Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript authors are reporting about the Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce. Authors generated CRISPR/Cas9-mediated LsSOC1 knockout (KO) lines and evaluated their phenotypes under high-temperature conditions. According to the claims, compared with wild-type (WT) plants, LsSOC1-KO lines exhibited delayed bolting by up to 18.6 days and reduced stem elongation by approximately 3.8 cm. Further authors performed transcriptome analysis to support their hypothesis. Finally, it was concluded that LsSOC1 is a promising genetic target for breeding heat-resilient leafy vegetables.

The manuscript is well prepared and needful is done to support the findings. There are a few concerns that must be addressed.

In abstract section the stem elongation of LsSOC1-KO lines must be provided in term of percentage change compared with the control for better understanding.

Line 86: The source and variety of Lettuce seeds must be mentioned here.

Line 116: A relevant citation must be mentioned regarding flowering time as authors mentioned that flowering was defined as the emergence of the first visible floral bud.

Section 2.7. Authors have used spectrophotometer to quantify total anthocyanin content. Whereas in the heading HPLC has been mentioned. It must be made clear which technique was used here.

Line 183: which method was used to observe the presence of frameshift-inducing mutations. Some details can be added with evidence for the ease of the readers.

Table 1: authors must revisit the statistical analysis regarding Days to flowering. It seems difficult for obtain P<0.001 for such a narrow range of treatment mean data.

 Section 3.2. data in agronomic traits must also be provided in term of percentage or fold change compared to control.

The same can be done for anthocyanin analysis in section 3.3.

Discussion is well prepared.

Author Response

Response of Review -1

Manuscript
Title: Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce

Manuscript ID
dna-3710721

 

We sincerely thank the reviewers for their valuable comments and suggestions. We have carefully addressed all the points raised and revised the manuscript accordingly. All changes made in the revised manuscript are highlighted in blue text.

 

In this manuscript authors are reporting about the Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce. Authors generated CRISPR/Cas9-mediated LsSOC1 knockout (KO) lines and evaluated their phenotypes under high-temperature conditions. According to the claims, compared with wild-type (WT) plants, LsSOC1-KO lines exhibited delayed bolting by up to 18.6 days and reduced stem elongation by approximately 3.8 cm. Further authors performed transcriptome analysis to support their hypothesis. Finally, it was concluded that LsSOC1 is a promising genetic target for breeding heat-resilient leafy vegetables.

The manuscript is well prepared and needful is done to support the findings. There are a few concerns that must be addressed.

 

In abstract section the stem elongation of LsSOC1-KO lines must be provided in term of percentage change compared with the control for better understanding.

• We appreciate the reviewer’s suggestion to provide the stem elongation data in terms of percentage change. While stem elongation in lettuce is closely associated with bolting and thus often reported in absolute values (cm) to reflect agronomic relevance, we agree that including the percentage change compared with WT can improve interpretability. Accordingly, we have revised the abstract to include both the absolute and relative values. The sentence now reads:
  line 18-20: SOC1-KO lines exhibited delayed bolting by up to 18.6 days and stem elongation was reduced by approximately 3.8 cm, which is equivalent to a 36.1% decrease compared to wild-type (WT) plants.

Line 86: The source and variety of Lettuce seeds must be mentioned here.

• We thank the reviewer for this helpful suggestion. In the revised manuscript, we have included the cultivar name and seed source to clarify the plant material used in this study. Specifically, the following sentence was added at the beginning of the Materials and Methods section:
  line 97-98: Lettuce (Lactuca sativa cv. Seonpung Plus; a Korean red romaine-type cultivar) seeds were obtained from Kwonnong Seed Co., Ltd. (Cheongju, Chungbuk, Korea)

Line 116: A relevant citation must be mentioned regarding flowering time as authors mentioned that flowering was defined as the emergence of the first visible floral bud.

• We thank the reviewer for this insightful comment. In the revised manuscript, we have replaced the previous statement with a more appropriate citation that clearly defines flowering time as the appearance of the first visible floral bud.

Section 2.7. Authors have used spectrophotometer to quantify total anthocyanin content. Whereas in the heading HPLC has been mentioned. It must be made clear which technique was used here.

