Morphological and Structural Responses of Albizia lebbeck to Different Lead and Nickel Stress Levels
, Zafran Khan 1, Nimra Gul 1, Iram Ijaz 4, Amir Bibi 1, Soha Pervaiz 5,†, Hesham F. Alharby 6, Muhammad Sayyam Tariq 5, Syed Riaz Ahmed 1,5,†, Khalid Rehman Hakeem 6,* and Daniel K. Y. Tan 7,*
Round 1
Reviewer 1 Report
This manuscript attempts to study the response of Albizia lebbeck, a plant that can potentially be used in soil phytoremediation, to stresses induced by increasing Pb or Ni levels. My main concern lies in the experimental design, for which a better explanation is required in the revised version.
1) How were the concentrations of Pb and Ni selected? Though the authors claim in section 2.1 that these concentrations are environmentally relevant by citing ref 23-25, I did not find convincing evidence in these references.
2) Were there repetitions in the anatomical study? Some micrographs are not clear. For example, cells are not distinguishable in Figures 6B and 6G. The section shown in Figure 6D may contain several layers of cells.
3) Given the unclear micrograph, how reliable are the values shown in Tables 1-2?
4) To compare the anatomy data among metal treatments (Tables 1-2), it is necessary to exclude the impact of other factors on the data. For example, the position or developmental stage of cryosections made on an organ could impact the parameters shown in Tables 1-2.
Besides experimental issues,
5) In the Introduction, a reference is required to support the sentence that "Lead and nickel are the most widespread heavy metals..."
6) The plants were treated by either Pb or Ni stress. There was no treatment mixing Pb and Ni. Therefore, "Pb and Ni treatment/stress" is misleading and should be revised to "Pb or Ni treatment/stress".
Author Response
Manuscript ID – Agriculture-2411542  
Title: Morphological and Structural Responses of Albizia lebbeck to Different Lead and Nickel stress levels
Thank you so much for the comments and suggestions. Each of your insights has served to strengthen our manuscript and we have made changes to reflect them. We have addressed the reviewers’ comments and updated the information in the new manuscript. Below is the point-by-point response to the comments outlined in the new manuscript.
Reviewer 1
This manuscript attempts to study the response of Albizia lebbeck, a plant that can potentially be used in soil phytoremediation, to stresses induced by increasing Pb or Ni levels. My main concern lies in the experimental design, for which a better explanation is required in the revised version.
The introduction part of the manuscript has been further improved by adding more information that will help the readers to better understand the theme of the manuscript and the importance of Pb and Ni. About 30 more relevant references have been added to the manuscript to improve the relevance of the citations. Moreover, the methodology, results, and conclusion portions have also been improved by making relevant changes.
Point 1: How were the concentrations of Pb and Ni selected? Though the authors claim in section 2.1 that these concentrations are environmentally relevant by citing ref 23-25, I did not find convincing evidence in these references.
Response: The presence of heavy metals in Pakistani soil is of great concern and has become an important issue here in Pakistan. The concentration of Pb and Ni across Pakistan (soil) varies greatly depending upon the region and city ranging from 0.01-150 mg kg-1 (Pb) and 0.02-500 mg kg-1 (Ni). The shortage of surface and groundwater in Pakistan has now forced farmers to use wastewater as a source of irrigation which contains abundant amounts of heavy metals. This use of wastewater has enhanced the concentration of these metals even more. Ni is present up to several thousands of mg kg-1 in serpentine soil worldwide. A few studies have even reported the presence of up to 2000 mg kg-1 Pb in soil. So keeping in view this scenario we decided to select a wide of concentrations for these metals to cover a wide range of these metals in Pakistan and worldwide. Moreover, we have also added new references (besides, 23-25, 75, 76,77,78,79) that have used both Pb and Ni from 50 mM to 150 mM.
Point 2: Were there repetitions in the anatomical study? Some micrographs are not clear. For example, cells are not distinguishable in Figures 6B and 6G. The section shown in Figure 6D may contain several layers of cells.
