Transcriptome Dynamics Reveal the Potential Roles of Long Non-Coding RNAs in Regulating Flower Color of Safflowers (Carthamus tinctorius)
, Lu Lv 1, Jian Wei 2, Zhaojun Wei 3, Jiao Liu 1, Hong Liu 1,* and Rui Qin 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsSafflower is an important medicinal plant widely used for pigment production. Flower color of plant is a key factor for pigment extract, but, the roles of the long non-coding RNAs (lncRNAs) in pigment accumulation hardly explored. In this manuscript, strand-specific RNA sequencing was performed on three safflower varieties with distinct flower colors and identified 4,851 lncRNAs, including 940 natural antisense transcript (NAT) pairs and showed that CtCHS.7 is a key involved in flavonoid modification, produced significant result. Thus, in this study, this manuscript address the key question is that long non-coding RNAs (lncRNAs) in pigment accumulation in Safflower with the this original finding and these finding will extend our understanding about roles of the long non-coding RNAs (lncRNAs) in Safflower. In summary, this manuscript was will written and results are novel and significant. I have only the following minor comments.
Minor comments:
- 7 should add to the key word;
- References need to be updated, there is only one reference cited from 2024-2026, there is no reference cited from 2026;
- For the methodology part, for validating RNA-seq results, four differentially expressed genes (CtYH8G243210,CtYH10G318070,CtYH11G3314 134 60,CtYH8G220570 ) were selected for qPCR analysis, why only choose these four genes, you should have an explanation for it;
- Page 4, AgriculturalSciences, there should be a space between these two word, please also check the text for the similar mistake;
- All the figures are not in the middle of the pages;
- Figure3, Figure4 and Figure5, there should be space between e and the number3, 4, 5.
Author Response
Responds to the reviewer’s comments:
We sincerely thank the reviewers for their careful evaluation of our manuscript and for the constructive and insightful comments. These suggestions have greatly helped us to improve the clarity, rigor, and biological relevance of our study. We have carefully revised the manuscript accordingly. Below, we provide a point-by-point response to all comments.
Reviewer #1:
Comment 1
1.7 should add to the key word;
Response:
Thank you for your suggestion.“CtCHS.7” has now been added to the Keywords section.
Comment 2:
References need to be updated, there is only one reference cited from 2024-2026, there is no reference cited from 2026;
Response:
Thank you for your valuable suggestion. We have updated the References section by incorporating several recent studies published between 2024 and 2026 to improve the timeliness and relevance of the manuscript.
Comment 3:
For the methodology part, for validating RNA-seq results, four differentially expressed genes(CtYH8G243210, CtYH10G318070, CtYH11G331460, and CtYH8G220570)were selected for qPCR analysis, why only choose these four genes, you should have an explanation for it;
Response
Thank you for your insightful comment. The four genes (CtYH8G243210, CtYH10G318070, CtYH11G331460, and CtYH8G220570) were selected for qPCR validation because they exhibited significant differential expression in the RNA-seq analysis and represented different expression patterns observed in the dataset. In addition, these genes were closely associated with pathways related to flavonoid biosynthesis and flower color formation. This explanation has now been added to the Methodology section.
Comment 4:
Page 4, AgriculturalSciences, there should be a space between these two word, please also check the text for the similar mistake;
Response
Thank you for pointing this out. We have corrected the formatting error by adding a space between “Agricultural” and “Sciences”. In addition, the entire manuscript has been carefully checked to correct similar spacing and formatting issues.
Comment 5:
All the figures are not in the middle of the pages;
Response
Thank you for your careful review. All figures have now been adjusted and aligned to the center of the pages to improve the overall formatting and presentation quality of the manuscript.
Comment 6:
Figure3, Figure4 and Figure5, there should be space between e and the number3, 4, 5.
Response
Thank you for your careful observation. The formatting issue in Figure 3, Figure 4, and Figure 5 has been corrected by adding the appropriate spaces between “Figure” and the corresponding numbers.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript entitled "Transcriptome Dynamics Reveal the Potential Roles of Long Non-Coding RNAs in Regulating Flower Color of Safflowers (Carthamus tinctorius)” has been submitted to the International Journal of Molecular Sciences.
The authors assessed the role of long non-coding RNAs (lncRNAs), particularly natural antisense transcript (NAT) pairs, in regulating flower color formation in safflower. They used strand-specific RNA sequencing and functional characterization of CtCHS. 7. The study is interesting, and the manuscript is written well. However, several issues need to be addressed.
