Recent Advances in Transcription Factor–Mediated Regulation of Salvianolic Acid Biosynthesis in Salvia miltiorrhiza

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article titled “Recent Advances in Transcription Factor–Mediated Regulation of Salvianolic Acid Biosynthesis in Salvia miltiorrhiza” has been reviewed.

 

Salvia miltiorrhiza is a significant medicinal plant, extensively studied for its therapeutic applications in Traditional Chinese Medicine. The key plant compounds, rosmarinic acid and salvianolic acid B, demonstrate multiple pharmacological bioactivities, including antibacterial, antioxidant, and cardio-cerebrovascular protective effects.

 

While the article discusses novel aspects in employing biotechnological approaches to improve SA biosynthesis in planta, the theme of the review is promising and highlights high scientific interests.

 

Some suggestions and queries for improvement are discussed as follows:

 

In the article, the authors summarize the recent progress facilitating improved understanding of salvianolic acid biosynthesis and its transcriptional regulation in planta. In medicinal plants, the specialized metabolites demonstrate potent pharmacological value; however, their presence in low concentrations hampers further use. Biotechnological progress aimed at improving the concentration of these important metabolites defines a promising and expanding area of research.

 

Abstract: Line 23-26: Key findings on transcription factor families…………control. In this section, it is important to provide examples of TF and case studies in the plant. In addition, line 26-29, it is suggested to discuss any challenges in research on understanding TF mediated regulation of Salvianolic acid biosynthesis and biotechnological approaches that provide feasible solutions, providing 1-2 examples.

 

Line 36-37: Please be consistent in the use of regular or italics form of the plant scientific names, here S. miltiorrhiza is normal while in italics form at other mentions.

Line 478: Arabidopsis plant, either use full plant name and regular/italics form consistently.

 

The material and method section should be included after the introduction. It is important to mention how the literature survey was carried out, the time period of execution, the criteria followed for screening literature, the inclusion and exclusion criteria, the objective and rationale of the review, key findings, etc.

(a) a PRISMA flow diagram (Page et al. 2021)

Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, Shamseer L, Tetzlaff JM, Akl EA, Brennan SE, Chou R, Glanville J, Grimshaw JM, Hróbjartsson A, Lalu MM, Li T, Loder EW, Mayo-Wilson E, McDonald S, McGuinness LA, Stewart LA, Thomas J, Tricco AC, Welch VA, Whiting P, Moher D. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021 Mar 29;372:n71. doi: 10.1136/bmj.n71. PMID: 33782057; PMCID: PMC8005924.

(b) Description of search strings, time periods, combination logic (e.g., AND/OR), or number of records screened at each stage.

 

 

 

 

 

 

Author Response

Comment 1 (Page 1-2, Lines 23-29): Abstract: Line 23-26: Key findings on transcription factor families…………control. In this section, it is important to provide examples of TF and case studies in the plant. In addition, line 26-29, it is suggested to discuss any challenges in research on understanding TF mediated regulation of Salvianolic acid biosynthesis and biotechnological approaches that provide feasible solutions, providing 1-2 examples.

Response 1 (Page 1-2, Lines 23-35): Thank you for this helpful suggestion. We have revised the Abstract to strengthen its specificity and practical relevance. Specifically, we now provide representative examples of transcription factors and case studies by naming well-characterized positive regulators (e.g., SmMYB111, SmMYC2 and SmTGA2) and negative regulators (e.g., SmMYB4/SmMYB39), together with their major pathway nodes/targets, rather than only summarizing TF families in general. In addition, we expanded the Abstract to explicitly outline key challenges in deciphering TF-mediated regulation of salvianolic acid biosynthesis (including regulatory redundancy and strong spatiotemporal specificity) and to highlight feasible biotechnological solutions, such as integrating spatial/single-cell omics with functional genomics approaches (e.g., genome editing and rational TF stacking) to enable predictive metabolic engineering. These changes have been incorporated in the revised Abstract.

Comment 2 (Page 1, Lines 36-37; Page 13, Lines 478): Line 36-37: Please be consistent in the use of regular or italics form of the plant scientific names, here S. miltiorrhiza is normal while in italics form at other mentions. Line 478: Arabidopsis plant, either use full plant name and regular/italics form consistently.

