Comparative Analysis of Alternative Splicing in Moso Bamboo and Its Dwarf Mutant, Phyllostachys edulis ‘Tubaeformis’

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Zenhua et al. describes the identification of alternative splicing events in the internodes of Mosa bamboo and its dwarf mutant variety, Shengyin bamboo. The transcriptomic study was complemented by phenotyping and electron microscopy. Further GO analysis revealed AS events in splicing factors (SR famly) and transcription factors (GRF family). The expreriment was designed properly, the results were clearly presented and the conclusions were supported by the results. In general, the manuscript is well-writen and comprehensive.  I have only two corrections/suggestions to the authors: 

- in line 193 and 372, I think that they mean 139,539 AS events (instead of 13,9539)

- I suggest that Figures 5 and 6 should be wider in order to  be more easily viewed by the reader. 

Author Response

The manuscript by Zenhua et al. describes the identification of alternative splicing events in the internodes of Mosa bamboo and its dwarf mutant variety, Shengyin bamboo. The transcriptomic study was complemented by phenotyping and electron microscopy. Further GO analysis revealed AS events in splicing factors (SR famly) and transcription factors (GRF family). The expreriment was designed properly, the results were clearly presented and the conclusions were supported by the results. In general, the manuscript is well-writen and comprehensive.  I have only two corrections/suggestions to the authors: 

- in line 193 and 372, I think that they mean 139,539 AS events (instead of 13,9539)

Answer: We revised it

- I suggest that Figures 5 and 6 should be wider in order to  be more easily viewed by the reader. 

Answer: We have widened Figures 5 and 6 according to your suggestion.

I consider the work of Zhenhua et al. to be a novel study with significant future potential. The paper is very well written and provides valuable datasets for the scientific community. However, I have some minor concerns and suggestions for improvement:

 

Reviewer 2 Report

Comments and Suggestions for Authors

I consider the work of Zhenhua et al. to be a novel study with significant future potential. The paper is very well written and provides valuable datasets for the scientific community. However, I have some minor concerns and suggestions for improvement:

 

Minor Concerns:

1. "Our sequencing data unveiled 13,9539 AS events, with retained 22 introns as the most prevalent events." -> It seems there is a typo in the number 13.9539. Please clarify the correct number.
2. Line 47: "Orchestrating post-transcriptional regulation of isoform diversity" -> Alternative splicing also affects cotranscriptional regulation. Please include this aspect.
3. Lines 60-62: "At the anatomical level, the difference in fiber length between Shengyin bamboo and Moso bamboo suggests that the shortening of internodes in Shengyin bamboo is due to the reduction in length of fiber cells." -> Please provide a citation for this data.

Methods:

1. "Were harvested at the Anji Bamboo Exposition Park, Zhejiang Province on April 8, 2022." -> As these are wild species, how was it ensured that environmental factors like light, shadow, or temperature didn’t affect the study?
2. Line 122: "Eukaryotic mRNA was selectively captured using Oligo(dT) beads." -> Please specify which poly(A) selection kit was used.
3. Line 135: "Kyoto Encyclopedia of Genes and Genomes (KEGG)" -> Specify which reference genome was used for the analysis.
   
   

Results:

1. Figure S1: The figure lacks axis labels and is unclear on its own. Please add appropriate labels and clarifications.
2. Figure 1C: Include a Y-axis label for better understanding.
3. Figure 2C: This could be moved to supplementary data. Also, clarify the number of samples used per species and whether the samples were taken from the same or different plants.
4. Figure 3: The figure is missing. Please include it or explain its omission.
5. Figure 4: This figure is not very informative. Specify whether the meta-analysis was done on wild-type or dwarf plants.
6. Figure 5A: Although well explained in lines 228, it would be clearer if Figure 5A included node numbers as reference points or a diagram indicating the parts of the plant from which samples were taken. Also, consider how many of the expressed genes are similar or different across nodes. Another point is that the expression of alternative spliced isoforms might vary with time of day or under different conditions, not just at the moment of sample extraction.
7. Figure 5B: This figure is difficult to read. I suggest moving it to supplementary data but keeping the explanation in the manuscript (lines 240).
   
   

Discussion:

1.     Have you found any genes up- or downregulated related to brassinosteroid synthesis or other hormones in your transcriptomes?

