Transcriptomic and Proteomic Integration Reveals Key Tapping-Responsive Factors for Natural Rubber Biosynthesis in the Rubber Tree Hevea brasiliensis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you very much for submitting the manuscript entitled "Integration of transcriptome and proteome highlights crucial tapping-responsive members involved in natural rubber biosynthesis in Heave rubber tree".

The study seems to be detailed, interesting for readers, and well-structured.

I suggest only minor corrections, which helps to improve the quality of this paper.

1. P. 1, Title

Please, correct "Heave rubber" to "Hevea rubber ".

2. P. 1, Abstract

The practical and scientific significance should be highlighted.

3. Section 1, Introduction

Please, add the schematic view of Hevea tree trunk structure (including laticiferous vessels) and the tapping process.

4. P. 2, Section 1 (Introduction), lines 60-61

I kindly recommend the Authors including the schemes of the cytosolic mevalonate (MVA) pathway and the chloroplasts 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

 

Author Response

Comments 1: P. 1, Title. Please, correct "Heave rubber" to "Hevea rubber ".

Response 1: Thank you for your suggestion. After careful consideration and discussion, we have revised our new title as: "Transcriptomic and proteomic integration reveals key tapping-responsive factors for natural rubber biosynthesis in the rubber tree Hevea brasiliensis" in the newly revised manuscript (Lines 2-4).

 

Comments 2: P. 1, Abstract. The practical and scientific significance should be highlighted.

Response 2: Thank you for your valuable feedback. Our results deepen the comprehension of the regulation mechanism underlying tapping and provides some candidate genes and proteins for improving latex production in the Hevea rubber tree in future. To better highlight the practical and scientific significance, we have thoroughly revised the Abstract section, and a new Abstract is presented in the newly revised version (Lines 14-28).

 

Comments 3: Section 1, Introduction. Please, add the schematic view of Hevea tree trunk structure (including laticiferous vessels) and the tapping process.

Response 3: Thank you for your constructive suggestion. We agree that a visual representation would significantly enhance the clarity and understanding of our study. We have added a schematic diagram (Figure 1A) that illustrates the anatomy of the Hevea tree trunk, and depicting the tapping process in the new version (Lines 234-239).

 

Comments 4: Section 1 (Introduction), lines 60-61. I kindly recommend the Authors including the schemes of the cytosolic mevalonate (MVA) pathway and the chloroplasts 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

Response 4: Thank you for your helpful suggestion. We agree that MVA and MEP pathways are crucial to understanding the biosynthetic processes involved in natural rubber production and should be clearly described in the Introduction section. In fact, in the original manuscript, we provided detailed representations of both the MVA and MEP pathways in the Figure 5 and Figure 6, and we also highlighted their roles for rubber biosynthesis in the Discussion section 4.1 (Lines 291-383 in the original version). Based on your suggestion, we have provided more descriptions for the MVA and MEP pathways in the Introduction section in our newly revised version. We appreciate your concern on this important point. The added description on is as following: Studies have indicated that tapping can enhance the expression of key enzymes in the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, thereby augmenting the production of isoprenoid precursors. Furthermore, tapping can also activate genes directly involved in rubber biosynthesis, such as rubber elongation factor (REF) and small rubber particle protein (SRPP). Following tapping, energy metabolism pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation, are also upregulated to support wound repair and rubber biosynthesis (Lines 65-72 in the new version).

Reviewer 2 Report

Comments and Suggestions for Authors

The article discusses a comprehensive analysis by integrating high-throughput transcriptomics and proteomics methods for rubber tree (Hevea brasiliensis) latex post-tapping to determine the effects of tapping on latex regeneration, total latex yield, and latex flow duration.

 Overall, the research and experiments are clearly described. These findings provide a better understanding of latex regeneration and flow mechanisms in rubber tree. However, here are some minor flaws in the manuscript listed below:

 - The manuscript contains minor grammar and punctuation errors.  The sentence in lines 89 and 90, “The virgin trees were planted for ten years and first subjected to the tapping.” can be replaced with “The virgin trees were planted for ten years and were first subjected to tapping.”

- I am confused about the number of samples used in these experiments. According to the materials and methods, specifically line 90,  a total of thirty trees were selected for all experiments. However, based on lines 92-97 of the experiments, only three samples were used for each treatment (T1, T2, T3, and T4), which would amount to only 12 trees being used. Further explanation is needed to address this discrepancy.

