Physiological and Histological Characterization of the ESB1 TILLING Mutant of Brassica rapa L.: Potential Use in Biofortification and Phytoremediation Programs

Round 1

Reviewer 1 Report

Congratulations. This is a nicely written manuscript. I have a few suggestions to add

Line 123 = identified as described by Lochlainn et al. [24] and Graham et al. [25].  A brief description will help.

Line 134 = When were they added to the solution and how

Line 164 = Briefly describe the process

Line 178 = When were these measurements taken (morning/evening/day/night) time range 

Author Response

Thank you very much for your suggestions, we have done all changes to improve this manuscript as you have commented us:

Line 123 = identified as described by Lochlainn et al. [24] and Graham et al. [25].  A brief description will help.

Response: Thanks for the suggestion. It was added to the manuscript (L112-114).

Line 134 = When were they added to the solution and how

Response: The mineral elements selenium (Se) and cadmium (Cd) were added to the nutrient solution at each irrigation (every three days) to both genotypes (wild-type and esb1 mutant) grown under Se and Cd conditions. It was added to the manuscript (L128).

Line 164 = Briefly describe the process

Response: Thanks for the suggestion. It was added to the manuscript (L153-156).

Line 178 = When were these measurements taken (morning/evening/day/night) time range 

Response: Thanks for the suggestion. It was added to the manuscript that the measurements were taken between 11.00 am and 12.00 pm (L169-170).

Reviewer 2 Report

Dear Authors,

Congratulations to all for all your hard and sincere efforts in bringing out this manuscript entitled “Physiological and Histological Characterization of ESB1 TILLING Mutant of Brassica rapa L: Potential Use in Biofortification and Phytoremediation Programs”. I have gone through it and found it quite interesting. You have very well described the physiological and histological characterization of esb1 mutant plants of Brassica rapa L. However, I have some critical observations regarding evaluating this mutant for selenium biofortification and/or cadmium phytoremediation.

1.       I would like to know why only these two concentrations with respect to         selenium and cadmium were used (20 μM of Na₂SeO₄ and 0.49 μM of          CdCl₂). Please explain the logic behind precisely using these                             concentrations of Se and Cd.

Besides this, I could find some minor corrections that need to be incorporated:

2.       At line no. 125, please correct the unit of resistivity as 18.2 MΩ cm                 instead of MV cm.

3.       At line no. 240, you have mentioned root surface area. However, that is         missing in Table 1; please incorporate it.

4.       At line no. 348, you have mentioned “will be studied” Please correct it.           You have already made the study.

5.       At line no. 454, you have mentioned “Qi et al.”; however, that is not               required. Please remove it.

Overall, I found your manuscript to be informative and well-written, and I appreciate the effort.

Author Response

Thank you very much for your suggestions, we have done all changes to improve this manuscript as you have commented us.

1. I would like to know why only these two concentrations with respect to selenium and cadmium were used (20μMof Na₂SeO₄ and 0.49 μM of CdCl₂). Please explain the logic behind precisely using these concentrations of Se and Cd.

Response: Thanks for your appreciation. Both concentrations (20 μM of Na₂SeO₄ and 0.49 μM of CdCl₂) were previously used in our research group. The optimal Se concentration for Se biofortification programs in Lactuca sativa L. was previously reported in our research group at 20 μM with different research papers published:

• Ríos, J. J., Rosales, M. A., Blasco, B., Cervilla, L. M., Romero, L., & Ruiz, J. M. (2008). Biofortification of Se and induction of the antioxidant capacity in lettuce plants. Scientia Horticulturae, 116(3), 248-255.
• Ríos, J. J., Blasco, B., Cervilla, L. M., Rosales, M. A., Sanchez‐Rodriguez, E., Romero, L., & Ruiz, J. M. (2009). Production and detoxification of H₂O₂ in lettuce plants exposed to selenium. Annals of Applied Biology, 154(1), 107-116.
• Rios, J. J., Blasco, B., Rosales, M. A., Sanchez‐Rodriguez, E., Leyva, R., Cervilla, L. M., ... & Ruiz, J. M. (2010). Response of nitrogen metabolism in lettuce plants subjected to different doses and forms of selenium. Journal of the Science of Food and Agriculture, 90(11), 1914-1919.

On the other hand, with respect to Cd concentration, 0.49 μM of CdCl₂ is a subtoxic dose that have been previously used in our group and it allows us to know how this element is distributed in the plant without causing toxicity. Besides, in esb1 mutants of Arabidopsis thaliana, Cd was also applied in nutrient solution at subtoxic dose:

• Baxter, I., Hosmani, P. S., Rus, A., Lahner, B., Borevitz, J. O., Muthukumar, B., ... & Salt, D. E. (2009). Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis. PLoS genetics, 5(5), e1000492.

Besides this, I could find some minor corrections that need to be incorporated:

2. At line no. 125, please correct the unit of resistivity as 18.2 MΩ cm  instead of MV cm.

Response: Thanks for your suggestion. It was included (L116).

3. At line no. 240, you have mentioned root surface area. However, that is missing in Table 1; please incorporate it.

Response: Thanks for your suggestion. It was included at lines 229-231: “However, Ranathunge and Schreiber [32]  observed an increase in root surface area, which does not agree with our results of root volume (Table 1).” In our work it was not measured the root surface area, that is why this root parameter does not appear in Table 1. However, it was measured the root volume which is similar to root surface area, and both parameters have a very similar trend.

4. At line no. 348, you have mentioned “will be studied” Please correct it. You have already made the study.

Response: Thanks for your appreciation. It was corrected (L345).

5. At line no. 454, you have mentioned “Qi et al.”; however, that is not required. Please remove it.

Response: Thanks for your suggestion. It was corrected (L451-452).

Overall, I found your manuscript to be informative and well-written, and I appreciate the effort.

Reviewer 3 Report

The authors show a physiological and histological characterization of esb1 mutant plants of Brassica rapa L. Authors analyzed the potential use of this mutant in selenium (Se) biofortification and cadmium (Cd) phytoremediation. The results of this study pave the way for the determine the potential use of this mutants in Fe and Cu biofortification and Cd phytoremediation programs. The subject of the paper fits the aims of the Journal and results could be of interest for the scientific community. The paper is well written.
Here are some major issues or points:
For abstract parts, too much background not so related to this manuscript were introduced. Also, the author did not show what the novelty and specific findings which can attract the readers.

Line 28-47 Commentary is basically academic knowledge. I suggest that you consider shortening this part of the manuscript to the bare minimum. Is all the information in this part necessary to understand the issue?
Why B. rapa was selected for homologous comparison, and the reason was not clarified.

Minor points:

Line: 70-71 Author please

Line 94-95 Author please

Author Response

Thank you very much for your suggestion. We have done all changes to improve it.

For abstract parts, too much background not so related to this manuscript were introduced. Also, the author did not show what the novelty and specific findings which can attract the readers.

Response: Thanks for the suggestion. The abstract background was reduced in the manuscript (L10-11). The end of the abstract has been modified to be more attractive for readers (L23-24).

Line 28-47 Commentary is basically academic knowledge. I suggest that you consider shortening this part of the manuscript to the bare minimum. Is all the information in this part necessary to understand the issue?

Response: Thanks for the suggestion. We have reduced the information to a minimum (L30-36).

Why B. rapa was selected for homologous comparison, and the reason was not clarified.

Response: Thanks for the suggestion. The information was added to the manuscript at lines 90-92.

Minor points:

Line: 70-71 Author please

Response: Done

Line 94-95 Author please

Response: Done