Comparative Analysis of the Expression of Genes Involved in Fatty Acid Synthesis Across Camelina Varieties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although the manuscript has limited data, it is beneficial for a newly introduced oil crop like camelina. You had concluded some findings that are not provable with the data you provided in this manuscript. it is better to correct them specially in discussion. I added some notes to the file which would improve the manuscript. 

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

We would like to express our gratitude for your valuable comments regarding the correction of the manuscript.
We have made the necessary corrections to the document, in line with your feedback and that of the other reviewers.

We are confident that it will meet your expectations.

Thank you for your time.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

Greetings,

Comments:

1. The title is not clear
2. The abstract needs to be rewritten
3. The introduction is not comprehensive enough, and the hypothesis is not clear
4. Table 2 and Figure 4 can be moved to the supplementary material
5. Construct the heat map for expression analysis and present it in fold change
6. It is interesting if authors provide the in silico expression pattern of all selected genes with a heat map along with their expressions under various environmental conditions in different developmental stages.
7. The discussion needs to be rewritten. They repeated mostly the results. The discussion section should provide a more in-depth analysis of the results obtained. A more comprehensive discussion linking the cross-talk between different genes enhances the quality of the discussion.

Author Response

2-1. The title is not clear

Thank you for your valuable comment regarding the clarity of the title. To improve precision and better reflect the scope of our study, we propose revising the title to:

“Comparative Analysis of the Expression of Genes Involved in Fatty Acid Synthesis Across Camelina Varieties”.

We believe this revised title more clearly conveys the comparative nature and specific focus of the research.

 

2-2. The abstract needs to be rewritten

Thank you for your suggestion. We have carefully revised the abstract to improve its clarity, conciseness, and structure. The new version provides a clearer summary of the study’s objectives, methodology, key findings, and relevance, in line with the journal’s expectations. We hope this revised abstract more effectively communicates the main contributions of our work.

Abstract: lines 16 to 36.

 

2.3- The introduction is not comprehensive enough, and the hypothesis is not clear

We appreciate the reviewer’s comment. In response, we now explicitly stated the hypothesis at the end of the introduction to clarify the aims of the study. We hope these change adequately address the reviewer’s concerns and improve the clarity and focus of the manuscript.

Hypothesis: lines 124 to 128.

 

2.4- Table 2 and Figure 4 can be moved to the supplementary material

Thank you for the suggestion. We agree that Table 2 and Figure 4 may be more appropriately placed in the supplementary material to streamline the main text and improve readability. We have therefore moved both items to the supplementary section, where they remain fully accessible to readers interested in the detailed data.

 

2.5- Construct the heat map for expression analysis and present it in fold change

We thank the reviewer for this valuable suggestion. In response, we have constructed a heat map to visualise the expression patterns of the analysed genes, presented in terms of fold change. This addition enhances the interpretability of the results and provides a clearer comparative overview of gene expression across varieties and developmental stages.

The heat map has been included as a new Figure 7 in the revised manuscript.

 

2.6- It is interesting if authors provide the in silico expression pattern of all selected genes with a heat map along with their expressions under various environmental conditions in different developmental stages.

We appreciate the reviewer’s interest in the in silico expression patterns of the selected genes. In response, we would like to clarify that detailed expression data are already provided in Table A1 of the supplementary material, which offers a visual and intuitive overview of gene expression across different tissues and silique developmental stages.

However, we do not currently have expression data for these genes under varying environmental conditions. Our study focuses specifically on gene expression throughout silique development and in different plant tissues under controlled conditions, which we believe offers valuable insights for understanding tissue-specific regulation.

We hope this clarification addresses the reviewer’s request and adds value to the presentation of our findings.

 

2.7-The discussion needs to be rewritten. They repeated mostly the results. The discussion section should provide a more in-depth analysis of the results obtained. A more comprehensive discussion linking the cross-talk between different genes enhances the quality of the discussion.

We are grateful for the reviewer’s constructive comment regarding the Discussion section. In response, we have substantially rewritten this section to avoid reiterating the Results and instead offer a deeper interpretation of the findings. Specifically, we now explore the cross-talk and coordinated regulation among genes involved in the fatty acid biosynthesis pathway, integrating our results with existing knowledge on their enzymatic roles, expression dynamics, and physiological significance.

Moreover, we have contextualised the observed expression patterns across varieties and developmental stages, identifying candidate varieties with potential application in nutritional, biofuel, and industrial sectors. We also discuss the implications of these results for breeding programmes and selection strategies in Camelina sativa under the current limitations of genetic modification in the European Union.

