Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Reviewer comments plants-4228993

The review manuscript entitled „Abiotic stress tolerance in foxtail millet (Setaria italica L.): From molecular mechanisms to climate-resilient breeding“ represents a useful overview on foxtail millet adaptations to a broad range of abiotic stresses including drought, heat, salinity, osmotic adjustment, nitrogen, phosphorus, zinc, and micronutrient deficiencies. The manuscript provides a comprehensive overview on morphophysiological, biochemical as well as „omics“ (transcriptomics, proteomics, metabolomics) approaches applied to study the responses of foxtail millet to abiotic stresses. I can thus recommend the review manuscript for publication in Plants.

However, I have one important comment on the present manuscript which relates to the absence of cold stress which is only mentioned in Figure 4 but is absent in the text. Foxtail millet is susceptible to cold so the impacts of cold stress treatments on foxtail millet plants have to be discussed.

Otherwise, I have only a few formal comments on English language and style which are provided below:

Line 190: Replace the word „higher“ with either „enhanced“ or „increased“ in the statment: „Millet exhibits enhanced water use efficiency under drought conditions…“

Line 339: Add a comma following the statement: „….with the key distinction that under drought,….“

Line 371: Correct the typing error in the heading: „5.1. Osmotic adjustment“ (not „Osmatic adjustment“).

Line 398: Replace the words „higher“ with „increased“ and „lower“ with „decreased“ in the statement: „Under stress, tolerant genotypes consistently exhibit increased SOD, CAT, and APX activities, along with decreased lipid peroxidation (MDA) levels, compared to sensitive varieties ….“

Line 468: Write „biosynthesis“ as a single word.

Line 536: Replace the word „higher“ with either „enhanced“ or „increased“ in the statement: „Stress-tolerant cultivars are able to maintain enhanced concentrations of osmoprotectants under adverse conditions,…“

Line 592: Add the word „tolerance“ and modify the 7.6. heading as follows: „7.6. Ionomics applications in tolerance enhancement under abiotic stress“.

Line 672: A relevant reference has to be added following the statement: „The mutagenesis frequency in the T0 generation was as high as 100%, and it was stably passed on to the next generation.“

Final recommendation: Accept after a minor revision.

Author Response

Response to Reviewer 1

We sincerely thank you for Reviewer 1, their thorough and constructive evaluation of our manuscript. The positive assessment and detailed suggestions have been invaluable in strengthening the quality of our work. We have carefully addressed each comment as follows.

Comment 1: However, I have one important comment on the present manuscript which relates to the absence of cold stress which is only mentioned in Figure 4 but is absent in the text. Foxtail millet is susceptible to cold so the impacts of cold stress treatments on foxtail millet plants have to be discussed.

Our Response: Thank the reviewer for this important observation. We agree and have now added a discussion of cold stress in the manuscript, highlighted in yellow for easy identification.

Comment 2: Otherwise, I have only a few formal comments on English language and style which are provided below:

Our Response: Thank you very much for your careful review of our manuscript. We have carefully re-reviewed the manuscript and have addressed all the mistakes highlighted in yellow.

Comment 3: Line 190: Replace the word „higher“ with either „enhanced“ or „increased“ in the statment: „Millet exhibits enhanced water use efficiency under drought conditions…“

Our Response: Thank the reviewer for this language correction. The word "higher" has been replaced with "enhanced" as suggested, highlighted in yellow Line no 160.

Comment 4: Line 339: Add a comma following the statement: „….with the key distinction that under drought,….“

Our Response: Thank you for this suggestion. The comma has been added as recommended, highlighted in yellow Line no 307.

Comment 5: Line 371: Correct the typing error in the heading: „5.1. Osmotic adjustment“ (not „Osmatic adjustment“).

Our Response: We thank the reviewer for catching this typographical error. We have corrected the heading from "Osmatic adjustment" to "Osmotic adjustment" as requested Line no 338.

Comment 6: Line 398: Replace the words „higher“ with „increased“ and „lower“ with „decreased“ in the statement: „Under stress, tolerant genotypes consistently exhibit increased SOD, CAT, and APX activities, along with decreased lipid peroxidation (MDA) levels, compared to sensitive varieties ….“

Our Response: Thank the reviewer for this language correction. Both terms have been corrected as suggested, highlighted in yellow, Line no 375 and 376.

Comment 7: Line 468: Write „biosynthesis“ as a single word.

Our Response: We have corrected this. "Bio synthesis" has been changed to "biosynthesis" as a single word and highlighted in yellow.

