Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Paper is very clear and well-written!

line 246: Title of Figure 2 should explain the distinction of A and B. This is done in the text but should be included in the title of the table as well. The same holds for Figure 3.

 

Author Response

Dear editors and reviewers:

Thank you very much for sending us the reviewers and editors’ comments regarding our manuscript entitled “Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera” (No. Forests-2696395). We were very pleased with overall constructive criticisms provided by the reviewers. Those comments are all valuable and very helpful for revising and improving our paper. We have revised the manuscript to accommodate the reviewers’ concerns. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously. All the authors listed have approved the manuscript. The main corrections and the responds to the reviewer’s comments are as followings:

 

To Reviewer #1:

1.comment: Paper is very clear and well-written!

line 246: Title of Figure 2 should explain the distinction of A and B. This is done in the text but should be included in the title of the table as well. The same holds for Figure 3.

Response: Firstly, thank you very much for your approval of our article. Secondly, Thank you very much for your comments. Such as, Figure 2 A means Al content in the leaves and the leaf cell wall of Camellia oleifera. B means distribution ratio of Al content in the cell wall. We answered this question in the reviewed manuscript as follows:

Lines 247-250: Figure 2. Al content in the leaves and the leaf cell wall of Camellia oleifera (A) and distribution ratio of Al content in the cell wall at three stages(B). The lowercase letters indicate a significant difference of 0.05 level between Camellia oleifera leaves of different ages.

Lines 266-269: Figure 3. Al content in pectin, hemicellulose, and cellulose (A) and their distribution ratio of Al content in the cell walls of Camellia oleifera leaves at three stages(B). The lowercase letters indicate a significant difference of 0.05 level between Camellia oleifera leaves of different ages.

Reviewer 2 Report

Comments and Suggestions for Authors

Review on “Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera”.

Generally, I think that the topic of the paper is very interesting and has great importance. However, the manuscript needs improvement. Please check the whole manuscript carefully (use period at the end of each sentences). Check the size and style of letter in the whole manuscript.

Keywords: Please arrange the keywords in alphabetical order. Try to use other words than are in the title e.g. use oil tree instead of Camellia oleifera

Materials and Methods:

Line 98-100: The presented data regarding to the soil analysis is not enough. Please provide a full soil analysis.

Line 171: Fresh, freeze dried, or frozen leaves were used?

Results:

Figure 4. Please use bigger pictures for a clearer view.

Figure 5. Figures are too small, no data can be seen.

Author Response

Dear editors and reviewers:

Thank you very much for sending us the reviewers and editors’ comments regarding our manuscript entitled “Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera” (No. Forests-2696395). We were very pleased with overall constructive criticisms provided by the reviewers. Those comments are all valuable and very helpful for revising and improving our paper. We have revised the manuscript to accommodate the reviewers’ concerns. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously. All the authors listed have approved the manuscript. The main corrections and the responds to the reviewer’s comments are as followings:

 

To Reviewer #2:

1.comment: Review on “Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera”. Generally, I think that the topic of the paper is very interesting and has great importance. However, the manuscript needs improvement. Please check the whole manuscript carefully (use period at the end of each sentences). Check the size and style of letter in the whole manuscript.

Response: Firstly, thank you very much for your approval of our article. Secondly, we apologize for our mistake, and thank you very much for your comments. We answered this question in the reviewed manuscript as follows:

Lines 41-44: However, when the soil pH is less than 5, stable Al dissolves in large quantities as Al³⁺ and Al(OH) ²⁺, with high mobility and increased phytotoxicity.

Lines 123-125: The digest was further diluted with distilled water and used to determine Al concentration by inductively coupled plasma-mass spectrometry (ICP-MS) (iCAP Q, Thermo Fisher Scientific, Waltham, MA, USA).

Lines 173-175: Finally, the transverse sections of these treated blades were coated with gold palladium by ion sputtering method then examined by SEM/EDS (SU3800/SU3900, Hitachi, Tokyo, Japan).

