Comprehensive Metabolomic–Transcriptomic Analysis of the Regulatory Effects of Armillaria mellea Source Differences on Secondary Metabolism in Gastrodia elata

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Han et al. describe on an integrated metabolomic and transcriptomic analysis of the symbiosis between Gastrodia elata and five different strains of Armillaria mellea. The Authors analyzed yield, secondary metabolite accumulation, and gene expression differences associated with fungal strain variation. While the experimental work is extensive and the dataset is valuable, the manuscript in its current form has important weaknesses in interpretation and discussion. Several conclusions are described too confidently, and key theoretical frameworks in plant biology are insufficiently addressed. Therefore, major revision is required before this manuscript can be considered for publication in Biology.

Major Comments:
1. Discussion: The results clearly show that strain A promotes high accumulation of gastrodin (GAS) and HBA but results in lower biomass, whereas strain B produces the highest yield with reduced secondary metabolite levels. This pattern is highly consistent with the well established growth–defense trade-off in plants. However, this important concept is only weakly discussed in the current manuscript. The authors should clearly introduce the growth–defense trade-off framework and clearly explain how their results fit into this model. Relevant references should be cited. Without this theoretical context, the biological meaning of the results remains insufficiently developed.
2. Discussion: The Authors discuss strain-specific symbiotic mechanisms mainly based on transcriptomic and metabolomic responses of G. elata. However, no direct data from A. mellea (genome, transcriptome, secreted metabolites) are presented. As a result, some mechanistic interpretations appear overstated. The authors should clearly state that their conclusions are host-centered and indirect. In addition, the lack of fungus-side data must be unambiguously described as a limitation, and future studies incorporating fungal omics analyses should be clearly proposed.

3. Data Availability: Accession numbers for the transcriptomic and metabolomic datasets are not provided. This is a serious omission. The authors must deposit the data in appropriate public repositories (e.g., NCBI SRA) and include the accession numbers in the Materials and Methods section and/or the Data Availability Statement.

Minor Comments:
1. Introduction (p.2, line 37): “sterides” should be corrected to “steroids” or “sterols”.

2. Fig. 4 caption: 207 genes expression levels” should be corrected to “207 gene expression levels”.
3. Fig. 7 caption: “Ko04075” should be corrected to “ko04075”.
4. Fig. 9 caption: “A:Nine-quadrant” should be revised to “A: Nine-quadrant”. Besides, “Ko04075” should be corrected to “ko04075”.
5. Unit notation: The Authors are using both, for example, “ug/mg” and “µg·mg⁻¹”. The unit notation should be unified using SI-compliant format. Also, ion charges (e.g., K⁺, Ca²⁺) should be written in superscript, and structural prefixes for chemical compounds, such as n (normal), must be italic.
6. In Reference 46, the DOI ends with a duplicated period. This formatting error should be corrected.

Author Response

Major Comments Comments 1: Discussion: The results clearly show that strain A promotes high accumulation of gastrodin (GAS) and HBA but results in lower biomass, whereas strain B produces the highest yield with reduced secondary metabolite levels. This pattern is highly consistent with the well established growth–defense trade-off in plants. However, this important concept is only weakly discussed in the current manuscript. The authors should clearly introduce the growth–defense trade-off framework and clearly explain how their results fit into this model. Relevant references should be cited. Without this theoretical context, the biological meaning of the results remains insufficiently developed.

Response 1: Thank you for your comprehensive review and insightful comments on our manuscript, your suggestions are of great value in enhancing the quality of the article. In the resubmitted manuscript, we discussed the theoretical framework that aligns with the research findings in the discussion section, while also citing relevant references. In the discussion section, Line 347-361: The widely recognized "Growth-Defense Trade-off" (GDT) theory in botany posits that due to limited resources available to plants (such as carbon, nitrogen, and phosphorus), they must allocate resources meticulously between two core biological activities: "growth" (e.g., increasing biomass) and "defense" (e.g., synthesizing chemical compounds). During the growth of GE, when A. mellea hyphae invade its cortex, the plant secretes enzymes to break down polysaccharides and proteins in the hyphae, converting them into carbon and nitrogen sources needed for its own growth [27]. In this study, Group A exhibited significantly higher accumulation of GAS, HBA, and parishin metabolites compared to other groups, although its yield was lower than that of Group B. Conversely, Group B achieved the highest yield but had lower concentrations of active compounds. This pattern aligns closely with the GDT theory. Group A likely prioritized defense over growth by downregulating genes involved in growth and increasing the secretion of defense proteins, directing carbon and nitrogen resources toward the production of defensive compounds [28]. This shift may explain Group A's superior performance in active compound accumulation. In the discussion section, we also mentioned that, CHS, as the first enzyme in flavonoid biosynthesis, can channel carbon flow from the phenylpropanoid pathway into flavonoid production (lines 383-384); Environmental stimuli (e.g., light, temperature) can activate 4CL, enhancing carbon flow toward flavonoid synthesis and thereby increasing metabolite yield [33] (lines 383-384). These also reinforce the conformity of the Gastrodia data from Group A and Group B with the growth-defense trade-off theoretical framework.