2.     Have the authors considered validating the transcriptome data with biochemical methods like qPCR or Western Blot?

 

I believe addressing some of these concerns will improve the clarity and impact of this important study.

Comments on the Quality of English Language

None.

Author Response

Minor Concerns:

1."Our sequencing data unveiled 13,9539 AS events, with retained 22 introns as the most prevalent events." -> It seems there is a typo in the number 13.9539. Please clarify the correct number.

Answer: We revised it

2. Line 47: "Orchestrating post-transcriptional regulation of isoform diversity" -> Alternative splicing also affects cotranscriptional regulation. Please include this aspect.

Answer: We included this aspect in revised manuscript.

 

3.Lines 60-62: "At the anatomical level, the difference in fiber length between Shengyin bamboo and Moso bamboo suggests that the shortening of internodes in Shengyin bamboo is due to the reduction in length of fiber cells." -> Please provide a citation for this data.

Answer: We provide a citation for this data in revised manuscript.
Methods:

1."Were harvested at the Anji Bamboo Exposition Park, Zhejiang Province on April 8, 2022." -> As these are wild species, how was it ensured that environmental factors like light, shadow, or temperature didn’t affect the study?

Answer: The planting and management conditions for Shengyin bamboo and Moso bamboo are consistent, with no slope, similar forest density, and no surrounding trees to block sunlight or growth. Furthermore, because both are planted in the same bamboo garden, they share the same environmental conditions, including similar precipitation and wind exposure.

2. Line 122: "Eukaryotic mRNA was selectively captured using Oligo(dT) beads." -> Please specify which poly(A) selection kit was used.

Answer: We added the information you mentioned in our revised manuscript.

3.Line 135: "Kyoto Encyclopedia of Genes and Genomes (KEGG)" -> Specify which reference genome was used for the analysis.
Answer: Thank you for your suggestion, the KEGG analysis were referenced to Oryza Sativa genome. We provided these information according to your suggestion.

Results:

1. Figure S1: The figure lacks axis labels and is unclear on its own. Please add appropriate labels and clarifications.

Answer: The X-axis represents the number of internodes counting up from the base, and the Y-axis represents the internode length. We revised the figure and figure legend according to your suggestion.

1. Figure 1C: Include a Y-axis label for better understanding.

Answer: We added it.

1. Figure 2C: This could be moved to supplementary data. Also, clarify the number of samples used per species and whether the samples were taken from the same or different plants.

Answer: We moved Figure 2C to supplementary data according to your suggestion.

1. Figure 3: The figure is missing. Please include it or explain its omission.

Answer: Sorry, we forgot to insert Figure 3 into the main text of the article, but Figure 3 is included in the uploaded image compression package. We have already inserted Figure 3 in the revised manuscript.

1. Figure 4: This figure is not very informative. Specify whether the meta-analysis was done on wild-type or dwarf plants.

Answer: Due to the lack of significant differences in gene structure and the number of homologous isoforms between Shengyin bamboo and Mao bamboo, we did not separate the two species for individual analysis.  Additionally, we have provided an explanation in the figure legend.

1. Figure 5A: Although well explained in lines 228, it would be clearer if Figure 5A included node numbers as reference points or a diagram indicating the parts of the plant from which samples were taken. Also, consider how many of the expressed genes are similar or different across nodes. Another point is that the expression of alternative spliced isoforms might vary with time of day or under different conditions, not just at the moment of sample extraction.

 

Answer: Thank you for your suggestions. Firstly, following your instructions, we have re-described our sampling procedure and have annotated the specific sampling locations in Figure S1. Secondly, we have analyzed the similarity and differences of differentially expressed genes between internodes using a Venn diagram. The analysis of differentially expressed genes in the comparisons of the three groups revealed that there were more common differentially expressed genes between the middle (M15 vs S15) and the base of the bamboo shoots (M6 vs S6), while at the top, there were more unique differentially expressed genes (M24 vs S24) (Figure 5B). The results of the analysis of differential alternative splicing events were similar to those of the differential expression gene analysis (Figure 5C).

Thirdly, the collection time for our experimental samples was around 10 a.m., and this sampling time has been explained in the revised manuscript.

