Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

Germinating nasturtium pollen (Tropaeolum majus) is shown to excrete an enzyme(s) which hydrolyzes all types of monomers from biosynthetically labeled cutin and p-nitrophenyl esters, which are model substrates for fungal cutinases. The pollen cutinase showed an optimum pH near 6.5 and was inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethyl maleimide but not by diisopropyl-fluorophosphate, an "active serine"-directed reagent indicating that the pollen enzyme is an "-SH cutinase" unlike the fungal enzyme which is a serine cutinase. Excretion of the pollen cutinase into the extracellular fluid was complete within 4 to 6 hours at 30 C. Since actinomycin D and cycloheximide showed little effect on the level of cutinase excreted, it appears that cutinase is an enzyme synthesized prior to germination. Release of cutinase into the medium did not require germination. Electron microscopy revealed the presence of a continuous cutin layer on mature stigma with extensive folds, which are proposed to play a role similar to that played by the cellular papillae found in the stigma of other plants. Chemical analysis of stigma cutin by depolymerization and combined gas-liquid chromatography and mass spectrometry showed that this cutin consists of mainly the C(16) family of acids. The major (70%) components were dihydroxy C(16) acids which consisted of 10,16- (64%), 9,16- (16%), 8,16- (12%), and 7,16- (8%) dihydroxy plamitic acid. Deuterium-labeling studies showed the presence of 16-oxo-9-hydroxy C(16) acid and 16-oxo-10-hydroxy C(16) acid in this cutin. The biochemical and ultrastructural studies indicate that the pollen tube may gain entry into stigma using cutinase excreted by the pollen.