We believe these improvements have enhanced the interpretative depth and scientific value of the Discussion section, as suggested by the reviewer.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

• Abstract should briefly summarize the findings
• The manuscript should highlight the novelty of the work more explicitly, as role of the studied genes in fatty acid synthesis is well known.
• Authors should justify the basis of selection of these 9 varieties.
• A list of the selected varieties, along with their detailed information should be provided.
• Statistical analysis needs to be described in greater detail.
• Resolution of Figure 6 needs to be improved
• In methods – the selection of candidate genes should be elaborated in detail
• In results – quantitative comparisons like fold change data should be provided
• A summary table/ heatmap comparing the expression of genes should be provided.
• Too much of repetition of results in discussion. Discussion should be interpretation of results.
• Changes in gene regulation – up and down regulation should be highlighted
• Fatty acid profile not provided, so making a strong claim without corroboration with biochemical data may be misleading.
• Abbreviations should be used after first mention
• Some typographic errors are there in the manuscript, which may be corrected.

Author Response

3-1: Abstract should briefly summarize the findings.

We appreciate your comment and agree with the need for the abstract to reflect the main findings concisely. We have reviewed the abstract to highlight the key results of the gene expression analysis, emphasising the significant differences observed in the expression levels of genes related to fatty acid metabolism.

Please refer to the abstract of the reviewed manuscript.

 

3-2: The manuscript should highlight the novelty of the work more explicitly, as role of the studied genes in fatty acid synthesis is well known.

We appreciate your observation and acknowledge that the role of genes involved in fatty acid synthesis is well documented. However, the primary aim of our study was to explore the possibility of using gene expression analyses to speed up genetic improvement programs. To this aim, we investigated the expression of these genes in nine Camelina varieties developed by a company (Camelina Company, SL) based on their agronomical performance in the central Iberian Peninsula.

In this regard, we have emphasised in both the introduction and discussion the novelty of our work, which lies in the methodological approach, specifically the use of qRT-PCR for the fast analyses of gene expression in different varieties throughout silique development. This approach aims to select varieties that best align with the objectives of various industries.

We have modified the abstract (line 16 to 36), introduction (line 114 to 124), discussion (line 389 to 392)  and conclusions according to your comments.

 

3-3: Authors should justify the basis of selection of these 9 varieties.

We appreciate your comment. The nine selected varieties were chosen based on the availability of previous data, albeit incomplete, allowing us to compare the current results with fatty acid profile data, once they are completed in future campaigns. The varieties were also selected due to their adaptation to the climate of the central Iberian Peninsula. Additionally, their agricultural relevance, in terms of performance, resistance to pests/diseases, and adaptability to different environmental conditions, makes them particularly suitable for research on lipid metabolism.

Introduction: lines 114 to 117.

 

3-4: A list of the selected varieties, along with their detailed information should be provided.

We appreciate your comment. We understand the importance of providing detailed information about the selected varieties. However, due to intellectual property restrictions imposed by Camelina Company S.L., we cannot share  detailed data on the varieties in this manuscript. We appreciate your understanding and are happy to discuss any relevant aspects of the varieties within the limits set by these restrictions.

Mat y Met:  lines 132 to 134.

 

3-5: Statistical analysis needs to be described in greater detail.

We have added further information of all the steps carried out for statistical analysis. Including the incorporation of a heatmap in Figure 7.

 

3-6: Resolution of Figure 6 needs to be improved.

We appreciate your comment regarding the resolution of Figure 6. We have improved the image quality and replaced the figure in the manuscript with a 500 dpi version to ensure that the details are clearer and sharper. The updated version is embebed in the reviewed manuscript PDF file.

 

3-7: In methods the selection of candidate genes should be elaborated in detail.

We thank the reviewer for this valuable comment. In response, we have revised the Candidate Gene Selection subsection of the Methods to provide a more detailed explanation of the criteria used for gene selection.

Mat y Met: lines 173 to 190.

 

3-8: In results, quantitative comparison like fold change data should be provided.

We appreciate the reviewer’s suggestion. In response, we have now included quantitative fold change (FC) values in the Results section to support our comparative analysis of gene expression among varieties and developmental stages. For simplicity, the FC (expressed as log₂FC) values are included in a new Figure 7, which also includes a heat-map representation of the expression values

 

3-9:  A summary table/heatmap comparing the expression of genes should be provided.

Please, see our response to comment 3-8.

 

3-10: Too much repetition of results in discussion. Discussion should be interpretation of results.

We appreciate your comment. We have thoroughly reviewed the Discussion section to adjust it to yours and the other reviewer's recommendations.

 

3-11: Changes in gene regulation- up and down regulation should be highlighted.

Please, see our response to comment 3-8.

