Accumulation and Effect of Heavy Metals on the Germination and Growth of Salsola vermiculata L. Seedlings

Round 1

Reviewer 1 Report

It is noticeable first that the manuscript is unbalanced: extremely long introduction (as planning a complex study aiming to describe a large amount of results), followed by very short description of only limited amount of data, and, finally, relatively long discussion organized by individual metals (mostly containing rather general information). Phenomenon of hyperaccumualtion, the main point of interest, is analyzed superficially, not even mentioning all hyperaccumulation criteria. Most importantly, hyperaccumulation assumes preferred accumulation of metal of interest in aboveground parts. Literature analysis is mostly useless in the experimental context, as in this study, the approach used does not allow to examine this criterion, as metal analysis was performed in the whole seedling. Consequently, there is no sense to discuss "hyperaccumulation".

Moreover, the experimental system used involves cultivation of plants without any added nutrients (growing only on seed reserves) for 30 days. These seedlings probably were under heavy nutrient starvation stress, therefore, not having physiologically normal responses. Only size measurements are performed, which is another limitation, as metal effects on biomass accumulation in different plant parts are the most important.

It needs to be concluded that this manuscript is neither scientifically sound nor brings new scientific knowledge.

Author Response

Huelva 6th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer:

Thank you very much for your revision of the above referred manuscript. We appreciate the constructive comments.

Please find enclosed the revised manuscript, in which the main changes are labeled in red in the text. On the other hand, according the suggestions of referees, English language has been revised again by a native English revision service (Avenida Alemania 27, 3C - 21002 Huelva, Spain). In addition, the title has been shortened following the Assitant Editor indications. Editing and typographical errors have been corrected. The illustrations are the same, except figure 1, in which the letters above bars indicating significant differences, has been redistributed for an easier reading. Finally, we revised our manuscript, according to referees’ comments, which deserves the following considerations:

Reviewer 1.

1.- “It is noticeable first that the manuscript is unbalanced: extremely long introduction (as planning a complex study aiming to describe a large amount of results), followed by very short description of only limited amount of data”

We have revised introduction and we consider that it does not include superfluous information.

2.- “Relatively long Discussion organized by individual metals (mostly containing rather general information).”

We agree with referee and some information of this section, that would be considered as of minor interest has been removed.

3.- “Phenomenon of hyperaccumualtion, the main point of interest, is analyzed superficially, not even mentioning all hyperaccumulation criteria. Most importantly, hyperaccumulation assumes preferred accumulation of metal of interest in aboveground parts. Literature analysis is mostly useless in the experimental context, as in this study, the approach used does not allow to examine this criterion, as metal analysis was performed in the whole seedling. Consequently, there is no sense to discuss "hyperaccumulation"”

We agree with referee and the use of this species as a hyperaccumulator has been removed from the manuscript and bibliography, changing the focusing on the interest of the species as a hypertolerant.

4.- “Moreover, the experimental system used involves cultivation of plants without any added nutrients (growing only on seed reserves) for 30 days. These seedlings probably were under heavy nutrient starvation stress, therefore, not having physiologically normal responses”

Along 30 days we only analyzed germination rates. Seedling were measured 15 days after their germination. In this period, seedling growth are supported by stored reserves and with water absortion (see as example Soriano, D., Huante, P., Gamboa-de Buen, A.,&Orozco-Segovia, A. (2013). "Seed reserve trans location and early seedling growth of eight tree species in a tropical deciduous forest in Mexico". Plant Ecology, 214(11), 1361-1375). So we studied, the effect of metals in the metabolism of reserves after imbibing.

This methodology is widely accepted by the scientific community, and have been used in many studies on the effects of heavy metals on germination of seeds, included some of our previous studies with no criticism (for example: Infante-Izquierdo, M. D., Polo-Ávila, A., Sanjosé, I., Castillo, J. M., Nieva, F. J. J., Grewell, B. J.,&Muñoz-Rodríguez, A. F. (2020). "Effects of heavy metal pollution on germination and early seedling growth in native and invasive Spartina cord grasses". Marine pollution bulletin, 158, 111376; and also Márquez-García, B., Márquez, C., Sanjosé, I., Nieva, F. J. J., Rodríguez-Rubio, P.,&Muñoz-Rodríguez, A. F. (2013). "The effects of heavy metals on germination and seedling characteristics in two halophyte species in Mediterranean marshes". Marine pollution bulletin, 70(1-2), 119-124).

