title: Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition.
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.
authors.affiliation: School of life and Environmental Sciences, Centre for Chemistry and Biotechnology, Deakin University, Geelong Campus at Waurn Ponds, Geelong, VIC, 3217, Australia.
authors.forename: Pavani P
authors.initials: PP
authors.lastname: Nadiminti
authors.affiliation: School of life and Environmental Sciences, Centre for Chemistry and Biotechnology, Deakin University, Geelong Campus at Waurn Ponds, Geelong, VIC, 3217, Australia.
authors.forename: James E
authors.initials: JE
authors.lastname: Rookes
authors.affiliation: Drug Delivery, Disposition and Dynamics Monash Institute of Pharmaceutical Sciences, Monash University Parkville Campus, 381 Royal Parade, Parkville, VIC, 3052, Australia.
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Boyd
authors.affiliation: School of life and Environmental Sciences, Centre for Chemistry and Biotechnology, Deakin University, Geelong Campus at Waurn Ponds, Geelong, VIC, 3217, Australia. david.cahill@deakin.edu.au.
authors.forename: David M
authors.initials: DM
authors.lastname: Cahill
mesh_terms: D002849:Chromatography, Gas; D007091:Image Processing, Computer-Assisted; D031297:Lupinus / Q000737:chemistry / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D019441:Plant Epidermis / Q000737:chemistry / Q000648:ultrastructure; D018515:Plant Leaves / Q000737:chemistry / Q000648:ultrastructure; D013194:Staining and Labeling; D014908:Triticum / Q000737:chemistry / Q000648:ultrastructure; D014885:Waxes / Q000032:analysis*; D003313:Zea mays / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D014885:Waxes
keywords: Confocal laser scanning microscopy (CLSM); Cuticle; Epicuticular waxes; Image analysis; Micromorphology; Optical microscopy