Water and Solute Permeability of Plant Cuticles: Measurement and ...

6.5 Diffusion in Reconstituted Isolated Cuticular Waxes 193
cuticular waxes of grape berries had previously been used by Grncarevic and Radler
(1967) to assess their effect cuticular transpiration. A refined method allowing the
measurement of diffusion coefficients of lipophilic molecules in cuticular wax was
developed with barley leaf wax (Schreiber and Schönherr 1993b).
6.5.1 Experimental Approach
Wax was extracted from barley leaves using chloroform. Aluminium disks (8 mm
diameter) were immersed in the wax solution for 1–2 s. After evaporation of the
solvent the solid wax adhered to the surface of the disks, and wax-covered aluminium
disks were heated to 100 ◦ C for 5 min. Thus, wax was reconstituted from
the melt and not from solution. This ensured a good adhesion of the wax to
the aluminium surface, which is necessary for the following desorption experiments.
Scanning electron microscopy revealed that wax-covered aluminium disks
had smooth surfaces, while barley-leaf surfaces are densely covered with epicuticular
wax platelets. Therefore, the term “reconstituted” is preferred instead of
“recrystallised” (Sect. 9.6).
Diffusion coefficients were determined by desorbing radiolabelled probes, which
had been added to the wax solution before reconstitution (Schreiber and Schönherr
1993b). In some experiments, reconstituted wax samples were loaded with solutes
by equilibrating them in solutions of the radio-labelled compound (Kirsch et al.
1997). This is the same method as described for the determination of wax/water
partition coefficients (Sect. 6.1). Adding the radio-labelled probe directly to the wax
solution offers the advantage that desorption experiments can be started directly
after reconstitution of the wax.
Diffusion in reconstituted wax takes place in the amorphous wax phase, and
therefore molecules must be soluble in this phase. No problems occur with lipophilic
molecules (Table 6.2), which are sufficiently soluble in the amorphous wax phase
when they are applied from aqueous solutions or if added directly to the wax before
reconstitution. With both methods of sample preparation, the same diffusion coefficients
are obtained in most cases (Schreiber and Schönherr 1993b). When highly
water soluble solutes are added directly to the wax solution before reconstitution,
they crystallise and are not homogeneously dissolved in the amorphous wax phase,
due to their low lipid solubility. Hence, diffusion of highly water soluble solutes in
cuticular waxes cannot be studied using reconstituted waxes.
Desorption of radio-labelled probes from reconstituted wax samples is initiated
by immersing them in vials (5 ml) containing an inert desorption medium (e.g.,
phospholipid suspension) which does not affect structure of the wax layer. The desorption
medium is replaced at each sampling time. The amounts desorbed and the
residual amount of the probe remaining in the wax at the end of the experiment
are determined by scintillation counting. The total amount of radio-labelled probe
M0 present in the wax at the beginning of the desorption experiment is calculated