Comments 2: Discussion: The Authors discuss strain-specific symbiotic mechanisms mainly based on transcriptomic and metabolomic responses of G. elata. However, no direct data from A. mellea (genome, transcriptome, secreted metabolites) are presented. As a result, some mechanistic interpretations appear overstated. The authors should clearly state that their conclusions are host-centered and indirect. In addition, the lack of fungus-side data must be unambiguously described as a limitation, and future studies incorporating fungal omics analyses should be clearly proposed.

Response 2: Thank you for your comprehensive review and insightful comments on our manuscript. This study exclusively measured and analyzed the omics data of Gastrodia elata, with the objective of indirectly inferring differences in secondary metabolic accumulation and regulation resulting Armillaria mellea from different sources. However, it lacks corresponding fungal data. In the revised manuscript, we have meticulously refined the language, critically re-evaluated certain overly speculative mechanistic explanations, and comprehensively restructured the discussion section to ensure linguistic precision and avoid overinterpretation. For example, in the discussion section, line 414-421: This study reveals the association between the origin of A. mellea and the metabolic and transcriptional responses of GE, suggesting that high-quality strains may regulate host metabolic pathways by secreting specific signaling molecules, influencing secondary metabolite accumulation [41]. However, this research lacks direct analysis of the A. mellea genome and its secreted metabolites, failing to clarify whether the strains exhibit genetic or geographical differences. The conclusions are host (GE)-centric and indirectly inferred, representing a limitation. Future research will incorporate fungal omics analysis for deeper investigation.   Comments 3: Data Availability: Accession numbers for the transcriptomic and metabolomic datasets are not provided. This is a serious omission. The authors must deposit the data in appropriate public repositories (e.g., NCBI SRA) and include the accession numbers in the Materials and Methods section and/or the Data Availability Statement. Response 3: Thank you for pointing this out. We sincerely apologize for the significant oversight due to the author's negligence. Here, We provide an explanation regarding the uploading status of the omics datasets. The transcriptome datasets were submitted to a public repository (NCBI SRA). Accession numbers range from SAMN54581423 to SAMN54581440, and the accession numbers have been added to the Data Availability Statement in the revised manuscript (Line 617-618). Due to confidentiality issues related to the sequencing platform, the authors have provided an Excel sheet of metabolite data for all samples (Supplementary Material, metabolome_sample_data) after data imputation and filtering in the supplementary materials. The methods for data imputation and filtering were as follows: missing values were filled with one-fifth of the minimum value for each row (metabolite), followed by calculating the coefficient of variation (CV) for QC samples, and only metabolites with a CV value less than 0.5 were retained. We greatly appreciate your feedback. Accession numbers of transcriptome datasets are as follows (Table 1): Table 1 Accession numbers of transcriptome datasets Accession Sample Name SPUID Organism Tax ID Isolate SAMN54581423 A1 A1 Gastrodia elata 91201 Plant SAMN54581424 A2 A2 Gastrodia elata 91201 Plant SAMN54581425 A3 A3 Gastrodia elata 91201 Plant SAMN54581426 B1 B1 Gastrodia elata 91201 Plant SAMN54581427 B2 B2 Gastrodia elata 91201 Plant SAMN54581428 B3 B3 Gastrodia elata 91201 Plant SAMN54581429 C1 C1 Gastrodia elata 91201 Plant SAMN54581430 C2 C2 Gastrodia elata 91201 Plant SAMN54581431 C3 C3 Gastrodia elata 91201 Plant SAMN54581432 D1 D1 Gastrodia elata 91201 Plant SAMN54581433 D2 D2 Gastrodia elata 91201 Plant SAMN54581434 D3 D3 Gastrodia elata 91201 Plant SAMN54581435 E1 E1 Gastrodia elata 91201 Plant SAMN54581436 E2 E2 Gastrodia elata 91201 Plant SAMN54581437 E3 E3 Gastrodia elata 91201 Plant SAMN54581438 CK-B1 CK-B1 Gastrodia elata 91201 Plant SAMN54581439 CK-B2 CK-B2 Gastrodia elata 91201 Plant SAMN54581440 CK-B3 CK-B3 Gastrodia elata 91201 Plant   Minor Comments: Comments 1: Introduction (p.2, line 37): “sterides” should be corrected to “steroids” or “sterols”. Response 1: Thank you for your comprehensive review and insightful comments on our manuscript. In the resubmitted manuscript, we have revised "sterides" to "steroids".   Comments 2: Fig. 4 caption: 207 genes expression levels” should be corrected to “207 gene expression levels”. Response 2: Thank you for pointing this out. We agree with this comment. In the resubmitted manuscript, we have revised "genes expression levels" to "gene expression levels" in Fig. 4 caption.   Comments 3: Fig. 7 caption: “ko04075” should be corrected to “ko04075”. Response 3: Thank you for pointing this out. We agree with this comment. In the resubmitted manuscript, we have revised "Ko04075" to "ko04075" in Fig. 7 caption.   Comments 4: Fig. 9 caption: “A:Nine-quadrant” should be revised to “A: Nine-quadrant”. Besides, “ko04075” should be corrected to “ko04075”. Response 4: Thank you for pointing this out. We agree with this comment. In the resubmitted manuscript, we have revised them.   Comments 5: Unit notation: The Authors are using both, for example, “ug/mg” and “µg·mg⁻¹”. The unit notation should be unified using SI-compliant format. Also, ion charges (e.g., K⁺, Ca²⁺) should be written in superscript, and structural prefixes for chemical compounds, such as n (normal), must be italic. Response 5: Thank you for pointing this out. We agree with this comment. In the resubmitted manuscript, we have unified the unit notation using SI-compliant format (Change ug/mg to µg·mg-1 uniformly), and standardized the notation of ion charges as well as the structural prefixes of chemical compounds.   Comments 6: In Reference 46, the DOI ends with a duplicated period. This formatting error should be corrected. Response 6: Thank you for pointing this out. We agree with this comment. In the resubmitted manuscript, we have revised it.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I really enjoyed reading this manuscript. It presents combined metabolomic and transcriptomic analysis to examine how different Armillaria mellea sources influence secondary metabolism, gene expression, and yield in Gastrodia elata. I believe that the topic is relevant to medicinal plant cultivation and quality control, and the authors employ a really comprehensive multi-omics workflow with a substantial dataset. The study has clear practical significance; however, I have noted a few bits related to interpretation, experimental clarity, and statistical reporting that should be addressed to strengthen rigour and ensure appropriate conclusions.

