Biosynthesis of Cutin and Suberin
Biosynthesis of the Monomers
The aliphatic monomers of cutin and suberin derive from the general fatty acid biosynthetic pathway, that is, from palmitic (16:0), stearic (18:0), and oleic (18:1) acids synthesized in the plastids of the epidermal cell.The biosynthetic pathway leading to the characteristic cutin monomers had been largely discovered by the group of Kolattukudy in the early 70s (Kolattukudy, 1981). The major cutin monomers are synthesized by multiple hydroxylation and epoxidation reactions. These reactions are catalyzed by oxygen and NADP-dependent enzyme systems that are inhibited by CO, a typical characteristics of cytochrome P450-dependent enzymes. The research on plant cytochrome P450 has advanced much during the recent years (Kahn and Durst, 2000). Different cytochrome P450-dependent enzymes have been characterized that catalyze the internal as well as the o-hydroxylation of fatty acids (Beneviste et al., 1998; Cabello-Hurtado et al., 1998; Pinot et al., 1992, 1998; Tijet et al., 1998). Several of these cytochrome P450-dependent monooxygenases have been cloned, including CYP86A1, CYP94A1, CYP81B1, CYP86A8 (LCR), and CYP86A2 (ATT1) (Beneviste et al., 1998; Cabello-Hurtado et al., 1998; Tijet et al., 1998; Wellesen et al., 2001; Xiao et al., 2004). A function in cutin biosynthsis has been confirmed for LCR and ATT1 (Yephremov, unpublished results) (Xiao et al., 2004). Mutations in ATT1 of Arabidsopsis lead to a 30% loss in cutin and a much looser cuticular ultrastructure (Xiao et al., 2004). Alteration in the monomer composition of residual-bound lipids has been found in lcr plants (Yephremov, unpublished results).
For formation of the cutin monomers, fatty acids leave the plastid after release from the fatty acid synthetase since cytochrome P450-dependent enzymes are located at the endoplasmic reticulum (ER) membrane. Precursors for the unsaturated cutin monomers of Arabidopsis are provided by phospholipids of the ER (Bonaventure et al., 2004). Further details on the mechanism of the hydroxylation reactions have not yet been elucidated, that is, the order of hydroxylations and the substrates for the different enzymes in vivo or other enzymes and cofactors involved. The acyl-CoA synthetase LACS2 has been found to be involved in the synthesis of the cuticular membrane, indicating that changes in the activation status of the precursors of cutin monomers are necessary during cutin biosynthesis (Schnurr et al., 2004). Recombinant LACS2 has a higher activity with 16-hydroxypalmitate than with palmitate (Schnurr et al., 2004).
The very-long-chain fatty acid derivatives of suberin are synthesized by fatty acid elongases that catalyze the elongation of the carbon chain of stearate to different lengths, as found in wax biosynthesis (Domergue et al., 1998). Rootspecific fatty acid elongases have been characterized from maize (Schreiber et al., 2000). The necessary hydroxylation steps may be introduced by cytochrome P450-dependent enzymes. The formation of α,ω-dicarboxylic acids from ω-hydroxyacids is catalyzed by a ω-hydroxy fatty acid dehydrogenase (Agrawal and Kolattukudy, 1978a,b). A cytochrome P450 that oxidizes fatty acids to the corresponding ω-alcohols and subsequently to the α,ω-dicarboxylic acids was described (Le Bouquin et al., 2001). Another possibility may be that HTH/ACE, or one of the closely related proteins, are involved in the formation of α,ω-dicarboxylic acids present in suberin (Kurdyukov et al., 2006a). While the major types of enzymes responsible for the synthesis of aliphatic suberin monomers have been identified, none of them have been shown to be directly involved in suberin biosynthesis.
Formation of the Polyesters
How the cutin and suberin monomers are transported to the place of polymerization is still to be elucidated. On the other hand, an ATP-transporter involved in the transport of wax molecules across the plasmalemma has been identified by cloning the CER5 gene of Arabidopsis (Kunst and Samuels, 2003; Pighin et al., 2004). The very-long-chain fatty acid derivatives of suberin may be transported by a similar mechanism. However, the transport mechanism of cutin monomers also remains to be discovered; cutin monomers have a shorter chain length and much lower hydrophobicity than wax molecules. For cutin formation, an additional transport through the cell wall is required, and this transport step may involve lipoproteins. This model was proposed after proteins with the activity to transport lipids in vitro (lipid-transfer proteins) were localized to the cell wall (Kader, 1996). Although lots of circumstantial evidence for this function of lipidtransfer proteins have been collected, the direct involvement of lipid-transfer proteins in cutin biosynthesis has not yet been substantiated (Hollenbach et al., 1997; Pyee and Kolattukudy, 1995). Instead, recent work has indicated that lipidtransfer proteins act in plant defense against pathogens (Garcia-Olemedo et al., 1995; Maldonado et al., 2002; Molina and Garcia-Olmedo, 1997).In order to form the three-dimensional structure of cutin and suberin, the respective monomers have to be linked together, in part by ester bonds.
