Cytochemical and histochemical characterization of cotyledonary bodies from Pharbitis nil seedlings

Cytological and histochemical characterization oi the structures from which an obscure substance is secreted via open stomata to the abaxial surface of Japanese morning glory (Pharbitis nil Choisy cv. Violet) cotyledons has been carried out. Observation of intact cotyledons using the light microscope revealed randomly distributed semi-transparent structures. These structures, which were shown to be the same as those previously described as giant oil cells are referred to here as cotyledonary bodies. These bodies can be easily isolated and purified after enzymatic digestion of the cotyledons. Using different staining procedure we have confirmed that each cotyledonary body originates from an individual mesophyll cell during embryo development. Purified bodies consist of (i) a thick shell-like envelope; (ii) a transparent, hydrophilic zone; (iii) a hydrophobic core. Hydrophobic contents of the bodies were readily extracted with methanol and shown to contain fatty acids and phenolic compounds using the gas chromatography/mass spectrometry (GC/MS) technique. Methanolic extracts of cotyledonary bodies showed high fluorescence with two excitation and emission maxima. Using a fluorescence microscope we have shown that the bodies isolated fi om seedlings grown in continuous light, conditions inductive for flowering, and those grown under conditions inductive for flowering (a single 16 h, long dark period) have different fluorescence emission spectra. Different levels of free Ca2+ inside cotyledonary bodies isolated from light-grown and single dark-period treated P. nil seedlings were found using the fluorescent calcium indicator dye Fluo-3 under a confocal scanning laser microscope. On the basis of these observations we speculate that cotyledonary bodies could be involved in floral induction.