 

 

1. Figure 5B: This figure is difficult to read. I suggest moving it to supplementary data but keeping the explanation in the manuscript (lines 240).
   Answer: Thank you for your suggestion,  we moved it to supplementary data according to your suggestion.
   
   

Discussion:

1. Have you found any genes up- or downregulated related to brassinosteroid synthesis or other hormones in your transcriptomes?

Answer: Indeed, we did identify some differentially expressed genes related to brassinosteroid biosynthesis and signaling transduction in the sequencing results, but the number was relatively small. Furthermore, the primary focus of this study was to explore the differences in alternative splicing between the two bamboos. The gene families that were selected and analyzed were those that exhibited certain patterns of differential splicing events between the two bamboos. In future research, we will integrate your suggestions with the dynamic changes in the content of major endogenous hormones in these two bamboos to discuss the regulatory role of several hormones, including brassinosteroids, on the internodal elongation growth of bamboo shoots.

2. Have the authors considered validating the transcriptome data with biochemical methods like qPCR or Western Blot?

Answer: Thank you for your suggestions. We will incorporate these experiments into our future research. 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled " Comparative analysis of alternative splicing in Moso bamboo and the dwarf mutant variety Phyllostachys edulis 'Tubaeformis'" focuses on the comparative analysis of alternative splicing (AS) events in Moso bamboo (Phyllostachys edulis) and its dwarf mutant variety, Shengyin bamboo (Phyllostachys edulis ‘Tubaeformis’). The research utilizes PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide AS events. The findings reveal 13,9539 AS events, with retained introns being the most prevalent. Differentially alternatively spliced (DAS) genes between Moso bamboo and Shengyin bamboo were significantly enriched in RNA splicing-related processes. The high expression of certain SR isoforms in the 24th internode of Moso bamboo leads to a greater number of AS events. AS modifies the functional domains of specific GRF isoforms, resulting in either domain loss or the acquisition of new domains, a phenomenon more common in Shengyin bamboo. These insights highlight the molecular mechanisms underlying the complex regulatory networks affecting the growth of Shengyin bamboo. The research offers significant insights into the molecular mechanisms of AS and its impact on the growth differences between Moso bamboo and Shengyin bamboo, particularly through the analysis of SR and GRF gene families. The findings have broad implications for understanding plant growth and development, providing valuable information that can be applied to other species and potentially informing genetic improvement programs in bamboo and other crops. The aims of this study are clear and the results are interest to me. I have some comments and suggestions in this manuscript:

First, the study is unclear in the sample size. It looks like the authors collect samples from one individual each and separate the six, 15, and 24 internodes to study. Please revise this part to make it clear.

Second, the study does not consider how environmental factors might influence AS events. Since environmental conditions can significantly impact gene expression and splicing, this is a notable omission that could affect the generalizability of the findings. More discussion will be help to solve this point.
Third, the study does not fully explore the downstream functional consequences of the identified isoform variability. Understanding how these isoforms impact cellular processes and overall plant physiology would provide a more comprehensive mechanistic understanding. I could understand it is a difficult part, however, more discussion will be good for reader to figure out this point.

Forth, the temporal dynamics of AS events during different growth stages are not deeply analyzed. A temporal analysis could reveal how AS regulation changes over the course of bamboo development and growth.

Fifth, the study focuses on SR and GRF gene families, potentially overlooking other important gene families involved in internode growth regulation. A broader discussion or mechanism from related papers including other transcription factors and regulatory proteins could provide a more comprehensive picture for reader to catch the ideas of this manuscript.

 

Comments on the Quality of English Language

For the English writing, the manuscript generally maintains a high level of clarity and precision in its descriptions. However, some sentences could be rephrased for better readability if polish by native English speaking. Some examples that I put below:

1. The title”Comparative analysis of alternative splicing in Moso bamboo and the dwarf mutant variety Phyllostachys edulis 'Tubaeformis'" suggested to replace "Comparative Analysis of Alternative Splicing in Moso Bamboo and Its Dwarf Mutant, Phyllostachys edulis 'Tubaeformis'"

2. Line 16-17 “The internode length is an important phenotypic trait of bamboo and a key indicator affecting the processing and utilization of bamboo materials." suggested to replace "Internode length is a crucial phenotypic trait of bamboo, significantly impacting its processing and utilization."

