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First published online 23 January 2008
doi: 10.1242/dev.018051
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Center for Molecular Genetics, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0368, USA.
* Author for correspondence (e-mail: wloomis{at}ucsd.edu)
Accepted 24 December 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Discadenine, Isopentenyl adenine, Zeatin, Histidine kinase, SDF-2, Sporulation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Loomis, 1975). As each spore
is surrounded by a cellulose reinforced protein coat, prespore cells cannot
move once they are encapsulated. Premature encapsulation results in spores
that cannot reach the top of the stalk. Therefore, the timing of sporulation
must be carefully controlled.
Sporulation of dispersed cells of a strain (KP) with partially constitutive
PKA activity has been shown to be density dependent when they are developed on
the bottoms of multi-test wells (Anjard et
al., 1997). At densities higher than 104
cells/cm2, a phosphopeptide of about 1.2 kDa accumulates in the
buffer. When this peptide, SDF-1, is purified and added back to KP cells
developing at lower density, it induces sporulation 90 minutes later in a
process that requires protein synthesis
(Anjard et al., 1997). SDF-1
accumulates in fruiting bodies of wild-type cells where it is found together
with several other factors that can also induce encapsulation of KP cells
developed at low density. GABA, produced by the enzyme glutamate decarboxylase
(GadA) induces sporulation at 1 nM concentration
(Anjard and Loomis, 2006). When
GABA binds its receptor GrlE, a G-protein-coupled receptor of the
GABAB metabotropic receptor-like family, it triggers the rapid
release of the precursor of a second peptide factor, SDF-2, that can also
induce encapsulation in test cells. SDF-2 is a 34 amino acid peptide cleaved
from the secreted precursor AcbA (acyl-CoA binding protein)
(Anjard and Loomis, 2005).
SDF-2 binds the receptor histidine kinase DhkA and inhibits phosphorelay to
the internal cAMP phosphodiesterase RegA, resulting in a decrease in its
activity. The internal concentration of cAMP can then increase and activate
PKA, which leads to rapid encapsulation of prespore cells
(Anjard and Loomis, 2005).
Strains in which the genes encoding either AcbA or DhkA are disrupted
sporulate poorly but can sometimes reach 60% of the wild-type level of spores,
suggesting that there may be other sporulation signals. Disruption of the gene
encoding another histidine kinase, DhkB, reduces the proportion of spores to
about one-third when culmination is first completed
(Zinda and Singleton, 1998).
The number of viable spores in dhkB- strains decreases
after 25 hours of development such that it is only 3% of wild-type levels by
72 hours, apparently because the spores germinate while still on top of the
stalk. Zinda and Singleton (Zinda and
Singleton, 1998) suggested that dhkB- mutant
cells were unable to respond to the germination inhibitor, discadenine, to
maintain dormancy. DhkB may also play a role in initiating sporulation as
double mutants lacking both DhkA and DhkB are much more impaired in spore
formation than either of the single mutants lacking only one of these
histidine kinases (Wang et al.,
1999). The almost complete lack of sporulation in
dhkA- dhkB- strains suggests that
sporulation may result from the combined activity of both histidine kinases.
The adenylate cyclase AcrA is also required for sporulation and spore dormancy
as the null strain is impaired in spore formation
(Soderbom et al., 1999). Most
of the spores that form in acrA- null strains fail to
remain dormant and rapidly germinate. AcrA is a membrane associated adenylyl
cyclase that carries a degenerate histidine kinase domain of unknown function.
The region of the domain involved in phospho-transfer in other histidine
kinases has multiple variations in key amino acids in AcrA and is probably not
functional, whereas the two receiver domains in AcrA are well conserved and
could accept a phosphate group transferred by one of the 13 functional
histidine kinases existing in Dictyostelium
(Anjard and Loomis, 2002).