Major Comments
(1) The authors proposed that MSTRG.28365 negatively regulates CtCHS.7, the conclusion is primarily based on inverse expression patterns and genomic overlap. Correlation alone is not sufficient to establish regulation. Functional validation, such as transient overexpression/silencing, RNA interference, CRISPR interference, or dual-luciferase assays, should be considered to support the lncRNA's regulatory role.
(2) The study infers that CtCHS.7 contributes to red flower coloration in Yunhong-7. However, no pigment quantification (anthocyanins, flavonoids, or carotenoids) was conducted.
(3) The authors report that CtCHS.7 produces a metabolite different from canonical naringenin chalcone and hypothesize functional divergence due to an extra exon. This interpretation should be supported with experimental evidence.
(4) The authors state that 18 transcriptome samples were analyzed, but the biological replication strategy is not clear. Please specify the number of biological replicates per treatment.
(5) The authors briefly mention possible transcriptional interference mechanisms of NATs but do not sufficiently discuss how convergent or enclosed NATs may function specifically in safflower. Authors should add a deeper mechanistic discussion and support with recent literature.
Minor Comments
(6) Several grammatical and formatting issues are present throughout the manuscript. Examples include (1) “Biosysthesis” should be corrected to "biosynthesis," (2) “lncRNA” and “LncRNA” capitalization are inconsistent, and (3) Several sentences are long and repetitive or grammatically not correct. Thoroughly revise the whole manuscript.
(7) The lncRNA identification pipeline is repeated in the Materials and Methods section. Please
(8) Gene names (e.g., CtCHS.7, CtUGT.144, CtCYP76C.2) should be consistently italicized according to journal style. Especially check the conclusion section
(9) Lines 48-50: “Among these, anthocyanins confer orange to blue hues, while flavones and flavonols act as co-pigments…”
Anthocyanins mainly contribute to red, purple, and blue, not simply “orange to blue." Orange coloration often involves carotenoids. Improve sentence structure.
Example: "Among these pigments, anthocyanins primarily contribute to red, purple, and blue coloration, whereas carotenoids are commonly associated with yellow to orange pigmentation. Flavones and flavonols often function as co-pigments that stabilize anthocyanin-based coloration.”
(10) Lines 51-56: "Variation in flavonoid biosynthetic gene expression underlies flower color diversity in lotus…”
The lotus example is repeated twice. Merge into one concise sentence,
Example: “For example, differential expression of flavonoid biosynthetic genes, including chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), contributes to flower color variation in lotus and other ornamental species.”
(11) Lines 98-122: The manuscript does not clearly report the number of biological replicates used for RNA-seq sampling. This is a major methodological concern because reproducibility cannot be assessed.
Add a sentence such as: “For each flower color and developmental stage, three independent biological replicates were collected, resulting in a total of 18 transcriptome libraries.”
(12) Lines 101-107: Text contains formatting/spacing errors
Line 102: “AgriculturalSciences”
Line 103 “isapproximately”
Line 104: “300-800 umolm~2.s1 duringsunny mornings”
(13) Lines 123-131: No kit name, RNA quality threshold, or RNA integrity information is provided.
(14) Lines 128-130: “Finally, the PCR products were PCR amplification was performed to construct the sequencing library…”
The sentence is not correct. Make corrections
Example: "Finally, PCR amplification was performed to construct strand-specific sequencing libraries.”
(15) Lines 145-166: The lncRNA prediction workflow is described twice.
First description: Lines 151-156 and repeated: Lines 162-166. Delete Lines 162–166 completely and keep one concise description.
(16) Lines 147 and 221
Line 147: "yunhong-7 reference genome”
Line 221: “Yunhong-7 reference genome”
Inconsistent capitalization: Use the “Yunhong-7 reference genome" consistently.
(17) Lines 189-192: Antibiotic concentration appears incorrect
Line 190: “50 μg/L Kan”
Line 192: “50 mg/mL Kan”
These values appear biologically unrealistic/inconsistent. Check and make corrections.
This is a major reviewer concern.
(18) Line 195: “resuspended in pre-cooled Buffer A." Add buffer composition.
Example: "Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole).”
(19) Lines 415-421: "lncNAT-regulated CtCHS.7 can produce downstream compounds..."
No evidence that enzyme activity is due to lncNAT regulation. Only CtCHS.7 enzyme activity was tested.
(20) Lines 399-401: “yunhong-7 variety”
Should be “Yunhong-7 variety.”
(21) Line 390: Functional Validation of Flavonoid Biosynthesis Genes Regulated by Lncnats
What is “Lncnats”? Check and make corrections.
