Genome-Wide Identification of the BREVIS RADIX Gene Family in Foxtail Millet: Function, Evolution, and Expression
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper devoted to identification and characterization of the BRX gene family in foxtail millet: including analysis of the function, evolution, and gene expression
Line 12: BRX - need full name during first mentioning.
Lines 56-57_ connection between parts are required.
Line 67: „gene family members influence the development of multiple organs“ – gene family members can not influence development itself.
Lines 71 – 88: very comprehensive description. However, it seems authrs didnnt consider complexity of plant developmnet. For exmaple, if some BRX gene expression increase capacity of auxin canalisation to the root, they, as next step, improve hormonal balance in shoot (more nutrinets uotake, moré hormine production) and increase overal plant health and productivity. Plant is a complex system, indeed,
Line 126: „All This study used“ ??Maybe All is redudndant?
The same for line 147.
Line 175: „The first interaction pair“ ? Ithink it sinteraction prediction. Interaction can be mentioned only after in situ prove.
Loines 211-213: „13 of the 15 SiBRX genes contained auxin-responsive elements, whereas SiBRX3, SiBRX4, SiBRX5, SiBRX8, SiBRX9, and SiBRX13 lacked these elements.“ ?? Please, check: 15 – 13 =2, not 6.
Line 223: the most importnat informtaion is cell-typespecifci, Organs you menationed contains many cell types.
Line 234: it will be great to linked this part with some physiological paraetrs as photosynthesis, for example. Plus with precise localization: mesophyll cell? Stomata cell? Etc. Stress treatments induce different (even opposite) effcet in different cell type. That’s why in situlocalisatiois a key step in elucidating mechnaism.
Line 390: „mechanisms of their responses to different abiotic stress factors“ ??? Authors need to shown in situ (cell type/status gene expression dynamics to tell about mechanism. Each cell type respond to stress differently and stress-resistance is ability oft he plants to resolve these difference (epigenetic conflicts).
https://academic.oup.com/jxb/article/73/7/2021/6481726
Lines 651, 657 etc : wrong citations. Gabor is the first name, not family name, for example. Please, re-check all citations.
Author Response
Dear reviewer,
I hope this message finds you well. We sincerely appreciate the time and effort you’ve dedicated to thoroughly reviewing our paper and offering such professional and highly valuable feedback. Your rigorous approach and profound insights have truly inspired us. These comments are of utmost importance in enhancing the quality of our paper. We hold each of your suggestions in high regard and have addressed them with great earnestness. Below is our detailed response to the issues you raised.
Comments1：Line 12: BRX - need full name during first mentioning.
Response1：Dear reviewer, thank you so much for your careful review and the highly valuable feedback. Regarding your comment on line 12 about the need to provide the full name of “BRX” during its first mention, I truly appreciate your keen - eyed observation. We’ve immediately rectified this oversight in the revised paper. At the first instance where “BRX” appears, we’ve now included its complete name, [BREVIS RADIX], to make the content more accessible and understandable for readers. Your input is extremely important to us as it helps us enhance the overall quality and clarity of the paper. Once again, thank you for your time and guidance.
Comments2：Lines 56-57_ connection between parts are required.
Response2: Dear reviewer, I sincerely appreciate your meticulous review and the constructive feedback regarding the lack of connection between parts in lines 56 - 57. I completely understand the significance of maintaining a logical and seamless flow in the text. To address this issue, I’ve carefully analyzed the context and have inserted well - crafted transitional elements. For instance, I’ve added a bridging sentence that clearly delineates the relationship between the preceding and following parts. The new sentence, not only elaborates on how the ideas in these parts are interconnected but also serves as a guide for the readers to follow the thought progression more easily. I’m confident that these revisions will enhance the overall coherence and readability of this section, and I’m grateful for your input which has helped in refining the paper to a higher standard.
Comment3:Line 67: „gene family members influence the development of multiple organs“ – gene family members can not influence development itself.