• We sincerely thank the reviewer for pointing out this inconsistency. We confirm that total anthocyanin content in this section was quantified using a spectrophotometric method, not HPLC. Accordingly, we have revised the section heading from “HPLC-Based Quantification of Anthocyanins” to:
  Line 172 :Anthocyanin Quantification

Line 183: which method was used to observe the presence of frameshift-inducing mutations. Some details can be added with evidence for the ease of the readers.

• We appreciate the reviewer’s helpful comment. In response, we have revised the Materials and Methods section to clarify the method used to identify target site mutations and frameshift events. Specifically, we added the following sentence:
• Line 122-125: To identify target site mutations, PCR amplicons were subjected to paired-end sequencing using the MiniSeq platform (Illumina, San Diego, CA, USA), and the resulting reads were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/#!, accessed on 12 January 2020).

Table 1: authors must revisit the statistical analysis regarding Days to flowering. It seems difficult for obtain P<0.001 for such a narrow range of treatment mean data.

• We thank the reviewer for the insightful comment. However, we would like to clarify that the statistical analysis in Table 1 was performed using one-way ANOVA followed by Tukey’s HSD test, with a sample size of n ≥ 12 per genotype, as described in the figure/table legend and Materials and Methods. Despite the relatively narrow differences in mean flowering time, the consistently low standard errors and clear group-wise separation contributed to the observed statistical significance (P < 0.001). We have double-checked the raw data and revalidated the analysis to ensure robustness. For clarity, we have now revised the table legend to explicitly mention the sample size and statistical test used.

Section 3.2. data in agronomic traits must also be provided in term of percentage or fold change compared to control.

• We appreciate the reviewer’s constructive suggestion. In response, we have revised the Results section to include percentage changes relative to the wild-type (WT) control for major agronomic traits.

The same can be done for anthocyanin analysis in section 3.3.

• Thank you for the suggestion. As requested, we have presented the quantitative differences in anthocyanin accumulation in Section 3.3 using both absolute values (mg/100 g DW) and relative changes expressed as percentage increases compared to WT. The fold-change values have already been clearly described in the revised text to facilitate interpretation. We believe this adequately addresses the reviewer's comment.

Discussion is well prepared.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript review: ‘Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce’.

This study presents a robust investigation into the role of the LsSOC1 gene in regulating the generative phase transition and heat stress response in lettuce (Lactuca sativa). The work is comprehensive and timely, making use of advanced genome editing tools (CRISPR/Cas9), transcriptomic analysis, and the assessment of phenotypic traits and metabolites. The results have potential relevance for breeding heat-tolerant varieties. However, some issues need to be corrected or clarified, both substantively and linguistically. 

The introduction is too encyclopaedic in places — some paragraphs resemble summaries of literature reviews, with no clear transition to the hypothesis. I would also suggest adding information on the importance of metabolites such as flavonoids and anthocyanins in delaying bolting.
The materials and methods section lacks information on which genotypes, varieties, or lines were studied. The term 'deep sequencing' should be clarified, as should the information it covers. A description of the bioinformatics analysis for RNA-seq is also missing.
The images in the 'Results' section lack a scale, making it difficult to assess the actual changes.

In the discussion, the authors cite the conservative function of SOC1 but do not provide any comparative data, such as effect size, expression, or localisation, which weakens their argument. There is also no mention of the functional differences of SOC1 in monocotyledonous and dicotyledonous plants, which could explain some of the specific effects observed in lettuce.
Furthermore, the authors only discuss selected genes and GO terms. In contrast, the list of DEGs in the supplements includes numerous genes with potentially important functions (e.g., WRKY, MYB, GST, and aquaporins) that are not mentioned in the text.
The text mentions an increase in flavonoid pathway gene expression but does not attempt to correlate this with phenotype or anthocyanin levels despite having taken HPLC measurements.
The authors indicate that LsSOC1 modulates the heat response but do not provide a mechanism. Does LsSOC1 expression increase under heat stress? Were HSP70 or other indicators modulated directly?
The discussion ends abruptly without a section on the study's limitations or directions for future work.

