A Dictyostelium cell lacking the single PLCδ gene shows no abberrant phenotype at laboratory conditions. Therefore we measured the kinetics and dose-dependencies for survival of the amoebae at various stress conditions, including temperature, pH, osmolarity, and removal of extracellular Ca²⁺. We never observed a significant differences between plc-null and control cells (data not shown), suggesting that Dd-PLCδ is not essential to protect the amoebae. This is in strong contrast to another second messenger enzyme, guanylyl cyclase, which protects Dictyostelium cells against osmotic stress 19.

A common theme in species that have only the PLCδ isoform is the formation of spores or seeds, which can survive extreme environmental conditions. Sporulation to survive stress comes to an evolutionary advantage when germination of spores occurs only at conditions that allow growth of the emerging organism. We investigated the role of PLC in sporulation and germination. During Dictyostelium development about two-thirds of the cells differentiates into viable spores, whereas one-third develops into dead stalk cells. We observed that the proportioning of stalk and spore cells is not different in plc-null fruiting bodies (data not shown). Next, plc-null cells with a Neo marker were mixed 1:1 with wild-type cells containing a Bsr marker conferring resistance to the antibiotics G418 and blasticidin, respectively. Spores were isolated from the resulting fruiting bodies, and inoculated in microtiter plates at low density such that about 40% of the cultures showed growth. Subsequently cultures were transferred to media with G418 or blasticidin, allowing growth of plc-null and wild-type cells, respectively. In three independent experiments we observed that from a total of 101 cultures, 29 did grow in G418, 31 grew in blasticidin, whereas 41 showed growth in both G418 and blasticidin. This experiment demonstrates that the spores have essentially the same plc-null/wild-type ratio as the 1:1 mixture of cells from which they were derived, suggesting that Dd-PLCδ does not play a role in the formation of spores.

Germination of spores is strictly controlled by environmental factors such as pH, osmolarity and temperature 21. Figure 1 shows that the germination rate is nearly identical for wild-type and plc-null spores at 22°C (half times 4.0 ± 0.2 h and 4.2 ± 0.2 h, respectively). Germination at 16°C is reduced considerably for wild-type spores as compared to 22°C (half-time 17.8 ± 2.6 h). In contrast, plc-null spores germinate with almost the same rate at 16°C as at 22°C (half-time 6.6 ± 0.5 h). Similar data were obtained at other temperatures (Table 1). Thus, whereas at 22°C both strains germinate at the same rate, at 13°C plc-null spores germinate 15-fold faster than wild-type spores.

Osmotic pressure and pH of the medium are other environmental factors known to affect spore germination. Germination of wild-type spores is more strongly inhibited by sucrose than germination of plc-null spores (Figure 1B). Addition of 0.3 M sucrose inhibits wild-type germination 7-fold while germination of plc-null spores is retarded only 2-fold (Table 1). At acidic pH, spore germination is inhibited about 25-fold at pH 4.7, 5-fold at pH 5.2, and about 2-fold at the mildly acidic pH of 5.5 (Table 1); no statistical significant differences can be observed between germination of wild-type and plc-null spores. At a slightly alkaline pH up to pH 7.8 spore germination is not strongly affected if compared to the optimal pH, but at more alkaline pH germination is inhibited. As with temperature and osmolarity, germination is significantly more affected for wild-type than for plc-null spores.

To evaluate the effect of PLCδ deletion on survival of the Dictyostelium species, we tested the growth rate of amoebae at stress conditions that influence spore germination. Amoebae grown under optimal conditions were incubated in growth medium at reduced temperature, alkaline or acid pH, or increased osmolarity. The results (Figure 2) show that growth of the amoebae was hardly affected at pH 5.2. In contrast, cells grew about 5-fold slower at 15°C compared to 22°C, while cells did not grow at pH 8.7 or in 0.2 M sucrose. The data reveal no differences in growth rates between wild-type and plc-null cells. A role of Dd-PLCδ in spore germination is consistent with the expression of the Dd-PLC gene, which is absent in the multicellular slug, but reappears to high levels during culmination and formation of spores 9.

A simple model can explain the effect of PLC disruption on spore germination: unfavourable conditions activate PLC resulting in a return to dormancy of food-activated spores; in plc-null cells PLC can not be activated, so activated spores can not return to dormancy. However, we have observed that amoebae lacking PLC activity have near-normal IP₃ levels due to an alternative route of IP₃ formation, obtained from the degradation of IP₅18. Therefore, we have measured IP₃ levels in wild-type and plc-null spores germinating at 22°C and 16°C.

The IP₃ content of plc-null spores is significantly 50% higher than the IP₃ content of wild-type spores (figure 3). The identity of IP₃ as the Ins(1,4,5)P₃ isomer was confirmed using its sensitivity to degradation by specific enzymes (see 18; data not shown). The IP₃ concentration of wild-type spores does not change much during germination at 22°C. However, at 16°C the IP₃ levels decrease 53% at three hours after incubating the spores in growth medium. Subsequently, IP₃ levels return to basal levels, which coincides with the return of activated spores to dormancy. In plc-null spores no significant alteration of the IP₃ concentration occurs during incubation of spores at 22°C or 16°C. These results suggest that unfavourable conditions in wild-type cells induces the inhibition of PLC activity, and that high IP₃ levels are essential to complete germination.

The observation that IP₃ levels decline in wild-type spores at 16°C, but remain constant in plc-null cells, predicts that addition of exogenous IP₃ will induce spore germination of wild-type cells at 16°C. Figure 4 describes the effect of IP₃ on germination of wild-type and plc-null spores at 22°C and 16°C. At a concentration of 100 μM, IP₃ significantly promotes the germination of wild-type spores at 16°C; it has no effect at 22°C or on the germination of plc-null spores. The effect of IP₃ is specific for the active isomer Ins(1,4,5)P₃, because Ins(1,3,4)P₃ and inositol have no effect at this concentration.

Two sets of Dictyostelium cells were used. HD10 (plc-null cells) and HD11 (wild-type control for HD10) are G418 resistant clones in an AX3 background 17. Clone 1.19 (plc-null cells) and 0-mut (PLC expressed in 1.19 using an actin 15 promotor) are transformants in a DH1 background 26. The experiments presented in this report were performed with the combination HD10/HD11; the experiments of figures 1 and 2 were reproduced with the combination 1.19/0-mut.

Spores were isolated from 1 to 3 days old fruiting bodies. After treatment with 0.5% NP-40 for 3 minutes to kill remaining amoebae, spores were washed three times with water, and inoculated at a final density of 1.5 × 10⁶ spores per ml in flasks containing 10 ml medium as indicated. The flasks were shaken (150 rpm) at 22°C or 16°C. At various time intervals the spores were observed by phase contrast microscopy and scored for unswollen spores and amoebae. Germination is defined as the fraction of spores that have emerged as amoebae.

Cell growth was measured in medium as indicated in figure 2 using freshly growing cells from a culture at 22°C in HG5 medium at a density of 2 × 10⁶ cells/ml.

Two days old spores were incubated at a density of 1.5 × 10⁸/ml in HG5 medium at 22°C or 16°C. At the times indicated in figure 3, 20 μl of the suspension was added to 20 μl ice-cold 3.5% (v/v) perchloric acid. After incubation in a sonication bath for 15 min, the lysates were neutralized with 10 μl KHCO₃ (50% saturated at 22°C). IP₃ levels were measured in the extracts using an isotope dilution assay 27.