Composition of Cutin and Suberin

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).