Cytokinins are N6 substituted adenine derivatives that affect
growth and development of plants by activating two-component phosphorelay
pathways (Mok and Mok, 2001;
Kakimoto, 2001;
Kakimoto, 2003;
Rashotte et al., 2006). There
are three different receptor histidine kinases in the mustard Arabidopsis
thaliana: AHK2, AHK3 and AHK4/CRE1
(Inoue et al., 2001; Nishumura
et al., 2004; Suzuki et al.,
2001; Yamada et al.,
2001). Cytokinins bind to a conserved extracellular loop of about
200 amino acids found in each of these receptors, referred to as the CHASE
domain (Anantharaman and Aravind,
2001; Heyl et al.,
2007). Discadenine is a derivative of the cytokinin isopentenyl
adenine, which is synthesized by condensation of isopentenylpyrophosphate and
5'AMP followed by removal of the ribose phosphate group
(Abe et al., 1976;
Taya et al., 1978). Both
isopentenyl adenine and discadenine can be extracted from
Dictyostelium fruiting bodies and shown to inhibit germination when
added to washed spores at levels above 1 µM
(Tanaka et al., 1978;
Ihara et al., 1980) (D.
Cotter, personal communication). Moreover, discadenine shows cytokinin
activity in an assay using tobacco callus cells
(Nomura et al., 1977). We have
found that both discadenine and isopentenyl adenine, as well as other
cytokinins, induce rapid sporulation in Dictyostelium in a process
that is dependent on DhkB and AcrA. The cytokinin signaling pathway is
independent of the SDF-2 pathway but both converge at the level of activation
of PKA through an increase of intracellular cAMP.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Zeatin and isopentenyl adenosine were purchased from Acros Organics (Geel,
Belgium). Isopentenyl adenine, other cytokinins and adenine were purchased
from Sigma (St Louis, MO). Synthetic DL-discadenine was a kind gift from Dr
David Cotter. Labeled N6-isopentenyl [2-3H]adenine, 1
TBq/mmol, >97% pure, was purchased from Isotope Laboratory of the Institute
of Experimental Botany (Prague, Czech Republic).
H89 and myristoylated PKI (14-22) were purchased from Calbiochem (San
Diego, CA). The catalytic subunit of D. discoideum PKA is sensitive
to these inhibitors (Anjard et al.,
1993; Anjard et al.,
1997).
Strains and bioassay
The wild-type strain AX4, the pkaC overexpressing strain KP and
its derivative dhkA-/K have been previously described
(Anjard et al., 1992;
Anjard et al., 1998a). The
acgA- strain was a kind gift from Pauline Schaap
(van Es et al., 1996). To
generate the dhkB-/K strain, KP cells were transformed
with the construct used in the original disruption of dhkB
(Zinda and Singleton, 1998).
After selection with blasticidin and sub-cloning, dhkB disruptants
were identified using specific primers. More than 90% of the clones presented
the expected pattern for dhkB disruption.
In order to disrupt iptA (DDB0233672; GenBank XP 642693), a 1.5 kb
fragment was amplified by PCR and cloned in the pGEMT vector (Promega A1360).
The BSR cassette from pBSR519 (Puta and
Zeng, 1998) was cloned into the unique EcoRI site located
at codon 35 of the iptA. For gene disruption, 10 µg of the plasmid
was linearized with NotI before electroporation into 107
AX4 cells. Disruption of the endogenous gene in transformants was confirmed by
PCR using primers located outside the cloned sequences.
The bioassay was carried out on KP cells and their derivatives after 18
hours development in monolayers as previously described
(Anjard et al., 1998a). Cells
were incubated in buffer (20 mM MES pH 6.2, 20 mM NaCl, 20 mM KCl, 1 mM
MgSO4, 1 mM CaCl2) at a density of
2x103 cells/cm2 in the wells of a 24-well dish at
23°C. After 18 hours incubation, samples or defined products were added
and the number of spores and undifferentiated cells were counted 1 hour
(SDF-2) or 2 hours (SDF-1) later. The amounts of SDF-1 and SDF-2 activity were
determined by serial dilution of the sample before addition to KP cells. One
unit corresponds to the lowest dilution, giving full induction of spore
formation. The number of units in the sample were standardized to
103 producing cells when applicable. The cell density of monolayers
of regA- cells in the wells had to be reduced to
5x102 cells/cm2 to reduce the level of spontaneous
sporulation in the absence of added signals.
