Zinc-Enhanced Regulation of the Ginkgo biloba L. Response and Secondary Metabolites

Round 1

Reviewer 1 Report

The authors studied the effects of Zn on the growth and biochemical traits of Ginkgo biloba. There is no novelty in this domain. Few parameters have been studied

Dear authors

The following modifications are required:

Title

The word stress should be removed from the title because it has no sense. So, the title should be improved

Abstract

ü  The researchers must characterize the problem in a single sentence

ü  The manuscript lacks details on the experimental design and its components.

ü  The data from all treatments should be displayed as a percentage increase or decrease compared to the control plants.

ü  The authors should include a definitive conclusion based on the research in the final line of the abstract, along with a brief mention of future prospects.

Introduction

ü  L33-37 (Some studies have shown that they can complement each other to inhibit several processes related to carcinogenesis during the development of tumors [9]. The most commonly used product for utilizing these tw o compounds is ginkgo leaf extract (EGb). Ginkgo EGb761, which contains 24% flavonoids and 6% TTL, is the most widely used standardized ginkgo extract.): These lines are not related to this topic and should be deleted

ü  Some lines about the positive and negative impacts of Zn on the growth and biochemical traits should be added

ü  The authors should include a brief discussion about the knowledge gap their research aimed to fill, in addition to the hypothesis statement.

ü  The authors should also provide a novelty statement at the conclusion. What novel findings or connections have the authors made in this study that differ from past research?

ü  The broad and specific objectives are inaccurately stated and need to be provided.

Materials and Methods

ü  The chemical and physical properties of soil should be stated

ü  All procedures should be supported by the references

ü  All procedures are not clearly stated. The authors are not presented clearly the condition of research

ü  Why did not the authors measure the antioxidant activity by the DPPH or ABTS methods?

ü  No information about the type of experimental design and its component is available in this manuscript. The authors should summarize the studied factors in a Table

ü  The manufacture of all materials used in this work should be stated

Results and discussion

ü  The results are not presented clearly. The authors should present their results in this sequence:

·         Effect of Zn amount on the studied characters

·         Effect of groups or month on the studied characters

·         Effect of interaction of Zn amount and group on the studied traits

ü  It is preferable to give the results as percentages of increase and decrease.

ü  The significant status should be mentioned for each measured trait

ü  The headline contents of the Results section should be expressed as a phrase, not as individual terms.

ü  All captions should be improved by adding the full name of all abbreviations presenting in the figures

ü  To better comprehend and analyses all collected data, it is advisable to generate a PCA plot instead of the heatmap plot shown in figure 9.

ü  Why did the researchers study the enzymatic antioxidant systems? 

ü  The explanation is not persuasive. Most sentences in the discussion repeat results rather than provide explanations, making it weak and requiring enhancement. The authors should explain how all of the findings from this study relate to their own findings. The authors should discuss the Zn on the growth and biochemical traits. The authors showed some interpretation under stress conditions and the authors did not mention the stress condition in this manuscript

Conclusion

ü  The authors should provide a concise summary of the most important discoveries as they have presented this part in a reader-friendly style.

ü  Future research should incorporate further studies.

Extensive correction is needed

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Journal: Forests

Title: Zinc-Enhanced Regulation of the Ginkgo biloba Stress Response and Secondary Metabolites

General Comments: The topic is interesting and valuable to our society regarding conservation and adaptability of this endangered plant species. However authors need to well-structured the manuscript. Especially the Materials and Methods section, for better understanding.  Discussion section should be more precise. As here is large no of references mention in the text, then I suggest that comments which are referred by more than one reference, the recent references should keep and the older references should remove. In Figure 10 images should be more prominent. Therefore, I suggest revising the manuscript.

Specific comments: My specific comments are given below:

Line No                                                           Remarks

11-13                                       Use simple sentence. Re-write it.

13-17                                       Use simple sentence. Re-write it.

22                                            Does not clearly express the meaning, re-write it.

23                                            Arrange alphabetically.

29, 95                                      Check it.

36                                            Abbreviation does not specify that it is a leaf extract.

120                                          Mention the full form of abbreviation ZR.

147, 214, 258                          Remove space bar.

357                                          It is a scientific name, so it should be mentioned properly.

366                                          Figure. 10 (a, b, c) should be more prominent.

392                                          Figure. 10 (a, b, c) should be more prominent.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript entitled "Zinc-Enhanced Regulation of the Ginkgo biloba Stress Response and Secondary Metabolites”, although it must be improved in the English language, deals with one important topic: the action of zinc on the physiological well-being of the ginkgo plant and the accumulation of secondary metabolites in the leaves. However, the manuscript suffers for the scarce information in the Materials and Methods section, that generate some questions: how were the soil and environmental conditions? What about light intensity, photoperiod, relative humidity, temperature, soil compositions, and they should also give information about the volume of pot, the water conditions of plants, and so on. All relevant information related which the functionality of photosynthetic systems and other important events related with the plant tolerance and adaptation to heavy metal. Another relevant aspect is related with the strategy of the research: have all parameters been detected on September 22? Which means 50 days after the last treatments? The authors have to explain this strategy, as also as they have to explant more clearly the sampling: how sampled the biological replication; is the biological sample a pool of leaves? How many leaves for plant and how many plants have been used to run photosynthetic and SPAD analysis? Moreover, it is not mentioned any of statistical ANOVA and the letters used to indicate the statistical meaning in the top of each histogram generate to big confusion: the authors have to take more attention of the insertion and use of the capitol and lowercase letters.   