Response3:Dear Reviewer,I am extremely grateful for your perceptive comment regarding Line 67. I deeply apologize for the imprecise wording in the original statement. You are absolutely correct in pointing out that gene family members cannot directly “influence the development” on their own.To address this issue and convey a more accurate biological understanding, I have thoroughly revised the sentence. Instead of stating “gene family members influence the development of multiple organs,” I have rephrased it to “the expression patterns and regulatory mechanisms of gene family members contribute significantly to the modulation of various molecular and cellular processes that are indispensable for the proper development of multiple organs.”This revised version better captures the complexity of how gene family members interact within biological systems. It acknowledges that their influence on organ development is indirect, mediated through a cascade of molecular events such as transcriptional regulation, post - translational modifications, and participation in signaling pathways. By emphasizing these underlying mechanisms, the new statement adheres to scientific rigor and provides a clearer picture of the actual biological phenomena at play.
Comment4:very comprehensive description. However, it seems authrs didnnt consider complexity of plant developmnet. For exmaple, if some BRX gene expression increase capacity of auxin canalisation to the root, they, as next step, improve hormonal balance in shoot (more nutrinets uotake, moré hormine production) and increase overal plant health and productivity. Plant is a complex system, indeed.
Response4: Thank you very much for your constructive feedback and for highlighting the complexity of plant development that we may have overlooked in our manuscript. We wholeheartedly agree that the plant is an intricate and highly interconnected system, and the influence of gene expression on different plant parts and overall plant health is far more complex than we initially presented.We deeply appreciate your example regarding the BRX gene. It serves as a perfect illustration of how the regulation of gene expression in one part of the plant, such as the root, can have far - reaching consequences for other parts, like the shoot, and ultimately impact the entire plant’s well - being and productivity.To address this concern, we have made significant revisions to our manuscript. First, we have added a new section in the introduction to emphasize the complexity of plant development and the inter - relatedness of different plant organs. In this section, we cite several recent studies that demonstrate how local gene expression changes can trigger systemic responses in plants.Regarding the specific case of the BRX gene, we have incorporated a detailed analysis in the results and discussion part. We describe how an increase in BRX gene expression in the root can enhance auxin canalization, which in turn affects hormonal balance in the shoot. We also discuss the potential molecular mechanisms behind these processes, such as the long - distance transport of signaling molecules and the cross - talk between different hormonal pathways.Furthermore, we have updated our conclusions to reflect a more comprehensive understanding of plant development. We now emphasize that any study of gene function in plants should take into account the complex network of interactions between different organs and the influence of gene expression on overall plant health and productivity.
Comment5: „All This study used“ ??Maybe All is redudndant? The same for line 147.
Response5: Dear Reviewer,We sincerely apologize for the oversight in our manuscript. It was entirely our fault, and we deeply appreciate you bringing this issue to our attention.We have already made the necessary revisions. After your valuable feedback, we carefully re - examined the relevant parts where the problem occurred, and made corrections to ensure the accuracy and comprehensiveness of the content. Moreover, we conducted a thorough review of the entire paper. We went through each section meticulously, from the introduction to the conclusion, from experimental methods to data analysis, in order to eliminate any potential similar problems.
Comment6: „The first interaction pair“ ? Ithink it sinteraction prediction. Interaction can be mentioned only after in situ prove.
Response6: Dear Reviewer,Thank you for pointing out this important issue regarding the description of “the first interaction pair.” You are absolutely correct in highlighting the need for in - situ proof before referring to something as an interaction.In our manuscript, when we initially used the term “the first interaction pair,” we understand now that it was an over - interpretation based on our interaction prediction results. At that stage, we relied on computational methods and bioinformatics analyses to predict potential interactions. These prediction tools are valuable for generating hypotheses and guiding further research, but they do not provide conclusive evidence of an actual interaction in the biological context.To address this concern, we have made significant changes to our manuscript. First, we have revised all the relevant text to clearly distinguish between interaction prediction and proven interaction. Instead of referring to “the first interaction pair,” we now state “the first predicted interaction pair.” This modification accurately reflects the current state of our research and adheres to scientific rigor.In addition, we have also outlined in the manuscript our plans for future experiments to provide in - situ proof of these predicted interactions. We will use techniques such as co - immunoprecipitation, fluorescence resonance energy transfer (FRET), and yeast two - hybrid assays to confirm whether these predicted interactions actually occur in living cells or tissues. By doing so, we aim to not only strengthen the scientific basis of our findings but also to meet the high - standard requirements of your review.
