Analysis of Extracellular Cell Wall Lipids: Wax, Cutin, and Suberin in Leaves, Roots, Fruits, and Seeds.

Extracellular lipids of plants can be analyzed using gas chromatography and mass spectrometry. Soluble waxes are extracted with chloroform and thus separated from the extracellular polymers cutin and suberin. Cutin and suberin have to be depolymerized using boron trifluoride-methanol or methanolic HCl before analysis. The released monomeric hydroxylated fatty acids are then extracted with chloroform or hexane. Prior to gas chromatography, all free polar functional groups (alcohols and carboxylic acids) are derivatized by trimethylsilylation. Internal standards, that is, long chain alkanes, are used for the quantification of wax molecules and cutin or suberin monomers. Lipids are quantified using gas chromatography coupled to flame ionization detection. Qualitative analysis is carried out by gas chromatography coupled to mass spectrometry. Thus, all wax molecules of chain lengths from C16 to C60 and different substance classes (fatty acids, alcohols, esters, aldehydes, alkanes, etc.) or all cutin or suberin monomers of chain lengths from C16 to C32 and different substance classes (hydroxylated fatty acids, diacids, etc.) can be analyzed from one sample.