Response 2 (Page 2, Line 43; Page 8, Lines 262-264; Page 10, Line 360; Page 14, Line 518; Page 15, Line 558): We apologize for the inconsistency in formatting the scientific names. We have now carefully revised the entire manuscript to ensure that all Latin binomials, including Salvia miltiorrhiza, S. miltiorrhiza, and Arabidopsis thaliana, are consistently presented in italics throughout the text, tables, and figure legends.

Comment 3: The material and method section should be included after the introduction. It is important to mention how the literature survey was carried out, the time period of execution, the criteria followed for screening literature, the inclusion and exclusion criteria, the objective and rationale of the review, key findings, etc.

(a) a PRISMA flow diagram (Page et al. 2021)

Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, Shamseer L, Tetzlaff JM, Akl EA, Brennan SE, Chou R, Glanville J, Grimshaw JM, Hróbjartsson A, Lalu MM, Li T, Loder EW, Mayo-Wilson E, McDonald S, McGuinness LA, Stewart LA, Thomas J, Tricco AC, Welch VA, Whiting P, Moher D. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021 Mar 29;372:n71. doi: 10.1136/bmj.n71. PMID: 33782057; PMCID: PMC8005924.

(b) Description of search strings, time periods, combination logic (e.g., AND/OR), or number of records screened at each stage.

Response 3 (Page 3, Line 82-114, Page 20, Lines 807-809): We thank the reviewer for this crucial suggestion. Following the PRISMA 2020 guidelines [1], we have added a new "2. Literature Search Strategy" section after the Introduction. This section details our systematic search methodology, including databases, search strings, time frame, and inclusion/exclusion criteria. A corresponding PRISMA 2020 flow diagram (Figure 2) has been included to visualize the screening process. We believe this addition greatly enhances the transparency, reproducibility, and systematic rigor of our review.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,
Thank you for submitting this review article for review!
This article examines the regulatory role of transcription factors in the biosynthesis of salvianolic acid in the tissues of the medicinal plant Salvia miltiorrhiza. I believe the article is too narrowly focused and is more appropriate for a genetics journal. This work will be understandable and of interest only to a limited readership. However, if the journal editors deem it appropriate here, I agree with them.
I suggest the authors devote more attention to disclosing the details of the transcription factors regulating salvianolic acid synthesis under various types of biotic and abiotic stress. Furthermore, when describing transcriptional regulation, I would like the authors to mention the organ and tissue level at which the processes described occur.
Minor comments:
1. In the article title and throughout the text, the plant species name should be written in italics. The first mention in the text is written in full, then abbreviated.
2. Keyword(s) should not duplicate words in the article title.
In my opinion, the article can be published in "Plants" after correction.

Respectfully yours, reviewer.
December 26, 2025

Author Response

Comment 1: I suggest the authors devote more attention to disclosing the details of the transcription factors regulating salvianolic acid synthesis under various types of biotic and abiotic stress. Furthermore, when describing transcriptional regulation, I would like the authors to mention the organ and tissue level at which the processes described occur.

Response 1 (Page 9, Line 327; Page 9, Lines 342-358; Page 11, Lines 373-377): We thank the reviewer for this suggestion. We have expanded Section 4.3 (“Hormone- and stress-induced signaling…”) to explicitly include how both abiotic (e.g., UV-B, drought) and biotic/elicitor cues converge on the transcriptional regulation of salvianolic acid biosynthesis. To address organ/tissue context, we have added a clarifying statement at the beginning of Section 5, noting that the functional evidence discussed primarily derives from S. miltiorrhiza hairy roots and root tissues (the major site of metabolite accumulation), with detailed expression patterns compiled in Table 1.

Comment 2: Minor comments: 1. In the article title and throughout the text, the plant species name should be written in italics. The first mention in the text is written in full, then abbreviated. 2. Keyword(s) should not duplicate words in the article title. In my opinion, the article can be published in "Plants" after correction.