Author Response
Reviewer #2:
Comment 1:
The authors proposed that MSTRG.28365 negatively regulates CtCHS.7, the conclusion is primarily based on inverse expression patterns and genomic overlap. Correlation alone is not sufficient to establish regulation. Functional validation, such as transient overexpression/silencing, RNA interference, CRISPR interference, or dual-luciferase assays, should be considered to support the lncRNA's regulatory role.
Response:
Thank you for this important and insightful suggestion. We agree that the current evidence mainly supports a correlation between MSTRG.28365 and CtCHS.7 expression. Due to the current limitations in establishing an efficient genetic transformation system in safflower, functional validation approaches such as transient overexpression/silencing, RNA interference, CRISPR interference, or dual-luciferase assays could not be completed in the present study. Therefore, we have carefully revised the manuscript to avoid overstatement and modified the relevant conclusions to indicate that MSTRG.28365 is potentially involved in regulating CtCHS.7 expression rather than definitively regulating it. In addition, we have added a discussion describing these experimental approaches as important future directions for further validation.
Comment 2:
The study infers that CtCHS.7 contributes to red flower coloration in Yunhong-7. However, no pigment quantification (anthocyanins, flavonoids, or carotenoids) was conducted.
Response:
Thank you for this important comment. We agree that pigment quantification would provide stronger evidence. In this study, three safflower lines with clearly distinct flower color phenotypes were compared (Figure 1). Given that previous studies have linked altered CHS expression to pigmentation changes, and given the significant differential expression of CtCHS.7 across these three phenotypes, we made a reasonable inference that CtCHS.7 may contribute to red coloration. We have now acknowledged the lack of pigment quantification as a limitation in the Discussion and stated that future metabolite analyses are needed for further validation.
Comment 3:
The authors report that CtCHS.7 produces a metabolite different from canonical naringenin chalcone and hypothesize functional divergence due to an extra exon. This interpretation should be supported with experimental evidence.
Response:
Thank you for this constructive suggestion. We agree that the proposed functional divergence associated with the additional exon remains speculative without further experimental evidence. In the revised manuscript, we have carefully moderated the interpretation and clarified that this hypothesis is preliminary and based on sequence structure and enzymatic assay observations. We have also emphasized that additional biochemical characterization and structural analyses will be required to confirm the functional effects of the extra exon.
Comment 4:
The authors state that 18 transcriptome samples were analyzed, but the biological replication strategy is not clear. Please specify the number of biological replicates per treatment.
Response:
Thank you for pointing this out. We have now clarified the biological replication strategy in the Materials and Methods section. Specifically, three independent biological replicates were collected for each flower color and developmental stage, resulting in a total of 18 transcriptome libraries.
Comment 5:
The authors briefly mention possible transcriptional interference mechanisms of NATs but do not sufficiently discuss how convergent or enclosed NATs may function specifically in safflower. Authors should add a deeper mechanistic discussion and support with recent literature.
Response:
Thank you for this insightful suggestion. We have substantially expanded the Discussion section to include a deeper mechanistic discussion regarding the potential functions of convergent and enclosed NATs in safflower. In addition, several recent studies related to NAT-mediated transcriptional regulation in plants have been incorporated to strengthen the discussion and improve the biological interpretation of our findings.
Minor Comments
Comment 6:
Several grammatical and formatting issues are present throughout the manuscript.
Response:
Thank you for your careful review. We have thoroughly revised the entire manuscript to correct grammatical, spelling, capitalization, and formatting issues. Specifically, “Biosysthesis” has been corrected to “biosynthesis,” the capitalization of “lncRNA” has been standardized throughout the manuscript, and several long or grammatically incorrect sentences have been carefully revised for clarity and readability.
Comment 7:
The lncRNA identification pipeline is repeated in the Materials and Methods section.
Response:
Thank you for your suggestion. We have revised the Materials and Methods section and removed the repeated description of the lncRNA identification pipeline to improve conciseness and readability.
Comment 8:
Gene names should be consistently italicized according to journal style.
Response:
Thank you for pointing this out. We have carefully checked the manuscript and standardized the formatting of all gene names, including CtCHS.7, CtUGT.144, and CtCYP76C.2, according to the journal style requirements. Particular attention was given to the Conclusion section.
Comment 9:
Anthocyanins mainly contribute to red, purple, and blue, not simply “orange to blue."
Response:
Thank you for this valuable correction. We have revised the sentence accordingly to improve scientific accuracy and clarity.
Comment 10:
The lotus example is repeated twice.
Response:
Thank you for your suggestion. We have revised and merged the repeated lotus example into one concise sentence to improve readability and avoid redundancy.
Comment 11:
The manuscript does not clearly report the number of biological replicates used for RNA-seq sampling.