Comments on the Quality of English Language

The manuscript is written in English and is generally comprehensible, but significant editorial improvements are needed at both the stylistic and grammatical levels. In many places, the text appears to have been machine-translated.

Author Response

Response of Review -2

Manuscript
Title: Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce

Manuscript ID
dna-3710721

 

We sincerely thank the reviewers for their valuable comments and suggestions. We have carefully addressed all the points raised and revised the manuscript accordingly. All changes made in the revised manuscript are highlighted in blue text.

 

This study presents a robust investigation into the role of the LsSOC1 gene in regulating the generative phase transition and heat stress response in lettuce (Lactuca sativa). The work is comprehensive and timely, making use of advanced genome editing tools (CRISPR/Cas9), transcriptomic analysis, and the assessment of phenotypic traits and metabolites. The results have potential relevance for breeding heat-tolerant varieties. However, some issues need to be corrected or clarified, both substantively and linguistically. 

The introduction is too encyclopaedic in places — some paragraphs resemble summaries of literature reviews, with no clear transition to the hypothesis.

• We appreciate the reviewer’s insight. To address this concern, we revised the latter part of the introduction to reduce encyclopedic descriptions and improve the logical flow toward the hypothesis. We also added a clear hypothesis statement to establish a stronger conceptual link between the background and our research objectives.

I would also suggest adding information on the importance of metabolites such as flavonoids and anthocyanins in delaying bolting.

• We thank the reviewer for the suggestion to incorporate the role of metabolites such as flavonoids and anthocyanins in delaying bolting. In response, we have added a dedicated sentence in the latter part of the introduction (line 75-80), discussing how these metabolites contribute to thermotolerance and reproductive delay under heat stress. This addition reinforces the importance of metabolic modulation in floral timing control.

The materials and methods section lacks information on which genotypes, varieties, or lines were studied.

• We thank the reviewer for this helpful suggestion. In the revised manuscript, we have included the cultivar name and seed source to clarify the plant material used in this study. Specifically, the following sentence was added at the beginning of the Materials and Methods section:
  line 97-98: Lettuce (Lactuca sativa cv. Seonpung Plus; a Korean red romaine-type cultivar) seeds were obtained from Kwonnong Seed Co., Ltd. (Cheongju, Chungbuk, Korea)

The term 'deep sequencing' should be clarified, as should the information it covers.

• We appreciate the reviewer’s helpful comment. In response, we have revised the Materials and Methods section to clarify the method used to identify target site mutations and frameshift events. Specifically, we added the following sentence:
• Line 122-125: To identify target site mutations, PCR amplicons were subjected to paired-end sequencing using the MiniSeq platform (Illumina, San Diego, CA, USA), and the resulting reads were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/#!, accessed on 12 January 2020).

A description of the bioinformatics analysis for RNA-seq is also missing.

• Thank you for pointing this out. We have now included a detailed description of the bioinformatics pipeline used for transcriptome analysis, including quality filtering, read alignment, transcript quantification, normalization, and differential expression analysis. These details have been added to the Materials and Methods section under the newly titled subsection “RNA-sequencing” (Lines 161-171).

The images in the 'Results' section lack a scale, making it difficult to assess the actual changes.

• Thank you for pointing this out. To address the concern, we have added a scale bar to Figure 2A in the revised manuscript to allow for accurate assessment of morphological differences.

In the discussion, the authors cite the conservative function of SOC1 but do not provide any comparative data, such as effect size, expression, or localisation, which weakens their argument. There is also no mention of the functional differences of SOC1 in monocotyledonous and dicotyledonous plants, which could explain some of the specific effects observed in lettuce.
Furthermore, the authors only discuss selected genes and GO terms. In contrast, the list of DEGs in the supplements includes numerous genes with potentially important functions (e.g., WRKY, MYB, GST, and aquaporins) that are not mentioned in the text.
The text mentions an increase in flavonoid pathway gene expression but does not attempt to correlate this with phenotype or anthocyanin levels despite having taken HPLC measurements.
The authors indicate that LsSOC1 modulates the heat response but do not provide a mechanism. Does LsSOC1 expression increase under heat stress? Were HSP70 or other indicators modulated directly?
The discussion ends abruptly without a section on the study's limitations or directions for future work.

