FINE STRUCTURE STUDIES OF PHLEUM ROOT MERISTEM CELLS. I. MITOCHONDRIA

Avers , Charlotte J. (Douglass Coll., Rutgers—The State U., New Brunswick, N.J.) Fine structure studies of Phleum root meristem cells. I. Mitochondria. Amer. Jour. Bot. 49(9): 996–1003. Illus. 1962.—Mitochondrial numbers were computed from thick sections embedded in paraffin and stained with iron hematoxylin, from methacrylate embedded, 0.5 μ-thick sections stained with osmium, and from electron micrographs of ultrathin sections fixed in KMnO₄. The mean mitochondrial counts per average cell (20 × 20 × 10μ) were: 121 ± 4 in paraffin sections, 600 ± 40 in methacrylate sections, and 991 ± 96 from electron micrographs. These numbers were higher than the Janus green B count of 91 ± 2 per cell found during an earlier study. In each case, arithmetical conversion factors were used to calculate total numbers of mitochondria per cell since section thicknesses were less than that of whole cell length or depth. Determinations of numbers of mitochondria probably varied depending on section thickness and specific staining procedures used, but the higher count from electron micrographs was assumed to be reasonable on a volumetric basis. The cytoplasmic volume of the average cell is about 2500 μ³ and the volume of a single average mitochondrion is about 0.1μ^3. On this basis, the variety of structures and their relatively sparse distribution seems possible despite the apparently high numbers found. In addition to mitochondria, the numbers of various cellular components per unit cytoplasmic area were determined. These data showed that mitochondria were about 6 times more numerous than proplastids, about 4.5 times more numerous than Golgi bodies, and even more frequent when compared with vesicles, dense bodies, or microbodies. No correlation was found between cytoplasmic area and numbers of organelles per unit area. Photographic evidence was presented for the occurrence in plant cells of the hepatic cell, polymorphic “lysosome” described by Ashford and Porter. The controversial nature of the lysosome is discussed.     

PHOSPHATASE ACTIVITY AND CELLULAR DIFFERENTIATION IN PHLEUM ROOT MERISTEM

Using 9 different organic phosphate substrates as alternatives in a standardized 5′-nucleotidase histochemical test system, enzyme activity patterns were recorded for timothy grass root epidermis. At least 4 different phosphatases were distinguished on the bases of substrate specificity, reaction rate, tissue distribution, and response to inhibitors. Except with adenosine-3′-monophosphate, all activities were restricted to the 300-mμ-long root tip meristem. These enzyme activities were associated with the earliest phases of differentiation of the epidermal hair and hairless cell initials. The distribution of activities was not associated with the same cell type in each part of the meristem. Little activity was found with most substrates in the undifferentiated cells of the 0-100μ zone; alternating active hairless and inactive hair cell initials predominated in the 100-200μ segment; and active hair–inactive hairless sister cells formed the principal pattern in the 200-300μ segment of the meristem. The data showed that a particular enzyme activity was associated with a specific cell type only in relation to that cell's position along the differentiation gradient of the entire tissue. But, within a meristem segment, a specific cell type might act differently from its neighbors, depending on its mitotic capacity. This complex of physiological dependence and independence of a cell type on tissue ontogeny was cited as a characteristic of the phenomenon of cellular differentiation superimposed on tissue differentiation gradients.     

STUDIES IN THE QUIESCENT ROOT MERISTEM OF THE BULB OF ALLIUM CEPA L.: FINE STRUCTURE AND DNA SYNTHESIS *

Study of the quiescent root meristem of Allium cepa L. bulbs has revealed that its histological organization does not differ significantly from the growing meristem, except for the fact that the cells are all arrested in interphase. Ultrastructure of the quiescent tissue is, however, different in the organization of the nucleolus and in the absence of prominent endoplasmic reticulum, microtubules and golgi complexes. A variety of lomasome-like structures, plasma membrane modifications and vacuoles have been recorded. Most of the cells except for the ones in the root cap and quiescent center are highly vacuolated; vacuolation is maximum in the cortical zone of the meristem. The pattern of ³H thymidine incorporation during early stages of sprouting indicates that asynchrony of the mitotic cycle, which is the characteristic of the growing meristem, is maintained during quiescence by the arrest of nuclei at different subphases of interphase.     

