Differentiation and Ultrastructure of the Protophloem Sieve Elements of Minor Veins in the Maize Leaves: Changes in the Protoplast

Protophloem sieve element differentiation in the minor veins of the maize ( Zea mays L. ) leaves was first evidenced as an increase of the wall thickness, which began in the comers of the cell and then extended to other parts of the wall, and the appearance of long rough endoplasmic reticulum cistemae distributed throughout the cytoplasm, and then the presence of characteristic crystalloid inclusions within the plastids. As differentiation progressed, long cisternae of rough endoplasmic reticulum appeared to transform into shorter forms and eventually aggregated into small stacks, losing their ribosomes during the process. The nuclei degenerated, although frequently persisted until very late in differentiation the stages of maturation, as darkly stained amorphous aggregates surrounded by double nuclear envelope or only inner membrane of nuclear envelope. Subsequently, the nuclear envelope collapsed and became discontinuous. At the beginning of nuclear degeneration the perinuclear spaces were partly dilated and sometimes the outer nuclear envelope in the dilated portions then ruptured, and was accompanied by the disappearance of the cytoplasmic portion near it. During the peried of nuclear degeneration, in addition to the endoplasmic reticulum, plastids and mitochondria underwent structural modification, while components such as ribosomes, cytoplasmic ground substances, vacuoles and dictyosomes disintegrated and disappeared. At maturity, the surviving protoplasmic components, including plasmalemma, mitochondria, small stacked smooth endoplasmic reticulum and P-type plastids with crystalloids, became parietal in position. As differentiation of adjacent metaphloem sieve elements proceeded, the protoplasmic components of the mature protophloem sieve elements progresively degenerated and finally obliterated.     

Abnormal structure of protophloem sieve-element cell wall in colchicine-treated roots of Triticum aestivum L.

The structural aberrations of the cell walls of protophloem sieve elements (PSEs) in roots of wheat (Triticum aestivum L. cv. Maris Huntsman) caused by the anti-microtubule drug colchicine were investigated by electron microscopy. The initial effect of the drug on cell wall development was found to be an exceptionally rough wall surface, presumably caused by an uncontrolled fusion of Golgi vesicles with the plasma membrane. Cellulose microfibrils, which in normal PSEs are aligned transversely to the long axis and parallel to the cortical microtubules, in colchicine-treated PSEs display a predominant longitudinal orientation. The pattern of wall development is disturbed by deposition of wall material also within the sieve pores of the sieve-pore/plasmodesmata complexes, resulting in evenly thickened walls instead of the normal uneven layers, and in narrowing the sieve pores to the size of plasmodesmata. In prolonged and continuous colchicine treatment, PSEs develop unusual wall ingrowths projecting deeply into the cytoplasm, creating an extraordinary cell type not found in normal roots. The results confirm the view of microtubule involvement in the proper deposition and orientation of cellulose microfibrils, and in the normal patterning of the cell wall thickenings of differentiating PSEs.Abbreviations c colchicine-treated - PSE protophloem sieve element The author is grateful to Dr. B. Galatis, Dr. P. Apostolakos and Dr. C. Katsaros, Institute of General Botany, University of Athens, Greece, for helpful discussions and suggestions, and for the generous gift of the colchicine used here. This work was carried out in the Department of Botany, University of Thessaloniki, Greece, while observations were also made in the Lehrstuhl für Zellenlehre, University of Heidelberg, Germany, and in the Department of Botany, University of Georgia, USA. The author is thankful to Prof. E. Schnepf (Zellenlehre, Heidelberg, Germany) and Prof. B.A. Palevitz (Department of Botany, University of Athens, Ga., USA), for generously providing access to their equipment and facilities. The work was financially supported in part by the Stiftung Volkswagenwerk and by the Research Committee, University of Thessaloniki (No 7537).     

Development of protophloem in roots ofAegilops comosa var.thessalica. I. Differential divisions and pre-prophase bands of microtubules

Summary The formative divisions of protophloem mother cells in roots of the grassAegilops comosa var.thessalica have been investigated by means of light and electron microscopy. Two successive differential divisions create protophloem poles consisting of a protophloem sieve element and two companion cells. Pre-prophase bands of microtubules appear in premitotic cells anticipating the plane of orientation of the cell plate and indicating the site where the daughter wall will join the parent one. Evidence is accumulated that the site previously occupied by pre-prophase band is bisected by the new wall. Structural asymmetries that could express polarity, like organelle displacement, were not observed in premitotic cells. A working hypothesis is proposed integrating the conclusions of the present study in a diagram correlating pre-prophase bands of microtubules and differential divisions.     

Sieve-element plastids ofTriticum andAegilops (Poaceae)

Fourteen taxa of the Triticum-Aegilops group have been investigated for their sieve-element plastids. At maturity they contain dense and thin crystalloid inclusions and are classified into the PIIc' plastid type; onlyAe. comosa var.biaristata lacked the thin crystalloids and thus conforms to the PII c type. The proteinaceous nature of the crystalloids was demonstrated by application of proteolytic enzymes. Ultrastructural evidence suggests that both kinds of crystalloid inclusions are involved in the sealing of sieve-plate pores of injured sieve tubes. Measurements and calculations of the spacings and angles carried out on crystalloid prints permitted the construction of a two- and three-dimensional pattern forT. aestivum thin crystalloids.     