Lines 242-243, lines 256-257, lines 275-276: The lowercase letters indicate a significant difference of 0.05 level between Camellia oleifera leaves of different ages.

Lines 492-507: As an accumulator, Camellia oleifera accumulates a large amount of Al in leaves, which increases with the maturity of leaves. Here, the cell wall is the primary site for storing Al, and the hemicellulose of the cell wall acts as the main binding material and reduces the damage due to Al in the protoplasts. The accumulation rate of Al in the cell wall also increased with the increase in leaf age. Detailed analysis revealed that the antioxidant system components showed a synergistic effect on the resistance of C. oleifera leaves to Al stress. The trend of POD activity was first increasing then decreasing, while the CAT activity, SOD activity, and TP content were opposite trend. Correlation analysis showed that a significant correlation between antioxidant substances and aluminum content in C. oleifera leaves. It suggests that enhancing antioxidant enzyme activity and accumulating Al in cell walls are important mechanisms of Al detoxification in C. oleifera. These findings provide a theoretical foundation for screening C. oleifera varieties resistant to Al stress in acidic soil.

 

2.comments: Keywords: Please arrange the keywords in alphabetical order. Try to use other words than are in the title e.g. use oil tree instead of Camellia oleifera

Response: Firstly, thank you for your comments.

Keywords: Al content; antioxidative enzymes; oil tea; cell wall; histochemical staining; SEM-EDS

 

3.comments: Materials and Methods:

Line 98-100: The presented data regarding to the soil analysis is not enough. Please provide a full soil analysis.

Response: We are very sorry for the incomplete soil analysis data, as our soil samples are no longer available and cannot be supplemented. Because camellia oleifera is the subject of our research, the lack of soil data does not affect our exploration of this scientific issue.

Line 171: Fresh, freeze dried, or frozen leaves were used?

frozen leaves

 

4.comments: Results:

Figure 4. Please use bigger pictures for a clearer view.

Figure 5. Figures are too small, no data can be seen.

Response: Thank you very much for your comments. We have made modifications in manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

the authors studied the characteristics of Aluminum Accumulation and  Distribution in Leaves of Different Ages in Camellia  oleifera. The present study investigated the  accumulation and distribution of Al and the changes in antioxidant components  in C. oleifera leaves at different stages of growth. The analysis revealed that the content of Al in young, mature, and old leaves was 1.53, 7.49, and 12.04 g kg-1  ,  respectively. their findings suggest that the increase in antioxidant properties and  modification of the cell wall contributed to Al detoxification in the Al  hyperaccumulator C. oleifera.

but i still have some comments

first the introduction need to be organised and rewrite it somothly 

results 

plz make your figs clear

plz organise your discussion and make it deep

 and connections between your aprts

check the ref

 

 

Comments on the Quality of English Language

its ok

Author Response

Dear editors and reviewers:

Thank you very much for sending us the reviewers and editors’ comments regarding our manuscript entitled “Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera” (No. Forests-2696395). We were very pleased with overall constructive criticisms provided by the reviewers. Those comments are all valuable and very helpful for revising and improving our paper. We have revised the manuscript to accommodate the reviewers’ concerns. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously. All the authors listed have approved the manuscript. The main corrections and the responds to the reviewer’s comments are as followings:

 

To Reviewer #3:

1.comment: the authors studied the characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera. The present study investigated the accumulation and distribution of Al and the changes in antioxidant components in C. oleifera leaves at different stages of growth. The analysis revealed that the content of Al in young, mature, and old leaves was 1.53, 7.49, and 12.04 g kg-1, respectively. their findings suggest that the increase in antioxidant properties and modification of the cell wall contributed to Al detoxification in the Al hyperaccumulator C. oleifera. but I still have some comments, first the introduction needs to be organized and rewrite it smoothly.

