Effects of Acid Rain Stress on the Physiological and Biochemical Characteristics of Three Plant Species
Round 1
Reviewer 1 Report
Comments on forests-2382086
· The manuscript deals with the effects of acid rain stress on the photosynthetic, physiological and biochemical characteristics of three plant species viz., Pinus massoniana, Phyllostachys edulis, and Cinnamomum camphora. But the point should be noted that the photosynthesis is also one of the most important physiological processes. So, the tile is not suitable. Please modify the title.
· Language quality is very poor. It should be improved.
· Line 10: Mention the name of the ‘physiological and biochemical indicators’ within the bracket.
· Please rewrite the abstract section. The novelty statement and the objective of the study are missing.
· Give a space between the word and the reference no., like, line 31: ‘……….urbanization [1-2]’. Rectify it throughout the manuscript.
· Line 65: Use the word ‘adverse’ in place of ‘adversity’.
· Line 74: Rewrite sentence as, ‘…….under acid rain stress’.
· All the scientific names should be written in italics, like, line 96. Rectify it throughout the manuscript.
· Methodology should be written properly and can be given as supplementary file with the manuscript.
· Rewrite the sentence: line 120-121.
· The legends/description of the figures and tables were not described properly. The quality of figure 3 should be improved.
· The authors designed their experiments based on some previous reports/data. The authors treated their samples with tap water. The tap water may also contain various kinds of elements. Therefore, it should be estimated before performing such kind of experiments.
· Line 156: Remove space between 25 and ºC.
· Define correction factor. How could the authors measure the correction factor in their experiments?
· Some texts were repetitive. Please check and rewrite the manuscript properly.
· Results sections is very poorly written. The observations were not described properly. Give percentage values of increment/reduction of the tested physiological and biochemical parameters, like SOD, CAT, POD, APX, soluble sugar and protein, over control. The results are inappropriate for the present conclusion.
· Statistical analyses are inadequate. Motivation and Justification of performing the RDA/PCA analyses and the interpretation of the results are not properly described. The authors should check this issue for their experiments to continue the validity of statistical analysis. Try to justify your observations with statistical analyses within the text. Such an approach undermines the importance of statistical analyses and must be avoided in group research.
· The authors repetitively stated within the text that ‘The higher the acidity, the stronger the inhibitory effect, which is consistent with the first hypothesis that different acidity levels have different effects on the physiological and biochemical characteristics of plant seedlings/This results is in line with our first hypothesis that differences exist in the photosynthetic properties of plant seedlings exposed to different acidities’. In own research articles, this kind of repetition must be avoided. It is already known in some previous literatures that the variation of physiological and biochemical parameters are in line with the variation of stress levels (any kinds of stress). So, what is the novelty of the statement, first hypotheses.
Thus, the manuscript requires above corrections/modifications/incorporations to improve its quality and there are several major concerns that need to be addressed before its publication in the journal - Forests. NOT RECOMMENDED for its publication in its present form.
The quality of english language must be improved.
Author Response
Response letter
Dear Editor and Reviewers,
On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript. We feel great thanks for your professional review work on our manuscript. We use this feedback to improve the quality of the manuscript. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous manuscript, and presented it in revised mode in the manuscript. Furthermore, the point-to-point responses to the reviewer's comments are provided below. We hope that these revisions successfully address your concerns and requirements. Should you have any questions, please contact us without hesitate. Looking forward to hearing from you soon.
Response to Reviewer 1 Comments
Point 1:
The manuscript deals with the effects of acid rain stress on the photosynthetic, physiological and biochemical characteristics of three plant species viz., Pinus massoniana, Phyllostachys edulis, and Cinnamomum camphora. But the point should be noted that the photosynthesis is also one of the most important physiological processes. So, the tile is not suitable. Please modify the title.
Response 1:
We sincerely appreciate the valuable comments. We think this is an excellent suggestion. We have changed the title to Effect of acid rain stress on the physiological and biochemical characteristics of three plants species (in the row 2 and 3).
Point 2:
Language quality is very poor. It should be improved.
Response 2:
We thank the reviewer for this valuable suggestion and the entire manuscript has been edited by professional language serve. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in the article with a revision pattern. We appreciate for Editors/Reviewers' warm work earnestly and hope that the correction will meet with approval. Here is the certificate of language editing.
Point 3:
Line 10: Mention the name of the ‘physiological and biochemical indicators’ within the bracket.
Response 3:
Thank you for your careful review. We have specified each physiological and biochemical index in brackets in the summary section (in the row 15 to 18). The specific indicators are as follows. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (AQY), dark respiration rate (Rd), soluble sugar, starch, soluble protein, proline, relative chlorophyll, malondialdehyde (MDA) and antioxidant enzyme activity.
Point 4:
Please rewrite the abstract section. The novelty statement and the objective of the study are missing.
Response 4:
Thank you for your careful review. We have re-written this part according to the Reviewer’s suggestion, added novelty statement and study objectives (in the row of 9 to 31).
Point 5:
Give a space between the word and the reference no., like, line 31: ‘……….urbanization [1-2]’. Rectify it throughout the manuscript.
Response 5:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the format harmonized within the whole manuscript.
Point 6:
Line 65: Use the word ‘adverse’ in place of ‘adversity’.
Response 6:
Thanks for your careful checks. We have changed the content of this section (in the row of 93). The specific corrections are as follows. The contents of proline, soluble sugars, starch, and soluble protein reflect the degree of plant stress because these substances provide stress resistance under adverse conditions.
Point 7：
Line 74: Rewrite sentence as, ‘…….under acid rain stress’.
Response 7:
Thanks for your careful checks. We have changed the content of this section (in the row of 107). The specific corrections are as follows. Therefore, we investigate the response of three typical tree species (Pinus massoniana, Phyllostachys edulis, and Cinnamomum camphora) under acid rain stress.
Point 8:
All the scientific names should be written in italics, like, line 96. Rectify it throughout the manuscript.
Response 8:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript.
Point 9:
Methodology should be written properly and can be given as supplementary file with the manuscript.
Response 9:
Thank you for your careful review. We have compiled the experimental methods in detail and uploaded them as a supplemental file in the last part of this document.
Point 10:
Rewrite the sentence: line 120-121.
Response 10:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have rewritten the sentence. The specific corrections are as follows. Therefore, a mixed acid rain experiment was conducted. The mother liquor was prepared according to the molar ratio of H2SO4:HNO3=1:1, and diluted it with distilled water to obtain acid rain solutions with pH values of 4.5, 3.5, and 2.5 (in the row of 164 to 166).
