Effects of Exogenous Antioxidant Melatonin on Physiological and Biochemical Characteristics of Populus cathayana × canadansis ‘Xin Lin 1’ under Salt and Alkaline Stress
, Haiyang Yu 1, Changjun Ding 2,* and Xiyang Zhao 1,3,*
Hélida Mara Magalhães
Round 1
Reviewer 1 Report
Page 2 row – 78 was this substrate purchased commercially or was it prepared? If it was prepared, what type of soil was it and what depth was it made? Do you have chemical analysis?
line 73 – Were cuttings made? When were these cuttings collected? At what time of year? Were they woody or semi-herbaceous? What was the color? The diameter? It is necessary to characterize the propagating material well.
line 83 – Where were these concentrations based? What was the starting point for choosing these concentrations?
line 85 – How was it sprayed? With which equipment, what was the volume of solution used? What was the flow?
row 87 – How was this concentration defined and why three days? What was the starting point for this treatment? Ideally, saline concentrations should be placed gradually so that the plants do not go into osmotic shock.
line 88 – Was it unclear what the total duration of the trial was and why the trial was conducted for 10 days? What would be the point of this?
line 129 – Images are small
line 111- I think it would be interesting to insert the days that the collections were carried out instead of a legend.
Author Response
Response Letter
Reviewer 1 of manuscript Forests-1804775
Forests Journal
November 25th, 2022
Dear Reviewer 1,
Thank you for your comments and the opportunity to revise our manuscript on Forests Journal. The comments offered by reviewer 1 has been immensely helpful, and we also appreciate all insightful comments on revising all aspects of the previous manuscript.
I have included the reviewer 1 comments immediately below this letter, indicating exactly how we addressed each concern or problem and describing the changes we have made. The revisions have been approved by all authors.
We hope the revised manuscript will better suit to reviewer 1 and Forests Journal but we are happy to consider further revisions. We thank you for your acceptance and interest in our research.
Sincerely,
Corresponding author
We hope the revised version of our manuscript will be better than previous manuscript.
1.Comments : Page 2 row – 78 was this substrate purchased commercially or was it prepared? If it was prepared, what type of soil was it and what depth was it made? Do you have chemical analysis?
Response: Thanks for the comments. I am so sorry that you are confused about soil matrix composition due to my unclear statement. In fact these substrate are the farm buy from the local nutrient soil factory. The farm will provide the relevant substrate formula, and the soil factory will be responsible for processing and production. After the farm obtains the soil, the professionals will move the soil into the plastic pots and then plant the seedlings and be responsible for the growth of the seedlings. If they believe that the soil is not fertile enough, they will add an appropriate amount of compound fertilizer to the pot based on experience.
Changes : Page 2, Materials and Methods
2.Comments : line 73 – Were cuttings made? When were these cuttings collected? At what time of year? Were they woody or semi-herbaceous? What was the color? The diameter? It is necessary to characterize the propagating material well.
Response: Thanks for the comments. Yes , When we select the poplar clone, we will make cuttings, and we will use hardwood cuttings. Choose annual or biennial, woody stems (0.8~1.0cm in diameter) without pests and diseases. Ensure that each cutting has double buds(color is tan) and cut them into 10cm branches in early April (there is still snow on the land in late March).
Changes : Page 2, Materials and Methods
3.Comments : line 83 – Where were these concentrations based? What was the starting point for choosing these concentrations?
Response: Thanks for the comments. In view of the present melatonin in forest experimental data is less. So we used a lot of experiments on crops to design concentration gradients. Because different plants have different levels of sensitivity to melatonin . To more efficiently confirm the optimal concentration for poplar adaptation. We chose the appropriate melatonin concentration with positive effects as the melatonin setting gradient in this experiment (Arabidopsis thaliana, Brassica napus, Beta vulgaris, Avena sativa, Gossypium, Camellia japonica, Actinidia chinensis, Abutilon theophrasti, Litchi chinensis) .
4.Comments : line 85 – How was it sprayed? With which equipment, what was the volume of solution used? What was the flow?
Response: Thanks for the comments. Melatonin was dissolved in alcohol and distilled water then set the concentration gradient to 6 gradients (0, 50, 100, 200, 400, 800 μM). We use small agricultural lithium battery high pressure swirl sprayer to let melatonin treatment for samples. And the sprayer select air atomizing nozzles, Dv0.5 < 500μM. The volume of the sprayer can reach 20L.
Changes : Page 2, Materials and Methods
5.Comments : row 87 – How was this concentration defined and why three days? What was the starting point for this treatment? Ideally, saline concentrations should be placed gradually so that the plants do not go into osmotic shock.
