Putative Upstream Regulators DoNF-YB3 and DoIDD12 Correlate with DoGSTF11 Expression and Anthocyanin Accumulation in Dendrobium officinale

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Congratulations on a well-executed and comprehensive study investigating the molecular mechanisms underlying anthocyanin accumulation in Dendrobium officinale. The integration of gene expression analysis, functional validation in heterologous systems, protein-protein interaction assays, and promoter binding studies presents a solid foundation for understanding the regulatory role of DoGSTF11, DoNF-YB3, and DoIDD12 in flavonoid metabolism. The manuscript is well-structured, and the experimental approaches are, for the most part, well-designed and clearly described. These findings are valuable for advancing metabolic engineering efforts in medicinal plants and contribute significantly to the field of plant secondary metabolism.

However, I am providing you with a few comments to make your work even better.

Major recommendations:

Limited Functional Characterization of Upstream Regulators - While DoNF-YB3 and DoIDD12 are identified as upstream regulators, their biological function is inferred only from correlation and yeast one-hybrid data. Further validation in planta (e.g., overexpression or silencing in D. officinale or model systems) would significantly strengthen the regulatory claims.

Although the study is detailed, the ecological or physiological implications of anthocyanin accumulation in D. officinale have not been sufficiently addressed. Consideration of this issue could increase the significance of the findings.

The gene nomenclature (e.g., DoGSTF11, DoGST31) should be consistently italicized and formatted throughout the text to distinguish genes from proteins. This inconsistency may confuse readers unfamiliar with the gene families.

Although P-values are reported, the manuscript lacks detailed descriptions of the statistical methods (e.g., type of test, software used, correction for multiple comparisons). This information is critical for reproducibility.

Also, the writing could benefit from language polishing to reduce redundancy (e.g., repeated phrases like “purple-red coloration”) and to improve clarity. A professional language editing service may be helpful.

Minor recommendations:

The images are not of sufficient resolution. I suggest that for Figure 1, the authors submit each panel separately or reprocess the figure in a way that preserves image quality and then compile them into a single composite image. Additionally, Figure 3 is fully bordered on all sides, whereas Figures 4, 5, and 6 are only partially bordered. I kindly request that the authors standardize the presentation format across all figures.

Also, the Figure legends could be more detailed, especially in indicating the statistical tests used.

The abbreviation list is missing; not all readers will be familiar with terms like BiFC or Y1H.

Comments on the Quality of English Language

The writing could benefit from language polishing to reduce redundancy (e.g., repeated phrases like “purple-red coloration”) and to improve clarity. A professional language editing service may be helpful.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Liu et al. entitled “DoNF-YB3 and DoIDD12 positively regulate DoGSTF11 and are involved in anthocyanidin accumulation in Dendrobium officinale” characterize a gene, DoGSTF11 cloned from Dendrobium officinale. The authors claim that the gene positively enhancing anthocyanin accumulation in Solanum lycopersicum is potentially regulated by DoNF-YB3 and DoIDD12. The DoIDD12 protein subcellular localized in cell membrane and nucleus of tobacco leaf epidermal cells.  The work is technically sound piece of research, and within the scope of Horticulturae, however, there are major issues need to be fixed before publication.

 

1. The title needs to be rewritten, should emphasize the gene DoGSTF11, not DoNF-YB3 and DoIDD12
2. Polish the text to be more logic.
3. Provide the sequences or public ACC. Num. of the genes, including reference genes sequence information that can judge the methods and data reasonable or not.
4. The materials part, the authors should describe the tomato and tobacco plants, for example, what species of the tobacco, Nicotiana tabacum? Nicotiana benthamiana?

Comments on the Quality of English Language

need to be polished

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

TITLE

Current: "DoNF-YB3 and DoIDD12 Positively Regulate DoGSTF11 and are Involved in Anthocyanidin Accumulation in Dendrobium officinale"

Comments:

The term “anthocyanidin” is not entirely accurate, as the analyses focus on total anthocyanins (glycosides). It is suggested to change it to “anthocyanin” accumulation.

