21st Century Guidebook to Fungi, SECOND EDITION, by David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 11: Exploiting fungi for food
2. Chapter 12: Development and morphogenesis
   1. Development and morphogenesis
   2. The formal terminology of developmental biology
   3. The observational and experimental basis of fungal developmental biology
   4. Ten ways to make a mushroom
   5. Competence and regional patterning
   6. The Coprinopsis fruit body: making hymenia
   7. Coprinopsis and Volvariella making gills (not forgetting how polypores make tubes)
   8. The Coprinopsis fruit body: making stems
   9. Co-ordination of cell inflation throughout the maturing fruit body
   10. Mushroom mechanics
   11. Metabolic regulation in relation to morphogenesis
   12. Developmental commitment
   13. Comparisons with other tissues and other organisms
   14. Genetic approaches to study development: through the classic to genomic systems analysis
   15. Senescence and death
   16. Basic principles of fungal developmental biology
   17. Relevance to commercially cultivated fungi
   18. Chapter 12 References and further reading
   19. Buy a PDF of Chapter 12
3. Chapter 13: Ecosystem mycology: saprotrophs, and mutualisms between plants and fungi

12.9 Co-ordination of cell inflation throughout the maturing fruit body

As you can see from the descriptions above, most of the changes in shape during fruit body development in basidiomycetes depend on cell inflation; this is typically a slow process in young primordial stages and characteristically more rapid during late maturation. Local cell inflation during fruit body development has often been described and Reijnders (1963) showed that the different zones of fruit bodies enlarge proportionally, so that different tissues mature without being impeded, compressed or distorted by the growth of other parts. Such co-ordination of differentiation of cells in relation to their location is one of the most important general principles of animal and plant morphogenesis. It is thought to be based upon the migration through the developing tissues of pattern-forming morphogenetic factor(s) or signals.

If this is true for fungi, the nature of the signals is generally obscure, though in the larger fruit body primordia of Ascomycota and Basidiomycota the mechanism would need to operate over distances of many millimetres, a similar scale to animal and plant hormone fields. However, apparent ‘co-ordination’ could also result if developmental events are arranged in a consequential series such that one (secondary) event is only instigated by the initiation or completion of an earlier (primary) event. For any conclusion to be reached on this issue what is needed is an holistic study of inflation over the whole fruit body and an assessment of the correlation between cell behaviour in widely separated locations against an accurate time frame. This was done by Hammad et al. (1993b) for the fruit body of Coprinopsis cinerea.

This study examined a sufficiently large sample of fruit bodies to establish the exact timing of major meiotic and sporulation events, and this provided the basic timeline to which other processes could be referenced simply by microscopic examination of a small piece of cap tissue. Removing small tissue fragments (‘biopsies’) for rapid microscopic examination does not interfere with fruit body development. This timeline is objective in the sense that it depends upon processes which are endogenously controlled. It is reliable because these processes are central to fruit body function and it is versatile since by examining slivers at known (real) time intervals the effects of any change in cultivation conditions or culture genotype become apparent.

In the cultivation conditions described by Hammad et al. (1993b), fruit body primordia were formed 5 d after inoculation of the culture medium and developed into mature fruit bodies within 2-3 days, during which the basidia underwent a sequence of morphologically and physiologically distinctive stages.

• The dikaryotic protobasidia underwent nuclear fusion (karyogamy) and then entered meiosis I, followed immediately by meiosis II.
• After completion of meiosis, four sterigmata emerged
• followed by formation of four basidiospore initials,
• nucleus migration,
• maturation and discharge of the spores.

All these events were completed within 18 h, and the overlap between different stages varied through development.

• In a ‘meiosis I specimen’ about 60% of the basidia had two nuclei;
• at meiosis II about 70% had four nuclei;
• when sterigmata first appeared about 90% of the  basidia were at the same stage.

Defining karyogamy as time zero, basidia took 5 h to reach meiosis I and meiosis II was completed after a further hour. From meiosis II to the emergence of sterigmata required 1.5 h, spores emerging 1.5 h after that. Spore formation continued for 1 h and then nuclear migration started. Spore pigmentation commenced 1 h after spore formation and spores matured and were discharged 7 h later.

Co-ordination of cell inflation was studied by measuring cell sizes in microscope sections of fruit bodies of Coprinopsis cinerea whose chronological development was defined by the stage they had reached in meiosis and sporulation.

