Regulation of multiple tip formation by caffeine in cellular slime molds

ABSTRACT: BACKGROUND: The multicellular slug in Dictyostelium has a single tip that acts as an organising centre patterning the rest of the slug. High adenosine levels at the tip are believed to be responsible for this tip dominance and the adenosine antagonist, caffeine overrides this dominance promoting multiple tip formation. RESULTS: Caffeine induced multiple tip effect is conserved in all the Dictyostelids tested. Two key components of cAMP relay namely, cAMP phosphodiesterase (Pde4) and adenyl cyclase-A (AcaA) levels get reduced during secondary tip formation in Dictyostelium discoideum. Pharmacological inhibition of cAMP phosphodiesterase also resulted in multiple tips. Caffeine reduces cAMP levels by 16.4, 2.34, 4.71 and 6.30 folds, respectively in D. discoideum, D. aureostipes, D. minutum and Polysphondylium pallidum. We propose that altered cAMP levels, perturbed cAMP gradient and impaired signalling may be the critical factors for the origin of multiple tips in other Dictyostelids as well. In the presence of caffeine, slug cell movement gets impaired and restricted. The cell type specific markers, ecmA (prestalk) and pspA (prespore) cells are not equally contributing during additional tip formation. During additional tip emergence, prespore cells transdifferentiate to compensate the loss of prestalk cells. CONCLUSION: Caffeine decreases adenyl cyclase--A (AcaA) levels and as a consequence low cAMP is synthesised altering the gradient. Further if cAMP phosphodiesterase (Pde4) levels go down in the presence of caffeine, the cAMP gradient breaks down. When there is no cAMP gradient, directional movement is inhibited and might favour re-differentiation of prespore to prestalk cells.     

Isolation and some physiological properties of natural plant growth inhibitors

Using paper chromatography and conventional methods of isolation, natural growth inhibitors were isolated from green leaves of different plants (Brassica oleracea, Zea mays, Pisum sativum andSalix rubra). All isolated inhibitors were found to be phenolic compounds and the chemical structure of most of them was determined; only the final structure of theBrassica inhibitor has not yet been ascertained. 500 mg of natural inhibitor ofPisum sativum was isolated from 1500 g of leaves and was identified as quercetin-glucosil-p-coumarate (QGC), described earlier byFuruya, Galston andStowe (1961). The structure of the natural inhibitor ofZea mays (4 mg from 100 g of leaves) was identical with p-coumaric acid and the chemical nature of the plant growth inhibitor fromSalix rubra (700 mg from 1,5 kg of leaves and young bark) was that of 2-chalconaringenin-glucoside or isosalipurposide, described earlier byCharaux andRabaté (1931) andHarborne (1966). All isolated substances had inhibiting properties in the straight growth test of wheat coleoptile sections and decreased the growth of isolated stem sections prepared from plants—donors of inhibitors. Thus, maximum growth inhibition (LG max) was attained, if wheat coleoptile sections were incubated with:Brassica inhibitor in the concentration of 0·5 mg/ml, withPisum inhibitor (QGC) in the concentration of 16 mg/ml, withZea inhibitor (p-coumaric acid)—0·35 mg/ml and with Salix inhibitor (isosalipurposide) in the concentration of 0·5 mg/ml. In small concentrations no mentioned substances were able to enhance the growth as actively as indolic auxins (on 250–300%); only slight growth activation in biotests was sometimes observed for low concentrations. Inhibition in p-coumaric acid was much more active in a free form than in the bound form as an acyl-rest of QGC. As a rule, the wheat coleoptile test was much more sensitive (3–5 times) to the plant growth inhibitors, than tests prepared from tissue and organs of plants—donors. The retardation activity of plant growth inhibitors is not correlated with their molecular weight. Dormin (or±abscissin II) was also tested on wheat coleoptile sections. In neither of the applied concentrations (10-0·05 μg/l range) was dormin able to depress straight growth of wheat coleoptile sections, but even in a 1·7 μg/l concentration it inhibited the IAA-activated growth of sections. However, additional experiments showed that dormin in higher concentrations (40 μg/l and more was able even to depress endogenous straight growth of wheat coleoptise sections. The differences between the properties of natural phenolic growth inhibitors and dormin were discussed.