Moderate Drought Stress Interferes with the Physiological State and Promotes the Accumulation of Isoflavone in Reproductive Iris domestica Rhizomes
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this study, the effects of drought stress and rehydration on the resistance physiological characteristics and secondary metabolism of two-year-old I. domestica during the vegetative and reproductive growth periods were investigated. While the manuscript is promising, it requires several revisions before it can be considered for publication in "Agronomy."
1. The abstract contains numerous abbreviations. For instance, "vegetative growth" is abbreviated as "V," yet the full term is used five more times. It would be preferable to introduce these abbreviations in the introduction if they are not used repeatedly in the abstract.
2. Line 25: Please consider replacing "changes of upstream genes" with "changes in the expression levels of upstream genes."
3. Line 67: The authors mentioned "baicalin and saikosaponin." Are these specific types of isoflavonoids? If so, it would be beneficial to clarify this.
4. Line 70: When the authors refer to "changes in the level of genes," do they mean "expression levels"? Clarification is needed.
5. Line 72: The example "drought improves expression levels of the glycyrrhizin biosynthesis pathway genes" is given. Is this pathway related to isoflavonoids? This should be discussed in the section on pathways and genes not studied.
6. Line 80: The authors mention a "key point" but do not explain it. This should be briefly stated.
7. Line 99: "Seedlings" should begin with a lowercase letter.
8. Line 126: The sentence "When I. domestica grew naturally to August 1, 2021, the same treatment as I. domestica of the vegetative growth period will be implemented" is unclear. Please revise for clarity.
9. Line 136: One instance of "after" should be deleted.
10. Line 139: "weigh" should be corrected to "weight."
11. Line 150: Please indicate the brands of the kits used.
12. Line 154: The abbreviation "U" for "unit" is introduced but not used. If abbreviations are not necessary, they should be omitted.
13. Line 156: Abscisic acid is mentioned for the first time here. It would be helpful to mention ABA in the abstract alongside physiological changes such as SOD and POX.
14. Line 162: The qRT-PCR section lacks information on primer design, primer efficiency, biological or technical replicates, melt curve analysis, and the method used (dye or probe). Please provide these details.
15. Line 185: "All results are represented as means ± SE." Does SE stand for standard error? This should be clearly stated. Additionally, the abbreviation SD is used in the results section (Lines 214, 228, 245, 288, 304). Does SD represent standard deviation? The authors should specify whether they used standard error or standard deviation when reporting mean results.
16. Line 197: The phrase "as the soil moisture content..." needs revision for clarity. Is this a new sentence or a continuation of the previous one?
17. Line 240: "the roots accumulated significantly at the beginning of the drought" should be revised to "the roots accumulated proline significantly at the beginning of the drought."
18. Line 250: The title for section 3.3 could be "Expression of the isoflavone-related key enzyme genes."
19. Lines 251-253: These lines should be moved to the Introduction, where the pathway genes should be detailed.
20. Line 252: The authors claim to have studied key enzyme genes. Please cite any publications that indicate these enzymes (PAL, C4H, 4CL, CHS, CHI, F6H, and IFR) are key in this pathway.
21. Line 262: The authors should perform statistical analysis on qPCR data. After analysis, results should be indicated with asterisks in Figure 5, and the qPCR results section should be rewritten accordingly.
22. Line 278: The statement "For the reproductive period rhizomes, the contents of each isoflavone compound except iridin were synthesized positively" is very general. Figure 6 shows that isoflavone amounts in rhizomes, especially under the D2 application, decreased significantly compared to the control in the reproductive phase. This section should be rewritten carefully for roots and rhizomes.
23. Line 293: The statement "Studies have shown that the I. domestica roots are also rich in isoflavones" requires citations.
24. The introduction should include general information about flavonoids, clearly stating which ones were studied in this research.
According to Figures 6 and 7, moderate drought stress (D1):
- inhibits all five types of isoflavones in the vegetative phase in rhizomes,
- promotes tectoridin, tectorigenin, irigenin, and irisflorentin in rhizomes,
- inhibits all five types of isoflavones in the vegetative phase in roots,
- promotes only iridin and irisflorentin in the reproductive phase in roots.
In this context, the title "Moderate drought stress interferes with the physiological state and promotes the accumulation of isoflavone in Iris domestica rhizomes and roots" does not seem accurate. Moderate drought stress does not promote the accumulation of isoflavones in Iris domestica. While no isoflavone accumulation is promoted in the vegetative phase, the only isoflavone co-promoted between roots and rhizomes in the reproductive phase is irisflorentin. The authors should revise the title and rewrite the discussion section accordingly.
