Growth, Physiological, and Transcriptome Analyses Reveal Mongolian Oak Seedling Responses to Shading

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.      L. No. 7; authors can use common name of the Mongolian oak instead of botanical name. Kindly correct this entire manuscript.

2.      L. No. 9; kindly include the full abbreviation of sck.

3.      No need to divide S1, S2, S3 and sck. It was very confused when. So, kindly follow, control, moderate, natural and severe

4.      What is the use of Figure 1 in the present manuscript?  How did authors beloved that the yellowish formed in he leaves of oak by severe light? Nutrients deficiency also induced yellowish colour in the leaves of any plants. Is right? And if plants leaves are old automatically scorching formed in the leaves. So, kindly clarify this.

5.      What cultivars authors used in the present manuscript? What is the purpose particularly selected the particular cultivar?

6.      What did you mean Sck?

7.      In the section 2.1; authors mentioned that from the July onwards the experimental cite is hottest area. Then, how did the authors conducted shading experiments during May to late September? What is the temperature inside the shading nets? Authors should mention this details inside the manuscript.

8.      What is the basis authors divided 40% (S1), 20% (S2), and 3% (S3) for this experiment?

9.      Did authors used any fertilizer during the experiment? If yes, authors should include all the necessary details appropriately.

10.  How many days old plant used for physiological, biochemical and transcriptome analysis?

11.  How did this present investigation help to future experiment? Authors should include these details in the conclusion section.

12.  How many days old plant used for gene expression analysis? What tissue used for gene expression analysis?

13.  Why authors did not analyse the eight DEGs related to photosynthesis and antioxidant enzymes genes by qRT-PCR analysis under S1, S2, S3 and S4 treatments? Authors have already tissues. Is right?

14.  What software used for creating phylogenetic tree?

15.  The supplementary figure 2 looks like a sorghum plant. Not oak tree. Did authors conduct the experiment for oak or sorghum? Authors should submit all the original images collected from the field as a supplemental figure. I need to verify.

16.  Authors should include the temperature and product size of each primer in the supplemental table 1.

17.  How did authors predicted DEGs directly from the transcriptome data of oak?

18.  Why authors did not analyse the contents of any nutrients in the present manuscript. For proper photosynthesis, phosphorus is more important. Hope authors know very well. When photosynthesis prohibited automatically the plants reduces growth and other biochemical parameters. This is commonly reported in many plants. So, authors should what is the novel of the present manuscript apart from the already available reports.

19.  Authors briefly discussed about the what are the transcriptome analysis already reported in the oak or similar plants in the introduction section. And how did your manuscript correlate with the already available reports. These details are more important to project the novelty of the present manuscript.

Comments on the Quality of English Language

Author Response

Dear reviewers or editors,

We would like to thank you for your careful reading, helpful comments, and constructive suggestions, which has significantly improved the presentation of our manuscript. We quite agree with your comments and revise our manuscript according to these suggestions as shown below. The manuscript has also been errors we found have been corrected. We hope our revised manuscript can be accepted for publication. If there are other problems with the manuscript, we are happy to revise it again.

1. No. 7; authors can use common name of the Mongolian oak instead of botanical name. Kindly correct this entire manuscript.

Thank you very much for your suggestion. We have made revisions in the manuscript.

2. L. No. 9; kindly include the full abbreviation of sck.

Thank you very much for your suggestion. We refer to the control treatment in the shading experiment, which is the full light treatment without shading, as Sck. We hope you can understand.

3. No need to divide S1, S2, S3 and sck. It was very confused when. So, kindly follow, control, moderate, natural and severe.

Thank you very much for your suggestion. We have provided explanations in the abstract and materials and methods, including full illumination (Sck), moderate shading treatment (40% illumination (S1)), moderate shading treatment (20% illumination (S2)), and severe shading treatment (3% illumination (S3)).

4. What is the use of Figure 1 in the present manuscript?  How did authors beloved that the yellowish formed in he leaves of oak by severe light? Nutrients deficiency also induced yellowish colour in the leaves of any plants. Is right? And if plants leaves are old automatically scorching formed in the leaves. So, kindly clarify this.

Thank you very much for your suggestion. Figure 1 shows the phenomenon of scorched edge yellow leaves in Mongolian oak seedlings under normal cultivation and management conditions in summer under natural light conditions. It is not due to poor nutrition and leaf aging that the leaves turn yellow. It is precisely because Mongolian oak seedlings exhibit scorched edge yellow leaves in summer that we conducted this study to explore the effects of different shading levels on the growth and development of Mongolian oak seedlings. Figure 1 makes our research more meaningful.

