating the type of ecological functions.
Responses 1: Thank you for pointing this out. We agree with this comment. The mixture of fodder soybean and silage corn is beneficial for improving biodiversity, but this statement mainly expresses that fodder soybean is excellent forage. Therefore, “with high nutrition and multiple ecological function” has been changed to “with high protein content and hay yield” (Page 1 Line 16-17).
Comments 2: Author are suggested to replace the phrase "increased plant height, leaf area, and root system" with "enhanced plant growth parameters.
Responses 2: Thank you for the careful comments. "increased plant height, leaf area, and root system" has been changed to "enhanced plant growth parameters (Page 1 Line 24-25).
Comments 3: Authors should specify which metabolic processes are being studied.
Responses 3: Thank you for pointing this out. We agree with this comment. “The specific changes in metabolic processes of fodder soybean during spraying MT and SL under drought stress remain elusive.” has been changed (Page 1 Line 20-22)
Comments 4: Authors should summarize the key findings without extensive methodological details.
Responses 4: Thank you for your advice. Some extensive methodological details have been deleted in Abstract part. (Page 1 Line 25-30, 32-33)
INTRODUCTION
Comments 5: Authors are suggested to avoid repetitive statements about the effects of drought stress and the role of MT and SL. Introduction should be concise and focused referring the research gaps and significance of the current study. Authors mentioned the research gaps but does not highlight the novelty of the research.
Responses 5: Thank you for your advice. We revised as suggested (Page 2 Line 45-47, 56-59, 82-84, 97-100).
Comments 6: MT is a widely present endogenous hormone in plants, with many important biological functions, MT not only has extensive functions in plant germination, flowering, root growth but also plays a crucial role in plant resistance to various biotic and abiotic stresses." This sentence is lengthy and repetitive. Simplify and clarify the roles of MT.
Responses 6: Thank you for your valuable comments. This sentence has been simplified. (Page 2 Line 55-56)
METHODOLOGY
Comments 7: Authors should simplify the methodology section by focusing on key aspects and moving detailed protocols as supplementary materials.
Responses 7: Thank you for your advice, the methodology section has been simplified (Page 4 Line 146-150、154-155).
Comments 8: Authors must provide the statistical models used to represent their findings. Authors should ensure all the claims should be supported by appropriate statistical model.
Responses 8: Thank you for your advice, we revised as suggested (Page 4 Line 187-193).
DISCUSSION
Comments 9: The discussion should focus more on highlighting the similarities and differences with their findings and provide a deeper analysis of the mechanisms by which MT and SL confer drought tolerance.
Responses 9: Thank you for your advice, we revised as suggested (Page 13 Line 414-416; Page 14 Line 434-439; 442-445).
Comments 10: Authors should enlist the limitations of the study and these limitations should be addressed.
Responses 10: Thank you for your advice, we revised as suggested (Page 15 Line 537-540).
Comments 11: Authors may provide the practical implications of the study.
Responses 11: Thank you for your advice, we revised as suggested (Page 16 Line 540-542).
Comments 12: Authors should ensure that all references updated and relevant.
Responses 12: We checked all references and deleted the references with low relevance.
Comments 13: Similarity index should be within acceptable limit (< 20%).
Responses 13: Thank you for your reminder. We revised the manuscript and tested the repetition rate, and finally controlled it within 20%. The duplicate checking rate is 12% (If reviewers need it, we can provide duplicate checking documents).
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
In the present study authors investigated effects of externally applied melatonin (MT) and strigolactone (ST) on the morphological, physiological and biochemical characteristics as well as the key gene expression levels of fodder soybean (Glycine max) seedlings under induced drought stress. They concluded that MT and SL treatments improved soybean plants growth and development, especially under severe drought stress. This was associated with improved photosynthetic performance and gas exchange parameters as well as with upregulation of a plethora biochemical parameters such as antioxidative enzymes and important non-enzymatic antioxidants. Alleviating effects of MT and ST treatments were further seen as decrease in oxidative stress parameters. Also, the balance of plant endogenous hormones was adjusted by MT and ST treatments in a manner to maintain normal plants growth and development. Further authors revealed that all described changes in morpho- physiological features caused by drought stress and MT/ST treatments interplay were closely associated with changes in the expression of genes that regulate the photosynthetic and antioxidant system as well as hormone signalling pathways.
