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.

Key Message Hybrid saplings were more reactive to soil water deficit than Japanese and European larch. European larch had hydraulically safer wood and anisohydric behavior, Japanese and hybrid larch showed isohydric strategy. Abstract Deciduous larch species could be an alternative to evergreen conifers in reforestation, but little is known about drought sensitivity of their saplings. The effect of an experimental drought on hydraulics and quantitative wood anatomy was tested on saplings of European larch (EL, Larix decidua), Japanese larch (JL, Larix kaempferi) and their hybrid (HL). Across species, biomass, transpiration rate and relative water content were higher in controls than in drought stressed trees, but transpiration efficiency was lower. JL had the highest transpiration efficiency under drought, and EL the lowest, coinciding with slower growth of EL. Wood of EL formed before drought was hydraulically safer as shown by higher wall/lumen ratio and lower pit cavity area. EL neither had a significant increase in transpiration efficiency nor a reduction in transpiration rate under drought, suggesting that the stomata remained open under soil water deficit. HL saplings were the most reactive to water shortage, indicated by intra-annual density fluctuations and a decrease in relative water content of the sapwood. Significant reduction in transpiration by HL suggested a higher stomatal sensitivity, while the same leaf surface area was maintained and radial growth was still similar to its best parent, the JL. The latter showed a significantly lower leaf surface area under drought than controls. EL, with its hydraulically safer wood, followed an anisohydric behavior, while JL and HL revealed an isohydric strategy. Altogether, our results suggest species dependent acclimations to drought stress, whereby HL followed the strategy of JL rather than that of EL.