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

1.2 Cutin Composition 7
cells had obtained their maximum area. Initially, cutan mass increased slowly up
to 8–9 cm, but later its mass increased more rapidly and at 19–20 cm it was higher
than the mass of ester-cutin. Ester cutin reached its maximum mass at 11–13 cm;
thereafter it decreased, showing that ester cutin was converted in part to cutan.
Following transesterification the lipophilic cutin monomers are recovered with
organic solvents like chloroform. Polar compounds released by transesterification
are lost, since they remain in the reaction residue, which is discarded. Due to this
experimental approach, it was overlooked in all previous analyses of cutin composition
that glycerol, a small and highly polar organic molecule, is also released and
forms an important cross-linker in the MX (Graca et al. 2002).
Polypeptides (Schönherr and Huber 1977), aromatic compounds (Hunt and Baker
1980) and carbohydrates (Wattendorff and Holloway 1980; Dominguez and Heredia
1999; Marga et al. 2001) are significant although minor constituents of the MX. The
question arises as to whether they have any specific functions in the MX or if their
presence is accidental. Nothing is known about the nature and the origin of the proteins.
Their presence in amounts of about 1% has only been shown by amino acid
analysis (Schönherr and Huber 1977) or CHN analysis (Schreiber et al. 1994) of
isolated cuticles. It is not known whether proteins in the MX are structural proteins
with functional stabilising properties like extensins in plant cell walls. Alternatively,
it can be suggested that enzymes involved in the polymerisation of the MX (cutin
esterases) were trapped during polymer formation in the MX.
More rational explanations are available for the presence in the MX of about
20–40% of carbohydrates, mainly pectin and cellulose. The outer epidermal cell
wall and the cuticle on top of it must be connected to each other in some way. There
is evidence that this connection can be by direct covalent links of sugar molecules
to cutin molecules (Fang et al. 2001), and in addition cellulose fibrils extending
into the MX network may contribute to this connection. It is obvious that a significant
amount of polar functional groups on the inner physiological side of the
MX is protected from enzymatic digestion by the cutin polymer. This can easily be
demonstrated by testing the wettability with water of the physiological outer and
inner surfaces of the MX. Contact angles on the physiological outer side are around
90 ◦ , indicating a surface chemistry of methyl and methylene groups, as should
be the case with a polymer mainly composed of aliphatic monomers (Holloway
1970). Quite different from the outer side, the physiological inner side of the MX is
wet by water, and droplets spread. This indicates a highly polar surface chemistry,
presumably composed of hydroxyl and carboxyl groups from cellulose and pectin
fibrils.
It is also evident that crystalline cellulose has a fundamental function within
the amorphous cutin polymer, acting as a stabiliser which strongly affects the
biomechanical properties of cuticles. Volume expansion of pure cutin, obtained by
hydrolysis of carbohydrates, is much higher than expansion of the MX (Schreiber
and Schönherr 1990). Unfortunately, immunological techniques with antibodies
directed versus specific epitopes of cell wall carbohydrates have not yet been carried
out. This type of study should allow mapping with high precision the chemical