5.- “Only size measurements are performed, which is another limitation, as metal effects on biomass accumulation in different plant parts are the most important.”

The measures of the different organs at different concentrations were carried out to see the effect of metals on the growth of these organs. The aims of measurements are to determine the effects of heavy metals on the growth of the different parts of the seedlings.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study used the plant seeds to test the germination and growth for the purpose of phytoremediation. The overall findings can be accepted. However, the quality of Table 1 should be revised: mover the caption to the top of table and use available digital number for all data, for instance, germination (%) 92 changes as 92.00, t50 1.3 should be as 1.30, something like these. Additionally, conclusion is very long and please shorten it only as implication of findings, no need to repeat results and discussion.

Author Response

Huelva 6th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer:

Thank you very much for your revision of the above referred manuscript. We appreciate the constructive comments.

Please find enclosed the revised manuscript, in which the main changes are labeled in red in the text. On the other hand, according the suggestions of referees, English language has been revised again by a native English revision service (Avenida Alemania 27, 3C - 21002 Huelva, Spain). In addition, the title has been shortened following the Assitant Editor indications. Editing and typographical errors have been corrected. The illustrations are the same, except figure 1, in which the letters above bars indicating significant differences, has been redistributed for an easier reading. Finally, we revised our manuscript, according to referees’ comments, which deserves the following considerations:

Reviewer 2.

1.- “Are the methods adequately described? Must be improved.”

We agree with the referee and this section had been revised and modify.

2.- “The quality of Table 1 should be revised: Move the caption to the top of table.”

We agree with the referee and the proposed modification has been done.

3.- “Use available digital number for all data, for instance, germination (%) 92 changes as 92.00, t50 1.3 should be as 1.30, something like these.”

The correction has been included.

4.- “Additionally, conclusion is very long and please shorten it only as implication of findings, no need to repeat results and Discussion.”

We agree with the referee and the Conclusions has been shortened following the indications.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript entitled „Accumulation and effect of heavy metals on the germination and development of Salsola vermiculata L. seedlings; a proposed hyperaccumulator for the restoration of contaminated saline soils” by Israel Sanjosé, Francisco Navarro-Roldán, María Dolores Infante-Izquierdo, Gloria Martínez-Segarra, Juan Antonio Devesa, Alejandro Polo, Sara Ramírez-Acosta, Enrique Sánchez-Gullón, Francisco Javier Jiménez-Nieva, and Adolfo Francisco Muñoz-Rodríguez describes the potential of salt-tolerant plant Salsola vermiculata for phytoremediation of heavy metals. The problems of enhanced soil salinity and the contamination of soils with heavy metals is common problem all over the word and for sure economical- and environmental-friendly approaches needs to be quickly developed. In this context the manuscript is important and timely. In general, the manuscript is well-written and clearly organised however I do have some questions/comments.

Figure 1/Table 1: please change “root” to “radicle” in table 1 or “radicle” to “root” in figure 1 (since in both you are describing the date for 15-day-old seedling so should be the same).

Figure 1: The letters above bars are very difficult to read and moreover it is not clear what is really compared (are cotyledons/hypocotyls/radicles compared only for one metal or for each metals?). Please add this information in the manuscript.

