Search

Division of the guard mother cell (GMC) in Allium cotyledons has been examined in epidermal slices viewed with Nomarski optics and electron microscopy. Special attention has been directed towards elucidating the process by which the dividing cell determines its plane of division. In normal development, the cell plate formed during GMC division ultimately lies along the longitudinal axis of the cotyledon, in contrast to the transverse planes formed in other epidermal divisions. Our observations reveal that the final plane of division is not determined by the orientation of the spindle at metaphase but instead is established during late anaphase-telophase as a result of directed reorientation movements of the spindle-phragmoplast and associated daughter nuclei. The metaphase plate may lie at an oblique angle, even as great as 90°, from the final plane of the plate. Thus, daughter chromosomes separate into opposite corners of the cell. During late anaphase-telophase, movement of the spindle is activated; the daughter nuclei move along the sides of the cell while the interzone rotates. Movement continues until daughter nuclei reach positions opposite each other along the sides of the cell and the midzone or cell plate is positioned in the longitudinal orientation. Movement requires 15–20 minutes for completion, is highly directional, and does not overshoot the correct alignment. Following movement cytokinesis proceeds to completion forming two young guard cells. Possible mechanisms for reorientation are discussed, including one that suggests that interzone microtubules may interact with a cortical site on the plasmalemma adjacent to the end and paradermal walls. Such a site may be related to and governed by the same properties which controlled the prior formation of the preprophase band of microtubules in these cells.

Microfilaments associated with cytoplasmic streaming in Nitella flexilis internodes can be decorated with heavy meromyosin (HMM) from rabbit, both in vitro in cytoplasmic suspensions, and in situ in glycerinated cell segments. The bound HMM consists of clearly discernible, polarized arrowheads in a regular repeat of 360-380 Å that are similar to those produced on F-actin. In contrast, similar arrowheads or decorations are not evident on P-protein filaments in sieve elements of glycerinated hypocotyl segments of Phaseolus vulgaris L. treated with HMM. Thus, these results contradict a recent claim that P-protein binds HMM and is actin-like. The mass of other evidence now available from diverse studies indicating that P-protein does not consist of actin or tubulin is discussed.

SUMMARY The dense granulation characteristic of parenchyma cells redifferentiating into tracheary xylem elements was found to be due to a concentration of organelles and associated vesicles in the cytoplasmic bands within which the secondary-wall thickenings form. Among these organelles are mitochondria, plastid-like bodies, elements of the endoplasmic reticulum, and Golgi-structures. Numerous small vesicles occur in the cytoplasm adjacent to developing wall bands and occasionally within the bands themselves. As the secondary-wall reticulations develop, they stain more intensely with permanganate, becoming increasingly more electron dense. The evidence suggests that this is due to their progressive lignification. The electron density appears also in the primary wall at the bases of the bands, and extends through it between opposite bands of adjacent cells.

The role of microtubules in tracheary element formation in cultured stem segments ofColeus has been investigated through the use of the antimicrotubule drug, colchicine. Colchicine treatment of the cultured stem segments produced a dual effect on xylem differentiation. If applied at the time of stem segment isolation or shortly thereafter, wound vessel member formation is almost completely blocked. However, if colchicine is applied after the third day of culture, it does not inhibit differentiation, but instead large numbers of xylem elements are formed which have highly deformed secondary walls. Both effects are related to colchicine's specific affinity for microtubules. In the first case it is shown that colchicine blocks mitosis, presumably by destroying the spindle apparatus, and thus inhibits divisions which are prerequisite for the initiation of xylem differentiation. While, if colchicine is applied after the necessary preparative divisions have taken place, it destroys specifically the cortical microtubules associated with the developing bands of secondary wall, thus causing aberrant wall deposition.

