Genome-Wide Identification of Peanut Pyruvate Kinase Gene Family and Their Potential Roles in Seed Germination and Drought Stress Responses
, Zhao Zheng 2,* and Bin Yang 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe article "Genome-wide identification of peanut pyruvate kinase gene family and their potential roles in seed germination and abiotic stress responses" is devoted to a detailed study of the pyruvate kinase gene family of such an important agricultural species as peanut. The work is divided into two parts: bioinformatics analysis - identification of genes of the pyruvate kinase family, their structural analysis, evolutionary relationships and comparison with similar genes in the genomes of Arabidopsis and rice. In addition, practical analysis of the expression level of a sample of genes in various parts of the plant, during seed germination and under two types of stress effects. Analysis of the expression level made it possible to come closer to determining the function of the genes being studied and to confirm the theoretically obtained data in practice. The manuscript is easy to read, the material is presented logically and consistently and is supplied with rich illustrative material. The work may be of interest to a wide range of readers. However, there are a few remarks.
1. The phrase on lines 26-27 is very general in nature - it seems obvious that proteins can interact with each other and with other important proteins. It needs to be specified.
2. Figures 1 and 2 can be combined into one figure of 2 parts. Since they are described in one section and follow each other directly, not separated by text.
3. Figure 4 A – The gray lines in the center represent all gene duplication events within the genome and are almost invisible.
4. Figures 9 and 10 can probably also be combined, since they are not separated by text and are described in one paragraph and both relate to the assessment of the expression level of the studied genes under stressful influences.
5. Why, of all the studied families of these genes in the genomes of other plants, besides Arabidopsis, was rice chosen for comparison, and not soybean, potato or cotton? Perhaps the rationale for this choice should be added to the manuscript.
6. In the Discussion, it is worth adding a couple of sentences about how much the obtained data on the increase in expression level in response to stress coincide with the data obtained earlier. Are pyruvate kinases involved in the response to drought stress in other plant species? And to other types of stress? Can the data obtained with PEG and ABA be generalized to the overall response to abiotic stress? As stated in the last sentence of the Discussion section.
Comments for author File: 
Comments.pdf
Author Response
Response to reviewer Comments
Dear reviewer,
We would like to thank you for providing us with your constructive and detailed review comments on our manuscript. We have answered all the questions and revised our manuscript carefully according to your good comments. The changes in the Microsoft Word file have been made with the "track changes" feature. Changed words and sentences in the revised manuscript are in red. My explanation to the comments point-by-point is as follow:
The article "Genome-wide identification of peanut pyruvate kinase gene family and their potential roles in seed germination and abiotic stress responses" is devoted to a detailed study of the pyruvate kinase gene family of such an important agricultural species as peanut. The work is divided into two parts: bioinformatics analysis - identification of genes of the pyruvate kinase family, their structural analysis, evolutionary relationships and comparison with similar genes in the genomes of Arabidopsis and rice. In addition, practical analysis of the expression level of a sample of genes in various parts of the plant, during seed germination and under two types of stress effects. Analysis of the expression level made it possible to come closer to determining the function of the genes being studied and to confirm the theoretically obtained data in practice. The manuscript is easy to read, the material is presented logically and consistently and is supplied with rich illustrative material. The work may be of interest to a wide range of readers. However, there are a few remarks.
Point 1: The phrase on lines 26-27 is very general in nature - it seems obvious that proteins can interact with each other and with other important proteins. It needs to be specified.
Response 1: Thank you for pointing this out. We have revised in our revised manuscript. Please see Page 1, Lines 32–33. Thank you for your helpful comments.
Point 2: Figures 1 and 2 can be combined into one figure of 2 parts. Since they are described in one section and follow each other directly, not separated by text.
Response 2: Thank you for your suggestion. We have combined both figure 1 and figure 2 together into one figure (Page 6).
Point 3: Figure 4 A – The gray lines in the center represent all gene duplication events within the genome and are almost invisible.
Response 3: All gray lines overlapping together caused this problem. Please see the following Figure for example. Thank you.
Point 4: Figures 9 and 10 can probably also be combined, since they are not separated by text and are described in one paragraph and both relate to the assessment of the expression level of the studied genes under stressful influences.
Response 4: Thank you for your suggestion. We have combined both figure 9 and figure 10 together into one figure (Page 13).
