PREreview of A spontaneous mutation in a key C₄pathway gene significantly alters leaf δ¹³C, uncoupling its relationship with WUE and photosynthetic performance inZea mays

Summary

This study utilises a Zea mays line with a negative stable carbon isotope ratio (δ¹³C) to identify a deletion in carbonic anhydrase1 (cah1), which surprisingly to the authors conferred significantly higher carbonic anhydrase activity in the lines with this mutant allele. The authors conclude that this decouples the relationship between δ¹³C and intrinsic water use efficiency (WUE_i).

General Remarks

This article gave a decent account of previous literature in the field, well describes what further understanding is needed into C₄ carbon isotope discrimination and has a detailed methods section that is readily reproducible. However, we raise significant concerns regarding both the content of the paper and the presentation of figures throughout, which we outline below. In addition, we were not able to access the accompanying supplemental figures to this paper and thus are unable to review those.

Introduction

·       The statement made on lines 90-92 regarding that further research is needed ‘before δ¹³C_leaf can be implemented as a proxy of WUE_i’ seems to undermine the overall claims of this study. We recommend that this sentence be re-phrased so as not to cause confusion to the readers.

·       The results and discussion focus on the deletion in carbonic anhydrase1, however carbonic anhydrase is not mentioned in the introduction. We recommend that the role of and the importance of carbonic anhydrase in the C₄ pathway is explained in the introduction, accompanied by a figure.

·       On line 95, the use of brackets is not necessary as the information encompassed within them can be included in the sentence itself.

Table 1

·       Table 1 does not provide additional information to what is already described in-text. We recommend this table be replaced with a bar chart instead.

·       The data included in this table is not informative without a wild type line to compare the values to. We recommend including this.

·       If the authors keep the table within this manuscript, the table legend needs to be moved above the table and be more descriptive as it should be able to stand-alone without the aid of the main text.

Figure 1 (and the associated text)

·       We recommend that this figure is separated into two figures, with the F2 individuals separated from the two parental lines for improved understanding and less confusion.

·       The statement on line 299 that the segregation ratio 4:3:1 is close to a 3:1 ratio is an incorrect claim. We recommend that this and the claim based on this in the rest of the sentence be removed.

Figure 2 (and the associated text)

·       The markers discussed on line 302 would be interesting to know, however as we are unable to access the supplemental materials that detail these we don’t know what they are. We recommend detailing these in the text or making the supplemental materials available.

·       The abbreviations of ‘QTL’ and ‘LOD’ are not defined in text and may require the readers to research this themselves to understand the findings. We recommend defining these within the paper.

Figure 3

·       The colours used in this figure are not meaningful and at first glance appear that panels A and C are the same. We recommend changing the colour of figure 3C to a different colour that is not used in 3A or 3B.

·       The labels above the peaks are difficult to read. We recommend enlarging this information or including it within the figure legend.

Figure 4 (and the associated text)

·       The midline of multiple box plots on this figure are unclear due to the thickness of the line. We recommend reducing the thickness of this to make it easier to read.

·       In the associated text to this figure, it is unclear as to what the authors are complementing. We recommend explaining why and what is being complemented more clearly, which may benefit from an accompanying schema detailing this.

·       On line 329, the authors reference a paper that seems to have previously investigated what the authors have done in this paper, undermining the novelty of this research. We recommend that the others explain how their research differs to that of these referenced authors so as to ensure the claims being made are accurate.

Figure 5 (and the associated text)

·       Although this figure provides a welcome visualisation of what regions of cah1 have been deleted in this line, we don’t believe that this is a figure on its own.

·       The figure and associated text describe which areas of the DNA sequence have been deleted, however the authors do not detail what is being deleted from the protein at this region, such as the specific motifs encoded there. We believe that this is very important in explaining the findings of this research and thus we recommend that these be included and discussed in the paper.

Figure 6 (and associated text)

·       On line 366, we recommend that the authors change the text to ‘Gas exchange measurements were collected.’

·       This figure contains lots of valuable and interesting data, however we recommend this figure be separated into multiple figures and discussed in more detail as we believe it encompasses the main findings of this paper.

·       As in table 1, we believe that this figure would largely benefit from containing wild type data as a comparison, therefore we recommend including such data.

Figure 7

·       We recommend including wild type data as a comparison in this figure.

Figure 8

·       We recommend including wild type data as a comparison in this figure.

Discussion

·       New data is given in the discussion on line 387 which is not provided in the results section. We recommend moving these results to the results section, and then only discussing them within the discussion section.

·       We expected the discussion to focus on WUE_i due to the large focus on this in the introduction, however it was barely discussed in this section. We recommend that this either be discussed in more detail here, or the importance of this for the rest of the paper be toned down in the introduction.

·       On line 450 the authors provide no evidence/analysis that these ‘new isoforms’ arise via spontaneous mutations. We understand that it is assumed based on the parental lines etc., however we recommend that analyses be done to back up this claim, or this claim not made.

·       Carbon isotope discrimination is mentioned on line 402, however unless the readers have previous knowledge on this, it is difficult to follow. We recommend this be explained in the introduction.

Additional points for consideration

·       In the description of δ¹³C_leaf tissue collection, ‘12-24 leaf punches per sample’ is a wide range and claims based off of 12 vs 24 samples may not be comparable.

·       The figures throughout the paper are not coherent in regard to font, font size, design and formatting. We recommend that these all be revised to fit one common theme/design.

·       We believe that the title of this paper is misleading as beyond the introduction, WUE_i is not a main focus and instead the focus shifts onto carbonic anhydrase. We recommend the title be revised.

Competing interests

The authors declare that they have no competing interests.