Response 2 (Page 1, Lines 36-38; Page 2, Lines 43; Page 8, Lines 262-264; Page 10, Line 360; Page 14, Line 518; Page 15, Line 558): Thank you for these suggestions. (1) We have standardized the formatting of the plant scientific name in the title and throughout the manuscript. The full name (Salvia miltiorrhiza Bunge) is provided at the first mention, and the abbreviated form (S. miltiorrhiza) is used consistently thereafter. (2) We have revised the keywords to avoid duplication with the title. The updated list now better reflects the enhanced focus of the manuscript, particularly on stress-responsive signaling and its integration into the transcriptional regulatory network governing secondary metabolism in S. miltiorrhiza.

Reviewer 3 Report

Comments and Suggestions for Authors In the manuscript named “Recent Advances in Transcription Factor–Mediated Regulation of Salvianolic Acid Biosynthesis in Salvia miltiorrhiza”, author had reviewed TFs in regulation of salvianolic acid biosynthesis process in Salvia miltiorrhiza, they had summarized numerous TFs functions, which would be helpful for medical modern development in future. However, there were some comments about it. 1. Many TFs from same TF families would be characterized with different expressional profiles or different functions, but authors had only listed them, please provide more precision description about them. For example, the bHLH family had been characterized with different roles, see Table 1, authors would distinguish reason of them, different clade? or others? 2. The review was primarily focusing on the “results” of TF-mediated regulation rather than the “processes”. Consequently, its contribution to precision metabolic engineering and rational molecular design was somewhat limited. To elevate the impact of this work, the authors should shift their focus towards the mechanistic nuances of TF-enzyme interactions, the key enzymes in biosynthetic pathway of ssalvianolic acids. Specifically, defining core regulatory circuits or hubs would provide much-needed clarity on how these TFs synergistically or antagonistically modulate the biosynthetic flux. 3. A significant omission in this review was the absence of a centralized regulatory model. To improve the manuscript’s quality and clarity, the authors should construct a schematic that illustrated the interplay between transcription factors, enzymes, and hormones. This visual integration was essential for forming a cohesive understanding of the molecular mechanisms from hormones, to TFs, to enzymes. 4. The Conclusion section was currently too verbose and lacks focus. It was recommended that the authors significantly condensed this section by moving away from descriptive accounts of hormone-TF interactions. Instead, the focus should be shifted toward synthesizing core regulatory modules that explicitly bridge the gap between TFs and key enzymes of salvianolic acid biosynthesis process. By reorganizing the conclusion around these functional hubs, the authors should more clearly articulate how these mechanisms provide a critical blueprint for molecular design breeding in Salvia miltiorrhiza, thereby elevating the practical value of this review for the improvement of traditional Chinese medicine. 5. “In S. miltiorrhiza, 110 MYB members….” (line 161), not MYB TF, they were R2R3-MYB TFs in ref24, please check it. 6. “the AP2/ERF family is commonly divided into five subgroups” in line 176, was not inaccurate, AP2/ERF family contains four subfamilies, AP2, ERF/DREB, RAV and Soloist, the ERF/DREB subfamily was divided into ERF and DREB subgroups, please reorganize words about it.

Author Response

Comment 1: “Many TFs from same TF families would be characterized with different expressional profiles or different functions, but authors had only listed them, please provide more precision description about them. For example, the bHLH family had been characterized with different roles, see Table 1, authors would distinguish reason of them, different clade? or others?”

Response 1 (Page 11, Lines 363-372): We thank the reviewer for the insightful suggestion to provide a deeper rationale for the functional divergence among transcription factors (TFs) within the same family. In direct response, we have added a dedicated opening paragraph at the beginning of Section 5 (“Transcriptional regulation of salvianolic acids by transcription factors”). This paragraph establishes that functional divergence often stems from evolutionary specialization into distinct clades or subfamilies, and is further mechanistically shaped by differences in transcriptional complex assembly, target gene selection, and tissue- or condition-specific expression. This framework provides the explanatory lens through which the specific regulatory roles of individual TFs, detailed in the subsequent sections (5.1‑5.7), are analyzed and understood.

