Isolation and Identification of Oligomers from Partial Degradation of Lime Fruit Cutin

Abstract

Complementary degradative treatments with low-temperature hydrofluoric acid and methanolic potassium hydroxide have been used to investigate the protective biopolymer cutin from Citrus aurantifolia (lime) fruits, augmenting prior enzymatic and chemical strategies to yield a more comprehensive view of its molecular architecture. Analysis of the resulting soluble oligomeric fragments with one- and two-dimensional NMR and MS methods identified a new dimer and three trimeric esters of primary alcohols based on 10,16-dihydroxyhexadecanoic acid and 10-oxo-16-hydroxyhexadecanoic acid units. Whereas only 10-oxo-16-hydroxyhexadecanoic acid units were found in the oligomers from hydrofluoric acid treatments, the dimer and trimer products isolated to date using diverse degradative methods included six of the seven possible stoichiometric ratios of monomer units. A novel glucoside-linked hydroxyfatty acid tetramer was also identified provisionally, suggesting that the cutin biopolymer can be bound covalently to the plant cell wall. Although the current findings suggest that the predominant molecular architecture of this protective polymer in lime fruits involves esters of primary and secondary alcohols based on long-chain hydroxyfatty acids, the possibility of additional cross-linking to enhance structural integrity is underscored by these and related findings of nonstandard cutin molecular architectures.

Apoplastic Diffusion Barriers in Arabidopsis

“…The cuticle forms a continuum with the cell wall and may be covalently linked to the cell wall (Sitte and Rennier, 1963;Jeffree, 2006;Tian et al, 2008).…”

“…In addition, partial polymerization followed by liquid state NMR or GC/MS provides information about the linkages between different monomers within the polymer (Graça et al, 2002;Graça and Santos, 2006;Tian et al, 2008;Graça and Lamosa, 2010). However, these techniques require quantities of material that cannot be easily obtained in Arabidopsis.…”

“…In cutin, the amount of aromatic components is very low, but this proportion is higher in suberin (Kolattukudy, 2001b;Pollard et al, 2008;Beisson et al, 2012). For both cutin and suberin, the presence of ester linkages between ω-hydroxyacids and glycerol has been confirmed by nuclear magnetic resonance (NMR)-and by gas chromatography-mass spectrometry studies (GC/MS)-based studies (Graça and Santos, 2006;Tian et al, 2008) In addition, linkages between polyhydroxy acids and cell wall sugars have been tentatively identified in cutin (Tian et al, 2008). Lignin, which is a polymer of aromatic components, is usually seen as a structural polymer that is deposited in secondary cell walls (Boerjan et al, 2003).…”

Regulation of cuticle formation during fruit development and ripening in ‘Newhall’ navel orange ( Citrus sinensis Osbeck) revealed by transcriptomic and metabolomic profiling

“…The previous study provides us important information of the genes putatively involved in cuticle formation in citrus fruits by cDNA microarray coupled with laser microdissection [24]. Besides, extensive studies have been focused on the component analysis of cuticle in citrus fruits [25,26]. However, cuticle is established during development and there has been no report of a comprehensive study of cuticle development in citrus fruits.…”

Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization

“…Linear dimers were identified after partial alkaline hydrolysis of tomato (Solanum lycopersicum) cutin (Osman et al, 1995). NMR and mass spectrometry analyses of oligomers released after partial depolymerization revealed primary and secondary ester linkages between cutin monomers (Graça et al, 2002;Stark and Tian, 2006) as well as covalent linkages between some cutin OH fatty acids and oligosaccharides (Tian et al, 2008). However, it has been shown that partial hydrolysis does not necessarily release all of the representative building blocks of the entire polymer (Deshmukh et al, 2003).…”