NUCLEOLAR VARIATION DURING DIFFERENTIATION OF PHLEUM ROOT EPIDERMIS

Striking differences in nucleolar volume were found between trichoblasts and hairless initials all along the differentiation gradient of the tissue. The larger nucleoli of the trichoblasts were evident from the onset of their differentiation in the meristem and remained so throughout the growing 1000μ of epidermis. At the same time, nucleoli of the alternating, hairless initials rapidly became reduced in volume and virtually disappeared in maturing cells. Nuclear volume was relatively constant throughout the epidermis, so that nuclear:nucleolar volume ratios clearly indicated the nucleolar differences between the shorter trichoblasts and longer hairless initials. Along with the first signs of nucleolar volume difference in the meristem, there were higher concentrations of RNA and ribonucleoprotein in nucleoli and cytoplasm of trichoblasts compared with hairless initials. Although these chemical differences occurred principally in meristem cells, protein content was much higher in trichoblasts than in the alternating hairless initials of the enlargement zone, 300–500μ from the root tip apex. There was no essential difference in protein content between the 2 cell types in the meristem or in older enlarging cells. The data suggested that the initial increase in nucleolar volume and content of the meristem trichoblasts led to their increased protein content and enzyme activities during the enlargement phase of their differentiation. The sharp reduction in nucleoli of the hairless initials, at the same time, led to their generally lower metabolism during tissue differentiation.     

POLYRIBOSOMES IN PROLIFERATING AND NONPROLIFERATING ROOT MERISTEM CELLS

Pea root meristem cells which are actively dividing contain higher proportions of polyribosomes than do cells whose progression through the mitotic cycle has been arrested by starvation. The initiation of cell division which follows the addition of sucrose to starved roots is accompanied by an increase in polyribosome content. This reformation of polyribosomes is insensitive to actinomycin D; it is suggested that preexisting mRNA molecules, synthesized prior to starvation, participate in polyribosome reformation, but that those mRNA species necessary for movement of cells through the mitotic cycle are gradually lost during the starvation period.     

THE ROOT APICAL MERISTEM OF ASPLENIUM BULBIFERUM: STRUCTURE AND DEVELOPMENT

The root apical meristem of Asplenium bulbiferum Forst. f. has a prominent four-sided pyramidal cell with its base in contact with the rootcap. Derivatives (merophytes) that contribute to the main body of the root are produced from the three proximal faces of the apical cell. The rootcap has its origin from the fourth (distal) face of the apical cell. The first division in a proximal merophyte is periclinal to the root surface, separating an outer cell and an inner cell. The outer cell is the origin of the outer part of the cortex and the epidermis; the larger inner cell is the origin of the inner cortex, endodermis, pericycle, and vascular tissue. After the establishment of the basic number of cells in a unilayered merophyte, the cells undergo transverse divisions forming longitudinal files of cells. The mitotic index of the apical cell indicates that it is not a quiescent cell. Also, the first plane of division in a newly formed merophyte dictates that the apical cell is the originator of merophytes.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

THE ROOT APICAL MERISTEM OF EQUISETUM DIFFUSUM: STRUCTURE AND DEVELOPMENT

The root apical meristem of Equisetum diffusum Don has a prominent four-sided pyramidal apical cell with its base (distal face) in contact with the root cap. Derivatives (merophytes) that contribute to the main body of the root are produced from the three proximal faces of the apical cell. The first division of a proximal merophyte is periclinal to the root surface separating a small inner cell from a larger outer cell. The inner cell is the precursor of the vascular cylinder. The larger outer cell is the precursor of the epidermis, cortex, endodermis, and pericycle. Radial sectors, established early in the development of the cortex, alternate with sectors in the vascular cylinder. These developmental steps show quite clearly that early root development in Equisetum is markedly different from that of most ferns.     

