Melatonin Application Induced Physiological and Molecular Changes in Carnation (Dianthus caryophyllus L.) under Heat Stress
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript presents a study on the effect of melatonin on vegetative development, enzymatic activity and molecular regulation of carnation seedlings under conditions of thermal stress. The results show a positive concentration-dependent effect of melatonin in preventing heat damage. The work is an important contribution to the management and propagation of the carnation.
The work has an adequate experimental design and standardized methodologies for biochemical and molecular evaluation.
The main concern I have about the work is the existence of multiple errors in writing, spelling, and formatting throughout the manuscript. In particular, it is suggested to review the wording of the abstract.
Additionally, it is observed that the response variables evaluated are presented in isolation and it would be advisable to expand the discussion of the relationship they have. For example, a significant decrease in the content of reactive oxygen species (ROS) is observed, while the content of MDA is reduced. These two results are linked and the relationship between them is not discussed.
Additionally, the following changes are suggested:
Page 1, lines 9-11. Review the wording of the opening sentence of the summary.
Page 1, lines 13-16. Check the wording of the sentence.
Page 1, line 34. Pull apart "[3, 4].The".
Page 1, line 37. Pull apart "worldwide[6]".
Page 1, line 38. Pull apart "world[3, 7]". I suggest checking the same error throughout the manuscript.
Page 2, Lines 51-57. Review the wording of the paragraph where the objectives of the research are indicated. The wording is redundant.
Page 3, line 87. Change the writing of "12000 g" to "12,000 x g".
Page 3, line 92. Change the writing of "10,000 g" to "10,000 x g".
Page 3, line 94. Write radical notation correctly (OH).
Page 3, lines 98-101. It is suggested to review the wording of this sentence.
Page 3, lines 104-106. It is suggested to review the wording of this sentence.
Page 3, lines 109-113. It is suggested to review the wording of this sentence.
Page 3, line 114. Change "depend" to "following".
Page 3, lines 117-118. It is not understood what they mean by "depend on decline of inhibition H2O2".
Page 3, line 123. Pull apart "2μmol".
Page 3, line 126. Change " " to "NBT".
Page 4, lines 153-154. In this sentence it is understood that they refer to melatonin. It is suggested to review the wording.
Page 4, line 157. Delete point.
Page 5, line 174. Separate and capitalize "periods.in addition".
Page 5-6, lines 175-184. Change O2 to superoxide.
Page 7, line 211. Change " 1Mm" to "1 mM".
Page 8, lines 239-251. It is suggested to check the wording and spelling of the paragraph.
Page 10, line 278. Change "dray" to "dry".
Page 10, lines 289-291. Check the wording of the sentence.
Author Response
The manuscript presents a study on the effect of melatonin on vegetative development, enzymatic activity and molecular regulation of carnation seedlings under conditions of thermal stress. The results show a positive concentration-dependent effect of melatonin in preventing heat damage. The work is an important contribution to the management and propagation of the carnation.
The work has an adequate experimental design and standardized methodologies for biochemical and molecular evaluation.
The main concern I have about the work is the existence of multiple errors in writing, spelling, and formatting throughout the manuscript. In particular, it is suggested to review the wording of the abstract.
Dear reviewer, we have carefully checked through the whole manuscript for grammatical problems. Also, we have recasted the language of the manuscript especially the grammatical mistakes according to an English native speaker. We attached the certificate that proved the manuscript was revised linguistically. Hope the revised version meets the requirements.
Additionally, it is observed that the response variables evaluated are presented in isolation and it would be advisable to expand the discussion of the relationship they have. For example, a significant decrease in the content of reactive oxygen species (ROS) is observed, while the content of MDA is reduced. These two results are linked and the relationship between them is not discussed.
Replay: We appreciate great favors from you. Thanks a lot for the helpful proposal for improving our manuscript. We have explained an entire part in the introduction section about the genes expression selection of in carnation and their relationship to the effect of melatonin and heat stress condition (line 51-61). In addition, we have included in the discussion part about the relationship between ROS and MDA under heat stress with our data (line 338-348).
Additionally, the following changes are suggested:
Page 1, lines 9-11. Review the wording of the opening sentence of the summary.
Replay: thanks, it was cheeked. (lines 9-11)
Page 1, lines 13-16. Check the wording of the sentence.
thanks, it was cheeked. (lines 13-16)
Page 1, line 34. Pull apart "[3, 4].The".