In my opinion, the manuscript is extensive and comprehensive. Results are generally clearly presented and the chapter Discussion is well written. However, I have to point out several weaknesses:
1. Although the aim of the study is well described, the hypothesis is completely omitted at the end of the chapter Introduction. It must be clearly stated. This must be accompanied with re-writing the part of the chapter Conclusion. In this way authors will be able to stress the real importance of their work.
2. In the chapter M&M some important data are missing. The detailed composition of the growth substrate (a mixture of the nutrient soil) should be given (or at least the manufacturer and product ID) in order to ensure the repeatability of the experiment to other investigators. Further, the volumes of distilled water, 100 μM melatonin and 1 μM strigolactone solutions, respectively, that were used for spraying the plants should be given for the same reason.
3. In the chapter Results there are some discrepancies that concerns data of the photosynthetic parameters. Although the precise numbers are not given in the text, it clearly seen from the Fig. 6A that values of the maximum quantum yield of photosystem II (Fv/Fm) are very low in NW treatment. It is obvious that values are lower than 0,75 rel. units which is considered as a boundary value for fully functional photosystem II (see Bolhar-Nordenkampf et al. 1989. Chlorophyll fluorescence as a probe of the photosynthetic competence of leaves in the field: a review of current instrumentation. Funct. Ecol. 3, 497–514). If this is the case in it recommendable to directly compare data obtained in different drought stress / treatments. So, this should be discussed.
4. I would suggest to include full terms for the described parameters in the figures and tables captions, not just abbreviations. Since there is a lot of parameters it is difficult to link the certain abbreviation with its full term for common reader.
Author Response
Responses to reviewer #2:
Comments 1: Although the aim of the study is well described, the hypothesis is completely omitted at the end of the chapter Introduction. It must be clearly stated. This must be accompanied with re-writing the part of the chapter Conclusion. In this way authors will be able to stress the real importance of their work.
Responses 1: We revised as suggested. The hypothesis was added at the end of the chapter introduction (Page 3 Line 103-112). We also rewrite the part of the conclusion (Page 15 Line 524-542).
Comments 2: In the chapter M&M some important data are missing. The detailed composition of the growth substrate (a mixture of the nutrient soil) should be given (or at least the manufacturer and product ID) in order to ensure the repeatability of the experiment to other investigators. Further, the volumes of distilled water, 100 μM melatonin and 1 μM strigolactone solutions, respectively, that were used for spraying the plants should be given for the same reason.
Responses 2: Thank you for your advice. The detailed composition of the growth substrate (a mixture of the nutrient soil) was given (Page 3 Line 122-124). The volumes of distilled water, 100 μM melatonin and 1 μM strigolactone solution for spraying plants have been given respectively. They are 40 mL per pot each time (Page 3 Line 131).
Comments 3: In the chapter Results there are some discrepancies that concerns data of the photosynthetic parameters. Although the precise numbers are not given in the text, it clearly seen from the Fig. 6A that values of the maximum quantum yield of photosystem II (Fv/Fm) are very low in NW treatment. It is obvious that values are lower than 0,75 rel. units which is considered as a boundary value for fully functional photosystem II (see Bolhar-Nordenkampf et al. 1989. Chlorophyll fluorescence as a probe of the photosynthetic competence of leaves in the field: a review of current instrumentation. Funct. Ecol. 3, 497–514). If this is the case in it recommendable to directly compare data obtained in different drought stress / treatments. So, this should be discussed.