• We thank the reviewer for the thoughtful and constructive feedback. In response, we have substantially revised the Discussion section to address all points raised, including clarification of LsSOC1 function, integration of additional DEGs, and improved interpretation of phenotypic and metabolic outcomes.

The manuscript is written in English and is generally comprehensible, but significant editorial improvements are needed at both the stylistic and grammatical levels. In many places, the text appears to have been machine-translated.

• Thank you for the comment. We have carefully revised the manuscript to improve grammar, style, and readability.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I read this manuscript submitted to the journal DNA. It proposes to investigate, among other related biotechnological aspects, the role of LsSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) in lettuce (Lactuca sativa L.), a process that is highly sensitive to elevated temperatures and leads to premature bolting and reduced crop quality and yield. The authors generated CRISPR/Cas9-mediated knockout (KO) lines of LsSOC1 and evaluated their phenotypes under high-temperature conditions. Ultimately, the results aim to provide molecular and physiological evidence that LsSOC1 is a promising genetic target for the improvement of heat-tolerant leafy vegetables. I also provide comments below:
Regarding the abstract it might be more precise—for example, by stating that some genes are common to both leaves and shoots (which are considered shoot tissues in the context of the transcriptome analysis) for KO lines S1 and S2. This would help avoid confusion about the scope of the main transcriptomic analysis. Although the abstract mentions that premature bolting leads to “reduced crop quality and yield,” it does not specify how the delayed bolting observed in KO lines impacts quality or yield. A brief mention or explanation that delayed bolting implies an extended harvest window or better crop quality could reinforce the practical impact of the findings; still, it is up to the authors how to improve this point.
Regarding the introduction, although the importance of temperature perception in both photosynthetic and reproductive organs is acknowledged, the introduction could further emphasize why it is critical to investigate transcriptional functions upstream of SOC1 beyond the meristem. One could argue (for example, and not exclusively) that, since systemic flowering signals such as FT originate in those tissues before reaching the SAM, understanding SOC1 activity in them is essential for a complete view of signal integration.
Materials and Methods: The experimental description in section 2.1 lacks critical information and clarity regarding the rationale for several methodological choices. For example, the lettuce cultivar used is not specified. The use of MS basal medium, the 16 h/8 h photoperiod, and the light intensity of 100 μmol·m⁻²·s⁻¹ are described, but no justification or reference is provided for these parameters, which may influence seedling development and stress responses. Moreover, the choice to use 3-week-old seedlings for heat treatment at 37 °C for 10 days is not explained or contextualized in the literature. It is also unclear whether a control group was maintained at 22 °C throughout the experiment. Furthermore, the phenotypic observation of bolting is vaguely described, with no indication of the criteria used, timing of observation, or whether quantitative measurements were taken.
On RNA-Seq and qRT-PCR, and regarding metabolite analysis: The difference in experimental conditions for different datasets (transcriptome under standard conditions vs. metabolites under heat stress conditions) is fundamental to data interpretation and should be more clearly highlighted at the beginning of the Methods section or even in the Introduction to better prepare the reader for this distinction. Currently, this becomes clear only after reading the specific methodological subsections. Later in the Discussion, a "primed stress state, even in the absence of external cues" is mentioned, which is based on RNA-Seq data, while the anthocyanin data (metabolites) refer to heat-stressed conditions. This should be made explicit to avoid apparent contradictions in the interpretation.
Results:
The text could better and more clearly distinguish whether the increase in HSP70 and other potential molecular components under non-stress conditions indicates constitutive activation of the stress response, whereas the increase in anthocyanins was specifically measured under heat stress conditions (i.e., outdoor summer conditions), and how this supports the thermotolerance phenotype.
As in the Results the Discussion must be extremely precise about the conditions under which the observations were made. The statement, for example, that "the constitutive upregulation of heat shock proteins and enhanced anthocyanin content in LsSOC1 KO lines under non-stress conditions implies a primed stress state, even in the absence of external cues" is problematic regarding anthocyanin content, since the methodology states indeed that metabolite samples were collected under "outdoor summer conditions (average daily temperature ~32 °C)," which represents heat stress. The Discussion should be rewritten to reflect that the basal expression of HSPs (from RNA-Seq under standard conditions) suggests a primed or baseline stress state, while the increase in anthocyanins (under heat stress) corroborates stress responsiveness in the presence of actual stress. This adjustment would strengthen the argument by avoiding internal inconsistency;
Have the RNA-Seq data been deposited in a public repository for scientific transparency?

