No abstract

Germinating nasturtium polen (Tropaeolum majus) is shown to excrete an enzyme(s) which hydrolyzes all types of monomers from biosyntheticafly labeled cutin andp-nitrophenyl esters, which are model substrates for fungal cutases. The poUen cutinase showed an optimum pH near 6.5 and was inhibited by thiol-directed reagents such as phydroxymercurnbenzoate and N-ethyl maleimide but not by diisopropylfluoropbosphate, an "active serine"-directed reagent indicating that the polen enzyme is an "-SH cutinase" unlike the fungal enzyme which is a serine cutinase. Excretion of the pollen cutinse into the extracelular fluid was complete within 4 to 6 bourn at 30 C. Since actinomycin D and cyclohexinide 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 celular papifine found in the stigma of other plants. Chemical analysis of stigma cutin by depolymerization and combined gas-liquid chromatogrphy and mass spectrometry showed that this cutin consists of mainly the C,6 family of acids. The major (70%) components were dihydroxy C,6 acids which consisted of 10,16-(64%), 9,16-(16%), 8,16-(12%), and 7,16-(8%) dihydroxy palmitic acid. Deuterium-labeling studies sbowed the presence of 16-oxo-9-hydroxy C,6 acid and 16-oxo-10-hydroxy C,6 add in this cutin. The biochemical and ultratctrl studies indicate that the poUen tube may gain entry into stigma using cutinase excreted by the pollen.tal evidence for the presence of cutinase in germinating pollen consisted of the demonstration that addition of cutin preparation to germinating pollen caused a slight increase in titratable acidity (7,18). With this increase as a measure of cutinase, it was found that the presence of cutinase in germinating pollen was a characteristic of plants which have ' cutinized" stigma. Since fungal extracts also catalyzed release of titratable acids in the presence of cutin, including a cutin preparation from stigma surface, it was suggested that cutinase of germinating pollen was "identical with" cutinase detected in molds (7). Cutinase from neither molds nor pollen had been isolated or characterized and, therefore, it was not possible to compare the enzymes from the two sources. Recently, fungal cutinases, isolated in homogeneous form, have been characterized (22, 23. 25). The products released from cutin by the germinating pollen were also not characterized and therefore it was difficult to determine whether the pollen enzyme released all types of monomers of cutin or only the peripheral fatty acids.In this paper we demonstrate, with the use of radioactive cutin, that germinating pollen from nasturtium excrete an enzyme(s) which releases all types of monomers from apple cutin. Results which suggest that the appearance of this cutinase activity...