 

Major comments

Interpretation of Armillaria mellea effects

The manuscript frequently implies direct regulatory control of G. elata metabolism by A. mellea. However, no molecular data from A. mellea (e.g., genomic, transcriptomic, or metabolomic analyses) are included, and all conclusions are inferred from host responses. I think it would be worthwhile to use differnt language throughout the manuscript and really clarify that the findings demonstrate associations between A. mellea source and G. elata metabolic and transcriptional responses. I might also be worth noting this as a limitation in the Discussion.

Characterisation of A. mellea sources

The five A. mellea strains are distinguished only by commercial origin, with no genetic, physiological, or ecological characterisation provided. Could it be clarified whether these strains are known to differ genetically or geographically, or whether they are defined solely by supplier. If additional information is unavailable, please include this as a limitation which could effect interpretation and reproducibility.

Experimental design and replication

The description of replication is inconsistent and confusing (e.g., five replicates for cultivation versus n = 3 for omics analyses). It is not very clear which samples were used for metabolomics, transcriptomics, and chemical analyses, and whether these are biological or technical replicates. A table or schematic summarising sample numbers, replicate types, and analyses performed could be useful. This will help improve clarity and reproducibility.

Statistical Analysis and multiple-testing correction

While commonly used thresholds (VIP ≥ 1, FC ≥ 2) are applied in metabolomic analyses, adjusted p-values are not consistently reported. In addition, the statistical correction applied during KEGG enrichment analysis is not clearly stated. Could the multiple-testing correction methods be described that are used for metabolomics, transcriptomics, and pathway enrichment analyses?

Hormone signalling and mechanistic claims

The discussion emphasises the role of jasmonic acid signalling (e.g., JA, JAZ, MYC2) in regulating quality differences. However, hormone measurements are limited and functional validation is not provided. Could moderate mechanistic claims be noted or could these pathways be framed as putative regulatory mechanisms? Additionally, validation experiments would be suggested in the text as future work.

 

Minor comments

Language and style:
Reduce repetitive phrasing and revise overly long sentences for improved clarity. Examples include the phrase “this study, for the first time” which is used repeatedly and could be reduced to improve conciseness. Several sentences combine results and interpretation and could be split into shorter statements for clarity eg “Through the combined analysis of metabolomics and transcriptomics, it was found that the accumulation of gastrodin, parishin, and p-hydroxybenzyl alcohol was significantly increased in Group A, which may be related to the differential expression of key genes involved in phenylpropanoid biosynthesis and plant hormone signal transduction pathways.”. Maybe something like “Combined metabolomic and transcriptomic analyses showed increased accumulation of gastrodin, parishin, and p-hydroxybenzyl alcohol in Group A. These changes were associated with differential expression of genes involved in phenylpropanoid biosynthesis and hormone signaling pathways.” Could work better?

