Touch-Induced Transcriptional Changes in Flower Buds of a Non-Model Horticultural Plant Dianthus hybrida
Round 1
Reviewer 1 Report
The MS ” Touch-induced transcriptional changes in flower buds of a non-model horticultural plant Dianthus hybrida’ measured the RNA-Seq of Dianthus hybrid after touch treatment. Although it help us understand the response of Dianthus hybrid to external stimuli, I can not got the point in the MS.
1. Please concise the abstract, present the important resultant of the RNA-Seq, do not put discussion in abstract.
2. In introduction, JA was mentioned but not in the results.
3. Please present the key results in the results part, I did not find the key point in the results.
Author Response
We sincerely appreciate your help with our manuscript. As per your suggestions, we have throughly revised our manuscript.
Point 1: Please concise the abstract, present the important resultant of the RNA-Seq, do not put discussion in abstract.
Response 1: Thank you for your kind comment. The most important resultant was that flowers and leaves utilized the same mechanism (i.e. phosphorelay signaling), but different players, in touch stimulus response. To clarify that, we modified the abstract (p1, ln23).
Point 2: In introduction, JA was mentioned but not in the results.
Response 2: Thank you for this very useful suggestion. We added mention of JA biosyntesis and signaling genes upregulated (p4, ln134).
Point 3: Please present the key results in the results part, I did not find the key point in the results.
Response 3: Thank you very much for your comment. The key result is that 32 genes for serine/threonine kinase were detected upon external stimuli specifically in flowers, not in leaves. To emphasized this point, we added a sentence in the result section (p4, ln158).
Reviewer 2 Report
General comments
Dear authors,
The study is interesting. In my view, the addition of new data linking the changes the authors found in gene regulation to changes in flower bud quality/development or/and in the production of new flower buds is needed.
Specific comments
Keywords
I encourage authors to use words that are not written in the title of the manuscript.
Introduction: I encourage authors to write the practical utility of the study.
Materials and methods
L79. I encourage authors to write the age of the plants and the number of days the plants were grown until the application of touch-treatment.
L81. I encourage authors to write the conditions during plant growth in the chamber (e.g. temperature day/night, relative humidity, light intensity, light wavelength)
L. 81. The number of flower buds in each treatment is very small. In my opinion, this represent a weakness in a research design that may influence the conclusions. Did the authors take the flower buds from the same plant?
Results and discussion
L132. In my opinion, there are difficulties in directly comparing the results of their study with the results of other experiments carried out with different plants species, in different environmental conditions.
Author Response
We sincerely appreciate your help with our manuscript. As per your suggestions, we have thoroughly revised our manuscript.
Point 1: Keywords: I encourage authors to use words that are not written in the title of the manuscript.
Response 1: We removed “flower bud” and “Dianthus hybrida” and added “ornamental plant” (p1, ln26).
Point 2: Introduction: I encourage authors to write the practical utility of the study.
Response 2: Our final goal is to utilize touch stimulus response to develop new varieties. To emphasize this point, we added a sentence to the last paragraph in the Introduction section (p2, ln74).
Point 3: Materials and methods: L79. I encourage authors to write the age of the plants and the number of days the plants were grown until the application of touch-treatment.
Response 3: Thank you for your comment. I’m afraid but the age is difficult to stipulate as the plants used in this study were propagated in plant culture.
Point 4: L81. I encourage authors to write the conditions during plant growth in the chamber (e.g. temperature day/night, relative humidity, light intensity, light wavelength).
Response 4: We added the growth condition details to the first paragraph in the Materials and Methods section (p2, ln85).
Point 5: L. 81. The number of flower buds in each treatment is very small. In my opinion, this represent a weakness in a research design that may influence the conclusions. Did the authors take the flower buds from the same plant?
Response 5: Thank you for your kind comments. We took the flower buds from different plants (We added this description in the Materials and Methods (p2, ln89)). Albeit we had only three biological replicates, PCA showed the intra-replicate variance is quite small compared to the inter-replicate one, which supported the reproducibility of the experiments.
Point 6: Results and discussion
L132. In my opinion, there are difficulties in directly comparing the results of their study with the results of other experiments carried out with different plants species, in different environmental conditions.
Response 6: Thank you for your suggestion. We agree with you. Our main goal is to show that, even in two different organs, overall expression profiles are not so different and a certain number of homologs were commonly up- and downregulated upon touch stimuli. We toned down by removing the sentence “organ-specific molecular mechanism controlled touch stimulus response in flowers and leaves” from the Abstrsct section (p1, ln22), and by declairing “it must be considered that these DEGs specific to one condition might reflect the difference between the two species and/or growth condition” in the Results and Discussion section (p4, ln159).