Respond: Firstly, thank you for your valuable comments. Secondly, we will describe the introduction more clearly. First of all, we briefly described the increasingly serious Al toxicity caused by soil acidification. It is very important to explore the methods of resistance to Al stress. Then, the specific harm of Al stress on plants was briefly introduced, and the urgent need to solve the problem of Al stress on plants was put forward. We found some plants with Al accumulators, and the former had made some research on their mechanism of resistance to Al stress. Camellia oleifera, as an important seedling in acid soil in South China, played an important role. We found that Camellia oleifera is also an Al hyperaccumulator, and its leaves also have high Al content, but the mechanism of its resistance to Al stress has not been studied. Combined with the research content of the former, we want to explore the detoxification mechanism of Camellia oleifera leaves through our experiment. It is hoped to provide help for the selection and cultivation of Al tolerant plants and the harm of Al stress caused by soil acidification. We answered this question in the reviewed manuscript as follows:

Lines 37-76: Aluminum (Al) is the most abundant element in the Earth's crust and exists in various stable forms in the soil, such as alumina and aluminosilicate. Under neutral or moderately acidic soil conditions, Al is present as insoluble aluminosilicate or Al oxide [1], which causes no adverse effect on plant growth. However, when the soil pH is less than 5, stable Al dissolves in large quantities as Al³⁺ and Al (OH) ²⁺, with high mobility and increased phytotoxicity [2-4]. About 60% of the soils in tropical and subtropical regions of the world are acidic [4], and the acidic soil is mainly distributed in the southern part of the Yangtze River in China, covering 14 provinces and cities. In recent years, with the large-scale use of nitrogen fertilizers in agricultural production, soil acidification has intensified on a large scale. However, extreme acidification combined with highly active Al in soil has become increasingly serious [4-8]. Therefore, exploring and understanding the method of Al stress resistance has gained immense attention.

The impact of Al stress on plants is also becoming increasingly severe. Firstly, Al stress affects the nutrient absorption by roots and the normal metabolic activities of plants [9,10]. It further affects other biological processes, hinders plant growth, and reduces crop yield [11-14]. In addition, Al stress can also cause abnormal antioxidant tolerance, inhibiting the activities of superoxide dismutase, peroxidase, and catalase, disrupting the balance between ROS generation and clearance, leading to excessive accumulation of ROS [12]. Therefore, there has been an urgent need to solve the Al stress, and various means have been tested to reduce the effects of soil acidification and Al toxicity on plant growth. For example, alkaline substances such as lime and sulfate are used in fields to increase soil pH and reduce active Al content [15,16]. However, this approach is unsuitable for large-scale improvement due to the large quantity required and the high cost. Many plants called Al accumulators have evolved mechanisms to resist acidic soil and alleviate toxic symptoms by absorbing large amounts of Al [17,18]. In these species, the accumulated Al gets stored in different tissues. For example, in Camellia sinensis and Conostegia xalapensis, Al is mainly stored in the leaf epidermis [19,20]. In buckwheat, Al gets stored in vacuoles as Al oxalate [21,22]. These mechanisms of chelating active Al to the extracellular plastids undoubtedly reduce Al toxicity in the plant. Therefore, cultivating Al-tolerant plants is a more practical and effective strategy for managing high Al and acidic soil. Understanding the Al tolerance mechanism will help select and breed plants resistant to Al.

Lines 102-103: It is hoped to provide help for the selection and cultivation of Al tolerant plants and the harm of Al stress caused by soil acidification.

 

2. comments: results: please make your figs clear

Response: Thank you very much for your comments. We have made modifications in manuscript.

 

3.comments: please organize your discussion and make it deep and connections between your parts

Response: Firstly, thank you very much for your comments. Secondly, we have further effectively connected various parts through correlation analysis. Are you satisfied? If there are any deficiencies, please be sure to contact us promptly. We answered this question in the reviewed manuscript as follows:

Lines 387-391: From the correlation analysis, it can be seen that the Al content in leaves is significantly positively correlated with the cell structure, indicating that the cell structure of C. oleifera leaves is an important mechanism for Al accumulation.

