Content uploaded by Patrick Gervais
Author content
All content in this area was uploaded by Patrick Gervais on Jan 05, 2016
Content may be subject to copyright.
A preview of the PDF is not available
Fungal Cells Turgor Pressure: Theoretical Approach and Measurement
Abstract and Figures
Hyphal growth has been examined from a mechanistic standpoint to understand the role of turgor pressure on hyphal extension rate. From a rigourous momentum balance it is shown that turgor pressure can be expressed as a sum of two components : (i) a static contribution, related to the membrane elastic properties and resolved by capillary probe measurements; and (ii) a dynamic part (ΔP), responsible for the hyphal extension rate and proportional to it. Both contributions add up and explain why some researchers have found hyphae growing at finite rates when the capillary probe measures no pressure difference at all; it is simply because the dynamic part of the turgor pressure cannot be resolved by such type of technique. The linear relationship between turgor pressure and hyphal extension rate means that two measurements need to be taken in order to completely characterize the microscopic parameters involved in this phenomenum. An experimental setup is proposed based on quick osmotic shocks upon the hyphae - to stop tip growth without inducing plasmolysis - to accomplish such a discrimination. This approach has been applied to Aspergillus orizae and an increasing relation between the dynamic part of turgor pressure (ΔP) and hyphal extension rate has been found.
Figures - uploaded by Patrick Gervais
Author content
All figure content in this area was uploaded by Patrick Gervais
Content may be subject to copyright.
ResearchGate has not been able to resolve any citations for this publication.
Reference to a dictionary reveals that an object under stress is subjected to “demand upon energy” or is “constrained.” It is precisely these two aspects of microbial ecology that I have considered in this review, the stress being imposed by some factor directly associated with the water regime of the immediate environment. Such a topic is relevant to many branches of microbiology. Thus, those working in microbial physiology, soil microbiology, plant pathology, food preservation, biodegradation, and marine and lacustrine microbiology all find water and its biological availability to be an important factor influencing microbial activity and hence microbial ecology.
When colonies of Fusarium oxysporum, growing on plates of mineral-sucrose agar, are flooded with the mineral-sucrose solution, without added agar, or with solutions of any of the constituents of the mineral-sucrose mixture at a concentration of 0.076 M the leading hyphal apices at the agar surface continue to grow on unchecked. If, however, the colonies are flooded with solutions of decreasing and increasing molarity from 0.076 M an increasing proportion of the leading hyphal apices at the agar surface stop growing, and branch subterminally. In distilled water about 50 per cent. of the apices branch and this branching is preceded by swelling, whereas in 0.5 M sucrose more than 90 per cent. of the apices branch and the branching is not accompanied by swelling. In the distilled water those hyphae which do not branch swell a little and grow on from the apex within 40 seconds.
When hyphal apices are flooded with distilled water for from 10 to 40 seconds and then transferred to mineral-sucrose solution more than 90 per cent, of the hyphal apices branch, whereas flooding with distilled water for 60 seconds or longer gives the same percentage of branched apices as does flooding with distilled water alone.
It is shown that swelling and branching of the hyphal apex are not causally related but that branching always occurs following arrestment of the hyphal apex for more than 60 seconds. It is suggested that the phenomena reported can be explained in terms of an irreversible change in the apical cap of the arrested hypha such that continued extension can no longer take place in this region and fresh outlets for growth must then be found subterminally. Such a mechanism, however triggered, could account for a wide variety of morphogenetic forms in the fungi.
The morphology of the colony and of individual cells of the hyphae of Neurospora crassa are described. The behaviour of the hyphal apices in solutions of differing molarity is described and this behaviour is related to the behaviour of Fusarium oxysporum. Measurements of the water potential of the apical cells are made and it is shown that this corresponds to the balancing-point in this fungus and in F. oxysporum. Measurements are then made of the water potential of cells at various points along the length of the hyphae, and of the osmotic potential of these cells, and turgor pressure is calculated. The lowest turgor pressure of 12.4 atmospheres is shown by the apical cells and the highest of 17.5 atmospheres by the basal cells.