The response of cells from strains that are not sporogenous to sporulation
inducers was measured following dissociation of culminants that had developed
on filters (Anjard and Loomis,
2005). Filters (25 mm diameter) were each spread with
107 cells and allowed to develop for 20 hours at 22°C. Each
filter was then examined under a dissecting microscope. Only those filters
where most of the structures were similar were used and any asynchronously
developing culminants were removed from these filters with a needle. The cells
were allowed to continue to develop and monitored every 15 minutes. When
stalks became apparent under the rising sori, the cells were collected by
vortexing the filters in 1 ml cAMP buffer followed by centrifugation at 4000
rpm for 1 minute in a microfuge. The cells were counted and diluted to
3.6x104/ml. Because the window of development during which
induction of sporulation can be assayed is only 15-30 minutes, only
preparations that contained between 10% and 20% spores were used. Suspension
(500 µl) was added to each well of a 24-well plate, resulting into a cell
density of 104/cm2. Inducing compounds were added at
various concentrations and the number of spores counted after 1 hour.
Expression of iptA in bacteria
The coding sequence of iptA was amplified by PCR using
oligonucleotides that included a NcoI restriction site at the
5' end and a XhoI site at the 3' end. The coding sequence
was cloned in pGEMT-EASY (Promega A1360) and sequenced. The
NcoI-XhoI fragment was cloned into pET32a using the
NcoI-XhoI restriction sites. This plasmid was introduced
into E. coli BL21 DE3 and transformants selected. An overnight
culture of BL21 DE3 transformed with pET32a-iptA or an empty vector were
diluted 1/100 into 50 ml LB containing 50 µg/ml carbenicillin and grown to
an OD600 of 0.4 before induction with 1 mM IPTG. The bacteria were
incubated on a shaker at 37°C for another 4 hours. For the bioassay, 1 ml
aliquots of the cultures were harvested and the bacteria pelleted by
centrifugation at 14,000 rpm in an Eppendorf Microfuge for 1 minute. The
supernatants were then tested for sporulation induction on the KP cells.
For determination of isopentenyl adenine production, 20 ml aliquots of the induced bacterial cultures were collected and mixed with 80 ml ethanol. Insoluble material was pelleted by centrifugation at 12,000 g for 30 minutes. The supernatants were dried under vacuum and resuspended in 10 ml water before addition of 1 ml of 50% Amberlite XAD-2 (Supelco, Bellafonte, PA). After 30 minutes incubation, the resin was spun down and washed twice with 10 ml water. Isopentenyl adenine was eluted by three successive additions of 2 ml of 30% ethanol. The eluates were pooled, dried under vacuum and resuspended in 100 µl methanol before analyses by HPLC/MS (see below).
Development and spore viability
Cell were grown in axenic media (HL5) at 22°C in shaking culture
(Sussman, 1987). Development
was initiated by harvesting exponentially growing cells at a density of 2-5
106/ml. Cells were washed with PDF buffer [20 mM Na/K phosphate (pH
6.5), 20 mM KCl, 1.2 mM MgSO4], centrifuged again at 1000 rpm for 5
minutes and resuspended at a density of 1-2x108 cells/ml in
PDF before being deposited on nitrocellulose filters placed on pads saturated
with PDF (Anjard and Loomis,
2005).
For spore viability assays, 107 washed cells were deposited on a
small filter and developed for 24 hours. Spores were collected by placing the
filter in an Eppendorf tube with 1 ml PDF containing 0.5% Triton X-100 and
briefly vortexed. Spores were incubated for at least 5 minutes in the buffer
containing 0.5% Triton X-100 and then centrifuged at 6000 rpm for 1 minute and
resuspended in 1 ml PDF. After counting and dilution, 50 spores were plated in
triplicate on SM plates with a K. aerogenes suspension
(Anjard and Loomis, 2005). The
number of plaques, corresponding to the number of viable spores, was scored
after 4-5 days of incubation at 22°C. Spore viability assays were repeated
at least three times.
