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

It is well established that waxes built 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 waxes can be divided in epicuticular (on the surface of the cuticular membrane) and intracuticular (embedded in the cutin polymer) waxes. Until today there are only limited investigations dealing with the question to what extent epi- or intracuticular waxes contribute to the formation of the transpiration barrier. For Prunus laurocerasus previous studies have shown that epicuticular waxes do not contribute to the formation of the transpiration barrier. 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 wax was mechanically removed using collodion from the surface of either isolated cuticular membranes or intact leaf discs of ten further plant species differing in total wax amounts, wax compositions 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 collodion were sufficient for a complete removal of epicuticular wax. The treated surfaces appeared smooth after removal. The total wax amounts removed with the two collodion treatments and the residual amount of waxes after collodion treatment were quantified by gas chromatography and mass spectrometry. This showed that epicuticular waxes essentially consisted of long-chain aliphatic molecules (e.g. alkanes, primary alcohols, fatty acids), whereas intracuticular wax was composed of both, triterpenoids and long-chain aliphatic molecules. Cuticular transpiration using combined replicates was measured before and after removal of surface wax. Results clearly indicated that two consecutive collodion treatments, or the corresponding solvent treatments (diethyl ether:ethanol) serving as control, did not increase cuticular transpiration of the ten further leaf species investigated. Our results lead to the conclusion that epicuticular wax does not contribute to the formation of the transpiration barrier of leaves.