Response: Thanks for the comments. We want to explain that our design days are based on one day of melatonin treatment, one day of saline-alkali stress treatment, and one day of sampling. So 3 days is a cycle. In order to better simulate the local soil environment of Xinmin (pH 7.5~8.0), we need to detect the pH of the soil in each pot. The detection site in the pot is 7 cm from the soil in the pot. 5 sites were set up in each pot to be assayed. Add distilled water to the test site until the bottom is in a muddy state. Insert the pH meter probe to check the soil pH in the pot. Wait until the average pH in each pot reaches 7.7. Soil is different from plant agar medium in the lab. We cannot guarantee a uniform distribution of related molecules. After the saline-alkali solution was applied in the plastic pot, the related saline-alkali molecules was easily retained on the soil surface(the reason why the surface of the soil polluted by salt and alkali turns white), making it difficult to simulate the local conditions of Xinmin. This is why we need to repeat the cycle processing several times before the first sampling while identifying the pH in plot. Moreover, the seedlings have been growing in the pots for nearly two months. For the first stress treatment, most of the relevant saline-alkali molecules will remain on the soil surface. This will have a diminishing effect on saline-alkali shock. At the same time, before the saline-alkali stress treatment, the seedlings have been treated with melatonin, which will reduce the effect of saline-alkali stress as much as possible.
Changes : Page 2, Materials and Methods
6.Comments :line 88 – Was it unclear what the total duration of the trial was and why the trial was conducted for 10 days? What would be the point of this?
Response: Thanks for the comments. Actually, our design is like this , first melatonin treatment, second saline-alkali stress treatment, finally sample. And we perform preliminary operations such as sample grouping and listing on June 1st.Next day The first melatonin treatment is coming. Then the June 10th just the third treatment cycle. After the previous pH measurement, the real environment has been simulated in the basin at this time. At the same time, the seedlings have also adapted to the relevant environment. It can be said that the sampling at this time is representative.
Changes : Page 2, Materials and Methods
7.Comments :line 129 – Images are small
Response: Thanks for the comments. I'm sorry for the inconvenience caused by little picture
Changes : Change all the pictures in the paper
8.Comments :line 111- I think it would be interesting to insert the days that the collections were carried out instead of a legend.
Response: I am very sorry that my previous words related to time such as 2day 3day caused trouble to your reading. The reason for these words is that I want to express the process of the first melatonin treatment, the second stress treatment, and finally the sampling. The subsequent sampling period was changed to once every 4 days, that is, the sampling period was suspended for one day, and the melatonin treatment was carried out again the next day. Because there are not many functional leaves of poplar cutting seedlings, 3 functional leaves were collected from each seedling each time. It affects the growth and development of seedlings. Therefore, extending the sampling period will increase the adaptability of seedlings on the one hand, and new functional leaves will also grow during the period. Thereby reducing the experimental error.And we have add a schematic diagram to make the reader easily to understand.
illustration about experiment’s method. The black flag means the day that the samples were treated by melatonin; The green flag means the day that the samples were treated by NaCl and Na2CO3; The red flag means the day that the samples are collected. And this illustration can be used for all melatonin gradients
Changes : Page 2, Materials and Methods
Again, we appreciate all your insightful comments. We worked hard to be responsive to them. We hope our revised manuscript meets your approval. Thank you for taking the time and energy to help us improve the paper.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript reports the application of melatonin on poplar plants to reduce saline and alkaline stress under different doses for 30 days. The following suggestions are described:
Introduction: Sufficient information.
Materials and methods:
1- It is very confusing how many melatonin applications were performed, please specify more clearly. on what basis was the number and period of applications of the treatments defined?
2- Why only 20 days of monitoring the plants after applying treatments? Is it enough for a forest plant? On what basis was the period to be evaluated defined?
3- How were the plants sprayed, by nebulization? What was the droplet size? The importance of droplet size in the application of treatments has been reported in several papers.
Results: The figures are very small and with a very low resolution. It is suggested to improve the quality of these.
Discussion: Sufficient information.
Conclusion: The writing is not like a conclusion, it looks like the summary of the summary, so it is suggested that the writing of the conclusion should focus on the resolution of the hypothesis and how the hypothesis was proved.
Good luck!
Author Response
Response Letter
Reviewer 1 of manuscript Forests-1804775
Forests Journal
November 25th, 2022
Dear Reviewer 2,
Thank you for your comments and the opportunity to revise our manuscript on Forests Journal. The comments offered by reviewer 1 has been immensely helpful, and we also appreciate all insightful comments on revising all aspects of the previous manuscript.
I have included the reviewer 1 comments immediately below this letter, indicating exactly how we addressed each concern or problem and describing the changes we have made. The revisions have been approved by all authors.
We hope the revised manuscript will better suit to reviewer 1 and Forests Journal but we are happy to consider further revisions. We thank you for your acceptance and interest in our research.
Sincerely,
Corresponding author
We hope the revised version of our manuscript will be better than previous manuscript.
1.Comments : It is very confusing how many melatonin applications were performed, please specify more clearly. on what basis was the number and period of applications of the treatments defined?