-According to the material and methods, line 148, nine DEGs involved in the NRB pathways were randomly selected. Why were nine DEGs randomly selected?

- What is the relationship between transcriptome and proteome data for latex post-tapping? I recommend determining this relationship using statistical methods.

 

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Comments 1: The manuscript contains minor grammar and punctuation errors.  The sentence in lines 89 and 90, “The virgin trees were planted for ten years and first subjected to the tapping.” can be replaced with “The virgin trees were planted for ten years and were first subjected to tapping.”

Response 1: Thank you. We have corrected “The virgin trees were planted for ten years and first subjected to the tapping.” to “The virgin trees were planted for ten years and were first subjected to tapping.” as suggested (Lines 119-120). Furthermore, the English language of the manuscript has been carefully polished by all the authors, especially the correspond author Prof. Xuchu Wang. In the revised manuscript, some typographical and grammar errors were corrected. The changed parts are marked in red color in the new version.

 

Comments 2: I am confused about the number of samples used in these experiments. According to the materials and methods, specifically line 90, a total of thirty trees were selected for all experiments. However, based on lines 92-97 of the experiments, only three samples were used for each treatment (T1, T2, T3, and T4), which would amount to only 12 trees being used. Further explanation is needed to address this discrepancy.

Response 2: We apologize for causing your confusion on the number of samples used in our experiments. Our experimental design involved in allocating these trees across multiple treatments (T1, T2, T3, and T4). Each treatment was conducted with three biological replicates and the latex of every five trees was pooled as one biological replicate. Therefore, totaling thirty trees for the experimental analysis. To ensure the clarity of our methodology, we have added more information in the revised the materials and methods section to explicitly outline how the selected thirty trees were distributed among different experiments (Lines 128-129).

 

Comments 3: According to the material and methods, line 148, nine DEGs involved in the NRB pathways were randomly selected. Why were nine DEGs randomly selected?

Response 3: The selection of nine DEGs was based on the following considerations: firstly, the DEGs selected were those that demonstrated statistically significant differential expression across the experimental conditions. Secondly, the selected nine DEGs were chosen with particular attention to their biological roles in the NRB pathway. Each gene was evaluated for its known involvement in key processes related to NRB pathway, allowing us to gain insightful perspectives on the regulatory mechanisms. In summary, while the nine DEGs were selected in part randomly, careful consideration was given to their statistical significance and biological relevance within the NRB pathway. Thank you for your attention to this detail, and we have expanded on this explanation in the revised manuscript to provide clarity to readers (Lines 204-205, 415-418).

 

Comments 4: What is the relationship between transcriptome and proteome data for latex post-tapping? I recommend determining this relationship using statistical methods.

Response 4: Thank you for your valuable suggestions. In this study, the relationships between the proteins and genes detected under tapping treatment were constructed using the OmicShare online platform (https://www.omicshare.com/tools/home/report/reportjxx.html) with the screening criteria q<0.05 which has adjusted by controlling for the FDR. We have expanded the Methods section to include specific information on the relationship between transcriptome and proteome data for latex post-tapping (Lines 197-201). 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled "Integration of transcriptome and proteome highlights crucial tapping-responsive members involved in natural rubber biosynthesis in Hevea rubber tree" aims to investigate the molecular mechanisms underlying latex regeneration and rubber biosynthesis in Hevea brasiliensis through transcriptomic and proteomic analyses. The study identifies key genes and proteins (such as SRPPs and REFs) that respond to tapping stimulation, providing insights into how mechanical wounding affects natural rubber production. The research makes significant contributions by integrating high-throughput omics data and identifying molecular targets for improving rubber yield. To support my proposal for reconsideration after major revisions, I highlighted specific examples from the manuscript that require significant attention:

 

The title is mostly clear, but "highlights crucial tapping-responsive members" could be more specific. Consider revising as: "Transcriptomic and proteomic integration reveals key tapping-responsive factors in natural rubber biosynthesis of the Hevea rubber tree."

 

Some phrases in the abstract are unclear. For instance, "significantly impacts latex production" could be refined to "has a significant impact on latex production." Similarly, in "molecular mechanisms that natural rubber production in response to tapping stimulation remain largely enigmatic," the authors might revise to "the molecular mechanisms underlying natural rubber production in response to tapping stimulation remain largely unknown."

