Physiological and Transcriptomic Analyses Reveal the Mechanisms of Ilex chinensis Response to Different Types of Simulated Acid Rain
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors> I have question Author's worke with simulated acid rain of sulfuric acid rain (SAR), mixed acid and nitric acid rain at different pH values to investigate their effects. My question is that Is it sure that Natural Acid Rain have the same acids as authors are using? What about different pH should explain it if it's acidic or akaline?
> In Abstract data is irregular should start from background why selected the acid rain then objective, after the showed explain about the experiment detial and after that results with coclusion and suggestion or key implications. It's better to clear the abstract with different part because abstract reflect your manuscript for reader.
> Keywords should the words that related to abstract or title not from abstract or title of the manuscript.
> Introduction wrote well and clear showed the paper direction. I like that but little bit more improve the objective part with your clear hypothesis.
> In whole manuscript anytime you are using the photosynthetic pigments name Chlorophyll a or Chlorophyll b please used the "a" and "b" in italic style.
> I don't know the format but the formulas for the Chlorophyll shoudl be in formula style or equation style.
> In figure two author's used the axis title unit of Photosynthetic rate and other gas exchange parameters with FW at the last. I think you can take gas exchange for living plant so don't need to add the FW at the last.
>In heatmap correlation figure 5 try to increase the size of axis title to make it more clear and readable.
> You added alot of figure in the manuscript my suggestion is to move some material in supplementry rather than main manuscript may be help to understand more about the paper. If you rate it with the value which part of the figure should go to supplementry information will be better to understand more.
Comments on the Quality of English LanguageI didn't check the english because I am not native english background country.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe submitted Manuscript "Physiological and transcriptomic analyses reveal the mecha-2 nisms of Ilex chinensis response to different types of simulated 3 acid rain" provides interesting results about the multiple effects of acid rain on Ilex chinensis plants. The Manuscript could be published but it requires revisions initially.
Major comments.
1) The plant material is not well described. The growth conditions are not given; the conditions of illumination, temperature are to be given.
2) The soil should be described better: what was the pH of the soil extract, how was it mixed (ratios), what was the source of the peat etc.
3) The experiments with the roots of the plants would be interesting to be done. Please, also describe better the volumes of the liquid used.
4) It would be good to use pure H2SO4 and HNO3 without mixing them also. To have clear effects.
5) The transcriptomics results would be good to have for later times with more effects of the acid treatment.
6) Please, add to the introduction the pH values of the collected rain water in the areas mentioned.
7) Figure 10. Especially, 10B: it's not possible to see the numbers at any magnification and the figure legends, it's not readable.
8) Please, take care of all the figures in the way.
9) Most of the figure legends at the axes are not well seen.
10) Figure 2, transpiration 60 days, N5/6: please, discuss why it's above the control values.
11) The good results could be pondered better and the discussion could be enriched by the considerations about drop in photosynthesis when stomatal conductance and transpiration did not change.
12) The Manuscript suffers from the physiological description of the events happening.
13) The data obtained are to be deposited to the databases. It's a common practice for the sort of research.
14) What happened to the growth and weight of the plants during the treatments? It's probably the main point to start with.
15) Abstract.
Further research on gene 22 regulatory network at the two time points showed that the key pathways of I. chinensis in response 23 to acid rain stress include plant-pathogen interaction, MAPK signaling pathway-plant, protein 24 processing in endoplasmic reticulum, ubiquitin mediated proteolysis, etc., in which 11 hub genes, 25 include TOGT1, CML38, RPM1, At1g50180, WRKY53, At4g27190, Mtco2, Mtnd5, EP3, MDH1, 26 GLN1-1 were identified.
The genes mentioned are not the genes of Ilex chinensis. To be corrected in the text also.
Minor comments are not limited by the mentioned ones below.
1) 2.5. Determination of itrogen metabolism-related products and enzymes 136
Nitrogen, not introgen.
2) Funding information Please, remove the colour marks.
3) Please, add all the abbreviations to all the figures. Alternatively, it's not clear what are the treatments.
4) The style of references should be changed according to the Journal requirements (numbers).
5) It might be reasonable to add chla/chlb ratio.
Comments on the Quality of English LanguageReadable.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe Authors made some positive changes in the text of the Manuscript. However, still the essential revisions are required.
Major issues.
The Reviewer is not able to recommend the Manuscript for publication before the transcriptomics results are deposited in the public databases.
Minor and major comments.