Comment 8: Line 536: Replace the word „higher“ with either „enhanced“ or „increased“ in the statement: „Stress-tolerant cultivars are able to maintain enhanced concentrations of osmoprotectants under adverse conditions,…“

Our Response: Thank the reviewer for this suggestion. The word "higher" has been replaced with "enhanced," highlighted in yellow, Line no 502.

Comment 9: Line 592: Add the word „tolerance“ and modify the 7.6. heading as follows: „7.6. Ionomics applications in tolerance enhancement under abiotic stress“.

Our Response: Thank you very much for the reviewer for this correction. The heading has been updated as recommended and highlighted in yellow, Line no 538.

Comment 10: Line 672: A relevant reference has to be added following the statement: „The mutagenesis frequency in the T0 generation was as high as 100%, and it was stably passed on to the next generation.“

Our Response: We thank the reviewer for pointing out this omission. An appropriate citation [163] has been added as suggested and highlighted in yellow, Line no 614.

Reviewer 2 Report

Comments and Suggestions for Authors

This review is well written.

I have a minor comment.

The author name of Setaria italica should be (L.) P.Beauv.

Setaria italica (L.) P.Beauv. should be used in the title, abstract and text. 

Author Response

Response to Reviewer 2

We sincerely thank Reviewer 2 for their positive assessment and constructive suggestion. We have addressed the comment as follows.

Comment 1: The author name of Setaria italica should be (L.) P.Beauv. Setaria italica (L.) P.Beauv. should be used in the title, abstract and text.

Our Response: We agree and have corrected this throughout the manuscript. All instances of Setaria italica in the title, abstract, and main text now include the correct author citation (L.) P.Beauv highlighted in yellow.

Reviewer 3 Report

Comments and Suggestions for Authors

In the manuscript “Abiotic Stress Tolerance in Foxtail Millet (Setaria italica L.): From Molecular Mechanisms to Climate-Resilient Breeding,” The authors have done impressive work synthesizing a large body of literature on foxtail millet stress tolerance. The multi-omics integration section is particularly strong, and the tables provide excellent reference material. I think this is suitable for the readers. I strongly recommend it for publication after minor revisions are addressed as follows.

 

1. First mention of Setaria (Line 18): The species name is italicized, good, but check that all subsequent mentions maintain italics (I saw a few instances where it wasn't).
2. Taxonomic placement (Line 71): The sentence "This cereal, a member of the Paniceae tribe (subfamily Panicoideae, family Poaceae)" feels a bit heavy where it sits. Consider moving this taxonomic detail to the first time foxtail millet is introduced (around Line 58), it would flow more naturally there.
3. Numbers (Line 60, and throughout): The journal likely follows the convention of spelling out numbers one through nine. So "8000 years" should be "eight thousand years" or "8,000 years" check the journal style guide.
4. Water use efficiency value (Line 170): "only 257 g of water are needed to produce 1 g of biomass" this is a striking number. It needs a citation. I'd suggest adding a reference here, or if this comes from the Zhang et al. 2012 genome paper, say so explicitly.
5. Fragmented sentence (Line 191-192): "compared to heat-sensitive grains, their pollen viability and spikelet fertilization capacity can be maintained" this is awkward. Better: "Unlike heat-sensitive grains, foxtail millet maintains pollen viability and spikelet fertilization capacity under high temperatures.
6. Root length value (Line 236): "46,752 cm of specific root length per gram of root dry weight" this is extremely precise. Unless the precision matters for the point you're making, consider rounding to "approximately 46,800 cm" or even "over 46,000 cm." It reads more naturally.
7. Missing definition (Line 539): "panicle resilience" is central to Section 8 but isn't clearly defined. I'd add a brief explanation early in that section, something like "panicle resilience (the ability to maintain reproductive development and grain set under stress).
8. Gene formatting consistency: some genes are in italics (good), but I found some were not, like SiMYB19 and SiMYB-56. Please check all.
9. CRISPR/GMO note (Line 575): The statement that CRISPRa offers "a non-GMO approach" is technically true in some regulatory frameworks (like the US) but not others (like the EU). If you want to make this claim, either specify the regulatory context or soften it to something like "may be considered non-GMO in some jurisdictions.

Author Response

Response to Reviewer 3

We sincerely thank Reviewer 3 for their thorough and constructive evaluation of our manuscript. The positive assessment and detailed suggestions have been invaluable in strengthening the quality of our work. We have carefully addressed each comment as follows.

Comment: 1: First mention of Setaria (Line 18): The species name is italicized, good, but check that all subsequent mentions maintain italics (I saw a few instances where it wasn't).