Response:
Thank you for your comment. The number of biological replicates has now been clearly stated in the revised manuscript. Three independent biological replicates were used for each treatment group, resulting in a total of 18 transcriptome libraries.
Comment 12:
Text contains formatting/spacing errors.
Response:
Thank you for your careful observation. The formatting and spacing errors, including “AgriculturalSciences,” “isapproximately,” and “300-800 umolm~2.s1 duringsunny mornings,” have been corrected. In addition, the entire manuscript has been carefully checked for similar formatting issues.
Comment 13:
No kit name, RNA quality threshold, or RNA integrity information is provided.
Response:
Thank you for your suggestion. We have added detailed information regarding the RNA extraction kit, RNA quality assessment criteria, and RNA integrity requirements in the Materials and Methods section.
Comment 14:
The sentence is not correct.
Response:
Thank you for pointing this out. The sentence has been revised to:
“Finally, PCR amplification was performed to construct strand-specific sequencing libraries.”
Comment 15:
The lncRNA prediction workflow is described twice.
Response:
Thank you for your careful review. The repeated description of the lncRNA prediction workflow has been removed, and only one concise description has been retained in the revised manuscript.
Comment 16:
Inconsistent capitalization of “Yunhong-7 reference genome”.
Response:
Thank you for your observation. The capitalization has been standardized throughout the manuscript to “Yunhong-7 reference genome.”
Comment 17:
Antibiotic concentration appears incorrect.
Response:
Thank you for carefully checking this issue. We have re-examined the antibiotic concentration information and corrected the unit errors in the revised manuscript.
Comment 18:
Add buffer composition.
Response:
Thank you for your suggestion. The composition of Buffer A has now been added to the Materials and Methods section.
Comment 19:
No evidence that enzyme activity is due to lncNAT regulation.
Response:
Thank you for this important comment. We agree that the enzymatic activity assay only demonstrated the catalytic activity of CtCHS.7 and did not directly demonstrate regulation by lncNATs. Therefore, we have revised the relevant statements to avoid overinterpretation and clarified this limitation in the revised manuscript.
Comment 20:
“yunhong-7 variety” should be “Yunhong-7 variety.”
Response:
Thank you for pointing this out. The capitalization has been corrected accordingly.
Comment 21:
What is “Lncnats”?
Response:
Thank you for your careful observation.
An lncNAT, which serves as the antisense lncRNA component, together with its corresponding sense transcript, constitutes a natural antisense transcript (NAT) pair.
“Lncnats” was a formatting/capitalization error. It has been corrected to “lncNATs” throughout the manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript presents an interesting study on lncRNA-mediated regulation in safflower flower development and pigmentation; however, it requires substantial revision. Methodological clarity should be improved, particularly regarding correlation interpretation, validation approaches, and statistical support. The manuscript also needs structural refinement, including removal of duplicated content, keyword reordering, and improved figure quality and labeling. Several biological statements require clearer justification or more appropriate contextualization, especially where evidence is limited or derived from unrelated species. Technical issues such as unit formatting, grammatical errors, and typographical mistakes should be corrected. In addition, some conclusions, particularly regarding functional roles of lncNATs, appear overstated and should be moderated. Overall, the study shows potential but needs major revision to improve clarity, rigor, and consistency.
Comment 1: Please incorporate the concise statement regarding correlation strength or validation approach that would improve the clarity and reliability of the proposed lncRNA-mediated regulation.
Comment 2: Please rearrange the keywords alphabetically, the word started from “A” should be first keyword, the word started from “B” should be second keyword and so on…
Comment 3: The statement “Native to regions including Egypt, India, and the Mediterranean coast” requires clarification and supporting evidence, because the center of origin of safflower remains debated in the literature. Please provide a more precise description of safflower origin and domestication history. Please see Lines 34–35.
Comment 4: The sentence describing anthocyanin accumulation in poppy appears disconnected from safflower biology. Please explain how this example specifically supports the rationale of flower color regulation in safflower or replace it with a more closely related example from Asteraceae species. Please see Lines 50–52.
Comment 5: The description of lncNATs and NAT pair classification is informative; however, the manuscript does not clearly explain why NAT-mediated regulation is particularly relevant to flavonoid biosynthesis in safflower. Please add a short and concise transition linking NATs with pigment biosynthetic pathways. Please refer to Lines 57–69.
Comment 6: The examples of lncRNA-mediated flavonoid regulation in Salvia miltiorrhiza and apple are useful, but the molecular mechanisms are described in excessive detail compared with the surrounding text. This section might be more precise to maintain better flow and focus on the relevance to flower pigmentation. Please see Lines 75–81.
