The Change in Growth, Osmolyte Production and Antioxidant Enzymes Activity Explains the Cadmium Tolerance in Four Tree Species at the Saplings Stage
, Fahad Rasheed 1,*, Waseem Razzaq Khan 3,4, Muhammad Mohsin 5, Muhammad Zahid Rashid 6, Mohamad Maulana Magiman 7, Zohaib Raza 1, Zamri Rosli 4, Shazia Afzal 8 and Fauziah Abu Bakar 9,*
Round 1
Reviewer 1 Report
Except for the Latin spelling errors of the plant species, there is no deficiency in the article. It is shown at attachement.
Populus × canadensis, known as Canadian poplar or Carolina poplar, is a naturally occurring hybrid of Populus nigra and Populus deltoides.nThis is must be explain in the text.
Populus × canescens, the grey poplar, is a hybrid between Populus alba (white poplar) and P. tremula (common aspen). It is not compare with P. x canadensis !
Comments for author File: 
Comments.pdf
Author Response
Response: Thank you for appreciating our work. The suggestions have been incorporated in the revised version of the manuscript.
Reviewer 2 Report
Dear Corresponding Author
Your paper is about phytoremediation and it is well-developed manuscript but please check again and correct some spelling errors such as italic names and spaces between 200mM, for instance.
Regards
Author Response
Response: Thank you for appreciating our work. The inconsistencies have been removed in the revised version of the manuscript.
Reviewer 3 Report
The manuscript entitled “The nexus between growth, osmolyte production and antioxidant enzymes activity explains the cadmium tolerance in four tree species at saplings stage” presents data on Cd uptake, ROS accumulation, contents of total amount of major metabolites and the activity of antioxidant enzymes upon treatment of young tree seedlings with cadmium chloride. The paper aims at indicating the woody species with a high potential for utilization as a phytoextractor of Cd showing the highest tolerance among the four species analyzed. The results are interesting, however the manuscript have several major drawbacks and should be greatly adjusted before reconsideration for publication in the Forests journal. Major issues were listed as follows:
11. Title: is an overestimation of the results since “the nexus” was not evaluated with statistical methods, e.g. PCA. Additional analysis is needed or change the title to more appropriate to the paper contents
22. Abstract: provide the age of plants in abstract and type of inorganic salt used for treatment
33. Introduction: describe the species of interest, describe the level of soil pollution with Cd and plant tolerance levels
44. Methods: provide details for chemical analyses, suppliers and names of instrumentation
55. Results: Add PCA or other analysis to evaluate the relations between investigated traits
66. Discuss results with woody species
77. Discussion repeats partly the results section, can be combined? Do not address figures and tables in discussion section
88. Lack of Conclusion section
Whole text:
19. The language should be checked because of plenty of errors that lower the value of the manuscript can be found
210. Change “contamination” to the word “pollution”
111. Change “better tolerance” to “higher”
412. Avoid repeating the parts of sentences
513. Distinguish metal concentration (for solutions), content (for tissue accumulation or level in soil)
614. Do not use capital c in the word cadmium
715. Plants do not absorb heavy metals rather uptake
816. The overproduction of ROS caused damage of membranes and further electrolyte leakage
917. MDA is not an antioxidant
118. Use term “Cd treatment” instead of “Cd stress” throughout the manuscript since stress is the effect of treatment and is different for the investigated species
119. Use simple past and simple present tenses where possible
120. 2.1. Plant material and growth conditions
121. Indicate dimeter and height of pots
122. 2.2 line 118-121 must be removed it’s basic chemical knowledge
123. Change plant sections to organs
124. How was the tissue homogenized or just “mixed” as written? Was it frozen when terminated in liquid nitrogen?
125. The authors measured the activity of antioxidant enzymes rather than concentration (this error occurs multiple times)
126. Move TF calculation from 2.8 to 2.5 subsection
127. Was TF calculated as shoot-to-root or leaf-to-root ratio? Was it average? If so the biomass of organs was taken into calculations? Provide formula in text
228. In results subsections remove “Cd stress” form titles or replace with “treatment”
229. Why authors performed post-hoc analysis for SxT effect if it was not significant in case of about half of parameters?
230. Put “n” value in figure captions and table legends
231. Remove italic for Fig 2 caption
232. Use figures for all investigated traits instead of tables
233. Divide biomass parameters from pigment contents into two figures, the same with ROS and Cd accumulation
234. Keep the order of figures accordingly to the text contents
Author Response
We are highly grateful for your time and suggestions to improve our manuscript. All the suggestions have been incorporated in the revised manuscript. Point wise response to the quarries and suggestions are listed below.
- Title: is an overestimation of the results since “the nexus” was not evaluated with statistical methods, e.g. PCA. Additional analysis is needed or change the title to more appropriate to the paper contents
Reponses: Thank you for pointing out. The addition of PCA can be a valuable. However, at this stage, we opted to change the title of the paper. Your suggestion will be taken in account in the future.
- Abstract: provide the age of plants in abstract and type of inorganic salt used for treatment
Response: The desired information has been added in the revised version.