The conformation and structure of an atypical crista found in a small percentage of the mitochondria in root tip cells of Phaseolus vulgaris L. have been studied electron microscopically in material fixed in glutaraldehyde followed by osmium tetroxide. In its transformation into an atypical crista, a normal crista elongates, broadens, and flattens, and the inner leaflets of its apposed unit membranes appear to fuse in a manner analogous to the formation of "tight junctions" between certain animal cells. The result is a large platelike, quintuple-layered structure, 240–260 A thick, whose long axis parallels that of the mitochondrion. The outer layers of the "plate," bordering on the mitochondrial matrix, are thickened and exhibit striking patterns in the micrographs. The structure of the plate is compared with that previously described for tight junctions between animal cells.

Electron microscope studies of the erythrocytic forms, including gametocytes and asexual schizonts, of the protozoa Plasmodium fallax, P. lophurae, and P. cathemerium, have revealed a "cytostome," a specialized organelle of the pellicular membrane which is active in the ingestion of host cell cytoplasm. In material fixed in glutaraldehyde and postfixed in OsO4, the cytostome appears in face view as a pore limited by two dense circular membranes and having an inside diameter of approximately 190 mµ. In cross-section, the cytostome is a cavity bounded on each side by two dense segments corresponding to the two dense circles observed in face view; its base consists of a single unit membrane. In the process of feeding, the cytostome cavity enlarges by expansion of its membrane, permitting a large quantity of red cell cytoplasm to come into contact with the cytostome wall. Subsequent digestion of erythrocyte cytoplasm occurs exclusively in food vacuoles which emanate from the cytostome invagination. As digestion progresses, the food vacuoles initially stain more densely and there is a marked build-up of hemozoin granules. In the final stage of digestion, a single membrane surrounds a cluster of residual pigment particles and very little of the original host cell cytoplasm remains. The cytostome in exoerythrocytic stages of P. fallax has been observed only in merozoites and does not seem to play the same role in the feeding mechanism.

A fine structure study of the phragmoplast and developing cell plate has been made on glutaraldehyde-osmium tetroxide-fixed, dividing, cultured cells of the liquid endosperm of Haemanthus katherinae Baker. The phragmoplast arises between the telophase nuclei, usually in association with a remnant strand of spindle elements, and consists of an accumulation of microtubules oriented at right angles to the plane of the future cell plate. The microtubules, which are 200–240 A in diameter, occur in small clusters spaced at approximately 0.2–0.3 µ intervals along the plate. Short interconnections interpreted as "cross-bridges" have been observed between individual microtubules. Within each cluster there is an electron-opaque zone about 0.3 µ in width which can be attributed in part to an overlap of microtubules from both sides of the plate and in part to a local accumulation of an amorphous electron-opaque material. During development these dense zones become aligned in a plane which itself defines the plane of the plate. Vesicles, commonly observed in long files, are derived from a cytoplasmic matrix rich in elements of the endoplasmic reticulum and sparse in dictyosomes. They aggregate between the clusters of microtubules and eventually coalesce to form the cell plate.

Peroxidase activity has been localized in the cell walls and cytoplasm of wound vessel elements of Coleus which had been fixed in glutaraldehyde, incubated in diaminobenzidine (DAB) and H2O2, and postfixed in OSO4. Electron microscopic investigations revealed prominent staining in the reticulate secondary wall and in the primary wall where the secondary thickenings attach. The stain in the secondary wall is finely textured and heavier towards its periphery than towards its core. The staining of the primary wall, however, is coarsely granular. In the cytoplasm of differentiating vessel elements electron-opaque deposits are observed in the plasmalemma, especially where it overlies the secondary thickening, and in the dictyosomes and their associated vesicles. Staining also occurs on the internal membranes of developing chloroplasts where it is most likely the result of photooxidation of DAB.Staining, except in chloroplasts, appears to be due specifically to peroxidase, since either removal of H2O2 or preincubation with KCN markedly reduces staining, whereas preincubation with aminotriazole, an inhibitor of catalase, does not. The similarity of localization of peroxidase and lignin in the walls of Coleus wound vessel elements supports the postulate that the enzyme participates in lignification.

