Comparison of modern and fossil plant cuticles by selective chemical extraction monitored by flash pyrolysis-gas chromatography-mass spectrometry and electron microscopy

Abstract

In order to investigate the preservation processes influencing the occurrence of plant cuticles (and hence leaves etc.) in the fossil record we have undertaken a comparative study of modern and fossil Ginkgo cuticles by chemical and microscopical methods. Cuticles are stripped or released from modern leaf tissue with hydrogen peroxide in aqueous acetic acid. The polysaccharide component and lignin can be selectively removed by acetyl bromide in acetic acid, and the cutin (polyester) by saponification. These treatments reveal the presence of a non-saponifiable residue of a resistant biomacromolecule with a characteristic dominantly aliphatic pyrolysis pattern as demonstrated and named cutan in the prototypical Agave americana cuticle. However, the same chemical treatments of recent Ginkgo biloba, the untreated cuticle of which shows an aliphatic signature upon pyrolysis, results in complete solubilisation of the sample with no resistant residue. The pyrolysis patterns can be clearly related to electron microscopic observations of the cuticles at different stages of chemical treatment. In particular the initial presence and extent of extracuticular cellular material, and its subsequent removal by the acetylation treatment can be visualised and explained. The saponifiable cutin polyester has a structural function even when associated with a resistant biomacromolecule in the Agave americana cuticle because electron microscopy shows that the resistant residue consists only of cuticle fragments. Fossil cuticles of Ginkgo huttonii were examined by Py-GC-MS and electron microscopy. These consist of extensive cuticle sheets on which SEM reveals gross morphology closely comparable to that in the modern cuticle whilst TEM shows that extra cuticular cellular material is lacking but that the outermost amorphous cuticle zone is preserved. An aliphatic pattern of alkene/alkane doublets has been identified in all of the samples. In addition, phenolic compounds have been found in the modern and fossil Ginkgo cuticle. The presence of a series of alkene/alkane doublets and some phenolic compounds in the fossil sample, combined with preservation of outer cuticle morphology and ultrastructure suggests that a less resistant, saponifiable polymer like that in modern Ginkgo cuticle is one possible source of the morphologically-preserved organic matter in the fossil. A highly resistant macromolecule is not responsible for the preservation of fossil Ginkgo cuticles.