Point 5: Why, of all the studied families of these genes in the genomes of other plants, besides Arabidopsis, was rice chosen for comparison, and not soybean, potato or cotton? Perhaps the rationale for this choice should be added to the manuscript.
Response 5: Thank you for raising this important point. We have revised the manuscript to clarify the rationale for selecting rice as a comparative species. The key reasons are as follows: Rice is a model monocot species, while peanut and Arabidopsis are eudicots. Including rice allowed us to compare PK gene family evolution across divergent plant lineages (monocots vs. eudicots), providing insights into lineage-specific adaptations. In contrast, soybean, potato, and cotton are also eudicots, and their inclusion would have limited the analysis to intra-clade comparisons. Rice’s phylogenetic divergence from peanut/Arabidopsis helps highlight conserved versus lineage-specific features of PK genes. Moreover, rice has a well-annotated genome with extensive functional studies on PK genes, enabling robust cross-species comparisons. While soybean, potato, and cotton PK genes have been studied, their genomic annotations or functional data are less comprehensive compared to rice. These points have been added to the Page 5, Lines 213–217. Thank you for your insightful suggestion, which has improved the clarity and depth of our manuscript.
Point 6: In the Discussion, it is worth adding a couple of sentences about how much the obtained data on the increase in expression level in response to stress coincide with the data obtained earlier. Are pyruvate kinases involved in the response to drought stress in other plant species? And to other types of stress? Can the data obtained with PEG and ABA be generalized to the overall response to abiotic stress? As stated in the last sentence of the Discussion section.
Response 6: We have expanded the Discussion to address these points: Previous studies have revealed several PKs involved in drought and ABA responses by combining multi‐omics data [51, 52]. Our findings align with these studies, demonstrating conserved roles for PKs in stress adaptation across species. As ABA also mediates responses to salinity and cold [53, 54], our data imply broader roles for AhPKs in multiple abiotic stresses. Future studies will validate these hypotheses under diverse stress conditions. We add several references to the discussion part. These additions appear in the revised Discussion (Page 16, Lines 493–498). Thank you for your constructive feedback, which has significantly strengthened our manuscript.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIn the manuscript, the authors identified 21 pyruvate kinase genes of (Arachis hypogaea L.) from the Peanut Genome Resources database. They have analysed the sequences in silico for understanding the variations in the sequences. Further they tried to identify the function. The manuscript is well written. Up to in silico part, the manuscript I fine. The connectivity of in silico work and the lab work need to revised better.
1. Provide the source of the peanut plant.
2. In figure 6 A, what the colour difference (00.00-10.00) shows?
3. In the analysis of cis-acting elements, the results need to be explained more clearly, highlighting the differences among the 21 sequences analyzed. Additionally, if the authors correlate the functions with the variations in the cis-acting elements, it may provide a better understanding.
4. Why and how the authors selected ACTIN as the endogenous internal standard for normalizing the expression?
5. Expand PEG and ABA in abstract.
6. Although the study involved a good in silico work, the authors did not provide any strong new information. The conclusion is too vague and lacks clarity. It should be more specific, summarizing key findings and their significance.
Comments on the Quality of English LanguageThe language may be improved for better understanding
Author Response
Response to reviewer Comments
Dear reviewer,
We would like to thank you for your detailed and thoughtful comments that have helped us revise our manuscript. We have revised our manuscript according to all the comments. The changes in the Microsoft Word file have been made with the "track changes" feature. Changed words and sentences in the revised manuscript are in red. My explanation to the comments point-by-point is as follow:
In the manuscript, the authors identified 21 pyruvate kinase genes of (Arachis hypogaea L.) from the Peanut Genome Resources database. They have analysed the sequences in silico for understanding the variations in the sequences. Further they tried to identify the function. The manuscript is well written. Up to in silico part, the manuscript I fine. The connectivity of in silico work and the lab work need to revised better.
Point 1: Provide the source of the peanut plant.
Response 1: Thanks for this suggestion. We add the source of the peanut plant: Seeds were obtained from the Shandong Institute of Peanuts (Qingdao, China). Please see Page 4, Lines 159–160.
Point 2: In figure 6 A, what the colour difference (00.00-10.00) shows?