Our Response: Thank you for the reviewer for this careful observation. We have reviewed the entire manuscript and corrected all instances to ensure consistent italicization, highlighted in yellow.

Comment: 2: Taxonomic placement (Line 71): The sentence "This cereal, a member of the Paniceae tribe (subfamily Panicoideae , family Poaceae)" feels a bit heavy where it sits. Consider moving this taxonomic detail to the first time foxtail millet is introduced (around Line 58), it would flow more naturally there.

Response: We agree with the reviewer. The taxonomic detail has been moved to the first introduction of foxtail millet as suggested, highlighted in yellow Line no 55-58.

Comment: 2: Numbers (Line 60, and throughout): The journal likely follows the convention of spelling out numbers one through nine. So "8000 years" should be "eight thousand years" or "8,000 years" check the journal style guide.

Our Response: We thank the reviewer for this important formatting note. Numbers have been corrected throughout the manuscript in accordance with the journal style guide, highlighted in yellow Line no 65.

Comment: 4: Water use efficiency value (Line 170): "only 257 g of water are needed to produce 1 g of biomass" this is a striking number. It needs a citation. I'd suggest adding a reference here, or if this comes from the Zhang et al. 2012 genome paper, say so explicitly.

Our Response: We thank the reviewer for pointing out this omission. We have added a citation [43], Line no 126.

Comment: 5: Fragmented sentence (Line 191-192): "compared to heat-sensitive grains, their pollen viability and spikelet fertilization capacity can be maintained" this is awkward. Better: "Unlike heat-sensitive grains, foxtail millet maintains pollen viability and spikelet fertilization capacity under high temperatures.

Our Response: Thank you for the reviewer for this correction. The sentence has been replaced with the reviewer suggested version, highlighted in yellow Line no 199-201.

Comment: 6: Root length value (Line 236): "46,752 cm of specific root length per gram of root dry weight" this is extremely precise. Unless the precision matters for the point you're making, consider rounding to "approximately 46,800 cm" or even "over 46,000 cm." It reads more naturally.

Our Response: We thank the reviewer for this suggestion. We have change and highlighted in yellow color Line no 244.

Comment: 7: Missing definition (Line 539): "panicle resilience" is central to Section 8 but isn't clearly defined. I'd add a brief explanation early in that section, something like "panicle resilience (the ability to maintain reproductive development and grain set under stress).

Our Response: We agree and brief definition has been added as recommended, highlighted in yellow Line no 537-538.

Comment: 8: Gene formatting consistency: some genes are in italics (good), but I found some were not, like SiMYB19 and SiMYB-56. Please check all.

Our Response: Thank you for the reviewer for this careful observation. All gene names have been reviewed and corrected to ensure consistent italicization throughout the manuscript, highlighted in yellow.

Comment: 9: CRISPR/GMO note (Line 575): The statement that CRISPRa offers "a non-GMO approach" is technically true in some regulatory frameworks (like the US) but not others (like the EU). If you want to make this claim, either specify the regulatory context or soften it to something like "may be considered non-GMO in some jurisdictions.

Our Response: We thank the reviewer for this important clarification. The statement has been revised to "may be considered non-GMO in some jurisdictions" to accurately reflect regulatory variability, highlighted in yellow Line no 594-597.

Reviewer 4 Report

Comments and Suggestions for Authors

1. There are many minor errors and problems in the language and sentences of the manuscript, which need to be further revised.
2. “Central to the response is ROS (H₂O₂) homeostasis, which is negatively regulated by SiCBL-5, SiCBL-4, and SiPLATZ-12, positively regulated by SiNCED-1, and modulated by transcription factors SiNF-Y-A1, SiNF-Y-B8, and SiFN-YC-2.” In Figure2, SiNCED-1 plays a positive regulatory role, but it has the same representation (blunt-ended lines represent inhibition) as other genes in the figure, which can easily lead to misunderstandings among readers.
3. Role in Stress Biology Research Note the subheading format
4. Root Architectural Modifications and Morphological Adaptations Under Nutrient Deficiency and Abiotic Stress，Does “abiotic stress” refer solely to drought stress?
5. Modified root architecture improves resource acquisition across nutrient deficiency and drought conditions, reduced leaf area and stomatal regulation minimize water loss under drought and salinity while enabling evaporative cooling under heat, osmotic adjustment through compatible solute accumulation functions across drought, salinity, and heat stress with each context adding a distinct protective dimension and cellular protection through membrane stabilization and antioxidant capacity is similarly recruited across all stress types. This is not reflected in the manuscript
6. Does the content in Section 5.1 contribute to the main argument of the article? It does not highlight the advantages of millet in Osmotic Adjustment.
7. Antioxidant Response-Based Abiotic Stress Tolerance Note the subheading format
8. Molecular Mechanisms of Stress Tolerance Note the subheading format
9. Transcription Factor Families Helps Understand Molecular Mechanisms Underlying Stress Tolerance. The logical structure of this section is somewhat confusing and needs to be revised.
10. Section 7.5? Does this section have anything to do with millet?
11. The manuscript should focus on the main theme of “Abiotic Stress Tolerance in Foxtail Millet (Setaria italica L.): From Molecular Mechanisms to Climate-Resilient Breeding,” rather than engaging in irrelevant or vague generalizations. The research background should not be overly lengthy; instead, it should clearly explain the manuscript’s content: Why was millet chosen as the subject of study? What are the advantages of millet?