It would be very relevant to perform and incorporate proteomic analyzes as well

Author Response

Response of Review -3

Manuscript
Title: Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce

Manuscript ID
dna-3710721

 

We sincerely thank the reviewers for their valuable comments and suggestions. We have carefully addressed all the points raised and revised the manuscript accordingly. All changes made in the revised manuscript are highlighted in blue text.

 

I read this manuscript submitted to the journal DNA. It proposes to investigate, among other related biotechnological aspects, the role of LsSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) in lettuce (Lactuca sativa L.), a process that is highly sensitive to elevated temperatures and leads to premature bolting and reduced crop quality and yield. The authors generated CRISPR/Cas9-mediated knockout (KO) lines of LsSOC1 and evaluated their phenotypes under high-temperature conditions. Ultimately, the results aim to provide molecular and physiological evidence that LsSOC1 is a promising genetic target for the improvement of heat-tolerant leafy vegetables. I also provide comments below:

Regarding the abstract it might be more precise—for example, by stating that some genes are common to both leaves and shoots (which are considered shoot tissues in the context of the transcriptome analysis) for KO lines S1 and S2. This would help avoid confusion about the scope of the main transcriptomic analysis. Although the abstract mentions that premature bolting leads to “reduced crop quality and yield,” it does not specify how the delayed bolting observed in KO lines impacts quality or yield. A brief mention or explanation that delayed bolting implies an extended harvest window or better crop quality could reinforce the practical impact of the findings; still, it is up to the authors how to improve this point.

• We appreciate the reviewer’s suggestion and would like to clarify that transcriptome analysis in our study was performed on leaf and bud tissues, not on general shoot tissues. The previous use of the term "shoot tissues" in the abstract was inaccurate and may have caused confusion.
• Additionally, we have addressed the practical significance of delayed bolting by noting that it may extend the harvest window and enhance postharvest quality, thereby emphasizing the agronomic relevance of our findings.

Regarding the introduction, although the importance of temperature perception in both photosynthetic and reproductive organs is acknowledged, the introduction could further emphasize why it is critical to investigate transcriptional functions upstream of SOC1 beyond the meristem. One could argue (for example, and not exclusively) that, since systemic flowering signals such as FT originate in those tissues before reaching the SAM, understanding SOC1 activity in them is essential for a complete view of signal integration.

• We appreciate the reviewer’s insightful comment regarding the need to emphasize transcriptional functions upstream of SOC1 beyond the SAM. We have revised the relevant paragraph to explicitly explain how systemic signals such as FT, produced in peripheral tissues, necessitate investigation of SOC1 activity at their site of origin (line 67-69). This provides a stronger rationale for our tissue-specific transcriptome analysis.

Materials and Methods: The experimental description in section 2.1 lacks critical information and clarity regarding the rationale for several methodological choices. For example, the lettuce cultivar used is not specified.

• We thank the reviewer for this helpful suggestion. In the revised manuscript, we have included the cultivar name and seed source to clarify the plant material used in this study. Specifically, the following sentence was added at the beginning of the Materials and Methods section:
  line 97-99: Lettuce (Lactuca sativa cv. Seonpung Plus; a Korean red romaine-type cultivar) seeds were obtained from Kwonnong Seed Co., Ltd. (Cheongju, Chungbuk, Korea)

 The use of MS basal medium, the 16 h/8 h photoperiod, and the light intensity of 100 μmol·m⁻²·s⁻¹ are described, but no justification or reference is provided for these parameters, which may influence seedling development and stress responses. Moreover, the choice to use 3-week-old seedlings for heat treatment at 37 °C for 10 days is not explained or contextualized in the literature. It is also unclear whether a control group was maintained at 22 °C throughout the experiment.