Isopentenyl adenine binding assay
Vegetative cells were centrifuged at 1200 rpm for 5 minutes and washed
twice in 10 ml binding buffer [50 mM phosphate buffer (pH 7.5), 200 mM NaCl]
per 108 cells. Developed cells were generated by depositing
5x107 to 108 cells on 4.5 cm diameter filters and
incubating at 20°C until the fruiting bodies reached the
early-mid-culmination stage (
22 hours). Fruiting bodies were collected on
a spatula and washed three times in binding buffer before the cells were
resuspended at density of 2.5x107/ml in binding buffer
containing 1.25 mM adenine. Protein concentration of the suspension was
determined using the BioRad reagent.
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Isopentenyl adenine and discadenine quantitation
Isopentenyl adenine and discadenine were purified from developed
Dictyostelium cells using a simplification of the protocol of Taya et
al. (Taya et al., 1980). Cells
(108) of each strain were developed on filters for the indicated
times, harvested with a spatula and resuspended in 10 ml water. Cells were
pelleted by centrifugation at 2000 rpm for 5 minutes and the supernatants were
incubated for 10 minutes with 1 ml of Amberlite XAD-2 resin. The resin was
collected by centrifugation at 1000 rpm for 5 minutes and washed twice in 10
ml water. Cytokinins were eluted from the resin by three successive additions
of 1 ml 30% ethanol which were pooled and dried under vacuum before being
resuspended in 100 µl of methanol. Between 2.5 and 25 µl of the samples
were loaded on a Majic C-18 Column (ID 1 mm x 150 mm) column using an
ultrafast HPLC apparatus (Microm BioResources) coupled to a LCQdeca-Mass
spectrometer with electrospray ionization source (ESI) under positive ion mode
(ThermoFinnigan). The LC mobile phase A was 2.5% methanol in water and the LC
mobile phase B was pure methanol. The LC flow rate was 50 µl/minute, and
the LC gradient was 10% B to 95% B in 20 minutes then held at 95% B for three
minutes. Isopentenyl adenine gives a characteristic [M+H+] peak at
m/z 204 and is eluted after about 17 minutes in this gradient. Discadenine
gives a [M+H+] peak at m/z 305 and is eluted after about 3 minutes.
The product identities were further confirmed by ESI-MS/MS analysis. Upon
ESI-MS/MS fragmentation, isopentenyl adenine gives characteristic daughter
peaks at m/z 136 and m/z 148 respectively, while discadenine gives a daughter
peak at m/z 204 (data not shown). Known amounts of isopentenyl adenine and
discadenine standards were run under identical conditions for quantitation of
the samples using select ion monitoring.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Taya et al.,
1980). It appears that discadenine and isopentenyl adenine not
only function to maintain spore dormancy but also act earlier to induce
sporulation. Zeatin, a plant cytokinin not found in Dictyostelium, induces rapid encapsulation but only when added to about 100-fold higher concentration (Fig. 1A). The artificial cytokinins, thidiazuron, kinetin and 6-benzyl-aminopurine, which are not found in either plants or Dictyostelium but are as effective as the natural cytokinins in plants, also induce rapid sporulation in Dictyostelium when present at more than 10 µM (Fig. 1B). Induction of sporulation by cytokinins appears to act through PKA as addition of either of the specific inhibitors, H89 or myristoylated PKI, blocks sporulation in response to isopentenyl adenine (Fig. 1A).