Comment 7: The environmental conditions are described in detail; however, the unit for light intensity is incorrectly formatted as “umolm~2.s1”. It should be revised to the standard scientific notation “μmol m⁻² s⁻¹”. Please check this information and throughout the manuscript for other units used. Lines 102–105.
Comment 8: The phrase “representing a physiological lymature stage” contains a typographical error (“lymature”). Please correct this to “mature stage” or “physiologically mature stage”. Lines 121.
Comment 9: The sentence “Finally, the PCR products were PCR amplification was performed to construct the sequencing library to construct the sequencing library” contains substantial grammatical repetition and should be rewritten for clarity and readability. Line 129-132.
Comment 10: Lines 145–166: The lncRNA identification procedure is described twice. The workflow involving UniProt filtering and prediction using PlantLncBoost, LncFinder-plant, and CPAT-plant in lines 152–156 is repeated again in lines 163–166. Please make a single consolidated coherent paragraph without duplication.
Comment 11: The quality of figures is not very clear. Please revise the figures to improve the quality and there must be a space between “figure3, Figure4 and Figure5” in figure legends.
Comment 12: The manuscript reports the number of identified and shared lncRNAs across developmental stages and varieties, but no statistical analysis is provided to support the conclusion of “increased variety-specific expression.” Please add information about quantitative measures such as expression specificity indices or clustering validation would strengthen this interpretation. Line 227-235.
Comment 13: The hypothesis that CtCHS.7 acquired an additional exon during evolution leading to functional specialization is interesting, but currently lacks phylogenetic or comparative genomic evidence. Inclusion of evolutionary analysis with homologous CHS genes from related species would strengthen this conclusion.
Comment 14: The Manuscript conclude that lncNATs “play a crucial role” in flower development and flower color formation; however, the study primarily provides transcriptomic correlations rather than direct functional validation. Please modify the wording should be moderated to avoid overstating the biological significance.
Comments on the Quality of English LanguageQuality of Manuscript writing could be improved.
Author Response
Reviewer #3:
Comment 1
Please incorporate the concise statement regarding correlation strength or validation approach that would improve the clarity and reliability of the proposed lncRNA-mediated regulation.
Response
Thank you for this valuable suggestion. We agree that correlation analysis alone is insufficient to establish direct regulatory relationships. Therefore, we have revised the manuscript to clearly state that the proposed lncRNA-mediated regulation is primarily based on transcriptomic correlation analysis and genomic association. We also added a concise statement indicating that further functional validation approaches, such as transient overexpression/silencing, RNA interference, CRISPR interference, or dual-luciferase assays, will be necessary to confirm the regulatory relationships in future studies.
Comment 2
Please rearrange the keywords alphabetically, the word started from “A” should be first keyword, the word started from “B” should be second keyword and so on.
Response
Thank you for the suggestion. We have rearranged all keywords in alphabetical order according to the journal requirement.
Comment 3
The statement “Native to regions including Egypt, India, and the Mediterranean coast” requires clarification and supporting evidence, because the center of origin of safflower remains debated in the literature. Please provide a more precise description of safflower origin and domestication history.
Response
Thank you for this valuable comment. We agree that the original description of safflower origin was overly broad and potentially misleading, as the domestication center of safflower remains controversial. Accordingly, we revised the relevant statement to reflect the current consensus that safflower most likely originated in the Near East or eastern Mediterranean region, while acknowledging that its precise domestication history is still under debate. Relevant references have also been added accordingly.
[1] Knowles, P. F. (1969).Centers of plant diversity and conservation of crop germplasm: safflower.Economic Botany, 23, 324–329.
[2] Chapman, M. A., Hvala, J., Strever, J., et al. (2010).Population genetic analysis of safflower (Carthamus tinctorius; Asteraceae) reveals a Near Eastern origin and five centers of diversity.American Journal of Botany, 97(5), 831–840.
Comment 4
The sentence describing anthocyanin accumulation in poppy appears disconnected from safflower biology. Please explain how this example specifically supports the rationale of flower color regulation in safflower or replace it with a more closely related example from Asteraceae species.
Response
Thank you for this constructive suggestion. We agree that the poppy example was not sufficiently connected to safflower biology. Therefore, we replaced this example with a more relevant example associated with pigment regulation in Asteraceae species to improve the biological relevance and coherence of the introduction.
Comment 5
The description of lncNATs and NAT pair classification is informative; however, the manuscript does not clearly explain why NAT-mediated regulation is particularly relevant to flavonoid biosynthesis in safflower. Please add a short and concise transition linking NATs with pigment biosynthetic pathways.
