4. CYTOCHEMICAL LOCALIZATION AND REGULATION OF ACID
4.3 RESULTS
4.3.6 Fluorescent microscopy
149 Table 4.3: Summary of analysis by EDX. Mycobionts were grown in different concentrations of Pi (0.05 vs. 10 mM). The cells were fixed in 2% glutaraldehyde as explained in Section 4.2.2. They were then cut into thin sections by ultra microtome. This Table corresponds with the scan obtained in Figures 4.5-4.6
control Area 1 Area 2
Element Weight
%
Atomic% Element Weight% Atomic% Element Weight
%
Atomic
%
C K 80.97 86.33 C K 76.69 84.15 C K 68.54 78.35
O K 16.32 13.06 O K 18.03 14.86 O K 19.82 17.01
Si K 0.26 0.12 Si K 0.28 0.13 Si K 0.19 0.09
P K 0.25 0.10 P K 0.67 0.29 P K 0.07 0.03
Cl K 0.75 0.27 Cl K 0.99 0.37 Cl K 0.79 0.31
Pb M 1.19 0.07 Pb M 3.34 0.21 Pb M 2.38 0.16
Totals 100.00 Totals 100.00 100.00 100.00
150 Figure 4.7: (A-E) Lichen thallus visualized using fluorescence microscope. Controls were included to differentiate between natural autofluorescence (blue) and ELF-97 (green) visualized under UV light excitation. (A) Lichen thallus viewed under UV light, (ELF-97 substrate omitted). (B) Lichen thallus with ELF-97 substrate. (C) Bright field image of algal cells (Trebouxia). (D) Mycelium with no substrate viewed under UV light. (E) Mycelium with ELF substrate (F) Algal cells with ELF-97, viewed under UV light.
The mycelium grown under different concentrations of Pi (0.05, 1.0, 3.0,10 and 100 mM) were incubated with ELF-97 substrate, using 100 mM CASC buffer, pH 2.5. It must be noted that no direct quantification was made amongst treatments, but the microscopy settings were similar.
This was done by mounting a small patch of mycelium consisting of all three treatments on the same slide. Treatments were not mixed. A strong fluorescence was detected in the axenic mycelium at the lowest concentrations (0.05, 1.0 and 3.0 mM) (Figure 4.8A-D). At the lowest concentration, it was observed that the bigger clusters of mycelium did not fluoresce but it appeared blue as did the untreated control (Figure 4.8A-B). At 10 mM Pi, a strong fluorescent signal was detected compared to 100 mM Pi, where ELF granules appeared very sparse under the microscopy settings. In addition, when 10 mM NaF was used as an inhibitor, no apases were C B
A
F E
D
151 detected, whereas a strong fluorescence was detected in the presence of 5.0 mM levimisole.
These results confirm that the phosphatase activity observed was due to an acid phosphatase rather than an alkaline phosphatase.
Figure 4.8: (A-F) Axenic mycelium grown under different concentration of Pi (0.05, 1.0, 3.0, 10 and 100 mM KH2PO4) incubated with ELF-97 viewed by fluorescence microscopy. (A) A control mycelium, devoid of ELF-97 substrate. (B) Fungi grown under 0.05 mM Pi, with smaller patch displaying strong intensity of ELF-97 signals. (C-D). Mycelium grown at 1.0 and 3.0 mM treatments displaying almost similar reactions to ELF-97. (E) Mycelium grown under 10 mM Pi displaying granules of ELF-97 substrates. (F) Mycelium grown at 100 mM with ELF-97 signals appearing as sparse granules. (G) Mycelium (0.05 mM) co-labelled with ELF-97 and propidium iodide. (H) Mycelium (0.05 mM) co- labelled with ELF-97 and acridine orange. (I) Mycelium (100 mM) co-labelled with ELF-97 and Propidium iodide.
A B C
I H
G
D E F
152 To ascertain the viability of the mycelium, ELF-97 was co-stained with either propidium iodide or acridine orange dye (usually use to measure apoptosis). Big clusters of mycelium grown under Pi-starved conditions, stained red (dead) with only a few patches remaining blue, indicating viability when propidium iodide was used (Figure 4.8G). The mycelium grown at 1.0, 3.0, 10 and 100 mM Pi also partially stained red but the bigger clusters remained blue. It was obvious that the mycelia were at different stages of growth. The fluorescence of ELF-97 could be detected after co-labelling as indicated by green precipitation (H-I). In addition, a similar reaction was observed with acridine orange, where dead cells were stained orange and live cells were blue (Figure 4.8H).
To further illustrate the localization of the enzyme, higher magnification was used in connection with co-staining with Hoechst dye and FM4-64. A strong fluorescence was observed but no defined structures could be observed using fluorescence microscopy. Furthermore, no nucleus differentiation was observed after staining with Hoechst dye but the mycelium appeared blue. When the mycelium was stained with FM4-64, the red colour was intensified when viewed under NIBA but no membrane organelles were observed.
The mycelia were observed to be at different stages of development in different treatments.
When viewed under UV light, mycelium grown at 100 mM Pi, displayed sparse ELF-97 granules compared to 10 mM Pi where the fluorescence was stronger (Figure 4.9A and B).
Using bright field settings (100x), it was observed the cells at 100 mM Pi were growing vigorously and were dividing (Figure 4.9B). Compartmentalization partitioned by septa was distinct (Figure 4.9B). A mature vacuole was observed in the compartment in the cellular region delimited by septa. Apical branching (AB) to form new tips in mature hyphae were clearly distinguished (Figure 4.9B). Enlarged and globular germlings (G) could be seen at the hyphal tip (Figure 4.9B). Hyphal swelling was often observed, formed on the edge of contiguous cells, resembling appressoria (Figure 4.9B). Under UV light, a stronger ELF-97 fluorescence was observed at 10 mM Pi compared to 100 mM Pi (Figure 4.9C). Mycelia grown under 10 mM were more coiled, but germlings and apical branching were observed (Figure 4.9D). The mycelium grown at lower Pi concentrations, appeared stunted, the cells were much smaller and some parts of the mycelium were not viable (Figure 4.9E-F).
153 Figure 4.9: (A-F) Axenic mycelium with under different concentration of Pi (100, 10 and 0.05 mM) incubated with ELF-97 viewed with fluorescent microscope. (A) Fungi grown in 100 mM Pi with ELF- 97 fluorescence appearing as sparse granules. Bar =20 µm. (B) Magnification of the zone in A, viewed under bright field, with mycelium grown at 100 mM, observed with germlings (G) and apical branching (AB). (C) Mycelium grown under 10 mM Pi, showing intense ELF-97 fluorescence, appearing as green granules. (D) Magnification of the zone in C, observed under bright field with mycelium grown at 10 mM. (E) Mycelium grown under 0.05 mM Pi, showing intense ELF-97 fluorescence, appearing as green granules. (F) Magnification of the zone in E, showing mycelium grown under low Pi, under bright field light. Bar =100 µm.
AB
G
B
A 20 µm
C D
E F
154 However, strong fluorescence was observed at 0.05 mM, mainly on the edge of the hyphae (Figure 4.7E). Because of the relatively low level of the resolution given by light microscopy, it was impossible to localize the reactions precisely in the mycobiont. Extending incubation time and increasing the ELF-97 did not enhance any staining pattern to allow any localization at the ultrastructural level. To gain a better understanding of the localization of the enzyme, the confocal microscope was used as an alternative.