Replay: thanks, it was cheeked. (lines 34)
Page 1, line 37. Pull apart "worldwide[6]".
Replay: thanks, it was changed and edit the sentence ((lines 37) .
Page 1, line 38. Pull apart "world[3, 7]". I suggest checking the same error throughout the manuscript.
Replay: thanks, it was changed. (lines 38) .
Page 2, Lines 51-57. Review the wording of the paragraph where the objectives of the research are indicated. The wording is redundant.
Replay: thanks, it was cheeked. (lines 63-67) .
Page 3, line 87. Change the writing of "12000 g" to "12,000 x g".
Replay: thanks, it was changed. (lines 98) .
Page 3, line 92. Change the writing of "10,000 g" to "10,000 x g".
Replay: thanks, it was changed. (lines 104) .
Page 3, line 94. Write radical notation correctly (OH).
Replay: thanks, it was changed. (lines 106) .
Page 3, lines 98-101. It is suggested to review the wording of this sentence.
Replay: thanks, it was changed. (lines 38) .
Page 3, lines 104-106. It is suggested to review the wording of this sentence.
Replay: thanks, it was changed. (114-117).
Page 3, lines 109-113. It is suggested to review the wording of this sentence.
Replay: thanks, it was changed. (lines 136-140) .
Page 3, line 114. Change "depend" to "following".
Replay: thanks, it was changed. According to your suggestion (lines 141) .
Page 3, lines 117-118. It is not understood what they mean by "depend on decline of inhibition H2O2".
It was revised to be (Catalase (CAT, EC. 1.11.1.6) activity was measured following the method of Góth [31]. The total volume of the reaction mixture was 3 mL; this consisted of 0.1 mL enzyme extract and 0.1 mL H2O2 (0.4%), in addition to 2.8 mL phosphate buffer (25 mM, pH 7.0). The reduction in absorbance was measured at 240 nm.)
Page 3, line 123. Pull apart "2μmol".
Replay: thanks, it was cheeked. (lines 147) .
Page 3, line 126. Change " " to "NBT".
Replay: thanks, it was changed. (lines 152) .
Page 4, lines 153-154. In this sentence it is understood that they refer to melatonin. It is suggested to review the wording.
Replay: thanks , It was changed to be (In addition, when treated with melatonin at a concentration of 5 mM, carnation microshoots exhibited levels of chlorophyll which were significantly higher than for any other concentration.) (lines 184-187) .
Page 4, line 157. Delete point.
Replay: thanks, it was deleted. (lines 190) .
Page 5, line 174. Separate and capitalize "periods.in addition".
Replay: thanks, it was changed. (lines 199) .
Page 5-6, lines 175-184. Change O2 to superoxide.
Replay: thanks, it was changed. (lines 200-211).
Page 7, line 211. Change " 1Mm" to "1 mM".
Replay: thanks, it was changed. (lines 234) .
Page 8, lines 239-251. It is suggested to check the wording and spelling of the paragraph.
Replay: thanks, it was cheeked. To be (Treatment of carnations with melatonin in vitro increased the relative expression of the Gols1 gene, compared with untreated plants. This increase in expression of the Gols1 relative gene was greater after 1 and 3 hours, compared with other time points .Similarly, the relative transcript level of MBF1c was upregulated in heat-stressed plants treated with melatonin, compared with control samples (lines 270-275) .
Page 10, line 278. Change "dray" to "dry".
Replay: thanks, it was changed. (lines 312) .
Page 10, lines 289-291. Check the wording of the sentence.
Replay: thanks, it was cheeked. (lines 323-326) .
.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors, congratulations on your research. I appreciate your results. The work is of theoretical and practical interest in the field of research. The argument is interesting but should be improved.
Suggestions and recommendations
- To better argue the need for such research.
- More detailed Introduction and better argued research objectives.
- The authors should elaborate more on their conclusions and recommendations.
- Review of technical editing requirements, slight oversights. For example, in Bibliography: line spacing, justify all the text.
I think that a careful revise can increase the scientific value of this paper considerably.
Author Response
Dear authors, congratulations on your research. I appreciate your results. The work is of theoretical and practical interest in the field of research. The argument is interesting but should be improved.
Respected reviewer,
We appreciate great favors from you. Thanks a lot for the helpful proposal for improving our manuscript. We have reviewed the comments carefully and revised accordingly. We provide this letter to explain, point by point, the details of our revisions in the manuscript and our responses to the reviewers’ comments as follows.