3. Line 19-22 "Here, we conducted PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and Shengyin Bamboo, and compare the differences in alternative splicing between these two bamboo shoots." suggested to replace "We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin Bamboo, and compared the differences between the two."

4. Line 31-33 "Our results provide new insights into the molecular mechanisms underlying the complex regulatory networks of Shengyin bamboo shoot growth." suggested to replace "These findings offer new insights into the molecular mechanisms regulating the growth of Shengyin bamboo shoots."

5. Line 37-38 "Moso bamboo (Phyllostachys edulis) is a highly adaptable plant with multiple uses across different industries, including food production, papermaking, and construction." suggested to replace "Moso bamboo (Phyllostachys edulis) is a highly adaptable plant utilized in various industries, including food production, papermaking, and construction."

6. Line 44-45 "So far, the regulatory mechanisms played by alternative splicing in the abnormal internode development of Phyllostachys edulis ‘Tubaeformis’ remain unclear." suggested to replace "The role of alternative splicing in the abnormal internode development of Phyllostachys edulis ‘Tubaeformis’ is not well understood."

7. Line 193-194 "In total, 13,9539 AS events were identified without transcriptome assembly, so any possible artificial result could be avoided (Supplementary table 3)." suggested to replace "In total, 13,9539 AS events were identified without transcriptome assembly, avoiding potential artificial results (see Supplementary Table 3)."

8. Line 202-203 "Isoform complexity stems from alternative transcription start and end sites, alongside alternative splicing (AS), all pivotal in gene expression regulation." suggested to replace "Isoform complexity arises from alternative transcription start and end sites, as well as alternative splicing (AS), all of which are crucial in gene expression regulation."

9. Line 212-214 "As the number of exons and the overall mRNA length increase in bamboo shoots, the probability of genes undergoing alternative splicing also increases accordingly (Figure 4A and Figure 4B)." suggested to replace "The likelihood of genes undergoing alternative splicing increases with the number of exons and the overall mRNA length in bamboo shoots (Figures 4A and 4B)."

10. Line 289-291 "As an important post-transcriptional regulatory mechanism, alternative splicing (AS) is widely involved in various biological processes in plants." suggested to replace "Alternative splicing (AS), an important post-transcriptional regulatory mechanism, is widely involved in various biological processes in plants."

11. Line 293-297 "Through the analysis of differential alternative splicing and differential gene expression in moso bamboo and Shengyin bamboo shoots, it was found that the vast majority of differentially alternatively spliced genes do not exhibit differential expression, indicating that alternative splicing and gene expression regulation are two independent regulatory mechanisms." suggested to replace "Analysis of differential alternative splicing and gene expression in Moso bamboo and Shengyin bamboo shoots revealed that most differentially alternatively spliced genes do not exhibit differential expression. This indicates that alternative splicing and gene expression regulation are independent mechanisms."

12. Line 351-353 "During the growth and development of Moso bamboo and Shengyin bamboo shoots, many GRF isoforms lost the WRC or QLQ domain due to alternative splicing." suggested to replace "During the growth and development of Moso bamboo and Shengyin bamboo shoots, many GRF isoforms lost the WRC or QLQ domains due to alternative splicing."

13. Line 369=370 "This study addresses the understudied post-transcriptional mechanisms of internode shortening in Shengyin bamboo." suggested to replace "This study explores the previously understudied post-transcriptional mechanisms underlying internode shortening in Shengyin bamboo."

 

 

Author Response

The manuscript titled " Comparative analysis of alternative splicing in Moso bamboo and the dwarf mutant variety Phyllostachys edulis 'Tubaeformis'" focuses on the comparative analysis of alternative splicing (AS) events in Moso bamboo (Phyllostachys edulis) and its dwarf mutant variety, Shengyin bamboo (Phyllostachys edulis ‘Tubaeformis’). The research utilizes PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide AS events. The findings reveal 13,9539 AS events, with retained introns being the most prevalent. Differentially alternatively spliced (DAS) genes between Moso bamboo and Shengyin bamboo were significantly enriched in RNA splicing-related processes. The high expression of certain SR isoforms in the 24th internode of Moso bamboo leads to a greater number of AS events. AS modifies the functional domains of specific GRF isoforms, resulting in either domain loss or the acquisition of new domains, a phenomenon more common in Shengyin bamboo. These insights highlight the molecular mechanisms underlying the complex regulatory networks affecting the growth of Shengyin bamboo. The research offers significant insights into the molecular mechanisms of AS and its impact on the growth differences between Moso bamboo and Shengyin bamboo, particularly through the analysis of SR and GRF gene families. The findings have broad implications for understanding plant growth and development, providing valuable information that can be applied to other species and potentially informing genetic improvement programs in bamboo and other crops. The aims of this study are clear and the results are interest to me. I have some comments and suggestions in this manuscript:

First, the study is unclear in the sample size. It looks like the authors collect samples from one individual each and separate the six, 15, and 24 internodes to study. Please revise this part to make it clear.

Answer: Following your instructions, we have re-described our sampling procedure and have annotated the specific sampling locations in Figure S1.

Second, the study does not consider how environmental factors might influence AS events. Since environmental conditions can significantly impact gene expression and splicing, this is a notable omission that could affect the generalizability of the findings. More discussion will be help to solve this point.

Answer: Shoot samples of  Shengyin bamboo and  Moso bamboo, were harvested at the Anji Bamboo exposition park, Zhejiang Province at 10 a.m. on April 8, 2022. The planting and management conditions for Shengyin bamboo and Moso bamboo are consistent, with no slope, similar forest density, and no surrounding trees to block sunlight or growth. We provided related information in our revised manuscript.
Third, the study does not fully explore the downstream functional consequences of the identified isoform variability. Understanding how these isoforms impact cellular processes and overall plant physiology would provide a more comprehensive mechanistic understanding. I could understand it is a difficult part, however, more discussion will be good for reader to figure out this point.

Answer: In the Results and Discussion sections, we added analyses of two gene families, E2F and NAM, exploring the relationships between the expression of these isoforms and the elongation of bamboo internodes, as well as the growth involving cell elongation and cell division. Additionally, we discussed events of domain acquisition and loss in Shengyin bamboo and Moso bamboo, and explored the connections between domain acquisition of paralogous genes and the genome duplication events in Moso bamboo.

Forth, the temporal dynamics of AS events during different growth stages are not deeply analyzed. A temporal analysis could reveal how AS regulation changes over the course of bamboo development and growth.

Answer: Thank you for your suggestions. This study primarily focuses on comparing different parts of Shengyin bamboo and Phyllostachys edulis shoots during the same developmental stage. We will take your recommendations into account in our future research endeavors.

Fifth, the study focuses on SR and GRF gene families, potentially overlooking other important gene families involved in internode growth regulation. A broader discussion or mechanism from related papers including other transcription factors and regulatory proteins could provide a more comprehensive picture for reader to catch the ideas of this manuscript.

In the revised manuscript, we have included the analysis of E2F and NAC genes and discussed the E2F and NAC family in the discussion section. Additionally, we have conducted a correlation analysis between AS-PSI (percent spliced in) and gene expression, thereby enriching the content of the paper. In addition, we also investigated other transcription factors related to plant growth and development, such as ARF, ap2, etc. Although there are certain differences in expression, but there was no significant difference in alternative splicing.

 

 

 

For the English writing, the manuscript generally maintains a high level of clarity and precision in its descriptions. However, some sentences could be rephrased for better readability if polish by native English speaking. Some examples that I put below:

Answer: Thank you for your suggestion, our paper has already undergone language editing by one of our Indian colleagues. If necessary, we will engage a professional editing service for further refinement in the next round of revisions.

1.The title”Comparative analysis of alternative splicing in Moso bamboo and the dwarf mutant variety Phyllostachys edulis 'Tubaeformis'" suggested to replace "Comparative Analysis of Alternative Splicing in Moso Bamboo and Its Dwarf Mutant, Phyllostachys edulis 'Tubaeformis'"

Answer: Thank you for your suggestion, we revised it.

2.Line 16-17 “The internode length is an important phenotypic trait of bamboo and a key indicator affecting the processing and utilization of bamboo materials." suggested to replace "Internode length is a crucial phenotypic trait of bamboo, significantly impacting its processing and utilization."

Answer: Thank you for your suggestion, we revised it.