 

Please, in the Introduction provide a more comprehensive review of prior work related to the roles of SRPPs, REFs, and tapping-induced physiological responses in rubber trees. Additional references should be included, especially from recent high-impact research in plant molecular biology and industrial crop physiology.

Lines 37-40: "Natural rubber (cis-1,4-polyisoprene), is indispensable to the global industry due to its exceptional physical properties." This is a well-known fact, but the introduction should connect this to more recent studies on molecular pathways and how they are modulated by tapping.

 

The methods are generally well-organized, describing both transcriptomic and proteomic analyses. Please, provide more details on RNA and protein extraction, including temperature, conditions, and specific instruments used.

Lines 92-94: "Latex samples collected from the plants at days 0, 1, 3, and 5 were termed T1, T2, T3, and T4, respectively." The manuscript does not explain whether these time points are based on previous studies, preliminary experiments, or standard practices. Justify the selection of the sampling time points (T1, T2, T3, T4). Clarify why these intervals were chosen and how they relate to rubber regeneration stages.

Ensure all protocols (especially those related to RNA/protein extraction and bioinformatic analyses) are described with enough precision to allow replication. Clearly state the threshold values for statistical significance in the differential gene/protein expression analyses and mention any software used for validation, such as DESeq2.

Lines 119-121: "Identification of differentially expressed genes (DEGs) was conducted using DESeq2 between each pair of groups, with the criterion fold change >2 or <0.5 (q < 0.05)." While fold changes are mentioned, more information about how the q-values were adjusted is needed.

The results are presented in a clear, structured manner with figures that support the analysis. In Figure 1 (A) and (D), the statistical differences currently presented do not accurately reflect the actual results. The authors will conduct a thorough reanalysis of the data using appropriate statistical tests to ensure the correct representation of significant differences between the groups. The revised figure will include accurate error bars and significance markers (e.g., p-values or asterisks) to clearly indicate statistically significant differences between time points. The authors will ensure that all results are presented with proper statistical interpretation.

Lines 199-202: “Genes exhibiting similar expression patterns based on relative expression abundances in T2/T1, T3/T1, and T4/T1 were identified through trend analysis, categorizing gene expression trends into 20 clusters.” This statement doesn't explain the method used for trend analysis, such as software, algorithms, or statistical models.

Figure 2 (Lines 217-220): "Trend analysis of the identified genes... shows stage-specific patterns." While trends are described, there is no indication of how significance was determined for these trends. Additionally, the trends and patterns described in text (e.g., the upregulation of SRPPs and REFs) could be reinforced with additional visuals or summaries.

 

Lines 274-277: “The most significantly enriched pathway was ‘Oxidative phosphorylation,’ encompassing 15 DEGs, followed by ‘Valine, leucine, and isoleucine degradation’ (7 DEGs).” These pathways are listed, but the functional importance of oxidative phosphorylation or amino acid degradation in rubber biosynthesis is not explained.

 

Lines 342-345: “The results indicate that ACAT3, HMGR3, and SRPP8 exhibit negative feedback in response to tapping injury. Moreover, MVK2, SRPP5, SRPP6, REF5, REF6, and REF7 show positive feedback.” This section lacks detail on how the selection of these specific genes was justified.

 

The discussion interprets the results effectively, linking SRPP and REF protein expression to latex production. Please, expand on the broader implications of the findings. How could the identification of SRPP6 and REF5 be applied practically to enhance rubber yield or improve tapping strategies? Can this data inform breeding or tapping methods?

 

Lines 360-363: “Studies indicate that tapping-induced injuries can induce changes in the expression of many genes and proteins in rubber tree laticifer cells, particularly leading to the upregulation of genes and proteins related to latex metabolism.” This is a generic statement that needs more detailed discussion on how injury might lead to both positive (increased latex production) and negative (tree health deterioration) outcomes.

 

Lines 373-375: "The MVA pathway is the primary contributor to this rubber precursor." This statement is not expanded with further discussion on why this pathway is more significant than others, such as MEP.

 

Avoid restating results and focus more on interpreting their significance. Discuss possible future applications, such as molecular breeding or improved industrial tapping methods.

 

The conclusions are generally supported by the results. Please, strengthen the practical applications of your findings by discussing how they could inform breeding programs or tapping techniques. Highlight any limitations of the study and propose future research directions. 

