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Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition

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Abstract

Electron microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been invaluable tools for the study of the micromorphology of plant cuticles. However, for electron microscopy, the preparation techniques required may invariably introduce artefacts in cuticle preservation. Further, there are a limited number of methods available for quantifying the image data obtained through electron microscopy. Therefore, in this study, optical microscopy techniques were coupled with staining procedures and, along with SEM were used to qualitatively and quantitatively assess the ultrastructure of plant leaf cuticles. Leaf cryosections of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) were stained with either fat-soluble azo stain Sudan IV or fluorescent, diarylmethane Auramine O and were observed under confocal laser scanning microscope (CLSM). For all the plant species tested, the cuticle on the leaf surfaces could be clearly resolved in many cases into cuticular proper (CP), external cuticular layer (ECL), and internal cuticular layer (ICL). Novel image data analysis procedures for quantifying the epicuticular wax micromorphology were developed, and epicuticular waxes of L. angustifolius were described here for the first time. Together, application of a multifaceted approach involving the use of a range of techniques to study the plant cuticle has led to a better understanding of cuticular structure and provides new insights into leaf surface architecture.

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Acknowledgments

This research was supported under Australian Research Council’s Linkage Projects funding scheme (project number LP0991494). Thank you to Dr Yao Da Dong, Monash University, and Mr Chad Sayer, Nufarm Australia, for early discussions on this project.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. School of life and Environmental Sciences, Centre for Chemistry and Biotechnology, Deakin University, Geelong Campus at Waurn Ponds, Geelong, VIC, 3217, Australia

    Pavani P. Nadiminti, James E. Rookes & David M. Cahill

  2. Drug Delivery, Disposition and Dynamics Monash Institute of Pharmaceutical Sciences, Monash University Parkville Campus, 381 Royal Parade, Parkville, VIC, 3052, Australia

    Ben J. Boyd

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  1. Pavani P. Nadiminti
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  2. James E. Rookes
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  4. David M. Cahill
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Correspondence to David M. Cahill.

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Fig. S1

Cuticular arrangement on leaf transverse sections of Z. mays. (a and b) Under bright-field microscopy, the Cuticle proper (cp) can be observed after Sudan IV staining as a bright pink to red layer on the epidermal cells. (c and d) After Auramine O staining and wide-field fluorescence microscopy, cp could be identified as a bright layer external to the epidermal cells. (e and f) CLSM of sections stained with Auramine O. External cuticular layer (ecl) with low fluorescence intensity was sandwiched between brightly fluorescent cp and internal cuticular layer (icl) on the epidermis of the leaf lamina. (g and h) Wide-field fluorescence microscopy performed after dichromatic staining could not completely resolve the cuticular layers from cell wall (cw) regions that are bright blue in colour. (i and j) CLSM on doubly stained Z. mays leaf cross sections could clearly resolve cw from ecl, icl and cp. Scale bar a, i = 20 μm; b, d, g, h = 5 μm; c, e = 4 μm; f, j = 2.5 μm (JPEG 693 kb)

Fig. S2

3D reconstruction of the cuticle on the leaf surfaces of T. aestivum, Z. mays and L. angustifolius. The z-stacks generated from the CLSM are reconstructed to show three different surfaces of the cuticle. The arrangement of the cuticle is similar for T. aestivum and Z. mays, where the anticlinal pegs (ap) extended into the anticlinal cell junctions and is indicated with arrows. Contrastingly, theap of L. angustifolius did not extend into cell junctions. Arrow heads represent the chloroplasts while the nucleus is labelled ‘n’. Scale bar = 5 μm (JPEG 570 kb)

Fig. S3

Scanning electron microscopy (SEM) on the leaf surface of T. aestivum, Z. mays, L. angustifolius and A. thaliana. For each row the magnification of the SEM images presented increases from left to right. (a) The oval on the top of the image shows the leaf region covered with a dense array of epicuticular waxes, while the lower oval indicates the regions of phylloplane devoid of epicuticular waxes. (b) The stomatal opening on T. aestivum leaf. (c) The arrangement of irregular crenate platelets (icp) on the surface of T. aestivum. (d) Unresolved epicuticular waxes on Z. mays leaf at low magnification. (e) Similar to T. aestivum, Z. mays also has regions densely covered with wax crystals (top oval) and regions devoid of any wax crystals (lower oval). The arrow head shows a stomatal opening. (f) Different types of epicuticular waxes can be clearly seen. For L. angustifolius the region around the stomatal openings are devoid of epicuticular waxes and appear as pits under (g) lower magnification. (h) Stomatal opening surrounded by region devoid of epicuticular waxes. (i) The wax crystals on the surface of L. angustifolius have membranous extensions and hence the name membranous platelets. (j, k and l) The phylloplane of A. thaliana is devoid of epicuticular wax crystals, stomatal opening indicated by an arrow head (k). (i) The epicuticular wax sheath could be clearly seen on the surface of A. thaliana. Scale bar a = 10 μm; b = 1 μm; c = 400 nm; d = 40 μm; e = 7 μm; f = 400 nm; g = μm; h = 5 μm; i = 400 nm; j = 20 μm; k = 5 μm; l = 1 μm (JPEG 1561 kb)

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Nadiminti, P.P., Rookes, J.E., Boyd, B.J. et al. Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition. Protoplasma 252, 1475–1486 (2015). https://doi.org/10.1007/s00709-015-0777-6

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