STUDIES ON THE FINE STRUCTURE OF ULTRACENTRIFUGED SPINAL GANGLION CELLS

The following structures were observed in electron micrographs of the mouse spinal ganglion cells: Nissl bodies composed of both aggregated rough-type, largely oriented, membranes of the endoplasmic reticulum and discrete particles; short rodlike mitochondria with well-developed transverse, obliquely or longitudinally arranged cristae, and a relatively typical Golgi complex. The components of ultracentrifuged ganglion cells (400,000 times gravity for 20 minutes) are stratified, the layers appearing in the order of their decreasing density as follows: (1) A microsomal or ergastoplasmic layer which may be further divided into three sublayers without sharp boundaries, namely, a discrete particle layer, a layer of discrete particles and highly distorted membranes of the endoplasmic reticulum, and a layer composed of relatively intact, but stretched membranes of the endoplasmic reticulum and discrete particles. (2) Mitochondria constitute a relatively broad layer. They are sometimes stretched; however, they retain most of their fine structure. The stratified nucleus is found within the mitochondrial layer. (3) A relatively wide layer of tightly packed vesicles. (4) At the centripetal end, resting against the cell membrane, are a few lipid vacuoles. A comparison is made between the ultrastructure of the stratified layers in situ and those described by others in differentially ultracentrifuged homogenates.     

HISTOCHEMICAL LOCALIZATION OF ENZYME ACTIVITIES IN ROOT MERISTEM CELLS

Avers , Charlotte J. (Douglass Coll., Rutgers—The State University, New Brunswick, N. J.) Histochemical localization of enzyme activities in root meristem cells. Amer. Jour. Bot. 48(2): 137–143. Illus. 1961,—Particle counts were made in epidermal cells of the root meristem of 2 grasses after exposure of living seedlings to various substrates involved in dehydrogenation reactions. Hydrolytic enzyme activities also were recorded for 1 of these species. The mean number of particles stained with Janus green B was about 90 for each species, but significantly lower counts were obtained in all the dehydrogenase tests. With lactate, pyruvate, glutamate, citrate, and isocitrate as substrates, Phleum cells showed about 25% of the Janus green count, while Panicum cells were about 33% active. These substrates are known to be oxidized by DPN-linked enzymes. The succinic dehydrogenase counts were about 50% of the Janus green total, and 60–70% particle activity was recorded with hexose-phosphate substrates which are probably oxidized in TPN-mediated reactions. In Phleum, particulate activity occurred in the adenosine triphosphatase and aryl sulfatase tests, but a non-particulate distribution characterized 5-nucleotidase activity. The particle counts in the ATPase tests were not significantly different from the Janus green counts, but the 85% particle activity in the aryl sulfatase tests was significantly lower than the Janus green results. These intracellular distributions were compared with those obtained by various authors using biochemical and histochemical techniques and were found to be in close agreement. It was suggested that the evidence indicated intracellular differentiation of at least one kind of cellular organelle, which in all probability was mitochondria.     

SECRETORY CELLS OF LILY PISTILS. I. FINE STRUCTURE AND FUNCTION

The fine structure of the cells which line the canal of Lilium longiflorum pistils confirms the secretory function which has been ascribed to them. The cells differ in structure from the secretory cells which cover the stigma surface and are therefore referred to as canal cells rather than stigmatoid cells. Their most striking feature is an elaborate wall, 8–14 μ in maximum depth, on the side facing the canal, which with associated structures, we term the secretion zone. Pollination, which triggers chemotropic activity in the style and secretory activity in the canal cells, is not correlated with marked changes in the fine structure of the canal cells. The canal cells appear to fit well into that category which Gunning and Pate have termed “transfer cells.”     