The title suggests direct regulation by DoNF-YB3 and DoIDD12, but direct transcriptional activator functions were not demonstrated, nor was regulatory function functionally assessed (e.g., via mutant or overexpression analysis of these TFs).

Suggested revision:

“Putative upstream regulators DoNF-YB3 and DoIDD12 correlate with DoGSTF11 expression and anthocyanin accumulation in Dendrobium officinale”

ABSTRACT

The abstract presents a clear overview of the study and mentions key results.

Comments:

Technical terms are used without definition for a broad audience (“Y2H”, “BiFC”, “Y1H”).

It is stated that “DoGSTF11 positively regulates anthocyanin accumulation,” though there is no direct validation of its function as a regulator—it more likely plays a role in transport.

Suggested partial rephrasing:

“Overexpression of DoGSTF11 in tomato enhanced anthocyanin accumulation, suggesting a role in anthocyanin sequestration or transport. Protein–protein interaction assays further revealed that DoGSTF11 interacts with DoGST31, while DoIDD12 and DoNF-YB3 are potential transcriptional regulators based on promoter-binding assays and expression correlation.”

INTRODUCTION

The literature review is adequate but incomplete:

The role of MYB, bHLH, and WDR genes, which are key regulators in anthocyanin metabolism, is not mentioned.

 

There are no specific references to previous studies on GSTs in orchids or other monocot ornamental/medicinal species.

Insufficient omics context:

Transcriptomics is only tangentially mentioned (line 57), with no detail on whether this work builds upon prior transcriptomic data (e.g., co-expression analysis, RNA-seq) for selecting DoGSTF11.

Hypothesis and objectives are not clearly stated:

The manuscript does not clearly state a hypothesis or define specific experimental objectives.

Questions missing and should be addressed in this section:

Why was DoGSTF11 chosen among the 11 differentially expressed GSTs?

What function is hypothesized for DoNF-YB3 and DoIDD12? Are they activators, repressors, cofactors?

Recommendation:

Reformulate the end of the introduction with an explicit hypothesis, for example:

“We hypothesized that DoGSTF11, a phi-type GST highly expressed in purple stems of D. officinale, may participate in anthocyanin transport, and that DoNF-YB3 and DoIDD12 are putative transcriptional regulators of its expression.”

MATERIALS AND METHODS

Add the following:

The number of independent biological replicates per group (variety and stage) is not reported

It is not specified whether the tissues used were pooled or sampled separately.

Missing: information on growth conditions, controlled environmental variables (temperature, light), method of anthocyanin quantification (spectrophotometry? HPLC?).

Gene expression analysis

RT-qPCR was performed, but reference genes used are not reported, nor is validation of their stability.

Statistical analysis methods (ΔΔCt, ANOVA, normalization, etc.) are not indicated.

Were technical duplicates used, and how many independent biological replicates?

Transformation in S. lycopersicum

The transformation procedure is overly descriptive (almost a lab manual) and should be simplified.

There is no molecular evidence presented for transgene integration (e.g., PCR, qPCR of transgenes).

Structural genes of the anthocyanin pathway are not validated as functional activation markers.

It is not possible to conclude that DoGSTF11 regulates biosynthesis without observing impacts on CHS, DFR, ANS, etc.

Interaction assays (Y2H, BiFC)

Technically well-conceived, but:

In BiFC: information is missing on fluorescence quantification, number of fields analyzed, percentage of cells with interaction.

Y1H for detecting upstream TFs

Promoter binding is identified, but transcriptional activation is not demonstrated.

No analysis of specific binding sites (motif, box, etc.).

No confirmation via EMSA, ChIP, or transcriptional activation assays in a dual-luciferase system.

RESULTS

Selection of DoGSTF11

Differential expression of 11 GSTs is reported, but:

The full list is not shown (a supplementary table would be useful).

Why DoGSTF11 and not another with equally differential expression?

