Spore germination and autoinhibitor control in the cellular slime mould Dictyostelium discoideum
Conditions were optimised for the production, germination and emergence of Dictyostelium discoideum V-12 and NC-4H spores, whereas Ax-2 spores failed to germinate under these conditions. The best method of activating spores for germination proved to be heat-shock treatment. Dimethyl- sulphoxide was less efficient; peptone was not effective in this study. Washed and unwashed V-12 and NC-4-H spores attained maximum levels of germination (90-100%), and emergence (80-100%), when incubated under "shake" conditions at concentrations up to 2-5x108 spores ml-1. From this approach there was no evidence for autoinhibitor activity with respect to spore crowding. At extremely high concentrations, 1.5x109 spores ml-1, oxygen deprivation resulted in inhibition of respiration and germination in both washed, and unwashed, NC-4H cultures. Addition back of washings to spores, at concentrations 30-fold higher than those found by extraction, failed to demonstrate any inhibition of germination of NC-4H spores. Inhibition of NC-4H spore germination (50%) was demonstrated with crude spore extract at levels of 10-15mg ml-1 and 100% inhibition of germination at 20mg ml-1. 3mg crude extract per 0.2ml assay inhibited spore germination (107 spores ml-1) by 50% (this is defined as 1 SG1 unit). Although certain physiological factors affected germination, e.g. osmotic pressure, ionic strength and extremes of pH, spore germination inhibitor (SG1) activity appeared to be spore concentration dependent, indicating the presence of a specific substance, rather than an environmental effect. In this study, the SG1 extract, isolated and taken through purification procedures, was characterised as follows :- 1) the substance was acidic in nature, 2) it showed U.V. absorbing characteristics around 260nm, 3) the Rf value was 0.06-0.20 using paper chromatography with n-butanol : acetic acid : water (4:1:2) solvent system, and 4) it was pale yellow in colour and showed slight fluorescence under U.V. (366nm). Purification profiles indicated the SG1 extract NOT 2 to be N2 -dimethylguanosine or discadenine. Although cytokinin activity was present in crude autoinhibitor extracts such activity was not always associated with the SGI on purification. Considering these findings the identity and precise role of autoinhibitors in germination is discussed.