Epicuticular wax on leaf cuticles does not establish the transpiration barrier, which is essentially formed by intracuticular wax

Abstract It is well established that waxesbuilt up the barrier properties of cuticles, since their extraction in organic solvent e.g. chloroform increases diffusion of water and organic compounds by 1–2 orders of magnitude. Leaf surface waxescan be divided in epicuticular (on the surface of the cuticular membrane) and intracuticular (embedded in the cutinpolymer) waxes. Until today there are only limited investigations dealing with the question to what extent epi- or intracuticular waxescontribute to the formation of the transpirationbarrier. For Prunus laurocerasusprevious studies have shown that epicuticular waxesdo not contribute to the formation of the transpirationbarrier. This approach successfully established for P. laurocerasus was applied to further species in order to check whether this finding also applies to a broader spectrum of species. Epicuticular waxwas mechanically removed using collodionfrom the surface of either isolated cuticular membranes or intact leaf discs of ten further plant species differing in total waxamounts, waxcompositions and transport properties. Scanning electron microscopy, which was performed to independently verify the successful removal of the surface waxes, indicated that two consecutive treatments with collodionwere sufficient for a complete removal of epicuticular wax. The treated surfaces appeared smooth after removal. The total waxamounts removed with the two collodiontreatments and the residual amount of waxesafter collodiontreatment were quantified by gas chromatography and mass spectrometry. This showed that epicuticular waxesessentially consisted of long-chain aliphatic molecules (e.g. alkanes, primary alcohols, fatty acids), whereas intracuticular waxwas composed of both, triterpenoids and long-chain aliphatic molecules. Cuticular transpirationusing combined replicates was measured before and after removal of surface wax. Results clearly indicated that two consecutive collodiontreatments, or the corresponding solvent treatments (diethyl ether:ethanol) serving as control, did not increase cuticular transpirationof the ten further leaf species investigated. Our results lead to the conclusion that epicuticular waxdoes not contribute to the formation of the transpirationbarrier of leaves.