Hope the revised version meets the requirements and look forward to hearing from you soon. Many thanks in advance!
Suggestions and recommendations
- To better argue the need for such research. - More detailed Introduction and better argued research objectives.
Answer: Dear reviewer, we mentioned in detail in the introduction section about needing to this research and how genes are expressed in carnation and how they relate to the effects of melatonin and heat stress. In addition, we used our data to discuss the link between antioxidant enzymes, genes, ROS and MDA during heat stress.
- The authors should elaborate more on their conclusions and recommendations.
Answer: Dear reviewer, more details about the conclusions have been provided in the recommendation part, including the entire research and what we have reached. In addition, what we have reached has been summarized in a graphical photo to demonstrate the mechanism of action of melatonin in reducing the detrimental effects of heat stress (figure 8). We also expanded our discussion section to include more recent literature. We added more than 15 referenced references between 2020 and 2023.
- Review of technical editing requirements, slight oversights. For example, in Bibliography: line spacing, justify all the text.
Answer: Dear reviewer, we carefully examined the entire manuscript for grammatical errors. In addition, we recasted the manuscript's language, particularly the grammatical errors, as reported by an English native speaker. We attached a certificate proving that the manuscript had been linguistically edited. I hope the new version fits the standards.
Reviewer 3 Report
Comments and Suggestions for Authors
After review, I suggest changes to the manuscript and offer the following comments:
Line 3: Please replace the capital letter of the word "Caryophyllus" with lower case letter.
Linе 8: Contact phone of the author is superfluous information, please delete it.
Abstract
Lines 9-26: The abstract contains all the recommended parts and pertinent overview of the work (the novelty of the content, background and objectives, treatments applied, the article's main findings and the main conclusions).
Keywords
Lines 27: I suggest that you list the keywords in alphabetical order. This is not provided for in the instructions for authors, but the text looks better.
Lines 30-341: Please distribute your text evenly between the margins (justify). In some places throughout the manuscript the spacing between words and punctuation is missing, please pay attention to this and correct it.
INTRODUCTION
Line 37: In this part of the sentence you have written the preposition "in" twice, delete one.
Lines 43-44: High temperature, salinity, and cold stress are abiotic stress. Please replace the word “biotic” with “abiotic”.
Line 50: The cited reference 17 (Mukherjee et al., 2014) is not suitable for the above text, as this work investigated the influence of salt stress on sunflower seedlings, and not heat stress as stated.
Lines 53-54: Please add the text "for the cultivation of carnation" to the existing sentence.
MATERIALS AND METHODS
Line 60: Please replace the capital letter of the word "Caryophyllus" with lower case letter.
Lines 64-65: Please replace the text “Subsequently, the glasses (250 mL jars) containing free MS media (30 g L−1 sucrose and 8 g L−1 agar (Difco Bacto TM Agar)) were used for seeds without adding any growth regulators to the media [18]” with “Subsequently, the glass jars (250 mL) containing MS media (30 g L−1 sucrose and 8 g L−1 agar (Difco Bacto TM Agar)) were used for seeds without adding any growth regulators to the media [18]”. Also, please add the details of the manufacturer of the agar (city, state).
Lines 66- 67: Please replace the text "The jars were incubated in growth place at 26 ± 1 °C supplemented with a 16 h photoperiod of 2500 lx light intensity" with the text “In vitro cultures were grown at 26 ± 1 °C under a long-day photoperiod (16/8 h light/dark) provided by lamps with a light intensity of 2500 lux.”.
Line 73: Since you are using the abbreviation MT for the first time in the text, write the word melatonin in front of it and put the abbreviation in brackets.
Lines 83-85: Please replace the text “Chlorophyll (Chl) measurement was calculated according to the way described by Frank et al. with some modification [19]. Shortly, it was taken (100 mg) and was ground with sterile pestle.” with “The content of chlorophyll (Chl) was determined according to Frank et al. with some modification [19]. Shortly, it was taken leaves of carnation (100 mg) and was ground with sterile pestle.”
Line 86: Please replace the word “moved” with “incubated”.
Lines 86-88: Please add the details of the manufacturer of the centrifuge and spectrophotometer and respect the uniformity of the text (manufacturer, city, state).
Line 89: Delete the dot at the end of the subtitle.
Line 91: How are shoots dissolved, were they not previously homogenized in liquid nitrogen?
Line 92: Please replace the text “TCA” with “trichloroacetic acid (TCA)”.