Point 11:
The legends/description of the figures and tables were not described properly. The quality of figure 3 should be improved.
Response 11:
Thank you for your careful review. We have restated the legend, added explanatory notes on the indicators and lowercase letters in the figure, and modified Figure 3 to make the data more clearly presented and thus increase the readability of the manuscript. If you have other specific comments on figure 3, please let us know.
Point 12:
The authors designed their experiments based on some previous reports/data. The authors treated their samples with tap water. The tap water may also contain various kinds of elements. Therefore, it should be estimated before performing such kind of experiments.
Response 12:
Thank you for your careful review. We feel sorry for our careleness. Because of a mistranslation, we expressed distilled water as tap water. However, we used distilled water throughout the experiment to complete our work. Therefore there is no situation where the various elements affect the experiment and we have changed the tap water to distilled water in the row of 177. We apologize again for our carelessness and thank you very much for your careful review.
Point 13:
Line 156: Remove space between 25 and ºC.
Response 13:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have removed the space between 25 and ℃ in order to make the formatting uniform throughout the manuscript (in the row of 204).
Point 14:
Define correction factor. How could the authors measure the correction factor in their experiments?
Response 14:
Thank you for your careful review. For this part of the light response curve, the corrected right-angle hyperbolic model proposed by Ye (2007) was used for curve fitting of optical response. The model equation is as follows:
(1)
(2)
(3)
where β is the correction factor (light suppression term), α is the initial slope of the photoresponse curve, i.e., the slope at point I=0. The factor γ (light saturation term ) is equal to the ratio of the initial slope of the photoresponse curve to Pmax, i.e., γ=α/Pmax, where I is the effective photosynthetic radiation, Pn is the net photosynthetic rate, and Rd is the dark respiration rate.
These coefficients were calculated automatically by the model, and we measured the values of each indicator when the light intensity was 0 µmol·m-2s-1 and we performed the dark response before the formal measurement. The model has the advantages that both the light saturation point and the maximum net photosynthetic rate in the fitted results are closer to the measured values, and it can fit the curve of photosynthetic rate with the decreasing phase of light intensity after saturation light intensity, so we chose it for the fitting process, which helps to obtain reliable results.
Point 15:
Some texts were repetitive. Please check and rewrite the manuscript properly.
Response 15:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have rechecked the whole manuscript, removed duplicate text and presented it in revised mode to increase the readability of the manuscript.
Point 16:
Results sections is very poorly written. The observations were not described properly. Give percentage values of increment/reduction of the tested physiological and biochemical parameters, like SOD, CAT, POD, APX, soluble sugar and protein, over control. The results are inappropriate for the present conclusion.
Response 16:
We apologize for the poor description of our manuscript. Based on your comments, we have redescribed the results and added incremental/reduction percentage values for each indicator to make the results more relevant to the conclusions (in the row of 289 to 311).
Point 17:
Statistical analyses are inadequate. Motivation and Justification of performing the RDA/PCA analyses and the interpretation of the results are not properly described. The authors should check this issue for their experiments to continue the validity of statistical analysis. Try to justify your observations with statistical analyses within the text. Such an approach undermines the importance of statistical analyses and must be avoided in group research.
Response 17:
Thank you for your careful review. The relationship between photosynthetic and physiological characteristics of plants and biochemical indicators was analyzed by RDA, and the differences between different species of plants were reflected by PCA. Based on your comments, We added descriptions of the PCA and RDA graphs, explained the relationships between the different factors, detailed the explanatory rates of the main factors, and added methods for interpreting the analysis in the figure notes to increase the validity of this analysis (in the row of 254 to 257, 340 to 342, 350 to 352, and 358-360).
Point 18:
The authors repetitively stated within the text that ‘The higher the acidity, the stronger the inhibitory effect, which is consistent with the first hypothesis that different acidity levels have different effects on the physiological and biochemical characteristics of plant seedlings/This results is in line with our first hypothesis that differences exist in the photosynthetic properties of plant seedlings exposed to different acidities’. In own research articles, this kind of repetition must be avoided. It is already known in some previous literatures that the variation of physiological and biochemical parameters are in line with the variation of stress levels (any kinds of stress). So, what is the novelty of the statement, first hypotheses.
Response 18:
Thanks for your careful checks. Based on your comments, we have reread the entire article, removed the repetitive parts of the article and presented it in revised mode to increase the readability of the manuscript. We think this is an excellent suggestion. We have modified the first scientific hypothesis to introduce the critical value of acid rain affecting each plant indicator into the scientific question, that is, the physiological, and biochemical characteristics of plant seedlings differ for different acidity levels of acid rain, and slightly acidic rain enhances the physiological and biochemical properties of plant seedlings. This study set up a natural rainfall treatment (NR). By simulating acid rain with different acidity, we found that compared with other experimental groups, the physiological and biochemical indexes of plants under NR were higher. This result is the highlight of the article.The relevant data showed that the natural rainfall pH of the test site is 5.42-5.83 (described in Article 2.3.1), that is, slightly acidic rain. The results of the study showed that the high values of the indicators under NR indicate that the plants in the Jinyun Mountain area of Chongqing have adapted to the current conditions. This study is important for the vegetation construction in the existing acid rain environment in Jinyun Mountain, Chongqing. Therefore, with your suggestion, the revised hypotheses are significant.
Sample collection and index determination method
- Determination of photosynthetic physiological indicators
A windless and sunny morning (8:00-11:00am) on September 3, 2021 was selected for measuring the photosynthetic parameters using a Li-6400 portable photosynthesis meter with a red and blue light source (Li-COR). The effective photosynthetic radiation intensity was 1600, 1400, 1200, 1000, 800, 600, 400, 200, 150, 100, 50, 20, and 0 µmol·m-2s-1 with 13 gradients based on the results of a pre-experiment and the solar radiation data from the Jinyun Mountain Nature Reserve in Beibei District, Chongqing, China. The leaf chamber temperature was 25℃, the CO2 concentration was 400 µmol·mol-1, the gas flow rate was 500µmol·s-1, and the relative humidity was 50%. Measurements were made from 8:00 to 11:00am on three healthy plants in each treatment. Three similar and mature, healthy leaves on the main tips were selected from each plant, and 5-7 needles were selected each time and placed side by side in the leaf chamber. We obtained three measurements and used the average as the result. During each measurement, the physiological indices were measured after 2 min of adaptation at each photosynthetically active radiation (PAR) intensity. The indices included the Pn, stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr).