Response: Yes, there were three replicates in the anatomical study. A total of three anatomical sections were made from each plant organ (root, stem, and leaf). A 2 cm piece of the thickest root (immediately after cutting the root cap), a 2 cm piece of stem (uppermost part), and a 2 cm piece of leaf from the leaf base were taken for sectioning. Figures 6B and 6G have been replaced with other micrographs that clearly distinguished the cells. Moreover, we have also attached several micrographs in the supplementary files as well. Figure 6D is a single layer and does not contain several layers. This was confirmed by Prof. Dr. Mansoor (research interest plant anatomy, University of Agriculture Faisalabad, Pakistan) and Dr. Mohsin (Associate Professor, research interest plant anatomy, University of Agriculture Faisalabad, Pakistan).
Point 3: Given the unclear micrograph, how reliable are the values shown in Tables 1-2?
Response: The values are reliable as they are the mean of three replicates (measured from sections of each replicate) using different micrographs. Moreover, the values were measured with utmost care and precision to avoid any type of experimental and human error. After analysis, the recorded values were checked and analyzed by experts in the field along with anatomical micrographs. We recorded the values for all the experiments (many of these have already been published in journals like Environmental and Experimental Botany, and Environmental Pollution, and many are on the way to being published) in the same way as for this article.
Point 4: To compare the anatomy data among metal treatments (Tables 1-2), it is necessary to exclude the impact of other factors on the data. For example, the position or developmental stage of cryosections made on an organ could impact the parameters shown in Tables 1-2.
Response: Yes you are absolutely right, all other factors that could lead to experimental error were controlled and the sections for each organ ( in each replicate) were made from the same developmental stage and the same plant organ. We have calculated the mean from a total of three anatomical sections made from each plant organ (root, stem, and leaf). A 2 cm piece of the thickest root (immediately after cutting root cap), a 2 cm piece of stem (uppermost part), and a 2 cm piece of leaf from the leaf base were taken for sectioning as detailed in the methodology section of the manuscript.
Point 5: In the Introduction, a reference is required to support the sentence that "Lead and nickel are the most widespread heavy metals
Response: Two references (81,82) have been added to support this sentence.
Point 6: The plants were treated by either Pb or Ni stress. There was no treatment mixing Pb and Ni. Therefore, "Pb and Ni treatment/stress" is misleading and should be revised to "Pb or Ni treatment/stress".
Response: Thank you for pointing out this error. We have made the changes accordingly throughout the manuscript as you suggested.
Regards,
Dr. Prof. Daniel KY Tan
Dr. Khalid Rehman Hakeem
Dr. Amir Bibi
Author Response File: 
Author Response.docx
Reviewer 2 Report
A lot of work has been done, a large amount of data has been processed, but some points require clarification, and possibly improvement.
1. Sources 24-25, indicated in the rationale for the choice of concentrations of lead and nickel solutions cannot explain the choice of concentrations. (p. 4: These concentrations are outcomes of comprehensive screening experiments and are also environmentally relevant [23-25].)
2. In the section where the model experiment is described, it is necessary to give characteristics (preferably the elemental composition, but at least the acidity and humus content) of the soils on which the experimental plants were grown.
3. Nowhere in the text of the article is it indicated how many anatomical sections were made from each organ of the experimental plants (root, stem, leaf) and in which part of the root and stem the anatomical sections were made. How many anatomical sections were analyzed statistically?
4. Figure 2D, 2C - is the biomass of a shoot or a whole plant with a root system indicated?
5. How could the number of leaves decrease in the 2nd phase of the experiment (as the plant grows) in comparison with the first one at a nickel nitrate concentration of 75 mM (Fig. 3B)?
What was the age of the plants from the beginning of the emergence of seedlings, if, according to the experiment data, they formed up to 20 or more leaves in the 1st phase of the experiment? (fig. 3 B) (if these were plants under the age of 1 month - it's hard to believe)...
6. How to explain the decrease in the length of leaflets and leaflet width as the plant grows (from phase 1 to phase 2) shown in fig. 4 A (nickel nitrate concentration 75 mM), fig. 4 B (lead nitrate concentration 50-75 mM; nickel nitrate control, and concentration 25 mM). On the leaves of which tier were the measurements taken? Is an experimental error possible (for example, if both middle-tier leaves and young growing ones were taken for measurement)?