Response 2: We are sorry for not describing it clearly. The numbers and gradient colors indicate the number of cis-acting elements in AhPKs. We add the description in the Figure legend. Please see Page 10, Lines 323–324.
Point 3: In the analysis of cis-acting elements, the results need to be explained more clearly, highlighting the differences among the 21 sequences analyzed. Additionally, if the authors correlate the functions with the variations in the cis-acting elements, it may provide a better understanding.
Response 3: Thank you for your constructive feedback. We have revised the cis-acting element analysis to better highlight differences among the 21 AhPK genes and explicitly link these variations to their potential functions. The key updates are as follows: Notably, the number of cis-elements in an AhPK member ranged from 13 to 36, with seven genes harboring over 30 elements. These findings underscore the functional diversity of AhPK promoters, suggesting their pivotal roles in regulating growth, developmental processes, and stress adaptation in peanut. These details are now explicitly described in Page 10, Lines 315–318. Thank you for your insightful suggestions, which have strengthened the clarity and biological relevance of our findings.
Point 4: Why and how the authors selected ACTIN as the endogenous internal standard for normalizing the expression?
Response 4: The ACTIN was chosen as the endogenous internal standard is based on the previous studies. We add this gene ID to the manuscript. Please see Page 4, Line 183. Thank you.
Reference 1: Bao G, Li S, Zhou Q, Ashraf U, Qiao J, Li X, Wan X, Zheng Y. Transcriptomic Analysis Provides Insights into the Differential Effects of Aluminum on Peanut (Arachis hypogaea L.). Genes (Basel). 2022 Oct 10;13(10):1830. doi: 10.3390/genes13101830.
Reference 2: Bao G, Zhou Q, Li S, Ashraf U, Huang S, Miao A, Cheng Z, Wan X, Zheng Y. Transcriptome Analysis Revealed the Mechanisms Involved in Ultrasonic Seed Treatment-Induced Aluminum Tolerance in Peanut. Front Plant Sci. 2022 Feb 8;12:807021.
Point 5: Expand PEG and ABA in abstract.
Response 5: This was an oversight on our part. We have revised it on revised manuscript. Thank you (Page 1, Lines 38–39).
Point 6: Although the study involved a good in silico work, the authors did not provide any strong new information. The conclusion is too vague and lacks clarity. It should be more specific, summarizing key findings and their significance.
Response 6: Thank you for this valuable feedback. We have revised the Conclusion to concisely highlight key findings and their implications: In this study, we conducted a comprehensive analysis of the PK gene family in peanut and identified 21 AhPK genes, phylogenetically classified into PKc and PKp subfamilies. Conserved domain patterns and motif compositions of the AhPK members in the same subfamily displayed universal similarities. Segmental duplication events and purifying selection drove the expansion and evolution of the AhPK family. Protein-protein interaction network analysis demonstrated the connections of AhPK proteins with other potential interactors. AhPK promoters are enriched with cis-elements related to light responses, stress, hormones, and development. Additionally, expression analysis revealed that AhPK genes may play important physiological regulatory roles in seed germination and drought stress responses. This study provides the first comprehensive characterization of PK genes in peanut, establishing a foundation for functional studies to enhance drought stress tolerance and seed vigor through genetic engineering or breeding. The revised Conclusion now appears on Page 16, Lines 502–517. Thank you for your constructive suggestion.
Point 7: The language may be improved for better understanding.
Response 7: We have used the English language editing service to edit this revised manuscript. Thank you for your suggestion.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis work is devoted to a comprehensive analysis of the Pyruvate kinase (PK) gene family in peanut. The authors identified 21 genes of the PK family, which were systematically analyzed for physicochemical properties of proteins, chromosome distribution, phylogenetic classification, protein motifs, gene structure, gene duplication, protein interaction network and cis-acting elements. In addition, expression analysis of some genes was conducted, which showed that AhPK genes may play an important physiological regulatory role in seed germination and drought stress tolerance. This paper is well written and provides interesting data for future studies on the functional characterization and regulatory mechanisms of PK genes in peanut. I recommend accepting this article for publication after making some edits:
1. The title of the article needs to be edited. Clarify the impact of abiotic stress (drought) in the title of the article.