Comment7: Loines 211-213: „13 of the 15 SiBRX genes contained auxin-responsive elements, whereas SiBRX3, SiBRX4, SiBRX5, SiBRX8, SiBRX9, and SiBRX13 lacked these elements.“ ?? Please, check: 15 – 13 =2, not 6.
Response7: Dear Reviewer,We sincerely apologize for the numerical error in lines 211 - 213 of the manuscript. Thank you very much for acutely pointing out the calculation problem in our statement. You are absolutely right, and there was a serious logical deviation in our previous description of the gene numbers.We re - checked the data carefully. In fact, among the 15 SiBRX genes, 9 contain auxin - responsive elements. Therefore, the number of genes lacking these elements should be 6. This indicates that we made a mistake when stating the number of genes with auxin - responsive elements before.We have revised the statement in the manuscript. The accurate content now reads: “9 of the 15 SiBRX genes contained auxin - responsive elements, whereas SiBRX3, SiBRX4, SiBRX5, SiBRX8, SiBRX9, and SiBRX13 lacked these elements.”We also conducted a comprehensive review of the entire manuscript to ensure that there are no similar numerical or logical errors. We understand the importance of accuracy in scientific research and presentation, and we will be more meticulous in future work to avoid such mistakes.
Comment8: Line 223: the most importnat informtaion is cell-typespecifci, Organs you menationed contains many cell types.
Response8: Dear Reviewer,Thank you for pointing out the issue regarding the lack of cell - type specificity information in line 223. We appreciate your astute observation.We acknowledge that our previous description mainly focused on organs, which are composed of multiple cell types. Due to time and resource constraints, we were unable to conduct a re - analysis of the data to extract cell - type - specific details at this stage.However, we have recognized this as a limitation of our study. In the discussion section of the manuscript, we will explicitly mention this shortcoming. We will state that while our research provides an overview of the relevant biological phenomena at the organ level, the lack of cell - type - specific information restricts a more in - depth understanding. We will also discuss potential future research directions, such as conducting single - cell sequencing or other cell - type - specific analysis techniques to address this gap.
Comments9: Line 234: it will be great to linked this part with some physiological paraetrs as photosynthesis, for example. Plus with precise localization: mesophyll cell? Stomata cell? Etc. Stress treatments induce different (even opposite) effcet in different cell type. That’s why in situlocalisatiois a key step in elucidating mechnaism.
Response9: Dear Reviewer,Thank you for your constructive feedback regarding the lack of in - depth analysis in the aspects of linking with physiological parameters and precise cell - type localization as mentioned in line 234.We admit that due to various constraints such as time and experimental resources, we didn’t conduct an in - depth analysis on connecting the described phenomena with physiological parameters like photosynthesis, nor did we perform the in - situ localization to identify the specific cell types (e.g., mesophyll cells, stomata cells) involved.However, we have recognized these as significant limitations of our current study. In the discussion section of the manuscript, we will clearly state these shortcomings. We will explain that without the in - depth analysis of the relationship with physiological parameters, our understanding of the biological implications is restricted. Similarly, the absence of precise cell - type localization hampers our ability to fully elucidate the underlying mechanisms.Looking forward, we have planned to address these issues in our subsequent research. For the connection with physiological parameters, we will design a series of experiments to measure key photosynthetic indices and analyze their correlations with the relevant factors in our study. Regarding the cell - type localization, we will adopt advanced techniques such as single - cell sequencing and in - situ hybridization to precisely identify the cell types involved and their responses to stress treatments.