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Response to Reviewer 4

We sincerely thank Reviewer 4 for their thorough and constructive evaluation of our manuscript.

Comment: 1: There are many minor errors and problems in the language and sentences of the manuscript, which need to be further revised.

Our Response: Thank you for carefully reviewing our manuscript. We have carefully reviewed the entire manuscript and corrected all minor errors in language and sentences. The revisions have been highlighted in yellow.

 

Comment: 2: Central to the response is ROS (H₂O₂) homeostasis, which is negatively regulated by SiCBL-5, SiCBL-4, and SiPLATZ-12, positively regulated by SiNCED-1, and modulated by transcription factors SiNF-Y-A1, SiNF-Y-B8, and SiFN-YC-2.” In Figure2, SiNCED-1 plays a positive regulatory role, but it has the same representation (blunt-ended lines represent inhibition) as other genes in the figure, which can easily lead to misunderstandings among readers.

Our Response: Thank you for pointing out this inconsistency. In our original Figure 2, SiNCED-1 was incorrectly connected to ROS H₂O₂ with a blunt-ended line (inhibition), contradicting our text which describes SiNCED-1 as a positive regulator. We have revised Figure 2 by changing this connection to a solid arrow (activation) to accurately represent the positive regulatory role of SiNCED-1. We apologize for any confusion and appreciate your careful review.

 

 

 

 

Comment: 3: Role in Stress Biology Research Note the subheading format

Our Response: Thank you for noting this formatting issue. We have reviewed all subheadings in the manuscript and standardized the format and highlighted in yellow.

 

Comment: 4:  Root Architectural Modifications and Morphological Adaptations Under Nutrient Deficiency and Abiotic Stress Does “abiotic stress” refer solely to drought stress?

Our Response: Thank you for this clarification. We have revised the heading of Section 4 to specify that "abiotic stress" includes drought, salinity, and heat stress, not solely drought and highlighted in yellow.

 

 

Comment: 5: Modified root architecture improves resource acquisition across nutrient deficiency and drought conditions, reduced leaf area and stomatal regulation minimize water loss under drought and salinity while enabling evaporative cooling under heat, osmotic adjustment through compatible solute accumulation functions across drought, salinity, and heat stress with each context adding a distinct protective dimension and cellular protection through membrane stabilization and antioxidant capacity is similarly recruited across all stress types. This is not reflected in the manuscript

Our Response: Thank you for this insightful comment. We have added a sentence to Section 4.1 acknowledging that similar root proliferation strategies operate under drought stress, and we have ensured cross-stress connections are explicitly stated throughout Section 4. These changes have been highlighted in yellow the revised manuscript.

 

Comment: 6: Does the content in Section 5.1 contribute to the main argument of the article? It does not highlight the advantages of millet in Osmotic Adjustment.

Our Response: Thank you for this important observation. We agree that the original Section 5.1 described general osmotic adjustment mechanisms without highlighting millets specific advantages. We have substantially revised this section to explicitly address highlighted in yellow Line no 346-356.

 

Comment: 7: Antioxidant Response-Based Abiotic Stress Tolerance Note the subheading format

Our Response: Thank you for noting this formatting issue. We have checked all subheadings in Section 5 and standardized them to use title case (major words in capitalized) and highlighted in yellow.

 

Comment: 8: Molecular Mechanisms of Stress Tolerance Note the subheading format

Our Response: Thank you for noting this formatting issue. We have reviewed all subheadings in the manuscript and standardized the format to title case (major words capitalized). The subheading "6.1 Molecular Mechanisms of Stress Tolerance" has been confirmed to be consistent with other subheadings, and all changes have been highlighted in yellow in the revised manuscript.