Lines 435-438: The distribution characteristics of Al in cells are similar to those in leaf tissue, indicating that the outer protective structure of C. oleifera is one of the important factors for C. oleifera to become an Al hyperaccumulating plant.

Lines 395-397: In plant protoplasts, Al³⁺ has a high affinity for O2-donors, such as DNA, RNA, ATP, and lipids [24], while the cell wall acts as a barrier for Al, and effectively binding Al³⁺ [45, 46].

Reference:

[45] Zheng S.J.; Lin X.Y.; Yang J.L.; Liu Q.; Tang C.X. The kinetics of Al adsorption and desorption by root cell walls of an Al resistant wheat (Triticum aestivum L.) cultivar. Plant Soil. 2004,261,85-90.

[46] Morita A.; Yanagisawa O.; Takatsu S.; Maeda S.; Hiradate S. Mechanism for the detoxification of aluminum in roots of tea plant (Camellia sinensis (L.) Kuntze). Phytochemistry. 2008,69,147-153.

Lines 432-434: This may be due to the transpiration of plants, which flows up and down the epidermis with material transportation, leading to the accumulation of Al [53].

Reference:

[53] Schroeder J.I.; Kwak J.M.; Allen G.J. Guard cell abscisic acid signalling and engineering drought hardiness in plants. Nature. 2001,410,327-330.

 

4.comments: check the reference

Response: Thank you very much for your comments. We have made modifications in manuscript. The following is a list of some modified content:

 

[1] Mukhopadyay, M.; Bantawa, P.; Das, A.; Sarkar, B.; Bera, B.; Ghosh, P.; Mondal, T.K. Changes of growth, photosynthesis and alteration of leaf antioxidative defence system of tea [Camellia sinensis (L.) O. Kuntze] seedlings under Al stress. BioMetals. 2012,25,1141-1154.

[2] Kopittke, P.M.; Moore, K.L.; Lombi, E.; Gianoncelli, A.; Ferguson, B.J.; Blamey, F.P.; Menzies NW, T. M.;Nicholson, B.A.; Wang, P.; Gresshoff, P.M.; et al. Identification of the primary lesion of toxic Al in plant roots. Plant Physiol. 2015,167,1402-1411.

[3] Hu, X.F.; Chen, F.S.; Wine, M.L.; Fang, X.M. Increasing acidity of rain in subtropical tea plantation alters Al and nutrient distributions at the root-soil interface and in plant tissues. Plant Soil. 2017,417,261-274.

[4] Kochian, L.V.; Hoekenga, O.A.; Piñeros, M.A. How do crop plants tolerate acid soils? Mechanisms of Al tolerance and phosphorous efficiency. Annu. Rev. Plant Biol. 2004,55,459-493.

[5] Cançado, G.M.A.; Loguercio, L.L.; Martins, P.R.; Parentoni, S.N.; Paiva, E.; Borém, A.; Lopes, M.A. Hematoxylin staining as a phenotypic index for Al tolerance selection in tropical maize (Zea mays L.). Theor. Appl. Genet. 1999,99,747-754.

[6] Wang, X.; Wu, M. H.; Xiao, D.; Huang, R. L.; Zhan, J.; Wang, A. Q.; He, L. F. Genome-wide identification and evolutionary analysis of RLKs involved in the response to aluminum stress in peanut. BMC plant biology. 2021,21,281.

[7] Xia, H.; Riaz, M.; Babar, S.; Yan, L.; Li, Y.; Wang, X.; Wang, J.; Jiang, C. " Assessing the impact of biochar on microbes in acidic soils: Alleviating the toxicity of Al and acidity". Journal of environmental management. 2023,345,118796.

[8] Li, X.W.; Li, Y.; Qu, M.; Xiao, H.; Feng, Y.; Liu, J.; Wu, L.; Yu, M. Cell wall pectin and its methyl-esterification in transition zone determine Al resistance in cultivars of Pea (Pisum sativum). Front. Plant Sci. 2016,00039.

