PREreview of A simple method to efficiently generate structural variation in plants

Summary

This article introduces a novel, time-efficient method for inducing heritable structural variation in Arabidopsis thaliana using the topoisomerase II inhibitor etoposide. Previously, etoposide has been used to study chromosome fragmentation in Arabidopsis; however, it has never been utilised as a mutagen. By incorporating etoposide into growth media, the authors induce a variety of heritable phenotypic changes, some of which include: variegation, virescence, dwarf-like features, and short internodes. These phenotypic changes are linked to structural variation caused by deletions, duplications, and inversions.  

This study effectively demonstrates that the application of etoposide can generate diverse mutant populations, presenting an innovative methodology that may be more readily accessible to the scientific community.

General remarks

This well-written and coherent manuscript effectively presents extensive, well-justified research. The methodologies are clearly detailed, and the findings are thoughtfully connected to their applications in academic research and potentially R&D. Below, we outline suggestions to enhance the flow and overall message, which we hope will be beneficial to the authors.

Figure 1 (and associated text):

• Figure 1B does not provide significant value. We recommend replacing it with a schematic illustrating the action of etoposide.

• There are inconsistencies between the etoposide concentrations mentioned in the text and those shown in Figure 1. The figure displays images of Arabidopsis treated with 0 μM, 80 μM, 160 μM, and 320 μM etoposide, however, the text does not mention the 320 μM treatment. Additionally, while the text references a 40 μM treatment, no images for this concentration are provided. We recommend ensuring consistency between the text and figures; if a concentration is mentioned in the text, it should be accompanied by a corresponding image in the figure, and vice versa.

• Throughout the manuscript, the concentration of etoposide is not consistently linked to the mutant phenotypes. For instance, in Figure 1C-F, it is unclear what concentration of etoposide the Arabidopsis mutants were exposed to.

• We believe additional quantifications are necessary to support the description of mutant phenotypes. We recommend including figures that show precise measurements, for example leaf size or leaf virescence, backed by statistical analysis.

• Lines 159-161 mention the generation of novel recessive and dominant phenotypes; however, we believe this is not directly aligned with the research presented, which only describes a partially dominant mutant phenotype. We recommend rewording this statement to more accurately reflect the results.

• Lines 129-131 refer to “light- and temperature-dependent variations,” but there is no supporting evidence or methodology provided related to these experiments. We recommend either including the relevant data and experimental details or revising the statement accordingly.

Figure S1:

• Here, we initially notice a consistent discrepancy when introducing different lines (e.g. the 35A line). These line names appear abruptly and take time to understand their origin. We recommend introducing the different lines and their relevance earlier in the text for clarity.

• Figures S1H-I are excellent, and additional figures like these would strengthen the presentation of the findings. However, Figure S1G-I is missing statistical testing. Additionally, we recommend an alternative colour for the non-dwarf relative box plot as this is confusingly similar to the DMSO-treated controls.

Figure S2:

• The grey bars used for "did not germinate" are difficult to distinguish from the background. We recommend changing the colour to improve visibility and make the figure clearer.

Figure S4 (and associated text):

• Here, a direct comparison is made between the control and etoposide-treated lines; however, the significant difference in the number of lines makes the comparison somewhat unbalanced. We recommend addressing this in the methodology or considering the inclusion of additional control lines.

• The text discusses differences in the copy numbers in controls lines and etoposide-treated lines, however, there is no statistical testing to support this. We recommend incorporating statistical analyses to substantiate the findings or adjusting the wording to avoid potential misinterpretation.

Figure S8:

• In Figure S8A, the lines are arranged by SNV count, which is misleading and inconsistent with ordering in Figure S5. We recommend maintaining a consistent presentation across figures and avoiding data reordering that could suggest trends that may not exist.

Additional points for consideration

• The abstract includes an unsubstantiated claim that this method for mutant generation may be applicable to other plant species. Since this statement is not supported or discussed further in the text, we recommend omitting it unless additional evidence is provided.

• The comparison between etoposide-induced mutagenesis and EMS is not well-justified, as it is only briefly mentioned in the introduction without supporting data or practical validation. In the discussion, comparisons are only made between etoposide-induced mutagenesis and irradiation. We recommend reconsidering this comparison unless further evidence is provided.

• In some instances, the text uses vague language, such as in line 155: “among other possibilities.” To enhance clarity, we recommend specifying these possibilities or removing the phrase to avoid ambiguity.

• Providing more specific and consistent names for the lines referenced in the text and figures would improve readability and maintain a clear narrative. Currently, there is a lack of cohesiveness between the use of line names and descriptive labels (e.g. "variegated"), making it difficult to interpret the figures. We recommend standardising these labels for better clarity.

• In places, statistical testing appears to be insufficient to fully support the results. While this may be due to a limited number of plants, given the importance of robust data analysis, we recommend incorporating further statistical tests where possible to strengthen the findings.

• In some instances, scale bars are missing, which are necessary to support the claims made.

Competing interests

The authors declare that they have no competing interests.