In the introduction and discussion section authors say clearly what values of metals inside plant tissues (mg/kg) need to be reached to consider a plant as a hyperaccumulator. However, I think that authors omit the second part of the definition of hyperaccumulator. The hyperaccumulator not only accumulate metals above threshold value but also translocate this metal from root to shoot and the accumulation of metals occurs in above-ground tissues (which is especially useful for phytoremediation purpose). In the presented study we do not actually know where metals are accumulated since the metal content was checked in the whole seedling. If plant accumulate more metals in roots than in shoots then is less suitable for phytoremediation purpose. Therefore, Authors show that S. vermiculata is metal-tolerant plant and the patterns of metal accumulation are different for Cu and Ni than for Mn and Zn however the potential for phytoremediation is less evident from the presented study. Some plants are hypertolerant but they are not hypeaccumulators.

I think that conclusion section needs to be shortened and large part of this section (e.g. lines 486-494) should be included in discussion. Moreover, the explanation why Cu, Ni, Zn and Mn have been selected for the study should not be the last sentence of the manuscript.

Line 495: Probably authors means ”……., and the first patters for Mn and Zn.” not  ”……., and the first patters for Mn and Ni.”    

Some typos and editing errors e.g. lines 119-120 (lack of space between value and the unit) lines 156-157, lines 192-193 (lack of subscript in chemical formulas), table caption should be above a table.

Author Response

Huelva 6th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer:

Thank you very much for your revision of the above referred manuscript. We appreciate the constructive comments.

Please find enclosed the revised manuscript, in which the main changes are labeled in red in the text. On the other hand, according the suggestions of referees, English language has been revised again by a native English revision service (Avenida Alemania 27, 3C - 21002 Huelva, Spain). In addition, the title has been shortened following the Assitant Editor indications. Editing and typographical errors have been corrected. The illustrations are the same, except figure 1, in which the letters above bars indicating significant differences, has been redistributed for an easier reading. Finally, we revised our manuscript, according to referees’ comments, which deserves the following considerations:

Reviewer 3.

1.- “Are the results clearly presented? Must be improved.

We agree with the referee and the figures have been optimized and corrected for the guidelines.

2.- “Figure 1/Table 1: please change “root” to “radicle” in table 1 or “radicle” to “root” in figure 1 (since in both you are describing the date for 15-day-old seedling so should be the same)”.

The complete text has been revised, including Figure 1 and Table 1, using "root" or "radicle" more homogeneously and according to the expression most appropriate to each context of the mauscript.

3.- “Figure 1: The letters above bars are very difficult to read and moreover it is not clear what is really compared (are cotyledons/hypocotyls/radicles compared only for one metal or for each metals?). Please add this information in the manuscript.”

We agree with referee in this observation and the letters above bars, in Figure 1, indicating significant differences, has been redistributed for an easier reading. Likewise, the corresponding text has been added at the foot of the figure.

4.- “In the introduction and Discussion section authors say clearly what values of metals inside plant tissues (mg/kg) need to be reached to consider a plant as a hyperaccumulator. However, I think that authors omit the second part of the definition of hyperaccumulator. The hyperaccumulator not only accumulate metals above threshold value but also translocate this metal from root to shoot and the accumulation of metals occurs in above-ground tissues (which is especially useful for phytoremediation purpose). In the presented study we do not actually know where metals are accumulated since the metal content was checked in the whole seedling. If plant accumulate more metals in roots than in shoots then is less suitable for phytoremediation purpose. Therefore, Authors show that S. vermiculata is metal-tolerant plant and the patterns of metal accumulation are different for Cu and Ni than for Mn and Zn however the potential for phytoremediation is less evident from the presented study. Some plants are hypertolerant but they are not hypeaccumulators.”

We agree with referee and we appreciate yours recommendations. We have removed all references in text to the use of this species as a hyperaccumulator, changing the focusing on the interest of the species as a hypertolerant as have rightly suggested.

5.- “I think that conclusion section needs to be shortened and large part of this section (e.g. lines 486-494) should be included in Discussion. Moreover, the explanation why Cu, Ni, Zn and Mn have been selected for the study should not be the last sentence of the manuscript.”

We agree with referee and some information of this section has been moved to the Discussion, and the recommendations, has been performed.

6.- Line 495: Probably authors means ”……., and the first patters for Mn and Zn.” not  ”……., and the first patters for Mn and Ni.”