The corrected right-angle hyperbolic model proposed by Ye (2007) (Eq. (1)) was used for curve fitting of the optical response. The maximum Pn (Pmax), light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (AQY), and dark respiration rate (Rd) were calculated using Equations (2) and (3).
The model is expressed as follows:
(1)
where β is the correction factor, α is the initial slope of the photoresponse curve, i.e., the slope at point I=0. The factor γ is equal to the ratio of the initial slope of the photoresponse curve to Pmax, i.e., γ=α/Pmax, where I is the effective photosynthetic radiation, Pn is the net photosynthetic rate, and Rd is the dark respiration rate.
The Pmax was calculated as follows:
(2)
The LSP was calculated as follows:
(3)
where LSP is the saturated light intensity of the plant, and Pmax is the maximum net photosynthetic rate corresponding to the saturated light intensity of the plant. The LCP, AQY, and Rd were obtained by model fitting.
- Determination of biochemical indicators
At the end of the acid rain treatment on September 6, three plants of approximately the same size were randomly selected for each treatment. We used 3-5 mature and fully expanded leaves of each plant and removed the main leaf veins to determine the physiological indices. Each measurement was repeated three times.
(1) The relative chlorophyll content
The relative chlorophyll content of the plant leaves was determined on May 1 and September 4, 2021, using a SPAD-502 chlorophyll meter. The leaves were placed inside the device, avoiding the leaf veins during the measurement. We selected 3-5 needles from the coniferous species and clamped them for the measurement. Three plants were randomly selected for each treatment; 3 mature leaves on the branch tips were selected from each plant, and the average value was used.
(2) The soluble sugar and starch contents were analyzed by the anthrone colorimetric method (Yemm and Willis, 1954).
Accurately weigh 1 g of the sample, add 25 ml of boiling water, boil in a water bath with a lid for 10 min, cool down and filter to fix the volume in a 50 ml volume. Aspirate 2 ml of the extract into another 50 ml volumetric flask and set aside with distilled water. Pipette 1 m of the diluted extract into a test tube, add 4.0 ml of anthrone reagent, and use the same amount of distilled water as the blank tube extract. Shake well and boil for 10 min in a boiling water bath, then cool down and colorimetric at 620 nm to find out the content from the drawn glucose standard curve.
The residue after extraction of soluble sugar was transferred into a 50 ml volumetric flask, 20 ml of hot distilled water was added, boiled in a boiling water bath for 30 min, then 2ml of 9.2 mol/L perchloric acid was added for 15 min, cooled, mixed, centrifuged at 4000 r/min for 10min, the residue was repeatedly extracted with 10ml of distilled water until the starch hydrolysis completeness test did not show blue color. Take 1.0 ml of the extract of the sample to be measured, add 5 ml of anthrone reagent, boil it for 10 min in a boiling water bath, cool it down, colorimetric at 620 nm wavelength, and find out the content from the glucose standard curve.
(3) The soluble protein contents were determined by colorimetry using Kemas Brilliant Blue G-250 (Bradford, 1976).
In this experiment, the soluble protein was determined with reference to the Bradford method, and the serum albumin solution was configured at the absorbance of 595 nm in order to draw the standard curve, and the soluble protein content in the leaves was determined at the absorbance of 595 nm, and the soluble protein content was found according to the standard curve of bovine serum albumin.
(4) The proline content was determined by the acidic ninhydrin method (Bates et al., 1973).
The proline extract was prepared with 3% sulfosalicylic acid solution, and the absorbance was read at 520 nm using toluene as a blank control. The proline content was calculated from the proline standard curve.
(5) The MDA content was obtained from the thiobarbituric acid method (Hodges et al., 1999).
Using thiobarbituric acid (TBA) heated under acidic conditions to produce a chromogenic reaction with malondialdehyde in tissues, the absorbance was measured at 532 nm, 600 nm and 450 nm to calculate the malondialdehyde content.
(6) Determination of antioxidant enzyme activity:
The POD content was measured by guaiacol. The SOD content was obtained by nitroblue tetrazolium (NBT) photochemical reduction, and the CAT and ascorbate peroxidase (APX) contents were determined by ultraviolet (UV) spectrophotometry (Debnath et al., 2018).
Preparation of extraction solution: take the sample and grind it into powder with liquid nitrogen, add 5 ml of potassium phosphate buffer, mix well, centrifuge at 4℃, 12000×g for 20 minutes, and store the supernatant at 4℃ for enzyme activity determination.
- Determination method of SOD activity:
The activity was determined by using the reaction of SOD inhibition of NBT reduction by O2-reduction in light, and the absorbance value was detected at 560 nm to determine the SOD activity.
- Determination method of POD activity:
POD activity was determined at 470 nm using guaiacol as a substrate.
- Determination of CAT activity:
The absorbance value of CAT at 240 nm was read on a UV-visible photometer using the reaction solution without H2O2 as a control.
- Determination method of APX activity:
The enzyme activity was determined by using the principle that APX reduces the amount of ascorbic acid in the presence of H2O2, and the absorbance value at 290 nm was read on the UV-visible photometer to determine the APX activity.
The above contents are our responses to your valuable questions or suggestions. Thank you very much for your patience! Your patience and high quality advice have helped us a lot, and we have learned a lot in the process of improving my manuscript. Thank you again! We wish you good health and happy life!
Thank you and best regards.
Sincerely,
Correspondi
Response letter
Dear Editor and Reviewers,
On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript. We feel great thanks for your professional review work on our manuscript. We use this feedback to improve the quality of the manuscript. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous manuscript, and presented it in revised mode in the manuscript. Furthermore, the point-to-point responses to the reviewer's comments are provided below. We hope that these revisions successfully address your concerns and requirements. Should you have any questions, please contact us without hesitate. Looking forward to hearing from you soon.
Response to Reviewer 1 Comments
Point 1:
The manuscript deals with the effects of acid rain stress on the photosynthetic, physiological and biochemical characteristics of three plant species viz., Pinus massoniana, Phyllostachys edulis, and Cinnamomum camphora. But the point should be noted that the photosynthesis is also one of the most important physiological processes. So, the tile is not suitable. Please modify the title.
Response 1:
We sincerely appreciate the valuable comments. We think this is an excellent suggestion. We have changed the title to Effect of acid rain stress on the physiological and biochemical characteristics of three plants species (in the row 2 and 3).
Point 2:
Language quality is very poor. It should be improved.