The number of spores started to increase within 10 minutes of addition of discadenine and reached maximum at 45 minutes (Fig. 2). There was no response to 1 mM adenine which is not a cytokinin. Simultaneous addition of discadenine and SDF-2 did not increase the proportion of spores (data not shown).
iptA encodes isopentenyl-transferase which generates cytokinin
The first step dedicated to the biosynthesis of cytokinins is catalyzed by
isopentenyl-transferase and results in the condensation of isopentenyl
pyrophosphate with AMP, ADP or ATP (Taya
et al., 1978; Ihara et al.,
1984). After dephosphorylation, the ribose moiety is removed to
generate isopentenyl adenine. Isopentenyl adenine is also generated by
post-transcriptional addition of an isopentenyl group to adenosine groups in
tRNA. Most bacteria and animals have a single gene encoding
isopentenyl-transferase to modify their tRNAs. Plants, however, have multiple
isopentenyl-transferases (Takei et al.,
2001; Miyawaki et al.,
2006). Dictyostelium has three isopentenyl-transferases
genes, iptA, iptB and iptC. A phylogenic analysis of the
corresponding proteins showed that IptB and IptC are closely related to the
eukaryotic and bacterial isopentenyl-transferases that modify tRNAs, while
IptA clusters with enzymes involved in cytokinin synthesis (data not
shown).
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), whereas the levels in iptA- cells were
less than 10%. Assuming that the cytokinins are uniformly distributed, we
calculated that the threshold concentration of 10 nM cytokinin, sufficient to
fully induce spore formation, corresponds to about 2 pmol/mg protein in our
assay. Wild-type cells reach this level between 22 and 24 hours of
development, whereas iptA- cells only reach it after 30
hours of development, long after encapsulation is usually completed. As less
than half of the iptA- spores were viable after detergent
treatment, whereas wild-type spores are completely detergent resistant (see
Table S1 in the supplementary material), it appears that these cytokinins play
significant roles in triggering efficient sporulation. The defect in iptA- cells appears to be non-cell autonomous as developing them together with an equal number of wild-type cells resulted in improved sporulation (see Table S1 in the supplementary material). However, developing them together with an equal number of acbA- cells, which cannot make the SDF-2 precursor, resulted in only a modest increase in the number of spores. All of these strains except acbA- accumulated comparable levels of SDF-2 in their sori (see Table S1 in the supplementary material).
Induction of sporulation by cytokinins depends on DhkB and AcrA
When spores are collected from wild-type fruiting bodies and incubated at
high density, they are inhibited from germinating by the presence of the
germination inhibitor (Nomura et al.,
1977). Disruption of the gene encoding the histidine kinase DhkB
was found to result in spores that germinate in the presence of discadenine
(Zinda and Singleton, 1998).
We found that dhkB- K cells, developing at low density,
fail to differentiate into spores in response to discadenine or other
cytokinins but are still able to respond to SDF-1, SDF-2 and GABA just as well
as wild-type KP cells (Fig.
4).
Histidine kinases relay phosphate to proteins carrying response regulator
regions resulting in modulation of activity. The adenylyl cyclase of late
development, AcrA, carries two response regulatory regions that might be
targets for DhkB (Anjard et al.,
2001). In support of this notion, we found that cells lacking AcrA
failed to respond to discadenine, isopentenyl adenine or zeatin, although they
responded normally to the other sporulation inducers
(Fig. 4). If the adenylyl
cyclase activity of AcrA is stimulated when its response regulatory regions
are phosphorylated, the resulting increase in cAMP would lead to high levels
of PKA activity necessary for rapid encapsulation.
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), we
introduced a construct that results in partially constitutive PKA activity
into an acaA- strain. The resulting
acaA-/K strain is able to make fruiting bodies but does
not have a sufficiently sporogenous phenotype that we could test cells of this
strain developing at low cell density for responses to cytokinins. Therefore,
we dissociated cells from early culminants that had developed on filter
supports and stimulated them with different spore inducing factors. Cells
lacking ACA responded to discadenine, zeatin, SDF-2 and GABA just as well as
cells dissociated from early culminants of wild-type AX4 cells
(Fig. 5). Likewise, cells of
the acgA- strain dissociated from early culminants
responded well to cytokinins (Fig.
5). It appears that DhkB-dependent induction of encapsulation by
cytokinins acts exclusively through the specific adenylyl cyclase that carries
response regulatory regions, AcrA, while SDF-2 induction of encapsulation is
not dependent on any specific source of cAMP. The response to SDF-1, however,
is dependent on ACG (Fig.