Some typos and editing errors e.g. lines 119-120 (lack of space between value and the unit) lines 156-157, lines 192-193 (lack of subscript in chemical formulas), table caption should be above a table.”

We agree with referee and all the corrections indicated have been incorporated into the new version of the manuscript.

 

Thank you in advance for your attention.

With best wishes,

Dr. Francisco Navarro

Prof. Cell Biology, Department of Integrated Sciences,

Faculty of Experimental Sciences,

University of Huelva,

21007 Huelva, Spain.

E-mail: [email protected]

Author Response File: Author Response.pdf

Reviewer 4 Report

Review comments on “Accumulation and effect of heavy metals on the germination and development of Salsola vermiculata L. seedlings; a proposed hyperaccumulator for the restoration of contaminated saline soils.” (Manuscript ID: diversity-1394173).

Generally, the topic and the results will be helpful for heavy metals remediation. Even though I think the paper is not ready to be publish, I have some major concerns:

- The title clearly reflects the contents.

- Introduction: in the present form is enough described but the originality and novelty of the paper need to be further clarified in the introduction section .

- The keywords, shouldn’t be the same as mentioned in the title

- The References style, tables and some parts of text do not fit with journal guidelines.

- Conclusions with elements of discussion is not recommended in reviewer opinion.

Therefore I recommend the manuscript for publication after major corrections.

Author Response

Huelva 6th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer:

Thank you very much for your revision of the above referred manuscript. We appreciate the constructive comments.

Please find enclosed the revised manuscript, in which the main changes are labeled in red in the text. On the other hand, according the suggestions of referees, English language has been revised again by a native English revision service (Avenida Alemania 27, 3C - 21002 Huelva, Spain). In addition, the title has been shortened following the Assitant Editor indications. Editing and typographical errors have been corrected. The illustrations are the same, except figure 1, in which the letters above bars indicating significant differences, has been redistributed for an easier reading. Finally, we revised our manuscript, according to referees’ comments, which deserves the following considerations:

Reviewer 4.

1.- Are the Conclusions supported by the results? Must be improved.

We agree with referee and the use of this species as a hyperaccumulator has been removed from the manuscript and bibliography, changing the focusing on the interest of the species as a hypertolerant.

2.- “Introduction: in the present form is enough described but the originality and novelty of the paper need to be further clarified in the introduction section.”

We have added, at the end of the introduction section, a paragraph stating the originality and novelty of this manuscript following the referee's recommendations.

3.- “The keywords, shouldn’t be the same as mentioned in the title.”

The title has been shortened following the Assitant Editor indications, and some keywords has been replaced.

4.- “The References style, tables and some parts of text do not fit with journal guidelines.”

The manuscript has been reviewed and adjusted to the journal's guidelines as indicated by the referee.

5.- “Conclusions with elements of Discussion is not recommended in reviewer opinion.”

We agree with the referee and the Conclusions has been shortened and the elements of Discussion removed from this section following the indications of referee.

Thank you in advance for your attention.

With best wishes,

Dr. Francisco Navarro

Prof. Cell Biology, Department of Integrated Sciences,

Faculty of Experimental Sciences,

University of Huelva,

21007 Huelva, Spain.

E-mail: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript indeed has been improved, but limitations of the experimental system still remain. I still have an opinion that (1) measuring only length of an organ is not a valid indication of effect on growth and (2) there is no sense to measure any concentration of element or substance in whole seedling or whole plant, as mass ratios between different parts could have changed as a result of treatments. Therefore, at least, experimental system used and its limitations need to be throughly discussed. I suggest that the part named "Conclusions" (which in fact are not conclusions) is renamed and included as a first subsection of Discussion, with necessary analysis added.

Some other comments follow.

Title needs to be rephrased, something like "Heavy metal accumulation and effect on germination and seedling growth of Salsola vermiculata seedlings", to avoid logical bias.

In the Abstract, results need to be described in the past tense.

In the Introduction, the aim needs to be reformulated. Studying something for the first time is good, but it is not the aim.