Responses 3: We agree with your point of view and have made changes in the results and discussion section (Page 9 Line 292-294; Page 15 Line 495-497).
Comments 4: I would suggest to include full terms for the described parameters in the figures and tables captions, not just abbreviations. Since there is a lot of parameters it is difficult to link the certain abbreviation with its full term for common reader.
Responses 4: Thank you for your careful review. The full terms for the described parameters in the figures and tables captions were added (Page 5 Line 214-216; Page 7 Line 251-252; Page 8 Line 270-272; Page 9 Line 307-308; Page 13 Line 400-402).
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsThe work titled “Exogenous application of melatonin and strigolactone by regulating morphophysiological responses and genes expression to improve drought resistance in fodder soybean seedlings” by Xie et al. showed the importance of spraying melatonin or strigolactone in protecting fodder soybean from hazardous effects of drought stress, more so with strigolactone, particularly under severe water deficit. Seedling growth, physiological parameters, and gene expression were analyzed under control and various levels of water deficit. The work is a confirmation of previously published studies as far as melatonin or strigolactone can improve different plant species' adaptation to water deficit. The manuscript is generally well-written and clear. However, the concerns below are important issues that must be addressed before accepting the manuscript for publication.
- Using the concentration of 100 µM melatonin and 1 µM strigolactone should be justified.
- The abbreviations in the abstract when first mentioned should be defined as they are not common abbreviations.
- The authors' conclusion at the end of the abstract does not hold as the experiment was not tested under natural field conditions. Therefore, the conclusion should be rewritten.
- Lines 58-59, citation [5] does not deal with strigolactone and hence it is not appropriate and sufficient to be cited here.
- One important growth condition is lacking, i.e. photosynthetic photon flux density (PPFD) in µmol m-2 s-1 should be provided for reproducibility purposes.
- The experiment had 5 replicates, but how many plants per replicate is not indicated. This should be given.
- Line 130 says “with 5 replicates per treatment”, but line 145 says “Each treatment was replicated 10 times”. Which one is correct?
- The subtitle “Measurement of Antioxidant Defense System Indexes” should be more clearly defined by dividing it into enzymatic (superoxide dismutase, peroxidase, and catalase) and nonenzymatic antioxidants (soluble sugars, soluble proteins, free proline, ascorbic acid, and glutathione) as it is currently mixed up even with incorrect unrelated items (see next concern).
- Relative electrical conductivity (measures ions’ level in the bathing medium and indirectly may probe changes in membrane integrity and is not an antioxidant index) and malondialdehyde (an indicator of membrane lipid peroxidation and is not an antioxidant index) are not antioxidant defense systems as stated by the authors. This should be corrected.
- Fig. 1 should be omitted since it adds nothing significant to the results.
- Lines 255-257, no interpretation of the data in the results section. The same is repeated in lines 275-279, 310-312, 325-328, 342-345, 359-360, 379-380, and 411-415. Omit or move to the discussion section. The interpretation of the data has never been included in the results section, it only executes and is detailed in the discussion section.
- The authors restricted nonenzymatic antioxidants to ascorbic acid and glutathione. They included soluble sugars, soluble proteins, and free proline only as osmolytes for osmotic adjustment. However, many published works reported them as efficient nonenzymatic antioxidants either under normal or stress conditions. Additionally, these solutes serve in osmotic adjustment only when accumulating at sufficiently high concentrations and when accumulating at low content they usually serve as antioxidants, which is the case in most studies. This issue needs to be considered and corrected in the sections of the results and discussion.
- Unit of soluble sugars is expressed in %, percentage of what? That of soluble proteins as mg/g, g of what? The same for proline expressed as µg, µg per what? The same is repeated with the units of catalase and superoxide dismutase. This is not detailed in the M & M section. Please correct.
- Line 351, change in EC is not only caused by oxidative stress but also can be caused by remodeling of membrane lipids and membrane architecture under stress. Please reconsider this and correct it.
