Compositional Analysis of Cutin in Maize Leaves

The cuticle is a lipid barrier that covers the air-exposed surfaces of plants. It consists of waxes and cutin, a cell wall-attached lipid polyester of oxygenated fatty acids and glycerol. Unlike waxes, cutin is insoluble in organic solvents, and its composition is typically studied by chemical depolymerization followed by monomer analysis by gas chromatography (GC). Here, we describe a method for the chemical depolymerization of cutin in maize leaves and subsequent compositional analysis of the constituent lipid monomers. The method has been adapted from protocols for cutin analysis developed for <i>Arabidopsis</i>, by both optimizing the amount of leaf tissue used and including a data analysis process specific to the monomers present in maize cutin. The approach uses base-catalyzed transmethylation, which produces fatty acid methyl esters, and silylation, which gives trimethylsilyl ether derivatives of hydroxyl groups for gas chromatographic analysis. For monomer identification, a few representative samples are first analyzed by GC-mass spectrometry (GC-MS). This is then followed by analysis of all replicates by gas chromatography coupled to a flame ionization detector (GC-FID) for monomer quantification, because the flame ionization detector provides a linear response over a wide mass range, is relatively simple to operate, and is more cost-effective to maintain compared to mass spectrometry detectors. Although the protocol bypasses time-consuming cuticle isolation steps by using whole-leaf samples, this means that a fraction of the compounds in the chromatographic profiles do not derive from cutin. Accordingly, we discuss some considerations for the interpretation of the resulting depolymerization products. Our protocol offers specific guidance on preparing maize leaf samples, ensuring reproducible results, and enabling the detection of subtle variations in cutin monomer composition among plant genotypes or developmental stages.