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

12.11 Metabolic regulation in relation to morphogenesis

We have emphasised the importance of cell inflation and tissue expansion in fruit body development, but there are several resources that must be provided to support cell inflation. Obviously, these demands include extensive synthesis of new cell wall (Chapter 6). However, wall synthesis alone will not inflate the cell:

• the volume enclosed by the wall needs to be filled with ‘cytoplasm’ and that usually means a large increase in the size, and perhaps number, of vacuoles.
• Vacuoles are filled with aqueous solutions, so this, in turn requires a considerable increase in uptake of water by the cells.
• And since water is driven across cytoplasmic and vacuolar membranes from a solution of high water potential (= low solute concentration) to one of low water potential (= high solute concentration) there is a consequential need to adjust metabolism so that osmotically active solutes are accumulated in the cells and cytoplasmic compartments that must be inflated.

Ultimately, therefore, cell inflation is driven by modifications to the pattern of primary (intermediary) metabolism (meaning specific increases or decreases in activity levels of existing enzymes and/or introduction or removal of particular enzymes) which are done as a crucial contribution to a differentiation process rather than as a response to nutritional conditions (CLICK HERE to view the Primary (intermediary) metabolism section of Chapter 10).

Quite a catalogue of changes in enzyme activities has been noted during the development of ink cap mushrooms:

• In Coprinopsis cinerea and Coprinus comatus development-related increases in the activities of glucosidase and glucanases, protease and chitinase have been noted during cap expansion (Iten, 1970; Iten & Matile, 1970; Bush, 1974) and have been identified in the ‘ink’ fluid (the autolysate); so degradation of the substance of the spent gill tissue is provided for by specific enzyme derepression.
• Specific chitinases and glucanases act synergistically in stipe wall extension of C. cinerea (Kang et al., 2019a; Zhou et al., 2019).
• In earlier stages, prior to the onset of autodigestion, a number of enzymes are specifically derepressed as tissues of the C. cinerea mushroom cap begin to form (Moore, 2013). Among these, NADP linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS), in particular have been found to derepress specifically in the Coprinopsis fruit body cap during development.

A vegetative mycelium also shows derepression of NADP‑GDH under specific conditions in vitro, and this has enabled experimental study of regulatory factors complementing observation of endogenously‑controlled events in the developing fruit body (for review, see Moore, 1984), to try to establish the function(s) of the enzymes that are so distinctively regulated. The crucial observations are as follows:

• derepression of NADP‑GDH in the mycelium involves de novo synthesis of the enzyme protein (Jabor & Moore, 1984) and is caused by transfer into a medium lacking any nitrogen source but rich in carbon (usually 100 mM pyruvate is used, but acetate, glucose and fructose are also suitable);
• inclusion of as little as 2 mM NH₄⁺ in the transfer medium prevents derepression of NADP‑GDH, among many other nitrogenous compounds tested only those able to generate ammonium (including glutamine) also inhibit derepression;
• a mutant unable to synthesise acetyl‑CoA failed to derepress NADP‑GDH (Moore, 2013);
• in the fruit body, derepression of NADP‑GDH occurs in the cap, but not in the stem (Stewart & Moore, 1974; Al-Gharawi and Moore, 1977) the accompanying NAD‑GDH also increases in activity (but in both tissues) as the fruit body develops. In the cap the ratio of the two enzymes alters from 10:1 (NAD‑GDH: NADP‑GDH) to 1.4:1, a considerable swing in favour of the enzyme with a 10‑fold greater affinity for ammonium;
• there is a high positive correlation between derepression of NADP‑GDH and derepression of glutamine synthetase in both fruit body cap and mycelium and co-ordinate regulation of four enzymes involved in arginine and urea synthesis metabolism in the cap (Ewaze, Moore & Stewart, 1978);
• both light and electron microscope observations demonstrated cytochemical localisation of NADP‑GDH to the developing basidia of the young hymenium in the fruit body cap (Elhiti, Butler & Moore, 1979);
• large quantities (up to 2 mg dry weight per fruit body) of glycogen are accumulated in fruit bodies and translocated specifically to the fruit body cap (Jirjis & Moore, 1976; Moore, Elhiti & Butler, 1979). Similarly, the metabolically inert amino acid analogue, α-amino-isobutyric acid, has been demonstrated to be directed to mushrooms of Agaricus bisporus when they started to develop, through mycelial cords which could show a 5 × increased rate of translocation to that observed in vegetative mycelium (Herman et al., 2020).
• derepression of NADP‑GDH occurs initially at karyogamy and is then re‑amplified postmeiotically. In Coprinopsis, unlike other agarics, the meiotic division is highly synchronised across the whole population of basidia. Observation of stained nuclei coupled with determination of activity of NADP-GDH and assay of glycogen in the same tissue showed that increase in enzyme activity was initiated as karyogamy became evident in normally developing fruit bodies. Glycogen utilisation is initiated towards the end of meiosis and is almost completely utilised within a few hours immediately following the meiotic division during which time basidiospore formation occurs (Moore, Liu & Kuhad, 1987).
• activity of the enzyme urease is absent from the cap though present in both stem and mycelium (Moore & Ewaze, 1976; Ewaze et al., 1978).  

