BBS Gene Expression and Its Diversity in the Genus Dendrobium

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

Dendrobium officinale is a medicinal plant that produces bibenzyl and phenanthrene derivatives. The production of these compounds can increase with fungal infection. Bibenzyl synthesis is regulated by bibenzyl synthase (BBS), but the regulation mechanisms in D. officinale are unclear. This study examined the effects of three mycorrhizal fungi on BBS gene expression and bibenzyl production. One fungus from the Tulasnellaceae family increased BBS gene expression and bibenzyl levels at specific times. Phylogenetic analysis of 19 BBS sequences from 12 Dendrobium species showed repeated gene duplication. The above results are of great significance for the further development of medicinal components in the Dendrobium. However, there are still several areas that can be improved:

1. Please provide the species names of the inoculated endophytic fungi, and if possible, include identification information.

2. Images of Dendrobium protocorms can be included in the main text.

3. It is recommended to briefly explain whether the selected endophytic fungi in this experiment increase secondary metabolite expression through interaction with the plant or by influencing the expression of secondary metabolites through the breakdown of organic or inorganic compounds in the environment.

4. In the phylogenetic tree of BBS genes, what criteria were used for the selection of species? For instance, if commonly used medicinal Dendrobium species were selected, please provide an explanation.

5. Some references on the phylogenetic relationships within the genus Dendrobium are outdated. Replacing them with more recent literature would enhance reliability.

 

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

Dear Reviewer

 

We really appreciate your positive and valuable comments. We revised our manuscript according to your comments.

 

Comment 1

1. Please provide the species names of the inoculated endophytic fungi, and if possible, include identification information.

 

Response to comment 1

These fungi are difficult to identify the species name even by phylogenetic analysis because the genus contains many unnamed taxonomic groups. In such cases, it is standard to define operational taxonomic groups (OTUs) by 97% sequence similarity for the internal transcribed spacer (ITS) region. The following sentence was added to Materials and Methods (page 3, line 108) to make it easier for readers to understand.

“The OTUs were defined at 97% sequence similarity for the internal transcribed spacer region.”

 

 

Comment 2

2. Images of Dendrobium protocorms can be included in the main text.

 

Response to comment 2

Images of Dendrobium protocorms were included in the main text as Figure 2, and the number of figures were reassigned.

 

 

Comment 3

3. It is recommended to briefly explain whether the selected endophytic fungi in this experiment increase secondary metabolite expression through interaction with the plant or by influencing the expression of secondary metabolites through the breakdown of organic or inorganic compounds in the environment.

 

Response to comment 3

The selected fungi in this study may increase secondary metabolite not through the breakdown of organic or inorganic compounds in the environment but through the interaction with the plant, because the expression of genes involved in bibenzyl biosynthesis were increased. We added the following sentences in Discussion.

“The results indicated that the fungi selected in this study increased specific secondary metabolites through interaction with the plant.” (page 11, line 418).

“Chen et al. showed that BBS gene expression was positively related to bibenzyl contents [17].” (page 12, line 479).

 

 

Comment 4

4. In the phylogenetic tree of BBS genes, what criteria were used for the selection of species? For instance, if commonly used medicinal Dendrobium species were selected, please provide an explanation.

 

Response to comment 4

The species selected in this study are important as medicinal species or representative horticultural species. To explain the reason for selection, we revised the sentence in Materials and Methods as follows (page 5 line193).

“In this study, we selected 12 important medicinal plants or representative horticultural species based on phylogenetic analysis [30–32], morphological classification of the genus Dendrobium [18,33], and the availability of plant specimens.”

 

 

Comment 5

5. Some references on the phylogenetic relationships within the genus Dendrobium are outdated. Replacing them with more recent literature would enhance reliability.

 

Response to comment 5

We added two recent literatures [31,32] on the phylogenetic relationships within the genus Dendrobium. Reference 30 was retained due to the importance in classification of genus Dendrobium.

31.    Wang, M.; Yu, W.; Yang, J.; Hou, Z.; Li, C.; Niu, Z.; Zhang, B.; Xue, Q.; Liu, W.; Ding, X. Mitochondrial genome comparison and phylogenetic analysis of Dendrobium (Orchidaceae) based on whole mitogenomes. BMC Plant Biol 2023, 23, 586.