[9] Inostroza-Blancheteau, C.; Rengel, Z.; Alberdi, M.; de la Luz Mora, M.; Aquea, F.; Arce-Johnson, P.; Reyes-Díaz, M. Molecular and physiological strategies to increase Al resistance in plants. Molecular biology reports. 2012,39,2069–2079.

[10] Ofoe, R.; Thomas, R. H.; Asiedu, S. K.; Wang-Pruski, G.; Fofana, B.; Abbey, L. Al in plant: Benefits, toxicity and tolerance mechanisms. Frontiers in plant science. 2023,13,1085998.

[11] Reis, A. R. D.; Lisboa, L. A. M.; Reis, H. P. G.; Barcelos, J. P. Q.; Santos, E. F.; Santini, J. M. K.; Venâncio Meyer-Sand, B. R.; Putti, F. F., Galindo; F. S., Kaneko; F. H.; Barbosa, J. Z.; Paixão, A. P.; Junior, E. F.; de Figueiredo, P. A. M.; Lavres, J. Depicting the physiological and ultrastructural responses of soybean plants to Al stress conditions. Plant physiology and biochemistry: PPB. 2018,130,377–390.

[12] Panda, S. K.; Matsumoto, H. Changes in antioxidant gene expression and induction of oxidative stress in pea (Pisum sativum L.) under Al stress. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 2010,23,753–762.

[13] Kochian, L.V.; Piñeros, M.A.; Liu, J.; Magalhaes, J.V. Plant adaptation to acid soils: the molecular basis for crop Al resistance. Ann. Rev. Plant Biol. 2015,66,571-598.

[14] Ligaba-Osena, A.; Fei, Z.; Liu, J.; Xu, Y.; Shaff, J.; Lee, S.C.; Luan, S.; Kudla, J.; Kochian, L.; Piñeros, M. Loss-of-function mutation of the calcium sensor CBL1 increases Al sensitivity in Arabidopsis. New Phytol. 2017, 214,830-841.

[15] Álvarez, E.; Fernández-Sanjurjo, M.J.; Núñez, A.; Seco, N.; Corti, G. Aluminium fractionation and speciation in bulk and rhizosphere of a grass soil amended with mussel shells or lime. Geoderma. 2011,12,015.

[16] Vera-Villalobos, H.; Lunario-Delgado, L.; Pérez-Retamal, D.; Román, D.; Leiva, J. C.; Zamorano, P.; Mercado-Seguel, A.; Gálvez, A. S.; Benito, C.; Wulff-Zottele, C. Sulfate nutrition improves short-term Al³⁺-stress tolerance in roots of Lolium perenne L. Plant physiology and biochemistry : PPB, 2020,148,103–113.

[17] Chen, R.F.; Shen, R.F.; Gu, P.; Wang, H.Y.; Xu, X.H. Investigation of Al-tolerant species in acid soils of South China. Commun. Soil Sci. Plant Anal. 2008,39,1493-1506.

[18] Huang, L.Y.; Yuan, J.; Wang, H.; Tan, X.F.; Niu, G.H. Al stress affects growth and physiological characteristics in Oil Tea. HortScience 2017, 52,1601-1607.

[19] Gonzalez-Santana, I.H.; Marquez-Guzman, J.; Cram-Heydrich, S.; Cruz-Ortega, R. Conostegia xalapensis (Melastomataceae): an Al accumulator plant. Physiol. Plant 2012,144,134-145.

[20] Uomori, S.Y.; Yamashita, H.; Saito, M.; Haruyama, Y.; Yasuda, K. Change of concentrations and distributions of Al and manganese in tea leaves. Int. J. PIXE. 2013,23,13-19.

Reviewer 4 Report

Comments and Suggestions for Authors

The topics of the research was excellent and authors did nice work and they concluded that an increase in antioxidant properties and modification of the cell wall contributed to Al detoxification in the Al hyperaccumulator C. oleifera. However, the following points needs to consider.