- Line 434, the sentence (… significantly upward levels of O2.-, MDA, and REC), the last part (REC) should be changed into (… and membrane leakage probed by REC).
- Lines 435-435, (… and destroying the integrity of membrane structure and function in fodder soybean.) should be changed into (… and impairing the membrane integrity and hence its function in fodder soybean). Then start a new sentence: However, the application of MT and SL markedly decreased these parameters and alleviated oxidative damage under MD and SD stress.
- Lines537-538, (... and improve cell membrane stability to alleviate oxidative damage caused by drought stress) should be changed into (... and improve cell membrane stability, thus oxidative damage was detoxified by MT and SL under drought stress).
- The conclusions need to be rewritten strongly and conclusively. The conclusion is currently very weak and repeats previous results and interpretations. Based on the last sentence of the abstract, the authors could not present a novel suggestion for future research rather than doing the same line of studies that are already been carried out.
Comments on the Quality of English Language
See above
Author Response
Responses to reviewer #3:
Comments 1: Using the concentration of 100 µM melatonin and 1 µM strigolactone should be justified.
Responses 1: In order to screen the concentrations of melatonin (MT) and strigolactone (SL), we conducted a preliminary experiment. Under drought stress, three concentrations of MT (50 μM, 100 μM and 150 μM) and three concentrations of SL (0.5 μM, 1 μM and 1.5 μM) were sprayed on the seedlings of fodder soybean. The changes of phenotypes and physiological indexes of fodder soybean at different concentrations were comprehensively analyzed. It was found that 100 μM MT and 1 μM SL had the most significant effect on alleviating the drought stress at the seedling stage. Therefore, we chose 100 µM melatonin and 1 µM strigolactone. We have added explanations in the manuscript (Page 3 Line 128-129).
Comments 2: The abbreviations in the abstract when first mentioned should be defined as they are not common abbreviations.
Responses 2: We have revised it according to your opinion (Page 1 Line 25-28).
Comments 3: The authors' conclusion at the end of the abstract does not hold as the experiment was not tested under natural field conditions. Therefore, the conclusion should be rewritten.
Responses 3: Thank you for your advice. The conclusion at the end of the abstract has been rewritten (Page 1 Line 35-36).
Comments 4: Lines 58-59, citation [5] does not deal with strigolactone and hence it is not appropriate and sufficient to be cited here.
Responses 4: We have revised it according to your opinion (Page 2 Line 50-52).
Comments 5: One important growth condition is lacking, i.e. photosynthetic photon flux density (PPFD) in µmol m-2 s-1 should be provided for reproducibility purposes.
Responses 5: The photosynthetic photon flux density (PPFD) was added (Page 3 Line 118).
Comments 6: The experiment had 5 replicates, but how many plants per replicate is not indicated. This should be given.
Responses 6: Thank you for your advice. The number of plants per replicate was given (Page 3 Line 127).
Comments 7: Line 130 says “with 5 replicates per treatment”, but line 145 says “Each treatment was replicated 10 times”. Which one is correct?
Responses 7: Line 130 “with 5 replicates per treatment” refers to the experimental treatment. Line 145 “Each treatment was replicated 10 times refers to each measurement index was repeated 10 times. In order not to be confused, we have modified it (Page 4 Line 151).
Comments 8: The subtitle “Measurement of Antioxidant Defense System Indexes” should be more clearly defined by dividing it into enzymatic (superoxide dismutase, peroxidase, and catalase) and nonenzymatic antioxidants (soluble sugars, soluble proteins, free proline, ascorbic acid, and glutathione) as it is currently mixed up even with incorrect unrelated items (see next concern).
Responses 8: We have revised it according to your opinion (Page 4 Line 160).
Comments 9: Relative electrical conductivity (measures ions’ level in the bathing medium and indirectly may probe changes in membrane integrity and is not an antioxidant index) and malondialdehyde (an indicator of membrane lipid peroxidation and is not an antioxidant index) are not antioxidant defense systems as stated by the authors. This should be corrected.