Response
Thank you for the insightful comment. We have added a concise transition paragraph in the Introduction to better connect NAT-mediated transcriptional regulation with flavonoid and pigment biosynthetic pathways. This revision improves the logical flow and highlights the potential importance of lncNATs in safflower flower color formation.
Comment 6
The examples of lncRNA-mediated flavonoid regulation in Salvia miltiorrhiza and apple are useful, but the molecular mechanisms are described in excessive detail compared with the surrounding text.
Response
Thank you for the suggestion. We have simplified and condensed this section to improve readability and maintain better focus on the relevance of lncRNA-mediated regulation to flower pigmentation rather than detailed molecular mechanisms unrelated to the main objective of this study.
Comment 7
The environmental conditions are described in detail; however, the unit for light intensity is incorrectly formatted as “umolm~2.s1”. It should be revised to the standard scientific notation “μmol m⁻² s⁻¹”.
Response
Thank you for the careful correction. We have revised the unit format to the standard scientific notation “μmol m⁻² s⁻¹”. In addition, we carefully checked the entire manuscript and corrected other unit formatting inconsistencies where necessary.
Comment 8
The phrase “representing a physiological lymature stage” contains a typographical error (“lymature”).
Response
Thank you for pointing out this typographical error. The phrase has been corrected to “representing a physiologically mature stage” in the revised manuscript.
Comment 9
The sentence “Finally, the PCR products were PCR amplification was performed to construct the sequencing library to construct the sequencing library” contains substantial grammatical repetition and should be rewritten for clarity and readability.
Response
Thank you for the careful review. The sentence has been revised as follows for improved clarity and readability:
“Finally, PCR amplification was performed to construct strand-specific sequencing libraries.”
Comment 10
The lncRNA identification procedure is described twice. Please make a single consolidated coherent paragraph without duplication.
Response
Thank you for the suggestion. We have removed the duplicated descriptions and reorganized the lncRNA identification workflow into a single concise and coherent paragraph to improve readability and manuscript structure.
Comment 11
The quality of figures is not very clear. Please revise the figures to improve the quality and there must be a space between “figure3, Figure4 and Figure5” in figure legends.
Response
Thank you for the valuable suggestion. We have improved the resolution and overall quality of all figures in the revised manuscript. In addition, spacing and formatting issues in figure legends, including “Figure 3”, “Figure 4”, and “Figure 5”, have been corrected according to the journal style requirements.
Comment 12
The manuscript reports the number of identified and shared lncRNAs across developmental stages and varieties, but no statistical analysis is provided to support the conclusion of “increased variety-specific expression.”
Response
Thank you for this important comment. We agree that additional quantitative support would strengthen the interpretation. Therefore, we revised the corresponding statements to avoid overinterpretation and clarified that the conclusion was primarily based on comparative transcriptomic observations. In addition, we added discussion regarding expression specificity analysis and clustering-based validation as important future directions for further investigation.
Comment 13
The hypothesis that CtCHS.7 acquired an additional exon during evolution leading to functional specialization is interesting, but currently lacks phylogenetic or comparative genomic evidence.
Response
Thank you for this insightful comment. To further address this concern, we performed an additional phylogenetic analysis using CtCHS.7 and representative chalcone synthase (CHS) proteins from multiple plant species. The resulting tree showed that CtCHS.7 clusters within the CHS-related clade rather than outside the canonical CHS lineage, and it forms a well-supported sister relationship with the CHS sequence from Helianthus debilis. This result indicates that CtCHS.7 is evolutionarily derived from the CHS family and retains clear homology with known plant CHS proteins. However, its relatively distinct branch position compared with several canonical CHSs suggests possible functional divergence after duplication or lineage-specific evolution. Therefore, we agree that CtCHS.7 may not represent a typical CHS enzyme, but the phylogenetic evidence supports its classification as a CHS-like member rather than a completely unrelated enzyme.
Figure 1. Phylogenetic placement of CtCHS.7 among plant CHS proteins.
Comment 14
The manuscript concludes that lncNATs “play a crucial role” in flower development and flower color formation; however, the study primarily provides transcriptomic correlations rather than direct functional validation.
Response
Thank you for this important comment. We fully agree that our current conclusions are primarily based on transcriptomic analyses and correlation evidence rather than direct functional validation. Therefore, we have carefully revised the relevant statements throughout the manuscript and moderated the wording accordingly. Specifically, expressions such as “play a crucial role” have been replaced with more cautious descriptions such as “may participate in” or “are potentially involved in” flower development and flower color formation. These revisions help avoid overstating the biological significance of the findings while maintaining the scientific value of the study.