Response 2:
We thank the reviewer for this valuable suggestion and the entire manuscript has been edited by professional language serve. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in the article with a revision pattern. We appreciate for Editors/Reviewers' warm work earnestly and hope that the correction will meet with approval. Here is the certificate of language editing.
Point 3:
Line 10: Mention the name of the ‘physiological and biochemical indicators’ within the bracket.
Response 3:
Thank you for your careful review. We have specified each physiological and biochemical index in brackets in the summary section (in the row 15 to 18). The specific indicators are as follows. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (AQY), dark respiration rate (Rd), soluble sugar, starch, soluble protein, proline, relative chlorophyll, malondialdehyde (MDA) and antioxidant enzyme activity.
Point 4:
Please rewrite the abstract section. The novelty statement and the objective of the study are missing.
Response 4:
Thank you for your careful review. We have re-written this part according to the Reviewer’s suggestion, added novelty statement and study objectives (in the row of 9 to 31).
Point 5:
Give a space between the word and the reference no., like, line 31: ‘……….urbanization [1-2]’. Rectify it throughout the manuscript.
Response 5:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the format harmonized within the whole manuscript.
Point 6:
Line 65: Use the word ‘adverse’ in place of ‘adversity’.
Response 6:
Thanks for your careful checks. We have changed the content of this section (in the row of 93). The specific corrections are as follows. The contents of proline, soluble sugars, starch, and soluble protein reflect the degree of plant stress because these substances provide stress resistance under adverse conditions.
Point 7：
Line 74: Rewrite sentence as, ‘…….under acid rain stress’.
Response 7:
Thanks for your careful checks. We have changed the content of this section (in the row of 107). The specific corrections are as follows. Therefore, we investigate the response of three typical tree species (Pinus massoniana, Phyllostachys edulis, and Cinnamomum camphora) under acid rain stress.
Point 8:
All the scientific names should be written in italics, like, line 96. Rectify it throughout the manuscript.
Response 8:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript.
Point 9:
Methodology should be written properly and can be given as supplementary file with the manuscript.
Response 9:
Thank you for your careful review. We have compiled the experimental methods in detail and uploaded them as a supplemental file in the last part of this document.
Point 10:
Rewrite the sentence: line 120-121.
Response 10:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have rewritten the sentence. The specific corrections are as follows. Therefore, a mixed acid rain experiment was conducted. The mother liquor was prepared according to the molar ratio of H2SO4:HNO3=1:1, and diluted it with distilled water to obtain acid rain solutions with pH values of 4.5, 3.5, and 2.5 (in the row of 164 to 166).
Point 11:
The legends/description of the figures and tables were not described properly. The quality of figure 3 should be improved.
Response 11:
Thank you for your careful review. We have restated the legend, added explanatory notes on the indicators and lowercase letters in the figure, and modified Figure 3 to make the data more clearly presented and thus increase the readability of the manuscript. If you have other specific comments on figure 3, please let us know.
Point 12:
The authors designed their experiments based on some previous reports/data. The authors treated their samples with tap water. The tap water may also contain various kinds of elements. Therefore, it should be estimated before performing such kind of experiments.
Response 12:
Thank you for your careful review. We feel sorry for our careleness. Because of a mistranslation, we expressed distilled water as tap water. However, we used distilled water throughout the experiment to complete our work. Therefore there is no situation where the various elements affect the experiment and we have changed the tap water to distilled water in the row of 177. We apologize again for our carelessness and thank you very much for your careful review.
Point 13:
Line 156: Remove space between 25 and ºC.
Response 13:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have removed the space between 25 and ℃ in order to make the formatting uniform throughout the manuscript (in the row of 204).
Point 14:
Define correction factor. How could the authors measure the correction factor in their experiments?
Response 14:
Thank you for your careful review. For this part of the light response curve, the corrected right-angle hyperbolic model proposed by Ye (2007) was used for curve fitting of optical response. The model equation is as follows:
(1)
(2)
(3)
where β is the correction factor (light suppression term), α is the initial slope of the photoresponse curve, i.e., the slope at point I=0. The factor γ (light saturation term ) is equal to the ratio of the initial slope of the photoresponse curve to Pmax, i.e., γ=α/Pmax, where I is the effective photosynthetic radiation, Pn is the net photosynthetic rate, and Rd is the dark respiration rate.
These coefficients were calculated automatically by the model, and we measured the values of each indicator when the light intensity was 0 µmol·m-2s-1 and we performed the dark response before the formal measurement. The model has the advantages that both the light saturation point and the maximum net photosynthetic rate in the fitted results are closer to the measured values, and it can fit the curve of photosynthetic rate with the decreasing phase of light intensity after saturation light intensity, so we chose it for the fitting process, which helps to obtain reliable results.
Point 15:
Some texts were repetitive. Please check and rewrite the manuscript properly.
Response 15:
Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have rechecked the whole manuscript, removed duplicate text and presented it in revised mode to increase the readability of the manuscript.
Point 16:
Results sections is very poorly written. The observations were not described properly. Give percentage values of increment/reduction of the tested physiological and biochemical parameters, like SOD, CAT, POD, APX, soluble sugar and protein, over control. The results are inappropriate for the present conclusion.
Response 16:
We apologize for the poor description of our manuscript. Based on your comments, we have redescribed the results and added incremental/reduction percentage values for each indicator to make the results more relevant to the conclusions (in the row of 289 to 311).
Point 17:
Statistical analyses are inadequate. Motivation and Justification of performing the RDA/PCA analyses and the interpretation of the results are not properly described. The authors should check this issue for their experiments to continue the validity of statistical analysis. Try to justify your observations with statistical analyses within the text. Such an approach undermines the importance of statistical analyses and must be avoided in group research.
Response 17:
Thank you for your careful review. The relationship between photosynthetic and physiological characteristics of plants and biochemical indicators was analyzed by RDA, and the differences between different species of plants were reflected by PCA. Based on your comments, We added descriptions of the PCA and RDA graphs, explained the relationships between the different factors, detailed the explanatory rates of the main factors, and added methods for interpreting the analysis in the figure notes to increase the validity of this analysis (in the row of 254 to 257, 340 to 342, 350 to 352, and 358-360).
Point 18:
The authors repetitively stated within the text that ‘The higher the acidity, the stronger the inhibitory effect, which is consistent with the first hypothesis that different acidity levels have different effects on the physiological and biochemical characteristics of plant seedlings/This results is in line with our first hypothesis that differences exist in the photosynthetic properties of plant seedlings exposed to different acidities’. In own research articles, this kind of repetition must be avoided. It is already known in some previous literatures that the variation of physiological and biochemical parameters are in line with the variation of stress levels (any kinds of stress). So, what is the novelty of the statement, first hypotheses.