7. How to explain the decrease in the area of leaflets presented in fig. 4 C in lead nitrate concentrations of 50 and 75 mM; in a nickel nitrate concentration of 25 mM from the first phase to the second?
8. Figure 6 F, G is fuzzy - does not give an idea of the size of cells and tissues. It must be replaced or removed. Rice. 6 H does not give insight into the structure of the epidermis. The guard cells of the stomata are not visible.
9. In fig. 6 B, C, D, E, F, G, H (or captions to them) there is no magnification of the microscope at which the photos were taken.
10. What do the letters ef, cd, bc, ab, c, etc. in tables 1 and 2 and above columns in figures 3, 4 and 5 mean?
Author Response
Manuscript ID – Agriculture-2411542  
Title: Morphological and Structural Responses of Albizia lebbeck to Different Lead and Nickel stress levels
Thank you so much for the comments and suggestions. Each of your insights has served to strengthen our manuscript and we have made changes to reflect them. We have addressed the reviewers’ comments and updated the information in the new manuscript. Below is the point by point response to the comments outlined in the new manuscript.
Reviewer 2
A lot of work has been done, a large amount of data has been processed, but some points require clarification, and possibly improvement.
Thirty more relevant references have been added to the manuscript to improve the relevance of the citations. Moreover, the methodology and result portions have also been improved by making relevant changes.
Point 1: Sources 24-25, indicated in the rationale for the choice of concentrations of lead and nickel solutions cannot explain the choice of concentrations. (p. 4: These concentrations are outcomes of comprehensive screening experiments and are also environmentally relevant [23-25].)
Response: The presence of heavy metals in Pakistani soil is of a great concern and has become an important issue here in Pakistan. The concentration of Pb and Ni across Pakistan (soil) varies greatly depending upon the region and city ranging from 0.01-150 mg kg-1 (Pb) and 0.02-500 mg kg-1 (Ni). The shortage of surface and ground water in Pakistan has now forced farmers to use wastewater as source of irrigation which contains abundant amounts of heavy metals. This use of wastewater has enhanced the concentration of these metals even more. Even through, Ni is present up to several thousands of mg kg-1 in serpentine soil worldwide. Few studies have even reported the presence of up to 2000 mg kg-1 Pb in soil. So keeping in view this scenario we decided to select a wide range of concentrations for these metals to cover a wide range of these metals in Pakistan and worldwide. Moreover, we have also added new references (besides, 23-25, 75, 76,77,78,79) that have used both Pb and Ni from 50 mM to 150 mM.
Point 2: In the section where the model experiment is described, it is necessary to give characteristics (preferably the elemental composition, but at least the acidity and humus content) of the soils on which the experimental plants were grown.
Response: Dear reviewer, thank you for your valuable suggestions. We have updated the information in the new manuscript to “All pots were uniformly filled with soil containing 60% sandy soil and 40% loamy soil (containing organic matter (0.74%), nitrogen (0.73%), phosphorus (8.1 mg kg-1), potassium (169 mg kg-1), electrical conductivity (EC 2.84 dS.m-1) and pH (7.60))”.
Point 3: Nowhere in the text of the article is it indicated how many anatomical sections were made from each organ of the experimental plants (root, stem, leaf) and in which part of the root and stem the anatomical sections were made. How many anatomical sections were analyzed statistically?
Response: We have updated the information in the revised manuscript as “A total of three anatomical sections were made from each plant organ (root, stem, and leaf). A 2 cm piece of the thickest root (immediately after cutting root cap), a 2 cm piece of stem (upper most part), and 2 cm piece of leaf from the leaf base were taken for sectioning”. Moreover, there were three replications from each organ (root, stem and leaf) and three sections (R1, R2 and R3) were made for each concentration of Pb and Ni stress and mean anatomical sections data of three replicates was statistically analysed.
Point 4: Figure 2D, 2C - is the biomass of a shoot or a whole plant with a root system indicated?
Response: Yes, Figure 2D, 2C indicates a whole plant biomass g plant-1 (root + shoot).
Point 5: How could the number of leaves decrease in the 2nd phase of the experiment (as the plant grows) in comparison with the first one at a nickel nitrate concentration of 75 mM (Fig. 3B)?