In the Materials and methods, Ni is not essential for plants (line 146), it is beneficial for legume symbiosis with N2-fixing bacteria and for plants growing on urea as a single source of N.

In the Discussion, exclude text from line 252 to line 253.

Author Response

Huelva 12th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer,

Thank you very much for your revision of the above referred manuscript. We appreciate your constructive comments.

Please find enclosed the revised manuscript (second round), in which the main changes are labeled in red in the text below.

1.- “Moderate English changes required”

The text has been reviewed by a professional native British and some misprints have been detected and corrected.

2.- “Is the research design appropriate? Must be improved.”

We agree with referee and some aspects have been clarified such as the use of the results of the concentrations of metals in the seedlings.

3.- “Are the methods adequately described? Can be improved.”

We have revised the Methods section and a description of the use made of accumulation in whole seedling has been added.

 

4.- “Are the conclusions supported by the results? Can be improved.”

Lines 252-253 have been removed from the Discussion section as it could be confusing. The last paragraph of the Discussion section has been removed and the explanation of why Mn, Zn, Ni and Cu where analyzed in this study has been included in the aims. We believe that these changes can contribute to a better understanding of the conclusions.

5.- “Measuring only length of an organ is not a valid indication of effect on growth.”

Stress induced by metals diminish meristematic cell growth and reduced activity of certain enzymes leading to lower seedling organs length. The same methodology that we use in the present manuscript is very widespread in the scientific literature, for example:

Nouri, M., El Rasafi, T., & Haddioui, A. (2020). Lepidium sativum L. hormesis induced by heavy metal stress for seed germination and seedling growth. Journal of Applied Science and Environmental Studies, 3(4), 3-4.

Akinci, I. E., & Akinci, S. (2010). Effect of chromium toxicity on germination and early seedling growth in melon (Cucumis melo L.). African Journal of Biotechnology, 9(29), 4589-4594.

Singh, D., Nath, K., & Sharma, Y. K. (2007). Response of wheat seed germination and seedling growth under copper stress. Journal of Environmental Biology, 28(2), 409.

Khan, M. A., Yamaguchi, S., & Kamiya, Y. (2005). Effects of heavy metals on seed germination and early seedling growth of Arabidopsis thaliana. Plant growth regulation, 46(1), 45-50.

6.- “There is no sense to measure any concentration of element or substance in whole seedling or whole plant, as mass ratios between different parts could have changed as a result of treatments. Therefore, at least, experimental system used and its limitations need to be throughly discussed.”

We agree with the referee but also the total accumulation may be indicative of increased accumulation when seedlings are exposed to increasing concentrations, especially in those concentrations in which there are no significant effects on the size of the different parts. Consequently, this comment has been included in the Materials and Methods section to clarify this aspect.

7.- “I suggest that the part named "Conclusions" (which in fact are not conclusions) is renamed and included as a first subsection of Discussion, with necessary analysis added.”

Actually the names of the sections are determined by the journal, for that reason and to create it, in the first version, we take a section of the Discussion as the referee indicates. However, we have modified that section as we have already indicated above, in point 4.

8.- “Title needs to be rephrased, something like "Heavy metal accumulation and effect on germination and seedling growth of Salsola vermiculata seedlings", to avoid logical bias.”

We agree with the referee and the proposed modification has been done.

9.- “In the Abstract, results need to be described in the past tense.”

We agree with the referee and the proposed modification has been done.

10.- “In the Introduction, the aim needs to be reformulated. Studying something for the first time is good, but it is not the aim.”

We believe that the line of work set out in the Introduction section as well as the justification for the study is clear in this section.

We must consider that:

- Halophytic plants are accepted as solutions for cleaning up coastal environments, acting as bioindicators or biomonitors to assess the extent of heavy metal contamination on sediments

- Chenopodiaceae family contributes the largest number of halophyte species, and they are dominant in Mediterranean tidal marsh vegetation.

- Many Chenopodiaceae species have been studied as potential accumulators of metals in saline soils, and they may be considered a valuable species for the phytoremediation of metal-polluted saline soils.