Response 18:
Thanks for your careful checks. Based on your comments, we have reread the entire article, removed the repetitive parts of the article and presented it in revised mode to increase the readability of the manuscript. We think this is an excellent suggestion. We have modified the first scientific hypothesis to introduce the critical value of acid rain affecting each plant indicator into the scientific question, that is, the physiological, and biochemical characteristics of plant seedlings differ for different acidity levels of acid rain, and slightly acidic rain enhances the physiological and biochemical properties of plant seedlings. This study set up a natural rainfall treatment (NR). By simulating acid rain with different acidity, we found that compared with other experimental groups, the physiological and biochemical indexes of plants under NR were higher. This result is the highlight of the article.The relevant data showed that the natural rainfall pH of the test site is 5.42-5.83 (described in Article 2.3.1), that is, slightly acidic rain. The results of the study showed that the high values of the indicators under NR indicate that the plants in the Jinyun Mountain area of Chongqing have adapted to the current conditions. This study is important for the vegetation construction in the existing acid rain environment in Jinyun Mountain, Chongqing. Therefore, with your suggestion, the revised hypotheses are significant.
Sample collection and index determination method
- Determination of photosynthetic physiological indicators
A windless and sunny morning (8:00-11:00am) on September 3, 2021 was selected for measuring the photosynthetic parameters using a Li-6400 portable photosynthesis meter with a red and blue light source (Li-COR). The effective photosynthetic radiation intensity was 1600, 1400, 1200, 1000, 800, 600, 400, 200, 150, 100, 50, 20, and 0 µmol·m-2s-1 with 13 gradients based on the results of a pre-experiment and the solar radiation data from the Jinyun Mountain Nature Reserve in Beibei District, Chongqing, China. The leaf chamber temperature was 25℃, the CO2 concentration was 400 µmol·mol-1, the gas flow rate was 500µmol·s-1, and the relative humidity was 50%. Measurements were made from 8:00 to 11:00am on three healthy plants in each treatment. Three similar and mature, healthy leaves on the main tips were selected from each plant, and 5-7 needles were selected each time and placed side by side in the leaf chamber. We obtained three measurements and used the average as the result. During each measurement, the physiological indices were measured after 2 min of adaptation at each photosynthetically active radiation (PAR) intensity. The indices included the Pn, stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr).
The corrected right-angle hyperbolic model proposed by Ye (2007) (Eq. (1)) was used for curve fitting of the optical response. The maximum Pn (Pmax), light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (AQY), and dark respiration rate (Rd) were calculated using Equations (2) and (3).
The model is expressed as follows:
(1)
where β is the correction factor, α is the initial slope of the photoresponse curve, i.e., the slope at point I=0. The factor γ is equal to the ratio of the initial slope of the photoresponse curve to Pmax, i.e., γ=α/Pmax, where I is the effective photosynthetic radiation, Pn is the net photosynthetic rate, and Rd is the dark respiration rate.
The Pmax was calculated as follows:
(2)
The LSP was calculated as follows:
(3)
where LSP is the saturated light intensity of the plant, and Pmax is the maximum net photosynthetic rate corresponding to the saturated light intensity of the plant. The LCP, AQY, and Rd were obtained by model fitting.
- Determination of biochemical indicators
At the end of the acid rain treatment on September 6, three plants of approximately the same size were randomly selected for each treatment. We used 3-5 mature and fully expanded leaves of each plant and removed the main leaf veins to determine the physiological indices. Each measurement was repeated three times.
(1) The relative chlorophyll content
The relative chlorophyll content of the plant leaves was determined on May 1 and September 4, 2021, using a SPAD-502 chlorophyll meter. The leaves were placed inside the device, avoiding the leaf veins during the measurement. We selected 3-5 needles from the coniferous species and clamped them for the measurement. Three plants were randomly selected for each treatment; 3 mature leaves on the branch tips were selected from each plant, and the average value was used.
(2) The soluble sugar and starch contents were analyzed by the anthrone colorimetric method (Yemm and Willis, 1954).
Accurately weigh 1 g of the sample, add 25 ml of boiling water, boil in a water bath with a lid for 10 min, cool down and filter to fix the volume in a 50 ml volume. Aspirate 2 ml of the extract into another 50 ml volumetric flask and set aside with distilled water. Pipette 1 m of the diluted extract into a test tube, add 4.0 ml of anthrone reagent, and use the same amount of distilled water as the blank tube extract. Shake well and boil for 10 min in a boiling water bath, then cool down and colorimetric at 620 nm to find out the content from the drawn glucose standard curve.
The residue after extraction of soluble sugar was transferred into a 50 ml volumetric flask, 20 ml of hot distilled water was added, boiled in a boiling water bath for 30 min, then 2ml of 9.2 mol/L perchloric acid was added for 15 min, cooled, mixed, centrifuged at 4000 r/min for 10min, the residue was repeatedly extracted with 10ml of distilled water until the starch hydrolysis completeness test did not show blue color. Take 1.0 ml of the extract of the sample to be measured, add 5 ml of anthrone reagent, boil it for 10 min in a boiling water bath, cool it down, colorimetric at 620 nm wavelength, and find out the content from the glucose standard curve.
(3) The soluble protein contents were determined by colorimetry using Kemas Brilliant Blue G-250 (Bradford, 1976).
In this experiment, the soluble protein was determined with reference to the Bradford method, and the serum albumin solution was configured at the absorbance of 595 nm in order to draw the standard curve, and the soluble protein content in the leaves was determined at the absorbance of 595 nm, and the soluble protein content was found according to the standard curve of bovine serum albumin.
(4) The proline content was determined by the acidic ninhydrin method (Bates et al., 1973).
The proline extract was prepared with 3% sulfosalicylic acid solution, and the absorbance was read at 520 nm using toluene as a blank control. The proline content was calculated from the proline standard curve.
(5) The MDA content was obtained from the thiobarbituric acid method (Hodges et al., 1999).
Using thiobarbituric acid (TBA) heated under acidic conditions to produce a chromogenic reaction with malondialdehyde in tissues, the absorbance was measured at 532 nm, 600 nm and 450 nm to calculate the malondialdehyde content.
(6) Determination of antioxidant enzyme activity:
The POD content was measured by guaiacol. The SOD content was obtained by nitroblue tetrazolium (NBT) photochemical reduction, and the CAT and ascorbate peroxidase (APX) contents were determined by ultraviolet (UV) spectrophotometry (Debnath et al., 2018).