Structure and development of sieve cells in the primary phloem ofPinus resinosa

Summary The primary phloem consists mostly of sieve cells. Procambial cells and very young sieve cells contain all the components characteristic of young nucleate cells. Increase in wall thickness, which is relatively limited, constitutes the first indication of sieve-cell differentiation. During the period of wall thickening, the plastids develop starch grains and then fibrillar inclusions. Eventually the internal lamellae of the plastids collapse. The plastids do not form crystalline inclusions. As the sieve cell approaches maturity, an extensive network of smooth, tubular endoplasmic reticulum (ER) appears and then becomes mostly parietal in distribution. At maturity, large aggregates of this ER occur at the sieve areas. These aggregates are interconnected longitudinally by the parietal network of ER. In addition to the ER, the mature, plasmalemma-lined primary sieve cell contains a degenerate nucleus, with intact nuclear envelope, plastids, and mitochondria. Dictyosomes, ribosomes, and vacuoles are lacking. P-protein is not present at any stage of development.This work was supported by U.S. National Science Foundation grants GB 8330 and GB 31417 to R. F.Evert.     

Postnatal differentiation of the Golgi apparatus and the dendrites of Purkinje cells of the rat cerebellum

Summary The morphology of postnatal differentiation of the Golgi apparatus, the nucleus, the perikaryon, and the dendrites was studied in Purkinje cells of the rat cerebellum for 30 days after birth using histochemical, histological, and electron microscopic methods.The Golgi apparatus during differentiation undergoes morphological and positional changes. From the 1st to 7th postnatal day, the Golgi apparatus is found in a supranuclear position, and is connected with the axes of differentiating primary dendrites by beam-like processes. From days 8 to 11 this connection disappears, and most of the Golgi apparatus assumes a lateronuclear and infranuclear position. After the 11th or 12th day, the Golgi apparatus is found in perinuclear and peripheral cytoplasmic positions. The formation of granular endoplasmic reticulum occurs in the vicinity of the perinuclear Golgi apparatus. The differentiation of cell and nuclear forms requires approximately 20 days. The morphological changes of differentiation are discussed in relation to the participation of the Golgi apparatus in the differentiation of dendrites and in the formation of the granular endoplasmic reticulum.     

Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh

Protoplast cultures of Vicia hajastana have a high division frequency. However, 20–40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole) and cell walls by Calcofluor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MT microtubule(s) - PB prophase band(s) - PNF perinuclear fluorescence - PPB pre-prophase band     

Reprogramming of cells following wounding in pea (Pisum sativum L.) roots. I. Cell division and differentiation of new vascular elements

Summary Normally quiescent cortical tissue of pea roots can be induced, by severing the adjacent vascular cylinder of the root, to undergo redifferentiation to form new files of tracheary and sieve elements which bridge the wound. The development of vascular transfer cells is also induced. Redifferentiation is normally accompanied by division of the original cortical cells. The planes of cell division, especially those preceding sieve element formation, are aligned very precisely in adjacent cells, to produce smooth files of cells. In roots wounded 3–4 mm from the apex, bands of microtubules in the periphery of the cells (pre-prophase bands) form at sites which correspond to the expected planes of cell division.     

AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells.     

Microtubule pattern and the occurrence of pre-prophase bands in embryogenic cultures of black spruce (Pieca mariana Mill.) and non-embryogenic cultures of jack pine (Pinus banksiana Lamb.)

Summary The organization of microtubules during interphase and prophase in embryogenic cultures of black spruce (Picea mariana) was investigated by indirect immunofluorescence. Somatic embryos of black spruce possessed an extensively branched and interconnecting network of fine interphase cortical microtubules. The development of pre-prophase bands (PPBs) in embryogenic black spruce cultures was compared with that in non-embryogenic cell cultures of jack pine (Pinus banksiana). PPBs in both species were initially arranged as a very broad array of microtubules, later (early to mid-prophase) becoming narrower and more intensely fluorescent. The occurrence of pre-prophase bands in relation to the number of phragmoplasts (i.e. PPB index) of black spruce somatic embryos was significantly higher (p<0.01) than that found for jack pine cells.     

Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5–3-m-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.     