Reviewer 3 Report
The manuscript ‘Touch-induced transcriptional changes in flower buds of a non-model horticultural plant Dianthus hybrida’ by Nishijima et al. focused on RNA sequencing of Dianthus hybrida to understand transcriptional regulations in touch-induced flower buds. The manuscript is well-planned, well-executed, and well-written. I have a few observations on the manuscript which may be useful for further improvement;
Introduction:
The introduction is well-written with rationale, justification, aim, and future implications. I appreciate it.
Materials and Methods
-The materials section is precisely written; however, I suggest the authors elaborate on the experimental setup with replications (biological-3 repn) and treatment details.
-Why the author used 40 min after touch treatment? Please justify.
-Please mention the make, region, and country name of all the equipment and reagents used in the study, like NovaSeq6000 (make, region/city, country).
Results and Discussion
-PCoA (fig 1b) showed a distinct variation among the touched samples. How do you correlate it? Is it an experimental error? Please justify.
-I would like to see the assemble statistics of the RNASeq data (supplementary), annotation summary, quantifiable differential summary, and top 10 GO in a figure.
-If not functional validation, at least a qPCR validation of the top up and down-regulated genes may be presented to justify their association with touch treatment.
Conclusions
-Please highlight the key up and down-regulated genes associated with touch therapy here.
-Future implications need more clarity.
Overall, it is a well-written manuscript and may be considered for publication with minor revisions.
Good luck with the revision.
Author Response
We sincerely appreciate your help with our manuscript. As per your suggestions, we have thoroughly revised our manuscript.
Materials and Methods
Point 1: The materials section is precisely written; however, I suggest the authors elaborate on the experimental setup with replications (biological-3 repn) and treatment details.
Response 1: Thank you for your kind comments. We added details of the sampling condition (p2, ln85).
Point 2: Why the author used 40 min after touch treatment? Please justify.
Response 2: We followed the conditions in a previous study on Arabidopsis leaves (Lee et al. 2015). We added a sentence in the materials section (p2, ln88).
Point 3: Please mention the make, region, and country name of all the equipment and reagents used in the study, like NovaSeq6000 (make, region/city, country).
Response 3: We added the equipment and reagent details as per your kind suggestion (p2, ln90; ln95; p3, ln96), thank you.
Results and Discussion
Point 4: PCoA (fig 1b) showed a distinct variation among the touched samples. How do you correlate it? Is it an experimental error? Please justify.
Response 4: Thank you for your helpful comment. We added “Genes with the highest loading values along the PC2 axis were a CEP1 homolog and three UDP-glycosyltransferase genes, which are involved in pollen development [33], indicating that the PC2 represented inflorescence developmental stage” (p3, ln120).
Point 5: I would like to see the assemble statistics of the RNASeq data (supplementary), annotation summary, quantifiable differential summary, and top 10 GO in a figure.
Response 5: We attached a supplementary file containing expression level, fold change, p-value and annotation summary of differentially expressed genes. For top GO terms, please refer to Figure 1D.
Point 6: If not functional validation, at least a qPCR validation of the top up and down-regulated genes may be presented to justify their association with touch treatment.
Response 6: We are very sorry but qPCR experiments were difficult to accomplish within seven days because of the following reason. To perform a precise analysis of qPCR, normalization by reference genes is required, and the choice of the reference genes should not solely rely on the gold standard used for the particular tissue type, organ type and plant species. These reference genes should always be systematically and experimentally validated. Unfortunately, there is still insufficient information on reliable reference genes in this plant. Alternatively, the pairwise Pearson correlation coefficients within touched and untouched plant replicates were 0.9 and 0.96, respectively, which we believe supported the reproducibility of the experiments. In view of this study, we didn’t focus on each component of touch-induced phosphorelay signaling. However, we plan to identify key players which control flower-specific touch stimulus responses in the future study, in which time-course qPCR experiments on top up and down-regulated genes will be required as the reviewer suggested. We really appreciate your kind and tender advice.
Conclusions
Point 7: Please highlight the key up and down-regulated genes associated with touch therapy here.
Response 7: We listed “upregulation of genes encoding protein kinases, xyloglucan endotransglucosylase, calmodulin, and JA biosynthetic and signaling components and downregulation of photosynthetic genes” (p5, ln172).
Point 8: Future implications need more clarity.
Response 8: Thank you for your kind suggestion. We focused on studies of the physiological mechanisms underlying touch stimulus response and breeding of new varieties by applying this mechanism (p5, ln180).
Round 2
Reviewer 1 Report
My concerns was addressed, I have no more questions.
Reviewer 2 Report
The authors made a good effort to improve the manuscript. The manuscript is acceptable in its present form.