As far as cell inflation was concerned (measured in terms of cell area in tissue sections), there was only a small increase before meiosis. Presumably, any fruit body expansion occurring in these early stages is due primarily to cell proliferation rather than cell inflation. In contrast, there was a large increase in cell profile area in sections of fruit bodies undergoing meiosis. The average length-to-width ratio of the cells in stems before meiosis (fruit bodies up to about 8 mm tall) was about 2, but this increased greatly after meiosis, to 10 in 48 mm tall fruit bodies, 20 in 55 mm tall fruit bodies and approximately 35 in fruit bodies that were 83 mm tall. The phase of most rapid stem elongation occupied the 5 h prior to spore discharge and started 8 h after karyogamy. Cap expansion started as spores matured, 14 h after karyogamy.

The most remarkable feature of these data was that rapid stem elongation was correlated with the ending of meiosis. Indeed, inflation of all the different cell types in the cap as well as elongation of cells of the stem began immediately after meiosis.

Elongation of the stem is necessary to raise the cap into the air for more effective spore dispersal; inflation of the different cell types in the gill tissue is also necessary for effective spore dispersal so co-ordination of cell inflation across the whole fruit body is clearly advantageous. The co-ordination that is observed may be achieved by some sort of signalling system that ‘reports’ the end of meiosis to spatially distant parts of the fruit body and initiates cell expansion. Indeed, stem elongation was significantly greater when the cap was left attached than when it was removed and leaving half of the cap in place was sufficient to ensure normal elongation.

This effect of surgical removal of cap segments on stem elongation has been observed with several species of mushroom over the years. Generally speaking, the elongating stem curves away from the side with the cap segment left intact (providing it contains gill tissues) and this has been interpreted as indicating that the gills are the source of a ‘growth hormone’. Unfortunately, the detailed information we have about hormonal effects in mushrooms is still sparse and unsatisfactory (Novak Frazer, 1996), with no definitive fungal growth hormone of any sort being chemically isolated even now; which is a remarkable statement for a kingdom of organisms which have been known for many years to include pathogenic/parasitic representatives that produce both animal and plant hormones to control their hosts.

However, some recently reported research has suggested that chemically-modified sugars, formed in the gill tissues and secreted into the mucilage that surrounds the hyphae making up the Coprinopsis fruit body, might be candidates for the signal that coordinates hyphal cell inflation across the whole fruit body (Moore & Novak Frazer, 2017).

This family of molecules (called Fungiflexes) was isolated during a study of gravitropism in Coprinopsis cinerea and were found to be most probably laevorotatory 6-deoxy hexoses, which could be substituted with amino and/or amido groups, then be N-acetylated, and possibly phosphorylated or sulfated to serve different signalling purposes. When applied asymmetrically to isolated fruit body stems, 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 (called Fungiflex 1) and the second (Fungiflex 2), after some hours, promoting hyphal lengthwise extension.

It is thought that the Fungiflex molecules are produced near 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 away from their source (which effectively means down the stipe) to regulate hyphal extension progressively. The assumption is that Fungiflex 1 inhibits stipe extension while the cap is being formed and, following-on several hours later, Fungiflex 2 enhances the extension of stipe hyphae to raise the cap above surrounding vegetation to facilitate the eventual spore release (Moore & Novak Frazer, 2017).

The mechanism that achieves this, suggested by Moore & Novak Frazer (2017), depends on the fact that Coprinopsis has a single hexose transporter; specifically, an allosteric ATP-binding cassette hexose transporter, which (a) couples hydrolysis of adenosine triphosphate (ATP) to the translocation of hexose across the hyphal membrane in a high-affinity configuration, which it assumes when sugar is in low supply; and (b) facilitates hexose transport as a glucose-proton symport by facilitated diffusion over a proton gradient in its low affinity configuration, which it assumes when extracellular glucose is abundant (Moore & Devadatham, 1979; Taj Aldeen & Moore, 1982).

Such carrier proteins are generally integral membrane proteins; meaning that they exist within, and span, the membrane across which they transport their substrates. Integral transporters have α-(alpha) helical structures in their membrane-spanning domains that contribute to substrate translocation across the membrane. Mutants selected in different ways were observed to be clustered in the recombination fine-structure gene map of this Coprinopsis transporter (Moore & Devadatham, 1975). The clusters are thought to correspond to the membrane-spanning domains of the transporter and that these regions of the protein would be responsible for, or at least take part in, the molecule-specific substrate binding. Observations with the Fungiflexes suggest that cytoskeletal microfilaments connected to the transporter’s membrane-spanning domains could distort those domains and alter the substrate affinities of the normal hexose transporter. This could provide a general metabolic control mechanism with different chemically-modified hexoses being transported into the cell in response to different cytoskeletal signals and modulating the activity of existing proteins, some of which could be transcription activators (Chang et al., 2018a).

Updated January, 2020