A modified method for enzymatic isolation of and subsequent wax extraction from Arabidopsis thaliana leaf cuticle
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ant Methods Open Access
METHODOLOGY
A modified method for enzymatic isolation of and subsequent wax extraction from Arabidopsis thaliana leaf cuticle Martina Vráblová1* , Daniel Vrábl1,2, Barbora Sokolová1, Dominika Marková1,3 and Marie Hronková4,5
Abstract Background: The plant cuticle represents one of the major adaptations of vascular plants to terrestrial life. Cuticular permeability and chemical composition differ among species. Arabidopsis thaliana is a widely used model for biochemical and molecular genetic studies in plants. However, attempts to isolate the intact cuticle from fresh leaves of Arabidopsis have failed so far. The goal of this study was to optimise an enzymatic method for cuticle isolation of species with a thin cuticle and to test it on several A. thaliana wild types and mutants. Results: We developed a method for isolation of thin cuticles that allows reducing the isolation time, the separation of abaxial and adaxial cuticles, and avoids formation of wrinkles. Optical microscopy was used for studying cuticle intactness and scanning electron microscopy for visualisation of external and internal cuticle structures after isolation. Wax extracts were analysed by GC–MS. Isolation of intact cuticle was successful for all tested plants. The wax compositions (very-long-chained fatty acids, alcohols and alkanes) of intact leaves and isolated cuticles of wild type Col-0 were compared. Conclusions: We conclude that the optimised enzymatic method is suitable for the isolation of A. thaliana adaxial and abaxial cuticles. The isolated cuticles are suitable for microscopic observation. Analysis of wax composition revealed some discrepancies between isolated cuticles and intact leaves with a higher yield of wax in isolated cuticles. Keywords: Arabidopsis thaliana, Leaf cuticle, Enzymatic isolation, Fatty acids, Wax Background The cuticular membrane covering the epidermis is an essential structure, protecting the aerial surface of living cells in all land plants. It is the lipophilic product of the epidermal cells of leaves, primary stems and fruits. Cuticles also occur internally in the lining of substomatal cavities. The primary function of the cuticle is to prevent uncontrolled water loss and regulation of gas exchange. Besides that, it works as a natural UV filter and as an infection barrier for viruses, bacteria and fungi. *Correspondence: [email protected] 1 VSB-Technical University of Ostrava, Institute of Environmental Technology, 17. listopadu 15, 708 00 Ostrava, Czech Republic Full list of author information is available at the end of the article
Moreover, the cuticle plays an important role as a border preventing organ fusion during organogenesis [23, 25, 29]. Cuticles consist of an insoluble matrix and wax soluble in organic solvents, which form layers above the cell wall or, in a new concept, are understood as a lipidised, chemically and structurally heterogeneous region of the epidermal cell wall [9]. The matrix is formed by cutin, possibly cutan and/or polysaccharides [26, 35]. T
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