The validity of the methods used is discussed.
β-glucan synthetases from particulate preparations of Saprolegnia monoica were partially solubilized by digitonin treatment. The enzymes which were still associated with the membrane fraction were increased in total and specific activities. Glucan synthetases assayed at low substrate concentration in the presence of Mg Cl2, were inhibited by trypsin. On the contrary, enzymes assayed at high substrate concentration in the absence of Mg Cl2, were stimulated by trypsin but inhibited by other proteases and by a trypsin inhibitor. Activation followed by inactivation of the synthetases was observed in the presence of high trypsin concentration.
Regulation of glucan synthetase activities is discussed in relation to hyphal morphogenesis.
With the exception of the unicellular yeasts, fungi typically grow by means of hyphae that extend only at their apices and ramify into a mycelium. This mode of growth provides fungi with a certain mobility and the ability to invade dead and living organic substrata. They are thus the main decomposers of plant residues but they also have established intricate symbiotic relationships with plants, both mutualistic and parasitic.
The process of apical growth of a hyphae requires the controlled expansion of the apical wall which must be transformed subsequently into a wall that resists turgor pressure and maintains the tubular shape of the hyphae. Although the driving force for hyphal extension is probably the turgor pressure, a subtle interplay between wall extension and cytoplasmic activity is necessary because only a precise gradient of wall-synthetic activity can maintain uniform wall thickness during expansion. Possibly, the presence in the plasma membrane of mechanico-sensitive proteins plays a role in conjunction with the cytoskeleton at the apex, particularly action. Although the major structural wall polysaccharides are probably manufactured directly on the expanding apical plasma membrane, proteins (and probably some wall components) are delivered to the growing surface by a continuous stream of exocytotic vesicles that fuse with the plasma membrane, at the same time extending its surface.
Our analyses of the chemistry of the fungal wall and its biosynthesis and assemblage have disclosed a simple mechanism (though complex in detail) that may explain the transition from a newly formed expandable wall at the apex to a more rigid wall at the base of the hyphal extension zone. Two individual wall polymers, chitin and β-glucan, extruded at the apex are modified within the domain of the wall. Among the modifications observed are the formation of covalent crosslinks between these two polymers and hydrogen bonds between the homologous polymer chains, leading to the formation of chitin microfibrils crosslinked to a glucan matrix. This process is thought to convert an initially plastic wall into a rigid wall as the polymers fall behind the advancing tip. We have called this the steady-state growth theory for apical wall extension because a steady-state amount of plastic wall is always maintained at the growing apex.
Excretion of lytic enzymes is a vital process in filamentous fungi because, in nature, they thrive on organic polymers which must be degraded extracellularly. Such enzymes are also necessary for infection processes. Cytological data suggest that such enzymes are extruded by the vesicles that continuously fuse with the plasma membrane at the growing apex. We have shown that a large portion of the excreted enzymes indeed leaves the hypha at the growing apex but another portion may be retained by the wall and is slowly released into the medium. In relation to the steady-state growth theory we hypothesize that enzymes can pass the wall at the apex by bulk flow, that is, by being carried by the flow of plastic wall material, making pores in the wall less important than previously thought.
Proteins excreted by filamentous fungi not only serve dissimilatory purposes but are also important for a variety of other activities of the whole mycelium, including morphogenesis. By cloning genes abundantly expressed during formation of aerial hyphae and fruit bodies, we have discovered a class of proteins, named hydrophobins, which are only produced when the mycelium has reached a certain stage of maturity. Whilst excreted by submerged hyphae as monomers into the medium, they self-assemble as insoluble complexes in the walls of emergent hyphae. In aerial hyphae a particular hydrophobin takes the form of rodlets which probably coat the hyphae with an impermeable layer. During fruit-body formation other hydrophobins are produced which may function in the aggregation of hyphae to form a multicellular tissue. Apart from such specific morphogenetic functions, the hydrophobins may play a general role in insulating hyphae from the environment, converting the differentiating structures into sinks for translocation of water and nutrients from the assimilating mycelium.