Line 93: Please replace the text “then it was merged with potassium phosphate buffer.” with “The supernatant was mixed with 10 mM potassium phosphate buffer”.
Line 98: Please add the details of the manufacturer of the water bath in the text (manufacturer, city, state).
Lines 100-101: Please briefly describe the method for detecting the concentration of the superoxide radical.
Line 102: Please replace the text “MDA” with “melondialdehyde”.
Lines 104-105: Please replace the text “0.1 M phosphate buffer (contained 2 mM EDTA + 1% PVP + pH 7.0 + at 4°C)” with “0.1 M phosphate buffer (pH 7.0, contained 2 mM EDTA + 1% PVP at 4°C).”.
Line 111: Please complete the existing text with details of the guaiacol (manufacturer, city, country).
Line 120: Please complete the existing text with details of the nitro blue tetrazolium (manufacturer, city, country).
Lines 128-130: The full names for TBA and TCA are not necessary as they are already written in the previous chapters.
Line 133: Please add to the existing text the method for RNA isolation, the qualitative and quantitative verification of the isolated RNA and the PCR cycle conditions.
DAB staining of leaves to localize H2O2 accumulation is not described in the Materials and methods section. Please complete the existing text with details of this method.
Line 136: Please complete the existing text with details of TB Green Premix Ex Taq and CFX Connect Real-Time System (manufacturer, city, country).
RESULTS
I consider it necessary to attach to the manuscript photos of experiments illustrating the effects of different melatonin concentrations and hеаt stress on the growth and morphology of the carnation.
Line 164: I recommend labeling the x-axis with "times of treatment" instead of "times after treatment" in Figure 1. Make the same changes to Figure 2, 4, 5 and 6. Please write the units in which the chlorophyll content is expressed on the y-axis in brackets. Make the same changes to Figure 4.
Line 171: Please replace the word “days” with “hours” and delete the word “Hydrogen peroxide”.
Line 177: Please delete the word “3,3'diaminobenzidine tetra-hydrochlorride”. When you complete in the section "Materials and methods" with a description of the dyes used for the detection of H2O2, also indicate which substance is designated by the abbreviation DAB.
Line 183: Please replace the words “all days” with “24 hours”.
Lines 181-193: Write the correct tags for ROS.
Lines 183-193: Please rephrase these sentences, as they all start the same way.
Line 204: Please replace the words “all tested days” with “all tested times”.
Line 209: Please replace the text “lowest values recorded” with “lowest value of SOD activity recorded”.
Line 212: Please replace the text “Figure 4A-C” with “Figure 4C”. Also, please replace the words “The application of high temperature” with “Treatment with high temperature”.
Line 214: Please replace the text “The melatonin treatments” with “Melatonin application”.
Line 219: Please delete the words “chilling”.
Lines 260 and 272: Please write letters in Figures 5 and 6 that indicate significant differences between the treatments.
Line 270: Please replace the text “medicago sativa” with “Medicago sativa”.
Line 289: Please replace the text “Dewir, Rangcai, Yanping, Ali and Paek” with “Dewir et al.,”.
Line 297: The Latin name of the eggplant must be written in the baskets.
Line 299: Please explain what is Fv/Fm.
In conclusion, I can recommend the authors to provide a small schematic of the mechanism of action of melatonin to reduce the negative effects of heat stress on seedling growth, chlorophyll content, ROS accumulation, MDA, ROS scavenging enzyme activity and gene expression. MDPI recommends that the research result of the last 5 years taken more into account. In view of the fact that recent literature is represented in your bibliography with 20%, I suggest that you increase this proportion by supplementing the discussion with more recent publications.
References
The names of the journals in which the cited papers were published are not listed consistently. Please correct them in the style of reference No1.
Comments on the Quality of English Language
Dear Editors and Authors,
I am pleased to inform you that I have completed the review of the manuscript with the full title: “Melatonin application induced physiological and molecular changes in carnation (Dianthus Caryophyllus L.) under heat stress” (manuscript ID: horticulturae-2796354) by Mohamed Elmongy and Mohaned Abdelbasеt.
Melatonin is a compound with antioxidant properties that occurs naturally in plant tissues and plays an important role in regulating various metabolic processes. Given its protective functions, melatonin has attracted increasing research attention in recent years due to the increasing detrimental effects of climate change. Over the past decade, melatonin has been widely used in various in vitro culture studies to reduce abiotic stress damage in plants. Temperature is one of the most important factors determining the growth, development and productivity of plants. Due to global climate change, which is leading to an increase in the global average temperature and greater temperature fluctuations, the survival of numerous plant species is under threat. Plants respond to high temperatures in complex ways at the molecular, biochemical, physiological and morphological levels.
