Local motion and conformational changes in the cuticle of Clivia miniata Regel

Abstract

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 mole...

Estimation of path lengths for diffusion of organic compounds through leaf cuticles

“…Extracted cuticles consist of cutin and polysaccharides and are called polymer matrix membranes (MX). The diffusion coefficients in MX and the lateral diffusion coefficients in leaf cuticles are of the same order as those observed for synthetic polymers (Wunderlich et al 1990). It has been suggested that the low mobilities in CM are due to the presence of highly ordered wax regions which increase the path length of diffusion across the cuticle (Schönherr & Baur 1994; Riederer & Schreiber 1995; Baur et al 1996; Merk, Blume & Riederer 1998).…”

Lognormal distribution of water permeability and organic solute mobility in plant cuticles

“…1) and (ii) solutes in tiny water filled pores (holes), which might potentially be produced by handling of the cuticles and which are too small for mass flow of PLS, will be desorbed immediately after the beginning of desorption. With diffusion coefficients in the cuticle below 10"'"^ m^ s~' (Schonherr & Riederer 1989;Wunderlich et al 1990) the amount of solute desorbed from the pores is not substituted by diffusion since the process is too slow over lateral distances of some 100 //m. The assumption of a normal distribution for the observed values is obviously not justified.…”

Mobilities of organic compounds in plant cuticles as affected by structure and molar volumes of chemicals and plant species

“…Apparent diffusion coefficients in the upper cuticles of Clivia leaves estimated from photo bleaching experiments (Wunderlich et al 1990) were only 1.5 x 10 -13 m2.s -1, which is larger by orders of magnitude than D measured in Citrus leaf cuticular membranes (Riederer and Sch6nherr 1985;Kerler and Sch6nherr 1988;Sch6nherr and Riederer 1989;Bauer and Sch6nherr 1992). Since Wunderlich et al (1990) measured lateral diffusion while all other estimates are based on transmembrane diffusion, the discrepancy could be caused by a highly tortuous diffusion path in transmembrane diffusion. In the photobleaching experiments, fluorescent pigments might have diffused parallel to the impermeable wax crystallites while in transmembrane diffusion solutes must travel around them.…”