32.    Burzacka-Hinz, A.; Dudek, M.; Szlachetko, D.L. Potential use of low-copy nuclear gene Xdh at lower taxonomic levels based on phylogenetic analysis of the nominal section of Dendrobium. Acta Societatis Botanicorum Poloniae 2024, 93, 1–13.

 

Our manuscript has undergone English revisions by MDPI's service.

Thank you for your consideration of our manuscript.

 

Yours sincerely,

 

Tomoko Takamiya, Ph.D.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is aimed to investigate the effect of three mycorrhizal fungi isolated from D. officinale in Japan on BBS gene expression and bibenzyl production. The article is well written and clearly illustrated. I think it can be published in its current form.

Comments on the Quality of English Language

English level is good.

Author Response

Dear Reviewer

 

We really appreciate your positive comments for this manuscript.

Our manuscript has undergone English revisions by MDPI's service.

 

Thank you for your consideration of our manuscript.

 

Yours sincerely,

 

Tomoko Takamiya, Ph.D.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is well-written, and the analyses are relevant. However, the analysis of fungal infection's induction of the expression of BBS genes does not correspond with the topic proposed in the issue, Distribution and Diversity of Orchids, or with the Biogeography and Macroecology section.

In any case, it would be a tangential effect of an ecological interaction. On the other hand, the phylogeny of the BBS genes in several species was analyzed. However, the functional biodiversity (BBS phylogeny) is disjointed with respect to the expression analysis. In other words, they are two related topics but could constitute separate, independent articles with greater affinity for the Genes magazine from the same MDPI publisher.

Due to the above, I consider that this article does not agree with the forum. A submission to another more related journal is suggested.

Comments on the Quality of English Language

Language quality is adequate

Author Response

Dear Reviewer

 

We wish to express our appreciation to the Reviewer for insightful comments, which have helped us significantly improve the paper.

 

Zhang L. et al. 2022 reported in Special Issue Distribution and Diversity of Orchids in Diversity, entitled [Symbiotic Culture of Three Closely Related Dendrobium Species Reveals a Growth Bottleneck and Differences in Mycorrhizal Specificity at Early Developmental Stages].

Our manuscript showed that the effect of mycorrhizal fungi studied in Zhang L. et al. 2022 on BBS gene expression and bibenzyl production in Dendrobium officinale, and these two reports are highly relevant. Our study provides insights into the diversity of orchid mycorrhizal specificity. Therefore, we consider that it is suitable for Distribution and Diversity of Orchids 2nd Edition.

 

The phylogenetic analysis of 31 BBS genes from 13 Dendrobium species provided knowledge on the phylogenetic relationships and evolution of the six BBS genes in D. officinale analyzed for expression. To describe these significances, the sentence in Introduction was revised as follows (page 2 lines 94).

“Additionally, we cloned BBS genes from 12 Dendrobium species, presenting a genetically diverse panel, and performed phylogenetic analysis to understand the similarity and duplication of the BBS genes in this genus, and the phylogenetic relationships among the six genes of BBS family in D. officinale.”

 

Our manuscript has undergone English revisions by MDPI's service.

 

Thank you for your consideration of our manuscript.

 

Yours sincerely,

 

Tomoko Takamiya, Ph.D.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been greatly improved after the revision and can be accepted now.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear author, I reiterate that your research is relevant to the induced expression of the orchid's BBS genes and mycorrhizae's specificity. Still, it is not a study of distribution or diversity but of tissue culture, induction of expression genetics, plant associations with mycorrhizae, biotechnological developments, and even practical management applications. It remains unclear to me how this work establishes a relationship with biogeography or macroecology. The only similar topic is the use of the genus Dendrobium; furthermore, its medicinal use is insisted upon as justification for the study and not the diversity of the species or the biogeographic distribution as the center of the topic. Do you consider that only three mycorrhizae isolated from a plant in the field are a macroecological association or sufficient diversity approach? It requires a population study and a representative sample size to establish the diversity of mycorrhizal species in one population, with an analysis of completeness or functional diversity.

The manuscript is good, with a lot of application, but it still does not have an adequate context for the magazine, the volume and the theme.