- Introduction: describe the species of interest, describe the level of soil pollution with Cd and plant tolerance levels
Response: The species have been briefly introduced and the level of Cd pollution has also been added the revised version.
- Methods: provide details for chemical analyses, suppliers and names of instrumentation
Response: The desired information has been added in the revised version.
- Discuss results with woody species
Response: The suggestion has been incorporated in the revised version.
- Discussion repeats partly the results section, can be combined? Do not address figures and tables in discussion section
Response: In the discussion section, the results were recalled briefly and were related to the previous studies. The reference of the tables and figures have been removed from the discussion section.
- Lack of Conclusion section
Response: The section has been added although it is not mandatory according to the journal guidelines.
- The language should be checked because of plenty of errors that lower the value of the manuscript can be found
Response: The language has been corrected as advised.
- Change “contamination” to the word “pollution”
Response: Corrected as advised.
- Change “better tolerance” to “higher”
Response: Corrected as advised.
- Avoid repeating the parts of sentences
Response: Corrected as advised.
- Distinguish metal concentration (for solutions), content (for tissue accumulation or level in soil)
Response: Corrected as advised.
- Do not use capital c in the word cadmium
Response: Corrected as advised.
- Plants do not absorb heavy metals rather uptake
Response: Corrected as advised.
- The overproduction of ROS caused damage of membranes and further electrolyte leakage
Response: Corrected as advised.
- MDA is not an antioxidant
Response: Corrected as advised.
- Use term “Cd treatment” instead of “Cd stress” throughout the manuscript since stress is the effect of treatment and is different for the investigated species
Response: Corrected as advised.
- 2.1. Plant material and growth conditions
Response: Corrected as advised.
- Indicate dimeter and height of pots
Response: The desired information has been added in the revised version.
- 2.2 line 118-121 must be removed it’s basic chemical knowledge
Response: Corrected as advised.
- Change plant sections to organs
Response: Corrected as advised.
- How was the tissue homogenized or just “mixed” as written? Was it frozen when terminated in liquid nitrogen?
Response: Samples were homogenized in the ice bath. The discrepancy has been corrected.
- The authors measured the activity of antioxidant enzymes rather than concentration (this error occurs multiple times)
Response: Corrected as advised.
- Move TF calculation from 2.8 to 2.5 subsection
Response: Corrected as advised.
Was TF calculated as shoot-to-root or leaf-to-root ratio? Was it average? If so the biomass of organs was taken into calculations? Provide formula in text
Response: Concentration of Cd in leaves, stem and roots were determined and TF was calculated as the ratio between Cd concentration in leaves+stem and roots. The total biomass of each organ was not taken into the calculation.
- In results subsections remove “Cd stress” form titles or replace with “treatment”
Response: Corrected as advised.
- Why authors performed post-hoc analysis for S xT effect if it was not significant in case of about half of parameters?
Response: The interaction effect was a part of the model where two factors were taken as fixed effect. Post-hoc was done to evidence the trend between each species.
- Put “n” value in figure captions and table legends
Response: The desired information has been added in the revised version.
- Remove italic for Fig 2 caption
Response: Corrected as advised.
- Use figures for all investigated traits instead of tables
Response: If we make figures for all the measured traits, the number of figures will increase way too much. Therefore, a mix of tables and figures were opted.
- Keep the order of figures accordingly to the text contents
Response: Corrected as advised.
Round 2
Reviewer 3 Report
The manuscript was partly adjusted to comments.
The text should be carrfully chacked for editing as well as language errors.
Still a few important issues needs correction:
1. distingush the content (in tissue, in soil) and concentration (in solution). I think authors may confuse the concentration which is measured in solution in AAS with the content of metal in tissue which is finally caluculated as a result
2. Why the sum of metal content in leaves and shoots is used fot TF calculation? Please explain or give literature referance. Why not to use mean value?
3. Do not mix physiological data with cadmium accumulation in one table.
4. Do not perform post-hoc test for not significant effect according to anova. Just show mean values and SE in chart/table. It's a serious methodological error. Needs correction.
Author Response
Thank you once again for your comments and suggestions for the improvement of our manuscript.
The manuscript was partly adjusted to comments.
The text should be carefully checked for editing as well as language errors.
Response: The text was checked for language errors.
Still a few important issues need correction:
- distinguish the content (in tissue, in soil) and concentration (in solution). I think authors may confuse the concentration which is measured in solution in AAS with the content of metal in tissue which is finally calculated as a result
Response: The error has been corrected throughout the manuscript as indicated.
- Why the sum of metal content in leaves and shoots is used fot TF calculation? Please explain or give literature reference. Why not to use mean value?
Response: The reference of the method has been added in the revised version.
- Do not mix physiological data with cadmium accumulation in one table.
Response: Technically, the suggestion is correct but to reduce the number of tables and figures we had to present both datas in a single table.
- Do not perform post-hoc test for not significant effect according to anova. Just show mean values and SE in chart/table. It's a serious methodological error. Needs correction.
Response: The correction has been made in both tables as suggested.