Responses 9: We have revised it according to your opinion (Page 4 Line 169).
Comments 10: Fig. 1 should be omitted since it adds nothing significant to the results.
Responses 10: We removed Fig. 1 from the text and put it in the supplementary materials.
Comments 11: Lines 255-257, no interpretation of the data in the results section. The same is repeated in lines 275-279, 310-312, 325-328, 342-345, 359-360, 379-380, and 411-415. Omit or move to the discussion section. The interpretation of the data has never been included in the results section, it only executes and is detailed in the discussion section.
Responses 11: We have revised it according to your opinion. (Line 209-210, 229-231, 266-268, 301-304, 322-324, 362-364, 394-398).
Comments 12: The authors restricted nonenzymatic antioxidants to ascorbic acid and glutathione. They included soluble sugars, soluble proteins, and free proline only as osmolytes for osmotic adjustment. However, many published works reported them as efficient nonenzymatic antioxidants either under normal or stress conditions. Additionally, these solutes serve in osmotic adjustment only when accumulating at sufficiently high concentrations and when accumulating at low content they usually serve as antioxidants, which is the case in most studies. This issue needs to be considered and corrected in the sections of the results and discussion.
Responses 12: This problem has been corrected in the results and discussion section (Page 10 Results 3.5. part, Discussion Page 14 Line 453-472).
Comments 13: Unit of soluble sugars is expressed in %, percentage of what? That of soluble proteins as mg/g, g of what? The same for proline expressed as µg, µg per what? The same is repeated with the units of catalase and superoxide dismutase. This is not detailed in the M & M section. Please correct.
Responses 13: We have revised it according to your opinion in the manuscript and figures.
Comments 14: Line 351, change in EC is not only caused by oxidative stress but also can be caused by remodeling of membrane lipids and membrane architecture under stress. Please reconsider this and correct it.
Responses 14: We have reconsidered this sentence and corrected it (Page11 Line 353).
Comments 15: Line 434, the sentence (… significantly upward levels of O2.-, MDA, and REC), the last part (REC) should be changed into (… and membrane leakage probed by REC).
Responses 15: We have reconsidered this sentence and corrected it (Page 13 Line 420-421).
Comments 16: Lines 435-435, (… and destroying the integrity of membrane structure and function in fodder soybean.) should be changed into (… and impairing the membrane integrity and hence its function in fodder soybean). Then start a new sentence: However, the application of MT and SL markedly decreased these parameters and alleviated oxidative damage under MD and SD stress.
Responses 16: Thank you for your advice, we revised as suggested (Page 13 Line 422-424).
Comments 17: Lines 537-538, (... and improve cell membrane stability to alleviate oxidative damage caused by drought stress) should be changed into (... and improve cell membrane stability, thus oxidative damage was detoxified by MT and SL under drought stress).
Responses 17: The conclusion has been rewritten, and this sentence was corrected.
Comments 18: The conclusions need to be rewritten strongly and conclusively. The conclusion is currently very weak and repeats previous results and interpretations. Based on the last sentence of the abstract, the authors could not present a novel suggestion for future research rather than doing the same line of studies that are already been carried out.
Responses 18: Thank you for your advice. The conclusion has been rewritten (Page 15 Line 524-542).
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors incorporated the suggested changes BUT iThenticate showed Percent match: 29% after revision which is higher than acceptable limit. Authors must reduce the similarity index without altering the quality of the article.
Author Response
Comments 1: Authors incorporated the suggested changes BUT iThenticate showed Percent match: 29% after revision which is higher than acceptable limit. Authors must reduce the similarity index without altering the quality of the article.
Responses 1: Thank you for your reminder again. We reduced the similarity index without altering the quality of the manuscript. We tested the repetition rate. This duplicate checking report has also been uploaded.
Author Response File: 
Author Response.pdf