Reviewer 4 Report
Comments and Suggestions for AuthorsDear authors:
Your work reports important results regarding the role of RNAs in the regulation of flower colors in Carthamus tinctorius. This research is novel and will undoubtedly be continued. However, it is important that you consider the following suggestions:
Title and Keywords: Please italicize the scientific name of the plant.
101-108: Please restructure this paragraph. You need to improve your grammar. The writing style you use makes me think you edited the manuscript with artificial intelligence, which is unethical. I perceive the same issue in lines 111-113.
122: In this section, you cite Figure 1b, but there is no prior description of Figure 1a. It is recommended that you please describe the material you present in an orderly fashion. That is, the correct approach is to first describe and cite Figure 1a, then Figure 1b, then Figure 1c, and so on. 123: Is the content of this line a subtopic or section? Please describe the information following the author guidelines.
124-132: Was this methodology performed using a kit? Or did you follow the methodology of a published paper? In either case, please cite the characteristics (if it was a kit: name, brand, country and city of manufacture, and batch number; if it was a scientific article: include the bibliographic citation).
183: Change the title of this section; propose a formal one (it is ambiguous). Please italicize the names of the microbial strains and plant species.
Figure 1: The figure contains valuable information, but unfortunately, its contribution to the description of sections 208-248 is not appreciated. The authors are encouraged to elaborate on this result, link it to the article title, and significantly reinforce the research's main finding. If necessary, divide the figure and expand the description of each attribute within each section.
Figure 2: It is recommended that the figure be enlarged and included in the supplementary material; it is too open, and its content is not readily apparent.
295-297: Please describe whether these results are related and if there is any precedent in similar reports.
Figure 3: Please describe the content of the figure with scientific rigor. The figure and its caption are excessive. Each section (3a, 3b, 3c, 3d, etc.) should be independent; it contains a significant amount of material that is not reflected in the description on lines 295-339.
352: Write an introductory paragraph to the presentation of this result; the connection to the previous results is not clear.
Discussion: I recommend merging the discussion with the results. If you choose to maintain the proposed style, please include a paragraph that specifically highlights whether the results were as expected and how they relate to the objectives stated in the introduction. Furthermore, it is crucial that you describe the limitations of the research and the future directions (what remains to be done) of the work.
Comments on the Quality of English LanguageAuthors must submit their work to a thorough language review; technical language contains grammatical errors that devalue the quality of the manuscript.
Author Response
Reviewer #4:
Comment 1
Title and Keywords: Please italicize the scientific name of the plant.
Response
Thank you for your careful suggestion. We have revised the title and keywords by italicizing the scientific name Carthamus tinctorius throughout the manuscript in accordance with journal formatting requirements.
Comment 2
101–108: Please restructure this paragraph. You need to improve your grammar. The writing style you use makes me think you edited the manuscript with artificial intelligence, which is unethical. I perceive the same issue in lines 111–113.
Response
We sincerely appreciate the reviewer’s concern regarding the writing quality and expression style. We have carefully revised and restructured the corresponding paragraphs (Lines 101–108 and 111–113) to improve grammatical accuracy, clarity, and academic readability. The revised text has been thoroughly edited by the authors to ensure a natural and professional scientific writing style consistent with journal standards.
Comment 3
122: In this section, you cite Figure 1b, but there is no prior description of Figure 1a. It is recommended that you please describe the material you present in an orderly fashion. That is, the correct approach is to first describe and cite Figure 1a, then Figure 1b, then Figure 1c, and so on.
Response
Thank you for this valuable suggestion. We have reorganized the description of Figure 1 to ensure that the panels are introduced sequentially and logically. The revised manuscript now first describes Figure 1a, followed by Figure 1b and subsequent panels in the correct order, thereby improving the coherence and readability of the Results section.
Comment 4
123: Is the content of this line a subtopic or section? Please describe the information following the author guidelines.
Response
Thank you for pointing out this formatting issue. We have revised the corresponding content according to the journal’s author guidelines and clarified the section structure to ensure consistency in formatting and presentation.
Comment 5
124–132: Was this methodology performed using a kit? Or did you follow the methodology of a published paper? In either case, please cite the characteristics (if it was a kit: name, brand, country and city of manufacture, and batch number; if it was a scientific article: include the bibliographic citation).
Response
We appreciate the reviewer’s important suggestion. We have revised the Materials and Methods section to provide complete methodological details. Specifically, we have added the relevant information regarding the experimental kits used, including manufacturer name, brand, city, country, and product details where applicable. In addition, appropriate references have been included for methodologies adopted from previously published studies.
