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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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References

  • Barthlott W, Neinhuis C (1997) Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta 202:1–8. doi:10.1007/s004250050096

    Article  CAS  Google Scholar 

  • Barthlott W, Neinhuis C, Cutler D, Ditsch F, Meusel I, Theisen I, Wilhelmi H (1998) Classification and terminology of plant epicuticular waxes. Bot J Linn Soc 126:237–260. doi:10.1111/j.1095-8339.1998.tb02529.x

    Article  Google Scholar 

  • Beattie GA, Marcell LM (2002) Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces. Plant Cell Environ 25:1–16. doi:10.1046/j.0016-8025.2001.00804.x

  • Broun P, Poindexter P, Osborne E, Jiang C-Z, Riechmann JL (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci U S A 101:4706–4711. doi:10.1073/pnas.0305574101

  • Buda G, Isaacson T, Matas A, Paolillo D, Rose J (2009) Three-dimensional imaging of plant cuticle architecture using confocal laser scanning microscopy. Plant J 60:378–385. doi:10.1111/j.1365-313X.2009.03960.x

    Article  CAS  PubMed  Google Scholar 

  • Buschhaus C, Herz H, Jetter R (2007) Chemical composition of the epicuticular and intracuticular wax layers on adaxial sides of Rosa canina leaves. Ann Bot 100:1557–1564. doi:10.1093/aob/mcm255

  • Cahill D, Rookes J, Michalczyk A, McDonald K, Drake A (2002) Microtubule dynamics in compatible and incompatible interactions of soybean hypocotyl cells with P hytophthora sojae. Plant Pathol 51:629–640. doi:10.1046/j.0032-0862.2002.00758.x

  • Calbiani F, Careri M, Elviri L, Mangia A, Pistarà L, Zagnoni I (2004) Development and in-house validation of a liquid chromatography–electrospray–tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli products. J Chromatogr 1042:123–130. doi:10.1016/j.chroma.2004.05.027

  • Carlsson K, Mossberg K (1992) Reduction of cross-talk between fluorescent labels in scanning laser microscopy. J Mic 167:23–37. doi:10.1111/j.1365-2818.1992.tb03216.x

    Article  Google Scholar 

  • Chaudhury AM et al (1994) Genetic control of male fertility in Arabidopsis thaliana: structural analysis of premeiotic developmental mutants. Sex Plant Reprod 7:17–28. doi:10.1007/BF00241884

    Article  Google Scholar 

  • Chen B, Schnoor JL (2009) Role of suberin, suberan, and hemicellulose in phenanthrene sorption by root tissue fractions of switchgrass (Panicum virgatum) seedlings. Environ Sci Technol 43:4130–4136. doi:10.1021/es803510u

  • Chen B, Johnson EJ, Chefetz B, Zhu L, Xing B (2005) Sorption of polar and nonpolar aromatic organic contaminants by plant cuticular materials: role of polarity and accessibility. Environ Sci Technol 39:6138–6146. doi:10.1021/es050622q

    Article  CAS  PubMed  Google Scholar 

  • Chen B, Li Y, Guo Y, Zhu L, Schnoor JL (2008) Role of the extractable lipids and polymeric lipids in sorption of organic contaminants onto plant cuticles. Environ Sci Technol 42:1517–1523. doi:10.1021/es7023725

    Article  CAS  PubMed  Google Scholar 

  • Czerednik A, Busscher M, Bielen BAM, Wolters-Arts M, de Maagd RA, Angenent GC (2012) Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes. J Exp Bot 63:2605–2617. doi:10.1093/jxb/err451

  • Domínguez E, López-Casado G, Cuartero J, Heredia A (2008) Development of fruit cuticle in cherry tomato (Solanum lycopersicum). Funct Plant Biol 35:403–411. doi:10.1071/FP08018

  • Falk RH, Guggenheim R, Schulke G (1994) Surfactant-induced phytotoxicity. Weed Technol 8:519–525

    CAS  Google Scholar 

  • Go YS, Kim H, Kim HJ, Suh MC (2014) Arabidopsis cuticular wax biosynthesis is negatively regulated by the DEWAX gene encoding an AP2/ERF-type transcription factor. The Plant Cell 26:1666–1680. doi:10.1105/tpc.114.123307

  • Gonthier PNG, Rettori A, Paoletti E, Gullino ML (2010) Testing Nerium oleander as a biomonitor for surfactant polluted marine aerosol. Int J Environ Res 4:1–10