 

3-12: Fatty acid profile not provided, so making a strong claim without corroboration with biochemical data may be misleading.

We appreciate your comment and understand the importance of supporting gene expression findings with corresponding biochemical data. We have added the following paragraph to the discussion section in order to make the text more comprehensible. Nonetheless, we have moderated the language in the manuscript to avoid making definitive claims not fully supported by current biochemical data, and we have addressed this limitation in the discussion section.

Discussion: lines 544 to 551.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “Comparative Gene Expression in Fatty Acid Synthesis Across Camelina Varieties” selected the native European oilseed crop Camelina as the research objects, and analyzed the expression pattern of 7 key enzymes involved in fatty acid biosynthesis in developing seeds from 9 accessions using qRT-PCR tests. The authors found only one of the studied genes, FAB2c, exhibiting a constitutive role in the production of MUFAs, but the others showed a variation pattern related to variety and silique developmental stage, which could provide some useful information for genetic breeding of high MUFs varieties. However, there are three major suggestions/comments here: One is that there is no data of MUFs contents for different Camelina Varieties, and I would like to strongly suggest an addition of these kind of data, so a correlation can be made since the key point is the MUFs, not just gene expression. The second would be the possibility to for the final selected 7 genes to quantify each individual homologous one (all three) using specific primers, instead of a pair of conserved primers, since it would provide more information about these key gene regulations. The expression of homologous genes is often a complex and dynamic process, and not all will necessarily exhibit the same expression pattern across all tissues or developmental stages. Thirdly, the experiment design did not seem to be a randomized block design based on the plot map, and please double check. Additionally, gene or gene expression is more likely a parameter measured, it might not be treated as a variable as variety and development stage, thus a reanalysis of the data would be suggested.

Other minor comments:

• Table 1. It is more like a figure, instead of a table
• spanned one intron site to minimize amplification 167 from contaminating genomic DNA in the cDNA preparations. This step is good, but might not be necessary if the DNA is treated well with DNAase.
• generalized linear mixed-effects model (glmer) using sample ID as a random factor  Usually abbreviation GLLM, it might be more proper to treat variety and development stage as the random factors.
• out within each gen using Tukey's test with Sidak correction gene?
• Only the genes that showed expression in 213 siliques for all three copies were selected for analysis by qRT-PCR. We could not proceed with FAB2b as align-221 ment issues prevented the design of a suitable sense and antisense primer pair in regions 222 common to the three gene copies. The selection criteria were not very proper as commented above.
• Different lower-case letters indicate statistically significant differences among treatments (interaction variety x silique), while capital letters indicate statistically significant differences among varieties. Why only figure 6 FAB2c showed significant differences among varieties, all the others showed only among treatments. To mark each panel with a number or something like that would make it clearer. And the treatment (interaction variety x silique) , is the variety x silique interaction significant? Suggested to use line instead to columns for the figures, which may help better present the gene trend across stages.
• Across varieties, the effect of silique classes differed as a function of the expression 239 of each gene (interaction gene × variety × silique; χ2=739; p<0.001). It would be proper for the claim here if a digit PCR was used to make comparison across genes. And as suggested above, gene might not be treated as a variable.

Author Response

4-1: There is no data of MUFs contents for different Camelina Varieties, and I would like to strongly suggest an addition of these kind of data, so a correlation can be made since the key point is the MUFs, not just gene expression.

We appreciate your valuable suggestion. We acknowledge that the content of MUFAs in the different Camelina varieties would provide an important additional layer of information to support the correlation with gene expression. Unfortunately, due to limitations in data availability for the current study, we were unable to include MUFA content for the varieties used this year. However, we would like to highlight that fatty acid profiles for some of these varieties were previously published in studies from the past two years [6, 7]. We believe these data, alongside our gene expression analysis, can still provide relevant insights into the relationship between gene expression and MUFA content.

In future work, we plan to include a direct analysis of MUFAs in these varieties to strengthen the correlation with gene expression and further elucidate the role of the studied genes in lipid metabolism. 

 

4-2: The second would be the possibility to for the final selected 7 genes to quantify each individual homoeologous one (all three) using specific primers, instead of a pair of conserved primers, since it would provide more information about these key gene regulations. The expression of homoeologous genes is often a complex and dynamic process, and not all will necessarily exhibit the same expression pattern across all tissues or developmental stages.

We appreciate your thoughtful suggestion regarding quantifying individual homoeologous genes using specific primers for each gene rather than a pair of conserved primers. We agree that this approach could provide more detailed information about the regulation of these key genes, especially given the complexity and dynamism of homologous gene expression across different tissues and developmental stages.