Long sentences with multiple subordinate clauses could be simplified to improve readability. Eg “The results demonstrated that under the influence of different A. mellea strains, the metabolic pathways related to secondary metabolism, including flavonoid biosynthesis, phenylpropanoid biosynthesis, and hormone signal transduction, showed significant differences, indicating that A. mellea may play an important regulatory role.” Could be alter to “Different A. mellea strains were associated with significant differences in secondary metabolic pathways, including flavonoid and phenylpropanoid biosynthesis as well as hormone signaling. These results suggest a potential regulatory role of A. mellea.”

Lastly, some results are reiterated verbatim in the Discussion. Could you focus the Discussion on interpretation rather than repeating of numerical outcomes.

Figures:
A lot of the figures are dense and difficult to interpret. Consider increasing label size or relocating detailed versions to supplementary materials. Figure 2, 3A, 4, 5A, 8, 9BCD,

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article "Comprehensive Metabolomic-Transcriptomic Analysis of the Regulatory Effects of Armillaria mellea Source Differences on Secondary Metabolism in Gastrodia elata" by Duo Han, Chengcui Yang, Liuyuan Bao, Li Dong, Haiyan He, Peng Tang, Yongzhi Zhang, Fen Xiong, Honggao Liu, and Shunqiang Yang presents the authors' data on the metabolomics and transcriptomics of interactions between two parasitically and symbiotically interdependent species.
This study is of particular interest for its approaches to identifying sensitive key points of interdependence, as these interactions remain an insurmountable obstacle to biotechnological production based on such interactions between species.
The manuscript is compiled in accordance with the guidelines and contains the necessary sections and illustrative material for visualizing the results. There are some problems associated with the fact that the authors did not specify the time ranges of the interactions, making it difficult to judge the reproducibility of this result due to the insufficient sample size. Cultivation details and volumes are not provided, making it difficult to evaluate the work. Differences between strains are not described, making it unclear why they were chosen. It's also unclear, for example, what it means to have tubers of the same size. How exactly can this be verified? The lack of photographic evidence in technological papers always seems somewhat inappropriate. After all, the authors do not provide evidence that they actually worked with the described specimens (anything can happen in laboratories: losses, accidental mix-ups, etc.). This is why a one-time experiment is always viewed with caution. And if the authors insist on reliability, readers should have no reason to distrust the methodology (which is described in a chaotic and incomplete manner). Another problem is the lack of logical controls—samples a priori excluded from the interaction chain.
In this regard, there is a question about the resulting scheme. Based on the positioning of the intake and exchange of metabolites between species, it would be logical to have a diagram clearly tracing which organism, in which situation, produced the "replacement synthesis" of a hormone or protein.
Even recognizing how difficult this is, it should be taken into account that this would qualify this work as complete.
I believe efforts should be made to revise the description of the methodology, taking into account differences in interactions by age and sampling time.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised version is now suitable for acceptance in Biology.

Author Response

Comments 1: The revised version is now suitable for acceptance in Biology.

Response 1: We are glad to receive your reply. we would like to thank the editor and the reviewer for your professional review and thoughtful suggestions on our article. All of these suggestions were invaluable and helpful in revising and improving our paper. Thank you once again.

Reviewer 3 Report

Comments and Suggestions for Authors

In the article "Comprehensive Metabolomic-Transcriptomic Analysis of the Regulatory Effects of Armillaria mellea Source Differences on Secondary Metabolism in Gastrodia elata," the authors made the necessary corrections and corrected inaccuracies.
However, while reviewing the manuscript, I had questions about the keywords and figure captions.
Please ensure that all abbreviations are explained in the caption and that the information is completely clear without reference to the text. Any names and abbreviations in figures should be clearly understood, as in open-access journals, figure information is often placed separately from the main text.
Regarding keywords, I believe that using "Active components" in this case does not provide any advantage in searching for this publication. I recommend adding "plant-fungus interaction" or something similar.

Author Response

Comments 1: While reviewing the manuscript, I had questions about the keywords and figure captions. Please ensure that all abbreviations are explained in the caption and that the information is completely clear without reference to the text. Any names and abbreviations in figures should be clearly understood, as in open-access journals, figure information is often placed separately from the main text. Regarding keywords, I believe that using "Active components" in this case does not provide any advantage in searching for this publication. I recommend adding "plant-fungus interaction" or something similar. Response 1: Thank you for your comprehensive review and insightful comments on our manuscript. We agree with you. In the resubmitted manuscript, we have annotated all abbreviations in the figures to ensure that the information can be presented and fully understood even when separated from the main text.  Regarding keywords, we agree with you very much. In the resubmitted manuscript, we have modified "Active components" to "Gastrodia-fungus interaction".