3.Line 19-22 "Here, we conducted PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and Shengyin Bamboo, and compare the differences in alternative splicing between these two bamboo shoots." suggested to replace "We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin Bamboo, and compared the differences between the two."

Answer: We revised it.

4.Line 31-33 "Our results provide new insights into the molecular mechanisms underlying the complex regulatory networks of Shengyin bamboo shoot growth." suggested to replace "These findings offer new insights into the molecular mechanisms regulating the growth of Shengyin bamboo shoots."

Answer: We revised it.

5.Line 37-38 "Moso bamboo (Phyllostachys edulis) is a highly adaptable plant with multiple uses across different industries, including food production, papermaking, and construction." suggested to replace "Moso bamboo (Phyllostachys edulis) is a highly adaptable plant utilized in various industries, including food production, papermaking, and construction."

Answer: We revised it.

6.Line 44-45 "So far, the regulatory mechanisms played by alternative splicing in the abnormal internode development of Phyllostachys edulis ‘Tubaeformis’ remain unclear." suggested to replace "The role of alternative splicing in the abnormal internode development of Phyllostachys edulis ‘Tubaeformis’ is not well understood."

Answer: We revised it.

7.Line 193-194 "In total, 13,9539 AS events were identified without transcriptome assembly, so any possible artificial result could be avoided (Supplementary table 3)." suggested to replace "In total, 13,9539 AS events were identified without transcriptome assembly, avoiding potential artificial results (see Supplementary Table 3)."

Answer: We revised it.

8.Line 202-203 "Isoform complexity stems from alternative transcription start and end sites, alongside alternative splicing (AS), all pivotal in gene expression regulation." suggested to replace "Isoform complexity arises from alternative transcription start and end sites, as well as alternative splicing (AS), all of which are crucial in gene expression regulation."

Answer: We revised it.

9.Line 212-214 "As the number of exons and the overall mRNA length increase in bamboo shoots, the probability of genes undergoing alternative splicing also increases accordingly (Figure 4A and Figure 4B)." suggested to replace "The likelihood of genes undergoing alternative splicing increases with the number of exons and the overall mRNA length in bamboo shoots (Figures 4A and 4B)."

Answer: We revised it.

10.Line 289-291 "As an important post-transcriptional regulatory mechanism, alternative splicing (AS) is widely involved in various biological processes in plants." suggested to replace "Alternative splicing (AS), an important post-transcriptional regulatory mechanism, is widely involved in various biological processes in plants."

Answer: We revised it.

11.Line 293-297 "Through the analysis of differential alternative splicing and differential gene expression in moso bamboo and Shengyin bamboo shoots, it was found that the vast majority of differentially alternatively spliced genes do not exhibit differential expression, indicating that alternative splicing and gene expression regulation are two independent regulatory mechanisms." suggested to replace "Analysis of differential alternative splicing and gene expression in Moso bamboo and Shengyin bamboo shoots revealed that most differentially alternatively spliced genes do not exhibit differential expression. This indicates that alternative splicing and gene expression regulation are independent mechanisms."

Answer: We revised it.

12.Line 351-353 "During the growth and development of Moso bamboo and Shengyin bamboo shoots, many GRF isoforms lost the WRC or QLQ domain due to alternative splicing." suggested to replace "During the growth and development of Moso bamboo and Shengyin bamboo shoots, many GRF isoforms lost the WRC or QLQ domains due to alternative splicing."

Answer: We revised it.

13.Line 369=370 "This study addresses the understudied post-transcriptional mechanisms of internode shortening in Shengyin bamboo." suggested to replace "This study explores the previously understudied post-transcriptional mechanisms underlying internode shortening in Shengyin bamboo."

 Answer: We revised it.

Reviewer 4 Report

Comments and Suggestions for Authors

The author provided datasets of long-read and short read sequencing of Moso bamboo and its dwarf mutant. They did some basic isoform detection and alternative splicing events discover. The methods seem fine. The sequenced datasets may useful for related researchers. 

 

Comments:

 

Major:

1. Line 237. The authors could not assume the Alternative splicing and gene expression are independent only by overlap analysis. Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) is an important pathway for gene expression regulation in eukaryotes, including plants. I suggest them do AS-NMD analysis and do AS-PSI (precent spliced in) to gene expression correlation analysis.