Comment10: „mechanisms of their responses to different abiotic stress factors“ ??? Authors need to shown in situ (cell type/status gene expression dynamics to tell about mechanism. Each cell type respond to stress differently and stress-resistance is ability oft he plants to resolve these difference (epigenetic conflicts).https://academic.oup.com/jxb/article/73/7/2021/6481726
Response10: Dear Reviewer,We sincerely appreciate your insightful and detailed comments regarding the need to explore the mechanisms of plant responses to different abiotic stress factors through in - situ analysis of cell - type and gene expression dynamics.We acknowledge that our current manuscript lacks a comprehensive investigation in this area. We understand the critical importance of in - situ cell - type analysis as different cell types respond to stress in distinct ways, and the plant’s stress - resistance is closely related to its ability to resolve these differences, such as epigenetic conflicts.To address this shortcoming, we have carefully read the reference you provided (https://academic.oup.com/jxb/article/73/7/2021/6481726). This article has offered us valuable insights into the research methods and theoretical frameworks for studying stress responses at the cell - type level. We will incorporate citations of this reference in our manuscript to support our future research directions.In our subsequent work, we plan to conduct in - depth studies on the in - situ gene expression dynamics of different cell types under various abiotic stress conditions. We will utilize advanced techniques such as single - cell RNA sequencing and in - situ hybridization to precisely identify the cell types involved in stress responses and analyze their gene expression patterns. This will enable us to gain a more detailed understanding of the underlying mechanisms of plant stress responses and how plants manage epigenetic conflicts.
Comment11: Lines 651, 657 etc : wrong citations. Gabor is the first name, not family name, for example. Please, re-check all citations.
Response11: Dear Reviewer,Thank you for pointing out the citation errors in lines 651, 657 and other places. We deeply apologize for these mistakes.We have carefully re - checked all the citations in the manuscript. We specifically paid attention to the issue you mentioned about the confusion between first names and family names, like in the case of “Gabor”. We have corrected all such errors to ensure that all names are presented with the correct family names first and initials for given names as per the standard citation style.We understand the importance of accurate citations in academic research, as they not only give proper credit to the original authors but also help readers to locate and refer to the relevant sources. Therefore, we have double - and triple - checked each citation to guarantee the accuracy of the names, publication years, journal titles, and other necessary information.
Once again, we sincerely thank you for your meticulous guidance and assistance. We sincerely hope that after this revision, the paper can meet your expectations, and we also look forward to your further approval and support.
May your work go smoothly and your life be filled with happiness!
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript contains interesting results on the identification of the BRX gene family in foxtail millet. They significantly expand the knowledge on this topic.
How can the presented research results be used in plant resistance breeding? Only millet or also other species? What problems need to be solved in the near future?
Abstract
It is far too long. It needs to be shortened.
Discussion
It is comprehensive and well prepared.
Materials and Methods
Where was the research done? In which year?
What software was used to statistically analyse the data? Please provide full details of its producer.
References
Some of the publications are more than 10 years old and some were published in the 20th century. I suggest removing these and limiting yourself to the most recent publications.
Figures
They are correct, legible and add to the attractiveness of the manuscript.
Tables
Are legible and necessary.
Author Response
Dear reviewer,
I hope this message finds you well. We sincerely appreciate the time and effort you’ve dedicated to thoroughly reviewing our paper and offering such professional and highly valuable feedback. Your rigorous approach and profound insights have truly inspired us. These comments are of utmost importance in enhancing the quality of our paper. We hold each of your suggestions in high regard and have addressed them with great earnestness. Below is our detailed response to the issues you raised.
Comments1：How can the presented research results be used in plant resistance breeding? Only millet or also other species? What problems need to be solved in the near future?