1.       In abstract, please one or two sentences about Al toxicity and its consequences in plants.

2.       Authors concluded about antioxidant enzyme activities help in Al detoxification, however, they did few enzymatic and one non-enzymatic antioxidant enzyme assay but it would be nice, if they can add a few more non antioxidant enzyme activities.

3.       Besides, if they have H2O2 content data, it would be nice to incorporate before MDA data.

4.       Authors need to carefully check the typo mistakes, such in different parts of manuscript was written in different font size. For example, formula 1, 414-421

 

5.       Please reduce the conclusion size, I mean it should me more precise and concise.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear editors and reviewers:

Thank you very much for sending us the reviewers and editors’ comments regarding our manuscript entitled “Characteristics of Aluminum Accumulation and Distribution in Leaves of Different Ages in Camellia oleifera” (No. Forests-2696395). We were very pleased with overall constructive criticisms provided by the reviewers. Those comments are all valuable and very helpful for revising and improving our paper. We have revised the manuscript to accommodate the reviewers’ concerns. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously. All the authors listed have approved the manuscript. The main corrections and the responds to the reviewer’s comments are as followings:

 

To Reviewer #3:

1.comment: The topics of the research was excellent and authors did nice work and they concluded that an increase in antioxidant properties and modification of the cell wall contributed to Al detoxification in the Al hyperaccumulator C. oleifera. However, the following points needs to consider.

1. In abstract, please one or two sentences about Al toxicity and its consequences in plants.

Response: Firstly, we would like to express our strong suggestions. Secondly, we would like to express that the increasingly serious impact of Al toxicity on plants is an important aspect of our discussion. Due to the harm caused by this, we must address this issue - the problem of aluminum stress in plants and the research on the mechanisms of resistance to aluminum stress.

 

2. Authors concluded about antioxidant enzyme activities help in Al detoxification, however, they did few enzymatic and one non-enzymatic antioxidant enzyme assay but it would be nice, if they can add a few more non antioxidant enzyme activities.

Response: First of all, thank you very much for your suggestion. However, our samples are not sufficient for us to measure these data. We apologize for our insufficient consideration in this regard. In future research, we will further complete our data. We apologize again for our shortcomings.

 

3. Besides, if they have H2O2 content data, it would be nice to incorporate before MDA data.

Response: Firstly, thank you very much for your suggestion. However, our samples are not sufficient for us to measure these data. We apologize for our insufficient consideration in this regard. In future research, we will further complete our data. We apologize again for our shortcomings.

 

4. Authors need to carefully check the typo mistakes, such in different parts of manuscript was written in different font size. For example, formula 1, 414-417

Response: We are very sorry for our mistake.

Lines 414-417: In this study, we used multiple methods to detect Al in leaf tissues, which allowed in-depth analysis. We stained the leaf sections with PCV, CAS, and hematoxylin, which have been widely used to reveal the distribution of Al in plant tissues.

 

5. Please reduce the conclusion size, I mean it should me more precise and concise.

Response: Firstly, thank you very much for your comments. Secondly, if there are any shortcomings, please contact us promptly. We answered this question in the reviewed manuscript as follows:

Lines 492-510: As an accumulator, Camellia oleifera accumulates a large amount of Al in leaves, which increases with the maturity of leaves. The accumulation rate of Al in the cell wall also increased with the increase in leaf age. Here, the cell wall is the primary site for storing Al, and the hemicellulose of the cell wall acts as the main binding material and reduces the damage due to Al in the protoplasts. Detailed analysis revealed that the antioxidant system components showed a synergistic effect on the resistance of C. oleifera leaves to Al stress. POD was mainly involved in the antioxidant response of mature leaves, while SOD, CAT, and TP were active in young and old leaves. Correlation analysis showed that a significant correlation between antioxidant substances and aluminum content in C. oleifera leaves. It suggests that enhancing antioxidant enzyme activity and accumulating Al in cell walls are important mechanisms of Al detoxification in C. oleifera. These findings provide a theoretical foundation for screening C. oleifera varieties resistant to Al stress in acidic soil.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

the authors did all suggestions