5).
Induction of sporulation by cytokinins is independent of DhkA, RdeA and RegA
SDF-2 induces rapid encapsulation by inhibiting the relay of phosphate from
DhkA to the cAMP phosphodiesterase RegA via the H2 intermediate RdeA
(Thomason et al., 1999;
Anjard and Loomis, 2005).
Cytokinins might be indirectly inducing sporulation by stimulating SDF-2
production, much as GABA induces sporulation by triggering the release of AcbA
and its processing to SDF-2 (Anjard and
Loomis, 2006). The resulting SDF-2-dependent inhibition of RegA,
rather than activation of the GABA signal transduction pathway, is responsible
for the rapid increase in PKA activity that leads to encapsulation. To
determine whether cytokinins also induce rapid sporulation by the SDF-2
pathway, we used dhkA- KP cells in the low density
monolayer assay. The mutant cells lack the SDF-2 receptor and so fail to
encapsulate in response to either SDF-2 or GABA, although they do respond to
SDF-1 (Anjard and Loomis, 2005;
Anjard and Loomis, 2006). The
responses to discadenine, isopentenyl adenine and zeatin were normal in
dhkA- KP cells, indicating that the cytokinins induce
rapid sporulation independently of SDF-2 signaling
(Fig. 6).
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Neither DhkB nor AcrA is responsible for binding isopentenyl adenine
Either DhkB or AcrA could be a cytokinin receptor as they are large
proteins with several potential transmembrane domains. AcrA has two predicted
extracellular loops of about 35 amino acids each flanked by transmembrane
domains near the N terminus that might bind cytokinins. However, DhkB has no
predicted extracellular loops of more than 10 amino acids separating the
transmembrane domains (Zinda and
Singleton, 1998). We determined the ability of whole cells to bind
3H-isopentenyl adenine in the presence of 1 mM adenine to reduce
non-specific background binding. Cells collected from wild-type culminants
were incubated at 20°C for 5 minutes with various concentrations of
3H-isopentenyl adenine in the absence or presence of 10,000 fold
excess unlabelled isopentenyl adenine. The cells bound
3H-isopentenyl adenine with an apparent Kd of 6 nM
(Fig. 7A). The concentration
dependence for binding was very similar to the concentration dependence in the
bioassay (Fig. 1). We then
tested vegetative and developed wild-type cells, as well as developed
acrA- and dhkB- cells at 10 nM
3H-isopentenyl adenine (Fig.
7B). Vegetative cells bound very little of the cytokinin,
indicating that the receptor is developmentally controlled. However, the
acrA- and dhkB- mutant cells bound as
much 3H-isopentenyl adenine as wild-type cells, indicating that
neither DhkB nor AcrA is likely to be the isopentenyl adenine receptor.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Nevertheless, Dictyostelium uses a histidine kinase, DhkB, in the
cytokinin signal transduction pathway leading to encapsulation of spores. The
use of cytokinins as hormones apparently predates the divergence between
Dictyostelium and plants over a billion years ago.
The cyanobacteria Anaebena species genome includes two isopentenyl
transferases, one being similar to IptA. Moreover, Anaebena encodes
an adenlylyl cyclase, cyaC, with the same unusual domain organization
as AcrA, starting with a receiver domain followed by a histidine kinase domain
and another receiver domain to finish with the adenylate cyclase domain
(Katayama and Ohmori, 1997).
This suggests that components of the cytokinin pathway might have appeared in
bacteria that predated eukaryotes but were lost in animal and fungal
lineages.
As the cytokinin response in Dictyostelium is mediated by the
histidine kinase DhkB and the adenylyl cyclase AcrA to the exclusion of other
histidine kinases or adenylyl cyclases, these proteins may have a close
association. Moreover, considering that the only known H2 component, RdeA, is
not required for the cytokinin response, DhkB may directly relay phosphate to
the aspartates in the response regulator regions of AcrA. Histidine kinases
are known to form dimers in which the phosphate on the active histidine is
transferred to an aspartate in the receiver domain
(Posas et al., 1996;
Wang et al., 1999). DhkB and
AcrA may form a heterodimer in which DhkB directly activates adenylyl cyclase
by phosphorelay. AcrA has a pseudo-histidine kinase domain in which the
replacement of the active histidine by aspartate precludes it from
autophosphorylation (Soderbom et al.,
1999). However, this domain may have been retained on the basis of
its ability to form a heterodimer with DhkB.