    CAS  Google Scholar 

  • Griffith MP, Magellan TM, Tomlinson PB (2014) Variation in leaflet structure in C ycas (Cycadales: cycadaceae): does anatomy follow phylogeny and geography? Int J Plant Sci 175:241–255. doi:10.1086/673884

  • Guzmán P, Fernández V, Khayet M, García ML, Fernández A, Gil L (2014a) Ultrastructure of plant leaf cuticles in relation to sample preparation as observed by transmission electron microscopy. Sci World J 2014:9 doi:10.1155/2014/963921

  • Guzmán P, Fernandez V, Graça J, Cabral V, Kayali N, Khayet M, Gil L (2014b) Chemical and structural analysis of Eucalyptus globulus and E. camaldulensis leaf cuticles: a lipidized cell wall region. Frontiers in Plant Science 5 doi:10.3389/fpls.2014.00481

  • Hannoufa A, McNevin J, Lemieux B (1993) Epicuticular waxes of eceriferum mutants of Arabidopsis thaliana. Phytochemistry 33:851–855. doi:10.1016/0031-9422(93)85289-4

  • Hardham A (2012) Confocal Microscopy in Plant–Pathogen Interactions. In: Bolton MD, Thomma BPHJ (eds) Plant Fungal Pathogens, vol 835. Methods in Molecular Biology. Humana Press, pp 295–309. doi:10.1007/978-1-61779-501-5_18

  • Herth W, Schnepf E (1980) The fluorochrome, calcofluor white, binds oriented to structural polysaccharide fibrils. Protoplasma 105:129–133. doi:10.1007/BF01279855

    Article  Google Scholar 

  • Jeffree CE (2007) The fine structure of the plant cuticle. In: Annual plant reviews volume 23: Biology of the plant cuticle. Blackwell Publishing Ltd, pp 11–125. doi:10.1002/9780470988718.ch2

  • Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA (1995) Leaf epicuticular waxes of the E ceriferum mutants in A rabidopsis. Plant Physiol 108:369–377. doi:10.1104/pp. 108.1.369

  • Jenks MA, Rashotte AM, Tuttle HA, Feldmann KA (1996) Mutants in Arabidopsis thaliana altered in epicuticular wax and leaf morphology. Plant Physiol 110:377–385. doi:10.1104/pp. 110.2.377

  • Jetter R, Schaffer S (2001) Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development. Plant Physiol 126:1725–1737. doi:10.1104/pp. 126.4.1725

  • Jetter R, Kunst L, Samuels AL (2007) Composition of plant cuticular waxes. In: Annual plant reviews volume 23: Biology of the plant cuticle. Blackwell Publishing Ltd, pp 145–181. doi:10.1002/9780470988718.ch4

  • Koch K et al (2006) Structural analysis of wheat wax (Triticum aestivum, c.v. ‘Naturastar’ L.): from the molecular level to three dimensional crystals. Planta 223:258–270. doi:10.1007/s00425-005-0081-3

    Article  CAS  PubMed  Google Scholar 

  • Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42:51–80. doi:10.1016/S0163-7827(02)00045-0

    Article  CAS  PubMed  Google Scholar 

  • Lequeu J, Fauconnier M-L, Chammaï A, Bronner R, Blée E (2003) Formation of plant cuticle: evidence for the occurrence of the peroxygenase pathway. Plant J 36:155–164. doi:10.1046/j.1365-313X.2003.01865.x

    Article  CAS  PubMed  Google Scholar 

  • Li Q, Chen B (2014) Organic pollutant clustered in the plant cuticular membranes: visualizing the distribution of phenanthrene in leaf cuticle using Two-photon confocal scanning laser microscopy. Environ Sci Technol 48:4774–4781. doi:10.1021/es404976c

    Article  CAS  PubMed  Google Scholar 

  • Lisek J, Habdas H, Gawroński S (2002) Relationship between selected morphological, anatomical and cytological characteristics of leaves and the level of tolerance to herbicides in strawberry cultivars. Acta Physiol Plant 24:371–378. doi:10.1007/s11738-002-0032-6

    Article  Google Scholar 

  • Ménard R, Verdier G, Ors M, Erhardt M, Beisson F, Shen W-H (2014) Histone H2B monoubiquitination is involved in the regulation of cutin and wax composition in Arabidopsis thaliana. Plant Cell Physiol 55:455–466. doi:10.1093/pcp/pct182