What was the age of the plants from the beginning of the emergence of seedlings, if, according to the experiment data, they formed up to 20 or more leaves in the 1st phase of the experiment? (fig. 3 B) (if these were plants under the age of 1 month - it's hard to believe)
Response: Thank you for pointing out the human data processing error. We checked the statistical data analysis and graph generated file for corrections and discovered that during bar graph generation we mistakenly put double “..” between the values that made errors and changes the values during graph generation. We checked all the graphs files again carefully and generated new graphs to correct all errors. The actual numbers of leaves per plant were ranging between 11 to 14 and the graph shows the mean values of the replicates.
Note: Statistical data analysis files were also re-checked and data were statistically analysed again.
Point 6: How to explain the decrease in the length of leaflets and leaflet width as the plant grows (from phase 1 to phase 2) shown in fig. 4 A (nickel nitrate concentration 75 mM), fig. 4 B (lead nitrate concentration 50-75 mM; nickel nitrate control, and concentration 25 mM). On the leaves of which tier were the measurements taken? Is an experimental error possible (for example, if both middle-tier leaves and young growing ones were taken for measurement)?
Response: We made a human error during graph formation. We made corrections to Fig 4A (nickel nitrate concentration 75 mM). Moreover, the reduction in leaflet width at 50 and 75 mM Pb was due to the severe stress due to drying of leaves edges after 14 days and leaves started shedding. We had mentioned in section 3.1 that “After 14 days of heavy metal stress, the symptoms were evident; including chlorotic circle spots on leaves and dryness of leaves edges”. Therefore, at the time of measurements, the width of leaves got reduced due to shedding. Moreover, the data shown in Fig 4B is the mean of three replications where plants were more badly affected by stress and showed more reduction in width. The same was the case observed with 25 mM of Ni. Whereas in control; we had made an error which has been corrected.
Point 7: How to explain the decrease in the area of leaflets presented in fig. 4 C in lead nitrate concentrations of 50 and 75 mM; in a nickel nitrate concentration of 25 mM from the first phase to the second?
Response: Due to the reduction in leaflet width at 50 and 75 mM Pb, and 25 mM Ni; the leaflet area was reduced because the leaflet area is calculated by multiplying leaflet length with leaflet width (L x W = A),
Where L is length, W is width and A is Area.
Point 8: Figure 6 F, G is fuzzy - does not give an idea of the size of cells and tissues. It must be replaced or removed. Rice. 6 H does not give insight into the structure of the epidermis. The guard cells of the stomata are not visible.
Response: Figure 6F, 6G and 6H have been replaced with new micrographs which are clear and not fuzzy.
Point 9: In fig. 6 B, C, D, E, F, G, H (or captions to them) there is no magnification of the microscope at which the photos were taken.
Response: The scale at which micrographs were taken has mentioned on each figure in the new manuscript and supplementary file.
Point 10: What do the letters ef, cd, bc, ab, c, etc. in tables 1 and 2 and above columns in figures 3, 4 and 5 mean?
Response: These alphabets indicate the significance and non-significance of the statistics. The different alphabets for example ab, efd, etc., indicate significance while the same alphabets such as aa, bb, or ff, etc., indicate non-significance at P = 0.05.
Regards,
Dr. Prof. Daniel KY Tan
Dr. Khalid Rehman Hakeem
Dr. Amir Bibi
Author Response File: Author Response.docx
Reviewer 3 Report
I have read the manuscript, and it deals with the effect of nickel and lead on the morphology and anatomy of the A. lebbeck plant, but to me, there are some notes that must be considered before publication, as follows:
1- In fact, the manuscript lacks novelty.
2- You must give the authority of the plant name when you write it for the first time. (Albizia lebbeck (L.) Benth.)
3- The name of the plant is mentioned for the first time in full, and then it is mentioned briefly as follows:
A. lebbeck.
4- The last sentence in the abstract should be changed to:
We also recommend that the genetic potential of A. lebbeck associated with its survival under heavy metal stress be investigated.
5- Why were nickel and lead chosen in this study? This must be clarified when stating the purpose of the study.
6- Figure 6(G): unclear
Is the scale of Figure 6 A the same for the rest of the figures. I do not think so.
7- It is preferable to show photos showing the effect of Ni and Pb on the morphology of A. lebbeck.