Author Response

Comments 1: The title is mostly clear, but "highlights crucial tapping-responsive members" could be more specific. Consider revising as: "Transcriptomic and proteomic integration reveals key tapping-responsive factors in natural rubber biosynthesis of the Hevea rubber tree."

Response 1: Thank you for pointing this out. After careful consideration and discussion, we have revised our new title as: "Transcriptomic and proteomic integration reveals key tapping-responsive factors for natural rubber biosynthesis in the rubber tree Hevea brasiliensis" (Lines 2-4).

 

Comments 2: Some phrases in the abstract are unclear. For instance, "significantly impacts latex production" could be refined to "has a significant impact on latex production." Similarly, in "molecular mechanisms that natural rubber production in response to tapping stimulation remain largely enigmatic," the authors might revise to "the molecular mechanisms underlying natural rubber production in response to tapping stimulation remain largely unknown."

Response 2: Thank you for your valuable comments. We have thoroughly revised the original Abstract section, and also carefully corrected grammatical issues and emphasized the practical and scientific significance of our work within the new Abstract in the newly revised version (Lines 14-28).

 

Comments 3: Please, in the Introduction provide a more comprehensive review of prior work related to the roles of SRPPs, REFs, and tapping-induced physiological responses in rubber trees. Additional references should be included, especially from recent high-impact research in plant molecular biology and industrial crop physiology.

Response 3: Thank you for your valuable comments. In the revised Introduction, we have expanded the literature review to include a thorough discussion on the SRPP and REF families, highlighting their known functions in rubber biosynthesis and the physiological responses invoked by tapping (Lines 86-104). In this new manuscript, we have cited six recent high-impact papers, they are:

Chao, J.; Wu, S.; Shi, M.; Xu, X.; Gao, Q.; Du, H.; Gao, B.; Guo, D.; Yang, S.; Zhang, S.; Li, Y.; Fan, X.; H.C.; Kou, L.; Zhang, J.; Wang, Z.; Li, Y.; Xue, W.; Xu, J.; Deng, X.; Huang, X.; Gao, X.; Zhang, X.; Hu, Y.; Zeng, X.; Li, W.; Zhang, L.; Peng, S.; Wu, J.; Hao, B.; Wang, X.; Yu, H.; Li, J.; Liang, C.; Tian, W.M. Genomic insight into domestication of rubber tree. Nat. Commun. 2023, 14, 4651-4663.

Fan, Y.J.; Qi, J.Y.; Xiao, X.H.; Li, H.P.; Lan, J.X.; Huang, Y.C.; Yang, J.H.; Zhang, Y.; Zhang, S.M.; Tao, J.; Tang, C.R. Transcript and protein profiling provides insights into the molecular mechanisms of harvesting-induced latex production in rubber tree. Front. Genet. 2022, 13, 756270-756284.

Fang, Y.J.; Xiao, X.H.; Lin, J.S.; Lin, Q.; Wang, J.; Liu, K.; Li, Z.H.; Xing, J.F.; Liu, Z.L.; Wang, B.Y.; Qi, Y.Y.; Long, X.Y.; Zeng, X.; Hu, Y.S.; Qi, J.Y.; Qin, Y.X.; Yang, J.H.; Zhang, Y.; Zhang, S.M.; Ye, D.; Zhang, J.S.; Liu, J.Q.; Tang, C.R. Pan-genome and phylogenomic analyses highlight Hevea species delineation and rubber trait evolution. Nat. Commun. 2024, 15, 7232.

Liu, J.; Shi, C.; Shi, C.C.; Li, W.D.; Zhang, Q.J.; Zhang, Y.; Li, K.; Lu, H.F.; Shi, C.; Zhu, S.T.; Xiao, Z.Y.; Nan, H.; Yue, Y.; Zhu, X.G.; Yu, W.; Hong, X.N.; Fan G.Y.; Tong, Y.; Zhang, D.; Mao, C.L.; Liu, Y.L.; Hao, S.J.; Liu, W.Q.; Lv, M.Q.; Zhang, H.B.; Liu, Y.; Hutang, G.R.; Wang, J.P.; Wang, J.H.; Sun, Y.H.; Ni, S.B.; Chen, W.B.; Zhang, X.C.; Jiao, Y.N.; Eichler, E.E.; Li, G.H.; Liu, X.; Gao, L.Z. The chromosome-based rubber tree genome provides new insights into spurge genome evolution and rubber biosynthesis. Mol. Plant. 2020, 13, 336-350.