5. 5. What cultivars authors used in the present manuscript? What is the purpose particularly selected the particular cultivar?

Thank you very much for your suggestion. The experimental materials for this manuscript are 3-year-old Mongolian oak seedlings propagated through sowing, and their seeds were collected from the Xiaoqinling Mountains in Sanmenxia City, Henan Province. As a variety of oak, Mongolian oak was chosen mainly due to the observation of scorched edges and yellow leaves in Mongolian oak seedlings under natural light in summer during production practice. Therefore, we began to explore the response mechanism of Mongolian oak seedlings to shading.

6. What did you mean Sck?

Thank you very much for your suggestion. Sck is the control treatment in the shading experiment, which is the full light treatment without shading.

7. In the section 2.1; authors mentioned that from the July onwards the experimental cite is hottest area. Then, how did the authors conducted shading experiments during May to late September? What is the temperature inside the shading nets? Authors should mention this details inside the manuscript.

Thank you very much for your suggestion. We started the shading experiment in May and ended it in September. Throughout the experiment, the shading net remained stationary, keeping the Mongolian oak seedlings in a shaded state. We selected 5 sunny days in June to monitor the temperature inside different shading nets, which is shown in Supplementary Table 2. We did not monitor the temperature inside the shading net in other months.

8. What is the basis authors divided 40% (S1), 20% (S2), and 3% (S3) for this experiment?

Thank you very much for your suggestion. The basis for dividing S1, S2, and S3 in this experiment is that under natural regeneration, the shading degree of most Mongolian oak stands is between 20% and 40%, and older stands may have higher shading degree.

9. Did authors used any fertilizer during the experiment? If yes, authors should include all the necessary details appropriately.

Thank you very much for your suggestion. We used fertilizer during the experiment, and applied 5g of nitro compound fertilizer (16N-16P-16K) per seedling per month during the seedling growth season (April to September). We have supplemented it in section 2.2 of the article.

10. 10. How many days old plant used for physiological, biochemical and transcriptome analysis?

Thank you very much for your suggestion. We collected leaves for physiological and biochemical indicators and transcriptome analysis from three year old Mongolian oak seedlings after 2 months of varying degrees of shading treatment. We have added this information in section 2.2 of the article.

11. How did this present investigation help to future experiment? Authors should include these details in the conclusion section.

Thank you very much for your suggestion. Thank you very much for your suggestion. We have made revisions in the manuscript.

12. How many days old plant used for gene expression analysis? What tissue used for gene expression analysis?

Thank you very much for your suggestion. We conducted transcriptome analysis on the leaf tissue of three year old Mongolian oak seedlings treated with varying degrees of shading for 2 months.

13. Why authors did not analyse the eight DEGs related to photosynthesis and antioxidant enzymes genes by qRT-PCR analysis under S1, S2, S3 and S4 treatments? Authors have already tissues. Is right?

Thank you very much for your suggestion. Compared with all natural light treatment, S1 and S2 treatments significantly improved the growth status of Mongolian oak seedlings. Therefore, significant differences in S1, S2, and Sck treated Mongolian oak leaves were selected for transcriptome analysis. Subsequently, only to verify the reliability of transcriptome data, Sck, S1, and S2 treatments were used for real-time fluorescence quantitative PCR validation.

14. What software used for creating phylogenetic tree?

Thank you very much for your suggestion. We used TBtools software to complete the clustering heatmap, which has been added in section 2.6 of the article.

15. The supplementary figure 2 looks like a sorghum plant. Not oak tree. Did authors conduct the experiment for oak or sorghum? Authors should submit all the original images collected from the field as a supplemental figure. I need to verify.

Thank you very much for your suggestion. Supplementary Figure 2 is a schematic diagram of the intercropping of Mongolian oak and corn. The higher one in the picture is corn, and the lower one is Mongolian oak seedlings. This is based on our experimental results. In production practice, by designing this intercropping mode of Mongolian oak and corn, the growth status of Mongolian oak seedlings has been improved, and the growth and development of Mongolian oak seedlings have been promoted.

16. Authors should include the temperature and product size of each primer in the supplemental table 1.

Thank you very much for your suggestion. We have added the annealing temperature and product size of primers in Supplementary Table 1.

17. How did authors predicted DEGs directly from the transcriptome data of oak?

Thank you very much for your suggestion. We used DESeq2 software to analyze the differences between different comparison groups, and identified significant DEGs between different comparison groups using | log2FC |>1 and FDR<0.05 as screening criteria. This has been supplemented in section 2.4 of the article.