1) Abstract.
reticulum, ubiquitin mediated proteolysis, etc., in which 6 hub genes were identified, including28 TRINITY_DN13584_c0_g1 (CML38), TRINITY_DN164_c0_g4 (At4g27190), TRINITY_DN654_c0_g129 (MT-CO2), etc
All the 6 genes have to be mentioned. Moreover, the mentioned genes are not the genes of the studied Ilex.
It’s better to remove the part of the sentence.
2) Westoredthe111 collected seeds in low-temperature sand and obtain the seedlings of I. chinensis through112 seedling cultivation.
Obtained, not obtain.
3) At the study site, themonthly119 average temperature ranged from 6℃ to 28℃, and the total solar radiationwasabout29.46 kcal/cm2 120 from September to November 2022
Please, indicate how the energy of solar radiation was measured; what was the time interval to get the values?
What was the temperature at the time of the experiments?
4) The plant material is still lacking description to reproduce the research. What was the thickness of the plants, any indications more? Weight? Number of leaves etc.
5) Comments 3: The experiments with the roots of the plants would be interesting to be done. Please, also describe better the volumes of the liquid used.
- Response 3: Thank you very much for pointing this out. We strongly concur that investigating the effect of acid rain on plant roots is interesting. Leaves are the main organs for photosynthesis in plants, and the effect of acid rain on leaves best reflects the growth and development status of plants. So our current study is primarily centered on the leaves of plants. We will take your valuable suggestion into account and plan to incorporate roots of plants into our future research endeavors.
The leaves are covered with wax and cuticle, leaf hairs etc., so the liquid could be reflected and the effects are likely be via roots anyway. Did you check the roots of the plants? Please, discuss if not done.
6) Still the quality of the figures did not increase sharply. Please, make more efforts.
Comments 7: Figure 10. Especially, 10B: it's not possible to see the numbers at any magnification and the figure legends, it's not readable.
- Response 7: We feel sorry for the unclear figures. In the resubmitted manuscript, we have modified the figures to improve readability, and thank you for your valuable suggestions.
7)
Comments 10: Figure 2, transpiration 60 days, N5/6: please, discuss why it's above the control values.
- Response 10: We greatly appreciate for pointing this out. In this study, the transpiration rate of chinensis after 60 days of NAR treatment at pH 5.6 was actually higher than that of CK. We speculate that due to the low acidity of NAR, Ilex. chinensis has adapted to this treatment after 60 days, and a certain concentration of NO3- requires Ilex. chinensis to enhance transpiration to promote its transport within the plant. Therefore, the transpiration rate was higher than CK. We have added this explanation in Section 3.1 (Page 5, Line 221-225).
We speculate that due to the low acidity of NAR, I.chinensis has adapted to this treatment after 60 days, and a certain concentrationofNO3-223 224 requires I. chinensis to enhance transpiration to promote its transport withintheplant.225 Therefore, the transpiration rate was higher than that of CK (Fig. 2).
It’s for the discussion part.
Why transpiration didn’t coincide with photosynthesis. Please, add more discussion points
8) Figure 4. Please, add time scale at the axis x.
9) Comments 14: What happened to the growth and weight of the plants during the treatments? It's probably the main point to start with.
- Response 14: Thank you very much for raising this out. In our complete study, we measured the height increment rate and specific leaf weight of Ilex chinensis after acid rain stress, and found that acid rain inhibited the growth of plant height and the specific leaf weight decreased. Due to the limitation of paper, we did not include these indicators in the manuscript, but we added a discussion of them in the second paragraph of Section 4.1 (Page 16-17, Line 418-427).
Please, provide the data available. The discussion refers to the Fu, X.P. The Effect of Simulated Acid Rain on Six kinds of Urban Greening Plant Seeding Physioecological characteristics. Central South705 University of Forestry and Technology. 2007.
which is not easy to find online. Please, discuss more.
10) 279 3.4. Illumina sequencing and correlation between Samples
Sample is small letter.
11) Quality of figures is not readable in the printed version.
12) In addition, TRINITY_DN13584_c0_g1 (CML38), TRINITY_DN164_c0_g4594 (At4g27190), TRINITY_DN654_c0_g1 (MT-CO2), TRINITY_DN13611_c1_g2(MT-ND5),595 and TRINITY_DN21290_c0_g2 (TOGT1)
Here are not the gens of Ilex, please, make corrections or explanations.
13) Could be more revisions required.
Comments on the Quality of English LanguageReadable without clarity.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
