Transcriptome Profiling of Cold-Stored Potato Tubers Revealed Similarities in the Regulation of Bud Dormancy Release, Tuberization, and Flowering Initiation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript conducted a comparative transcriptomic analysis of potato tubers stored at low temperature for 0.5, 3.5, and 6.5 months to investigate the transition from endodormancy to ecodormancy. Moreover, RNA-seq and RT-qPCR were employed to identify differentially expressed genes associated with hormonal signaling, metabolic pathways, and vernalization-related transcription factors. The manuscript provides valuable insights into the molecular mechanisms underlying tuber dormancy release and sprouting suppression under cold conditions.
However, here are some concerns to be improved for this article.
Major concerns:
- Basis for sampling time points:The rationale for selecting the specific sampling intervals (0.5, 5, and 6.5 months) requires clarification. Please provide relevant literature or physiological explanations in the "Materials and Methods" section to justify these time points.
- Sufficiency of biological replicates:The use of only two biological replicates (n=2) for RNA-seq may limit statistical robustness. To ensure data reliability, please provide a Pearson correlation analysis between replicates and cite relevant studies supporting this experimental design.
- Inclusion of phenotypic images: The manuscript lacks direct morphological evidence to complement the extensive transcriptomic data. It is recommended to include representative images of tuber eye-buds at each stage to visually verify their physiological states and strengthen the conclusions.
Minor concerns:
- Line 108: The expression "0.5 month" is inconsistent with the plural form "0.5 months" used in other parts of the manuscript. Please unify the unit notation to maintain consistency.
- Line 190: The notation "DGES" is inconsistent with "DEGs" used elsewhere in the manuscript. It is recommended to standardize the notation consistently as "DEGs".
- Line 313: There is a spelling error in "releated" -the correct spelling should be "related".
- Figures 4-8: A large number of gene IDs are used in the figures, and many IDs are highly similar. This makes it difficult for readers to quickly identify key genes. It is suggested to appropriately highlight or label the key genes.
- Figure 2: The information is overly dense with overlapping elements, which affects readability. It is recommended to appropriately optimize the layout to enhance clarity.
- Line 950: Query the capitalization of "micrornas" – the standard terminology is "microRNAs" or its abbreviation "miRNAs". Please unify it with the terminology used elsewhere in the manuscript.
- Figure 9: The font size in the figure is too small and lacks clarity. It is suggested to optimize the figure to facilitate reader comprehension.
- Figure 10: In addition to the small font size, the error bars are too light in color. It is recommended to adjust the error bar color and optimize overall visualization for better readability.
Author Response
Comments 1: This manuscript conducted a comparative transcriptomic analysis of potato tubers stored at low temperature for 0.5, 3.5, and 6.5 months to investigate the transition from endodormancy to ecodormancy. Moreover, RNA-seq and RT-qPCR were employed to identify differentially expressed genes associated with hormonal signaling, metabolic pathways, and vernalization-related transcription factors. The manuscript provides valuable insights into the molecular mechanisms underlying tuber dormancy release and sprouting suppression under cold conditions.
Response 1: We are sincerely grateful to the Reviewer 1 for important corrections and advices.
Comments 2: However, here are some concerns to be improved for this article. Major concerns: Basis for sampling time points:The rationale for selecting the specific sampling intervals (0.5, 5, and 6.5 months) requires clarification. Please provide relevant literature or physiological explanations in the "Materials and Methods" section to justify these time points.
Response 2: Explanations are given in the Materials and Methods (lines 109-112).
Comments 3: Sufficiency of biological replicates:The use of only two biological replicates (n=2) for RNA-seq may limit statistical robustness. To ensure data reliability, please provide a Pearson correlation analysis between replicates and cite relevant studies supporting this experimental design.