Preparation of extraction solution: take the sample and grind it into powder with liquid nitrogen, add 5 ml of potassium phosphate buffer, mix well, centrifuge at 4℃, 12000×g for 20 minutes, and store the supernatant at 4℃ for enzyme activity determination.
- Determination method of SOD activity:
The activity was determined by using the reaction of SOD inhibition of NBT reduction by O2-reduction in light, and the absorbance value was detected at 560 nm to determine the SOD activity.
- Determination method of POD activity:
POD activity was determined at 470 nm using guaiacol as a substrate.
- Determination of CAT activity:
The absorbance value of CAT at 240 nm was read on a UV-visible photometer using the reaction solution without H2O2 as a control.
- Determination method of APX activity:
The enzyme activity was determined by using the principle that APX reduces the amount of ascorbic acid in the presence of H2O2, and the absorbance value at 290 nm was read on the UV-visible photometer to determine the APX activity.
The above contents are our responses to your valuable questions or suggestions. Thank you very much for your patience! Your patience and high quality advice have helped us a lot, and we have learned a lot in the process of improving my manuscript. Thank you again! We wish you good health and happy life!
Thank you and best regards.
Sincerely,
Corresponding author: Yunqi Wang
Institution and address: Beijing Forestry University
Email: [email protected]
ng author: Yunqi Wang
Institution and address: Beijing Forestry University
Email: [email protected]
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear Authors,
I reviewed your article and I found some points of the article which are not clearly presented. Please see the attached file below for detailed comments.
Comments for author File: Comments.docx
Author Response
Response letter
Dear Editor and Reviewers,
On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript. We feel great thanks for your professional review work on our manuscript. We use this feedback to improve the quality of the manuscript. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous manuscript, and presented it in revised mode in the manuscript. Furthermore, the point-to-point responses to the reviewer's comments are provided below. We hope that these revisions successfully address your concerns and requirements. Should you have any questions, please contact us without hesitate. Looking forward to hearing from you soon.
Response to Reviewer 2 Comments
Point 1:
The manuscript focuses the effect of acid rain on three different tree species. The main idea is good and as well as important, but the article contains some logically mistakes. These may come from that the important information are not well describe. These are making the article difficult to follow. So, my suggestion is to explain the highlighted sentences for better understanding.
Response 1:
Thank you for your careful review of the manuscript. Your affirmation of the article topic is our motivation to continue our in-depth research. We have revised the article based on your suggestions to increase the readability of the article. Our responses to each of your suggestions are presented below. Thank you again for your valuable suggestions on the manuscript.
Point 2:
Specific comments:
Abstract: The first statement is hard to imagine at first reading. Does natural precipitation increase chlorophyll content? Is this the main finding of the article? This is very strange in a Q1 journal. I would rather focus on the fact that moderate acidity rain has not caused significant damage.
Response 2:
We sincerely appreciate the valuable comments. This study sets up natural rainfall treatment (NR). By simulating acid rain with different acidity, we found that compared with other experimental groups, the physiological and biochemical indexes of plants under the natural rainfall treatment group (NR) were higher. This result is the highlight of the article. The relevant data showed that the natural rainfall pH of the test site is 5.42-5.83 (described in Article 2.3.1), that is, slightly acidic rain, Therefore, as you think, moderate acid rain can promote the growth of plant indicators. Thank you again for your question, and the authors have revised the wording in the abstract (in the row of 19) to increase the readability of the article.
Point 3:
Specific comments:
Introduction is well written; it contains relevant information the effect of acid rainfall. However, it is not so clear for me, why the basic hypothesis is that the slightly acidic rain improves the growth of plant seedlings if the whole introduction is about how acid rain is harmful.
Response 3:
Thank you for your careful review. We think this is an excellent suggestion. Our topic is the effect of pH changes of acid rain in the region on plants. Therefore, in the introduction, we introduce the effect of acid rain on plants. Based on your suggestions, we have reorganized the introductory section of the article to present the positive and negative effects of acid rain on plants, respectively (in the row of 80). Thus, it is pointed out that acid rain acidity is an important factor affecting all indicators of plant seedlings.
Point 4:
Material and methods:
In 97. Row: “The soil types are rich”. And yellow soil, yellow-brown soil, brown soil. These expressions are not a good sound of the scientific terminology. There are many soil classification methods, I very suggest using that type of expressions. For example, French Soil Reference System or World Reference Base for Soil Resources.
Response 4:
Thank you for your valuable comments. We think this is an excellent suggestion. The authors have re-described soil types by consulting the World Reference Base (IUSS Working Group WRB, 2022), as described in section 2.1 of the article. We have changed yellow soil into Cambisols, Cambisols into Cambisols, brown soil into Luvisols, paddy soil into Anthrosols, and Cambisols into Cambisols (in the row of 131 to 133).
[1] IUSS Working Group WRB. 2022. World Reference Base for Soil Resources. International soil classification system for naming soils and creating legends for soil maps. 4th edition. International Union of Soil Sciences (IUSS), Vienna, Austria.
Point 5:
Material and methods:
In 114-115: You mentioned that the soil in the pots was yellow soil with the pH 5.37±0.1. However, in the row of 98-99, you wrote that the yellow soil usually has 3.5-4.5 pH. How to explain this?
Response 5:
Thanks for your careful checks. As you think, the pH of the soil used in the experiment was 5.37 ± 0.1. The Cambisols (yellow soil) with pH 3.5-4.5 introduced in 2.1 Study area profile is the soil of the Three Gorges reservoir area. The following literature is cited “Zheng, Y.; Wang, Y.; Zheng, Y.; Li, Y. Effects of Simulated Acid Rain on Soil Enzyme Activity and Related Chemical Indexes in Woodlands. Forests 2022, 13, 860. https://doi.org/10.3390/f13060860”. But the soil used in this experiment originated from Jinyun Mountain in Chongqing, there are some differences between them. Thank you again for your careful review. In order to better introduce the soil profile of the study area, we corrected the original description and restated the soil profile of the study area (Jinyun Mountain). The specific corrections are as follows. The pH range of the soil is 4.5-5.5in Jinyun Mountain (in the row of 134). The following literature is cited “Acid Deposition Monitoring Network in East Asia. https://monitoring.eanet.asia/document/public/index. 2021”.
Point 6:
Figure 1. You describe the geographical location of the study area. But the second picture would be better placed later, because it displays information about the research design, not the location.