 

Minor:

1.     Line 129, a typo of fastpp?. And you’d better cite the published paper rather than its preprint if it already published.  https://doi.org/10.1002/imt2.107

 

1.     Fig. 1C, please add length unit, statistic method and sample numbers (n) in figure legend.

 

2.     Line 171. Suggest the author use full name of FLNC when it first appears, they explained it when it expressed second in Line 172. Why the number of FLNC reads not showed in Table S1? Please add titles on the supplementary tables.

 

3.     Table S2. Please add titles. Is the column name incorrect? Unmapped reads rates were 99%+ ?

 

4.     Fig. 2B Used the reverse color legend for the novel/annotated genes? The figure 2B legend said the blue and gray bar, while the figure used gray and black.

 

5.     Fig. 3B. It showed M24 in figure, why the authors used M22 in main text (Line 198). And the Figure 2C also used M22.

 

6.     Line 200, which statistic method the authors used to get it is not significant different?

 

7.     Figure 5A, please add the comparison name in figure or in figure legend.

 

8.     Line 256. Is it M24 rather than M22?

 

 

Author Response

Major:

1. Line 237. The authors could not assume the Alternative splicing and gene expression are independent only by overlap analysis. Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) is an important pathway for gene expression regulation in eukaryotes, including plants. I suggest them do AS-NMD analysis and do AS-PSI (precent spliced in) to gene expression correlation analysis.

 Answer: Thank you for your suggestion. We have removed these statements. We also appreciate your suggestion to conduct AS-NMD and AS-PSI analyses in our paper. Regarding the AS-NMD analysis, we have tried several software tools but have not obtained satisfactory results. We are currently attempting the analysis using GATK HaplotypeCaller software. Due to the journal's deadline for revisions having passed, we are unable to include AS-NMD results in the revised manuscript. If possible, we plan to incorporate these analyses in future studies. Additionally, the differential alternative splicing analysis conducted in this study was filtered using the percent spliced in (PSI) criteria you mentioned, with filtering thresholds of FDR ≤ 0.05 and ∣IncLevelDiff∣ ≥ 0.2. Additionally, we have also plotted a volcano plot for differential PSI, as shown in Figure 4S.

Minor:

1. Line 129, a typo of fastpp?. And you’d better cite the published paper rather than its preprint if it already published. https://doi.org/10.1002/imt2.107

        Answer: Fastp is correct, and we revised it. In addition, we have re-cited a previously published reference according to your suggestion.

2. Fig. 1C, please add length unit, statistic method and sample numbers (n) in figure legend.

 Answer: We added length unit in revised Fig. 1c. For each individual, 10 fields of view were observed, and within each field of view, 5 cells were examined. In total, the lengths of 150 cells were observed for each type of bamboo. p < 0.01 indicates a statistically significant difference, marked as** in figure. In the revised figure legend, we have described the method for measuring cell length according to your suggestion.

3. Line 171. Suggest the author use full name of FLNC when it first appears, they explained it when it expressed second in Line 172. Why the number of FLNC reads not showed in Table S1? Please add titles on the supplementary tables.

 Answer: Thank you for your reminding. We explained FLNC when it first appears in new version manuscript. Besides ,we added FLNC reads as well as titles of table S1 in revised manuscript.

4. Table S2. Please add titles. Is the column name incorrect? Unmapped reads rates were 99%+ ?

Answer: Thank you for your reminding.  In the revised manuscript, we have included titles. In the previous version, we reversed the column headers of the two sets of data in the table S2, and this has been corrected in the revised manuscript.

5. Fig. 2B Used the reverse color legend for the novel/annotated genes? The figure 2B legend said the blue and gray bar, while the figure used gray and black.

 Answer: Thank you for your reminding, the black column representing annotated genes and the grey column representing newly identified genes, we revised the figure legend.

6. Fig. 3B. It showed M24 in figure, why the authors used M22 in main text (Line 198). And the Figure 2C also used M22.

 Answer:We have made modifications to our error standards, and the correct one is M24.

7. Line 200, which statistic method the authors used to get it is not significant different?

 Answer: We have added differential expression gene screening parameters according to your suggestions.

8. Figure 5A, please add the comparison name in figure or in figure legend.

 Answer: We added it.

9. Line 256. Is it M24 rather than M22?

Answer: We revised it.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

No more comments.