Cytochemical and histochemical characterization of cotyledonary bodies from Pharbitis nil seedlings

Abstract

Cytological and histochemical characterization of 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 eas- ily isolated and purified after enzymatic digestion of the cotyledons. Using different staining procedures we have confirmed that each cotyledonary body originates from an individual mesophyll cell dur- ing embryo development. Purified bodies consist of (i) a thick shell- like envelope; (ii) a transparent, hydrophilic zone; (iii) a hydropho- bic core. Hydrophobic contents of the bodies were readily extracted with methanol and shown to contain fatty acids and phenolic com- pounds 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 flu- orescence microscope we have shown that the bodies isolated from seedlings grown in continuous light, conditions non-inductive for flowering, and those grown under conditions inductive for flowering (a single 16 h, long dark period) have different fluorescence emis- sion spectra. Different levels of free Ca 2+ inside cotyledonary bodies isolated from light-grown and single dark-period treated P. nil seed- lings 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 in- volved in floral induction.