Carnations are an important horticultural species grown throughout the world. In this study, the effect of melatonin at different concentrations on mitigating or neutralizing the effects of heat stress on the growth and development of carnations was investigated.
I agree with the authors that the article contains important new findings on the effect of melatonin use on alleviating high temperature stress in carnations. However, the manuscript also has its weaknesses. Firstly, the authors did not pay enough attention to the technical arrangement of the manuscript. E.g. 1. The text is not aligned between the two margins, 2. In some places in the manuscript, spaces are missing between two words or words and punctuation marks, 3. The authors have written the first word in the sentence in lower case or capitalised both words in the name of the plant species, 4. In the Figure 4, the units expressing enzyme activity are not put in brackets, 5. In Figures 5 and 6, the statistical significance is not entered, 6. In the bibliography, the names of the journals are not consistently written.
My main criticism is that the introductory section does not clarify which genes are monitored for expression and why they are important for regulating the response to heat stress. I also think that the authors omitted the description of the method for histochemical localization of hydrogen peroxide (DAB staining of leaves), the method for RNA isolation, the qualitative and quantitative verification of the isolated RNA, and the PCR cycling conditions. In addition, I consider it necessary to attach to the manuscript photos of experiments illustrating the effects of different melatonin concentrations and hеаt stress on the growth and morphology of the carnation.
Thank you for inviting me to participate in the journal's manuscript review process. As the authors will see from the comments below, this manuscript may become acceptable for publication in Horticulturae after major revision.
Kind regards,
Author Response
Respected reviewer,
We appreciate great favors from you. Thanks a lot for the helpful proposal for improving our manuscript. We have reviewed the comments carefully and revised accordingly. We provide this letter to explain, point by point, the details of our revisions in the manuscript and our responses to the reviewers’ comments as follows.
Hope the revised version meets the requirements and look forward to hearing from you soon. Many thanks in advance!
Answer: Dear reviewer, we have carefully checked through the whole manuscript for grammatical problems. Also, we have recasted the language of the manuscript especially the grammatical mistakes according to an English native speaker. We attached the certificate that proved the manuscript was revised linguistically. Hope the revised version meets the requirements.
Kind Regards,
Comment 1: Line 3: Please replace the capital letter of the word "Caryophyllus" with lower case letter.
Answer: it was changes at manuscript (page 1 line 3).
Linе 8: Contact phone of the author is superfluous information, please delete it.
Answer: it was removed from manuscript
Abstract
Lines 9-26: The abstract contains all the recommended parts and pertinent overview of the work (the novelty of the content, background and objectives, treatments applied, the article's main findings and the main conclusions).
Thank you very much for your comment
Keywords
Lines 27: I suggest that you list the keywords in alphabetical order. This is not provided for in the instructions for authors, but the text looks better.
Reply: thank you for your suggestion. We already checked the keywords and we ordered in alphabetical method (Lines 26).
Lines 30-341: Please distribute your text evenly between the margins (justify). In some places throughout the manuscript the spacing between words and punctuation is missing, please pay attention to this and correct it.
Reply: Dear reviewer, we are so sorry for this mistake because we used the journal template. We have carefully checked through the whole manuscript for this problems.
INTRODUCTION
Line 37: In this part of the sentence you have written the preposition "in" twice, delete one.
Answer: it was deleted at manuscript (line 36).
Lines 43-44: High temperature, salinity, and cold stress are abiotic stress. Please replace the word “biotic” with “abiotic”.
Answer: it was replaced at manuscript (line 43).
Line 50: The cited reference 17 (Mukherjee et al., 2014) is not suitable for the above text, as this work investigated the influence of salt stress on sunflower seedlings, and not heat stress as stated.
Answer: Thank you for this comment. We added the corrected reference which are suitable for using melatonin under heat stress. (Line 50).
Reference number 17 ( Hu, D.; Zhang, X.; Xue, P.; Nie, Y.; Liu, J.; Li, Y.; Wang, C.; Wan, X. Exogenous melatonin ameliorates heat damages by regulating growth, photosynthetic efficiency and leaf ultrastructure of carnation. Plant Physiol. Biochem 2023, 198, 107698.)
