Raman imaging reveals in-situ microchemistry of cuticle and epidermis of spruce needles

BACKGROUND: The cuticle is a protective layer playing an important role in plant defense against biotic and abiotic stresses. So far cuticle structure and chemistry was mainly studied by electron microscopy and chemical extraction. Thus, analysing composition involved sample destruction and the link between chemistry and microstructure remained unclear. In the last decade, Raman imaging showed high potential to link plant anatomical structure with microchemistry and to give insights into orientation of molecules. In this study, we use Raman imaging and polarization experiments to study the native cuticle and epidermal layer of needles of Norway spruce, one of the economically most important trees in Europe. The acquired hyperspectral dataset is the basis to image the chemical heterogeneity using univariate (band integration) as well as multivariate data analysis (cluster analysis and non-negative matrix factorization). RESULTS: Confocal Raman microscopy probes the cuticle together with the underlying epidermis in the native state and tracks aromatics, lipids, carbohydrates and minerals with a spatial resolution of 300 nm. All three data analysis approaches distinguish a waxy, crystalline layer on top, in which aliphatic chains and coumaric acid are aligned perpendicular to the surface. Also in the lipidic amorphous cuticle beneath, strong signals of coumaric acid and flavonoids are detected. Even the unmixing algorithm results in mixed endmember spectra and confirms that lipids co-locate with aromatics. The underlying epidermal cell walls are devoid of lipids but show strong aromatic Raman bands. Especially the upper periclinal thicker cell wall is impregnated with aromatics. At the interface between epidermis and cuticle Calcium oxalate crystals are detected in a layer-like fashion. Non-negative matrix factorization gives the purest component spectra, thus the best match with reference spectra and by this promotes band assignments and interpretation of the visualized chemical heterogeneity. CONCLUSIONS: Results sharpen our view about the cuticle as the outermost layer of plants and highlight the aromatic impregnation throughout. In the future, developmental studies tracking lipid and aromatic pathways might give new insights into cuticle formation and comparative studies might deepen our understanding why some trees and their needle and leaf surfaces are more resistant to biotic and abiotic stresses than others.