Endoplasmic Reticulum to Golgi Trafficking in Multinucleated Skeletal Muscle Fibers

The organization of membrane trafficking between endoplasmic reticulum and Golgi within multinucleated muscle fibers was analyzed. We found that markers for the compartment involved in endoplasmic reticulum to Golgi trafficking exhibited perinuclear as well as interfibrillar localization. Furthermore, these markers showed prominent colocalization with microtubules. To analyze membrane trafficking, we followed the temperature-controlled transport of the G protein of the mutant vesicular stomatitis virus, tsO45, in isolated myofibers. Perinuclear and cross-striated staining were seen at 39°C, while at 15°C a diffuse staining component appeared along a subset of interfibrillar microtubules. At 20°C, bright Golgi spots were seen to be associated with microtubules that appeared as circumnuclear rings and longitudinal bundles. Beneath the motor end plate, however, the organization of the Golgi elements and microtubules was found to be distinctive. Retrograde trafficking induced by brefeldin A resulted in the disappearance of the Golgi spots throughout the myofibers and the appearance of staining along microtubules. Thus, interfibrillar membranes seem to be active in protein export, and trafficking between endoplasmic reticulum and Golgi elements occurred throughout the myofibers. The results suggest that microtubules served as tracks for the two-way trafficking between the endoplasmic reticulum and the Golgi compartment.     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells

Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation. microtubules; confocal immunofluorescence microscopy; extracellular matrix; osteoblast differentiation     

Monoplastidic mitosis in the mossTimmiella barbuloides (Bryophyta)

Summary Mitotic cell division of monoplastidic sporogones was investigated in the mossTimmiella barbuloides (Brid.) Moenk. (Pottiales, Bryophyta) by TEM. Division polarity of sporogones is established by the interphase position of the single oblong cup-shaped plastid, which is orientated with its long axis parallel to one of the cell walls. In preprophase the plastid elongates and its extremities bend at right angles. Plastid growth is directed by microtubules and accompanied by plastid tubules. The plastid begins the process of duplication by constricting centrally in the plane of the future cytokinetic septum. There is no preprophase band of microtubules at the division site. The large central nucleus becomes fusiform and aligned parallel to the main plastid axis. By the end of prophase the daughter plastids are positioned at the opposite poles of the nucleus where they probably function as nucleating or organizing centres for the spindle microtubules. Metaphase and anaphase spindles contain long sheets of ER. Cytokinesis involves the formation of a well developed phragmoplast.Abbreviations TEM transmission electron microscopy - PPB preprophase band of microtubules - ER endoplasmic reticulum     

Ultrastructural features of differentiating protophloem sieve elements in adventitious roots of Salix viminalis

The differentiation of the protophloem in 9- to 14-day-old adventitious roots of Salix viminalis was studied. Ultrastructural observations were mainly made on longitudinal serial sections through an uninterrupted file of 32 differentiating sieve elements. The first cell in the file was located about 50 μm from the apical meristem. At an early stage the nucleus was lobed in outline, and in older cells the nucleoplasm became electron lucent. In the first or second cell from the first mature sieve element the nuclear envelope broke open. The nucleoli decreased gradually in size and disappeared finally. From the 9th cell the plastids contained starch and grew somewhat in size. ER increased in amount and began to form stacks in the 20th cell. These stacks moved to a peripheral position. Callose platelets were first observed on the transverse walls in cell 18. Flattened ER-cisternae covered the sieve pore sites. Gradually the middle lamella was dissolved and the callose aggregations formed cylinders around the pores of the sieve plate. Aggregations of tubular P-protein were present from cell 15. P-protein bodies were also present in parenchyma cells adjoining mature sieve elements. The only cell components remaining in mature sieve elements were plastids, mitochondria, stacked ER, the plasmalemma, remnants of other membranes and bodies consisting of P-protein and of an unidentified granular material. The sieve elements had no ontogenetically related companion cells. At a level where both metaphloem and metaxylem had matured the first formed protophloem sieve elements remained intact.     

Ultrastructural Observations of Papaver rhoeas Mature Pollen Grains

The mature pollen grain of Papaver rhoeas is bicellular. The vegetative cell contains numerous mitochondria; endoplasmic reticulum is not very extensive and there are few ribosomes and plastids. Golgi bodies are in a very active state. The generative cell is lobed and spindle-shaped. The cytoplasm contains many, generally longitudinally arranged, bundles of microtubules. Other organelles are few in number, and include mitochondria, Golgi bodies and short cisternae of endoplasmic reticulum.     

Presence and absence of the preprophase band of microtubules in moss protonemata: a clue to understanding its function?

Summary InFunaria protonemata, preprophase bands (PPBs) of microtubules do not develop when the tip cell divides, when side branches are initiated or in intercalary regeneration divisions. We report here that PPBs do, however, develop when a tmema cell is formed. In the former cases, cell division is not coupled with an expansion of the mother cell wall at the site where the cell plate will attach. In the latter case, the mother cell wall ruptures at that site and the tmema cell elongates. This observation and the findings on presence and absence of the PPB in other cell types indicate a connection between PPB occurrence and mother cell wall expansion. They support the idea that the PPB might be involved in the local secretion of cell wall material. We extend this notion, suggesting that the microtubules of the PPB control the oriented deposition of a thin layer of cellulose microfibrils at the mother cell wall which supports the firm attachment of the cell plate when the mother cell wall expands.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - MT microtubule - PPB preprophase band of microtubules - TC tmema cell     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band