Response 3: The text was updated by a correlation analysis data performed on transcriptome data (lines 197-201). The pairwise Pearson’s correlation coefficients (r) were found to be 0.9892, 0.9891, and 0.9949 for pairs of the 0.5, 3.5, and 6.5 months transcriptomes, respectively, indicating high repeatability of the sequencing data.
Comments 4: Inclusion of phenotypic images: The manuscript lacks direct morphological evidence to complement the extensive transcriptomic data. It is recommended to include representative images of tuber eye-buds at each stage to visually verify their physiological states and strengthen the conclusions.
Response 4: We included photographs of potato tuber buds at time 0 (immediately after removal from cold) (a) and after exposure to room temperature (20°C) for 1 (b), 7 (c), and 9 (d) days (Supplementary Figure S2).
Comments 5: Minor concerns: Line 108: The expression "0.5 month" is inconsistent with the plural form "0.5 months" used in other parts of the manuscript. Please unify the unit notation to maintain consistency.
Response 5: Corrected (L108)
Comments 6: Line 190: The notation "DGES" is inconsistent with "DEGs" used elsewhere in the manuscript. It is recommended to standardize the notation consistently as "DEGs".
Response 6: Corrected (L214)
Comments 7: Line 313: There is a spelling error in "releated" -the correct spelling should be "related".
Response 7: Corrected (L343).
Comments 8: Figures 4-8: A large number of gene IDs are used in the figures, and many IDs are highly similar. This makes it difficult for readers to quickly identify key genes. It is suggested to appropriately highlight or label the key genes.
Response 8: Corrected. In Figures 4-7, the names of the proteins they encode have been added to the identifiers of a number of key genes. The heat map in Figure 8 reflects the expression dynamics of too many genes, so we have supplemented the figure caption with the phrase “details are provided in Supplementary Table S13.”
Comments 9: Figure 2: The information is overly dense with overlapping elements, which affects readability. It is recommended to appropriately optimize the layout to enhance clarity.
Response 9: Optimized.
Comments 10: Line 950: Query the capitalization of "micrornas" – the standard terminology is "microRNAs" or its abbreviation "miRNAs". Please unify it with the terminology used elsewhere in the manuscript.
Response 10: Corrected (page 21).
Comments 11: Figure 9: The font size in the figure is too small and lacks clarity. It is suggested to optimize the figure to facilitate reader comprehension.
Response 11: Figure 9 have been updated by eight more genes taken to the analysis, for a total of 16 genes. Font size was increased.
Comments 12: Figure 10: In addition to the small font size, the error bars are too light in color. It is recommended to adjust the error bar color and optimize overall visualization for better readability.
Response 12: Optimized.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study explores the potential molecular mechanisms underlying dormancy release and tuberization of potato tubers during cold storage at the transcriptomic level, demonstrating high application value. However, several issues need to be addressed:
1.The manuscript clearly describes the expression changes of lipoxygenase genes at different stages (lines 272-275), but these genes are not explicitly labeled in Figure 4a. Additionally, there appears to be an inconsistency between the textual description and the data presented in Table S12. The text states that "2 and 3 of them were upregulated in 3.5- and 6.5- vs 0.5-mo samples," but Table S12 shows that 3 genes (LOC102577615, LOC102596122, LOC102588373) were upregulated in 3.5- vs 0.5-mo samples, while only 2 genes (LOC102596122, LOC102588373) were upregulated in 6.5- vs 0.5-mo samples. The authors are advised to carefully verify and correct this discrepancy.
2.The authors only randomly selected 8 genes for RT-qPCR to validate the RNA-seq results, which seems insufficient. It is recommended to increase the number of validated genes to enhance the reliability of the transcriptomic data.
3.The authors selected 17 genes related to dormancy release for gene expression analysis (lines 517-521), but no clear rationale for the selection of these genes is provided in the manuscript. Furthermore, only 16 gene names are listed, and excluding StSP6A (which was not analyzed), the expression changes of only 15 genes are ultimately presented. The authors should clarify the selection criteria for these candidate genes and correct the inconsistency in the number of genes.
