|Content of Pathways for the Synthesis of Polyesters in Plants: Cutin, Suberin, and Polyhydroxyalkanoates
Cutin and suberin consist of fatty acid derivatives, phenolic compounds, and
glycerol. In most plants, cutin consists mainly of hydroxy- and epoxy-hydroxy
fatty acids of 16 and 18 carbons as well as a very small portion of phenols.
In contrast, suberin also contains very long-chain fatty acid derivatives, a high
proportion of dicarboxylic acids, and a large fraction of phenols (Kolattukudy,
1981). The main components of suberin are largely defined to different subdomains
in the polymer, a polyphenol domain that is part of the primary cell wall
and an aliphatic domain close to the plasmalemma (Bernards and Lewis, 1998).
It has been suggested that only the aliphatic polyester domain should be called
suberin (Grac¸a and Pereira, 1997).
Cutin can be obtained in relative pure form by separation of the cuticular
membrane from the cell wall by enzyme digestion and subsequent solvent extractions
to remove the wax fraction. Suberin, as part of the primary cell wall, cannot
be isolated in pure form, except from cork oaks (Rocha et al., 2001). The polyesters
can be depolymerized by typical procedures cleaving ester bonds, for example,
alkaline hydrolysis, transesterification with methanol containing boron trifluoride
or sodium methoxide, as well as reductive cleavage with lithium aluminum
hydride (Kolattukudy, 1981; Walton and Kolattukudy, 1972). The liberated monomers
may either be first methylated or are directly converted into trimethylsilyl
derivatives before subjecting them to gas chromatography/mass spectrometry
(GC/MS). The monomers are identified by their characteristic fragmentation
pattern (Walton and Kolattukudy, 1972).
Cutin may be formed by either hydroxylated C16 fatty acids (C16 class), or by
epoxy or hydroxy C18 fatty acids (C18 class), with many cuticles having a mixed
composition with different proportions of both monomer classes. The characteristic
cutin monomers of the C16 class are 9,16- or 10,16-dihydroxypalmitic acids.
Other C16 monomers
present in cutin are palmitic acid, ω-hydroxypalmitic acid,
and dihydroxypalmitic acid having the mid-chain hydroxy group at other positions.
The characteristic monomers of the C18 cutin are 9,10,18-trihydroxystearic
acid and 9,10-epoxy,18-hydroxystearic acid. Other cutin monomers of this type are
stearic acid, o-hydroxystearic acid, and some unsaturated isologs of these monomers
(Kolattukudy, 1981). Minor monomers may also be other fatty acids, fatty
alcohols, aldehydes, ketones, dicarboxylic acids as well as hydroxycinnamic acids.
However, the cutin of Arabidopsis was found to be rich in dicarboxylic acids, in
particular unsaturated C18-dicarboxylic acids, and 2-hydroxy acids up to 26
carbons in length, revealing a monomer composition that is closer to that of
suberin than to that of a canonical cutin (Bonaventure et al., 2004; Franke et al., 2005; Xiao et al., 2004). Cutin may thus have a larger plasticity in composition
within the plant kingdom than earlier expected (Nawrath, 2006). Glycerol is
present in cutin to varying amounts between 1% and 14% (Graça et al., 2002).
Partial depolymerization by calcium oxide-catalyzed methanolysis led to the
identification of 1- and 2-monoacylglyceryl esters (Graça et al., 2002). Interestingly,
the different types of glyceryl esters found do not always correspond to the
relative proportions of the hydroxylated fatty acids present in the polyester.
Some monomers also seem to be excluded from the glyceryl esters’ formation,
for example, epoxy fatty acids (Graça et al., 2002). Thus, glycerol may contribute
substantially to the three-dimensional structure of cutin, implying that the
previous models based primarily on the inter-esterification of hydroxy and
epoxy-hydroxy fatty acids need to be revised.
A non-hydrolysable core remains after the hydrolysis of cutin. This non-ester
fraction contains a network of aliphatic compounds linked by ether bonds in
which linolenic acid is preferentially incorporated (Villena et al., 1999). Whether
this fraction should still be called cutin or should be named cutan is still under
discussion (Kolattukudy, 1996).
Suberin contains significant amounts (roughly one third) of monomeric hydroxycinnamic
acids, such as ferulic, cinnamic, p-coumaric, or caffeic acids, in addition to aliphatic compounds and, in some species, (poly)hydroxycinnamates, like
feruloyl tyramine (Bernards, 2002; Bernards and Lewis, 1998; Kolattukudy, 1981;
Schreiber et al., 1999). The aliphatic portion of the polymer consists of five
dominant substance classes: o-hydroxy fatty acids (C16–C28), α, ω-dicarboxylic
acids (C16–C26), very-long-chain carboxylic acids, primary alcohols (C18–C30),
and 2-hydroxy fatty acids (Kolattukudy, 1981; Schreiber et al., 1999). Glycerol is
a principal monomer (20%) of suberin in oak, cotton, and potato (Graça and
Pereira, 2000a,b; Moire et al., 1999). Partial methanolysis with calcium oxide as
catalyst has identified that glycerol may be present as mono-acylglycerol esters of alkanoic acids, α,ω-diacids, and ferulic acid, as well as diglycerol esters being
linked to a α,ω-diacid at both ends (Graça and Pereira, 2000b,c). Thus, the
hypothesis has been proposed that glycerol and a,o-diacids may form the backbone
of the suberin polymer, implicating that suberin is a poly-(acylglycerol)-
polyester (Graça and Pereira, 2000c). Glycerol may also cross-link the aromatic
and aliphatic suberin components, while aliphatic and aromatic suberin monomers
may only form a linear polymer on their own (Moire et al., 1999). A revised
model for suberin has been developed including the new compositional and
structural data obtained for potato suberin (Bernards, 2002). That work also
gives an excellent overview of the synthesis of the polyphenol domain of suberin,
which is not subject of the present chapter (Bernards, 2002).