 

Comment: 9: Transcription Factor Families Helps Understand Molecular Mechanisms Underlying Stress Tolerance. The logical structure of this section is somewhat confusing and needs to be revised.

Our Response: Thank you for this valuable feedback. We have substantially revised Section 6 to improve logical structure. The section now follows a clear organization: introduction to transcription factor families, followed by specific subsections on AP2/ERF, NAC/DREB (distinguishing ABA-dependent and ABA-independent mechanisms), and NF-Y families. The heading has also been corrected for clarity and grammar. All changes have been highlighted in yellow in the revised manuscript.

 

Comment: 10: Section 7.5? Does this section have anything to do with millet?

Our Response: Thank you very much to the reviewer for this critical observation. We acknowledge that the original Section 7.5 did not focus on foxtail millet (Setaria italica L.). We have now improved this section especially for foxtail millet, and the changes are highlighted in yellow Line no 524-537.

 

Comment: 11: The manuscript should focus on the main theme of “Abiotic Stress Tolerance in Foxtail Millet (Setaria italica L.): From Molecular Mechanisms to Climate-Resilient Breeding,” rather than engaging in irrelevant or vague generalizations. The research background should not be overly lengthy; instead, it should clearly explain the manuscript’s content: Why was millet chosen as the subject of study? What are the advantages of millet?

Our Response: Thank you so much for the reviewer for this valuable suggestion. We have revised the Introduction accordingly: (i) Removed repetitive and vague generalizations that were not directly relevant to the manuscript's main theme. (ii) Added a clear and explicit justification for choosing foxtail millet, highlighting its key advantages: superior abiotic stress tolerance, ability to grow on marginal lands, compact and fully sequenced genome, and phylogenetic proximity to major C₄ cereals. (iii) Shortened the background to ensure it remains concise and focused on the manuscript's central theme of abiotic stress tolerance in foxtail millet from molecular mechanisms to climate-resilient breeding and has been highlighted in yellow in the revised manuscript.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Following the author’s revisions to the manuscript, its quality has improved considerably, but Root Architectural Modifications and Morphological Adaptations Under Nutrient Deficiency and Abiotic Stress Does “abiotic stress” refer solely to drought stress？and Modified root architecture improves resource acquisition across nutrient deficiency and drought conditions, reduced leaf area and stomatal regulation minimize water loss under drought and salinity while enabling evaporative cooling under heat, osmotic adjustment through compatible solute accumulation functions across drought, salinity, and heat stress with each context adding a distinct protective dimension and cellular protection through membrane stabilization and antioxidant capacity is similarly recruited across all stress types. This is not reflected in the manuscript. The subheadings in the manuscript are Root Architectural Modifications and Morphological Adaptations Under Nutrient Deficiency, Drought, Salinity, and Heat Stress. However, this section only addresses drought stress; other abiotic stresses are not mentioned. Expanding on the content relating to these other stresses might help to improve the quality of the manuscript.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Response to Reviewer 5

Comment 1:Following the author’s revisions to the manuscript, its quality has improved considerably, but Root Architectural Modifications and Morphological Adaptations Under Nutrient Deficiency and Abiotic Stress Does “abiotic stress” refer solely to drought stress？and Modified root architecture improves resource acquisition across nutrient deficiency and drought conditions, reduced leaf area and stomatal regulation minimize water loss under drought and salinity while enabling evaporative cooling under heat, osmotic adjustment through compatible solute accumulation functions across drought, salinity, and heat stress with each context adding a distinct protective dimension and cellular protection through membrane stabilization and antioxidant capacity is similarly recruited across all stress types. This is not reflected in the manuscript. The subheadings in the manuscript are Root Architectural Modifications and Morphological Adaptations Under Nutrient Deficiency, Drought, Salinity, and Heat Stress. However, this section only addresses drought stress; other abiotic stresses are not mentioned. Expanding on the content relating to these other stresses might help to improve the quality of the manuscript.

Our Response: We thank the reviewer for this valuable observation. The reviewer correctly identified that Section 4, despite its heading referencing nutrient deficiency, drought, salinity, and heat stress, only addressed drought stress in its content. We have revised the manuscript accordingly by adding two new subsections: Section 4.5 (Root Adaptations Under Salinity Stress) and Section 4.6 (Root Adaptations Under Heat Stress). These additions ensure that all four stress types named in the section heading are now fully addressed in the manuscript content and highlighted in yellow.

Comments on the Quality of English Language

Comment: 2 The English could be improved to more clearly express the research.

Our Response: Thank you for your careful review of our manuscript. We have carefully re-reviewed the manuscript and have addressed all the mistakes highlighted in yellow.