Local motion and conformational changes in the cuticle of Clivia miniata Regel : A microfluorescence and spin-label study.

The temperature dependence of the local diffusion of fluorescent molecular probes of various polarities (alkane, long-chain fatty acid, short-chain alcohol and fatty acid), all labelled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl in the cuticle of Clivia miniata Regel was studied by the technique of fluorescence recovery after photobleaching. The technique yields the coefficient of diffusion, D, in the plane of the cuticle over distances of some 10 μm and the fraction, R, of mobile reporter molecules. The inner (more hydrophilic) and the outer (more hydrophobic) faces of the cuticle were studied separately by appropriate incubation. The value of D was found to depend sensitively on the polarity of the probe, the temperature and the position within the cuticle (outer hydrophobic or inner hydrophilic side). Depending on the type of probe, D increased (in the case of the alkane) or decreased (in the case of the alcohol) after removal of the (monomeric) waxes. The electron-spin-resonance (ESR) spectra of incorporated spin-labelled fatty-acid probes measured in the intact cuticle contained a major component similar to the spectrum recorded from the polymerized matrix from which waxes had been extracted, and a second component similar to the spectrum from the monomeric waxes. At low temperatures, the ESR spectra from labels at two different chain positions corresponded to chain motion which was slow on the ESR timescale. At high temperatures, the spectral component from the monomeric waxes indicated chain motions in the motional narrowing regime which were of an essentially isotropic nature.No evidence was found for a liquid-crystalline lipid phase such as found for the polar lipids in cell membranes, nor was there evidence for a sharp, thermotropic, lipid-phase transition either in the cuticle or in the waxes. Experiments with oriented samples did not demonstrate the presence of large domains with a uniform orientation of the lipid chains relative to the cuticular layers. The diffusion measurements and spin-label studies provide evidence for conformational changes of the cuticle extending over the whole temperature range studied (10° C to 70° C). These conformational changes are attributed to phase-separation processes within the cuticle. The phase separation in extracted waxes extended over a similar broad temperature range. This indicates that the transitions in the cuticle are largely determined by these components. At higher temperatures, however, the chain mobility in the regions of monomeric wax was considerably greater than that in the polymerized matrix. The experimental results strongly indicate that all three layers of the Clivia cuticle exhibit a multilamellar structure of alternatingly stacked, highly hydrophobic layers of welldefined thickness (5±0.5 nm) and more disordered layers of variable (4 to 15 nm) thickness. The lamellae are wellordered and extend over the whole leaf in the cuticle proper but are split-up into small domains in the inner and the external cuticular layer. Furthermore, changes of the molecular transport properties caused by the influence of ozone exerted during the growth of the plant were studied. We found that the diffusion coefficient increased both in the outer and the inner layer of the cuticle. A particularly large increase, by about a factor of three, was found for alkane diffusion in the hydrophobic outer face, pointing to defects in the polymerized matrix.