4.The authors used mature tubers of similar size as experimental materials, but it is questionable whether similar tuber size can fully ensure material consistency. It is recommended to determine relevant physiological indicators of the tubers to confirm the uniformity of the experimental materials.
5.It is suggested to simultaneously detect key physiological indicators during tuber dormancy (e.g., starch/soluble sugar content) and establish a correlation network between physiological indicators and gene expression, which will strengthen the persuasiveness of the conclusions.
Author Response
Comments 1: (x) The English could be improved to more clearly express the research.
Response 1: The English language in the paper was corrected by a native speaker.
Comments 2: This study explores the potential molecular mechanisms underlying dormancy release and tuberization of potato tubers during cold storage at the transcriptomic level, demonstrating high application value. However, several issues need to be addressed:
Response 2: We are sincerely grateful to the Reviewer 2 for valuable comments, the answers to which obviously helped to make the manuscript more understandable and thoughtful.
Comments 3: 1.The manuscript clearly describes the expression changes of lipoxygenase genes at different stages (lines 272-275), but these genes are not explicitly labeled in Figure 4a. Additionally, there appears to be an inconsistency between the textual description and the data presented in Table S12. The text states that "2 and 3 of them were upregulated in 3.5- and 6.5- vs 0.5-mo samples," but Table S12 shows that 3 genes (LOC102577615, LOC102596122, LOC102588373) were upregulated in 3.5- vs 0.5-mo samples, while only 2 genes (LOC102596122, LOC102588373) were upregulated in 6.5- vs 0.5-mo samples. The authors are advised to carefully verify and correct this discrepancy.
Response 3: We are grateful to the reviewer for finding the mistake. This has been checked and corrected (line 299).
Comments 4: 2.The authors only randomly selected 8 genes for RT-qPCR to validate the RNA-seq results, which seems insufficient. It is recommended to increase the number of validated genes to enhance the reliability of the transcriptomic data.
Response 4: Eight more genes were added to the analysis, for a total of 16 genes. Figure 9 and text (lines 543, 548-549) have been updated.
Comments 5: 3.The authors selected 17 genes related to dormancy release for gene expression analysis (lines 517-521), but no clear rationale for the selection of these genes is provided in the manuscript. Furthermore, only 16 gene names are listed, and excluding StSP6A (which was not analyzed), the expression changes of only 15 genes are ultimately presented. The authors should clarify the selection criteria for these candidate genes and correct the inconsistency in the number of genes.
Response 5: Corrected: 15 genes (line 552). The criteria for selecting candidate genes have been clarified (lines 557-570).
Comments 6: 4.The authors used mature tubers of similar size as experimental materials, but it is questionable whether similar tuber size can fully ensure material consistency. It is recommended to determine relevant physiological indicators of the tubers to confirm the uniformity of the experimental materials.
Response 6: The harvested tubers were similar in size and weight. We supplemented the text with data on the dynamics of moisture, starch, glucose, and fructose content at each of the three analyzed time points (Supplementary Figure S1). Each biological replicate included two tuber samples, both for biochemical analysis and for constructing mRNA libraries. The text has been updated (lines 113-124, 135, 141-143).
Comments 7: 5.It is suggested to simultaneously detect key physiological indicators during tuber dormancy (e.g., starch/soluble sugar content) and establish a correlation network between physiological indicators and gene expression, which will strengthen the persuasiveness of the conclusions.
Response 7: The dynamics of starch, glucose, and fructose content was analyzed at each of the three time points (Supplementary Figure S1). Analysis of possible correlations between the content of starch/hexoses with the level of expression of carbohydrate metabolism DEGs showed the absence of significant correlations. The text has been updated (lines 412-415).
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI have carefully reviewed the revised version of the manuscript and am satisfied with the authors’ responses to all previously raised comments and the corresponding revisions made.
