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

20 1 Chemistry and Structure of Cuticles as Related to Water and Solute Permeability
which developed rapidly. In contrast to intracuticular wax, where interference by
the cutin polymer with crystallisation is probable, this problem does not arise when
wax layers form on the surface of the cuticle proper. Stripping of surface wax with
epoxy glue in the study by Koch et al. (2004) did not appear to damage the cuticle
or the leaf, and the method seems to be suitable to study effects of surface wax on
permeability. Barrier properties of lipid monolayers are addressed in Sect. 4.6.
1.4.2.3 Transmission Electron Microscopy
Before cuticle cross-sections can be viewed with the TEM, leaf tissue is fixed with
glutaraldehyde, stained en bloc with OsO4 or KMnO4, dehydrated with ethanol or
acetone, infiltrated with epoxy resins (Epon-Aradite) and then polymerised at 60 ◦ C.
From the block, ultra-thin sections are cut, which are usually contrasted with aqueous
uranyl acetate and lead citrate. It is not very likely that cuticular waxes survive
these procedures without change in structure or dislocation. The resin monomers
may dissolve waxes, and when the resin is cured at 60 ◦ C most waxes will become
fluid and redistribute. Epicuticular waxes or remnants of them are rarely seen in
the TEM, indicating that they have disappeared. Rather strangely, solvent properties
and melting behaviour of waxes in Epon-Araldite seem not to have been investigated
so far, and localisation of waxes in thin sections should not be attempted.
Hence, fine structure seen with the TEM is that of cutin, cutan, polysaccharides and
polypeptides.
Unstained cuticles appear in the TEM similar to the embedding media, both of
them being polyester polymers composed mainly of carbon, hydrogen and oxygen.
Staining is necessary to obtain micrographs with fine structure. The stains contain
atoms with higher masses, and they absorb electrons much more effectively.
Stains either bind selectively to functional groups or they react with them. In either
case, they must diffuse into the tissue when staining is en bloc or in the embedding
medium during section staining.
Glutaraldehyde is used routinely to preserve cytological details. It is not known
if it affects fine structure of cuticles. OsO4 is a non-ionic and lipophilic stain. At
25 ◦ C solubility in water is small (7.24 g/100 g water), while in carbon tetrachloride
375 g/100 g can be dissolved. OsO4 is used as a buffered aqueous solution.
It is an oxidising agent, and converts olefins to glycols. It also oxidises peroxides
(Budavary 1989). Cutin acids with double bonds do not occur frequently in cutin
monomers (Holloway 1982b), but in cutin from Clivia leaves, 18% of the identified
cutin acids was 18-hydroxy-9-octadecenoic acid (Riederer and Schönherr 1988).
In cuticles lacking unsaturated cutin acids, the contrast after en bloc staining with
OsO4 is probably caused by sorption in cutin, while in Clivia cuticle oxidation of
double bonds may contribute to fine structure. We could not find any study dealing
with changes in chemistry of cutin acids following treatment with OsO4.
KMnO4 is a salt; it has a high water solubility and is used as aqueous solution. It
is a very strong oxidising agent which breaks double bounds and converts alcoholic