18. Why authors did not analyse the contents of any nutrients in the present manuscript. For proper photosynthesis, phosphorus is more important. Hope authors know very well. When photosynthesis prohibited automatically the plants reduces growth and other biochemical parameters. This is commonly reported in many plants. So, authors should what is the

Thank you very much for your suggestion. We also completely agree with your statement. At that time, due to limited experimental materials, we did not analyze the impact of shading on the nutritional content of Mongolian oak. We hope you can understand. Most of the existing reports are based on the effects of shading on plant growth and development at the growth and physiological levels. However, this study is based on growth, physiological indicators, and transcriptome sequencing to reveal the response mechanisms of Mongolian oak seedlings to different shading levels at the growth, physiological, and molecular levels, providing scientific theoretical basis and technical support for the cultivation and large-scale production of Mongolian oak seedlings.

19. Authors briefly discussed about the what are the transcriptome analysis already reported in the oak or similar plants in the introduction section. And how did your manuscript correlate with the already available reports. These details are more important to project the novelty of the present manuscript.

Thank you very much for your suggestion. The literature cited in the introduction is based on the growth and physiological aspects to explain the impact of shading on plant growth and development, indicating that weak or strong light is not conducive to plant growth. However, this study investigates the adaptive characteristics of Mongolian oak under different light intensities at the growth, physiological, and molecular levels, which can provide scientific theoretical basis and practical reference for efficient seedling cultivation of Mongolian oak in plain areas.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the physiological response of Mongolian oak (Quercus mongolica) using differing light intensity, coupled with transcriptome analysis. It concluded that under moderate shading conditions, plants obtained maximal growth and produced the highest number of DEG’s.

The introduction provides sufficient details for the study background and methods are described fully.

It is recommended to use Tukey HSD, or other post-hoc test and not Duncans as this test does not make acceptable error rate adjustments.

It would be good to further detail why the particular province of Q. mongolica was chosen in the paper.

Results

Line 86- “a gradually decreasing trend as the degree of shading increased” this would indicate a linear regression and not a treatment x year comparison. Consider removing use of ‘trend” and replace with “higher” or “lower” or “greater response in year x compared to x”.

Consider making this change throughout the results section. There is no need to say it “increased and then decreased”, as it is unclear which treatments are being compared in each of these statements. It should be explained which treatments is higher or lower compared to the other treatments.

Line 228- “The effects of different degrees of shading on leaf photosynthetic parameters are shown in Table 1”. This is unnecessary as the sentence is basically repeated in the Table heading.

Line 240-242 “again this sentence is repeating the table 2 heading.

Line 308-Figure 8. Please reorder so that each graph presented in part b is the same as in part a i.e. profile 0 graph in b should be shown first.

Line 324-Figure 9. Please add the meaning of GO and KEGG in the figure caption.

Line 362-Figure 10. It is too difficult to read the legend located in the centre of each subfigure.

Discussion

Line 429-438. There is no reference to the results here, and as such, it is reading as information that should be removed and placed in the literature review. Either move this into the introduction or explain how it relates to the results, and not only talking about results at lie 439.

Line 457-460. Again, reading as though it is part of the introduction.

Line 488-497. This is just restating introduction and results.

The next paragraph Lines 499-542, although a little long, does well to relate the studies results to theory being tested and results of other literature and the previous two paragraphs would benefit from this structure.

Comments on the Quality of English Language

some editing required, particularly in the discussion

Author Response

Dear reviewers or editors,

We would like to thank you for your careful reading, helpful comments, and constructive suggestions, which has significantly improved the presentation of our manuscript. We quite agree with your comments and revise our manuscript according to these suggestions as shown below. The manuscript has also been errors we found have been corrected. We hope our revised manuscript can be accepted for publication. If there are other problems with the manuscript, we are happy to revise it again.

1. This study investigated the physiological response of Mongolian oak (Quercus mongolica) using differing light intensity, coupled with transcriptome analysis. It concluded that under moderate shading conditions, plants obtained maximal growth and produced the highest number of DEG’s.

Thank you very much for your suggestion.

2. The introduction provides sufficient details for the study background and methods are described fully.

Thank you very much for your suggestion.

3. It is recommended to use Tukey HSD, or other post-hoc test and not Duncans as this test does not make acceptable error rate adjustments.

Thank you very much for your suggestion. We conducted post hoc multiple comparisons using Tukey's method again.

It would be good to further detail why the particular province of Q. mongolica was chosen in the paper.

