Synchrotron Based X-ray Microtomography Reveals Cellular Morphological Features of Developing Wheat Grain

Round 1

Reviewer 1 Report

Review of the manuscript

‘Synchrotron based X-ray microtomography reveals cellular morphological features of developing wheat grain’

The manuscript applsci-1627285 describing the use of Xray-microtomography for analysyes of wheat grain. The methods have been well chosen, and results presented are supported by exhaustive discussion. Also, conclusion meets the objective taken. The graphic part of the article is interesting, and useful for imaging of conducted research and obtained results.

Carried out analysis with the pioneering Xray approach are extremely well done, which proves the authors' great experience. I’ve got only one comment corresponding to anatomical/embryological part. In my opinion it is a little too general. Results allow to analyse mainly morphological features, and still for detailed description of developmental stages, the staining techniques for optical microscopy are needed. Using images of Xray- we do not have detailed information about particular stages in development program of grain, because of still too low resolution, magnifications. Sometimes even very easy staining allow to get information about polysaccharides content etc, such as ruthenium red, PAS staining method. Current work would be more valuable if it included microscopic results, it would be interesting to compare the results from the two methods. Another option for the future studies, to complete the research would be a step-by-step analysis of the progressive grain development process using standard microscopic methods. And then, Xray analysis of the same grains to see if it is possible to see these changes using microtomography.

However, conclusions provide a clear message based in the results obtained in the ms, and emphasize that these are the results of preliminary research into a new method. In this way, the paper is correct and logical. Due to the high importance of the research conducted with new tool, the manuscript should be accepted for publication as a first report about possibility to use Xray in embryological studies. My comments are mainly editorial, therefore, in my opinion the manuscript can be accepted for publication in its current form.

Author Response

We thank the reviewer for the very positive review !

We totally agree with the fact the microscopy provides information that cannot be obtained from tomography. One of our initial objectives was to combine precise anatomical and chemical information provided by microscopy with the global 3D anatomical information provided by tomography. We updated the manuscript to provide more comments in the comparison between information that can be obtained by tomography and microscopy, and updated the discussion. We also still plan to progress in the fusion of the images obtained by both modalities.

Reviewer 2 Report

Taking advantage of the phase-contrast tomography provided by synchrotron radiation, Legland et al. reconstructed 3D images of wheat grains at various developmental stages, using the low- and high resolution settings. Compared with classical laboratory microtomography, the synchrotron based X-ray microtomography showed an enhanced level of contrast and thus provided more details of the outer and inner compartments of a wheat grain. The authors also compared the low- and high resolution images. They showed that low resolution setup provided high-quality 3D images of the whole grains, whereas high resolution setup showed more tissue layers and structures. At last, the authors discussed the advantages and disadvantages of the synchrotron-based tomography, and it compared to other approaches. Overall, this manuscript is well written and shows interests to the relevant fields.

My main criticism is that the compartments and structures of wheat grain are not properly labeled. For example, in Fig.4G, the embryo was covered by the label; in fig.5, it is ambiguous which layers are ip, ne, t and al, respectively. I also have some other comments:

1, Were the samples for sectioning (Fig5C&D) at the same age to those for tomography? There was no endosperm cavities observable in fig5.A and B, which were visible in fig.5C and D. In addition, aleurone layer is not clear in fig.5D but evident in fig.5b.

2, I would suggest the author provide magnified figures to show the details described in Line282-302. For example, “Cell layers of the inner pericarp (cross cells and tube cells) can be recognized between the endosperm and the pericarp parenchyma”, which are cross cells and tube cells? Similarly, a magnified figure will be helpful for understanding the statement “Cells surrounding the vascular bundle were less defined and the testa was not revealed by the X-rays.”

3, The figures showed in Fig.6 were not fully cited in the relevant paragraph.

4, line 332, re-mains >> remains

Author Response

We thank the reviewer for the positive comments.

We updated the figures (in particular Figure 4 and Figure 5) to better visualize anatomical structures.

1. Were the samples for sectioning (Fig5C&D) at the same age to those for tomography?

Yes, samples used for microscopy and tomography were sampled at the same age. Some differences may exist due to the biological variability between grains, and because of preparation artifacts for the microscopy.

There was no endosperm cavities observable in fig5.A and B, which were visible in fig.5C and D.

Yes, there is an “endosperm cavity” in fig5. B where it is labelled “ec” (not much visible in fig 5A). Although it is called cavity, this structure is filled in vivo (Chateigner-Boutin et al., 2021). Sample preparation and sectioning before microscopy observations induced deformations and emptied the cavity.

In addition, aleurone layer is not clear in fig.5D but evident in fig.5b.

The aleurone layer in fig 5 B can be guessed between the nucellar epidermis and the starchy endosperm because it makes a layer with no starch grains. in fig 5D we can see it but we agree that a magnified figure improves the distinction of the different layers. We therefore have added a zoomed figure (new figure 6) where the different layers are more visible. We can see the aleurone layer in figure 6 B and D. Sample preparation for microscopy has also damaged the nucellar epidermis which appears crushed in figure 6 B and D (except in the top of figure 6 D) compared to the tomography images figure 6 A and B.

2, I would suggest the author provide magnified figures to show the details described in Line282-302. For example, “Cell layers of the inner pericarp (cross cells and tube cells) can be recognized between the endosperm and the pericarp parenchyma”, which are cross cells and tube cells?

We have now provided a magnified figure 6 where cross cells and tube cells are visible and labeled in fig 6A and 6C

Similarly, a magnified figure will be helpful for understanding the statement “Cells surrounding the vascular bundle were less defined and the testa was not revealed by the X-rays.”

We have now provided a magnified figure 6B and D zoomed in the vascular bundle region

3. The figures showed in Fig.6 were not fully cited in the relevant paragraph.

we improved the references to figures within the text.

4. line 332, re-mains >> remains

Thanks, this was fixed.