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First published online 23 January 2008
doi: 10.1242/dev.018051

Development 135, 819-827 (2008)
Published by The Company of Biologists 2008
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Cytokinins induce sporulation in Dictyostelium

Christophe Anjard and William F. Loomis*

Center for Molecular Genetics, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0368, USA.


Figure 1
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  		Fig. 1. Sporulation induced by cytokinins. (A) Developed KP cells were incubated with various concentrations of isopentenyl adenine (triangles), discadenine (filled circles) or zeatin (squares). Cells were treated with PKA inhibitors 10 µM H89 (open circles) or 5 µM myristoylated PKI (crosses) 45 minutes prior to addition of isopentenyl adenine. Spores were counted after 1 hour. (B) Developed KP cells were incubated with various concentrations of the artificial cytokinins, thidiazuron (squares), kinetin (open circles) and 6-benzyl-aminopurine (triangles), and the number of spores determined after 1 hour. Each experiment was repeated three to five times.

 

Figure 2
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  		Fig. 2. Time course of spore induction by discadenine. KP cells that had developed at low density in buffer containing cAMP were treated with 100 nM discadenine (circles) or 1 mM adenine (squares). The proportion of cells that became ellipsoid phase-bright spores was determined microscopically over the following hour. The experiments were repeated three times.

 

Figure 3
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  		Fig. 3. Isopentenyl adenine and discadenine production in wild-type and iptA-null strains. Isopentenyl adenine (squares) and discadenine (circles) recovered from wild-type cells (filled symbols) and mutant cells (open symbols) at the indicated times of development. The horizontal line indicates the approximate level of cytokinin required to induce spore formation fully.

 

Figure 4
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  		Fig. 4. Response of sporogenous strains to spore inducers. Mutant strains overexpressing the catalytic subunit of PKA as the result of being transformed with the KP construct were developed as monolayers at 2x103 cells/cm2 for 20 hours. Cells of the indicated mutant strains were then treated with no addition (none), 100 nM discadenine (disc), 100 nM isopentenyl adenine (iP), 1 µM zeatin, 10 pM synthetic SDF-1, 10 pM synthetic SDF-2 or 1 µM GABA. Spores were counted after 1 hour. Cells treated with SDF-1 were scored after 90 minutes. Each experiment was repeated three times. Error bars correspond to one standard deviation.

 

Figure 5
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  		Fig. 5. Response to spore inducers in culminants. Cells of the indicated wild-type and mutant strains were developed on filters and harvested at the mid-culmination stage. The fruiting bodies were dissociated and the cells washed before plating a density of 104 cells/cm2 in cAMP buffer. The cells were then treated with no addition (none), 100 nM discadenine (disc), 100 nM isopentenyl adenine (iP), 1 µM zeatin, 10 pM synthetic SDF-1, 10 pM synthetic SDF-2 or 1 µM GABA. Spores were counted after 1 hour for most factors. Cells treated with SDF-1 were scored after 90 minutes.

 

Figure 6
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  		Fig. 6. Consequences of loss of components in the SDF-2 pathway. Cells of the dhkA-/K, rdeA-/K strains were developed as monolayers at 2x103 cells/cm2 for 20 hours. regA- cells were developed as monolayers at 5x102 cells/cm2 for 20 hours. They were then treated with no addition (none), 100 nM discadenine (disc), 100 nM isopentenyl adenine (iP), 1 µM zeatin, 10 pM synthetic SDF-1, 10 pM synthetic SDF-2 or 1 µM GABA. Spores were counted after 1 hour. Cells treated with SDF-1 were scored after 90 minutes. Each experiment was repeated three times. Error bars correspond to one standard deviation.

 

Figure 7
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  		Fig. 7. Isopentenyl adenine binding to whole cells. (A) Cells of strain AX4 were developed on filters for 22 hours, dissociated, washed and suspended at 2.5x107/ml in binding buffer containing 1.25 mM adenine. 3H-isopentenyl adenine was added to the indicated concentrations and the amount of specific binding determined after 5 minutes at 20°C. (B) Vegetative and 22 hour developed AX4, acrA- and dhkB- cells were suspended at 2.5x107/ml in binding buffer containing 1.25 mM adenine. 3H-isopentenyl adenine (10 nM) was added and the amount of specific binding determined after 5 minutes at 20°C. All experiments were repeated at least three times.

 

Figure 8
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  		Fig. 8. Model for induction of sporulation by cytokinin and SDF-2. Cytokinins activate the histidine kinase DhkB and the adenylyl cyclase AcrA in prespore cells, while SDF-2 binds to the histidine kinase DhkA, such that the cAMP phosphodiesterase RegA is no longer activated. As a result, cAMP accumulates and activates PKA, which triggers encapsulation. PKA activity also leads to the release of the SDF-2 precursor AcbA which is processed by the prestalk specific protease TagC. Glutamate blocks the effect of PKA on AcbA. GABA competes with glutamate for the receptor GrlE. The kinases PI3K and PKBR 1 are essential components of the GABA signal transduction pathway leading to release of AcbA [adapted, with permission, from Anjard and Loomis (Anjard and Loomis, 2006Go)].

 

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