Lines 53-54: Please add the text "for the cultivation of carnation" to the existing sentence.
Reply: thank you for your suggestion. It was added (line 67-68)
MATERIALS AND METHODS
Line 60: Please replace the capital letter of the word "Caryophyllus" with lower case letter.
Reply: thanks, it was replaced. (line 71)
Lines 64-65: Please replace the text “Subsequently, the glasses (250 mL jars) containing free MS media (30 g L−1 sucrose and 8 g L−1 agar (Difco Bacto TM Agar)) were used for seeds without adding any growth regulators to the media [18]” with “Subsequently, the glass jars (250 mL) containing MS media (30 g L−1 sucrose and 8 g L−1 agar (Difco Bacto TM Agar)) were used for seeds without adding any growth regulators to the media [18]”. Also, please add the details of the manufacturer of the agar (city, state).
Reply: thanks, we replaced the text according to your suggestion. Also, we added the manufacturer of the agar (city, state) (line75-79).
Lines 66- 67: Please replace the text "The jars were incubated in growth place at 26 ± 1 °C supplemented with a 16 h photoperiod of 2500 lx light intensity" with the text “In vitro cultures were grown at 26 ± 1 °C under a long-day photoperiod (16/8 h light/dark) provided by lamps with a light intensity of 2500 lux.”.
Reply: thanks, we replaced it. (line75-79).
Line 73: Since you are using the abbreviation MT for the first time in the text, write the word melatonin in front of it and put the abbreviation in brackets.
Reply: it was corrected. (line 83).
Lines 83-85: Please replace the text “Chlorophyll (Chl) measurement was calculated according to the way described by Frank et al. with some modification [19]. Shortly, it was taken (100 mg) and was ground with sterile pestle.” with “The content of chlorophyll (Chl) was determined according to Frank et al. with some modification [19]. Shortly, it was taken leaves of carnation (100 mg) and was ground with sterile pestle.”
Reply: it was replaced. With small modification hope to find your request (line94-97).
Line 86: Please replace the word “moved” with “incubated”.
Reply: it was corrected. (line 97).
Lines 86-88: Please add the details of the manufacturer of the centrifuge and spectrophotometer and respect the uniformity of the text (manufacturer, city, state).
Reply: Dear reviewer, all details have been added to the devices and chemicals that are used in the manuscript to be (the supernatant was assayed by spectrophotometer (Shimadzu, Kyoto, Japan) at 665 nm and 649 nm wavelengths.) (line 98-99).
Line 89: Delete the dot at the end of the subtitle.
Reply: it was deleted. (line 100)
Line 91: How are shoots dissolved, were they not previously homogenized in liquid nitrogen?
Reply: Shoots were grounded in liquid nitrogen, then dissolved in 5.0 mL of 0.1% trichloroacetic acid (TCA).and we add this data to manuscript (line 102)
Reply: The sentence was changed to be “The shoots were grounded in liquid nitrogen then it were dissolved in 5.0 mL of 0.1% TCA.
Line 92: Please replace the text “TCA” with “trichloroacetic acid (TCA)”.
Reply: it was replaced. (line 103)
Line 93: Please replace the text “then it was merged with potassium phosphate buffer.” with “The supernatant was mixed with 10 mM potassium phosphate buffer”.
Reply: thanks, we replaced the text according to your suggestion. (line 104)
Line 98: Please add the details of the manufacturer of the water bath in the text (manufacturer, city, state).
Reply: we added the manufacturer of the water bath (city, state) the text according to your suggestion to be ((Fisher Scientific Isotemp Digital-Control Water Bath- Malaysia) (line 110-111)
Lines 100-101: Please briefly describe the method for detecting the concentration of the superoxide radical.
Reply: we have add describe the method for detecting the concentration of the superoxide radical.
(line 113-117)
Line 102: Please replace the text “MDA” with “melondialdehyde”.
Reply: it was replaced (line 129).
Lines 104-105: Please replace the text “0.1 M phosphate buffer (contained 2 mM EDTA + 1% PVP + pH 7.0 + at 4°C)” with “0.1 M phosphate buffer (pH 7.0, contained 2 mM EDTA + 1% PVP at 4°C).”
Reply: it was replaced. (line 132)
Line 111: Please complete the existing text with details of the guaiacol (manufacturer, city, country).