Thank you very much for your suggestion. We did not choose Mongolian oak from a specific province. When we conducted the experiment, the forest farm only had Mongolian oak seedlings from Sanmenxia City, Henan Province, China. We did not choose, and we hope you can understand.

Results

4. Line 186- “a gradually decreasing trend as the degree of shading increased” this would indicate a linear regression and not a treatment x year comparison. Consider removing use of ‘trend” and replace with “higher” or “lower” or “greater response in year x compared to x”.

Thank you very much for your suggestion. We have made revisions in the manuscript.

5. Consider making this change throughout the results section. There is no need to say it “increased and then decreased”, as it is unclear which treatments are being compared in each of these statements. It should be explained which treatments is higher or lower compared to the other treatments.

Thank you very much for your suggestion. We have made revisions in the manuscript.

6. Line 228- “The effects of different degrees of shading on leafphotosynthetic parameters are shown in Table 1”. This is unnecessary as the sentence is basically repeated in the Table heading.

Thank you very much for your suggestion. We have removed this sentence from the manuscript.

7. 240-242 “again this sentence is repeating the table 2 heading.

Thank you very much for your suggestion. We have removed this sentence from the manuscript.

8. Line 308-Figure 8. Please reorder so that each graph presented in part b is the same as in part a i.e. profile 0 graph in b should be shown first.

Thank you very much for your suggestion. We have made modifications to Figure 8.

9. Line 324-Figure 9. Please add the meaning of GO and KEGG in the figure caption.

Thank you very much for your suggestion. We have added the meanings of GO and KEGG in Figure 9.

10. Line 362-Figure 10. It is too difficult to read the legend located in the centre of each subfigure.

Thank you very much for your suggestion. We have made modifications to Figure 10 again.

Discussion

10. Line 429-438. There is no reference to the results here, and as such, it is reading as information that should be removed and placed in the literature review. Either move this into the introduction or explain how it relates to the results, and not only talking about results at line

Thank you very much for your suggestion. We have made revisions in the manuscript.

11. Line 457-460. Again, reading as though it is part of the introduction.

Thank you very much for your suggestion. We have made revisions in the manuscript.

12. Line 488-497. This is just restating introduction and results.

Thank you very much for your suggestion. We have made revisions in the manuscript.

13. The next paragraph Lines 499-542, although a little long, does well to relate the studies results to theory being tested and results of other literature and the previous two paragraphs would benefit from this structure.

    Thank you very much for your suggestion. We have made revisions in the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

MS is in good frame with sufficient methodology. Though, some of the lacuna's observed in Introductory part. Authors need to go through the attached file and attend it.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

No comments

Author Response

Dear reviewers or editors,

We would like to thank you for your careful reading, helpful comments, and constructive suggestions, which has significantly improved the presentation of our manuscript. We quite agree with your comments and revise our manuscript according to these suggestions as shown below. The manuscript has also been errors we found have been corrected. We hope our revised manuscript can be accepted for publication. If there are other problems with the manuscript, we are happy to revise it again.

MS is in good frame with sufficient methodology. Though, some of the lacuna's observed in Introductory part. Authors need to go through the attached file and attend it.

Thank you very much for your suggestion. We have made revisions in the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all the comments properly and the revised manuscript is suitable for publications. 

Comments on the Quality of English Language

Minor English correction is required before publish the manuscript. 

Author Response

Dear reviewers or editors,

We would like to thank you for your careful reading, helpful comments, and constructive suggestions, which has significantly improved the presentation of our manuscript. We quite agree with your comments and revise our manuscript according to these suggestions as shown below. The manuscript has also been errors we found have been corrected. We hope our revised manuscript can be accepted for publication. If there are other problems with the manuscript, we are happy to revise it again.

Regarding the issue of English language in articles，We have contacted a professional polishing agency for two rounds of polishing, The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

  http://www.textcheck.com/certificate/zgjnMb

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks for attending the comments. Good luck

Comments on the Quality of English Language

NA

Author Response

Dear reviewers or editors,

We would like to thank you for your careful reading, helpful comments, and constructive suggestions, which has significantly improved the presentation of our manuscript. We quite agree with your comments and revise our manuscript according to these suggestions as shown below. The manuscript has also been errors we found have been corrected. We hope our revised manuscript can be accepted for publication. If there are other problems with the manuscript, we are happy to revise it again.

Regarding the issue of English language in articles，We have contacted a professional polishing agency for two rounds of polishing, The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

  http://www.textcheck.com/certificate/zgjnMb

Best wishes！