Integrated Transcriptome and Metabolome to Elucidate the Mechanism of Aluminum-Induced Blue-Turning of Hydrangea Sepals
Juliana Degenhardt-Goldbach
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript titled "Integrated Transcriptome and Metabolome to Elucidate the Mechanism of Aluminum-Induced Blue-Turning of Hydrangea Sepals" demonstrates scientific merit, and the results are intriguing, revealing novel insights into how aluminum affects the formation of blue coloration in Hydrangea macrophylla, both from molecular and metabolic perspectives.
However, the text is confusing and requires revision. While the Discussion section is clear and provides good contextualization and explanation of the observed results, other sections need to be improved.
Starting with the abstract itself, which is unclear and fails to summarize the findings, with statements such as "Aluminum ion only induced the significant up-regulation of the upstream gene 4CL." Additionally, the abstract does not mention the relationship between the metabolome and transcriptome.
The Introduction section also lacks clarity in do not properly presents the research of the work.
In the Materials and Methods section, it is mentioned that UHPLC was used to evaluate metabolites, but in the results they are presented as GC-MS results. Furthermore, the metabolome data processing are not clearly described.
In the transcriptome section of the methodology, it is unclear which samples were used. Additionally, in the qPCR analysis, the genes evaluated are not mentioned until the results section.
On line 158, I believe that the Nanodrop should have been used to evaluate the quality of cDNA, not the library as stated.
Regarding the results, the manuscript presents abundant and relevant findings; however, the presentation hinders understanding of the article. Section 3.4 is particularly confusing, making it difficult to follow the results presented, such as the mentioned profiles. It is unclear what these profiles represent in terms of flower development stages.
The explanations between lines 398 and 400 are insightful but are more suited for discussion rather than results. If left in the results section, references should be included.
In conclusion, the authors have valuable results for a paper to be accepted by this journal; however, they should revise the text presentation, especially in the abstract, introduction, materials and methods, and results sections.
Author Response
Comments 1: Starting with the abstract itself, which is unclear and fails to summarize the findings, with statements such as "Aluminum ion only induced the significant up-regulation of the upstream gene 4CL." Additionally, the abstract does not mention the relationship between the metabolome and transcriptome.
Response 1: Agreed, we have rewritten the 'abstract' according to the reviewer 's suggestion. Firstly, the effect of aluminum treatment on the flower color of Hydrangea macrophylla was expounded from the physiological level, and the relationship between aluminum ion, delphinidin and flower color was clarified. Secondly, using metabolomics and transcriptomics, taking anthocyanin and aluminum ion as the key points, from the perspective of anthocyanin synthesis, transport and aluminum ion transport, the mechanism of anthocyanin synthesis and transport induced by aluminum and the formation mechanism of 'blue complex' were further explored. Changes have been marked in red and their position in the revised manuscript - page number: 1; line number: 11-35.
Comments 2: The Introduction section also lacks clarity in do not properly presents the research of the work.
Response 2: Agreed. As suggested by the reviewers, we have reorganized our thinking and revised the introduction. Firstly, the characteristics of Hydrangea macrophylla were introduced. Secondly, the formation of flower color is affected by many factors, including core anthocyanin types, anthocyanin transport methods, anthocyanin accumulation and metal ions. Therefore, the work content of this study is cut from these parts, and the introduction of the latter part is also written according to this logic. Changes have been marked in red and their position in the revised manuscript - page number: 2; line number: 45-110.
Comments 3: In the Materials and Methods section, it is mentioned that UHPLC was used to evaluate metabolites, but in the results they are presented as GC-MS results. Furthermore, the metabolome data processing are not clearly described.
Response 3: We are very sorry for our incorrect writing, and the 'UHPLC-MS / MS' used in 'Materials and Methods' is the correct content. We corrected the writing errors in the results and further supplemented the information of metabolite extraction, analysis and data processing in 'Materials and Methods'. Changes have been marked in red and their position in the revised manuscript - page number: 4, 5; line number: 157-180, 231-233.
Comments 4: In the transcriptome section of the methodology, it is unclear which samples were used. Additionally, in the qPCR analysis, the genes evaluated are not mentioned until the results section.
Response 4: We are sorry that we did not introduce the materials used clearly. We have supplemented the sample information used in the transcriptome and metabolome sections of the method according to the reviewer 's recommendations (the samples used were sepals at three stages of flower development in CK group and 3 g/L aluminum treatment group. Each sample included three biological replicates). Changes have been marked in red and their position in the revised manuscript - page number: 4; line number: 157-159,183-184.