Comment 2: “The review was primarily focusing on the ‘results’... rather than the ‘processes’. ... should shift focus towards the mechanistic nuances of TF-enzyme interactions... defining core regulatory circuits or hubs...”

Response 2 (Page 18-19, Lines 735-752): The reviewer’s point is well taken. The revised manuscript strengthens the mechanistic perspective by adding a new subsection (Section 5.8) that synthesizes core regulatory circuits and hubs underlying TF-mediated flux control. This section highlights process-level features, including hormone-gated activation and de-repression, transcriptional complex assembly, and convergent regulation of bottleneck enzymatic nodes, and it outlines practical design principles for rational TF stacking in precision metabolic engineering.

Comment 3: “A significant omission... was the absence of a centralized regulatory model. ... should construct a schematic that illustrated the interplay between transcription factors, enzymes, and hormones.”

Response 3 (Page 2, Lines 76-81): Thank you for this constructive suggestion. Following your advice, we have added a centralized schematic that integrates the interplay among upstream signals, regulatory modules/TF complexes, promoter cis-elements, key biosynthetic genes, and metabolite outputs. Specifically, we prepared a new integrated model (now presented as Figure 1) summarizing how phytohormones and environmental cues (e.g., JA/MeJA, SA, ABA, GA, UV-B, drought, and biotic elicitors) converge on representative regulatory modules (e.g., the JA-gated JAZ–MYC2 module and the MYB–bHLH–WD40 ternary complex), which then act on characteristic cis-elements in promoter regions to coordinate transcriptional regulation of key enzyme genes (PAL, C4H, 4CL, TAT, HPPR, RAS, and CYP98A14) and ultimately modulate rosmarinic acid and salvianolic acid B accumulation. We also revised the Introduction to explicitly introduce this framework and guide the subsequent family-by-family discussion.

Comment 4 (Page 16-17, Lines 655-695): “The Conclusion section was currently too verbose and lacks focus. It was recommended that the authors significantly condensed this section by moving away from descriptive accounts of hormone-TF interactions. Instead, the focus should be shifted toward synthesizing core regulatory modules that explicitly bridge the gap between TFs and key enzymes of salvianolic acid biosynthesis process. By reorganizing the conclusion around these functional hubs, the authors should more clearly articulate how these mechanisms provide a critical blueprint for molecular design breeding in Salvia miltiorrhiza, thereby elevating the practical value of this review.”

Response 4 (Page 19, Lines 754-778): We thank the reviewer for this constructive suggestion. In response, we have completely rewritten the Conclusion (Section 6) to focus squarely on synthesizing the core regulatory hubs and outlining how they provide a rational blueprint for molecular design breeding in S. miltiorrhiza. The revised conclusion is now concise and forward-looking, emphasizing actionable design principles derived from the regulatory network rather than descriptive summaries, thereby significantly elevating the practical value of the review.

Comment 5 (page5, Lines 161-162): “In S. miltiorrhiza, 110 MYB members….” (line 161), not MYB TF, they were R2R3-MYB TFs in ref24, please check it.

Response 5 (Page 6-7, Lines 208-211): We agree and corrected the text to specify that the MYB members reported in Ref. [24] are R2R3-MYB transcription factors. We also added a brief statement noting that most functionally characterized MYB regulators involved in specialized metabolism in S. miltiorrhiza reported to date belong to the R2R3-MYB subgroup.

Comment 6 (Page 5, Lines 175-183): “the AP2/ERF family is commonly divided into five subgroups” in line 176, was not inaccurate, AP2/ERF family contains four subfamilies, AP2, ERF/DREB, RAV and Soloist, the ERF/DREB subfamily was divided into ERF and DREB subgroups, please reorganize words about it.

Response 6 (Page 7, Lines 224-232): Thank you for this correction. We revised the description of AP2/ERF classification. The manuscript now states that the AP2/ERF superfamily comprises four subfamilies (AP2, ERF/DREB, RAV, and Soloist), and clarifies that the ERF/DREB subfamily can be further subdivided into ERF and DREB groups.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks for authors revised manuscript, most of my comments were well addressed, there was no new comment about it.