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Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development

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Abstract

Main conclusion

The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes.

The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as ‘amorphous,’ lacking in ultrastructural features, and is often observed as a thin (~80–100 nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.

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Abbreviations

FS:

Freeze-substitution

HPF:

High-pressure freezing

TEM:

Transmission electron microscopy

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Acknowledgments

We are grateful to J. K. C. Rose and R. Jetter for valuable discussions and encouragement. We are further grateful to R. Jetter for reviewing the manuscript. This work was funded by NSERC Discovery grant (309189) awarded to DAB and (Grant No 229548) to ALS. We also extend our thanks for technical support from the UBC bioimaging facility. ALS and DAB conceived and designed the research. SJS and DAB conducted experiments. DAB analyzed the data and wrote the manuscript. All authors read and approved the manuscript.

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Authors and Affiliations

  1. Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada

    Sarah J. Shumborski & A. Lacey Samuels

  2. Department of Biology, Mount Royal University, Calgary, AB, T3E 6K6, Canada

    David A. Bird

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  1. Sarah J. Shumborski
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  2. A. Lacey Samuels
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  3. David A. Bird
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Corresponding author

Correspondence to David A. Bird.

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425_2016_2549_MOESM1_ESM.tif

Supplementary material 1 Fig. S1 Arabidopsis stem sections prepared by HPF/FS and low temperature embedding. a 300-nm section stained with toluidine blue O stain. The embedding resin often separated from the tissue (arrow) and the epidermal cell wall was not properly preserved. Gaps in the resin between the cell wall and cytosol are often observed as well (asterisk). b TEM of a region similar to the box in a. The cuticle was not preserved in the section and gaps in the resin are often seen where the cuticle would be present (asterisk). Scale bars 5 μm (a) and 500 nm (b) (TIFF 2380 kb)

Supplementary material 2 (DOCX 55 kb)

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Shumborski, S.J., Samuels, A.L. & Bird, D.A. Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development. Planta 244, 843–851 (2016). https://doi.org/10.1007/s00425-016-2549-8

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  • DOI: https://doi.org/10.1007/s00425-016-2549-8

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