PROBABILITY OF DIVISION OF CELLS IN THE EPIDERMIS OF THE PHLEUM ROOT

Erickson , Ralph O. (U. Pennsylvania, Philadelphia.) Probability of division of cells in the epidermis of the Phleum root. Amer. Jour. Bot. 48(3): 268–274. Illus. 1961.—Photomicrographic records of the growth of a Phleum root, made at R. H. Goodwin's laboratory, in which individual epidermal cells can be identified, have been analyzed to provide estimates of the probability that cells at various distances from the apex will divide. In the apical part of the meristem, from 0μ to about 100μ from the apex, all cells divide (prob. = 1.0). From about 100μ to 275μ, the probability of division falls progressively to 0.0. The relationship of these estimates to rates of cell division and elongation in the same root is discussed.     

FINE STRUCTURE OF THE DIATOM AMPHIPLEURA PELLUCIDA. II. CYTOPLASMIC FINE STRUCTURE AND FRUSTULE FORMATION

The general arrangement of cytoplasmic organelles in Amphipleura pellucida Kutz. is similar to that in other naviculoid diatoms. The chromatophores are parietal with a single, non-membrane-limited, pyrenoid. The pyrenoid is crossed by several double-disc lamellar bands which are occasionally interrupted by less dense areas containing convoluted tubules. Similar areas also interrupt the three-disc bands of the chromatophores. The nucleus is irregular in shape. The outer membrane of the porous nuclear envelope outfolds around the chromatophore. A perinuclear dictyosome complex is present. Amorphous dense bodies are formed in elaborations of the dictyosomes. Vesicles, both with and without dense inclusions, are formed by the dictyosomes during cell division and a role is suggested for these vesicles in both cytokinesis and frustule development. The first evidence of frustule formation is the deposition of the siliceous median rib within a membranous sac. This sac expands laterally to form the silica deposition vesicle which appears to serve as a mold for the formation of the valve. After the valve is formed, the membranes and the small amount of cytoplasm external to it are sloughed off.     

QUANTITATIVE STUDIES OF THE ROOT APICAL MERISTEM OF EQUISETUM SCIRPOIDES

An investigation was made of the meristematic activity of the apical cell, its immediate derivatives (merophytes), and of other selected cell populations of the root of Equisetum scirpoides Michx. The plane of the first division of a derivative of the apical cell is radiallongitudinal, which provides evidence that merophytes immediately adjacent to the apical cell cannot be the ultimate root initials. The apical cell is as active mitotically in roots 20–40 mm long as it is in roots that are 0.25–1 mm in length. The mitotic activity of the apical cell and of other cell populations was determined from the mitotic index, and from determination of the durations of the cell cycle and of mitosis of the apical cell by using the colchicine method of metaphase accumulation. Microspectrophotometric measurements of DNA content indicated that there was no consistent increase in DNA (endopolyploidy) in the apical cell or in the other meristematic cells as roots increased in length. Conclusion: there is no evidence that the apical cell becomes quiescent or undergoes endopolyploidy as a root increases in length.     

THE FINE STRUCTURE OF BONE CELLS

An electron microscopic study of Araldite-embedded, undecalcified human woven and chick lamellar bone is presented. The fine structure of the cells of bone in their normal milieu is described. Active osteoblasts possess abundant granular endoplasmic reticulum, numerous small vesicles, and a few secretion droplets. Their long cytoplasmic processes penetrate the osteoid. The transition of osteoblasts into osteoid osteocytes and then into osteocytes is traced and found to involve a progressive reduction of cytoplasmic organelles. Adjoining the osteocytes and their processes is a layer of amorphous material which is interposed between the cell surfaces and the bone walls of their respective cavities. Osteoclasts contain numerous non-membrane-associated ribosomes, abundant mitochondria, and little granular endoplasmic reticulum, thus differing markedly from other bone cells. The brush border is a complex of cytoplasmic processes adjacent to a resorption zone in bone. No unmineralized collagen is seen at resorption sites and it appears that collagen is removed before or at the time of mineral solution. All bone surfaces are covered by cells, some of which lack distinctive qualities and are designated endosteal lining cells. The structure of osteoid, bone, and early mineralization sites is illustrated and discussed.