Raman micro-spectroscopy uncovers complex structural and chemical adaptations of alpine azalea leaf surface

• Raman imaging found alkanes, triterpenoids, and phenolics within the cutin matrix. • Flavonoids occurred on the adaxial leaf surface, cinnamic acids on the abaxial surface. • Triterpenoid levels and composition varied between cuticle layers. • The trichome basis contained alkanes, while triterpenoids dominated in distal parts. • Carotenoids and unsaturated lipids were found in subsidiary cells of the stomata. Cuticles are layered multicomponent structures covering plant surfaces. They have numerous functions, and their composition varies between species, plant organs and even within cuticle layers. Therefore, microchemical methods are needed to investigate cuticle structures with the necessary spatial and chemical resolution. Raman micro-spectroscopy allows such non-destructive, in-situ analysis with a lateral resolution of 300 nm. After cutting microsections from the alpine azalea ( Kalmia procumbens ) leaves, we acquired position-resolved Raman spectra from the adaxial and abaxial surfaces (including trichomes and stomata). Multivariate data analysis of the hyperspectral datasets revealed various cuticle layers and the epidermal cell walls, with characteristic composition of alkanes, triterpenoids, phenolics and carbohydrates. Alkanes were found as thin cuticle layers and inside cells at the basis of trichomes, while triterpenoids were present in diverse cuticle locations in varying amounts and compositions. In particular, amyrins were detected on the abaxial cuticle, while ursolic and oleanolic acid impregnated the adaxial cuticle and the distal part of the trichomes. On the adaxial side of the leaf, flavonoids were detected throughout the cuticle and as clusters within the epidermal cell lumen as well as tiny spines on the outer surface. In contrast, flavonoids were less prominent in the abaxial cuticle, but characteristic Raman bands could instead be attributed to cinnamic acids. The trichomes and thick cuticle across both leaf surfaces, and their impregnation with triterpenoids and aromatic components may help K. procumbens withstand the extreme environmental conditions of its habitat.