Nazri, A.Z.; Ghaffar, M.A.A. Response of the clone RRIM 3001 (Hevea brasiliensis) to three ethephon stimulation treatments and the identification of differentially expressed transcription factors for a water-based stimulant. J. Rubber Res. 2024, 27(1), 103-114.

Qin, Y.X.; Wang, J.; Fang, Y.J; Lu, J.L.; Shi, X.Y.; Yang, J.H.; Xiao X.H.; Luo X.H.; Long X.Y. Anaerobic metabolism in Hevea brasiliensis laticifers is relevant to rubber synthesis when tapping is initiated. Ind. Crops Prod. 2022, 178, 114663.

 

Comments 4: Lines 37-40: "Natural rubber (cis-1,4-polyisoprene), is indispensable to the global industry due to its exceptional physical properties." This is a well-known fact, but the introduction should connect this to more recent studies on molecular pathways and how they are modulated by tapping.

Response 4: Thank you for your valuable comments. We have revised the Introduction to provide a more comprehensive review of studies on molecular pathways related to tapping in rubber trees (Lines 57-72).

 

 Comments 5: The methods are generally well-organized, describing both transcriptomic and proteomic analyses. Please, provide more details on RNA and protein extraction, including temperature, conditions, and specific instruments used.

Response 5: Thank you for your valuable suggestions. We have expanded the Methods section to include more details on the RNA and protein extraction protocols (Lines 143-147, 162-173).

 

Comments 6: Lines 92-94: "Latex samples collected from the plants at days 0, 1, 3, and 5 were termed T1, T2, T3, and T4, respectively." The manuscript does not explain whether these time points are based on previous studies, preliminary experiments, or standard practices. Justify the selection of the sampling time points (T1, T2, T3, T4). Clarify why these intervals were chosen and how they relate to rubber regeneration stages.

Response 6: Thank you for your valuable feedback. The time points for latex sample collection (days 0, 1, 3, and 5) were selected based on a combination of our previous study and standard practices (S/2 d3 harvesting system: tapping half of the spiral once in three days frequency) in rubber tree tapping. We have revised the manuscript to include this justification and provide a clearer context for our sampling strategy (Lines 121-123).

 

Comments 7: Ensure all protocols (especially those related to RNA/protein extraction and bioinformatic analyses) are described with enough precision to allow replication. Clearly state the threshold values for statistical significance in the differential gene/protein expression analyses and mention any software used for validation, such as DESeq2.

Response 7: Thank you for your valuable feedback. In our revised manuscript, we ensure that all protocols related to RNA and protein extraction, as well as bioinformatic analyses, were detailed with sufficient precision to facilitate replication. We clearly specify the threshold values for statistical significance and include the software used for validation to enhance the transparency and reproducibility of our research. We have addressed these points in our revisions (Lines 143-201).

 

Comments 8: Lines 119-121: "Identification of differentially expressed genes (DEGs) was conducted using DESeq2 between each pair of groups, with the criterion fold change >2 or <0.5 (q < 0.05)." While fold changes are mentioned, more information about how the q-values were adjusted is needed.

Response 8: Thank you for your valuable feedback. In our analysis, the resulting p values were adjusted by controlling for the false discovery rate (FDR), which is a widely accepted approach in multiple hypothesis testing. We have revised the manuscript to include a detailed explanation of the q-value adjustment process (Lines 156-159).

 

Comments 9: The results are presented in a clear, structured manner with figures that support the analysis. In Figure 1 (A) and (D), the statistical differences currently presented do not accurately reflect the actual results. The authors will conduct a thorough reanalysis of the data using appropriate statistical tests to ensure the correct representation of significant differences between the groups. The revised figure will include accurate error bars and significance markers (e.g., p-values or asterisks) to clearly indicate statistically significant differences between time points. The authors will ensure that all results are presented with proper statistical interpretation.

Response 9: Thank you for your constructive feedback. In response to your comments, we have conducted a thorough re-analysis of the data using the Student’s t test to accurately reflect the significant differences between the groups. The revised Figure 1 will include updated error bars and significance markers (asterisks) to clearly indicate statistically significant differences between time points (Lines 234-239).