Additional Comments:
1. The qRT-PCR methodology is well designed, with appropriate controls and reference genes. However, the selection of primers and the efficiency of the PCR reactions should be discussed in more detail.
2. The authors should provide more information on the methodology of cDNA synthesis, including how they prevented genomic DNA contamination (e.g., DNase treatments, choosing primers on exon junctions).
3. The experimental setup, including the division of plants into control and treatment groups, is well structured. However, the rationale behind the specific soil water content percentages (14.0% and 6.5%) chosen for moderate and severe drought stress should be better justified with references to previous studies or preliminary data.
Comments on the Quality of English LanguageThe abstract contains numerous abbreviations. For instance, "vegetative growth" is abbreviated as "V," yet the full term is used five more times. It would be preferable to introduce these abbreviations in the introduction if they are not used repeatedly in the abstract.
2. Line 25: Please consider replacing "changes of upstream genes" with "changes in the expression levels of upstream genes."
3. Line 67: The authors mentioned "baicalin and saikosaponin." Are these specific types of isoflavonoids? If so, it would be beneficial to clarify this.
4. Line 70: When the authors refer to "changes in the level of genes," do they mean "expression levels"? Clarification is needed.
5. Line 72: The example "drought improves expression levels of the glycyrrhizin biosynthesis pathway genes" is given. Is this pathway related to isoflavonoids? This should be discussed in the section on pathways and genes not studied.
6. Line 80: The authors mention a "key point" but do not explain it. This should be briefly stated.
7. Line 99: "Seedlings" should begin with a lowercase letter.
8. Line 126: The sentence "When I. domestica grew naturally to August 1, 2021, the same treatment as I. domestica of the vegetative growth period will be implemented" is unclear. Please revise for clarity.
9. Line 136: One instance of "after" should be deleted.
10. Line 139: "weigh" should be corrected to "weight."
11. Line 150: Please indicate the brands of the kits used.
12. Line 154: The abbreviation "U" for "unit" is introduced but not used. If abbreviations are not necessary, they should be omitted.
13. Line 156: Abscisic acid is mentioned for the first time here. It would be helpful to mention ABA in the abstract alongside physiological changes such as SOD and POX.
14. Line 162: The qRT-PCR section lacks information on primer design, primer efficiency, biological or technical replicates, melt curve analysis, and the method used (dye or probe). Please provide these details.
15. Line 185: "All results are represented as means ± SE." Does SE stand for standard error? This should be clearly stated. Additionally, the abbreviation SD is used in the results section (Lines 214, 228, 245, 288, 304). Does SD represent standard deviation? The authors should specify whether they used standard error or standard deviation when reporting mean results.
16. Line 197: The phrase "as the soil moisture content..." needs revision for clarity. Is this a new sentence or a continuation of the previous one?
17. Line 240: "the roots accumulated significantly at the beginning of the drought" should be revised to "the roots accumulated proline significantly at the beginning of the drought."
18. Line 250: The title for section 3.3 could be "Expression of the isoflavone-related key enzyme genes."
19. Lines 251-253: These lines should be moved to the Introduction, where the pathway genes should be detailed.
20. Line 252: The authors claim to have studied key enzyme genes. Please cite any publications that indicate these enzymes (PAL, C4H, 4CL, CHS, CHI, F6H, and IFR) are key in this pathway.
21. Line 262: The authors should perform statistical analysis on qPCR data. After analysis, results should be indicated with asterisks in Figure 5, and the qPCR results section should be rewritten accordingly.
22. Line 278: The statement "For the reproductive period rhizomes, the contents of each isoflavone compound except iridin were synthesized positively" is very general. Figure 6 shows that isoflavone amounts in rhizomes, especially under the D2 application, decreased significantly compared to the control in the reproductive phase. This section should be rewritten carefully for roots and rhizomes.
23. Line 293: The statement "Studies have shown that the I. domestica roots are also rich in isoflavones" requires citations.
24. The introduction should include general information about flavonoids, clearly stating which ones were studied in this research.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsL107-108: The repeated cycles of wetting and drying is done in the experimental pots for bringing soil to natural level of compaction. Irrespective of the number of days the water is added for this purpose, the addition of water is done based on the moisture availability in the soil, and it is never same (150 ml each day), how you arrived at the figure of 150 ml per day? The addition of water is done to reach the field capacity, have you checked field capacity of the pot soil before adding water?