In our current study, we opted for conserved primers to ensure broad amplification and comparability across the varieties. However, we recognise that this could mask potentially important variations in the expression of individual homologous genes.

We plan to explore this more specific approach in future studies, where individual homoeologous genes will be quantified separately using gene-specific primers, to gain a more precise understanding of their differential regulation in the context of fatty acid biosynthesis.

In addition, the design of allele-specific PCR-tests can be technically challenging. In our hands, the outcome of using allele-specific primers is somehow unpredictable. We found in other projects that primer pairs differing in up to three nucleotides at the 3’ end could still amplify cloned copies of the other alleles.

Discusion: lines 414 to 420.

4-3: Thirdly, the experiment design did not seem to be a randomized block design based on the plot map, and please double check. Additionally, gene or gene expression is more likely a parameter measured, it might not be treated as a variable as variety and development stage, thus a reanalysis of the data would be suggested.

We appreciate your observation regarding the experimental design. The design does not correspond to a completely randomised design (CRD), as the replications were not entirely distributed randomly across the field. Instead, we implemented a blocked layout with four blocks, each containing one replication of every variety. This approach was intended to control for field variability, notably positional effects, by ensuring that each block served as a spatial replicate.  

While this does not represent a classical randomised block design in the strict statistical sense, it does incorporate the principles of blocking to account for environmental heterogeneity. We will revise the description of the experimental design in the Materials and Methods section to reflect this more accurately.

Mat and Meth. Experimental Design: The field experiment was arranged in four blocks to account for spatial variability. Each block contained one replicate of each of the nine Camelina varieties, ensuring that environmental variation across the field (e.g., soil composition, exposure) was controlled. Although the replications were not fully randomised across the entire field, this layout allowed for effective blocking, and the data were analysed accordingly.

Initially we carried out a first analysis considering gene expression as dependent variable and as independent variables gene, variety and silique classes. But also we carried out a statistical analysis for each considering the gene expression of the specific gene as dependent variable and variety and silique classes as independent variables. We have eliminated the first analysis considering the reviewer recommendation. We have also described the statistical analysis in more detail.

Due to the low data availability (n=6) it was impossible include block and plot nested to the block and id nested to the plot as random variables and obtain convergent models. Both blocks were really close from each other and in a terrain with little differences, same legacy, geological and edaphic origin and obviously same climatology. Consistently plots within blocks had minor differences. Thus we can consider that differences among plots/blocks are minimal and data can be considered independent. In this sense, we choose to include id of the sample as random factor that include intrinsically information of the plot and block.

Plant Material and Experimental Design: lines 133 to 143.

 Statistical Analysis: lines 228 to 237.

 

Other minor comments:

4-4 :Table 1. It is more like a figure, instead of a table.

We understand your suggestion and have renamed Table 1 as Figure 4

 

4-5: spanned one intron site to minimize amplification from contaminating genomic DNA in the cDNA preparations. This step is good, but might not be necessary if the DNA is treated well with DNAase.

We appreciate your comment regarding including an intronic region in the primer design to minimize amplification from contaminating genomic DNA. Before cDNA synthesis, all RNA samples were treated with DNase to effectively eliminate genomic DNA contamination. The use of an intronic region in the primers further ensured that any potential genomic DNA amplification was minimized.

In our hands, no method ensures the complete elimination of genomic DNA in RNA preparations. While this is not noticed when the expression of moderately abundant genes is analysed (CT <25), it could be an issue when the target genes are low-abundant ones (CT>30), such as in null-mutant analyses. Hence, we became used to including intron span sites in our designs.  

4-6: generalized linear mixed-effects model (glmer) using sample ID as a random factor  Usually abbreviation GLLM, it might be more proper to treat variety and development stage as the random factors.

Glmer is not the acronym of the model is the function used in R. We have clarified the statistical analysis in the present version. (Kolde R (2019). _pheatmap: Pretty Heatmaps_. R package version 1.0.12, https://CRAN.R-project.org/package=pheatmap)

 

4-7: Post hoc analysis was carried out within each gen using Tukey's test with Sidak correction gene?

We have corrected the statistic methods description.

Mat y Met: lines 231 to 237.

 

4-8: Only the genes that showed expression in siliques for all three copies were selected for analysis by qRT-PCR. We could not proceed with FAB2b as alignment issues prevented the design of a suitable sense and antisense primer pair in regions common to the three gene copies. The selection criteria were not very proper as commented above.

We thank the reviewer for this observation. The selection of genes for qRT-PCR analysis was based primarily on two criteria:

• detectable expression in siliques across all three gene copies, and
• the feasibility of designing a specific primer pair targeting conserved regions.