 

Comments 10: Lines 199-202: “Genes exhibiting similar expression patterns based on relative expression abundances in T2/T1, T3/T1, and T4/T1 were identified through trend analysis, categorizing gene expression trends into 20 clusters.” This statement doesn't explain the method used for trend analysis, such as software, algorithms, or statistical models.

Response 10: Thank you for your insightful comment. In our manuscript, we utilized the OmicShare platform's analytical tools (https://www.omicshare.com/tools/home/report/reporttrend.html) to conduct the trend analysis. We have revised our manuscript to include additional information about the trend analysis method (Lines 186-190).

 

Comments 11: Figure 2 (Lines 217-220): "Trend analysis of the identified genes... shows stage-specific patterns." While trends are described, there is no indication of how significance was determined for these trends. Additionally, the trends and patterns described in text (e.g., the upregulation of SRPPs and REFs) could be reinforced with additional visuals or summaries.

Response 11: Thank you for your insightful comments and suggestions. we have revised methods section where we detailed the methods used to assess the significance of the trends analysis. Figure 6 of our manuscript have depicted the stage-specific expression patterns of the identified genes related to the natural rubber biosynthesis (NRB) pathway, such as the upregulation of SRPPs and REFs. These visuals not only highlight the trends but also provide a more comprehensive view of the data. We have also included a summary table (Table S3) that presented the expression level of genes related to NRB, making it easier to grasp the patterns (Lines 186-190, 411-413, 426-428).

 

 Comments 12: Lines 274-277: “The most significantly enriched pathway was ‘Oxidative phosphorylation,’ encompassing 15 DEGs, followed by ‘Valine, leucine, and isoleucine degradation’ (7 DEGs).” These pathways are listed, but the functional importance of oxidative phosphorylation or amino acid degradation in rubber biosynthesis is not explained.

Response 12: Thank you for your valuable feedback. Oxidative phosphorylation is a critical process in cellular respiration, in which energy released from the electron transport chain can be used to synthesize ATP. In NRB, the ATP generated through oxidative phosphorylation serves as the primary energy source for the synthesis of isoprene units, the building blocks of natural rubber. The upregulation of genes involved in NRB pathway suggests an increased energy demand during the rubber biosynthesis process, highlighting the importance of maintaining an efficient energy supply for optimal rubber production. Furthermore, the “valine, leucine, and isoleucine degradation” pathway is involved in the catabolism of branched-chain amino acids, which are known to be precursors for the synthesis of isoprenoids. The degradation of these amino acids generates intermediates that can be channeled into the isoprenoid biosynthetic pathway, thus contributing to the supply of precursors for rubber production. The enrichment of DEGs in this pathway indicates a potential upregulation of isoprenoid biosynthesis, further supporting the role of these amino acids in rubber biosynthesis. We have revised the manuscript to provide a more detailed discussion on the relevance of these pathways. We hope that these revisions provide a clearer understanding of the functional importance of these pathways in rubber biosynthesis and strengthen the overall narrative of our study. (Lines 338-351).

 

Comments 13: Lines 342-345: “The results indicate that ACAT3, HMGR3, and SRPP8 exhibit negative feedback in response to tapping injury. Moreover, MVK2, SRPP5, SRPP6, REF5, REF6, and REF7 show positive feedback.” This section lacks detail on how the selection of these specific genes was justified.

Response 13: Thank you for your valuable feedback. The selection of nine DEGs was based on the following considerations: firstly, the DEGs selected were those that demonstrated statistically significant differential expression across the experimental conditions. Secondly, the selected nine DEGs were chosen with particular attention to their biological roles in the NRB pathway. Each gene was evaluated for its known involvement in key processes related to NRB pathway, allowing us to gain insightful perspectives on the regulatory mechanisms. In summary, while the nine DEGs were selected in part randomly, careful consideration was given to their statistical significance and biological relevance within the NRB pathway. Thank you for your attention to this detail, and we have expanded on this explanation in the revised manuscript to provide clarity to readers (Lines 415-418).

 

Comments 14: The discussion interprets the results effectively, linking SRPP and REF protein expression to latex production. Please, expand on the broader implications of the findings. How could the identification of SRPP6 and REF5 be applied practically to enhance rubber yield or improve tapping strategies? Can this data inform breeding or tapping methods?