• We appreciate the reviewer’s valuable comments. In response, we have revised the Materials and Methods section to justify the growth conditions and heat treatment protocol by citing relevant literature. We also clarified that a control group was maintained at 22 °C and that plants were monitored for bolting-related phenotypes following heat treatment. Please refer to lines [100-102] in the revised manuscript.

Furthermore, the phenotypic observation of bolting is vaguely described, with no indication of the criteria used, timing of observation, or whether quantitative measurements were taken.

• We thank the reviewer for this comment. In the revised manuscript, we have clarified the criteria and timing for bolting and flowering phenotypes, as well as the quantitative parameters measured (stem and internode length) and number of biological replicates (n ≥ 12). These details are now provided in the Materials and Methods section (lines 151-154).

On RNA-Seq and qRT-PCR, and regarding metabolite analysis: The difference in experimental conditions for different datasets (transcriptome under standard conditions vs. metabolites under heat stress conditions) is fundamental to data interpretation and should be more clearly highlighted at the beginning of the Methods section or even in the Introduction to better prepare the reader for this distinction. Currently, this becomes clear only after reading the specific methodological subsections. Later in the Discussion, a "primed stress state, even in the absence of external cues" is mentioned, which is based on RNA-Seq data, while the anthocyanin data (metabolites) refer to heat-stressed conditions. This should be made explicit to avoid apparent contradictions in the interpretation.

• We agree with the reviewer that clarifying the experimental condition differences between transcriptome and metabolite datasets is important for accurate interpretation. We have now explicitly stated that RNA-seq samples were collected from plants grown under standard conditions, while metabolite profiling was conducted using heat-stressed plants. This clarification has been added both to the Materials and Methods section (Lines 156-158).
• We appreciate the reviewer’s careful reading. We acknowledge that the initial sentence in section 2.6 Anthocyanin Quantification may have caused confusion due to an inaccurate description of the sampling conditions. To address this, we have revised the sentence to more accurately reflect the experimental procedure as follows:
• Line 173-175: Leaf tissues were collected from WT and LsSOC1 KO plants at the onset of bolting, defined by stem elongation of ≥1.0 cm, under controlled chamber conditions (22 °C, 16 h light/8 h dark, 100 µmol·m⁻²·s⁻¹).

Results:
The text could better and more clearly distinguish whether the increase in HSP70 and other potential molecular components under non-stress conditions indicates constitutive activation of the stress response, whereas the increase in anthocyanins was specifically measured under heat stress conditions (i.e., outdoor summer conditions), and how this supports the thermotolerance phenotype.As in the Results the Discussion must be extremely precise about the conditions under which the observations were made. The statement, for example, that "the constitutive upregulation of heat shock proteins and enhanced anthocyanin content in LsSOC1 KO lines under non-stress conditions implies a primed stress state, even in the absence of external cues" is problematic regarding anthocyanin content, since the methodology states indeed that metabolite samples were collected under "outdoor summer conditions (average daily temperature ~32 °C)," which represents heat stress. The Discussion should be rewritten to reflect that the basal expression of HSPs (from RNA-Seq under standard conditions) suggests a primed or baseline stress state, while the increase in anthocyanins (under heat stress) corroborates stress responsiveness in the presence of actual stress. This adjustment would strengthen the argument by avoiding internal inconsistency;

• As previously explained, we acknowledge that the first sentence in Section 2.6, "Anthocyanin Quantification," may have caused confusion due to an inaccurate description of sampling conditions. We have revised the relevant sentence to address this issue, and this has been noted in the above response.

Have the RNA-Seq data been deposited in a public repository for scientific transparency?

• We appreciate the reviewer’s concern regarding data transparency. However, the RNA-Seq data generated in this study will not be deposited in a public repository due to institutional restrictions and internal data management policies. Nonetheless, all relevant processed data (e.g., DEG lists, expression values, pathway analyses) are thoroughly included in the manuscript and supplementary materials to ensure reproducibility and scientific rigor. We are also happy to provide the raw data upon reasonable request.

