Suberin depolymerization

The only thorough characterization of mixtures of depolymerized suberin components (dep-suberin) was carried out by us in a comprehensive investigation of this material. The opaque pasty samples were obtained by the alkaline methanolysis of Quercus suber L. cork [8,76]. Under the conditions used for the depolymerization, most of the carboxylic acid functions were therefore converted into the corresponding methyl esters. 

Fig. 6.4.5. Methods used to analyze the aliphatic components of suberin. Top left chemical methods used to depolymerize suberin. Top right gas-liquid chromatogram of the mixture of monomers generated by LiAlD4 treatment of suberin from the chalazal region of the inner seed coat of Citrus paradisi the components are trimethylsilyl ethers of 1,16-dihydroxyhexadecane (1), 1,18-dihydroxy-octadecene (2), 1,9,18-trihydroxyoctadecene (3), 1,9,18-trihydroxyoctadecane (4), 1,9,10,18-tetrahy-droxyoctadecane (5), 1,22-dihydroxydocosane (6), 1,24-dihydroxytetracosane (7). Bottom mass spectrum of component 2 from gas chromatogram (111)...

Depolymerization techniques that cleave ester bonds release the indicated aliphatic monomers and phenolic components from suberin. 

High-resolution 13C NMR studies have been conducted on intact cuticles from limes, suberized cell walls from potatoes, and insoluble residues that remain after chemical depolymerization treatments of these materials. Identification and quantitation of the major functional moieties in cutin and suberin have been accomplished with cross-polarization magic-angle spinning as well as direct polarization methods. Evidence for polyester crosslinks and details of the interactions among polyester, wax, and cell-wall components have come from a variety of spin-relaxation measurements. Structural models for these protective plant biopolymers have been evaluated in light of the NMR results. 

Suberized Cell Walls. An analogous set of CPMAS experiments is presented for suberin in Figure 6. Because this polymer is an integral part of the plant cell wall, the 13C NMR spectrum had contributions from both polysaccharide and polyester components. Chemical-shift assignments, summarized in Table IV, demonstrated the feasibility of identifying major polyester and sugar moieties despite serious spectral overlap. Semiquantitative estimates for the various carbon types indicated that, as compared with cutin, the suberin polyester had dramatically fewer aliphatic and more aromatic residues. A similar observation was made previously for the soluble depolymerization products of these plant polymers (1,8,11). 

Figure 6. 31.94 MHz 13C NMR spectra for suberized cell walls from potatoes, before (bottom) and after (top) depolymerization treatment. The experimental parameters were as in Figure 4. Chemical-shift assignments and relative numbers of carbons for the untreated material are found in Table IV. Delayed-decoupling experiments left some (CH2) signal intensity in the spectrum of intact suberin, but the analogous signals were drastically attenuated in the NMR spectrum of the depolymerization residue.

Preliminary structural studies of cutin and suberin breakdown involved examination of 13C NMR spectra for insoluble residues that were resistant to chemical depolymerization. In cutin samples, flexible CH2 moieties in particular were removed by such treatments, but CHOCOR crosslinks and polysaccharide impurities were retained preferentially. A concomitant narrowing of NMR spectral lines suggested that the treatments produced more homogeneous polyester structures in both cases. Our current studies of cu-ticular breakdown also employ selective depolymerization strategies with appropriate enzymes (1,28). 

Suberin is tightly associated with the cell wall and cannot be removed in a pure form. The outer layer is usually removed physically and then treated with pectinase, cellulase and organic solvents to yield an enriched fraction. The crude suberin must then be subjected to chemical treatment in order to depolymerize it for chemical analysis (references in Kolattukudy, 1980). Crude suberin preparations have also been isolated from endodermis (Robards et al.y 1976) and hypodermis-epidermis (Clarkson et al.y 1978) but the amounts obtained from these sources are usually too small for meaningful analyses to be performed. 