Some early studies showed that the cutin monomers bound to CoA as cofactors are transferred to free hydroxyl groups present in the cutin polymer (Croteau and Kolattukudy, 1973, 1975; Kolattukudy, 1981). An hydroxyl-CoA:cutin transacylase activity has been detected in a crude extract that needs ATP for the reaction as well as cutin polymer as a primer. However, the transacylase has not been purified and no gene encoding the enzyme has been identified. A putative acyl-CoA:cutin transferase has been claimed to be purified from Agave epidermis (Reina and Heredia, 2001). After partial protein sequencing, a gene was isolated that encodes a novel small valine-rich protein with a putative HxxxE domain present in other acyltransferases (Reina and Heredia, 2001).No confirmation exists to date, however, that this protein has the proposed function.
BODYGUARD, an enzyme of the α,β hydrolase family, has been found to be critically involved in the formation of the cuticular membrane of Arabidopsis (Kurdyukov et al., 2006b). In the bdg mutant, the cuticular membrane is disrupted and the outer extracellular matrix is disorganized, with polysaccharides coming to the surface and polyester also deposited within the cell wall.
Mutants Affected in Cutin Deposition
The best means for linking enzyme activities and the corresponding proteins and genes to their respective functions is by mutation. The Sorghum bicolor bloomless (bm) mutant was the first mutant identified having a thinner cuticuler membrane as well as a reduced wax deposition. The bm mutant exhibits a higher conductance to water vapor and an increased susceptibility to the fungal pathogen Exserohilum turcicum ( Jenks et al., 1994). Since several aspects of the cuticle were altered in bm, it could not be determined which cuticular component contributes which feature of the cuticle. Furthermore, Sorghum is not a species well suited for map-based cloning of genes.A phenotype that was at first unexpected but found to be related to cuticular changes was organ fusion (Lolle and Cheung, 1993; Lolle and Pruitt, 1999; Lolle et al., 1997, 1998). Support for the idea that a disrupted cuticular membrane structure and/or less cutin lead to organ fusions was originally obtained by an indirect approach using transgenic Arabidopsis plants expressing and secreting a fungal cutinase and therefore degrading their own cutin (Sieber et al., 2000). These transgenic plants show an altered ultrastructure and a higher permeability of the cuticle. When organs having a disrupted cuticular membrane are in close contact early during development, fusions form most likely by cross polymerization. These organ fusions are very strong so that organs do not separate during further growth, leading to distortions of the growth habit of the plant (Sieber et al., 2000). A number of organ fusion mutants were shown to be altered in the cuticular polyester (Kurdyukov et al., 2006a,b). Organ fusions are still used as a selection criterion for mutants having changes in cuticular structure or composition (Yephremov and Schreiber, 2005).
The increasing number of well-characterized Arabidopsis plants having alterations in the cuticular membrane enables some phenotype comparisons to be made. The organ fusion mutant bdg shares most of the phenotypes with cutinaseexpressing plants, such as an increased permeability of the cuticle, higher wax accumulation, ectopic pollen germination, stunted growth, altered trichome formation, and increased resistance to Botrytis cinerea (Kurdyukov et al., 2006b; Sieber et al., 2000). The characteristic difference in the structure of the cuticular membranes between cutinase-expressing plants and bdg mutants, namely, that bdg accumulates, in addition, large amounts of osmophilic material deeper within the cell wall, lead to the hypothesis that BDG acts directly in the formation of the extracellular matrix, as discussed above (Kurdyukov et al., 2006b).
The analysis of the molecular basis underlaying the obvious differences in composition, structure, and function will surely be of great interest during the next years (Nawrath, 2006).
WAX2/YORE-YORE is an Arabidopsis protein with six-membrane spanning domains having homology to the sterol desaturase family at the N-terminus and the short-chain dehydrogenase/reductase family at the C-terminus as well as having an overall homology to CER1, a protein required for wax deposition of unknown function in Arabidopsis (Chen et al., 2003; Kurata et al., 2003). In contrast to the other mutants having a looser cuticular membrane structure and loss of cuticular membrane material, wax2/yore-yore has, in addition, a reduced wax deposition (Chen et al., 2003; Kurata et al., 2003). Other phenotypes of the wax2/ yore-yore mutants are typical for cutin mutants, such as an increased permeability of the cuticularmembrane, disorders in the development of epidermal cell types, and organ fusions (Chen et al., 2003). Thus,WAX2 plays a critical role in the synthesis of both cuticular components, cutin and wax.
ABNORMAL LEAF SHAPE (ALE1) is a subtilisin-like protease that is involved in the regulation of the formation of the cuticle in embryos and juvenile plants in Arabidopsis (Tanaka et al., 2001). ale1 mutants have a disrupted cuticular membrane in embryos, cotyledons, and juvenile leaves. The leaves of ale1 are crinkled, often have organ fusions, and are very susceptible to low humidity, resulting in conditional lethality. Interestingly, ALE1 is expressed in certain endosperm cells adjacent to the embryo, as well as in the young embryo, and may be essential for the separation of the two entities (Tanaka et al., 2001).
CRINKLY4 (CR4) is a receptor kinase with homology to tumor-necrosis factor receptors that is involved in proper epidermal formation that has first been identified in maize (Becraft et al., 1996). CR4 mutants have organ fusions as well as abnormal epidermal cell wall and cuticle deposition (Jin et al., 2000). ACR4, the CR4 homologue in Arabidopsis that is expressed in the outer cell layers of embryos and mature plants, has similar function in epidermal differentiation and cuticle development as CR4 (Tanaka et al., 2002; Watanabe et al., 2004). ALE1 and ACR4 affect synergistically the differentiation and function of the epidermis, since ale1/acr4 double mutants have a stronger phenotype than do both single mutants (Watanabe et al., 2004).