Non-Enzymatic and Enzymatic Antioxidant Responses of Hypericum perforatum L. Hairy Roots upon Photooxidative Stress
Round 1
Reviewer 1 Report
The work is interesting and deserves publication.
Some minor corrections could be done:
-Conclusion section is missing;
-A graphical abstract can be added;
Did the author confirm their phytochemical resluts through chromatographic analyses?
Author Response
Dear Reviewer
At first, we want to thank to the reviewers for their suggestions in order to improve our manuscript. We have prepared a novel version as suggested in your mail of the 14.03.2023. We have tried to follow all the recommendations and we indicate further, in the order of the reviewer's suggestions, the changes introduced into the text.
MODIFICATIONS ACCORDING TO REVIEWER 1:
- Some minor corrections could be done:
- Conclusion section is missing.
Modifications in the section Conclusions
Pg. 16: Line 604-616
The section Conclusions is added at the end of manuscript.
- Conclusions
This is the first study for the evaluation of non-enzymatic and enzymatic antioxidant responses of fifteen Hypericum perforatum L. dark-grown (HR1 А-HR1 O) and photoperiod-exposed (HR2 А-HR2 O) hairy root clones. The growth inhibition of photoperiod-exposed HR2 clones was related to their regenerative potential and usage of photosynthetic assimilates for shoot organogenesis. These cultures grown under photoperiod were proposed as an alternative source for accumulation of hypericins as lipophilic antioxidants. On the other hand, dark-grown HR1 clones represented an efficient system for production of flavonoids, flavan-3-ols and anthocyanins with strong hydrogen-donating capacity. The up-regulation of antioxidant enzymatic system in HR2 cultures suppressed photooxidative stress and lipid peroxidation processes. Further studies will be focused on the regeneration of H. perforatum hairy roots into trans-genic plants as a novel biotechnological tool for evaluation of naphthodianthrones as biological active compounds.
- A graphical abstract can be added.
Graphical abstract is copied as the last page of revised manuscript.
- Did the author confirm their phytochemical results through chromatographic analyses?
We have already published the HPLC profile of dark-grown and photoperiod-exposed HR clones (Tusevski et al., 2013). Those results corresponded to present data for phenolic compounds production.
Tusevski, O.; Petreska Stanoeva, J.; Stefova, M.; Simic, S.G. Phenolic profile of dark-grown and photoperiod-exposed Hypericum perforatum L. hairy root cultures. Sci. World J. 2013, 2013, 1-9.
Reviewer 2 Report
The author investigated the difference of the biomass, the content of TP, TFA, TA, TH, and enzymatic antioxidant responses in fifteen Hyperi-cum perforatum L. hairy roots, which are very useful for the optimized of higher production cultured. Therefore, i suggested this paper could be accepted after minor revision. The comment was followed.
1. Non-enzymatic was not mentioned in the results and discussion section, i strong suggested that this word should be replaced by other word.
2. The difference condition of A, B,.....O should be mentioned in this paper.
3. Do you mean the significant differences between the NTR and A using the symbol a after 7.10±0.71 in table 1 ? it look like that the NTR and B also have the significant differences. Please confirmed the other date in all of the table.
Author Response
Dear Reviewer
At first, we want to thank to the reviewers for their suggestions in order to improve our manuscript. We have prepared a novel version as suggested in your mail of the 14.03.2023. We have tried to follow all the recommendations and we indicate further, in the order of the reviewer's suggestions, the changes introduced into the text.
MODIFICATIONS ACCORDING TO REVIEWER 2:
The author investigated the difference of the biomass, the content of TP, TFA, TA, TH, and enzymatic antioxidant responses in fifteen Hypericum perforatum L. hairy roots, which are very useful for the optimized of higher production cultured. Therefore, I suggested this paper could be accepted after minor revision. The comment was followed.
- Non-enzymatic was not mentioned in the results and discussion section, I strong suggested that this word should be replaced by other word.
The term non-enzymatic antioxidant activity was connected to the following assays: cupric reducing antioxidant activity (CUPRAC), DPPH radical scavenging activity, ferrous chelating activity and lipid peroxidation inhibition, while the term enzymatic antioxidant activity was related to the guaiacol peroxidase, superoxide dismutase and catalase activities.
Modification in the section Materials and methods
Pg. 4: Line 174
2.5. Non-enzymatic antioxidant capacity assays
Modification in the section Results
Pg. 9: Line 332
3.3. Non-enzymatic antioxidant activities of dark-grown and photoperiod-exposed hairy roots
Modification in the section Discussion
Pg. 15: Line 542
4.3. Non-enzymatic antioxidant activities in dark-grown and photoperiod-exposed hairy roots
- The difference condition of A, B,.....O should be mentioned in this paper.
The suggestion is accepted and it is included in the section Materials and methods.
Modifications in the section Materials and methods
Pg. 3: Line 99-101
Solid-grown HR clones have been previously established by A. rhizogenes A4-mediated transformation of H. perforatum [19,25]. Fifteen HR clones derived from independent transformation events were denoted with capital letters (HR A-HR O).
- Do you mean the significant differences between the NTR and A using the symbol a after 7.10±0.71 in table 1? it look like that the NTR and B also have the significant differences. Please confirmed the other date in all of the table.
The significant differences between HR clones were presented in the note at the end of each Table in the manuscript.
For example: Pg. 7: Line 286-291
The values in one column marked with different lower-case letters denoted significant differences at p<0.05 between clones (Duncan's multiple range test). The values in one row marked with asterisk (*) denoted significant differences at p<0.05 between particular HR1 and HR2 clone (Student's t test).
Reviewer 3 Report
Remarks
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer
At first, we want to thank to the reviewers for their suggestions in order to improve our manuscript. We have prepared a novel version as suggested in your mail of the 14.03.2023. We have tried to follow all the recommendations and we indicate further, in the order of the reviewer's suggestions, the changes introduced into the text.
MODIFICATIONS ACCORDING TO REVIEWER 3:
Introduction and Material and methods are too long. Needs some reduction.
Our intention in the writing of Introduction was to cover the most relevant previously published data in order to reach the novelty and scope of this study.
The Material and methods shortly described the protocols for analyzed parameters. The extensive section of Material and methods could be ascribed to the large number of assays that have been performed in this study.