Chromosome motion during mitosis can be explained by combining the facts available about the equilibrium between the mitotic spindle and its subunits with the postulate that the recently described cross-bridges between spindle microtubules are capable of sliding adjacent tubules over one another.

The fine structure of dividing root tip cells of Phaseolus vulgaris L. has been studied in material fixed in glutaraldehyde followed by osmium tetroxide and poststained in uranyl acetate and lead citrate. Microtubules, observed in both longitudinal and transverse sections of the root tip, are abundant during the early stages of cell plate formation when vesicles from the dictyosomes are aggregating and beginning to fuse in the plate region, but disappear in later stages during which fusion continues and the vesicular aggregates grow laterally. The distribution of microtubules among the vesicles is observable in transverse sections in which the young plate is seen in face view. No consistent grouping of microtubules around the vesicles or structural association between microtubules and vesicles has been detected. Analysis of progressive stages of plate development made from transverse sections reveals that the fusing vesicles subsequently form conspicuous stellate or branched bodies, the prominent linear arms of which protrude at sharp angles from the central mass. It appears that the arms are the sites of further fusion and growth, and that the growth is confined to the plane of the plate. In later stages the branched bodies continue to grow and fuse, creating large interconnected expanses of cell plate material.

The fine structure of the exoerythrocytic cycle of an avian malarial parasite, Plasmodium fallax, has been analyzed using preparations grown in a tissue culture system derived from embryonic turkey brain cells which were fixed in glutaraldehyde-OsO4. The mature merozoite, an elongated cell 3- to 4-µ long and 1- to 2-µ wide, is ensheathed in a complex double-layered pellicle. The anterior end consists of a conoid, from which emanate two lobed paired organelles and several closely associated dense bodies. A nucleus is situated in the mid portion of the cell, while a single mitochondrion wrapped around a spherical body is found in the posterior end. On the pellicle of the merozoite near the nucleus a cytostomal cavity, 80 to 100 mµ in diameter, is located. Based on changes in fine structure, the subsequent sequence of development is divided into three phases: first, the dedifferentiation phase, in which the merozoite loses many complex structures, i.e. the conoid, paired organelles, dense bodies, spherical body, and the thick inner layers of the pellicle, and transforms into a trophozoite; second, the growth phase, which consists of many nuclear divisions as well as parallel increases in mitochondria, endoplasmic reticulum, and ribosomes; and third, the redifferentiation and cytoplasmic schizogony phase, in which the specialized organelles reappear as the new merozoites bud off from the mother schizont.

Bundles of microfilaments very similar in appearance to actin are present in cytoplasmic suspensions obtained from Nitella flexilis . The microfilaments bind rabbit heavy meromyosin in arrowhead arrays similar to those produced on muscle actin. The arrowheads are removed with ATP. The results provide evidence that actin is present in green plants, probably in the form of microfilaments thought to be involved in cytoplasmic streaming.

The mitotic inhibitor isopropyl N-phenylcarbamate (IPC) effects changes in the ultrastructure of the dividing endosperm cells of Haemanthus katherinae. In cells treated with 10 p.p.m. IPC for 0·5–2 h the microtubules lose their parallel alignment and become oriented in radial arrays. These radial arrays are interpreted as micropoles of the spindle apparatus and are thought to be the focal points for the chromosomes as they aggregate into micronuclei. Thus, IPC has a markedly different effect on the structure of the spindle apparatus than colchicine, which is observed at concentrations of 50 p.p.m. for 1 h to destroy the microtubules in Haemanthus. The structure of the microtubules appears unaffected by IPC since those from control and treated cells are similar in diameter (22 nm), in degree of staining, and in sensitivity to the fixation. In addition, the chromosomes and cytoplasmic components such as mitochondria, plastids, dictyosomes, elements of the endoplasmic reticulum, and ribosomes are not structurally modified by treatment with IPC.