Reply: we added the manufacturer of guaiacol (city, state) the text according to your suggestion to be (Peroxidase (POD, EC 1.11.1.7) was detected using guaiacol (Sigma-Aldrich- USA) in a mixture of 3 mL) (line 136-137)
Line 120: Please complete the existing text with details of the nitro blue tetrazolium (manufacturer, city, country).
Reply: we added the manufacturer of nitro blue tetrazolium (city, state) the text according to your suggestion to be (Superoxide dismutase (SOD, EC 1.15.1.1) enzyme activity was checked by measuring its inhibition of the amount of nitro blue tetrazolium (NBT) photochemical reduction (Sigma-Aldrich- USA) (line 145-147)
Lines 128-130: The full names for TBA and TCA are not necessary as they are already written in the previous chapters.
Reply: it was removed. (line 152-153)
Line 133: Please add to the existing text the method for RNA isolation, the qualitative and quantitative verification of the isolated RNA and the PCR cycle conditions.
Reply: the method of RNA isolation was by Using Trizol reagent and it was added to manuscript (line 161-164). In addition it was added the PCR cycle conditions (line 167-172).
DAB staining of leaves to localize H2O2 accumulation is not described in the Materials and methods section. Please complete the existing text with details of this method.
Reply: dear reviewer, we explained this method with the caption of figure 3 which we showed that Carnation leaves treated DAB solution to measure the hydrogen peroxide, then it were transferred in darkness one day. And the total method was added as a section in material and methods. Under title (2.5 Detection and quantification of ROS) (line 118-128).
Line 136: Please complete the existing text with details of TB Green Premix Ex Taq and CFX Connect Real-Time System (manufacturer, city, country).
Reply: we added the existing text with details of TB Green Premix Ex Taq and CFX (city, state) the text according to your suggestion. (line 167).
RESULTS
I consider it necessary to attach to the manuscript photos of experiments illustrating the effects of different melatonin concentrations and hеаt stress on the growth and morphology of the carnation.
Answer: Dear reviewer thank you for this comment. The photos of effect of melatonin on morphological traits was added to the manuscript at the part of results (Figure 2) (line 219).
Line 164: I recommend labeling the x-axis with "times of treatment" instead of "times after treatment" in Figure 1. Make the same changes to Figure 2, 4, 5 and 6. Please write the units in which the chlorophyll content is expressed on the y-axis in brackets. Make the same changes to Figure 4.
Reply: we changed the labeling of the x-axis to "times after treatment" in all figures. And we write the units of all tested data expressed on the y-axis in brackets. (Figure1, 3, 4, 5 and 6).
Line 171: Please replace the word “days” with “hours” and delete the word “Hydrogen peroxide”.
Reply: it was corrected. (Figure1, 3, 4, 5 and 6).
Line 177: Please delete the word “3,3'diaminobenzidine tetra-hydrochlorride”. When you complete in the section "Materials and methods" with a description of the dyes used for the detection of H2O2, also indicate which substance is designated by the abbreviation DAB.
Reply: we have add the method and we deleted the full word, and leave the abbreviations. Under title (2.5 Detection and quantification of ROS) (line 118-128).
Line 183: Please replace the words “all days” with “24 hours”.
Reply: it was replaced. (line 207).
Lines 181-193: Write the correct tags for ROS.
Reply: it was write. (line 197-211).
Lines 183-193: Please rephrase these sentences, as they all start the same way.
Thanks, this paragraph was re-write again. (line 197-211).
Line 204: Please replace the words “all tested days” with “all tested times”.
Reply: it was replaced. (line 229).
Line 209: Please replace the text “lowest values recorded” with “lowest value of SOD activity recorded”.
Reply: it was replaced. (line 231).
Line 212: Please replace the text “Figure 4A-C” with “Figure 4C”. Also, please replace the words “The application of high temperature” with “Treatment with high temperature”.
Reply: it was replaced. (line 235).
Line 214: Please replace the text “The melatonin treatments” with “Melatonin application”.
Reply: it was replaced.
Line 219: Please delete the words “chilling”.
Reply: it was DELETED. (line 242).
Lines 260 and 272: Please write letters in Figures 5 and 6 that indicate significant differences between the treatments.
Reply: it was added to Figures 5 and 6
Line 270: Please replace the text “medicago sativa” with “Medicago sativa”.
Reply: it was corrected. . (line 308).
Line 289: Please replace the text “Dewir, Rangcai, Yanping, Ali and Paek” with “Dewir et al.,”.