    Article  PubMed  Google Scholar 

  • Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L (1999) CUT1, an arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11:825–838. doi:10.1105/tpc.11.5.825

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nadiminti PP, Dong YD, Sayer C, Hay P, Rookes JE, Boyd BJ, Cahill DM (2013) Nanostructured liquid crystalline particles as an alternative delivery vehicle for plant agrochemicals. ACS Appl Mater & Interfaces 5:1818–1826. doi:10.1021/am303208t

  • Pathan AK, Bond J, Gaskin RE (2008) Sample preparation for scanning electron microscopy of plant surfaces—Horses for courses. Micron 39:1049–1061. doi:10.1016/j.micron.2008.05.006

    Article  CAS  PubMed  Google Scholar 

  • Peirson B, Owen H, Feldmann K, Makaroff C (1996) Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis. Sex Plant Reprod 9:1–16. doi:10.1007/BF00230361

  • Poulios I, Avranas A, Rekliti E, Zouboulis A (2000) Photocatalytic oxidation of Auramine O in the presence of semiconducting oxides. J Chem Technol Biotechnol 75:205–212. doi:10.1002/(SICI)1097-4660(200003)75:3<205::AID-JCTB201>3.0.CO;2-L

    Article  CAS  Google Scholar 

  • Rascio A, Rascio N, Rinaldi M, Valentini M (2014) Functional, histological and biomechanical characterization of wheat water-mutant leaves Physiol Plant:n/a-n/a doi:10.1111/ppl.12280

  • Rashotte AM, Jenks MA, Thanh DN, Feldmann KA (1997) Epicuticular wax variation in ecotypes of Arabidopsis thaliana. Phytochemistry 45:251–255. doi:10.1016/S0031-9422(96)00792-3

  • Riederer M (2007) Introduction: Biology of the Plant Cuticle. In: Annual plant reviews volume 23: Biology of the plant cuticle. Blackwell Publishing Ltd, pp 1–10. doi:10.1002/9780470988718.ch1

  • Ruberti C et al. (2014) Mitochondria Change Dynamics and Morphology during Grapevine Leaf Senescence PLoS One 9:e102012 doi:10.1371/journal.pone.0102012

  • Samaha MA, Tafreshi HV, Gad-el-Hak M (2012) Superhydrophobic surfaces: from the lotus leaf to the submarine. Comptes Rendus Mécanique 340:18–34. doi:10.1016/j.crme.2011.11.002

  • Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. doi:10.1038/nmeth.2089

    Article  CAS  PubMed  Google Scholar 

  • Serrano M, Coluccia F, Torres M, L’Haridon F, Metraux J (2014) The cuticle and plant defense to pathogens front. Plant Sci 5:274. doi:10.3389/fpls.2014.00274

    Google Scholar 

  • Tamura H, Knoche M, Bukovac MJ (2001) Evidence for surfactant solubilization of plant epicuticular wax. J Agric Food Chem 49:1809–1816. doi:10.1021/jf000608r

    Article  CAS  PubMed  Google Scholar 

  • Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7:1716–1727. doi:10.1038/nprot.2012.096

    Article  CAS  PubMed  Google Scholar 

  • Xue Y, Xiao S, Kim J, Lung S-C, Chen L, Tanner JA, Suh, MC, Chye, M-L (2014) Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation. J Exp Bot doi:10.1093/jxb/eru304

  • Yeats TH, Rose JKC (2013) The formation and function of plant cuticles. Plant Physiol 163:5–20. doi:10.1104/pp. 113.222737

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yeats TH, Martin LBB, Viart HMF, Isaacson T, He Y, Zhao L, Matas AJ, Buda GJ, Domozych DS, Clausen MH, Rose JKC (2012) The identification of cutin synthase: formation of the plant polyester cutin Nat Chem Biol 8:609–611 doi:http://www.nature.com/nchembio/journal/v8/n7/abs/nchembio.960.html#supplementary-information

  • Zhou X, Jenks M, Liu J, Liu A, Zhang X, Xiang J, Zou J, Peng Y, Chen X (2014) Overexpression of transcription factor OsWR2 regulates wax and cutin biosynthesis in rice and enhances its tolerance to water deficit. Plant Mol Biol Rep 32:719–731. doi:10.1007/s1-013-0687-8

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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.

Conflict of interest

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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  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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