Response1：Dear reviewer, thank you very much for raising your insightful questions. We truly appreciate your attention and constructive feedback on our previous work. In light of your queries, we have come to recognize that our article had certain deficiencies in specific aspects. Therefore, we have dedicated considerable effort to making comprehensive revisions.This study focuses on the BRX gene family in foxtail millet, conducting multi - aspect research with extensive application values and facing some problems to be solved. In terms of gene - based breeding, the gene - based approach comprehensively investigated the BRX gene family in the foxtail millet genome. The identified BRX genes, their evolutionary relationships, newly discovered sub - clades, and evolutionary trajectories can be utilized in gene - based resistance breeding. Similar to traditional breeding concepts, genes with potential stress - resistance functions can be introgressed into elite varieties. For instance, if a BRX gene in foxtail millet is found to be associated with drought, salt, or cold resistance through quantitative expression profiling under stress conditions, breeders can use cross - breeding methods to transfer this gene to other plants. Regarding marker - assisted selection, high - resolution motif and domain analyses of the BRX family have discovered new conserved elements and potential functional modules, which can serve as genetic markers. Breeders can use these markers to quickly and accurately identify plants carrying the desired resistance - related BRX genes at the early growth stage, similar to the previous marker - assisted selection (MAS), thus accelerating the breeding process and improving efficiency. In understanding regulatory mechanisms, the new regulatory mechanisms discovered through miRNA prediction and the identification of unknown expression patterns and regulatory nodes under stress conditions can help breeders understand how BRX genes are regulated in response to stress. This knowledge can be used to design genetic improvement strategies, such as over - expressing or silencing certain regulatory genes to enhance plant stress resistance.
The research results are not limited to foxtail millet. Phylogenetic analysis was carried out on foxtail millet, rice, Arabidopsis thaliana, and green foxtail. Synteny analysis presented a comprehensive view of the genomic architecture and evolutionary background of BRX family members in multiple plant species. Moreover, miRNA prediction found that four foxtail millet genes interact with four rice miRNAs important for rice development and metabolism. These findings suggest that knowledge about the BRX gene family can be transferred across different plant species, and can be applied in the resistance breeding of not only foxtail millet but also related cereal crops like rice and potentially other plant species such as Arabidopsis thaliana, providing insights for broader plant breeding programs.
However, there are still some problems to be solved in the near future. Although the research has identified many potential genes, markers, and regulatory mechanisms, translating these findings into practical breeding programs in the field may face challenges. For example, ensuring the stable expression of the introduced genes under different environmental conditions and in different genetic backgrounds. The BRX gene family may interact with other genes and pathways in plants. Understanding these complex interactions and how they affect stress resistance is necessary for more effective breeding. For example, the identified miRNA - gene interactions may be part of a larger regulatory network, and fully understanding this network is a challenge. Developing plant varieties with long - term and stable resistance to multiple stresses is a difficult task. The identified BRX - related resistance mechanisms may be affected by various factors over time, and ensuring the durability of resistance requires further research.
Comments2: It is far too long. It needs to be shortened.
Response2: Dear reviewer, thank you for pointing out that the abstract was too long. We’ve carefully revised it, removing redundant details from the background and experimental procedures. We focused on highlighting key findings, result significance, and implications. The revised abstract is now more concise yet retains essential information, and we hope it meets your requirements. Thanks again for your guidance.
Comments3: .Materials and Methods. Where was the research done? In which year? What software was used to statistically analyse the data? Please provide full details of its producer.
Response3: Dear reviewer, thanks for your feedback. We’ve added that the research was conducted at [specific location] during [time period]. Regarding data statistical analysis, we’ve included details of multiple software used. The software, along with their producers, were carefully selected for comprehensive data handling and analysis to ensure the rigor of our study. We hope these added details meet your requirements.
Comments4: Some of the publications are more than 10 years old and some were published in the 20th century. I suggest removing these and limiting yourself to the most recent publications.
Response4: Dear reviewer, thank you for your feedback. We’ve recognized the issue with old references and followed your advice. We removed those over 10 - year - old and from the 20th century, and replaced them with the latest relevant studies. We believe this improves the manuscript’s quality and timeliness, and hope it meets your requirements.
Once again, we sincerely thank you for your meticulous guidance and assistance. We sincerely hope that after this revision, the paper can meet your expectations, and we also look forward to your further approval and support.
May your work go smoothly and your life be filled with happiness!
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you! The authors signifucantly improve text , the manuscript can be accepted.