2. In the text of the article, at the first mention, it is necessary to decipher the abbreviations used: PEG and ABA.
3. The introduction needs to be expanded. Explain why you decided to use the ABA and PEG treatment in your work.
4. Conduct a statistical analysis of Figure 7. Describe how you did this in paragraph 2.8. Also indicate the age of the plants when they were fixed for the expression of 10 selected AhPK genes in various tissues in Fig. 7.
5. In section 2.8, describe how many experiments were conducted and how many biological replicates you took in each experiment.
6. Line 145-146 indicate the Actin gene ID.
7. Line 129. "at different developmental stages". Please expand on this phrase in paragraph 2.6.
8. Line 421-422. "and abiotic stress tolerance" - specify to what stress.
Author Response
Response to reviewer Comments
Dear reviewer,
We would like to take this opportunity to thank you for your thoughtful critiques and constructive comments that helped us to improve our manuscript. We have answered all the questions and revised our manuscript carefully according to your professional comments. The changes in the Microsoft Word file have been made with the "track changes" feature. Changed words and sentences in the revised manuscript are in red. My explanation to the comments point-by-point is as follow:
This work is devoted to a comprehensive analysis of the Pyruvate kinase (PK) gene family in peanut. The authors identified 21 genes of the PK family, which were systematically analyzed for physicochemical properties of proteins, chromosome distribution, phylogenetic classification, protein motifs, gene structure, gene duplication, protein interaction network and cis-acting elements. In addition, expression analysis of some genes was conducted, which showed that AhPK genes may play an important physiological regulatory role in seed germination and drought stress tolerance. This paper is well written and provides interesting data for future studies on the functional characterization and regulatory mechanisms of PK genes in peanut. I recommend accepting this article for publication after making some edits:
Point 1: The title of the article needs to be edited. Clarify the impact of abiotic stress (drought) in the title of the article.
Response 1: Thank you for your suggestion. We have revised the title to emphasize the study’s focus on drought stress, as PEG-induced osmotic stress and ABA signaling are key components of drought response mechanisms. The updated title now reads: "Genome-wide identification of peanut pyruvate kinase gene family and their potential roles in seed germination and drought stress responses". This modification better reflects the specific abiotic stress (drought) investigated in our work while retaining the broader context of seed germination.
Point 2: In the text of the article, at the first mention, it is necessary to decipher the abbreviations used: PEG and ABA.
Response 2: This was an oversight on our part. We have revised it on revised manuscript (Page 1, Lines 38–39). Thank you.
Point 3: The introduction needs to be expanded. Explain why you decided to use the ABA and PEG treatment in your work.
Response 3: We appreciate the reviewer’s suggestion. In the revised manuscript, we have expanded the Introduction to clarify the rationale for selecting ABA and PEG treatments. Previous studies have shown that the PK promoters share common cis-acting elements related to drought and ABA. However, the expression pattern and the functional roles of peanut PK genes in stress responses, including drought and ABA, remain poorly understood. This rationale has now been explicitly stated in the revised Introduction (Page 2–3, Lines 92–95).
Point 4: Conduct a statistical analysis of Figure 7. Describe how you did this in paragraph 2.8. Also indicate the age of the plants when they were fixed for the expression of 10 selected AhPK genes in various tissues in Fig. 7.
Response 4: Statistical analysis has now been included in new Figure 6, as requested (Page 11). We also add the statistical method into Method Section (Page 5, Line 192). The age of the plants was added as you requested (Page 4, Lines 162–164; Page 11, Lines 343–345). Thank you for your constructive suggestion.
Point 5: In section 2.8, describe how many experiments were conducted and how many biological replicates you took in each experiment.
Response 5: Thank you for your constructive suggestion. We add this point in the manuscript. Please see Page 5, Lines 189–190. Thank you.
Point 6: Line 145-146 indicate the Actin gene ID.
Response 6: We add this gene ID to the manuscript. Please see Page 4, Line 183. Thank you.
Point 7: Line 129. "at different developmental stages". Please expand on this phrase in paragraph 2.6.
Response 7: We are sorry for not describing it clearly. We have added a description of sampling to the Methods. lease see Page 4, Lines 162–164. Thank you.
Point 8: Line 421-422. "and abiotic stress tolerance" - specify to what stress.
Response 8: We have revised it on revised manuscript. Please see Page 16, Lines 514–515. Thank you.
Author Response File: Author Response.pdf