Response 6:
Thank you for your valuable comments. The authors moved the second image of Figure 1 from Figure 1 to Figure 2.
Point 7:
Experimental design:
Why has NR treatment been used? Where did they get the NR irrigation water from if NR was also under the shelter? Or were the NR pots located outside the shelter and watered by natural rainfall? This is not clearly written part. Please specify it.
Response 7:
Thank you for your careful review. For your first question, our responses are as follows. Previous simulation experiments only set acid rain with different acidity and concluded that acid rain with different acidity affects plants. In this experiment, natural rainfall (NR) was taken into account and compared with other experimental groups to evaluate whether plants in the study area could adapt to the existing natural environment, so as to reveal the impact of acid rain on the ecological environment. Therefore, the natural rainfall (NR) set up in this article is the highlight of the article.
For your second question, our responses are as follows. To ensure that all plant growth could receive all environmental factors (light, humidity, temperature, etc.) in the study area. A temporary rain shelter consisting of stainless steel with a sliding roof was built at the experimental site, and used it for shade adjustment according to climatic conditions. In case of rain during the experiment, the natural rainfall (NR) was placed in an open environment to receive natural rainfall drenching, while the pH 4.5, 3.5, 2.5 and CK treatments were placed in a rain shelter, using the shelter to shield the natural rainfall, with ventilation around the shelter without shade to ensure plant growth in environmental conditions other than natural rainfall. If there was no rainfall during the experiment, the shelter was pulled open to simulate the field environment. As suggested by the reviewer, we have provided additional descriptions in 2.2 Experimental Materials (in the row of 139 to 144).
Point 8:
Spray application:
This part is about everything BUT not about the application timing and amount. It is not clear whether the water was used as an irrigation or a foliar application of acid water?
Response 8:
Thank you for your careful review. This experiment uses the high spray method of the sprinkler system to create the effect of spraying to maximize the simulation of the natural form of rainfall, i.e., the top-down rainfall pattern. We have added a note in section 2.3.2 of the paper (in the row of 186 to 187). The specific corrections are as follows. The spray was applied to the top of the plants to distinguish the degree of stress resulting from different acidities and simulate the natural rainfall pattern.
Point 9:
Timing.
The plants were transplanted on 14th of March (112. Row), and the seedlings were placed under the rain shelter for 1.5 months (132. Row). The experiment started from May (135. Row). This means that the plants are put out of the rain shelter. If this happened how to prevent the plants from natural rainfall? But if you leave them under the shelter than the sentence in 132nd row is false. Please correct this part.
Response 9:
Thank you for your careful review. In this experiment, the test seedlings were placed in a temporary stainless steel rain shelter to slow down for 1.5 months before the simulated acid rain. The acid rain simulation experiment was formally started in May, when all treatments were placed in the open air, and the spraying volume was simulated with different acid rain gradients according to the monthly rainfall and pattern of Jinyun Mountain for many years (at the beginning of the experiment, there was no natural rainfall). In case of rain, the pH 4.5, 3.5, 2.5 and CK treatments were placed in a rain shelter, using the shelter to shield them from natural rainfall, while the natural rainfall treatment (NR) was placed in an open environment to receive natural rainfall drenching. Therefore, the pH 4.5, 3.5, 2.5 and CK treatments were not affected by natural rainfall.
Point 10:
Table 2:
it is not clear this amount of water is the precipitation amount or irrigated/spraying amount. The footnote of the table is too short. Self-explanatory figures must be clear and easy to understand! Pleas modify it.
Response 10:
Thank you for your careful review. The data in Table II are monthly simulated acid rain sprays, which were calculated based on multi-year monthly surface rainfall and patterns in the study area. This experiment follows the data in the table for the monthly simulated spraying of plants. Thank you for your care in pointing out the problem, which we have added in the manuscript (in the row of 194 to 195).
Point 11:
Sample collection:
It is also not clear when did take the measurements. Because you wrote the application of acid water were once a week for four months (138th row). But in the 2.4.1 parts you did not mention the time of photosynthetic measurements. Were there only two SPAD measurements? And in the case of enzyme activity?
Response 11:
Thank you for your careful review. As you believe, acid rain is sprayed once a week for 4 months (i.e. May-August). The measurement of indicators was started in September, and the photosynthetic indicators were measured in the morning (8:00-11:00) on September 3, 2021 (windless and sunny), and the relative chlorophyll content was measured before (May 1) and after (September 4) the simulation experiment, i.e., once in May and twice in September. The plant samples were collected on September 6 for the determination of other biochemical indicators such as antioxidant enzyme activity were measured. All indicators were measured in September. Thank you again for your question, which we have added in section 2.4 (in the row of 198, 232, 233, 238).
Point 12:
Results:
In Figures and Tables, you don't use letters consistently for significance levels. You add ‘a’ to the highest value and sometimes ‘a’ indicates the lowest values. Please choose one way and use consistently.
Response 12:
Thank you for your careful review. We have rechecked the labeling of all significance levels and explained the meaning of the letters in the figure notes (in the row of 284, 285, 318, 319, 326 and 327).
Point 13:
Discussion and Conclusion:
In 428: “The NR and low acidity treatments improved the growth and development of the plants” But you did not measure the growth parameters. Please correct the sentence.
This statement above is very dangerous, because you did not clarify when did you applied the treatment and when did you took the measurements. The photosynthetic activity and the level of the antioxidant enzymes are influenced by a lot of environmental factors and time also. So, if you measured the parameters only at the end of the experiment (in September), you cannot make this kind of summary. In this case you have to rewrite the whole Result and discussion.
Response 13:
Thank you for your careful review. We have corrected the inappropriate language in the text and have marked it with a revision pattern in the conclusion section (in the row of 529 and 530). The sentence has been changed to " The NR and low acidity treatments enhanced the physiological and biochemical characteristics of the plants. "
The specific schedules of this experiment are as follows. Acid rain was sprayed once a week for 4 months (i.e., May-August). The measurements of the indicators were started in September, and photosynthetic indicators were measured in the morning (8:00-11:00 am) on September 3, 2021 (windless and sunny), relative chlorophyll content was measured before and after the simulation experiment, i.e., once on May 1 and twice on September 4. The plant samples were collected on September 6 for the determination of biochemical indicators (antioxidant enzyme activity, malondialdehyde, soluble sugars, soluble proteins, proline and starch). All indicators were measured in September.
