9. And now some speculations
So where does all this lead us? In the 1990s we showed that what we now believe to be modified sugars could be extracted by the simplest procedure of water-infusion of fruit body stipes or caps. When applied asymmetrically to isolated stipes, hyphal cells in the immediate region of the application changed their growth pattern in response to at least two components, one promoting immediate hyphal lengthwise contraction (Fungiflex 1) and the second (Fungiflex 2), after some hours promoting hyphal lengthwise extension.
One obvious interpretation is that the Fungiflex molecules are produced in the vicinity of the stipe apex, perhaps in what constitutes cap tissue near or at the junction of stipe and cap. They are then released into the extracellular matrix of the stipe to diffuse downwards to regulate hyphal extension progressively. The expectation is that Fungiflex 1 inhibits stipe extension while the cap forms and, following several hours later, Fungiflex 2 enhances the extension of stipe hyphae to raise the cap above surrounding vegetation to facilitate the eventual spore release.
In the 1970s we demonstrated that modified sugars caused grossly abnormal construction of the hyphal wall; the exact nature of the effect was dependent on the molecular nature of the modified sugar. The wall abnormalities themselves caused mycelial growth inhibitions and all mutants selected for resistance to those growth inhibitions were found to have mutations in the organism’s one and only hexose transporter.
The clear-cut interpretation of these facts is that normal wall construction is controlled by endogenously produced sterically or chemically modified sugars. When such sugars are applied externally experimentally (under which circumstances, location and concentration are undoubtedly abnormal), they interfere with wall growth, resulting in an altered, and also abnormal, hyphal growth response.
Arguably, the modified sugars must be transported into the intracellular environment to be effective; in normal circumstances, it is the single hexose transporter that translocates normal and sterically or chemically-modified hexose sugars.
Which all leads to the overall conclusion: the Fungiflexes are examples of the endogenously-produced modified sugars that function as control molecules for normal hyphal wall construction.
We think Fungiflexes are specifically-modified sugar molecules that are externalised at source (presumed to be the apex of the stipe) and diffuse through the extracellular matrix surrounding the hyphae. They are effective in modifying the activity of the hyphal wall construction apparatus after being translocated to the intracellular environment. We showed above that Fungiflex 1 was present at all stages of mushroom development and the appearance of Fungiflex 2 activity was correlated with differentiation of the cap region in primordia (preceding meiosis).
We interpret the normal function of Fungiflex 1 as an immediate down-regulator of the extension growth of stipe hyphae to allow time for the cap to differentiate.
Fungiflex 2 activity promotes hyphal extension growth and appears some hours after Fungiflex 1; remember, though, this late Fungiflex 2 activity is from the same extract application in which the more immediate Fungiflex 1 activity appears. There are a number of plausible implications of this:
- Fungiflex 2 (the ‘up-regulator’) could be a derivative, produced spontaneously or enzymically, of Fungiflex 1 (the ‘down-regulator’);
- Fungiflex 2 could be a separate molecule that is transported with such a low Vmax for translocation across the membrane that it takes several hours for the intracellular concentration to reach activation levels;
- Plausibly, both Fungiflex 1 and Fungiflex 2 may be transported equally into the intracellular compartment but their targets for regulation are separated in time. That is, the target of Fungiflex 1 is immediately accessible to down-regulation, but the regulatory target of Fungiflex 2 may need to be freshly synthesised or enzymatically modified, either to overcome the down-regulation of Fungiflex 1 or to remove/replace permanently repressed Fungiflex 1 targets.
This interpretation would make Fungiflex 2 (specifically) a candidate for the signal that coordinates hyphal cell inflation across the fruit body as a whole (Hammad, Ji, Watling & Moore, 1993a; Hammad, Watling & Moore, 1993b).
These two studies were the first, and so far only, quantitative hyphal analyses in which computer-aided image analysis was used to measure and enumerate cell types at different stages of development in light microscope sections of fruit bodies of Coprinopsis cinerea. The quantitative analyses enabled a dynamic understanding of fruit body development in this organism. Essentially, early development involves cell proliferation and late development involves cell elongation and expansion (see the following information box for details).
The stipe of Coprinopsis cinerea comprises two cell populations: large and small diameter hyphae. Cell inflation is accentuated in cells occupying a specific zone just beneath the ‘epidermis’ of the stipe; differential expansion of cells in this zone readily explains how the fruit body stipe changes from a solid cylinder to a hollow tube during its development.
Cell length does not increase during early stages, between a 3 mm and an 8 mm tall fruit body (both at pre-meiotic developmental stages). Presumably any stipe elongation occurring at these stages is due primarily to cell proliferation rather than cell elongation.
In contrast, there is a large increase in cell length between stipes of an 8 mm pre-meiotic fruit body and that of a 25 mm fruit body undergoing meiosis. Initially the cells in the basal and middle regions of the stipe lengthen. Cells in the extreme basal and apical regions are always shorter than those in other regions of elongated stipes. For example, cells near the cap/stipe junction at the extreme apex of an 83 mm fruit body (fully elongated) have a typical length of 150 μm compared to an average of 313 μm for the whole of the apical section examined (about 10 mm long).
The most elongated cells are found in the upper mid-region of the stipe. Ratios of length to width are about 2 in pre-meiotic stipes (3 mm and 8 mm fruit bodies), but increase after meiosis, particularly in the upper middle regions, to 10, 20 and approximately 35 in 48 mm, 55 mm and 83 mm tall fruit bodies respectively.
Overall, therefore, developmental stipe extension of C. cinerea involves increase in length and cross-sectional area of inflated hyphae and recruitment of narrow hyphae into the inflated population. [CLICK on the two citations that follow to download into a new window a (free) PDF of the original publication (Hammad, Ji, Watling & Moore, 1993a; Hammad, Watling & Moore, 1993b)].
Significant for the present discussion, expansion of all the different cell types in the fruit body cap as well as inflation of cells of the fruit body stipe coincides with completion of meiosis. Hammad et al. (1993a) suggested that coordination between cap and stipe could be achieved by some sort of signalling system that ‘reports’ the end of meiosis to spatially distant parts of the fruit body [CLICK on the following citation to download into a new window a (free) PDF of the original publication Hammad, Ji, Watling & Moore, 1993a].
The extracellular matrix, which surrounds all the cells of the fruit body, could provide an aqueous continuum through which such a reporter molecule could diffuse and provide signalling over both short (less than μm-length) and long (more than mm-length) distances (Moore, 1993). We suggest that Fungiflex 2 may be, or may generate, that cell-expansion-coordinating reporter signal.
Copyright © David Moore & Lily Novak-Frazer 2016