In the case of FAB2b, alignment inconsistencies among the three homologues hindered the design of a suitable primer pair that could accurately amplify all copies, leading to its exclusion from further qRT-PCR analysis.

We acknowledge that this selection strategy, while technically driven, may appear limited, and we will clarify this rationale in the manuscript. We also plan to explore a more specific approach in future studies, where individual homoeologous genes will be quantified separately using gene-specific primers, to gain a more precise understanding of their differential regulation in the context of fatty acid biosynthesis.

Metodology: lines 196 to 199.

4-9: Different lower-case letters indicate statistically significant differences among treatments (interaction variety x silique), while capital letters indicate statistically significant differences among varieties. Why only figure 6 FAB2c showed significant differences among varieties, all the others showed only among treatments. To mark each panel with a number or something like that would make it clearer. And the treatment (interaction variety x silique), is the variety x silique interaction significant? Suggested to use line instead to columns for the figures, which may help better present the gene trend across stages.

We thank the reviewer for these helpful suggestions and observations regarding Figure 6.

First, with regard to the statistical differences, we confirm that FAB2c was the only gene for which a statistically significant effect was found among varieties independently (as indicated by the capital letters), while for the other genes, significant differences were observed only for the interaction between variety and silique developmental stage (indicated by lower-case letters). This reflects differences in the underlying expression patterns and regulation among the studied genes. We have reinforced the statistical significance in each gene in the results text.

We include a table with the obtained χ2 and p-values to make it easier for you to check the changes in the text.

Table X. Chi square (χ2) and p values of the generalized linear mixed-effects model of the expression of each gene as a function of the Variety of Camelina sativa and the silique class and their interaction. Bold values indicate statistical significance while Ɨ- indicate marginal statistical significance.

Gene     Variety  Silique class       Variety × Silique class

ADS2     χ2=266; p<0.001              χ2=13.9; p<0.001             χ2=26.7; p=0.045

FAB1      χ2=57.6; p<0.001             χ2=6.37; p=0.041             χ2=24.7; p=0.076 Ɨ

FAB2a   χ2=53.6; p<0.001             χ2=38.8; p<0.001             χ2=23.8; p=0.094 Ɨ

FAB2c    χ2=57.6; p<0.001             χ2=98.5; p<0.001             χ2=9.5; p=0.89

FAD2     χ2=115; p<0.001              χ2=27.3; p<0.001             χ2=52.5; p<0.001

FAD3     χ2=260; p<0.001              χ2=27.7; p<0.001             χ2=2669; p<0.001

FAE1      χ2=319; p<0.001              χ2=38.8; p<0.001             χ2=35.5; p=0.0034

 

Regarding the clarity of the figure, we have tried to label the panels as suggested, but found that the Figure XX and his legend became more complex to interpret.   

Regarding the figure format, we acknowledge that line graphs are helpful for visualising trends; however, we chose bar plots because they allow for more precise comparison of expression levels across varieties and developmental stages within each gene. This format better suited the type of variation we intended to highlight.

 

4-10 Across varieties, the effect of silique classes differed as a function of the expression of each gene (interaction gene × variety × silique; χ2=739; p<0.001). It would be proper for the claim here if a digit PCR was used to make comparison across genes. And as suggested above, gene might not be treated as a variable.

We thank the reviewer for this valuable comment.

As indicated previously to the reviewer 3: Initially we carried out a first analysis considering gene expression as dependent variable and as independent variables gene, variety and silique classes. But also we carried out a statistical analysis for each considering the gene expression of the specific gene as dependent variable and variety and silique classes as independent variables. We have eliminated the first analysis considering the reviewers recommendation. We have also described the statistical analysis in more detail and clarify the results section for each gene.

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Since most of the previous comments have been answered, and the authors stated they will measure MUFA contents directly in some selected varieties in future study, and I would believe so. Here I would like to recommend an acceptance contingent some minor improvements, such as making some long sentences to be shorter/clearer "Our results highlight V18 as particularly promising, displaying elevated expression of genes associated with the synthesis of PUFAs and very-long-chain fatty acids (VLCFAs), key traits for food, biofuel, and industrial applications. " "These findings, alongside the qRT-PCR assays developed, provide valuable tools for selecting Camelina varieties with tailored genetic profiles underscoring the potential to exploit natural transcriptional diversity for cultivar selection and improvement in agricultural and industrial contexts. "

Author Response

Dear reviewer,

Thank you for your valuable comments. Please find below the updated manuscript abstract, which has been revised in line with your instructions.

It is our sincere hope that the new version will prove to be satisfactory.

Best regards.

Author Response File: Author Response.pdf