Reply: it was corrected. . (line 324).
Line 297: The Latin name of the eggplant must be written in the baskets.
Reply: it was corrected. . (line 331).
Line 299: Please explain what is Fv/Fm.
Dear reviewer we calculated the Fv/Fm according to the (Minimal fluorescence (F0) and maximal fluorescence (Fm). Maximal variable fluores-cence (Fv = Fm - F0) and the photochemical efficiency of PSII (Fv/Fm)) and we added this part to discussion section. . (line 134-238).
In conclusion, I can recommend the authors to provide a small schematic of the mechanism of action of melatonin to reduce the negative effects of heat stress on seedling growth, chlorophyll content, ROS accumulation, MDA, ROS scavenging enzyme activity and gene expression. MDPI recommends that the research result of the last 5 years taken more into account. In view of the fact that recent literature is represented in your bibliography with 20%, I suggest that you increase this proportion by supplementing the discussion with more recent publications.
Reply: thank for this recommendation we made a graphical photo indicated the mechanism of action of melatonin to reduce the negative effects of heat stress (figure 8). Also, we increased the recent literature in our discussion part. We have added more than 15 cited references during the years from 2020-2023.
We have explained an entire part in the introduction section about the genes expression selection of in carnation and their relationship to the effect of melatonin and heat stress condition. In addition, we have included in the discussion part about the relationship between ROS and MDA under heat stress with our data.
References
The names of the journals in which the cited papers were published are not listed consistently. Please correct them in the style of reference No1.
Reply: in fact we used the endnote program to create the reference and we checks all bibliography throughout our manuscript, Hope the revised version meets the requirements.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors made the suggested changes
Author Response
Dear reviewer, thank you very much for your review of the manuscript, and thank you very much for your comments, which contributed to raising the manuscript quality.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Editors and Authors,
I would like to inform you that I have thoroughly read the new version of the manuscript with the full title: “Melatonin application induced physiological and molecular changes in carnation (Dianthus caryophyllus L.) under heat stress” (manuscript ID: horticulturae-2796354) by Mohamed Elmongy and Mohaned Abdelbasеt and the authors' letter. I would like to thank the authors for taking into account all my suggestions and for taking great pains to correct the text of the manuscript. I believe that the newly added photographs of the morphological characteristics of carnations under various treatments and the citation of recent literature from the studied topic have contributed significantly to the quality of the manuscript. In addition, by presenting the mechanism of the role of melatonin in increasing the heat tolerance of carnations, the realization of the aim of the experiment has been clarified.
I would like to ask you to make some minor corrections to the text, after which the manuscript will be become acceptable for publication in the journal Horticulturae.
Kind regards,
I propose the following minor changes:
- Line 111: Please complete the text with the name of the city in Malaysia where the water bath was made.
Line 123: Please complete the text with the full name of DAB (3,3'diaminobenzidine tetra-hydrochlorride) and the name of the author whose method was used for the coloration.
- Line 131: Please delete the word “rpm”, it is superfluous.
- Line 136: Please complete the text with the name of the city where the guaiacol was produced.
- Line 147: Please complete the text with the name of the city where the NBT was produced.
- Line 256: Please change the text “Mm” to “mM”.
Author Response
Dear reviewer, we thank you deeply for your comments, which greatly improved our manuscript. We thank you for the effort expended in reviewing, which yielded great results in improving the quality of our manuscript. We hope that the changes will be satisfactory to you.
the following minor changes:
- Line 111: Please complete the text with the name of the city in Malaysia where the water bath was made.
Replay: it was completed to be (Fisher Scientific Isotemp Digital-Control Water Bath- Malaysia- kuala lambor) line 111
Line 123: Please complete the text with the full name of DAB (3,3'diaminobenzidine tetra-hydrochlorride) and the name of the author whose method was used for the coloration.
Thanks it was added and the method of Fukao et al. [reference number 29] was used line 120
- Line 131: Please delete the word “rpm”, it is superfluous.
Replay; thanks it was deleted line 133
- Line 136: Please complete the text with the name of the city where the guaiacol was produced.
It was added to the manuscript to be (Sigma-Aldrich- USA- Burlington, Massachusetts) Line 136.
- Line 147: Please complete the text with the name of the city where the NBT was produced.
It was added to the manuscript to be (Sigma-Aldrich- USA- Burlington, Massachusetts)
- Line 256: Please change the text “Mm” to “mM”.
Replay; thanks it was changed line 256