- Salsola vermiculata is a shrub of wide distribution and ecological amplitude, and is an important structural element in the vegetation of the arid and coastal zones.

For all the above, we consider that the objective of the study is clear.

11.- “In the Materials and methods, Ni is not essential for plants (line 146), it is beneficial for legume symbiosis with N2-fixing bacteria and for plants growing on urea as a single source of N.”

As you will remember, some of this information was included in the first version of the manuscript, but it was deleted at your recommendation as it was considered "general information". Certainly, urease was the only enzyme in higher plants that had been reported to possess Ni as an integral component.

However, some authors consider this element as essential, having observed that its presence is decisive for other enzyme activities, maintaining proper cellular redox state and various other biochemical, physiological and growth responses (see for example Fabiano, C., Tezotto, T., Favarin, J. L., Polacco, J. C.,&Mazzafera, P. (2015). Essentiality of nickel in plants: a role in plant stresses. Frontiers in plant science, 6, 754: The references to these authors are included in the citations that are included in the text of the manuscript).

In the present version, we recognize the essentiality of nickel by following authors such as Yusuf et al. (2011) (reference 98), who stated that Ni could be considered a essential nutrient, because it fulfills essentiality criteria, showing vital roles in a wide range of physiological processes, from seed germination, through vegetative growth culminating ultimately to the seed development.

12.- “In the Discussion, exclude text from line 252 to line 253”

We agree with the referee and those lines have been removed.

Thank you in advance for your attention.

With best wishes,

Dr. Francisco Navarro

Prof. Cell Biology, Department of Integrated Sciences,

Faculty of Experimental Sciences,

University of Huelva,

21007 Huelva, Spain.

E-mail: [email protected]

 

Author Response File: Author Response.pdf

Reviewer 3 Report

I appreciate all changes that have been made by authors to improve their manuscript. In my opinion those changes significantly improve the manuscript. Now only a few minor issues should be corrected/changed prior to publication.

Page 7 lines 252-253 – Please delete or explain why you decided to discuss metals separately (which in fact in my opinion is a good idea). Moreover, it is not clear what you mean „There is also a general discussion in the Conclusions.”

Page 10, lines 419-421 – I really think that explanation why Mn, Zn, Ni and Cu where analysed in this study should be included in the aim.

Author Response

Huelva 12th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer,

Thank you very much for your revision of the above referred manuscript. We appreciate your constructive comments.

Please find enclosed the revised manuscript (second round), in which the main changes are labeled in red in the text below.

1.- “English language and style are fine/minor spell check required.”

Thank you. The text has been reviewed by a professional native British and some misprints have been detected and corrected.

2.- “Page 7 lines 252-253 – Please delete or explain why you decided to discuss metals separately (which in fact in my opinion is a good idea). Moreover, it is not clear what you mean “There is also a general discussion in the Conclusions.””

We agree with the referee and those lines have been removed.

3.- “Page 10, lines 419-421 – I really think that explanation why Mn, Zn, Ni and Cu where analysed in this study should be included in the aim.”

We agree with the referee and the correction has been included following the indications.

 

Thank you in advance for your attention.

With best wishes,

Dr. Francisco Navarro

Prof. Cell Biology, Department of Integrated Sciences,

Faculty of Experimental Sciences,

University of Huelva,

21007 Huelva, Spain.

E-mail: [email protected]

Author Response File: Author Response.pdf

Reviewer 4 Report

Great revisions from authors. The manuscript has been improved significantly.

Author Response

Huelva 12th of October 2021

Manuscript ID: Diversity-1394173

Dear Reviewer,

Thank you very much for your revision of the above referred manuscript. We appreciate your constructive comments. We think the manuscript has been substantially improved.

With best wishes,

Dr. Francisco Navarro

Prof. Cell Biology, Department of Integrated Sciences,

Faculty of Experimental Sciences,

University of Huelva,

21007 Huelva, Spain.

E-mail: [email protected]

 

Author Response File: Author Response.pdf