Comments 5: On line 158, I believe that the Nanodrop should have been used to evaluate the quality of cDNA, not the library as stated.
Response 5: According to the reviewer 's suggestion, we checked the content of the 158 line and consulted the method, and corrected the ' library ' to ' cDNA library ', but we were sorry that the ' Agilent Bioanalyzer 2100 system ' was used to evaluate the quality of cDNA in the method provided by the sequencing company, so this was not modified. Changes have been marked in red and their position in the revised manuscript - page number: 4; line number: 187-191.
Comments 6: Regarding the results, the manuscript presents abundant and relevant findings; however, the presentation hinders understanding of the article. Section 3.4 is particularly confusing, making it difficult to follow the results presented, such as the mentioned profiles. It is unclear what these profiles represent in terms of flower development stages.
Response 6: We have revised the textual presentation of the results in each section as suggested by the reviewers. In Section 3.4, each profile represents a cluster, genes with similar temporal kinetic patterns are classified into the same cluster, and the profile trend represents the overall direction of gene expression over time in that cluster, representing the trend of gene expression during flower development. We have improved the description of the results and the figure in this section to make it easier to understand. Changes have been marked in red and their position in the revised manuscript - page number: 8; line number:291-313.
Comments 7: The explanations between lines 398 and 400 are insightful but are more suited for discussion rather than results. If left in the results section, references should be included.
Response 7: Agreed. As suggested by the reviewer, we have further revised this explanation and cited references. Changes have been marked in red and their position in the revised manuscript - page number: 12; line number: 410-413.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIt was interesting that they evaluated the treatments from germination (growth and development); however, it is missing that the substrate used from germination and if it was the same during the development of the plant is indicated.
The times of germination, development and phenological stages; if possible, explain chronologically; and consider the environmental conditions in which the plants were developed.
The third level titles of the materials and methods do not coincide with those presented in the results.
No results are observed from the UHPLC-MS / MS system (coupled to masses).
It is explained moderately and requires further explanation, related to the physiology of the plant; the metabolic route of aluminum.
At higher concentrations it accumulates in the root, why.
Author Response
Comments 1: It was interesting that they evaluated the treatments from germination (growth and development); however, it is missing that the substrate used from germination and if it was the same during the development of the plant is indicated.
Response 1: Agree, so we have completed the use of matrix formula supplement. During the experiment after planting, there was no pot change treatment, so the matrix used was the same matrix. Changes have been marked in red and their position in the revised manuscript - page number: 3; line number: 113-115.
Comments 2: The times of germination, development and phenological stages; if possible, explain chronologically; and consider the environmental conditions in which the plants were developed.
Response 2: According to the reviewer 's suggestion, we redefined the three periods as S1 stage (bud stage), S2 stage (discoloration stage) and S3 stage (full-bloom stage) according to the time sequence and flower development morphological characteristics. Changes have been marked in red and their position in the revised manuscript - page number: 3; line number: 122-123.
Comments 3: The third level titles of the materials and methods do not coincide with those presented in the results.
Response 3: We have made a unified modification of the title format according to the format of the standard template.
Comments 4: No results are observed from the UHPLC-MS / MS system (coupled to masses).
Response 4: We are sorry to miswrite 'UHPLC-MS / MS' as 'GC-MS', and we have corrected it. In fact, this part is the result of UHPLC-MS / MS analysis.
Changes have been marked in red and their position in the revised manuscript - page number: 5-6; line number: 231-261.
Comments 5: It is explained moderately and requires further explanation, related to the physiology of the plant; the metabolic route of aluminum.
Response 5: In order to cope with aluminum toxicity, plants have developed a variety of aluminum tolerance mechanisms, including external exclusion mechanisms and internal tolerance mechanisms. In Hydrangea macrophylla, aluminum ions can be absorbed and transported to the vacuole of the sepals for storage, thereby avoiding damage to the cytoplasm and other organelles. It can also bind to delphinidin and promote the formation of blue sepals. Therefore, this article mainly introduces the related protein genes involved in aluminum ion transport.
Comments 6: At higher concentrations it accumulates in the root, why.
Response 6: The most sensitive site of plants to aluminum toxicity is the root, and aluminum ions can be retained in the cell wall by binding to the components in the root cell wall (such as gum, cellulose, etc.); in addition, aluminum ions have high mobility in soil solution and are easy to reach plant roots through mass flow and diffusion. In summary, when the concentration of aluminum ions is high, aluminum accumulates in plant roots.
Author Response File: Author Response.pdf