Functional evidence for DoGSTF11

The increase in anthocyanins in transgenic tomato is consistent, but it does not prove the gene regulates biosynthesis—it may facilitate transport/sequestration.

Intracellular localization of anthocyanins is not analyzed.

A function of “positive regulation of synthesis” is interpreted without evidence of activation of biosynthetic pathway structural genes.

DISCUSSION

The possibility that DoGSTF11 is a vacuolar carrier rather than a transcriptional regulator is completely omitted.

Study limitations are not discussed, such as:

Lack of functional validation of upstream TFs.

No KO, RNAi, or CRISPR lines used for DoGSTF11.

DoGSTF11–DoGST31 interactions are presented as key without discussing whether this interaction functionally affects transport.

Why was no comparison made with known anthocyanin transport genes like MATE or ABC transporters?

CONCLUSIONS

This study proposes that transcription factors DoNF-YB3 and DoIDD12 act as positive regulators of DoGSTF11, implicated in anthocyanin accumulation in Dendrobium officinale. However, the evidence presented to support direct transcriptional regulation is still preliminary and insufficient to establish a robust causal relationship.

First, the authors employ a yeast one-hybrid (Y1H) assay using the DoGSTF11 promoter as the “bait” sequence in the pHIS2 vector, co-transformed with a D. officinale cDNA library. This approach identified 21 potential interacting proteins, including DoNF-YB3 and DoIDD12, suggesting both may bind to the promoter region of the target gene.

Second, spatiotemporal expression analysis revealed similar expression patterns among DoGSTF11, DoNF-YB3, and DoIDD12, with higher expression in stem and leaf tissues of purple phenotypes and in later developmental stages. This correlation is supported by statistical analyses of gene expression levels and anthocyanin content.

Nevertheless, these results are merely correlative and, while valuable as preliminary evidence, do not allow for the conclusion of direct transcriptional regulation. To strengthen this claim, additional functional validation experiments are required, including:

1. i) dual-luciferase assays to quantify the activating capacity of DoNF-YB3 and DoIDD12 on the DoGSTF11 promoter in a heterologous plant system;
2. ii) ChIP-qPCR assays to demonstrate direct binding of these factors to the promoter in vivo within the chromatin context; and

iii) gain- and loss-of-function studies (via overexpression or silencing) to assess whether manipulation of DoNF-YB3 or DoIDD12 significantly alters DoGSTF11 expression and, consequently, anthocyanin accumulation.

In summary, although the article provides an interesting foundation for future research into anthocyanin regulation in D. officinale, the authors are advised not to overinterpret the Y1H and expression correlation results as conclusive evidence of direct regulation. It would be advisable to include at least one direct functional validation before making definitive claims about the proposed regulatory architecture.

Therefore, we recommend reformulating the manuscript’s wording to avoid categorical statements about direct regulation. Instead, more precise and cautious expressions should be used, such as:

“DoNF-YB3 and DoIDD12 are candidate transcriptional regulators of DoGSTF11, whose interaction with the promoter was suggested by Y1H assays and supported by similar expression patterns. Although these results point to a potential regulatory relationship, additional functional studies are required to confirm direct transcriptional regulation.”

This rewording will enhance the interpretative strength of the manuscript and align the claims with the actual scope of the data obtained, avoiding overinterpretations that could be challenged during the scientific review process.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The work has been significantly improved and advanced.

However, several sections could be improved to strengthen the clarity, reproducibility, and impact of the study.

DoGSTF11 localization is observed in both nucleus and membrane, but its specific role in these compartments is not discussed. Try to discuss potential functional implications of DoGSTF11’s dual localization—whether it indicates multiple roles beyond anthocyanin transport.

 

If it is possible for you, consider using ANOVA where appropriate for multiple comparisons (Figure 6C-D), but it is not necessary. 

 

Ensure figure legends are fully self-explanatory (some abbreviations like “OE3” or “NE/CE” are not defined immediately).

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

No comments

Author Response

Please see the attachment.

Author Response File: Author Response.pdf