In the following section are reported other suggestions, corrections and criticisms.

Results

Line 174-176: For the SOD, If the capital letters indicate significant differences between time groups, then what you claim is not true from the statistics point of view. Check careful the letter of T2 and T4 of Group B and C, because CK is A!

Line 186-200: For greater clarity, describe each parameter separately from each other.

Line 188-189: If they are significant greater why the capital letter is only A for all?????

Line 194: Correct from T2 to T3

Line 194-196: “… The MDA content peaked in the T?? treatment, with a significant increase of 27.65% compared with that in the CK treatment (P<0.05).” In which group???

Line 201: Correct in “The H2O2 content of the ginkgo (Figure 4) …”

Line 234-236: “… The TP content in ginkgo leaves fluctuated among the different time points (Figure 6b), with small peaks observed in the T2 and T4 treatments.” What are these time points? Rewrite this sentence more clearly.

Line 255-257: “… The maximum values reached 4.16 mg g-1DW and 13.71 mg g-1DW in the T2 treatment, which were 35.95% and 24.30% greater than those in the CK treatment, respectively (P<0.05).” Respectively of what? Rewrite this sentence more clearly. In group B, the TTL

Authors consider that Each Figure should be independent and Use only one criterion when assigning significance letters for each histogram. If you use A for the highest value, then all other values below the highest must follow B and C, if they are significantly different. Check the capital letters of all parameters; therefore, added to each caption from Figure 2 to 8: The capital letters indicate significant differences between time treatment groups (P<0.05), while the lowercase letters indicate significant differences between different treatment concentrations within the same time group (P<0.05).

Discussion

Line 354-357: “… The study revealed that the ZR, IAA, and ABA contents of ginkgo leaves in group B were generally greater than those in groups A and C, indicating that zinc spraying in August had a significant impact on the endogenous hormone levels of ginkgo plants.” Group C plants were also treated in August; so how can the difference be explained?

Line 358-359: “… This decrease may be attributed to the accumulation of secondary metabolites in ginkgo plants under zinc stress.” It would be necessary for you to indicate the position on the plant of the sampled leaves, i.e. at which node from the apex of the shoot leader they were at the time of sampling. This is very important to understand the accumulation of IAA in the leaves under the different treatments. See Materials and Methods

Line 368-369: “… Zinc has been shown to influence plant sugar transport and protein anabolism through its involvement in starch metabolism.” Also, auxin affect the sugar transport.

Line 371: “… Group A exhibited greater FAA and SS contents in ginkgo leaves than groups B and C.” It is not completely the true for FAA: the contest is higher than that detected in CK-plants in most of the treatments of group B and C; although the statical meaning is ambiguous!

Line 374: “… was also observed following the spraying of ZnSO4 in June. “ Is the spraying of June represented by the Group A?

Line 374-377: “Moreover, the over-all photosynthetic efficiency of ginkgo plants was greater in June than in August, suggesting that the application of zinc fertilizer in August may not support the transport and accumulation of sugar in ginkgo plants.” How can formulate this hypothesis you do not have information on transport and accumulation in other organs of plants!

Line 386-387: Correct in “In this study, the contents of TTL and total flavonoids in group A increased at T2 and then decreased at the T4 zinc application.”

Line 388-391: “Overall, spraying 12 mmol/L ZnSO4 in June has been found to have the most significant impact on           increasing the content of medicinal ingredients in ginkgo leaves.”

The figure indicates that you have detected higher content in other treatment in Group B and C. Consider the data and rewrite the sentence. 

What about the analysis of PCA and correlation analysis? Discuss the results!

Moreover, the manuscript needs a language revision.

The manuscript cannot be publishable under this form. The authors will give the requested information and revised the critical points. Therefore, I will recommend considering for the publication the manuscript after the revisions and corrections, and the necessary clarifications requested.  

More appropriate language should be use

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors should mention the type of experimental design in statistical data analysis section like Two ANOVA-CRD or Two ANOVA-RCBD. In addition, The levels of each factors should be added

Moderate correction is needed

Author Response

Reviewer #1: The authors should mention the type of experimental design in statistical data analysis section like Two ANOVA-CRD or Two ANOVA-RCBD. In addition, The levels of each factors should be added.

Answer: Thank you for your thoughtful suggestions. We are very pleased to be asked to submit a revision. We have made modifications in the statistical data analysis section, and the corresponding modification is “The experimental data were organized using Excel 2019 software and analyzed with IBM SPSS Statistics 26.0 software (IBM Corp., NY, USA).  ANOVA was conducted on three zinc -treated time groups labeled as A, B, and C. Two-way ANOVA was conducted on zinc application time (with treatment levels A, B, and C) and different zinc spray concentrations (with treatment levels CK, T1, T2, T3, and T4)”. In addition, we also focused on polishing the language part of the manuscript.

Reviewer 3 Report

Now is ready for pubblication

Author Response

Reviewer #3: Now is ready for pubblication.

Answer: I am very honored to receive your approval of the manuscript and thank you very much for your valuable comments on the manuscript.