M. H. Lopes

A 13C SOLID STATE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPIC STUDY OF CORK CELL WALL STRUCTURE : THE EFFECT OF SUBERIN REMOVAL

Solid state 13C NMR measurements of cork, before and after suberin removal, showed that aliphatic suberin is spatially separated from carbohydrate and lignin and experiences higher motional freedom. Two types of chain methylenes, differing in chemical shift and in dynamic properties, were identified in aliphatic suberin. Experimental evidence indicated that the more motionally hindered methylenes are those situated nearer the linkages of aliphatic suberin to the cell wall. These linkages were shown to involve -CH2O- groups, probably engaged in ester linkages to phenylpropane units and carbohydrate C6 carbons. Spectral intensity changes indicated that, during the first steps of alkaline desuberization, these linkages are broken and the shorter aliphatic suberin chains removed. Longer chains require hydrolysis of the ester linkages within the chains and are removed upon stronger alkaline treatment. T1(C), T1 rho (H) and T1 rho (C) relaxation times have shown that the removal of suberin from cork leads to a motionally restricted and more compact environment, on the megahertz and mid-kilohertz timescales. The properties of cork suberin showed that suberin organization in cork is distinct from that in potato tissue.

Spectral editing of 13C CP/MAS NMR spectra of complex systems: application to the structural characterisation of cork cell walls

A mathematical method of obtaining 13C CP/MAS subspectra of single components of a complex system is presented and applied to three- and four-component systems. The method is based on previously reported work that exploits different proton relaxation properties for different domains of an heterogeneous system. However, unlike the original method that obtained subspectra through a trial-and-error approach, the method here presented solves the problem mathematically, thus avoiding the time-consuming and non-rigorous trial-and-error step. The method is applied to mixtures of three and four polymers and to a more complex system: cork cell walls. As expected, as the number of components increases, the sharing of relaxation properties between different components is increasingly probable, either due to incidental coincidence of relaxation times or to specific interactions and intimate mixing of compounds. While this hinders the calculation of the subspectra of single chemical components, it may provide useful information about inter-component interactions. This possibility was demonstrated by the application of this method to cork cell walls. Both three-component and four-component approaches showed that three domains exist in cork cell walls: carbohydrate/lignin matrix, mobile suberin close to (probably bonded to) lignin groups (about 42% w/w) and hindered suberin close to (probably bonded to) carbohydrate-OCH2O groups (about 4% w/w).

An NMR microscopy study of water absorption in cork

NMR Microscopy is used to measure the imbibition of water into natural cork, extractives-free cork and desuberised cork. The results clearly indicate that suberin is the key constituent which determines the ability of cork to resist water uptake. Furthermore, a particular suberin with distinct spectral properties as viewed by 13C NMR is shown to be the component responsible for cork resistance to water absorption. Laser confocal microscopy suggests that this function is associated with the role of suberin in preserving cell wall structure but the highly hydrophobic nature of suberin may also play an important role. The NMR microscopy study shows that the water absorbed by natural cork, after soaking for three days, is confined to the lenticels, narrow channels on the order of 1000 to 1500 μm in diameter. One incidental outcome is the observation of a clear down-field shift in NMR frequency for water near the cut transverse surfaces of the cork, an effect~associated with susceptibility inhomogeneity.

Very high-resolution 1H MAS NMR of a natural polymeric material

The use of ultrafast magic angle spinning (> 30 kHz) in tandem with delayed echo acquisition is shown to yield very high-resolution lH MAS NMR spectra of complex natural organic materials. For the first time, very high-resolution 1H MAS NMR spectra are reported for cork and wood components, two natural materials with great economic importance. The effect of the spinning rate on the 1H NMR spectra was evaluated with single-pulse acquisition and delayed-echo acquisition. The delayed-echo acquisition spectra presented linewidths as sharp as 67 and 25 Hz. The narrow peaks, characterised by proton spin-spin and spin-lattice relaxation, were assigned to the isotropic chemical shifts and the general spectral features were shown to correlate with the sample chemical structure. The tentative assignments of cork 1H MAS NMR signals were presented.

Langmuir monolayers of fractions of cork suberin extract

The wide variability in composition and molecular weight of natural polymers has hampered understanding of their physicochemical properties and ultimately their use in noble applications, especially in the cases where surface properties need to be probed at the molecular level. A useful approach to analyse data from surface monolayers of complex mixtures is to try distinguishing the effects from the distinct fractions in such mixtures. The cork suberin extract investigated here is known to contain aliphatic monomers with terminal carboxylic acid and methyl ester groups as well as long esterified aliphatic chains dispersed in a polymeric aliphatic matrix. The role of such terminal groups was studied and the results showed that depending on the nature of the terminal groups the monolayers present distinct isotherms due to the different interactions with the water subphase. Fractionation strategies based on different solubilities of the cork suberin components in chloroform were also employed to probe their effect on the monolayer characteristics. From the two sets of experiments it is clear that the presence of monomers with terminal carboxylic acids in the suberin extract affects considerably the monolayer-forming ability. This approach may be used as a complementary, relatively simple route to assess suberin genetic engineering strategies towards resistance to environmental stress.