At the beginning of the experimental design, we reviewed a large amount of literature, and through our reading, we found that measuring the parameters at the end of the experiment could reflect the response of the plant to the stressful environment. For example, [1] Li, D.; Guo, L.; Tian, M.; Miao, L.; Xiang, L.; Yang, F. Effects of Waterlogging Stress on the Neighboring Relationships between Cleistocalyx operculatus (Roxb.) Merr. and Dalbergia odorifera T. Chen Saplings. Forests 2023, 14, 377. https://doi.org/10.3390/f14020377. [2] Sun, Z.; Yin, Y.; Zhu, W.; Zhou, Y. Morphological, Physiological, and Biochemical Composition of Mulberry (Morus spp.) under Drought Stress. Forests 2023, 14, 949. https://doi.org/10.3390/f14050949. [3] Lei, X.; Wu, H.; Yin, M.; Zhang, X.; Yang, H.; Huang, X.; Zhu, P. Comparative Evaluation of Physiological Response and Drought Tolerance between Cunninghamia unica and C. lanceolata Seedlings under Drought Stress. Forests 2023, 14, 464. https://doi.org/10.3390/f14030464.
All of the above-mentioned literature measured physiological, biochemical, and other indicators at the end of the experiment and derived the results that environmental stress changed the physiological and biochemical characteristics of plants, thus evaluating the mechanisms by which plants face stress and providing basic data for subsequent studies. At the same time, the experiment was designed to strictly control the consistency of variables, except for the acid rain treatment, to ensure that plants in each treatment group received consistent other environments (light, humidity, temperature, etc.) to the greatest extent possible, and the experimental treatment time was consistent, so that the influence of environmental factors and time on each index was maximally eliminated. Therefore, the present experiment was able to conclude that NR and low acidity acid rain enhanced the physiological and biochemical indices of plants.
The above contents are our responses to your valuable questions or suggestions. Thank you very much for your patience! Your patience and high quality advice have helped us a lot, and we have learned a lot in the process of improving my manuscript, Thank you again! We wish you good health and happy life!
Thank you and best regards.
Sincerely,
Corresponding author: Yunqi Wang
Institution and address: Beijing Forestry University
Email: [email protected]
Author Response File: Author Response.docx
Reviewer 3 Report
The research topic addressed by the authors is one of interest nowadays when in many places around the world there is an increasing presence of acid rain, due to the existing pollution. In the present paper, researchers evaluated the photosynthetic, physiological, and biochemical traits of plants by simulating acid rain at various pH levels. Plant photosynthesis was impacted by the simulated acid rain by altering physiologic and biochemical processes. However, the evaluated period is rather brief given the life duration of individuals from the three species tested, as the authors have already pointed out as one of the study's limitations. Because aging and consequently the physiological changes that take place at the individual level might have a considerable impact on the results, I advise the authors to extend the research period to include a longer time of the species investigated. Nevertheless, I think the study can be used as a starting point for more extensive investigation. Finally, I kindly request that the authors thoroughly review again the manuscript after the authors' guide.
Author Response
Response letter
Dear Editor and Reviewers,
On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript. We feel great thanks for your professional review work on our manuscript. We use this feedback to improve the quality of the manuscript. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous manuscript, and presented it in revised mode in the manuscript. Furthermore, the point-to-point responses to the reviewer's comments are provided below. We hope that these revisions successfully address your concerns and requirements. Should you have any questions, please contact us without hesitate. Looking forward to hearing from you soon.
Response to Reviewer 3 Comments
Point 1:
The research topic addressed by the authors is one of interest nowadays when in many places around the world there is an increasing presence of acid rain, due to the existing pollution. In the present paper, researchers evaluated the photosynthetic, physiological, and biochemical traits of plants by simulating acid rain at various pH levels. Plant photosynthesis was impacted by the simulated acid rain by altering physiologic and biochemical processes. However, the evaluated period is rather brief given the life duration of individuals from the three species tested, as the authors have already pointed out as one of the study's limitations. Because aging and consequently the physiological changes that take place at the individual level might have a considerable impact on the results, I advise the authors to extend the research period to include a longer time of the species investigated. Nevertheless, I think the study can be used as a starting point for more extensive investigation. Finally, I kindly request that the authors thoroughly review again the manuscript after the authors' guide.
Response 1:
Thank you for your careful review of the manuscript. As you believe, the evaluated period is rather brief given the life duration of individuals from the three species tested, it is one of the limitations of the study. Because aging and consequently the physiological changes that take place at the individual level might have a considerable impact on the results, it is necessary to extend the study period. However, we found the same opinion as yours from reading the literature when designing our experiments that short duration simulations can also reflect the effects of stress on plants to some extent. For example, [1] Debnath, B., Irshad, M., Mitra, S. et al. Acid Rain Deposition Modulates Photosynthesis, Enzymatic and Non-enzymatic Antioxidant Activities in Tomato. Int J Environ Res 12, 203–214 (2018). https://doi.org/10.1007/s41742-018-0084-. [2] Zhang, C.Y.; Yi, X.Q.; Gao, X.Z.; Wang, M.H.; Shao, C.Y.; Lv, Z.D.; Chen, J.J.; Liu, Z.H.; Shen, C.W. Physiological and biochemical responses of tea seedlings (Camellia sinensis) to simulated acid rain conditions. Ecotoxicol. and Environ. Saf. 2020, 192, 110315. DOI: 10.1016/j.ecoenv.2020.110315. Thus, it can be used as a starting point for more extensive investigation. Meanwhile, the following conclusions are obtained from the study: A comprehensive comparison of the indicators revealed that NR and mild acid rain enhanced the plant seedlings' physiological and biochemical characteristics. A pH of 3.5 was the threshold where acid rain had an adverse effect on Pinus massoniana, Phyllostachys edulis, and Cinnamomum camphora. The high indicator values for NR indicate that these tree species have adapted to current conditions in the Jinyun Mountain area of Chongqing. These results of this manuscript provide new information for selecting tree species adapted to the acid rain environment in Jinyun Mountain, Chongqing. This limitation will be taken into account in future studies of our group in order to obtain more in-depth results. Also, we have checked the manuscript again based on your suggestions and would appreciate if you could review it again.
The above contents are our responses to your valuable questions or suggestions. Thank you very much for your patience! Your patience and high quality advice have helped us a lot, and we have learned a lot in the process of improving my manuscript, Thank you again! We wish you good health and happy life!
Thank you and best regards.
Sincerely,
Corresponding author: Yunqi Wang
Institution and address: Beijing Forestry University
Email: [email protected]
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors carefully improved the manuscript as per suggestions.
Recommended for its publication in the journal - Forests.