Response 14: Thank you for your valuable feedback. we have revised the discussion section to expand on the broader implications of our findings, particularly focusing on the practical applications of the identification of SRPP6 and REF5 in enhancing rubber yield and improving tapping strategies (Lines 502-522).

 

 Comments 15: Lines 360-363: “Studies indicate that tapping-induced injuries can induce changes in the expression of many genes and proteins in rubber tree laticifer cells, particularly leading to the upregulation of genes and proteins related to latex metabolism.” This is a generic statement that needs more detailed discussion on how injury might lead to both positive (increased latex production) and negative (tree health deterioration) outcomes.

Response 15: We appreciate your insightful comment. To address this, we have expanded the discussion to elaborate on the dual effects of tapping-induced injuries. While such injuries can indeed result in the upregulation of genes and proteins associated with latex metabolism, fostering increased latex production, it is equally important to acknowledge the potential adverse effects on overall tree health. Specifically, tapping can lead to cellular stress and subsequent physiological disturbances, which may impair the tree’s growth and resilience over time. We have included a more detailed analysis in the revised manuscript to capture this complexity (Lines 434-452).

 

Comments 16: Lines 373-375: "The MVA pathway is the primary contributor to this rubber precursor." This statement is not expanded with further discussion on why this pathway is more significant than others, such as MEP.

Response 16: Thank you for your insightful comments. MVA pathway predominantly occurs in cytosol, which is the primary site for rubber biosynthesis in the laticifer cells of Hevea rubber tree. This spatial proximity allows for efficient utilization of the pathway's products for rubber synthesis [13]. While our both transcriptomic and proteomic data in this study demonstrated that MEP pathway can also contribute to isoprenoid synthesis for NRB, although it is traditionally localized in the plastids and is more associated with the production of isoprenoids for photosynthesis and other plastid-related processes [4]. In the laticifer cells of Hevea rubber tree, MVA pathway is major supplier of isoprenoid precursors due to its localization and regulatory features. We have revised the manuscript to provide a more comprehensive explanation (Lines 463-471).

 

 Comments 17: Avoid restating results and focus more on interpreting their significance. Discuss possible future applications, such as molecular breeding or improved industrial tapping methods.

Response 17: Thank you for your valuable comments, we have revised the discussion section to expand on the broader implications of our findings, particularly focusing on the practical applications and improving tapping strategies (Lines 502-522).

 

 Comments 18: The conclusions are generally supported by the results. Please, strengthen the practical applications of your findings by discussing how they could inform breeding programs or tapping techniques. Highlight any limitations of the study and propose future research directions. 

Response 18: Thank you for your insightful comment. We have revised conclusions section, incorporating the reviewer's suggestions regarding practical applications, limitations, and future research directions. Specifically: However, this study has also obvious limitations, including the potential influence of environmental factors for the gene and protein expression. Additionally, our analysis primarily focused on specific time points post-tapping, a more comprehensive temporal study could yield deeper insights into the dynamics of latex metabolism. Future study should include the exploration of the functional roles of the identified DEGs and DEPs through gene-editing technology or overexpression of target genes into the Hevea rubber tree to confirm their roles in latex regeneration and rubber yield. Overall, this study deepen our understanding of latex regeneration mechanisms in the latex of Hevea rubber tree and sets the groundwork for future research aimed at improving rubber yield through informed breeding and tapping strategies. (Lines 558-582).

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors:

Thank you for addressing the comments with such careful attention to detail. I appreciate the revisions you've made, which significantly improve the clarity and scientific rigor of the manuscript. The title is now much clearer and more specific, reflecting the key points of your study. The updated abstract and introduction are more comprehensive and do a better job of framing the study within the context of current research, especially regarding SRPPs, REFs, and tapping-induced responses in Hevea brasiliensis.

The additional references and expansion on recent high-impact studies are valuable contributions. The methodological details you've added for RNA and protein extraction, as well as the inclusion of more specific information about statistical analyses, will enhance the reproducibility of your work.

Furthermore, your justification for the selection of sampling time points is well explained, and the reanalysis of Figure 1, with clearer error bars and significance markers, makes the results more robust. The enhanced explanation of key pathways, such as oxidative phosphorylation and amino acid degradation, also strengthens the link between the data and natural rubber biosynthesis.

Congratulations. Overall, I find the revisions satisfactory, and I believe the manuscript has been significantly improved. Thank you for your diligence in responding to the feedback.

With kind regards,