Author Response
Manuscript ID – Agriculture-2411542  
Title: Morphological and Structural Responses of Albizia lebbeck to Different Lead and Nickel stress levels
Thank you so much for the comments and suggestions. Each of your insights has served to strengthen our manuscript and we have made changes to reflect them. We have addressed the reviewers’ comments and updated the information in the new manuscript. Below is the point by point response to the comments outlined in the new manuscript.
Reviewer 3
I have read the manuscript, and it deals with the effect of nickel and lead on the morphology and anatomy of the A. lebbeck plant, but to me, there are some notes that must be considered before publication, as follows:
The introduction part of the manuscript has further improved by adding more information that will help the readers to better understand the theme of the manuscript and the importance of Pb and Ni. About 30 more relevant references have been added to the manuscript to improve the relevance of the citations. Moreover, the methodology, results, and conclusion portions have also been improved by making relevant changes.
Point 1:- In fact, the manuscript lacks novelty.
Response: To the best of our knowledge, the effect of both Pb and Ni on anatomical modifications has not yet been explored in the species A. lebbeck. Keeping in view the significance of A. lebbeck as an important means of phytoremediation (for removal of heavy metals including Pb and Ni) it is the opportune time to explore the effect of heavy metals on A. lebbeck anatomy and understand the tolerance behaviour of A. lebbeck to heavy metals stress. In Pakistan, heavy metals are emerging as an alarming threat to the agriculture sector (including Pb and Ni) and A. lebbeck is a major natural source used for phytoremediation and therefore it is important to explore and understand the modification that occurs at anatomical stages of A. lebbeck.
Point 2: You must give the authority of the plant name when you write it for the first time. (Albizia lebbeck (L.) Benth.)
Response: Thank you so much for your valuable suggestion. The change has been made accordingly.
Point 3: The name of the plant is mentioned for the first time in full, and then it is mentioned briefly as follows:
- lebbeck.
Response: Thank you for your valuable suggestion. The corrections have made throughout the manuscript as you suggested.
Point 4: The last sentence in the abstract should be changed to:
We also recommend that the genetic potential of A. lebbeck associated with its survival under heavy metal stress be investigated.
Response: We have made the changes and corrected to “We also recommend that the genetic potential of A. lebbeck associated with its survival under heavy metal stress be investigated”.
Point 5: Why were nickel and lead chosen in this study? This must be clarified when stating the purpose of the study.
Response: The presence of heavy metals in Pakistani soil is of a great concern and has become an important issue here in Pakistan. The concentration of Pb and Ni across Pakistan (soil) varies greatly depending upon the region and city ranging from 0.01-150 mg kg-1 (Pb) and 0.02-500 mg kg-1 (Ni). The shortage of surface and ground water in Pakistan has now forced farmers to use wastewater as source of irrigation which contains abundant amounts of heavy metals. This use of wastewater has enhanced the concentration of these metals even more. Ni is present up to several thousands of mg kg-1 in serpentine soil worldwide. Few studies have even reported the presence of Pb up to 2000 mg kg-1 in soil. So keeping in view this scenario we decided to select a wider range of concentrations for these metals to cover a wide range of these metals in Pakistan and worldwide. Moreover, we have also added new references (besides, 23-25, 75, 76,77,78,79) that have used for both Pb and Ni from 50 mM to 150 mM.
Point 6. Figure 6(G): unclear
Is the scale of Figure 6 A the same for the rest of the figures. I do not think so.
Response: Figure 6 G has been replaced with a clearer micrograph and the scalehas corrected to be consistent with the other micrographs.
Point 7. It is preferable to show photos showing the effect of Ni and Pb on the morphology of A. lebbeck.
Response: We do not have photos showing the effect of Ni and Pb on the morphology of A. lebbeck.
Regards,
Dr. Prof. Daniel KY Tan
Dr. Khalid Rehman Hakeen
Dr. Amir Bibi
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I think the manuscript reads much clear now and is suitable for publication.
Author Response
I think the manuscript reads much clear now and is suitable for publication.
Response: Thank you
Author Response File: Author Response.docx
Reviewer 3 Report
Thanks for your response
Author Response
Comment: Thanks for your response
Response: Thank you
Author Response File: Author Response.docx