In interpreting these observations the first important point we want to make is that such modifications of metabolism do not represent a novelty in terms of the reaction sequences involved; much of what occurs in the fruit body can be shown to occur in mycelia. However, the fruit body, and the cap in particular, does present a very special regulatory picture, because these metabolic changes which are a normal a part of the development of the fruit body, can only be induced in vegetative cultures by exposing them to particular (and in some cases most peculiar) synthetic media. What is specific to the fruit body, then, is that at the outset of its development there is set in train such a fundamental change in the emphasis of the metabolism of its constituent cells that cap cells become metabolically quite distinct from those of either the stem or the parent mycelium. To understand better the following discussion you might find it useful to refer to the section of Chapter 10 that deals with Primary (intermediary) metabolism, especially Fig. 9. showing oxidation of pyruvate, the tricarboxylic acid cycle and glutamate decarboxylation loop; the flow chart illustrating pathways of nitrogen redistribution of Fig. 12 in Chapter 10; and the urea cycle shown in Fig. 13.

The observations listed above show that arginine synthesis and urea formation via the ornithine cycle occurred in the Coprinopsis cinerea fruit body cap and stem (Fig. 13 in Chapter 10). Although urease occurred at high activity in mycelium and stem, it was not detectable in extracts of cap tissue, but arginine biosynthesis was specifically amplified during development of the cap as judged from metabolism of isotopically labelled substrates and increased enzyme activities. Four enzymes, NADP‑linked glutamate dehydrogenase, glutamine synthetase, ornithine acetyltransferase and ornithine carbamoyltransferase, were highly depressed in developing caps while remaining low (or declining) in activity in the stems supporting those caps. As an aside, we should say that metabolic differences between caps and stems are not a unique feature of Coprinopsis. Comparative proteomic analysis of Pleurotus ostreatus has revealed numerous proteins involved in carbohydrate metabolism as well as sphingolipid signalling and metabolism, that are differentially expressed between the cap and stipe (Zhu et al., 2019). Studies of metabolites in Coprinopsis showed that arginine, alanine and glutamate accumulated in the cap as a result of amplified arginine biosynthesis. More significantly, the quantity of urea in the cap doubled during development, although the concentration of urea remained unchanged, which suggests a causal relationship between urea accumulation and water influx into developing tissues. So we conclude that urea is the osmotically-active metabolite in C. cinerea and metabolism is adapted to enhance the flow of nitrogen towards the urea cycle, and all of this is devoted to driving water into the cap tissue to enable its expansion, accounting for the changes in form through which the cap progresses as maturation proceeds; but that’s neither the end, nor is it the whole, of the story.

Urea is an ideal osmotic metabolite, being a low-molecular weight, non-toxic, nitrogenous excretion product, so it is easy to understand how tissues which are about to enter an autodigestive phase, in which the whole emphasis is shifting from anabolism to catabolism, can have their metabolism altered to yield large quantities of urea rapidly (Fig. 12 in Chapter 10), and the decline in activity of urease exclusively in the cap ensures that the urea will accumulate there.

In this system, however, coregulation of NADP-glutamate dehydrogenase and glutamine synthetase (GS) depends on a circuit involving build up of acetyl-CoA in tissues where ammonium is limiting; and this does not fit well into the ‘urea-accumulation through catabolism story’. In other circumstance (and many other organisms), NADP-GDH and GS both have very high affinities for ammonium and together contribute to an ammonium scavenging system. This is an anabolic process, alleviating nitrogen deprivation and creating amino acids needed for protein synthesis. In C. cinerea cap tissue, amino-nitrogen must be obtainable readily from autodigested proteins, so it is unlikely that the ammonium‑scavenging GDH and GS activities are needed to assimilate nitrogen at this late stage in fruit body maturation.

However, a consequence of enzymatic scavenging is that the scavenged substrate is very effectively removed from the cytoplasm, so an alternative interpretation is that these two enzymes are derepressed to maintain an ammonium‑free environment in cells committed to processes which are inhibited by ammonium. Now, NADP-GDH activity has been specifically localised to basidia in C. cinerea cap tissue, so the cells we are talking about are basidia, which are specialised for meiosis. And, indeed, it seems that proper progress of meiosis in eukaryotes generally may depend on ammonium‑sensitive processes. Glycogen breakdown, DNA synthesis, and extensive RNA and protein breakdown occur together uniquely in fungal meiocytes; and all of these processes seem to be inhibited by ammonium ions (Moore, Horner & Liu, 1987 and references therein). This is true even in yeast:

• degradation of accumulated glycogen is not observed in yeast cells incubated in medium supplemented with ammonium;
• treatment with ammonium delays degradation of proteins at the onset of meiosis and inhibits protein and DNA synthesis;
• DNA replication is arrested by ammonium after initiation and continued incubation in the presence of ammonium leads to massive DNA degradation.

The interpretation is, therefore, that NADP-GDH and GS activities are specifically enhanced in basidia of C. cinerea to protect those cells from the inhibitory effects of ammonium ions, and in the next Section we will describe how meiosis and sporulation were shown to be sensitive to inhibition by ammonium ions by direct experiments in vitro and in vivo.

Updated January, 2021