It would be very relevant to perform and incorporate proteomic analyzes as well

• We thank the reviewer for the valuable suggestion. We agree that incorporating proteomic analyses would provide deeper insights into the molecular mechanisms underlying the observed phenotypes. Although proteomic analysis was beyond the scope of the current study, we consider it a highly relevant direction for future research and will incorporate it in subsequent investigations.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript by Kim et. al. entitled “Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce” reports that CRISPR/Cas9-mediated LsSOC1 knockout lines were generated, and phenotype of the knockout lines were evaluated under high-temperature conditions. The authors claim that ‘Compared with wild-type (WT) plants, LsSOC1-KO lines exhibited delayed bolting by up to 18.6 days and reduced stem elongation by approximately 3.8 cm’; and some abiotic genes are regulated. The authors conclude that ‘LsSOC1 is a key integrator of developmental and thermal cues, orchestrating both bolting and stress-responsive transcriptional programs’. The work is technically sound piece of research, and within the scope of DNA, but it requires minor revision before its acceptance for publication.

 

1. Many cas9s in crispr system, please point out which cas9
2. Provide more references that related to respond of heat stresses
3. In ‘Phenotypic Characterization and Bolting Assay’ and ‘Delayed Bolting and Reduced Stem Elongation in LsSOC1 KO Lines’, the descriptions of the growth conditions are not agreed with each other, the authors need clarify the statements

Comments on the Quality of English Language

generally OK

Author Response

Response of Review -4

Manuscript
Title: Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce

Manuscript ID
dna-3710721

 

We sincerely thank the reviewers for their valuable comments and suggestions. We have carefully addressed all the points raised and revised the manuscript accordingly. All changes made in the revised manuscript are highlighted in blue text.

 

The manuscript by Kim et. al. entitled “Loss of LsSOC1 Function Delays Bolting and Reprograms Transcriptional and Metabolic Responses in Lettuce” reports that CRISPR/Cas9-mediated LsSOC1 knockout lines were generated, and phenotype of the knockout lines were evaluated under high-temperature conditions. The authors claim that ‘Compared with wild-type (WT) plants, LsSOC1-KO lines exhibited delayed bolting by up to 18.6 days and reduced stem elongation by approximately 3.8 cm’; and some abiotic genes are regulated. The authors conclude that ‘LsSOC1 is a key integrator of developmental and thermal cues, orchestrating both bolting and stress-responsive transcriptional programs’. The work is technically sound piece of research, and within the scope of DNA, but it requires minor revision before its acceptance for publication.

1. Many cas9s in crispr system, please point out which cas9          

• Thank you for the comment. In this study, we used the Streptococcus pyogenes Cas9 (SpCas9) variant, which is the most widely used and well-characterized Cas9 in plant genome editing. This has been clarified in the revised Methods section accordingly.

1. Provide more references that related to respond of heat stresses

• We appreciate the reviewer’s suggestion. Additional references related to plant responses to heat stress have been included in the revised Discussion to support our interpretations.

1. In ‘Phenotypic Characterization and Bolting Assay’ and ‘Delayed Bolting and Reduced Stem Elongation in LsSOC1 KO Lines’, the descriptions of the growth conditions are not agreed with each other, the authors need clarify the statements

• We appreciate the reviewer’s careful observation. We have reviewed the relevant sections and confirmed that all bolting and flowering-related phenotypic analyses, including stem and internode measurements, were consistently conducted under the same controlled environmental conditions (22 °C, 16 h light/8 h dark photoperiod, 100 µmol·m⁻²·s⁻¹). To avoid potential confusion, we have revised the Materials and Methods section to more explicitly state that the same growth conditions were uniformly applied to all experiments related to bolting, flowering, and stem morphology (Lines 136-138,174-175).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Figures 1b and 6a still lack a scale.

Author Response

We sincerely thank the reviewer for the thoughtful review. The scale bars have been added accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been improved.

Author Response

While we appreciate the reviewer’s note that the manuscript has been improved, we observed that all evaluation items were marked as 'Not applicable'. We would like to clarify whether this response accurately reflects the reviewer’s overall evaluation.