TLC is commonly used for the separation of different classes of wax components or for analysis of monomers from cutin and suberin depolymerization. A typical separation is shown in Fig. 6.12. By such methods, it is possible to separate hydrocarbons, wax esters, primary alcohols, secondary alcohols and /8-diketones from plant waxes (von Wettstein-Knowles, 1979). Products of hydrogenolysis from cutin can be separated by TLC into alkan-l-ols, alkane-a,ft>-diols, Cis triols, Ci6 triols and Cis tetrols (Kolattukudy, 1980). Unsaturated components can be resolved by argentation-TLC (Tulloch, 1976) and threo or erythro diastereoisomers separated by boric acid/silica gel TLC (Eglinton and Hunneman, 1968). Straight-chain compounds can be preferentially removed from branched compounds as their urea complexes (Kolattukudy, 1980). 

Hydroxy acids are major aliphatic components of cutin and suberin and these are readily identified by GLC-mass spectrometry. Major ions generated from the usual cutin components are listed in Kolattukudy (1977). The position of the hydroxyl group in the chain is easily seen because cleavage occurs on either side of the substituent (Fig. 6.13). The rather simple phenolic compounds yielded by reductive depolymerization of cutin and suberin are also very... 

The present chapter will, therefore, first give a general overview of the properties and applications of cork, as well as of its utilization as a starting material for the synthesis of liquid polyols, before dealing with the macro-molecular structure of suberin, its depolymerization methods, and the composition and applications of the ensuing fragment mixtures. 

The suberin polymeric structure cannot be defined in terms of a monomer repeating unit, since the spatial arrangement of these moieties cannot be accurately defined, even when their relative abundance is known. Additionally, the latter aspect depends on the depolymerization methods used to isolate the aliphatic fraction and, moreover, as far as the aromatic domain is concerned, its identification/quantification is also quite complex because of its macromolecular nature and structural similarity with lignin [1, 42-45]. Notwithstanding these difficulties, several models have been proposed to illustrate the suberin macromolecular structure in suberized cell walls [46, 47]. In 2002, Bernards proposed a model for suberin from potato periderm, which summarized the state of the art on the structural data related to this macromolecular component [1]. 

The depolymerization of suberin through ester cleavage is in general a key step both for composition analysis (usually carried out by GC-MS), and to isolate monomers for further chemical transformation. Depending either... 

Little has been published on the use of the suberin depolymerization products as monomers for the synthesis of novel macromolecular materials, which has so far concentrated on polyurethanes and polyesters using the mixture of aliphatic monomers. 

Benitez et al. [82], recently reported the synthesis of a polyester resembling cutin, a natural polymer whose structure is close to that of aliphatic suberin [83], by a circular approach, which consisted in depolymerizing cutin through ester cleavage and then submitting the ensuing monomer mixture to a chemical polyesterification process. 

Fernando G, Zimmermann W, Kolattukudy PE (1984) Suberin-grown Fusarium solani f. sp. pisi generates a cutinase like esterase which depolymerizes the aliphatic components of suberin. Plant Pathol 24 143-155... 

Table 6.4.6. Major components released by the depolymerization of suberin polymers from the bark of various trees and shrubs. 

Family Genus/species Suberin content Depolymerization products Reference(s)... 

Fig. 6.4.8. Cross-polarization/magic-angle spinning NMR spectra of cutin from the fruit peel of Malus pumila suberin from the bark of Pseudotsuga menziesiiy suberin from the periderm of the tuber of Solarium tuberosum and the residue left after L1A1H4 depolymerization of S. tuberosum suberin. (Spectra courtesy of Regional NMR Center, Fort Collins, Colorado.)...

Suberin is laid down in various internal locations in order to seal off specific regions of the plant (234). In the primary development of plant roots, suberin is deposited in the Casparian band of endodermal cells (45, 103, 162, 317, 364, 367, 369, 474, 483). The presence of suberin in the Casparian band of the endodermis of Sorghum bicolor was shown chemically by depolymerization (244). Suberized layers are also found in the mestome sheath and bundle sheath cell walls of grasses (52, 61, 108, 118, 171, 172, 331, 368), and these layers have been chemically characterized in Zea mays and Secale cereale (114, 146). 

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