Comment 6
183: Change the title of this section; propose a formal one (it is ambiguous). Please italicize the names of the microbial strains and plant species.
Response
Thank you for the constructive comment. We have revised the section title to make it more formal and scientifically precise. Additionally, all scientific names of plant species and microbial strains have been carefully checked and italicized throughout the manuscript according to standard taxonomic conventions.
Comment 7
Figure 1: The figure contains valuable information, but unfortunately, its contribution to the description of sections 208–248 is not appreciated. The authors are encouraged to elaborate on this result, link it to the article title, and significantly reinforce the research's main finding. If necessary, divide the figure and expand the description of each attribute within each section.
Response
We sincerely thank the reviewer for this insightful suggestion. We have substantially expanded the description and interpretation of Figure 1 in the revised manuscript to better emphasize its biological significance and its direct relevance to the main objective of the study. The relationship between transcriptome dynamics, lncRNA regulation, and flower color formation has now been more explicitly discussed. In addition, we reorganized parts of the figure presentation and improved the corresponding explanations to enhance clarity and scientific impact.
Comment 8
Figure 2: It is recommended that the figure be enlarged and included in the supplementary material; it is too open, and its content is not readily apparent.
Response
Thank you for your suggestion. We have significantly improved the resolution, size, and layout of Figure 2 to enhance its clarity. However, we prefer to keep Figure 2 in the main text, as it is crucial for the logical coherence of the manuscript. We hope these improvements are acceptable, and we thank the reviewer for the constructive comment.
Comment 9
295–297: Please describe whether these results are related and if there is any precedent in similar reports.
Response
We appreciate this important comment. In the revised manuscript, we have expanded the discussion of these results and clarified their potential relationships. Furthermore, we added relevant references to previous studies reporting similar observations in pigment biosynthesis and lncRNA-mediated regulatory pathways, thereby strengthening the scientific context and interpretation of our findings.
Comment 10
Figure 3: Please describe the content of the figure with scientific rigor. The figure and its caption are excessive. Each section (3a, 3b, 3c, 3d, etc.) should be independent; it contains a significant amount of material that is not reflected in the description on lines 295–339.
Response
Thank you for this detailed and constructive suggestion. We have carefully revised Figure 3 and its corresponding description. Specifically, the figure legend has been simplified and reorganized, and each panel (3a, 3b, 3c, 3d, etc.) is now independently described with clearer scientific interpretation. We also expanded the relevant Results section to ensure that all presented data are adequately explained and discussed in the main text.
Comment 11
352: Write an introductory paragraph to the presentation of this result; the connection to the previous results is not clear.
Response
We thank the reviewer for identifying this issue. A new introductory transition paragraph has been added before this section to improve the logical flow between the previous findings and the newly presented results. This revision enhances the continuity and readability of the manuscript.
Comment 12
Discussion: I recommend merging the discussion with the results. If you choose to maintain the proposed style, please include a paragraph that specifically highlights whether the results were as expected and how they relate to the objectives stated in the introduction. Furthermore, it is crucial that you describe the limitations of the research and the future directions (what remains to be done) of the work.
Response
We sincerely thank the reviewer for this valuable and thoughtful suggestion. After careful consideration, we decided to retain the separate “Results” and “Discussion” sections in accordance with the journal format and to maintain clarity in data presentation and interpretation. However, following the reviewer’s recommendation, we have substantially revised the Discussion section.
Specifically, we added a new paragraph summarizing how the obtained results align with the original objectives and hypotheses proposed in the Introduction. We also clarified which findings were consistent with our expectations and discussed their biological implications in the context of flower color regulation.
In addition, we have now explicitly addressed the limitations of the current study, particularly the lack of direct functional validation of candidate lncRNAs. Finally, we included a dedicated section describing future research directions, including functional experiments such as gene overexpression, RNA interference, CRISPR-based validation, and comparative regulatory analyses to further elucidate the molecular mechanisms underlying flower color formation in Carthamus tinctorius.
We once again sincerely thank the reviewer for the constructive comments and professional suggestions, which have greatly improved the quality and scientific rigor of our manuscript. We hope that the revised version satisfactorily addresses all concerns.
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript can be accepted in present form.
Comments on the Quality of English LanguageQuality of Manuscript writing could be improved (if possible).
Reviewer 4 Report
Comments and Suggestions for AuthorsDear authors,
Your manuscript has been sufficiently improved, and you may proceed with editing. As a final recommendation, please carefully review the figures' dimensions, as they extend beyond the file boundaries.
Authors must submit their work to a thorough language review; technical language contains grammatical errors that devalue the quality of the manuscript.