The cytokinin receptors are known in plants to be histidine kinases that
bind cytokinins through an extracellular CHASE domain
(Anantharaman and Aravind,
2001). This domain is found in only two Dictyostelium
proteins, DhkA and ACG, but neither appears to be a cytokinin receptor as null
mutants lacking these proteins respond normally to cytokinins (Figs
5,
6). Because neither DhkB nor
AcrA appear to account for isopentenyl adenine binding, further studies will
be required to recognize the Dictyostelium cytokinin receptor.
The cyctokinin response could be an artifact of the bioassay as we used
cells that overexpress PKA developed as monolayers or cells dissociated from
culminants to assess rapid sporulation. However, iptA-
cells, in which synthesis of isopentenyl adenine is compromised in an
otherwise wild-type background, are impaired in sporulation, indicating the
importance of cytokinin signaling during normal development. Moreover, DhkB
has been shown to play a direct role in determining the intracellular levels
of cAMP during culmination as cAMP levels have been found to be significantly
reduced in dhkB- spores
(Zinda and Singleton,
1998).
Cytokinins can be produced either through a dedicated pathway or from the
degradation products of tRNA (Kakimoto,
2003). Primitive plants like the moss Physcomitrella
patens seem to use only the tRNA-IPT pathway for cytokinin production
(Yevdakova and von Schwartzenberg,
2007). In Dictyostelium, the inactivation of
iptA results into a 5- to 10-fold reduction in isopentenyl adenine
during culmination. The remaining isopentenyl adenine is probably generated
from degraded tRNA. Discadenine levels are even lower in
iptA- cells (Fig.
3). However, when iptA- cells are allowed to
form fruiting bodies within clearings of a bacterial lawn, they can accumulate
25% as much discadenine as wild-type spores (data not shown). As these cells
had been actively ingesting bacteria under these conditions, discadenine may
have been derived from the degradation products of bacterial tRNA.
SDF-2 stimulates spore formation at about the same time as cytokinins
(Anjard et al., 1998b).
However, SDF-2 acts by removing the activating phosphates from RegA in the
pathway involving RdeA and DhkA (Fig.
8). As the RegA cAMP phosphodiesterase activity drops, cAMP can
accumulate and stimulate PKA. At the same time cytokinins activate an
independent pathway such that cAMP is rapidly synthesized by AcrA. This dual
control is similar to pushing the accelerator and releasing the brake on cAMP
accumulation (Fig. 8).
As both SDF-2 and cytokinins are released at about the same time of
development (t=22 hours), it is possible that their production is
coordinated. However, cytokinins do not seem to play an essential role in
SDF-2 production as iptA- cells accumulate SDF-2 normally
(see Table S1 in the supplementary material). Moreover, addition of cytokinins
to developed KP cells does not result in the accumulation of SDF-2. Likewise,
SDF-2 is not essential for cytokinin release as acbA-
cells produce as much cytokinin as wild-type cells (data not shown). Cytokinin
production is dependent on the activity of isopentenyl-transferase, which
appears only during culmination (Ihara et
al., 1980). Thus, the timing of cytokinin production may be
mediated by the regulation of iptA. The timing of SDF-2 production
may be set by the time of accumulation of GABA, which triggers release of the
SDF-2 precursor AcbA (Anjard and Loomis,
2006). The enzyme that synthesizes GABA, GadA, accumulates only in
prespore cells late in development and its regulation may determine the time
of appearance of SDF-2. No matter how accumulation of SDF-2 and cytokinin are
coordinated, both pathways need to be activated at about the same time to
obtain maximal efficiency of sporulation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/5/819/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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