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Hyphal tips of fungi representing OQmycetes, Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes were examined by light and electron microscopy and compared with respect to their protoplasmic organization. In all fungi studied, there is a zone at the hyphal apex which is rich irn cytoplasmic vesicles but nearly devoid of other cell components. Some vesicle profiles are continuous with the plasma membrane at the apices of these tip-growing cells. The subapical zones of hyphae contain an endomembrane system which includes smooth-surfaced cisternae associated with small clusters of vesicles. The findings are consistent with the hypothesis that vesicles produced by the endomembrane system in the subapical region become concentrated in the apex where they are incorporated at the expanding surface. Septate fungi (Ascomycetes, Basidiomycetes, and Deuteromycetes) have an apical body (Spitzenkorper) which is associated with growing hyphal tips. In electron micrographs of these fungi, an additional specialized region within the accumulation of apical vesicles is shown for the first time. This region corresponds on the bases of distribution among fungi, location in hyphae, size, shape and boundary characteristics to the Spitzenkorper seen by light microscopy. This structure is not universally associated with tip growth, whereas apical vesicles are widespread among tip-growing systems.

A membrane-bound adenosine triphosphatase (EC 3.6.1.3) that requires Mg++ and that is stimulated by monovalent ions has been purified 7-to 8-fold from homogenates of oat (Avena sativa L. Cult. Goodfield) roots by discontinuous sucrose-gradient centrifugation. The enzyme was substrate specific; adenosine triphosphate was hydrolyzed 25 times more rapidly than other nucleoside triphosphates. The membrane fraction containing adenosine triphosphatase was enriched in plasma membranes, which were identified by the presence of a glucan synthetase (EC 2.4.1.12), a high sterol to phospholipid ratio, and by a stain consisting of periodic acid, chromic acid, and phosphotungstic acid that is specific for plant plasma membranes. Oat-root plasma membranes and the associated adenosine triphosphatase were purified on either a 6-layer discontinuous sucrose gradient or on a simplified gradient consisting of only two sucrose layers.These results represent the first demonstration that plant plasma membranes contain an adenosine triphosphatase that is activated by monovalent ions, and this finding further implicates the enzyme in the absorption of inorganic ions by plant roots.Absorption of inorganic ions bylplant-root cells is an energyrequiring process dependent on aerobic respiration (1, 2). Furthermore, adenosine triphosphate (ATP) appears to be the energy source, since ion absorption by plant roots is inhibited by dinitrophenol (3,4), arsenate (4), and oligomycin (5-7). The mechanism of energy transfer from ATP to the ion-transport system is unknown, however, and this phenomenon represents one of the major unresolved aspects of the ion-absorption process in plants.We have suggested (8, 9) that the energy transduction process involved in ion transport of plant cells involves an adenosine triphosphatase (ATPase; EC 3.6.1.3) similar to the "transport" ATPase of animal cells (10). Plant ATPase is associated with membranes, requires MIg++, and is further activated by monovalent ions (8, 9, 11). A high correlation exists between the KCl-or RbCl-activated component of the ATPase and K+ or Rb+ absorption by root tissue (9). Also, the kinetics of monovalent-ion transport into roots and the kinetics of monovalent ion-stimulated ATPase are similar (8, 9). However, in order for this ATPase to be involved in energy transduction for ion transport, it should be associated with one or both of the membranes involved in active ion transport (i.e., either the plasma membrane or tonoplast), and this has not been demonstrated.It is difficult to isolate and identify the membrane system containing the ion-stimulated ATPase because of the ubiquity of membrane-associated ATPases in plants (12)(13)(14) and the paucity of known membrane "markers" for plant cells (14). We have recently found, however, that the membrane system containing the monovalent ion-stimulated ATPase can be separated from nearly all the other membranes on either continuous or discontinuous sucrose gradients (14). In this paper, we show that this membrane system has a high ste...

Somatic fungal hyphae are generally assumed to elongate at steady linear rates when grown under constant environmental conditions with ample nutrients. However, patterns of pulsed hyphal elongation were detected during apparent steady growth of hyphal tips in fungi from several major taxonomic groups (Oomycetes, Pythium aphanidermatum and

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The fine structure of isolated chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) particles (chitosomes) from Mucor rouxii and the elaboration of chitin microfibrils were studied by electron microscopy. Chitosomes are spheroidal, but often polymorphic, structures, mostly 40-70 nm in diameter. Their appearance after negative staining varies. Some reveal internal granular structure enclosed by a shell measuring 6-12 nm thick; others do not show internal structure but have a pronounced depression of the external surface. In thin sections, isolated chitosomes appear as microvesicular structures with a tripartite shell 6.5-7.0 nm thick. Morphologically similar structures can be seen in intact cells of M. rouxii. Isolated chitosomes undergo a seemingly irreversible series of transformations when substrate and activators are added. The internal structure changes, and a coiled microfibril (fibroid) appears inside the chitosome. The shell of the chitosome is opened or shed, and an extended microfibril arises from the fibroid particle. During prolonged incubation, the fibroid coils become less common and extended microfibrils appear thicker. We regard the chitosome as the cytoplasmic container and conveyor of chitin synthetase en route to its destination at the cell surface. Isolated chitosomes are well suited for integrated ultrastructural-biochemical studies of microfibril biogenesis in vitro. Chitin microfibrils can be assembled in vitro by chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-j3-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) preparations isolated from the yeast form bf the fungus Mucor rouxii (1, 2). Electron microscopy of shadow cast specimens showed that the fibrils were formed from particles ("granules") smaller than 100 nm in diameter (2). These particles, which we now call chitosomes, contain a chitin synthetase complex capable of forming a microfibril by collective synthesis of the polysaccharide chains. The biochemical properties of chitosomal chitin synthetase will be reported elsewhere. An understanding of the underlying molecular mechanism of microfibril biogenesis requires knowledge of the detailed fine structure of the enzyme complex and the way in which microfibrils are assembled. Here, we show the first evidence of the morphological complexity of chitosomes as well as the striking transformations they undergo during the course of microfibril elaboration.MATERIALS AND METHODS Isolation and Purification of Chitin Synthetase. Mucor rouxii strain IM-80 was grown in liquid medium (0.3% yeast extract, 1% peptone, 2% glucose, pH 4.5) at 28°for 13 hr in a N2:CO2 (30%:70%) atmosphere (2). Cells were harvested, mixed with 50 mM KH2PO4-NaOH buffer, pH 6.5, and 10 mM MgCl2, and broken in a Braun model MSK cell homogenizer (2). All subsequent manipulations were done in this buffer.After cell walls were removed at 1000 X g for 5 min, the cellfree extracts were centrifuged at 54,000 X g (Rav) for 45 min.The supernatant was ...