Distribution of Cutan in Modern Leaves - Biopolymers: A Molecular Paleontology Approach - page 21

Fresh leaves

Crushing in liquid N 2

Crushed leaves

Lipid extraction for 8 h in 2:1 CH 2 Cl 2 :CH 3 OH

Py-GC/MS
Microscopy

Extracted Residue 1

Saponification for 24 h
at 70 ° C in 1M Methanolic NaOH

Py-GC/MS
Microscopy

Saponified Residue 2

Acid hydrolysis for 1 h in
CH 3 COOH/ Acetyl bromide*

Acidified Residue 3

Py-GC/MS

*Applied to Agave, Pinus, and Acer to test the acid resistance of constituent biopolymers

Fig. 2.1 Analytical protocol used to detect the presence of cutan in leaves

of NaOH in 0.5 ml double distilled water and 9.5 ml methanol. This solution was
added to Residue 1, which was then refl uxed at 70 °C for 24 h in a reactitherm
(Mösle et al. 1997 ). Thus, the resultant residue (Residue 2) is devoid of constituents
that can be hydrolysed under basic conditions (e.g. cutin). Residue 2 from Agave
americana , Pinus sylvestris , and Acer campestre was further subjected to acid
hydrolysis (Mösle et al. 1997 ) as a control to test the acid resistance of Agave , one
gymnosperm and one angiosperm, as cutan by defi nition should be resistant to acid
treatment as well. The resultant residue (Residue 3) is devoid of constituents hydro-
lysable under basic and acidic conditions (e.g. lignin, cutan) and hence is resistant
to both base and acid hydrolysis.
Untreated leaf, Residue 1 and Residue 2 of modern Quercus robur and Pinus
sylvestris were studied using transmission electron microscopy (as described in
Collinson et al. 1998 ) to determine the effects of base hydrolysis (saponifi cation) on
the leaf cuticle.
Flash pyrolysis-GC-MS was conducted on the residues of the extant leaves after
lipid extraction (Residue 1), and after base hydrolysis (Residue 2) and, where pres-
ent, after acid hydrolysis (Residue 3). Flash pyrolysis involves the thermal fragmen-
tation of the chemical constituents of the sample at high temperatures in an inert gas
stream. These fragments are then separated and identifi ed by gas chromatography-
mass spectrometry. Flash pyrolysis reveals bulk macromolecular information and it
has been used extensively in the molecular characterisation of both modern and
fossil plant tissues (see van Bergen 1999 for review). Samples were analysed with a
Perkin Elmer GC/MS. A CDS (Chemical Data System) AS-2500 Pyroprobe pyroly-
sis unit was used with both the injector and interface temperature at 290 °C. 100-
150

μ

g of tissue sample was introduced into quartz tubes and pyrolysed at 610 °C.