L119-120: Same mistake during the experiment, how did you arrived at the figure of 150 ml per day, present the moisture loss data from pots and how field capacity was calculated?
L109: Do you have a factorial design?
L141-160: How SOD and POD activity was calculated, which formula was used?
L169: Why only 1 reference gene was used?
The gene expression data was analysed by parametric or non-parametric analysis ?
L189-190: Data was analysed by one way ANOVA or two way ANOVA
Resolution of graphs are poor
Figure 2: The data is two factorial experiment, but the analysis is not a factorial analysis
L189-193: How LSD and Duncans are used together ? Either of the multiple comparison can be used at one time
Data interpretation need to be changed. In graphs, used either LSD or Duncans, and how factorial data was compared ?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors1. Please ensure that the Abstract has a total of about 200 words maximum.
2. Introduction: Please reduce then length of the Introduction. Please change the phrase “The effects on sickness” with a more appropriate scientific phrase, such as “pharmacologic effects”.
3. In the chapter 2. Materials and Methods, please provide a subchapter detailing the standards used, and their provenance.
4. In the chapter 2. Materials and Methods, please provide a subchapter detailing the technical details of the devices used in the study (country, manufacturer, model, etc.).
5. Subchapter 2.2. Experimental design: I recommend offering the studied parameters as a table in order to provide a better and quicker understanding on behalf of the reader.
6. Subchapter 2.6. Determination of isoflavones concentration: the experimental conditions for the HPLC analysis were not clearly explained; please provide HPLC results with concentrations of bioactive compounds corresponding to the different periods of hydration, for the respective plant materials, as a table.
7. Please use the phrase “plant bioactive compounds” for the general naming of the compounds of interest.
8. Please ensure that the italicized binomial name of the plant species is provided throughout the entire text. Alongside the taxonomic authority, e.g. Iris domestica (L.) Goldblatt & Mabb., Bupleurum chinense DC.
Comments on the Quality of English LanguageSome English orthography needs to be improved as there are multiple errors in the body of text.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsModerate drought stress interferes with the physiological state and promotes the accumulation of isoflavone in Iris domestica rhizomes and roots
General Comments: The article is attractive in terms of secondary metabolite production related to drought stress. The work somehow draw an attention that how drought stress effect on yield of Iris domestica. Though, the field works done only in one month. Such short exposure creates more chances to give erroneous result. So, be cautious before conclude the result. Manuscript should be more precise and suggest using simple sentence. Few errors are present in references section, follow the author’s guideline. I like to suggest minor revisions before acceptance.
Detail views mention below.
Line No Remarks
12 It is true for all plants not only for medicinal plants
34 Arrange alphabetically
102 Mention the name of the place
130 Not needed
133 Is it -80 degree centigrade? Mention appropriate symbol
136, 260 Delete it
139 Mention the time span
185, 375-376 Check it
226, 243, 287, 303 Figure should be clearer and enlarge
314 Delete one
342 This is not only for medicinal plants; it is true for all economically important plants
361-364, 365-369 Use simple sentence for better understanding
364 Italicized
378 Check it
398-405, 441-444 Use simple sentence for better understanding
434 Use subscript (H2O2)
470, 485, Check it
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for your resubmission and the opportunity to review your manuscript again. I appreciate the effort you have put into addressing the feedback provided during the initial review.
However, the qRT-PCR section still lacks crucial details necessary for the repeatability and robustness of the study. Specifically, the following issues remain inadequately addressed:
Primer Design and Efficiency: The manuscript does not provide sufficient information on the design of primers for qRT-PCR. Although you referenced a study on Scutellaria baicalensis Georgi under drouhgt stress, you did not explain how the gene sequences (PAL, C4H, 4CL, CHS, CHI, F6H, and IFR) for Iris domestica were obtained to design these primers. It is not clear whether primers specifically designed for Iris sequences were used in the study. Additionaly, there is no information on the efficiency of the primers, which should fall between 90-110% for SYBR Green analysis.
Given the imprtance of these methodological details for the validity and reproducibility of your findings, I am afraid I must reject the manuscript in its current form.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have incorporated the suggested chnages
Author Response
Thank you, we have finished all the changes.
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for your detailed response. I recommend allowing additional time to submit the requested data on primer efficiency, as it is essential for a thorough review.
Author Response
Please see the attachment。
Author Response File: Author Response.pdf