Response: Thanks for the comments. I am so sorry that you are confused about this. In this experiment, the hardwood cutting technique was used. At the beginning of April, one or two-year-old branches with no pests and diseases and better lignification were selected, cut into 10cm spikes, transplanted into plastic pots, and taken care of by farm staff until early July. Relevant seedlings can be transplanted to woodland for afforestation. In this experiment, potted plants with a seedling height of more than 60 cm were selected as the research objects from June 1st, and group experiments and preliminary preparations for related experiments were carried out. The original intention of this experimental design is the first step of melatonin treatment, the second step of stress treatment, and the third step of sampling. Therefore, we take the above three steps as a process. In view of the fact that the soil is different from the plant colloid medium in the laboratory, the related molecules cannot be evenly distributed in the matrix, so in order to better simulate the local environment of Xinmin (soil pH 7.5~8), we set the pH of the soil at 7 cm in the pot. Identify from time to time (after applying saline-alkali solution in plastic pots, related saline-alkali molecules are easily retained on the soil surface (the reason why the surface of saline-alkali-contaminated soil turns white), and it is difficult to simulate the local situation in Xinmin. This is why we need to Repeat the cycle a few times before the first sampling while determining the pH in the pot). We started the first process on June 2, and June 10 happened to be the sampling period for the third process. At this point, after the previous pH measurement, the real environment has been simulated in the basin. At the same time, the seedlings also adapted to the relevant environment. It can be said that the sampling at this time is representative. At the same time, collecting 3 functional leaves from each seedling will have an impact on the growth and development of the seedling. Therefore, extending the sampling period will increase the adaptability of the seedling on the one hand, and new functional leaves will grow during the period, which can reduce the experimental effect. error. In order to ensure that the relevant seedlings can be transplanted for afforestation in July, it is convenient for follow-up research and observation. We promise to capture as much as possible without changing the processing flow. Therefore, we collected every four days after the first sampling. And we have add a illustration to make the reader easily to understand. And this illustration can be used for all melatonin gradients
illustration about experiment’s method. The black flag means the day that the samples were treated by melatonin; The green flag means the day that the samples were treated by NaCl and Na2CO3; The red flag means the day that the samples are collected.
Changes : Page 2, Materials and Methods
2.Comments : Why only 20 days of monitoring the plants after applying treatments? Is it enough for a forest plant? On what basis was the period to be evaluated defined?
Response: Thanks for the comments. And I think the response is the same with the comment1
Changes : Page 2, Materials and Methods
3.Comments : 3- How were the plants sprayed, by nebulization? What was the droplet size? The importance of droplet size in the application of treatments has been reported in several papers.
Response: Thanks for the comments. Melatonin was dissolved in alcohol and distilled water then set the concentration gradient to 6 gradients (0, 50, 100, 200, 400, 800 μM). We use small agricultural lithium battery high pressure swirl sprayer to let melatonin treatment for samples. And the sprayer select air atomizing nozzles, Dv0.5 < 500μM. The volume of the sprayer can reach 20L.
Changes : Page 2, Materials and Methods
4.Comments :Results: The figures are very small and with a very low resolution. It is suggested to improve the quality of these.
Response: Thanks for the comments. I'm sorry for the inconvenience caused by little picture
Changes : Change all the pictures in the paper
5.Comments :Conclusion: The writing is not like a conclusion, it looks like the summary of the summary, so it is suggested that the writing of the conclusion should focus on the resolution of the hypothesis and how the hypothesis was proved.
Response:
Thanks for the comments. I have changed my conclusion as follows:
Saline-alkali stress can inhibit the growth and development of forest trees, especially poplar, a fast-growing and high-yield timber species. Although melatonin has been shown to alleviate recent salinity stress in crops, detailed data are still lacking in forestry related research. Therefore, the melatonin-mediated alleviation of ophthalmic alkali stress in poplar was explored. In this study, the experimental group and the control group were set up to be stressed with a solution of 120mM/L NaCl and Na2CO3, and 6 concentration gradients were set at the same time. After analyzing eight common physiological indicators and five hormonal indicators, we found that melatonin had a significant dose effect in the process of alleviating saline-alkali stress. In the face of oxidative damage caused by saline-alkali stress, the regulation effect of low concentrations of melatonin (50 μM and 100 μM) was more significant. This dose is therapeutic, the levels of osmotic regulators and MDA in leaves will be significantly reduced, and the activities of related antioxidant enzymes (SOD, CAT) will also be significantly increased. In contrast, the induction effect of high concentrations of melatonin was not obvious. In terms of hormone regulation, melatonin not only promotes the secretion of ABA. Furthermore, IAA secretion was also induced under low-concentration treatment conditions. Therefore, this study provides new clues in using different levels of melatonin to alleviate the salinity-alkali stress of forest trees. Our findings contribute to the development of a practical approach to sustainable forestry in saline lands.
Changes : Conclusion
Again, we appreciate all your insightful comments. We worked hard to be responsive to them. We hope our revised manuscript meets your approval. Thank you for taking the time and energy to help us improve the paper.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Dear editor,
I received and re-evaluated the manuscript there are aspects of the methodology that could still be improved based on my previous suggestions. But as most of it has been resolved, I recommend publishing it.
